Early RSV infection aggravates asthma-related Th2 responses by increasing the number of CD4 + TRM cells through upregulation of PLZF

Abstract

Respiratory syncytial virus (RSV) infection is correlated with the chronic pathogenesis and exacerbation of asthma. However, the mechanism remains unclear. In this study, acute and memory (Mem) asthma models with early RSV infection are established to explore the persistence of the effects of RSV infection on asthma. Intravascular injection of an anti-CD45 antibody is performed to define CD4 ⁺ TRM cells accurately. RSV infection has a sustained impact on asthma exacerbation for at least six weeks, with high Th2 cytokine secretion in lung tissue instead of IgE response-related B cells. CD45 ^-CD4 ⁺ TRM cells are positively correlated with RSV-related asthma exacerbation and severe airway inflammation. Mechanistically, overexpression of the transcription factor PLZF in vitro increases the number of CD4 ⁺ TRM cells, and conditional knockout of Zbtb16 (encoding PLZF) can decrease the number of CD4 ⁺ TRM cells to aggravate allergic inflammation and reduce Th2 responses. This study provides evidence for potential combined strategies that might benefit asthma patients.

Keywords: PLZF; asthma; respiratory syncytial virus infection; tissue-resident memory T cell.

Figure 1

Early RSV infection aggravates allergic lung inflammation and elevates Th2 cytokine levels (A) Establishment of the RSV + asthma and asthma model. (B) Representative microphotographs of lung histological sections from control, RSV + asthma and asthma mice at objective magnifications of 1× and 29×. (C) The gene expression levels of Il4,Il5 and Il13 in the whole lung were measured by real-time qPCR. (D) Plasma IgE levels were detected by ELISA. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant (unpaired two-tailed Student’s t test). The results are from one representative experiment of at least three independent experiments in (C,D) (n = 3–6 in each group).

Figure 2

The aggravation of asthma caused by early RSV infection is maintained for at least six weeks (A) Establishment of the RSV-infected asthma-Mem and asthma-Mem models. (B) Representative microphotographs of histological lung sections from control-Mem, RSV-infected asthma-Mem and asthma-Mem mice at objective magnifications of 1× and 29×. (C) Gene expression levels of Il4,Il5 and Il13 in the whole lung were measured by real-time qPCR. (D) Plasma IgE levels were detected by ELISA. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant (unpaired two-tailed Student’s t test). The results are from one representative experiment of at least three independent experiments in (C,D) (n = 3–6 in each group).

Figure 3

CD4 ⁺CD44 ⁺ T cells are greater in the RSV-infected asthma group than in the asthma group (A) Flow cytometric plots of CD4+CD44+ T cells gated from live CD4+ T cells in the lungs of control, asthma, and RSV + asthmatic mice. (B,C) Comparison of the proportions of CD4+CD44+ T cells among live cells in the lungs and spleens of control, asthma, and RSV + asthma mice detected by flow cytometry. (D) Flow cytometric plots of CD4+CD44+ T cells gated from live CD4+ T cells in the lungs of control-Mem, asthma-Mem, and RSV + asthma-Mem mice. (E,F) Comparison of the proportions of CD4+CD44+ T cells among live cells in the lungs and spleens of control-Mem, asthma-Mem, and RSV+asthma-Mem mice detected by flow cytometry. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant (unpaired two-tailed Student’s t test). The results are from one representative experiment of at least three independent experiments in(B,C,E,F) (n = 3–5 in each group).

Figure 4

Early RSV-infected asthma induces persistently high expression of CD4+ TRMs (A) Flow cytometric plots of CD4+ TCMs (CD62L+CD69–), CD4+ TEM (CD62L–CD69–) cells and CD4+ TRM (CD62L+CD69+) cells gated from live CD4+CD44+ T cells in the lungs of control, asthma, and RSV + asthma mice. (B) Comparison of CD4+ TCMs, CD4+ TEMs and CD4+ TRMs in the lungs of control, asthma, and RSV+asthmatic mice. (C) Flow cytometric plots of CD4+ TCMs (CD62L+CD69–), CD4+ TEM (CD62L–CD69–) cells and CD4+ TRM (CD62L+CD69+) cells gated from live CD4+CD44+ T cells in the lungs of control-Mem, asthma-Mem, and RSV + asthma-Mem mice. (D) Comparison of CD4+ TCMs, CD4+ TEMs and CD4+ TRMs in the lungs of control-Mem, asthma-Mem, and RSV + asthma-Mem mice. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant (unpaired two-tailed Student’s t test). The results are from one representative experiment of at least three independent experiments in (B,D) (n = 3-6 in each group).

Figure 5

CD45 ^-CD4 ⁺ TRMs dominantly contributes to the upregulation of CD4 ⁺CD44 ⁺ memory T cells (A,B) General panel logic of CD45+ and CD45– cells detected by flow cytometry. (C,D) Comparison of CD45–CD4+ TRMs in the lungs of control, asthma, and RSV+asthma mice. (E,F) Comparison of CD45–CD4+ TRMs in the lungs of control-Mem, asthma-Mem, and RSV + asthma-Mem mice. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant (unpaired two-tailed Student’s t test). The results are from one representative experiment of at least three independent experiments in (D,F) (n = 3–6 in each group).

Figure 6

Overexpression of Zbtb16 in vitro increases the number of CD4 ⁺ TRMs (A) Comparison of pulmonary PLZF+CD4+ TRMs in the control, asthma and RSV-infected asthma groups. (B) Comparison of pulmonary PLZF+CD4+ TRMs in the lungs of Control-Mem, asthma-Mem and RSV-infected asthma-Mem mice. (C) Workflow of Zbtb16 overexpression and OVA-Beas2b stimulation of the splenic lymphocytes of BALB/c mice. (D) Validation of the overexpression of Zbtb16 (encoding PLZF) via real-time qPCR and flow cytometry. (E) Changes in CD4+CD44+ T cells among live cells stimulated with OVA-Beas2b. (F,G) Comparison of CD4+ TCM-like, CD4+ TEM-like and CD4+ TRM-like cells among live cells stimulated with OVA-Beas2b. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant (unpaired two-tailed Student’s t test). The results are from one representative experiment of at least three independent experiments in (A,B,D,E–G) (n = 3–6 in each group).

Figure 7

Conditional knockout of Zbtb16 in CD4 ⁺ T cells in mice alleviates allergic lung inflammation aggravated by early RSV infection, accompanied by decreased Th2 responses (A) Establishment of RSV-infected asthma models in wild-type (WT) and cKO mice. (B) The knockout of Zbtb16 in CD4+ T cells in cKO mice was validated by flow cytometry. (C) Representative microphotographs of histological lung sections from cKO and WT RSV + asthmatic mice at objective magnifications of 1×, 20× and 40×. (D) BALF of cKO and WT RSV + asthmatic mice at objective magnification 10×. (E) Comparison of Th2-related cytokines and transcription factors in the lungs of WT and cKO mice. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant (unpaired two-tailed Student’s t test). The results are from one representative experiment of at least three independent experiments in (E) (n = 3–4 in each group).

Figure 8

Comparison of pulmonary CD45 ^-CD4 ⁺ TRM in wild-type and cKO mice (A,B) The comparison of proportion of CD4+CD44+ T cells and subtype memory T cells in live cells detected by flow cytometry. (C,D) The comparison of proportion of CD4+ TCM, CD4+ TEM and CD4+ TRMs in CD4+CD44+ T cells detected by flow cytometry. (E) General panel logic of CD45–CD4+ TRMs detected by flow cytometry. (F) The comparison of proportion of CD45–CD4+ TRMs in lungs of cKO and WT RSV + asthma mice. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant (unpaired two-tailed Student’s t test). The results are from one representative experiment of at least three independent experiments in (B,D,F) (n = 3–5 in each group).