Natural antibodies are required for clearance of necrotic cells and recovery from acute liver injury

Abstract

Background & Aims

Hepatocellular necrosis is common in both acute and chronic liver injury and may evolve to fibrosis and liver failure. Injury leads to accumulation of necrotic cell debris in the liver, which drives persistent inflammation and poor recovery. This study investigated the role of natural antibodies (NAbs) in the clearance of necrotic cells in the injured liver, their impact on tissue regeneration and their potential as a therapy for acute liver injury.

Methods

We used murine models of drug-induced liver injury and focal thermal injury in immunocompetent and antibody-deficient mice (Rag2^-/- and IgMi). Intravital microscopy was used to investigate the role of NAbs in the phagocytosis of necrotic cells in the liver in vivo. Immunostainings were used to quantify the extent of liver necrosis (fibrin), antibody deposition (IgM and IgG) and cellular proliferation (Ki67).

Results

Both IgM and IgG NAbs bound necrotic liver areas and opsonized multiple debris molecules released during hepatocellular necrosis such as DNA, histones, actin, phosphoinositides and mitochondrial cardiolipin, but not phosphatidylserine. Rag2^-/- and IgMi mice presented impaired recovery from liver injury, which was correlated to the sustained presence of necrotic debris in the tissue, prolonged inflammation and reduced hepatocellular proliferation. These defects were rescued by treating mice with NAbs after the induction of injury. Mechanistically, in vitro and in vivo, phagocytosis of necrotic debris was dependent on NAbs via Fcγ receptors and CD11b. Moreover, NAb-mediated phagocytosis of necrotic cell debris occurs in two waves, firstly driven by neutrophils and then by recruited monocytes. Importantly, supplementation of immunocompetent mice with NAbs also improved liver regeneration significantly, demonstrating the therapeutic potential of natural IgM and IgG.

Conclusion

NAbs drive the phagocytosis of necrotic cells in liver injury and promote liver regeneration and recovery.

Impact and implications

Treatment with natural antibodies after acute liver injury improved recovery by increasing the clearance of necrotic debris and by improving cellular proliferation in the liver. This preclinical study provides a basis for the development of an immunotherapy for patients with early-stage, reversible, liver injury that aims to prevent disease chronification into fibrosis and liver failure.

Graphical abstract

Highlights

• •Natural antibodies opsonize exposed self-antigens during hepatocellular necrosis.
• •Phagocytosis of necrotic hepatocytes requires natural antibodies, FcγRs and CD11b.
• •Natural antibodies drive hepatocellular proliferation and tissue regeneration after liver injury.
• •Treatment with natural antibodies improves the recovery from liver injury in both immunodeficient and immunocompetent mice.

Introduction

According to a recent report from the EASL-Lancet commission, a current problem in tackling hepatic illnesses has been the clinical focus on end-stage liver diseases whereas early-stage, still reversible, liver diseases are often disregarded.¹ Clearly, there is a need for therapeutic strategies to resolve acute and early-stage liver disorders, preventing their worsening and chronification.

Many hepatic disorders are characterized by mitochondrial dysfunction that drives hepatocyte death by necrosis.² In fact, hepatocellular necrosis is common in both acute and chronic liver diseases and the worst prognosis in patients with acute liver failure (ALF) is for those with significant hepatocellular necrosis.³ Necrosis results in plasma membrane rupture and release of intracellular contents,⁴ which are deposited as cellular debris and recognized as damage-associated molecular patterns, acting as powerful inducers of inflammation. The prolonged presence of necrotic debris in the liver aggravates inflammation, causing its chronification and driving the evolution of disease.⁵ Thus, therapies that accelerate the removal of hepatic cell debris during liver injury would reduce inflammation and collateral tissue damage, while simultaneously allowing for the replicative potential of hepatocytes to regenerate the tissue. Recent work demonstrated that monocyte-derived macrophages play a key role in repairing necrotic injuries in a model of ConA-induced liver injury by inducing a wall of cell death-resistant SOX9+ hepatocytes around the lesion and by activating hepatic stellate cells to constrict the necrotic lesion to facilitate its repair.⁶ However, the mechanisms underlying the clearance of necrotic debris from the liver are poorly understood, preventing its therapeutic use in acute liver disorders such as drug-induced liver injury, ischemia-reperfusion injury and liver graft dysfunction.

Natural antibodies (NAbs) are circulating polyreactive immunoglobulins that arise without exogenous antigenic stimulation. NAbs are mostly IgM and IgG3 isotypes produced by B-1 cells,⁷ being characterized as low affinity antibodies that, due to their polyreactivity, bind to different classes of self and exogenous molecules including proteins, nucleic acids, carbohydrates and lipids.⁸ In contrast to adaptive antibodies that can bind to virtually any epitope, NAbs have germline-encoded variable regions which bias their recognition capacity to phylogenetically conserved molecules.^9,10 In fact, natural IgM and IgG harvested from human cord blood or germ-free mice can bind to common molecules such as DNA, fibrin, actin, tubulin, lysophosphatidylcholine and oxidized phospholipids.[11], [12], [13], [14] Importantly, these are all debris molecules that will become exposed upon hepatocellular necrosis, which must be cleared from the hepatic tissue to allow hepatocytes to regenerate. Thus, we hypothesized that NAbs opsonize hepatic necrotic debris and act as adaptors for their recognition and clearance by recruited phagocytes.

To test this hypothesis, we utilized in vitro and in vivo approaches to understand how necrotic cells are cleared from injury sites and to determine their impact in liver regeneration. Protein and lipid blots were performed using purified debris to determine that multiple cellular components in necrotic debris were recognized by both IgM and IgG NAbs. Using liver intravital microscopy, we investigated the phagocytosis of necrotic cell debris in real time, assessing the participation of NAbs in both immunocompetent and immunodeficient (Rag2^-/- and IgMi) mice.

Materials and methods

An extended description of the materials and methods used is provided in the supplementary information.

Mice

C57BL/6J and C57BL/6NRj mice were purchased from Janvier Labs. C57BL/6N-Rag2Tm1/CipheRj (Rag2^-/-) were bred in specific pathogen-free conditions at the Animal Facility of the Rega Institute (KU Leuven). C57BL/6-Ly 5.1 and IgMi mice were provided by Ari Waisman. All mice used in this study were between 10-12 weeks old and both male and female mice were equally distributed across experiments (no phenotypic differences between genders were observed). All experiments were approved by the Animal Ethics Committee from KU Leuven (registry number: P125/2019).

Acetaminophen-induced liver injury model

Mice were fasted for 15 h before a single oral gavage of vehicle or acetaminophen (APAP: 600 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) dissolved in warm PBS. Fasting was performed to guarantee full APAP absorption and to increase reproducibility among the experiments. After 6, 12, 24, 48 or 72 h, mice were sacrificed under anesthesia containing ketamine (80 mg/kg) and xylazine (4 mg/kg) for liver and blood harvesting.

Image analysis

Liver cryosections were imaged using an Andor Dragonfly 200 spinning-disk confocal microscope equipped with a 25X objective. The measurement of fibrin(ogen), IgG, IgM and Ki67 areas was performed using FIJI software. The positive area for each fluorophore was determined by thresholding the minimum and maximum pixel values. Then, the positive area was represented as a percentage of the total area of the image. Each dot in the graphs is the averaged quantification of at least 10 random images of one mouse liver.

In vivo phagocytosis assay

Mice were anesthetized by a subcutaneous injection of 80 mg/kg ketamine and 4 mg/kg xylazine. Then, a small midline incision was made in the abdominal area to expose the liver. With a hot needle (26G), a liver burn injury of approximately 1 mm³ was made on which a droplet of pHrodo Red succinimidyl ester (4 μM; Thermo Fisher Scientific) was administered. The incision was closed and after 6 h mice were again anaesthetized for imaging of the injury site by intravital microscopy. For the restitution of NAbs, Rag2^-/- mice were treated with purified IgM and IgG antibodies (100 μg each) intravenously 30 min prior the focal thermal injury (FTI). Intravital imaging was performed as previously described^15,16

Statistical analysis

All statistical analysis was performed in GraphPad Prism v9.3.1. Significance between two groups was analyzed by Student’s t test and between multiple groups with one-way ANOVA. Differences were considered significant if p ≤0.05. Grubb’s test (extreme studentized deviate) was applied to determine whether extreme values were significant outliers from the rest. Data were represented as ± SEM.

Results

Natural IgM and IgG are rapidly deposited on necrotic cell debris following liver injury

To evaluate the role of NAbs in clearance of necrotic cell debris, we used a well-described and clinically relevant model of acute liver injury, namely APAP-induced liver injury.^16,17 Consistent with previous work, mice that received 600 mg/kg of APAP orally developed extensive and time-dependent hepatocellular necrosis starting 6 h post-overdose, peaking at 24 h and resolving from 48 h onwards, as assessed by fibrin(ogen) deposition in liver cryosections (Figs 1A,B and Fig. S1). Fibrin(ogen) staining was used as the main method for identification and measurement of necrotic areas in liver cryosections. Liver injury was confirmed by increased levels of alanine transaminase (ALT) in murine sera after the APAP challenge (Fig. 1C), which followed similar kinetics to the centrilobular necrosis observed in cryosections. Liver injury resulted in recruitment of neutrophils to the liver after as little as 6 h (Fig. 1D), followed by significant recruitment of monocytes 24 h after APAP intoxication (Fig. 1E). The recruitment of both types of phagocytes peaked at 24 h, however, the percentage of monocytes remained elevated until 72 h, further into the resolution phase of injury (Fig. 1D, E).

Fig. 1.

Natural IgM and IgG are rapidly deposited on necrotic cells following injury.

(A) Representative images showing immunostaining of liver cryosections from control mice and mice gavaged with APAP (600 mg/kg) for 24 and 48 h. Gray: fibrin(ogen), red: IgM, green: IgG, orange: merged IgM and IgG. Scale bar = 100 μm. See full panel from 6 to 72 h in Fig. S1. (B) Quantification of the fibrin(ogen) positive area fraction in liver cryosections. (C) Serum ALT in APAP-challenged mice. (D) Flow cytometry of liver NPCs showing the percentage of neutrophils (Ly6G⁺). (E) Flow cytometry of liver NPCs showing the percentage of monocytes (Ly6G^-/CCR2⁺). (F) Quantification of the IgM positive area fraction in liver cryosections. (G) Quantification of the IgG positive area fraction in liver cryosections. (H) Quantification of debris-reactive serum IgM after APAP challenge. (I) Quantification of debris-reactive serum IgG after APAP challenge. Data are from mice 6-72 h after an oral gavage of 600 mg/kg APAP (A-I) and are represented as mean ± SEM. Each dot in the graph represents a single mouse (n ≥4) (B–I). Image quantifications were pooled from 10 random pictures per liver. (B, F, G). ∗p ≤0.05 compared to control group (one-way ANOVA). ALT, alanine aminotransferase; APAP, acetaminophen; NPCs, non-parenchymal cells.

Once the kinetics of hepatic necrosis was ascertained, we investigated if NAbs were deposited in the injured liver. Liver cryosections were stained with fluorescently labeled anti-mouse IgM and anti-mouse IgG antibodies, which showed that both natural IgM and IgG were bound to the necrotic areas identified by the fibrin(ogen) staining (Figs 1A and Fig. S1). Interestingly, accumulation of IgM and IgG in necrotic areas occurred in parallel to the development of liver injury, also peaking at 24 h and decreasing with tissue recovery 48-72 h after injury (Figs 1A,F,G and Fig. S1). The staining in necrotic regions was not due to unspecific labeling or autofluorescence as confirmed in unstained liver cryosections (Fig. S2A). We also found that the deposition of NAbs in the necrotic liver led to the reduction of circulating IgM and IgG that is reactive to necrotic debris (Fig. 1H, I). While both NAb isotypes were reduced in the bloodstream 6 h after APAP, the reduction of natural IgG was more pronounced and persistent during liver injury. This shows that these antibodies were sequestered from the bloodstream as they became deposited in injury sites.

To visualize the binding of NAbs to necrotic cells in living tissue, confocal intravital microscopy (IVM) was performed 24 h after the APAP challenge. Prior to imaging, mice were injected intravenously (i.v.) with Sytox green, a membrane-impermeable DNA dye, and anti-mouse IgM and IgG to label the necrotic cells and deposited NAbs, respectively. Using this approach, we confirmed the binding of both natural IgM and IgG to necrotic cells in vivo (Fig. 2A and movie S1). Moreover, the plasma membrane of dying hepatocytes was often positive for IgM/IgG labeling even before these cells became positive for Sytox green, indicating that the binding of NAbs is fast and independent of the full demise of the cells (Fig. S2B and movie S2). Altogether, these data demonstrate that NAbs leave the systemic circulation and become deposited within the necrotic areas during liver injury. Binding of NAbs to necrotic debris followed the kinetics of liver injury, decreasing progressively towards 72 h after the APAP challenge. These observations suggested that recognition and binding of NAbs to necrotic debris may have a role in necrotic cell clearance and tissue repair.

Fig. 2.

Natural IgM and IgG antibodies bind multiple self-antigens exposed upon necrotic cell death.

(A) Representative confocal intravital microscopy images showing IgM (red) and IgG (cyan) deposition in dead/dying hepatocytes (sytox green⁺) 24h after APAP intoxication (600 mg/kg). Scale bar = 50 μm. See also supplementary videos 1 and 2. (B) Binding of serum natural IgM (red) and IgG (cyan) to a necrotic hepatocyte debris spot. DNA is in blue and F-actin in green. Scale bar = 100 μm. (C,D) MFI of IgM and IgG labeling after pre-treatment of debris spot for 15 min with DNase and/or trypsin before adding serum. (E) Dot blots showing the reactivity of purified IgM and IgG to 4 μg of purified DNA, histones and actin. (F,G) Lipid blot showing the reactivity of purified IgM and IgG to lipids (100 pmol). Data are represented as mean ± SEM. ∗p ≤0.05 compared to the untreated sample (one-way ANOVA). APAP, acetaminophen; CL, cardiolipin; DAG, diacylglycerol; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; MFI, mean fluorescence intensity; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PI(3)P, PI 3-phosphate; PI(4)P, PI 4-phosphate; PI(5)P, PI 5-phosphate; PI(3,4)P2, PI 3,4-bisphosphate; PI(4,5)P2, PI 4,5-bisphosphate; PI(3,4,5)P3, PI 3,4,5-trisphosphate; PS, phosphatidylserine; S1P, sphingosine 1-phosphate; SM, sphingomyelin; TAG, triacylglycerol.

Supplementary video related to this article can be found at https://doi.org/10.1016/j.jhepr.2024.101013.

The following is the supplementary data related to this article:

Video S1

4

Natural IgM and IgG antibodies bind multiple self-antigens exposed during hepatocellular necrosis

We next sought to investigate if NAbs bound specifically to necrotic debris or if they were trapped non-specifically in injury sites. For this purpose, hepatocytes from healthy mouse livers were purified and crushed mechanically to expose their intracellular contents, which were then spotted onto coverslips. The necrotic debris spots were incubated with mouse serum and labeled with anti-mouse IgM and IgG. We observed that the hepatocyte debris spots, shown by DNA and F-actin staining, were bound by IgM and IgG from healthy mouse serum (Fig. 2B). The binding of antibodies was antigen specific, since incubation with secondary antibodies alone yielded no labeling of the debris (Fig. S2C). Next, the types of antigens that these polyreactive NAbs recognized in the necrotic debris were investigated. To distinguish whether NAbs were preferentially bound to nucleic acids or proteins, we pre-treated the debris spots with DNase and/or trypsin for 15 min. Treatment with DNase and trypsin was sufficient to degrade DNA and F-actin substantially, but not completely (Fig. S2D). Pre-incubation of debris spots with either of the enzymes reduced both IgM and IgG labeling significantly, suggesting that NAbs bind DNA and protein epitopes (Fig. 2C, D). Combined treatment with DNAse and trypsin resulted in a cumulative reduction of IgM and IgG binding. To further investigate the specificity of IgM and IgG NAbs towards necrotic cell debris, dot blots with purified DNA, histones and actin were performed and incubated with mouse serum. We found that serum NAbs indeed bind directly to histones, actin and DNA, albeit with lower avidity for the latter (Fig. S2E). In order to avoid interference from other possible debris-binding molecules present in serum, we also performed dot blots using purified murine IgM or IgG and we found a similar binding pattern (Fig. 2E).

To assess if the recognition of necrotic cell molecules by NAbs also encompassed lipids, we performed assays with lipid strips containing a variety of cellular phospholipids, phosphoinositides and their intermediates. We observed that IgM and IgG NAbs recognized several phosphoinositides such as phosphatidylinositol 3 phosphate [PI(3)P], PI(4)P, PI(5)P, PI(3,5)P2, PI(4,5)P2 (Fig. 2F) as well as phosphatidic acid, PI(3,4,5)P3 and the mitochondrial phospholipid cardiolipin (Fig. 2G). Importantly, we observed no binding of IgM nor IgG NAbs to phosphatidylserine,¹⁸ phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, PI, sphingomyelin, sphingosine-1-phosphate, diacylglycerol, lysophosphatidic acid, lysophosphocholine, triacylglycerol or cholesterol. These data indicate that NAbs are capable of recognizing specific categories of membrane lipids, e.g. phosphoinositides and cardiolipin, which are restricted to the cytoplasmic leaflet of the plasma membrane and to intracellular organelles such as endosomes, Golgi, endoplasmic reticulum and mitochondria (for cardiolipin).^19,20 These results further show that both IgM and IgG NAbs bind to multiple classes of cellular components that are exposed after hepatocellular necrosis including DNA, actin, histones and phospholipids. Yet, natural IgG was able to bind essentially to the same antigens as natural IgM, suggesting a redundant recognition of necrotic cell debris for the two types of NAbs.

Rag2^-/- mice exhibit impaired cell debris clearance, prolonged inflammation and delayed recovery from liver injury

Once we determined that NAbs recognize necrotic cell debris and are present within the necrotic areas in vivo, we investigated the hypothesis that NAbs play a role in debris clearance and recovery from liver injury. For this, we challenged Rag2^-/- mice, which lack mature lymphocytes and consequently antibodies, with APAP for 24 and 48 h. We chose to work with these timepoints to evaluate if there were differences in the peak of injury (24 h) and to assess if defective tissue repair occurs (48 h).

Immunostaining of liver cryosections of APAP-challenged mice were performed to confirm the absence of NAb deposition in the injured livers of Rag2^-/- mice. As expected, no IgM nor IgG was present in Rag2^-/- livers 24 or 48 h after APAP intoxication, in contrast to wild-type (WT) Rag2^+/+ samples (Fig. S3A). To evaluate the hepatic necrotic areas directly, we performed IVM of livers of WT and Rag2^-/- mice after APAP intoxication. The necrotic debris was identified with Sytox green, since DNA is a common debris component found in the liver following hepatocellular necrosis.⁴ With this approach, we found that the DNA debris in livers of WT mice had already been largely cleared 48 h after APAP, however, Rag2^-/- livers still presented a significant amount of extracellular DNA in the tissue, indicating an impairment in DNA debris clearance (Fig. 3A,B). Fibrin(ogen) staining showed very similar results, in which Rag2^-/- mice presented significantly larger necrotic areas 48 h post injury (Fig. 3C).

Fig. 3.

Rag2^-/- mice have impaired cell debris clearance, prolonged inflammation and delayed recovery from liver injury.

(A) Representative intravital microscopy images of WT and Rag2^-/- mice showing the deposition of DNA (sytox green⁺) in the liver 24 and 48 h after APAP overdose (600 mg/kg). Scale bar = 100 μm. (B), Quantification of the sytox green⁺ area in the liver. (C) Quantification of the fibrin(ogen)⁺ area fraction in liver cryosections. See Fig. S3A for IgM and IgG stainings. (D) Serum ALT levels in WT and Rag2^-/- mice 24 and 48 h after APAP overdose. Data are from WT and Rag2^-/- mice 24 and 48 h after an oral gavage of 600 mg/kg APAP (A-D) and are represented as mean ± SEM. Each dot in the graph represents a single mouse (B-D). Image quantifications were pooled from 10 random pictures per liver (B–C). ∗p ≤0.05 (Student’s t test). ALT, alanine aminotransferase; APAP, acetaminophen; Rag2, recombination activating gene 2; WT, wild-type.

It is important to mention that the absence of NAbs did not cause excessive liver injury, as shown by similar ALT levels between WT and Rag2^-/- mice 24 h after APAP (Fig. 3D). Instead, Rag2^-/- mice presented ALT levels that remained as high at 48 h as they were at the peak of injury, showing that liver injury is essentially not resolving. Fibrin(ogen) staining of Rag2^-/- livers reproduced the observations with DNA debris by IVM, in which trends towards an accumulation of necrotic debris and larger necrotic areas 24 h after APAP challenge became significantly increased after 48 h (Fig. 3A,C,D). Altogether, Rag2^-/- mice presented a severe delay in liver recovery 48 h post injury, as shown by DNA debris accumulation in vivo, larger fibrin(ogen)⁺ necrotic areas still present in the liver, and persistently elevated ALT levels (Fig. 3B-D). Corroborating the prolonged liver injury of Rag2^-/- mice, we also observed a significantly higher recruitment of neutrophils and monocytes, indicating that Rag2^-/- livers are proportionally more inflamed compared to WT livers (Fig. 3E,F). Altogether, this suggests that Rag2^-/- mice have impaired necrotic debris clearance and a delayed resolution of liver injury.

Mice that lack soluble antibodies (IgMi) have a defective regenerative response to necrotic liver injury

To refine our observations with Rag2^-/- mice, we investigated APAP-induced liver injury in IgMi mice. These mice develop mature lymphocytes, including B cells that express membrane IgM as the B cell receptor, however, they are unable to perform class switch and to differentiate into plasma cells.²¹ The resultant phenotype is that IgMi mice have mature lymphocytes but do not secrete soluble antibodies.²² To confirm the absence of NAbs in IgMi mice, we performed liver cryosections 24 h and 48 h after APAP overdose and stained them with fluorescent anti-IgM and anti-IgG antibodies. As in Rag2^-/- mice, we did not detect any IgM or IgG bound to the necrotic areas (Fig. 4A). IgMi mice presented larger fibrin(ogen)⁺ necrotic areas in the liver 24 h after APAP, which remained larger at 48 h when compared to WT mice (Fig. 4A,B). Moreover, serum ALT levels were not different between IgMi and WT mice 24 h after injury, however, ALT levels in IgMi mice remained significantly elevated at the 48 h timepoint (Fig. 4C), indicating that the necrotic injury is prolonged comparably in IgMi mice and Rag2^-/- mice.

Fig. 4.

Mice that lack soluble antibodies (IgMi) have a defective regenerative response to necrotic liver injury.

(A) Representative images showing immunostaining of liver cryosections from WT and IgMi mice, 24 and 48 h after oral gavage with APAP (600 mg/kg). White squares indicate the area zoomed. Gray: fibrin(ogen), red: IgM and green: IgG. Scale bar = 100 μm. (B) Quantification of fibrin(ogen)⁺ area fraction in liver cryosections. (C) Serum ALT levels in WT and IgMi mice challenged with APAP. (D) Flow cytometry of liver NPCs showing the percentage of neutrophils (Ly6G⁺). (E) Flow cytometry of liver NPCs showing the percentage of monocytes (Ly6G^-/CCR2⁺). (F) Percentage of neutrophils expressing CD11b by flow cytometry. (G) Serum levels of CXCL1 in control mice or after receiving APAP for 24 and 48 h. (H) Serum levels of TNF-α as in G. See also Fig. S4 for IL-6 and IL-10 levels. (I) Quantification of Ki67⁺ hepatocytes in liver cryosections of control and APAP-challenged mice. Data are from WT and IgMi mice 24 and 48 h after an oral gavage with 600 mg/kg APAP (A-I) and are represented as mean ± SEM. Each dot in the graph represents a single mouse (B–I). Image quantifications were pooled from 10 random pictures per liver (B, I). ∗p ≤0.05 (Student’s t test). ALT, alanine aminotransferase; APAP, acetaminophen; NPCs, non-parenchymal cells; WT, wild-type.

We also analyzed the recruitment of phagocytes to the livers of IgMi mice using flow cytometry. The migration of neutrophils to IgMi livers was significantly higher at both 24 and 48 h post injury in comparison to WT (Fig. 4D). Monocytes were also recruited to the livers of WT and IgMi mice throughout injury, but no differences were observed between the mouse strains (Fig. 4E). Interestingly, almost 100% of neutrophils in the livers of IgMi mice expressed surface CD11b 24 h after injury, compared to only 40% in WT mice. CD11b expression is used as a marker of neutrophil activation, which indicated that hepatic neutrophils were more activated in IgMi mice during liver injury. Although CD11b expression decreased at 48 h in both groups, it remained significantly higher in IgMi neutrophils (Fig. 4F). In addition, we found increased levels of circulating CXCL1, TNF-α, IL-6 and IL-10 in IgMi mice, confirming that the absence of NAbs during necrotic liver damage promotes a severe dysregulation of the immune response to injury (Figs 4G,H and Fig. S4A,B). Of note, CXCL1 and TNF-α were significantly elevated during the peak of liver injury and CXCL1 remained elevated after up to 48 h in IgMi mice, which correlates with disproportionate neutrophil recruitment and demonstrates the excessive pro-inflammatory state of mice lacking NAbs during injury.

We also hypothesized that besides restricting the extent of hepatic damage and inflammation during injury, NAbs had a beneficial effect on liver regeneration.²³ To evaluate this, we assessed the levels of cellular proliferation in the injured liver via immunostaining of Ki67 in cryosections of WT and IgMi mice. We found a significant increase in cellular proliferation 48 h post injury (regenerative phase) in WT mice, which was essentially absent in the livers of NAb-deficient IgMi mice (Fig. 4I). This showed that NAbs drive hepatocellular proliferation after injury, which is required for liver regeneration and recovery. Considering that we could not observe cells proliferating in the livers from IgMi mice at 48 h, we investigated whether the cell proliferation was only delayed or if it was completely blocked in the liver of mice lacking soluble antibodies. In this way, we performed an APAP challenge for 72 h in IgMi mice. At this timepoint, IgMi mice still had fibrin(ogen) deposition around the centrilobular veins (Fig. S4C) and presented higher ALT levels than WT mice, whose levels were already back to baseline (Fig. S4D). Interestingly, we observed cells proliferating in IgMi livers at the 72 h timepoint (Fig. S4E), indicating that their hepatocellular proliferation is delayed by approximately 1 day, not prevented. Altogether, the data on IgMi mice corroborated our findings in Rag2^-/- mice and confirmed that the defective liver recovery is mainly due to the absence of NAbs. The lack of NAbs worsens several critical events during liver injury, including delaying the clearance of necrotic debris and the resolution of inflammation, and impairing the proliferation of hepatocytes.

Restitution of NAbs to Rag2^-/- mice improves the recovery from liver injury

To confirm the role of NAbs in the recovery from liver injury, we subjected Rag2^-/- mice to APAP-induced liver injury and after 4 h they received an i.v. transfer of Rag2^+/+ (WT) or Rag2^-/- serum (lacking NAbs). Treatment with WT but not with Rag2^-/- serum successfully restored the deposition of natural IgM and IgG in necrotic areas in the liver (Fig. 5A). The restitution of NAbs did not affect the initial injury significantly, as evidenced by similar fibrin(ogen)⁺ areas and serum ALT levels after 24 h (Fig. 5A-C). It is important to mention that the serum ALT levels and fibrin(ogen) areas followed a similar pattern to what we observed in the previous experiment when we compared WT with Rag2^-/- mice (Fig. 3C,D). In accordance with the similar levels of injury, the serum transfer did not affect the recruitment of neutrophils nor monocytes to the liver (Fig. S5A,B), nor the production of CXCL1, TNF-α, IL-6 and IL-10, except for an increase in IL-10 48 h after injury in mice treated with Rag2^-/- serum (Fig. S5C-F). This indicates that the induction of injury and inflammation in the liver was not altered significantly by the serum transfer and that NAbs do not regulate leukocyte recruitment to the necrotic liver.

Fig. 5.

Restitution of NAbs to Rag2^-/- mice improves the recovery from liver injury.

(A) Representative images showing immunostaining of liver cryosections from APAP-challenged Rag2^-/- mice treated with 150 μl of WT or Rag2^-/- serum. Gray: fibrin(ogen), red: IgM, green: IgG. Scale bar = 100 μm. (B) Quantification of the fibrin(ogen)⁺ area fraction in liver cryosections. (C) Serum ALT levels at 24 and 48 h after APAP administration. (D) Quantification of Ki67⁺ hepatocytes in liver cryosections of Rag2^-/- mice after serum transfer. (E) Quantification of Ki67⁺ hepatocytes in liver cryosections of Rag2^-/- mice treated with 100 μg (i.v.) of purified IgM and IgG. See also figure S5G for Ki67 staining. Data are from Rag2^-/- mice that received an intravenous injection of WT serum or Rag2^-/- serum (A-D) or 100 μg purified IgG and IgM (E) 4 h after an oral gavage with 600 mg/kg APAP and were sacrificed 24 and 48 h after the APAP challenge. Each dot in the graph represents a single mouse (B-E). Image quantifications were pooled from 10 random pictures per liver (B, D and E). ∗p ≤0.05 (Student’s t test). ALT, alanine aminotransferase; APAP, acetaminophen; NAbs, natural antibodies; Rag2, recombination activating gene 2; WT, wild-type.

However, Rag2^-/- mice injected with WT serum displayed an improved liver recovery 48 h after injury, with significantly less necrotic fibrin(ogen)⁺ areas than mice treated with Rag2^-/- serum (Fig. 5A,B). Interestingly, the transfer of WT sera to Rag2^-/- mice was sufficient to increase the proliferation of hepatic cells 48 h after injury, which was significantly elevated in comparison to Rag2^-/- mice that received Rag2^-/- serum (Figs 5D and Fig. S5G). To confirm that the improvement of liver regeneration by NAb restitution was not due to the presence of other serum components, Rag2^-/- mice were treated with total IgM and IgG antibodies purified from WT sera. Similarly to the serum transfer experiment, treatment with purified antibodies alone significantly improved hepatocellular proliferation in the injured liver in comparison to Rag2^-/- mice treated with vehicle (Fig. 5E). These data confirm that NAbs are central to drive liver repair after necrotic injury and that sera containing NAbs are sufficient to rescue the poor recovery from liver injury observed in Rag2^-/- mice.

Natural antibodies drive the phagocytosis of hepatic cell debris through FcγRs and CD11b

As NAbs improved the recovery from necrotic liver injury, we further investigated the mechanism by which they exert their role in injury resolution. Antibody-opsonized antigens are known to be phagocytosed in an FcR-dependent manner, therefore, NAb opsonization of necrotic cell debris likely promotes its clearance by phagocytosis. To test this hypothesis, we developed an in vitro assay of necrotic debris phagocytosis by feeding necrotic cell debris to murine macrophage-like RAW 264.7 cells. Debris was prepared by crushing HepG2 cells and labeling them covalently with pHrodo Red succinimidyl ester, a pH-sensitive dye that emits increased fluorescence in acidified compartments such as phagosomes. This approach allowed us to discern the debris that was phagocytosed and matured in phagosomes from debris bound to or in proximity to cells. Labeled necrotic debris was left non-opsonized (PBS) or opsonized with mouse serum or with heat-inactivated (HI) serum (lacking complement activation). Opsonization with serum led to a significant increase in the phagocytosis of necrotic debris by RAW cells compared to non-opsonized (PBS) control samples (Fig. S6A). Remarkably, necrotic debris phagocytosis was essentially identical if debris were opsonized with native or HI serum, indicating that the complement cascade was not required in these conditions. In addition, incubation of RAW cells with latrunculin B, an inhibitor of actin polymerization known to prevent phagocytosis,²⁴ completely blocked the internalization of necrotic debris.

We then investigated the FcRs expressed by RAW cells and found that they expressed constitutively CD64 (FcγRI), CD32 (FcγRII), CD16 (FcγRIII) and, at lower level, CD16a (FcγRIV). However, the expression of the two FcRs that recognize IgM-coated targets, CD351 (Fcα/μR) and Faim3 (FcμR) was absent (Fig. S6B). Stimulation of RAW cells with NAb-opsonized necrotic debris for 6 h increased the expression of CD64 and CD16a but reduced the expression of CD32 (Fig. S6C-F). Even after stimulation with NAb-opsonized debris, we could not detect mRNA for CD351 and Faim3 in RAW cells (data not shown). The phagocytosis of NAb-coated debris increased the expression of IL-10 while it did not alter the expression of TNF-α, suggesting an anti-inflammatory role of NAb-mediated phagocytosis (Fig. S6G,H).

After validating our methodology, we investigated necrotic debris phagocytosis in primary mouse neutrophils. Similar to our findings with RAW cells, serum-opsonized debris were efficiently phagocytosed by murine neutrophils and heat inactivation of serum did not alter the phagocytosis rate (Fig. 6A), suggesting that NAbs but not complement are required in necrotic debris clearance. Importantly, the internalization of serum-opsonized debris is also completely inhibited by latrunculin B, indicating that phagocytosis is indeed the main process of internalization (Fig. 6A). Our next step was to identify the receptors involved in the phagocytosis of NAbs-debris immunocomplexes. First, we performed a general blockade of FcγRs by treating neutrophils with 100 μg/ml of purified mouse IgG, as previously described,²⁵ and we found a 50% reduction in phagocytosis efficiency of debris opsonized with normal serum or HI serum, suggesting that FcγRs and natural IgG are driving at least half of necrotic debris phagocytosis (Fig. 6A). Considering that mouse neutrophils might not express Fc receptors for IgM,[26], [27], [28] we hypothesized that CD11b could have a role in the phagocytosis of IgM-coated debris, since complement receptor 3 has been implicated in the internalization of IgM/IgA-opsonized targets.²⁹ Thus, we performed a combined blockade of FcγRs and CD11b, which led to an even greater reduction of phagocytosis of serum-opsonized debris. Conversely, CD11b blockade did not alter the rate of phagocytosis of HI-opsonized debris (Fig. 6A).

Fig. 6.

Natural antibodies drive the phagocytosis of necrotic cell debris through FcγRs and CD11b.

(A) Phagocytosis by primary mouse neutrophils 3 h after adding debris opsonized with normal or HI serum. FcγRs were blocked by adding 100 μg/ml IgG to the cells. CD11b was blocked with 10 μg/ml anti-mouse CD11b. Phagocytosis was blocked with 10 μM Latrunculin B. (B,C) Phagocytosis by primary mouse neutrophils 3 h after adding IgG- or IgM-opsonized debris. Receptors were blocked using 10 μg/ml anti-CD16.2, anti-CD16/CD32, anti-CD11b or 100 μg/ml purified mouse IgG. (D) Phagocytosis by primary human neutrophils 3 h after adding debris opsonized with normal or HI serum. FcγRs were blocked with 100 μg/ml IgG and Latrunculin B was used at 10 μM. (E) Representative confocal images from human neutrophils containing pHrodo⁺ phagosomes. FcγRs were blocked with 100 μg/ml purified human IgG. Scale bar = 10 μm. See also supplementary video 6. (F) Representative image from immunostaining of liver cryosections from WT and Rag2^-/- mice 12 h after FTI showing deposition of IgM (red) and IgG (cyan). Scale bar = 200 μm. (G) Representative intravital microscopy images showing neutrophils (Ly6G⁺) phagocytosing necrotic debris as in H. Scale bar = 20 μm. Yellow square represents the area zoomed. Also see supplementary videos 3-5. (H) Quantification of the percentage of neutrophils (Ly6G⁺, green) phagocytosing necrotic debris 6 h after a FTI in the liver of WT, Rag2^-/- mice. A separate group of Rag2^-/- mice were restituted with total purified Abs (100 μg/mouse) (n ≥4). The FTI was labeled with a droplet of 4 μM pHrodo Red succinimidyl ester. Each dot in the graph represents a mouse. (I) Quantification of Ki67⁺ cells in liver cryosections of WT, Rag2^-/- and Rag2^-/- mice treated with purified IgM and IgG (100 μg/mouse). Mice were treated with purified antibodies 30 min prior to the FTI (n ≥4). Data are represented as mean ± SEM. ∗p ≤0.05 (one-way ANOVA for panels A, B, D, H and I); (Student’s t test for panel C). Abs, antibodies; FTI, focal thermal injury; HI, heat-inactivated; Rag2, recombination activating gene 2; WT, wild-type.

To better dissect the role of each immunoglobulin isotype, as well as to identify the FcγR involved in the recognition of IgG-coated debris, we performed the phagocytosis assay using necrotic debris opsonized with purified antibodies. Similar to our findings with serum-opsonized debris, the blockade of FcγRs using excess IgG led to a significant reduction in the phagocytosis of IgG-coated debris. Surprisingly, the combined blockade of FcγRII, FcγRIII and FcγRIV did not affect phagocytosis, indicating that FcγRI might be the predominant phagocytic receptor for IgG-coated debris (Fig. 6B). Using purified IgM-coated necrotic debris, CD11b blockade also resulted in an approximately 50% reduction in phagocytosis, supporting a role for this integrin in the clearance of IgM-coated debris (Fig. 6C). These data suggest that FcγRs are driving IgG-mediated phagocytosis of necrotic debris, whereas IgM-mediated phagocytosis is occurring at least in part via CD11b.

To investigate the mechanisms of NAb-dependent necrotic debris phagocytosis in a more translational manner, we also used blood-derived human neutrophils. Primary neutrophils were also proficient in the phagocytosis of necrotic debris opsonized with native serum, a process that was once more prevented by latrunculin B (Fig. 6D). Of note, no differences were observed in phagocytosis efficiency if necrotic debris was opsonized with donor-matched serum or not, corroborating the notion that NAbs share broad recognition of conserved molecules between individuals (data not shown). The phagocytosis of necrotic debris, quantified as the percentage of neutrophils containing pHrodo-positive events, was quite similar between normal and HI serum, confirming that the clearance of necrotic debris occurs independently of the complement cascade (Fig. 6D). Similarly to murine neutrophils, FcγR blocking in human neutrophils reduced the phagocytosis of necrotic debris by more than 50%, an inhibitory effect that was equal when debris was opsonized with native or HI serum (Fig. 6D,E). Using 3D confocal imaging 3 h after adding serum-opsonized debris, we observed that neutrophils carried multiple pHrodo⁺ phagosomes and confirmed that the positive events were inside the cells (Fig. 6E and movie S3). This indicates that, similarly to our data on mouse cells, the phagocytosis of necrotic debris by human neutrophils is complement-independent, while FcγRs drive a substantial fraction of the phagocytosis. Blockage of CD11b in human neutrophils, however, had no effect on the phagocytosis level (Fig. S6I). Altogether, these findings show that the mechanisms of NAb-mediated necrotic debris phagocytosis are shared between murine and human leukocytes. We also demonstrated that the phagocytosis of necrotic debris requires IgG and IgM NAbs and depends largely on FcγRs expressed on leukocytes.

Supplementary video related to this article can be found at https://doi.org/10.1016/j.jhepr.2024.101013.

The following is the supplementary data related to this article:

Video S3

6

Natural antibodies are required for optimal debris phagocytosis at sites of necrotic liver injury

Considering the role of NAbs in the phagocytosis of necrotic cells and the defective recovery response observed in immunodeficient mice after liver injury, we sought to investigate if the absence of NAbs in these mice interfered with necrotic debris clearance in the liver. The experiments involved IVM imaging of a FTI in the liver in which the whole necrotic area was labeled with a droplet of pHrodo Red succinimidyl ester (Fig. S6J). The deposition of IgM and IgG in this model of necrotic injury was confirmed by performing cryosections of WT livers following FTI and staining for IgM and IgG (Fig. 6F). As expected, we could not find IgM or IgG deposition in FTI in Rag2^-/- mice (Fig S2F). After 6 h, mice were injected i.v. with AF488-labeled anti-Ly6G to identify neutrophils migrating in the focal injury site. Approximately 66% of neutrophils in the injury site in WT mice contained pHrodo⁺ phagosomes, whereas Rag2^-/- mice presented a significant reduction in the number of pHrodo⁺ neutrophils (Fig. 6G,H). To test if the impaired phagocytosis of necrotic debris in Rag2^-/- mice was mainly due to the lack of NAbs, these mice were treated i.v. with 100 μg of purified IgM and 100 μg IgG 30 min prior to FTI. Restitution of NAbs to Rag2^-/- mice completely rescued the phagocytosis of necrotic debris by neutrophils to levels observed in WT mice (Fig. 6G, H and movies S4-6). Importantly, no differences were found in the amount of recruited neutrophils inside necrotic areas between WT and Rag2^-/- mice (Fig. S6K), and parameters of neutrophil migration including displacement, total distance, directionality, circularity and cell size were similar between the groups (Fig. S6L-Q).

Supplementary video related to this article can be found at https://doi.org/10.1016/j.jhepr.2024.101013.

The following is the supplementary data related to this article:

Video S4

7

We next evaluated the role of other phagocytes in the clearance of necrotic cell debris in the liver FTI model. Considering that neutrophils predominated in the injured area 6 h after FTI (Fig. S4F), we decided to perform IVM 12 h post FTI to assess the distribution and role of Kupffer cells and monocytes. Kupffer cells were still absent from the necrotic injury at this timepoint and were present only in the surrounding healthy area (Fig. S7B). Additionally, we did not observe phagocytosis of necrotic debris by Kupffer cells. On the other hand, monocytes were abundantly recruited to the site of injury, and, in contrast to Kupffer cells, were clearly phagocytosing necrotic debris (Fig. S7C,D). Quantification of monocytes carrying pHrodo+ phagosomes showed that about 80% of monocytes phagocytosed necrotic debris in WT mice. When using Rag2^-/- mice, there was a small but significant reduction in the percentage of pHrodo+ monocytes that phagocytosed debris (Fig. S7D). These data suggest that KCs are not involved in necrotic debris clearance at least until 12 h post FTI. Moreover, recruited monocytes become substantial at this timepoint and contribute to clearing necrotic debris in a manner partially dependent on NAbs.

We also assessed whether NAbs are required for tissue repair following FTI as in the APAP model. We found that Rag2^-/- mice have impaired cell proliferation in the area surrounding the focal injury site when compared to WT mice (Figs 6I and Fig. S3B). Interestingly, restitution of purified antibodies to Rag2^-/- mice also restored the Ki67⁺ cellular proliferation in the liver, confirming that NAbs are also required for effective tissue repair in this model (Figs 6I and Fig S3B). In order to support the FTI model data, we confirmed that necrotic debris phagocytosis occurred in the liver of APAP-challenged mice (Fig. S7A). Altogether, these data suggest that NAbs are required for efficient clearance of necrotic cell debris by phagocytes in vivo, but not for leukocyte recruitment and migration in necrotic injury sites. In addition, it indicates that the delayed tissue repair in NAb-deficient mice is correlated with impaired necrotic debris phagocytosis in the injured liver. Finally, these data show that the mechanisms involving NAbs and healing are shared between the APAP and the FTI models.

Treatment with NAbs increases cellular proliferation and tissue regeneration after liver injury in immunocompetent mice

We evaluated whether the administration of NAbs could also have protective effects in immunocompetent mice. To assess this, WT mice were treated i.v. with a combination of 100 μg IgM and 100 μg IgG 4 h after the APAP challenge and were evaluated 48 h post injury. We observed that supplementation with NAbs significantly decreased the fibrin(ogen)⁺ necrotic areas in the liver (Fig. 7A,B). Serum ALT levels were also reduced in the WT mice treated with NAbs (Fig. 7C). Immunostaining of Ki67 in liver cryosections revealed that the treatment with NAbs significantly increased hepatocellular proliferation 48 h after APAP (Fig. 7D,F). In addition, during necrotic injury, the actin cytoskeleton labeling is lost within necrotic liver areas. However, as cells proliferate to regenerate the liver, the actin cytoskeleton scaffolds return, providing evidence of repair. By measuring the intensity of F-actin staining in centrilobular areas, we obtained further evidence that treatment with NAbs significantly improved the regeneration of liver injury in WT mice (Fig. 7E,F). In summary, treatment of immunocompetent mice with NAbs improved the resolution of injury and liver regeneration, suggesting that this approach could have therapeutic applications in immunocompetent individuals.

Fig. 7.

Treatment with NAbs increases cellular proliferation and tissue regeneration after liver injury in immunocompetent mice.

(A) Representative images showing immunostaining of liver cryosections from WT mice treated with 100 μg/mouse of purified IgM and IgG, or isotype. Mice were treated 4 h after the APAP challenge. Gray: fibrin(ogen), red: IgM, green: IgG. Scale bar = 100 μm. (B) Quantification of the fibrin(ogen)⁺ area in cryosections. (C) Serum ALT levels 48 h after APAP administration. (D) Quantification of the Ki67⁺ area in WT mice treated with purified IgM and IgG or isotype (100 μg/mouse, i.v.) 48 h after APAP challenge. (E) Mean fluorescence intensity of phalloidin staining around the centrilobular veins in WT mice treated with purified IgM and IgG or isotype (100 μg/mouse, i.v.) 48 h after APAP. (F) Representative images of liver cryosections from WT mice showing parenchymal cell proliferation (Ki67⁺, red), F-actin (phalloidin, green) and nuclei (Hoechst, blue). Mice were treated with purified IgM and IgG (100 μg/mouse, i.v.). Scale bar = 100 μm. Data are represented as mean ± SEM. ∗p ≤0.05 (Student’s t test). ALT, alanine aminotransferase; APAP, acetaminophen; NAbs, natural antibodies; WT, wild-type.

Discussion

Hepatocellular necrosis is a key driver of poor outcomes of both acute and chronic liver diseases;³⁰ thus, increasing the removal of necrotic cell debris from liver tissue is a promising approach to prevent chronification of injury, scarring, hepatocellular carcinoma and liver failure. Our work identified an unexplored physiological function of NAbs: to act as “eat-me” signals for the phagocytosis of necrotic cell debris in sites of liver injury. We found that IgM and IgG NAbs rapidly bound necrotic debris and promoted their clearance partially via FcγRs and CD11b. We also provided direct evidence that the administration of NAbs to immunocompetent mice improves recovery from necrotic liver injury, showing the potential of NAbs as therapeutic agents for liver diseases.

An important implication of our research is that NAbs may be an accessible and safe therapy for a variety of acute liver disorders, once further validation of effectiveness in humans can be determined. Interestingly, our data showed that a single dose of NAbs – at a concentration 250-fold lower than that used in humans^31,32 – is sufficient to reduce the severity of liver injury in mice. Moreover, the effectiveness of NAbs if given post injury and their naturally long half-life in circulation are interesting advantages in comparison to N-acetylcysteine, the gold-standard treatment for APAP-induced liver injury.³³ No disease-modifying therapies are available for patients suffering from ischemic liver damage.³³

The abundance, immediate availability, polyreactivity and targeting of conserved endogenous antigens makes NAbs ideal agents to clean up the disordered and heterogeneous debris released upon hepatocellular necrosis. Herein, we confirmed that NAbs opsonize diverse molecules including DNA, actin, histones, phosphoinositides and cardiolipin, all of which are normally present only within cells. The neutralization of necrotic debris is critical since they are known to exacerbate inflammation and disease severity in conditions ranging from ischemia-reperfusion injury, to alcohol-related liver disease and even fibrosis.³⁰ We further expanded the list of necrotic debris targets that are recognized by NAbs and showed the existence of natural IgG that binds to essentially the same targets as natural IgM. On the other hand, molecules that are found either in the outer leaflet of the plasma membrane or extracellularly such as phosphatidylethanolamine, phosphatidylcholine, cholesterol and triglycerides were not recognized by NAbs. A notable exception is PS, which although restricted to the plasma membrane inner leaflet in living cells, was not recognized by IgM or IgG NAbs. These observations indicate that NAbs discriminate between necrotic debris and apoptotic bodies, and suggest that the immune system has evolved different mechanisms to deal with distinct types of cellular remnants.

The role of natural IgM in sterile liver injury has been investigated in previous works. Marshall et al. suggested that natural IgM participates in both the induction of liver ischemia-reperfusion injury and liver regeneration after 70% hepatectomy.²³ They evaluated two monoclonal IgM antibodies: B4, against annexin IV³⁴ and C2, against a subset of phospholipids.³⁵ In our work, however, restitution of NAbs to immunodeficient mice did not increase injury. We believe this difference stems from our approach of reconstituting mice with the entire natural antibody repertoire, including natural IgG which was disregarded in previous literature. Nevertheless, in accordance with their data, we also observed NAb-dependent hepatocyte proliferation, which can be explained by several mechanisms: first, NAb-dependent removal of debris will clear room for new cells to proliferate and reconstitute the dead parenchyma; second, NAb-dependent phagocytosis can promote the release of pro-resolving mediators by phagocytes; third, natural IgM and IgG can activate the complement cascade in the liver. Liver recovery is dependent on the activation of complement, as shown by impaired hepatic regeneration in C3-deficient mice, which was restored by C3a administration.³⁶ In C5-deficient mice, hepatocytes showed a marked delay in entering the cell cycle (S phase) and reduced mitotic activity.³⁷ Thus, the mechanisms by which NAbs are beneficial may be multiple and synergistic, including liver regeneration but also resolution of inflammation.

Altogether, our study provides new insights on the clearance of necrotic remnants from the liver, highlighting NAbs as adaptors for the phagocytosis of these potentially dangerous self-antigens. Considering that phagocytes play a key role in the clearance of necrotic debris, immunosuppressants and therapies that inhibit leukocyte recruitment to the liver must be carefully evaluated. Conversely, a therapy based on NAbs may arise as a pro-resolving alternative by increasing debris clearance and liver repair, rather than preventing inflammation from occurring.

Financial support

MSM was supported by an EILF-EASL Sheila Sherlock Post-graduate Fellowship. SV and SS hold PhD fellowships from FWO-Vlaanderen (SB1S56521N and 1116922N, respectively). PEM is supported by a Marie Sklodowska-Curie Fellowship (MSCA–IF–2018-839632) and the Rega Foundation. AW and NH were supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project number 318346496 – SFB1292/2 and project number 490846870 –TRR355/1. This work was supported by the Research Foundation of Flanders (FWO-Vlaanderen) Junior Research Grants (G058421N and G025923N).

Authors’ contributions

MSM and PEM designed the experiments. MSM, SV and PEM wrote the manuscript; MSM, SV, SS, LF and NH conducted the experiments; AW, MSM and PEM provided reagents, analyzed and discussed data.

Data availability statement

Any additional information required to reanalyze the data reported here, is available from the corresponding author upon request.

Conflict of interest

The authors have no conflicts to disclose.

Please refer to the accompanying ICMJE disclosure forms for further details.

Supplementary data

The following are the supplementary data to this article: