Study On The Role And Mechanism Of Macrophages In Primary Cutaneous Amyloidosis

Background and objectivePrimary cutaneous amyloidosis（PCA）is a chronic pruritic disease with the characteristic of amyloid deposition in the superficial dermis of previously normal skin without invasion of other organs.It is believed that amyloid is mainly derived from keratinocytes.Studies have found that OSMR missense mutations exist in patients with PCA.OSMR encodes the oncostatin M receptor β（OSMR-β）which works as a subunit of OSM type II receptor that transduces the signal activated by its ligand,OSM.Previous studies have shown that OSMR mutations inhibits the response of keratinocytes to OSM and IL-31,and cause intracellular JAK/STAT and PI3K/AKT signaling activation disorders,which might decrease cytokine secretion and boost apoptosis.Other studies have suggested that the deposition of amyloid may be related to the incapability of monocytes/macrophages to degrade it.In this study,multiple technical methods including immunohistochemistry,flow cytometry,Western Blot and RNA sequencing were performed to analyze the immune status of skin and peripheral circulation of PCA patients.We aim to elucidate the role of OSM/OSMR system in the pathogenesis of PCA.Methods1.Distribution and quantity of macrophages in healthy skin and PCA lesions were detected by scanning electron microscope and immunohistochemistry;2.Flow cytometry was used to detect the proportion of CD14+monocytes in PBMCs from healthy people and PCA patients;3.Dextran uptake experiments and flow cytometry were performed to analyze the function and phenotypes of monocyte-derived macrophages（MDMs）of healthy people and PCA patients;4.Immunofluorescence and Western Blot were performed to detect the expression level of JAK/STAT3 and PI3K/AKT signaling protein in skin tissue from PCA patients and healthy people;5.Stimulated normal human primary keratinocytes and MDMs with OSM.Detected p-STAT3 in keratinocytes by Western Blot,immunocytochemistry and analyzed the function and phenotypes of MDMs by Dextran uptake experiment and flow cytometry.6.Differentially expressed genes between PCA patients and healthy people were identified by RNA sequencing.7.Immunofluorescence was used to observe the infiltration of CD4+T cells in normal skin and PCA lesions.CD4+ T cells subsets in PBMCs were analyzed by flow cytometry.8.Flow cytometry was performed to analyze the phenotypes of MDMs after cocultured with autologous CD4+T cells.Results1.Macrophages were trying to engulf amyloid in PCA lesions.There was no significant difference in the proportion of peripheral CD 14+monocytes between patients and normal people.While the quantity of CD68+macrophages in the PCA lesions was significantly higher than that in healthy skin.There was no difference in phagocytic function and phenotypes of MDMs between patients and healthy people.2.RNA sequencing showed that CD4+T cell receptor signaling was downregulated,and T cells chemokines were reduced in PCA patients’ skin.Besides,after cocultured with autologous CD4+T cells,MDMs expressed more MHC-Ⅱand CD86 than before.3.The expression of OSMR protein in the lesions and non-lesion skin of PCA patients was significantly lower than that in healthy skin.And p-STAT3 and pAKT in samples with low expression of OSMR were also reduced.After stimulated with OSM,the expression of p-STAT3 was apparently improved in keratinocytes.While no significant change was found in phagocytosis function and phenotypes of MDMs stimulated with OSM.ConclusionMacrophages from PCA patients did not present abnormal functions and phenotypes in vitro.But certain changes about the immune microenvironment had occurred locally in the PCA lesions,which may cause the dysfunction of macrophages and thus make it difficult to degrade the amyloid.