Establishing Immune Molecules And Cells Signatures From Different Types Of Leprosy Granulomas Via Laser Capture Microdissection And RNA-seq

Background:Leprosy,also known as Hansen's Disease,is a chronic granulomatous infectious disease caused by Mycobacterium leprae in susceptible individuals.It mainly affects the skin and peripheral nerves and,in later stages,can cause irreversible disability.Due to differential response of the host's immune system to M.leprae,leprosy clinically manifests as five general categories,the spectrum of which is continuous.Similarly,the leprosy granulomas corresponding to each category exhibit a spectrum of morphological features.The leprosy granuloma is a characteristic structure produced by the individual's innate and adaptive immune systems' recognition and resistance responses to M.leprae.Presently,our understanding of the fine structure and formation mechanisms of leprosy granulomas is limited,particularly with respect to the various categories of leprosy.This,in turn,has significantly hindered research work in understanding the mechanisms underlying leprosy pathogenesis.Objectives:To characterize the different categories of leprosy by mapping the molecular and cellular structures of their granulomas.Methods:1. 5 paraffin-embedded samples classified as either tuberculoid(TT)or borderline tuberculoid(BT)as well as 5 classified as lepromatous(LL)or borderline lepromatous(BL)were examined using immunohistochemical staining to label critical immune cells.Images of these were then compiled into maps showing the fine structure of each type of granuloma.2. 4 fresh tissue samples classified as either TT or BT and 4 classified as either BL or LL were examined using antibody microarrays to investigate differential cytokine expression.In vitro models of leprosy granulomas were then constructed using these varying cytokine levels and further examined for their bactericidal capacities.3. 5 granuloma samples classified as either TT or BT,5 samples classified as either BL or LL,and 4 samples of cutaneous tuberculosis granulomas were isolated using laser capture microdissection for total RNA extraction.Each sample was then RNA sequenced(mRNA,LncRNA,CircRNA),and their expression and regulatory profiles were analyzed.Results:1.Leprosy granulomas are characterized by infiltration mainly by macrophages,and minorly by other cells,e.g.T cells,B cells,NK cells,etc.In T-lep,T cells occupy a relatively high proportion and show a dominant CD8+T profile.On the other hand,L-lep has relatively few T cells and shows a dominant CD4+T profile.In addition,CD68+CD163+ macrophages are notably present in L-lep.2.In T-lep,the abundance of cytokines IL-15/cystatinC/Lipocalin-2/MIP-1? is greater than that in L-lep;in L-lep,the abundance of cytokines ICAM-1/IL-10 is greater than that in T-lep.In in vitro leprosy granuloma models,IL-15 promoted cell aggregation and enhanced bactericidal capacity of the granuloma;on the other hand,IL-10 inhibited its bactericidal capacity.3.In tandem with analysis of cell composition and immune cell genetics,mRNA sequencing data can accurately determine the ratio of immune cells in leprosy granulomas.Further,gene subset expression patterns and KEGG pathway analyses showed that the lysosome pathway gene is particularly active in L-lep.4.Expression profiles from circRNA and IncRNA sequencing show that T-lep and CTB are relatively similar,while T-lep and L-lep are relatively different.Further,the differentially expressed ncRNAs between T-lep and L-lep groups may be involved in the formation and sustenance mechanisms of granulomas.Conclusions:1.There are clear differences in immune cell composition and characteristics between tuberculoid and lepromatous granulomas.2.IL-15 promotes cell aggregation and enhances macrophage killing and can be used to construct in vitro granuloma models for TT and BT leprosy.On the other hand,IL-10 can be used to inhibit macrophage killing.3.Differential mRNA expression between the different classes of leprosy granulomas is directly related to their varying formation and sustenance mechanisms.4.Bioinformatical analysis revealed that various IncRNAs and circRNAs participate in the regulation of the formation and sustenance mechanisms used by the different classes of leprosy granulomas.