Mesenchymal stromal cells and their small extracellular vesicles in allergic diseases: From immunomodulation to therapy.

Mesenchymal stromal cells (MSCs) have long been considered a potential tool for treatment of allergic inflammatory diseases, owing to their immunomodulatory characteristics. In recent decades, the medical utility of MSCs has been evaluated both in vitro and in vivo, providing a foundation for therapeutic applications. However, the existing limitations of MSC therapy indicate the necessity for novel therapies. Notably, small extracellular vesicles (sEV) derived from MSCs have emerged rapidly as candidates instead of their parental cells. The acquisition of abundant and scalable MSC-sEV is an obstacle for clinical applications. The potential application of MSC-sEV in allergic diseases has attracted increasing attention from researchers. By carrying biological microRNAs or active proteins, MSC-sEV can modulate the function of various innate and adaptive immune cells. In this review, we summarise the recent advances in the immunomodulatory properties of MSCs in allergic diseases, the cellular sources of MSC-sEV, and the methods for obtaining high-quality human MSC-sEV. In addition, we discuss the immunoregulatory capacity of MSCs and MSC-sEV for the treatment of asthma, atopic dermatitis, and allergic rhinitis, with a special emphasis on their immunoregulatory effects and the underlying mechanisms of immune cell modulation.

Mesenchymal stromal cells-derived small extracellular vesicles modulate DC function to suppress Th2 responses via IL-10 in patients with allergic rhinitis.

Mesenchymal stromal cells (MSCs) are well known for their immunoregulatory roles on allergic inflammation particularly by acting on T cells, B cells, and dendritic cells (DCs). MSC-derived small extracellular vesicles (MSC-sEV) are increasingly considered as one of the main factors for the effects of MSCs on immune responses. However, the effects of MSC-sEV on DCs in allergic diseases remain unclear. MSC-sEV were prepared from the induced pluripotent stem cells (iPSC)-MSCs by anion-exchange chromatography, and were characterized with the size, morphology, and specific markers. Human monocyte-derived DCs were generated and cultured in the presence of MSC-sEV to differentiate the so-called sEV-immature DCs (sEV-iDCs) and sEV-mature DCs (sEV-mDCs), respectively. The phenotypes and the phagocytic ability of sEV-iDCs were analyzed by flow cytometry. sEV-mDCs were co-cultured with isolated CD4+ T cells or peripheral blood mononuclear cells (PBMCs) from patients with allergic rhinitis. The levels of Th1 and Th2 cytokines produced by T cells were examined by ELISA and intracellular flow staining. And the following mechanisms were further investigated. We demonstrated that MSC-sEV inhibited the differentiation of human monocytes to iDCs with downregulation of the expression of CD40, CD80, CD86, and HLA-DR, but had no effects on mDCs with these markers. However, MSC-sEV treatment enhanced the phagocytic ability of mDCs. More importantly, using anti-IL-10 monoclonal antibody or IL-10Rα blocking antibody, we identified that sEV-mDCs suppressed the Th2 immune response by reducing the production of IL-4, IL-9, and IL-13 via IL-10. Furthermore, sEV-mDCs increased the level of Treg cells. Our study identified that mDCs treated with MSC-sEV inhibited the Th2 responses, providing novel evidence of the potential cell-free therapy acting on DCs in allergic airway diseases.

Small extracellular vesicles derived from human mesenchymal stem cells prevent Th17-dominant neutrophilic airway inflammation via immunoregulation on Th17 cells.

Type 17 helper T cells (Th17)-dominant neutrophilic airway inflammation is critical in the pathogenesis of steroid-resistant airway inflammation such as severe asthma. Small extracellular vesicles (sEV) derived from human mesenchymal stem cells (MSCs) display extensive therapeutic effects and advantages in many diseases. However, the role of MSC-sEV in Th17-dominant neutrophilic airway inflammation and the related mechanisms are still poorly studied. Here we found that MSC-sEV significantly alleviated the infiltration of inflammatory cells in peribronchial interstitial tissues and reduced levels of inflammatory cells, especially neutrophils, in bronchoalveolar lavage fluids (BALF) of mice with neutrophilic airway inflammation. Consistently, MSC-sEV significantly decreased levels of IL-17A in BALF and Th17 in lung tissues. Furthermore, we found that labelled MSC-sEV were taken up by human CD4+ T cells most obviously at 12 h after incubation, and distributed mostly in mouse lungs. More importantly, potential signaling pathways involved in the MSC-sEV mediated inhibition of Th17 polarization were found using RNA sequencing. Using Western blot, JAK2-STAT3 pathway was identified as an important role in the inhibition of Th17 polarization by MSC-sEV. We found that proteins in MSC-sEV were mostly involved in the therapeutic effects of MSC-sEV. In total, our study suggested that MSC-sEV could be a potential therapeutic strategy for the treatment of neutrophilic airway inflammation.

BDNF-enriched small extracellular vesicles protect against noise-induced hearing loss in mice.

Noise-induced hearing loss (NIHL) is one of the most prevalent acquired sensorineural hearing loss etiologies and is characterized by the loss of cochlear hair cells, synapses, and nerve terminals. Currently, there are no agents available for the treatment of NIHL because drug delivery to the inner ear is greatly limited by the blood-labyrinth barrier. In this study, we used mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) as nanoscale vehicles to deliver brain-derived neurotrophic factor (BDNF) and evaluated their protective effects in a mouse model of NIHL. Following intravenous administration, BDNF-loaded sEVs (BDNF-sEVs) efficiently increased the expression of BDNF protein in the cochlea. Systemic application of sEVs and BDNF-sEVs significantly attenuated noise-induced cochlear hair cell loss and NIHL in CBA/J mice. BDNF-sEVs also alleviated noise-induced loss of inner hair cell ribbon synapses and cochlear nerve terminals. In cochlear explants, sEVs and BDNF-sEVs effectively protected hair cells against H2O2-induced cell loss. Additionally, BDNF-sEVs remarkably ameliorated H2O2-induced oxidative stress, cell apoptosis, and cochlear nerve terminal degeneration. Transcriptomic analysis revealed that many mRNAs and miRNAs were involved in the protective actions of BDNF-sEVs against oxidative stress. Collectively, our findings reveal a novel therapeutic strategy of MSC-sEVs-mediated BDNF delivery for the treatment of NIHL.

Dendritic cells mediated by small extracellular vesicles derived from MSCs attenuated the ILC2 activity via PGE2 in patients with allergic rhinitis.

BACKGROUND: Mesenchymal stromal cells-derived small extracellular vesicles (MSC-sEVs) have recently attracted considerable attention because of their therapeutic potential in various immune diseases. We previously reported that MSC-sEVs could exert immunomodulatory roles in allergic airway inflammation by regulating group 2 innate lymphoid cell (ILC2) and dendritic cell (DC) functions. Therefore, this study aimed to investigate the indirect effects of MSC-sEVs on ILC2s from patients with allergic rhinitis (AR) via DCs. METHODS: Here, we isolated sEVs from induced pluripotent stem cells-MSCs using anion-exchange chromatography and mature DCs (mDCs) were treated with MSC-sEVs. sEV-mDCs were co-cultured with peripheral blood mononuclear cells from patients with AR or purified ILC2s. The levels of IL-13 and GATA3 in ILC2s were examined by flow cytometry. Bulk RNA sequence for mDCs and sEV-mDCs was employed to further probe the potential mechanisms, which were then validated in the co-culture systems. RESULTS: sEV-mDCs showed impaired capacity in priming the levels of IL-13 and GATA3 in ILC2s when compared with mDCs. Furthermore, there was higher PGE2 and IL-10 production from sEV-mDCs, and the blockade of them especially the former one reversed the inhibitory effects of sEV-mDCs. CONCLUSIONS: We demonstrated that MSC-sEVs were able to dampen the activating effects of mDCs on ILC2s in patients with AR. Mechanismly, the PGE2-EP2/4 axis played an essential role in the immunomodulatory effects of sEV-mDCs on ILC2s. Herein, we provided new insights into the mechanism underlying the therapeutic effects of MSC-sEVs in allergic airway inflammation.

Mesenchymal stem cells overexpressing interleukin-10 prevent allergic airway inflammation.

BACKGROUNDS: Allergic airway inflammation is prevalent worldwide and imposes a considerable burden on both society and affected individuals. This study aimed to investigate the therapeutic advantages of mesenchymal stem cells (MSCs) overexpressed interleukin-10 (IL-10) for the treatment of allergic airway inflammation, as both IL-10 and MSCs possess immunosuppressive properties. METHODS: Induced pluripotent stem cell (iPSC)-derived MSCs were engineered to overexpress IL-10 via lentiviral transfection (designated as IL-10-MSCs). MSCs and IL-10-MSCs were administered intravenously to mice with allergic inflammation induced by ovalbumin (OVA), and the features of allergic inflammation including inflammatory cell infiltration, Th cells in the lungs, and T helper 2 cell (Th2) cytokine levels in bronchoalveolar lavage fluid (BALF) were examined. MSCs and IL-10-MSCs were co-cultured with CD4+ T cells from patients with allergic rhinitis (AR), and the levels of Th2 cells and corresponding type 2 cytokines were studied. RNA-sequence was performed to further investigate the potential effects of MSCs and IL-10-MSCs on CD4+ T cells. RESULTS: Stable IL-10-MSCs were established and characterised by high IL-10 expression. IL-10-MSCs significantly reduced inflammatory cell infiltration and epithelial goblet cell numbers in the lung tissues of mice with allergic airway inflammation. Inflammatory cell and cytokine levels in BALF also decreased after the administration of IL-10-MSCs. Moreover, IL-10-MSCs showed a stronger capacity to inhibit the levels of Th2 after co-cultured with CD4+ T cells from patients with AR. Furthermore, we elucidated lower levels of IL-5 and IL-13 in IL-10-MSCs treated CD4+ T cells, and blockade of IL-10 significantly reversed the inhibitory effects of IL-10-MSCs. We also reported the mRNA profiles of CD4+ T cells treated with IL-10-MSCs and MSCs, in which IL-10 played an important role. CONCLUSION: IL-10-MSCs showed positive effects in the treatment of allergic airway inflammation, providing solid support for the use of genetically engineered MSCs as a potential novel therapy for allergic airway inflammation.

Sanhuang Shu'ai decoction alleviates DSS-induced ulcerative colitis via regulation of gut microbiota, inflammatory mediators and cytokines.

BACKGROUND: Sanhuangshu'ai decoction (SH), a traditional Chinese medicine (TCM) prescription, has been safely used to treat diarrhea, dysentery and other inflammatory diseases with little side effect and low cost for thousands of years. However, its mechanism remains elusive. This study was designed to investigate the anti-ulcerative colitis (UC) activity of SH and mechanism by detecting its anti-inflammatory, anti-oxidative, and intervention effects of intestinal flora with the dextran sodium sulfate (DSS)-induced colitis mice. METHODS: The DSS-induced colitis mice was orally administered SH for 1 week with 0.8 or 1.6 g kg-1 d-1 dosage. A clinical disease activity score was evaluated daily. The colonic tissues of the mice were collected and prepared to detect its anti-inflammatory, anti-oxidative, intervention effects of intestinal flora and hydrogen peroxide(H2O2) in vivo, cytotoxicity and ROS influencing effects in vitro. Histological colitis severity and expression of cytokines were also determined. RESULTS: Oral administration of SH significantly prevented the development of colitis. It reduced the expression of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α in the colon. Moreover, SH administration alleviated the oxidative stress in the colon of DSS-induced colitis mice, evidenced by the decrease of myeloperoxidase (MPO) activity and malondialdehyde (MDA) level, and increase of ROS level. Furthermore, SH can prevent the decrease ofLactobacillus sp. and population abundance of intestinal flora caused by DSS. CONCLUSION: SH significantly ameliorates the symptoms of DSS-induced colitis mice and the potential mechanism of SH may involve in multiple kinds of metabolic pathway including the regulation of gut microbiota, inflammatory mediators and cytokines.

Small extracellular vesicles derived from human MSCs prevent allergic airway inflammation via immunomodulation on pulmonary macrophages.

Allergic airway inflammation is a major public health disease that affects up to 300 million people in the world. However, its management remains largely unsatisfactory. The dysfunction of pulmonary macrophages contributes greatly to the development of allergic airway inflammation. It has been reported that small extracellular vesicles derived from mesenchymal stromal cells (MSC-sEV) were able to display extensive therapeutic effects in some immune diseases. This study aimed to investigate the effects of MSC-sEV on allergic airway inflammation, and the role of macrophages involved in it. We successfully isolated MSC-sEV by using anion exchange chromatography, which were morphologically intact and positive for the specific EV markers. MSC-sEV significantly reduced infiltration of inflammatory cells and number of epithelial goblet cells in lung tissues of mice with allergic airway inflammation. Levels of inflammatory cells and cytokines in bronchoalveolar lavage fluid were also significantly decreased. Importantly, levels of monocytes-derived alveolar macrophages and M2 macrophages were significantly reduced by MSC-sEV. MSC-sEV were excreted through spleen and liver at 24 h post-administration in mice, and were able to be taken in by macrophages both in vivo and in vitro. In addition, proteomics analysis of MSC-sEV revealed that the indicated three types of MSC-sEV contained different quantities of proteins and shared 312 common proteins, which may be involved in the therapeutic effects of MSC-sEV. In total, our study demonstrated that MSC-sEV isolated by anion exchange chromatography were able to ameliorate Th2-dominant allergic airway inflammation through immunoregulation on pulmonary macrophages, suggesting that MSC-sEV were promising alternative therapy for allergic airway inflammation in the future.

Suppressive Effect of Huzhentongfeng on Experimental Gouty Arthritis: An In Vivo and In Vitro Study.

BACKGROUND: Huzhentongfeng (HZTF) is an extract from four Chinese medical herbs for treating gout. This study aims to evaluate its antigout activity and preliminary explore its mechanism in vivo and in vitro. METHODS: The rats were intragastrically administered with HZTF for 5 days and then injected 0.1 ml (10 mg) of MSU crystals to their joints for generating a gout model to analyze the paw volume and histopathology of joint synovial tissues of rats with different doses. We also investigated the antioxidant capacity of HZTF in vitro using indication including lipid peroxidation, DPPH·, and ABTS+ radical-scavenging capacity; besides, we used qRT-PCR to measure the effect of HZTF on interleukin (IL)-1ß, caspase-1, NLRP3, and NQO1 expression in hydrogen peroxide-stimulated RAW264.7 macrophages and IL-1ß, IL-6, and tumor necrosis factor (TNF)-α in MSU crystal-induced THP-1 monocytes. Confocal microscopy analysis was used to observe the dimerization of ASC adapter proteins. In addition, we also established quality standard of HZTF by using the high-performance liquid chromatography (HPLC) method. RESULTS: HZTF could significantly suppress the paw swelling and neutrophil infiltration induced by MSU intra-articular injection in rats compared with the control group. HZTF also showed inhibition effects of inflammatory cytokines (IL-1ß, IL-6, and TNF-α) secretion at 25.00 and 50.00 µg/ml in MSU-induced THP-1 cells but showed no effects of IL-1ß, IL-6, and TNF-α mRNA expression in MSU-induced THP-1 cells. Furthermore, confocal microscopy analysis showed that HZTF could prevent the oligomerization of ASC. Moreover, HZTF also showed effects in cell-free and cell-base tests of antioxidant capacity. CONCLUSION: The results prove that HZTF possessed the potential preventive effect against gout arthritis, and the effect may be attributed to its preventing effect on neutrophil infiltration and proinflammatory cytokines secretion such as IL-1ß, IL-6, and TNF-α which were caused by the activation of inflammasome.