The Mycobacterium marinum ESX-1 system mediates phagosomal permeabilization and type I interferon production via separable mechanisms.

Following mycobacterial entry into macrophages the ESX-1 type VII secretion system promotes phagosomal permeabilization and type I IFN production, key features of tuberculosis pathogenesis. The current model states that the secreted substrate ESAT-6 is required for membrane permeabilization and that a subsequent passive leakage of extracellular bacterial DNA into the host cell cytosol is sensed by the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway to induce type I IFN production. We employed a collection of Mycobacterium marinum ESX-1 transposon mutants in a macrophage infection model and show that permeabilization of the phagosomal membrane does not require ESAT-6 secretion. Moreover, loss of membrane integrity is insufficient to induce type I IFN production. Instead, type I IFN production requires intact ESX-1 function and correlates with release of mitochondrial and nuclear host DNA into the cytosol, indicating that ESX-1 affects host membrane integrity and DNA release via genetically separable mechanisms. These results suggest a revised model for major aspects of ESX-1-mediated host interactions and put focus on elucidating the mechanisms by which ESX-1 permeabilizes host membranes and induces the type I IFN response, questions of importance for our basic understanding of mycobacterial pathogenesis and innate immune sensing.

Streptococcal M protein promotes IL-10 production by cGAS-independent activation of the STING signaling pathway.

From an evolutionary point of view a pathogen might benefit from regulating the inflammatory response, both in order to facilitate establishment of colonization and to avoid life-threatening host manifestations, such as septic shock. In agreement with this notion Streptococcus pyogenes exploits type I IFN-signaling to limit detrimental inflammation in infected mice, but the host-pathogen interactions and mechanisms responsible for induction of the type I IFN response have remained unknown. Here we used a macrophage infection model and report that S. pyogenes induces anti-inflammatory IL-10 in an M protein-dependent manner, a function that was mapped to the B- and C-repeat regions of the M5 protein. Intriguingly, IL-10 was produced downstream of type I IFN-signaling, and production of type I IFN occurred via M protein-dependent activation of the STING signaling pathway. Activation of STING was independent of the cytosolic double stranded DNA sensor cGAS, and infection did not induce detectable release into the cytosol of either mitochondrial, nuclear or bacterial DNA-indicating DNA-independent activation of the STING pathway in S. pyogenes infected macrophages. These findings provide mechanistic insight concerning how S. pyogenes induces the type I IFN response and identify a previously unrecognized macrophage-modulating role for the streptococcal M protein that may contribute to curb the inflammatory response to infection.

ESX-1 exploits type I IFN-signalling to promote a regulatory macrophage phenotype refractory to IFNγ-mediated autophagy and growth restriction of intracellular mycobacteria.

The ability of macrophages to eradicate intracellular pathogens is normally greatly enhanced by IFNγ, a cytokine produced mainly after onset of adaptive immunity. However, adaptive immunity is unable to provide sterilizing immunity against mycobacteria, suggesting that mycobacteria have evolved virulence strategies to inhibit the bactericidal effect of IFNγ-signalling in macrophages. Still, the host-pathogen interactions and cellular mechanisms responsible for this feature have remained elusive. We demonstrate that the ESX-1 type VII secretion systems of Mycobacterium tuberculosis and Mycobacterium marinum exploit type I IFN-signalling to promote an IL-12(low) /IL-10(high) regulatory macrophage phenotype characterized by secretion of IL-10, IL-27 and IL-6. This mechanism had no impact on intracellular growth in the absence of IFNγ but suppressed IFNγ-mediated autophagy and growth restriction, indicating that the regulatory phenotype extends to function. The IFNγ-refractory phenotype was partly mediated by IL-27-signalling, establishing functional relevance for this downstream cytokine. These findings identify a novel macrophage-modulating function for the ESX-1 secretion system that may contribute to suppress the efficacy of adaptive immunity and provide mechanistic insight into the antagonistic cross talk between type I IFNs and IFNγ in mycobacterial infection.

Detection of Inflammasome Activation in Murine Bone Marrow-Derived Macrophages Infected with Group A Streptococcus.

Inflammasomes are large multiprotein complexes that assemble mainly in innate immune cells after detection of microbial or sterile insults. Activation of inflammasomes is a key proinflammatory event during infection, and many pathogens have evolved specific evasion mechanisms to evade or inhibit inflammasome activation. One such pathogen is the common bacterium group A Streptococcus (GAS), which causes a wide range of diseases of varying severity. GAS secretes a multitude of virulence factors whereof the pore-forming protein streptolysin O (SLO) is the main inflammasome activation determinant. Here we provide a protocol for reliable evaluation of inflammasome activation in murine bone marrow-derived macrophages (BMDM) infected with GAS, including instructions for generating BMDMs and growing the bacterium. This protocol can easily be modified to other bacterial pathogens, or human macrophages.

Interplay between human STING genotype and bacterial NADase activity regulates inter-individual disease variability.

Variability in disease severity caused by a microbial pathogen is impacted by each infection representing a unique combination of host and pathogen genomes. Here, we show that the outcome of invasive Streptococcus pyogenes infection is regulated by an interplay between human STING genotype and bacterial NADase activity. S. pyogenes-derived c-di-AMP diffuses via streptolysin O pores into macrophages where it activates STING and the ensuing type I IFN response. However, the enzymatic activity of the NADase variants expressed by invasive strains suppresses STING-mediated type I IFN production. Analysis of patients with necrotizing S. pyogenes soft tissue infection indicates that a STING genotype associated with reduced c-di-AMP-binding capacity combined with high bacterial NADase activity promotes a 'perfect storm' manifested in poor outcome, whereas proficient and uninhibited STING-mediated type I IFN production correlates with protection against host-detrimental inflammation. These results reveal an immune-regulating function for bacterial NADase and provide insight regarding the host-pathogen genotype interplay underlying invasive infection and interindividual disease variability.

The Secreted Virulence Factor NADase of Group A Streptococcus Inhibits P2X7 Receptor-Mediated Release of IL-1ß.

The common human pathogen Group A Streptococcus (GAS) causes superficial as well as invasive, life-threatening diseases. An increase in the occurrence of invasive GAS infection by strains of the M1 and M89 serotypes has been correlated with increased expression of the genetically and functionally linked virulence factors streptolysin O (SLO) and ß-NAD+-glycohydrolase (NADase). NADase affects host cells differently depending on its location: its SLO-dependent translocation into the cytosol can lead to cell death through ß-NAD+ depletion, while extracellularly located NADase inhibits IL-1ß release downstream of Nlrp3 inflammasome activation. In this study, we use a macrophage infection model to investigate the NADase-dependent inhibition of IL-1ß release. We show that bacteria expressing a functional NADase evade P2X7 activation, while infection with a NADase-deficient GAS strain leads to a P2X7-mediated increase in IL-1ß. Further, our data indicate that in the absence of NADase, IL-1ß is released through both P2X7-dependent and -independent pathways, although the precise mechanisms of how this occur are still unclear. This study adds information about the mechanism by which NADase regulates inflammasome-dependent IL-1ß release, which may in part explain why increased NADase expression correlates with bacterial virulence.

Plasma Profiles of Inflammatory Markers Associated With Active Tuberculosis in Antiretroviral Therapy-Naive Human Immunodeficiency Virus-Positive Individuals.

BACKGROUND: Diagnosis of tuberculosis (TB) in human immunodeficiency virus (HIV)-coinfected individuals is challenging. We hypothesized that combinations of inflammatory markers could facilitate identification of active TB in HIV-positive individuals. METHODS: Participants were HIV-positive, treatment-naive adults systematically investigated for TB at Ethiopian health centers. Plasma samples from 130 subjects with TB (HIV+/TB+) and 130 subjects without TB (HIV+/TB-) were tested for concentration of the following markers: CCL5, C-reactive protein (CRP), interleukin (IL)-6, IL12-p70, IL-18, IL-27, interferon-γ-induced protein-10 (IP-10), procalcitonin (PCT), and soluble urokinase-type plasminogen activator receptor (suPAR). Analyzed markers were then assessed, either individually or in combination, with regard to infection status, CD4 cell count, and HIV ribonucleic acid (RNA) levels. RESULTS: The HIV+/TB+ subjects had higher levels of all markers, except IL12p70, compared with HIV+/TB- subjects. The CRP showed the best performance for TB identification (median 27.9 vs 1.8 mg/L for HIV+/TB+ and HIV+/TB-, respectively; area under the curve [AUC]: 0.80). Performance was increased when CRP was combined with suPAR analysis (AUC, 0.83 [0.93 for subjects with CD4 cell count <200 cells/mm3]). Irrespective of TB status, IP-10 concentrations correlated with HIV RNA levels, and both IP-10 and IL-18 were inversely correlated to CD4 cell counts. CONCLUSIONS: Although CRP showed the best single marker discriminatory potential, combining CRP and suPAR analyses increased performance for TB identification.

Streptolysin O Induces the Ubiquitination and Degradation of Pro-IL-1ß.

Group A Streptococcus (GAS) is a common and versatile human pathogen causing a variety of diseases. One of the many virulence factors of GAS is the secreted pore-forming cytotoxin streptolysin O (SLO), which has been ascribed multiple properties, including inflammasome activation leading to release of the potent inflammatory cytokine IL-1ß from infected macrophages. IL-1ß is synthesized as an inactive pro-form, which is activated intracellularly through proteolytic cleavage. Here, we use a macrophage infection model to show that SLO specifically induces ubiquitination and degradation of pro-IL-1ß. Ubiquitination was dependent on SLO being released from the infecting bacterium, and pore formation by SLO was required but not sufficient for the induction of ubiquitination. Our data provide evidence for a novel SLO-mediated mechanism of immune regulation, emphasizing the importance of this pore-forming toxin in bacterial virulence and pathogenesis.

Inhibition of Inflammasome-Dependent Interleukin 1ß Production by Streptococcal NAD+-Glycohydrolase: Evidence for Extracellular Activity.

Group A Streptococcus (GAS) is a common human pathogen and the etiologic agent of a large number of diseases ranging from mild, self-limiting infections to invasive life-threatening conditions. Two prominent virulence factors of this bacterium are the genetically and functionally linked pore-forming toxin streptolysin O (SLO) and its cotoxin NAD+-glycohydrolase (NADase). Overexpression of these toxins has been linked to increased bacterial virulence and is correlated with invasive GAS disease. NADase can be translocated into host cells by a SLO-dependent mechanism, and cytosolic NADase has been assigned multiple properties such as protection of intracellularly located GAS bacteria and induction of host cell death through energy depletion. Here, we used a set of isogenic GAS mutants and a macrophage infection model and report that streptococcal NADase inhibits the innate immune response by decreasing inflammasome-dependent interleukin 1ß (IL-1ß) release from infected macrophages. Regulation of IL-1ß was independent of phagocytosis and ensued also under conditions not allowing SLO-dependent translocation of NADase into the host cell cytosol. Thus, our data indicate that NADase not only acts intracellularly but also has an immune regulatory function in the extracellular niche.IMPORTANCE In the mid-1980s, the incidence and severity of invasive infections caused by serotype M1 GAS suddenly increased. The results of genomic analyses suggested that this increase was due to the spread of clonal bacterial strains and identified a recombination event leading to enhanced production of the SLO and NADase toxins in these strains. However, despite its apparent importance in GAS pathogenesis, the function of NADase remains poorly understood. In this study, we demonstrate that NADase inhibits inflammasome-dependent IL-1ß release from infected macrophages. While previously described functions of NADase pertain to its role upon SLO-mediated translocation into the host cell cytosol, our data suggest that the immune regulatory function of NADase is exerted by nontranslocated enzyme, identifying a previously unrecognized extracellular niche for NADase functionality. This immune regulatory property of extracellular NADase adds another possible explanation to how increased secretion of NADase correlates with bacterial virulence.

Targeting oxidized LDL improves insulin sensitivity and immune cell function in obese Rhesus macaques.

Oxidation of LDL (oxLDL) is a crucial step in the development of cardiovascular disease. Treatment with antibodies directed against oxLDL can reduce atherosclerosis in rodent models through unknown mechanisms. We demonstrate that through a novel mechanism of immune complex formation and Fc-γ receptor (FcγR) engagement, antibodies targeting oxLDL (MLDL1278a) are anti-inflammatory on innate immune cells via modulation of Syk, p38 MAPK phosphorylation and NFκB activity. Subsequent administration of MLDL1278a in diet-induced obese (DIO) nonhuman primates (NHP) resulted in a significant decrease in pro-inflammatory cytokines and improved overall immune cell function. Importantly, MLDL1278a treatment improved insulin sensitivity independent of body weight change. This study demonstrates a novel mechanism by which an anti-oxLDL antibody improves immune function and insulin sensitivity independent of internalization of oxLDL. This identifies MLDL1278a as a potential therapy for reducing vascular inflammation in diabetic conditions.