The inflammatory response in the CNS begins with the movement of leukocytes across the blood-brain barrier in a multistep process that requires cells to pass through a perivascular space before entering the parenchyma. The molecular mechanisms that orchestrate this movement are not known. The chemokine CXCL12 is highly expressed throughout the CNS by microendothelial cells under normal conditions, suggesting it might play a role maintaining the blood-brain barrier. We tested this hypothesis in the setting of experimental autoimmune encephalomyelitis (EAE) by using AMD3100, a specific antagonist of the CXCL12 receptor CXCR4. We demonstrate that the loss of CXCR4 activation enhances the migration of infiltrating leukocytes into the CNS parenchyma. CXCL12 is expressed at the basolateral surface of CNS endothelial cells in normal spinal cord and at the onset of EAE. This polarity is lost in vessels associated with an extensive parenchymal invasion of mononuclear cells during the peak of disease. Inhibition of CXCR4 activation during the induction of EAE leads to loss of the typical intense perivascular cuffs, which are replaced with widespread white matter infiltration of mononuclear cells, worsening the clinical severity of the disease and increasing inflammation. Taken together, these data suggest a novel anti-inflammatory role for CXCL12 during EAE in that it functions to localize CXCR4-expressing mononuclear cells to the perivascular space, thereby limiting the parenchymal infiltration of autoreactive effector cells.

MD-2 is associated with Toll-like receptor 4 (TLR4) on the cell surface and enables TLR4 to respond to LPS. We tested whether MD-2 enhances or enables the responses of both TLR2 and TLR4 to Gram-negative and Gram-positive bacteria and their components. TLR2 without MD-2 did not efficiently respond to highly purified LPS and LPS partial structures. MD-2 enabled TLR2 to respond to nonactivating protein-free LPS, LPS mutants, or lipid A and enhanced TLR2-mediated responses to both Gram-negative and Gram-positive bacteria and their LPS, peptidoglycan, and lipoteichoic acid components. MD-2 enabled TLR4 to respond to a wide variety of LPS partial structures, Gram-negative bacteria, and Gram-positive lipoteichoic acid, but not to Gram-positive bacteria, peptidoglycan, and lipopeptide. MD-2 physically associated with TLR2, but this association was weaker than with TLR4. MD-2 enhanced expression of both TLR2 and TLR4, and TLR2 and TLR4 enhanced expression of MD-2. Thus, MD-2 enables both TLR4 and TLR2 to respond with high sensitivity to a broad range of LPS structures and to lipoteichoic acid, and, moreover, MD-2 enhances the responses of TLR2 to Gram-positive bacteria and peptidoglycan, to which the TLR4-MD-2 complex is unresponsive.

During the process of cross presentation, viral or tumor-derived antigens are presented to CD8+ T cells by the Batf3-dependent CD8α+/XCR1+ classical dendritic cell (cDC1). We designed a functional CRISPR screen for novel regulators of cross presentation, and identified the BEACH-domain containing protein WDFY4 as essential for cross-presentation of cell-associated antigens by cDC1. WDFY4 was not, however, required for MHC class II presentation or for cross-presentation by monocyte-derived DCs. In contrast to Batf3−/− mice, Wdfy4−/− mice have normal lymphoid and non-lymphoid cDC1 populations that produce IL-12 and protect against Toxoplasma gondii infection. However similar to Batf3−/− mice, Wdfy4−/− mice fail to prime virus- specific CD8+ T cells in vivo or induce tumor rejection, revealing a critical role for cross-presentation in anti-viral and anti-tumor immunity.

Summary Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using “air-liquid interface” (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro . ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro , demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

The intracellular parasite Toxoplasma gondii enjoys a wide host range and is adept at surviving in both naive and activated macrophages. Previous studies have emphasized the importance of the active serine-threonine protein kinase rhoptry protein 18 (ROP18), which targets immunity-related GTPases (IRGs), in mediating macrophage survival and acute virulence of T. gondii in mice. Here, we demonstrate that ROP18 exists in a complex with the pseudokinases rhoptry proteins 8 and 2 (ROP8/2) and dense granule protein 7 (GRA7). Individual deletion mutant Δgra7 or Δrop18 was partially attenuated for virulence in mice, whereas the combined Δgra7Δrop18 mutant was avirulent, suggesting these proteins act together in the same pathway. The virulence defect of the double mutant was mirrored by increased recruitment of IRGs and clearance of the parasite in IFN-γ-activated macrophages in vitro. GRA7 was shown to recognize a conserved feature of IRGs, binding directly to the active dimer of immunity-related GTPase a6 in a GTP-dependent manner. Binding of GRA7 to immunity-related GTPase a6 led to enhanced polymerization, rapid turnover, and eventual disassembly. Collectively, these studies suggest that ROP18 and GRA7 act in a complex to target IRGs by distinct mechanisms that are synergistic.pathogenesis | innate immunity | cooperative polymerization T he apicomplexan parasite Toxoplasma gondii has a remarkable host range and is capable of infecting most warm-blooded animals (1). The cellular life cycle of this opportunistic pathogen involves active invasion of nucleated host cells and establishment of an intracellular niche within the parasitophorous vacuole. This compartment is demarcated by the parasitophorous vacuole membrane (PVM), which avoids fusion with the host endomembrane system, although being studded with many parasite proteins (2). PVM-localized proteins derive from two major organelles: the rhoptries (ROP proteins) and the dense granules (GRA proteins), which are sequentially secreted upon invasion (3). The strategic location of GRAs and ROPs on the PVM positions them to play important roles in interacting with the host.ROP proteins are secreted directly into the host cell cytosol at the time of invasion, after which they target to the PVM or other locations within the cell (4). Although many ROP proteins contain a kinase fold, nearly half of these are predicted to be pseudokinases because they lack the critical catalytic residues that are normally required for phosphate transfer (5). Often, these pseudokinases (i.e., ROP5, ROP8/2, ROP4/7) exist as tandem gene duplications and show evidence of positive selection (5). Following secretion, ROP2 family members are targeted to the cytoplasmic face of the PVM via a series of amphipathic α-helical regions in their N termini (6, 7).ROP proteins generated renewed interest when it became evident that some ROPs confer critical strain-specific virulence in mice (8-12). In particular, the active kinase ROP18 and the pseudokinase ROP5 defend the parasite vacuole by blocking the ...

Innate immunity is an early line of host defense against pathogens. Both gram-positive and gram-negative bacteria and their cell wall components activate the innate immune system of the host and induce secretion of proinflammatory molecules, mainly chemokines and cytokines (1, 5-7, 21, 32). These inflammatory molecules are the main mediators of the pathological effects induced by bacteria, including inflammation, fever, hypotension, leukocytosis, decreased appetite, and arthritis (1,(5)(6)(7)21).We have recently discovered that chemokines are the main proinflammatory mediators induced in monocytes by bacteria and peptidoglycan (PGN) and lipopolysaccharide (LPS), the main cell wall components of gram-positive and gram-negative bacteria, respectively (32). The gene for the chemokine interleukin-8 (IL-8) is the gene most highly induced by all bacterial stimulants out of 600 genes studied (32). However, the mechanism of this induction, i.e., the receptors, signal transduction pathways, and transcription factors involved in the transcriptional activation of the gene for IL-8, are unknown.Gram-positive bacteria and PGN activate cells through the pattern recognition receptors CD14 and Toll-like receptor 2 (TLR2), which results in the activation of the transcription factor NF-B (8,11,27,(29)(30)(31)36). NF-B is a ubiquitous transcription factor that regulates the transcription of various genes involved in immune responses. LPS from gram-negative bacteria also induces activation of NF-B through CD14 and TLR (16,34), and this activation requires the signal transduction molecules myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase (IRAK), tumor necrosis factor (TNF) receptor-associated kinase 6 (TRAF6), NF-B-inducing kinase (NIK), and IB kinase (IKK) (16,30,34,37). LPS stimulation of this pathway results in the activation of IKK (10), which then phosphorylates IB, resulting in its degradation and the subsequent release and translocation of NF-B to the nucleus, where NF-B activates various genes. However, the signal transduction pathway that is activated by gram-positive bacteria and PGN and results in the activation of NF-B is not known and furthermore, the role of NF-B in the induction of the gene for IL-8 (and the genes for other chemokines and cytokines) is also unknown.Therefore, the objectives of this study were to determine whether gram-positive bacteria and their PGN component (i) induce TLR2-dependent transcription of IL-8, (ii) activate the TLR23MyD883IRAK3TRAF63NIK3IKK3NF-B signal transduction pathway, and (iii) activate the gene for IL-8 through this TLR2-mediated pathway. Soluble PGN (sPGN), a polymeric un-cross-linked PGN (average M r ϭ 125,000) released from Staphylococcus aureus grown in the presence of penicillin, was purified by vancomycin affinity chromatography and analyzed as described before (25). Micrococcus luteus ATCC 4698 (obtained from Sigma, St. Louis, Mo.) was used as a prototypic gram-positive nonpathogenic (i.e., easily eliminated by the host innate immune system) bacteri...

Innate lymphoid cells (ILCs) were originally classified based on their cytokine profiles, placing natural killer (NK) cells and ILC1s together, but recent studies support their separation into different lineages at steady-state. However, tumors may induce NK cell conversion into ILC1-like cells that are limited to the tumor microenvironment and whether this conversion occurs beyond this environment remains unknown. Here, we describe Toxoplasma gondii infection converts NK cells into ILC1-like cells that are distinct from both steady-state NK cells and ILC1s in uninfected mice. These cells were Eomes-dependent, indicating that NK cells can give rise to Eomes– Tbet-dependent ILC1-like cells that circulate widely and persist independent of ongoing infection. Moreover, these changes appear permanent, as supported by epigenetic analyses. Thus, these studies markedly expand current concepts of NK cells, ILCs, and their potential conversion.

Toxoplasma gondii is sensitive to bulky pyrazolo [3,4-d] pyrimidine (PP) inhibitors due to the presence of a Gly gatekeeper in the essential calcium dependent protein kinase 1 (CDPK1). Here we synthesized a number of new derivatives of 3-methyl-benzyl-PP (3-MB-PP, or 1). The potency of PP analogs in inhibiting CDPK1 enzyme activity in vitro (low nM IC50 values) and blocking parasite growth in host cell monolayers in vitro (low μM EC50 values) were highly correlated and occurred in a CDPK1-specific manner. Chemical modification of the PP scaffold to increase half-life in the presence of microsomes in vitro led to identification of compounds with enhanced stability while retaining activity. Several of these more potent compounds were able to prevent lethal infection with T. gondii in the mouse model. Collectively the strategies outlined here provide a route for development of more effective compounds for treatment of toxoplasmosis, and perhaps related parasitic diseases.