Study on the cytokines related to SARS-Cov-2 in testicular cells and the interaction network between cells based on scRNA-seq data

Abstract

Inflammatory cytokine storms (CS) in COVID-19 patients are associated with elevated levels of 13 specific cytokines, potentially impacting male fertility by causing testicular cell damage and disrupting the immune microenvironment. Some patients present with scrotal discomfort and orchitis. However, few studies have explored cytokine expression in testicular cells and their role in cell-to-cell communication. In this study, we integrated single-cell sequencing data sets of testicular cells, annotating 20 cell clusters using marker genes and the Human Cell Landscape database. We constructed cell pseudo-chronological trajectories, hub genes, and analyzed the cytokine interaction network between sperm cells using CellChat. Our findings identified 12 types of testicular cells, with four cytokines (IL8, CCL2, CCL3, and TNF) potentially involved in immune processes. Pseudo-chronological trajectory analysis indicated IL8 and CCL3's essential roles in testicular macrophages and endothelial cell development, affecting the immune microenvironment. We determined eight key cytokines (IL1, IL2, IL4, IL6, CCL, CSF3, TNF, and IFN-II) functions in cell interaction networks. Network analysis of exogenous cytokines directly acting on testicular cells showed IL2 potentially affecting all testicular cells, suggesting a vital role in cell communication. This research offers valuable insights into CSs effects on testicular cells and their potential impact on male fertility during COVID-19 infection.

Keywords: COVID-19; SARS-CoV-2; cytokine; single-cell RNA-Seq; testicular cell.

Figure 1

Single-cell sequencing data integrated by Seurat: (a) distribution of 10 data sets, (b) 6 cell groups merged from the data sets, (c) an integrated data set containing 20 cell clusters, and (d) the stacked graph showing the proportion of six cell groups in the cell clusters.

Figure 2

Identification of testicular cell clusters and cytokine expression: (a) heat map of cell clustering marker genes, (b) heat map of the correlation of each cell cluster, (c) the distribution of 12 cell clusters, (d) the heat map of the six cytokines selected in the integrated data set, and (e) the distribution of 6 cytokines in each cell cluster.

Figure 3

Pseudotime time analysis of testicular cells: (a) Scatter plot of gene expression levels. The black dots are genes that are abnormally mutated between cells for cluster analysis. (b) The pseudo-chronological analysis of testicular cells shows that a branch appears during the development of testicular cells. (c and d) The results of pseudo-time sequence analysis show that there are three states in total. (e and f) The specific positions of 20 cell clusters in state, among which Macrophage and Endothelial cells are mainly enriched in state 1. (g) Heat map of the differential genes among the three state cells. The CCL4 and IL8 genes show a trend from high expression to low expression in state 1. (h and i) The expression of CCL4 and IL8 genes in the developmental trajectory of testicular cells.

Figure 4

Key cytokines that play a central role in testicular cells. (a) According to function classification, eight key cytokines out of 13 cytokines play a role in testicular cells (circled part). (b) IL1 single pathway network of testicular cells, (c) IL2 single pathway network of testicular cells, (d) IL4 single pathway network of testicular cells, (e) IL6 single pathway network of testicular cells, (f) CCL single pathway network of testicular cells, (g) TNF single pathway network of testicular cells, (h) CSF3 single pathway network of testicular cells, and (i) IFN-Ⅱ single pathway network of testicular cells.

Figure 5

Recognition of cytokines that can affect all testicular cells: (a) The mulberry chart showing that all testicular cells are divided into five patterns according to the functional classification, and IL2 directly acts on the cells of pattern 5. (b) The bubble chart showing the testicular cells that eight key cytokines directly affect. The imported IL2 can directly affect all testicular sperm cells. (c) The network diagram showing that the IL2 single pathway between testicular cells can affect all testicular cells. (d) The heat map showing that the elongated spermatid and sperm1 participate in the sender, receiver, and mediator of the IL2 signaling pathway. (e) The bar plot showing the relative contribution of the ligand-receptor pairs for cell interaction.