The Influenced Interaction Between Dendritic Cells And T Lymphocytes By Circadian Rhythms And Temperature Changes And The Relevant Regulation Of Traditional Chinese Medicine

ObjectiveDendritic cells (DCs) are the most important and professional antigen presenting cells.Serving as the important guard of mucosal barrier, DC is also a bridge between the innate andadaptive immune response. DCs sensor the stimulus signals by its pattern recognitionreceptors(PRR) on the surface. And with the help of various kinds of functional andco-stimulating molecules, DCs pass the message to na ve T lymphocytes and mediate thepolarization of various kinds of T subsets. In consideration of the important roles of DCs andT cells, they can be also easily influenced by the outside world. The signal transmissionbetween DC and T cells is so important that it serves as the basis of the results of immuneresponse. The classical Traditional Chinese Medicine Yu-Ping-Feng(YPF) has been widelyused for thousands of years, based on its anti-microbial and anti-viral functions. Thus, ourstudy is focused on the influences of circadian rhythms and temperature changes on thephenotypes of DCs and the interaction between T lymphocytes. And we also investigate themodulating mechanisms of Yu-Ping-Feng on low temperatures induced abnormal interactionbetween DCs and T cells. Methods1. The classical Traditional Chinese Medicine Yu-Ping-Feng(YPF) is strictly combinedby Fang-feng and Huang-qi and Bai-zhu in a proportion of1:2:2. YPF is easily prepared bywater decoction and alcohol deposit.2. The circadian rhythm experiment is carried out by separating the mononuclear cells ofdifferent organs (peripheral blood, spleen, lung and lymph node) at the time point of8AM,12AM,4PM,8PM and12PM. The expressions of MHC class II molecules and co-stimulatorymolecules CD86on DCs are detected by labeling specific fluorescence antibodies in flowcytometry; the intracellular and extracellular double staining of flow cytometry are used tolabel T cells subsets, such as Th1(CD3+CD4+IFN-γ+)、Th2(CD3+CD4+IL-4+)、Th17(CD3+CD4+IL-17+) and Treg (CD4+CD25+foxp3+).3. The BALB/c mice were randomly divided into the groups of control group of25℃,model group at low temperature of10℃, small dose, middle dose and large dose of YuPing-Feng. And the mononuclear cells of spleen, peripheral blood, lungs and lymph nodeswere separated. The expressions of MHC class II molecules, co-stimulatory molecules CD86and chemokine receptor CCR7on DCs are detected by labeling specific fluorescenceantibodies in flow cytometry; the intracellular and extracellular double staining of flowcytometry are used to label T cells subsets: Th1(CD3+CD4+IFN-γ+)、Th2(CD3+CD4+IL-4+)、Th17(CD3+CD4+IL-17+) and Treg(CD4+CD25+foxp3+).4. The transcription levels of CD86, CCR7, IL-4, IL-6, IL-12, transforming growthfactor–β (TGF-β) mRNA were measured by reverse transcription-polymerase chain reaction(RT-PCR).Results1. There exists a diurnal rhythm phenomenon of the molecules on mice DCs and TsubsetsThe results detected by flow cytometry at different time points (8AM，12AM,4PM,8PMand12PM) suggest that the expression of MHC-II, co-stimulating molecule CD86presents adaily circadian phenomenon: they have a base level at8AM, and between8AM and12AM,their expressions actually increase and peaked at12AM. While as time goes on, theexpression has a declining trend and presents a fluctuation within a narrow range at the base levels. All T cell subsets Th1, Th2, Th17and Treg also take on a diurnal rhythm phenomenonand have an accordance with the expression of MHC-Ⅱ and CD86: between8AM and12AM, their expressions actually increase and peaked at12AM. Then it is followed by agradual decline and a low-level of rhythmic fluctuations.2. The expression of molecules on mice DCs and T subsets are decreased at lowtemperatureCompared with the control groups at room temperature, the rectal temperature of modelgroups at low temperature is lower (p<0.05); in all the organs of spleen, peripheral blood,lungs and lymph nodes the expression of MHC-II, CCR7, CD86on the surface of DCs aredecreased (p<0.05), and so do the mRNA level of CCR7, CD86, IL-4, IL-6andTGF-β(p<0.05); the percentage of Th1, Th2, Th17, Treg subsets and the ratios of Th1/Th2,Th17/Treg are also decreased (p<0.05).3. The abnormal interaction between DCs and T cells induced by low temperature can besignificantly reversed by Yu-Ping-FengCompared with the model groups, Chinese traditional medicine Yu-Ping-Feng(YPF)could enhance the expression of MHC-II on DCs, and so do the expression of CCR7,CD86and the corresponding mRNA levels(p<0.05), while the levels of IL-6, IL-12and TGF-βmRNA have no significant difference except for IL-4; YPF has no significant influence onTh1subset, but it can return the ratio of Th1/Th2to normal by lowering the proportion of Th2subset (p<0.05); and furthermore, YPF can increase the proportion of Th17subset butdecrease regulatory T cell Treg subset(p<0.05).Conclusions1. There exists a diurnal rhythm phenomenon of the interaction between mice DCs and Tcells: the expression levels of T cells subsets and phenotypic and functional molecules on DCsin the morning are higher than that in the afternoon. And they go to the peak at12o ’clock,followed by a low-level of rhythmic fluctuations.2. At low temperatures, the expression levels of T cells subsets and the phenotypic andfunctional molecules such as MHC-Ⅱ,CCR7and CD86on DCs are decreased, interactionsbetween DCs and T cell subsets are suppressed. 3. The abnormal interaction between DCs and T cells induced by low temperature can besignificantly reversed by Yu-Ping-Feng. YPF can strengthen the interactions throughincreasing the expression of MHC-II, CCR7, CD86on DCs and the ratio of Th1/Th2,boosting Th17subset, reducing the proportion of Treg.