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.

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.

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.

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.

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.

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.

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.

Green Tea Epigallocatechin-3-Gallate Suppresses Autoimmune Arthritis Through Indoleamine-2,3-Dioxygenase Expressing Dendritic Cells and the Nuclear Factor, Erythroid 2-Like 2 Antioxidant Pathway.

BACKGROUND: The activity of one of the major catechins in Green Tea, the polyphenol (-)-epigallocatechin-3-gallate (EGCG), has been shown to have a variety of health benefits. Recent studies suggest that EGCG can modulate both the innate and adaptive arms of the immune system. The goal of the current studies was to examine the immunomodulatory effects and mechanisms of action of EGCG on experimental arthritis in mice. METHODS: EGCG (10 mg/kg) was administered by oral gavage after CIA induction, while control mice were administered phosphate buffered saline (PBS). Disease mechanisms were studied in both groups of mice. Phenotypes were examined using repeated measure analysis of variance (ANOVA) and data from in vitro and ex vivo experiments were analyzed for significance using the Mann-Whitney U test. RESULTS: EGCG treatment ameliorated clinical symptoms and reduced histological scores in arthritic mice. Serum type-II collagen-specific immunoglobulin (Ig) IgG2a antibodies were significantly lower in EGCG-fed mice compared to PBS-treated mice. EGCG significantly suppressed T cell proliferation and relative frequencies of CD4 T cells, CD8 T cells and B cell subsets including marginal zone B cells, T1 and T2 transitional B cells, while increasing the frequency of CD4(+) Foxp3(+) regulatory T cells (Tregs) and indoleamine-2,3-dioxygenase (IDO) expression by CD11b(+) dendritic cells (DC). Splenic CD11b(+) DC from EGCG fed mice induced an increased frequency of Tregs via an IDO-dependent mechanism in in vitro cultures. Importantly, joint homogenates from EGCG-fed mice exhibited significantly increased levels of Nuclear Factor, Erythroid 2-Like 2 (Nrf-2) and Heme oxygenase-1 (HO-1) compared with PBS-fed mice. CONCLUSIONS: This is the first report of upregulation of the Nrf-2 antioxidant pathway in EGCG-mediated immunoregulation. EGCG ameliorated experimental arthritis in mice by eliciting IDO-producing DCs, increasing frequencies of T regs and inducing the activation of the Nrf-2 antioxidant pathway. It remains to be established whether EGCG is useful for the prevention and treatment of rheumatoid arthritis and other inflammatory disorders.

Prevention of murine lupus nephritis by targeting multiple signaling axes and oxidative stress using a synthetic triterpenoid.

OBJECTIVE: Current treatment options for lupus are far from optimal. Previously, we reported that phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin, MEK-1/ERK-1,2, p38, STAT-3, STAT-5, NF-κB, multiple Bcl-2 family members, and various cell cycle molecules were overexpressed in splenic B cells in an age-dependent and gene dose-dependent manner in mouse strains with spontaneous lupus. Since the synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) has been shown to inhibit AKT, MEK-1/2, and NF-κB, and to induce caspase-mediated apoptosis, we tested the therapeutic potential of this agent in murine lupus nephritis. METHODS: The synthetic triterpenoid CDDO-Me or placebo was administered to 2-month-old B6.Sle1.Sle3 mice or MRL/lpr mice, which develop spontaneous lupus. All mice were phenotyped for disease. RESULTS: CDDO-Me-treated mice exhibited significantly reduced splenic cellularity, with decreased numbers of both CD4+ T cells and activated CD69+/CD4+ T cells compared to the placebo-treated mice. These mice also exhibited a significant reduction in serum autoantibody levels, including anti-double-stranded DNA (anti-dsDNA) and antiglomerular antibodies. Finally, CDDO-Me treatment attenuated renal disease in mice, as indicated by reduced 24-hour proteinuria, blood urea nitrogen, and glomerulonephritis. At the mechanistic level, CDDO-Me treatment dampened MEK-1/2, ERK, and STAT-3 signaling within lymphocytes and oxidative stress. Importantly, the NF-E2-related factor 2 pathway was activated after CDDO-Me treatment, indicating that CDDO-Me may modulate renal damage in lupus via the inhibition of oxidative stress. CONCLUSION: These findings underscore the importance of AKT/MEK-1/2/NF-κB signaling in engendering murine lupus. Our findings indicate that the blockade of multiple signaling nodes and oxidative stress may effectively prevent and reverse the hematologic, autoimmune, and pathologic manifestations of lupus.

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.

Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation.

INTRODUCTION: Oxidative stress is implicated in tissue inflammation, and plays an important role in the pathogenesis of immune-mediated nephritis. Using the anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) mouse model, we found that increased expression of glutathione S-transferase Mu 2 (GSTM2) was related to reduced renal damage caused by anti-GBM antibodies. Furthermore, mesenchymal stem cell (MSC)-based therapy has shed light on the treatment of immune-mediated kidney diseases. The aim of this study was to investigate if MSCs could be utilized as vehicles to deliver the GSTM2 gene product into the kidney and to evaluate its potential therapeutic effect on anti-GBM-GN. METHODS: The human GSTM2 gene (hGSTM2) was transduced into mouse bone marrow-derived MSCs via a lentivirus vector to create a stable cell line (hGSTM2-MSC). The cultured hGSTM2-MSCs were treated with 0.5 mM H2O2, and apoptotic cells were measured by terminal dUTP nick-end labeling (TUNEL) assay. The 129/svj mice, which were challenged with anti-GBM antibodies, were injected with 106 hGSTM2-MSCs via the tail vein. Expression of hGSTM2 and inflammatory cytokines in the kidney was assayed by quantitative PCR and western blotting. Renal function of mice was evaluated by monitoring proteinuria and levels of blood urea nitrogen (BUN), and renal pathological changes were analyzed by histochemistry. Immunohistochemical analysis was performed to measure inflammatory cell infiltration and renal cell apoptosis. RESULTS: MSCs transduced with hGSTM2 exhibited similar growth and differentiation properties to MSCs. hGSTM2-MSCs persistently expressed hGSTM2 and resisted H2O2-induced apoptosis. Upon injection into 129/svj mice, hGSTM2-MSCs migrated to the kidney and expressed hGSTM2. The anti-GBM-GN mice treated with hGSTM2-MSCs exhibited reduced proteinuria and BUN (58% and 59% reduction, respectively) and ameliorated renal pathological damage, compared with control mice. Mice injected with hGSTM2-MSCs showed alleviated renal inflammatory cell infiltration and reduced expression of chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1ß and IL-6 (53%, 46% and 52% reduction, respectively), compared with controls. Moreover, hGSTM2-MSCs increased expression of renal superoxide dismutase and catalase, which may associate with detoxifying reactive oxygen species to prevent oxidative renal damage. CONCLUSIONS: Our data suggest that the enhanced protective effect of GSTM2-transduced MSCs against anti-GBM-GN might be associated with inhibition of oxidative stress-induced renal cell apoptosis and inflammation, through over-expression of hGSTM2 in mouse kidneys.

Kallikrein transduced mesenchymal stem cells protect against anti-GBM disease and lupus nephritis by ameliorating inflammation and oxidative stress.

Previously we have shown that kallikreins (klks) play a renoprotective role in nephrotoxic serum induced nephritis. In this study, we have used mesenchymal stem cells (MSCs) as vehicles to deliver klks into the injured kidneys and have measured their therapeutic effect on experimental antibody induced nephritis and lupus nephritis. Human KLK-1 (hKLK1) gene was transduced into murine MSCs using a retroviral vector to generate a stable cell line, hKLK1-MSC, expressing high levels of hKLK1. 129/svj mice subjected to anti-GBM induced nephritis were transplanted with 10(6) hKLK1-MSCs and hKLK1 expression was confirmed in the kidneys. Compared with vector-MSCs injected mice, the hKLK1-MSCs treated mice showed significantly reduced proteinuria, blood urea nitrogen (BUN) and ameliorated renal pathology. Using the same strategy, we treated lupus-prone B6.Sle1.Sle3 bicongenic mice with hKLK1-MSCs and demonstrated that hKLK1-MSCs delivery also attenuated lupus nephritis. Mechanistically, hKLK1-MSCs reduced macrophage and T-lymphocyte infiltration into the kidney by suppressing the expression of inflammation cytokines. Moreover, hKLK1 transduced MSCs were more resistant to oxidative stress-induced apoptosis. These findings advance genetically modified MSCs as potential gene delivery tools for targeting therapeutic agents to the kidneys in order to modulate inflammation and oxidative stress in lupus nephritis.

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.

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.

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.

Bone marrow T cells are superior to splenic T cells to induce chimeric conversion after non-myeloablative bone marrow transplantation.

BACKGROUND/AIMS: The bone marrow functions not only as the primary B-lymphocyte-producing organ but also as a secondary lymphoid organ for CD4 and CD8 cell responses and a site of preferential homing and persistence for memory T cells. Bone marrow T (BM-T) cells are distinguished from peripheral blood T cells by surface phenotype, cytokine secretion profile, and immune functions. In this study, we evaluated the alloreactive potential of donor lymphocyte infusion (DLI) using BM-T cells in mixed chimerism compared to that using spleen T (SP-T) cells. METHODS: Cells were prepared using established procedures. BM-T cells were obtained as a by-product of T-cell depletion in BM grafting and then cryopreserved for subsequent DLI. We performed DLI using BM-T cells in allogeneic mixed chimera mice on post-BMT day 21. RESULTS: When the same dose of T cells, 5-10x10(5) (Thy1.2+), fractionated from BM and spleen were administered into mixed chimeras, the BM-T group showed complete chimeric conversion, with self-limited graft-versus-host disease (GVHD) and no pathological changes. However, the SP-T group showed persistent mixed chimerism, with pathological signs of GVHD in the liver and intestine. CONCLUSIONS: Our results suggest that DLI using BM-T cells, even in small numbers, is more potent at inducing chimeric conversion in mixed chimerism than DLI using SP-T cells. Further study is needed to determine whether cryopreserved BM-T cells are an effective cell source for DLI to consolidate donor-dominant chimerism in clinical practice without concerns about GVHD.

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.

Dysfunctional interferon-alpha production by peripheral plasmacytoid dendritic cells upon Toll-like receptor-9 stimulation in patients with systemic lupus erythematosus.

BACKGROUND: It is well known that interferon (IFN)-alpha is important to the pathogenesis of systemic lupus erythematosus (SLE). However, several reports have indicated that the number of IFN-alpha producing cells are decreased or that their function is defective in patients with SLE. We studied the function of plasmacytoid dendritic cells (pDCs) under persistent stimulation of Toll-like receptor (TLR)9 via a TLR9 ligand (CpG ODN2216) or SLE serum. METHODS: The concentrations of IFN-alpha were determined in serum and culture supernatant of peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy controls after stimulation with CpG ODN2216 or SLE serum. The numbers of circulating pDCs were analyzed by fluoresence-activated cell sorting analysis. pDCs were treated with CpG ODN2216 and SLE serum repeatedly, and levels of produced IFN-alpha were measured. The expression of IFN-alpha signature genes and inhibitory molecules of TLR signaling were examined in PBMCs from SLE patients and healthy control individuals. RESULTS: Although there was no significant difference in serum concentration of IFN-alpha and number of circulating pDCs between SLE patients and healthy control individuals, the IFN-alpha producing capacity of PBMCs was significantly reduced in SLE patients. Interestingly, the degree which TLR9 ligand-induced IFN-alpha production in SLE PBMCs was inversely correlated with the SLE serum-induced production of IFN-alpha in healthy PMBCs. Because repeated stimulation pDCs with TLR9 ligands showed decreased level of IFN-alpha production, continuous TLR9 stimulation may lead to decreased production of IFN-alpha in SLE PBMCs. In addition, PBMCs isolated from SLE patients exhibited higher expression of IFN-alpha signature genes and inhibitory molecules of TLR signaling, indicating that these cells had already undergone IFN-alpha stimulation and had become desensitized to TLR signaling. CONCLUSION: We suggest that the persistent presence of endogenous IFN-alpha inducing factors induces TLR tolerance in pDCs of SLE patients, leading to impaired production of IFN-alpha.