Microparticles Enriched With Oxidized Cholesterol Target Alveolar Macrophages For The Treatment Of SARS-CoV-2 Infectio

Objective:SARS-CoV-2 is an emerging coronavirus,which has caused more than 490 million infections in the world.To date,vaccines,neutralizing antibody and anti-viral drugs are the main therapeutic strategies.However,currently available vaccines can prevent viral spread,they are unable to treat infected patients and the effect of neutralizing antibody remains uncertainty.On the other hand,the anti-viral drugs have not been applied widely.Thus,this suggests that anti-viral drugs need more clinical data to validate their safety and efficacy and pricing obstacles lead to difficult widespread application.Thus,exploration of strategies for treatment of COVID-19 patients is needed.SARS-CoV-2 mainly invades the alveoli,causing dry cough and hypoxia.Alveolar macrophages(AMs)are the most abundant immune cells residing in the alveoli,which are responsible for the defense against foreign microbes.However,how the AMs response to SARS-CoV-2 is unclear.Microparticles(MPs),an extracelluar particle with mean size of 500 nm,are used to deliver chemotherapeutic drugs for cancer treatment as new biocarrier.Furthermore,macrophage is a target for MPs therapy and this therapeutic method is safe,low-cost and simple.Thus,we are pleased to explore the role of AMs in SARS-CoV-2 infection and modify MPs for a new treatment of COVID-19.Methods:1.AMs isolated from ICR mice were polarized into M1 and M2,then the efficacy of taking up virus by the two phenotypes of AMs was analyzed.To test whether ACE2 affected the phagocytosis of AMs,we generated hACE2-overexpressing AMs or knocked down ACE2 in AMs by siRNAs.2.The virus was incubated with different phenotypes of AMs,and then the viral load was detected in time points to compare the SARS-CoV-2 replication in two phenotypes of AMs.To validate whether replicated virus was released,the supernatants for AMs were collected to infect Vero E6.3.The pH of endosome and lysosome for Ml and M2 AMs was determined by pH-sensor probe to explain the diverse replication in AMs.4.We modified A549 cells into A549-ACE2 overexpressing cells or ACE2-konckout cells by lentiviral system and CRISPR-Cas9,respectively.Then,AO-MPs(ACE2-overexpressing A549 cells-derived microparticles)and AD-MPs(ACE2-knockout A549 cells-derived microparticles)were extracted to validate their capacity for adsorbing virus.5.AO-MPs binding virus were given to mice by intranasal administration to test whether MPs could be taken up by AMs first.In vitro,AMs and ATII cells isolated from ICR mice were infected with SARS-CoV-2 to detect virus was taken up by AMs first.6.AO-MPs binding virus were incubated with AMs,and viral load was detected at different time points to determine the fate after the virus was phagocytosed by AMs.To test how the AMs degrade virus with the assistance of MPs,the endosomal and lysosomal pH in AO-MPs-treated AMs was detected.Further,we validated that whether oxized cholesterol in AO-MPs caused the change of endosomal pH.7.We validated the phenotype of AO-MPs-treated AMs.8.The animal model of SARS-CoV-2 infection was established using hACE2-transgenic mice and the therapeutic effect of AOMPs on infected mice was verified in vivo by intranasal administration once a day for five days.Results:1.The viral load in M1 AMs was higher than that in M2 with a short phagocytosis in an ACE2-independent manner.2.With the prolonged time for incubation AMs with virus,virus load in M1 was higher than M2 ones.In addition,the supernatants for culturing M1 AMs showed more infective for Vero E6.3.The endosomal acidity of M1 AMs is more acidic than M2,while lysosomal pH of M2 AMs was lower than M1.Thus,the colocalization of NP and endosome or lysosome was rare in M1 AMs.4.AO-MPs had a stronger ability to adsorb viruses than A-MPs and AD-MPs.5.AO-MPs adsorbed virus were taken up by AMs first and the adsorbed virus was delivered to AMs for degradation.6.AO-MPs were rich in oxidized cholesterol 25-hydroxycholesterol(25-HC),which inhibited endosomal acidification by interfering v-ATPase and decreased lysosomal pH,further preventing the virus from escaping into cytoplasm and promoting the virus to be degraded by AMs.7.AO-MPs could impede the cytokines upregulation by virus infection.however,the production of IFNs was not altered.8.AO-MPs-treated mice show less viral load,slight inflammatory cytokines(IL-1β,TNF-α,IL-6)and reduced pathological damage.Conclusions:AMs take up SARS-CoV-2 in a ACE2-independent manner and M1 AMs have a higher capacity for viral phagocytosis than M2 ones.However,endosomes are more acidic in M1 AMs than their M2 counterparts;whereas,the lysosomes are more acidic in M2 AMs.Thus,virus in M1 is apt to replicate and to degrade in M2.Based on this mechanism,modified AO-MPs effectively adsorb SARS-CoV-2 and then virus is delivered into AMs.Meanwhile,oxidized cholesterol in AO-MPs interfering with endosomal proton pumps increases endosomal pH to prevent viral RNA releasing into cytoplasm.In addition,AO-MPs could not induce hyper inflammation during virus degradation.Thus,AO-MPs treatment is a promising strategy for COVID-19.