Disease Risk-Associated Genetic Variants in STAT1 and STAT4 Function in a Complementary Manner to Increase Pattern-Recognition Receptor-Induced Outcomes in Human Macrophages

Abstract

STAT proteins can regulate both pro- and anti-inflammatory cytokine signaling. Therefore, identifying consequences of modulating expression of a given STAT is ultimately critical for determining its potential as a therapeutic target and for defining the mechanisms through which immune-mediated disease variants in STAT genes contribute to disease pathogenesis. Genetic variants in the STAT1/STAT4 region are associated with multiple immune-mediated diseases, including inflammatory bowel disease (IBD). These diseases are characterized by dysregulated cytokine secretion in response to pattern-recognition receptor (PRR) stimulation. We found that the common IBD-associated rs1517352 C risk allele increased both STAT1 and STAT4 expression in human monocyte-derived macrophages (MDMs). We therefore hypothesized that the STAT1/STAT4 variant might regulate PRR-initiated responses in a complementary and cooperative manner because of the important role of autocrine/paracrine cytokines in modulating PRR-initiated signaling. STAT1 and STAT4 were required for PRR- and live bacterial-induced secretion of multiple cytokines. These outcomes were particularly dependent on PRR-initiated autocrine/paracrine IL-12-induced STAT4 activation to generate IFN-γ, with autocrine IFN-γ then signaling through STAT1. STAT1 and STAT4 also promoted bacterial-induced cytokines in intestinal myeloid cells and PRR-enhanced antimicrobial pathways in MDMs. Importantly, MDMs from rs1517352 C IBD risk allele carriers demonstrated increased TLR4-, IFN-γ- and IL-12-induced STAT1 and STAT4 phosphorylation and cytokine secretion and increased TLR4-enhanced antimicrobial pathways. Taken together, STAT1 and STAT4 expression is coregulated by a shared genetic region, and STAT1 /STAT4-immune disease-associated variants modulate IFN-γ- and IL-12-associated outcomes, and in turn, PRR-induced outcomes, highlighting that these genes cooperate to regulate pathways relevant to disease pathogenesis.

FIGURE 1.

MDMs from rs1517352 CC disease risk carriers secrete increased levels of PRR-induced cytokines relative to AA carrier MDMs. Human MDMs from rs1517352 CC, CA, and AA carriers (n = 10 per genotype) were treated for 24 h with the indicated doses of (A) lipid A (TLR4 ligand), or (B) Pam3Cys (TLR2), poly(I:C) (TLR3), or CpG DNA (TLR9). Cytokine secretion + SEM. One-way ANOVA with Tukey posttest. *p < 0.05, **p < 0.01, ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵.

FIGURE 2.

MDMs from rs1517352 C disease risk carriers express higher levels of STAT1 and STAT4 and show increased TLR4-induced STAT1 and STAT4 activation relative to A carriers. (A–D) Human MDMs from rs1517352 CC, CA, and AA carriers were assessed for the following: (A) STAT1 and (B) STAT4 mRNA expression (n = 10 per genotype) or (C) STAT1 and (D) STAT4 protein expression with (left) representative flow cytometry and mean fluorescence intensity (MFI) values shown and (right) summarized data (n = 8 per genotype). (E and F) MDMs from rs1517352 CC, CA, and AA carriers (n = 10 per genotype) were treated for 30 min with 0.1 μg/ml lipid A. Fold (E) STAT1 and (F) STAT4 phosphorylation was assessed with representative flow cytometry and summarized data. Mean + SEM. One-way ANOVA with Tukey posttest. *p < 0.05, **p < 0.01, ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵.

FIGURE 3.

STAT1 and STAT4 are required for optimal secretion of cytokines in MDMs upon stimulation through a broad range of PRRs. (A, C, and D) Human MDMs were transfected with scrambled siRNA or with STAT1 or STAT4 siRNA, alone or in combination, and then treated for 24 h with the following: (A) 0.1 μg/ml lipid A (TLR4) (n = 6, similar results seen in an independent n = 8), (C) 10 μg/ml Pam3Cys (TLR2), 100 μg/ml poly(I:C) (TLR3), or 10 μg/ml CpG DNA (TLR9) (n = 6, similar results seen in an independent n = 8), or (D) S. Typhimurium (n = 6). (B) MDMs were pretreated for 1 h with a STAT1 inhibitor (fludarabine) or a STAT4 inhibitor (lisofylline), alone or in combination, or with vehicle (DMSO) and then with 0.1 μg/ml lipid A for 24 h (n = 4). (A–D) Mean cytokine secretion + SEM. Significance is shown compared with stimulated, scrambled siRNA–transfected cells or stimulated, vehicle control-treated cells. t test with Bonferroni–Holm correction. **p < 0.01, ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. scr, scrambled; Tx, treatment.

FIGURE 4.

Autocrine/paracrine IL-12 and IFN-γ secretion promotes TLR4-induced cytokine secretion. Human MDMs (n = 6, similar results in an additional six donors) were transfected with scrambled, IFNGR1, IFNAR, or IL-12Rβ2 siRNA and then treated with 0.1 μg/ml lipid A for 24 h. Mean cytokine secretion + SEM. Significance is shown compared with scrambled siRNA–transfected, lipid A–treated cells. ***p < 0.001, ^†p < 1 × 10⁻⁴. scr, scrambled; Tx, treatment.

FIGURE 5.

IFN-γ and IL-12 activate STAT1 and STAT4, respectively, and this activation contributes to subsequent cytokine secretion. (A) Human MDMs (n = 6; similar results in an additional six donors) were treated with 10 ng/ml IFN-γ or 10 ng/ml IL-12 for 60 min. Fold STAT1 and STAT4 phosphorylation. 0.1 μg/ml lipid A is shown as a control. (B) MDMs (n = 4) were transfected with scrambled, IFNGR1, or IL-12Rγ2 siRNA, and then treated with 0.1 μg/ml lipid A for 30 min. Fold STAT1 and STAT4 phosphorylation. (C) MDMs were transfected with scrambled, STAT1, or STAT4 siRNA. Cells were then treated with 10 ng/ml IFN-γ, IFN-α, or IL-12 for 24 h. Cytokine secretion (n = 8; similar results in an additional eight donors). Mean + SEM. Significance with t test in (A) and with Bonferroni-Holm correction in (B) and (C). *p < 0.05, **p < 0.01, ***p < 0.001, _†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. scr, scrambled; Tx, treatment.

FIGURE 6.

IL-12 and IFN-γ activate MAPK and NF-κB pathways. (A) MDMs (n = 6, similar results in an additional six donors) were treated with 10 ng/ml IFN-γ or 10 ng/ml IL-12 for 30 min. (Left) Representative flow cytometry of phosphoproteins with mean fluorescence intensity (MFI) values shown. (Right) Summary of fold phosphorylation of MAPKs and IκBα. (B) MDMs (n = 6, similar results in an additional eight donors) were transfected with scrambled or the indicated siRNA. Cells were treated with 10 ng/ml IFN-γ or 10 ng/ml IL-12 for 24 h. Cytokine secretion. Mean + SEM. Significance is shown compared with cytokine-treated, scrambled siRNA-transfected cells. *p < 0.05, **p < 0.01, ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. scr, scrambled; Tx, treatment.

FIGURE 7.

STAT1 and STAT4 promote S. Typhimurium–induced cytokine secretion in human intestinal myeloid cells. Human intestinal myeloid cells (n = 6 donors) or peripheral MDMs (n = 6 donors) were preincubated for 1 h with either fludarabine (STAT1 inhibitor) or lisofylline (STAT4 inhibitor), alone or in combination, and then cocultured with S. Typhimurium (S. Typhim) at MOI 10:1 for 24 h. Cytokine secretion + SEM. Significance with t test with Bonferroni–Holm correction. ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. Inh, inhibitor.

FIGURE 8.

MDMs from rs1517352 CC disease risk carriers demonstrate increased IFN-γ– and IL-12–induced signaling and cytokines relative to AA carrier MDMs. (A) Human MDMs from rs1517352 CC, CA, and AA carriers were treated with 10 ng/ml IFN-γ or 10 ng/ml IL-12. (A) Fold STAT1 and STAT4 phosphorylation at 60 min (n = 8 per genotype). (B) Cytokine secretion at 24 h (n = 12 per genotype). Mean + SEM. Significance with one-way ANOVA with Tukey posttest. *p < 0.05, **p < 0.01, ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵.

FIGURE 9.

JAK1 and TYK2 regulate TLR4-induced cytokines in a manner distinct to STAT1 and STAT4. (A) MDMs were transfected with scrambled, STAT1 and STAT4, or IL10RA (to block autocrine IL-10), alone or in combination, and then treated with 0.1 μg/ml lipid A for 24 h. Cytokine secretion (n = 6). (B–E) MDMs were pretreated with vehicle (DMSO) or the indicated doses of either (B and C) Upadacitinib (Upa; JAK1 inhibitor) or (D and E) BMS-986165 (BMS; TYK2 inhibitor) for 1 h. (B and D) Fold induction of the indicated phosphoproteins with 0.1 μg/ml lipid A for 15 min (n = 5; similar results in an additional n = 4) (earlier time point assessed to minimize signaling through autocrine/paracrine cytokine loops). (C and E) Cells were treated with 0.1 μg/ml lipid A or 10 ng/ml IFN-γ or IL-12. Cytokines at 24 h (n = 6; similar results in an additional n = 4 for lipid A). (F) MDMs were transfected with scrambled, JAK1, TYK2, or STAT3 siRNA, and then treated with 0.1 μg/ml lipid A for 24 h. Cytokine secretion (n = 6). Mean + SEM. Significance is to scrambled siRNA–transfected, lipid A–treated cells for (F), to vehicle and cytokine- or lipid A–treated cells in (C) and (E), or as indicated. t test for (A) combined with Bonferroni–Holm correction for (B)–(F). *p < 0.05, **p < 0.01, ***p < 0.001, ^†p < 1 × 10⁻⁴. scr, scrambled; Tx, treatment.

FIGURE 10.

STAT1 and STAT4 are required for optimal TLR4-induced bacterial uptake. MDMs were transfected with scrambled siRNA or with STAT1 or STAT4 siRNA, alone or in combination (comb). Cells were then treated for 48 h with 0.1 μg/ml lipid A, 10 ng/ml IFN-γ, or 10 ng/ml IL-12. Uptake of S. Typhimurium–GFP or E. coli–FITC was assessed (n = 10 from two independent experiments) as per Materials and Methods. Representative flow cytometry and summary graphs. Mean fluorescence intensity (MFI) + SEM. t test with Bonferroni–Holm correction. ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. NT, no treatment; scr, scrambled; Tx, treatment.

FIGURE 11.

STAT1 and STAT4 are required for optimal intracellular bacterial clearance. MDMs were transfected with scrambled siRNA or with STAT1 or STAT4 siRNA, alone or in combination (comb). Cells were then (A) left untreated (n = 6) or (B) treated with 0.1 μg/ml lipid A, 10 ng/ml IFN-γ, or 10 ng/ml IL-12 for 48 h (n = 6; similar results in an additional n = 4). Intracellular bacterial clearance. Mean CFU + SEM. Significance with t test with Bonferroni–Holm correction. **p < 0.01, ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. scr, scrambled; Tx, treatment.

FIGURE 12.

STAT1 and STAT4 promote TLR4-induced antimicrobial pathways. MDMs were transfected with scrambled siRNA or with STAT1 or STAT4 siRNA, alone or in combination (comb). Cells were then left untreated or treated with 0.1 μg/ml lipid A, 10 ng/ml IFN-γ or 10 ng/ml IL-12 for 48 h (n = 6) and assessed by flow cytometry for the following: (A) ROS production, (B) NOS2 expression, or (C) LC3II expression. Representative flow cytometry and summary graphs (mean fluorescence intensity [MFI]). Mean + SEM. Significance with t test with Bonferroni–Holm correction. ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. NT, no treatment; scr, scrambled; Tx, treatment.

FIGURE 13.

MDMs from rs1517352 CC disease risk carriers secrete increased levels of S. Typhimurium–induced cytokines relative to AA carrier MDMs. Human MDMs from rs1517352 CC, CA, and AA carriers (n = 10 per genotype) were treated for 24 h with (A) S. Typhimurium or (B) 0.1 μg/ml lipid A. Cytokine secretion + SEM. Significance with one-way ANOVA with Tukey posttest. *p < 0.05, **p < 0.01, ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. Tx, treatment.

FIGURE 14.

MDMs from rs1517352 CC disease risk carriers demonstrate increased bacterial uptake relative to AA carrier MDMs. Human MDMs from rs1517352 CC, CA, and AA carriers (n = 10 per genotype) were left untreated or treated for 48 h with 0.1 μg/ml lipid A, and then assessed for bacterial uptake. (Left) Representative flow cytometry with mean fluorescence intensity (MFI). (Right) Summary of S. Typhimurium or E. coli uptake. Mean + SEM. Significance with one-way ANOVA with Tukey posttest. *p < 0.05, **p < 0.01, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. NT, no treatment; Tx, treatment.

FIGURE 15.

MDMs from rs1517352 CC disease risk carriers demonstrate increased intracellular bacterial clearance relative to AA carrier MDMs. Human MDMs from rs1517352 CC, CA, and AA carriers (n = 10 per genotype) were left untreated or treated for 48 h with 0.1 μg/ml lipid A. (A) ROS production. (B) NOS2 expression. (C) LC3II expression. Representative flow cytometry and summary graphs (mean fluorescence intensity [MFI]). (D) Intracellular bacterial clearance (CFU). Mean + SEM. Significance with one-way ANOVA with Tukey posttest. *p < 0.05, **p < 0.01, ***p < 0.001, ^†p < 1 × 10⁻⁴, ^††p < 1 × 10⁻⁵. NT, no treatment; Tx, treatment.