Multi-Drug Resistance and Breast Cancer Progression via Toll-Like Receptors (TLRs) Signaling.

Toll-like receptors (TLRs) are essential receptors involved in inflammation and innate immunity. Various types of cancer cells, as well as innate immune cells, express TLRs. There is mounting proof that TLRs are critical to the development and spread of cancer as well as metabolism. In breast cancer, up-regulated levels of TLRs have been linked to the aggressiveness of the diseases, worse treatment outcomes, and the emergence of therapeutic resistance. Patients with advanced non-resectable, recurring, and metastatic breast cancer currently have few available treatment choices. An intriguing new strategy is an innate immunity-mediated anticancer immunotherapy, either used alone or in conjunction with existing treatments. In fact, several TLR agonists and antagonists have been used in clinical studies for anti-cancer immunotherapy. Consequently, TLRs serve as critical targets for controlling the course of breast cancer and treatment resistance in addition to being implicated in immune responses against pathogen infection and cancer immunology. In this review, we deliver an overview of the most current findings on TLR involvement in the development of breast cancer and treatment resistance.

E3 Ubiquitin Ligase RNF13 Suppresses TLR Lysosomal Degradation by Promoting LAMP-1 Proteasomal Degradation.

As a highly organized system, endo-lysosomes play a crucial role in maintaining immune homeostasis. However, the mechanisms involved in regulating endo-lysosome progression and subsequent inflammatory responses are not fully understood. By screening 103 E3 ubiquitin ligases in regulating endo-lysosomal acidification, it is discovered that lysosomal RNF13 inhibits lysosome maturation and promotes inflammatory responses mediated by endosomal Toll-like receptors (TLRs) in macrophages. Mechanistically, RNF13 mediates K48-linked polyubiquitination of LAMP-1 at residue K128 for proteasomal degradation. Upon TLRs activation, LAMP-1 promotes lysosomes maturation, which accelerates lysosomal degradation of TLRs and reduces TLR signaling in macrophages. Furthermore, peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA) show increased RNF13 levels and decreased LAMP-1 expression. Accordingly, the immunosuppressive agent hydroxychloroquine (HCQ) can increase the polyubiquitination of RNF13. Taken together, the study establishes a linkage between proteasomal and lysosomal degradation mechanisms for the induction of appropriate innate immune response, and offers a promising approach for the treatment of inflammatory diseases by targeting intracellular TLRs.

Participation of TLRs in cancer immunopathogenesis and drug resistance via interacting with immunological and/or non-immunological signaling pathways as well as lncRNAs.

Toll-like receptors (TLRs) have a convoluted role in cancer even though they are crucial to the immune system. By bridging the innate immune system and cancer, TLRs have a very complex impact on the formation of tumors and the effectiveness of anti-cancer treatments. TLR signaling links the innate and adaptive immune systems and initiates direct pathogen eradication. In cancer immunopathogenesis and treatment resistance, long non-coding RNAs (lncRNAs) modify TLR signaling linkages with immunological and non-immunological pathways. We identified lncRNAs that positively and negatively control TLR signaling, impacting immunological response and drug sensitivity. These results highlight the complex interactions between long non-coding RNAs and TLRs that influence the start of cancer and its response to treatment. Targeting specific lncRNAs is a practical way to control TLR signaling and perhaps enhance anti-tumor immunity while overcoming medication resistance. We provide a framework for developing novel immunotherapeutic regimens and customized medicine approaches for cancer treatment. The exact mechanisms by which lncRNAs regulate TLR signaling pathways should be defined by further research, and these findings should be validated in clinical situations. This finding makes future research of lncRNA-based drugs in combination with existing cancer treatments feasible.

Interactions between toll-like receptors signaling pathway and gut microbiota in host homeostasis.

BACKGROUND: Toll-like receptors (TLRs) are a family of fundamental pattern recognition receptors in the innate immune system, constituting the first line of defense against endogenous and exogenous antigens. The gut microbiota, a collection of commensal microorganisms in the intestine, is a major source of exogenous antigens. The components and metabolites of the gut microbiota interact with specific TLRs to contribute to whole-body immune and metabolic homeostasis. OBJECTIVE: This review aims to summarize the interaction between the gut microbiota and TLR signaling pathways and to enumerate the role of microbiota dysbiosis-induced TLR signaling pathways in obesity, inflammatory bowel disease (IBD), and colorectal cancer (CRC). RESULTS: Through the recognition of TLRs, the microbiota facilitates the development of both the innate and adaptive immune systems, while the immune system monitors dynamic changes in the commensal bacteria to maintain the balance of the host-microorganism symbiosis. Dysbiosis of the gut microbiota can induce a cascade of inflammatory and metabolic responses mediated by TLR signaling pathways, potentially resulting in various metabolic and inflammatory diseases. CONCLUSION: Understanding the crosstalk between TLRs and the gut microbiota contributes to potential therapeutic applications in related diseases, offering new avenues for treatment strategies in conditions like obesity, IBD, and CRC.

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.

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.

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.

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.

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.

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.

Revitalizing antitumor immunity: Leveraging nucleic acid sensors as therapeutic targets.

Nucleic acid sensors play a critical role in recognizing and responding to pathogenic nucleic acids as danger signals. Upon activation, these sensors initiate downstream signaling cascades that lead to the production and release of pro-inflammatory cytokines, chemokines, and type I interferons. These immune mediators orchestrate diverse effector responses, including the activation of immune cells and the modulation of the tumor microenvironment. However, careful consideration must be given to balancing the activation of nucleic acid sensors to avoid unwanted autoimmune or inflammatory responses. In this review, we provide an overview of nucleic acid sensors and their role in combating cancer through the perception of various aberrant nucleic acids and activation of the immune system. We discuss the connections between different programmed cell death modes and nucleic acid sensors. Finally, we outline the development of nucleic acid sensor agonists, highlighting how their potential as therapeutic targets opens up new avenues for cancer immunotherapy.

Investigate the Therapeutic Effect of Ibandronate Sodium on Knee Osteoarthritis Based on TLRs/MyD88/NF-κB Signaling Pathway in Vitro and in Vivo.

BACKGROUND: For decades, bisphosphonates have primarily found application in clinical practice for the treatment and prevention of bone metastases associated with malignant tumors and various bone metabolic disorders. However, third-generation bisphosphonates like ibandronate have demonstrated significant utility in addressing conditions like osteoporosis (OA) and other bone metabolism-related ailments. Ibandronate, distinguished by its high effectiveness, low toxicity, and ease of administration, has garnered attention for its potential applications in the treatment of rheumatoid arthritis, OA, and orthopedic concerns. In recent years, the utilization of ibandronate sodium in these contexts has sparked considerable interest. Research has pointed to a possible connection between ibandronate and the Toll-like receptors (TLRs), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) signaling pathway, particularly in the context of inflammation and immunological regulation. Consequently, this study is designed to investigate the therapeutic impact of ibandronate on in vitro and in vivo models of knee osteoarthritis, while also delving into its influence on the TLRs/MyD88/NF-κB pathway. METHOD: Various dosages of ibandronate sodium, including low (10 g/kg), medium (20 g/kg), and high (30 g/kg), were administered following the establishment of both in vivo and in vitro models of knee osteoarthritis (KOA). Post-intervention, an in-depth quantitative analysis of bone tissue microstructure was conducted. The morphology of articular cartilage tissue was observed in vivo, and the modified Mankin score was subsequently calculated. In the in vitro setting, cartilage was entirely isolated, and mRNA and total protein were extracted to measure the expression levels of TLR4, MyD88, and NF-κB at both the mRNA and protein levels. Furthermore, the study explored the effects of Interleukin-1 beta (IL-1ß) on cell proliferation, apoptosis, stromal decomposition enzyme activity, ossification, and the expression of TLR4, MyD88, and NF-κB. RESULT: In the results of the in vivo experiments, several noteworthy findings emerged. The knee curvature, gait score, Mankin score, pathological knee joint injury degree, cartilage protein loss, and trabecular separation within the model group exhibited significant elevations compared to both the sham operation group and the blank control group (p < 0.05). Conversely, bone density, bone volume fraction, and trabecular thickness in the model group displayed lower values in comparison to the sham operation and blank control groups (p < 0.05). Following the administration of ibandronate sodium, there was a progressive improvement in these parameters, with the medium and high-dose groups demonstrating the most favorable outcomes (p < 0.05). Additionally, the model group exhibited the highest expression levels of TLR4, MyD88, and NF-κB, while the ibandronate sodium intervention group displayed reduced expression levels of these markers, with the high-dose group registering the most significant changes (p < 0.05). Turning to the in vitro experiments, it was observed that the cell proliferation capacity and ossification degree of the IL-1ß-induced group experienced declines, concomitant with an increase in stromal decomposition enzyme activity and cell apoptosis rate (p < 0.05). However, post-intervention with ibandronate sodium, all these indicators gradually returned to normal, with the medium-dose group exhibiting the most notable improvements. The expression levels of TLR4, MyD88, and NF-κB in the IL-1ß-induced group showed an increase, while the expression levels in the ibandronate sodium intervention group displayed a decrease, particularly in the high-dose group (p < 0.05). CONCLUSIONS: Ibandronate sodium demonstrates a protective effect on articular chondrocytes and exhibits the potential to decelerate the pathological progression of knee osteoarthritis (KOA) in rats. This mechanism is likely achieved through the inhibition of the TLRs/MyD88/NF-κB signaling pathway.

The expression of Nrf2 and TLRs in ear effusion in children with different types of otitis media and their relationship with inflammatory factors.

OBJECTIVE: This study aimed to analyze the differences in the expression of Toll-like receptors (TLRs) and nuclear factor erythroid 2-related factor 2 (Nrf2) in ear effusion in children with different types of otitis media (OM), to elaborate the relationship between the expression of TLRs and Nrf2 in ear effusion and the pathogenesis of OM, and to explore the relationship between the two indicators and pro-inflammatory cytokines in children with OM, thereby laying a scientific foundation for revealing the underlying molecular mechanisms of the progression of different types of OM. METHODS: A total of 73 children with OM who were treated in our hospital from March 2019 to July 2021 were selected as the study subjects. By using the cross-sectional investigation method, participants were divided into three groups according to the different pathological types, including the secretory OM group (30 cases), the chronic suppurative OM group (27 cases), and the cystic lesional OM group (16 cases). The levels of Nrf2, TLR2, TLR4 and proinflammatory cytokines [interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), transforming growth factor-ß 1(TGF-ß1), procalcitonin (PCT) and interleukin-1ß (IL-1ß)] were detected in ear effusion of children with different types of OM. Linear regression was used to analyze the correlation between the Nrf2, TLR2 and TLR4 expression levels and pro-inflammatory cytokines in ear effusion. RESULTS: The expression levels of TNF-α and PCT in the ear effusion of the children under 3 years old were significantly higher than that of the children between 3 and 5 years old and that of the children between 6 and 8 years old (all P < 0.001). The mRNA levels of Nrf2, TLR2 and TLR4 in the ear effusion of the children from the chronic suppurative OM group were higher than these from the secretory OM group (P < 0.001, P = 0.008 and P = 0.021). The mRNA levels of Nrf2, TLR2, and TLR4 in the ear effusion of the children from the cystic lesional OM group were higher than those from the chronic suppurative OM group (P < 0.001, P = 0.029 and P = 0.018). A prominent increase in the concentrations of IFN-γ, TNF-α, TGF-ß1, PCT and IL-1ß was found in the ear effusion of children from the chronic suppurative OM group compared to these from the secretory OM group (P = 0.021, P = 0.044, P = 0.048, P = 0.004 and P = 0.001). The concentrations of IFN-γ, TNF-α, TGF-ß1, PCT and IL-1ß in the ear effusion of the children from the cystic lesional OM group were markedly increased as compared with these from the chronic suppurative OM group (P < 0.001, P = 0.004, P = 0.003, P < 0.001 and P < 0.001). Nrf2, TLR2 and TLR4 were taken as independent variables, and inflammatory indexes, including IFN-γ, TNF-α, TGF-ß1, PCT and IL-1ß were used as dependent variables for the linear regression analysis. The results showed that Nrf2, TLR2 and TLR4 were positively correlated with the secretion levels of pro-inflammatory cytokines after adjusting for age, sex, course and the OM classification (all P < 0.05). CONCLUSION: The expressions of Nrf2, TLR2 and TLR4 in the ear effusion of children with different types of OM gradually increased with the severity of the disease, these were significantly positively correlated with the pro-inflammatory cytokines of the children. Nrf2/TLR signaling pathway maintained chronic inflammation in OM, induced damage of middle ear tissue, and promoted the transition from acute OM to chronic OM.

Leptospiral lipopolysaccharide dampens inflammation through upregulation of autophagy adaptor p62 and NRF2 signaling in macrophages.

Leptospira interrogans are pathogenic bacteria responsible for leptospirosis, a worldwide zoonosis. All vertebrates can be infected, and some species like humans are susceptible to the disease whereas rodents such as mice are resistant and become asymptomatic renal carriers. Leptospires are stealth bacteria that are known to escape several immune recognition pathways and resist killing mechanisms. We recently published that leptospires may survive intracellularly in and exit macrophages, avoiding xenophagy, a pathogen-targeting form of autophagy. Interestingly, the latter is one of the antimicrobial mechanisms often highjacked by bacteria to evade the host immune response. In this study we explored whether leptospires subvert the key molecular players of autophagy to facilitate infection. We showed in macrophages that leptospires triggered a specific accumulation of autophagy-adaptor p62 in puncta-like structures, without altering autophagic flux. We demonstrated that Leptospira-induced p62 accumulation is a passive mechanism depending on the leptospiral virulence factor LPS signaling via TLR4/TLR2. p62 is a central pleiotropic protein, also mediating cell stress and death, via the translocation of transcription factors. We demonstrated that Leptospira-driven accumulation of p62 induced the translocation of transcription factor NRF2, a key player in the anti-oxidant response. However, NRF2 translocation upon Leptospira infection did not result as expected in antioxydant response, but dampened the production of inflammatory mediators such as iNOS/NO, TNF and IL6. Overall, these findings highlight a novel passive bacterial mechanism linked to LPS and p62/NRF2 signaling that decreases inflammation and contributes to the stealthiness of leptospires.

Probiotic Bacillus spp. enhance TLR3-mediated TNF signalling in macrophages.

Probiotics have been reported to have immunomodulatory properties in the context of infectious disease and inflammation, although the underlying mechanisms are not fully understood. Here, we aimed to determine how different probiotic bacterial strains modulated macrophage function during TLR3 stimulation mimicking viral infection. We screened 14 different strains for their ability to modulate TNF-α, IL-6 IL-10, IFN-α, IFN-ß and IFN-γ secretion in RAW 264.7 macrophages with or without poly(I:C) stimulation. Seven strains were selected for further analysis using primary porcine alveolar macrophages. In-depth transcriptomic analysis on alveolar macrophages was conducted for two strains. Most strains induced a synergistic effect when co-incubated with poly(I:C) resulting in increased levels of IL-6 and TNF-α secretion from RAW 264.7 cells. This synergistic effect was found to be TLR2 independent. Only strains of Bacillus spp. could induce this effect in alveolar macrophages. Transcriptomic analysis indicated that the increased TNF-α secretion in alveolar macrophages after co-incubation with poly(I:C) correlated with significant upregulation of TNF and IL23A-related pathways. Collectively, our data show that probiotic bacteria possess strain-dependent immunomodulatory properties that may be harnessed to enhance innate immune responses to pathogens.

Chlamydia trachomatis T3SS Effector CT622 Induces Proinflammatory Cytokines Through TLR2/TLR4-Mediated MAPK/NF-κB Pathways in THP-1 Cells.

BACKGROUND: The pathogenesis of Chlamydia trachomatis is associated with the induction of the host inflammatory response; however, the precise underlying molecular mechanisms remain poorly understood. METHODS: CT622, a T3SS effector protein, has an important role in the pathogenesis of C trachomatis; however, whether CT622 can induce a host inflammatory response is not understood. Our findings demonstrate that CT622 induces the expression of interleukins 6 and 8 (IL-6 and IL-8). Mechanistically, these effects involve the activation of the MAPK/NF-κB signaling pathways (mitogen-activated protein kinase/nuclear factor κB). RESULTS: Interestingly, we demonstrated that the suppression of toll-like receptor 4 using small interfering RNA markedly reduced the phosphorylation of ERK, p38, JNK, and IκBα, concomitant with a significant decrease in IL-6 and IL-8 secretion. Conversely, disruption of toll-like receptor 2 abrogated the CT622-induced upregulation of IL-8 and activation of ERK, whereas IL-6 expression and p38, JNK, and IκBα phosphorylation were unaffected. CONCLUSIONS: Taken together, these results indicate that CT622 contributes to the inflammatory response through the toll-like receptor 2/4-mediated MAPK/NF-κB pathways, which provides insight into the molecular pathology of C trachomatis infection.

Paracrine Responses of Cardiosphere-Derived Cells to Cytokines and TLR Ligands: A Comparative Analysis.

Cardiosphere-derived cells (CDCs) are currently being evaluated in clinical trials as a potential therapeutic tool for regenerative medicine. The effectiveness of transplanted CDCs is largely attributed to their ability to release beneficial soluble factors to enhance therapeutic effects. An emerging area of research is the pretreatment of stem cells, including CDCs, with various cytokines to improve their therapeutic properties. This strategy aims to enhance their survival, proliferation, differentiation, and paracrine activities after transplantation. In our study, we investigated the differential effects of various cytokines and TLR ligands on the secretory phenotype of human CDCs. Using a magnetic bead-based immunoassay, we analyzed the CDCs-conditioned media for 41 cytokines and growth factors and detected the presence of 21 cytokines. We found that CDC incubation with lipopolysaccharide, a TLR4 ligand, and the cytokine combination of TNF/IFN significantly increased the secretion of most of the cytokines detected. Specifically, we observed an increased secretion and gene expression of IP10, MCP3, IL8, and VEGFA. In contrast, the TLR3 ligand polyinosinic-polycytidylic acid and TGF-beta had minimal effects on CDC cytokine secretion. Additionally, TNF/IFN, but not LPS, enhanced ICAM1 expression. Our findings offer new insights into the role of cytokines in potentially modulating the biology and regenerative potential of CDCs.

Cbl-b negatively regulates TLR/MyD88-mediated anti-Toxoplasma gondii immunity.

IMPORTANCE: This is the first report that a human E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), functions as a host dependency factor for the intracellular protozoan Toxoplasma gondii and the mechanism for how T. gondii infection inhibits the TLR/MyD88 innate immunity pathway through MyD88 degradation mediated by Cbl-b. This finding is an impactful contribution for understanding the host cell immunity against T. gondii infection.

Toll-like receptor-guided therapeutic intervention of human cancers: molecular and immunological perspectives.

Toll-like receptors (TLRs) serve as the body's first line of defense, recognizing both pathogen-expressed molecules and host-derived molecules released from damaged or dying cells. The wide distribution of different cell types, ranging from epithelial to immune cells, highlights the crucial roles of TLRs in linking innate and adaptive immunity. Upon stimulation, TLRs binding mediates the expression of several adapter proteins and downstream kinases, that lead to the induction of several other signaling molecules such as key pro-inflammatory mediators. Indeed, extraordinary progress in immunobiological research has suggested that TLRs could represent promising targets for the therapeutic intervention of inflammation-associated diseases, autoimmune diseases, microbial infections as well as human cancers. So far, for the prevention and possible treatment of inflammatory diseases, various TLR antagonists/inhibitors have shown to be efficacious at several stages from pre-clinical evaluation to clinical trials. Therefore, the fascinating role of TLRs in modulating the human immune responses at innate as well as adaptive levels directed the scientists to opt for these immune sensor proteins as suitable targets for developing chemotherapeutics and immunotherapeutics against cancer. Hitherto, several TLR-targeting small molecules (e.g., Pam3CSK4, Poly (I:C), Poly (A:U)), chemical compounds, phytocompounds (e.g., Curcumin), peptides, and antibodies have been found to confer protection against several types of cancers. However, administration of inappropriate doses of such TLR-modulating therapeutics or a wrong infusion administration is reported to induce detrimental outcomes. This review summarizes the current findings on the molecular and structural biology of TLRs and gives an overview of the potency and promises of TLR-directed therapeutic strategies against cancers by discussing the findings from established and pipeline discoveries.