Impact of K pneumoniae infection and/or exogenous surfactant protein-A2 (1A0) on alveolar macrophage miRNome in SP-A-knockout mice.

Human SP-A1 and SP-A2, encoded by SFTPA1 and SFTPA2 and their genetic variants differentially impact alveolar macrophage (AM) functions and regulation, including the miRNome. single dose of SP-A exogenous treatment of SP-A-KO mice prior to infection, after infection, or at the time of infection significantly improved survival. we investigated the role of exogenous SP-A protein treatment on the regulation of AM miRNome in SP-A-KO mice at the time of infection. Towards this, SP-A-KO male and female mice were infected with K. pneumoniae alone or in combination with exogenous SP-A2 (1A0) protein for 6 h, and the expression levels of AM miRNAs, target mRNAs of the significant miRNAs, and pathways involved were studied. We found (i) significant differences in AM miRNome of KO in terms of sex and exposure; (ii) the expression of the overwhelming majority of miRNA targets in KO males were increased in response to infection and exogenous SP-A2 (1A0) protein treatment at the time of infection; (iii) miRNA-mRNA targets were involved in the pro-inflammatory response, anti-apoptosis, cell cycle, cellular growth and proliferation pathways. These data may assist in studying molecular mechanisms of exogenous SP-A mediated the AM miRNome regulation and potentially identify novel therapeutic targets for K. pneumoniae infection. Overall design: Twelve-week-old, SP-A-knockout (KO) mice, were used in this study. The male and female mice used in this study were raised and maintained in a pathogen-free environment. The females were synchronized with regards to the estrous cycle by group housing and exposure to the bedding from male mice. A total of 28 mice (16 for miRNA analysis and 12 for target gene validation by qRT-PCR analysis) were used in the present study. All the procedures involving animals were approved by The Penn State Hershey Medical Center Institutional Animal Care and Use Committee (IACUC). SP-A (KO) male and female mice (n = 4/group for miRNA analysis and 3/group for qRT-PCR validation) were anesthetized with a mixture of ketamine and xylazine and infected with K. pneumoniae (~ 450 CFU/mouse) in 50 µl of PBS oropharyngeal. SP-A-KO male and female mice were anesthetized and the mice were infected with K. pneumoniae (~450 CFU/mouse) as described above, and/or in combination with 10 µg of purified SP-A2 (1A0) protein expressed by stably transfected CHO cells as described [50] in 50 µl of PBS oropharyngeal. The mice were monitored for 6 h after infection. Mouse AM were obtained from both male and female mice by bronchoalveolar lavage (BAL) at 6 h, after infection alone and/or in combination with SP-A2(1A0) protein.Total RNA extraction from AM cells, library construction, and sequencing were performed. The differentially expressed miRNAs between infection alone and/or in combination with SP-A2 (1A0) protein in 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.