Loss of GTPase of immunity-associated protein 5 (Gimap5) promotes pathogenic CD4+ T-cell development and allergic airway disease

Abstract

Background: GTPase of immunity-associated protein 5 (GIMAP5) is essential for lymphocyte homeostasis and survival. Recently, human GIMAP5 single nucleotide polymorphisms have been linked to an increased risk for asthma, whereas loss of Gimap5 in mice has been associated with severe CD4⁺ T cell-driven immune pathology.

Objective: We sought to identify the molecular and cellular mechanisms by which Gimap5 deficiency predisposes to allergic airway disease.

Methods: CD4⁺ T-cell polarization and development of pathogenic CD4⁺ T cells were assessed in Gimap5-deficient mice and a human patient with a GIMAP5 loss-of-function (LOF) mutation. House dust mite-induced airway inflammation was assessed by using a complete Gimap5 LOF (Gimap5^sph/sph) and conditional Gimap5^fl/flCd4^Cre/ert2 mice.

Results: GIMAP5 LOF mutations in both mice and human subjects are associated with spontaneous polarization toward pathogenic T_H17 and T_H2 cells in vivo. Mechanistic studies in vitro reveal that impairment of Gimap5-deficient T_H cell differentiation is associated with increased DNA damage, particularly during T_H1-polarizing conditions. DNA damage in Gimap5-deficient CD4⁺ T cells could be controlled by TGF-β, thereby promoting T_H17 polarization. When challenged with house dust mite in vivo, Gimap5-deficient mice displayed an exacerbated asthma phenotype (inflammation and airway hyperresponsiveness), with increased development of T_H2, T_H17, and pathogenic T_H17/T_H2 cells.

Conclusion: Activation of Gimap5-deficient CD4⁺ T cells is associated with increased DNA damage and reduced survival that can be overcome by TGF-β. This leads to selective survival of pathogenic T_H17 cells but also T_H2 cells in human subjects and mice, ultimately promoting allergic airway disease.

Keywords: Allergic airway disease; DNA damage response; GIMAP5; T(H)2/T(H)17 cells; T-cell polarization; TGF-β; house dust mite; immune homeostasis; lung disease; primary immune deficiency.

FIG 1.

Enhanced In vivo polarization of Gimap5-deficent mouse and human CD4⁺ T cells. (A) Number of CD4⁺ T cells in the spleen of 8wk WT (black bars) and Gimap5^sph/sph (red bars) mice. (B) frequency of splenic CD4⁺ T cells expressing Foxp3, Tbet, Gata3, or RORγt (n=7). (C-G) Expression of IFNγ, IL-17A, and/or IL-13 in splenic CD4⁺ T cells after PMA/ionomycin stimulation (n=4). (H) Number of CD4⁺ T cells in the mesenteric lymph node of 8wk mice. (I) frequency of CD4⁺ T cells expressing Foxp3, Tbet, Gata3, or RORγt in the mesenteric lymph node (n=7). (J-N) Expression of IFNγ, IL-17A, and/or IL-13 in CD4⁺ T cells from the mesenteric lymph node after PMA/ionomycin stimulation (n=4). Bars represent mean values ± SD. Statistical significance is determined by Student’s two-tailed test. (O-S) Frequency of CD4⁺ T cells producing IFNγ, IL-17A, and/or IL-13 upon PMA/ionomycin stimulation. T cells isolated from the PBMCs of a GIMAP5 patient (GIMAP5^204-Pro/Pro), his heterozygous parent, and unrelated controls (n=2, 2, and 7, respectively). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

FIG 2.

Preferential expression of RORγt by surviving Gimap5^sph/sph CD4⁺ T cells upon TCR stimulation. (A) Proliferation of WT and Gimap5^sph/sph CD4⁺ T cells after 3 days stimulation with αCD3 (1 μg/ml) + αCD28 (2 μg/ml). (B) Survival CD4⁺ T cells after 3d stimulation. (C) Expression of RORγt in live CD4⁺ T cells after 3d stimulation with αCD3/αCD28. Plots depict live cells (C), while bar graphs (B) represent mean values ± SD (n=4). All experiments were performed at least three times. Statistical significance is determined by Student’s two-tailed test. ****P<0.0001

FIG 3.

Preferential survival of Gimap5^sph/sph CD4⁺ T cells in T_H17 and iTreg polarizing conditions is associated with TGFβ. (A-B) Proliferation and survival of naïve CD4⁺ T cells from WT and Gimap5^sph/sph mice stimulated 3d in non-polarizing conditions (T_H0) or T_H1, T_H2, T_H17, or iTreg polarizing conditions. Blue histograms represent dead cells; red histograms show live cells. Hatched bars represent non-proliferated cells; solid bars represent proliferated cells. (C) Proliferation of live/dead CD4⁺ T cells after 3d stimulation in different T_H17 polarizing conditions. (D) γH2AX expression (DNA damage) in live CD4⁺ T cells in cycle after 3d stimulation in different polarizing conditions. Data represent mean values ± SD (n=4). All experiments were performed at least three times. Statistical significance is determined by ANOVA followed by Sidak’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

FIG 4.

TGFβ1 rescues survival and proliferation of naïve Gimap5^sph/sph CD4⁺ T cells. Proliferation and survival of naïve WT and Gimap5^sph/sph CD4⁺ T cells after 3d stimulation in (A) T_H0 or (B) T_H2 polarizing conditions ± 2 ng/mL TGFβ1. Blue histograms represent dead cells; red histograms show live cells. Survival, proliferation, and γH2AX expression of naïve CD4⁺ T cells stimulated in (C-D) T_H0, (E-F) T_H1, (G-H) or T_H2 polarizing conditions. At 1 or 2 days after initial stimulation, 2 ng/ml TGFβ1 was added to the indicated culture. Data represent mean values ± SD (n=4). Statistical significance is determined by ANOVA followed by Sidak’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

FIG 5.

Gimap5^sph/sph mice exhibit increased AHR, CD4⁺ T cell polarization, and airway pathology upon HDM sensitization. C57BL/6 WT (black bars) and Gimap5^sph/sph (red bars) mice were administered 25 μg HDM 9 times IT over three weeks. (A) Airway hyper-responsiveness (AHR) in response to methacholine challenge 1 day after the last HDM challenge (saline: n=3; HDM: n=5–6). (B-D) Number of macrophages, neutrophils, and eosinophils in the BAL fluid (saline: n=3; HDM: n=4–7). (E-F). Number of (E) total lung-infiltrating cells and (F) CD4⁺ T cells (in 2 lobes). (G-J) Percentage of lung-infiltrating CD4⁺CD3⁺ T cells expressing IL-13, IL-17A, IL-13 and IL-17A, or IFNγ upon restimulation with PMA/ionomycin for 6h (saline: n=3; HDM: n=6–7). (K-L) Representative (K) H&E and (L) PAS images of lung tissue from WT and Gimap5^sph/sph mice given saline or HDM. Data represents mean values ± SD. Statistical significance is determined by ANOVA followed by Sidak’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

FIG 6.

Impaired survival and increased DNA damage associated with Gimap5-deficiency is CD4⁺ T cell intrinsic. (A) Expression of Gimap5 in CD4⁺ T cells isolated from WT and Gimap5^fl/flCd4^cre-ert2 mice after tamoxifen-induced deletion of Gimap5. Immunoblot shows three independent cKO mice. (B-C) Proliferation and survival of naïve CD4⁺ T cells from WT and Gimap5^fl/flCd4^cre-ert2 mice stimulated 3d in nonpolarizing conditions (T_H0) or T_H1, T_H2, T_H17, or iTreg polarizing conditions. Blue histograms represent dead cells; red histograms show live cells. (D) γH2AX expression in live, cycling CD4⁺ T cells after 3d stimulation in T_H0, T_H1, T_H2, T_H17, or iTreg polarizing conditions. Data represent mean values ± SD (n=4–5). Statistical significance is determined by ANOVA followed by Sidak’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

FIG 7.

Gimap5-deficiency in CD4⁺ T cells is sufficient to drive increased AHR, CD4⁺ T cell polarization, and airway pathology upon HDM sensitization. C57BL/6 WT (black data points) and Gimap5^fl/flCd4^cre-ert2 (red data points) mice were administered 50 μg HDM 9 times IT over three weeks. (A) Airway hyper-responsiveness (AHR) in response to methacholine challenge 1 day after the last HDM challenge (saline: n=4–5; HDM: n=7–8). (B-D) Number of macrophages, neutrophils, and eosinophils in the BAL fluid (saline: n=4–5; HDM: n=8). (E-F). Number of (E) total lung-infiltrating cells (in 2 lobes) and (F) frequency of CD4⁺CD3⁺ T cells therein. (G) Frequency of CD4⁺CD3⁺ T cells expressing an activated (CD44^hi) phenotype. (H-K) Percentage of lung-infiltrating CD4⁺CD3⁺ T cells expressing IL-13, IL-17A, IL-13 and IL-17A, or IFNγ upon restimulation with PMA/ionomycin for 6h (saline: n=4–5; HDM: n=8). (L-M) Representative (L) H&E and (M) PAS images of lung tissue from WT and Gimap5^fl/flCd4^cre-ert2 mice given saline or HDM. Data represents mean values ± SD. Statistical significance is determined by ANOVA followed by Sidak’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001