Fig 2: Loss of NAC1 enhances the functional activity of Tregs.Purified CD4+ Tregs from the pooled LNs and spleen of WT or NAC1-/- mice were stimulated with anti-CD3 plus CD28 antibodies in the presence of rIL-2 for various times. (A) OCR. (B) ECAR. (C) Cytokine production by intracellular staining. (D) Cytokine secretion by ELISA. (E) In vitro suppressive assay. (F to I) T cell transfer model of colitis. (F) Changes of body weight. (G) Representative hematoxylin and eosin (H&E)–stained sections of gut tissues. (H) Treg frequencies/numbers in the adoptive transfer–induced colitis model. (I and J to M) Lipopolysaccharide (LPS)–mediated colitis. (I) Representative colon images of the LPS-induced colitis. (J) Colon lengths in LPS-induced colitis. (K) Representative H&E-stained sections of images of proximal colon cross section. (L and M) Numbers of transferred Tregs and FoxP3 MFI (bottom), gating on Thy1.2+ populations (top). (N to P) In vivo analyses of Tregs. WT and NAC1-/- Tregs (Thy1.2+) were labeled with CFSE and intravenously injected into mice. On days 2 and 5, the transferred Th1.2+ Tregs were analyzed by flow cytometry. (N) Proliferation by CFSE. (M) FoxP3 expression. (P) Apoptosis. (Q and R) In vivo cotransfer of CFSE-labeled WT (Thy1.1+) and NAC1-/- (Thy1.2+) at 1:1 ratio into recipients and examined the NAC1-/-/WT ratios of Tregs on days 2 and 5. Data shown are the means ± SD of three independent experiments and are the representative of three identical experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s unpaired t test.

Fig 3: Loss of NAC1 enhances the induction of iTregs but not cell division and survival.The naive CD4+CD25- T cells from the pooled spleen and LNs of WT or NAC1-/- mice were induced to iTregs in vitro in the presence of TGF-ß for 5 days (A to C), or purified CD4+ Tregs from the pooled LNs and spleen of WT or NAC1-/- mice were stimulated with anti-CD3 plus CD28 antibodies in the presence of rIL-2 for various times (D to F). (A) Expression of NAC1 in naïve CD4 and iTregs of WT T cells detected by immunoblots. (B) Expression of CD25 and FoxP3 in iTregs generated from WT and NAC1-/- CD4+CD25- T cells by flow cytometry. Data shown are the representative of three identical experiments. (C) MFI of FoxP3 in iTregs generated from WT and NAC1-/- CD4+CD25- T cells as analyzed by flow cytometry. Results shown are the means ± SD of three identical experiments. **P < 0.01, Student’s unpaired t test. (D) Cell proliferation/division by carboxyfluorescein diacetate succinimidyl ester (CFSE)–based flow cytometry. Data shown are the representative of three identical experiments. (E) Percentage and (F) numbers of cell recovery on various days were examined by trypan blue exclusion. The numbers of T cells present on day 0 were assigned a value of 100%, and numbers surviving on various days were used to calculate the percentage recovery relative to day 0. Data shown represent the means ± SEM of percentage change or numbers of three independent experiments. All P > 0.05, Student’s unpaired t test.

Fig 4: NAC1-/- mice are tolerant to induction of autoimmunity.WT or NAC1-/- mice were challenged with either bovine type II collagen in complete Freund’s adjuvant by one intradermal immunization at two sites in the base and slightly above of the tail on day 0, or by oral ingestion of 3% DSS (MP Biomedicals) in drinking water for 5 days. (A to C) Arthritis: The histology of the joints (A), arthritis incidence (B), and clinical score (C) were evaluated by examining the paws. Values are the means ± SEM of three independent experiments (n = 10). ****P < 0.0001 in (B) and (C), simple linear regression. (D to H) Colitis: The severity of colitis activity was graded on the designated dates. Histology of colon (D), animal body weight change (E), survival (F), animal size and colon length (G), and the resultant IBD disease activity index (H) were determined. Values are the means ± SEM of three independent experiments (n = 10). ****P < 0.0001 in (E) and (H), simple linear regression. ****P < 0.0001 in (F), survival curve comparison **P < 0.01.

Fig 5: Up-regulation of NAC1 by proinflammatory cytokines breaks immune tolerance via down-regulation of FoxP3.(A) NAC1 and FoxP3 in Tregs from the FIR reporter mice by immunoblots. (B) NAC1 and FoxP3 mRNA of WT Tregs generated in vitro and with ectopic expression of NAC1 by reverse transcription polymerase chain reaction. ***P < 0.001. (C) FoxP3 protein of the sorted Tregs generated in vitro and with ectopic expression of NAC1 by immunoblots. (D) Immunofluorescence staining of 4',6-diamidino-2-phenylindole, NAC1, and FoxP3 in Tregs generated in vitro. (E) Expressions of FoxP3 and NAC1 in Tregs from WT and NAC1-/- mice by immunoblots. (F) FoxP3 was immunoprecipitated and examined for its acetylation of lysine. (G) WT Tregs were cultured in the presence of various cytokines for 12 hours, and then the expressions of FoxP3 and NAC1 were analyzed by immunoblots. (H) FoxP3 from WT Tregs treated in (G) was immunoprecipitated and examined for its acetylation. (I) WT or NAC1-/- Tregs were treated with cycloheximide (150 µg/ml) and chased for the indicated hours. FoxP3 protein level was analyzed by immunoblotting. (J) HDAC expression. (K) HDAC9 and FoxP3 of the sorted WT or NAC1-/- Tregs generated in vitro and with ectopic expression of either the control Mig vector or human HDAC9 vector were analyzed by immunoblots. All the data shown are the representative of three identical experiments. (L) Proposed model of regulation of FoxP3 by NAC1 in immunity.