Dendritic cells transfected with DNA constructs encoding CCR9, IL-10, and type II collagen demonstrate induction of immunological tolerance in an arthritis model

Abstract

Introduction: Restoring immune tolerance is a promising area of therapy for autoimmune diseases. One method that helps restore immunological tolerance is the approach using tolerogenic dendritic cells (tolDCs). In our study, we analyzed the effectiveness of using dendritic cells transfected with DNA constructs encoding IL-10, type II collagen, and CCR9 to induce immune tolerance in an experimental model of arthritis.

Methods: Dendritic cell cultures were obtained from bone marrow cells of Balb/c mice. Dendritic cells (DCs) cultures were transfected with pmaxCCR9, pmaxIL-10, and pmaxCollagen type II by electroporation. The phenotype and functions of DCs were studied using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Migration of electroporated DCs was assessed in vitro. Induction of antigen-collagen induced arthritis (ACIA) was carried out according to the protocol in Balb/c mice. DCs were then administered to ACIA mice. The development of arthritis was monitored by measuring paw swelling with a caliper at different time points. The immunological changes were assessed by analyzing the content of antibodies to type II collagen using enzyme immunoassay. Additionally, a histological examination of the joint tissue was conducted, followed by data analysis.

The results are as follows: DCs were obtained, characterized by reduced expression of CD80, CD86, and H-2Db (MHC class I), increased expression of CCR9, as well as producing IL-10 and having migratory activity to thymus cells. Transfected DCs induced T-regulatory cells (T-reg) and increased the intracellular content of IL-10 and TGF-β in CD4⁺T cells in their co-culture, and also suppressed their proliferative activity in response to antigen. The administration of tolDCs transfected with DNA constructs encoding type II collagen, IL-10, and CCR9 to mice with ACIA demonstrated a reduction in paw swelling, a reduction in the level of antibodies to type II collagen, and a regression of histological changes.

Conclusion: The study presents an approach by which DCs transfected with DNA constructs encoding epitopes of type II collagen, IL-10 and CCR9 promote the development of antigen-specific tolerance, control inflammation and reduce the severity of experimental arthritis through the studied mechanisms: induction of T-reg, IL-10, TGF-β.

Keywords: DNA-constructs; T-regulatory cells; antigen-specific dendritic cells; experimental arthritis; immunological tolerance; tolerant dendritic cells.

Figure 1

(A-C) Expression analysis of maturation markers of dendritic cells transfected with different plasmids: (A) relative content of DCs expressing CD80; (B) relative content of DCs expressing CD86; (C) relative content of DCs expressing H-2D^b, (n = 8). * – statistically significant differences from the non-EP DC group (p ≤ 0.01). (D) CCR9 expression analysis of cultured DCs after transfection with different plasmids (n = 8). * – statistically significant differences from the group of non-EP DC, DCp5, DCpIL-10, DCCII and DCpIL10+pCII (p ≤ 0.001). (E) Analysis of the IL-10 content in the conditioned DC culture medium after transfection with different plasmids (n = 8). * – statistically significant differences from the group of non-EP DCs and DCp5 (p ≤ 0.001). Note: non-EP DC – DC culture that was not electroporated, DCp5 – DC culture electroporated with control non-coding plasmid (p5), DCpIL10 – DC culture electroporated with the experimental plasmid (pIL10), DCpCII – DC culture electroporated with the experimental plasmid (pCII), DCpCCR9 – DC culture electroporated with the experimental plasmid (pCCR9), DCpIL10+pCII – DC culture electroporated with the experimental plasmids (pIL10 and pCII), DCpCCR9+pCII+pIL10 – DC culture electroporated with the experimental plasmids (pCCR9, pCII and pIL10), DCpCCR9+pCII – DC culture electroporated with the experimental plasmids (pCCR9 and pCII).

Figure 2

(A) Analysis of the relative content of T-reg (CD25⁺FoxP3⁺) in the co-culture of the studied DCs and splenocytes, (n = 8). * – statistically significant differences groups DCpIL-10 and DCpIL-10+pCII+pCCR9 from the DCp5 group, # – statistically significant differences of DCpIL10+pCII+pCCR9 from all other groups (one-way ANOVA, p ≤ 0.0001). Analysis of the intracellular IL-10 (B) and TGF-β (C) content in DC and CD4⁺splenocytes co-culture, (n = 8). * – statistically significant differences of the DCpIL-10, DCpIL-10+pCII, and DCpIL-10+pCII+pCCR9 groups from the other groups (one-way ANOVA, p ≤ 0.0001). (D) Evaluation of the proliferative activity of CD4⁺splenocytes in co-culture with T-reg upon the addition of type II collagen, (n = 6). CD4 alone – CD4⁺splenocytes without addition of T-reg and type II collagen, CD4+CII – CD4⁺splenocytes with addition of type II collagen, AgT-reg+CD4+CII – antigen-specific T-reg, CD4⁺splenocytes and type II collagen, non-AgT-reg +CD4+CII – nonspecific T-reg, CD4⁺splenocytes and type II collagen. Brackets indicate statistically significant differences (one-way ANOVA, **** - P < 0.0001).

Figure 3

(A) Evaluation of dendritic cell migration ability, (n = 6). DCs are dendritic cells unaffected by electroporation, DCpCCR9 are DCs transfected with the CCR9 plasmid. Medians and interquartile range. * – statistically significant differences from the DC group (two-way ANOVA, P < 0.0001). (B) Analysis of T-reg content in co-culture of DCs and thymocytes, (n = 6). * – statistically significant differences of the DCpCCR9, DCpCCR9+pCII, and DCpIL-10+pCII+pCCR9 groups from the control groups Thymocytes and DCp5 (p ≤ 0.0001).

Figure 4

Comparison of paw swellings of laboratory animals on days 7 (A) and 14 (B) after the initiation of therapy (n = 8–12). ACIA without treatment and DCp5 are the control groups. Medians and interquartile range. # - statistically significant differences from the control group ACIA without treatment, * - statistically significant differences from the control group DCp5, & - statistically significant differences from the DCpCCR9+pCII+pIL10 group (one-way ANOVA, P < 0.0001). Comparison of antibody levels to type II collagen in laboratory animals on days 7 (C) and 14 (D) after treatment (n = 8–12). ACIA without treatment and DCp5 are the control groups. Medians and interquartile range. * – statistically significant differences from the control groups, # – statistically significant differences the DCpCCR9+pCII+pIL10 group (one-way ANOVA, P < 0.001).

Figure 5

Data of histologic analysis of mice joint tissues (n = 4–6). On day 7 after treatment: group 1 – pronounced lymphocytic infiltration of the synovial stroma, formation of a subchondral cyst (A); group 2 – pannus formation, weak inflammatory infiltration of the synovial membrane (B); group 3 – thickening of the synovial membrane with weak lymphocytic infiltration, formation of synovial villi (C); group 4 – moderate infiltration of the synovial membrane, penetration of the synovial membrane into the articular cartilage (D); group 5 – synovial membrane ingrowth into the bone tissue near the articular cartilage, moderate lymphocytic infiltration of the synovial membrane with its thickening and formation of villi (E); group 6 – weak inflammatory infiltration of synovia, pannus is visualized (F). On day 14 after treatment: group1 – infiltration of the synovial membrane by lymphocytes (G); group 2 – moderate infiltration of the synovial membrane of the joint, proliferation of fibroblasts (H); group 3 – no visible pathologic changes were detected (I); group 4 – moderate lymphocytic infiltration of the subsynovial stroma (J); group 5 – thickening and numerous outgrowths (villi) of the synovial membrane, small piles of leukocytes in the joint cavity (K); group 6 – synovial membrane overgrowth with pannus formation, lymphocytic infiltration of the subsynovial stroma (L).