Mechanism Of β-Glucan Upregulation Macrophage LC3-Associated Autophagy Clearance Talaromyces Marneffei

【Background】Talaromycosis（TSM）is a serious invasive fungal disease caused by Talaromyces marneffei（TM）,which is distributed in Southeast Asian countries and southern provinces of China.With the increase in the incidence of AIDS since the last century,the infection rate has also increased year by year and has become a clinical diagnostic indication for AIDS.However,a growing number of studies have shown that the incidence of non-AIDS TSM is increasing significantly year by year,its epidemic area is expanding,and its clinical manifestations are complex and lack specificity compared to those of AIDS-hosted TSM.Macrophages（Mac）play a key role in the immune response against TM and the monocyte-macrophage system is the main site of TM invasion into the body.After TM invasion,Macs in the host can recognize,phagocytose and present antigens through pattern recognition receptors,which is a key step in the induction of adaptive immune response and clearance of pathogens.Therefore,it is important to study and elucidate the mechanism of how TM is cleared by macrophages in the organism to find potential new therapeutic approaches and improve clinical efficacy.β-glucan is an immunomodulatory agent that fights infection by driving the immune effect of macrophages,a phenomenon that lasts for weeks to months.β-glucan in fungal and yeast cell walls causes phosphorylation of macrophage NADPH oxidase complexes,culminating in the production of ROS（e.g.superoxide anion）and an oxidative burst reaction.β-glucan can significantly stimulate macrophage proliferation in a range of concentrations,and also significantly increases intracellular free calcium and c AMP.LC3-associated phagocytosis(LC3-associated phagocytosis（LAP）is an efficient pathway for host cell phagocytosis and degradation of pathogens,and has an important role in the clearance of pathogenic microbial infections such as Salmonella,Mycobacterium tuberculosis,Aspergillus,and Candida albicans.β-glucan activates LC3-associated phagocytosis through Dectin-1 recognition ofβ-glucan-containing particles on phagocytes LC3 recruitment enhances phagosome maturation.Therefore,there is a theoretical basis for studying and elucidating the regulation of LC3-related phagocytosis byβ-glucan in macrophages.【Object】1.To elucidate the correlation between theβ-glucan expression level of TM and the severity of TSM disease.2.To elucidate the effect and mechanism ofβ-glucan of TM on the function of macrophage phagocytosis and TM clearance.【Methods】1.Correlation study betweenβ-glucan expression levels and clinical prognosis of TSM patients:clinical information（G test results,clinical prognosis）andβ-glucan expression levels of clinical TM pathogenic strains were collected from TSM patients,and after detecting and analyzing the expression levels ofβ-glucan genes（FKSP and RHO1）of TM strains in patients by transomics and q RT-PCR;the above results were combined to The clinical TM pathogenic strains were classified into G-positive(TM^G+)and G-negative(TM^G-)strains.2.β-glucan expression differences of TM andβ-glucan-mediated Mac phagocytosis and functional study of TM clearance:single cell sequencing to detect the ratio of PBMC cell subpopulations in non-AIDS TSM patients and healthy human;THP-1 differentiated macrophages were co-cultured with TM standard strain andβ-glucanase-eluting TM standard strain yeast phase,and Mac was observed under laser confocal microscopy and transmission electron microscopy phagocytosis of TM;TM and Mac were co-cultured and then inoculated to observe the bacterial load of TM in each group.3.β-glucan-mediated Mac phagocytosis and TM clearance mechanism study:single cell sequencing to detect and analyze the expression levels and differences of key molecules such as macrophage Dectin-1,LC3,Rubicon,ATF6,CEBP-β,DAPK1,LAP pathway in the peripheral blood of TM^G+strain infection,TM^G-strain infection of TSM patients and healthy population;usingβ-glucanase was used to establish aβ-glucan-eluting TM strain model,and the effects ofβ-glucan on the expression levels of key molecules of Dectin-1 receptor and LAP pathway on the surface of Mac and the functions of Mac on TM phagocytosis and clearance were investigated by cellular in vitro experiments.q RT-PCR and Western blot assays and molecular macrophages with TM expressing differentβ-glucan levels The expression levels and differences of key molecules of macrophage Dectin-1,LC3II/LC3I,Rubicon,LAP pathway（ATF6,CEBP-β,DAPK1）after co-culture with TM expressing different levels ofβ-glucan were detected by q RT-PCR and Western blot.【Results】1.The clinical prognosis of TSM patients is closely related to the level ofβ-glucan expressed by the corresponding TM strain:（1）transcriptomics found that yeast phase（pathogenic phase）TM,β-glucan genes（RHO1 and FKSP）of G experimental positive TM clinical strain were significantly lower than those G negative TM clinical strain;（2）q RT-PCR verified and confirmed that the strains corresponding to patients with positive clinical G test TSM had significantly higher levels ofβ-glucan（RHO1）m RNA levels than those of patients with negative G test TSM（P<0.05）;（3）The prognosis of patients with positive G test was better than that of patients with negative G test,mainly because their persistent infection rate（9.7%）and mortality rate（6.5%）were significantly lower than those in the negative G test group（18.4%,10.2%）（P<0.05）.2.Mac participates in the phagocytosis and clearance of TM and is an important immune cell for the body’s defense TM:The results of PBMC single-cell sequencing of human peripheral blood showed that the proportion and absolute value of Mac in non-AIDS TSM patients were significantly higher than those in healthy people,confirming that Mac plays an important role in the body’s defense TM.3.The difference between Mac phagocytosis and clearance of TM is closely related to the difference in TM expressionβ-glucan:（1）TM spores in Mac could be formed and phagocytosed after 4 hours of co-culture between clinical TM^G+strain and Mac macrophages,and the number of intravesicle spores was basically cleared and the number was significantly reduced after 24 hours,while the clinical TM^G-strain and Mac macrophages were co-cultured for 4 hours,although Phagocytosis TM spores were visible in Mac,but no monolamellar membrane vesicles were seen.and after 24 hours,TM spores in the cells multiplied in large numbers;（2）At 37°C,after usingβglucanase to establishβdextran-eluting TM standard strain,it was found that compared with the uneluting group,the expression level ofβdextran in the TM strain of theβdextran eluting group decreased significantly（P<0.05）;（3）After establishing the co-culture system of TM strain and THP-1before and afterβglucanase elution,the ability of Mac to engulf and clear TM was observed,and it was found that compared with the uneluting group,the TM phagocytosis ability of Mac toβdextran elution was significantly reduced（P<0.05）,and the bacterial load of the TM strain was significantly higher than that in the uneluting group（P<0.05）,indicating that the decrease in the content ofβdextran in TM strain could significantly weaken the phagocytosis and clearance of TM by Mac.4.β-glucan may upregulate the phagocytosis and clearance of TM by Mac’s LAP by activating the ATF6/CEBP-β/DAPK1 pathway:（1）Single-cell sequencing and analysis of PBMC in non-AIDS G test positive TSM patients,G test negative TSM patients,and healthy people found that CLEC7A（Dectin-1）,ATF6,CEBP-β,DAPK1,MAPLC3A（LC3）,The expression of RUBINN（Rubicon）was significantly higher than that in healthy people and patients with negative G test TSM;（2）After observing the co-culture of Mac-positive and negative TM strains with G test for 4 hours and 24 hours under immunofluorescence microscopy,it was found that the fluorescence expression of LC3 was visible in the TMG+group for 4 hours,and its fluorescence intensity was significantly enhanced after 24 hours,and there was no obvious LC3 fluorescence expression in the TMG-group for 4 hours and 24hours;（3）After establishing the co-culture system of TM strain and THP-1 before and afterβdextran elution respectively,it was found that the m RNA and protein levels of Dectin-1,LC3,CEBP-βand DAPK1 in theβ-dextran uneluting group were significantly increased（P<0.05）.【Conclusion】The clinical prognosis of TSM patients and the molecular mechanism of TM differential clearance by Mac are closely related to the level ofβ-glucan expression and LAP activation,and the specific molecular mechanism may be related to the activation of ATF6/CEBP-β/DAPK1 pathway byβ-glucan and upregulation of phagocytosis and clearance of TM by LAP of Mac.