Card9/neutrophil signalling axis promotes IL-17A-mediated ankylosing spondylitis.

OBJECTIVE: Polymorphisms in the antifungal signalling molecule CARD9 are associated with ankylosing spondylitis (AS). Here, we investigated the cellular mechanism by which CARD9 controls pathogenic Th17 responses and the onset of disease in both experimental murine AS and patients. METHODS: Experiments in SKG, Card9-/-SKG, neutrophil-deplete SKG mice along with in vitro murine, neutrophil and CD4+ T cell cocultures examined Card9 function in neutrophil activation, Th17 induction and arthritis in experimental AS. In AS patients the neutrophil: Bath Ankylosing Spondylitis Functional Index relationship was analysed. In vitro studies with autologous neutrophil: T cell cocultures examined endogenous CARD9 versus the AS-associated variant (rs4075515) of CARD9 in T cellular production of IL-17A. RESULTS: Card9 functioned downstream of Dectin-1 and was essential for induction of Th17 cells, arthritis and spondylitis in SKG mice. Card9 expression within T cells was dispensable for arthritis onset in SKG mice. Rather, Card9 expression controlled neutrophil function; and neutrophils in turn, were responsible for triggering Th17 expansion and disease in SKG mice. Mechanistically, cocultures of zymosan prestimulated neutrophils and SKG T cells revealed a direct cellular function for Card9 within neutrophils in the potentiation of IL-17 production by CD4+ T cells on TCR-ligation. The clinical relevance of the neutrophil-Card9-coupled mechanism in Th17-mediated disease is supported by a similar observation in AS patients. Neutrophils from HLA-B27+ AS patients expanded autologous Th17 cells in vitro, and the AS-associated CARD9S12N variant increased IL-17A. CONCLUSIONS: These data reveal a novel neutrophil-intrinsic role for Card9 in arthritogenic Th17 responses and AS pathogenesis. These data provide valuable utility in our future understanding of CARD9-specific mechanisms in spondyloarthritis .

Orientia tsutsugamushi selectively stimulates the C-type lectin receptor Mincle and type 1-skewed proinflammatory immune responses.

Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. The lung is a major target organ of infection, displaying type 1-skewed proinflammatory responses. Lung injury and acute respiratory distress syndrome are common complications of severe scrub typhus; yet, their underlying mechanisms remain unclear. In this study, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in mice, we performed pulmonary differential expression analysis with NanoString. Of 671 genes examined, we found 312 significantly expressed genes at the terminal phase of disease. Mincle (Clec4e) was among the top 5 greatest up-regulated genes, accompanied with its signaling partners, type 1-skewing chemokines (Cxcr3, Ccr5, and their ligands), as well as Il27. To validate the role of Mincle in scrub typhus, we exposed murine bone marrow-derived macrophages (MΦ) to live or inactivated O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that while heat-killed bacteria stimulated transitory Mincle expression, live bacteria generated a robust response in MΦ, which was validated by indirect immunofluorescence and western blot. Notably, infection had limited impact on other tested CLRs or TLRs. Sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27) was induced by live, but not inactivated, bacteria; infected Mincle-/- MΦ significantly reduced proinflammatory responses compared with WT cells. Together, this study provides the first evidence for a selective expression of Mincle in sensing O. tsutsugamushi and suggests a potential role of Mincle- and IL-27-related pathways in host responses to severe infection. Additionally, it provides novel insight into innate immune recognition of this poorly studied bacterium.

The innate immune receptor Nlrp12 suppresses autoimmunity to the retina.

BACKGROUND: Nod-like receptors (NLRs) are critical to innate immune activation and induction of adaptive T cell responses. Yet, their role in autoinflammatory diseases of the central nervous system (CNS) remains incompletely defined. The NLR, Nlrp12, has been reported to both inhibit and promote neuroinflammation in an animal model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), where its T cell-specific role has been investigated. Uveitis resulting from autoimmunity of the neuroretina, an extension of the CNS, involves a breach in immune privilege and entry of T cells into the eye. Here, we examined the contribution of Nlrp12 in a T cell-mediated model of uveitis, experimental autoimmune uveitis (EAU). METHODS: Mice were immunized with interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP1-20) emulsified in Complete Freund's adjuvant, CFA. Uveitis was evaluated by clinical and histopathological scoring, and comparisons were made in WT vs. Nlrp12-/- mice, lymphopenic Rag1-/- mice reconstituted with WT vs. Nlrp12-/- CD4+ T cells, or among bone marrow (BM) chimeric mice. Antigen-specific Th-effector responses were evaluated by ELISA and intracellular cytokine staining. Cellular composition of uveitic eyes from WT or Nlrp12-/- mice was compared using flow cytometry. Expression of Nlrp12 and of cytokines/chemokines within the neuroretina was evaluated by immunoblotting and quantitative PCR. RESULTS: Nlrp12-/- mice developed exacerbated uveitis characterized by extensive vasculitis, chorioretinal infiltrates and photoreceptor damage. Nlrp12 was dispensable for T cell priming and differentiation of peripheral Th1 or Th17 cells, and uveitis in immunodeficient mice reconstituted with either Nlrp12-/- or WT T cells was similar. Collectively, this ruled out T cells as the source of Nlrp12-mediated protection to EAU. Uveitic Nlrp12-/- eyes had more pronounced myeloid cell accumulation than uveitic WT eyes. Transplantation of Nlrp12-/- BM resulted in increased susceptibility to EAU regardless of host genotype, but interestingly, a non-hematopoietic origin for Nlrp12 function was also observed. Indeed, Nlrp12 was found to be constitutively expressed in the neuroretina, where it suppressed chemokine/cytokine induction. CONCLUSIONS: Our data identify a combinatorial role for Nlrp12 in dampening autoimmunity of the neuroretina. These findings could provide a pathway for development of therapies for uveitis and potentially other autoinflammatory/autoimmune diseases of the CNS.

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 ß-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.

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.

Blau syndrome-associated Nod2 mutation alters expression of full-length NOD2 and limits responses to muramyl dipeptide in knock-in mice.

The biochemical mechanism by which mutations in nucleotide-binding oligomerization domain containing 2 (NOD2) cause Blau syndrome is unknown. Several studies have examined the effect of mutations associated with Blau syndrome in vitro, but none has looked at the implication of the mutations in vivo. To test the hypothesis that mutated NOD2 causes alterations in signaling pathways downstream of NOD2, we created a Nod2 knock-in mouse carrying the most common mutation seen in Blau syndrome, R314Q (corresponding to R334Q in humans). The endogenous regulatory elements of mouse Nod2 were unaltered. R314Q mice showed reduced cytokine production in response to i.p. and intravitreal muramyl dipeptide (MDP). Macrophages from R314Q mice showed reduced NF-κB and IL-6 responses, blunted phosphorylation of MAPKs, and deficient ubiquitination of receptor-interacting protein 2 in response to MDP. R314Q mice expressed a truncated 80-kDa form of NOD2 that was most likely generated by a posttranslational event because there was no evidence for a stop codon or alternative splicing event. Human macrophages from two patients with Blau syndrome also showed a reduction of both cytokine production and phosphorylation of p38 in response to MDP, indicating that both R314Q mice and cells from patients with Blau syndrome show reduced responses to MDP. These data indicate that the R314Q mutation when studied with the Nod2 endogenous regulatory elements left intact is associated with marked structural and biochemical changes that are significantly different from those observed from studies of the mutation using overexpression, transient transfection systems.

Aberrant interleukin-1 signalling does not increase susceptibility of mice to NOD2-dependent uveitis.

BACKGROUND: NOD2 is the genetic cause of Blau syndrome, an autoinflammatory disease that manifests as coincident uveitis and arthritis. Since dysregulation of IL-1 signalling is considered a pathogenic mechanism in a number of related autoinflammatory conditions, we examined the extent to which unimpeded interleukin (IL)-1 signalling influences NOD2-dependent inflammation of the eye versus the joint. METHODS: Mice deficient for IL-1R antagonist (IL-1Ra) were administered the NOD2 agonist muramyl dipeptide (MDP) by systemic (intraperitoneal) or local (intraocular and/or intra-articular) injections. NOD2-deficient mice received an intraocular injection of recombinant IL-1ß. Uveitis was evaluated by intravital videomicroscopy and histopathology, and arthritis was assessed by near-infrared imaging and histopathology. Ocular levels of IL-1α, IL-1ß and IL-1Ra were quantified by enzyme-linked immunosorbent assay. RESULTS: IL-1Ra deficiency did not render mice more responsive to systemic exposure of MDP. Despite the increased production of IL-1R agonists IL-1α and IL-1ß in response to intraocular injection of MDP, deficiency in IL-1Ra did not predispose mice to MDP-triggered uveitis, albeit intravascular cell rolling and adherence were exacerbated. NOD2 expression was dispensable for the potential of IL-1 to elicit uveitis. However, we find that IL-1Ra does play an important protective role in arthritis induced locally by MDP injection in the joint. CONCLUSIONS: Our findings highlight the complexity of NOD2 activation and IL-1 signalling effects that can be compounded by local environmental factors of the target organ. These observations may impact how we understand the molecular mechanisms by which NOD2 influences inflammation of the eye versus joint, and consequently, treatment options for uveitis versus arthritis.

Interferon-γ regulates discordant mechanisms of uveitis versus joint and axial disease in a murine model resembling spondylarthritis.

OBJECTIVE: The spondylarthritides (such as ankylosing spondylitis) are multisystem inflammatory diseases that frequently result in uveitis. Despite the common co-occurrence of uveitis with arthritis, there has been no explanation for the susceptibility of the eye to inflammation. Using an innovative intravital videomicroscopic approach, we discovered the coexistence of uveitis with axial and peripheral joint inflammation in mice immunized with cartilage proteoglycan (PG). The aim of this study was to elucidate the characteristics of uveitis and test the impact of interferon-γ (IFNγ) deficiency on the eye versus the joint and spine. METHODS: Female T cell receptor (TCR)-transgenic mice or IFNγ-knockout mice crossed to TCR-transgenic mice were immunized with PG. Uveitis was assessed by intravital videomicroscopy and histology. The clinical and histopathologic severity of arthritis and spondylitis were evaluated. The bone remodeling process within the spine was assessed by whole-body near-infrared imaging. Immunoblotting and immunofluorescence staining were used to examine the expression of PG and ADAMTS-5 and to examine the cellular composition of eyes with uveitis. RESULTS: PG neoepitopes along with the aggrecanase ADAMTS-5 were present in the eye, as they were the joint. Anterior uveitis developed in response to PG immunization. The cellular infiltrate consisted mainly of neutrophils and eosinophils. Unexpectedly, IFNγ deficiency markedly exacerbated uveitis while ameliorating joint and spine disease, indicating divergent mechanisms that drive diseases in the eye versus the joints and spine. CONCLUSION: This study provides the first detailed description of a murine disease model in which uveitis coincides with arthritis and spondylitis. Our observations provide a great opportunity for understanding the pathogenesis of a relatively common but poorly understood disease.

Impact of IL-1 signalling on experimental uveitis and arthritis.

BACKGROUND: Uveitis, or inflammatory eye disease, is a common extra-articular manifestation of many systemic autoinflammatory diseases involving the joints. Anakinra (recombinant interleukin (IL)-1 receptor antagonist (Ra)) is an effective therapy in several arthritic diseases; yet, few studies have investigated the extent to which IL-1 signalling or IL-1Ra influences the onset and/or severity of uveitis. OBJECTIVE: To seek possible links between arthritis and uveitis pathogenesis related to IL-1 signalling. METHODS: The eyes of IL-1Ra-deficient BALB/c mice were monitored histologically and by intravital videomicroscopy to determine if uveitis developed along with the expected spontaneous arthritis in ankles and knees. Expression levels of IL-1R and its negative regulators (IL-1Ra, IL-1RII, IL-1RAcP and single Ig IL-1R-related molecule) in eye and joint tissues were compared. Differences in uveitis induced by intraocular injection of lipopolysaccharide (LPS) in mice lacking IL-1R or IL-1Ra were assessed. RESULTS: Deficiency in IL-1Ra predisposes to spontaneous arthritis, which is exacerbated by previous systemic LPS exposure. The eye, however, does not develop inflammatory disease despite the progressive arthritis or LPS exposure. Organ-specific expression patterns for IL-1Ra and negative regulators of IL-1 activity were observed that appear to predict predisposition to inflammation in each location in IL-1Ra knockout mice. The eye is extremely sensitive to locally administered LPS, and IL-1Ra deficiency markedly exacerbates the resulting uveitis. CONCLUSION: This study demonstrates that IL-1Ra plays an important role in suppressing local responses in eyes injected with LPS and that there is discordance between murine eyes and joints in the extent to which IL-1Ra protects against spontaneous inflammation.

The NLRP3 inflammasome is active but not essential in endotoxin-induced uveitis.

OBJECTIVE: The inflammasome complex involving caspase-1 and nucleotide-binding domain, leucine-rich repeat containing protein (NLRP)3, also known as NALP3 or cryopyrin is important for host responses to microbial pathogens and several autoinflammatory diseases. We investigated the extent to which NLRP3 and caspase-1 control ocular interleukin (IL)-1ß production and severity of uveitis (intraocular inflammatory disease) in an established, acute inflammatory uveitis model, endotoxin-induced uveitis (EIU). METHODS: Expression of NLRP3, its adaptor molecule ASC, also known as PYCARD (PYD and CARD domain containing), and caspase-1 were examined by immunoblotting. IL-1ß production was measured by enzyme-linked immunosorbent assay (ELISA). Using knockout mice, roles for caspase-1 and NLRP3 were examined in uveitis induced by intraocular injection of Escherichia coli lipopolysaccharide (LPS). RESULTS: NLRP3, ASC, and caspase-1 proteins are constitutively expressed in eye tissue. During EIU, IL-1ß protein production increases; this requires the presence of both caspase-1 and NLRP3. However, severity of EIU is not altered by deficiency in either caspase-1 or NLRP3, as assessed by both intravital microscopy and histology. CONCLUSIONS: These data identify the importance of the NLRP3 inflammasome for IL-1ß production in the eye, yet indicate that its participation in EIU is nonessential.

Dectin-1 and NOD2 mediate cathepsin activation in zymosan-induced arthritis in mice.

OBJECTIVE: Activation of pattern recognition receptors (PRR) may contribute to arthritis. Here, we elucidated the role of NOD2, a genetic cause of inflammatory arthritis, and several other PRR in a murine model of inflammatory arthritis. METHODS: The roles of CR3, TLR2, MyD88, NOD1, NOD2, Dectin-1 and Dectin-2 were tested in vivo in arthritis elicited by intra-articular injections of zymosan, the fungal cell wall components curdlan, laminarin and mannan, and the bacterial cell wall peptidoglycan. RESULTS: Dectin-1, and to a lesser extent Dectin-2, contributed to arthritis. TLR2, MyD88 and CR3 played non-essential roles. Observations based on injection of curdlan, laminarin or mannan supported the dominant role of the Dectin-1 pathway in the joint. We demonstrated differential roles for NOD1 and NOD2 and identified NOD2 as a novel and essential mediator of zymosan-induced arthritis. CONCLUSIONS: Together, Dectin-1 and NOD2 are critical, sentinel receptors in the arthritogenic effects of zymosan. Our data identify a novel role for NOD2 during inflammatory responses within joints.

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.

T cell-intrinsic role for Nod2 in protection against Th17-mediated uveitis.

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome, an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established, yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here, we report a non-conventional, T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2-/- CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous, Rip2-independent mechanism for Nod2 in uveitis. In naive animals, Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly, CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity, and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome.

NOD2, the gene responsible for familial granulomatous uveitis, in a mouse model of uveitis.

PURPOSE: NOD2 plays an important role in the recognition of intracellular bacteria through its ability to sense the components of bacterial peptidoglycan (PGN), namely muramyl dipeptide (MDP) and muramyl tripeptide (MTP). Specific mutations in the human NOD2 gene cause Blau syndrome, an autosomal dominant form of uveitis, arthritis, and dermatitis. As a first step toward understanding the role of NOD2 in the pathogenesis of uveitis, the authors developed a mouse model of MDP-dependent uveitis. METHODS: BALB/c mice and mice deficient in L-selectin or NOD2 received intravitreal injection of MDP, MTP, or PGN. The intravascular response within the iris and cellular infiltration was quantified by intravital microscopy and histologic assessment. RESULTS: MDP induced an acute, ocular inflammatory response, wherein rolling and adhering leukocytes within the vasculature were significantly increased within 6 hours after MDP treatment. A minor increase in cellular infiltration occurred at 12 hours after MDP treatment. The adhesion molecule L-selectin participated in MDP-induced vascular inflammation because L-selectin knockout mice showed a significant decrease in the number of rolling cells. Importantly, NOD2 plays an essential role in ocular inflammation induced by MDP, as indicated by the fact that uveitis did not develop in Nod2 knockout mice in response to MDP. Nod2 knockout mice also showed abolished ocular inflammation in response to MTP but not to PGN treatment. CONCLUSIONS: These findings demonstrate a novel mouse model of uveitis, wherein NOD2 plays an essential role in inflammation induced by the minimal components of PGN. Thus, innate immune responses mediated by NOD2 may participate in the development of uveitis in response to bacterial products.

Uveitis secondary to bacterial products.

Bacteria are suspected contributors to several forms of immune-mediated, noninfectious forms of uveitis including that associated with ankylosing spondylitis, sarcoidosis, Behçet's disease and inflammatory bowel disease. Endotoxin (lipopolysaccharide)-induced uveitis has been a widely used model for more than 2 decades. Both rats and mice develop a transient, bilateral anterior uveitis after a systemic injection of endotoxin. Inflammation posterior to the lens is generally milder than anterior segment inflammation. The uveitis is severer if the lipopolysaccharides are injected intraocularly. The model has been invaluable in helping to identify mediators induced in the inflamed eye and in testing pharmacologic approaches to reduce eye inflammation. Muramyl dipeptide is another bacterial cell component that can induce uveitis in laboratory animals. Muramyl dipeptide is especially intriguing as a cause of uveitis because it activates the intracellular protein, Nod2, and mutations in the NOD2 gene are the cause of the autosomal dominant form of uveitis that is characteristic of Blau syndrome. Since a mutation in a gene that codes for a protein which senses a bacterial product consistently results in uveitis, it is critical to understand more fully the mechanisms by which bacterial products cause uveitis in laboratory animals.

Endotoxin preconditioning protects against the cytotoxic effects of TNFalpha after stroke: a novel role for TNFalpha in LPS-ischemic tolerance.

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury. Tumor necrosis factor-alpha (TNFalpha) is protective in LPS-induced preconditioning yet exacerbates neuronal injury in ischemia. Here, we define dual roles of TNFalpha in LPS-induced ischemic tolerance in a murine model of stroke and in primary neuronal cultures in vitro, and show that the cytotoxic effects of TNFalpha are attenuated by LPS preconditioning. We show that LPS preconditioning significantly increases circulating levels of TNFalpha before middle cerebral artery occlusion in mice and show that TNFalpha is required to establish subsequent neuroprotection against ischemia, as mice lacking TNFalpha are not protected from ischemic injury by LPS preconditioning. After stroke, LPS preconditioned mice have a significant reduction in the levels of TNFalpha (approximately threefold) and the proximal TNFalpha signaling molecules, neuronal TNF-receptor 1 (TNFR1), and TNFR-associated death domain (TRADD). Soluble TNFR1 (s-TNFR1) levels were significantly increased after stroke in LPS-preconditioned mice (approximately 2.5-fold), which may neutralize the effect of TNFalpha and reduce TNFalpha-mediated injury in ischemia. Importantly, LPS-preconditioned mice show marked resistance to brain injury caused by intracerebral administration of exogenous TNFalpha after stroke. We establish an in vitro model of LPS preconditioning in primary cortical neuronal cultures and show that LPS preconditioning causes significant protection against injurious TNFalpha in the setting of ischemia. Our studies suggest that TNFalpha is a twin-edged sword in the setting of stroke: TNFalpha upregulation is needed to establish LPS-induced tolerance before ischemia, whereas suppression of TNFalpha signaling during ischemia confers neuroprotection after LPS preconditioning.

Neuroprotection by osteopontin in stroke.

Osteopontin (OPN) is a secreted extracellular phosphoprotein involved in diverse biologic functions, including inflammation, cell migration, and antiapoptotic processes. Here we investigate the neuroprotective potential of OPN to reduce cell death using both in vitro and in vivo models of ischemia. We show that incubation of cortical neuron cultures with OPN protects against cell death from oxygen and glucose deprivation. The effect of OPN depends on the Arg-Gly-Asp (RGD)-containing motif as the protective effect of OPN in vitro was blocked by an RGD-containing hexapeptide, which prevents integrin receptors binding to their ligands. Osteopontin treatment of cortical neuron cultures caused an increase in Akt and p42/p44 MAPK phosphorylation, which is consistent with OPN-inducing neuroprotection via the activation of these protein kinases. Indeed, the protective effect of OPN was reduced by inhibiting the activation of Akt and p42/p44 MAPK using LY294002 and U0126, respectively. The protective effect of OPN was also blocked by the protein synthesis inhibitor cycloheximide, suggesting that the neuroprotective effect of OPN required new protein synthesis. Finally, intracerebral ventricular administration of OPN caused a marked reduction in infarct size after transient middle cerebral artery occlusion in a murine stroke model. These data suggest that OPN is a potent neuroprotectant against ischemic injury.

Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states.

BACKGROUND: Molecular mechanisms of neuroprotection that lead to ischaemic tolerance are incompletely understood. Identification of genes involved in the process would provide insight into cell survival and therapeutic approaches for stroke. We developed a mouse model of neuroprotection in stroke and did gene expression profiling to identify potential neuroprotective genes and their associated pathways. METHODS: Eight mice per condition were subjected to occlusion of the middle cerebral artery for 15 min (preconditioning), 60 min (injurious ischaemia), or preconditioning followed 72 h later by injurious ischaemia. RNA was extracted from the cortical regions of the ischaemic and non-ischaemic hemispheres. Three pools per condition were generated, and RNA was hybridised to oligonucleotide microarrays for comparison of ischaemic and non-ischaemic hemispheres. Real-time PCR and western blots were used to validate results. Follow-up experiments were done to address the biological relevance of findings. FINDINGS: Microarray analysis revealed changes in gene expression with little overlap among the conditions of injurious ischaemia, ischaemic preconditioning, or both. Injurious ischaemia induced upregulation of gene expression; 49 (86%) of 57 genes regulated showed increased expression in the ischaemic hemisphere. By contrast, preconditioning followed by injurious ischaemia resulted in pronounced downregulation; 47 (77%) of 61 regulated genes showed lower expression. Preconditioning resulted in transcriptional changes involved in suppression of metabolic pathways and immune responses, reduction of ion-channel activity, and decreased blood coagulation. INTERPRETATION: Preconditioning reprogrammes the response to ischaemic injury. Similar changes reported by others support an evolutionarily conserved endogenous response to decreased blood flow and oxygen limitation such as seen during hibernation.

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

INTRODUCTION: Systemic 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. METHODS: SKG 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. RESULTS: Exposure 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. CONCLUSIONS: This 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.

Endotoxin preconditioning prevents cellular inflammatory response during ischemic neuroprotection in mice.

BACKGROUND AND PURPOSE: Tolerance to ischemic brain injury is induced by several preconditioning stimuli, including lipopolysaccharide (LPS). A small dose of LPS given systemically confers ischemic protection in the brain, a process that appears to involve activation of an inflammatory response before ischemia. We postulated that LPS preconditioning modulates the cellular inflammatory response after cerebral ischemia, resulting in neuroprotection. METHODS: Mice were treated with LPS (0.2 mg/kg) 48 hours before ischemia induced by transient middle cerebral artery occlusion (MCAO). The infarct was measured by 2,3,5-triphenyltetrazolium chloride staining. Microglia/macrophage responses after MCAO were assessed by immunofluorescence and flow cytometry. The effect of MCAO on white blood cells in the brain and peripheral circulation was measured by flow cytometry 48 hours after MCAO. RESULTS: LPS preconditioning induced significant neuroprotection against MCAO. Administration of low-dose LPS before MCAO prevented the cellular inflammatory response in the brain and blood. Specifically, LPS preconditioning suppressed neutrophil infiltration into the brain and microglia/macrophage activation in the ischemic hemisphere, which was paralleled by suppressed monocyte activation in the peripheral blood. CONCLUSIONS: LPS preconditioning induces neuroprotection against ischemic brain injury in a mouse model of stroke. LPS preconditioning suppresses the cellular inflammatory response to ischemia in the brain and circulation. Diminished activation of cellular inflammatory responses that ordinarily exacerbate ischemic injury may contribute to neuroprotection induced by LPS preconditioning.