| Objective Persistent immune inflammation is a major feature of HIV/AIDS patients that cannot be resolved by antiretroviral therapy(ART).Inflammatory M1-type polarization of macrophages upon HIV-1 infection is closely related to long-term inflammatory activation in patients,and immunometabolism may be involved in the progress.But little studies have been conducted so far.This subject aims to explore the relationship and molecular mechanism between the enhanced glycolysis and the M1 polarization in macrophage with HIV-1 infection from the perspective of immune metabolic reprogramming.Hoping to provide a new idea for resolving persistent immune activation in clinical AIDS patients.Methods The morphological changes of macrophages were observed and photographed by optical microscope.The gene and protein expression of M1 polarization markers TNF-α,IL-1β and IL-6 were measured by q PCR and ELISA,respectively.The effect of HIV-1 on the metabolic profile of macrophages was analyzed by non-target metabolomics using LC-MS/MS.The m RNA of polarization markers,key enzymes of glycolysis and aerobic oxidation,COX-2and JNK were quantified by q PCR.The content of lactic acid in cell culture supernatant was detected using colorimetric method.The 2-NBDG Glucose Uptake Assay Kit was used to detect the glucose uptake capacity of macrophages.The protein expression levels of COX-2,p-JNK,p-ERK,p-p38,JNK,ERK and p38 were quantified by Western blot.Results(1)After HIV-1 infection for 1 to 3 days,THP-1-derived macrophages extended longer pseudopods and irregularly shaped.Expression level of polarization markers were up-regulated in general,while M1 markers TNF-α,IL-1β,IL-6 had greater rise than M2 markers CD163 and CD206.(2)MDMs exposed to HIV-1 in vitro for 3 days had similar morphological characteristics to MDMs of HIV/AIDS patients,showing irregular shape with the spindly pseudopod extends outward.Meanwhile,q PCR and ELISA results showed that TNF-α,IL-1β and IL-6 were significantly increased after HIV-1 infection(P < 0.001).While MDMs from healthy controls were flat and round with short pseudopod.(3)The results of non-target metabolomics were screened for differential metabolites according to P-value<0.05,VIP>1,and 681 differential metabolites between the HIV-1 group and the Control group were obtained,14 of which matched the database information.The differential metabolites obtained by metabolomics were enriched by pathways and the results showed that glycolysis and galactose metabolism got the highest score.(4)After HIV-1 infection,the level of lactic acid rose,and the glucose uptake capacity enhanced in MDMs.While m RNA of ACAT1,IDH1,IDH2,MDH1,MDH2,SCO2,UQCRB,SDHB and other key enzymes of aerobic oxidation were significantly down-regulated(P<0.05).(5)Expression of HIF-1α and its downstream target genes such as LDHA,PDK1,HK2 and PKM2 were significantly up-regulated in MDMs with HIV-1 infection(P<0.05).The up-regulation of TNF-α,IL-1β and IL-6 by HIV-1 were reversed using 2-DG,which is an inhibitor of glycolysis(P<0.05).(6)With HIV-1infection,the m RNA and protein expression of COX-2 were increased by51.85±1.98 times and 45.38±5.22 times compared with control group,respectively(P<0.001).The increased lactate release level and expression of HIF-1α induced by HIV-1 was inhibited by meloxicam via COX-2(P<0.05).Besides,the M1 polarization marker TNF-α decreased by about 1.3 times(P<0.05),IL-1βdecreased by about 1.5 times(P<0.05)and IL-6 decreased by 2.6 times(P<0.001)with meloxicam treatment.(7)After HIV-1 infection in vitro,JNK in MDMs was apparently increased.m RNA was increased by about 1.5 times(P<0.05),and the phosphorylation level of JNK protein was increased by about 1.7 times(P<0.05).(8)Under SP600125 treatment,the expression of COX-2 activated by HIV-1 was inhibited,m RNA and protein expression were decreased by 27.8 times(P<0.01)and 7 times(P<0.001),respectively.The elevated lactic acid release and M1polarization(high expression of TNF-α,IL-1β,and IL-6)caused by HIV-1infection were also significantly inhibited by SP600125(P<0.001).Conclusion(1)After infected with HIV-1,macrophages will be induced to polarize to M1 status,releasing inflammatory factors and play a pro-inflammatory role.(2)At the same time,intracellular glucose metabolism is reprogrammed,the process of glycolysis is active while TCA cycle and oxidative phosphorylation are inhibited.This process also affect the immune function of macrophages.(3)HIV-1 may activate the JNK/COX-2/HIF-1α axis to strengthen the downstream glycolysis process then promote the M1 polarization in macrophages. |
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