Liver injury in COVID-19 and IL-6 trans-signaling-induced endotheliopathy

Abstract

Background and aims: COVID-19 is associated with liver injury and elevated interleukin-6 (IL-6). We hypothesized that IL-6 trans-signaling in liver sinusoidal endothelial cells (LSECs) leads to endotheliopathy (a proinflammatory and procoagulant state) and liver injury in COVID-19.

Methods: Coagulopathy, endotheliopathy, and alanine aminotransferase (ALT) were retrospectively analyzed in a subset (n = 68), followed by a larger cohort (n = 3,780) of patients with COVID-19. Liver histology from 43 patients with COVID-19 was analyzed for endotheliopathy and its relationship to liver injury. Primary human LSECs were used to establish the IL-6 trans-signaling mechanism.

Results: Factor VIII, fibrinogen, D-dimer, von Willebrand factor (vWF) activity/antigen (biomarkers of coagulopathy/endotheliopathy) were significantly elevated in patients with COVID-19 and liver injury (elevated ALT). IL-6 positively correlated with vWF antigen (p = 0.02), factor VIII activity (p = 0.02), and D-dimer (p <0.0001). On liver histology, patients with COVID-19 and elevated ALT had significantly increased vWF and platelet staining, supporting a link between liver injury, coagulopathy, and endotheliopathy. Intralobular neutrophils positively correlated with platelet (p <0.0001) and vWF (p <0.01) staining, and IL-6 levels positively correlated with vWF staining (p <0.01). IL-6 trans-signaling leads to increased expression of procoagulant (factor VIII, vWF) and proinflammatory factors, increased cell surface vWF (p <0.01), and increased platelet attachment in LSECs. These effects were blocked by soluble glycoprotein 130 (IL-6 trans-signaling inhibitor), the JAK inhibitor ruxolitinib, and STAT1/3 small-interfering RNA knockdown. Hepatocyte fibrinogen expression was increased by the supernatant of LSECs subjected to IL-6 trans-signaling.

Conclusion: IL-6 trans-signaling drives the coagulopathy and hepatic endotheliopathy associated with COVID-19 and could be a possible mechanism behind liver injury in these patients.

Lay summary: Patients with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection often have liver injury, but why this occurs remains unknown. High levels of interleukin-6 (IL-6) and its circulating receptor, which form a complex to induce inflammatory signals, have been observed in patients with COVID-19. This paper demonstrates that the IL-6 signaling complex causes harmful changes to liver sinusoidal endothelial cells and may promote blood clotting and contribute to liver injury.

Keywords: SARS-CoV-2; coagulopathy; endothelial cell dysfunction; thrombosis.

Graphical abstract

Fig. 1

Procoagulant factors and IL-6 are significantly increased in COVID-19 patients with liver injury. (A) Serum levels of fibrinogen (n = 57), factor VIII activity (n = 65), and factor II activity (n = 65) in patients with liver injury in an initial sample of patients with COVID-19 (n = 68). (B,C) Retrospective analysis using a large database of COVID-19 inpatients from 3/10/2020 to 12/1/2020 (n = 3,780). (B) Serum levels of fibrinogen (n = 3,344), D-dimer (n = 3,478), vWF activity (n = 129), vWF Ag (n = 131), and hsCRP (n = 2,828) in patients with COVID-19. (C) Initial IL-6 levels in patients with COVID-19. ALT <3x (n = 1,110) and ≥3x (n = 481). (D) Correlation between serum IL-6 and vWF antigen (n = 119), factor VIII activity (n = 126), and D-dimer (n = 1,600). Means compared with 2-tailed Student’s t test for (A) and medians compared with 2-tailed Mann-Whitney U test for (B), (C). Correlation in (D) assessed with Pearson correlation. Patients excluded in each analysis if necessary data not available. ALT, alanine aminotransferase; hsCRP, high-sensitivity C-reactive protein; IL-6, interleukin-6; vWF, von Willebrand factor; vWF Ag, VWF antigen.

Fig. 2

Expression of vWF in the liver is associated with increased platelet adhesion and liver injury in COVID-19. (A) Representative liver histological features in patients with COVID-19 and controls. Scale bars:100 μm. (B) Summary of histological features (H&E staining) of the livers from patients with COVID-19 (n = 43) and controls (n = 12). #: Neutrophils per high-power field (C) Summary of histological features associated with liver injury: ALT <3x (n = 29) and ≥3x (n = 12). (D) Immunohistochemistry for vWF and CD61 in the liver from patients with COVID-19 and controls. Scale bars:100 μm. Comparisons of vWF and CD61 positive areas in the liver from patients with COVID-19 (n = 43) and controls (n = 5) (Upper and lower left graphs), as well as those from patients with liver injury (n = 12) and those without (n = 29) (upper and lower right graphs). (E) Correlation between vWF and CD61 positive areas in the livers of patients with COVID-19 (n = 43). (F) Correlation between intralobular neutrophils and CD61-positive area (n = 43) and vWF-positive area (n = 43) in the livers of patients with COVID-19. (G) Immunofluorescence of vWF and CD41 (a platelet marker) in the livers from patients with COVID-19 and controls. Scale bars: 25 μm. In patients with COVID-19, vWF is expressed mainly on LSECs, and CD41-positive platelets attach to these LSECs (arrow head). A lower level of vWF is expressed on CD41-positive platelets (arrow). (a) and (b) are different fields from the same patient. (H) Correlation between IL-6 and vWF-positive area in COVID-19 patients. Comparisons between 2 groups were obtained using Welch’s t test for continuous variables, Chi-squared test was used for categorical variables. Pearson’s correlation co-efficients were calculated to examine the correlation among vWF area, CD61 area, and intralobular neutrophils. Spearman correlation was used to examine the correlation between serum IL-6 level and vWF area. Biochemical liver injury in 2 patients with COVID-19 could not be classified (no available ALT value). Data are mean ± SEM. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. ALT, alanine aminotransferase; DIC, differential interference contrast; IL-6, interleukin-6; LSECs, liver sinusoidal endothelial cells; vWF, von Willebrand factor.

Fig. 3

IL-6 trans-signaling induces endotheliopathy and increases cell surface levels of vWF and platelet attachment to LSECs. (A) Schema of IL-6 trans-signaling and blocking by soluble gp130 or ruxolitinib. (B,C) qPCR of markers for (B) procoagulant and (C) proinflammatory endotheliopathy. Human primary LSECs were incubated with control, human recombinant IL-6 (20 ng/ml), human recombinant sIL-6R (20 ng/ml) or IL-6/sIL-6R complex (20 ng/ml) for 1 hour. Graphs show the fold-change (control is set to 1). n = 6. (D) Flow cytometry for cell surface levels of vWF in LSECs treated with control or IL-6/sIL-6R complex (20 ng/ml) for 6 hours. n = 3. (E) Platelet attachment to LSECs. Phalloidin (Green, F-actin for cell structure), CD41 (Red, platelet). LSECs were treated with control or IL-6/sIL-6R complex (20 ng/ml) for 4 hours and incubated with platelets for 2 hours. Scale bar = 10 μm n = 10. Data are mean ± SEM of at least 3 experiments. One-way ANOVA with Tukey’s test for multiple groups, or 2-tailed unpaired t test for 2 groups was used. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. CXCL, chemokine (C-X-C motif) ligand; ICAM1, intercellular adhesion molecule 1; IL-6, interleukin-6; LSECs, liver sinusoidal endothelial cells; qPCR, quantitative PCR; sIL-6R, soluble IL-6 receptor; vWF, von Willebrand factor.

Fig. 4

Soluble gp130 blocks JAK1 and STAT3 phosphorylation induced by IL-6 trans-signaling in LSECs. Human primary LSECs were treated with control, IL-6 (20 ng/ml) or IL-6/sIL-6R complex (20 ng/ml) in the presence or absence of sgp130 Fc (100 ng/ml) for 15 minutes (western blot) and 1 hour (qPCR). (A) A representative blot. β-actin (loading control). n = 3. (B) Activation (phosphorylation) of JAK1, JAK2, STAT3 and STAT1. The fold-change for quantification of western blot. (complex without sgp130 is set to 1). n = 3. qPCR of markers for (C) procoagulant and (D) proinflammatory endotheliopathy in LSECs with or without sgp130 Fc (100 ng/ml). n = 6. Control is set to 1 for each experiment and data presented as fold-change vs. complex. Data are mean ± SEM of at least 3 experiments. One-way ANOVA with Tukey’s test for multiple groups, or 2-tailed unpaired t test for 2 groups was used. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. CXCL, chemokine (C-X-C motif) ligand; gp130, glycoprotein 130; ICAM1, intercellular adhesion molecule 1; IL-6, interleukin-6; JAK, Janus kinase; LSECs, liver sinusoidal endothelial cells; qPCR, quantitative PCR; sgp130, soluble gp130; sIL-6R, soluble IL-6 receptor; STAT, signal transducer and activator of transcription.

Fig. 5

JAK inhibitor ruxolitinib blocks JAK1/STAT3 induction by IL-6 trans-signaling and blocks procoagulant endotheliopathy in LSECs. Human primary LSECs were treated with control or IL-6/sIL-6R complex (20 ng/ml) in the presence or absence of ruxolitinib (2 μM) for 15 minutes (western blot) and 1 hour (qPCR). Cells were incubated with ruxolitinib for 20 minutes before treatment with complex. (A) Representative western blot. (B) Quantification of western blot. Control is set to 1 for each experiment and data presented as fold-change vs. complex. n = 3. qPCR of markers for (C) procoagulant and (D) proinflammatory endotheliopathy in LSECs with or without ruxolitinib. The graphs show the fold-change (control is set to 1). n = 6. (E) Representative western blot and its quantification. LSECs were treated with siRNA-STAT1 & 3 [STAT1 (10 nM), STAT3 (20 nM) for 64 hours] and then incubated with control or IL-6/sIL-6R complex for 24 hours. n = 3. Data are mean ± SEM of at least 3 experiments. Two-tailed unpaired t test was used. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. IL-6, interleukin-6; JAK, Janus kinase; LSECs, liver sinusoidal endothelial cells; qPCR, quantitative PCR; sIL-6R, soluble IL-6 receptor; STAT, signal transducer and activator of transcription.

Fig. 6

Fibrinogen levels are increased in livers of patients with COVID-19 by IL-6 signaling amplified by LSEC-hepatocyte crosstalk. (A) Representative immunolabeling of fibrinogen (Red) in livers from patients with COVID-19. Scale bar: 50 μm. Quantification of fibrinogen positive area (right panel) in patients with COVID-19 (n = 43) and controls (n = 6). (B) Correlation between fibrinogen area and ALT (n = 41), neutrophil infiltration (n = 43), and CD61 (n = 43) in livers of patients with COVID-19. (C) Western blot analyses in primary hepatocytes and primary LSECs treated with control, IL-6 alone (20 ng/ml) or IL-6/sIL-6R complex (20 ng/ml). Representative western blot (left) and its quantification (right panel). n = 3. Complex is set to 1 for each experiment and data presented as fold-change. (D) Western blot analyses in mouse primary hepatocytes treated with control, IL-6 alone (20 ng/ml) or IL-6/sIL-6R complex (20 ng/ml) in the presence or absence of ruxolitinib (2 μM) for 15 minutes. Ruxolitinib was added 20 minutes before treatment with IL-6 alone or complex. n = 3. The graphs show the fold-change (control is set to 1). (E) Experimental scheme for (F & G). (F) mRNA expression of fibrinogen. n = 9. The graphs show the fold-change (control is set to 1). (G) mRNA expression of fibrinogen in mouse primary hepatocytes treated with control medium or supernatant from LSECs treated with IL-6/sIL-6R complex for 24 hours in the presence or absence of ruxolitinib. n = 3. The graphs show the fold-change (control is set to 1). Data are the mean ± SEM of at least 3 experiments. Pearson’s correlation coefficients were calculated to examine the correlation among fibrinogen area, ALT, neutrophil infiltration and CD61 area. One-way ANOVA with Tukey’s test for multiple groups, or two-tailed unpaired t test for 2 groups was used. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. ALT, alanine aminotransferase; IL-6, interleukin-6; LSECs, liver sinusoidal endothelial cells; sIL-6R, soluble IL-6 receptor.