Subconjunctival injection of human umbilical cord mesenchymal stem cells alleviates experimental allergic conjunctivitis via regulating T cell response

doi:10.21203/rs.3.rs-3082933/v1

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Research Article

Subconjunctival injection of human umbilical cord mesenchymal stem cells alleviates experimental allergic conjunctivitis via regulating T cell response

https://doi.org/10.21203/rs.3.rs-3082933/v1

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Journal Publication

published 02 Oct, 2023

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Background

T helper 2 (Th2) cells are thought to play critical roles in allergic conjunctivitis (AC). They release inflammatory cytokines to promote an allergic response in AC. Due to individual heterogeneity and long-term chronic management, current therapies do not always effectively control AC. Mesenchymal stem cells (MSCs) have been shown to be effective in treating allergy-related disorders, but it is unclear how exactly the Th2-mediated allergic response is attenuated. This study aims to elucidate the therapeutic effect and mechanism of the human umbilical cord MSCs (hUCMSCs) in a mouse model of experimental AC (EAC).

Methods

A mouse EAC model was established by inoculating short ragweed (SRW) pollen. After the SRW pollen challenge, the mice received a single subconjunctival or tail vein injection of 2 x 10⁶ hUCMSCs; subsequent scratching behavior and clinical symptoms were assessed. Immunostaining and flow cytometry were carried out to show allergic reactions and the activation of CD4 + T cell subsets in the conjunctiva and cervical lymph nodes (CLNs). Gene expression was determined by RNA-seq and further verified by qRT-PCR and Western blot. Co-culture assays were performed to explore the regulatory role of hUCMSCs in the differentiation of CD4 + Th2 cells.

Results

Subconjunctival administration of hUCMSCs resulted in fewer instances of scratching and lower inflammation scores in EAC mice compared to the tail-vein delivery and control groups. Subconjunctival administration of hUCMSCs reduced the number of activated mast cells and infiltrated eosinophils in the conjunctiva, as well as decreased the number of Th2 cells in CLNs. After pretreatment with EAC mouse serum in vitro to mimic the in vivo milieu, hUCMSCs were able to inhibit the differentiation of naive T cells (Th0) into Th2 cells. Further evidence demonstrated that repression of Th2 cell differentiation by hUCMSCs is mediated by CRSPLD2 through downregulation of STAT6 phosphorylation. Additionally, hUMCSCs were able to promote the differentiation of Th0 cells into regulatory T cells (Treg) in CLNs of EAC mice.

Conclusions

Subconjunctival injection of hUCMSCs suppressed the Th2-allergic response and alleviated clinical symptoms. This study provides not only a potential therapeutic target for the treatment of AC but also other T cell-mediated diseases.

Allergic conjunctivitis

Human umbilical mesenchymal stem cells

Immunomodulatory

Subconjunctival injection

Th2 cells

Allergic conjunctivitis (AC) has been characterized as a bilateral ocular surface inflammatory response resulting from exposure to allergens such as pollen, house dust mites (HDM), animal dander, or some molds (1). There are four types of allergic conjunctivitis: vernal and atopic keratoconjunctivitis (chronic), and perennial and seasonal allergic conjunctivitis (acute). The latter two are the most common types, leaving 30%-40% of the general population with diminished quality of life (2, 3). Refractory and chronic cases may lead to vision-threatening ocular surface complications. They may also deprive corneal allografts of immune privileges and increase immune rejection (4–6). The main symptoms of AC are itching, redness, and increased tear production.

Of the several immune cells implicated in the occurrence or progression of allergic diseases (e.g., mast cells, T cells, B cells, dendritic cells), the inappropriate activation of T helper 2 (Th2) cells has been recognized as a critical mechanism in various allergic reactions (7). After being exposed to a conjunctival allergen, dendritic cells (DCs) migrate into the cervical lymph nodes (CLNs) to initiate the proliferation of naive T cells (Th0) and differentiation into Th2 cells. Cytokines produced by Th2 cells, namely interleukin (IL)-4, IL-5, and IL-13, favor the activation and stimulation of B cells to produce IgE. Mast cells (MCs) and basophils have high-affinity IgE receptors which, when bound with IgE, “sensitizes” the cells (8). Upon re-exposure to the allergen, bound IgEs on a sensitized MC undergo crosslinking and leads to the degranulation of the cell. The subsequent release of histamine, cytokines, lipid mediators, and chemokines, contribute to the clinical symptoms of AC (9). In addition, there is growing evidence of other pathways contributing to Th2-dominant allergic inflammation, such as Th17 cell-derived IL-17 (10, 11), as well as innate immunity. It was recently reported that, during AC pathogenesis, elevated group 2 innate lymphoid cells produce cytokines such as thymic stromal lymphopoietin, IL-25, and IL-33 that enhance Th2 response and induce inflammation (12). On the other hand, regulatory T cells (Treg) cells have been acknowledged as a suppressor of Th2-dependent allergic inflammation and mast cell degranulation, facilitated by the production of IL-10 and transforming growth factor-β (TGF-β). Dysregulation of Treg cells could lead to allergic sensitization and allergic disease (13).

Generally, pharmacological treatments for acute and chronic forms of AC include a variety of topical and oral agents (e.g., short-acting beta-agonists, antihistamines, leukotriene receptor antagonists, and corticosteroids). However, these compounds only provide temporary symptomatic relief, since they cannot directly address the root cause. Furthermore, they are limited by systemic side effects and drug resistance (14). Other potential treatments, such as allergen-specific immunotherapy, JAK/STAT inhibitors, and Bruton’s tyrosine kinase (BTK) inhibitors, are being evaluated in clinical trials (15, 16). Mesenchymal stem cells (MSCs) have emerged as a promising alternative for various allergic and autoimmune diseases (17). Su et al. found that the conditioned medium (CM) from bone marrow-derived MSCs stimulated by tumor necrosis factor-a (TNF-a) alleviates experimental AC (EAC) by suppressing the activation of MCs, reducing the number of inflammatory cells, and the levels of related factors (e.g., TNF-α, IL-4, NF-κB) (18). Using a mouse model of ovalbumin-induced airway inflammation, Kavanagh and Mahon et al. reported that systemic administration of allogeneic MSCs inhibited IL-13 and IL-4 production, and activated Treg cells (19). Shin et al. demonstrated that human umbilical cord-derived MSCs (hUCMSCs) attenuated inflammation in mouse asthma models and downregulated IL-5 and IL-13 production in Th2 cells from mice (20). The available evidence indicates that MSCs have the capacity to suppress Th2 function in Th2-dominated inflammation (21).

However, the mechanisms underlying the regulation of Th2 cells by MSCs in AC are still poorly understood. In addition, there is limited literature demonstrating the role and secretion profile of MSCs in AC. Herein, we investigated the therapeutic functions of hUCMSCs in an SRW-induced mouse EAC model and explored the mechanisms behind the suppression of T lymphocyte subset differentiation by hUCMSCs. In addition, the different delivery routes of hUCMSCs were also evaluated. Our results provide new insights into the immunoregulatory mechanism mediated by MSCs in AC and other T cell-mediated allergic diseases.

Animal Preparation

Six-week-old female BALB/c mice (Laboratory Animal Center of Tongji University) were used in this study. All experiments on animals were approved by the animal experimentation ethics committee of Tongji University (Approval NO. TJAA09620101). Principles of animal research conformed to the guidelines of ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All mice were maintained on a regular diurnal lighting cycle (12:12 light: -dark) with access to food and water. Mice were randomly allocated to experimental groups and no blinding method was used for cell transplantation. There was no animal exclusion criterion.

hUCMSCs culture and stimulation

The hUCMSCs were obtained from the Eastern Union Stem Cell and Gene Engineering Co., Ltd. (China). Informed consent was obtained by Eastern Union Stem Cell and Gene Engineering Co., Ltd. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Gibco, California, USA) containing 10% FBS (Applied StemCell, California, USA) at 37°C, 5% CO₂. The cells have been identified according to our previous study (22). For clarifying the immunoregulatory effect of hUCMSCs, the cells were pretreated with serum obtained from normal or EAC mice with 10% concentration in DMEM/F12 medium supplemented with penicillin and streptomycin (P/S) antibiotic solution (Gibco, Grand Island, NM) for 24h at 37°C in a 5% CO₂ humidified atmosphere.

Establishment of mouse EAC model and administration of hUCMSCs

Animals were randomized using a computer based random order generator and divided into four groups (six animals/group); For the establishment of the EAC model, BALB/c mice were immunized with 50 µg of short ragweed (SRW) pollen (Greer Lab, Lenoir, NC USA) by footpad injection at day 0 and a secondary injection at day 5. Then, the mice were injected intraperitoneally with pentobarbital sodium (1% v/v) for anesthesia. Next, by using eye drops, 1.5 mg of SRW pollen dissolved in 10 µL of PBS was administered to each eye once a day from day 10 to 15. For the experimental group, at day-10 following the SRW-pollen challenge, 2 × 10⁶ cells were delivered in 30 µl of PBS via subconjunctival injection, or in 200 µl of PBS via intravenous injection.

For each animal, three investigators were involved for different roles respectively and animals that failed the injection were excluded in the analysis; The signs of immediate hypersensitivity responses and instances of scratching in the 20 minutes after the topical-SRW-pollen challenge were evaluated. Total clinical signs were scored based on chemosis, conjunctival redness, lid edema, and tearing, and each was graded from a score of 0 to 3 (0 = absent; 1 = mild; 2 = moderate and 3 = severe) (23). Twenty-four hours after the last challenge, the mice were euthanized by cervical dislocation after CO2 anesthesia, and the blood of EAC mice was collected and centrifugated, the supernatant was labeled as EAC mouse serum and stored at − 80°C for pretreatment of hUCMSCs in vitro.

To evaluate whether the intravenously injected hUCMSCs migrated to the conjunctiva, shControl-hUCMSCs expressing exogenous ZsGreen were intravenously injected into the mouse EAC model. Twenty-four hours later, conjunctivas were collected and cryosections were prepared. Samples were observed under fluorescence microscope (Olympus IX73). Samples with subconjunctival injection of shControl-hUCMSCs were used as the positive control.

Histologic Analysis of conjunctiva and CLNs

The eyes with eyelids and conjunctival tissue attached, and the CLNs were collected on day-15 following the SRW-pollen challenge. Tissues were fixed with 4% paraformaldehyde and embedded in optimal cutting temperature compound (VWR, Suwanee, GA). Cryosections (10 µm thickness) of eyeballs and CLNs were prepared with a cryostat (Leica CM 1950, Germany). The infiltrated eosinophils were detected by Giemsa staining (Beyotime, #C0131), and MCs were stained by toluidine blue staining (Solarbio, #G3670). The sections were visualized with a microscope system (Olympus IX73; Olympus, Tokyo, Japan).

For immunostaining, sections were incubated with primary antibodies against CD4, IL-4, IL-5, IL-10, IL-17, IL-13, FOXP3, and IFN-γ, then incubated with Alexa Fluor 488 or 555–conjugated anti-rabbit and mouse IgG antibody (Jackson ImmunoResearch Labs). Antibodies are listed in Supplementary Table 1. Afterward, nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA). The samples were then examined by fluorescence microscope (Olympus IX73, Tokyo, Japan).

RNA isolation and quantitative real-time PCR

Total RNA was extracted and reverse transcription was performed using Primescript™ RT Master Mix kit (Takara, Shiga, Japan). qRT-PCR was performed in a Chromo4 instrument cycler (Bio-Rad, Hercules, USA) using Superreal Premix plus kit (Tiangen Biotech, Beijing, China). PCR amplification was carried out with the following cycling parameters: denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s. Primer sequences (Synthesized by Sangon Biotech Co., Ltd., Shanghai, China) were listed in Supplementary Table 2.

Flow cytometry

Fresh CLNs were dissociated, resuspended in FACS buffer, and filtered through a 70-µm mesh cell strainer (Corning, NY, USA). The single-cell suspensions were labeled with anti-CD4-FITC (eBioscience 11-0041-81) or anti-CD25-PE-Cyanine7(eBioscience, 25-0251-81) antibodies. Cells were washed with pre-chilled PBS to remove unbound antibodies. Fixation and permeabilization were performed with the fixation and permeabilization kit (Cat. No. 88-8824-00, eBioscience). After washing once in PBS and twice in buffer, cells were incubated with anti-IFN-γ-PE (eBioscience, 12-7311-82) or anti-IL-4-APC (eBioscience, 17-7041-81) antibodies. FOXP3 was labeled with Brilliant Violet 421™ anti-mouse FOXP3 Antibody in True-Nuclear™ Transcription Factor Buffer (Cat No.126419, Biolegend). Flow cytometric analysis was performed on a flow cytometer (Beckman Coulter, CA, USA), and data were analyzed with Flow Jo 10.7.1 software or CytoExpert software (Beckman Coulter).

RNA sequencing

Following the Illumina mRNA-seq protocol, pooled RNA libraries of the cells were established, with 50ng of RNA from hUCMSCs treated with serum from EAC mice or normal mice. Sequencing was performed by the MAJORBIO company (Shanghai, China). Filtering and quality control of the raw reads from RNA-seq was carried-out using FastQC. The clean reads were mapped to reference sequences using SOAP2 aligner. Gene expression levels were calculated using the TPM method. Log₂ fold change (FC) of TPM (hUCMSCs treated with EAC serum) / TPM (hUCMSCs treated with normal serum) was used to identify differentially expressed genes (DEGs) between these two groups. Only those genes indicating |FC| > 1.5 and adjusted p < 0.05 were regarded as DEGs.

Assembly of lentivirus to knockdown crispld2

Lentiviral pLVX-shRNA2-ZsGreen1 (Takara) vector was used to prepare lentivirus. The packaging plasmids psPAX2 and pMD2.G were used. The shRNA target sequences were as follows: CRISPLD2-RNAi-1: CGTCAGATGTGACACCAAGAT; CRISPLD2-RNAi-2: GCAGCTGCAGGAACAACTTGT; CRISPLD2-RNAi-3: GGAGTACATGACCTGGGATGA. N;on-targeting sequence construct was used as the control: GCGCGATAGCGCTAATAATTT. HEK293FT cells were transfected with vectors. Individual supernatants containing the assembled virus were harvested at 48h and used to infect hUCMSCs. The positively transfected cells were sorted by FACS based on ZsGreen expression. Reduced expression of target gene CRISPLD2 at the transcript level was determined by qRT-PCR. The most effective shRNA sequence out of the three was selected.

T cell proliferation assay

CD4 + T cells from spleen cells of BALB/c mice were isolated with a negative-selection magnetic cell separation (MACS) approach. MACS separation was performed using the Dynabeads™ Untouched™ CD4 + T Cell Isolation Kit (Invitrogen™,11415D) and LS column (Miltenyi Biotec, cat no. 130-042-401). 1 × 10⁵ hUCMSCs were seeded in a 6-well culture plate overnight. Isolated T cells were labeled with Carboxy-fluorescein succinimidyl ester (CFSE) and co-cultured with hUCMSCs in the presence of CD3/CD28 activation beads. The proliferation index of T cells was assessed based on CFSE dye dilution rate after 3 days of co-culturing.

Differentiation of Th0 cells

Th0 cells (CD4 + CD25-CD62Lhigh CD44low) from lymph nodes and spleen of BALB/c mice were isolated and purified by flow cytometry. Then, in 12-well plates with humidified 5% CO2 at 37°C, Th0 cells (2×10⁶/ml) were treated with Dynabeads™ Mouse T-Activator CD3/CD28 (Gibco™, 11456D) in the presence of IL-2 (10 ng/ml). hUCMSCs were co-cultured with T cells at a ratio of 1:5. For Th2 differentiation, anti-IFN-γ antibody (10 ug/ml) and IL-4 (20 ng/ml) were added at the start of co-culture. For Treg differentiation, anti-IFN-γ antibody (10 ug/ml) and TGF-β1(20 ng/ml) were added at the start of co-culture. To assess the polarization of Th2, cells were stimulated with a cell activation cocktail containing PMA, ionomycin, and Brefeldin A (Cat No. 423303, Biolegend) for 6h. The cells were then fixed and permeabilized with Cyto-Fast™ Fix/Perm Buffer Set (Cat No.426803, Biolegend) and stained with anti-CD4-FITC, anti-IFN-γ-PE, or anti-IL-4-APC antibodies (eBioscience). Treg cells were fixed and permeabilized with True-Nuclear™ Transcription Factor Buffer Set and stained with anti-CD4-FITC, anti-CD25-PE-Cyanine7, and anti-FOXP3- Brilliant Violet 421™ antibodies (Cat No.126419, Biolegend). After staining, cells were evaluated by flow cytometry.

Western blot analyses

The CD4 + T cells co-cultured with hUCMSCs were lysed in RIPA lysis buffer (Beyotime, China) containing protease and phosphatase inhibitors. A total of 10 µg protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 3% BSA in PBS for 2h, the membrane was incubated with primary antibodies against phospho-signal transducer and activator of transcription (STAT)6 (1:1000, Abcam, ab263947), STAT6 (1:1000, Proteintech, Cat No. 10253-2-AP), or GAPDH (1:5000, Proteintech, Cat No. 60004-1-Ig) for 12h at 4℃, then incubated with anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:5000, Proteintech, Cat No. SA00001-2) for 1h at room temperature. The blots were visualized with a chemiluminescence imaging system (Tanon 5200, Shanghai, China) and quantified using ImageJ (Version 1.48v).

Statistical analysis

GraphPad Prism (Version 9.3.0) was used for statistical analysis. All quantitative data were recorded as mean ± SD. Statistical differences between two datasets were evaluated using Student’s t-test. Multi-group comparisons were analyzed by one-way ANOVA and post hoc Bonferroni’s test. P-values less than 0.05 was considered significant. All experiments were repeated in triplicates.

Subconjunctival injection of hUCMSCs alleviates EAC

To investigate whether different routes of administration affected the therapeutic effects of hUCMSCs, we injected hUCMSCs (2×10⁶ cells) subconjunctivally or intravenously (IV) in the mouse EAC model at day 10 post-immunization (Fig. 1A). We observed typical allergic symptoms and scratching behavior induced in the EAC mice after the SRW-pollen challenge. Treatment by subconjunctival injection of hUCMSCs significantly alleviated scratching behavior and allergy-related symptoms such as eyelid swelling, conjunctival edema, redness, and tearing (Fig. 1B-C). However, IV administration of hUCMSCs did not attenuate the symptoms of EAC.

The severity of EAC was graded by modified Magone scoring standard (24), including conjunctival redness (0–3), chemosis (0–3), lid edema (0–3), and tearing (0–3; total score: 0–12) (Fig. 1D). As compared with the EAC groups, the subconjunctival injection of hUCMSCs significantly reduced clinical scores of allergy at all-time points. The IV infusion of hUCMSCs had lower scores than the EAC groups at several time points, but the differences were not significant (Fig. 1E). The above results demonstrate that subconjunctival injection of hUCMSCs significantly attenuated allergy scores and instances of scratching, while IV infusion of hUCMSCs did not yield therapeutic effects. We further confirmed that IV-infused hUCMSCs did not migrate to the conjunctiva (Supplementary Fig. 1). Therefore, we focused on the subconjunctival injection route in subsequent experiments.

Subconjunctival injection of hUCMSCs reduced the inflammatory cells in EAC mice

Activated MCs and infiltrated eosinophils in the conjunctiva release inflammatory mediators that aggravate allergy symptoms in the late phases of AC (25). We performed histopathological tests to evaluate whether hUCMSCs can reduce these cells in the conjunctiva. Histological analysis revealed a higher number of activated degranulated MCs in the conjunctiva of EAC mice compared to the normal group (Fig. 2A, B), whereas the number of degranulated MCs was significantly decreased in the hUCMSC treatment group. In addition, giemsa staining showed that the number of eosinophils in the conjunctiva was reduced in hUCMSC-treated mice compared to the EAC PBS group (Figs. 2C, D). These findings indicate that hUCMSC treatment significantly reduced the presence of degranulated MCs and infiltrated eosinophils in the conjunctiva of EAC mice.

hUCMSCs reduced Th2 cells in CLNs of EAC mice

In the early phase of AC, the excessive activation of Th2 cells has been recognized to play a critical role. Th0 cells proliferate and differentiate into Th2 cells, with the latter causing eosinophilic infiltration by migrating to the conjunctiva and producing IL-4, IL-5, and IL-13. To further investigate the immunomodulatory effects of hUCMSCs on T cell phenotypes in the draining CLNs of EAC mice, we assessed the Th1 and Th2 cell populations and the number of Treg cells by flow cytometry. Results showed a lower ratio of CD4 + IFN-γ + Th1 cells, and a high percentage of CD4 + IL-4 + Th2 cells and CD4 + CD25 + FOXP3 + Treg cells in the CLNs in EAC mice compared with the control group. However, after treatment with hUCMSCs, an increased ratio of Th1, a reduced ratio of Th2, and an increased population of CD4 + CD25 + FOXP3 + Treg cells were observed in draining CLNs (Figs. 3A-C).

Immunofluorescent staining showed increased IL-4+, IL-5+, and IL-13 + T cells in the draining CLNs in EAC mice, demonstrating stronger Th2-dominant inflammation. In addition, Th17 cells derived from Th0 cells produce IL-17, which aggravates Th2-dominant allergic inflammation in allergic eye disease (26). The number of L-17 + T cells was increased in CLNS in EAC mice (Fig. 4A.B). Subconjunctival injection of hUMCSCs markedly reduced the number of Th2 and TH17 cells. Furthermore, IFN-γ + Th1 cells were decreased in EAC mice and significantly increased with hUCMSC treatment. In addition, the levels of the transcription factor FOXP3, cytokine IL-10, and Treg cell markers were increased in the draining CLNs of EAC mice and further increased in the hUCMSC treatment group. The expression levels of cytokines were confirmed by qRT-PCR (Fig. 4C). These findings collectively indicate that hUCMSCs markedly reduced the number of Th2 and Th17 immune cells and elevated the number of Treg cells in CLNs, thereby decreasing the levels of inflammatory factors, and increasing the levels of anti-inflammatory factors in draining CLNs.

We further examined the expression levels of Th2 marker gata3 and Th17 marker rorc in conjunctiva tissues by real-time PCR and found that these two genes were upregulated in EAC mice compared to the control. Meanwhile, the expression of anti-inflammatory factors foxp3 and tgf-β were increased in EAC mice. Administration of hUCMSC significantly reduced the expression of gata3 and rorc, and further increased the levels of anti-inflammatory factors foxp3 and tgf-β (Fig. 4D). These results suggest that the local subconjunctival administration of hUCMSCs is able to reduce the number of Th2 and Th17 cells, and augment Treg cell population.

hUCMSCs use CRISPLD2 to suppress the development of Th2 cells

It has been demonstrated that, in a pro-inflammatory environment, MSCs will exhibit an anti-inflammatory phenotype and secrete immunosuppressive factors (27). To mimic the in vivo EAC milieu, we treated hUCMSCs with serum from EAC mice to determine whether hUCMSCs can regulate the differentiation and cytokine production of Th2 cells directly. We pretreated hUCMSCs with different kinds of sera and co-cultured them with freshly isolated Th0 cells. Five days later, Th2 (CD4 + IL-4+) and Treg (CD4 + CD25 + Foxp3+) cells generated in co-cultures were evaluated by FACS analysis. The results show that hUCMSCs treated with EAC mouse serum (EMS-hUCMSCs) repressed Th0 differentiation into Th2 lymphocytes (Fig. 5A, B). In addition, EMS-hUCMSCs also increased the generation of anti-inflammatory Treg lymphocytes versus hUCMSCs pretreated with FBS (FBS-hUCMSCs) or normal mouse serum (NMS-hUCMSCs) (Fig. 5C, D). These results indicate that hUCMSCs attenuate the severity of EAC through direct regulation of T cell differentiation.

Previous studies have identified several immunosuppressive factors in MSCs, including TSG6, PGE2, IDO, and TGF-β [1, 23]. To identify more factors that mediate the immunoregulatory capacity of hUCMSCs, we performed RNA sequencing of EMS-hUCMSCs and NMS-hUCMSCs. Of the 703 DEGs identified based on the threshold criteria, 281 genes were downregulated and 422 genes were upregulated in EMS-hUCMSCs versus NMS-hUCMSCs (Fig. 6.A). GO annotations show that genes related to immune processes were enriched from the DEGs (Fig. 6.B), represented by the heat map (Fig. 6.C). Among them, six genes with high TPM values were verified by qRT-PCR (Fig. 6.D). We decided to focus on CRISPLD2, as it demonstrated the greatest difference between the two groups. CRISPLD2 is a cystine-rich secretory protein (CRISP) that can inhibit LPS-mediated inflammation. Its features include a secretory signal and two LCCL domains (28). The higher levels of CRISPLD2 in EMS-hUCMSCs compared to NMS-hUCMSCs were confirmed by Western blot (Fig. 6E, F).

To investigate the biochemical mechanisms behind the regulation of Th2 differentiation by CRISPLD2, hUCMSCs were transfected with lentiviral particles to establish CRISPLD2-knockdown cells. We constructed three specific shRNA vectors targeting CRISPLD2 and verified their knockdown efficiency by qRT-PCR (Fig. 7A). Based on the results, we selected shCrispld2-1 and transfected hUCMSCs (shCrispld2-hUCMSCs) for further investigations. The effective knockdown of shCrispld2-hUCMSCs was further confirmed by Western blot and qRT-PCR (Fig. 7B-D). To investigate the role of CRISPLD2 in hUCMSC-mediated immunomodulation, we co-cultured Th0 cells for 5 days, in the presence of Th2 differentiation conditions, with shCrispld2-hUCMSCs pretreated with EAC mouse serum. FACS results show that the IL-4 + CD4 + Th2 cell population was markedly increased when co-cultured with shCrispld2-hUCMSCs compared to the group co-cultured with shControl-hUCMSCs (Fig. 7E-F). These findings demonstrate that CRISPLD2 mediates the suppression of Th2 differentiation by hUCMSCs.

In order to explore the role of CRISPLD2 in hUCMSC-mediated immunosuppression, an in-vitro T-cell proliferation experiment was performed. Sorted CD4 + T cells were cultured in the presence of CD3/CD28 activation beads and labeled with CFSE. After co-cultured with hUCMSCs, shControl-hUCMSC, or shCrispld2-hUCMSCs pretreated with EAC mouse serum for 3 days, the cells were collected and analyzed by flow cytometry. We found that hUCMSC and shControl-hUCMSC strongly suppressed the proliferation of activated CD4 + T cells (Fig. 7G, H), while shCrispld2-hUCMSCs showed less suppressive capacity. This further confirms that the immunosuppression function of hUCMSCs is partly mediated by CRISPLD2.

IL-4 is thought to promote Th2 cell differentiation by activating the STAT6 signaling pathway. To assess whether CRISPLD2 represses Th2 T cell differentiation by inhibiting the STAT6 pathway, we performed Western blot analysis of phosphorylated STAT6 in activated T cells pretreated with EAC mouse serum and co-cultured with either shCrispld2-hUCMSCs or shControl-hUCMSCs (Fig. 7I, J). Compared with the shControl-hUCMSCs, shCrispld2-hUCMSCs markedly increased the expression of phosphorylated STAT6 induced by IL-4. This finding suggests that CRISPLD2 mediates the differentiation of Th2 cells by inhibiting STAT6 phosphorylation.

In this study, we found that subconjunctival injection of hUCMSCs strongly suppressed conjunctival inflammation and alleviated allergy symptoms compared to intravenous injection of hUCMSCs in a mouse EAC model. The therapeutic effects of hUCMSCs involve significant inhibition of Th2 cell differentiation in conjunctival tissue and draining CLNs, mediated by CRISPLD2.

Although the most common route for MSC application is intravenous injection in the treatment of systemic diseases, local administration avoids the ‘first-pass’ accumulation of MSCs in the lungs. Conjunctival injection delivers a high effective cell concentration into the space between the conjunctiva and Tenon’s capsule with fewer systemic side effects, and immediately produces paracrine signaling factors to maintain tissue homeostasis (29). Studies have reported that among topical, subconjunctival, intravenous, and intraperitoneal routes, subconjunctival administration is the most effective for ocular repair (30). In our mouse EAC model, most of the intravenously infused hUCMSCs may have accumulated in the lungs and left nearly no cells to migrate to the conjunctiva, thereby yielding no therapeutic effect. Topical and intraperitoneal routes may also fail to accumulate in the conjunctiva. Therefore, subconjunctival administration of hUCMSCs demonstrated better therapeutic outcomes for AC treatment.

Th0 cells, under different disease conditions, differentiate into Th1, Th2, Th17, and Treg cells. Meanwhile, MSCs are involved in regulating T cell differentiation by producing a wide array of cytokines and growth factors. For instance, MSCs inhibit Th17 differentiation by IDO and PD-1, promote Treg differentiation by TGF-β and IL-10, and inhibit T-cell proliferation by HLA-G1, TGF-β, and HGF (31–33). Th2 cells play an important role in allergic diseases. Peng et al. demonstrated that MSC-derived small extracellular vesicles suppressed Th2 responses in patients with allergic rhinitis (34). However, IDO was reported to induce the differentiation and maturation of Th2 cells. Furthermore, we found that hUCMSCs effectively promote Th2 cell differentiation when co-cultured with Th0 cells. Nonetheless, pretreatment with the serum of EAC mice enabled hUCMSCs to inhibit Th2 cell differentiation. This finding coincides with a previous report that serum from asthmatic mice potentiates the therapeutic effects of MSCs in experimental allergic asthma and increased the expression of various mediators, including TGF-β, IFN-γ, IL-10, TSG-6, IDO-1, and IL-1RN (35). These results confirm that certain conditions are required for MSCs to inhibit allergic inflammation. For instance, the CM from bone marrow-derived MSCs stimulated by TNF-α was able to alleviate EAC (18). Indeed, hUCMSCs pretreated with EAC mouse serum not only repressed Th2 cell differentiation, but also promoted Th1 cell differentiation, which suggests that a suitable ratio of Th1/Th2 is important for maintaining tissue homeostasis.

Notably, serum from EAC mice led to higher expression of CRISPLD2 in hUCMSCs compared to those unstimulated or stimulated with serum from normal mice. CRISPLD2, a cystine-rich secretory protein with a secretory signal and two LCCL domains, competitively inhibits LPS binding to its original target receptor TLR4, thereby reducing LPS-derived inflammation. Endogenous CRISPLD2 in healthy human serum was found to downregulate LPS-induced TNF-α production in vitro (36), and CRISPLD2 was found to protect mice from endotoxic shock. In addition, CRISPLD2 also exhibits a broad range of anti-inflammatory properties in a series of inflammation-related diseases, including asthma and obesity. Dexamethasone and IL1-β can also elevate CRISPLD2 levels, which then modulate the expression of inflammatory genes IL-6 and IL-8 (28). Another study reported that CRISPLD2 attenuates pro-inflammatory cytokines TNF-α, IL-6, IL-8, and MCP-1 production in HMGB1-stimulated monocytes and septic mice (38). The co-culturing of hUCMSCs with Th0 cells demonstrates that CRISPLD2 was able to inhibit Th2 differentiation.

Several reports have shown that, following activation of Janus-kinases (JAKs) in allergic conjunctivitis, IL-4 and IL-13 induce the expression of specific genes by activating STATs (38, 39). STAT6 signaling has been recognized as a necessary part of developing the Th2 response and allergic inflammation (40). The inhibition of STAT6 activities by CRISPLD2 suggests a potential mechanism through which the suppression of the JAK/STAT signaling pathway results in the repression of Th2 cell differentiation. However, more efforts are needed to reveal the target and mechanism of CRISPLD2 in regulating T cell differentiation.

This study is limited by the wide scope of synergistic actions between reported factors and novel mediators from hUCMSCs that mediate therapeutic functions, rather than one or two modulators. Among the other upregulated molecules (Fig. 6D), Phosphoinositide-3-Kinase Regulatory Subunit 2 (PIK3R2) has the capacity to repress T cell proliferation in mice (41, 42); Formyl peptide receptor 1 (FPR1) are expressed in various immune cells and has a complex role in pathogenic processes (43). Tumor necrosis factor-induced protein 6(TNFAIP6) secreted by bone marrow–derived MSCs attenuates inflammatory bowel disease by modulating follicular helper T cells and follicular regulatory T cells balance in mice (44). Hence, further studies are needed to identify and characterize factors in the hUCMSC secretome. These factors have the potential to be utilized as topical immunosuppressive formulations for AC.

Our results demonstrate that subconjunctival injection of hUCMSCs significantly suppressed conjunctival inflammation and alleviated allergy symptoms in an SRW-induced mouse EAC model. After pretreatment with serum from EAC mice, hUCMSCs repressed the differentiation of Th2 cells, modulating the Th1/Th2 balance. In addition, EAC mouse serum-exposed hUCMSCs were found to be potent promoters of Treg cell differentiation and activation. We further confirmed that these immunomodulatory effects of hUCMSCs were mediated, at least partly, by CRISPLD2. This study not only provides a potential therapeutic target for the treatment of allergic conjunctivitis but also other T cell-mediated diseases.

AC Allergic conjunctivitis

ARVO Association for Research in Vision and Ophthalmology

CFSE Carboxy-fluorescein succinimidyl ester

CLNs Cervical lymph nodes

CRISPLD2 Cystine-rich secretory protein (CRISP) and two LCCL domains

DMEM Dulbecco’s modified Eagle medium

EDTA Ethylene diamine tetraacetic Acid

EAC Experimental allergic conjunctivitis

FOXP3 Forkhead box protein 3

hUCMSCs Human umbilical cord mesenchymal stem cells

MCs Mast cells

MACS Magnetic cell separation

SRW Short ragweed pollen

STAT6 Signal transducer and activator of transcription 6

TNF-a Tumor necrosis factor

Acknowledgements

We would like to acknowledge our colleagues for their helpful assistance in this study.

Author Contributions

D.L. and Q.O. designed the study, performed the experiments, drafted the paper; performed the experiments; Q.S. and M.M.Z.L. carried out statistical analysis and interpretation of data; J.Y.X., C.J., F.G., J.W., J.Z., J.L. discussed the experiments, reviewed, and edited the manuscript; L.L., G.T.X. and H.T., led and funded the project, overall supervised the project, and designed some of the experiments, discussed and revised and finalized the manuscript as well as proved the submission. All authors read and approved the final manuscript.

Funding

This work is supported by grants obtained from the Ministry of Science and Technology of China (2020YFA0113102 and 2017YFA0104100), National Natural Science Foundation of China (81770942). The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files. The raw data of RNA-seq have been deposited in the NCBI Sequence Read Archive (SRA) database under accession number: PRJNA931840.

Ethics approval and consent to participate

All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Tongji University on August 12, 2020(Approved Number. TJAA09620101). The title of ethical approved project:” The comparison of therapeutic effects and mechanism of different tissue-derived mesenchymal stem cell conditioned medium for experimental allergic conjunctivitis”. Principles of animal research conformed to the guidelines of ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing commercial and financial interests related to the studies described.

Author details

1 Department of Ophthalmology of Tongji Hospital and Laboratory of Clinical and Visual Sciences of Tongji Eye Institute, Tongji University School of Medicine, Shanghai 200065, China; 2 Department of Ophthalmology, Shanghai General Hospital (Shanghai First People’s Hospital), Shanghai Jiao Tong University, Shanghai 200080, China; 3 Department of Physiology and Pharmacology, TUSM, Shanghai 200092, China; 4 The Collaborative Innovation Center for Brain Science, Tongji University, Shanghai 200092, China.

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Supplementalfig.tif
Supplementary figure1A. hUCMSCs are not able to migrate to the conjunctiva by intravenous infusion. (a) hUCMSCs (2×10⁶cells) were intravenously or subconjunctivally injected into the mouse EAC model. 24 hours later, samples were collected and used for preparing cryosections. hUCMSCs did not migrate to the conjunctiva after intravenous infusion. Scare bar = 50μm.
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published 02 Oct, 2023

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Subconjunctival injection of human umbilical cord mesenchymal stem cells alleviates experimental allergic conjunctivitis via regulating T cell response

Status:

Journal Publication

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Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and methods

Animal Preparation

hUCMSCs culture and stimulation

Establishment of mouse EAC model and administration of hUCMSCs

Histologic Analysis of conjunctiva and CLNs

RNA isolation and quantitative real-time PCR

Flow cytometry

RNA sequencing

Assembly of lentivirus to knockdown crispld2

T cell proliferation assay

Differentiation of Th0 cells

Western blot analyses

Statistical analysis

Results

Subconjunctival injection of hUCMSCs alleviates EAC

Subconjunctival injection of hUCMSCs reduced the inflammatory cells in EAC mice

hUCMSCs reduced Th2 cells in CLNs of EAC mice

hUCMSCs use CRISPLD2 to suppress the development of Th2 cells

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1