No abstract

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.

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.

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury. Tumor necrosis factor-a (TNFa) is protective in LPS-induced preconditioning yet exacerbates neuronal injury in ischemia. Here, we define dual roles of TNFa 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 TNFa are attenuated by LPS preconditioning. We show that LPS preconditioning significantly increases circulating levels of TNFa before middle cerebral artery occlusion in mice and show that TNFa is required to establish subsequent neuroprotection against ischemia, as mice lacking TNFa are not protected from ischemic injury by LPS preconditioning. After stroke, LPS preconditioned mice have a significant reduction in the levels of TNFa (Bthreefold) and the proximal TNFa 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 (B2.5-fold), which may neutralize the effect of TNFa and reduce TNFamediated injury in ischemia. Importantly, LPS-preconditioned mice show marked resistance to brain injury caused by intracerebral administration of exogenous TNFa 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 TNFa in the setting of ischemia. Our studies suggest that TNFa is a twin-edged sword in the setting of stroke: TNFa upregulation is needed to establish LPS-induced tolerance before ischemia, whereas suppression of TNFa signaling during ischemia confers neuroprotection after LPS preconditioning.

Uveitis, which occurs in association with systemic immunological diseases, presents a considerable medical challenge due to incomplete understanding of its pathogenesis. The signals that initiate T cells to target the eye, which may be of infectious or non-infectious origin, are poorly understood. Experimental autoimmune uveoretinitis (EAU) develops in mice immunized with the endogenous retinal protein interphotoceptor retinoid binding protein (IRBP) 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 antigen-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.

In vertebrates, the innate and adaptive immune systems have evolved seamlessly to protect the host by rapidly responding to danger signals, eliminating pathogens and creating immunological memory as well as immunological tolerance to self. The innate immune system harnesses receptors that recognize conserved pathogen patterns and alongside the more specific recognition systems and memory of adaptive immunity, their interplay is evidenced by respective roles during generation and regulation of immune responses. The hallmark of adaptive immunity which requires engagement of innate immunity is an ability to discriminate between self and non-self (and eventually between pathogen and symbiont) as well as peripheral control mechanisms maintaining immunological health and appropriate responses. Loss of control mechanisms and/or regulation of either the adaptive or the innate immune system lead to autoimmunity and autoinflammation respectively. Although autoimmune pathways have been largely studied to date in the context of development of non-infectious intraocular inflammation, the recruitment and activation of innate immunity is required for full expression of the varied phenotypes of non-infectious uveitis. Since autoimmunity and autoinflammation implicate different molecular pathways, even though some convergence occurs, increasing our understanding of their respective roles in the development of uveitis will highlight treatment targets and influence our understanding of immune mechanisms operative in other retinal diseases. Herein, we extrapolate from the basic mechanisms of activation and control of innate and adaptive immunity to how autoinflammatory and autoimmune pathways contribute to disease development in non-infectious uveitis patients.

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.

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 wildtype controls were given an intraarticular injection of muramyl dipeptide (MDP), peptidoglycan (PG; a metabolite of which is MDP), or palmitoyl-3-cysteineserine-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.The nucleotide-binding oligomerization domain (NOD)-like receptor family plays a critical role in innate immunity. The members of the NOD-like receptor family share many functional and structural characteristics and are thought to cooperate with Toll-like receptors (TLRs) in host defense. While much of the focus has been on the role of TLRs and their involvement in autoinflammatory diseases such as arthritis, the NODlike receptor family is emerging as an important participant in inflammation, perhaps even more so than TLRs in light of the association of the many NOD-like receptor family members with inflammatory diseases (1,2). One NOD-like receptor family member in particular, NOD-2 (also known as NOD-like receptor C2 or