Aberrant Pregnancy Adaptations in the Peripheral Immune Response in Type 1 Diabetes: A Rat Model

Abstract

Introduction: Despite tight glycemic control, pregnancy complication rate in type 1 diabetes patients is higher than in normal pregnancy. Other etiological factors may be responsible for the development of adverse pregnancy outcome. Acceptance of the semi-allogeneic fetus is accompanied by adaptations in the maternal immune-response. Maladaptations of the immune-response has been shown to contribute to pregnancy complications. We hypothesized that type 1 diabetes, as an autoimmune disease, may be associated with maladaptations of the immune-response to pregnancy, possibly resulting in pregnancy complications.

Methods: We studied pregnancy outcome and pregnancy-induced immunological adaptations in a normoglycemic rat-model of type 1 diabetes, i.e. biobreeding diabetes-prone rats (BBDP; 5 non-pregnant rats, 7 pregnant day 10 rats and 6 pregnant day 18 rats) , versus non-diabetic control rats (i.e. congenic non-diabetic biobreeding diabetes-resistant (BBDR; 6 non-pregnant rats, 6 pregnant day 10 rats and 6 pregnant day 18 rats) and Wistar-rats (6 non-pregnant, 6 pregnant day 10 rats and 5 pregnant day 18 rats)).

Results: We observed reduced litter size, lower fetal weight of viable fetuses and increased numbers of resorptions versus control rats. These complications are accompanied by various differences in the immune-response between BBDP and control rats in both pregnant and non-pregnant animals. The immune-response in non-pregnant BBDP-rats was characterized by decreased percentages of lymphocytes, increased percentages of effector T-cells, regulatory T-cells and natural killer cells, an increased Th1/Th2-ratio and activated monocytes versus Wistar and BBDR-rats. Furthermore, pregnancy-induced adaptations in BBDP-rats coincided with an increased Th1/Th2-ratio, a decreased mean fluorescence intensity CD161a/NKR-P1b ratio and no further activation of monocytes versus non-diabetic control rats.

Conclusion: This study suggests that even in the face of strict normoglycemia, pregnancy complications still occur in type 1 diabetic pregnancies. This adverse pregnancy outcome may be related to the aberrant immunological adaptations to pregnancy in diabetic rats.

Figure 1. Flowchart of the experimental design.

Figure 2. An representative example of a FACS analysis procedure to evaluate lymphocytes subpopulations.

[A] A forward/sideward (FSC/SSC) scatterplot of all events in which leukocytes were identified. [B] The leukocyte gate was copied to a FSC/SSC scatterplot in which the lymphocytes are identified. [C] These lymphocytes are copied to a SSC/CD3-PerCP scatterplot to distinguish T-lymphocytes (CD3⁺). [D] The lymphocytes are further divided into helper (CD3⁺/CD4⁺) and cytotoxic (CD3^+/CD4⁺; ct) T-cells in a CD3 PerCP/CD4 Alexa 700 scatterplot. [E] A CD3 PerCP/CD25 FITC scatterplot in which CD3⁻ cells were copied to assess the gate for FITC-positivity. [F] This gate for FITC positivity was copied to a CD4 Alexa 700/CD25 FITC scatterplot to identify CD25 positive and negative cells; i.e. effector (CD3⁺/CD4⁺/CD25⁺) and naive helper T-cells (CD3⁺/CD4⁺/CD25⁻). [G] A CD25 FITC/FoxP3 Alexa 647 scatterplot to identify the regulatory T-cell (CD3⁺/CD4⁺/CD25⁺/FoxP3⁺; Treg) positive population.

Figure 3. Lymphocyte subpopulations in non-pregnant rats.

[A] The percentages of T-lymphocytes (of the total leukocyte population), the ratio of T-helper cells (Th) and cytotoxic T-cells (Tc), percentages effector T-cells (of Th; Teff), regulatory T-cells (of Th; Treg), NK-cells (of total lymphocyte population) and the ratio of MFI CD161a/NKR-P1b of NK-cells of the three non-pregnant rat-strains. [B] The Th1/Th2-mRNA ratio and mRNA expression of ROR-C of the three non-pregnant rat-strains. Values are expressed as median with range (Q₁–Q₃), mRNA was expressed as Fold Change (2^∧−ΔCT). ‘*’ significant difference, Mann-Whitney U-Test, p<0.05.

Figure 4. Monocyte subpopulations in non-pregnant rats.

The ratio of non-classical/classical monocytes [A], the percentage of CD4⁺ classical monocytes [B], the MFI of CD4 on classical monocytes [C], the percentage of CD4⁺ non-classical monocytes [D] and the MFI of CD4 on non-classical monocytes [E] in non-pregnant animals of each strain. Values expressed as median with range (Q₁–Q₃), ‘*’ significant difference, Mann-Whitney U-Test, p<0.05.

Figure 5. Lymphocyte subpopulations in pregnant rats.

Values of day 10 or 18 of pregnancy are recalculated regarding the median values of non-pregnant animals (which was set at 100%) to express (as median (Q₁–Q₃)) pregnancy-induced changes. [A] The adaptations to pregnancy in percentage of T-lymphocytes, ratio of T-helper cells (Th) and cytotoxic T-cells (Tc), percentages of effector T-cells (Teff), regulatory T-cells (Treg), NK-cells and the ratio of MFI CD161a/NKR-P1b of NK-cells in all three strains during pregnancy. [B] The adaptations to pregnancy of the Th1/Th2-mRNA ratio and ROR-C mRNA expression in all three strains during pregnancy. d10/d18: day 10 or 18 of pregnancy. ∞slope significantly different from zero (Regression-analysis, ANCOVA, p<0.05). *slope significant different between the marked strains (Regression-analysis, ANCOVA, p<0.05).

Figure 6. Monocyte subpopulations in pregnant rats.

Values of day 10 or 18 of pregnancy are recalculated regarding the median values of non-pregnant animals (which was set at 100%) to express (as median (Q₁–Q₃)) pregnancy-induced changes. [A] The adaptations to pregnancy in the ratio non-classical/classical monocytes, [B] the percentage of CD4⁺ classical monocytes, [C] the MFI of CD4 on classical monocytes, [D] the percentage of CD4⁺ non-classical monocytes and [E] the MFI of CD4 on non-classical monocytes of each strain during pregnancy. d10/d18: day 10 or 18 of pregnancy. ∞slope significantly different from zero (Regression-analysis, ANCOVA, p<0.05). *slope significant different between the marked strains (Regression-analysis, ANCOVA, p<0.05).