Vitamin D3 Controls TLR4- and TLR2-Mediated Inflammatory Responses of Endometrial Cells.

OBJECTIVES: Vitamin D has potent immunoregulatory features and modulates innate and adaptive immune responses. There is a significant association between intrauterine infection-associated inflammatory responses and pregnancy complications such as abortion and preterm labor. Here, we investigated how 1,25 (OH)2 D3 could modulate inflammatory responses of endometrial cells. DESIGN: This is an in vitro experimental study. Endometrial stromal cells (ESCs) and whole endometrial cells (WECs) were collected from 15 apparently normal women, and the immunomodulatory effects of 1,25 (OH)2 D3 on lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated ESCs and WECs were investigated. Participants/Materials, Setting, and Methods: Women with no history of abortion, infertility, endometriosis, or sign of vaginal infection were enrolled in this study. Endometrial samples were collected by gynecologists using a Pipelle pipette in the proliferative phase of the menstrual cycle. WECs and ESCs were collected and treated with either LPS or LTA. The levels of IL-6, IL-8, and TNF-α in culture supernatants were quantified using the ELISA technique. TLR2, TLR4, and MyD88 expressions were assessed by RT-qPCR. TLR4 expression at the protein level was studied by the Western blot technique. RESULTS: 1,25 Dihydroxycholecalciferol (1,25 (OH)2 D3) significantly reduced TNF-α production in LPS-activated ESCs and TNF-α and IL-6 production by LTA-stimulated WECs. In contrast, 1,25 (OH)2 D3 pretreatment increased the production of IL-8 by LPS- and LTA-stimulated endometrial cells. 1,25 (OH)2 D3 pretreatment markedly reduced LPS-induced TLR4 protein expression by ESCs. LPS treatment of ESCs significantly induced MyD88 gene expression. This effect was reversed when these cells were pretreated with 1,25 (OH)2 D3 before stimulation with LPS. LIMITATIONS: Because of the small size of samples, doing experiments all together on some samples was not feasible. Confirmation of the results obtained here needs well-designed in vivo studies. CONCLUSIONS: 1,25 (OH)2 D3 is an immunomodulatory molecule essential for maintaining endometrial immune homeostasis by controlling potentially harmful inflammatory responses associated with female reproductive tract infections.

The impact of the level and distribution of methyl-esters of pectins on TLR2-1 dependent anti-inflammatory responses.

Pectins have anti-inflammatory effects via Toll-like receptor (TLR) inhibition in a degree of methyl-esterification-(DM)-dependent manner. However, pectins also vary in distribution of methyl-esters over the galacturonic-acid (GalA) backbone (Degree of Blockiness - DB) and impact of this on anti-inflammatory capacity is unknown. Pectins mainly inhibit TLR2-1 but magnitude depends on both DM and DB. Low DM pectins (DM18/19) with both low (DB86) and high DB (DB94) strongly inhibit TLR2-1. However, pectins with intermediate DM (DM43/DM49) and high DB (DB60), but not with low DB (DB33), inhibit TLR2-1 as strongly as low DM. High DM pectins (DM84/88) with DB71 and DB91 do not inhibit TLR2-1 strongly. Pectin-binding to TLR2 was confirmed by capture-ELISA. In human macrophages, low DM and intermediate DM pectins with high DB inhibited TLR2-1 induced IL-6 secretion. Both high number and blockwise distribution of non-esterified GalA in pectins are responsible for the anti-inflammatory effects via inhibition of TLR2-1.

Hypoxic-conditioned medium from adipose tissue mesenchymal stem cells improved neuroinflammation through alternation of toll like receptor (TLR) 2 and TLR4 expression in model of Alzheimer's disease rats.

Microglia have a pivotal role to initiate immune responses in AD brains through toll-like receptors and induce neuroinflammation. Adipose tissue mesenchymal stem cells (ATSCs) secret many neurotrophic and anti-inflammatory factors called conditioned medium (CM). Many studies have demonstrated that CM of mesenchymal stem cells facilitate regeneration and attenuates inflammation in many disorders. To this purpose, the effect of ATSCs-conditioned medium (ATSC-CM) on brain inflammation and the role of toll-like receptors were investigated in this study. Seventy-two rats were randomly divided into 6 groups: control, sham, sham+ATSC-CM: 200µl ATSC-CM once a day intraperitoneally for 8 days, AD group injected the Aß1-40 intra-hippocampal, AD+ASC-CM, which was injected Aß1-40 intra-hippocampal and 200µl ATSC-CM once a day intraperitoneally for 8 days and AD+ rivastigmine: was injected Aß1-40 intra-hippocampal and received rivastigmine (0.6 mg/kg) orally once a day for 2 weeks. Memory and learning were measured by Morris water maze and novel object recognition tests. For detection of beta-amyloid plaque, Congo red staining was used, and neuronal survival was assessed by Nissl staining. Expression of TLR2 and TLR4 was measured by real-time PCR, and finally, to assess inflammation markers (IL-1ß and TNF-α) in the hippocampus, ELISA kits were used. In treatment group spatial and recognition memory significantly was improved. ATSC-CM administration decreased beta amyloid plaques and enhanced neuronal survival in AD brain rats. In addition, TLR2 and TLR4 expression decreased in treatment group. Results also showed that ATSC-CM reduced IL-1ß and TNF-α as inflammation markers. ATSC-CM improved memory deficit, decreased beta amyloids formation, increased neuron survival, and attenuated inflammation by reducing the expression of TLRs.

Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia.

Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

The modulatory effect of Artemisia annua L. on toll-like receptor expression in Acanthamoeba infected mouse lungs.

The genus Acanthamoeba, which may cause different infections in humans, occurs widely in the environment. Lung inflammation caused by these parasites induces pulmonary pathological changes such as pulmonary necrosis, peribronchial plasma cell infiltration, moderate desquamation of alveolar cells and partial destruction of bronchial epithelial cells, and presence of numerous trophozoites and cysts among inflammatory cells. The aim of this study was to assess the influence of plant extracts from Artemisia annua L. on expression of the toll-like receptors TLR2 and TLR4 in lungs of mice with acanthamoebiasis. A. annua, which belongs to the family Asteraceae, is an annual plant that grows wild in Asia. In this study, statistically significant changes of expression of TLR2 and TLR4 were demonstrated. In the lungs of infected mice after application of extract from A. annua the expression of TLRs was observed mainly in bronchial epithelial cells, pneumocytes (to a lesser extent during the outbreak of infection), and in the course of high general TLR expression. TLR4 in particular was also visible in stromal cells of lung parenchyma. In conclusion, we confirmed that a plant extract of A. annua has a modulatory effect on components of the immune system such as TLR2 and TLR4.

Ceftaroline modulates the innate immune and host defense responses of immunocompetent cells exposed to cigarette smoke.

BACKGROUND: Cigarette smoke, the principal risk factor for chronic obstructive pulmonary disease (COPD), negatively influences the effectiveness of the immune system's response to a pathogen. The antibiotic ceftaroline exerts immune-modulatory effects in bronchial epithelial cells exposed to cigarette smoke. AIMS AND METHODS: The present study aims to assess the effects of ceftaroline on TLR2 and TLR4 expression, LPS binding and TNF-α and human beta defensin (HBD2) release in an undifferentiated and PMA-differentiated human monocyte cell line (THP-1) exposed or not to cigarette smoke extracts (CSE). TLR2, TLR4, and LPS binding were assessed by flow cytometry, TNF-α and HBD2 release were evaluated by ELISA. RESULTS: The constitutive expression of TLR2 and TLR4 and LPS binding were higher in differentiated compared to undifferentiated THP-1 cells. In undifferentiated THP-1 cells, CSE increased TLR2 and TLR4 protein levels, LPS binding and TNF-α release and reduced HBD2 release and ceftaroline counteracted all these effects. In differentiated THP-1, CSE did not significantly affect TLR2 and TLR4 expression and LPS binding but reduced HBD2 release and increased TNF-α release. Ceftaroline counteracted the effects of CSE on HBD2 release in differentiated THP-1. CONCLUSION: Ceftaroline counteracts the effect of CSE in immune cells by increasing the effectiveness of the innate immune system. This effect may also assist in reducing pathogen activity and recurrent exacerbations in COPD patients.

MiR-344b-1-3p targets TLR2 and negatively regulates TLR2 signaling pathway.

OBJECTIVES: COPD is an abnormal inflammatory response characterized by decreased expression of TLR2 in patients, which is suggested to induce invasive pulmonary aspergillosis (IPA). MicroRNAs (miRNAs) have been shown to play important roles in the pathogenesis of human respiratory system disorders. Therefore, the aim of this study was to identify the miRNAs involved in the regulation of TLR2 signaling in COPD. MATERIALS AND METHODS: miRNA microarray analysis was performed to screen for the dysregulated miRNAs in alveolar macrophages (AMs) isolated from COPD rats. The interaction between these miRNAs and TLR2 gene was predicted using miRBase and validated using dual luciferase assay. Based on the analysis, a novel miR-344b-1-3p was identified as a novel modulator of TLR2 gene. Then, the mechanism through which miR-344b-1-3p regulated TLR2 expression was explored using cigarette smoke extract (CSE)-pretreated NR8383 cells. Moreover, by subjecting CSE-pretreated NR8383 cells to Pam3CSK4, the effect of miR-344b-1-3p on NF-κB activity and other important mediators of COPD, including IRAK-1, ERK, TNF-α, IL-1ß, and MIP-2, was also assessed. RESULTS: COPD rat model was successfully induced by smoke inhalation. Among the 11 upregulated miRNAs in AMs from COPD rats, miR-344b-1-3p was predicted to be a novel miRNA targeting TLR2 gene. In the CSE pretreated NR8383 cells exposed to Pam3CSK4, miR-344b-1-3p inhibition increased the expression levels of TLR2, TNF-α, and IL-1ß and decreased the expression levels of MIP-2. In addition, the phosphorylation of IRAK-1, IκBα, and IRK was augmented by miR-344b-1-3p inhibition. CONCLUSION: Findings outlined in this study suggest that miR-344b-1-3p was an effective modulator of TLR2 gene, which can be employed as a promising therapeutic and preventive target of IPA in COPD patients.

Ribavirin attenuates the respiratory immune responses to influenza viral infection in mice.

Ribavirin is a broad-spectrum antiviral agent that is used against RNA and DNA viruses and has been reported to inhibit infection by influenza A and B virus in vitro and in vivo. Studies have shown that ribavirin can lower convalescent antibody titers in young children hospitalized with influenza. Here, we report that ribavirin administration in juvenile mice significantly attenuated respiratory immune responses, production of total IgA and hemagglutinin (HA)-specific secretory IgA responses on the mucosal surface. In contrast, systemic IgG and IgA responses were not affected. Ribavirin significantly suppressed toll-like receptor 2 and 4 expression in the lung and decreased the level of IL-1ß, IL-6, TNF-α, and IFN-γ in lung tissues of mice infected with influenza virus. Our findings suggest ribavirin appears to be able to inhibit viral replication and, as a result, TLR and cytokine expression are not up-regulated, attenuating inflammation as well as the respiratory tract's immune response.

Synthesis and anti-inflammatory evaluation of novel paclitaxel analogs.

A series of paclitaxel analogs modified at C-3'-N and C-7 positions were synthesized from baccatin III and their structures were confirmed by 1H-NMR, 13C-NMR, HR-MS. Compound 7e exhibited potent ability to decrease TNFα (tumor necrosis factor α) in the LPS-activated RAW264.7 murine macrophage-like cell line. The preliminary data indicated that the anti-inflammatory effects may be related to MD-2 and Toll-like receptor 4 (TLR4), rather than Toll-like receptor 2 (TLR2).

Interleukin-23 Secreted by Activated Macrophages Drives γδT Cell Production of Interleukin-17 to Aggravate Secondary Injury After Intracerebral Hemorrhage.

BACKGROUND: Neuroinflammation plays a key role in intracerebral hemorrhage (ICH)-induced secondary brain injury, but the specific roles of peripheral inflammatory cells such as macrophages and lymphocytes remain unknown. The purpose of this study was to explore the roles of macrophages, T lymphocytes, and the cytokines they secrete as potential targets for treating secondary brain injury after ICH. METHODS AND RESULTS: Our results showed that peripheral macrophages and T lymphocytes successively infiltrated the brain, with macrophage counts peaking 1 day after ICH and T-lymphocyte counts peaking after 4 days. These peaks in cellular infiltration corresponded to increases in interleukin (IL)-23 and IL-17 expression, respectively. We found that hemoglobin from the hematoma activated IL-23 secretion by infiltrating macrophages by inducing the formation of toll-like receptor (TLR) 2/4 heterodimer. This increased IL-23 expression stimulated γδT-cell production of IL-17, which increased brain edema and neurologic deficits in the model mice as a proinflammatory factor. Finally, we found that sparstolonin B (SsnB) could ameliorate brain edema and neurologic deficits in ICH model mice via inhibition of TLR2/TLR4 heterodimer formation, and notably, SsnB interacted with myeloid differentiation factor 88 Arg196. CONCLUSIONS: Together, our results reveal the importance of the IL-23/IL-17 inflammatory axis in secondary brain injury after ICH and thus provide a new therapeutic target for ICH treatment.

Paeoniflorin inhibits high glucose-induced macrophage activation through TLR2-dependent signal pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin(PF), extracted from the root peeled of Paeonia lactiflora Pall(Family: Ranunculaceae), has therapeutic potential in many animal models of inflammatory diseases. AIM OF THE STUDY: Although the anti-inflammatory efficacy of PF has been well illustrated in several animal models, whether it could attenuate diabetic nephropathy and detailed mechanisms are still obscure. Till now, accumulating evidence has proposed the pivotal role of toll-like receptors (TLRs) in renal inflammation in diabetic patients. In this setting, the current study aimed to investigate the effects and underlying mechanism of PF on high glucose-induced activation of toll like-receptor 2 (TLR2) signaling in macrophages. MATERIALS AND METHODS: Bone marrow-derived macrophages (BMDM) were isolated from male Tlr2tm1kir (TLR2-/-) mice and wild-type littermates (C57BL/6JWT). The level of TLR2 and activation of downstream signaling were evaluated in response to 30mmol/L high glucose (HG)-containing medium. Macrophages behaviors, which include cell viability, migration and inflammatory cytokines production, were also determined. RESULTS: PF suppressed HG-induced production of TLR2, activation of downstream signaling and synthesis of inducible nitric oxide synthase (iNOS). PF could further inhibit MyD88-dependent pathway in HG-induced models in which TLR2 was knocked out. Moreover, deletion of TLR2 inhibited the HG-induced activation of MyD88-dependent pathway, but not TIR domain containing adapter inducing interferon-ß (Trif) signal pathway in BMDMs. As HG stimulation polarizes macrophages into M1 phenotype, treatment of PF or knockout of TLR2 significantly reduces M1 markers on the membrane of macrophages. Additionally, levels of inflammatory cytokines and iNOS were remarkably reduced in response to PF or TLR2 deficiency. CONCLUSION: Collectively, these data demonstrated that HG activated macrophages primarily through TLR2-dependent mechanisms which aggravated the severity of renal inflammation and eventually contributed to DN. Additionally, PF might be applied as a potential therapeutic agent in the battle against progressive DN.

Anti-ApoA-1 IgG serum levels predict worse poststroke outcomes.

BACKGROUND: Autoantibodies to apolipoprotein A-1 (anti-ApoA-1 IgG) were shown to predict major adverse cardiovascular events and promote atherogenesis. However, their potential relationship with clinical disability and ischaemic lesion volume after acute ischaemic stroke (AIS) remains unexplored. MATERIALS AND METHODS: We included n = 76 patients admitted for AIS and we investigated whether baseline serum anti-ApoA-1 IgG levels could predict (i) AIS-induced clinical disability [assessed by the modified Rankin Scale (mRS)], and (ii) AIS-related ischaemic lesion volume [assessed by Computed Tomography (CT)]. We also evaluated the possible pro-apoptotic and pro-necrotic effects of anti-ApoA-1 IgG on human astrocytoma cell line (U251) using flow cytometry. RESULTS: High levels of anti-ApoA-1 IgG were retrieved in 15·8% (12/76) of patients. Increased baseline levels of anti-ApoA-1 IgG were independently correlated with worse mRS [ß = 0·364; P = 0·002; adjusted odds ratio (OR): 1·05 (95% CI 1·01-1·09); P = 0·017] and CT-assessed ischaemic lesion volume [ß = 0·333; P < 0·001; adjusted OR: 1·06 (95% CI 1·01-1·12); P = 0·048] at 3 months. No difference in baseline clinical, biochemical and radiological characteristics was observed between patients with high vs. low levels of anti-ApoA-1 IgG. Incubating human astrocytoma cells with anti-ApoA-1 IgG dose dependently induced necrosis and apoptosis of U251 cells in vitro. CONCLUSION: Anti-ApoA-1 IgG serum levels at AIS onset are associated with poorer clinical recovery and worse brain lesion volume 3 months after AIS. These observations could be partly explained by the deleterious effect of anti-ApoA-1 IgG on human brain cell survival in vitro and may have clinical implication in the prediction of poor outcome in AIS.

Structural Change in Microbiota by a Probiotic Cocktail Enhances the Gut Barrier and Reduces Cancer via TLR2 Signaling in a Rat Model of Colon Cancer.

BACKGROUND: Structural change in the gut microbiota is implicated in cancer. The beneficial modulation of the microbiota composition with probiotics and prebiotics prevents diseases. AIM: We investigated the effect of oligofructose-maltodextrin-enriched Lactobacillus acidophilus, Bifidobacteria bifidum, and Bifidobacteria infantum (LBB), on the gut microbiota composition and progression of colorectal cancer. METHODS: Sprague Dawley rats were acclimatized, given ampicillin (75 mg/kg), and treated as follows; GCO: normal control; GPR: LBB only; GPC: LBB+ 1,2-dimethylhydrazine dihydrochloride (DMH); and GCA: DMH only (cancer control). 16S V4 Pyrosequencing for gut microbiota analysis, tumor studies, and the expression of MUC2, ZO-1, occludin, TLR2, TLR4, caspase 3, COX-2, and ß-catenin were conducted at the end of experiment. RESULTS: Probiotic LBB treatment altered the gut microbiota. The relative abundance of genera Pseudomonas, Congregibacter, Clostridium, Candidactus spp., Phaeobacter, Escherichia, Helicobacter, and HTCC was decreased (P < 0.05), but the genus Lactobacillus increased (P < 0.05), in LBB treatment than in cancer control. The altered gut microbiota was associated with decreased tumor incidence (80 % in GPC vs. 100 % in GCA, P = 0.0001), tumor volume (GPC 84.23 (42.75-188.4) mm(3) vs. GCA 243 (175.5-344.5) mm(3), P < 0.0001) and tumor multiplicity/count (GPC 2.92 ± 0.26 vs. GCA 6.27 ± 0.41; P < 0.0001). The expression of MUC2, ZO-1, occludin, and TLR2 was increased, but expression of TLR4, caspase 3, Cox-2, and ß-catenin was decreased by LBB treatment than in cancer control GCA (P < 0.05). CONCLUSION: Administration of LBB modulates the gut microbiota and reduces colon cancer development by decreasing tumor incidence, multiplicity/count, and volume via enhanced TLR2-improved gut mucosa epithelial barrier integrity and suppression of apoptosis and inflammation.

Effects of budesonide on toll-like receptor expression in alveolar macrophages from smokers with and without COPD.

INTRODUCTION: Alveolar macrophages (AMs) are equipped with innate immune receptors such as toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4). In primary bronchial epithelial cells, exposure of toll-like receptor (TLR) ligands or tumor necrosis factor-alpha (TNF-α) increased TLR2 mRNA expression and reduced interleukin-8 (IL-8) release when coincubated with glucocorticosteroids. The aim of this study was to compare TLR2 and TLR4 expression levels and the effect of a glucocorticosteroid after stimulation with TLR ligands on AMs from smokers with and without COPD compared with the healthy controls. SUBJECTS AND METHODS: Bronchoalveolar lavage was performed, and AMs were isolated from smokers with (n=10) and without COPD (n=11) and healthy controls (n=10) and stimulated ex vivo with peptidoglycan (PGN), lipopolysaccharide (LPS), or TNF-α ± budesonide (Bud). Blocking antibodies to TLR2 or TLR4 were added before stimulation with LPS or PGN ± Bud, respectively. The release of proinflammatory cytokine (TNF-α), chemoattractant (CXCL8), and TLR expression was analyzed by enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. RESULTS: LPS, PGN, and TNF-α induced an increased release of IL-8 and TNF-α in the AMs in all the groups independent of smoking or disease. These responses were inhibited by a glucocorticosteroid (Bud) in all the three groups, except PGN-induced IL-8 secretion in smokers without COPD. Bud increased TLR2 expression in the healthy controls and smokers without COPD. Costimulation of TLR ligands and Bud significantly enhanced TLR2 mRNA expression in both groups of smokers compared with TLR ligands alone. In smokers, costimulation with PGN and Bud significantly increased TLR2 expression when compared with Bud alone. On stimulation with the TLR4 agonist, LPS downregulated TLR4 mRNA expression in all the three groups. CONCLUSION: The combination of glucocorticosteroids with TLR ligands can increase TLR2 expression, thereby improving host defense in smokers. Also this combination can decrease the secretion of proinflammatory cytokines and chemokines as an anti-inflammatory response. Our findings indicate that glucocorticosteroid therapy strengthens immune defense pathways, which may have implication during exacerbation caused by microorganisms.

Argon mediates protection by interleukin-8 suppression via a TLR2/TLR4/STAT3/NF-κB pathway in a model of apoptosis in neuroblastoma cells in vitro and following ischemia-reperfusion injury in rat retina in vivo.

Argon has recently come into scientific focus as a neuroprotective agent. The underlying neuroprotective mechanism remains unknown although toll-like receptors were recently suggested to play an important role. We hypothesized that TLR-associated downstream transcription factors are responsible for argon's effects, leading to anti-apoptotic and anti-inflammatory properties. Apoptosis was induced in human neuroblastoma cells. Immediately afterwards, argon treatment (75 Vol% for 2 h) was initiated. Cells were analyzed, measuring mitochondrial membrane potential, reactive-oxygen-species, annexin-V/propidium iodide staining, transcription factor phosphorylation and binding activity as well as protein and mRNA expression of interleukins. Argon's in vivo effects were analyzed by quantification of retinal ganglion cell density, mRNA expression, serum cytokine analysis and immunohistochemistry after retinal ischemia reperfusion injury (IRI) in rats. Argon diminished rotenone-induced kappa-light-chain-enhancer' of activated B-cells (NF-κB) and signal transducer and activator of transcription 3 (STAT3) but not STAT5 or cAMP-response element-binding protein (CREB) phosphorylation and DNA-binding activity. Argon treatment attenuated apoptosis by preservation of mitochondrial membrane potential and decline in reactive oxygen species (ROS) generation. NF-κB and STAT3 inhibition, as well as TLR2 and TLR4 inhibition reversed argon's effects on IL-8 mRNA expression. Argon attenuated rotenone-induced IL-8 protein and mRNA expression in vitro. Inhibition of TLR2 and 4 attenuated argon's protective effect in vivo reducing IRI driven retinal IL-8 expression. IL-8 expression was found in the retina in co-localization with Müller cells and retinal ganglion cells. Argon mediates its neuroprotective effects by TLR-mediated regulation of transcription factors NF-κB and STAT3, thus decreasing interleukin-8 expression in vitro and in vivo. These findings may open up new opportunities to effectively treat cerebral ischemia and reperfusion injury through the inhalation of argon. Argon exerts its protective effects in vitro and in vivo via toll-like receptors TLR2 and TLR4 signaling, followed by alteration of downstream enzymes. In conclusion, argon mediates its beneficial effects by suppression of STAT3 and NF-κB phosphorylation and subsequent suppression of interleukin IL-8 protein expression. These novel findings may open up opportunities for argon as a therapeutic agent, particularly in the treatment of neuronal injury. Cover image for this issue: doi: 10.1111/jnc.13334.

Female sex hormones modulate Porphyromonas gingivalis lipopolysaccharide-induced Toll-like receptor signaling in primary human monocytes.

BACKGROUND AND OBJECTIVE: Female sex hormones are elevated and are potential host response modifiers during pregnancy. Modulation of immune responses by estrogen and progesterone may be responsible for periodontal inflammation. Therefore, we aimed to investigate the role of ß-estradiol and progesterone in human monocyte immune responses, at cellular and molecular levels, to identify their role as a possible immunological link between pregnancy and periodontal disease. MATERIAL AND METHODS: Primary human monocytes were purified from human peripheral blood mononuclear cells by adherent method. Expression of Toll-like receptor (TLR) 2, 4 and CD14 was analyzed by flow cytometry. TLR2, TLR4, cyclooxygenase-2 (COX2), nuclear factor-kappa B (NF-κB) and NF-κB inhibitor-alpha mRNA expressions were measured using real-time reverse transcriptase-polymerase chain reaction and prostaglandin E2 secretion was assayed by enzyme-linked immunosorbent assay. NF-κB expression was also examined by immunofluorescence. Western blotting was performed to determine the activation of mitogen-activated protein kinase pathway. RESULTS: We report herein that both ß-estradiol and progesterone significantly reduced TLR2 expression at both protein and mRNA levels but had less of an effect on TLR4 expression in primary human monocytes. We also found that the hormones decreased monocyte cell surface protein expression of CD14. Significantly, ß-estradiol and progesterone dose-dependently downregulated monocyte expression of COX2 mRNA. Pretreatment monocytes with ß-estradiol or progesterone reduced effects of Porphyromonas gingivalis lipopolysaccharide (LPS) on COX2 mRNA expression and decreased prostaglandin E2 secretion by the monocytes. Furthermore, we demonstrated that both ß-estradiol and progesterone inhibited P. gingivalis LPS-induced NF-κB signaling pathway through the upregulation of NF-κB inhibitor-alpha expression. However, neither ß-estradiol nor progesterone altered the phosphorylation of the p38, the extracellular signal-regulated kinase 1/2 and the c-Jun N-terminal activated kinase in P. gingivalis LPS-stimulated monocytes. Thus, the inhibitory effects of these hormones on the response of human monocytes to P. gingivalis LPS appear to be independent on mitogen-activated protein kinase signaling pathway. CONCLUSION: The results of the present study suggest that ß-estradiol and progesterone could influence the immune response of human monocytes to periodontal pathogens and this process may have a role in the clinical manifestations of periodontal disease associated with pregnancy.

Antitumor and immunomodulatory effects of recombinant fusion protein rMBP-NAP through TLR-2 dependent mechanism in tumor bearing mice.

The pro-inflammatory and immunomodulatory properties of Helicobacter pylori neutrophil activating protein (Hp-NAP) not only make it to play an important role in disease pathogenesis but also make it to be a potential candidate for therapeutic applications, including vaccine and drug development. Our previous work demonstrated that the recombinant Hp-NAP fused with the maltose binding protein of Escherichia coli (rMBP-NAP) play an important role in regulating the differentiation of Th1 cells. In this study, we investigated the ability of rMBP-NAP to induce antitumor immunity using two murine models of hepatoma H22 and sarcoma S180. Subcutaneous administration of mice with rMBP-NAP resulted in an about 40%-50% decrease of tumor growth compared with that of the control mice. Splenocytes from the tumor-bearing mice treated with rMBP-NAP showed a significant accumulation of CD4(+) IFN-γ-secreting cells, which is a cytokine profile of Th1 cells. Furthermore, intravenous injection of T2.5, toll like receptor (TLR) 2 blocking antibody, significantly recede the antitumor effect of rMBP-NAP and the production of IFN-γ induced by rMBP-NAP. Our findings indicate that potentiality of rMBP-NAP to be a candidate for the development of immunomodulatory antitumoral drugs.

CD14 as a Mediator of the Mineralocorticoid Receptor-Dependent Anti-apolipoprotein A-1 IgG Chronotropic Effect on Cardiomyocytes.

In vitro and animal studies point to autoantibodies against apolipoprotein A-1 (anti-apoA-1 IgG) as possible mediators of cardiovascular (CV) disease involving several mechanisms such as basal heart rate interference mediated by a mineralocorticoid receptor-dependent L-type calcium channel activation, and a direct pro-inflammatory effect through the engagement of the toll-like receptor (TLR) 2/CD14 complex. Nevertheless, the possible implication of these receptors in the pro-arrhythmogenic effect of anti-apoA-1 antibodies remains elusive. We aimed at determining whether CD14 and TLRs could mediate the anti-apoA-1 IgG chronotropic response in neonatal rat ventricular cardiomyocytes (NRVC). Blocking CD14 suppressed anti-apoA-1 IgG binding to NRVC and the related positive chronotropic response. Anti-apoA-1 IgG alone induced the formation of a TLR2/TLR4/CD14 complex, followed by the phosphorylation of Src, whereas aldosterone alone promoted the phosphorylation of Akt by phosphatidylinositol 3-kinase (PI3K), without affecting the chronotropic response. In the presence of both aldosterone and anti-apoA-1 IgG, the localization of TLR2/TLR4/CD14 was increased in membrane lipid rafts, followed by PI3K and Src activation, leading to an L-type calcium channel-dependent positive chronotropic response. Pharmacological inhibition of the Src pathway led to the decrease of L-type calcium channel activity and abrogated the NRVC chronotropic response. Activation of CD14 seems to be a key regulator of the mineralocorticoid receptor-dependent anti-apoA-1 IgG positive chronotropic effect on NRVCs, involving relocation of the CD14/TLR2/TLR4 complex into lipid rafts followed by PI3K and Src-dependent L-type calcium channel activation.

Protection of Gastrointestinal Mucosa from Acute Heavy Alcohol Consumption: The Effect of Berberine and Its Correlation with TLR2, 4/IL1ß-TNFα Signaling.

The purpose of the present study is to confirm the protective effect of berberine (BBR) on gastrointestinal injury caused by acute heavy alcohol exposure, an effect that has not been reported previously. Our research details how BBR protects against gastrointestinal injuries from acute alcohol exposure using both in vivo and in vitro experiments. Acute high alcohol concentrations lead to obvious damage to the gastrointestinal mucosa, resulting in necrosis of the intestinal mucosa. Oral administration of BBR was able to significantly reduce this alcohol-induced damage, inhibit increases of alcohol-induced TNFα and IL-1ß expression in gastrointestinal mucosa as well as their upstream signals TLR2 and TLR4, and regulate cytokines that modulate tight junctions. Alcohol consumption is a popular human social behavior worldwide, and the present study reports a comprehensive mechanism by which BBR protects against gastrointestinal injuries from alcohol stress, providing people with a novel application of BBR.

Lactobacillus sakei K040706 evokes immunostimulatory effects on macrophages through TLR 2-mediated activation.

Lactobacillus sakei K040706 is the most populous lactic acid bacteria (LAB) in over ripened Doenjang, a traditional Korean fermented soybean paste. In this study, we investigated the immunostimulating effects of L. sakei K040706 (K040706) in macrophages and in cyclophosphamide-induced immunosuppressed mice. Upon exposure to K040706, significant increases in phagocytic activity and in the productions of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were observed in rIFN-γ-primed RAW 264.7 macrophages. K040706 also increased the expressions of inducible nitric oxide synthase (iNOS) at the protein and promoter binding levels, and the expressions of iNOS, TNF-α, and IL-6, at the mRNA level. In addition, K040706 significantly increased the transcriptional activities and DNA binding of nuclear factor-κB (NF-κB), which was accompanied by parallel enhancement of the nuclear translocation of p65 via the phosphorylations inhibitory kappa B-α (IκB-α) and IκB-kinase (IKK). On the other hand, pretreatment with NF-κB inhibitors reduced K040706-induced NO production in IFN-γ-primed RAW 264.7 macrophages. Furthermore, K040706 induced-NO production was completely abolished by anti-Toll-like receptor 2 (TLR2) antibody. In our cyclophosphamide-induced immunosuppressed mouse model, administration of K040706 restored thymus and spleen indices. Taken together, our findings suggest that K040706 improves immune function by regulating immunological parameters, such as, the productions of NO, TNF-α, and IL-6 via NF-κB activation, and by activating TLR2 in rIFN-γ-primed macrophages. In parallel, K040706 restored immunological parameters in cyclophosphamide-induced immunosuppressed mice and may warrant further evaluations as potential immunomodulatory agent.