Humanized transgenic (hTG) mice carrying both gene variants, SP-A1/SP-A2 (6A²/1A⁰, co-ex), 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 (59). The animals used in this study were raised and maintained in a pathogen-free environment, at the Penn State College of Medicine animal facility as described previously (43). Both males and females were used. The females were synchronized with regard to the estrous cycle as described previously (43). A total of 44 mice (32 for miRNA analysis and 12 for qRT-PCR analysis) were used in the present study. All the procedures were approved by The Penn State Hershey Medical Center Institutional Animal Care and Use Committee (IACUC).

The animals were exposed to FA or O₃ in parallel as described previously (60). A group of 4 animals (males, females) were exposed to FA or O₃ for 3 h, and alveolar macrophages (AM) were isolated after 4 h of recovery as described (61).

Total RNA was extracted from AMs using mirVana kit (#AM1560, Ambion, Waltham, MA). The extracted RNAs were quantified and quality checked using a BioAnalyzer RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA). Small RNA-seq libraries were generated by NEXTflex Small RNA Library Prep Kit v3 for Illumina (BioO Scientific, Austin, TX), followed by deep sequencing on an Illumina HiSeq 2500 as per the manufacturer's instructions. Briefly, 1-2 ng of total RNA was ligated with chemically modified 3′- and 5′- adapters that can specifically bind to mature micro RNAs, followed by reverse transcription and PCR amplification. Unique index sequence tags were introduced during PCR to enable multiplexed sequencing. Each library was assessed for the presence of desired micro RNA population and approximate library quantity by Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled libraries were denatured and loaded onto a TruSeq Rapid flow cell on an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe according to the manufacturer's instructions. De-multiplexed sequencing reads passed the default purify filtering of Illumina CASAVA pipeline (released version 1.8) and were quality trimmed/filtered using The FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit). The filtered reads were further trimmed with both 5′ and 3′ adapter sequences and subjected to Chimira suite to align and count miRNA expression (62). The differentially expressed miRNAs (DEG) between FA to O₃, males and females were identified by using the edgeR (63) and the TCC v1.14.0 R package (64) with false discovery rate (FDR) adjusted p-value of 0.1 as a significance cutoff.

We successfully identified 307 miRNAs with good correlation between mice (3 out of 4, Supplementary Table 1). We used two different parameters (methods) to analyze the identified miRNA expression (fold change). (a) The expression levels (fold change) of miRNAs from co-ex (FA to O₃ for males and females) and KO (FA to O₃ for males and females) were analyzed and differentially expressed miRNAs in co-ex and KO males and females were identified in response to FA or O₃. (b) The changes in miRNA expression in co-ex were calculated by normalizing to KO, i.e., the level of expression of each individual experimental miRNA in co-ex males and females exposed to FA or O₃ was divided by the corresponding miRNA in the KO. Next, the differentially expressed miRNAs between co-ex males and females were determined by dividing a specific individual male miRNA by the corresponding female miRNA (Supplementary Table 1).

The expression levels of CCND1, CCND2, CCNE1, CDK7, IL-6, IL-10, TLR2, TLR3, STAT3, MYD88, IL-4, IL2RG, EGR2, PTEN, TNFSF12, MDTH, JUN, E2F3, BCL2, TNF, CDK2, SMAD2, MMP2, ARG1, AKT1, PPARA, and MYC genes at mRNA level in the female and male co-ex and KO AM, were validated by qRT-PCR as described previously (41). The RT2 qPCR Primer assays were purchased from Qiagen. The AM cell samples [3 animals/treatment (FA or O₃)] were analyzed in triplicates/animal and quantified relative to GAPDH mRNA.

Statistical differences between miRNA expression levels (fold change) in FA vs. O₃ and male vs. female were evaluated by two-tailed t-test and nonparametric Mann-Whitney test. For multiple comparison analysis one-way analysis of variance (ANOVA) was employed followed by Bonferroni multiple comparisons. The p-values <0.05 were considered to be significant. All the data points are means ± standard deviation, and analyses were performed using Graph-Pad Prism software version 5.0 (Graph-Pad Software, San Diego, USA).

We further looked into the AM miRNAs, whose expression compared to control miRNAs is altered ≥2 fold in response to FA or O₃ from co-ex and KO males and females, and compared them between males and females.

We performed IPA to further understand the role of significantly changed miRNAs in co-ex males and females under the studied conditions. After Bonferroni correction, expression of miRNAs in co-ex females exhibited significant differences in response to OxS, but not in males (Figure 2C). However, the IPA analyses before and after the Bonferroni correction were identical because the miRNA data input in IPA was same. We were able to identify biological functions and regulatory network targets of these differentially expressed miRNAs. The targets shown in Figure 4 are involved in anti-apoptosis, cell cycle, cellular growth and proliferation, as well as pro-inflammatory response are affected by differential expression of miRNAs in response to O₃ exposure (Supplementary Table 1). These targets include CCND1, CCND2, CCNE1, CDK7, IL-6, IL-10, TLR2, TLR3, STAT3, TNFSF12, MYD88, IL-4, IL2RG, EGR2, PTEN, MDTH, JUN, E2F3, BCL2, TNF, CDK2, MYC, SMAD2, MMP2, ARG1, AKT1, and PPARA mRNAs. The miRNAs that were changed significantly in co-ex females and males in response to O₃ exposure and their targets are listed in Table 1.

In general, we observed largely a downregulation of miRNAs in both males and females and upregulation of their targets in co-ex males but not in females (Figures 4A,B). For example, a significant downregulation of miR-191-5p, miR-155-5p, and miR-92a-3p expression was observed in response to O₃ in both co-ex males and females but their target IL-6 mRNA is upregulated in males but downregulated in females. Though most of the miRNAs are downregulated in both males and females, the expression of miR-340-5p, miR455-3p, miR-143-3p, and miR-503-5p in females, and miR1195 and miR101b-3p in males is upregulated (Figures 4A,B).

To measure the expression levels of target genes by differentially expressed miRNA in response to FA or O₃, we performed qRT-PCR analysis on AM cell samples isolated from co-ex and KO males and females after FA or O₃ exposure (Figure 5).

In response to OxS, the expression level of all target genes studied, with the exception of SMAD2 that remained similar in both sexes under OxS, was significantly upregulated in co-ex males compared to females (Figure 5A, Supplementary Table 2). These included CCND1, CCND2, CCNE1, CDK7, IL-6, IL-10, TLR2, TLR3, STAT3, MYD88, IL-4, IL2RG, EGR2, PTEN, TNFSF12, MTDH, JUN, E2F3, BCL2, TNF, CDK2, MMP2, ARG1, AKT1, PPARA, and MYC. A similar analysis in KO males and females resulted in significant upregulation of CCND1, CCND2, CCNE1, CDK7, IL-6, TLR2, TLR3, STAT3, MYD88, PTEN, TNFSF12, MTDH, JUN, E2F3, TNF, CDK2, SMAD2, and AKT1 in KO males (Figure 5B, Supplementary Table 2), whereas the expression level of IL-10, MMP2, ARG1, and PPARA was significantly upregulated in KO females (Figure 5B). The levels of IL-4, IL2RG, EGR2, BCL2, and MYC remained similar in both sexes under OxS (Figure 5B).

In Summary the collective information of the significantly changed miRNAs and their targets indicate sex specific differences. Females (unlike males) largely failed to upregulate the target genes as expected when regulatory miRNAs are downregulated.

The expression of miR-191-5p, miR-155-5p and miR-92a-3p were decreased significantly in response to O₃ in both co-ex males and females (Figures 4A,B). These miRNAs target pro- and anti-inflammatory IL-6 cytokine (41, 65–67). The IL-6 level decreased significantly in co-ex females, whereas this increased significantly in co-ex males (Figures 4A,B) compared to the control mRNA (GAPDH). The latter is consistent with our previous observation with SP-A2 males in response to OxS (41).

IL-6 plays a crucial role in the activation of STAT3 (68–70). In response to OxS, STAT3 gets phosphorylated which results in the activation of genes involved in inflammation and injury (71). miR-17-5p, and miR-1195 that were significantly altered in response to OxS, are known to interact with STAT3 (Figures 4A,B). In co-ex females and males miR-17-5p is downregulated whereas miR-1195 is downregulated in co-ex females but upregulated in co-ex males. Although, the downregulation of these miRNAs in females should have resulted in an increase in the expression of STAT3, the opposite was observed, indicating that either the regulation of STAT3 by these (and perhaps other) miRNAs is dysfunctional or mechanisms other than miRNAs are involved. The downregulation of STAT3 in females was associated with decreased levels of TNF, TNFSF12, IL-6, IL-10, and IL-4 (Figure 4B). In contrast, in male co-ex, although miR-17-5p is decreased, and miR-1195 is increased, STAT3, which is target for both, is increased, as well as the levels of the pro-inflammatory cytokines were increased as one may expect. Also in males the levels of a number of target genes (ARG1, EGR2, and IL2RG) that contribute, via STAT3, to the upregulation of pro-inflammatory cytokines were upregulated. Although, the details of the underlying mechanisms are unknown currently, these data show a disconnect between miRNA expression and target gene expression in females. The emerging picture is that pro-inflammatory cytokines are downregulated in females and upregulated in males (Figure 4A).

Toll like receptors (TLRs) are a family of membrane bound proteins that recognize pathogen-associated molecular patterns and mediate innate immune response (72). SP-A differentially regulates TLR expression (73). We found four co-ex regulated miRNAs (miR-92a-3p, miR-125b-5p, miR-155-5p, and miR-191-5p) that target TLR2, and TLR3. The expression of these miRNAs is significantly downregulated in our study in both males and females, and this is associated with increased mRNA levels of TLR2 and TLR3 in co-ex males but a decrease in co-ex females. Both miR-125b-5p and miR-155-5p are shown in several studies to regulate TLRs (74–77). TLR2 engages the ubiquitous intracellular adaptor MyD88 (myeloid differentiation primary response 88) and TLR3 engages TRIF (TIR-domain containing adaptor protein). In the current study, the level of miR-125b-5p, miR155-5p and miR130b-5 targeting MyD88 are downregulated and the mRNA level of MyD88 is upregulated in co-ex males but decreased in co-ex females. The involvement of TLR2, TLR3, and activation of MyD88 in co-ex males may result in the recruitment of other genes involved in the activation of NF-kB (78, 79), and this may result in the transcription of pro-inflammatory genes, such as TNF, TNFSF12, IL-10, and IL-4, as observed in the present study (Figures 4A,B). Previous studies have provided evidence that SP-A activates NF-kB (80) either through accumulation of inhibitory IkBa (81, 82) or via direct interaction with TLR2 and TLR4 (73, 80, 83–85) or SIRPα and CD91 (86). It has also been shown that, SP-A is unable to activate NF-kB in response to O₃ as assessed by the lack of changes in the nuclear p65 subunit and the cytoplasmic IkBa levels as it would have been expected in the classical NF-kB pathway (87). Moreover, it has been shown that decreased levels of MTDH expression attenuate NF-kB signaling (88) and that TNFSF12 regulates NF-kB activity (89). Upregulation of TNFSF12 and MTDH as it occurs in co-ex males in the current study may alter NF-kB signaling, enhance its translocation to the nucleus to facilitate the transcription of pro-inflammatory genes in co-ex males but not in females. The present data support the idea that NF-kB and STAT3-mediated pathways play a role in the pro-inflammatory gene expression in co-ex males, and that these pathways in females are compromised in response to OxS.

The expression of two miRNAs miR-16-5p and miR-21a-5p (TargetScan) that bind BCL2 (90–93) was significantly downregulated in both males and females in response to OxS and the BCL2 mRNA levels were increased in co-ex males but decreased in females. An increase in BCL2 is likely to result in the inhibition of apoptosis and cell proliferation in males, but not in co-ex females (Figures 4A,B). These findings together indicate that OxS differentially affects anti-apoptotic pathways in co-ex males and females, and that females seem to have a disconnect between miRNA expression and target gene expression.

A number of miRNAs whose expression was for the most part downregulated significantly in response to OxS were predicted to target CCND1, CCND2, CCNE1, CDK2, CDK7, and E2F3 (Figures 4A,B). miR-16-5p and miR-17-5p, predicted to bind CCND1, CCND2, CCNE1, and E2F3 mRNAs (TargetScan), have been shown in several studies that these genes are regulated by these two miRNAs (94–97). The expression of all the target genes followed a similar pattern as described above, there was an increase in males and decrease in females. These genes are involved in cell cycle, and growth and proliferation, indicating that ozone differentially affects expression of molecules involved in cell cycle and proliferation pathways in co-ex male and female mice. Of interest significant differences in survival have been observed with females being more affected than males in several lung diseases (53–56). In our animal studies, we observed a significant difference in survival after infection and O₃ exposure. Females were more susceptible to oxidative stress than males and exhibited lower survival (7, 9). Sex hormones were shown to play a role in the observed survival differences (57).

AKT, PPARA, PTEN, and JUN involved in the MAPK pathway were significantly upregulated in co-ex males but not in females in response to OxS. The miRNAs that targeted these genes, miR-21-5p, miR-103-3p, let-7a-5p, miR-130b-3p, miR-151-5p, and miR-92-3p, were significantly downregulated in co-ex males and females (Figures 4A,B). The upregulation of MAPK pathway genes has the potential to regulate the genes involved in cell cycle, growth and proliferation (as shown in Figure 4), as well as pro-inflammatory and anti-apoptotic genes (not shown) in co-ex males but not in co-ex females in response to OxS.

We have previously studied the effect of a single, SP-A1 or SP-A2 gene, on the AM miRNome after OxS, and found that SP-A2 (but not SP-A1) had a significant impact on AM in males (41). To gain further insight into the contributions of SP-A2 vs. the co-ex on AM miRNome, we compared the data from two studies. This comparison is shown diagrammatical in Figure 6. A number of observations are readily evident. Broadly these show (1) the miRNAs that changed (≥2 fold) significantly in response to OxS were decreased in male SP-A2 and co-ex male and female after normalization to KO. (2) In males (SP-A2 & co-ex) the expression of all validated target genes (except SMAD2), of the significant miRNAs, identified by IPA, is increased but decreased in co-ex females. (3) The common pathways include pro-inflammatory response, and anti-apoptosis. However the SP-A2 males include ROS-homeostasis processes and the co-ex include cell cycle, growth, and proliferation processes. These data show that SP-A2 alone or in combination with SP-A1 may regulate the expression of pro-inflammatory genes via the STAT3-NF-kB pathway and anti-apoptotic genes in response to OxS (Figure 4A). Furthermore, this indicated that these pathways may in part (if not in their entirety) be regulated or driven by SP-A2 and that the presence of SP-A1 does not negatively affect this. However, although SP-A1 by itself did not have any significant effect on AM, in presence of SP-A2 is shown to regulate genes involved in cell cycle, growth and proliferation, whereas SP-A2 alone regulates genes involved in homeostasis of ROS (41).

Although the data of this study are largely in line with our previous observations, the current study has a few limitations: (a) the study is carried out at single time point, (b) validation analysis was performed only for genes which are targeted by significantly changed miRNAs in co-ex males and females after normalizing to KO, and we did not look at the protein levels of the targeted mRNAs, (c) we did not study the molecular mechanisms of the identified pathways, (d) we did not differentiate the impact of varying amount SP-A1 and SP-A2 on miRNA expression in response to OxS. It has been shown that the ratio of SP-A1 to SP-A differs in lung diseases (23, 24), and this may have functional consequences given the varying activities of SP-A1 and SP-A2, (e) we did not study the impact of gonadectomy on the expression of AM miRNAs from co-ex and KO males and females. However, we have previously studied the effect of a single, SP-A1, or SP-A2 gene, on the AM miRNome after OxS, and found that SP-A2 (but not SP-A1) had a significant impact on AM in males (41). In this study, we observed that the regulation of the miRNome of the SP-A2 male mice compared to that of female mice in response to OxS is significantly altered after gonadectomy (41). It has also been shown that different stages of the estrous cycle have significant impact on the lung miRNA expression (98). In addition, a role of sex hormones on survival after Klebsiella pneumoniae infected wildtype (SP-A) mice with or without exposure to ozone has been observed (57). This study indicated that (1) after removal of gonadal hormones, differences in survival in animals after infection, and oxidative stress are minimized in males, and eliminated in females. (2) Treatment of gonadectomized females with DHT and males with E2 resulted in a similar kind of survival compared to the intact male and female animals, respectively. This further supports a role of DHT and E2 in survival after infection and oxidative stress. Based on these observations, we speculate that in co-ex males and females sex hormones play a significant role in the regulation of AM miRNome. However, the result of this study advances our knowledge of the differential impact of co-expressed SP-A1/SP-A2 and sex on the AM miRNome.

We postulate that in males in response to OxS, SP-A2 via its activity in ROS-homeostasis provides some protection from the injurious ROS in its microenvironment. Whereas, the co-ex males via cell cycle, growth, and proliferation process may promote cellular recovery, perhaps a more sustained recovery. We further postulate that in co-ex females the disconnect between the downregulation of miRNAs and the expression of their target genes is responsible or contributes to the reduced ability in females to enhance phagocytosis by AM as well as to the poorer survival we observed in females after OxS and infection (9). Although the details of the underlying mechanisms are unknown, the AM miRNome appears to play a significant role in OxS.

In summary, SP-A1/SP-A2 (6A²/1A⁰, co-ex) regulate miRNAs that play a role in pathways involved in inflammatory responses, anti-apoptosis, cell cycle, growth, and proliferation. Both gene products are needed to alleviate the deleterious effects of OxS in males and promote cellular recovery. However, in females even in the presence of both SP-A1 and SP-A2 genes, expression of target genes to mitigate the OxS injury is lacking, indicating that other hormone dependent mechanisms are involved. Because the innate immune molecules, SP-A1 and SP-A2 appear to play a differential role in the outcome of males and females after OxS, the potential impact on health of innate immune genetics should be considered separately in males and females.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.