TAG, a splice variant of the adaptor TRAM, negatively regulates the adaptor MyD88-independent TLR4 pathway.

Toll-like receptor 4 (TLR4) signals the induction of transcription factor IRF3-dependent genes from the early endosome via the adaptor TRAM. Here we report a splice variant of TRAM, TAG ('TRAM adaptor with GOLD domain'), which has a Golgi dynamics domain coupled to TRAM's Toll-interleukin 1 receptor domain. After stimulation with lipopolysaccharide, TRAM and TAG localized to late endosomes positive for the GTPase Rab7a. TAG inhibited activation of IRF3 by lipopolysaccharide. Knockdown of TAG with small interfering RNA enhanced induction of the chemokine CCL5 (RANTES), but not of interleukin 8, by lipopolysaccharide in human peripheral blood mononuclear cells. TAG displaced the adaptor TRIF from TRAM. TAG is therefore an example of a specific inhibitor of the adaptor MyD88-independent pathway activated by TLR4. Targeting TAG could be useful in the effort to boost the immunostimulatory effect of TLR4 without causing unwanted inflammation.

Functional and genetic evidence that the Mal/TIRAP allele variant 180L has been selected by providing protection against septic shock.

Adequate responses by our innate immune system toward invading pathogens were of vital importance for surviving infections, especially before the antibiotic era. Recently, a polymorphism in Mal (Ser180Leu, TIRAP rs8177374), an important adaptor protein downstream of the Toll-like receptor (TLR) 2 and 4 pathways, has been described to provide protection against a broad range of infectious pathogens. We assessed the functional effects of this polymorphism in human experimental endotoxemia, and we demonstrate that individuals bearing the TIRAP 180L allele display an increased, innate immune response to TLR4 and TLR2 ligands, but not to TLR9 stimulation. This phenotype has been related to an increased resistance to infection. However, an overshoot in the release of proinflammatory cytokines by TIRAP 180L homozygous individuals suggests a scenario of balanced evolution. We have also investigated the worldwide distribution of the Ser180Leu polymorphism in 14 populations around the globe to correlate the genetic makeup of TIRAP with the local infectious pressures. Based on the immunological, clinical, and genetic data, we propose that this mutation might have been selected in West Eurasia during the early settlement of this region after the out-of-Africa migration of modern Homo sapiens. This combination of functional and genetic data provides unique insights to our understanding of the pathogenesis of sepsis.

The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation.

The Krebs cycle-derived metabolite itaconate is highly upregulated in inflammatory macrophages and exerts immunomodulatory effects through cysteine modifications on target proteins. The NLRP3 inflammasome, which cleaves IL-1ß, IL-18, and gasdermin D, must be tightly regulated to avoid excessive inflammation. Here we provide evidence that itaconate modifies NLRP3 and inhibits inflammasome activation. Itaconate and its derivative, 4-octyl itaconate (4-OI), inhibited NLRP3 inflammasome activation, but not AIM2 or NLRC4. Conversely, NLRP3 activation was increased in itaconate-depleted Irg1-/- macrophages. 4-OI inhibited the interaction between NLRP3 and NEK7, a key step in the activation process, and "dicarboxypropylated" C548 on NLRP3. Furthermore, 4-OI inhibited NLRP3-dependent IL-1ß release from PBMCs isolated from cryopyrin-associated periodic syndrome (CAPS) patients, and reduced inflammation in an in vivo model of urate-induced peritonitis. Our results identify itaconate as an endogenous metabolic regulator of the NLRP3 inflammasome and describe a process that may be exploited therapeutically to alleviate inflammation in NLRP3-driven disorders.

Caspase-11 promotes allergic airway inflammation.

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E2 (PGE2) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE2 suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE2 is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE2 production with indomethacin enhances, whereas the prostaglandin E1 analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE2 as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma.

Genetic variation of innate immune genes in HIV-infected african patients with or without oropharyngeal candidiasis.

BACKGROUND: The occurrence of oropharyngeal candidiasis (OPC) in combination with HIV disease progression is a very common phenomenon. However, not all HIV-infected patients develop OPC, even when they progress to low CD4 T-cell counts. Because T-cell immunity is defective in AIDS, the innate defence mechanisms are likely to have a central role in antifungal immunity in these patients. We investigated whether genetic variations in the innate immune genes DECTIN-1, TLR2, TLR4, TIRAP, and CASPASE-12 are associated with the presence of OPC in HIV-infected subjects from East Africa. METHODS: A total of 225 HIV patients were genotyped for several single nucleotide polymorphisms (SNPs), and this was correlated with the occurrence of OPC in these patients. In addition, primary immune cells obtained from individuals with different genotypes were stimulated with Candida albicans, and cytokine production was measured. RESULTS: The analysis revealed that no significant differences in the polymorphism frequencies could be observed, although a tendency toward a protective effect on OPC of the DECTIN-1 I223S SNP was apparent. Furthermore, interferon gamma production capacity was markedly lower in cells bearing the DECTIN-1 SNP I223S. It could also be demonstrated that the 223S mutated form of the DECTIN-1 gene exhibits a lower capacity to bind zymosan. CONCLUSIONS: These data demonstrate that common polymorphisms of TLR2, TLR4, TIRAP, and CASPASE-12 do not influence susceptibility to OPC in HIV-infected patients in East Africa but suggest an immunomodulatory effect of the I223S SNP on dectin-1 function and possibly the susceptibility to OPC in HIV patients.

NF-kappaB activation by the Toll-IL-1 receptor domain protein MyD88 adapter-like is regulated by caspase-1.

Toll-like receptors (TLRs)-2 and -4 are important proteins in innate immunity, recognizing microbial products and eliciting host defense responses. Both use the adapter proteins MyD88 and MyD88 adapter-like (Mal) to activate signaling pathways. Here we report that Mal but not MyD88 interacts with caspase-1, the enzyme that processes the precursors of the proinflammatory cytokines IL-1beta and IL-18. The interaction was found in a yeast two-hybrid screen and was confirmed by reciprocal GST pull-downs and coimmunoprecipitation of endogenous proteins. We were unable to implicate Mal in regulating caspase-1 activation. However, we found that Mal was cleaved by caspase-1 and that inhibition of caspase-1 activity blocked TLR2- and TLR4-mediated NF-kappaB and p38 MAP kinase activation but not IL-1 or TLR7 signaling, which are Mal independent. These responses, and the induction of TNF, were also attenuated in caspase-1-deficient cells. Finally, unlike wild-type Mal, a mutant Mal, which was not cleaved by caspase-1, was unable to signal and acted as a dominant negative inhibitor of TLR2 and TLR4 signaling. Our study therefore reveals a role for caspase-1 in the regulation of TLR2 and TLR4 signaling pathways via an effect on Mal. This functional interaction reveals an important aspect of the coordination between TLRs and caspase-1 during the innate response to pathogens.