The association between TLR2/4 and clinical outcome in intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Toll-like receptors (TLRs) are involved in innate immunity and inflammatory responses in various diseases. Our study aimed to investigate the association between the levels of soluble TLR4 (sTLR4) and soluble TLR2 (sTLR2) and clinical outcomes following intracerebral hemorrhage (ICH). METHODS: Patients admitted to department of Neurology with acute ICH were included. Plasma levels of sTLR4 and sTLR2 after ICH were measured by enzyme-linked immunosorbent assay. Poor clinical outcome was defined as a modified Rankin score (mRS) of 3-6 at 3-month and 12-month after onset. RESULTS: All 207 patients with ICH and 100 non-stroke controls were included in our analysis. The mean sTLR4 level was 4.53±1.51 ng/ml and mean sTLR2 level was 3.65±0.72 ng/ml. There was significant trend towards worse clinical outcomes with increasing sTLR4 and sTLR2 terciles at 3 and 12 months. According to receiver operating curve (ROC), the sTLR4 was reliable predictor for poor clinical outcome at 3 months (ROC=0.75) and 12 months (ROC=0.74). The sTLR2 was less reliable predictor for poor clinical outcome at 3 months (ROC=0.64) and 12 months (ROC=0.65). The level of sTLR4 was an independent predictor of poor clinical outcome at 12-month (OR 1.24, 95 % CI 1.16-1.80; P=0.019). CONCLUSIONS: The sTLR4 quantification may provide accurate prognostic information after ICH.

Calprotectin Impairs Platelet Survival in Patients With Primary Antiphospholipid Syndrome.

OBJECTIVE: While thrombosis and pregnancy loss are the best-known clinical features of antiphospholipid syndrome (APS), many patients also exhibit "extra-criteria" manifestations, such as thrombocytopenia. The mechanisms that drive APS thrombocytopenia are not completely understood, and no clinical biomarkers are available for predicting antiphospholipid antibody (aPL)-mediated thrombocytopenia. Calprotectin is a heterodimer of S100A8 and S100A9 that is abundant in the neutrophil cytoplasm and released upon proinflammatory neutrophil activation. Here, we sought to evaluate the presence, clinical associations, and potential mechanistic roles of circulating calprotectin in a cohort of primary APS and aPL-positive patients. METHODS: Levels of circulating calprotectin were determined in plasma by the QUANTA Flash chemiluminescent assay. A viability dye-based platelet assay was used to assess the potential impact of calprotectin on aPL-mediated thrombocytopenia. RESULTS: Circulating calprotectin was measured in 112 patients with primary APS and 30 aPL-positive (without APS criteria manifestations or lupus) patients as compared to patients with lupus (without APS), patients with unprovoked venous thrombosis (without aPL), and healthy controls. Levels of calprotectin were higher in patients with primary APS and aPL-positive patients compared to healthy controls. After adjustment for age and sex, calprotectin level correlated positively with absolute neutrophil count (r = 0.41, P < 0.001), positively with C-reactive protein level (r = 0.34, P = 0.002), and negatively with platelet count (r = -0.24, P = 0.004). Mechanistically, we found that calprotectin provoked aPL-mediated thrombocytopenia by engaging platelet surface toll-like receptor 4 and activating the NLRP3-inflammasome, thereby reducing platelet viability in a caspase-1-dependent manner. CONCLUSION: These data suggest that calprotectin has the potential to be a functional biomarker and a new therapeutic target for APS thrombocytopenia.

Elevated expression of Toll-like receptor 4 and cytokines in both serum and myometrium at term may serve as promising biomarkers for uterine activation preceding labor.

Objective: Increased inflammation and cytokine levels are considered risk factors and promoters of preterm birth (PTB). However, the regulatory mechanism of pregnancy-related inflammation remains unclear. Toll-like receptor 4 (TLR4) plays a critical role in inflammatory responses in various diseases. Therefore, our study aimed to investigate whether TLR4 is involved in the inflammatory responses during uterine activation for labor, with the goal of identifying potential biomarkers for uterine activation at term. Materials and methods: We used flow cytometry to detect TLR4 expression on CD14+ maternal blood monocytes in the first, second, and third trimesters. ELISA was employed to measure TLR4 and cytokines levels in the maternal serum of term non-labor (TNL), term labor (TL) women and LPS induced preterm labor and PBS injected controls. TLR4siRNA was transfected into the human myometrial smooth muscle cells (HMSMCs), which were subsequently treated with IL-1ß. The mRNA and protein levels of TLR4, uterine contraction-related protein connexin 43 (CX43), oxytocin receptor (OTR), MAPK/NF-κB signaling pathway, and cytokines were analyzed using qRT-PCR, western blotting, and immunohistochemistry. Results: The study revealed TLR4 expression on CD14+ maternal blood monocytes was higher in the third trimester group compared to the first and second trimester groups (p<0.001). Maternal serum concentrations of TLR4 and cytokines were significantly higher in the TL group than the TNL group (p<0.001). TLR4, OTR, CX43, activated MAPK/NF-κB expression, and cytokines levels were upregulated in TL group, and similarly significantly higher in the LPS-induced preterm group than in the control group. Using the HMSMCs we demonstrated that TLR4siRNA transfection suppressed contractility. Interfering with TLR4 expression reduced the expression of OTR, CX43, cytokines, and MAPK/NF-κB activation. There was a significant positive relationship between TLR4 expression and the inflammatory status in the myometrium. ROC analysis indicated that TLR4 and cytokines may serve as potential biomarkers for predicting uterine activation for labor. Conclusion: Our data suggest that TLR4 and cytokines can act as stimulators of uterine activation for labor at term. Furthermore, the MAPK/NF-κB pathway appears to be one of the potential signaling pathways mediating TLR4's regulation of parturition initiation.

Correlation of HMGB1, TLR2 and TLR4 with left ventricular diastolic dysfunction in sepsis patients.

Left ventricular diastolic dysfunction (LVDD) is a common consequence of sepsis due to dysregulated inflammatory responses. Here we aim to investigate high mobility group box 1 (HMGB1), toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) as serum biomarkers to assess LVDD risk of patients with sepsis. We recruited 120 patients with sepsis, among which 52 had ultrasonically confirmed LVDD and 68 were without LVDD. Blood samples were collected, and enzyme-linked immunosorbent assay (ELISA) was used to analyse levels of HMGB1, TLR2 and TLR4 in serum. Multivariate analysis was performed to assess the odds ratio of the serum biomarkers. Spearman's correlation analysis was conducted to evaluate the correlation between the serum biomarkers to B-type natriuretic peptide (BNP) and cardiac troponin I (cTnl) levels and the ratios of early diastolic mitral inflow velocity to early diastolic mitral annulus velocity (E/e' ratios) in ultrasound. Receiver operating curve was used to measure the sensitivity and specificity of HMGB1, TLR2 and TLR4 individually and in combination as diagnostic markers. Elevated HMGB1, TLR2 and TLR4 had significant values in predicting LVDD suggested by high odds ratio (all P < .05). A significant correlation was found between these values and cTnl, the current gold standard for LVDD analysis. HMGB1, TLR2 and TLR4 also showed a high diagnostic sensitivity and specificity in ROC analysis. HMGB1, TLR2 and TLR4 are potentially valuable in predicting LVDD risk among patients with sepsis, providing additional tools with the capability of potentially assisting the clinical management of patients with sepsis.

The Effects of 16 Weeks of Exercise Training on Neutrophil Functions in Breast Cancer Survivors.

Following therapy, breast cancer survivors (BCS) have an increased risk of infections because of age and cancer dysregulation of inflammation and neutrophil functions. Neutrophil functions may be improved by exercise training, although limited data exist on exercise and neutrophil functions in BCS.Sixteen BCS [mean age: 56 (SD 11) years old] completed 16 weeks of community-based exercise training and a 45-minute acute bout of cycling before (Base) and after (Final) the exercise training program. Exercise training consisted of 3 x 40 - 60 minute mixed mode aerobic exercises, comprising 10 - 30 minutes aerobic and 30 minutes resistance training. At Base and Final, we took BCS blood samples before (PRE), immediately after (POST), and 1 hour after (1Hr) acute exercise to determine neutrophil counts, phenotype, bacterial killing, IL-6, and IL-8 levels. Eleven healthy, age- and physical activity levels-matched women (Control) completed the acute bout of exercise once as a healthy response reference. Resting Responses. BCS and Controls had similar Base PRE absolute neutrophil counts [mean (SD): 3.3 (1.9) v 3.1 (1.2) x 109/L, p=0.801], but BCS had lower bacterial phagocytosis [3991 (1233) v 4881 (417) MFI, p=0.035] and higher oxidative killing [6254 (1434) v 4709 (1220) MFI, p=0.005], lower CD16 [4159 (1785) v 7018 (1240) MFI, p<0.001], lower CXCR2 [4878 (1796) v 6330 (1299) MFI, p=0.032] and higher TLR2 [98 (32) v 72 (17) MFI, p=0.022] expression, while IL-6 [7.4 (5.4) v 4.0 (2.7) pg/mL, p=0.079] levels were marginally higher and IL-8 [6.0 (4.7) v 7.9 (5.0) pg/mL, p=0.316] levels similar. After 16 weeks of training, compared to Controls, BCS Final PRE phagocytosis [4510 (738) v 4881 (417) MFI, p=0.146] and TLR2 expression [114 (92) v 72 (17) MFI, p=0.148] were no longer different. Acute Exercise Responses. As compared to Controls, at Base, BCS phagocytic Pre-Post response was lower [mean difference, % (SD): 12% (26%), p=0.042], CD16 Pre-Post response was lower [12% (21%), p=0.016] while CD16 Pre-1Hr response was higher [13% (25%), p=0.022], TLR2 Pre-Post response was higher [15% (4%) p=0.002], while IL-8 Pre-Post response was higher [99% (48%), p=0.049]. As compared to Controls, following 16 weeks of training BCS phagocytic Pre-Post response [5% (5%), p=0.418], CD16 Pre-1Hr response [7% (7%), p=0.294], TLR2 Pre-Post response [6% (4%), p=0.092], and IL-8 Pre-Post response [1% (9%), p=0.087] were no longer different. Following cancer therapy, BCS may have impaired neutrophil functions in response to an acute bout of exercise that are partially restored by 16 weeks of exercise training. The improved phagocytosis of bacteria in BCS may represent an exercise-induced intrinsic improvement in neutrophil functions consistent with a reduced risk of infectious disease. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03760536.

Esculetin Alleviates Acute Liver Failure following Lipopolysaccharide/D-Galactosamine in Male C57BL/6 Mice.

Background: Acute liver failure (ALF) is a fatal clinical situation that rapidly leads to the loss of normal liver function. Esculetin is a natural herbal compound used for the management of various diseases such as cardiovascular and renal disorders. In this study, we evaluated the protective effects of esculetin in a mouse model of ALF. Methods: This article is a report on an experimental study that was conducted at Iran University of Medical Sciences in 2019. Forty-eight male C57BL/6 mice were randomly divided into control, LPS/D-Gal, and LPS/D-Gal+Esculetin (40 mg/kg) groups (n=16 per group). ALF was induced with an intraperitoneal injection of lipopolysaccharide (LPS)/D-galactosamine (D-Gal).The LPS/D-Gal group received a mixture of LPS (50 µg/kg) and D-Gal (400 mg/kg). The LPS/D-Gal+Esculetin group received esculetin by gavage 24 hours and one hour before receiving LPS/D-Gal. Six hours after LPS/D-Gal injection, the mice were sacrificed. Liver injury markers, including alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP), were measured in the serum. Oxidative stress indices and inflammatory markers such as interleukin-1 beta (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-α) were measured in hepatic tissue. The histopathology of liver tissue was also assessed. The data were analyzed using one-way ANOVA, followed by the post hoc Tukey test. Results: Esculetin lowered oxidative stress and myeloperoxidase activity (P<0.001); reduced the serum levels of ALT (P=0.037), AST (P=0.032), and ALP (P=0.004); and decreased the hepatic levels of IL-1ß (P=0.002), IL-6 (P=0.004), toll-like receptor 4 (P<0.001), TNF-α (P=0.003), and nuclear factor-kappa B (P<0.001) as compared with LPS/D-Gal. Additionally, esculetin ameliorated hepatic tissue injury following LPS/D-Gal challenge. Conclusion: Esculetin can reduce liver injury through the mitigation of oxidative burden, inflammation, and neutrophil infiltration and also exerts hepatoprotective effects against ALF.

The elevated expression of TLR4 and MMP9 in human abdominal aortic aneurysm tissues and its implication.

BACKGROUND: Toll-like receptor 4 (TLR4) and matrix metalloproteinase 9 (MMP9) have been investigated to play significant roles in the formation of abdominal aortic aneurysm (AAA). But the reports on the expression pattern of TLR4 and MMP9 in human AAA specimens were relatively scant. The aim of this study was to make a detailed analysis of TLR4 and MMP9 expression in situ and their association with clinical parameters involved in human AAA. METHODS: 40 AAA specimens were obtained from full-thickness aneurysmal tissues at the maximal dilation area during the open surgical repair, and 8 non-aneurysmal abdominal aortas from transplant donors served as controls. Expression of TLR4 and MMP9 protein was determined by immunohistochemistry. RESULTS: There were increased levels of TLR4 and MMP9 expression in human AAA tissues. Compared with macrophages or SMCs, lymphocytes showed a higher positive rate of TLR4 and MMP9 staining, and an elevated ratio of high MMP9 expression (all P < 0.05). There existed a significant association between TLR4 and MMP9 expression (r = 0.767, P < 0.001), and both TLR4 and MMP9 levels were statistically related to circulating CRP. Moreover, TLR4 expression in situ indicated a positive correlation with its serum level (r = 0.654, P = 0.006). Multiple analysis revealed that high TLR4 expression in situ was associated with the risk of large AAA (OR = 6.211, 95%CI = 1.226-31.480, P = 0.027), while high MMP9 expression was correlated to the presence of thrombus within AAA (OR = 5.494, 95%CI = 1.181-25.562, P = 0.030), separately compared with their low expression. CONCLUSIONS: This study confirmed the overexpression of TLR4 and MMP9 in human AAA tissues, and their close relationship implying in the pathogenesis of AAA. We further provided evidence that TLR4 had a potential effect on AAA size and MMP9 could influence the occurrence of thrombus within AAA.

The Inflammasome Signaling Pathway Is Actively Regulated and Related to Myocardial Damage in Coronary Thrombi from Patients with STEMI.

BACKGROUND: The Nod-Like-Receptor-Protein-3 (NLRP3) inflammasome and the Interleukin-6 (IL-6) pathways are central mechanisms of the inflammatory response in myocardial reperfusion injury. Expanding our knowledge about the inflammasome signaling axis is important to improve treatment options. In a cross-sectional study, we aimed to study presence, localization, and genetic expression of inflammasome- and IL-6- signaling-related proteins in coronary thrombi and circulating leukocytes from ST-elevation myocardial infarction (STEMI) patients, with relation to myocardial injury and time from symptoms to PCI. METHODS: Intracoronary thrombi were aspirated from 33 STEMI patients. Blood samples were drawn. mRNA of Toll-Like-Receptor-4 (TLR4), NLRP3, caspase 1, Interleukin-1ß (IL1-ß), Interleukin-18 (IL-18), IL-6, IL-6-receptor (IL-6R), and glycoprotein 130 (gp130) were isolated from thrombi and circulating leukocytes and relatively quantified by RT-PCR. A part of each thrombus was embedded in paraffin for histology and immunohistochemistry analyses. RESULTS: Genes encoding the 8 markers were present in 76-100% of thrombi. Expression of TLR4 in thrombi significantly correlated to troponin T (r = 0.455, p = 0.013), as did NLRP3 (r = 0.468, p = 0.024). Troponin T correlated with expression in circulating leukocytes of TLR4 (r = 0.438, p = 0.011), NLRP3 (r = 0.420, p = 0.0149), and IL-1ß (r = 0.394, p = 0.023). IL-6R expression in thrombi correlated significantly to troponin T (r = 0.434, p = 0.019), whereas gp130 was inversely correlated (r = -0.398, p = 0.050). IL-6 in circulating leukocytes correlated inversely to troponin T (r = -0.421, p = 0.015). There were no significant correlations between genes expressed in thrombi and time from symptom to PCI. CONCLUSIONS: The inflammasome signaling pathway was actively regulated in coronary thrombi and in circulating leukocytes from patients with STEMI, in association with myocardial damage measured by troponin T. This supports the strategy of medically targeting this pathway in treating myocardial infarction and contributes to sort out optimal timing and targets for anti-inflammatory treatment. The study is registered at clinicaltrials.gov with identification number NCT02746822.

Endotoxin Acts Synergistically With Clostridioides difficile Toxin B to Increase Interleukin 1ß Production: A Potential Role for the Intestinal Biome in Modifying the Severity of C. difficile Colitis.

BACKGROUND: Inflammation is a crucial driver of host damage in patients with Clostridioides difficile colitis. We examined the potential for the intestinal microbiome to modify inflammation in patients with C. difficile colitis via the effects of gut-derived endotoxin on cytokine production. METHODS: Endotoxin from Escherichia coli and Pseudomonas aeruginosa as well as stool-derived endotoxin were tested for their ability to enhance interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α) production by toxin B-stimulated peripheral blood mononuclear cells. Inflammasome and Toll-like receptor 4 (TLR4) blocking studies were done to discern the importance of these pathways, while metagenomic studies were done to characterize predominant organisms from stool samples. RESULTS: Endotoxin significantly enhanced the ability of C. difficile toxin B to promote IL-1ß production but not TNF-α. The magnitude of this effect varied by endotoxin type and was dependent on combined inflammasome and TLR4 activation. Stool-derived endotoxin exhibited a similar synergistic effect on IL-1ß production with less synergy observed for stools that contained a high proportion of γ-proteobacteria. CONCLUSIONS: The ability of endotoxin to enhance IL-1ß production highlights a manner by which the microbiome can modify inflammation and severity of C. difficile disease. This information may be useful in devising new therapies for severe C. difficile colitis.

Monocyte markers correlate with immune and neuronal brain changes in REM sleep behavior disorder.

Synucleinopathies are neurodegenerative diseases with both central and peripheral immune responses. However, whether the peripheral immune changes occur early in disease and their relation to brain events is yet unclear. Isolated rapid-eye-movement (REM) sleep behavior disorder (iRBD) can precede synucleinopathy-related parkinsonism and provides a prodromal phenotype to study early Parkinson's disease events. In this prospective case-control study, we describe monocytic markers in a cohort of iRBD patients that were associated with the brain-imaging markers of inflammation and neuronal dysfunction. Using 11C-PK11195 positron emission tomography (PET), we previously showed increased immune activation in the substantia nigra of iRBD patients, while 18F-DOPA PET detected reduced putaminal dopaminergic function. Here we describe that patients' blood monocytic cells showed increased expression of CD11b, while HLA-DR expression was decreased compared to healthy controls. The iRBD patients had increased classical monocytes and mature natural killer cells. Remarkably, the levels of expression of Toll-like receptor 4 (TLR4) on blood monocytes in iRBD patients were positively correlated with nigral immune activation measured by 11C-PK11195 PET and negatively correlated with putaminal 18F-DOPA uptake; the opposite was seen for the percentage of CD163+ myeloid cells. This suggesting a deleterious role for TLR4 and, conversely, a protective one for the CD163 expression. We show an association between peripheral blood monocytes and brain immune and dopaminergic changes in a synucleinopathy-related disorder, thus suggesting a cross-talk among periphery and brain during the disease.

Integrated traditional Chinese medicine alleviates sciatica while regulating gene expression in peripheral blood.

BACKGROUND: Although integrated traditional Chinese medicine (TCM) has long been indicated to be effective in the treatment of sciatica and is widely used in the management of this condition, the mechanism by which integrated TCM alleviates sciatica has not yet been fully defined, and the effect of integrated TCM on gene expression in the peripheral blood of patients with sciatica is still unknown. We performed this study to investigate the effect of integrated TCM on peripheral blood gene expression in patients with sciatica and to explore new clues for studying the mechanism of integrated TCM in alleviating sciatica. METHODS: We used a microarray to identify differentially expressed genes (DEGs) in the peripheral blood of patients with sciatica and healthy controls (DEGs-baseline), bioinformatic analysis to reveal the characteristics of DEGs-baseline, and the key genes that contribute to the gene dysregulation. A microarray was also used to identify DEGs in the peripheral blood of patients with sciatica after integrated TCM treatment compared with those at baseline, and the expression levels of DEGs were validated by qRT-PCR. RESULTS: We identified 153 DEGs-baseline, which included 131 upregulated genes and 22 downregulated genes. Bioinformatic analysis revealed that most of the DEGs-baseline were related to immunity and the inflammatory response and that TLR4, MMP9, MPO, CAMP, RETN, TLR5, and IL1RN were key genes involved in the dysregulation of genes in the peripheral blood of patients with sciatica. The expression levels of TLR5, IL1RN, SLC8A1, RBM20, GPER1, IL27, SOCS1, and GRTP1-AS1 were decreased in the peripheral blood of patients after integrated TCM treatment compared with that at baseline, which was accompanied by relief of pain. CONCLUSION: Integrated TCM treatment relieved pain while regulating the gene expression of TLR5, IL1RN, SLC8A1, RBM20, GPER1, IL27, SOCS1, and GRTP1-AS1 in the peripheral blood of patients with sciatica. Our study provides new clues for studying the mechanism of TCM in treating sciatica.

TLR4 rs1927914 polymorphism contributes to serum TLR4 levels in patients with aortic aneurysm.

Toll-like receptor 4 (TLR4) is a crucial regulator of inflammatory reactions and vascular remodeling. Elevated TLR4 expression has been proved to be correlated with an increased risk of aortic aneurysm (AA). This study aimed to explore the influence of TLR4 gene polymorphisms on TLR4 expression levels and its probable functional significance in AA disease. A total of 294 AA patients and 285 controls were enrolled in the study and serum TLR4 levels were detected by ELISA. All the participants were genotyped for two tag-SNPs in TLR4 (rs1927914 in the promoter region and rs11536889 in the 3'-untranslated region) using the KASP method. Relative luciferase activity was measured by the dual-luciferase reporter assay system. The rs1927914 TC, TC/CC genotypes and C allele showed associations with increased serum TLR4 levels in the total population and AA patients (all P<0.05). Further stratified analysis demonstrated that AA subjects with TC or TC/CC genotype of rs1927914 had significantly higher serum levels of TLR4 than those with TT genotype in male, age>60y, hypertension, diabetes, TAA type and size>5.0 cm subgroups (all P<0.05). In binary logistic analysis, rs1927914 TC genotype and dominant model presented significant associations with high TLR4 levels (OR = 1.579 and 1.431, P = 0.020 and 0.049, respectively) after adjusting age, hypertension and diabetes. However, rs11536889 polymorphism had no significant influence on serum TLR4 levels. Regarding rs1927914, luciferase activity of the C allele construct was significantly increased in comparison with the T allele construct (0.589 ± 0.004 vs. 0.340 ± 0.014, P<0.001). Our results provided evidence that rs1927914 polymorphism contributed to serum TLR4 levels, possibly by influencing promoter activity of TLR4, and could be a novel genetic factor in the formation of AA.

Changes in inflammatory factors in the Brown Norway rat model of food allergy.

BACKGROUND: The role of serum S100A8/A9 in intestinal inflammation has been confirmed, and its role in food allergy is currently being investigated. OBJECTIVE: To explore the levels of S100A8/A9 and inflammatory factors, including Toll-like receptors 4 (TLR4), Nuclear transcription factors (NF-κB) and Tumor necrosis factor α (TNF-α), in mild food allergies. METHODS: Eighty 3-week-old male Brown Norway rats were used. Forty rats were randomly assigned to the ovalbumin-sensitized experimental group, while 40 rats were assigned to the normal saline sham-sensitized control group. Body weight and length and the levels of serum ovalbumin-specific IgE (OVA-IgE), histamine, Th1-associated and Th2-associated factors, S100A8/A9 and inflammation-associated cytokines were compared. RESULTS: Through the evaluation of OVA-IgE level and Th1/Th2 balance in the experimental group, a successful IgE-mediated food allergy model was constructed. Compared with the control group, the experimental group had higher serum S100A8/A9 levels on days 21, 28, 35 and 42 (all P < 0.05); higher TLR4 levels on days 28, 35 and 42 (all P < 0.05); higher TNF-α levels on days 28, 35 and 42 (all P < 0.05); higher NF-κB levels on days 35 and 42 (all P < 0.05); and higher IL-1ß and IL-6 levels on days 7 to 42 (all P < 0.05). Moreover, positive correlations were found between the serum levels of S100A8/A9 and inflammation-associated cytokines [TNF-α: r = 0.378, P = 0.039; IL-1ß: r = 0.679, P = 0.000; IL-6: r = 0.590, P = 0.001]. CONCLUSION: S100A8/A9 and inflammatory-related factors, including TLR4, NF-κB, TNF-α, IL-6 and IL-1ß, is closely related to food allergies. Moreover, immune and inflammatory factors interact with each other in food allergies, which may provide insight into food allergy causes and treatments.

Lipidomic analysis of local inflammation models shows a specific systemic acute phase response to lipopolysaccharides.

Toll-like receptors (TLR) are crucial for recognizing bacterial, viral or fungal pathogens and to orchestrate the appropriate immune response. The widely expressed TLR2 and TLR4 differentially recognize various pathogens to initiate partly overlapping immune cascades. To better understand the physiological consequences of both immune responses, we performed comparative lipidomic analyses of local paw inflammation in mice induced by the TLR2 and TLR4 agonists, zymosan and lipopolysaccharide (LPS), respectively, which are commonly used in models for inflammation and inflammatory pain. Doses for both agonists were chosen to cause mechanical hypersensitivity with identical strength and duration. Lipidomic analysis showed 5 h after LPS or zymosan injection in both models an increase of ether-phosphatidylcholines (PC O) and their corresponding lyso species with additional lipids being increased only in response to LPS. However, zymosan induced stronger immune cell recruitment and edema formation as compared to LPS. Importantly, only in LPS-induced inflammation the lipid profile in the contralateral paw was altered. Fittingly, the plasma level of various cytokines and chemokines, including IL-1ß and IL-6, were significantly increased only in LPS-treated mice. Accordingly LPS induced distinct changes in the lipid profiles of ipsilateral and contralateral paws. Here, oxydized fatty acids, phosphatidylcholines and phosphatidylethanolamines were uniquely upregulated on the contralateral side. Thus, both models cause increased levels of PC O and lyso-PC O lipids at the site of inflammation pointing at a common role in inflammation. Also, LPS initiates systemic changes, which can be detected by changes in the lipid profiles.

Aberrant miR-219-5p is correlated with TLR4 and serves as a novel biomarker in patients with multiple organ dysfunction syndrome caused by acute paraquat poisoning.

This study aimed to investigate the clinical significance of serum microRNA-219-5p (miR-219-5p) in patients with multiple organ dysfunction syndrome (MODS) caused by acute paraquat (PQ) poisoning, and its correlation with Toll-like Receptor 4 (TLR4). Luciferase reporter assay was used to investigate in vitro the correlation of miR-219-5p with TLR4. Serum miR-219-5p levels were evaluated by quantitative real-time polymerase chain reaction. Serum levels of TLR4, IL-1ß, and TNF-α were measured by Enzyme-linked immune sorbent assay (ELISA). ROC analysis was performed to assess the diagnostic significance, Kaplan-Meier survival curves and Cox regression analysis were used to evaluate the prognostic value of miR-219-5p in MODS patients. TLR4 was a target gene of miR-219-5p and was increased in MODS patients. Serum miR-219-5p level was decreased and negatively correlated with TLR4 level in MODS patients (r = -0.660, P < 0.001), which had important diagnostic value and negatively correlated with APACHE II score in MODS patients. The miR-219-5p expression was markedly associated with the WBC, ALT, AST, PaCO2, Lac, and APACHE II score. Non-survivals had more patients with low miR-219-5p expression. Patients with low miR-219-5p expression had shorter survival time. MiR-219-5p and APACHE II score were two independently prognostic factors for 28-day survival. MiR-219-5p was negatively correlated with, while TLR4 was positively correlated with the levels of IL-1ß and TNF-α. The serum miR-219-5p level may be a potential biomarker for acute PQ-induced MODS diagnosis and prognosis. Furthermore, miR-219-5p may be associated with the progression of MODS by regulating TLR4-related inflammatory response.

Anti-inflammatory Activity of Mollugin on DSS-induced Colitis in Mice.

We aimed to explore the anti-inflammatory activity of mollugin extracted from Rubia cordifolia L, a traditional Chinese medicine, on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. Thirty C57BL/6 mice were divided into a control group (n=6), a model group (n=6), and three experimental groups (40, 20, 10 mg/kg of mollugin, n=6 each). DSS solution (3%) was given to mice in the model group and experimental groups from day 4 to day 10 to induce the mouse UC model. Mice in the experimental groups were intragastrically administrated mollugin from day 1 to day 10. Animals were orally given distilled water in the control group for the whole experiment time and in the model group from day 1 to day 3. The changes in colon pathology were detected by hematoxylin and eosin (HE) staining. Interleukin-1ß (IL-1ß) in the serum, and tumor necrosis factor-α (TNF-α) and interferon-γ (IFN) in the tissues were measured by enzyme linked immunosorbent assay. Expression levels of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 in the colon tissues were detected by immunohistochemistry. Results showed that mollugin could significantly reduce weight loss and the disease activity index in the DSS-induced UC mouse model. HE examinations demonstrated that mollugin treatment effectively improved the histological damage (P<0.05). The overproduction of IL-1ß and TNF-α was remarkably inhibited by mollugin treatment at doses of 20 and 40 mg/kg (P<0.05). Additionally, the levels of TLR4 in colon tissues were significantly reduced in mollugin-treated groups compared with the DSS group. Our findings demonstrated that mollugin ameliorates DSS-induced UC by inhibiting the production of pro-inflammatory chemocytokines.

TLR4/AP-1-Targeted Anti-Inflammatory Intervention Attenuates Insulin Sensitivity and Liver Steatosis.

Insulin resistance has been shown to be the common pathogenesis of many metabolic diseases. Metainflammation is one of the important characteristics of insulin resistance. Macrophage polarization mediates the production and development of metainflammation. Toll-like receptor 4 (TLR4) mediates macrophage activity and is probably the intersection of immunity and metabolism, but the detailed mechanism is probably not fully understood. Activated protein 1 (AP1) signaling pathway is very important in macrophage activation-mediated inflammation. However, it is unclear whether AP1 signaling pathway mediates metabolic inflammation in the liver. We aimed to investigate the effects of macrophage TLR4-AP1 signaling pathway on hepatocyte metabolic inflammation, insulin sensitivity, and lipid deposition, as well as to explore the potential of TLR4-AP1 as new intervention targets of insulin resistance and liver steatosis. TLR4 and AP1 were silenced in the RAW264.7 cells by lentiviral siRNA transfection. In vivo transduction of lentivirus was administered in mice fed with high-fat diet. Insulin sensitivity and inflammation were evaluated in the treated cells or animals. Our results indicated that TLR4/AP-1 siRNA transfection alleviated high-fat diet-induced systemic and hepatic inflammation, obesity, and insulin resistance in mice. Additionally, TLR4/AP-1 siRNA transfection mitigated palmitic acid- (PA-) induced inflammation in RAW264.7 cells and metabolic abnormalities in cocultured AML hepatocytes. Herein, we propose that TLR4-AP1 signaling pathway activation plays a crucial role in high fat- or PA-induced metabolic inflammation and insulin resistance in hepatocytes. Intervention of the TLR4 expression regulates macrophage polarization and metabolic inflammation and further alleviates insulin resistance and lipid deposition in hepatocytes.

Gestational diabetes mellitus in women increased the risk of neonatal infection via inflammation and autophagy in the placenta.

BACKGROUND: Gestational diabetes mellitus (GDM) produces numerous problems for maternal and fetal outcomes. However, the precise molecular mechanisms of GDM are not clear. METHODS: In our study, we randomly assigned 22 pregnant women with fasting glucose concentrations, 1 hour oral glucose tolerance test (1H-OGTT) and 2 hour oral glucose tolerance test (2H-OGTT), different than 28 normal pregnant women from a sample of 107 pregnant women at the First Affiliated Hospital of Jinan University in China. Lipopolysaccharide (LPS), interleukin 1 alpha (IL-1α), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) were measured from blood plasma of pregnant women and umbilical arteries using ultraviolet spectrophotometry. Hematoxylin & Eosin (H&E), Periodic acid-Schiff (PAS) or Masson staining were performed to examine whether diabetes mellitus altered the morphology of placenta. Quantitative PCR (Q-PCR), western blotting and immunofluorescent staining were performed to examine whether diabetes mellitus and autophagy altered the gene expressions of the placental tissue. RESULTS: We found that women with GDM exhibited increased placental weight and risk of neonatal infection. The concentrations of IL-6 protein and IL-8 protein in GDM were increased in both maternal and umbilical arterial blood. H&E, Masson and PAS staining results showed an increased number of placental villi and glycogen deposition in patients with GDM, but no placental sclerosis was found. Q-PCR results suggested that the expression levels of HIF-1α and the toll like receptor 4 (TLR4)/ myeloid differential protein-88 (MyD88)/ nuclear factor kappa-B (NF-κB) pathway were increased in the GDM placenta. Through Western Blotting, we found that the expression of NF-kappa-B inhibitor alpha (IKBα) and Nuclear factor-κB p65 (NF-κB p65) in GDM placenta was significantly enhanced. We also showed that the key autophagy-related genes, autophagy-related 7 (ATG7) and microtubule-associated protein 1A/1B-light chain 3 (LC3), were increased in GDM compared with normal pregnant women. CONCLUSIONS: Our results suggest that women with GDM exhibit an increased risk of neonatal infection via inflammation and autophagy in the placenta.

The Effect of TLR4 Blockade on Some Indicators of Systemic Inflammatory Response to Proteus mirabilis LPS in Rats.

The effects of TLR4 blocker on blood cell morphology, concentrations proinflammatory cytokines, and functional state of the liver and kidneys were studied in outbred male rats (n=60) after intravenous injection of 20 mg/kg LPS isolated from opportunistic Proteus mirabilis strain ATCC 51393. TLR4 blocker TLR4-IN-C34 was injected intravenously in a dose of 1 mg/kg/day over 3 days. Systemic inflammatory reaction induced by LPS was characterized by elevation of serum TNFα, IL-1ß, IL-6, erythrocyte sedimentation rate, leukocytosis, and thrombocytosis. Increased activity of hepatocyte enzymes (ALT, alkaline phosphatase, and lactate dehydrogenase), retention of nitrogen metabolites (urea and creatinine), elevated content of protein oxidation products, and enhanced protein catabolism were also observed. Administration of TLR4 blocker reduced parameters of inflammatory reaction and prevented the development of hypercatabolic syndrome; endotoxicosis and kidney function indicators approached the normal levels.

Phenotypical and functional abnormalities of circulating neutrophils in patients with ß-thalassemia.

ß-Thalassemia is an inherited single gene disorder related to reduced synthesis of the ß-globin chain of hemoglobin. Patients with ß-thalassemia present variable clinical severity ranging from asymptomatic trait to severe transfusion-dependent anemia and multiple organs complications. Moreover, multiple immune abnormalities are a major concern in ß-thalassemia patients. Aberrant neutrophil effector function plays a pivotal role in infection susceptibility in these patients. In severe and persistent inflammation, immature neutrophils are released from the bone marrow and are functionally different compared with mature ones. Despite some abnormalities reported for thalassemia patient's immune system, few data exist on the characterization of human neutrophils in ß-thalassemia. The aim of this study was to investigate the phenotype and function of circulating neutrophil subsets in patients with ß-thalassemia major and with ß-thalassemia intermedia divided in transfusion-dependent and non-transfusion-dependent. By the use of immunochemical and cytofluorimetric analyses, we observed that patients' CD16+ neutrophils exhibit abnormalities in their phenotype and functions and the abnormalities vary according to the clinical form of the disease and to the neutrophil subset (CD16bright and CD16dim). Abnormalities include altered surface expression of the innate immune receptor CD45, Toll-like receptor 4, and CD32, reduced ability to produce an oxidative burst, and elevated levels of membrane lipid peroxidation, especially in patients with a more severe form of the disease. Overall, our results indicating the occurrence of an immuno-senescent phenotype on circulating neutrophils from thalassemia patients suggest the usefulness of neutrophil feature assessment as a tool for better clinical management of ß-thalassemia.