analysis of HCQ showing preferential protection for early target cell types in the lung, consistent with the increased efficacy seen with early treatment.
Authors analyze various cell types in bronchoalveolar lavage fluid from COVID-19 patients, providing ultrastructural evidence that prophylactic HCQ can reduce SARS-CoV-2 infection in certain lung epithelial cell types like ciliated epithelium and type II pneumocytes, while no protection was seen in alveolar macrophages, neutrophils, and enucleated granulocyte fragments, although enucleated granulocyte fragments exhibited an enhanced ability to phagocytize and destroy mature, infectious SARS-CoV-2 virus.
Previous studies have shown HCQ preferentially accumulates in lung epithelial cells compared to immune cells, aligning with the cell-specific antiviral effects seen here. The differential impact likely contributes to heterogeneous clinical outcomes based on timing, severity, and patient factors.
Reduced SARS-CoV-2 infection in lung epithelial cells may help limit disease severity and progression for early treatment, while the lack of benefit to immune cells with the same dosage could explain poor results for late-stage infection involving immune hyperactivation.
The typical sequence of SARS-CoV-2 infection of the lung cells here is likely to be:
- Ciliated epithelium - cells lining the airways are likely the initial site of virus exposure and replication after inhalation of respiratory droplets.
- Type II pneumocytes - the virus spreads to infect type II pneumocytes underlying the epithelium.
- Alveolar macrophages - resident macrophages in the alveoli encounter and phagocytose infected dead cells and viruses, becoming infected themselves. As antigen presenting cells, they also trigger an inflammatory immune response.
- Neutrophils - circulating neutrophils are recruited as part of the inflammatory response, entering the lungs and also becoming infected.
By preferentially protecting early target cell types, HCQ may be most beneficial early in infection.
One theory for the antiviral activity of HCQ is alteration of endosomal pH Al-Bari, Shang
which may inhibit replication of endosomal low pH dependent viruses like SARS-CoV-2 Al-Bari, Kreutzberger
. The endosomal pH altering effects of HCQ may differ between cell types in a dose-dependent manner based on inherent biological properties of each cell lineage. For example, HCQ accumulation, endosomal pH dynamics, reliance on endosomes for viral entry vary across cell types like lung epithelial cells and immune cells, base levels of endosomal/lysosomal pH, and expression levels of key proteins involved in endosomal acidification. HCQ's effect on endosomal pH may also vary over time, with some studies showing an initial increase followed by a later decrease, which may explain the differential efficacy of early treatment vs. prophylaxis.
This study underscores the complexity of pharmacological responses in different cell types during viral infection, and provides further rationale for multi-drug combination therapies, and for early treatment in general before progression to other tissues, as used by the most successful physicians.
Ruiz et al. Ruiz
and this study confirm that therapeutic levels of HCQ are obtained and provide protection in the lung focused on cells corresponding to earlier infection.
Chaudhary et al., 5 Aug 2023, India, peer-reviewed, 9 authors.
Impact of prophylactic hydroxychloroquine on ultrastructural impairment and cellular SARS-CoV-2 infection in different cells of bronchoalveolar lavage fluids of COVID-19 patients
Scientific Reports, doi:10.1038/s41598-023-39941-6
Many drugs were recommended as antiviral agents for infection control and effective therapy to reduce the mortality rate for COVID-19 patients. Hydroxychloroquine (HCQ), an antimalarial drug, has been controversially recommended for prophylactic use in many countries, including India, to control SARS-CoV-2 infections. We have explored the effect of prophylactic HCQ from the cells of bronchoalveolar lavage fluids from COVID-19-induced acute respiratory distress syndrome patients to determine the level of infection and ultrastructural alterations in the ciliated epithelium, type II pneumocytes, alveolar macrophages, neutrophils, and enucleated granulocytes. Ultrastructural investigation of ciliated epithelium and type II pneumocytes showed lesser infections and cellular impairment in the prophylactic HCQ + group than HCQ -group. However, macrophages and neutrophils displayed similar infection and ultrastructural alterations in both patient groups. The enucleated fragments of granulocytes showed phagocytosis of the matured virus in HCQ + groups. The present report unveils the ultrastructural proof to complement the paradox regarding the role of prophylactic HCQ in COVID-19 patients. The COVID-19 outbreak caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly propagated with nearly half a billion infected human beings globally 1 . There were negligible reports on specific and effective treatments for this deadly infection. Due to sudden outbreaks and very high mortality (29%) 2 by delta variant, many random trials and repurposing of existing drugs were conducted to control and cure the COVID-19 disease 2,3 . Hydroxychloroquine (HCQ), an antimalarial drug, has gained significant attention in the initial phase of COVID-19 from May 2020 onwards 4,5 . This drug was earlier reported to be effective (in vitro) in reducing viral internalization (by blocking proteolytic activation of S-protein) and replication (increasing the acidic environment of the endosome to inhibit viral assembly), including the SARS-CoV-2 and MERS-CoV 6-8 . The anti-SARS-CoV-2 effect of HCQ (by inhibiting internalization and proliferation) was proposed due to its ability to increase endosomal acidification, reduction of cathepsin L activation, interference with ACE-2 terminal glycosylation, proteolytic self-activation of furin, and the blockage of clathrin-mediated endocytosis 4,    . The immunomodulatory effects, alkalinization of vacuolar pH, Zinc ionophores, and binding ability of HCQ to sialic acids were proposed to inhibit the COVID-19 infection in vitro non-specifically 9,12-14 . Many clinical trials were initiated in various countries to investigate the effect of HCQ in the control and cure of COVID-19 disease    . It was reported that HCQ was very effective in reducing the multiplication of the SARS-COV-2 virus under in vitro culture conditions using Vero E6 cells with 6.90 µM concentration (EC) 90 18 .
Author contributions S.C. performed the experiments for sample processing, PAP and IF imaging standardization, electron microscopy, interpretation of the data, management of clinical records, and generation of the microscopy figures. P.R. performed the immunofluorescence imaging, recorded the clinical data, and helped S.C. with electron microscopyrelated experiments. A.J. performed the PAP imaging. K.S. collected the sample from the ICU wards. S.K. was involved in designing the study and standardizing the sample collection strategies. A.R.M. and T.C.N. helped interpret the results and the images and correction of manuscripts. A.R.M. and U.B. also helped in the correction of the manuscript. S.C.Y. designed the study, performed, and supervised COVID-19 patient experiments, analyzed the data, generated the figures, wrote the manuscript, and directed the project. All authors read and approved the final draft of this manuscript.
Competing interests The authors declare no competing interests.
Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-023-39941-6. Correspondence and requests for materials should be addressed to S.C.Y. Reprints and permissions information is available at www.nature.com/reprints. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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