TLR4-dependent Manner Research Articles

Anemoside A3 activates TLR4-dependent M1-phenotype macrophage polarization to represses breast tumor growth and angiogenesis

The polarization of macrophages has been previously demonstrated to be closely related to immune and inflammatory processes in the tumorigenesis and progression of breast cancer. In the present study, Anemoside A3 (A3), an active compound from Pulsatilla saponins, was screened out and polarized M0 macrophages into the classically activated macrophages (M1-phenotype). We found that A3 is an activator of TLR4/NF-κB/MAPK signaling pathway. A3 increased the expression of CD86+ (a marker of M1 macrophage) in M0 macrophage, and increased the typical M1 macrophage pro-inflammatory cytokines TNF-α, and IL-12 expression in a TLR4-dependent manner. A macrophage-cancer cell co-culture system was established to evaluate whether A3 can could switch tumor-associated macrophages (TAMs) to the M1-phenotype. In the co-culture system, A3 increased the expression of IL-12 in macrophages, whereby suppressing MCF-7 breast cancer cell line proliferation and VEGF-mediated angiogenesis. Moreover, A3 induced M1 macrophage polarization in the 4 T1 murine breast cancer model and effectively inhibited tumor growth and tumor angiogenesis. Collectively, these findings indicated that A3 induced M1 macrophages polarization to repress breast tumorigenesis via targeting the TLR4/NF-κB/MAPK signaling pathway. This study provides a rationale for utilizing traditional Chinese medicine extracts in the immunotherapy of breast cancer.

Branched chain fatty acid synthesis drives tissue-specific innate immune response and infection dynamics of Staphylococcus aureus.

Fatty acid-derived acyl chains of phospholipids and lipoproteins are central to bacterial membrane fluidity and lipoprotein function. Though it can incorporate exogenous unsaturated fatty acids (UFA), Staphylococcus aureus synthesizes branched chain fatty acids (BCFA), not UFA, to modulate or increase membrane fluidity. However, both endogenous BCFA and exogenous UFA can be attached to bacterial lipoproteins. Furthermore, S. aureus membrane lipid content varies based upon the amount of exogenous lipid in the environment. Thus far, the relevance of acyl chain diversity within the S. aureus cell envelope is limited to the observation that attachment of UFA to lipoproteins enhances cytokine secretion by cell lines in a TLR2-dependent manner. Here, we leveraged a BCFA auxotroph of S. aureus and determined that driving UFA incorporation disrupted infection dynamics and increased cytokine production in the liver during systemic infection of mice. In contrast, infection of TLR2-deficient mice restored inflammatory cytokines and bacterial burden to wildtype levels, linking the shift in acyl chain composition toward UFA to detrimental immune activation in vivo. In in vitro studies, bacterial lipoproteins isolated from UFA-supplemented cultures were resistant to lipase-mediated ester hydrolysis and exhibited heightened TLR2-dependent innate cell activation, whereas lipoproteins with BCFA esters were completely inactivated after lipase treatment. These results suggest that de novo synthesis of BCFA reduces lipoprotein-mediated TLR2 activation and improves lipase-mediated hydrolysis making it an important determinant of innate immunity. Overall, this study highlights the potential relevance of cell envelope acyl chain repertoire in infection dynamics of bacterial pathogens.

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4.

Extracellular cold-inducible RNA-binding protein (eCIRP), a new damage-associated molecular pattern (DAMP), has been recently shown to play a critical role in promoting the development of bleomycin-induced pulmonary fibrosis. Although fibroblast activation is a critical component of the fibrotic process, the direct effects of eCIRP on fibroblasts have never been examined. We studied eCIRP’s role in the induction of inflammatory phenotype in pulmonary fibroblasts and its connection to bleomycin-induced pulmonary fibrosis in mice. We found that eCIRP causes the induction of proinflammatory cytokines and differentially expression-related pathways in a TLR4-dependent manner in pulmonary fibroblasts. Our analysis further showed that the accessory pathways MD2 and Myd88 are involved in the induction of inflammatory phenotype. In order to study the connection of the enrichment of these pathways in priming the microenvironment for pulmonary fibrosis, we investigated the gene expression profile of lung tissues from mice subjected to bleomycin-induced pulmonary fibrosis collected at various time points. We found that at day 14, which corresponds to the inflammatory-to-fibrotic transition phase after bleomycin injection, TLR4, MD2, and Myd88 were induced, and the transcriptome was differentially enriched for genes in those pathways. Furthermore, we also found that inflammatory cytokines gene expressions were induced, and the cellular responses to these inflammatory cytokines were differentially enriched on day 14. Overall, our results show that eCIRP induces inflammatory phenotype in pulmonary fibroblasts in a TLR4 dependent manner. This study sheds light on the mechanism by which eCIRP induced inflammatory fibroblasts, contributing to pulmonary fibrosis.

Giardia duodenalis Induces Proinflammatory Cytokine Production in Mouse Macrophages via TLR9-Mediated p38 and ERK Signaling Pathways.

Giardia duodenalis, also known as Giardia lamblia or Giardia intestinalis, is an important opportunistic, pathogenic, zoonotic, protozoan parasite that infects the small intestines of humans and animals, causing giardiasis. Several studies have demonstrated that innate immunity-associated Toll-like receptors (TLRs) are critical for the elimination of G. duodenalis; however, whether TLR9 has a role in innate immune responses against Giardia infection remains unknown. In the present study, various methods, including reverse transcriptase–quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, immunofluorescence, inhibitor assays, and small-interfering RNA interference, were utilized to probe the role of TLR9 in mouse macrophage-mediated defenses against G. lamblia virus (GLV)–free or GLV-containing Giardia trophozoites. The results revealed that in G. duodenalis–stimulated mouse macrophages, the secretion of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-12 p40, was enhanced, concomitant with the significant activation of TLR9, whereas silencing TLR9 attenuated the host inflammatory response. Notably, the presence of GLV exacerbated the secretion of host proinflammatory cytokines. Moreover, G. duodenalis stimulation activated multiple signaling pathways, including the nuclear factor κB p65 (NF-κB p65), p38, ERK, and AKT pathways, the latter three in a TLR9-dependent manner. Additionally, inhibiting the p38 or ERK pathway downregulated the G. duodenalis–induced inflammatory response, whereas AKT inhibition aggravated this process. Taken together, these results indicated that G. duodenalis may induce the secretion of proinflammatory cytokines by activating the p38 and ERK signaling pathways in a TLR9-dependent manner in mouse macrophages. Our in vitro findings on the mechanism underlying the TLR9-mediated host inflammatory response may help establish the foundation for an in-depth investigation of the role of TLR9 in the pathogenicity of G. duodenalis.

Up-regulation of AIM2 and TLR4 and down-regulation of NLRC4 are associated with septicemia

PurposeInnate immunity receptors play key roles in recognition of bacterial associated molecular patterns. Inflammasomes and toll like receptors (TLRs) are the important innate immunity receptors. In this project transcription levels of TLR4, a TLR member, absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4), as inflammasomes, in the patients suffering from septicemia. MethodsAIM2, NLRC4 and TLR4 mRNA levels were evaluated in the 40 patients suffering from septicemia and 40 healthy controls using Real-Time PCR technique. ResultsData analysis revealed that, although NLRC4 expression decreased, TLR4 and AIM2 levels significantly increased in the patients suffering from septicemia. Gender and infection with various bacteria did not affect expression of AIM2, NLRC4 and TLR4. ConclusionsIt appears that septicemia can be limited by immune responses in AIM2 and TLR4 dependent manner. The potential roles played by bacteria to down-regulation of NLRC4 need to be evaluated by further investigations.

Role of neutrophil extracellular traps in radiation resistance of invasive bladder cancer

Radiation therapy (RT) is used in the management of several cancers; however, tumor radioresistance remains a challenge. Polymorphonuclear neutrophils (PMNs) are recruited to the tumor immune microenvironment (TIME) post-RT and can facilitate tumor progression by forming neutrophil extracellular traps (NETs). Here, we demonstrate a role for NETs as players in tumor radioresistance. Using a syngeneic bladder cancer model, increased NET deposition is observed in the TIME of mice treated with RT and inhibition of NETs improves overall radiation response. In vitro, the protein HMGB1 promotes NET formation through a TLR4-dependent manner and in vivo, inhibition of both HMGB1 and NETs significantly delays tumor growth. Finally, NETs are observed in bladder tumors of patients who did not respond to RT and had persistent disease post-RT, wherein a high tumoral PMN-to-CD8 ratio is associated with worse overall survival. Together, these findings identify NETs as a potential therapeutic target to increase radiation efficacy.

Tolerance Induction by Exopolysaccharide from the Probiotic Bacillus subtilis

Abstract Intestinal microbiota can lessen the severity of inflammatory diseases, although the mechanisms by which this occur are poorly understood. We showed that exopolysaccharide (EPS) from the probiotic Bacillus subtilis induces anti-inflammatory macrophages and protects mice from enteric infection and sepsis. Here, we examined the molecular mechanisms by which EPS induces an anti-inflammatory response. In co-cultures of OVA-loaded bone marrow-derived dendritic cells (BMDC) and OT-II T cells, EPS inhibits T cell activation in a TLR4-dependent manner. We hypothesize that EPS engages TLR4 and activates the noncanonical NF-kB signaling pathway, which up-regulates the expression of indoleamine 2,3-dioxygenase (IDO). IDO degrades the essential amino acid tryptophan into kynurenine. The subsequent tryptophan depletion and kynurenine formation leads to suppression of T-cell activation and induction of tolerance. Western blot analysis of BMDC treated with EPS showed nuclear translocation of the transcriptional factor RelB, which is central to the noncanonical pathway, and upregulation of IDO. Inhibition of IDO using the inhibitor, 1-methyl tryptophan and deletion of IDO in BMDCs reverses the effect of EPS. We conclude that EPS inhibition of T cell activation and proliferation is mediated by IDO.

TLR4 Transactivates CD8+ T Lymphocytes upon Acute Sterile Tissue Injury.

Acute major tissue injury induces immune dysregulation that is characterized by the development of systemic sterile inflammation and an increased risk for opportunistic infections. Although the contribution of the innate immune system has been examined in detail, research on the impact of acute sterile tissue damage on the T cell compartment remains limited. In the current study, we used a clinically relevant mouse model for traumatic skeletal muscle injury to investigate the impact of sterile tissue damage on diverse subpopulations of CD4+ Th and CD8+ cytotoxic T cells in systemic and local lymphoid organs. For the first time, to our knowledge, we provide evidence that injury selectively induced the expression of the activation marker CD69 on naive and central/virtual memory CD8+ T cells in the lymph nodes but not in the spleen of male mice. CD4+ Th cells remained unaffected in both organs. The activation of CD8+ T cells was dependent on signaling through TLR4. Within a few hours, injury triggered the expression of IL-12 in the lymph nodes in a TLR4-dependent manner. Blocking of IL-12 prevented the activation of naive and central memory CD8+ T cells after injury. Thus, early after traumatic tissue damage, TLR4 transactivates naive and central/virtual memory CD8+ T cells through innate cytokines in local lymph nodes, where they might modulate forthcoming local immune responses.

Amelioration of Graft-versus-Host Disease by Exopolysaccharide from a Commensal Bacterium.

Acute graft-versus-host disease (aGvHD) is a severe, often lethal, complication of hematopoietic stem cell transplantation, and although prophylactic regimens are given as standard pretransplantation therapy, up to 60% of these patients develop aGvHD, and require additional immunosuppressive intervention. We treated mice with a purified probiotic molecule, exopolysaccharide (EPS) from Bacillus subtilis, shortly before and after induction of aGvHD and found that, whereas only 10% of control mice survived to day 80, 70% of EPS-treated mice survived to 80 d. EPS treatment of donor-only mice resulted in ∼60% survival. Using a biosensor mouse model to assess inflammation in live mice during aGvHD, we found that EPS prevented the activation of alloreactive donor T cells. In vitro, EPS did not affect T cells directly but, instead, induced bone marrow-derived dendritic cells (BMDCs) that displayed characteristics of inhibitory dendritic cells (DCs). Development of these BMDCs required TLR4 signaling through both MyD88 and TRIF pathways. Using BMDCs derived from IDO knockout mice, we showed that T cell inhibition by EPS-treated BMDCs was mediated through the suppressive effects of IDO. These studies describe a bacterial molecule that modulates immune responses by inducing inhibitory DCs in a TLR4-dependent manner, and these cells have the capacity to inhibit T cell activation through IDO. We suggest that EPS or EPS-treated DCs can serve as novel agents for preventing aGvHD.

Baicalein Binds to TLR4, Inhibits Colorectal Cancer Growth and Metastasis via the TLR4/HIF/VEGF Axis

Background: Lipopolysaccharide(LPS) inserts into the hydrophobic binding pocket of the myeloid differentiation protein 2(MD-2) that bound to toll-like receptor 4(TLR4). TLR4 antagonists have been designed based on the structure of lipid A, the innermost regions of LPS. However, these antagonists have poor bioavailability and may be toxic. Furthermore, the presence of lipid A moiety is not necessary for driving TLR4 activation. Therefore, compound directly binds to TLR4 is a better strategy to inhibit TLR4 activity. Methods: Biophysical techniques were used to examine the direct binding between baicalein and TLR4. With colorectal cancer(CRC) as models, coupled with drug-target-disease network, protein-protein interaction network and functional studies, the effect and the associated underlying mechanism of action upon the binding baicalein to TLR4 were examined. Findings: We evidently show a direct physical binding of baicalein to TLR4 in molecular docking, surface plasmon resonance biosensor analysis and bio-layer interferometry analysis. With CRC cells as model, we demonstrate the binding of baicalein to TLR4 with cellular thermal shift assay; which interrupts the binding of LPS to TLR4-MD-2 in the cells. Baicalein inhibits TLR4 activity, reduces CRC cell viability and migration in TLR4-dependent manner. Drug–targets–disease network and protein-protein interaction network highlight hypoxia inducible factor(HIF-1α) and vascular endothelial growth factor(VEGF) as downstream of TLR4, which are further validated in cell models. Involvement of TLR4/HIF-1α/VEGF axis in the anti-CRC and anti-angiogenic effect of baicalein is demonstrated in CRC-bearing mice and chick-chorioallantoic-membrane assay, respectively. Our study provides evidence to support the translation of baicalein into TLR4-targeting therapeutics. Funding Information: This work was partially supported by Research Grant Council of HKSAR HKBU-22103017-ECS, Innovation & Technology Commission #PRP/015/19FX, National Natural Science Foundation of China #SCM-2016-NSFC-003 and Natural Science Foundation of Guangdong Province #2018A0303130122 to HYK; the National Natural Science Foundation of China (82074019 and 81703705), Characteristic innovation projects of universities in Guangdong Province (2020KTSCX030), the Opening Project of Zhejiang Provincial Preponderant and Characteristic Subject of Key University (Traditional Chinese Pharmacology), Zhejiang Chinese Medical University (ZYAOX2018010) to ST. Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: Animal experiments were approved by the Guangzhou University of Chinese Medicine Animal Care and Use Committee (Guangzhou, China), and conducted according to the ethical standards and national guidelines.

Multiple Viral microRNAs Regulate Interferon Release and Signaling Early during Infection with Epstein-Barr Virus.

Epstein-Barr virus (EBV), a human herpesvirus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN-α in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the prelatent phase of infection. The presence of epigenetically naive, non-CpG methylated viral DNA was essential to induce IFN-α secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plasmacytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN-α in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the prelatent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV.IMPORTANCE Acute antiviral functions of all nucleated cells rely on type I interferon (IFN-I) pathways triggered upon viral infection. Host responses encompass the sensing of incoming viruses, the activation of specific transcription factors that induce the transcription of IFN-I genes, the secretion of different IFN-I types and their recognition by the heterodimeric IFN-α/β receptor, the subsequent activation of JAK/STAT signaling pathways, and, finally, the transcription of many IFN-stimulated genes (ISGs). In sum, these cellular functions establish a so-called antiviral state in infected and neighboring cells. To counteract these cellular defense mechanisms, viruses have evolved diverse strategies and encode gene products that target antiviral responses. Among such immune-evasive factors are viral microRNAs (miRNAs) that can interfere with host gene expression. We discovered that multiple miRNAs of Epstein-Barr virus (EBV) control over a dozen cellular genes that contribute to the antiviral states of immune cells, specifically B cells and plasmacytoid dendritic cells (pDCs). We identified the viral DNA genome as the activator of IFN-α and question the role of abundant EBV EBERs, that, contrary to previous reports, do not have an apparent inducing function in the IFN-I pathway early after infection.

Gut-liver axis modulation of Panax notoginseng saponins in nonalcoholic fatty liver disease

Background and aimsNonalcoholic fatty liver disease (NAFLD) is an obesity-related comorbidity, and it is characterized as a spectrum of liver abnormalities, including inflammation, steatosis, and fibrosis. The gut-liver axis is implicated in the pathogenesis and development of NAFLD. A promising drug agent targeting the gut-liver axis is expected to reverse NAFLD.MethodsWe utilized high-fat diet (HFD)-induced obese mice and obesity-prone Lepob mice to examine the gut-liver regulation of the natural medicine Panax Notoginseng Saponins (PNS) on NAFLD.ResultsPNS exhibited potent anti-lipogenesis and anti-fibrotic effects in NAFLD mice, that was associated with the TLR4-induced inflammatory signalling pathway in liver. More strikingly, PNS treatment caused a deceleration of gut-to-liver translocation of microbiota-derived short chain fatty acids (SCFAs) products. PNS-induced TLR4 inhibition and restoration of Claudin-1 and ZO-1 proteins in the gut-liver axis contributed to the reverse of leaky gut, which in turn abolished by the addition of lipopolysaccharide (LPS), an agonist of TLR4. Specifically, hepatic steatosis in HFD-treated mice was attenuated by PNS through regulating AMPKα, but restored by the replenishment of LPS. Meanwhile, the anti-fibrotic effect of PNS was abolished by LPS stimulation via the overproduction of collagen I/IV and α-SMA.ConclusionPNS exerted hepatoprotection against NAFLD in both ob/ob and HFD-induced obese mice, primarily by mediating the gut-liver axis in a TLR4-dependent manner.Graphic abstractPanax notoginseng saponins (PNS) ameliorated hepatic steatosis and fibrosis, and gut-liver axis-mediated pathogenesis of NAFLD is proposed to occur in a TLR4-dependent manner.

Toll-like receptor 2 signaling deficiency in cardiac cells ameliorates Ang II-induced cardiac inflammation and remodeling

Activation of the innate immune system represents a vital step in inflammation during cardiac remodeling induced by the angiotensin II (Ang II). This study aimed to explore the role of Toll-like receptors 2 (TLR2) in Ang II-induced cardiac remodeling. We investigated the effect of TLR2 deficiency on Ang II-induced cardiac remodeling by utilizing TLR2 knockout mice, bone marrow transplantation models, and H9C2 cells. Though TLR2 deficiency had no effect on body weight change, cardiac Ang II content and blood pressure, it significantly ameliorated cardiac hypertrophy, fibrosis and inflammation, as well as improved heart function. Further bone marrow transplantation studies showed that TLR2-deficiency in cardiac cells but not bone marrow-derived cells prevented Ang II-induced cardiac remodeling and cardiac dysfunction. The underlying mechanism may involve increased TLR2-MyD88 interaction. Further in vitro studies in Ang II-treated H9C2 cells showed that TLR2 knockdown by siRNA significantly decreased Ang II-induced cell hypertrophy, fibrosis and inflammation. Moreover, Ang II significantly increased TLR2-MyD88 interaction in H9C2 cells in a TLR4-independent manner. TLR2 deficiency in cardiac cells prevents Ang II-induced cardiac remodeling, inflammation and dysfunction through reducing the formation of TLR2-MyD88 complexes. Inhibition of TLR2 pathway may be a therapeutic strategy of hypertensive heart failure.

Generation of αCD11b-CpG antibody conjugates for the targeted stimulation of myeloid cells

CpG oligonucleotides are short single-stranded synthetic DNA molecules. Upon binding to Toll-like receptor 9 (TLR9), CpG activates immune cells in humans and mice. This results in robust Th1 type immunity potentially resulting in clearance of pathogens, reduction of allergy and anti-tumor immunity. However, the effectiveness of CpG as an adjuvant depends on its administration route, with only strong effects seen when CpG is administered locally. As local administration is not always feasible, we generated conjugates to specifically deliver CpG to myeloid cells often abundantly present in tumors. For this we coupled CpG (3′-Thiol-modified phosphorothioate (PTO) CpG-ODN1826 type B (5′-tccatgacgttcctgacgtt-3′)) to monoclonal antibodies (mAbs) directed against the myeloid cell marker CD11b using maleimide-thiol coupling. The CD11b-CpG mAb (αCD11b-CpG) conjugates contained about four CpG molecules/conjugate and displayed binding and internalization characteristics similar to unconjugated CD11b mAbs (αCD11b). The αCD11b-CpG conjugates readily induced maturation of murine dendritic cells (DCs) in a TLR9-dependent manner in vitro. Following intravenous injection, αCD11b-CpG conjugates efficiently targeted CD11b+ immune cells in the blood, lymph nodes and spleen. Finally, injection of αCD11b-CpG conjugates, but not untargeted conjugates, induced maturation of CD11b+ cell subsets in vivo. In conclusion, conjugating CpG to αCD11b enabled specific targeting and activation of myeloid cells in vivo.

An Exopolysaccharide Produced by Bifidobacterium longum 35624® Inhibits Osteoclast Formation via a TLR2-Dependent Mechanism.

The probiotic Bifidobacterium longum subsp. longum 35624® (B. longum 35624®), with its surface exopolysaccharide (EPS624), has previously been demonstrated to induce immunoregulatory responses in the host and may, therefore, be a novel approach to prevent bone loss in inflammatory conditions such as post-menopausal osteoporosis (PMO). The aim of this study was to investigate the effect of EPS624 on osteoclast and osteoblast differentiation and to assess the potential of B. longum 35624® to prevent bone loss in vivo. In vitro cell assays were used to assess the impact of EPS624 on osteoclast and osteoblast differentiation. The potential of two probiotic B. longum 35624® strains, including an EPS-deficient strain, for preventing ovariectomy (Ovx)-induced bone loss was assessed in a murine model. EPS624 prevented osteoclast formation from murine bone marrow precursors under both normal and TNFα-induced inflammatory conditions and modestly increased mineralized matrix deposition in osteogenic cell cultures. However, in the presence of an anti-TLR2 blocking antibody, or in MyD88-/-osteoclast precursors, the inhibitory effect of EPS624 on osteoclast formation was diminished or completely prevented, respectively. Moreover, EPS624 induced IL-10 production in osteoclast precursors in a TLR2-dependent manner, although IL-10 was dispensable in the EPS624-mediated inhibition of osteoclast formation. In addition, EPS624-producing B. longum 35624® partially prevented bone loss in Ovx mice when administered by oral gavage. This study introduced EPS624 as a potential anti-resorptive therapy, although optimal in vivo delivery of the probiotic strain for treating low-grade inflammatory diseases such as PMO remains to be determined.

Synthesis of the pentasaccharide repeating unit from Ruminococcus gnavus and measurement of its inflammatory properties.

The roles played by the gut microbiome in human health are increasingly recognized, and the prevalence of specific microorganisms has been correlated with different diseases. For example, blooms of the Gram-positive bacterium Ruminococcus gnavus have been correlated with inflammatory bowel disease, and recently a polysaccharide produced by this organism was shown to stimulate release of inflammatory cytokines. This stimulation was proposed to signal through toll-like receptor 4 (TLR4). We have synthesized the pentasaccharide repeating unit of this polysaccharide and showed that it stimulates TNF-α and IL-6 release from bone marrow-derived dendritic cells (BMDCs) in a TLR4-dependent manner. A related glycan does not stimulate significant cytokine release, demonstrating TLR4 selectivity in glycan recognition.

Morphine-3-glucuronide upregulates PD-L1 expression via TLR4 and promotes the immune escape of non-small cell lung cancer.

Objective:Patients with cancer pain are highly dependent on morphine analgesia, but studies have shown a negative correlation between morphine demand and patient outcomes. The long-term use of morphine may result in abnormally elevated serum morphine-3-glucuronide (M3G) levels. Hence, the effects of M3G on tumor progression are worth studying.Methods:The effects of M3G on PD-L1 expressions in human non-small cell lung cancer (NSCLC) cell lines were first evaluated. Activation of TLR4 downstream pathways after M3G treatment was then determined by Western blot. The effects of M3G on human cytotoxic T lymphocytes (CTL) cytotoxicity and INF-γ release was also detected. Finally, the LLC murine lung adenocarcinoma cell line were used to establish a murine lung cancer model, and the effects of M3G on tumor growth and metastasis were determined.Results:M3G promoted the expressions of PD-L1 in the A549 and H1299 cell lines in a TLR4-dependent manner (P < 0.05). M3G activated the PI3K and the NFκB signaling pathways, and this effect was antagonized by a TLR4 pathway inhibitor. A PI3K pathway inhibitor reversed the M3G-mediated PD-L1 upregulation. M3G inhibited the cytotoxicity of CTL on A549 cells and decreased the level of INF-γ. Repeated M3G intraperitoneal injections promoted LLC tumor growth and lung metastasis through the upregulation of tumor expressed PD-L1 and the reduction of CTL in the tumor microenvironment.Conclusions:M3G specifically activated TLR4 in NSCLC cells and upregulated PD-L1 expression through the PI3K signaling pathway, thereby inhibiting CTL cytotoxicity and finally promoting tumor immune escape.

Mycolactone toxin induces an inflammatory response by targeting the IL-1β pathway: Mechanistic insight into Buruli ulcer pathophysiology.

Mycolactone, a lipid-like toxin, is the major virulence factor of Mycobacterium ulcerans, the etiological agent of Buruli ulcer. Its involvement in lesion development has been widely described in early stages of the disease, through its cytotoxic and immunosuppressive activities, but less is known about later stages. Here, we revisit the role of mycolactone in disease outcome and provide the first demonstration of the pro-inflammatory potential of this toxin. We found that the mycolactone-containing mycobacterial extracellular vesicles produced by M. ulcerans induced the production of IL-1β, a potent pro-inflammatory cytokine, in a TLR2-dependent manner, targeting NLRP3/1 inflammasomes. We show our data to be relevant in a physiological context. The in vivo injection of these mycolactone-containing vesicles induced a strong local inflammatory response and tissue damage, which were prevented by corticosteroids. Finally, several soluble pro-inflammatory factors, including IL-1β, were detected in infected tissues from mice and Buruli ulcer patients. Our results revisit Buruli ulcer pathophysiology by providing new insight, thus paving the way for the development of new therapeutic strategies taking the pro-inflammatory potential of mycolactone into account.

Human Gut Commensal Membrane Vesicles Modulate Inflammation by Generating M2-like Macrophages and Myeloid-Derived Suppressor Cells.

Immunomodulatory commensal bacteria modify host immunity through delivery of regulatory microbial-derived products to host cells. Extracellular membrane vesicles (MVs) secreted from symbiont commensals represent one such transport mechanism. How MVs exert their anti-inflammatory effects or whether their tolerance-inducing potential can be used for therapeutic purposes remains poorly defined. In this study, we show that MVs isolated from the human lactic acid commensal bacteria Pediococcus pentosaceus suppressed Ag-specific humoral and cellular responses. MV treatment of bone marrow-derived macrophages and bone marrow progenitors promoted M2-like macrophage polarization and myeloid-derived suppressor cell differentiation, respectively, most likely in a TLR2-dependent manner. Consistent with their immunomodulatory activity, MV-differentiated cells upregulated expression of IL-10, arginase-1, and PD-L1 and suppressed the proliferation of activated T cells. MVs' anti-inflammatory effects were further tested in acute inflammation models in mice. In carbon tetrachloride-induced fibrosis and zymosan-induced peritonitis models, MVs ameliorated inflammation. In the dextran sodium sulfate-induced acute colitis model, systemic treatment with MVs prevented colon shortening and loss of crypt architecture. In an excisional wound healing model, i.p. MV administration accelerated wound closure through recruitment of PD-L1-expressing myeloid cells to the wound site. Collectively, these results indicate that P. pentosaceus-derived MVs hold promise as therapeutic agents in management/treatment of inflammatory conditions.

Anti-HSV-1 effect of dihydromyricetin from Ampelopsis grossedentata via the TLR9-dependent anti-inflammatory pathway

ObjectivesHerpes simplex virus 1 (HSV-1) is one of the most prevalent viruses in humans worldwide. Owing to limited therapeutic options mainly with acyclovir (ACV) and analogues and the emergence of ACV-resistant strains, new drugs with different modes of action and low toxicity are required. The aim of this study was to determine the anti-HSV-1 effect and mechanism of action of the flavonoid compound dihydromyricetin (DHM) from Ampelopsis grossedentata. MethodsThe HSV-1 inhibitory effect of DHM was evaluated by measuring plaque formation and generation of progeny virus as well as expression of HSV-1-related genes in Vero cells. The molecular mechanism of the antiviral activity of DHM against HSV-1 was explored by real-time quantitative PCR and ELISA. ResultsDHM presented a significant inhibitory effect on HSV-1 plaque formation and generation of progeny virus, with an EC50 (50% effective concentration) of 12.56 μM in Vero cells. Furthermore, expression of HSV-1 immediate-early genes (ICP4 and ICP22), early genes (ICP8 and UL42) and late genes (gB, VP1/2) was decreased by DHM at concentrations of 16 μM and 32 μM. DHM specifically suppressed mRNA levels of Toll-like receptor 9 (TLR9), leading to inhibition of the inflammatory transcriptional factor NFκB and a decrease in TNFα. ConclusionThese findings indicate that the effective inhibitory activity of DHM was achieved by suppressing TNFα production in a TLR9-dependent manner. Although further studies are needed to better characterise the activity of DHM in vivo, the results suggest this extract as a promising new anti-HSV-1 agent.