Toll-like Receptor Ligands Research Articles

EXTH-40. IMMUNOPHENOTYPING OF RWTC-MBTA AUTOLOGOUS CANCER VACCINE IN PRECLINICAL GLIOBLASTOMA MODELS

Abstract Despite advances in immunotherapy, glioblastoma multiforme (GBM) remains challenging to treat due to its low inherent immunogenicity and a suppressive tumor microenvironment. Converting “cold” GBMs to “hot” tumors is crucial for effective immune activation and improved patient outcomes. We comprehensively characterized the rWTC-MBTA autologous cancer vaccine, consisting of Mannan-BAM-anchored irradiated whole tumor cells, Toll-like receptor ligands (LTA, Poly(I:C), R-848), and an anti-CD40 agonistic antibody, in preclinical GL261 and SB28 GBM models. A substantial number of vaccinated mice exhibited complete regression of GBM tumors in a T-cell-dependent manner, with no significant toxicity. Long-term tumor-specific immune memory was confirmed upon tumor rechallenge. In the vaccine-draining lymph nodes of the SB28 model, rWTC-MBTA vaccination triggered a major rise in cDC1 12 hours post-treatment, followed by an increase in cDC2, moDC, and pDC on Days 5 and 13. Enhanced cytotoxicity of CD4+ and CD8+ T cells in vaccinated mice was verified in co-culture with tumor cells. Comprehensive immunophenotyping in the GL261 and SB28 GBM microenvironments confirmed decreased regulatory T cells and increased CD4+ and CD8+ T cell infiltration in vaccinated mice. T-cell exhaustion analyses revealed an increase in the Tim3+CD8+ T cells. No significant differences were observed in the quantification of M-MDSC and PMN-MDSC between control and vaccinated mice, but PD-L1 expression significantly increased in M2 macrophages in vaccinated mice. Our findings demonstrate that rWTC-MBTA induces potent and long-term adaptive immune responses against GBM, warranting further investigation of combinations with other immunotherapies for enhanced efficacy.

Cell damage shifts the microRNA content of small extracellular vesicles into a Toll-like receptor 7-activating cargo capable to propagate inflammation and immunity

BackgroundThe physiological relevance of cell-to-cell communication mediated by small extracellular vesicle-encapsulated microRNAs (sEV-miRNAs) remains debated because of the limiting representativity of specific miRNAs within the extracellular pool. We hypothesize that sEV-miRNA non-canonical function consisting of the stimulation of Toll-like receptor 7 (TLR7) may rely on a global shift of the sEV cargo rather than on the induction of one or few specific miRNAs. Psoriasis represents an ideal model to test such hypothesis as it is driven by overt activation of TLR7-expressing plasmacytoid dendritic cells (pDCs) following keratinocyte damage.MethodsTo mimic the onset of psoriasis, keratinocytes were treated with a cocktail of psoriatic cytokines or UV-irradiated. SmallRNA sequencing was performed on sEVs released by healthy and UV-treated keratinocytes. sEV-miRNAs were analyzed for nucleotide composition as well as for the presence of putative TLR7-binding triplets. Primary human pDCs where stimulated with sEVs +/- inhibitors of TLR7 (Enpatoran), of sEV release (GW4869 + manumycin) and of TLR7-mediated pDC activation (anti-BDCA-2 antibody). Secretion of type I IFNs and activation of CD8+T cells were used as readouts. qPCR on psoriatic and healthy skin biopsies was conducted to identify induced miRNAs.ResultssEV-miRNAs released by damaged keratinocytes revealed a significantly higher content of TLR7-activating sequences than healthy cells. As expected, differential expression analysis confirmed the presence of miRNAs upregulated in psoriatic skin, including miR203a. More importantly, 76.5% of induced miRNAs possessed TLR7-binding features and among these we could detect several previously demonstrated TLR7 ligands. In accordance with this in silico analysis, sEVs from damaged keratinocytes recapitulated key events of psoriatic pathogenesis by triggering pDCs to release type I interferon and activate cytotoxic CD8+T cells in a TLR7- and sEV-dependent manner.DiscussionOur results demonstrate that miR203a is just one paradigmatic TLR7-activating miRNA among the hundreds released by UV-irradiated keratinocytes, which altogether trigger pDC activation in psoriatic conditions. This represents the first evidence that cell damage shifts the miRNA content of sEVs towards a TLR7-activating cargo capable to propagate inflammation and immunity, offering strong support to the physiological role of systemic miRNA-based cell-to-cell communication.

Primary sensory neuron-derived miR-let-7b underlies stress-elicited psoriasis

Psoriasis, a chronic autoimmune skin condition with significant global morbidity, badly impairs patients’ quality of life. Stress has been identified as a prominent trigger for psoriasis, and effectively management of stress can ameliorate its pathological manifestations. However, the precise mechanisms by which stress influences psoriasis remain elusive. In this study, we found that mice subjected to chronic social defeat stress (CSDS) exhibit severer imiquimod (IMQ)-induced psoriasis with increased epidermal scaling, epidermal hyperplasia, number of epidermal ridges, itch, and skin inflammation than control mice. Mechanistic study reveals that CSDS leads to an elevated release of miR-let-7b, an endogenous ligand of Toll-like receptor 7 (TLR7), from the peripheral terminal of dorsal root ganglia (DRG) neurons into the skin. This process can stimulate skin-resident macrophages to release cytokines (such as IL-6 and TNF-a) and chemokines (such as MCP-1), subsequently promoting the recruitment of additional macrophages into the skin. Notably, the specific blockade of miR-let-7b in DRG neurons effectively relieve stress-induced exacerbations of psoriasis. Furthermore, intradermal injection of synthetic miR-let-7b can induce a psoriasis-like phenotype in wildtype mice, a phenomenon that can be countered by the application of a TLR7 antagonist. Additionally, microfluidic chamber coculture assays demonstrated that miR-let-7b released by DRG neurons activates macrophages via TLR7 expressed on these immune cells. Totally, this study found that stress-induced upregulation and release of miR-let-7b from DRG neurons stimulates macrophages to secrete more inflammatory cytokines and chemokines, thereby exacerbating skin inflammation and the psoriatic phenotype. These findings provide a potential therapeutic strategy targeting the miR-let-7b/TLR7 pathway to alleviate stress-induced exacerbation of psoriasis.

Toll-like receptors ligand immunomodulators for the treatment congenital diaphragmatic hernia

BackgroundCongenital diaphragmatic hernia (CDH) is a rare disease that affects the development of the diaphragm, leading to abnormal lung development. Unfortunately, there is no established therapy for CDH. Retinoic acid pathways are implicated in the ethology of CDH and macrophages are known to play a role in repairing organ damage.MethodsWe have analyzed the effect of several Toll like receptor (TLR) ligands in the nitrofen-induced CDH model in pregnant rats widely used to study this disease and in the G2-GATA4Cre;Wt1fl/fl CDH genetic mice model. Morphometric and histological studies were carried out. Immune cell infiltration was assayed by immunochemistry and immunofluorescence and retinoic pathway gene expression analyzed in vivo and in vitro in macrophages.ResultsWe found that administering a single dose of atypical TLR2/4 ligands (CS1 or CS2), 3 days after nitrofen, cured diaphragmatic hernia in 73% of the fetuses and repaired the lesion with complete diaphragm closure being on the other hand nontoxic for the mothers or pups. Moreover, these immunomodulators also improved pulmonary hypoplasia and alveolar maturation and vessel hypertrophy, enhancing pulmonary maturity of fetuses. We also found that CS1 treatment rescued the CDH phenotype in the G2-GATA4Cre;Wt1fl/fl CDH genetic mice model. Only 1 out of 11 mutant embryos showed CDH after CS1 administration, whereas CDH prevalence was 70% in untreated mutant embryos. Mechanistically, CS1 stimulated the infiltration of repairing M2 macrophages (CD206+ and Arg1+) into the damaged diaphragm and reduced T cell infiltration. Additionally, those TLR ligands induced retinol pathway genes, including RBP1, RALDH2, RARα, and RARβ, in the affected lungs and the diaphragm and in macrophages in vitro.ConclusionsOur research has shown that TLR ligand immunomodulators that influence anti-inflammatory macrophage activation can be effective in treating CDH, being nontoxic for the mothers or pups suggesting that those TLR ligands are a promising solution for CDH leading to orphan drug designation for CS1. The immune system of the fetus would be responsible for repairing the damage and closure of the hernia in the diaphragm and enhanced proper lung development after CS1 treatment.

Salmonella Typhimurium infection inhibits macrophage IFNβ signaling in a TLR4-dependent manner.

Type I Interferons (IFNs) generally have a protective role during viral infections, but their function during bacterial infections is dependent on the bacterial species. Legionella pneumophila, Shigella sonnei and Mycobacterium tuberculosis can inhibit type I IFN signaling. Here we examined the role of type I IFN, specifically IFNβ, in the context of Salmonella enterica serovar Typhimurium (STm) macrophage infections and the capacity of STm to inhibit type I IFN signaling. We demonstrate that IFNβ has no effect on the intracellular growth of STm in infected bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice. STm infection inhibits IFNβ signaling but not IFNγ signaling in a murine macrophage cell line. We show that this inhibition is independent of the type III and type VI secretion systems expressed by STm and is also independent of bacterial phagocytosis. The inhibition is Toll-like receptor 4 (TLR4)-dependent as the TLR4 ligand, lipopolysaccharide (LPS), alone is sufficient to inhibit IFNβ-mediated signaling. Cells downregulated their surface levels of IFNα/β receptor 1 (IFNAR1) in response to LPS, which may be mediating our observed inhibition. Lastly, we examined this inhibition in the context of TLR4-deficient BMDMs as well as TLR4 RNA interference and we observed a loss of inhibition with LPS stimulation as well as STm infection. In summary, we show that macrophages exposed to STm have reduced IFNβ signaling via crosstalk with TLR4 signaling, which may be mediated by reduced host cell surface IFNAR1, and that IFNβ signaling does not affect cell-autonomous host defense against STm.

Mono-palmitoyl-N-alkylurea ligands as specific activators of human Toll-like receptor 2/6 heterodimer.

Ligands for Toll-like-receptor 2 (TLR2) have demonstrated significant potential as immune-stimulating components in synthetic vaccines. Activation of TLR2 relies on the formation of dimeric complexes with either TLR1 or TLR6 and the nature of these dimers can impact therapeutic outcomes. The lipopeptide-based TLR2 ligands Pam3CysSK4 and Pam2CysSK4 have been extensively studied, and their recognition by different TLR-receptor heterodimers, TLR2/TLR1 and TLR2/TLR6, respectively, has been established. However, the high lipophilicity of these ligands, containing multiple palmitoyl residues, can result in solubility issues when used as vaccine adjuvants. To address this, we previously synthesized a less lipophilic ligand containing a single palmitoyl chain called mini-UPam, which effectively stimulates human moDC maturation. We here probe the receptor-dimer specificity of several mini-Upam derivatives and reveal that these mini-UPam are hTLR2/TLR6 selective ligands and that the introduction of longer urea alkyl chains does not shift the binding specificity to hTLR2/TLR1 heterodimers, in contrast to their Pam2CysSK4 and Pam3CysSK4 counterparts, pointing to a different binding mode of the UPam ligands.

Downregulation of type I interferon signalling pathway by urate in primary human PBMCs.

Type I interferons (IFN1s) mediate innate responses to microbial stimuli and regulate interleukin (IL)-1 and IL-1 receptor antagonist (Ra) production in human cells. This study explores interferon-stimulated gene (ISG) alterations in the transcriptome of patients with gout and stimulated human primary cells in vitro in relation to serum urate concentrations. Peripheral blood mononuclear cells (PBMCs) and monocytes of patients with gout were primed in vitro with soluble urate, followed by lipopolysaccharide (LPS) stimulation. Separately, PBMCs were stimulated with various toll-like receptor (TLR) ligands. RNA sequencing and IL-1Ra cytokine measurement were performed. STAT1 phosphorylation was assessed in urate-treated monocytes. Cytokine responses to IFN-β were evaluated in PBMCs cultured with or without urate and restimulated with LPS and monosodium urate (MSU) crystals. Transcriptomics revealed suppressed IFN-related signalling pathways in urate-exposed PBMCs or monocytes which was supported by diminishment of phosphorylated STAT1. The stimulation of PBMCs with IFN-β did not modify the urate-induced inflammation. Interestingly, in vivo, serum urate concentrations were inversely correlated to in vitro ISG expression upon stimulations with TLR ligands. These findings support a deficient IFN1 signalling in the presence of elevated serum urate concentrations, which could translate to increased susceptibility to infections.

Intranasal immunization with poly I:C and CpG ODN adjuvants enhances the protective efficacy against Helicobacter pylori infection in mice

Helicobacter pylori (H. pylori) infection is a serious public health issue, and development of vaccines is a desirable preventive strategy for H. pylori. Toll-like receptor (TLR) ligands have shown potential as vaccine adjuvants that induce immune responses, but polyinosinic-polycytidylic acid (poly I:C), a nucleic acid-based TLR9 ligand, is less well studied in H. pylori vaccine research. Here, we evaluated the effects of poly I:C and CpG oligodeoxynucleotide (CpG ODN), a nucleic acid TLR3 ligand, as adjuvants in combination with the H. pylori recombinant proteins LpoB and UreA to protect against H. pylori infection. For analysis of specific immune responses, the levels of specific antibodies and splenic cytokines were measured in the immunized mice. Compared with CpG ODN, poly I:C could induce mucosal sIgA antibody responses and reduce H. pylori colonization. Additionally, the combination of poly I:C and CpG ODN caused greater immunoprotection and significantly reduced gastritis, exerting synergistic effects. Analysis of splenic cytokines revealed that poly I:C mainly triggered a mixed Th1/Th2/Th17 immune response, whereas the combination of CpG ODN and poly I:C induced a Th1/Th17 immune response. Our findings indicated that increased levels of mucosal sIgA antibodies and a robust splenic Th1/Th17 immune response were associated with reduced H. pylori colonization in vaccinated mice. This study identified a potential TLR ligand adjuvant for developing more effective H. pylori vaccines.

B cell αv integrin regulates germinal center derived lung-resident IgA B cell responses following influenza virus infection.

Emerging studies have highlighted the importance of tissue-resident B cells in the lungs, for protective immunity against respiratory viruses. However, the mechanisms controlling generation and maintenance of such tissue-resident B cells at respiratory sites remain obscure. We have previously shown that αv integrins limit B cell responses to antigens containing Toll-like receptor ligands, and that deletion of B cell αv integrins, in mice, enhances germinal center (GC)-derived long-lived B cell responses after systemic immunization with viral antigens. Here we investigated whether αv also regulates B cell responses at the respiratory tract during viral infection. Our data show that αv integrin restricts tissue-resident B cell responses in the airway, and that deletion of B cell αv promotes generation of lung-resident IgA B cell responses following influenza A virus (IAV) infection. Investigating the mechanism for this, we found that loss of B cell αv, promotes persistence of GC reactions locally in the lungs, which leads to increases in lung-resident IgA+ memory B cells, cross-reactive to antigenic variants. Thus, these studies reveal how IgA B cells are maintained in the lungs and point to a new strategy to improve the durability of lung-resident IgA B cell responses for IAV vaccine efficacy.

Toll-like Receptors: Therapeutic Potential in Life Threatening Diseases-Cardiac Disorders.

Toll-like receptors (TLRs) belong to the innate immune system. TLRs identify and respond to invading pathogens by recognizing certain molecular patterns associated with the infections. TLRs are crucial for the host's defence against these diseases. TLRs are capable of detecting several endogenous chemicals through the recognition of damage-associated molecular patterns, which are generated in response to various harmful situations. Recent animal studies have shown that TLR signaling has a significant role in the development of serious heart diseases, such as ischemia myocardial damage, myocarditis, and septic cardiomyopathy, where inflammation of the heart muscle is a key factor. This manuscript examines the animal research findings on (1) TLRs, TLR ligands, and the signal transduction system, and (2) the significant involvement of TLR signaling in these crucial cardiac diseases.

Cylindromatosis lysine 63 deubiquitinase (CYLD) suppress TLR3-mediated CCL5 expression in human renal proximal tubular epithelial cells.

One of the causes of tubulointerstitial nephritis is viral infection, with innate immune responses affecting its pathogenesis. Toll-like receptor 3 (TLR3) recognizes viral infections and acts antivirally by activating signaling to produce inflammatory cytokines/chemokines, including C-C motif chemokine ligand 5 (CCL5) and interferon-β (IFN-β). Although cylindromatosis lysine 63 deubiquitinase (CYLD) is known to be associated with tubulointerstitial nephritis and renal function, its role in the antiviral innate immune response in tubular epithelial cells remains unknown. In this study, we investigated the association between CYLD and TLR3-mediated CCL5 production in cultured human renal proximal tubular epithelial cells (hRPTECs). Polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 ligand, was used to stimulate hRPTECs. mRNA expression was measured using reverse transcription-quantitative polymerase chain reaction. Protein expression was assayed using western blotting or an enzyme-linked immunosorbent assay. Knockdown of IFN-β, nuclear factor-kappa B (NF-κB) p65, and CYLD was performed by transfecting cells with specific small interfering RNAs. The intracellular localization of CYLD in hRPTECs was analyzed using immunofluorescence. Poly IC induced CCL5 expression in a time- and concentration-dependent manner, and knockdown of either IFN-β or p65 reduced poly IC-induced CCL5 expression. CYLD knockdown increased the poly IC-induced CCL5, phosphorylated IκB kinase α/β (IKK complex), and phosphorylated p65 expression. The CYLD protein was localized in the cytoplasm, and poly IC did not alter its expression. CYLD may prevent excessive inflammation due to an antiviral innate immune response by suppressing IKK complex and NF-κB activation downstream of TLR3 in hRPTECs.

Comparison of antigen-specific B cell responses reveals disparity in immunogenicity and memory B cell formation across COVID-19 vaccine platforms

Various vaccine technologies have been employed in the coronavirus disease 2019 (COVID-19) vaccines, including whole inactivated virus (WIV), recombinant protein, mRNA, and nanoparticle vaccines. To elucidate the cellular mechanisms underlying the immune responses elicited by different vaccines, we examined and compared antigen-specific B cell responses targeting the receptor-binding domain (RBD) of the viral spike protein. We found that the nanoparticle vaccine pathogen-like antigens-RBD (PLA-RBD) and the mRNA vaccine demonstrated superior immunogenicity compared with the WIV vaccine and the RBD-dimer, a recombinant protein vaccine. Interestingly, the WIV vaccine contains toll-like receptor ligands that enhance IgG2a/c class-switching. For the mRNA vaccine, although it induces robust germinal center responses and T follicular helper (Tfh) cells, it has limited ability to induce memory B cells and long-lived plasma cells. These results indicate that vaccine formats significantly influence both the quantity and quality of immune responses, providing valuable insights for the future development of vaccines.

Damage sensing through TLR9 Regulates Inflammatory and Antiviral Responses During Influenza Infection.

Host response aimed at eliminating the infecting pathogen, as well as the pathogen itself, can cause tissue injury. Tissue injury leads to the release of a myriad of cellular components including mitochondrial DNA, which the host senses through pattern recognition receptors. How the sensing of tissue injury by the host shapes the anti-pathogen response remains poorly understood. In this study, we utilized mice that are deficient in toll-like receptor-9 (TLR9), which binds to unmethylated CpG DNA sequences such as those present in bacterial and mitochondrial DNA. To avoid direct pathogen sensing by TLR9, we utilized the influenza virus, which lacks ligands for TLR9, to determine how damage sensing by TLR9 contributes to anti-influenza immunity. Our data show that TLR9-mediated sensing of tissue damage promotes an inflammatory response during early infection, driven by the epithelial and myeloid cells. Along with the diminished inflammatory response, the absence of TLR9 led to impaired viral clearance manifested as a higher and prolonged influenza components in myeloid cells including monocytes and macrophages rendering them highly inflammatory. The persistent inflammation driven by infected myeloid cells led to persistent lung injury and impaired recovery in influenza-infected TLR9-/- mice. Further, we show elevated TLR9 activation in the plasma samples of patients with influenza and its association with the disease severity in hospitalized patients, demonstrating its clinical relevance. Overall, we demonstrate an essential role of damage sensing through TLR9 in promoting anti-influenza immunity and inflammatory response.

Inflammatory disease status and response to TNF blockade are associated with mechanisms of endotoxin tolerance

The mechanisms of endotoxin tolerance (ET), which down-regulate inflammation, are well described in response to exogenous toll-like receptor ligands, but few studies have focused on ET-associated mechanisms in inflammatory disease. As blocking TNF can attenuate the development of ET, the effect of anti-TNF on the expression of key ET-associated molecules in inflammatory auto-immune disease was measured; changes in inflammatory gene expression were confirmed using an ET bioassay. The expression of immunomodulatory molecules was measured in a murine model of arthritis treated with anti-TNF and the expression of ET-associated molecules was measured in whole blood in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients, before and after therapy. The expression of ET-associated genes was also measured in RA patient monocytes before and after therapy, in anti-TNF responders and non-responders. Tnfaip3, Ptpn6 and Irak3 were differentially expressed in affected paws, spleens, lymph nodes and circulating leucocytes in experimental murine arthritis treated with anti-TNF. Prior to therapy, the expression of TNFAIP3, INPP5D, PTPN6, CD38 and SIGIRR in whole blood differed between human healthy controls and RA or AS patients. In blood monocytes from RA patients, the expression of TNFAIP3 was significantly reduced by anti-TNF therapy in non-responders. Prior to therapy, anti-TNF non-responders had higher expression of TNFAIP3 and SLPI, compared to responders. Although the expression of TNFAIP3 was significantly higher in RA non-responders prior to treatment, the post-treatment reduction to a level similar to responders did not coincide with a clinical response to therapy.

The activation of TLR4-MyD88 signaling promotes hepatic dysfunction and fibrotic changes in SD rats resulting from prolonged exposure to sodium arsenite

Arsenic, a poisonous metalloid element, is linked to liver diseases, but the exactmechanisms for this process are not yet to be completely elucidated. Toll like receptor 4 (TLR4), acting as a pathogenic pattern recognition receptor, plays a pivotal role in various inflammatory diseases via the myeloid differentiation factor 88 (MyD88) pathway. This study aims to investigate the involvement of the TLR4-MyD88 signaling pathway in liver injury induced by prolonged exposure to sodium arsenite (NaAsO2) in Sprague-Dawley rats. Our research findings demonstratethe activation of TLR4-MyD88 signaling pathway in long-term NaAsO2-exposed rat liver tissues, leading to a significant release of inflammatory factors, which suggests its potential involvement in the pathogenesis of NaAsO2-induced liver injury. We further administered lipopolysaccharide (LPS), a natural ligand of TLR4, and TAK-242, a specific inhibitor of TLR4, to rats in order to validate the specific involvement of the TLR4-MyD88 signaling pathway in NaAsO2-induced liver injury. The results showed that, 1 mg/kg.bw LPS treatment significantly activated TLR4-MyD88 signalling pathway and its mediated pro-inflammatory factors, leading to up-regulation of activation indicators in hepatic stellate cells (HSCs) as well as increased secretion levels of extracellular matrix (ECM) in the liver, and ultimately induced liver fibrosis and dysfunction in rats. Relevantly, subsequent administration of 0.5 mg/kg.bw TAK-242 significantly attenuated the expression levels of TLR4 and its associated proteins, mitigated collagen deposition, and partially improved liver fibrosis and dysfunction caused by NaAsO2 in rats. Our study fully confirms the pivotal role of the TLR4-MyD88 signaling in promoting liver injury induced by NaAsO2, thereby providing a novel molecular target for preventing and treating patients with arsenic poisoning-related liver injury.

Hepatokines: unveiling the molecular and cellular mechanisms connecting hepatic tissue to insulin resistance and inflammation.

Insulin resistance arising from Non-Alcoholic Fatty Liver Disease (NAFLD) stands as a prevalent global ailment, a manifestation within societies stemming from individuals' suboptimal dietary habits and lifestyles. This form of insulin resistance emerges as a pivotal factor in the development of type 2 diabetes mellitus (T2DM). Emerging evidence underscores the significant role of hepatokines, as hepatic-secreted hormone-like entities, in the genesis of insulin resistance and eventual onset of type 2 diabetes. Hepatokines exert influence over extrahepatic metabolism regulation. Their principal functions encompass impacting adipocytes, pancreatic cells, muscles, and the brain, thereby playing a crucial role in shaping body metabolism through signaling to target tissues. This review explores the most important hepatokines, each with distinct influences. Our review shows that Fetuin-A promotes lipid-induced insulin resistance by acting as an endogenous ligand for Toll-like receptor 4 (TLR-4). FGF21 reduces inflammation in diabetes by blocking the nuclear translocation of nuclear factor-κB (NF-κB) in adipocytes and adipose tissue, while also improving glucose metabolism. ANGPTL6 enhances AMPK and insulin signaling in muscle, and suppresses gluconeogenesis. Follistatin can influence insulin resistance and inflammation by interacting with members of the TGF-β family. Adropin show a positive correlation with phosphoenolpyruvate carboxykinase 1 (PCK1), a key regulator of gluconeogenesis. This article delves into hepatokines' impact on NAFLD, inflammation, and T2DM, with a specific focus on insulin resistance. The aim is to comprehend the influence of these recently identified hormones on disease development and their underlying physiological and pathological mechanisms.

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

BackgroundColorectal 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.AimTo 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.MethodsFrom 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.ResultsOur 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.ConclusionMicroRNAs could act as antagonistic ligands of TLRs limiting the inflammatory tumor microenvironment.

TLR Agonists Modify NK Cell Activation and Increase Its Cytotoxicity in Acute Lymphoblastic Leukemia.

Natural killer (NK) cells play a crucial role in innate immunity, particularly in combating infections and tumors. However, in hematological cancers, NK cells often exhibit impaired functions. Therefore, it is very important to activate its endosomal Toll-like receptors (TLRs) as a potential strategy to restore its antitumor activity. We stimulated NK cells from the peripheral blood mononuclear cells from children with acute lymphoblastic leukemia and NK cells isolated, and the NK cells were stimulated with specific TLR ligands (Poly I:C, Imiquimod, R848, and ODN2006) and we evaluated changes in IFN-γ, CD107a, NKG2D, NKp44 expression, Granzyme B secretion, cytokine/chemokine release, and cytotoxic activity. Results revealed that Poly I:C and Imiquimod enhanced the activation of both immunoregulatory and cytotoxic NK cells, increasing IFN-γ, CD107a, NKG2D, and NKp44 expression. R848 activated immunoregulatory NK cells, while ODN2006 boosted CD107a, NKp44, NKG2D, and IFN-γ secretion in cytotoxic NK cells. R848 also increased the secretion of seven cytokines/chemokines. Importantly, R848 and ODN 2006 significantly improved cytotoxicity against leukemic cells. Overall, TLR stimulation enhances NK cell activation, suggesting TLR8 (R848) and TLR9 (ODN 2006) ligands as promising candidates for antitumor immunotherapy.

Immunostimulatory effects of Toll-like receptor ligands as adjuvants in establishing a novel mouse model for pemphigus vulgaris.

The meticulous selection of appropriate vaccine adjuvants is crucial for optimizing immune responses. Traditionally, pemphigus vulgaris (PV), an autoimmune disorder, has been modelled using complete Freund's adjuvant (CFA). In this study, we aimed to discern potential variations in immune responses elicited by Toll-like receptor (TLR) ligands as compared to CFA. A comprehensive investigation was conducted, comparing the effects of these adjuvants in conjunction with ovalbumin or desmoglein-3. Flow cytometry was employed to analyse distinct cell subsets, while enzyme-linked immunosorbent assay quantified antigen-specific antibodies and cytokine levels. Histological examination of harvested skin tissues and transcriptome analysis of skin lesions were performed to identify differentially expressed genes. TLR ligands demonstrated efficacy in inducing PV-like symptoms in wild-type mice, in contrast to CFA. This underscored the substantial impact of the adjuvant on self-antigen tolerance. Furthermore, we proposed an enhanced method for establishing a PV model through adoptive transfer, substituting CFA with TLR ligands. Our results revealed that in contrast to the perception that CFA being the most potent immunopotentiator reported, CFA promoted regulatory T cells (Treg), follicular regulatory T cells and IL-10-producing neutrophils, whereas TLR ligands downregulated CCL17 and IL-10. This suggested potential implications for the recruitment and activation of Treg subsets. While B cell and CD8+ T cell responses exhibited similarity, CFA induced less activation in dendritic cell subsets. A novel mouse model of PV and systemic comparison of immunostimulatory effects of adjuvants were provided by this study. The systematic comparison of CFA and TLR ligands shed light on the distinctive properties of these adjuvants, presenting innovative mouse models for the investigation of pemphigus. This study significantly contributes to adjuvant research and advances our understanding of PV pathogenesis. Immunization with desmoglein 3 and Toll-like receptor (TLR) ligands effectively induces pemphigus symptoms in wild-type mice, whereas complete Freund's adjuvant (CFA) fails. TLR ligands heightened the autoreactivity of donor cells in the adoptive transfer pemphigus model. CFA promoted regulatory T cells and IL-10-producing neutrophils, whereas TLR ligands downregulated CCL17 and IL-10, leading to more effective immune responses.

Candida and Long Covid: Mannan Not from Heaven

The pandemic has supercharged growing awareness of the gut microbiome as a critical determinant of human health. “Long haulers” share microbiomes similar to those seen in myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia, all frequently associated with Candida overgrowth (CO). Candida can synthesize its own IDO, altering tryptophan metabolism (ATM). Zonulin, a circulating protein that increases intestinal and endothelial permeability, has emerged as a central player. Candida hyphal walls express proteins analogous to gliadin/gluten, e.g., celiac disease (CeD), and mannans, e.g., Crohn’s disease (CrD), that may trigger antigliadin and anti-Gq coupled GPCR auto-antibodies linked to their lectin binding domain respectively. Hyphal mannan may induce auto-antibodies to AT1Rs, α1-ARs, mAChRs, and β2-ARs, prominent in LC, and regulate T cell receptors (TCRs) and regulatory B cell function, compromised in not only LC (vitiligo, psoriasis, alopecia) but also SLE, RA, and many other autoimmune diseases. All are Gq coupled GPCRs. The spike protein S on SARS CoV2 can attach to both the ACE2 receptor (required for tryptophan absorption) and Toll-like receptor4 (TLR4) bearing endothelial cells and enterocytes. Spike protein S is persistent in most with LC and, as a ligand for TLR4, can also activate zonulin. S can also activate the NLRP3 inflammasome, as can candidalysin. This inflammasome is directly connected to dementia, cancer, autoimmunity and obesity. Candidalysin causes hypercitrullination, instrumental in creating ACPAs (anti-citrullinated peptide antibodies) linked to LC, MCAS (mast cell activation syndrome), HSD (hypermobility spectrum disorder), and APS (antiphospholipid syndrome). A hypothetical pathophysiologic model is proposed implicating pre-existing CO, aggravated by Covid-19, in not only the genesis of LC but also that of autoimmune disease, dementia, cancer, many chronic diseases, and aging.