To kill a tachyzoite: assault and battery.

Polymeric guanylate-binding proteins (GBPs) physically dismember the vacuole membrane formed by Toxoplasma gondii while nitric oxide (NO) poisons and inhibits parasite replication within interferon (IFN)-γ activated macrophages. Zhao et al. report a novel mechanism for synergy between these classical microbicidal and microbistatic effectors in cell-autonomous immunity to the intracellular parasites.

Growth differentiation factor-15 is an IFN-γ regulated mediator of infection-induced weight loss and the hepatic FGF21 response.

Infections are often accompanied by weight loss caused by alterations in host behavior and metabolism, also known as sickness behaviors. Recent studies have revealed that sickness behaviors can either promote or impede survival during infections depending on factors such as the type of infectious pathogen. Nevertheless, we have an incomplete understanding of the underlying mechanisms of sickness behaviors. Furthermore, although the host immune responses to infections have long been known to contribute to the induction of sickness behaviors, recent studies have identified emerging cytokines that are also key regulators of host metabolism during infection and inflammation, such as growth differentiation factor 15 (GDF-15). GDF-15 is a distant member of the TGF-ß superfamily that causes weight loss by suppressing appetite and food consumption and causing emesis. These effects require activation of neurons that express the only known GDF-15 receptor, the GFRAL receptor. GDF-15 also functions in the periphery including the induction of ketogenesis and immunoregulation. Nevertheless, the functions and regulation of GDF-15 during live infections is not yet known. Murine infection with avirulent Toxoplasma gondii is an established model to understand infection-induced weight loss. Past studies have determined that acute T. gondii infection causes weight loss due to diminished food consumption and increased energy expenditure through unknown mechanisms. Additionally, our lab previously demonstrated that T. gondii causes upregulation in serum GDF-15 in an IFN-γ-dependent manner during the post-acute phase of the infection. In this study, we interrogated the in-vivo functions and immune regulation of GDF-15 during Toxoplasma gondii infection. First, we found that in wild-type mice, acute T. gondii infection caused a significant weight loss that is preceded by elevation of serum levels of IFN-γ and GDF-15. To determine whether IFN-γ regulates GDF-15, we neutralized IFN-γ on days 5 and 6 and measured GDF-15 on day 7 and found that serum but not tissue levels of GDF-15 decreased after IFN-γ neutralization. Additionally, exogenous IFN-γ was sufficient to elevate serum GDF-15 in the absence of infection. Next, we compared the outcomes of T. gondii infection between WT and Gdf15-/- mice. We observed that the weight trajectories were declining in WT mice while they were increasing in Gdf15-/-mice during the acute phase of the infection. This difference in trajectories extended throughout the chronic infection resulting to an overall weight loss relative to initial weights in WT mice but not Gdf15-/-mice. Then, we determined that GDF-15 is not essential for survival and immunoregulation during T. gondii infection. We also demonstrated that GDF-15 is required for the induction of FGF21, stress-induced cytokine with prominent roles in regulating host metabolism. Finally, we discovered a cytokine cascade IFN-γ-GDF-15-FGF21 that is likely involved in the regulation of host metabolism. Overall, our study provides evidence that IFN-γ contributes to the regulation of host metabolism during infection by inducing GDF-15 and FGF21. GDF-15 orchestrates changes in host metabolism that supports the host immune response in clearing the infection. These physiological alterations induce FGF21, which in turn, orchestrates the adaptive responses to the effects of GDF-15, which can be detrimental when protracted.

"AHR-ming" host defense against cryptosporidiosis.

Globally, cryptosporidiosis is a leading cause of childhood diarrheal disease and is a major risk factor for malnutrition and impairment of growth and cognitive development. In this issue of Cell Host & Microbe, Maradana et al. identify a target for dietary enhancement of innate immune defenses against cryptosporidiosis.

Emerging Roles of Growth Differentiation Factor 15 in Immunoregulation and Pathogenesis.

Growth differentiation factor 15 (GDF-15) is a cytokine that is widely used as a biomarker for the severity of diverse disease states. It also has been shown to play a protective role after tissue injury and to promote a negative energy balance during obesity and diabetes. In addition to its metabolic effects, GDF-15 also regulates the host's immune responses to infectious and noninfectious diseases. GDF-15 can suppress a type 1 and, in contrast, promote a type 2 inflammatory response. In this brief review, we discuss how GDF-15 affects the effector function and recruitment of immune cells, the pathways that induce its expression, and the diverse mechanisms by which it is regulated during inflammation and infection. We further highlight outstanding questions that should be the focus of future investigations in this emerging field.

Macrophage to dendritic cell transitioning induced by Toxoplasma.

Toxoplasma gondii exploits the migratory properties of monocytes and dendritic cells to promote tissue dissemination. Recently, ten Hoeve et al. reported that the parasite effector protein GRA28 conspires with host chromatin modifiers to confer dendritic cell-like features that convert sessile macrophages into migratory cells that transport infection to distal organs.

The Impact of Helminth Coinfection on Innate and Adaptive Immune Resistance and Disease Tolerance during Toxoplasmosis.

More than 2 billion people worldwide are infected with helminths. Thus, it is possible for individuals to experience concomitant infection with helminth and intracellular microbes. Although the helminth-induced type 2 response can suppress type 1 proinflammatory responses required for the immunity against intracellular pathogens in the context of a coinfection, conflicting evidence suggest that helminth infection can enhance antimicrobial immunity. Using a coinfection model with the intestinal helminth Heligmosomoides polygyrus followed by infection with Toxoplasma gondii in Mus Musculus, we showed that the complex and dynamic effect of helminth infection is highly suppressive during the innate phase (days 0-3) of T. gondii infection and less stringent during the acute phase (d10). Helminth coinfection had a strong suppressive effect on the neutrophil, monocytic, and early IFN-γ/IL-12 responses. The IFN-γ response was later restored by compensatory production from T cells despite decreased effector differentiation of T. gondii-specific CD8 T cells. In accordance with the attenuated IFN-γ response, parasite loads were elevated during the acute phase (d10) of T. gondii infection but were transiently controlled by the compensatory T cell response. Unexpectedly, 40% of helminth-coinfected mice exhibited a sustained weight loss phenotype during the postacute phase (d14-18) that was not associated with T. gondii outgrowth, indicating that coinfection led to decreased disease tolerance during T. gondii infection. Our work uncovers the dynamic nature of the helminth immunomodulatory effects on concomitant infections or immune responses and unveils a loss of disease tolerance phenotype triggered by coinfection with intestinal helminth.

Avirulence: an essential feature of the parasitic lifestyle.

The host plays an essential role in parasite transmission. The viability of the host-parasite relationship depends upon development of immune resistance and the induction of disease tolerance. Here I propose that pathogen coevolution of avirulence factors promoting host disease tolerance is an essential feature of the parasitic lifestyle.

Adenosine metabolized from extracellular ATP promotes type 2 immunity through triggering A2BAR signaling in intestinal epithelial cells.

Intestinal nematode parasites can cross the epithelial barrier, causing tissue damage and release of danger-associated molecular patterns (DAMPs) that may promote host protective type 2 immunity. We investigate whether adenosine binding to the A2B adenosine receptor (A2BAR) on intestinal epithelial cells (IECs) plays an important role. Specific blockade of IEC A2BAR inhibits the host protective memory response to the enteric helminth, Heligmosomoides polygyrus bakeri (Hpb), including disruption of granuloma development at the host-parasite interface. Memory T cell development is blocked during the primary response, and transcriptional analyses reveal profound impairment of IEC activation. Extracellular ATP is visualized 24 h after inoculation and is shown in CD39-deficient mice to be critical for the adenosine production mediating the initiation of type 2 immunity. Our studies indicate a potent adenosine-mediated IEC pathway that, along with the tuft cell circuit, is critical for the activation of type 2 immunity.

A Role for Basigin in Toxoplasma gondii Infection.

The role of specific host cell surface receptors during Toxoplasma gondii invasion of host cells is poorly defined. Here, we interrogated the role of the well-known malarial invasion receptor, basigin, in T. gondii infection of astrocytes. We found that primary astrocytes express two members of the BASIGIN (BSG) immunoglobulin family, basigin and embigin, but did not express neuroplastin. Antibody blockade of either basigin or embigin caused a significant reduction of parasite infectivity in astrocytes. The specific role of basigin during T. gondii invasion was further examined using a mouse astrocytic cell line (C8-D30), which exclusively expresses basigin. CRISPR-mediated deletion of basigin in C8-D30 cells resulted in decreased T. gondii infectivity. T. gondii replication and invasion efficiency were not altered by basigin deficiency, but parasite attachment to astrocytes was markedly reduced. We also conducted a proteomic screen to identify T. gondii proteins that interact with basigin. Toxoplasma-encoded cyclophilins, the protein 14-3-3, and protein disulfide isomerase (TgPDI) were among the putative basigin-ligands identified. Recombinant TgPDI produced in E. coli bound to basigin and pretreatment of tachyzoites with a PDI inhibitor decreased parasite attachment to host cells. Finally, mutagenesis of the active site cysteines of TgPDI abolished enzyme binding to basigin. Thus, basigin and its related immunoglobulin family members may represent host receptors that mediate attachment of T. gondii to diverse cell types.

Microglia and astrocyte responses to neuropathogenic protozoan parasites.

Cerebral toxoplasmosis and cerebral malaria are two important neurological diseases caused by protozoan parasites. In this review, we discuss recent findings regarding the innate immune responses of microglia and astrocytes to Toxoplasma and Plasmodium infection. In both infections, these tissue-resident glial cells perform a sentinel function mediated by alarmin crosstalk that licenses adaptive type 1 immunity in the central nervous system. Divergent protective or pathogenic effects of type 1 activation of these astrocytes and microglia are revealed depending on the inherent lytic potential of the protozoan parasite.

Cutting Edge: CD36 Mediates Phagocyte Tropism and Avirulence of Toxoplasma gondii.

Resistance and tolerance are vital for survivability of the host-pathogen relationship. Virulence during Toxoplasma infection in mice is mediated by parasite kinase-dependent antagonism of IFN-γ-induced host resistance. Whether avirulence requires expression of parasite factors that induce host tolerance mechanisms or is a default status reflecting the absence of resistance-interfering factors is not known. In this study, we present evidence that avirulence in Toxoplasma requires parasite engagement of the scavenger receptor CD36. CD36 promotes macrophage tropism but is dispensable for the development of resistance mechanisms. Instead CD36 is critical for re-establishing tissue homeostasis and survival following the acute phase of infection. The CD36-binding capacity of T. gondii strains is negatively controlled by the virulence factor, ROP18. Thus, the absence of resistance-interfering virulence factors and the presence of tolerance-inducing avirulence factors are both required for long-term host-pathogen survival.

Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine.

Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.

Trichinella spiralis-induced mastocytosis and erythropoiesis are simultaneously supported by a bipotent mast cell/erythrocyte precursor cell.

Anti-helminth responses require robust type 2 cytokine production that simultaneously promotes worm expulsion and initiates the resolution of helminth-induced wounds and hemorrhaging. However, how infection-induced changes in hematopoiesis contribute to these seemingly distinct processes remains unknown. Recent studies have suggested the existence of a hematopoietic progenitor with dual mast cell-erythrocyte potential. Nonetheless, whether and how these progenitors contribute to host protection during an active infection remains to be defined. Here, we employed single cell RNA-sequencing and identified that the metabolic enzyme, carbonic anhydrase (Car) 1 marks a predefined bone marrow-resident hematopoietic progenitor cell (HPC) population. Next, we generated a Car1-reporter mouse model and found that Car1-GFP positive progenitors represent bipotent mast cell/erythrocyte precursors. Finally, we show that Car1-expressing HPCs simultaneously support mast cell and erythrocyte responses during Trichinella spiralis infection. Collectively, these data suggest that mast cell/erythrocyte precursors are mobilized to promote type 2 cytokine responses and alleviate helminth-induced blood loss, developmentally linking these processes. Collectively, these studies reveal unappreciated hematopoietic events initiated by the host to combat helminth parasites and provide insight into the evolutionary pressure that may have shaped the developmental relationship between mast cells and erythrocytes.

Helminth Infections Induce Tissue Tolerance Mitigating Immunopathology but Enhancing Microbial Pathogen Susceptibility.

Helminths are ubiquitous and have chronically infected vertebrates throughout their evolution. As such helminths have likely exerted considerable selection pressure on our immune systems. The large size of multicellular helminths and their limited replicative capacity in the host necessarily elicits different host protective mechanisms than the immune response evoked by microbial pathogens such as bacteria, viruses and intracellular parasites. The cellular damage resulting from helminth migration through tissues is a major trigger of the type 2 and regulatory immune responses, which activates wound repair mechanisms that increases tissue tolerance to injury and resistance mechanisms that enhance resistance to further colonization with larval stages. While these wound healing and anti-inflammatory responses may be beneficial to the helminth infected host, they may also compromise the host's ability to mount protective immune responses to microbial pathogens. In this review we will first describe helminth-induced tolerance mechanisms that develop in specific organs including the lung and the intestine, and how adaptive immunity may contribute to these responses through differential activation of T cells in the secondary lymphoid organs. We will then integrate studies that have examined how the immune response is modulated in these specific tissues during coinfection of helminths with viruses, protozoa, and bacteria.

Transcription factor Etv6 regulates functional differentiation of cross-presenting classical dendritic cells.

An IRF8-dependent subset of conventional dendritic cells (cDCs), termed cDC1, effectively cross-primes CD8+ T cells and facilitates tumor-specific T cell responses. Etv6 is an ETS family transcription factor that controls hematopoietic stem and progenitor cell (HSPC) function and thrombopoiesis. We report that like HSPCs, cDCs express Etv6, but not its antagonist, ETS1, whereas interferon-producing plasmacytoid dendritic cells (pDCs) express both factors. Deletion of Etv6 in the bone marrow impaired the generation of cDC1-like cells in vitro and abolished the expression of signature marker CD8α on cDC1 in vivo. Moreover, Etv6-deficient primary cDC1 showed a partial reduction of cDC-specific and cDC1-specific gene expression and chromatin signatures and an aberrant up-regulation of pDC-specific signatures. Accordingly, DC-specific Etv6 deletion impaired CD8+ T cell cross-priming and the generation of tumor antigen-specific CD8+ T cells. Thus, Etv6 optimizes the resolution of cDC1 and pDC expression programs and the functional fitness of cDC1, thereby facilitating T cell cross-priming and tumor-specific responses.

Paneth Cell Multipotency Induced by Notch Activation following Injury.

Paneth cells are post-mitotic intestinal epithelial cells supporting the stem cell niche and mucosal immunity. Paneth cell pathologies are observed in various gastrointestinal diseases, but their plasticity and response to genomic and environmental challenges remain unclear. Using a knockin allele engineered at the mouse Lyz1 locus, we performed detailed Paneth cell-lineage tracing. Irradiation induced a subset of Paneth cells to proliferate and differentiate into villus epithelial cells. RNA sequencing (RNA-seq) revealed that Paneth cells sorted from irradiated mice acquired a stem cell-like transcriptome; when cultured in vitro, these individual Paneth cells formed organoids. Irradiation activated Notch signaling, and forced expression of Notch intracellular domain (NICD) in Paneth cells, but not Wnt/ß-catenin pathway activation, induced their dedifferentiation. This study documents Paneth cell plasticity, particularly their ability to participate in epithelial replenishment following stem cell loss, adding to a growing body of knowledge detailing the molecular pathways controlling injury-induced regeneration.

Toxoplasma gondii Inactivates Human Plasmacytoid Dendritic Cells by Functional Mimicry of IL-10.

Plasmacytoid dendritic cells (pDCs) are the major producers of IFN-α, an antiviral cytokine involved in immunomodulation and control of HIV type 1 replication, whereas Toxoplasma gondii is a life-threatening opportunistic infection in AIDS patients. During infection with HIV type 1, human pDCs decrease in circulation and remaining pDC produce lower amounts of IFN-α in response to viral stimulation. In this study, we investigated the impact of coinfection with T. gondii on the innate virus-directed responses of human pDCs. Using intracellular flow cytometry and fluorescence microscopy, we determined that T. gondii invaded but did not induce IFN-α or TNF-α in human pDC. However, T. gondii inhibited IFN-α and TNF-α produced in response to HSV and HIV, thus functionally inactivating pDC. IFN-α production was inhibited only in cells infected by T. gondii, which inhibited neither uptake of GFP-HSV nor localization of TLR9 in CD71+ endosomes, directing us to investigate downstream events. Using imaging flow cytometry, we found that both T. gondii and IL-10 inhibited virus-induced nuclear translocation, but not phosphorylation, of IFN response factor 7. Blockade of IFN response factor 7 nuclear translocation and inhibition of the IFN-α response was partially reversed by a deficiency in the T. gondii-derived ROP16 kinase, known to directly phosphorylate STAT3, a critical mediator of IL-10's anti-inflammatory effects. Taken together, our results indicate that T. gondii suppresses pDC activation by mimicking IL-10's regulatory effects through an ROP16 kinase-dependent mechanism. Our findings further imply a convergent mechanism of inhibition of TLR signaling by T. gondii and IL-10 and suggest potential negative consequences of HIV/T. gondii coinfection.

FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry.

We describe a flow-cytometry-based protocol for intracellular mRNA measurements in nonadherent mammalian cells using fluorescence in situ hybridization (FISH) probes. The method, which we call FISH-Flow, allows for high-throughput multiparametric measurements of gene expression, a task that was not feasible with earlier, microscopy-based approaches. The FISH-Flow protocol involves cell fixation, permeabilization and hybridization with a set of fluorescently labeled oligonucleotide probes. In this protocol, surface and intracellular protein markers can also be stained with fluorescently labeled antibodies for simultaneous protein and mRNA measurement. Moreover, a semiautomated, single-tube version of the protocol can be performed with a commercially available cell-wash device that reduces cell loss, operator time and interoperator variability. It takes ∼30 h to perform this protocol. An example of FISH-Flow measurements of cytokine mRNA induction by ex vivo stimulation of primed T cells with specific antigens is described.

Cutting Edge: Helminth Coinfection Blocks Effector Differentiation of CD8 T Cells through Alternate Host Th2- and IL-10-Mediated Responses.

Concurrent helminth infection potently inhibits T cell immunity; however, whether helminthes prevent T cell priming or skew clonal recruitment and effector differentiation is not known. Using coinfection with two natural mouse pathogens, Heligmosomoides polygyrus and Toxoplasma gondii, to investigate the negative impact of helminthes on the CD8 T cell response, we demonstrate helminth-induced suppression of IL-12-dependent differentiation of killer-like receptor G1+ effector CD8 T cells and IFN-γ production. Nevertheless, reversal of helminth suppression of the innate IL-12 response of CD8α+ dendritic cells, which occurred in STAT6-deficient mice, was not sufficient to normalize CD8 T cell differentiation. Instead, a combined deficiency in IL-4 and IL-10 was required to reverse the negative effects of helminth coinfection on the CD8 T cell response. Monoclonal T. gondii-specific CD8 T cells adoptively transferred into coinfected mice recapitulated the spectrum of helminth-induced effects on the polyclonal CD8 T response, indicating the lack of requirement for clonal skewing.