Role of the Innate Immune Response in Glomerular Disease Pathogenesis: Focus on Podocytes.

Accumulating evidence indicates that inflammatory and immunologic processes play a significant role in the development and progression of glomerular diseases. Podocytes, the terminally differentiated epithelial cells, are crucial for maintaining the integrity of the glomerular filtration barrier. Once injured, podocytes cannot regenerate, leading to progressive proteinuric glomerular diseases. However, emerging evidence suggests that podocytes not only maintain the glomerular filtration barrier and are important targets of immune responses but also exhibit many features of immune-like cells, where they are involved in the modulation of the activity of innate and adaptive immunity. This dual role of podocytes may lead to the discovery and development of new therapeutic targets for treating glomerular diseases. This review aims to provide an overview of the innate immunity mechanisms involved in podocyte injury and the progression of proteinuric glomerular diseases.

Genetic association and functional implications of TLR4 rs1927914 polymorphism on colon cancer risk.

BACKGROUND: Colon cancer remains a major health concern worldwide, with genetic factors playing a crucial role in its development. Toll-like receptors (TLRs) has been implicated in various cancers, but their role in colon cancer is not well understood. This study aims to identify functional polymorphisms in the promoter and 3'UTR regions of TLRs and evaluate their association with colon cancer susceptibility. METHODS: We conducted a case-control study involving 410 colon cancer patients and 410 healthy controls from the Chinese population. Genotyping of polymorphisms in TLR3, TLR4, TLR5 and TLR7 was performed using PCR-RFLP and TaqMan MGB probes. Using logistic regression analysis, we evaluated the association of TLRs polymorphisms and the susceptibility to colon cancer. To understand the biological implications of the TLR4 rs1927914 polymorphism, we conducted functional assays, including luciferase reporter assay and electrophoretic mobility shift assay (EMSA). RESULTS: Our results demonstrated that the G-allele of the TLR4 rs1927914 polymorphism is significantly associated with a decreased risk of colon cancer (OR = 0.68, 95%CI = 0.50-0.91). Stratified analysis showed that TLR4 rs1927914 AG or GG genotype contributed to a decreased risk of colon cancer among younger individuals (OR = 0.52, 95%CI = 0.34-0.81), males (OR = 0.58, 95%CI = 0.38-0.87), non-smokers (OR = 0.58, 95%CI = 0.41-0.83) and non-drinker with OR (95%CI) of 0.66 (0.46-0.93). Functional assays demonstrated that in HCT116 and LOVO colon cancer cells, the luciferase activity driven by the TLR4 promoter with the rs1927914A allele was 5.43 and 2.07 times higher, respectively, compared to that driven by the promoter containing the rs1927914G allele. Electrophoretic mobility shift assay (EMSA) results indicated that the rs1927914G allele enhanced transcription factor binding. Using the transcription factor prediction tool, we found that the G allele facilitates binding of the repressive transcription factor Oct1, while the A allele does not. CONCLUSION: The TLR4 rs1927914 polymorphism influence the susceptibility to colon cancer, with the G allele offering a protective effect through modulation of gene expression. These insights enhance our understanding of the genetic determinants of colon cancer risk and highlight TLR4 as a promising target for cancer prevention strategies.

Schisandrin B alleviates angiotensin II-induced cardiac inflammatory remodeling by inhibiting the recruitment of MyD88 to TLRs in mouse cardiomyocytes.

Cardiac tissue remodeling is characterized by altered heart tissue architecture and dysfunction, leading to heart failure. Sustained activation of the renin-angiotensin-aldosterone system (RAAS) greatly promotes the development of myocardial remodeling. Angiotensin II (Ang II), which is the major component of RAAS, can directly lead to cardiac remodeling by inducing an inflammatory response. Schisandrin B (Sch B), the active component extracted from the fruit of Schisandra chinensis (Turcz.) Baill has been shown to exhibit anti-inflammatory activity through its ability to target TLR4 and its adaptor protein, MyD88. In this study, we explored whether Sch B alleviates Ang II-induced myocardial inflammation and remodeling via targeting MyD88. Sch B significantly suppressed Ang II-induced inflammation as well as increased the expression of several genes of tissue remodeling (ß-Mhc, Tgfb, Anp, α-Ska) both in vivo and in vitro. These protective effects of Sch B were due to the inhibition of recruitment of MyD88 to TLR2 and TLR4, suppressing the Ang II-induced NF-κB activation and reducing the following inflammatory responses. Moreover, the knockdown of Myd88 in cardiomyocytes abrogated the Ang II-induced increases in the production of inflammatory cytokines and expression of remodeling genes. These findings provide new evidence that the mechanism of Sch B protection was attributed to selective inhibition of MyD88 signaling. This finding could pave the way for novel therapeutic strategies for myocardial inflammatory diseases.

Flagellin conjugated Per a 10 and its T cell peptides attenuate airway inflammation and restore cellular function.

Adjuvants were used to modulate response towards relevant immune cells. The present study aims to investigate FlaA-conjugated Per a 10 and T cell peptides in amelioration of allergic airway disease in mice. Mice given Per a 10 showed allergic features with higher cellular infiltration, IgE, Th-2 cytokines and alarmins. Fusion protein treatment reduced lung inflammation (p < 0.0001) and cellular infiltrates (p < 0.001) with higher IgG2a/IgE indicating resolution of disease. Immunotherapy with FPT1 and FPT3 reduces IL-4, IL-5 and IL-13 levels (p < 0.0001) with a fourfold increase in IFN-γ secretion in BALF. FPT1- and FPT3-treated mice have increased IL-10 and TGF-ß levels (p < 0.001) with CD4+Foxp3+ T cells (p < 0.01) indicating Treg response. There was enhanced expression of claudin-1 (1.7-fold) and occludin (fourfold) in lungs of FPT1- and FPT3-treated mice with reduced TSLP (p < 0.01) and IL-33 (p < 0.0001) secretion in BALF indicating recovery of epithelial function. Peptide-conjugated FlaA proteins showed protective immunity in mice and have potential for immunotherapy with restoration of cellular function.

C5AR1-induced TLR1/2 pathway activation drives proliferation and metastasis in anaplastic thyroid cancer.

This study aimed to elucidate the role and mechanisms of Complement C5a receptor 1 (C5AR1) in driving the malignant progression of anaplastic thyroid carcinoma (ATC). C5AR1 expression was assessed in ATC tissues and cell lines. Functional assays evaluated the effects of C5AR1 knockdown on the malignant features of ATC cells. The interaction between C5AR1 and miR-335-5p was confirmed using a luciferase reporter assay and Fluorescence in situ hybridization, and the impact of C5AR1 knockdown on the Toll-like receptor (TLR) 1/2 signaling pathway was examined. In vivo studies evaluated the effects of C5AR1 modulation on tumor growth and metastasis. C5AR1 levels were elevated in ATC tumor samples and associated with poor survival in ATC patients. C5AR1 knockdown impeded ATC cell proliferation, migration, and invasion in vitro. MiR-335-5p was identified as an upstream regulator of C5AR1, which negatively modulates C5AR1 expression. C5AR1 knockdown diminished TLR1, TLR2, and myeloid differentiation primary response 88 (MyD88) levels, while C5AR1 overexpression activated this pathway. Blocking TLR1/2 signaling abrogated the oncogenic effects of C5AR1 overexpression. C5AR1 silencing inhibited tumor growth and lung metastasis of ATC cells in nude mice. C5AR1 contributes to ATC tumorigenesis and metastasis by activating the TLR1/2 pathway, and is negatively regulated by miR-335-5p. Targeting the miR-335-5p/C5AR1/TLR1/2 axis represents a potential therapeutic strategy for ATC.

[Intervention Effect and Mechanism of Regulating MiR-155 on Young Rats with Dysfunction of Blood Coagulation].

OBJECTIVE: To investigate the intervention effect and mechanism of regulating miR-155 on young rats with dysfunction of blood coagulation. METHODS: Twenty-six healthy and clean SD male rats were selected to establish the coagulopathy models. Twenty-four rats successfully established models and were randomly divided into three groups: model group, up-regulated miR-155 group and down-regulated miR-155 group, with 8 rats in each group. The expression of miR-155 was detected by real-time fluorescence quantitative polymerase chain reaction. The changes of coagulation factors and coagulation indicators were observed. Liver pathological tissues were observed by HE staining. The expressions of HMGB1-RAGE/TLRs-NF-κB signaling pathway related proteins were detected by Western blot. RESULTS: Compared with model group, the expressions of HMGB1, RAGE, TLR2, TLR4 and NF-κB were significantly increased in up-regulated miR-155 group (all P < 0.05), while decreased in down-regulated miR-155 group (all P < 0.05). Compared with model group, the expressions of coagulation factor Ⅱ, Ⅶ, Ⅸ, and Ⅹ were significantly decreased in up-regulated miR-155 group (all P < 0.05), while increased in down-regulated miR-155 group (P < 0.05). There was no significant difference in the expression of coagulation factor Ⅺ among the three groups (P >0.05). Compared with model group, the levels of prothrombin time (PT) and activated partial thromboplastin time (APTT) were lower and fibrinogen (FIB) was higher in up-regulated miR-155 group (all P < 0.05), while in the down-regulated miR-155 group they were opposite. CONCLUSION: Down-regulation of miR-155 can effectively improve coagulation factors and coagulation indexes and inhibit inflammation in young rats with dysfunction of blood coagulopathy, and the mechanism may be related to HMGB1-RAGE/TLRs-NF-κB signaling pathway.

IL-1 receptor-associated kinase family proteins: An overview of their role in liver disease.

The interleukin-1 receptor-associated kinase (IRAK) family is a group of serine-threonine kinases that regulates various cellular processes via toll-like receptor (TLR)/interleukin-1 receptor (IL1R)-mediated signaling. The IRAK family comprises four members, including IRAK1, IRAK2, IRAK3, and IRAK4, which play an important role in the expression of various inflammatory genes, thereby contributing to the inflammatory response. IRAKs are key proteins in chronic and acute liver diseases, and recent evidence has implicated IRAK family proteins (IRAK1, IRAK3, and IRAK4) in the progression of liver-related disorders, including alcoholic liver disease, non-alcoholic steatohepatitis, hepatitis virus infection, acute liver failure, liver ischemia-reperfusion injury, and hepatocellular carcinoma. In this article, we provide a comprehensive review of the role of IRAK family proteins and their associated inflammatory signaling pathways in the pathogenesis of liver diseases. The purpose of this study is to explore whether IRAK family proteins can serve as the main target for the treatment of liver related diseases.

Bacillus Calmette-Guérin immunotherapy induces an efficient antitumor response to control murine melanoma depending on MyD88 signaling.

Bacillus Calmette-Guérin (BCG) is the first line treatment for bladder cancer and it is also proposed for melanoma immunotherapy. BCG modulates the tumor microenvironment (TME) inducing an antitumor effective response, but the immune mechanisms involved still poorly understood. The immune profile of B16-F10 murine melanoma cells was assessed by infecting these cells with BCG or stimulating them with agonists for different innate immune pathways such as TLRs, inflammasome, cGAS-STING and type I IFN. B16-F10 did not respond to any of those stimuli, except for type I IFN agonists, contrasting with bone marrow-derived macrophages (BMDMs) that showed high production of proinflammatory cytokines. Additionally, we confirmed that BCG is able to infect B16-F10, which in turn can activate macrophages and spleen cells from mice in co-culture experiments. Furthermore, we established a subcutaneous B16-F10 melanoma model for intratumoral BCG treatment and compared wild type mice to TLR2-/-, TLR3-/-, TLR4-/-, TLR7-/-, TLR3/7/9-/-, caspase 1-/-, caspase 11-/-, IL-1R-/-, cGAS-/-, STING-/-, IFNAR-/-, MyD88-/-deficient animals. These results in vivo demonstrate that MyD88 signaling is important for BCG immunotherapy to control melanoma in mice. Also, BCG fails to induce cytokine production in the co-culture experiments using B16-F10 and BMDMs or spleen cells derived from MyD88-/- compared to wild-type (WT) animals. Immunotherapy with BCG was not able to induce the recruitment of inflammatory cells in the TME from MyD88-/- mice, impairing tumor control and IFN-γ production by T cells. In conclusion, MyD88 impacts on both innate and adaptive responses to BCG leading to an efficient antitumor response against melanoma.

NLRC5 senses NAD+ depletion, forming a PANoptosome and driving PANoptosis and inflammation.

NLRs constitute a large, highly conserved family of cytosolic pattern recognition receptors that are central to health and disease, making them key therapeutic targets. NLRC5 is an enigmatic NLR with mutations associated with inflammatory and infectious diseases, but little is known about its function as an innate immune sensor and cell death regulator. Therefore, we screened for NLRC5's role in response to infections, PAMPs, DAMPs, and cytokines. We identified that NLRC5 acts as an innate immune sensor to drive inflammatory cell death, PANoptosis, in response to specific ligands, including PAMP/heme and heme/cytokine combinations. NLRC5 interacted with NLRP12 and PANoptosome components to form a cell death complex, suggesting an NLR network forms similar to those in plants. Mechanistically, TLR signaling and NAD+ levels regulated NLRC5 expression and ROS production to control cell death. Furthermore, NLRC5-deficient mice were protected in hemolytic and inflammatory models, suggesting that NLRC5 could be a potential therapeutic target.

TLR2/4 are novel activating receptors for SARS-CoV-2 spike protein on NK cells.

Background: In early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s). Methods: SP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs. Results: SPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals. Conclusions: TLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID-19 subjects.

HBeAg induces neutrophils activation impairing NK cells function in patients with chronic hepatitis B.

BACKGROUND: The role of neutrophils in hepatitis B virus (HBV) infection has been a subject of debate due to their involvement in antiviral responses and immune regulation. This study aimed to elucidate the neutrophil characteristics in patients with chronic hepatitis B (CHB). METHODS: Through flow cytometry and ribonucleic acid-sequencing analysis, the phenotypes and counts of neutrophils were analyzed in patients with CHB. Moreover, the effects of HBeAg on neutrophils and the corresponding pattern recognition receptors were identified. Simultaneously, the cross-talk between neutrophils and natural killer (NK) cells was investigated. RESULTS: Neutrophils were activated in patients with CHB, characterized by higher expression levels of programmed death-ligand 1 (PD-L1), cluster of differentiation 86, and interleukin-8, and lower levels of CXC motif chemokine receptor (CXCR) 1 and CXCR2. Hepatitis B e antigen (HBeAg) partially induces neutrophil activation through the Toll-like receptor 2 (TLR2). A consistent upregulation of the TLR2 and HBeAg expression was observed in patients with CHB. Notably, the genes encoding molecules pivotal for NK-cell function upon NK receptor engagement enriched in neutrophils after HBeAg activation. The HBeAg-activated neutrophils demonstrated the ability to decrease the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in NK cells, while the PD-1 and PD-L1 pathways partially mediated the immunosuppression. CONCLUSIONS: The immunosuppression of neutrophils induced by HBeAg suggests a novel pathogenic mechanism contributing to immune tolerance in patients with CHB.

Differential TLR-ERK1/2 activity promotes viral ssRNA and dsRNA mimic-induced dysregulated immunity in macrophages.

RNA virus induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as 'dysregulated immunity,' is caused by viral single-stranded RNA (ssRNA) and double-stranded-RNA (dsRNA) mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We show that murine bone marrow-derived macrophages (BMDMs) stimulated with TLR3 and TLR7 agonists induce differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified a significantly higher cell-surface and endosomal TLR7 expression compared to TLR3, which further correlated with early and robust MAPK (pERK1/2 and p-P38) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2, p38, and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA-mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG responses, and blocking ERK1/2 activity would mitigate viral-RNA/TLR-induced dysregulated immunity.

Transcriptomics Reveals Effect of Pulsatilla Decoction Butanol Extract in Alleviating Vulvovaginal Candidiasis by Inhibiting Neutrophil Chemotaxis and Activation via TLR4 Signaling.

The Pulsatilla decoction is a well-known herbal remedy used in clinical settings for treating vulvovaginal candidiasis (VVC). However, the specific mechanism that makes it effective is still unclear. Recent studies have shown that in cases of VVC, neutrophils recruited to the vagina, influenced by heparan sulfate (HS), do not successfully engulf Candida albicans (C. albicans). Instead, they release many inflammatory factors that cause damage to the vaginal mucosa. This study aims to understand the molecular mechanism by which the n-butanol extract of Pulsatilla decoction (BEPD) treats VVC through transcriptomics. High-performance liquid chromatography was used to identify the primary active components of BEPD. A VVC mouse model was induced using an estrogen-dependent method and the mice were treated daily with BEPD (20 mg/kg, 40 mg/kg, and 80 mg/kg) for seven days. The vaginal lavage fluid of the mice was analyzed for various experimental indices, including fungal morphology, fungal burden, degree of neutrophil infiltration, and cytokines. Various assessments were then performed on mouse vaginal tissues, including pathological assessment, immunohistochemistry, immunofluorescence, Western blot (WB), quantitative real-time PCR, and transcriptome assays. Our results showed that BEPD reduced vaginal redness and swelling, decreased white discharge, inhibited C. albicans hyphae formation, reduced neutrophil infiltration and fungal burden, and attenuated vaginal tissue damage compared with the VVC model group. The high-dose BEPD group even restored the damaged vaginal tissue to normal levels. The medium- and high-dose groups of BEPD also significantly reduced the levels of IL-1ß, IL-6, TNF-α, and LDH. Additionally, transcriptomic results showed that BEPD regulated several chemokine (CXCL1, CXCL3, and CXCL5) and S100 alarmin (S100A8 and S100A9) genes, suggesting that BEPD may treat VVC by affecting chemokine- and alarmin-mediated neutrophil chemotaxis. Finally, we verified that BEPD protects the vaginal mucosa of VVC mice by inhibiting neutrophil recruitment and chemotaxis in an animal model of VVC via the TLR4/MyD88/NF-κB pathway. This study provides further evidence to elucidate the mechanism of BEPD treatment of VVC.

Role of pattern recognition receptors in the development of MASLD and potential therapeutic applications.

Metabolic dysfunction-associated steatotic liver disease (MASLD) has become one of the most prevalent liver diseases worldwide, and its occurrence is strongly associated with obesity, insulin resistance (IR), genetics, and metabolic stress. Ranging from simple fatty liver to metabolic dysfunction-associated steatohepatitis (MASH), even to severe complications such as liver fibrosis and advanced cirrhosis or hepatocellular carcinoma, the underlying mechanisms of MASLD progression are complex and involve multiple cellular mediators and related signaling pathways. Pattern recognition receptors (PRRs) from the innate immune system, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), RIG-like receptors (RLRs), and DNA receptors, have been demonstrated to potentially contribute to the pathogenesis for MASLD. Their signaling pathways can induce inflammation, mediate oxidative stress, and affect the gut microbiota balance, ultimately resulting in hepatic steatosis, inflammatory injury and fibrosis. Here we review the available literature regarding the involvement of PRR-associated signals in the pathogenic and clinical features of MASLD, in vitro and in animal models of MASLD. We also discuss the emerging targets from PRRs for drug developments that involved agent therapies intended to arrest or reverse disease progression, thus enabling the refinement of therapeutic targets that can accelerate drug development.

Immune profiling reveals umbilical cord blood mononuclear cells from South India display an IL-8 dominant, CXCL-10 deficient polyfunctional monocyte response to pathogen-associated molecular patterns (PAMPs) that is distinct from adult blood cells.

Neonate responses to pathogen-associated molecular patterns (PAMPS) differ from adults; such understanding is poor in Indian neonates, despite recognised significant infectious risk. Immune profiling analysis was undertaken of ten secreted mediators contextualised with cellular source induced by six PAMPs in umbilical cord (CB; n=21) and adult-blood (PBMC, n=14) from a tertiary care hospital in South India. Differential cytokine expression analysis (minimum log2-fold difference; adj p-value<0.05) identified bacterial PAMPs induced higher concentrations of IL-1ß, IL-10, TNF-α in adults versus IL-8, GM-CSF, IFN-γ and IL-2 in CB. CB responded to poly I:C and SARS-CoV-2 lysate with a dominant IL-8 response, whereas, in PBMC, CXCL-10 dominated poly I:C, but not SARS-CoV-2, responses, highlighting potential IL-8 importance, in absence of Type I Interferons, in antiviral CB immunity. Candida albicans was the only PAMP to uniformly induce higher secretion of effectors in CB. The predominant source of IL-8/IL-6/TNF-α/IL-1ß in both CB and PBMC was polyfunctional monocytes and IFN-γ /IL-2/IL-17 from innate lymphocytes. Correlation matrix analyses revealed IL-8 to be the most differentially regulated, correlating positively in CB versus negatively in PBMC with IL-6, GM-CSF, IFN-γ, IL-2, consistent with more negatively regulated cytokine modules in adults, potentially linked to higher anti-inflammatory IL-10. Cord and adult blood from India respond robustly to PAMPs with unique effector combinations. These data provide a strong foundation to monitor, explore, mechanisms that regulate such immunity during the life course, an area of significant global health importance given infection-related infant mortality incidence.

A Cell Specific Effect of MBOAT7 MAFLD-risk Variant on Immune Cells.

BACKGROUND: Disease risk variants are likely to affect gene expression in a context- and cell-type specific manner. The membrane bound O-acyltransferase domain containing 7 (MBOAT7) rs8736 metabolic-dysfunction-associated fatty liver disease (MAFLD)-risk variant was recently reported to be a negative regulator of toll-like receptors (TLRs) signalling in macrophages. Whether this effect is generic or cell-type specific in immune cells is unknown. METHODS: We investigated the impact of modulating TLR signaling on MBOAT7 expression in peripheral blood mononuclear cells (PBMCs). We also examined whether the rs8736 polymorphism in MBOAT7 regulates this effect. Furthermore, we measured the allele-specific expression of MBOAT7 in various immune cell populations under both unstimulated and stimulated conditions. RESULTS: We show that MBOAT7 is down-regulated by TLRs in PBMCs. This effect is modulated by the MBOAT7 rs8736 polymorphism. Additionally, we provide evidence that MBOAT7 acts primarily as a modulator of TLR signalling in mononuclear phagocytes. CONCLUSION: Our results highlight the importance of studying Genome-Wide Association Studies (GWAS) signals in the specific cell types in which alterations of gene expression are found.

Immune targeting of filarial glutaredoxin through a multi-epitope peptide-based vaccine: A reverse vaccinology approach.

Despite the efforts of global programme to eliminate lymphatic filariasis (GPELF), the threat of lymphatic filariasis (LF) still looms over humanity in terms of long-term disabilities, and morbidities across the globe. In light of this situation, investigators have chosen to focus on the development of immunotherapeutics targeting the physiologically important filarial-specific proteins. Glutaredoxin (16.43 kDa) plays a pivotal role in filarial redox biology, serving as a vital contributor. In the context of the intra-host survival of filarial parasites, this antioxidant helps in mitigating the oxidative stress imposed by the host immune system. Given its significant contribution, the development of a vaccine targeting glutaredoxin holds promise as a new avenue for achieving a filaria-free world. Herein, multi-epitope-based vaccine was designed using advanced immunoinformatics approach. Initially, 4B-cell epitopes and 6 T-cell epitopes (4 MHC I and 2 MHC II) were identified from the 146 amino acid long sequence of glutaredoxin of the human filarid, Wuchereria bancrofti. Subsequent clustering of these epitopes with linker peptides finalized the vaccine structure. To boost TLR-mediated innate immunity, TLR-specific adjuvants were incorporated into the designed vaccine. After that, experimental analyses confirm the designed vaccine, Vac4 as anefficient ligand of human TLR5 to elicit protective innate immunity against filarial glutaredoxin. Immune simulation further demonstrated abundant levels of IgG and IgM as crucial contributors in triggering vaccine-induced adaptive responses in the recipients. Hence, to facilitate the validation of immunogenicity of the designed vaccine, Vac4 was cloned in silico in pET28a(+) expression vector for recombinant production. Taken together, our findings suggest that vaccine-mediated targeting of filarial glutaredoxin could be a future option for intervening LF on a global scale.

The E3 Ubiquitin Protein Ligase LINCR Amplifies the TLR-Mediated Signals through Direct Degradation of MKP1.

Toll-like receptors (TLRs) induce innate immune responses through activation of intracellular signaling pathways, such as MAP kinase and NF-κB signaling pathways, and play an important role in host defense against bacterial or viral infections. Meanwhile, excessive activation of TLR signaling leads to a variety of inflammatory disorders, including autoimmune diseases. TLR signaling is therefore strictly controlled to balance optimal immune response and inflammation. However, its balancing mechanisms are not fully understood. In this study, we identified the E3 ubiquitin ligase LINCR/ NEURL3 as a critical regulator of TLR signaling. In LINCR-deficient cells, the sustained activation of JNK and p38 MAPKs induced by the agonists for TLR3, TLR4, and TLR5, was clearly attenuated. Consistent with these observations, TLR-induced production of a series of inflammatory cytokines was significantly attenuated, suggesting that LINCR positively regulates innate immune responses by promoting the activation of JNK and p38. Interestingly, our further mechanistic study identified MAPK phosphatase-1 (MKP1), a negative regulator of MAP kinases, as a ubiquitination target of LINCR. Thus, our results demonstrate that TLRs fine-tune the activation of MAP kinase pathways by balancing LINCR (the positive regulator) and MKP1 (the negative regulator), which may contribute to the induction of optimal immune responses.

Infection of Human Macrophage-Like Cells by African Swine Fever Virus.

BACKGROUND: The African swine fever (ASF) virus (ASFV) and ASF-like viral sequences were identified in human samples and sewage as well as in different water environments. Pigs regularly experience infections by the ASFV. The considerable stability of the virus in the environment suggests that there is ongoing and long-term contact between humans and the ASFV. However, humans exhibit resistance to the ASFV, and the decisive factor in developing infection in the body is most likely the reaction of target macrophages to the virus. Therefore, this study aimed to characterize the responses of human macrophages to the virus and explore the distinct features of the viral replication cycle within human macrophages. METHODS: The ASFV Armenia/07 strain was used in all experiments. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the ASFV gene expression; flow cytometry analysis was performed to evaluate the effects of the inactive and active ASFV (inASFV and aASFV) treatments on the phenotype of THP-1-derived macrophages (Mφ0) and inflammatory markers. Moreover, other methods such as cell viability and apoptosis assays, staining techniques, phagocytosis assay, lysosome-associated membrane protein (LAMP-1) cytometry, and cytokine detection were used during experiments. RESULTS: Our findings showed that the virus initiated replication by entering human macrophages. Subsequently, the virus shed its capsid and initiated the transcription of numerous viral genes, and at least some of these genes executed their functions. In THP-1-derived macrophages (Mφ0), the ASFV implemented several functions to suppress cell activity, although the timing of their implementation was slower compared with virus-sensitive porcine alveolar macrophages (PAMs). Additionally, the virus could not complete the entire replication cycle in human Mφ0, as indicated by the absence of viral factories and a decrease in infectious titers of the virus with each subsequent passage. Overall, the infection of Mφ0 with the ASFV caused significant alterations in their phenotype and functions, such as increased TLR2, TLR3, CD80, CD36, CD163, CXCR2, and surface LAMP-1 expression. Increased production of the tumor necrosis factor (TNF) and interleukin (IL)-10 and decreased production of interferon (IFN)-α were also observed. Taken together, the virus enters human THP-1-derived macrophages, starts transcription, and causes immunological responses by target cells but cannot complete the replicative cycle. CONCLUSION: These findings suggest that there may be molecular limitations within human macrophages that at least partially restrict the complete replication of the ASFV. Understanding the factors that hinder viral replication in Mφ0 can provide valuable insights into the host-virus interactions and the mechanisms underlying the resistance of human macrophages to the ASFV.

Genome-wide identification of toll-like receptors in Octopus sinensis and expression analysis in response to different PAMPs stimulation.

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation.