key: cord-0784817-98qu6nte authors: Hu, Wei; Song, Xiang; Yu, Haibo; Zhao, Laura; Zhao, Yeqian; Zhao, Yong title: Further comments on the role of ACE‐2 positive macrophages in human lung date: 2021-08-06 journal: Cytometry A DOI: 10.1002/cyto.a.24484 sha: 0343a7863780e85dc90fad03a549f484cfd3054c doc_id: 784817 cord_uid: 98qu6nte nan Since our previous publication [1] , emerging evidence has demonstrated the actions of lung macrophages in the pathogenesis of COVID-19. However, we understand that there were some inconsistencies in the figures and some of the Data S1 in our original paper. Therefore, we wish to address these by adding Data S1. The ACE2 expression on the surface of macrophages have been well established by studies such as ACE2 on alveolar macrophages [2] [3] [4] [5] [6] , blood monocyte-derived macrophages [7] [8] [9] , and macrophages of atherosclerotic carotid arteries [10] . Specifically, the ACE2 protein expression on alveolar macrophages has been demonstrated by different studies with lung tissues from normal human and COVID-19 patients (Table 1) . Recently, Wang et al. demonstrated that alveolar macrophages derived from COVID-19 patients displayed ACE2 receptor, which can be infected by SARS-CoV-2 [4] . Furthermore, there was high ACE2 expression on normal lung macrophages that enables binding to the S protein [4] . Taken together, these publications support the conclusion and hypothesis regarding the action of alveolar macrophages in the pathogenesis of COVID-19 [1, [17] [18] [19] . There were inconsistent results regarding the ACE2 mRNA expression in alveolar macrophages reported by RNA-seq analysis. Contrasted with the reports [14] [15] [16] , the ACE2 mRNA expression in alveolar macrophages were indicated by other groups [5, 11, 13] . In agreement with these studies, our current studies have demonstrated ACE2 protein expression on alveolar, lipopolysaccharide (LPS)treated and other tissue macrophages [1] . These findings are consistent with other reports [2, [4] [5] [6] [7] . For alveolar macrophages, there are different subpopulations with different phenotypes [20, 21] . To isolate macrophages, all procedures need to be performed on ice with cold phosphate buffered saline (PBS) to avoid the adherence of macrophages to vessels. Therefore, some macrophages may be potentially lost during the cDNA library preparations for bulk or single cell RNAseq. The transcriptomic data from bulk or single cell RNA-seq requires further validation due to the poor correlation between transcriptomic analysis and protein abundance [22] . There were some inconsistencies in the controls for three figures (e.g., Figure 1 (A-C)) in our previous publication [1] . Figure 2C in our original publication [1] has been revised with their associated isotype-matched IgG controls in the overlay histograms ( Figure 1(A) ). The conclusion remains the same, with up-regulations of CD206 and CD209 on the IL-4-treated M2 macrophages, not on the LPS-treated M1 macrophages (Figure 1 (A)) [1] . Figure 2D in our original publication [1] has been revised with two additional panels displaying the ACE2 expressions on M1 and M2 macrophages with their associated second Ab controls respectively ( Figure 1 (B)). The results were consistent with our previous data. The level of ACE2 expression was higher on the LPS-activated M1 macrophages than that of IL-4-treated M2 macrophages ( Figure 1 (B)) [1] . To confirm the ACE2 expression on M1 and M2 macrophages and to circumvent any autofluorescence in the green channel, we performed the immunocytochemistry by using a Cy5-conjugated donkey anti-mouse second antibody (Jackson ImmunoResearch Lab, catalog No. 711-175-150). Figure 2G in our original publication [1] has been revised with M1-and M2-associated second Ab controls respectively for COVID-19 are still lacking. Human pulmonary system is primarily organ targeted by SARS-CoV-2 through ACE2, which has been recognized as the primary entry receptor for SARS-CoV-2 infecting host cells [24] . Current work and previous studies [1] [2] [3] [4] [5] [6] [25] [26] [27] . Therefore, it is critical to improve lung macrophage function, eradicate the viral infections, and stop the transmission. Lung macrophages, as essential antigen-presenting cells, can be derived from yolk sac and bone marrow-derived circulating monocytes [28] . To date, overreactions of monocytes/macropahges have been recognized in the hyperinflammation or cytokine storm of severe COVID-19 [18, 19, 29] . Little to no expression of angiotensin-converting enzyme-2 on most human peripheral blood immune cells but highly expressed on tissue macrophages Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts Alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe COVID-19 patients Angiotensin-converting enzyme 2 (ACE2) expression increases with age in patients requiring mechanical ventilation Factors associated with the expression of ACE2 in human lung tissue: pathological evidence from patients with Normal FEV1 and FEV1/FVC Gamliel-Lazarovich A. ACE2 activity is increased in monocyte-derived macrophages from prehypertensive subjects Mineralocorticoid receptor blocker increases angiotensin-converting enzyme 2 activity in congestive heart failure patients No evidence for human monocyte-derived macrophage infection and antibody-mediated enhancement of SARS-CoV-2 infection Angiotensin-converting enzyme 2 (ACE2) expression and activity in human carotid atherosclerotic lesions Single-cell RNA expression profiling of ACE2, the receptor of SARS-CoV-2 The protein expression profile of ACE2 in human tissues Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics Human lungs show limited permissiveness for SARS-CoV-2 due to scarce ACE2 levels but strong virus-induced immune activation in alveolar macrophages. Sneak Peek Expression pattern of the SARS-CoV-2 entry genes ACE2 and TMPRSS2 in the respiratory tract SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes The lung macrophage in SARS-CoV-2 infection: a friend or a foe? Front Immunol Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages Contribution of monocytes and macrophages to the local tissue inflammation and cytokine storm in COVID-19: lessons from SARS and MERS, and potential therapeutic interventions Characterisation of lung macrophage subpopulations in COPD patients and controls Identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties Integrative proteomics reveals an increase in non-degradative ubiquitylation in activated CD4(+) T cells Human cord blood stem cell-modulated regulatory T lymphocytes reverse the autoimmune-caused type 1 diabetes in nonobese diabetic (NOD) mice Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity BNT162b2-elicited neutralization of B.1.617 and other SARS-CoV-2 variants Developmental origin of lung macrophage diversity The many faces of the anti-COVID immune response Pharmacologic treatments for coronavirus disease 2019 (COVID-19): a review Should chloroquine and hydroxychloroquine be used to treat COVID-19? A rapid review Early safety indicators of COVID-19 convalescent plasma in 5000 patients Reversal of type 1 diabetes via islet beta cell regeneration following immune modulation by cord blood-derived multipotent stem cells Stem cell educator therapy and induction of immune balance Targeting insulin resistance in type 2 diabetes via immune modulation of cord bloodderived multipotent stem cells (CB-SCs) in stem cell educator therapy: phase I/II clinical trial Released Exosomes contribute to the immune modulation of cord blood-derived stem cells Differentiation of monocytes into phenotypically distinct macrophages after treatment with human cord blood stem cell (CB-SC)-derived exosomes Effective treatment of severe COVID-19 patients with tocilizumab SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of this article. How to cite this article The peer review history for this article is available at https://publons. com/publon/10.1002/cyto.a.24484. https://orcid.org/0000-0002-1571-954X