Toll-like Receptor 4 Deficient Mice Research Articles

Spinal HMGB1 participates in the early stages of paclitaxel-induced neuropathic pain via microglial TLR4 and RAGE activation.

Chemotherapy-induced neuropathic pain (CINP) is one of the main adverse effects of chemotherapy treatment. At the spinal level, CINP modulation involves glial cells that upregulate Toll-like receptor 4 (TLR4) and signaling pathways, which can be activated by pro-inflammatory mediators as the high mobility group box-1 (HMGB1). To evaluate the spinal role of HMGB1 in the paclitaxel-induced neuropathic pain via receptor for advanced glycation end products (RAGE) and TLR4 activation expressed in glial cells. Male C57BL/6 Wild type and TLR4 deficient mice were used in the paclitaxel-induced neuropathic pain model. The nociceptive threshold was measured using the von Frey filament test. In addition, recombinant HMGB1 was intrathecally (i.t.) injected to confirm its nociceptive potential. To evaluate the spinal participation of RAGE, TLR4, NF-kB, microglia, astrocytes, and MAPK p38 in HMGB1-mediated nociceptive effect during neuropathic pain and recombinant HMGB1-induced nociception, the drugs FPS-ZM1, LPS-RS, PDTC, minocycline, fluorocitrate, and SML0543 were respectively administrated by i.t. rout. Microglia, astrocytes, glial cells, RAGE, and TLR4 protein expression were analyzed by Western blot. ELISA immunoassay was also used to assess HMGB1, IL-1β, and TNF-α spinal levels. The pharmacological experiments demonstrated that spinal RAGE, TLR4, microglia, astrocytes, as well as MAPK p38 and NF-kB signaling are involved with HMGB1-induced nociception and paclitaxel-induced neuropathic pain. Furthermore, HMGB1 spinal levels were increased during the early stages of neuropathic pain and associated with RAGE, TLR4 and microglial activation. RAGE and TLR4 blockade decreased spinal levels of pro-inflammatory cytokines during neuropathic pain. Taken together, our findings indicate that HMGB1 may be released during the early stages of paclitaxel-induced neuropathic pain. This molecule activates RAGE and TLR4 receptors in spinal microglia, upregulating pro-inflammatory cytokines that may contribute to neuropathic pain.

Defining TLR4-independent molecular pathways activated during endotoxemia

Abstract Sepsis is the leading cause of nosocomial mortality, accounting for almost 20% of annual global deaths. Patients who recover from septic shock maintain a significantly increased risk for mortality due to secondary infection. Toll-like receptor 4 (TLR4) recognizes Gram-negative bacterial lipopolysaccharides (LPS) and initiates robust immune activation that can be detrimental to the host. Towards the end of septicemia, cells self-regulate and remove surface TLR4 which contributes to immune ataxia. TLR4 deficient (TLR4 −/−) C57BL6 mice are used as a model for the immune refractory period following septic shock and compared to wildtype (WT) mice. In the serum of both WT and TLR4 −/−mice, 51 proteins were previously found to be dysregulated over the course of endotoxemia. We hypothesize that these 51 proteins represent TLR4 independent activation mechanisms. This study further investigates protein and mRNA distribution during endotoxemia. Liver, spleen, kidney, heart, and brain were collected at 0, 6, 12, and 18 hours post-intraperitoneal injection of 30 mg/kg E. coli LPS. Using qPCR and Western Blot to quantify gene and protein expression, we observed down regulation of genes involved with metabolism (Adsl, Mdh1) and erythrocyte cytoskeleton stabilization (Dmtn). In the same samples we observe activation of genes and proteins involved in T-cell function (Coro1a), and the complement cascades (Masp1, C1qb, Cfd, F5). These results further define TLR4 independent activation during endotoxemia as a model for immune ataxia that occurs after primary septic shock. This project could provide proof of principle to support the development of more targeted treatments to address immune activation during secondary infection after septic shock. This project was supported by a Towson University FDRC grant to EMH and a Fisher College of Science and Mathematics undergraduate research award to JCH, HA, PC, and AA.

Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice

ObjectiveToll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2−/−) mice to gain insight into TLR2 function in biological circumstances without microbial infection. MethodsEight-week-old male wild-type and TLR2−/− mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis. ResultsTLR2−/− tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2−/− osteoclasts reduced cell fusion and bone resorption activity. ConclusionIt seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.

Hepatocyte Toll-Like Receptor 4 Mediates Alcohol-Induced Insulin Resistance in Mice.

Accumulating evidence has demonstrated the association between alcohol overconsumption and the development of insulin resistance. However, the underlying mechanisms are not completely understood. To investigate the requirement and sufficiency of hepatocyte toll-like receptor 4 (TLR4) in alcohol-induced insulin resistance, we used two mouse models (Tlr4fl/fl and Tlr4LoxTB) that allow ablation of TLR4 only in hepatocytes (Tlr4LKO) and restoration of endogenous TLR4 expression in hepatocytes on a TLR4-null background (Tlr4LoxTB × Alb-Cre), respectively. A Lieber-DeCarli feeding model was used to induce glucose intolerance and insulin resistance in mice. Glucose tolerance test, insulin tolerance test, and insulin signaling experiments were performed to examine systemic and tissue-specific insulin sensitivity. We found that alcohol-fed hepatocyte TLR4 deficient mice (Tlr4LKO) had lower blood glucose levels in response to intraperitoneal injection of insulin. Moreover, increased phosphorylation of glycogen synthase kinase-3β (GSK3β) was observed in the liver of Tlr4LKO mice after chronic alcohol intake. In contrast, when hepatic TLR4 was reactivated in mice (Tlr4LoxTB × Alb-Cre), alcohol feeding caused glucose intolerance in these mice compared with littermate controls (Tlr4LoxTB). In addition, AKT phosphorylation was dramatically reduced in the liver and epididymal white adipose tissue (eWAT) of alcohol-fed Tlr4LoxTB × Alb-Cre mice, which was similar to that of mice with whole-body TLR4 reactivation (Tlr4LoxTB × Zp3-Cre). Collectively, these findings suggest that hepatocyte TLR4 is both required and sufficient in the development of insulin resistance induced by alcohol overconsumption.

Targeting Immunosuppressive Macrophages in the Cutaneous T-Cell Lymphoma Microenvironment

Introduction: Immunosuppressive M2-like macrophages contribute to malignancy progression in cutaneous T-cell lymphoma (CTCL). Hence targeting M2-like macrophage differentiation and function may have potential therapeutic utility in CTCL. Targetable signaling pathways in M2 macrophages include the mannose receptor CD206 and the pattern recognition receptor Toll-like Receptor 4 (TLR4). Objective: To evaluate TLR4 and CD206 function in CTCL-associated macrophages and their therapeutic potential as targets of immunomodulation. Methods: We assessed TLR4 and CD206 expression in human CTCL lesional skin biopsies and in transwell co-cultures containing macrophages and CTCL cells. Using an established murine model of CTCL, we quantified tumor burden of mouse MBL2 cells after transplantation into immunocompetent mice with and without manipulating TLR4 or CD206 function. To abrogate TLR4 function we used TLR4 deficient mice and to stimulate CD206 signaling we used a novel CD206 binding peptide, RP-832c. Results: Human CTCL lesional skin biopsies and MBL2 tumors displayed co-localized expression of CD206 and TLR4 on macrophages. CTCL-associated macrophages in co-cultures exhibited an M2-like polarized phenotype as well as increased TLR4 expression. In contrast, when these co-cultures were treated with RP-832c, macrophages displayed the M1-like phenotype. In mice lacking TLR4, CTCL tumor growth was reduced (Figure 1) and M1 to M2 macrophage ratios were significantly higher compared to that of wild type. Similarly, CTCL tumors in mice treated with RP-832c displayed a shift from M2 macrophage phenotype to M1 and exhibited attenuated tumor growth (Figure 2). Conclusion: Our data suggest that both TLR4 inhibition and CD206 activation in the CTCL tumor microenvironment inhibit tumor growth by inducing a phenotypic shift from immunosuppressive M2-like to anti-tumor M1-like macrophages. Therefore, topical or systemic formulations of CD206 binding peptide, RP-832c, and small molecule TLR4 inhibitors present promising immunomodulatory therapeutic options in CTCL. Figure 1: MBL2 cells were injected s.c. into flanks of C57BL/6 and TLR4-/- recipients (5 mice /grp.) and tumor volumes monitored overtime. Shown are the mean and standard error for each group. Figure 2: MBL2 cells were injected s.c. into flanks of C57BL/6 recipients (5 mice /grp.) and mice were treated daily with i.p. injections of RP832c or saline starting 10 days post inoculation. Tumor volumes were monitored overtime. Shown are the mean and standard error for each group. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bio-P-06 - Toll-like receptor 4 activity promotes immunosuppression in the cutaneous T-cell lymphoma microenvironment

How microenvironmental factors such as skin extracellular matrix (ECM) and resident immune cells contribute to cutaneous T-cell lymphoma (CTCL) is not well understood. Here we report a ligand-receptor interaction connecting the ECM and immunosuppressive macrophages in the CTCL microenvironment. We find that expression of a fibronectin splice variant encoding extra domain A (EDA), a hallmark of ECM dysregulation in cancer, occurs in human CTCL lesions and in mouse CTCL tumors. Notably, EDA is a ligand for toll-like receptor 4 (TLR4), a pattern recognition receptor with roles in regulating immunity within tumor microenvironments. We find that CD206+ immunosuppressive macrophages within the lesional skin of CTCL patients express high levels of TLR4. Similarly, tumor-localized CD206+ macrophages are TLR4-high in our immune-competent CTCL mouse model. Lastly, we observe the abrogation of CTCL tumor growth in TLR4 deficient mice compared to wild type. Together, these data suggest that TLR4 activity in the CTCL microenvironment promotes tumor growth, and EDA-TLR4 interactions may drive immunosuppressive macrophages. Our results uncover a link between the extracellular matrix and anti-tumor immune response that may reveal new therapeutic targets in CTCL. How microenvironmental factors such as skin extracellular matrix (ECM) and resident immune cells contribute to cutaneous T-cell lymphoma (CTCL) is not well understood. Here we report a ligand-receptor interaction connecting the ECM and immunosuppressive macrophages in the CTCL microenvironment. We find that expression of a fibronectin splice variant encoding extra domain A (EDA), a hallmark of ECM dysregulation in cancer, occurs in human CTCL lesions and in mouse CTCL tumors. Notably, EDA is a ligand for toll-like receptor 4 (TLR4), a pattern recognition receptor with roles in regulating immunity within tumor microenvironments. We find that CD206+ immunosuppressive macrophages within the lesional skin of CTCL patients express high levels of TLR4. Similarly, tumor-localized CD206+ macrophages are TLR4-high in our immune-competent CTCL mouse model. Lastly, we observe the abrogation of CTCL tumor growth in TLR4 deficient mice compared to wild type. Together, these data suggest that TLR4 activity in the CTCL microenvironment promotes tumor growth, and EDA-TLR4 interactions may drive immunosuppressive macrophages. Our results uncover a link between the extracellular matrix and anti-tumor immune response that may reveal new therapeutic targets in CTCL.

060 Toll-like receptor 4 activity in the tumor microenvironment promotes cutaneous T-cell lymphoma

How microenvironmental factors such as skin extracellular matrix (ECM) and resident immune cells contribute to cutaneous T-cell lymphoma (CTCL) is not well understood. Here we report a ligand-receptor interaction connecting the ECM and immunosuppressive macrophages in the CTCL microenvironment. We find that expression of a fibronectin splice variant encoding extra domain A (EDA), a hallmark of ECM dysregulation in cancer, occurs in mouse CTCL tumors, and we confirm this finding in human CTCL lesions. Notably, EDA is a ligand for toll-like receptor 4 (TLR4), a pattern recognition receptor with roles in regulating immunity within tumor microenvironments. We find that CD206+ immunosuppressive macrophages within the lesional skin of CTCL patients express high levels of TLR4. Similarly, tumor-localized CD206+ macrophages are TLR4-high in our immune-competent CTCL mouse model. Lastly, we observe the abrogation of CTCL tumor growth in TLR4 deficient mice compared to wild-type. Together, these data suggest that TLR4 activity in the CTCL microenvironment promotes tumor growth, and EDA-TLR4 interactions may drive immunosuppressive macrophages. Our results uncover a link between the extracellular matrix and anti-tumor immune response that may reveal new therapeutic targets in CTCL.

Anti-High Mobility Group Box 1 Neutralizing-Antibody Ameliorates Dextran Sodium Sulfate Colitis in Mice.

High mobility group box 1 (HMGB1) is a ubiquitous nuclear protein in mammals. When released into the extracellular space, it acts as a damage-associated molecular pattern. This study investigates whether increased HMGB1 levels are found in the intestinal mucosa of ulcerative colitis (UC) patients, and whether an anti-HMGB1 neutralizing-antibody (HnAb) can inhibit the intestinal inflammation elicited by dextran sulfate sodium (DSS) in mice. Because toll-like receptor 4 (TLR4) is implicated in HMGB1-mediated immune cell activation, DSS colitis was also elicited in TLR4-deficient mice in the presence and absence of HnAb. The expression of HMGB1 in UC patients was examined. HnAb was administered via intraperitoneal injection to TLR4 deficient mice and their wild-type littermates, both being induced to colitis with DSS. Finally, the protective effect of HnAb and TLR4 deficiency were evaluated. In UC patients, HMGB1 was up-regulated in the inflamed colon. When administered during DSS application, HnAb alleviated the severity of colitis with a lower disease activity index, limited histological damages, and reduced production of proinflammatory cytokines. This antibody also limited colonic barrier loss, decreased colonic lamina propria macrophages and partially reversed the DSS treatment-associated dysbiosis. The protective effect of this antibody was enhanced in TLR4-deficient mice in some aspects, indicating that both additional HMGB1-mediated as well as TLR4-mediated inflammatory signaling pathways were involved in the induction of colitis by DSS. HnAb ameliorated colitis via macrophages inhibition and colonic barrier protection. It may therefore be a novel treatment option in colitis.

Toll-like receptor 4 differentially regulates adult hippocampal neurogenesis in an age- and sex-dependent manner.

Toll-like receptor 4 (TLR4) is primarily responsible for initiating an immune response following pathogen recognition. However, TLR4 is also expressed on neural progenitor cells and has been reported to regulate hippocampal neurogenesis as young male TLR4 knockout mice show increases in cell proliferation and doublecortin positive cells. Whether these effects occur in both sexes and are sustained with normal aging is currently unknown. The present study evaluated whether TLR4 deficiency alters adult hippocampal neurogenesis in young (3-4 months) and aged (18-20 months), male and female, TLR4 deficient (TLR4-/-; B6.B10ScN-Tlr4lps-del/JthJ) and wild type (WT) mice. Additionally, neurogenesis within the dorsal and the ventral hippocampal subdivisions was evaluated to determine if TLR4 has differential effects across the hippocampus. Bromodeoxyuridine (BrdU) was administered to quantify new cell survival as well as cell differentiation. Ki-67 was measured to evaluate cell proliferation. Results show that young TLR4-/- females had higher rates of proliferation and neuronal differentiation in both the dorsal and ventral hippocampus relative to WT females. Young TLR4-/- males show elevated proliferation and neuronal differentiation mainly in the ventral hippocampus. While young TLR4-/- mice show enhanced neurogenesis compared to young WT mice, the increase was not apparent in the aged TLR4-/- mice. Both aged WT and TLR4-/- mice showed a decrease in proliferation, new cell survival, and neuronal differentiation compared to young WT and TLR4-/- mice. The data collectively indicate that TLR4 regulates hippocampal neurogenesis in young adults, but that these effects are region-specific in males and that females show broader changes in neurogenesis throughout the hippocampus.

Toll-like receptor 2-deficiency on bone marrow-derived cells augments vascular healing of murine arterial lesions

AimsNeointimal hyperplasia contributes to arterial restenosis after percutaneous transluminal coronary angioplasty or vascular surgery. Neointimal thickening after arterial injury is determined by inflammatory processes. We investigated the role of the innate immune receptor toll-like receptor 2 (TLR2) in neointima formation after arterial injury in mice. Materials and methodsCarotid artery injury was induced by 10% ferric chloride in C57Bl/6J wild type (WT), TLR2 deficient (B6.129-Tlr2tm1Kir/J, TLR2−/−) and WT mice treated with a TLR2 blocking antibody. 21 days after injury, carotid arteries were assessed histomorphometrically and for smooth muscle cell (SMC) content. To identify the contribution of circulating cells in mediating the effects of TLR2-deficiency, arterial injury was induced in WT/TLR2−/−-chimeric mice and the paracrine modulation of bone marrow-derived cells from WT and TLR2−/− on SMC migration compared in vitro. Key findingsTLR2−/− mice and WT mice treated with TLR2 blocking antibodies exhibited reduced neointimal thickening (23.7 ± 4.2 and 6.5 ± 3.0 vs. 43.1 ± 5.9 μm, P < 0.05 and P < 0.01), neointimal area (5491 ± 1152 and 315 ± 76.7 vs. 13,756 ± 2627 μm2, P < 0.05 and P < 0.01) and less luminal stenosis compared to WT mice (8.5 ± 1.6 and 5.0 ± 1.3 vs. 22.4 ± 2.2%, both P < 0.001n = 4–8 mice/group). The phenotypes of TLR2−/− vs. WT mice were completely reverted in WT/TLR2−/− bone marrow chimeric mice (5.9 ± 1.5 μm neointimal thickness, 874.2 ± 290.2 μm2 neointima area and 2.7 ± 0.6% luminal stenoses in WT mice transplanted with TLR2−/− bone marrow vs. 23.6 ± 5.1 μm, 3555 ± 511 μm2 and 12.0 ± 1.3% in WT mice receiving WT bone marrow, all P < 0.05, n = 6/group). Neointimal lesions of WT and WT mice transplanted with TLR2−/− bone marrow chimeric mice showed increased numbers of SMC (10.8 ± 1.4 and 12.6 ± 1.4 vs. 3.8 ± 0.9 in TLR2−/− and 3.5 ± 1.1 cells in WT mice transplanted with TLR2−/− bone marrow, all P < 0.05, n = 6). WT bone marrow cells stimulated SMC migration more than TLR2-deficient bone marrow cells (1.7 ± 0.05 vs. 1.3 ± 0.06-fold, P < 0.05, n = 7) and this effect was aggravated by TLR2 stimulation and diminished by TLR2 blockade (1.1 ± 0.03-fold after stimulation with TLR2 agonists and 0.8 ± 0.02-fold after TLR2 blockade vs. control treated cells defined as 1.0, P < 0.05, n = 7). SignificanceTLR2-deficiency on hematopoietic but not vessel wall resident cells augments vascular healing after arterial injury. Pharmacological blockade of TLR2 may thus be a promising therapeutic option to improve vessel patency after iatrogenic arterial injury.

Toll-Like Receptor 4 Promotes Th17 Lymphocyte Infiltration Via CCL25/CCR9 in Pathogenesis of Experimental Autoimmune Encephalomyelitis.

Toll-like receptor 4 (TLR4) is a key component in innate immunity and has been linked to central nervous system (CNS) inflammation diseases, such as multiple sclerosis (MS), an inflammatory disorder induced by autoreactive Th17 cells. In our study, we found that TLR4 deficient (TLR4-/-) mice were inadequate to induce experimental autoimmune encephalomyelitis (EAE), characterized by low clinic score and weight loss, alleviative demyelinating, as well as decreased inflammatory cell infiltration in the spinal cord. In the lesion area of EAE mice, loss of TLR4 down-regulated the secretion of inflammatory cytokines and chemokine CCL25. Furthermore, the expression of CCR9 was decreased and chemotactic migration was attenuated in TLR4-/- Th17 cells. Our results demonstrate that TLR4 may mediate Th17 infiltration through CCL25/CCR9 signal during pathogenesis of EAE. Graphical Abstract Immunofluorescent staining of RORγt (green) and CCR9 (red) in spinal cords. TLR4 deficiency down-regulates CCR9 expression in infiltrating lymphocytes.

Toll-like receptor 4 is necessary for glucose-dependent glucagon-like peptide-1 secretion in male mice

Lipopolysaccharide (LPS), a natural toll-like receptor 4 (TLR4) ligand, can induce the secretion of glucagon-like peptide-1 (GLP-1) in both animal models and humans consistent with the notion that TLR4 may influence physiological process of GLP-1 secretion. Here, we explored the possible role of TLR4 in the process of glucose-dependent GLP-1 secretion. Wild-type and TLR4 knockout mice were used to observe GLP-1 expression and secretion, as well as the cytokines tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) levels in plasma and ileum after glucose load. TLR4 deficient mice also were given an injection of TNF-α and IL-6, respectively, to confirm the key role of TLR4-mediated inflammatory cytokines in the process of glucose-induced GLP-1 secretion. We found that the TLR4 deficiency impaired the glucose-induced GLP-1 release and prevented an increase in IL-6 and TNF-α levels in plasma and ileum following glucose stimulation. Importantly, injection of TLR4 deficient mice with either TNF-α and IL-6 partly restored the glucose-induced secretion of GLP-1. In conclusion, the production of pro-inflammatory cytokines downstream of TLR4 promotes glucose-dependent GLP-1 secretion from intestinal L-cells in mice.

Young and aged TLR4 deficient mice show sex-dependent enhancements in spatial memory and alterations in interleukin-1 related genes

Toll-like receptor-4 (TLR4) is a transmembrane receptor that initiates an immune response following a bacterial infection or host derived molecules associated with cellular distress. Beyond triggering inflammation, TLR4 has been implicated in modulating behavioral and cognitive processes in a physiologically normal state, as young adult TLR4 deficient mice show learning enhancements in select tasks. Currently unknown is whether these benefits are present in both sexes and persist with aging. The present study evaluated spatial memory, anxiety-like behavior, and central levels of pro- and anti-inflammatory molecules in young (4–5 months) and aged (18–19 months) TLR4 deficient (TLR4−/−) and wild-type (WT) male and female mice. Results confirmed that TLR4−/− mice show enhanced spatial memory compared to WT mice. These effects were age- and sex-specific, as memory retention was superior in the TLR4−/− young males and aged females. While TLR4−/− mice showed age-related changes in behavior, these changes were attenuated relative to aged WT mice. Further, aged TLR4−/− mice showed differential expression of molecules involved in interleukin (IL)-1 signaling in the hippocampus. For instance, aged TLR4−/− females showed heightened expression of IL-1 receptor antagonist (IL-1ra) and the IL-1 accessory proteins AcP and AcPb. Collectively, these data provide the initial evidence that TLR4 deficiency enhances cognitive function and modulates the inflammatory profile of the hippocampus in a sex- and age-dependent manner.

Hepatocyte toll-like receptor 4 deficiency protects against alcohol-induced fatty liver disease

ObjectiveRecent studies have suggested a critical role for toll-like receptor 4 (TLR4) in the development of alcoholic liver disease. As TLR4 is widely expressed throughout the body, it is unclear which TLR4-expressing cell types contribute to alcohol-induced liver damage. MethodsWe selectively ablated TLR4 in hepatocytes and myeloid cells. Male mice were fed a liquid diet containing either 5% alcohol or pair-fed a control diet for 4 weeks to examine chronic alcohol intake-induced liver damage and inflammation. In addition, mice were administered a single oral gavage of alcohol to investigate acute alcohol drinking-associated liver injury. ResultsWe found that selective hepatocyte TLR4 deletion protected mice from chronic alcohol-induced liver injury and fatty liver. This result was in part due to decreased expression of endogenous lipogenic genes and enhanced expression of genes involved in fatty acid oxidation. In addition, mice lacking hepatocyte TLR4 exhibited reduced mRNA expression of inflammatory genes in white adipose tissue. Furthermore, in an acute alcohol binge model, hepatocyte TLR4 deficient mice had significantly decreased plasma alanine transaminase (ALT) levels and attenuated hepatic triglyceride content compared to their alcohol-gavaged control mice. In contrast, deleting TLR4 in myeloid cells did not affect the development of chronic-alcohol induced fatty liver, despite the finding that mice lacking myeloid cell TLR4 had significantly reduced circulating ALT concentrations. ConclusionsThese findings suggest that hepatocyte TLR4 plays an important role in regulating alcohol-induced liver damage and fatty liver disease.

Linking Toll‐like Receptor 4, Gut Microbiota, and Doxycycline in the Hypertensive Kidney

Hypertension is one of the leading causes of chronic kidney disease (CKD) and affects approximately one‐third of the United States population. Toll‐like receptor 4 (TLR4) plays a role in the innate immune system through induction of NF‐κB‐mediated inflammatory response, which if unresolved, can lead to tissue damage. Activation of TLR4 is through recognition of foreign antigens, such as lipopolysaccharide (LPS), which is a product of gram‐negative bacteria. Several recent studies have revealed the gut microbiome can influence multiple physiological processes, such as metabolism, immunity, and blood pressure (BP) regulation. Doxycycline is routinely prescribed for a variety of illnesses and infections, however, the exact effects on gut microbiome composition and blood pressure regulation and kidney function have not been evaluated. In this study, we sought to determine the link between TLR4, gut microbiota and the kidney during hypertension and whether the broad‐spectrum antibiotic doxycycline could reverse the effects to mitigate renal dysfunction. C3H/HeJ (TLR4 deficiency) and C3H/HeOuJ (normal TLR4) mice were treated without or with Ang‐II (1000ng/kg−1/min−1) via mini osmotic pump for 4 weeks. Initial results indicated a blunted response in TLR4 deficient mice treated with Ang‐II as observed by a lower mean blood pressure compared to mice with functional TLR4. In addition, kidney injury marker (KIM‐1) and lactate dehydrogenase (LDH) were reduced in the tubules and glomeruli of the kidney, respectively, in TLR4‐deficient mice. Moreover, TLR4‐deficient mice were protected from inflammation‐mediated damage as evidenced by a decrease in IL‐6 and IL‐1β expression. Kidney function of mice lacking TLR4 treated with Ang‐II was improved as observed by a higher glomerular filtration rate and a lower resistive index of cortex and renal artery compared to controls. Hypertensive mice with normal TLR4 function were also found to have a decrease in plasma hydrogen sulfide (H2S) levels as well as hypermethylation of the H2S converting enzyme 3‐mercaptopyruvate sulfurtransferase compared to deficient TLR4 mice. Expression of zonulin, claudin 1, and occluding, tight‐junction proteins involved in the maintenance and integrity of the gut‐epithelial barrier, were found to be similar in Ang‐II TLR4‐deficient mice compared to untreated mice. In contrast, normal TLR4 mice showed lack of tight junction protein expression, indicating a compromised gut‐epithelial barrier. This result was supported by increased levels of plasma FITC‐dextran that was administered to mice at the end of the treatment period to assess gut leakage, as well as an increase in 16S bacterial expression in the kidney. In conclusion, our data suggests TLR4‐deficient mice have improved renal function and an intact gut‐epithelial barrier as well as normalized H2S levels. Current efforts are aimed at profiling the microbiome of these mice as well as assess the effects of doxycycline on gut microbiota composition and whether this antibiotic affects blood pressure regulation.Support or Funding InformationNIH Grant DK104653 to Utpal SenAHA Grant 15SDG25840013 to Sathnur PushpakumarThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Toll-like receptor 4 on β2-glycoprotein I-induced splenic T cell subsets differentiation

Our previous study demonstrated that beta 2-glycoprotein I (β2GPI) stimulation promotes bone marrow derived dendritic cells (BMDCs) maturation and T cell proliferation in a Toll-like receptor 4 (TLR4) dependent manner. However, β2GPI induced T cell differentiation and the role of TLR4 in this process have rarely been reported. In the present study, we focused on the differentiation of splenic T cells in β2GPI immunized Balb/c, C3H/HeN and C3H/HeJ mice. According to our results, Th2 dominated differentiation was observed in β2GPI immunized Balb/c and C3H/HeN mice than in those treated with normal saline (NS), namely the up-regulated levels of Th2 markers GATA3 and IL-4 (p < 0.05). Meanwhile, reduced Th1 markers T-bet and IFN-γ, and Treg marker Foxp3 were observed in β2GPI immunized mice (p < 0.05). C3H/HeJ mice have the same gene background with C3H/HeN mice except a functional mutant in TLR4 gene. However, the described Th2 differentiation was not detected in these TLR4 deficient mice, indicating the importance of TLR4 in immune response against β2GPI. In addition, we found that β2GPI-induced Th2 differentiation could be strengthened by cytokines secreted by dendritic cells (DCs) and DCs-T cells interaction. However, DCs-T cells contact was indispensable during this process because of its unique role in suppressing Th1 function. Furthermore, this Th2 biased differentiation pattern was more noticeable in mice received 4 times β2GPI immunization than those received 2 times, suggesting the amplifying effects of anti-β2GPI Ab on β2GPI induced Th2 response. These findings may partly explain the immune imbalance in APS patient through the view angle of T cell differentiation and anti-β2GPI antibody production.

Bacterial amyloid curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9.

Bacterial biofilms are associated with numerous human infections. The predominant protein expressed in enteric biofilms is the amyloid curli, which forms highly immunogenic complexes with DNA. Infection with curli-expressing bacteria or systemic exposure to purified curli-DNA complexes triggers autoimmunity via the generation of type I interferons (IFNs) and anti-double-stranded DNA antibodies. Here, we show that DNA complexed with amyloid curli powerfully stimulates Toll-like receptor 9 (TLR9) through a two-step mechanism. First, the cross beta-sheet structure of curli is bound by cell-surface Toll-like receptor 2 (TLR2), enabling internalization of the complex into endosomes. After internalization, the curli-DNA immune complex binds strongly to endosomal TLR9, inducing production of type I IFNs. Analysis of wild-type and TLR2-deficient macrophages showed that TLR2 is the major receptor that drives the internalization of curli-DNA complexes. Suppression of TLR2 internalization via endocytosis inhibitors led to a significant decrease in Ifnβ expression. Confocal microscopy analysis confirmed that the TLR2-bound curli was required for shuttling of DNA to endosomal TLR9. Structural analysis using small-angle X-ray scattering revealed that incorporation of DNA into curli fibrils resulted in the formation of ordered curli-DNA immune complexes. Curli organizes parallel, double-stranded DNA rods at an inter-DNA spacing that matches up well with the steric size of TLR9. We also found that production of anti-double-stranded DNA autoantibodies in response to curli-DNA was attenuated in TLR2- and TLR9-deficient mice and in mice deficient in both TLR2 and TLR9 compared to wild-type mice, suggesting that both innate immune receptors are critical for shaping the autoimmune adaptive immune response. We also detected significantly lower levels of interferon-stimulated gene expression in response to purified curli-DNA in TLR2 and TLR9 deficient mice compared to wild-type mice, confirming that TLR2 and TLR9 are required for the induction of type I IFNs. Finally, we showed that curli-DNA complexes, but not cellulose, were responsible elicitation of the immune responses to bacterial biofilms. This study defines the series of events that lead to the severe pro-autoimmune effects of amyloid-expressing bacteria and suggest a mechanism by which amyloid curli acts as a carrier to break immune tolerance to DNA, leading to the activation of TLR9, production of type I IFNs, and subsequent production of autoantibodies.

Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's Amyloid-β Oligomers in Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the main cause of dementia in the world. Brain accumulation of amyloid-β oligomers (AβOs) is a major feature in the pathogenesis of AD. Although clinical and epidemiological data suggest a strong connection between AD and depression, the underlying mechanisms linking these two disorders remain largely unknown. Here, we report that aberrant activation of the brain innate immunity and decreased serotonergic tonus in the brain are key players in AβO-induced depressive-like behavior in mice. Our findings may open up new possibilities for the development of effective therapeutics for AD and depression aimed at modulating microglial function.

Effects of conventional tobacco smoke and nicotine-free cigarette smoke on airway inflammation, airway remodelling and lung function in a triple allergen model of severe asthma.

Patients with asthma who smoke have reduced lung function, increased exacerbation rates and increased steroid resistance compared to non-smoking asthmatics. In mice, cigarette smoke has been reported to have both pro- and anti-Th2 response effects. We hypothesized that combining tobacco cigarette smoke (tCS) with allergen exposure increases inflammation, airway remodelling and lung function in mice. To test this hypothesis, we combined a severe triple allergen model with tCS exposure and investigated whether effects were due to Toll-like receptor 4 signalling and/or nicotine and also observed when nicotine-free cigarettes were used. Mice were sensitized with ovalbumin, cockroach and house dust mite allergen in alum followed by intratracheal challenges with allergen twice a week for 6 weeks or additionally exposed to tCS during the allergen challenge period. Nicotine or nicotine-free herbal cigarette smoke was also applied to allergen challenged mice. tCS significantly reduced eosinophil numbers, IL-4 and IL-5 concentrations in the lung, total and allergen-specific IgE in serum, improved lung function and reduced collagen I levels. With the exception of collagen I all parameters reduced by tobacco cigarette smoke were also reduced in Toll-like receptor 4-deficient mice. Nicotine-free cigarette smoke also had significant anti-inflammatory effects on eosinophils, IL-4 and IL-5 concentrations in the lung and reduced airway hyperreactivity, albeit weaker than tobacco smoke. Applying nicotine alone also reduced Th2 cytokine levels and eosinophil numbers in the airways. Our experiments show that tCS exposure reduces allergen-induced Th2 response in the lung and associated collagen I production and development of airway hyperreactivity. With the exception on collagen I formation, these effects were not dependent on Toll-like receptor 4. The observed anti-Th2 effects of both nicotine and nicotine-free herbal cigarette smoke together suggests that tCS reduces the Th2 responses through nicotine and other products released by burning tobacco.

High glucose induces and activates Toll-like receptor 4 in endothelial cells of diabetic retinopathy.

BackgroundHyperglycemia-induced inflammation causes the dysfunction of blood vessels, and Toll-like receptor 4 (TLR4) plays a key role in inflammation-induced angiogenesis. However, the impact of TLR4 on the pathogenesis of diabetic retinopathy (DR) is poorly understood. In this study, we examined the expression of TLR4 in retinal vascular endothelial cells of patients with DR and diabetic mice, and explored the role of TLR4 in mediating inflammatory responses by human microvascular endothelial cells (HMEC-1) under high-glucose condition.MethodsThe expression of TLR4 in retinal vascular endothelial cells of patients with proliferative diabetic retinopathy and diabetic mice induced by streptozotocin was examined using immunofluorescence. HMEC-1 cells were cultured and the expression of TLR4, MyD88 and Interleukin-1β (IL-1β) was examined under high-glucose condition. Endothelial cells with TLR4 silencing and antagonist of TLR4 as well as endothelial cells from TLR4 deficient mice were used to study the effect of activated TLR4 on inflammation induced by high-glucose treatment.ResultsWe observed that TLR4 was detected in CD31-labled human retinal vascular endothelia and its expression was markedly increased in fibrovascular membranes from DR patients and in retinal vascular endothelial cells of diabetic mice. The expression of TLR4, MyD88 and IL-1β was enhanced by high glucose in cultured HMEC-1 and the expression of TLR4 and IL-1β was inhibited by TLR4 siRNA knock-down and TLR4 antagonist. The expression of IL-1β by endothelial cells from TLR4 deficient mice under high glucose condition was decreased.ConclusionsOur results revealed that hyperglycemia induced overexpression and activation of TLR4 in endothelial cells. This effect may lead to inflammatory responses contribute to the pathogenesis of diabetic retinopathy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13098-015-0086-4) contains supplementary material, which is available to authorized users.