Yu-Ping-Feng-San Constrains Allergic Inflammation By Dampening The "Memory-Like" And Interorgan Migration Properties Of ILC2s

Allergic diseases like allergic asthma are induced by systemic type 2 inflammatory reaction to allergens resulting from the complex interplay between our immune system and environmental factors.Previously,our group has found that the traditional Chinese prescription Yu-Ping-Feng-San(YPFS),as well as the active ingredients like cimifugin,are capable of alleviating house dust mite(HDM)-induced asthma recurrence and fluorescein isothiocyante(FITC)-induced allergic contact dermatitis(ACD)recurrence.And the therapeutic effect of YPFS administered during remission is even more efficient than some clinically used drugs like corticosteroid.Furthermore,YPFS ameliorates allergic inflammatory reactions partially by restoring epithelial barrier,which is the very first step to exert its potency.Thus,the next question we need to address is what the targeting effector cells are involved in the unique therapeutic efficacy of YPFS.Generally,antigen-specific memory T cells are considered as the pivotal effector cells during the relapse of allergic inflammation.More recent works have further expanded immune memory to antigen non-specific innate immune cells,namely group 2 innate lymphoid cells(ILC2).ILC2s could be rapidly activated by epithelial cell-derived cytokines including IL-33,IL-25 and thymic stromal lymphopoietin(TSLP)upon allergen stimulation,triggering large release of type 2 cytokines like IL-5 and IL-13.Recently,it becomes increasingly controversial on which subset of ILC2 possess memory-like and interorgan migration properties.And the focus is mainly on the surface markers IL-33R/ST2,IL-17RB/IL-25R,as well as killer cell lectin-like receptor subfamily G member 1(KLRGI).Our group has confirmed that ILC2s,rather than Th2s,are abundant in draining lymph nodes during the remission of ACD Furthermore,these memory-like ILC2s could be significantly reduced by YPFS,indicating that constraining ILC2s might play an important role for YPFS to exert its potency against the relapse of allergic inflammationIn this study,we explored the mechanism of YPFS against allergic inflammation based on the memory-like and interorgan migration properties as follows:(1)First of all,we evaluated which subset of ILC2s possess the memory-like and migration properties based on two allergic inflammation mice models.And the regulation of YPFS on ILC2s was analyzed as well.? we analyzed which subset of ILC2s might migrate from draining lymph nodes to peripheral sites based on an FITC-induced ACD model;?Subsequently,we established an HDM-induced asthma model,and YPFS was administered during remission(D24-30).Cells collected from trachea,lung,mediastinal lymph node,small intestine lamina propria(siLP),mesenteric lymph node(MLN)and peripheral blood(PB)were stained with several markers.We determined the distribution and number of different subsets of ILC2s during rerrussion with or without YPFS administration,and delta mean fluorescence intensity(?MFI)of several markers were compared as well;? Then we determined the existing time of Lin-KLRGl+IL-25R+ILC2s in different sites of asthmatic mice after elicitation to confirm if these cells possess "memory-like" capacity.(2)We then evaluated the existence of "memory-like" ILC2s in peripheral sites of several clinical allergic asthmatic subjects,as well as the regulation of YPFS and cimifugin on these ILC2s in vitro.?We firstly compared the number and function of "memory-like" ILC2s in peripheral sites of patients and healthy subjects;? Afterwards,we utilized HDM or recombinant IL-25 with or without YPFS to treat Lin" cells isolated by magnetic separation,and analyzed the allergic responses of primary ILC2s derived from allergic asthmatic and healthy subjects;? Afterwards,we performed another elicitation(from D31 to D33)post remission of an HDM-induced asthma model,and analyzed the dynamic changes of Lin-KLRGl+IL-25R+ILC2 in trachea,lung,mediastinal lymph node,siLP,MLN and PB at the following time points:D23-6h,D24,D27,D30,D34,D37 and D40.(3)We next further explored the interorgan migration property of "memory-like" ILC2s cells,and whether YPFS could restrain this process.? To determine which markers are indispensable for the migration capability of these cells,we analyzed CCR9,a4p7 and SI PR expression on them and the corresponding ligand including CCL25,MAdCAM-1 and SIP levels in different sites;? Then,we transplanted leukocytes derived from siLP,lung,spleen or bone marrow of asthmatic CD45.1 murine to peripheral sites of CD45.2 mice,and asthmatic responses of CD45.2 mice were evaluated followed by airway stimulation.By this way we could determine which figure out the source of "memory-like" ILC2s;?Subsequently,we evaluated the efficacy of YPFS against ILC2s migration based on a parabiotic model of CD45.1 and CD45.2 mice by detecting the number of Lin-CD45.1+KLRGl+IL-25R+ILC2 in trachea,lung,mediastinal lymph node,siLP,MLN and PB(4)To farther verify the significance of "memory-like,ILC2s mediating allergic inflamlation,? we isolated the leukocytes from siLP of asthmatic mice with CD3+ cell depleted and transplanted into Ragl-/-mice.After treatment with IL-25 or KLRG1 antibody,we evaluated the allergic responses of recipients after single intranasal stimulation and the efficacy of YPFS as well;?We performed intranasal stimulation on FITC-induced ACD mice which is analogous to the concept atopic march(AM)clinically.And we could verify the importance of ILC2s for YPFS exerting its potency against allergic inflammation by determining the efficacy of YPFS alleviating pulmonary responses(5)Finally,we evaluated the interactions between ILC2s and epithelial cells.Briefly,we isolated the primary ILC2 cells from both healthy or asthmatic patients,and cocultured them with stratified human bronchial epithelial cells post air-liquid interface(ALI)culture.The proliferation and function of "memory-like"ILC2s as well as the epithelial barrier integrity were analyzed under the treatment of HDM with or without YPFS.The results showed that:(1)? Lin-CD25+ST2-instead of ST2+ILC2 significantly reduced in draining lymph nodes and increased in PB of ACD mice,which suggested that ST2-ILC2 might be capable of trafficking to peripheral region from local sites.Furthermore,we determined it was the Lin-KLRG1+IL-25R+subset of ILC2s who might possess the migration capability;?The number of Lin-KLRG1+IL-25R+ILC2 remain much higher in lung and siLP but not PB,mediastinal lymph nodes,trachea and MLN of HDM-induced asthmatic mice during the remission phase,which indicated that these cells might be the "memory-like" subset possessing the capability to migrate gradually from lung to small intestine after acute response stage.And YPFS could markedly reduce the number and the IL-13 producing capability of these cells.? We also found the distribution and number of these“memory-like,ILC2s in trachea,lung,mediastinal lymph node,siLP,MLN and PB changes dynamically,which further explained the previous results.(2)?The percentage of Lin-CRTH2+KLRG1+IL-25R+ILC2 in PBMC and IL-13 expression in plasma of allergic asthma patients was significantly higher than healthy subjects.?ILC2s isolated from patients were more sensitive to HDM or IL-25 treatment in vitro.YPFS at 100 ?g/mL dramatically depleted the number of these cells and reduced IL-13 level in culture medium;? We also found the distribution and number of these "memory-like" ILC2s in trachea,lung,mediastinal lymph node,siLP,MLN and PB changes dynamically,which further explained the previous results obtained from clinical subjects.(3)? Surface markers including CCR9,?4?7 and S1PR were highly expressed on the"memory-like" ILC2s,and CCL25 and MAdCAM-1 protein levels were also much higher in small intestine of mice during remission while S1P expression elevated in plasma after another elicitation.These data suggested that "memory-like,ILC2s migrate in vivo utilizing specific tissue homing receptors and SIP signaling;? Only the cells isolated from siLP of asthmatic CD45.1 mouse gave rise to the high number of rapidly proliferating Lin-KLRG1+IL-25R+ILC2 in the lungs of CD45.2 mice and mediating the asthmatic responses like airway hyperresponsiveness and pulmonary inflammation of the recipient mice,indicating that the"memory-like,ILC2s might reside in small intestine during remission stage of allergic inflammation;?These CD45.1 positive "memory-like" ILC2s were identified in CD45.2 mice,suggesting the migrating capability of them.And also,YPFS administered only on CD45.1 murine could remarkably reduce the number of these cells in both mice of parabiotic pairs,which indicated YPFS could also limit the migration of them.(4)HDM stimulation intranasally on Rag1-/-mice transplanted with CD3-leucocytes isolated from asthmatic mice led to dramatic type 2 responses.In sharp contrast,mice treated with IL-25R or KLRG1 antibody failed to response to HDM,indicating an indispensable role of the "memory-like" ILC2s.Besides,the efficacy of YPFS against asthma was reversed by subsequent transplantation of CD3-leucocytes isolated from asthmatic mice.Most importantly,treatment of IL-25R or KLRG1 antibody on these leucocytes before transplantation failed to restrain the potency of YPFS;?YPFS could dramatically limit the "memory-like" ILC2s and alleviate the pulmonary in:flammation,suggesting that YPFS exerts anti-allergy potency by regulating the antigen non-specific "memory-like" ILC2s.(5)The epithelial barrier compromised and IL-25 elevated in culture medium after treatment of HDM.ILC2s cultured in the lower chamber could be activated and release type 2 cytokines,and further decreased the integrity of epithelial barrier.ILC2s derived from asthma patients displayed stronger responses compared with those from healthy subjects,indicating the interactions between the "memory-like" ILC2s and epithelial cells might play a pivotal role in allergic inflammation process.Collectively,ILC2s proliferate rapidly upon antigen stimulation,and the "memory-like"Lin"KLRG1+IL-25R?subset acquire migrating capability.Utilizing specific tissue homing receptors,they may traffic in vivo and reside in small intestine during remission phase,and rapidly move to the effector sites upon another elicitation of same or different antigens.The interactions between the "memory-like" ILC2s and epithelial cells might be the key mechanism of antigen non-specific allergic inflammation pathology.Logically,YPFS possess its advantage over the clinical using medications against allergic inflammation by both regulating epithelial barrier and the "memory-like" ILC2s.