The putative Na+/H+(NH4+) antiporter in the pillar cell flange apical membranes [35,39] could lead proportionally much more to overall Na+ uptake in adult when compared to juvenile M. amazonicum, which seem to depend much more on V(H+)ATPase-dependent Na+ uptake [32]. 1905481-36-8The presence of a gill (Na+, K+)-ATPase displaying specific kinetic attributes that favor synergistic stimulation by NH4+ plus K+ could be related to diverse epithelial permeabilities in the different ontogenetic stages of M. amazonicum. Zoea I and decapodid III are generally found in reasonably saline waters while juveniles and older people inhabit refreshing drinking water. Reduced entire body surface area permeabilities most likely have accompanied the profession of brackish and freshwater habitats by maritime species: to illustrate, gills of the marine crab Cancer pagurus show substantial permeabilities and constitute minor or no barrier to NH4+ influx in contrast, freshwater-tailored Eriocheir sinensis gills exhibit a 63-fold lower ionic permeability, which could decrease passive NH4+ influx [103]. Active ammonia excretion by C. pagurus is two-fold better than in E. sinensis, reflecting its leaky epithelium, suggesting that an productive system of energetic ammonia excretion compensates for ammonia inflow [103].Elevated exterior ammonia may possibly cause substitution of K+ by NH4+, leading to a reduce in intracellular K+ [ninety two], even so acute exposure of the estuarine crab Neohelice ( = Chasmagnathus) granulata to ammonia reveals hemolymph ammonia to be much less than ambient ammonia [43], suggesting a system for NH4+ excretion against its gradient [96,103]. The lively excretion of NH4+ across the gill epithelium against elevated exterior ammonia concentrations in the crabs Carcinus maenas, Cancer pagurus and Eriocheir sinensis [ninety six,103] corroborates the idea that the (Na+, K+)-ATPase is immediately included in the translocation of NH4+ from the hemolymph. An extra, magnesium-inhibitable pumping power for NH4+ extrusion, represented by a 2nd NH4+-binding site, appears when the pump is fully saturated by K+ [forty five]. Carcinus maenas uncovered to large NH4+ titers (two mM) maintains lower hemolymph NH4+ concentrations (<100 mM) [103]. These findings suggest that most ammonia is regulated by transport proteins [93]. Some palaemonids like M. rosenbergii and M. olfersi exhibit accelerated oxidative deamination of free amino acids, which results in higher hemolymph ammonia concentrations and increased ammonia excretion rates [104]. Hemolymph NH4+, transported into the gill ionocyte cytosol by the basal (Na+, K+)ATPase, can be exchanged for Na+ via an apical Na+/NH4+ antiporter, contributing to Na+ uptake [10507]. This deamination mechanism may operate in adult M. amazonicum and, as proposed for M. olfersi [87], synergistic stimulation of the gill (Na+,K+)-ATPase by NH4+ plus K+ may constitute a valuable physiological adaptation, coupling NH4+ excretion and Na+ uptake in dilute media. However, cation and NH4+ fluxes across the gill cuticle of Carcinus maenas are inhibited by amiloride [97,108] thus, findings on Na+-dependent NH4+ transport should be interpreted cautiously since Na+ uptake and ammonia excretion may not be directly linked given this limiting cuticular component [95]. Concluding, we have already shown that the crustacean gill (Na+, K+)-ATPase exhibits species-specific synergistic stimulation by NH4+ plus K+ [44,45,66,850]. Our current findings for M. amazonicum demonstrate changes during ontogeny, suggesting that the kinetic behavior of the gill (Na+, K+)-ATPase may be both species- and stage-specific, possibly correlating with the biochemical adjustment of each ontogenetic stage to the optimal salinity found in its natural environment. Further, sensitivity to NH4+ decreases during the ontogeny of M. amazonicum, which together with the synergistic stimulation by NH4+ plus K+ seen in adults, may underlie a novel regulatory mechanism for the crustacean (Na+, K+)-ATPase.Crohn's disease (CD) and ulcerative colitis (UC) are two forms of inflammatory bowel disease (IBD) in man. The etiology of IBD remains unclear, but evidence indicates that it results from an interaction between genetic and environmental factors, which eventually lead to an excessive and poorly controlled mucosal inflammatory response directed against components of the normal microflora and mucosal constituents of the gut [1]. Studies over the last 2 decades have shown that different T cell differentiation patterns determine disease progression [3]. For example, it is known that CD is linked to a predominantly T helper cell (Th1) immune response (e.g., secretion of IFN-c, TNF-a, and IL-12). Accordingly, therapeutic strategies targeting these cytokines have been widely investigated. Antibody against TNF-a attenuates colitis in IBD patients, but more than one third of IBD patients do not respond to anti-TNF-a therapy [5-6]. These observations suggest the need to identify novel targets for therapeutic intervention in IBD. In addition to the classical Th1/Th2 pathways, a new pathway, the Th17 pathway, has been discovered as a result of the identification of a novel CD4 T cell subset, the Th17 cell [7]. It is now known that IL-17A has pro-inflammatory effects on a wide range of cellular targets, such as epithelium, endothelium, and monocytes/macrophages [80], and plays pathogenic roles in some organ-specific autoimmune diseases, such as rheumatoid arthritis (RA) and multiple sclerosis, as well as IBD [11]. Because of this, the therapeutic effects of an IL-17 neutralizing antibody, secukinumab (AIN457T), in RA are now being evaluated in phase II clinical trials [12]. As regards IBD, IL-17A is produced in the healthy gut, but high IL-17A mRNA expression is seen in inflamed colonic mucosa [13-14], suggesting a pathogenic role of IL-17A in the progression of IBD. Accordingly, IL-17A has been examined as a target for reducing autoimmune damage in IBD [15]. Unfortunately, clinical trials targeting IL-17A in IBD failed to show an effect, indicating that further studies are needed on its role in IBD. It is now known that there is a complex and active interplay between IL-17A and colonic epithelial cells (CECs) during the progression of IBD. After stimulation by IL-17A, CECs release a wide range of pro-inflammatory cytokines and chemokines, e.g., CXCL8 for neutrophil chemotaxis and CCL20 to attract Th17 cells, further amplifying the gut inflammation [16]. On the other hand, IL-17A also has protective effects on the gut epithelial barrier, e.g., by upregulating the expression of antimicrobial peptides [17]. Recent data have also shown that IL-17A, by directly binding to its receptor (IL-17R) expressed on Th1 cells,inhibits Th1 cell-mediated colonic inflammation [18].Together, these data suggest that IL-17A plays both a pro-inflammatory and an anti-inflammatory role in IBD, which might explain the failure of the clinical trial targeting IL-17A. To explore more effective intervention strategies, the mechanisms by which IL-17A mediates its pathogenic or protective effects, especially the latter, need to be investigated. In most target cells, IL-17A signaling activates the MAPK and NF-kB pathways through IL-17RA and increases the expression of inflammatory cytokines [16]. Act1 has been identified as an essential adaptor molecule in IL-17 signaling [19]. In addition, the results of a microarray screen suggested the involvement of the CCAAT/enhancer binding protein transcription factors C/EBPb and C/EBPd in the IL-17A-induced signaling cascade [20], while another report showed that the PI3K pathway is involved in IL17A signaling, mainly in an Act1-independent manner [21], but the underlying mechanisms remain largely unclear. Further investigation of the signaling mechanisms of IL-17A will shed light on its biological functions and help in understanding and treating inflammatory diseases. Our previous data suggested that IL-17A signaling inhibited the function of Th1 cell in IBD [22]. However, the underlying mechanisms remain largely unclear. Although some data suggest that IL-17A suppresses the development of colonic inflammation by directly inhibiting the differentiation of Th1 cells [18], we argue that other mechanisms may exist, since IL-17A binds to multiple target cells and stimulates complex intracellular cascades. In this study, CECs were used as the target for IL-17A and we demonstrated, for the first time, that IL-17A signaling in CECs can also trigger anti-inflammatory mechanisms by activating the PI3K-AKT and ERK-CEBP/b pathways in an Act1-dependent manner, finally leading to inhibition of TNF-a-induced expression of IL-12P35 and of a Th1 cell chemokine, CXCL11, and of Th1 cell function. This is the first report demonstrating the involvement of the Act1-PI3K-AKT pathway in the IL-17A-triggered signaling cascade. Further investigation of this pathway should shed new light on therapeutic strategies against many IL-17Aelated clinical diseases conditions were an initial denaturation step at 95uC for 3 min 40 cycles at 95uC for 10 s, annealing at 60uC for 15 s, and extension at 72uC for 10 s and 71 cycles at 60uC for 30s. The sequences of the primers used, produced by Assays-by-Design Service for Gene Expression Assays (Biomics Biotechnologies Co. Ltd., China), are listed in Table 1. At the end of the PCR cycles, the specificity of the amplification products was checked by dissociation curve analysis. mRNA levels in each sample were determined using the gene-specific threshold cycle (Ct) for each sample (gCt) corrected by subtracting the Ct for the GAPDH housekeeping gene. Untreated controls were used as the reference samples and the gCt for all experimental samples was subtracted from the gCt for the control samples (ggCt). The magnitude of the change in levels of the test gene mRNA was expressed as 2-ggCt. Each measurement was performed in duplicate.Western blotting was performed to evaluate levels of ERK, AKT, phospho-ERK, phospho-AKT, phospho-C/EBPb, PI3K p110c, Act1, and GAPDH. Briefly, 30 ug of protein was electrophoretically separated on a 12% sodium dodecyl sulfatepolyacrylamide gel and transferred to a polyvinylidene difluoride membrane, which was then blocked by incubation for 1 h at room temperature in 5% fat-free dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). The blots were then incubated overnight at 4uC with rabbit antibodies against human ERK (1:1000), AKT (1:1000), phospho-ERK (1:1000), phosphoAKT (1:1000), phospho-C/EBP(1:1000), or PI3K p110c(1:1000) (Cell Signaling Technology, USA), rat antibodies against human Act1 (eBiosciences, San Diego, CA), or mouse antibodies against GAPDH (1:5000) (Tianjin Sungene Biotech Co. Ltd) diluted in TBST containing 5% BSA, washed for 25 min with TBST, and incubated for 1 h at room temperature with alkaline phosphataseconjugated anti-rabbit, anti-mouse, or anti-rat IgG antibodies (KPL, Gaithersburg, MD, USA) (1:2000 in TBST containing 5% BSA).To directly examine whether Act1 was involved in the IL-17 signaling pathway, Act1 gene expression in HT-29 cells was Table 1. Sequences of the primers used for real-time PCR.HT-29 human colorectal cancer cells (ATCC) were cultured in McCoy's 5A medium (ATCC) supplemented with 10% fetal bovine serum (FBS), penicillin (10 U/ml), and streptomycin (10 mg/ml) (all from Sigma-Aldrich). For tests, they were plated in 12-well plates at a density of 36105 cells per well in McCoy's 5A medium containing 10% FBS and antibiotics. Before cytokine treatment, the cells were incubated overnight in McCoy's 5A medium containing 0.5% FBS and antibiotics, then were incubated for 6 h with different dose of TNF-a (R & D Systems) and/or of IL-17 (eBiosciences, San Diego, CA). Here 0.5 ng/ml of TNF-a (suboptimal dose from which we can see the effects of IL17A) and/or 50 ng/ml of IL-17 were used for in vitro cell stimulation. The cells were then harvested and RNA prepared using Trizol reagent (Invitrogen). RNA samples (2 mg) were then reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (New England Biolabs) and real-time PCR performed using SYBR Green (TOYOBO) and a standard curve for quantization, as described previously [23]. The relative expression of cytokine mRNAs was evaluated by real-time PCR. The real-time PCR reaction mixture consisted of 10 ml of 26SYBR green Master Mix, 0.5 ml of 10 pM primers, and 2 ml of cDNA in a total volume of 20 ml. The thermal cycling blocked using short-hair RNA (shRNA). Three non-overlapping shRNA duplexes (Biomics Biotechnologies Co. Ltd, China) were individually tested for maximal knockdown of gene expression. The duplex sequences were CCATAGACACGGGATATGA (shRNA1), CCCTGAAACTTGCAAATC A (shRNA2), CTGCAATTGACATATTTGA (shRNA3), and TTCTCCGAACGTGTCACGT. (negative control (NC)). 8206995These sequences were inserted into the pRNAT-U6.1/Neo vector, then the purified recombinant vectors were transfected into HT-29 cells using Lipofectamine 2000TM (Invitrogen) according to the manufacturer’s protocol. The shRNA duplex giving maximal knockdown was identified and HT-29 cell clones stably express Act1 shRNA selected using G418 (Gibco) and analyzed for Act1 expression by Western blotting and RTCR.HT-29 cells were plated in 24-well plates at a density of 1.56105 cells/well in McCoy’s 5A medium containing 10% FBS and antibiotics and incubated for 24 h, then were treated with IL-17 (50 ng/ml eBiosciences) and/or TNF- a(0.5 ng/ml eBiosciences) for 24 h. Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and added to the culture in a ratio of 1 HT-29 cells to 10 PBMCs. The co-cultures were then stimulated for 24 h by a combination of monoclonal antibodies (mAbs) against CD3 (3 mg/ml) and CD28 (3 mg/ml) ( eBiosciences) with or without IL-12 (12.5 ng/ml eBiosciences), then non-adherent PBMCs and adherent HT-29 cells were harvested separately for analysis. The human PBMC used in this study have been described in our previous publication [22], and the study protocol was approved by the Ethics Committee of the General Hospital of the Air Force of the PLA, Beijing, China.placed in a 150 ml conical flask containing 20 ml of 15 mM HEPES, 5 mM EDTA, 10% FBS, and 100 mg/ml of gentamycin and incubated at 37uC with shaking for 30 min. The sample was then filtered at room temperature through a 200 mesh filter, then the filtrates from three collections were combined and centrifuged at 850 g for 10 min at 37uC and the pellets (CECs) resuspended in phosphate-buffered saline (PBS). For the collection of lymphocytes from colonic lamina propria, colon tissue removed of CECs was further incubated with collagenase D (Roche) (0.6 mg/ml) in 20 ml RPMI-1640 medium at 37uC for about 3 hours.
