Splenectomy during the late stage of disease shifts serum proteins toward regeneration
The pathogenesis and pathophysiology of AIH are still largely unknown. Likewise, the roles of different lymphocyte populations are still under discussion. Therefore, we induced emAIH with an adenovirus (Ad) encoding FTCD (Ad-FTCD) and removed the spleen at week twelve, which was during the late stage of the disease [9]. After 6 weeks, the mice were sacrificed (Fig. 1A). Serum samples were collected and analyzed, and we quantified 92 different proteins within them with Olink technology (Fig. 1B). In total, 9 of the proteins were differentially regulated with clustering within the two groups (Fig. 1C), with three exhibiting significant changes (p < 0.001) (Fig. 1D). Protein analyses demonstrated the downregulation of the inflammatory proteins chemokine (C–C motif) ligand 3 (CCL3), interleukin (IL)-1a and cadherin (Cdh)6 as well as Axin1 and quinoid dihydropteridine reductase (Qdpr). On the other hand, proteins related to regeneration such as Matrilin (Matn)-2, Glial cell-derived neurotrophic factor (Gdnf) and the myokine IL‐6 were upregulated. Notably, the chemokine Cxcl9 was upregulated and thought to be an inflammatory chemokine.
We also analyzed TH1-, TH2-, TH17- and fibrosis-related markers in liver tissue by quantitative PCR, but we could not find any differentially regulated genes in a set of 21 genes (data not shown).
In conclusion, splenectomy during the late stage of a chronic inflammatory disease shaped regeneration at the molecular level.
Biochemical remission was induced by regenerative processes
We previously showed that splenectomy does not affect hepatic histology [1, 2]. Nonetheless, AIH is a chronic and progressive disease. Thus, the pathology at the histological and biochemical levels after 18 weeks was completely unknown with respect to the observed regenerative molecular pattern.
Therefore, liver sections were produced and analyzed by microscopy. No obvious differences in histology could be observed at 6 weeks after splenectomy between splenectomized and non-splenectomized mice with emAIH (Fig. 2A). The same was true for the mHAI, which was higher than in comparison to the scores 6 weeks earlier from other studies [5, 9] but comparable between the two groups (Fig. 2B). Additionally, the average size of lymphatic infiltrates was just slightly smaller (Fig. 2C), while the liver weight was unchanged (Fig. 2D). However, biochemical analysis of the serum revealed a consequence of the regenerative molecular pattern observed. While the levels of aspartate transaminases (ASTs) were not prominently altered (Fig. 2E), the levels of alanine transaminases (ALTs) were significantly reduced by approximately 40% (Fig. 2F).
In conclusion, splenectomy induced biochemical remission of AIH.
Increase in intrahepatic Tregs after splenectomy in the inflamed liver
The regenerative molecular pattern and biochemical remission of AIH after splenectomy were revealed, but the cellular mechanism remained unknown.
Therefore, we analyzed the cellular composition of IHLs. In contrast to the changes observed in other models, the removal of the spleen, which resulted in the loss of a lymphocyte site, was compensated by an increase in lymphocytes in the liver (Fig. 3A). Here, the relative numbers of B cells were increased after splenectomy (Fig. 3B). Consequently, the relative number of T cells decreased (Fig. 3C). The IgG serum level did not subsequently change (Fig. 3D).
Given that the spleen was removed, the increase in B cells (Fig. 3E) and T cells in absolute numbers was not surprising as the liver is a harbor for lymphocytes (Fig. 3F). To investigate the outcome of the regenerative serum protein pattern and cause of biochemical remission, other cells were evaluated. Analyses revealed that both the relative and absolute numbers of intrahepatic Tregs were increased (Fig. 3G). This supraproportional increase even within the enlarged T-cell compartment of the immune regulatory and tissue-repairing cell subpopulation was meaningful. Consequently, the number of activated CD8+ T cells was decreased (Fig. 3H). In summary, the most prominent immunoregulatory cell population was supraproportionally increased in the liver.
In addition to all intrahepatic lymphocytes, we analyzed the local population within the portal inflammation. For this purpose, we prepared cryosections from liver tissue. To reduce the immense background, the organs were treated with sucrose before embedding. Frozen organs were sectioned to 4 µm and stained for CD4, CD8 and Foxp3 (Fig. 4A). CD4 (blue) T cells with Foxp3 (red) within the nuclei are easily visible, which are Tregs. In the animals with emAIH that were additionally splenectomized, there was an even more significant increase in Tregs than in the flow cytometric analysis of all IHLs (Fig. 4B).
An increase in intrahepatic Tregs correlates with a reduction in ALT
Given that the CD4/CD8 ration is decreased in other liver diseases if the disease is worsening, we analyzed this in our splenectomy emAIH model.
As in other models and diseases, the ratio of CD4+ to CD8+ T cells was slightly increased within the shrunken T-cell compartment (Figs. 4C and 5A). The same was true for the ratio of Tregs to Teffs (Fig. 5B). We also noted a slight increase in the Treg/Teff ratio, but this increase was not significant due to the normal error distribution. In contrast, the correlation analysis of Tregs and ALT showed a good correlation between an increase in Tregs and a decrease in ALT (Fig. 5C).
The observed increase in intrahepatic Tregs might have been the cause of the observed biochemical remission.