TRI microparticles prevent inflammatory arthritis in a collagen-induced arthritis model

Abstract

Despite recent progress in the treatment of rheumatoid arthritis (RA), many patients still fail to achieve remission or low disease activity. An imbalance between auto-reactive effector T cells (Teff) and regulatory T cells (Treg) may contribute to joint inflammation and damage in RA. Therefore, restoring this balance is a promising approach for the treatment of inflammatory arthritis. Accordingly, our group has previously shown that the combination of TGF-β-releasing microparticles (MP), rapamycin-releasing MP, and IL-2-releasing MP (TRI MP) can effectively increase the ratio of Tregs to Teff in vivo and provide disease protection in several preclinical models. In this study TRI MP was evaluated in the collagen-induced arthritis (CIA) model. Although this formulation has been tested previously in models of destructive inflammation and transplantation, this is the first model of autoimmunity for which this therapy has been applied. In this context, TRI MP effectively reduced arthritis incidence, the severity of arthritis scores, and bone erosion. The proposed mechanism of action includes not only reducing CD4+ T cell proliferation, but also expanding a regulatory population in the periphery soon after TRI MP administration. These changes were reflected in the CD4+ T cell population that infiltrated the paws at the onset of arthritis and were associated with a reduction of immune infiltrate and inflammatory myeloid cells in the paws. TRI MP administration also reduced the titer of collagen antibodies, however the contribution of this reduced titer to disease protection remains uncertain since there was no correlation between collagen antibody titer and arthritis score.

Fig 1. TRI MP administration reduces incidence and severity of CIA onset.

A) Survival curve indicating percentage of mice that remained arthritis free (score of 0 for all paws). B) Arthritis scores over time. A two-way ANOVA was performed, followed by Tukey post-hoc analysis to compare the mean of every group with the mean of every other group at each time point. Significance labels apply to all time points from Day 32 on. C-D) Average number of paws per mouse with arthritis score greater than or equal to specified threshold of 1 (C) or 3 (D) at Day 40. n = 24 mice per group, data presented as mean ± SEM, and the following cutoffs were used for significance: * p < 0.05, ** p < 0.01, **** p < 0.0001.

Fig 2. Lower arthritis score correlates with less bone erosion.

A) Representative 3D reconstructions of micro-CT scans showing a paw without bone erosion (top) and a paw with severe bone erosion (bottom). Joint masks indicated in cyan were the regions used to calculate joint bone volume. B) Joint bone volume versus arthritis score at Day 40 for individual hind paws. Color coded based on treatment group: black–PBS, red–Blank MP, blue–TRI MP. Spearman correlation coefficient and p value for correlation are indicated. C) Average joint bone volume by treatment group. D) Joint bone surface area to volume ratio versus arthritis score at Day 40 for individual hind paws. Color coded based on treatment group: black–PBS, red–Blank MP, blue–TRI MP. Spearman correlation coefficient and p value for correlation are indicated. E) Average joint bone surface area to volume ratio by treatment group. n = 24 paws (12 mice) per group, data presented as mean ± SEM, and the following cutoffs were used for significance: ** p < 0.01.

Fig 3. TRI MP administration lowers level of anti-collagen II IgG antibodies.

A) Representative serial dilution curves with each curve corresponding to a single mouse, black–PBS treated mouse, red–Blank MP treated mouse, blue–TRI MP treated mouse, green–monoclonal anti-collagen II (CII) antibody (Ab) (Clone 2B1.5) used as standard. B) Normalized anti-CII IgG Ab titer versus arthritis score. Ab titer was defined as the dilution corresponding to the half-maximal absorbance in the linear section of the dilution curve, or the IC50 value using a non-linear four parameter regression. Normalized titer was calculated by dividing the titer by that of the 2B1.5 Ab standard for a given plate. Color coded based on treatment group: black–PBS, red–Blank MP, blue–TRI MP. Spearman correlation coefficient and p value for correlation are indicated. C) Average normalized anti-CII IgG Ab titer by treatment group. n = 12 mice per group, data presented as mean ± SEM, and the following cutoffs were used for significance: * p < 0.05.

Fig 4. TRI MP leads to more CD25⁺FoxP3^- T cells, which express elevated levels of LAP, CTLA-4, and CD73.

A) Representative pseudocolor plots of CD25 expression versus FoxP3 expression for CD4⁺ cells from the iLN (top row) or spleen (bottom row) of mice treated with PBS (left column), Blank MP (center column), or TRI MP (right column). B-E) Quantification of plots from A, showing percentage of CD4⁺ cells that are FoxP3⁺CD25⁺ (B, E) or FoxP3^-CD25⁺ (C, D) by treatment group for the iLN (B, C) and spleen (D, E). F) Representative histogram plots of LAP (left), CTLA-4 (middle), and CD73 (right) expression for the isotype control (shaded gray), the FoxP3^-CD25^- population (black), the FoxP3^-CD25⁺ population (orange), and the FoxP3⁺CD25⁺ (cyan) population (from a PBS treated mouse). G-L) Quantification of the percentage of CD4⁺ T cell populations that are LAP⁺ (G,J), CTLA-4⁺ (H, K), or CD73⁺ (I, L) relative to isotype control. Presented by CD4⁺ T cell population (FoxP3^-CD25^-, FoxP3^-CD25⁺, and FoxP3⁺CD25⁺) for the iLN (G-I) and spleen (J-L). n = 6 mice per treatment group and n = 18 mice per CD4⁺ T cell population group, data presented as mean ± SEM, and the following cutoffs were used for significance: ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 5. TRI MP reduces conventional T cell expansion and expands regulatory population not only for the draining lymph node, but also for a non-arthritic immunization in the contralateral lymph node.

A) Number of live cells in draining iLN as determined using counting beads. B) Percentage of CD4⁺ T cells expressing the proliferation marker Ki67 in the the draining LN. C) Percentage of CD4⁺ T cells that are FoxP3^-CD25⁺ in the draining LN. D-F) Same as A-C, but for the contralateral LN instead of the draining LN. n = 6 mice per group, data presented as mean ± SEM, and the following cutoffs were used for significance: ** p < 0.01, *** p < 0.001.

Fig 6. Lower arthritis score correlates with less CD4+ T cells, a higher proportion of regulatory cells, and less inflammatory innate immune cells in the paws.

A,B,D-H) Indicated parameter of the paw immune infiltrate versus arthritis score (Day 40–42). Spearman correlation coefficient and p value for correlation are indicated. n = 6–12 mice per group. These include the number of CD4⁺ T cells (A), the percentage of CD4⁺ T cells that are FoxP3^- and CD25⁺ (B), the number of CD45⁺ immune cells (D), the percentage of CD45⁺ cells that are monocytes/macrophages (CD11b⁺Ly-6G^-Ly-6C⁺) (E), the percentage of CD45⁺ cells that are neutrophils (CD11b⁺Ly-6G⁺) (F), the number of TNF-α expressing monocytes/macrophages (G), and the number of TNF-α expressing neutrophils (H). C) Percentage of CD4⁺ T cells that are FoxP3^-CD25⁺ by treatment group. n = 12 mice per group, data presented as mean ± SEM.