The Ultrastructure of Hepatic Stellate Cell–Macrophage Intercellular Crosstalk as a New Morphological Insight into Phenomenon of Fibrogenesis in Pediatric Autoimmune Hepatitis

The aim of the study was the pioneering retrospective ultrastructural evaluation of respective forms of hepatic stellate cells (HSCs) and analysis of their crosstalk with other adjacent nonparenchymal cells (NPCs), especially Kupffer cells/macrophages (KCs/MPs), in pediatric autoimmune hepatitis (AIH). Methods: Ultrastructural assessment of the HSC population and NPCs was performed in transmission electron microscopy (TEM) using pretreatment liver biopsies from 25 children (8 boys and 17 girls) aged 4–17 with clinic-pathologically diagnosed untreated AIH. Results: Submicroscopic evaluation allowed easy identification of numerous HSCs in the form of transitory cells, i.e., T-HSCs, accompanied by signs of fibrosis. T-HSCs included cells with features of activation initiation (iHSCs) and activation perpetuation (pHSCs), indicating high HSC activation plasticity. The pHSCs were markedly elongated and mainly showed a distinct loss of lipid cytoplasmic material, expanded and dilated channels of granular endoplasmic reticulum, and linear bundles of microfilaments beneath the cell membrane. They were surrounded by usually mature collagen fibers. Frequently activated KCs/MPs adhered directly to T-HSCs. Between them, tight intercellular junctions were formed by means of point desmosomes. Conclusions: Our qualitative TEM observations indicate a key role of T-HSCs in liver fibrogenesis in pediatric AIH, with the essential involvement of activated KCs/MPs that directly adhere to them. Tight intercellular junctions, being the ultrastructural exponent of the specific cellular mechanisms of the crosstalk between NPCs, can play a vital role in hepatic collagen fibroplasia. A better understanding of HSC population morphology at the ultrastructural level in AIH seems important not only to improve the disease morphological diagnostics but to also provide new insights into therapeutic interventions for the phenomenon of liver fibrogenesis.


Introduction
Autoimmune hepatitis (AIH) is an immune-mediated autodestructive inflammatory disease affecting both children and adults, with female predominance, in the absence of a known etiology. Its diagnosis remains difficult and should involve clinical manifestations, laboratory evaluation, histopathology, and exclusion of other more common liver diseases. supported by repeated schematic drawings without presenting the electron microscopic documentation of this phenomenon.
Up to now, literature data on the sequence of morphological events observed at the ultrastructural level in the pathogenesis of liver fibrosis have been limited to adult patients and experimental studies. Similar reports on pediatric patients are lacking.
Taking that into consideration, the aim of the current study was to provide, for the first time in the hepatological literature, a descriptive qualitative morphological research characterizing the ultrastructural profile of the respective forms of hepatic stellate cells and adhering hepatic macrophages in untreated children with AIH, in relation to the process of fibrogenesis, based on retrospective biopsy material. Also interesting is the submicroscopic analysis of the intercellular crosstalk between HSCs and adjacent nonparenchymal hepatic cells, especially KCs/MPs, in the same pediatric patients with AIH. The elucidation of the ultrastructure characteristics of this intercellular communication of cell-cell interaction in HSC activation may be crucial for research on novel potential antifibrotic therapies in AIH.
The current study is a continuation of our many years of morphological and clinical research, especially ultrastructural studies in chronic liver diseases in children, such as chronic hepatitis B (chB) [28,29,[45][46][47], nonalcoholic steatohepatitis (NASH) [48,49], pediatric AIH [3,50,51], and also in the formation of biliary fibrosis in an animal experimental model [44]. It is noteworthy submicroscopic and immunohistochemical observations with a focus on the role of the population of NPCs and HPCs in the morphogenesis of pediatric AIH [3,50,51].

Study Patients' Profile
Retrospective ultrastructural investigations were conducted using tissue material embedded in epon blocks, including pretreatment needle liver biopsy specimens obtained from 25 untreated children (8 boys and 17 girls) aged 4-17 hospitalized in the Department of Pediatrics, Gastroenterology, Hepatology, Nutrition and Allergology, Medical University of Bialystok, with clinic-pathologically diagnosed AIH.
The laboratory tests revealed markedly increased serum levels of aspartate and alanine aminotransferase in all study patients. Immunological and serological disturbances in the blood serum were manifested by substantially elevated IgG levels and the presence of autoantibodies-antinuclear antibodies (ANAs) and/or smooth muscle antibodies (SMAs). Differential diagnostics excluded, among others, infectious liver diseases (HBV, HCV, CMV, and Toxoplasma gondii), some metabolic disorders (Wilson's disease, cystic fibrosis, and alpha-1 antitrypsin deficiency), and celiac disease. The clinical data, including immunological and serological disturbances in blood serum and differential diagnostics of patients, were reported in our earlier papers [3,50].
The collected material was subjected to pathomorphological investigations, i.e., histological, histochemical, immunohistochemical (for alpha-SMA and desmin, CK7 and CK19), and ultrastructural analyses using TEM in the Department of Medical Pathomorphology, Medical University of Bialystok.
It should be added that as regards the identification of two subpopulations of liver macrophages (KCs/MPs) carried out in the present study at the level of electron microscopy, it was easy to distinguish KCs, i.e., liver resident macrophages, from the others, i.e., liverrecruited macrophages. KCs showed their characteristic location within the lumen of the liver sinusoides and also the submicroscopic image. In contrast, for the remaining liver macrophages, similar to other authors dealing with the ultrastructure of this cell population and to our previous research on KCs/MPs in AIH [3], we treated them as liver-recruited macrophages.
Hepatic necroinflammatory injuries and fibrosis had been previously assessed histologically using routine Mayer's hematoxylin and eosin (H&E) stain. Liver fibrosis staining for connective tissue stroma components was determined by a panel of histochemical stains (for collagen fibers, by Sirius red, Masson' trichrome blue, the Azan method, according to Masson-Goldner, and for reticulin fibers, according to Gomori) and assessed by a single hepatopathologist blinded to patient clinical data. The study showed typical histological features of AIH, i.e., interface and lobular hepatitis, moderate/severe in nature with portal infiltration of lymphocytes and plasma cells, severe necroinflammatory reaction, and rosette formation of hepatocytes. The alterations were frequently accompanied by portal, periportal, and bridging fibrosis [3,50,51]. The stage of fibrosis (staging-S; range: 0-4) and inflammation grade (grading-G; range: 0-4) in liver biopsies were retrospectively scored using the semiquantitative scoring system according to Batts and Ludwig [52] and, in some cases, complemented by the scoring system proposed by Ishak [53]. In the group of 25 children, we identified 5 patients with advanced liver fibrosis (corresponding to S-3) but not with liver cirrhosis and 7 with mild (S-1) and 13 with moderate (S-2) liver fibrosis.
It should be notice that, for obvious bioethical and procedural reasons, no control groups consisting of healthy children subjected to liver biopsy could be formed as liver biopsy is an invasive procedure and cannot be performed in control, healthy children. This is clearly stated by the ESPGHAN Hepatology Committee in the guidelines of 2015 for pediatric liver biopsy [4]. Thus, we compared the ultrastructure of the respective morphological forms of HSCs in pediatric AIH to the same cell population in other chronic liver diseases in children investigated at our center, i.e., chronic hepatitis B [29,45,47] and nonalcoholic steatohepatitis (NASH) [48,49].
Informed consent was obtained from the parents of each patient included in the study. The current research was approved by the Ethical Committee, Medical University of Bialystok (R-I-002/410/2016).

Liver Tissue Processing for Transmission Electron Microscopy
For TEM, fresh small tissue blocks (1 mm 3 volume) obtained from liver biopsy material were primarily fixed in Karnovsky's fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4) for 12 h. Next, they were postfixed in 2% osmium tetroxide (OsO4) in 0.1 M cacodylate buffer (pH 7.4) for 1 h. Then, the material was dehydrated through a graded series of ethanols and propylene oxide, embedded in Epon 812 or Gycid ether 100 for electron microscopy, and sectioned on a Reichert ultramicrotome (Reichert Ultracut S). The obtained semithin sections were stained with 1% methylene blue in 1% sodium borate. The ultrathin sections (approximately 70 nm) were subsequently cut with the same Reichert ultramicrotome, mounted on copper grids, contrasted with uranyl acetate and lead citrate, examined under an Opton EM 900 electron microscope (Oberkochen, Germany), and photographed with a TRS camera (CCD-camera for TEM, 2K inside). The same procedure had been used in our earlier ultrastructural investigations of the liver in children [3,29,50,51].
The ultrastructural profile of the HSC population and the surrounding cells in relation to the process of fibrogenesis was determined by a microscopist who was blinded to the clinical information.

Results
The retrospective ultrastructural analysis of the liver affected by AIH from children with coexisting mild fibrosis (S-1) showed the transformation of resting forms of stellate cells, i.e., Q-HSCs, normally found in a healthy liver, into a transitory form of hepatic stellate cells (T-HSCs) in the phase of activation initiation (i-HSCs) ( Figure 1A,B). Interestingly, T-HSCs being in the phase of activation perpetuation, i.e., pHSCs were also found, although more seldom, in AIH coexisting with mild fibrosis (Figure 2A,B). However, the analogous electron microscopic study using pretreatment liver biopsies obtained from children with AIH and accompanying advanced fibrosis (S-2, S-3) showed almost a complete lack of resting forms of stellate cells and their replacement with transitory hepatic stellate cells presenting features of pHSCs ( Figures 3A,B, 4A,B, 5 and 6).
analogous electron microscopic study using pretreatment liver biopsies obtained from children with AIH and accompanying advanced fibrosis (S-2, S-3) showed almost a complete lack of resting forms of stellate cells and their replacement with transitory hepatic stellate cells presenting features of pHSCs ( Figures 3A,B, 4A,B, 5 and 6).  The cell nucleus of iHSCs contained a distinct nucleolus/nucleoli, while the cytoplasm showed an abundance of lipid material and slightly dilated channels of granular endoplasmic reticulum (ger), which is well demonstrated in Figure 1A,B.
It should be noted that TEM examination allowed easy identification of numerous second-type T-HSCs, i.e., in the phase of activation perpetuation, which clearly predominated in the whole population of the hepatic stellate cells studied.       The subpopulation of pHSCs showed either perisinusoidal location (Figures 2A,B and 3A,B) or was observed outside the space of Disse, in the spaces between hepatocytes (Figures 4A,B, 5 and 6). These cells were markedly elongated (Figures 2A,B, 3A,B and 6). Many times, their substantially elongated and thinned cytoplasmic processes reached neighboring hepatocytes (Figure 2A,B). Interestingly, pHSCs showed a substantial loss of lipid cytoplasmic material (Figures 2A,B, 3A,B and 6) and frequently contained only a single drop or very tiny lipid droplet, with a volume smaller than 10% of the cytoplasmic volume ( Figures 3A,B and 6). The cytoplasm of such cells contained expanded and markedly dilated channels of ger with the formation of cistern-like structures filled up with delicate flocculent material, segmentally deprived of ribosomes ( Figures 4A,B and 6). Greater degranulation of ger channels resulted in the formation of cytoplasm areas filled with free ribosomes (Figure 4A,B). Sometimes, dilated canals of the Golgi apparatus were observed. Cytoskeleton components were observed as linearly arranged bundles of microfilaments, usually around 5 nm in diameter, beneath the cell membrane of pHSCs ( Figure 4A,B).  Many times, pHSCs were surrounded by abundant quantities of thick bundles of mostly mature collagen fibers that frequently adhered directly to them (Figures 3A,B, 5 and  6). However, apart from mature collagen fibers, some images also showed a microfibrillar, flocculent, and condensed material in the cytoplasm of pHSCs (within the markedly dilated channels of ger and loosely laying outside them) and in very close vicinity of these cells, which can be referred to as a morphological precursor of collagen fibers ( Figure 6).
Interestingly, activated KCs/MPs directly adhered to T-HSCs (both to iHSCs and to pHSCs). These cells were markedly enlarged and contained numerous primary and secondary phagosomes that varied in size and were filled up with the phagocytized extracellular material (Figures 1A,B, 2A,B, 4A,B and 5).
The adhering T-HSCs and activated KCs/MPs formed direct cell-cell contacts, which was ultrastructurally demonstrated by the presence of tight intercellular junctions (point desmosomes) (Figures 1A,B, 2A,B and 4A,B). Figure 6. An electronogram of a fragment of a pHSC situated between thick bundles of mature lagen fibers (c). Its cytoplasm shows markedly dilated ger channels (->); visible areas of accumu ing microfibrillar, flocculent material within substantially dilated ger channels, loosely laying o side them, and in some places, directly adhering to pHSC (+), which may correspond to deposit immature collagen components; Li-fine single lipid droplets. Scale bar 0.5 μm, original magnif tion ×20,000.
The cell nucleus of iHSCs contained a distinct nucleolus/nucleoli, while the cy plasm showed an abundance of lipid material and slightly dilated channels of granu endoplasmic reticulum (ger), which is well demonstrated in Figure 1A,B.
It should be noted that TEM examination allowed easy identification of numero second-type T-HSCs, i.e., in the phase of activation perpetuation, which clearly predo nated in the whole population of the hepatic stellate cells studied.
The subpopulation of pHSCs showed either perisinusoidal location (Figures 2A  and 3A,B) or was observed outside the space of Disse, in the spaces between hepatocy ( Figures 4A,B, 5 and 6). These cells were markedly elongated (Figures 2A,B, 3A,B and Many times, their substantially elongated and thinned cytoplasmic processes reach neighboring hepatocytes (Figure 2A,B). Interestingly, pHSCs showed a substantial loss lipid cytoplasmic material (Figures 2A,B, 3A,B and 6) and frequently contained onl single drop or very tiny lipid droplet, with a volume smaller than 10% of the cytoplasm volume ( Figures 3A,B and 6). The cytoplasm of such cells contained expanded and ma edly dilated channels of ger with the formation of cistern-like structures filled up w delicate flocculent material, segmentally deprived of ribosomes ( Figures 4A,B and   Figure 6. An electronogram of a fragment of a pHSC situated between thick bundles of mature collagen fibers (c). Its cytoplasm shows markedly dilated ger channels (->); visible areas of accumulating microfibrillar, flocculent material within substantially dilated ger channels, loosely laying outside them, and in some places, directly adhering to pHSC (+), which may correspond to deposits of immature collagen components; Li-fine single lipid droplets. Scale bar 0.5 µm, original magnification ×20,000.
It should be added that the comparison of the current findings presenting the ultrastructural profile of the respective morphologic forms of HSCs in relation to the process of fibrogenesis in pediatric AIH with the same cell population in other chronic liver diseases in children assessed in our earlier studies in TEM revealed that it was not specific to this disease, since it considerably resembled the ultrastructure of these cells in the course of chronic hepatitis B [29,45,47] and NASH [48,49] in pediatric patients hospitalized at our center.

Discussion
The currently conducted, for the first time in hepatology, electron microscopic study of a population of stellate cells of the liver in pediatric AIH coexisting with fibrogenesis and fibrosis progression revealed almost complete replacement of Q-HSCs, i.e., vitamin Astoring cells, by T-HSCs, which were in two activation phases: initiation and perpetuation, indicating high plastic properties of this cell population.
The pHSCs, which were the most commonly observed in our study, were markedly elongated and showed substantial loss of lipid cytoplasmic material and the presence of markedly delated channels of ger, segmentally deprived of ribosomes, within the cytoplasm.
Beneath the cell membrane, there were linearly arranged bundles of microfilaments. These cells were frequently surrounded by abundant, mostly mature collagen fibers forming bundles. Many times, activated KCs/MPs directly adhered to or were in close vicinity of T-HSCs. Between the adhering T-HSCs and liver macrophages, there were direct cellcell contacts, i.e., intercellular junctions constituting the morphological sign of cell-cell interactions.
Interestingly, the submicroscopic picture of the HSC population and fibrogenesis in pediatric AIH did not show features of morphological specificity to this pathology since its ultrastructure considerably resembled the ones observed in chB [29,45,47] and NASH [48,49] in children.
Generally, HSC activation occurs in two phases: initiation and perpetuation. In the initiation period, Q-HSCs receive molecular signals from adjacent cells, which secrete profibrogenic cytokines and growth factors, and then they demonstrate profibrogenic transcriptional and secretory properties. If activated HSCs proliferate and constantly secrete ECM proteins, leading to the accumulation of ECM and the formation of scar tissue, they are in the phase of perpetuation. The perpetuation of HSCs involves a number of cell-cell interactions and wound-healing responses [16][17][18]21,27,29,32]. If fibrosis begins to regress, the activated HSCs may develop a potential third phase, i.e., resolution, where they may either undergo apoptosis or revert to the senescent/quiescent phenotype [17,18,23,31,32,37].
It should be emphasized that HSC activation is a dynamic process that mainly depends on the interaction with other adjacent cells, including hepatocytes, hepatic macrophages, liver sinusoidal endothelial cells, natural killer/natural killer T (NK/NKT) cells, biliary epithelial cells, heptic progenitor cells (HPCs), and platelets, which can precisely "talk" to each other [17,21,22,27,29,31,45].
The exact ultrastructural picture of activated KCs/MPs, LSECs, and HPCs that may help in the diagnosis of pediatric AIH was presented in our earlier manuscripts devoted to this pathology [3,50,51].
Hepatic macrophages, in a simplified way and taking into account their ontogenes, can be divided into liver resident macrophages, termed Kupffer cells (KCs), and bloodor bone-marrow-derived macrophages recruited to the liver, called monocyte-derived macrophages (MPs). KCs are localized within the lumen of the liver sinusoids, accounting for about 30% of sinusoidal cells [30].
It is currently assumed that Kupffer cells, which adhere to the sinusoidal endothelial layer, can be activated by circulating diverse stimuli of blood and secreting various proinflammatory cytokines and chemokines, including TGF-β1, TNF-α, MCP-1, CCL3, and CCL5, and other soluble mediators, inducing a physiological response to other liver cells [21,27,31,42,[56][57][58][59]. There is clear evidence from in vitro and in vivo studies that KCs can activate HSCs into the transitional form of HSCs, i.e., T-HSCs, the major collagenproducing cell type in liver fibrogenesis. KCs activate hepatic stellate cells via paracrine mechanisms, likely involving the potent profibrotic and mitogenic cytokines TGF-β and PDGF [18,56].
It is worth noting that although it is well established that hepatic macrophages, including Kupffer cells and recruited macrophages, play a crucial role in the process of fibrogenesis and in the progression of liver fibrosis, the underlying mechanisms remain largely elusive and poorly understood. Therefore, the interesting research conducted by Pradere and Colleagues on the mechanisms of macrophage-HSC crosstalk deserves special attention here, which clearly showed that hepatic macrophages bilaterally regulate the activation of HSCs [60]. The authors demonstrated, for the first time in hepatology, that hepatic macrophages enhance liver fibrosis by promoting the survival of activated HSCs in a nuclear factor kappa B (NF-kB)-dependent manner. Moreover, they showed that macrophage-induced activation of NF-kB in HSCs in vitro and in vivo was mediated by interleukin (IL)-1 and tumor necrosis factor. They emphasized that IL-1 and TNF did not promote HSC activation but promoted the survival of activated HSCs in vitro as well as in vivo and thereby increased liver fibrosis. However, the neutralization of these proinflammatory cytokines in coculture experiments and genetic ablation of their receptors in mouse models led to increased apoptosis of HSCs and decreased fibrosis [60].
It is also worth adding that inhibition of NF-kB (via inhibition of the inhibitor of kB kinase/nuclear factor-kB pathway) with sulfasalazine induced apoptosis of activated rat and human HSCs, exerting a potential antifibrotic effect [61].
Like other authors, we believe that it is necessary to identify the key triggers between KC activity and HSC activation in future research, which may contribute to more effective interventions for KCs in liver fibrosis [18,21,22,27,31,58,62,63]. It is assumed that some important proteins can regulate the cooperation between KCs and HSCs [21,31,[57][58][59]62,63], which may benefit the therapy of liver fibrosis. For instance, Oncostatin M (OSM), a member of the IL-6 family cytokines, triggers collagen production in HSCs depending on the cellular source of fibrogenic cytokines from hepatic macrophages [62]. When macrophage regulation is blocked on OSM, collagen secretion of HSCs is inhibited, thus leading to reduced fibrosis.
Although the results of some experimental studies seem to be optimistic, showing that liver fibrosis is reversible and even that cirrhosis may regress in some cases, there is no effective antifibrotic therapy [27,[36][37][38][40][41][42]64,65]. Responsible for this situation is probably a relatively small number of morphological studies, especially submicroscopic investigations, in this field. It is, therefore, important to identify the vital pathological mechanisms underlying liver fibrogenesis, which could hopefully develop more efficient antifibrotic therapies to improve the quality of life of patients with liver fibrosis, including AIH. Deciphering the morphogenesis of the specific cellular mechanisms, taking into account the ultrastructure of cell-cell interactions in HSC activation, may provide new insights into therapeutic strategies for hepatic fibrosis. We still do not know what plays the leading role in the regulation of HSC activation in the process of liver fibrosis.
As the intercellular crosstalk in liver fibrogenesis in pediatric AIH is poorly understood, improved knowledge of the submicroscopic picture of cellular communication, i.e., cellcell interactions between HSCs and the surrounding cells, especially NPCs, could more efficiently promote fibrosis resolution and liver regeneration in the future. We hope that our current ultrastructural qualitative study, being the first to investigate the process of fibrogenesis in children with AIH, constitutes interesting comparative material for similar observations in children with this pathology. Further morphological research on the phenomenon of liver fibrogenesis in pediatric AIH will be also extended to quantitative analyses at our center.

Conclusions
The current ultrastructural qualitative assessment of hepatic stellate cells in children with clinic-pathologically diagnosed AIH allowed the identification of numerous transitional HSCs in the place of normal quiescent HSCs, with accompanying marked features of fibrogenesis. T-HSCs showed features of initiation and perpetuation of activation (i.e., iH-SCs and, the most predominant, pHSCs), which may indicate high properties of fibroblastic plasticity of this cell population. Many times, the activated KCs/MPs directly adhered to T-HSCs by means of the so-called point desmosomes. Tight intercellular junctions, being the ultrastructural component of the specific cellular mechanisms of crosstalk between NPCs, can play a vital role in the process of liver fibrogenesis in AIH.
In our opinion, further clarification of these cellular mechanisms in the population of hepatic stellate cells in children with AIH at the level of TEM, including cell-cell interaction in HSC activation, seems crucial not only for thorough microscopic diagnostics of the disease that is also extended with morphogenesis and fibrosis progression but also for providing new insights into therapeutic interventions for the phenomenon of liver fibrosis.