Human TLR4 and noncanonical inflammasome differ in their ability to respond to distinct lipid A variants

Detection of Gram-negative bacterial lipid A by the extracellular sensor, MD-2/TLR4 or the intracellular inflammasome sensors, CASP4 and CASP5, induces robust inflammatory responses. The chemical structure of lipid A, specifically the phosphorylation and acylation state, varies across and within bacterial species, potentially allowing pathogens to evade or suppress host immunity. Currently, it is not clear how distinct alterations in the phosphorylation or acylation state of lipid A affect both human TLR4 and CASP4/5 activation. Using a panel of engineered lipooligosaccharides (LOS) derived from Yersinia pestis with defined lipid A structures that vary in their acylation or phosphorylation state, we identified that differences in phosphorylation state did not affect TLR4 or CASP4/5 activation. However, the acylation state differentially impacted TLR4 and CASP4/5 activation. Specifically, all of the examined tetra-, penta-, and hexa-acylated LOS variants activated CASP4/5-dependent responses, whereas TLR4 responded to penta- and hexa-acylated LOS but did not respond to tetra-acylated LOS or penta-acylated LOS lacking the secondary acyl chain at the 3' position. As expected, lipid A alone was sufficient for TLR4 activation; however, human macrophages required both lipid A and the core oligosaccharide to mount a robust CASP4/5 inflammasome response. Our findings show that human TLR4 and CASP4/5 detect both shared and non-overlapping LOS/lipid A structures, which enables the innate immune system to recognize a wider range of bacterial LOS/lipid A, thereby constraining the ability of pathogens to evade innate immune detection.

Regulation of TLR4 signaling through the TRAF6/sNASP axis by reversible phosphorylation mediated by CK2 and PP4

Recognition of invading pathogens by Toll-like receptors (TLRs) activates innate immunity through signaling pathways that involved multiple protein kinases and phosphatases. We previously demonstrated that somatic nuclear autoantigenic sperm protein (sNASP) binds to TNF receptor–associated factor 6 (TRAF6) in the resting state. Upon TLR4 activation, a signaling complex consisting of TRAF6, sNASP, interleukin (IL)-1 receptor–associated kinase 4, and casein kinase 2 (CK2) is formed. CK2 then phosphorylates sNASP to release phospho-sNASP (p-sNASP) from TRAF6, initiating downstream signaling pathways. Here, we showed that protein phosphatase 4 (PP4) is the specific sNASP phosphatase that negatively regulates TLR4-induced TRAF6 activation and its downstream signaling pathway. Mechanistically, PP4 is directly recruited by phosphorylated sNASP to dephosphorylate p-sNASP to terminate TRAF6 activation. Ectopic expression of PP4 specifically inhibited sNASP-dependent proinflammatory cytokine production and downstream signaling following bacterial lipopolysaccharide (LPS) treatment, whereas silencing PP4 had the opposite effect. Primary macrophages and mice infected with recombinant adenovirus carrying a gene encoding PP4 (Ad-PP4) showed significant reduction in IL-6 and TNF-α production. Survival of Ad-PP4–infected mice was markedly increased due to a better ability to clear bacteria in a sepsis model. These results indicate that the serine/threonine phosphatase PP4 functions as a negative regulator of innate immunity by regulating the binding of sNASP to TRAF6.

SARS-CoV-2 Spike Glycoprotein S1 Induces Neuroinflammation in BV-2 Microglia

In addition to respiratory complications produced by SARS‐CoV‐2, accumulating evidence suggests that some neurological symptoms are associated with the disease caused by this coronavirus. In this study, we investigated the effects of the SARS‐CoV‐2 spike protein S1 stimulation on neuroinflammation in BV-2 microglia. Analyses of culture supernatants revealed an increase in the production of TNF-α, IL-6, IL-1β and iNOS/NO. S1 also increased protein levels of phospho-p65 and phospho-IκBα, as well as enhanced DNA binding and transcriptional activity of NF-κB. These effects of the protein were blocked in the presence of BAY11-7082 (1 µM). Exposure of S1 to BV-2 microglia also increased the protein levels of NLRP3 inflammasome and enhanced caspase-1 activity. Increased protein levels of p38 MAPK was observed in BV-2 microglia stimulated with the spike protein S1 (100 ng/ml), an action that was reduced in the presence of SKF 86,002 (1 µM). Results of immunofluorescence microscopy showed an increase in TLR4 protein expression in S1-stimulated BV-2 microglia. Furthermore, pharmacological inhibition with TAK 242 (1 µM) and transfection with TLR4 small interfering RNA resulted in significant reduction in TNF-α and IL-6 production in S1-stimulated BV-2 microglia. These results have provided the first evidence demonstrating S1-induced neuroinflammation in BV-2 microglia. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-κB and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2.

TLR 4 / MD2 Complex inhibitors potential therapeutic prospective for asthma and asthma exacerbation

The feedback of our body to foreign particles is initially initiated by the innate immunity where the likes Toll- Like Receptors (TLRs), play an important role in the identification process. The identification of the trouble through the interaction of the receptor is the initial step to propagate the protective agents throughout the body. These interact action with the attacking pathogens like viruses and bacteria (PAMPs, pathogen-associated molecular patterns) or substances produced cells of the body which are injured (DAMPs, danger-associated molecular patterns) helping in the identification. However, the innate and adaptive immunity of both of them gets stimulated through the release of various substances such as cytokines and chemokines due to TLR activation. In part of the progress, many reports have been identified as the activation of the TLR4 complex, a receptor of the innate immune system that may underpin the pathophysiology of many human diseases including asthma, cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory and psychiatric disorders. Substances of synthetic and natural origin have been discussed here and have been found to produce the anti-inflammatory effect by targeting pathways of TLR4 activation. In the present review, our focus is to develop a conclusion about the TL4 complex inhibition for the betterment of asthma patients and combine the reports about the progressing of TLR signalling pathway modulators.

TLR4-interactor with leucine-rich repeats (TRIL) is involved in diet-induced hypothalamic inflammation

Obesity and high-fat diet (HFD) consumption result in hypothalamic inflammation and metabolic dysfunction. While the TLR4 activation by dietary fats is a well-characterized pathway involved in the neuronal and glial inflammation, the role of its accessory proteins in diet-induced hypothalamic inflammation remains unknown. Here, we demonstrate that the knockdown of TLR4-interactor with leucine-rich repeats (Tril), a functional component of TLR4, resulted in reduced hypothalamic inflammation, increased whole-body energy expenditure, improved the systemic glucose tolerance and protection from diet-induced obesity. The POMC-specific knockdown of Tril resulted in decreased body fat, decreased white adipose tissue inflammation and a trend toward increased leptin signaling in POMC neurons. Thus, Tril was identified as a new component of the complex mechanisms that promote hypothalamic dysfunction in experimental obesity and its inhibition in the hypothalamus may represent a novel target for obesity treatment.

TLR4 signaling in the development of colitis-associated cancer and its possible interplay with microRNA-155

Ulcerative colitis (UC) has closely been associated with an increased risk of colorectal cancer. However, the exact mechanisms underlying colitis-associated cancer (CAC) development remain unclear. As a classic pattern-recognition receptor, Toll like receptor (TLR)4 is a canonical receptor for lipopolysaccharide of Gram-negative bacteria (including two CAC-associated pathogens Fusobacterium nucleatum and Salmonella), and functions as a key bridge molecule linking oncogenic infection to colonic inflammatory and malignant processes. Accumulating studies verified the overexpression of TLR4 in colitis and CAC, and the over-expressed TLR4 might promote colitis-associated tumorigenesis via facilitating cell proliferation, protecting malignant cells against apoptosis, accelerating invasion and metastasis, as well as contributing to the creation of tumor-favouring cellular microenvironment. In recent years, considerable attention has been focused on the regulation of TLR4 signaling in the context of colitis-associated tumorigenesis. MicroRNA (miR)-155 and TLR4 exhibited a similar dynamic expression change during CAC development and shared similar CAC-promoting properties. The available data demonstrated an interplay between TLR4 and miR-155 in the context of different disorders or cell lines. miR-155 could augment TLR4 signaling through targeting negative regulators SOCS1 and SHIP1; and TLR4 activation would induce miR-155 expression via transcriptional and post-transcriptional mechanisms. This possible TLR4-miR-155 positive feedback loop might result in the synergistic accelerating effect of TLR4 and miR-155 on CAC development.

SARS-CoV-2 infection activates dendritic cells via cytosolic receptors rather than extracellular TLRs

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infectious disease characterized by strong induction of inflammatory cytokines, progressive lung inflammation and potentially multi-organ dysfunction. It remains unclear whether SARS-CoV-2 is sensed by pattern recognition receptors (PRRs) leading to immune activation. Several studies suggest that the Spike (S) protein of SARS-CoV-2 might interact with Toll-like receptor 4 (TLR4) and thereby activate immunity. Here we have investigated the role of TLR4 in SARS-CoV-2 infection and immunity. Neither exposure of isolated S protein, SARS-CoV-2 pseudovirus nor a primary SARS-CoV-2 isolate induced TLR4 activation in a TLR4-expressing cell line. Human monocyte-derived dendritic cells (DCs) express TLR4 but not ACE2, and DCs were not infected by a primary SARS-CoV-2 isolate. Notably, neither S protein nor the primary SARS-CoV-2 isolate induced DC maturation or cytokines, indicating that both S protein and SARS-CoV-2 virus particles do not trigger extracellular TLRs, including TLR4. Ectopic expression of ACE2 in DCs led to efficient infection by SARS-CoV-2. Strikingly, infection of ACE2-positive DCs induced type I IFN and cytokine responses, which was inhibited by antibodies against ACE2. These data strongly suggest that not extracellular TLRs but intracellular viral sensors are key players in sensing SARS-CoV-2. These data imply that SARS-CoV-2 escapes direct sensing by TLRs, which might underlie the lack of efficient immunity to SARS-CoV-2 early during infection.

Leptin Induced TLR4 Expression via the JAK2-STAT3 Pathway in Obesity-Related Osteoarthritis

Obesity is considered as a risk factor of osteoarthritis (OA), but the precise relationship is still poorly understood. Leptin, one of the most relevant factors secreted by adipose tissues, plays an important role in the pathogenesis of OA. Our aim was to investigate the regulation and molecular mechanism of the leptin signaling pathway in obesity-related OA. SD rats were fed with a high-fat diet (HFD) for 5, 15, and 27 weeks. The levels of leptin in serum increased from W5, while in the synovial fluid increased from W15. The histological evaluation showed that the pathological changes of OA occurred at 27 weeks rather than 5 or 15 weeks. We also found that leptin induced CD14/TLR4 activation by the JAK2-STAT3 signaling pathway to promote OA. Moreover, silencing SOCS3 enhanced leptin-induced JAK2-STAT3-CD14/TLR4 activation in rat primary chondrocytes. Our findings indicated that leptin may be one of the initiating factors of obesity-related OA. TLR4 is at least partially regulated by leptin through the JAK2-STAT3-CD14 pathway. Meanwhile, SOCS3 acting as a negative feedback inhibitor of leptin signaling presented a potential therapeutic prospect for obesity-related OA. Our study provided new evidence suggesting the key role of leptin in mediating obesity-related OA process and its underlying mechanisms.