Induction and functional role of type I interferon in bacterial infection of macrophages

Infection represents an evolutionary arms race between pathogen and host. Thus, scientists find that pathogenic microbes have evolved elegant strategies to manipulate or avoid our immune system, allowing them to persist within the human population. This thesis is largely focused on how bacteria regulate and exploit the type I interferon (IFN) response in infected macrophages – a processes of central importance to cellular immunity and bacterial pathogenesis. More specifically, we have been interested in understanding how Streptococcus pyogenes and mycobacteria – representing evolutionarily distant pathogens causing acute and chronic infections, respectively – interact with macrophages to induce and functionally exploit the type I IFN response. Our studies have identified a specific streptococcal component, the surface M protein, and the mechanisms underlying induction of the type I IFN response in infected macrophages. In the case of mycobacterial infection, we provide new insight into the genetic requirements and mechanisms for ESX-1-mediated type I IFN induction, and describe a macrophage-modulating role for induced type I IFN that can be exploited by the bacteria to avoid being cleared by the immune system

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

Summary: 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 Mycobacteriummarinum exploit type I IFN-signalling to promote an IL-12low/IL-10high 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

A novel bacterial resistance mechanism against human group IIA-secreted phospholipase A2: role of Streptococcus pyogenes sortase A.

International audienceHuman group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is a bactericidal molecule important for the innate immune defense against Gram-positive bacteria. In this study, we analyzed its role in the host defense against Streptococcus pyogenes, a major human pathogen, and demonstrated that this bacterium has evolved a previously unidentified mechanism to resist killing by sPLA(2)-IIA. Analysis of a set of clinical isolates demonstrated that an ~500-fold higher concentration of sPLA(2)-IIA was required to kill S. pyogenes compared with strains of the group B Streptococcus, which previously were shown to be sensitive to sPLA(2)-IIA, indicating that S. pyogenes exhibits a high degree of resistance to sPLA(2)-IIA. We found that an S. pyogenes mutant lacking sortase A, a transpeptidase responsible for anchoring LPXTG proteins to the cell wall in Gram-positive bacteria, was significantly more sensitive (~30-fold) to sPLA(2)-IIA compared with the parental strain, indicating that one or more LPXTG surface proteins protect S. pyogenes against sPLA(2)-IIA. Importantly, using transgenic mice expressing human sPLA(2)-IIA, we showed that the sortase A-mediated sPLA(2)-IIA resistance mechanism in S. pyogenes also occurs in vivo. Moreover, in this mouse model, we also showed that human sPLA(2)-IIA is important for the defense against lethal S. pyogenes infection. Thus, we demonstrated a novel mechanism by which a pathogenic bacterium can evade the bactericidal action of sPLA(2)-IIA and we showed that sPLA(2)-IIA contributes to the host defense against S. pyogenes infection

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

Selective induction of IL-10 is not explained by M5 protein-dependent regulation of MyD88 activation.

<p>Wild type (B6) and MyD88-KO macrophages were infected as indicated. Culture supernatants were collected 24 hpi and assayed for indicated cytokines. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of three independent experiments. ANOVA (*<0.033; **<0.002; ***<0.001).</p

<i>S</i>. <i>pyogenes</i> induces the STING signaling pathway via an active process that is independent of cGAS.

<p><b>A)</b> B6 macrophages were infected with live or heat-killed M5 bacteria, as indicated. Cytokines were assayed 24 hpi. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of three independent experiments. <b>B)</b> B6, cGAS-KO and STING-KO macrophages were infected with M5 for 4 hours, and IFNβ transcripts were measured by RTqPCR. mRNA levels are presented as fold-change relative to UI B6 macrophages. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of three independent experiments. <b>C)</b> B6, cGAS-KO and STING-KO macrophages were infected as indicated, and cytokine output was assayed 24 hpi. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of three independent experiments. <b>D)</b> B6 macrophages were infected with wild type M5 bacteria in the presence (SVPD) or absence (Ctrl.) of snake venom phosphodiesterase (1.3 U/ml final concentration), as indicated. Similarly treated UI macrophages were analyzed as control. Cytokines were assayed 24 hpi. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of two independent experiments. ANOVA (*<0.033; **<0.002; ***<0.001).</p

<i>S</i>. <i>pyogenes</i> promotes IL-10 secretion from infected macrophages in an M5 protein-dependent manner.

<p><b>A)</b> Wild type C57BL/6 (B6) bone marrow-derived macrophages were infected with wild type M5, ΔM5 or ΔM5/pM5 <i>S</i>. <i>pyogenes</i> or uninfected (UI), as indicated. Culture supernatants were collected 24 hours post infection (hpi) and assayed for indicated cytokines. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of three independent experiments. <b>B)</b> Indicated bacterial strains were analyzed for reactivity with a rabbit antiserum against the N-terminal region of the M5 protein by flow cytometry. As control we analyzed binding to M5 bacteria of a preimmune serum from the same rabbit. Left panel: Shown is a representative histogram. Right panel: Geometrical mean (mean and SD) of three independent experiments. <b>C)</b> Human monocyte-derived macrophages were infected as indicated. Culture supernatants were collected 24 hpi and assayed for indicated cytokines. Results shown (mean and SD; <i>n</i> = 3 per group) are representative of two independent experiments. <b>D)</b> Kinetic analysis of cytokine secretion from B6 macrophages infected as indicated. Results shown (mean; <i>n</i> = 2 per group) are representative of two independent experiments. ANOVA (*<0.033; **<0.002; ***<0.001).</p