TLR4-mutant Mice Research Articles

Toll-like receptor 4 mutation mitigates gut microbiota-mediated hypertensive kidney injury

Hypertension-associated dysbiosis is linked to several clinical complications, including inflammation and possible kidney dysfunction. Inflammation and TLR4 activation during hypertension result from gut dysbiosis-related impairment of intestinal integrity. However, the contribution of TLR4 in kidney dysfunction during hypertension-induced gut dysbiosis is unclear. We designed this study to address this knowledge gap by utilizing TLR4 normal (TLR4N) and TLR4 mutant (TLR4M) mice. These mice were infused with high doses of Angiotensin-II for four weeks to induce hypertension. Results suggest that Ang-II significantly increased renal arterial resistive index (RI), decreased renal vascularity, and renal function (GFR) in TLR4N mice compared to TLR4M. 16 S rRNA sequencing analysis of gut microbiome revealed that Ang-II-induced hypertension resulted in alteration of Firmicutes: Bacteroidetes ratio in the gut of both TLR4N and TLR4M mice; however, it was not comparably rather differentially. Additionally, Ang-II-hypertension decreased the expression of tight junction proteins and increased gut permeability, which were more prominent in TLR4N mice than in TLR4M mice. Concomitant with gut hyperpermeability, an increased bacterial component translocation to the kidney was observed in TLR4N mice treated with Ang-II compared to TLR4N plus saline. Interestingly, microbiota translocation was mitigated in Ang-II-hypertensive TLR4M mice. Furthermore, Ang-II altered the expression of inflammatory (IL-1β, IL-6) and anti-inflammatory IL-10) markers, and extracellular matrix proteins, including MMP-2, −9, −14, and TIMP-2 in the kidney of TLR4N mice, which were blunted in TLR4M mice. Our data demonstrate that ablation of TLR4 attenuates hypertension-induced gut dysbiosis resulting in preventing gut hyperpermeability, bacterial translocation, mitigation of renal inflammation and alleviation of kidney dysfunction.

Microbial neuraminidase induces TLR4-dependent long-term immune priming in the brain.

Innate immune memory explains the plasticity of immune responses after repeated immune stimulation, leading to either enhanced or suppressed immune responses. This process has been extensively reported in peripheral immune cells and also, although modestly, in the brain. Here we explored two relevant aspects of brain immune priming: its persistence over time and its dependence on TLR receptors. For this purpose, we used an experimental paradigm consisting in applying two inflammatory stimuli three months apart. Wild type, toll-like receptor (TLR) 4 and TLR2 mutant strains were used. The priming stimulus was the intracerebroventricular injection of neuraminidase (an enzyme that is present in various pathogens able to provoke brain infections), which triggers an acute inflammatory process in the brain. The second stimulus was the intraperitoneal injection of lipopolysaccharide (a TLR4 ligand) or Pam3CSK4 (a TLR2 ligand). One day after the second inflammatory challenge the immune response in the brain was examined. In wild type mice, microglial and astroglial density, as well as the expression of 4 out of 5 pro-inflammatory genes studied (TNFα, IL1β, Gal-3, and NLRP3), were increased in mice that received the double stimulus compared to those exposed only to the second one, which were initially injected with saline instead of neuraminidase. Such enhanced response suggests immune training in the brain, which lasts at least 3 months. On the other hand, TLR2 mutants under the same experimental design displayed an enhanced immune response quite similar to that of wild type mice. However, in TLR4 mutant mice the response after the second immune challenge was largely dampened, indicating the pivotal role of this receptor in the establishment of immune priming. Our results demonstrate that neuraminidase-induced inflammation primes an enhanced immune response in the brain to a subsequent immune challenge, immune training that endures and that is largely dependent on TLR4 receptor.

TLR4 mutation protects neurovascular function and cognitive decline in high-fat diet-fed mice

BackgroundMetabolic syndrome (MS) is defined as a low-grade proinflammatory state in which abnormal metabolic and cardiovascular factors increase the risk of developing cardiovascular disease and neuroinflammation. Events, such as the accumulation of visceral adipose tissue, increased plasma concentrations of free fatty acids, tissue hypoxia, and sympathetic hyperactivity in MS may contribute to the direct or indirect activation of Toll-like receptors (TLRs), specifically TLR4, which is thought to be a major component of this syndrome. Activation of the innate immune response via TLR4 may contribute to this state of chronic inflammation and may be related to the neuroinflammation and neurodegeneration observed in MS. In this study, we investigated the role of TLR4 in the brain microcirculation and in the cognitive performance of high-fat diet (HFD)-induced MS mice.MethodsWild-type (C3H/He) and TLR4 mutant (C3H/HeJ) mice were maintained under a normal diet (ND) or a HFD for 24 weeks. Intravital video-microscopy was used to investigate the functional capillary density, endothelial function, and endothelial–leukocyte interactions in the brain microcirculation. Plasma concentrations of monocyte chemoattractant protein-1 (MCP-1), adipokines and metabolic hormones were measured with a multiplex immunoassay. Brain postsynaptic density protein-95 and synaptophysin were evaluated by western blotting; astrocytic coverage of the vessels, microglial activation and structural capillary density were evaluated by immunohistochemistry.ResultsThe HFD-induced MS model leads to metabolic, hemodynamic, and microcirculatory alterations, as evidenced by capillary rarefaction, increased rolling and leukocyte adhesion in postcapillary venules, endothelial dysfunction, and less coverage of astrocytes in the vessels, which are directly related to cognitive decline and neuroinflammation. The same model of MS reproduced in mice deficient for TLR4 because of a genetic mutation does not generate such changes. Furthermore, the comparison of wild-type mice fed a HFD and a normolipid diet revealed differences in inflammation in the cerebral microcirculation, possibly related to lower TLR4 activation.ConclusionsOur results demonstrate that TLR4 is involved in the microvascular dysfunction and neuroinflammation associated with HFD-induced MS and possibly has a causal role in the development of cognitive decline.

TLR4-Mediated Inflammatory Responses Regulate Exercise-Induced Molecular Adaptations in Mouse Skeletal Muscle.

Endurance exercise induces various adaptations that yield health benefits; however, the underlying molecular mechanism has not been fully elucidated. Given that it has recently been accepted that inflammatory responses are required for a specific muscle adaptation after exercise, this study investigated whether toll-like receptor (TLR) 4, a pattern recognition receptor that induces proinflammatory cytokines, is responsible for exercise-induced adaptations in mouse skeletal muscle. The TLR4 mutant (TLR4m) and intact TLR4 control mice were each divided into 2 groups (sedentary and voluntary wheel running) and were housed for six weeks. Next, we removed the plantaris muscle and evaluated the expression of cytokines and muscle regulators. Exercise increased cytokine expression in the controls, whereas a smaller increase was observed in the TLR4m mice. Mitochondrial markers and mitochondrial biogenesis inducers, including peroxisome proliferator-activated receptor beta and heat shock protein 72, were increased in the exercised controls, whereas this upregulation was attenuated in the TLR4m mice. In contrast, exercise increased the expression of molecules such as peroxisome proliferator-activated receptor-gamma coactivator 1-alpha and glucose transporter 4 in both the controls and TLR4m mice. Our findings indicate that exercise adaptations such as mitochondrial biogenesis are mediated via TLR4, and that TLR4-mediated inflammatory responses could be involved in the mechanism of adaptation.

Toll-like Receptor 4 Mediates Reflux-Induced Inflammation in a Murine Reflux Model

Dysregulation of toll-like receptor (TLR) signaling within the gastrointestinal epithelium has been associated with uncontrolled inflammation and tumorigenesis. We sought to evaluate the role of TLR4 in the development of gastroesophageal reflux-mediated inflammation and mucosal changes of the distal esophagus. Verified human esophageal Barrett's cells with high grade dysplasia (CPB) and esophageal adenocarcinoma cells (OE33) were treated with deoxycholic acid for 24 hours. Cells were pretreated with a TLR4-specific inhibitor peptide 2 hours prior to deoxycholic acid treatment. Inflammatory markers were evaluated using immunoblotting and enzyme-linked immunosorbent assay. A surgical reflux mouse model was generated by performing a side-to-side anastomosis between the second portion of the duodenum and the gastroesophageal junction. Control animals underwent laparotomy with incision and closure of the esophagus superior to the gastroesophageal junction (sham procedure). Esophageal sections were evaluated using hematoxylin and eosin staining and immunohistochemistry. Deoxycholic acid increased expression of inflammatory markers including intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and interleukin 8. Pretreatment with a TLR4 inhibitor significantly decreased deoxycholic acid-induced inflammatory marker expression. C3H/HeNCrl mice demonstrated a significant increase in mucosal hyperplasia and proliferation following DGEA compared to sham procedure. TLR4 mutant mice (C3H/HeJ) undergoing DGEA demonstrated an attenuated hyperplastic and proliferative response compared to C3H/HeNCrl mice. Inhibition of TLR4 signaling attenuates reflux-induced inflammation in vivo. These findings identify TLR4 inhibition as a potential therapeutic target to halt the progression of pathologic esophageal changes developing in the setting of chronic gastroesophageal reflux disease.

Citrullinated vimentin mediates development and progression of lung fibrosis.

The mechanisms by which environmental exposures contribute to the pathogenesis of lung fibrosis are unclear. Here, we demonstrate an increase in cadmium (Cd) and carbon black (CB), common components of cigarette smoke (CS) and environmental particulate matter (PM), in lung tissue from subjects with idiopathic pulmonary fibrosis (IPF). Cd concentrations were directly proportional to citrullinated vimentin (Cit-Vim) amounts in lung tissue of subjects with IPF. Cit-Vim amounts were higher in subjects with IPF, especially smokers, which correlated with lung function and were associated with disease manifestations. Cd/CB induced the secretion of Cit-Vim in an Akt1- and peptidylarginine deiminase 2 (PAD2)-dependent manner. Cit-Vim mediated fibroblast invasion in a 3D ex vivo model of human pulmospheres that resulted in higher expression of CD26, collagen, and α-SMA. Cit-Vim activated NF-κB in a TLR4-dependent fashion and induced the production of active TGF-β1, CTGF, and IL-8 along with higher surface expression of TLR4 in lung fibroblasts. To corroborate ex vivo findings, mice treated with Cit-Vim, but not Vim, independently developed a similar pattern of fibrotic tissue remodeling, which was TLR4 dependent. Moreover, wild-type mice, but not PAD2-/- and TLR4 mutant (MUT) mice, exposed to Cd/CB generated high amounts of Cit-Vim, in both plasma and bronchoalveolar lavage fluid, and developed lung fibrosis in a stereotypic manner. Together, these studies support a role for Cit-Vim as a damage-associated molecular pattern molecule (DAMP) that is generated by lung macrophages in response to environmental Cd/CB exposure. Furthermore, PAD2 might represent a promising target to attenuate Cd/CB-induced fibrosis.

Catechin-Rich Green Tea Extract and the Loss-of-TLR4 Signaling Differentially Alter the Hepatic Metabolome in Mice with Nonalcoholic Steatohepatitis.

Catechin-rich green tea extract (GTE) limits inflammation in nonalcoholic steatohepatitis (NASH) consistent with a Toll-like receptor 4 (TLR4)-dependent mechanism. It is hypothesized that GTE supplementation during NASH will shift the hepatic metabolome similar to that attributed to the loss-of-TLR4 signaling. Wild-type (WT) and loss-of-function TLR4-mutant (TLR4mut ) mice are fed a high-fat diet containing 0% or 2% GTE for 8 weeks prior to performing untargeted mass spectrometry-based metabolomics on liver tissue. The loss-of-TLR4 signaling and GTE shift the hepatic metabolome away from that of WT mice. However, relatively few metabolites are altered by GTE in WT mice to the same extent as the loss-of-TLR4 signaling in TLR4mut mice. GTE increases acetyl-coenzyme A precursors and spermidine to a greater extent than the loss-of-TLR4 signaling. Select metabolites associated with thiol metabolism are similarly affected by GTE and the loss-of-TLR4 signaling. Glycerophospholipid catabolites are decreased by GTE, but are unaffected in TLR4mut mice. Conversely, the loss-of-TLR4 signaling but not GTE increases several bile acid metabolites. GTE limitedly alters the hepatic metabolome consistent with a TLR4-dependent mechanism. This suggests that the anti-inflammatory activities of GTE and loss-of-TLR4 signaling that regulate hepatic metabolism to abrogate NASH are likely due to distinct mechanisms.

Differential Regulation of Hepatic UDP‐glucuronosyltransferase (UGT) 1A1 by Toll‐like Receptors during Irinotecan‐induced Steatosis

Irinotecan (CAMPTOSAR®) has been widely used in preoperative chemotherapy for colorectal cancer liver metastases. However, irinotecan‐induced steatosis and steatohepatitis increases mortality of liver surgery. Up to date the mechanism underlying this hepatoxicity remains largely unknown. It is known that in human irinotecan is hydrolyzed to SN‐38, a reactive metabolite, by carboxylesterases in liver. SN‐38 is then rapidly detoxified by enzyme, UDP‐glucuronosyltransferase (UGT1A1) to generate its glucuronide form. Our preliminary study indicated that irinotecan‐induced steatosis is associated with activation of Toll‐like receptors (TLRs) signaling. Previously we reported that activation of TLRs decreased multiple drug metabolism enzymes in liver, including UGT1A1. Hereby, we hypothesize that TLRs are involved in the regulation of irinotecan detoxification. TLR2wt(C57BL/6J), TLR2−/−( B6.129‐Tlr2tm1Kir/J), TLR4wt(C3HeB/FeJ) and TLR4mutant (C3H/HeJ) mice (all male, 5‐weeks old) were treated with 50mg/kg irinotecan via intraperitoneal injection once daily for nine days. Wildtype mice were sacrificed on day 2 and day 9, while TLR‐deficient mice were sacrificed on day 9. Blood and liver were collected for further analysis. We found that in C57BL/6J mice, after two days irinotecan did not cause any changes in liver. However, after nine days constitutive injection in wildtype mice, irinotecan increased proinflammatory cytokines and reduced ugt1a1 expression. Also, it decreased UGT1A1 activity in liver. Compared to wildtype mice, deficiency of either TLR2 or TLR4 signaling alleviated irinotecan‐induced proinflammatory response. However, histology evidences showed that only in TLR2−/− mice but not TLR4mutant mice, irinotecan‐induced intracellular vacuoles in liver became less and smaller than in wildtype mice. Additionally, ugt1a1 downregulation by irinotecan was attenuated in TLR2−/− mice. Pharmacokinetics study indicated that knockout of TLR2 significantly decreased the systemic exposure of SN‐38, the toxic metabolite of irinotecan; no such effect was observed in TLR4mutant mice. Based on the results, we confirm that TLRs are involved in irinotecan metabolism and associated steatosis. Although both TLR2 and TLR4 share the same adaptors to regulate similar downstream pathways, TLR2 probably plays more important role in reducing ugt1a1 during irinotecan‐induced steatosis. Hereby, we conclude that TLR2 is preferentially activated during irinotecan hepatotoxicity and such activation suppress irinotecan detoxification. We characterize a novel feature of TLRs signaling in irinotecan‐induced liver injury. It provides valuable information to address drug safety issues for public health.

Vascular Expression of Toll‐Like Receptor 4 and Myeloid Differentiation Factor‐88 Mediate Endothelial Dysfunction in Response to Lipopolysaccharide Independent of Circulating Immune Cells

The goal of this study was to test the hypothesis that vascular expression of toll‐like receptor 4 (TLR4) and its primary downstream signaling pathway, myeloid‐differentiation factor‐88 (MyD88), mediate endothelial dysfunction in response to lipopolysaccharide (LPS). Carotid arteries from wild‐type (C57Bl/6), Tlr4‐Lps‐d (Tlr4 mutant mice), and Myd88‐deficient (Myd88−/−) mice were incubated with either vehicle (saline) or LPS (0.05–0.50 μg/ml) for 22 hrs. LPS, but not vehicle, treatment produced concentration‐dependent impairment (P<0.05) of endothelium‐dependent relaxation to acetylcholine (ACh) in wild‐type mice. In contrast, in carotid arteries from Tlr4‐Lps‐d or Myd88−/− mice endothelial responses to ACh following LPS incubation were found to be similar (P>0.05) to responses in wild‐type mice incubated with vehicle. LPS has no effect (P>0.05) on vascular response to sodium nitroprusside in any of the six experimental groups. Both basal and NADPH‐stimulated superoxide levels were selectively increased (P<0.05) in vessels from wild‐type, but not Tlr4‐Lps‐d or Myd88−/−, mice treated with LPS. RNA expression revealed that both TLR4 and MyD88 are indeed expressed in the vascular wall under basal conditions and that LPS induces differential expression of a number of inflammatory gene products, including Tlr4, Myd88, and Nfkb1. Pathway analysis revealed a linkage between TLR4 and MyD88 in the vessels wall with IL‐6 and downstream targets such as Nox2 and Nox4. These findings serve to link activation of TLR4 and downstream signaling through MyD88 and NFkB1 with increases in vascular superoxide and functional impairment of endothelial function in response to LPS. Taken together, these findings demonstrate that both Tlr4 and Myd88 expression and activity within the vascular wall contributes to LPS‐induced impairment of endothelial function. The fact that experiments were performed ex vivo, demonstrate that the effects of LPS on endothelial function occur directly within the vascular wall independently of circulating immune cell activation.Support or Funding InformationSupported in part by NIH/NIBIB R01‐EB2006.

Critical role of TLR-4/NF-κB pathway in upregulation mechanism of cerebral ischemia reperfusion injury of mouse

The objective of this study is to investigate the expression of TLR-4/ NF-κB pathway in the cerebral ischemia injury of male adult mice. TLR-4 mutant (HeJ) mice and TLR-4 wild (HeN) mice were divided into the stroke model group and sham group. The stroke model was made by transient cerebral ischemia through middle cerebral artery occlusion (MCAO), while mice in the sham group were manipulated in the same way, but the MCA was not occluded. The brain tissues were collected for the morphological observation. The immunohistochemistry was employed to detect the expression of NF-κB, Western blot was used to detect the expression of TLR-4 and ELISA to detect the expression of serum pro-inflammatory mediators. The expression of NF-κB in HeJ mice after the cerebral I/R was significantly lower than that in HeN mice. For the stroke model group and sham group, the expression of TLR-4 in HeN mice was all significantly higher than that in HeJ mice, while the expression of TLR-4 in HeJ mice in the stroke model group was significantly higher than that in sham group. The expression of all pro-inflammatory mediators with HeJ was significantly lower than that in HeN mice. Together, these data concluded that TLR-4/NF-κB pathway is closely related to the cerebral I/R injury, which plays its role through the release of pro-inflammatory cytokines..

Binding of the polysaccharide from Acanthopanax giraldii Harms to toll-like receptor 4 activates macrophages

Ethnopharmacological relevanceThe traditional Chinese medicine, Acanthopanax giraldii Harms, is commonly used to treat arthralgia due to wind, cold and dampness, as well as weakness in the feet and knees. Its other reported effects include eliminating flatulence, strengthening muscles and bones, and delaying aging. The polysaccharides in A. giraldii Harms are the major bioactive substances that confer the herb's antioxidant properties as well as anticancer and antiviral effects. Aims of the studyTo elucidate the underlying mechanism and signaling cascade involved in the homogeneous A. giraldii Harms polysaccharide II (AHP–II)–mediated immunomodulation of mice macrophages. Materials and methodsThe phagocytosis of neutral red and the production of nitric oxide, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), were measured to determine AHP–II–induced macrophage activation. Confocal microscopy and flow cytometry were used to confirm the binding of AHP-II to macrophages. The involvement of Toll-like receptor (TLR) 4 in AHP–II–induced macrophage activation was demonstrated using antibody blocking and macrophages from C3H/HeJ TLR4-mutant mice. Western blotting was used to map AHP–II–induced downstream signaling pathways. ResultsAHP-II increased the phagocytosis of macrophages and the release of nitric oxide, IL-6 and TNF-α cytokines. Direct, saturable and reversible binding of AHP-II to macrophages was observed, while it can be inhibited by the anti-TLR4 antibody. In addition, the presence of the anti-TLR4 antibody inhibited AHP–II–induced macrophage IL-6 and TNF-α production in the peritoneal macrophages of C3H/HeJ mice. Moreover, AHP–II–TLR4-stimulated macrophages activate the downstream intracellular ERK and JNK/nuclear factor (NF)-κB signaling pathways. In addition, the AHP–II–mediated regulation of IL-6 and TNF-α production from macrophages was greatly affected by specific ERK, JNK and NF-κB inhibitors. ConclusionOur study elucidated the immunomodulatory mechanism of AHP-II in macrophage activation and identified TLR4 as the main receptor coordinating AHP-II binding. Our findings suggest AHP-II may be used as a novel immunopotentiator for medical purposes.

Focal TLR4 activation mediates disturbed flow-induced endothelial inflammation

Disturbed blood flow at arterial branches and curvatures modulates endothelial function and predisposes the region to endothelial inflammation and subsequent development of atherosclerotic lesions. Activation of the endothelial Toll-like receptors (TLRs), in particular TLR4, contributes to vascular inflammation. Therefore, we investigate whether TLR4 can sense disturbed flow (DF) to mediate the subsequent endothelial inflammation. En face staining of endothelium revealed that TLR4 expression, activation, and its downstream inflammatory markers were elevated in mouse aortic arch compared with thoracic aorta, which were absent in Tlr4mut mice. Similar results were observed in the partial carotid ligation model where TLR4 signalling was activated in response to ligation-induced flow disturbance in mouse carotid arteries, and such effect was attenuated in Tlr4mut mice. DF in vitro increased TLR4 expression and activation in human endothelial cells (ECs) and promoted monocyte-EC adhesion, which were inhibited in TLR4-knockdown ECs. Among endogenous TLR4 ligands examined as candidate mediators of DF-induced TLR4 activation, fibronectin containing the extra domain A (FN-EDA) expressed by ECs was increased by DF and was revealed to directly interact with and activate TLR4. Our findings demonstrate the indispensable role of TLR4 in DF-induced endothelial inflammation and pinpoint FN-EDA as the endogenous TLR4 activator in this scenario. This novel mechanism of vascular inflammation under DF condition may serve as a critical initiating step in atherogenesis.

Increased TLR4 Expression Aggravates Sepsis by Promoting IFN-γ Expression in CD38-/- Mice.

Gram-negative bacterial sepsis accounts for up to 50% worldwide sepsis that causes hospital mortality. Acute kidney injury (AKI), a common complication of Gram-negative bacterial sepsis, is caused by Toll-like receptor 4 (TLR4) activation. Lipopolysaccharide (LPS) is an endotoxin in Gram-negative bacteria and is recognized specifically by TLR4, which initiates innate immune response. Also, TLR4 signaling pathway activation is essential in response to LPS infection. CD38 is one of the well-known regulators of innate immunity, whose dysregulation contributes to sepsis. Many studies have proven that an attenuated Gram-positive bacterium induces sepsis in a CD38-blocking model. However, the pathogenesis of Gram-negative bacteria-induced sepsis in a CD38−/− mouse model remains unclear. The aim of this study is to investigate whether kidney injury is still attenuated in a LPS-induced CD38−/− sepsis model and identify the potential mechanism. We assess the severity of kidney injury related to proinflammatory cytokine expressions (IFN-γ, TNF-α, IL-1β, and IL-6) in WT and CD38−/− mice. Our results showed more aggravated kidney damage in CD38−/− mice than in WT mice, accompanied with an increase of proinflammatory cytokine expression. In addition, compared with CD38−/−TLR4mut mice, we found an increase of TLR4 expression and mRNA expression of these cytokines in the kidney of CD38−/− mice, although only increased IFN-γ level was detected in the serum. Taken together, these results demonstrated that an increased TLR4 expression in CD38−/− mice could contribute to the aggravation of AKI through boosting of the production of IFN-γ.

Toll-like receptor 4 plays an important role to enhance bacterial clearance from the nose in synergy with triggering receptor expressed on myeloid cells (TREM)-1 expression on polymorphonuclear neutrophils

ObjectiveAcute rhinosinusitis (ARS) is among the most common infectious diseases. Neutrophils play a major role in innate host defenses against pathogenic microorganisms such as fungi and bacteria. Recently, in neutrophils, ligation of the triggering receptor expressed on myeloid cells (TREM)-1 was found to activate the full spectrum of neutrophil effector mechanisms, including the release of inflammatory mediators, degranulation, phagocytosis, and oxidative burst in synergy with Toll-like receptors (TLRs). In this study, we investigated the effect of TREM-1 on the functions of neutrophils in relation to TLR4 in a nasal and nasopharyngeal inflammation mouse model via nontypeable Haemophilus influenzae (NTHi) intranasal inoculation. MethodsWe used C3H/HeJ (TLR4-deficient) mice, which arose spontaneously and have non-functional TLR4 protein, and normal wild-type (WT) C3H/HeN mice. Mice were inoculated intranasally with NTHi (107 cfu/mouse) to investigate the effects of TLR4 on the function of Neutrophils. We examined the kinetics of bacterial clearance and inflammatory cell infiltration in nasal washes at 6, 12, 24, and 72 h after inoculation. The expression of TREM-1 on neutrophils, and TREM-1 mRNA expression in neutrophils in the nasal washes were examined by flow cytometric analysis and RT-PCR. ResultsBacterial counts of NTHi from nasal washes were significantly lower in WT mice than in TLR4-mutant mice after inoculation. The numbers of inflammatory cells in nasal washes were significantly higher in WT mice at 6 h, 12 h, and 24 h after inoculation than in TLR4-deficient mice. The expression of TREM-1 protein on neutrophils and the mRNA levels were greater in WT mice than in TLR4-mutant mice. The concentrations of soluble TREM-1 in WT nasal washes were also significantly higher than in those of TLR4-deficient mice. ConclusionTREM-1 may play an important role together with TLR4 in the nasopharyngeal clearance of NTHi by neutrophils. Further studies will need to clarify the innate immune responses of neutrophils via TLR4 to prevent NTHi infection.

Reduced inflammatory factor expression facilitates recovery after sciatic nerve injury in TLR4 mutant mice

Toll-like receptors (TLRs) are extremely significant pattern recognition receptors. When nerve injury occurs, a variety of inflammatory factors are generated, leading to an exceedingly complex micro-environment. TLRs recognize damage-associated molecular patterns. To investigate the correlation between TLR4 and recovery after sciatic nerve injury, the model of sciatic nerve injury was conducted using TLR4-mutated mice (C3H/HeJ) and wild mice (C3H/HeN). Our goal was to identify short-stage and long-stage changes after sciatic nerve injury, mainly by checking the expression changes of inflammation factors in the short-stage and the differences in the recovery of the injured sciatic nerve in the long-stage. The results show that the increase of changes in the HeN group of IL-1β, IL-6, TNF-α and MCP-1 are more obvious than in the HeJ group, with caspase1 expression higher and Nlrp3 expression lower in the former group. Further results reveal intense inflammation occurred in the HeN group showing more neutrophils and macrophages. Nlrp3 and caspase1 showed little difference by Immunohistochemistry, with Nlrp6 expression differing between the HeJ group and the HeN group. The results led us to conclude that better recovery of the injured sciatic nerve occurred in the HeJ group because the expression of GAP-43 and p75NTR was higher and had a better SFI figure. TLR4 mutation can decrease the expression of inflammatory factors and enhance the speed of recovery after sciatic nerve injury. The changes in the expression of Nlrp6, which are related to the TLR4 mutation, may influence recovery of the injured sciatic nerve. Further studies will be conducted to confirm these results.

Green tea extract protects against hepatic NFκB activation along the gut-liver axis in diet-induced obese mice with nonalcoholic steatohepatitis by reducing endotoxin and TLR4/MyD88 signaling

Green tea extract (GTE) reduces NFκB-mediated inflammation during nonalcoholic steatohepatitis (NASH). We hypothesized that its anti-inflammatory activities would be mediated in a Toll-like receptor 4 (TLR4)-dependent manner. Wild-type (WT) and loss-of-function TLR4-mutant (TLR4m) mice were fed a high-fat diet containing GTE at 0 or 2% for 8 weeks before assessing NASH, NFκB-mediated inflammation, TLR4 and its adaptor proteins MyD88 and TRIF, circulating endotoxin, and intestinal tight junction protein mRNA expression. TLR4m mice had lower (P<.05) body mass compared with WT mice but similar adiposity, whereas body mass and adiposity were lowered by GTE regardless of genotype. Liver steatosis, serum alanine aminotransferase, and hepatic lipid peroxidation were also lowered by GTE in WT mice, and were similarly lowered in TLR4m mice regardless of GTE. Phosphorylation of the NFκB p65 subunit and pro-inflammatory genes (TNFα, iNOS, MCP-1, MPO) were lowered by GTE in WT mice, and did not differ from the lowered levels in TLR4m mice regardless of GTE. TLR4m mice had lower TLR4 mRNA, which was also lowered by GTE in both genotypes. TRIF expression was unaffected by genotype and GTE, whereas MyD88 was lower in mice fed GTE regardless of genotype. Serum endotoxin was similarly lowered by GTE regardless of genotype. Tight junction protein mRNA levels were unaffected by genotype. However, GTE similarly increased claudin-1 mRNA in the duodenum and jejunum and mRNA levels of occludin and zonula occluden-1 in the jejunum and ileum. Thus, GTE protects against inflammation during NASH, likely by limiting gut-derived endotoxin translocation and TLR4/MyD88/NFκB activation.

Common Transcriptomic Changes Regulated by Innate Immune Receptors and Nuclear Factor NF-kappa-B in Mice Exposed to Ozone

Ozone (O3) is a predominant oxidant in air pollution and has been linked to diminished lung function, respiratory inflammation, and worsening of preexisting airway diseases. Tumor necrosis factor (TNF)-α and toll-like receptor 4 (TLR4) play critical roles in innate immune responses. Signaling through TNF receptor (TNFR) and TLR4 activates nuclear factor NF-kappa-B (NF-κB), leading to the transcriptional regulation of immunity genes. The injurious roles of TNF-α, TLR4, and NF-κB in O3-induced lung pathogenesis have been determined in mice. The current study was designed to examine lung transcriptome changes and downstream mechanisms regulated by these innate immune response factors. Mice lacking Tnfr (Tnfr-/-) or Nfkb (Nfkb-/-), Tlr4 mutant mice (C3H/HeJ), and their wild-types (Tnfr+/+, Nfkb+/+, C3H/HeOuJ) were exposed to air or 0.3 ppm O3. Lung RNAs were isolated for cDNA microarray analysis. Significantly varied genes between the wild-type and gene knockout mice after O3 exposure were determined (2-way ANOVA) to elucidate commonly changed genes between the TNFR-NF-κB, TLR4-NF-κB, and TNFR-TLR4 pathways (Venn Diagram analysis). Downstream mechanisms affected by the common differentially regulated genes and chemicals/drugs that may act on O3-responsive genes were predicted by bioinformatics analysis tools. qPCR, ELISA, Western blotting, and chromatin immunoprecipitation assay validated the results. Tnfr- and Nfkb-dependently changed genes by O3, including matrix metalloproteinase 9 (Mmp9) and endothelin 1 (Edn1), were involved in cell death, connective tissue injury, and inflammation. Interleukin 12 (IL-12) and transforming growth factor (TGF)-β1 were suggested as upstream regulators. Genes changed in common by Tlr4 and Nfkb during O3 exposure (e.g. D-box binding PAR BZIP transcription factor, Dbp; lipocalin 2, Lcn2; serum amyloid A 3, Saa3) were related to lipid derangement, cell morphology, and lymphoid tissue hyperplasia. Common gene transcripts regulated by TNFR and TLR4 such as fatty acid binding protein 1 (Fabp1) and tissue inhibitor of metalloproteinase 1 (Timp1) contributed to the chemotaxis and fatty acid metabolism. In conclusion, the TNFR-NF-κB and TLR4-NF-κB pathways modulated distinct transcriptomes during the development of O3-induced lung injury in mice. Results will provide significant insights into the pulmonary O3 pathogenesis and therapeutic interventions.

The Regulatory Roles of Toll-Like Receptor 4 in Secretions of Type 1/Type 2 Relative Cytokines by Splenocytes and Dendritic Cells Exposed to Clonorchis sinensis Excretory/Secretory Products.

The roles of TLR4 in mediation of innate immune response and in regulation of adaptive immune responses triggered by Clonorchis sinensis remain unknown. In the present study, splenocytes derived from C3H/HeN (TLR4 wild ) and C3H/Hej mice (TLR4 mut ) that were infected with 45 metacercariae of C. sinensis were harvested, then stimulated by C. sinensis excretory/secretory products (ESP) or medium (control) for 48h, respectively. Meanwhile, bone marrow-derived dendritic cells (BMDCs) from normal C3H/HeN and C3H/Hej mice were prepared and stimulated with medium, ESP, LPS, or ESP+LPS for 24h, respectively. The supernatants were collected, and the concentrations of type 1 and type 2 relative cytokines were determined by ELISA. The maturation of BMDCs indicated by surface markers of CD80, CD86, and MHC II was evaluated by flow cytometry. The results showed that the levels of IFN-γ, IL-6, TNF-α, and IL-10 in the splenocytes from C. sinensis-infected TLR4 mut mice were significantly lower than those from TLR4 wild mice when they were further exposed to ESP. For BMDCs, the productions of the cytokines IL-12p70 and IL-10, but not IL-4, in the BMDCs from TLR4 mutation mice were predominantly decreased compared with those from TLR4 wild mice when the BMDCs were co-stimulated by ESP combined with LPS. Flow cytometry analysis showed that ESP could significantly decrease the high levels of CD80, CD86, and MHC II which were elevated by LPS. In conclusion, these data suggest that TLR4 may play a regulatory role in type 1 immune responses during C. sinensis infection.

Critical role of TLR-4/NF-κB pathway in upregulation mechanism of cerebral ischemia reperfusion injury of mouse

The objective of this study is to investigate the expression of TLR-4/ NF-κB pathway in the cerebral ischemia injury of male adult mice. TLR-4 mutant (HeJ) mice and TLR-4 wild (HeN) mice were divided into the stroke model group and sham group. The stroke model was made by transient cerebral ischemia through middle cerebral artery occlusion (MCAO), while mice in the sham group were manipulated in the same way, but the MCA was not occluded. The brain tissues were collected for the morphological observation. The immunohistochemistry was employed to detect the expression of NF-κB, Western blot was used to detect the expression of TLR-4 and ELISA to detect the expression of serum pro-inflammatory mediators. The expression of NF-κB in HeJ mice after the cerebral I/R was significantly lower than that in HeN mice. For the stroke model group and sham group, the expression of TLR-4 in HeN mice was all significantly higher than that in HeJ mice, while the expression of TLR-4 in HeJ mice in the stroke model group was significantly higher than that in sham group. The expression of all pro-inflammatory mediators with HeJ was significantly lower than that in HeN mice. Together, these data concluded that TLR-4/NF-κB pathway is closely related to the cerebral I/R injury, which plays its role through the release of pro-inflammatory cytokines..

The roles of Toll-like receptor 4 in the pathogenesis of pathogen-associated biliary fibrosis caused by Clonorchis sinensis

Pathogen-associated biliary fibrosis (PABF) is a type of liver fibrosis characterized by injuries of cholangiocytes and extra cellular matrix (ECM) deposition around bile ducts caused by various bacteria, fungi, virus and parasites. Recent studies show that TLR4 plays an important role in several other types of liver fibrosis, but the mechanism of TLR4 in PABF is yet really unclear. In the present study, a PABF mouse model was established by a trematode infection-Clonorchis sinensis which dwells in the bile ducts and causes severe biliary fibrosis of mice. The results showed that the levels of collagen depositions, α-SMA and hydroxyproline (Hyp) contents in TLR4mut mice infected by C. sinensis were significantly lower than in those of TLR4wild ones. Furthermore, we found that the activation of TGF-β signaling was impaired in the TLR4mut mice, compared with wild mice when they were challenged to the same dose of C. sinensis metacercariae. Moreover, the mice with TLR4 mutation showed a decreased activation of hepatic stellate cells indicated by the expression of α-SMA, when compared with TLR4wild mice. These data demonstrate that TLR4 contributes to PABF caused by C. sinensis and TLR4 signaling may be a potential medical target for treatment of PABF.