Host defence peptides in pregnancy: influences on the microbiome and preterm labour

Abstract

Although inflammation is a crucial mechanism in response to injury and pathogen clearance, inappropriate or excessive induction of the inflammatory response in pregnancy can cause initiation of the labour cascade and subsequent preterm delivery. Host Defence Peptides (HDPs) have important anti-microbial properties but are also implicated as multifunctional modulators of immunity and infection. They are predominantly secreted by mucosal epithelial cells and released by leukocytes. The specific HDPs that are the focus of this thesis are Human beta-defensin 3 (hBD3) and Human Cathelicidin (hCAP-18/LL-37). The immunomodulatory effect of HDPs in reproductive tissues in response to infection/inflammation has not been well studied. In a pregnant state, the hypothesis of this thesis is that HDPs have a dual role in preventing ascending infection, but also preventing an exacerbated inflammatory response that can cause preterm birth by initiation of the labour cascade. To explore this I determine whether bacterial stimuli can regulate HDPs expression in pregnancy tissues. I also explore what interactions HDPs have on the production/induction of important cytokines that are vital to the inflammatory response. With the aid of HDP knockout mice, the role of these peptides in infection/inflammation and continuation of pregnancy is investigated in a mouse-model of induced preterm-labour. To understand how ascending infection might be controlled by HDPs in pregnancy, I explore how HDPs regulate commensal and pathogenic bacteria. This is achieved by interrogating the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene and next generation sequencing. Results Placental explants respond to Lipopolysaccharide (LPS) challenge by increasing production of pro-inflammatory cytokines. LL-37 but not hBD3 peptide was able to modulate this inflammation by inhibiting the release of these pro-inflammatory cytokines. To establish whether HDPs are critical in the continuation of pregnancy I use a LPS induced mouse–model of preterm labour in animals lacking the genes for the HDPs, Defb14 (Defb14-/-), or Camp (Camp-/-). Intrauterine injection of LPS induced preterm labour in wildtype mice. However, the Defb14-/- and Camp-/- mice do not have an increased rate of preterm labour. Key inflammatory mediators are increased in response to LPS-induced PTL. Camp-/- animals have a similar inflammatory response to wildtype mice when given LPS during pregnancy. To understand how ascending infection might be controlled by HDPs, I interrogated the maternal microbiome at mucosal sites in HDP knockout animals, utilising the bacterial 16S rRNA gene. I established a workflow for 16S rRNA gene sequencing on next-generation sequencing platforms and a bioinformatic pipeline for data analysis. Using this approach I was able to show the mucosal microbiome of Camp-/- animals were significantly different to that of wildtype controls, showing increased diversity in the microbes present. In murine pregnancy, there were very little global cumulative or progressive shifts in bacteria, with the exception of Candidatus arthromitus, which significantly increases with gestation compared to non-pregnancy This thesis has demonstrated that Host Defence Peptides are expressed in pregnancy tissues and have anti-inflammatory properties in response to bacterial stimuli. It is not clear whether the HDPs, hBD3 and LL-37 are fundamental to the immune defence in pregnancy by preventing excessive inflammation, Although, I have shown LL-37 may have a role in modulation of the maternal microbiota.