EP4543914A1 - Use of natural antibodies in necrotic cell debris clearance and liver repair during necrotic liver injury - Google Patents

Abstract

The invention relates to natural antibodies and their use in the treatment of liver injury.

Description

Use of natural antibodies in necrotic cell debris clearance and liver repair during necrotic liver injury

Field of the invention

The invention relates to liver injury and treatment thereof.

The invention related to the use of natural antibodies in treatment of liver repair.

Background of the invention

Necrosis is common in both acute and chronic liver diseases and, if persistent, can progress to liver fibrosis. In fact, in patients with acute liver failure (ALF), the worst prognosis is for those with significant liver necrosis. Drug-induced liver injury (DILI) is a world-wide concern, being a common etiology for liver necrosis and one of the most prevalent causes of liver transplantation (LT). Among the hepatotoxic drugs, acetaminophen (APAP) poisoning accounts for more than 50% of ALF cases in Europe and United States. Nowadays, the only available treatment for APAP poisoning is the administration of N-acetyl cysteine in the first 12 hours and, if this early deadline is not met, LT becomes the only lifesaving alternative. APAP overdosage causes hepatocyte death by necrosis, a catastrophic event characterized by plasma membrane rupture and deposition of intracellular contents (debris) in the tissue, where it acts as a powerful inducer of inflammation. Prolonged permanence of debris in the liver can lead to excessive inflammation, further tissue damage and impairment of liver regeneration. One critical step towards liver repair is the clearance of necrotic cell debris, creating room for new hepatocytes, shaping the immune response towards resolution and rescuing liver function. Despite its relevance in liver diseases, the mechanisms underlying the clearance of necrotic debris in vivo are poorly understood. Marshall etal. (2018) Hepatology 67, 721-735, disclose that Natural Immunoglobulin M initiates an inflammatory response important for both hepatic ischemia reperfusion injury and regeneration in mice.

Tak et al. (2020) ACG case reports 7, e00337 disclose the administration of intravenous immunoglobulin in a severe alcoholic hepatitis patient with a bacterial infection.

It is postulated that debris are phagocytosed by innate leukocytes, however, owing to its varied composition, the recognition and internalization of necrotic debris is still elusive. Summary of the invention

The present invention illustrates that polyreactive Natural Antibodies (NAbs) act as adaptors for recognition and phagocytosis of necrotic debris.

The invention is further summarized in the following statements:

1. A composition comprising natural antibodies fur use in the treatment of liver injury, typically acute liver injury.

2. The composition comprising natural antibodies for use according to statement 1, wherein the liver injury is drug induced liver injury, an ischemia-reperfusion liver injury or an ischemia induced injury during liver transplantation.

3. The composition comprising natural antibodies for use according to statement 2, wherein the drug induced liver injury, is an acetaminophen/paracetamol induced liver injury.

4. The composition comprising natural antibodies for use according to any one of statements 1 to 3, wherein the composition is full plasma or serum.

5. The composition comprising natural antibodies for use according to any one of statements 1 to 3, wherein the composition is a preparation of purified IgM and/or purified IgG.

6. The composition comprising natural antibodies for use according to any one of statements 1 to 3, wherein the composition is a preparation of one of more of purified IgGl, IgG2 and IgG3.

7. The composition comprising natural antibodies for use according to any one of statements 1 to 6, in the treatment of a human individual.

8. The composition comprising natural antibodies for use according to statement 7, wherein the dose of administration is between 5 mg natural antibodies /kg and 400 mg natural antibodies /kg.

Natural antibodies (NAbs) are circulating polyreactive immunoglobulins that bind endogenous and exogenous antigens. Here, we investigated the role of NAbs in driving the clearance of necrotic celldebris from injury sites. Using mouse models of liver injury, we observed that IgM and IgG Nabs opsonize necrotic debris in vivo by recognizing common self-molecules such as histones, actin, phosphoinositides and cardiolipin, but not phosphatidylserine. Importantly, mice lacking Nabs presented impaired recovery from liver injury, which was correlated to sustained presence of necrotic debris in the tissue, prolonged inflammation and reduced hepatocellular proliferation. Mechanistically, necrotic debris phagocytosis was dependent on NAbs in vitro and in vivo, and restitution with total immunoglobulins rescued the defective recovery from liver injury in immunodeficient mice. In summary, t NAbs opsonize necrotic cell debris and act as "eat-me" signals for engulfment through FcyRs and CDllb, driving the recovery from tissue injury.

Detailed description

Figure 1: (A) Quantification of the fibrin(ogen) deposition in liver cryosections. (B) ALT levels in the serum after APAP overdosage (600 mg/Kg). (C) Quantification of the IgM or (D) IgG positive area in liver cryosections. (E) Flow cytometry evidencing the percentage of neutrophils (Ly6G+) and (F) monocytes (Ly6G-/CCR2+) relative to the total non-parenchymal cells in the liver.. Relative change in (G) natural IgM and (H) IgG levels in mouse serum after challenge with APAP. Cryosections quantification were performed in 10 images per liver. * = p < 0.05.

Figure 2: Both natural IgM and IgG can opsonize the most abundant hepatocytes necrotic debris. Dot Blot showing that both serum and purified natural IgM and IgG bind into purified DNA (DNA sodium salt from calf thymus, Sigma), Histones (Histone from calf thymus, Sigma ) and Actin (Actin from bovine muscle, Sigma).

Figure 3: Mice that lack Nabs have impaired necrotic debris clearance and delayed recovery of the liver. (A) Quantification of the Sytox green positive area, 10 fields per liver, n=3. (B) Quantification of fibrinogen deposition in liver cryosections of RAG2 -/- mice after APAP intoxication. (C) Serum ALT levels. (D) Recruitment of neutrophils (LY6G+); (E) macrophages (Ly6G-/CCR2-/F4/80+) and (F) monocytes (Ly6G-/CCR2+) to RAG2 -/- mice liver following APAP challenge. Images are presented as a stitching of 4 pictures taken using 25X objective. Scale bars = 100 pm. * = p < 0.05.

Figure 4: IgMi mice have impaired debris clearance, prolonged inflammation and delayed liver recovery. (A) Quantification of the fibrin(ogen) deposition in liver cryosections. (B) ALT levels in the serum after APAP overdosage (600 mg/Kg). Flow cytometry evidencing the percentage of (C) neutrophils (Ly6G+), (D) activated neutrophils (Ly6G+/CDllb+), (E) monocytes (Ly6G-/CCR2+), (F) monocytes expressing CDllb (Ly6G-/CCR2+/CDllb+), (G) macrophages (Ly6G-/CCR2- /F4/80+) and (H) macrophages expressing CDllb (Ly6G-/CCR2- /F4/80+/CDllb+) (relative to the total non-parenchymal cells in the liver. Cryosections quantifications were performed in 10 images per liver. * = p < 0.05.

Figure 5: Adoptive transfer of WT serum to RAG2 -/- mice rescue the deposition of NAbs in the necrotic areas and improve liver recovery. RAG2 -/- mice were challenged with APAP (600 mg/Kg) and, 4 hours afterwards, were treated with WT serum or RAG2 -/- serum. (A) Quantification of the fibrin(ogen) deposition in liver cryosections. (B) ALT levels in the serum after APAP overdosage (600 mg/Kg). Flow cytometry evidencing the percentage of (C) neutrophils (Ly6G+), (D) monocytes (Ly6G- /CCR2+) and (I)inflammatory monocytes (Ly6G-/CCR2+/Ly6C+) relative to the total non-parenchymal cells in the liver. ELISAs showing (G) CCL2 and (E) CXCL1 chemokines levels. Cytokines levels in the liver (H) IL-10, (E) GM-CSF and (F) TNF- a. Cryosections quantification were performed in lOimages per liver. ELISAs values were obtained from 100 mg of liver. * = p < 0.05.

Figure 6: Natural IgM and IgG antibodies bind multiple self-antigens exposed upon necrotic cell death. (A and B) Mean fluorescence intensity (MFI) of IgM and IgG labeling after pre-treatment of debris spot for 15 minutes with DNase and/or Trypsin before adding serum. Data are represented as mean ± SEM. *p< 0.05 compared to untreated samples. (C) Dot blots showing the reactivity of purified IgM and IgG to 4 pg of purified DNA, histones and actin. (D and E) Lipid blot showing the reactivity of purified IgM and IgG to lipids (100 pmol). CL: cardiolipin; DAG: diacylglycerol; LPA: lysophosphatidic acid; LPC: lysophosphatidylcholine; 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; SIP: sphingosine 1-phosphate; SM: sphingomyelin; TAG: triacylglycerol Data are represented as mean ± SEM. *p< 0.05 compared to the untreated sample.

Figure 7: Natural antibodies drive the phagocytosis of necrotic cell debris through FcyRs and CDllb. (A) Phagocytosis by primary mouse neutrophils 3 hours after adding debris opsonized with normal or heat inactivated (HI) serum. FcyRs were blocked by adding 100 pg/mL IgG to the cells. CDllb was blocked with 10 pg/mL anti-mouse CDllb. Phagocytosis was blocked with 10 pM Latrunculin B. (B and C) Phagocytosis by primary mouse neutrophils 3 hours after adding IgG- or IgMopsonized debris. Receptors were blocked using 10 pg/ml anti-CD16.2, anti- CD16/CD32, anti-CDllb or 100 pg/ml purified mouse IgG. (D) Phagocytosis by primary human neutrophils 3 hours after adding debris opsonized with normal or heat inactivated serum. FcyRs were blocked with 100 pg/mL IgG and Latrunculin B was used at 10 pM. (E) Quantification of the percentage of neutrophils (Ly6G+, green) phagocytosing necrotic debris 6 hours after a focal burn injury in the liver of WT, RAG2-/- mice. v

RECTIFIED SHEET (RULE 91) ISA/EP Figure 8: Treatment with NAbs increases cellular proliferation and tissue regeneration after liver injury in immunocompetent mice. (A) Quantification of the fibrin(ogen)+ area in cryosections. (B) Serum ALT levels 48 hours after APAP administration. (C) Quantification of the Ki67+ area in WT mice treated with purified IgM and IgG or isotype (100 pg/mouse, i.v.) 48h after APAP challenge. (D) Mean fluorescence intensity of phalloid in staining around the centrilobular veins in WT mice treated with purified IgM and IgG or isotype (100 pg/mouse, i.v.) 48h after APAP.

Considering the massive recruitment of phagocytes to the liver during APAP-induced liver injury, it is assumed that necrotic cell debris are phagocytosed by these leukocytes. Our data indicate that neutrophils phagocytose necrotic debris in vivo. However, owing to its biochemical nature and varied composition (e.g. DNA, histones, actin, lipids, etc), the recognition and removal of necrotic debris represents a challenging task for leukocytes, whose mechanism is still very elusive. The present invention unravels how phagocytes bind and phagocytose the myriad of necrotic debris. Instead of requiring several different receptors, phagocytes may take advantage of polyreactive Natural Antibodies (NAbs) as adaptors for recognition and phagocytosis of necrotic hepatocyte debris.

NAbs are circulating polyreactive immunoglobulins, mostly of IgM, IgA and IgG3 isotypes that arise early in life in the absence of exogenous antigenic stimulation, being produced mainly by CD5⁺ B-l cells. NAbs can react against altered self-antigens found in all cell types, which may comprise proteins, nucleic acids, carbohydrates, lipids or combinations thereof. In this sense, both IgM and IgG NAbs were found to bind to molecules exposed on necrotic cells in vitro. Upon recognition, antigens are opsonized by NAbs, forming an immunocomplex which is recognized by phagocytes through Fc receptors (FcRs). The family of FcRs for IgG, FcyRs, are key in the phagocytosis of IgG-coated particles by macrophages in vitro. IgM can potentially assist the clearance of necrotic debris through Fc-o/p receptor, which has been shown to mediate phagocytosis of IgM-coated microorganisms.

RECTIFIED SHEET (RULE 91) ISA/EP NAbs are characterized as low affinity antibodies that, due to their polyreactivity, bind to different classes of self and exogenous molecules including proteins, nucleic acids, carbohydrates, lipids or combinations thereof. In contrast to adaptive antibodies that can bind virtually to any epitope, NAbs have germline encoded variable regions which restrict their recognition capacity to phylogenetically conserved molecules.

Natural IgM recognizes several evolutionarily conserved intracellular components, such as actin, tubulin, single-stranded DNA and double-stranded DNA. IgM was also shown to bind to lysophosphatidylcholine and to oxidized phospholipids on necrotic cells. Worth mentioning is that, during drug-induced liver injury, deposition of IgM occurs onto necrotic sites early after injury and that IgM deposits disappeared with liver repair both in mice and humans. The present invention demonstrates that RAG- 1 KO mice, which lack mature lymphocytes and do not produce NAbs, have increased DNA debris deposits in the liver after APAP-induced injury. Importantly, RAG-1 KO mice tend to have even less liver injury than wild-type mice, indicating that the increased DNA deposits are not due to excessive injury. Instead, it is likely due to impaired debris clearance in the RAG-1 KO mice. This result supports the concept that NAbs have a role in necrotic debris clearance in the liver.

Necrotic cell death is inexorably connected to human life as a result of our daily behavior, since we are constantly exposed to stresses such as burns, traumas and intoxications that culminate in necrotic injuries. Necrotic cell death, accidental or programmed, and regardless of the wide spectrum of necrosis-initiating events, converges in plasma membrane rupture and the consequent release of cellular content (debris) in the tissue. Once exposed, necrotic debris are recognized as damage associated molecular patterns (DAMPs), acting as powerful inducers of inflammation. The generation and longevity of necrotic debris in tissues are associated with chronic inflammation and worsening of atherosclerosis, arthritis, liver injury, systemic lupus erythematosus and neurodegenerative disorders. In order to avoid this grim prospect, necrotic debris must be efficiently cleared from tissues.

The present invention investigates the physiological role of polyreactive NAbs in necrotic debris recognition and removal by phagocytes in the liver, and aims to: describe the molecular composition of necrotic debris; Describe the distribution of NAbs within the necrotic liver; unveil which antigens NAbs recognize in the necrotic liver; conduct in vivo time-lapse imaging of NAbs-dependent debris phagocytosis; evaluate the efficacy of IgM and IgG3 to induce necrotic debris phagocytosis; describe which Fc receptors in phagocytes recognize these immunocomplexes; test the potential of Fc-engineered NAbs to improve necrotic debris clearance, resolution of inflammation and hepatic repair.

WT mice challenged with APAP rapidly developed liver injury, as assessed by increased levels of serum ALT, extensive centrilobular necrosis and massive liver inflammation, including neutrophil (6h-48h) and monocyte (24h-72h) recruitment. The data demonstrate that both IgM and IgG NAbs become deposited in the necrotic areas early after liver injury (6h), and in necrotic hepatocytes in vitro. The antibody deposits disappear with liver recovery (72h). In both Rag2 -/- and IgMi -/- mice (which lack NAbs), significantly increased liver injury was observed, hepatic inflammation and impairment of liver repair, as compared to their WT littermates. Adoptive transfer of WT serum to these mice rescued the deposition of NAbs in the necrotic areas, causing also a reduction in serum ALT levels, centrilobular necrosis, and improvement in liver recovery.

Both natural IgM and IgG are deposited within necrotic areas following liver injury Mice challenged with APAP developed time-dependent necrotic liver injury, peaking at 24h and already recovering at 48h, as assessed by fibrin(nogen) deposition in liver cryosections and by increased levels of serum ALT (Figure 1A and B ). The liver injury induced a massive recruitment of neutrophils as early as 6h (Figure IE) followed by recruitment of monocytes, starting at 12 hours after APAP intoxication (Figure IF). It was also found that both natural IgM and IgG accumulates within the necrotic areas as soon as the injury starts and disappear with tissue recovery (Figure 10 & D). It was also found that the deposition of NAbs in the necrotic liver, causes the reduction of IgM and, mainly, IgG in the circulation (Figure 1 G 8i H). Considering that the injury peaks at 24 hours and 48 hours there is already a significant recovering, these two timepoints were followed in order to assess liver recovery.

IgM and IgG NAbs rapidly bind necrotic debris and promotedtheir clearance partially via FcyRs and CDllb. NAbs-mediated clearance of necrotic cells increased hepatocellular proliferation and tissue recovery. In mice, it is estimated that around 80% of serum IgM is natural IgM derived from Bl cells. Its abundance, immediate availability, polyreactivity and targeting of conserved endogenous antigens makes NAbs ideal agents to clean up the disordered and heterogeneous necrotic cell debris. NAbs opsonize diverse conserved molecules including DNA, actin, histones, phosphoinositides and cardiolipin, all of which are normally present only within cells. On the other hand, molecules that are found either in the outer leaflet of the plasma membrane or extracellularly such as PE, PC, 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. Heat-inactivation of serum did not impact the efficiency of phagocytosis, suggesting that clearance of necrotic debris is independent of complement. This may be due to multiple reasons, the first is that NAbs are sufficient to drive debris phagocytosis, thus, the absence of complement activation is easily overcome in vitro. Second, other phagocytic pathways will likely occur simultaneously, such as PS-dependent phagocytosis of necrotic cells. Lastly, complement may require a higher concentration to opsonize necrotic debris effectively. By opsonizing debris in 20% serum, the role of the complement cascade may have been underestimated. Nevertheless, it has been shown that complement-mediated phagocytosis induces upregulation of genes encoding proinflammatory cytokines in mouse macrophages such as TNF-042, while our NAbs-dependent phagocytosis of necrotic debris did not alter TNF-o expression but upregulated IL-10. We showed that the phagocytosis of necrotic debris immunocomplexes is largely dependent of FcyRs, since blockage of these receptors in neutrophils reduced phagocytosis by 50%. Dual blockage of FcyRs and CDllb decreased phagocytosis even further (70%), suggesting also a role for CDllb likely via IgM recognition. However, CDllb blockage did not affect the phagocytosis of Hi-opsonized necrotic debris. This may be due to the heat inactivation process also affecting the polygonal structure of IgM, thereby reducing its binding to antigen and recognition by receptors. Recent work suggested that heatinactivation reduces IgM values in serum in a temperature-dependent manner. Moreover, a well characterized method to produce multimeric IgG is by heat-induced aggregation, which increases both the avidity to antigens and recognition by FcRs in macrophages. Thus, heat inactivation may favor the role of IgG in promoting phagocytosis in vitro. Although the phagocytosis of necrotic debris is mainly driven by NAbs, the remaining phagocytosis can be occurring via other opsonins, e.g. PS- binding proteins and calreticulin. In vivo, the reduction of phagocytosis in RAG2-/- was approximately 40%. Especially in these experiments, it is most likely that RAG2- /- develop compensatory mechanisms for the lack of NAbs. Since the complement cascade was shown to be activated during several types of liver injury, it is possible that the complement cascade contributes to the clearance of necrotic debris observed in immunodeficient mice. Altogether, the present inventions provides a new insight on the clearance of necrotic remnants from tissues, highlighting NAbs as adaptors for the phagocytosis of these potentially dangerous self-antigens. A therapy based on NAbs by increasing debris clearance and tissue repair is provided, rather than preventing inflammation from occurring.

Natural IgM and IgG can bind to hepatocyte necrotic debris

An important question that arose is if NAbs can bind to necrotic debris or if they are only getting stuck within the necrotic areas. It was investigated if NAbs were able to bind to the most abundant hepatocytes necrotic debris. For this purpose, hepatocytes were purified from mouse liver and were mechanically crushed these cells to expose the intracellular content. The debris was spotted into coverslips, incubated with mouse serum for 15 minutes and stained for DNA, Actin, IgM and IgG. Both IgG and IgM were able to bind to hepatocytes debris. To further investigate if there is difference in the avidity of IgM or IgG to opsonize the different necrotic debris, a dot blot was performed using purified debris (DNA, Histone & Actin) opsonized with normal mouse serum or with purified total mouse IgG or IgM. It was found that both natural IgM and IgG can bind to all necrotic debris tested, being IgM slightly better to opsonize DNA compared to IgG (Figure 2). These results show that both Natural IgM and IgG can, indeed, opsonize the most common necrotic debris generated upon hepatocytes death.

Mice that lack NAbs have impaired debris clearance, prolonged inflammation and delayed liver recovery.

To evaluate if NAbs play a role in the clearance of necrotic debris from the injured liver, RAG2 -/- mice were challenged, which lacks mature lymphocytes and, consequently, NAbs, with APAP during 24 and 48h. Intravital microscopy of the liver was performed and increased DNA deposition was found in the tissue and impaired liver recovery of RAG2 -/- mice if compared with their WT littermates (Figure 3A ). RAG2-/- mice, as expected, had no deposition of IgM and IgG within the necrotic areas (data not shown). The quantification of fibrin(ogen) deposition in liver cryosections, as well as serum ALT levels, confirmed the impaired liver repair at 48h (Figure 3B & C). RAG2 -/- mice also showed increased number of inflammatory cells in the liver compared to WT mice (Figure 3D-F). However, this phenotype could be due to the lack of lymphocytes rather the lack of NAbs. To supersede this caveat, it was decided to challenge IgMi mice with APAP. IgMi mice has all the mature lymphocytes but lacks soluble antibodies. IgMi mice showed impaired liver regeneration as evidenced by increased Fibrin(ogen), but no IgM or IgG, deposition (Figure 4A ) and increased serum ALT levels at 48h (Figure 4B). Similar to RAG2 -/- mice, the immune response in the liver of IgMi mice also need longer period to return to its homeostasis, once these mice had increased number of neutrophils in the liver, which express more CDllb, and reduced number of liver macrophages (Figure 4 C- H).

Adoptive transfer of WT serum to RAG2 -/- mice improves liver recovery

To confirm the role of NAbs in the clearance of necrotic debris, RAG2 -/- mice were challenged with APAP and, after 4 hours, adoptive transfer of WT serum was performed for a group of RAG2 -/- mice while the remaining RAG2 -/- mice were treated with the same amount of RAG2 -/- serum as a control. Treatment with WT, but not RAG2 -/-, serum successfully rescued the deposition of natural IgM and IgG in the necrotic liver, which resulted in efficient clearance of debris and tissue repair 48 hours after APAP intoxication, when compared with the mice that received RAG2 -/- serum (Figure 5A). The treatment with WT or RAG2 -/- serum do not alters the serum levels of ALT, chemokines or the number of infiltrating neutrophils or monocytes (Figures 5B, C, D, G, H), indicating that NAbs has no role in the onset of inflammatory response or in preventing or increasing injury but, rather, it has a role in the tissue repair. Mice treated with WT serum, showed lower number of inflammatory monocytes (Ly6C+), but increased levels of IL-10 and GM-CSF 48h after APAP, indicating that NAbs are also key to shape immune response towards resolution (Figure 51).

The binding of NAbs to the necrotic cell debris in the liver is central for a quick and effective liver repair, and to reduce the inflammatory response to injury. These data indicate that NAbs are potential therapeutical options to improve liver recovery by allowing the effective removal of necrotic debris and, consequently, avoiding prolonged inflammation and additional collateral damage.

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

It was further investigated 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 labelled with anti-mouse IgM and IgG. The hepatocyte debris spots, shown by DNA and F-actin staining, were bound by IgM and IgG from healthy mouse serum. The binding of antibodies was antigen specific, since incubation with secondary antibodies alone yielded no labelling of the debris. 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, the debris spots were pre-treated with DNase and/or trypsin for 15 minutes. Treatment with DNase and trypsin was sufficient to degrade DNA and F- actin substantially, but not completely. Pre-incubation of debris spots with either of the enzymes reduced both IgM and IgG labelling significantly, suggesting that NAbs bind DNA and protein epitopes (Figure 60 and 6D). 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. It was found that serum NAbs indeed bind directly to histones, actin and DNA, the latter albeit with lower avidity. In order to avoid interference from other possible debris-binding molecules present in serum, dot blots were also performed using purified murine IgM or IgG and a similar binding pattern was found (Figure 6C).

To assess if the recognition of necrotic cell molecules by NAbs also encompassed lipids, assays were performed with lipid strips containing a variety of cellular phospholipids, phosphoinositides and their intermediates. 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 (Figure 6D) as well as phosphatidic acid (PA), PI(3,4,5)P3 and the mitochondrial phospholipid cardiolipin (CL) (Figure 6E). Importantly, no binding of IgM nor IgG NAbs was observed to phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), sphingomyelin (SM), sphingosine-l-phosphate (SIP), diacylglycerol (DAG), lysophosphatidic acid (LPA), lysophosphocholine (LPC), the triglyceride glyceryl tripalmitate (TAG) 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). These results further show that both IgM and IgG NAbs bind to multiple classes of cellular components that are exposed after necrotic cell death including DNA, actin, histones and phospholipids. Yet, IgM and IgG are able to bind essentially to the same antigens, suggesting a redundant role for the two types of NAbs.

Natural antibodies drive the phagocytosis of necrotic cells debris through FcyRs and CDllb

As NAbs improved the recovery from necrotic liver injury, the mechanism by which they exert their role in injury resolution was further investigated. Antibody-opsonized antigens are known to be phagocytosed in an FcR-dependent manner, therefore, NAbs opsonization of necrotic cell debris likely promotes its clearance by phagocytosis. To test this hypothesis an in vitro assay of necrotic debris phagocytosis was developed by feeding necrotic cell debris to murine macrophage-like RAW 264.7 cells. Debris was prepared by crushing HepG2 cells and labelling it covalently with pHrodo Red succinimidyl ester, a pH-sensitive dye that emits increased fluorescence in acidified compartments such as phagosomes. This approach allowed to discern the debris that was phagocytosed and matured in phagosomes from debris bound to or in proximity to cells. Labelled 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. Remarkably, necrotic debris phagocytosis was essentially identical if debris were opsonized with native or heat-inactivated 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, completely blocked the internalization of necrotic debris. The FcRs expressed by RAW cells were investigated and found that they expressed constitutively the genes CD64 (FcyRI), CD32 (FcyRII), CD16 (FcyRIII) and, at lower level, CD16a (FcyRIV). However, the expression of the two FcRs that recognize IgM- coated targets, CD351 (Fco/pR) and Faim3 (FcpR) was absent. Stimulation of RAW cells with NAbs-opsonized necrotic debris for 6h increased the expression of CD64 and CD16a, but reduced the expression of CD32. Even after stimulation with NAbs- opsonized debris, mRNA for CD351 and Faim3 in RAW cells could not be detected (data not shown). The phagocytosis of NAbs-coated debris increased the expression of IL-10 while it did not alter the expression of TNF-o, suggesting an antiinflammatory role of NAbs-mediated phagocytosis. After validating this methodology, necrotic debris phagocytosis was investigated in primary mouse neutrophils. Similar to the findings with RAW cells, serum-opsonized debris were efficiently phagocytosed by murine neutrophils and heat inactivation of serum did not alter the phagocytosis rate (Figure 7A), suggesting that NAbs but not complement is 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 (Figure 7A). The next step was to identify the receptors involved in the phagocytosis of NAbs-debris immunocomplexes. First, a general blockage of FcyRs was performed by treating neutrophils with 100 pg/mL purified mouse IgG, as previously described, and a 50% reduction in phagocytosis efficiency was found of debris opsonized with normal serum or HI serum, suggesting that FcyRs and natural IgG are driving at least half of necrotic debris phagocytosis (Figure 7A). Considering that mouse neutrophils do not express Fc receptors for IgM33,34, CDllb could have a role in the phagocytosis of IgM-coated debris, since CR3 has been implicated in the internalization of IgM/IgA-opsonized targets35. Thus, a combined blockage of FcyRs and CDllb was performed, which led to even further reduction of phagocytosis of serum-opsonized debris. Conversely, CDllb blockage did not alter the phagocytosis rate of Hi-opsonized debris phagocytosis (Figure 7A). To better dissect the role of each immunoglobulin isotype, as well as to identify the FcyR involved in the recognition of IgG-coated debris, the phagocytosis assay was performed using necrotic debris opsonized with purified antibodies. Similar to the findings with serum-opsonized debris, the blockage of FcyRs using excessive IgG led to a significant reduction in the phagocytosis of IgG-coated debris. Surprisingly, the combined blockage of FcyRII, FcyRIII and FcyRIV did not affect phagocytosis, indicating that FcyRI might be the predominant phagocytic receptor for IgG-coated debris (Figure 7B). Using purified IgM-coated necrotic debris, CDllb blockage also resulted in a reduction of approximately 50% of phagocytosis, supporting a role for this integrin in the clearance of IgM-coated debris (Figure 7C). These data indicate that FcyRs are driving IgG-mediated phagocytosis of necrotic debris, whereas IgM mediated phagocytosis is occurring at least in part via CDllb. To investigate the mechanisms of NAbs-dependent necrotic debris phagocytosis in a more translational manner, blood-derived human neutrophils were also used. 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 (Figure 7D). 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. The phagocytosis of necrotic debris, quantified as the percentage of neutrophils containing pHrodo positive events, was quite similar between normal and heat-inactivated serum, confirming that the clearance of necrotic debris occurs independently of the complement cascade (Figure 67). Similarly to murine neutrophils, FcyR 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 heat inactivated serum (Figure 7D). Using 3D confocal imaging 3h after adding serum-opsonized debris, neutrophils carried multiple pHrodo+ phagosomes and confirmed that the positive events were inside the cells. This indicates that, similarly to the data on mouse cells, the phagocytosis of necrotic debris by human neutrophils is complement independent, while FcyRs drive a substantial fraction of the phagocytosis. Blockage of CDllb in human neutrophils, however, had no effect on the phagocytosis level. Altogether, these findings show that the mechanisms of NAbs-mediated necrotic debris phagocytosis are shared between murine and human leukocytes. This demonstrates that the phagocytosis of necrotic debris requires IgG and IgM NAbs and depends largely on FcyRs expressed on leukocytes.

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

Considering the role of NAbs in the phagocytosis of necrotic cells and the defective recovery response observed in immunodeficient mice after liver injury, it was further investigated if the absence of NAbs in these mice interfered with necrotic debris clearance in the liver. The experiments involved IVM imaging of a focal burn injury in the liver in which the whole necrotic area was labelled with a droplet of pHrodo Red succinimidyl ester. After 6h, 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 (Figure 7E). To prove that 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 pg of purified IgM and 100 pg IgG 30 min prior 344 to the focal burn injury. Restitution of NAbs to RAG2-/- mice completely rescued the phagocytosis of necrotic debris by neutrophils to levels observed in WT mice (Figure 7E). Importantly, no differences were found in the amount of recruited neutrophils inside necrotic areas between WT and RAG2-/- mice, and parameters of neutrophil migration including displacement, total distance, directionality, circularity and cell size were similar between the groups. These data indicate that NAbs are required for efficient clearance of necrotic cells 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 NAbs- deficient mice is correlated with impaired necrotic debris phagocytosis.

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

It was evaluated whether the administration of NAbs could have protective effects also in normal mice. To assess this, WT mice were treated i.v. with a combination of 100 pg IgM and 100 pg IgG 4h after the APAP challenge and were evaluated 48h post injury. Supplementation with NAbs significantly decreased the fibrin(ogen)+ necrotic areas in the liver (Figure 8B). Serum ALT levels were also reduced in the WT mice treated with NAbs (Figure 8B). Immunostaining of Ki67 in liver cryosections revealed that the treatment with NAbs significantly increased the hepatocellular proliferation 48h after APAP (Figure 8C). In addition, during necrotic injury the actin cytoskeleton labelling is lost within necrotic liver areas. However, as cells proliferate to regenerate the liver, the actin cytoskeleton scaffolds return, evidencing the repairing injury. By measuring the intensity of actin staining in centrilobular areas, further evidence was obtained that the treatment with NAbs improved significantly the regeneration of liver injury in WT mice (Figure 8D). 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.

EXAMPLES

Example 1. Methods

C57BL/6J wild-type (WT), Rag2-/- and IgMi-/- mice received an oral overdose of APAP (600 mg/Kg). Liver injury/repair, deposition of natural antibodies, and inflammation were assessed by immunofluorescence, intravital microscopy and flow cytometry, an in vitro hepatocyte debris assay was used to evaluate binding of NAbs to hepatocyte debris. C57BL/6J wild-type (WT), C57BL/6N-Rag2Tml -/- (Rag2 -/-) and IgMi mice, aged 8-10 weeks, received an overdose of APAP (600 mg/Kg) by oral route. IgMi mice have mature B cells that express IgM BCR but cannot secrete antibodies and were also kept under C57BL/6 background. Liver injury/repair, deposition of natural antibodies, and inflammation were assessed by immunofluorescence, intravital microscopy and flow cytometry. An in vitro hepatocyte debris assay was used to evaluate binding of NAbs to cell debris directly.

Cytokines in the liver were measured by ELISA.

Mice

C57BL/6J and C57BL/6NRj mice were purchased from Janvier Labs. C57BL/6N- Rag2Tml/CipheRj (RAG2-/-) were bred in specific pathogen-free conditions at the Animal Facility of the Rega Institute (KU Leuven). All mice used in this study were between 10-12 weeks old and both male and female mice were equally distributed 468 across experiments (no phenotypic differences between genders were observed). Mice were housed in acrylic filtertop cages (5 mice per cage) with an enriched environment (bedding, toys and small houses), at the Animal Facility of the Rega institute (KU Leuven). Water and food were provided ad libitum and mice were kept under a controlled dark/light cycle (12/12 h) at 21 °C. All experiments were approved and performed following the guidelines of the Animal Ethics Committee from KU Leuven (registry number: P125/2019).

Cell Lines RAW 264.7 is a monocyte/macrophage like cell that was derived from a tumor induced in a male mouse with the Albeston murine leukemia virus. HepG2 cell is a hepatocyte-like cell derived from a hepatocellular carcinoma of a 15-year-old male human. The cells were cultured at 37 °C and 5% CO2 atmosphere in high glucose Dulbecco's Modified Eagle Medium (DMEM) with GlutaMAX (Thermo Fisher Scientific), supplemented with 10% FBS (Sigma-Aldrich), 1 mM sodium pyruvate (Thermo Fisher Scientific) and 0.12% sodium bicarbonate (Thermo Fisher Scientific).

Acetaminophen (APAP)-induced liver injury model

Mice were fasted for 15 hours before a single oral gavage of vehicle or 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 the reproducibility amongst the experiments. After 6, 12, 24, 48 or 72 hours, mice were sacrificed under anesthesia containing ketamine (80 mg/kg) and xylazine (4 mg/kg) whereafter liver and blood were harvested.

Alanine aminotransferase (ALT) assay

Liver injury was indirectly assessed by monitoring levels of serum ALT utilizing a kinetic test (Infinity, Thermo Fisher Scientific, Waltham, MA, USA). Briefly, blood samples were harvested and centrifuged for 10 minutes at 1500 x g and then serum was harvested. Pure serum and three different dilutions (1: 10, 1:20, 1:30) were added to a 96-well plate, then, the substrate (HEPES buffer pH 7.8, LDH, L494 ine, NaCI) and coenzymes (alfacetoglutarate and NADH) were added to the serum samples at 37 °C. The reaction was monitored every minute (for a total of 3 minutes) by measuring the rate of decrease in absorbance at 340 nm (CLARIOstar, BMG Labtech, Cary, NC) due to the oxidation of NADH to NAD.

Liver cryosectioning and immunostaining

To perform liver immunostainings, the left lobe was harvested, embedded in frozen mounting medium (PolyFreeze, Sigma-Aldrich, St. Louis, MO) and snap-frozen in liquid nitrogen. Cryosections of 14 pm thickness were generated using a cryostat (Microm Cryo-Star HM560, Thermo Fisher Scientific, Waltham, MA, USA). The sections were fixed for 1 hour with 4% paraformaldehyde (PFA) in Hank's Balanced Salt Solution (HBSS, Gibco, Waltham, MA, pH 7.2) supplemented with 0.1% Bovine Serum Albumin (BSA, Albumin Fraction V, protease-free, Carls Roth, Karlsruhe, DE) at room temperature (RT). Then, the sections were washed with HBSS and permeabilized with 0.1% Triton X-100 in HBSS for 1 hour at RT. Livers sections were washed again and blocked using 1% Fc Block (MACS, Miltenyi Biotec, Auburn, CA, USA) and 5% BSA in HBSS during 1 hour at RT. To visualize the necrotic areas in liver cryosections, we stained for fibrin(ogen), once the deposition of fibrin(ogen) within areas of hepatocellular necrosis is well described48,49. After another washing step, the primary polyclonal rabbit anti-human/mouse fibrin(ogen) (10 pg/mL in HBSS, Agilent Dako, Glostrup, DK) antibody was added overnight at 4°C. The sections were washed and the secondary antibodies Alexa Fluor 511 488 donkey anti512 rabbit, Rhodamine RED-X (RRX) donkey anti-mouse IgM and Alexa Fluor 647 goat anti-mouse IgG (all at 10 pg/mL, Jackson ImmunoResearch, West Grove, PA, USA) were added for 3 hours at RT. After another washing step, mounting medium was applied (ProLong Diamond, Thermo Fisher Scientific) and the sections were imaged using an Andor Dragonfly 200 spinning-disk confocal microscope equipped with a 25X objective, and analyzed using FIJI.

Purification of liver non-parenchymal cells (NPCs)

The purification of liver NPCs was performed as previously described26. Briefly, the caudate and median liver lobes were harvested in RPMI-1640 medium (Biowest Riverside, MO, US) and mechanically minced using the gentleMACS Dissociator (Miltenyi Biotec, Auburn, CA, USA). Then, RPMI-1640 medium was added to the liver homogenate to complete 30 ml and centrifuged at 300 x g for 5 minutes at 4 °C. The supernatant was discarded and more RPMI-1640 medium was added to complete 30 ml. After homogenization, the cells were centrifuged at 60 x g for 3 minutes at 4 °C and the supernatant, containing the NPCs, was harvested and filtered through a 40 pm cell strainer in order to remove undigested tissue. The cells were centrifuged again at 300 x g for 5 minutes at 4 °C and the supernatant was discarded. The pellet was resuspended, transferred to a 15 mL tube and centrifuged again (300 xg, 5 minutes at 4 °C). The supernatant was discarded and red blood cells were lysed with 2 ml of ACK Lysing Buffer (Gibco, Grand Island, NY, US) for 5 minutes on ice. ACK was washed away by adding PBS until 10 ml and centrifuged at 300 x g for 5 minutes at 4 °C. The final pellet, containing the liver NPCs, was reconstituted for further analyses.

Flow Cytometry

For flow cytometry, 5 x 10⁵ NPCs were labeled with viability dye marker (Zombie Aqua, Biolegend, San Diego, CA, USA) and blocked with PBS supplemented with 1% Fc Block (MACS, Miltenyi Biotec) and 0.5% BSA for 15 minutes at 4 °C. The cells were washed with 500 pL FACS buffer (PBS supplemented with 0.5% BSA and 2mM EDTA) and labeled with different antibodies (see Key resources Table) for 30 minutes at 4 °C in the dark. After labeling, the samples were washed by adding 500 pL FACS buffer, centrifuged at 300 x g for 5 min at 4 °C and resuspended in 300 pl FACS buffer. Cells were immediately read in a Fortessa X20 (BD Biosciences, Franklin Lakes, NJ US). The data was analyzed using FlowJo X (FlowJo 10.8.0, LLC, Asland, OR USA). The gate strategy can be seen at supplementary figure 7.

Hepatocyte debris spot

Livers from healthy mice were harvested in RPMI-1640 medium (Biowest Riverside, MO, US) and mechanically minced using a gentleMACS Dissociator. Then, RPMI-1640 medium was added to the liver homogenate to complete 30 ml and centrifuged at 300 x g for 5 minutes at 4 °C. The supernatant was discarded and more RPMI-1640 medium was added to complete 30 ml and centrifuged at 60 x g for 3 min at 4 °C. The supernatant was discarded and the liver homogenate was transferred to a new 50 mL tube and filtered through a 40 pm cell strainer in order to remove the undigested tissue. Samples were centrifuged again at 300 x g for 5 minutes at 4 °C. The supernatant was discarded and red blood cells were lysed by adding ACK to the pellet (containing the hepatocytes) for 5 minutes on ice. The cells were then washed with HBSS and centrifuged at 300 x g for 5 min at 4 °C. 1 x 107 hepatocytes were mechanically disrupted with a pellet mixer (VWR Radnor, PA, USA) for 5 minutes. After crushing the cells, the debris spot was generated by adding 6 pL of the solution containing the necrotic hepatocytes into an 8-well chamber slide (Nunc, Rochester, NY, USA) and drying for 2 hours in the laminar flow. The hepatocyte debris spot was then blocked with HBSS supplemented with 1% Fc Block and 0.5% BSA for 15 min at RT. After washing three times with HBSS, healthy mouse serum (1: 10 in HBSS) was added for 30 min at 37 °C. The hepatocyte debris spots were then washed three times and stained with Hoechst (10 pg/mL), Phalloidin (66 nM) and secondary antibodies Rhodamine RED-556 X (RRX) rabbit anti557 mouse IgM and Alexa Fluor 647 goat anti-mouse IgG (both 10 pg/mL, Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at RT. After another washing step, the hepatocytes debris spots were imaged using a Zeiss Axiovert200M microscope and analyzed with FIJI. Images were taken using an 25X objective.

Measurement of natural antibodies in the serum

To assess the levels of circulating NAbs during APAP-induced liver injury, serum was collected from healthy mice and from mice 6, 12, 24, 48 and 72 hours after APAP administration. Then, hepatocyte debris spots were incubated with the serum for 30 min at 37 °C. The hepatocyte debris spots were then washed three times and stained with Hoechst (10 pg/mL), phalloidin (66 nM) and secondaries antibodies Rhodamine RED-X (RRX) rabbit anti-mouse IgM and Alexa Fluor 647 goat anti-mouse IgG (both 10 pg/mL, Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at RT. After another washing step, the hepatocyte debris spots were imaged using a Zeiss Axiovert200M microscope and the mean fluorescence intensity (MFI) of IgM and IgG were determined using FIJI. The data are represented as the relative change in the MFI compared to the control (healthy mouse serum) group.

Serum adoptive transfer and treatment with purified IgG and IgM

For the serum adoptive transfer, WT or RAG2-/- mice were euthanized under anesthesia and blood was harvested. Serum was collected by centrifugationat 1500 x g for 10 min at 4 °C. Next, RAG2-/- mice were injected with either 150 pL of WT serum or with the same amount of RAG2-/- serum. For the purified antibodies treatment, mice received 100 pg of purified IgG and 100 pg of purified IgM diluted in sterile PBS in a final volume of 200 pL. As control for the purified antibodies injection, control mice received an injection with the same amount of IgG and IgM monoclonal isotype control (see key resources table). The serum or purified antibodies were administered intravenously, only once, through the retro-orbital sinus 4 hours after APAP challenge. During the injection, mice were anaesthetized with isoflurane.

Dot blot and lipid blot assays

For the dot blot assay, purified DNA (Sigma-Aldrich), actin (Sigma-Aldrich) and histones (Cytoskeleton, Inc.) were diluted in Milli-Q water to a final concentration of 2 mg/mL. Then, 2 or 4 pg of purified debris was spotted on nitrocellulose membranes and dried for 1 hour at RT. For the lipid blot, PIP Strips and Membrane Lipid Strips (Echelon Biosciences, Salt Lake City, UT, USA) containing different lipids at 100 pmol per spot were used. Unspecific labeling was blocked by adding washing buffer (Tris 20 mM, NaCI 150 mM and Tween 20 0.1%) supplemented with 5% BSA for 1 hour at RT under constant agitation. The membrane was washed three times for 5 minutes with washing buffer. After washing, the membrane was incubated with mouse serum (1: 10 in washing buffer) or 5 pg/mL of purified IgG and IgM (Rockland, Limerick, PA, USA) in washing buffer overnight at 4°C under agitation. The membrane was washed again three times for 5 minutes each, and incubated with the secondary goat antimouse IgM IRDye 680RD and goat anti-mouse IgG IRDye 800CW (1: 10,000, LI-COR Biosciences, Lincoln, NE, USA) for 3 hours in the dark at RT with agitation. The membrane was washed again as aforementioned and imaged with an Odyssey Fc Imaging System (LI-COR Biosciences) at 800 and 700 nm. The images were analyzed with Image Studio lite (LI-COR Biosciences).

Analysis of chemokines and cytokines

Levels of cytokines and chemokines were assessed in serum samples by sandwich ELISA using commercial kits (DuoSet R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol. The absorbance was determined using a spectrophotometer (CLARIOstar, BMG Labtech, 599 Cary, NC) at 490 nm. Results were represented as pg/mL of cytokines or chemokines.

Neutrophil purification

Human neutrophils were purified from blood of healthy volunteers with the EasySep neutrophil isolation kit (StemCell Technologies, Vancouver, Canada) following the manufacturer's instructions. Mouse bone marrow (BM) neutrophils were extracted from femurs and tibias of C57BL/6J mice by flushing the bones with 5 mL cold RPMI- 1640 medium using a 26 gauge needle. Cells were filtered through a 70 pm nylon strainer and further purified with the EasySep mouse neutrophil enrichment kit (StemCell Technologies, Vancouver, Canada), following the manufacturer's instructions.

Human IgG purification

10 ml of human plasma from 4 healthy volunteers was pooled, filtered (0.45 pm) and diluted 1: 1 in PBS. A 5 ml protein G Sepharose 4 Fast flow column (GE Healthcare) was used to extract total human IgG. Sample was loaded (1 ml/minute) and washed with 4 column volumes PBS. Bound antibodies were eluted using 0.1 M glycine (pH 2.3), and 1 M Tris-HCI (pH 8.0) was used to neutralize the eluted fractions. The antibody concentration was determined by extinction coefficient with an absorbance at 280 nm of 1.4 being equal to a concentration of 1.0 mg of IgG.

In vitro neutrophil phagocytosis assay

For the phagocytosis assay, purified human neutrophil or mouse BM-derived neutrophils were stimulated with 10-7M N-formyl-Met-Leu-Phe (fMLF; Sigma-Aldrich) or 1 pM WKYMV (Phoenix Pharmaceuticals, Germany), respectively, labeled with 1 pM calcein AM viability dye (Invitrogen) and seeded in a 48-well plate at 50 x 10³ cells per well. Receptors were blocked by adding 100 pg/ml purified human IgG, 100 pg/ml purified mouse IgG (Sigma-Aldrich), 10 pg/ml anti-mouse CD16.2 (BioCell), 10 pg/ml anti-mouse CD16/CD32 (BD Pharmingen), 10 pg/ml anti-human/mouse CDllb (MI/70 clone, Biolegend) antibody or 10 pg/ml purified rat IgG2b isotype (Biolegend), 10 minutes prior adding opsonized debris. Necrotic debris was generated by mechanical disruption of HepG2 cells with a pellet mixer for 5 minutes. The debris was washed with PBS (5 min, 13000 x g, RT) and labeled for 1 hour with pHrodo Red succinimidyl ester (Thermo Fisher Scientific) with 2 pL of a 10 mM solution per 10xl0⁶ cells, in 0.1 M sodium bicarbonate at pH 8.4. The unbound pHrodo was washed away (5 min, 13000 x g, RT), whereafter the debris was opsonized with 20% fresh mouse/human serum, mouse/human heat629 inactivated serum (30 min, 56°C) in PBS, 10 pg/mL purified mouse IgG, or 10 pg/mL purified mouse IgM (Mouse IgG and IgM whole molecule, Rockland, Limerick, PA, USA) for Ih at 37°C. The debris was washed with PBS (5 min, 13000 x g, RT) and added to the neutrophils in a 1: 10 (cells/debris) ratio. 10 pM latrunculin B, an actin polymerization inhibitor (Sigma- Aldrich), was used to block phagocytosis. To ensure that the necrotic debris would reach the cells in a fast and homogenous way, the plate was centrifuged for 5 min, 300 x g at 4°C. Images were taken every 30 min with the Incucyte Live-Cell Analysis (Sartorius).

In vitro RAW cell phagocytosis assay

50 x 10³ RAW 264.7 cells were seeded in a 48-well plate (Corning) overnight at 37°C. Living RAW 264.7 cells were labeled with 1 pM calcein acetoxymethyl ester (AM) viability dye (Invitrogen) for 20 min at 37°C in FBS free medium. Necrotic debris was generated by mechanical disruption of HepG2 cells with a pellet mixer for 5 minutes. The debris was washed with PBS (5 min, 13 000 x g, RT) and labeled for 1 hour with pHrodo Red succinimidyl ester (Thermo Fisher Scientific) with 2 pL of a 10 mM stock per 10xl0⁶ cells, in 0.1 M sodium bicarbonate at pH 8.4. The unbound pHrodo was washed 642 away with PBS (5 min, 13 000 x g, RT) whereafter the debris was opsonized with 20% fresh mouse serum, heat inactivated (30 min, 56°C) serum in PBS, 10 pg/mL purified IgG or 10 pg/mL purified IgM (Mouse IgG and IgM whole molecule, Rockland, Limerick, PA, USA) for Ih at 37°C. The debris was washed with PBS (5 min, 13 000 x g, RT) and added to the RAW cells in a 1 : 10 (cells/debris) ratio. 10 pM latrunculin B (Sigma-Aldrich) was used to block phagocytosis. After centrifugation (5 min, 300 x g, 4°C), images were taken every 30 min with the Incucyte Live-Cell Analysis (Sartorius).

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 pM; Thermo Fisher Scientific) was administered. The incision was stitched and after 6h mice were again anaesthetized with ketamine and xylazine for imaging of the burn site by intravital microscopy. For the restitution of NAbs, RAG2-/- mice were treated with purified IgM and IgG antibodies (100 pg each) intravenously 30 minutes prior the focal burn injury.

RNA extraction and qPCR

After the phagocytosis experiments, cells were harvested and total RNA was extracted using a Rneasy Plus Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. After extraction, total RNA was quantified using a Nanodrop and 1 pg of RNA was used to perform the reverse transcription using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, US). 50 ng of the resulting cDNA was amplified in a 7500 Real-Time PCR system (Applied Biosystems) using IDT primers (See key resource table) and the TaqMan Gene Expression Master Mix (Applied Biosystems). RT-qPCR data were expressed as 2-AACT relative to gene expression of cells in steady-state. CT values were obtained using Cdknla as housekeeper gene.

Statistical analysis

All statistical analysis was performed in GraphPad Prism v9.3.1. Significance between 683 two groups was analyzed with Student 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 in the data in order to determine whether extreme values were significant outliers from the rest. Data were represented as ± SEM.

Example 2. Distribution and molecular composition of necrotic debris.

For the distribution of NAbs within the necrotic liver, to unveil the molecular composition of necrotic debris, and to determine which antigens NAbs recognize in the dead tissue, s, a mouse model of APAP-induced liver injury is used to induce liver necrosis. Briefly, wild type (WT) C57BL/6J, aging 8-10 weeks, receive an overdose of APAP (400-600 mg/Kg) orally, dissolved in warm saline. Liver injury is evaluated directly by morphometry of the necrotic areas (histology and intravital microscopy) and indirectly by serum levels of alanine aminotransferase (ALT). Liver function is monitored by the depuration rate of indocyanine green (ICG) injected intravenously. To unveil the molecular composition of the necrotic debris in tissue, mass spectrometry imaging (MSI) of the necrotic livers is performed. MSI is a label free, multiplex technique that is used to visualize the two- and three-dimensional molecular distribution of lipids, proteins, peptides and drugs. WT mice are challenged with APAP 24 hours prior to MSI analysis. Then, mice are anesthetized (ketamine 60mg/Kg and xylazine 15mg/Kg in saline, s.c.) and blood and liver is harvested. The liver is snap frozen in liquid nitrogen and embedded in optimal cutting temperature media. Liver cryo-sectioning is performed in a cryostat to generate sections of 14 pm thickness and thaw-mounted onto Tin Oxide-coated glass slides. MSI data are acquired with a RapifleX tissue Typer. For multiple labeling and detection of intracellular antigens, immunostainings of liver cryo-sections are also performed. Example 3. In vivo imaging of NAbs-dependent phagocytosis.

Alongside C57BL/6J, IgMi mice are used to dissect the physiological importance of NAbs in the clearance of necrotic debris. IgMi mice have normal B-cell development but cannot produce any soluble antibodies, as a consequence, they lack NAbs and one can reconstitute mice with specific immunoglobulins to test its physiological role in debris clearance and liver repair.

IVM are used to characterize the hepatic debris clearance in vivo. Briefly, mice are anesthetized and receive an intravenous injection of fluorescently-labeled antibodies (anti-IgM;-IgG; -Ly6G; -CCR2; -CX3CR1; -F4/80; -F-actin) and DNA dyes (Sytox green; Hoechst; Propidium iodide) according to the experiment. Then, a midline laparotomy is performed to expose the liver for imaging for up to 4 hours. An optimal timepoint for further evaluations is defined in a time-response curve of APAP-induced injury from 6 to 72 hours after the challenge. WT and IgMi mice (reconstituted or not with purified IgM or IgG3) are challenged with APAP at the chosen timepoint. Inflammation and necrosis scores, tracking of leukocytes, necrotic debris phagocytosis, NAbs distribution within necrotic sites and liver repair are imaged in real time by IVM. After IVM, liver and serum are harvested for further analysis of serum ALT and NAbs, cytokines and chemokines (e.g. CXCL1, CXCL2, GM-CSF, CCL2, IL-6, IL-ip, TNF-o, IFNY, TGF- (3). Altered secretion of these mediators provides additional insights on how NAbs indirectly affect leukocyte activation and recruitment, necrotic debris clearance and liver repair.

Example 4. Evaluation of NAbs-coated necrotic debris phagocytosis.

The cellular response triggered by FcRs comprises both activating and inhibitory effects. To identify which FcRs in phagocytes promote the phagocytosis of NAbs- coated necrotic debris, freshly purified human phagocytes are treated with blocking antibodies against FcyR (anti- CD32a; -CD32b; -CD32c; CD16; CD16.2) or Fc-o/pR (anti-CD351) and incubated with fluorescent IgM- or IgG-coated debris during 1 hour. The phagocytosis rate and ROS production (CellRox) in leukocytes is assessed by confocal microscopy and flow cytometry; the supernatants are harvested for measuring cytokines and chemokines production. Example 5. Development of NAbs-based therapies for necrotic liver injury.

For this purpose, treatment with Fc-engineered NAbs with improved affinity to the FcRs that were found as key in debris phagocytosis (e.g. FcyR2a and Fc-o/pR) are given to mice intravenously 4-6 hours after the APAP challenge. The therapeutic efficacy of blocking inhibitory FcRs, such as FcyR2b, is tested once it improves the recognition of immunocomplexes by effector (phagocytosis-inducing) FcRs. The effectiveness of these approaches is explored using NAbs-coated phagocytosis of cell debris in vitro, and by measuring liver function (ICG) and repair (morphometry, ALT and enhanced necrotic debris phagocytosis) in vivo.

Claims

1. A composition comprising polyreactive immunoglobulins for use in the treatment of acute liver injury.

2. The composition comprising polyreactive immunoglobulins for use according to claim 1, wherein the liver injury is drug-induced liver injury, an ischemiareperfusion liver injury or an ischemia induced injury during liver transplantation.

3. The composition comprising polyreactive immunoglobulins for use according to claim 2, wherein the drug-induced liver injury, is an acetaminophen/paracetamol-induced liver injury.

4. The composition comprising polyreactive immunoglobulins for use according to any one of claims 1 to 3, for the removal of necrotic hepatocyte debris in acute liver injury.

5. The composition comprising polyreactive immunoglobulins for use according to any one of claims 1 to 4, wherein the composition is full blood plasma or blood serum.

6. The composition comprising polyreactive immunoglobulins for use according to any one of claims 1 to 4, wherein said composition is a preparation of purified IgM and/or purified IgG.

7. The composition comprising polyreactive immunoglobulins for use according to any one of claims 1 to 4, wherein the composition is a preparation of one or more of purified IgGl, purified IgG2 and purified IgG3.

8. The composition comprising polyreactive immunoglobulins for use according to any one of claims 1 to 5, in the treatment of a human individual. The composition comprising polyreactive immunoglobulins for use according to claim 8, wherein the dose of administration is between 5 mg polyreactive immunoglobulins /kg body weight and 400 mg polyreactive immunoglobulins /kg body weight.