Innate Immune Complement System and Developmental Programming of Functional β Cell Mass

妊娠期间胎盘功能不全会导致宫内生长受限，并使后代容易患上2型糖尿病，并导致功能性β细胞团减少。了解子宫内β细胞质量减少的机制对于制定预防或逆转它的治疗策略至关重要。补体系统是一种先天性免疫放大系统，对宿主防御、炎症和胎儿存活至关重要，但补体过度激活与妊娠并发症有关，包括先兆子痫合并IUGR。在胎盘功能不全大鼠子宫胎盘灌流压降低(RUPP)模型中，胚胎14天，妊娠晚期，流向子宫胎盘单位的血流受到机械干扰，导致胎盘缺血、DAM和IUGR高血压。利用这一模型，我们发表的研究表明，生长受限胎儿的E19胰岛β细胞面积减少，细胞凋亡明显增加，母体补体激活增加是RUPP DAM高血压的关键。我们的初步研究表明，RUPP后代E19胰岛中C3减少，巨噬细胞标志物增加。长期目标：确定在宫内或出生后操纵补体或巨噬细胞以减轻宫内发育迟缓后代β细胞量减少的治疗效用。目的：探讨补体C3、C3a和巨噬细胞在RUPP模型中对β细胞质量和存活的影响。中心假设：胎盘缺血最终导致子代胰岛C3/C3a减少，巨噬细胞数量或M1/M2表型改变 导致β细胞量减少，细胞凋亡率增加。目的1：确定胎盘缺血诱导胚胎胰腺β细胞质量减少和细胞凋亡是否需要C3和/或C3a的减少(Aim 1)或巨噬细胞数量或表型的改变(Aim 2)。C3、C3a、C3a受体和巨噬细胞变化的时间进程将在出生后15-12周测定。在正常妊娠中(宫内或出生后)，通过siRNA降低C3或拮抗C3aR对β细胞面积或质量、细胞凋亡和巨噬细胞的影响将被确定。相反，将评估在RUPP后代中通过给予C3aR激动剂来拯救β细胞团而增加C3a的能力。我们将评估宫内或出生后使用氯屈膦酸脂质体去除胎儿巨噬细胞对伴有和不伴有胎盘缺血的胎儿胰岛补体、β细胞质量和细胞凋亡的影响。这项研究具有创新性，因为它首次研究了补体在胰腺发育中的机制作用及其对IUGR应激的反应，并将胰岛生物学的学科内专业知识与补体和RUPP IUGR模型的专业知识相结合。这一贡献将具有重大意义，因为它将确定在胰腺发育早期以补体系统或巨噬细胞为靶点是否是一种可行的治疗策略，以在胎盘缺血的情况下保留胰腺β细胞团，从而防止成年后的长期后果。

Placental insufficiency during pregnancy causes intrauterine growth restriction (IUGR) and predisposes offspring to Type 2 diabetes with reductions in functional β-cell mass. Understanding the mechanism of this decrease in β-cell mass in utero is essential to developing treatment strategies to prevent or reverse it. The complement system is an innate immune amplification system essential for host defense, inflammation and fetal survival, but excessive complement activation is associated with pregnancy complications including preeclampsia with IUGR. In the reduced uteroplacental perfusion pressure (RUPP) model of placental insufficiency in rat, blood flow to uteroplacental unit is mechanically disrupted at embryonic day (e)14, beginning of third trimester, resulting in placental ischemia, high blood pressure in dam and IUGR. Using this model, our published studies demonstrated β cell area is reduced and increased apoptosis evident in e19 islet of growth restricted fetus, and increased maternal complement activation is critical for hypertension in RUPP dam. Our preliminary studies demonstrate decreased C3 and increased macrophage marker in e19 islets of RUPP offspring. Long-term goal: Determine therapeutic utility of manipulating complement or macrophages in utero or postnatally to mitigate reduced β cell mass in IUGR offspring. Objective: Assess contribution of C3, C3a and macrophages to β cell mass and survival in RUPP model. Central hypothesis: Placental ischemia results in decreased C3/C3a and change in macrophage numbers or M1/M2 phenotype in islets of offspring, ultimately leading to reduced β cell mass and increased apoptosis. Aim 1: Determine whether decrease in C3 and/or C3a (Aim 1) or change in macrophage numbers or phenotype (Aim 2) are required for placental ischemia-induced reduction in β cell mass and apoptosis in developing fetal pancreas. Time course of changes in C3, C3a, C3a receptor, and macrophages will be determined from e15 to 12 wk postnatal. Effect of decreasing C3 by siRNA or antagonizing C3aR in normal pregnancy (in utero or postnatally) on β cell area or mass, apoptosis and macrophages will be determined. Conversely, ability of increasing C3a by administration of a C3aR agonist in RUPP offspring to rescue β cell mass will be evaluated. The effect of fetal macrophage depletion using clodronate liposomes in utero or postnatally on complement, β cell mass and apoptosis in fetal islets with and without placental ischemia will be assessed. This research is innovative because it is the first to examine the mechanistic role of complement in pancreatic development and its response to IUGR stress, and combines intradisciplinary expertise in islet biology with expertise in complement and RUPP IUGR model. This contribution will be significant because it will determine if targeting complement system or macrophages early in pancreatic development is a feasible therapeutic strategy for preserving pancreatic β cell mass in the face of placental ischemia and thus preventing long term consequences in adulthood.