Your privacy, your choice

We use essential cookies to make sure the site can function. We also use optional cookies for advertising, personalisation of content, usage analysis, and social media.

By accepting optional cookies, you consent to the processing of your personal data - including transfers to third parties. Some third parties are outside of the European Economic Area, with varying standards of data protection.

See our privacy policy for more information on the use of your personal data.

for further information and to change your choices.
Skip to main content
Download PDF

Semaphorin 7a is protective through immune modulation during acetaminophen-induced liver injury

Journal of Inflammation volume 22, Article number: 13 (2025) Cite this article

Abstract

Background and Aim

Acetaminophen (APAP) induced acute liver injury (ALI), the leading cause acute liver failure in the western world, has limited treatment options. APAP toxicity results in massive hepatic necrosis and secondary infiltrating monocytes and neutrophils, which contribute to pathogenesis. Semaphorin 7a (Sema7a), a chemoattractant and modulator of monocytes and neutrophils, is a potential therapeutic target in other conditions, but its role in APAP-ALI is unexplored.

Methods

Wild-type (WT) and Sema7a knockout (KO) mice were examined during APAP-ALI. Serum liver function tests, histological analysis and cellular localisation of Sema7a and its receptors, Plexin C1 and Integrin β1, were examined. Serum cytokines were quantified, tissue macrophages and neutrophils were localised, and in vivo phenotype, including phagocytosis, was assessed by immunohistochemistry and flow cytometry.

Results

Sema7a was expressed by HNF4α + peri-necrotic hepatocytes circumferentially during APAP-ALI injury phases, and serum concentrations were increased, and correlated with hepatic injury. Sema7a KO mice had increased circulating inflammatory cytokines and significantly less hepatic F4/80 + macrophages, a cell type required for hepatic repair. Sema7a KO mice had higher necrotic area neutrophils, and increased neutrophil chemoattractant CXCL1. Without Sema7a expression, mice displayed increased necrosis and liver injury markers compared to Sema7a WT mice. Without peri-necrotic hepatocyte Sema7a expression, we also identified increased cell death and hepatic cellular stress outside of necrosis.

Conclusion

We have identified a novel protective role of Sema7a during injury phases of APAP-ALI. Without peri-necrotic hepatocyte Sema7a expression and secretion, there is increased inflammation, time specific worsened hepatic necrosis and increased hepatic cell stress and death outside of the necrotic zone.

Graphical Abstract

Synopsis

Semaphorin 7a (Sema7a) is expressed on hepatocytes in a circular ring surrounding necrotic cells during acute liver injury, and circulatory levels correlate with damage. Without peri-necrotic Sema7a and secretion, there is increased hepatic inflammation, and worsened necrosis, cell stress and death.

Background

Acetaminophen (APAP) is a widely used analgesic and antipyretic medication, which is safe at therapeutic doses but overdose causes 46% of acute liver failure (ALF) in Western countries, and it remains the commonest cause of ALF-induced death [1,2,3]. APAP-induced acute liver injury (APAP-ALI) causes systemic inflammation, which can lead to multiorgan dysfunction and sepsis [4, 5]. The current pharmacological therapy for APAP-ALI, N-acetylcysteine (NAC), is effective if given within 10 h of APAP overdose ingestion [6]. Otherwise, only supportive care is available. Liver transplantation is considered for patients that develop liver failure, but suitable donor organs are not always available [7, 8]. Therefore, alternative therapeutic strategies are required to limit hepatic injury and promote recovery. One potential therapeutic avenue is to manipulate the innate immune system to limit damage and promote tissue recovery.

Semaphorins are a diverse family of highly conserved signalling proteins, that perform a variety of functions from neurogenesis axon guidance to bone homeostasis and immune responses [9,10,11,12]. Semaphorin 7a (Sema7a) is an immune semaphorin that modulates immune processes such as inflammatory infiltration, immune cell interactions, activation and suppression [10, 13, 14]. Sema7a is the only semaphorin connected to the membrane via a glycosylphosphatidylinositol linker, and it is expressed by neurones, immune cells and activated hepatic stellate cells [9, 15, 16]. Sema7a has two receptors Integrin β1 and Plexin C1 [17, 18], and promotes immune cell migration, such as facilitating neutrophil pulmonary transmigration [18, 19], or acting as a chemoattractant for monocytes and macrophages [20, 21]. Sema7a has cumulative evidence for involvement in autoimmune and inflammatory diseases [15, 20, 22] and anti-Sema7a treatment has shown efficacy for some of these conditions [23, 24]. More recent reports indicate a role in reducing inflammation in acute tissue damage [25, 26] but its actions have not been evaluated in APAP-ALI.

Despite conflicting literature, there is now substantial evidence that macrophages play a key role in resolving injury and inflammation following ALI [27,28,29,30], with poor outcomes from APAP-ALI correlating with blood monocytopenia [31]. Macrophages are highly plastic cells which acquire distinct phenotypes depending on molecular cues in their microenvironment [32, 33]. Ly6Chi macrophages show a pro-inflammatory phenotype, whereas Ly6Clo macrophages are considered restorative and promote restoration of the liver after CCl4 injury [34]. Sema7a can influence macrophage behaviour in a receptor-dependent manner and both pro-reparative and proinflammatory phenotypes have been documented [15, 22, 26].

APAP-ALI causes hepatocyte necrosis and the release of danger-associated-molecular patterns (DAMPs). DAMPs activate Kupffer cells (KCs), the liver resident macrophage, to release cytokines, which activate the innate immune system. This causes monocytes, macrophages and neutrophils to infiltrate the liver [33]. During APAP-ALI, KCs are depleted and monocyte-derived macrophages infiltrate the liver in large numbers [32]. Restorative Ly6Clo macrophages have been shown to be crucial for appropriate liver repair via phagocytosis [35, 36] and secretion of IL-10 to reduce inflammation [28]. Macrophage ablation delays repair [29, 32, 37] and stimulating macrophage proliferation, or delivering alternatively-activated macrophages can promote liver regeneration [38, 39]. Neutrophils have controversial roles in APAP-ALI, with reports indicating they contribute to injury, or tissue repair [40,41,42,43,44]. Since Sema7a influences macrophage phenotype and has involvements in neutrophil migration, we tested whether Sema7a has a hepatoprotective role during acute liver injury and repair.

Using a genetic ablation approach, we describe the local and temporal effects of Sema7a during APAP-ALI, detailing Sema7a expression on peri-necrotic hepatocytes during injury and repair, and its importance to limit hepatocyte damage and reduce inflammation. We report that Sema7a KO mice have reduced hepatic F4/80 + macrophages, higher numbers of neutrophils within necrotic areas, and increased pro-inflammatory serum cytokine concentrations, alongside increased spread of tissue damage. In conclusion, we report that Sema7a has a protective role in APAP-ALI and modulates innate immune cells to limit the damage caused by APAP-ALI.

Methods

All authors had access to the study data and had reviewed and approved the final manuscript.

In vivo experiments

Animals

C57BL6/J or Sema7a−/− mice (Jackson Laboratory, C57BL6/J background) were housed in specific pathogen-free environment and kept under standard conditions with a 12 h day/night cycle and access to food and water ad libitum. Mouse genotyping was performed by a commercial laboratory (Transnetyx). All animal experiments were carried out under procedural guidelines, severity protocols and with ethical permission from the University of Edinburgh Animal Welfare and Ethical Review Body and the Home Office (UK), licence numbers 70/7847 and P231C5F81.

APAP experiments

9–12 week old male mice were fasted for 12 h then intra-peritoneally (i.p.) injected with 350 mg/kg APAP (Sigma)), dissolved in sterile saline, n ≥ 6 / group. Controls were injected with an equivalent volume of sterile saline. Mice were kept in a 28 °C heat box, and closely monitored for the duration of the experiment.

Mice were humanely euthanised and blood was collected by cardiac puncture, into EDTA tubes for flow cytometry analysis, or clotted overnight at 4 °C then centrifuged for 10 min, 8,000 × g at 4 °C and serum collected. Livers were perfused with PBS and snap frozen or fixed in formalin overnight and paraffin embedded (FFPE).

Non-haemolysed serum liver function tests (LFTs) were performed by Dr Forbes Howie at the QMRI, University of Edinburgh according to manufacturer instructions (Alpha Laboratories). Kit instruction were adapted for use in the Cobas Fara or Cobas Mira analyser (Roche).

Immunohistochemistry

Frozen sections were fixed for 20 min with ice cold Methanol: Acetone. 4 µm-thick FFPE sections were dewaxed and rehydrated before 15 min heat mediated antigen retrieval then permeabilised in PBS 0.1% Tween 20 (PBST). For IF sections were blocked for 30 min at RT with Protein Block (Spring Bio), then incubated overnight with the primary antibody at 4 °C, then incubated with secondary antibodies for 1 h RT. Sections were stained with DAPI (1:1,000) and mounted with fluoromount (Southern Biotech).

For 3,3'-diaminobenzidine (DAB) stains, FFPE sections were sequentially blocked at RT with Bloxall (Vector) for 15 min, then Avidin and Biotin (Invitrogen) for 10 min each, followed by 30 min Protein Block. Sections were stained overnight at 4 °C with the primary antibody, followed by the secondary antibody for 1 h at RT. Slides were blocked with R.T.U. VECTASTAIN Elite ABC reagent (Vector) for 30 min, before detection with DAB (DAKO). Slides were counterstained with haematoxylin before dehydration and mounting. All primary antibodies, their required antigen retrieval and dilution is provided in Supplementary Table 1 & 2. Isotype controls can be seen in Supplementary Fig. 1, Supplementary Materials & Methods Figs. 1 and 2.

Haematoxylin and Eosin (H&E) staining and block processing was performed by SuRF Histology at the QMRI, University of Edinburgh. Slides were scanned with the Vectra Polaris multispectral slide scanner, and necrotic areas were segmented and quantified using inForm 2.4 (Perkin Elmer) software after tissue specific training, using eight 10 × FOV per mouse liver, at 1 μm resolution, as shown in Supplementary Materials & Methods Fig. 3.

Microscopy and image analysis

Fluorescent and brightfield images were acquired using a Nikon Eclipse e600 microscope fitted with a Retiga 2000R camera (Q-Imaging, Image Pro premier software). Images were contrasted and analysed using Fiji ImageJ (ImageJ version 1.52e software for Windows (ImageJ Software, National Institutes of Health, USA, available at: http://rsb.info.nih.gov/ij/)). The isotype control acted as the negative reference. All images from one batch of staining were treated equally.

Numbers of Ly6G + cells were counted manually on at least five 10 × FOV per slide. Number of F4/80 + cells were quantified using a macroinstruction on six 20 × FOV per slide. Number of Ly6G + or F4/80 + cells were divided by the necrotic area of the respective slide to give number of cells per necrotic area. Separation of the inner and outer necrotic area is demonstrated in Supplementary Materials & Methods Fig. 5. Percentage area of Sema7a positivity on DAB staining was quantified using a Fiji image J macroinstruction on six 20 × FOV per slide.

Confocal microscopy was performed with an inverted Leica TCS SP8 Confocal microscope. High magnification images used the Nyquist criterion to give the maximum resolution using 60 × objective lens. Images were converted from Z- stacks to Maximum Intensity Projections, and contrast adjusted using Fiji ImageJ.

TUNEL assay

Terminal deoxynucleotidyl transferase–mediated biotinylated deoxy-uridine triphosphate nickend labelling (TUNEL) assay, was performed on 4 µm FFPE tissue according to manufacturer instructions (In Situ Cell Death Detection Kit, TMR Red, Roche). TUNEL + DAPI + nuclei were imaged and quantified using the Perkin Elmer Operetta high content imaging system and Columbus software (Supplementary Materials & Methods Fig. 4).

Protein quantification

Protein extraction

60 mg liver tissue was homogenised with a Tissue Tearor (Biospec Products) in cold Meso Scale Diagnostics (MSD) lysis buffer (150 mM NaCl, 20 mM Tris pH7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1X Halt Protease inhibitor Cocktail (Thermo Scientific)). Homogenates were slowly mixed for 30 min, then centrifuged for 10 min 20,000 × g at 4 °C. The aqueous supernatant was removed, and the protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific).

Quantification of cytokines in mouse serum and liver

10 cytokines were multiplexed (IFN-γ, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-6, CXCL1, TNF) in 25 µL mouse serum using the V-PLEX Pro-inflammatory Panel 1’ mouse plate (MSD), following the manufacturer instructions.

The plate was read on the QuickPlex SQ 120 analyser (MSD). Standard curves were used to quantify the concentration of the respective cytokine.

Sema7a enzyme-linked immunosorbent assay (ELISA)

The Mouse LS Bio sandwich ELISA kit was used to quantify Sema7a in mouse liver protein homogenate, diluted to 0.5 mg/ml, or mouse serum, diluted 1:5. Samples were diluted in sample diluent and assayed in duplicate by following the manufacturer’s instructions (Manufacturer; LS-F6958).

Quantitative reverse transcriptase PCR (qRT-PCR)

40 mg of liver tissue was homogenised in 500 µL Qiazol (Qiagen). Homogenates were mixed with 100 µL chloroform, incubated at RT for 3 min, and then centrifuged at 4 °C 1200 × g, for 15 min. The aqueous supernatant was removed and mixed in an equal volume of 70% ethanol. RNA was extracted using an RNAeasy Kit according to manufacturer instructions (Qiagen). Reverse Transcription and Real Time-qPCR was performed using Qiagen Quantitect and Quantifast reagents on a LightCycler 480 II (Roche). Commercial primers (peptidylprolyl isomerase A (PPIA), Sema7a, QT00173488) from Qiagen’s Quantitect range were used. Gene expression was normalised to the housekeeping gene PPIA. Samples were run in technical triplicate.

Statistics

GraphPad Prism 8 Software was used for all statistical analysis. Data are presented as mean ± SD. Each datapoint represents a mouse. Normality was determined by a Shapiro-Wilks test. To test two sample groups a two-tailed unpaired t-test, with a Welch correction applied if required, or Mann Whitney test was used to compare parametric or non-parametric data respectively. Sample size was based on a power calculation where α = 0.05, desired power = 0.8, or from investigator experience.

For further information on flow cytometry, imaging, and other materials and methods, please refer to the CTAT table and supplementary materials and methods.

Results

Sema7a is expressed in peri-necrotic hepatocytes during APAP-ALI

Sema7a protein expression, assessed by immunostaining during a time course of APAP-ALI, (Fig. 1A and Supplementary Fig. 1) was not detected in healthy liver tissue or during the early stages of APAP-ALI (0–8 h). As hepatic necrosis and inflammation progressed from 12 to 24 h post APAP-ALI, Sema7a expression was localised to peri-necrotic cells, with peak expression at 24 h post-APAP-ALI. Sema7a expression diminished as necrosis resolved (36—60 h post APAP-ALI) (Fig. 1B, C).

Fig. 1
figure 1

Sema7a is expressed on peri-necrotic cells during APAP-ALI. A Experimental schematic; WT mice received 350 mg/kg APAP i.p. and tissues collected during injury (< 24 h) and recovery/repair (> 24 h). B Sema7a expression during the APAP time course study. Black dashes outline necrotic areas. Yellow dashes outline Sema7a positive peri-necrotic cells. (C) Percentage area of Sema7a positivity, calculated from Image J, averaged from 6 fields of view/ mouse, 24 h expression highest p = 0.0005 ANOVA, Tukey's multiple comparisons test 0 vs 24 h p = 0.0057. Each datapoint represents an individual mouse. Scale bars 100 µm. CV, central vein; PT, portal tract; S7a, Sema7a. n ≥ 3 mice/time point

Full size image

Since peak Sema7a expression occurs at 24 h post-APAP, we further characterised Sema7a expression at this time point. Sema7a/Hnf4α dual staining identified that peri-necrotic hepatocytes express Sema7a (Fig. 2A). In perfused whole liver, Sema7a mRNA expression significantly increased with APAP-ALI compared to healthy mice (p = 0.0053, Fig. 2B). However, hepatic Sema7a protein levels did not significantly change (Fig. 2C). This total hepatic difference between transcribed and translated quantification may reflect release of the protein into circulation. Circulating Sema7a significantly increased in mouse serum at 12 and 24 h post-APAP (p = 0.0127 and p = 0.007, respectively) (Fig. 2D), and circulating Sema7a correlated with a serum marker of hepatocellular injury, alanine aminotransferase (ALT) (Pearson coefficient (r) = 0.728, Fig. 2E).

Fig. 2
figure 2

Sema7a is expressed by hepatocytes and correlates with serum ALT activity (A) Experimental schematic; WT mice received 350 mg/kg APAP i.p and tissues collected during injury (12 and 24 h) and repair (42 h) (B) Representative immunofluorescent labelled hepatic sections, Sema7a (red) and Hnf4α (green) from healthy mice (top) and at 24 h APAP-ALI (bottom). Yellow arrows, Sema7a + Hnf4a + hepatocytes. White dashed line and * indicate necrotic areas. C Whole liver lysate expression of Sema7a qRT-PCR, t-test, Welch’s correction (p = 0.0053); and (D) ELISA, unpaired t-test (p = 0.13). E Sema7a ELISA on serum during an APAP-ALI time course, Brown-Forsythe ANOVA (P = 0.0007), Dunnett’s comparisons Saline vs 12 h (p = 0.0149), Saline vs 24 h (p = 0.0089). F Correlation of Sema7a levels and ALT in serum. Linear Regression R2 = 0.53, P = 0.0001. Scale bars 100 µm. Each datapoint represents an individual mouse. * p < 0.05; ** p < 0.01. n ≥ 6 mice/ time point

Full size image

As Sema7a is localised to peri-necrotic hepatocytes, we ensured Sema7a does not label dying cells. Sema7a + hepatocytes are negative for both active caspase 3 and TUNEL + DAPI + nuclei (Supplementary Fig. 1) demonstrated by serial stained sections. Therefore, Sema7a is expressed by the viable hepatocytes, which surround necrosis.

Sema7a expressing hepatocytes directly contact cells expressing Sema7a receptors

The predominant Sema7a receptors are Integrin β1 and Plexin C1. Integrin β1 is ubiquitously expressed across the liver [45] and Plexin C1 is expressed by hepatic stellate cells (HSCs), (Supplementary Fig. 2). Integrin β1 is upregulated by HSCs during fibrosis and Sema7a binding is known to contribute to TGF-beta mediated fibrosis [16]. Sema7a also effects spiny stellate cell function [46], but the impact of APAP-ALI on Sema7a receptor expression and their proximity to Sema7a expressing hepatocytes is unknown. To assess this relationship, we completed a dual stain for Sema7a during APAP-ALI. Integrin β1 + and Plexin C1 + HSCs were detected between the Sema7a + hepatocytes at peak Sema7a expression, suggesting a localised signalling interaction (Fig. 3A&B). At 24 h post APAP-ALI, hepatic Integrin β1 mRNA expression and Plexin C1 protein expression increased compared to healthy mice (Supplementary Fig. 3). In Sema7a KO mice, Plexin C1 but not Integrin β1 expression was reduced at 24 h post APAP (Supplementary Fig. 4, p = 0.0208), suggesting Sema7a promotes the expression of Plexin C1.

Fig. 3
figure 3

Expression of Sema7a and its Receptors PlexinC1 and Integrin β1 Results from 24 h post 350 mg/kg APAP or saline treated WT mice, Scale bars 100 µm. DAPI counterstain, n ≥ 8 mice/group, (A)Representative immunofluorescent labelled hepatic sections Sema7a (red) and Integrin β1 (green) with insert showing proximity of cell to cell expression, but no colocalization. B Representative immunofluorescent labelled hepatic sections Sema7a (red) and Plexin C1 (green) with insert showing proximity of cell to cell expression, but no colocalization

Full size image

Sema7a KO mice have more liver injury during APAP-ALI

To investigation of the importance of peri-necrotic hepatocyte Sema7a expression in APAP-ALI we completed experiments in Sema7a KO mice. We first confirmed deficiency of Sema7a expression in Sema7a KO mice both with hepatic qPCR analysis and immunohistochemistry at 24 h post APAP-ALI (Fig. 4). We also compared LFTs, and liver histology (Fig. 5A-C. & Supplementary Fig. 5) of healthy WT and Sema7a KO mice and showed no significant differences compared to WT mice (Fig. 5A-C. & Supplementary Fig. 5).

Fig. 4
figure 4

Sema7a KO mice do not express Sema7a Results from WT and Sema7A KO mice 24 h post saline or 350 mg/kg APAP. A Hepatic lysate qPCR, Sem7a mRNA expression relative to housekeeper PPIA1. WT APAP-ALI treated mice have significantly elevated expression at 24 h post APAP compared to all other groups (ANOVA, p < 0.0001, Tukey’s multiple comparisons, p ≤ 0.0001). n = 4. B Representative hepatic Sema7a (DAB) labelled sections from WT and Sema7a mice 24 h post APAP, showing no expression in WT mice

Full size image
Fig. 5
figure 5

Sema7a KO mice have more liver necrosis during APAP-ALI. A Experimental schematic; WT and Sema7a KO mice treated with 350 mg/kg APAP, assessed at 12, 24 and 42 h. B Representative liver histological stains from Sema7a WT, and Sema7a KO mice. Necrotic areas outlined with black dashed line. Scale bars 100 µm. C-F Necrosis quantification, necrotic areas were segmented and quantified using inForm 2.4 (Perkin Elmer) software using inform tissue training for consistent changes; enlarged cell organelles, membrane rupture and cell lysis at 0 h (C) 12 h, t-test (p = 0.0391) (D), 24 h (E) and 42 h (F) post 350 mg/kg APAP injection. Each datapoint represents a mouse. * p < 0.05; ** p < 0.01, n ≥ 8 mice/group

Full size image

Comparing Sema7a KO and WT mice during a time course of 350 mg/kg APAP-ALI identified that Sema7a KO mice exhibit significantly more hepatic necrosis at 12 h post APAP-ALI (Fig. 5D-F,p = 0.0391). This was supported by higher serum ALT (p = 0.0095) and aspartate aminotransferase (AST) (p = 0.0107, Fig. 6A&B) at 12 h post APAP-ALI. Although increased necrosis was not evident at 24 h post APAP-ALI and only a trended increase was present in LFTs, Sema7a KO mice had higher serum bilirubin at 24 h, which can increase with reduced liver function, (p = 0.0109, Supplementary Fig. 5).

Fig. 6
figure 6

Sema7a KO mice have more liver damage during APAP-ALI. Serum markers of liver injury (LFTs), ALT (A) and AST (B) were higher at 12 h post 350 mg/kg APAP in Sema7a KO mice compared to WT mice during the time course of APAP-ALI, (t-test, Welch’s correction, p = 0.0095 and p = 0.0107 respectively). Each datapoint represents a mouse. * p < 0.05; ** p < 0.01, n ≥ 7 mice/group. During recovery, at 42 h post APAP-ALI, Sema7a KO mice and Sema7a WT mice exhibited similar necrosis (Fig. 5F) and proliferation (Supplementary Fig. 6). This suggests Sema7a does not directly influence recovery from APAP-ALI. However, Sema7a KO mice had raised ALP at this time point (p = 0.0306, Supplementary Fig. 5)

Full size image

Absence of Sema7a on peri-necrotic hepatocytes results in diffuse cell death during APAP-ALI

Alongside the documented elevated hepatocellular injury and necrosis, deficiency of Sema7a in peri-necrotic hepatocytes was associated with alterations in cell death distribution. At 24 h post APAP-ALI, during peak Sema7a expression, Sema7a KO mice had the same percentage of TUNEL + nuclei as WT mice, but TUNEL + nuclei were not contained within the centrilobular necrotic areas and there were TUNEL + cells in the surrounding healthy parenchyma (Fig. 7, p = 0.0151), suggesting Sema7a plays a localised role to limit the extent of liver tissue damage at 24 h post APAP-ALI.

Fig. 7
figure 7

Absence of Sema7a on peri-necrotic hepatocytes results in diffuse cell death during APAP-ALI. A TUNEL assay (yellow), DAPI (blue), in Sema7a WT and Sema7a KO mice 24 h post APAP-ALI. Necrotic areas outlined with white lines and *. Red arrows are TUNEL + nuclei outside the necrotic area. B Percentage of TUNEL + DAPI + nuclei per FOV. C Number of TUNEL + DAPI + nuclei outside the necrotic area, per FOV (t-test, p0.0151). FOV, fields of view. Scale bars 100 µm. Each datapoint represents a mouse. n = 8 mice/group. *p < 0.05, **p < 0.01

Full size image

To further assess if Sema7a KO mice have more local tissue inflammation and cell stress during APAP-ALI, High Mobility Group Box 1 (HMGB1) localisation was examined. In healthy mice, HMGB1 is retained in the nucleus, but is released from necrotic cells, during injury, therefore non-nuclear or cytosolic HMGB1 is a sign of cell stress [47]. Healthy Sema7a WT and Sema7a KO mice displayed similar nuclear HMGB1 staining (Fig. 8). However, at 24 h post APAP-ALI, Sema7a KO mice had significantly more peri-necrotic hepatocytes with HMGB1 negative nuclei (p = 0.0058, Fig. 8).

Fig. 8
figure 8

Absence of Sema7a on peri-necrotic hepatocytes results in higher cell stress during APAP-ALI. Results from WT and Sema7a KO mice 24 h post saline or 350 mg/kg APAP. A Representative HMGB1 (DAB) labelled hepatic sections from Sema7a WT (top) and Sema7a KO (bottom) mice with indicated treatments. Inset, area for 40x. Yellow arrowheads, show HMGB1 negative nuclei. B Average number of HMGB1- nuclei per FOV quantified in healthy mice. C Average number of HMGB1- nuclei per FOV following APAP-ALI were higher in Sema7a KO mice (t-test, p = 0.0058). FOV, fields of view. Scale bars 100 µm. Each datapoint represents a mouse. n ≥ 4 mice/group, **p < 0.01

Full size image

Sema7a KO mice have more inflammation during APAP-ALI

Inflammation is known to contribute to APAP induced injury, and Sema7a is known as an immunomodulator, so to start assessing the effect of Sema7a deficiency on inflammation during APAP-ALI, a panel of pro-inflammatory cytokines were quantified in the serum of Sema7a WT and Sema7a KO mice throughout the time course. Sema7a KO mice had more CXCL1 (p = 0.0459) and IL-6 (p = 0.0078) at 24 h post APAP-ALI (Fig. 9), indicative of a higher systemic pro-inflammatory response.

Fig. 9
figure 9

Sema7a KO mice have increased serum pro-inflammatory cytokines. Serum cytokines in Sema7a WT and Sema7a KO mice following 350 mg/kg APAP. A CXCL1, a neutrophil chemoattractant, was higher in Sema7a KO mice serum at 24 h (t-test, Welch’s correction, p = 0.0459). B IL-6 was higher in Sema7a KO mice serum at 24 h (t-test, Welch’s correction, p = 0.0078)

Full size image

Sema7a KO mice have more neutrophils within necrosis during APAP-ALI

HMGB1 and CXCL1 are known neutrophil chemo-attractants. We therefore quantified numbers of Ly6G + neutrophils in liver tissue using both immunohistochemistry (Fig. 10 A-B) and flow cytometry, and in whole blood by flow cytometry. Sema7a WT and Sema7a KO mice had the same total number of hepatic and circulating neutrophils, throughout the APAP-ALI time course (Fig. 10 A-B, Supplementary Fig. 7–9). However, Sema7a KO mice had a greater density of neutrophils within centrilobular necrotic areas at 24 h post APAP-ALI, when Sema7a expression was otherwise highest in WT mice (Fig. 10 C; p = 0.0182). WT mice hepatic neutrophils do not express Sema7a, but express both known receptors for Sema7a, Plexin C1 and Integrin αvβ1 (Supplementary Fig. 10).

Fig. 10
figure 10

Sema7a KO mice have increased neutrophils infiltrating necrotic areas. Results from Sema7a WT and Sema7a KO mice 24 h post 350 mg/kg APAP. A Representative Ly6G labelled hepatic sections in Sema7a WT (left) and Sema7a KO mice (right). B Average number of Ly6G neutrophils per FOV. C The average number of neutrophils within necrosis was higher in Sema7a KO mice (t-test, p = 0.0182). E Ly6G (green) and active Caspase 3 (aCsp3, red) expression in WT (centre) and Sema7a KO mice (right) at 24 h post-APAP-ALI. Quantified (right) as percentage of apoptotic neutrophils (aCsp3 + , Ly6G +) in the total Ly6G population. Unpaired t test with Welch's correction, p = 0.0129. Scale bars 100 µm. Each datapoint represents a mouse. Unpaired t-test, unless stated. *p < 0.05, **p < 0.01

Full size image

Neutrophils rapidly migrate into liver after APAP-ALI [48] and we recently identified hepatic neutrophil cleaved caspase 3 expression is markedly reduced, consistent with increased activation and survival [49]. At 24 h post APAP-ALI, we show only 1.6% of neutrophils expressed active caspase 3 in Sema7a WT mice consistent with this finding, and this was significantly lower in Sema7a KO mice (0.7%, Fig. 10D-E, p = 0.0129), indicating less neutrophil apoptosis. Although this reduction is small, we believe this is significant in this already activated cell population. Further reduction in this marker of cell death could indicate increased neutrophil activation in the Sema7a KO mice or altered phagocytosis/clearance of apoptotic neutrophils. Macrophages remove apoptotic neutrophils by phagocytosis [50,51,52,53] but this has yet to be shown in vivo after APAP-ALI. We investigated the effect of Sema7a on phagocytosis with an in vivo PKH assay during APAP-ALI and found no differences between Sema7a KO and WT mice for monocytes, macrophages, or neutrophils (Supplementary Fig. 11). Therefore, Sema7a does not directly impact this form of phagocytosis, though leucocytosis clearance is mediated by Sema7a in other models [25].

Sema7a KO mice have less F4/80 + macrophages during APAP-ALI

To examine if a Sema7a deficiency impacted liver macrophages, a F4/80 stain was performed (Fig. 11A). Healthy Sema7a WT and Sema7a KO mice have comparable numbers of F4/80 + macrophages. However, at 12 h post APAP-ALI, total hepatic F4/80 + macrophages numbers were significantly lower in Sema7a KO mice (p = 0.0315, Fig. 11B), with a reduced density within the necrotic area (p = 0.044, Fig. 11C) indicating a possible exaggeration of the previously recognised KC depletion [30, 32].

Fig. 11
figure 11

Sema7a KO mice show less F4/80 + macrophages at 12 h APAP-ALI, with aberrant localisation. Results are from WT and Sema7a KO mice receiving 350 mg/kg APAP or saline. A Representative images of liver F4/80 + macrophages in WT (left) and Sema7a KO mice (right) at the indicated timepoints. B Average number of F4/80 + macrophages per FOV, (12 h t-test, p = 0.0315). C Average number of F4/80 + macrophages in necrotic areas during the time course of APAP-ALI (12 h t-test, p = 0.044). D Location of F4/80 + macrophages in the necrotic area at 24 h APAP-ALI. The necrotic area was separated into outer and central necrotic zones. Average number of F4/80 + macrophages per necrotic zones was calculated in Sema7a WT and Sema7a KO mice (12 h t-test, p = 0.0366). Scale bars 100 μm. Each datapoint represents an individual mouse. n ≥ 6 mice/group. *p < 0.05

Full size image

From 12 to 24 h post APAP-ALI the hepatic F4/80 + population increased in both Sema7a WT and Sema7a mice, consistent with previous literature of infiltrating macrophages replenishing the liver macrophage pool [32]. At 24 h post APAP-ALI total hepatic macrophages were not different (Fig. 11B Supplementary Fig. 12), however, Sema7a KO mice had less central necrosis macrophages (p = 0.0366, Fig. 11D), suggesting Sema7a has a localised role which facilitates F4/80 + macrophage migration into the necrotic area. During recovery, total hepatic F4/80 + macrophages numbers declined similarly in both Sema7a WT and Sema7a KO mice (Fig. 11B).

Discussion

Novel strategies to limit the extent of necrosis, or to promote healthy liver repair from APAP-ALI are urgently required for APAP-overdose patients [2, 5]. Understanding APAP-ALI and secondary inflammation are crucial to identify potential targets for patient therapy. Here, we report a novel role of the immunomodulator Sema7a during APAP-ALI.

Sema7a is expressed by hepatocytes, shown here and previously [54]. It’s highly localised, and increased expression by peri–necrotic hepatocytes during APAP-ALI is a novel finding. Serum concentrations of Sema7a correlate with serum injury biomarkers as injury progresses, consistent with the findings in human paediatric patients with acute inflammatory abdominal disease [26]. Sema7a deficiency in this APAP-ALI model was detrimental, leading to increased hepatocellular damage, demonstrated by raised LFTs, more non-nuclear HMGB1 cells, higher necrosis, and TUNEL positivity in liver parenchyma. This finding is in contrast to various non-hepatic models, where Sema7a promotes tissue injury and fibrosis [15, 55, 56]. This protective finding is also different to chronic hepatic injury models, such as CCl4-induced liver injury, where Sema7a promotes hepatic fibrosis and Il6 and Ccl2 expression [16]. More recent hepatobiliary focussed publications identified mutations in Sema7a causing a gain of function, progressing disease; non-alcoholic fatty liver disease (NAFLD) and intrahepatic cholestasis [54, 57]. Paradoxical to these studies, in acute sepsis and peritonitis, a protective role of Sema7a has been identified through immunomodulation [25, 26]. Here we show a protective action of Sema7 in ALI that has not previously been recognised.

We identified that Plexin C1 was reduced in Sema7a KO mice, at 24 h APAP-ALI (Supplementary Fig. 6), suggesting Sema7a promotes Plexin C1 expression on HSCs. This increased expression of receptor Plexin C1 has also been identified in paediatric systemic vasculitis [58]. As both receptors are present on neutrophils this difference may influence their migration and activation.

Alongside higher hepatic injury and altered receptor expression, Sema7a KO mice had more inflammation during APAP-ALI with higher CXCL1 and IL-6 at 24 h. This finding also coincided with alterations in inflammatory cell populations, with both reduced central necrotic zone F4/80 + macrophages and higher neutrophils. As Sema7a is a macrophage chemoattractant [20], its lack of expression on peri-necrotic hepatocytes may have reduced monocyte derived macrophage migration, which are known to replenish depleted F4/80 macrophages after in APAP-ALI [32, 59].

As well as hepatic necrosis macrophage reduction we identified that Sema7a KO resulted in higher necrosis neutrophils. Neutrophils are attracted to the liver during injury by DAMPs, such as passively released HMGB1 [42,43,44] or cytokines such as CXCL1 [60]. In Sema7a KO mice, the hepatic necrotic area neutrophil increase may be due to the documented HMGB1 and CXCL1 elevations increasing local neutrophil migration or retention. Alternatively, increased numbers may be due to increased survival and potentially activation during APAP-induced inflammation [61]. Körner et al. identified Sema7a dampened neutrophil recruitment and was crucial for inflammation resolution, including leukocyte clearance in murine peritonitis. Furthermore, Sema7a deficiency may also delay neutrophil migration out of the necrotic tissue, as endothelial Sema7a has been shown to facilitate neutrophil transmigration during pulmonary injury, [18, 29]. Further interrogations of how Sema7a impacts in vivo neutrophil migration in APAP-ALI are warranted. Sema7a promotes extracellular matrix remodelling factors including Cathepsin S [56] and neutrophils secrete cathepsins to degrade basement membranes in sterile thermal liver injury, enabling them to exit the damaged tissue [62]. The increased numbers of necrotic area neutrophils may be one of the reasons for higher hepatic necrosis, consistent with a pro-inflammatory role postulated by several authors [21, 42].

The higher hepatic damage in the Sema7a KO mice and higher inflammation in this study may also by due to altered immune cell function. Macrophages are highly plastic cells which acquire distinct phenotypes depending on molecular cues in their microenvironment [32, 33] and Sema7a is a recognised cue of macrophage and monocyte activity [15, 63]. We did not find evidence of deficiency of innate immune cell PKH phagocytosis, so reduced efferocytosis of hepatic neutrophils, an important component of inflammation resolution [35, 64], is unlikely in APAP-ALI, despite this finding in peritonitis [26]. Therefore, the increased neutrophils in the necrotic area at 24 h APAP-ALI in Sema7a KO mice could be a result of reduced macrophage migration [35, 65]. Sema7a has been shown as crucial for the resolution of severe inflammation, through polarization of macrophages to a pro-resolving phenotype in acute peritonitis [26]. This would be an important future direction for research of Sema7a in acute liver injuries, given the knowledge that restorative Ly6Clo macrophages have been shown to be crucial for appropriate liver repair via phagocytosis [35, 36] and secretion of IL-10 to reduce inflammation [28].

Conclusion

In conclusion, we have identified a novel hepatoprotective role of Sema7a in acute liver injury, with associated innate immunity changes, highlighting a potential immunomodulatory role behind this phenotype. Hepatic Sema7a expression increased during the injury phase of APAP-ALI and peak expression was localised to the peri-necrotic area. Without peri-necrotic Sema7a there was more hepatic necrosis during injury (12 h) and cellular damage (TUNEL + and HMGB1-) was not restricted the necrotic region. Sema7a deficiency resulted in more inflammation at 24 h post APAP-ALI, with increased IL6 and CXCL1 quantification. There were more neutrophils within necrosis, with lower cleaved caspase 3, indicating increased activation. There were also lower necrotic area macrophages, key cells for liver repair following APAP-ALI. Future work to further clarify the mechanisms behind the hepatoprotective effect of Sema7a in APAP-ALI are warranted. Better understanding of these mechanisms might identify this as a novel therapeutic target to limit the spread of necrosis and reduce inflammation during acute tissue injury.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

APAP:

Acetaminophen

APAP-ALI:

Acetaminophen-induced liver injury

ALF:

Acute liver failure

ALT:

Alanine aminotransferase

APAP-ALI:

APAP-induced acute liver injury

AST:

Aspartate aminotransferase

CXCL1:

Chemokine (C-X-C motif) ligand 1

DAMPs:

Danger-associated-molecular patterns

HSCs:

Hepatic stellate cells

HMGB1:

High mobility group box 1

IL:

Interleukin

KCs:

Kupffer cells

KO:

Knockout

LFTs:

Liver function tests

NAC:

N-acetylcysteine

NAFLD:

Non-alcoholic fatty liver disease

Sema7a:

Semaphorin 7a

WT:

Wild-type

References

  1. Bernal W, Auzinger G, Dhawan A, Wendon J. Acute liver failure. The Lancet. 2010;376:190–201.

    Article  Google Scholar 

  2. Reuben A, Tillman H, Fontana RJ, Davern T, Mcguire B, Stravitz RT, Durkalski V, Larson AM, Liou I, Fix O, Schilsky M, Mccashland T, Hay JE, Murray N, Shaikh OS, Ganger D, Zaman A, Han SB, Chung RT, Smith A, Brown R, Crippin J, Harrison ME, Koch D, Munoz S, Reddy KR, Rossaro L, Satyanarayana R, Hassanein T, Hanje AJ, Olson J, Subramanian R, Karvellas C, Hameed B, Sherker AH, Robuck P, Lee WM. Outcomes in adults with acute liver failure between 1998 and 2013: An observational cohort study. Ann Intern Med. 2016;164:724–32.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Karvellas CJ, Leventhal TM, Rakela JL, Zhang J, Durkalski V, Reddy KR, Fontana RJ, Stravitz RT, Lake JR, Lee WM, Parekh JR. Outcomes of patients with acute liver failure listed for liver transplantation: A multicenter prospective cohort analysis. Liver Transpl. 2023;29:318–30.

    Article  PubMed  Google Scholar 

  4. Rolando N, Wade J, Davalos M, Wendon J, Philpott-Howard J, Williams R. The systemic inflammatory response syndrome in acute liver failure. Hepatology. 2000;32:734–9.

    Article  CAS  PubMed  Google Scholar 

  5. Stravitz RT, Fontana RJ, Karvellas C, Durkalski V, McGuire B, Rule JA, Tujios S, Lee WM. Acute Liver Failure Study Group. Future directions in acute liver failure. Hepatology 2023;78:1266–1289.

  6. Prescott LF, Critchley JAJH, Proudfoot AT, Illingworth RN, Stewart MJ, Adam RD. Intravenous N-acetylcysteine: The treatment of choice for paracetamol poisoning. Br Med J . Epub ahead of print 1979. https://doi.org/10.1136/bmj.2.6198.1097.

  7. Lee WM. Acetaminophen (APAP) hepatotoxicity-Isn’t it time for APAP to go away? J Hepatol. 2017;67:1324–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Simpson KJ, Bates CM, Henderson NC, Wigmore SJ, Garden OJ, Lee A, Pollok A, Masterton G, Hayes PC. The utilization of liver transplantation in the management of acute liver failure: Comparison between acetaminophen and non-acetaminophen etiologies. Liver Transpl. 2009;15:600–9.

    Article  PubMed  Google Scholar 

  9. Pasterkamp RJ, Peschon JJ, Spriggs MK, Kolodkin AL. Semaphorin 7A promotes axon outgrowth through integrins and MAPKs. Nature. 2003;424:398–405.

    Article  PubMed  Google Scholar 

  10. Song Y, Wang L, Zhang L, Huang D. The involvement of semaphorin 7A in tumorigenic and immunoinflammatory regulation. J Cell Physiol. 2021;236:6235–48.

    Article  CAS  PubMed  Google Scholar 

  11. Köhler D, Granja T, Volz J, Koeppen M, Langer HF, Hansmann G, Legchenko E, Geisler T, Bakchoul T, Eggstein C, Häberle HA, Nieswandt B, Rosenberger P. Red blood cell-derived semaphorin 7A promotes thrombo-inflammation in myocardial ischemia-reperfusion injury through platelet GPIb. Nat Commun;11 . Epub ahead of print December 1, 2020. https://doi.org/10.1038/s41467-020-14958-x.

  12. Carulli D, de Winter F, Verhaagen J. Semaphorins in Adult Nervous System Plasticity and Disease. Frontiers in Synaptic Neuroscience;13 . Epub ahead of print May 11, 2021. https://doi.org/10.3389/fnsyn.2021.672891.

  13. Nishide M, Kumanogoh A. The role of semaphorins in immune responses and autoimmune rheumatic diseases. Nat Rev Rheumatol. 2018;14:19–31.

    Article  CAS  PubMed  Google Scholar 

  14. Worzfeld T, Offermanns S. Semaphorins and plexins as therapeutic targets. Nat Rev Drug Discov. 2014;13:603–21.

    Article  CAS  PubMed  Google Scholar 

  15. Suzuki K, Okuno T, Yamamoto M, Pasterkamp RJ, Takegahara N, Takamatsu H, Kitao T, Takagi J, Rennert PD, Kolodkin AL, Kumanogoh A, Kikutani H. Semaphorin 7A initiates T-cell-mediated inflammatory responses through alpha1beta1 integrin. Nature. 2007;446:680–4.

    Article  CAS  PubMed  Google Scholar 

  16. De Minicis S, Rychlicki C, Agostinelli L, Saccomanno S, Trozzi L, Candelaresi C, Bataller R, Millán C, Brenner D a., Vivarelli M, Mocchegiani F, Marzioni M, Benedetti A, Svegliati-Baroni G. Semaphorin 7A contributes to TGF-β-mediated liver fibrogenesis. Am J Pathol 2013;183:820–30.

  17. Liu H, Juo ZS, Shim AHR, Focia PJ, Chen X, Garcia KC, He X. Structural Basis of Semaphorin-Plexin Recognition and Viral Mimicry from Sema7A and A39R Complexes with PlexinC1. Cell. 2010;142:749–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Morote-Garcia JC, Napiwotzky D, Köhler D, Rosenberger P, Kohler D, Rosenberger P, Köhler D, Rosenberger P, Kohler D, Rosenberger P, Köhler D, Rosenberger P, Kohler D, Rosenberger P. Endothelial Semaphorin 7A promotes neutrophil migration during hypoxia. Proc Natl Acad Sci U S A. 2012;109:14146–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Granja T, Köhler D, Mirakaj V, Nelson E, König K, Rosenberger P. Crucial role of Plexin C1 for pulmonary inflammation and survival during lung injury. Mucosal Immunol. 2014;7:879–91.

    Article  CAS  PubMed  Google Scholar 

  20. Elder AM, Tamburini BAJ, Crump LS, Black SA, Wessells VM, Schedin PJ, Borges VF, Lyons TR. Semaphorin 7A promotes macrophage-mediated lymphatic remodeling during postpartum mammary gland involution and in breast cancer. Cancer Res. 2018;78:6473–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Holmes S, Downs AM, Fosberry A, Hayes PD, Michalovich D, Murdoch P, Moores K, Fox J, Deen K, Pettman G, Wattam T, Lewis C. Sema7A is a potent monocyte stimulator. Scand J Immunol. 2002;56:270–5.

    Article  CAS  PubMed  Google Scholar 

  22. Kang S, Okuno T, Takegahara N, Takamatsu H, Nojima S, Kimura T, Yoshida Y, Ito D, Ohmae S, You D-J, Toyofuku T, Jang MH, Kumanogoh A. Intestinal epithelial cell-derived semaphorin 7A negatively regulates development of colitis via αvβ1 integrin. J Immunol. 2012;188:1108–16.

    Article  CAS  PubMed  Google Scholar 

  23. Mizutani N, Nabe T, Yoshino S. Semaphorin 7A plays a critical role in IgE-mediated airway inflammation in mice. Eur J Pharmacol. 2015;764:149–56.

    Article  CAS  PubMed  Google Scholar 

  24. Chen X, Wang H, Jia K, Wang H, Ren T. Anti-Semaphorin-7A single chain antibody demonstrates beneficial effects on pulmonary inflammation during acute lung injury. Exp Ther Med. 2018;15:2356–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Körner A, Köhler D, Schneider M, Roth JM, Granja TF, Eggstein C, Mirakaj V, Rosenberger P. Semaphorin 7A is protective during inflammatory peritonitis through integrin receptor signaling. Front Immunol;14 . Epub ahead of print 2023. https://doi.org/10.3389/fimmu.2023.1251026.

  26. Körner A, Bernard A, Fitzgerald JC, Alarcon-Barrera JC, Kostidis S, Kaussen T, Giera M, Mirakaj V. Sema7A is crucial for resolution of severe inflammation. Proc Natl Acad Sci. 2021;118: e2017527118.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Laskin DL, Gardner CR, Price VF, Jollow DJ. Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology. 1995;21:1045–50.

    Article  CAS  PubMed  Google Scholar 

  28. Ju C, Reilly TP, Bourdi M, Radonovich MF, Brady JN, George JW, Pohl LR. Protective role of kupffer cells in acetaminophen-induced hepatic injury in mice. Chem Res Toxicol. 2002;15:1504–13.

    Article  CAS  PubMed  Google Scholar 

  29. You Q, Holt M, Yin H, Li G, Hu CJ, Ju C. Role of hepatic resident and infiltrating macrophages in liver repair after acute injury. Biochem Pharmacol. 2013;86:836–43.

    Article  CAS  PubMed  Google Scholar 

  30. Mossanen JC, Krenkel O, Ergen C, Govaere O, Liepelt A, Puengel T, Heymann F, Kalthoff S, Lefebvre E, Eulberg D, Luedde T, Marx G, Strassburg CP, Roskams T, Trautwein C, Tacke F. Chemokine (C-C motif) receptor 2-positive monocytes aggravate the early phase of acetaminophen-induced acute liver injury. Hepatology;64 . Epub ahead of print 2016. https://doi.org/10.1002/hep.28682.

  31. Moore JK, MacKinnon AC, Man TY, Manning JR, Forbes SJ, Simpson KJ. Patients with the worst outcomes after paracetamol (acetaminophen)-induced liver failure have an early monocytopenia. Aliment Pharmacol Ther. 2017;45:443–54.

    Article  CAS  PubMed  Google Scholar 

  32. Zigmond E, Samia-Grinberg S, Pasmanik-Chor M, Brazowski E, Shibolet O, Halpern Z, Varol C. Infiltrating Monocyte-Derived Macrophages and Resident Kupffer Cells Display Different Ontogeny and Functions in Acute Liver Injury. J Immunol. 2014;193:344–53.

    Article  CAS  PubMed  Google Scholar 

  33. Wen Y, Lambrecht J, Ju C, Tacke F. Hepatic macrophages in liver homeostasis and diseases-diversity, plasticity and therapeutic opportunities. Cell Mol Immunol. 2021;18:45–56.

    Article  CAS  PubMed  Google Scholar 

  34. Ramachandran P, Pellicoro A, Vernon MA, Boulter L, Aucott RL, Ali A, Hartland SN, Snowdon VK, Cappon A, Gordon-Walker TT, Williams MJ, Dunbar DR, Manning JR, Van Rooijen N, Fallowfield JA, Forbes SJ, Iredale JP. Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis. Proc Natl Acad Sci U S A;109 . Epub ahead of print 2012. https://doi.org/10.1073/pnas.1119964109.

  35. Triantafyllou E, Pop OT, Possamai LA, Wilhelm A, Liaskou E, Singanayagam A, Bernsmeier C, Khamri W, Petts G, Dargue R, Davies SP, Tickle J, Yuksel M, Patel VC, Abeles RD, Stamataki Z, Curbishley SM, Ma Y, Wilson ID, Coen M, Woollard KJ, Quaglia A, Wendon J, Thursz MR, Adams DH, Weston CJ, Antoniades CG. MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure. Gut. 2018;67:333–47.

    Article  CAS  PubMed  Google Scholar 

  36. Campana L, Starkey Lewis PJ, Pellicoro A, Aucott RL, Man J, O’Duibhir E, Mok SE, Ferreira-Gonzalez S, Livingstone E, Greenhalgh SN, Hull KL, Kendall TJ, Vernimmen D, Henderson NC, Boulter L, Gregory CD, Feng Y, Anderton SM, Forbes SJ, Iredale JP. The STAT3–IL-10–IL-6 Pathway Is a Novel Regulator of Macrophage Efferocytosis and Phenotypic Conversion in Sterile Liver Injury. J Immunol. 2018;200:1169–87.

    Article  CAS  PubMed  Google Scholar 

  37. Holt MP, Cheng L, Ju C. Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury. J Leukoc Biol. 2008;84:1410–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Stutchfield BM, Antoine DJ, Mackinnon AC, Gow DJ, Bain CC, Hawley CA, Hughes MJ, Francis B, Wojtacha D, Man TY, Dear JW, Devey LR, Mowat AM, Pollard JW, Park BK, Jenkins SJ, Simpson KJ, Hume DA, Wigmore SJ, Forbes SJ. CSF1 Restores Innate Immunity after Liver Injury in Mice and Serum Levels Indicate Outcomes of Patients with Acute Liver Failure. Gastroenterology. 2015;149:1896–909.

    Article  CAS  PubMed  Google Scholar 

  39. Lewis PS, Campana L, Aleksieva N, Cartwright JA, Mackinnon A, O’Duibhir E, Kendall T, Vermeren M, Thomson A, Gadd V, Dwyer B, Aird R, Man T-Y, Rossi AG, Forrester L, Park BK, Forbes SJ. Alternatively activated macrophages promote resolution of necrosis following acute liver injury. J Hepatol 2020;1–12.

  40. Yang W, Tao Y, Wu Y, Zhao X, Ye W, Zhao D, Fu L, Tian C, Yang J, He F, Tang L. Neutrophils promote the development of reparative macrophages mediated by ROS to orchestrate liver repair. Nat Commun 2019;1–14.

  41. Guo H, Chen S, Xie M, Zhou C, Zheng M. The complex roles of neutrophils in APAP-induced liver injury. Cell Prolif;54 . Epub ahead of print 2021. https://doi.org/10.1111/cpr.13040.

  42. Marques PE, Amaral SS, Pires DA, Nogueira LL, Soriani FM, Lima BHFF, Lopes GAOO, Russo RC, Ávila TV, Melgaço JG, Oliveira AG, Pinto MA, Lima CX, De Paula AM, Cara DC, Leite MF, Teixeira MM, Menezes GB, Avila TV, Melgaço JG, Oliveira AG, Pinto MA, Lima CX, De Paula AM, Cara DC, Leite MF, Teixeira MM, Menezes GB, Ávila TV, Melgaço JG, Oliveira AG, Pinto MA, Lima CX, De Paula AM, Cara DC, Leite MF, Teixeira MM, Menezes GB, Avila TV, Melgaço JG, Oliveira AG, Pinto MA, Lima CX, De Paula AM, Cara DC, Leite MF, Teixeira MM, Menezes GB, Ávila TV, Melgaço JG, Oliveira AG, Pinto MA, Lima CX, De Paula AM, Cara DC, Leite MF, Teixeira MM, Menezes GB, Avila TV, Melgaço JG, Oliveira AG, Pinto MA, Lima CX, De Paula AM, Cara DC, Leite MF, Teixeira MM, Menezes GB. Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure. Hepatology. 2012;56:1971–82.

    Article  CAS  PubMed  Google Scholar 

  43. Huebener P, Pradere JP, Hernandez C, Gwak GY, Caviglia JM, Mu X, Loike JD, Jenkins RE, Antoine DJ, Schwabe RF. The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis. J Clin Investig. 2015;125:539–50.

    Article  PubMed  Google Scholar 

  44. Marques PE, Oliveira AG, Pereira RV, David BA, Gomides LF, Saraiva AM, Pires DA, Novaes JT, Patricio DO, Cisalpino D, Menezes-Garcia Z, Leevy WM, Chapman SE, Mahecha G, Marques RE, Guabiraba R, Martins VP, Souza DG, Mansur DS, Teixeira MM, Leite MF, Menezes GB. Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice. Hepatology. 2015;61:348–60.

    Article  CAS  PubMed  Google Scholar 

  45. Speicher T, Siegenthaler B, Bogorad RL, Ruppert R, Petzold T, Padrissa-Altes S, Bachofner M, Anderson DG, Koteliansky V, Fässler R, Werner S. Knockdown and knockout of β1-integrin in hepatocytes impairs liver regeneration through inhibition of growth factor signalling. Nat Commun. 2014;5:3862.

    Article  CAS  PubMed  Google Scholar 

  46. Carcea I, Patil SB, Robison AJ, Mesias R, Huntsman MM, Froemke RC, Buxbaum JD, Huntley GW, Benson DL. Maturation of cortical circuits requires Semaphorin 7A. Proc Natl Acad Sci U S A. 2014;111:13978–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bianchi ME, Crippa MP, Manfredi AA, Mezzapelle R, Rovere Querini P, Venereau E. High-mobility group box 1 protein orchestrates responses to tissue damage via inflammation, innate and adaptive immunity, and tissue repair. Immunol Rev. 2017;280:74–82.

    Article  CAS  PubMed  Google Scholar 

  48. Williams CD, Lynn M, Sharpe MR, Mcgill MR, Farhood A, Jaeschke H, Bajt ML, Sharpe MR, Mcgill MR, Farhood A, Jaeschke H. Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans. Toxicol Appl Pharmacol. 2014;275:122–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Dubey M, Nagarkoti S, Awasthi D, Singh AK, Chandra T, Kumaravelu J, Barthwal MK, Dikshit M. Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism. Cell Death Dis. 2016;7: e2348.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Loison F, Zhu H, Karatepe K, Kasorn A, Liu P, Ye K, Zhou J, Cao S, Gong H, Jenne DE, Remold-O’Donnell E, Xu Y, Luo HR. Proteinase 3-dependent caspase-3 cleavage modulates neutrophil death and inflammation. Journal of Clinical Investigation 2014;124:4445–4458.

  51. Fadok VA, Savill JS, Haslett C, Bratton DL, Doherty DE, Campbell PA, Henson PM. Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. The Journal of Immunology 1992;149:4029 LP – 4035.

  52. Shi J, Fujieda H, Kokubo Y, Wake K. Apoptosis of neutrophils and their elimination by Kupffer cells in rat liver. Hepatology. 1996;24:1256–63.

    Article  CAS  PubMed  Google Scholar 

  53. Roth JM, Köhler D, Schneider M, Granja TF, Rosenberger P. Semaphorin 7A Aggravates Pulmonary Inflammation during Lung Injury. PLoS ONE. 2016;11: e0146930.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Zhao N, Zhang X, Ding J, Pan Q, Zheng M-H, Liu W-Y, Luo G, Qu J, Li M, Li L, Cheng Y, Peng Y, Xie Q, Wei Q, Li Q, Zou L, Ouyang X, Cai S-Y, Boyer JL, Chai J. SEMA7AR148W mutation promotes lipid accumulation and NAFLD progression via increased localization on the hepatocyte surface. JCI Insight;7 . Epub ahead of print August 2022. https://doi.org/10.1172/jci.insight.154113.

  55. Kang H-R, Lee CG, Homer RJ, Elias J a. Semaphorin 7A plays a critical role in TGF-beta1-induced pulmonary fibrosis. J Exp Med 2007;204:1083–93.

  56. Pan Q, Luo G, Qu J, Chen S, Zhang X, Zhao N, Ding J, Yang H, Li M, Li L, Cheng Y, Li X, Xie Q, Li Q, Zhou X, Zou H, Fan S, Zou L, Liu W, Deng G, Cai S-Y, Boyer JL, Chai J. A homozygous R148W mutation in Semaphorin 7A causes progressive familial intrahepatic cholestasis. EMBO Mol Med. 2021;13: e14563.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Huang J, Zhao C, Zhang S. Semaphorin 7A promotes endothelial permeability and inflammation via plexin C1 and integrin β1 in Kawasaki disease. BMC Pediatr;24 . Epub ahead of print December 1, 2024. https://doi.org/10.1186/s12887-024-04766-3.

  58. Dal-Secco D, Wang J, Zeng Z, Kolaczkowska E, Wong CHY, Petri B, Ransohoff RM, Charo IF, Jenne CN, Kubes P. A dynamic spectrum of monocytes arising from the in situ reprogramming of CCR2+ monocytes at a site of sterile injury. J Exp Med. 2015;212:447–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I, Waterhouse CCM, Beck PL, Muruve DA, Kubes P. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science. 1979;2010(330):362–6.

    Google Scholar 

  60. Ward C, Dransfield I, Chilvers ER, Haslett C, Rossi AG. Pharmacological manipulation of granulocyte apoptosis: Potential therapeutic targets. Trends Pharmacol Sci. 1999;20:503–9.

    Article  CAS  PubMed  Google Scholar 

  61. Wang J, Hossain M, Thanabalasuriar A, Gunzer M, Meininger C, Kubes P. Visualizing the function and fate of neutrophils in sterile injury and repair. Science. 2017;358:111–6.

    Article  CAS  PubMed  Google Scholar 

  62. Ishida Y, Kondo T, Kimura A, Tsuneyama K, Takayasu T, Mukaida N. Opposite roles of neutrophils and macrophages in the pathogenesis of acetaminophen-induced acute liver injury. Eur J Immunol. 2006;36:1028–38.

    Article  CAS  PubMed  Google Scholar 

  63. De Oliveira S, Rosowski EE, Huttenlocher A. Neutrophil migration in infection and wound repair: Going forward in reverse. Nat Rev Immunol. 2016;16:378–91.

    Article  PubMed  PubMed Central  Google Scholar 

  64. Tauzin S, Starnes TW, Becker FB, Lam P ying, Huttenlocher A. Redox and Src family kinase signaling control leukocyte wound attraction and neutrophil reverse migration. Journal of Cell Biology 2014;207:589–598.

  65. Bouchery T, Harris N. Neutrophil-macrophage cooperation and its impact on tissue repair. J Immunol Cell Biol. 2019;97:289–98.

Download references

Acknowledgements

We thank F. Rossi and C. Cryer with fluorescence-activated cell sorting (FACS)

Funding

Funding is acknowledged from the MRC Centre for regenerative Medicine PhD Studentship MR/L501426/1 and the Wellcome Trust [108906/Z/15/Z] JAC.

Author information

Author notes

  1. Eilidh J. Livingstone and Jennifer Ann Cartwright Joint first author.

Authors and Affiliations

  1. Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK

    Eilidh J. Livingstone, Lara Campana, Philip J. Starkey Lewis, Benjamin J. Dwyer, Rhona Aird, Tak Yung Man, Matthieu Vermeren & Stuart John Forbes

  2. Centre for Inflammation Research, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK

    Jennifer A. Cartwright & Adriano Giorgio Rossi

  3. The Royal (Dick) School of Veterinary Studiesand Theaq , Roslin Institute, University of Edinburgh, Edinburgh, UK

    Jennifer A. Cartwright

  4. MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, EH4 2XU, UK

    Luke Boulter

Authors
  1. Eilidh J. Livingstone
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Jennifer A. Cartwright
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Lara Campana
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Philip J. Starkey Lewis
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Benjamin J. Dwyer
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Rhona Aird
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Tak Yung Man
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Matthieu Vermeren
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Adriano Giorgio Rossi
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Luke Boulter
    View author publications

    You can also search for this author inPubMed Google Scholar

  11. Stuart John Forbes
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Authors' contributions: Conceptualisation and design (E.J.L., J.A.C., L.C., P.S.L., B.J.D., L.B., S.J.F.). Data generation (E.J.L., J.A.C., L.C., P.S.L., R.A., T.-Y.K., M.V.). Data analysis and interpretation (E.J.L., J.A.C., L.C., B.J.D., P.S.L., M.V., L.B., S.J.F., A.G.R.). Manuscript preparation (E.J.L., J.A.C., L.C., P.S.L., S.J.F.). Review and editing (E.J.L., J.A.C., B.J.D., M.V., L.C., P.S.L., S.J.F., A.G.R), Funding acquisition (S.J.F.).

Corresponding author

Correspondence to Jennifer A. Cartwright.

Ethics declarations

Competing interests

There was no competing interests at the time of experiments, however L.C., B.J.D, and S.J.F are shareholders of Resolution Therapeutics Ltd. a macrophage cell therapy developer. S.J.F is a scientific adviser and L.C. is an employee of Resolution Therapeutics.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Livingstone, E.J., Cartwright, J.A., Campana, L. et al. Semaphorin 7a is protective through immune modulation during acetaminophen-induced liver injury. J Inflamm 22, 13 (2025). https://doi.org/10.1186/s12950-025-00429-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12950-025-00429-x

Keywords

Journal of Inflammation

ISSN: 1476-9255

Contact us