Human Mincle Binds to Cholesterol Crystals and Triggers Innate Immune Responses

C-type lectin receptors (CLRs) are an emerging family of pattern recognition receptors that recognizes pathogens or damaged tissue to trigger innate immune responses. However, endogenous ligands for CLRs are not fully understood. In this study, we sought to identify an endogenous ligand(s) for human macrophage-inducible C-type lectin (hMincle). A particular fraction of lipid extracts from liver selectively activated reporter cells expressing hMincle. MS analysis determined the chemical structure of the active component as cholesterol. Purified cholesterol in plate-coated and crystalized forms activates reporter cells expressing hMincle but not murine Mincle (mMincle). Cholesterol crystals are known to activate immune cells and induce inflammatory responses through lysosomal damage. However, direct innate immune receptors for cholesterol crystals have not been identified. Murine macrophages transfected with hMincle responded to cholesterol crystals by producing pro-inflammatory cytokines. Human dendritic cells expressed a set of inflammatory genes in response to cholesterol crystals, and this was inhibited by anti-human Mincle. Importantly, other related CLRs did not bind cholesterol crystals, whereas other steroids were not recognized by hMincle. These results suggest that cholesterol crystals are an endogenous ligand for hMincle and that they activate innate immune responses.     

Soluble Interleukin-6 Receptor-Mediated Innate Immune Response to DNA and RNA Viruses

The interleukin-6 (IL-6) receptor, which exists as membrane-bound and soluble forms, plays critical roles in the immune response. The soluble IL-6 receptor (sIL6R) has been identified as a potential therapeutic target for preventing coronary heart disease. However, little is known about the role of this receptor during viral infection. In this study, we show that sIL6R, but not IL-6, is induced by viral infection via the cyclooxygenase-2 pathway. Interestingly, sIL6R, but not IL-6, exhibited extensive antiviral activity against DNA and RNA viruses, including hepatitis B virus, influenza virus, human enterovirus 71, and vesicular stomatitis virus. No synergistic effects on antiviral action were observed by combining sIL6R and IL-6. Furthermore, sIL6R mediated antiviral action via the p28 pathway and induced alpha interferon (IFN-α) by promoting the nuclear translocation of IFN regulatory factor 3 (IRF3) and NF-κB, which led to the activation of downstream IFN effectors, including 2′,5′-oligoadenylate synthetase (OAS), double-stranded RNA-dependent protein kinase (PKR), and myxovirus resistance protein (Mx). Thus, our results demonstrate that sIL6R, but not IL-6, plays an important role in the host antiviral response.     

Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and β-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.     

Retinoid-X-Receptors (α/β) in Melanocytes Modulate Innate Immune Responses and Differentially Regulate Cell Survival following UV Irradiation

Understanding the molecular mechanisms of ultraviolet (UV) induced melanoma formation is becoming crucial with more reported cases each year. Expression of type II nuclear receptor Retinoid-X-Receptor α (RXRα) is lost during melanoma progression in humans. Here, we observed that in mice with melanocyte-specific ablation of RXRα and RXRβ, melanocytes attract fewer IFN-γ secreting immune cells than in wild-type mice following acute UVR exposure, via altered expression of several chemoattractive and chemorepulsive chemokines/cytokines. Reduced IFN-γ in the microenvironment alters UVR-induced apoptosis, and due to this, the survival of surrounding dermal fibroblasts is significantly decreased in mice lacking RXRα/β. Interestingly, post-UVR survival of the melanocytes themselves is enhanced in the absence of RXRα/β. Loss of RXRs α/β specifically in the melanocytes results in an endogenous shift in homeostasis of pro- and anti-apoptotic genes in these cells and enhances their survival compared to the wild type melanocytes. Therefore, RXRs modulate post-UVR survival of dermal fibroblasts in a “non-cell autonomous” manner, underscoring their role in immune surveillance, while independently mediating post-UVR melanocyte survival in a “cell autonomous” manner. Our results emphasize a novel immunomodulatory role of melanocytes in controlling survival of neighboring cell types besides controlling their own, and identifies RXRs as potential targets for therapy against UV induced melanoma.     

Endoplasmic Reticulum Aminopeptidase-1 Functions Regulate Key Aspects of the Innate Immune Response

Endoplasmic reticulum aminopeptidase-1 (ERAP1) is a multifunctional, ubiquitously expressed enzyme whose peptide-trimming role during antigen processing for presentation by MHC I molecules is well established, however, a role for ERAP1 in modulating global innate immune responses has not been described to date. Here we demonstrate that, relative to wild type mice, mice lacking ERAP1 exhibit exaggerated innate immune responses early during pathogen recognition, as characterized by increased activation of splenic and hepatic NK and NKT cells and enhanced production of pro-inflammatory cytokines such as IL12 and MCP1. Our data also revealed that ERAP1 is playing a critical role in NK cell development and function. We observed higher frequencies of terminally matured NK cells, as well as higher frequencies of licensed NK cells (expressing the Ly49C and Ly49I receptors) in ERAP1-KO mice, results that positively correlated with an enhanced NK activation and IFNγ production by ERAP1-KO mice challenged with pro-inflammatory stimuli. Furthermore, during pathogen recognition, ERAP1 regulates IL12 production by CD11c⁺ DCs specifically, with increases in IL12 production positively correlated with an increased phagocytic activity of splenic DCs and macrophages. Collectively, our results demonstrate a previously unrecognized, more central role for the ERAP1 protein in modulating several aspects of both the development of the innate immune system, and its responses during the initial stages of pathogen recognition. Such a role may explain why ERAP1 has been implicated by GWAS in the pathogenesis of autoimmune diseases that may be precipitated by aberrant responses to pathogen encounters.     

IAPs Regulate Distinct Innate Immune Pathways to Co-ordinate the Response to Bacterial Peptidoglycans

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斑马鱼TAK1正向调控IRF7介导的天然免疫反应

在鱼类中关于转化生长因子β-激活激酶1(TAK1)在天然免疫反应中的功能研究较少。为了探究TAK1在斑马鱼天然免疫中的功能,本文克隆并获得了一种斑马鱼tak1剪接异构体(Drtak1),其开放阅读框含有1737个核苷酸,编码578个氨基酸,其中包括N端的丝氨酸/苏氨酸蛋白激酶结构域和C端的卷曲螺旋结构部。通过免疫荧光试验,证实DrTAK1是一种胞质蛋白。双荧光素酶报告试验显现在EPC细胞中单转DrTAK1不能诱导IFN的产生,但与IRF7共转时能显著提高其诱导干扰素启动子表达的能力。本文研究结果首次在斑马鱼中发现TAK1能正向调控IRF7介导的天然免疫反应,为后续DrTAK1功能研究奠定了基础。     

Nonlinear Oscillations in Models of Immune Responses to Persistent Viruses

This paper compares several population models for HIV dynamics. The effect of perfect or nonperfect drugs proposed by Ho, Perelson, and others are considered in the more complicated models of Nowak and Bangham for immune responses to persistent viruses. It is pointed out that two models proposed by Nowak and Bangham have different dynamical behaviors. One of them can have sustained oscillations via the Hopf bifurcation and the other does not. To overcome the difficulty in symbolic computations for complicated systems, we use parameter transforms and introduce a semisymbolic method. This method is very efficient in finding Hopf bifurcation parameters and can be used for other models.     

MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses

CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4⁺ and CD8⁺ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.     

Inhibition of Innate Immune Responses Is Key to Pathogenesis by Arenaviruses

Mammalian arenaviruses are zoonotic viruses that cause asymptomatic, persistent infections in their rodent hosts but can lead to severe and lethal hemorrhagic fever with bleeding and multiorgan failure in human patients. Lassa virus (LASV), for example, is endemic in several West African countries, where it is responsible for an estimated 500,000 infections and 5,000 deaths annually. There are currently no FDA-licensed therapeutics or vaccines available to combat arenavirus infection. A hallmark of arenavirus infection (e.g., LASV) is general immunosuppression that contributes to high viremia. Here, we discuss the early host immune responses to arenavirus infection and the recently discovered molecular mechanisms that enable pathogenic viruses to suppress host immune recognition and to contribute to the high degree of virulence. We also directly compare the innate immune evasion mechanisms between arenaviruses and other hemorrhagic fever-causing viruses, such as Ebola, Marburg, Dengue, and hantaviruses. A better understanding of the immunosuppression and immune evasion strategies of these deadly viruses may guide the development of novel preventative and therapeutic options.     

NFATc1 Mediates Toll-Like Receptor-Independent Innate Immune Responses during Trypanosoma cruzi Infection

Host defense against the intracellular protozoan parasite Trypanosoma cruzi depends on Toll-like receptor (TLR)-dependent innate immune responses. Recent studies also suggest the presence of TLR-independent responses to several microorganisms, such as viruses, bacteria, and fungi. However, the TLR-independent responses to protozoa remain unclear. Here, we demonstrate a novel TLR-independent innate response pathway to T. cruzi. Myd88 ^−/− Trif ^−/− mice lacking TLR signaling showed normal T. cruzi-induced Th1 responses and maturation of dendritic cells (DCs), despite high sensitivity to the infection. IFN-γ was normally induced in T. cruzi-infected Myd88 ^−/− Trif ^−/− innate immune cells, and further was responsible for the TLR-independent Th1 responses and DC maturation after T. cruzi infection. T. cruzi infection induced elevation of the intracellular Ca²⁺ level. Furthermore, T. cruzi-induced IFN-γ expression was blocked by inhibition of Ca²⁺ signaling. NFATc1, which plays a pivotal role in Ca²⁺ signaling in lymphocytes, was activated in T. cruzi-infected Myd88^−/−Trif^−/− innate immune cells. T. cruzi-infected Nfatc1 ^−/− fetal liver DCs were impaired in IFN-γ production and DC maturation. These results demonstrate that NFATc1 mediates TLR-independent innate immune responses in T. cruzi infection.     

Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection

Background

Burkholderia pseudomallei, a facultative intracellular pathogen, causes systemic infection in humans with high mortality especially when infection occurs through an infectious aerosol. Previous studies indicated that the epithelial cells in the lung are an active participant in host immunity. In this study, we aimed to investigate the innate immune responses of lung epithelial cells against B. pseudomallei.

Methodology and Principal Findings

Using a murine lung epithelial cell line, primary lung epithelial cells and an inhalational murine infection model, we characterized the types of innate immunity proteins and peptides produced upon B. pseudomallei infection. Among a wide panel of immune components studied, increased levels of major pro-inflammatory cytokines IL-6 and TNFα, chemokine MCP-1, and up-regulation of secretory leukocyte protease inhibitor (SLPI) and chemokine (C-C motif) ligand 20 (CCL20) were observed. Inhibition assays using specific inhibitors suggested that NF-κB and p38 MAPK pathways were responsible for these B. pseudomallei-induced antimicrobial peptides.

Conclusions

Our findings indicate that the respiratory epithelial cells, which form the majority of the cells lining the epithelial tract and the lung, have important roles in the innate immune response against B. pseudomallei infection.     

Proteomic Profiling of Endothelial Invasion Revealed Receptor for Activated C Kinase 1 (RACK1) Complexed with Vimentin to Regulate Focal Adhesion Kinase (FAK)

Angiogenesis is critical for many physiological and pathological processes. To identify molecules relevant to angiogenesis, we performed a proteomic screen comparing invading versus non-invading endothelial cells in three-dimensional collagen matrices. We found up-regulated levels of receptor for activated C kinase 1 (RACK1) and the intermediate filament protein vimentin that correlated with increased endothelial cell invasion. Because both RACK1 and vimentin have been linked to focal adhesion kinase (FAK), we investigated whether this pathway regulated invasion. RACK1 depletion reduced invasion responses, and this was associated with attenuated activation of FAK. Knockdown of vimentin significantly decreased levels of phosphorylated and total FAK. Treatment with a pharmacological inhibitor of FAK dose-dependently reduced invasion, indicating a crucial role for FAK activity during invasion. Because RACK1 and vimentin were both up-regulated with sphingosine 1-phosphate treatment, required for invasion, and regulated FAK, we tested whether they complexed together. RACK1 complexed with vimentin, and growth factors enhanced this interaction. In addition, RACK1, vimentin, and FAK formed an intermolecular complex in invading endothelial cultures in three dimensions in response to stimulation by sphingosine 1-phosphate and growth factors. Moreover, depletion of RACK1 decreased the association of vimentin and FAK, suggesting that RACK1 was required for stabilizing vimentin-FAK interactions during sprouting. Silencing of vimentin and RACK1 decreased cell adhesion and focal contact formation. Taken together, these results demonstrate that proangiogenic signals converge to enhance expression and association of RACK1 and vimentin, which regulated FAK, resulting in successful endothelial sprout formation in three-dimensional collagen matrices.     

Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.