TLR4 signalling in ischemia/reperfusion injury: a promising target for linking inflammation, oxidative stress and programmed cell death to improve organ transplantation outcomes.

Transplantations represent the principal therapeutic interventions for terminal organ failure, a procedure that has salvaged myriad lives annually. Ischemia/reperfusion injury (IRI) is frequently correlated with an unfavourable prognosis and is relevant for early graft dysfunction and graft survival. IRI constitutes a complex pathological state influenced by a series of factors such as oxidative stress, metabolic stress, leukocytic infiltration, programmed cell death pathways, and inflammatory immune responses. Reducing ischemia/reperfusion injury is one of the main directions of transplantation research. Toll-like receptors (TLRs) are important pattern-recognition receptors expressed on various organs that orchestrate the immune responses upon recognising PAMPs and DAMPs. Targeting the TLR4 signalling has recently been suggested as a promising approach for alleviating IRI by affecting inflammation, oxidative stress and programmed cell death (PCD). In this minireview, we summarise the role of TLR4 signalling in regulating inflammation, oxidative stress and PCD in organ transplantation and discuss their interactions during IRI. A detailed understanding of the multiple functions of TLR4 in IRI provides novel insights into developing therapies to improve organ transplantation outcomes.

miRNAs: possible regulators of toll like receptors and inflammatory tumor microenvironment in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is ranked as the third most commonly diagnosed cancer and the third cause of cancer related deaths. CRC is greatly attributed to genetic and epigenetic mutations and immune dysregulation. Tumor aberrant expression of Toll-like Receptors (TLRs) can contribute to tumorigenesis. Recent studies suggested that microRNAs act as direct ligands of TLRs altering their expression and signaling pathways. AIM: To prove our concept that specific miRNA mimics may act as antagonists of their specific toll like receptors inhibiting their expression that could limit the release of pro-inflammatory and pro-tumorigenic cytokines leading to apoptosis of tumor cells. METHODS: From public microarray databases, we retrieved TLRs and miRNAs related to CRC followed by in silico docking of the selected miRNA ligands into the TLRs. Clinical validation after co-immunoprecipitation of TLRs and their interacting miRNA ligands was done. Expression of TLRs 1, 7,8 was determined by ELISA while miRNAs was measured by RT-qPCR. In addition, microRNA mimics of the down regulated miRNAs were transfected into human CRC cell lines. RESULTS: Our data demonstrate that TLRs 1, 7, 8 are up regulated in CRC compared to controls. Further, three miRNAs (-122, -29b and -15b) are relatively downregulated, while 4 miRNAs (-202, miRNA-98, -21 and -let7i) are upregulated in CRC patients compared to those with benign tumor and healthy controls. Transfection of down regulated miRNA mimics into CRC cell lines resulted in a significant reduction of the number and viability of cells as well as down regulating the expression of TLRs 1, 7 and 8 with ultimate reduction of downstream effector IL6 protein, suggesting that these miRNAs are negative regulators of carcinogenesis. CONCLUSION: MicroRNAs could act as antagonistic ligands of TLRs limiting the inflammatory tumor microenvironment.

Study of gene polymorphisms in Toll-like receptor 2 in patients with acute lymphoblastic leukemia.

Objectives: Acute Lymphoblastic Leukemia (ALL) is a multifactorial disease that results from the interaction between multiple genetic factors. ALL is characterized by uncontrolled production of hematopoietic precursor cells of the lymphoid progenitors within the bone marrow. The development of hematological malignancies has been associated with malignant-like cells that express low levels of immunogenic surface molecules, thus, facilitating their escape from cellular antineoplastic immune responses. This risk may be partly influenced by variations in polymorphic genes that control immune function and regulation. Toll-like receptors (TLRs) are well known pattern recognition receptors playing key role in innate immune response. Abnormal expression and dysregulation of TLRs will provide an opportunity for cancer cells to escape from the immune system and enhance their proliferation and angiogenesis. Toll-like receptor 2 (TLR2) play an essential role in innate immunity. Single nucleotide polymorphisms (SNPs) are present in a number of TLR genes and have been associated with various disorders. Methods: In this study, 265 subjects have been divided into two groups included 150 patients with ALL and115 healthy volunteers. All subjects were genotyped using TaqMan PCR techniques. In total, Five SNPs were statistically evaluated in the TLR2 (rs1898830 A/G, rs3804099 T/C, rs3804100 T/C, rs1339 T/C, and rs1337 C/G), which may influence the susceptibility of ALL. Minor allele frequency and genotype distribution were compared across the study groups, and the relative risk and differences between patients and controls were estimated. Moreover, the mRNA expression level was evaluated in patients with ALL and the matched healthy individuals by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Results: TLR2 rs1898830 A/G; rs3804099 T/C; rs3804100 T/C; rs1339 T/C, were significantly decrease the risk in our population, overall and for certain subtypes and ALL samples exhibited significant increase in the mRNA levels of TLR2. Conclusions: This study shows that TLR2 could be an independent prognostic factor of ALL risks in the Saudi population. Suggesting that genetic variation in genes associated with an immune response may be important in the etiology of ALL. In addition, the results herein revealed that TLR2 overexpression is associated with ALL and has diverse biological significance in the context of the complex relationship between inflammation and cancer development. Therefore, these data could open further studies to explore the possible clinical relevance of TLRs as pathological markers for Leukemia and enhance the strategies regarding hematological malignancies prevention based on their gene expression.

Subcutaneous checkpoint inhibition is equivalent to systemic delivery when combined with nelitolimod delivered via pressure-enabled drug delivery for depletion of intrahepatic myeloid-derived suppressor cells and control of liver metastases.

BACKGROUND: Toll-like receptor 9 (TLR9) agonists induce inflammatory responses that promote the killing of infectious micro-organisms, cancer cells and develop adaptive immune responses. Their ability as immunomodulators to enhance the activity of checkpoint inhibitors (CPI) in treating liver tumors is limited in part by the distinctive biology of intrahepatic myeloid-derived suppressor cells (MDSC) and challenges with tumor-specific therapeutic delivery. We have shown that the regional delivery of type C TLR9 agonist via pressure-enabled drug delivery (PEDD) system improves delivery to the tumor, enhances depletion of MDSCs and overall, stimulates the immune system in combination with or without CPI. Currently, CPIs are delivered intravenously, although there is a growing interest in its subcutaneous (SQ) administration. We compared nelitolimod formerly known as SD-101 administered using PEDD in combination with systemic (Sys) or SQ CPI in murine liver metastases (LM). METHODS: The LM model was developed by injecting MC38-Luc cells via the spleen of 8-12 week old male C57/BL6 mice followed by splenectomy. After a week, fluorescently labeled nelitolimod (10 µg/mouse) was delivered via PEDD and co-administered anti-programmed cell death-1 (α-PD-1) either via Sys or SQ. Tumor burden was monitored by in vivo imaging system. Serum cytokine levels were analyzed by Luminex. Tissues were harvested on Day 3 (D3) or Day 10 (D10) post-PEDD to enrich CD45+ cells and were analyzed via NanoString targeted transcriptomics (D3) or flow cytometry (FC, D10) to interrogate immune cell populations (D10). For NanoString analysis, the innate immune panels were selected, and for FC, MDSCs (CD11b+Gr1+), B cells (B220+), dendritic cells (DC, CD11c+), T (CD3+) cells, and M1-like macrophages (F4/80+CD38+Egr2-) were quantified. RESULTS: Nelitolimod delivered via PEDD resulted in changes in innate and adaptive immune cells within LM, including depletion of liver MDSC and increased M1-like macrophages in the liver, which are supportive of antitumor immunity. While CPI monotherapy failed to control tumor progression, nelitolimod and CPI combination improved LM control, survival and antitumor immunity beyond the nelitolimod monotherapy effect, irrespective of CPI delivery route. CONCLUSION: The SQ route of CPI delivery was equivalent to Sys in combination with nelitolimod, suggesting SQ-CPI may be a rational choice in combination with PEDD of nelitolimod for liver tumor treatment.

Toll-like receptor 13-mediated signaling protects against the development of colon cancer.

Appropriate host-microbiota interactions are essential for maintaining intestinal homeostasis; hence, an imbalance in these interactions leads to inflammation-associated intestinal diseases. Toll-like receptors (TLRs) recognize microbial ligands and play a key role in host-microbe interactions in health and disease. TLR13 has a well-established function in enhancing host defenses against pathogenic bacteria. However, its role in maintaining intestinal homeostasis and controlling colitis-associated colon cancer (CAC) is largely unknown. This study aimed to investigate the involvement of TLR13-mediated signaling in intestinal homeostasis and colonic tumorigenesis using ex vivo cell and in vivo CAC animal model. Tlr13-deficient mice were prone to dextran sodium sulfate (DSS)-induced colitis. During the early stages of the CAC regimen (AOM/DSS-treated), Tlr13 deficiency led to severe ulcerative colitis. Moreover, Tlr13-deficient mice exhibited increased intestinal permeability, as evidenced by elevated levels of fluorescein isothiocyanate (FITC)-dextran, endotoxins, and bacterial translocation. Enhanced cell survival and proliferation of colonic intestinal cells were observed in Tlr13-deficient mice. A transcriptome analysis revealed that Tlr13 deficiency is associated with substantial changes in gene expression profile of colonic tumor tissue. Tlr13-deficient mice were more susceptible to CAC, with increased production of interleukin (IL)-6, IL-12, and TNF-α cytokines and enhanced STAT3, NF-κB, and MAPK signaling in colon tissues. These findings suggest that TLR13 plays a protective role in maintaining intestinal homeostasis and controlling CAC. Our study provides a novel perspective on intestinal health via TLR13-mediated signaling, which is crucial for deciphering the role of host-microbiota interactions in health and disease.

MyD88 Signaling Accompanied by Microbiota Changes Supports Urinary Bladder Carcinogenesis.

Urinary bladder cancer (BC) inflicts a significant impairment of life quality and poses a high mortality risk. Schistosoma haematobium infection can cause BC, and the urinary microbiota of BC patients differs from healthy controls. Importantly, intravesical instillation of the bacterium Bacillus Calmette-Guerin stands as the foremost therapy for non-muscle invasive BC. Hence, studying the receptors and signaling molecules orchestrating bacterial recognition and the cellular response in the context of BC is of paramount importance. Thus, we challenged Toll-like receptor 4 (Tlr4) and myeloid differentiation factor 88 (Myd88) knock-out (KO) mice with N-butyl-N-(4-hydroxylbutyl)-nitrosamine (BBN), a well-known urinary bladder carcinogen. Gut microbiota, gene expression, and urinary bladder pathology were followed. Acute exposure to BBN did not reveal a difference in bladder pathology despite differences in the animal's ability to recognize and react to bacteria. However, chronic treatment resulted in reduced cancer invasiveness among Myd88KO mice while the absence of functional Tlr4 did not influence BC development or progression. These differences correlate with a heightened abundance of the Faecalibaculum genus and the lowest microbial diversity observed among Myd88KO mice. The presented data underscore the important role of microbiota composition and MyD88-mediated signaling during bladder carcinogenesis.

Moderate Prenatal Alcohol Exposure Increases Toll-like Receptor Activity in Umbilical Cord Blood at Birth: A Pilot Study.

The prevalence of prenatal alcohol exposure (PAE) is increasing, with evidence suggesting that PAE is linked to an increased risk of infections. PAE is hypothesized to affect the innate immune system, which identifies pathogens through pattern recognition receptors, of which toll-like receptors (TLRs) are key components. We hypothesized that light-to-moderate PAE would impair immune responses, as measured by a heightened response in cytokine levels following TLR stimulation. Umbilical cord samples (10 controls and 8 PAE) from a subset of the Ethanol, Neurodevelopment, Infant and Child Health Study-2 cohort were included. Peripheral blood mononuclear cells (PMBCs) were stimulated with one agonist (TLR2, TLR3, TLR4, or TLR9). TLR2 agonist stimulation significantly increased pro-inflammatory interleukin-1-beta in the PAE group after 24 h. Pro- and anti-inflammatory cytokines were increased following stimulation with the TLR2 agonists. Stimulation with TLR3 or TLR9 agonists displayed minimal impact overall, but there were significant increases in the percent change of the control compared to PAE after 24 h. The results of this pilot investigation support further work into the impact on TLR2 and TLR4 response following PAE to delineate if alterations in levels of pro- and anti-inflammatory cytokines have clinical significance that could be used in patient management and/or attention to follow-up.

SKLB023 protects against inflammation and apoptosis in sepsis-associated acute kidney injury via the inhibition of toll-like receptor 4 signaling.

Sepsis-associated acute kidney injury (SA-AKI) is one of common critical illnesses with high morbidity and mortality. At present, effective therapeutic drugs for SA-AKI are remain lacking. SKLB023 is a synthetic small-molecule compound which exerts potent anti-inflammatory effects in our previous studies. Here, this study aimed to characterize the protective effect of SKLB023 on SA-AKI and explore its underlying mechanism. The SA-AKI experimental models have been established by cecum ligation/puncture (CLP) and lipopolysaccharide (LPS) injection in male C57BL/6J mice. SKLB023 was administered by gavage (50 or 25 mg/kg in CLP model and 50 mg/kg in LPS model) daily 3 days in advance and 30 min earlier on the day of modeling. Our results confirmed SKLB023 treatment could improve the survival of SA-AKI mice and ameliorate renal pathological injury, inflammation, and apoptosis in the two types of septic AKI mice. Mechanically, SKLB023 deceased the expression of TLR4 in LPS-triggered renal tubular epithelial cells, and inhibited the activation of downstream pathways including NF-κB and MAPK pathways. Our study suggested that SKLB023 is expected to be a potential drug for the prevention and treatment of septic AKI.

Cellular and microenvironmental cues that promote macrophage fusion and foreign body response.

During the foreign body response (FBR), macrophages fuse to form foreign body giant cells (FBGCs). Modulation of FBGC formation can prevent biomaterial degradation and loss of therapeutic efficacy. However, the microenvironmental cues that dictate FBGC formation are poorly understood with conflicting reports. Here, we identified molecular and cellular factors involved in driving FBGC formation in vitro. Macrophages demonstrated distinct fusion competencies dependent on monocyte differentiation. The transition from a proinflammatory to a reparative microenvironment, characterised by specific cytokine and growth factor programmes, accompanied FBGC formation. Toll-like receptor signalling licensed the formation of FBGCs containing more than 10 nuclei but was not essential for cell-cell fusion to occur. Moreover, the fibroblast-macrophage crosstalk influenced FBGC development, with the fibroblast secretome inducing macrophages to secrete more PDGF, which enhanced large FBGC formation. These findings advance our understanding as to how a specific and timely combination of cellular and microenvironmental factors is required for an effective FBR, with monocyte differentiation and fibroblasts being key players.

The processed Euphorbia lathyris L. alleviates the inflammatory injury via regulating LXRα/ABCA1 expression and TLR4 positioning to lipid rafts.

Euphorbia lathyris L. (EL) is a traditional poisonous herbal medicine used to treat dropsy, ascites, amenorrhea, anuria and constipation. Processing to reduce toxicity of EL is essential for its safe and effective application. However, there is little known regarding the molecular mechanism of reducing toxicity after EL processing. This research aimed to screen the differential markers for EL and PEL, explore the differential mechanisms of inflammatory injury induced by EL and processed EL (PEL) to expound the mechanism of alleviating toxicity after EL processing. The results showed that 15 potential biomarkers, mainly belonging to diterpenoids, were screened to distinguish EL from PEL. EL promoted the expressions of TLR4, NLRP3, NF-κB p65, IL-1ß and TNF-α, increased lipid rafts abundance and promoted TLR4 positioning to lipid rafts. Meanwhile, EL decreased LXRα and ABCA1 expression, and reduced cholesterol efflux. In contrast to EL, the effects of PEL on these indicators were markedly weakened. In addition, Euphorbia factors L1, L2, and L3 affected LXRα, ABCA1, TLR4, NLRP3, NF-κB p65, TNF-α and IL-1ß expression, influenced cholesterol efflux and lipid rafts abundance, and interfered with the colocalization of TLR4 and lipid rafts. The inflammatory injury caused by processed EL was significantly weaker than that caused by crude EL, and reduction of Euphorbia factors L1, L2, and L3 as well as attenuation of inflammatory injury participated in processing-based detoxification of EL. Our results provide valuable insights into the attenuated mechanism of EL processing and will guide future research on the processing mechanism of toxic traditional Chinese medicine.

Novel insights into paclitaxel's role on tumor-associated macrophages in enhancing PD-1 blockade in breast cancer treatment.

BACKGROUND: Triple-negative breast cancer (TNBC) poses unique challenges due to its complex nature and the need for more effective treatments. Recent studies showed encouraging outcomes from combining paclitaxel (PTX) with programmed cell death protein-1 (PD-1) blockade in treating TNBC, although the exact mechanisms behind the improved results are unclear. METHODS: We employed an integrated approach, analyzing spatial transcriptomics and single-cell RNA sequencing data from TNBC patients to understand why the combination of PTX and PD-1 blockade showed better response in TNBC patients. We focused on toll-like receptor 4 (TLR4), a receptor of PTX, and its role in modulating the cross-presentation signaling pathways in tumor-associated macrophages (TAMs) within the tumor microenvironment. Leveraging insights obtained from patient-derived data, we conducted in vitro experiments using immunosuppressive bone marrow-derived macrophages (iBMDMs) to validate if PTX could augment the cross-presentation and phagocytosis activities. Subsequently, we extended our study to an in vivo murine model of TNBC to ascertain the effects of PTX on the cross-presentation capabilities of TAMs and its downstream impact on CD8+ T cell-mediated immune responses. RESULTS: Data analysis from TNBC patients revealed that the activation of TLR4 and cross-presentation signaling pathways are crucial for the antitumor efficacy of PTX. In vitro studies showed that PTX treatment enhances the cross-presentation ability of iBMDMs. In vivo experiments demonstrated that PTX activates TLR4-dependent cross-presentation in TAMs, improving CD8+ T cell-mediated antitumor responses. The efficacy of PTX in promoting antitumor immunity was elicited when combined with PD-1 blockade, suggesting a complementary interaction. CONCLUSIONS: This study reveals how PTX boosts the effectiveness of PD-1 inhibitors in treating TNBC. We found that PTX activates TLR4 signaling in TAMs. This activation enhances their ability to present antigens, thereby boosting CD8+ T cell antitumor responses. These findings not only shed light on PTX's immunomodulatory role in TNBC but also underscore the potential of targeting TAMs' antigen presentation capabilities in immunotherapy approaches.

Association between Toll-like receptor 2 rs4696483 and rs1898830 polymorphisms and the risk of triple-negative breast cancer.

PURPOSE: Breast cancer (BC) is heterogeneous in clinical manifestation, of which the triple-negative (TNBC) subtype is the most aggressive. This study examines the associations between Toll-Like Receptor (TLR)-2 polymorphisms and the susceptibility to BC and TNBC. METHODS: Genotyping of TLR-2 rs1898830 and rs4696483 polymorphisms was done by real-time PCR in 488 women with BC (130 TNBC, 358 non-TNBC) and 476 cancer-free control women. RESULTS: The minor allele frequency (MAF) of rs4696483 was significantly lower in BC cases compared to controls, and significantly lower frequencies of rs4696483 C/T and higher frequencies of rs1898830 G/G genotypes were seen in BC cases. Significantly higher MAF of rs4696483 and higher C/T and T/T rs4696483 genotypes frequencies were seen in TNBC than in non-TNBC cases. Considering the prevalent AC haplotype as a reference, 2-locus TLR-2 haplotype analysis did not identify any 2-locus TLR-2 haplotype associated with an altered risk of BC or TNBC. Positive associations of rs1898830 and rs4966483 were seen with the histological type in TNBC and negatively with distant metastasis and HR status in TNBC and non-TNBC rs1898830 carriers. In addition, rs4696483 was positively correlated with hormonotherapy and surgery in non-TNBC cases, while rs1898830 was negatively associated with hormonotherapy. Furthermore, rs1898830 was negatively and positively correlated with BMI in TNBC and TNBC cases, respectively, but positively with Ki-67 status. CONCLUSIONS: Our study highlights the association between TLR-2 genetic polymorphisms and BC and TNBC susceptibility, suggesting these variants' diagnostic/prognostic capacity in BC patients and patient subgroups.

Naltrexone accelerated oral traumatic ulcer healing and downregulated TLR-4/NF-kB pathway in Wistar rats.

OBJECTIVE: To assess the effect of naltrexone on oral mucosal healing using a traumatic ulcer model DESIGN: Wistar rats (n = 112) received distilled water (control) or naltrexone (0.5, 10, or 50 mg/kg/day). Ulcers were induced on the buccal mucosa using a round skin biopsy punch (diameter 6 mm). Euthanasia was performed on days 1, 3, 7, and 14. Healing was assessed by ulcer area, histological scores, histomorphometric analysis (number of polymorphonuclears, mononuclears, and fibroblasts), and collagen percentage. Immunohistochemistry for TLR-2, TLR-4, NF-kB, and CD31 was evaluated. Nociceptive threshold was measured daily. RESULTS: The 50 mg/kg group showed reduced ulcer area on days 1 (p < 0.001), 3 (p < 0.05), and 14 (p < 0.01). In this group, there was, on day 14, an increase in the percentage of reepithelization (p = 0.043) and collagen (p < 0.05), an increase in connective tissue maturation (p = 0.016), and on day 7 an increase in fibroblasts (p < 0.001). The 10 mg/kg dose reduced the ulcer area on day 1 (p < 0.001). The 50 mg/kg group showed lower expression of TLR-4 (p < 0.001) on day 1, NF-kB on days 1 (p < 0.05) and 14 (p < 0.05), and CD31 on day 14 (p < 0.05). The 0.5 and 10 mg/kg doses reduced TLR-4 expression on day 1 (p < 0.05; p < 0.01, respectively). Nociceptive threshold increased in the 50 mg/kg group (p < 0.01). CONCLUSION: Naltrexone enhanced traumatic oral ulcer healing by reducing TLR-4/NF-kB signaling and promoting fibroblast proliferation and collagen deposition. Additionally, naltrexone reduced pain in rats.

Toll-like Receptor Homologue CD180 Ligation of B Cells Upregulates Type I IFN Signature in Diffuse Cutaneous Systemic Sclerosis.

Type I interferon (IFN-I) signaling has been shown to be upregulated in systemic sclerosis (SSc). Dysregulated B-cell functions, including antigen presentation, as well as antibody and cytokine production, all of which may be affected by IFN-I signaling, play an important role in the pathogenesis of the disease. We investigated the IFN-I signature in 71 patients with the more severe form of the disease, diffuse cutaneous SSc (dcSSc), and 33 healthy controls (HCs). Activation via Toll-like receptors (TLRs) can influence the IFN-I signaling cascade; thus, we analyzed the effects of the TLR homologue CD180 ligation on the IFN-I signature in B cells. CD180 stimulation augmented the phosphorylation of signal transducer and activator of transcription 1 (STAT1) in dcSSc B cells (p = 0.0123). The expression of IFN-I receptor (IFNAR1) in non-switched memory B cells producing natural autoantibodies was elevated in dcSSc (p = 0.0109), which was enhanced following anti-CD180 antibody treatment (p = 0.0125). Autoantibodies to IFN-Is (IFN-alpha and omega) correlated (dcSSc p = 0.0003, HC p = 0.0192) and were present at similar levels in B cells from dcSSc and HC, suggesting their regulatory role as natural autoantibodies. It can be concluded that factors other than IFN-alpha may contribute to the elevated IFN-I signature of dcSSc B cells, and one possible candidate is B-cell activation via CD180.

Mechanisms of interleukin-10 induction in murine spleen and RAW264 cells by Latilactobacillus curvatus K4G4 isolated from fermented Brassica rapa L.

Lactic acid bacteria (LAB) are commonly used in fermented foods, and some LAB modulate the immune response. We aimed to investigate the mechanism by which LAB isolates from fermented Brassica rapa L. induce the production of anti-inflammatory interleukin (IL)-10 by the murine spleen and RAW264 cells. Spleen cells from BALB/c mice or the mouse macrophage cell line RAW264 were cultured with heat-killed LAB isolated from fermented B. rapa L., and the IL-10 level in the supernatant was measured. Latilactobacillus curvatus K4G4 provided the most potent IL-10 induction among 13 isolates. Cell wall components of K4G4 failed to induce IL-10, while treatment of the bacteria with RNase A under a high salt concentration altered K4G4 induction of IL-10 by spleen cells. In general, a low salt concentration diminished the IL-10 induction by all strains, including K4G4. In addition, chloroquine pretreatment and knock down of toll-like receptor 7 through small interfering RNA suppressed K4G4 induction of IL-10 production by RAW264 cells. Our results suggest that single-stranded RNA from K4G4 is involved, via endosomal toll-like receptor 7, in the induction of IL-10 production by macrophages. K4G4 is a promising candidate probiotic strain that modulates the immune response by inducing IL-10 from macrophages.

[Effect of oxymatrine on Cryptosporidium parvum infection in mice based on the HMGB1-TLR2/TLR4-NF-κB pathway].

OBJECTIVE: To investigate the involvement of the high mobility group box protein B1 (HMGB1)-Toll-like receptor 2 (TLR2)/TLR4-nuclear factor κB (NF-κB) pathway in the intestinal mucosal injury induced by Cryptosporidium parvum infection, and to examine the effect of oxymatrine (OMT) on C. parvum infection in mice. METHODS: Forty SPF 4-week-old BALB/c mice were randomly divided into four groups, including the control group, infection group, glycyrrhizin (GA) group and OMT group. Each mouse was orally administered with 1 × 105 C. parvum oocysts one week in the infection, GA and OMT groups following dexamethasone-induced immunosuppression to model C. parvum intestinal infections in mice. Upon successful modeling, mice in the GA group were intraperitoneally injected with GA at a daily dose of 25.9 mL/kg for successive two weeks, and animals in the OMT group were orally administered OMT at a daily dose of 50 mg/kg for successive two weeks, while mice in the control group were given normal food and water. All mice were sacrificed two weeks post-treatment, and proximal jejunal tissues were sampled. The pathological changes of mouse intestinal mucosal specimens were observed using hematoxylin-eosin (HE) staining, and the mouse intestinal villous height, intestinal crypt depth and the ratio of intestinal villous height to intestinal crypt depth were measured. The occludin and zonula occludens protein 1 (ZO1) expression was determined in mouse intestinal epithelial cells using immunohistochemistry, and the relative expression of HMGB1, TLR2, TLR4, myeloid differentiation primary response gene 88 (MyD88) and NF-κB p65 mRNA was quantified in mouse jejunal tissues using quantitative real-time PCR (qPCR) assay. RESULTS: HE staining showed that the mouse intestinal villi were obviously atrophic, shortened, and detached, and the submucosal layer of the mouse intestine was edematous in the infection group as compared with the control group, while the mouse intestinal villi tended to be structurally intact and neatly arranged in the GA and OMT groups. There were significant differences among the four groups in terms of the mouse intestinal villous height (F = 6.207, P = 0.000 5), intestinal crypt depth (F = 6.903, P = 0.000 3) and the ratio of intestinal villous height to intestinal crypt depth (F = 37.190, P < 0.000 1). The mouse intestinal villous height was lower in the infection group than in the control group [(321.9 ± 41.1) µm vs. (399.5 ± 30.9) µm; t = 4.178, P < 0.01] and the GA group [(321.9 ± 41.1) µm vs. (383.7 ± 42.7) µm; t = 3.130, P < 0.01], and the mouse intestinal crypt depth was greater in the infection group [(185.0 ± 35.9) µm] than in the control group [(128.4 ± 23.6) µm] (t = 3.877, P < 0.01) and GA group [(143.3 ± 24.7) µm] (t = 2.710, P < 0.05). The mouse intestinal villous height was greater in the OMT group [(375.3 ± 22.9) µm] than in the infection group (t = 3.888, P < 0.01), and there was no significant difference in mouse intestinal villous height between the OMT group and the control group (t = 1.989, P > 0.05). The mouse intestinal crypt depth was significantly lower in the OMT group [(121.5 ± 27.3) µm] than in the infection group (t = 4.133, P < 0.01), and there was no significant difference in mouse intestinal crypt depth between the OMT group and the control group (t = 0.575, P > 0.05). The ratio of the mouse intestinal villous height to intestinal crypt depth was significantly lower in the infection group (1.8 ± 0.2) than in the control group (3.1 ± 0.3) (t = 10.540, P < 0.01) and the GA group (2.7 ± 0.3) (t = 7.370, P < 0.01), and the ratio of the mouse intestinal villous height to intestinal crypt depth was significantly higher in the OMT group (3.1 ± 0.2) than in the infection group (t = 15.020, P < 0.01); however, there was no significant difference in the ratio of the mouse intestinal villous height to intestinal crypt depth between the OMT group and the control group (t = 0.404, P > 0.05). Immunohistochemical staining showed significant differences among the four groups in terms of occludin (F = 28.031, P < 0.000 1) and ZO1 expression (F = 14.122, P < 0.000 1) in mouse intestinal epithelial cells. The proportion of positive occluding expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.3 ± 4.5)% vs. (28.3 ± 0.5)%; t = 3.810, P < 0.01], and the proportions of positive occluding expression were significantly higher in mouse intestinal epithelial cells in the GA group [(30.3 ± 1.3)%] and OMT group [(25.8 ± 1.5)%] than in the infection group (t = 7.620 and 5.391, both P values < 0.01); however, there was no significant differences in the proportion of positive occluding expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 1.791 and 2.033, both P values > 0.05). The proportion of positive ZO1 expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.4 ± 1.8)% vs. (24.2 ± 2.8)%; t = 4.485, P < 0.01], and the proportions of positive ZO1 expression were significantly higher in mouse intestinal epithelial cells in the GA group [(24.1 ± 2.3)%] (t = 5.159, P < 0.01) and OMT group than in the infection group [(22.5 ± 1.9)%] (t = 4.441, P < 0.05); however, there were no significant differences in the proportion of positive ZO1 expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group (t = 0.037 and 0.742, both P values > 0.05). qPCR assay showed significant differences among the four groups in terms of HMGB1 (F = 21.980, P < 0.000 1), TLR2 (F = 20.630, P < 0.000 1), TLR4 (F = 17.000, P = 0.000 6), MyD88 (F = 8.907, P = 0.000 5) and NF-κB p65 mRNA expression in mouse jejunal tissues (F = 8.889, P = 0.000 7). The relative expression of HMGB1 [(5.97 ± 1.07) vs. (1.05 ± 0.07); t = 6.482, P < 0.05] 、TLR2 [(5.92 ± 1.29) vs. (1.10 ± 0.14); t = 5.272, P < 0.05] 、TLR4 [(5.96 ± 1.50) vs. (1.02 ± 0.03); t = 4.644, P < 0.05] 、MyD88 [(3.00 ± 1.26) vs. (1.02 ± 0.05); t = 2.734, P < 0.05] and NF-κB p65 mRNA [(2.33 ± 0.72) vs. (1.04 ± 0.06); t = 2.665, P < 0.05] was all significantly higher in mouse jejunal tissues in the infection group than in the control group. A significant reduction was detected in the relative expression of HMGB1 (0.63 ± 0.01), TLR2 (0.42 ± 0.10), TLR4 (0.35 ± 0.07), MyD88 (0.70 ± 0.11) and NF-κB p65 mRNA (0.75 ± 0.01) in mouse jejunal tissues in the GA group relative to the control group (t = 8.629, 5.830, 11.500, 4.729 and 6.898, all P values < 0.05), and the relative expression of HMGB1, TLR2, TLR4, MyD88 and NF-κB p65 mRNA significantly reduced in mouse jejunal tissues in the GA group as compared to the infection group (t = 7.052, 6.035, 4.084, 3.165 and 3.274, all P values < 0.05). In addition, the relative expression of HMGB1 (1.14 ± 0.60), TLR2 (1.00 ± 0.24), TLR4 (1.14 ± 0.07), MyD88 (0.96 ± 0.25) and NF-κ B p65 mRNA (1.12 ± 0.17) was significantly lower in mouse jejunal tissues in the OMT group than in the infection group (t = 7.059, 5.320, 3.510, 3.466 and 3.273, all P values < 0.05); however, there were no significant differences between the OMT and control groups in terms of relative expression of HMGB1, TLR2, TLR4, MyD88 or NF-κB p65 mRNA in mouse jejunal tissues (t = 0.239, 0.518, 1.887, 0.427 and 0.641, all P values > 0.05). CONCLUSIONS: C. parvum infection causes intestinal inflammatory responses and destruction of intestinal mucosal barrier through up-regulating of the HMGB1-TLR2/TLR4-NF-κB pathway. OMT may suppress the intestinal inflammation and repair the intestinal mucosal barrier through inhibiting the activity of the HMGB1-TLR2/TLR4-NF-κB pathway.

Interplay of TLR4 and SARS-CoV-2: Unveiling the Complex Mechanisms of Inflammation and Severity in COVID-19 Infections.

The late 2019 emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, caused profound and unprecedented disruption to the global socio-economic structure, negatively affecting millions of lives worldwide. A typical hallmark of severe COVID-19 is hyper inflammation due to aberrant cytokine release (cytokine storm) by innate immune cells. Recent studies have revealed that SARS-CoV-2, through its spike (S) protein, can activate the body's innate immune cells via Toll-Like Receptors (TLRs), particularly TLR4. In silico studies have demonstrated that the S protein binds with high affinity to TLR4, triggering downstream signaling processes that result in pro-inflammatory cytokine release. Compared to other TLRs, such as TLR2, TLR4 plays a more significant role in initiating and sustaining the inflammatory response associated with severe COVID-19. Furthermore, interactions between the virus and target cells can enhance the cellular expression of TLR4, making cells more susceptible to viral interactions and subsequent inflammation. This increased expression of TLR4 upon viral entry creates a feedback loop, where heightened TLR4 levels lead to amplified inflammatory responses, contributing to the severity of the disease. Additionally, TLR4's potent activation of inflammatory pathways sets it apart from other TLRs, underscoring its pivotal role in the pathogenesis of COVID-19. In this review, we thoroughly explore the multitude of regulatory signaling pathways that SARS-CoV-2 employs to incite inflammation. We specifically focus on the critical impact of TLR4 activation compared to other TLRs, highlighting how TLR4's interactions with the viral S protein can exacerbate the severity of COVID-19. By delving into the mechanisms of TLR4-mediated inflammation, we aim to shed light on potential therapeutic targets that could mitigate the inflammatory damage caused by severe COVID-19. Understanding the unique role of TLR4 in the context of SARS-CoV-2 infection could pave the way for novel treatment strategies that specifically inhibit this receptor's activity, thereby reducing the overall disease burden and improving patient outcomes.

Evaluation of the adjuvant effect of imiquimod and CpG ODN 1826 in chimeric DNA vaccine against Japanese encephalitis.

Vaccines represent a significant milestone in the history of human medical science and serve as the primary means for controlling infectious diseases. In recent years, the geographical distribution of Japanese encephalitis viruses (JEV) of various genotypes has become increasingly complex, which provides a rationale for the development of safer and more effective vaccines. The advent of subunit and nucleic acid vaccines, especially propelled by advancements in genetic engineering since the 1980s, has accelerated the application of novel adjuvants. These novel vaccine adjuvants have diversified into toll-like receptor (TLR) agonists, complex adjuvants, nanoparticles and so on. However, the efficacy of adjuvant combinations can vary depending on the host system, disease model, or vaccine formulation, sometimes resulting in competitive or counteractive effects. In our previous study, we constructed a pJME-LC3 chimeric DNA vaccine aimed at inducing an immune response through autophagy induction. Building on this, we investigated the impact of the TLR7/8 agonist imiquimod (IMQ) and the TLR9 agonist CpG ODN 1826 as adjuvants on the immunogenicity of the Japanese encephalitis chimeric DNA vaccine. Our findings indicate that the combination of the pJME-LC3 vaccine with IMQ and CpG ODN 1826 adjuvants enhanced the innate immune response, promoting the maturation and activation of antigen-presenting cells in the early immune response. Furthermore, it played a regulatory and optimizing role in subsequent antigen-specific immune responses, resulting in effective cellular and humoral immunity and providing prolonged immune protection. The synergistic effect of IMQ and CpG ODN 1826 as adjuvants offers a novel approach for the development of Japanese encephalitis nucleic acid vaccines.