Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1

Coordination among multiple signaling pathways ensures an appropriate immune response, where a signaling pathway may impair or augment another signaling pathway. Here, we report a negative feedback regulation of signaling through the key innate immune mediator MyD88 by inflammasome-activated caspase-1. NLRP3 inflammasome activation impaired agonist- or infection-induced TLR signaling and cytokine production through the proteolytic cleavage of MyD88 by caspase-1. Site-specific mutagenesis was used to identify caspase-1 cleavage site within MyD88 intermediary segment. Different cleavage site location within MyD88 defined the functional consequences of MyD88 cleavage between mouse and human cells. LPS/monosodium urate–induced mouse inflammation model corroborated the physiological role of this mechanism of regulation, that could be reversed by chemical inhibition of NLRP3. While Toll/interleukin-1 receptor (TIR) domain released by MyD88 cleavage additionally contributed to the inhibition of signaling, Waldenström’s macroglobulinemia associated MyD88L265P mutation is able to evade the caspase-1-mediated inhibition of MyD88 signaling through the ability of its TIRL265P domain to recruit full length MyD88 and facilitate signaling. The characterization of this mechanism reveals an additional layer of innate immunity regulation.

Extracellular HMGB1 Induced Glomerular Endothelial Cell Injury via TLR4/MyD88 Signaling Pathway in Lupus Nephritis

Previously, our study showed that HMGB1 was significantly elevated in the blood and located in the glomerular endothelium in LN patients. But whether extracellular HMGB1 is involved in the injury of glomerular endothelial cells (GECs) in LN still needs further investigation. Firstly, we detected the levels of SDC-1, VCAM-1, and proteinuria in LN patients and MRL/lpr mice and analyzed their correlations. Then, HMGB1 and TLR4/MyD88 were inhibited to observe the shedding of glycocalyx and injury of GECs in vivo and in vitro. Our results showed that HRGEC injury and SDC-1 shedding played an important role in the increase of permeability and proteinuria formation in LN. Additionally, inhibition of extracellular HMGB1 and/or downstream TLR4/MyD88/NF-κB/p65 signaling pathway also alleviated GEC monolayer permeability, reduced the shedding of the glomerular endothelial glycocalyx, improved the intercellular tight junction and cytoskeletal arrangement, and downregulated the NO level and VCAM-1 expression. These results suggested that extracellular HMGB1 might involve in GEC injury by activating the TLR4/MyD88 signaling pathway in LN, which provided novel insights and potential therapeutic target for the treatment of lupus nephritis.

Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here we show that in COVID-19 patients, circulating plasmacytoid dendritic cells (pDCs) decline early after symptom onset and this correlated with COVID-19 disease severity. This transient depletion coincides with decreased expression of antiviral type I IFNα and the systemic inflammatory cytokines CXCL10 and IL-6. Importantly, COVID-19 disease severity correlated with decreased pDC frequency in peripheral blood. Using an in vitro stem cell-based human pDC model, we demonstrate that pDCs directly sense SARS-CoV-2 and in response produce multiple antiviral (IFNα and IFNλ1) and inflammatory (IL-6, IL-8, CXCL10) cytokines. This immune response is sufficient to protect epithelial cells from de novo SARS-CoV-2 infection. Targeted deletion of specific sensing pathways identified TLR7-MyD88 signaling as being crucial for production of the antiviral IFNs, whereas TLR2 is responsible for the inflammatory IL-6 response. Surprisingly, we found that SARS-CoV-2 engages the neuropilin-1 receptor on pDCs to mitigate the antiviral IFNs but not the IL-6 response. These results demonstrate distinct sensing pathways used by pDCs to elicit antiviral vs. immunopathological responses to SARS-CoV-2 and suggest that targeting neuropilin-1 on pDCs may be clinically relevant for mounting TLR7-mediated antiviral protection.

B Cell Recognition of Candida albicans Hyphae via TLR 2 Promotes IgG1 and IL-6 Secretion for TH17 Differentiation

Candida albicans is usually a benign member of the human gut microbiota, but can become pathogenic under certain circumstances, for example in an immunocompromised host. The innate immune system, in particular neutrophils and macrophages, constitutes a crucial first line of defense against fungal invasion, however adaptive immunity may provide long term protection and thus allow vaccination of at risk patients. While TH1 and TH17 cells are important for antifungal responses, the role of B cells and antibodies in protection from C. albicans infection is less well defined. In this study, we show that C. albicans hyphae but not yeast, as well as fungal cell wall components, directly activate B cells via MyD88 signaling triggered by Toll- like receptor 2, leading to increased IgG1 production. While Dectin-1 signals and specific recognition by the B cell receptor are dispensable for B cell activation in this system, TLR2/MyD88 signals cooperate with CD40 signals in promoting B cell activation. Importantly, recognition of C. albicans via MyD88 signaling is also essential for induction of IL-6 secretion by B cells, which promotes TH17 polarization in T-B cell coculture experiments. B cells may thus be activated directly by C. albicans in its invasive form, leading to production of antibodies and T cell help for fungal clearance.

Blockade of Myd88 Signaling by Novel MyD88 Inhibitor Prevents Colitis-associated Colorectal Cancer From Development by Impairing Myeloid-Derived Suppressor Cells

Background: Myeloid-derived suppressor cells (MDSCs) are currently confirmed to be due to escaping the host immune system of tumor by developing a highly suppressive environment. MDSC elimination provides an ideal target for tumor immunotherapy. However, little is still known about the exact molecular mechanism behind MDSC-mediated tumor immune evasion. Methods: We used a novel myeloid differentiation factor 88 (MyD88) inhibitor TJ-M2010-5 to block the MyD88 signaling and prevent colitis-associated colorectal cancer (CAC) development in mice. Results: We showed that CAC growth inhibition was involved in diminished MDSC generation, expansion, and suppressive function, and that the MDSC-mediated immune escape was dependent on the MyD88 signaling pathway activation. The MyD88 inhibitor treatment decreased accumulation of CD11b+Gr1+ MDSC in mice with CAC, thereby reducing cytokine (GM-CSF, G-CSF, IL-1β, IL-6 and TGF-β) secretion associated with MDSC accumulation, and reducing expression of molecules (iNOS, Arg-1 and IDO) associated with the suppressive capacity of MDSC. In addition, the MyD88 inhibitor treatment reduced the differentiation of MDSC from myeloid cells and the suppressive capacity of MDSC on proliferation of activated CD4+ T cell in vitro. Conclusion: Therefore, MDSC is a primary cellular target of the novel MyD88 inhibitor during CAC development. Our findings prove that MyD88 signaling is involved in the regulation of the immunosuppressive functions of MDSC. Our novel MyD88 inhibitor, TJ-M2010-5, is a new and effective agent that modulates MyD88 signaling to overcome MDSC suppressive functions and to enable the development of a successful antitumor immunotherapy.

Cytotoxic CD4+ T cells driven by T-cell intrinsic IL-18R/MyD88 signaling predominantly infiltrate Trypanosoma cruzi-infected hearts

Increasing attention has been directed to cytotoxic CD4+ T cells (CD4CTLs) in different pathologies, both in humans and mice. The impact of CD4CTLs in immunity and the mechanisms controlling their generation, however, remain poorly understood. Here, for the first time, we showed that CD4CTLs abundantly differentiate during mouse infection with an intracellular parasite. CD4CTLs appear in the spleen in parallel to Th1 cells, display pathogen-derived peptide-specific cytotoxicity against antigen-presenting cells and express immunoregulatory and/or exhaustion markers. We demonstrated that CD4CTL absolute numbers and activity are severely reduced in both Myd88-/- and Il18ra-/- mice. Of note, the infection of mixed-bone marrow chimeras revealed that WT, but not Myd88-/-, cells transcribe the CD4CTL gene signature and that Il18ra-/-CD4+ phenocopy Myd88-/-CD4+ T cells. Moreover, the adoptive transfer of WT CD4+GzB+ T cells to susceptible Il18ra-/- mice increased their survival. Importantly, cells expressing the CD4CTL phenotype predominate among CD4+ T cells infiltrating the infected cardiac tissue, are increased in the circulation of Chagas patients and their frequency correlates with severe cardiomyopathy. Our findings describe CD4CTLs as a major player in immune response to a relevant human pathogen and disclose T-cell intrinsic IL-18R/MyD88 signaling as a key pathway controlling the magnitude of the CD4CTL response.

Role of TLR4/MyD88 Signaling Pathway in the Occurrence and Development of Uremia-Induced Myocardial Hypertrophy and Possible Mechanism

The morbidity and mortality of cardiovascular disease (CVD) are relatively high. Studies have shown that most patients with chronic kidney disease (CKD) die from cardiovascular complications. Clinically, the pathophysiological state in which heart disease and kidney disease are causal and influence each other is called cardiorenal syndrome (CRS). Myocardial hypertrophy is the key stage of the heart structure changing from reversible to irreversible. It is an important pathophysiological basis for heart failure. Therefore, this study intends to start with the end-stage uremic phase of CKD to construct an animal model of uremia in rats to study the relationship between uremia, TLR4/MyD88 signaling pathway, and myocardial hypertrophy. The results showed that the uremic rats showed slow weight gain and were thinner. At 12 weeks (w), the serum creatinine and urea nitrogen of the uremic rats increased, and the global hypertrophy index increased. Detecting the expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor (MyD88) in blood samples of rats, we found that the expression of TLR4 and MyD88 increased at 12 w in the uremia group; pathological observation showed that at 4 weeks of uremia model rats, renal tissue compensatory hypertrophy, renal fibrous membrane proliferation, renal parenchyma atrophy, a large number of fibrous proliferation and inflammatory cell infiltration in the interstitium, and protein casts in the renal tubules were observed. Myocardial cells were obviously hypertrophy and disordered. At 12 w, renal tubules were obviously expanded, the epithelium was flat, the brush border disappeared, and the interstitial fibrous connective tissue of the myocardial tissue was proliferated. The detection of TLR4 and MyD88 in kidney tissue and myocardial tissue revealed that the positive expression of TLR4 and MyD88 gradually increased over time. Therefore, the final result of the study is that uremia can gradually lead to myocardial hypertrophy and TLR4 and MyD88 are highly expressed in serum, kidney, and myocardial tissues of uremic rats, suggesting that TLR4 and MyD88 may be related to the degree of uremic disease and the myocardium caused by it. Hypertrophy is related.

Maternal B Cell-Intrinsic MyD88 Signaling Mediates LPS-Driven Intrauterine Fetal Death

Immunological networks balance tolerance towards paternal alloantigens during pregnancy with normal immune response to pathogens. Subclinical infections can impact this balance and lead to preterm birth or even intrauterine fetal death (IUFD). We recently showed that loss of maternal B cells renders murine fetuses susceptible to IUFD after LPS exposure. Since the signaling pathway involved in this B-cell mediated response remains unclear, we aimed to understand the participation of MyD88 in this response using B-cell-specific MyD88-deficient (BMyD88-/-) mice. B cells isolated from wild-type (WT), BMyD88-/-, CD19-/- and MyD88-/- dams on gestational day (gd) 10 responded differently to LPS concerning cytokine secretion. In vivo LPS challenge on gd 10 provoked IUFD in CD19-/- mothers with functional MyD88, while fetuses from BMyD88-/- and MyD88-/- mice were protected. These outcomes were associated with altered cytokine levels in the maternal serum and changes in CD4+ T-cell responses. Overall, the loss of MyD88 signaling in maternal B cells prevents the activation of cytokine release that leads to IUFD. Thus, while MyD88 signaling in maternal B cells protects the mother from infection, it ultimately kills the fetus. Understanding the cellular mechanisms underlying infection-driven pregnancy complications is the first step to designing powerful therapeutic strategies in the future.