Varney, M. E. | Search bioRxiv

bioRxiv2021

Prenatal cadmium exposure alters proliferation in mouse CD4+ T cells via LncRNA Snhg7

ObjectivePrenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA hostgene 7 (lncSnhg7) in T cell proliferation. MethodsRNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. ResultsWe identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. ConclusionPrenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

immunology↗

bioRxiv2019

Characterizing the innate and adaptive responses of immunized mice to Bordetella pertussis infection using in vivo imaging and transcriptomic analysis

Bordetella pertussis (B. pertussis) is the causative agent of pertussis (whooping cough). Since the 1990s, pertussis has re-emerged in the United States despite an estimated 95% vaccine coverage. Our goal was to characterize neutrophil responses and gene expression profiles of murine lungs in the context of vaccination and B. pertussis challenge. We utilized a bioluminescent neutrophil mouse model (NECre luc) to track neutrophil recruitment. NECre luc mice were immunized with whole cell vaccine (WCV), acellular vaccine (ACV), or a truncated adenylate cyclase toxoid (RTX) vaccine. Neutrophil recruitment was measured in live mice across time and corroborated by flow cytometry and other data. WCV immunized mice showed signs of neutrophilia in response to B. pertussis challenge. Mice immunized with either ACV or WCV cleared the challenge infection; however immunization with RTX alone was not protective. RNA sequencing revealed distinctive gene expression profiles for each immunization group. We observed an increase in expression of genes associated with responses to infection, and changes in expression of distinct genes in each vaccine group, providing a complex view of the immune response to B. pertussis infection in mice. This study suggests that combination of immunological analysis with transcriptomic profiling can facilitate discovery of pre-clinical correlates of protection for vaccine development.

immunology↗