Pulmonary IFN-γ Causes Lymphocytic Inflammation and Cough Hypersensitivity by Increasing the Number of IFN-γ-Secreting T Lymphocytes

Abstract

Purpose: Respiratory viral infection increases the number of lung-resident T lymphocytes, which enhance cough sensitivity by producing interferon-γ (IFN-γ). It is poorly understood why IFN-γ-secreting T lymphocytes persist for a long time when the respiratory viruses have been removed.

Methods: Repeated pulmonary administration of IFN-γ and intraperitoneal injection with different inhibitors were used to study the effects of pulmonary IFN-γ in mice and guinea pigs.

Results: IFN-γ administration caused the increasing of IFN-γ-secreting T lymphocytes in both lung and blood, followed by the elevated physiological level of IFN-γ in the lung, the airway inflammation and the airway epithelial damage. IFN-γ administration also enhanced the cough sensitivity of guinea pigs. IFN-γ activated the STAT1 and extracellular signal-regulated kinase (ERK) pathways in lung tissues, released IFN-γ-inducible protein 10 (IP-10), and resulted in F-actin accumulation in lung-resident lymphocytes. The CXC chemokine receptor 3 (CXCR3) inhibitor potently suppressed all the IFN-γ-induced inflammatory changes. The STAT1 inhibitor mitigated IFN-γ-secreting T lymphocytes infiltration by inhibiting T lymphocytes proliferation. F-actin accumulation and the ERK1/2 pathway contributed to pulmonary IFN-γ-induced augmentation of the airway inflammation and increasing of IFN-γ-secreting T lymphocytes in blood.

Conclusions: High physiological levels of IFN-γ in the lung may cause pulmonary lymphocytic inflammation and cough hypersensitivity by increasing the number of IFN-γ-secreting T lymphocytes through the IP-10 and CXCR3 pathways.

Keywords: CXC chemokine receptor 3; Interferon-gamma; T lymphocytes; cough.

Fig. 1. (A) The experimental protocol for the mice that were treated with IFN-γ and different inhibitors. All the mice were sacrificed 2 days after the last treatment. (B) Effects of IFN-γ and different inhibitors on the total protein concentration in the supernatant of BALF (n = 5 per group). (C) Effects of IFN-γ and different inhibitors on the uric acid concentration in the supernatant of BALF (n = 5 per group). (D-E) Representative figures of pathological changes in hematoxylin and eosin-stained lung sections from the group of “Control” (D) and “IFN-γ model” (E). The symbol of “↑” marks the airway epithelial damage. Scale bars are 20 µm for the electron micrographs. Mice in different groups were treated as follows: the control group (i.n. with PBS + i.p. with the vehicle solution), IFN-γ model group (i.n. with 50 µg/kg IFN-γ + i.p. with the vehicle solution), JAK1 inhibitor group (i.n. with 50 µg/kg IFN-γ + i.p. with 1.5 mg/kg Filgotinib), STAT1 inhibitor group (i.n. with 50 µg/kg IFN-γ + i.p. with 15 mg/kg Fludarabine), CXCR3 inhibitor group (i.n. with 50 µg/kg IFN-γ + i.p. with 5 mg/kg AMG487), Actin polymerization inhibitor group (i.n. with 50 µg/kg IFN-γ + i.p. with 0.75 mg/kg Cytochalasin D), and ERK1/2 inhibitor group (i.n. with 50 µg/kg IFN-γ + i.p. with 5 mg/kg Ravoxertinib). Data are shown as mean ± SD.

i.n., intranasal; PBS, phosphate-buffered saline; IFN-γ, interferon-γ; Sac., sacrifice; BALF, bronchoalveolar lavage fluid; CXCR3, CXC chemokine receptor 3; ERK, extracellular signal-regulated kinase; i.p., intraperitoneal. ^*P < 0.05, compared with the “Control”; ^†P < 0.05, compared with the “IFN-γ model.”

Fig. 2. Effects of IFN-γ and different inhibitors on the proportion of lymphocytes in BALF and White Blood Cells, and the F-actin level in lung-resident lymphocytes. (A) Summarized data showing the proportion of lymphocytes in BALF are representative of three independent experiments. Mice in different groups were treated as follows: the control group (Intranasally instilled with PBS, n = 9), low IFN-γ group (intranasally instilled with 3.125 µg/kg IFN-γ, n = 9), medium IFN-γ group (intranasally instilled with 12.5 µg/kg IFN-γ, n = 10) and high IFN-γ group (intranasally instilled with 50 µg/kg IFN-γ, n = 10). (B) Group data showing the effects of IFN-γ instillation and different inhibitors on the proportion of lymphocytes in BALF (n = 5 per group). (C) Group data showing the effects of IFN-γ instillation and different inhibitors on the proportion of lymphocytes in White Blood Cells (n = 5 per group). (D) Summarized data representing the fluorescence value of F-actins in lung-resident lymphocytes (n = 5 per group). Mice in different groups were treated as the same as that in Fig. 1. Data are shown as mean ± SD.

BALF, bronchoalveolar lavage fluid; IFN-γ, interferon-γ; CXCR3, CXC chemokine receptor 3; ERK, extracellular signal-regulated kinase. ^*P < 0.05, compared with the “Control”; ^†P < 0.05, compared with the “IFN-γ model.”

Fig. 3. Effects of IFN-γ and different inhibitors on lung-resident T lymphocytes and blood T lymphocytes. (A) Summarized data showing the proportion of CXCR3⁺CD8⁺ T lymphocytes in lung-resident T lymphocytes from the “Control” group (n = 9) and the “IFN-γ model” group (n = 10). (B) Summarized data showing the proportion of IFN-γ⁺ T lymphocytes in lung-resident T lymphocytes (n = 5 per group). (C) Summarized data showing the proportion of CD8⁺ T lymphocytes in blood T lymphocytes from the “Control” group (n = 9) and the “IFN-γ model” group (n = 10). (D) Summarized data showing the proportion of CXCR3⁺CD8⁺ T lymphocytes in blood T lymphocytes from the “Control” group (n = 9) and the “IFN-γ model” group (n = 10). (E) Summarized data showing the proportion of IFN-γ⁺ T lymphocytes in blood T lymphocytes (n = 5 per group). (F) Summarized data showing the proportion of CD4⁺IFN-γ⁺ T lymphocytes in blood T lymphocytes (n = 5 per group). Mice in different groups were treated as the same as that in Fig. 1. Data are shown as mean ± SD.

CXCR3, CXC chemokine receptor 3; IFN-γ, interferon-γ; ERK, extracellular signal-regulated kinase. ^*P < 0.05, compared with the “Control”; ^†P < 0.05, compared with the “IFN-γ model.”

Fig. 4. Effects of IFN-γ and different inhibitors on the fluorescence intensity of the Phospho-JAK1 level. (A-G) Representative lung tissues stained with anti-phospho-JAK1 (magenta) and Hoechst 33342 (blue) and imaged by the fluorescence microscope. Scale bar = 50 µm. (H) Summarized data showing the fluorescence intensity of the Phospho-JAK1 level in different groups. The images are representatives of 3 independent experiments. Mice in different groups were treated as the same as that in Fig. 1. Data are shown as mean ± SD. n = 5 per group.

IFN-γ, interferon-γ; CXC chemokine receptor 3; ERK, extracellular signal-regulated kinase. ^*P < 0.05, compared with the “Control”; ^†P < 0.05, compared with the “IFN-γ model.”

Fig. 5. (A-C) Effects of IFN-γ on the fluorescence intensity of the Phospho-Stat1 level. Lung tissues stained with anti-phospho-STAT1 (green) and Hoechst 33342 (blue) and imaged by a fluorescence microscope. Scale bar = 50 µm. n = 5 per group. (D-E) Effects of IFN-γ and different inhibitors on the expression of phospho-JAK1, phospho-STAT1 and phospho-ERK1/2 as indicated by the western blot. There are representative blots of phospho-JAK1, phospho-STAT1, phospho-ERK1/2 and GAPDH in lung tissues from seven different groups (D). Densitometric analyses are presented as the relative ratio of the targeted protein to GAPDH (E). The images are representatives of 3 independent experiments. n = 5 per group. Mice in different groups were treated as the same as that in Fig. 1. Data are shown as mean ± SD.

IFN-γ, interferon-γ; CXC chemokine receptor 3; ERK, extracellular signal-regulated kinase. ^*P < 0.05, compared with the “Control”; ^†P < 0.05, compared with the “IFN-γ model.”

Fig. 6. (A) Summarized data showing the ratio of IP-10 concentrations to the total protein concentrations in homogenized lung tissues are representative of three independent experiments. Mice in different groups were treated as follows: the control group (Intranasally instilled with PBS, n = 10), low IFN-γ group (Intranasally instilled with 3.125 µg/kg IFN-γ, n = 9), medium IFN-γ group (Intranasally instilled with 12.5 µg/kg IFN-γ, n = 7) and high IFN-γ group (Intranasally instilled with 50 µg/kg IFN-γ, n = 10). (B-C) Group data showing the effects of IFN-γ instillation and different inhibitors on the ratio of IP-10 concentrations (B) and the IFN-γ concentrations (C) to the total protein concentrations in homogenized lung tissues (n = 5 per group). (D-E) Group data showing the effects of IFN-γ instillation on the IP-10 concentrations (D) and the IL-10 concentrations (E) in the supernatant of BALF (n = 10 per group). Results shown are representative of three independent experiments. Mice in different groups were treated as the same as that in Fig. 1. Data are shown as mean ± SD.

IFN-γ, interferon-γ; CXC chemokine receptor 3; ERK, extracellular signal-regulated kinase. ^*P < 0.05, compared with the “Control”; ^†P < 0.05, compared with the “IFN-γ model.”

Fig. 7. IFN-γ instillation enhanced the cough sensitivity and the total number of leukocytes in BALF of guinea pigs. It shows the effects of IFN-γ instillation on the number of cough events (A) and time to the first cough (B) within 10 minutes of exposing the animals to aerosolised citric acid (0.3 M). (C) Group data showing the effects of IFN-γ instillation on the total number of leukocytes in BALF. Guinea pigs in the control group (n = 8) or the IFN-γ group (n = 7) were intratracheally instilled with PBS or 12.5 µg/kg recombinant guinea pig IFN-γ once a day for 7 consecutive days. Results shown (mean ± SD) are representative of three independent experiments.

IFN-γ, interferon-γ; BALF, bronchoalveolar lavage fluid; PBS, phosphate-buffered saline. ^*P < 0.05, compared with the “Control.”