Brucella abortus RNA does not polarize macrophages to a particular profile but interferes with M1 polarization

Monocytes and macrophages play a central role in chronic brucellosis. Brucella abortus (Ba) is an intracellular pathogen that survives inside these cells. On the other hand, macrophages could be differentiated into classical (M1), alternative (M2) or other less-identified profiles. We have previously shown that Ba RNA (a bacterial viability-associated PAMP or vita-PAMP) is a key molecule by which Ba can evade the host immune response. However, we did not know if macrophages could be polarized by this vita-PAMP. To assess this, we used two different approaches: we evaluated if Ba RNA per se was able to differentiate macrophages to M1 or M2 or, given that Ba survives inside macrophages once a Th1 response is established (i.e., in the presence of IFN-γ), we also analysed if Ba RNA could interfere with M1 polarization. We found that Ba RNA alone does not polarize to M1 or M2 but activates human macrophages instead. However, our results show that Ba RNA does interfere with M1 polarization while they are being differentiated. This vita-PAMP diminished the M1-induced CD64, and MHC-II surface expression on macrophages at 48 h. This phenomenon was not associated with an alternative activation of these cells (M2), as shown by unchanged CD206, DC-SIGN and CD163 surface expression. When evaluating glucose metabolism, we found that Ba RNA did not modify M1 glucose consumption or lactate production. However, production of Nitrogen Reactive Species (NRS) did diminish in Ba RNA-treated M1 macrophages. Overall, our results show that Ba RNA could alter the proper immune response set to counterattack the bacteria that could persist in the host establishing a chronic infection.


Introduction
Macrophages play a key role in immunity responses against pathogens. Not only do these cells have phagocytic functions but also discriminate self from non-self antigens. Once outside the bone marrow, monocytes can migrate to different tissues and differentiate into macrophages exhibiting a wide array of phenotypic and functional characteristics. Initially, two macrophage subpopulations had been described: classical macrophages (or M1; CD14 ++ CD16 -) and alternative macrophages (or M2; CD14 + CD16 ++ ). More recently, other profiles have been incorporated, such as tumour-associated macrophages (or TAM), CD169 + macrophages, which are implicated in immune tolerance and antigen presentation and TCR + macrophages [1,2]. M1 macrophages secrete pro-inflammatory cytokines, mediate resistance against pathogens and show potent microbicidal properties. However, they also contribute to the destruction of tissues. M2 macrophages are essential in parasitic infections, tissue remodelling, allergies, and angiogenesis [1,2].
Brucellosis is an infectious disease driven by bacteria from genus Brucella. Brucella abortus (Ba) is an intracellular pathogen whose preferential niches to persist are monocytes, macrophages, and trophoblastic cells. The infection with Ba triggers the innate and adaptive immunity towards Th1 and activation of CD8 + T cells [3][4][5]. Despite eliciting an immune response, this bacterium relies on several strategies to persist inside cells, evading host defence. Previous results from our laboratory demonstrate that infection of monocytes/macrophages with Ba diminishes MHC-I and MHC-II IFN-γ-induced surface expressions, impairing antigen presentation to T cells [6][7][8][9]. Moreover, a pathogen-associated molecular pattern (PAMP) associated with bacterial viability (vita-PAMP), the Ba RNA, sensed by Toll like receptors (TLR) 7/8 [10] is involved in both phenomena [11,12]. MHC-I and II down-modulation cause a reduction in antigen presentation of RNA-treated macrophages to CD8 + and CD4 + T cells, respectively [11,12]. However, we could not determine whether macrophages are differentiated to a particular profile or whether other macrophage functions were also affected. In line with this, Saha et al. (2017) demonstrated that TLR7/8 signalling triggered by Hepatitis C virus (HCV) promotes the differentiation of monocytes and polarization of macrophages towards an M2 profile [13]. We also demonstrated that RNA from other microorganisms (Escherichia coli, Klebsiella pneumoniae, Trypanosoma cruzi and Salmonella typhimurium) is also able to diminish MHC-I and II IFN-γ-induced surface expression on human monocytes [11,12]. Moreover, other pathogens activate macrophages via recognition of their RNA. The micro RNA 3 encoded by the Hepatitis B virus (HBV-miR-3) triggers M1 polarization on macrophages [14]. Streptococcus pyogenes RNA is sensed by TLR13 in murine bone marrow-derived macrophages and induce an immune response in vitro [15].
Regarding metabolic pathways in macrophages, M1 cells oxidise glucose towards aerobic glycolysis and lactate production (rapid fuel of energy) while M2 shifts the glucose consumption towards tricarboxylic acid and oxidative phosphorylation [16][17][18]. So, we also wondered if Ba RNA was also interfering with metabolic pathways on macrophages.
Hence, considering the evidence and the events that occur on other infectious processes, we hypothesized that Ba RNA favours the differentiation of macrophages to an M1 profile at early time points, but it interferes with M1 profile and/or change towards M2 at later time points. To test our hypothesis, we aimed to determine the effect of Ba RNA on macrophage polarization per se or during the M1 differentiation program.

Ethics statement
Human monocytes were isolated exclusively from healthy adult donors according to the rules of the Ethics Committee of the Instituto de Medicina Experimental (IMEX)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Academia Nacional de Medicina. All donors signed an informed consent prior to the study. The donation was anonymous, and all the information of the donors is protected. Bone marrow derived murine macrophages (BMM) were generated by differentiation of bone marrow progenitors from female C57BL/6 mice (age: 2-3 months). All procedures were approved and carried out under the conditions of the Institutional Committee on the Care and Use of Laboratory Animals (CICUAL: Comité Institucional para el Cuidado y Uso de Animales de Laboratorio) of the IMEX.

Bacteria strains
Brucella abortus S2308, was grown on triptein soy agar medium, supplemented with yeast extract (Merck Millipore). The number of bacteria in the stationary phase cultures (in triptein soy broth) was determined by comparing the optical density at 600 nm with a standard curve. The cultures in liquid media of Ba were carried out in the Biosafety Level 3 laboratory (BSL3) of the Operational Unit Center for Biological Containment (UOCCB) of the National Administration of Laboratories and Health Institutes 'Dr. Carlos G. Malbrán' (ANLIS-Malbrán), thanks to an agreement established in 2017.

Cell cultures
Peripheral blood mononuclear cells (PBMCs) were obtained by centrifugation in Ficoll-Hypaque gradients (GE Healthcare) from human blood collected from healthy adult donors. Human monocytes were obtained after centrifugation of the PBMCs in a Percoll gradient (GE Healthcare) and then resuspended in standard medium. The purity of the isolated CD14 + monocytes was greater than 80%, determined by flow cytometry. Cell viability was measured by the trypan blue exclusion test and was greater than 95% in all experiments. BMMs were generated by differentiation of bone marrow progenitors obtained from C57BL/6 mice, as previously described [19].
All the experiments were carried out in an incubator at 37˚C in an atmosphere with 5% CO 2 . The standard medium used was composed of RPMI 1640 supplemented with 25mM Hepes, 2mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS, Gibco), 100U of penicillin.ml −1 and 100 μg of streptomycin.ml −1 . 5 x 10 9 CFU of Ba were resuspended in 1 ml of TRIzol (Invitrogen) and their RNA was purified using Quick-RNA DirectZol columns (Zymo Research) according to the manufacturer's instructions. The quantification and purity of the Ba RNA was determined using a DeNovix DS-11 spectrophotometer (DeNovix Inc.). In all cases, the absorbance ratio 260/280 was higher than 2.0 and the ratio 260/230 was higher than 1.8.

Viability assay
For viability assays, human peripheral blood (PB) derived macrophages were treated with Ba RNA in the presence or absence of IFN-γ and LPS for 24 and 48 h. Cells treated with 2% paraformaldehyde (PFA) were included as a positive control of the technique. At both times, cells were washed and incubated with Annexin V-FITC and Propidium Iodide (IP) (BD Biosciences) for 10 minutes on ice and in the dark. After staining, cells were analysed with FACSCalibur (BD Biosciences) or Partec CyFlow (LabSystems) flow cytometers. Cells were evaluated in the Annexin V + /IP -(early apoptosis), Annexin V + /IP + (late apoptosis) and Annexin V -/IP + (necrosis) quadrants. Data analysis was performed with FlowJo 7.6.2 software (FlowJo, LLC).

In vitro stimulation
In experiments with murine macrophages, BMMs were treated with Ba RNA in the presence of 10 ng/ml of recombinant murine IFN-γ (mIFN-γ, PeProtech) and E. coli LPS for different times. In experiments with human macrophages, 2.5 x 10 5 PB-human monocytes were stimulated with GM-CSF for 5-7 days prior to stimulation with Ba RNA in the presence or absence of IFN-γ and E. coli LPS. Cell cultures were incubated for 48 h at 37˚C in a 5% CO 2 atmosphere. In all cases, the expression of the different cell markers was evaluated by flow cytometry, as described in the following paragraph.

Flow cytometry
Macrophages from PB-purified monocytes were stained with the anti-human MHC-II conjugated to PE (clone L243, BD) or anti-human CD86 conjugated to PE (clone IT2.2; BD) or antihuman CD64 conjugated to FITC (clone 10.1; BD) or anti-human CD206 conjugated to PE (clone 19.2, BD) or anti-human DC-SIGN conjugated to FITC (clone DCN46, BD) or antihuman CD163 conjugated to PerCP (clone GHI61, Biolegend) or anti-mouse MHC-II conjugated to FITC (I-A/I-E, clone M5/114.15.2, e-Bioscience) or the corresponding isotype control antibodies and then evaluated by flow cytometry at different times post-stimulation. When possible, cells were washed and incubated with 7-AAD (BD Biosciences) for 10 minutes on ice and in the dark. The expression of the different surface markers was evaluated within the viable cell population (7-AAD negative). After labelling, the cells were analysed on a FACSCalibur flow cytometer (BD Biosciences) or Partec CyFlow (LabSystems) and the data were processed with the FlowJo 7.6.2 or vX.0.7 applications (FlowJo, LLC). Data was normalized to untreated cells, i.e., in each set of experiments, for each marker, the MFI of treatments (from the % of positive cells for each marker) was divided by MFI of media condition (from the % of positive cells for each marker).

Determination of cytokine concentration
The concentration of different cytokines and chemokines corresponding to the M1 profile (TNF-α, IL-1β and IL-8) and M2 (IL-10) was quantified in supernatants (SNs) from human macrophages stimulated with Ba RNA for 24 and 48 h by ELISA sandwich, according to the manufacturer's instructions. The following detection kits were used: TNF-α (e-Bioscience), IL-1β (e-Bioscience), IL-8 (BioLegend) and IL-10 (BioLegend).

Determination of Nitrogen Reactive Species (NRS)
SNs were harvested from Ba RNA-treated BMMs in the presence of mIFN-γ and LPS for 24 and 48 h. The content of NRS was measured by the determination of nitrites with the Griess reagent (Reagent A: sulfanilic acid, Reagent B: α-naphthylamine) by comparison with a standard curve of OD at 550 nm, as described in [20]. The results were expressed as micromoles of nitrite.
The determination of the inducible NO synthase was done as described before [21]. Briefly, treated BMMs were washed with PBS, fixed with 0.05% paraformaldehyde. Then cells were treated with 0.25% Triton X-100 for 15 min, blocked with 3% bovine serum albumin (BSA) for 1 hr and stained with an anti-iNOS antibody (clone 6, BD Biosciences) for 30 min. An isotypematched antibody was assayed in parallel, and fluorescence was determined by BD FACSCalibur on 10,000 events per sample.

Lactate production and glucose concentration
Lactate production and glucose concentrations in the culture medium was measured using the spectrophotometric assays Lactate Kit and Glicemia Enzimatica AA Kit both from Wiener (Argentina) as previously described [18]. Basically, the kits are based on the oxidation of lactate or glucose, respectively, and the subsequent production of hydrogen peroxide. The consumption of glucose was determined by assessing the diminution of glucose levels in culture supernatants in comparison with RPMI 10% FBS. The absorbance was read using a Biochrom Asys UVM 340 Microplate Reader and software.

Statistical analysis
Results were analysed with one-way ANOVA followed by post hoc Tukey test or two-way ANOVA followed by post hoc Bonferroni test with GraphPad Prism software, 8.4.3 version.

Ba RNA activates but does not polarize macrophages towards M1
We first evaluated whether Ba RNA per se was able to polarize human macrophages towards M1. Human monocytes were differentiated to macrophages with GM-CSF for 5-7 days and then stimulated with Ba RNA (5 μg/ml) for 24 or 48 h. MHC-II, CD86 and CD64 (M1 markers) expression was then assessed by flow cytometry. (IFN-γ + LPS)-treated macrophages were used as a positive control of M1 macrophages. Ba RNA did not significantly modify the surface expression of MHC-II and CD86 (Fig 1A and 1B) neither at 24 nor 48 h. However, there was a reduction in CD64 expression in Ba RNA-treated macrophages at 48 h ( Fig 1C). Supernatants at each time were harvested and IL-1β, TNF-α and IL-8 were quantified by ELISA sandwich. Ba RNA induced the secretion of all pro-inflammatory cytokines by macrophages, except for IL-8, at 24 h but not at 48 h (Fig 2A-2C).
We also performed an Annexin assay to evaluate whether there was a loss of cell viability in Ba RNA stimulated cultures. This treatment did not induce early and late apoptosis or necrosis (S1 Fig).
These results indicate that Ba RNA activates -temporarily though-macrophage immune responses but is not able to polarize per se those cells to M1 macrophages.

Ba RNA activates but does not polarize macrophages towards M2
Next, we evaluated whether Ba RNA was able to polarize macrophages towards M2. We performed the same experiments as described before, and CD206, DC-SIGN and CD163 expressions were measured as typical M2 markers. IL-4-treated macrophages were used as a positive control for CD206 and DC-SIGN expressions and IL-10-treated macrophages for CD163. No significant differences among DC-SIGN or CD163 were observed in Ba RNAtreated macrophages in comparison with untreated macrophages at both times (Fig 3B and  3C). On the contrary, CD206 expression was reduced in Ba RNA-treated macrophages at both times ( Fig 3A).
Supernatants were harvested and the anti-inflammatory cytokine IL-10 was quantified. Ba RNA induced the secretion of this cytokine at 24 and 48 h (Fig 4). Hence, this PAMP can activate macrophages but does not polarize these cells towards a define profile.

Ba RNA interferes with the expression of pro-inflammatory markers during M1 macrophage polarization
We showed that Ba RNA does not polarize macrophages per se. It is already elucidated that Ba infection activates Th1 cells with the consequent secretion of IFN-γ and induction of CD8 + T cells. Moreover, we have previously demonstrated that Ba RNA diminishes the IFN-γ and LPS-induced MHC-II surface expression on human monocytes altering antigen presentation to CD4 + T cells [12]. So, we wondered whether Ba RNA affects M1 polarization while it is occurring, i.e., in macrophages cultivated in the presence of IFN-γ and LPS.
To evaluate whether Ba RNA was able to modify macrophage inflammatory profile while they are being polarized to M1, human monocytes were differentiated to macrophages as mentioned above and then stimulated with Ba RNA (5 μg/ml) in the presence of IFN-γ + LPS for 24 and 48 h. The expression of MHC-II, CD86 and CD64 was assessed by flow cytometry. At 24 h there were no differences in the expression of MHC-II and CD86 in cells treated with Ba RNA + (IFN-γ and LPS). CD64 expression was significantly reduced though (Fig 5C). At 48 h there was a significant reduction in the expression of MHC-II and CD64 in Ba RNA + (IFN-γ and LPS)-treated macrophages (Fig 5A and 5C). There were no changes in CD86 expression at any evaluated time points in comparison with IFN-γ and LPS-treated cells (Fig 5B).
We also performed an Annexin assay to evaluate whether there was a loss of cell viability in Ba RNA + (IFN-γ + LPS)-stimulated cultures. This treatment did not induce early or late apoptosis or necrosis (S2 Fig). Moreover, we determined the ability of Ba RNA to induce the secretion of pro-inflammatory cytokines from macrophages during M1 polarization. IL-1β, TNF-α and IL-8 were PLOS NEGLECTED TROPICAL DISEASES quantified in the supernatants of Ba RNA plus IFN-γ and LPS-treated macrophages at 24 and 48 h post-stimulation. There were no significant changes in the secretion of cytokines at any evaluated time point (Fig 6). We also calculated the ratio of pro and anti-inflammatory cytokines. At 48 h, there was a reduction tendency -non-significant though-in the ratio TNF-α/IL-10 in Ba RNA-treated macrophages during M1 polarization (S3 Fig), implying that there could be a predominant anti-inflammatory cytokine context at later times post-stimulation.
As consequence, at least phenotypically, Ba RNA interferes with M1 polarization.

Ba RNA does not modulate M2 markers under M1 polarizing conditions
Since Ba RNA interferes with M1 polarization, we asked whether this event was accompanied with a differentiation to an M2 profile. To evaluate this, the expression of M2 markers (CD163, CD206 and DC-SIGN) was determined during M1 polarizing conditions at 24 and 48 h post-stimulation. Also, in culture supernatants we quantified the secretion of IL-10. As observed, there were neither no significant changes on CD206 and DC-SIGN surface expression nor in the IL-10 secretion mediated by Ba RNA in the presence of IFN-γ and LPS. However, there was a transient induction of CD163 surface expression on Ba RNA-treated macrophages in the presence of IFN-γ and LPS at 24 h (Fig 7A-7D). So, these results altogether show that Ba RNA interferes with M1 polarization, but not because of macrophages turning into an M2 profile.

Ba RNA induces glucose consumption at 24 h and lactate production at 48 h
Given the fact that metabolic features of M1 macrophages are increased glucose consumption to produce lactate and diminished OXPHOS metabolic pathway, we determined the glucose consumption of Ba RNA or Ba RNA + (IFN-γ + LPS)-treated macrophages at 24 and 48 h post-stimulation. As demonstrated in Fig 8A, Ba RNA induced glucose consumption at 24 h and lactate production at 48 h, confirming its pro-inflammatory properties, at least transiently. On the contrary, Ba RNA + (IFN-γ + LPS)-treated macrophages did not change glucose consumption and lactate production compared to (IFN-γ + LPS)-treated macrophages (Fig 8A  and 8B). Hence, this phenotypic pathway of M1 metabolism is not altered by Ba RNA.

Ba RNA diminishes the production of Nitrogen Reactive Species (NRS) during M1 polarization at 48 h
Since we previously showed that Ba RNA diminishes the antigen presentation capacity of macrophages to CD8 + and CD4 + T cells [11,12], we wondered if microbicidal capacity (as a key feature of M1 profile) of Ba RNA-treated macrophages in the presence of IFN-γ and LPS was altered as well. So, we performed two types of experiments: Griess assay and intracellular iNOS expression. To perform these experiments, we used murine BMM. Firstly, we evaluated whether Ba RNA was able to interfere with M1 polarization in BMM. Ba RNA diminished the (mIFN-γ + LPS)-induced MHC-II surface expression at 48 h (S4 Fig). Then, we determined NRS in culture supernatants of Ba RNA-treated BMM during M1 polarization and the intracellular expression of iNOS.
As observed, at 24 h, Ba RNA-treated BMMs in the presence of mIFN-γ and LPS did not induce or reduce the secretion of NRS (detected as nitrite oxide in supernatants) or the expression of iNOS. However, at 48 h NRS secretion diminished in BMMs treated with Ba RNA in the presence of mIFN-γ and LPS (Fig 9A). Although not significant, this could be related with a decreased iNOS expression at least at 48 h (Fig 9B).

Discussion
Brucellosis is a zoonotic disease, caused by bacteria from Brucella genus. It is distributed worldwide, especially affecting livestock from Africa, Latin America, New Zealand and Australia, India, Saudi Arabia, and Mediterranean countries among others. The main pathogenic species for livestock are B. abortus, responsible for bovine brucellosis, B. melitensis, the main aetiological agent of brucellosis in both small ruminants and humans, B. suis, responsible for swine brucellosis and B. ovis, responsible for sheep brucellosis. The disease affects male and female livestock's fertility and causes abortions. Despite eradication programmes, brucellosis continues to be a big issue for public health and for economy policies due to the loss of money

PLOS NEGLECTED TROPICAL DISEASES
driven by the need of 'brucellosis free'-status livestock. Hence, to design mitigation strategies and diminish economic burden, there is a common need of understanding the pathogenesis of this disease in humans and livestock.
Brucella spp., like most intracellular bacteria, display strategies to evade or counterattack the host immune responses. By achieving this, a chronic infection could be established. These bacteria can live intracellularly in macrophages, their quintessential replicative niche [22].
Monocytes and macrophages play a key role against infections mainly through phagocytosis and antigen presentation [23]. Peripheral blood monocytes migrate inside the body tissues and polarize to macrophages of different subclasses depending on the microenvironment. Among the subclasses of macrophages, M1 (formerly known as 'classical macrophages') and M2 (formerly known as 'alternative macrophages') are the two main subpopulations. IFN-γ and LPS differentiate unpolarized (M0) macrophages to M1 while IL-4 and IL-13, IL-10 or corticosteroids differentiate M0 to M2. Basically, M1 cells eliminate pathogens but also damage the tissues while M2 cells suppress inflammation and mediate tissue healing. Both populations are necessary to solve the infectious process [2].
What is particularly interesting for Ba is that monocytes and macrophages constitute the first line of defense against the bacteria but also are the place where they reside [24].
Results from our group have shown that a Ba PAMP associated with bacterial viability (vita-PAMP) is responsible for the IFN-γ-induced MHC-I and MHC-II down-modulation on monocytes/macrophages. This vita-PAMP is Ba RNA [11,12]. We have shown that Toll-like receptor 8 (TLR8) is, at least, one receptor by which Ba RNA is sensed [11]. Besides, stimulation of human monocytes by Ba RNA or synthetic TLR8 ligands promotes the early secretion of proinflammatory mediators (TNF-α e IL-1β) [11]. All these results and the importance of monocytes/macrophages in the resolution of Ba infection laid the ground for this work. Therefore, we aimed to study the role of Ba RNA during the polarization of macrophages.
Considering that infection with Ba starts with the activation of innate immunity [30] we wondered whether Ba RNA per se could constitute enough stimulus for macrophages to be polarized to M1 or M2. Our results show that Ba RNA can activate macrophages into a proinflammatory profile -at least for a short time-early on during infection. In line with this, Saha et al. demonstrated that TLR7/8 signalling during Hepatitis C infection promotes the differentiation of monocytes and polarization of macrophages to M2, although with a combined M1 and M2 pattern of cytokine secretion [13]. Our results establish that Ba RNA activates but does not polarize macrophages. This makes sense if we consider that Brucella spp. infection does not generate an inflammatory pathology as massive as other microorganisms [31]. Hence, Ba RNA as a PAMP is not as potent as other immunomodulatory PAMPs such as E. coli LPS.
In order to control the progress of the disease, two main components of adaptive immunity play significant roles: IFN-γ secreted by CD4 + and CD8 + T cells [32,33] and cytotoxic CD8 + T cells which eliminate Brucella infected cells [34]. Hence, next we performed experiments to evaluate whether Ba RNA was capable of modulating or interfering with M1 polarization of M0 macrophages, i.e., in an established Th1 environment. At 24 h, Ba RNA diminished M1-induced CD64 expression and at 48 h, Ba RNA could also down modulate M1 induced-CD64 and MHC-II expressions. This, however, was not accompanied by augmented M2 makers. Cytokine secretion was not altered in M1 macrophages treated with Ba RNA despite finding a tendency towards an anti-inflammatory cytokine context. In literature, IL-8 secretion could be increased in M1 as well as in M2 macrophages [35]. Also, nowadays, there are much more than M1 and M2 macrophages [2,36] and it is being discovered a wide spectrum between M1 and M2 [37].
We have already shown that Ba-infected or Ba RNA-treated macrophages show a diminished antigen presentation capacity to CD4 + T cells [8,9,12]. In addition, we and others have demonstrated that phagocytic capacity of Ba-infected monocytes is also altered [25,38]. This was also supported in this study, since Ba RNA diminishes CD64 expression (type I receptor for the Fc portion of IgG, FcγRI). Therefore, this vita-PAMP may be another component besides lipoproteins of Ba to contribute to change key microbicidal macrophage actions.
Therefore, our results indicate that Ba RNA does not polarize macrophages to a particular profile, but 'interferes' with M1 polarization instead, at least phenotypically.
This kind of M1 interference has already been seen in other infectious pathologies: Mycobacterium tuberculosis inhibits transcription of IFN-γ target genes and contributes to the development of the typical pleural effusion of tuberculosis patients [39]. However, granulomatose macrophages express both pro-and anti-inflammatory markers, indicating that polarization is a kind of spectrum more than binary. Also, in Salmonella typhimurium infections, e.g., diverse activation status of macrophages between M1 and M2 have been observed [40]. Regarding Brucella spp. in vivo, there are no determinant results according to macrophage polarization so far. Monocytes from brucellosis patients are not able to polarize to M1 and M2 defined profiles [24].
On the other hand, Ba RNA-treated macrophages diminished the IFN-γ-induced production of NRS at 48 h. In concordance with this, Hu et al. show that infection of murine macrophages with Ba down modulates TXNIP (thioredoxin-interacting protein) expression which promotes survival of the bacteria in those cells through reduction of NRS and Reactive Oxygen Species [41]. Moreover, Kerrinnes et al. showed that alternatively activated macrophages infected with Ba shifted the production of NRS towards polyamines, favouring the establishment of a chronic infection in mice [42]. We saw no effects on glucose consumption and lactate production on M1 macrophages by Ba RNA compared to untreated M1 cells, except for an augmented -although non-significant-lactate production at 48 h in Ba RNA-treated M1 macrophages. Czyz et al. demonstrated that Ba infected macrophages display a metabolic pathway characterized by an augmented lactate production that acts as the only source of carbon and energy [43]. Some Brucella virulence factors modulate the host metabolism as reviewed in [44], perhaps it is not the case for Ba RNA, or at least, not in this model.
An issue that merits discussion is the differences observed between the two experimental times points evaluated. In our first set of experiments, the results show that Ba RNA induces the secretion of pro-inflammatory cytokines (IL-1β and TNF-α) at 24 h and the anti-inflammatory cytokine IL-10 at 24 h and 48 h. However, in our second set of experiments under polarizing conditions, Ba RNA interferes with the expression of M1 markers, and it decreases NRS production at 48 h. These results are in line with those previously observed in our laboratory in which at early post-stimulation time points (24 h) either infection with Ba, its RNA or synthetic TLR8 agonists activate immune parameters of dendritic cells (DCs) or monocytes. Particularly, we previously published results showing that Ba infection induces DCs maturation, as evidenced by the up-regulation of CD86, CD80, CCR7, CD83, MHC-II, MHC-I and CD40 at 24 h post-infection [45]. Several years later, we also demonstrated that Ba RNA induces MHC-II expression on DCs at 24 h post-stimulation [12]. Moreover, we have previously published results demonstrating that the stimulation of human monocytes with Ba RNA or synthetic TLR8 agonists for 24 h induce the secretion of the pro-inflammatory cytokines TNF-α and IL-1β [11]. On the other hand, we previously demonstrated that at later poststimulation time points (48 h), either infection with Ba or stimulation with its RNA inhibit the expression of MHC-II and MHC-I in monocytes/macrophages and the consequent antigen presentation to CD4 + and CD8 + T cells, respectively [6,9,11,12].
The apparent discrepancies between the up-regulation of MHC-I and MHC-II in DCs and the downregulation of both molecules in monocytes/macrophages could be explain in terms of the kinetics of Ba infection. One explanation is that activation of DCs with Ba is likely relevant at the onset of immune response, when Th1 and T CD8 + responses are triggered. At later time points, Ba might be able to circumvent these responses to establish a chronic infection by means of different evasion mechanism such as the down-modulation of MHC-I and MHC-II [33] and the consequent interference with M1 polarization of macrophages, where it dwells.
Overall, the results of this study shed light into a relevant aspect whereby Ba evades immune host surveillance: the interference with M1 macrophage polarization. Our findings demonstrate that the vita-PAMP Ba RNA constitute a novel virulence factor whereby this bacterium interferes with M1 polarization of human macrophages, not only by modification of their phenotype but also with their killing activity, specifically by diminishing the production of nitrogen reactive species. This provides new insights about how Ba can persist in the presence of Th1 responses, i.e., in the presence of IFN-γ, establishing a chronic infection.
These results also lay the ground for studying more deeply the modulatory properties of microbial RNA in the context of brucellosis, other intracellular infections, and tumours.

Limitations
This study has some limitations. We designed a model using purified human monocytes differentiated to macrophages with GM-CSF. Nevertheless, other models include differentiation of monocytes with M-CSF (which we used for BMM). Moreover, we could not perform an in vivo analysis with blood samples from brucellosis patients (to purify monocytes and differentiate them with GM-CSF as we did for monocytes from healthy blood donors) due to COVID-19 pandemic and the fact that these patients are sub-diagnosed in Argentina. In future research, we would like to obtain a representative number of these samples and deep our understanding of the pathology. We also should design an in vivo model with Ba infected mice to obtain monocytes and differentiate them to macrophages and evaluate their profile and functionality. Furthermore, we did not deeply evaluate the mechanisms downstream the signalling pathway of Ba RNA-stimulation of M1 macrophages. Evaluation of the receptor/s and metabolic pathways of M1-interefered macrophages could be interesting studies to make a better understanding of the disease. Finally, when understanding the intracellular pathway triggered by Ba RNA, we will be able to propose molecules or drugs to revert the interference in M1 macrophages and provide perhaps some new treatments to be tested for human and animal brucellosis.