Surfactant protein A (SP-A) variants and sex-specific regulation of gene expression networks in alveolar macrophages in response to Klebsiella pneumoniae-induced lung infection

Surfactant protein A (SP-A) in addition to its surfactant-related functions interacts with alveolar macrophages (AM), the guardian cells of innate immunity, and regulates many of its functions under basal condition and in response to various insults, such as infection and oxidative stress. The human SP-A locus consists of two functional genes, SFTPA1 and SFTPA2, and one pseudogene. The functional genes encode human SP-A1 and SP-A2 proteins, respectively, and each has been shown to have several genetic variants. It has been shown that SP-A variants differ in their ability to regulate lung function mechanics and survival in response to bacterial infection. Here, we investigated the effect of hSP-A variants on the AM gene expression profile in response to K. pneumoniae (Kp) infection. We used four humanized transgenic (hTG) mice that each carried SP-A1 (6A2, 6A4) or SP-A2 (1A0, 1A3), and KO. The gene expression profiling of AM was performed after 6 h post-infection. We found: a) significant sex differences in the expression of AM genes between males and females; b) in response to infection, 858 (KO), 196 (6A2), 494 (6A4), 276 (1A0) and 397 (1A3) genes were identified (P < 0.05) and some of these were differentially expressed with ≥ 2 fold, specific to either males or females; c) significant gene-specific differences between the SP-A1 and SP-A2 variants; d) via Ingenuity Pathway Analysis (IPA) key pathways and molecules were identified that had direct interaction with TP53, TNF, and cell cycle signaling nodes; e) validation of key molecules exhibited significant differences in the expression between males and females; f) IPA for SP-A1 and SP-A2 variants and KO revealed, top diseases and bio-functions for males and females that contained a high number of genes involved in the inflammatory response, immunological disease, infectious diseases, respiratory diseases, cancer, and others. These results reveal for the first time a large number of biologically relevant functional pathways influenced by sex and SP-A variant in response to K. pneumoniae infection. These data may assist in studying molecular mechanisms of SP-A-mediated AM gene regulation and potentially identify novel therapeutic targets for K. pneumoniae infection. Humanized transgenic (hTG) mice that carried SP-A1 (6A2, 6A4), SP-A2 (1A0, 1A3) variant, as well as SP-A knockout (KO) mice were used in this study. They were 12 weeks old. hTG mice were generated on the C57BL/6J SP-A (KO) background. The animals were exposed to K. peumoniae for 6hrs. A group of 4 animals (males, females) were exposed and alveolar macrophages (AM) were isolated after 6 h of recovery. Total RNA was extracted, RNA-seq libraries were generated followed by deep sequencing to identiy mRNA expression. The differentially expressed miRNAs (DEG) between males and females were identified by using the edgeR and the TCC v1.14.0 R package with false discovery rate (FDR) adjusted p value of 0.1 as a significance cutoff.