IL-2R Expression Research Articles

Abstract 3864: Targeting IL-6/STAT3 signaling in EGFR-mutant drug tolerant persister cells

Abstract While the majority of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients initially respond to treatment with an EGFR tyrosine kinase inhibitor (TKI), most will eventually develop acquired resistance to these agents. The evolution of tumor cells with acquired resistance to targeted agents initially involves the persistence of a drug-tolerant subpopulation of tumor cells which display a transient and flexible resistant state. These persister cells can remain quiescent for prolonged periods of time, but eventually give rise to fully drug resistant tumor cells that resume growth and metastatic spread. The signaling pathways that allow persister cells to remain viable or to re-activate expansive growth are poorly understood. We and others have reported that EGFR TKI resistance that occurs independent of secondary EGFR mutations or MET amplification is associated with an epithelial to mesenchymal transition (EMT). Our analysis of tumor cells that had undergone EMT-mediated resistance to the EGFR TKI osimertinib revealed that interleukin-6 (IL-6) was highly upregulated in resistant cells as compared to parental cells. We find that in the setting of EGFR mutant NSCLC, IL-6 suppresses the anti-tumor cell activity of T cells and NK cells. To assess the role of IL-6 in the early events of EGFR TKI resistance, we generated drug-tolerant persister cells by treating EGFR mutant HCC4006 and HCC827 NSCLC cells with the EGFR TKI osimertinib for 10 days and then assessed IL-6 expression. We observed that IL-6 was upregulated at both the RNA and protein levels in EGFR-TKI persister cells as compared to parental cells. While the cell surface expression of IL6R was not significantly increased in EGFR-TKI persister cells as compared to parental cells, we observed increased secretion of soluble IL6R in persister cells by ELISA assay. We next assessed changes in protein expression and activation in drug tolerant persister cells by reverse phase protein array (RPPA) and observed that p-STAT3, a key component of the IL-6 signaling pathway, was significantly elevated in persister cells as compared parental cells or cells treated with osimertinib for 24 hours. Using genetically engineered mouse models (GEMM) of EGFRL858R mutant lung cancer, we also observed a similar upregulation of both IL-6 and p-STAT3 expression at the RNA and protein levels in EGFR-mutant tumors treated with osimertinib for 2 or 4 weeks. Additionally, circulating levels of IL-6 also are elevated in EGFRL858R GEMMs treated with osimertinib for 2 or 4 weeks. Collectively, our data indicates that upregulation of IL-6 is an early event in the evolution of drug resistance. Given the role of IL-6 in promoting EGFR TKI resistance, further studies investigating whether targeting of IL-6 signaling may impair the survival or outgrowth of persister populations are warranted. Citation Format: Sonia A. Patel, Monique Nilsson, Yan Yang, Li Shen, Jing Wang, Alissa Poteete, Xiaoxing Yu, Xiaoyang Ren, Xiuning Le, John Heymach. Targeting IL-6/STAT3 signaling in EGFR-mutant drug tolerant persister cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3864.

Combined therapy with methotrexate nanoconjugate and dendritic cells with downregulated IL-10R expression modulates the tumor microenvironment and enhances the systemic anti-tumor immune response in MC38 murine colon carcinoma.

Understanding the negative impact of the tumor microenvironment on the creation of an effective immune response has contributed to the development of new therapeutic anti-cancer strategies. One such solution is combined therapy consisting of chemotherapeutic administration followed by dendritic cell (DC)-based vaccines. The use of cytostatic leads to the elimination of cancer cells, but can also modulate the tumor milieu. Moreover, great efforts are being made to increase the therapeutic outcome of immunotherapy, e.g. by enhancing the ability of DCs to generate an efficient immune response, even in the presence of immunosuppressive cytokines such as IL-10. The study aimed to determine the effectiveness of combined therapy with chemotherapeutic with immunomodulatory potential - HES-MTX nanoconjugate (composed of methotrexate (MTX) and hydroxyethyl starch (HES)) and DCs with downregulated expression of IL-10 receptor stimulated with tumor antigens (DC/shIL-10R/TAg) applied in MC38 murine colon carcinoma model. With the use of lentiviral vectors the DCs with decreased expression of IL-10R were obtained and characterized. During in vivo studies MC38-tumor bearing mice received MTX or HES-MTX nanoconjugate as a sole treatment or combined with DC-based immunotherapy containing unmodified DCs or DCs transduced with shRNA against IL-10R (or control shRNA sequence). Tumor volume was monitored during the experiment. One week after the last injection of DC-based vaccines, tumor nodules and spleens were dissected for ex vivo analysis. The changes in the local and systemic anti-tumor immune response were estimated with the use of flow cytometry and ELISA methods. In vitro studies showed that the downregulation of IL-10R expression in DCs enhances their ability to activate the specific anti-tumor immune response. The use of HES-MTX nanoconjugate and DC/shIL-10R/TAg in the therapy of MC38-tumor bearing mice resulted in the greatest tumor growth inhibition. At the local anti-tumor immune response level a decrease in the infiltration of cells with suppressor activity and an increase in the influx of effector cells into MC38 tumor tissue was observed. These changes were crucial to enhance the effective specific immune response at the systemic level, which was revealed in the greatest cytotoxic activity of spleen cells against MC38 cells.

Piceatannol induces regulatory T cells and modulates the inflammatory response and adipogenesis

The beneficial effects of the polyphenolic compound piceatannol (PC) has been reported for metabolic diseases, antiproliferative, antioxidant, and anti-cancer properties. Despite its beneficial effects on inflammatory diseases, little is known about how PC regulates inflammatory responses and adipogenesis. Therefore, this study was designed to determine the effects of PC on the inflammatory response and adipogenesis. The effect of PC on splenocytes, 3T3-L1 adipocytes, and RAW264.7 macrophages was analyzed by flow cytometry, qRT-PCR, morphometry, and western blot analysis. PC induced apoptosis in activated T cells in a dose-dependent manner using stimulated splenocytes and reduced the activation of T cells, altered T cell frequency, and interestingly induced the frequency of regulatory T (Treg) cells as compared to controls. PC suppressed the expression of TNF-α, iNOS, IL-6R, and NF-κB activation in RAW264.7 macrophages after lipopolysaccharides (LPS)-induction as compared to the control. Interestingly, PC altered the cell morphology of 3T3-L1 adipocytes with a concomitant decrease in cell volume, lipid deposition, and TNF-α expression, but upregulation of leptin and IL-1β. Our findings suggested that PC induced apoptosis in activated T cells, decreased immune cell activation and inflammatory response, and hindered adipogenesis. This new set of data provides promising hope as a new therapeutic to treat both inflammatory disease and obesity.

Increased expression of serum IL-18 and IL-18R in newly diagnosed type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a heterogeneous metabolic disorder in which genetic, sedentary lifestyle, obesity, and environmental factors come together to produce insulin resistance in target tissues, leading to hyperglycemia. Evidence reveals that inflammation may play an essential role in the pathogenesis of T2DM. Interleukin-18 (IL-18), a proinflammatory cytokine, plays a crucial role in the acute and chronic inflammatory process. The association of IL-18 levels with IL-18R expression in T2DM has not been investigated so far. The aim of this study was to compare the peripheral changes in serum IL-18 levels and its receptor (IL18R) expression in newly diagnosed T2DM and healthy controls. A total of 35 newly diagnosed type 2 diabetic cases and 35 non-diabetic controls were enrolled after obtaining informed consent. Venous whole blood was taken under aseptic conditions. Biochemical parameters were estimated in an auto-analyzer. Serum IL-18 levels were calculated using ELISA, whereas IL-18R expression was determined via RT-PCR. GAPDH was used as an internal control. When compared to non-diabetic controls, the serum IL-18 levels were significantly higher in T2DM patients (P=0.010) along with a significant upregulation of IL18R (P=0.0018). Serum IL-18 levels in T2DM and non-diabetic controls were 669.5 (445) and 498.3 (404.9) pg/mL respectively, and IL-18R showed a fold change of 10.33. Both serum IL-18 and its receptor IL-18R is significantly higher in newly diagnosed T2DM patients.

Screening of core genes prognostic for sepsis and construction of a ceRNA regulatory network

ObjectiveTo screen out core genes potentially prognostic for sepsis and construct a competing endogenous RNA (ceRNA) regulatory network.MethodsSubjects included in this project were 23 sepsis patients and 10 healthy people. RNA-seq for lncRNA, miRNA and mRNA was performed in the peripheral blood samples. Differentially expressed RNAs (DER) were screened out for further analysis. GO annotation and GSEA functional clustering were performed to view the functional enrichment of DEmRNAs. Core genes of prognostic significance were screened out with the weighted correlation network analysis (WGCNA). Meta-analysis and Survival analysis was devised in different microarray datasets. RT-qPCR was conducted to validate these core genes. A ceRNA network was accordingly constructed according to the correlation analysis and molecular interaction prediction.ResultsRNA-seq and differential analysis screened out 1,044 DEmRNAs, 66 DEmiRNAs and 155 DElncRNAs. The GO and GSEA analysis revealed that DEmRNAs are mainly involved in inflammatory response, immune regulation, neutrophil activation. WGCNA revealed 4 potential core genes, including CD247, IL-2Rβ, TGF-βR3 and IL-1R2. In vitro cellular experiment showed up-regulated expression of IL-1R2 while down-regulated of CD247, IL-2Rβ, TGF-βR3 in sepsis patients. Correspondingly, a ceRNA regulatory network was build based on the core genes, and multiple lncRNAs and miRNAs were identified to have a potential regulatory role in sepsis.ConclusionThis study identified four core genes, including CD247, IL-1R2, IL-2Rβ and TGF-βR3, with potential to be novel biomarkers for the prognosis of sepsis. In the meantime, a ceRNA network was constructed aiming to guide further study on prognostic mechanism in sepsis.

Interferon-related gene array in predicting the efficacy of interferon therapy in chronic hepatitis B

This study aims to clarify host factors of IFN treatment in the treatment of chronic hepatitis B (CHB) patients by screening the differentially expressed genes of IFN pathway CHB patients with different response to interferon (IFN) therapy. Three cases were randomly selected in IFN-responding CHB patients (Rs), non-responding CHB patients (NRs) and healthy participants, respectively. The human type I IFN response RT 2 profiler PCR array was used to detect the expression levels of IFN-related genes in peripheral blood monocytes (PBMCs) from healthy participants and CHB patients before and after Peg-IFN-α 2a treatment. The results showed that more differentially expressed genes appeared in Rs group than NRs group after IFN treatment. Comparing with healthy participants, IFNG, IL7R, IRF1, and IRF8 were downregulated in both Rs and NRs group before IFN treatment; CXCL10, IFIT1, and IFITM1 were upregulated in the Rs; IL13RA1 and IFI35 were upregulated in the NRs, while IFRD2, IL11RA, IL4R, IRF3, IRF4, PYHIN1, and ADAR were downregulated. The expression of IL15, IFI35 and IFI44 was downregulated by 4.09 ( t = 10.58, P < 0.001), 5.59 ( t = 3.37, P = 0.028) and 10.83 ( t = 2.8, P = 0.049) fold in the Rs group compared with the NRs group, respectively. In conclusion, IFN-response-related gene array is able to evaluate IFN treatment response by detecting IFN-related genes levels in PBMC. High expression of CXCL10, IFIT1 and IFITM1 before treatment may suggest satisfied IFN efficacy, while high expression of IL13RA1, IL15, IFI35 and IFI44 molecules and low expression of IFRD2, IL11RA, IL4R, IRF3, IRF4, PYHIN1 and ADAR molecules may be associated with poor IFN efficacy.

Decoy Receptors Regulation by Resveratrol in Lipopolysaccharide-Activated Microglia.

Resveratrol is a polyphenol that acts as antioxidants do, protecting the body against diseases, such as diabetes, cancer, heart disease, and neurodegenerative disorders, such as Alzheimer's (AD) and Parkinson's diseases (PD). In the present study, we report that the treatment of activated microglia with resveratrol after prolonged exposure to lipopolysaccharide is not only able to modulate pro-inflammatory responses, but it also up-regulates the expression of decoy receptors, IL-1R2 and ACKR2 (atypical chemokine receptors), also known as negative regulatory receptors, which are able to reduce the functional responses promoting the resolution of inflammation. This result might constitute a hitherto unknown anti-inflammatory mechanism exerted by resveratrol on activated microglia.

Acute IL-6 exposure triggers canonical IL6Ra signaling in hiPSC microglia, but not neural progenitor cells

BackgroundPrenatal exposure to elevated interleukin (IL)-6 levels is associated with increased risk for psychiatric disorders with a putative neurodevelopmental origin, such as schizophrenia (SZ), autism spectrum condition (ASC) and bipolar disorder (BD). Although rodent models provide causal evidence for this association, we lack a detailed understanding of the cellular and molecular mechanisms in human model systems. To close this gap, we characterized the response of human induced pluripotent stem cell (hiPSC-)derived microglia-like cells (MGL) and neural progenitor cells (NPCs) to IL-6 in monoculture. ResultsWe observed that human forebrain NPCs did not respond to acute IL-6 exposure in monoculture at both protein and transcript levels due to the absence of IL6R expression and soluble (s)IL6Ra secretion. By contrast, acute IL-6 exposure resulted in STAT3 phosphorylation and increased IL6, JMJD3 and IL10 expression in MGL, confirming activation of canonical IL6Ra signaling. Bulk RNAseq identified 156 up-regulated genes (FDR < 0.05) in MGL following acute IL-6 exposure, including IRF8, REL, HSPA1A/B and OXTR, which significantly overlapped with an up-regulated gene set from human post-mortem brain tissue from individuals with schizophrenia. Acute IL-6 stimulation significantly increased MGL motility, consistent with gene ontology pathways highlighted from the RNAseq data and replicating rodent model indications that IRF8 regulates microglial motility. Finally, IL-6 induces MGLs to secrete CCL1, CXCL1, MIP-1α/β, IL-8, IL-13, IL-16, IL-18, MIF and Serpin-E1 after 3 h and 24 h. ConclusionOur data provide evidence for cell specific effects of acute IL-6 exposure in a human model system, ultimately suggesting that microglia-NPC co-culture models are required to study how IL-6 influences human cortical neural progenitor cell development in vitro.

The effect of microRNA-663b in the inhibition of interleukin-1-induced nucleus pulposus cell apoptosis and inflammatory response.

The aim of this study was to explore the role and pathological mechanism of microRNA-663b in interleukin-1beta (IL-1β)-induced inflammation and apoptosis of nucleus pulposus cells. First, the best concentration and time to construct the nucleus pulposus cell inflammation model was screen out. Overexpression or inhibition of miR-663b expression was performed by adding microRNA-663b mimic or microRNA-663b inhibitor. 293T cells were transfected according to experimental requirements. The luciferase activity of each group was detected to analyze the targeted regulation of microRNA-663b on interleukin-1 receptor (IL1R1). Compared with the mimic negative control (NC) group, the expression of inflammatory factors in the microRNA-663b overexpression group was inhibited (P<0.05), and the expression of type 2 collagen and polysaccharide protein increased (P<0.05), and the apoptosis of nucleus pulposus cells was inhibited (P<0.01), and the number of TUNEL-positive cells decreased significantly (P<0.01), and the microRNA and protein expression of IL1R1, the ratio of P-P65/P65 and phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (P-IκBα)/nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) protein expression were significantly decreased (P<0.05). The expression of inflammatory factors in the miR-663b inhibitor group was significantly higher than that in the inhibitor NC group (P<0.01), and the expression of type 2 collagen and polysaccharide protein was significantly decreased (P<0.01), and the number of apoptosis cells and TUNEL staining positive cells increased (p<0.01). The expression of IL1R1 gene and protein was significantly increased (P<0.01). The ratio of P-P65/P65 and P-IκBα/IκBα protein expression increased (P<0.05). IL1R1 is a downstream target gene of microRNA-663b. MicroRNA-663b may down-regulate the expression of IL1R1 at the transcriptional level by targeting IL1R1, inhibit the inflammatory response of nucleus pulposus cells, and slow down the degeneration of nucleus pulposus cells.

The effect of microRNA-663b in the inhibition of interleukin-1-induced nucleus pulposus cell apoptosis and inflammatory response.

The aim of this study was to explore the role and pathological mechanism of microRNA-663b in interleukin-1beta (IL-1β)-induced inflammation and apoptosis of nucleus pulposus cells. First, the best concentration and time to construct the nucleus pulposus cell inflammation model was screen out. Overexpression or inhibition of miR-663b expression was performed by adding microRNA-663b mimic or microRNA-663b inhibitor. 293T cells were transfected according to experimental requirements. The luciferase activity of each group was detected to analyze the targeted regulation of microRNA-663b on interleukin-1 receptor (IL1R1). Compared with the mimic negative control (NC) group, the expression of inflammatory factors in the microRNA-663b overexpression group was inhibited (P<0.05), and the expression of type 2 collagen and polysaccharide protein increased (P<0.05), and the apoptosis of nucleus pulposus cells was inhibited (P<0.01), and the number of TUNEL-positive cells decreased significantly (P<0.01), and the microRNA and protein expression of IL1R1, the ratio of P-P65/P65 and phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (P-IκBα)/nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) protein expression were significantly decreased (P<0.05). The expression of inflammatory factors in the miR-663b inhibitor group was significantly higher than that in the inhibitor NC group (P<0.01), and the expression of type 2 collagen and polysaccharide protein was significantly decreased (P<0.01), and the number of apoptosis cells and TUNEL staining positive cells increased (p<0.01). The expression of IL1R1 gene and protein was significantly increased (P<0.01). The ratio of P-P65/P65 and P-IκBα/IκBα protein expression increased (P<0.05). IL1R1 is a downstream target gene of microRNA-663b. MicroRNA-663b may down-regulate the expression of IL1R1 at the transcriptional level by targeting IL1R1, inhibit the inflammatory response of nucleus pulposus cells, and slow down the degeneration of nucleus pulposus cells.

Follicular fluid progesterone downregulated HPGD and COX2 in granulosa cells via suppressing NF-ĸB in endometriosis†.

Increasing evidences showed that ovulatory dysfunction, possibly caused by luteinized unruptured follicular follicle syndrome (LUFS), is one of the reasons for endometriosis-related infertility. The present study was conducted to explore the potential effect of elevated progesterone in follicular fluid (FF) on ovulation in endometriosis. A prospective study including 50 ovarian endometriosis patients and 50 control patients with matched pairs design was conducted with alterations in FF and peritoneal fluid (PF) components identified by metabolomics analyses and differentially expressed genes in granulosa cells (GCs) identified by transcriptome analysis. Patients with endometriosis exhibited a significantly higher progesterone level in serum, FF, and PF. Granulosa cells from endometriosis patients revealed decreased expression of HPGD, COX-2, and suppressed NF-ĸB signaling. Similarly, progesterone treatment in vitro downregulated HPGD and COX2 expression and suppressed NF-ĸB signaling in granulosa tumor-like cell line KGN (Bena Culture Collection, China) and primarily cultured GCs, as manifested by decreased expressions of IL1R1, IRAK3, reduced pIĸBα/IĸBα ratio, and nucleus translocation of p65. On the contrary, TNF-α treatment increased expression of IL1R1, IRAK3, pIĸBα, p65, and HPGD in GCs. One potential p65 binding site was identified in the promoter region of HPGD by chromatin immunoprecipitation. In conclusion, we found that intrafollicular progesterone might downregulate HPGD and COX-2 in GCs via suppressing the NF-ĸB signaling pathway, shedding light on the mechanism underlying the endometriosis-related ovulatory dysfunction.

P721 Treatment of Ulcerative Colitis with OSE-127 (lusvertikimab), a Strict IL-7Rα Antagonist, Non-Cytotoxic Monoclonal Antibody

Abstract Background Key transcripts of the IL-7R pathway were shown to accumulate in inflamed colon tissues of severe IBD patients not responding to conventional or biologics therapies. Furthermore, high expression of both IL7R and IL-7R signaling signature in the colon before treatment is strongly associated with non-responsiveness to anti-TNF therapy. We have designed a humanized monoclonal antibody targeting the IL-7Rα chain, named OSE-127 (lusvertikimab), which displays strict antagonist activity without cytotoxicity nor internalizing properties. In an ex vivo culture system using colon biopsies from UC patients, OSE-127 altered synthesis of interferon γ by Teff. In addition, IL-7R blockade by OSE-127 in humanized mouse models reduced human T cell homing to the gut and colonic inflammation (Belarif L et al, 2019). Methods A first-in-human, phase I, randomized, double-blind, placebo-controlled, single-center study was carried out to determine the safety, pharmacokinetics and pharmacodynamics of OSE-127 administration. Results Sixty-three healthy subjects were randomly assigned to OSE-127 or placebo with 45 of them exposed to the active product in 9 groups: 6 single ascending dose groups with intravenous (IV) administration (0.002-10 mg/kg), 1 subcutaneous treatment group (1 mg/kg) and 2 double IV infusion groups (6 or 10 mg/kg). OSE-127 was well tolerated, with no evidence of cytokine-release syndrome and no significant alteration of blood lymphocyte counts or subset populations. OSE-127’s pharmacokinetic half-life after a single dose increased from 4.6 days (1mg/kg) to 11.7 days (10 mg/kg) and, after a second dose, from 12.5 days (6 mg/kg) to 16.25 days (10 mg/kg). Receptor occupancy was ≥ 95% at doses ≥ 0.02 mg/kg and this saturation level was maintained up to &amp;gt;100 days after 2 IV infusions at 10 mg/kg. Furthermore, a differential gene expression signature performed in peripheral leukocytes, pre- and post-OSE-127 administration, identified six IL-7 pathway-related genes (BCL2, CISH, PTGER2, DPP4, SOCS2 and FLT3LG). Interestingly, decreased expression of this signature was sustained overtime in accordance with the systemic drug exposure. The differential expression of 4 of these 6 genes (BCL2, CISH, PTGER2, DPP4) has been individually validated by quantitative RT-qPCR, and this novel 4-gene signature appears to follow a dose-dependent expression. Conclusion Our data provide evidence that IL-7 receptor can be blocked in humans without inducing serious adverse events. The signature identified in the peripheral leukocytes of subjects having received OSE-127 represents a robust and simple means to monitor target engagement in the clinical setting. OSE-127 is currently being evaluated in a Phase 2b study in UC patients (CoTikiS study: NCT04882007).

Impact of SIV infection on mycobacterial lipid-reactive T cell responses in Bacillus Calmette-Guérin (BCG) inoculated macaques.

Although BCG vaccine protects infants from tuberculosis (TB), it has limited efficacy in adults against pulmonary TB. Further, HIV coinfection significantly increases the risk of developing active TB. In the lack of defined correlates of protection in TB disease, it is essential to explore immune responses beyond conventional CD4 T cells to gain a better understanding of the mechanisms of TB immunity. Here, we evaluated unconventional lipid-reactive T cell responses in cynomolgus macaques following aerosol BCG inoculation and examined the impact of subsequent SIV infection on these responses. Immune responses to cellular lipids of M. bovis and M. tuberculosis were examined ex vivo in peripheral blood and bronchioalveolar lavage (BAL). Prior to BCG inoculation, innate-like IFN-γ responses to mycobacterial lipids were observed in T cells. Aerosol BCG exposure induced an early increase in frequencies of BAL γδT cells, a dominant subset of lipid-reactive T cells, along with enhanced IL-7R and CXCR3 expression. Further, BCG exposure stimulated greater IFN-γ responses to mycobacterial lipids in peripheral blood and BAL, suggesting the induction of systemic and local Th1-type response in lipid-reactive T cells. Subsequent SIV infection resulted in a significant loss of IL-7R expression on blood and BAL γδT cells. Additionally, IFN-γ responses of mycobacterial lipid-reactive T cells in BAL fluid were significantly lower in SIV-infected macaques, while perforin production was maintained through chronic SIV infection. Overall, these data suggest that despite SIV-induced decline in IL-7R expression and IFN-γ production by mycobacterial lipid-reactive T cells, their cytolytic potential is maintained. A deeper understanding of anti-mycobacterial lipid-reactive T cell functions may inform novel approaches to enhance TB control in individuals with or without HIV infection.

Carnosol Alleviates Collagen-Induced Arthritis by Inhibiting Th17-Mediated Immunity and Favoring Suppressive Activity of Regulatory T Cells.

Current approaches are incurable for rheumatoid arthritis (RA). Regulatory T (Treg) cells and T helper cells (Th1 and Th17) are crucial in controlling the process of RA, which is characterized by inflammatory cell infiltration and bone destruction. Carnosol is an orthodiphenolic diterpene that has been extensively applied in traditional medicine for the treatment of multiple autoimmune and inflammatory diseases. Herein, we indicate that administration of carnosol dramatically alleviated the severity of collagen-induced arthritis (CIA) model with a decreased clinical score and inflammation reduction. Cellular mechanistically, carnosol inhibits the Th17 cell differentiation and maintains Treg cell suppressive function in vitro and in vivo. Meanwhile, it also restrains Treg cells from transdifferentiation into Th17 cells under inflammatory milieu. Furthermore, carnosol modulates the function of Th17 and Treg cells possibly via limiting IL-6R (CD126) expression. Collectively, our results suggest that carnosol can alleviate the severity of CIA via hiding Th17 cell differentiation and maintain the stability of Treg cells. Administration of carnosol can be applied as a potential therapy for patients with RA.

Mechanisms of Panax Ginseng on Treating Sepsis by RNA-Seq Technology.

To explore the potential active targets and mechanisms of Panax Ginseng in the treatment of sepsis using network pharmacology and RNA-seq technology. Patients with sepsis and healthy volunteers were collected according to SEPSIS 3.0, and their peripheral blood was used for RNA-seq analysis. The active ingredients and targets of Panax Ginseng were obtained using the TCMSP database, PPI and GO analysis were performed for disease-drug intersection targets. Then, we used Meta-analysis to screen core genes. Finally, single-cell RNA-seq was used to perform cell localization analysis on core genes. RNA-seq analysis collected 4521 sepsis-related genes, TCMSP database obtained 86 Panax Ginseng active ingredients and their 294 active targets. PPI and GO analysis showed intersection targets were closely linked, and mainly involved in cellular response to chemical stress, response to drug and molecule of bacterial origin, etc. Then, core targets, IL1B, ALOX5, BCL2 and IL4R, were sorted by Meta-analysis, and all four genes have high expression in the sepsis survivor group compared to the sepsis non-survivor group; single-cell RNA-seq revealed that IL1B was mainly localized in macrophages, ALOX5 was mainly localized in macrophages and B cells, BCL2 was mainly localized in natural killer cells, T cells and B cells, IL4R was widely distributed in immune cells. Finally, according to the correspondence between the active ingredients and targets of Panax Ginseng in TCMSP database, we found that Ginsenoside rh2 regulates the expression of IL1B, Ginsenoside rf regulates the expression of IL1B and IL4R, Kaempferol regulates the expression of ALOX5 and BCL2, and β-sitosterol regulates the expression of BCL2. Ginsenoside rh2, Ginsenoside rf, Kaempferol and β-sitosterol may produce anti-sepsis effects by regulating the expression of IL1B, ALOX5, BCL2 and IL4R, thus improving the survival rate of sepsis patients.

The Mechanism of Resveratrol on Acute T-Lymphocyte Leukemia through IL-7-Mediated JAK / STAT Signaling Pathway

To explore the roles and relative mechanism of resveratrol against T-ALL through detecting the signaling molecules in IL-7 and JAK/STAT pathway. In vitro experiments, Molt4 cells were divided into 3 groups, including the control group, the DMSO group and resveratrol-treated group (Res group). The control group cells without any treatment, the DMSO group cells treated with 0.05% DMSO for 48 hours, the Res group cells treated with 200 μmol/L resveratrol for 48 hours. In vivo experiments, female C57BL/6J mice (6-8 weeks) were randomly divided into the control group, the T-ALL model group (T-ALL group), and Res treatment group (Res group). The control group mice treated with 0.05% DMSO by intragastric treatment, the T-ALL group mice treated with 0.05% DMSO by intragastric treatment, and the Res group mice treated with 10 mg/ml resveratrol. Expression of IL-7, IL-7R and Pim1 mRNA in the cells and mice spleen tissues were detected by RT-qPCR. Cell proliferation ability was detected by CCK-8. The expression of JAK1, JAK3, STAT5, phosphorylated JAK1 (p-JAK1), phosphorylated JAK3 (p-JAK3), phosphorylated STAT5 (p-STAT5) and Pim1 were detected by Western blot. ELISA was used to detect the IL-7 and IL-7R in the cells and mice serum of each groups. Resveratrol could inhibit the proliferation ability of Molt4 cells, decrease the relative levels of p-JAK1, p-JAK3, p-STAT5, Pim1 protein, and the expression levels of Pim1, IL-7 and IL-7R mRNA in cells and mice spleens, reduce the IL-7 and IL-7R in Molt4 cells and mice serum. Resveratrol may inhibite IL-7-medicated JAK/STAT signaling pathway to reduce the expression of target protein Pim1 to further exert its anti-T-ALL effects.

IL-1β enhances cell viability and decreases 5-FU sensitivity in novel colon cancer cell lines derived from African American patients.

In the U.S., African Americans (AAs) present with the highest incidence and mortality rates for Colorectal Cancer (CRC). When compared to Caucasian American (CA) patients, AAs also have reduced response to the first line standard of care chemotherapeutic agent 5-Fluorouracil (5-FU). Previously, we observed differential gene expression between the two populations, suggesting that colon tumors from AA patients display a decreased antitumor immune response and an increased expression of genes encoding proteins involved in inflammatory processes, such as Interleukin-1β (IL-1β). Here, we investigate the role of IL-1β in modifying chemotherapeutic response and altering expression of proteins in novel AA and well-established CA colon cancer cell lines. RNA sequencing analysis was performed to detect expression of genes involved in inflammation in AA and CA colon cancer cells. The effects of IL-1β on 5-FU response was evaluated by assessing cell viability (MTS assay) and apoptosis (flow cytometry analysis) following treatment with 5-FU alone or in combination with the cytokine. Further, we used an IL-1 receptor antagonist (IL-1Ra) to inhibit IL-1β-induced effects on 5-FU sensitivity and NF-kB pathway activation. AA colon cancer cell lines present significant increase in expression of genes IL1R2 (373-fold change (FC), IRAK1 (3.24 FC), IKBKB, (5.33 FC) NF-KB IA (5.95 FC), MYD88, (3.72 FC), IRAK3 (161 FC), TRAF5 (4.1 FC). A significant decrease in the response to 5-FU treatment, as well as a significant increase in phosphorylation of IκBα and secretion of IL-8, was seen following IL-1β treatment, in both AA and CA cell lines. Finally, treatment with IL-1Ra was able to reverse the effects induced by IL-1β, by increasing the cells sensitivity to 5-FU. IL-1Ra also inhibited phosphorylation of IκBα and IL-8 secretion. Our results suggest a differential expression of inflammatory genes and proteins that might regulate the different response to IL-1β between AA and CA colon cancer cell lines. Our data also demonstrates that IL-1β is involved in modulating 5-FU response in both AA and CA colon cancer cell lines. Further investigation of these mechanisms might help elucidate the differences seen in incidence, mortality and response to therapy in AA colon cancer patients.

5-Fluorouracil Induces an Acute Reduction in Neurogenesis and Persistent Neuroinflammation in a Mouse Model of the Neuropsychological Complications of Chemotherapy.

The neuropsychological symptoms associated with chemotherapy treatment remain a major challenge with their prevention hampered by insufficient understanding of pathophysiology. While long-term neuroimmune changes have been identified as a hallmark feature shared by neurological symptoms, the exact timeline of mechanistic events preceding neuroinflammation, and the relationship between the glial cells driving this neuroinflammatory response, remain unclear. We therefore aimed to longitudinally characterize the neuroimmunological changes following systemic 5-fluorouracil (5-FU) treatment to gain insight into the timeline of events preceding the well-documented chronic neuroinflammation seen following chemotherapy. Eighteen female C57Bl/6 mice received a single intraperitoneal dose of 5-FU and groups were killed at days 1 and 2 (acute timepoint), days 4 and 8 (subacute timepoint), and days 16 and 32 (chronic timepoint). A further six mice were administered with vehicle control with tissues collected from three mice on day 1 and day 32 of the study. The expression of key genes of interest, BCL2, BDNF, TIMP1, MMP-9, MMP-2, TNFα, IL-1β, and IL-6R were assessed using real time polymerase chain reaction. Levels of neurogenesis were determined through immunofluorescent staining of doublecortin (DCX). The density of microglia and astrocytes were assessed using immunofluorescence staining of Iba1 and GFAP respectively. 5-FU treatment caused significant decreases to DCX staining at acute timepoints (p = 0.0030) which was positively correlated with BCL2 expression levels. An increase to microglial density was observed in the prefrontal cortex (p = 0.0256), CA3 region (p = 0.0283), and dentate gyrus (p = 0.0052) of the hippocampus at acute timepoints. 5-FU caused increases to astrocyte density, across multiple brains regions, at subacute and chronic timepoints which were positively correlated with TNFα, TIMP-1, MMP-2, and IL-6R expression. This study has identified acute objective neuroinflammatory changes suggesting that the role of early intervention should be explored to prevent the development of neuropsychological deficits in the longer-term following chemotherapy.

In patients with primary Sjögren's syndrome innate-like MAIT cells display upregulated IL-7R, IFN-γ, and IL-21 expression and have increased proportions of CCR9 and CXCR5-expressing cells.

Mucosal-associated invariant T (MAIT) cells might play a role in B cell hyperactivity and local inflammation in primary Sjögren's syndrome (pSS), just like previously studied mucosa-associated CCR9+ and CXCR5+ T helper cells. Here, we investigated expression of CCR9, CXCR5, IL-18R and IL-7R on MAIT cells in pSS, and assessed the capacity of DMARDs to inhibit the activity of MAIT cells. Circulating CD161+ and IL-18Rα+ TCRVα7.2+ MAIT cells from pSS patients and healthy controls (HC) were assessed using flow cytometry, and expression of CCR9, CXCR5, and IL-7R on MAIT cells was studied. Production of IFN-γ and IL-21 by MAIT cells was measured upon IL-7 stimulation in the presence of leflunomide (LEF) and hydroxychloroquine (HCQ). The numbers of CD161+ and IL-18Rα+ MAIT cells were decreased in pSS patients compared to HC. Relative increased percentages of CD4 MAIT cells in pSS patients caused significantly higher CD4/CD8 ratios in MAIT cells. The numbers of CCR9 and CXCR5-expressing MAIT cells were significantly higher in pSS patients. IL-7R expression was higher in CD8 MAIT cells as compared to all CD8 T cells, and changes in IL-7R expression correlated to several clinical parameters. The elevated production of IL-21 by MAIT cells was significantly inhibited by LEF/HCQ treatment. Circulating CD161+ and IL-18Rα+ MAIT cell numbers are decreased in pSS patients. Given their enriched CCR9/CXCR5 expression this may facilitate migration to inflamed salivary glands known to overexpress CCL25/CXCL13. Given the pivotal role of IL-7 and IL-21 in inflammation in pSS this indicates a potential role for MAIT cells in driving pSS immunopathology.

IL1R1 Expression Predicts the Benefit from Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia and Intermediate-Risk Cytogenetics

Introduction: Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy with variable clinical outcomes depending on baseline patient and disease characteristics. After achieving complete remission (CR) with induction chemotherapy, eligible patients are treated with consolidation chemotherapy or allogeneic hematopoietic stem cell transplantation (HSCT) to prevent relapse. In patients with intermediate-risk cytogenetics (IRC) without adverse-risk mutations, the decision for post-remission therapy is challenging because of the significant risks of morbidity and mortality associated with HSCT which can offset its benefit in preventing relapse. Therefore, novel biomarkers are needed to support the decision to recommend HSCT in first CR (CR1) in patients with IRC AML. Methods: We used the Leucegene AML prognostic cohort composed of patients diagnosed with de novo IRC AML and treated with intensive induction chemotherapy (N = 316) to identify biomarkers that are predictive for the benefit of HSCT in CR1. Whole-transcriptome sequencing was performed on diagnostic patients' samples. Gene expression data was normalized in transcripts per million (TPM) then log transformed and standardized into Z-scores. We used univariable and multivariable Cox proportional hazards (CPH) regression models for overall survival (OS) and relapse-free survival (RFS) to evaluate the association between gene expression and clinical outcomes. For multivariable analyses (MVA), we included age and white blood cell (WBC) count at diagnosis and NPM1, FLT3-ITD, DNMT3A, biallelic CEBPA, ASXL1 and RUNX1 mutations as covariables. To identify genes predictive of the benefit of HSCT in CR1, we evaluated interaction terms between gene expression and HSCT in CR1 as a time-dependent variable (HSCT-TD) in extended CPH regression models for OS and RFS. We dichotomized the expression of genes based on optimal cutoffs for p value and hazard ratio (HR) for OS and RFS while optimizing their clinical utility to predict the benefit of HSCT. Results: We identified IL1R1 as the top gene associated with RFS in MVA and with a significant interaction with HSCT-TD (p < 0.05). Based on an optimal cutoff of 2.0 TPM, 193 (61%) and 123 (39%) patients had low (< 2.0 TPM, IL1R1low) and high (≥ 2.0 TPM, IL1R1high) expression of IL1R1, respectively. Patients with IL1R1high were older (median age 59 years with IL1R1high vs 54 years with IL1R1low, p < 0.01), had a higher WBC count (44 vs 31 x 109/L, p = 0.06), and a higher frequency of myelomonocytic or monocytic differentiation (43% vs 25%, p < 0.01). FLT3-ITD (54% vs 33%, p < 0.01) and RUNX1 (18% vs 9%, p = 0.04) mutations were more frequent in patients with IL1R1high whereas biallelic CEBPA mutations (1% vs 6%, p=0.04) were more frequent in patients with IL1R1low. High expression of IL1R1 was associated with worse clinical outcomes. The CR rate was 74% and 85% in patients with IL1R1high and IL1R1low, respectively (p = 0.02). The 5-year OS rate was 10% in patients with IL1R1high vs 38% in patients with IL1R1low (HR 2.27, p < 0.01). The 5-year cumulative incidence of relapse (CIR) was 17% higher in patients with IL1R1high (76% vs 59%, p < 0.01). In MVA, high expression of IL1R1 was independently associated with OS (HR 1.80, p < 0.01) and RFS (HR 1.81, p < 0.01). HSCT in CR1 significantly improved OS in patients with IL1R1high (HR for HSCT-TD 0.26, p < 0.01), but not in patients with IL1R1low (HR 0.70, p = 0.17). With a 6-month landmark analysis, the 5-year OS rates were 67% vs 27% among patients with IL1R1high and 62% vs 54% among patients with IL1R1low in patients who underwent HSCT in CR1 or not, respectively (Figure 1). Specifically among patients without FLT3-ITD mutations, patients with IL1R1high benefited from HSCT in CR1 (HR 0.41, p = 0.04) whereas patients with IL1R1low did not (HR 1.03, p = 0.92). Among patients who have undergone HSCT in CR1, the 5-year post-HSCT OS was 60% vs 56% in patients with IL1R1high and IL1R1low, respectively (HR 0.85, p = 0.68), confirming that HSCT may abrogate the adverse impact of high IL1R1 expression. Conclusion: In patients with IRC AML, high expression of IL1R1 is independently associated with worse OS and RFS which may be overcome by HSCT in CR1. Among patients without FLT3-ITD mutations who account for ~60% of IRC AML, those with IL1R1high benefit from HSCT in CR1, as opposed to those with IL1R1low. IL1R1 expression may guide the decision to proceed with HSCT in CR1 in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal