Yasmín Ayelén Bertinat

Argentina

Ba RNA induces early activation but reduces proliferation and promotes apoptosis in lymphocytes

Yasmín Ayelén Bertinat1, Agustina Serafino1, Federico Birnberg Weiss1, Joselyn Castro1, María Ayelén Milillo2, Paula Barrionuevo1

1. Laboratorio de Fisiología de los Procesos Inflamatorios, IMEX-CONICET, Academia Nacional de Medicina, Buenos Aires, Argentina.
2. Instituto de Estudios en Ciencia, Tecnología, Cultura y Desarrollo (CITECDE), Universidad
Nacional de Río Negro Sede Andina-CONICET, Rio Negro, Argentina.

Abstract

Background

In order to survive inside the host, Brucella abortus (Ba) must trigger
different strategies to evade the adaptive T cell response it elicits. We have recently
demonstrated that Ba RNA (a PAMP related to pathogens’ viability or vita-PAMP) increases
the expression of the early activation marker CD69 on peripheral blood mononuclear cells
(PBMCs) independently of plate-bound anti-CD3 stimulation at 24 h. However, Ba RNA
decreases the expression of the senescence marker CD28 only in anti-CD3 pre-stimulated
PBMCs at 4 days. The aim of this study was to deepen this dual modulation of Ba
RNA-mediated lymphocyte response.

Methods

We first evaluated whether Ba RNA could reproduce this modulation in T
lymphocytes. T lymphocytes were obtained from PBMCs using a CD3+ T lymphocyte
negative isolation kit. T-cells previously stimulated or not with plate-bound anti-CD3 were
incubated with Ba RNA for 24, 48, and 96 h, the expression of CD69, PD-1, and CD28 was
evaluated by flow cytometry, respectively. We evaluated the proliferation and apoptosis on
PBMCs previously stimulated or not with anti-CD3 and treated with Ba RNA for 24, 48, 72
and 96 h. Proliferation was measured by MTT colorimetric assay. For apoptosis assays, cells were stained with Annexin V and Propidium Iodide and then evaluated by flow cytometry (N>4 in all experiments).

Results

Ba RNA increased the expression of CD69 at 24 h but decreased the expression of
CD28 at 4 days, only in anti-CD3 pre-stimulated T-cells. However, Ba RNA decreases the
proliferative response of PBMCs unstimulated or pre-stimulated at 72 and 96 h. More
importantly, Ba RNA increased the expression of PD-1 in unstimulated or pre-stimulated
T-cells at 48 h. In line with this, Ba RNA slightly increased the apoptosis (Annexin V+ cells) in
unstimulated cells at 48, 72 and 96 h but did not affect apoptosis in pre-stimulated PBMCs.

Conclusions

Overall, our results show that Ba RNA initially activates lymphocytes, but later
reduces proliferation and promotes apoptosis, which may favor the establishment of a
chronic infection. These results also lay the ground for studying more deeply the modulatory
properties of bacterial RNA in the context of brucellosis, other intracellular infections, and
inflammatory diseases.