Interleukin-17 (IL-17) Enhancement Of The Positive Feedback Loop Of IL-6 In Astrocytes

BackgroundAstrocytes are the major glial cell type within the CNS and are critical for CNS homeostasis. Astrocytes regulate neuronal function by releasing neurotrophic factors, guiding neuronal development, contributing to neurotransmitter metabolism, and participating in the formation of the blood-brain-barrier. Astrocytes can serve as a bridge between the CNS and the immune system. In particular, astrocytes produce a wide array of chemokines and cytokines that act as immune mediators in cooperation with those produced by microglia. Aberrant expression of chemokines and cytokines accompanies CNS disorders such as Multiple Sclerosis (MS), Alzheimer's Disease, HIV-1-Associated Dementia, and brain injury/trauma. However, the mechanisms by which astrocytes contribute to these disorders remains unclear.IL-6 is a regulator of inflammatory and immunological responses and acts on target cells through a receptor complex composed of an IL-6-binding subunit, the IL-6 receptor (IL-6R), and the signal transducing receptor gp130. Initiation of IL-6 signaling occurs when IL-6 binds to the IL-6R, leading to an association with gp130. This leads to activation of gp130-associated Janus Kinases (JAKs), and various signaling pathways such as JAK/STAT, MAPK, and NF-κB. The IL-6R is found in both membrane-bound and soluble (sIL-6R) forms. Astrocytes are the major source of IL-6 in CNS injury and neuroinflammation. We previously determined that the IL-6/sIL-6R complex plays an important role in regulation of IL-6 expression by astrocytes, particularly in conjunction with the proinflammatory cytokines TNF-a and IL-1p.Th17 cells produce IL-17A (IL-17), IL-17F, IL-21 and IL-22, and are required for the induction of several autoimmune diseases, including collagen-induced arthritis, experimental autoimmune encephalomyelitis (EAE), and inflammatory bowel disease. Th17 cells are generated as a discrete lineage following priming in the presence of TGF-βand IL-6, and expansion in the presence of IL-23 and IL-1β. IL-6 not only functions upstream of IL-17 but also acts as a critical downstream target of IL-17, and IL-17 together with IL-6 triggers a positive-feedback loop of IL-6 expression through the activation of NF-κB and STAT-3 in fibroblasts. IL-17 signals through a heteromeric receptor complex consisting of IL-17 receptor A (IL-17RA) and IL-17RC, which are single-pass transmembrane proteins expressed by a variety of cells. Evidence indicates that NF-κB and MAPK pathways are involved in IL-17RA and IL-17RC signaling, however, little is known about its signaling in cells of the CNS.Suppressor Of Cytokine Signaling (SOCS) proteins function in a negative feedback loop to terminate signal transduction through the JAK/STAT pathway. One member, SOCS3, is expressed by immune cells and cells of CNS, and regulates inflammatory cytokine and chemokine production, activation of microglia, macrophages and astrocytes, immune cell infiltration and autoimmunity. The predominant function of SOCS3 is inhibition of signaling by the IL-6 family of cytokines. However, SOCS3 exerts a much broader effect on immune responses by inhibiting signaling of additional mediators such as LPS, type I and type II IFNs, IL-2 and IL-12. Furthermore, SOCS3 inhibits the NF-κB pathway, antagonizes cAMP-mediated signaling and enhances signaling through the Ras pathway. We recently found that TGF-βinhibits IL-6-and IL-21-induced SOCS3 expression, thus enhancing as well as prolonging STAT-3 activation in naive CD4+CD25- T cells and promoting Th17 cell development. These results suggest that SOCS3 may participate in the regulation of Th17 cell differentiation and IL-17 functions.An important question is how IL-17 contributes to autoimmune diseases and/or inflammation in the CNS. Thus, identification of the downstream targets of IL-17 in the CNS should advance our understanding of the mechanisms underlying IL-17-mediated autoimmune diseases. In this study, we demonstrate that astrocytes are a target of IL-17. IL-17 enhances the IL-6/sIL-6R (IL-6/R) signaling cascade and positive-feedback loop of IL-6 expression in astrocytes. SOCS3 participates in the regulation of IL-6/R plus IL-17 enhanced IL-6 expression in astrocytes, and plays an important role in down-regulating the IL-6 signaling cascade, indicating that SOCS3 participates in IL-17 functions in the CNS as a negative feedback regulator.PartⅠIL-17 Enhances IL-6-induced IL-6 Expression in Primary Astrocytes.ObjectiveTo investigate whether IL-17RA and IL-17RC genes are expressed in astrocytes, we prepared primary astrocytes and cultured RAW264.7 cells as positive control. To detect the effect of IL-17 on IL-6/sIL-6R-mediated IL-6 expression in primary astrocytes.Methods1. IL-17RA and IL-17RC mRNA expression was determined by RT-PCR in RAW264.7 cells (positive control), and primary astrocytes, in duplicate.2. Primary astrocytes were treated with medium (UN), IL-6, sIL-6R or IL-17 in various combinations for 4 h, and levels of IL-6 mRNA expression were determined by QRT-PCR.3. Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2-48 h, and supernatants were analyzed for IL-6 protein using ELISA.Results1. IL-17RA and IL-17RC mRNA was expressed in both astrocytes and RAW264.7 cells.2. IL-17 enhances IL-6/R-induced IL-6 expression at the mRNA levels in astrocytes.3. Added with IL-6/R, the expression of IL-6 protein was significantly enhanced at all time pointsConclusion1. Astrocytes are a target of Th17 cells and IL-17 in the CNS2. IL-17 enhances IL-6-induced IL-6 expression in primary astrocytes. Part II Investigation of the Mechanism on IL-17 Enhancement of IL-6-induced IL-6 Expression in Primary Astrocytes.ObjectiveTo study the transfection efficiency of SIM2-s siRNA in T98G and U251 cell lines. To detect the silencing potency of SIM2-s siRNA in T98G and U251 cell lines. To detect the effect of silencing SIM2-s expression in GBM cell proliferation and invasion.Methods1. Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 15 and 30 min, and then cell lysates were immunoblotted with antibodies.2. Astrocytes were cultured in absence or presence of IL-17 followed by IL-6/R treatment for 4 h. Actinomycin D (5 ng/ml) was then added, and cells harvested at 0, 30,60,120, and 240 min after addition. The abundance of IL-6 mRNA was determined by QRT-PCR.3. BAY 11 (5μM), U0126 (10μM), SB203580 (10μM) or JNKi II (10μM) were added to cultures 1 h before cytokine addition, and then astrocytes were incubated with medium, IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h. Levels of IL-6 mRNA expression were determined by QRT-PCR.4. Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 and then cells were cross-linked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with Abs against p65, P-p65, c-Fos, c-Jun, histone acetylation (Ac-H3 and Ac-H4), p300, CBP, RNA Pol II or normal rabbit IgGResults1. IL-17 effect is not mediated by stabilization of IL-6 mRNA.2. IL-17 alone induces activation of the NF-κB, ERK1/2 and p38 pathways, and synergizes with IL-6/R for enhancement of NF-κB, ERK1/2, p38 and JNK signaling.3. The NF-κB pathway inhibitor BAY11, JNK MAPK inhibitor II, and p38 MAPK inhibitor SB203580 suppressed the synergistic effect of IL-6 and IL-17 on IL-6 mRNA induction.4. IL-6/R plus IL-17 enhance phospho-p65, c-Jun, c-Fos, RNA Pol II, and the coactivators CBP and p300 to the IL-6 promoter and modification of H3 and H4 acetylation recruitment to initiate transcription of the IL-6 gene. Conclusion1. The synergetic effect of IL-17 and IL-6 on IL-6 gene expression involved numerous signaling pathways including NF-κB, JNK MAPK and p38 MAPK.2. IL-17 synergizes with IL-6 to enhance the recruitment of activated NF-κB p65, c-Fos, c-Jun, and the histone acetyltransferases CBP, p300 and RNA Pol II to the IL-6 promoter to induce transcription of IL-6 in astrocytes. This was accompanied by enhanced acetylation of Histones H3 and H4 on the IL-6 promoter.PartⅢSOCS3 is a Negative Regulator of the Synergistic Effect of IL-6/R and IL-17.ObjectiveTo detect the effect of SOCS3 in the regulation of synergistic effect of IL-6/R and IL-17 and valuate NF-κB and MAPK activation in the absence or presence of SOCS3 to investigate the mechanism of the regulation.Methods1. Astrocytes were transfected with SOCS3 siRNA (100 nM) or siRNA control (100 nM) for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 4 h. Levels of SOCS3 and IL-6 mRNA expression was determined by QRT-PCR and supernatants analyzed for IL-6 protein by ELISA2. SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, the cells were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h, and levels of IL-6 mRNA expression determined by QRT-PCR and supernatants analyzed for IL-6 protein by ELISA3. SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were treated with medium (UN) or IL-6/R plus IL-17, and then cell lysates were immunoblotted with antibodies against phospho-p65 Ser 536, p65, phospho-p38, p38, phospho-JNK, JNK or GAPDH.Results1. SOCS3 mRNA induced by IL-6/R plus IL-17 was inhibited approximately 49% by siSOCS3. SOCS3 mRNA expression indicated more than 90% removal of SOCS3 in GFP-Cre infected SOCS3 floxed astrocytes compared with GFP infected SOCS3 floxed astrocytes at each treatment conditions 1. In the GFP-Cre infected SOCS3 flox astrocytes, we observed a highly increase of IL-6 expression in all conditions including treatment of medium (UN), IL-6/R, IL-17 or IL-6/R and IL-17, and the enhanced of IL-6 expression still existed in sIL-6R plus IL-17 treatment compared to sIL-6R or IL-17 alone2. The expression of IL-6 protein was dramatically increased in SOCS3 knockout astrocytes compared to the GFP infected cells in the treatment of medium (UN), IL-6 with SIL-6R, IL-17 or IL-6 with sIL-6R and IL-17.3. Activation of the NF-κB, p38 and JNK MAPK pathways pathway following exposure to IL-6/R plus IL-17 is enhanced in GFP-Cre infected SOCS3 flox astrocytes.ConclusionSOCS3 siRNA knockdown and SOCS3 deletion in astrocytes enhances the synergistic effect of IL-6 and IL-17 on IL-6 gene expression, which is due to an enhancement of activation of the NF-κB and MAPK pathways.