TLR4 Knockout Mice Research Articles

Spinal TLR4/P2X7 Receptor-Dependent NLRP3 Inflammasome Activation Contributes to the Development of Tolerance to Morphine-Induced Antinociception.

BackgroundLong-term use of morphine induces antinociceptive tolerance and limits its clinical efficacy. Neuroinflammation in the spinal cord is thought to play a pivotal role in the development of morphine tolerance. Toll-like receptor 4 (TLR4) and P2X7 receptor (P2X7R) are key modulators of neuroinflammation. Recent studies show that the Nod-like receptor protein 3 (NLRP3) inflammasome play a crucial role in microglia-mediated neuroinflammation. Thus far, the mechanism underlying NLRP3 inflammasome activation during morphine-induced tolerance is not yet fully understood. Therefore, we sought to investigate the mechanisms of NLRP3 inflammasome activation and its role in the development of morphine-induced tolerance.MethodsRepeated morphine treatment through intrathecal injection (15 μg once daily for 7 days) was given to establish antinociceptive tolerance in mice. Tail-flick latency was used to evaluate morphine-induced antinociception. NLRP3 knockout mice were used to assess the role of NLRP3 inflammasome in morphine tolerance. TLR4 knockout mice and A438079, a P2X7R antagonist, were used to assess the role of TLR4 and P2X7R in chronic morphine-induced NLRP3 inflammasome activation. Western blot and immunofluorescence were used for quantitative comparison.ResultsRepeated morphine treatment increased the expression of NLRP3. Knockout of NLRP3 attenuated morphine-induced tolerance and suppressed morphine-induced activation of microglia. Knockout of TLR4 alleviated morphine tolerance and chronic morphine-induced upregulation of spinal NLRP3. Inhibition of spinal P2X7R with A438079 not only prevented the development of morphine-induced tolerance but also inhibited repeated morphine treatment-induced upregulation of spinal NLRP3. Furthermore, spinal NLRP3, TLR4 and P2X7R were collectively colocalized with the microglia marker Iba1.ConclusionThis study demonstrates that the NLRP3 inflammasome in microglia plays a crucial role in morphine tolerance and that both TLR4- and P2X7R-dependent pathways are required for NLRP3 inflammasome activation over the course of the development of morphine-induced tolerance. Our results provide a new perspective for the targeted treatment of morphine-induced tolerance.

Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling

Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll-like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection via activation of TLR9. We performed studies with human colonocytes and monocytes and macrophages from wild-type and TLR9 knockout mice incubated with TcdA or its antagonist (ODN TTAGGG) or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with enzyme-linked immunosorbent assay. We studied a transduction domain of TcdA (TcdA57-80), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle X-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight to length ratios. A TLR9 antagonist was tested in mice infected with C difficile. We incubated human colon explants with an antagonist of TLR9 and measured TcdA-induced production of cytokines. The TcdA57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNA entered human colonocytes. TLR9 was required for production of cytokines by cultured cells and in human colon explants incubated with TcdA. TLR9 was required in TcdA-induced mice intestinal secretions and in the survival of mice infected by C difficile. Even in a protease-rich environment, in which only fragments of TcdA exist, the TcdA57-80 domain organized DNA into a geometrically ordered structure that activated TLR9. TcdA from C difficile can bind and organize bacterial DNA to activate TLR9. TcdA and TcdA fragments remodel membranes, which allows them to access endosomes and present bacterial DNA to and activate TLR9. Rather than inactivating the ability of DNA to bind TLR9, TcdA appears to chaperone and organize DNA into an inflammatory, spatially periodic structure.

Production of IFN-γ by splenic dendritic cells during innate immune responses against Francisella tularensis LVS depends on MyD88, but not TLR2, TLR4, or TLR9.

Production of IFN-γ is a key innate immune mechanism that limits replication of intracellular bacteria such as Francisella tularensis (Ft) until adaptive immune responses develop. Previously, we demonstrated that the host cell types responsible for IFN-γ production in response to murine Francisella infection include not only natural killer (NK) and T cells, but also a variety of myeloid cells. However, production of IFN-γ by mouse dendritic cells (DC) is controversial. Here, we directly demonstrated substantial production of IFN-γ by DC, as well as hybrid NK-DC, from LVS-infected wild type C57BL/6 or Rag1 knockout mice. We demonstrated that the numbers of conventional DC producing IFN-γ increased progressively over the course of 8 days of LVS infection. In contrast, the numbers of conventional NK cells producing IFN-γ, which represented about 40% of non-B/T IFN-γ-producing cells, peaked at day 4 after LVS infection and declined thereafter. This pattern was similar to that of hybrid NK-DC. To further confirm IFN-γ production by infected cells, DC and neutrophils were sorted from naïve and LVS-infected mice and analyzed for gene expression. Quantification of LVS by PCR revealed the presence of Ft DNA not only in macrophages, but also in highly purified, IFN-γ producing DC and neutrophils. Finally, production of IFN-γ by infected DC was confirmed by immunohistochemistry and confocal microscopy. Notably, IFN-γ production patterns similar to those in wild type mice were observed in cells derived from LVS-infected TLR2, TLR4, and TLR2xTLR9 knockout (KO) mice, but not from MyD88 KO mice. Taken together, these studies demonstrate the pivotal roles of DC and MyD88 in IFN-γ production and in initiating innate immune responses to this intracellular bacterium.

Toll-like receptor-9 (TLR-9) deficiency alleviates optic nerve injury (ONI) by inhibiting inflammatory response in vivo and in vitro

Traumatic optic neuropathy is a common clinical problem. Damage to the optic nerve leads to shear stress and triggers secondary swelling within the optic canal. The study aims to explore the role of the inflammatory response following optic nerve injury (ONI) in toll-like receptor-9 knockout mice (TLR-9-/-) compared to wild-type mice (WT). At first, TLR-9-/- and WT mice were subjected to ONI. We then found that ONI significantly up-regulated TLR-9 expression levels in retinal tissues of WT mice. The retinal degeneration after ONI was alleviated in TLR-9-/- mice, as evidenced by the increased number of retinal ganglion cells (RGCs) and thickness of inner retinal layer (IRL). TUNEL staining and immunofluorescence staining of BRN3A indicated that TLR-9 knockout effectively improved the survival of RGCs. ONI-enhanced expression of Iba-1 and TMEM119 was markedly reduced in TLR-9-/- mice, indicating the suppression of microglial activation. Moreover, production of pro-inflammatory regulators, including inducible nitric oxide synthase (iNOS), macrophage chemo-attractant protein (MCP)-1, cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-18 and tumor necrosis factor-α (TNF-α), was significantly decreased in TLR-9-/- mice following ONI. TLR-9 knockout-attenuated inflammation was mainly through repressing myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase 4 (IRAK4). Furthermore, ONI greatly up-regulated the protein expression levels of phosphorylated (p)-IKKα, p-IκBα and p-nuclear factor (NF)-κB, whereas being repressed in TLR-9-/- mice. The effects of TLR-9 on ONI were verified in lipopolysaccharide (LPS)-stimulated retinal microglial cells transfected with small interfering RNA TLR-9 (siTLR-9). As expected, promoting TLR-9 with its agonist markedly restored inflammation in TLR-9 knockdown cells stimulated by LPS. Therefore, all findings above suggested that suppressing TLR-9 showed neuroprotective effects against ONI through reducing inflammatory response, and TILR-9 might be a promising therapeutic target to develop effective strategies for the treatment of optic neuropathies.

TLR2 favors OVA-induced allergic airway inflammation in mice through JNK signaling pathway with activation of autophagy

AimsNumerous studies indicate that toll-like receptor 2 (TLR2) led to divergent effects in asthma. The occurrence of autophagy in asthma pathogenesis is still incompletely understood. Here, we aimed to investigate the role of TLR2 and the underlying mechanisms in allergic airway inflammation and autophagy activation. Main methodsC57BL/6 and TLR2 knockout (TLR2−/−) mice were subjected to an ovalbumin (OVA)-immunized allergic airway model, and were treated with SP600125. Differential cell counts in bronchoalveolar lavage fluid were determined by Wright's staining. Histological analysis of airway inflammation was determined by haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining. The levels of OVA-specific immunoglobulin E (IgE), tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) were detected by enzyme-linked immunosorbent assay (ELISA). Proteins expression in lung tissues was detected by western blot, expression of TLR2 was further observed by immunofluorescence. Autophagy activation was determined by western blot and transmission electron microscopy (TEM). Key findingsTLR2 expression was increased upon OVA challenge, and TLR2 deficiency was associated with decreased allergic airway inflammation. Meanwhile, TLR2 deficiency weakened autophagy activation. Moreover, inhibition of c-Jun N-terminal kinase (JNK) by SP600125 also suppressed OVA-induced allergic airway inflammation and autophagy activation. Interestingly, treating TLR2−/− mice with SP600125 showed similar OVA-induced allergic airway inflammation and autophagy activation compared to that in vehicle-treated TLR2−/− mice. SignificanceTLR2 might contribute to the maintenance of allergic airway inflammation through JNK signaling pathway accompanying with autophagy activation. These findings may provide a novel signal target for prevention of allergic airway inflammation.

1674-P: Stearate Potentiates MIP-1a Production with TNF-a via TLR4/TBK1/IKKe /IRF3 Signal Axis: Significance for Insulin Resistance

Increased levels of plasma free fatty acids (FAs) are an important contributor to chronic inflammation, insulin resistance and other obesity-related complications. However, the molecular mechanisms by which individual FAs contribute to obesity-associated inflammation and disorders remain incompletely understood. Here we report that dietary saturated FAs specifically stearate led to a profound production of macrophage inflammatory protein (MIP)-1α (CCL3) in the presence of TNF-α in monocytes, macrophages and adipose tissues. Using pharmacologic and genetic approaches, we identified the involvement of TLR4/TBK1/IKKε/IRF3 signal axis in this production of MIP-1α in response to stearate and TNF-α. Consistent with this, cultured adipose tissues from TLR4 knockout (KO) mice revealed that stearate and TNF-α treatment did not induce MIP-1α compared with TLR2 KO or wild type (C57BL/6) mice. Moreover, C57BL/6 mice fed a high fat diet (HFD) for 16 weeks showed elevated levels of plasma fatty acids, TNF-α and MIP-1α along with insulin resistance. MIP-1α was significantly correlated with TNF-α. Our human data shows that expression of TLR4/p-IRF3 was elevated in the PBMCs of obese individuals as compared to lean. Furthermore, elevated MIP-1α expression levels in obese fat tissues were significantly correlated with TNF-α and macrophage markers. In conclusion, these findings provide a mechanistic link between stearate and TNF-α for the overproduction of obesity associated MIP-1α, which could be used as a new target for the treatment of obesity-related chronic inflammation/insulin resistance. Disclosure S.P. Kochumon: None. F. Almulla: None. Funding Kuwait Foundation for the Advancement of Science (RA-AH-2016-007)

TLR4 knockout upregulates the expression of Mfn2 and PGC-1α in a high-fat diet and ischemia-reperfusion mice model of liver injury

AimsPatients with nonalcoholic fatty liver disease (NAFLD) have less tolerance to ischemia-reperfusion injury (IRI) of the liver than those with the healthy liver; hence have a higher incidence of severe complications after surgery. This study aimed to investigate the dynamics of the liver and mitochondrial damage and the impact of TLR4 knockout (TLR4KO) on Mfn2 expression in the composite model of NAFLD and IRI. Main methodsWe performed high-fat diet (HFD) feeding and ischemia reperfusion (IR) on wild type (WT) and TLR4 knockout TLR4KO mice. Key findingsThe degree of structural and functional injuries to the liver and mitochondria (NAFLD and IRI) is greater than that caused by a single factor (NAFLD or IRI) or a simple superposition of both. The IL-6 and TNF-α expressions were significantly suppressed (P < .05), while PGC-1α and Mfn2 expressions were up-regulated considerably (P < .05) after TLR4KO. Furthermore, mitochondrial fusion increased, while ATP consumption and ROS production decreased significantly after TLR4KO (P < .05). The degree of reduction of compound injury by TLR4KO is more significant than the reduction degree of single factor injury. Also, TNF-α and IL-6 levels can be used predictive markers for mitochondrial damage and liver tolerance to NAFLD and IRI. SignificanceTLR4KO upregulates the expression of Mfn2 and PGC-1α in the composite model of NAFLD and IRI. This pathway may be related to IL-6 and TNF-α. This evidence provides theoretical and experimental basis for the subsequent Toll-like receptor 4 (TLR4) receptor targeted therapy.

Mitochondrial Double-Stranded RNA in Exosome Promotes Interleukin-17 Production Through Toll-Like Receptor 3 in Alcohol-associated Liver Injury.

Mitochondrial double-stranded RNA (mtdsRNA) and its innate immune responses have been reported previously; however, mtdsRNA generation and its effects on alcohol-associated liver disease (ALD) remain unclear. Here, we report that hepatic mtdsRNA stimulates toll-like receptor 3 (TLR3) in Kupffer cells through the exosome (Exo) to enhance interleukin (IL)-17A (IL-17A) production in ALD. Following binge ethanol (EtOH) drinking, IL-17A production primarily increased in γδ T cells of wild-type (WT) mice, whereas the production of IL-17A was mainly facilitated by CD4+ T cells in acute-on-chronic EtOH consumption. These were not observed in TLR3 knockout (KO) or Kupffer cell-depleted WT mice. The expression of polynucleotide phosphorylase, an mtdsRNA-restricting enzyme, was significantly decreased in EtOH-exposed livers and hepatocytes of WT mice. Immunostaining revealed that mtdsRNA colocalized with the mitochondria in EtOH-treated hepatocytes from WT mice and healthy humans. Bioanalyzer analysis revealed that small-sized RNAs were enriched in EtOH-treated Exos (EtOH-Exos) rather than EtOH-treated microvesicles in hepatocytes of WT mice and humans. Quantitative real-time PCR and RNA sequencing analyses indicated that mRNA expression of mitochondrial genes encoded by heavy and light strands was robustly increased in EtOH-Exos from mice and humans. After direct treatment with EtOH-Exos, IL-1β expression was significantly increased in WT Kupffer cells but not in TLR3 KO Kupffer cells, augmenting IL-17A production of γδ T cells in mice and humans. EtOH-mediated generation of mtdsRNA contributes to TLR3 activation in Kupffer cells through exosomal delivery. Consequently, increased IL-1β expression in Kupffer cells triggers IL-17A production in γδ T cells at the early stage that may accelerate IL-17A expression in CD4+ T cells in the later stage of ALD. Therefore, mtdsRNA and TLR3 may function as therapeutic targets in ALD.

Effect of Toll-like receptor 4 deficiency on clinical severity and expression of Th1/Th2/Th17-associated cytokines in a murine model of experimental autoimmune neuritis.

The aim was to observe the effect of Toll-like receptor 4 (TLR4) deficiency on clinical severity and expression of Th1/Th2/Th17-associated cytokines in experimental autoimmune neuritis (EAN). We selected C57BL/10 wild type (WT) mice and TLR4 knockout (KO) mice with the C57BL/10 background for induction of the EAN model by immunizing mice twice (days 0 and 8) via subcutaneous injection of 180 μg P0 peptide 180-199 emulsion in 25 μl of PBS and 0.5 mg Mycobacterium tuberculosis (Difco, USA) in 25 μl of Freund's incomplete adjuvant into the back of mice. The concentrations of serum cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) were determined using the Ms Th1/Th2/Th17 CBA kit. We found that TLR4 deficiency could attenuate the clinical severity and delay the onset of EAN. Moreover, our data showed that the sera levels of IFN-γ, TNF, IL-6 and IL-17A were elevated in the WT mice with EAN when compared with the naive WT mice, but only the production of IL-17A was significantly lower in the TLR4 KO mice with EAN than in their WT counterparts. Based on these findings, TLR4 may contribute to the pathogenesis of EAN by regulating Th17 cells and the production of Th17-associated factors. However, the exact mechanism remains unclear and more evidence is needed to elucidate its role in EAN.

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.

TLR2 and caspase-1 signaling are critical for bacterial containment but not clearance during craniotomy-associated biofilm infection

BackgroundA craniotomy is required to access the brain for tumor resection or epilepsy treatment, and despite precautionary measures, infectious complications occur at a frequency of 1–3%. Approximately half of craniotomy infections are caused by Staphylococcus aureus (S. aureus) that forms a biofilm on the bone flap, which is recalcitrant to antibiotics. Our prior work in a mouse model of S. aureus craniotomy infection revealed a critical role for myeloid differentiation factor 88 (MyD88) in bacterial containment and pro-inflammatory mediator production. Since numerous receptors utilize MyD88 as a signaling adaptor, the current study examined the importance of Toll-like receptor 2 (TLR2) and TLR9 based on their ability sense S. aureus ligands, namely lipoproteins and CpG DNA motifs, respectively. We also examined the role of caspase-1 based on its known association with TLR signaling to promote IL-1β release.MethodsA mouse model of craniotomy-associated biofilm infection was used to investigate the role of TLR2, TLR9, and caspase-1 in disease progression. Wild type (WT), TLR2 knockout (KO), TLR9 KO, and caspase-1 KO mice were examined at various intervals post-infection to quantify bacterial burden, leukocyte recruitment, and inflammatory mediator production in the galea, brain, and bone flap. In addition, the role of TLR2-dependent signaling during microglial/macrophage crosstalk with myeloid-derived suppressor cells (MDSCs) was examined.ResultsTLR2, but not TLR9, was important for preventing S. aureus outgrowth during craniotomy infection, as revealed by the elevated bacterial burden in the brain, galea, and bone flap of TLR2 KO mice concomitant with global reductions in pro-inflammatory mediator production compared to WT animals. Co-culture of MDSCs with microglia or macrophages, to model interactions in the brain vs. galea, respectively, also revealed a critical role for TLR2 in triggering pro-inflammatory mediator production. Similar to TLR2, caspase-1 KO animals also displayed increased S. aureus titers coincident with reduced pro-inflammatory mediator release, suggestive of pathway cooperativity. Treatment of caspase-1 KO mice with IL-1β microparticles significantly reduced S. aureus burden in the brain and galea compared to empty microparticles, confirming the critical role of IL-1β in limiting S. aureus outgrowth during craniotomy infection.ConclusionsThese results demonstrate the existence of an initial anti-bacterial response that depends on both TLR2 and caspase-1 in controlling S. aureus growth; however, neither pathway is effective at clearing infection in the WT setting, since craniotomy infection persists when both molecules are present.

Effect of TLR2 on the proliferation of inflammation-related colorectal cancer and sporadic colorectal cancer

BackgroundColitis-associated cancer (CAC) is a complication of inflammatory bowel disease (IBD) with a poor prognosis because it is often diagnosed in advanced stages with local progression or metastasis. Compared with the more common polyp-induced sporadic colorectal cancer (sCRC), CAC has different molecular mechanisms. Toll-like receptor 2 (TLR2) expression is not limited to cells related to inflammation and immune function. High levels of TLR2 expression in tumor tissues of colorectal cancer (CRC) patients have been reported. This report is to investigate the effects of knockout and knockdown of the TLR2 gene on the proliferation of CAC and sCRC.MethodsTwelve C57BL/6 J wild-type mice (WT) and 12 TLR2 knockout mice (TLR2-/-) were used to rapidly establish a colitis-associated cancer (CAC) model via the 1,2-dimethylhydrazine-dextran sodium sulfate (DMH-DSS) method and were divided into the normal WT control group (NC), TLR2 knockout control group (KC), normal wild-type tumor modeling group (NT), and TLR2 knockout tumor modeling group (KT), with 6 mice in each group. The general performance of the mice during modeling, the gross changes of the colon and the rectum, and the pathological score of HE staining were used to observe tumor growth. The expression of TLR2 was detected by immunohistochemistry, and tumor proliferation was detected by Ki67 labeling. Lentivirus carrying TLR2-RNAi was used to stably infect colorectal cancer cells (HCT116 and HT29) to knock down TLR2 gene expression. The experimental groups included the uninfected control group, negative control group, and gene knockdown group. After infection, the expression of TLR2 protein was detected by Western blot, and cell proliferation and the cell cycle were detected by the CCK-8 method and fluorescence-activated cell sorting. Western blot was used to detect the expression levels of p- NF-κβ, cyclin D1 and cyclin D3 protein in each group of cells.ResultsTLR2 knockout in the CAC model resulted in greater changes in body weight and more severe diarrhea and colorectal hemorrhage. However, knocking out the TLR2 gene reduced the shortening of colorectal length, the number of tumors, and the total tumor volume and inhibited the growth of CAC. Knocking out the TLR2 gene also reduced the pathological score and tumor severity. TLR2 was localized in the cell membrane of the colorectal epithelium of the NC group and of the colorectal tumors of the NT group and was highly expressed in the NT group, while antigen Ki67 was localized in the nucleus of the colorectal tumor cells of the NT group and the KT group, and its expression was reduced in the KT group. In an in vitro sporadic colorectal cancer cell experiment, TLR2 protein in the TLR2 knockdown group was significantly downregulated, and TLR2 knockdown significantly inhibited the proliferation of HCT116 and HT29 colorectal cancer cells, resulting in G1 phase arrest. The expression levels of p-NF-κβ, cyclin D1 and cyclin D3 proteins in TLR2 gene knockdown group cells were significantly reduced.ConclusionKnockout and knockdown of TLR2 can inhibit the proliferation of inflammation-related colorectal cancer and sporadic colorectal cancer.

Abrogation of Toll-Like Receptor 4 Mitigates Obesity-Induced Oxidative Stress, Proinflammation, and Insulin Resistance Through Metabolic Reprogramming of Mitochondria in Adipose Tissue.

Aims: Obesity-induced excessive visceral fat (VF) accumulation is associated with insulin resistance (IR), systemic oxidative stress, and chronic inflammation. As toll-like receptor 4 (TLR4) plays an important role in innate immunity, we herein investigate the effect of TLR4 knockout (T4KO) in a high-fat high-sucrose diet (HFD)-induced obesity mouse model. Results: C57BL6 wild-type (WT) and T4KO mice were fed with either control diet (CD) or HFD for 12 months, rendering four experimental groups: WT+CD, WT+HFD, T4KO+CD, and T4KO+HFD. Compared with WT+CD, WT+HFD demonstrated significant increase in VF accumulation, oxidative damage, M1/M2 macrophage ratio, chronic inflammation, and development of IR. Compared with WT+HFD, T4KO+HFD presented increased BW and body fat with higher subcutaneous fat (SF)/VF ratio, but lower body temperature, as well as decreased oxidative damage, M1/M2 macrophage ratio, chronic inflammation, and IR. Unlike WT+HFD, T4KO+HFD exhibited an increase in mitochondrial electron transport chain activity but a decrease of uncoupling protein 2 (UCP2) level. While T4KO hindered HFD-induced increasing mitochondrial oxygen consumption rate, a shift toward a higher extracellular acidification rate in VF was observed. Notably, T4KO inhibits HFD-induced mitochondrial translocation of nuclear factor of activated T cells 2 (NFATC2), which contributed to mitochondrial metabolic reprogramming. Both fat distribution shifting from VF to SF and mitochondrial metabolic reprogramming may alleviate systemic oxidative stress and chronic inflammation. Innovation and Conclusion: Abrogation of TLR4 contributes to reduction of oxidative stress through metabolic reprogramming of mitochondria in VF, mitigating obesity-induced IR. The study provides critical insight into associating innate immunity-mitochondria interplay with prevention of diabetes.

Toll-like receptor 4 deficiency ameliorates β2-microglobulin induced age-related cognition decline due to neuroinflammation in mice

Toll-like receptor 4 (TLR4) is a crucial receptor in neuroinflammation and apoptotic neuronal death, and increasing evidences indicated that β2-microglobulin (B2M) is thought to be a major contributor to age-related cognitive decline. In present study, we designed to investigate the effects of TLR4 on B2M-induced age-related cognitive decline. Wild-type (WT) C57BL/6, TLR4 knockout (TLR4 -KO) mice and hippocampal neurons from the two type mice were respectively divided into two groups: (1) Veh group; (2) B2M-treated group. The behavioral responses of mice were measured using Morris Water Maze. Hippocampal neurogenesis and neuronal damage, inflammatory response, apoptosis, synaptic proteins and neurotrophic factors, and TLR4/MyD88/NF-κB signaling pathway proteins were examined using molecular biological or histopathological methods. The results showed that WT mice received B2M in the DG exhibited age-related cognitive declines, increased TLR4 mRNA expression and high levels of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and apoptotic neuronal death in the hippocampus, which were partially attenuated in TLR4-KO mice. Moreover, in absence of TLR4, B2M treatment improved hippocampus neurogenesis and increased synaptic related proteins. Our cell experiments further demonstrated that deletion of TLR4 could significantly increase synaptic related protein, decrease neuroinflammatory fators, inhibited apoptotic neuronal death, and regulated MyD88/NF-κB signal pathway after B2M treatment. In summary, our results support the TLR4 contributes to B2M-induced age-related cognitive decline due to neuroinflammation and apoptosis through TLR4/MyD88/NF-κB signaling pathway via a modulation of hippocampal neurogenesis and synaptic function. This may provide an important neuroprotective mechanism for improving age-related cognitive decline.

Melatonin controls microbiota in colitis by goblet cell differentiation and antimicrobial peptide production through Toll-like receptor 4 signalling

Microbial dysbiosis has long been postulated to be associated with the pathogenesis of inflammatory bowel disease (IBD). Although evidence supporting the anti-colitic effects of melatonin have been accumulating, it is not clear how melatonin affects the microbiota. Herein, we investigated the effects of melatonin on the microbiome in colitis and identified involvement of Toll-like receptor (TLR) 4 signalling in the effects. Melatonin improved dextran sulfate sodium (DSS)-induced colitis and reverted microbial dysbiosis in wild-type (WT) mice but not in TLR4 knockout (KO) mice. Induction of goblet cells was observed with melatonin administration, which was accompanied by suppression of Il1b and Il17a and induction of melatonin receptor and Reg3β, an antimicrobial peptide (AMP) against Gram-negative bacteria. In vitro, melatonin treatment of HT-29 intestinal epithelial cells promotes mucin and wound healing and inhibits growth of Escherichia coli. Herein, we showed that melatonin significantly increases goblet cells, Reg3β, and the ratio of Firmicutes to Bacteriodetes by suppressing Gram-negative bacteria through TLR4 signalling. Our study suggests that sensing of bacteria through TLR4 and regulation of bacteria through altered goblet cells and AMPs is involved in the anti-colitic effects of melatonin. Melatonin may have use in therapeutics for IBD.

Ruscogenin alleviates LPS-induced pulmonary endothelial cell apoptosis by suppressing TLR4 signaling

Acute lung injury (ALI) or its most advanced form, acute respiratory distress syndrome (ARDS) is a severe inflammatory pulmonary process triggered by varieties of pathophysiological factors, among which apoptosis of pulmonary endothelial cells plays a critical role in the progression of ALI/ARDS. Ruscogenin (RUS) has been found to exert significant protective effect on ALI induced by lipopolysaccharides (LPS), but there is little information about its role in LPS-induced pulmonary endothelial cell apoptosis. The aim of the present study was to investigate the underlying mechanism in which RUS attenuates LPS-induced pulmonary endothelial cell apoptosis. Mice were challenged with LPS (5 mg/kg) by intratracheal instillation for 24 h to induce apoptosis of pulmonary endothelial cells in model group. RUS (three doses: 0.1, 0.3, and 1 mg/kg) was administrated orally 1 h prior to LPS challenge. The results showed that RUS could attenuate LPS-induced lung injury and pulmonary endothelial apoptosis significantly. And we observed that RUS inhibited the activation of TLR4/MYD88/NF-κB pathway in pulmonary endothelium after LPS treatment. In murine lung vascular endothelial cells (MLECs) we further confirmed that RUS (1 μmol/L) markedly ameliorated MLECs apoptosis by suppressing TLR4 signaling. By using TLR4 knockout mice we found that TLR4 was essential for the RUS-mediated eff ;ect on LPS-stimulated pulmonary endothelial apoptosis. Collectively, our results indicate that RUS plays a protective role against LPS-induced endothelial cell apoptosis via regulating TLR4 signaling, and may be a promising agent in the management of ALI.

PUMA-mediated epithelial cell apoptosis promotes Helicobacter pylori infection-mediated gastritis

The molecular mechanism responsible for Helicobacter pylori infection-mediated gastritis and carcinogenesis is not yet clear. Increased evidence suggests that chronic gastritis and elevated gastric epithelial cell (GEC) apoptosis are crucial events during stomach carcinoma transformation. PUMA is a potent proapoptotic Bcl-2 protein and mediates acute tissue injury. In this study, we aimed to investigate the role of PUMA in GEC apoptosis and inflammation induced by H. pylori infection. As a result, we found that PUMA expression was elevated in gastritis tissues compared with uninvolved tissues, and it was correlated with the severity of apoptosis and gastritis. In mice, PUMA mRNA and protein were markedly induced in GECs upon induction of gastritis by H. pylori. PUMA-deficient mice were highly resistant to apoptosis and gastritis induced by H. pylori. Furthermore, the transcription factor NF-κB p65 binds to PUMA promoter to activate PUMA transcription after H. pylori infection. In addition, NF-κB inhibitor could rescue H. pylori-induced apoptosis and gastritis. Finally, H. pylori-induced activation of p-p65 and PUMA was mediated via Toll-like receptor 2 (TLR2) and blocked in TLR2 knockout mice. Taken together, these results verified the pro-inflammatory effect of PUMA in H. pylori-infected gastric tissue. Moreover, TLR2/NF-κB-mediated transcriptional regulation of PUMA contributes to the pathogenesis of H. pylori-infected gastritis.

Role of TLR2 and TLR4 in regulation of articular chondrocyte homeostasis

Toll-like receptor (TLR)-mediated catabolic responses are implicated to contribute to osteoarthritis (OA). However, deficiency of TLRs has little chondroprotection in mice in vivo. Here, we studied the effect of deficiency of TLR2 and TLR4 in articular chondrocytes on cellular stress responses in vitro. Chondrocytes isolated from TLR2 and TLR4 double knockout (TLR2/4dKO) and wild type (WT) mice and recombinant HMGB1 (rHMGB1) and LPS were used. Expression of anti-oxidant and DNA repair enzymes including SOD1, SOD2 and OGG1, and phosphorylation of H2AX (a marker for DNA damage) were examined by Western blotting. MitoSOX Red staining was used for assessing mitochondrial superoxide generation. Autophagic activity was monitored by flow cytometry analysis of mean fluorescence intensity (MFI) of GFP and RFP in chondrocytes transfected with a tandem GFP-mRFP-LC3 plasmid, and by Western blot analysis of expression of LC3 and p62, a selective autophagy adaptor. Basal expression of SOD2 but not SOD1 was largely reduced in TLR2/4dKO compared to WT chondrocytes, correlated with significantly enhanced menadione-induced mitochondrial superoxide generation (2.85-3.92 and 3.39 to 8.97 with mean difference 3.39 and 6.18 for 25 and 50μM menadione, respectively) and phosphorylation of H2AX. LPS and rHMGB1 induced expression of SOD2, OGG1 and p62 in WT but not TLR2/4dKO chondrocytes. Autophagy flux was impaired in TLR2/4dKO chondrocytes after acute nutrient stress and by LPS and rHMGB1. TLR2 and TLR4 deficiency appears to reduce chondrocyte anti-oxidative stress and autophagy flux capacity, which may compromise cartilage homeostasis as a result of chondrocyte dysfunction.

Hyperglycaemia and Ischaemia Impair Wound Healing via Toll-like Receptor 4 Pathway Activation in vitro and in an Experimental Murine Model

Diabetes mellitus has reached epidemic proportions. Foot ulceration is a multifactorial complication of diabetes associated with marked morbidity and mortality. Innate immune Toll-like receptor 4 (TLR4) mediated inflammation has been implicated in the systemic pathogenesis of diabetes and may contribute to impairment of wound healing. This study investigates the effect of high glucose and hypoxic conditions on TLR4 activation and signalling in vitro and in vivo. Fibroblasts cultured at physiological glucose concentration (5.5 mM) were exposed to glucose concentrations from 0 mM to 25 mM, with duplicates placed in a hypoxic chamber. TLR4 inhibition was assessed in the 25 mM glucose groups. Diabetes was induced in wild type (WT) and TLR4 knockout (KO) C57BL/6 mice by intraperitoneal injection of low dose streptozocin (STZ). Hindlimb ischaemia was induced by femoral artery ligation four weeks post streptozocin, and a full thickness 4 mm skin wound inflicted below the knee. Wound healing was assessed via digital planimetry on days 3, 7, and 14 post surgery. Hypoxic and high glucose (25 mM) conditions led to an increase in TLR4 protein expression, apoptosis, and interleukin (IL)-6 release. Inhibition with a TLR4 neutralising antibody and specific TLR4 antagonist ameliorated the effects of high glucose and ischaemia (p < .05). In vivo, wound healing was significantly impaired in the diabetic ischaemic group at day 14 (p < .05). Diabetic ischaemic wounds in TLR4 KO mice exhibited significantly improved healing rates compared with those in WT mice at all time points. Hypoxia stimulates upregulation of TLR4 protein expression and this effect is exaggerated by hyperglycaemia. In TLR4 KO mice, there is a significant improvement in the healing of diabetic ischaemic wounds compared with WT. It is suggested that a synergistic effect between hypoxia and hyperglycaemia impairing wound healing exists, through TLR4 mediated inflammation.

2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione, isolated from the root of Averrhoa carambola L., protects against diabetic kidney disease by inhibiting TLR4/TGFβ signaling pathway

ObjectiveDiabetic kidney disease (DKD) is the leading cause of death and disability of diabetes mellitus. However, there is still a lack of specific drugs for the treatment of DKD. The chief aim of this research is to investigate the role and mechanism of 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) for DKD. MethodsWild type and TLR4 knockout mice were induced to diabetes. After 4-week treatment with DMDD, blood sugar, renal function, blood lipid and pathological changes were assessed. Real-time PCR, western blotting, and immunohistochemistry were employed to detect the expressions of TLR4, TGFβ1 and Smad2/3 in the renal tissue. ResultsDMDD improved the serum lipid and decreased fasting blood glucose levels in diabetic mice. CysC and urinary albumin levels increased markedly in the diabetic group, and they were obviously decreased after 4 weeks of DMDD treatment. Compared with the WT diabetic mice, the urinary albumin and CysC in the TLR4-/- mice were expressed at lower levels. HE and Masson’s staining revealed that DMDD clearly ameliorated pathological changes and renal fibrosis. When TLR4 gene was knock out, the pathological was improved. Mechanistically, TLR4, TGF-β1 and Smad2/3 were obvious up-regulation in the renal tissues of diabetic mice. The expressions of these proteins were significantly down-regulated after DMDD treatment (p < 0.05). In the TLR4-/- mice, mRNA and protein levels of TGF-β1 and Smad2/3 were obviously lower than those in the WT mice. In addition, IHC revealed that a strong in situ expressions of TLR4, TGF-β1 and Smad2/3 were seen in the kidney tissues of diabetic mice, which were distinctly weakened in the DMDD-treated mice. In the TLR4-/- mice, however, expressions of TGF-β1 and Smad2/3 were not remarkable increase in the diabetic mice compared with normal mice. ConclusionsThese results strongly indicate that TLR4 is essential for DMDD protection against renal dysfunction in diabetic mice. Its hypoglycemic and anti-fibrosis effects were likely mediated by the TLR4/TGFβ signaling pathway.