DR. JULIO LUIS HANG

Note: This part of the results section, i.e. “The pre-delivery inflammatory reaction is mitigated by MO in C57BL/6N mice”, was published in the Journal of Reproductive Immunology in the article “Maternal obesity attenuates predelivery inflammatory reaction in C57BL/6N mice” [79]. Sarah Appel, Merle Schulze-Edinghausen and Tobias Kretschmer contributed equally to this work with the help of several others:

Animal handling and tissue collections was performed by Ruth Janoschek, Inga Bae-Gartz, Marion Handwerk and Sarah Appel. The qPCR assays were prepared and performed by Merle Schulze- Edinghausen, Tobias Kretschmer and Maria Wohlfarth, while data calculation was done by Merle Schulze-Edinghausen and Sarah Appel. Histological stainings (IHC for CD45) were performed by Tobias Kretschmer, while picture processing for cell counting was done by Merle Schulze-Edinghausen and Tobias Kretschmer. Lipid peroxidation assay was performed by Merle Schulze-Edinghausen and Maria Wohlfarth. Malte Heykants performed multiplex cytokine assays. Statistical analyses were performed by Merle Schulze-Edinghausen, Sarah Appel and Tobias Kretschmer. The project was supervised and supported by technical input and advice by Kai-Dietrich Nüsken, Eva Hucklenbruch- Rother, Esther Mahabir, Jörg Dötsch and Sarah Appel.

4.1.1 Inflammation, leukocyte infiltration and oxidative stress in placental tissue and egWAT

Two time-points of murine gestation, E15.5 which represents the beginning of the third trimester of pregnancy and E18.5 which represents the end of the third trimester shortly before parturition, were investigated on placental and egWAT level. While SD dams usually gave birth around E19, HFD dams took about an extra day before giving birth (Figure 4.1.1.2 D). Gene expression of IL-6 and TNFα, two pro-inflammatory cytokines, was significantly increased in placentas of SD dams on E18.5 compared to E15.5. MCP1 and the murine IL-8 homologue CXCL1 were not significantly altered and CXCL10, IL-1α and IL-1β were down-regulated without reaching statistical significance (Figure 4.1.1.1 B). On E18.5 in placentas of obese dams, TNFα was significantly up-regulated compared to E15.5 and IL-6, MCP1, and IL-1β were unaltered in expression. CXCL1, CXCL10 and IL-1α on the other hand appeared down- regulated, but without reaching statistical significance (Figure 4.1.1.1 C). Concomitantly, with an increase in IL-6 and TNFα gene expression at E18.5 compared to E15.5 in SD dams, there was significant infiltration of leukocytes (CD45-positive) in the Lz. This increase in leukocytes towards the end of pregnancy was not observed in HFD dams (Figure 4.1.1.1 A). Further, in both, SD and HFD placentas, lipid peroxidation, a marker for oxidative stress, as assessed by MDA assay was significantly increased at E18.5 compared to E15.5, however MDA level were also significantly higher in HFD placentas at E18.5 compared to SD (Figure 4.1.1.1 D). Anti-oxidative genes GPX and CAT were significantly down-regulated at E18.5 compared to E15.5 in SD placentas, whereas pro-oxidative stress genes remained largely unaltered (Figure 4.1.1.1 E).

4.RESULTS

The pro-oxidative genes CYBB, NCF1 and NCF2 were significantly up-regulated in placentas of obese dams at E18.5 compared to E15.5 and the anti-oxidative CAT gene expression was significantly reduced (Figure 4.1.1.1 F).

Figure 4.1.1.1: Inflammation, leukocyte infiltration and oxidative stress measurements in placentas at E15.5 and E18.5. (A) Quantitation of CD45-positive leukocytes in the Lz of the placenta of obese and lean dams at E15.5

and E18.5, n = 5 placentas for both groups and both gestations days. (B) Gene expression measurement by qPCR of pro-inflammatory factors in placentas of SD dams normalized to ACTB at E15.5 and E18.5. Expression was set to 1 for E15.5 and the fold change on E18.5 relative to E15.5 is shown. For SD at E15.5 n = 16 placentas from 5 dams, at E18.5 n = 25 placentas from 5 dams. (C) Gene expression measurement by qPCR of pro-inflammatory factors in placentas of HFD dams normalized to ACTB at E15.5 and E18.5. Expression was set to 1 for E15.5 and the fold change on E18.5 relative to E15.5 is shown. For HFD at E15.5 n = 21 placentas from 5 dams, at E18.5

n = 25 placentas from 5 dams. (D) Lipid peroxidation assay to determine oxidative stress level as assessed by

change in MDA in placentas of SD and HFD dams at E15.5 and E18.5. For SD n = 11 placentas from 11 dams of E15.5 and n = 9 placentas from 9 dams of E18.5. For HFD n = 10 placentas from 10 dams of E15.5 and n = 10 placentas from 10 dams of E18.5. (E) Gene expression measurement by qPCR of oxidative stress markers in placentas of SD dams normalized to ACTB at E15.5 and E18.5. Expression was set to 1 for E15.5 and the fold change on E18.5 relative to E15.5 is shown. For SD at E15.5 n = 21 placentas from 5 dams, at E18.5 n = 25 placentas from 5 dams. (F) Gene expression measurement by qPCR of oxidative stress markers in placentas of HFD dams normalized to ACTB at E15.5 and E18.5. Expression was set to 1 for E15.5 and the fold change on E18.5 relative to E15.5 is shown. For HFD at E15.5 n = 21 placentas from 5 dams, at E18.5 n = 25 placentas from 5 dams. ns = not significant; * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, calculated by two-tailed Student’s

t test (for normally distributed data) or a Mann-Whitney test (for non-normally distributed data). Graphs show

mean ± SEM. Figure was published in Appel, Schulze-Edinghausen, Kretschmer et al. [79] Figure 1 and was modified.

4.RESULTS

We further investigated inflammatory markers in the egWAT of both diet groups and gestation days, since we hypothesized that the egWAT might produce higher amounts of inflammatory markers that could affect uterine tissues due to its proximity to these tissues. Significantly elevated expression of pro-inflammatory genes IL-6, TNFα, CXCL1, CXCL10, MCP1 and IL-1β were measured in egWAT of SD dams at E18.5 compared to E15.5 (Figure 4.1.1.2 B). By contrast, these genes remained unaltered in egWAT of obese dams (Figure 4.1.1.2 C). Additionally, the amount of leukocytes was not significantly altered in egWAT in either diet group at E15.5 or E18.5 (Figure 4.1.1.2 A).

Figure 4.1.1.2: Leukocyte infiltration, inflammatory factor expression in egWAT at E15.5 and E18.5, and duration of gestation. (A) Quantitation of CD45-positive leukocytes in the egWAT of SD and HFD dams at E15.5

and E18.5, n = 5 egWAT for both groups and both gestation days. (B) Gene expression measurement by qPCR of pro-inflammatory factors in egWAT of SD dams normalized to ACTB at E15.5 and E18.5. Expression was set to 1 for E15.5 and the fold change on E18.5 relative to E15.5 is shown. For SD at E15.5 n = 8, at E18.5 n = 5. (C) Gene expression measurement by qPCR of pro-inflammatory factors in egWAT of HFD dams normalized to ACTB at E15.5 and E18.5. Expression was set to 1 for E15.5 and the fold change on E18.5 relative to E15.5 is shown. For SD at E15.5 n = 8, at E18.5 n = 8. (D) Duration of gestation expressed as the “birth date at Gx” in SD and HFD dams. n = 8 for SD and n = 11 for HFD. ns = not significant; * p <0.05, ** p <0.01, calculated by two-tailed Student’s

t test (for normally distributed data) or a Mann-Whitney test (for non-normally distributed data). Graphs show

mean ± SEM. Figure was published in Appel, Schulze-Edinghausen, Kretschmer et al. [79] Figure 1 and was modified.

4.RESULTS

4.2 MO affects EC homeostasis and causes elevated IL-6 serum level which could cause EC