Endometrial factors similarly induced by IFNT2 and IFNTc1 through transcription factor FOXS1

<div><p>In ruminants, Interferon tau (IFNT) is the pregnancy recognition protein produced by the mononuclear trophectoderm of the conceptus, and is secreted into the uterine lumen during the peri-attachment period. In our previous study, the high-throughput RNA sequencing (RNA-seq) data obtained from bovine conceptuses during the peri-attachment period identified two <i>IFNT</i> mRNAs, <i>IFNT2</i> and <i>IFNTc1</i>. However, how each of these IFNT variants regulates endometrial gene expression has not been characterized. Using RNA-seq analysis, we evaluated how IFNT2 and IFNTc1 affected transcript expression in primary bovine endometrial epithelial cells (EECs). IFNT treatment induced 348 differentially expressed genes (DEGs); however, there are few DEGs in IFNT2 or IFNTc1 treated EECs, indicating that IFNT2-induced DEGs were similar to those induced by IFNTc1 treatment. In in silico analysis, we identified four IFNT2- and IFNTc1-induced pathways: 1) type II interferon signaling, 2) proteasome degradation, 3) type III interferon signaling, and 4) DNA damage response. We further demonstrated that IFNT2 and IFNTc1 up-regulated several transcription factors, among which forkhead box S1 (<i>FOXS1</i>) was identified as the most highly expressed gene. Furthermore, the knockdown of <i>FOXS1</i> in IFNT2- or IFNTc1-treated EECs similarly down-regulated 9 genes including <i>IRF3</i> and <i>IRF9</i>, and up-regulated 9 genes including <i>STAT1</i>, <i>STAT2</i>, and <i>IRF8</i>. These represent the first demonstration that effects of each IFNT on EECs were studied, and suggest that endometrial response as well as signaling mechanisms were similar between two IFNT variants existed in utero.</p></div

Identification of gene expression induced by IFNTs in EECs.

<p>EECs were incubated without (Ctrl) or with IFNT2 or IFNTc1 (2 x 10⁵ cells/5000 IU/well) for 24 h. RNA was extracted from the EECs and subjected to real-time PCR analysis to determine gene expression related to type II interferon, proteasome degradation, type III interferon, and DNA damage response signaling in Ctrl, IFNT2-, or IFNTc1-treated EECs (n = 3 each group). <i>GAPDH</i> mRNA was used as an internal control for RNA integrity. ^aP < 0.01, ^bP<0.05 vs. Ctrl. Value represent the mean ± SEM from three independent experiments in each treatment.</p

Diagram illustrating the potential role of IFNT through FOXS1 in EECs.

<p>IFNT2 and IFNTc1 bind to their receptor and then activate STAT1 or STAT2. Activated STATs up-regulate FOXS1 expression, which down-regulates STATs expression, resulting in a negative feedback loop between STATs and FOXS1.</p

Transcriptional factors up-regulated by IFNTs in EECs.

<p>Transcriptional factors up-regulated by IFNTs in EECs.</p

Differential gene expression in bovine endometrial epithelial cells treated with IFNT2 or IFNTc1.

<p>(A) Venn diagram shows the number of gene with 1.5-fold changes among Control (Ctrl), IFNT2, and IFNTc1 treatment groups, and right table lists increased or decreased genes in IFNT2 vs. IFNTc1 group, which overlap with Ctrl vs. IFNT2 or Ctrl vs. IFNTc1 group. (B) EECs were incubated without (Ctrl) or with IFNT2 or IFNTc1 (2 x 10⁵ cells/5000 IU/well) for 24 h. RNA was extracted from the EECs and subjected to real-time PCR analysis on mRNA expression with overlapping IFNT2 vs. IFNTc1 group with other groups. <i>GAPDH</i> mRNA was used as an internal control for RNA integrity. Value represent the mean ± SEM from three independent experiments in each treatment. (C) these diagrams show pair plots comparison among Ctrl, IFNT2, and IFNTc1, and density plots in each groups. Figures show correlation coefficient among Ctrl, IFNT2, and IFNTc1.</p

Effects of FOXS1 knockdown on the expression of IFNTs downstream factors.

<p>(A-C) EECs were transfected with non-targeting control (Ctrl: 200 nM) or <i>FOXS1</i> siRNA (#1 or #2: 200 nM) for 48h, and then incubated with IFNT2 (B) or IFNTc1 (C) (2 x 10⁵ cells/5000 IU/well) for 24 h. RNA was extracted from the EECs and subjected to real-time PCR analysis (n = 3 each group). <i>GAPDH</i> mRNA was used as an internal control for RNA integrity. Values represent the mean ± SEM from three independent experiments in each treatment.</p

Genes related to IFNTs-induced enriched pathways in EECs.

<p>Genes related to IFNTs-induced enriched pathways in EECs.</p

Determination of IFNTs’ downstream transcription factors.

<p>EECs were incubated without (Ctrl) or with IFNT2 or IFNTc1 (2 x 10⁵ cells/5000 IU/well) for 24 h. RNA was extracted from the EECs and subjected to real-time PCR analysis to determine the expression of transcription factors in Ctrl, IFNT2-, or IFNTc1-treated EECs (n = 3 each group). <i>GAPDH</i> mRNA was used as an internal control for RNA integrity. ^aP < 0.01, ^bP<0.05 vs. Ctrl. Value represent the mean ± SEM from three independent experiments in each treatment.</p

Increase in complement iC3b is associated with anti-inflammatory cytokine expression during late pregnancy in mice

<div><p>Immunological tolerance between fetal allograft and mother is crucial for pregnancy establishment and maintenance; however, these mechanisms particularly those during the latter part of pregnancy have not been definitively elucidated. The aim of this study was to examine the presence and potential function of innate immunity characteristic to the middle to late pregnancy. We first characterized up-regulated proteins in decidua from day 11 pregnant (P11) mice using 2D-PAGE, followed by MALDI-TOF/MS analysis. These analyses identified increased complement component 3 (C3) and its derivatives in P11 decidua. We then found that in the decidual tissues, <i>C3</i> mRNA increased on P15 and remained high on P19. C3 is converted to C3b and then iC3b by complement component factor I (<i>Cfi</i>) and complement receptor 1-like protein (<i>Crry</i>), both of which were present in P19 placentas. In addition, iC3b proteins and its receptor CR3 (<i>Cd11b/Cd18</i>) in decidual and placental tissues increased toward the latter phase of pregnancy. Moreover, CR3 subunit CD11b protein was predominantly localized to spongiotrophoblast layer in the P19 placenta. Because iC3b is known to induce anti-inflammatory cytokine production, the analysis was extended to examine changes in pro- and anti-inflammatory cytokines, <i>Il12</i>, <i>Il10</i>, and <i>Tgfb1</i>. <i>Il12</i> expression decreased in P15 and P19 placenta, while high mRNA expression of <i>Il10</i> and <i>Tgfb1</i> was found in P19 placental tissues. Furthermore, placental <i>Il10</i> and <i>Tgfb1</i> mRNAs were down-regulated when pregnant mice were treated with an anti-C3 antibody, detecting C3, C3b and iC3b. These results indicated that C3 derivatives, in particular, iC3b and its receptor CR3 were up-regulated at the fetal-maternal interface, and suggest that iC3b may regulate the placental expression of anti-inflammatory cytokines, IL10 and TGFB1, during the latter phase of pregnancy.</p></div

Expression and localization of iC3b receptor CR3 in the uterine decidua and placenta.

<p>(A) Changes in <i>CR3</i> (<i>Cd11b</i> or <i>Cd18</i>) mRNA levels in P11, P15, or P19 decidua (solid bar) and P11, P15, or P19 placenta (white bar) (n = 3 mice each day). <i>Actb</i> mRNA was used as an internal control for RNA integrity. Values represent the mean ± SEM from three animals with duplicate samples. Different letters indicate statistically significant differences in mRNA levels (p < 0.05) when compared to that of P11. (B) Western blot analysis of CD11b expression in P11, P15, or P19 decidual and P11, P15, or P19 placental tissues. ACTB was used as an internal control. A representative data from three independent experiments containing protein samples from three different animals is shown. (C) Immunohistochemical detection of TPBPA and CD11b in P19 mouse placenta. Tissue sections were immunostained for a spongiotrophoblast specific TPBPA (a, c, and e) using anti-TPBPA antibody (a and c) or normal rabbit IgG (e) as a negative control. Boxed area in (a) is shown at a higher magnification in (c). Tissue sections were immunostained for iC3b receptor subunit CD11b (b, d, and f) using anti-CD11b antibody (b and d) or normal rabbit IgG (f) as a negative control. Boxed area in (b) is shown at a higher magnification (100 x) in (d). Scale bar = 200 μm (a, b, e, and f), or 100 μm (c and d), respectively. A representative immunostaining from three independent experiments is shown.</p