The immune system is heavily involved in tissue repair as a major regulatory entity complementary to nervous and endocrine elements. Clear anatomical connections between certain immune and non-immune organs ensure coherent reactions to infections, traumas, toxic damage, etc. It is primarily about the relationship between the spleen and the liver within the so-called hepatosplenic axis (Tarantino et al., 2013; Li et al., 2017). The basis of this interaction is the portal circulation linking the spleen and liver, as well as the presence of common barrier and immune functions (Tarantino et al., 2013; Li et al., 2017). Despite the direct connection of the liver and spleen, the specific mechanisms of mutual influence of these organs, including reparative processes, remain poorly understood (Yamada et al., 2016; Liu et al., 2018; Elchaninov et al., 2022).

The hepatosplenic axis was extensively studied in connection with cirrhosis in clinical and experimental models. Accordingly, the emphasis was made on spleen-derived factors affecting Ito cells of the liver implicated in the excessive fibrous matrix production (Romanelli and Stasi, 2016; Higashi et al., 2017). In hepatitis and cirrhosis, i.e., under conditions of diffuse damage to liver parenchyma, macrophages of the splenic red pulp produce increased amounts of TGFβ1 spreading to the liver via the portal vein and activating Ito cells—this effect has been confirmed in various settings, both experimental (Akahoshi et al., 2002) and clinical (Asanoma et al., 2014). By contrast, under conditions of sheer massive loss in liver volume, splenic influences appear beneficial. Increased expression of certain interleukins and hepatocyte growth factor (HGF) by the spleen after experimental liver resections (Kono et al., 1992; Yin et al., 2011) indicates a humoral support deserving a dedicated investigation.

The spleen is a plausible source of i) cytokines and growth factors that regulate inflammation and repair; and ii) migratory leukocytes. Deposition of monocytes in the spleen and their release towards the damage foci have been demonstrated in models of myocardial infarction and ischemic stroke (Swirski et al., 2009; Blomster et al., 2013; Kim et al., 2014; Rizzo et al., 2020). The immigration of monocytes-macrophages and other leukocytes in the liver after hepatotoxic injury or surgical resection is well documented (Goh et al., 2013; Zigmond et al., 2014; Elchaninov et al., 2021), but their sources and contribution of the spleen to this process are unknown.

The spleen is the largest site of T- and B-lymphocyte differentiation in adult mammals. Almost any organ in the body has its own lymphocyte population with varying degree of splenic connections (Marquez-Medina et al., 2012; Li and Hua, 2017). A decrease in CD4⁺ splenic pools has been observed in mouse model of cirrhosis (Tanabe et al., 2015) and migration to the liver as a possible cause cannot be excluded.

Reactions of splenic lymphocytes to hepatectomy have not been addressed experimentally. Moreover, in liver resection models, the hepatosplenic axis status and retrieval are often neglected. Here we address this issue using a transcriptomic approach focused on inflammation- and repair-related secreted molecules. We also assess the dynamics of monocyte-macrophage and lymphocyte populations of the spleen after 70% hepatectomy in mouse model.

The modulatory effects of the immune system on liver regeneration are a major focus in biomedical research. The spleen, which is the largest site for antigen-dependent maturation of immune cells and coordination of immune responses in the body, is close to the liver both physically and functionally, and connected to it via the portal vein. Negative effects of the spleen on functional status of the liver were demonstrated in experimental models of hepatitis and liver cirrhosis, where splenectomy in the model animals alleviated the degree of fibrotic changes to the liver (Tarantino et al., 2013; Li et al., 2017). The effect was explained as follows. Hepatitis, the leading cause of liver fibrosis, is accompanied by pervasive cell death of hepatocytes. The products of decay enter systemic circulation and eventually reach the spleen where they stimulate pro-inflammatory ad pro-fibrotic cytokine and growth factor secretion (Akahoshi et al., 2002; Asanoma et al., 2014; Tang et al., 2016). Straightforward delivery of these factors to the liver via the portal vein closes the loop; splenectomy cancels the feedback altogether (Elchaninov et al., 2022).

Splenic influences in liver regeneration after 70% resection are less evident. Although removal of the spleen was reported to negatively affect the rates of liver repair after 70% resection (Babaeva and Zotikov, 1987), mechanisms of this effect have not been elucidated. Several assumptions made in this regard can be reduced to two main possibilities: 1) the spleen is a site for the synthesis of biologically active substances that enter the regenerating liver with the bloodstream and modulate repair processes and 2) the spleen can act as a source of immune cells (monocytes, lymphocytes, etc.) migrating to the liver and contributing to its regeneration on the spot.

Studies assuming model 1) were designed based on current knowledge of secretomic influences of immune cells on tissue repair, ultimately focusing on a selection of few biologically active secreted molecules as plausible candidates. One study showed induction of hepatocyte growth factor gene expression in the spleen (Kono et al., 1992; Yin et al., 2011). Another study showed that IL17a or IL6 secreted by the spleen activate hepatocyte proliferation in the liver (Furuya et al., 2013; Behnke et al., 2018). We used a more general approach to avoid assumptions and studied the spleen transcriptome in order to comprehensively cover a variety of factors that can be produced in the spleen and regulate liver regeneration. We observed hepatectomy-induced increases in expression of serine protease inhibitor-encoding genes Serpina3n, Stfa2 and Stfa2l1which may favorably influence the repair processes in the liver.

The role of protease inhibitors in tissue repair is a recent focus in experimental studies. In paracetamol-induced hepatotoxic injury, increased levels of Serpina3n alleviated the degree of necrotic and inflammatory changes in the liver (Tran et al., 2023) and a similar trend was observed for experimental ischemic stroke (Zhang et al., 2022). The elevated splenic levels of Serpina3n may spread via the portal vein to exert a healing, anti-inflammatory effect in the regenerating liver.

The other two differentially expressed protease inhibitors, Stfa2 and Stfa2l1, can further mitigate the inflammatory response by inhibiting cysteine endo- and exopeptidases, notably cathepsins L and S involved in antigen presentation (Mihelič et al., 2006). The inhibition of cathepsin S in experimental Sjögren syndrome reduced both the autoantibody production and the lymphocytic infiltration of salivary and lacrimal glands (Saegusa et al., 2002; Roper et al., 2003).

In this section, we would like to point out some limitations of our research. When studying the spleen transcriptome during liver resection, only the spleen of intact animals was used as a control. In our opinion, this is justified by identical data obtained for intact and sham-operated animals by other analytical tools used in this study, including RT PCR, Western blot analysis and flow cytometry. As the increased expression levels of serine protease inhibitor genes Serpina3n, Stfa2 and Stfa2l1 in the spleen of hepatectomized animals is sustained for up to 7 days after the intervention, association of these changes with sheer opening-suturing of the abdominal cavity is unlikely.

Another possible mechanism of splenic influence on repair processes in the liver is based on immune cell recruitment from the spleen to the site of injury, as described for other models (Swirski et al., 2009; Blomster et al., 2013; Kim et al., 2014; Rizzo et al., 2020).

Apart from the transcriptomic influences, hepatectomy significantly altered the cytological architecture and leukocyte subpopulation balance of the spleen. The Ki-67 positivity-measured proliferation activity of splenic parenchyma was reduced on days 1 and 3 and returned to normal by day 7 post-intervention. Expression of cyclin genes followed these dynamics, albeit not at the protein level; a similar discrepancy for cyclin mRNA and protein expression levels has been reported previously (Albrecht et al., 1995). The hepatectomy-induced suppression of splenic hemopoiesis may unleash some compensatory mechanisms, as indicated by increased expression of megakaryocyte stimulating factor Prg4 known to stimulate myelo-and lymphopoiesis (Novince et al., 2011).

The efflux of monocytes from the spleen after liver resection is indirectly indicated by decrease in splenic monocyte counts. The hepatectomy-induced changes in the monocyte-macrophage subpopulation balance of the spleen include a reduction in CD115+ and Ly6C + relative cell counts. These data are consistent with previously reported dynamics attributed to recruitment of splenic monocytes to damaged organs (Swirski et al., 2009; Blomster et al., 2013; Kim et al., 2014; Rizzo et al., 2020). Accordingly, the spleen is a likely source of monocytes that colonize the remnant liver during its compensatory growth and functional recovery. In this regard, it is interesting to note the induced splenic expression of Sparc (osteonectin) involved in regulation of cell adhesion, extracellular matrix remodeling and differentiation/homing of immune cells at particular locations in the body (Riley and Bradshaw, 2020). The plausible migration of splenic monocytes to regenerating liver should be regarded as a beneficial influence, as the post-hepatectomy inhibition of the monocyte-macrophage influx to the liver impedes the regeneration (Constandinou et al., 2008; You et al., 2013).

Apart from the signs of monocyte-macrophage efflux from the spleen, we observed changes in their functional status indicated by the reduced CD86⁻, CD163-and CD206-positivity among cells expressing the M-CSF receptor CD115. The trend apparently reflects the reaction of macrophages to pro-inflammatory stimuli. A competing anti-inflammatory stimulation of splenic milieus may be indicated by increased expression of Cd5l known to encode a protein which stimulates the anti-inflammatory macrophage polarization (Sanjurjo et al., 2018).

The observed effects of liver resection on splenic lymphocyte populations were limited to a significant decrease in the proportion of cytotoxic T cells on day 7. Cytotoxic T cells are important for liver regeneration after hepatectomy: they produce lymphotoxins which stimulate IL-6 synthesis and hepatocyte proliferation (Tumanov et al., 2009). The observed decrease in the cytotoxic T cell counts inside the spleen may indicate their migration to the liver or reflect the activation of Treg cells shown to increase in number inside the spleen after liver resection. Treg cells can exert anti-inflammatory effects by several mechanisms (Li et al., 2018). They produce anti-inflammatory interleukins (IL4, IL10, IL13, IL35) and stimulate production of the same molecules by neutrophils and macrophages (Hawrylowicz and O’Garra, 2005; Taams et al., 2005; Collison et al., 2007). The anti-inflammatory cytokines inhibit the activity of CD8⁺ and CD4⁺ lymphocytes thereby supporting repair processes (Weirather et al., 2014). The anti-inflammatory effect of Treg cells apparently underlies their favorable effect on liver regeneration after resection (Wang et al., 2023).

Overall, the accumulated evidence confirms both the positive and the negative roles of the spleen in liver repair. A dominance of either negative or positive influences under particular circumstances can be explained by the nature of liver damage—whether it involves a toxicity component. In situations of pervasive hepatocyte death, exemplified by hepatitis and liver fibrosis, the spleen becomes an extra source of damage-aggravating cytokines; accordingly, its removal stimulates repair processes in the liver. In situations of massive loss and zero extraneous toxicity within the remnant, the rates of hepatocyte cell death are minimal (Elchaninov et al., 2016); accordingly, the spleen stays non-intoxicated by decay products and imposes no extra cytokine burden on the regenerating liver. Moreover, splenectomy under these conditions will negatively affect the compensatory growth of the healthy remnant by cancelling the influx of monocytes and biologically active molecules including the extracellular protease inhibitors and HGF. The negative impact of splenectomy on healthy liver tissue, both intact and regenerating after 70% resection of its volume, was demonstrated experimentally (Babaeva and Zotikov, 1987; Babaeva, 1989; Makarova et al., 2012).

The liver exchange with the bloodstream is enormous, and it is difficult to identify specific substances addition or subtraction of which by means of hepatectomy will influence gene expression profiles of splenic cells. A likely candidate is bacterial endotoxin (lipopolysachcaride, LPS) produced in the gut. The liver is the major site of LPS utilization; accordingly, endotoxin levels in the blood of hepatectomized animals are increased (Michalopoulos and DeFrances, 1997) and may promote pro-inflammatory activation of macrophages and other TLR4-expressing cells in the spleen, eventually counteracted by anti-inflammatory cascades associated with increased production of serine protease inhibitors.

Another possible mechanism of hepatic influence on other organs involves bile acids. The circulation of bile acids between the liver and the small intestine is affected by hepatectomy, as the remnant liver cannot cope with bile acid recycling and their blood levels may increase. Macrophages express bile acid receptors including the farnesoid X receptor (FXR) (Renga et al., 2009) activation of which promotes the anti-inflammatory M2 polarization of macrophages while blocking the TH1/TH17 lymphocyte inflammatory polarization (Shi et al., 2022; Jaroonwitchawan et al., 2023).

Liver resection causes significant changes in the spleen, affecting its transcriptomic profiles and leukocyte homeostasis. The early stages of liver repair are accompanied by induction of certain anti-inflammatory markers, notably the protease inhibitor genes Stfa2l1, Stfa2 and Serpina3n and partial repression of cyclin genes in the spleen. Leukocyte populations of the spleen react to hepatectomy by decreased proliferation rates and shifted subpopulation balance. Significant reduction in relative counts of CD115+ and F4/80+ cells in the spleen may indicate an increase in the monocyte-macrophage efflux.