Sharon A. McCracken

A balancing act: mechanisms by which the fetus avoids rejection by the maternal immune system.

Successful pregnancy requires strict temporal regulation of maternal immune function to accommodate the growing fetus. Early implantation is facilitated by inflammatory processes that ensure adequate vascular remodeling and placental invasion. To prevent rejection of the fetus, this inflammation must be curtailed; reproductive immunologists are discovering that this process is orchestrated by the fetal unit and, in particular, the extravillous trophoblast. Soluble and particulate factors produced by the trophoblast regulate maternal immune cells within the decidua, as well as in the periphery. The aim of this review is to discuss the action of recently discovered immunomodulatory factors and mechanisms, and the potential effects of dysregulation of such mechanisms on the maternal immune response that may result in pregnancy loss or preeclampsia.

Pregnancy-Specific Down-Regulation of NF-κB Expression in T Cells in Humans Is Essential for the Maintenance of the Cytokine Profile Required for Pregnancy Success

It is accepted that human pregnancy is associated with a shift away from Th1 type and a bias toward Th2-type immune responses. The molecular mechanisms that regulate this shift are as yet unknown. We assessed the expression and activity of NF-κB, a transcription factor that plays a central role in regulating immune responses. We isolated T cells from PBMCs from nonpregnant and pregnant females and demonstrated that the NF-κB/IκB signaling pathway is down-regulated in T cells in pregnancy. Using Western blotting, high levels of NF-κB (p65) were detected in all nuclear fractions of T cells from nonpregnant females. In contrast, low levels of p65 were detected in nuclear fractions from T cells from pregnant females. Levels of IκBα and -β were also higher in cytoplasmic fractions from T cells from nonpregnant than from pregnant females. The reduction in p65 levels in pregnancy was reflected in the activity of NF-κB in EMSA; T cells from pregnant females contain less active NF-κB than from nonpregnant females. Stimulation of T cells from nonpregnant females with PMA/ionomycin resulted in IκBα degradation, p65 translocation, and subsequent production of the Th1 cytokines IFN-γ and IL-2. In contrast, PMA stimulation had no effect on NF-κB activity in T cells from pregnant females, and this was reflected in reduced Th1 cytokine production. Using the inhibitor of NF-κB activity, SN50, we were able to show that NF-κB activity was essential for the production of Th1 cytokines, suggesting that specific down-regulation of NF-κB in T cells throughout gestation is paramount to pregnancy success through specific regulation of cytokine production.

Depletion of Protease-Activated Receptor 2 but Not Protease-Activated Receptor 1 May Confer Protection Against Osteoarthritis in Mice Through Extracartilaginous Mechanisms

To explore the involvement of protease‐activated receptor 1 (PAR‐1) and PAR‐2 in the pathologic processes of osteoarthritis (OA) and to identify the cells/tissues primarily affected by ablation of PAR‐1 or PAR‐2 in mice.

Pregnancy is associated with suppression of the nuclear factor kappa B/I kappa B activation pathway in peripheral blood mononuclear cells

Modulations of maternal immune cell function are critical for successful growth and development of an antigenically distinct fetus. It has been proposed that pregnancy is associated both with suppression of the adaptive immune system and a generalised maternal inflammatory response with changes in immune function resembling those associated with septicemia, and these changes are more exaggerated when pregnancies are complicated with pre-eclampsia. The nuclear factor (NF)-kappaB family of transcription factors play a significant role in immune regulation. We hypothesised therefore that if pregnancy is associated with activation of the maternal immune system, this would be supported by the activation of NF-kappaB and degradation of IkappaBalpha and beta in peripheral blood mononuclear cells (PBMCs). We demonstrate the contrary: NF-kappaB activity is suppressed in PBMCs from pregnant females and more in pre-eclampsia. The inhibition of NF-kappaB activation in pregnancy is not attributed to over-expression of IkappaBalpha or beta. In contrast, levels of IkappaBalpha and beta in cytoplasmic extracts from PBMCs in pregnancy are decreased compared with non-pregnant controls, and IkappaBalpha levels are decreased more so in pre-eclampsia. We have shown that activation of NF-kappaB in PBMCs from patients with septicemia follows the classical pathway. This pathway is differentially regulated in pregnancy. Alterations in NF-kappaB nuclear binding and IkappaBalpha levels were reproducible by culturing PBMCs in pooled pregnant serum. Taken together, these data indicate that pregnancy-specific factors exist to regulate expression of NF-kappaB/IkappaB in a pregnancy-specific manner, and may underlie one mechanism by which the fetus avoids maternal rejection throughout pregnancy.

NF-κB-regulated suppression of T-bet in T cells represses Th1 immune responses in pregnancy

The molecular mechanisms that suppress Th1 immune responses in pregnancy are unknown. We assessed the expression of the Th1 cytokine transcription factor T‐bet. We isolated PBMC and T cells from non‐pregnant and pregnant women and demonstrated that T‐bet is specifically down‐regulated in pregnancy under basal and stimulated conditions. Low levels of T‐bet protein were detected in the nuclear fraction of unstimulated PBMC from non‐pregnant, but not pregnant women. Nuclear levels of T‐bet increased in response to PMA/ionomycin in PBMC from non‐pregnant, but not pregnant women. T‐bet expression was greater in whole cell lysates of stimulated CD3+ T cells from non‐pregnant relative to pregnant women. NF‐κB is specifically down‐regulated in T cells in pregnant women, resulting in suppressed expression of Th1 cytokines IL‐2, IFN‐γ and TNF‐α. In this study, down‐regulation of NF‐κB also resulted in diminished expression of T‐bet. PMA induces NF‐κB translocation, T‐bet expression and IL‐2, IFN‐γ and TNF‐α production. Conversely, pre‐incubation with SN50, and NF‐κB oligodeoxyribonucleotide decoys suppressed PMA‐induced NF‐κB translocation and gene transcription, respectively, resulting in diminished T‐bet expression and Th1 cytokine production. Therefore, maintenance of the cytokine environment for pregnancy success is mediated via strict regulation of Th1 immune responses, more specifically through control of NF‐κB and T‐bet transcription.

Exosomes: Mechanisms of Uptake

Exosomes are 30–100 nm microvesicles which contain complex cellular signals of RNA, protein and lipids. Because of this, exosomes are implicated as having limitless therapeutic potential for the treatment of cancer, pregnancy complications, infections, and autoimmune diseases. To date we know a considerable amount about exosome biogenesis and secretion, but there is a paucity of data regarding the uptake of exosomes by immune and non-immune cell types (e.g., cancer cells) and the internal signalling pathways by which these exosomes elicit a cellular response. Answering these questions is of paramount importance.

Anti-IgD antibody attenuates collagen-induced arthritis by selectively depleting mature B-cells and promoting immune tolerance.

Membrane (m)IgD forms a major part of B-cell receptor complexes. Its wider role in the immune system has been enigmatic. Stimulation of mIgD with an antibody (anti-IgD) can activate B-cells and elicit a broad immune response in vivo. Given the role of B-cells in autoimmune diseases and the profound impact of anti-IgD on B-cells, the potential effects of anti-IgD on autoimmune conditions are intriguing and yet to be explored. Here we report a novel therapeutic effect of anti-IgD in the collagen-induced arthritis (CIA) mouse model. Administration of anti-IgD at the onset of early clinical symptoms as a therapeutic intervention, but not as a prophylactic treatment, significantly ameliorates disease severity and joint pathology. Anti-IgD treatment selectively depletes mature B cells while it spares regulatory B-cell subsets. This results in a significant reduction of autoantibody levels but does not affect antibody responses to a T-cell-dependent antigen. Therapeutic treatment with anti-IgD increases the numbers of regulatory B-cells and regulatory T-cells whilst it augments adaptive Th1/Th2 responses in vivo. In human PBMC samples, anti-IgD also promotes adaptive Th1/Th2 responses and modulates the innate responses toward an anti-inflammatory Th2-biased response. Collectively, anti-IgD treatment may offer a selective approach to B-cell depletion that also promotes immune tolerance and anti-inflammatory tendencies without compromising the general adaptive B-cell and T-cell responses. The multiple mechanisms of action by anti-IgD treatment suggest a wider clinical application for a number of chronic inflammatory and autoimmune conditions.

Evaluation of the clinical usefulness of isolation of fetal DNA from the maternal circulation

Objective: To assess the reliability of isolating free fetal DNA from maternal plasma and to evaluate its clinical usefulness.

Regulated suppression of NF-κB throughout pregnancy maintains a favourable cytokine environment necessary for pregnancy success

Th1 immune responses are suppressed in pregnancy, but the temporal regulation and the mechanism(s) underlying this immune alteration are unknown. We assessed the expression of Th1 cytokines IFNγ, IL-2 and TNFα in response to stimulation in isolated T-cells from pregnant women throughout gestation. Using flow cytometry we demonstrated an early and sustained reduction in IFNγ and IL-2 production in CD3+ T-cells, but TNFα levels are not reduced until the third trimester. We assessed the expression of NF-κB and T-bet, transcription factors that play a central role in Th1 immune responses, throughout pregnancy. In isolated T-cells levels of available p65 were suppressed early in pregnancy, but T-bet expression was suppressed only in the third trimester. In contrast to p65, T-bet expression was transcriptionally regulated, with diminished T-bet mRNA in third-trimester samples. Re-expression of p65 in T-cells from third-trimester pregnant women resulted in an induction of T-bet expression in response to PMA stimulation and a concomitant increase in the production of IL-2 and IFNγ. The suppressive effect of pregnancy was ameliorated as early as 72h post-partum when p65 levels returned to normal as did the level of inducible IFNγ and IL-2. TNFα levels in post-partum women were significantly increased relative to non-pregnant controls. The pregnancy-specific suppression of p65 and subsequent loss of cytokine production suggest that this transcription factor acts specifically to regulate the cytokine environment that is required for pregnancy success.

Variation in the reliability of RHD antenatal genotyping using the polymerase chain reaction and targeting multiple exons of the RHD gene

Antibodies to Rh(D) [anti-Rh(D)] are potent, and, despite preventative treatment with anti-Rh(D) immunoglobulin, remain the most frequent cause of haemolytic disease of the fetus and newborn. It is now well accepted that a minute number of copies (as low as 35 copies/ml) of cell-free fetal RHD DNA in the maternal plasma [1] can be utilized as a target for non-invasive genotyping of the fetus. The aim of this study was to compare the reliability of antenatal genotyping procedures, reported for several exons of the RHD gene, when examined by conventional polymerase chain reaction (PCR), using fetal DNA fragments recovered from maternal plasma as the PCR target. Specimens were obtained from 47 pregnant Rh(D)-negative women (32 were 18–30 weeks, and 15 were 30–40 weeks of gestation). DNA was extracted from 2 ml of plasma by using a QIAamp® blood mini kit. For primer pairs located in exons 4 [2], 5 [3] and 10 [4] of the RHD gene, PCR was carried out using procedures as previously published [2–4]. For each neonate, the genotyping results were compared with the Rh(D) phenotype (36 positive and 11 negative), determined using serological techniques, after delivery. The sensitivity [Rh(D)-positive fetuses correctly genotyped divided by the total number of Rh(D)-positive neonates, expressed as a percentage] and specificity [Rh(D)-negative fetuses correctly genotyped divided by the total number of Rh(D)-negative neonates, expressed as a percentage] of genotyping were calculated (Table 1). For the exon 5 PCR the sensitivity was low, 50%; however, the specificity was 100% [no products amplified for any pregnancy with an Rh(D)-negative fetus]. With the exon 4 PCR, the sensitivity and specificity were similar to the results obtained for exon 5 (62% sensitivity and 91% specificity). Contrasting results were obtained for the exon 10 PCR. The sensitivity was higher, 94%, but the specificity was low, 36% (Table 1). For the exon 10 protocol, a product of the predicted size was consistently amplified from plasma samples from seven of the 11 Rh(D)-negative mothers with an Rh(D)-negative fetus, possibly as a result of failure of the selectivity of binding of the RHD -specific reverse primer. When genotypes were determined on the basis of at least two results concordant, the sensitivity rose to 100%, but the specificity remained at 91% (Table 1). For pregnancies of < 30 weeks, 60% of the genotypes determined were concordant with the postdelivery phenotype. For pregnancies > 30 weeks, the concordance was considerably higher, at 85%. These data highlight the importance of examining more than one exon of the RHD gene when attempting antenatal genotyping [3] and that conventional PCR, even when multiple exons are examined, may be unsuitable for diagnostic use, particularly in pregnancies of < 30 weeks of gestation. Improved sensitivity of antenatal genotyping for the RHD gene, as reported by Finning et al . [1], requires concordant results for multiple replicates and that several exons are tested. As others have noted [1,5], real-time PCR procedures are most easily adapted to replicate testing of this type.