MOG autoantibodies trigger a tightly-controlled FcR and BTK-driven microglia proliferative response.

Autoantibodies are a hallmark of numerous neurological disorders, including multiple sclerosis, autoimmune encephalitides and neuromyelitis optica. Whilst well understood in peripheral myeloid cells, the pathophysiological significance of autoantibody-induced Fc receptor signalling in microglia remains unknown, in part due to the lack of a robust in vivo model. Moreover, the application of therapeutic antibodies for neurodegenerative disease also highlights the importance of understanding Fc receptor signalling in microglia. Here, we describe a novel in vivo experimental paradigm that allows for selective engagement of Fc receptors within the CNS by peripherally injecting anti-myelin oligodendrocyte glycoprotein (MOG) monoclonal antibodies into normal wild-type mice. MOG antigen-bound immunoglobulins were detected throughout the CNS and triggered a rapid and tightly regulated proliferative response in both brain and spinal cord microglia. This microglial response was abrogated when anti-MOG antibodies were deprived of Fc receptor effector function or injected into Fcγ receptor knockout mice and was associated with the downregulation of Fc receptors in microglia, but not peripheral myeloid cells, establishing that this response was dependent on central Fc receptor engagement. Downstream of the Fc receptors, BTK was a required signalling node for this response, as microglia proliferation was amplified in BtkE41K knock-in mice expressing a constitutively active form of the enzyme and blunted in mice treated with a CNS-penetrant small molecule inhibitor of BTK. Finally, this response was associated with transient and stringently regulated changes in gene expression predominantly related to cellular proliferation, which markedly differed from transcriptional programs typically associated with Fc receptor engagement in peripheral myeloid cells. Together, these results establish a physiologically-meaningful functional response to Fc receptor and BTK signalling in microglia, while providing a novel in vivo tool to further dissect the roles of microglia-specific Fc receptor and BTK-driven responses to both pathogenic and therapeutic antibodies in CNS homeostasis and disease.

Differential accumulation of storage bodies with aging defines discrete subsets of microglia in the healthy brain.

To date, microglia subsets in the healthy CNS have not been identified. Utilizing autofluorescence (AF) as a discriminating parameter, we identified two novel microglia subsets in both mice and non-human primates, termed autofluorescence-positive (AF+) and negative (AF-). While their proportion remained constant throughout most adult life, the AF signal linearly and specifically increased in AF+ microglia with age and correlated with a commensurate increase in size and complexity of lysosomal storage bodies, as detected by transmission electron microscopy and LAMP1 levels. Post-depletion repopulation kinetics revealed AF- cells as likely precursors of AF+ microglia. At the molecular level, the proteome of AF+ microglia showed overrepresentation of endolysosomal, autophagic, catabolic, and mTOR-related proteins. Mimicking the effect of advanced aging, genetic disruption of lysosomal function accelerated the accumulation of storage bodies in AF+ cells and led to impaired microglia physiology and cell death, suggestive of a mechanistic convergence between aging and lysosomal storage disorders.

Microglia are a unique type of immune cell found in the brain and spinal cord. Their job is to support neurons, defend against invading microbes, clear debris and remove dying neurons by engulfing them. Despite these diverse roles, scientists have long believed that there is only a single type of microglial cell, which adapts to perform whatever task is required. But more recent evidence suggests that this is not the whole story. Burns et al. now show that we can distinguish two subtypes of microglia based on a property called autofluorescence. This is the tendency of cells and tissues to emit light of one color after they have absorbed light of another. Burns et al. show that about 70% of microglia in healthy mouse and monkey brains display autofluorescence. However, about 30% of microglia show no autofluorescence at all. This suggests that there are two subtypes of microglia: autofluorescence-positive and autofluorescence-negative. But does this difference have any implications for how the microglia behave? Autofluorescence occurs because specific substances inside the cells absorb light. In the case of microglia, electron microscopy revealed that autofluorescence was caused by structures within the cell called lysosomal storage bodies accumulating certain materials. The stored material included fat molecules, cholesterol crystals and other substances that are typical of disorders that affect these compartments. Burns et al. show that autofluorescent microglia contain larger amounts of proteins involved in storing and digesting waste materials than their non-autofluorescent counterparts. Moreover, as the brain ages, lysosomal storage material builds up inside autofluorescent microglia, which increase their autofluorescence as a result. Unfortunately, this accumulation of cellular debris also makes it harder for the microglia to perform their tasks. Increasing evidence suggests that the accumulation of waste materials inside the brain contributes to diseases of aging. Future work should examine how autofluorescent microglia behave in animal models of neurodegenerative diseases. If these cells do help protect the brain from the effects of aging, targeting them could be a new strategy for treating aging-related diseases.

NOD2 modulates immune tolerance via the GM-CSF-dependent generation of CD103+ dendritic cells.

Four decades ago, it was identified that muramyl dipeptide (MDP), a peptidoglycan-derived bacterial cell wall component, could display immunosuppressive functions in animals through mechanisms that remain unexplored. We sought to revisit these pioneering observations because mutations in NOD2, the gene encoding the host sensor of MDP, are associated with increased risk of developing the inflammatory bowel disease Crohn's disease, thus suggesting that the loss of the immunomodulatory functions of NOD2 could contribute to the development of inflammatory disease. Here, we demonstrate that intraperitoneal (i.p.) administration of MDP triggered regulatory T cells and the accumulation of a population of tolerogenic CD103+ dendritic cells (DCs) in the spleen. This was found to occur not through direct sensing of MDP by DCs themselves, but rather via the production of the cytokine GM-CSF, another factor with an established regulatory role in Crohn's disease pathogenesis. Moreover, we demonstrate that populations of CD103-expressing DCs in the gut lamina propria are enhanced by the activation of NOD2, indicating that MDP sensing plays a critical role in shaping the immune response to intestinal antigens by promoting a tolerogenic environment via manipulation of DC populations.

Acute microglia ablation induces neurodegeneration in the somatosensory system.

Previous studies have reported that microglia depletion leads to impairment of synapse formation and these cells rapidly repopulate from CNS progenitors. However, the impact of microglia depletion and repopulation in the long-term state of the CNS environment has not been characterized. Here, we report that acute and synchronous microglia depletion and subsequent repopulation induces gray matter microgliosis, neuronal death in the somatosensory cortex and ataxia-like behavior. We find a type 1 interferon inflammatory signature in degenerating somatosensory cortex from microglia-depleted mice. Transcriptomic and mass cytometry analysis of repopulated microglia demonstrates an interferon regulatory factor 7-driven activation state. Minocycline and anti-IFNAR1 antibody treatment attenuate the CNS type 1 interferon-driven inflammation, restore microglia homeostasis and reduce ataxic behavior. Neither microglia depletion nor repopulation impact neuropathology or T-cell responses during experimental autoimmune encephalomyelitis. Together, we found that acute microglia ablation induces a type 1 interferon activation state of gray matter microglia associated with acute neurodegeneration.

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

Constitutive induction of intestinal Tc17 cells in the absence of hematopoietic cell-specific MHC class II expression.

The enteric pathogen Citrobacter rodentium induces a mucosal IL-17 response in CD4(+) T helper (Th17) cells that is dependent on the Nod-like receptors Nod1 and Nod2. Here, we sought to determine whether this early Th17 response required antigen presentation by major histocompatibility complex class II (MHCII) for full induction. At early phases of C. rodentium infection, we observed that the intestinal mucosal Th17 response was fully blunted in irradiated mice reconstituted with MHCII-deficient (MHCII(-/-) →WT) hematopoietic cells. Surprisingly, we also observed a substantial increase in the relative frequency of IL-17(+) CD8(+) CD4(-) TCR-ß(+) cells (Tc17 cells) and FOXP3(+) CD8(+) CD4(-) TCR-ß(+) cells in the lamina propria and intraepithelial lymphocyte compartment of MHCII(-/-) →WT mice compared with that in WT→WT counterparts. Moreover, MHCII(-/-) →WT mice displayed increased susceptibility, increased bacterial translocation to deeper organs, and more severe colonic histopathology after infection with C. rodentium. Finally, a similar phenotype was observed in mice deficient for CIITA, a transcriptional regulator of MHCII expression. Together, these results indicate that MHCII is required to mount early mucosal Th17 responses to an enteric pathogen, and that MHCII regulates the induction of atypical CD8(+) T-cell subsets, such as Tc17 cells and FOXP3(+) CD8(+) cells, in vivo.

Nod1 and Nod2 signaling does not alter the composition of intestinal bacterial communities at homeostasis.

Patients with inflammatory bowel diseases (IBD) harbour intestinal bacterial communities with altered composition compared with healthy counterparts; however, it is unknown whether changes in the microbiota are associated with genetic susceptibility of individuals for developing disease or instead reflect other changes in the intestinal environment related to the disease itself. Since deficiencies in the innate immune receptors Nod1 and Nod2 are linked to IBD, we tested the hypothesis that Nod-signaling alters intestinal immune profiles and subsequently alters bacterial community structure. We used qPCR to analyze expression patterns of selected immune mediators in the ileum and cecum of Nod-deficient mice compared with their Nod-sufficient littermates and assessed the relative abundance of major bacterial groups sampled from the ileum, cecum and colon. The Nod1-deficient ileum exhibited significantly lower expression of Nod2, Muc2, α- and ß-defensins and keratinocyte-derived chemokine (KC), suggesting a weakened epithelial barrier compared with WT littermates; however, there were no significant differences in the relative abundance of targeted bacterial groups, indicating that Nod1-associated immune differences alone do not promote dysbiosis. Furthermore, Nod2-deficient mice did not display any changes in the expression of immune markers or bacterial communities. Shifts in bacterial communities that were observed in this study correlated with housing conditions and were independent of genotype. These findings emphasize the importance of using F2 littermate controls to minimize environmental sources of variation in microbial analyses, to establish baseline conditions for host-microbe homeostasis in Nod-deficient mice and to strengthen models for testing factors contributing to microbial dysbiosis associated with IBD.

Identification of a synthetic muramyl peptide derivative with enhanced Nod2 stimulatory capacity.

Muramyl peptides (MPs) represent the building blocks of bacterial peptidoglycan, a critical component of bacterial cell walls. MPs are well characterized for their immunomodulatory properties, and numerous studies have delineated the role of MPs or synthetic MP analogs in host defense, adjuvanticity and inflammation. More recently, Nod1 and Nod2 have been identified as the host sensors for specific MPs, and, in particular, Nod2 was shown to detect muramyl dipeptide (MDP), a MP found in both Gram-positive and Gram-negative bacterial cell walls. Because mutations in Nod2 are associated with the etiology of Crohn's disease, there is a need to identify synthetic MP analogs that could potentiate Nod2-dependent immunity. Here, we analyzed the Nod2-activating property of 36 MP analogs that had been tested previously for their adjuvanticity and anti-infectious activity. Using a luciferase-based screen, we demonstrate that addition of a methyl group to the second amino acid of MDP generates a MDP derivative with enhanced Nod2-activating capacity. We further validated these results in murine macrophages, human dendritic cells and in vivo. These results offer a basis for the rational development of synthetic MPs that could be used in the treatment of inflammatory disorders that have been associated with Nod2 dysfunction, such as Crohn's disease.

Phenotyping of Nod1/2 double deficient mice and characterization of Nod1/2 in systemic inflammation and associated renal disease.

It is indispensable to thoroughly characterize each animal model in order to distinguish between primary and secondary effects of genetic changes. The present study analyzed Nod1 and Nod2 double deficient (Nod1/2 DKO) mice under physiological and inflammatory conditions. Nod1 and Nod2 are members of the Nucleotide-binding domain and Leucine-rich repeat containing Receptor (NLR) family. Several inflammatory disorders, such as Crohn's disease and asthma, are linked to genetic changes in either Nod1 or Nod2. These associations suggest that Nod1 and Nod2 play important roles in regulating the immune system.Three-month-old wildtype (Wt) and Nod1/2 DKO mice were sacrificed, body and organ weight were determined, and blood was drawn. Except for lower liver weight in Nod1/2 DKO mice, no differences were found in body/organ weight between both strains. Leukocyte count and composition was comparable. No significant changes in analyzed plasma biochemical markers were found. Additionally, intestinal and vascular permeability was determined. Nod1/2 DKO mice show increased susceptibility for intestinal permeability while vascular permeability was not affected. Next we induced septic shock and organ damage by administering LPS+PGN intraperitoneally to Wt and Nod1/2 DKO mice and sacrificed animals after 2 and 24 hours. The systemic inflammatory and metabolic response was comparable between both strains. However, renal response was different as indicated by partly preserved kidney function and tubular epithelial cell damage in Nod1/2 DKO at 24 hours. Remarkably, renal inflammatory mediators Tnfα, KC and Il-10 were significantly increased in Nod1/2 DKO compared with Wt mice at 2 hours.Systematic analysis of Nod1/2 DKO mice revealed a possible role of Nod1/2 in the development of renal disease during systemic inflammation.

Nod-like receptors in the control of intestinal inflammation.

The Nod-like receptor (NLR) family of intracellular pattern recognition molecules plays critical roles in the control of inflammation through the modulation of different signalling pathways, including those dependent on NF-κB and caspase-1-mediated cleavage of interleukin (IL)-1ß and IL-18. A number of NLRs or NLR-associated proteins have been genetically associated with susceptibility to inflammatory bowel disease (IBD), either Crohn's disease or ulcerative colitis. Accordingly, recent studies have examined the role of NLR proteins in chemical-induced or bacteria-induced murine models of colitis. In this review, we will discuss the genetic associations of NLRs with IBD and the research using NLR-deficient mice in different colitis models.

Examining host-microbial interactions through the lens of NOD: From plants to mammals.

Nod-like receptors (NLRs) for detecting microbial invaders are features of many plant and animal families. Although broadly similar in form and function, intimate co-evolutionary events with environmental microbes have shaped specific classes of NLRs in different types of hosts. Details of the roles of different NLRs in signaling cellular immune responses to invading microbes are only beginning to emerge. This review will discuss the current understanding of NLRs in plants, invertebrates, and mammals, with emphasis on their role in regulating NF-κB and inflammasome activity in mammals.

Innate IL-17 and IL-22 responses to enteric bacterial pathogens.

With the identification of T helper (Th)17 cells, a specific subset of CD4 T cells expressing interleukin (IL)-17 and IL-22, research on the function of these cytokines initially largely focused on traditional adaptive immune responses. However, IL-17 and IL-22 enhance basic innate barrier defenses at mucosal surfaces, such as antimicrobial peptide production and neutrophil recruitment; both events that occur rapidly and precede adaptive phase immunity. At the intestinal mucosal surface, it is now clear that innate lymphoid cells are also important sources of IL-17 and IL-22 during early phases of infection. Here, we discuss the function of innate IL-17- and IL-22-producing lymphocytes during enteric bacterial infection and their regulation by the intestinal microbiota, Toll-like receptors (TLRs) and Nod-like receptors (NLRs).

Nucleotide oligomerization domain-containing proteins instruct T cell helper type 2 immunity through stromal activation.

Although a number of studies have examined the development of T-helper cell type 2 (Th2) immunity in different settings, the mechanisms underlying the initiation of this arm of adaptive immunity are not well understood. We exploited the fact that immunization with antigen plus either nucleotide-binding oligomerization domain-containing proteins 1 (Nod1) or 2 (Nod2) agonists drives Th2 induction to understand how these pattern-recognition receptors mediate the development of systemic Th2 immune responses. Here, we show in bone-marrow chimeric mice that Nod1 and Nod2 expression within the stromal compartment is necessary for priming of effector CD4(+) Th2 responses and specific IgG1 antibodies. In contrast, sensing of these ligands by dendritic cells was not sufficient to induce Th2 immunity, although these cells contribute to the response. Moreover, we determined that CD11c(+) cells were the critical antigen-presenting cells, whereas basophils and B cells did not affect the capacity of Nod ligands to induce CD4(+) Th2 effector function. Finally, we found that full Th2 induction upon Nod1 and Nod2 activation was dependent on both thymic stromal lymphopoietin production by the stromal cells and the up-regulation of the costimulatory molecule, OX40 ligand, on dendritic cells. This study provides in vivo evidence of how systemic Th2 immunity is induced in the context of Nod stimulation. Such understanding will influence the rational design of therapeutics that could reprogram the immune system during an active Th1-mediated disease, such as Crohn's disease.

Identification of an innate T helper type 17 response to intestinal bacterial pathogens.

Interleukin 17 (IL-17) is a central cytokine implicated in inflammation and antimicrobial defense. After infection, both innate and adaptive IL-17 responses have been reported, but the type of cells involved in innate IL-17 induction, as well as their contribution to in vivo responses, are poorly understood. Here we found that Citrobacter and Salmonella infection triggered early IL-17 production, which was crucial for host defense and was mediated by CD4(+) T helper cells. Enteric innate T helper type 17 (iT(H)17) responses occurred principally in the cecum, were dependent on the Nod-like receptors Nod1 and Nod2, required IL-6 induction and were associated with a decrease in mucosal CD103(+) dendritic cells. Moreover, imprinting by the intestinal microbiota was fully required for the generation of iT(H)17 responses. Together, these results identify the Nod-iT(H)17 axis as a central element in controlling enteric pathogens, which may implicate Nod-driven iT(H)17 responses in the development of inflammatory bowel diseases.