Eva Medina

Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation.

These days it has been increasingly recognized that mast cells (MCs) are critical components of host defense against pathogens. In this study, we have provided the first evidence that MCs can kill bacteria by entrapping them in extracellular structures similar to the extracellular traps described for neutrophils (NETs). We took advantage of the ability of MCs to kill the human pathogen Streptococcus pyogenes by a phagocytosis-independent mechanism in order to characterize the extracellular antimicrobial activity of MCs. Close contact of bacteria and MCs was required for full antimicrobial activity. Immunofluorescence and electron microscopy revealed that S pyogenes was entrapped by extracellular structures produced by MCs (MCETs), which are composed of DNA, histones, tryptase, and the antimicrobial peptide LL-37. Disruption of MCETs significantly reduced the antimicrobial effect of MCs, suggesting that intact extracellular webs are critical for effective inhibition of bacterial growth. Similar to NETs, production of MCETs was mediated by a reactive oxygen species (ROS)-dependent cell death mechanism accompanied by disruption of the nuclear envelope, which can be induced after stimulation of MCs with phorbol-12-myristate-13-acetate (PMA), H(2)O(2), or bacterial pathogens. Our study provides the first experimental evidence of antimicrobial extracellular traps formation by an immune cell population other than neutrophils.

Use of live bacterial vaccine vectors for antigen delivery : potential and limitations

Most infectious agents are restricted to the mucosal membranes or their transit through the mucosa constitutes a critical step in the infection process. Therefore, the elicitation of an efficient immune response, not only at systemic, but also at mucosal level, after vaccination is highly desirable, representing a significant advantage in order to prevent infection. This goal can be only achieved, when the vaccine formulation is administered by the mucosal route. However, soluble antigens given by this route are usually poorly immunogenic. Among the available approaches to stimulate efficient mucosal responses, the use of bacterial carriers to deliver vaccine antigens, probably, constitutes one of the most successful strategies. The potential and limitations of the most extensively studied bacterial carrier systems will be discussed.

Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection

Staphylococcus aureus is a frequent cause for serious, chronic and therapy‐refractive infections in spite of susceptibility to antibiotics in vitro. In chronic infections, altered bacterial phenotypes, such as small colony variants (SCVs), have been found. Yet, it is largely unclear whether the ability to interconvert from the wild‐type to the SCV phenotype is only a rare clinical and/or just laboratory phenomenon or is essential to sustain an infection. Here, we performed different long‐term in vitro and in vivo infection models with S. aureus and we show that viable bacteria can persist within host cells and/or tissues for several weeks. Persistence induced bacterial phenotypic diversity, including SCV phenotypes, accompanied by changes in virulence factor expression and auxotrophism. However, the recovered SCV phenotypes were highly dynamic and rapidly reverted to the fully virulent wild‐type form when leaving the intracellular location and infecting new cells. Our findings demonstrate that bacterial phenotype switching is an integral part of the infection process that enables the bacteria to hide inside host cells, which can be a reservoir for chronic and therapy‐refractive infections.

Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production

Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

Protective Immune Response against Streptococcus pyogenes in Mice after Intranasal Vaccination with the Fibronectin-Binding Protein SfbI

Despite the significant impact on human health of Streptococcus pyogenes, an efficacious vaccine has not yet been developed. Here, the potential as a vaccine candidate of a major streptococcal adhesin, the fibronectin-binding protein SfbI, was evaluated. Intranasal immunization of mice with either SfbI alone or coupled to cholera toxin B subunit (CTB) triggered efficient SfbI-specific humoral (mainly IgG) and lung mucosal (14% of total IgA) responses. CTB-immunized control mice were not protected against challenge with S. pyogenes (90%-100% lethality), whereas SfbI-vaccinated animals showed 80% and 90% protection against homologous and heterologous challenge, respectively. Multiple areas of consolidation with diffused cellular infiltrates (macrophages and neutrophils) were observed in lungs from control mice; the histologic structure was preserved in SfbI-vaccinated animals, which occasionally presented focal infiltrates confined to the perivascular, peribronchial, and subpleural areas. These results suggest that SfbI is a promising candidate for inclusion in acellular vaccines against S. pyogenes.

Immunological Mechanisms Underlying the Genetic Predisposition to Severe Staphylococcus aureus Infection in the Mouse Model

Host genetic variations play a significant role in conferring predisposition to infection. In this study, we examined the immune mechanisms underlying the host genetic predisposition to severe Staphylococcus aureus infection in different mouse strains. Whereas C57BL/6 mice were the most resistant in terms of control of bacterial growth and survival, A/J, DBA/2, and BALB/c mice were highly susceptible and succumbed to infection shortly after bacterial inoculation. Other strains (C3H/HeN, CBA, and C57BL/10) exhibited intermediate susceptibility levels. Susceptibility of mice to S. aureus was associated with an inability to limit bacterial growth in the kidneys and development of pathology. Resistance to S. aureus in C57BL/6 mice was dependent on innate immune mechanisms because Rag2-IL2Rgamma(-/-) C57BL/6 mice, which are deficient in B, T, and NK cells, were also resistant to infection. Indeed, neutrophil depletion or inhibition of neutrophil recruitment rendered C57BL/6 mice completely susceptible to S. aureus, indicating that neutrophils are essential for the observed resistance. Although neutrophil function is not inhibited in A/J mice, expression of neutrophil chemoattractants KC and MIP-2 peaked earlier in the kidneys of C57BL/6 mice than in A/J mice, indicating that a delay in neutrophil recruitment to the site of infection may underlie the increased susceptibility of A/J mice to S. aureus.

The expanding world of extracellular traps: not only neutrophils but much more

The release of extracellular traps (ETs) is a recently described mechanism of innate immune response to infection. Although ETs have been intensely investigated in the context of neutrophil antimicrobial effector mechanisms, other immune cells such as mast cells, eosinophils, and macrophages can also release these structures. The different ETs have several features in common, regardless of the type of cells from which they originated, including a DNA backbone with embedded antimicrobial peptides, proteases, and histones. However, they also exhibit remarkable individual differences such as the type of sub-cellular compartments from where the DNA backbone originates (e.g., nucleus or mitochondria), the proportion of responding cells within the pool, and/or the molecular mechanism/s underlying the ETs formation. This review summarizes the knowledge accumulated in recent years regarding the complex and expanding world of ETs and their role in immune function with particular emphasis on the role of other immune cells rather than on neutrophils exclusively.

Role of macrophages in host resistance to group A streptococci

ABSTRACT Macrophages provide the first line of defense against invading pathogens. The aim of this study was to determine the role of macrophages during infection with group A streptococci (Streptococcus pyogenes) in mice. Here, we report that resident macrophages can efficiently take up and kill S. pyogenes during in vivo infection, as demonstrated by immunofluorescence and electron microscopy, as well as colony counts. To evaluate the contribution of macrophages to the resolution of experimental infection with S. pyogenes, we compared the susceptibility of BALB/c mice rendered macrophage deficient by treatment with carrageenan with that of intact mice. The results show that depletion of macrophages enhanced the susceptibility of BALB/c mice to S. pyogenes infection, as evidenced by 100% mortality of macrophage-depleted mice compared to 90% survival of nondepleted control animals. The in vivo depletion of macrophages strongly enhanced bacterial loads in the blood and systemic organs. Resistance to S. pyogenes can be restored in macrophage-depleted mice by adoptive transfer of purified macrophages. The in vivo blocking of the macrophage phagocytic function by treatment with gadolinium III chloride also resulted in enhanced susceptibility to S. pyogenes. Interestingly, depletion of macrophages prior to or during the first 24 h of infection decreased survival dramatically; in contrast, no mortality was observed in infected nondepleted animals or mice depleted after 48 h of infection. These results emphasize the important contribution of macrophages to the early control of S. pyogenes infection.

Survival of Streptococcus pyogenes within Host Phagocytic Cells: A Pathogenic Mechanism for Persistence and Systemic Invasion

Streptococcus pyogenes is generally an extracellular pathogen that can survive and persist within the host by circumventing the host defense mechanisms. To achieve this, S. pyogenes has developed a number of strategies to circumvent the host immune system (e.g., virulence factors directed to prevent phagocytosis). By use of a murine model of skin infection, it was shown that survival within host phagocytic cells constitutes an additional strategy used by S. pyogenes to evade the host defenses and disseminate. Viable microorganisms were isolated from mouse phagocytic cells after in vitro or during in vivo infection. The capacity of intracellularly located bacteria to establish infection was demonstrated by the efficiency of gentamicin-treated neutrophils isolated from infected mice to transfer infection when injected intravenously into naive mice. The ability of S. pyogenes to exploit the inflammatory response of the host by surviving inside phagocytic cells may constitute an additional virulence mechanism of this pathogen.

Neutrophil Extracellular Traps: A Strategic Tactic to Defeat Pathogens with Potential Consequences for the Host

Recent investigations have highlighted new roles for neutrophils in the biology of infection and inflammation. Neutrophils are one of the main players in the innate immune system and actively contribute to host defense by killing pathogens. Added to their ability to eliminate microorganisms by phagocytosis, neutrophils can also kill microbes by capturing them in extracellular structures consisting of granule proteins and DNA called neutrophil extracellular traps (NETs). This review summarizes the recent advancements regarding the structure, production and biological relevance of NETs.