Abstract Background Numerous studies have demonstrated that NLRP3 is involved in the pathogenesis of inflammatory bowel disease (IBD). Mesenchymal stem cells (MSCs) have been highlighted as new candidates for treating IBD based on their immunomodulatory properties, including regulation of the NLRP3 inflammasome. However, the role of NLRP3 in shaping the immunoregulatory function of MSCs remains unclear. In this study, we demonstrated that NLRP3 influences the therapeutic effect of MSCs on colitis in an inflammasome-independent manner. Methods MSCs were isolated from the femurs of male C57/B6 mice or Nlrp3 KO mice, identified by osteogenic adipogenic differentiation assay and flow cytometry. In vitro, WT and Nlrp3 KO MSCs were treated with LPS, then the supernatant was collected; WT MSCs were stimulated with different concentrations of NLRP3-specific inhibitor MCC950, and the level of IL-10 was detected in the supernatant; Detect gene expression levels by RNA sequencing; Use Seahorse to detect cellular oxidative phosphorylation (OXPHOS) and glycolysis levels; The expression of glucose transporter 1 ( Glut1 ) in MSCs was detected by Western blot; WT MSCs were stimulated with different concentrations of Glut1 inhibitor BAY876, and the level of IL-10 was detected. In vivo, DSS-induced colitis was treated with WT or Nlrp3 KO MSCs, body weight changes were monitored daily, measure colon length and collect colons for further evaluation; Then DSS-induced colitis was treated with pretreated MSCs, such as BAY876 pretreated WT MSCs or Glut1-overexpressed Nlrp3 KO MSCs (OE-Glut1), then the experimental operation is as above. Results Nlrp3 knockout did not alter MSC phenotypes, but promoted osteogenic differentiation. Further investigation indicated that deletion of Nlrp3 reduced the production of the IL-10 in MSCs, leading to an impaired protective effect on DSS-induced colitis. Mechanistically, the deficiency of Nlrp3 downregulated Glut1 expression and glycolysis activation in MSCs, which reduced IL-10 production. Subsequently, overexpressing Glut1 in Nlrp3 KO MSCs recovered their therapeutic effect dampened due to Nlrp3 deletion. Conclusions These results improve the current understanding of the molecular mechanisms underlying the therapeutic effectiveness of MSCs and provide a basis for optimizing MSC-based therapeutic strategies for immune-mediated diseases.
The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.livres.2017.07.001. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal
Mutations in the genes encoding two proteins of the retinal rod phototransduction cascade, opsin and the beta subunit of rod cGMP phosphodiesterase, cause retinitis pigmentosa (RP) in some families. Here we report defects in a third member of this biochemical pathway in still other patients with this disease. We screened 94 unrelated patients with autosomal dominant RP and 173 unrelated patients with autosomal recessive RP for mutations in the gene encoding the alpha subunit of the rod cGMP-gated cation channel. Five mutant sequences cosegregated with disease among four unrelated families with autosomal recessive RP. Two of these were nonsense mutations early in the reading frame (Glu76End and Lys139End) and one was a deletion encompassing most if not all of the transcriptional unit; these three alleles would not be expected to encode a functional channel. The remaining two mutations were a missense mutation (Ser316Phe) and a frameshift [Arg654(1-bp del)] mutation truncating the last 32 aa in the C terminus. The latter two mutations were expressed in vitro and found to encode proteins that were predominantly retained inside the cell instead of being targeted to the plasma membrane. We conclude that the absence or paucity of functional cGMP-gated cation channels in the plasma membrane is deleterious to rod photoreceptors and is an uncommon cause of RP.
Background: The deleterious side effects of calcineurin inhibitors (CNIs) such as nephrotoxicity, cardiovascular events, infection and malignancy, have impaired patients long-term survival. New immunotherapy regimens with minimization or elimination of CNIs are required to improve transplant outcome. Mesenchymal stem cells (MSCs) represent a unique cell population with potent immunosuppressive function, and can prolong allograft survival in experimental organ transplant models. In this present study, MSCs combined with sparing tacrolimus (TAC) were administered to de novo living related kidney transplant (KTx) recipients to prevent acute rejection, and post-transplant immune profile was monitored. Patients and methods: Twelve patients were enrolled in this prospective study. Six patients received donor-derived MSCs combined with minimized TAC, and the other six without MSCs were assigned as control. MSCs were infused into renal allograft via renal artery prior to vessel anastomosis (5×106 cells), and were subsequently given intravenously one month later (2×106 cells/kg). Safety of MSCs infusion, renal function and complications were observed. Donor-specific mixed lymphocyte reaction (MLR) was performed prior to KTx (day 0) and three (mon 3) or six months (mon 6) post-transplant. Chimerism was examined. Immune cells including CD4+ and CD8+ T cell, CD4+CD25+CD127lowFoxp3+ regulatory T cell (Tregs), NK cell, and CD19+CD27+ memory B cell were monitored by flow cytometry. Intracellular cytokines including IFN-γ, TNF-α, IL-4 and IL-10 were examined. Results: Tacrolimus dose in MSCs group was significantly reduced compared to control group (0.047 ± 0.005 vs 0.073 ± 0.013 mg/kg). One acute rejection occurred in control group, and was reversed by antithymocyte globulin and methylprednisolone therapy. None acute rejection was observed in MSCs group. All patients survived with stable renal function at mon 6. There was no significant different in MLR proliferation analysis at mon 3 and mon 6 between two groups. The proportion and absolute count of peripheral blood lymphocyte was not significantly different between two groups. Total T cells, CD4+ T cells, CD8+ T cells, and CD4+/CD8+ ratio were significantly different neither. Tregs proportion in MSCs group increased at mon 3 and returned at mon 6, compared to Tregs at day 0. However, Tregs significantly decreased at mon 3 in control group. B cells proportion in MSCs group increased from 9.5% (day 0) to 16.7% at mon 3, and returned at mon 6. While in control group, B cells constantly decreased within six months (3.8% vs 7.9%). The proportion of memory B cells increased by 27.9% at mon 3 in MSCs group, in contrast to 23% of decrease in control group. NK cells proportion in control group continually increased post KTx, while it showed transient decrease at mon 3 in MSCs group. No chimerism was detected in both groups. The proportion of IL-10 producing cells showed constant increase tendency within six months after KTx in MSCs group, while it transiently decreased at mon 3 in control group. Conclusions: Donor-derived MSCs may guarantee efficient hypo-responsiveness to allograft while minimizing calcineurin inhibitors in living related kidney transplantation. The long-term therapeutic effect and its mechanism of action need to be further investigated.
// Guang-hui Pan 1,* , Zheng Chen 1,* , Lu Xu 1 , Jing-hui Zhu 1 , Peng Xiang 2 , Jun-jie Ma 1 , Yan-wen Peng 2 , Guang-hui Li 1 , Xiao-yong Chen 2 , Jia-li Fang 1 , Yu-he Guo 1 , Lei Zhang 1 and Long-shan Liu 3 1 The Transplantation Centre, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, China 2 Center for Stem Cell Biology and Tissue Engineering, SunYat-sen University, Guangzhou, Guangdong, China 3 Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China * These authors contributed to the work equally and should be regarded as co-first authors Correspondence to: Guang-hui Pan, email: // Keywords : acute rejection, calcineurin inhibitors, graft survival, mesenchymal stem cells (MSCs), nephrotoxicity, Pathology Section Received : September 18, 2015 Accepted : January 29, 2016 Published : February 25, 2016 Abstract Calcineurin inhibitors, including tacrolimus, are largely responsible for advances in allotransplantation. However, the nephrotoxicity associated with these immunosuppressants impairs patients’ long-term survival after renal allograft. Therefore, novel regimens that minimize or even eliminate calcineurin inhibitors could improve transplantation outcomes. In this pilot study, we investigated the use of low-dose tacrolimus in combination with mesenchymal stem cells (MSCs), which are immunosuppressive and prolong allograft survival in experimental organ transplant models. Donor-derived, bone marrow MSCs combined with a sparing dose of tacrolimus (0.04-0.05 mg/kg/day) were administered to 16 de novo living-related kidney transplant recipients; 16 other patients received a standard dose of tacrolimus (0.07-0.08 mg/kg/day). The safety of MSC infusion, acute rejection, graft function, graft survival, and patient survival were evaluated over ≥24 months following kidney transplantation. All patients survived and had stable renal function at the 24 month follow-up. The combination of low-dose tacrolimus and MSCs was as effective as standard dose tacrolimus in maintaining graft survival at least 2 years after transplantation. In addition, both groups had similar urea, urine protein, urinary RBC, urinary WBC, 24-h urine protein, and creatinine clearance rates from 7 days to 24 months after transplantation. Furthermore, no differences in the proportion of lymphocytes, CD19, CD3, CD34, CD38, and natural killer cells were detected between the control and experimental groups. None of the MSC recipients experienced immediate or long-term toxicity from the treatment. This preliminary data suggests that the addition of MSCs permits the use of lower dosages of nephrotoxic calcineurin inhibitors following renal transplantation.
Cytoskeletal proteins and associated regulatory proteins are essential for maintaining cell structure and growth. β-actin is a major component of the cytoskeleton, and β-actin remodeling is involved in lymphocyte migration, infiltration and apoptosis. However, little is known about whether changes in β-actin expression affect lymphocyte cell fate, particularly during acute rejection after liver transplantation in a rat model. In our studies, grafts were harvested on days 5, 7 or 9 after xenogeneic rat liver transplantation. The acute rejection grade was histopathologically evaluated. Recipient-derived CD8+ T lymphocytes gradually infiltrated into liver allografts in cases of severe acute rejection. The apoptotic rate of CD8+ T lymphocytes peaked on day 7 and then decreased. Moreover, changes in β-actin expression were consistent with the apoptotic rate of CD8+ T lymphocytes in both allografts and peripheral blood based on western blotting and immunohistochemistry results. Additionally, jasplakinolide (an actin-stabilizing drug) evoked CD8+ T lymphocyte apoptosis. In conclusion, our study is the first to describe the fluctuating expression levels and dynamics of the cytoskeletal protein β-actin and its potential roles in the pathogenesis of acute rejection following rat liver transplantion. Our results enhance the understanding of the roles of CD8+ T lymphocytes during acute rejection and suggest that β-actin regulation leads to apoptosis.
Mesenchymal stromal cells (MSCs) have shown significant heterogeneity in terms of therapeutic efficacy for inflammatory bowel disease (IBD) treatment, which may be due to an insufficient number of MSCs homing to the damaged tissue of the colon. Engineering MSCs with specific chemokine receptors can enhance the homing ability by lentiviral transduction. However, the unclear specific chemokine profile related to IBD and the safety concerns of viral-based gene delivery limit its application. Thus, a new strategy to modify MSCs to express specific chemokine receptors using mRNA engineering is developed to evaluate the homing ability of MSCs and its therapeutic effects for IBD. We found that CXCL2 and CXCL5 were highly expressed in the inflammatory colon, while MSCs minimally expressed the corresponding receptor CXCR2. Transient expression of CXCR2 in MSC was constructed and exhibited significantly enhanced migration to the inflamed colons, leading to a robust anti-inflammatory effect and high efficacy. Furthermore, the high expression of semaphorins7A on MSCs were found to induce the macrophages to produce IL-10, which may play a critical therapeutic role. This study demonstrated that the specific chemokine receptor CXCR2 mRNA-engineered MSCs not only improves the therapeutic efficacy of IBD but also provides an efficient and safe MSC modification strategy.
The deleterious side effects of calcineurin inhibitors have impaired long-term survival after renal allograft. New immunotherapy regimens that minimize or even eliminate calcineurin inhibitors are required to improve transplantation outcome. Mesenchymal stem cells (MSCs) represent a unique cell population with immunosuppressive function and prolong allograft survival in experimental organ transplant models.In this pilot study, donor-derived bone marrow MSCs combined with a sparing dose of tacrolimus (50% of standard dose) were administered to six de novo living-related kidney transplant recipients. Six other patients who received a standard dose of tacrolimus were enrolled as a control. The safety of MSC infusion, acute rejection, graft function, and patient and graft survival within 12 months after kidney transplantation were observed. The immune profiles were analyzed at different time points after transplantation.None of the MSC recipients experienced immediate or long-term toxic side effects associated with MSC infusion. The tacrolimus dose (0.045±0.002 mg/kg) in the MSC group was significantly reduced compared with the control group (0.077±0.005 mg/kg). One acute rejection occurred only in the control group. All patients survived with stable renal function at month 12 and no chimerism was detectable at month 3. Patients in the MSC group showed significantly higher B-cell levels than the control group at month 3.These preliminary data suggest that the use of MSCs could provide potential benefits in renal transplantation by reducing the dosage of conventional immunosuppressive drug that is required to maintain long-term graft survival and function.
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