ABSTRACT Toxoplasma gondii is an intracellular parasite that reconfigures its host cell to promote pathogenesis. One consequence of Toxoplasma parasitism is increased migratory activity of host cells, which facilitates dissemination. Here, we show that Toxoplasma triggers the unfolded protein response (UPR) in host cells through calcium release from the endoplasmic reticulum (ER). We further identify a novel role for the host ER stress sensor protein IRE1 in Toxoplasma pathogenesis. Upon infection, Toxoplasma activates IRE1, engaging its noncanonical role in actin remodeling through the binding of filamin A. By inducing cytoskeletal remodeling via IRE1 oligomerization in host cells, Toxoplasma enhances host cell migration in vitro and dissemination of the parasite to host organs in vivo. Our study has identified novel mechanisms used by Toxoplasma to induce dissemination of infected cells, providing new insights into strategies for treatment of toxoplasmosis. IMPORTANCE Cells that are infected with the parasite Toxoplasma gondii exhibit heightened migratory activity, which facilitates dissemination of the infection throughout the body. In this report, we identify a new mechanism used by Toxoplasma to hijack its host cell and increase its mobility. We further show that the ability of Toxoplasma to increase host cell migration involves not the enzymatic activity of IRE1 but rather IRE1 engagement with actin cytoskeletal remodeling. Depletion of IRE1 from infected host cells reduces their migration in vitro and significantly hinders dissemination of Toxoplasma in vivo. Our findings reveal a new mechanism underlying host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent infection around the body.

Signal transducer and activator of transcription 4 (STAT4) is expressed in hematopoietic cells and plays a key role in the differentiation of T helper 1 cells. Although STAT4 is required for immunity to intracellular pathogens, the T cell-independent protective mechanisms of STAT4 are not clearly defined. In this report, we demonstrate that STAT4-deficient mice are acutely sensitive to methicillin-resistant Staphylococcus aureus (MRSA) infection. We show that STAT4 is expressed in neutrophils and activated by IL-12 via a Jak2-dependent pathway. We demonstrate that STAT4 is required for multiple neutrophil functions including IL-12-induced ROS production, chemotaxis, and production of the neutrophil extracellular traps.Importantly, myeloid-specific and neutrophil-specific deletion of STAT4 results in enhanced susceptibility to MRSA, demonstrating the key role of STAT4 in the in vivo function of these cells. Thus, these studies identify STAT4 as an essential regulator of neutrophil functions and a component of innate immune responses in vivo.

Peanut-induced allergy is an immunoglobulin E (IgE)–mediated type I hypersensitivity reaction that manifests symptoms ranging from local edema to life-threatening anaphylaxis. Although there are treatments for symptoms in patients with allergies resulting from allergen exposure, there are few preventive therapies other than strict dietary avoidance or oral immunotherapy, neither of which are successful in all patients. We have previously designed a covalent heterobivalent inhibitor (cHBI) that binds in an allergen-specific manner as a preventive for allergic reactions. Building on previous in vitro testing, here, we developed a humanized mouse model to test cHBI efficacy in vivo. Nonobese diabetic–severe combined immunodeficient γc-deficient mice expressing transgenes for human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3 developed mature functional human mast cells in multiple tissues and displayed robust anaphylactic reactions when passively sensitized with patient-derived IgE monoclonal antibodies specific for peanut Arachis hypogaea 2 (Ara h 2). The allergic response in humanized mice was IgE dose dependent and was mediated by human mast cells. Using this humanized mouse model, we showed that cHBI prevented allergic reactions for more than 2 weeks when administered before allergen exposure. cHBI also prevented fatal anaphylaxis and attenuated allergic reactions when administered shortly after the onset of symptoms. cHBI impaired mast cell degranulation in vivo in an allergen-specific manner. cHBI rescued the mice from lethal anaphylactic responses during oral Ara h 2 allergen–induced anaphylaxis. Together, these findings suggest that cHBI has the potential to be an effective preventative for peanut-specific allergic responses in patients.

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26Toxoplasma gondii is an intracellular parasite that reconfigures its host cell to promote 27 pathogenesis. One consequence of Toxoplasma parasitism is increased migratory activity of host 28 cells, which facilitates dissemination. Here we show that Toxoplasma triggers the unfolded 29 protein response (UPR) in host cells through calcium release from the endoplasmic reticulum 30 (ER). We further found that host IRE1, an ER stress sensor protein activated during Toxoplasma 31 infection, also plays a noncanonical role in actin remodeling by binding filamin A in infected 32 cells. By inducing cytoskeletal remodeling via IRE1 oligomerization in host cells, Toxoplasma 33 enhances host cell migration in vitro and dissemination of the parasite to host organs in vivo. Our 34 study identifies novel mechanisms used by Toxoplasma to induce dissemination of infected cells, 35 providing new insights into strategies for treatment of toxoplasmosis. 36 37 interactions 39 3 Importance 40Cells that are infected with the parasite Toxoplasma gondii exhibit heightened migratory 41 activity, which facilitates dissemination of the infection throughout the body. In this study, we 42 identify a new mechanism used by Toxoplasma to hijack its host cell and increase its mobility. 43 We further show that the ability of Toxoplasma to increase host cell migration does not involve 44 the enzymatic activity of IRE1, but rather IRE1 engagement with actin cytoskeletal remodeling. 45 Depletion of IRE1 from infected host cells reduces their migration in vitro and significantly 46 hinders dissemination of Toxoplasma in vivo. Our findings reveal a new mechanism underlying 47 host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent 48 infection around the body. 49 50 93 leading to IRE1 oligomerization, association with filamin A, and enhanced cell migration.94 Importantly, the IRE1-associated migration is a crucial determinant for successful dissemination 95 of toxoplasmosis in a mouse model of infection. 96 6 Results 97 Induction of the UPR in Toxoplasma-infected host cells 98 It is currently unclear why intracellular tachyzoites recruit host ER to the parasite PV. To 99 address whether Toxoplasma perturbs host ER homeostasis, we infected mouse embryonic 100 fibroblast (MEF) cells with RH strain parasites and measured three primary markers of the host 101 UPR over a 36-hour time course. Within 12 h of infection, Toxoplasma increased activation of 102 PERK as measured by its self-phosphorylation (PERK-P), induced expression of ATF6 and 103 formation of its cleavage product ATF6-N, and increased levels of the IRE1-derived spliced 104 variant of XBP1 (XBP1s) ( Fig. 1A). It is noteworthy that whereas expression of ATF6-N and 105 XBP1s were transient, with increased amounts of the proteins appearing between 12 to 20 h post-106 infection (hpi), PERK-P increased throughout the 36 h of infection (Fig. 1A). These results 107 indicate that Toxoplasma infection causes ER stress that activates each of the sensory prote...