Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of caspase activation. Expression of IcaA in L. pneumophila inhibited the caspase-11 activation in macrophages. Moreover, icaA- mutants of C. burnetii failed to suppress the caspase-11-mediated inflammasome activation induced by L. pneumophila. Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot/Icm type IV secretion system and interferes with the caspase-11-induced, non-canonical activation of the inflammasome.

dCoxiella burnetii replicates within permissive host cells by employing a Dot/Icm type IV secretion system (T4SS) to translocate effector proteins that direct the formation of a parasitophorous vacuole. C57BL/6 mouse macrophages restrict the intracellular replication of the C. burnetii Nine Mile phase II (NMII) strain. However, eliminating Toll-like receptor 2 (TLR2) permits bacterial replication, indicating that the restriction of bacterial replication is immune mediated. Here, we examined whether additional innate immune pathways are employed by C57BL/6 macrophages to sense and restrict NMII replication. In addition to the known role of TLR2 in detecting and restricting NMII infection, we found that TLR4 also contributes to cytokine responses but is not required to restrict bacterial replication. Furthermore, the TLR signaling adaptors MyD88 and Trif are required for cytokine responses and restricting bacterial replication. The C. burnetii NMII T4SS translocates bacterial products into C57BL/6 macrophages. However, there was little evidence of cytosolic immune sensing of NMII, as there was a lack of inflammasome activation, T4SS-dependent cytokine responses, and robust type I interferon (IFN) production, and these pathways were not required to restrict bacterial replication. Instead, endogenous tumor necrosis factor (TNF) produced upon TLR sensing of C. burnetii NMII was required to control bacterial replication. Therefore, our findings indicate a primary role for TNF produced upon immune detection of C. burnetii NMII by TLRs, rather than cytosolic PRRs, in enabling C57BL/6 macrophages to restrict bacterial replication.T o initiate innate immune defense against bacterial pathogens, infected host cells utilize pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) (1-3). Toll-like receptors (TLRs) located at the cell surface and within endosomes detect extracellular PAMPs such as bacterial lipoproteins and lipopolysaccharide (LPS) (4). Downstream of TLRs, the adaptor proteins MyD88 and Trif activate several signaling pathways, including NF-B, mitogen-activated protein kinases (MAPKs), and interferon (IFN) regulatory factor 3 (IRF3), which direct the expression of proinflammatory cytokines and other antimicrobial effectors (4). For intracellular bacterial pathogens, cytosolic PRRs, such as those of the nucleotide binding domain/ leucine-rich repeat (NLR) and RIG-I-like receptor (RLR) families, often are critical for host defense as they respond to PAMPs introduced into the host cell cytosol by bacterial pore-forming toxins or specialized secretion systems (5-8). In addition, cytosolic sensing can lead to the assembly of a multiprotein complex termed the inflammasome, which activates the host proteases caspase-1 and caspase-11, resulting in the release of IL-1 family cytokines and a form of cell death known as pyroptosis (9-16). These innate immune pathways collaborate to restrict intracellular bacterial infection through both cell-intrinsic and -extrinsic mechanisms (17)(18...

Inflammasomes are multimeric protein complexes that initiate inflammatory cascades. Their activation is a hallmark of many infectious or inflammatory diseases. Their composition and activity are specified by proinflammatory stimuli. For example, the NLRP3 inflammasome is activated in response to cell damage and K efflux, whereas the AIM2 inflammasome is activated in response to cytosolic DNA. We used Legionella pneumophila, an intracellular bacterial pathogen that activates multiple inflammasomes, to elucidate the molecular mechanisms regulating inflammasome activation during infection. Upon infection, the AIM2 inflammasome engaged caspase-1 to induce pore formation in the cell membrane, which then caused K-efflux-mediated activation of NLRP3. Thus, the AIM2 inflammasome amplifies signals of infection, triggering noncanonical activation of NLRP3. During infection, AIM2 and caspase-11 induced membrane damage, which was sufficient and essential for activating the NLRP3 inflammasome. Our data reveal that different inflammasomes regulate one another's activity to ensure an effective immune response to infection.

bCoxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. Q fever is an atypical pneumonia transmitted through inhalation of contaminated aerosols. In mammalian lungs, C. burnetii infects and replicates in several cell types, including alveolar macrophages (AMs). The innate immunity and signaling pathways operating during infection are still poorly understood, in part because of the lack of relevant host cell models for infection in vitro. In the study described here, we investigated and characterized the infection of primary murine AMs by C. burnetii phase II in vitro. Our data reveal that AMs show a pronounced M2 polarization and are highly permissive to C. burnetii multiplication in vitro. Murine AMs present an increased susceptibility to infection in comparison to primary bone marrow-derived macrophages. AMs support more than 2 logs of bacterial replication during 12 days of infection in culture, similar to highly susceptible host cells, such as Vero and THP-1 cells. As a proof of principle that AMs are useful for investigation of C. burnetii replication, we performed experiments with AMs from Nos2 ؊/؊ or Ifng ؊/؊ mice. In the absence of gamma interferon and nitric oxide synthase 2 (NOS2), AMs were significantly more permissive than wild-type cells. In contrast, AMs from Il4 ؊/؊ mice were more restrictive to C. burnetii replication, supporting the importance of M2 polarization for the permissiveness of AMs to C. burnetii replication. Collectively, our data account for understanding the high susceptibility of alveolar macrophages to bacterial replication and support the use of AMs as a relevant model of C. burnetii growth in primary macrophages.

Os sistemas agroflorestais (SAFs) além de geração de renda e conservação da biodiversidade pode ser uma alternativa para a estocagem de carbono. O trabalho teve como objetivo caracterizar três sistemas agroflorestais (SAFs) e estimar os estoques de carbono na fitomassa aérea, na serapilheira e no solo. Os SAFs e suas respectivas vegetações nativas adjacentes foram caracterizados quanto ao número, classificação botânica,distribuição diamétrica e estratificação vertical dos indivíduos arbóreose. Em cada um dos sistemas, foram estimados os estoques de carbono na fitomassa aérea, na serapilheira e no solo. Os SAFs,implantados a partir do manejo da vegetação nativa, apresentaram uma biodiversidade vegetal e estoque de carbono na fitomassa, na serapilheira e no solo semelhantes à área de vegetação nativa adjacente. Por outro lado, os estoques de carbono no SAF implantado em área anteriormente cultivada com culturas anuais apresentou menor diversidade de espécies arbóreas e estoques de carbono na fitomassa, serapilheira e no solo, em relação à vegetação nativa adjacente. Sistemas agroflorestais bem manejados se assemelham à vegetação nativa em estoque de carbono e conservação da biodiversidade dos recursos naturais.