| BackgroundWHO had pointed "allergic disease would be one of the biggest challenges for human health in21-century". Allergic rhinitis belongs to allergic disease. The pathophysiology of allergic rhinitis is occurred in nasal mucosa and originated from systemic immune abnormalities. After in contact with allergens, TH2type immune responses were started by IgE mediated eosinophil reaction and dominated by a variety of inflammatory cells and cytokines involved in. The common features of allergic disease were so-called TH2type immune response.In our knowledge of TH2type immune responds, many kinds of cells take part in this sophisticated network of immune responds. There are included T cell, B cell, Eosinophils, Basophil, APC, innate helper cell and so on. Type2responses are characterized by the induction of CD4+T helper (TH)2cells, which secrete cytokines such as interleukin-4(IL-4), IL-5, IL-9, and IL-13. TH2cells promote B cell responses and immunoglobulin E (IgE) secretion through their production of IL-4. IgE immune complexes bind to high-affinity IgE receptors (FceRl) on basophils and mast cells, leading to their activation and secretion of several cytokines and inflammatory mediators such as histamine, heparin, and serotonin. These factors mediate a range of effector functions characteristic of type2inflammation, including recruitment of alternatively activated macrophages and granulocytes, smooth muscle contractility, and mucus hypersecretion. There are four stages in the courses of the formation of TH2type immune responses in allergic rhinitis. There are so-called immune recognition, sensitization, immune responses and immune maintenance. At the same time, mucosa immune and system immune are all involved in TH2type immune responses. A marked feature of type2responses is their diversity. There is diversity in terms of the array of stimuli that trigger type2responses, the mechanisms by which the innate immune system senses such stimuli, and the cellular and molecular pathways that orchestrate the response. Indeed, there even appears to be diversity in the cytokine profiles of responding cells in a TH2cell response. In summary, although many attentions have been pay in the field of TH2type immune responses, but the mechanisms that initiate and control type2responses remain enigmatic.Allergen-specific immunotherapy has been used more than100years as a desensitizing therapy for allergic diseases and represents the potentially curative and specific method of treatment. Specific immunotherapy is the only treatment modality with capacity of change the natural course of allergic diseases. According to the guideline of ARIA, allergen specific immunotherapy is referred to consistent moderate-severe allergic rhinitis. Now days, specific immunotherapy are applied in clinic treatment for allergic rhinitis with two ways, subcutaneous immunotherapy and sublingual immunotherapy. Although many big samples randomized double-blind placebo-controlled trails have confirmed the clinic efficiency of SIT in patients with allergic rhinitis. But the mechanism of SIT is unclear. Many conclusions about SIT were originated from SCIT. SLIT was first applied in clinic treatment in1986. Compared with the consensus about SCIT, many disputations are still existed about SLIT. The mainly parts are clinic efficiency and mechanism. Allergen specific sublingual immunotherapy has recently received much attention around the world as a treatment for allergic rhinitis and is now widely used to replace the subcutaneous route. To date, many randomized double-blind placebo-controlled trials have confirmed the clinic efficiency of SLIT for patients with allergic rhinitis.The knowledge of SLIT is mainly originated from Europe study team and the patients received SLIT are mainly with seasonal allergic rhinitis sensitive to grass or pollen allergen. Although clinic efficiency of SLIT have already been confirmed in seasonal allergic rhinitis. But for consistent moderate-severe allergic rhinitis, whether house dust mite sublingual immunotherapy would possess the same result or not were uncertain. On account of new progressions of SLIT and the finds of innate immune system, increasing interest in SLIT has immerged in recent years.The recent identification of previously unrecognized innate cell populations, termed innate helper cell. Studies show that innate helper cell has ability to induce TH2type immune responses by directly secreting TH2type cytokine. Thymic stromal lymphopoietin (TSLP), a distant paralog of IL-7, is a type I cytokine that is part of the IL-2cytokine family. TSLP are produced primarily by epithelial cell in airway during airway’s allergic inflammation. Recently study shows that TSLP can induce a robust expansion of human TH2memory cells while maintaining their central memory phenotype and TH2commitments. OX40L and its receptor OX40are members of the TNF and TNF receptor super families, respectively. OX40L is expressed by APCs, endothelial cells and T cells, whereas OX40is found mainly on activated T cells. The expression of OX40L on dendritic cells is mainly induced by thymic stromal lymphopoietin. Many factors of promoting mature of APC could improve the expression of OX40L. TSLP has ability to induce naive T cell difference into TH2cell by polarizing dendritic cells and this role is accomplice by inducing the expression of OX40L on the surface of dendritic cells. New results demonstrated the critical roles of OX40/OX40L interaction on the maintenance and re-activation of TH2memory responses during TSLP-mediated allergic responses. Targeting the TSLP-induced inflammatory TH2cells or allergen-specific TH2memory cells by blocking OX40/OX40L interaction may be one of the new therapeutic approaches for the prevention and treatment of human-allergic diseases. The important roles of TSLP-OX40L signal in TH2type immune responses have been reported in many allergic disease trials. The previous studies had demonstrated that TSLP-OX40L signal play a crucial role in pathogenesis of allergic disease. Until now, there is no research to concern the influence of house dust mite sublingual immunotherapy in TSLP-OX40L mediated TH2type Immanuel responds in patient with allergic rhinitis. Memory TH2cell is main cell mass that could maintain chronic inflammation stage in allergic responses and insure allergic responses reappearance when allergen was exposure again. The expression of TSLP can conduce to maintain the function of memory TH2cell. So TSLP-OX40L signal would become a potential molecular target in treatment of allergic diseases.In conclusion, TSLP-OX40L signal has become a hot spot in treatment of allergic diseases. The traditional medicine treatment strategy is mainly focused on the effector phase of allergic responses. Its role is mainly on been activated cells, such as T cell, Eosinophils and Basophil. Since the ability of SLIT to regulate TH2-type immune response is characterized by mucosal route, and TH2-type immune response mediated by TSLP-OX40L signaling pathway is also in the mucosal start-up phase, thereby causing the body’s immune cascade series. Whether there exist interaction between house dust mite allergen SLIT and TH2type immune response mediated by TSLP-OX40L signal or not, is worth the wait. This is the main purpose of this study to investigate. But until now, no related reports were published.ObjectiveTo investigate the impact of house dust mite allergen sublingual immunotherapy on TSLP-OX40L mediated TH2type immune responds in patient with moderate to severe allergic rhinitis, including changes in local mucosal immune of nasal mucosa and the system immune. Combined with changes in the patients clinic symptoms of observation and assessment, further analysis of the impact of dust mite allergen SLIT on TH2-type immune response in patients with moderated to severe allergic rhinitis and mechanism were carried out. We look forward to provide guidance and assistance for the clinical treatment of dust mite SLIT and the research of mechanism of SLIT.MethodsIn this study, according to the guideline of ARIA,46cases of allergic rhinitis patients in southern China were enrolled in our study. All of the patients had moderate to severe persistent allergic rhinitis with or without asthma. All patients received house dust mite allergen specific sublingual immunotherapy. The cut-off point of trials is12months immunotherapy. The case-control study is employed in trials. Each patient was treated for a12-month period. Mixture extracts of D.f. and D.p.(Zhejiang Wolwo Bio-Pharmaceutical Co., Ltd., China) were used in our study. Changes of TH2-type immune response and clinical symptoms in patients with allergic rhinitis in our trials were dynamic observed and analyses respectively at the times before the start of immune therapy and immunotherapy for12months. First, the characteristics and rules of changes related to clinical symptoms were summarized within12months’dust mite allergen SLIT; On the basis of the progress in TH2-type immune response and sublingual allergen-specific immunotherapy, TSLP-OX40L signaling pathway was selected as a starting point for further analysis of the changes both occurred in the nasal mucosa immune and system immune by impact of SLIT. There are included the expressions of IL-4, IL-13, INF-γ and TSLP in nasal lavage detected by ELISA; The expressions of T-bet mRNA, GATA-3mRNA and OX40L mRNA in blood detected by QRT-PCR; The expressions of OX40L in PBMC detected by flow cytometric analysis; The expression of TSLP detected by immunohistochemistry and the counts of EOS in nasal secretion smear detected by HE staining. From changes in the above objective indicators, we can deduce whether TH2-type immune response mediated by TSLP-OX40L signaling pathway would be changed or not, including the mucosa immune response and system immune response. Finally, the results of clinical observations and laboratory tests are analyzed.ResultsOutcomes of syndrome scoreAfter12-month treatment, TNSS, medication score and all INSS (nasal rhinorrhea, itching, sneezing and nasal congestion) significantly decreased compared with baseline value by overall evaluation (P<0.001). TNSS significantly decreased after12-month SLIT compared with the baseline value from9.065±1.162to4.413±0.717(X2=82.667, P<0.001); Medication score decreased from2.870±0.341to1.109±0.315(X2=79.590, P<0.001). Among all INSS, itching score decreased from2.391±0.493to1.022±0.257(X2=86.681, P<0.001); nasal congestion score decreased from2.000±0.633to1.196±0.401(X2=60.057,P<0.001); sneezing score decreased from2.457±0.504to1.109±0.315(X2=74.146,P<0.001);nasal rhinorrhea score decreased from2.217±0.417to1.087±0.354(X2=83.532, P<0.001). All show significant difference. The results showed that a significant improvement of clinical symptoms for patients with moderate to severe persistent allergic rhinitis had achieved after12-month dust mite allergen SLIT treatment. The dependence on drugs for all patients was reduced significantly. No serious adverse events and no symptoms aggravated case were reported during12-month SLIT treatment, symptoms aggravated case report.Additionally, by dynamic observation of clinic syndromes, the changes of TNSS showed a gradual decline trend during12-month dust mite allergen SLIT. Among this trend, TNSS decreased significantly after1-month dust mite allergen SLIT from9.065±1.162to7.132±1.554(Z=-5.264, P<0.001). During the first6-month dust mite allergen SLIT, TNSS declined obviously and showed a steep drop trend from9.065±1.162to4.935±1.063. The difference was statistically significant(Z=-5.962, P <0.001). But during the second6-month SLIT, the changes of TNSS were relatively stable and the trend of TNSS presented a platform-type curve. The difference was not statistically significant(Z=-2.449, P=0.014>0.0125)(The significance level in here needed to be adjusted to P<0.0125for the statistical significant difference). Until to the endpoint of12-month SLIT, TNSS had been declined50%compared with baseline. The changes of INSS during12-month dust mite allergen SLIT were basically as same as TNSS. Only the differences of nasal congestion score and medicine score compared at the time of6-month SLIT and12-month SLIT were statistical significant(P<0.0125)(significance level was adjusted). The study proved the safety and efficiency of dust mite allergen SLIT.The results also showed that the trend of syndromes improvement was gradual and wavy during12-month dust mite allergen SLIT period. The wavy or the transient rebound of syndromes presented mainly mainly at the times after3-7months SLIT treatment. All of the transient rebounds of TNSS were less than baseline.Outcomes of laboratory parameter HE staining:The counts of EOS in nasal secretion smear were decreased significantly by overall evaluation(Cochran’s Q=23.330, P<0.001) after12-month dust mite allergen SLIT. After that, pair-wise comparisons of the counts of EOS in nasal secretion smear were carried respectively at the three time points (baseline,6-month and12-month) to evaluate the differences. The results showed that the counts of EOS in nasal secretion smear at the times of6-month SLIT and12-month SLIT decreased significantly compared with baseline(6-month SLIT: Z=-3.162, P<0.0125;12-month SLIT:Z=-3.873, P<0.001). But there were no statistical difference of the counts of EOS in nasal secretion smear between6-month SLIT with12-month SLIT(Z=-2.236,P>0.0125).ELISA:The levels of IL-4, IL-13, INF-y and TSLP in nasal lavage were decreased significantly after12-month dust mite allergen SLIT (P<0.05). The level of IL-4decreased from76.010±9.394(pg/ml) to32.910±8.752(pg/ml)(t=27.010, P <0.05); The level of IL-13decreased from55.68±12.86(pg/ml) to30.000±9.453(pg/ml)(t=14.050, P<0.05); The level of INF-y decreased from30.600±6.755(pg/ml) to21.710±5.457(pg/ml)(t=8.087, P<0.05); The level of TSLP decreased from36.390±6.610(pg/ml) to21.780±4.231(pg/ml)(t=11.110, P<0.05). The ratio of IL-4/INF-γ decreased from2.584±0.581tol.611±0.593. The difference was statistically significant (t=8.987, P<0.05).The expressions of TIgE and sIgE in serum didn’t showed significant changes compared with baseline (TIgE:423.900±29.990(kU/L) vs420.200±39.110(kU/L); slgE:54.920±5.217(kU/L)vs56.790±6.750(kU/L)(P>0.05).QRT-PCR:The expressions of T-bet mRNA in blood significantly increased from3.458±0.6202to5.389±1.228fold after12-month SLIT compared with baseline (t=12.230, P<0.05). The expressions of GATA-3mRNA in blood significantly decreased from12.440±2.536fold to7.246±1.953fold after12-month SLIT compared with baseline (t=11.920, P<0.05). The ratio of T-bet mRNA/GATA-3mRNA increased significantly from0.288±0.071to0.785±0.251(t=13.890, P<0.05). The level of OX40L mRNA in blood decreased significantly after12-month SLIT from1.401±0.361fold to0.772±0.301fold compared with baseline (t=12.300, P<0.05). The negative associations of the levels of OX40L mRNA and the ratios of T-bet mRNA/GATA-3mRNA in blood were observed before and after12-month SLIT(before SLIT:r=-0.440, P<0.05;after12-month SLIT:r=-0.565, P<0.05). The difference was statistically significant. The positive associations of the levels of OX40L mRNA in blood and the levels of TSLP in nasal lavage fluid were observed before and after12-month SLIT(before SLIT:r=0.491, P<0.05; after12-month SLIT:r=0.548, P<0.05). The difference was statistically significant.Flow cytometry:The expressions of OX40L on the surface of peripheral blood mononuclear cells after12-month SLIT were decreased significantly (t=13.100, P<0.05), from before treatment (2.712±0.511)%, down to12-month treatment (1.630±0.471)%. The expressions of OX40L on the surface of CDllc+CD86+cells in peripheral blood mononuclear cells also significantly decreased after12-month SLIT from the former (5.839±2.841)%, reduced to (2.098±0.870)%. The difference was statistically significant (t=9.946, P<0.05).Immunohistochemical staining:There were6cases of nasal mucosa biopsies be obtained to study the HE staining and the expression of TSLP before and after12-month SLIT. The results showed that positive staining of TSLP in nasal mucosa was weakened after12-month SLIT compared with baseline from83.33%to33.33%. From the HE staining, the results showed that epithelial cells arranged closely and eosinophil infiltration under the epithelium decreased after12-month SLIT.Conclusion 1. Dust mite allergen SLIT treatment for patients with moderate to severe persistent allergic rhinitis has significant clinical efficacy and safety. After12-month dust mite allergen SLIT, the TNSS was about to decrease50%compared with before immunotherapy.2. During12-month dust mite allergen SLIT, the remission of clinical symptoms showed a gradual improvement trend. An obviously clinic improvement was found in the first6-month dust mite allergen SLIT. The changes of clinic symptoms showed a steep drop trend in the first six months. During the second six months, the changes of clinic symptoms were relatively stable and showed a platform-type curve. The transient rebounds of syndromes were presented in some patients mainly at the times after3-7months SLIT treatment. All of the transient rebounds of TNSS were less than baseline. Overall clinic symptoms showed a waving decline trend.3. The significant clinical efficacy could be achieved in early stage of dust mite allergen SLIT (one month), earlier than the onset time being expected.4. TH2type immune responses in nasal mucosa immune were down-regulated after12-month dust mite allergen SLIT in patients with moderate to severe persistent allergic rhinitis. It was showed that the intensity of TH2type immune responses was weakened and the balance of TH1/TH2immune responses was shifted to TH1.5. TH2type immune responses in system immune were down-regulated after12-month dust mite allergen SLIT in patients with moderate to severe persistent allergic rhinitis. It was showed that the level of OX40L which was in charge of immune memory of TH2type immune responses was down-regulated and the balance of TH1/TH2immune responses was shifted to TH1.6. The down-regulation of OX40L in system immune, TSLP in mucosa immune and TH2type immune responses in both of system and mucosa system implied the signaling pathway conducted by TSLP-OX40L in TH2type immune responses was down-regulated after12-month dust mite allergen SLIT.7. TH2type immune responses both in mucosa immune and in system immune were down-regulated by12-month dust mite allergen SLIL in patients with moderate to severe persistent allergic rhinitis.8. The achievement of clinic efficiency in early stages of dust mite allergen SLIT was derived from the down-regulation of TH2immune responses in nasal mucosa immune. While the maintaining the clinical efficacy was derivered from the down-regulation of OX40L in system immune.9. The expressions of TSLP could be a predictor of the down-regulation of TH2type immune responses in system immune responses for patients with moderate to severe persistent allergic rhinitis after being received house dust mite SLIT. |
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