ISM1 suppresses LPS-induced acute lung injury and post-injury lung fibrosis in mice

Abstract

Background: Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are clinical syndromes characterized by acute lung inflammation, pulmonary edema and hypoxemia, with up to 50% mortality rate without effective pharmacological therapy. Following the acute inflammation, repair and remodeling occurs which in some cases resulting in lung fibrosis. The pathophysiology of ALI/ARDS remains incompletely understood. Lipopolysaccharide (LPS)-induced ALI in mice have been widely used as a model to study human ALI/ARDS. Isthmin 1 (ISM1) is a secreted protein highly abundant in mouse lung. We have previously reported that upon intratracheal LPS instillation, ISM1 expression in the lung is further upregulated. Recently, we also reported that ISM1 is an anti-inflammatory protein in the lung with Ism1^-/- mice presenting spontaneous chronic low-grade lung inflammation and obvious emphysema at young adult stage. However, what role ISM1 plays in ALI/ARDS and lung fibrosis remain unclear.

Methods: Using Ism1^-/- mice and intratracheal LPS-induced ALI, and local delivery of recombinant ISM1 (rISM1), we investigated the role ISM1 plays in ALI and post-ALI lung fibrosis using flow cytometry, Western blot, antibody array, immunohistochemistry (IHC), immunofluorescent and other histological staining.

Results: We reveal that ISM1 deficiency in mice led to an intensified acute lung inflammation upon intratracheal LPS challenge, with a heightened leukocyte infiltration including neutrophils and monocyte-derived alveolar macrophages, as well as upregulation of multiple pro-inflammatory cytokines/chemokines including tumor necrosis factor α (TNF-α). Although innate immune cells largely subsided to the baseline by day 7 post-LPS challenge in both wild-type and Ism1^-/- mice, Ism1^-/- lung showed increased post-ALI fibrosis from day 9 post-LPS treatment with increased myofibroblasts, excessive collagen accumulation and TGF-β upregulation. The heightened lung fibrosis remained on day 28 post-LPS. Moreover, intranasal delivered recombinant ISM1 (rISM1) effectively suppressed LPS-induced acute lung inflammation and ALI, and rISM1 suppressed LPS-induced NF-κB activation in cultured mouse alveolar macrophages.

Conclusion: Together with our previous report, this work further established ISM1 as an endogenous anti-inflammation protein in the lung, restraining excessive host inflammatory response to LPS-triggered ALI and suppressing post-ALI lung fibrosis likely through suppressing NF-κB activation and pro-inflammatory cytokine/chemokine production.

Keywords: Acute lung injury; ISM1; Inflammation; LPS; Pulmonary fibrosis.

Fig. 1

ISM1 deficiency leads to increased leukocyte infiltration in the lung under ambient condition. a Increased immune cell infiltration in the lungs of Ism1^−/− mice at 2 months via H&E staining. b Increased macrophages and neutrophils in the lungs of Ism1^−/− mice at 2 months detected by IHC staining for CD68 and NIMP-R14, respectively. c–e Differential immune cell count showed increased total leukocytes, alveolar macrophages, and neutrophils in Ism1^−/− lungs. f Analysis of the peripheral blood of Ism1^−/− mice showed increased total white blood cells, neutrophils and lymphocytes at 2 months. K/µl count per µl, WBC white blood cell, NE neutrophil, LY lymphocyte, MO monocyte, EO eosinophil, BA basophil. *represents p < 0.05; **represents p < 0.01. For a-e, n = 4 mice per group; for f, n = 7 mice per group

Fig. 2

ISM1 deficiency leads to heightened immune responses to LPS in mouse lung. a–e Time-course of lung immune response to LPS challenge. Ism1^−/− lungs showed heightened immune responses to LPS via differential cell count by flow cytometry. LPS (2 mg/kg) was intratracheally administered once, followed by isolation of single-cell suspension from the whole lungs at day 1, 3, 5 and 7. f LPS induced higher pulmonary permeability in Ism1^−/− mice at 24 h post LPS challenge indicated by total BALF protein content. g Representative images of lung tissue sections displaying the extent of the acute inflammation in H&E stained (left) and IF stained NIMP-R14⁺ neutrophils (right) at 24 h post LPS challenge. *represents p < 0.05; **represents p < 0.01. For (a–e and g), n = 3 mice per group; for (f), n = 6 for WT and n = 5 for Ism1^−/− mice

Fig. 3

Increase of AMs in Ism1^−/− mouse under LPS is mainly due to increase of Mo-AMs. BALF of 8–9 weeks old WT and Ism1^−/− mice was collected at day 3 and day 5 after intratracheal administration of 2 mg/kg LPS (n = 4–5). After BALF collection, cells were isolated, erythrocytes were lysed, and stained with Viobility fixable dye for live/dead cells, various marker antibodies and Annexin-V and analyzed by flow cytometry. Total cells from BALF were presented in panel a-d. The flow cytometry gating strategy is shown in Additional file 1: Fig. S5. a Quantification of the amount of TR-AM and Mo-AM. b Percentage of early apoptotic Mo-AM as identified as Annexin-V⁺ in Mo-AM population. c Percentage of early apoptotic TR-AM as identified as Annexin-V⁺ cells in TR-AM population. d Percentage of early apoptotic neutrophils as identified as Annexin-V⁺ cells in neutrophil population. e Representative histograms showing the percentage of Annexin-V⁺ cells in Mo-AM, RT-AM, and neutrophil population on day 5 post LPS. Statistical analysis was conducted by Student's t-test. “*” represents P < 0.05; “***” represents P < 0.001

Fig. 4

Intranasal rISM1 reduces LPS-induced inflammatory responses in mouse lung. a A schematic diagram of rISM1 treatment regimen in intratracheal LPS-treated mice. Each mouse was pre-treated with 50 µg rISM1 via intranasal delivery one day before receiving one dose of 2 mg/kg LPS. The mouse was continuously treated with 50 µg rISM1 daily till day 3. BALF was isolated and separated into the BAL fluid protein and cell components. b BALF protein levels were reduced in rISM1-treated mice at 24 h post LPS challenge. Proteins were measured by Bradford protein dye. c–g Differential immune cell quantification of BALF cells using flow cytometry. Total cells in BALF are presented. rISM1 suppressed immune cell infiltration induced by LPS in the lung. *represents p < 0.05; ** represents p < 0.01. For b–g, n = 5 mice for LPS-treated groups; n = 3 for non-LPS-treated groups

Fig. 5

ISM1 deficiency enhances lung fibrosis post LPS-triggered ALI. a Representative images of H & E stained lung tissue sections showing the extent of fibrosis in wild-type (WT) and Ism1^−/− mice at day 9 post LPS challenge. b, c Collagen deposition, detected by Picro-sirius staining, was higher in the lungs of Ism1^−/− mice as compared to those of WT mice. d, e Increased presence of contractile myofibroblasts within fibrotic foci (detected by α-SMA staining) in Ism1^−/− lungs as compared with those of WT. ** represents p < 0.01. n = 3 mice per group, 2 sections per lung, 5 microscopic fields per section were analyzed

Fig. 6

ISM1 deficiency increases proliferation of alveolar epithelial type 2 cells (AT2) and elevates profibrotic TGF-β in mouse lung. a Representative images of SP-C and PCNA double IF stained lung sections of Ism1^−/− and WT mice at day 9 post LPS challenge. b Quantification of the percentage of SP-C⁺PCNA⁺ cells in the lung of Ism1^−/− and WT mice at day 9 post LPS challenge. Increased number of proliferating AT2 cells in the lungs of Ism1^−/− mice were shown. *** represents p < 0.001. n = 3 mice per group, 2 sections per lung, 5 microscopic fields per section were analyzed. c Representative images of IF stained TGF-β lung sections of Ism1^−/− and WT mice at day 9 post LPS challenge. n = 3 animals per group, 2 sections per lung, 5 microscopic fields per section were analyzed. d, e TGF-β protein level in the lung of Ism1^−/− and WT mice at day 9 post LPS challenge by Western blot of whole lung lysate d and quantification (e). ** represents p < 0.01. n = 3 mice per group, 1 total lung lysate per mouse

Fig. 7

ISM1 deficiency increased proinflammatory cytokine/chemokine production in lung and promoted LPS-induced NF-κB activation. a Lung cytokine/chemokine profile determined by an inflammatory cytokine antibody array using lung homogenates at 24 h post LPS challenge. * represents p < 0.05. n = 4 mice per group, 1 total lung lysate per mouse. b, c Increased levels of TNF-α in Ism1^−/− lung 24 h post LPS challenge shown by Western blot of whole lung lysate. ** represents p < 0.01. n = 3 mice per group, 1 total lung lysate per mouse. d, e IF staining showing increased p65 NF-κB nuclear translocation in Ism1^−/− lung. Representative images showing increased signal co-localization of the p65 NF-κB (red) and the DAPI nucleus (blue) in the lung sections of Ism1^−/− mice d and the quantification of nuclear p65 NF-κB in panel d, showing the percentage of cells with nuclear localized p65 in WT and Ism1^−/− lung (e). n = 5 mice per group, 2 sections per lung, 5 microscopic fields per section. f rISM1 dose-dependently suppresses LPS-induced NF-κB p65 phosphorylation at S276 and S536 in cultured mouse MH-S alveolar macrophage cells. Cells are harvested at 30 min post LPS treatment (100 ng/ml). Based on two independent experiments