Mechanistic Study On TRAF1/NOS2 In Macrophage Immunity Against Sporothrix Induced By Hyperthermia

Objective:Sporotrichosis is a kind of chronic granulomatous disease,which is a deep mycosis that global distributed.Sporotrichosis is caused by sporotrichix complex infection,mainly causing dermal infection,but mucosal,subcutaneous tissue and nearby lymphatic system also could be involved.It can also cause systemic damage to blood and lymphatic system.Currently,common drugs including potassium iodide and antifungal drugs such as amphotericin are used to treat sporotrichosis.However,these treatments have disadvantages such as long treatment cycle and many adverse reactions.Local hyperthermia has the advantages of less special contraindications and painless while ensuring effective treatment rate,which has become an effective mean to treat sporotrichosis.Both innate and adaptive immune responses are involved in the process of the body’s resistance to sporotrichx,in which macrophages play an important role.Swallowing phages and producing reactive oxygen species by macrophages and neutrophils is a potent weapon of the innate immune response,by way of the recognition of bacterial antigens by pattern recognition receptors(PRRs)and pathogen-associated molecular patterns(PAMPs).Macrophages also have a strong antigen-presenting function and produce many cytokines that act as a bridge between innate and adaptive immunity.In this study,the influence of hyperthermia on macrophages in sporotrichx infection was explored,as well as the potential mechanisms involved in,in the aim to provide new strategy to improving the curative effect,shortening the treatment cycle and reducing recurrence rate in the treatment of sporotrichosis.Methods:In this study,ANA-1 macrophage line,mouse bone marrow derived macrophages(BMDM),fixed type sporotrichosis mouse model and clinical sporotrichosis biopsied tissue blocks were selected as the research objects.293 T cells were introduced as tool cells during the immunoprecipitation and ubiquitination assays.Single cell-RNA sequencing was used to detected composition of cells in tissue and expression of genes in specific cells.Multiple immunofluorescence staining was performed on sporotrichosis sections of patients and mice to explore the expression of protein in macrophages.The m RNA and protein expression in cells were detected by RT-PCR,Western Blot and immunofluorescence.Lentivirus was used to construct stable overexpressed and knockdown gene cell lines,and plasmids were used for transfection tool cells.Immunoprecipitation,GST-pulldown,fluorescence co-location and truncation tests were used to detect proteins interaction.The ubiquitin assay was used to detect the ubiquitin level of the protein.Gene site-specific mutation was used to explore the regulatory sites of protein ubiquitination.Fungal colony formation assay was used to detect the effects of TRAF1 and NOS2 on the killing of sporotrichx by macrophages.Adeno-associated viruses were injected into mice to overexpress TRAF1 in macrophages.student’s t test or one-way ANOVA were used for statistics,and the inspection level was P<0.05.Graph Pad7 was used to plot the data,and Photoshop 2022 was used to typeset and combine the pictures.Results: 1)Single cell-RNA analysis integrated sporotrichosis tissue and normal skin tissue of mice,cells were annotated to 10 clusters,which were mononuclearmacrophages,dendritic cells,T cells,fibroblasts,endothelial cells,pericytes,three kinds of keratinocytes and melanocytes.TRAF1 was mainly expressed in mononuclear macrophages,dendritic cells and T cells.In mononuclear macrophages,expression of TRAF1 in sporotrichosis lesions was significantly higher than that in normal skin.NOS2 was mainly expressed in mononuclear macrophages in sporotrichosis lesions,but hardly expressed in normal skin.2)TRAF1 and NOS2 were mainly expressed in the dermis,with rare expression in the epidermis.TRAF1 and NOS2 were mainly expressed in granuloma lesions inducedby sporothrichx,and their expressions in skin were significantly higher than those in the uninfected skin.TRAF1 and NOS2 were mainly expressed in mononuclear macrophages and lymphocytes at the site of granuloma,among which,the expression of TRAF1 and NOS2 in CD68-positive macrophages was higher than that in the uninfected skin.TRAF1 and NOS2 were also co-localized in macrophages.The above results were also observed in sporotrichosis lesions in mice,and the expressions of TRAF1 and NOS2 in macrophages were down-regulated after local hyperthermia treatment.3)Both TRAF1 and NOS2 were upregulated after sporothrichx infection with macrophages,and the change of TRAF1 expression was faster,there was no significant difference of expression level 2 and 6 hours after infection.The change of NOS2 expression was slower,and it still showed a low level 2 hours after infection,and a high level 6 hours after infection.Six hours after hyperthermia treatment,the expressions of both were significantly reduced.4)Macrophage cell lines with stable overexpression and knockdown of TRAF1 were constructed with high efficiency.In both TRAF1 overexpression and knockdown macrophages,TRAF1 can positively regulate the expression of NOS2 protein,but has no effect on the expression of NOS2 m RNA,and the overexpression of TRAF1 can recover the decreased expression of NOS2 caused by the decreased expression level of TRAF1.NO is the catalytic product of NOS2,and its release can represent the enzyme activity of NOS2.TRAF1 can also positively regulate the release of NO in macrophages.TRAF1 and NOS2 proteins bind in macrophages,showing co-localization.In vitro experiments showed that TRAF1 and NOS2 protein had direct interaction.NOS2 binds to the 258-409 amino acid region of TRAF1,which has TRAF1’s MACH domain.5)TRAF1 can slow down the degradation of NOS2 protein,proteasome inhibitors can reverse the decreased expression of NOS2 caused by the decreased expression of TRAF1,which proves that TRAF1 can increase the stability of NOS2 protein,reduce its degradation by proteasome,and thus increase the expression of NOS2 protein.Ubiquitination experiments demonstrated that TRAF1 inhibited the ubiquitination of NOS2,and this inhibition was achieved through the lysine 19 site of NOS2.6)Overexpression of TRAF1 in macrophages delayed the healing of sporotrichosis in mice.At the fourth week after infection with Sporotrichx,lesions of mice in the control group were mostly healed and the ulcers scabs or even disappeared,while the skin lesions of mice in the TRAF1 overexpression group still existed and showed obvious ulcers.The killing effect of macrophages overexpressing TRAF1 against Sporothrix was weakened,but the killing effect of macrophages against sporothrix was recovered when the macrophages overexpressed TRAF1 and knocked down NOS2 at the same time.TRAF1 positively regulates the expression of IL1βand IL6 in macrophages.Conclusions: 1)Hyperthermia down-regulated the expression of TRAF1 and NOS2 in sporotrichx infected macrophages and sporotrichosis lesions,suggesting that the treatment of sporotrichosis by hyperthermia may be achieved by regulating the expression of TRAF1/NOS2.2)TRAF1 could positively regulate expression of NOS2 protein,but had no regulating effect on the expression of NOS2 m RNA.TRAF1 increases the stability of NOS2 protein by inhibiting the degradation of NOS2 by ubiquitin-proteasome,TRAF1 and NOS2 proteins have direct interaction in vivo and in vitro.NOS2 binds to the MACH domain of TRAF1 protein;TRAF1 inhibits the ubiquitination level of NOS2 protein by inhibiting the ubiquitination of lysine 19 site.3)Overexpression of TRAF1 in macrophages can delay the healing of sporotrichosis lesions in mice.