The Effect And Mechanism Of NLRP3 In Allergic Contact Dermatitis Chronic Itch

Background and ObjectiveItch(pruritus)serves as a defense mechanism for the body as patients can remove the irritant by scratching.However,chronic pruritus costs the patients a lot,as well as the society,and damaged the quality of the patients’life.However,as the conventional drugs for itch,antihistamines,are ineffective or have a mild effect on chronic itch,and the mechanism and novel therapy of chronic itch are required for the patients.Allergic contact dermatitis(ACD)is a skin disease characterized by redness,swelling,papules and blisters in the early phase and followed by scales and scars.A recent study had shown that the predominant role of Th2 immunity response to ACD with a significantly increased expression of Th2 cytokines like thymic stromal lymphopoietin(TSLP),interleukin(IL)-4,IL-5,and IL-13,and also with elevated immunoglobulin(Ig)E production and mast cell/eosinophil activation.Meanwhile,transient receptor potential ankyrin1(TRPA1)and transient receptor potential vanilloid 1(TRPV1)were also upregulated in dorsal root ganglion(DRG)in mice with chronic itch or pain.NLRP3(NOD-,LRR-and pyrin domain-containing protein 3)served as the most notable(Nucleotide-binding oligomerization domain,NOD)like-receptors(NLRs)has been described in many immune or autoimmune skin diseases,such as acne,dermatophytosis,and psoriasis.It has been reported that NLR treatment can alleviate the symptoms of allergic diseases,such as atopic dermatitis,allergic contact dermatitis or allergic asthma in mice model,which indicates that NLR has the potential to be a target for allergic diseases.However,the role of NLRP3 inflammasome in chronic itching induced by ACD has not been reported.Our study was going to use NLRP3 knockout mice to clarify the role of NLRP3 in ACD and expected to provide evidence for new therapeutic targets for chronic itch.Methods:Animal experiments were performed on 8-week-old male C57BL/6J wild-type(WT),NLRP3 knockout(NLRP3-/-)and caspase1/11 knockout(CASP1/11-/-)mice.The acute itch model was induced by subcutaneously injection of pruritogens:chloroquine,histamine,serotonin,SLIGRL polypeptide,endothelin-1,and compound 48/80,respectively,on the back of the mice’s nape,and intrathecally(i.t.)injection of neuromedin B(NMB)and gastrin-releasing peptide(GRP),respectively,in mice.As for establishing a chronic itch mice model,hapten 1-fluoro-2,4-dinitrobenzene(DNFB)was painting on the mice’s neck for inducing chronic ACD.Intraperitoneal(i.p.)injection of NLRP3 inflammasome specific inhibitor MCC950 was performed for suppress the activation of inflammasome.RT-PCR was used to detect the transcription expression of the vital molecules in the skin and in DRG,western blot(WB)and immunohistochemistry(IHC)were used to detect the protein expression of NLRP3,caspase-1 cleaved P20,and IL-18 in the skin.The levels of Ig E in serum and mature IL-1βand IL-4 in skin tissue were measured by ELISA.The number of mast cells in the skin,the expression and location of NLRP3 and IL-1βin the skin,IL-4R and TRPA1 in DRG were performed by immunofluorescence.Results:1.The expression of NLRP3,caspase-1,mature IL-1β,and IL-18 were significantly increased in the skin of DNFB induced chronic itch mice,according to the results of RT-PCR,WB,IHC,and ELISA.2.The scratching number of histamine,serotonin,compound 48/80 was significantly increased in NLRP3-/-mice than that in WT mice,but not significant difference in chloroquine,NMB and GRP induced acute itch in both group mice.3.In DNFB induced ACD model,the number of scratching was significantly increased in NLRP3-/-mice compared to that in WT mice.4.In the DNFB model of NLRP3-/-mice,the number of mast cells in the skin wassignificantly increased,and the serum Ig E was also elevated but had no statistical significance;the expression of IL-4 and TSLP were upregulated in the skin while IL-4R and TRPA1 were significantly increased in DRG but not TRPV1.5.In the CASP-/-mice with ACD,the scratching number and serum Ig E level were significantly higher than those of WT mice,but there were no significant difference in the thickness of epidermis and in the number of mast cells infiltrated into dermis.The m RNA levels of IL-4 and TSLP in the skin of CASP-/-mice were significantly decreased,but no difference in protein.6.Applied MCC950 reduced the serum Ig E level but had no effect on the number of scratching,on the number of mast cells and on epidermal thickness in DNFB induced ACD mice.7.The majority of IL-4R were located in TRPA1~+neurons.Conclusion:1.NLRP3 inflammasome is obviously activated in the skin of DNFB induced chronic itching mice.2.NLRP3 genetic deletion would aggravate histaminergic itch but had no influence on non-histaminergic itch.3.The scratching number was aggravated in NLRP3 knockout mice after DNFB induced ACD model,and the mechanism is related to the activation and infiltration of mast cells in the skin,the up-regulated expression of IL-4 and TSLP,and the significantly increased of TRPA1 in dorsal root ganglion.4.NLRP3 inflammasome specific inhibitor MCC950 has no effect on the severity of chronic itching,while CASP1/11-/-genetic deletion aggravates chronic itching independent of IL-4 and TSLP.5.NLRP3 genetic deletion aggravated ACD chronic itching by increasing the expression of IL-4 and TSLP in the skin and elevated the activation of TRPA1 in DRG which was responded in an inflammasome-independent manner.