Laurel L. Lenz

MPYS Is Required for IFN Response Factor 3 Activation and Type I IFN Production in the Response of Cultured Phagocytes to Bacterial Second Messengers Cyclic-di-AMP and Cyclic-di-GMP

Cyclic-di-GMP and cyclic-di-AMP are second messengers produced by bacteria and influence bacterial cell survival, differentiation, colonization, biofilm formation, virulence, and bacteria–host interactions. In this study, we show that in both RAW264.7 macrophage cells and primary bone marrow-derived macrophages, the production of IFN-β and IL-6, but not TNF, in response to cyclic-di-AMP and cyclic-di-GMP requires MPYS (also known as STING, MITA, and TMEM173). Furthermore, expression of MPYS was required for IFN response factor 3 but not NF-κB activation in response to these bacterial metabolites. We also confirm that MPYS is required for type I IFN production by cultured macrophages infected with the intracellular pathogens Listeria monocytogenes and Francisella tularensis. However, during systemic infection with either pathogen, MPYS deficiency did not impact bacterial burdens in infected spleens. Serum IFN-β and IL-6 concentrations in the infected control and MPYS−/− mice were also similar at 24 h postinfection, suggesting that these pathogens stimulate MPYS-independent cytokine production during in vivo infection. Our findings indicate that bifurcating MPYS-dependent and -independent pathways mediate sensing of cytosolic bacterial infections.

Induction of IFN-αβ enables Listeria monocytogenes to suppress macrophage activation by IFN-γ

Production of type I interferon (IFN; IFN-αβ) increases host susceptibility to Listeria monocytogenes, whereas type II IFN (IFN-γ) activates macrophages to resist infection. We show that these opposing immunological effects of IFN-αβ and IFN-γ occur because of cross talk between the respective signaling pathways. We found that cultured macrophages infected with L. monocytogenes were refractory to IFN-γ treatment as a result of down-regulation of the IFN-γ receptor (IFNGR). The soluble factor responsible for these effects was identified as host IFN-αβ. Accordingly, macrophages and dendritic cells (DCs) showed reduced IFNGR1 expression and reduced responsiveness to IFN-γ during systemic infection of IFN-αβ–responsive mice. Furthermore, the increased resistance of mice lacking the IFN-αβ receptor (IFNAR−/−) to L. monocytogenes correlated with increased expression of IFN-γ–dependent activation markers by macrophages and DCs and was reversed by depletion of IFN-γ. Thus, IFN-αβ produced in response to bacterial infection and other stimuli antagonizes the host response to IFN-γ by down-regulating the IFNGR. Such cross talk permits prioritization of IFN-αβ–type immune responses and may contribute to the beneficial effects of IFN-β in treatment of inflammatory diseases such as multiple sclerosis.

Transient Receptor Potential Melastatin 2 (TRPM2) ion channel is required for innate immunity against Listeria monocytogenes

The generation of reactive oxygen species (ROS) is inherent to immune responses. ROS are crucially involved in host defense against pathogens by promoting bacterial killing, but also as signaling agents coordinating the production of cytokines. Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca2+-permeable channel gated via binding of ADP-ribose, a metabolite formed under conditions of cellular exposure to ROS. Here, we show that TRPM2-deficient mice are extremely susceptible to infection with Listeria monocytogenes (Lm), exhibiting an inefficient innate immune response. In a comparison with IFNγR-deficient mice, TRPM2−/− mice shared similar features of uncontrolled bacterial replication and reduced levels of inducible (i)NOS-expressing monocytes, but had intact IFNγ responsiveness. In contrast, we found that levels of cytokines IL-12 and IFNγ were diminished in TRPM2−/− mice following Lm infection, which correlated with their reduced innate activation. Moreover, TRPM2−/− mice displayed a higher degree of susceptibility than IL-12–unresponsive mice, and supplementation with recombinant IFNγ was sufficient to reverse the unrestrained bacterial growth and ultimately the lethal phenotype of Lm-infected TRPM2−/− mice. The severity of listeriosis we observed in TRPM2−/− mice has not been reported for any other ion channel. These findings establish an unsuspected role for ADP-ribose and ROS-mediated cation flux for innate immunity, opening up unique possibilities for immunomodulatory intervention through TRPM2.

Antagonistic crosstalk between type I and II interferons and increased host susceptibility to bacterial infections.

Type I and II interferons (IFNs αβ and γ) have opposing effects on immune resistance to certain pathogenic bacteria. While IFNγ generally plays a protective role, IFNαβ exacerbates Listeria monocytogenes and Mycobacterium tuberculosis infections. Our findings provided evidence that this increased susceptibility reflects a novel antagonistic cross talk between IFNαβ and IFNγ. Macrophages infected with L. monocytogenes strains that induce IFNαβ production responded poorly to IFNγ, as measured by reduced phosphorylation of STAT1 and reduced IFNγ-dependent gene expression. The impaired responsiveness to IFNγ correlated with reduced expression of its receptor, IFNGR, by both infected and bystander macrophages. Down regulation of IFNGR was dependent on responsiveness to IFNγ and mimicked by recombinant IFNβ. Mice lacking responsiveness to IFNαβ (IFNAR1-/-) retained high IFNGR expression, developed higher expression of MHC-II on macrophages and DCs, and were more resistant to systemic L. monocytogenes infection - but only in the presence of IFNγ. Thus, the ability of IFNαβ to down regulate IFNGR provides an explanation for its ability to reduce responsiveness to IFNγ and to increase host susceptibility to bacterial infection. It remains to be determined whether and how such antagonistic interferon crosstalk benefits the host.

A Breakthrough: Macrophage-Directed Cancer Immunotherapy.

Successful immunotherapy of cancer is becoming a reality aided by the realization that macrophages play an important role in the growth or regression of tumors. Specifically, M2/repair-type macrophages predominate in human cancers and produce growth-promoting molecules that actively stimulate tumor growth in much the same way they help wounds heal. However, modulating M2/repair-type macrophages to M1/kill-type can slow or stop cancer growth. The effects involve direct activity of M1 kill-type as well as the ability of M1-type macrophages to stimulate Th1-type cytotoxic T cells and other effector cells. Macrophage responses can also predict cancer susceptibility; individuals with a high M1/kill to M2/repair ratio are less prone. That macrophages/innate immunity can be modulated to play a central role in directly or indirectly combating cancer is a breakthrough that seems likely to finally make successful immunotherapy of cancer a reality.

Bacterial Peptidoglycan-Degrading Enzymes and Their Impact on Host Muropeptide Detection

Peptidoglycan (PGN) is a major component of the bacterial cell envelope in both Gram-positive and Gram-negative bacteria. These muropeptides can be produced or modified by the activity of bacterial glycolytic and peptidolytic enzymes referred to as PGN hydrolases and autolysins. Some of these bacterial enzymes are crucial for bacterial pathogenicity and have been shown to modulate muropeptide release and/or host innate immune responses. The ability of muropeptides to modulate host responses is due to the fact that eukaryotes do not produce PGN and have instead evolved numerous strategies to detect intact PGN and PGN fragments (muropeptides). Here we review the structure of PGN and introduce the various bacterial enzymes known to degrade or modify bacterial PGN. Host factors involved in PGN and muropeptide detection are also briefly discussed, as are examples of how specific bacterial pathogens use PGN degradation and modification to subvert host innate immunity.

Distinct Licensing of IL-18 and IL-1β Secretion in Response to NLRP3 Inflammasome Activation

Inflammasome activation permits processing of interleukins (IL)-1β and 18 and elicits cell death (pyroptosis). Whether these responses are independently licensed or are “hard-wired” consequences of caspase-1 (casp1) activity has not been clear. Here, we show that that each of these responses is independently regulated following activation of NLRP3 inflammasomes by a “non-canonical” stimulus, the secreted Listeria monocytogenes (Lm) p60 protein. Primed murine dendritic cells (DCs) responded to p60 stimulation with reactive oxygen species (ROS) production and secretion of IL-1β and IL-18 but not pyroptosis. Inhibitors of ROS production inhibited secretion of IL-1β, but did not impair IL-18 secretion. Furthermore, DCs from caspase-11 (casp11)-deficient 129S6 mice failed to secrete IL-1β in response to p60 but were fully responsive for IL-18 secretion. These findings reveal that there are distinct licensing requirements for processing of IL-18 versus IL-1β by NLRP3 inflammasomes.

IL-15 AND TYPE I INTERFERON ARE REQUIRED FOR ACTIVATION OF TUMORICIDAL NK CELLS BY VIRUS-INFECTED DENDRITIC CELLS

There is increasing evidence that natural killer (NK) cells play an important role in antitumor immunity following dendritic cell (DC) vaccination. Little is known, however, about the optimal stimulation of DCs that favors NK activation in tumor-bearing hosts. In this study, we demonstrate that treatment with toll-like receptor (TLR) ligands and infection with a mutant vesicular stomatitis virus (VSV-ΔM51) both induced DC maturation. Further, inoculation of these DCs led to robust NK-mediated protection against tumor challenge. Strikingly, only VSV-ΔM51-infected DCs were capable of suppressing the growth of established tumors, suggesting that additional signals provided by viral infection may be required to activate tumoricidal NK cells in tumor-bearing hosts. VSV-ΔM51 infection of DCs induced greater type I interferon (IFN I) production than TLR ligand treatment, and disruption of the IFN I pathway in DCs eliminated their ability to induce NK activation and tumor protection. However, further studies indicated that IFN I alone was not sufficient to activate NK cells, especially in the presence of a tumor, and DC-derived IL-15 was additionally required for tumoricidal NK activation. These results suggest that induction of IFN I by VSV-ΔM51 allows DCs to overcome tumor-associated immunosuppression and facilitate IL-15-mediated priming of tumoricidal NK cells. Thus, the mode of DC maturation should be carefully considered when designing DC-based cancer immunotherapies.

Activation of Naive NK Cells in Response to Listeria monocytogenes Requires IL-18 and Contact with Infected Dendritic Cells

The mechanisms for NK cell activation during infection by intracellular bacterial pathogens are not clearly defined. To dissect how Listeria monocytogenes infection elicits NK cell activation, we evaluated the requirements for activation of naive splenic NK cells by infected bone marrow-derived dendritic cells (BMDCs). We found that NK cell activation in this setting required infection of BMDCs by live wild type bacteria. NK cells were not activated when BMDCs were infected with a live hemolysin deficient (Δhly) strain. Neutralization of IL-12, TNF-α, or caspase-1 each dramatically reduced NK cell IFN-γ production in response to live wt L. monocytogenes infection. Addition of recombinant IL-18, but not IL-1β, reversed the effects of caspase-1 inhibition. Recombinant IL-18 also restored NK cell activation by BMDCs infected with Δhly L. monocytogenes, which produced IL-12 but not IL-18. IL-18 acted on NK cells because MyD88 expression was required in responding NK cells, but not infected BMDC. However, secreted cytokines were not sufficient for activation of naive NK cells by infected BMDCs. Rather, NK cell activation additionally required contact between infected BMDCs and NK cells. These data suggest that the activation of NK cells during L. monocytogenes infection requires both secreted cytokines and ligation of NK activating receptors during direct contact with infected DCs.

Expression of the p60 Autolysin Enhances NK Cell Activation and Is Required for Listeria monocytogenes Expansion in IFN-γ-Responsive Mice

Both peptidoglycan and muropeptides potently modulate inflammatory and innate immune responses. The secreted Listeria monocytogenes p60 autolysin digests peptidoglycan and promotes bacterial infection in vivo. Here, we report that p60 contributes to bacterial subversion of NK cell activation and innate IFN-γ production. L. monocytogenes deficient for p60 (Δp60) competed well for expansion in mice doubly deficient for IFNAR1 and IFN-γR1 or singly deficient for IFN-γR1, but not in wild-type, IFNAR1−/−, or TLR2−/− mice. The restored competitiveness of p60-deficient bacteria suggested a specific role for p60 in bacterial subversion of IFN-γ-mediated immune responses, since in vivo expansion of three other mutant L. monocytogenes strains (ΔActA, ΔNamA, and ΔPlcB) was not complemented in IFN-γR1−/− mice. Bacterial expression of p60 was not required to induce socs1, socs3, and il10 expression in infected mouse bone marrow macrophages but did correlate with enhanced production of IL-6, IL-12p70, and most strikingly IFN-γ. The primary source of p60-dependent innate IFN-γ was NK cells, whereas bacterial p60 expression did not significantly alter innate IFN-γ production by T cells. The mechanism for p60-dependent NK cell stimulation was also indirect, given that treatment with purified p60 protein failed to directly activate NK cells for IFN-γ production. These data suggest that p60 may act on infected cells to indirectly enhance NK cell activation and increase innate IFN-γ production, which presumably promotes early bacterial expansion through its immunoregulatory effects on bystander cells. Thus, the simultaneous induction of IFN-γ production and factors that inhibit IFN-γ signaling may be a common strategy for misdirection of early antibacterial immunity.