Antibiotic use during pregnancy is linked to offspring gut microbial dysbiosis, barrier disruption, and altered immunity along the gut–lung axis

Antibiotic use during pregnancy is associated with increased asthma risk in children. Since approximately 25% of women use antibiotics during pregnancy, it is important to identify the pathways involved in this phenomenon. We investigate how mother-to-offspring transfer of antibiotic-induced gut microbial dysbiosis influences immune system development along the gut–lung axis. Using a mouse model of maternal antibiotic exposure during pregnancy, we immunophenotyped offspring in early life and after asthma induction. In early life, prenatal-antibiotic exposed offspring exhibited gut microbial dysbiosis, intestinal inflammation (increased fecal lipocalin-2 and IgA), and dysregulated intestinal ILC3 subtypes. Intestinal barrier dysfunction in the offspring was indicated by a FITC-dextran intestinal permeability assay and circulating lipopolysaccharide. This was accompanied by increased T-helper (Th)17 cell percentages in the offspring's blood and lungs in both early life and after allergy induction. Lung tissue additionally showed increased percentages of RORγt T-regulatory (Treg) cells at both time points. Our investigation of the gut–lung axis identifies early-life gut dysbiosis, intestinal inflammation, and barrier dysfunction as a possible developmental programming event promoting increased expression of RORγt in blood and lung CD4+ T cells that may contribute to increased asthma risk.Fil: Alhasan, Moumen M.. Universität zu Berlin; AlemaniaFil: Hölsken, Oliver. Freie Universität Berlin; AlemaniaFil: Duerr, Claudia. Freie Universität Berlin; AlemaniaFil: Helfrich, Sofia. Freie Universität Berlin; AlemaniaFil: Branzk, Nora. Freie Universität Berlin; AlemaniaFil: Philipp, Alina. Freie Universität Berlin; AlemaniaFil: Leitz, Dominik. Freie Universität Berlin; AlemaniaFil: Duerr, Julia. Freie Universität Berlin; AlemaniaFil: Almousa, Yahia. Freie Universität Berlin; AlemaniaFil: Barrientos, Gabriela Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Hospital Aleman; ArgentinaFil: Mohn, William W.. University of British Columbia; CanadáFil: Gamradt, Stefanie. Freie Universität Berlin; AlemaniaFil: Conrad, Melanie L.. Freie Universität Berlin; Alemani

Chromosomal Instability Estimation Based on Next Generation Sequencing and Single Cell Genome Wide Copy Number Variation Analysis

<div><p>Genomic instability is a hallmark of cancer often associated with poor patient outcome and resistance to targeted therapy. Assessment of genomic instability in bulk tumor or biopsy can be complicated due to sample availability, surrounding tissue contamination, or tumor heterogeneity. The Epic Sciences circulating tumor cell (CTC) platform utilizes a non-enrichment based approach for the detection and characterization of rare tumor cells in clinical blood samples. Genomic profiling of individual CTCs could provide a portrait of cancer heterogeneity, identify clonal and sub-clonal drivers, and monitor disease progression. To that end, we developed a single cell Copy Number Variation (CNV) Assay to evaluate genomic instability and CNVs in patient CTCs. For proof of concept, prostate cancer cell lines, LNCaP, PC3 and VCaP, were spiked into healthy donor blood to create mock patient-like samples for downstream single cell genomic analysis. In addition, samples from seven metastatic castration resistant prostate cancer (mCRPC) patients were included to evaluate clinical feasibility. CTCs were enumerated and characterized using the Epic Sciences CTC Platform. Identified single CTCs were recovered, whole genome amplified, and sequenced using an Illumina NextSeq 500. CTCs were then analyzed for genome-wide copy number variations, followed by genomic instability analyses. Large-scale state transitions (LSTs) were measured as surrogates of genomic instability. Genomic instability scores were determined reproducibly for LNCaP, PC3, and VCaP, and were higher than white blood cell (WBC) controls from healthy donors. A wide range of LST scores were observed within and among the seven mCRPC patient samples. On the gene level, loss of the <i>PTEN</i> tumor suppressor was observed in PC3 and 5/7 (71%) patients. Amplification of the androgen receptor (<i>AR</i>) gene was observed in VCaP cells and 5/7 (71%) mCRPC patients. Using an <i>in silico</i> down-sampling approach, we determined that DNA copy number and genomic instability can be detected with as few as 350K sequencing reads. The data shown here demonstrate the feasibility of detecting genomic instabilities at the single cell level using the Epic Sciences CTC Platform. Understanding CTC heterogeneity has great potential for patient stratification prior to treatment with targeted therapies and for monitoring disease evolution during treatment.</p></div

Prostate cancer cell line single cell CNV profiles, genomic instability scores and AR, PTEN copy number status.

<p>Whole genome copy number plots from prostate cancer cell lines (A) LNCaP, (B) PC3, and (C) VCaP, and (D) WBC controls. (E) Absolute Pearson correlation values (0–100%) were calculated across samples and viewed using Circos Table Viewer (<a href="http://circos.ca/presentations/articles/vis_tables1/" target="_blank">http://circos.ca/presentations/articles/vis_tables1/</a>). For visualization purposes, the top 25% highest correlations are displayed. Each color-coded segment represents a cell line replicate. Correlations between replicates are denoted by links or ribbons, the width of which is proportional to the degree of correlation. Much higher correlations were observed in intra-cell line comparisons than inter-cell line comparisons, indicating that the assay has good reproducibility regardless of cell line used. (F) Box-whisker plot of LST scores for prostate cancer cell lines and WBCs. All 3 cell lines had high LST scores compared to the WBCs, with PC3 and VCaP having the highest scores. (G) Box-whisker plot of log2 normalized DNA copy number in <i>AR</i>. Amplification of the <i>AR</i> gene was observed in the VCaP single cells reproducibly (5/5, 100%). This amplification was not observed in PC3 (0/7), LNCaP (0/8), or WBC controls (0/3). (H) Box-whisker plot of log2 normalized DNA copy number in <i>PTEN</i>. The VCaP cell line has non-deleted <i>PTEN</i> (0/5, 0%), while <i>PTEN</i> loss was detected in PC3 (6/7, 86%), LNCaP (1/8, 13%), and 1/3 WBC controls (1/3, 33%).</p

<i>PTEN</i> status detected by CNV and FISH in patient CTCs.

<p><i>PTEN</i> status detected by CNV and FISH in patient CTCs.</p

Patient demographics.

<p>Patient demographics.</p

Representative images of CTCs identified.

<p>Representative CTC images from cell lines (A) LNCaP, (B) PC3, and (C) VCaP. Slides were stained with CK, AR, CD45, and DAPI. Individual CTCs were identified by Epic Sciences’ algorithm and visually confirmed.</p

Prostate Cancer Cell Line Genomic Instability Scores.

<p>Prostate Cancer Cell Line Genomic Instability Scores.</p