Clustering Analysis of Splenocyte MicroRNAs in Pig Reveals the Key Signal Regulators in Immune Modulation of the Host During the Acute and Chronic Toxoplasma Gondii Infection
Authors: Hou Zhaofeng , Zhang Hui , Xu Kangzhi , Zhu Shifan , Wang Lele , Su Dingzeyang , Liu Jiantao , Su Shijie , Liu Dandan , Huang Siyang , Xu Jinjun , Pan Zhiming , Tao Jianping 
Published: November 2021

Abstract
Background: Toxoplasma gondii is an obligatory intracellular protozoan parasite that can cause a geographically widespread zoonosis. Our previous splenocyte microRNA profiles analyses of pig infected with T. gondii revealed that the coordination of a large number of miRNAs regulates the host immune response during infection. However, the functions of more miRNAs involved to the immune regulation during T. gondii infection are not yet known.Methods: Clustering analysis was performed by K-means, self-organizing map (SOM) and Hierarchical clustering, respectively, to obtain miRNA groups with the similar expression patterns. Then, the target genes of miRNA group in each subcluster were further analyzed for function enrichment by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway to recognize the key signaling molecules and the regulatory signatures of the innate and adaptive immune responses of the host during T. gondii infection.Results: A total of 252 miRNAs were successfully divided into 22 subclusters by K-means clustering (named by K1~K22), 29 subclusters by SOM clustering (named by SOM1~SOM29) and 6 subclusters by Hierarchical clustering (named by H1~H6) based on their dynamic expression levels in the different infection stages. A total of 634, 660 and 477 GO terms, 15, 26 and 14 KEGG pathways, and 16, 15 and 7 Reactome pathways were significantly enriched by K-means, SOM and Hierarchical clustering, respectively. Of note, up to 22 miRNAs mainly showing the downregulated expression at 50 DPI were identified into one subcluster (namely subcluster H3-K17-SOM1) through the three algorithms. Functional analysis revealed that a large group of immunomodulatory signaling molecules were controlled by the different miRNA groups to regulate multiple immune processes, for instance, IL-1-mediated cellular response and Th1/Th2 cell differentiation partly depending on Notch signaling transduction for subclusters K1 and K2, innate immune response involving to Neutrophil degranulation and TLR4 cascade signaling for subcluster K15, B cell activation for subclusters SOM17, SOM1 and SOM25, leukocyte migration and chemokine activity for subcluster SOM9, Cytokine-cytokine receptor interaction for subcluster H2, and interleukin production, chemotaxis of immune cells, Chemokine signaling pathway and C-type lectin receptor signaling pathway for subcluster H3-K17-SOM1.Conclusions: Clustering analysis of splenocyte microRNAs in pig reflected the key regulatory properties of subcluster miRNA molecules, as well as the important features in the immune regulation induced by acute and chronic infections of T. gondii. These results contribute to new insight into the identification of physiologic immune responses and maintenance of tolerance in pig spleen tissues during T. gondii infection.