Sexual dimorphism in Hofbauer cells
Keywords: Hofbauer cells
proteomics
sexual dimorphism
single cell isolation
next generation sequencing

Sexual dimorphism is defined as the existing differences between individuals of different genders in features other than gender itself. In addition to well described differences in physical traits, reproductive organs, or hormones the term also indicates the differences which are present in cognition, immunity, and the biological responses to various medical conditions.
Regarding sexual dimorphism in the placenta; although it is known that male foetuses have more severe cases of preeclampsia and cases of gestational diabetes mellitus, on the other hand, female foetuses suffer more HIV infections transmitted from the mother, however, the detailed mechanisms underlying to explain these intriguing facts are unknown.
Hofbauer cells are immune cells located in the placenta that are of fetal origin and can be identified from an early stage of pregnancy - only after 18 days of gestation. They are known to function as macrophages including phagocytosis, antigen presentation, angiogenesis and vasculogenesis, and also known to be involved in several medical conditions during pregnancy such as villitis of unknown etiology and preeclampsia. In spite of their important roles, research focused on Hofbauer cells has been limited, largely due to difficulties in their isolation.
I successfully established a new methodology to isolate Hofbauer cells from whole term placenta with high yield and purity and subjected Hofbauer cells isolated from 6 normal male placentae and 6 normal female placentae to mass spectrometry analysis. From the result of spectrometry analysis, 86 out of 5426 proteins were found to be significantly differentially expressed between male and female placentae (p<0.05). These 86 proteins were bioinformatically analysed utilising STRING® and found to connectively form four different pathways engaged in immunity, mitochondrial function, extracellular vesicle functions and transcriptional regulation function. Results from mass spectrometry were validated further using western blotting, which corroborated the proteomics differences.
Furthermore, I extracted RNAs from the cells that were subjected to proteomics and next-generation sequencing was performed. Of 6844 included transcripts, 26 transcripts were significantly up-regulated in male HBCs samples and 23 transcripts were significantly up-regulated in female HBCs samples (adjusted p value<0.05). To identify the signalling pathways between differentially expressed genes, results from RNA-sequencing data was applied to KEGG (Kyoto Encyclopedia of Genes and Genomics) and “cytokine- cytokine receptor interaction pathway” was most related to the functions of Hofbauer cells as immune cells and had the smallest p value (0.001) and adjusted p value (0.073). From the results, it was clear that although there are some genes which are related to immune functions and are located on X chromosomes, such as IL2RG, are highly up-regulated in female HBCs, male HBCs manage their immunocompetence by regulating the expressions of chemokines, chemokine receptors and several cytokines, cytokine receptors including IL-11, IL23A, IL-1 (IL1A, IL1B, IL1RN and IL1F5), IL22RA1, IL34 and IL2RB, as well as members from the TNF and TGF-β family. Some of them are heavily involved in the pathogenesis of pregnancy related conditions represented by preeclampsia and GDM.
In summary, HBCs exhibit significant differences in proteomic and genetic signature in male and female placentae. It is likely these differences confer functional alterations in immune cell function which may impact a number of critical functions.