Toll like receptor 4 mediates the inhibitory effect of SARS-CoV-2 spike protein on proximal tubule albumin endocytosis.

Tubular proteinuria is a common feature in COVID-19 patients, even in the absence of established acute kidney injury. SARS-CoV-2 spike protein (S protein) was shown to inhibit megalin-mediated albumin endocytosis in proximal tubule epithelial cells (PTECs). Angiotensin-converting enzyme type 2 (ACE2) was not directly involved. Since Toll-like receptor 4 (TLR4) mediates S protein effects in various cell types, we hypothesized that TLR4 could be participating in the inhibition of PTECs albumin endocytosis elicited by S protein. Two different models of PTECs were used: porcine proximal tubule cells (LLC-PK1) and human embryonic kidney cells (HEK-293). S protein reduced Akt activity by specifically inhibiting of threonine 308 (Thr308) phosphorylation, a process mediated by phosphoinositide-dependent kinase 1 (PDK1). GSK2334470, a PDK1 inhibitor, decreased albumin endocytosis and megalin expression mimicking S protein effect. S protein did not change total TLR4 expression but decreased its surface expression. LPS-RS, a TLR4 antagonist, also counteracted the effects of the S protein on Akt phosphorylation at Thr308, albumin endocytosis, and megalin expression. Conversely, these effects of the S protein were replicated by LPS, an agonist of TLR4. Incubation of PTECs with a pseudovirus containing S protein inhibited albumin endocytosis. Null or VSV-G pseudovirus, used as control, had no effect. LPS-RS prevented the inhibitory impact of pseudovirus containing the S protein on albumin endocytosis but had no influence on virus internalization. Our findings demonstrate that the inhibitory effect of the S protein on albumin endocytosis in PTECs is mediated through TLR4, resulting from a reduction in megalin expression.

Co-Culture of P. gingivalis and F. nucleatum Synergistically Elevates IL-6 Expression via TLR4 Signaling in Oral Keratinocytes.

Periodontitis, characterized by persistent inflammation in the periodontium, is intricately connected to systemic diseases, including oral cancer. Bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, play a pivotal role in periodontitis development because they contribute to dysbiosis and tissue destruction. Thus, comprehending the interplay between these bacteria and their impacts on inflammation holds significant relevance in clinical understanding and treatment advancement. In the present work, we explored, for the first time, their impacts on the expressions of pro-inflammatory mediators after infecting oral keratinocytes (OKs) with a co-culture of pre-incubated P. gingivalis and F. nucleatum. Our results show that the co-culture increases IL-1ß, IL-8, and TNF-α expressions, synergistically augments IL-6, and translocates NF-kB to the cell nucleus. These changes in pro-inflammatory mediators-associated with chronic inflammation and cancer-correlate with an increase in cell migration following infection with the co-cultured bacteria or P. gingivalis alone. This effect depends on TLR4 because TLR4 knockdown notably impacts IL-6 expression and cell migration. Our study unveils, for the first time, crucial insights into the outcomes of their co-culture on virulence, unraveling the role of bacterial interactions in polymicrobial diseases and potential links to oral cancer.

Probiotic supplementation for 24 weeks in patients with non-alcoholic steatohepatitis: the PROBILIVER randomized clinical trial.

Background and aim: Considering the increasing prevalence of non-alcoholic steatohepatitis (NASH) and treatment gaps, this study aimed to evaluate the effect of probiotic supplementation on liver function markers, nutritional status, and clinical parameters. Methods: This double-blind, randomized clinical trial (ClinicalTrials.gov ID: NCT0346782) included adult outpatients with biopsy-proven NASH. The intervention consisted of 24 weeks of supplementation with the probiotic mix Lactobacillus acidophilus (1 × 109 CFU) + Lactobacillus rhamnosus (1 × 109 CFU) + Lactobacillus paracasei (1 × 109 CFU) + Bifidobacterium lactis (1 × 109 CFU), or placebo, twice a day. The following parameters were evaluated: demographic and clinical data, transient elastography (FibroScan), liver enzymes, NAFLD fibrosis score, fatty liver index, laboratory assessment, serum concentration of toll-like receptor-4 (sTLR-4) and cytokeratin 18 (CK-18), anthropometric data, dietary intake, and physical activity. Regarding data analysis, the comparison between the groups was based on the delta of the difference of each variable analyzed (value at the end of treatment minus the baseline value) using the t-test for independent samples or the Mann-Whitney U-test. Results: Forty-four patients with NASH completed the trial (51.4 ± 11.6 years). At baseline, 87% of participants had a mild liver fibrosis degree on biopsy, normal values of liver enzymes, transient elastography values consistent with grade 1 fibrosis in both groups, increased waist circumference (WC), a BMI of 30.97 kg/m2, and 76% presented with metabolic syndrome (MetS). After the intervention, no differences were observed between the probiotic and placebo groups in terms of MetS, WC, BMI scores, or liver enzyme levels (p > 0.05 for all). The elastography values remained consistent with grade 1 fibrosis in both groups. Although CK-18 was reduced in both groups, a larger effect size was noted in the probiotic group (D = 1.336). sTLR-4 was also reduced in both groups, with no difference between groups (p = 0.885). Conclusion: Intervention with probiotics in the early stages of NASH demonstrated no significant change in hepatic and clinical parameters. Clinical trial registration: ClinicalTrials.gov, identifier NCT0346782.

Association of COX-2, TNF-α, TLR4, and IKKα With Survival of Patients With Oral Squamous Cell Carcinomas: A Systematic Review.

BACKGROUND: This review investigated the association of COX-2, TNF-α, TLR4, and IKKα with the survival of patients with oral squamous cell carcinoma (SCC). METHODS: A systematic search was conducted in the databases PUBMED, Web of Science, LILACS, EMBASE, Scopus, and Cochrane Library. The studies should assess the expression of those proteins in the tumor and survival outcomes. RESULTS: Twenty-one articles were included. The meta-analysis results leaned towards an association of COX-2 overexpression with a lower overall survival. The estimated hazard ratio was 1.51 (95% CI 0.97, 2.33), but not statistically significant (p=0.07). A low heterogeneity was observed (I2=0%). Regarding TNF-α, TLR4, and IKKα, statistically significant results for the association with survival were presented, but there was not enough data to a meta-analysis. CONCLUSION: COX-2 overexpression may be associated with a poorer prognosis in oral SCC. The insufficiency of studies about TNF-α, TLR4, and IKKα restrained their validation as predictors of prognosis.

The ecotin-like peptidase inhibitor of Trypanosoma cruzi prevents TMPRSS2-PAR2-TLR4 crosstalk downmodulating infection and inflammation.

Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.

Spinal alarmin HMGB1 and the activation of TLR4 lead to chronic stress-induced nociceptive hypersensitivity in rodents.

Chronic stress affects millions of people around the world, and it can trigger different behavioral disorders like nociceptive hypersensitivity and anxiety, among others. However, the mechanisms underlaying these chronic stress-induced behavioral disorders have not been yet elucidated. This study was designed to understand the role of high-mobility group box-1 (HMGB1) and toll-like receptor 4 (TLR4) in chronic stress-induced nociceptive hypersensitivity. Chronic restraint stress induced bilateral tactile allodynia, anxiety-like behaviors, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) and activation of spinal microglia. Moreover, chronic stress enhanced HMGB1 and TLR4 protein expression at the dorsal root ganglion, but not at the spinal cord. Intrathecal injection of HMGB1 or TLR4 antagonists reduced tactile allodynia and anxiety-like behaviors induced by chronic stress. Additionally, deletion of TLR4 diminished the establishment of chronic stress-induced tactile allodynia in male and female mice. Lastly, the antiallodynic effect of HMGB1 and TLR4 antagonists were similar in stressed male and female rats and mice. Our results suggest that chronic restraint stress induces nociceptive hypersensitivity, anxiety-like behaviors, and up-regulation of spinal HMGB1 and TLR4 expression. Blockade of HMGB1 and TLR4 reverses chronic restraint stress-induced nociceptive hypersensitivity and anxiety-like behaviors and restores altered HMGB1 and TLR4 expression. The antiallodynic effects of HMGB1 and TLR4 blockers in this model are sex independent. TLR4 could be a potential pharmacological target for the treatment of the nociceptive hypersensitivity associated with widespread chronic pain.

Cannabidiol reduces lipopolysaccharide-induced nociception via endocannabinoid system activation.

Bacterial infections are often accompanied by fever and generalized muscle pain. However, the treatment of pain with an infectious aetiology has been overlooked. Thus, we investigated the impact of cannabidiol (CBD) in bacterial lipopolysaccharide (LPS)-induced nociception. Male Swiss mice received intrathecal (i.t.) LPS injection, and the nociceptive threshold was measured by the von Frey filaments test. Spinal involvement of the cannabinoid CB2 receptor, toll-like receptor 4 (TLR4), microglia and astrocytes were evaluated by i.t. administration of their respectively antagonists or inhibitors. Western blot, immunofluorescence, ELISA and liquid chromatography-mass spectrometry were used to assess Cannabinoid CB2 receptors and TLR4 spinal expression, proinflammatory cytokines and endocannabinoid levels. CBD was administered intraperitoneally at 10 mg/kg. The pharmacological assay demonstrated TLR4 participation in LPS-induced nociception. In addition, spinal TLR4 expression and proinflammatory cytokine levels were increased in this process. CBD treatment prevented LPS-induced nociception and TLR4 expression. AM630 reversed antinociception and reduced CBD-induced endocannabinoids up-regulation. Increased spinal expression of the cannabinoid CB2 receptor was also found in animals receiving LPS, which was accompanied by reduced TLR4 expression in CBD-treated mice. Taken together, our findings indicated that CBD is a potential treatment strategy to control LPS-induced pain by attenuating TLR4 activation via the endocannabinoid system.

Toll-like Receptor 4 in Acute Kidney Injury.

Acute kidney injury (AKI) is a common and devastating pathologic condition, associated with considerable high morbidity and mortality. Although significant breakthroughs have been made in recent years, to this day no effective pharmacological therapies for its treatment exist. AKI is known to be connected with intrarenal and systemic inflammation. The innate immune system plays an important role as the first defense response mechanism to tissue injury. Toll-like receptor 4 (TLR4) is a well-characterized pattern recognition receptor, and increasing evidence has shown that TLR4 mediated inflammatory response, plays a pivotal role in the pathogenesis of acute kidney injury. Pathogen-associated molecular patterns (PAMPS), which are the conserved microbial motifs, are sensed by these receptors. Endogenous molecules generated during tissue injury, and labeled as damage-associated molecular pattern molecules (DAMPs), also activate pattern recognition receptors, thereby offering an understanding of sterile types of inflammation. Excessive, uncontrolled and/or sustained activation of TLR4, may lead to a chronic inflammatory state. In this review we describe the role of TLR4, its endogenous ligands and activation in the inflammatory response to ischemic/reperfusion-induced AKI and sepsis-associated AKI. The potential regeneration signaling patterns of TLR4 in acute kidney injury, are also discussed.

Toll-Like Receptor 4 Plays a Significant Role in the Biochemical and Neurological Alterations Observed in Two Distinct Mice Models of Huntington's Disease.

Toll-like receptors (TLRs) are central players in innate immunity responses. They are expressed in glial cells and neurons, and their overactivation leads to the production of proinflammatory molecules, neuroinflammation, and neural damage associated with many neurodegenerative pathologies, such as Huntington's disease (HD). HD is an inherited disorder caused by a mutation in the gene coding for the protein Huntingtin (Htt). Expression of mutated Htt (mHtt) causes progressive neuronal degeneration characterized by striatal loss of GABAergic neurons, oxidative damage, neuroinflammatory processes, and impaired motor behavior. The main animal models to study HD are the intrastriatal injection of quinolinic acid (QA) and the transgenic B6CBA-Tg (HDexon1)61Gpb/1 J mice (R6/1). Those models mimic neuronal damage and systemic manifestations of HD. The objective of this work was to study the participation of TLR4 in the manifestations of neuronal damage and HD symptoms in the two mentioned models. For this purpose, C57BL6/J and TLR4-KO mice were administered with QA, and after that motor activity, and neuronal and oxidative damages were measured. R6/1 and TLR4-KO were mated to study the effect of low expression of TLR4 on the phenotype manifestation in R6/1 mice. We found that TLR4 is involved in motor activity, and neurological and oxidative damage induced by intrastriatal injection of QA, and the low expression of TLR4 causes a delay in the onset of phenotypic manifestations by the mHtt expression in R6/1 mice. Our results show that TLR4 is involved in both models of HD and focuses then as a therapeutic target for some deleterious reactions in HD.

Genetic ablation of Toll-like Receptor 4 seems to activate the apoptosis pathway in the skeletal muscle of mice after acute physical exercise.

Many conditions, such as inflammation and physical exercise, can induce endoplasmic reticulum (ER) stress. Toll-like Receptor 4 (TLR4) can trigger inflammation and ER stress events. However, there are still no data in the literature regarding the role of TLR4 in ER stress during exercise in skeletal muscle. Therefore, the current investigation aimed to verify the responses of ER stress markers in wild-type (WT) and Tlr4 global knockout (KO) mice after acute and chronic physical exercise protocols. Eight-week-old male WT and KO mice were submitted to acute (moderate or high intensity) and chronic (4-week protocol) treadmill exercises. Under basal conditions, KO mice showed lower performance in the rotarod test. Acute high-intensity exercise increased eIF2α protein in the WT group. After the acute high-intensity exercise, there was an increase in Casp3 and Ddit3 mRNA for the KO mice. Acute moderate exercise increased the cleaved Caspase-3/Caspase-3 in the KO group. In response to chronic exercise, the KO group showed no improvement in any performance evaluation. The 4-week chronic protocol did not generate changes in ATF6, CHOP, p-IRE1α, p-eIF2α/eIF2α, and cleaved Caspase-3/Caspase-3 ratio but reduced BiP protein compared with the KO-Sedentary group. These results demonstrate the global deletion of Tlr4 seems to have the same effects on UPR markers of WT animals after acute and chronic exercise protocols but decreased performance. The cleaved Caspase-3/Caspase-3 ratio may be activated by another pathway other than ER stress in Tlr4 KO animals.

Antinociceptive effect of ultra-low dose naltrexone in a pre-clinical model of postoperative orofacial pain.

Painmanagement after oral surgeries is essential to enhance recovery, reduce negative outcomes and improve the experience of the patient. Naltrexone (NTX) is a non-selective opioid receptor antagonist that has been shown to modulate neuro-inflammation when employed in low to ultra-low doses. In addition, ultra-low dose naltrexone (ULDN) has been shown to potentiate opioids' analgesia and to have opioid-sparing effects. Herein it was investigated the effect of ULDN in a model of postoperative orofacial pain in rats, and it was tested the hypothesis that blockade of TLR4-signalling pathway contributes to its antinociceptive effect. Systemic NTX reduced heat hyperalgesia in female rats and heat and mechanical hyperalgesia in male rats after incision surgery. Combined treatment with NTX and morphine, both at ineffective doses, resulted in a significant reduction of heat hyperalgesia in male rats. NTX injection at the incision site failed to change heat hyperalgesia, but injection at the trigeminal ganglion (TG) or subnucleus caudalis (Sp5C) caused a significant reduction in heat hyperalgesia. At these sites, blockade of TLR4 impeded NTX effect. Lipopolysaccharide (LPS) injection in the intraoral mucosa resulted in facial heat hyperalgesia an increase in IL-1ß levels in the TG, which were reduced by systemic NTX. Stimulation of macrophages with LPS resulted in increase of nitric oxide, IL-1ß and CXCL-2 levels which were reduced by NTX. Altogether, these results provide evidence for an antinociceptive effect of ULDN in postoperative orofacial pain and suggest that blockade of TLR4 and downstream signaling pathway contribute to its effect.

Morphine promotes migration and lung metastasis of mouse melanoma cells.

BACKGROUND: Morphine is an analgesic agent used for cancer pain management. There have been recent concerns that the immunosuppressant properties of morphine can also promote cancer metastasis. Morphine is an agonist for toll like receptor 4 (TLR4) that has a dual role in cancer development. The promotor or inhibitor role of morphine in cancer progression remains controversial. We investigated the effects of morphine on migration and metastasis of melanoma cells through TLR4 activation. METHODS: Mouse melanoma cells (B16F10) were treated with only morphine (0, 0.1, 1, and 10 µM) or in combination with a TLR4 inhibitor (morphine10 µM +CLI-095 1µM) for either 12 or 24 hours. Migration of cells was analyzed by transwell migration assays. Twenty C57BL/6 male mice were inoculated with B16F10 cells via the left ventricle of the heart and then randomly divided into two groups (n = 10 each) that received either morphine (10 mg.kg-1, sub-q) or PBS injection for 21 days (control group). Animals were euthanized and their lungs removed for evaluation of metastatic nodules. RESULTS: Morphine (0.1, 1, and 10 µM) increased cell migration after 12 hours (p < 0.001) and after 24 hours of treatment with morphine (10 µM) (p < 0.001). Treatment with CLI-095 suppressed migration compared to cells treated with morphine alone (p < 0.001). Metastatic nodules in the morphine-treated group (64 nodules) were significantly higher than in the control group (40 nodules) (p < 0.05). CONCLUSION: Morphine increases the migration and metastasis of mouse melanoma cells by activating TLR4.

Morphine promotes migration and lung metastasis of mouse melanoma cells

Abstract Background Morphine is an analgesic agent used for cancer pain management. There have been recent concerns that the immunosuppressant properties of morphine can also promote cancer metastasis. Morphine is an agonist for toll like receptor 4 (TLR4) that has a dual role in cancer development. The promotor or inhibitor role of morphine in cancer progression remains controversial. We investigated the effects of morphine on migration and metastasis of melanoma cells through TLR4 activation. Methods Mouse melanoma cells (B16F10) were treated with only morphine (0, 0.1, 1, and 10 μM) or in combination with a TLR4 inhibitor (morphine10 μM +CLI-095 1μM) for either 12 or 24 hours. Migration of cells was analyzed by transwell migration assays. Twenty C57BL/6 male mice were inoculated with B16F10 cells via the left ventricle of the heart and then randomly divided into two groups (n = 10 each) that received either morphine (10 mg.kg−1, sub-q) or PBS injection for 21 days (control group). Animals were euthanized and their lungs removed for evaluation of metastatic nodules. Results Morphine (0.1, 1, and 10 μM) increased cell migration after 12 hours (p < 0.001) and after 24 hours of treatment with morphine (10 μM) (p < 0.001). Treatment with CLI-095 suppressed migration compared to cells treated with morphine alone (p < 0.001). Metastatic nodules in the morphine-treated group (64 nodules) were significantly higher than in the control group (40 nodules) (p < 0.05). Conclusion Morphine increases the migration and metastasis of mouse melanoma cells by activating TLR4.

The relationship between 896A/G (rs4986790) polymorphism of TLR4 and infectious diseases: A meta-analysis.

Toll-like Receptors (TLRs), such as the TLR4, are genes encoding transmembrane receptors of the same name, which induce a pro- or anti-inflammatory response according to their expression as the host's first line of defense against pathogens, such as infectious ones. Single nucleotide polymorphisms (SNPs) are the most common type of mutation in the human genome and can generate functional modification in genes. The aim of this article is to review in which infectious diseases there is an association of susceptibility or protection by the TLR4 SNP rs4986790. A systematic review and meta-analysis of the literature was conducted in the Science Direct, PUBMED, MEDLINE, and SciELO databases between 2011 and 2021 based on the dominant genotypic model of this SNP for general and subgroup analysis of infectious agent type in random effect. Summary odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated for genotypic comparison. I2 statistics were calculated to assess the presence of heterogeneity between studies and funnel plots were inspected for indication of publication bias. A total of 27 articles were included, all in English. Among the results achieved, the categories of diseases that were most associated with the SNP studied were in decreasing order of number of articles: infections by bacteria (29.63%); caused by viruses (22.23%); urinary tract infection-UTI (7.4%), while 11 studies (40.74%) demonstrated a nonsignificant association. In this meta-analysis, a total of 5599 cases and 5871 controls were finalized. The present meta-analysis suggests that there is no significant association between TLR4-rs4986790 SNP and infections (OR = 1,11; 95% CI: 0,75-1,66; p = 0,59), but in the virus subgroup it was associated with a higher risk (OR = 2,16; 95% CI: 1,09-4,30; p = 0,03). The subgroups of bacteria and parasites did not show statistical significance (OR = 0,86; 95% CI: 0,56-1,30; p = 0,47, and no estimate of effects, respectively). Therefore, it has been shown that a diversity of infectious diseases is related to this polymorphism, either by susceptibility or even severity to them, and the receptor generated is also crucial for the generation of cell signaling pathways and immune response against pathogens.

Caffeine Inhibits NLRP3 Inflammasome Activation by Downregulating TLR4/MAPK/NF-κB Signaling Pathway in an Experimental NASH Model.

Caffeine elicits protective effects against liver diseases, such as NASH; however, its mechanism of action involving the pyrin domain-containing-3 (NLRP3) inflammasome signaling pathway remains to be elucidated. This study aimed to evaluate the effect of caffeine on the NLRP3 inflammasome signaling pathway in a rat model of NASH. NASH was induced by feeding rats a high-fat, -sucrose, and -cholesterol diet (HFSCD) for 15 weeks along with a weekly low dose (400 mg/kg, i.p.) of CCl4. Caffeine was administered at 50 mg/kg p.o. The effects of HFSCD+CCl4 and caffeine on the liver were evaluated using biochemical, ultrastructural, histological, and molecular biological approaches. The HFSCD+CCl4-treated rats showed fat accumulation in the liver, elevated levels of inflammatory mediators, NLRP3 inflammasome activation, antioxidant dysregulation, and liver fibrosis. Caffeine reduced necrosis, cholestasis, oxidative stress, and fibrosis. Caffeine exhibited anti-inflammatory effects by attenuating NLRP3 inflammasome activation. Moreover, caffeine prevented increases in toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) protein levels and mitigated the phosphorylation of mitogen-activated protein kinase (MAPK). Importantly, caffeine prevented the activation of hepatic stellate cells. This study is the first to report that caffeine ameliorates NASH by inhibiting NLRP3 inflammasome activation through the suppression of the TLR4/MAPK/NF-κB signaling pathway.

Porphyromonas gingivalis-Helicobacter pylori co-incubation enhances Porphyromonas gingivalis virulence and increases migration of infected human oral keratinocytes.

Background: Porphyromonas gingivalis is part of the subgingival biofilm and a keystone species in the development of periodontitis. Interactions between P.gingivalis and other bacteria in biofilms have been shown to affect bacterial virulence. Helicobacter pylori also inhabits the subgingival biofilm, but the consequences of interactions there with P.gingivalis remain unknown. Here, we investigated how the pre-incubation of P.gingivalis with H.pylori affects P.gingivalis virulence. Methods: We assayed P.gingivalis internalization by oral keratinocytes (OKs), hemagglutination and biofilm formation to identify alterations in virulence after pre-incubation with H. pylori. Also, we evaluated viability and migration of OKs infected with P. gingivalis, as well as the role of toll-like receptor 4 (TLR4).   In addition, we quantified the mRNA of genes associated with P.gingivalis virulence. Results: Pre-incubation of P.gingivalis with H.pylori enhanced P.gingivalis biofilm formation, bacterial internalization into OKs and hemagglutination. Infection with pre-incubated P.gingivalis increased OK migration in a manner dependent on the O-antigen and linked to  increased expression of the gingipain RgpB. Also, OK TLR4 participates in these events, because upon TLR4 knock-down, pre-incubated P.gingivalis no longer stimulated OK migration. Discussion: We provide here for the first time insight to the consequences of direct interaction between P.gingivalis and H.pylori. In doing so, we shed light on the mechanism by which H. pylori presence in the oral cavity increases the severity or progression of periodontitis.

Caloric restriction in mice improves short-term recognition memory and modifies the neuroinflammatory response in the hippocampus of male adult offspring.

Restrictive diets (RD) can influence the inflammatory phenotype of dams and their offspring. Thus, this study aimed to evaluate the effects of caloric restriction on the neuroinflammatory profile in the hippocampus and the short-term recognition memory of male offspring from RD-fed dams. Mice dams received standard diet ad libitum (CONT) or restrictive diet (RD; 30% reduction of CONT consumption) during pregnancy and lactation. Male pups were weaned at 21 days and randomly divided into two groups that received CONT or RD; groups were named according to maternal/offspring diets: CONT/CONT, CONT/RD, RD/CONT, and RD/RD. At 90 days old, short-term memory was assessed by the object recognition test (ORT); the inflammatory state of the hippocampus was analyzed by gene expression of sirtuin-1 (Sirt1) and inflammasome Nlrp3; and by protein expression of toll-like receptor-4 (TLR-4) and zonula occludens-1 (ZO-1). Our results showed an improvement in short-term memory in RD-fed offspring. The expression of Sirt1 was higher in RD/CONT compared to CONT/CONT and decreased in RD/RD compared to CONT/RD. Nlrp3 gene expression showed an offspring effect, being decreased in RD-fed mice. TLR-4 expression was higher in RD/CONT compared to CONT/CONT, similarly to ZO-1 expression. However, ZO-1 also showed a maternal diet effect and increased expression in the offspring of RD dams. Our findings demonstrate that caloric restriction improved short-term recognition memory. However, a restrictive diet should be applied with caution; depending on the offspring's diet, it may not benefit the neuroinflammatory phenotype or cognition.

Epididymal embryonic development harbors TLR4/NFKB signaling pathway as a morphogenetic player.

The Wolffian duct (WD) is an embryonic tissue that undergoes androgen-induced morphological changes to become the epididymis. Toll-like receptor 4 (TLR4)- and nuclear factor kB (NFKB)-induced effectors are expressed in the adult epididymis and represent important players in epididymal innate immune responses. TLR4/NFKB signaling pathway is evolutionarily conserved and plays a critical morphogenetic role in several species; however, its function during WD morphogenesis is unknown. We hypothesized that TLR4/NFKB pathway plays a role during WD development. Here we examined TLR4 expression and regulation of TLR4-target genes during rat WD morphogenesis between embryonic days (e) 17.5-20.5. The functionality of TLR4/NFKB signaling was examined using WD organotypic cultures treated with lipopolysaccharide (LPS) from E. coli (TLR4 agonist) and PDTC (NFKB inhibitor). TLR4 was detected at mRNA level in e17.5 (uncoiled duct) and e20.5 (coiled duct) WDs, and spatio-temporal changes in TLR4 immunoreactivity were observed between these two time points. Expression level analysis of a subset of TLR4-regulated genes showed that TLR4/NFKB pathway was activated after exposure of cultured WD to LPS (4 h), an event that was abrogated by PDTC. Long-term exposure of cultured WDs to LPS (96 h) resulted in dysregulations of morphogenetic events and LAMA1 immunodistribution changes, suggesting the extracellular matrix at the intersection between WD morphogenesis and balance of innate immune components. Our results unveil the epididymal morphogenesis as an event equipped with TLR4/NFKB signaling components that may serve developmental functions, and eventually transition to host defense function when the fetus is exposed to an infectious or noninfectious threat.

Mechanism of dexmedetomidine preconditioning on spinal cord analgesia in rats with functional chronic visceral pain

Purpose: To analyze the effect and mechanism of dexmedetomidine (DEX) analgesia pretreatment on functional chronic visceral pain in rats. Methods: Rats were divided into six groups: W1, W2, W3, W4, W5, and W6. The behavioral changes and electrophysiological indexes of rats in each group before and after DEX treatment were detected. Results: The levels of abdominal withdrawal reflex (AWR) in W5 and W6 groups were significantly lower than those in group W3, while the levels of thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were significantly higher than those in group W3 (p < 0.05). The electromyographic signals of W1, W5, and W6 groups showed little fluctuation, while those of groups W2, W3, and W4 showed obvious fluctuation. TLR4 mRNA expression, IRF3, P65, and phosphorylation levels in W4, W5, and W6 groups were significantly lower than those in group W2 (p < 0.05). Conclusions: Dexmedetomidine epidural anesthesia pretreatment could significantly inhibit visceral pain response in rats with functional chronic visceral pain, and its mechanism was related to the activation of TLR4 in spinal dorsal horn tissue of rats and the activation inhibition of IRF3 and P65 in the downstream key signals.

Resveratrol and Quercetin as Regulators of Inflammatory and Purinergic Receptors to Attenuate Liver Damage Associated to Metabolic Syndrome.

Nonalcoholic fatty liver disease (NAFLD) is considered a manifestation of metabolic syndrome (MS) and is characterized by the accumulation of triglycerides and a varying degree of hepatic injury, inflammation, and repair. Moreover, peroxisome-proliferator-activated receptors (PPARs) play a critical role in the pathophysiological processes in the liver. There is extensive evidence of the beneficial effect of polyphenols such as resveratrol (RSV) and quercetin (QRC) on the treatment of liver pathology; however, the mechanisms underlying their beneficial effects have not been fully elucidated. In this work, we show that the mechanisms underlying the beneficial effects of RSV and QRC against inflammation in liver damage in our MS model are due to the activation of novel pathways which have not been previously described such as the downregulation of the expression of toll-like receptor 4 (TLR4), neutrophil elastase (NE) and purinergic receptor P2Y2. This downregulation leads to a decrease in apoptosis and hepatic fibrosis with no changes in hepatocyte proliferation. In addition, PPAR alpha and gamma expression were altered in MS but their expression was not affected by the treatment with the natural compounds. The improvement of liver damage by the administration of polyphenols was reflected in the normalization of serum transaminase activities.