Severe acute lung injury induced by LPS plus high-volume mechanical ventilation is associated with NETs formation in the alveoli. LPS or normal saline (NS) were intratracheally instilled into C57BL/6 mice and, after 120 minutes, the animals were anesthetized and placed on mechanical ventilation (MV) for 180 minutes with the tidal volumes of 30 mL/kg, high-volume ventilation (HVV), or 10 mL/kg low-volume ventilation (LVV) (A). Arterial blood partial pressure of oxygen (PaO2) was measured at 30 and 150 minutes after starting MV (B). Absolute counts of neutrophils (C) and macrophages (D) were determined in BALF. The levels of albumin (E), IL-1β (F), IL-1⍺ (G), CXCL2 (H), MPO (I) and NE (J) in BALF collected from euthanized animals after 180 minutes of MV were determined by ELISA. Cell death in BALF was evaluated by measuring histone-DNA complexes (K). NETs formation was evaluated by the detection of MPO-DNA complex (L). ****, ***, and ** indicate p<0.0001, p<0.001, and p<0.01, respectively, determined by two-way ANOVA followed by Tukey’s multiple comparisons test; ns, non-significant; the absence of asterisks means non-significant between all the groups; **** on B indicate that the group is different from all the other groups; values are the mean ± SEM; n=7-12.

Neutrophils are required for the development of severe acute lung injury in the LPS+HVV model.

Eighteen hours before starting mechanical ventilation (MV), the anti-neutrophil monoclonal antibody (αLy6G [1A8]) or the control IgG were administered i.p. to C57BL/6 mice. Sixteen hours later, LPS was instilled i.t. in the mice and, after 120 minutes, they were anesthetized and placed on HVV for 180 minutes (A). Arterial blood partial pressure of oxygen (PaO2) was measured at 30 and 150 minutes after starting MV (B). Absolute counts of total cells (C), neutrophils (D) and macrophages (E) in BALF. The concentration of albumin (F), IL-1β (G), IL-6 (H), TNF⍺ (I), CXCL2 (J), MPO (K) and NE (L) were measured in the BALF by ELISA. Cell death and NETs formation in the BALF were evaluated by histone-DNA (M), and MPO-DNA (N) respectively. ****, ***, **, and * indicate p<0.0001, p<0.001, p<0.01, and p<0.05, respectively, determined by two-way ANOVA followed by Tukey’s multiple comparisons test or unpaired two-tailed Student’s t test; ns, non-significant; values are the mean ± SEM; n=5-12. Illustrations in the image are from BioRender.com/s52z652.

NETs contribute to the development of severe hypoxemia in the LPS-HVV-induced ALI.

Cl-amidine or vehicle were injected i.p. to C57BL/6 mice. 180 minutes later LPS was instilled i.t and, after 120 minutes, the animals were placed on mechanical ventilation (MV) for 180 minutes (A). Arterial blood partial pressure of oxygen (PaO2) was measured at 30 and 150 minutes after starting MV (B). Absolute counts of neutrophils (C) and the levels of albumin (D) and IL-1β (E) were measured in BALF. Cell death and NETs formation in BALF were evaluated by measuring histone-DNA (F), and MPO-DNA (G) respectively. LPS was instilled to neutrophil specific PAD4 deficient (Padi4Δ/Δ S100A8cre) or the controls (Padi4fl/fl) mice, which were then placed on MV (H). PaO2 was measured at 30 and 150 minutes after starting MV (I). Neutrophils (J), albumin (K), IL-1β (L), histone-DNA (M) and MPO-DNA (N) were measured in BALF. LPS and DNase I were instilled i.t. to C57BL/6 mice, mice were placed on MV (O). PaO2 was measured at 30 and 150 minutes after starting MV (P). Neutrophils (Q), albumin (R), IL-1β (S), histone-DNA (T) and MPO-DNA (U) were measured in BALF. ****, ***, **, and * indicate p<0.0001, p<0.001, p<0.01, and p<0.05, respectively, determined by two-way ANOVA followed by Tukey’s multiple comparisons test or unpaired two-tailed Student’s t test; ns, non-significant; values are the mean ± SEM; n=7-11. Illustrations in the image are from BioRender.com/d58i426.

IL-1R1 signaling is required for NETs formation in LPS+HVV-induced ALI.

LPS was instilled i.t. into wild type (WT) and Il1r1-/- mice and, after 120 minutes, the animals were anesthetized and placed on HVV for 180 minutes, followed by sacrifice (A). Arterial blood partial pressure of oxygen was measured at 30 and 150 minutes after starting MV (B). Absolute counts of neutrophils (C) and macrophages (D) in BALF were determined. The levels of albumin (E), IL-1β (F), IL-6 (G), TNF⍺ (H), CXCL2 (I), MPO (J), NE (K) were measured in the bronchoalveolar lavage fluid (BALF) by ELISA. Cell death and NETs formation in the BALF were evaluated by measuring histone-DNA (L), and MPO-DNA (M) respectively. ****, **, and * indicate p<0.0001, p<0.01, and p<0.05, respectively, determined by two-way ANOVA followed by Tukey’s multiple comparisons test or unpaired two-tailed Student’s t test; ns, non-significant; values are the mean ± SEM; n=7-12. Illustrations in the image are from BioRender.com/w36u811.

IL-1β enhances ionomycin-induced NETs formation in vitro. Hoechst-stained bone marrow neutrophils (BMN) or alveolar neutrophils (AN) were incubated for 1 hour prior to stimulation at 37°C. At time zero the different stimuli were added, and the cells were incubated for 4 hours then stained with Sytox green and the images were captured under microscope (A). BMN (B) and AN (E) were stimulated with several concentrations of lipopolysaccharide (LPS, 30-100,000 ng/mL), IL-1β (0.3-1000 ng/mL), and ionomycin (ION) (10-100,000 nM). For combined stimulation, BMN (C and D) and AN (F and G) were first incubated with LPS or IL-1β 30 min prior ION. The NETs forming neutrophils was analyzed as elongated-shaped Sytox green positive cells and expressed as percentage (%). The Sytox green and Hoechst positive cells are represented by green and white colors, respectively, on the representative images. ****, ***, and ** indicate p<0.0001, p<0.001 and p<0.01, respectively, determined by two-way ANOVA followed by Tukey’s multiple comparisons test; ns, non-significant; values are the mean ± SEM; representative of 3 independent experiments. Illustrations in the image are from BioRender.com/y06f775.

Hypothermia inhibits macrophage IL-1β release by modulating NLRP3 inflammasome-induced gasdermin D cleavage.

BMDM were primed with LPS for 3h at 37 ℃, then incubated at 37 ℃ or 32 ℃ for 30 min prior to ATP or nigericin (NIG) treatments for another 30 min (A). In the supernatant, IL-1β concentration was determined by ELISA (B). The supernatant (C) and cell lysate (D) were used for western blotting (WB) analysis, and the resulting membranes were stained for IL-1β, caspase-1 and gasdermin D (GSDMD)(C). WB analysis were also made in the cell lysate, were we investigated the expression of IL-1β and GSDMD. The protein distribution in the cell lysate samples was certified by ponceau staining. BMDM were stained with anti-GSDMD (red) and DAPI (blue), as shown in the representative images. The GSDMD area was analyzed and normalized by DAPI area (E). To evaluate caspase-1 activity, the cells were stained with FAM-YVAD-FMK FLICA and analyzed under the microscope (F). ****, ***, and * indicate p<0.0001, p<0.001 and p<0.05, respectively, determined by two-way ANOVA followed by Tukey’s multiple comparisons test; ns, non-significant; values are the mean ± SEM; representative of 3 independent experiments. Illustrations in the image are from BioRender.com/b55u111.

Hypothermia protects against LPS+HVV-induced severe acute lung injury by controlling IL-1β, GSDMD and NETs in the alveoli.

LPS was instilled i.t. to C57BL/6 mice and, after 120 minutes, the animals were anesthetized and placed on high-volume ventilation (HVV) for 180 min under controlled body temperature of 37±1 ℃ or 32±1 ℃, designated as normothermia and hypothermia, respectively (A). The body temperature for each group were monitored (B). Arterial blood partial pressure of oxygen was measured at 30 and 150 minutes after starting MV (C). Absolute counts of neutrophils (D) and macrophages (E) in the BALF collected from euthanized after 180 minutes of MV. The levels of albumin (F), IL-1β (G), IL-6 (H), TNF⍺ (I), MPO (J), NE (K) soluble gasdermin D (GSDMD) (L) in the BALF were determined by ELISA. Cell death and NETs formation in the BALF were evaluated by histone-DNA (M), and MPO-DNA (N) respectively. ****, ***, **, and * indicate p<0.0001, p<0.001, p<0.01, and p<0.05, respectively, determined by two-way ANOVA followed by Tukey’s multiple comparisons test or unpaired two-tailed Student’s t test; ns, non-significant; values are the mean ± SEM, n=6-12 mice/group. Illustrations in the image are from BioRender.com/m67j875.