| ObjectiveCryptococcosis is an infectious fungal disease caused by Cryptococcus spp.,of them Cryptococcus neoformans is most common in cryptococcosis.Due to the peculiar neurotropism of C.neoformans,it often causes fatal cryptococcal meningitis,which is lethal to the patients.At present,in the treatment of cryptococcal meningitis,antifungal drugs such as amphotericin B are mainly used,which not only have serious drug-related side effects,but also are prone to problems such as fungal resistance.This article aims to investigate the effect of FCN2/A protein in exosomes on macrophages and mice against C.neoformans.to provide a new theoretical basis and scheme for the immune adjuvant therapy of patients with cryptococcosis.Method1.The C.neoformans H99 infection mouse model and the PBS control mouse model were constructed by the nasal inhalation method,and the mouse plasma exosomes were extracted(H99-EXO: plasma exosomes of the C.neoformans infected mouse group;PBSEXO: plasma exosomes from PBS-treated mice),morphological identification of exosomes was carried out by nanoflow cytometry;the uptake of exosomes of different mouse plasma by macrophages was verified by confocal microscopy;H99-EXO and PBS-EXO were incubated with mouse macrophages(Raw264.7 macrophage cell line and BMDM primary macrophage cell)for 24 hours,the treated macrophages were co-incubated with the standard strain of C.neoformans H99.By observing the related phenotypes of the interaction between macrophages and C.neoformans: killing,phagocytosis,IPR(intracellular proliferation rate)and the release of inflammatory factors and nitric oxide in the cell supernatant of H99-EXO and PBS-EXO cocultured mouse macrophage were detected to explore the effect of H99-EXO on macrophages against C.neoformans infection;H99-EXO and PBS-EXO were injected into healthy mice by intraperitoneal injection,and 24 hours later they were infected with C.neoformans and observed: mouse survival time,tissue colony load(lung,brain),HE and PAS tissue stained(lung,brain),tissue inflammatory factor release(lung,brain),to explore the effect of H99-EXO on the anti-C.neoformans infection ability of mice.2.8 patients with cryptococcal meningitis and 8 healthy controls during the same period were included.Plasma exosomes from patients with cryptococcal meningitis and healthy controls were extracted by using a plasma exosome extraction kit,and exosome proteomics was used to explore the differentially expressed protein.The differential protein expression between patients and healthy control was analyzed,and Western-Blot was used to verify the differential protein FCN2 in plasma exosomes of cryptococcal meningitis patients,healthy control,and FCN2 mouse homologous protein FCNA in plasma exosomes of C.neoformans infected,PBS treated group;H99-EXO and PBS-EXO were incubated with mouse macrophages for 24 hours,and then the m RNA expression levels of FCNA in each group were verified by real-time quantitative PCR;The expression level of FCNA protein were analyzed to explore whether FCNA in H99-EXO and PBS-EXO could be taken up by macrophages.3.Using Lipofectamine RNA i MAX transfection reagent,FCNA-negative control and FCNA-small interfering RNA were transfected into mouse Raw264.7 macrophages,and BMDM of FCNA knockout mice and wild-type mice was extracted.The cells were coincubated with C.neoformans H99 standard strain,and the phenotypes related to the interaction between macrophages and C.neoformans were observed: killing,phagocytosis,IPR(intracellular proliferation rate)and the release of inflammatory factors and nitric oxide in the cell supernatant of knockdown and knockout FCNA macrophages were detected.was to explore the effect of FCNA protein on the function of macrophages against C.neoformans.And mouse survival time,HE、PAS tissue stained(lung,brain)were used to explore the effect of FCNA knockout mice on the ability of anti-C.neoformans infection.4.Transcriptomic analysis was performed on mouse BMDM macrophages coincubated with H99-EXO and PBS-EXO and FCNA knockout and wild-type mouse BMDM macrophages,and the same differential genes in the two groups of transcriptomics was selected.RT PCR was done to confirm the selected differentially expressed genes,to initially reveal the possible targets of FCNA protein in mouse plasma exosomes in regulating macrophage killing and phagocytosis of C.neoformans.Results1.Firstly,different infection time gradients were set up.It was found that compared with the PBS-treated group,the plasma exosomes extracted after 14 days of infection mouse with the C.neoformans H99 were different in diameter and concentration;Macrophages could directly take up the exosomes derived from different mouse plasma,and the uptake of exosomes by macrophages gradually increased with time.The phagocytosis and killing of C.neoformans were enhanced,the intracellular proliferation ability of C.neoformans in macrophages was weakened,the release of pro-inflammatory inflammatory factors was increased,and there was no significant difference in the release of nitric oxide;Healthy mice were reinfused with H99-EXO and PBS-EXO and then infected with C.neoformans.Mice reinfused with H99-EXO had prolonged survival,decreased lung and brain fungal load,increased protein expression of IL10 and MCP-1 in brain tissue,and alleviated lung damage.2.After proteomic analysis of plasma exosomes from patients with cryptococcal meningitis and healthy controls,a total of 158 differential proteins were found,of which 144 were down-regulated and 14 were up-regulated.Five differential proteins were screened out and verified by Western-Blot experiments.Combined with the verification results and literature review,it was finally determined that FCN2/A protein was the main research object in this study.We found that FCN2 protein was lowly expressed in the plasma exosomes of patients with cryptococcosis,and the FCN2 mouse homologous protein FCNA was also lowly expressed in the plasma exosomes of C.neoformans infected mice;H99-EXO and PBS-EXO were co-incubated with murine macrophages,it was found that compared with the blank control group(without exosome co-incubation)there was no significant difference in the m RNA expression of FCNA,but the FCNA protein expression was higher than that in the blank control group,proving that FCNA in exosomes were taken up by macrophages.3.After Comparison with the FCNA-NC group,the FCNA knockdown mouse Raw264.7 was found to have enhanced phagocytosis and killing of C.neoformans weakened intracellular proliferation ability of C.neoformans in macrophage,increased release of proinflammatory inflammatory factors.The ability of anti-C.neoformans of macrophage may be positively correlated with the efficiency of FCNA gene knockout.Compared with the wild-type group,BMDM of FCNA knockout mice has enhanced phagocytosis and killing of C.neoformans,weakened intracellular proliferation ability of C.neoformans in macrophage,and pro-inflammatory inflammatory factors release increases.There was no obvious nitrogen release in the supernatant of macrophage cells in the above two groups.The FCNA knockout and wild-type mice model of C.neoformans infection was constructed,and it was found that the survival time of mice of FCNA knockout group was prolonged and the lung damage was alleviated compared with the wild-type group.4.Transcriptomic analysis of the BMDM of H99-EXO and PBS-EXO co-incubated mice and the BMDM of FCNA knockout and wild-type mice showed that there were 20 differentially expressed genes in common between the two group,of which 4 were identical.After verification by Real-time quantitative PCR,it was found that the expression of C1ql3,Pcdhb3,and Shroom1 genes were up regulated in the macrophages of the two groups,which was consistent with the transcriptomic results of the two groups.Based on the previous results,it is suggested that the ability of FCNA protein in mouse plasma exosomes to regulate macrophages against Cryptococcal infection may be related to the changes in the expression of C1ql3,Pcdhb3 and Shroom1 genes in macrophage.ConclusionThe ability of FCNA protein in mouse plasma exosomes to regulate macrophage resistance to cryptococcal infection may be related to the changes in the expression of C1ql3,Pcdhb3 and Shroom1 genes in macrophages. |
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