Interactions between Gut Microbiota and Immunomodulatory Cells in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases caused by abnormal immune activation and immune tolerance. Immunomodulatory cells (ICs) play a critical role in the maintenance and homeostasis of normal immune function and in the pathogenesis of RA. The human gastrointestinal tract is inhabited by trillions of commensal microbiota on the mucosal surface that play a fundamental role in the induction, maintenance, and function of the host immune system. Gut microbiota dysbiosis can impact both the local and systemic immune systems and further contribute to various diseases, such as RA. The neighbouring intestinal ICs located in distinct intestinal mucosa may be the most likely intermediary by which the gut microbiota can affect the occurrence and development of RA. However, the reciprocal interaction between the components of the gut microbiota and their microbial metabolites with distinct ICs and how this interaction may impact the development of RA are not well studied. Therefore, a better understanding of the gut microbiota, ICs, and their interactions might improve our knowledge of the mechanisms by which the gut microbiota contribute to RA and facilitate the further development of novel therapeutic approaches. In this review, we have summarized the roles of the gut microbiota in the immunopathogenesis of RA, especially the interactions between the gut microbiota and ICs, and further discussed the strategies for treating RA by targeting/regulating the gut microbiota.

Expression and function of phosphoinositide 3-kinase delta in mesenchymal stromal cells from normal and leukaemic bone marrow.

Within lymphoid tissues, chronic lymphocytic leukaemia (CLL) cells interact with mesenchymal stromal cells (MSC). Inhibitors of phosphoinositide 3-kinase delta (PI3Kδ) cause release of CLL cells from lymphoid tissues into blood. PI3Kδ inhibitors are thought to target only CLL and other immune cells because PI3Kδ expression is restricted to haematopoietic cells. We found that PI3Kδ is unexpectedly expressed in primary MSC derived from CLL patients and healthy donors. PI3Kδ inhibition in MSC using idelalisib or duvelisib significantly reduced their ability to support CLL migration and adhesion. These observations provide the first evidence that PI3Kδ is expressed and functional in CLL MSC.

Inducible indoleamine 2,3-dioxygenase 1 and programmed death ligand 1 expression as the potency marker for mesenchymal stromal cells.

AIM: Establishment of a potency assay in the manufacturing of clinical-grade mesenchymal stromal cells (MSCs) has been a challenge due to issues of relevance to function, timeline and variability of responder cells. In this study, we attempted to develop a potency assay for MSCs. METHODS: Clinical-grade bone marrow-derived MSCs were manufactured. The phenotype and immunosuppressive functions of the MSCs were evaluated based on the International Society for Cellular Therapy guidelines. Resting MSCs licensed by interferon (IFN)-γ exposure overnight were evaluated for changes in immune suppression and immune-relevant proteins. The relationship of immune-relevant protein expression with immunosuppression of MSCs was analyzed. RESULTS: MSC supressed third-party T-lymphocyte proliferation with high inter-donor and inter-test variability. The suppression of T-lymphocyte proliferation by IFN-γ-licensed MSCs correlated with that by resting MSCs. Many cellular proteins were up-regulated after IFN-γ exposure, including indoleamine 2,3-dioxygenase 1 (IDO-1), programmed death ligand 1 (PD-L1), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and bone marrow stromal antigen 2 (BST-2). The expression levels of IDO-1 and PD-L1 on licensed MSCs, not VCAM-1, ICAM-1 or BST-2 on licensed MSCs, correlated with MSC suppression of third-party T-cell proliferation. CONCLUSION: A flow cytometry-based assay of MSCs post-IFN-γ exposure measuring expression of intracellular protein IDO-1 and cell surface protein PD-L1 captures two mechanisms of suppression and offers the potential of a relevant, rapid assay for MSC-mediated immune suppression that would fit with the manufacturing process.

Interferon γ induced compositional changes in human bone marrow derived mesenchymal stem/stromal cells.

BACKGROUND: Mesenchymal stem/stromal cells (MSC) display a range of immunoregulatory properties which can be enhanced by the exposure to cytokines such interferon γ (IFN-γ). However the compositional changes associated with the 'licensing' of these cells have not been clearly defined. The present study was undertaken to provide a detailed comparative proteomic analysis of the compositional changes that occur in human bone marrow derived MSC following 20 h treatment with IFN-γ. METHODS: 2D LC MSMS analysis of control and IFN-γ treated cells from 5 different healthy donors provided confident identification of more than 8400 proteins. RESULTS: In total 210 proteins were shown to be significantly altered in their expression levels (≥|2SD|) following IFN-γ treatment. The changes for several of these proteins were confirmed by flow cytometry. STRING analysis determined that approximately 30% of the altered proteins physically interacted in described interferon mediated processes. Comparison of the list of proteins that were identified as changed in the proteomic analysis with data for the same proteins in the Interferome DB indicated that ~35% of these proteins have not been reported to be IFN-γ responsive in a range of cell types. CONCLUSIONS: This data provides an in depth analysis of the proteome of basal and IFN-γ treated human mesenchymal stem cells and it identifies a number of novel proteins that may contribute to the immunoregulatory capacity if IFN-γ licensed cells.

Recent Advances: The Imbalance of Cytokines in the Pathogenesis of Inflammatory Bowel Disease.

Cytokines play an important role in the immunopathogenesis of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, where they drive and regulate multiple aspects of intestinal inflammation. The imbalance between proinflammatory and anti-inflammatory cytokines that occurs in IBD results in disease progression and tissue damage and limits the resolution of inflammation. Targeting cytokines have been novel strategies in the treatment of IBD. Recent studies show the beneficial effects of anticytokine treatments to IBD patients, and multiple novel cytokines are found to be involved in the pathogenesis of IBD. In this review, we will discuss the recent advances of novel biologics in clinics and clinical trials, and novel proinflammatory and anti-inflammatory cytokines found in IBD with focusing on IL-12 family and IL-1 family members as well as their relevance to the potential therapy of IBD.

rAed a 4: A New 67-kDa Aedes aegypti Mosquito Salivary Allergen for the Diagnosis of Mosquito Allergy.

BACKGROUND: Accurate diagnosis of mosquito allergy has been hampered by the laborious task of obtaining mosquito salivary allergens. We have previously studied 3 recombinant (r) Aedes aegypti mosquito salivary allergens: rAed a 1, rAed a 2 and rAed a 3. Here, we report the expression, purification, identification and evaluation of rAed a 4, a 67-kDa α-glucosidase. METHODS: rAed a 4 was expressed using a baculovirus/insect cell system, purified by a combination of anion- and cation-exchange chromatography, and identified by immunoblotting. A. aegypti saliva extract was prepared in our laboratory. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to measure rAed a 4-specific immunoglobulin E (IgE) and IgG antibodies in sera from 13 individuals with a positive mosquito-bite test from a laboratory-reared mosquito. Sera from 18 individuals with a negative bite test served as controls. RESULTS: Purified rAed a 4 bound to the IgE in mosquito-allergic sera, as detected by ELISA and immunoblotting. The binding of rAed a 4 to IgE could be inhibited in a dose-dependent manner by the addition of an A. aegypti extract. Mosquito-allergic individuals had significantly higher mean levels of rAed a 4-specific IgE and IgG than controls. Using the mean of the controls ± 2 SD as a cut-off level, 46% of the 13 allergic individuals had a positive IgE, while none of the controls was positive (p < 0.001). CONCLUSIONS: Aed a 4 is a major allergen in mosquito saliva. Its recombinant form has the hydrolase function and can be used for the diagnosis of mosquito allergy.

Functional Myeloid-Derived Suppressor Cell Subsets Recover Rapidly after Allogeneic Hematopoietic Stem/Progenitor Cell Transplantation.

Myeloid-derived suppressor cells (MDSCs) are regulatory cell populations that have the ability to suppress effector T cell responses and promote the development of regulatory T cells (Tregs). They are a heterogeneous population of immature myeloid progenitors that include monocytic and granulocytic subsets. We postulated that given the rapid expansion of myeloid cells post-transplant, these members of the innate immune system may be important contributors to the early immune environment post-transplant. To evaluate the kinetics of recovery and function of MDSCs after allogeneic hematopoietic stem cell transplant (HSCT), 26 patients undergoing allogeneic HSCT were studied at 6 time points in the first 3 months after HSCT. Both MDSC subsets recovered between 2 and 4 weeks, well before the recovery of T and B lymphocytes. MDSC subset recovery positively correlated with T, B, and/or double-negative T cell numbers after HSCT. MDSCs isolated from patients post-transplant were functional in that they suppressed third-party CD4(+) T cell proliferation and Th1 differentiation and promoted Treg development. In conclusion, functional MDSC are present early after HSCT and likely contribute to the regulatory cell population post-transplant.

The Interactions of T Cells with Myeloid-Derived Suppressor Cells in Peripheral Blood Stem Cell Grafts.

The interaction of myeloid-derived suppressor cells (MDSCs) with T cells within G-CSF-mobilized peripheral blood stem cell (PBSC) grafts in patients undergoing autologous or allogeneic hematopoietic stem cell transplantation remains to be elucidated. Through studying allo- and auto-PBSC grafts, we observed grafts containing large numbers of T cells and MDSCs with intergraft variability in their percentage and number. T cells from autologous grafts compared to allografts expressed relative higher percentages of inhibitory receptors PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and BTLA. Autograft T cells had decreased cell proliferation and IFN-γ secretion, which supported the possible presence of T cell exhaustion. On the contrary, graft monocytic MDSCs (M-MDSCs) expressed multiple inhibitory receptor ligands, including PD-L1, CD86, Galectin-9, HVEM and CD155. The expression of inhibitory receptor ligands on M-MDSCs was correlated with their corresponding inhibitory receptors on T cells in the grafts. Isolated M-MDSCs had the ability to suppress T cell proliferation and IFN-γ secretion and/or promote Treg expansion. Blocking the PD-L1-PD-1 signaling pathway partially reversed the functions of M-MDSCs. Taken together, our data indicated that T cells and M-MDSCs in PBSC grafts express complementary inhibitory receptor-ligand pairing, which may impact the quality of immune recovery and clinical outcome post transplantation.

Sustained suppression of IL-13 by a vaccine attenuates airway inflammation and remodeling in mice.

We previously reported that a recombinant IL-13 peptide-based virus-like particle vaccine significantly suppressed murine acute airway allergic inflammatory responses. The impact of this strategy on the development of chronic airway inflammation and remodeling has not been investigated. We evaluated whether the vaccine-mediated sustained suppression of IL-13 attenuates features of chronic airway inflammation and remodeling in mice repeatedly challenged with allergen. BALB/c mice received two intraperitoneal sensitizing injections of ovalbumin (OVA) and alum, followed by six consecutive 2-day intranasal OVA challenges at 12-day intervals and then a 4-week recovery period. Anti-IL-13 antibodies were induced with a vaccine before (preventive experiments) or after (interventional experiments) the OVA challenge commenced. Respiratory mechanics were assessed using low-frequency forced oscillation with a small animal ventilator. Cytokine concentrations in bronchoalveolar lavage fluid (BALF) and lung histology were also assessed. In the preventive experiments, vaccination significantly suppressed IL-13 concentrations, the accumulation of inflammatory cells in BALF, lung mucus production, and collagen deposition. Furthermore, vaccination significantly attenuated OVA challenge-induced increases in airway resistance, tissue resistance, and tissue elastance, both acutely and after a 4-week recovery from allergen challenges. In the interventional experiments, vaccination decreased IL-13, TGF-ß1, and IL-12p40 concentrations in BALF, as well as mucus production and collagen deposition. Chronic inflammation and sustained airway hyperresponsiveness were not significantly reversed. The persistent suppression of IL-13 with a vaccine inhibits chronic airway inflammation and the development of several key components of airway remodeling, and this intervention is more effective at early stages than later during chronic inflammation.

Targeting IL-12/IL-23 by employing a p40 peptide-based vaccine ameliorates TNBS-induced acute and chronic murine colitis.

Interleukin (IL)-12 and IL-23 both share the p40 subunit and are key cytokines in the pathogenesis of Crohn's disease. Previously, we have developed and identified three mouse p40 peptide-based and virus-like particle vaccines. Here, we evaluated the effects and immune mechanisms of the optimal vaccine in downregulating intestinal inflammation in murine acute and chronic colitis, induced by intrarectal administrations of trinitrobenzene sulfonic acid (TNBS). Mice were injected subcutaneously with vaccine, vaccine carrier or saline three times, and then intrarectally administered TNBS weekly for 2 wks (acute colitis) or 7 wks (chronic colitis). The severity of colitis was evaluated by body weight, histology and collagen and cytokine levels in colon tissue. Th1 and Th17 cells in mesenteric lymph nodes (MLN) were determined. Our results showed the vaccine induced high level and long-lasting specific IgG antibodies to p40, IL-12 and IL-23. After administrations of TNBS, vaccinated mice had significantly less body weight loss and a significant decrease of inflammatory scores, collagen deposition and expression of p40, IL-12, IL-23, IL-17, TNF, iNOS and Bcl-2 in colon tissues, compared with carrier and saline groups. Moreover, vaccinated mice exhibited a trend to lower percentages of Th1 cells in acute colitis and of Th17 cells in chronic colitis in MLN than in controls. In summary, administration of the vaccine induced specific antibodies to IL-12 and IL-23, which was associated with improvement of intestinal inflammation and fibrosis. This suggests that the vaccine may provide a potential approach for the long-term treatment of Crohn's disease.

Novel anti-inflammatory action of 5-aminoimidazole-4-carboxamide ribonucleoside with protective effect in dextran sulfate sodium-induced acute and chronic colitis.

AMP-activated protein kinase (AMPK) is an important cellular energy sensor that is responsible for maintaining systemic and cellular energy balance. Its role in intestinal inflammation remains unclear. Recent studies indicate that AMPK activation initiated by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) participates in modulating inflammatory responses. Inflammatory bowel disease (IBD) has been characterized by sustained intestinal mucosa inflammation, caused mainly by excessive macrophage activation and T helper type 1 (Th1) and Th17 immune responses. Thus, we sought to determine the effect of AICAR on inflammatory responses of murine models of IBD. Mice with acute or chronic colitis induced by dextran sulfate sodium (DSS) were treated with or without AICAR. Body weight and colon inflammation were evaluated, and production of proinflammatory cytokines in colon tissues was determined. Nuclear factor kappaB (NF-kappaB) activation in colon tissues was assayed, and Th1 and Th17 cell responses were also evaluated. By inducing AMPK activation, AICAR had a therapeutic effect in ameliorating acute and chronic DSS-induced murine colitis as shown by reduced body weight, loss and significant attenuation in clinical symptoms, and histological inflammation. Moreover, AICAR treatment inhibited NF-kappaB activation in macrophages, reduced levels of Th1- and Th17-type cytokines in colon tissues, and down-regulated Th1 and Th17 cell responses during the progress of acute and chronic experimental colitis. AICAR acts as a central inhibitor in immune responses of experimental colitis. Our data show that AICAR-initiated AMPK activation may represent a promising alternative to our current approaches to suppress intestinal inflammation in IBD.

Manufacturing autologous myoblast for regenerative medicine applications.

BACKGROUND: Autologous myoblasts have been tested in the treatment of muscle-related diseases. However, the standardization of manufacturing myoblasts is still not established. Here we report a flask and animal-free medium-based method of manufacturing clinical-grade myoblast together with establishing releasing criteria for myoblast products under Good Manufacturing Practice (GMP). METHODS: Quadriceps muscle biopsy samples were donated from three patients with myogenic ptosis. After biopsy samples were digested through enzymatic dissociation, the cells were grown in T175 flasks (passage 0) and hyperflasks (passage 1) in the animal-free SkGMTM-2 skeletal muscle cell growth medium containing 5% human platelet lysate for 15-17 days. The harvested cells were released based on cell morphology, cell dose, viability, sterility, endotoxin, mycoplasma and immunophenotype. Myotube differentiation was also evaluated. RESULTS: 400 to 500 million myoblast cells were manufactured within 15 to 17 days by the end of passage 1, which met pre-determined releasing criteria. The manufactured myoblast cells could differentiate and fuse into myotubes in vitro, with the possible trend that the donor age may impact the differentiation ability of myoblasts. CONCLUSIONS: The present study establishes a flask-based method of manufacturing myoblast in the animal-free medium together with releasing criteria, which is simple, robust, inexpensive and easily reproducible. This study will serve as the validation for a planned phase 1 clinical trial to assess the use of autologous myoblast transplants for the treatment of myogenic ptosis and other myogenic diseases.

A Comprehensive Review and Update on the Pathogenesis of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic and life-threating inflammatory disease of gastroenteric tissue characterized by episodes of intestinal inflammation. The pathogenesis of IBD is complex. Recent studies have greatly improved our knowledge of the pathophysiology of IBD, leading to great advances in the treatment as well as diagnosis of IBD. In this review, we have systemically reviewed the pathogenesis of IBD and highlighted recent advances in host genetic factors, gut microbiota, and environmental factors and, especially, in abnormal innate and adaptive immune responses and their interactions, which may hold the keys to identify novel predictive or prognostic biomarkers and develop new therapies.

Sustained suppression of IL-18 by employing a vaccine ameliorates intestinal inflammation in TNBS-induced murine colitis.

AIM: To develop IL-18 peptide-based virus-like particle vaccines that elicit autoantibodies against IL-18 and to evaluate the in vivo effects of the vaccines in murine colitis. METHODS: Recombinant IL-18 vaccines were constructed, and the effects of the vaccines were evaluated in trinitrobenzene sulfonic acid-induced acute and chronic colitis in mice. RESULTS: Two murine IL-18 peptide-based vaccines (A and D) were developed, which induced relative long-lasting specific antibodies against IL-18. Vaccine-immunized mouse antisera could partially block IL-18-induced IFN-γ production in vitro. Mice receiving vaccine D, not vaccine A, had a significant decrease in intestinal inflammation, collagen deposition and pro-inflammatory cytokine levels in colon tissue. CONCLUSION: IL-18 vaccine may provide a potential therapeutic approach in the treatment of Crohn's disease.

A standardized immune phenotyping and automated data analysis platform for multicenter biomarker studies.

The analysis and validation of flow cytometry-based biomarkers in clinical studies are limited by the lack of standardized protocols that are reproducible across multiple centers and suitable for use with either unfractionated blood or cryopreserved PBMCs. Here we report the development of a platform that standardizes a set of flow cytometry panels across multiple centers, with high reproducibility in blood or PBMCs from either healthy subjects or patients 100 days after hematopoietic stem cell transplantation. Inter-center comparisons of replicate samples showed low variation, with interindividual variation exceeding inter-center variation for most populations (coefficients of variability <20% and interclass correlation coefficients >0.75). Exceptions included low-abundance populations defined by markers with indistinct expression boundaries (e.g., plasmablasts, monocyte subsets) or populations defined by markers sensitive to cryopreservation, such as CD62L and CD45RA. Automated gating pipelines were developed and validated on an independent data set, revealing high Spearman's correlations (rs >0.9) with manual analyses. This workflow, which includes pre-formatted antibody cocktails, standardized protocols for acquisition, and validated automated analysis pipelines, can be readily implemented in multicenter clinical trials. This approach facilitates the collection of robust immune phenotyping data and comparison of data from independent studies.

Autologous Bone Marrow Stem Cell Therapy in Patients With ST-Elevation Myocardial Infarction: A Systematic Review and Meta-analysis.

BACKGROUND: Randomized controlled trials (RCTs) on bone marrow stem cell (BMSC) therapy in ST-elevation myocardial infarction (STEMI) patients have reported conflicting results. Our main objective was to critically appraise and meta-analyze best-available evidence on efficacy and safety of intracoronary administration of autologous BMSC therapy in STEMI patients after primary percutaneous coronary intervention. METHODS: We conducted a search of MEDLINE, PubMed, EMBASE, CENTRAL, Global Health, CINAHL, and conference proceedings in February 2017. Our primary outcome was all-cause mortality. Secondary and safety outcomes included cardiac death, heart failure, arrhythmias, repeat myocardial infarction, or target vessel revascularizations; or improved health-related quality of life, left ventricular ejection fraction, or infarct size. Summary relative and absolute risks were obtained using random effects models. We also evaluated the strength of evidence. RESULTS: A comprehensive database search identified 42 RCTs (3365 STEMI patients). BMSC therapy did not significantly decrease mortality (risk ratio, 0.71; 95% confidence interval, 0.45-1.11; I2, 0%; absolute risk reduction, 0.1%; 95% confidence interval, -0.71 to 0.91; 40 trials; 3289 participants; I2, 0%; low strength of evidence). BMSC therapy had no effect on secondary or adverse outcomes. Trial sequential analysis for all-cause mortality showed no evidence of a clinically important difference, with a very low probability that future studies can change the current conclusion. CONCLUSIONS: On the basis of evidence from 42 RCTs published in the past 15 years, we provide conclusive evidence for a lack of beneficial effect for autologous BMSC therapy in patients with STEMI.