Effect of Papaya Leaves (Carica papaya L.) Extract on Immune Response (TLR-7, TLR-9) and Inflammation (COX-2) in Rats Induces DMBA (7,12-Dimethylbenz[a]antrasen).

BACKGROUND AND OBJECTIVE: TLR is known to regulate the immune system in cancer. TLR-7 and TLR-9 can enhance the antitumor immune system in many types of solid tumors. Cyclooxygenase-2 (COX-2) is a biomarker of inflammation. This study aimed to investigate the effect of papaya leaves extract on immune response (TLR 7, TLR 9) and inflammation (COX-2) in rats induced DMBA. MATERIALS AND METHODS: This experimental study used Sprague dawley female rats of age more less 50 days. Rats were divided into 4 groups: Negative Control (NC), Positive Control (PC), Cancer Drug Doxorubicin (DOXO) and Papaya Leaves Extract (PLE). The study was conducted for 13 weeks. DMBA induction performed for 5 weeks with administration of 2 times per week. RESULTS: the expression of TLR-7 of PLE and DOXO was higher than PC groups significantly different (p<0.05). The expression of TLR-9 of PLE was higher than NC, PC and DOXO groups but not significantly different (p>0.05) while the expression of COX-2 of PLE and DOXO groups was lower than NC and PC groups but not significantly different (p>0.05). CONCLUSION: It can be concluded that papaya leaves extract can improve the immune system and reduce inflammation. It shows that papaya leaves extract has potent as anti-cancer.

Toll-like receptor 9 and the inflammatory response to surgical trauma and cardiopulmonary bypass.

OBJECTIVES: Cardiac surgery can lead to post-operative end-organ complications secondary to activation of systemic inflammatory response. We hypothesize that surgical trauma or cardiopulmonary bypass (CPB) may initiate systemic inflammatory response via release of mitochondrial DNA (mtDNA) signaling Toll-like receptor 9 (TLR9) and interleukin-6 production (IL-6). MATERIALS AND METHODS: The role of TLR9 in systemic inflammatory response in cardiac surgery was studied using a murine model of sternotomy and a porcine model of sternotomy and CPB. mtDNA and IL-6 were measured with and without TLR9-antagonist treatment. To study ischemia-reperfusion injury, we utilized an ex-vivo porcine kidney model. RESULTS: In the rodent model (n = 15), circulating mtDNA increased 19-fold (19.29 ± 3.31, p < 0.001) and plasma IL-6 levels increased 59-fold (59.06 ± 14.98) at 1-min post-sternotomy compared to pre-sternotomy. In the murine model (n = 11), administration of TLR-9 antagonists lowered IL-6 expression post-sternotomy when compared to controls (59.06 ± 14.98 vs. 5.25 ± 1.08) indicating that TLR-9 is a positive regulator of IL-6 after sternotomy. Using porcine models (n = 10), a significant increase in circulating mtDNA was observed after CPB (Fold change 29.9 ± 4.8, p = 0.005) and along with IL-6 following renal ischaemia-reperfusion. Addition of the antioxidant sulforaphane reduced circulating mtDNA when compared to controls (FC 7.36 ± 0.61 vs. 32.0 ± 4.17 at 60 min post-CPB). CONCLUSION: CPB, surgical trauma and ischemic perfusion injury trigger the release of circulating mtDNA that activates TLR-9, in turn stimulating a release of IL-6. Therefore, TLR-9 antagonists may attenuate this response and may provide a future therapeutic target whereby the systemic inflammatory response to cardiac surgery may be manipulated to improve clinical outcomes.

Toll-like receptor 9 negatively related to clinical outcome of AML patients.

BACKGROUND: Acute myeloid leukemia (AML) can modulate toll-like receptor-9 (TLR9) expression and activation. This study was conducted to elucidate the expression of TLR9 in AML patients and its relation to the prognosis of the disease. RESULTS: The study included 40 newly diagnosed AML patients managed in the hospital in addition to 20 sex and age matched normal volunteers as control. TLR9 expression assay was conducted on peripheral blood samples of AML cases before the start of treatment as well as the controls by immunophenotyping. TLR9 expression was ranging from 0.10 to 2.40% in AML patients with higher expression among the control, ranging from 0.94 to 8.25%. The median TLR9 expression in AML patients was significantly lower with advanced cytogenetic risk score. It is not significantly differing in relation to patients' sex, age group, and FAB type of AML. However, significant lower median expression was found in relation to clinical outcome. TLR9 expression ≤ 1% showed lower median overall survival time when compared to those with > 1% expression. CONCLUSION: This study concluded that AML patients express TLR9 in leukemic cells with very low percentage. This expression was negatively related to the clinical outcome.

Toll-like receptor 2 and 9 expression on circulating neutrophils is associated with increased mortality in critically ill patients.

BACKGROUND: Toll-like receptors (TLRs) play an important role in inflammatory processes in critically ill patients by binding to pathogen-associated molecular patterns and danger-associated molecular patterns (DAMPs). Whether neutrophil or monocyte TLR expression patterns are associated with outcome in critical illness is unknown. OBJECTIVES: To answer this question, we conducted a prospective, observational study including 215 consecutive patients admitted to a medical ICU at a tertiary care center. METHODS: Blood was drawn at admission and expression of TLR-2, TLR-4, and TLR-9 on neutrophils and monocytes were analyzed by flow cytometry. RESULTS: Median Acute Physiology and Chronic Health Evaluation II (APACHE II) score was 19, and 30-day mortality was 26%. TLR-2 expression on neutrophils was associated with APACHE II, Simplified Acute Physiology Score II, and Sepsis-related Organ Failure Assessment score. TLR-2 (P < 0.001) and TLR-9 (P < 0.05) expression on neutrophils was significantly higher in nonsurvivors. In contrast, neutrophil TLR-4 expression and monocyte TLR expression were not associated with survival. Neutrophil TLR-2 (odds ratio 3.8; 95% confidence interval 1.4-10.2; P < 0.05) and TLR-9 (odds ratio 4.0; 95% confidence interval 2.0-8.1; P < 0.001) expression in the third tertile predicted mortality independent from APACHE II, serum lactate, serum creatinine, and procalcitonin, respectively. CONCLUSION: We provide evidence for prognostic properties of neutrophil TLR-2 and TLR-9 expression regarding 30-day mortality in unselected critically ill patients, independent from baseline clinical characteristics, and laboratory values. These findings suggest that specific TLR-dependent activation of the innate immune system via neutrophils possibly caused by cell damage and release of otherwise intracellular components may play a significant role in the pathophysiology of critical illness.

Mitochondrial­DNA­associated TLR9 signalling is a potential serological biomarker for non­small cell lung cancer.

Mitochondrial (mt) DNA has been long suggested to contribute to carcinogenesis, and a variety of mutations in mtDNA have been confirmed to be related to various early stages of cancers; these data revealed that the detection of mtDNA in clinical samples may be a promising approach for cancer diagnosis. In the present study, the serum mtDNA in healthy donors and groups of patients with cancer was detected. It was revealed that patients with lung cancer without metastasis had more mtDNA in serum compared to patients with metastasis. Moreover, TLR9­associated signalling was activated in vitro after treatment with a synthetic CpG oligodeoxyribonucleotide (ODN) called ODN­M362. In addition, our data revealed that TLR9 and its adaptor protein, MyD88, were induced by ODN­M362 in a dose­dependent manner. A human cytokine array to evaluate stimulation of cytokine secretion by ODN­M362 was also used. Our findings may identify the role that TLR9 and mtDNA play in lung cancer progression and metastasis.

Impact of ketamine intervention for acute lung injury on RAGE and TLR9.

OBJECTIVE: The purpose of this study was to explore the benefits of ketamine intervention for acute lung injury (ALI) and its effects on the receptor for advanced glycation end-product (RAGE) and toll-like receptor 9 (TLR9). MATERIALS AND METHODS: Lipopolysaccharide (LPS, 3 mg/kg) was used to induce ALI rat model. Forty healthy Sprague-Dawley rats (6-8 weeks) were assigned into control, model, low ketamine (5 mg/kg), and high ketamine (50 mg/kg) groups. After 24 h, these rats were sacrificed and lungs were collected. RESULTS: The pathological score, lung W/D ratio, the percentage of leukocytes and epithelial in bronchoalveolar lavage fluids (BALF), the expression levels of RAGE, TLR9, and other inflammation markers in serum and lungs were significantly higher in the Model group, indicating a good ALI model. Ketamine intervention restored all these parameters, with more benefits in the High dose group. CONCLUSIONS: The high dose ketamine decreased the degree of ALI by inhibiting the expression of RAGE, TLR9, TNF-α, NF-κB, IL-6 and MPO in tissues.

Higher levels of free plasma mitochondrial DNA are associated with the onset of chronic GvHD.

Toll-like receptor-9 (TLR9) responsive B cells have previously been associated with the onset of extensive chronic graft-versus-host disease (cGvHD). We hypothesized that the onset of cGvHD associated with a higher level of plasma-free mitochondrial DNA (mtDNA), a putative TLR9 agonist. Plasma cell-free mtDNA levels were measured in 39 adult patients post-HSCT with and without cGvHD. mtDNA was isolated from plasma and quantified by Q-PCR amplification. We correlated B cell responsiveness to CpG-DNA, a prototypical TLR9 agonist, and previously identified cGVHD biomarkers with mtDNA levels. Free plasma mtDNA were elevated in patients post-HSCT without cGvHD compared to normal non-HSCT adults. There was a significantly higher level of free plasma mtDNA associated with the onset of cGvHD (3080 ± 1586 versus 1834 ± 1435 copies/µL; p = 0.02) compared to 6 months post-HSCT controls. Free mtDNA levels post-HSCT correlated with B cell responsiveness to CpG-DNA and known cGvHD biomarkers: CXCL10 (p = 0.003), ICAM-1 (p = 0.007), CXCL9 (p = 0.03), sCD25 (p = 0.05) and sBAFF (p = 0.05), and percentage of CD21low B cells. Plasma levels of free mtDNA are increased in cGvHD and may represent an endogenous inflammatory stimulus for TLR9 expressing B cells.

Red Blood Cells Homeostatically Bind Mitochondrial DNA through TLR9 to Maintain Quiescence and to Prevent Lung Injury.

RATIONALE: Potentially hazardous CpG-containing cell-free mitochondrial DNA (cf-mtDNA) is routinely released into the circulation and is associated with morbidity and mortality in critically ill patients. How the body avoids inappropriate innate immune activation by cf-mtDNA remains unknown. Because red blood cells (RBCs) modulate innate immune responses by scavenging chemokines, we hypothesized that RBCs may attenuate CpG-induced lung inflammation through direct scavenging of CpG-containing DNA. OBJECTIVES: To determine the mechanisms of CpG-DNA binding to RBCs and the effects of RBC-mediated DNA scavenging on lung inflammation. METHODS: mtDNA on murine RBCs was measured under basal conditions and after systemic inflammation. mtDNA content on human RBCs from healthy control subjects and trauma patients was measured. Toll-like receptor 9 (TLR9) expression on RBCs and TLR9-dependent binding of CpG-DNA to RBCs were determined. A murine model of RBC transfusion after CpG-DNA-induced lung injury was used to investigate the role of RBC-mediated DNA scavenging in mitigating lung injury in vivo. MEASUREMENTS AND MAIN RESULTS: Under basal conditions, RBCs bind CpG-DNA. The plasma-to-RBC mtDNA ratio is low in naive mice and in healthy volunteers but increases after systemic inflammation, demonstrating that the majority of cf-mtDNA is RBC-bound under homeostatic conditions and that the unbound fraction increases during inflammation. RBCs express TLR9 and bind CpG-DNA through TLR9. Loss of TLR9-dependent RBC-mediated CpG-DNA scavenging increased lung injury in vivo. CONCLUSIONS: RBCs homeostatically bind mtDNA, and RBC-mediated DNA scavenging is essential in mitigating lung injury after CpG-DNA. Our data suggest a role for RBCs in regulating lung inflammation during disease states where cf-mtDNA is elevated, such as sepsis and trauma.

cfDNA correlates with endothelial damage after cardiac surgery with prolonged cardiopulmonary bypass and amplifies NETosis in an intracellular TLR9-independent manner.

Cardiopulmonary bypass (CPB) provokes inflammation culminating in organ dysfunction and increased mortality. Recently, neutrophil extracellular traps (NETs) have been found to be involved in a variety of cardiovascular diseases promoting tissue and organ injury. Here, we aimed to elaborate the proinflammatory potential of circulating cell-free (cf)DNA in patients undergoing cardiac surgery with CPB. Plasma was collected pre- and postoperatively as well as at d1, d3, d5 and d8 after surgery. At d1, we found circulating cfDNA levels to be significantly increased in patients with prolonged CPB duration (>100 min) when compared to those with shorter CPB times (CPB < 100 min). Increased CPB duration yielded in higher levels of circulating mitochondrial (mt)DNA, soluble thrombomodulin (sCD141) and ICAM-1, reflecting endothelial damage. Positive correlation between cfDNA and sCD141 was demonstrated at all time points. Plasma and cfDNA from patients with CPB > 100 min induced NETs release by neutrophils from healthy donors which was not suppressed by inhibitors of intracellular toll-like receptor (TLR)9. DNA binding to neutrophils' surface (s)TLR9 has been evidenced. Altogether, we demonstrate that elevated plasma cfDNA might be useful to assess CPB-mediated detrimental effects, including endothelial damage, in cardiac surgical patients with prolonged CPB duration. cfDNA-triggered NETosis is independent of classical TLR9 signaling.

TLR9 gene polymorphism -1486T/C (rs187084) is associated with uterine cervical neoplasm in Mexican female population.

PURPOSE: The aim of this work was to evaluate the association of single nucleotide polymorphisms in TLR9 (-1486 T/C [rs187084], -1237T/C [rs5743836] and G2848A [rs352140]) with HPV infection, squamous intraepithelial lesions, and uterine cervical neoplasm in a Mexican population. Additionally, the peripheral expression of TLR9 was evaluated to evaluate the differences in the TLR9 expression associated with every genotype in the locus -1486 of the TLR9 gene. The serum concentration of TLR9 was evaluated in a randomly selected subsample. METHODS: Genotyping was performed using predesigned 5' endonuc lease assays and the association of the polymorphisms with the diagnosis groups were assessed by performing multinomial regression models. The relative expression of TLR9 in peripheral blood mononuclear cells was evaluated by real-time polymerase chain reaction and the association of the level of TLR9 expression with the diagnosis was evaluated by performing multinomial regression models. The serum concentration of TLR9 was evaluated in a subsample of patients diagnosed with uterine cervical neoplasm by ELISA. RESULTS: The results showed that genotype TT in the -1486 locus of TLR9 was significantly associated with HPV infection (OR = 3.25, 95% CI 1.12-9.46), squamous intraepithelial cervical lesion (OR = 3.76, 95% CI 1.36-10.41), and uterine cervical neoplasm (OR = 5.30, 95% CI 1.81-15.55). Moreover, the highest level of TLR9 expression was significantly associated with a greater risk for developing squamous intraepithelial cervical lesion and uterine cervical neoplasm. The serum TLR9 concentration was higher in patients with uterine cervical cancer than in controls. CONCLUSION: Our findings indicate that genotype TT in the -1486 locus of the TLR9 gene could comprise a risk genotype for HPV infection, squamous intraepithelial cervical lesion, and uterine cervical neoplasm in Mexican female population. Further studies with larger samples are needed to evaluate if the peripheral expression of TLR9 could be used as a biomarker of uterine cervical neoplasm progression.

Excessive activation of the TLR9/TGF-ß1/PDGF-B pathway in the peripheral blood of patients with systemic lupus erythematosus.

BACKGROUND: Our aim is to study the existence of the TLR9/TGF-ß1/PDGF-B pathway in healthy humans and patients with systemic lupus erythematosus (SLE), and to explore its possible involvement in the pathogenesis of lupus nephritis (LN). METHODS: Protein levels of the cytokines were detected by ELISA. mRNA levels of the cytokines were analyzed by real-time PCR. MTT assay was used to test the proliferation of mesangial cells under different treatments. RESULTS: Compared to healthy controls (N Control = 56), levels of Toll-like receptor (TLR)9, transforming growth factor (TGF)-ß1, and platelet-derived growth factor B (PDGF-B) were increased significantly in the peripheral blood of SLE patients (N SLE = 112). Significant correlations between the levels of TLR9, TGF-ß1, and PDGF-B were observed in both healthy controls and SLE patients. The levels of TGF-ß1 and PDGF-B were greatly enhanced by TLR9 activation in primary cell cultures. The proliferation of mesangial cells induced by the plasma of SLE patients was significantly higher than that induced by healthy controls; PDGF-B was involved in this process. The protein levels of PDGF-B homodimer correlated with the levels of urine protein in SLE patients with LN (N LN =38). CONCLUSIONS: The TLR9/TGF-ß1/PDGF-B pathway exists in humans and can be excessively activated in SLE patients. High levels of PDGF-B may result in overproliferation of mesangial cells in the kidney that are involved in the development of glomerulonephritis and LN. Further studies are necessary to identify TLR9, TGF-ß1, and PDGF-B as new therapeutic targets to prevent the development of glomerulonephritis and LN.

Low expression of Toll-like receptors in peripheral blood mononuclear cells of pediatric patients with acute lymphoblastic leukemia.

Cancer is the second most common cause of death among children aged 1-14 years. Leukemia accounts for one-third of all childhood cancers, 78% of which is acute lymphoblastic leukemia (ALL). The development of cancer has been associated with malignant cells that express low levels of immunogenic molecules, which facilitates their escape from the antineoplastic immune response. It is thought that it may be possible to rescue the antineoplastic immune response through the activation of recognition receptors, such as Toll-like receptors (TLRs), which activate the innate immune system. TLRs are type I membrane glycoproteins expressed mainly in immune system cells such as monocytes, neutrophils, macrophages, dendritic cells, T, B and natural killer cells. The aim of the present study was to evaluate the expression of TLR1, TLR3, TLR4, TLR7 and TLR9 in peripheral blood mononuclear cells (PBMCs) in patients with ALL and prior to any treatment. PBMCs were obtained from 50 pediatric patients diagnosed with ALL and from 20 children attending the ophthalmology and orthopedics services. The mean fluorescence intensity was obtained by analysis of immunofluorescence. We found lower expression levels of TLR1, TLR3, TLR4, TLR7 and TLR9 in PBMCs from patients with ALL compared with those from control patients. We also observed that the PBMCs from patients with Pre-B and B ALL had lower TLR4 expression than controls and patients with Pro-B, Pre-B, B and T ALL had lower TLR7 expression than controls. The present study is the first to demonstrate reduced expression of TLRs in PBMCs from pediatric patients with ALL. This finding is of great relevance and may partly explain the reduction in the antineoplastic immune response in patients with ALL.

[TLR9 expression is positively correlated with the levels of CD38, HLA-DR and CD95 on peripheral blood mononuclear cells in chronic HBV infected patients].

OBJECTIVE: To explore the relationship between the expression of TLR9 and the levels of CD38, HLA-DR and CD95 on peripheral blood mononuclear cells (PBMCs) of chronic hepatitis B virus (HBV) infected patients. METHODS: 70 chronic HBV infected patients and 12 healthy donors were enrolled in this study, and density gradient centrifugation was used to isolate PBMCs from peripheral blood with EDTA for anticoagulation. Flow cytometry was used to detect the levels of TLR9, CD38, HLA-DR and CD95 on PBMCs. RESULTS: Compared to the healthy donors, chronic HBV infected patients with low viral load or high viral load had significantly higher levels of TLR9, HLA-DR and CD95 on PMBCs. Furthermore, the co-expression rates of TLR9 and CD38, HLA-DR, CD95 on PBMCs were obviously higher than those of the healthy donors. Correlation analysis showed that the expression of TLR9 was positively correlated with CD38 (r=0.345), HLA-DR (r=0.334), CD95 (r=0.227) on PBMCs in the patients with chronic HBV infection. CONCLUSION: The expression of TLR9 increased and was positively associated with CD38, HLA-DR and CD95 on PBMCs during chronic HBV infection.

Leishmania donovani influenced cytokines and Toll-like receptors expression among Sudanese visceral leishmaniasis patients.

Leishmaniasis remains a serious health problem. The outcome of Leishmania infection depends on the early innate response. In this study, whole blood samples of 40 patients with visceral leishmaniasis (VL), 10 leishmanin skin test-negative (LST-ve) controls and 10 leishmanin skin test-positive (LST+ve) controls were stimulated by live L. donovani promastigotes. Also, THP1 human cell line was infected with L. donovani. The production of interleukin 10 (IL-10), tumour necrosis factor alpha (TNF) and interferon gamma (IFNG) cytokines was measured, and the expression of Toll-like receptors (TLR2, TLR4 and TLR9) was done in the blood samples and also in the THP1 cell line. IL-10 was found to be higher in LST+ve controls compared with VL patients. TNF was moderately produced with no variation between patients, controls and THP1 cells. IFNG was higher in LST+ve controls also in THP1 cells. TLR4 and TLR9 were found to be highly expressed in patients with VL. L. donovani increases the expression of TLR4 and TLR9 in patients with VL and TLR2 in THP1 cells, suggesting a TLRs relation in induction of a mixed cytokine response. TLR9 was markedly recognized by L. donovani DNA.

The expression of TLR-9, CD86, and CD95 phenotypes in circulating B cells of patients with chronic viral hepatitis B or C before and after antiviral therapy.

AIMS: This study aimed to assess the differential expression of specific B cell subtypes in patients with chronic viral hepatitis. METHODS: The frequencies of differential expression of specific B cell subtypes in patients with chronic viral hepatitis and healthy controls were assessed by flow cytometry using monoclonal antibodies specific for CD38, CD27, CD86, CD95, TLR-9, and IgD. The effect of adefovir treatment on B cell subsets in HBV patients was determined. The values of clinical parameters in the patients were also measured. RESULTS: The frequency of CD86+ B cells was not significantly different in chronic HBV patients but was higher in HCV patients compared with that in healthy controls. CD95 and IgD levels were lower in HBV and HCV patients than in healthy controls. A significant negative correlation occurred between the proportion of CD95+ B cells and HBV DNA viral load. The frequency of TLR-9 on the B cells in HBV and HCV patients was higher compared with that of healthy controls. After treatment with adefovir, the frequency of CD95 and IgD expressed on B cells was increased in HBV patients. CONCLUSIONS: Activated B cells and exhausted B cells homeostasis were commonly disturbed in HBV and HCV patients.

Correlation of serum toll like receptor 9 and trace elements with lipid peroxidation in the patients of breast diseases.

Toll-like receptors are recognized as redox sensitive receptor proteins and have been implicated in cellular response to oxidative stress. Altered pro-oxidant-antioxidant balance leads to an increased oxidative damage and consequently play an important role in breast diseases. The study was designed to access the oxidative stress status by quantification of byproducts generated during lipid peroxidation and inadequate trace elements during oxidative damage and its effects on the toll like receptor (TLR) activity in patients of breast diseases. Decreased levels of selenium, copper, zinc, magnesium and iron with elevated levels of malondialdehyde (marker of lipid peroxidation) were accompanied by decreased TLR activity in patients of benign breast diseases as well as breast carcinoma. A similar pattern was observed with the advancement of disease and its subsequent progression in breast carcinoma patients. Results of multinomial regression analysis suggest benign breast disease patients are at higher risk of developing breast cancer with high odds ratio of lipid damage.

Expression of serum toll-like receptor 9 and oxidative damage markers in benign and malignant breast diseases.

The intracellular redox environment plays an important role in the maintenance of proper cellular homeostasis and functions. Disturbances in redox equilibrium of cells result in pro-inflammatory conditions, and these inflammatory conditions can induce carcinogenesis or increase the malignant potential of the tumor. Oxidative stress or tissue damage can trigger toll-like receptor (TLR) family of receptors that are involved in altering the innate immune system. The present study was aimed at evaluating the level of oxidative damage markers in breast diseases by measuring the 8-hydroxydeoxyguanosine (8-OHdG), protein carbonyl (PC), malondialdehyde (MDA), and total antioxidant status (TAS) alterations in relation to expression of TLR-9. A significant increase in the level of oxidative damage markers was observed in breast carcinoma patients in comparison to benign and normal controls, which was accompanied by a significant decrease in TAS and expression of TLR-9 concentrations. 8-OHdG, PC, and MDA were negatively correlated with expression of TLR-9 and TAS levels. Altered levels of biomarkers of oxidative stress and TLR-9 among the malignant, benign, and controls suggest a correlation of oxidative stress and TLR signaling in the progression of disease in breast carcinoma patients. Receiver operating characteristic analysis depicts that expression of TLR-9 is a good indicator for distinguishing cancer patients from benign and normal controls. High accuracy, specificity, and sensitivity of oxidative stress markers and expression of TLR-9 can be used as discriminatory marker/s for efficient diagnosis.

Evaluation of Toll-like, chemokine, and integrin receptors on monocytes and neutrophils from peripheral blood of septic patients and their correlation with clinical outcomes

Recognition of pathogens is performed by specific receptors in cells of the innate immune system, which may undergo modulation during the continuum of clinical manifestations of sepsis. Monocytes and neutrophils play a key role in host defense by sensing and destroying microorganisms. This study aimed to evaluate the expression of CD14 receptors on monocytes; CD66b and CXCR2 receptors on neutrophils; and TLR2, TLR4, TLR5, TLR9, and CD11b receptors on both cell types of septic patients. Seventy-seven septic patients (SP) and 40 healthy volunteers (HV) were included in the study, and blood samples were collected on day zero (D0) and after 7 days of therapy (D7). Evaluation of the cellular receptors was carried out by flow cytometry. Expression of CD14 on monocytes and of CD11b and CXCR2 on neutrophils from SP was lower than that from HV. Conversely, expression of TLR5 on monocytes and neutrophils was higher in SP compared with HV. Expression of TLR2 on the surface of neutrophils and that of TLR5 on monocytes and neutrophils of SP was lower at D7 than at D0. In addition, SP who survived showed reduced expression of TLR2 and TLR4 on the surface of neutrophils at D7 compared to D0. Expression of CXCR2 for surviving patients was higher at follow-up compared to baseline. We conclude that expression of recognition and cell signaling receptors is differentially regulated between SP and HV depending on the receptor being evaluated.

Immunoregulatory profile of monocytes from cutaneous leishmaniasis patients and association with lesion size.

Leishmaniasis is an important tropical disease composed of several clinical forms that adversely affect millions of people globally. Critical cells involved in the host-Leishmania interaction are monocytes and macrophages, which act to protect against infections due to their ability to both control intracellular infections and regulate the subsequent adaptive immune response. Both soluble factors and cell surface receptors are keys in directing the immune response following interaction with pathogens such as Leishmania. Toll-like receptors (TLRs) have an essential role in immune responses against infections, but little is known about their role in human infection with Leishmania braziliensis. In this work, we evaluated peripheral blood CD14+ monocytes for the expression of immunoregulatory cytokines, co-stimulatory molecules and TLR9 from cutaneous leishmaniasis patients infected with L. braziliensis and noninfected individuals. Our results showed that patients present decreased expression of co-stimulatory molecules such as CD80 and CD86 following culture with media alone or after stimulus with soluble Leishmania antigen. Interestingly, TLR9 expression was higher after culture with soluble Leishmania antigen (SLA), suggesting a role of this molecule in immunoregulation of active disease. Lastly, higher frequencies of TLR9+ monocytes were correlated with greater lesion size. These findings demonstrate a peripheral monocytes profile compatible with important immunoregulatory potential.

Toll-like receptor-9 polymorphisms in sarcoidosis and chronic obstructive pulmonary disease.

The etiology of inflammatory diseases of the lung like sarcoidosis and chronic obstructive pulmonary disease (COPD) is multifactorial. The main trigger for developing a COPD is tobacco smoking while exogenous factors causing sarcoidosis are unclear. In both diseases there is an underlying genetic susceptibility determining both the onset and the course of the diseases. Toll-like receptor (TLR)-9 plays an important role in innate immunity by recognizing bacterial CpG-DNA motifs. It is unclear whether single nucleotide polymorphisms (SNPs) in TLR-9 are able to alter the course of sarcoidosis or COPD, or raise the susceptibility for developing one of the disorders. We examined two SNPs in the promoter region of the TLR-9 gene (T1486C and T1237C) in 175 COPD patients (59% with a stable course of the disease, 41% with an instable course with more than 3 exacerbations over the last 3 years) and 166 sarcoidosis patients (19% with an acute and 81% with a chronic course of the disease lasting >2 years) comparing each group to 233 healthy controls. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis was used for genotyping. The C-allele frequency of T1486C was significantly elevated in COPD patients (p = 0.008). For T1237C there were no significant associations comparing the COPD cohort with the controls. In the sarcoidosis cohort, we could observe a significantly higher prevalence of the C-allele for T1237C in the chronic sarcoidosis cohort in comparison to the control group (p = 0.026). For T1486 there no statistical association was observed. This is the first study showing an association between a SNP (T1486C) in the TLR-9 gene and the onset of COPD. Moreover, we could demonstrate that T1237C is able to alter the course of sarcoidosis as a disease-modifying gene. This study underlines that SNPs in TLR-9 might be involved in acquiring and maintaining lung diseases such as sarcoidosis and COPD.