TLR4 KO Research Articles

Morphine-induced intestinal microbial dysbiosis drives TLR-dependent IgA targeting of gram-positive bacteria and upregulation of CD11b and TLR2 on a sub-population of IgA+ B cells

ABSTRACT IgA binding dictates the composition of the intestinal microbiome and reflects dysbiotic states during chronic disease. Both pathogenic and commensal bacteria differentially bind to IgA with varying outcomes. Little is known regarding IgA dynamics immediately following microbial dysbiosis. Recent work shows that morphine treatment rapidly induces microbial dysbiosis within hours of administration. This microbial shift is characterized by the expansion of pathogenic bacteria with a concurrent decrease in commensal bacteria. Because of this rapid microbial shift, a murine model of chronic morphine treatment was used to gain insight on the host IgA response during early microbial disruption. Within 24 h, morphine treatment induces microbial dysbiosis which disrupts IgA-bacterial homeostasis, resulting in an increased concentration of unbound IgA with a corresponding decrease in the frequency of IgA-bound bacteria. Additionally, the increased concentration of unbound IgA is dependent on the microbiome, as microbial depletion abolishes the increase. At 48 h of morphine treatment, the frequency of IgA-bound bacteria increases and IgA-seq reveals increased IgA targeting of gram-positive bacteria. Both a whole-body TLR2 KO and treatment with the TLR inhibitor OxPAPC resulted in abrogation of IgA binding to bacteria, implicating modulation of IgA binding through TLR signaling. Finally, we identify that a sub-population of IgA+ B cells in the intestinal lamina propria has increased CD11b and TLR2 expression at 24 h of morphine treatment which could be a potential source of the observed IgA that targets gram-positive bacteria. Together, we demonstrate for the first time the role of TLR2 in IgA targeting of intestinal bacteria, and this study sheds light on the IgA dynamics during the initial hours of microbial dysbiosis.

Macrophages, IL-10, and nitric oxide increase, induced by hyperglycemic conditions, impact the development of murine melanoma B16F10-Nex2

Epidemiological studies show a strong correlation between diabetes and the increased risk of developing different cancers, including melanoma. In the present study, we investigated the impact of a streptozotocin (STZ)-induced hyperglycemic environment on B16F10-Nex2 murine melanoma development. Hyperglycemic male C57Bl/6 mice showed increased subcutaneous tumor development, partially inhibited by metformin. Tumors showed increased infiltrating macrophages, and augmented IL-10 and nitric oxide (NO) concentrations. In vivo neutralization of IL-10, NO synthase inhibition, and depletion of macrophages reduced tumor development. STZ-treated TLR4 KO animals showed delayed tumor development; the transfer of hyperglycemic C57Bl/6 macrophages to TLR4 KO reversed this effect. Increased concentrations of IL-10 present in tumor homogenates of hyperglycemic mice induced a higher number of pre-angiogenic structures in vitro, and B16F10-Nex2 cells incubated with different glucose concentrations in vitro produced increased levels of IL-10. In summary, our findings show that a hyperglycemic environment stimulates murine melanoma B16F10-Nex2 primary tumor growth, and this effect is dependent on tumor cell stimulation, increased numbers of macrophages, and augmented IL-10 and NO concentrations. These findings show the involvement of tumor cells and other components of the tumor microenvironment in the development of subcutaneous melanoma under hyperglycemic conditions, defining novel targets for melanoma control in diabetic patients.

LPS-TLR4 pathway exaggerates alcoholic hepatitis via provoking NETs formation

BackgroundIntrahepatic infiltration of neutrophils is a character of alcoholic hepatitis (AH) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. The gut-liver axis has been thought to play a critical role in many liver diseases also including AH. However, whether NETs appear in AH and play role in AH is still unsure. MethodsSerum samples from AH patients were collected and LPS and MPO-DNA were detected. WT, NE KO, and TLR4 KO mice were used to build the AH model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AH-related markers were detected. ResultsThe serum MPO-DNA and LPS concentration was increased in AH patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AH mice. NETs formation decreased with antibiotic intervention and restored with antibiotic intervention plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AH-related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with a combined treatment with antibiotics plus LPS. But the AH-related markers did show a difference in TLR4 KO mice when they received the same treatment. ConclusionIntestinal-derived LPS promotes NETs formation in AH through the TLR4 pathway and further accelerates the AH process by NETs.

Evaluation of the TLR3 involvement during Schistosoma japonicum-induced pathology

BackgroundDespite the functions of TLRs in the parasitic infections have been extensively reported, few studies have addressed the role of TLR3 in the immune response to Schistosoma japonicum infections. The aim of this study was to investigate the properties of TLR3 in the liver of C57BL/6 mice infected by S. japonicum.MethodsThe production of TLR3+ cells in CD4+T cells (CD4+CD3+), CD8+T cells (CD8+CD3+), γδT cells (γδTCR+CD3+), NKT cells (NK1.1+CD3+), B cells (CD19+CD3−), NK (NK1.1−CD3+) cells, MDSC (CD11b+Gr1+), macrophages (CD11b+F4/80+), DCs (CD11c+CD11b+) and neutrophils (CD11b+ Ly6g+) were assessed by flow cytometry. Sections of the liver were examined by haematoxylin and eosin staining in order to measure the area of granulomas. Hematological parameters including white blood cell (WBC), red blood cell (RBC), platelet (PLT) and hemoglobin (HGB) were analyzed. The levels of ALT and AST in the serum were measured using biochemical kits. The relative titers of anti-SEA IgG and anti-SEA IgM in the serum were measured by enzyme-linked immunosorbent assay (ELISA). CD25, CD69, CD314 and CD94 molecules were detected by flow cytometry.ResultsFlow cytometry results showed that the expression of TLR3 increased significantly after S. japonicum infection (P < 0.05). Hepatic myeloid and lymphoid cells could express TLR3, and the percentages of TLR3-expressing MDSC, macrophages and neutrophils were increased after infection. Knocking out TLR3 ameliorated the damage and decreased infiltration of inflammatory cells in infected C57BL/6 mouse livers.,The number of WBC was significantly reduced in TLR3 KO-infected mice compared to WT-infected mice (P < 0.01), but the levels of RBC, platelet and HGB were significantly increased in KO infected mice. Moreover, the relative titers of anti-SEA IgG and anti-SEA IgM in the serum of infected KO mice were statistically decreased compared with the infected WT mice. We also compared the activation-associated molecules expression between S.japonicum-infected WT and TLR3 KO mice.ConclusionsTaken together, our data indicated that TLR3 played potential roles in the context of S. japonicum infection and it may accelerate the progression of S. japonicum-associated liver pathology.

The adjuvant BcfA activates antigen presenting cells through TLR4 and supports TFH and TH1 while attenuating TH2 gene programming.

Adjuvants added to subunit vaccines augment antigen-specific immune responses. One mechanism of adjuvant action is activation of pattern recognition receptors (PRRs) on innate immune cells. Bordetella colonization factor A (BcfA); an outer membrane protein with adjuvant function, activates TH1/TH17-polarized immune responses to protein antigens from Bordetella pertussis and SARS CoV-2. Unlike other adjuvants, BcfA does not elicit a TH2 response. To understand the mechanism of BcfA-driven TH1/TH17 vs. TH2 activation, we screened PRRs to identify pathways activated by BcfA. We then tested the role of this receptor in the BcfA-mediated activation of bone marrow-derived dendritic cells (BMDCs) using mice with germline deletion of TLR4 to quantify upregulation of costimulatory molecule expression and cytokine production in vitro and in vivo. Activity was also tested on human PBMCs. PRR screening showed that BcfA activates antigen presenting cells through murine TLR4. BcfA-treated WT BMDCs upregulated expression of the costimulatory molecules CD40, CD80, and CD86 and produced IL-6, IL-12/23 p40, and TNF-α while TLR4 KO BMDCs were not activated. Furthermore, human PBMCs stimulated with BcfA produced IL-6. BcfA-stimulated murine BMDCs also exhibited increased uptake of the antigen DQ-OVA, supporting a role for BcfA in improving antigen presentation to T cells. BcfA further activated APCs in murine lungs. Using an in vitro TH cell polarization system, we found that BcfA-stimulated BMDC supernatant supported TFH and TH1 while suppressing TH2 gene programming. Overall, these data provide mechanistic understanding of how this novel adjuvant activates immune responses.

Nucleic acid sensing Toll-like receptors 3 and 9 play complementary roles in the development of bacteremia after nasal colonization associated with influenza co-infection.

Streptococcus pneumoniae can cause mortality in infant, elderly, and immunocompromised individuals owing to invasion of bacteria to the lungs, the brain, and the blood. In building strategies against invasive infections, it is important to achieve greater understanding of how the pneumococci are able to survive in the host. Toll-like receptors (TLRs), critically important components in the innate immune system, have roles in various stages of the development of infectious diseases. Endosomal TLRs recognize nucleic acids of the pathogen, but the impact on the pneumococcal diseases of immune responses from signaling them remains unclear. To investigate their role in nasal colonization and invasive disease with/without influenza co-infection, we established a mouse model of invasive pneumococcal diseases directly developing from nasal colonization. TLR9 KO mice had bacteremia more frequently than wildtype in the pneumococcal mono-infection model, while the occurrence of bacteremia was higher among TLR3 KO mice after infection with influenza in advance of pneumococcal inoculation. All TLR KO strains showed poorer survival than wildtype after the mice had bacteremia. The specific and protective role of TLR3 and TLR9 was shown in developing bacteremia with/without influenza co-infection respectively, and all nucleic sensing TLRs would contribute equally to protecting sepsis after bacteremia.

LPS-TLR4 pathway exaggerates alcoholic hepatitis via provoking NETs formation

BackgroundIntrahepatic infiltration of neutrophils is a character of alcoholic hepatitis (AH) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. The gut-liver axis has been thought to play a critical role in many liver diseases also including AH. However, whether NETs appear in AH and play role in AH is still unsure. MethodsSerum samples from AH patients were collected and LPS and MPO-DNA were detected. WT, NE KO, and TLR4 KO mice were used to build the AH model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AH-related markers were detected. ResultsThe serum MPO-DNA and LPS concentration was increased in AH patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AH mice. NETs formation decreased with antibiotic intervention and restored with antibiotic intervention plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AH-related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with a combined treatment with antibiotics plus LPS. But the AH-related markers did show a difference in TLR4 KO mice when they received the same treatment. ConclusionIntestinal-derived LPS promotes NETs formation in AH through the TLR4 pathway and further accelerates the AH process by NETs.

Probiotic-derived exopolysaccharide interferes with T H2 priming and inhibits allergic inflammation

Abstract Allergic asthma is an increasing burden in industrialized countries, where treatments are limited to targeting symptoms and end-stage effectors rather than root causes. In recent years, the use of prophylactic, inhaled probiotic cocktails has shown promise in preventing allergic asthma. However, the mechanisms by which such probiotics prevent allergic symptoms and the exact bacterial product(s) responsible for probiotic activity remain to be elucidated. In this study, we focused on exopolysaccharide (EPS) from Bacillus subtilis, a ubiquitous soil bacterium that is commonly found as a dietary supplement. We found that in a mouse model of allergic airway disease, EPS treatment, prior to allergic sensitization and challenge, limits lung mucus hypersecretion and eosinophilia. Furthermore, EPS treatment inhibited accumulation of T H2, but not T H1 and T H17 subsets. To determine whether EPS was affecting T H2 cell priming by dendritic cells (DCs), we compared the composition and phenotype of DCs in the lung and mediastinal lymph nodes (medLN) after EPS treatment. Surprisingly, pre-treatment with EPS induced DC maturation as measured by an increase in costimulatory molecules and a decrease in the ability to uptake allergen and migrate to the medLN. DC maturation by EPS was TLR4-dependent, as TLR4 KO DCs did not respond to EPS. Together, our data suggest that EPS inhibits allergic sensitization by inducing DC maturation prior to allergen exposure, thereby leading to decreased T H2 priming and allergic inflammation. Thus, we provide a potential mechanism by which inhalation of a single probiotic-derived molecule can prevent the development of allergic inflammation. Supported by grants from NIH (3R01 AI125644-05S1, 1F31 HL156659-01A1)

Roxadustat protect mice from DSS-induced colitis in vivo by up-regulation of TLR4

BackgroundThe incidence of inflammatory bowel disease (IBD) is growing in the population. At present, the etiology of inflammatory bowel disease remains unclear, and there is no effective and low-toxic therapeutic drug. The role of the PHD-HIF pathway in relieving DSS-induced colitis is gradually being explored. MethodsWild-type C57BL/6 mice were used as a model of DSS-induced colitis to explore the important role of Roxadustat in alleviating DSS-induced colitis. High-throughput RNA-Seq and qRT-PCR methods were used to screen and verify the key differential genes in the colon of mice between normal saline (NS) and Roxadustat groups. ResultsRoxadustat could alleviate DSS-induced colitis. Compared with the mice in the NS group, TLR4 were significantly up-regulated in the Roxadustat group. TLR4 KO mice were used to verify the role of TLR4 in the alleviation of DSS-induced colitis by Roxadustat. ConclusionRoxadustat has a repairing effect on DSS-induced colitis, and may alleviate DSS-induced colitis by targeting the TLR4 pathway and promote intestinal stem cell proliferation.

Zymosan-A promotes the regeneration of intestinal stem cells by upregulating ASCL2

Intestinal stem cells (ISCs) are responsible for intestinal tissue homeostasis and are important for the regeneration of the damaged intestinal epithelia. Through the establishment of ionizing radiation (IR) induced intestinal injury model, we found that a TLR2 agonist, Zymosan-A, promoted the regeneration of ISCs in vivo and in vitro. Zymosan-A improved the survival of abdominal irradiated mice (81.82% of mice in the treated group vs. 30% of mice in the PBS group), inhibited the radiation damage of intestinal tissue, increased the survival rate of intestinal crypts and the number of ISCs after lethal IR in vivo. Through organoid experiments, we found that Zymosan-A promoted the proliferation and differentiation of ISCs after IR. Remarkably, the results of RNA sequencing and Western Blot (WB) showed that Zymosan-A reduced IR-induced intestinal injury via TLR2 signaling pathway and Wnt signaling pathway and Zymosan-A had no radioprotection on TLR2 KO mice, suggesting that Zymosan-A may play a radioprotective role by targeting TLR2. Moreover, our results revealed that Zymosan-A increased ASCL2, a transcription factor of ISCs, playing a core role in the process of Zymosan-A against IR-induced intestinal injury and likely contributing to the survival of intestinal organoids post-radiation. In conclusion, we demonstrated that Zymosan-A promotes the regeneration of ISCs by upregulating ASCL2.

FG-4592 protects the intestine from irradiation-induced injury by targeting the TLR4 signaling pathway

BackgroundSevere ionizing radiation (IR)-induced intestinal injury associates with high mortality, which is a worldwide problem requiring urgent attention. In recent years, studies have found that the PHD-HIF signaling pathway may play key roles in IR-induced intestinal injury, and we found that FG-4592, the PHD inhibitor, has significant radioprotective effects on IR-induced intestinal injury.MethodsIn the presence or absence of FG-4592 treatment, the survival time, pathology, cell viability, cell apoptosis, and organoids of mice after irradiation were compared, and the mechanism was verified after transcriptome sequencing. The data were analyzed using SPSS ver. 19 software.ResultsOur results show that FG-4592 had significant radioprotective effects on the intestine. FG-4592 improved the survival of irradiated mice, inhibited the radiation damage of intestinal tissue, promoted the regeneration of intestinal crypts after IR and reduced the apoptosis of intestinal crypt cells. Through organoid experiments, it is found that FG-4592 promoted the proliferation and differentiation of intestinal stem cells (ISCs). Moreover, the results of RNA sequencing and Western blot showed that FG-4592 significantly upregulated the TLR4 signaling pathway, and FG-4592 had no radioprotection on TLR4 KO mice, suggesting that FG-4592 may play protective role against IR by targeting TLR4.ConclusionOur work proves that FG-4592 may promote the proliferation and regeneration of ISCs through the targeted regulation of the TLR4 signaling pathway and ultimately play radioprotective roles in IR-induced injury. These results enrich the molecular mechanism of FG-4592 in protecting cells from IR-induced injury and provide new methods for the radioprotection of intestine.

TLR3 deletion inhibits programmed necrosis of brain cells in neonatal mice with sevoflurane-induced cognitive dysfunction.

This research aimed to explore the influence of TLR deletion on sevoflurane-induced postoperative cognitive dysfunction in neonatal mice. Herein, WT and TLR3 KO neonatal mice, each with 24, were randomly divided into control group, sevoflurane group, and TLR3−/−+sevoflurane group. The hippocampal neurons of WT, TLR3 KO and RIP3 KO neonatal mice in C group, SEV group, TLR3−/−+SEV group and RIP3−/−+SEV group were extracted for in vitro experiments. The results revealed the degeneration and necrosis of nerve cells in SEV group. Microscopic findings indicated that nerve cells showed shrinkage and nuclear hyperchromatism, along with lessening or even disappearance of nuclei and enlargement of cell spaces, and apoptotic cells in the brain tissues were evidently increased. Compared with SEV group, TLR3−/−+SEV group displayed reductions in these phenomena. Additionally, SEV group showed the reduced SHP2 expression and the increased expressions of proteins associated with TLR signaling pathway and apoptosis. Furthermore, there were no obvious differences in the expressions of such proteins in hippocampal neurons between RIP3−/−+SEV and TLR3−/−+SEV groups. The results confirmed that inhibiting RIP3 phosphorylation and suppressing TLR3 expressions exerted the same influence on the expressions of these proteins in the hippocampus of neonatal mice with sevoflurane-induced cognitive dysfunction. Based on these, it is speculated that TLR3 influences neonatal mice with sevoflurane-induced cognitive dysfunction probably by regulating RIP3 phosphorylation.

Effects of repetitive training on learning and memory performance of TLR2 KO mice

Current antipsychotics have limited effects on the cognitive deficits of schizophrenia patients, therefore, cognitive remediation has been applied to schizophrenia patients to ameliorate cognitive dysfunction. However, the neurobiological mechanisms of cognitive training programs have not been well studied because established animal models are not suitable or because repetitive training has not been introduced in such animal models. In the present study, we employed Toll-like receptor 2 knockout (TLR2 KO) mouse as a schizophrenia mouse model and evaluated the effects of repetitive training as cognitive remediation therapy for schizophrenia. TLR2 KO mice could fully learn the Barnes maze paradigm through repetitive training to improve memory retrieval and reversal learning ability, although the learning speed was slower than that of wild-type (WT) animals. In addition, highly repetitive training activated the neuronal cells in the prefrontal cortex, hippocampal CA3 and hippocampal DG regions of TLR2 KO mice, similar to WT mice. These results indicated that TLR2 KO mouse would be a useful tool for studying the neurobiological mechanisms of cognitive remediation in schizophrenia.

TLR4 deletion increases basal energy expenditure and attenuates heart apoptosis and ER stress but mitigates the training-induced cardiac function and performance improvement

Strategies capable of attenuating TLR4 can attenuate metabolic processes such as inflammation, endoplasmic reticulum (ER) stress, and apoptosis in the body. Physical exercise has been a cornerstone in suppressing inflammation and dysmetabolic outcomes caused by TRL4 activation. Thus, the present study aimed to evaluate the effects of a chronic physical exercise protocol on the TLR4 expression and its repercussion in the inflammation, ER stress, and apoptosis pathways in mice hearts. Echocardiogram, RT-qPCR, immunoblotting, and histological techniques were used to evaluate the left ventricle of wild-type (WT) and Tlr4 knockout (TLR4 KO) mice submitted to a 4-week physical exercise protocol. Moreover, we performed a bioinformatics analysis to expand the relationship of Tlr4 mRNA in the heart with inflammation, ER stress, and apoptosis-related genes of several isogenic strains of BXD mice. The TLR4 KO mice had higher energy expenditure and heart rate in the control state but lower activation of apoptosis and ER stress pathways. The bioinformatics analysis reinforced these data. In the exercised state, the WT mice improved performance and cardiac function. However, these responses were blunted in the KO group. In conclusion, TLR4 has an essential role in the inhibition of apoptosis and ER stress pathways, as well as in the training-induced beneficial adaptations.

Abstract MP49: Macrophage-mediated Extracellular Digestive Exophagy Of Aggregated LDL Is Responsible For The Formation Of Cholesterol Crystals In Atherosclerotic Plaques

Atherosclerosis is a disease that develops overtime through dyslipidemia and chronic inflammation. Formation of cholesterol crystals in the atherosclerotic plaques is a major contributor to the inflammatory response. However, how these crystals form in the atherosclerotic plaques remained poorly understood. We have observed that macrophage-mediated extracellular digestive exophagy of aggregated LDL releases a large amount of free cholesterol in the extracellular space. Therefore, we hypothesized that this release of cholesterol may play a major role in formation of cholesterol crystals in plaques. We have shown previously that TLR4 knockout in macrophages compromises digestive exophagy of aggregated LDL. Thus, to evaluate the role of digestive exophagy in cholesterol crystal formation, we examined cryosections of aortic plaques from Ldlr -/- mice that were gamma-irradiated and reconstituted with WT or Tlr4 -/- bone marrows. Interestingly, TLR4 KO in macrophages reduce both free cholesterol and cholesterol crystal in plaques compared to WT macrophages, suggesting a role of digestive exophagy in cholesterol crystal formation. To investigate whether digestive exophagy of aggregated LDL results in inflammatory response, we cultured bone-marrow derived macrophages expressing the inflammasome marker ASC-citrine with aggregated LDL over time. We observed a gradual increase in number of inflammasomes over a 24 h time period. We also observed extracellular cholesterol crystal formation as early as 2 h after the addition of aggregated LDL. Moreover, treating bone marrow-derived macrophages with acetylated LDL did not result in the formation of extracellular cholesterol crystals, suggesting this process is specific to a subset of modified LDL in the vessel wall. Overall, our data provide evidence that macrophage-mediated digestive exophagy of aggregated LDL is a major contributor to the formation of extracellular cholesterol crystal found in atherosclerotic lesions.

Abstract 2447: TLR4 expression as a determinant of EMT and stress response gene expression in UV exposed human keratinocytes

Abstract Toll-like receptor 4 (TLR4) is gaining attention for its role in several cancer-associated signaling pathways, including the regulation of inflammation, DNA repair and epithelial-mesenchymal transition (EMT). There is evidence that TLR4 contributes to ultraviolet (UV) radiation-induced stress signaling in skin cells, including keratinocytes. Blockade of TLR4 activity using the pharmacological antagonist resatorvid (TAK-242) inhibits not only acute UV-induced signaling in vitro and in vivo, but blocks UV-induced skin carcinogenesis in mouse models. In order to genetically explore the role of TLR4 in keratinocyte responses to UV-induced stress, we recently utilized CRISPR/Cas9 techniques to delete TLR4 from the genome of human HaCaT keratinocyte cells in culture. First, using differential gene expression analysis (employing the EMTRT2 Profiler PCR array technology) it was observed that TLR4 KO status is associated with pronounced downregulation of EMT-related gene expression (MMP9, VIM, FN1, SNAI1, TWIST1) confirmed by independent RT-qPCR analysis. TLR4 was also a determinant of UV-induced inflammatory enzyme expression including PTGS2. Phenotypically, TLR4 KO cells displayed an attenuation of Matrigel transwell migration. TLR4 KO cells demonstrated increased sensitivity to UV-induced apoptosis as detected by flow cytometry of annexin V/propidium iodide stained cells, and by immunodetection of increased PARP cleavage. Moreover, NanoString nCounter expression analysis of murine skin chronically exposed to tumorigenic UV indicated that topical resatorvid treatment was associated with MMP9 downregulation. Future studies will employ this novel TLR4 KO keratinocyte cell line as a mechanistic tool to dissect the role of TLR4 in inflammatory signaling and EMT response in the context of skin photodamage and carcinogenesis. Supported by NIH grants ES029579, R03CA230830, P01CA229112. Citation Format: Viktoria Kirschnerova, Prajakta Vaishampayan, Maria Khawam, Clara Curiel-Lewandrowski, Georg T. Wondrak, Sally E. Dickinson. TLR4 expression as a determinant of EMT and stress response gene expression in UV exposed human keratinocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2447.

TLR2 Deficiency Exacerbates Imiquimod-Induced Psoriasis-Like Skin Inflammation through Decrease in Regulatory T Cells and Impaired IL-10 Production.

Emerging evidence has demonstrated that Toll-like receptors (TLRs) are associated with autoimmune diseases. In this study, we investigated the role of TLR2 in psoriasis using imiquimod-induced psoriasis-like dermatitis. Although TLR2 signaling is known to play a critical role in the induction of proinflammatory cytokines by immune cells, such as dendritic cells (DCs), macrophages, and monocytes, TLR2 deficiency unexpectedly exacerbated psoriasiform skin inflammation. Importantly, messenger RNA (mRNA) levels of Foxp-3 and IL-10 in the lesional skin were significantly decreased in TLR2 KO mice compared with wild-type mice. Furthermore, flow cytometric analysis of the lymph nodes revealed that the frequency of regulatory T cells (Tregs) among CD4-positive cells was decreased. Notably, stimulation with Pam3CSK4 (TLR2/1 ligand) or Pam2CSK4 (TLR2/6 ligand) increased IL-10 production from Tregs and DCs and the proliferation of Tregs. Finally, adoptive transfer of Tregs from wild-type mice reduced imiquimod-induced skin inflammation in TLR2 KO mice. Taken together, our results suggest that TLR2 signaling directly enhances Treg proliferation and IL-10 production by Tregs and DCs, suppressing imiquimod-induced psoriasis-like skin inflammation. Enhancement of TLR2 signaling may be a new therapeutic strategy for psoriasis.

Hepatic Surgical Stress Promotes Systemic Immunothrombosis That Results in Distant Organ Injury.

Innate immunity can initiate platelet activation during the development of thrombosis through a process, termed immunothrombosis. Neutrophils form neutrophil extracellular traps (NETs) that have been shown to interact directly with platelets and play pro-coagulant roles in a variety of infectious and sterile inflammatory settings. Hepatic surgical stress initiated by ischemia/reperfusion (I/R) injury has wide systemic consequences on distant organs. However, the mechanisms of this remote injury phenomenon are not well-understood. Here, we sought to determine the role of NETs in causing systemic immunothrombosis and distant organ injury following a local inflammatory insult with liver I/R. Postoperative thromboelastographic revealed that the speed of clot formation (alpha-angle) was significantly increased whereas time to clot formation (R-time) were decreased by in patients undergoing liver resection, indicating a hypercoagulable state after surgery. In mice subjected to liver I/R, circulating platelet activation and platelet-neutrophil aggregates were significantly increased. Injured distant organs such as the lung and kidney displayed NETs and platelet-rich micro-thrombi in the microvasculature following liver I/R. The immune-thrombi and organ damage were dramatically decreased when NETs were inhibited by DNase treatment. Depletion of Tlr4 on platelets limited NET-induced activation of platelets but had no effect on NET formation. Furthermore, platelet-specific TLR4 KO mice had significantly reduced distant organ injury with decreased circulating platelet activation, platelet-neutrophil aggregates following liver I/R in comparison to their control counterparts. These data establish that after an acute local inflammatory process, NET-activated platelets can lead to a systemic pro-coagulant state with resultant remote organ injury by immunothrombosis.

TRPV4 Mediates Acute Bladder Responses to Bacterial Lipopolysaccharides.

Urinary tract infections (UTI) affect a large proportion of the population, causing among other symptoms, more frequent and urgent micturition. Previous studies reported that the gram-negative bacterial wall component lipopolysaccharides (LPS) trigger acute epithelial and bladder voiding responses, but the underlying mechanisms remain unknown. The cation channel TRPV4 is implicated in the regulation of the bladder voiding. Since TRPV4 is activated by LPS in airway epithelial cells, we sought to determine whether this channel plays a role in LPS-induced responses in urothelial cells (UCs). We found that human-derived UCs display a fast increase in intracellular Ca2+ concentration upon acute application of Escherichia coli LPS. Such responses were detected also in freshly isolated mouse UCs, and found to be dependent on TRPV4, but not to require the canonical TLR4 signaling pathway of LPS detection. Confocal microscopy experiments revealed that TRPV4 is dispensable for LPS-induced nuclear translocation of NF-κB in mouse UCs. On the other hand, quantitative RT PCR determinations showed an enhanced LPS-induced production of proinflammatory cytokines in TRPV4-deficient UCs. Cystometry experiments in anesthetized wild type mice revealed that acute intravesical instillation of LPS rapidly increases voiding frequency. This effect was not observed in TRPV4-deficient animals, but was largely preserved in Tlr4 KO and Trpa1 KO mice. Our results suggest that activation of TRPV4 by LPS in UCs regulates the proinflammatory response and contributes to LPS-induced increase in voiding frequency. These findings further support the concept that TRP channels are sensors of LPS, mediating fast innate immunity mechanisms against gram-negative bacteria.

TLR2 Signaling Promoted Tumor Development through Enhancing Inflammation and Regulatory T cells to Suppress Anti-Tumor Immunity

Abstract Inflammation is a relevant physiologic process necessary for immune protection, tissue repair and remodeling. However, chronic inflammation is known to contribute tumor development, including tumor initiation, promotion and progression. Recent studies have shown that up-regulation of Toll-like receptors (TLRs) in tumor microenvironment induced the inflammatory responses. Here, we hypothesized that the TLR2 signaling within tumor microenvironment elicits an inflammatory response and such facilitates tumor development. A murine B16F10 melanoma model was utilized to investigate the role of endogenous TLR2 signaling in tumor development. Anti-tumor immunity and the presence of tumor-associated inflammatory mediators were analyzed by flow cytometry and ELISA in the TLR2 knockout (TLR2 KO) and wild-type (WT) tumor-bearing animals. When compared to the TLR2 deficient mice, WT mice had a significantly aggressive-appearing pathology, such as elicited tumor growth and metastasis. When the splenocytes cultured with tumor-conditional medium, WT produced elevated levels of pro-inflammatory cytokines, whereas TLR2KO or the WT receiving anti-TLR2 antibody treatment blocked the inflamed-like phenomenon and consequently showed a better anti-tumor IFN-γ response. Additionally, tumors from WT mice exhibited comparably higher infiltration levels of CD4+CD25+Foxp3+ regulatory T cells, but much less CD8+ T cells. These results support that cancer cells-derived components through TLR2 signaling generate an inflammatory microenvironment hospitable for tumor growth, and it indicates TLR2 as a potential therapeutic target for cancer immunotherapy.