Heather A. Bruns

Stat4 Regulates Multiple Components of IFN-γ-Inducing Signaling Pathways

Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-γ, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-γ induction has not been established, experiments were conducted to examine Stat4 activation of IFN-γ and other genes required for cytokine-induced expression of IFN-γ. We first examined IL-12 signaling components. Basal expression of IL-12Rβ1 and IL-12Rβ2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-γ production are defective in the absence of Stat4. IL-18 induces minimal IFN-γ production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-κB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-γ production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-γ.

Th1 Cells Regulate Hematopoietic Progenitor Cell Homeostasis by Production of Oncostatin M

Regulation of hematopoietic progenitor cell homeostasis is crucial for maintenance of innate immunity and the ability of the body to respond to injury and infection. In this report, we demonstrate that progenitor cell numbers and cycling status in vivo are dramatically increased in mice deficient in Stat6 and decreased in mice deficient in Stat4, targeted mutations which also alter T helper cell polarization. Experiments using mice that have T cell restricted transgenic expression of Stat4 or Stat6 or have been in vivo depleted of T cell subsets demonstrate that CD4(+) T cells regulate progenitor cell activity. Injection of the Th1 cytokine Oncostatin M but not other cytokines into Stat4-deficient mice recovers progenitor cell activity to wild-type levels. Thus, T helper cells actively regulate hematopoietic progenitor cell homeostasis.

Expression of a Constitutively Active Stat6 In Vivo Alters Lymphocyte Homeostasis with Distinct Effects in T and B Cells

IL-4 is a critical cytokine in the regulation of immune responses and genesis of atopy. Engagement of the IL-4R activates multiple signaling pathways, including the transcription factor Stat6. Stat6-deficient mice demonstrate the importance of this factor in lymphocyte proliferation, gene expression, and Th cell differentiation. Recently, a mutant Stat6 (Stat6VT) was generated that is transcriptionally active independent of IL-4 stimulation. To determine the ability of a constitutively active Stat6 to mimic IL-4-stimulated responses, we have generated transgenic mice expressing Stat6VT under control of the CD2 locus control region, restricting expression to lymphoid populations. The phenotype of Stat6VT transgenic mice is similar, but not identical, to IL-4 transgenic mice, suggesting a critical role for Stat6-independent signaling pathways in the generation of some IL-4 responses in vivo. The expression of a constitutively active Stat6 in vivo increases surface expression of IL-4-induced genes and increases serum levels of IgG1 and IgE, compared with nontransgenic mice. Stat6VT expression increases Th2 differentiation in vivo and in vitro. Stat6VT expression also dramatically alters homeostasis of peripheral lymphocyte populations resulting in decreased CD3+ cells and increased B220+ cells, compared with nontransgenic littermates. Altered T and B cell populations correlate with an activated phenotype and increased cell death in transgenic T cell, but not B cell, populations. Together these results suggest that expression of a constitutively active Stat6 has distinct effects on B and T lymphocytes.

IL-4 Is a Critical Determinant in the Generation of Allergic Inflammation Initiated by a Constitutively Active Stat6

IL-4 is required for the pathogenesis of atopic diseases and immune regulation. Stat6 is critical for IL-4-induced gene expression and Th cell differentiation. Recently, we have generated mice expressing a mutant Stat6 (Stat6VT) under control of the CD2 locus control region that is transcriptionally active independent of IL-4 stimulation. To determine whether active Stat6 in T cells is sufficient to alter immune regulation in vivo, we mated Stat6VT transgenic mice to IL-4-deficient mice. Stat6VT expression in IL-4-deficient lymphocytes was sufficient to alter lymphocyte homeostasis and promote Th2 differentiation in vitro. HyperTh2 levels in Stat6 transgenic mice correlated with an atopic phenotype that manifested as blepharitis and pulmonary inflammation with a high level of eosinophilic infiltration. In the absence of endogenous IL-4, Stat6VT transgenic mice were protected from allergic inflammation. Thus, in mice with hyperTh2 immune responses in vivo, IL-4 is a critical effector cytokine.

IL-4-stimulated NF-κB activity is required for Stat6 DNA binding

IL‐4 is a critical cytokine in the regulation of immune responses. In B lymphocytes, IL‐4 signaling promotes the Stat6‐dependent cell surface expression of several proteins including MHC Class II and CD86. However, the requirement for other transcription factors in IL‐4‐induced B cell gene expression has not been studied extensively. Here, we show that IL‐4 induces NF‐κB p100 processing to NF‐κB p52 in B cells but not in T cells or macrophages. IL‐4 induced NF‐κB p52 production requires PI‐3K activity and correlates with IκB kinase phosphorylation and TNF receptor‐associated factor 3 degradation. Blocking NF‐κB activity eliminates IL‐4‐stimulated gene expression in B cells by reducing IL‐4‐induced DNA binding but not phosphorylation or nuclear localization of Stat6. These results describe a novel role for NF‐κB in IL‐4‐induced signaling and gene expression.

Th17 Cells Demonstrate Stable Cytokine Production in a Proallergic Environment

Th17 cells are critical for the clearance of extracellular bacteria and fungi, but also contribute to the pathology of autoimmune diseases and allergic inflammation. After exposure to an appropriate cytokine environment, Th17 cells can acquire a Th1-like phenotype, but less is known about their ability to adopt Th2 and Th9 effector programs. To explore this in more detail, we used an IL-17F lineage tracer mouse strain that allows tracking of cells that formerly expressed IL-17F. In vitro–derived Th17 cells adopted signature cytokine and transcription factor expression when cultured under Th1-, Th2-, or Th9-polarizing conditions. In contrast, using two models of allergic airway disease, Th17 cells from the lungs of diseased mice did not adopt Th1, Th2, or Th9 effector programs, but remained stable IL-17 secretors. Although in vitro–derived Th17 cells expressed IL-4Rα, those induced in vivo during allergic airway disease did not, possibly rendering them unresponsive to IL-4–induced signals. However, in vitro–derived, Ag-specific Th17 cells transferred in vivo to OVA and aluminum hydroxide–sensitized mice also maintained IL-17 secretion and did not produce alternative cytokines upon subsequent OVA challenge. Thus, although Th17 cells can adopt new phenotypes in response to some inflammatory environments, our data suggest that in allergic inflammation, Th17 cells are comparatively stable and retain the potential to produce IL-17. This might reflect a cytokine environment that promotes Th17 stability, and allow a broader immune response at tissue barriers that are susceptible to allergic inflammation.

Host cell invasion by Staphylococcus aureus stimulates the shedding of microvesicles.

During severe sepsis, microvesicles that are positive for tissue factor (TF) are at increased levels within blood and in pulmonary lavage. These microvesicles potentially disperse TF, the major initiator of the coagulation cascade, throughout multiple organ systems, initiating fibrin deposition and resultant ischemia. The source of these microvesicles has remained incompletely defined. Although TF(+) microvesicles are shed from cells that express nascent TF transcript in response to injury, recent findings revealed that circulating, full-length TF protein is detectable prior to these nascent transcripts. This finding suggested that the protein is released from constitutive sources as an acute response. We examined whether Staphylococcus aureus, the Gram-positive bacteria that is emerging as one of the most common etiologic agents in sepsis, is capable of stimulating the release of TF(+) microvesicles from a pulmonary cell line that constitutively expresses TF protein. We found that host cell invasion stimulated an acute release of TF(+) microvesicles and that these microvesicles mediated the transfer of the protein to TF-negative endothelial cells. We also found that transfer was inhibited by cholesterol-lowering simvastatin. Taken together, our findings reveal that S. aureus pathogenesis extends to the acute release of TF(+) microvesicles and that inhibiting dispersal by this mechanism may provide a therapeutic target.

Wheezing and itching: The requirement for STAT proteins in allergic inflammation

The development of allergic inflammation requires the orchestration of gene expression from the inflamed tissue and from the infiltrating immune cells. Since many of the cytokines that promote allergic inflammation signal through hematopoietin family receptors, the Signal Transducer and Activator of Transcription (STAT) family have obligate roles in pro-allergic cytokine-induced gene regulation in multiple cell types. In this review, we summarize work defining the contribution of each of the STAT family members to the development of allergic inflammation, using data from mouse models of allergic inflammation, studies on patient samples and correlations with single nucleotide polymorphisms in STAT genes.

GTPase activating protein function of p85 facilitates uptake and recycling of the β1 integrin

Beta1-containing adhesions at the plasma membrane function as dynamic complexes to provide bidirectional communication between the cell and its environment, yet commonly are used by pathogens to gain host cell entry. Recently, the cholesterol-lowering drug simvastatin was found to inhibit host invasion through beta1-containing adhesion complexes. To better understand the regulatory mechanisms controlling adhesion formation and uptake and the use of these complexes by Staphylococcus aureus, the primary etiologic agent in sepsis, bacteremia and endocarditis, we investigated the mechanism of inhibition by simvastatin. In response to simvastatin, adhesion complexes diminished as well as beta1 trafficking to the plasma membrane required to initiate adhesion formation. Simvastatin stimulated CDC42 activation and coupling to p85, a small-guanosine triphosphatase (GTPase) activating protein (GAP), yet sequestered CDC42 coupled to p85 within the cytosol. Loss of p85 GAP activity through use of genetic strategies decreased host cell invasion as well as beta1 trafficking. From these findings, we propose a mechanism whereby p85 GAP activity localized within membrane compartments facilitates beta1 trafficking. By sequestering p85 within the cytosol, simvastatin restricts the availability and uptake of the receptor used by pathogenic strains to gain host cell entry.

Small Molecule Inhibitors Limit Endothelial Cell Invasion by Staphylococcus aureus

Staphylococcus aureus is a leading causative agent in sepsis, endocarditis, and pneumonia. An emerging concept is that prognosis worsens when the infecting S. aureus strain has the capacity to not only colonize tissue as an extracellular pathogen, but to invade host cells and establish intracellular bacterial populations. In previous work, we identified host CDC42 as a central regulator of endothelial cell invasion by S. aureus. In the current work, we report that ML 141, a first-in-class CDC42 inhibitor, decreases invasion and resultant pathogenesis in a dose-dependent and reversible manner. Inhibition was found to be due in part to decreased remodeling of actin that potentially drives endocytic uptake of bacteria/fibronectin/integrin complexes. ML 141 decreased binding to fibronectin at these complexes, thereby limiting a key pathogenic mechanism used by S. aureus to invade. Structural analogs of ML 141 were synthesized (designated as the RSM series) and a subset identified that inhibit invasion through non-cytotoxic and non-bactericidal mechanisms. Our results support the development of adjunctive therapeutics targeting host CDC42 for mitigating invasive infection at the level of the host.