Browsing by Author "Alakhras, Nada S."

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    Cell-Type Specific Function of STAT4 in an Animal Model of Multiple Sclerosis
    (2023-12) Alakhras, Nada S.; Kaplan, Mark H.; Cook-Mills, Joan; Dong, X. Charlie; Quilliam, Lawrence A.
    Signal transducer and activator of transcription 4 (STAT4) is a critical regulator of inflammation. STAT4 promotes protective immunity and autoimmunity downstream of pro-inflammatory cytokines including IL-12 and IL-23. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), germ-line deletion of STAT4 in mice results in resistance to the development of inflammation and paralysis. In parallel, genome-wide association studies (GWAS) have identified polymorphisms in the STAT4 gene associated with susceptibility to several autoimmune diseases including MS demonstrating a potential role for STAT4 in human autoimmunity. Here, we examined cell-type requirements for STAT4 in EAE. Using conditional Stat4 mutant mice, we found that mice lacking Stat4 in T cells and CD11c+-expressing cells are resistant to EAE, while mice lacking Stat4 in Lyz2+-expressing cells are susceptible to EAE. STAT4 is expressed and activated in CD11c+ dendritic cells (DCs) in the CNS during peak disease severity. Stat4fl/flCD11cCre mice exhibit significantly decreased classical dendritic cell (cDC) expansion in the CNS and this correlates with diminished numbers of infiltrated T cells in the CNS and decreased inflammatory cytokine production. Adoptive transfer of wild type but not Stat4-/- or Il23r-/- DCs into Stat4fl/flCD11cCre rescues the development of EAE. Transferred Il23r-/- DCs were retained in the lymph nodes suggesting that IL-23-STAT4 signaling promotes their migration to and expansion in the CNS. Single-cell RNA-seq analyses of CNS DCs from WT and Stat4fl/flCD11cCre mice identified cDC populations with STAT4-dependent gene expression and migratory phenotypes. Collectively, our results demonstrate that STAT4 in cDCs is required for expansion in the CNS, the development of encephalitogenic T cells, and the clinical symptoms of EAE. Thus, our study reveals previously unrecognized functions of STAT4 in cDCs that provide mechanistic insight into CNS autoimmunity and provide a foundation for identifying new therapeutic targets for the disease.
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    Peanut Allergen-specific Inhibition of Anaphylaxis in a Humanized Mouse Model
    (American Association for the Advancement of Science, 2023) Alakhras, Nada S.; Shin, Jaeho; Smith, Scott A.; Sinn, Anthony L.; Zhang, Wenwu; Hwang, Gyoyeon; Sjoerdsma, Jenna; Bromley, Emily K.; Pollok, Karen E.; Bilgicer, Basar; Kaplan, Mark H.; Biochemistry and Molecular Biology, School of Medicine
    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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    STAT3 Signaling Heterogeneity Underlies Cytokine-Expressing Fate in Th17 Cultures
    (The American Association of Immunologists, 2023) Niese, Michelle L.; Glosson-Byers, Nicole; Moreira Serezani, Ana Paula; Alakhras, Nada S.; Kaplan, Mark H.; Microbiology and Immunology, School of Medicine
    The polarization of naive Th cells into differentiated subsets in vitro was a powerful approach to define the development and function of Th cells in vivo. Th cell cultures identified cytokines that promote polarization and defined the phenotype and stability of differentiated cells. One of the limitations of this approach is the heterogeneity of the differentiated culture, essentially with regard to what proportion of the culture is secreting the hallmark cytokine of interest. This heterogeneity has always been puzzling because all cells in the culture have been exposed to identical culture conditions. We examined this phenomenon using an Il17f lineage-tracing allele (Cost, Cre on seventeen transcript) crossed to stop-flox Rosa-YFP (yellow fluorescent protein) mice. We found that less than half of the cells in a Th17 culture become lineage-positive during a differentiation culture and that it is primarily cells that are lineage-positive that produce cytokines when cultures are restimulated after differentiation. We sorted and analyzed YFP-positive and YFP-negative cells and found similar expression of many Th17 transcription factors, although YFP-negative cells had increased expression of other lineage-defining transcription factors. We observed that YFP-negative cells had diminished expression of Stat3 and Il6ra, as well as decreased STAT3 activation. YFP-negative cells transduced with active STAT3 had significant increases in IL-17A expression, without increases in Th17 transcription factors. Taken together, these data suggest that there is a threshold of STAT3 activation that is required for efficient Th17 differentiation, and that even in a culture of homogeneous naive T cells there is heterogeneity in the receipt of early cytokine signals.
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    STAT4 is expressed in neutrophils and promotes antimicrobial immunity
    (American Society for Clinical Investigation, 2021-07-22) Mehrpouya-Bahrami, Pegah; Moriarty, Alina K.; De Melo, Paulo; Keeter, W. Coles; Alakhras, Nada S.; Nelson, Andrew S.; Hoover, Madeline; Barrios, Maria S.; Nadler, Jerry L.; Serezani, C. Henrique; Kaplan, Mark H.; Galkina, Elena V.; Microbiology and Immunology, School of Medicine
    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 were acutely sensitive to methicillin-resistant Staphylococcus aureus (MRSA) infection. We show that STAT4 was expressed in neutrophils and activated by IL-12 via a JAK2-dependent pathway. We demonstrate that STAT4 was 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 resulted 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.
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    Toxoplasma gondii Co-opts the Unfolded Protein Response To Enhance Migration and Dissemination of Infected Host Cells
    (American Society for Microbiology, 2020-07-07) Augusto, Leonardo; Martynowicz, Jennifer; Amin, Parth H.; Alakhras, Nada S.; Kaplan, Mark H.; Wek, Ronald C.; Sullivan, William J., Jr.; Biochemistry and Molecular Biology, School of Medicine
    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.