FGF21 ameliorates septic liver injury by restraining proinflammatory macrophages activation through the autophagy/HIF-1α axis

Abstract

Introduction: Sepsis, a systemic immune syndrome caused by severe trauma or infection, poses a substantial threat to the health of patients worldwide. The progression of sepsis is heavily influenced by septic liver injury, which is triggered by infection and cytokine storms, and has a significant impact on the tolerance and prognosis of septic patients. The objective of our study is to elucidate the biological role and molecular mechanism of fibroblast growth factor 21 (FGF21) in the process of sepsis.

Objectives: This study was undertaken in an attempt to elucidate the function and molecular mechanism of FGF21 in therapy of sepsis.

Methods: Serum concentrations of FGF21 were measured in sepsis patients and septic mice. Liver injury was compared between mice FGF21 knockout (KO) mice and wildtype (WT) mice. To assess the therapeutic potential, recombinant human FGF21 was administered to septic mice. Furthermore, the molecular mechanism of FGF21 was investigated in mice with myeloid-cell specific HIF-1α overexpression mice (LyzM-Cre^DIO-HIF-1α) and myeloid-cell specific Atg7 knockout mice (Atg7^△mye).

Results: Serum level of FGF21 was significantly increased in sepsis patients and septic mice. Through the use of recombinant human FGF21 (rhFGF21) and FGF21 KO mice, we found that FGF21 mitigated septic liver injury by inhibiting the initiation and propagation of inflammation. Treatment with rhFGF21 effectively suppressed the activation of proinflammatory macrophages by promoting macroautophagy/autophagy degradation of hypoxia-inducible factor-1α (HIF-1α). Importantly, the therapeutic effect of rhFGF21 against septic liver injury was nullified in LyzM-Cre^DIO-HIF-1α mice and Atg7^△mye mice.

Conclusions: Our findings demonstrate that FGF21 considerably suppresses inflammation upon septic liver injury through the autophagy/ HIF-1α axis.

Keywords: Autophagy; FGF21; HIF-1α; Liver injury; Macrophage; Sepsis.

Graphical abstract

Fig. 1

The expression of FGF21 in macrophages and hepatocytes is upregulated in liver of septic mice. (A) The serum level of FGF21 in healthy volunteers (n = 5) and sepsis patients (n = 9). (B) The serum level of FGF21 in mice with CLP operation (n = 4). (C) Relative protein level of FGF21 analyzed by western blotting in different organs (liver, heart, iWAT, pancreas and muscle) from mice with CLP operation (n = 4). (D) Representative immunofluorescence of in situ hybridization of FGF21 in liver from mice with CLP operation. DAPI (blue), FGF21(red), and Albumin (green). Scale bars: 50 μm. (E) Representative immunofluorescence of in situ hybridization of FGF21 in liver from mice with CLP operation. DAPI (blue), FGF21(red), and F4/80 (green). White arrows indicate FGF21-expressed macrophages. Scale bars: 50 μm. (F) The expression of FGF21 in primary hepatocytes after stimulated with LPS for various periods of time, and normalized to β-actin (n = 4). (G) The protein level of FGF21 in culture medium of primary hepatocytes after stimulated with LPS for 12 h (n = 4). (H) Real-time PCR analysis for fgf21 in primary hepatocytes with LPS stimulation for 12 h (n = 3). (I) The expression of FGF21 in BMDMs after stimulated with LPS for various periods of time, and normalized to β-actin (n = 4). (J) The protein level of FGF21 in culture medium of BMDMs after stimulated with LPS for 12 h (n = 4). (K) Real-time PCR analysis for fgf21 in BMDMs with LPS stimulation for 12 h (n = 3). All values are presented as mean ± SEM. Statistical significance was measured using the unpaired 2-tailed Student t test for two experimental groups and one way ANOVA test for multiple groups. ns = no significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

FGF21 protects mice suppresses inflammation to against septic liver injury. (A) Short-term survival CLP model (n = 8). (B, C) Representative image and quantification of hepatic TUNEL staining of mice in indicated groups (n = 5). Scale bars: 50 μm. (D-F) Liver tissue samples from indicated groups were subjected to H&E staining, and infiltrated cells number around large vessel and small vessel was counted (n = 5). Scale bars: 50 μm. The region enclosed by the dashed lines represents the area infiltrated by inflammatory cells. (G, H) Representative image and quantification of hepatic MPO staining of mice in indicated groups (n = 5). Scale bars: 50 μm. (I) The Serum levels of ALT and AST from mice in indicated groups (n = 5). (J) Elisa analysis for IL-1β, IL-6, and TNF-α in serum of mice in indicated groups (n = 5). (K) The Serum level of FGF21 from mice in indicated groups (n = 5). (L) The expression of FGF21 in liver lysates from wildtype mice and FGF21 KO mice, and normalized to β-actin (n = 4). (M, N) Representative image and quantification of hepatic TUNEL staining of mice in indicated groups (n = 5). Scale bars: 50 μm. (O-Q) Liver tissue samples from indicated groups were subjected to H&E staining, and infiltrated cells number around large vessel and small vessel was counted (n = 5). Scale bars: 50 μm. The region enclosed by the dashed lines represents the area infiltrated by inflammatory cells. (R, S) Representative image and quantification of hepatic MPO staining of mice in indicated groups (n = 5). Scale bars: 50 μm. (T) The Serum levels of ALT and AST from mice in indicated groups (n = 5). (U) Elisa analysis for IL-1β, IL-6, and TNF-α in serum of mice in indicated groups (n = 5). Data are presented as mean ± SEM of each group. Statistical difference in mortality were analyzed by log-rank test. And one-way ANOVA test is using to assess the statistical differences in multiple groups. ns = no significant.

Fig. 3

LPS impaired autophagic flux in macrophages. (A, B) Representative immunofluorescence and quantification of iNOS-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), iNOS (red), and F4/80 (green). White arrows indicate iNOS-F4/80 double positive macrophages. Scale bars: 50 μm. (C, D) Representative immunofluorescence and quantification of LC3 puncta in F4/80-positive macrophages in liver from mice in indicated group. DAPI (blue), LC3β (red), and F4/80 (green) (n = 5). Scale bars: 50 μm. (E, F) Representative immunofluorescence and quantification of p62-F4/80 double positive macrophages in liver from mice in indicated groups. DAPI (blue), p62 (red), and F4/80 (green) (n = 5). White triangles indicate p62-F4/80 double positive macrophages. Scale bars: 50 μm. (G) The expression of LC3 and p62 in lysates of BMDMs in “control-group” and “LPS-group”, and normalized to β-actin (n = 4). (H) The expression of LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). And Bafilomycin A1 was added for last 6 h. (I, J) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-mCherry-LC3B (n = 10). Scale bars: 20 μm. (K) The expression of LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). 3-MA was given as pretreatment for 12 h before LPS administration. (L, M) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-LC3B in indicated groups (n = 8). Scale bars: 10 μm. (N) The expression of LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). U0126, SP600125 and SB203580 were pretreated to BMDMs for 12 h. (O, P) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-LC3B in indicated groups (n = 8). Scale bars: 20 μm. (Q, R) Representative confocal images and quantification of pHLys Red in indicated groups (n = 5). Scale bars: 50 μm. (J) Real-time PCR analysis for lamp1, ctsa, ctsb, and ctsf from BMDMs in indicated group. All values are presented as mean ± SEM. Statistical significance was measured using the unpaired 2-tailed Student t test for two experimental groups and one way ANOVA test for multiple groups. ns = no significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

rhFGF21 impaired autophagic flux in macrophages. (A, B) Representative immunofluorescence and quantification of iNOS-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), iNOS (red), and F4/80 (green). White arrows indicate iNOS-F4/80 double positive macrophages. Scale bars: 50 μm. (C, D) Representative immunofluorescence and quantification of LC3 puncta in F4/80-positive macrophages in liver from mice in indicated group. DAPI (blue), LC3β (red), and F4/80 (green) (n = 5). Scale bars: 50 μm. (E, F) Representative immunofluorescence and quantification of p62-F4/80 double positive macrophages in liver from mice in indicated groups. DAPI (blue), p62 (red), and F4/80 (green) (n = 5). White triangles indicate p62-F4/80 double positive macrophages. Scale bars: 50 μm. (G) Heatmaps and quantitative results of mRNA level of inflammatory genes in BMDMs. (H) The expression of iNOS, LC3 and p62 in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). And Bafilomycin A1 was added for last 6 h. (I, J) Representative confocal images and quantification of Raw264.7 transduced with Lenti-GFP-mCherry-LC3B (n = 10). Scale bars: 20 μm. (K, L) Representative electron micrographs and quantification from BMDMs in indicated group. Scale bars: 0.5 μm. Black arrows indicate autophagosome, and red arrows indicate autolysosome (n = 5). (M, N) Representative confocal images and quantification of pHLys Red in indicated groups (n = 5). Scale bars: 50 μm. (O) Real-time PCR analysis for lamp1, ctsa, ctsb, and ctsf from BMDMs in indicated group. Data are presented as mean ± SEM of each group. And one-way ANOVA test is using to assess the statistical differences in multiple groups. ns = no significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

The protective effect of rhFGF21 in septic liver injury is invalid in Atg7^△mye mice. (A, B) Representative immunofluorescence and quantification of iNOS-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), iNOS (red), and F4/80 (green). White arrows indicate iNOS-F4/80 double positive macrophages. Scale bars: 50 μm. (C) Elisa analysis for IL-1β, IL-6, and TNF-α in serum of mice in indicated groups (n = 5). (D) The Serum levels of ALT and AST from mice in indicated groups (n = 5). (E-G) Liver tissue samples from indicated groups were subjected to H&E staining, and infiltrated cells number around large vessel and small vessel was counted (n = 5). Scale bars: 50 μm. The region enclosed by the dashed lines represents the area infiltrated by inflammatory cells. (H, I) Representative image and quantification of hepatic MPO staining of mice in indicated groups (n = 5). Scale bars: 50 μm. (J, K) Representative image and quantification of hepatic TUNEL staining of mice in indicated groups (n = 5). Scale bars: 50 μm. Data are presented as mean ± SEM of each group. And one-way ANOVA test is using to assess the statistical differences in multiple groups. ns = no significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

rhFGF21 inhibits proinflammatory activation of macrophage via downregulating HIF-1α. (A, B) Representative immunofluorescence and quantification of HIF-1α-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), HIF-1α (red), and F4/80 (green). White triangles indicate HIF-1α-F4/80 double positive macrophages. Scale bars: 50 μm. (C) The expression of HIF-1α in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). (D, E) Representative immunofluorescence and quantification of iNOS-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), iNOS (red), and F4/80 (green). White arrows indicate iNOS-F4/80 double positive macrophages. Scale bars: 50 μm. (F) Elisa analysis for IL-1β, IL-6, and TNF-α in serum of mice in indicated groups (n = 5). (G) The Serum levels of ALT and AST from mice in indicated groups (n = 5). (H-J) Liver tissue samples from indicated groups were subjected to H&E staining, and infiltrated cells number around large vessel and small vessel was counted (n = 5). Scale bars: 50 μm. The region enclosed by the dashed lines represents the area infiltrated by inflammatory cells. (K, L) Representative image and quantification of hepatic MPO staining of mice in indicated groups (n = 5). Scale bars: 50 μm. Data are presented as mean ± SEM of each group. And one-way ANOVA test is using to assess the statistical differences in multiple groups. ns = no significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

rhFGF21 promotes autophagic degradation of HIF-1α. (A, B) Representative immunofluorescence and quantification of colocalization of HIF-1α and Lamp1 in BMDMs from indicated group (n = 5). DAPI (blue), HIF-1α (red), and Lamp1 (green). Scale bars: 20 μm. (C) The expression of HIF-1α in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 5). Bafilomycin A1 was added for the last 6 h. (D) BMDMs were treated as indicated, and the cell lysates were subjected to immunoprecipitation with HIF-1α or p62 antibody, followed by immunoblotting with the HIF-1α or p62 antibody. MG132 (10 μM) and Bafilomycin A1 (100 nM) were added for the last 6 h. (E) The expression of HIF-1α in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 5). BMDMs were transfected with Si-scramle (20 nM) and Si-sqstm1 (20 nM) for 48 h, then BMDMs were treated with single LPS or LPS + rhFGF21 for 12 h (n = 4). (F) The expression of p-ULK1 (Ser757) and p-p62 (Ser405) in lysates of BMDMs in indicated groups, and normalized to ULK1 and p62 (n = 4). (G, H) Representative immunofluorescence and quantification of HIF-1α-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), HIF-1α (red), and F4/80 (green). White triangles indicate HIF-1α-F4/80 double positive macrophages. Scale bars: 50 μm. (I) The expression of HIF-1α, LC3 and p62 in Kupffer cells isolated from liver of mice in indicated groups (n = 5). β-actin was used as the standard for verifying equivalent loading. (J) Real-time PCR analysis for hk2, pkm2, and ldha in liver from mice in indicated groups. Data are presented as mean ± SEM of each group. And one-way ANOVA test is using to assess the statistical differences in multiple groups. ns = no significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

PF-05231023 protects mice against septic liver injury. (A) The expression of iNOS in lysates of BMDMs in indicated groups, and normalized to β-actin (n = 4). (B) Short-term survival CLP model (n = 8). (C) The Serum levels of ALT and AST from mice subjected to mild-grade CLP in indicated groups (n = 5). (D) Elisa analysis for IL-1β, IL-6, and TNF-α in serum of mice in indicated groups (n = 5). (E-G) Liver tissue samples from mice in indicated groups were subjected to H&E staining, and infiltrated cells number around large vessel and small vessel was counted (n = 5). Scale bars: 50 μm. The region enclosed by the dashed lines represents the area infiltrated by inflammatory cells. (H, I) Representative immunofluorescence staining of MPO in liver section from mice in indicated groups (n = 5). Scale bars: 50 μm. (J, K) Representative immunofluorescence of iNOS-F4/80 double positive macrophages in liver from mice in indicated groups (n = 5). DAPI (blue), iNOS (red), and F4/80 (green). White arrows indicate iNOS-F4/80 double positive macrophages. All values are presented as mean ± SEM. Statistical significance was measured using one way ANOVA test for multiple groups. ns = no significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)