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Maternal and fetal pregnancy outcomes related to placental function vary based on fetal sex, which may be due to sexually dimorphic epigenetic regulation of RNA expression. We identified sexually dimorphic miRNA expression throughout gestation in human placentae. Next-generation sequencing identified miRNA expression profiles in first and third trimester uncomplicated pregnancies using tissue obtained at chorionic villous sampling (n = 113) and parturition (n = 47). Sequencing analysis identified 986 expressed mature miRNAs from female and male placentae at first and third trimester (baseMean>10). Of these, 11 sexually dimorphic (FDR < 0.05) miRNAs were identified in the first and 4 in the third trimester, all upregulated in females, including miR-361-5p, significant in both trimesters. Sex-specific analyses across gestation identified 677 differentially expressed (DE) miRNAs at FDR < 0.05 and baseMean>10, with 508 DE miRNAs in common between female-specific and male-specific analysis (269 upregulated in first trimester, 239 upregulated in third trimester). Of those, miR-4483 had the highest fold changes across gestation. There were 62.5% more female exclusive differences with fold change>2 across gestation than male exclusive (52 miRNAs vs 32 miRNAs), indicating miRNA expression across human gestation is sexually dimorphic. Pathway enrichment analysis identified significant pathways that were differentially regulated in first and third trimester as well as across gestation. This work provides the normative sex dimorphic miRNA atlas in first and third trimester, as well as the sex-independent and sex-specific placenta miRNA atlas across gestation, which may be used to identify biomarkers of placental function and direct functional studies investigating placental sex differences.

Aim: To understand miRNA changes across gestation in healthy human placentae. This is essential before miRNAs can be used as biomarkers or prognostic indicators during pregnancy. Materials & methods: Using next-generation sequencing, we characterize the normative human placenta miRNome in first (n = 113) and third trimester (n = 47). Results & conclusion: There are 801 miRNAs expressed in both first and third trimester, including 182 with similar expression across gestation (p ≥ 0.05, fold change ≤2) and 180 significantly different (false discovery rate <0.05, fold change >2). Of placenta-specific miRNA clusters, chromosome 14 miRNA cluster decreases across gestation and chromosome 19 miRNA cluster is overall highly expressed. Chromosome 13 clusters are upregulated in first trimester. This work provides a rich atlas of healthy pregnancies to direct functional studies investigating the epigenetic differences in first and third trimester placentae.

Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice (Ro52(-/-)), which appear phenotypically normal if left unmanipulated. However, Ro52(-/-) mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52(-/-) mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.

Toll-like receptors (TLRs)-2 and -4 are important proteins in innate immunity, recognizing microbial products and eliciting host defense responses. Both use the adapter proteins MyD88 and MyD88 adapterlike (Mal) to activate signaling pathways. Herewereport that Mal but not MyD88 interacts with caspase-1, the enzyme that processes the precursors of the proinflammatory cytokines IL-1B and IL-18. The interaction was found in a yeast two-hybrid screen and was confirmed by reciprocal GST pull-downs and coimmunoprecipitation of endogenous proteins. We were unable to implicate Mal in regulating caspase-1 activation. However, we found that Mal was cleaved by caspase-1 and that inhibition of caspase-1 activity blocked TLR2- and TLR4-mediated NF-kB and p38 MAP kinase activation but not IL-1 or TLR7 signaling, which are Mal independent. These responses, and the induction of TNF, were also attenuated in caspase-1-deficient cells. Finally, unlike wild-type Mal, a mutant Mal, which was not cleaved by caspase-1, was unable to signal and acted as a dominant negative inhibitor of TLR2 and TLR4 signaling. Our study therefore reveals a role for caspase-1 in the regulation of TLR2 and TLR4 signaling pathways via an effect on Mal. This functional interaction reveals an important aspect of the coordination between TLRs and caspase-1 during the innate response to pathogens.

Toll-like receptors (TLRs)-2 and -4 are important proteins in innate immunity, recognizing microbial products and eliciting host defense responses. Both use the adapter proteins MyD88 and MyD88 adapterlike (Mal) to activate signaling pathways. Herewereport that Mal but not MyD88 interacts with caspase-1, the enzyme that processes the precursors of the proinflammatory cytokines IL-1B and IL-18. The interaction was found in a yeast two-hybrid screen and was confirmed by reciprocal GST pull-downs and coimmunoprecipitation of endogenous proteins. We were unable to implicate Mal in regulating caspase-1 activation. However, we found that Mal was cleaved by caspase-1 and that inhibition of caspase-1 activity blocked TLR2- and TLR4-mediated NF-kB and p38 MAP kinase activation but not IL-1 or TLR7 signaling, which are Mal independent. These responses, and the induction of TNF, were also attenuated in caspase-1-deficient cells. Finally, unlike wild-type Mal, a mutant Mal, which was not cleaved by caspase-1, was unable to signal and acted as a dominant negative inhibitor of TLR2 and TLR4 signaling. Our study therefore reveals a role for caspase-1 in the regulation of TLR2 and TLR4 signaling pathways via an effect on Mal. This functional interaction reveals an important aspect of the coordination between TLRs and caspase-1 during the innate response to pathogens.

Signal transduction processes activated by Toll-like receptors (TLRs) include the important transcription factor NF-kappaB and 2 MAP kinases, p38 and Jun N-terminal kinase. These signals ultimately give rise to increased expression of a multitude of pro-inflammatory proteins. Receptor-proximal proteins involved in signalling by all TLRs include the adapter MyD88, 3 IRAKs (IRAK-4, IRAK and IRAK-2), Tollip, Traf-6 and TAK-1. Differences between signals generated by TLRs are emerging, with both TLR4 and TLR2 signalling requiring an additional adapter termed MyD88-adapter-like (Mal; also known as TIRAP). MyD88 and Mal both have a homologous Toll/IL-1 receptor (TIR) domain although they differ in their N-termini, with MyD88 possessing a death domain. In addition, structural models reveal marked differences in surface charges which, when taken with surface charge differences between TLR2 and TLR4 TIR domains, may indicate that TLR4 but not TLR2 recruits Mal directly. Another difference is that Mal can become phosphorylated. Future studies on Mal will reveal specificities in signal transduction by different TLRs, which may ultimately provide molecular explanations for specificities in the innate immune response to infection.