IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality

Abstract

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis-associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI.

Figure 1. IL-6 levels correlate with the extent of pulmonary damage and lethality during SAP.

(A) Schematic model for SAP. (B and C) Histological sections of H&E-stained pancreatic and lung tissue of C57BL/6 mice at the indicated time points. Note the increase of edema (asterisk) and necrosis (white arrowheads) in the pancreas after 8 hours and 3 days and the first signs of regeneration of damaged pancreatic tissue (black arrowheads) after 3 days. Lung damage continued to increase after 3 days, as demonstrated by alveolar wall thickening and collapse (see higher-magnification views of boxed regions at far right; enlarged ×3). (D) MPO activity in lung tissue of C57BL/6 mice (n = 5). (E) Flow cytometry analysis of CD11b⁺/Gr-1⁺ cells in total lung tissue (n = 6) and cytospin preparation of BALF in C57BL/6 mice. (F) Lung permeability, evaluated by FITC-dextran clearance. (G) Interstitial fluid accumulation, measured as capillary-alveolar membrane thickness (n = 10). (H–K) Total cell count (H), total protein concentration (I), BALF CXCL1 (J), and BALF IL-6 (K) (n = 1–3 per condition; 4 independent experiments). (L and M) Serum levels of IL-6 (L) and CXCL1 (M) in C57BL/6 mice (n > 5). (N) Kaplan-Meier curves of cerulein-treated Il6^–/– mice (green; n = 5) and of C57BL/6 mice treated with cerulein (black; n = 6), cerulein plus 5 μg/d recombinant IL-6 (blue; n = 5), or NaCl sham plus 5 μg/d recombinant IL-6 (purple; n = 5). Results represent mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.001. Scale bars: 50 μm.

Figure 2. IL-6 is required to link pancreatic damage to pulmonary damage during AP.

C57BL/6 and Il6^–/– mice were subjected to 8 hours of AP. (A) Morphological analysis of H&E-stained pancreatic tissue. (B and C) Amylase and lipase levels (n = 4). (D) Histological sections of lung tissue. Note the increased alveolar wall thickening and collapse in C57BL/6 mice. (E) Capillary-alveolar membrane thickness (n = 10). (F) MPO activity in lung tissue (n > 5). (G) Serum CXCL1 (n = 4). (H) Pancreatic tissue was isolated at the indicated time points and homogenized to detect p-STAT3^Y705 and p-STAT1^Y701. ERK1/2 served as loading control (representative blot; n = 4 per time point). (I) IHC staining of p-STAT3^Y705 in pancreatic tissue. Only C57BL/6 mice showed STAT3 activation in acinar cells; Il6^–/– mice showed phosphorylation of STAT3 in immune cells (black arrowhead), but not in acinar cells (white arrowhead). Results represent mean ± SD. *P < 0.05, ***P < 0.001. Scale bars: 50 μm. Boxed regions are shown at higher magnification at right (enlarged ×3).

Figure 3. IL-6 trans-signaling via STAT3 mediates ALI during SAP.

(A) Acinar cells were incubated with indicated concentrations of IL-6 or hyper–IL-6 for 2 hours. Protein lysates from incubated acinar cells were homogenized and blotted with p-STAT3^Y705, p-STAT1^Y701, and STAT3. β-Actin served as loading control (representative blot; n = 3). (B) Morphological analysis of representative H&E stains revealed less alveolar collapse and thickness in opt_sgp130Fc compared with C57BL/6 mice. (C) Interstitial fluid accumulation, measured as capillary-alveolar membrane thickness (n = 5). (D) Lung tissue was removed to measure MPO activity (n = 5). (E) Serum IL-6 concentration (n = 4). (F) Pancreatic tissue was isolated at the indicated times and homogenized to detect p-STAT3^Y705. β-Actin served as loading control (representative blot; n = 4 per time point). (G) Morphological analysis of representative H&E stains revealed less pancreatic injury in opt_sgp130Fc compared with C57BL/6 mice. (H and I) Serum analysis showed significantly lower levels of amylase and lipase after 4 and 8 hours in opt_sgp130Fc versus C57BL/6 mice. Results represent mean ± SD. *P < 0.05, **P < 0.005. Scale bars: 50 μm. Boxed regions are shown at higher magnification at right (enlarged ×3).

Figure 4. Classical IL-6 signaling and IL-6 trans-signaling activate different pathways in the pancreas during inflammation.

At 0, 4, and 8 hours, pancreatic tissue from C57BL/6, Il6^–/–, and opt_sgp130Fc mice was isolated and homogenized to detect p-STAT3^Y705, p-STAT3^S727, p-RelA, IκBα, and IκBβ. ERK1/2 served as loading control (representative blot; n = 4 per time point).

Figure 5. Myeloid cells secrete IL-6 in a NF-κB–dependent manner.

(A and B) Immunohistochemical analyses were used to localize p-IκBα (A) and RelA/p65 (B) in pancreas and lung tissues 8 hours after the first injection of cerulein. Positive results for p-IκBα and RelA/p65 in the pancreas (black arrowheads) were mainly restricted to inflammatory cells. Acinar cells remained negative (asterisk). (A) Alveolar cells showed weak activation of p-IκBα. (B) Bronchial epithelium (arrowhead) and infiltrating cells (circle) in the lung harbored nuclear RelA/p65. (C) IHC analyses were used to localize IL-6 in the pancreas and lung. Positive results for IL-6 in the pancreas were strictly restricted to inflammatory cells (black arrows). Alveolar macrophages expressed IL-6 in the lung (white arrows). (D) Myeloid-specific abrogation of RelA/p65 in bone marrow–derived macrophages (BMDM) of Rela^F/F and RelA^Δmye mice. (E) Pancreatic nuclear protein extracts (10 μg) were subjected to gel retardation assays with an NF-κB consensus binding site (representative EMSA; n = 4). (F) Lung tissue was removed to measure MPO activity (n = 4). (G–I) Serum was removed for IL-6 evaluation (G), and levels of Il6 (H) and Cxcl1 (I) mRNA of total pancreatic mRNA were determined, in Rela^F/F and RelA^Δmye mice. Fold change values (± SD) were normalized to cyclophilin mRNA (n = 4). (J) Pancreases were harvested and homogenized to detect p-STAT3^Y705. STAT3 and β-actin served as loading controls (representative blot; n = 4). Results represent mean ± SD. *P < 0.05, **P < 0.005. Scale bars: 50 μm. Boxed regions are shown at higher magnification below (enlarged ×3).

Figure 6. Pancreas-specific p-STAT3^Y705 modulates the severity of local inflammation during AP.

(A) Macroscopic images of X-gal–stained liver, pancreas, and small bowel. Microscopic images of nuclear lacZ activity in sections of pancreas, liver, and lung. (B) Pancreases were isolated to detect p-STAT3^Y705 during AP. Note the absence of STAT3 activation in Stat3^Δpanc mice. STAT3 served as loading control (representative blot; n = 5). Truncated STAT3 protein in the pancreas (STAT3^Δpanc) is indicated. (C) Pancreases were subjected to Western blot analysis using a p-STAT3^Y705–specific antibody. Note the increased p-STAT3^Y705 in Socs3^Δpanc mice. STAT3 served as a loading control (representative blot; n = 5 per time point). (D) Morphological analysis of pancreases after 8 hours of AP in the indicated mice. (E) Serum was removed for amylase measurement at the indicated time points. Note the increased release of amylase into the serum in Socs3^Δpanc mice, while levels remained lower in Stat3^Δpanc mice compared with controls (Stat3^F/F and Socs3^F/F). (F) Levels of Cxcl1 mRNA of total pancreatic mRNA in control, Stat3^Δpanc, and Socs3^Δpanc mice. Fold change values (± SD) were normalized to cyclophilin mRNA (n > 3). (G) Early trypsin activation was independent of STAT3 activation (n = 8). (H) Serum IL-6 levels at the indicated time points. Values are mean ± SD for independent animals (n = 5). *P < 0.05, **P < 0.005. Scale bars: 100 μm.

Figure 7. Phosphorylation of STAT3 in the pancreas contributes to systemic complications.

(A) Histological sections of lung tissue from control, Stat3^Δpanc, and Socs3^Δpanc mice revealed marked hemorrhage and alveolar collapse in Socs3^Δpanc mice. (B) MPO activity in lung tissue of control, Stat3^Δpanc, and Socs3^Δpanc mice at the indicated time points during AP (n = 6). (C) Lung permeability, determined by injection of EBD in the right femoral artery and measurement of dye concentration in lung tissue at 0 and 8 hours (n = 4). (D) Interstitial fluid accumulation, determined by capillary-alveolar membrane thickness. Values represent mean ± SD (n = 10). (E) Lung edema, determined indirectly by the increase in pulmonary fluid accumulation (n = 8). Animals were killed at 8 hours, and the left lung was removed in order to determine the wet/dry ratio (n = 8). (F–H) Protein concentration (F), IL-6 (G), and CXCL1 (H) measured in BALF taken from control and experimental animals (n = 4; 1–3 BALF/animal). Note that BALF could not be taken from Socs3^Δpanc mice (n.a.), since all mice died due to SAP. (I) p-STAT3^Y705 was linked to SAP-induced lethal ALI. Kaplan-Meier curves of control (n = 6), Stat3^Δpanc (n = 9), and Socs3^Δpanc (n = 5) mice during SAP. Values represent mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.001. Scale bars: 50 μm.

Figure 8. Pharmacological inhibition of STAT3, CXCR2, CXCL1, and IL-6 trans-signaling attenuates SAP-induced lethal ALI.

(A) Kaplan-Meier curves of C57BL/6 mice (black; n = 11) and C57BL/6 mice treated with the CXCR2 antagonist SB225002 (green; n = 12), the STAT3 inhibitor S3I-201 (red; n = 7), recombinant sgp130Fc (blue; n = 8), or an anti-CXCL1 antibody (orange; n = 5) during SAP. (B and C) Serum was removed for amylase and lipase analyses at the indicated time points (n = 4). (D) MPO activity in lung tissue of C57BL/6 mice or treated mice 8 hours after the first cerulein injection (n = 4). (E and F) Histological sections of pancreatic and lung tissue. Note the decrease in lung injury in treated versus C57BL/6 mice. **P < 0.005 versus control. Scale bars: 50 μm (E); 100 μm (F).

Figure 9. IL-6 trans-signaling in patients with AP.

(A) Sampling of blood in individuals with mild AP and SAP after the onset of symptoms. (B and C) Serum IL-6 and sIL-6R levels in control patients and patients with mild AP and SAP. (D) IL-6/sIL-6R ratio. (E) Serum IL-8 levels in control patients and patients with mild AP and SAP. (F) Central role of IL-6 trans-signaling during SAP-associated ALI. Results represent mean ± SD (n > 6). *P < 0.05, **P < 0.005, ***P < 0.001.