Beneficial role of rapamycin in experimental autoimmune myositis

Abstract

Introduction: We developed an experimental autoimmune myositis (EAM) mouse model of polymyositis where we outlined the role of regulatory T (Treg) cells. Rapamycin, this immunosuppressant drug used to prevent rejection in organ transplantation, is known to spare Treg. Our aim was to test the efficacy of rapamycin in vivo in this EAM model and to investigate the effects of the drug on different immune cell sub-populations.

Methods: EAM is induced by 3 injections of myosin emulsified in CFA. Mice received rapamycin during 25 days starting one day before myosin immunization (preventive treatment), or during 10 days following the last myosin immunization (curative treatment).

Results: Under preventive or curative treatment, an increase of muscle strength was observed with a parallel decrease of muscle inflammation, both being well correlated (R(2) = -0.645, p<0.0001). Rapamycin induced a general decrease in muscle of CD4 and CD8 T cells in lymphoid tissues, but spared B cells. Among T cells, the frequency of Treg was increased in rapamycin treated mice in draining lymph nodes (16.9 ± 2.2% vs. 9.3 ± 1.4%, p<0.001), which were mostly activated regulatory T cells (CD62L(low)CD44(high): 58.1 ± 5.78% vs. 33.1 ± 7%, treated vs. untreated, p<0.001). In rapamycin treated mice, inhibition of proliferation (Ki-67(+)) is more important in effector T cells compared to Tregs cells (p<0.05). Furthermore, during preventive treatment, rapamycin increased the levels of KLF2 transcript in CD44(low) CD62L(high) naive T cell and in CD62L(low) CD44(high) activated T cell.

Conclusions: Rapamycin showed efficacy both as curative and preventive treatment in our murine model of experimental myositis, in which it induced an increase of muscle strength with a parallel decrease in muscle inflammation. Rapamycin administration was also associated with a decrease in the frequency of effector T cells, an increase in Tregs, and, when administered as preventive treatment, an upregulation of KFL2 in naive and activated T cells.

Trial registration: ClinicalTrials.gov NCT00525889.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Preventive administration of rapamycin permits to decrease severity of EAM.

A: strength (time to fall) improvement in rapamycin-treated mice. B: gastrocnemius, quadriceps, and triceps muscle inflammatory infiltrates evaluated by histological grading after hematoxylin-eosin staining as illustrated in C. C: the first two upper images represent a gastrocnemius section of an EAM mouse untreated with a histological score of 4 (HE, ×20). The middle picture also shows an invaded/tunnelized fiber (arrow, HE, ×40). The third image panel represents a gastrocnemius section of a EAM mouse treated with 3 mg/kg/day of rapamycin, displaying a histological score of 1 (HE, ×20). D: correlation between muscle strength (time to fall) and histological grade of inflammation.

Figure 2. Rapamycin treatment induced a T cells lymphopenia sparing B cells.

A: absolute lymphocyte number in draining lymph nodes, spleen, and non-draining lymph nodes is decreased in rapamycin-treated mice compare to controls in a dose-dependent fashion. B: quantification of the different sub-populations of lymphocytes: B lymphocytes (B, CD19+B220+), T (CD3+), T CD4+ (CD3+CD4+) and T CD8+ (CD3+CD8+) cells. * p<0.05, *** p<0.001. White histogram bars: control mice, black histogram bars: rapamycin treated mice (3 mg/kg/day). C: percent of T or B lymphocytes in draining lymph nodes. D: Representative dot plot of flow cytometry analysis of draining lymph nodes for percentage of pre-activated CD4+ T cell (CD3+CD4+FoxP3−CD69+) and percentage of pre-activated CD4+ T cell (CD3+CD4+FoxP3−CD69+) in controls and rapamycin (3 mg/kg/day) treated mice.

Figure 3. Effect of rapamycin on Treg cells.

Representative dot plot of flow cytometry analysis of draining lymph nodes for percent of Treg (CD4+CD25+FoxP3+) in CD4+ (A) and in CD4+CD44high (B) in controls and rapamycin (3 mg/kg/day) treated mice. Percent of Treg (CD4+CD25+FoxP3+) in CD4+ (A, right) and in CD4+CD44high (B, right) in controls and rapamycin (3 mg/kg/day) treated mice. C: Histogram representative of the difference in activation of Treg (CD62Llow cell) and naive Treg (CD62Lhigh) in controls and rapamycin (3 mg/kg/day) treated mice. D: Suppressive test. Horizontal lines indicate means. Suppressive activity of sorted CD4+CD25+ T cells (Treg) from controls (white bars) and rapamycin (3 mg/kg/day, black bars) mice on the proliferation of autologous CD4+CD25− T cells (responders) stimulated with irradiated (15 Gy) splenocytes. Proliferation of responder cells (Teff) was measured by 3H-Tymidine incorporation (counts per minute, cpm). Results are indicated as percentage inhibition (±SD) compared to a max (ratio 1∶0). Different Teff∶Treg ratios were tested. E: Percent of Ki-67positive cells (i.e. proliferative cells) in activated effector (aT) and activated regulatory (aTreg) T cells in controls and rapamycin (3 mg/kg/day) treated mice.

Figure 4. Rapamycin treatment did not alter muscle infiltrates composition.

A: Gastrocnemius muscle fiber membranes are stained with an anti-laminin antibody (green). Nuclei were counterstained with DAPI (blue). Cells of muscular inflammatory infiltrates are stained with anti-CD4 (yellow) and with anti-FoxP3 (red) antibodies. B: Percent of Tregs (CD4+FoxP3+) in muscle inflammatory infiltrates.

Figure 5. Change in KLF2 pathway induced by rapamycin treatment.

A: Histogram plot of the shift of CCR7 expression in controls and rapamycin-treated (3 mg/kg/day) mice showing naive T cell subset (nT:CD62LhighCD44low). B: Shift of CCR7 expression in controls and rapamycin-treated (3 mg/kg/day) mice in naive T cells (nT:CD62LhighCD44low) an activated T cells (eT:CD62LlowCD44high). C: Q-RT-PCR of KLF2 in naive T cells (nT:CD62highCD44low), activated T cells (aT: CD62low CD44high), naive regulatory T cells (nTreg:CD62high), activated Treg cells (aTreg: CD62low) in controls and rapamycin treated (3 mg/kg/day) mice.

Figure 6. Beneficial effect of rapamycin (3 mg/kg/day) in curative treatment of EAM.

Rapamycin (or water for control animals) was given orally for 10 days at a dose of 3 mg/kg. A: strength of mice evaluated by inverted screen test (time to fall in seconds). B: Gastrocnemius muscle inflammatory infiltrates evaluated by histological grading after haematoxylin-eosin staining. C: Percent of Treg cells (CD3+CD4+CD25+FoxP3+) in draining lymph nodes from controls and rapamycin-treated (3 mg/kg/day) mice.