Literatura académica sobre el tema "Leukocyte trafficking, Anti-integrins therapies"

Background: Inflammatory bowel diseases (IBD) are a group of heterogeneous conditions, characterized by immune-mediated inflammation of the gastrointestinal tract. Traditionally, medical management of these disorders has been based on use of systemic immunosuppressives. The development of new drugs that selectively inhibit leukocyte trafficking to the gut has the potential to reduce inflammation and minimize systemic toxicities. Key Messages: In this article, we review the immunology of the gut and the mechanism of action these emerging therapies for IBD. Natalizumab, a monoclonal antibody to the α4 integrin, was approved for the treatment of multiple sclerosis and showed promise in Crohn's disease (CD), however it is encumbered by the risk of progressive multifocal leukoencephalopathy. Vedolizumab inhibits the α4β7 integrin to induce clinical remission in patients with both ulcerative colitis and CD. Long-term safety data on this agent is not yet available. We also review agents in the pipeline. Finally, we discuss the positioning of therapies and potential alterations to therapeutic algorithms as new medications emerge. Conclusions: New therapies are emerging for IBD; however, long-term data are pending. The positioning of these agents in algorithms will evolve.

Adhesion molecules regulate the influx of leukocytes in normal and inflamed gut. They are also involved in local lymphocyte stimulation and antigen presentation within the intestinal mucosa. In intestinal inflammation, many adhesion molecules are upregulated, but α4-integrins most likely hold a key position in directing leukocytes into the inflamed bowel wall. Therapeutic compounds directed against trafficking of leukocytes have been designed and are being developed as a novel class of drugs in the treatment of Crohn's disease and ulcerative colitis. This review deals with the immunological aspects of leukocyte trafficking focused on gut homing of T cells. Second, the changes in adhesion molecules and T cell trafficking during intestinal inflammation are discussed. Finally, we review the clinical data that have been gathered with respect to the therapeutic potential and the safety of antiadhesion molecule treatment. Antegren, or natalizumab, a humanized anti-α4 integrin IgG4 antibody, has been most extensively evaluated and may be close to registration. A more specific humanized α4β7-integrin MLN-02 has shown preliminary clinical efficacy in ulcerative colitis, and both antergren and MLN-02 appear to be very safe. Trials with the anti-ICAM-1 antisense oligonucleotide ISIS-2302 in steroid refractory Crohn's disease have provided conflicting efficacy data. In the near future, some of these novel biological agents may prove valuable therapeutic tools in the management of refractory inflammatory bowel disease, although it is too early to define the patient population that will benefit most from these agents.

Immunotherapy has emerged as a valuable strategy for the treatment of many cancer types. Interleukin-15 (IL-15) promotes the growth and function of cytotoxic CD8+ T and natural killer (NK) cells. It also enhances leukocyte trafficking and stimulates tumor-infiltrating lymphocytes expansion and activity. Bioactive IL-15 is produced in the body as a heterodimeric cytokine, comprising the IL-15 and the so-called IL-15 receptor alpha chain that are together termed “heterodimeric IL-15” (hetIL-15). hetIL-15, closely resembling the natural form of the cytokine produced in vivo, and IL-15:IL-15Rα complex variants, such as hetIL-15Fc, N-803 and RLI, are the currently available IL-15 agents. These molecules have showed favorable pharmacokinetics and biological function in vivo in comparison to single-chain recombinant IL-15. Preclinical animal studies have supported their anti-tumor activity, suggesting IL-15 as a general method to convert “cold” tumors into “hot”, by promoting tumor lymphocyte infiltration. In clinical trials, IL-15-based therapies are overall well-tolerated and result in the expansion and activation of NK and memory CD8+ T cells. Combinations with other immunotherapies are being investigated to improve the anti-tumor efficacy of IL-15 agents in the clinic.

Abstract Cell-mediated immune responses are highly dependent on environmental context, thus making in vivo studies an important complement to in vitro and molecular approaches. Two-photon microscopy (2PM) is a fluorescence based imaging approach that allows single-cell dynamics to be studied directly in their 3D native tissue context. 2PM is an ideal approach for analyzing leukocyte trafficking dynamics quantitatively and testing cellular immune mechanisms in vivo. Several example applications will be presented where 2PM has uncovered novel immunological phenomena and provided fresh insight into immune responses to infection, autoimmunity and cancer. While 2P imaging has been used extensively to study immune cell trafficking and function in mice, progress is being made to use this imaging technique on clinical biopsy specimens to acquire a multi-dimensional picture of human tissue pathology. We used in vivo 2PM in pre-clinical models of arthritis and bacterial infection to compare and contrast the role of monocytes on neutrophil recruitment. The rapid recruitment of neutrophils and monocytes is critical to early host immune responses to bacterial infection. However, leukocyte recruitment also contributes to chronic inflammatory diseases such as human rheumatoid arthritis. Understanding how cell recruitment is regulated in different inflammatory contexts is crucial for developing safe and effective anti-inflammatory therapies. We found that monocyte depletion with clodronate-liposomes prevented arthritis development in a modified K/BxN serum transfer arthritis model. This protective effect was associated with significantly reduced neutrophil transendothelial migration efficiency. Furthermore, single-cell tracking of a minor population of extravasated neutrophils showed that neutrophil migration and chemotaxis in interstitial tissues was disrupted, contributing to decreased cell localization at phalangeal joints. Similar results were obtained when CCR2+ monocytes were depleted selectively using the monoclonal antibody MC-21, thus implicating CCR2+ monocytes as key regulators of neutrophil extravasation during arthritis initiation. In contrast, neutrophil recruitment to subcutaneous bacterial challenge remained intact and neutrophil extravasation and chemotaxis to sites of infection was not significantly different as compared to non-depleted controls. We also examined whether neutrophil extravasation during acute pulmonary inflammation required monocytes. Neutrophil recruitment in vivo was assessed in a mouse lung transplant-mediated ischemia reperfusion injury model. Similar to the results in the arthritis model, neutrophil recruitment in response to ischemia reperfusion injury was also monocyte dependent. In addition, Ccr2 knockout recipient mice were protected for ischemia reperfusion injury. Results from these complementary mouse models implicate CCR2+ monocytes as key regulators of neutrophil extravasation and chemotaxis in under conditions of aseptic inflammation and further suggest that the cell recruitment signals that that operate during bacterial infection may be quantitatively and/or qualitatively distinct. These studies raise the intriguing possibility that targeting monocytes during chronic inflammatory diseases such as rheumatoid arthritis or acute inflammatory conditions such as ischemia reperfusion injury might provide safer and more selective anti-inflammatory therapies than those that target neutrophils directly. Disclosures: No relevant conflicts of interest to declare.

Abstract Lupus nephritis (LN) is a serious end organ complication of systemic lupus erythematosus (SLE) in which T cells are thought to play an essential role. CD6 is a co-stimulatory receptor on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation and trafficking, and increased levels of CD6 are associated with pathogenic T cell responses. To assess the role of the CD6-ALCAM pathway in LN pathogenesis, we tested a monoclonal antibody against CD6 in a short-term, validated, inducible murine model of lupus nephritis known as nephrotoxic serum nephritis (NTN). NTN mice were treated 3× per week with an anti-CD6 mAb (10D12, 60ug/dose, n=23) or with vehicle control (n=23). Healthy mice were also included as a control (n=12). Mice treated with the anti-CD6 mAb displayed decreased levels of proteinuria (p<0.001) and significantly improved BUN levels (p<0.01) compared to vehicle control mice. Histology also significantly improved with anti-CD6 treatment (p<0.05). RT-PCR revealed significantly decreased levels of VCAM and RANTES in the kidneys of treated mice, while anti-inflammatory IL-10 was increased, compared to vehicle control mice. Flow cytometry analysis indicated decreased accumulation of both renal-infiltrating activated T cells (CD4+CD25+CD69+, p <0.01) inflammatory macrophages (p<0.05). Overall, these results indicate that the CD6-ALCAM pathway is an important driver of inflammation and pathology in LN and, thus, a promising therapeutic option that is more selective than the immunosuppressive therapies currently offered.

Abstract Background A role for gut microbes in IBD has been suspected since the early descriptions of potential infectious agents. IBD is clearly associated with intestinal dysbiosis, that is the imbalance in structures and functions of gut microbiota that disrupts host-microbe and immune homeostasis. Members of the commensal gut microbiota may play a role in etiopathogenesis of IBD. New therapies with diverse mechanisms of action inhibiting leukocyte trafficking or blocking inflammatory cytokines have revolutionized therapeutic approach. A less diverse microbiome, greater abundance of pro-inflammatory and depletion of species with protective mechanisms, such as short-chain fatty acid production, is associated with intestinal inflammation in IBD. Given that intestinal dysbiosis is a hallmark of IBD patients, we investigate the potential role of biologic drugs on both gut microbial and serum metabolomic in the restoration of microbial homeostasis. Methods In 2020-2021, 238 IBD patients (CD=145, UC=93) and 45 healthy controls of the North Italy were enrolled. To define the characteristics of metabolic profile of IBD patients, three different machine learning models based on serum metabolic signatures were developed and applied for the random forest analysis of anti-TNF or anti-integrin users. Stool sample from anti-integrin (N=18) or anti-TNF (N=19)-treated IBD patients was profiled by 16S rRNA gene sequencing, to identify putative associations between medication and microbial composition. Results In IBD patients untargeted metabolomics on serum samples confirmed that both most of the IBD patients (70% of anti-TNF and 100% of vedolizumab users) harbor a metabolic profile enriched of anti-inflammatory and antioxidant molecules. Noteworthy, vedolizumab-treated patients showed a higher microbial diversity than anti-TNF users along with an enrichment of both Bacteroidetes and Proteobacteria. Despite the prevalence of Firmicutes species (60,85-62,97%) in IBD patients, the Firmicutes/Bacteroidetes ratio of vedolizumab (2,9:1) is lower than anti-TNF (5,7:1) users, suggesting that anti-integrin-treated patients have an improved microbial composition. Conclusion The gut microbiome is a key determinant of initiation and propagation of luminal inflammation in IBD. We describe the microbial composition and structure from a large cohort of IBD patients with different biologic therapies, and their metabolomic profile. We found a correlation between an improvement of dysbiosis and a more anti-oxidant and anti-inflammatory metabolic profile in vedolizumab-treated patients. Further experimental studies are important to mechanistically determine how biologic drugs differentially influence gut microbiota and metabolomic and how this may impact IBD course.

Abstract Th17 cells have been implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis (RA). Chemokine receptor CCR2 mediates leukocyte trafficking to sites of inflammation and is considered to be an important proinflammatory factor in RA. However, as an unexpected paradox, mice lacking CCR2 (CCR2-/-) develop an accelerated and enhanced arthritis in the collagen induced arthritis model (CIA). To investigate the underlying mechanism(s), we examined the role of Th17 cells in CCR2-/- mice with CIA. We found that the number of Th17 cells was increased significantly in draining lymph nodes of CCR2-/- CIA mice (845±144) compared to WT controls (289±136, p=0.017). In contrast, Th1 cells were not significantly different between groups (485±171 vs. 516±86 for WT, p=0.88). Consistently, CCR2-/- CIA mice had elevated levels of sera IL-17 (84.4±15.5 pg/ml) and IL-17 mRNA in arthritic joints (3.43±0.61 fold) compared to WT mice (45±9.7 pg/ml in the serum and 0.21±0.05 fold in the joint, p=0.04 and 0.009 respectively). Furthermore, Th17-favored cytokines IL-6 and IL-1β were up-regulated in the serum of CCR2-/- animals (IL-6: 366.1±54.5 pg/ml vs. 58.7±13.3 pg/ml for WT, p=0.0004; IL-1β: 189.6±21.9 pg/ml vs. 106.4±21.3 pg/ml for WT, p=0.01). These data suggest that Th17 cells contribute to the pathogenesis of the aggravated phenotype of autoimmune arthritis in CCR2-/- mice. Mice deficient in CCR2 may serve as a model for evaluations of anti-Th17 therapies against RA.

Abstract Transfusion-related acute lung injury (TRALI) is a leading cause of blood transfusion-related fatalities without available therapies. The pulmonary endothelium is damaged in TRALI, through incompletely understood pathogenic mechanisms, resulting in pulmonary edema. Generally, anti-leukocyte antibodies or biological response modifiers in the transfusion product, in combination with predisposing risk factors in the transfused recipient (e.g. inflammation), are responsible for initiation of TRALI. Remarkably, not all anti-leukocyte antibodies cause TRALI. In a previous in vitro study, we identified increased Fc-mediated complement activation to be a key feature of murine TRALI-inducing antibody 34-1-2S (anti-H-2K d) compared to TRALI-resistant antibody SF1.1.10 (anti-H-2K d) (Zeeuw van der Laan et al, Blood Adv 2020). In the current study, we further explored antibody-mediated TRALI mechanisms in vivo using our previously established TRALI mouse model in which mice are pre-depleted of protective CD4+ T cells and primed with LPS, followed by infusion of antibody 34-1-2S (Kapur et al, Blood 2017, Blood Adv 2018, Blood 2019). A key read-out for TRALI was the lung wet/dry weight ratio (Lung W/D, measure for pulmonary edema). We found that in vitro antibody-mediated complement activation was associated with in vivo antibody-mediated TRALI. 34-1-2S caused severe TRALI (complement activation: +++, Lung W/D: 7.4 ± 0.21), while SF1.1.10 did not cause TRALI (complement activation: +, Lung W/D: 4.68 ± 0.16). Furthermore, Fc-deglycosylated 34-1-2S still caused significant TRALI (complement activation: ++, in vivo Lung W/D: 5.16 ± 0.52). TRALI development was fully Fc-dependent as 34-1-2S-Fab did not cause any TRALI (no complement activation, Lung W/D: 4.35 ± 0.18). Importantly, we found significantly increased levels of complement C1q-C4 complexes in plasma samples of TRALI patients (n=46) compared to healthy controls (n=25): 6.49 ± 5.27 vs 3.84 ± 2.27 AU/ml, respectively, P: 0.0005,***. Similarly, C5a levels were significantly elevated in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 2.52 ± 2.17 vs 1.37 ± 1.08 ng/ml, respectively, P: 0.0006,***. To further dissect the effect of the Fc-part of 34-1-2S in TRALI-induction, we aim to investigate the contribution of Fc-mediated complement activation vs Fc-receptor interaction. We therefore successfully generated chimeric variants of 34-1-2S with a humanized IgG1 Fc-domain containing mutations making them functionally complement dead (K322A), Fc-receptor dead (dG236) or both complement and Fc-receptor dead (PG LALA). We are currently investigating the effects of these variants on in vivo TRALI induction. We next investigated how the in vivo murine TRALI reaction was related to numbers of macrophages, monocytes and neutrophils in blood and lungs. We found that 34-1-2S-TRALI was associated with significantly decreased levels of macrophages in the lungs and increased levels in blood, compared to mice infused with PBS, SF1.1.10 or 34-1-2S-Fab, suggesting that Fc-mediated complement activation and TRALI induction is related to macrophage trafficking from lungs to blood. We did not observe any significant differences between blood and lung neutrophil levels of 34-1-2S-TRALI mice compared to SF1.1.10 TRALI-resistant mice. We hypothesized that in TRALI neutrophils undergo formation of neutrophil extracellular traps (NETs) induced by complement. We observed that C5a enabled potent neutrophil-chemotaxis in vitro (P: 0.0048,**). In addition, using direct immunofluorescence staining of extracellular DNA and Citrullinated histone H3, we observed that both LPS and C5a on their own could induce NET formation in vitro, which was synergistically increased with a combination of both LPS and C5a (P: 0.0417,*), as occurring during TRALI. Furthermore, we found increased levels of NETs to be present in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 1.64 ± 0.97 vs 0.80 ± 0.34 MPO-DNA OD, respectively, P: 0.0002,***. Finally, we targeted the C5a-receptor (C5aR) using a C5aR-antagonist in our TRALI mouse model. Surprisingly, this did not prevent but even worsened TRALI (P: 0.0398,*), with elevated levels of blood monocytes and macrophages. This suggests that an approach directly targeting complement components may be a more promising therapeutic strategy to explore in combatting TRALI. Disclosures No relevant conflicts of interest to declare.

AbstractTick saliva contains anti-inflammatory and immunosuppressive substances that facilitate blood feeding and enhance tick-vectored pathogen transmission, including Anaplasma phagocytophila,an etiologic agent of granulocytic ehrlichiosis. As such, inflammation at a tick-feeding site is strikingly different than that typically observed at other sites of inflammation. Up-regulation of CD11b/CD18 occurs in host granulocytes following interaction or infection withA phagocytophila, and the absence of CD11b/CD18 results in early increases in bacteremia. We hypothesized that β2 integrin–dependent infection kinetics and leukocyte extravasation are important determinants of neutrophil trafficking to, and pathogen acquisition at, tick-feeding sites.A phagocytophila infection kinetics were evaluated in CD11a/CD18, CD11b/CD18, and CD18 knock-out mice using quantitative polymerase chain reaction (PCR) of blood, ticks, and skin biopsies in conjunction with histopathology. A marked increase in the rate ofA phagocytophila infection of neutrophils and pathogen burden in blood followed tick feeding. Infection kinetics were modified by β2 integrin expression and systemic neutrophil counts. Significant neutrophil-pathogen trafficking was observed to both suture and tick sites. Despite the prominent role for β2 integrins in neutrophil arrest in flowing blood, successful pathogen acquisition by ticks occurred in the absence of β2 integrins. Establishment of feeding pools that rely less on leukocyte trafficking and more on small hemorrhages may explain the ready amplification of A phagocytophila DNA from ticks infested on CD11/CD18-deficient mouse strains.

Abstract In the 9 years since the last review on leukocyte and endothelial interactions was published in this journal many of the critical structures involved in leukocyte adherence to and migration across endothelium have been elucidated. With the advent of cell and molecular biology approaches, investigations have progressed from the early descriptions by intravital microscopy and histology, to functional and immunologic characterization of adhesion molecules, and now to the development of genetically deficient animals and the first phase I trial of “anti-adhesion” therapy in humans. The molecular cloning and definition of the adhesive functions of the leukocyte integrins, endothelial members of the Ig gene superfamily, and the selectins has already provided sufficient information to construct an operative paradigm of the molecular basis of leukocyte emigration. The regulation of these adhesion molecules by chemoattractants, cytokines, or chemokines, and the interrelationships of adhesion pathways need to be examined in vitro and, particularly, in vivo. Additional studies are required to dissect the contribution of the individual adhesion molecules to leukocyte emigration in various models of inflammation or immune reaction. Certainly, new adhesion structures will be identified, and the current paradigm of leukocyte emigration will be refined. The promise of new insights into the biology and pathology of the inflammatory and immune response, and the potential for new therapies for a wide variety of diseases assures that this will continue to be an exciting area of investigation.

Alzheimer’s disease (AD) is characterized by severe, progressive decline of cognition due to neuronal loss in brain regions involved in learning and memory. Two main pathophysiological hallmarks of AD are well characterized: amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein. Significant evidence obtained over the last decade has shown that neuroinflammation is also associated with AD pathology. Furthermore, vascular leakage and endothelial activation were reported in AD brains, suggesting a role for vascular inflammation and leukocyte trafficking in the pathogenesis of AD. However, the inflammation mechanisms involved in AD pathogenesis remain largely unknown and a better understanding of the role of inflammation in AD may help to develop new therapeutic approaches to slow the progression of this disorder. Bloodderived leukocyte subpopulations, including lymphocytes, monocytes, and neutrophils, have been identified in the brains of patients with AD and in corresponding animal models, but their role in disease pathogenesis is unclear. We have recently reported an active inflammatory process taking place during AD, which includes up-regulation of adhesion molecules on cerebrovascular endothelium and leukocyte trans-endothelial migration into the brain of AD-like disease mice. Notably, neutrophil depletion during the early phase of disease led to an amelioration of cognitive deficits and neuropathological condition in mouse models of AD, suggesting their contribution to the pathology. Indeed, the inhibition of neutrophil function strongly reduced microglial activation and Aβ deposition, suggesting that neutrophils play a key role in disease progression. The GOAL of the present study was to investigate the role of LFA-1 and VLA-4 integrins in the pathogenesis of AD-like disease. LFA-1, the predominant β2-integrin expressed on leukocytes, is known to play a key role in leukocyte adhesion on inflamed endothelium. Instead VLA-4, the predominant α4-integrin expressed on the surface of lymphocytes, is expressed only by approximately 3% of circulating neutrophils, but several studies demonstrated it represents an alternative pathway for neutrophil migration to inflammatory sites. Interestingly, in addition to neutrophil infiltration we found T cells infiltrating the brain in mouse models of AD at different time points of disease. Therefore, different leukocyte subpopulations migrate into the brain of 3xTg-AD mice, suggesting that neutrophils and T cells may play a role in disease evolution. We performed our experiments in 3xTg-AD mice, which reproduces AD-like cerebral amyloidosis and tangle pathology, and closely resemble the cognitive and behavioral alterations reported in human disease. We first evaluated by two-photon microscopy the effect of monoclonal antibodies known to block the integrin LFA-1 on neutrophil thus affecting adhesion and extravasation in the central nervous system (CNS). Our results showed that LFA-1 integrin blockade prevents neutrophil adhesion, extravasation, and inhibits intraparenchymal motility in the brain of 3xTg-AD mice. In addition, we demonstrate that both oligomeric and fibrillary Aβ are able - 4 - to trigger rapid LFA-1- dependent neutrophil adhesion to its counterligand ICAM-1, with oligomeric Aβ preparation being more effective. Subsequently, we assessed the blockade of neutrophil trafficking by an anti-LFA–1 integrin antibody treatment in 3xTg-AD mice at early stages of the disease confirming that the integrin LFA-1 is fundamental for brain infiltration of neutrophils in AD-like mice. We next studied the effects of LFA-1 ablation on 3xTg-ADxItgal-/- mice lacking LFA-1 integrin. We found that they show improved memory in cognitive tests compared to wild-type animals. Our results demonstrated a reduced of cognitive dysfunctions in 3xTg-ADxItgal-/- compared to age-matched 3xTg-AD mice. These findings were supported by neuropathological data showing a lower density and activation state of microglia in the CA1 and DG and a reduction of Ab deposition and tau hyperphosphorylation in 3xTg-ADxItgal-/- compared to 3xTg-AD aged-matched controls. Taken together, these results suggest that the inhibition of neutrophil trafficking through the blockade of LFA-1 integrin may represent a new therapeutic strategy for AD. In addition, our results showed that treated 3xTg-AD mice with an anti-a4 integrin antibody improved memory function compared to control treated mice in behavioral tests. Notably, restoration of cognitve function in mice that received anti-a4 treatment during early stages of disease was also maintained at late time points in aged animals, suggesting that therapeutic blockade of leukocyte adhesion during the early stages of disease has a longterm beneficial effect on cognition in older mice. In support of the results obtained in cognitive tests, neuropathological studies showed a reduction of amyloid beta deposition, tau hyperphosphorylated and microglial activation. Therefore, VLA-4 integrin may also play a key role in the induction of cognitive deficit and progression of AD-like disease. In summary, the results obtained in the present study show that LFA-1 and VLA-4 integrins contribute to disease pathogenesis and may represent novel therapeutic targets in AD.