In this study, PHx was performed in a mouse model of subcutaneous E. multilocularis infection to explore the alterations in circulatory and local related cytokines and their possible influence on the post-hepatic resection regenerative process. This study is the first to report that E. multilocularis infection could delay the remnant liver regeneration process and that the immune-tolerance milieu induced by E. multilocularis infection might be a determining factor during this process.
Based on both experimental and clinical studies, it is well-known that E. multilocularis infection could actively modulate the host immune system14,15,16. Among the modulators, pro- and anti- inflammatory cytokines play a vital role in parasite clearance and growth17,18. Type 1 helper T (Th1) cell immunity, which functions via its related effector cytokines TNF-α and IFN-γ, plays a protective role during the early stage, while regulatory cytokines, such as IL-10 and TGF-β, which are mainly secreted by Th2 subsets, are associated with infiltration of metacestodes and disease susceptibility. Our previous study showed that IL-6 and TNF-α are actively involved in disease progression and granuloma formation19,20. Increasing evidence suggests that Th17 and Treg cells are closely related to immune tolerance phenomenon during E. multilocularis infection21. More recently, we reported that IL-17 and IL-23 are associated with disease progression in both experimental and clinical studies14,22.
The liver has a high regenerative capacity, and the immune patterns are thought to perform pivotal functions during the initiation and modulation process after injury or resection23. Evidence from studies involving humans or mammals suggests that immunological factors, including a series of extrinsic and intrinsic cytokines (i.e., TNF-α, IL-6, etc.), multiple cell-signalling pathways (i.e., LPS/TLRs/MyD88) and their downstream cascades (i.e., STAT3) interact and play a critical role in the liver regeneration process24,25. Both peripheral and regional remnant hepatic TNF-α levels are elevated, resulting in the further activation of NF-κB and IL-6 induction after PHx24,26,27. The IL-6/IL-6R complex could activate STAT3, further stimulate liver cells, and enhance the hepatocyte protection ability and survival28,29,30. Both IL-6 and TNF-α knockout mice exhibit severe deficits in liver regeneration after PHx29,30,31,32. In the present study, the rapid decrease in the TNF-α and IL-6 relative expression levels at 96 h may be due to the beginning of the termination phase of the liver regeneration process.
Since an interaction exists between cytokine alterations after E. multilocularis infection and liver regeneration, we developed a model to investigate whether E. multilocularis infection has any impact on liver regeneration. Interestingly, the results demonstrated delayed regeneration after PHx. To the best of our knowledge, the first report investigating parasitic infection and liver regeneration was based on Teixeira et al.’s33 pioneering research. Costa et al.34 postulated that the pro-inflammatory cytokines TNF-α and IFN-γ, which are produced by Th1 cells during the acute phase of Schistosoma mansoni infection, may contribute to the enhanced liver regeneration after PHx. In our clinical practice, based on more than 400 AE hepatic resections, hypertrophy of the unaffected liver parenchyma occurs frequently. This finding might be partially attributed to the rearrangement of portal blood flow caused by parasitic portal obliteration in the diseased lobe. Previous studies conducted by our team have shown extensive liver fibrosis in both human and murine echinococcosis, but the exact mechanism has not been clearly uncovered to date35,36,37. Lin et al.35 observed heavy liver fibrosis in tissues near AE lesions compared with tissues in distant areas, suggesting that E. multilocularis infection might induce or accelerate liver fibrosis. These authors concluded that TGF-β plays a significant role leading to immune tolerance in this immune-pathologic injury. Simultaneously, Zhang et al.38 observed distinct anti-apoptotic and hepatocyte proliferative characteristics during the early and middle stages of E. multilocularis infection in vivo.
The TLR and MyD88-mediated pathways are responsible for the recognition of and responsiveness to conserved pathogen-associated molecular patterns (PAMPs), such as E. multilocularis infections, and most importantly, these pathways could also influence subsequent acquired immunity by preferentially inducing Th1 cell-derived responses39. Our previous studies observed both regional and systematic elevation in TLRs in patients with hepatic AE, suggesting that TLRs actively interact and may play a significant role during infection14. Intriguingly, the current study demonstrated a relatively delayed (48 h after surgery) increase in the TLR4 mRNA expression levels in the regenerating liver tissues. In addition, the high MyD88 mRNA expression levels were altered at 96 h but remained significantly higher at the other time points, and compared with the control group, the protein levels were elevated after 48 h in the E. multilocularis infected group (Figs. 8–9). Subsequently, the TLR and MyD88 pathways in the non-parenchymal liver cells mediated the induction of the Th1 cell type cytokine TNF-α, which is believed to play a pivotal promotive role during the priming and progressing stages of liver regeneration; however, in our results, their patterns were restricted during the priming phase (Fig. 7), which might be caused by the eradication of the Th2/Treg cell type predominant immune response during the E. multilocularis chronic phase.
TSP-1 is considered a negative regulator of liver regeneration40. Clinical observational studies have indicated the predictive potential of TSP-1 in liver dysfunction after hepatectomy41,42. Elevated circulatory TPS-1 levels are associated with delayed recovery and even poor outcomes in patients who underwent partial resection43. TSP-1 can convert latent TGF-β1 into biologically active TGF-β1, which is also called the TSP-1/TGF-β axis, contributing to the blockade of hepatocyte proliferation40,42. TGF-β mRNA has been shown to begin to release immediately after PHx, reach a plateau at approximately 24 h, and remain steady for at least 4 days after surgery27,44,45. Our data from the E. multilocularis infected mice revealed a blunt increase with a peak 96 h after surgery. Studies have shown that TGF-β can maintain hepatocytes in the G0 phase through an anti-growth factor effect. The elimination of TGF-β and its receptors accelerates DNA synthesis, further enhances regeneration and prolongs hepatocyte proliferation46,47. In the present study, consistent with above paradigm, the TGF-β mRNA levels in the liver tissues began to increase 24 h after surgery and continued to elevate until 48 h. Approximately 96 h after PHx, TGF-β was increased, especially in the non-infected mice and infected mice at 168 h, which may be indicative of the termination phase of liver regeneration with some delay in the latter group. However, the plasma TGF-β concentrations reached the peak levels in the non-infected mice; the highest delayed points were observed 96 h after surgery. We assume that the persistent stimulus and chronic inflammation responses caused by E. multilocularis infection may be responsible for this delayed “crest”.
Additionally, as an important chromatin protein, HMGB1 performs multiple functions. In the cell nucleus, HMGB1 facilitates the transcription of genes that could interact with transcription factors, such as NF-κB48. In injured tissues, HMGB1 can also trigger inflammation, which is often accompanied by tissue repair49,50. In the liver, HMGB1 is secreted by various types of immune cells and acts as a mediator cytokine of inflammation51. Studies have revealed that the above action of HMGB1 is often closely related to its binding or interactions with LPS-TLR4, further leading to TLR4 activation, and its binding MyD88 results in signal transduction and downstream cascades52. In the present study, the plasma HMGB1 levels were increased early after surgery in the control mice but exhibited a delayed peak at 96 h in the E. multilocularis infected mice.
Previous results from our basic and clinical studies have shown that during the late stage of E. multilocularis infection, the Treg/Th17 ratio was significantly elevated, resulting in a Treg-dominant suppressive immune response. After liver parenchymal resection, LPS could strongly stimulate Kupffer cells to commence the cascade responsible for regeneration. However, in E. multilocularis infection, LPS fails to “wake-up” the immune response because of the immune tolerance formed by the continuous metacestode stimulation. Consistent with our expectations, after E. multilocularis infection, the host Th2-type immune responses may attenuate Th1 cell related immune patterns and cytokine secretion and then weaken the regeneration ability. In addition, as shown in our and others’ previous reports, the late stage of AE is characterized by a Th17/Treg imbalance that plays crucial roles in the formation of the host’s immune tolerance. Thus, the decreased reactivity of the immune system to pathological stimulators (e.g., LPS) may be another potential reason for the delayed regeneration in the E. multilocularis infected subjects.
Although this study aimed to reveal the potential impact of E. multilocularis infection on post-operative liver regeneration, several inherent limitations should be considered. First, this study was based on a murine model; thus, the findings of the current study may not fully translate to human subjects. In addition, only MyD88 related liver regeneration was considered, and other important pathways might have been omitted in the current work and require further in-depth investigation.