Trp53 negatively regulates autoimmunity via the STAT3-Th17 axis

Abstract

Emerging evidence suggests that the tumor suppressor p53 is also a crucial regulator for many physiological processes. Previous observations indicate that p53 suppresses inflammation by inhibiting inflammatory antigen-presenting cells. To investigate the potential role of p53 in autoimmune effector T cells, we generated p53(null)CD45.1 mice by crossing p53(null)CD45.2 and CD45.1 mice. We demonstrate that p53(null)CD45.1 mice spontaneously developed autoimmunity, with a significant increase in IL-17-producing Th17 effectors in their lymph nodes (4.7 ± 1.0%) compared to the age-matched counterparts (1.9 ± 0.8% for p53(null)CD45.2, 1.1 ± 0.2% for CD45.1, and 0.5 ± 0.1% for CD45.2 mice). Likewise, p53(null)CD45.1 mice possess highly elevated serum levels of inflammatory cytokines IL-17 and IL-6. This enhanced Th17 response results largely from an increased sensitivity of p53(null)CD45.1 T cells to IL-6-induced STAT3 phosphorylation. Administration of STAT3 inhibitor S31-201 (IC50 of 38.0 ± 7.2 μM for IL-6-induced STAT3 phosphorylation), but not PBS control, to p53(null)CD45.1 mice suppressed Th17 effectors and alleviated autoimmune pathology. This is the first report revealing that p53 activity in T cells suppresses autoimmunity by controlling Th17 effectors. This study suggests that p53 serves as a guardian of immunological functions and that the p53-STAT3-Th17 axis might be a therapeutic target for autoimmunity.

Figure 1.

p53^nullCD45.1 mice manifest splenomegaly, lymphadenopathy, and autoimmune pathology at an early age. A) Lymphoid organs of 14-wk-old p53^nullCD45.1 mice were compared with those of age-matched CD45.2, CD45.1, and p53^nullCD45.2 mice. SP, spleen; LN, lymph node; Thy, thymus. B) Lymphoid tissue cellularity of 14-wk old mice of all 4 genotypes was compared. C) Thymocyte surface expression of CD4 and CD8 and subpopulation composition of 14-wk old mice of all 4 genotypes were compared. D) Progressive alterations in size of lymphoid organs of 6–14 wk old p53^nullCD45.1 mice were examined. E) Kidney pathology of 6–14 wk-old p53^nullCD45.1 mice was examined via trichrome staining and compared to age-matched counterparts. An overt mesangial expansion (yellow arrowheads) with increased collagen deposits (blue stain) in the glomeruli was observed in the kidneys of 14 wk-old p53^nullCD45.1 mice. F) Kidneys of 14 wk-old mice of all 4 genotypes were used for immunofluorescence staining to determine IgG deposition in the glomeruli. Scale bars = 5 mm (A, D); 50 μm (E); 100 μm (F). Data are means ± se, representative of 3–5 mice from 3 independent experiments. *P < 0.05 vs. age-matched CD45.2 mice; 2-tailed Student's t test.

Figure 2.

CD4 T cells in p53^nullCD45.1 mice are pathogenic. A) Composition of lymphoid and myeloid subpopulations in the spleens of 14-wk-old CD45.2, CD45.1, p53^nullCD45.2, and p53^null CD45.1 mice was examined via FACS, and the absolute number of each subset was calculated. Data are presented as means ± se of 5 mice from 3 independent experiments. B, C) Representative FACS plots of the relative percentage of CD4⁺ and CD8⁺ T cells and their activation status (B) and Gr-1⁺CD11b⁺ myeloid subpopulation (C) in the spleen of 14-wk-old mice of all 4 genotypes. D) Kidney pathology was examined via trichrome staining of CD45.1 mice, which were transferred adoptively with 1 × 10⁷ total splenocytes or 3 × 10⁶ of sorted CD4⁺ cells, CD8⁺ T cells, or CD19⁺ B cells from 14-wk-old p53^nullCD45.1 mice for 6 wk. In a control group, CD45.1 mice received 3 × 10⁶ sorted CD44^hi T cells (CD3⁺) from syngeneic CD45.1 mice. Scale bars = 50 μm. Images are representative of 3 mice/group in 2 independent experiments. *P < 0.05 vs. age-matched CD45.2 mice; 2-tailed Student's t test.

Figure 3.

Increases in Th17 and Th1 effectors in lymphoid and nonlymphoid tissues of p53^nullCD45.1 mice correlate to autoimmune pathology. A) Spleens and peripheral lymph nodes (LN) of mice of different genotypes were examined for the percentage of Th17 and Th1 cells via FACS. B) Summarization of data from panel A. C) CD4 T cells enriched from the kidneys of all 4 genotypes of mice were analyzed for the existence of Th17 and Th1 cells via FACS. D) Summarization of data from C. Data are presented as means ± sem of 5 mice from 3 independent experiments. FACS plots are representative of 3 to 5 mice/genotype from 3 independent experiments. *P < 0.05 vs. age-matched CD45.2 mice; 2-tailed Student's t test.

Figure 4.

Serum levels of inflammatory cytokines, including IL-6 and IL-17, are highly elevated in p53^nullCD45.1 mice associated with autoimmune pathology. Serum of 12- to 24-wk old age-matched CD45.2, CD45.1, p53^nullCD45.2, and p53^nullCD45.1 mice was collected to determine cytokine levels via the Millipore multiplex array. Representative inflammatory cytokines, such as IL-17 (A), IL-6 (B), TNF-α (C), and IL-12 (D), and Th2 cytokine IL-5 (E) were compared. Bars represent average values of each group of mice; n mice/group. *P < 0.05 vs. age-matched CD45.2 mice; 2-tailed Student's t test.

Figure 5.

Naive CD4 T cells from p53^nullCD45.1 mice are more sensitive to IL-6-mediated Th17 polarization and expansion. Naive CD4⁺CD62L⁺ T cells from 6- to 8-wk-old age-matched CD45.2, CD45.1, p53^nullCD45.2, and p53^nullCD45.1 mice were enriched and activated under Th17 polarization conditions in the presence of 0, 1 ng/ml, or 5 ng/ml IL-6 for 3 d. A) Representative FACS analyses of IL-17A- and IFN-γ-producing CD4 T cells. B, C) Number of viable CD4 T cells (B) and relative percentage of Th17 cells (C) among cultured CD4 T cells from each genotype were normalized against the percentage of Th17 cells among CD45.2 CD4 T cells cultured in the presence of 5 ng/ml IL-6. D) Total IL-17 produced in the supernatant of polarized CD4 T cells during 3 d of culture was determined via multiplex cytokine array. E, F) RoRα (E) and RoRγt (F) mRNA levels were determined via real-time RT-PCR from naive CD4 T cells activated for 24 h in the absence or presence of 1 ng/ml IL-6. Data represent means ± se of 2 independent experiments from 3–4 measurements/genotype. *P < 0.05 vs. corresponding CD45.2 T cells, ⁺P < 0.05 vs. −IL-6 base level; 2-tailed Student's t test.

Figure 6.

STAT3 and NF-κB/RelA are hyperphosphorylated in naive p53^nullCD45.1 and p53^nullCD45.2 CD4 T cells, which are enhanced further by IL-6 stimulation and Th17 differentiation. A) Fresh splenocytes were harvested from CD45.2, CD45.1, p53^nullCD45.2, and p53^nullCD45.1 mice to determine STAT3 phosphorylation status via Western blotting. B) Naive CD4 T cells of each genotype were cultured in serum-free medium for 4 h, followed by addition of 0, 1, or 5 ng/ml IL-6 for 15 min. STAT3 phosphorylation and total STAT3 from the cell lysates were examined via Western blotting. C) Fresh CD4 T cells were harvested from each genotype to determine NF-κB/RelA phosphorylation status via Western blotting. D) Naive CD4 T cells from each genotype were enriched and activated under Th17 polarization conditions in the presence of 1 ng/ml. Intensity of each signal was quantified and normalized against that of β-actin of the control sample in the absence of IL-6. Data are representative of ≥3 independent experiments.

Figure 7.

STAT3 hyperphosphorylation and autoimmune pathology of p53^nullCD45.1 mice was alleviated by STAT3 inhibitor treatment in vivo. A, B) Enriched CD4 T cells were cultured for 4 h, followed by the treatment of STAT3 inhibitor S31-201 at various concentrations for 1 h. They were then stimulated with 5 ng/ml IL-6 for 15 min (A) or 24 h (B), and the level of STAT3 phosphorylation was determined via Western blotting. Intensity of each signal was quantified and normalized against that of β-actin of the control sample in the absence of S31-201 and IL-6. Data are representative of ≥3 independent experiments. C–F) Eight- to 10-wk-old p53^nullCD45.1 mice were treated with i.v. injection of S31-201 3×/wk at 5 mg/kg, using PBS injection as controls. C) At 4 wk after treatment, spleens of those mice were examined for STAT3 phosphorylation via Western blotting. Signal intensity was quantified and normalized against that of β-actin of a PBS treated mouse (#529). D, E) Lymph nodes (D) and kidneys (E) of those treated mice were examined for IL-17A- and IFN-γ-producing cells via FACS. F) Kidney pathology was examined via trichrome staining. Scale bars = 50 μm. Data are representative of ≥3 (A, B) or 2 (C–F) independent experiments and presented as means ± se of 3–4 mice/treatment/experiment. *P < 0.05 vs. PBS treatment; 2-tailed Student's t test.