Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE.

Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE.

The lupus susceptibility locus Sle1 facilitates the peripheral development and selection of anti-DNA B cells through impaired receptor editing.

Systemic lupus erythematosus is characterized by the spontaneous production of IgG autoantibodies in patients and lupus-prone mice. In this study, we investigated the effect of the Sle1 lupus susceptibility locus on the peripheral development of 56R(+) anti-DNA transgenic B cells by tracking 56R(+) B cells in mice without (B6.56R) or with (B6.Sle1.56R) the Sle1 locus. Compared with B6.56R mice, B6.Sle1.56R mice exhibited increased class-switched IgG2a anti-DNA Abs in their serum, encoded by the transgene. Interestingly, within the spleen, Sle1 facilitated the development of these cells into clusters of IgG2a class-switched B cells juxtaposed to CD4(+) T cells within extrafollicular sites. Through sequence analysis of B cell hybridomas, we also found that B cells from B6.Sle1.56R mice are inefficient at Ig H and L chain editing. Thus, the Ig H chains in Sle1.56R(+) B cells are partnered more often with cationic L chains that facilitate DNA binding. Taken together, these findings indicate that the Sle1 lupus-susceptibility locus may facilitate the emergence of anti-DNA B cells by subduing BCR revision and possibly by shaping the extrafollicular development of effector B cells, although the precise molecular mechanisms await further study.

Human intestinal organoids from Cronkhite-Canada syndrome patients reveal link between serotonin and proliferation.

Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.

Peritoneal catheter implantation elicits IL-10-producing immune-suppressor macrophages through a MyD88-dependent pathway.

Catheters are implanted into the peritoneal cavity during the process of peritoneal dialysis. Though these catheters may be effective and beneficial, the impact of catheters on the immune system is poorly understood. Catheters and other devices implanted in the peritoneal cavity elicit a foreign body reaction. However, the immunological consequences of this remain uncharacterized. To model this, catheters were implanted into the peritoneal cavity of healthy mice. Catheter implantation induced rapid cellular changes within the peritoneal cavity. Whereas B-cells and T-cells were reduced, catheter implantation was associated with the rapid expansion of F4/80-low-positive, CD11b-positive macrophages that elaborated IL-10, and suppressed T-cell division and Th1 skewing in co-culture assays. Peritoneal catheter elicited macrophages had increased Jmjd3 but reduced NF-κB activation, and their emergence was MyD88-dependent. Collectively, these studies indicate that foreign body implantation into the peritoneal cavity is associated with the expansion of suppressor macrophages. Whether peritoneal cavity catheter implantation may have systemic immunoregulatory roles remains to be explored.

A distinct tolerogenic subset of splenic IDO(+)CD11b(+) dendritic cells from orally tolerized mice is responsible for induction of systemic immune tolerance and suppression of collagen-induced arthritis.

In oral tolerance, locally instigated tolerance in the gut propagate to systemic tolerance. In order to investigate the mechanism, we analyzed indoleamine 2,3-dioxygenase (IDO) expression in splenic dendritic cell (DC) subsets and tested whether DCs suppress collagen-induced arthritis (CIA) by inducing regulatory T cells (Tregs). The proportion of IDO-expressing cells was higher in the CD11b(+) subset of splenic DCs from orally tolerized CIA mice. These DCs suppressed type II collagen-specific T cell proliferation and promoted Treg induction from CD4(+)CD25(-) T cells using transforming growth factor-ß. These DCs also increased the expression of cytotoxic T lymphocyte antigen-4 and programmed death-1 on Tregs. When adoptively transferred, spenic IDO-expressing CD11b(+) DCs from tolerized animals suppressed the development of arthritis, increased the Treg/Th17 cell ratio, and decreased the production of inflammatory cytokines in the spleen. Taken together, a distinct subset of splenic IDO(+)CD11b(+)DCs is responsible for the systemic immune regulation in oral tolerance.

Transforming growth factor ß-transduced mesenchymal stem cells ameliorate experimental autoimmune arthritis through reciprocal regulation of Treg/Th17 cells and osteoclastogenesis.

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (MSCs) can prevent various autoimmune diseases. We examined the therapeutic potential of transforming growth factor ß (TGFß)-transduced MSCs in experimental autoimmune arthritis, using an accepted animal model of collagen-induced arthritis (CIA). METHODS: DBA/1J mice with CIA were treated with syngeneic TGFß-induced MSCs, whereas control mice received either vehicle or MSCs alone. Arthritis severity was assessed by clinical and histologic scoring. TGFß-transduced MSCs were tested for their immunosuppressive ability and differential regulation in mice with CIA. T cell responses to type II collagen were evaluated by determining proliferative capacity and cytokine levels. The effects of TGFß-transduced MSCs on osteoclast formation were analyzed in vitro and in vivo. RESULTS: Systemic infusion of syngeneic TGFß-transduced MSCs prevented arthritis development and reduced bone erosion and cartilage destruction. Treatment with TGFß-transduced MSCs potently suppressed type II collagen-specific T cell proliferation and down-regulated proinflammatory cytokine production. These therapeutic effects were associated with an increase in type II collagen-specific CD4+FoxP3+ Treg cells and inhibition of Th17 cell formation in the peritoneal cavity and spleen. Furthermore, TGFß-transduced MSCs inhibited osteoclast differentiation. CONCLUSION: TGFß-transduced MSCs suppressed the development of autoimmune arthritis and joint inflammation. These data suggest that enhancing the immunomodulatory activity of MSCs and modulating T cell-mediated immunity using gene-modified MSCs may be a gateway for new therapeutic approaches to clinical rheumatoid arthritis.

Live probiotic bacteria administered in a pathomimetic Leaky Gut Chip ameliorate impaired epithelial barrier and mucosal inflammation.

Here, we report a pathomimetic Leaky Gut Chip that recapitulates increased epithelial permeability and intestinal inflammation to assess probiotic intervention as live biotherapeutics. We leveraged a mechanodynamic human gut-on-a-chip (Gut Chip) that recreates three-dimensional epithelial layers in a controlled oxygen gradient and biomechanical cues, where the addition of a cocktail of pro-inflammatory cytokines, TNF-α and IL-1ß, reproducibly induced impaired epithelial barrier followed by intestinal inflammation. This inflamed leaky epithelium was not recovered for up to 3 days, although the cytokine treatment ceased. However, when probiotic bacteria, either Lactobacillus rhamnosus GG or a multi-species mixture (VSL#3), were respectively administered on the leaky epithelium, bacterial cells colonized mucosal surface and significantly improved barrier function, enhanced the localization of tight junction proteins such as ZO-1 and occludin, and elevated mucus production. In addition, inflammatory markers, including p65, pSTAT3, and MYD88, that were highly expressed in the germ-free control were significantly reduced when probiotic bacteria were co-cultured in a Leaky Gut Chip. Probiotic treatment also significantly reduced the production of secretory pro-inflammatory cytokines. Hence, our pathomimetic Leaky Gut Chip may offer a translational strategy to dissect the therapeutic mechanism of live biotherapeutic products and validate their clinical potential by incorporating patient-derived organoids.

A Bioprinted Tubular Intestine Model Using a Colon-Specific Extracellular Matrix Bioink.

Tremendous advances have been made toward accurate recapitulation of the human intestinal system in vitro to understand its developmental process, and disease progression. However, current in vitro models are often confined to 2D or 2.5D microarchitectures, which is difficult to mimic the systemic level of complexity of the native tissue. To overcome this problem, physiologically relevant intestinal models are developed with a 3D hollow tubular structure using 3D bioprinting strategy. A tissue-specific biomaterial, colon-derived decellularized extracellular matrix (Colon dECM) is developed and it provides significant maturation-guiding potential to human intestinal cells. To fabricate a perfusable tubular model, a simultaneous printing process of multiple materials through concentrically assembled nozzles is developed and a light-activated Colon dECM bioink is employed by supplementing with ruthenium/sodium persulfate as a photoinitiator. The bioprinted intestinal tissue models show spontaneous 3D morphogenesis of the human intestinal epithelium without any external stimuli. In consequence, the printed cells form multicellular aggregates and cysts and then differentiate into several types of enterocytes, building junctional networks. This system can serve as a platform to evaluate the effects of potential drug-induced toxicity on the human intestinal tissue and create a coculture model with commensal microbes and immune cells for future therapeutics.

IL-23 induces receptor activator of NF-kappaB ligand expression on CD4+ T cells and promotes osteoclastogenesis in an autoimmune arthritis model.

IL-23, a clinically novel cytokine, targets CD4(+) T cells. Recent IL-1Ra(-/-) mouse studies have demonstrated that IL-23 indirectly stimulates the differentiation of osteoclast precursors by enhancing IL-17 release from CD4(+) T cells. IL-17, in turn, stimulates osteoclastogenesis in osteoclast precursor cells. In this study, we found that IL-23 up-regulates receptor activator of NF-kappaB ligand expression by CD4(+) T cells, and thus contributes to osteoclastogenesis. This indirect pathway is mediated by NF-kappaB and STAT3. We have also demonstrated that IL-23 can influence osteoclastogenesis positively under the special conditions in the IL-1-dominant milieu of IL-1Ra(-/-) mice. We propose that IL-23-enhanced osteoclastogenesis is mediated mainly by CD4(+) T cells. The results of this study show that IL-23 is a promising therapeutic target for the treatment of arthritis-associated bone destruction.

Microphysiological Engineering of Immune Responses in Intestinal Inflammation.

The epithelial barrier in the gastrointestinal (GI) tract is a protective interface that endures constant exposure to the external environment while maintaining its close contact with the local immune system. Growing evidence has suggested that the intercellular crosstalk in the GI tract contributes to maintaining the homeostasis in coordination with the intestinal microbiome as well as the tissue-specific local immune elements. Thus, it is critical to map the complex crosstalks in the intestinal epithelial-microbiome-immune (EMI) axis to identify a pathological trigger in the development of intestinal inflammation, including inflammatory bowel disease. However, deciphering a specific contributor to the onset of pathophysiological cascades has been considerably hindered by the challenges in current in vivo and in vitro models. Here, we introduce various microphysiological engineering models of human immune responses in the EMI axis under the healthy conditions and gut inflammation. As a prospective model, we highlight how the human "gut inflammation-on-a-chip" can reconstitute the pathophysiological immune responses and contribute to understanding the independent role of inflammatory factors in the EMI axis on the initiation of immune responses under barrier dysfunction. We envision that the microengineered immune models can be useful to build a customizable patient's chip for the advance in precision medicine.

Spatiotemporal Gradient and Instability of Wnt Induce Heterogeneous Growth and Differentiation of Human Intestinal Organoids.

In a conventional culture of three-dimensional human intestinal organoids, extracellular matrix hydrogel has been used to provide a physical space for the growth and morphogenesis of organoids in the presence of exogenous morphogens such as Wnt3a. We found that organoids embedded in a dome-shaped hydrogel show significant size heterogeneity in different locations inside the hydrogel. Computational simulations revealed that the instability and diffusion limitation of Wnt3a constitutively generate a concentration gradient inside the hydrogel. The location-dependent heterogeneity of organoids in a hydrogel dome substantially perturbed the transcriptome profile associated with epithelial functions, cytodifferentiation including mucin 2 expression, and morphological characteristics. This heterogeneous phenotype was significantly mitigated when the Wnt3a was frequently replenished in the culture medium. Our finding suggests that the morphological, transcriptional, translational, and functional heterogeneity in conventional organoid cultures may lead to a false interpretation of the experimental results in organoid-based studies.

Three-Dimensional Regeneration of Patient-Derived Intestinal Organoid Epithelium in a Physiodynamic Mucosal Interface-on-a-Chip.

The regeneration of the mucosal interface of the human intestine is critical in the host-gut microbiome crosstalk associated with gastrointestinal diseases. The biopsy-derived intestinal organoids provide genetic information of patients with physiological cytodifferentiation. However, the enclosed lumen and static culture condition substantially limit the utility of patient-derived organoids for microbiome-associated disease modeling. Here, we report a patient-specific three-dimensional (3D) physiodynamic mucosal interface-on-a-chip (PMI Chip) that provides a microphysiological intestinal milieu under defined biomechanics. The real-time imaging and computational simulation of the PMI Chip verified the recapitulation of non-linear luminal and microvascular flow that simulates the hydrodynamics in a living human gut. The multiaxial deformations in a convoluted microchannel not only induced dynamic cell strains but also enhanced particle mixing in the lumen microchannel. Under this physiodynamic condition, an organoid-derived epithelium obtained from the patients diagnosed with Crohn's disease, ulcerative colitis, or colorectal cancer independently formed 3D epithelial layers with disease-specific differentiations. Moreover, co-culture with the human fecal microbiome in an anoxic-oxic interface resulted in the formation of stochastic microcolonies without a loss of epithelial barrier function. We envision that the patient-specific PMI Chip that conveys genetic, epigenetic, and environmental factors of individual patients will potentially demonstrate the pathophysiological dynamics and complex host-microbiome crosstalk to target a patient-specific disease modeling.

Robust Formation of an Epithelial Layer of Human Intestinal Organoids in a Polydimethylsiloxane-Based Gut-on-a-Chip Microdevice.

Polydimethylsiloxane (PDMS) is a silicone polymer that has been predominantly used in a human organ-on-a-chip microphysiological system. The hydrophobic surface of a microfluidic channel made of PDMS often results in poor adhesion of the extracellular matrix (ECM) as well as cell attachment. The surface modification by plasma or UV/ozone treatment in a PDMS-based device produces a hydrophilic surface that allows robust ECM coating and the reproducible attachment of human intestinal immortalized cell lines. However, these surface-activating methods have not been successful in forming a monolayer of the biopsy-derived primary organoid epithelium. Several existing protocols to grow human intestinal organoid cells in a PDMS microchannel are not always reproducibly operative due to the limited information. Here, we report an optimized methodology that enables robust and reproducible attachment of the intestinal organoid epithelium in a PDMS-based gut-on-a-chip. Among several reported protocols, we optimized a method by performing polyethyleneimine-based surface functionalization followed by the glutaraldehyde cross linking to activate the PDMS surface. Moreover, we discovered that the post-functionalization step contributes to provide uniform ECM deposition that allows to produce a robust attachment of the dissociated intestinal organoid epithelium in a PDMS-based microdevice. We envision that our optimized protocol may disseminate an enabling methodology to advance the integration of human organotypic cultures in a human organ-on-a-chip for patient-specific disease modeling.

Recapitulation of the accessible interface of biopsy-derived canine intestinal organoids to study epithelial-luminal interactions.

Recent advances in canine intestinal organoids have expanded the option for building a better in vitro model to investigate translational science of intestinal physiology and pathology between humans and animals. However, the three-dimensional geometry and the enclosed lumen of canine intestinal organoids considerably hinder the access to the apical side of epithelium for investigating the nutrient and drug absorption, host-microbiome crosstalk, and pharmaceutical toxicity testing. Thus, the creation of a polarized epithelial interface accessible from apical or basolateral side is critical. Here, we demonstrated the generation of an intestinal epithelial monolayer using canine biopsy-derived colonic organoids (colonoids). We optimized the culture condition to form an intact monolayer of the canine colonic epithelium on a nanoporous membrane insert using the canine colonoids over 14 days. Transmission and scanning electron microscopy revealed a physiological brush border interface covered by the microvilli with glycocalyx, as well as the presence of mucin granules, tight junctions, and desmosomes. The population of stem cells as well as differentiated lineage-dependent epithelial cells were verified by immunofluorescence staining and RNA in situ hybridization. The polarized expression of P-glycoprotein efflux pump was confirmed at the apical membrane. Also, the epithelial monolayer formed tight- and adherence-junctional barrier within 4 days, where the transepithelial electrical resistance and apparent permeability were inversely correlated. Hence, we verified the stable creation, maintenance, differentiation, and physiological function of a canine intestinal epithelial barrier, which can be useful for pharmaceutical and biomedical researches.

Oral administration of type-II collagen suppresses IL-17-associated RANKL expression of CD4+ T cells in collagen-induced arthritis.

The receptor activator of nuclear factor kappaB ligand (RANKL) is an osteoclastogenic mediator, which is mainly expressed by stromal cells and osteoblast. However, T cells can also be an important provider for RANKL in special condition such as autoimmune arthritis. We examined the RANKL expression of hyporesponsive CD4+ T cells induced by oral feeding with type II collagen in collagen-induced arthritis (CIA) mice. The potential of RANKL expression in CD4+ T cells was downregulated in tolerance, as compared with CIA. One of possible explanations for this phenomenon is that CII-specific T cell activation was intrinsically impaired in oral tolerance, which caused suppression of RANKL expression of CD4+ T cells. We also investigated the extrinsic role of cytokine in this process. IL-17, well-known pro-inflammatory cytokine was upregulated in CIA and downregulated in tolerance. IL-17 had a potential to stimulate T cells to express RANKL in dose-dependent manner. IL-17-associated RANKL expression of CD4+ T cells was downregulated in oral tolerance, suggesting that the induction of tolerance ameliorates IL-17-induced RANKL expression of T cells in murine CIA. We also discovered that CIA - T cells could enhance osteoclastogenesis but not oral tolerance - T cells. Oral tolerance might be promising therapeutic option in viewpoints of modulating autoreactivity of CII which can induce not only IL-17 production but also RANKL expression in CD4+ T cells.

Type II collagen autoimmunity in a mouse model of human rheumatoid arthritis.

Type II collagen (CII) is expressed exclusively in the joint articular. Although the relationship between anti-CII immunity and human rheumatoid arthritis (RA) has been studied for a long time, definitive conclusions have not been reached. CII, as an autoantigen, has been studied extensively in small animal models, such as mice, and the collagen-induced arthritis (CIA) model has increased our understanding of the pathogenesis of human RA. In the present report, we summarize the available information on anti-CII immunity and discuss recent updates regarding pathogenesis in the CIA model, including the role of Th17 cells.

Human rheumatoid synovial fibroblasts promote osteoclastogenic activity by activating RANKL via TLR-2 and TLR-4 activation.

The interplay between the innate immune system and inflammatory bone destruction in the joints of individuals with rheumatoid arthritis (RA) remains unclear. This study was undertaken to explore the effect of toll-like receptor (TLR) signaling in fibroblast-like synoviocytes (FLS) on the expression of RANKL and induction of osteoclastogenic activity. The levels of RANKL mRNA and protein were measured using RT-PCR, real-time PCR, and immunostaining. Monocytes were cocultured with RA -FLS that had been stimulated with TLR ligands in fresh media and subsequently stained for tartrate-resistant acid phosphatase (TRAP) activity. Osteoclast molecule markers were measured using real-time PCR. Expression of TLR-2 and TLR-4 was higher in RA-FLS than in OA-FLS and normal skin fibroblasts. TLR-2 and TLR-4 ligands induced RANKL expression in RA-FLS. TLR stimulation of RA-FLS also induced the production of IL-1beta and TNF-alpha to a lesser extent; however, it had no effect on IL-17 production. Inhibition of TLR induced IL-1beta production, which partially reversed the upregulation of RANKL induced by TLR ligands. RA-FLS stimulated by TLR-2 and TLR-4 ligands and cocultured with human monocytes induced high levels of expression of TRAP, RANK, cathepsin K, calcitonin receptor, and matrix metalloproteinase-9, suggesting that RA-FLS promote osteoclast differentiation. Our results suggest that the TLR signaling pathway, through TLR-2 and TLR-4, induces RANKL expression in RA-FLS and the expression of RANKL promotes the differentiation of osteoclasts in RA synovium. Targeting specific TLRs may be a promising approach to prevent inflammatory bone destruction in the pathogenesis of RA.

CD8alpha+ dendritic cells enhance the antigen-specific CD4+ T-cell response and accelerate development of collagen-induced arthritis.

To investigate the role of CD8alpha(+) DCs in the development of collagen-induced arthritis (CIA). The immunogenic properties of CD8alpha(+) and CD8alpha(-) DC subsets were investigated by mixed-lymphocyte reaction and cytokine enzyme-linked immunoassay. CII-pulsed CD8alpha(+) DCs or CD8alpha(-) DCs with CD4(+) T cells from CIA mice were adoptively transferred onto the hind footpad of DBA mice. The onset of arthritis and the arthritis index were examined for 14 weeks after adoptive transfer. Expression of MHC-II and CD80 but not CD86 and CD40 was higher in CD8alpha(+) DCs than in CD8alpha(-) DCs from the spleens of CIA mice. Culturing CD8alpha(+) DCs with CD4(+) T cells significantly increased the proliferative response of CD4(+) T cells in the presence of CII. The production of interleukin (IL)-12p70, IL-17, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha was slightly increased in CD8alpha(+) DCs than in CD8alpha(-) DCs. DBA/1 mice that were adoptively transferred with CII-pulsed CD8alpha(+) DCs and CD4(+) T cells into the footpads showed accelerated onset of CIA compared to control group. By contrast, CD8alpha(-) DCs showed a partial inhibitory effect on CIA. These findings show that CD8alpha(+) DCs accelerated the onset of CIA when aoptively transferred with CD4(+) T cells and that CD8alpha(+) DCs provoke the development of CIA probably by stimulating the immune responses of CII-reactive CD4(+) T cells and by increasing the production of inflammatory cytokines.

Cyclosporine A inhibits IL-15-induced IL-17 production in CD4+ T cells via down-regulation of PI3K/Akt and NF-kappaB.

Cyclosporine A (CSA) has various biological effects on T cells, including inhibition of interleukin (IL)-15-induced IL-17 production in CD4+ T cells from patients with rheumatoid arthritis (RA). However, the mechanism underlying this effect is not fully understood. Here, we tried to investigate the mechanism of CSA to inhibit IL-17 production induced by IL-15 in CD4+ T cells. Synovial fluid and serum levels of IL-15 and IL-17 were determined by ELISA. CD4+ T cells from RA patients were treated with IL-15 in the presence of CSA or several signal inhibitors. The concentration of IL-17 in culture supernatants was measured by ELISA and IL-17 mRNA expression was determined by RT-PCR. NF-kappaB binding activity for IL-17 transcription was assessed by electrophoretic mobility shift assay. IL-15 induced IL-17 production by CD4+ T cells in dose- and time-dependent manner. IL-15-stimulated IL-17 production and mRNA expression were inhibited by CSA in CD4+ T cells. Moreover PI3K/Akt inhibitor, NF-kappaB inhibitor, and FK506 significantly inhibited IL-15-induced IL-17 production in CD4+ T cells. Inhibition studies revealed the requirement of PI3K/Akt and NF-kappaB signal pathway for IL-15-induced IL-17 production. CSA down-regulated the phosphorylation of Akt and IkappaB. CSA inhibited binding of NF-kappaB to IL-17 promoter. The inhibitory effect of CSA on IL-15 induced IL-17 production partially depended on the increase in IL-10, since neutralizing anti-IL-10 antibodies were able to partially reverse this inhibition. CSA inhibits IL-17 production by CD4+ T cells and this effect is mediated by IL-15-activated NF-kappaB pathway in CD4+ T cells, which is possible mechanism of CSA in treating RA as NF-kappaB targeting strategy.

Transforming growth factor beta 1(TGF-beta1) down-regulates TNFalpha-induced RANTES production in rheumatoid synovial fibroblasts through NF-kappaB-mediated transcriptional repression.

Transforming growth factor (TGF)-beta1 is a pleiotropic cytokine with many functions, including those related to growth modulation, immunosuppression, and pro-inflammation, in a wide variety of cell types. In this study, we investigated the ability of TGF-beta1 to regulate RANTES production by activated rheumatoid synovial fibroblasts. Fibroblast-like synoviocytes (FLS) were cultured in the presence of TGF-beta1 and IL-1beta, IL-15, TNFalpha, or IL-17, and the secretion of RANTES into culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Expression of RANTES encoded mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR), and NF-kappaB binding activity for RANTES transcription was determined by electrophoretic mobility shift assay (EMSA). We found that the concentrations of RANTES in synovial fluid (SF) from rheumatoid arthritis (RA) patients were lower than in SF from osteoarthritis (OA) patients, whereas the concentrations of TGF-beta1 were higher in RA SF than in OA SF. TGF-beta1 dose-dependently inhibited TNFalpha-induced production of RANTES protein and mRNA from RA FLS. Addition of RA SF with high-level TGF-beta1 mimicked the effect of TGF-beta1 on TNFalpha-induced RANTES production, which was inhibited by treatment with anti-TGF-beta1 neutralizing antibody. TGF-beta1 blocked the degradation of cytosolic IkappaB-alpha and the translocation of activated NF-kappaB to the nucleus. EMSA showed that the inhibitory effect of TGF-beta1 was associated with decreased binding of NF-kappaB to the RANTES promoter. These results suggest that elevated TGF-beta1 in rheumatoid synovial tissue may suppress joint inflammation by inhibiting RANTES secretion from synovial fibroblasts, thus blocking the infiltration of immune cells. These findings may provide an explanation for the mechanism by which TGF-beta1 regulates immune function in RA.