Cell-in-cell: an Emerging Player in COVID-19 and Immune Disorders

: Cell-in-cell is a unique phenomenon mostly documented in human cancer tissues. A recent study demonstrated that cell-in-cell might promote lymphopenia by internalizing and killing immune cells in COVID-19, which implicates cell-in-cell as an emerging player in a broader spectrum of pathological processes, such as immune dysregulation

other CIC models as well, promoted the conception that CIC formation may constitute a cell death program that kills the internalized cells in an unconventional way [9]. The formation of entotic CIC structures is tightly controlled by a set of molecular machinery that consists of three core elements (adherens junction, mechanical ring, and contractile actomyosin) and a group of regulatory factors [10][11][12][13][14][15][16][17][18][19], on which readers are referred to a recent review [20] for detail. Following CIC formation, the internalized cells were dead and cleared in an acidified huge lysosomal compartment [21], which was regulated by the unconventional autophagic signaling [22]. It was found that homotypic CIC structures were important players of multiple important biological processes, such as tumor evolution as a mechanism of cell competition [16,23], epithelial homeostasis as a mechanism of mitotic surveillance [24], embryonic development by eliminating unwanted cells [25,26], and genome instability by interfering cytokinesis [27,28]. Along with the studies on homotypic CIC structures by entosis, considerable progress was made on heterotypic CIC structures ( Figure 1).

HETEROTYPIC CIC STRUCTURES IN COVID-19
Recently, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread all over the world, leading to the pandemic of coronavirus disease 19 (COVID-19) [29]. A remarkable clinical feature of patients with COVID-19 is the reduced lymphocyte count, or lymphopenia, which was a critical factor associated with unfavorable prognosis. However, the underlying mechanisms are poorly understood. By examining a panel of post-mortem autopsies, we found that the multinucleated syncytia were readily detected in the lung tissues of patients with severe COVID-19 [30]. Remarkably, the majority of the syncytia contained CD45 + lymphocytes that were mostly CD8 + within their cytoplasm, morphologically resembling heterotypic CIC structures. Moreover, the number of the heterotypic CIC structures in COVID-19 autopsies had a negative correlation with the number of lymphocytes in the periphery blood, suggesting a negative impact of CIC structures on circulating lymphocytes [30]. In agreement with this idea, subsequent co-culture experimentations showed that syncytia could effectively internalize periphery blood mononucleated cells (PBMC) to form heterotypic CIC structures, and promoted the death of PBMCs, in particular those that are CD8 + [30]. These data fit well with a working model that the multinucleated syncytia, induced by SARS-CoV-2 infection, may serve as a disastrous unity to eliminate lymphocyte via CIC-mediated death [31], and targeting syncytia and/or CIC formation holds the promise to treat COVID-19 and syncytia-related diseases. It is noted that syncytia are common in various of virological diseases. For example, the infection of respiratory syncytial virus was known to cause syncytia formation of infected cells, the same was true for other pathogenic viruses, such as human immunodeficiency virus and highly contagious flu viruses H5N3, H7N1 and the like [30,32]. The formation of syncytia was ascribed to the fusogenic glycoproteins expressing on the surface of viral particles that mediate membrane fusion and host entry of the viral genomes [32]. Interestingly, the victims of virus infection frequently developed lymphopenia. It therefore remains to be explored whether syncytia formation is correlated with lymphocyte loss in these patients, and to what extend syncytia formation may contribute to lymphopenia, and furthermore to what extends blocking the formation of syncytia and/or CIC structures may relieve the related virological diseases, including COVID-19 and the like.

HETEROTYPIC CIC STRUCTURES IN CANCERS
Similar to syncytia in virological diseases, multinucleated cells are common in human cancer, where immune edition was identified as one of the key drivers promoting tumor development and progression [33]. Multiple lines of evidence supported the idea that heterotypic CIC formation may contribute to immune evasion and cancer malignancy. First, the formation of heterotypic CIC structures with cancer cells internalizing immune cells was shown to be an independent prognostic factor that associated with shorter overall survival time (8 months vs 15 months) in two independent cohorts of patients with pancreatic ductal adenocarcinoma [4]; second, multinucleated cancer cells were demonstrated in vitro to internalize more immune cells and in a higher frequency as compare with mononucleate cancer cells [30]; third, experimental models of emperitosis or cell cannibalism showed that the formation of heterotypic CIC structures could result in the death of the internalized NK or CD8 + T cells [34,35], either apoptotically or non-apoptotically. Nevertheless, it remains to be confirmed further in vivo in animal models and in human cancer samples whether a physiological relevance and a causal link existed between heterotypic CIC formation and immune evasion. To this end, a systemic deciphering of the molecular mechanisms controlling heterotypic CIC formation in a contextdependent way would be helpful.

HETEROTYPIC CIC STRUCTURES IN IMMUNE DISORDERS
In addition to virological diseases and cancers, heterotypic CIC structures were also implicated in the Natl. Sci. Open, 2022, Vol. 1,2021004 immune disorders related to inflammations. Benseler et al. reported that hepatocytes could internalize selfreactive CD8 + T cells for destruction. This was believed to help maintain immune homeostasis to avoid autoimmune hepatitis. Consistent with this notion, blocking the formation of heterotypic CIC structures by Wortmannin, an inhibitor of myosin light chain kinase, led to an accumulation of autoreactive CD8 + T cells in the liver and breach of tolerance, with the development of autoimmune hepatitis [36]. Conversely, there were studies that proposed that penetration of CD8 + T cells into hepatocytes was positively associated with autoimmune hepatitis [37] and chronic hepatitis B [38]. Though these studies are descriptive and the quantification of CIC structures warrants calibration, the proposed conclusions suggest that heterotypic CIC formation may be either an effect secondary to inflammation, or a driver/promoter of inflammation, or a vicious circle with inflammation. A recent study actually touched this issue by claiming that CD44/p-ERM/ F-actin pathway mediates the penetration of CD8 + T cells, unfortunately, the conclusion was not solidly supported by the experimental data presented [39]. Hence, this issue remains to be explored for future heterotypic CIC studies. Other than T cells, NK cells were also reported to be internalized to form heterotypic CIC structures during the development of liver cirrhosis associated with chronic hepatitis B [40], whereas, the outer cells were not hepatocytes, but activated hepatic stellate cells. Under this context, TGF-β seemed to be a promoter that facilitates penetration of NK cells into hepatic stellate cells for apoptotic death, leading to enhanced liver fibrosis associated with chronic hepatitis B.

CONCLUSION REMARKS
Together, the existing data suggest complicated roles for heterotypic CIC structure in various contexts, including virological diseases, cancers, and inflammatory disorders, and the fourth (Figure 1). On top of these is the regulation of cellular immunity by targeting immune cells for internalization into different host cells, which was followed by altered cell fates or behaviors of all the cells, inner and outer, engaged in the CIC structures. Currently, the study on heterotypic CIC structures is still in its infancy with different models being developed. In the short future, much effort may be endeavored on deciphering the key molecules that control the CIC formation and the fates of cells involved, the progress on which may dictate the development of potential therapeutic strategies for different immune-related diseases.

DATA AVAILABILITY
All data generated during this study are included in this published article.