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Plasmacytoid dendritic cells (pDC) are the major producer of type 1 IFN in response to TLR7 agonists. Aberrant TLR7 activation and type 1 IFN expression by pDCs are linked to the pathogenesis of certain types of autoimmune diseases, including systemic lupus erythematosus (SLE). This study investigated the underlying mechanisms for TLR7-mediated cytokine expression by pDCs using a late endosome trafficking inhibitor, EGA (4-bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone). We found that EGA treatment decreased IFNα expression by pDCs stimulated with imiquimod (R837), single-stranded RNA40, and influenza virus. EGA also decreased TNFα expression and secretion by R837-stimulated pDCs. Mechanistically, EGA treatment decreased phosphorylation of IKKα/β, STAT1, and p38, and prolonged degradation of IκBα. Furthermore, EGA treatment decreased the colocalization of 3F, a substituted adenine TLR7 agonist, with LAMP1+ compartments in pDCs. EGA was also capable of diminishing IFNα expression by SLE pDCs treated with R837 or live PR8/A/34 influenza viruses. Therefore, we concluded that trafficking of TLR7 agonists to LAMP1+ compartments is important for IFNα expression by pDCs. Data from this study support additional examinations of the potential benefits of EGA in treating type 1 IFN-associated inflammatory diseases in the future.

The bacterium Brucella abortus is an intracellular pathogen that modulates autophagy in host cells. Here, the authors identify two B. abortus effectors that interact with host protease SENP3, thus promoting cytoplasmic accumulation of nucleolar proteins associated with ribosomal biogenesis and facilitating intracellular replication of the pathogen

Summary: Severe acute malnutrition (SAM) is a multifactorial disease affecting millions of children worldwide. It is associated with changes in intestinal physiology, microbiota, and mucosal immunity, emphasizing the need for multidisciplinary studies to unravel its full pathogenesis. We established an experimental model in which weanling mice fed a high-deficiency diet mimic key anthropometric and physiological features of SAM in children. This diet alters the intestinal microbiota (less segmented filamentous bacteria, spatial proximity to epithelium), metabolism (decreased butyrate), and immune cell populations (depletion of LysoDC in Peyer’s patches and intestinal Th17 cells). A nutritional intervention leads to a fast zoometric and intestinal physiology recovery but to an incomplete restoration of the intestinal microbiota, metabolism, and immune system. Altogether, we provide a preclinical model of SAM and have identified key markers to target with future interventions during the education of the immune system to improve SAM whole defects.

Plasmacytoid dendritic cells (pDCs) exhibit bifurcated cytokine responses to TLR9 agonists, an IRF7-mediated type 1 IFN response or a pro-inflammatory cytokine response via the activation of NF-κB. This bifurcated response has been hypothesized to result from either distinct signaling endosomes or endo-lysosomal trafficking delay of TLR9 agonists allowing for autocrine signaling to affect outcomes. Utilizing the late endosome trafficking inhibitor, EGA, we assessed the bifurcated cytokine responses of pDCs to TLR9 stimulation. EGA treatment of pDCs diminished both IFNα and pro-inflammatory cytokine expression induced by CpG DNAs (D- and K-type), CpG-DNAs complexed with DOTAP, and genomic DNAs complexed with LL37. Mechanistically, EGA suppressed phosphorylation of IKKα/β, STAT1, Akt, and p38, and decreased colocalization of CpG oligodeoxynucleotides with LAMP+ endo-lysosomes. EGA also diminished type 1 IFN expression by pDCs from systemic lupus erythematosus patients. Therefore, our findings help understand mechanisms for the bifurcated cytokine responses by pDCs and support future examination of the potential benefit of EGA in treating type 1 IFN-associated inflammatory diseases in the future.

IntroductionThe emergence of several SARS-CoV-2 variants during the COVID pandemic has revealed the impact of variant diversity on viral infectivity and host immune responses. While antibodies and CD8 T cells are essential to clear viral infection, the protective role of innate immunity including macrophages has been recognized. The aims of our study were to compare the infectivity of different SARS-CoV-2 variants in monocyte-derived macrophages (MDM) and to assess their activation profiles and the role of ACE2 (Angiotensin-converting enzyme 2), the main SARS-CoV-2 receptor. We also studied the ability of macrophages infected to affect other immune cells such as γδ2 T cells, another partner of innate immune response to viral infections.ResultsWe showed that the SARS-CoV-2 variants α-B.1.1.7 (United Kingdom), β-B.1.351 (South Africa), γ-P.1 (Brazil), δ-B.1.617 (India) and B.1.1.529 (Omicron), infected MDM without replication, the γ-Brazil variant exhibiting increased infectivity for MDM. No clear polarization profile of SARS-CoV-2 variants-infected MDM was observed. The β-B.1.351 (South Africa) variant induced macrophage activation while B.1.1.529 (Omicron) was rather inhibitory. We observed that SARS-CoV-2 variants modulated ACE2 expression in MDM. In particular, the β-B.1.351 (South Africa) variant induced a higher expression of ACE2, related to MDM activation. Finally, all variants were able to activate γδ2 cells among which γ-P.1 (Brazil) and β-B.1.351 (South Africa) variants were the most efficient.ConclusionOur data show that SARS-CoV-2 variants can infect MDM and modulate their activation, which was correlated with the ACE2 expression. They also affect γδ2 T cell activation. The macrophage response to SARS-CoV-2 variants was stereotypical.

Brucellosis is considered one of the major zoonoses worldwide, constituting a critical livestock and human health concern with a huge socio-economic burden. Brucella genus, its etiologic agent, is composed of intracellular bacteria that have evolved a prodigious ability to elude and shape host immunity to establish chronic infection. Brucella’s intracellular lifestyle and pathogen-associated molecular patterns, such as its specific lipopolysaccharide (LPS), are key factors for hiding and hampering recognition by the immune system. Here, we will review the current knowledge of evading and immunosuppressive mechanisms elicited by Brucella species to persist stealthily in their hosts, such as those triggered by their LPS and cyclic β-1,2-d-glucan or involved in neutrophil and monocyte avoidance, antigen presentation impairment, the modulation of T cell responses and immunometabolism. Attractive strategies exploited by other successful chronic pathogenic bacteria, including Mycobacteria, Salmonella, and Chlamydia, will be also discussed, with a special emphasis on the mechanisms operating in brucellosis, such as granuloma formation, pyroptosis, and manipulation of type I and III IFNs, B cells, innate lymphoid cells, and host lipids. A better understanding of these stratagems is essential to fighting bacterial chronic infections and designing innovative treatments and vaccines.

The lipopolysaccharide (LPS) is a major virulence factor of Brucella, a facultative intracellular pathogenic Gram-negative bacterium. Brucella LPS exhibits a low toxicity and its atypical structure was postulated to delay the host immune response, favouring the establishment of chronic disease. Here we carried out an in-depth in vitro and in vivo characterisation of the immunomodulatory effects of Brucella LPS on different dendritic cell (DC) subpopulations. By using LPSs from bacteria that share some of Brucella LPS structural features, we demonstrated that the core component of B. melitensis wild-type (Bm-wt) LPS accounts for the low activation potential of Brucella LPS in mouse GM-CSF-derived (GM-) DCs. Contrary to the accepted dogma considering Brucella LPS a poor TLR4 agonist and DC activator, Bm-wt LPS selectively induced expression of surface activation markers and cytokine secretion from Flt3-Ligand-derived (FL-) DCs in a TLR4-dependent manner. It also primed in vitro T cell proliferation by FL-DCs. In contrast, modified LPS with a defective core purified from Brucella carrying a mutated wadC gene (Bm-wadC), efficiently potentiated mouse and human DC activation and T cell proliferation in vitro. In vivo, Bm-wt LPS promoted scant activation of splenic DC subsets and limited recruitment of monocyte- DC like cells in the spleen, conversely to Bm-wadC LPS. Bm-wadC live bacteria drove high cytokine secretion levels in sera of infected mice. Altogether, these results illustrate the immunomodulatory properties of Brucella LPS and the enhanced DC activation ability of the wadC mutation with potential for vaccine development targeting Brucella core LPS structure.

Initially intended for nutrient uptake, phagocytosis represents a central mechanism of debris removal and host defense against invading pathogens through the entire animal kingdom. In vertebrates and also many invertebrates, macrophages (MFs) and MF-like cells (e.g., coelomocytes and hemocytes) are professional phagocytic cells that seed tissues to maintain homeostasis through pathogen killing, efferocytosis and tissue shaping, repair, and remodeling. Some MF functions are common to all species and tissues, whereas others are specific to their homing tissue. Indeed, shaped by their microenvironment, MFs become adapted to perform particular functions, highlighting their great plasticity and giving rise to high population diversity. Interestingly, the gut displays several anatomic and functional compartments with large pools of strikingly diversified MF populations. This review focuses on recent advances on intestinal MFs in several species, which have allowed to infer their specificity and functions.

T-bet is a transcription factor known to initiate and coordinate the gene expression program during Th1 differentiation, which is crucial for clearance of intracellular pathogens. Q fever is a worldwide zoonosis caused by Coxiella burnetii. This bacterium is transmitted to humans by aerosol. Indeed, the inhibition of the Coxiella-specific adaptive Th1 immune response leads to persistent infection and organ injury. How deficiency of T-bet affects host infection by C. burnetii has not been investigated. Here, using mice with a deletion of the T-bet gene and an airborne mode of infection to reproduce the natural conditions of C. burnetii infection, we show that infected T-bet–/– mice were more affected than wild-type mice. The lack of T-bet leads to defective bacterial control, intense replication, persistent infection, and organ injury manifesting as an increased number of granulomas. The absence of T-bet was also associated with an impaired immune response. Indeed, the production of the immunomodulatory cytokines interleukin (IL)-6 and IL-10 was increased, whereas the expression of microbicidal genes by splenocytes was impaired. Moreover, the absence of T-bet exhibited impaired production of interferon-γ, the principal cytokine released by Th1 effector cells. Thus, our study highlights the key role of T-bet in the control of C. burnetii infection in mice and leads to a reappraisal of granulomas in the pathogenesis of Q fever disease.

Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

Summary: The monocyte-derived phagocytes termed LysoDCs are hallmarks of Peyer’s patches, where their main function is to sample intestinal microorganisms. Here, we study their differentiation pathways in relation with their sampling, migratory, and T cell-priming abilities. Among four identified LysoDC differentiation stages displaying similar phagocytic activity, one is located in follicles, and the others reside in subepithelial domes (SED), where they proliferate and mature as they get closer to the epithelium. Mature LysoDCs but not macrophages express a gene set in common with conventional dendritic cells and prime naive helper T cells in vitro. At steady state, they do not migrate into naive T cell-enriched interfollicular regions (IFRs), but upon stimulation, they express the chemokine receptor CCR7 and migrate from SED to the IFR periphery, where they strongly interact with proliferative immune cells. Finally, we show that LysoDCs populate human Peyer’s patches, strengthening their interest as targets for modulating intestinal immunity. : Wagner et al. dissect the differentiation pathways of the Peyer’s patch monocyte-derived dendritic cells termed LysoDCs. They show that LysoDCs mature as they get closer to the epithelium. Mature LysoDCs migrate to the periphery of the T cell zone, where proliferation of immune cells occurs only when stimulated. Keywords: intestinal immunity, Peyer’s patches, phagocytes, dendritic cells, monocyte-derived cells, LysoDC, cell differentiation, single-cell RNA sequencing, antigen sampling, naive T cell priming

Salmonella enterica serotype Typhimurium is a bacterium that causes gastroenteritis and diarrhea in humans. The genome of S. Typhimurium codes for diverse virulence factors, among which are the toxin-antitoxin (TA) systems. SehAB is a type II TA, where SehA is the toxin and SehB is the antitoxin. It was previously reported that the absence of the SehB antitoxin affects the growth of S. Typhimurium. In addition, the SehB antitoxin can interact directly with the SehA toxin neutralizing its toxic effect as well as repressing its own expression. We identified conserved residues on SehB homologous proteins. Point mutations were introduced at both N- and C-terminal of SehB antitoxin to analyze the effect of these changes on its transcription repressor function, on its ability to form homodimers and on the virulence of S. Typhimurium. All changes in amino acid residues at both the N- and C-terminal affected the repressor function of SehB antitoxin and they were required for DNA-binding activity. Mutations in the amino acid residues at the N-terminal showed a lower capacity for homodimer formation of the SehB protein. However, none of the SehB point mutants were affected in the interaction with the SehA toxin. In terms of virulence, the eight single-amino acid mutations were attenuated for virulence in the mouse model. In agreement with our results, the eight amino acid residues of SehB antitoxin were required for its repressor activity, affecting both homodimerization and DNA-binding activity, supporting the notion that both activities of SehB antitoxin are required to confer virulence to Salmonella enterica.

Whooping cough is a severe, highly contagious disease of the human respiratory tract, caused by Bordetellapertussis. The pathogenicity requires several virulence factors, including pertussis toxin (PTX), a key component of current available vaccines. Current vaccines do not induce mucosal immunity. Tissue-resident memory T cells (Trm) are among the first lines of defense against invading pathogens and are involved in long-term protection. However, the factors involved in Trm establishment remain unknown. Comparing two B.pertussis strains expressing PTX (WT) or not (ΔPTX), we show that the toxin is required to generate both lung CD4+ and CD8+ Trm. Co-administering purified PTX with ΔPTX is sufficient to generate these Trm subsets. Importantly, adoptive transfer of lung CD4+ or CD8+ Trm conferred protection against B. pertussis in naïve mice. Taken together, our data demonstrate for the first time a critical role for PTX in the induction of mucosal long-term protection against B. pertussis.

Brucella is an intracellular bacterium that causes abortion, reproduction failure in livestock and leads to a debilitating flu-like illness with serious chronic complications if untreated in humans. As a successful intracellular pathogen, Brucella has developed strategies to avoid recognition by the immune system of the host and promote its survival and replication. In vivo, Brucellae reside mostly within phagocytes and other cells including trophoblasts, where they establish a preferred replicative niche inside the endoplasmic reticulum. This process is central as it gives Brucella the ability to maintain replicating-surviving cycles for long periods of time, even at low bacterial numbers, in its cellular niches. In this review, we propose that Brucella takes advantage of the environment provided by the cellular niches in which it resides to generate reservoirs and disseminate to other organs. We will discuss how the favored cellular niches for Brucella infection in the host give rise to anatomical reservoirs that may lead to chronic infections or persistence in asymptomatic subjects, and which may be considered as a threat for further contamination. A special emphasis will be put on bone marrow, lymph nodes, reproductive and for the first time adipose tissues, as well as wildlife reservoirs.

Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific T cells. Delivering antigen to DCs via surface receptors is thus an appealing strategy to evoke cellular immunity. Nonetheless, which DC surface receptor to target to yield the optimal CD8+ and CD4+ T cell responses remains elusive. Herein, we report the superiority of CD40 over 9 different lectins and scavenger receptors at evoking antigen-specific CD8+ T cell responses. However, lectins (e.g., LOX-1 and Dectin-1) were more efficient than CD40 at eliciting CD4+ T cell responses. Common and distinct patterns of subcellular and intracellular localization of receptor-bound αCD40, αLOX-1 and αDectin-1 further support their functional specialization at enhancing antigen presentation to either CD8+ or CD4+ T cells. Lastly, we demonstrate that antigen targeting to CD40 can evoke potent antigen-specific CD8+ T cell responses in human CD40 transgenic mice. This study provides fundamental information for the rational design of vaccines against cancers and viral infections.

The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life.

The gut represents a potential entry site for a wide range of pathogens including protozoa, bacteria, viruses, or fungi. Consequently, it is protected by one of the largest and most diversified population of immune cells of the body. Its surveillance requires the constant sampling of its encounters by dedicated sentinels composed of follicles and their associated epithelium located in specialized area. In the small intestine, Peyer’s patches (PPs) are the most important of these mucosal immune response inductive sites. Through several mechanisms including transcytosis by specialized epithelial cells called M-cells, access to the gut lumen is facilitated in PPs. Although antigen sampling is critical to the initiation of the mucosal immune response, pathogens have evolved strategies to take advantage of this permissive gateway to enter the host and disseminate. It is, therefore, critical to decipher the mechanisms that underlie both host defense and pathogen subversive strategies in order to develop new mucosal-based therapeutic approaches. Whereas penetration of pathogens through M cells has been well described, their fate once they have reached the subepithelial dome (SED) remains less well understood. Nevertheless, it is clear that the mononuclear phagocyte system (MPS) plays a critical role in handling these pathogens. MPS members, including both dendritic cells and macrophages, are indeed strongly enriched in the SED, interact with M cells, and are necessary for antigen presentation to immune effector cells. This review focuses on recent advances, which have allowed distinguishing the different PP mononuclear phagocyte subsets. It gives an overview of their diversity, specificity, location, and functions. Interaction of PP phagocytes with the microbiota and the follicle-associated epithelium as well as PP infection studies are described in the light of these new criteria of PP phagocyte identification. Finally, known alterations affecting the different phagocyte subsets during PP stimulation or infection are discussed.

Peyer's patches (PPs) are primary inductive sites of mucosal immunity. The PP mononuclear phagocyte system, which encompasses both dendritic cells (DCs) and macrophages, is essential for the initiation of the mucosal immune response. We recently developed a method to isolate each mononuclear phagocyte subset of PP (Bonnardel et al., 2015). We performed a transcriptional analysis of three of these subsets: the CD11b+ conventional DC, the lysozyme-expressing monocyte-derived DC termed LysoDC and the CD11chi lysozyme-expressing macrophages. Here, we provide details of the gating strategy we used to isolate each phagocyte subset and show the quality controls and analysis associated with our gene array data deposited into Gene Expression Omnibus (GEO) under GSE65514.

Peyer’s patches (PPs) are primary inductive sites of mucosal immunity. Defining PP mononuclear phagocyte system (MPS) is thus crucial to understand the initiation of mucosal immune response. We provide a comprehensive analysis of the phenotype, distribution, ontogeny, lifespan, function, and transcriptional profile of PP MPS. We show that monocytes give rise to macrophages and to lysozyme-expressing dendritic cells (LysoDCs), which are both involved in particulate antigen uptake, display strong innate antiviral and antibacterial gene signatures, and, upon TLR7 stimulation, secrete IL-6 and TNF, but neither IL-10 nor IFNγ. However, unlike macrophages, LysoDCs display a rapid renewal rate, strongly express genes of the MHCII presentation pathway, and prime naive helper T cells for IFNγ production. Our results show that monocytes differentiate locally into LysoDCs and macrophages, which display distinct features from their adjacent villus counterparts.

Brucella is a Gram-negative facultative intracellular bacterium responsible for a chronic disease known as brucellosis, the most widespread re-emerging zoonosis worldwide. Establishment of a T helper 1 (Th1)-mediated immune response characterized by the production of IL-12 and IFNγ is essential to control the disease. Leukotrienes derived from arachidonic acid (AA) metabolism are known to negatively regulate a protective Th1 immune response against bacterial infections. Here, using genomics approaches we demonstrate that Brucella abortus strongly stimulates the prostaglandin pathway in dendritic cells (DC). We also show an induction of AA production by infected cells. This correlates with the expression of Ptgs2, a gene encoding the downstream cyclooxygenase-2 (COX-2) enzyme in infected DC. By comparing different infection routes (oral, intradermal, intranasal and conjunctival), we identified the intradermal inoculation route as the more potent in inducing Ptgs2 expression but also in inducing a local inflammatory response in the draining cervical lymph nodes (CLN). NS-398, a specific inhibitor of COX-2 enzymatic activity decreased B. melitensis burden in the CLN after intradermal infection. This effect was accompanied by a decrease of Il10 and a concomitant increase of Ifng expression. Altogether these results suggest that Brucella has evolved to take advantage of the prostaglandin pathway in the harsh environment of the CLN in order to persist and subvert immune responses. This work also proposes that novel strategies to control brucellosis may include the use of COX-2 inhibitors.

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.

The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1β signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1β antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1β and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the α-hemolysin toxin inhibits IL-1β secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia.

Cervical lymph nodes (CLN) are the first lymph nodes encountered by material taking the oral route. To study their role in orally acquired infections, we analyzed 307 patients of up to 14 years treated in the university clinic of Skopje, Macedonia, for brucellosis, a zoonotic bacterial disease frequently acquired by ingestion of contaminated dairy products. From these children, 36% had lymphadenopathy. Among orally infected children, lymphadenopathy with CLN being the only lymph nodes affected was significantly more frequent as compared to those infected by contact with animals (83% vs. 63%), suggesting a possible involvement of CLN during orally acquired human brucellosis. Using a murine model where bacteria are delivered into the oral cavity, we show that Brucella quickly and selectively colonize the CLN where they proliferate and persist over long periods of time for up to 50 days post-infection. A similar efficient though less specific drainage to CLN was found for Brucella, Salmonella typhimurium and fluorescent microspheres delivered by gavage, a pathway likely representing a mixed infection mode of intragastric and oral infection, suggesting a central pathway of drained material. Microspheres as well as bacteria drained to CLN predominately reside in cells expressing CD68 and no or low levels of CD11c. Even though no systemic response could be detected, Brucella induced a locally restricted inflammatory reaction with increased expression levels of interferon γ, interleukin (IL)-6, IL-12, granzyme B and a delayed induction of Nos2. Inflammation led to pronounced lymphadenopathy, infiltration of macrophages/monocytes expressing high levels of major histocompatibility complex II and to formation of epitheloid granulomas. Together, these results highlight the role of CLN in oral infections as both, an initial and efficient trap for bacterial invaders and as possible reservoir for chronic pathogens. They likewise cast a new light on the significance of oral routes for means of vaccination.

Dendritic cells (DCs) orchestrate host defenses against microorganisms. In infectious diseases due to intracellular bacteria, the inefficiency of the immune system to eradicate microorganisms has been attributed to the hijacking of DC functions. In this study, we selected intracellular bacterial pathogens with distinct lifestyles and explored the responses of monocyte-derived DCs (moDCs). Using lipopolysaccharide as a control, we found that Orientia tsutsugamushi, the causative agent of scrub typhus that survives in the cytosol of target cells, induced moDC maturation, as assessed by decreased endocytosis activity, the ability to induce lymphocyte proliferation and the membrane expression of phenotypic markers. In contrast, Coxiella burnetii, the agent of Q fever, and Brucella abortus, the agent of brucellosis, both of which reside in vacuolar compartments, only partly induced the maturation of moDCs, as demonstrated by a phenotypic analysis. To analyze the mechanisms used by C. burnetii and B. abortus to alter moDC activation, we performed microarray and found that C. burnetii and B. abortus induced a specific signature consisting of TLR4, TLR3, STAT1 and interferon response genes. These genes were down-modulated in response to C. burnetii and B. abortus but up-modulated in moDCs activated by lipopolysaccharide and O. tsutsugamushi. This transcriptional alteration was associated with the defective interferon-β production. This study demonstrates that intracellular bacteria specifically affect moDC responses and emphasizes how C. burnetii and B. abortus interfere with moDC activation and the antimicrobial immune response. We believe that comparing infection by several bacterial species may be useful for defining new pathways and biomarkers and for developing new treatment strategies.

Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.

Toxin-antitoxin (TA) modules are widely prevalent in both bacteria and archaea. Originally described as stabilizing elements of plasmids, TA modules are also widespread on bacterial chromosomes. These modules promote bacterial persistence in response to specific environmental stresses. So far, the possibility that TA modules could be involved in bacterial virulence has been largely neglected, but recent comparative genomic studies have shown that the presence of TA modules is significantly associated with the pathogenicity of bacteria. Using Salmonella as a model, we investigated whether TA modules help bacteria to overcome the stress conditions encountered during colonization, thereby supporting virulence in the host. By bioinformatics analyses, we found that the genome of the pathogenic bacterium Salmonella Typhimurium encodes at least 11 type II TA modules. Several of these are conserved in other pathogenic strains but absent from non-pathogenic species indicating that certain TA modules might play a role in Salmonella pathogenicity. We show that one TA module, hereafter referred to as sehAB, plays a transient role in virulence in perorally inoculated mice. The use of a transcriptional reporter demonstrated that bacteria in which sehAB is strongly activated are predominantly localized in the mesenteric lymph nodes. In addition, sehAB was shown to be important for the survival of Salmonella in these peripheral lymphoid organs. These data indicate that the transient activation of a type II TA module can bring a selective advantage favouring virulence and demonstrate that TA modules are engaged in Salmonella pathogenesis.

CD4(+) T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4(+) T cells (CD4(+) CTL) during Brucella abortus infection. These CD4(+) CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4(+) CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4(+) T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4(+) CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.

Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. We investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4(+) T and CD8(+) T cell responses and, importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, our data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.

The brucellae are α-Proteobacteria facultative intracellular parasites that cause an important zoonosis. These bacteria escape early detection by innate immunity, an ability associated to the absence of marked pathogen-associated molecular patterns in the cell envelope lipopolysaccharide, lipoproteins and flagellin. We show here that, in contrast to the outer membrane ornithine lipids (OL) of other Gram negative bacteria, Brucella abortus OL lack a marked pathogen-associated molecular pattern activity. We identified two OL genes (olsB and olsA) and by generating the corresponding mutants found that olsB deficient B. abortus did not synthesize OL or their lyso-OL precursors. Liposomes constructed with B. abortus OL did not trigger IL-6 or TNF-α release by macrophages whereas those constructed with Bordetella pertussis OL and the olsB mutant lipids as carriers were highly active. The OL deficiency in the olsB mutant did not promote proinflammatory responses or generated attenuation in mice. In addition, OL deficiency did not increase sensitivity to polymyxins, normal serum or complement consumption, or alter the permeability to antibiotics and dyes. Taken together, these observations indicate that OL have become dispensable in the extant brucellae and are consistent within the trend observed in α-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity.

Salmonella enterica serovar Typhimurium is a Gram-negative bacterial pathogen causing gastroenteritis in humans and a systemic typhoid-like illness in mice. The capacity of Salmonella to cause diseases relies on the establishment of its intracellular replication niche, a membrane-bound compartment named the Salmonella-containing vacuole (SCV). This requires the translocation of bacterial effector proteins into the host cell by type three secretion systems. Among these effectors, SifA is required for the SCV stability, the formation of Salmonella-induced filaments (SIFs) and plays an important role in the virulence of Salmonella. Here we show that the effector SopD2 is responsible for the SCV instability that triggers the cytoplasmic release of a sifA(-) mutant. Deletion of sopD2 also rescued intra-macrophagic replication and increased virulence of sifA(-) mutants in mice. Membrane tubular structures that extend from the SCV are the hallmark of Salmonella-infected cells. Until now, these unique structures have not been observed in the absence of SifA. The deletion of sopD2 in a sifA(-) mutant strain re-established membrane trafficking from the SCV and led to the formation of new membrane tubular structures, the formation of which is dependent on other Salmonella effector(s). Taken together, our data demonstrate that SopD2 inhibits the vesicular transport and the formation of tubules that extend outward from the SCV and thereby contributes to the sifA(-) associated phenotypes. These results also highlight the antagonistic roles played by SopD2 and SifA in the membrane dynamics of the vacuole, and the complex actions of SopD2, SifA, PipB2 and other unidentified effector(s) in the biogenesis and maintenance of the Salmonella replicative niche.

BACKGROUND: Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs) within autophagosomes. METHODOLOGY/PRINCIPAL FINDINGS: A 10-fold increase of CFU is found between 2 h and 6 h p.i. in H. pylori-infected BMDCs. Autophagy is induced around the bacterium and participates at late time points of infection for the clearance of intracellular H. pylori. As a consequence of infection, LC3, LAMP1 and MHC class II molecules are retained within the H. pylori-containing vacuoles and export of MHC class II molecules to cell surface is blocked. However, formalin-fixed H. pylori still maintain this inhibitory activity in BMDC derived from wild type mice, but not in from either TLR4 or TLR2-deficient mice, suggesting the involvement of H. pylori-LPS in this process. TNF-alpha, IL-6 and IL-10 expression was also modulated upon infection showing a TLR2-specific dependent IL-10 secretion. No IL-12 was detected favoring the hypothesis of a down modulation of DC functions during H. pylori infection. Furthermore, antigen-specific T cells proliferation was also impaired upon infection. CONCLUSIONS/SIGNIFICANCE: H. pylori can infect and replicate in BMDCs and thereby affects DC-mediated immune responses. The implication of this new finding is discussed for the biological life cycle of H. pylori in the host.

BACKGROUND: The two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions. METHODOLOGY/PRINCIPAL FINDINGS: A total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d), lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others) were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ) were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered. CONCLUSIONS/SIGNIFICANCE: All these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the "Brucella-containing vacuole" (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

"Nanobacteria" are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of "nanobacteria" susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of "nanobacteria" as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call "nanons." The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor.

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.

BACKGROUND: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. METHODOLOGY/PRINCIPAL FINDINGS: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

Brucellosis is a zoonotic bacterial infection that may persist for long periods causing relapses in antibiotic-treated patients. The ability of Brucella to develop chronic infections is linked to their capacity to invade and replicate within the mononuclear phagocyte system, including the bone marrow (BM). Persistence of Brucella in the BM has been associated with hematological complications such as neutropenia, thrombocytopenia, anemia, and pancytopenia in human patients. In the mouse model, we observed that the number of Brucella abortus in the BM remained constant for up to 168 days of postinfection. This persistence was associated with histopathological changes, accompanied by augmented numbers of BM myeloid GMP progenitors, PMNs, and CD4+ lymphocytes during the acute phase (eight days) of the infection in the BM. Monocytes, PMNs, and GMP cells were identified as the cells harboring Brucella in the BM. We propose that the BM is an essential niche for the bacterium to establish long-lasting infections and that infected PMNs may serve as vehicles for dispersion of Brucella organisms, following the Trojan horse hypothesis. Monocytes are solid candidates for Brucella reservoirs in the BM.