Lower Abundance and Impaired Function of CD71+ Erythroid Cells in Inflammatory Bowel Disease Patients During Pregnancy

Abstract

Background and aims: CD71+ erythroid cells are enriched during pregnancy with immuno suppressive properties. We investigated the frequency and functionality of CD71+ erythroid cells in peripheral blood, cord blood, and placenta of inflammatory bowel disease [IBD] patients versus healthy controls [HCs]. We aimed to determine their role in IBD pathogenesis during pregnancy.

Methods: Peripheral blood was collected at preconception, the first, second and third trimesters, and postpartum. Cord blood and placental tissues were collected at the time of birth. Cells from different specimens were subjected to immune-phenotyping and functional assays. CD71+ erythroid cells were purified for quantitative polymerase chain reaction [qPCR] analysis. Using an allogeneic mouse model of pregnancy, the effects of CD71+ erythroid cells depletion on intestinal homeostasis and dysbiosis was studied.

Results: IBD patients had lower CD71+ erythroid cells during pregnancy compared with HCs. Placenta and cord blood CD71+ erythroid cells from IBD patients exhibited impaired functionality and expressed lower inhibitory molecules including VISTA, TGF-β, and reactive oxygen species [ROS]. Lower CD71+ erythroid cells were correlated with reduced regulatory T cells and increased immune-activation in IBD patients. Depletion of CD71+ erythroid cells in an allogeneic pregnancy model resulted in upregulation of TLRs, IL-6, and CXCL-1, and enhanced production of TNF-α, in intestinal tissues. In contrast, TGF-β gene expression was reduced. Excessive inflammatory response in the gut [e.g. TNF-α] affects intestinal integrity and CD71+ erythroid cells impact on the gut's bacterial composition.

Conclusions: Reduced frequency and/or impaired functionality of CD71+ erythroid cells during pregnancy may predispose IBD patients to a more pro-inflammatory milieu in their gastrointestinal tract, characterised by lower Tregs, higher IL-6, and TNF-α, and dysbiosis.

Figures

Figure 1.

Expansion of CD71+ erythroid cells in human pregnancy. [A] Representative dot plots showing gating strategy and frequency of CD71+ erythroid cells in blood of pregnant versus non-pregnant women. [B] Cumulative data indicating percentages of CD71+ erythroid cells in preconception [PC], first trimester [T1], second trimester [T2], third trimester [T3], and postpartum [PP]. [C] Representative dot plots showing frequency of CD71+ erythroid cells in placenta tissues of a healthy woman. [D] Percentages of CD71+ erythroid cells in placental tissues of healthy women. [E] Representative dot plots showing proliferation of CD3+ T cells as measured by carboxyfluorescein succinimidyl ester[CFSE] dye in peripheral blood mononuclear cells [PBMCs] in the presence or absence of CD71+ erythroid cells. [F] Percentage of CD3+ T cells proliferation in PBMCs in the presence or absence of CD71+ erythroid cells. [G] Representative dot plots showing IL-6 and TNF-α production among CD11b+ cells in PBMCs in the presence or absence of CD71+ erythroid cells. [H] Percentages of CD11b+ cells producing IL-6, TNF-α or both cytokines, in the presence or absence of CD71+ erythroid cells. [I] Representative dot plots showing proliferation of CD4+ and CD8+ T cells in the presence or absence of CD71+ erythroid cells. [J] Percentages of proliferated CD4+ and CD8+ T cells in placenta cells in the presence or absence of CD71+ erythroid cells.

Figure 2.

Lower frequency of CD71+ erythroid cells in IBD patients during pregnancy. [A] Representative dot plots showing frequency of CD71+ erythroid cells in the peripheral blood of ulcerative colitis [UC], Crohn’s disease [CD]. and healthy controls [HC] in the third trimester [T3]. [B] Percentages of CD71+ erythroid cells in the peripheral blood of pregnant women [T3], either those with UC or CD, or HCs. [C] Percentages of Tregs at T3 in inflammatory bowel disease [IBD] versus HCs. [D] Correlation of % Tregs with % CD71+ cells in IBD patents. [E] Representative dot plots showing how Tregs are defined. [F] Representative dot plots showing frequency of CD71+ erythroid cells in placental tissues of UC, CD, and HC. [G] Percentages of CD71+ erythroid cells in placental tissues of UC, CD or HCs. [H] Correlation of activated cells [CD71+ cells lymphocytes] with frequency of CD71+ erythroid cells in placenta. [I] Percentages of caesarean section [C-section] in HC versus IBD pregnancies in the cohort. [J] Percentages of preterm deliveries by HC versus IBD mothers in the cohort. [K] Detected levels of serum erythropoietin [EPO] by ELISA at T3 in IBD versus HCs.

Figure 3.

Lack of immunosuppression by CD71+ erythroid cells in inflammatory bowel disease [IBD] patients. [A] Representative dot plots showing proliferation of CD4+ T cells in peripheral blood mononuclear cells [PBMCs] of an ulcerative colitis [UC] patient. [B] Percentages of CD4+ and CD8+ T cells proliferation in PBMCs of UC patients in the presence or absence of CD71+ erythroid cells. [C] Representative dot plots showing proliferation of CD4+ T cells in PBMCs of a Crohn’s disease [CD] patient. [D] Percentages of CD4+ and CD8+ T cells proliferation in PBMCs of CD patients in the presence or absence of CD71+ erythroid cells. [E] TNF-α production by PBMCs of UC patients; or [F] CD patients. [G] TNF-α production by PBMCs of UC patients; or [H] CD patients following in vitro stimulation with anti-CD3 as measured by ELISA. [I] Correlation of CD71+ erythroid cells frequency in mothers in the third trimester [T3] with faecal calprotectin [FCP] levels in meconium.

Figure 4.

Impaired functionality of CD71+ erythroid cells from cord blood of newborns to inflammatory bowel disease [IBD] mothers. [A] Representative dot plots showing frequency of CD71+ erythroid cells in cord blood mononuclear cells [CBMCs] of infants born to ulcerative colitis [UC], Crohn’s disease [CD], and healthy control [HC] mothers. [B] Percentages of CD71+ erythroid cells in CBMCs of infants born to UC, CD, and HC mothers. [C] Representative dot plots showing proliferation of CD4+ T cells in the presence or absence of CD71+ erythroid cells among CBMCs of an infant born to an HC mother. [D] Percentages of proliferated CD4+ T cells and CD8+ T cells in the presence or absence of CD71+ erythroid cells among CBMCs of infants born to HC mothers following stimulation with anti-CD3 in vitro. [E] Representative dot plots showing proliferation of CD4+ T cells in the presence or absence of CD71+ erythroid cells among CBMCs of an infant born to a UC mother. [F] Percentages of proliferated CD4+ T cells and CD8+ T cells in the presence or absence of CD71+ erythroid cells among CBMCs of infants born to UC mothers. [G] Representative dot plots showing proliferation of CD4+ T cells in the presence or absence of CD71+ erythroid cells among CBMCs of an infant born to a CD mother. [H] Percentages of proliferated CD4+ T cells and CD8+ T cells in the presence or absence of CD71+ erythroid cells among CBMCs of infants born to CD mothers. [I] TNF-α and [J] IL-6 production by stimulated CBMCs with anti-CD3 antibody obtained from infants born to HC mothers in the presence or absence of CD71+ erythroid cells. [K] TNF-α and [L] IL-6 production by stimulated CBMCs with anti-CD3 antibody obtained from infants born to UC mothers. [M] TNF-α and [N] IL-6 production by stimulated CBMCs with anti-CD3 antibody obtained from infants born to CD mothers in the presence or absence of CD71+ erythroid cells as measured by ELISA.

Figure 5.

Differential gene expression of inhibitory and stimulatory molecules by CD71+ erythroid cells of inflammatory bowel disease [IBD] patients versus healthy controls [HCs]. [A] Representative dot plots showing production of TGF-β by neonatal mice CD71+ erythroid cells. [B] Expression of TGF-β gene by CD71+ erythroid cells from cord blood or [C] placenta of HC compared with ulcerative colitis [UC] and Crohn’s disease [CD] patients. [D] Expression of PD-1H gene by CD71+ erythroid cells from cord blood or [E] placenta of HC compared with UC and CD patients. [F] Expression of arginase-2 gene by CD71+ erythroid cells from cord blood or [G] placenta of HC compared with UC and CD patients. [H] Representative plots showing expression of reactive oxygen species [ROS] among CD71+ erythroid cells from cord blood [pink] or placenta [orange] compared with isotype control [grey] of an HC. [I] Cumulative data showing percentages and [J] mean fluorescence intensity [MFI] of ROS+CD71+ erythroid cells in cord blood versus placenta of HCs. [K] Representative plots showing expression ROS among CD71+ erythroid cells from cord blood [pink] or placenta [orange] compared with isotype control [grey] of an IBD patient. [L] Cumulative data showing percentages of ROS+CD71+ erythroid cells in cord blood and [M] placenta of IBD versus HCs. [N] Expression of VEGF-α gene by CD71+ erythroid cells from cord blood or [O] placenta of HC compared with UC and CD patients. Data are obtained from a minimum of 5–7 patients/group.

Figure 6.

CD71+ erythroid cells may impact on gut haemostasis during pregnancy. [A] Representative dot plots showing percentages of CD71+ erythroid cells in placenta and [B] spleen of mice before and after anti-CD71 antibody treatment. [C] Expression of TLR-1, [D] TLR-4, [E] TLR-9, [F] TLR-2, [G] CXCL-1, [H] IL-6 and [I] TGF-β genes by gut tissues of non-pregnant compared with pregnant mice treated with rat IgG isotype control or anti-CD71 antibody three days after treatment. [J] Representative plots and [K] cumulative data showing IL-6 and TNF-α production by intestinal CDllc+ cells. [L] Representative plots and [M] cumulative data showing IL-6 and TNF-α production by intestinal CDllb+ cells. [N] Expression of VEGFα gene in placenta tissues. [O] Treated mice were subjected to 16S rRNA-based polymerase chain reaction [PCR] for total bacteria, [P] Bacteroides-Prevotella-Porphyromonas group [Q] Enterobacteriaceae group, [R] Clostridium cluster XIVa, [S] Clostridium cluster 1, and [T] Clostridium cluster IV. [U] Levels of fluorescein isothiocyanate labelled dextran [FITC-dextran] in the blood of IgG versus anti-CD71 treated mice. Data are obtained from a minimum of five mice/group and at least two independent experiments.