Emily E. Vance

Hypothesis: Sarcoidosis is a STAT1-mediated disease

Immunologic pathways involved in sarcoidosis pathogenesis are largely unknown. We hypothesized that patients with sarcoidosis have characteristic mRNA profiles. Microarray analysis of gene expression was done on peripheral blood (12 patients, 12 controls), lung (6 patients, 6 controls) and lymph node (8 patients, 5 controls). Comparing peripheral blood from patients with sarcoidosis to controls, 872 transcripts were upregulated and 1039 were downregulated at >1.5-fold change and a significant q value. Several transcripts associated with interferon and STAT1 were upregulated. Lung and lymph node analyses also showed dramatic increases in STAT1 and STAT1-regulated chemokines. Granulomas in lymph nodes of patients with sarcoidosis expressed abundant STAT1 and phosphorylated STAT1. STAT1 might play an important role in sarcoidosis. This novel hypothesis unites seemingly disparate observations with regard to sarcoidosis including implication of a casual role for interferons, a suspected infectious trigger, T(H)1 predominating lymphocytes in bronchoalveolar lavage, and the association with hypercalcemia.

Nucleotide-binding oligomerization domain 2 and Toll-like receptor 2 function independently in a murine model of arthritis triggered by intraarticular peptidoglycan.

OBJECTIVE Blau syndrome is an autoinflammatory disease resulting from mutations in the NOD2 gene, wherein granulomatous arthritis, uveitis, and dermatitis develop. The mechanisms by which aberrant NOD2 causes joint inflammation are poorly understood. Indeed, very few studies have addressed the function of nucleotide-binding oligomerization domain 2 (NOD-2) in the joint. This study was undertaken to investigate NOD-2 function in an experimental model of arthritis and to explore the potential interplay between Toll-like receptor 2 (TLR-2) and NOD-2 in joint inflammation. METHODS Mice deficient in TLR-2, myeloid differentiation factor 88 (MyD88), or NOD-2 and their wild-type controls were given an intraarticular injection of muramyl dipeptide (MDP), peptidoglycan (PG; a metabolite of which is MDP), or palmitoyl-3-cysteine-serine-lysine-4 (Pam(3)CSK(4)), a synthetic TLR-2 agonist. Joint inflammation was assessed by near-infrared fluorescence imaging and histologic analysis. RESULTS Locally administered PG resulted in joint inflammation, which was markedly reduced in mice deficient in either TLR-2 or the TLR signaling mediator MyD88. In addition to TLR-2 signaling events, NOD-2 mediated joint inflammation, as evidenced by the fact that mice deficient in NOD-2 showed significantly reduced PG-induced arthritis. TLR-2 or MyD88 deficiency did not influence arthritis induced by the specific NOD-2 agonist MDP. In addition, NOD-2 deficiency did not alter the TLR-2-dependent joint inflammation elicited by the synthetic TLR-2 agonist Pam(3)CSK(4). CONCLUSION Whereas NOD-2 and TLR-2 are both critical for the development of PG-induced arthritis, they appear to elicit inflammation independently of each other. Our findings indicate that NOD-2 plays an inflammatory role in arthritis.

Mincle Activation and the Syk/Card9 Signaling Axis Are Central to the Development of Autoimmune Disease of the Eye

Uveitis, which occurs in association with systemic immunological diseases, presents a considerable medical challenge because of incomplete understanding of its pathogenesis. The signals that initiate T cells to target the eye, which may be of infectious or noninfectious origin, are poorly understood. Experimental autoimmune uveoretinitis (EAU) develops in mice immunized with the endogenous retinal protein interphotoreceptor retinoid binding protein in the presence of the adjuvant CFA. EAU manifests as posterior ocular inflammation consisting of vasculitis, granulomas, retinal damage, and invasion of self-reactive T cells, which are key clinical features of human uveitis. Our studies uncover Card9 as a critical genetic determinant for EAU. Card9 was responsible for Th17 polarization and Th17-associated Ag-specific responses, but not Th1-associated responses. Nonetheless, Card9 expression was essential for accumulation of both lineages within the eye. Consistent with its recently identified role as an intracellular signaling mediator for C-type lectin receptors (CLRs), a Card9-dependent transcriptional response in the neuroretina was observed involving genes encoding the CLRs Dectin-1, Dectin-2, and Mincle. Genetic deletion of these individual CLRs revealed an essential role for Mincle. Mincle activation was sufficient to generate the EAU phenotype, and this required activation of both Syk and Card9. In contrast, Dectin-1 contributed minimally and a possible repressive role was shown for Dectin-2. These findings extend our understanding of CLRs in autoimmune uveitis. The newly identified role of Mincle and Syk/Card9-coupled signaling axis in autoimmune uveitis could provide novel targets for treatment of patients with ocular inflammatory disease.

NOD2 deficiency results in increased susceptibility to peptidoglycan-induced uveitis in mice.

PURPOSE The innate immune receptor NOD2 is a genetic cause of uveitis (Blau syndrome). Intriguingly, in the intestine where polymorphisms of NOD2 predispose to Crohns disease, NOD2 reportedly suppresses inflammation triggered by the bacterial cell wall component, peptidoglycan (PGN). Whether NOD2 exerts a similar capacity in the regulation of ocular inflammation to PGN has not been explored. METHODS NOD2, NOD1, or MyD88 knockout (KO) mice and their wild-type (WT) controls were administered an intravitreal injection of PGN (a metabolite of which is the NOD2 agonist, muramyl dipeptide), or synthetic TLR2/1 and TLR2/6 agonists, Pam₃CSK4 and FSL-1. Ocular inflammation was assessed by intravital microscopy and histopathology. Cytokine production in eye tissue homogenates was measured by ELISA. RESULTS PGN triggered uveitis in mice. This inflammation was abolished in the absence of the TLR signaling mediator MyD88. NOD2 exerted a negative regulatory role because PGN-triggered eye inflammation was exacerbated in NOD2 KO mice. Increased intravascular response coincided with enhanced leukocytes within the aqueous and vitreous humors. The enhanced susceptibility of NOD2 KO mice to PGN uveitis coincided with increased cytokine production of IL-12p40, IL-17, and IL-23 but not IL-12p70, TNFα, or IFNγ. NOD1 deficiency did not result in the same sensitivity to PGN. Ocular inflammation induced by synthetic TLR2 agonists required MyD88 but not NOD2 or NOD1. CONCLUSIONS NOD2 may serve differential roles in the eye to promote inflammation while also tempering cell responses to PGN akin to what has been reported in colitis.

Fungal-derived cues promote ocular autoimmunity through a Dectin-2/Card9-mediated mechanism

Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic–environmental interactions that break immune tolerance to generate eye‐specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria‐containing complete Freunds adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4+ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye‐specific T cells remain poorly defined. In this study, we found that fungal‐derived antigens possess autoimmune uveitis‐promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP‐triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C‐type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9‐activating CLRs. Using single receptor‐deficient mice we identified Dectin‐2, but not Mincle or Dectin‐1, as a predominant mediator of fungal‐promoted uveitis. Conversely, Dectin‐2 activation by α‐mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9‐coupled signalling axis and interleukin (IL)‐17 production. Taken together, this report relates the potential of the Dectin‐2/Card9‐coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell‐mediated autoimmunity, it also reveals a previously unappreciated ability of fungal‐derived signals to promote autoimmunity.

Investigation of the relationship between the onset of arthritis and uveitis in genetically predisposed SKG mice

IntroductionSystemic rheumatic conditions are often accompanied by intraocular inflammatory disease (termed uveitis). Despite the frequent manifestation of uveitis with arthritis, very little is understood of the underlying mechanisms that mediate the eye’s susceptibility to disease. The genetically susceptible SKG mouse strain develops arthritis that arises from an inherent mutation that disrupts T-cell antigen receptor signal transduction and thymic selection. The ensuing T-cell–mediated disease is further modulated through exposure to microbial triggers. The purpose of this study was to elucidate how a genetically determined shift in the T-cell repertoire toward self-reactive T cells that drive arthritis influences uveitis in SKG mice.MethodsSKG mice (BALB/c mice that harbor the W163C point mutation in zeta-chain-associated protein kinase 70 [i.e., ZAP-70]) were housed under arthritis-resistant, specific pathogen–free conditions. Arthritis was induced by intraperitoneal injection with fungal glucans (zymosan or curdlan). Arthritis onset and severity were evaluated by clinical scoring, histopathology and infrared imaging within the joints. Periocular traits involving blepharoconjunctivitis were evaluated by clinical scoring and histology. Eyes were evaluated for signs of anterior uveitis using intravital videomicroscopy to document cell-trafficking responses within the iris vasculature and stroma and by histology to detect inflammatory infiltrate and tissue damage within the anterior and posterior eye segments.ResultsExposure to zymosan resulted in the predicted arthritic, sexually dimorphic phenotype in SKG mice. The eyes of SKG mice exhibited episodic intravascular cellular responses to zymosan or curdlan as indicated by significant increases in leukocyte–endothelium interactions akin to ocular vasculitis. However, despite the significant increase in early cell-trafficking responses, cellular infiltration into the iris stroma was not observed and histopathological signs indicative of a sustained uveitis were absent. Instead, eyes of SKG mice developed blepharoconjunctivitis that coincided with arthritis and exhibited sexual dimorphism.ConclusionsThis study underscores the complexity surrounding the pathogenesis of uveitis and its relationship with arthritis. The findings suggest that distinct mechanisms exist by which pathogenic autoimmune T cells target the eyes versus joints, which likely involves the environmental context but nonetheless should be taken into account in the identification and development of effective therapies for each organ.

Nod2 Deficiency Augments Th17 Responses and Exacerbates Autoimmune Arthritis

Arthritis in a genetically susceptible SKG strain of mice models a theoretical paradigm wherein autoimmune arthritis arises because of interplay between preexisting autoreactive T cells and environmental stimuli. SKG mice have a point mutation in ZAP-70 that results in attenuated TCR signaling, altered thymic selection, and spontaneous production of autoreactive T cells that cause arthritis following exposure to microbial β-glucans. In this study, we identify Nod2, an innate immune receptor, as a critical suppressor of arthritis in SKG mice. SKG mice deficient in Nod2 (Nod2−/−SKG) developed a dramatically exacerbated form of arthritis, which was independent of sex and microbiota, but required the skg mutation in T cells. Worsened arthritis in Nod2−/−SKG mice was accompanied by expansion of Th17 cells, which to some measure coproduced TNF, GM-CSF, and IL-22, along with elevated IL-17A levels within joint synovial fluid. Importantly, neutralization of IL-17A mitigated arthritis in Nod2−/−SKG mice, indicating that Nod2-mediated protection occurs through suppression of the Th17 response. Nod2 deficiency did not alter regulatory T cell development or function. Instead, Nod2 deficiency resulted in an enhanced fundamental ability of SKG CD4+ T cells (from naive mice) to produce increased levels of IL-17 and to passively transfer arthritis to lymphopenic recipients on a single-cell level. These data reveal a previously unconsidered role for T cell–intrinsic Nod2 as an endogenous negative regulator of Th17 responses and arthritogenic T cells. Based on our findings, future studies aimed at understanding a negative regulatory function of Nod2 within autoreactive T cells could provide novel therapeutic strategies for treatment of patients with arthritis.