MyD88 is critically involved in immune tolerance breakdown at environmental interfaces of Foxp3-deficient mice

Abstract

Tregs expressing the transcription factor Foxp3 suppress self-reactive T cells, prevent autoimmunity, and help contain immune responses to foreign antigens, thereby limiting the potential for inadvertent tissue damage. Mutations in the FOXP3 gene result in Treg deficiency in mice and humans, which leads to the development of a multisystem autoimmune inflammatory disease. The contribution of dysregulated innate immune responses to the pathogenesis of Foxp3 deficiency disease is unknown. In this study, we examined the role of microbial signals in the pathogenesis of Foxp3 deficiency disease by studying Foxp3 mutant mice that had concurrent deficiencies in TLR signaling pathways. Global deficiency of the common TLR adaptor MyD88 offered partial protection from Foxp3 deficiency disease. Specifically, it protected from disease at the environmental interfaces of the skin, lungs, and gut. In contrast, systemic disease, in the form of unrestrained lymphoproliferation, continued unabated. The effect of MyD88 deficiency at environmental interfaces involved the disruption of chemokine gradients that recruit effector T cells and DCs, resulting in their entrapment in secondary lymphoid tissues. These results suggests that Tregs have a key role in maintaining tolerance at host-microbial interfaces by restraining tonic MyD88-dependent proinflammatory signals. Moreover, microbial factors may play a substantial role in the pathogenesis of human autoimmune disease resulting from Treg deficiency.

Figure 1. Concurrent MyD88 deficiency protects against the inflammatory skin manifestations and runting of Foxp3^mut mice.

(A) Gross appearance of mutant mice compared with a WT control. (B) Myd88^–/– and Myd88^–/–Foxp3^mut littermates around 30 days of age. (C) Phenotypic characteristics of WT and mutant mice at 30 days of age, including weight, alopecia, skin hemorrhage, and dryness. (D) MyD88 deficiency protects against skin inflammation in Foxp3-deficient mice. Shown are H&E and Trichrome staining. Original magnification, ×200. Results for C and D are representative of 4–21 mice per group, derived from 4–13 independent experiments. *P < 0.05; ***P < 0.001.

Figure 2. MyD88 deficiency downregulates markers of immune activation in the skin of Foxp3^mut mice.

(A and B) Downregulation of NF-κB activation and ICAM-1 expression. Immunohistochemistry staining of phospho–NF-κB p65 subunit (A) and ICAM-1 (B) in the skin of WT and mutant mice. Original magnification, ×200. Results are representative of 4 mice per group from 2 independent experiments. (C) Downregulation of cytokines associated with the adaptive and innate immune responses. Transcript expression of the respective cytokines was determined by real-time PCR analysis. Each point represents results obtained from 1 individual mouse. Results are from 4–21 mice per group, derived from 4–13 independent experiments. *P < 0.05; **P < 0.01.

Figure 3. Concurrent MyD88 deficiency protects from tissue inflammation in the lungs and small intestines of Foxp3^mut mice.

(A) Histopathology (H&E staining) of lung, small intestine, liver, pancreas, and salivary gland tissues of WT, Foxp3^mut, and Myd88^–/–Foxp3^mut mice. Arrowheads denote inflammatory infiltrates in Foxp3^mut mouse small intestine. Original magnification, ×200. (B) Inflammation scores. Each point represents results obtained from 1 individual mouse. (C and D) Flow cytometric analysis (C) and enumeration (D) of IFN-γ and IL-17a expression in CD4⁺ T cells in lung and small intestinal tissues (LPL fraction) of WT and mutant mice. Results are from 4–11 mice per group, derived from 2–3 independent experiments. *P < 0.05; **P < 0.01.

Figure 4. Effect of antibiotic therapy on immunopathology of Foxp3^mut mice.

(A) Weight at 30 days of age in WT and mutant mice treated or not with a dual antibiotic (Abx) regimen (doxycycline and cotrimoxazole). Results are representative of 5–9 mice per group, derived from 3–5 independent experiments. (B) Tissue histology (H&E staining) of WT and antibiotic-treated Foxp3^mut mice. Original magnification, ×200. (C) Tissue inflammatory scores in WT, Foxp3^mut, and antibiotic-treated Foxp3^mut mice. (D) Expression of Il6 and Il17a transcripts, as evaluated by real-time PCR, in skin of WT, Foxp3^mut, and antibiotic-treated Foxp3^mut mice. (E) Total lymphocyte counts and absolute number of CD4⁺ effector memory T cells (CD4⁺CD62L^loCD44^hi), CD8⁺ T cells, and B220⁺ B cells in the draining lymph nodes. Results are representative of 4–19 mice per group, derived from 3–12 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. MyD88 deficiency does not restrain the lymphoproliferative disease of Foxp3 deficiency.

(A) Photomicrograph of spleen and draining (axillary and inguinal) lymph nodes of 30-day-old WT, Myd88^–/–, Foxp3^mut, and Myd88^–/–Foxp3^mut mice. (B) Total lymphocyte counts and absolute number of CD4⁺ effector memory T cells, CD8⁺ T cells, and B220⁺ B cells in the draining lymph nodes. (C) Percent BrdU-labeled cells in different cell populations in the draining lymph nodes. (D) Flow cytometric analysis of IL-4, IFN-γ, and IL-17a expression in CD4⁺ T cells in draining lymph node. Numbers in quadrants indicate percentage of gated cells present. (E) Absolute number of cytokine-secreting CD4⁺ T cells in D. Each dot represents the absolute cell number (×10⁶) from a single mouse; columns denote means. Results in A–E are representative of 3–5 experiments involving 5–10 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. The protective effects of MyD88 deficiency are not caused by curtailed DC function.

(A) Flow cytometric analysis shows percent CD11c⁺I-A^b+ DCs present in the lungs and draining lymph nodes of WT, Myd88^–/–, Foxp3^mut, and Myd88^–/–Foxp3^mut mice. Expression of CD40, CD80, and CD86 on gated CD11c⁺I-A^b+ DCs from the draining lymph nodes is also shown. (B) Absolute number of CD11c⁺I-A^b+ DCs in the lungs and draining lymph nodes. Each dot represents the absolute cell number from a single mouse; columns denote means. (C) DC antigen-presenting function. DCs were pulsed with OVA, then cultured with OVA_323–339 peptide–specific OT-II TCR transgenic T cells. (D) Total, CD4⁺ T cell, CD8⁺ T cell, and B cell counts in draining lymph nodes and spleens of WT, Myd88^fl/flFoxp3^mut, and Cd11c-Cre⁺Myd88^Δ/ΔFoxp3^mut mice. (E) IFN-γ and IL-17a expression in CD4⁺ T cells in the lungs, small intestines (LPL fraction), draining lymph nodes, and spleens. Results are representative of 3–5 experiments involving 5–10 mice per group. *P < 0.05; **P < 0.01.

Figure 7. MyD88 deficiency restricts the recruitment of effector T cells to the lungs of Foxp3^mut mice.

(A and B) Flow cytometric analysis of CD45.1/CD45.2 congenically marked (A) or CFSE-labeled (B) CD4⁺CD44^hiCD62^lo T cells derived from Foxp3^mut or Myd88^–/–Foxp3^mut draining lymph nodes and transferred into the indicated recipient mice. At 72 hours after transfer, lung tissues were collected and examined to assess the presence or absence of the transferred T cells. Numbers denote percent transferred T cells in the boxed regions. n = 3–5 mice per group. (C) Absolute number of congenically marked donor T cells in the lungs and draining lymph nodes of recipient mice. (D) Flow cytometric analysis of CFSE dilution in transferred T cells isolated from the lungs and draining lymph nodes of recipient mice. Percentage of cells undergoing at least 1 cellular division is shown within histograms. (E) Percent proliferating cells, based on CFSE dilution analysis (D), in donor T cells isolated from recipient mice. Results are representative of 3–5 independent experiments. **P < 0.01; ***P < 0.001.

Figure 8. Protective function of tissue MyD88 deficiency against inflammatory bowel disease induced by Foxp3^mut lymphocytes.

(A) Weight change after adoptive transfer of 1 × 10⁶ unfractionated draining lymph node cells from Foxp3^mut or Myd88^–/–Foxp3^mut mice into Rag1^–/– or Myd88^–/–Rag1^–/– mice. n = 3–5 per group. (B) Gut inflammation scores for mice as in A. (C) Representative histological sections (H&E staining) from the small intestines of mice as in A. Original magnification, ×200. (D) Weight change after adoptive transfer of 1 × 10⁶ unfractionated draining lymph node cells from Foxp3^mut mice into Rag1^–/– or Myd88^–/–Rag1^–/– radiation chimeric recipients of BM transplants from the indicated mice. n = 3–5 per group. (E) Myd88 mRNA expression in BM cells of mice as in D. Results in A–C and in D and E are each representative of 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 9. MyD88 deficiency disrupts chemokine gradients involved in the homing of effector T cells and DCs to the lungs, skin, and small intestine of Foxp3^mut mice.

Shown are transcript levels of Cxcl9, Cxcl10, Ccl22, Ccl20, and Cxcl3 in lungs (A), skin (B), small intestines (C), and draining lymph nodes (D) of WT and mutant mice. mRNA levels were determined by real-time PCR analysis. Each point represents results obtained from 1 individual mouse. Results are representative of 4–10 mice per group, derived from 3–5 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.