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Morrison, Robert J., Nicolas-George Katsantonis, Kevin M. Motz, Alexander T. Hillel, C. Gaelyn Garrett, James L. Netterville, Christopher T. Wootten, et al. "Pathologic Fibroblasts in Idiopathic Subglottic Stenosis Amplify Local Inflammatory Signals." Otolaryngology–Head and Neck Surgery 160, no. 1 (October 16, 2018): 107–15. http://dx.doi.org/10.1177/0194599818803584.
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Objective To characterize the phenotype and function of fibroblasts derived from airway scar in idiopathic subglottic stenosis (iSGS) and to explore scar fibroblast response to interleukin 17A (IL-17A). Study Design Basic science. Setting Laboratory. Subjects and Methods Primary fibroblast cell lines from iSGS subjects, idiopathic pulmonary fibrosis subjects, and normal control airways were utilized for analysis. Protein, molecular, and flow cytometric techniques were applied in vitro to assess the phenotype and functional response of disease fibroblasts to IL-17A. Results Mechanistically, IL-17A drives iSGS scar fibroblast proliferation ( P < .01), synergizes with transforming growth factor ß1 to promote extracellular matrix production (collagen and fibronectin; P = .04), and directly stimulates scar fibroblasts to produce chemokines (chemokine ligand 2) and cytokines (IL-6 and granulocyte-macrophage colony-stimulating factor) critical to the recruitment and differentiation of myeloid cells ( P < .01). Glucocorticoids abrogated IL-17A-dependent iSGS scar fibroblast production of granulocyte-macrophage colony-stimulating factor ( P = .02). Conclusion IL-17A directly drives iSGS scar fibroblast proliferation, synergizes with transforming growth factor ß1 to promote extracellular matrix production, and amplifies local inflammatory signaling. Glucocorticoids appear to partially abrogate fibroblast-dependent inflammatory signaling. These results offer mechanistic support for future translational study of clinical reagents for manipulation of the IL-17A pathway in iSGS patients.
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Swieboda, Dominika, Erica Johnson, Ioanna Skountzou, and Rana Chakraborty. "Baby’s First Macrophage: How do placental macrophages (Hofbauer Cells, HCs) contribute to immune tolerance and infection response during pregnancy?" Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 126.28. http://dx.doi.org/10.4049/jimmunol.202.supp.126.28.
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Abstract Placental immunity is dichotomous: tolerance of the semiallogenic fetus is balanced with limiting transmission of maternal pathogens. HCs are the major fetal immune cell at the placenta, but mechanisms responsible for maintaining immune homeostasis while preventing infection require elucidation. We determined the phenotype of human HCs throughout gestation and analyzed stimulation response. Activated HCs were present in early pregnancy and reduced in number by term while maintaining similar phenotypes. Tolerant HC numbers were highest in midgestation, after a relatively intolerant phenotype early in gestation. We saw a significant shift in macrophage polarization as gestation progressed. Transcription of ARG2 exceeded iNOS at all points, reaching 10-fold higher at term. Following treatment with IL4+IL13 or IFNγ+LPS midgestation HCs underwent significant phenotype change and activation. Basal expression of antiviral IFN stimulated genes (ISGs) was lowest at midgestation and was enhanced by IFN-α and IFN-λ1 with a 10-fold stronger response to IFN-α. RIG-I agonism induced HC activation, 10-fold iNOS upregulation and enhanced transcription of IFNs, MDA5, RIG-I, and ISGs. Response to stimulation by IFNγ+LPS, IL4+IL13, IL1β+HAGG was limited to loss of tolerance at term. IL-10 treatment increased numbers of CD163+ HCs, and IFNγ+LPS caused loss of discernible polarization patterns. Basal expression of RIG-I, MDA5 and ISGs was highest in term HCs, but IFN-α did not activate them. RIG-I agonism reduced markers of tolerance. HCs are variable macrophages, with phenotype and immune capacity strongly dependent on gestational age. Understanding placental immunobiology is fundamental to addressing key pregnancy morbidities.
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Gluchowski, Marcel, Xiaoqiong Yu, Bernard Abrenica, Samantha Yao, Joshua Kimani, Renée N. Douville, Terry Blake Ball, and Ruey-Chyi Su. "Transient Increases in Inflammation and Proapoptotic Potential Are Associated with the HESN Phenotype Observed in a Subgroup of Kenyan Female Sex Workers." Viruses 14, no. 3 (February 25, 2022): 471. http://dx.doi.org/10.3390/v14030471.
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Interferon (IFN) -stimulated genes (ISGs) are critical effectors of IFN response to viral infection, but whether ISG expression is a correlate of protection against HIV infection remains elusive. A well-characterized subcohort of Kenyan female sex workers, who, despite being repeatedly exposed to HIV-1 remain seronegative (HESN), exhibit reduced baseline systemic and mucosal immune activation. This study tested the hypothesis that regulation of ISGs in the cells of HESN potentiates a robust antiviral response against HIV. Transcriptional profile of a panel of ISGs with antiviral function in PBMC and isolated CD4+ T cells from HESN and non-HESN sex worker controls were defined following exogenous IFN-stimulation using relative RT-qPCR. This study identified a unique profile of proinflammatory and proapoptotic ISGs with robust but transient responses to exogenous IFN-γ and IFN-α2 in HESN cells. In contrast, the non-HESN cells had a strong and prolonged proinflammatory ISG profile at baseline and following IFN challenge. Potential mechanisms may include augmented bystander apoptosis due to increased TRAIL expression (16-fold), in non-HESN cells. The study also identified two negative regulators of ISG induction associated with the HESN phenotype. Robust upregulation of SOCS-1 and IRF-1, in addition to HDM2, could contribute to the strict regulation of proinflammatory and proapoptotic ISGs in HESN cells. As reducing IRF-1 in the non-HESN cells resulted in the identified HESN ISG profile, and decreased HIV susceptibility, the unique HESN ISG profile could be a correlate of protection against HIV infection.
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Gupta, Sarthak, Shuichiro Nakabo, Luz P. Blanco, Liam J. O’Neil, Gustaf Wigerblad, Rishi R. Goel, Pragnesh Mistry, et al. "Sex differences in neutrophil biology modulate response to type I interferons and immunometabolism." Proceedings of the National Academy of Sciences 117, no. 28 (June 29, 2020): 16481–91. http://dx.doi.org/10.1073/pnas.2003603117.
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Differences between female and male immunity may contribute to variations in response to infections and predisposition to autoimmunity. We previously reported that neutrophils from reproductive-age males are more immature and less activated than their female counterparts. To further characterize the mechanisms that drive differential neutrophil phenotypes, we performed RNA sequencing on circulating neutrophils from healthy adult females and males. Female neutrophils displayed significant up-regulation of type I IFN (IFN)-stimulated genes (ISGs). Single-cell RNA-sequencing analysis indicated that these differences are neutrophil specific, driven by a distinct neutrophil subset and related to maturation status. Neutrophil hyperresponsiveness to type I IFNs promoted enhanced responses to Toll-like receptor agonists. Neutrophils from young adult males had significantly increased mitochondrial metabolism compared to those from females and this was modulated by estradiol. Assessment of ISGs and neutrophil maturation genes in Klinefelter syndrome (47, XXY) males and in prepubescent children supported that differences in neutrophil phenotype between adult male and female neutrophils are hormonally driven and not explained by X chromosome gene dosage. Our results indicate that there are distinct sex differences in neutrophil biology related to responses to type I IFNs, immunometabolism, and maturation status that may have prominent functional and pathogenic implications.
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Jurczyszak, Denise, Lara Manganaro, Sofija Buta, Conor Gruber, Marta Martin-Fernandez, Justin Taft, Roosheel S. Patel, et al. "ISG15 deficiency restricts HIV-1 infection." PLOS Pathogens 18, no. 3 (March 25, 2022): e1010405. http://dx.doi.org/10.1371/journal.ppat.1010405.
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Type I interferons (IFN-Is) are a group of potent inflammatory and antiviral cytokines. They induce IFN stimulated genes (ISGs), which act as proinflammatory mediators, antiviral effectors, and negative regulators of the IFN-I signaling cascade itself. One such regulator is interferon stimulated gene 15 (ISG15). Humans with complete ISG15 deficiency express persistently elevated levels of ISGs, and consequently, exhibit broad spectrum resistance to viral infection. Here, we demonstrate that IFN-I primed fibroblasts derived from ISG15-deficient individuals are more resistant to infection with single-cycle HIV-1 compared to healthy control fibroblasts. Complementation with both wild-type (WT) ISG15 and ISG15ΔGG (incapable of ISGylation while retaining negative regulation activity) was sufficient to reverse this phenotype, restoring susceptibility to infection to levels comparable to WT cells. Furthermore, CRISPR-edited ISG15ko primary CD4+ T cells were less susceptible to HIV-1 infection compared to cells treated with non-targeting controls. Transcriptome analysis of these CRISPR-edited ISG15ko primary CD4+ T cells recapitulated the ISG signatures of ISG15 deficient patients. Taken together, we document that the increased broad-spectrum viral resistance in ISG15-deficiency also extends to HIV-1 and is driven by a combination of T-cell-specific ISGs, with both known and unknown functions, predicted to target HIV-1 replication at multiple steps.
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Hancock, Meaghan H., Karen L. Mossman, and James R. Smiley. "Cell Fusion-Induced Activation of Interferon-Stimulated Genes Is Not Required for Restriction of a Herpes Simplex Virus VP16/ICP0 Mutant in Heterokarya Formed between Permissive and Restrictive Cells." Journal of Virology 83, no. 17 (June 17, 2009): 8976–79. http://dx.doi.org/10.1128/jvi.00142-09.
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ABSTRACT Herpes simplex virus VP16 and ICP0 mutants replicate efficiently in U2OS osteosarcoma cells but are restricted in other cell types. We previously showed that the restrictive phenotype is dominant in a transient cell fusion assay, suggesting that U2OS cells lack an antiviral mechanism present in other cells. Recent data indicate that unscheduled membrane fusion events can activate the expression of interferon-stimulated genes (ISGs) in fibroblasts, raising the possibility that our earlier results were due to a fusion-induced antiviral state. However, we show here that the permissive phenotype is also extinguished following fusion with Vero cells in the absence of ISG induction.
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Chan, Jennie, Peter Liehl, Rosane DeOliveira, Shruti Sharma, Douglas Golenbock, Maria Mota, and Katherine Fitzgerald. "Dual role of type I IFN during plasmodium infection (P3056)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 125.6. http://dx.doi.org/10.4049/jimmunol.190.supp.125.6.
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Abstract The molecular mechanisms regulating the inflammatory response during malaria are still poorly defined. Inflammatory cytokines and type I IFNs induced when innate immune sensors recognize components of the malaria parasite can contribute to clearance of the parasite and in some circumstances these same effectors lead to experimental cerebral malaria (ECM). Infection of C57BL/6 mice with Plasmodium berghei ANKA (PbA) leads to ECM where animals succumb to infection and die. Upon infection with PbA infected red blood cells (iRBCs), C57/Bl6 mice succumb to death 6-12 days post-infection. Recently, we have found that mice lacking the type I IFN receptor, IFNAR-/-, are protected from ECM-mediated death, implicating an important role of type I IFNs in exacerbating the ECM phenotype. Alternatively, when mice are infected with liver-tropic PbA sporozoites, a type I IFN response is induced while the parasites develop inside hepatocytes. This host response is responsible for upregulating interferon stimulated genes (ISGs) and limit the parasite load in the liver. The expression of ISGs are abrogated in IFNAR-/- mice. This protective phenotype is dependent on IRF3/7 and the adaptor MAVS suggesting that parasite RNA is recognized by host cells. Collectively, these findings reveal a dual role of type I IFNs that contribute to ECM-mediated death during PbA blood stage infections, but are also responsible for reducing parasite load during a liver stage infection.
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Thomas, Emmanuel, Mazen Noureddin, Yaron Rotman, and T. Jake Liang. "Mechanism of induction and genotype-phenotype correlation of IL28B and ISG expression in HCV-infected primary human hepatocytes and liver (P1383)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 57.3. http://dx.doi.org/10.4049/jimmunol.190.supp.57.3.
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Abstract Background: IL28B gene polymorphisms associated with hepatitis C virus (HCV) clearance suggests a role for type III interferons (IFNs. However, the intrinsic innate immune response following HCV infection remains poorly understood. Methods: Immunofluorescence staining of innate immune molecules (IRF3, ISG15) were performed on HCV infected PHHs. PHHs were also studied for correlation of IL28 genotype with type III IFN and ISG expression. In addition, RNA was extracted from 60 pre-treatment archived liver biopsies and hepatic expression of IFNs was measured by qPCR. Rs12979860 genotype was determined in PHHs and patients. Results: Primary human hepatocytes infected with HCV, but not contaminating non-parenchymal cells, demonstrate activated nuclear IRF3, supporting a direct anti-viral response to HCV pathogen associated molecular pattern (PAMP). In the patient cohort, there was a significant correlation between the hepatic expression of type III IFNs (IL28B & IL29, r=0.60, P <0.0001). ISGs expression was positively correlated with Type III IFNs (e.g. for ISG115 r=0.55, p<0.001). Conclusions: Our study demonstrates that HCV infection results in a rapid and robust intrinsic innate response in the liver. Although non-parenchymal cells may subsequently participate in this response, it is the infected hepatocyte that signals the initial danger signal. Finally, the hepatic expression of ISGs in HCV patients is mainly associated with type III IFNs.
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Foxman, Ellen F., James A. Storer, Megan E. Fitzgerald, Bethany R. Wasik, Lin Hou, Hongyu Zhao, Paul E. Turner, Anna Marie Pyle, and Akiko Iwasaki. "Temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells." Proceedings of the National Academy of Sciences 112, no. 3 (January 5, 2015): 827–32. http://dx.doi.org/10.1073/pnas.1411030112.
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Most isolates of human rhinovirus, the common cold virus, replicate more robustly at the cool temperatures found in the nasal cavity (33–35 °C) than at core body temperature (37 °C). To gain insight into the mechanism of temperature-dependent growth, we compared the transcriptional response of primary mouse airway epithelial cells infected with rhinovirus at 33 °C vs. 37 °C. Mouse airway cells infected with mouse-adapted rhinovirus 1B exhibited a striking enrichment in expression of antiviral defense response genes at 37 °C relative to 33 °C, which correlated with significantly higher expression levels of type I and type III IFN genes and IFN-stimulated genes (ISGs) at 37 °C. Temperature-dependent IFN induction in response to rhinovirus was dependent on the MAVS protein, a key signaling adaptor of the RIG-I–like receptors (RLRs). Stimulation of primary airway cells with the synthetic RLR ligand poly I:C led to greater IFN induction at 37 °C relative to 33 °C at early time points poststimulation and to a sustained increase in the induction of ISGs at 37 °C relative to 33 °C. Recombinant type I IFN also stimulated more robust induction of ISGs at 37 °C than at 33 °C. Genetic deficiency of MAVS or the type I IFN receptor in infected airway cells permitted higher levels of viral replication, particularly at 37 °C, and partially rescued the temperature-dependent growth phenotype. These findings demonstrate that in mouse airway cells, rhinovirus replicates preferentially at nasal cavity temperature due, in part, to a less efficient antiviral defense response of infected cells at cool temperature.
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Ganguly, Payal, Agata Burska, Charlotte Davis, Jehan J. El-Jawhari, Peter V. Giannoudis, and Elena Jones. "Intrinsic Type 1 Interferon (IFN1) Profile of Uncultured Human Bone Marrow CD45lowCD271+ Multipotential Stromal Cells (BM-MSCs): The Impact of Donor Age, Culture Expansion and IFNα and IFNβ Stimulation." Biomedicines 8, no. 7 (July 15, 2020): 214. http://dx.doi.org/10.3390/biomedicines8070214.
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Skeletal aging is associated with reduced proliferative potential of bone marrow (BM) multipotential stromal cells (MSCs). Recent data suggest the involvement of type 1 interferon (IFN1) signalling in hematopoietic stem cell (HSC) senescence. Considering that BM-HSCs and BM-MSCs share the same BM niche, we investigated IFN1 expression profile in human BM-MSCs in relation to donor age, culture-expansion and IFN1 (α and β) stimulation. Fluorescence-activated cell sorting was used to purify uncultured BM-MSCs from younger (19–41, n = 6) and older (59–89, n = 6) donors based on the CD45lowCD271+ phenotype, and hematopoietic-lineage cells (BM-HLCs, CD45+CD271−) were used as controls. Gene expression was analysed using integrated circuits arrays in sorted fractions as well as cultured/stimulated BM-MSCs and Y201/Y202 immortalised cell lines. IFN1 stimulation led to BM-MSC growth arrest and upregulation of many IFN1-stimulated genes (ISGs), with IFNβ demonstrating stronger effects. Uncultured MSCs were characterised by a moderate-level ISG expression similar to Y201 cells. Age-related changes in ISG expression were negligible in BM-MSCs compared to BM-HLCs, and intracellular reactive oxygen species (ROS) levels in BM-MSCs did not significantly correlate with donor age. Antiaging genes Klotho and SIRT6 correlated with more ISGs in BM-MSCs than in BM-HLCs. In patients with osteoarthritis (OA), BM-MSCs expressed considerably lower levels of several ISGs, indicating that their IFN1 signature is affected in a pathological condition. In summary, BM-MSCs possess homeostatic IFN1 gene expression signature in health, which is sensitive to in vitro culture and external IFN1 stimulation. IFN signalling may facilitate in vivo BM-MSC responses to DNA damage and combating senescence and aberrant immune activation.
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Dorrington, Michael G., Sinu John, and Iain D. C. Fraser. "Type I IFN mediates cell-intrinsic host protective effects during acute gram-negative bacterial infection of macrophages." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 123.7. http://dx.doi.org/10.4049/jimmunol.198.supp.123.7.
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Abstract Many bacteria require entrance into host immune cells, such as macrophages, in order to replicate. These intracellular bacteria have evolved complex strategies for avoiding the antimicrobial programming of these immune cells to create replicative niches. While we know a great deal about various virulence factors deployed by intracellular bacteria as well as how cells try to kill these pathogens, how bacteria and host cells modulate each others’ responses during infection is less well understood. Using the Gram-negative bacteria Burkholderia cenocepacia, we have uncovered a negative relationship between type I IFN signaling and the replicative potential of invading bacteria. Firstly, cells infected with these bacteria produce significant levels of IFN as well as the products of IFN-stimulated genes (ISGs). Cells pre-treated with IFNβ are less permissive to bacterial replication, while cells from mice lacking the type I IFN receptor (IFNAR) have increased levels of bacterial replication compared to wild-type cells. Interestingly, this phenotype is type I IFN-specific, as pre-treatment with IFNγ has no effect on bacterial replication. Additionally, siRNA knockdown of select ISGs, such as IFIT1, leads to increased bacterial replication within macrophages. A rigorous, concurrent transcriptomic profiling of both infected cells and intracellular bacteria is providing insight into how the bacteria and their host cells interact with one another during acute infection, providing a basis for targeted CRISPR-based screening experiments to unravel critical regulators of the host-pathogen interaction.
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Mar, Katrina Bockying, Jennifer Eitson, and John Schoggins. "Interferon-stimulated gene LY6E enhances entry of diverse RNA viruses." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 217.7. http://dx.doi.org/10.4049/jimmunol.196.supp.217.7.
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Abstract The type I interferon (IFN) response is the primary cellular defense mechanism against viruses. IFN protects against viral infection by activating the expression of hundreds of interferon-stimulated genes (ISGs), some of which have potent antiviral effector activity. Our recent screening efforts to identify novel antiviral ISGs unexpectedly uncovered a small subset of genes that enhanced viral infectivity. The goals of this project are to characterize the mechanism of LY6E, an ISG that enhanced unrelated viruses from multiple families. Here we show that ectopic expression of LY6E enhances cellular infection by a subset of enveloped RNA viruses from diverse families, including yellow fever virus, influenza A virus, vesicular stomatitis virus, and O’nyong’nyong virus. The enhancement phenotype is dependent on cell lineage, with the strongest effects seen in fibroblast and monocytic cell lines. Ablation of endogenous LY6E by CRISPR/Cas9 reduces but does not abrogate viral infectivity, indicating that LY6E is important for optimal infection. Loss of LY6E does not alter antiviral protection by IFN, suggesting that the enhancing effects may be direct. Mechanistic studies reveal that LY6E enhances infectivity at an early step in the viral life cycle, after viral binding but prior to the establishment of replication. Visualization of single-cell infections by ImageStream confirms that LY6E enhances viral entry. We conclude that LY6E enhances viral uptake at the population level, which may be important for initiation of the cell-mediated antiviral immune response. Understanding the mechanism and significance of LY6E to the antiviral interferon response may aid in the development of novel antiviral therapeutics.
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Lucas, Tiffany M., Justin M. Richner, and Michael S. Diamond. "The Interferon-Stimulated GeneIfi27l2aRestricts West Nile Virus Infection and Pathogenesis in a Cell-Type- and Region-Specific Manner." Journal of Virology 90, no. 5 (December 23, 2015): 2600–2615. http://dx.doi.org/10.1128/jvi.02463-15.
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ABSTRACTThe mammalian host responds to viral infections by inducing expression of hundreds of interferon-stimulated genes (ISGs). While the functional significance of many ISGs has yet to be determined, their cell type and temporal nature of expression suggest unique activities against specific pathogens. Using a combination of ectopic expression and gene silencing approaches in cell culture, we previously identifiedIfi27l2aas a candidate antiviral ISG within neuronal subsets of the central nervous system (CNS) that restricts infection by West Nile virus (WNV), an encephalitic flavivirus of global concern. To investigate the physiological relevance of Ifi27l2a in the context of viral infection, we generatedIfi27l2a−/−mice. Although adult mice lackingIfi27l2awere more vulnerable to lethal WNV infection, the viral burden was greater only within the CNS, particularly in the brain stem, cerebellum, and spinal cord. Within neurons of the cerebellum and brain stem, in the context of WNV infection, a deficiency of Ifi27l2a was associated with less cell death, which likely contributed to sustained viral replication and higher titers in these regions. Infection studies in a primary cell culture revealed thatIfi27l2a−/−cerebellar granule cell neurons and macrophages but not cerebral cortical neurons, embryonic fibroblasts, or dendritic cells sustained higher levels of WNV infection than wild-type cells and that this difference was greater under conditions of beta interferon (IFN-β) pretreatment. Collectively, these findings suggest that Ifi27l2a has an antiviral phenotype in subsets of cells and that at least some ISGs have specific inhibitory functions in restricted tissues.IMPORTANCEThe interferon-stimulatedIfi27l2agene is expressed differentially within the central nervous system upon interferon stimulation or viral infection. Prior studies in cell culture suggested an antiviral role for Ifi27l2a during infection by West Nile virus (WNV). To characterize its antiviral activityin vivo, we generated mice with a targeted gene deletion ofIfi27l2a. Based on extensive virological analyses, we determined that Ifi27l2a protects mice from WNV-induced mortality by contributing to the control of infection of the hindbrain and spinal cord, possibly by regulating cell death of neurons. This antiviral activity was validated in granule cell neurons derived from the cerebellum and in macrophages but was not observed in other cell types. Collectively, these data suggest that Ifi27l2a contributes to innate immune restriction of WNV in a cell-type- and tissue-specific manner.
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Garau, Jessica, Vanessa Cavallera, Marialuisa Valente, Davide Tonduti, Daisy Sproviero, Susanna Zucca, Domenica Battaglia, et al. "Molecular Genetics and Interferon Signature in the Italian Aicardi Goutières Syndrome Cohort: Report of 12 New Cases and Literature Review." Journal of Clinical Medicine 8, no. 5 (May 26, 2019): 750. http://dx.doi.org/10.3390/jcm8050750.
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Aicardi-Goutières syndrome (AGS) is a genetically determined early onset encephalopathy characterized by cerebral calcification, leukodystrophy, and increased expression of interferon-stimulated genes (ISGs). Up to now, seven genes (TREX1, RNASEH2B, RNASEH2C, RNASEH2A, ADAR1, SAMHD1, IFIH1) have been associated with an AGS phenotype. Next Generation Sequencing (NGS) analysis was performed on 51 AGS patients and interferon signature (IS) was investigated in 18 AGS patients and 31 healthy controls. NGS identified mutations in 48 of 51 subjects, with three patients demonstrating a typical AGS phenotype but not carrying mutations in known AGS-related genes. Five mutations, in RNASEH2B, SAMHD1 and IFIH1 gene, were not previously reported. Eleven patients were positive and seven negatives for the upregulation of interferon signaling (IS > 2.216). This work presents, for the first time, the genetic data of an Italian cohort of AGS patients, with a higher percentage of mutations in RNASEH2B and a lower frequency of mutations in TREX1 than those seen in international series. RNASEH2B mutated patients showed a prevalence of negative IS consistent with data reported in the literature. We also identified five novel pathogenic mutations that warrant further functional investigation. Exome/genome sequencing will be performed in future studies in patients without a mutation in AGS-related genes.
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von Recum-Knepper, Jessica, Anne Sadewasser, Viola K. Weinheimer, and Thorsten Wolff. "Fluorescence-Activated Cell Sorting-Based Analysis Reveals an Asymmetric Induction of Interferon-Stimulated Genes in Response to Seasonal Influenza A Virus." Journal of Virology 89, no. 14 (April 22, 2015): 6982–93. http://dx.doi.org/10.1128/jvi.00857-15.
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ABSTRACTInfluenza A virus (IAV) infection provokes an antiviral response involving the expression of type I and III interferons (IFN) and IFN-stimulated genes (ISGs) in infected cell cultures. However, the spatiotemporal dynamics of the IFN reaction are incompletely understood, as previous studies investigated mainly the population responses of virus-infected cultures, although substantial cell-to-cell variability has been documented. We devised a fluorescence-activated cell sorting-based assay to simultaneously quantify expression of viral antigens and ISGs, such as ISG15, MxA, and IFIT1, in IAV-infected cell cultures at the single-cell level. This approach revealed that seasonal IAV triggers an unexpected asymmetric response, as the major cell populations expressed either viral antigen or ISG, but rarely both. Further investigations identified a role of the viral NS1 protein in blocking ISG expression in infected cells, which surprisingly did not reduce paracrine IFN signaling to noninfected cells. Interestingly, viral ISG control was impaired in cultures infected with avian-origin IAV, including the H7N9 virus from eastern China. This phenotype was traced back to polymorphic NS1 amino acids known to be important for stable binding of the polyadenylation factor CPSF30 and concomitant suppression of host cell gene expression. Most significantly, mutation of two amino acids within the CPSF30 attachment site of NS1 from seasonal IAV diminished the strict control of ISG expression in infected cells and substantially attenuated virus replication. In conclusion, our approach revealed an asymmetric, NS1-dependent ISG induction in cultures infected with seasonal IAV, which appears to be essential for efficient virus propagation.IMPORTANCEInterferons are expressed by infected cells in response to IAV infection and play important roles in the antiviral immune response by inducing hundreds of interferon-stimulated genes (ISGs). Unlike many previous studies, we investigated the ISG response at the single-cell level, enabling novel insights into this virus-host interaction. Hence, cell cultures infected with seasonal IAV displayed an asymmetric ISG induction that was confined almost exclusively to noninfected cells. In comparison, ISG expression was observed in larger cell populations infected with avian-origin IAV, suggesting a more resolute antiviral response to these strains. Strict control of ISG expression by seasonal IAV was explained by the binding of the viral NS1 protein to the polyadenylation factor CPSF30, which reduces host cell gene expression. Mutational disruption of CPSF30 binding within NS1 concomitantly attenuated ISG control and replication of seasonal IAV, illustrating the importance of maintaining an asymmetric ISG response for efficient virus propagation.
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Duong, Francois H. T., Gaia Trincucci, Tujana Boldanova, Diego Calabrese, Benedetta Campana, Ilona Krol, Sarah C. Durand, et al. "IFN-λ receptor 1 expression is induced in chronic hepatitis C and correlates with the IFN-λ3 genotype and with nonresponsiveness to IFN-α therapies." Journal of Experimental Medicine 211, no. 5 (April 21, 2014): 857–68. http://dx.doi.org/10.1084/jem.20131557.
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The molecular mechanisms that link IFN-λ3 genotypes to differential induction of interferon (IFN)-stimulated genes (ISGs) in the liver of patients with chronic hepatitis C (CHC) are not known. We measured the expression of IFN-λ and of the specific IFN-λ receptor chain (IFN-λR1) in 122 liver biopsies of patients with CHC and 53 control samples. The IFN-λ3 genotype was not associated with differential expression of IFN-λ, but rather IFN-λR1. In a series of 30 primary human hepatocyte (PHH) samples, IFN-λR1 expression was low but could be induced with IFN-α. IFN-α–induced IFN-λR1 expression was significantly stronger in PHHs carrying the minor IFN-λ3 allele. The analysis of liver biopsies of patients with CHC revealed a strong association of high IFN-λR1 expression with elevated ISG expression, with IFN-λ3 minor alleles, and with nonresponse to pegylated IFN-α and ribavirin. The findings provide a missing link between the IFN-λ3 genotype and the associated phenotype of treatment nonresponse.
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Wagner, Allison, Chi Weindel, Robert Watson, and Kristin Patrick. "Alternative splicing of Bax controls cell death and innate immune responses in macrophages." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 97.01. http://dx.doi.org/10.4049/jimmunol.206.supp.97.01.
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Abstract To defend against invading pathogens, the macrophage innate immune response requires rapid, robust gene expression reprogramming. Innate immune genes that are induced as part of this response are regulated at many steps. While transcriptional activation of innate immune genes has been extensively studied, the mechanisms driving their post-transcriptional regulation (i.e. alternative splicing) needs further investigation. To begin learning how pre-mRNA splicing changes impact the innate immune response in macrophages, we examined how loss of a splicing regulatory protein called SRSF6 (serine arginine rich splicing factor 6) impacts gene expression. We discovered a novel phenotype whereby loss of SRSF6 increased basal expression of many interferon stimulated genes (ISGs). Consistent with mitochondrial dysfunction, a phenotype that we and others have previously associated high basal ISG expression, we measured an increase in apoptosis and macrophage depolarization in SRSF6 knockdown macrophages compared to scramble controls. Subsequent bioinformatic analysis of RNA-seq data from SRSF6 knockdown macrophages identified an alternative splicing event in the apoptosis activation factor BAX. While Bax isoforms have previously been linked to cell death regulation, no one has reported a role for Bax isoforms in modulating mtDNA-driven innate immune responses. Our data is consistent with a model whereby upregulation of a Bax isoform promotes pore formation in mitochondria which 1) causes leakage of mtDNA into the cytosol where it is detected by cGAS, eliciting type I interferon production and 2) primes macrophages to undergo programmed cell death.
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Wang, Qingde, Xiaoni Li, Ruofan Qi, Wenshuo Zhang, Pengyuan Zheng, and Timothy R. Billiar. "RNA editing is required for innate immune homeostasis through inhibiting cytosolic RNA receptor MDA-5 activated by cellular endogenous RNAs." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 169.3. http://dx.doi.org/10.4049/jimmunol.200.supp.169.3.
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Abstract Cytosolic RNA receptors, RLRs, play an essential role in antiviral infections that specifically detect the invaded viral RNAs leading to IFN and ISG expression and antiviral reactions through RNA sensing signaling pathways. This viral RNA sensing mechanism tolerate cellular “self” RNA in normal cells. However, how RLRs distinguish viral and self RNA molecules remains ambiguous. Our recent study showed that insufficient RNA editing by editing enzyme ADAR1 enables the cellular endogenous RNAs to stimulate the RLRs and activate the innate immune signaling pathway that lead to type I IFN and ISGs expression. Specific deletion of ADAR1 in the hepatocytes in a mouse model causes profound inflammation in the liver. As a result, the mortality rate is dramatically increased in the young animals. However, deletion of MDA-5 in these ADAR1 KO mice completely rescues the mice, and all the inflammatory phenotype was reversed. This finding indicated that RNA editing is necessary for the innate immune homeostasis, and MDA-5 plays a critical role in sensing endogenous self RNAs. Studies on this endogenous RNA sensing signaling pathway, its regulation and potential involvement in autoimmune and inflammatory diseases is warranted.
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Talukdar, Priyanka, Joshua T. Mattila, and Beth A. Fallert Junecko. "Type III interferons are expressed in tuberculosis granulomas and promote an inflammatory phenotype in macrophages that differs from type I interferon." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 149.15. http://dx.doi.org/10.4049/jimmunol.204.supp.149.15.
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Abstract Humans and non-human primates express four subtypes of type III interferons (IFNλs; IFNλ1-IFNλ4). Unlike type I interferons, which have been thoroughly investigated in tuberculosis (TB), the role of IFNλs and their effects on immunity in TB remain unknown, especially at the disease site. To better understand the role of IFNλs in TB, we examined IFNλ1 and IFNλ4 expression in cynomolgus macaques with TB and investigated the effects of IFNα/β, IFNλ1 and IFNλ4 signaling on macaque macrophages. Using IHC, we identified differential IFNλ1 and IFNλ4 expression in macrophages and neutrophils, including IFNλ4 localization in the nuclei of epithelioid macrophages. CD206+ macrophages and CD3+ cells in the airway expressed IFNλR while CD11c+ macrophages in the lymphocyte cuff of the granuloma expressed IFNλR. To measure IFNλ1 and IFNλ4’s effect on macrophage gene expression and compare these cytokines against IFNα/β, we used NanoString transcriptional profiling and Ingenuity Pathway Analysis (IPA) on cytokine-stimulated macrophages to identify differentially regulated pathways. We found that IFNα/β upregulated the greatest number of ISGs, followed by IFNλ1, whereas IFNλ4 stimulation had minimal effect on gene expression. Interestingly, pro-inflammatory genes including IL-1β, IL-8, TLR1, BATF were upregulated by IFNλ1 while IL-1β and TLR1 were downregulated by IFNα/β. To determine the effects of IFNλs on anti-mycobacterial macrophage responses, we used a reporter Mtb strain to determine how IFNλ1 and IFNλ4 effect the viability of Mycobacterium tuberculosis. Our data suggest that IFNλs have non-redundant properties with type 1 interferons that may promote macrophage activation, inflammation, and antibacterial activity in TB.
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Scott, Haley M., Allison R. Wagner, Kelsi West, Robert Watson, and Kristin L. Patrick. "Serine Arginine Splicing Factor 7 (SRSF7) is a critical regulator of innate immune activation in macrophages." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 163.29. http://dx.doi.org/10.4049/jimmunol.208.supp.163.29.
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Abstract Macrophages are immune cells that receive signals from pathogens and respond by reprogramming gene expression to activate an innate immune response. We currently have a poor understanding of how this response is fine-tuned at a post-transcriptional level. To address this, we turned to recent proteomics studies that identified mRNA processing as a pathway enriched for differentially phosphorylated proteins in macrophages infected with the important human pathogen Mycobacterium tuberculosis (Mtb). We became interested in one of these mRNA processing proteins, a splicing regulatory factor called SRSF7 (formerly known as 9G8), that was differentially phosphorylated at three serine residues during early Mtb infection. Transcriptomics analysis of SRSF7 knockdown RAW 264.7 macrophage-like cells revealed that SRSF7 is required for proper expression of a group of innate immune transcripts including the antiviral genes Mx1 and Ifit3 and the cytosolic DNA sensor Zbp1. Consistent with this phenotype, SRSF7 KD macrophages are permissive to Vesicular Stomatitis Virus (VSV) hyper replication. On the other hand, overexpression of SRSF7 in RAW 264.7 cells leads to elevated ISG abundance in resting cells and enhanced ability to restrict VSV replication. Curiously, it appears that certain ISGs are more reliant on SRSF7 than others, suggesting that SRSF7 acts at the level of the RNA itself. We have started to implicate phosphorylation of SRSF7 in these phenotypes. Together, these studies provide some of the best evidence to date that pathogen sensing cascades can functionalize RNA binding proteins during macrophage activation and highlight a previously unappreciated role for SRSF7 in controlling macrophage antiviral responses. Supported by grants from 1F31GM143893-01 NIH Predoctoral Fellowship and R35GM133720 from NIH/NIGMS.
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Li, Jian, Josephine Mun-Yee Ko, Wei Dai, Valen Zhuoyou Yu, Hoi Yan Ng, Jean-Sébastien Hoffmann, and Maria Li Lung. "Depletion of DNA Polymerase Theta Inhibits Tumor Growth and Promotes Genome Instability through the cGAS-STING-ISG Pathway in Esophageal Squamous Cell Carcinoma." Cancers 13, no. 13 (June 26, 2021): 3204. http://dx.doi.org/10.3390/cancers13133204.
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Overexpression of the specialized DNA polymerase theta (POLQ) is frequent in breast, colon and lung cancers and has been correlated with unfavorable clinical outcomes. Here, we aimed to determine the importance and functional role of POLQ in esophageal squamous cell carcinoma (ESCC). Integrated analysis of four RNA-seq datasets showed POLQ was predominantly upregulated in ESCC tumors. High expression of POLQ was also observed in a cohort of 25 Hong Kong ESCC patients and negatively correlated with ESCC patient survival. POLQ knockout (KO) ESCC cells were sensitized to multiple genotoxic agents. Both rH2AX foci staining and the comet assay indicated a higher level of genomic instability in POLQ-depleted cells. Double KO of POLQ and FANCD2, known to promote POLQ recruitment at sites of damage, significantly impaired cell proliferation both in vitro and in vivo, as compared to either single POLQ or FANCD2 KOs. A significantly increased number of micronuclei was observed in POLQ and/or FANCD2 KO ESCC cells. Loss of POLQ and/or FANCD2 also resulted in the activation of cGAS and upregulation of interferon-stimulated genes (ISGs). Our results suggest that high abundance of POLQ in ESCC contributes to the malignant phenotype through genome instability and activation of the cGAS pathway.
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Farina, Giuseppina Alessandra, Antonella Farina, Mara Cirone, Michael York, Stefania Lenna, Cristina Padilla, Sarah Mclaughlin, Alberto Faggioni, Maria Trojanowska, and Robert Lafyatis. "Epstein-Barr virus infection induces aberrant TLR/MyD88 activation pathway and fibroblast-myofibroblast conversion in systemic sclerosis. (P6322)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 182.13. http://dx.doi.org/10.4049/jimmunol.190.supp.182.13.
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Abstract Activated fibroblasts, mainly myofibroblasts are considered the principal mediators of fibrogenesis in systemic sclerosis (SSc), although the origin of persistent activation of fibroblasts and myofibroblasts is unclear. Activation of Interferon by Toll-like receptors in immune cells may play a role in the pathogenesis of inflammation in many autoimmune diseases, including SSc. We investigated whether fibroblasts persistently activated by innate immune and infectious stimuli might induce IFNs and TGFβ, two major markers of inflammation and fibrosis implicated in SSc pathogenesis. Since Epstein-Barr virus has been a leading candidate in triggering several autoimmune diseases, we developed methodology for infecting SSc fibroblasts with this virus. We found that EBV signals through the TLR/MyD88-pathway in infected-fibroblasts, activating a distinct innate immune response characterized by the expression of selected IFN- and TGFβ-inducible genes. Intriguingly, activation of the TLR/MyD88-pathway by CpGODN2006 or R837 ligand, significantly induced IFN-regulated genes, but no expression of TGFβ-regulated genes was detected in TLR/MyD88-stimulated fibroblasts. EBV-TLR/MyD88 aberrant activation induces the expression of selected IRFs, ISGs, TGFβ1 and several markers of fibroblast activation, such as smooth-muscle-actin and Endothelin-1, and all of these genes play a key role in determining the pro-fibrotic phenotype in SSc-fibroblasts.
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Kim, Min-Jung, Sun-Young Hwang, Tadaatsu Imaizumi, and Joo-Yeon Yoo. "Negative Feedback Regulation of RIG-I-Mediated Antiviral Signaling by Interferon-Induced ISG15 Conjugation." Journal of Virology 82, no. 3 (December 5, 2007): 1474–83. http://dx.doi.org/10.1128/jvi.01650-07.
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ABSTRACT RIG-I senses intracellular virus-specific nucleic acid structures and initiates an antiviral response that induces interferon (IFN) production, which, in turn, activates the transcription of RIG-I to increase RIG-I protein levels. Upon intracellular poly(I:C) stimulation, however, the levels of RIG-I protein did not correlate with the expression patterns of RIG-I transcripts. When the ISG15 conjugation system was overexpressed, ISG15 was conjugated to RIG-I and cellular levels of the unconjugated form of RIG-I decreased. The ISGylation of RIG-I reduced levels of both basal and virus-induced IFN promoter activity. Levels of unconjugated RIG-I also decreased when 26S proteasome activity was blocked by treatment with MG132, ALLN, or Lactacystin. In the presence of MG132, ISG15 conjugation to RIG-I increased, and hence, the unconjugated form of RIG-I was reduced. In Ube1L−/− cells, which lack the ability to conjugate ISG15, basal levels of both RIG-I protein and transcripts were increased compared to those in wild-type cells. As a result, enhanced production of ISGs and enhanced IFN promoter activity in Ube1L−/− cells were observed, and the phenotype was restored to that of wild-type cells by the overexpression of Ube1L. Based on these results, we propose a novel negative feedback loop which adjusts the strength of the RIG-I-mediated antiviral response and IFN production through the regulation of RIG-I protein by IFN-induced ISG15 conjugation.
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Dai, Manman, Shibing Li, Keyi Shi, Jiayu Liao, Hui Sun, and Ming Liao. "Systematic Identification of Host Immune Key Factors Influencing Viral Infection in PBL of ALV-J Infected SPF Chicken." Viruses 12, no. 1 (January 16, 2020): 114. http://dx.doi.org/10.3390/v12010114.
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Although research related to avian leukosis virus subgroup J (ALV-J) has lasted for more than a century, the systematic identification of host immune key factors against ALV-J infection has not been reported. In this study, we establish an infection model in which four-week-old SPF chickens are infected with ALV-J strain CHN06, after which the host immune response is detected. We found that the expression of two antiviral interferon-stimulated genes (ISGs) (Mx1 and IFIT5) were increased in ALV-J infected peripheral blood lymphocytes (PBL). A significant CD8+ T cell response induced by ALV-J appeared as early as seven days post-infection (DPI), and humoral immunity starting from 21 DPI differed greatly in the time scale of induction level. Meanwhile, the ALV-J viremia was significantly decreased before antibody production at 14 DPI, and eliminated at 21 DPI under a very low antibody level. The up-regulated CD8+ T cell in the thymus (14DPI) and PBL (7 DPI and 21 DPI) was detected, indicating that the thymus may provide the output of CD8+ T cell to PBL, which was related to virus clearance. Besides, up-regulated chemokine CXCLi1 at 7 DPI in PBL was observed, which may be related to the migration of the CD8+ T cell from the thymus to PBL. More importantly, the CD8 high+ T cell response of the CD8αβ phenotype may produce granzyme K, NK lysin, or IFN-γ for clearing viruses. These findings provide novel insights and direction for developing effective ALV-J vaccines.
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Zeng, Hui, Cynthia Goldsmith, Pranee Thawatsupha, Malinee Chittaganpitch, Sunthareeya Waicharoen, Sherif Zaki, Terrence M. Tumpey, and Jacqueline M. Katz. "Highly Pathogenic Avian Influenza H5N1 Viruses Elicit an Attenuated Type I Interferon Response in Polarized Human Bronchial Epithelial Cells." Journal of Virology 81, no. 22 (September 12, 2007): 12439–49. http://dx.doi.org/10.1128/jvi.01134-07.
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ABSTRACT The unparalleled spread of highly pathogenic avian influenza A (HPAI) H5N1 viruses has resulted in devastating outbreaks in domestic poultry and sporadic human infections with a high fatality rate. To better understand the mechanism(s) of H5N1 virus pathogenesis and host responses in humans, we utilized a polarized human bronchial epithelial cell model that expresses both avian alpha-2,3- and human alpha-2,6-linked sialic acid receptors on the apical surface and supports productive replication of both H5N1 and H3N2 viruses. Using this model, we compared the abilities of selected 2004 HPAI H5N1 viruses isolated from humans and a recent human H3N2 virus to trigger the type I interferon (IFN) response. H5N1 viruses elicited significantly less IFN regulatory factor 3 (IRF3) nuclear translocation, as well as delayed and reduced production of IFN-β compared with the H3N2 virus. Furthermore, phosphorylation of Stat2 and induction of IFN-stimulated genes (ISGs), such as MX1, ISG15, IRF7, and retinoic acid-inducible gene I, were substantially delayed and reduced in cells infected with H5N1 viruses. We also observed that the highly virulent H5N1 virus replicated more efficiently and induced a weaker IFN response than the H5N1 virus that exhibited low virulence in mammals in an earlier study. Our data suggest that the H5N1 viruses tested, especially the virus with the high-pathogenicity phenotype, possess greater capability to attenuate the type I IFN response than the human H3N2 virus. The attenuation of this critical host innate immune defense may contribute to the virulence of H5N1 viruses observed in humans.
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Gibb, David R., Emaan Madany, Najwa El Kadi, Sumaarg Pandya, Kessandra Ng, and Caroline Anne Jefferies. "Type 1 interferons promote allo-antibody responses to RBC transfusion in a lupus mouse model." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 219.19. http://dx.doi.org/10.4049/jimmunol.204.supp.219.19.
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Abstract Red blood cell (RBC) transfusion exposes recipients to hundreds of unmatched minor RBC antigens. This exposure can lead to production of alloantibodies that can cause potentially fatal hemolytic events. Prior studies have reported that inflammatory states, including autoimmune disease, in the transfusion recipient can promote alloimmunization. Recent studies reported that pro-inflammatory type 1 interferons (IFNα/β) regulated RBC alloimmunization in multiple mouse transfusion models. Interestingly, patients with systemic lupus erythematosus (SLE) have an increased frequency of alloimmunization and express an IFNα/β gene signature. Thus, we tested the hypothesis that IFNα/β activation in lupus promotes RBC alloimmunization by utilizing the pristane-induced lupus mouse model. Pristane significantly induced serum IFNα and expression of multiple interferon-stimulated genes (ISG) in peritoneal fluid cells, including inflammatory macrophages. Following transfusion with allogeneic RBCs expressing the KEL antigen, pristane-treated WT mice produced significantly elevated levels of anti-KEL IgM and anti-KEL IgG, compared to untreated mice. Pristane-treated mice lacking the IFNα/β receptor (IFNAR1−/−) or IFNα/β-inducing transcription factors (IRF3/7−/−) mice failed to produce ISGs and produced significantly lower levels of anti-KEL IgG compared to WT mice. In conclusion, pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens and the contribution of IFNα/β responses to the increased frequency of alloimmunization in patients with SLE.
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Wagner, Allison, Chi Weindel, Kelsi West, Robert Watson, and Kristin Patrick. "The splicing factor SRSF6 controls Mycobacterium tuberculosis replication through its role in maintaining mitochondrial homeostasis." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 50.13. http://dx.doi.org/10.4049/jimmunol.208.supp.50.13.
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Abstract Macrophages are the primary host cell that supports the survival and replication of the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). We are interested in how macrophage proteins are functionalized during Mtb infection. Global proteomics analysis of Mtb-infected primary macrophages found that many RNA binding proteins of the Serine/Arginine rich splicing family (SRSF) can be differentially phosphorylated, suggesting that these factors regulate splicing decisions during Mtb infection. Knockdown of one such SRSF protein, called SR6, results in basal upregulation of interferon stimulated genes (ISGs) in macrophages. We hypothesized that this phenotype was driven by mitochondrial damage that leaks mitochondria DNA into the cytosol and activates cGAS. Consistent with this idea, we measured increased mitochondrial stress and increased cell death in SR6 KD macrophages. Transcriptomics analysis of these cells revealed upregulation of an alternatively spliced (AS) isoform of the apoptotic factor BAX, which contributes to mitochondrial dysfunction, mtDNA release, and basal ISG expression. While BAX isoforms are known to regulate cell death, this is the first report of BAX AS regulating the type I IFN response. Lastly, we found that SR6 KD macrophages are permissive to Mtb replication, suggesting that controlling cell death at the level of BAX isoform abundance is an important but unappreciated component of the macrophage antibacterial repertoire. Together, our data support a model whereby SR6-dependent AS of Bax triggers mitochondria stress causing mtDNA leakage and type I IFN expression, metabolic dysfunction that primes cells to undergo cell death, and increased susceptibility to Mtb infection.
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Mountz, John D., Shanrun Liu, Qi Wu, PingAr Yang, W. Alex Essman, Oluwagbemiga A. Ojo, Bao Luo, et al. "Type I IFN and IL-4R define B cell checkpoint defects in systemic lupus erythematosus." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 179.5. http://dx.doi.org/10.4049/jimmunol.202.supp.179.5.
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Abstract The development of pathogenic autoantibody producing B cells is normally limited at the early transitional (Tr) stage and a later T-dependent developmental checkpoint. The precise molecular mechanisms underlying defective autoreactive B cell deletion/anergy at each checkpoint have been difficult to define. We utilized a combined single-cell B cell transcriptome and high dimensional flow cytometry analysis approach for a parallel understanding of B-cell developmental checkpoint defects at multiple stages in SLE. We found that while high levels of B-cell endogenous interferon-β (IFNβ) is an important risk factor for autoantibody positive African American (AA) SLE patients, low expression of IL-4R was a risk factor of both AA and European-Americans (EA) patients for development of pathogenic autoantibodies. The molecular signature of autoreactive B cells that escape deletion at the Tr stage include type I IFN stimulated genes (ISGs), including IRF7, ISG15, ISG20, MX1, and STAT1. The molecular signature of autoreactive B cells that escape the second T-dependent checkpoint, include low expression of IL4R and the transcriptional repressor BACH2, and high levels of TBX21, ITGAX, FCRL5 and IFI30. Interestingly, in vitro culture stimulation confirmed that the presence of IL-4 signaling significantly suppressed the development of pathogenic CD21−CD27−IgD−CD11c+T-bet+ double negative 2 (DN2) B cells in SLE. These results suggest that B-cell checkpoint defects can be used to stratify SLE patients according to high type I IFN or low IL-4R and their response signatures. These immune phenotype and transcriptome signatures offer the potential to predict therapy responses to type I IFN blockade or other therapies.
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Erlandsson, M., E. Malmhäll-Bah, K. M. Andersson, S. Töyrä Silfverswärd, R. Pullerits, and M. I. Bokarewa. "POS0702 ANTI-RHEUMATIC TREATMENT ALTERS PFKFB3 EXPRESSION, KEY OF GLYCOLYSIS, IN CD14+ MONOCYTES OF RA PATIENTS, WHICH CONTRIBUTES TO DISSIMILARITIES OF THE IFNg-SIGNATURE." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 631.1–631. http://dx.doi.org/10.1136/annrheumdis-2022-eular.4562.
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BackgroundInterferon-gamma (IFNαnterferon-gamma (IFNRheumatology clinic, Gothenburg, Swedenh, Göteborg, Swedenmonocytes of RA patients, which contributes to dissimilarities of the IFNg-signature Peter C. Taylor Consultant of: AbbVie, Biogen, Eli Lilly and Company, Fresenius, Galapagos, mulation is energy consuming and often demands metabolic adaptation of a cell by switching glucose metabolism from the tricarboxylic acid cycle (TCA) to the pentose phosphate pathway (PPP) alternative of glycolysis that dramatically increases glucose intake. Fructose-2,6-biphosphatase 3 (coded by PFKFB3) has been identified as the rate-limiting regulator of glucose metabolism suppressed in leukocytes of RA patients (2).ObjectivesTo study effect of anti-rheumatic treatment on the cellular energy metabolism of CD14+ mononuclear cells and its connection to the phenotype and IFNαmononuclear.MethodsCD14+ cells were sorted from the peripheral blood of the randomly recruited 60 RA patients (mean age 59.6y, DD 13.8y). The cells were LPS activated for 2h and submitted to RNA sequencing (RNAseq, IluminaNextseq). The patients were divided by actual DMARD treatment into those who had no DMARDs (n=8), methotrexate (MTX) only (n=15), biologics (MTX+aTNF n=12, MTX+RTX n=4) and JAK-inhibitors (JAKi, n=24). Differentially expressed gene (DEG) analysis between the groups was performed in R-studio, Bioconductor package, DESeq2. Reported IFN signatures (1) were combined in a set of 51 IFN signature genes (ISG) and analyzed in relation to the transcriptomic profile behind the cellular energy metabolism and DMARD treatment.ResultsMTX and JAKi but not biologics make a significant and opposing contribution of the transcriptomic of energy metabolism in CD14+ cells. MTX-treated patients had significantly higher levels of the rate-limiting enzyme of glycolysis PFKFB3 and PGAM1 compared to those with no DMARDS, which normalized aerobic glycolysis by increasing expression of the pyruvate dehydrogenase complex proteins PDHA1, PHDX and PDK3 linking glycolysis with TCA and decreasing PPP enzymes PGLS, RPIA and TKT. In contrast, PFKFB3 was suppressed in patients treated with JAK-inhibitors compared to those treated with MTX (cor.p=1.32e-8), which significantly activated glycolysis downstream of PFKFB3 and shunting metabolism to PPP inducing expression of G6PD (cor.p=5.0e-92) and PGLS (cor.p=3.1e-46) and increased the major glucose transporter SLC2A1 (cor.p=1.11e-24).These differences in glucose metabolism were linked to divergent phenotype of CD14+ cells being short-lived CD14intCD11chi cells and IL6 producing in MTX-treated patients and long-lived mature CD14hiCD11bhiCX3CR1hi cells and IFNαcells and IFN producing in MTX-treated pa+ cells of JAKi-treated patients expressed low levels of STAT1 and ISGs compared to MTX-treated patients.ConclusionDMARD treatment har divergent effect on glycolysis of CD14+ cells, acting through PFKFB3. This has significant impact on the phenotype of CD14+ cells and inflammatory ability.References[1]Lamot L. Et.al. Clin Exp Rheumatol 2019, 37, 1077.[2]Yang Z. et al, Science Translational Medicine 2016, 8, 331, 331ra38.Disclosure of InterestsNone declared
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Netterwald, James, Shaojun Yang, Weijia Wang, Salena Ghanny, Michael Cody, Patricia Soteropoulos, Bin Tian, Walter Dunn, Fenyong Liu, and Hua Zhu. "Two Gamma Interferon-Activated Site-Like Elements in the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer Are Important for Viral Replication." Journal of Virology 79, no. 8 (April 15, 2005): 5035–46. http://dx.doi.org/10.1128/jvi.79.8.5035-5046.2005.
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ABSTRACT Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.
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EL Kadi, Najwa, Emaan Madany, Kessandra NG, Sumaarg Pandya, Caroline Jefferies, and David R. Gibb. "Type 1 Interferon Promotes RBC Alloimmunization in a Lupus Mouse Model." Blood 134, Supplement_1 (November 13, 2019): 101. http://dx.doi.org/10.1182/blood-2019-130903.
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Background: Red blood cell (RBC) alloimmunization can follow exposure to foreign RBC antigens during transfusion therapy and during pregnancy/delivery. Alloantibodies against RBC antigens can cause potentially fatal hemolytic transfusion reactions and hemolytic disease of the newborn. Recent studies indicate that inflammatory states, including autoimmunity, promote RBC alloimmunization. For example, patients with systemic lupus erythematosus (SLE) have an elevated risk of RBC alloimmunization. Approximately 50% of SLE patients have a pro-inflammatory type 1 interferon (IFNα/β) gene signature, defined as the expression of interferon stimulated genes (ISGs), that is associated with disease severity. Recent studies in mouse transfusion models indicate that IFNα/β significantly promotes RBC alloimmunization. Thus, we hypothesized that IFNα/β produced in a lupus mouse model would promote RBC alloimmunization following transfusion. Methods: To test this hypothesis, we utilized the pristane-induced lupus mouse model, known to manifest lupus autoantibodies and a lupus-like phenotype. Mice lacking the IFNα/β receptor (IFNAR1-/-), mice lacking transcription factors required for IFNα/β production (IRF3/7-/-) and wildtype (WT) mice were administered an intraperitoneal injection of pristane 14 days prior to transfusion with transgenic RBCs expressing the KEL antigen. At the time of transfusion, serum IFNα levels were measured by ELISA, and expression of interferon stimulated gene 15 (ISG15) was measured by quantitative PCR. The mean fluorescence intensity (MFI) of the anti-KEL IgM and IgG responses were measured by flow cytometry 5 days and 7-28 days, respectively, following transfusion. A Mann-Whitney U test and a Kruskall-Wallis test with a Dunn's post-test was used to compare 2 or more than 2 groups, respectively. Results: Pristane induced serum IFNα in WT mice (Day 14 post-pristane, mean IFNα=127.3 Units/ml ± 28.21 standard error of the mean, SEM, p<0.01), which was undetectable in untreated mice. Pristane treatment also resulted in a non-significant trend toward upregulated expression of ISG15, post-pristane ISG15 =2.94 ± 1.16 SEM; no pristane ISG15 = 1.4 ± 0.2 SEM). Following transfusion, untreated mice did not form anti-KEL IgG. However, pristane-treated WT mice produced significantly elevated levels of anti-KEL IgM and anti-KEL IgG in 3 out of 3 experiments (untreated IgM MFI = 79.1 ± 13.5, pristane-treated IgM MFI = 142.7 ± 21.6, p<0.01; untreated IgG MFI = 10.7± 4.6, pristane-treated IgG MFI= 203.9 ± 99.6, p<0.05, Figure 1). Pristane-treated IFNAR1-/- and IRF3/7-/- mice produced significantly lower levels of anti-KEL IgG (IFNAR1-/- MFI = 39.0 ± 35.6, p<0.05; IRF3/7-/- MFI = 36.1 ± 16.3, p<0.02) compared to WT mice (MFI = 361 ± 148). Conclusion: Pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens, and the investigation of the contribution of IFNα/β responses to increased frequency of alloimmunization in patients with SLE. Disclosures No relevant conflicts of interest to declare.
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Dorer, D. R., M. A. Cadden, B. Gordesky-Gold, G. Harries, and A. C. Christensen. "Suppression of a lethal trisomic phenotype in Drosophila melanogaster by increased dosage of an unlinked locus." Genetics 134, no. 1 (May 1, 1993): 243–49. http://dx.doi.org/10.1093/genetics/134.1.243.
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Abstract One of the most extreme examples of gene dosage sensitivity is the Triplo-lethal locus (Tpl) on the third chromosome of Drosophila melanogaster, which is lethal when present in either one or three copies. Increased dosage of an unlinked locus, Isis, suppresses the triplo-lethal phenotype of Tpl, but not the haplo-lethal phenotype. We have mapped Isis to the X chromosome region 7E3-8A5, and shown that the suppression is a gene dosage effect. Altered dosage of Isis in the presence of two copies of Tpl has no obvious effects. By examining the interactions between Isis dosage and Tpl we suggest that Isis does not directly repress Tpl expression, but acts downstream on the triplo-lethal phenotype of Tpl.
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Huschka, Henriette, and Sabine Mihm. "Hepatic IFNL4 Gene Activation in Hepatocellular Carcinoma Patients with Regard to Etiology." International Journal of Molecular Sciences 22, no. 15 (July 21, 2021): 7803. http://dx.doi.org/10.3390/ijms22157803.
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Hepatocellular carcinoma (HCC) is a malignancy with a leading lethality. The etiology is quite diverse, ranging from viral infections to metabolic disorders or intoxications, and associates with specific somatic mutational patterns and specific host immunological phenotypes. Particularly, hepatitis C virus (HCV)-infected liver is featured by an activation of interferon (IFN)-stimulated genes (ISGs; IFN signature), which we suppose is driven by type III IFNL4. Taking advantage of the TCGA collection of HCC patients of various different etiologies, this study aimed at validating our previous findings on hepatic IFNL4 gene activation in HCV infection in an independent and larger cohort of patients with advanced liver disease. In a cohort of n = 377 cases, the entirety of the sequencing data was used to assess the IFNL genotypes, and the cases were stratified for etiology. The number of IFNL4 transcripts within nonmalignant and malignant tissues was found to be more abundant in patients with HCV or HCV/HBV infections when compared to other risk factors. Moreover, in patients with HCV infection as a risk factor, a close, positive relationship was found between ISG activation and the number of functional IFNL4 transcripts. Data on this independent TCGA sample support the concept of an IFNL4-dependent HCV-driven activation of hepatic ISGs. In addition to that, they add to the understanding of etiology-related host immunological phenotypes in HCC.
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You, Eunae, Luli Zou, Patrick Danaher, Ildiko E. Phillips, Michael J. Raabe, Bidish Patel, Amaya Pankaj, et al. "Abstract C038: Repeat RNA dysregulation of cellular states in the pancreatic cancer microenvironment." Cancer Research 82, no. 22_Supplement (November 15, 2022): C038. http://dx.doi.org/10.1158/1538-7445.panca22-c038.
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Abstract Aberrant transcription of the repeat RNAs is a common feature in epithelial cancers including PDAC, but the function of these non-coding RNAs in cancer development is relatively unexplored. We have found that these repeat RNAs are sensed and replicate like retroviruses, and now have identified the ability of these viral-like elements to be transmitted from cancer cells through extracellular vesicles (EVs). PDAC-derived EVs applied to cancer-associated fibroblasts (CAFs) activates interferon-stimulated genes (ISGs) and is able to drive CAFs towards an inflammatory CAF (iCAF) phenotype with concordant loss of myofibroblast CAF (myCAF) marker genes. Using in-vitro transcription, we demonstrate that individual repeat RNAs (HSATII, HERVK (env), LINE-1 5’UTR and LINE-1 3’UTR) are sufficient to induce ISG response in CAFs with HSATII and HERVK (env) having the most potent ISG response. In contrast, PDAC cells were found to induce epithelial-mesenchymal transition (EMT) with loss of epithelial gene expression. To determine the potential sensor of HSATII repeat RNAs, we utilized CRISPR/Cas9 knockout of the viral RNA sensors RIG-I, MDA5, and MAVS in PDAC and CAF cells. Notably, these sensors were important for PDAC repeat RNA sensing and response, but not in CAF cells. Evaluating the innate immune pathway further downstream, we used genetic knockout of IRF3 with CRISPR/Cas9 knockout and find significant downregulation of key EMT genes that are shared with myCAF markers (ACTA2, FN1, SERPINE1). Interestingly, HSATII RNA activated IRF3 dependent EMT genes in PDAC cells, but induced IRF3 degradation in CAF cells that results in loss of myCAF gene expression. Furthermore, we found that conditioned media from HSATII transfected CAF activates EMT-related gene expression (ACTA2, FN1, SERPINE1) in PDAC cell lines, which indicates an cell extrinsic mechanism to augment EMT induction in PDAC cells. We utilized next generation spatial transcriptomic platforms NanoString GeoMx and CosMx to understand the spatial distribution of repeat RNAs in human PDAC tumors. We find that repeat RNAs can be found as a gradient from PDAC cells to the surround tumor microenvironment consistent with delivery of these RNA species. Analysis of over 300,000 individual cells in 3 PDAC tumor specimens, we find that high repeat PDAC cells have lost epithelial gene expression and high repeat CAFs have lost myCAF gene expression. Altogether, these findings support the “infection” of repeat RNAs disrupts cellular identity in both tumor cells and the CAF microenvironment as a mechanism for tumor progression. Citation Format: Eunae You, Luli Zou, Patrick Danaher, Ildiko E. Phillips, Michael J. Raabe, Bidish Patel, Amaya Pankaj, Khrystyna North, Sean Kim, Youngmi Kim, Martin Aryee, David T. Ting. Repeat RNA dysregulation of cellular states in the pancreatic cancer microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C038.
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Johnson, Deborah C., Mihaela-Carmen Unciuleac, and Dennis R. Dean. "Controlled Expression and Functional Analysis of Iron-Sulfur Cluster Biosynthetic Components within Azotobacter vinelandii." Journal of Bacteriology 188, no. 21 (August 25, 2006): 7551–61. http://dx.doi.org/10.1128/jb.00596-06.
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ABSTRACT A system for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon was developed. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis, the scrX gene, contained within the sucrose catabolic regulon, was replaced by the contiguous A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, and iscX genes, resulting in duplicate genomic copies of these genes: one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA, and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in a null growth phenotype only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted of HscBA could be partially reversed by culturing cells under conditions of low oxygen. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or whose replacement results in a partial or complete dominant-negative growth phenotype were also identified using this system.
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Ho, Manh Tin, Jiongming Lu, Paula Vazquez-Pianzola, and Beat Suter. "α-Phenylalanyl tRNA synthetase competes with Notch signaling through its N-terminal domain." PLOS Genetics 18, no. 4 (April 29, 2022): e1010185. http://dx.doi.org/10.1371/journal.pgen.1010185.
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The alpha subunit of the cytoplasmic Phenylalanyl tRNA synthetase (α-PheRS, FARSA in humans) displays cell growth and proliferation activities and its elevated levels can induce cell fate changes and tumor-like phenotypes that are neither dependent on the canonical function of charging tRNAPhe with phenylalanine nor on stimulating general translation. In intestinal stem cells of Drosophila midguts, α-PheRS levels are naturally slightly elevated and human FARSA mRNA levels are elevated in multiple cancers. In the Drosophila midgut model, elevated α-PheRS levels caused the accumulation of many additional proliferating cells resembling intestinal stem cells (ISCs) and enteroblasts (EBs). This phenotype partially resembles the tumor-like phenotype described as Notch RNAi phenotype for the same cells. Genetic interactions between α-PheRS and Notch suggest that their activities neutralize each other and that elevated α-PheRS levels attenuate Notch signaling when Notch induces differentiation into enterocytes, type II neuroblast stem cell proliferation, or transcription of a Notch reporter. These non-canonical functions all map to the N-terminal part of α-PheRS which accumulates naturally in the intestine. This truncated version of α-PheRS (α-S) also localizes to nuclei and displays weak sequence similarity to the Notch intracellular domain (NICD), suggesting that α-S might compete with the NICD for binding to a common target. Supporting this hypothesis, the tryptophan (W) residue reported to be key for the interaction between the NICD and the Su(H) BTD domain is not only conserved in α-PheRS and α-S, but also essential for attenuating Notch signaling.
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Zhou, Jianbiao, Jonathan Adam Scolnick, Stacy Xu, Melissa Ooi, Priscella Shirley Chia, Sabrina Hui-Min Toh, Kalpnaa Balan, et al. "Single-Cell Multi-Omic Analysis Uncovers Comprised Immune Function and Primary Resistance Mechanism in Acute Myeloid Leukemia." Blood 138, Supplement 1 (November 5, 2021): 378. http://dx.doi.org/10.1182/blood-2021-149022.
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Abstract Background: Approximately 20% of AML patients do not respond to induction chemotherapy (primary resistance) and 40-60% of patients develop secondary resistance, eventually leading to relapse followed by refractory disease (RR-AML). Diversified molecular mechanisms have been proposed for drug resistance and RR phenotype. However, we still cannot predict when relapse will occur, nor which patients will become resistant to therapy. Single-cell multi-omic (ScMo) profiling may provide new insights into our understanding of hematopoietic stem cell (HSC) differentiation trajectories, tumor heterogeneity and clonal evolution. Here we applied ScMo to profile bone marrow (BM) from AML patients and healthy controls. Methods: AML samples were collected at diagnosis with institutional IRB approval. Cells were stained with a panel of 62 DNA barcoded antibodies and 10x Genomics Single Cell 3' Library Kit v3 was used to generate ScMo data. After normalization, clusters were identified using Uniform Manifold Approximation and Projection (UMAP) and annotated using MapCell (Koh and Hoon, 2019). We analyzed 23,933 cells from 4 adult AML BM samples, and 39,522 cells from 2 healthy adults and 3 sorted CD34+ normal BM samples. Gene set enrichment analysis (GSEA) and Enrichr program were used to examine underlying pathways among differentially expressed genes between healthy and AML samples. Results: We identified 16 cell types between the AML and normal samples (Fig 1a) amongst 45 clusters in the UMAP projection (Fig 1b). Comparative analysis of the T cell clusters in AML samples with healthy BM cells identified an "AML T-cell signature" with over-expression of genes such as granzymes, NK/T cell markers, chemokine and cytokine, proteinase and proteinase inhibitor (Fig 2a). Among them, IL32 is known to be involved in activation-induced cell death in T cells and has immunosuppressive role, while CD8+ GZMB+ and CD8+ GZMK+ cells are considered as dysfunctional or pre-dysfunctional T cells. Indeed, Enrichr analysis showed the top rank of phenotype term - "decreased cytotoxic T cell cytolysis". We next examined whether NK cells, are similarly dysfunctional in the AML ecosystem. The "AML NK cell signature" includes Fc Fragment family, IFN-stimulated genes (ISGs), the effector protein-encoding genes and other genes when compared to normal NK cells (Fig 2b). GSEA analysis revealed "PD-1 signalling" among the top 5 ranked pathways in AML-NK cells, though no increase in PD-1 protein nor PDCD1 gene were identified in these cells. Inhibitory receptor CD160 was expressed higher in AML samples along with exhaustion (dysfunction) associated genes TIGIT, PRF1 and GZMB (Fig 2c). Enrichr analysis uncovered enrichment of "abnormal NK cell physiology and "impaired natural killer cell mediated cytotoxicity". Similarly, the "AML monocyte signature" was significantly enriched with genes in "Tumor Infiltrating Macrophages in Cancer Progression and Immune Escape" and "Myeloid Derived Suppressor Cells in Cancer Immune Escape". We also analyzed HSPC component in one pair of cytogenetically matched, untreated complete remission (CR) /RR AML pair (Fig 2d). Notably, half of the 10 genes overexpressed in RR-AML, CXCR4, LGALS1, S100A8, S100A9, SRGN (Serglycin), regulate cell-matrix interaction and play pivotal roles in leukemic cells homing bone marrow niche. The first 4 of these genes have been demonstrated as prognostic indicators of poor survival and associated with chemo-resistance and anti-apoptotic function. Furthermore, single-cell trajectory analysis of this CR/RR pair illustrated a change in differentiation pattern of HSPCs in CR-AML to monocytes in RR-AML. We are currently analyzing more AML samples to validate these findings. Conclusions: Our ScMo analysis demonstrates that the immune cells are systematically reprogrammed and functionally comprised in the AML ecosystem. Upregulation of BM niche factors could be the underlying mechanism for RR-AML. Thus, reversing the inhibited immune system is an important strategy for AML therapy and targeting leukemic cell-BM niche interaction should be considered for cases with high expression of these molecules on AML HSPCs. Note: J.Z. and J.A.S. share co-first authorship. Figure 1 Figure 1. Disclosures Scolnick: Proteona Pte Ltd: Current holder of individual stocks in a privately-held company. Xu: Proteona Pte Ltd: Current Employment. Ooi: Jansen: Honoraria; Teva Pharmaceuticals: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Lovci: Proteona Pte Ltd: Current Employment. Chng: Aslan: Research Funding; Takeda: Honoraria; Johnson & Johnson: Honoraria, Research Funding; BMS/Celgene: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria.
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Amcheslavsky, Alla, Naoto Ito, Jin Jiang, and Y. Tony Ip. "Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells." Journal of Cell Biology 193, no. 4 (May 9, 2011): 695–710. http://dx.doi.org/10.1083/jcb.201103018.
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Intestinal stem cells (ISCs) in the adult Drosophila melanogaster midgut can respond to damage and support repair. We demonstrate in this paper that the tuberous sclerosis complex (TSC) plays a critical role in balancing ISC growth and division. Previous studies have shown that imaginal disc cells that are mutant for TSC have increased rates of growth and division. However, we report in this paper that loss of TSC in the adult Drosophila midgut results in the formation of much larger ISCs that have halted cell division. These mutant ISCs expressed proper stem cell markers, did not differentiate, and had defects in multiple steps of the cell cycle. Slowing the growth by feeding rapamycin or reducing Myc was sufficient to rescue the division defect. The TSC mutant guts had a thinner epithelial structure than wild-type tissues, and the mutant flies were more susceptible to tissue damage. Therefore, we have uncovered a context-dependent phenotype of TSC mutants in adult ISCs, such that the excessive growth leads to inhibition of division.
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Tessmann, Josiane Weber, Murilo Ramos Rocha, Renata Ivo Vasconcelos, Danielle Lazarin-Bidoia, Raphael Rodrigues Corrêa, Rafael Soares Lindoso, Paulo Emílio Corrêa Leite, Celso Vataru Nakamura, and Jose Andres Morgado-Diaz. "Abstract 211: Hypofractionated radiation therapy promotes distinct resistant cellular fates as a survival mechanism in two colorectal cell lines: senescence or transient dormancy followed by heterogeneous repopulation." Cancer Research 82, no. 12_Supplement (June 15, 2022): 211. http://dx.doi.org/10.1158/1538-7445.am2022-211.
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Abstract Radiotherapy (RT) is an effective non-surgical treatment used in colorectal cancer (CRC), which is one of the major causes of cancer mortality. In patients with advanced rectal cancer, this approach is used as neoadjuvant therapy to reduce local recurrence risk. However, RT has a small impact on metastasis formation, which remains an obstacle in clinical practice, being responsible for the high mortality rates. Also, local tumor recurrence after RT tends to be more aggressive, with shorter survival expectancy. This suggests that RT surviving cells develop resistance mechanisms that allow their survival and increase metastatic potential. Despite efforts in this field, the knowledge concerning the post-irradiation behavior and the radioresistant cancer cells phenotype is still limited. Therefore, it is important to elucidate the mechanisms that contribute to the acquisition of this resistant phenotype to improve therapeutic success. In this study, we investigated the characteristics of surviving cells that were submitted to a clinically relevant treatment protocol of hypofractionated irradiation. The cell lines were chosen due to the different levels of genomic instability and genetic mutations and were exposed to 25 Gy delivery in 5 daily fractions. Irradiation surviving cells (ISC) from both cell lines, SW-480 and HCT-116, gain resistance to re-irradiation, increasing their autophagic process and assuming a polyploid giant cancer cell phenotype. In addition, HCT-116 ISC became senescent, while SW-480 ISC show an increase in mesenchymal markers and a decrease in epithelial markers. Long-term evaluation of the ISCs confirmed senescence in HCT-116, while SW-480 exhibited a transient dormancy state, resuming its growth after 14 days. SW-480 ISC also generates a heterogeneous progeny with three distinct phenotypes: rounded and highly proliferative cells, elongated mesenchymal cells, and polyploid giant cancer cells. Furthermore, both ISC enhance the secretion of extracellular vesicles and cytokines (like IL-8, MCP-1, VEGF, and GM-CSF) in a cell-specific manner. Based on these results and the identification of proteins and pathways differentially regulated in ISCs we can contribute to the identification of therapeutic targets for neoadjuvant therapies, in order to improve the prognosis of CRC patients. Citation Format: Josiane Weber Tessmann, Murilo Ramos Rocha, Renata Ivo Vasconcelos, Danielle Lazarin-Bidoia, Raphael Rodrigues Corrêa, Rafael Soares Lindoso, Paulo Emílio Corrêa Leite, Celso Vataru Nakamura, Jose Andres Morgado-Diaz. Hypofractionated radiation therapy promotes distinct resistant cellular fates as a survival mechanism in two colorectal cell lines: senescence or transient dormancy followed by heterogeneous repopulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 211.
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Hiramatsu, Yukiko, Akihisa Fukuda, Satoshi Ogawa, Norihiro Goto, Kozo Ikuta, Motoyuki Tsuda, Yoshihide Matsumoto, et al. "Arid1a is essential for intestinal stem cells through Sox9 regulation." Proceedings of the National Academy of Sciences 116, no. 5 (January 11, 2019): 1704–13. http://dx.doi.org/10.1073/pnas.1804858116.
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Inactivating mutations of Arid1a, a subunit of the Switch/sucrose nonfermentable chromatin remodeling complex, have been reported in multiple human cancers. Intestinal deletion of Arid1a has been reported to induce colorectal cancer in mice; however, its functional role in intestinal homeostasis remains unclear. We investigated the functional role of Arid1a in intestinal homeostasis in mice. We found that intestinal deletion of Arid1a results in loss of intestinal stem cells (ISCs), decreased Paneth and goblet cells, disorganized crypt-villous structures, and increased apoptosis in adult mice. Spheroids did not develop from intestinal epithelial cells deficient for Arid1a. Lineage-tracing experiments revealed that Arid1a deletion in Lgr5+ ISCs leads to impaired self-renewal of Lgr5+ ISCs but does not perturb intestinal homeostasis. The Wnt signaling pathway, including Wnt agonists, receptors, and target genes, was strikingly down-regulated in Arid1a-deficient intestines. We found that Arid1a directly binds to the Sox9 promoter to support its expression. Remarkably, overexpression of Sox9 in intestinal epithelial cells abrogated the above phenotypes, although Sox9 overexpression in intestinal epithelial cells did not restore the expression levels of Wnt agonist and receptor genes. Furthermore, Sox9 overexpression permitted development of spheroids from Arid1a-deficient intestinal epithelial cells. In addition, deletion of Arid1a concomitant with Sox9 overexpression in Lgr5+ ISCs restores self-renewal in Arid1a-deleted Lgr5+ ISCs. These results indicate that Arid1a is indispensable for the maintenance of ISCs and intestinal homeostasis in mice. Mechanistically, this is mainly mediated by Sox9. Our data provide insights into the molecular mechanisms underlying maintenance of ISCs and intestinal homeostasis.
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Wang, Xiaohang, Wei Li, Juan Chen, Sheng Zhao, Shanhu Qiu, Han Yin, Vladmir Carvalho, et al. "A Transcriptional Sequencing Analysis of Islet Stellate Cell and Pancreatic Stellate Cell." Journal of Diabetes Research 2018 (2018): 1–8. http://dx.doi.org/10.1155/2018/7361684.
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Background. Our previous studies have shown that islet stellate cell (ISC), similar to pancreatic stellate cell (PSC) in phenotype and biological characters, may be responsible for the islet fibrosis in type 2 diabetes. To further identify the differences between PSC and ISC and for better understanding of the physiological function of ISC, we employed genome-wide transcriptional analysis on the PSCs and ISCs of Wistar rats. Method. PSCs and ISCs from each rat were primarily cultured at the same condition. Genome-wide transcriptional sequence of stellate cells was generated. The identified differentially expressed genes were validated using RT-PCR. Results. 32 significant differentially expressed genes between PSCs and ISCs were identified. Moreover, collagen type 11a1 (COL11A1), was found to be expressed 2.91-fold higher in ISCs compared with PSCs, indicating that COL11A1 might be a potential key gene modulating the differences between PSC and ISC. Conclusions. Our study identified and validated the differences between PSC and ISC in genome-wide transcriptional scale, confirming the assumption that ISC and PSC are similar other than identical. Moreover, our data might be instrumental for further investigation of ISC and islet fibrosis, and some differential expressed genes may provide an insight into new therapeutic targets for type 2 diabetes.
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Goveas, Neha, Claudia Waskow, Kathrin Arndt, Julian Heuberger, Qinyu Zhang, Dimitra Alexopoulou, Andreas Dahl, et al. "MLL1 is required for maintenance of intestinal stem cells." PLOS Genetics 17, no. 12 (December 3, 2021): e1009250. http://dx.doi.org/10.1371/journal.pgen.1009250.
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Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.
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Gao, Daxing, Tuo Li, Xiao-Dong Li, Xiang Chen, Quan-Zhen Li, Mary Wight-Carter, and Zhijian J. Chen. "Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases." Proceedings of the National Academy of Sciences 112, no. 42 (September 14, 2015): E5699—E5705. http://dx.doi.org/10.1073/pnas.1516465112.
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TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi–Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1−/− mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1−/− mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1−/− mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII−/− mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1−/− and DNaseII−/− mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA.
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Masuzawa, Mikio, Takao Fujimura, Mikihiko Tsubokawa, Shigeo Nishiyama, Kensei Katsuoka, Eiji Terada, Satoshi Kunita, Yoshinori Sakurai, and Hideki Kato. "Establishment of a new murine-phenotypic angiosarcoma cell line (ISOS-1)." Journal of Dermatological Science 16, no. 2 (January 1998): 91–98. http://dx.doi.org/10.1016/s0923-1811(97)00032-7.
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Van Landeghem, Laurianne, M. Agostina Santoro, Adrienne E. Krebs, Amanda T. Mah, Jeffrey J. Dehmer, Adam D. Gracz, Brooks P. Scull, Kirk McNaughton, Scott T. Magness, and P. Kay Lund. "Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation." American Journal of Physiology-Gastrointestinal and Liver Physiology 302, no. 10 (May 15, 2012): G1111—G1132. http://dx.doi.org/10.1152/ajpgi.00519.2011.
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Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.
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Mountz, John D., Min Gao, Shanrun Liu, Zechen Chong, Qi Wu, PingAr Yang, W. Winn Chatham, and Hui-Chen Hsu. "Dysregulation of type I IFN and IL-4R signaling regulates the development of BCR VH and VL repertoire encoding anti-Sm autoantibodies in SLE." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 219.12. http://dx.doi.org/10.4049/jimmunol.204.supp.219.12.
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Abstract The most specific predictor for systemic lupus erythematosus (SLE) and lupus nephritis (LN) is the presence of anti-Smith (Sm) autoAb. Mechanisms leading to selection bias for anti-Sm development is unclear. We found that anti-Sm levels in SLE correlated with increased endogenous IFN-β and interferon-stimulated genes (ISGs) in combination with low (IL-4R) network genes. Using flow cytometry analysis of 47 SLE patients, we found that anti-Sm+ patients exhibited an increased ratio of IFN-β+ activated naive (aNAV) B cells and a decrease in IL-4R+ resting naive (rNAV) B cells. Using a combined 10X single-cell transcriptomics and BCR usage analysis, anti-Sm+LN+ISGhiIL4Rlopatients (Sm+) were compared with anti-Sm−LN− ISGloIL4Rhi patients (Sm−). BCR heavy chain analysis showed that while B cells from Sm+ patients exhibit greater usage of IGHV1-2, IGHV4-34, and IGHV4-39 genes, Sm− SLE patients exhibit greater usage of IGHV3-30, IGHV3-33, and IGHV4-31 genes. Preferential Ig light chain usage for ISGhi B cells was IGLV1-51, IGLV3-21, IGLV6-57, IGKV1-5, IGKV3-15 and IGKV 6-21. Dominant heavy-light chain pair usages for the Sm+ patients were IGHV4/IGKV1 and IGHV4/IGKV3. IGHV4-34, which is intrinsically autoreactive and the target of the 9G4 anti-idiotype Ab, was found to be expressed by the ISGhiIL4Rlo B cells derived from the same Sm+ patients. The results indicated that increased levels of ISGs and decreased levels of IL-4R pathway genes are the two dominant signatures and the underlying mechanisms leading to selection and activation of anti-Sm+ B cells. Verification that the B-cell IFN-β and IL-4R phenotypes are associated with race (AA vs. EA), repertoire (anti-Sm+) and disease (LN+) would lay the basis for targeted rational therapies.
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Cotticelli, M. Grazia, Fabio Acquaviva, Shujuan Xia, Avinash Kaur, Yongping Wang, and Robert B. Wilson. "Phenotypic Screening for Friedreich Ataxia Using Random shRNA Selection." Journal of Biomolecular Screening 20, no. 9 (August 18, 2015): 1084–90. http://dx.doi.org/10.1177/1087057115600433.
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Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix and regulates the iron–sulfur cluster (ISC) assembly complex. ISCs are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain. Decreased expression of frataxin is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. In media with beta-hydroxybutyrate (BHB) as carbon source, primary FRDA fibroblasts grow poorly and/or lose viability over several days. We screened a random, short-hairpin-RNA (shRNA)-expressing library in primary FRDA fibroblasts and identified two shRNAs that reverse the growth/viability defect in BHB media. One of these two clones increases frataxin expression in primary FRDA fibroblasts, either as a vector-expressed shRNA or as a transfected short-interfering RNA (siRNA).
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White, Michael G., Hussain R. Al-Turaifi, Graham N. Holliman, Ali Aldibbiat, Aiman Mahmoud, and James A. M. Shaw. "Pluripotency-associated stem cell marker expression in proliferative cell cultures derived from adult human pancreas." Journal of Endocrinology 211, no. 2 (August 12, 2011): 169–76. http://dx.doi.org/10.1530/joe-11-0123.
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The source of new β-cells in adult human pancreas remains incompletely elucidated with recent studies on rodents providing evidence for neogenesis from progenitor cells in addition to self-replication. The aim of this study was to investigate the expression of pluripotency-associated stem cell markers in proliferative cultures derived from adult human pancreas. Human pancreatic tissue was obtained from deceased donors following ethical approval and relative consent. Islet-enriched fraction was separated from the retrieved organ by digestion and density gradient centrifugation. Dissociated cells were seeded in adherent culture forming proliferative ‘islet survivor cells’ (ISCs). These were characterised at fifth passage by RT-PCR, immunofluorescence staining, FACS, western blot and transfection studies with an OCT4 promoter-driven reporter. Nuclear expression of the pluripotency-associated stem cell marker complex OCT4/SOX2/NANOG was confirmed in ISCs. The phenotype constituted ∼8% of the overall population. OCT4 biosynthesis was confirmed by western blot and activation of an exogenous OCT4 promoter. Co-expression of pluripotency-associated markers has been confirmed in proliferative primary cells derived from adult human pancreas. Further studies are required to elucidate whether these cells possess functional stem cell characteristics and assess potential for differentiation into pancreatic cell lineages including new β-cells.
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Ardiel, Evan L., Troy A. McDiarmid, Tiffany A. Timbers, Kirsten C. Y. Lee, Javad Safaei, Steven L. Pelech, and Catharine H. Rankin. "Insights into the roles of CMK-1 and OGT-1 in interstimulus interval-dependent habituation in Caenorhabditis elegans." Proceedings of the Royal Society B: Biological Sciences 285, no. 1891 (November 14, 2018): 20182084. http://dx.doi.org/10.1098/rspb.2018.2084.
Full textAbstract:
Habituation is a ubiquitous form of non-associative learning observed as a decrement in responding to repeated stimulation that cannot be explained by sensory adaptation or motor fatigue. One of the defining characteristics of habituation is its sensitivity to the rate at which training stimuli are presented—animals habituate faster in response to more rapid stimulation. The molecular mechanisms underlying this interstimulus interval (ISI)-dependent characteristic of habituation remain unknown. In this article, we use behavioural neurogenetic and bioinformatic analyses in the nematode Caenorhabiditis elegans to identify the first molecules that modulate habituation in an ISI-dependent manner. We show that the Caenorhabditis elegans orthologues of Ca 2+ /calmodulin-dependent kinases CaMK1/4, CMK-1 and O-linked N-acetylglucosamine (O-GlcNAc) transferase, OGT-1, both function in primary sensory neurons to inhibit habituation at short ISIs and promote it at long ISIs. In addition, both cmk-1 and ogt-1 mutants display a rare mechanosensory hyper-responsive phenotype (i.e. larger mechanosensory responses than wild-type). Overall, our work identifies two conserved genes that function in sensory neurons to modulate habituation in an ISI-dependent manner, providing the first insights into the molecular mechanisms underlying the universally observed phenomenon that habituation has different properties when stimuli are delivered at different rates.
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Takashima, Shuichiro, Maria L. Martin, Suze A. Jansen, Ya-Yuan Fu, Anastasiya Egorova, Chen Liu, Richard Kolesnick, Caroline A. Lindemans, and Alan M. Hanash. "T-Cell Derived Interferon Gamma Directly Targets Intestinal Epithelium to Induce Stem Cell Apoptosis in GI Gvhd." Blood 134, Supplement_1 (November 13, 2019): 585. http://dx.doi.org/10.1182/blood-2019-130811.
Full textAbstract:
Crypt base intestinal stem cells (ISCs) marked by Lgr5 and Olfm4 maintain the intestinal epithelium, and Paneth cells (PCs) provide an epithelial niche for ISCs in the small bowel. ISCs are reduced during gastrointestinal (GI) GVHD, but the precise mechanisms including the role of niche injury are unknown. Additionally, the specific effects of Interferon-γ (IFNγ) on intestinal epithelium in GVHD remain ill-defined. We evaluated kinetics of ISC loss by histology after allogeneic (allo) BMT using Lgr5-LacZ reporter mice. In both MHC- and miHA-mismatched models (LP>B6 and B6>BDF1), ISC numbers quickly recovered from pretransplant TBI conditioning in recipients of T-cell-depleted (TCD) BMT by day +10, but ISCs failed to recover in recipients of allo T cells. T-cell-induced ISC reduction was functionally validated by genetic marking of stem cell progeny and by culturing intestinal organoids from crypts isolated post-BMT. Similar to the kinetics of ISC loss, ISC-dependent organoid-forming capacity was impaired in recipients of allo T cells compared with TCD BMT recipients on day +10 (p<0.05). Likewise, BMT into Olfm4 reporter mice showed significantly reduced lineage tracing from ISCs in recipients of allo T cells (Fig. 1). To better understand the potential for T cells to interact with the ISC compartment, we performed whole-mount 3-D microscopy to distinguish T cell localization within the intraepithelial and lamina propria compartments post-transplant. We found that donor T cells invading the small bowel after BMT were mostly in the crypt region, and infiltration within the lamina propria adjacent to the ISC compartment was much greater than invasion within the epithelium itself (Fig. 2). We next established an ex vivo co-culture system, to model interactions between intestinal epithelium and donor T cells and investigate mechanisms of T-cell-mediated ISC injury. Screening of effector pathways revealed no impact of perforin or FasL, but identified IFNγ as a principal mediator of ISC injury. Culture with allo T cells significantly reduced viable human and mouse intestinal organoid numbers, and this was inhibited by IFNγ neutralization. IFNγ receptor knockout (IFNγR-/-) organoids were resistant to T cells. IFNγ increased expression of Bak1 and decreased expression of Bcl2 in organoids, and induced ISC apoptosis defined by Annexin+Dapi-Lgr5+ phenotype. ISC killing was mediated by intraepithelial JAK/STAT signaling, as JAK1- and STAT1-deficient organoids were resistant, and it was inhibited by Ruxolitinib. Investigating the role of IFNγ in vivo, FACS analysis confirmed donor T cells to be the primary producers of IFNγ in crypt lamina propria, and BMT with IFNγ-/- donor T cells reduced crypt apoptosis, and preserved ISC frequencies. Moreover, BMT with recipients lacking IFNγR specifically in the intestinal epithelium significantly protected ISCs, reduced crypt apoptosis, and ameliorated GI GVHD pathology (Fig.3). Furthermore, ISCs were also protected by epithelial deletion of STAT1 and by Ruxolitinib treatment. As specific genetic manipulation of ISCs in vivo is not possible because genetic targeting of ISCs results in the same changes in their progeny, we utilized ex vivo models to determine if IFNγ kills ISCs by directly inducing their apoptosis or by damaging the PC niche. Manipulation of the niche by culturing wild-type ISCs with IFNγR-/- PCs was not protective to allo T cells. Using a niche-independent high-purity Lgr5+ ISC culture system based on combined GSK3β and HDAC inhibition, IFNγ directly induced cleaved-caspase-3+ ISC apoptosis and substantial ISC colony death, which were inhibited by Bak/Bax deficiency and by the pan-caspase inhibitor QVD. These results confirmed that IFNγ can directly induce ISC apoptosis independent of other cytotoxic effector molecules and independent of injury to the PC niche. In summary, T cells migrating to the GI tract primarily infiltrated the lamina propria adjacent to the ISC compartment, and T-cell-derived IFNγ directly targeted intestinal epithelium via JAK1/STAT1 signaling to induce ISC apoptosis in a PC-independent manner. ISC reduction and GI GVHD pathology were prevented by inhibiting the IFNγR/JAK1/STAT1 axis within the intestinal epithelium, indicating that in addition to their effects on T cells, JAK inhibitors may treat GVHD by inhibiting pathologic cytokine signaling within target organs and shielding them from allo T cells. Disclosures Hanash: Nexus Global Group LLC: Consultancy.