Synaptopodin is necessary for Shigella flexneri intercellular spread.

For many intracellular pathogens, their virulence depends on an ability to spread between cells of an epithelial layer. For intercellular spread to occur, these pathogens deform the plasma membrane into a protrusion structure that is engulfed by the neighboring cell. Although the polymerization of actin is essential for spread, how these pathogens manipulate the actin cytoskeleton in a manner that enables protrusion formation is still incompletely understood. Here, we identify the mammalian actin binding protein synaptopodin as required for efficient intercellular spread. Using a model cytosolic pathogen, Shigella flexneri , we show that synaptopodin contributes to organization of actin around bacteria and increases the length of the actin tail at the posterior pole of the bacteria. We show that synaptopodin presence enables protrusions to form and to resolve at a greater rate, indicating that greater stability of the actin tail enables the bacteria to push against the membrane with greater force. We demonstrate that synaptopodin recruitment around bacteria requires the bacterial protein IcsA, and we show that this recruitment is further enhanced in a type 3 secretion system dependent manner. These data establish synaptopodin as required for intracellular bacteria to reprogram the actin cytoskeleton in a manner that enables efficient protrusion formation and enhance our understanding of the cellular function of synaptopodin. Authors Summary: Intercellular spread is essential for many cytosolic dwelling pathogens during their infectious life cycle. Despite knowing the steps required for intercellular spread, relatively little is known about the host-pathogen interactions that enable these steps to occur. Here, we identify a requirement for the actin binding protein synaptopodin during intercellular spread by cytosolic bacteria. We show synaptopodin is necessary for the stability and recruitment of polymerized actin around bacteria. We also demonstrate synaptopodin is necessary to form plasma membrane structures known as protrusions that are necessary for the movement of these bacteria between cells. Thus, these findings implicate synaptopodin as an important actin-binding protein for the virulence of intracellular pathogens that require the actin cytoskeleton for their spread between cells.

Dichotomous effects of cellular expression of STAT3 on tumor growth of HNSCC.

STAT3 signaling has been shown to regulate cellular function and cytokine production in the tumor microenvironment (TME). Within the head and neck squamous cell carcinoma (HNSCC) TME, we previously showed that therapeutic targeting of STAT3 in combination with radiation resulted in improved tumor growth delay. However, given the independent regulatory effects STAT3 has on anti-tumor immunity, we aimed to decipher the effects of individually targeting STAT3 in the cancer cell, regulatory T cells (Tregs), and natural killer (NK) cell compartments in driving tumor growth and resistance to therapy in HNSCCs. We utilized a CRISPR knockout system for genetic deletion of STAT3 within the cancer cell as well as two genetic knockout mouse models, FoxP3-Cre/STAT3 fl and NKp46-Cre/STAT3 fl, for Tregs and NK cell targeting, respectively. Our data revealed differences in development of resistance to treatment with STAT3 CRISPR knockout in the cancer cell, driven by differential recruitment of immune cells. Knockout of STAT3 in Tregs overcomes this resistance and results in Treg reprogramming and recruitment and activation of antigen-presenting cells. In contrast, knockout of STAT3 in the NK cell compartment results in NK cell inactivation and acceleration of tumor growth. These data underscore the complex interplay between the cancer cell and the immune TME and carry significant implications for drug targeting and design of combination approaches in HNSCCs.

Targeting Treg-Expressed STAT3 Enhances NK-Mediated Surveillance of Metastasis and Improves Therapeutic Response in Pancreatic Adenocarcinoma.

PURPOSE: Metastasis remains a major hurdle in treating aggressive malignancies such as pancreatic ductal adenocarcinoma (PDAC). Improving response to treatment, therefore, requires a more detailed characterization of the cellular populations involved in controlling metastatic burden. EXPERIMENTAL DESIGN: PDAC patient tissue samples were subjected to RNA sequencing analysis to identify changes in immune infiltration following radiotherapy. Genetically engineered mouse strains in combination with orthotopic tumor models of PDAC were used to characterize disease progression. Flow cytometry was used to analyze tumor infiltrating, circulating, and nodal immune populations. RESULTS: We demonstrate that although radiotherapy increases the infiltration and activation of dendritic cells (DC), it also increases the infiltration of regulatory T cells (Treg) while failing to recruit natural killer (NK) and CD8 T cells in PDAC patient tissue samples. In murine orthotopic tumor models, we show that genetic and pharmacologic depletion of Tregs and NK cells enhances and attenuates response to radiotherapy, respectively. We further demonstrate that targeted inhibition of STAT3 on Tregs results in improved control of local and distant disease progression and enhanced NK-mediated immunosurveillance of metastasis. Moreover, combination treatment of STAT3 antisense oligonucleotide (ASO) and radiotherapy invigorated systemic immune activation and conferred a survival advantage in orthotopic and metastatic tumor models. Finally, we show the response to STAT3 ASO + radiotherapy treatment is dependent on NK and DC subsets. CONCLUSIONS: Our results suggest targeting Treg-mediated immunosuppression is a critical step in mediating a response to treatment, and identifying NK cells as not only a prognostic marker of improved survival, but also as an effector population that functions to combat metastasis.

FLT3L Release by Natural Killer Cells Enhances Response to Radioimmunotherapy in Preclinical Models of HNSCC.

PURPOSE: Natural killer (NK) cells are type I innate lymphoid cells that are known for their role in killing virally infected cells or cancer cells through direct cytotoxicity. In addition to direct tumor cell killing, NK cells are known to play fundamental roles in the tumor microenvironment through secretion of key cytokines, such as FMS-like tyrosine kinase 3 ligand (FLT3L). Although radiotherapy is the mainstay treatment in most cancers, the role of radiotherapy on NK cells is not well characterized. EXPERIMENTAL DESIGN: This study combines radiation, immunotherapies, genetic mouse models, and antibody depletion experiments to identify the role of NK cells in overcoming resistance to radiotherapy in orthotopic models of head and neck squamous cell carcinoma. RESULTS: We have found that NK cells are a crucial component in the development of an antitumor response, as depleting them removes efficacy of the previously successful combination treatment of radiotherapy, anti-CD25, and anti-CD137. However, in the absence of NK cells, the effect can be rescued through treatment with FLT3L. But neither radiotherapy with FLT3L therapy alone nor radiotherapy with anti-NKG2A yields any meaningful tumor growth delay. We also identify a role for IL2 in activating NK cells to secrete FLT3L. This activity, we show, is mediated through CD122, the intermediate affinity IL2 receptor, and can be targeted with anti-CD25 therapy. CONCLUSIONS: These findings highlight the complexity of using radio-immunotherapies to activate NK cells within the tumor microenvironment, and the importance of NK cells in activating dendritic cells for increased tumor surveillance.

Pulmonary insults exacerbate susceptibility to oral Listeria monocytogenes infection through the production of IL-10 by NK cells.

Most individuals who consume foods contaminated with the bacterial pathogen Listeria monocytogenes (Lm) develop mild symptoms, while others are susceptible to life-threatening systemic infections (listeriosis). Although it is known that the risk of severe disease is increased in certain human populations, including the elderly, it remains unclear why others who consume contaminated food develop listeriosis. Here, we used a murine model to discover that pulmonary coinfections can impair the host's ability to adequately control and eradicate systemic Lm that cross from the intestines to the bloodstream. We found that the resistance of mice to oral Lm infection was dramatically reduced by coinfection with Streptococcus pneumoniae (Spn), a bacterium that colonizes the respiratory tract and can also cause severe infections in the elderly. Exposure to Spn or microbial products, including a recombinant Lm protein (L1S) and lipopolysaccharide (LPS), rendered otherwise resistant hosts susceptible to severe systemic Lm infection. In addition, we show that this increase in susceptibility was dependent on an increase in the production of interleukin-10 (IL-10) from Ncr1+ cells, including natural killer (NK) cells. Lastly, the ability of Ncr1+ cell derived IL-10 to increase disease susceptibility correlated with a dampening of both myeloid cell accumulation and myeloid cell phagocytic capacity in infected tissues. These data suggest that efforts to minimize inflammation in response to an insult at the respiratory mucosa render the host more susceptible to infections by Lm and possibly other pathogens that access the oral mucosa.

Myeloid Cell CK2 Regulates Inflammation and Resistance to Bacterial Infection.

Kinase activity plays an essential role in the regulation of immune cell defenses against pathogens. The protein kinase CK2 (formerly casein kinase II) is an evolutionarily conserved kinase with hundreds of identified substrates. CK2 is ubiquitously expressed in somatic and immune cells, but the roles of CK2 in regulation of immune cell function remain largely elusive. This reflects the essential role of CK2 in organismal development and limited prior work with conditional CK2 mutant murine models. Here, we generated mice with a conditional (floxed) allele of Csnk2a, which encodes the catalytic CK2α subunit of CK2. When crossed to Lyz2-cre mice, excision of Csnk2a sequence impaired CK2α expression in myeloid cells but failed to detectably alter myeloid cell development. By contrast, deficiency for CK2α increased inflammatory myeloid cell recruitment, activation, and resistance following systemic Listeria monocytogenes (Lm) infection. Results from mixed chimera experiments indicated that CK2α deficiency in only a subset of myeloid cells was not sufficient to reduce bacterial burdens. Nor did cell-intrinsic deficiency for CK2α suffice to alter accumulation or activation of monocytes and neutrophils in infected tissues. These data suggest that CK2α expression by Lyz2-expressing cells promotes inflammatory and anti-bacterial responses through effects in trans. Our results highlight previously undescribed suppressive effects of CK2 activity on inflammatory myeloid cell responses and illustrate that cell-extrinsic effects of CK2 can shape inflammatory and protective innate immune responses.

IL-10-producing NK cells exacerbate sublethal Streptococcus pneumoniae infection in the lung.

Lung inflammation is tightly controlled to balance microbial clearance with the tissue damage that accompanies this response. Bacterial pathogens including Streptococcus pneumoniae (S. pneumoniae) modulate immune regulation by promoting secretion of the anti-inflammatory cytokine IL-10. The important cellular sources of IL-10 that impact protection against different bacterial infections are not well characterized. We find that S. pneumoniaeactivates IL-10 secretion from natural killer (NK) cells in the lung, which restrict host protection in a mouse model of sublethal infection. Direct transfer of wild-type NK cells into the lungs of IL-10-deficient mice drives bacterial expansion, identifying NK cells as a critical source of IL-10 promoting S. pneumoniae infection. The S. pneumoniae virulence protein Spr1875 was found to elicit NK cell IL-10 production in purified cells and in the lungs of live animals. These findings reveal therapeutic targets to combat bacterial-driven immune regulation in the lung.

Inflammation as a Modulator of Host Susceptibility to Pulmonary Influenza, Pneumococcal, and Co-Infections.

Bacterial and viral pathogens are predominant causes of pulmonary infections and complications. Morbidity and mortality from these infections is increased in populations that include the elderly, infants, and individuals with genetic disorders such as Down syndrome. Immune senescence, concurrent infections, and other immune alterations occur in these susceptible populations, but the underlying mechanisms that dictate increased susceptibility to lung infections are not fully defined. Here, we review unique features of the lung as a mucosal epithelial tissue and aspects of inflammatory and immune responses in model pulmonary infections and co-infections by influenza virus and Streptococcus pneumoniae. In these models, lung inflammatory responses are a double-edged sword: recruitment of immune effectors is essential to eliminate bacteria and virus-infected cells, but inflammatory cytokines drive changes in the lung conducive to increased pathogen replication. Excessive accumulation of inflammatory cells also hinders lung function, possibly causing death of the host. Some animal studies have found that targeting host modulators of lung inflammatory responses has therapeutic or prophylactic effects in these infection and co-infection models. However, conflicting results from other studies suggest microbiota, sequence of colonization, or other unappreciated aspects of lung biology also play important roles in the outcome of infections. Regardless, a predisposition to excessive or aberrant inflammatory responses occurs in susceptible human populations. Hence, in appropriate contexts, modulation of inflammatory responses may prove effective for reducing the frequency or severity of pulmonary infections. However, there remain limitations in our understanding of how this might best be achieved-particularly in diverse human populations.

Selective Loss of Responsiveness to Exogenous but Not Endogenous Cyclic-Dinucleotides in Mice Expressing STING-R231H.

Stimulator of interferon genes (STING) plays a central role in innate immune responses to viral and intracellular bacterial infections, and cellular damage. STING is a cytosolic sensor of cyclic dinucleotides (CDNs) including those produced by pathogenic bacteria and those arising endogenously as products of the DNA sensor cGAS (e.g., 2'3' cGAMP). The two most common alternative allelic variants of STING in humans are STING-R71H-G230A-R293Q (STING-HAQ) and STING-R232H that are found in 20.4% and 13.7-17.6% of the population, respectively. To determine the biologic consequences of these genotypic variations, we generated knock-in mice containing the murine equivalents of each variant and studied their responsiveness to CDNs. Homozygous STING-HAQ (R71H-I229A-R292Q) and STING-R231H mice were found to be unresponsive to all exogenous CDNs tested (ci-di-GMP, ci-di-AMP, 3'3' cGAMP and Rp,Rp-CDA). Responses of homozygous STING-HAQ mice to endogenous 2'3' cGAMP was also greatly impaired. However, homozygous STING-R231H mice are fully responsive to 2'3' cGAMP. Analysis of heterozygous mice revealed reduced responsiveness to exogenous and endogenous CDNs in mice carrying a single copy of STING-HAQ, while STING-R231H heterozygous mice exhibit reduced responsiveness to exogenous but not endogenous CDNs. These findings confirm and extend previous reports by demonstrating differing impact of allelic variation of STING on the ability to sense and respond to exogenous vs. endogenous CDNs. Finally, the STING-R231H variant mouse represents a useful tool with which to examine the relative contributions of STING sensing of exogenous and endogenous CDNs in the context of bacterial infections and CDN-based cancer immunotherapeutics.

Myeloid cell responsiveness to interferon-gamma is sufficient for initial resistance to Listeria monocytogenes.

The type II interferon (IFNγ) promotes resistance to intracellular pathogens. Most immune and somatic cells also express the IFNγ receptor (IFNGR) and respond to IFNγ. While myeloid cell have been implicated as important targets of IFNγ, it remains unknown if IFNγ signaling to myeloid cell types suffices for resistance to infection. Here, we addressed this question by generating mice in which IFNGR1 is selectively expressed by myeloid cells. These "MSGR1" (myeloid selective IFNGR1) mice express an epitope-tagged Ifngr1 transgene (fGR1) from the myeloid-specific c-fms promoter in a background lacking endogenous Ifngr1. IFNGR staining was selectively observed on myeloid cells in the MSGR1 mice and correlated with responsiveness of these cells to IFNγ. During systemic infection by the bacterium Listeria monocytogenes, activation marker staining was comparable on monocytes from MSGR1 and control B6 mice. Bacterial burdens and survival were also equivalent in MSGR1 and wildtype B6 animals at a timepoint when B6.Ifngr1 -/- mice began to succumb. These data confirm that activation of inflammatory monocytes and neutrophils is a key mechanism by which IFNγ promotes innate anti-bacterial immunity and suggest that IFNγ targeting of myeloid cells is largely sufficient to mediate protection against systemic L. monocytogenes.

IFNγ receptor down-regulation facilitates Legionella survival in alveolar macrophages.

Legionella pneumophila is an opportunistic human pathogen and causative agent of the acute pneumonia known as Legionnaire's disease. Upon inhalation, the bacteria replicate in alveolar macrophages (AM), within an intracellular vacuole termed the Legionella-containing vacuole. We recently found that, in vivo, IFNγ was required for optimal clearance of intracellular L. pneumophila by monocyte-derived cells (MC), but the cytokine did not appear to influence clearance by AM. Here, we report that during L. pneumophila lung infection, expression of the IFNγ receptor subunit 1 (IFNGR1) is down-regulated in AM and neutrophils, but not MC, offering a possible explanation for why AM are unable to effectively restrict L. pneumophila replication in vivo. To test this, we used mice that constitutively express IFNGR1 in AM and found that prevention of IFNGR1 down-regulation enhanced the ability of AM to restrict L. pneumophila intracellular replication. IFNGR1 down-regulation was independent of the type IV Dot/Icm secretion system of L. pneumophila indicating that bacterial effector proteins were not involved. In contrast to previous work, we found that signaling via type I IFN receptors was not required for IFNGR1 down-regulation in macrophages but rather that MyD88- or Trif- mediated NF-κB activation was required. This work has uncovered an alternative signaling pathway responsible for IFNGR1 down-regulation in macrophages during bacterial infection.

Ligand-induced IFNGR1 down-regulation calibrates myeloid cell IFNγ responsiveness.

The type II IFN (IFNγ) enhances antimicrobial activity yet also drives expression of genes that amplify inflammatory responses. Hence, excessive IFNγ stimulation can be pathogenic. Here, we describe a previously unappreciated mechanism whereby IFNγ itself dampens myeloid cell activation. Staining of monocytes from Listeria monocytogenes-infected mice provided evidence of type I IFN-independent reductions in IFNGR1. IFNγ was subsequently found to reduce surface IFNGR1 on cultured murine myeloid cells and human CD14+ peripheral blood mononuclear cells. IFNγ-driven reductions in IFNGR1 were not explained by ligand-induced receptor internalization. Rather, IFNγ reduced macrophage Ifngr1 transcription by altering chromatin structure at putative Ifngr1 enhancer sites. This is a distinct mechanism from that used by type I IFNs. Ligand-induced reductions in IFNGR1 altered myeloid cell sensitivity to IFNγ, blunting activation of STAT1 and 3. Our data, thus, reveal a mechanism by which IFNGR1 abundance and myeloid cell sensitivity to IFNγ can be modulated in the absence of type I IFNs. Multiple mechanisms, thus, exist to calibrate macrophage IFNGR1 abundance, likely permitting the fine tuning of macrophage activation and inflammation.

NK Cell IL-10 Production Requires IL-15 and IL-10 Driven STAT3 Activation.

Natural killer (NK) cells can produce IFNγ or IL-10 to regulate inflammation and immune responses but the factors driving NK cell IL-10 secretion are poorly-defined. Here, we identified NK cell-intrinsic STAT3 activation as vital for IL-10 production during both systemic Listeria monocytogenes (Lm) infection and following IL-15 cytokine/receptor complex (IL15C) treatment for experimental cerebral malaria (ECM). In both contexts, conditional Stat3 deficiency in NK cells abrogated production of IL-10. Initial NK cell STAT3 phosphorylation was driven by IL-15. During Lm infection, this required capture or presentation of IL-15 by NK cell IL-15Rα. Persistent STAT3 activation was required to drive measurable IL-10 secretion and required NK cell expression of IL-10Rα. Survival-promoting effects of IL-15C treatment in ECM were dependent on NK cell Stat3 while NK cell-intrinsic deficiency for Stat3, Il15ra, or Il10ra abrogated NK cell IL-10 production and increased resistance against Lm. NK cell Stat3 deficiency did not impact production of IFNγ, indicating the STAT3 activation initiated by IL-15 and amplified by IL-10 selectively drives the production of anti-inflammatory IL-10 by responding NK cells.

Author Correction: The gut microbiota in infants of obese mothers increases inflammation and susceptibility to NAFLD.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The gut microbiota in infants of obese mothers increases inflammation and susceptibility to NAFLD.

Maternal obesity is associated with increased risk for offspring obesity and non-alcoholic fatty liver disease (NAFLD), but the causal drivers of this association are unclear. Early colonization of the infant gut by microbes plays a critical role in establishing immunity and metabolic function. Here, we compare germ-free mice colonized with stool microbes (MB) from 2-week-old infants born to obese (Inf-ObMB) or normal-weight (Inf-NWMB) mothers. Inf-ObMB-colonized mice demonstrate increased hepatic gene expression for endoplasmic reticulum stress and innate immunity together with histological signs of periportal inflammation, a histological pattern more commonly reported in pediatric cases of NAFLD. Inf-ObMB mice show increased intestinal permeability, reduced macrophage phagocytosis, and dampened cytokine production suggestive of impaired macrophage function. Furthermore, exposure to a Western-style diet in Inf-ObMB mice promotes excess weight gain and accelerates NAFLD. Overall, these results provide functional evidence supporting a causative role of maternal obesity-associated infant dysbiosis in childhood obesity and NAFLD.

Crosstalk between type I and II interferons in regulation of myeloid cell responses during bacterial infection.

Type I and type II interferons (IFNα/ß and IFNγ) are cytokines that play indispensable roles in directing myeloid cell activity during inflammatory and immune responses. Each IFN type binds a distinct receptor (IFNAR or IFNGR) to transduce signals that reshape gene expression and function of myeloid and other cell types. In the context of murine models and human bacterial infections, production of IFNγ generally promotes resistance while production of IFNα/ß is associated with increased host susceptibility. Here, we review mechanisms of crosstalk between type I and II IFNs in myeloid cells and their impact on myeloid cell activation and anti-microbial function.

A Batf3/Nlrp3/IL-18 Axis Promotes Natural Killer Cell IL-10 Production during Listeria monocytogenes Infection.

The bacterial pathogen Listeria monocytogenes (Lm) capitalizes on natural killer (NK) cell production of regulatory interleukin (IL)-10 to establish severe systemic infections. Here, we identify regulators of this IL-10 secretion. We show that IL-18 signals to NK cells license their ability to produce IL-10. IL-18 acts independent of IL-12 and STAT4, which co-stimulate IFNγ secretion. Dendritic cell (DC) expression of Nlrp3 is required for IL-18 release in response to the Lm p60 virulence protein. Therefore, mice lacking Nlrp3, Il18, or Il18R fail to accumulate serum IL-10 and are highly resistant to systemic Lm infection. We further show that cells expressing or dependent on Batf3 are required for IL-18-inducing IL-10 production observed in infected mice. These findings explain how Il18 and Batf3 promote susceptibility to bacterial infection and demonstrate the ability of Lm to exploit NLRP3 for the promotion of regulatory NK cell activity.

Altered Natural Killer Cell Function in HIV-Exposed Uninfected Infants.

OBJECTIVES: HIV-exposed uninfected (HEU) infants have higher rates of severe and fatal infections compared with HIV-unexposed (HUU) infants, likely due to immune perturbations. We hypothesized that alterations in natural killer (NK) cell activity might occur in HEU infants and predispose them to severe infections. DESIGN: Case-control study using cryopreserved peripheral blood mononuclear cells (PBMCs) at birth and 6 months from HEU infants enrolled from 2002 to 2009 and HUU infants enrolled from 2011 to 2013. METHODS: NK cell phenotype and function were assessed by flow cytometry after 20-h incubation with and without K562 cells. RESULTS: The proportion of NK cells among PBMCs was lower at birth in 12 HEU vs. 22 HUU (1.68 vs. 10.30%, p < 0.0001) and at 6 months in 52 HEU vs. 72 HUU (3.09 vs. 4.65%, p = 0.0005). At birth, HEU NK cells demonstrated increased killing of K562 target cells (p < 0.0001) and increased expression of CD107a (21.65 vs. 12.70%, p = 0.047), but these differences resolved by 6 months. Stimulated HEU NK cells produced less interferon (IFN)γ at birth (0.77 vs. 2.64%, p = 0.008) and at 6 months (4.12 vs. 8.39%, p = 0.001), and showed reduced perforin staining at 6 months (66.95 vs. 77.30%, p = 0.0008). Analysis of cell culture supernatants indicated that lower NK cell activity in HEU was associated with reduced interleukin (IL)-12, IL-15, and IL-18. Addition of recombinant human IL-12 to stimulated HEU PBMCs restored IFNγ production to that seen in stimulated HUU cultures. CONCLUSION: NK cell proportion, phenotype, and function are altered in HEU infants. NK cell cytotoxicity and degranulation are increased in HEU at birth, but HEU NK cells have reduced IFNγ and perforin production, suggesting an adequate initial response, but decreased functional reserve. NK cell function improved with addition of exogenous IL-12, implicating impaired production of IL-12 by accessory cells. Alterations in NK cell and accessory cell function may contribute to the increased susceptibility to infection in HEU infants.

Down regulation of macrophage IFNGR1 exacerbates systemic L. monocytogenes infection.

Interferons (IFNs) target macrophages to regulate inflammation and resistance to microbial infections. The type II IFN (IFNγ) acts on a cell surface receptor (IFNGR) to promote gene expression that enhance macrophage inflammatory and anti-microbial activity. Type I IFNs can dampen macrophage responsiveness to IFNγ and are associated with increased susceptibility to numerous bacterial infections. The precise mechanisms responsible for these effects remain unclear. Type I IFNs silence macrophage ifngr1 transcription and thus reduce cell surface expression of IFNGR1. To test how these events might impact macrophage activation and host resistance during bacterial infection, we developed transgenic mice that express a functional FLAG-tagged IFNGR1 (fGR1) driven by a macrophage-specific promoter. Macrophages from fGR1 mice expressed physiologic levels of cell surface IFNGR1 at steady state and responded equivalently to WT C57Bl/6 macrophages when treated with IFNγ alone. However, fGR1 macrophages retained cell surface IFNGR1 and showed enhanced responsiveness to IFNγ in the presence of type I IFNs. When fGR1 mice were infected with the bacterium Listeria monocytogenes their resistance was significantly increased, despite normal type I and II IFN production. Enhanced resistance was dependent on IFNγ and associated with increased macrophage activation and antimicrobial function. These results argue that down regulation of myeloid cell IFNGR1 is an important mechanism by which type I IFNs suppress inflammatory and anti-bacterial functions of macrophages.