Type 2 cannabinoid receptor expression on microglial cells regulates neuroinflammation during graft-versus-host disease.

Neuroinflammation is a recognized complication of immunotherapeutic approaches such as immune checkpoint inhibitor treatment, chimeric antigen receptor therapy, and graft versus host disease (GVHD) occurring after allogeneic hematopoietic stem cell transplantation. While T cells and inflammatory cytokines play a role in this process, the precise interplay between the adaptive and innate arms of the immune system that propagates inflammation in the central nervous system remains incompletely understood. Using a murine model of GVHD, we demonstrate that type 2 cannabinoid receptor (CB2R) signaling plays a critical role in the pathophysiology of neuroinflammation. In these studies, we identify that CB2R expression on microglial cells induces an activated inflammatory phenotype that potentiates the accumulation of donor-derived proinflammatory T cells, regulates chemokine gene regulatory networks, and promotes neuronal cell death. Pharmacological targeting of this receptor with a brain penetrant CB2R inverse agonist/antagonist selectively reduces neuroinflammation without deleteriously affecting systemic GVHD severity. Thus, these findings delineate a therapeutically targetable neuroinflammatory pathway and have implications for the attenuation of neurotoxicity after GVHD and potentially other T cell-based immunotherapeutic approaches.

Brain Maturation as a Fundamental Factor in Immune-Neurovascular Interactions in Stroke.

Injuries in the developing brain cause significant long-term neurological deficits. Emerging clinical and preclinical data have demonstrated that the pathophysiology of neonatal and childhood stroke share similar mechanisms that regulate brain damage, but also have distinct molecular signatures and cellular pathways. The focus of this review is on two different diseases-neonatal and childhood stroke-with emphasis on similarities and distinctions identified thus far in rodent models of these diseases. This includes the susceptibility of distinct cell types to brain injury with particular emphasis on the role of resident and peripheral immune populations in modulating stroke outcome. Furthermore, we discuss some of the most recent and relevant findings in relation to the immune-neurovascular crosstalk and how the influence of inflammatory mediators is dependent on specific brain maturation stages. Finally, we comment on the current state of treatments geared toward inducing neuroprotection and promoting brain repair after injury and highlight that future prophylactic and therapeutic strategies for stroke should be age-specific and consider gender differences in order to achieve optimal translational success.

Interleukin-9 production by type 2 innate lymphoid cells induces Paneth cell metaplasia and small intestinal remodeling.

Paneth cell metaplasia (PCM) typically arises in pre-existing gastrointestinal (GI) diseases; however, the mechanistic pathway that induces metaplasia and whether PCM is initiated exclusively by disorders intrinsic to the GI tract is not well known. Here, we describe the development of PCM in a murine model of chronic myelogenous leukemia (CML) that is driven by an inducible bcr-abl oncogene. Mechanistically, CML induces a proinflammatory state within the GI tract that results in the production of epithelial-derived IL-33. The binding of IL-33 to the decoy receptor ST2 leads to IL-9 production by type 2 innate lymphoid cells (ILC2) which is directly responsible for the induction of PCM in the colon and tissue remodeling in the small intestines, characterized by goblet and tuft cell hyperplasia along with expansion of mucosal mast cells. Thus, we demonstrate that an extra-intestinal disease can trigger an ILC2/IL-9 immune circuit, which induces PCM and regulates epithelial cell fate decisions in the GI tract.

MICROGLIAL CELL EXPRESSION OF THE TYPE 2 CANNABINOID RECEPTOR REGULATES IMMUNE-MEDIATED NEUROINFLAMMATION.

Neuroinflammation is a recognized complication of immunotherapeutic approaches such as immune checkpoint inhibitor treatment, chimeric antigen receptor therapy, and graft versus host disease (GVHD) occurring after allogeneic hematopoietic stem cell transplantation. While T cells and inflammatory cytokines play a role in this process, the precise interplay between the adaptive and innate arms of the immune system that propagates inflammation in the central nervous system remains incompletely understood. Using a murine model of GVHD, we demonstrate that type 2 cannabinoid receptor (CB2R) signaling plays a critical role in the pathophysiology of neuroinflammation. In these studies, we identify that CB2R expression on microglial cells induces an activated inflammatory phenotype which potentiates the accumulation of donor-derived proinflammatory T cells, regulates chemokine gene regulatory networks, and promotes neuronal cell death. Pharmacological targeting of this receptor with a brain penetrant CB2R inverse agonist/antagonist selectively reduces neuroinflammation without deleteriously affecting systemic GVHD severity. Thus, these findings delineate a therapeutically targetable neuroinflammatory pathway and has implications for the attenuation of neurotoxicity after GVHD and potentially other T cell-based immunotherapeutic approaches.

Global sphingosine-1-phosphate receptor 2 deficiency attenuates neuroinflammation and ischemic-reperfusion injury after neonatal stroke.

Arterial ischemic stroke is common in neonates-1 per 2,300-5,000 births-and therapeutic targets remain insufficiently defined. Sphingosine-1-phosphate receptor 2 (S1PR2), a major regulator of the CNS and immune systems, is injurious in adult stroke. Here, we assessed whether S1PR2 contributes to stroke induced by 3 h transient middle cerebral artery occlusion (tMCAO) in S1PR2 heterozygous (HET), knockout (KO), and wild type (WT) postnatal day 9 pups. HET and WT of both sexes displayed functional deficits in Open Field test whereas injured KO at 24 h reperfusion performed similarly to naives. S1PR2 deficiency protected neurons, attenuated infiltration of inflammatory monocytes, and altered vessel-microglia interactions without reducing increased cytokine levels in injured regions at 72 h. Pharmacologic inhibition of S1PR2 after tMCAO by JTE-013 attenuated injury 72 h after tMCAO. Importantly, the lack of S1PR2 alleviated anxiety and brain atrophy during chronic injury. Altogether, we identify S1PR2 as a potential new target for mitigating neonatal stroke.

CXCL13 expressed on inflamed cerebral blood vessels recruit IL-21 producing TFH cells to damage neurons following stroke.

BACKGROUND: Ischemic stroke is a leading cause of mortality worldwide, largely due to the inflammatory response to brain ischemia during post-stroke reperfusion. Despite ongoing intensive research, there have not been any clinically approved drugs targeting the inflammatory component to stroke. Preclinical studies have identified T cells as pro-inflammatory mediators of ischemic brain damage, yet mechanisms that regulate the infiltration and phenotype of these cells are lacking. Further understanding of how T cells migrate to the ischemic brain and facilitate neuronal death during brain ischemia can reveal novel targets for post-stroke intervention. METHODS: To identify the population of T cells that produce IL-21 and contribute to stroke, we performed transient middle cerebral artery occlusion (tMCAO) in mice and performed flow cytometry on brain tissue. We also utilized immunohistochemistry in both mouse and human brain sections to identify cell types and inflammatory mediators related to stroke-induced IL-21 signaling. To mechanistically demonstrate our findings, we employed pharmacological inhibitor anti-CXCL13 and performed histological analyses to evaluate its effects on brain infarct damage. Finally, to evaluate cellular mechanisms of stroke, we exposed mouse primary neurons to oxygen glucose deprivation (OGD) conditions with or without IL-21 and measured cell viability, caspase activity and JAK/STAT signaling. RESULTS: Flow cytometry on brains from mice following tMCAO identified a novel population of cells IL-21 producing CXCR5+ CD4+ ICOS-1+ T follicular helper cells (TFH) in the ischemic brain early after injury. We observed augmented expression of CXCL13 on inflamed brain vascular cells and demonstrated that inhibition of CXCL13 protects mice from tMCAO by restricting the migration and influence of IL-21 producing TFH cells in the ischemic brain. We also illustrate that neurons express IL-21R in the peri-infarct regions of both mice and human stroke tissue in vivo. Lastly, we found that IL-21 acts on mouse primary ischemic neurons to activate the JAK/STAT pathway and induce caspase 3/7-mediated apoptosis in vitro. CONCLUSION: These findings identify a novel mechanism for how pro-inflammatory T cells are recruited to the ischemic brain to propagate stroke damage and provide a potential new therapeutic target for stroke.

Scavenger receptor CD36 governs recruitment of myeloid cells to the blood-CSF barrier after stroke in neonatal mice.

BACKGROUND: Ischemic stroke induces the activation and recruitment of peripheral leukocytes to the injured brain. These cells can infiltrate the brain through multiple routes, either by penetrating blood-brain barrier or via blood-CSF barriers at the meninges or the choroid plexus (CP). We previously showed that myeloid cell trafficking via the CP occurs early after neonatal arterial stroke and modulates injury. CD36 is a receptor that mediates function of endothelial cells and cells of the monocyte lineage under various neurodegenerative conditions and can influence brain injury after neonatal stroke. Here we asked whether CD36 impacts injury by altering leukocyte trafficking through the CP in neonatal mice subjected to transient middle cerebral artery occlusion (tMCAO). METHODS: In neonatal mice with intact or globally disrupted CD36 signalling (CD36 KO), we characterized the phenotypes of myeloid cells by flow cytometry and the underlying gene expression signatures in the CPs contralateral and ipsilateral to tMCAO by RNA sequencing analyses, focussing on early post-reperfusion time window. RESULTS: Flow cytometry in the isolated CPs revealed that CD36 mediates stepwise recruitment of myeloid cells to the CP ipsilateral to tMCAO early after reperfusion, with a predominant increase first in inflammatory monocyte subsets and neutrophils followed by patrolling monocytes. RNA sequencing analyses demonstrated marked changes in gene expression in the CP ipsilateral compared to the CP contralateral to tMCAO in wild type mice. Changes were further modified by lack of CD36, including distinction in several clusters of genes involved in inflammatory, metabolic and extracellular matrix signalling in the CP ipsilateral to tMCAO. CONCLUSION: Altogether, our data suggest cooperation between blood-CSF-brain interface via the CP through CD36-mediated signalling following neonatal stroke with a key role for inflammatory monocytes and neutrophils.

Translational Clinical Strategies for the Prevention of Gastrointestinal Tract Graft Versus Host Disease.

Graft versus host disease (GVHD) is the major non-relapse complication associated with allogeneic hematopoietic stem cell transplantation (HSCT). Unfortunately, GVHD occurs in roughly half of patients following this therapy and can induce severe life-threatening side effects and premature mortality. The pathophysiology of GVHD is driven by alloreactive donor T cells that induce a proinflammatory environment to cause pathological damage in the skin, gastrointestinal (GI) tract, lung, and liver during the acute phase of this disease. Recent work has demonstrated that the GI tract is a pivotal target organ and a primary driver of morbidity and mortality in patients. Prevention of this complication has therefore emerged as an important goal of prophylaxis strategies given the primacy of this tissue site in GVHD pathophysiology. In this review, we summarize foundational pre-clinical studies that have been conducted in animal models to prevent GI tract GVHD and examine the efficacy of these approaches upon subsequent translation into the clinic. Specifically, we focus on therapies designed to block inflammatory cytokine pathways, inhibit cellular trafficking of alloreactive donor T cells to the GI tract, and reconstitute impaired regulatory networks for the prevention of GVHD in the GI tract.

Viral mimetic triggers cerebral arteriopathy in juvenile brain via neutrophil elastase and NETosis.

Stroke is among the top ten causes of death in children but has received disproportionally little attention. Cerebral arteriopathies account for up to 80% of childhood arterial ischemic stroke (CAIS) cases and are strongly predictive of CAIS recurrence and poorer outcomes. The underlying mechanisms of sensitization of neurovasculature by viral infection are undefined. In the first age-appropriate model for childhood arteriopathy-by administration of viral mimetic TLR3-agonist Polyinosinic:polycytidylic acid (Poly-IC) in juvenile mice-we identified a key role of the TLR3-neutrophil axis in disrupting the structural-functional integrity of the blood-brain barrier (BBB) and distorting the developing neurovascular architecture and vascular networks. First, using an array of in-vivo/post-vivo vascular imaging, genetic, enzymatic and pharmacological approaches, we report marked Poly-IC-mediated extravascular leakage of albumin (66kDa) and of a small molecule DiI (∼934Da) and disrupted tight junctions. Poly-IC also enhanced the neuroinflammatory milieu, promoted neutrophil recruitment, profoundly upregulated neutrophil elastase (NE), and induced neutrophil extracellular trap formation (NETosis). Finally, we show that functional BBB disturbances, NETosis and neuroinflammation are markedly attenuated by pharmacological inhibition of NE (Sivelestat). Altogether, these data reveal NE/NETosis as a novel therapeutic target for viral-induced cerebral arteriopathies in children.

Microglia-leucocyte axis in cerebral ischaemia and inflammation in the developing brain.

Development of the Central Nervous System (CNS) is reliant on the proper function of numerous intricately orchestrated mechanisms that mature independently, including constant communication between the CNS and the peripheral immune system. This review summarizes experimental knowledge of how cerebral ischaemia in infants and children alters physiological communication between leucocytes, brain immune cells, microglia and the neurovascular unit (NVU)-the "microglia-leucocyte axis"-and contributes to acute and long-term brain injury. We outline physiological development of CNS barriers in relation to microglial and leucocyte maturation and the plethora of mechanisms by which microglia and peripheral leucocytes communicate during postnatal period, including receptor-mediated and intracellular inflammatory signalling, lipids, soluble factors and extracellular vesicles. We focus on the "microglia-leucocyte axis" in rodent models of most common ischaemic brain diseases in the at-term infants, hypoxic-ischaemic encephalopathy (HIE) and focal arterial stroke and discuss commonalities and distinctions of immune-neurovascular mechanisms in neonatal and childhood stroke compared to stroke in adults. Given that hypoxic and ischaemic brain damage involve Toll-like receptor (TLR) activation, we discuss the modulatory role of viral and bacterial TLR2/3/4-mediated infection in HIE, perinatal and childhood stroke. Furthermore, we provide perspective of the dynamics and contribution of the axis in cerebral ischaemia depending on the CNS maturational stage at the time of insult, and modulation independently and in consort by individual axis components and in a sex dependent ways. Improved understanding on how to modify crosstalk between microglia and leucocytes will aid in developing age-appropriate therapies for infants and children who suffered cerebral ischaemia.

Neonatal Stroke and TLR1/2 Ligand Recruit Myeloid Cells through the Choroid Plexus in a CX3CR1-CCR2- and Context-Specific Manner.

Neonatal stroke is as frequent as stroke in the elderly, but many pathophysiological injury aspects are distinct in neonates, including immune signaling. While myeloid cells can traffic into the brain via multiple routes, the choroid plexus (CP) has been identified as a uniquely educated gate for immune cell traffic during health and disease. To understand the mechanisms of myeloid cell trafficking via the CP and their influence on neonatal stroke, we characterized the phenotypes of CP-infiltrating myeloid cells after transient middle cerebral artery occlusion (tMCAO) in neonatal mice of both sexes in relation to blood-brain barrier permeability, injury, microglial activation, and CX3CR1-CCR2 signaling, focusing on the dynamics early after reperfusion. We demonstrate rapid recruitment of multiple myeloid phenotypes in the CP ipsilateral to the injury, including inflammatory CD45+CD11b+Ly6chighCD86+, beneficial CD45+CD11b+Ly6clowCD206+, and CD45+CD11b+Ly6clowLy6ghigh cells, but only minor leukocyte infiltration into acutely ischemic-reperfused cortex and negligible vascular albumin leakage. We report that CX3CR1-CCR2-mediated myeloid cell recruitment contributes to stroke injury. Considering the complexity of inflammatory cascades triggered by stroke and a role for TLR2 in injury, we also used direct TLR2 stimulation as an independent injury model. TLR2 agonist rapidly recruited myeloid cells to the CP, increased leukocytosis in the CSF and blood, but infiltration into the cortex remained low over time. While the magnitude and the phenotypes of myeloid cells diverged between tMCAO and TLR2 stimulation, in both models, disruption of CX3CR1-CCR2 signaling attenuated both monocyte and neutrophil trafficking to the CP and cortex.SIGNIFICANCE STATEMENT Stroke during the neonatal period leads to long-term disabilities. The mechanisms of ischemic injury and inflammatory response differ greatly between the immature and adult brain. We examined leukocyte trafficking via the choroid plexus (CP) following neonatal stroke in relation to blood-brain barrier integrity, injury, microglial activation, and signaling via CX3CR1 and CCR2 receptors, or following direct TLR2 stimulation. Ischemia-reperfusion triggered marked unilateral CX3CR1-CCR2 dependent accumulation of diverse leukocyte subpopulations in the CP without inducing extravascular albumin leakage or major leukocyte infiltration into the brain. Disrupted CX3CR1-CCR2 signaling was neuroprotective in part by attenuating monocyte and neutrophil trafficking. Understanding the migratory patterns of CP-infiltrating myeloid cells with intact and disrupted CX3CR1-CCR2 signaling could identify novel therapeutic targets to protect the neonatal brain.

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.

Neuroinflammation-induced lymphangiogenesis near the cribriform plate contributes to drainage of CNS-derived antigens and immune cells.

There are no conventional lymphatic vessels within the CNS parenchyma, although it has been hypothesized that lymphatics near the cribriform plate or dura maintain fluid homeostasis and immune surveillance during steady-state conditions. However, the role of these lymphatic vessels during neuroinflammation is not well understood. We report that lymphatic vessels near the cribriform plate undergo lymphangiogenesis in a VEGFC - VEGFR3 dependent manner during experimental autoimmune encephalomyelitis (EAE) and drain both CSF and cells that were once in the CNS parenchyma. Lymphangiogenesis also contributes to the drainage of CNS derived antigens that leads to antigen specific T cell proliferation in the draining lymph nodes during EAE. In contrast, meningeal lymphatics do not undergo lymphangiogenesis during EAE, suggesting heterogeneity in CNS lymphatics. We conclude that increased lymphangiogenesis near the cribriform plate can contribute to the management of neuroinflammation-induced fluid accumulation and immune surveillance.

Regional Distribution of CNS Antigens Differentially Determines T-Cell Mediated Neuroinflammation in a CX3CR1-Dependent Manner.

T cells continuously sample CNS-derived antigens in the periphery, yet it is unknown how they sample and respond to CNS antigens derived from distinct brain areas. We expressed ovalbumin (OVA) neoepitopes in regionally distinct CNS areas (Cnp-OVA and Nes-OVA mice) to test peripheral antigen sampling by OVA-specific T cells under homeostatic and neuroinflammatory conditions. We show that antigen sampling in the periphery is independent of regional origin of CNS antigens in both male and female mice. However, experimental autoimmune encephalomyelitis (EAE) is differentially influenced in Cnp-OVA and Nes-OVA female mice. Although there is the same frequency of CD45high CD11b+ CD11c+ CX3CL1+ myeloid cell-T-cell clusters in neoepitope-expressing areas, EAE is inhibited in Nes-OVA female mice and accelerated in CNP-OVA female mice. Accumulation of OVA-specific T cells and their immunomodulatory effects on EAE are CX3C chemokine receptor 1 (CX3CR1) dependent. These data show that despite similar levels of peripheral antigen sampling, CNS antigen-specific T cells differentially influence neuroinflammatory disease depending on the location of cognate antigens and the presence of CX3CL1/CX3CR1 signaling.SIGNIFICANCE STATEMENT Our data show that peripheral T cells similarly recognize neoepitopes independent of their origin within the CNS under homeostatic conditions. Contrastingly, during ongoing autoimmune neuroinflammation, neoepitope-specific T cells differentially influence clinical score and pathology based on the CNS regional location of the neoepitopes in a CX3CR1-dependent manner. Altogether, we propose a novel mechanism for how T cells respond to regionally distinct CNS derived antigens and contribute to CNS autoimmune pathology.

Immune responses in stroke: how the immune system contributes to damage and healing after stroke and how this knowledge could be translated to better cures?

Stroke is one of the leading causes of death and disability worldwide. The long-standing dogma that stroke is exclusively a vascular disease has been questioned by extensive clinical findings of immune factors that are associated mostly with inflammation after stroke. These have been confirmed in preclinical studies using experimental animal models. It is now accepted that inflammation and immune mediators are critical in acute and long-term neuronal tissue damage and healing following thrombotic and ischaemic stroke. Despite mounting information delineating the role of the immune system in stroke, the mechanisms of how inflammatory cells and their mediators are involved in stroke-induced neuroinflammation are still not fully understood. Currently, there is no available treatment for targeting the acute immune response that develops in the brain during cerebral ischaemia. No new treatment has been introduced to stroke therapy since the discovery of tissue plasminogen activator therapy in 1996. Here, we review current knowledge of the immunity of stroke and identify critical gaps that hinder current therapies. We will discuss advances in the understanding of the complex innate and adaptive immune responses in stroke; mechanisms of immune cell-mediated and factor-mediated vascular and tissue injury; immunity-induced tissue repair; and the importance of modulating immunity in stroke.

Intrauterine inflammation induces sex-specific effects on neuroinflammation, white matter, and behavior.

Exposure to inflammation during pregnancy has been linked to adverse neurodevelopmental consequences for the offspring. One common route through which a developing fetus is exposed to inflammation is with intrauterine inflammation. To that end, we utilized an animal model of intrauterine inflammation (IUI; intrauterine lipopolysaccharide (LPS) administration, 50µg, E15) to assess placental and fetal brain inflammatory responses, white matter integrity, anxiety-related behaviors (elevated zero maze, light dark box, open field), microglial counts, and the CNS cytokine response to an acute injection of LPS in both males and females. These studies revealed that for multiple endpoints (fetal brain cytokine levels, cytokine response to adult LPS challenge) male IUI offspring were uniquely affected by intrauterine inflammation, while for other endpoints (behavior, microglial number) both sexes were similarly affected. These data advance our understanding of sex-specific effects of early life exposure to inflammation in a translationally- relevant model.

Contrasting roles of immune cells in tissue injury and repair in stroke: The dark and bright side of immunity in the brain.

Despite considerable efforts in research and clinical studies, stroke is still one of the leading causes of death and disability worldwide. Originally, stroke was considered a vascular thrombotic disease without significant immune involvement. However, over the last few decades it has become increasingly obvious that the immune responses can significantly contribute to both tissue injury and protection following stroke. Recently, much research has been focused on the immune system's role in stroke pathology and trying to elucidate the mechanism used by immune cells in tissue injury and protection. Since the discovery of tissue plasminogen activator therapy in 1996, there have been no new treatments for stroke. For this reason, research into understanding how the immune system contributes to stroke pathology may lead to better therapies or enhance the efficacy of current treatments. Here, we discuss the contrasting roles of immune cells to stroke pathology while emphasizing myeloid cells and T cells. We propose that focusing future research on balancing the beneficial-versus-detrimental roles of immunity may lead to the discovery of better and novel stroke therapies.

CCR7 deficient inflammatory Dendritic Cells are retained in the Central Nervous System.

Dendritic cells (DC) accumulate in the CNS during neuroinflammation, yet, how these cells contribute to CNS antigen drainage is still unknown. We have previously shown that after intracerebral injection, antigen-loaded bone marrow DC migrate to deep cervical lymph nodes where they prime antigen-specific T cells and exacerbate experimental autoimmune encephalomyelitis (EAE) in mice. Here, we report that DC migration from brain parenchyma is dependent upon the chemokine receptor CCR7. During EAE, both wild type and CCR7-/- CD11c-eYFP cells infiltrated into the CNS but cells that lacked CCR7 were retained in brain and spinal cord while wild type DC migrated to cervical lymph nodes. Retention of CCR7-deficient CD11c-eYFP cells in the CNS exacerbated EAE. These data are the first to show that CD11chigh DC use CCR7 for migration out of the CNS, and in the absence of this receptor they remain in the CNS in situ and exacerbate EAE.

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.