The Selenoprotein Thioredoxin Reductase 1 Is Crucial for B-1 Cell but Dispensable for B-2 Cell Development.
Authors: Kumar Mandal Pankaj , Zimber-Strobl Ursula , Bornkamm Georg W. , Gerbitz Armin , Conrad Marcus 
Published: October 2019

Abstract
Abstract 3691
Poster Board III-627

Introduction
A growing body of evidence has implicated the role of reactive oxygen species (ROS) as second messengers in the signaling of various cytokines and growth factors that govern innate immunity and inflammation. For example, ROS is involved in TLR4-mediated activation of MAPK pathways. However, oxidative stress resulting from elevated ROS levels is involved in the etiology of many pathological conditions. Although immune cells generate ROS for signaling and microbicidal activity, they are extremely sensitive towards oxidative stress and therefore maintain high levels of antioxidants. The immunomodulatory and antioxidant function of selenium is in part due to the incorporation of selenium into selenoproteins, which are an important component of the antioxidant system. The selenoprotein thioredoxin reductase 1 (Txnrd1) and its major substrate thioredoxin 1 are essential for embryogenesis and are implicated in sustaining high proliferation rates due to provision of electrons for ribonucleotide reductase. The rapid turnover of immune cells upon activation and the extreme sensitivity towards ROS prompted us to address the role of Txnrd1 in B cell development and physiology.

Methods
Using the Cre-loxP system, we addressed the role of Txnrd1 in B cell development. B cell–specific Txnrd1 knockout mice were generated by breeding conditional Txnrd1 knockout mice (Txnrd1fl/fl; fl= loxP flanked allele) with mb-1 cre mice, in which expression of cre recombinase is driven by the murine mb-1 locus. Cells from bone marrow, spleen, lymph node and peritoneal cavity were isolated and analyzed by FACS using combinations of cell surface markers. For in vitro stimulation experiments, untouched B cells were isolated from spleen by MACS using CD43 microbeads and treated with various stimulants (α-CD40, α-CD40+IL4, LPS, LPS+IL4 and α-IgM).

Results
B-cell specific ablation of Txnrd1 leads to severly impaired development of peritoneal cavity B cells (PercB or B-1). The dramatic reduction in B-1 cells in mb-1 Cre; Txnrd1fl/fl mice indicated that Txnrd1 is required for the development and/or self-renewal of B-1 cells. Analysis of B220+ cells from bone marrow, spleen and lymph node revealed a normal distribution of different subpopulation of B-2 cells, demonstrating the dispensable role of Txnrd1 in B-2 cell development. In vitro stimulation of Txnrd1 deficient B-2 cells with various stimuli (stated above) revealed normal activation as shown by increased expression of activation markers CD86 and CD95. However, a higher percentage of dead cells was detected in Txnrd1 null B-2 cells as compared to control cells. This suggests that either Txnrd1 deficient B-2 cells fail to proliferate in response to various stimuli or undergo activation-induced cell death.

Conclusions
Our data provide first evidence that Txnrd1 is indispensable for the development and/or self-renewal of B-1 cells. As peritoneal cavity B-cells are responsible for producing natural antibodies, it is tempting to speculate that the compromised Txnrd1 function may lead to an increased susceptibility towards pathogens. Although Txnrd1 deficiency has little impact on the development of B-2 cells, they show compromised proliferation or activation-induced cell death upon stimulation. These findings could either be due to the essential role of Txnrd1 in sustaining high proliferation rates or the induction of cell death in response to massive oxidative stress resulting from receptor activation-induced signaling. Further studies will provide intriguing insight in to the downstream signaling cascades and in the immune response upon immunization.

Disclosures:
No relevant conflicts of interest to declare.