Preliminary Study On The Allergic Response And Molecular Mechanism Of Dendritic Cells To Recombinant German Blattella

Studies on asthmatic patients and animal models of allergy have demonstratedthat Th2cells produce cytokines IL-4, IL-5and IL-13, and thus contribute todevelopment of asthma, including airway inflammation, mucus hypersecretion, andairway hyperresponsiveness. However, the mechanisms through which naive T cellsdifferentiate to Th2cells in response to allergens remain unclear.Dendritic cells (DCs) are a group of cells which possess the ability in dictatingna ve CD4+T cells to differentiate to either Th1or Th2cells depending on ambientmicroenvironment. It is still an enigma how antigen-specific Th2cells get skewedpolarization and how they maintain a dominant status in allergy. Recent studies haveshowed that DCs express T cell immunoglobulin mucin domain (TIM)4that ligatesTIM1on Th2cells to promote Th2cell development. The report has shed a new lighton the study of the mechanism of allergic diseases.TIM is a new family of cell surface proteins that are potentially involved in theregulation of effector T cell responses. Accumulated data suggest that several TIMmolecules play critical roles in the regulation of Th1and Th2immune responses.Among them, DC-derived TIM4has been found to be able to drive CD4+T cells into Th2cells. It has also been observed that the expression of TIM4can be up-regulateddramatically upon activation, but little is known of actions of allergens in up-regulationof expression of TIM4in DCs. Toll-like receptors are a crucial family of conserved pattern recognition receptors wellknown for their roles in recognizing specific microbial patterns and allowing the hostcells to distinguish between self and non-self molecules. Several Toll-like receptors(TLR) such as TLR2, TLR4are expressed by DCs. The activation of TLRs bymicrobial ligands triggers innate immune responses and primes antigen-specific adaptiveimmunity toward exogenous pathogens. Relatively recent appreciation of the ability ofTLRs to link innate and adaptive immunity offers a new prospect to consider agonists thatengage TLR signaling in allergy development.Cockroaches have been described to trigger allergy and especially asthma indifferent parts of the world. The most common cockroach species, which arefrequently found in homes, are B. germanica and Periplaneta Americana (P.Americana). Over the past decade, seven allergens of B. germanica (Bla g1, Bla g2,Bla g4, Bla g5, Bla g6, Bla g7and Bla g8) have been cloned. Among them, Bla g7is a tropomyosin, and a highly cross-reactive pan-allergen with serum IgE. SinceBla g7has very similar structure to Per a7, a tropomyosin from P. Americana, whichhas previously been reported to activate the Th2cytokine IL-4and IL-13secretionfrom P815mast cell line, we anticipated Bla g7may also be able to stimulate Th2cytokine release from other immune cells. As DCs are the most powerful antigenpresenting cells, which can dictate the polarization of Th2cells and lead to intestinalallergy, we investigated the effect of Bla g7on DCs and its potential mechanisms. The aim of the study is to demonstrate the likelihood that Bla g7might act on DCs topromote TIM4expression in DCs, and subsequently drive na ve CD4+T cells todifferentiate into Th2cells.PART1Cockroach allergen Bla g7promotes TIM4expressionin dendritic cells leading to Th2PolarizationBackground: As a major source of indoor allergens, cockroach causes perennialrhinitis and asthma, but the mechanisms of cockroach allergy remain obscure.Methods: To investigate effects of Bla g7on TIM4expression and cytokinerelease from Dendritic cellss and the immune-modulatory effects of Bla g7on DCscontrolling Th cell polarization. DCs were challenged by recombinant Bla g7(rBla g7), and expression of TIM4mRNA and protein was assessed mainly by real timePCR and Western blotting. The polarization of CD4+T cells in co-culture with rBla g7-activated DCs was assessed by flow cytometry analysisResults: The results showed that expression of TIM4, CD80and CD86wasupregulated when DCs were incubated with recombinant Bla g7(rBla g7). rBla g7induced secretion of IL-13from DCs through TIM4, CD80and CD86dependentmechanism. Co-culture of rBla g7-activated DCs with isolated CD4+T cells inducedTh2polarization of Th cells as showed by enhanced ratio of IL-4+cells vs. IFN-γ+ cells on flow cytometry analysis, and elevated level of IL-13in the culturesupernatant. Antibodies against TIM4, CD80and CD86inhibited rBla g7-inducedenhancement of ratio of IL-4+vs. IFN-γ+cells and IL-13release, indicating rBla g7-induced Th2polarization is also via TIM4, CD80and CD86dependent mechanism.Conclusion: Bla g7plays an important role in the development of cockroachallergy through modulation of cytokine release from DCs, and induction ofDC-dictated Th2polarization of CD4+T cells. PART2Toll-like Receptor4Is Essential for Cockroach Allergen blag7to Activate Dendritic Cells and Induce cytokines releaseThrough MAPK Signaling Pathways.Background: Cockroaches have been described to trigger allergy and especiallyasthma. Toll-like receptors (TLRs) play important roles in initiation of innate andadaptive immune responses. We hypothesis that Bla g7could play a TLR4agonistand interact with TLRs to play a role in allergy. This study aimed to investigate thethe underlying mechanism between Bla g7and TLR4on the allergy.Methods: The purified recombinant Bla g7(rBla g7) was used to challenge DCs at various concentrations and the expression of CD80, CD86, HLA-DR andTLR4was determined by using flow cytometry. The protein level of TLR4wasmeasured by Western-blotting. T cells co-cultured with Bla g7activated DCs andproliferation was assessed by CCK-8. The release of cytokines was detected withELISA. Signaling pathways of Mitogenactivated protein kinases (MAPKs) which arenecessary for transmitting a TLR-triggered immune were measured by Western-blotting.The involvement of MAPKs was studied using specific inhibitors.Results: Separated DCs were cultured in the presence of rBla g7at gradient doses.Exposure to rBla g7significantly increased the MFI of MHC II and costimulatorymolecules. Approximately up to40.5%up-regulated expression of TLR4wasobserved when cells were incubated with rBla g7. Co-immunoprecipitationdemonstrated TLR4expressed on the DCs is capable of binding rBla g7.And we foundthat Bla g7binds directly to TLR4on the surface of DCs, activates MAPK and NF-κBpathways, and promotes production of IL-4，IL-5以及IL-13Conclusion: These results demonstrate that TLR4is a key receptor mediating theinteraction of Bla g7with DCs and subsequently induced cytokine production.