Agustina Serafino

Conference 2022 Live Talk

 

Talk title

Brucella abortus RNA interferes with M1 polarization of human macrophages

 

Authors and Affiliations

Agustina Serafino1; José Luis Marin Franco1; Aldana Trotta1; Melanie Genoula1; Luis A. Castillo1; Federico Birnberg Weiss1, Luciana Balboa1; Paula Barrionuevo*1 and M. Ayelén Milillo*1.

1. Instituto de Medicina Experimental-CONICET, Academia Nacional de Medicina; Buenos Aires, Argentina.
*Both authors contributed equally to this work

 

Abstract

Background

Brucellosis is a zoonotic disease caused by Brucella spp bacteria. These pathogens can survive inside macrophages, persisting inside the host. Monocytes and macrophages play a central role in chronic brucellosis. Macrophages could be differentiated into classical (M1 -microbicidal, inflammatory) or alternative (M2-or tissue healers) profiles, among others. We previously demonstrated that Brucella abortus (Ba) RNA is a PAMP involved in the immune evasion mediated by Ba. One of the mechanisms displayed by this bacterium is the down-modulation of MHC molecules when Th1 response is being held, i.e., in the presence of IFN-gamma, a condition that allows macrophages to polarize to M1. So, besides MHC down-modulation, we aimed to evaluate if Ba RNA could interfere with M1 polarization.

Methods

For this, human primary monocytes were differentiated to macrophages and treated with Ba RNA (5 μg/ml) under M1 conditions (IFN-gamma +LPS) for 24 and 48 h in parallel with untreated and positive controls. Afterwards, M1 (CD64, MHC-II and CD86) or M2 (CD206, CD163 and DC-SIGN) markers were assessed by flow cytometry. Moreover, pro (IL-1beta, TNF-alpha and IL-8) and anti-inflammatory (IL-10) cytokines were quantified by ELISA in supernatants of treated macrophages. Last, Nitrogen Reactive Species were quantified by Griess reaction (N>5 in all experiments).

Results

At 24 h, no M1 marker was modified by Ba RNA in (IFN-gamma + LPS)-treated cells. We observed that at 48 h Ba RNA diminished the (IFN-gamma + LPS)-induced MHC-II and CD64 surface expression. Secretion of pro and anti-inflammatory cytokines did not change at 24 h. However, at 48 h, IL-8 was decreased in supernatants of Ba RNA + (IFN-gamma + LPS)-treated cells. The down-modulation of M1 markers at 48 h was not associated with an alternative activation of these cells (M2), as shown by unchanged DC-SIGN, CD163 and CD206 surface expression at neither time. More importantly, secretion of Nitrogen Reactive Species -hallmark of M1 activation- was diminished in Ba RNA-treated M1 macrophages.

Conclusions

Overall, our results show that Ba RNA could alter the proper immune response set to counterattack the bacteria which could persist in the host establishing a chronic infection, basically by interfering with M1 polarization. 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 tumors.