Complement Modulation Mitigates Inflammation-Mediated Preterm Birth and Fetal Neural Inflammation

Abstract

Preterm birth and the neonatal pathological sequelae that follow spontaneous preterm labor are closely associated with maternal and fetal inflammatory activation. Previous studies have indicated a role for the complement system in this inflammatory response. Utilizing an LPS inflammation-induced model of preterm birth, we investigated various delivery outcomes and their correlation with complement activation products within cervical, uterine, and fetal brain tissue after administration of LPS. We provide further evidence that complement-mediated inflammation within cervical and uterine tissue contributes to aberrant cellular changes and an increase in preterm delivery. We additionally show that a targeted complement inhibitor that specifically targets to sites of complement activation (CR2-Crry) mitigates the effects of LPS-induced pathology and preterm birth. Complement inhibition increased latency to delivery, mean gestational age at delivery, and average number of viable pups. Furthermore, the improved delivery outcomes seen with CR2-Crry treatment correlated with a reduced inflammatory response in maternal tissue and in fetal brain tissue in terms of reduced complement activation, reduced pro-inflammatory cytokines, and reduced macrophage recruitment. These data indicate that complement inhibition represents a potential therapeutic strategy for preventing preterm birth. The localization of complement inhibition by a site-targeting approach reduces the possibility of unwanted off-target effects.

Keywords: complement modulation; complement system; fetal inflammation; maternal-fetal interface; preterm birth.

Figure 1

Deposition of complement (C3) in murine cervical tissue after LPS administration. Representative 40× immunofluorescent imaging of C3, captured at different timepoints following LPS administration; no quantitative analysis performed. (a) Naïve cervical tissue showing minimal C3 deposition. (b) Cervical tissue collected 1 h after LPS administration showing minimal, but increased C3 deposition compared to naive control tissue. (c) Cervical tissue collected 9 h after LPS administration showing increased C3 deposition compared to both naive control and 1 h post-LPS administration tissue.

Figure 2

Complement inhibition increases latency to delivery and completion of full-term deliveries. Following LPS administration and subsequent treatment with CR2-Crry or vehicle, multiple parameters of length and completion of successful term deliveries were compared. CR2-Crry (a) increased latency to delivery, (b) mean gestational age at delivery, and (c) average number of viable pups. (d) Summary of pregnancy outcomes. n = 7 vehicle and n = 7 CR2-Crry-treated. Hours to delivery comparison made with Welch’s t-test. Gestational age at delivery and number of viable pups’ comparison made with Mann–Whitney test. ** p< 0.01, *** p< 0.001, **** p< 0.0001. Error bar = mean ± SD. (e) Kaplan–Meier curve showing that 50% of CR2-Crry-treated dams were pregnant at 100 h after LPS administration while 0 vehicle-treated dams remained pregnant.

Figure 3

CR2-Crry treatment reduces complement deposition and macrophage recruitment in cervical tissue. Sections stained by immunofluorescence for C3 and macrophages (Iba1). (a) Quantification of C3 deposition following LPS administration showing significance increase in vehicle-treated vs. CR2-Crry-treated animals. (b) Quantification of macrophage recruitment following LPS administration showing significant increase in vehicle-treated vs. CR2-Crry-treated animals, which correlated with levels of C3 deposition. (c) 40× representative images of C3 (red) and Iba1 (teal) in maternal dam cervical tissue. n = 10 vehicle and n = 11 CR2-Crry-treated. Student’s t-test. * p < 0.05, ** p < 0.01. Error bars = mean ± SEM.

Figure 4

CR2-Crry treatment reduces pro-inflammatory maternal and fetal response. Cytokine levels within homogenized maternal uterine tissue and fetal brain tissue for IL-1b, IL-6, IL-10, MCP-1, TNFa. (a) Quantification of pro-inflammatory cytokines in maternal uterine tissue comparing vehicle-treated (n = 7) with CR2-Crry-treated (n = 7) maternal dams following LPS administration. There was a significant reduction in IL-6 and MCP-1 levels with CR2-Crry treatment. All comparisons made with Mann–Whitney test and Error bars = median ± SD, with exception of MCP-1 where comparison was made with Welch’s t-test. * p < 0.05, ** p < 0.01. (b) Quantification of pro-inflammatory cytokines in fetal brain tissue comparing. Fetal brains were collected from naïve (n = 8), vehicle-treated (n = 13), or CR2-Crry-treated (n = 9) dams following administration of maternal LPS administration. A significant reduction was found in IL-6 and MCP-1 levels in fetal pups from maternal dams treated with CR2-Crry with levels similar to that seen in full-term naïve pups. All comparisons made with Mann–Whitney test and Error bars = median ± SD, with exception of IL-1b (CR2-Crry and Naive) where comparison was made with Welch’s t-test. * p < 0.05, ** p < 0.01. Error bars = mean ± SD.