Global analyses of Chromosome 17 and 18 genes of lung telocytes compared with mesenchymal stem cells, fibroblasts, alveolar type II cells, airway epithelial cells, and lymphocytes

Telocytes (TCs) is an interstitial cell with extremely long and thin telopodes (Tps) with thin segments (podomers) and dilations (podoms) to interact with neighboring cells. TCs have been found in different organs, while there is still a lack of TCs-specific biomarkers to distinguish TCs from the other cells. We compared gene expression profiles of murine pulmonary TCs on days 5 (TC5) and days 10 (TC10) with mesenchymal stem cells (MSCs), fibroblasts (Fbs), alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8+ T cells from bronchial lymph nodes (T-BL), and CD8+ T cells from lungs (T-LL). The chromosome 17 and 18 genes were extracted for further analysis. The TCs-specific genes and functional networks were identified and analyzed by bioinformatics tools. 16 and 10 of TCs-specific genes were up-regulated and 68 and 22 were down-regulated in chromosome 17 and 18, as compared with other cells respectively. Of them, Mapk14 and Trem2 were up-regulated to indicate the biological function of TCs in immune regulation, and up-regulated MCFD2 and down-regulated E4F1 and PDCD2 had an association with tissue homeostasis for TCs. Over-expressed Dpysl3 may promote TCs self-proliferation and cell-cell network forming. The differential gene expression in chromosomes 17 and 18 clearly revealed that TCs were the distinctive type of interstitial cells. Our data also indicates that TCs may play a dual role in immune surveillance and immune homoeostasis to keep from immune disorder in acute and chronic pulmonary diseases. TCs also participated in proliferation, differentiation and regeneration. This article was reviewed by Qing Kay Li and Dragos Cretoiu.


Background
Telocytes (TCs) have been proposed as a new type of interstitial cells with extremely long telopodes (Tps), which were recognized in different organs, such as trachea and lung [1,2], oesophagus [3], intestine [4], liver [5], kidney [6], heart [7,8], skin [9], eyes [10], urinary tract [11], bladder [12], uterus [13], prostate gland [14], and euromuscular spindles [15], as documented on www.telocytes. com. TCs were found to form a three-dimensional network in close contacts with blood vessels, nerve bundles and cells of the local immune system [8,13,[16][17][18]. There is growing evidence to support that TCs have close associations with stem cells and will be a critical player in regeneration medicine [10,19]. TCs may also take part in immune regulation, proliferation, cellular reparation and fibrosis. However, there are no specific biomarkers to distinguish TCs from other tissue-resident cells. The reliable way to identify TCs is dependent on the specific ultrastructural feature -a small nucleated body and extremely long Tps with podomers and podoms by transmission electron microscopy (TEM) [20]. TCs present positive CD34, c-kit and vimentin by immunohistochemistry [19] and other markers like iNOS, caveolin-1, VEGF-D and PDGFRα were also identified to indicate various immunophetypes of TCs existed in different organs [4,21].
Our previous study initially approved the existence of TCs in the trachea and lung tissues of the mouse and human [1,22,23]. We compared genes expression profiles of murine TCs with mesenchymal stem cells (MSCs) and fibroblasts (Fbs) and proteomic profiles of human lung TCs and Fbs to explore TC-specific markers and their biological function [24,25]. MicroRNA signature was also used to differentiate TCs from other interstitial cells [26]. Furthermore, we revealed features and patterns of genes in chromosomes 1 by comparing gene expression profiles of murine pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8 + T cells from bronchial lymph nodes (T-BL), and CD8 + T cells from lungs (T-LL) by global analyses [27]. The similar work has been done to detect patterns of TCs-specific genes in chromosome 2 and 3, and showed that TCs played an important role in tissue injury and aging, inhibition of tissue inflammation, tumor promotion, and development of pulmonary fibrosis and other interstitial lung diseases [28]. However, features and patterns of TCs-specific genes in other chromosomes have not been uncovered, and potential function of TCs still remained unclear.
The present study aimed at investigating features and patterns of TCs-specific gene profiles and exploring potential function of TCs by focusing in chromosomes 17 and 18. The significant difference in gene expression profiles of murine pulmonary TCs on days 5 (TC5) and days 10 (TC10) with the other cells, MSCs, Fbs, ATII, ABCs, PACs, T-BL and T-LL were compared to identify TCs-specific genes and the functional networks were identified by bioinformatics tools.

Data mining and preprocessing
The gene expression profiles of murine lung TCs, MSCs and Fbs from our previous study contained 23861 probes. About 13236 probes and 11532 genes were defined after eliminating the probes without corresponding official symbols. Gene expression of ATII, ABCs, PACs, T-BL, and T-LL from GEO originally included 45,101 probes. 5684 probes without corresponding official symbols were eliminated and 39,417 probes and 21,680 genes were obtained. Data integration and genes detected in all the samples were selected to analysis, of which 11532 genes were elected finally. Totally, 582 genes of the chromosome 17 and 267 genes of the chromosome 18 were focused and furthermore analyzed in the present study.

Identification of differentially expressed genes
Gene expression data were normalized and imported into Agilent GeneSpring GX software (version 11.5.1) for further analysis. Differentially expressed genes between two samples were identified through Fold Change filtering as our previous research [27]. The propensity of functional changes was reflected in different levels of the gene expression in each cell types. The genes in TCs on days 5 and 10, which were up-or down-regulated more than onefold compared with other cells, were identified as TCsspecific genes in the present study. Up-or down-regulated folds of TCs genes were calculated by comparing with other cells, after the averages of gene expression in cells were obtained from the raw data of multi-databases, as shown in Additional files 1 and 2.
Hierarchical clustering of genes in chromosomes 17 and 18 was performed by TIGR Multi-experiment Viewer (MeV v4.9), respectively ( Figure 1A and B). The physical and functional interaction of specific genes was further Table 3 The up-regulated fold of specific genes in TCs as compared with others in chromosome 17

Discussion
Chromosome 17 represents between 2.5 and 3 percent of human genome while chromosome 18 approximately 2.7% [30,31]. It has been reported that chromosomes 17 and 18 contained many disease-associated genes [32][33][34][35][36][37]. The present study initially demonstrated TCs-specific genes, of which 16 and 10 up-regulated and 68 and 22 downregulated, in chromosomes 17 and 18, respectively. Of them, data from the present study indicated Mapk14, Trem2, MCFD2, or Dpysl3 as the representative of up-regulated genes and E4F1 and PDCD2 as the representative of downregulated genes, though the further study is needed.     Mapk14 is the encoding gene of p38 to regulate different cellular functions, promote the production of proinflammatory cytokines, and be involved in the response to stress and metabolic pathways. p38 plays important roles in the maintenance of homoeostasis and related pathologies [38][39][40]. Trem2 is the member of Trem family which is highly conserved in evolution in different specials [41,42]. Trem2 negatively regulates cytokine synthesis and plays an inhibitory regulator in inflammatory response [43,44]. Telocytes were found to connect with immune cells and regulate the immune response [19]. Over-expressed Mapk14 gene in TCs may promote the production and secretion of cytokines or other signal materials to induce inflammation, while up-regulated Trem2 gene tends to inhibit inflammatory cytokine synthesis to counteract the activation of p38. It seems that TCs play a complicated and dual role in immune surveillance and immune regulation. During the physical process, TCs can be activated to maintain homeostasis to induce proliferation, differentiation and tissue regeneration. On the other hand, TCs initiate the tissue inflammation to induce pathogenesis under some challenges.
MCFD2 encodes proteins involved in the transport of FV and FVIII from the endoplasmic reticulum to the Golgi apparatus [45]. It is suggested that TCs play an important role as an autocrine/paracrine factor in maintaining stem cell potential and self-renewal [46,47]. TCs were recently discovered to join the stem cells in the regeneration and repair from myocardial infarction [48]. We speculate that MCFD2 up-regulated in TCs may promote positive interaction and intercommunication between TCs and stem cells, and contribute to tissue injury and repair by maintaining tissue homoeostasis. CRMP4 protein encoded by Dpysl3 is strongly expressed in the developing nervous system, and play a critical role in neuronal outgrowth and polarity, axon guidance and axonal protection, and regeneration [49,50]. The features of neural axon have great similarity with Tps of TCs on morphology and biological function. It is possible that up-regulated Dpysl3 is indispensable for TCs self-proliferation and cell-cell network forming.
E4F1 controls mammalian embryonic and somatic cell proliferation and survival, and is a key posttranslational regulator of p53, which modulates its effector functions involved in cell growth arrest or apoptosis [51,52]. The low expression of E4F1 in TCs has a positive effect on cell proliferation and differentiation and maintains the viability and activity of TCs in tissues. Programmed cell death 2 (PDCD2) is a highly conversed protein and expressed in Table 9 The number of genes specifically up-or down-regulated in pulmonary telocytes, as compared with other cells respectively in chromosome 17 embryonic and adult tissues widely. The transfection of a construct expressing PDCD2 could induce apoptosis in human cell lines [53]. Equally, TCs down-regulate the PDCD2 to balance apoptosis and proliferation to maintain the homeostasis.

Conclusion
The study globally analyzed the variation of genes in chromosome 17 and 18 among pulmonary TCs, MSCs, Fbs, ATII, ABCs, PACs, T-BL, and T-LL for the first time.
Approximately 15% and 12% genes in chromosomes 17 and 18 were identified as TCs-specific genes. The specific up-regulated genes, i.e. Mapk14, Trem2, MCFD2 and Dpysl3, and specific down-regulated genes, i.e. E4F1 and PDCD2, in chromosome 17 and 18 made us have a deeper insight into biological features and functions of TCs. It has been found that TCs present morphological contact with immune cells to form a cellular interaction network to participate in immune regulation [19]. Our study revealing variation of immune-associated genes in chromosome 17 and 18 gave an additional support to the essential role of TCs in immune surveillance and immune homeostasis which may protect from immune disorder in acute and chronic pulmonary diseases. TCs also played a vital role in tissue proliferation, differentiation and regeneration to maintain the tissue homeostasis.

Reviewer 1: Dragos Cretoiu
This topic is original and the study provides new cues within the field of the telocytes (TCs), a distinct type of stromal cells. There are some minor issues which, if addressed, could improve the manuscript. 1) Introduction -There are more papers already published regarding the presence of telocytes in different organs, which might be worth mentioning. Some characteristics of the immunophenotype and ultrastructure should also be described.
Authors' response: We have now described the presence of TCs in different organs and the characteristics of the immunophenotype and ultrastructure in introduction paragraph.
The aim of the study is not clearly stated and therefore a strong justification for the purpose of this study must be included. Also, some discussion about the chromosome 2 and 3 gene expression profile in TCs should be mentioned.
Authors' response: We revised and highlighted the aim of our study to investigate features and patterns of TCs-specific gene profiles and explore potential function of TCs by focusing in chromosomes 17 and 18. It was not only the first time but also a new method to mine the TCs-specific gene profiles in chromosome 17 and 18. We now also have made a discussion about the study for the chromosome 2 and 3 gene expression profiles in TCs, which had not been published before we submitted this article.
2) Results -It would be useful for the readers to describe name of gene (or the encoded protein) at least for the most significant ones.
3) Conclusion -I would suggest that this paragraph should be re-written pointing out the specific novelty of this study and the major findings leading to some functional hypotheses. Anyway, phrases like "Data from the present study demonstrated TC-specific genes in chromosomes 17 and 18, although the mechanism of TCs-specific genes in biological process of TCs needs to be furthermore explored" must be removed since is hampering the clarity and the significance of the study. TCs diversity according to organ location is quite intriguing, even considering the proposed roles of this cell subset in immune surveillance/tissue homeostasis. The authors should comment more on this.
Authors' response: We thank the reviewer's advice and have re-written the conclusion paragraph to highlight the specific novelty of our study and TCs-specific genes identification with its potential function in chromosomes 17 and 18. Also, negative phrases like "Data from the present study demonstrated TC-specific genes in chromosomes 17 and 18, although the mechanism of TCs-specific genes in biological process of TCs needs to be furthermore explored" have been removed.
Besides, TCs was isolated from murine lung tissue in this study. We have described the proposed roles of TCs in immune surveillance/tissue homeostasis which may protect from immune disorder in acute and chronic pulmonary diseases. We think it's really meaning to differentiate the proposed roles of TCs subsets from different organ location in further study, as reviewer suggested.

4) An
English language revision is mandatory. Many sentences should be rephrased. For example "The novel feature of telocytes under TEM has extremely long TPs with podomers and podoms, to contact with neighboring or distant effectors like axinal of neuron".
Quality of written English: Needs some language corrections before being published.
Authors' response: We have revised the article by an English native specialist. I hope you are satisfied with the revised version of the manuscript and hope it suitable for the publication. Although telocytes (TCs) have been found in a variety of organs, TCs-specific biomarkers still need to be evaluated. This manuscript studied the gene expression profiles of murine pulmonary TCs and compared their profiles with a variety of cell types, including mesenchymal stem cells, fibroblasts, alveolar type II cells, airway basal cells, proximal airway cells, CD8+ T cells from bronchial lymph nodes, and CD8+ T cells from the lung. In the study, genes of chromosome 17 and 18 were extracted and analyzed using bioinformatics tools, in order to identify TCs-specific genes and their functional networks. They found that 16 and 10 of TCs-specific genes were upregulated, and 68 and 22 were down-regulated in the chromosome 17 and 18, respectively. Particularly, Mapk14, Trem2, MCFD2 and Dpysl3 were up-regulated, whereas the E4F1 and PDCD2 were down-regulated in TCs. Their data demonstrated that the differential expression of subset of genes in chromosomes 17 and 18 were unique features of pulmonary TCs, and they might be used as biomarkers to distinguish TCs from the other type of cells. Their findings were also suggestive of that TCs might play critical roles in immune surveillance and other intracellular functions.
Quality of written English: Acceptable. Authors' response: We appreciated Dr. Qing Kay Li's encouraging comment on this article.