Neutrophil-specific Shp1 loss results in lethal pulmonary hemorrhage in mouse models of acute lung injury

Abstract

Acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality, and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain-containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1-KO mice, suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (SC43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

Keywords: Bacterial infections; Immunology; Neutrophils; Phosphoprotein phosphatases; Pulmonology.

Figure 1. Shp1 deletion in neutrophils leads to severe pulmonary hemorrhage and increased inflammation after LPS-induced lung injury.

(A–D) Gross lung and bronchoalveolar lavage (BAL) findings after intratracheal LPS in (A and B) Ptpn6^fl/fl and (C and D) Ptpn6^fl/fl S100A8(Cre⁺) mice. Quantitative analysis of BAL indicates (E) alveolar hemorrhage, (F) alveolar neutrophilia, (G) increased vascular permeability, and (H) increased BAL NETs in Ptpn6^fl/fl S100A8(Cre⁺) mice compared to Ptpn6^fl/fl. (I) Decreased survival in Ptpn6^fl/fl S100A8(Cre⁺) mice after LPS. (J and K) H&E staining of lung from (J) Ptpn6^fl/fl and (K) Ptpn6^fl/fl S100A8(Cre⁺) mice showing increased inflammation and alveolar hemorrhage after LPS with the loss of Shp1 in neutrophils. (L and M) Intravital image after LPS challenge with Evans Blue (plasma stain) and Sytox Green (NET stain) in (L) Ptpn6^fl/fl and (M) Ptpn6^fl/fl S100A8(Cre⁺) indicating exacerbated vascular leak and NETs in Ptpn6^fl/fl S100A8(Cre⁺) mice. Scale bars: (A and B) 50 mm; (J and K) 500 μm; (L and M) 50 μm. P values are from unpaired 2-tailed t tests on log₁₀-transformed data (E–H) and log-rank test (I). **P < 0.01, ****P < 0.0001.

Figure 2. Intravascular neutrophil clusters and perivascular inflammation with loss of neutrophil Shp1.

(A–D) Immunofluorescence imaging of fixed lung tissue with staining for S100A8 (neutrophils), Ter119 (red blood cells), laminin, and smooth muscle actin (SMA) at 48 hours after LPS challenge from (A and C) Ptpn6^fl/f and (B and D) Ptpn6^fl/fl S100A8(Cre⁺) indicating (B) intravascular neutrophil clusters (green arrowheads) and (D) perivascular inflammation with preferential alveolar hemorrhage near pulmonary arterioles (red arrowhead). Live intravital lung imaging of (E) Ptpn6^fl/fl and Ptpn6^fl/fl S100A8(Cre⁺) with anti-Ly6G antibody (neutrophils, red) and plasma albumin labeled by Evan’s blue (cyan) with a large intravascular neutrophil cluster (yellow arrowhead) 48 hours after LPS challenge. (F) Histogram of neutrophil cluster size observed over 15 minutes of intravital imaging 48 hours after LPS challenge, indicating presence of large clusters. (G) Increase in neutrophil cluster size between Ptpn6^fl/fl and Ptpn6^fl/fl S100A8(Cre⁺) 48 hours after LPS challenge using the Mann-Whitney nonparametric test. ****P < 0.0001. Scale bars: 100 μm.

Figure 3. Shp1 deletion in neutrophils leads to a disorganized innate immune response, alveolar hemorrhage, and impaired bacterial clearance after P. aeruginosa infection.

(A) Pulmonary hemorrhage in Ptpn6^fl/fl S100A8(Cre⁺) in comparison with Ptpn6^fl/fl, with similar (B) alveolar neutrophilia and (C) alveolar protein leak but increased (D) BAL NETs in Ptpn6^fl/fl S100A8(Cre⁺). (E) Increased BAL bacteria, (F) spleen bacteria, and (G) bacteremia in Ptpn6^fl/fl S100A8(Cre⁺) with associated (H) decreased survival. H&E stained lung tissue after P. aeruginosa induced ALI from (I and J) Ptpn6^fl/fl and (K and L) Ptpn6^fl/fl S100A8(Cre⁺) mice with perivascular inflammation (black arrowhead). P values are from unpaired 2-tailed t tests on log₁₀-transformed data (A–G) and log-rank test (H). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: (I and K) 500 μm; (J and L) 50 μm.

Figure 4. Deletion of Shp1 in neutrophils produces more severe lung injury after SARS-CoV-2 (MA-10) infection.

MA-10 infection in Ptpn6^fl/fl S100A8(Cre⁺) mice produces increased (A) weight loss, (B) alveolar hemorrhage, (C) alveolar inflammation, (D) alveolar neutrophilia, (E) similar protein leak, (F) increased NE-DNA complexes, and (G) similar CitH3-DNA complexes compared with control mice. H&E stained lung tissue from (H) Ptpn6^fl/fl (control) and (I) Ptpn6^fl/fl S100A8(Cre⁺) indicating increased peribronchial inflammation. P values are from unpaired 2-tailed t tests (A), and unpaired 2-tailed t tests on log₁₀-transformed data (B–G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars: 500 μm.

Figure 5. Lung injury from neutrophil Shp1 deletion is dependent on Syk kinase signaling.

(A) Alveolar hemorrhage and (B) BAL neutrophilia is dependent on Syk kinase signaling. (C) Increased BAL neutrophil pSyk in Ptpn6^fl/fl S100A8(Cre⁺) compared with Ptpn6^fl/fl(control). (D) Reduced BAL albumin, (E) NE-DNA, and (F) CitH3-DNA complexes in Ptpn6^fl/fl Syk^fl/fl S100A8(Cre⁺) mice compared with Ptpn6^fl/fl S100A8(Cre⁺). H&E-stained lung tissue in (G and H) Ptpn6^fl/fl S100A8(Cre⁺), (I and J) Ptpn6^fl/fl Syk^fl/fl S100A8(Cre⁺) indicating perivascular inflammation is dependent on Syk kinase signaling. Log₁₀ transformed data were analyzed using 1-way ANOVA with Tukey’s test for multiple comparisons (A, B, and D–F). Log₁₀ transformed data were analyzed using Student’s t test for comparison of pSyk MFI (C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars: (G and I) 500 μm; (H and J) 50 μm.

Figure 6. Shp1 activation with SC43 reduces inflammation in LPS-induced lung injury.

(A) Reduced alveolar neutrophils and (C) CitH3-DNA complexes (NETs), with similar (B) alveolar protein leak, (D) NE-DNA complexes (NETs). Lung H&E stained tissue in (E and F) DMSO control and (G and H) SC43 treated mice indicating reduced perihilar inflammation with SC43 administration. (I and J) Dose-dependent reduction in LPS-induced ROS (superoxide release) production with SC43 versus DMSO (vehicle) control (n = 3). P values are from unpaired 2-tailed t tests on log₁₀-transformed data (A–D). **P < 0.01. Scale bars: 500 μm.