SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved

Abstract

Background: Human innate host defense molecules, surfactant protein A1 (SP-A1), and SP-A2 differentially affect the function and proteome of the alveolar macrophage (AM). We hypothesized that SP-A genes differentially regulate the AM miRNome.

Methods: Humanized transgenic mice expressing SP-A1 and SP-A2 were subjected to O₃-induced oxidative stress (OxS) or filtered air (FA), AMs were isolated, and miRNA levels were measured.

Results: In SP-A2 males, we found significant changes in miRNome in terms of sex and sex-OxS effects, with 11 miRNAs differentially expressed under OxS. Their mRNA targets included BCL2, CAT, FOXO1, IL6, NF-kB, SOD2, and STAT3. We followed the expression of these transcripts as well as key cytokines, and we found that (a) the STAT3 mRNA significantly increased at 4 h post OxS and returned to baseline at 18 h post OxS. (b) The anti-oxidant protein SOD2 level significantly increased, but the CAT level did not change after 4 h post OxS compared to control. (c) The anti-apoptotic BCL2 mRNA increased significantly (18 h post OxS), but the levels of the other transcripts were decreased. The presence of the SP-A2 gene had a protective role in apoptosis of AMs under OxS compared to mice lacking SP-A (knockout, KO). (d) Pro-inflammatory cytokine IL-6 protein levels were significantly increased in SP-A2 mice compared to KO (4 and 18 h post OxS), which signifies the role of SP-A2 in pro-inflammatory protein expression. (e) SOD2 and CAT mRNAs changed significantly in OxS indicating a plausible role of SP-A2 in the homeostasis of reactive oxygen species. (f) Gonadectomy of transgenic mice showed that sex hormones contribute to significant changes of the miRNome expression.

Conclusions: We conclude that SP-A2 influences the miRNA-mediated sex-specific differences in response to OxS. In males, these differences pertain to inflammatory, anti-apoptotic, and anti-oxidant pathways.

Keywords: IL-6; Ozone; STAT3; Sex differences; Surfactant protein A.

Fig. 1

Volcano plots indicating the statistical significance of SP-A1 AM miRNome expression levels compared to SP-A KO under FA or O₃ exposure for males and females. The x-axis plots the log₂ of the fold changes, while the y-axis plots the −log₁₀ of their p values based on t test of the replicate raw Ct data (see the “Methods” section). Each plot has 3 vertical lines. The middle vertical line that is graded corresponds to 0 changes. The lines on either side represent ≥ 2-fold differences. Dots in the volcano plots above the blue horizontal line identify fold changes with statistical significance of at least the Bonferroni corrected p < 0.0166. The red and green dots represent miRNAs that were upregulated ≥ 2-fold and downregulated ≤ − 2-fold, respectively, compared to KO. Black dots signify miRNAs that were regulated less than 2-fold times (i.e., − 2 ≤ x ≤ 2; x is the fold change). a Male SP-A1 mice compared to KO exposed to FA. b Female SP-A1 mice compared to KO exposed to FA. c Male SP-A1 mice compared to KO after O₃ exposure. d Female SP-A1 mice compared to KO after O₃ exposure. The shaded gray areas show the differences in the miRNAs that are highly and significantly regulated between the two sexes and the two conditions compared to KO. The shaded gray areas with a broken outline are to highlight the significantly changed miRNAs in SP-A1 male O₃ exposed mice and SP-A2 male O₃ exposed mice (Fig. 2). The x-axis on a is broken to allow comparison between all panels

Fig. 2

Volcano plots (as described in Fig. 1) indicating the statistical significance of SP-A2 AM miRNome expression levels compared to SP-A KO under FA or O₃ exposure for males and females. a Male SP-A2 mice compared to KO exposed to FA. b Female SP-A2 mice compared to KO after FA exposure. c Male SP-A2 mice compared to KO after O₃ exposure. d Female SP-A2 mice compared to KO after O₃ exposure. The shaded gray areas with a broken outline compare the significantly changed miRNAs in SP-A2 male O₃ exposed mice and SP-A1 male O₃ exposed mice (Fig. 1)

Fig. 3

Venn diagrams showing the distribution of differentially expressed SP-A1 and SP-A2 AM miRNAs that changed ≥ 2-fold compared to SP-A KO AM, which are unique or common (overlapping areas) in FA and O₃ exposure. Also, the figure shows the effect of sex on the expression of miRNAs after FA and O₃ exposure. Venn diagrams are showing unique and commonly identified miRNAs among SP-A1 and SP-A2 male and female hTG mice. Out of the total 226 miRNAs identified in male and female SP-A1, 49 identified miRNAs are unique to male and 24 are unique to female; 153 miRNAs are identified in common after O₃ exposure. In the case of SP-A2, out of the 166 miRNAs identified, 48 miRNAs are unique to male and 100 are unique to female; 18 miRNAs were identified in common after O₃ exposure. However, under control conditions, more miRNAs (n = 122) were in common in the SP-A1 male and female mice than in the SP-A2 (n = 24)

Fig. 4

The effect of gonadectomy and OxS on AM miRNA expression profiles of SP-A2 mice. The differentially expressed miRNAs in SP-A2 non-gonadectomized (NGx) and gonadectomized (Gx) mice were identified after normalizing to corresponding NGx and Gx KO. NGx shows the miRNAs (n = 37) that changed significantly (≥ 2-fold) in OxS when males were compared to females. Gx depicts the comparison of Gx values (male vs female) to NGx (male vs female). Out of the same 37 miRNAs (found to have their levels increased in NGx), 2 miRNAs (5.4%) showed a significant increase (≥ 2-fold) and 35 miRNAs (94.59%) showed a significant decrease (≤ 2-fold). Inset depicts the comparison, of the 37 differentially expressed miRNAs, between Gx male and female. Out of 37 miRNAs studied, 17 miRNAs (45.9%) are significantly increased in female (≥ 2-fold), and 7 miRNAs (18.9%) are significantly increased in male (≥ 2-fold)

Fig. 5

Effect of O₃ in males. a mRNA levels of GAPDH, SOD2, CAT, IL-6, STAT3, BCL2, FOXO1, FOXO3, BECN1, IKKβ, and NF-kB-p65 genes were measured in AM of male SP-A2 mice 4 h after O₃ exposure. mRNA levels were measured by qRT-PCR and normalized to GAPDH. STAT3 mRNA levels were significantly increased by 5-fold (p < 0.05), while the levels of other mRNAs did not change significantly compared to FA exposure. Inset depicts protein expression levels of STAT3, NF-kB-p65, FOXO1, CAT, and SOD2 in AM of male SP-A2 mice 4 h after O₃ exposure. Protein levels were measured by Western blot analysis and normalized to GAPDH. b mRNA levels of the above mentioned genes were measured by qRT-PCR 18 h post exposure. In male SP-A2, O₃ exposure significantly increased BCL2 mRNA levels compared to FA (p < 0.05). The mRNA levels of SOD2, CAT, IL-6, FOXO3, BECN1, and IKKβ were significantly decreased compared to FA (p < 0.05). c In KO, mRNA levels of SOD2 and CAT were significantly increased (p < 0.05) 18 h post exposure, while the levels of other mRNAs did not change significantly compared to FA. Data were generated using 3 animals/group, n = 18, and 3 replicate/animal

Fig. 6

Effect of O₃ on IL-6, TNF-α, and IL-1β protein levels in the bronchoalveolar lavage of male SP-A2 and KO mice compared to FA exposure. a Protein levels were measured by ELISA 4 h post exposure. OxS significantly increased IL-6 levels in SP-A2 male mice compared to FA (control) SP-A2 mice (p < 0.05). b Protein levels were measured at 18 h post exposure in SP-A2 and KO. OxS significantly increased IL-6, IL-1β, and TNF-α in SP-A2 mice (p < 0.05) and significantly decreased in KO mice (p < 0.05). Data were generated using 3 animals/group, n = 18 and 3 replicate/animal

Fig. 7

Effect of O₃ on AM apoptosis by TUNEL assay. The TUNEL assay was performed at 18 h post exposure to ozone in SP-A2 and KO mice by using AM cells (70,000 cells/well). Data were generated using 3 animals/group (n = 12) and 4 replicate/animal (p < 0.05)

Fig. 8

Schematic representation for the role of miRNAs in mediating regulation of pathways involved in anti-apoptotic, inflammatory, and reactive oxygen species on AM of SP-A2 mice compared to KO mice in response to O₃. The miRNAs and genes studied in the present study are highlighted with yellow. Up (↑) and down (↓) arrows indicate increase or decrease