Vimentin modulates regulatory T cell receptor-ligand interactions at distal pole complex, leading to dysregulated host response to viral pneumonia

Abstract

Forkhead box P3 (Foxp3)⁺ regulatory T cells (Tregs) resolve acute inflammation and repair the injured lung after viral pneumonia. Vimentin is a critical protein in the distal pole complex (DPC) of Tregs. This study reveals the inhibitory effect of vimentin on the suppressive and reparative capacity of Tregs. Treg-specific deletion of vimentin increases Helios⁺interleukin-18 receptor (IL-18R)⁺ Tregs, suppresses inflammatory immune cells, and enhances tissue repair, protecting Vim^fl/flFoxp3^YFP-cre mice from influenza-induced lung injury and mortality. Mechanistically, vimentin suppresses the induction of amphiregulin, an epidermal growth factor receptor (EGFR) ligand necessary for tissue repair, by sequestering IL-18R to the DPC and restricting receptor-ligand interactions. We propose that vimentin in the DPC of Tregs functions as a molecular switch, which could be targeted to regulate the immune response and enhance tissue repair in patients with severe viral pneumonia.

Keywords: AREG; CP: Immunology; IL-18R; Tregs; distal pole complex; epidermal growth factor receptor; lung injury; lung repair; regulatory T cells; vimentin; viral pneumonia.

Figure 1.. Vimentin limits Tregs’ suppressive function in vivo and in vitro

(A) Weight change of Rag1^−/− mice following adoptive transfer of 4 × 10⁵ Tcon cells (n = 5) alone or with 0.5 × 10⁵ WT or Vim^−/− Tregs. Data are mean ± SEM. (B) H&E staining of distal colon from Rag1^−/− mice 45 days post-transfer. Scale bar, 100 μm. (C) Histological scores of colon tissue from (B). (D) Vimentin expression in WT and Vim^−/− nTregs. Scale bar, 5 μm. (E) In vitro suppression assay by co-culturing Tcon cells with WT or Vim^−/− nTregs, showing proliferating cell percentage (left) and suppression percentage (right) (n = 3). Data are mean ± SD, n = 3. (F) Expression of Foxp3, CD25, Helios, and CD73 on CD4⁺Foxp3⁺ cells from WT and Vim^−/− spleens. Data are mean ± SD, n = 3–6 mice per group. p values were calculated via one-way ANOVA (C), two-way ANOVA (E), and two-tailed unpaired Student’s t test (F). NS, not significant (p > 0.05), *p < 0.05, **p < 0.005, and ***p < 0.0005.

Figure 2.. Treg-specific deletion of vimentin protects the host from IAV-induced lung injury and mortality

WT (Vim^+/+Foxp3^YFP-Cre) and vimentin cKO (Vim^fl/flFoxp3^YFP-Cre) mice were intratracheally infected with lethal (60 plaque-forming units [PFUs]) (A) or a sublethal (30 PFUs) (A–K) dose of IAV. (A) Survival curve for WT (n = 15, lethal; n = 9, sublethal) and vimentin cKO (n = 9, lethal; n = 8, sublethal) mice using log rank (Mantel-Cox) test. (B) Percentage of weight loss from baseline in WT (n = 8) and vimentin cKO (n = 10) mice, analyzed by mixed-effects model with Sidak’s multiple comparisons test. Data are mean ± SEM. (C) Representative H&E lung staining at 21 dpi. Scale bar, 100 μm. (D) Lung injury score from H&E sections. (E) CD45 staining of lungs at 21 dpi. Scale bar, 100 μm. (F) Flow cytometry quantification of CD45⁺ cells from whole lung at 21 dpi. (G) Krt5 and Foxp3 staining of lungs at 21 dpi. Scale bars, 100 μm (bright field) and 50 μm (fluorescence). (H and I) Quantification of Krt5 expression (H) and frequency (I) using TissueGnostics HistoQuest. (J) IFN-β levels in BALF by multiplex immunoassay (n = 3–8 mice per group). (K) Viral titer of whole-lung homogenates at 5 dpi. Data are mean ± SD, n = 4–6 mice per group. p values were calculated by two-way ANOVA (B and J) and two-tailed unpaired Student’s t test (D, F, H, I, and K). NS, not significant (p > 0.05), *p < 0.05, **p < 0.005, and ****p < 0.0001.

Figure 3.. Treg-specific deletion of vimentin promotes the resolution of inflammation after influenza infection

(A and B) Treg frequency and number in WT and vimentin cKO lungs following influenza infection. (C and D) MoAM frequency within the CD45⁺ population and MoAM absolute number in WT and vimentin cKO lungs following influenza infection. (E–I) IL-18, TNF-α, IL-6, IFN-γ, and IL-33 levels in BALF measured by multiplex assay. Data are mean ± SD, n = 3–8 mice per group. p values were calculated by two-way ANOVA. NS, not significant (p > 0.05), *p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001.

Figure 4.. Vimentin deficiency induces enhanced CD73-adenosine signaling in Tregs

(A) Nt5e, Entpd1, and Vim expression in WT and Vim-deficient splenic nTregs (n = 3). (B) CD73⁺ Treg frequency among lung Tregs after influenza infection. (C) CD73 expression on lung Tregs after influenza infection. (D) CD39⁺CD73⁺ Treg frequency in WT or Vim^−/− iTregs (n = 6). (E) Adenosine levels in supernatants from WT and Vim^−/− iTregs treated with 5 mM ATP for 1 h (n = 3). (F and G) WT and vimentin cKO mice were intratracheally infected with IAV and vimentin cKO mice received daily anti-CD73 (100 μg/mouse, intraperitoneal) or PBS from 1 dpi for 1 week. (F) Weight loss. (G) Representative H&E lung staining at 21 dpi. Scale bar, 500 μm. Data are mean ± SD, n = 3–5 mice per group. p values were calculated by two-way ANOVA (A–C) and two-tailed unpaired Student’s t test (D and E). *p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001.

Figure 5.. Vimentin deficiency augments IL-18/IL-18R/Areg signaling in Tregs

(A) Flow cytometry contour plots and quantification of IL-18R⁺ Treg frequency among lung Tregs of WT and vimentin cKO mice after influenza infection. (B) IL-18R expression on lung Tregs after influenza infection (n = 4–5 mice per group). (C) IL-18R⁺ Treg number in whole lung after influenza infection. (D) BALF Areg levels measured by ELISA (n = 3–8 mice per group). (E) Areg expression in lung Treg by qPCR at 10 dpi. (F) CD4⁺CD25⁺ lung Tregs cultured with IL-2, CD3/CD28 beads, and 100 ng/mL IL-18 for 4 days, with Areg levels assessed in supernatant by ELISA. (G–I) WT and vimentin cKO mice infected with IAV; vimentin cKO mice received anti-Areg (50 μg/mouse, intraperitoneal) at 5 dpi, 4 times in 3 day intervals, or vehicle control. Lung histology at 21 dpi. Scale bar, 500 μm. (J and K) Representative contour plots (J) and Ki67⁺IL-18R⁺ Treg frequency (K) among lung Tregs after influenza infection. Data are mean ± SD, n = 3–5 mice per group. p values were calculated by two-way ANOVA (A–F and K) and two-way ANOVA (I). *p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001.

Figure 6.. Vimentin deficiency in Tregs leads to accumulation and persistence of Helios⁺IL-18R⁺ Tregs in the lung after influenza infection

(A) Flow cytometry contour plots and quantification of Helios⁺ Treg frequency among lung Tregs in WT and vimentin cKO mice after influenza infection. (B) Helios expression on lung Tregs of WT and vimentin cKO after influenza infection. (C) Helios⁺ Treg number in whole lung after influenza infection. (D and G) Heatmap of differentially expressed genes (p < 0.05). (E, F, H, I, and N) Normalized expression of Nt5e, Entpd1, Il18r1, Areg, and Prkcq in Helios⁺ and Helios⁻ Tregs. (J) Frequency of Helios⁺IL-18R⁺, Helios⁺IL-18R⁻, and Helios⁻IL-18R⁺ cells among lung Tregs after influenza infection. (K and L) Frequency and absolute number of Helios⁺IL-18R⁺ Tregs in the whole lung tissue after influenza infection. (M) Relative mRNA expression of Il18r1, Areg, and Vim in WT and Vim-deficient splenic nTregs. Data are mean ± SD, n = 4–5 mice/group. p values were calculated by two-way ANOVA (A–C and K–M) and two-tailed unpaired Student’s t test (E, F, H, I, and N). *p < 0.05, ***p < 0.0005, and ****p < 0.0001.

Figure 7.. Vimentin spatially sequesters IL-18R at the DPC of Tregs

(A and B) Confocal images of vimentin and CD43 or ezrin in WT iTregs treated with DMSO (vehicle control) or WFA and Vim^−/− iTregs. Scale bar, 5 μm. (C and D) Confocal images showing uniform IL-18R and CD73 distribution on vimentin-deficient nTregs. Scale bar, 5 μm. (E) Confocal images of vimentin and IL-18R in WT nTregs treated with vehicle control or 1 μM WFA for 24 h. Scale bar, 5 μm. (F) Vimentin expression in WT iTregs treated with 1 μM WFA or vehicle control for 24 h. Scale bar, 5 μm. (G) Carboxyfluorescein succinimidyl ester (CFSE) dilution in CD4⁺CD25⁻ Tcon cells co-cultured with WT iTregs treated with WFA or vehicle control (n = 3). (H) CD4⁺CD25⁺ lung Tregs from WT mice were cultured with IL-2, CD3/CD28 beads, and 100 ng/mL IL-18 for 3 days, followed by 1 μM WFA treatment for 24 h. Supernatants were collected for Areg measurement by ELISA (n = 6–7 per group). Data are presented as mean ± SD. p values were calculated by two-way ANOVA (G) and two-tailed unpaired Student’s t test (H). **p < 0.005, ***p < 0.0005, and ****p < 0.0001.