Beth Holder

Dysfunctional CD39(POS) Regulatory T Cells and Aberrant Control of T-Helper Type 17 Cells in Autoimmune Hepatitis

Autoimmune hepatitis (AIH) is an important cause of severe liver disease and is associated with both quantitative and qualitative regulatory T‐cell (Treg) impairments. Tregs express CD39, an ectonucleotidase responsible for extracellular nucleotide hydrolysis, culminating in the production of immunosuppressive adenosine. Here, we describe multiple CD39pos Treg defects that potentially contribute to the impaired immunoregulation that is characteristic of AIH. We have examined the frequency and phenotype of CD39pos Tregs by flow cytometry and measured their ectonucleotidase activity. The capacity of CD4posCD25high, CD4posCD25highCD39pos, and CD4posCD25highCD39neg subsets to suppress both proliferation of effector T cells and interleukin (IL)‐17 production was evaluated. In AIH, CD39pos Tregs are decreased in frequency, exhibit limited adenosine triphosphate/adenosine diphosphate hydrolysis activity, and fail to suppress IL‐17 production by effector CD4 T cells. Moreover, these CD39pos Tregs display a more proinflammatory profile in AIH, which is characterized by elevated CD127 positivity, and a greater propensity to produce interferon‐gamma or IL‐17 upon challenge with proinflammatory stimuli. Conclusions: In AIH, CD39pos Tregs are decreased in number, fail to adequately hydrolyze proinflammatory nucleotides and do not efficiently suppress IL‐17 production by effector CD4 T cells. CD39pos Tregs show plasticity and are unstable upon proinflammatory challenge, suggesting that defective immunoregulation in AIH might result not only from reduced Treg number and function, but also from increased conversion of Tregs into effector cells. (Hepatology 2014;59:1007–1015)

Inhibition of Interleukin-17 Promotes Differentiation of CD25– Cells Into Stable T Regulatory Cells in Patients With Autoimmune Hepatitis

BACKGROUND & AIMS Patients with autoimmune hepatitis (AIH) have reduced numbers and function of CD4+CD25(high)FOXP3+ T regulatory cells (Tregs). Tregs can be generated from CD25⁻ (ngTreg) cells, which suppress the immune response less efficiently than Tregs. We investigated whether their differentiation into T-helper (Th)17 cells, an effector subset that has the same CD4+ progenitors as Tregs, accounts for the reduced suppressive functions of ngTregs. We investigated whether blocking interleukin (IL)-17 increased the immunosuppressive activity of Tregs. METHODS ngTregs were generated from 36 patients with AIH and 23 healthy subjects (controls). During Treg differentiation, expression of IL-17 was inhibited by physical removal of IL-17-secreting cells, exposure to recombinant transforming growth factor β or neutralizing antibodies against IL-6 and IL-1β (to promote differentiation of ngTregs vs Th17 cells), small inhibitory RNAs specific for the Th17 transcription factor RORC, or a combination of all these approaches. RESULTS ngTregs from patients with AIH contained greater proportions of IL-17+ and RORC+ cells than Tregs from controls. All approaches to inhibit IL-17 increased expression of FOXP3 by ngTregs and their suppressive functions. Inhibition of IL-17 led to development of ngTregs that were phenotypically stable and did not acquire proinflammatory properties after exposure to IL-6 and IL-1β. CONCLUSIONS Blocking Th17 allows ngTregs to differentiate into functionally stable immune inhibitory cells; this approach might be developed for therapy of patients with AIH.

The impaired immune regulation of autoimmune hepatitis is linked to a defective galectin‐9/tim‐3 pathway

In autoimmune hepatitis (AIH), liver‐damaging CD4 T cell responses are associated with defective CD4posCD25pos regulatory T cells (T‐regs). Galectin‐9 (Gal9), a β‐galactosidase–binding protein expressed by T‐regs, is key to their function, inhibiting T helper 1 immune responses by binding T cell immunoglobulin and mucin domain 3 (Tim‐3) on CD4 effector cells. We investigated whether impaired immunoregulation in AIH results from reduced expression of Gal9 in T‐regs and/or Tim‐3 on CD4 effector cells. Circulating Gal9posCD4posCD25pos and Tim‐3posCD4posCD25neg T cell phenotype was assessed by flow cytometry in 75 AIH patients. To evaluate whether Tim‐3 expression renders CD4posCD25neg T cells amenable to T‐reg control, purified CD4posCD25negTim‐3pos (Tim‐3pos) and CD4posCD25negTim‐3neg (Tim‐3neg) cells were cocultured with T‐regs. To determine whether Gal9 expression is essential to function, T‐regs were treated with small interfering RNA (siRNA) to repress Gal‐9 translation; T‐reg suppressor function was assessed by proliferation. In AIH, Tim‐3pos cells within CD4posCD25neg cells and their T‐betpos and RORCpos subsets were fewer and contained higher numbers of interferon‐γ (IFNγ)pos and interleukin (IL)‐17pos cells than healthy subjects (HS). In AIH and HS, Tim‐3pos cells proliferated less vigorously and were more susceptible to T‐reg control than Tim‐3neg cells. In AIH, Gal9posT‐regs were fewer and contained less FOXP3pos, IL‐10pos, and transforming growth factor βpos and more IFNγpos and IL‐17pos cells than HS. siRNA treatment of Gal‐9pos T‐regs drastically reduced T‐reg ability to suppress CD4posCD25neg and Tim‐3pos cell proliferation in AIH and HS. Tim‐3pos cell percentage correlated inversely with aminotransferase and CD25negT‐betpos cell values. Conclusion: Reduced levels of Tim‐3 on CD4posCD25neg effector cells and of Gal9 in T‐regs contribute to impaired immunoregulation in AIH by rendering effector cells less prone to T‐reg control and T‐regs less capable of suppressing. (HEPATOLOGY 2012)

Immune cell activation by trophoblast-derived microvesicles is mediated by syncytin 1

Envelope glycoproteins of human endogenous retrovirus (HERV), such as syncytin 1 (HERV‐W), are highly expressed in the placenta and some family members have immunomodulatory properties. Placental microvesicles (MV), which are shed into the maternal circulation during pregnancy, have been demonstrated to induce immune cell activation. Therefore, the aim of this study was to investigate the immunological properties of the highly expressed placental HERV‐W protein, syncytin 1, and its potential involvement in placental MV modulation of immune cell activity. The MV shed from first trimester, normal term and pre‐eclamptic term placentas, and from the BeWo trophoblast cell line, all contain syncytin 1. Recombinant syncytin 1 and syncytin 1‐positive BeWo trophoblast MV both induced peripheral blood mononuclear cell (PBMC) activation, indicated through production of cytokines and chemokines. Reducing syncytin 1 content in BeWo MV inhibited PBMC activation. Recombinant syncytin 1 and syncytin‐1‐positive BeWo MV dampened PBMC responses to lipopolysaccharide challenge. Our findings suggest that syncytin 1 is shed from the placenta into the maternal circulation in association with MV, and modulates immune cell activation and the responses of immune cells to subsequent lipopolysaccharide stimulation. These studies implicate placental MV‐associated HERV in fetal regulation of the maternal immune system.

Heightened Pro-Inflammatory Effect of Preeclamptic Placental Microvesicles on Peripheral Blood Immune Cells in Humans

ABSTRACT Normal pregnancy is associated with the presence of circulating placental microvesicles (MVs). Increased MV shedding and altered immune activation are seen in patients with preeclampsia, suggesting that placental MVs may play a role in the pathophysiology of this disease. Therefore, the aim of this study was to investigate the activation of peripheral blood mononuclear cells (PBMCs) by MVs shed by first-trimester, normal term, and preeclamptic term placenta. First-trimester and preeclamptic term, but not normal term, placental-derived MVs activated PBMCs, as evidenced by elevated IL1B. Significant changes were also seen with several other cytokines and chemokines, and in general when compared to normal term MVs, preeclamptic MVs induced a greater pro-inflammatory response in PBMCs. Pretreatment of PBMCs with first-trimester or normal term placental MVs resulted in a dampened IL1B response to a subsequent lipopolysaccharide (LPS) challenge. In contrast, treatment of PBMCs with preeclamptic term placental MVs exacerbated the LPS response. This was also the case for several other cytokines and chemokines. These studies suggest that placental MVs can modulate basal peripheral immune cell activation and responsiveness to LPS during normal pregnancy, and that in preeclampsia this effect is exacerbated.

In autoimmune hepatitis type 1 or the autoimmune hepatitis–sclerosing cholangitis variant defective regulatory T‐cell responsiveness to IL‐2 results in low IL‐10 production and impaired suppression

Defective immune regulation plays a permissive role enabling effector cells to initiate and perpetuate tissue damage, eventually resulting in autoimmune disease. Numerical and functional regulatory T‐cell (Treg) impairment has been previously reported in autoimmune liver disease (AILD; including autoimmune hepatitis and autoimmune sclerosing cholangitis ASC). However, in these early reports, Tregs were phenotypically defined as CD4+CD25+ or CD4+CD25high cells. In the current study, we reexamined phenotypic and functional properties of Tregs by adopting a more refined definition of these cells that also includes negativity or low level of expression of CD127. We studied 43 AILD patients and 22 healthy subjects (HSs) and found that CD4+CD25+CD127− Tregs were decreased in the former. This decrease was more marked in patients with active disease than in those in remission. In AILD, Treg frequencies correlated inversely with parameters of disease activity and were not affected by immunosuppressive treatment. We also document, for the first time, that, in AILD, bona‐fide Tregs produce less interleukin (IL)−10 and are impaired in their ability to suppress CD4+CD25− target cell proliferation, a feature that in HSs, but not in AILDs, is dependent, at least in part, on IL‐10 secretion. Decreased IL‐10 production by Tregs in AILD is linked to poor responsiveness to IL‐2 and phospho signal transducer and activator of transcription 5 up‐regulation. Conclusion: Tregs are numerically impaired in AILD, this impairment being more prominent during active disease. Notably, defective IL‐10 production, resulting from low Treg responsiveness to IL‐2, contributes to Treg functional impairment. (Hepatology 2015;62:863–875)

What determines uptake of pertussis vaccine in pregnancy? A cross sectional survey in an ethnically diverse population of pregnant women in London

INTRODUCTION Following the major outbreak of pertussis and 14 infant deaths across England in 2012, the Department of Health (DH) introduced the UKs first maternal pertussis vaccination programme. Data published by Public Health England (PHE) suggest uptake of the vaccine varies considerably across the country. The reasons for this heterogeneity need to be addressed to optimise the impact of the programme. OBJECTIVE To assess uptake of antenatal pertussis and influenza vaccine in a leading NHS Trust in London and to explore awareness and attitudes of pregnant women towards the pertussis vaccination programme. DESIGN A cross sectional survey was conducted in an ethnically diverse group of 200 pregnant women accessing antenatal care at Imperial Healthcare NHS Trust. Quantitative data was tabulated and content analysis was carried out on the free text. Qualitative data was divided into themes for accepting or declining the vaccine. RESULTS Awareness of the programme was 63% (126/200) with actual uptake of the vaccine only 26.0% (52/200). Women had received information from multiple sources, primarily General Practitioners (GP) and midwives. 34.0% (68/200) of women were offered the vaccine at their GP practice, only 24% reported a meaningful discussion with their GP about it. Uptake differed by up to 15.0% between ethnicities. Qualitative data showed that uptake could be significantly enhanced if vaccination was recommended by a familiar healthcare professional. Feeling uninformed, lack of professional encouragement and uncertainties of risk and benefit of the vaccine were the greatest barriers to uptake. CONCLUSION Vaccine uptake in this cohort of pregnant women was poor. Understanding the target audience and engaging with key groups who influence womens decision-making is essential. Knowledgeable health care professionals need to recommend the vaccine and provide accurate and timely information to increase success of this important programme.

Cytomegalovirus infection induces T-cell differentiation without impairing antigen-specific responses in Gambian infants

Cytomegalovirus (CMV) infection induces profound differentiation of T cells, and is associated with impaired responses to other immune challenges. We therefore considered whether CMV infection and the consequent T‐cell differentiation in Gambian infants was associated with impaired specific responses to measles vaccination or polyclonal responses to the superantigen staphylococcal enterotoxin B (SEB). While the concentration of undifferentiated (CD27+ CD28+ CCR7+) T‐cells in peripheral blood was unaffected by CMV, there was a large increase in differentiated (CD28− CD57+) CD8 T‐cells and a smaller increase in differentiated CD4 cells. One week post‐vaccination, the CD4 cell interferon‐γ (IFN‐γ) response to measles was lower among CMV‐infected infants, but there were no other differences between the cytokine responses, or between the cytokine or proliferative responses 4 months post‐vaccination. However, the CD8 T cells of CMV‐infected infants proliferated more in response to SEB and the antibody response to measles correlated with the IFN‐γ response to CMV, indicating that CMV infection actually enhances some immune responses in infancy.

Syncytin 1 in the human placenta

This study characterises HERV-W (syncytin 1) expression in normal and pathologic placenta and in BeWo cells. HERV-W mRNA levels were higher in the first trimester than at term, and similar patterns were observed with another retrovirally-derived mRNA species, ERV-3. N-glycosylated syncytin 1 precursor (73 kDa) is cleaved to surface-associated (SU) and transmembrane (TM) subunits. Both were evident in villous trophoblast, where perinuclear and punctate cytoplasmic deposits were observed, and linear TM subunit immunoreactivity was seen at the syncytial microvillous membrane. Punctate immunoreactivity was seen in BeWo cells with antibodies to SU and TM, and the two were co-localised. SU immunoreactivity was observed in association with fetal endothelium, and this effect was increased in tissue from pre-eclamptic placentas, which also showed a higher level of total SU protein. Absence of the TM subunit from endothelium suggests it is not a biosynthetic source. We suggest that SU is released from trophoblast into fetal circulation where it may bind vascular endothelium.

Macrophage Exosomes Induce Placental Inflammatory Cytokines: A Novel Mode of Maternal-Placental Messaging

During pregnancy, the placenta forms the interface between mother and fetus. Highly controlled regulation of trans‐placental trafficking is therefore essential for the healthy development of the growing fetus. Extracellular vesicle‐mediated transfer of protein and nucleic acids from the human placenta into the maternal circulation is well documented; the possibility that this trafficking is bi‐directional has not yet been explored but could affect placental function and impact on the fetus. We hypothesized that the ability of the placenta to respond to maternal inflammatory signals is mediated by the interaction of maternal immune cell exosomes with placental trophoblast. Utilizing the BeWo cell line and whole placental explants, we demonstrated that the human placenta internalizes macrophage‐derived exosomes in a time‐ and dose‐dependent manner. This uptake was via clathrin‐dependent endocytosis. Furthermore, macrophage exosomes induced release of proinflammatory cytokines by the placenta. Taken together, our data demonstrates that exosomes are actively transported into the human placenta and that exosomes from activated immune cells modulate placental cytokine production. This represents a novel mechanism by which immune cells can signal to the placental unit, potentially facilitating responses to maternal inflammation and infection, and thereby preventing harm to the fetus.