The TNF-family ligand TL1A and its receptor DR3 promote T cell-mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9-producing T cells.

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

Efficient delivery of the toll-like receptor agonists polyinosinic:polycytidylic acid and CpG to macrophages by acetalated dextran microparticles.

To enhance the immune activity of vaccine adjuvants polyinosinic:polycytidylic acid (poly I:C) and CpG acetalated dextran (Ac-DEX) microparticles can be used. Ac-DEX is a biodegradable and water-insoluble polymer that degrades significantly faster at pH 5.0 (phagosomal pH) than at pH 7.4 and has tunable degradation rates that can range from hours to months. This is an ideal characteristic for delivery of an antigen and adjuvant within the lysosomal compartment of a phagocytic cell. We evaluated poly I:C and CpG encapsulated in Ac-DEX microparticles using RAW macrophages as a model antigen-presenting cell. These cells were cultured with poly I:C or CpG in their free form, encapsulated in a fast degrading Ac-DEX, in slow degrading Ac-DEX, or in the Food and Drug Administration-approved polymer poly(lactic-co-glycolic acid) (PLGA). Ac-DEX had higher encapsulation efficiencies for both poly I:C and CpG than PLGA. Furthermore, poly I:C or CpG encapsulated in Ac-DEX also showed, in general, a significantly stronger immunostimulatory response than PLGA and unencapsulated CpG or poly I:C, which was indicated by a higher rate of nitric oxide release and increased levels of cytokines such as TNF-α, IL-6, IL-10, and IFN-γ. Overall, we have illustrated a method for enhancing the delivery of these vaccine adjuvants to further enhance the development of Ac-DEX vaccine formulations.

Activation of the latent PlcR regulon in Bacillus anthracis.

Many genes in Bacillus cereus and Bacillus thuringiensis are under the control of the transcriptional regulator PlcR and its regulatory peptide, PapR. In Bacillus anthracis, the causative agent of anthrax, PlcR is inactivated by truncation, and consequently genes having PlcR binding sites are expressed at very low levels when compared with B. cereus. We found that activation of the PlcR regulon in B. anthracis by expression of a PlcR-PapR fusion protein does not alter sporulation in strains containing the virulence plasmid pXO1 and thereby the global regulator AtxA. Using comparative 2D gel electrophoresis, we showed that activation of the PlcR regulon in B. anthracis leads to upregulation of many proteins found in the secretome of B. cereus, including phospholipases and proteases, such as the putative protease BA1995. Transcriptional analysis demonstrated expression of BA1995 to be dependent on PlcR-PapR, even though the putative PlcR recognition site of the BA1995 gene does not exactly match the PlcR consensus sequence, explaining why this protein had escaped recognition as belonging to the PlcR regulon. Additionally, while transcription of major PlcR-dependent haemolysins, sphingomyelinase and anthrolysin O is enhanced in response to PlcR activation in B. anthracis, only anthrolysin O contributes significantly to lysis of human erythrocytes. In contrast, the toxicity of bacterial culture supernatants from a PlcR-positive strain towards murine macrophages occurred independently of anthrolysin O expression in vitro and in vivo.

In vitro analysis of acetalated dextran microparticles as a potent delivery platform for vaccine adjuvants.

Toll-like receptor (TLR) agonists induce potent innate immune responses and can be used in the development of novel vaccine adjuvants. However, access to TLRs can be challenging as exemplified by TLR 7, which is located intracellularly in endosomal compartments. To increase recognition and subsequent stimulatory effects of TLR 7, imiquimod was encapsulated in acetalated dextran (Ac-DEX) microparticles. Ac-DEX, a water-insoluble and biocompatible polymer, is relatively stable at pH 7.4, but degrades rapidly under acidic conditions, such as those found in lysosomal vesicles. To determine the immunostimulatory capacity of encapsulated imiquimod, we compared the efficacy of free versus encapsulated imiquimod in activating RAW 264.7 macrophages, MH-S macrophages, and bone marrow derived dendritic cells. Encapsulated imiquimod significantly increased IL-1 beta, IL-6, and TNF-alpha cytokine expression in macrophages relative to the free drug. Furthermore, significant increases were observed in classic macrophage activation markers (iNOS, PD1-L1, and NO) after treatment with encapsulated imiquimod over the free drug. Also, bone marrow derived dendritic cells produced significantly higher levels of IL-1 beta, IL-6, IL-12p70, and MIP-1 alpha as compared to their counterparts receiving free imiquimod. These results suggest that encapsulation of TLR ligands within Ac-DEX microparticles results in increased immunostimulation and potentially better protection from disease when used in conjunction with vaccine formulations.

Bone marrow stromal cells use TGF-beta to suppress allergic responses in a mouse model of ragweed-induced asthma.

Bone marrow stromal cells [BMSCs; also known as mesenchymal stem cells (MSCs)] effectively suppress inflammatory responses in acute graft-versus-host disease in humans and in a number of disease models in mice. Many of the studies concluded that BMSC-driven immunomodulation is mediated by the suppression of proinflammatory Th1 responses while rebalancing the Th1/Th2 ratio toward Th2. In this study, using a ragweed induced mouse asthma model, we studied if BMSCs could be beneficial in an allergic, Th2-dominant environment. When BMSCs were injected i.v. at the time of the antigen challenge, they protected the animals from the majority of asthma-specific pathological changes, including inhibition of eosinophil infiltration and excess mucus production in the lung, decreased levels of Th2 cytokines (IL-4, IL-5, and IL-13) in bronchial lavage, and lowered serum levels of Th2 immunoglobulins (IgG1 and IgE). To explore the mechanism of the effect we used BMSCs isolated from a variety of knockout mice, performed in vivo blocking of cytokines and studied the effect of asthmatic serum and bronchoalveolar lavage from ragweed challenged animals on the BMSCs in vitro. Our results suggest that IL-4 and/or IL-13 activate the STAT6 pathway in the BMSCs resulting in an increase of their TGF-beta production, which seems to mediate the beneficial effect, either alone, or together with regulatory T cells, some of which might be recruited by the BMSCs. These data suggest that, in addition to focusing on graft-versus-host disease and autoimmune diseases, allergic conditions--specifically therapy resistant asthma--might also be a likely target of the recently discovered cellular therapy approach using BMSCs.

The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases.

DR3 (TRAMP, LARD, WSL-1, TNFRSF25) is a death-domain-containing tumor necrosis factor (TNF)-family receptor primarily expressed on T cells. TL1A, the TNF-family ligand for DR3, can costimulate T cells, but the physiological function of TL1A-DR3 interactions in immune responses is not known. Using DR3-deficient mice, we identified DR3 as the receptor responsible for TL1A-induced T cell costimulation and dendritic cells as the likely source for TL1A during T cell activation. Despite its role in costimulation, DR3 was not required for in vivo T cell priming, for polarization into T helper 1 (Th1), Th2, or Th17 effector cell subtypes, or for effective control of infection with Toxoplasma gondii. Instead, DR3 expression was required on T cells for immunopathology, local T cell accumulation, and cytokine production in Experimental Autoimmune Encephalomyelitis (EAE) and allergic lung inflammation, disease models that depend on distinct effector T cell subsets. DR3 could be an attractive therapeutic target for T cell-mediated autoimmune and allergic diseases.

IL-13Ralpha2 and IL-10 coordinately suppress airway inflammation, airway-hyperreactivity, and fibrosis in mice.

Development of persistent Th2 responses in asthma and chronic helminth infections are a major health concern. IL-10 has been identified as a critical regulator of Th2 immunity, but mechanisms for controlling Th2 effector function remain unclear. IL-10 also has paradoxical effects on Th2-associated pathology, with IL-10 deficiency resulting in increased Th2-driven inflammation but also reduced airway hyperreactivity (AHR), mucus hypersecretion, and fibrosis. We demonstrate that increased IL-13 receptor alpha 2 (IL-13Ralpha2) expression is responsible for the reduced AHR, mucus production, and fibrosis in BALB/c IL-10(-/-) mice. Using models of allergic asthma and chronic helminth infection, we demonstrate that IL-10 and IL-13Ralpha2 coordinately suppress Th2-mediated inflammation and pathology, respectively. Although IL-10 was identified as the dominant antiinflammatory mediator, studies with double IL-10/IL-13Ralpha2-deficient mice illustrate an indispensable role for IL-13Ralpha2 in the suppression of AHR, mucus production, and fibrosis. Thus, IL-10 and IL-13Ralpha2 are both required to control chronic Th2-driven pathological responses.

Differential modulation of allergic eye disease by chronic and acute ascaris infection.

PURPOSE: To assess alterations in allergic ocular responses to nonparasite antigens in an experimental system in which mice were skewed toward a Th2 cytokine profile by helminth infection. METHODS: Mice were inoculated with Ascaris suum (A. suum) eggs concurrent with ragweed (RW) sensitization (RW/acute) or by repeated inoculation before RW sensitization (RW/chronic). Control subjects were divided into RW, A. suum, and sham-sensitized groups. Animals were RW-challenged in the eye and examined for changes in ocular responses, inflammatory cell infiltrates, and in vitro assessment of cytokines after antigen restimulation. In subsequent experiments, CD4(+)/CD25+ T regulatory and CD4(+)/CD25- control T cells were adoptively transferred into mice before ocular challenge. RESULTS: RW sensitization and challenge increased ocular symptoms and eosinophil infiltration into the conjunctiva over PBS control eyes. Acute A. suum infection significantly increased RW-induced clinical symptoms and eosinophil infiltrates in the conjunctiva (P = 0.0001) and resulted in the development of anterior uveitis. In contrast, RW/chronic infection provided protection from allergic responses to RW with significantly fewer eosinophils in the eye and reduced eotaxin levels. Transfer of CD4(+)/CD25+ T cells from RW/chronic mice into RW/acute animals also decreased disease intensity, suggesting that T regulatory cells may contribute to protection from allergic eye disease. CONCLUSIONS: The current studies suggest acute parasitic infections exacerbate allergic symptoms, whereas chronic infections offer protection and provide possible explanations for the role of parasitic infection in susceptibility and resistance to nonparasite allergens.

IgE(+), Kit(-), I-A/I-E(-) myeloid cells are the initial source of Il-4 after antigen challenge in a mouse model of allergic pulmonary inflammation.

BACKGROUND: IL-4 is generated within hours after antigen lung challenge and influences events that take place early in the induction of pulmonary inflammation. However, the cells responsible for this early IL-4 production in the lung are unknown. OBJECTIVES: We sought to characterize the initial inflammatory events in the lung after antigen challenge and to identify cells responsible for producing IL-4 at early time points. METHODS: Mice were sensitized with ovalbumin or passive IgE and challenged intranasally. Histologic measures of inflammation were used, and lung tissue cytokine production was analyzed by means of RT-PCR. Cells producing IL-4 were characterized by means of in situ hybridization and flow cytometry. RESULTS: IL-4 mRNA was detectable 100 minutes after challenge in sensitized animals. Blockade of this early IL-4 downregulated vascular cell adhesion molecule 1 mRNA expression and attenuated the early recruitment of eosinophils to the lung. CD4-depleted and mast cell-deficient mice both expressed early IL-4. Cellular analysis revealed the presence of IL-4 protein at 100 minutes exclusively in IgE(+) myeloid cells that did not express CD3, Kit, or I-A/I-E. Moreover, IL-4 production induced by means of passive IgE sensitization and abrogated in FcR gamma-chain-deficient mice supports the conclusion that this IL-4 production is dependent on IgE/gamma-chain interaction. CONCLUSION: IL-4 production by an IgE/gamma-chain-dependent mechanism occurs rapidly after allergen challenge. At these early time points, IL-4 is produced by a myeloid cell with the characteristics of a mouse basophil (IgE(+), Kit(-), I-A/I-E(-)). These data thus suggest that strategies targeting basophils should be considered in the treatment of early lung inflammation.