TLR7 Activation in M-CSF-Dependent Monocyte-Derived Human Macrophages Potentiates Inflammatory Responses and Prompts Neutrophil Recruitment.

Toll-like receptor 7 (TLR7) is an endosomal pathogen-associated molecular pattern (PAMP) receptor that senses single-stranded RNA (ssRNA) and whose engagement results in the production of type I IFN and pro-inflammatory cytokines upon viral exposure. Recent genetic studies have established that a dysfunctional TLR7-initiated signaling is directly linked to the development of inflammatory responses. We present evidence that TLR7 is preferentially expressed by monocyte-derived macrophages generated in the presence of M-CSF (M-MØ). We now show that TLR7 activation in M-MØ triggers a weak MAPK, NFκB, and STAT1 activation and results in low production of type I IFN. Of note, TLR7 engagement reprograms MAFB+ M-MØ towards a pro-inflammatory transcriptional profile characterized by the expression of neutrophil-attracting chemokines (CXCL1-3, CXCL5, CXCL8), whose expression is dependent on the transcription factors MAFB and AhR. Moreover, TLR7-activated M-MØ display enhanced pro-inflammatory responses and a stronger production of neutrophil-attracting chemokines upon secondary stimulation. As aberrant TLR7 signaling and enhanced pulmonary neutrophil/lymphocyte ratio associate with impaired resolution of virus-induced inflammatory responses, these results suggest that targeting macrophage TLR7 might be a therapeutic strategy for viral infections where monocyte-derived macrophages exhibit a pathogenic role.

Interactions of the Streptococcus pneumoniae Toxin-Antitoxin RelBE Proteins with Their Target DNA.

Type II bacterial toxin-antitoxin (TA) systems are found in most bacteria, archaea, and mobile genetic elements. TAs are usually found as a bi-cistronic operon composed of an unstable antitoxin and a stable toxin that targets crucial cellular functions like DNA supercoiling, cell-wall synthesis or mRNA translation. The type II RelBE system encoded by the pathogen Streptococcus pneumoniae is highly conserved among different strains and participates in biofilm formation and response to oxidative stress. Here, we have analyzed the participation of the RelB antitoxin and the RelB:RelE protein complex in the self-regulation of the pneumococcal relBE operon. RelB acted as a weak repressor, whereas RelE performed the role of a co-repressor. By DNA footprinting experiments, we show that the proteins bind to a region that encompasses two palindromic sequences that are located around the -10 sequences of the single promoter that directs the synthesis of the relBE mRNA. High-resolution footprinting assays showed the distribution of bases whose deoxyriboses are protected by the bound proteins, demonstrating that RelB and RelB:RelE contacted the DNA backbone on one face of the DNA helix and that these interactions extended beyond the palindromic sequences. Our findings suggest that the binding of the RelBE proteins to its DNA target would lead to direct inhibition of the binding of the host RNA polymerase to the relBE promoter.

CD28 is expressed by macrophages with anti-inflammatory potential and limits their T-cell activating capacity.

CD28 expression is generally considered to be T lymphocyte specific. We have previously shown CD28 mRNA expression in M-CSF-dependent anti-inflammatory monocyte-derived macrophages (M-MØ), and now demonstrate that CD28 cell surface expression is higher in M-MØ than in GM-CSF-dependent macrophages, and that macrophage CD28 expression is regulated by MAFB and activin A. In vivo, CD28 was found in tumor-associated macrophages and, to a lower extent, in pro-inflammatory synovial fluid macrophages from rheumatoid arthritis patients. Analysis of mouse macrophages confirmed Cd28 expression in bone-marrow derived M-MØ. Indeed, anti-CD28 antibodies triggered ERK1/2 phosphorylation in mouse M-MØ. At the functional level, Cd28KO M-MØ exhibited a significantly higher capacity to activate the OVA-specific proliferation of OT-II CD4+ T cells than WT M-MØ, as well as enhanced LPS-induced IL-6 production. Besides, the Cd28KO M-MØ transcriptome was significantly different from WT M-MØ regarding the expression IFN response, inflammatory response, and TGF-ß signaling related gene sets. Therefore, defective CD28 expression in mouse macrophages associates to changes in gene expression profile, what might contribute to the altered functionality displayed by Cd28KO M-MØ. Thus, CD28 expression appears as a hallmark of anti-inflammatory macrophages and might be a target for immunotherapy.

MAFB and MAF Transcription Factors as Macrophage Checkpoints for COVID-19 Severity.

Defective IFN production and exacerbated inflammatory and pro-fibrotic responses are hallmarks of SARS-CoV-2 infection in severe COVID-19. Based on these hallmarks, and considering the pivotal role of macrophages in COVID-19 pathogenesis, we hypothesize that the transcription factors MAFB and MAF critically contribute to COVID-19 progression by shaping the response of macrophages to SARS-CoV-2. Our proposal stems from the recent identification of pathogenic lung macrophage subsets in severe COVID-19, and takes into consideration the previously reported ability of MAFB to dampen IFN type I production, as well as the critical role of MAFB and MAF in the acquisition and maintenance of the transcriptional signature of M-CSF-conditioned human macrophages. Solid evidences are presented that link overexpression of MAFB and silencing of MAF expression with clinical and biological features of severe COVID-19. As a whole, we propose that a high MAFB/MAF expression ratio in lung macrophages could serve as an accurate diagnostic tool for COVID-19 progression. Indeed, reversing the macrophage MAFB/MAF expression ratio might impair the exacerbated inflammatory and profibrotic responses, and restore the defective IFN type I production, thus becoming a potential strategy to limit severity of COVID-19.

Growth Hormone Reprograms Macrophages toward an Anti-Inflammatory and Reparative Profile in an MAFB-Dependent Manner.

Growth hormone (GH), a pleiotropic hormone secreted by the pituitary gland, regulates immune and inflammatory responses. In this study, we show that GH regulates the phenotypic and functional plasticity of macrophages both in vitro and in vivo. Specifically, GH treatment of GM-CSF-primed monocyte-derived macrophages promotes a significant enrichment of anti-inflammatory genes and dampens the proinflammatory cytokine profile through PI3K-mediated downregulation of activin A and upregulation of MAFB, a critical transcription factor for anti-inflammatory polarization of human macrophages. These in vitro data correlate with improved remission of inflammation and mucosal repair during recovery in the acute dextran sodium sulfate-induced colitis model in GH-overexpressing mice. In this model, in addition to the GH-mediated effects on other immune cells, we observed that macrophages from inflamed gut acquire an anti-inflammatory/reparative profile. Overall, these data indicate that GH reprograms inflammatory macrophages to an anti-inflammatory phenotype and improves resolution during pathologic inflammatory responses.

Serotonin (5-HT) Shapes the Macrophage Gene Profile through the 5-HT2B-Dependent Activation of the Aryl Hydrocarbon Receptor.

Macrophages can either promote or resolve inflammatory responses, and their polarization state is modulated by peripheral serotonin (5-hydroxytryptamine [5-HT]). In fact, pro- and anti-inflammatory macrophages differ in the expression of serotonin receptors, with 5-HT2B and 5-HT7 expression restricted to M-CSF-primed monocyte-derived macrophages (M-MØ). 5-HT7 drives the acquisition of profibrotic and anti-inflammatory functions in M-MØ, whereas 5-HT2B prevents the degeneration of spinal cord mononuclear phagocytes and modulates motility of murine microglial processes. Because 5-HT2B mediates clinically relevant 5-HT-related pathologies (valvular heart disease, pulmonary arterial hypertension) and is an off target of anesthetics, antiparkinsonian drugs, and selective serotonin reuptake inhibitors, we sought to determine the transcriptional consequences of 5-HT2B engagement in human macrophages, for which 5-HT2B signaling remains unknown. Assessment of the effects of specific agonists and antagonist revealed that 5-HT2B engagement modifies the cytokine and gene signature of anti-inflammatory M-MØ, upregulates the expression of aryl hydrocarbon receptor (AhR) target genes, and stimulates the transcriptional activation of AhR. Moreover, we found that 5-HT dose dependently upregulates the expression of AhR target genes in M-MØ and that the 5-HT-mediated activation of AhR is 5-HT2B dependent because it is abrogated by the 5-HT2B-specific antagonist SB204741. Altogether, our results demonstrate the existence of a functional 5-HT/5-HT2B/AhR axis in human macrophages and indicate that 5-HT potentiates the activity of a transcription factor (AhR) that regulates immune responses and the biological responses to xenobiotics.

The Streptococcus pneumoniaeyefM-yoeB and relBE Toxin-Antitoxin Operons Participate in Oxidative Stress and Biofilm Formation.

Type II (proteic) toxin-antitoxin systems (TAs) are widely distributed among bacteria and archaea. They are generally organized as operons integrated by two genes, the first encoding the antitoxin that binds to its cognate toxin to generate a harmless protein⁻protein complex. Under stress conditions, the unstable antitoxin is degraded by host proteases, releasing the toxin to achieve its toxic effect. In the Gram-positive pathogen Streptococcus pneumoniae we have characterized four TAs: pezAT, relBE, yefM-yoeB, and phD-doc, although the latter is missing in strain R6. We have assessed the role of the two yefM-yoeB and relBE systems encoded by S. pneumoniae R6 by construction of isogenic strains lacking one or two of the operons, and by complementation assays. We have analyzed the phenotypes of the wild type and mutants in terms of cell growth, response to environmental stress, and ability to generate biofilms. Compared to the wild-type, the mutants exhibited lower resistance to oxidative stress. Further, strains deleted in yefM-yoeB and the double mutant lacking yefM-yoeB and relBE exhibited a significant reduction in their ability for biofilm formation. Complementation assays showed that defective phenotypes were restored to wild type levels. We conclude that these two loci may play a relevant role in these aspects of the S. pneumoniae lifestyle and contribute to the bacterial colonization of new niches.

The Activin A-Peroxisome Proliferator-Activated Receptor Gamma Axis Contributes to the Transcriptome of GM-CSF-Conditioned Human Macrophages.

GM-CSF promotes the functional maturation of lung alveolar macrophages (A-MØ), whose differentiation is dependent on the peroxisome proliferator-activated receptor gamma (PPARγ) transcription factor. In fact, blockade of GM-CSF-initiated signaling or deletion of the PPARγ-encoding gene PPARG leads to functionally defective A-MØ and the onset of pulmonary alveolar proteinosis. In vitro, macrophages generated in the presence of GM-CSF display potent proinflammatory, immunogenic and tumor growth-limiting activities. Since GM-CSF upregulates PPARγ expression, we hypothesized that PPARγ might contribute to the gene signature and functional profile of human GM-CSF-conditioned macrophages. To verify this hypothesis, PPARγ expression and activity was assessed in human monocyte-derived macrophages generated in the presence of GM-CSF [proinflammatory GM-CSF-conditioned human monocyte-derived macrophages (GM-MØ)] or M-CSF (anti-inflammatory M-MØ), as well as in ex vivo isolated human A-MØ. GM-MØ showed higher PPARγ expression than M-MØ, and the expression of PPARγ in GM-MØ was found to largely depend on activin A. Ligand-induced activation of PPARγ also resulted in distinct transcriptional and functional outcomes in GM-MØ and M-MØ. Moreover, and in the absence of exogenous activating ligands, PPARγ knockdown significantly altered the GM-MØ transcriptome, causing a global upregulation of proinflammatory genes and significantly modulating the expression of genes involved in cell proliferation and migration. Similar effects were observed in ex vivo isolated human A-MØ, where PPARγ silencing led to enhanced expression of genes coding for growth factors and chemokines and downregulation of cell surface pathogen receptors. Therefore, PPARγ shapes the transcriptome of GM-CSF-dependent human macrophages (in vitro derived GM-MØ and ex vivo isolated A-MØ) in the absence of exogenous activating ligands, and its expression is primarily regulated by activin A. These results suggest that activin A, through enhancement of PPARγ expression, help macrophages to switch from a proinflammatory to an anti-inflammatory polarization state, thus contributing to limit tissue damage and restore homeostasis.

Serotonin drives the acquisition of a profibrotic and anti-inflammatory gene profile through the 5-HT7R-PKA signaling axis.

Peripheral serotonin (5-hydroxytryptamine, 5-HT) regulates cell growth and differentiation in numerous cell types through engagement of seven types of cell surface receptors (HTR1-7). Deregulated 5-HT/HTR levels contribute to pathology in chronic inflammatory diseases, with macrophages being relevant targets for the physio-pathological effects of 5-HT. In fact, 5-HT skews human macrophage polarization through engagement of 5-HT2BR and 5-HT7R receptors. We now report that 5-HT primes macrophages for reduced pro-inflammatory cytokine production and IFN type I-mediated signaling, and promotes an anti-inflammatory and pro-fibrotic gene signature in human macrophages. The acquisition of the 5-HT-dependent gene profile primarily depends on the 5-HT7R receptor and 5-HT7R-initiated PKA-dependent signaling. In line with the transcriptional results, 5-HT upregulates TGFß1 production by human macrophages in an HTR7- and PKA-dependent manner, whereas the absence of Htr7 in vivo results in diminished macrophage infiltration and collagen deposition in a mouse model of skin fibrosis. Our results indicate that the anti-inflammatory and pro-fibrotic activity of 5-HT is primarily mediated through the 5-HT7R-PKA axis, and that 5-HT7R contributes to pathology in fibrotic diseases.

Atypical Activin A and IL-10 Production Impairs Human CD16+ Monocyte Differentiation into Anti-Inflammatory Macrophages.

Human CD14(++)CD16(-) and CD14(+/lo)CD16(+) monocyte subsets comprise 85 and 15% of blood monocytes, respectively, and are thought to represent distinct stages in the monocyte differentiation pathway. However, the differentiation fates of both monocyte subsets along the macrophage (Mϕ) lineage have not yet been elucidated. We have now evaluated the potential of CD14(++) CD16(-) and CD16(+) monocytes to differentiate and to be primed toward pro- or anti-inflammatory Mϕs upon culture with GM-CSF or M-CSF, respectively (subsequently referred to as GM14, M14, GM16, or M16). Whereas GM16 and GM14 were phenotypic and functionally analogous, M16 displayed a more proinflammatory profile than did M14. Transcriptomic analyses evidenced that genes associated with M-CSF-driven Mϕ differentiation (including FOLR2, IL10, IGF1, and SERPINB2) are underrepresented in M16 with respect to M14. The preferential proinflammatory skewing of M16 relative to M14 was found to be mediated by the secretion of activin A and the low levels of IL-10 produced by M16. In fact, activin A receptor blockade during the M-CSF-driven differentiation of CD16(+) monocytes, or addition of IL-10-containing M14-conditioned medium, significantly enhanced their expression of anti-inflammatory-associated molecules while impairing their acquisition of proinflammatory-related markers. Thus, we propose that M-CSF drives CD14(++)CD16- monocyte differentiation into bona fide anti-inflammatory Mϕs in a self-autonomous manner, whereas M-CSF-treated CD16(+) monocytes generate Mϕs with a skewed proinflammatory profile by virtue of their high activin A expression unless additional anti-inflammatory stimuli such as IL-10 are provided.

Macrophages from the synovium of active rheumatoid arthritis exhibit an activin A-dependent pro-inflammatory profile.

Rheumatoid arthritis (RA) is a chronic inflammatory disease whose pathogenesis and severity correlates with the presence of macrophage-derived pro-inflammatory cytokines within the inflamed synovium. Macrophage-derived cytokines fuel the pathological processes in RA and are targets of clinically successful therapies. However, although macrophage polarization determines cytokine production, the polarization state of macrophages in RA joints remains poorly defined. To dissect the molecular basis for the tissue-damaging effects of macrophages in RA joints, we undertook the phenotypic and transcriptomic characterization of ex vivo isolated CD14(+) RA synovial fluid (RA-SF) macrophages. Flow cytometry and gene profiling indicated that RA-SF macrophages express pro-inflammatory polarization markers (MMP12, EGLN3, CCR2), lack expression of markers associated with homeostatic and anti-inflammatory polarization (IGF1, HTR2B) and exhibit a transcriptomic profile that resembles the activin A-dependent gene signature of pro-inflammatory in vitro-generated macrophages. In fact, high levels of Smad-activating activin A were found in RA-SF and, accordingly, the Smad signalling pathway was activated in ex vivo-isolated RA-SF macrophages. In vitro experiments on monocytes and macrophages indicated that RA-SF promoted the acquisition of pro-inflammatory markers (INHBA, MMP12, EGLN3, CCR2) but led to a significant reduction in the expression of genes associated with homeostasis and inflammation resolution (FOLR2, SERPINB2, IGF1, CD36), thus confirming the pro-inflammatory polarization ability of RA-SF. Importantly, the macrophage-polarizing ability of RA-SF was inhibited by an anti-activin A-neutralizing antibody, thus demonstrating that activin A mediates the pro-inflammatory macrophage-polarizing ability of RA-SF. Moreover, and in line with these findings, multicolour immunofluorescence evidenced that macrophages within RA synovial membranes (RA-SM) also express pro-inflammatory polarization markers whose expression is activin A-dependent. Altogether, our results demonstrate that macrophages from RA synovial fluids and membranes exhibit an MMP12(+) EGLN3(+) CCR2(+) pro-inflammatory polarization state whose acquisition is partly dependent on activin A from the synovial fluid.

CCL2 shapes macrophage polarization by GM-CSF and M-CSF: identification of CCL2/CCR2-dependent gene expression profile.

The CCL2 chemokine mediates monocyte egress from bone marrow and recruitment into inflamed tissues through interaction with the CCR2 chemokine receptor, and its expression is upregulated by proinflammatory cytokines. Analysis of the gene expression profile in GM-CSF- and M-CSF-polarized macrophages revealed that a high CCL2 expression characterizes macrophages generated under the influence of M-CSF, whereas CCR2 is expressed only by GM-CSF-polarized macrophages. Analysis of the factors responsible for this differential expression identified activin A as a critical factor controlling the expression of the CCL2/CCR2 pair in macrophages, as activin A increased CCR2 expression but inhibited the acquisition of CCL2 expression by M-CSF-polarized macrophages. CCL2 and CCR2 were found to determine the extent of macrophage polarization because CCL2 enhances the LPS-induced production of IL-10, whereas CCL2 blockade leads to enhanced expression of M1 polarization-associated genes and cytokines, and diminished expression of M2-associated markers in human macrophages. Along the same line, Ccr2-deficient bone marrow-derived murine macrophages displayed an M1-skewed polarization profile at the transcriptomic level and exhibited a significantly higher expression of proinflammatory cytokines (TNF-α, IL-6) in response to LPS. Therefore, the CCL2-CCR2 axis regulates macrophage polarization by influencing the expression of functionally relevant and polarization-associated genes and downmodulating proinflammatory cytokine production.

Aryl hydrocarbon receptor contributes to the MEK/ERK-dependent maintenance of the immature state of human dendritic cells.

Dendritic cells (DCs) promote tolerance or immunity depending on their maturation state, which is enhanced or accelerated upon MEK-ERK signaling pathway inhibition. We have determined the contribution of MEK-ERK activation to the profile of gene expression of human immature monocyte-derived dendritic cells (MDDCs) and peripheral blood myeloid DCs. ERK inhibition altered the expression of genes that mediate Chemokine (C-C motif) ligand 19 (CCL19)-directed migration (CCR7) and low-density lipoprotein (LDL) binding (CD36, SCARB1, OLR1, CXCL16) by immature DCs. In addition, ERK upregulated CCL2 expression while impairing the expression of DC maturation markers (RUNX3, ITGB7, IDO1). MEK-ERK-regulated genes exhibited an overrepresentation of cognate sequences for the aryl hydrocarbon receptor (AhR) transcription factor, whose transcriptional and DNA-binding activities increased in MDDCs upon exposure to the MEK1/2 inhibitor U0126. Therefore, the MEK-ERK signaling pathway regulates antigen capture, lymph node homing, and acquisition of maturation-associated genes, and its contribution to the maintenance of the immature state of MDDCs and myeloid DCs is partly dependent on the activity of AhR. Since pharmacologic modulation of the MEK-ERK signaling pathway has been proposed as a potential therapeutic strategy for cancer, our findings indicate that ERK inhibitors might influence antitumor responses through regulation of critical DC effector functions.

Serotonin skews human macrophage polarization through HTR2B and HTR7.

Besides its role as a neurotransmitter, serotonin (5-hydroxytryptamine, 5HT) regulates inflammation and tissue repair via a set of receptors (5HT(1-7)) whose pattern of expression varies among cell lineages. Considering the importance of macrophage polarization plasticity for inflammatory responses and tissue repair, we evaluated whether 5HT modulates human macrophage polarization. 5HT inhibited the LPS-induced release of proinflammatory cytokines without affecting IL-10 production, upregulated the expression of M2 polarization-associated genes (SERPINB2, THBS1, STAB1, COL23A1), and reduced the expression of M1-associated genes (INHBA, CCR2, MMP12, SERPINE1, CD1B, ALDH1A2). Whereas only 5HT(7) mediated the inhibitory action of 5HT on the release of proinflammatory cytokines, both 5HT(2B) and 5HT(7) receptors mediated the pro-M2 skewing effect of 5HT. In fact, blockade of both receptors during in vitro monocyte-to-macrophage differentiation preferentially modulated the acquisition of M2 polarization markers. 5HT(2B) was found to be preferentially expressed by anti-inflammatory M2(M-CSF) macrophages and was detected in vivo in liver Kupffer cells and in tumor-associated macrophages. Therefore, 5HT modulates macrophage polarization and contributes to the maintenance of an anti-inflammatory state via 5HT(2B) and 5HT(7), whose identification as functionally relevant markers for anti-inflammatory/homeostatic human M2 macrophages suggests their potential therapeutic value in inflammatory pathologies.

The prolyl hydroxylase PHD3 identifies proinflammatory macrophages and its expression is regulated by activin A.

Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163(+) lung macrophages under basal homeostatic conditions, whereas PHD3(+) macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn's disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro.

The toxin-antitoxin proteins relBE2Spn of Streptococcus pneumoniae: characterization and association to their DNA target.

The chromosome of the pathogenic Gram-positive bacterium Streptococcus pneumoniae contains between six to 10 operons encoding toxin-antitoxin systems (TAS). TAS are widespread and redundant in bacteria and archaea and their role, albeit still obscure, may be related to important aspects of bacteria lifestyle like response to stress. One of the most abundant TAS is the relBE family, being present in the chromosome of many bacteria and archaea. Because of the high rates of morbility and mortality caused by S. pneumoniae, it has been interesting to gain knowledge on the pneumococcal TAS, among them the RelBE2Spn proteins. Here, we have analyzed the DNA binding capacity of the RelB2Spn antitoxin and the RelB2Spn-RelE2Spn proteins by band-shift assays. Thus, a DNA region encompassing the operator region of the proteins was identified. In addition, we have used analytical ultracentrifugation and native mass spectrometry to measure the oligomerization state of the antitoxin alone and the RelBE2Spn complex in solution bound or unbound to its DNA substrate. Using native mass spectrometry allowed us to unambiguously determine the stoichiometry of the RelB2Spn and of the RelBE2Spn complex alone or associated to its DNA target.

Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae.

We report the construction of a plasmid vector designed for regulated gene expression in Streptococcus pneumoniae. The new vector, pLS1ROM, is based on the replicon of the streptococcal promiscuous rolling circle replication (RCR) plasmid pMV158. We inserted the controllable promoter P(M) of the S. pneumoniaemalMP operon, followed by a multi-cloning site sequence aimed to facilitate the insertion of target genes. The expression from P(M) is negatively regulated by the transcriptional repressor MalR, which is released from the DNA operator sequence by growing the cells in maltose-containing media. To get a highly regulated expression of the target gene, MalR was provided in cis by inserting the malR gene under control of the constitutive P(tet) promoter, which in pMV158 directs expression of the tetL gene. To test the functionality of the system, we cloned the reporter gene gfp from Aequorea victoria, encoding the green fluorescent protein (GFP). Pneumococcal cells harboring the recombinant plasmid rendered GFP fluorescence in a maltose-dependent mode with undetectable background levels in the absence of the inducer. The new vector, pLS1ROM, exhibits full structural and segregational stability and constitutes a valuable tool for genetic manipulation and regulated gene expression in S. pneumoniae.

Genetic regulation of the yefM-yoeB toxin-antitoxin locus of Streptococcus pneumoniae.

Type II (proteic) toxin-antitoxin systems (TAS) are ubiquitous among bacteria. In the chromosome of the pathogenic bacterium Streptococcus pneumoniae, there are at least eight putative TAS, one of them being the yefM-yoeB(Spn) operon studied here. Through footprinting analyses, we showed that purified YefM(Spn) antitoxin and the YefM-YoeB(Spn) TA protein complex bind to a palindrome sequence encompassing the -35 region of the main promoter (P(yefM2)) of the operon. Thus, the locus appeared to be negatively autoregulated with respect to P(yefM2), since YefM(Spn) behaved as a weak repressor with YoeB(Spn) as a corepressor. Interestingly, a BOX element, composed of a single copy (each) of the boxA and boxC subelements, was found upstream of promoter P(yefM2). BOX sequences are pneumococcal, perhaps mobile, genetic elements that have been associated with bacterial processes such as phase variation, virulence regulation, and genetic competence. In the yefM-yoeB(Spn) locus, the boxAC element provided an additional weak promoter, P(yefM1), upstream of P(yefM2) which was not regulated by the TA proteins. In addition, transcriptional fusions with a lacZ reporter gene showed that P(yefM1) was constitutive albeit weaker than P(yefM2). Intriguingly, the coupling of the boxAC element to P(yefM1) and yefM(Spn) in cis (but not in trans) led to transcriptional activation, indicating that the regulation of the yefM-yoeB(Spn) locus differs somewhat from that of other TA loci and may involve as yet unidentified elements. Conservation of the boxAC sequences in all available sequenced genomes of S. pneumoniae which contained the yefM-yoeB(Spn) locus suggested that its presence may provide a selective advantage to the bacterium.

Activin A skews macrophage polarization by promoting a proinflammatory phenotype and inhibiting the acquisition of anti-inflammatory macrophage markers.

M-CSF favors the generation of folate receptor ß-positive (FRß⁺), IL-10-producing, immunosuppressive, M2-polarized macrophages [M2 (M-CSF)], whereas GM-CSF promotes a proinflammatory, M1-polarized phenotype [M1 (GM-CSF)]. In the present study, we found that activin A was preferentially released by M1 (GM-CSF) macrophages, impaired the acquisition of FRß and other M2 (M-CSF)-specific markers, down-modulated the LPS-induced release of IL-10, and mediated the tumor cell growth-inhibitory activity of M1 (GM-CSF) macrophages, in which Smad2/3 is constitutively phosphorylated. The contribution of activin A to M1 (GM-CSF) macrophage polarization was evidenced by the capacity of a blocking anti-activin A antibody to reduce M1 (GM-CSF) polarization markers expression while enhancing FRß and other M2 (M-CSF) markers mRNA levels. Moreover, an inhibitor of activin receptor-like kinase 4/5/7 (ALK4/5/7 or SB431542) promoted M2 (M-CSF) marker expression but limited the acquisition of M1 (GM-CSF) polarization markers, suggesting a role for Smad2/3 activation in macrophage polarization. In agreement with these results, expression of activin A and M2 (M-CSF)-specific markers was oppositely regulated by tumor ascites. Therefore, activin A contributes to the proinflammatory macrophage polarization triggered by GM-CSF and limits the acquisition of the anti-inflammatory phenotype in a Smad2-dependent manner. Our results demonstrate that activin A-initiated Smad signaling skews macrophage polarization toward the acquisition of a proinflammatory phenotype.

The relBE2Spn toxin-antitoxin system of Streptococcus pneumoniae: role in antibiotic tolerance and functional conservation in clinical isolates.

Type II (proteic) chromosomal toxin-antitoxin systems (TAS) are widespread in Bacteria and Archaea but their precise function is known only for a limited number of them. Out of the many TAS described, the relBE family is one of the most abundant, being present in the three first sequenced strains of Streptococcus pneumoniae (D39, TIGR4 and R6). To address the function of the pneumococcal relBE2Spn TAS in the bacterial physiology, we have compared the response of the R6-relBE2Spn wild type strain with that of an isogenic derivative, Delta relB2Spn under different stress conditions such as carbon and amino acid starvation and antibiotic exposure. Differences on viability between the wild type and mutant strains were found only when treatment directly impaired protein synthesis. As a criterion for the permanence of this locus in a variety of clinical strains, we checked whether the relBE2Spn locus was conserved in around 100 pneumococcal strains, including clinical isolates and strains with known genomes. All strains, although having various types of polymorphisms at the vicinity of the TA region, contained a functional relBE2Spn locus and the type of its structure correlated with the multilocus sequence type. Functionality of this TAS was maintained even in cases where severe rearrangements around the relBE2Spn region were found. We conclude that even though the relBE2Spn TAS is not essential for pneumococcus, it may provide additional advantages to the bacteria for colonization and/or infection.