PMC

Casz1 controls T helper lineage differentiation depending on the cytokine milieu. (A–C) Naïve CD4⁺ cells isolated from WT (left) or knockout (right) mice were stimulated with 1 µg/ml of plate-bound α-CD3 and 2 µg/ml of soluble α-CD28 antibodies for 4 days (A,B) or for indicated times and subjected to flow cytometry. The percentage of divided cells is indicated as % Prol. in (A). (C) Averages from 3 independent experiments are plotted. *P < 0.05, as determined by unpaired Student’s t-test. (D,E) Cells were stimulated as in (A), in the presence of indicated cytokines for 4 days before the intracellular staining and flow cytometry. Data represent one of the five independent experiments showing similar results. Gates were drawn using unstain or isotype controls.

Casz1 promotes the expression of Th17 genes by affecting chromatin modifications in Th17 lineage genes. Casz1^+/−WT (blue) or Casz1 knockout (red) naïve cells were fixed (A), or stimulated as in Figure B for 4–5 days before fixation (B), for chromatin immunoprecipitation using α-H3K9me3, α-H3K27me3, α-H3K4me3, α-H4K12Ac, or control IgG antibodies and qPCR primer pairs for the indicated genes. Casz1^+/−heterozygous (blue) or Casz1 knockout (red) naïve CD4⁺ cells were stimulated under Th17 conditions as in Figure B for 4 days in the presence of dimethyl sulfoxide or the indicated inhibitors. The frequency of IL-17A⁺ cells was determined by flow cytometry (gated on CD4⁺CD44^high cells) (C) Antigen presenting cells were removed by MACS purification of CD4⁺ T cells before determining the mRNA levels of IL-17A (left) and RAR-related orphan receptor-γt (right) in CD4⁺ T cells by qPCR (D). Results represent 3 independent experiments. *P < 0.05, as determined by unpaired Student’s t-test.

RNA sequencing (RNA-seq) analysis shows that Casz1 favors Th17 specification program. Volcano plot [(A), top], Scatter plot [(A), bottom], and heat maps of genes encoding literature curated Th17 signature proteins (B) and Th1 regulatory proteins (C), derived from RNA-seq analysis comparing partially polarized CD4-cre Casz1^fl/fl Th17 cells and CD4-cre Casz1^wt/wt Th17 cells. Th17 and Th1 signature genes were defined based on the published literature. In (B,C), the upper dendrograms illustrate the results of unsupervised hierarchical clustering of all six samples based on the expression of these Th17 signature genes. (D) Gene set enrichment analysis (GSEA) was performed using the whole gene list generated from RNA-seq. This whole gene list was pre-ranked based on T-Score, then uploaded to GSEA software. Abbreviations: Nes, normalized enrichment score; Nom, nominal; FDR, false discovery rate. Th17 signature gene list that was used for GSEA is depicted in Figure S10B in Supplementary Material. Data are representative of three independent biological replicates from cells derived from three independent experiments.

Loss of Casz1 ameliorates Th17 inflammation during experimental autoimmune encephalomyelitis (EAE) in vivo. (A) EAE was induced in 8- to 10-week-old Casz1^+/+WT (blue; n = 7) or Casz1 knockout (red; n = 6) mice using myelin oligodendrocyte glycoprotein (MOG) peptide. Some Casz1^+/+ WT control mice were left untreated (white; n = 6; No EAE). EAE scores were recorded in masked fashion. Mean scores ± SEM are plotted. ***P = 0.002 was determined by two-way ANOVA t-test. (B) On day 10 after EAE induction, pooled axillary/inguinal lymph nodes (LN), or spinal cords (SC) were harvested for re-stimulation with MOG peptide and intracellular staining of Foxp3 (X axis) and IL-17A (Y axis) (B), or IFN- γ (X axis) and IL-17A (Y axis) (C) (Gated on CD4⁺ cells). (D–I) The frequencies and cell counts of cytokine and Foxp3 expressing cells in LN or SC are plotted (gated on CD4⁺ cells). Mean ± SEM of each group is shown. Two-way ANOVA with Bonferroni’s post hoc analyses were performed to determine significance. These data represent three independent experiments. Gates were drawn using unstain or isotype controls.

Casz1 mRNA expression in CD4⁺ T lymphocytes. RNA was isolated from fluorescence-activated cell sorting (FACS) sorted CD4⁺CD25⁻GFP⁻CD44^low (Naïve) cells, CD4⁺CD25⁺GFP⁺ (regulatory T cells, T_regs), CD4⁺CD44^high (effector/memory) cells pooled from spleen (SPLN) and lymph nodes (LN) of six Foxp3^GFP mice and used for Quantitative PCR (A) Naïve cells were stimulated with 1 µg/ml of plate-bound α-CD3 and 2 µg/ml of soluble α-CD28 antibodies for 3 days (activated) (A), or for indicated times [(B), upper panel]. Human FACS sorted CD4⁺CD25⁻ CD44^low CD45RA naïve cells were activated for indicated times [(B), lower panel]. Phenotype of CD4 conditional Casz1 knockout mice (C–E). (C) Naïve CD4⁺ cells from WT (CD4-cre Casz1^wt/wt) (blue), or knockout (CD4-cre Casz1^fl/fl) (red) mice were stimulated for indicated times. (D) Single cell suspensions from thymus, SPLN, and LN from WT (blue) or knockout (red) mice (D), or SPLN and LN pooled (E), were used for flow cytometry staining in ex vivo [Gated on CD4⁺ T cells in second–fourth panels in (E)]. Two-way ANOVA multiple comparison tests (A,B), Student’s t-tests (C), Two-way ANOVA multiple comparisons test (D) were performed to determine the significance. Data from one of three independent experiments are shown.

Loss of Casz1 impairs IL-17A production and Th17 recall responses during Candida infection in mice. (A) 8- to 10-week-old CD4-cre Casz1^wt/fl (n = 5) or CD4-cre Casz1^fl/fl (n = 5) were infected with Candida as described in Methods. Some control mice received PBS (Sham; n = 5) or immunosuppressed with Cortisone injection (Cort; n = 3). Pooled axillary and cervical draining lymph nodes (LN) and the tongues were harvested and restimulated with PMA/Ionomycin on day 3 after primary infection for IFN-γ (X-axis) and IL-17A (Y-axis) staining (gated on CD4⁺ cells). Data on day 5 showed similar results (not shown). (B) Percent weight change on indicated days after primary infection. (C,D) Mice were infected as in (A), and re-infected on d21 after primary infection. Two days after reinfection, spleen (SPLN), draining LN, and tongues were processed for flow cytometry. (E) Percent weight change on indicated days after re-infection. (F) On day 5 after reinfection, histology sections of the tongue were stained with periodic acid schiffs to detect the fungus. Microscopic images of the slides that were viewed at 200× magnification, with insets showing further magnification [arrows show Candida (pink)]. At least five independent experiments, comparing heterozygous with knockout animals, as well as WT with knockout animals showed similar results. Two-way ANOVA test was performed to determine the significance. Gates were drawn using unstain or isotype controls.

Casz1 promotes IL-17A expression in Th17 cultures. (A) Casz1^+/+WT or Casz1 knockout naïve CD4⁺ T cells were cultured under Th1 (top), Th2 (second panel), iT_reg (third panel), or Th17 (bottom panel) conditions for 5 days. Th1, Th2, Th17 cells were stimulated with antigen presenting cells (APC), and iT_regs without APC (Figure S6 in Supplementary Material). Intracellular cytokine staining was performed in PMA/ionomycin re-stimulated cells. Data from flow cytometric analyses (gated on CD4⁺ cells) showing the proliferation (carboxy-fluorescein-succinimidyl-ester, X axis), percentage of IFN-γ (top), IL-4 (second panel), Foxp3 (third panel), and IL-17A (bottom panel) positive cells (Y-axis). Gates were drawn based on unstained and un-stimulated controls (not shown). G.M of IFN-γ staining in Th1 cells (top panel) is indicated at the bottom of the plots. (B) Casz1^+/+WT or Casz1 knockout naïve CD4⁺ cells were stimulated under Th17 skewing conditions in the presence of APC for indicated days before flow cytometry. (C) Casz1^+/+WT (blue) or Casz1 knockout (red) naïve CD4⁺ cells were stimulated under Th17 conditions as in (B) in five independent experiments, and the frequency of IL-17A⁺ cells was determined by flow cytometry on day 4 (*P < 0.05 was determined by Wilcoxon matched-pairs signed rank test). (D) Supernatants were collected for enzyme linked immunosorbent assay at indicated time-points. (E) Casz1^+/+WT (blue) or Casz1 knockout (red) naïve CD4⁺ cells were stimulated under Th17 conditions for indicated time-points as in (B), and APCs were removed by MACS before determining the mRNA levels of Casz1 (top), RAR-related orphan receptor-γt (middle), and IL-17A (bottom) in CD4⁺ T cells by qPCR. (F) Casz1^+/+WT (blue) or Casz1 knockout (red) cells were stimulated as in (B) for 3 days and were stained for indicated proteins (gated on CD4⁺ cells). For phosphorylated STAT-3 (pSTAT3) and pSTAT5 staining, cells were washed and re-stimulated with IL-6 or IL-2, respectively for 30 min before fixation. The results are representative of at least three independent experiments (*P < 0.05, as determined by unpaired Student’s t-test or Mann–Whitney test). Gates were drawn using unstain or isotype controls.