Background
Alveolar echinococcosis, caused by the larval stage of
E. multilocularis, continues to be a real world-wide public health issue. It is prevalent mainly in Western China, Middle East and as well as Central Europe [
1]. China harbors nearly 90% of economic burden of AE around the world and thus sustained efforts have been made on prevention, control and management of this disease [
2].
E. multilocularis infection predominantly target itself in the host’s liver and reside itself with incoming infiltrative growth and consequently lead to the critical involvement of vasculature [
3]. Although, tremendous improvement has been made in the field of hepatic surgery including radical resection, liver transplantation and ex vivo liver resection and autotransplantation with promising clinical outcome [
3]. Of note, nearly 90% mortality rate was reported within 10~15 years after initial diagnosis if untreated or insufficiently treated [
4‐
6]. The attempt to unveil the underlined mechanism of such an infiltrative disease, regarded as “parasitic cancer”, is vital important.
To date, AE is considered as immune related parasitic infection with very intriguing and diversified immune cross-talk between host and parasite depending on the stage of the disease [
7]. It is reported the
E. multilocularis infection modulate Th cell subsets to maintain a high Th1 in early stage while Th2 dominant immune profile in both peripheral and regional milieu [
8]. Our recent studies have demonstrated the potential importance of the remaining Th subsets such as Th17 [
9], Treg [
10] and Th9 [
11] in
E. multilocularis infection. Besides, our data indicated T-cell tolerance and exhaustion during clearance of
E. multilocularis [
12]. CD4
+T and CD8
+T cells present the major source of T cells in early and late stage of
E. multilocularis infection, respectively [
13]. Other studies indicate that the early infective stage of
E. multilocularis is a strong inducer of tolerance in dendritic cells (DCs) [
14], and the proliferative potential of the parasite metacestode tissue is dependent on the peri-parasitic immune-mediated processes of the host [
7].
The both adaptive and innated immunity is pivotal importance to the parasite infection [
15]. As an active member of innate immunity, NK cells compose approximately 20–30% of liver-resident lymphocytes with the far lower percentage in peripheral blood [
16]. The contact-dependent signals provided by DCs, monocyte/macrophages, CD4
+T cells as well as secreted cytokines activate NK cells during various infections [
17]. It causes death of virus-infected cells [
18,
19], tumor cells [
20], and limit the progression of intracellular and extracellular parasites [
21‐
25]. It is also reported that, the liver fibrosis and carcinogenesis formation process is hugely limited in the presence of NK cells in hepatitis [
26]. Preliminary data showed the inhibited activation and proliferation of NK cells in
E. multilocularis vesicular fluid co-culture and indicated its possible role in tolerative pathogen-host interaction [
27].
Although, a plenty of work has been done in the field of immune interaction in E. multilocularis infection, however, very few is known regarding the possible role of innate immunity, especially NK cells in E. multilocularis infection. Herein, we are aiming to explore the expression of NK cells and its relative molecules, its potential impact on the disease progression, if any, in murine model of portal vein inoculation of E. multilocularis.
Discussion
Our results demonstrated the profound decrease of liver NK cells population but sharp increase of NKG2A expression in E. multilocularis infected mice. Such alteration might result in low cytotoxic activity through decreased IFN-γ secretion, as well as the disease progression with its potentiality in the immune tolerance milieu in infected liver. To the best of our knowledge, this is the first report on hepatic NK cells and their related molecules in murine model of E. multilocularis infection.
NK cells play significant role against intracellular viruses and bacteria, tumors, protozoa, parasites infection [
33]. Immune tolerance due to compromised NK cells’ cytotoxic activity has been frequently observed both in experimental and human study with tumor and chronic infection [
31,
34]. Since NK cell population play a key role in the process of tumor and chronic infection development, many recent studies support the importance of NK cells during parasitic infection.
Toxoplasma gondii infection impairs NK cell recognition of target cells and cytokine release, then independently enhances its survival [
35]. NK cells can limit malaria infection by producing IFN-γ and killing infected cells before being superseded by the adaptive immune response [
22]. Moreover, reduction of NK cell numbers and impaired NK cells response observed in patients with acute cutaneous
leishmaniasis [
36]. NK cells population and its function changed in
Angiostrongylus cantonensis infected mice, suggesting their involvement in pathogenesis of the infection [
24]. Activated NK cells is reported to negatively regulate egg-induced liver fibrosis via producing IFN-γ, and killing activated stellate cells in
Schistosoma japonicum infection [
25]. Like other metacestode infection, AE is featured by chronic granuloses formation and present various immunopathological processes. Available data showed inhibited activation of NK cells after co-culture with
E. multilocularis vesicular [
27]. Human study demonstrated decreased NK cells frequency in peripheral blood in AE patients and concluded the possible involvement of NK cells during AE infection [
37]. Herein, our results displayed decrease in NK cell percentage and IFN-γproduction in hepatic tissue of
E. multilocularis infected mice. It is known that CD49a
+DX5
− and CD49a
−DX5
+ are the two major hepatic NK subsets. CD49a
+DX5
− NK cells is liver resident NK cells and possesses memory potential [
38]. In current study, lower percentage of CD49a
+DX5
− NK cells were observed in infected liver. And most intriguingly, significant increase of parasite load but decrease of peri-parasitic fibrosis was also observed after the depletion of NK cells.
Commonly, the ingestion of
E. multilocularis metacestode initiate cross-talk with the host’s immune system and induces recruitment and infiltration of various immune cell types. The fibrotic layer, secondary to the immune response, successfully separates the parasite and limits its growth [
39,
40]. In the context of NK cell depletion, the process of cross-talk is not “smooth” and lead the continuous growth of the parasite and failure of limitation by fibrotic tissues. Besides, the over expression of TGF-β [
40], and IL-10 [
41] after
E. multilocularis infection might contribute to NK cell inhibition, resulting into NK cell activating receptors’ imbalance, consequently reducing IFN-γ secretion and impairing the NK cell immune surveillance.
NK cell function is tightly regulated by activating and inhibitory molecules [
33]. Either the downregulation of activating receptors such as NKG2D, NKp30, NKp46, or upregulation of inhibitory receptors like Tim-3, NKG2A, PD-1 lead to NK cell dysfunction [
26]. Upregulation of NKG2A in chronic HBV and HCV infection is purportedly associated with NK cell exhaustion [
42,
43]. NKG2A
high status contributes to NK cell exhaustion and predicts poor prognosis in liver cancer patients [
12]. In addition, newest research demonstrated that anti-NKG2A mouse antibody is an immune checkpoint inhibitor that promotes anti-tumor immunity by unleashing both T and NK cells [
44] . In this study, our result showed that NKG2A is upregulated at all designated time points in experimental
E. multilocularis infection. Such change is related with decreased numeric and functional NK cells at early and middle stages, and it might be responsible for the increased parasite load and extensive infection. However, at the late stage, NK cells’ functional decrease has been reversed, which may be related to server T cell exhaustion led to upregulation of other activated molecules on NK cells in very late stage of infection. Thus, further studies need to work on its explicit mechanism.
NK cells exert its function on pathogens, tumor cells, stressed hepatocytes, and HSCs via the production of cytokines (IFN-γ, TNF-α, IL-10, IL-12, IL-22, etc.) and cytotoxic molecules (granzyme B, Perforin, etc.) both in direct or indirect fashion [
26]. The over-expressed NKG2A in hepatitis and HCC patients showed markedly reduction of IFN-γ secretion. Moreover, the NKG2A blockade resulted with significant boosts of IFN-γ production and interdependent of NK and CD8
+ T cell functions to prevent in hepatitis and HCC patients [
44,
45]. Besides, NKG2A downregulation increases the anti-tumor activity of NK cells and infusions in a subset patient with HCC [
46]. Human liver-derived CXCR6
+ NK cells are predominantly educated through NKG2A and show reduced cytokine production [
47]. NKG2A downregulation enhanced NK cytotoxicity and accelerates effective treatment responses in patients with chronic myeloid leukemia [
48]. Notably, IFN-γ produced by NK cells plays predominant role during anti-viral [
19], anti-fibrosis [
49], and anti-tumor [
50], anti-parasitic process [
24]. And, its secretion is inhibited by over expression of TGF-β and IL-10 and therefore related with immune tolerance [
51]. High levels of plasma IL-10 is related with over expression of NKG2A and lead to decreased secretion of IFN-gamma and cytotoxicity [
12]. In line with abovementioned results, treatment with anti- NKG2A resulted with increased production of NK cells secreted IFN-γ in vivo study [
44]. Da-Zhong Shi reported the possible curative effect of IFN-γ both in human and experimental study and indicated the importance of IFN-γ during
E. multilocularis infection [
52]. Further, our results showed the significant reduction of regional levels of NK cell secreted IFN-γ in
E. multilocularis infection and negative correlation with NKG2A expression.
There are still some inherent limitations of current study should be stated. First, our results were based upon experimental model that might not be the best option but the result currently available. The failure to access commercialized antibody of NKG2A kept us from deeper understanding the possible role of it during E. multilocularis infection, however, this would be done in our future work in support to current findings.
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