Hepatic fibrosis is a reversible wound-healing response to liver injury, which is a major feature of any form of the chronic liver disease progressing to cirrhosis and liver failure (1). The main manifestations of hepatic fibrosis are chronic liver injury and accumulation of extracellular matrix (ECM) proteins (2). The continuous accumulation of ECM proteins could disrupt the normal function of the liver, and persistent hepatic fibrosis will progress to liver cirrhosis and even hepatocellular carcinoma (3). However, no effective drugs have been approved to target hepatic fibrosis. The therapeutic approaches to alleviate hepatic fibrosis mainly include: controlling or removing the underlying causes of hepatic fibrosis, preventing the activation and proliferation of HSCs, inhibiting the overexpression of pro-fibrotic cytokine TGF-β, and promoting fibrous tissue degradation. An emerging stem cell therapy is that the stem cells can transform into functionally active hepatocyte-like cells and participate in liver function repair and reconstruction (4).

It has been confirmed that hepatic fibrosis is driven by the activation of HSCs (5), which is the main source of ECM (6). HSCs are located in the perisinusoidal space and maintain a non-proliferative and quiescent phenotype in the normal liver tissue. The HSCs will transdifferentiate into proliferative fibrotic myofibroblasts, acquiring pro-inflammatory and pro-fibrotic properties under the chronic injury caused by many various pathogenic factors such as pathogenic microorganisms, alcohol, chemical drugs, and lipid deposition (7). The myofibroblasts transformed from HSCs account for about 82%-96% of myofibroblasts in various chronic liver diseases (8). Therefore, activated HSCs are often regarded as the main therapeutic target.

HIME is a dynamic network system composed of a variety of immune cells, immune cytokines, and related ECM (9), and it closely affects the onset and progression of hepatic fibrosis. Various immune cells are enriched in liver tissue and involved in the physiological and pathological process of hepatic metabolism and disease (10). Immune cells in the liver are diverse at a stable state and evolve further during the development of chronic liver disease, directly affecting the severity of the disease (11, 12). The recruitment and activation of immune cells in the diseased liver tissue are often initiated by the massive release and activation of cytokines and chemokines. Hepatic kuffer cells (KCs) are activated by various damage-associated molecular patterns (DAMPs, such as free DNA, ATP, high mobility group box 1) and pathogen-associated molecular patterns (such as LPS or viral DNA) that interact with Toll-like receptors (TLRs) or recombinant purinergic receptor P2X, ligand-gated ion channel 7 when hepatocytes are damaged (13, 14). This leads to the activation of the inflammasome, and the release of interleukin-1β (IL-1β), IL-18, and various other pro-inflammatory cytokines as well as chemokines. It will promote the recruitment of circulating leukocytes (monocytes and neutrophils), and activate adaptive immune T cells function. Once the infection or injury is controlled, the KCs and macrophages transform into anti-inflammatory and tissue repair phenotypes to control excessive tissue-damaging inflammatory responses (15). However, the persistent injurious stimulus in the liver will result in inflammatory cytokines continuously releasing that promote HSCs-mediated fibrous tissue hyperproliferation and excessive repair, which causes hepatic fibrosis development and progression to cirrhosis (16).

Hepatic fibrosis is an intermediate link and reversible stage in the process of chronic liver disease developing into liver cirrhosis. Intervention in this link will determine the prognostic direction of the various liver diseases. However, there is no efficient treatment strategy for hepatic fibrosis, which is mainly because profound awareness of the changes in the microenvironment of hepatic fibrosis still lacks, and no targeting and efficient intervention on the hepatic fibrosis microenvironment. Exploring the changes in local immune homeostasis and microenvironment caused by immune response under this pathological state could greatly enrich our understanding of liver disease reversal, chronicity, and even deterioration to liver sclerosis. Therefore, it is of great significance to elucidate the HIME for the study of hepatic fibrosis and the development of therapeutic strategies. In this review, we first summarized the critical components of HIME, the alteration and function of different sub-type immune cells, and their related mechanisms according to their effect on the development of progression of hepatic fibrosis. The specific changes and the related mechanisms of HIME in different chronic liver diseases were also reviewed and analyzed. Finally, we also summarized the therapeutic approaches and targets for HIME to reverse the progression of hepatic fibrosis, and we aimed to provide new research ideas and the possibility of exploring the therapy for hepatic fibrosis.

The immune cells are central members of the immune microenvironment in the progression of hepatic fibrosis, and they can mediate the immune response by synthesizing and secreting different chemical inflammatory mediators. The immune cells affect the function and number of HSCs mainly through direct or indirect effects, thus influencing the progression of hepatic fibrosis. Simultaneously, they constitute a dynamically evolving microenvironment and interact as well as restrict each other, forming a complex network system, which jointly affects the process of hepatic fibrosis.

Hepatic macrophages include liver-resident KCs and monocyte-derived macrophages (MDMs) (17). The MDMs, with high heterogeneity and plasticity, can differentiate into functionally distinct macrophage subsets. In mice, the circulating macrophages can be divided into the Ly6C^hi CCR2^hi CX3CR1^lo monocytes which are considered to be potent pro-inflammatory cells, and Ly6C^lo CCR2^lo CX3CR1^hi monocytes, which with an anti-inflammatory patrolling function (18). Ly6C^hi monocytes in response to liver chronic injury are recruited to the liver and differentiate into MDM. These Ly6C^hi MDMs have been shown to promote liver fibrosis, and they could promote HSCs activation as well as proliferation and ECM production by expressing a large number of pro-fibrotic mediators. The MDMs are also capable of differentiating into Ly6C^lo restorative macrophage phenotypes, which promote ECM degradation and fibrosis resolution (19). KCs, the settled macrophages in the liver, have been identified as key regulators of hepatic inflammation. The DAMPs released from damaged hepatocytes can activate KCs and promote circulating macrophage infiltration in the liver (12). The activated macrophages have been reported to possess a bidirectional effect on the onset, progression, and reversal of hepatic fibrosis (17). Activated KCs could activate the HSCs by secreting large amounts of pro-inflammatory factors such as TGF-β1, IL-1β, and tumor necrosis factor-α (TNF-α) as well as the chemokines (20, 21). The TGF-β1 and platelet-derived growth factor (PDGF) are the most important inflammatory mediators in hepatic fibrosis. TGF-β1 regulates the activation of HSCs through the Smad pathway and inhibits the degradation of the ECM (22). PDGF activates HSCs and promotes their proliferation through the subsequent phosphatidylinositol 3 kinase (PI3K) and extracellular signal-regulated kinase pathways (23). Additionally, macrophages also mediate the reversal of fibrosis. Firstly, macrophages secrete a series of matrix metallic-proteinases (MMPs), which specifically degrade collagen and non-collagen extracellular matrix (24). Secondly, macrophages mediate HSCs apoptosis by expressing TNF-related apoptosis-inducing ligand (25), leading to the reduction of ECM and alleviation of hepatic fibrosis (26). Relaxin is an anti-fibrotic peptide hormone that can directly reverse the activation of HSCs to promote the resolution of hepatic fibrosis (27). A study showed that hepatic macrophages express primary relaxin receptors and they switch from a profibrogenic to the pro-resolution phenotype upon binding with relaxin. The latter releases exosomes that promote relaxin-mediated quiescence of activated hepatic astrocytes via miR-30a-5p (28). This indicates that macrophages have great potential in anti-fibrosis research and can be used as a target for anti-fibrosis therapy or reversal of fibrosis.

Dendritic cells (DCs) are the specialized antigen-presenting cells, with the function of transforming immune tolerance to immune activation, regulating the direction of the immune response in HIME of hepatic fibrosis (29). During hepatic fibrosis, DCs proliferate and undergo phenotypic changes. They stimulate adjacent T cells and natural killer(NK) cells through TNF-α, and up-regulate intercellular adhesion molecule-1 and CD40 expression of HSCs, thus, promoting the HSCs proliferation and activation (30, 31). DCs also aggravate liver inflammation by inhibiting infiltration of Treg cells and activating TLRs to produce a large number of cytokines (32). And the DCs depletion completely abrogates the elevated levels of many inflammatory mediators that are produced in the fibrotic liver (33). A similar study found knockout of DCs reduced the clearance of activated HSCs and delayed the fibrotic reversal process during the reversal phase of CCL4-induced hepatic fibrosis in mice (34).However, it also reported that although DCs can promote the activation of HSCs, the depletion of DCs does not affect the evolution of liver fibrosis in CCL4-induced hepatic fibrosis (35). Moreover, the application of FMS-liketyrosinekinase3 ligand induced the proliferation of DCs, accompanied by increased MMP-9 secretion from DCs. And the MMP-9 not only directly degrades collagen but also recruits innate immune cells such as macrophages and neutrophils, secreting MMP-8 and MMP-13 to degrade collagen, facilitating the reversal of hepatic fibrosis (34). Besides, long-term (12 weeks) alcohol intake can specifically recruit plasmacytoid dendritic cells to the liver in female mice. These plasmacytoid dendritic cells are characterized by secreting IFN-α and with anti-fibrosis function (36). These results suggest the effect of DCs in hepatic fibrosis is still controversial and need to be revealed and defined in the future research.

NK cells in liver were subdivided into CD49a⁺DX5 ^– liver-resident natural killer (lrNK) cells and CD49a^- DX5⁺ conventional natural killer (cNK) cells. Both of them could kill activated HSCs in a tumor necrosis factor-related apoptosis-inducing ligand dependent manner (37). Compared to cNK cells, lrNK cells are less mature at a rest statue, but it have enhanced activity upon pathogenic stimuli and exhibit higher cytotoxicity (38). It suggested that the enrichment of lrNK cells in the liver may be to prepare for a special function against liver injury. It also was reported that lrNK cells can highly express CD107a and perforin, work together with a variety of activated receptors, release interferon-γ (INF-γ) while splitting infected hepatocytes, and inhibit the activation of HSCs in the human viral hepatitis (39). In the early stage of hepatic fibrosis, cNK cells infiltrate into the liver parenchyma and alleviate or reverse hepatic fibrosis by directly killing or inducing apoptosis of HSCs through degranulation and TNF-related apoptosis-inducing ligand expression (40). Moreover, the activated NK cells release IFN-γ through the JAK-STAT pathway to antagonize hepatic fibrosis by exerting a killing effect on the activated HSCs (41, 42). The activated NK cells can also secrete IL-10 and IL-22,which promote the senescence of activated HSCs and alleviate liver fibrosis through signal transducer-activator 3 (STAT3)/p53/p21 pathway (43, 44). And the activation of metatropic gluamate receptor 5 in NK cells can also reduce liver fibrosis by increasing their cytotoxicity and IFN-γ production (45).The NKp46(+)NK· cells were reported to attenuate metabolism-induced hepatic fibrosis by regulating macrophage activation in mice (46). In patients with chronic HVB infection, the terminally differentiated NK cells often highly express CD57 and DNAM-1, while NKp46 and NKG2A expression at low levels. These NK cells can kill activated HCSs through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) dependent and CD44-osteopontin dependent manners (47). In contrast, during the progressive phase of hepatic fibrosis, persistent activation of HSCs will inhibit NK cell activity and weaken its anti-fibrotic effect. It is attributed to the increased metabolism of vitamin A. Its metabolites, retinoic acid, and retinol can inhibit the activation of IFN-γ on the transcriptional STAT-1 pathway by secreting the suppressor of cytokine signaling 1 (SOCS1) (41, 48).

NKT cells, a subset of T cells, possess NK and T cell receptors and display both NK cell and T cell properties. Similar to the NK cells, NKT cells also inhibit hepatic fibrosis by directly killing the activated HSC cells or producing IFN-γ to exert the inhibiting effect on HSCs. In the CCL4-induced acute liver injury mouse model, the hepatic NKT can slow down the onset of acute liver injury and liver inflammation by inhibiting activated HSCs (49). However, the anti-fibrotic effect of NKT cells is limited to the acute liver injury period, and in persistent chronic liver injury, the anti-fibrotic effect of NKT is diminished due to functional failure of NKT and immune tolerance of the liver (50). Additionally, Park et al. found that α-galactosyl neurosphingosamine overactivated NKT cells will accelerate CCL4-induced acute liver injury, inflammation, and fibrosis (51). In the progression of the alcoholic liver, NKT cell activation can exacerbate liver injury and promote an inflammatory response (52). Another study found that NKT cells from viral-infected patients could secrete more pro-inflammatory factors IL-4 and IL-13 compared with those from non-infected patients. It suggests that NKT cells may promote fibrosis by producing pro-inflammatory factors IL-4 and IL-13 to promote the development of fibrosis in patients with chronic viral infection (53).

Different subtypes of T cells have different effects on hepatic fibrosis. Th1 and Th2 cells play the core role in this process, and they tend to exhibit mutually antagonistic effects on hepatic fibrosis. IFN-γ secreted by Th1 cells regulates the balance of MMP and tissue inhibitor of metalloproteinase (TIMP), which can inhibit HSCs activation and proliferation, induce HSCs apoptosis, and inhibit TGF-β expression in a variety of cells (e.g., hepatocytes, KCs, HSCs) to exert anti-fibrotic effects (54, 55). IL-13 secreted by Th2 induces the production of collagen I, collagen III, α-smooth muscle actin, and TIMP-1 to induce TGF-β secretion, activate HSCs, and inhibit HSCs apoptosis thus promoting the progression of hepatic fibrosis (56). Moreover, IL-4, IL-5, and IL-13 secreted by Th2 cells reduce the liver inflammatory response, but also inhibit the Th1-mediated cellular immune response and prevent the clearance of viruses and parasites, leading to the persistence of viruses and inflammation (57). In addition, Th1 and Th2 cells regulate cellular collagen synthesis by antagonistically regulating nitric oxide synthase 2/arginase activity (58). In a non-alcoholic steatohepatitis (NASH) mouse model, mice lacking the IFN-γ (typical Th1 cytokine) showed significant protection against liver damage and fibrosis (59).

CD8⁺ T cells mainly produce cytotoxic molecules such as IFN-γ, TNF, and perforin (60). During NASH in mice, the number of CD8⁺ T cells in the liver was increased, and lipid-conditioned CXCR6⁺ CD8⁺ T cells induce hepatocyte killing in a perforin-independent, FasL (CD95L)-dependent manner (61). Consequently, in a diet-induced mouse model of NASH, CD8⁺ T cell depletion blunted liver injury (62). Thus, CD8⁺ T cells are likely to promote hepatic injury during NASH. But the mechanism by which T cell subsets are activated and interact to promote liver inflammation is not well understood. And more research is needed to investigate the specific mechanism and how to target these pathways without compromising immune defenses (63). Additionally, hepatic tissue-resident memory T cells (TRM cells) stably occupy tissues and participate in hepatic fibrosis progression differing from T cells in the circulation. CD8⁺T cells characterized by CD69 and CD103 are defined as CD8⁺ TRM cells (64, 65). Koda Y et al. found that CD8⁺ TRM cells were able to attract HSCs in a CCR5-dependent manner and mediated apoptosis of activated HSCs through the Fas- FasL pathway (66). Although little latest work had been done to verify the exist of CD4⁺TRM fraction in human liver, its relationship with liver fibrosis has not been systematically studied and documented (67).

Gamma-delta T cells(γδ T cells), as liver tissue-resident T cells, are characterized by the gamma and delta chains of T cell receptors (68, 69). Hepatic γδ T cells account approximately for 3% - 5% of the hepatic lymphocytes and 15% - 25% of the hepatic T cells, which are identified as liver-resident cells with predominant production of IL-17as well as the IFN-γ (70). And the IFN-γ will activate macrophages to release IL-15, prompting γδ T cells to accumulate at the infectious site and participate in local anti-inflammatory and anti-fibrotic effects, whereas IL-17, a major pro-inflammatory factor, often plays a pro-fibrotic effect on the fibrosis progress (70, 71). In the CCl4-induced hepatic fibrosis murine model, γδ T cells could activate the mTOC2 signaling pathway in response to macrophages, promoted CXCR3 transcription, and drove γδ T cell accumulation in the liver (72). It also documented that hepatic γδ T cells could kill the activated HSCs in a TRAIL-or FasL-dependent manner and secrete IFN-γ to inhibit differentiation of pro-fibrotic Th 17 cells to relieve hepatic fibrosis. And it also promoted HSC lysis by enhancing the cytotoxicity of conventional NK cells and liver-resident NK cells on activated HSCs (37). However, hepatocyte-derived exosomes mediated TLR3 activation in HSCs and increased IL-17 production from γδ T cells, and the IL-17 strongly stimulated the expression of a-SMA, TGF-β, IL-6, and collagen type I-α1 in HSCs and aggravated liver fibrosis (73).

B cells are classified as B-1 cells, B-2 cells, effector B cells, and regulatory B cells (Bregs) based on their surface molecular, localization, and functional characteristics. Relatively few studies indicated that B cells can influence the development and progression of hepatic fibrosis in an antibody-independent manner (74). It has been demonstrated that B cells knockout mice given CCL4 injections had significantly lower hepatic fibrosis than normal control mice, suggesting that B cells have a pro-fibrotic effect and this effect is independent of antibodies and T cells (75). Conversely, B cells can also exacerbate hepatic fibrosis by indirectly affecting T lymphocyte function and contact with fibroblasts, phagocytes, and NK cells through the secretion of IL-1, IL-6, and IL-4 (76). Secondly, it has been found by retrospective analysis that the number of B lymphocytes was significantly lower in the cirrhotic group than in the hepatitis group and healthy individuals, which also implies that B lymphocytes are affected in the progression of cirrhosis and are involved in the development of cirrhosis (5). In addition, similarly to T cells, regulatory B cells (Bregs) also secrete cytokines inducing immune tolerance and maintaining environmental homeostasis. Bregs from patients with chronic hepatitis B or hepatitis B virus(HBV)-associated hepatic fibrosis can inhibit the function of Th1 and Th17 through intercellular contact and secretion of IL-10. Bregs can also induce the conversion of CD4⁺CD25^-T cells to Tregs, resulting in inflammatory response suppression and inflammatory repair (77).

In summary, the immune cells directly act on HSCs through the interaction of ligands and receptors or by secreting cytokines to regulate the activity of HSCs. Additionally, immune cells play an immunomodulatory role or indirect effect on HSCs regulating the inflammatory response through the synthesis and secretion of different cytokines and chemokines, thus promoting or inhibiting the formation and progression of hepatic fibrosis. We depicted how the main sub-types of immune cells in the HIME influence the HSCs in Figure 1 .

The importance of immune cell populations in the development of chronic liver disease is self-evident. However, most traditional treatments for liver fibrosis are still focused on inhibiting the activation of HSCs. The role of HIME in liver fibrosis should not be underestimated. Therefore, blocking the infiltration of immune cells in the blood or altering the function of infiltrating immune cells or cytokines may be a potential target for the treatment of liver fibrosis. Table 1 summarized the details of the effects of traditional Chinese medicine(TCM) in modulating the HIME for preventing hepatic fibrosis of chronic liver disease. In four preclinical studies, researchers regulated the functions of HSCs (130, 132), macrophages or KCs (25, 131, 134–136, 138), regulatory T cells (133), DCs (30), and NK cells (25, 137) by different treatment approach in the CCl4 or bile duct ligation -induced liver fibrosis murine model. And these interventions can effectively alleviate hepatic fibrosis and tissue inflammation. Another study found inhibiting the chemokines CCR2/5 could significantly reduce circulating Ly6C^hi monocytes and hepatic monocyte-derived macrophage infiltration in murine models of NASH, and attenuate hepatic inflammation and fibrosis (140). A chemical agent, dopamine receptor D2 antagonism, was screened to target the Yes-associated protein signaling pathway in macrophages. It antagonizes Yes-associated protein-dependent fibrotic crosstalk between selectively targeted macrophages and CTGF + VCAM1 + vascular niche, blocks fibrosis, and restores liver architecture in rodent and large animal models of NASH (139).

The occurrence and progression of liver fibrosis often involve a variety of cells and multiple signaling pathways. Clinical TCM treatments are advanced in action on multi-cellular and multi-target, which might be a new choice for alleviating liver fibrosis. Ginsenoside (KD), the active ingredient in the TCM ginseng, was reported could reduce hepatic histopathological damage, proinflammatory cytokine release, and extracellular matrix deposition in CCl4-induced hepatic fibrosis. The KD restrained the hepatic fibrosis-driven rise in CD86, MHC-II, and CCR7 levels, while upregulated PD-L1 expression on DCs, which blocked CD8⁺ T cell activation. Additionally, KD reduced DC glycolysis, maintained DCs immature, and was accompanied by an IL-12 decrease in DCs. These effects disturbed the communication of DCs and HSCs with the expression or secretion of α-smooth muscle actin and Col-I declined in the liver (30). Lycium barbarum polysaccharides (LBPs) supplementation was reported to reduce CCl4-induced oxidative injury, inflammatory response, and TLRs/NF-kB signaling pathway expression, which alleviated CCl4-induced liver fibrosis (132). Moreover, two Chinese herbal formulas Si-Wu-Tang (134)and Yi Guan Jian (131) were reported to regulate the activation and polarization of macrophage, which significantly improved liver fibrosis and inflammatory responses. Additionally, Si-Wu-Tang also can inhibit the M1 from inhibiting the infiltration of neutrophils and promote the CD8⁺ organization to reside memory T cells cytotoxic effect inducing the HSCs apoptosis (134). The Yi guan Jian can enhance the therapeutic effects of fetal liver stem/progenitor cells that promoted the liver regeneration differentiation of fetal liver stem/progenitor cells into hepatocytes (131). Collectively the data supports the notion that multi-target therapeutics may be an effective option for t liver fibrosis treatment ( Table 1 ).

The immune cells and HSCs could synthesize and secrete several chemical mediators under chronic liver injury, and they constitute a dynamic HIME of an immune cells-cytokines-chemokines network system that affects the processes of hepatic fibrosis. It can be seen the HIME of hepatic fibrosis is complex not only because there are a variety of cells involved, but also because there are different sub-types of immune cells, which play different roles at different stages of liver disease progression. Moreover, in addition to local immune cells in the liver, a variety of immune cells in the circulation also can be chemotactic into the liver tissue and differentiate into different cell subtypes, exerting an effect on the initiation, progression, and regression of hepatic fibrosis. According to the summary and analysis in this review, most sub-types of immune cells have bidirectional effects on the development of hepatic fibrosis, because the immune cells interact with each other and also with other stromal cells in the microenvironment through diverse and complex cytokines networks, and eventually achieve a balance. While this balance will be broken with the persistence or elimination of the disease’s causes, then these cells will form a new balance through the other signaling pathways and exert a promoting or inhibiting role in hepatic fibrosis to a different degree. Therefore, it is necessary not only to explore the function of a certain group of immune cells, but also to explore cells’ diversity, and accurately analyze the cells’ specific phenotype and role in different liver diseases, different stages of the same disease, and different HIME caused by different pathogenic factors stimulus. Recently, single-cell sequencing can clarify the specific role of single cells. If this detection method is applied to the investigation of the immune microenvironment of hepatic fibrosis, it may greatly enrich the existing immune network and provide comprehensive and accurate ideas for the study of hepatic fibrosis. Additionally, the specific mechanisms for the immune cells of a specific phenotype to play different roles at different stages of chronic liver disease need to be elucidated, so that a precise intervention can be performed. However, the study on specific mechanisms is currently a major vacancy for the effect of HIME on hepatic fibrosis, thus, future studies need to focus on a deeper and more specific regulatory pathway to achieve an accurate intervention target, thereby delaying or avoiding the occurrence and progression of hepatic fibrosis.

NZ, HY, and ZZ conceived of the subject matter and wrote the logic of this review, searched, categorized, and summarized all relevant literature, and wrote the initial draft. ZL, XC, YZ, and RJ helped to check relevant information check the content of the initial draft. JH and HP edited the second draft and composed the manuscript. XL and YL reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.