Work Description

Date: 2023-12-08

Dataset Title: Supplementary Materials for The influence of maternal high fat diet during lactation on offspring hematopoietic priming

Dataset Creators: Singer, Kanakadurga

Dataset Contact: ksinger@umich.edu

Research Overview:
Obesity and metabolic diseases are rising in women of reproductive age, increasing offspring metabolic risk. Maternal nutritional interventions during lactation present an opportunity to modify offspring outcomes. We previously demonstrated in mice that adult male offspring have metabolic impairments and increased adipose tissue macrophages (ATM) when dams are fed high fat diet (HFD) during the postnatal lactation window (HFD PN). We sought to understand the effect of HFD during lactation on early life inflammation.

HFD PN offspring were evaluated at postnatal day 16-19 for tissue weight and gene expression. Profiling of adipose tissue and bone marrow immune cells was conducted through lipidomics, in vitro myeloid colony forming unit assays, and flow cytometry.

HFD PN mice had more visceral gonadal white adipose tissue (GWAT) and subcutaneous fat. Adipose tissue RNA sequencing demonstrated enrichment of inflammation, chemotaxis, and fatty acid metabolism and concordant changes in GWAT lipidomics. Bone marrow (BM) of both HFD PN male and female offspring had increased monocytes (CD45+Ly6G-CD11b+CD115+) and B-cells (CD45+Ly6G-CD11b-CD19+). Similarly, serum from HFD PN offspring enhanced in vitro BM myeloid colonies in a toll-like receptor 4-dependent manner. We identified that male HFD PN offspring had increased GWAT pro-inflammatory CD11c+ ATMs (CD45+CD64+).

Maternal exposure to HFD alters milk lipids enhancing adiposity and myeloid inflammation even in early life. Future studies are needed to understand the mechanisms driving this pro-inflammatory state of both BM and ATMs, the causes of the sexually dimorphic phenotypes and the feasibility of intervening in this window to improve metabolic health.

Methodology:
C57BL/6J male and female mice were purchased from the Jackson Laboratories. Breeding started at 10 weeks of age after a 2-week acclimation periods. Cages were inspected daily for litters 12 days after breeding started. After delivery of pups was detected, mice were either maintained on normal chow diet (5L0D, 13.5% fat, Lab Diets) or started on high fat diet (HFD, D12492:60% fat, Research Diets) until weaning. Offspring were termed control postnatal (Ctrl PN) or those exposed to HFD (HFD PN).

Instrument and/or Software specifications: NA

Files contained here:

093023 Supplementary Fig 1.pdf: Example gating scheme for bone marrow mature leukocytes and hematopoietic progenitors. To stain for mature leukocytes antibodies used were against CD45, Ly6G, CD11b, CD115, CD19, and CD3e. All CD45+ cells were gated first. Neutrophils were defined as Ly6G+CD11b+, monocytes were defined as Ly6G-CD11b+CD115+ (17,18), B cells were defined as Ly6G-CD11b-CD19+, and T cells were defined as Ly6G-CD11b-CD3e+. To stain for hematopoietic stem and progenitor cells antibodies used were against lineage panel (B220, Gr1, TER119, CD11b, CD4, CD8), cKit, Sca1, CD48, CD150, CD16/32, and CD105. HSCs were defined as Lin-Sca1+cKit+CD48-CD150+, MPPs were defined as Lin-Sca1+cKit+CD48-CD150-, HPC1 were defined as Lin-Sca1+cKit+CD48+CD150-, HPC2 were defined as Lin-Sca1+cKit+CD48+CD150+, GMP were defined as Lin-Sca1-cKit+CD150-CD16/32+, PreGM were defined as Lin-Sca1+cKit+CD150-CD105-, preMegE were defined as Lin-Sca1+cKit+CD150+CD105-, and PreCFUe were defined as Lin-Sca1+cKit+CD150+CD105+.

093023 Supplementary Fig 2.pdf: Hematopoietic stem and progenitor cell frequency by flow cytometry as a percentage of CD45 bone marrow cells in male and female Ctrl and HFD PN offspring. Hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), Pre-Granulocyte Macrophage (Pre-GM), granulocyte monocyte precursors (GMP), Pre-Megakaryocyte-Erythroid Precursors (Pre-MegE) and erythroid precursors (Pre-CFUE). Analyses were Student’s t-test ang gating per Supplementary Figure 1.

Supplementary Table 1 DESeq2 Results.csv: Differential gene expression between Ctrl and HFD PN male gonadal white adipose tissue (GWAT) from postnatal day 16. The significant gene expression differences were determined by the DESeq2 package for R Studio. Sequencing The RNA was extracted from adipose tissue using Trizol LS (Life Technologies) by Qiagen RNeasy Mini Kit (74106) according to the manual. The RNA was sent to the University of Michigan Advanced Genomics Core for RNA-sequencing. For RNAseq studies, gonadal white adipose tissue 3’ QuantSeq single-end poly-A mRNA libraries were generated (Lexogen). These were sequenced to a depth of 10-20M reads on Illumina NovaSeq platform. Data are available from GEO at accession number GSE227337.

Supplementary Table 2.csv: Differentially expressed genes between Ctrl and HFD PN male gonadal white adipose tissue (GWAT) from postnatal day 16 that are significant after correction for false discovery rate were determined by the DESeq2 package for R Studio.

Related publication(s):
Kim K, Varghese M, Sun H, Abrishami S, Bowers E, Bridges D, Meijer JL, Singer K* and Gregg B*. The influence of maternal high fat diet during lactation on offspring hematopoietic priming. Endocrinology. PMID 38048597.

Use and Access:
This data set is made available under a Creative Commons Public Domain license (CC0 1.0).

To Cite Data:
Singer, K. Supplementary Materials for The influence of maternal high fat diet during lactation on offspring hematopoietic priming [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/2nfz-3k25