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

Up-regulated proteins in P11 decidual tissues.

<p>SYPRO Ruby stained 2D-PAGE gel images of endometrial tissue from day 11 pseudopregnant (Left panel) mice and decidual tissue from P11 (Right panel) mice. Proteins in the circled spot in P11 decidual tissues were up-regulated compared to those in day 11 pseudopregnant endometrial tissues, which were subjected to MALDI-TOF/MS analysis. Protein migration patterns were reproducible from animal to animal, and a representative 2D gel from three independent experiments is shown.</p

Expression of pro- and anti-inflammatory cytokines, <i>Il12</i>, <i>Il10</i>, and <i>Tgfb1</i>.

<p>Changes in <i>Il12a</i>, <i>Il12b</i>, <i>Il10</i>, or <i>Tgfb1</i> mRNAs in P11, P15, or P19 decidua (solid bar) and P11, P15, or P19 placenta (white bar) (n = 3 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 small or capital letters indicate statistically significant differences in mRNA levels (p < 0.05) when compared to that of P11.</p

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