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Olfactory ecto-mesenchymal stem cell-derived exosomes ameliorate murine Sjögren’s syndrome via suppressing Tfh cell response

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Published/Copyright: December 31, 2022
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Rheumatology and Immunology Research
From the journal Rheumatology and Immunology Research Volume 3 Issue 4

Abstract

Objectives

To investigate the effect of olfactory ecto-mesenchymal stem cell-derived exosomes (OE-MSC-Exos) on T follicular helper (Tfh) cell response and their implication in treating experimental Sjögrens syndrome (ESS).

Methods

C57BL/6 mice were immunized with salivary glands (SG) proteins to induce ESS mouse model. OE-MSC-Exos were added to the Tfh cell polarization condition, and the proportion of Tfh cells was detected by FCM. The PD-L1 of OE-MSCs was silenced with small interfering RNA to extract siPD-L1-OE-MSC-Exos.

Results

We found that transfer of OE-MSC-Exos markedly attenuated disease progression and reduced Tfh cell response in mice with ESS. In culture, OE-MSC-Exos potently inhibited the differentiation of Tfh cells from naïve T cells. Moreover, OE-MSC-Exos expressed high level of the ligand for the programmed cell death protein 1 (PD-L1), knocking down PD-L1 expression in OE-MSC-Exos significantly decreased their capacity to suppress Tfh cell differentiation in vitro. Consistently, transfer of OE-MSC-Exos with PD-L1 knockdown exhibited profoundly diminished therapeutic effect in ESS mice, accompanied with sustained Tfh cell response and high levels of autoantibody production.

Conclusion

Our results suggest that OE-MSC-Exos may exert their therapeutic effect in ameliorating ESS progression via suppressing Tfh cell response in a PD-L1-dependent manner.

Keywords: exosomes; mesenchymal stem cells; PD-L1, Sjögren’s syndrome; Tfh cells

Introduction

Sjögren’s syndrome (SS) is a chronic autoimmune disease that mainly causes dysfunction of exocrine glands, including salivary glands (SGs) and lacrimal glands.[1] In SS, infiltration of lymphocytes and tissue inflammation in exocrine glands, with the deposition of autoantibodies (auto-Abs), lead to impaired secretory functions of the exocrine glands, resulting in clinical manifestations of dry eyes and dry mouth.[2] Although significant progress has been made in understanding the pathophysiology of SS, the etiology of this disease is still unknown, with possible contributing factors including environmental, hormonal, and genetic factors.[3] Accumulated studies have demonstrated the involvement of many types of immune cells in the pathogenesis of SS, including type 1 T helper (Th1), Th2, Th17, and T follicular helper (Tfh) cells, regulatory T cells (Tregs), and regulatory B cells.[4–6] Currently, the treatment of SS mainly focuses on relieving the symptoms of xerostomia and xerophthalmia with local moistening agents, such as artificial tears and mouthwash.[7,8] As symptom relief–based therapies have limited efficacy, it is necessary to explore new therapies for SS.

Mesenchymal stem cells (MSCs) are a group of stromal cells with the potential of self-renewal and pluripotent differentiation, which can be purified from various tissues and organs for expansion in culture.[9,10] In addition to their strong differentiation ability, MSCs also have attracted much attention for their therapeutic potential with their immunomodulatory function, which can inhibit immune responses through intercellular contact or secretion of cytokines.[11] Currently, the clinical applications of MSC therapy have been widely investigated in various autoimmune diseases and inflammatory diseases.[12] Although MSC-based therapy is considered a promising strategy for treating autoimmune diseases, the applications of MSCs in clinical treatment still have many limitations.[12] Due to the large size of MSCs, intravascular administration may possibly lead to vascular obstruction in severe cases.[13] In addition, MSCs age rapidly and are costly for mass production and storage.[14] In particular, the immunomodulatory function of MSCs is susceptible to the influence of the local microenvironment, whereas high levels of inflammatory cytokines in patients with autoimmune disorders may affect the therapeutic effect of MSCs in vivo.[15,16] Recent studies have shown that MSC-secreted exosomes are more resistant to influence by microenvironmental factors and can deliver effector molecules steadily, which may serve as a promising therapeutic approach for the treatment of autoimmune diseases.

Exosomes are extracellular vesicles composed of lipid bilayer membranes with a diameter of 40–200 nm, which are released into the extracellular environment after budding from the plasma membrane.[17] Exosomes carry abundant bioactive substances, including various proteins, nucleic acids, metabolites, and lipid membrane components, which play an important role in regulating the physiological and pathological reactions of the body.[17,18] Increasing evidence has shown that MSC-derived exosomes (MSC-Exos) usually exhibit similar effects as MSCs, including immunomodulatory functions and repair of damaged tissue, but MSC-Exos are more functionally stable and considerably smaller in size.[19] Therefore, MSC-Exos may serve as a replacement for cell-based therapy; in addition, they have significant advantages over MSCs in the treatment of autoimmune diseases.

Olfactory ecto-MSCs (OE-MSCs) are a relatively new type of resident mesenchymal stem cells with high proliferation rate, self-renewal ability, and multilineage differentiation potential.[20] OE-MSCs are mainly derived from neural crest cells and are located in the olfactory lamina propria.[20,21] Previous studies have shown that OE-MSCs can induce neurogenesis and promote the recovery of hippocampal neuronal networks.[22] It has also been reported that OE-MSCs exhibit their powerful immunosuppressive ability by inhibiting the proliferation of effector T cells and increasing the expansion of Tregs.[23] Our recent studies have demonstrated that OE-MSC-derived exosomes (OE-MSC-Exos) possess potent immunosuppressive function and have strong potential in the treatment of autoimmune diseases.[24,25]

During autoimmune pathogenesis, Tfh cells, a subset of effector cluster of differentiation-4-positive (CD4+) T cells, play a pivotal role in driving germinal center (GC) reaction and high-affinity antibody (Ab) production.[26] Tfh cells are characterized by high expression levels of C-X-C motif chemokine receptor family (CXCR5), inducible T-cell costimulator (ICOS), B-cell lymphoma 6 protein (Bcl-6), and interleukin-21 (IL-21). These characteristics facilitate the migration of Tfh cells and their interaction with GC B cells, thereby driving B cells to differentiate into memory B cells and plasma cells.[26] However, abnormal activation and expansion of Tfh cells can interfere with GC B cell selection and generate autoreactive Abs, contributing to the development of various autoimmune diseases.[27] Increased frequencies of Tfh cells have been detected in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and SS.[28–30] Many studies have reported that programmed cell death protein 1 (PD-1) and its ligand PD-L1 are involved in inhibiting the activation and function of autoreactive T cells under autoimmunity.[31,32] Notably, PD-1 is expressed on activated Tfh cells, which may mediate the inhibitory signal in Tfh cell response.[32]

Materials and Methods

Mice

Female C57BL/6 mice between 6 and 8 weeks of age were purchased from the Experimental Animal Center of Yangzhou University (Jiangsu, China) and maintained under specific pathogen-free conditions in the Jiangsu University Animal Center. All animal experiments were performed following an institutionally approved protocol in line with the guidelines of the Jiangsu University Animal Ethics and Research Committee.

Isolation and Culture of OE-MSCs

OE-MSCs were prepared as previously described.[23] Briefly, olfactory epithelial tissue was carefully removed from the nasal cavity of wild C57BL/6 mice and cut into small pieces and cultured in flasks with Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12) supplemented with 15% fetal calf serum (Gibco, USA). After 7 days, isolated OE-MSCs were further expanded for 3 generations.

Isolation of OE-MSC-Exos

OE-MSCs were washed with phosphate-buffered saline (PBS) 3 times and cultured in conditioned medium containing fetal bovine serum. After 48 hours, supernatant fractions were collected for isolating exosomes. Briefly, the culture supernatant was subjected to differential centrifugation to remove cells and debris, and then, exosomes were isolated from the supernatant by ultracentrifugation at 100,000×g (Beckman Coulter, USA) for 1 hour at 4℃. After the supernatant was removed, the exosome particles were washed in PBS and centrifuged at 100,000×g at 4℃ for 1 hour and then resuspended in PBS and frozen at −80℃. OE-MSC-Exos protein concentration was quantified using a micro-bicinchoninic acid (BCA) protein detection kit.

To prepare OE-MSC-Exos with PD-L1 knockdown, PD-L1 small interfering RNA (siRNA) (RiboBio Co., Guangzhou, China) was transfected into OE-MSCs, and exosomes were extracted from transfected cells following the manufacturer’s protocol. The sequence of the PD-L1 siRNA is described below: GCCACAGCGAATGATGTTT.

Flow Cytometric Analysis

For surface markers, the prepared single-cell suspensions were identified with the following fluorochrome-conjugated monoclonal antibodies (mAbs): anti-CD4 (clone RM4-5), anti-PD-1 (clone 29F.1A12), anti-CXCR5 (clone SPRCL5), anti-B220 (clone RA3-6B2), anti-CD138 (clone 281-2), anti-CD19 (clone 6D5), anti-GL-7 (clone GL7), and anti-Fas (clone SA367H8). Intranuclear staining was performed with anti-Bcl-6 (clone 7D1) according to the instructions on the Foxp3 Staining Buffer Set (eBioscience). The stained cells were analyzed with a BD FACSCanto II flow cytometer (Becton Dickinson, NJ, USA), and the acquired data were analyzed with FlowJo software (TreeStar, Ashland, OR, USA). To detect exosome surface markers, latex microspheres (4 μm; Thermo) were mixed with OE-MSC-Exos according to the manufacturer’s instruction and then stained with the relevant fluorochrome-conjugated mAbs, viz., anti-PD-L1 (clone MIH5).

ESS Induction and OE-MSC-Exos Treatment

The ESS models were induced in 8-week-old female mice using our previously described protocol.[33,34] Each mouse was immunized with 200 μg of SG proteins, which were emulsified with complete Freund’s adjuvant (CFA) (Sigma-Aldrich, St. Louis, MO, USA) via subcutaneous injection in the neck on Days 0 and 7. On Day 14, a booster injection was administered with SG proteins (1 mg/mL) emulsified in incomplete Freund’s adjuvant (IFA) (Sigma-Aldrich). To explore the effects of OE-MSC-Exos treatment, mice were administered PBS or exosomes (100 μg/mouse) through the tail vein on Days 18 and 25 after the first immunization. Similarly, ESS mice were injected with siPD-L1-OE-MSC-Exos or negative control (NC) siNC-OE-MSC-Exos (100 μg per mouse) by tail vein.

Induction of Tfh Differentiation

Cells were enriched for naïve CD4+ T cells using the naïve CD4+ T cell Isolation Kit (Stem Cell, Canada) according to the manufacturer’s instruction. Purified murine naive CD4+ T cells (1.75×106/mL) were seeded in a culture plate precoated with anti-CD3 (1 μg/mL, clone 17A2; BioLegend) and anti-CD28 (1 μg/mL, clone E18; BioLegend) Abs under Tfh polarization conditions for 3 days. Cytokines for Tfh cell subset differentiation were as follows: recombinant murine IL-6 (25 ng/mL) and IL-21 (20 ng/mL); anti-interferon-gamma (anti-IFN-γ) (5 μg/mL, clone AN-18), anti-IL-4 (5 μg/mL, clone 11B11), and anti-transforming growth factor beta (anti-TGF-β) (5 μg/mL, clone TW7-20B9) neutralizing Abs. In order to explore the effects of PD-L1 molecule carried by the OE-MSC-Exos on Tfh differentiation, the proportion of Tfh cells was detected after adding siPD-L1-OE-MSC-Exos into the Tfh induction system.

Detection of Serum Auto-Abs

The serum levels of auto-Abs, anti-SG and anti-M3 muscarinic receptor (M3R), and anti-SS-related antigen A (SSA) were examined with a sandwich enzyme-linked immunosorbent assay (ELISA), as previously described.[25] SSA antigenic peptide (QEMPLTALLRNLGKMT) and SSA (AVALREYRKKMDIPA) were synthesized by the solid-phase chemical method and purified using high-performance liquid chromatography (HPLC) (SBS Genetec Co., Ltd, China).

Saliva Collection and Measurement

The salivary flow rate was measured as described in our previous study.[25] In brief, intraperitoneal injection of pilocarpine (Sigma-Aldrich, USA) at a dose of 5 mg/kg body weight induced salivary production in mice anesthetized with phenobarbital. Then, saliva was collected from the mouth for 15 minutes with the tip of a 20 μL pipette at room temperature.

Histopathological Assessment

The histological evaluation of glandular destruction in each group was performed on tissue sections of SGs with hematoxylin and eosin (H&E) staining 15 weeks after first immunization. Then, these tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The SG tissues were sectioned at 4 μm thickness and stained with H&E for morphologic examination. The severity of salivary tissue damage was assessed by a scoring system based on the size and extent of lymphocyte infiltration into the tissue.[35]

Statistical Analysis

All data were analyzed using Student’s t-test or 1-way analysis of variance (ANOVA). P-values <0.05 were considered to be statistically significant. Graphical generation of data was presented with GraphPad Prism software (San Diego, St Louis, MO, USA).

Results

Adoptive Transfer of OE-MSC-Exos Alleviates Disease Progression in ESS Mice

To evaluate the therapeutic effect of OE-MSC-Exos in ESS, we adoptively transferred OE-MSC-Exos into ESS mice on Days 18 and 25 after the first immunization (Figure 1A). Compared to the control ESS mice, OE-MSC-Exos-treated mice displayed smaller spleen and cervical lymph nodes (CLNs) (Figure 1B), and the cell numbers in these secondary immune organs were also significantly decreased (Figure 1C). As shown in Figure 1D, the reduction in salivary flow rate was markedly ameliorated in ESS mice with OE-MSC-Exos treatment. Histopathologic assessment showed reduced pathological changes in the submandibular glands of ESS mice treated with OE-MSC-Exos (Figure 1E). Moreover, serum levels of serum auto-Abs against SG, M3R, and SSA in OE-MSC-Exos-treated mice were significantly decreased when compared with control ESS mice without treatment (Figure 1F–H). Together, these data demonstrated that OE-MSC-Exos treatment could effectively attenuate disease progression in ESS mice.

Figure 1 OE-MSC-Exos alleviate disease progression in ESS mice. (A) A graphic scheme of ESS induction and OE-MSC-Exos administration. ESS mice were immunized with SG/CFA on Days 0 and 7 and boosted with SG/IFA on Day 14. The treatment groups were intravenously injected with OE-MSC-Exos on Days 18 and 25 after the first immunization. Mice were sacrificed on Day 42 (n = 6 per group). (B) Representative micrographs show the sizes of spleen and CLNs and SGs from each group. (C) The cell numbers in these secondary immune organs were analyzed. (D)The saliva flow rates were detected in each group. (E) The histological evaluation of glandular destruction in each group was performed with H&E staining on tissue sections of submandibular glands from mice 15 weeks post first immunization. (F–H) Autoantibodies against SG antigens (F), M3R (G), and SSA (H) were measured in the serum of ESS mice treated with OE-MSC-Exos or PBS. Data are shown as the mean ± SD of 3 independent experiments. **P < 0.01, *P < 0.05. CFA, complete Freund’s adjuvant; CLN, cervical lymph node; ESS, experimental Sjögren’s syndrome; H&E, hematoxylin and eosin; IFA, incomplete Freund’s adjuvant; LN, lymph node; M3R, M3 muscarinic receptor; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PBS, phosphate-buffered saline; SD, standard deviation; SG, salivary gland; SP, spleen; SSA, Sjögren’s syndrome-related antigen A.
Figure 1

OE-MSC-Exos alleviate disease progression in ESS mice. (A) A graphic scheme of ESS induction and OE-MSC-Exos administration. ESS mice were immunized with SG/CFA on Days 0 and 7 and boosted with SG/IFA on Day 14. The treatment groups were intravenously injected with OE-MSC-Exos on Days 18 and 25 after the first immunization. Mice were sacrificed on Day 42 (n = 6 per group). (B) Representative micrographs show the sizes of spleen and CLNs and SGs from each group. (C) The cell numbers in these secondary immune organs were analyzed. (D)The saliva flow rates were detected in each group. (E) The histological evaluation of glandular destruction in each group was performed with H&E staining on tissue sections of submandibular glands from mice 15 weeks post first immunization. (F–H) Autoantibodies against SG antigens (F), M3R (G), and SSA (H) were measured in the serum of ESS mice treated with OE-MSC-Exos or PBS. Data are shown as the mean ± SD of 3 independent experiments. **P < 0.01, *P < 0.05. CFA, complete Freund’s adjuvant; CLN, cervical lymph node; ESS, experimental Sjögren’s syndrome; H&E, hematoxylin and eosin; IFA, incomplete Freund’s adjuvant; LN, lymph node; M3R, M3 muscarinic receptor; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PBS, phosphate-buffered saline; SD, standard deviation; SG, salivary gland; SP, spleen; SSA, Sjögren’s syndrome-related antigen A.

Treatment with OE-MSC-Exos Suppresses Tfh Cell Response in ESS Mice

It has been recognized that Tfh cells can provide essential help for B cell activation during GC reactions and are critically involved in driving the pathogenesis of ESS.[29] Compared with control ESS mice treated with PBS, frequencies of both Tfh cells and GC B cells in the spleen and CLN were significantly decreased in ESS mice after the administration of OE-MSC-Exos (Figure 2A,B). Moreover, the proportions of plasma cells in spleen, CLN, and bone marrow were also markedly reduced in OE-MSC-Exos-treated ESS mice (Figure 2C). Taken together, these results indicated that OE-MSC-Exos could efficiently inhibit Tfh cell response and suppress plasma cell generation in ESS mice.

Figure 2 OE-MSC-Exos inhibit Tfh cell response in ESS mice. (A–C) Proportions of CD4+ PD-1+CXCR5+Bcl-6+ Tfh cells (A), CD19+Fas+GL-7+ GC B cells (B), and B220-CD138+ plasma cells (C) in the spleen, CLN, and BM of ESS mice treated with OE-MSC-Exos or PBS were analyzed by flow cytometry (n = 4). Data are shown as the mean ± SD of 3 independent experiments. ***P < 0.001, *P < 0.05. Bcl-6, B-cell lymphoma 6 protein; BM, bone marrow; CD4+, cluster of differentiation-4-positive; CXCR5, C-X-C motif chemokine receptor family; ESS, experimental Sjögren’s syndrome; LN, lymph node; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PBS, phosphate-buffered saline; PD-1, programmed cell death protein 1; SD, standard deviation; SG, salivary gland; SP, spleen; Tfh cells, T follicular helper cells.
Figure 2

OE-MSC-Exos inhibit Tfh cell response in ESS mice. (A–C) Proportions of CD4+ PD-1+CXCR5+Bcl-6+ Tfh cells (A), CD19+Fas+GL-7+ GC B cells (B), and B220-CD138+ plasma cells (C) in the spleen, CLN, and BM of ESS mice treated with OE-MSC-Exos or PBS were analyzed by flow cytometry (n = 4). Data are shown as the mean ± SD of 3 independent experiments. ***P < 0.001, *P < 0.05. Bcl-6, B-cell lymphoma 6 protein; BM, bone marrow; CD4+, cluster of differentiation-4-positive; CXCR5, C-X-C motif chemokine receptor family; ESS, experimental Sjögren’s syndrome; LN, lymph node; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PBS, phosphate-buffered saline; PD-1, programmed cell death protein 1; SD, standard deviation; SG, salivary gland; SP, spleen; Tfh cells, T follicular helper cells.

OE-MSC-Exos Inhibit Tfh Cell Differentiation Mainly Dependent upon PD-L1

To delineate the regulatory function of OE-MSC-Exos involved in modulating T cell response, we detected high levels of PD-L1 expression on OE-MSC-Exos by flow cytometry (Figure 3A). To investigate whether PD-L1 expressed on OE-MSC-Exos was involved in mediating the suppressive effect on Tfh response, we first knocked down the expression of PD-L1 on OE-MSC-Exos (Figure 3A). Then, we performed Tfh cell differentiation from splenic naïve CD4+ T cells of wild-type mice under Tfh cell polarization conditions in the presence of OE-MSC-Exos or OE-MSC-Exos with PD-L1 knockdown in culture. As shown in Figure 3B, OE-MSC-Exos treatment markedly inhibited the differentiation of Tfh cells in culture, whereas the suppressive effect of OE-MSC-Exos with PD-L1 knockdown was profoundly decreased on inhibiting Tfh cell differentiation. These results indicated that OE-MSC-Exos may exert the suppressive effect mainly dependent upon PD-L1.

Figure 3 OE-MSC-Exos-derived PD-L1 suppresses Tfh cell differentiation in vitro. (A) Specific short RNA was used to knock down PD-L1 on OE-MSC-Exos, and the knockdown efficiency was analyzed by flow cytometry. (B) Under Tfh cell polarization conditions, the percentage of Tfh cells was detected in each group. Data are shown as the mean ± SD of 3 independent experiments. *P < 0.05. CD4+, cluster of differentiation-4-positive; CTRL, control; Iso, Isotype; CXCR5, C-X-C motif chemokine receptor family; NC, negative control; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PD-1, programmed cell death protein 1; PD-L1, ligand for the programmed cell death protein 1 (PD-1); SD, standard deviation; siRNA, small interfering RNA; Tfh cells, T follicular helper cells.
Figure 3

OE-MSC-Exos-derived PD-L1 suppresses Tfh cell differentiation in vitro. (A) Specific short RNA was used to knock down PD-L1 on OE-MSC-Exos, and the knockdown efficiency was analyzed by flow cytometry. (B) Under Tfh cell polarization conditions, the percentage of Tfh cells was detected in each group. Data are shown as the mean ± SD of 3 independent experiments. *P < 0.05. CD4+, cluster of differentiation-4-positive; CTRL, control; Iso, Isotype; CXCR5, C-X-C motif chemokine receptor family; NC, negative control; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PD-1, programmed cell death protein 1; PD-L1, ligand for the programmed cell death protein 1 (PD-1); SD, standard deviation; siRNA, small interfering RNA; Tfh cells, T follicular helper cells.

Knockdown of PD-L1 Expression on OE-MSC-Exos Diminishes Their Immunosuppressive Function in vivo

To further determine the role of PD-L1 in mediating the immunoregulatory function of OE-MSC-Exos in vivo, we adoptively transferred the siPD-L1-OE-MSC-Exos into ESS mice on Days 18 and 25 after the first immunization (Figure 4A). The immunoregulatory effect of OE-MSC-Exos with PD-L1 knockdown in alleviating ESS development was almost abolished when compared with OE-MSC-Exos-treated ESS mice. As shown in Figure 4B,C, the sizes of spleen and CLN remained enlarged and cell numbers were increased in ESS mice treated with siPD-L1-OE-MSC-Exos. Moreover, both the reduction of salivary flow rate and the elevation of serum auto-Abs did not show any significant improvement in ESS mice with treatment of OE-MSC-Exos with PD-L1 knockdown (Figure 4D,E). Furthermore, histological analysis showed pronounced lymphocytic infiltration in the submandibular glands in the siPD-L1-OE-MSC-Exos group (Figure 4F). Collectively, these data suggest that PD-L1 is involved in the OE-MSC-Exos-mediated suppression of ESS development in vivo.

Figure 4 Knockdown of PD-L1 attenuates the effect of OE-MSC-Exos in alleviating the ESS. (A) A graphic scheme of ESS induction and OE-MSC-Exos (siPD-L1) administration. ESS mice were immunized with SG/CFA on Days 0 and 7 and boosted with SG/IFA on Day 14. The treatment groups were intravenously injected with siPD-L1-OE-MSC-Exos/siNC-OE-MSC-Exos on Days 18 and 25 after the first immunization. Mice were sacrificed on Day 42 (n = 6 per group). (B) Representative micrographs show the sizes of the spleen and CLNs of ESS mice in each group on Day 42 after the first immunization. (C) The cell numbers in these secondary immune organs were analyzed. (D,E) The saliva flow rates and autoantibodies (anti-SG, anti-M3R, and anti-SSA antibodies) were measured in each group. (F) The histological evaluation of glandular destruction in each group was performed on tissue sections of submandibular glands with H&E staining 15 weeks after first immunization. Data are shown as the mean ± SD of 3 independent experiments. ***P < 0.001, **P < 0.01. CFA, complete Freund’s adjuvant; CLN, cervical lymph node; ESS, experimental Sjögren’s syndrome; H&E, hematoxylin and eosin; IFA, incomplete Freund’s adjuvant; IgG, immunoglobulin G; i.v., intravenous; LN, lymph node; M3R, M3 muscarinic receptor; NC, negative control; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PD-L1, ligand for the programmed cell death protein 1 (PD-1); SD, standard deviation; SG, salivary gland; siRNA, small interfering RNA; SP, spleen; SSA, Sjögren’s syndrome-related antigen A.
Figure 4

Knockdown of PD-L1 attenuates the effect of OE-MSC-Exos in alleviating the ESS. (A) A graphic scheme of ESS induction and OE-MSC-Exos (siPD-L1) administration. ESS mice were immunized with SG/CFA on Days 0 and 7 and boosted with SG/IFA on Day 14. The treatment groups were intravenously injected with siPD-L1-OE-MSC-Exos/siNC-OE-MSC-Exos on Days 18 and 25 after the first immunization. Mice were sacrificed on Day 42 (n = 6 per group). (B) Representative micrographs show the sizes of the spleen and CLNs of ESS mice in each group on Day 42 after the first immunization. (C) The cell numbers in these secondary immune organs were analyzed. (D,E) The saliva flow rates and autoantibodies (anti-SG, anti-M3R, and anti-SSA antibodies) were measured in each group. (F) The histological evaluation of glandular destruction in each group was performed on tissue sections of submandibular glands with H&E staining 15 weeks after first immunization. Data are shown as the mean ± SD of 3 independent experiments. ***P < 0.001, **P < 0.01. CFA, complete Freund’s adjuvant; CLN, cervical lymph node; ESS, experimental Sjögren’s syndrome; H&E, hematoxylin and eosin; IFA, incomplete Freund’s adjuvant; IgG, immunoglobulin G; i.v., intravenous; LN, lymph node; M3R, M3 muscarinic receptor; NC, negative control; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PD-L1, ligand for the programmed cell death protein 1 (PD-1); SD, standard deviation; SG, salivary gland; siRNA, small interfering RNA; SP, spleen; SSA, Sjögren’s syndrome-related antigen A.

PD-L1 on OE-MSC-Exos Mediates the Suppression of Tfh Cell Response in ESS

To determine whether the changes of Tfh cells in OE-MSC-Exos-treated ESS mice was dependent on PD-L1 expression, we performed flow cytometric analysis on Tfh cell frequencies in mice treated with siPD-L1-OE-MSC-Exos. As shown in Figure 5A, the markedly increased Tfh cell response was sustained in ESS mice treated with OE-MSC-Exos with knocking down of PD-L1 expression. Moreover, the frequencies of GC B cells and plasma cells remained high in siPD-L1-OE-MSC-Exos-treated mice (Figure 5B,C). Together, these results suggest that the immunoregulatory effect of OE-MSC-Exos in inhibiting Tfh cell responses in ESS mice was predominantly dependent on PD-L1 expression.

Figure 5 Knockdown of PD-L1 abolishes the effect of OE-MSC-Exos in suppressing Tfh cell response in ESS. Proportions of Tfh cells (A), GC B cells (B), and plasma cells (C) in the spleen, cervical lymph node, and bone marrow of ESS mice treated with siPD-L1-OE-MSC-Exos or siNC-OE-MSC-Exos were analyzed by flow cytometry (n = 4 per group). Data are shown as the mean ± SD of 3 independent experiments. *P < 0.05. BM, bone marrow; CD4+, cluster of differentiation-4-positive; CXCR5, C-X-C motif chemokine receptor family; ESS, experimental Sjögren’s syndrome; GC, germinal center; LN, lymph node; NC, negative control; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PD-1, programmed cell death protein 1; PD-L1, ligand for the programmed cell death protein 1 (PD-1); SD, standard deviation; SG, salivary gland; siRNA, small interfering RNA; SP, spleen; Tfh cells, T follicular helper cells.
Figure 5

Knockdown of PD-L1 abolishes the effect of OE-MSC-Exos in suppressing Tfh cell response in ESS. Proportions of Tfh cells (A), GC B cells (B), and plasma cells (C) in the spleen, cervical lymph node, and bone marrow of ESS mice treated with siPD-L1-OE-MSC-Exos or siNC-OE-MSC-Exos were analyzed by flow cytometry (n = 4 per group). Data are shown as the mean ± SD of 3 independent experiments. *P < 0.05. BM, bone marrow; CD4+, cluster of differentiation-4-positive; CXCR5, C-X-C motif chemokine receptor family; ESS, experimental Sjögren’s syndrome; GC, germinal center; LN, lymph node; NC, negative control; OE-MSC-Exos, olfactory ecto-mesenchymal stem cell-derived exosomes; PD-1, programmed cell death protein 1; PD-L1, ligand for the programmed cell death protein 1 (PD-1); SD, standard deviation; SG, salivary gland; siRNA, small interfering RNA; SP, spleen; Tfh cells, T follicular helper cells.

Discussion

In this study, we have demonstrated that transfer of OE-MSC-Exos exhibited significantly therapeutic effect in ESS mice, in which both Tfh cell response and plasma cell generation were markedly suppressed after OE-MSC-Exos treatment. Moreover, we have found that PD-L1, an inhibitor of the checkpoint protein in MSCs, is particularly enriched in OE-MSC-Exos and is critically involved in mediating the immunoregulatory function of OE-MSC-Exos in inhibiting Tfh cell differentiation both in vitro and in vivo. Collectively, our results reveal that OE-MSC-Exos can exert their immunosuppressive function in restraining Tfh cell response and ameliorating ESS progression mainly via a PD-L1-dependent manner.

MSCs have the potential of self-renewal and multidirectional differentiation and are widely distributed in various tissues in the body, such as bone marrow, adipose tissue, muscle, and umbilical cord.[36] Our previous studies have demonstrated that OE-MSCs not only possess typical characteristics of stem cells but also exhibit strong immunosuppressive function.[23] Accumulating evidences suggest that MSC-mediated immune modulation occurs mainly through paracrine effects, especially via exosomes.[37] Exosomes derived from MSCs are spherical vesicles that contain abundant anti-inflammatory compounds with immunomodulatory properties.[38] Recent studies have reported the immunomodulatory properties of MSC-Exos in animal models of autoimmune disease. Here, we show that OE-MSC-Exos treatment significantly alleviates the disease severity of ESS, with improved salivary flow rate and decreased lymphocytic infiltration in SGs. Notably, Tfh cells were remarkably reduced in spleen and draining lymph nodes after OE-MSC-Exos administration. These data support the notion that OE-MSC-Exos may exert their immunosuppressive effect by restraining Tfh cell response and thus ameliorating the disease progression. Although available studies have reported the functional abnormality of autologous MSCs from patients with autoimmune diseases, it is currently unclear whether both OE-MSCs and OE-MSC-derived exosomes from diseased ESS mice show any functional changes in modulating T cell responses, which warrants further investigation.

It has been recognized that MSC-Exos can carry abundant regulatory molecules, such as PD-L1.[39] Previous reports have shown that PD-1, the PD-L1 receptor, is upregulated on activated CD4+ T cells, while the PD-1/PD-L1 pathway plays an important role in exosome-mediated immune regulation.[40] The PD-1/PD-L1 pathway acts by either promoting Treg function or inhibiting activation of effector T cells, thus restraining autoimmune pathogenesis.[41] Thus, targeting the PD-1/PD-L1 axis has been considered a promising strategy in the treatment of autoimmune disorders.[42] We have previously observed that OE-MSCs could suppress the proliferation of CD4+ T cells by PD-L1.[23] Here, we find that PD-L1 is particularly enriched in OE-MSC-Exos, whereas knocking down PD-1 in the exosomes of OE-MSCs profoundly diminishes the immunoregulatory function of OE-MSC-Exos in inhibiting Tfh cell differentiation in vitro and their therapeutic effect in vivo. Therefore, these results indicate a critical role of exosomecarried PD-L1 in mediating the immunosuppressive function of OE-MSCs in inhibiting Tfh response, which may facilitate the further development of OE-MSC-based therapy for potential application for treating SS patients.

In summary, our findings have demonstrated that OE-MSC-Exos exert their immunomodulatory function in ameliorating disease severity in ESS mice possibly by suppressing Tfh cell response. In addition, we provide strong evidence that exosome-carried PD-L1 further enhances the potential application of OE-MSC-Exos in treating autoimmune diseases through the PD-L1/PD-1 axis in immune regulation. Taken represent a new generation of cell-free therapeutics for the together, these results suggest that OE-MSC-Exos may treatment of SS and other autoimmune diseases.

#Ke Rui and Ziwei Shen have contributed equally to this work.

Department of Immunology, Jiangsu Key Laboratory of Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang 212003, Jiangsu Province, China.
Department of Pathology, The University of Hong Kong, Hong Kong 999077, China.

Funding statement: This work was supported by Shenzhen Science and Technology Program (YCYJ20210324114602008), Chongqing International Institute for Immunology (2020YJC10), and National Natural Science Foundation of China (Nos. 81971542, 82171771, 82071817, and 82271854).

  1. Author Contributions

    KR, ZS, and NP performed the experiments, analyzed the data, and wrote the paper; SL, XX and CL performed the experiments; FZ, YT, and LW analyzed the data; LL and JT designed the study and revised the paper. All authors read and approved the final manuscript.

  2. Informed Consent

    None declared.

  3. Ethical Statement

    All the experiments were approved by the Committee on the Use of Live Animal in Research and Teaching of Jiangsu University (UJS-IACUC-AP-20190307016).

  4. Conflict of Interest

    Liwei Lu is an Editorial Board Member of the journal. The article was subject to the journal’s standard procedures, with peer review handled independently of the editor and his research groups.

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Received: 2022-07-29
Accepted: 2022-11-13
Published Online: 2022-12-31

© 2022 Ke Rui, Ziwei Shen, Na Peng, Futao Zhao, Yuan Tang, Shiyi Liu, Xinyi Xu, Chang Liu, Ling Wu, Jie Tian, Liwei Lu, published by De Gruyter

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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https://doi.org/10.2478/rir-2022-0035
Keywords for this article
exosomes; mesenchymal stem cells; PD-L1, Sjögren’s syndrome; Tfh cells
Creative Commons
BY-NC-ND 4.0

Articles in the same Issue

  1. Editorial
  2. Editorial for “Social Media in Rheumatology”
  3. Social media and its impact on rheumatology
  4. Commentary
  5. How Twitter use in rheumatology has evolved
  6. Perspective
  7. Social media and the patient – on education and empowerment
  8. An overview of social media within APLAR rheumatology societies
  9. Review
  10. #RheumTwitter – The rise of social media in rheumatology: Research, collaboration, education, and engagement
  11. Social media for research discourse, dissemination, and collaboration in rheumatology
  12. Mini Review
  13. Social media and rheumatology societies: Strategic insights
  14. Communication
  15. RheumCloud App: A novel mobile application for the management of rheumatic diseases patients in China
  16. Original Article
  17. The therapeutic effect of tacrolimus in a mouse psoriatic model is associated with the induction of myeloid-derived suppressor cells
  18. Olfactory ecto-mesenchymal stem cell-derived exosomes ameliorate murine Sjögren’s syndrome via suppressing Tfh cell response
  19. Case Report
  20. Successful treatment with bortezomib for refractory thrombotic thrombocytopenic purpura associated with systemic lupus erythematosus
  21. Images
  22. Bilateral ureterohydronephrosis after intestinal pseudo-obstruction in a patient with systemic lupus erythematosus
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