| Treatment of fungal infections in the central nerve system exhibits pronounced challenge with high mortality.Cryptococcus meningoencephalitis predominates among all the fungal infections in the CNS.As with more and more application of immune suppressive therapies for autoimmune diseases and the pandemic of HIV/AIDS,the morbidity of cryptococcosis is increasing year by year.It is estimated that 1 million meningoencephalitis cases occur globally each year in patients with AIDS,resulting in 625 thousand deaths.Cryptococcosis arises primarily by inhaling fungal spores into the lung,causing lung infection and pneumonia.Fungal spores could disseminate through the blood brain barrier(BBB)in the absence of an effective immune response,causing meningoencephalitis,with symptoms of headache,fever,blindness and altered mental state.Pulmonary infection is the first step for cryptococcus to intrude the host,and pulmonary innate phagocytes,including pulmonary macrophages and dendritic cells are pioneer sentinels of the pulmonary immune system,combating intruding pathogens at the first line of the immune defense system.The interaction between cryptococcus and macrophages could directly influence the clinical outcome of the cryptococcosis.Macrophage activating phenotype could directly influence its antifungal effects,with M1 polarisation killing the fungi and M2 polarisation facilitating fungi's survival,replication,immune escape and traffic through the BBB.This study mainly focuses on the macrophage-cryptococcus interactions with the treatment of Azithromycin(AZM).AZM is a macrolide antibiotic combating community-acquired bacterial or mycoplasma infection,which could drive macrophages to M2 polarisation and display immunomodulatory anti-inflammatory effects.Early,hard-to-diagnose cryptococcal pneumonia is often treated systematically with AZM,because it is misdiagnosed as community-acquired pneumonia.Although previous study by Rossato L.have demonstrated that amphotericin B(AMB)and AZM forms a synergistic antifungal effects against cryptococcus in vitro,our data have demonstrated that AZM dampens the antifungal ability of the immune system,facilitating the dissemination of cryptococcus,and increasing the fungal burden of mice infected with cryptococcus in vivo.Our study also suggests that in vitro drug sensitive test may not reflect the real picture of the host-pathogen interaction.Given that C.neoformans is a facultative intracellular pathogen and its interaction with macrophages directly results in the clinical outcome of infection,we postulated that AZM might exert an adverse influence on the macrophage antifungal ability.Former studies have demonstrated that AZM could promote macrophage transition towards M2 phenotype.Macrophage activation status is closely related to metabolic reprogramming.In this study,we report AZM reprograms glutamine metabolism by upregulating GPT2,resulting in macrophage M2 polarisation and impaired fungicidal activity of macrophages,eventually leading to dissemination and worsening of cryptococcus infection.Specifically,we use parallel integrated high-throughput metabolic-transcriptional profiling to reveal the overall systemic changes in AZM treated murine macrophage cell lines,uncovering a whole picture of the variable metabolic modules in macrophages with AZM treatment.By integrating transcriptional and metabolic analysis,we identify both variant functional modules and genes specific to AZM treatment rather than the involved pathway.Surprisingly,we validate the critical role of glutamine metabolism and GPT2 in AZM-induced M2 polarisation,which are consistent with the previous discovery of Jha AK.By depriving the medium of glutamine or knocking down GPT2 gene expression,AZM-treated or IL-4 treated macrophage cell lines show impaired M2 polarisation.The results can also be interpreted that GPT2 knockdown macrophages are more resistant to glutamine deprivation than the control macrophages as reflected by the ratio of ARG1/INOS,which provide compelling evidence that glutamine metabolism plays an important role in AZM/IL-4 induced M2 polarisation.In addition,our findings also demonstrate that the upregulation of GPT2 plays a central role in M2 polarisation.GPT2 is an aminotransaminase that catalyses glutamate to pyruvate and ?-ketoglutarate(?-KG).Targeted metabolome validates that amounts of ?-KG are higher in the AZM treated macrophages than in the control group,which is consistent with our postulation.Furthermore,addition of ?-KG can remedy the deprivation of glutamine in the induction of macrophage M2 activation,suggesting that?-KG is a critical metabolite in M2 polarisation.Taken together,these data give a strong evidence that the upregulation of GPT2 by AZM produces more?-KG from glutamine to drive macrophage M2 polarisation.We further validated the upregulation of GPT2 expression at the 7th day postinfection through the inhalational model of mice,indicating that GPT2 may act as an early biomarker for diagnosis of cryptococosis.By collecting PBMCs from blood samples of 12 patients with cryptococosis and 12 healthy people,we found that expression of GPT2 in patients with cryptococosis were higher than the healthy people by q PCR,which further verified our postulation that GPT2 may act as the biomarker for early detection of cryptococosisIn summary,we have revealed a novel mechanism through which AZM upregulates glutamine metabolism and drives macrophages to M2 polarisation and more sensitive to glutamine deprivation.We have identified that AZM enhances ATF4 expression to activate GPT2 transcription,converting more glutamine to ?-KG.This study has important clinical significance.Firstly,AZM should not be the first systematic treatment for community-acquired pneumonia with unknown pathogens.Secondly,our results suggest GPT2 is a potential immune modulatory therapeutic target for systematic fungal infections,which may also provide us a deeper understanding of defective antifungal host responses to identify the people at risk for systematic fungal infections. |
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