IRAK1 mRNA Research Articles

Upregulated IRAK1 and IRAK4 promoting the production of IFN-γ and IL-17 in Behcet's disease.

To investigate the expression and function of IRAK1 and IRAK4 involved in the development of Behcet's disease. Twenty-eight Behcet's patients and thirty-two normal subjects were involved in this study. The mRNA levels of IRAK1 and IRAK4 from active Behcet's patients, inactive Behcet's patients and normal controls were detected using real-time quantitative PCR. CD4+T cells were extracted from peripheral blood mononuclear cells of active Behcet's patients and normal controls. After coculturing IRAK1/4 inhibitor with CD4+T cells in the presence of rIL-18 protein or rIL-1β protein for 3days, the proliferation of CD4+T cells was measured using a modified MTT assay. Meanwhile, the levels of IFN-γ and IL-17 were detected by enzyme-linked immunosorbent assay. The mRNA levels of IRAK1 and IRAK4 were both significantly increased in active Behcet's patients compared with those of inactive Behcet's patients and normal subjects. However, there was no difference of IRAK1 mRNA level or the IRAK4 mRNA level between the inactive Behcet's patients and normal controls. After coculturing with IRAK1/4 inhibitor, the proliferation of the CD4+T cells was inhibited both in active Behcet's patients and in normal controls. Meanwhile, the expression of IFN-γ and IL-17 was also suppressed by IRAK1/4 inhibitor both in active Behcet's patients and in normal subjects. The high mRNA levels of IRAK1 and IRAK4 were correlated with the development of Behcet's disease, which suggested that IRAK1 and IRAK4 might participate in the pathogenesis of Behcet's disease. The inhibitory function of IRAK1/4 inhibitor prompts that it may be a new therapeutic target for treating this blindness disease.

Forsythoside A Controls Influenza A Virus Infection and Improves the Prognosis by Inhibiting Virus Replication in Mice

Objective: The objective of this study was to observe the effects of forsythoside A on controlling influenza A virus (IAV) infection and improving the prognosis of IAV infection. Methods: Forty-eight SPF C57BL/6j mice were randomly divided into the following four groups: Group A: normal control group (normal con); Group B: IAV control group (V con); Group C: IAV+ oseltamivir treatment group (V oseltamivir; 0.78 mg/mL, 0.2 mL/mouse/day); Group D: IAV+ forsythoside A treatment group (V FTA; 2 μg/mL, 0.2 mL/mouse/day). Real-time fluorescence quantitative PCR (RT-qPCR) was used to measure mRNA expression of the TLR7, MyD88, TRAF6, IRAK4 and NF-κB p65 mRNA in TLR7 signaling pathway and the virus replication level in lung. Western blot was used to measure TLR7, MyD88 and NF-κB p65 protein. Flow cytometry was used to detect the proportion of the T cell subsets Th1/Th2 and Th17/Treg. Results: The body weight began to decrease after IAV infection, while FTA and oseltamivir could reduce the rate of body weight loss. The pathological damages in the FTA and oseltamivir group were less serious. TLR7, MyD88, TRAF6, IRAK4 and NF-κB p65 mRNA were up-regulated after virus infection (p < 0.01) while down-regulated after oseltamivir and FTA treatment (p < 0.01). The results of TLR7, MyD88 and NF-κB p65 protein consisted with correlative mRNA. Flow cytometry showed the Th1/Th2 differentiated towards Th2, and the Th17/Treg cells differentiated towards Treg after FTA treatment. Conclusions: Our study suggests forsythoside A can control influenza A virus infection and improve the prognosis of IAV infection by inhibiting influenza A virus replication.

Tracheal overexpression of IL-1β, IRAK-1 and TRAF-6 mRNA in obese-asthmatic male Wistar rats.

Human and animal studies have shown a close relationship between obesity and asthma severity. Here, we examined the effects of diet-induced obesity (DIO) on the expression levels of IL-1β, IRAK-1 and TRAF-6 mRNA in male Wistar rats tracheal after sensitization with ovalbumin (OVA). Twenty male Wistar rats divided to four groups, included, control group with normal diet (C+ND), OVA-sensitized group with normal diet (S+ND), control group with high-fat diet (C+HFD), and OVA-sensitized group with high-fat diet (S+HFD). All animals fed for 8 weeks with standard pelts or high-fat diet, and then were sensitized and challenged with OVA or saline for another 4 weeks with designed regimens. At the end of study, trachea isolated and examined for expression levels of IL-1β, IRAK-1 and TRAF-6 mRNA with RT-PCR method. Diet-induced obesity groups developed increased weight, obesity indexes and lipid profiles (P<0.05 to P<0.001). The expression levels of IL-1β mRNA in OVA-sensitization groups (S+ND and S+HFD) showed a significantly increased when compared with control group. Also in S+HFD group, expression level of TRAF-6 mRNA was higher than other groups (P<0.001). IRAK-1 expression level was high in S+HFD compared with control group.IL-1β and TRAF-6 mRNA correlated positively with obesity indexes. The results showed that DIO causes overexpression of IL-1β, IRAK-1 and TRAF-6 mRNA in an experimental model of asthma. Our results suggested that in obese-asthmatic conditions locally production and activation of pro-inflammatory agents can be increased. These findings showed that possible mechanism for obesity-asthma relationships.

Increased expression of the interleukin-1 receptor-associated kinase (IRAK)-1 is associated with adipose tissue inflammatory state in obesity.

BackgroundThe emerging role of TLR2/4 as immuno-metabolic receptors points to key involvement of TLR/IL-1R/MyD88 pathway in obesity/type-2 diabetes (T2D). IL1R-associated kinase (IRAK)-1 is a critical adapter protein (serine/threonine kinase) of this signaling pathway. The changes in adipose tissue expression of IRAK-1 in obesity/T2D remain unclear. We determined modulations in IRAK-1 gene/protein expression in the subcutaneous adipose tissues from lean, overweight and obese individuals with or without T2D.MethodsA total of 49 non-diabetic (22 obese, 19 overweight and 8 lean) and 42 T2D (31 obese, 9 overweight and 2 lean) adipose tissue samples were obtained by abdominal subcutaneous fat pad biopsy and IRAK-1 expression was determined using real-time RT-PCR, immunohistochemistry, and confocal microscopy. IRAK-1 mRNA expression was compared with adipose tissue proinflammatory mediators (TNF-α, IL-6, IL-18), macrophage markers (CD68, CD11c, CD163), and plasma markers (CCL-5, C-reactive protein, adiponectin, and triglycerides). The data were analyzed using t test, Pearson’s correlation, and multiple stepwise linear regression test.ResultsIn non-diabetics, IRAK-1 gene expression was elevated in obese (P = 0.01) and overweight (P = 0.04) as compared with lean individuals and this increase correlated with body mass index (r = 0.45; P = 0.001) and fat percentage (r = 0.36; P = 0.01). In diabetics, IRAK-1 mRNA expression was also higher in obese as compared with lean subjects (P = 0.012). As also shown by immunohistochemistry/confocal microscopy in non-diabetics and by immunohistochemistry in diabetics, IRAK-1 protein expression was higher in obese than overweight and lean adipose tissues. IRAK-1 gene expression correlated positively/significantly with mRNAs of TNF-α (r = 0.46; P = 0.0008), IL-6 (r = 0.30; P = 0.03) and IL-18 (r = 0.31; P = 0.028) in non-diabetics; and only with TNF-α (r = 0.32; P = 0.03) in diabetics. IRAK-1 expression also correlated positively/significantly with CD68 (r = 0.32; P = 0.02), CD11c (r = 0.30; P = 0.03), and CD163 (r = 0.43; P = 0.001) in non-diabetics; and only with CD163 (r = 0.34; P = 0.02) in diabetics. IRAK-1 mRNA levels also correlated with plasma markers including CCL-5 (r = 0.39; P = 0.02), C-reactive protein (r = 0.48; P = 0.005), adiponectin (r = −0.36; P = 0.04), and triglycerides (r = 0.40; P = 0.02) in non-diabetics; and only with triglycerides (r = −0.36; P = 0.04) in diabetics. IRAK-1 expression related with TLR2 (r = 0.39; P = 0.007) and MyD88 (r = 0.36; P = 0.01) in non-diabetics; and MyD88 (r = 0.52; P = 0.0003) in diabetics.ConclusionsThe elevated IRAK-1 expression in obese adipose tissue showed consensus with local/circulatory inflammatory signatures and represented as a tissue marker for metabolic inflammation. The data have clinical significance as interventions causing IRAK-1 suppression may alleviate meta-inflammation in obesity/T2D.Electronic supplementary materialThe online version of this article (doi:10.1186/s13098-015-0067-7) contains supplementary material, which is available to authorized users.

Altered immunometabolism at the interface of increased endoplasmic reticulum (ER) stress in patients with type 2 diabetes.

The mechanism of perturbed immune function in patients with T2DM is poorly understood. Recent studies imply a role for ER stress in linking immune-system alterations and metabolism. Here, we investigated whether ER stress markers and its downstream effector signals are altered in patients with type 2 diabetes along with proinflammatory augmentation. In our study, gene and protein expression of ER stress markers (GRP-78, PERK, IRE1α, ATF6, XBP-1 and CHOP) was elevated significantly (P < 0.05) in PBMCs from T2DM patients compared with control subjects. The mRNA expression of both the proinflammatory cytokines (TNF-α and IL-6) and oxidative stress markers (p22(phox), TXNIP, and TRPC-6; P < 0.05) was also increased in PBMCs from patients with T2DM. SOCS3 mRNA expression was reduced significantly (P < 0.05) in diabetes patients. mRNA expression of most of the ER stress markers from PBMCs correlated significantly and positively with poor glycemic control, dyslipidemia, IR, and inflammatory and oxidative stress markers. Chronic ER stress in PBMCs from patients with T2DM was evident from the increased caspase-3 activity (P < 0.01), which is an executioner of apoptosis. Along with an impairment of miR-146a levels, the downstream targets of miR-146a, viz., IRAK1 and TRAF6 mRNA levels, were also elevated significantly (P < 0.01) in patients with T2DM. There was an inverse relationship among miR-146a levels and ER stress markers, inflammatory markers, and glycemic control. We demonstrate evidence of increased ER stress markers with impaired miR-146a levels and increased proinflammatory signals in patients with type 2 diabetes.

Effects of microRNA-155 on interleukin-1 receptor-associated kinase 1 and 4 mRNA expression in liver injury of endotoximia mice

Objective To explore the protective effect of microRNA (miRNA)-155 inhibitor on interleukin-1 receptor-associated kinase (IRAK)-1 mRNA and IRAK-4 mRNA in endotoximia induced liver injury in mice. Methods One hundred and twenty male BALB/c mice were randomly divided into healthy control group (n=40), endotoximia group (n=40) and miRNA-155 inhibitor group (n=40). Each group were divided into 6 h, 12 h, 24 h, 48 h subgroups, each of which consisted of 10 mice. The mice in miRNA-155 inhibitor group were administered with miRNA-155 inhibitor[80 mg (kg·d)] via tail vein injection before lipopolysaccharide (LPS) administration while the other two groups treated with normal saline, following 24 hours, model of endotoximia mice was produced by injection of LPS intraperitoneally. At 6 h, 12 h, 24 h, 48 h after LPS exposure, the experimental mice were sacrificed and the liver tissue samples were collected. Histopathological changes, the expression of miRNA-155, IRAK-1 mRNA, IRAK-4 mRNA, tumor necrosis factor (TNF)-α, IL-1, IL-10 were detected. Results LPS exposure resulted in increase of miRNA-155, IRAK-1 mRNA, IRAK-4 mRNA, TNF-α, IL-1 and IL-10 in both endotoximia group and miRNA-155 inhibitor group compared to the control group. miRNA-155 inhibitor resulted in decrease of miRNA-155, IRAK-1 mRNA, IRAK-4 mRNA, TNF-α, IL-1 and IL-10 in miRNA-155 inhibitor group compared to the endotoximia group. There were significant differences of miRNA-155 expression at 12 h, 24 h, 48 h after LPS exposure among 3 groups (P<0.05). Both IRAK-1 mRNA and IRAK-4 mRNA showed significant differences at 12 h, 24 h, 48 h. Turning to inflammation factors, differences were found among 3 groups at all time points (P<0.05). At light-scope, there was improvement in sepsis associated liver injury in miRNA-155 inhibitor group compared to endotoximia group. Conclusion miRNA-155 inhibitor administration appears to down regulate IRAK-1 mRNA and IRAK-4 mRNA expression and further deduce the excessive inflammatory and anti-inflammatory reaction, which may alleviate liver injury in endotoximia mice. Key words: MicroRNA-155; Interleukin-1 receptor-associated kinase mRNA; Interleukin-4 receptor-associated kinase mRNA; Liver injury; Endotoxin; RNA interference; Mice

Inhibition of IRAK1/4 sensitizes T cell acute lymphoblastic leukemia to chemotherapies.

Signaling via the MyD88/IRAK pathway in T cells is indispensable for cell survival; however, it is not known whether this pathway functions in the progression of T acute lymphoblastic leukemia (T-ALL). Here, we determined that compared with thymic and peripheral T cells, T-ALL cells from patients have elevated levels of IRAK1 and IRAK4 mRNA as well as increased total and phosphorylated protein. Targeted inhibition of IRAK1 and IRAK4, either with shRNA or with a pharmacological IRAK1/4 inhibitor, dramatically impeded proliferation of T-ALL cells isolated from patients and T-ALL cells in a murine leukemia model; however, IRAK1/4 inhibition had little effect on cell death. We screened several hundred FDA-approved compounds and identified a set of drugs that had enhanced cytotoxic activity when combined with IRAK inhibition. Administration of an IRAK1/4 inhibitor or IRAK knockdown in combination with either ABT-737 or vincristine markedly reduced leukemia burden in mice and prolonged survival. IRAK1/4 signaling activated the E3 ubiquitin ligase TRAF6, increasing K63-linked ubiquitination and enhancing stability of the antiapoptotic protein MCL1; therefore, IRAK inhibition reduced MCL1 stability and sensitized T-ALL to combination therapy. These studies demonstrate that IRAK1/4 signaling promotes T-ALL progression through stabilization of MCL1 and suggest that impeding this pathway has potential as a therapeutic strategy to enhance chemotherapeutic efficacy.

Herb-Partitioned Moxibustion Regulates the TLR2/NF-κB Signaling Pathway in a Rat Model of Ulcerative Colitis.

The TLR2/NF-κB signaling pathway plays an important role in the pathomechanism of ulcerative colitis (UC); acupuncture and moxibustion can improve the damage in colonic tissues of UC, but the regulatory mechanism remains unknown. This study observed the effect of moxibustion on the TLR2/NF-κB signaling pathway at the Tianshu (ST25) and Qihai (CV6) acupuncture points in the UC rat. The result shows that TLR2, IRAK1, and IKK-b mRNA and protein levels in the colonic mucosa were significantly higher in the UC rats than in the control rats. Herb-partitioned moxibustion reduced the expression of TLR2, IRAK1, and IKK-b mRNA and proteins in the UC rats. Similarly, the expression of NF-κB was significantly increased and IFN-β and IL-10 were significantly decreased in the colonic mucosa of UC rats, but herb-partitioned moxibustion reduced the expression of IFN-β and upregulating the expression of IFN-β and IL-10 significantly. It indicates that herb-partitioned moxibustion can inhibit the expression of multiple signaling molecules of the TLR2 pathway effectively, and it may modulate the excessive local immune response by inhibiting TLR2 signaling, thereby promoting the repair of damaged colonic mucosa.

Mir-146a and Mir-146b Regulate Human Dendritic Cell Apoptosis and Cytokine Production By Targeting of TRAF6 and IRAK1

MicroRNA (miRNA) regulation of dendritic cell (DC) development and function is not fully understood. We have previously reported 27 differentially expressed miRNAs during human monocyte differentiation into immature DCs (imDCs) and mature DCs (mDCs). Here, we aimed at uncovering the functional role of miR-146a and miR-146b (miR-146a/b) during this differentiation process.To investigate miR-146a and miR-146b expression during human monocyte differentiation into imDCs and mDCs, monocytes were differentiated into imDCs with GM-CSF and IL-4 and matured with IL-1β, IL-6, TNF-α, and PGE2. We found by qRT-PCR that expression of miR-146a/b was dramatically increased upon monocyte differentiation into imDCs (miR-146a, 10-fold; miR-146b, 37-fold at day6; n=4; p value of Student t test<0.05) and mDCs (miR-146a, 51-fold; miR-146b, 79-fold; n=4; p<0.005). Up-regulation of miR-146a/b in mDCs was predominantly mediated by IL-1β but not IL-6, TNF-a, or PGE2 (n=3; p<0.01).DC apoptosis is important for self-tolerance and immunity. We then evaluated the effect of altering miR-146a/b expression levels on DC apoptosis by Annexin V/PI staining. Silencing of miR-146a, miR-146b or both in imDCs (Fig. 1A) and mDCs significantly prevented DC from apoptosis (Fig. 1A; miR-146a, 24%±3.1; miR-146b, 26%±1.7; miR-146a/b; 23%±2.8 vs control, 46%±2.3 of Annexin V+ populations; n=8; p<0.005) whereas overexpression of miR-146a, miR-146b or both in imDCs (Fig. 1B) and mDCs significantly increased the proportion of apoptotic cells (Fig. 1B; 46%±1.9, 47%±2.1, 45%±2.3 vs 36%±3.3; n=6; p<0.05). These results indicate that miR-146a/b may function as pro-apoptotic regulators during human monocyte differentiation into imDCs and mDCs.It is known that the NF-κB pathway regulates DC development, function and survival, and that TRAF6 and IRAK1 are major signal transducers in the NF-κB pathway. In addition, both TRAF6 and IRAK1 are known target genes of miR-146a. Indeed, miR-146a/b expression in imDCs and mDCs was inversely correlated with TRAF6 and IRAK1 mRNA and protein expression (n=4; p<0.05). Furthermore, siRNA silencing of TRAF6 and/or IRAK1 in imDCs and mDCs significantly enhanced DC apoptosis (n=4; p<0.05). By contrast, lentivirus overexpression of TRAF6 and/or IRAK1 promoted DC survival compared to control lentivirus transduced cells (n=2; p<0.05). To confirm that miR-146a/b-induced human DC apoptosis is involved in suppression of the NF-κB pathway, at least in part through down regulation of the NF-κB signaling transducers TRAF6 and IRAK1, we examined the protein level of IκB as a negative regulator of NF-κB and Bcl-2 as a known downstream anti-apoptotic molecule of the NF-κB pathway. Silencing of miR-146a/b in imDCs and mDCs significantly decreased IκBα and increased Bcl-2 expression whereas overexpression of miR-146a and/or miR-146b or silencing of TRAF6 and/or IRAK1 significantly increased IκBα and decreased Bcl-2 expression in imDCs and mDCs (n=4; p<0.01). These results indicate that miR-146a/b modulate DC apoptosis through inhibition of NF-κB activation via targeting TRAF6 and IRAK1.Next, we investigated whether miR-146a/b regulates pro-inflammatory cytokine production in DCs. We found that IL-12p70, IL-6 and TNF-α production were significantly enhanced after miR-146 and/or miR-146b silencing during DC maturation (n≥2; p<0.05), although altering miR-146a/b expression had little effect on DC maturation (n=6). By contrast, IL-12p70, IL-6 and TNF-α production was highly reduced after miR-146a and/or miR-146b overexpression (n≥2; p<0.05).In conclusion, we have demonstrated three important findings in this report. First, expression of both miR-146a and miR-146b is up-regulated during human monocyte differentiation into imDCs and mDCs. Secondly, although miR-146a and miR-146b do not appear to play a role in DC maturation, they may be critical regulators of DC apoptosis and cytokine production. Thirdly, mechanistically, miR-146a/b targets TRAF6 and IRAK1, leading to inhibition of NF-κB and reduced expression of Bcl-2. We thus demonstrate for the first time that miR-146a/b regulates human DC apoptosis and cytokine production, uncovering a new negative feedback mechanism for miR-146 in controlling overstimulation of the immune responses (Fig. 2). [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Role of Interleukin-1 Receptor-Associated Kinases in Vogt-Koyanagi-Harada Disease

PurposeTo explore whether IRAK1 and IRAK4 are involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease.MethodsThirty-nine VKH patients and thirty-two healthy controls were included in this study. The mRNA levels of IRAK1 and IRAK4 from active VKH patients, inactive VKH patients, and normal controls in peripheral blood mononuclear cells (PBMCs) were detected using real-time quantitative PCR. CD4+T cells were purified from PBMCs obtained from active VKH patients and normal controls. The effect of IRAK1/4 inhibition on CD4+T cell proliferation following stimulation with IL-18 or IL-1β was measured using a modified MTT assay. CD4+T cell expression of IFN-γ and IL-17 were detected by flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA). The effect of IRAK1/4 inhibition on NF-κB, STAT1, and STAT3 activation was detected by FCM.ResultsThe mRNA levels of IRAK1 and IRAK4 were both significantly increased in active VKH patients compared to inactive VKH patients and healthy controls. No difference in the IRAK1 or IRAK4 mRNA level could be detected between inactive patients and healthy controls. After incubation with IRAK1/4 inhibitor, the proliferation of CD4+T cells was inhibited both in the active VKH patients and in the healthy controls. IRAK1/4 inhibition was also associated with a decreased expression of IFN-γ and IL-17. Phosphorylation of NF-κB, STAT1, and STAT3 in CD4+T from healthy controls was significantly decreased after inhibition of IRAK1/4.ConclusionsHigh mRNA levels of IRAK1 and IRAK4 correlated with VKH disease activity. IRAK1 and IRAK4 play a role in the activation and proliferation of CD4+T cells and the higher expression observed in VKH may contribute to the pathogenesis of this blinding condition.

Distinct IRAK4 Expression in Normal and B-CLL Lymphocytes

Toll-like receptors (TLR) signaling plays an important role in the B cell biology. It induces the maturation, proliferation and antibody production in response to pathogen recognition. Interleukin-1 Receptor-associated Kinase-4 (IRAK-4) is TLR downstream molecule which is of strategic importance in starting a cascade of NF-kappaB (Nuclear factor-kappaB) and MAPK (Mitogen-activated protein kinase) intracellular signaling pathways. The aim of the study was to identify the differences in IRAK-4 expression in normal and chronic lymphocytic leukemia (CLL) lymphocytes. We have also studied the IRAK-4 expression alteration upon TLR2/4 ligand stimulation in leukemic and normal B cells. We carried out Real-Time PCR and Western blot analysis of IRAK4 mRNA (RQ) and protein expression. The study were performed on the isolated CD19+ normal B lymphocytes and CLL cells (CD5+CD19+) before and 30 min after (Lipopolysaccharide) LPS and (Staphylococcus aureus strain Cowan I) SAC stimulation. IRAK-4 mRNA expression was significantly higher in normal CD19+ lymphocytes as compared to leukemic CD5+CD19+ subpopulation (RQ median 0.90 vs 0.54; p=0.012). In normal lymphocytes, LPS and SAC treatment led to significant IRAK-4 down-regulation both at mRNA (RQ median 0.90 vs 0.53 vs 0.40; p=0.03, p=0.01) and protein level, whereas generally there were no significant changes in IRAK-4 expression neither on mRNA nor protein level. No trend or significant differences in IRAK-4 expression in leukemic lymphocytes after LPS and SAC stimulation may imply the presence of alleged defect of immune tolerance in CLL.

Study on effect and mechanism of volatile oil of Schizonepetae Herba and its essential components against influenza virus

To observe the effect of volatile oil of Schizonepetae Herba (VOSH), and its essential components-menthone and pulegone against anti-influenza virus A/PR/8/34 (H1N1) in vivo and in vitro, as well as the signaling mechanism of its toll-like receptor/interferon (TLR/IFN). The lung-adapted PR-8 virus model was prepared in mice. They were administered with preventive and therapeutic drugs, and the hemagglutination titer of model animals was determined to evaluate in vivo effect against H1N1. ELISA test was conducted to observe the effect on IFN-alpha, IFN-beta, IL-2, IL-6 and TNF-alpha in serum, as well as IFN-beta secretion in H1N1 infected MDCK supernatant. Real-time RT-PCR was employed to observe the expression levels of IRAK4 and TLR3 mRNA. The in vivo experiment shows that the hemagglutination titer was significantly decreased when the mice were treated with VOSH (0.266 mg x kg(-1)), menthone(0.5 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) in therapeutic way; VOSH (0.226 mg x kg(-1)) had a significant effect on increasing serum levels of IFN-alpha, IL-2; Methone (0.5 mg x kg(-1)) had a significant effect on increasing serum levels of IFN-beta; Methone (0.5 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) had a significant effect on decreasing serum levels of IL-6; VOSH (0.452, 0.226 mg x kg(-1)) and pulegone (0.19 mg x kg(-1)) had a significant effect on decreasing serum levels TNF-alpha. The in vitro experiment showed that the expression levels of IRAK4 mRNA and IFN-beta were significantly increased in VOHS (0.1 g x L(-1)) and pulegone (0.1 g x L(-1)) groups; and the menthone (0.25 g x L(-1)) group showed a significant rise in the expression levels of IRAK4 mRNA, but a notable decline in TLR3 mRNA. The administration with VOSH, methone and pulegone in therapeutic way can significantly decrease the hemagglutination titer, which demonstrates the anti-virus effect of the administration in therapeutic way, but no notable efficacy of the administration in preventive way. The in vivo anti-virus mechanism is related to regulation of IFN-alpha, IFN-beta and IL-2.

Alveolar macrophage phagocytic activity is enhanced with LPS priming, and combined stimulation of LPS and lipoteichoic acid synergistically induce pro-inflammatory cytokines in pigs

The objective of the present study was to investigate LPS and lipoteichoic acid (LTA)-induced TLRs, associated signaling molecules and inflammatory mediators, as well as to compare their combined effect in porcine alveolar macrophages. Macrophages were incubated for 24 h with various concentrations of LPS, LTA, LPS + LTA or control. Multiple concentrations of LPS elicited marked up-regulation in mRNA for TLR2 and TLR4, CD14, MD2, MyD88, IRAK-4 and TRAF6 compared with the control. LTA had no effect on TLR4 and MD2; only higher doses up-regulated TLR2, CD14, MyD88, IRAK-4 and TRAF6 mRNA. LPS-activated cells released IL1-β, IL12-β, TNF-α, IL-6, IL-8, IFN-γ and IL-10 in a dose-dependent manner, while LTA had no effect on IL-1β, IL-6 and IFN-γ. Higher doses of LTA induced IL-12β, TNF-α, IL-8 and IL-10. Combined stimulation augmented TLR2, CD14 and MyD88 mRNA, and subsequently produced elevated levels of IL-6, TNF-α and IL-8 when compared with LPS and LTA alone. Additionally, phagocytosis of macrophages was significantly increased following low concentration of LPS treatment. Only low levels of NO (nitric oxide) were detected in the LPS group. Overall, compared with LPS, LTA was a relatively weak inducer, and co-stimulation accelerated gene and cytokine production associated with pulmonary innate immune function.

IRAK1, a potential therapeutic target for rheumatoid arthritis?

Rheumatoid arthritis (RA) is a chronic inflammatory disorder with hypertrophy, hyperplasia and angiogenesis of synovial tissue, which contribute to inflammatory joint destruction [1]. Although the exact causes of RA remain unknown, immunological dysregulation by inflammatory cytokines has been shown to be involved in driving the inflammation and synovial cell proliferation that characterize joint destruction in RA patients. Recently, Zhang et al. [2] found that IRAK1 rs3027898 C/A polymorphism was associated with risk of RA in a Chinese population. When the IRAK1 rs3027898 CC homozygote genotype was used as the reference group, the AA genotype was associated with significantly increased risk of RA. A significantly increased risk of RA associated with the IRAK1 rs3027898 AA genotype was more evident among anti-CCP positive patients compared with the IRAK1 rs3027898 CC/CA genotypes. Similarly, in a study in Greek, strong statistically significant difference was observed in IRAK1 rs3027898 A [ C polymorphism distribution between RA patients and controls, which was higher comparing the distribution of allele A versus allele C between the studied groups [3]. IRAK1 is a serine/threonine protein kinase implicated in the signaling cascade of the Toll/interleukin-1 receptor (TIR) family [4]. On Toll-like receptor (TLR) stimulation, IRAK1 is recruited and phosphorylated into the TLR signaling complex. Moreover, activation of MyD88 leads to phosphorylation of the IRAK1 in RA synovial fibroblasts (RASFs) [5]. After simulation with a TLR-2 ligand, PGN, increased mRNA expressions of IRAK1 and MyD88 were observed in RA fibroblast-like synoviocytes (RA-FLS). When treated with an anti-TLR-2 neutralizing antibody, IRAK1 and MyD88 mRNAs returned to baseline levels [6]. Repression of IRAK1 in THP-1 cells could also result in up to an 86 % reduction in tumor necrosis factor-a (TNF-a) production in RA patients [7]. Indeed, TNF-a was found to be associated with susceptibility to RA and with the presence of erosion in RA patients [8]. In addition, siRNA knockdown in monocytes reduced IRAK1 expression by *75 %. The siRNA-mediated reduction of IRAK1 impaired the ability of monocytes to produce TNF-a in response to IL-1b. Concomitantly, siRNA targeting of IRAK1 reduced IRAK1 mRNA levels by *65 % in Th17 cells, cytokine production of Th17 cells was even more profoundly affected [9]. Taken together, available evidence suggests a potential association of IRAK1 with RA. However, further studies are required to comprehensively explore the role of IRAK1 in RA, and the development of therapeutic agents targeting IRAK1 might result in important new, innovative therapies for RA.

Abstract 407: Loss of Smad3 alters host-microbial interactions, predisposing the colonic epithelium to inflammation

Abstract Introduction: The TGFβ pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Smad3−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 signaling and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 signaling on colonic bacterial composition we used the Smad3−/− GEM. We designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on cecal DNA extracted from Smad3−/-− and Smad3+/+ mice. To further examine the host-microbial interaction we extracted RNA and protein from the cecum of Smad3−/− and Smad3+/+ mice and ran Real Time-PCR or Western Blot for different Toll-like Receptor Pathway components. Results: qRT-PCR showed that Smad3−/-− mice have a significantly lower quantity of Bacteroides sp; the introduction of Helicobacter hepaticus results in a significant increase in B. distasonis and a significant decrease in B. thetaiotaomicron in Smad3−/− mice. These data suggest that loss of SMAD3 and the addition of Helicobacter hepaticus alters the colonic microflora. Previous studies illustrated the importance of epithelial signaling in microbial induced inflammation. To examine if Smad3 is altering the Toll-like receptor pathway we examined RT-PCR of the cecum and found a significant increase in Tlr4, Irak4, Cd14, Myd88, Nfkb, Cox2, and Nos2 mRNA in the Smad3−/−− mice compared to Smad3+/+ mice. Western blots confirmed these data by showing that cecal tissue from Smad3−/− mice has increased IRAK4 and pNF-κB. Conclusion: Smad3−/− mice show heightened Toll-like receptor pathway activity which leads to a hyper-inflammatory response of the mucosa to endogenous microbes. Further, loss of SMAD3 results in dysbiosis of the gut microbiota. These findings suggest that SMAD3 plays a role in microbial-host homeostasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 407. doi:1538-7445.AM2012-407

Inhibition of IRAK1 As a Novel Therapeutic Strategy in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Abstract 612Recent work has shown that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients exhibit downregulation of miR-146a, a miRNA that negatively regulates the innate immune pathway by targeting IRAK1 and TRAF6. Mice lacking miR-146a show elevated IRAK1 protein expression, and develop AML and MDS-like features resembling the human diseases. Prior to this study, the role of IRAK1 in human myeloid malignancies was unknown. We conducted a comparison of gene expression profiles of 136 cases of MDS CD34+ cells with 17 normal CD34+ cells obtained from ArrayExpress (E-GEOD-19429; Pellagatti et al., Leukemia, 2010). According to this data set, we observed IRAK1 overexpression in MDS patients (P = 0.017). IRAK1 is a serine/threonine kinase, and after phosphorylation on threonine-209 (T209), its kinase activity is induced, thus allowing for subsequent activation of TRAF6 and eventually NF-kB. Interestingly, we observed higher basal levels of phospho-IRAK1 at T209 in MDS and AML samples as compared to normal human CD34+ cells. To investigate the potential role of IRAK1 in AML and MDS, we used genetic and pharmacological approaches to suppress IRAK1 activity in MDS/AML cell lines and bone marrow cells from MDS patients. RNAi-mediated knockdown of IRAK1 in MDS and AML samples resulted in impaired growth of malignant hematopoietic stem/progenitor cells in methylcellulose assays and rapid apoptosis in vitro. In addition, we used a small-molecule inhibitor (benzimidazole analog; Amgen Inc.) to potently inhibit IRAK1 kinase activity. MDS/AML cell lines and MDS patient samples cultured with the IRAK1 inhibitor exhibited impaired growth and increased apoptosis, which coincided with decreased phospho-IRAK1 at T209, and active versions of TRAF6 and NF-kB. Importantly, the inhibition of IRAK1 kinase function is selectively detrimental to MDS and AML samples while preserving normal CD34+ cell viability and function. Given this novel requirement of IRAK1 in MDS and AML, we examined whether Lenalidomide or Bortezomib, two treatment options for MDS/AML and reported immunosuppressors, exhibit anti-leukemic activity in part by targeting IRAK1. We observed that Bortezomib, but not Lenalidomide, inhibits IRAK1 mRNA and protein expression in MDS/AML cells. The cytotoxic effect of Bortezomib can be partly rescued by forced expression of IRAK1 in these cells. To determine the molecular and cellular basis of cell death following loss of IRAK1 function or expression, we applied microarrays to MDS cells treated with IRAK1 inhibitor or transduced with a lentiviral vector encoding an shRNA targeting IRAK1. An overlap of commonly deregulated genes imposed by loss of IRAK1 expression or by the IRAK1 inhibitor revealed unique pathways relevant to the survival of MDS and AML cells. In summary, these findings are the first to implicate IRAK1 in the maintenance of myeloid malignancies and describe the effectiveness of an IRAK1 inhibitor on suppressing MDS and AML viability. Disclosures:Oliva:Celgene: Consultancy.

Effect of Dureping injection on TIR signal pathway on Ana-1 cells

To investigate the influence of Dureping injection to the murinal celiac macrophage Ana-1 on TIR signal pathway. Ana-1 cell line was infected by influenza virus FM1 strain and treated with the Dureping injection in different concentrations (10.1 mg x L(-1) group) for 12 h and 24 h. Then we collected the cells, extracted mRNA and measured the expressions of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 respectively by RT-PCR. Dureping injection down-regulated the expression of TLR7, MyD88, IRAK4, TRAF6 and NF-kappaB p65 mRNA in Ana-1 cell line infected by influenza virus, in a dose-dependent manner significantly. Dureping injection has an obvious effect against influenza virus FM1 strain by regulating the TIR signal pathway.

Toll-like receptor 2 ligand mediates the upregulation of angiogenic factor, vascular endothelial growth factor and interleukin-8/CXCL8 in human rheumatoid synovial fibroblasts

Rheumatoid arthritis (RA) is characterized by infiltrations of inflammatory cells accompanied by neovascularization in the joint. We hypothesized that cell activation via the toll-like receptor (TLR) may be involved in the induction of angiogenic molecules, which are relevant to the pathogenesis of RA. RA fibroblast like synoviocytes (FLS) were stimulated with TLR-2 ligand bacterial peptidoglycan (PGN), TLR-4 ligand lipopolysaccharide (LPS) and various cytokines. Vascular endothelial growth factor (VEGF) and IL-8 were measured by ELISA in culture supernatants; mRNA levels were assessed by RT-PCR and real time PCR. The levels of TLR-2, VEGF and IL-8 were analyzed by dual immunohistochemistry in RA synovium and compared with osteoarthritis (OA). Regulation of MyD88, IRAK4, IRAK1, IRAK-M and TRAF-6 mRNA expression levels by PGN were analyzed by RT-PCR. Phosphorylation of IκBα was evaluated by western blotting. Levels of VEGF and IL-8 were upregulated in culture supernatants of RA FLS stimulated with PGN, similar to the levels of IL-1β and IL-17 stimulation. Neutralization of TLR-2 with a blocking monoclonal antibody significantly reduced both VEGF and IL-8 levels ( P < 0.05), which reflected the functional relevance of TLR-2 activation to the induction of VEGF and IL-8 production. Downstream intracellular signaling following TLR-2 stimulation involved MyD88-IRAK-4-TRAF-6 pathways, resulting in NF-κB activation. Thus, TLR-2 activation in RA FLS by microbial constituents could be involved in the induction of VEGF and IL-8 and thereby promote inflammation either directly or via angiogenesis. This possibly contributes to the perpetuation of synovitis in patients with RA.

The Intestinal Intraepithelial Lymphocytes with T Cell Receptor αβ Express Toll-Like Receptor 4 and Are Responsive to Lipopolysaccharide

Background: Intestinal intraepithelial lymphocytes (iIELs) play an important role in intestinal innate immunity and oral immune tolerance. To compare the differences in gene expression between murine iIELs and splenic T lymphocytes, we established the cDNA subtractive library of iIELs and analyzed the iIELs special genes. Our study focused on the relationship between Toll-like receptor 4 (TLR4), TLR5 and iIELs. Methods: Ninety percent purified iIELs and splenic T lymphocytes were isolated by density-gradient centrifugation in a Percoll and nylon column, respectively. We then established the cDNA subtractive library of iIELs via improved subtractive hybridization. The special expressed sequence tags of iIELs were screened by reverse Northern blot. The expressions of TLR4 and TLR5 were analyzed by RT-PCR and fluorescence staining. The proliferation of T cells was determined by ³H-TdR incorporation. Results: TLR4, but not TLR5, was detected in iIELs by RT-PCR and fluorescence staining. However, TLR4 was only found in αβ iIELs. Furthermore, iIELs were observed to proliferate in response to lipopolysaccharide in vitro, with upregulation of IRAK-1 mRNA expression. Conclusion: αβ iIELs can recognize lipopolysaccharide via TLR4, which may play an important role in the intestinal innate immunity.

Bacterial lipoprotein-induced self-tolerance and cross-tolerance to LPS are associated with reduced IRAK-1 expression and MyD88-IRAK complex formation

Tolerance to bacterial cell-wall components may represent an essential regulatory mechanism during bacterial infection. We have demonstrated previously that the inhibition of nuclear factor (NF)-kappaB and mitogen-activated protein kinase activation was present in bacterial lipoprotein (BLP) self-tolerance and its cross-tolerance to lipopolysaccharide (LPS). In this study, the effect of BLP-induced tolerance on the myeloid differentiation factor 88 (MyD88)-dependent upstream signaling pathway for NF-kappaB activation in vitro was examined further. When compared with nontolerant human monocytic THP-1 cells, BLP-tolerant cells had a significant reduction in tumor necrosis factor alpha (TNF-alpha) production in response to a high-dose BLP (86+/-12 vs. 6042+/-245 ng/ml, P < 0.01) or LPS (341+/-36 vs. 7882+/-318 ng/ml, P < 0.01) stimulation. The expression of Toll-like receptor 2 (TLR2) protein was down-regulated in BLP-tolerant cells, whereas no significant differences in TLR4, MyD88, interleukin-1 receptor-associated kinase 4 (IRAK-4), and TNF receptor-associated factor 6 expression were observed between nontolerant and BLP-tolerant cells, as confirmed by Western blot analysis. The IRAK-1 protein was reduced markedly in BLP-tolerant cells, although IRAK-1 mRNA expression remained unchanged as revealed by real-time reverse transcriptase-polymerase chain reaction analysis. Furthermore, decreased MyD88-IRAK immunocomplex formation, as demonstrated by immunoprecipitation, was observed in BLP-tolerant cells following a second BLP or LPS stimulation. BLP pretreatment also resulted in a marked inhibition in total and phosphorylated inhibitor of kappaB-alpha (IkappaB-alpha) expression, which was not up-regulated by subsequent BLP or LPS stimulation. These results demonstrate that in addition to the down-regulation of TLR2 expression, BLP tolerance is associated with a reduction in IRAK-1 expression, MyD88-IRAK association, and IkappaB-alpha phosphorylation. These findings further elucidate the molecular mechanisms underlying bacterial peptide tolerance.