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Systemic lupus erythematosus (SLE) is a complex autoimmune disease with genetic and environmental contributions. Hallmarks of the disease are the appearance of immune complexes (IC) containing autoreactive Abs and TLR-activating nucleic acids, whose deposition in kidney glomeruli is suspected to promote tissue injury and glomerulonephritis (GN). Here, using a mouse model based on the human SLE susceptibility locus TNFAIP3-interacting protein 1 (TNIP1, also known as ABIN1), we investigated the pathogenesis of GN. We found that GN was driven by TLRs but, remarkably, proceeded independently of ICs. Rather, disease in 3 different mouse models and patients with SLE was characterized by glomerular accumulation of patrolling monocytes (PMos), a cell type with an emerging key function in vascular inflammation. Consistent with such function in GN, monocyte-specific deletion of ABIN1 promoted kidney disease, whereas selective elimination of PMos provided protection. In contrast to GN, PMo elimination did not protect from reduced survival or disease symptoms such as IC generation and splenomegaly, suggesting that GN and other inflammatory processes are governed by distinct pathogenic mechanisms. These data identify TLR-activated PMos as the principal component of an intravascular process that contributes to glomerular inflammation and kidney injury.

The immune system uses members of the toll-like receptor (TLR) family to recognize a variety of pathogen- and host-derived molecules in order to initiate immune responses. Although TLR-mediated, pro-inflammatory immune responses are essential for host defense, prolonged and exaggerated activation can result in inflammation pathology that manifests in a variety of diseases. Therefore, small molecule inhibitors of the TLR signaling pathway might have promise as anti-inflammatory drugs. We have previously identified a class of triaryl pyrazole compounds that inhibit TLR signaling by modulation of the protein-protein interactions essential to the pathway. We have now systematically examined the structural features essential for inhibition of this pathway, revealing characteristics of compounds that inhibited all TLRs tested (pan-TLR signaling inhibitors) as well as compounds that selectively inhibited certain TLRs. These findings reveal interesting classes of compounds that could be optimized for particular inflammatory diseases governed by different TLRs.

Toll-like receptors (TLRs) recognize various pathogen- and host tissue-derived molecules and initiate inflammatory immune responses. Exaggerated or prolonged TLR activation, however, can promote etiologically diverse diseases, such as bacterial sepsis, metabolic and autoimmune diseases, or stroke. Despite the apparent medical need, no small molecule drugs against TLR-pathways are clinically available. This may be because of the complex signaling mechanisms of TLRs, which are governed by a series of protein interactions initiated by Toll-interleukine-1 receptor (TIR)-domains found in TLRs and the cytoplasmic adapter proteins TIRAP and MyD88. Activation/oligomerization of TLRs and consecutive oligomerization of MyD88 and/or TIRAP leads to the recruitment of IRAK family members and the ubiquitin ligase TRAF6. Here, we developed a phenotypic drug screening system based on inducible dimerization of TIRAP, MyD88 and TRAF6, that ranked hits according to the position of action in the hierarchy of the TLR signaling cascade. From a bioactive compound library, we identified methyl-piperidino-pyrazole (MPP) as a TLR-specific inhibitor. Structure-activity relationship analysis, quantitative proteomics, protein interaction assays, and cellular thermal shift assays strongly suggest that MPP targets the TIR domain of MyD88. Chemical evolution of the original MPP-scaffold generated compounds with selectivity for distinct TLRs that interfered with specific TIR interactions. Administration of a MPP-analog to mice protected against LPS-mediated TNFα release. These results validate this phenotypic screening approach and implicate the MPP scaffold as starting point for anti-inflammatory drug development.

Psoriasis is a chronic inflammatory skin disease with a clear genetic contribution, characterized by keratinocyte proliferation and immune cell infiltration. Various closely interacting cell types, including innate immune cells, T cells, and keratinocytes, are known to contribute to inflammation. Innate immune cells most likely initiate the inflammatory process by secretion of IL-23. IL-23 mediates expansion of T helper 17 (Th17) cells, whose effector functions, including IL-17A, activate keratinocytes. Keratinocyte activation in turn results in cell proliferation and chemokine expression, the latter of which fuels the inflammatory process through further immune cell recruitment. One question that remains largely unanswered is how genetic susceptibility contributes to this process and, specifically, which cell type causes disease due to psoriasis-specific genetic alterations. Here we describe a mouse model based on the human psoriasis susceptibility locus TNIP1, also referred to as ABIN1, whose gene product is a negative regulator of various inflammatory signaling pathways, including the Toll-like receptor pathway in innate immune cells. We find that Tnip1-deficient mice recapitulate major features of psoriasis on pathological, genomic, and therapeutic levels. Different genetic approaches, including tissue-specific gene deletion and the use of various inflammatory triggers, reveal that Tnip1 controls not only immune cells, but also keratinocyte biology. Loss of Tnip1 in keratinocytes leads to deregulation of IL-17-induced gene expression and exaggerated chemokine production in vitro and overt psoriasis-like inflammation in vivo. Together, the data establish Tnip1 as a critical regulator of IL-17 biology and reveal a causal role of keratinocytes in the pathogenesis of psoriasis.

The adaptor ASC contributes to innate immunity through the assembly of inflammasome complexes that activate the cysteine protease caspase-1. Here we demonstrate that ASC has an inflammasome-independent, cell-intrinsic role in cells of the adaptive immune response. ASC-deficient mice showed defective antigen presentation by dendritic cells (DCs) and lymphocyte migration due to impaired actin polymerization mediated by the small GTPase Rac. Genome-wide analysis showed that ASC, but not the cytoplasmic receptor NLRP3 or caspase-1, controlled the mRNA stability and expression of Dock2, a guanine nucleotide-exchange factor that mediates Rac-dependent signaling in cells of the immune response. Dock2-deficient DCs showed defective antigen uptake similar to that of ASC-deficient cells. Ectopic expression of Dock2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating Dock2-dependent Rac activation and actin polymerization in DCs and lymphocytes.

Caspase-mediated cleavage of the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP1) is a hallmark of apoptosis. However, it remains unclear whether PARP1 is processed during pyroptosis, a specialized cell-death program that occurs upon activation of caspase-1 in inflammasome complexes. In this article, we show that activation of the Nlrp3 and Nlrc4 inflammasomes induces processing of full-length PARP1 into a fragment of 89 kDa in a stimulus-dependent manner. Macrophages deficient for caspase-1 and those lacking the inflammasome adaptors Nlrp3, Nlrc4, and ASC were highly resistant to cleavage, whereas macrophages lacking the downstream inflammasome effector caspase-7 were partially protected. A modest, but statistically significant, reduction in Nlrp3 inflammasome-induced pyroptosis was observed in PARP1 knockout macrophages. Thus, protease-mediated inactivation of PARP1 is a shared feature of apoptotic, necrotic, and pyroptotic cells.

Hepatitis E virus (HEV) infection is an important cause of acute viral hepatitis in several developing countries but has recently been shown to cause chronic hepatitis in immunosuppressed persons. Other hepatotropic viruses that cause chronic infection have been shown to infect peripheral blood mononuclear cells (PBMCs) and to persist in those cells. We therefore decided to look for evidence of replication of HEV in PBMCs obtained from patients with acute hepatitis E, using strand-specific assays for positive and negative HEV RNA. Of the 44 patients with acute hepatitis E during an outbreak in India, including 27 with detectable IgM anti-HEV and 19 with detectable serum HEV RNA, 11 had detectable HEV RNA in their PBMCs. However, of the six PBMC specimens with strong HEV RNA signal, none had detectable negative-strand HEV RNA, a marker of viral replication. These findings indicate the presence of HEV RNA but the absence of its replication in PBMCs from patients with acute hepatitis E.

Contains fulltext : 88045.pdf (Publisher’s version ) (Open Access) Rheumatoid arthritis is an autoimmune disease with 1% prevalence in the industrialized world. The contributions of the inflammasome components Nlrp3, ASC, and caspase-1 in the pathogenesis of collagen-induced arthritis have not been characterized. Here, we show that ASC(-/-) mice were protected from arthritis, whereas Nlrp3(-/-) and caspase-1(-/-) mice were susceptible to collagen-induced arthritis. Unlike Nlrp3(-/-) and caspase-1(-/-) mice, the production of collagen-specific antibodies was abolished in ASC(-/-) mice. This was due to a significantly reduced antigen-specific activation of lymphocytes by ASC(-/-) dendritic cells. Antigen-induced proliferation of purified ASC(-/-) T cells was restored upon incubation with wild type dendritic cells, but not when cultured with ASC(-/-) dendritic cells. Moreover, direct T cell receptor ligation with CD3 and CD28 antibodies induced a potent proliferation of ASC(-/-) T cells, indicating that ASC is specifically required in dendritic cells for antigen-induced T cell activation. Therefore, ASC fulfills a hitherto unrecognized inflammasome-independent role in dendritic cells that is crucial for T cell priming and the induction of antigen-specific cellular and humoral immunity and the onset of collagen-induced arthritis.

Persistent hepatitis E virus (HEV) infection has been reported among solid-organ transplant recipients in nonendemic areas. Such chronic infections have all been related to genotype 3 HEV, which is prevalent in these areas. Whether persistent infection occurs with genotype 1 HEV, prevalent in areas where the infection is hyperendemic, is unclear. We therefore tested sera from renal transplant recipients receiving immunosuppressive agents in India, where genotype 1 HEV infection is endemic, for alanine aminotransferase levels, and presence of IgM and IgG anti-HEV antibodies and HEV RNA. Of the 205 subjects studied [aged 16-65 (median, 38) years, 182 male], 46 (22.4%) had abnormal ALT levels (>40 IU/mL). IgG anti-HEV was detected in 52 (20.5%) and IgM anti-HEV was detected in 14 (6.8%) subjects, including four who had IgG anti-HEV; antibody positivity had no relation with serum ALT or serum creatinine. All the sera tested were negative for HEV RNA. These findings suggest that chronic infection with genotype 1 HEV is infrequent.

Outbreaks of acute hepatitis E, associated with consumption of contaminated drinking water, are frequent in India. Sewage is a major source for contamination of surface water. Data on the presence of hepatitis E virus (HEV) in sewage in India are limited. The aim of this study was to look for the presence of HEV RNA in concentrates of sewage specimens collected from a major open sewage drain in Lucknow, India during August 2004 to July 2006, by the polymerase chain reaction, using primers specific for human HEV (genotype 1) or Indian swine HEV (genotype 4). Of the 192 sewage specimens tested, 79 (41%) showed presence of human HEV RNA. The positivity rate was higher during the second year (52/103 [51%]) than during the first year (27/89 [30%]; P = 0.005). The seasonal pattern of HEV RNA positivity was as follows: winter months (November to February): 28 of 61 (46%); summer months (March to June): 36 of 66 (55%); and, monsoon months (July to October) 15 of 65 (23%). There was no reported outbreak of hepatitis E in the city during the study period. Swine HEV RNA was not detected in any of the 69 specimens tested. Repeat testing confirmed the reproducibility of the results. In addition, nucleic acid sequencing of six sewage isolates showed that these belonged to HEV genotype 1. The study suggests that HEV infection and fecal viral excretion may be common in HEV-endemic regions throughout the year even during non-epidemic periods.