Context-specific anti-inflammatory roles of type III interferon signaling in the lung in nonviral injuries

Abstract

Type III interferons (λ1, λ2, and λ3) are potent antiviral cytokines in the lung. However, their roles in nonviral lung injuries are less well understood. This study investigates the activation of type III interferon signaling in three distinct models of lung injuries caused by diverse stimuli: the bacterial pathogen Pseudomonas aeruginosa, bacterial endotoxin LPS, and the chemotherapeutic agent bleomycin. Our data show that, despite inducing a potent inflammatory response, Pseudomonas and LPS did not increase IFNλ secretion. In contrast, bleomycin instillation increased secretion of IFNλ in the airways at both early and late time points. Consistent with limited secretion, type III interferon signaling had a minimal role in the host response to both Pseudomonas and LPS, as measured by pathogen burden, inflammatory response, and lung injury. Conversely, a deficiency in type III interferon signaling led to increased inflammatory signaling and elevated acute lung injury in the bleomycin model on day 3. This elevated early injury resulted in impaired recovery in IFNLR1 knockout mice, as evidenced by their recovery from bleomycin-induced weight loss. Taken together, these data suggest a context-specific activation of type III interferon signaling, where it plays an anti-inflammatory role in the lung.

Keywords: bleomycin; fibrosis; lung inflammation; lung injury; type III interferons.

FIGURE 1

Stimuli specific elevation of type III interferons in the lung: BAL samples were obtained from wild type (C57/B6) and co‐housed age‐ and sex‐matched IFNLR1 KO mice that were either intratracheally infected with Pseudomonas aeruginosa (12 h) or administered with LPS (12 h) or bleomycin (3 or 14 days). BAL samples from mock (PBS) administered mice were obtained as controls. Type III interferon levels were measured by ELISA assays. Data are obtained from two independent experiments performed for each stimulus. Statistically significant p values are indicated using two‐way ANOVA and Sidak's multiple comparisons test. N = 4 wild type, 3 IFNLR1 KO for PBS, N = 9 wild type, 8 IFNLR1KO for bleomycin 3 days, N = 9 wild type, 12 IFNLR1KO for bleomycin 14 days, N = 10 wild type, 8 IFNLR1KO for Pseudomonas, N = 9 wild type, 8 IFNLR1KO for LPS. The data are plotted as box that shows 25th and 75th percentile and the middle line indicating median and whiskers that show spread of the data. This is consistent throughout all the figures.

FIGURE 2

A minimal role of type III interferon signaling in Pseudomonas aeruginosa infection: Wild type and IFNLR1 KO mice were infected with 2.5 × 10⁶ CFUs of PA01 strain of Pseudomonas aeruginosa (PA) for 12 h. Serial dilutions of BAL samples (a) and homogenized left lung (b) were plated on minimal agar to enumerate the bacterial numbers. WBCs were counted using Coulter counter (c). ELISA assays were performed on the BAL samples of Pseudomonas or mock infected mice using Duoset ELISA kits for KC and IL‐1β (d and e). Total protein levels were measured by BCA assay (f). Red blood cells were counted by Coulter counter (g). Hereafter, in all figures, wild type mice are indicated by solid circles in red boxes and IFNLR1 KO mice are indicated in solid squares in blue boxes. Data are pooled from three independent experiments (N = 15 wild type and 13 IFNLR1 KO) except for total protein (f) and cytokines, which are from pooled from two independent experiments (N = 10 wild type and 8 INFLR1 KO). p values are indicated using unpaired t‐test or two‐way ANOVA and Sidak's multiple comparisons test. For baseline samples, N = 3 for each genotype in (c) and (g), N = 4 (e), or 5 (f) wild type and 3 INFLR1 KO for PBS samples. p values are indicated on top of the graph.

FIGURE 3

Dispensable role of type III interferon signaling in LPS model of lung injury: BAL samples were obtained from wild type and IFNLR1 KO mice that were intratracheally administered 5 μg of E. coli LPS for 12 h. Levels of total WBC were measured in the BAL (a) and the level of cytokines were measured in cell free BAL for KC and IL‐1β (b and c). Total protein content in the cell free BAL (d) and RBCs were measured (e). N = 18 wild type and 15 IFNLR1 KO mice for (a) and (e) pooled from four different experiments, N = 9 wild type and 8 IFNLR1 KO mice pooled from two independent experiments for (b), (c), and (d). p values are indicating on the graph using student's t‐test.

FIGURE 4

Anti‐inflammatory role of type III interferon signaling in bleomycin‐induced acute lung injury: Wild type and IFNLR1 KO mice were intratracheally administered 1.5 U/kg bleomycin by intratracheal route and euthanized on day 3 post infection. WBC counts (a), macrophages (b), and neutrophils (c), cytokines KC (d), IL‐1β (e), IL‐I (f), and TNFα (g). Total protein content (h) and red blood cell counts (i) in the BAL as markers of lung injury. N = 9 wild type and 8 IFNLR1 KO mice for all the figures from two independent experiments except for (b) and (c) where n = 4 wild type and 5 INFLR1 KO mice. Data are analyzed using t‐test and p values are indicated on top of the graph.

FIGURE 5

Role of type III interferon signaling in bleomycin‐induced chronic lung injury, fibrosis, and recovery: Wild type and IFNLR1 KO mice were administered 1.5 U/kg bleomycin or PBS by intratracheal route and followed up to 14 days with their body weight measured every other day (a). WBC (b), levels of KC (c), IL‐1β (d), IL‐6 (e), and TNFα (f) were measured to estimate inflammatory responses. Total protein in the BAL (g) and levels of RBCs in the BAL (h) were measured for lung injury. Collagen levels were measured on day 14 post infection in the left lung (i) and by trichrome staining of lung tissue (j). Data are pooled from at least two experiments for each measurement except collagen measurement, which was performed on one of the representative experiments. p values are indicated on the top of the graph using two‐way ANOVA with Tukey's multiple comparisons for (a), student's t‐test for (b)–(h), and two‐way ANOVA with Sidak's multiple comparison test for (i). For figure (a), N = 5 in PBS group for both genotypes, N = 16 wild type and 13 IFNLR1 KO mice from three independent experiments. N = 20 wild type and 21 IFNL1 KO mice from four independent experiments for (b) and (h). Cytokines were measured in two independent experiments N = 9 wild type and 11 IFNLR1 KO mice as shown in (c)–(f). N = 14 wild type and 12 INFLR1 KO from three independent experiments for (g). N = 4 wild type and 3 IFNLR1 KO mice for PBS and N = 6 wild type and 6 IFNLR1 KO each for bleomycin group in (i).