Ulrika Holmlund

Expression and regulation of the pattern recognition receptors Toll‐like receptor‐2 and Toll‐like receptor‐4 in the human placenta

The placenta constitutes a physical and immunological barrier against invading infectious agents and has been suggested to be a pregnancy‐specific component of the innate immune system. The aim of this study was to investigate the presence and regulation of Toll‐like receptors‐2 and ‐4 (TLR2 and TLR4) in the human placenta, because these receptors are believed to be important for immune responses against pathogens. Twenty‐eight placentas from normal term pregnancies were analysed with immunohistochemistry, which showed a strong immunoreactivity for TLR2 and TLR4 in the villous and the intermediate trophoblasts. The regulation of TLR2 and TLR4 by microbial stimulus was assessed by incubating explants of term chorionic villi with zymosan or lipopolysaccharide (LPS) and analysed with real‐time reverse transcriptase–polymerase chain reaction. Stimulation with zymosan and LPS readily induced interleukin (IL)‐6 and IL‐8 cytokine production in the placenta cultures, whereas TLR2 and TLR4 mRNA and protein expression remained at the same high level as in unstimulated explants. These data suggests a novel mechanism for the fetoplacental unit to interact with micro‐organisms.

The novel inflammatory cytokine high mobility group box protein 1 (HMGB1) is expressed by human term placenta

High mobility group box protein 1 (HMGB1) was previously considered a strict nuclear protein, but lately data are accumulating on its extranuclear functions. In addition to its potent proinflammatory capacities, HMGB1 has a prominent role in a number of processes of specific interest for the placenta. Our overall aim was to investigate the expression of HMGB1 in human term placenta and elucidate a potential difference in HMGB1 expression comparing vaginal deliveries with elective Caesarean sections. In addition, placentas from normal pregnancies were compared with placentas from pregnancies complicated by pre‐eclampsia. Twenty‐five placentas, 12 from normal term pregnancies and 13 from pregnancies complicated by pre‐eclampsia were analysed with immunohistochemistry for HMGB1 and its putative receptors; receptor for advanced glycation end‐products (RAGE), Toll‐like receptor 2 (TLR2) and TLR4. We present the novel finding that in addition to a strong nuclear HMGB1 expression in almost all cells in investigated placentas, an individual variation of cytoplasmic HMGB1 expression was detected in the syncytiotrophoblast covering the peripheral chorionic villi, by cells in the decidua and in amnion. Production of HMGB1 was confirmed by in situ hybridization. Although labour can be described as a controlled inflammatory‐like process no differences in HMGB1 expression could be observed comparing active labour and elective Caesarean sections. However, a tendency towards a higher expression of cytoplasmic HMGB1 in the decidua from women with pre‐eclampsia was demonstrated. The abundant expression of the receptors RAGE, TLR2 and TLR4 implicates a local capability to respond to HMGB1, although the precise role in the placenta remains to be elucidated.

IgE is expressed on, but not produced by, fetal cells in the human placenta irrespective of maternal atopy.

The prevalence of atopic diseases in children has increased during the last decades. Atopic symptoms usually appear early in life. This implies an early priming for atopic disease, possibly even at the fetal level. We therefore compared the presence and production of IgE in the local in utero environment during pregnancy in atopic and non‐atopic women. Eighty‐six women were included in the study. Fifty women were demonstrated to be atopics, based on clinical symptoms of atopic disease together with a positive Phadiatop and/or skin prick test. Placentas from these term pregnancies were obtained. Slices covering the full thickness of the placenta were cut clockwise around the umbilical cord and were analysed with immunohistochemistry. Surprisingly, numerous IgE+ cells, located primarily in the fetal villous stroma, were detected in a majority of the investigated placentas irrespective of the atopy of the mother or maternal or fetal total serum IgE levels. The placental IgE could not be demonstrated to be bound to IgE receptors, but was shown to be bound to fetal macrophages, possibly via FcγRI. No evidence was found for local fetal IgE production, although cells producing epsilon transcripts were occasionally detected in the decidua. We describe here the novel finding of numerous IgE+ cells in the human placenta, suggesting an hitherto unknown role for IgE in a successful pregnancy outcome, irrespective of whether or not the mother is atopic.

Maternal country of origin, breast milk characteristics and potential influences on immunity in offspring

Breast milk contains pro‐ and anti‐inflammatory cytokines and chemokines with potential to influence immunological maturation in the child. We have shown previously that country of birth is associated with the cytokine/chemokine profile of breast milk. In this study we have investigated how these differences in breast milk affect the cellular response of cord blood mononuclear cells (CBMCs) and intestinal epithelial cells (IECs, cell line HT‐29) to microbial challenge. Ninety‐five women were included: 30 from Mali in West Africa, 32 Swedish immigrants and 33 native Swedish women. CBMCs or IECs were stimulated in vitro with breast milk, alone or in combination with lipopolysaccharide (LPS) or peptidoglycan (PGN). Breast milk in general abrogated the LPS‐induced down‐regulation of surface CD14 and Toll‐like receptor (TLR)‐4 expression on CB monocytes, while inhibiting the PGN‐induced TLR‐2 up‐regulation. However, breast milk from immigrant women together with LPS induced a lower CBMC release of interleukin (IL)‐6 (P = 0·034) and CXCL‐8/IL‐8 (P = 0·037) compared with breast milk from Swedish women, while breast milk from Swedish women and Mali women tended to increase the response. The same pattern of CXCL‐8/IL‐8 release could be seen after stimulation of IECs (HT‐29). The lower CBMC and IEC (HT‐29) responses to microbial compounds by breast milk from immigrant women could be explained by the fact that breast milk from the immigrant group showed a divergent pro‐ and anti‐inflammatory content for CXCL‐8/IL‐8, transforming growth factor‐β1 and soluble CD14, compared to the other two groups of women. This may have implications for maturation of their childrens immune responses.

Impaired Toll-like receptor 2 signalling in monocytes from 5-year-old allergic children

The relative composition of the two major monocytic subsets CD14+CD16− and CD14+CD16+ is altered in some allergic diseases. These two subsets display different patterns of Toll‐like receptor levels, which could have implications for activation of innate immunity leading to reduced immunoglobulin E‐specific adaptive immune responses. This study aimed to investigate if allergic status at the age of 5 years is linked to differences in monocytic subset composition and their Toll‐like receptor levels, and further, to determine if Toll‐like receptor regulation and cytokine production upon microbial stimuli is influenced by the allergic phenotype. Peripheral blood mononuclear cells from 5‐year‐old allergic and non‐allergic children were stimulated in vitro with lipopolysaccharide and peptidoglycan. Cells were analysed with flow cytometry for expression of CD14, Toll‐like receptors 2 and 4 and p38‐mitogen‐activated protein kinase (MAPK). The release of cytokines and chemokines [tumour necrosis factor, interleukin (IL)‐1β, IL‐6, IL‐8, IL‐10, IL‐12p70] into culture supernatants was measured with cytometric bead array. For unstimulated cells there were no differences in frequency of the monocytic subsets or their Toll‐like receptor levels between allergic and non‐allergic children. However, monocytes from allergic children had a significantly lower up‐regulation of Toll‐like receptor 2 upon peptidoglycan stimulation. Further, monocytes from allergic children had a higher spontaneous production of IL‐6, but there were no differences between the two groups regarding p38‐MAPK activity or cytokine and chemokine production upon stimulation. The allergic subjects in this study have a monocytic population that seems to display a hyporesponsive state as implicated by impaired regulation of Toll‐like receptor 2 upon peptidoglycan stimulation.

Maternal country of birth and previous pregnancies are associated with breast milk characteristics

Populations in high infectious exposure countries are at low risk of some immune‐mediated diseases such as Crohn’s disease and allergy. This low risk is maintained upon immigration to an industrialized country, but the offspring of such immigrants have a higher immune‐mediated disease risk than the indigenous population. We hypothesize that early life exposures in a developing country shape the maternal immune system, which could have implications for the offspring born in a developed country with a low infectious load. The aim of this study was to investigate if exposures in childhood (indicated by country of origin) and subsequent exposures influence immunologic characteristics relevant to stimulation of offspring. Breast milk components among 64 mothers resident in Sweden, 32 of whom immigrated from a developing country, were examined using the ELISA and Cytometric Bead Array methods. Immigrants from a developing country had statistically significantly higher levels of breast milk interleukin‐6 (IL‐6), IL‐8 and transforming growth factor‐β1. A larger number of previous pregnancies were associated with down‐regulation of several substances, statistically significant for soluble CD14 and IL‐8. The results suggest that maternal country of birth may influence adult immune characteristics, potentially relevant to disease risk in offspring. Such a mechanism may explain the higher immune‐mediated disease risk among children of migrants from a developing to developed country. Older siblings may influence disease risk through the action of previous pregnancies on maternal immune characteristics.

CD14 and development of atopic disease at 2 years of age in children with atopic or non‐atopic mothers

Background CD14, a myeloid cell marker and LPS receptor has been acclaimed to play a role in development and manifestation of atopic allergy, as the gene encoding CD14 is located in a chromosomal region linked to total IgE levels and atopic disease.

Maternal allergy influences p38-mitogen-activated protein kinase activity upon microbial challenge in CD14+ monocytes from 2-year-old children

Background The development of allergic diseases is dependent on genetic and environmental factors. It has been shown previously that cord blood mononuclear cells (CBMCs) from infants with parental allergy have altered cytokine profiles upon bacterial encounter; it might be possible that such impairment persists during the early years of childhood.

Staphylococcus aureus-derived factors induce IL-10, IFN-γ and IL-17A-expressing FOXP3+CD161+ T-helper cells in a partly monocyte-dependent manner

Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4+FOXP3+ T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10, IFN-γ and IL-17A in FOXP3+ cells. Further, CD161 and HELIOS separated the FOXP3+ cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3+ cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3+ cells. Together, these data show that S. aureus potently induces FOXP3+ cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3+ cells to inflammation and repression of immune-suppression upon encounter with S. aureus.

Peripheral monocyte expression of the chemokine receptors CCR2, CCR5 and CXCR3 is altered at parturition in healthy women and in women with systemic lupus erythematosus.

Monocytes are precursors of macrophages and recruited to the uterus throughout pregnancy to perform important immunological functions. In this study, we hypothesized that pregnant women have reduced peripheral monocyte expression of chemokine receptors and alterations in PBMC responses to microbial stimuli as an adaption to pregnancy and that these changes are less pronounced in women with autoimmunity. We therefore investigated the chemokine receptor expression, migratory behaviour and responses to microbial stimulation of peripheral monocytes from pregnant women at parturition (n = 13) and from non‐pregnant women (n = 9). In addition, we compared healthy pregnant women with women suffering from SLE (n = 5), a condition with pronounced systemic inflammation increasing the risk for pregnancy complications. We demonstrate that peripheral monocytes are affected by pregnancy with reduced percentages of CCR2+, CCR5+ and CXCR3+ monocytes of both classical (CD16−) and inflammatory (CD16+) subsets and that the trophoblast‐secreted chemokine CCL2/MCP‐1 recruited monocytes of both subsets in vitro. Further, PBMCs from pregnant women had a divergent response to microbial stimulation with lower CCL5/RANTES and higher CCL2/MCP‐1 secretion compared with non‐pregnant women. In addition, pregnant women had lower basal PBMC‐secretion of CCL5/RANTES and higher basal secretion of IL‐10 and CCL2/MCP‐1. Interestingly, the women with SLE responded similar to pregnancy as did healthy women with lower percentages of CCR2+, CCR5+ and CXCR3+ monocytes. However, they had increased expression of CCR5 on CD16+ monocytes and heightened PBMC‐secretion of CCL5/RANTES. In conclusion, our data indicate that monocyte chemokine receptor expression and the chemokine milieu during pregnancy are tightly regulated to support pregnancy.