Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Mar 5.
Published in final edited form as: Hepatology. 2024 Jan 24;81(3):947–961. doi: 10.1097/HEP.0000000000000762

Neutrophil extracellular traps promote MASH fibrosis by metabolic reprogramming of HSC

Yujia Xia 1,2, Yu Wang 2,3, Qi Xiong 3, Jiayi He 2,4, Han Wang 1,2, Mozaffarul Islam 2, Xinyu Zhou 5, Alex Kim 2, Hongji Zhang 5, Hai Huang 6, Allan Tsung 5
PMCID: PMC11881075  NIHMSID: NIHMS2045173  PMID: 38266270

Abstract

Background and Aims:

Metabolic dysfunction–associated steatohepatitis (MASH) fibrosis is a reversible stage of liver disease accompanied by inflammatory cell infiltration. Neutrophils extrude a meshwork of chromatin fibers to establish neutrophil extracellular traps (NETs), which play important roles in inflammatory response regulation. Our previous work demonstrated that NETs promote HCC in MASH. However, it is still unknown if NETs play a role in the molecular mechanisms of liver fibrosis.

Approach and Results:

Following 12 weeks of Western diet/carbon tetrachloride, MASH fibrosis was identified in C57BL/6 mice with increased NET formation. However, NET depletion using DNase I treatment or mice knocked out for peptidyl arginine deaminase type IV significantly attenuated the development of MASH fibrosis. NETs were demonstrated to induce HSCs activation, proliferation, and migration through augmented mitochondrial and aerobic glycolysis to provide additional bioenergetic and biosynthetic supplies. Metabolomic analysis revealed markedly an altered metabolic profile upon NET stimulation of HSCs that were dependent on arachidonic acid metabolism. Mechanistically, NET stimulation of toll-like receptor 3 induced cyclooxygenase-2 activation and prostaglandin E2 production with subsequent HSC activation and liver fibrosis. Inhibiting cyclooxygenase-2 with celecoxib reduced fibrosis in our MASH model.

Conclusions:

Our findings implicate NETs playing a critical role in the development of MASH hepatic fibrosis by inducing metabolic reprogramming of HSCs through the toll-like receptor 3/cyclooxygenase-2/cyclooxygenase-2 pathway. Therefore, NET inhibition may represent an attractive treatment target for MASH liver fibrosis.

INTRODUCTION

Liver fibrosis is a common pathological feature of most types of end-stage liver diseases.[1] Metabolic dysfunction–associated steatotic liver disease (MASLD)[2] is a rising cause of liver fibrosis worldwide.[3] Metabolic dysfunction–associated steatohepatitis (MASH) is a severe form of MASLD characterized by steatosis, hepatocyte damage, and inflammation leading to fibrosis. MASH has become the third most common indication for liver transplantation and the second leading cause of HCC requiring liver transplantation in the United States.[4] The degree of liver fibrosis serves as a prognostic indicator of liver-related morbidity or overall mortality and the onset of severe liver diseases in biopsy-proven MASLD.[5] While hepatic fibrosis is reversible, the resulting structural damage to liver lobules and vasculature leads to cirrhosis, which is irreversible.[6] Therefore, interventions to prevent and reverse liver fibrosis are imperative to treat MASLD and to prevent cirrhosis and MASH-HCC complications.

HSCs are identified as the major contributors to liver fibrogenesis.[7] In MASH, HSCs become activated, proliferative, and transdifferentiating from vitamin A–storing cells to myofibroblasts in response to the injury, including lipid-derived mediators and profibrotic factors.[8,9] Activated HSCs express α-smooth muscle actin (α-SMA) and produce collagen proteins, resulting in extracellular matrix (ECM) accumulation that contributes to liver fibrosis.[7] HSCs that become activated need an uninterrupted energy supply to maintain the enhanced ECM synthesis/secretion.[10,11] Demand for energy provides a new opportunity to interfere with the function of activated HSCs.

Neutrophil infiltration within portal tracts has been identified as a contributing factor to liver fibrosis and ductular reaction in human MAFLD.[12,13] However, how neutrophil infiltration affects the progression of MASLD-associated fibrosis remains uncertain. An undescribed property of neutrophils has been demonstrated that neutrophils extrude a meshwork of chromatin fibers to establish neutrophil extracellular traps (NETs),[14] which are involved in immune and inflammatory response regulation.[15] Our work demonstrated that NET formation promotes the development of HCC in MASH.[16] Strategies aimed at eliminating NETs hold promise in reducing the risk of HCC development in MASLD.[17] However, the specific role of NETs in the molecular mechanisms governing liver fibrosis remains elusive. Therefore, we hypothesize that NETs can selectively target metabolic reprogramming in the activation of HSCs and contribute to the progression of liver fibrosis.

METHODS

Human samples

Formalin-fixed paraffin-embedded liver samples of 5 patients with no symptoms and 9 patients with MASH were retrospectively selected from Tongji Hospital between January 2020 and July 2022. None of the patients had hepatitis virus infection, excessive alcohol consumption, drug abuse, Wilson’s disease, autoimmune-related disorders, or cancers. This study was approved by the Ethics Committee of Tongji Hospital, Huazhong University of Science and Technology. Written informed consent was obtained from each participant.

Animals

Male C57BL/6 mice and toll-like receptor 3 (TLR3) KO (B6;129S1-Tlr3tm1Flv/J, 005217) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Peptidyl arginine deiminase type IV knockout (PAD4−/−) mice were a gift from Dr Yanming Wang, Pennsylvania State University, PA. All procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee of the Ohio State University and the experiments were performed in adherence to NIH guidelines for the use of laboratory.

Dietary, CCl4 interventions, and celecoxib and CU CPT 4a treatment

The MASH fibrosis mouse model was established as described.[18] Celecoxib or CU CPT 4a was administered with the diet administration.[19,20] Please refer to Supplementary Methods.

Histological analysis

Liver histology and fibrosis were assessed using hematoxylin and eosin stains, Sirius Red, and Masson stains using established methodology.[21] MASLD activity score (MAS) and fibrosis stage were evaluated by 2 pathologists who were blinded about the study.[22]

Immunofluorescence

Frozen liver tissue was incubated with anti-myeloperoxidase (MPO), anti-Ly6G, anti-CollagenⅠ, anti-CK18, anti-α-SMA, anti-CD34, or anti-Collagen IV antibody. HSCs were incubated with anti-COX 2 and anti-α-SMA antibodies. The detailed procedures are given in Supplementary Methods.

Identification/quantification of NETs

Western blot assays were performed with total protein from mouse liver tissues. Membranes were incubated with anti-citrullinated histone-3 (CitH3, 1:1000, Abcam) as a primary antibody. MPO associated with DNA was performed as described.[23] Liver samples were incubated with primary antibodies: anti-CitH3 (1:50, Abcam, USA) and anti-Ly6G (1:100, BD Bioscience, USA). Hoechst 33342 (Sigma-Aldrich, USA) and phalloidin (Thermo Fisher Scientific, USA) were used for nuclear and cell wall delineation, respectively.

NET inhibition with DNase I

To block NET formation, we treated mice with DNase I as described.[24] The detailed procedures are given in Supplementary Methods.

NETs’ structure generation and isolation

Bone marrow was flushed from the femur and tibia of male wild-type mice. Neutrophils were sorted with a BD-Aria-Plus sorter. Isolated neutrophils were stimulated with 5 μm of selective calcium ionophore A23187 (Sigma-Aldrich, USA), allowed NET formation, and obtained a NET-rich supernatant.[25] Please refer to Supplementary Methods for the detailed procedures.

HSC isolation and culture

Primary HSCs were isolated from livers of 16-week male WT and TLR3 KO mice, as described[26] with modifications. Please refer to Supplementary Methods. HSCs were treated with isolated NETs or Celecoxib (Sigma-Aldrich, USA) on day 2 after isolation.

RNA sequencing

Total RNAs were isolated from HSCs with or without NET treatment using the RNeasy mini kit (Qiagen, MD, USA) and sequenced by Novogene Bioinformatics Technology Co. (Beijing, China) based on the Illumina MiSeq PE300 platform. Please refer to Supplementary Methods.

Metabolic profiling of HSCs after NET treatment

Liquid chromatography-mass spectroscopy–based metabolic profiling was performed on the HSC extracts with or without NET treatment with established methods.[27]

Seahorse analyses

HSCs were plated on Seahorse XFe 24-well plates for 1–2 days to achieve 80% confluence. Seahorse XFe24 Analyzer (Agilent, CA, USA) measures the Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live HSCs. Glycolytic rate assay and real-time ATP rate assay were also analyzed with a Seahorse XFe24 Analyzer (Agilent).

Other techniques

Please consult the Supplementary Methods: flow cytometry, immunohistochemistry, liver damage and metabolic assessment, CCK-8 assay, cell migration, ELISA, real-time PCR, and western blot.

Statistical analysis

Experimental results are presented as mean ± SEM. Differences were considered statistically significant at p < 0.05. Please refer to Supplementary Methods for the detailed procedures.

Results

Neutrophil infiltration and NET formation during the development of MASH fibrosis

We explored NET formation in patients with MASH with liver fibrosis. MPO+ cells colocalized with CitH3, a marker of NET formation.[28] We found that the colocalization of CitH3 and MPO was significantly increased in MASH livers with grade 2 or 3 fibrosis compared with normal (Figure 1A, B). Moreover, this colocalization of CitH3 and MPO was markedly higher in patients with MASH with significant (F ≥ 2) or advanced fibrosis (F ≥ 3) as compared with patients with MASH with early-stage liver fibrosis (Figure 1A, B), suggesting a positive correlation between NET formation and the degree of fibrosis in patients with MASH. Recently, a new murine MASH fibrosis model has been established, which closely replicates the key metabolic and histologic features of human MASH fibrosis.[18,29] A rapid and severe progression of steatohepatitis with extensive fibrosis can be achieved in mice within 12 weeks, fed with Western diet (WD) and carbon tetrachloride (CCl4). In our present study, representative pictures of hematoxylin and eosin, Sirius Red, and Masson staining from mice of normal diet (ND)/Oil, WD/Oil, ND/CCl4, and WD/CCl4 are shown (Supplemental Fig. 1A-B, http://links.lww.com/HEP/I222). The increased ratio of liver/body weight in WD/CCl4-treated mice was accompanied by elevations of serum ALT and AST at 12 weeks (Supplemental Fig. 1C-D, http://links.lww.com/HEP/I222). To confirm the results in the WD/CCl4 model, we used the methionine/choline-deficient (MCD) diet, a classic approach inducing histological features of MASH.[30] After eating an MCD diet for 8 weeks, the MASH and fibrosis model was successfully established (Supplemental Fig. 1E-F, http://links.lww.com/HEP/I222).

FIGURE 1.

FIGURE 1

Open in a new tab

Neutrophil infiltration and NET formation during the development of MASH fibrosis. (A, B) Immunofluorescence staining for MPO, CitH3, and nucleus in the liver of normal patients (n = 5) and patients with MASH fibrosis (n = 9). The overlap coefficient is shown in bar graph format. Green = MPO. Red = CitH3. Blue = nucleus. Scale bar = 50 μm. (C, D, E) The proportion and number of infiltrating CD11b+Ly6G+ neutrophils in ND/Oil, WD/Oil, and WD/CCl4 groups were detected by flow cytometry (n = 4). (F-G) Immunofluorescence staining for Ly6G, CitH3, actin, and nucleus in the livers. The overlap coefficient is shown in bar graph format (n = 3). Green = Ly6G. Red = CitH3. Grey = actin. Blue = nucleus. Scale bar = 50 μm. (H-I) The proportion of infiltrating Ly6G+CitH3+ neutrophils in the spleen of ND/Oil and WD/CCl4 groups were detected by flow cytometry (n = 4). (J-K) Immunofluorescence staining for MPO, CitH3, and nucleus in the livers (n = 3). The overlap coefficient is shown in bar graph format. Green = MPO. Red = CitH3. Blue = nucleus. Scale bar = 50 μm. (L) Serum MPO-DNA levels were compared in ND/Oil and WD/CCl4 groups (n = 9). (M) The level of the NET marker CitH3 in livers was detected by western blot (n = 3). (N, O) The serum levels of IL-6 and TNF-α were increased in the WD/CCl4 group at 12 weeks (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations: CCl4, carbon tetrachloride; CitH3, citrullinated histone-3; MASH, Metabolic dysfunction–associated steatohepatitis; MPO, myeloperoxidase; MPO-DNA, MPO associated with DNA; ND, normal diet; NET, neutrophil extracellular traps; WD, Western diet.

To assess the recruitment of neutrophils into the liver in our MASH fibrosis models, we examined the percentage of CD45+CD11b+Ly6G+ neutrophils by flow cytometry (Supplemental Fig. 2A, http://links.lww.com/HEP/I223). We showed the onset of neutrophil infiltration as early as 2 weeks and a gradual increase up to 12 weeks (Supplemental Fig. 2B, http://links.lww.com/HEP/I223). There was a significant increase of neutrophil infiltration in the liver of WD/CCl4-treated mice at 12 weeks (Figure 1C–E), and MCD-treated mice at 8 weeks (Supplemental Fig. 2C-E, http://links.lww.com/HEP/I223). We then explored NET formation in our MASH-associated fibrosis mouse models. Ly6G-positive cells colocalized with CitH3, a marker of NET formation,[31] in the liver, indicating NET formation in response to WD/CCl4 treatment (Figure 1F, G). In addition, we examined the percentage of CD45+Ly6G+CitH3+ neutrophils by flow cytometry[32] (Supplemental Fig. 2A, http://links.lww.com/HEP/I223). There was a significant increase of Ly6G+-CitH3+ cells in WD/CCl4-treated mice compared with the ND/Oil controls at 12 weeks (Figure 1H, I). Immunofluorescence staining revealed an increased MPO+CitH3+ and Ly6G+ neutrophils population in the livers of MASH fibrosis groups compared with ND/Oil controls (Figure 1J, K, Supplemental Fig. 2F-G, http://links.lww.com/HEP/I223). Notably, many MPO+ neutrophils are found to be recruited predominantly surrounding steatotic hepatocytes and activated HSCs were found to be closely associated with MPO+ neutrophils in MASH fibrosis groups (Supplemental Fig. 2H-M, http://links.lww.com/HEP/I223). Furthermore, a nonelectron dense basement membrane–like structure with reticular Collagen IV is present in the liver with the space of Disse.[33] Immunofluorescence showed that MPO+ neutrophils infiltrating into the space of Disse presented with reticular Collagen IV in WD/CCl4-treated mice (Supplemental Fig. 2N-O, http://links.lww.com/HEP/I223). This suggested that MPO+ neutrophils are recruited predominantly surrounding steatotic hepatocytes and activated HSCs are closely associated with MPO+ neutrophils in MASH fibrosis groups. Serum levels of MPO associated with DNA, a marker for circulating NETs,[23] were also found to be significantly elevated in the WD/CCl4 group (Figure 1L). Western blot confirmed the increase of CitH3 in the liver of WD/CCl4 mice compared with the ND/Oil controls (Figure 1M). Additionally, increased levels of the inflammatory cytokines IL-6 and TNF-α coincided with the time point when infiltrating neutrophils became more numerous (Figure 1N, O). These results indicate the prominent role of neutrophil accumulation and NET formation in the inflammation of MASH-associated fibrosis.

NET depletion alleviates the development of MASH-associated fibrosis

DNase I is an endonuclease that selectively cleaves the DNA scaffold of NETs.[34] To inhibit NET formation, WD/CCl4 mice were injected with DNase I intraperitoneally throughout the experimental period (Figure 2A). In addition, PAD4 KO mice that are incapable of forming NETs were also treated with the WD/CCl4 model[35] (Supplemental Fig. 3A, http://links.lww.com/HEP/I224). DNase I treatment or genetic depletion of PAD4 markedly reduced the levels of CitH3 and attenuated liver inflammation in mice undergoing WD/CCl4 diet compared with control treatment (Figure 2B, C, Supplemental Fig. 3B-C, http://links.lww.com/HEP/I224). Mice treated with DNase I or PAD4 KO mice also exhibited a significant improvement in MAS, lower body weight, and decreased serum hepatocellular enzyme levels compared to the WD/CCl4 group (Supplemental Fig. 3D-H, http://links.lww.com/HEP/I224). Serum levels of inflammatory mediators IL-6 and TNF-α in the WD/CCl4 group were also markedly higher than those in the ND/Oil group but significantly reduced in the serum of DNase I-treated (Figure 2D, E) or PAD4 KO WD/CCl4 groups (Supplemental Fig. 3I-J, http://links.lww.com/HEP/I224).

FIGURE 2.

FIGURE 2

Open in a new tab

NET depletion alleviates the development of MASH-associated fibrosis. (A) Experiment design of WD, CCl4, and DNase I treatment in mice for 12 weeks. (B) The protein level of CitH3 in the WD/CCl4 group with or without DNase I treatment (n = 3). (C) H&E, Sirius Red, and Masson staining of WD/CCl4 mice treated with or without DNase I (n = 5). Scale bar = 100 μm. (D, E) The serum levels of IL-6 and TNF-α in the WD/CCl4 group with or without DNase I (n = 5). (F) Quantification of Sirius Red-positive area (n = 5) and (G) Masson staining-positive area (n = 5). (H) Western blot for Fibronectin, Collagen I, and α-SMA in extracted livers of WD/CCl4 mice simultaneously without or with DNase I (n = 3). *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. Abbreviations: α-SMA, alpha-smooth muscle actin; CCl4, carbon tetrachloride; CitH3, citrullinated histone-3; H&E, hematoxylin and eosin; MASH, Metabolic dysfunction–associated steatohepatitis; NET, neutrophil extracellular traps; WD, Western diet.

Importantly, inhibition of NET formation protected against MASH fibrosis with the attenuation of liver bridging fibrosis and collagen deposition in DNase I-treated mice and PAD4 KO mice (Figure 2F, G, Supplemental Fig. 3K-L, http://links.lww.com/HEP/I224). Moreover, compared with WD/CCl4 controls, the expressions of predominant fibrotic proteins, including α-SMA, collagen I, and fibronectin,[1] were significantly decreased in DNase I-treated mice (Figure 2H). Similarly, the progression of liver fibrosis by DNase I-depleting NETs was also found markedly delayed in the MCD model. (Supplemental Fig. 3M-N, http://links.lww.com/HEP/I224).

Since the liver constitutes an essential organ in lipid metabolism, the disruption of lipid metabolism precipitates the retention of fat within the liver and the subsequent development of MASLD.[36] We thus aimed to determine whether NETs affect lipid metabolism and observed a significant increase in serum total cholesterol following WD/CCl4 treatment compared with ND/Oil treatment. DNase I injection and PAD4 KO in WD/CCl4-treated mice significantly lowered the serum total cholesterol (Supplemental Fig. 3O, http://links.lww.com/HEP/I224). These data demonstrated that NET depletion can improve lipid metabolic dysfunction, protect against liver inflammation and injury, and ameliorate liver fibrosis in MASH models. However, the MCD diet has limitations as it induces weight loss and lacks the induction of features associated with MS (obesity and metabolic syndrome), a critical risk factor for MASLD. Therefore, we did not continue to use the MCD model in the subsequent study.

NETs induce the activation, proliferation, and migration of HSCs

HSCs play an essential role in the initiation and progression of liver fibrosis by secreting fibrogenic factors.[8] Activated HSCs exhibit a high level of α-SMA and collagen expressions, representing the typical profibrogenic phenotype. To elucidate the role of NETs in HSC activation, we isolated primary HSCs of mice (Supplemental Fig. 4, http://links.lww.com/HEP/I225) and found that the expression of α-SMA dramatically increased in WD/CCl4 primary HSCs compared with ND/Oil primary HSCs (Figure 3A). HSCs treated with NETs demonstrated increased expression of α-SMA compared with controls (Figure 3B). NETs also induced the mRNA expression of the fibrogenic genes, such as Collagen Type I Alpha 1, Collagen Type III Alpha 1, Actin Alpha 2, and Tissue inhibitor metalloproteinase 1, along with the protein expression of α-SMA in primary HSCs (Figure 3C–E). Further, NETs significantly increased HSCs proliferation in a time-dependent manner after treatment (Figure 3F). Similarly, the migration speed and the wound healing rate were remarkedly increased in HSCs stimulated with NETs as compared to the untreated group (Figure 3G–J). Collectively, these data demonstrate that NETs promote the activation, proliferation, and migration of HSCs.

FIGURE 3.

FIGURE 3

Open in a new tab

NETs induce the activation, proliferation, and migration of HSCs. (A) The protein level of α-SMA in HSCs extracted from WD/CCl4 and ND/Oil groups detected by western blot (n = 3). (B) Western blot for α-SMA in HSCs treated with NETs by different doses (n = 3). (C) RT-PCR for Col1a1, Col3a1, Acta2, and Timp1 in HSCs treated with or without NETs (n = 3). (D, E) Immunofluorescence staining for α-SMA and nucleus in HSCs treated with or without NETs (n = 3). Green = α-SMA. Blue = nucleus. Scale bar = 50 μm. (F) The cell viability of HSCs treated with NETs for 24 hours, 48 hours, and 72 hours were detected by CCK-8 assay (n = 5). (G, H) The migratory capacities of HSCs treated with or without NETs were evaluated by transwell assays (n = 3). Scale bar = 100 μm. (I) The migratory capacities of HSCs treated with or without NETs were detected by wound healing assay (n = 3). (J) Wound healing analysis of the migratory capacities of HSCs (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001. Abbreviations: α-SMA, alpha-smooth muscle actin; CCl4, carbon tetrachloride; CCK8, Cell Counting Kit 8; NET, neutrophil extracellular traps; WD, Western diet.

NETs drive metabolic changes in HSC activation

Activated HSCs are dependent on intracellular ATP supply to be responsible for ECM synthesis and accumulation.[10,11] To assess the metabolic state of HSCs, we evaluated the OCR as a measure of mitochondrial respiration, and the ECAR to assess glycolytic flux. Analysis of mitochondrial respiration revealed a significant elevation in OCR, ATP-coupled respiration, and ECAR of primary HSCs from WD/CCl4 mice compared to the ND/Oil mice (Figure 4A–E). After DNase I treatment, both the OCR and ECAR markedly decreased. In vitro, HSCs treated with NETs exhibited a substantial increase in mitochondrial respiration and ATP production (Figure 4F, G). This increase in the mitochondrial activity of HSCs after NET treatment was also confirmed by MitoTracker Deep Red staining (Figure 4H, I). These results demonstrate that NETs upregulate ATP production from mitochondria in primary HSCs.

FIGURE 4.

FIGURE 4

Open in a new tab

NETs drive metabolic changes in HSC activation. (A) OCR rate, (B) OCR analysis, (C) ECAR rate, (D) ECAR analysis, and (E) the ratio of basal OCR and ECAR of HSCs extracted from WD/CCl4-treated with or without DNase I assessed by Seahorse (n = 4). (F) OCR rate and OCR analysis (G) of wild-type HSCs treated with or without NETs (n = 4). (H) Mitochondrial capacity of HSCs treated with or without NETs was stained with the MitoTracker fluorescent probe (an indicator of mitochondrial function) and fluorescence quantitative analysis (I) (n = 3). Red = active mitochondrial. Blue = nucleus. Scale bar = 50 μm. (J) ECAR rate, (K) ECAR analysis, and the ratio of basal OCR and ECAR (L) of HSCs treated with or without NETs assessed by Seahorse (n = 4). (M) Total metabolite profiling in HSCs treated with or without NETs was determined by the metabolomics assay based on LC-MS/MS and assessed by PCA (n = 3). (N, O) Pathway-enrichment analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001. Abbreviations: CCl4, carbon tetrachloride; ECAR, extracellular acidification rate; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NET, neutrophil extracellular traps; OCR, oxygen consumption rate; PCA, principal component analysis; WD, Western diet.

Activated HSCs exhibit augmented aerobic glycolysis regarding an additional bioenergetic and biosynthetic supply, even in oxygen-rich conditions.[37] We further assessed whether glycolysis plays a crucial role in NET-induced HSC activation. HSCs treated with NETs for 24 hours demonstrated a significant increase in ECAR compared to control HSCs (Figure 4J). In the glycolytic rate assay, compensatory glycolysis refers to the ability of the cell to increase glycolysis after oxidative phosphorylation has been inhibited with rotenone and antimycin A. To confirm specificity, the glycolysis inhibitor 2-deoxyglucose was added to inhibit glycolytic acidification. We detected significant increases in basal and compensatory glycolytic proton efflux rate of HSCs after NET treatment when compared to control HSCs (Figure 4K, L). These above findings show that the activation of HSCs induced by NETs is dependent on both mitochondrial respiration and aerobic glycolysis.

To further explore the relationship between NETs and metabolic disturbances in HSCs, we employed metabolomics to measure the metabolic composition and principal component analysis to assess the metabolic changes in HSCs treated with or without NETs. The results of principal component analysis score points revealed a markedly altered metabolic profile upon NET stimulation (Figure 4M). Functional pathway analysis facilitating further biological interpretation revealed 8 potential metabolites that were important following NET treatment. (Figure 4N). Mummichog pathway mapper for metabolomics data analysis indicated that the metabolic switch in HSCs after NET treatment during HSC activation involves arachidonic acid metabolism. (Figure 4O).

NETs modulate HSC metabolism through the arachidonic acid pathway

Cyclooxygenases (COX) and lipoxygenases are wellrecognized key enzymes in the metabolic pathway of arachidonic acid.[38] Since our results indicated arachidonic acid metabolism as the pathway being affected most, we then assessed the potential effects of COX and lipoxygenases in HSCs. In untreated control HSCs, low levels of COX-2 mRNA were detected but NET treatment upregulated COX-2 expression. However, COX-1 expression dramatically decreased after NET treatment in HSCs and 5-lipoxygenases showed no significant differences between groups (Figure 5A). Further, immunofluorescence staining showed that COX-2 and α-SMA protein expressions were upregulated in HSCs treated with NETs (Figure 5B, C). As a result, we then assessed the effect of celecoxib, a selective COX-2 inhibitor,[19] on the inactivation of HSCs. Treatment with 2 μM celecoxib significantly inhibited the expression of COX-2 and α-SMA (Figure 5D–G). The analysis of mitochondrial respiration showed that celecoxib treatment led to a striking decrease of OCR, ATP-coupled respiration, and glycolytic rates in HSCs, effects that were reversed by NET stimulation (Figure 5H–L). Celecoxib also inhibited cell proliferation and migration of HSCs (Supplemental Fig. 5A-F, http://links.lww.com/HEP/I226), while cotreatment of celecoxib with NETs demonstrated the reversal of suppressive effects induced by celecoxib on proliferation and migration of HSCs. (Supplemental Fig. 5B-F, http://links.lww.com/HEP/I226). Prostaglandin E2 (PGE2) secretion, the downstream product of COX-2, [39] was significantly increased after NET treatment. The inhibitory effects of celecoxib on PGE2 secretion of HSCs were reversed by NETs (Figure 5M). These results indicate that NET-mediated metabolic changes, proliferation, and migration of HSCs are dependent on the COX-2/PGE2 pathway.

FIGURE 5.

FIGURE 5

Open in a new tab

NETs regulate the metabolic change of HSCs dependent on arachidonic acid metabolism. (A) RT-PCR for COX-2, COX-1, and 5-LOX in HSCs treated with or without NETs (n = 3). (B, C) Immunofluorescence staining for COX-2 and α-SMA in HSCs treated with or without NETs for 72 hours (n = 3). The mean intensity of α-SMA and COX-2 were shown in bar graph format (n = 3). Green = α-SMA. Red = COX-2. Blue = Nucleus. Scale bar = 50 μm. (D) Western blot for COX-2 in HSCs treated with NETs, celecoxib, or co-treated with celecoxib and NETs (n = 3). (E) Western blot for COX-2 and α-SMA in HSCs treated with NETs, celecoxib, or co-treated with celecoxib and NETs (n = 3). (F, G) Western blot analysis of COX-2 and α-SMA in HSCs (n = 3). (H) OCR rate of HSCs assessed by Seahorse (n = 4). (I) OCR analysis of HSCs (n = 4). (J) ECAR rate of HSCs assessed by Seahorse (n = 4). (K) ECAR analysis of HSCs (n = 4). (L) The ratio of basal OCR and ECAR in HSCs treated with NETs, celecoxib, or co-treated with celecoxib and NETs (n = 4). (M) PGE2 secretion of HSCs (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, no significance. Abbreviations: α-SMA, alpha-smooth muscle actin; COX-2, cyclooxygenase-2; ECAR, extracellular acidification rate; LOX, lipoxygenases; NET, neutrophil extracellular traps; OCR, oxygen consumption rate; PGE2, prostaglandin E2.

The accelerated progression of liver fibrosis induced by NETs is dependent on COX-2

COX-2 (Figure 6A, B) and PGE2 (Figure 6C) expressions were induced in the livers and serum of mice treated with WD/CCl4 for 12 weeks, whereas DNase I treatment resulted in a significant decrease in both COX-2 and PGE2 in WD/CCl4 mice. Celecoxib (20 mg/kg/day) effectively inhibited the expression of COX-2 in wild-type mice (Supplemental Fig. 6A-B, http://links.lww.com/HEP/I227). Liver tissue morphometry after Sirius Red and Masson staining demonstrated that celecoxib significantly decreased liver fibrosis induced by WD/CCl4 (Figure 6D–F). Compared with WD/CCl4 group without celecoxib, the expressions of the key fibrotic genes and proteins, including α-SMA, collagen Ⅰ, collagen Ⅲ, and fibronectin were significantly decreased in celecoxib-treated mice (Figure 6G–J). The production of PGE2 also dramatically decreased in celecoxib-treated mice combined with WD/CCl4 (Figure 6K) with significant improvement in MAS, ratio of liver/body weight, and body weight compared to the WD/CCl4 controls (Figure 6L, M, Supplemental Fig. 6C, http://links.lww.com/HEP/I227). Furthermore, celecoxib treatment led to a significant decrease in liver enzymes, total cholesterol, and inflammatory cytokines in WD/CCl4-treated mice compared with control treatment (Supplemental Fig. 6D-H, http://links.lww.com/HEP/I228). These data indicate that celecoxib can protect against NET-mediated MASH liver inflammation and fibrosis.

FIGURE 6.

FIGURE 6

Open in a new tab

The accelerated development of liver fibrosis by NETs is dependent on COX-2. (A, B) RT-PCR and western blot for COX-2 in extracted livers of ND/Oil, WD/CCl4, and WD/CCl4 simultaneously with DNase I mice (n = 3). (C) Serum PGE2 of ND/Oil, WD/CCl4, and WD/CCl4 simultaneously with DNase I mice (n = 5). (D) H&E staining, Sirius Red staining, and Masson staining of extracted livers in WD/CCl4 mice simultaneously without or with celecoxib after 12 weeks (n = 5). Scale bar = 100 μm. (E) Quantification of Sirius red-positive area and (F) Masson staining-positive area of extracted livers in WD/CCl4 mice simultaneously without or with celecoxib (n = 5). (G-H) RT-PCR for Coll1a1 and Coll3a1 in extracted livers of WD/CCl4 mice simultaneously without or with celecoxib (n = 3). (I) Western blot for COX-2, Fibronectin, Collagen I, and α-SMA in extracted livers of WD/CCl4 mice simultaneously without or with celecoxib (n = 3). (J) Western blot analysis of extracted livers in WD/CCl4 mice simultaneously without or with celecoxib (n = 3). (K) Serum PGE2, (L) MASLD activity score, and (M) Liver per body weight ratio of WD/CCl4 mice simultaneously without or with celecoxib (n = 5). *p < 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. Abbreviations: α-SMA, alpha-smooth muscle actin; COX-2, cyclooxygenase-2; CCl4, carbon tetrachloride; H&E, hematoxylin and eosin; MASLD, metabolic dysfunction–associated steatotic liver disease; NET, neutrophil extracellular traps; PGE2, prostaglandin E2; WD, Western diet.

NETs activate COX-2 and PGE2 through toll-like receptor 3

To explore the molecular mechanisms by which NETs activate COX-2 signaling in HSCs, we conducted a comparative gene expression analysis in HSCs with or without NET treatment. A total of 7806 genes were differentially regulated in HSCs treated with NETs (log2 fold change > 1 and adjusted p value < 0.05), including 4093 upregulated genes and 3713 down-regulated genes (Supplemental Fig. 7A-B, http://links.lww.com/HEP/I229). GO analysis indicated that many upregulated genes were involved in defense responses to foreign pathogens and viruses as well as responses to interferon-beta and collagen-containing extracellular matrix (Supplemental Fig. 7C, http://links.lww.com/HEP/I229). Kyoto Encyclopedia of Gene and Genomes analysis demonstrated upregulated genes’ significant enrichment in several cascades, such as the toll-like receptor signaling pathway, retinoic acid-inducible gene I-like receptor signaling pathway, NOD-like receptor signaling pathway, and cytokine-cytokine receptor interaction (Supplemental Fig. 7D, http://links.lww.com/HEP/I229). Given that the toll-like receptor signaling pathway has been associated with NET formation,[40,41] we examined it as a candidate receptor of NET-mediated COX-2 activation in HSCs.

Since only TLR2, TLR3, TLR7, TLR8, and TLR13 were upregulated in HSCs after NET stimulation, we focused on these 5 TLRs to elucidate the specific TLR associated with NET-mediated COX-2 activation in HSCs (Figure 7A). We found that TLR3 was the most highly upregulated toll-like receptor in HSCs treated with NETs as compared to control HSCs (Figure 7B). Utilizing TLR3 KO HSCs combined with NETs, the previously increased expression of COX-2, α-SMA, and the production of PGE2 seen in wild-type HSC was inhibited (Figure 7C–G, Supplemental Fig. 7E, http://links.lww.com/HEP/I229). Mice treated with selective TLR3 inhibitor CU CPT 4a[20] showed a significant improvement in MAS compared to the WD/CCl4 group (Figure 7H). Furthermore, liver tissue morphometry after Sirius Red and Masson staining indicated that CU CPT 4a significantly decreased liver fibrosis induced by WD/CCl4 (Figure 7I–K). Taken together, these results show that NETs stimulate COX-2 activation and PGE2 production through the activation of TLR3 in HSCs.

FIGURE 7.

FIGURE 7

Open in a new tab

NETs activate COX-2 and PGE2 through toll-like receptor 3 in HSCs. (A) Heat map of the top receptor (padj < 0.05) in HSCs treated with or without NETs for 72 hours (n = 3). (B) RT-PCR for differentially regulated genes of the toll-like receptor signaling pathway in HSCs treated with or without NETs for 72 hours (n = 3). (C) Western blot for COX-2 and α-SMA in TLR3WT and TLR3 KO HSCs treated with or without NETs for 72 hours (n = 3). (D, E) Western blot analysis of COX-2 and α-SMA in TLR3WT and TLR3KO HSCs treated with or without NETs for 72 hours (n = 3). (F, G) Immunofluorescence staining for COX-2 and α-SMA in TLR3KO HSCs treated with or without NETs for 72 hours (n = 3). The mean intensity of α-SMA and COX-2 were shown in bar graph format. (H) MASLD activity score of WD/CCl4 mice treated without or with CU CPT 4a (n = 5). (I) H&E, Sirius Red, and Masson staining of extracted livers in WD/CCl4 mice simultaneously without or with CU CPT 4a (n = 5). (J) Quantification of Sirius Red-positive area and (K) Masson staining-positive area of extracted livers in WD/CCl4 mice simultaneously without or with CU CPT 4a (n = 5). Scale bar = 100 μm. Green = α-SMA. Red = COX-2. Blue = Nucleus. Scale bar = 50 μm. *p< 0.05, **p <0.01, ***p< 0.001. Abbreviations: α-SMA, alpha-smooth muscle actin; COX-2, cyclooxygenase-2; CCl4, carbon tetrachloride; H&E, hematoxylin and eosin; MASLD, metabolic dysfunction–associated steatotic liver disease; NET, neutrophil extracellular traps; ns, no significance; PGE2, prostaglandin E2; TLR3, toll-like receptor 3; WD, Western diet.

DISCUSSION

The presence of inflammation is considered as one of the strongest predictors for the progression of MASH into advanced fibrosis.[42] This MASH fibrosis transition, characterized by HSC activation, involves cross-talk interactions between HSCs and other cells in the liver.[43] In our present study, we provide compelling evidence to support the essential role of NETs in the pathogenesis of MASH fibrosis. We found that the presence of neutrophil recruitment and NET formation promoted HSC activation and subsequent development of fibrosis in the livers of mice with MASH. Notably, we also demonstrated that the mechanism by which NETs mediated this effect involves the regulation of metabolic reprogramming in HSCs.

Neutrophil infiltration in the liver is a key histopathological feature of MASH.[44] Our work demonstrated the critical role of NETs in establishing a chronic inflammatory liver microenvironment in a MASH STAM model that is favorable to the development of HCC.[16] However, MASH STAM mice exhibit type 1 diabetes and display very mild fibrosis.[45] In addition, the major disadvantage of high-fat diet or WD-based MASH models is that they do not fully progress to severe steatohepatitis and advanced fibrosis even after longterm feeding.[46,47] The present experiments employed a WD with weekly dosing of CCl4 mice, resulting in a MASH model with rapid progression of advanced fibrosis that mimics features of human MASH.[18] In both our WD/CCl4 and MCD NAFLD models, we observed a prominent accumulation of neutrophils and NETs during the development of MASH compared to control mice.

NETs that consist of DNA, histones, and neutrophil enzymes have been implicated in host cell damage due to their cytotoxic, pro-inflammatory, and prothrombotic activity.[15] In vitro, purified NET components include DNA, histones, and neutrophil granular enzymes. Several studies have used purified NET to investigate the mechanisms by which NET promotes different diseases.[31,48] DNase I, a recognized endonuclease, holds therapeutic potential in diseases associated with excessive NET formation.[49] DNase I can completely degrade the extracellular DNA of NETs but does not influence the activity of neutrophil elastase and histones.[50] Sahar et al added a protease inhibitor cocktail to determine whether neutrophil elastase can act as a cofactor in DNase I-mediated degradation of NETs. The presence of the protease inhibitor cocktail did not affect the activity of DNase I, as the NETs were fully degraded. In addition, they also demonstrated that PAD4-mediated citrullination of histones weakens DNA-histone interactions, thus rendering the nucleosome component of NETs more susceptible to degradation by DNase I. However, histone and elastase alone in NET fragments were not responsible for the enhanced maximum clot formation velocity.[51,52] In our study, neutrophils were centrifuged and NET-DNA was quantified in the supernatants as described.[24] We found that inhibition of NET formation protected against MASH fibrosis with the attenuation of liver bridging fibrosis and collagen deposition in DNase I-treated mice and PAD4 KO mice. However, further investigation is warranted to elucidate the specific NET component driving MASH-associated fibrosis.

To prevent liver-related mortality, the reversal of advanced fibrosis or prevention of progression to cirrhosis is paramount.[5] Activated HSCs drive most of the architectural changes that characterize the fibrotic or cirrhotic MASH liver, in particular, the deposition of type I collagen-rich ECM proteins.[53] The activation of HSCs involves a transition from a quiescent to a proliferative, migratory, and fibrogenic phenotype.[7] Zhou et al found that coculture of HSCs with neutrophils induced HSC activation and prolonged neutrophil survival.[54] However, prior to our findings, the role of NETs in HSC activation was unknown. Metabolic alterations have long been identified to be an important event during HSC activation. Metabolic pathways that enable HSCs to satisfy the increased bioenergetics and biosynthetic demands of the myofibroblastic phenotype are attractive therapeutic candidates for inhibiting HSC activation. Our findings suggest that NETs cause significant metabolic pathway changes in HSCs to promote activation. Chen et al have reported that the reprogramming of quiescent HSCs into myofibroblastic HSCs is dependent on the induction of aerobic glycolysis,[55] a process similar to the Warburg effect described in cancer cells.[56] Our study demonstrates that NET treatment increases mitochondrial respiration and glycolysis, and NET abrogation impairs the ability of the HSCs to activate glycolysis and mitochondrial respiration significantly.

Our study finds that NETs induce metabolic reprogramming and activation of HSCs through its effects on arachidonic acid metabolism. The selective COX-2 inhibitor celecoxib has been extensively applied in the therapy of rheumatic diseases,[57] as well as ameliorating portal hypertension and liver fibrosis through suppressing inflammation in animal models.[58,59] In our study, celecoxib not only suppressed the proliferation, migration, OCR, ATP-coupled respiration, glycolytic rates, and PGE2 production in vitro, but also displayed a significant improvement in MAS, body weight, and the ratio of liver/body weight in vivo. Moreover, these suppressive effects on HSCs could be reversed by NET stimulation.

TLRs are a class of pattern recognition receptors with an integral role in the mediation of the identification of pathogen-associated molecular patterns or damage-associated molecular patterns.[60] To date, several functional TLRs have been identified in mammals, including 10 in humans and 12 in mice.[61,62] One of the TLR members, TLR3, an intracellular receptor that recognizes foreign nucleic acids,[63] has been reported to play a pivotal role in chronic liver diseases.[64,65] In our study, TLR3 emerged as the most highly upregulated TLR in response to NET stimulation in HSCs. Considering that NET-associated RNA is an abundant component within NETs and may function as an additional immunostimulatory NET component,[41] it is reasonable to presume that TLR3 may serve as an endogenous sensor of NET-associated RNA and signaling through TLR3 might be involved in cellular responses to NET stimulation. We found that signaling through TLR3 on HSCs could induce the expressions of COX-2 and α-SMA protein and the production of PGE2 to promote liver inflammation and injury to accelerate MASH-associated liver fibrosis. However, TLR3 KO HSCs were found to lose the ability to activate within NET stimulation and its absence could attenuate liver fibrosis after MASH development.

In conclusion, our findings implicate that NETs play a key role in the development of MASH-related hepatic fibrosis by inducing metabolic reprogramming of HSCs through the TLR3/COX-2/PGE2 pathway. Therefore, NET inhibition may represent a novel therapeutic strategy for the treatment of MASH liver fibrosis.

Supplementary Material

Supplemental Fig. 6
Supplemental Fig. 7
Supplemental Fig. 8
Supplemental Fig. 5
Supplemental Fig. 4
Supplemental Fig. 3
Supplemental Fig. 2
Supplemental Fig. 1

ACKNOWLEDGMENTS

The authors thank Prof. Zhiwei Hu, Dr. Xinghua Liao, and Pengyan Fa for their professional advice and technical support.

FUNDING INFORMATION

This work was supported by the National Institute of Health grants R01-CA214865 and R01-GM95566 to Allan Tsung. State funding within the UVA Comprehensive Cancer Center “IDEA-Cancer pilot award” and “Cancer Therapeutics (CRX) pilot award” to Hongji Zhang. National Natural Science Foundation of China Grant Number 82270651 and 81700515 to Yujia Xia.

Abbreviations:

α-SMA

alpha-smooth muscle actin

CCl4

carbon tetrachloride

CitH3

citrullinated histone-3

COX-2

cyclooxygenase-2

ECAR

extracellular acidification rate

ECM

extracellular matrix

MAS

MASLD activity score

MASH

metabolic dysfunction–associated steatohepatitis

MASLD

metabolic dysfunction–associated steatotic liver disease

MCD

methionine/choline-deficient

MPO

myeloperoxidase

ND

normal diet

NETs

neutrophil extracellular traps

OCR

oxygen consumption rate

PAD4

peptidyl arginine deiminase type IV

PGE2

prostaglandin E2

TLR3

toll-like receptor 3

WD

Western diet

Footnotes

Supplemental Digital Content is available for this article. Direct URL citations are provided in the HTML and PDF versions of this article on the journal’s website, www.hepjournal.com.

CONFLICTS OF INTEREST

The authors have no conflicts to report.

REFERENCES

  • 1.Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005;115: 209–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Rinella ME, Lazarus JV, Ratziu V, Francque SM, Sanyal AJ, Kanwal F, et al. A multisociety Delphi consensus statement on new fatty liver disease nomenclature. Hepatology. 2023;78:1966–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Sayiner M, Koenig A, Henry L, Younossi ZM. Epidemiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in the united states and the rest of the world. Clin Liver Dis 2016; 20:205–14. [DOI] [PubMed] [Google Scholar]
  • 4.Wong RJ, Cheung R, Ahmed A. Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S. Hepatology. 2014;59:2188–95. [DOI] [PubMed] [Google Scholar]
  • 5.Hagström H, Nasr P, Ekstedt M, Hammar U, Stål P, Hultcrantz R, et al. Fibrosis stage but not NASH predicts mortality and time to development of severe liver disease in biopsy-proven NAFLD. J Hepatol 2017;67:1265–73. [DOI] [PubMed] [Google Scholar]
  • 6.Brenner DA. Reversibility of liver fibrosis. Gastroenterol Hepatol (N Y). 2013;9:737–9. [PMC free article] [PubMed] [Google Scholar]
  • 7.Friedman SL. Hepatic stellate cells: Protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 2008;88:125–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 2017;14: 397–411. [DOI] [PubMed] [Google Scholar]
  • 9.Schwabe RF, Tabas I, Pajvani UB. Mechanisms of fibrosis development in nonalcoholic steatohepatitis. Gastroenterology. 2020;158:1913–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ganapathy-Kanniappan S, Karthikeyan S, Geschwind JF, Mezey E. Is the pathway of energy metabolism modified in advanced cirrhosis? J Hepatol 2014;61:452. [DOI] [PubMed] [Google Scholar]
  • 11.Nishikawa T, Bellance N, Damm A, Bing H, Zhu Z, Handa K, et al. A switch in the source of ATP production and a loss in capacity to perform glycolysis are hallmarks of hepatocyte failure in advance liver disease. J Hepatol 2014;60:1203–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Gao B, Tsukamoto H. Inflammation in alcoholic and nonalcoholic fatty liver disease: Friend or foe? Gastroenterology. 2016;150: 1704–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Gadd VL, Skoien R, Powell EE, Fagan KJ, Winterford C, Horsfall L, et al. The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease. Hepatology. 2014;59: 1393–405. [DOI] [PubMed] [Google Scholar]
  • 14.Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–5. [DOI] [PubMed] [Google Scholar]
  • 15.Papayannopoulos V Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol 2018;18:134–47. [DOI] [PubMed] [Google Scholar]
  • 16.van der Windt DJ, Sud V, Zhang H, Varley PR, Goswami J, Yazdani HO, et al. Neutrophil extracellular traps promote inflammation and development of hepatocellular carcinoma in nonalcoholic steatohepatitis. Hepatology. 2018;68:1347–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Wang H, Zhang H, Wang Y, Brown ZJ, Xia Y, Huang Z, et al. Regulatory T cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis. J Hepatol 2021;75:1271–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Tsuchida T, Lee YA, Fujiwara N, Ybanez M, Allen B, Martins S, et al. A simple diet- and chemical-induced murine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancer. J Hepatol 2018;69:385–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Cunha NV, de Abreu SB, Panis C, Grassiolli S, Guarnier FA, Cecchini R, et al. Cox-2 inhibition attenuates cardiovascular and inflammatory aspects in monosodium glutamateinduced obese rats. Life Sci 2010;87:375–81. [DOI] [PubMed] [Google Scholar]
  • 20.Sardana S, Singh KP, Saminathan M, Vineetha S, Panda S, Dinesh M, et al. Effect of inhibition of Toll-like receptor 3 signaling on pathogenesis of rabies virus in mouse model. Acta Trop 2022;234:106589. [DOI] [PubMed] [Google Scholar]
  • 21.Lattouf R, Younes R, Lutomski D, Naaman N, Godeau G, Senni K, et al. Picrosirius red staining: A useful tool to appraise collagen networks in normal and pathological tissues. J Histochem Cytochem 2014;62:751–8. [DOI] [PubMed] [Google Scholar]
  • 22.Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–21. [DOI] [PubMed] [Google Scholar]
  • 23.Maruchi Y, Tsuda M, Mori H, Takenaka N, Gocho T, Huq MA, et al. Plasma myeloperoxidase-conjugated DNA level predicts outcomes and organ dysfunction in patients with septic shock. Crit Care. 2018;22:176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Albrengues J, Shields MA, Ng D, Park CG, Ambrico A, Poindexter ME, et al. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. Science. 2018;361:eaao4227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Najmeh S, Cools-Lartigue J, Rayes RF, Gowing S, Vourtzoumis P, Bourdeau F, et al. Neutrophil extracellular traps sequester circulating tumor cells via β1-integrin mediated interactions. Int J Cancer. 2017;140:2321–30. [DOI] [PubMed] [Google Scholar]
  • 26.Mederacke I, Dapito DH, Affò S, Uchinami H, Schwabe RF. High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers. Nat Protoc 2015;10:305–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Knobloch TJ, Ryan NM, Bruschweiler-Li L, Wang C, Bernier MC, Somogyi A, et al. Metabolic regulation of glycolysis and AMP activated protein kinase pathways during black raspberry-mediated oral cancer chemoprevention. Metabolites. 2019;9: 140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Li RHL, Johnson LR, Kohen C, Tablin F. A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy. BMC Vet Res 2018;14:210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Scagliola A, Miluzio A, Ventura G, Oliveto S, Cordiglieri C, Manfrini N, et al. Targeting of eIF6-driven translation induces a metabolic rewiring that reduces NAFLD and the consequent evolution to hepatocellular carcinoma. Nat Commun 2021;12: 4878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Mridha AR, Wree A, Robertson AAB, Yeh MM, Johnson CD, Van Rooyen DM, et al. NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. J Hepatol 2017;66:1037–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Noubouossie DF, Whelihan MF, Yu YB, Sparkenbaugh E, Pawlinski R, Monroe DM, et al. In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps. Blood. 2017;129:1021–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Perdomo J, Leung HHL, Ahmadi Z, Yan F, Chong JJH, Passam FH, et al. Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia. Nat Commun 2019;10:1322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Häussinger D, Kordes C. Space of Disse: A stem cell niche in the liver. Biol Chem 2019;401:81–95. [DOI] [PubMed] [Google Scholar]
  • 34.Warnatsch A, Ioannou M, Wang Q, Papayannopoulos V. Inflammation. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis. Science. 2015;349:316–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lewis HD, Liddle J, Coote JE, Atkinson SJ, Barker MD, Bax BD, et al. Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat Chem Biol 2015;11:189–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Bechmann LP, Hannivoort RA, Gerken G, Hotamisligil GS, Trauner M, Canbay A. The interaction of hepatic lipid and glucose metabolism in liver diseases. J Hepatol 2012;56: 952–64. [DOI] [PubMed] [Google Scholar]
  • 37.Bernard K, Logsdon NJ, Ravi S, Xie N, Persons BP, Rangarajan S, et al. Metabolic reprogramming is required for myofibroblast contractility and differentiation. J Biol Chem 2015;290: 25427–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kutil Z, Temml V, Maghradze D, Pribylova M, Dvorakova M, Schuster D, et al. Impact of wines and wine constituents on cyclooxygenase-1, cyclooxygenase-2, and 5-lipoxygenase catalytic activity. Mediators Inflamm 2014;2014:178931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Nakanishi M, Rosenberg DW. Multifaceted roles of PGE2 in inflammation and cancer. Semin Immunopathol 2013;35: 123–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Agarwal S, Loder SJ, Cholok D, Li J, Bian G, Yalavarthi S, et al. Disruption of neutrophil extracellular traps (NETs) links mechanical strain to post-traumatic inflammation. Front Immunol 2019;10:2148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Herster F, Bittner Z, Archer NK, Dickhöfer S, Eisel D, Eigenbrod T, et al. Neutrophil extracellular trap-associated RNA and LL37 enable self-amplifying inflammation in psoriasis. Nat Commun 2020;11:105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Argo CK, Northup PG, Al-Osaimi AMS, Caldwell SH. Systematic review of risk factors for fibrosis progression in non-alcoholic steatohepatitis. J Hepatol 2009;51:371–9. [DOI] [PubMed] [Google Scholar]
  • 43.Hardy T, Oakley F, Anstee QM, Day CP. Nonalcoholic fatty liver disease: Pathogenesis and disease spectrum. Annu Rev Pathol 2016;11:451–96. [DOI] [PubMed] [Google Scholar]
  • 44.Brunt EM. Histological assessment of nonalcoholic fatty liver disease in adults and children. Clin Liver Dis (Hoboken). 2012;1: 108–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Shalapour S, Lin XJ, Bastian IN, Brain J, Burt AD, Aksenov AA, et al. Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity. Nature. 2017;551:340–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Machado MV, Michelotti GA, Xie G, de Almeida TP, Boursier J, Bohnic B, et al. Mouse models of diet-induced nonalcoholic steatohepatitis reproduce the heterogeneity of the human disease. PLoS One. 2015;10:e0127991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Charlton M, Krishnan A, Viker K, Sanderson S, Cazanave S, McConico A, et al. Fast food diet mouse: Novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition. Am J Physiol Gastrointest Liver Physiol 2011;301:G825–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Guimarães-Costa AB, Nascimento MTC, Froment GS, Soares RPP, Morgado FN, Conceição-Silva F, et al. Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps. Proc Natl Acad Sci U S A 2009;106:6748–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Jiménez-Alcázar M, Rangaswamy C, Panda R, Bitterling J, Simsek YJ, Long AT, et al. Host DNases prevent vascular occlusion by neutrophil extracellular traps. Science. 2017;358: 1202–6. [DOI] [PubMed] [Google Scholar]
  • 50.Sohrabipour S, Muniz VS, Sharma N, Dwivedi DJ, Liaw PC. Mechanistic Studies of DNase I activity: Impact of heparin variants and PAD4. Shock. 2021;56:975–87. [DOI] [PubMed] [Google Scholar]
  • 51.Podolska MJ, Mahajan A, Hahn J, Knopf J, Maueröder C, Petru L, et al. Treatment with DNases rescues hidden neutrophil elastase from aggregated NETs. J Leukoc Biol 2019;106:1359–66. [DOI] [PubMed] [Google Scholar]
  • 52.Jeffery U, LeVine DN. Canine neutrophil extracellular traps enhance clot formation and delay lysis. Vet Pathol 2018;55: 116–23. [DOI] [PubMed] [Google Scholar]
  • 53.Mederacke I, Hsu CC, Troeger JS, Huebener P, Mu X, Dapito DH, et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun 2013;4:2823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Zhou Z, Xu MJ, Cai Y, Wang W, Jiang JX, Varga ZV, et al. Neutrophil-hepatic stellate cell interactions promote fibrosis in experimental steatohepatitis. Cell Mol Gastroenterol Hepatol 2018;5:399–413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Chen Y, Choi SS, Michelotti GA, Chan IS, Swiderska-Syn M, Karaca GF, et al. Hedgehog controls hepatic stellate cell fate by regulating metabolism. Gastroenterology. 2012;143:1319–329. e11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Warburg O, Wind F, Negelein E. The metabolism of tumors in the body. J Gen Physiol 1927;8:519–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Fidahic M, Jelicic Kadic A, Radic M, Puljak L. Celecoxib for rheumatoid arthritis. Cochrane Database Syst Rev 2017;6: CD012095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Wen SL, Gao JH, Yang WJ, Lu YY, Tong H, Huang ZY, et al. Celecoxib attenuates hepatic cirrhosis through inhibition of epithelial-to-mesenchymal transition of hepatocytes. J Gastroenterol Hepatol 2014;29:1932–42. [DOI] [PubMed] [Google Scholar]
  • 59.Paik YH, Kim JK, Lee JI, Kang SH, Kim DY, An SH, et al. Celecoxib induces hepatic stellate cell apoptosis through inhibition of Akt activation and suppresses hepatic fibrosis in rats. Gut 2009;58:1517–27. [DOI] [PubMed] [Google Scholar]
  • 60.Fitzgerald KA, Kagan JC. Toll-like receptors and the control of immunity. Cell. 2020;180:1044–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Rehli M Of mice and men: Species variations of Toll-like receptor expression. Trends Immunol 2002;23:375–8. [DOI] [PubMed] [Google Scholar]
  • 62.Beutler B Inferences, questions and possibilities in toll-like receptor signalling. Nature. 2004;430:257–63. [DOI] [PubMed] [Google Scholar]
  • 63.Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 2001;413:732–8. [DOI] [PubMed] [Google Scholar]
  • 64.Wang Y, Li J, Wang X, Ye L, Zhou Y, Ho W. Induction of interferon-λ contributes to Toll-like receptor-3-activated hepatic stellate cell-mediated hepatitis C virus inhibition in hepatocytes. J Viral Hepat 2013;20:385–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Byun JS, Suh YG, Yi HS, Lee YS, Jeong WI. Activation of toll-like receptor 3 attenuates alcoholic liver injury by stimulating Kupffer cells and stellate cells to produce interleukin-10 in mice. J Hepatol 2013;58:342–9. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Fig. 6
NIHMS2045173-supplement-Supplemental_Fig__6.tif (247KB, tif)
Supplemental Fig. 7
NIHMS2045173-supplement-Supplemental_Fig__7.tif (247KB, tif)
Supplemental Fig. 8
NIHMS2045173-supplement-Supplemental_Fig__8.tif (453.3KB, tif)
Supplemental Fig. 5
NIHMS2045173-supplement-Supplemental_Fig__5.tif (942.4KB, tif)
Supplemental Fig. 4
NIHMS2045173-supplement-Supplemental_Fig__4.tif (104.1KB, tif)
Supplemental Fig. 3
NIHMS2045173-supplement-Supplemental_Fig__3.tif (1.1MB, tif)
Supplemental Fig. 2
NIHMS2045173-supplement-Supplemental_Fig__2.tif (636.3KB, tif)
Supplemental Fig. 1
NIHMS2045173-supplement-Supplemental_Fig__1.tif (1.7MB, tif)

RESOURCES