The poliovirus receptor (PVR) is a ubiquitously expressed glycoprotein involved in cellular adhesion and immune response. It engages the activating receptor DNAX accessory molecule (DNAM)-1, the inhibitory receptor TIGIT, and the CD96 receptor with both activating and inhibitory functions. Human cytomegalovirus (HCMV) down-regulates PVR expression, but the significance of this viral function in vivo remains unknown. Here, we demonstrate that mouse CMV (MCMV) also down-regulates the surface PVR. The m20.1 protein of MCMV retains PVR in the endoplasmic reticulum and promotes its degradation. A MCMV mutant lacking the PVR inhibitor was attenuated in normal mice but not in mice lacking DNAM-1. This attenuation was partially reversed by NK cell depletion, whereas the simultaneous depletion of mononuclear phagocytes abolished the virus control. This effect was associated with the increased expression of DNAM-1, whereas TIGIT and CD96 were absent on these cells. An increased level of proinflammatory cytokines in sera of mice infected with the virus lacking the m20.1 and an increased production of iNOS by inflammatory monocytes was observed. Blocking of CCL2 or the inhibition of iNOS significantly increased titer of the virus lacking m20.1. In this study, we have demonstrated that inflammatory monocytes, together with NK cells, are essential in the early control of CMV through the DNAM-1-PVR pathway.

The recognized diversity of innate lymphoid cells (ILCs) is rapidly expanding. Three ILC classes have emerged, ILC1, ILC2 and ILC3, with ILC1 and ILC3 including several subsets. The classification of some subsets is unclear, and it remains controversial whether natural killer (NK) cells and ILC1 cells are distinct cell types. To address these issues, we analyzed gene expression in ILCs and NK cells from mouse small intestine, spleen and liver, as part of the Immunological Genome Project. The results showed unique gene-expression patterns for some ILCs and overlapping patterns for ILC1 cells and NK cells, whereas other ILC subsets remained indistinguishable. We identified a transcriptional program shared by small intestine ILCs and a core ILC signature. We revealed and discuss transcripts that suggest previously unknown functions and developmental paths for ILCs.

Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii. Importance: Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T. gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.

Apicomplexan parasites actively invade host cells using a mechanism predicted to be powered by a parasite actin-dependent myosin motor. In the model apicomplexan Toxoplasma gondii, inducible knockout of the actin gene, ACT1, was recently demonstrated to limit but not completely abolish invasion. This observation has led to the provocative suggestion that T. gondii possesses alternative, ACT1-independent invasion pathways. Here, we dissected the residual invasive ability of Δact1 parasites. Surprisingly, we were able to detect residual ACT1 protein in inducible Δact1 parasites as long as 5 days after ACT1 deletion. We further found that the longer Δact1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed. Both findings are consistent with the quantity of residual ACT1 retained in Δact1 parasites being responsible for their invasive ability. Furthermore, invasion by the Δact1 parasites was also sensitive to the actin polymerization inhibitor cytochalasin D. Finally, there was no clear defect in attachment to host cells or moving junction formation by Δact1 parasites. However, Δact1 parasites often exhibited delayed entry into host cells, suggesting a defect specific to the penetration stage of invasion. Overall, our results support a model where residual ACT1 protein retained in inducible Δact1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion.

Viral infections and type 2 immune responses are thought to be critical for the development of chronic respiratory disease, but the link between these events needs to be better defined. Here, we study a mouse model in which infection with a mouse parainfluenza virus known as Sendai virus (SeV) leads to long-term activation of innate immune cells that drive IL-13-dependent lung disease. We find that chronic postviral disease (signified by formation of excess airway mucus and accumulation of M2-differentiating lung macrophages) requires macrophage expression of triggering receptor expressed on myeloid cells-2 (TREM-2). Analysis of mechanism shows that viral replication increases lung macrophage levels of intracellular and cell surface TREM-2, and this action prevents macrophage apoptosis that would otherwise occur during the acute illness (5-12 d after inoculation). However, the largest increases in TREM-2 levels are found as the soluble form (sTREM-2) long after clearance of infection (49 d after inoculation). At this time, IL-13 and the adapter protein DAP12 promote TREM-2 cleavage to sTREM-2 that is unexpectedly active in preventing macrophage apoptosis. The results thereby define an unprecedented mechanism for a feed-forward expansion of lung macrophages (with IL-13 production and consequent M2 differentiation) that further explains how acute infection leads to chronic inflammatory disease.

The microglial triggering receptor expressed on myeloid cells 2 (TREM2) signals via the activatory membrane adaptor molecule TYROBP. Genetic variants or mutations of TREM2 or TYROBP have been linked to inflammatory neurodegenerative diseases associated with aging. The typical aging process goes along with microglial changes and mild neuronal loss, but the exact contribution of TREM2 is still unclear. Aged TREM2 knock-out mice showed decreased age-related neuronal loss in the substantia nigra and the hippocampus. Transcriptomic analysis of the brains of 24 months old TREM2 knock-out mice revealed 211 differentially expressed genes mostly downregulated and associated with complement activation and oxidative stress response pathways. Consistently, 24 months old TREM2 knock-out mice showed lower transcription of microglial (Aif1 and Tmem119), oxidative stress markers (Inos, Cyba, and Cybb) and complement components (C1qa, C1qb, C1qc, C3, C4b, Itgam, and Itgb2), decreased microglial numbers and expression of the microglial activation marker Cd68, as well as accumulation of oxidized lipids. Cultured microglia of TREM2 knock-out mice showed reduced phagocytosis and oxidative burst. Thus, microglial TREM2 contributes to age-related microglial changes, phagocytic oxidative burst, and loss of neurons with possible detrimental effects during physiological aging.

While signals that activate group 3 innate lymphoid cells (ILC3s) have been described, the factors that negatively regulate these cells are less well understood. Here we found that the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor κB ligand (RANKL) suppressed ILC3 activity in the intestine. Deletion of RANKL in ILC3s and T cells increased C-C motif chemokine receptor 6 (CCR6)+ ILC3 abundance and enhanced production of interleukin-17A (IL-17A) and IL-22 in response to IL-23 and during infection with the enteric murine pathogen Citrobacter rodentium. Additionally, CCR6+ ILC3s produced higher amounts of the master transcriptional regulator RORγt at steady state in the absence of RANKL. RANKL-mediated suppression was independent of T cells, and instead occurred via interactions between CCR6+ ILC3s that expressed both RANKL and its receptor, RANK. Thus, RANK-RANKL interactions between ILC3s regulate ILC3 abundance and activation, suggesting that cell clustering may control ILC3 activity.

Phagocytic cells are the first line of innate defense against intracellular pathogens, and yet Toxoplasma gondii is renowned for its ability to survive in macrophages, although this paradigm is based on virulent type I parasites. Surprisingly, we find that avirulent type III parasites are preferentially cleared in naive macrophages, independent of gamma interferon (IFN-γ) activation. The ability of naive macrophages to clear type III parasites was dependent on enhanced activity of NADPH oxidase (Nox)-generated reactive oxygen species (ROS) and induction of guanylate binding protein 5 (Gbp5). Macrophages infected with type III parasites (CTG strain) showed a time-dependent increase in intracellular ROS generation that was higher than that induced by type I parasites (GT1 strain). The absence of Nox1 or Nox2, gp91 subunit isoforms of the Nox complex, reversed ROS-mediated clearance of CTG parasites. Consistent with this finding, both Nox1-/- and Nox2-/- mice showed higher susceptibility to CTG infection than wild-type mice. Additionally, Gbp5 expression was induced upon infection and the enhanced clearance of CTG strain parasites was reversed in Gbp5-/- macrophages. Expression of a type I ROP18 allele in CTG prevented clearance in naive macrophages, suggesting that it plays a role counteracting Gbp5. Although ROS and Gbp5 have been linked to activation of the NLRP3 inflammasome, clearance of CTG parasites did not rely on induction of pyroptosis. Collectively, these findings reveal that not all strains of T. gondii are adept at avoiding clearance in macrophages and define new roles for ROS and Gbps in controlling this important intracellular pathogen.IMPORTANCEToxoplasma infections in humans and other mammals are largely controlled by IFN-γ produced by the activated adaptive immune system. However, we still do not completely understand the role of cell-intrinsic functions in controlling Toxoplasma or other apicomplexan infections. The present work identifies intrinsic activities in naive macrophages in counteracting T. gondii infection. Using an avirulent strain of T. gondii, we highlight the importance of Nox complexes in conferring protection against parasite infection both in vitro and in vivo We also identify Gbp5 as a novel macrophage factor involved in limiting intracellular infection by avirulent strains of T. gondii The rarity of human infections caused by type III strains suggests that these mechanisms may also be important in controlling human toxoplasmosis. These findings further extend our understanding of host responses and defense mechanisms that act to control parasitic infections at the cellular level.

Innate lymphoid cells (ILCs) are important regulators of early infection at mucosal barriers. ILCs are divided into three groups based on expression profiles, and are activated by cytokines and neuropeptides. Yet, it remains unknown if ILCs integrate other signals in providing protection. We show that signaling through herpes virus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor superfamily, in ILC3 is important for host defense against oral infection with the bacterial pathogen Yersinia enterocolitica. HVEM stimulates protective interferon-γ (IFN-γ) secretion from ILCs, and mice with HVEM-deficient ILC3 exhibit reduced IFN-γ production, higher bacterial burdens and increased mortality. In addition, IFN-γ production is critical as adoptive transfer of wild-type but not IFN-γ-deficient ILC3 can restore protection to mice lacking ILCs. We identify the TNF superfamily member, LIGHT, as the ligand inducing HVEM signals in ILCs. Thus HVEM signaling mediated by LIGHT plays a critical role in regulating ILC3-derived IFN-γ production for protection following infection. VIDEO ABSTRACT.

Tubulo-interstitial damage is a common finding in the chronically diseased kidney and is characterized by ongoing inflammation and fibrosis leading to renal dysfunction and end-stage renal disease. Upon kidney injury, endogenous ligands can be released which are recognized by innate immune sensors to alarm innate immune system. A new family of innate sensors is the family of TREM (triggering receptor expressed on myeloid cell). TREM1 is an activating receptor and requires association with transmembrane adapter molecule DAP12 (DNAX-associated protein 12) for cell signaling. TREM1-DAP12 pathway has a cross-talk with intracellular signaling pathways of several Toll-like receptors (TLRs) and is able to amplify TLR signaling and thereby contributes to the magnitude of inflammation. So far, several studies have shown that TLRs play a role in obstructive nephropathy but the contribution of TREM1-DAP12 herein is unknown. Therefore, we studied TREM1 expression in human and murine progressive renal diseases and further investigated the role for TREM1-DAP12 by subjecting wild-type (WT), TREM1/3 double KO and DAP12 KO mice to murine unilateral ureter obstruction (UUO) model. In patients with hydronephrosis, TREM1 positive cells were observed in renal tissue. We showed that in kidneys from WT mice, DAP12 mRNA and TREM1 mRNA and protein levels were elevated upon UUO. Compared to WT mice, DAP12 KO mice displayed less renal MCP-1, KC and TGF-β1 levels and less influx of macrophages during progression of UUO, whereas TREM1/3 double KO mice displayed less renal MCP-1 level. Renal fibrosis was comparable in WT, TREM1/3 double KO and DAP12 KO mice. We conclude that DAP12, partly through TREM1/3, is involved in renal inflammation during progression of UUO.

Recent genome-wide association studies linked variants in TREM2 to a strong increase in the odds of developing Alzheimer's disease. The mechanism by which TREM2 influences the susceptibility to Alzheimer's disease is currently unknown. TREM2 is expressed by microglia and is thought to regulate phagocytic and inflammatory microglial responses to brain pathology. Given that a single allele of variant TREM2, likely resulting in a loss of function, conferred an increased risk of developing Alzheimer's disease, we tested whether loss of one functional trem2 allele would affect Aβ plaque deposition or the microglial response to Aβ pathology in APPPS1-21 mice.

Apicomplexan parasites contain a conserved protein CelTOS that, in malaria parasites, is essential for traversal of cells within the mammalian host and arthropod vector. However, the molecular role of CelTOS is unknown because it lacks sequence similarity to proteins of known function. Here, we determined the crystal structure of CelTOS and discovered CelTOS resembles proteins that bind to and disrupt membranes. In contrast to known membrane disruptors, CelTOS has a distinct architecture, specifically binds phosphatidic acid commonly present within the inner leaflet of plasma membranes, and potently disrupts liposomes composed of phosphatidic acid by forming pores. Microinjection of CelTOS into cells resulted in observable membrane damage. Therefore, CelTOS is unique as it achieves nearly universal inner leaflet cellular activity to enable the exit of parasites from cells during traversal. By providing novel molecular insight into cell traversal by apicomplexan parasites, our work facilitates the design of therapeutics against global pathogens.

Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-β pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.

DNAX accessory molecule-1 (DNAM-1, also known as CD226) is an activating receptor expressed on subsets of natural killer (NK) and T cells, interacts with its ligands CD155 or CD112, and has co-varied expression with inhibitory receptors. Since inhibitory receptors control NK-cell activation and are necessary for MHC-I-dependent education, we investigated whether DNAM-1 expression is also involved in NK-cell education. Here we show an MHC-I-dependent correlation between DNAM-1 expression and NK-cell education, and an association between DNAM-1 and NKG2A that occurs even in MHC class I deficient mice. DNAM-1 is expressed early during NK-cell development, precedes the expression of MHC-I-specific inhibitory receptors, and is modulated in an education-dependent fashion. Cd226-/- mice have missing self-responses and NK cells with a normal receptor repertoire. We propose a model in which NK-cell education prevents or delays downregulation of DNAM-1. This molecule endows educated NK cells with enhanced effector functions but is dispensable for education.

IFN-γ activates cells to restrict intracellular pathogens by upregulating cellular effectors including the p65 family of guanylate-binding proteins (GBPs). Here we test the role of Gbp1 in the IFN-γ-dependent control of T. gondii in the mouse model. Virulent strains of T. gondii avoided recruitment of Gbp1 to the parasitophorous vacuole in a strain-dependent manner that was mediated by the parasite virulence factors ROP18, an active serine/threonine kinase, and the pseudokinase ROP5. Increased recruitment of Gbp1 to Δrop18 or Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages in vitro, a process dependent on the autophagy protein Atg5. The increased susceptibility of Δrop18 mutants in IFN-γ-activated macrophages was reverted in Gbp1(-/-) cells, and decreased virulence of this mutant was compensated in Gbp1(-/-) mice, which were also more susceptible to challenge with type II strain parasites of intermediate virulence. These findings demonstrate that Gbp1 plays an important role in the IFN-γ-dependent, cell-autonomous control of toxoplasmosis and predict a broader role for this protein in host defense.

Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with excellent analytical tools available.

Apicomplexan parasites such as Toxoplasma gondii rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of T. gondii in vitro. Structural modeling of chondramide A (2) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (2b-k) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC₅₀ values that ranged from 0.3 to 1.3 μM. In vitro polymerization assays using highly purified recombinant actin from T. gondii verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites.

Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.

DAP12 (KARAP) is a transmembrane signaling adaptor for a family of innate immunoreceptors that have been shown to activate granulocytes and monocytes/macrophages, amplifying production of inflammatory cytokines. Contrasting with these data, recent studies suggest that DAP12 signaling has an inhibitory role in the macrophage response to microbial products (Hamerman, J.A., N.K. Tchao, C.A. Lowell, and L.L. Lanier. 2005. Nat. Immunol. 6:579-586). To determine the in vivo role for DAP12 signaling in inflammation, we measured the response of wild-type (WT) and DAP12-/- mice to septic shock. We show that DAP12-/- mice have improved survival from both endotoxemia and cecal ligation and puncture-induced septic shock. As compared with WT mice, DAP12-/- mice have decreased plasma cytokine levels and a decreased acute phase response during sepsis, but no defect in the recruitment of cells or bacterial control. In cells isolated after sepsis and stimulated ex vivo, DAP12 signaling augments lipopolysaccharide-mediated cytokine production. These data demonstrate that, during sepsis, DAP12 signaling augments the response to microbial products, amplifying inflammation and contributing to mortality.

Natural killer (NK) cells express multiple activating receptors that initiate signaling cascades through DAP10- or immunoreceptor tyrosine-based activation motif-containing adapters, including DAP12 and FcRgamma. Among downstream signaling mediators, the guanine nucleotide exchange factor Vav1 carries out a key role in activation. However, whether Vav1 regulates only some or all NK cell-activating pathways is matter of debate. It is also possible that two other Vav family molecules, Vav2 and Vav3, are involved in NK cell activation. Here, we examine the relative contribution of each of these exchange factors to NK cell-mediated cytotoxicity using mice lacking one, two, or all three Vav proteins. We found that Vav1 deficiency is sufficient to disrupt DAP10-mediated cytotoxicity, whereas lack of Vav2 and Vav3 profoundly impairs FcRgamma- and DAP12-mediated cytotoxicity. Our results provide evidence that these three Vav proteins function specifically in distinct pathways that trigger NK cell cytotoxicity.