IL-17A mRNA Research Articles

IMQ-induced skin inflammation in mice is dependent on IL-1R1 and MyD88 signaling but independent of the NLRP3 inflammasome.

The pathogenesis of inflammatory skin diseases such as psoriasis involves the release of numerous proinflammatory cytokines, including members of the IL-1 family. Here we report overexpression of IL-1α, IL-1β, and IL-1 receptor antagonist mRNA, associated to expression of IL-23p19, IL-17A, and IL-22 in skin cells, upon topical application of the TLR7 agonist imiquimod (IMQ) in C57BL/6J mice. IMQ-induced skin inflammation was partially reduced in mice deficient for both IL-1α/IL-1β or for IL-1 receptor type 1 (IL-1R1), but not in IL-1α- or IL-1β-deficient mice, demonstrating the redundant activity of IL-1α and IL-1β for skin inflammation. NLRP3 or apoptosis-associated Speck-like protein containing a Caspase recruitment domain-deficient mice had no significant reduction of skin inflammation in response to IMQ treatment, mainly due to the redundancy of IL-1α. However, IMQ-induced skin inflammation was abolished in the absence of MyD88, the adaptor protein shared by IL-1R and TLR signaling pathways. These results are consistent with the TLR7 dependence of IMQ-induced skin inflammation. Thus, IL-1R1 contributes to the IMQ-induced skin inflammation, and disruption of MyD88 signaling completely abrogates this response.

IL-17A and its homologs IL-25/IL-17E recruit the c-RAF/S6 kinase pathway and the generation of pro-oncogenic LMW-E in breast cancer cells.

Pro-inflammatory IL-17 cytokines were initially described for their pathogenic role in chronic inflammatory diseases and subsequent accumulating evidence indicated their involvement in carcinogenesis. In the present study we report that IL-17A and IL-17E receptors subunits mRNA expressions are upregulated in breast cancers versus normal samples. IL-17E, which is undetectable in most normal breast tissues tested, seems more expressed in some tumors. Investigation of the molecular signaling following stimulation of human breast cancer cell lines with IL-17A and IL-17E showed that both cytokines induced the phosphorylation of c-RAF, ERK1/2 and p70 S6 Kinase were involved in the proliferation and survival of tumor cells. Accordingly, IL-17A and IL-17E promoted resistance to Docetaxel and failed to induce apoptosis as previously reported for IL-17E. Interestingly, we also revealed that both cytokines induced the generation of tumorogenic low molecular weight forms of cyclin E (LMW-E), which high levels correlated strongly with a poor survival in breast cancer patients. These results show for the first time some of the molecular pathways activated by IL-17A and IL-17E that may participate to their pro-oncogenic activity in breast cancers.

Expression of T helper type 17 (Th17)-associated cytokines and toll-like receptor 4 and their correlation with Foxp3 positive cells in rectal biopsies of horses with clinical signs of inflammatory bowel disease

Inflammatory bowel disease (IBD) in horses is an idiopathic disorder, encompassing different types of chronic intestinal inflammation. The pathogenesis of the disease remains to be established, but it has been suggested that an imbalance between regulatory T cells (Tregs) and T helper 17 (Th17)-associated cytokines and altered toll-like receptor 4 (TLR4) expression is associated with intestinal inflammation in other species. The aim of the present study was to quantify Tregs in rectal biopsies from horses affected with IBD by immunohistochemistry and to evaluate expression of genes encoding interleukin (IL)-12p40, IL-17A, IL-23p19 and TLR4 by real-time quantitative PCR.Rectal biopsies from 11 healthy horses and 11 horses with clinical signs of IBD, showing inflammation classified as chronic simple proctitis (CSP) or chronic active simple proctitis (CASP), were evaluated. Expression of IL-17A mRNA was greater in horses affected with CASP compared with horses with CSP or healthy horses. In contrast, expression of IL-12p40 was lower in horses with CSP compared with horses with CASP or healthy horses. TLR4 expression was greater in horses with CASP compared with healthy horses. A positive correlation was seen between the numbers of Tregs and expression of IL-17A and IL-23p19. An association was demonstrated between the histopathological pattern of inflammation, cytokine profile and number of infiltrating Tregs. The research findings suggest that Th17 cells are involved in active IBD, possibly through recruitment of neutrophils via IL-17A, in combination with inadequate suppression of the inflammatory response by Tregs.

Interleukin-1α deficiency attenuates endoplasmic reticulum stress-induced liver damage and CHOP expression in mice

Background & AimsER stress promotes liver fat accumulation and induction of inflammatory cytokines, which contribute to the development of steatohepatitis. Unresolved ER stress upregulates the pro-apoptotic CHOP. IL-1α is localized to the nucleus in apoptotic cells, but is released when these cells become necrotic and induce sterile inflammation. We investigated whether IL-1α is involved in ER stress-induced apoptosis and steatohepatitis. MethodsWe employed WT and IL-1α-deficient mice to study the role of IL-1α in ER stress-induced steatohepatitis. ResultsLiver CHOP mRNA was induced in a time dependent fashion in the atherogenic diet-induced steatohepatitis model, and was twofold lower in IL-1α deficient compared to WT mice. In the ER stress-driven steatohepatitis model, IL-1α deficiency decreased the elevation in serum ALT levels, the number of apoptotic cells (measured as caspase-3-positive hepatocytes), and the expression of IL-1β, IL-6, TNFα, and CHOP, with no effect on the degree of fatty liver formation. IL-1α was upregulated in ER-stressed-macrophages and the protein was localized to the nucleus. IL-1β mRNA and CHOP mRNA and protein levels were lower in ER-stressed-macrophages from IL-1α deficient compared to WT mice. ER stress induced the expression of IL-1α and IL-1β also in mouse primary hepatocytes. Recombinant IL-1α treatment in hepatocytes did not affect CHOP expression but upregulated both IL-1α and IL-1β mRNA levels. ConclusionWe show that IL-1α is upregulated in response to ER stress and IL-1α deficiency reduces ER stress-induced CHOP expression, apoptosis and steatohepatitis. As a dual function cytokine, IL-1α may contribute to the induction of CHOP intracellularly, while IL-1α released from necrotic cells accelerates steatohepatitis via induction of inflammatory cytokines by neighboring cells.

Resveratrol ameliorates imiquimod-induced psoriasis-like skin inflammation in mice.

BackgroundThe polyphenol resveratrol has anti-inflammatory effects in various cells, tissues, animals and human settings of low-grade inflammation. Psoriasis is a disease of both localized and systemic low-grade inflammation. The Sirtuin1 enzyme thought to mediate the effects of resveratrol is present in skin and resveratrol is known to down regulate NF-κB; an important contributor in the development of psoriasis. Consequently we investigated whether resveratrol has an effect on an Imiquimod induced psoriasis-like skin inflammation in mice and sought to identify candidate genes, pathways and interleukins mediating the effects.MethodsThe study consisted of three treatment groups: A control group, an Imiquimod group and an Imiquimod+resveratrol group. Psoriasis severity was assessed using elements of the Psoriasis Area Severity Index, skin thickness measurements, and histological examination. We performed an RNA microarray from lesional skin and afterwards Ingenuity pathway analysis to identify affected signalling pathways. Our microarray was compared to a previously deposited microarray to determine if gene changes were psoriasis-like, and to a human microarray to determine if findings could be relevant in a human setting.ResultsImiquimod treatment induced a psoriasis-like skin inflammation. Resveratrol significantly diminished the severity of the psoriasis-like skin inflammation. The RNA microarray revealed a psoriasis-like gene expression-profile in the Imiquimod treated group, and highlighted several resveratrol dependent changes in relevant genes, such as increased expression of genes associated with retinoic acid stimulation and reduced expression of genes involved in IL-17 dependent pathways. Quantitative PCR confirmed a resveratrol dependent decrease in mRNA levels of IL-17A and IL-19; both central in developing psoriasis.ConclusionsResveratrol ameliorates psoriasis, and changes expression of retinoic acid stimulated genes, IL-17 signalling pathways, IL-17A and IL-19 mRNA levels in a beneficial manner, which suggests resveratrol, might have a role in the treatment of psoriasis and should be explored further in a human setting.

Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis.

The response of psoriasis to antibodies targeting the interleukin (IL)-23/IL-17A pathway suggests a prominent role of T-helper type-17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate-to-severe psoriasis receiving 3 different intravenous dosing regimens of the anti-IL-17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL-17 and may store the cytokine preformed, as IL-17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c-positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest-dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil–keratinocyte axis in psoriasis that may involve neutrophil-derived IL-17 and is an early target of IL-17A-directed therapies such as secukinumab.

Abstract 65: T cell Differentiation in Human Kawasaki Disease and a Murine Model of Coronary Arteritis

The role of T cell differentiation in the immunopathogenesis of Kawasaki disease (KD) remains unclear. The aim of this study is to elucidate the role of T cell subsets in coronary artery lesion (CAL) of KD. Peripheral blood was obtained in 10 patients with acute KD before and 1 week after intravenous gamma-immunoglobulin (IVIG) treatment and in 20 patients with past history of KD for more than 5~20 years. Meanwhile, induction of coronary arteritis was performed on wild type BALB/c mice by Lactobacillus casei cell wall extract (LCWE). Human peripheral blood leukocytes were analyzed by using flow cytometric analysis and murine hearts were examined for immunofluorescence study and for RNA expression levels. Results: Compared to the febrile controls, KD patients prior to IVIG treatment had increased percentage of CD3 + /CD4 + /interferon-γ + (T helper 1, Th1) cells and CD3 + /CD4 + /interleukin-17A + (Th17) cells (mean ± SD, 1.36% ± 1.39% and 0.51% ± 0.25%, respectively) among Th cells (CD3 + /CD4 + ). Both increases declined after IVIG treatment (0.71% ± 0.74% and 0.33% ± 0.18%) despite no statistically difference by Mann-Whitney test. None of these 10 acute KD patients developed CAL after IVIG treatment. However, patients with previous KD and definite CALs (n= 11) seemed to have higher percentage of Th17 cells (0.50% ± .025% versus 0.35% ± 0.23%) but similar level of Th1 cells (0.93% ± 0.51% versus 1.05% ± 0.64%) when compared to those without CAL (n= 9). Murine cardiac tissues displayed the presence of Th1 (double-stained with CD3 and T-bet) and Th17 cells (double-stained with CD3 and RORγt) during days 7 and 14 after LCWE treatment but not in PBS-treated mice. Compatible with these, cardiac mRNA levels showed both increased levels of IFN-γ and IL17A mRNA in LCWE-treated mice. Conclusions: Our initial data suggests that specific T cell differentiation into Th1 and Th17 cells occurred in both human KD and mice stimulated with LCWE. IVIG treatment was associated with the recovery of such T cell differentiation. However, the clinical application to predict IVIG responsiveness and future CAL development by such increase in the peripheral Th17 remains unclear. Further studies to elucidate the detailed immune regulation of these T subsets on CAL are warrant by using this LCWE murine model.

Randomized Clinical Trial: Impact of Oral Administration of Saccharomyces boulardii on Gene Expression of Intestinal Cytokines in Patients Undergoing Colon Resection.

When intestinal microbiota is imbalanced, a patient becomes more vulnerable to infectious complications; intervention with beneficial probiotics may help lower risk for infection. The aim of this study was to measure levels of inflammatory cytokine messenger RNA (mRNA) in surgical samples of intestinal mucosal tissues from patients who were given the probiotic Saccharomyces boulardii before undergoing colon surgery. Thirty-three patients undergoing colon resection were randomly assigned to receive at least 7-day preoperative probiotic treatment (n = 15) or conventional (n = 18) treatment. Probiotic treatment consisted of oral lyophilized S boulardii Cytokine mRNA levels (interleukin [IL]-10, IL-1β, IL-23A, tumor necrosis factor [TNF]-α, IL-12B, interferon-γ [INF-γ], and IL-17A) were measured in samples obtained during the operation. Postoperative infections were also assessed. Patients who received probiotics had significantly lower mucosal IL-1β, IL-10, and IL-23A mRNA levels than the control group (P = .001, P = .04, and P = .03, respectively). However, mRNA expression of other cytokines did not differ between the 2 groups (P > .05). The incidence of postoperative infectious complications was 13.3% and 38.8% in probiotic and control groups, respectively (P > .05). There was no perioperative mortality in either group. The mean total length of hospital stay was similar between the groups (P > .05). Probiotic treatment with S boulardii downregulates both pro- and anti-inflammatory cytokines in the intestinal colonic mucosa with no statistical impact on postoperative infection rates.

MyD88-Dependent Signaling Decreases the Antitumor Efficacy of Epidermal Growth Factor Receptor Inhibition in Head and Neck Cancer Cells.

EGFR is upregulated in the majority of head and neck squamous cell carcinomas (HNSCC). However, many patients with HNSCC respond poorly to the EGFR inhibitors (EGFRI) cetuximab and erlotinib, despite tumor expression of EGFR. Gene expression analysis of erlotinib-treated HNSCC cells revealed an upregulation of genes involved in MyD88-dependent signaling compared with their respective vehicle-treated cell lines. We therefore investigated whether MyD88-dependent signaling may reduce the antitumor efficacy of EGFRIs in HNSCC. Erlotinib significantly upregulated IL6 secretion in HNSCC cell lines, which our laboratory previously reported to result in reduced drug efficacy. Suppression of MyD88 expression blocked erlotinib-induced IL6 secretion in vitro and increased the antitumor activity of erlotinib in vivo. There was little evidence of Toll-like receptor or IL18 receptor involvement in erlotinib-induced IL6 secretion. However, suppression of IL1R signaling significantly reduced erlotinib-induced IL6 production. A time-dependent increase of IL1α but not IL1β was observed in response to erlotinib treatment, and IL1α blockade significantly increased the antitumor activity of erlotinib and cetuximab in vivo. A pan-caspase inhibitor reduced erlotinib-induced IL1α secretion, suggesting that IL1α was released because of cell death. Human HNSCC tumors showed higher IL1α mRNA levels compared with matched normal tissue, and IL1α was found to be negatively correlated with survival in patients with HNSCC. Overall, the IL1α/IL1R/MYD88/IL6 pathway may be responsible for the reduced antitumor efficacy of erlotinib and other EGFRIs, and blockade of IL1 signaling may improve the efficacy of EGFRIs in the treatment of HNSCC.

Lack of IL-17 signaling decreases liver fibrosis in murine schistosomiasis japonica.

Accumulating evidence has identified the profibrogenic properties of IL-17A in organ fibrosis. However, the role of IL-17A signal in liver fibrosis induced by Schistosoma japonicum infection remains unclear. In this study, we investigated liver fibrosis in wild-type (WT) and IL-17RA(-/-) mice upon S. japonicum infection. Hepatic IL-17A, IL-17C, IL-17E (IL-25), IL-17F, IL-17RA, IL-17RB and IL-17RC transcript levels were determined by RT-PCR. IL-17A(+) cells were analyzed by flow cytometry and confocal microscopy among granuloma cells. Immunostaining of IL-17R was performed on liver sections. Collagen deposition was assessed by Van Gieson's staining. IL-17A, IL-17C, IL-17E, IL-17F, IL-17RA and IL-17RC mRNA levels were dramatically increased in fibrotic livers. Among granuloma cells, CD3(+) and CD3(-) lymphocytes, neutrophils and macrophages were found to express IL-17A. Compared to WT, IL-17RA(-/-) mice displayed attenuated granulomatous inflammation, liver fibrosis, improved liver function and high survival. Meanwhile, α-smooth muscle actin staining and the expression of fibrogenic genes (transforming growth factor β, IL-13 and collagen-I) as well as IL-17A-induced proinflammatory mediators (IL-1β, IL-6, tumor necrosis factor α, CXCL1 and CXCL2) and proteinases (MMP3 and TIMP1) involved in fibrosis were markedly reduced in IL-17RA(-/-) mice. In addition, Th2 cytokines IL-4 and IL-17E (IL-25) were also decreased in IL-17RA(-/-) mice. These results indicated that IL-17A signal contributes to the pathogenesis of liver fibrosis in murine schistosomiasis. This effect was induced possibly by activating hepatic stellate cells and stimulating the release of proinflammatory cytokines and chemokines. Furthermore, the Th2 response was also enhanced by IL-17A signals. Our data demonstrate that IL-17A may serve as a promising target for antifibrotic therapy.

Macrophage Phenotype in the Ocular Surface of Experimental Murine Dry Eye Disease.

To evaluate the phenotype of macrophages in the cornea and conjunctiva of C57BL/6 mice with induced experimental dry eye. C57BL/6 mice exposed to desiccating stress (DS) were evaluated at 1, 5, and 10 days and C57BL/6 mice maintained in non-stressed environment were used as controls. Whole eyes and adnexa were excised for histology or used for gene expression analysis. Location and phenotype of macrophages infiltrating the cornea and conjunctiva was evaluated by immunofluorescence analysis. Quantitative polymerase chain reaction evaluated macrophage markers and T cell-related and inflammatory cytokine expression in cornea and conjunctiva. Immunofluorescence staining demonstrated that macrophages reside in the conjunctiva of control and dry eye mice and their number did not change with DS. Real-time RT-PCR demonstrated that the level of M1 macrophage marker, iNOS, increased prominently in the conjunctiva at DS 10 days. In contrast, there was a non-significant decrease of the M2 marker Arg1 with DS. The levels of inflammatory cytokine, IL-12a mRNA transcript in the conjunctiva increased significantly at DS1 and decreased at DS5, while levels of IL-18 were significantly increased at DS 10. Macrophages reside in the ocular surface tissues of C57BL/6 mice. Although the number of macrophages in the conjunctiva does not change, evidence of inflammatory M1 activation after desiccating stress was observed. Better understanding of phagocyte diversity and activation in dry eye disease provide a basis for the development of phagocyte-targeted therapeutic strategies.

Effects of Bu Shen Yi Sui Capsule on Th17/Treg cytokines in C57BL/6 mice with experimental autoimmune encephalomyelitis.

BackgroundT helper (Th) 17 and regulatory T (Treg) cells play a critical role in the pathogenesis of multiple sclerosis (MS) disease. Bu Shen Yi Sui Capsule (BSYSC), a traditional Chinese medicine formula, has been used clinically for the treatment of MS patients in China.MethodsTo evaluate the neuroprotective effects and the underlying mechanisms of BSYSC on MS, experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice was induced with myelin oligodendrocyte glycoprotein (MOG) 35–55. Th17 and Treg cells and the related cytokines were detected by flow cytometry, ELISA, real-time quantitative reverse transcription PCR, western blot and immunohistochemistry.ResultsWe found that BSYSC improved neurological function, reduced inflammatory cell infiltration and damage to the axons and myelin in the brain and spinal cord. BSYSC down-regulated markedly the ratio of CD4 + IL-17+/CD4 + CD25 + FoxP3+ T cells in the spleen, decreased the cytokines of IL-17A, IL-6, IL-23, TGF-beta1 in the brain, and dropped the ratio of IL-17A and FoxP3 mRNA and protein in the brain or spinal cord at different stages.ConclusionsThe study demonstrated that BSYSC had a strong neuroprotective effect on EAE mice. The protective mechanisms of BSYSC might be associated with mediating the regulation of Th17/Treg cells.

Involvement of IL-1 genes in the cellular responses to carbon nanotube exposure

The interleukin-1 (IL-1) family has been implicated in cellular responses to nanoparticles including carbon nanotubes (CNTs). IL-1α and β are key proinflammatory cytokines important in inflammatory and oxidative stress responses. The aim of this study was to characterize the role of IL-1 in cellular responses of CNTs in cells from IL-1α/β wild type (IL1-WT) mice and cells with reduced inflammatory potential from IL-1α/β deficient (IL1-KO) mice. Two multi-walled CNTs, CNT-1 containing long and thick fibers and CNT-2 containing short and thin fibers, were compared to UICC crocidolite asbestos fibers. Upon CNT exposure toxicity and apoptosis were affected differently in IL1-WT and IL1-KO cells. Upregulation of TNFα and IL-1α mRNA expression in IL1-WT cells was dependent on the type of CNT. On the contrary precursor IL-1α protein was downregulated after 24h. The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) was activated in IL1-KO cells and regulated by CNTs, whereas no significant changes of extracellular regulated kinase (ERK) were observed when comparing IL1-WT and IL1-KO cells. In summary, the results presented here indicate that IL-1 contributes to the cellular and molecular effects of CNT exposure and that the type of CNT has an important effect on the cellular response.

Dimethyl fumarate suppresses Theiler's murine encephalomyelitis virus-induced demyelinating disease by modifying the Nrf2-Keap1 pathway.

Dimethyl fumarate (DMF) is a modifier of the nuclear factor (erythroid-derived 2)-2 (Nrf2)-kelch-like ECH-associated protein 1 (Keap1) pathway. DMF treatment in the effector phase significantly suppressed the development of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) both clinically and histologically. DMF treatment leads to an enhanced Nrf2 antioxidant response in TMEV-IDD mice. DMF treatment in the effector phase significantly suppressed the level of IL-17A mRNA. DMF is known to inhibit differentiation of T helper 17 (Th17) cells via suppressing NF-κB. Taken together, our data suggest that DMF treatment in the effector phase may suppress TMEV-IDD not only via enhancing the antioxidant response but also via suppressing IL-17A.

Abstract B29: The role of MYD88-dependent signaling in the anti-tumor efficacy of the EGFR inhibitor erlotinib in head and neck cancer cells

Abstract Epidermal growth factor receptor (EGFR) is upregulated in the majority of head and neck squamous cell carcinomas (HNSCC). Unfortunately, the incorporation of EGFR inhibitors (EGFRIs) into the management of HNSCC has not improved long-term survival rates in patients with advanced stage HNSCC tumors. Therefore, the identification and understanding of strategies that will improve the efficacy of EGFRIs may potentially improve patient treatment and survival. Using microarray analysis, our laboratory has observed a profound increase in genes involved in MYD88-dependent pro-inflammatory pathways in EGFRI-treated HNSCC cell lines compared to their respective vehicle-treated cell lines. Additionally, we have previously shown that EGFRIs induce production of the inflammatory cytokine interleukin-6 (IL-6), leading to the reduced efficacy and acquired resistance to EGFRIs in HNSCC cells. Since MYD88-dependent signaling may lead to IL-6 production and secretion, the purpose of these studies is to determine if MYD88-dependent pathways such as Toll-like receptor (TLR), interleukin-1 receptor (IL-1R) and interleukin-18 receptor (IL-18R) pathways are responsible for the IL-6 production induced by EGFRIs. We found that knockdown of MYD88 (using siRNA and shRNA) significantly inhibited IL-6 secretion induced by the EGFRI erlotinib in Cal-27 and SQ20B HNSCC cells in vitro, increased the anti-tumor activity of erlotinib in vitro and enhanced the anti-tumor efficacy of erlotinib in a HNSCC xenograft mouse model. These results suggested that MYD88-dependent signaling was involved in IL-6 production and in the reduced efficacy of erlotinib. In order to elucidate which MYD88-dependent pathway was involved in erlotinib-induced IL-6 production, we investigated the involvement of TLR, IL-1R or IL-18R-mediated signaling. Although TLRs and the IL-18R were expressed and active on our HNSCC cells, we found little to no evidence of their involvement in erlotinib-induced IL-6 production. However, inhibition of the IL-1R through pharmacologic (anakinra) or genetic (siRNA and shRNA transfection) methods significantly reduced erlotinib-induced IL-6 production and increased HNSCC cell sensitivity to erlotinib in vitro. These observations point to the IL-1R/MYD88 pathway being involved in IL-6 production and in the reduced efficacy of erlotinib. We observed a time-dependent increase of IL-1 alpha (IL-1α) but not IL-1 beta (IL-1β) in response to erlotinib treatment implicating IL-1α as the damage associated molecular pattern (DAMP) responsible for activating the IL-1R. Furthermore, suppression of cell death with a pan-caspase inhibitor reduced erlotinib-induced IL-1α secretion. Finally, human HNSCC tumors were found to have higher IL-1α mRNA levels than matched normal tissue, and comparison of high and low IL-1α expressing HNSCC tumors (from the TCGA PANCAN databank) showed IL-1α to be negatively correlated with survival. Altogether, erlotinib-induced cell death may result in the release of IL-1α which activates IL-1R on adjacent surviving cells resulting in MYD88-mediated IL-6 secretion and the development of acquired resistance to erlotinib. In summary, the IL-1α/IL-1R/MYD88/IL-6 pathway may be responsible for the reduced anti-tumor efficacy of erlotinib and other EGFRIs; and blockade of the IL-1 pathway may improve the efficacy of EGFRIs in the treatment of HNSCC. This work was supported by the grants NIH K01CA134941 and ACS IRG7700434. Citation Format: Andrean L. Simons, Adam T. Koch, Laurie Love-Homan, Aditya Stanam. The role of MYD88-dependent signaling in the anti-tumor efficacy of the EGFR inhibitor erlotinib in head and neck cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B29.

Effect of Hangeshashinto on calprotectin expression in human oral epithelial cells.

Oral epithelial cells produce antimicrobial peptides (AMPs) to prevent microbial infection. Calprotectin (S100A8/S100A9) is one of these AMPs in oral epithelial cells, the expression of which is up-regulated by interleukin-1α (IL-1α). Hangeshashinto (HST) is a traditional Japanese herbal medicine that has anti-inflammatory effects. The purpose of this study was to investigate the effect of HST on the expression of calprotectin through the regulation of IL-1α in oral epithelial cells. Human oral epithelial cells (TR146) were cultured with HST in the presence or absence of anti-IL-1α antibody or IL-1 receptor antagonist, or with six major components of HST (3,4-dihydroxybenzaldehyde, baicalin, ginsenoside Rb1, glycyrrhizin, oleanolic acid and berberine). The expression of S100A8, S100A9, other AMPs and cytokine mRNAs was examined by RT-PCR and quantitative real-time PCR. Calprotectin expression and IL-1α secretion were investigated by ELISA. HST (6 μg/ml) increased the expression of S100A8/S100A9 mRNAs and calprotectin protein, and also up-regulated β-defensin 2 (DEFB4) and S100A7 expression. The expression of IL-1α mRNA and its protein was slightly but significantly increased by HST. A neutralizing antibody against IL-1α and IL-1 receptor antagonist inhibited HST-up-regulated S100A8/S100A9 mRNA expression. Although 3,4-dihydroxybenzaldehyde, baicalin and ginsenoside Rb1 as HST components increased S100A8/S100A9 expression, oleanolic acid and berberine decreased their expression. These results suggest that HST increases the expression of calprotectin, DEFB4 and S100A7 in oral epithelial cells. In response to HST, up-regulation of calprotectin expression may be partially induced via IL-1α.

Using Bayesian modelling to investigate factors governing antibiotic-induced Candida albicans colonization of the GI tract.

Receipt of broad-spectrum antibiotics enhances Candida albicans colonization of the GI tract, a risk factor for haematogenously-disseminated candidiasis. To understand how antibiotics influence C. albicans colonization, we treated mice orally with vancomycin or a combination of penicillin, streptomycin, and gentamicin (PSG) and then inoculated them with C. albicans by gavage. Only PSG treatment resulted in sustained, high-level GI colonization with C. albicans. Furthermore, PSG reduced bacterial diversity in the colon much more than vancomycin. Both antibiotic regimens significantly reduced IL-17A, IL-21, IL-22 and IFN-γ mRNA levels in the terminal ileum but had limited effect on the GI fungal microbiome. Through a series of models that employed Bayesian model averaging, we investigated the associations between antibiotic treatment, GI microbiota, and host immune response and their collective impact on C. albicans colonization. Our analysis revealed that bacterial genera were typically associated with either C. albicans colonization or altered cytokine expression but not with both. The only exception was Veillonella, which was associated with both increased C. albicans colonization and reduced IL-21 expression. Overall, antibiotic-induced changes in the bacterial microbiome were much more consistent determinants of C. albicans colonization than either the GI fungal microbiota or the GI immune response.

Filaggrin deficiency promotes the dissemination of cutaneously inoculated vaccinia virus

Eczema vaccinatum is a life-threatening complication of smallpox vaccination in patients with atopic dermatitis (AD) characterized by dissemination of vaccinia virus (VV) in the skin and internal organs. Mutations in the filaggrin (FLG) gene, the most common genetic risk factor for AD, confer a greater risk for eczema herpeticum in patients with AD, suggesting that it impairs the response to cutaneous viral infections. We sought to determine the effects of FLG deficiency on the response of mice to cutaneous VV inoculation. VV was inoculated by means of scarification of unsensitized skin or skin topically sensitized with ovalbumin in FLG-deficient flaky tail (ft/ft) mice or wild-type (WT) control mice. The sizes of primary and satellite skin lesions were measured, and hematoxylin and eosin staining was performed. VV genome copy numbers and cytokine mRNA levels were measured by using quantitative PCR. VV inoculation in unsensitized skin of ft/ft mice, independent of the matted hair mutation, resulted in larger primary lesions, more abundant satellite lesions, heavier viral loads in internal organs, greater epidermal thickness, dermal cellular infiltration, and higher local Il17a, Il4, Il13, and Ifng mRNA levels than in WT control mice. VV inoculation at sites of topical ovalbumin application amplified all of these features in ft/ft mice but had no detectable effect in WT control mice. The number of satellite lesions and the viral loads in internal organs after cutaneous VV inoculation were significantly reduced in both unsensitized and topically sensitized ft/ftxIl17a(-/-) mice. FLG deficiency predisposes to eczema vaccinatum. This is mediated primarily through production of IL-17A.

Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ

The retinoic acid receptor-related orphan receptors α and γ (RORα and RORγ), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. In this study, we investigated the effects of isoflavones on RORα/γ activity and the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In doxycycline-inducible CHO stable cell lines, we found that four isoflavones, biochanin A (BA), genistein, formononetin, and daidzein, enhanced RORα- or RORγ-mediated transcriptional activity in a dose-dependent manner. In an activation assay of the Il17a promoter using Jurkat cells, these compounds enhanced the RORα- or RORγ-mediated activation of the Il17a promoter at concentrations of 1 × 10(-6)M to 1 × 10(-5)M. In mammalian two-hybrid assays, the four isoflavones enhanced the interaction between the RORα- or RORγ-ligand binding domain and the co-activator LXXLL peptide in a dose-dependent manner. In addition, these isoflavones potently enhanced Il17a mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin, but showed slight enhancement of Il17a gene expression in RORα/γ-knockdown EL4 cells. Immunoprecipitation and immunoblotting assays also revealed that BA enhanced the interaction between RORγt and SRC-1, which is a co-activator for nuclear receptors. Taken together, these results suggest that the isoflavones have the ability to enhance IL-17 gene expression by stabilizing the interactions between RORα/γ and co-activators. This also provides the first evidence that dietary chemicals can enhance IL-17 gene expression in immune cells.

Periapical Cytokine Expression in Sickle Cell Disease

IntroductionSickle cell anemia (SCA) is the most prevalent genetic disease worldwide. Patients with SCA exhibit increased levels of proinflammatory mediators as part of a permanently activated immunoinflammatory status. MethodsThe aim of this study was to evaluate the mRNA expression levels of the cytokines interferon (IFN-γ), tumor necrosis factor, interleukin (IL-1β, IL-17A, IL-10), receptor activator for nuclear factor kappa B ligand, and the chemokines CCL2/MCP-1 and CCL5 in the periapical interstitial fluid from SCA individuals compared with healthy individuals. Samples were collected from 12 teeth of SCA patients and 12 non-SCA patients with apical periodontitis. In addition, 12 teeth were sampled from the periapical region of healthy patients with vital pulp (control). The expression of cytokine mRNA was detected by using real-time polymerase chain reaction. ResultsThe expression of mRNA for the Th1-associated cytokines IFN-γ, tumor necrosis factor-α, and IL-1β were significantly higher in SCA individuals than in the control individuals (P < .05). Among Th1-associated cytokines, only IFN-γ was significantly increased in non-SCA compared with control patients (vital pulp). The expression of IL-17A mRNA was significant higher in SCA cases than in control samples (P < .05), whereas the IL-10 mRNA expression was significantly increased in SCA and non-SCA individuals when compared with the control group. Similar levels of receptor activator for nuclear factor kappa B ligand, CCL2, and CCL5 mRNA expression were observed in all samples. However, no significant differences were observed in the expression of cytokine or chemokine mRNA between SCA and non-SCA individuals (P > .05). ConclusionsThe results were able to demonstrate that SCA patients presented prone proinflammatory ability, despite the fact that any differences in periapical immune responses between SCA and non-SCA individuals were not observed.