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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.

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.