T Cell Interactions in Mycobacterial Granulomas: Non-Specific T Cells Regulate Mycobacteria-Specific T Cells in Granulomatous Lesions.

Infections with pathogenic mycobacteria are controlled by the formation of a unique structure known as a granuloma. The granuloma represents a host-pathogen interface where bacteria are killed and confined by the host response, but also where bacteria persist. Previous work has demonstrated that the T cell repertoire is heterogenous even at the single granuloma level. However, further work using pigeon cytochrome C (PCC) epitope-tagged BCG (PCC-BCG) and PCC-specific 5CC7 RAG-/- TCR transgenic (Tg) mice has demonstrated that a monoclonal T cell population is able to control infection. At the chronic stage of infection, granuloma-infiltrating T cells remain highly activated in wild-type mice, while T cells in the monoclonal T cell mice are anergic. We hypothesized that addition of an acutely activated non-specific T cell to the monoclonal T cell system could recapitulate the wild-type phenotype. Here we report that activated non-specific T cells have access to the granuloma and deliver a set of cytokines and chemokines to the lesions. Strikingly, non-specific T cells rescue BCG-specific T cells from anergy and enhance the function of BCG-specific T cells in the granuloma in the chronic phase of infection when bacterial antigen load is low. In addition, we find that these same non-specific T cells have an inhibitory effect on systemic BCG-specific T cells. Taken together, these data suggest that T cells non-specific for granuloma-inducing agents can alter the function of granuloma-specific T cells and have important roles in mycobacterial immunity and other granulomatous disorders.

Current concepts on communication between the central nervous system and peripheral immunity via lymphatics: what roles do lymphatics play in brain and spinal cord disease pathogenesis?

The central nervous system (CNS) lacks conventional lymphatics within the CNS parenchyma, yet still maintains fluid homeostasis and immunosurveillance. How the CNS communicates with systemic immunity has thus been a topic of interest for scientists in the past century, which has led to several theories of CNS drainage routes. In addition to perineural routes, rediscoveries of lymphatics surrounding the CNS in the meninges revealed an extensive network of lymphatics, which we now know play a significant role in fluid homeostasis and immunosurveillance. These meningeal lymphatic networks exist along the superior sagittal sinus and transverse sinus dorsal to the brain, near the cribriform plate below the olfactory bulbs, at the base of the brain, and surrounding the spinal cord. Inhibition of one or all of these lymphatic networks can reduce CNS autoimmunity in a mouse model of multiple sclerosis (MS), while augmenting these lymphatic networks can improve immunosurveillance, immunotherapy, and clearance in glioblastoma, Alzheimer's disease, traumatic brain injury, and cerebrovascular injury. In this review, we will provide historical context of how CNS drainage contributes to immune surveillance, how more recently published studies fit meningeal lymphatics into the context of CNS homeostasis and neuroinflammation, identify the complex dualities of lymphatic function during neuroinflammation and how therapeutics targeting lymphatic function may be more complicated than currently appreciated, and conclude by identifying some unresolved questions and controversies that may guide future research.

Neuroinflammation-Driven Lymphangiogenesis in CNS Diseases.

The central nervous system (CNS) undergoes immunosurveillance despite the lack of conventional antigen presenting cells and lymphatic vessels in the CNS parenchyma. Additionally, the CNS is bathed in a cerebrospinal fluid (CSF). CSF is continuously produced, and consequently must continuously clear to maintain fluid homeostasis despite the lack of conventional lymphatics. During neuroinflammation, there is often an accumulation of fluid, antigens, and immune cells to affected areas of the brain parenchyma. Failure to effectively drain these factors may result in edema, prolonged immune response, and adverse clinical outcome as observed in conditions including traumatic brain injury, ischemic and hypoxic brain injury, CNS infection, multiple sclerosis (MS), and brain cancer. Consequently, there has been renewed interest surrounding the expansion of lymphatic vessels adjacent to the CNS which are now thought to be central in regulating the drainage of fluid, cells, and waste out of the CNS. These lymphatic vessels, found at the cribriform plate, dorsal dural meninges, base of the brain, and around the spinal cord have each been implicated to have important roles in various CNS diseases. In this review, we discuss the contribution of meningeal lymphatics to these processes during both steady-state conditions and neuroinflammation, as well as discuss some of the many still unknown aspects regarding the role of meningeal lymphatics in neuroinflammation. Specifically, we focus on the observed phenomenon of lymphangiogenesis by a subset of meningeal lymphatics near the cribriform plate during neuroinflammation, and discuss their potential roles in immunosurveillance, fluid clearance, and access to the CSF and CNS compartments. We propose that manipulating CNS lymphatics may be a new therapeutic way to treat CNS infections, stroke, and autoimmunity.

Mycobacterium bovis Bacillus Calmette-Guérin-Infected Dendritic Cells Induce TNF-α-Dependent Cell Cluster Formation That Promotes Bacterial Dissemination through an In Vitro Model of the Blood-Brain Barrier.

CNS tuberculosis (CNSTB) is the most severe manifestation of extrapulmonary tuberculosis infection, but the mechanism of how mycobacteria cross the blood-brain barrier (BBB) is not well understood. In this study, we report a novel murine in vitro BBB model combining primary brain endothelial cells, Mycobacterium bovis bacillus Calmette-Guérin-infected dendritic cells (DCs), PBMCs, and bacterial Ag-specific CD4+ T cells. We show that mycobacterial infection limits DC mobility and also induces cellular cluster formation that has a similar composition to pulmonary mycobacterial granulomas. Within the clusters, infection from DCs disseminates to the recruited monocytes, promoting bacterial expansion. Mycobacterium-induced in vitro granulomas have been described previously, but this report shows that they can form on brain endothelial cell monolayers. Cellular cluster formation leads to cluster-associated damage of the endothelial cell monolayer defined by mitochondrial stress, disorganization of the tight junction proteins ZO-1 and claudin-5, upregulation of the adhesion molecules VCAM-1 and ICAM-1, and increased transmigration of bacteria-infected cells across the BBB. TNF-α inhibition reduces cluster formation on brain endothelial cells and mitigates cluster-associated damage. These data describe a model of bacterial dissemination across the BBB shedding light on a mechanism that might contribute to CNS tuberculosis infection and facilitate treatments.

Current concepts in granulomatous immune responses.

Persistent irritants that are resistant to innate and cognate immunity induce granulomas. These macrophage-dominated lesions that partially isolate the healthy tissue from the irritant and the irritant induced inflammation. Particles, toxins, autoantigens and infectious agents can induce granulomas. The corresponding lesions can be protective for the host but they can also cause damage and such damage has been associated with the pathology of more than a hundred human diseases. Recently, multiple molecular mechanisms underlying how normal macrophages transform into granuloma-inducing macrophages have been discovered and new information has been gathered, indicating how these lesions are initiated, spread and regulated. In this review, differences between the innate and cognate granuloma pathways are discussed by summarizing how the dendritic cell - T cell axis changes granulomatous immunity. Granuloma lesions are highly dynamic and depend on continuous cell replacement. This feature provides new therapeutic approaches to treat granulomatous diseases.

A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood-Brain Barrier Mouse Model.

In vitro culture models of the blood-brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to test Mycobacterium tuberculosis (Mtb) dissemination across brain endothelial cells. One-third of the global population is infected with Mtb, and in 1%-2% of cases bacteria invade the CNS through a largely unknown process. The "Trojan horse" theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB-granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of primary mouse brain astrocytes and endothelial cells Basic Protocol 2: Isolation of primary mouse bone marrow-derived dendritic cells Support Protocol 1: Validation of dendritic cell purity by flow cytometry Basic Protocol 3: Isolation of primary mouse peripheral blood mononuclear cells Support Protocol 2: Isolation of primary mouse spleen cells Support Protocol 3: Purification and validation of CD4+ T cells from PBMCs and spleen cells Basic Protocol 4: Isolation of liver granuloma supernatant and determination of organ load Support Protocol 4: In vivo and in vitro infection with mycobacteria Basic Protocol 5: Assembly of the BBB co-culture model Basic Protocol 6: Assembly of the combined in vitro granuloma and BBB model.

VEGF-A from Granuloma Macrophages Regulates Granulomatous Inflammation by a Non-angiogenic Pathway during Mycobacterial Infection.

Many autoimmune and infectious diseases are characterized by the formation of granulomas which are inflammatory lesions that consist of spatially organized immune cells. These sites protect the host and control pathogens like Mycobacterium tuberculosis (Mtb), but are highly inflammatory and cause pathology. Using bacille Calmette-Guerin (BCG) and Mtb infection in mice that induce sarcoid or caseating granulomas, we show that a subpopulation of granuloma macrophages produces vascular endothelial growth factor (VEGF-A), which recruits immune cells to the granuloma by a non-angiogenic pathway. Selective blockade of VEGF-A in myeloid cells, combined with granuloma transplantation, shows that granuloma VEGF-A regulates granulomatous inflammation. The severity of granuloma-related inflammation can be ameliorated by pharmaceutical or genetic inhibition of VEGF-A, which improves survival of mice infected with virulent Mtb without altering host protection. These data show that VEGF-A inhibitors could be used as a host-directed therapy against granulomatous diseases like tuberculosis and sarcoidosis, thereby expanding the value of already existing and approved anti-VEGF-A drugs.

Mycobacterium-Infected Dendritic Cells Disseminate Granulomatous Inflammation.

The disappearance and reformation of granulomas during tuberculosis has been described using PET/CT/X-ray in both human clinical settings and animal models, but the mechanisms of granuloma reformation during active disease remains unclear. Granulomas can recruit inflammatory dendritic cells (iDCs) that can regulate local T-cell responses and can carry bacteria into the lymph nodes, which is crucial for generating systemic T-cell responses against mycobacteria. Here, we report that a subset of mycobacterium-infected iDCs are associated with bacteria-specific T-cells in infected tissue, outside the granuloma, and that this results in the formation of new and/or larger multi-focal lesions. Mycobacterium-infected iDCs express less CCR7 and migrate less efficiently compared to the non-infected iDCs, which may support T-cell capture in granulomatous tissue. Capture may reduce antigen availability in the lymph node, thereby decreasing systemic priming, resulting in a possible regulatory loop between systemic T-cell responses and granuloma reformation. T-cell/infected iDCs clusters outside the granuloma can be detected during the acute and chronic phase of BCG and Mtb infection. Our studies suggest a direct role for inflammatory dendritic cells in the dissemination of granulomatous inflammation.

Lymphangiogenesis is induced by mycobacterial granulomas via vascular endothelial growth factor receptor-3 and supports systemic T-cell responses against mycobacterial antigen.

Granulomatous inflammation is characteristic of many autoimmune and infectious diseases. The lymphatic drainage of these inflammatory sites remains poorly understood, despite an expanding understanding of lymphatic role in inflammation and disease. Here, we show that the lymph vessel growth factor Vegf-c is up-regulated in Bacillus Calmette-Guerin- and Mycobacterium tuberculosis-induced granulomas, and that infection results in lymph vessel sprouting and increased lymphatic area in granulomatous tissue. The observed lymphangiogenesis during infection was reduced by inhibition of vascular endothelial growth factor receptor 3. By using a model of chronic granulomatous infection, we also show that lymphatic remodeling of tissue persists despite resolution of acute infection and a 10- to 100-fold reduction in the number of bacteria and tissue-infiltrating leukocytes. Inhibition of vascular endothelial growth factor receptor 3 decreased the growth of new vessels, but also reduced the proliferation of antigen-specific T cells. Together, our data show that granuloma-up-regulated factors increase granuloma access to secondary lymph organs by lymphangiogenesis, and that this process facilitates the generation of systemic T-cell responses to granuloma-contained antigens.

CCR2-dependent dendritic cell accumulation in the central nervous system during early effector experimental autoimmune encephalomyelitis is essential for effector T cell restimulation in situ and disease progression.

Dendritic cells (DCs)--although absent from the healthy CNS parenchyma--rapidly accumulate within brain and spinal cord tissue during neuroinflammation associated with experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis). Yet, although DCs have been appreciated for their role in initiating adaptive immune responses in peripheral lymphoid organ tissues, how DCs infiltrate the CNS and contribute to ongoing neuroinflammation in situ is poorly understood. In this study, we report the following: 1) CD11c(+) bone marrow-derived DCs and CNS-infiltrating DCs express chemokine receptor CCR2; 2) compared with CCR2(+/+) cells, adoptively transferred CCR2(-/-) bone marrow-derived DCs or DC precursors do not accumulate in the CNS during EAE, despite abundance in blood; 3) CCR2(-/-) DCs show less accumulation in the inflamed CNS in mixed bone marrow chimeras, when compared with CCR2(+/+) DCs; and 4) ablation of CCR2(+/+) DCs during EAE clinical onset delays progression and attenuates cytokine production by infiltrating T cells. Whereas the role of CCR2 in monocyte migration into the CNS has been implicated previously, the role of CCR2 in DC infiltration into the CNS has never been directly addressed. Our data suggest that CCR2-dependent DC recruitment to the CNS during ongoing neuroinflammation plays a crucial role in effector T cell cytokine production and disease progression, and signify that CNS-DCs and circulating DC precursors might be key therapeutic targets for suppressing ongoing neuroinflammation in CNS autoimmune diseases.

Mapping the accumulation of co-infiltrating CNS dendritic cells and encephalitogenic T cells during EAE.

Evidence from experimental autoimmune encephalomyelitis (EAE) suggests that CNS-infiltrating dendritic cells (DCs) are crucial for restimulation of coinfiltrating T cells. Here we systematically quantified and visualized the distribution and interaction of CNS DCs and T cells during EAE. We report marked periventricular accumulation of DCs and myelin-specific T cells during EAE disease onset prior to accumulation in the spinal cord, indicating that the choroid plexus-CSF axis is a CNS entry portal. Moreover, despite emphasis on spinal cord inflammation in EAE and in correspondence with MS pathology, inflammatory lesions containing interacting DCs and T cells are present in specific brain regions.

Deficient import of acetyl-CoA into the ER lumen causes neurodegeneration and propensity to infections, inflammation, and cancer.

The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the N(ε)-lysine acetylation of ER-resident and ER-transiting proteins. A point-mutation (S113R) in AT-1 has been associated with a familial form of spastic paraplegia. Here, we report that AT-1S113R is unable to form homodimers in the ER membrane and is devoid of acetyl-CoA transport activity. The reduced influx of acetyl-CoA into the ER lumen results in reduced acetylation of ER proteins and an aberrant form of autophagy. Mice homozygous for the mutation display early developmental arrest. In contrast, heterozygous animals develop to full term, but display neurodegeneration and propensity to infections, inflammation, and cancer. The immune and cancer phenotypes are contingent on the presence of pathogens in the colony, whereas the nervous system phenotype is not. In conclusion, our results reveal a previously unknown aspect of acetyl-CoA metabolism that affects the immune and nervous systems and the risk for malignancies.

Immune privilege of the CNS is not the consequence of limited antigen sampling.

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Monocyte-derived inflammatory dendritic cells in the granuloma during mycobacterial infection.

The monocyte-derived, inflammatory dendritic cell subset plays an important role during immune responses against infections. This review will focus on the complex, changing role of this subset during mycobacterial infection. Studies demonstrate that in addition to sustaining a systemic anti-mycobacterial response, the inflammatory dendritic cell subset present in Mycobacterium-induced granulomas also influences local immune regulation within the granuloma over the course of infection. This review will also survey the literature on how similar and different inflammatory dendritic cell subsets during other infections.

Granuloma transplantation: an approach to study mycobacterium-host interactions.

The host-pathogen biology during infection with Mycobacterium tuberculosis is incredibly complex and despite accelerating progress in research, remains poorly understood. Our limited understanding hinders the development of new drugs, next generation vaccines, and novel therapies. The granuloma is the site where mycobacteria are both controlled and allowed to persist, but it remains one of the least studied aspects of the host-pathogen relationship. Here, we review the development, application, potential uses, and limitations of a novel model of granuloma transplantation as a tool to study specific host-pathogen interactions that have been difficult to probe. Application of this new model has already contributed to our understanding of granuloma cell traffic, repopulation, and the relationship between systemic immunity and mycobacteria-containing granulomas. The data collected highlight the dynamic interaction between systemic and local immune processes and support a paradigm that defines the granuloma as a highly dynamic structure. Granuloma transplantation also has special potential as a novel latency model that can contribute to our understanding of host protection factors and bacterial mutants, and serve as a platform for drug testing.

The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations.

Phagocytosis and intracellular processing of mycobacteria by macrophages are complex cellular processes that require spatial and temporal coordination of particle uptake, organelle movement, activation of signaling pathways, and channel-mediated ionic flux. Recent work demonstrated that human macrophage NaV1.5, an intracellular voltage-gated sodium channel expressed on late endosomes, enhances endosomal acidification and phagocytosis. Here, using bacillus Camille-Guerin (BCG) as a model of mycobacterial infection, we examined how this channel regulates phagocytosis and phagosome maturation in human macrophages. Knockdown of NaV1.5 reduced high capacity uptake of labeled BCG. BCG-containing, NaV1.5-expressing cells demonstrated localization of NaV1.5 and Rab-7 positive endosomes and mitochondria to periphagosome regions that was not observed in NaV1.5-deficient cells. Knockdown of the channel reduced the initial calcium response following bacterial challenge and prevented the generation of prolonged and localized calcium oscillations during phagosome maturation. Inhibition of the mitochondrial Na(+) /Ca(2+) exchanger also prevented prolonged calcium oscillations during phagosome maturation. These results suggest that NaV1.5 and mitochondrial-dependent calcium signaling regulate mycobacteria phagocytosis and phagosome maturation in human macrophages through spatial-temporal coordination of calcium signaling within a unique subcellular region.

Inflammatory dendritic cells migrate in and out of transplanted chronic mycobacterial granulomas in mice.

An estimated one-third of the world's population is infected with Mycobacterium tuberculosis, although most affected individuals maintain a latent infection. This control is attributed to the formation of granulomas, cell masses largely comprising infected macrophages with T cells aggregated around them. Inflammatory DCs, characterized as CD11c+CD11b+Ly6C+, are also found in granulomas and are an essential component of the acute immune response to mycobacteria. However, their function during chronic infection is less well understood. Here, we report that CD11c+ cells dynamically traffic in and out of both acute and chronic granulomas induced by Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) in mice. By transplanting Mycobacterium-induced granulomas containing fluorescently labeled CD11c+ cells and bacteria into unlabeled mice, we were able to follow CD11c+ cell trafficking and T cell activation. We found that half of the CD11c+ cells in chronic granulomas were exchanged within 1 week. Compared with tissue-resident DC populations, CD11c+ cells migrating out of granuloma-containing tissue had an unexpected systemic dissemination pattern. Despite low antigen availability, systemic CD4+ T cell priming still occurred during chronic infection. These data demonstrate that surveillance of granulomatous tissue by CD11c+ cells is continuous and that these cells are distinct from tissue-resident DC populations and support T cell priming during both stages of Mycobacterium infection. This intense DC surveillance may also be a feature of Mycobacterium tuberculosis infection and other granuloma-associated diseases.

CC chemokine receptor 4 contributes to innate NK and chronic stage T helper cell recall responses during Mycobacterium bovis infection.

Cysteine-cysteinyl chemokine receptor 4 (CCR4) is expressed by a variety of T-cell subsets and leukocytes. This study examined the participation of CCR4 in response to pulmonary infection with Mycobacterium bovis Bacille-Calmette-Guerin (BCG). Constitutive and induced CCR4 agonist expression was detected among large mononuclear cells. The course of infection and mobilization of effector cell populations were then analyzed in CCR4 knockout (CCR4(-/-)) mice. Compared with controls, CCR4(-/-) mice displayed delayed innate stage (<2 weeks) bacterial clearance and reduced late stage inflammation. Innate impairment was associated with reduced natural killer cell activation. In the adaptive phase, CCR4(-/-) mice generated effector T cells in draining lymph nodes and accumulated effector T cells in lungs, which resulted in normal adaptive stage bacterial elimination at 2 to 4 weeks. However, during the late stage, CCR4(-/-) mice had reduced interferonγ+CD4(+)α/ß+ (Th1) and interleukin (IL)-17+CD4(+)α/ß+ (Th17) T helper cells in lungs. In contrast, IL-17+ γ/δ T cells in lungs were unaffected. When challenged with mycobacterial antigen- (Ag-) Ag-coated beads to elicit a recall granulomatous response, CCR4(-/-) mice displayed abrogated recall granuloma formation and reduced interferon γ+ Th1 cells. These findings indicate that CCR4 supports innate natural killer cell activation and sustains later CD4(+) Th effector/memory antimycobacterial responses in the lung but is redundant in the early adaptive elimination phase.

Cysteine-cysteinyl chemokine receptor 6 mediates invariant natural killer T cell airway recruitment and innate stage resistance during mycobacterial infection.

This study examined the contribution of cysteine-cysteinyl chemokine receptor 6 (CCR6) to the innate pulmonary antimycobacterial immune response. Using a mouse model of Mycobacterium bovis BCG airway infection, we detected maximal induction of the CCR6 agonist CCL20 in lungs at 1 week after infection. Infected CCR6 knockout (CCR6-/-) mice displayed an early impairment of bacterial clearance, but ultimately eliminated the attenuated organisms with the onset of adaptive immunity. Flow-cytometric analyses of bronchoalveolar lavages and dispersed lungs revealed a 60% reduction in TCR-α/ß+ T cells in airways but no compromise of TCR-γ/δ+ T cells. The subset of CD1d-restricted, CD8-TCR-α/ß+ natural killer cells, which mediate innate mycobacterial resistance, was profoundly reduced (90%). Analysis of the adaptive response using ovalbumin-specific transgenic TCR T cell (OT-II) transfer combined with infection with recombinant M. bovis BCG producing ovalbumin peptide indicated no impairment of adaptive T cell activation in CCR6-/- mice. There was also no impairment of the induction of cytokine-producing cells in draining lymphoid tissue of CCR6-/- mice. Taken together, our findings indicate that CCR6 is not required for induction of the adaptive antimycobacterial response, but is likely critical to airway compartment mobilization of TCR-α/ß+CCR6+ innate and adaptive effector T cells.

Granuloma formation and host defense in chronic Mycobacterium tuberculosis infection requires PYCARD/ASC but not NLRP3 or caspase-1.

The NLR gene family mediates host immunity to various acute pathogenic stimuli, but its role in chronic infection is not known. This paper addressed the role of NLRP3 (NALP3), its adaptor protein PYCARD (ASC), and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1beta secretion, and this requires the inflammasome components PYCARD, caspase-1, and NLRP3. However, in vivo Mtb aerosol infection of Nlrp3(-/-), Casp-1(-/-), and WT mice showed no differences in pulmonary IL-1beta production, bacterial burden, or long-term survival. In contrast, a significant role was observed for Pycard in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of Pycard(-/-) mice. Decreased survival of Pycard(-/-) animals was associated with defective granuloma formation. These data demonstrate that PYCARD exerts a novel inflammasome-independent role during chronic Mtb infection by containing the bacteria in granulomas.