Добірка наукової літератури з теми "Immune subversion"

Une caractéristique majeure de l’infection par Toxoplasma gondii est le contrôle rapide de la population parasitaire par une réponse immunitaire engageant des cellules résidentes et recrutées ainsi que des cytokines pro- et anti-inflammatoire. Dans ce contexte, l’IFNγ active une multitude d’activité anti- T. gondii des cellules immunes et non-immunes, mais peut aussi contribuer à l’immunopathologie. T. gondii a élaboré des mécanismes pour contrer les défenses de l’hôte en interférant avec la transcription des gènes stimulés par l’IFNγ. Nous avons identifié TgIST (T. gondii inhibitor of STAT1 transcriptional activity) comme un interrupteur moléculaire exporté par les parasites intracellulaires et qui est localisé dans le noyau des cellules hôtes, où il inhibe l’expression des gènes pro-inflammatoires dépendants de STAT1. Nous avons montré que TgIST séquestre STAT1 à des sites spécifiques, et promeut la formation de chromatine non permissive grâce à sa capacité à recruter le remodeleur chromatinien NuRD. Nous avons montré que durant l’infection aiguë en souris, les parasites déficients pour TgIST sont rapidement éliminés par les monocytes pro-inflammatoires GR1+, ce qui montre le rôle protecteur de TgIST contre les défenses médiées par l’IFNγ. En révélant les fonctions de TgIST, cette étude montre de nouvelles évidences sur la façon dont T.gondii a élaboré une arme moléculaire de choix pour prendre le contrôle sur la réponse immune, de façon à promouvoir le parasitisme à long terme
An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii–targeting activities in immune and nonimmune cells but can also con- tribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ–stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1+ inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ–mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism

The obligate intracellular protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, is a highly ubiquitous pathogen that infects virtually any warm-blooded animal. Although the infection generally remains asymptomatic in healthy individuals, the parasite invariably encysts. It has been shown that T. gondii is able to achieve this goal at least by interfering with MHC-II antigen presentation to dampen the development of the CD4+ T helper cell response and gain a head start on the host adaptive immune system. Previous reports have shown that T. gondii inhibits transcription of MHC-II and several other related genes, but the causative inhibitory molecules have yet to be identified.In an attempt to identify these molecules, a forward genetic screening strategy was initially elaborated, with a genome-wide insertional mutagenesis carried out in order to disrupt the genes encoding for the inhibitory molecules followed. By specifically isolating mutants unable to inhibit MHC-II expression and antigen presentation by flow cytometry, isolation of the disrupted loci and database mining could have enabled the identification by of the encoded inhibitory molecules. However, the screen was not carried out due to experimental limitations.Biochemical analyses were employed to further characterize the MHC-II inhibitory activity, revealing that this activity segregated with the high-speed supernatant (HSS) prepared from sonicated parasites and was enriched with increasing centrifugal speeds. The inhibitory activity was protein dose-dependent and was completely abrogated when the HSS was treated with a broad-spectrum protease. Subcellular fractionation revealed that the inhibitory activity was found in fractions enriched with secretory organelles, specifically rhoptries (ROP) and/or dense granules (GRA). Furthermore, excreted-secreted antigens (ESA) from freshly egressed tachyzoites displayed inhibitory activity. Proteins from ESA preparations were separated by a two-step fractionation using ion exchange chromatography followed size-exclusion chromatography, and analyzed by tandem mass spectrometry (MS/MS). Database mining of the MS/MS results generated a list of possible candidates, the majority of which originated from secretory organelles.Although MHC-II expression was inhibited, low levels of MHC-II molecules were still detected in infected or lysate-treated cells. Experimental results argued that the first layer of transcriptional regulation has to be complemented by a post-translational layer of interference in the host cell endocytic pathway, involving the MHC-II associated invariant chain (Ii), and peptide editor H2-DM. Ii mRNA and protein levels were induced in T. gondii-infected cells, while MHC-II and H2-DM IFN-induced expression was down-regulated. Ii accumulated in infected cells from 20 hour post-infection until host cell lysis, mainly in the ER. In Ii KO cells, the absence of Ii restored the ability of infected bone marrow-derived dendritic cells to present a parasite antigen in the context of MHC-II, arguing that Ii acts as a dominant negative on MHC-II-restricted antigen presentation of endogenously acquired parasite antigens. Keys host proteases, namely legumain, and cathepsins L and S, and the acidification of endosomal compartments were modulated by the parasite, pointing toward a wider manipulation of the host endocytic pathway by T. gondii. Opposing expression patterns of Ii and H2-DM had a drastic effect in vivo not only on parasite dissemination towards lymphoid organs, CD4+ T cell activation, and IFNγ production during acute infection, but also on cyst numbers and survival at the chronic phase of the infection. Altogether, these findings reveal a broader manipulation of host cell processes by T. gondii, and shed new light on the intricate interactions between intracellular pathogens and host cells, and their ability to subvert immune functions to establish successful infections.
Le parasite protozoaire intracellulaire obligatoire Toxoplasma gondii, l'agent causant la toxoplasmose, est un pathogène ubiquitaire capable d'infecter tout animal à sang chaud. En dépit du fait que l'infection reste généralement asymptomatique chez les individus en bonne santé, le parasite s'enkyste inévitablement. Il a été démontré que T. gondii est capable d'atteindre ce but en partie en interférant avec la présentation d'antigène par le complexe majeur d'histocompatibilité (CMH)-II pour diminuer le développement de la réponse des cellules T CD4+ et pour ainsi devancer la réponse adaptative du système immunitaire. Il a été démontré que T. gondii inhibe la transcription de CMH-II et autres gènes liés, mais les molécules inhibitrices causatives restent inconnues.Pour identifier ces molécules, une stratégie pour un criblage génétique avait été élaborée. Une mutagénèse insertionelle à travers le génome devait être conduite pour perturber les gènes codant pour ces molécules inhibitrices, suivie d'un tri par cytométrie en flux de cellules infectées avec des mutants. En isolant les mutants incapables d'inhiber l'expression de CMH-II, l'isolement des locus génétiques et l'exploration des bases de données auraient pu permettre l'identification des molécules codées. Cependant, ce criblage ne fut complété dû à des limitations expérimentales. Des analyses biochimiques ont démontré que l'activité inhibitrice se retrouvait dans le surnageant d'haute-vitesse (HSS) préparé à partir de parasites soniqués, et était enrichie avec des vitesses de centrifugation croissantes. L'activité inhibitrice était dose-dépendante de protéines et était complètement abrogée lorsque le SHV était préalablement traité avec une protéase. Un fractionnement subcellulaire révéla que l'activité se retrouvait dans les fractions enrichies d'organelles sécrétoires (rhoptries et granules denses). Aussi, des antigènes excrétés-sécrétés (ESA) obtenus de tachyzoïtes fraîchement lysés possédaient une activité inhibitrice. Les protéines d'ESA furent séparées par fractionnement en deux étapes, commençant par une chromatographie par échange d'ions, suivi par une chromatographie d'exclusion par taille et analysées par spectrométrie de masse en tandem (MS/MS). Les résultats obtenus furent comparés aux bases de données, et une liste fut dressée avec de candidats potentiels, la majorité d'entre eux provenant d'organelles de sécrétion.Malgré l'expression réduite, quelques molécules de CMH-II étaient détectés dans les cellules infectées ou traitées. Nos résultats démontrent que la régulation transcriptionelle doit être complémentée par une interférence au niveau post-traductionel dans la voie endocytique de la cellule hôte qui implique la chaîne invariante associée au CMH-II (Ii) et l'éditeur de peptide H2-DM. Les niveaux d'ARNm et de protéines d'Ii étaient induits dans les cellules infectées, alors que ceux de CMH-II et d'H2-DM étaient inhibés. Ii s'accumulait dans les cellules infectées dès 20 heures post-infection, principalement dans le RE. Dans les cellules Ii KO, l'absence d'Ii rétablit la capacité de cellules dendritiques à présenter un antigène du parasite dans le contexte de CMH-II, proposant ainsi qu'Ii agit comme un dominant négatif sur la présentation d'antigènes endogènes provenant du parasite. Des protéases de l'hôte (légumaine, cathepsines L et S) et l'acidification des compartiments endosomaux étaient modulées par le parasite, révélant une manipulation plus étendue de la voie endocytique. Les modes d'expression opposés d'Ii et H2-DM avaient un effet in vivo sur la dissémination des parasites vers des organes lymphoïdes, l'activation des cellules T CD4+, la production d'IFN, le nombre de kystes et la survie. Collectivement, nos résultats montrent l'étendue des processus de manipulation par T. gondii, révélant de complexes interactions avec l'hôte et sa capacité de subvertir les fonctions immunitaires pour établir une infection chronique.

Les cellules dendritiques (DCs), comprenant les cDC2s BDCA1+, les cDC1s BDCA3+ et les pDCs BDCA2+ sont les chefs d’orchestre des réponses immunitaires. Leur plasticité joue un rôle crucial dans l’orientation des réponses immunitaires, notamment en contexte de cancer. Cependant, l’échappement à la surveillance immunitaire est une étape essentielle dans le développement des tumeurs. En contexte de mélanome, les DCs trouvées dans la tumeur ont une fonctionnalité altérée, influençant négativement l’évolution clinique des patients. Les mécanismes employés par le mélanome pour moduler l’immunité ne sont que partiellement élucidés. L’immuno-métabolisme émerge comme un facteur décisif pour l’orientation des réponses immunes en contexte de cancer. Parallèlement, les cellules tumorales présentent à leur surface une glycosylation aberrante des protéines et lipides qui peut être reconnue par les lectines, récepteurs exprimés par les DCs. Parmi eux, les récepteurs lectines de type C (CLR) sont cruciaux pour la plasticité des DCs et le façonnement des réponses immunitaires, et leur expression est perturbée sur les DCs des patients mélanome. De plus, le glycocode des cellules tumorales de mélanome est corrélé avec la fonction des DCs et l’évolution clinique des patients. Néanmoins, l’impact des différents motifs de glycosylation dans le mélanome sur le phénotype, la fonction et le métabolisme des DCs n’est pas connu.Nous avons étudié les interactions des sous-types de DCs avec six glycans présents à la surface des cellules de mélanome (Gal, Man, GalNAc, s-Tn, Fuc, GlcNAc). Nous avons analysé l’impact de ces glycans sur le phénotype (état d’activation, points de contrôle immunitaires (ICP)) et la fonction (cytokines/chimiokines) des DCs. Afin de mieux comprendre la dérégulation de la fonction des DCs dans le mélanome, nous avons exploré leur métabolisme chez les patients grâce à la technique SCENITH, et mis en relation leur profil métabolique avec leur phénotype, leur fonction et l’évolution clinique des patients. Nous avons aussi évalué l’impact des cellules tumorales et du glycocode sur le métabolisme des DCs, et évalué la possibilité de moduler les voies métaboliques afin de réverser l’impact des glycans sur la fonction des DCs.Les DCs sont capables d’interagir avec et d’internaliser les glycans étudiés, à différentes intensités selon le sous-type de DCs et la nature du glycan. Le fucose induit un remodelage de l’expression des ICPs et une augmentation des molécules d’activation, et provoque la sécrétion de cytokines/chimiokines associées à l’inflammation et à la progression tumorale. Après activation des DCs, leur sécrétome est complètement remodelé par l’exposition aux glycans, particulièrement avec le fucose. Parallèlement, nous avons mis en évidence des perturbations métaboliques majeures des DCs dans le sang et la tumeur des patients mélanome par rapport aux donneurs sains. L’expression de marqueurs d’activation et d’ICPs par les DCs ainsi que l’évolution clinique des patients sont liés au profil métabolique des DCs. De plus, le métabolisme des DCs en co-culture avec des cellules de mélanome est corrélé avec l’expression de certains glycans tumoraux. En accord avec ces résultats, les glycans étudiés modulent directement le métabolisme des DCs en plus de leur phénotype et de leur fonction. Le blocage du transporteur de lactate MCT-1 permet de restaurer la fonctionnalité des DCs altérée par les glycans.Cette étude révèle l’importance des motifs glycosylés dans la modulation et la régulation des DCs. L’axe glycan-lectine-DC émerge comme un nouveau point de contrôle immunitaire dans le mélanome, lié au métabolisme et qui pourrait permettre la restauration de l’immunité anti-tumorale en empêchant les interactions des DCs avec les glycans ou en modulant leur métabolisme. Cet axe ouvre la voie au développement de nouvelles stratégies thérapeutiques afin d’améliorer l’évolution clinique des patients atteints de mélanome
Dendritic cells (DCs), mostly consisting of BDCA1+ cDC2s, BDCA3+ cDC1s, and BDCA2+ pDCs are the conductors of immune responses. Their plasticity plays a crucial role in the orientation of immune responses, especially in the context of cancer. However, escape from immune surveillance is a key step for tumor development. In the context of melanoma, tumor-infiltrating and circulating DCs harbor an altered functionality, negatively linked with the clinical outcome of patients. The mechanisms employed by melanoma to modulate immunity are only partially deciphered. Immuno-metabolism emerges as a decisive factor for the orientation of immune responses in cancer. In parallel, tumor cells display aberrant glycans on surface protein and lipids that can be recognized by lectin receptors, expressed by DCs. Among them, C-type lectin receptors (CLRs) are crucial for DCs’ plasticity and the modeling of immune responses, and their expression is perturbed on DCs from melanoma patients. In addition, the tumor cells’ glycocode correlates with DC function and clinical outcome of patients. Nevertheless, influence of the various glycosylation motifs on immunity remains unknown in melanoma.We investigated the interactions of DC subsets with six glycans present on the surface of melanoma tumor cells (Gal, Man, GalNAc, s-Tn, Fuc, GlcNAc). We analyzed the effect of these glycans on the phenotype (activation status, immune checkpoints (ICP)), and the function (cytokines/chemokines) of DCs. In order to better understand DCs dysregulation in melanoma, we explored their metabolism among patients thanks to the SCENITH technique, and analyzed the correlation with their phenotype, their function and the clinical outcome of patients. We also assessed the impact of tumor cells and their glycocode on DCs’ metabolism, and we evaluated the possibility to modulate metabolic pathways with the aim of reverting the impact of glycans on DCs’ function.DCs are able to interact with and to internalize the studied glycans, at different intensities according to the DC subset and to the nature of the glycan. Fucose induces a remodeling of ICP expression and increases activation molecules, in addition to trigger the secretion of pro-inflammatory and pro-tumoral cytokines/chemokines. After activation, DC’s secretome is completely reshaped by glycan exposure, particularly with fucose. In parallel, we highlight major metabolic disturbances in DCs from patients’ blood and tumor compared to healthy donors. The expression of activation markers and ICPs by DCs as well as the clinical outcome of patients are linked with the metabolic profile of DCs. Moreover, DCs’ metabolism in co-culture with melanoma cells correlates with the expression of particular tumor glycans. Coherently, the studied glycans directly modulate DCs’ metabolism in addition to their phenotype and function. The blockade of the MCT-1 lactate transporter allows restoring DCs’ function altered by glycans.This study unveils the importance of glycan motifs in the modulation and regulation of DCs. The glycan-lectin-DC axis emerges as a new immune checkpoint in melanoma, linked with metabolism, and which could enable the restoration of anti-tumor immunity by preventing DC-glycan interactions or by acting on their metabolism. This axis opens the way for the development of new therapeutic strategies with the aim of improving clinical success for melanoma patients

.Les pDCs et Tγδ ont des rôles cruciaux dans l’initiation et l’orientation des réponses immunitaires. Leurs fonctions uniques, leur grande plasticité et leur capacité d’interagir avec de nombreux acteurs immunitaires leur permettent de créer un lien entre l’immunité innée et l’immunité adaptative. Elles contribuent donc grandement aux réponses immunitaires protectrices et pathogéniques, et sont de ce fait très prometteuses pour le développement d’immunothérapies anti-tumorales, autant en tant que vecteurs que cibles. Cependant, les lymphocytes Tγδ n’ont pas été étudiés de manière approfondie en contexte de mélanome, et les interactions entre pDCs et Tγδ n’ont été explicitées ni en contexte sain, ni en contexte mélanome, ou le contrôle immunitaire de la tumeur ‡ long terme est encore un défi. Nous avons réalisé une étude détaillée du phénotype et de la fonction des lymphocytes Tγδ circulant et infiltrant le mélanome, et analysé leur impact sur l’évolution clinique. Nous avons aussi caractérisé les interactions bidirectionnelles entre les pDCs et les Tγδ issus de sang de donneurs sains, et de sang ou de tumeur de patients. Nous avons mis en évidence que le mélanome détourne les fonctions effectrices des Tγδ dans le but d’échapper au contrôle immunitaire, et que les caractéristiques des Tγδ issus de sang ou de tumeurs de patients peuvent étre des bio-marqueurs prometteurs d’évolution clinique. Nous avons également montré que les interactions entre pDCs et Tγδ sont médiées par les IFNs de type I et II et par le récepteur BTN3A, essentiel pour l’activation des Td2+, et sont dérégulées en contexte de mélanome. L’administration de cytokines et d’anticorps ciblant les points de contrôle immunitaire peut rétablir des interactions fonctionnelles entre les deux populations cellulaires. De façon intéressante, nous avons observé une augmentation de l’expression de BTN3A sur les pDCs et Tγδ issus de sang de patients ou de tumeurs, tout en soulignant une potentielle dysfonction de cette molécule. Notre étude révèle que le mélanome détourne les interactions entre pDCs et Tgd notamment via la dérégulation de BTN3A. De tels résultats motivent l’exploitation de ces effecteurs immunitaires ainsi que de leur synergie, pour développer de nouvelles approches thérapeutiques exploitant leur potentiel anti-tumoral tout en évitant leur détournement par la tumeur pour améliorer l’évolution clinique des patients. Nos découvertes soutiennent l’exploitation de ces partenaires puissants et prometteurs pour élaborer de nouvelles stratégies thérapeutiques et restaurer des réponses immunes appropriées en contexte de cancer, infections et auto-immunité
Both pDCs and γδT cells harbor critical roles in immune responses induction and orientation. Their unique features, high functional plasticity and ability to interact with many immune cell types allow them to bridge innate and adaptive immunity. They actively contribute to protective and pathogenic immune responses, which render them very attractive both as targets and vectors for cancer immunotherapy. Yet, γδT cells have not been extensively explored in melanoma, and despite strategic and closed missions, cross-talks between pDCs and γδT cells have not been deciphered yet, neither in healthy context nor in cancers, especially in melanoma where the long-term control of the tumor still remains a challenge. We provided here a detailed investigation of the phenotypic and functional properties of circulating and tumor-infiltrating γδT cells in melanoma patients, as well as their impact on clinical evolution. We also characterized the bidirectional cross-talks between pDCs and γδT cells both from healthy donor’s blood, patient’s blood and tumor micro-environment. Our study highlighted that melanoma hijacked γδT cells to escape from immune control, and revealed that circulating and tumor-infiltrating γδT cell features are promising potential biomarkers of clinical evolution. We also demonstrated crucial bidirectional interactions between these key potent immune players though type I and II IFN and BTN3A that are dysfunctional in the context of melanoma. Reversion of the dysfunctional bidirectional cross-talks in melanoma context could be achieved by specific cytokine administration and immune checkpoint targeting. We also revealed an increased expression of BTN3A on circulating and tumor-infiltrating pDCs and γδT cells from melanoma patients but stressed out its potential functional impairment.Thus, our study uncovered that melanoma hijacked pDCs/ γδT cells bidirectional interplay to escape from immune control, and pointed out BTN3A dysfunction. Such understanding will help harnessing and synergizing the power of these potent immune cells to design new therapeutic approaches exploiting their antitumor potential while counteracting their skewing by tumors to improve patient outcomes. Our findings pave the way to manipulate these potent and promising cell partners to design novel immunotherapeutic strategies and restore appropriate immune responses in cancers, infections and autoimmune diseases

Pathogenic mycobacteria are among the most dreadful pathogens known to mankind as one third of the world’s population is latently infected with M. tuberculosis, the causative agent of pulmonary tuberculosis. Host immune responses to mycobacterial infections is characterized by increased cell-mediated immunity constituted by CD4+, CD8+, γδT cells, macrophages, and dendritic cells (DCs). The effector mechanisms of these cells are chiefly mediated by the molecules such as IFN-γ, TNF-α, IL-12, IL-23, lymphotoxins, prostaglandins, reactive oxygen species (ROS) and reactive nitrogenous species (RNI) which are secreted during infection. These effector molecules trigger series of events that culminate into containment of the infection. Despite such robust immune responses elicited, mycobacteria still survive within the host. The success of mycobacteria for immune evasion lies in its ability to regulate the functions of sentinels of the immune system like macrophages and DCs. Modulation of the host cellular signaling cascades is one of the strategies that are employed by mycobacteria to significantly reprogram the innate immune cells for their benefit. Therefore, the key to control mycobacterial growth and limit pathogenesis lies in the understanding of the initial events of the mycobacterial infection including interaction between Mycobacterium and primary responders like macrophages and DCs. In this context, the role of pattern recognition receptors (PRRs) in orchestrating the host mediated immune responses attains central importance. The host cell surface/ intracellularly expressed PRRs play a vital role in shaping the immune response to the mycobacterial infections. Of these PRRs, Toll-Like Receptors (TLRs) are the predominant interacting partners on macrophages for mycobacteria that activate the initial signaling cascades which eventually lead to containment or elimination of the pathogen. Of the TLRs, TLR2 constitute the key immune surveillance mechanism for recognition as well as control of mycobacterial infection. Mycobacteria contain complex lipid rich cell wall that is constituted by several natural ligands which can trigger various PRRs on the host immune cells. Lipo-arabinomannan (LAM), Lipomannan (LM), Phosphotidyl Inositol Mannosides (PIM) and 19-kDa antigens of Mycobacteria are known to interact with TLR2 and induce pro-inflammatory responses. Recent reports have shown that, PE_PGRS antigens of M. tuberculosis interact with TLR2 to regulate DC maturation and functions as well as to regulate DC mediated activation of T cells. Rv0754, Rv0978c, Rv0980c that belong to PE family of genes of mycobacteria have been implicated in modulation of human DCs. In another study, it has been reported that antigen processing and presentation by host immune cells can be inhibited by 19-kDa protein, LpqH (Rv3763) and LprG (Rv1411c) by utilizing TLR2. Surprisingly, recognition as well as amplification of the pathogen specific signaling events play important role not only in mounting immune response to infection but also to regulate explicit immune responses. In this scenario, integration of key signaling networks which modulate host immunity to pathogenic mycobacterial infections remains to be explored. In accordance with these above observations, signal transducing pathways that act downstream of TLRs play crucial role in modulation of host cell gene expression in terms of immune modulatory cytokines and chemokines which bring in the primary responders like neutrophils, NK cells, T cells, etc including macrophages to the site of infection. This emphasizes the decisive role of TLRs in programming host immune cell function. However, during the process of ensuing immunity against invading pathogens, beside TLR-mediated signaling, many other signaling cascades takes place as an effector cascade of TLR or in parallel to TLR signaling pathway to execute specific functions in divergent cellular contexts. Recent studies from our laboratory have shown the role for TLR2 dependent activation of Notch1 pathway in regulating the expression of an immune-modulator, SOCS-3 in macrophages during mycobacterial infection. Very recently, it has been also shown that activation of Sonic Hedgehog signaling pathway plays a crucial role in fine-tuning the TLR2-mediated gene expression upon mycobacterial infection in macrophages. The current study primarily focuses on the role for Wnt signaling pathway in TLR2-mediated regulation of macrophage gene expression and its functional significance. The current investigation demonstrates the differential activation of Wnt-β-Catenin and Notch1 signaling in response to pathogen-specific activation of TLR2 during infection with M. bovis BCG, S. typhimurium and S. aureus. Among these pathogens, M. bovis BCG triggered robust expression of Wnt5a, FzD4, LRP5 including a heightened stabilization and nuclear translocation of β-Catenin. Thus, effectuating the transcriptional activation of Jagged1 forming functional overlap between Wnt-β-Catenin and Notch1 signaling. Bringing correlation with the clinical manifestations of M. tuberculosis infection in vivo, we could detect augmented expression of signaling cohorts of Wnt-β-Catenin-Notch1 cascade as well as SOCS-3, a Notch1 responsive gene, in PBMC of pulmonary tuberculosis patients or brain samples derived from TBM patients. Induced expression of SOCS-3 acts as a significant factor in influencing the initiation and strength of the mounted innate immune response. SOCS-3, a negative regulator of multiple cytokines and TLR-induced signaling, is often associated with down modulation of proinflammatory responses during infection with pathogenic microbes. During intensive interplay between signaling pathways, nitric oxide (NO) serves as a pathological link that modulates direct cooperation of TLR2 with Notch1 signaling to regulate specific components of TLR2 responses. Significantly, NO was shown to regulate Wnt-mediated responses in colitis. In the view of these observations, we explored whether TLR2 triggered activation of Wnt-β-Catenin signaling could include the capacity of inducible nitric oxide synthase (iNOS)/NO to regulate Notch1 responses. We show that stabilization of β-Catenin in wild type macrophages but not in iNOS-/- could trigger the activation of Notch1 signaling as evidenced by activation of γ-secretase complex as well as expression of Notch1 ligand, Jagged1 and Notch1 target gene product SOCS-3. Our study identified Wnt-β-Catenin as critical regulator of pathogen-specific TLR2 responses which in conjunction with Notch1 controls the macrophage gene expression. As mentioned, among several PRRs, TLRs especially TLR2 has been reported to interact with Mycobacterium and regulate gene expression in macrophages. For efficient activation or fine tuning of the TLR mediated immune responses, TLR may be complemented or even may relay on PRRs that belong to divergent families. Interestingly, it has been reported that, mycobacterial infections lead to induced expression of IL-12, a key cytokine that mount efficient inflammatory response and drives infected DCs to draining lymphnode for antigen presentation independent of TLR2. In this regard, C-type lectin receptors (CLR) like Dectin-1 has been implicated in both opsonic and nonopsonic binding and internalization of pathogens and is therefore thought to be important player in mouthing effective immune responses to infections. Dectin-1 being the important PRR that recognize fungi and fungal cell wall components like β 1- 3 glucan linked carbohydrates can directly induce respiratory burst in macrophages and IL-12, IL-10 as well as phagocytosis in DCs. Despite the growing literature on the biology of Dectin-1 in fungal immunity, there remains scarcity of information on the function of Dectin-1 in host recognition of other pathogens like mycobacteria. In this study, we assessed the possible involvement of Dectin-1 in Mycobacterium mediated immune responses in murine macrophages. Though mycobacterium is shown to interact with Dectin-1, the components of mycobacteria that induce the signal are yet to be identified. We utilized Curdlan, a specific ligand to Dectin-1 to address the downstream signaling pathways which are dependent or independent of TLR2 and their role in fine tuning TLR mediated inflammatory responses. Signaling perturbation studies as well as the existing literature clearly suggest that there is activation of SyK kinase immediate downstream of Dectin-1 receptor that induces respiratory burst. The current investigation demonstrated that activation of Dectin-1 receptor stabilized cytosolic β-Catenin and induced expression of Wnt5a in macrophages. Pharmacological signaling intervention as well as genetic knock out studies clearly implicated the critical role of ROS in stabilization of β-Catenin and Wnt5a expression. Activation of Ca+2/CAMKII-mediated Wnt pathway by Wnt5a and its interaction with several signaling cohorts eventually led to induction of members of two cytokine regulatory families PIAS-1 and SOCS-1 respectively. Surprisingly, activation of TLRs and Dectin-1 lead to counteraction of signaling cascades and down regulated the TLR-induced inflammatory gene expression. Competitive ligand mediated inhibition or dominant negative approach or pharmacological inhibitor studies that block Dectin- 1 signaling relieved the Dectin-1 inhibitory effect over TLR signaling. Ectopic expression of Dectin-1-induced genes such as PIAS-1 and SOCS-1 led to modulation of the protein levels of MyD88, IRAK1 and IRAK4, which are the key adaptor molecules for generating TLR responses. Thus, the present study highlights the decisive role for Wnt signaling pathway activated by Dectin-1 in fine-tuning the TLR mediated inflammatory responses. In perspective of these studies, sequential and coordinated activation of TLR2-driven signal transduction pathways influence the overall strength of innate immune responses. As described, TLR2 exhibits dominant role in sensing various agonists and pathogen associated molecular patterns of microbes at the cell surface and considered to be a major effectuator of proinflammatory responses. Mycobacterial cell wall antigens that belong to PE and PPE family such as Rv0978c, Rv0980c and Rv0754 are shown to induce maturation of human DCs and drive the T cell mediated immune responses towards Th1 or Th2. In addition to this, our laboratory data has shown that DC maturation was rescued upon treatment with TLR2-agoninsts like Rv0754 in the presence of immunosuppressive conditions exhibited by CTLA-4 and TGF-β in a coordinated fashion along with other PRR family receptors NOD1 and NOD2. TLR2 and NOD dependent activation of Notch/PI3K kinase pathway is implicated in maturation of human DCs under immunosuppressive milieu. In the current investigation, we demonstrated that TLR2 receptor triggering by mycobacterial cell wall antigens like Rv0754 and Rv1917c induce maturation of human DCs and activation of T cells under Programmed Death-1 (PD-1) induced immune suppressive condition. During this process, Wnt signaling pathway plays a decisive role and matures the human DCs in coordination with other host cell signaling cascades. Altogether, our findings establish the understanding of the conceptual framework of regulation of TLR responses by Wnt signaling pathway. Integration of PRRs signaling by Wnt signaling led to fine tuning of TLR-mediated inflammatory gene expression. This study underscores the role for Wnt responsive molecular regulators like PIAS-1 and SOCS-1 in deciding the macrophage cell-fate during immune responses elicited by multiple pathogenic infections. Importantly, our results also suggest the critical role of Wnt in rescuing the human DC maturation under immunosuppressive conditions induced by PD-1.

Pathogenic Mycobacteria are among the most unrelenting pathogens known to mankind as one-third of the world population is latently infected with Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis. Despite many species of mycobacteria elicits robust host T cell responses as well as production of cytokines like interferon-γ (IFN- γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. One potential mechanism by which mycobacteria may achieve a state of long-term persistence amid a robust host immune response is by modulating the signaling cascades leading to macrophage activation. Activation of proinflammatory responses by the host macrophages upon infection with mycobacteria requires the involvement of a variety of signaling events. Studies have indicated that macrophages infected with pathogenic mycobacteria produce significantly less tumor necrosis factor (TNF)-α and other proinflammatory molecules compared with infection with nonpathogenic mycobacteria, which likely play a role in enhancing mycobacterial survival in vivo. Furthermore, macrophages infected with mycobacteria become refractory to many cytokines including IFN-γ and modulation of host cell signaling responses is critical for the suppression of a generalized inflammatory response which might influence the persistence of mycobacteria within the host. In this context, Suppressor of cytokine signaling (SOCS) 3, a member of SOCS family function as negative regulators of multiple cytokine and toll like receptor induced signaling. The SOCS3 has been shown to specifically inhibit signaling by IFN-γ, IL-6 family of cytokines and can act as a negative regulator of inflammatory responses. In this regard, many species of mycobacteria including M. bovis BCG triggers the inducible expression of SOCS3. Further, it has been suggested that M. bovis BCG triggered SOCS3 and SOCS1 proteins leads to the inhibition of IFN- γ stimulated JAK/STAT signaling in macrophages. Albeit JAK/STAT signaling pathway is generally believed to be involved, STAT-independent signals are suggested to take part in the induction of SOCS proteins in many systems signifying the involvement of multiple signal pathways in regulation of SOCS expression. Further little is known about the early, receptor proximal signaling mechanisms underlying mycobacteria-mediated induction of SOCS3. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like LAM, PIM, TDM, PE family antigens etc are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extra cellular environment in the form of exocytosed vesicles. In this context, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. PIM are suggested to be the common anchor of LM and LAM as PIM, LM, and LAM originate from identical biosynthetic pathway. PIM are present in virulent M. tuberculosis H37Rv as well as in M. bovis BCG and a number of biological functions have been recently credited to PIM2. PIM2 is suggested to trigger the activation of cells via Toll like receptor (TLR)-2 and stimulation resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. PIM2 induces proinflammatory stimuli such as TNF-α and IL-12 in murine and human macrophages in a TLR2 dependent manner. PIM exhibited pulmonary granuloma-forming activities as well as was shown to be responsible for the recruitment of NKT cells to granulomas. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether M. bovis BCG or novel cell surface antigens like PIM2 or Rv0978c, a PE-PGRS protein with unknown function can contribute to M. bovis BCG triggered molecular signaling events leading to SOCS3 expression in macrophages. Our studies clearly demonstrated that M. bovis BCG can trigger SOCS3 expression in macrophages. The inception of signaling by M. bovis BCG is TLR2-MyD88 dependent, but not TLR4 dependent. The perturbation of TLR2 signaling and the downregulation of MyD88 resulted in significant decrease in SOCS3 expression implicating the role of TLR2-MyD88 axis in M. bovis BCG triggered signaling. Experiments with cycloheximide and neutralizing antibodies to IL-10 evinced that M. bovis BCG triggered SOCS3 expression is a primary response and requires direct activation of signaling cascades. In the current study, we show for the first time that infection of macrophages with M. bovis BCG activates NOTCH1 signaling events, which leads to expression of SOCS3. The perturbation of NOTCH signaling in infected macrophages either by siRNA mediated down regulation of NOTCH1 or RBP-Jk or by inhibition with pharmacological inhibitor gamma secretase-I, resulted in the marked reduction in the expression of SOCS3. Further, the enforced expression of the NOTCH1 intracellular domain (NICD) in RAW264.7 macrophages induces the expression of SOCS3, which can be further potentiated by M. bovis BCG. Furthermore, the inhibition of TLR2 signaling by a TLR2 dominant-negative construct resulted in inhibition of NOTCH1 activation. Additionally, our results demonstrates for the first time that physical association of TLR2 with both Phosphoinositide-3 Kinase (PI3K) and NOTCH1, which suggest the significant role of TLR2 triggering by of M. bovis BCG in the activation of PI3K and NOTCH1. More importantly, signaling perturbations data suggest the involvement of cross-talk among the members of PI3K and MAPK cascades with NOTCH1 signaling in SOCS3 expression. In addition, SOCS3 expression requires the NOTCH1 mediated recruitment of CSL/RBP-Jk and Nuclear Factor-B (NF-B) to the SOCS3 promoter. A number of biological functions triggered by mycobacteria are often attributed to many of the cell wall antigens. As part of our current investigation, we explored whether two novel cell wall associated antigens namely PIM2 and a PE-PGRS antigen, Rv0978c could play as significant or crucial cell wall ingredients which imparts ability to M. bovis BCG to trigger activation of NOTCH signaling leading to SOCS3 expression. Akin to M. bovis BCG, PIM2 activates NOTCH1 signaling resulting NICD formation which leads to the expression of SOCS3 in a TLR2-MyD88 dependent manner. PIM2 mediated NOTCH1 activation, both directly influences the SOCS3 expression by serving as coactivator in RBP-Jk complex and indirectly triggers SOCS3 expression by activating PI3K-MAPK-NF-κB cascade. One important outcome of the genome sequencing project of M. tuberculosis was the discovery of two new multigene families designated PE and PPE, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many PE and PPE proteins are composed only of PE or PPE homologous domains. However, in other proteins, the PE domain is often linked to a unique domain of various lengths that is rich in alanine and glycine amino acids, termed the PGRS domain (PE-PGRS subfamily). PE family genes were suggested to play roles in the virulence of the pathogen and many members of PE family proteins are reported be localized on the surface of M. tuberculosis bacilli. Some of the PE proteins may play a role in immune evasion and antigenic variation or may be linked to virulence. Additionally, it has been suggested that the PE-PGRS subfamily of PE genes is enriched in genes with a high probability of being essential for M. tuberculosis. The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. In this context, we have chosen to study Rv0978c as a typical member of PE-PGRS family based on the following observations. Rv0978c was upregulated in TB bacilli upon infection of macrophages. Rv0978c was demonstrated to be a member of a group of genes called in vivo-expressed genomic island, which were shown to be upregulated in M. tuberculosis bacilli during infection of mice. Rv0978c was also shown to be upregulated, at least eightfold, in human brain microvascular endothelial cell-associated M. tuberculosis infection, suggesting a role for endothelial cell invasion and intracellular survival. In the current investigation, we have demonstrated that Rv0978c is hypoxia responsive gene based on promoter analysis and upregulated in M. tuberculosis during the infection of macrophages. Further, Rv0978c is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. In this perspective, our results clearly demonstrate that Rv0978c triggers SOCS3 expression by activating PI3K-ERK1/2-NF-B cascade in mouse macrophages. Additionally, Rv0978c elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0978c is an immunodominant antigen demonstrating significant T cell and humoral reactivites. These observations clearly advocate that Rv0978c protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0978c is immunogenic. These results clearly describe the cross-talk of NOTCH1 signaling with signaling pathways like PI3K and MAPK pathways during infection of macrophages with M. bovis BCG eventually resulting in regulation of specific gene expressions, such as SOCS3. These observations lead to a possibility of differential effects of NOTCH1 signaling activated upon infection by an intracellular bacillus, which could be involved in modulation of macrophage functions depending on a local immunological milieu. Taken together, our findings suggest that, induction of Suppressors of Cytokine Signaling 3 molecule by M. bovis BCG or by its cell wall antigens represents a crucial immune subversion mechanism in order to suppress or attenuate host responses to cytokines to generate the conditions that favor survival of the mycobacteria.

Pathogenic Mycobacteria are among the most unrelenting pathogens known to mankind as one-third of the world population is latently infected with Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis. Despite many species of mycobacteria elicits robust host T cell responses as well as production of cytokines like interferon-γ (IFN- γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. One potential mechanism by which mycobacteria may achieve a state of long-term persistence amid a robust host immune response is by modulating the signaling cascades leading to macrophage activation. Activation of proinflammatory responses by the host macrophages upon infection with mycobacteria requires the involvement of a variety of signaling events. Studies have indicated that macrophages infected with pathogenic mycobacteria produce significantly less tumor necrosis factor (TNF)-α and other proinflammatory molecules compared with infection with nonpathogenic mycobacteria, which likely play a role in enhancing mycobacterial survival in vivo. Furthermore, macrophages infected with mycobacteria become refractory to many cytokines including IFN-γ and modulation of host cell signaling responses is critical for the suppression of a generalized inflammatory response which might influence the persistence of mycobacteria within the host. In this context, Suppressor of cytokine signaling (SOCS) 3, a member of SOCS family function as negative regulators of multiple cytokine and toll like receptor induced signaling. The SOCS3 has been shown to specifically inhibit signaling by IFN-γ, IL-6 family of cytokines and can act as a negative regulator of inflammatory responses. In this regard, many species of mycobacteria including M. bovis BCG triggers the inducible expression of SOCS3. Further, it has been suggested that M. bovis BCG triggered SOCS3 and SOCS1 proteins leads to the inhibition of IFN- γ stimulated JAK/STAT signaling in macrophages. Albeit JAK/STAT signaling pathway is generally believed to be involved, STAT-independent signals are suggested to take part in the induction of SOCS proteins in many systems signifying the involvement of multiple signal pathways in regulation of SOCS expression. Further little is known about the early, receptor proximal signaling mechanisms underlying mycobacteria-mediated induction of SOCS3. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like LAM, PIM, TDM, PE family antigens etc are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extra cellular environment in the form of exocytosed vesicles. In this context, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. PIM are suggested to be the common anchor of LM and LAM as PIM, LM, and LAM originate from identical biosynthetic pathway. PIM are present in virulent M. tuberculosis H37Rv as well as in M. bovis BCG and a number of biological functions have been recently credited to PIM2. PIM2 is suggested to trigger the activation of cells via Toll like receptor (TLR)-2 and stimulation resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. PIM2 induces proinflammatory stimuli such as TNF-α and IL-12 in murine and human macrophages in a TLR2 dependent manner. PIM exhibited pulmonary granuloma-forming activities as well as was shown to be responsible for the recruitment of NKT cells to granulomas. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether M. bovis BCG or novel cell surface antigens like PIM2 or Rv0978c, a PE-PGRS protein with unknown function can contribute to M. bovis BCG triggered molecular signaling events leading to SOCS3 expression in macrophages. Our studies clearly demonstrated that M. bovis BCG can trigger SOCS3 expression in macrophages. The inception of signaling by M. bovis BCG is TLR2-MyD88 dependent, but not TLR4 dependent. The perturbation of TLR2 signaling and the downregulation of MyD88 resulted in significant decrease in SOCS3 expression implicating the role of TLR2-MyD88 axis in M. bovis BCG triggered signaling. Experiments with cycloheximide and neutralizing antibodies to IL-10 evinced that M. bovis BCG triggered SOCS3 expression is a primary response and requires direct activation of signaling cascades. In the current study, we show for the first time that infection of macrophages with M. bovis BCG activates NOTCH1 signaling events, which leads to expression of SOCS3. The perturbation of NOTCH signaling in infected macrophages either by siRNA mediated down regulation of NOTCH1 or RBP-Jk or by inhibition with pharmacological inhibitor gamma secretase-I, resulted in the marked reduction in the expression of SOCS3. Further, the enforced expression of the NOTCH1 intracellular domain (NICD) in RAW264.7 macrophages induces the expression of SOCS3, which can be further potentiated by M. bovis BCG. Furthermore, the inhibition of TLR2 signaling by a TLR2 dominant-negative construct resulted in inhibition of NOTCH1 activation. Additionally, our results demonstrates for the first time that physical association of TLR2 with both Phosphoinositide-3 Kinase (PI3K) and NOTCH1, which suggest the significant role of TLR2 triggering by of M. bovis BCG in the activation of PI3K and NOTCH1. More importantly, signaling perturbations data suggest the involvement of cross-talk among the members of PI3K and MAPK cascades with NOTCH1 signaling in SOCS3 expression. In addition, SOCS3 expression requires the NOTCH1 mediated recruitment of CSL/RBP-Jk and Nuclear Factor-B (NF-B) to the SOCS3 promoter. A number of biological functions triggered by mycobacteria are often attributed to many of the cell wall antigens. As part of our current investigation, we explored whether two novel cell wall associated antigens namely PIM2 and a PE-PGRS antigen, Rv0978c could play as significant or crucial cell wall ingredients which imparts ability to M. bovis BCG to trigger activation of NOTCH signaling leading to SOCS3 expression. Akin to M. bovis BCG, PIM2 activates NOTCH1 signaling resulting NICD formation which leads to the expression of SOCS3 in a TLR2-MyD88 dependent manner. PIM2 mediated NOTCH1 activation, both directly influences the SOCS3 expression by serving as coactivator in RBP-Jk complex and indirectly triggers SOCS3 expression by activating PI3K-MAPK-NF-κB cascade. One important outcome of the genome sequencing project of M. tuberculosis was the discovery of two new multigene families designated PE and PPE, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many PE and PPE proteins are composed only of PE or PPE homologous domains. However, in other proteins, the PE domain is often linked to a unique domain of various lengths that is rich in alanine and glycine amino acids, termed the PGRS domain (PE-PGRS subfamily). PE family genes were suggested to play roles in the virulence of the pathogen and many members of PE family proteins are reported be localized on the surface of M. tuberculosis bacilli. Some of the PE proteins may play a role in immune evasion and antigenic variation or may be linked to virulence. Additionally, it has been suggested that the PE-PGRS subfamily of PE genes is enriched in genes with a high probability of being essential for M. tuberculosis. The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. In this context, we have chosen to study Rv0978c as a typical member of PE-PGRS family based on the following observations. Rv0978c was upregulated in TB bacilli upon infection of macrophages. Rv0978c was demonstrated to be a member of a group of genes called in vivo-expressed genomic island, which were shown to be upregulated in M. tuberculosis bacilli during infection of mice. Rv0978c was also shown to be upregulated, at least eightfold, in human brain microvascular endothelial cell-associated M. tuberculosis infection, suggesting a role for endothelial cell invasion and intracellular survival. In the current investigation, we have demonstrated that Rv0978c is hypoxia responsive gene based on promoter analysis and upregulated in M. tuberculosis during the infection of macrophages. Further, Rv0978c is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. In this perspective, our results clearly demonstrate that Rv0978c triggers SOCS3 expression by activating PI3K-ERK1/2-NF-B cascade in mouse macrophages. Additionally, Rv0978c elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0978c is an immunodominant antigen demonstrating significant T cell and humoral reactivites. These observations clearly advocate that Rv0978c protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0978c is immunogenic. These results clearly describe the cross-talk of NOTCH1 signaling with signaling pathways like PI3K and MAPK pathways during infection of macrophages with M. bovis BCG eventually resulting in regulation of specific gene expressions, such as SOCS3. These observations lead to a possibility of differential effects of NOTCH1 signaling activated upon infection by an intracellular bacillus, which could be involved in modulation of macrophage functions depending on a local immunological milieu. Taken together, our findings suggest that, induction of Suppressors of Cytokine Signaling 3 molecule by M. bovis BCG or by its cell wall antigens represents a crucial immune subversion mechanism in order to suppress or attenuate host responses to cytokines to generate the conditions that favor survival of the mycobacteria.

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.

This chapter discusses the continued decline of amateurism during the late 1960s and 1970s. Soaked in the countercultural spirit of the era, movements around the world challenged social norms and social order, often through radical and subversive efforts. The sustained push for civil rights along racial, gender, and social lines powerfully exposed the system of inequality in capitalist societies. Amateur sport was not immune to emerging cultural movements that challenged exploitation and threatened the status quo. Hair gradually lengthened as athletes questioned the authority of coaches and administrators. The sociologist Harry Edwards founded the Olympic Project for Human Rights in 1967, which also protested racial discrimination in both sport and society at large. Even sportswomen mobilized in their push for greater inclusion and pay equity, particularly as television and commercial marketing transformed elite sport into lucrative commodities. The International Olympic Committee suddenly found itself caught between the pillars of tradition and modernity. Under the leadership of its aging president, Avery Brundage, it struggled to keep pace with the shifting sporting landscape.

The complex interplay between immune cells and tumor cells within the tumor microenvironment (TME) can lead to disease progression. Specifically, signals generated in the TME can cause immunosuppression, promoting angiogenesis and immune evasion, which leads to tumor development. The interplay of M1 and M2 macrophage populations that coincide with these tumor markers is particularly important in the TME. Triple-negative breast cancer (TNBC) often presents as advanced disease, and these tumors are also often bereft of recognized molecular targets that can be found in other subtypes, limiting their therapeutic options. However, tumor-associated macrophages (TAMs) infiltration in TNBC is frequently observed. Moreover, a high density of TAMs, particularly M2 macrophages, is associated with poorer outcomes in various cancers, including TNBC. This provides a strong basis for exploiting TAMs as potential therapeutic targets. Specifically, efforts to increase M2 to M1 repolarization are promising therapeutic approaches in TNBC, and four recent studies wherein divergent approaches to target the M2-rich macrophage population and reverse immune subversion are described. These and similar efforts may yield promising diagnostic or therapeutic options for TNBC, a great clinical need.

Ceremonies and patriotic pageants are typically immune to symbolic challenge, but the rankling of political and corporate elites over the Colin Kaepernick-led protests that saw a number of National Football League (NFL) players kneeling during the traditional pre-game playing of the “Star-Spangled Banner” suggests that a line has been drawn as it pertains to the nation’s anthem. Despite safeguards embedded in the U.S. Constitution regarding freedom of speech and expression, Kaepernick, a former NFL quarterback who steered the San Francisco 49ers to the 2012 Super Bowl, has since the initial August 2016 protests been collectively (albeit unofficially) barred from participation following the end of that season while subsequently emerging as one of the primary voices in the social justice movements of the early twenty-first century. Challenging the presumption that silently ‘taking a knee’ during the anthem is tantamount to subversion, Brian Carroll explores the racial implications of this ongoing controversy that he contends mirrors the contemporary embrace of ‘reality television’ and is enveloped in a debased form of patriotism.