key: cord-0720934-dihslblj
authors: Boumaza, Asma; Gay, Laetitia; Mezouar, Soraya; Diallo, Aïssatou Bailo; Michel, Moise; Desnues, Benoit; Raoult, Didier; Scola, Bernard La; Halfon, Philippe; Vitte, Joana; Olive, Daniel; Mege, Jean-Louis
title: Monocytes and macrophages, targets of SARS-CoV-2: the clue for Covid-19 immunoparalysis
date: 2020-09-17
journal: bioRxiv
DOI: 10.1101/2020.09.17.300996
sha: eb3aea1a7ad15fa07b7ae81f5ce2c624af63e2df
doc_id: 720934
cord_uid: dihslblj

To date, the Covid-19 pandemic affected more than 18 million individuals and caused more than 690, 000 deaths. Its clinical expression is pleiomorphic and severity is related to age and comorbidities such as diabetes and hypertension. The pathophysiology of the disease relies on aberrant activation of immune system and lymphopenia that has been recognized as a prognosis marker. We wondered if the myeloid compartment was affected in Covid-19 and if monocytes and macrophages could be infected by SARS-CoV-2. We show here that SARS-CoV-2 efficiently infects monocytes and macrophages without any cytopathic effect. Infection was associated with the secretion of immunoregulatory cytokines (IL-6, IL-10, TGF-β) and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In addition, we found that in vitro macrophage polarization did not account for the permissivity to SARS-CoV-2, since M1-and M2-type macrophages were similarly infected. Finally, in a cohort of 76 Covid-19 patients ranging from mild to severe clinical expression, all circulating monocyte subsets were decreased, likely related to massive emigration into tissues. Monocytes from Covid-19 patients exhibited decreased expression of HLA-DR and increased expression of CD163, irrespective of the clinical status. Hence, SARS-CoV-2 drives circulating monocytes and macrophages inducing immunoparalysis of the host for the benefit of Covid-19 disease progression.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-n-54 CoV) emerged in Wuhan (China) at the end of 2019 and caused the coronavirus disease of 55 2019 (Covid-19) pandemic in a few weeks, affecting more than 18 million people and killing 56 more than 690,000 to date (1). The disease is characterized by a strikingly heterogeneous 57 clinical presentation and prognosis. Most patients are pauci-symptomatic or have fever, cough 58 and fatigue, while a minority experience progression to an acute respiratory distress syndrome 59 or other critically severe conditions. The severity of the disease is related to underlying 60 conditions such as hypertension, diabetes, coronary heart diseases or obesity (2). The 61 mechanisms of the disease remain elusive at this stage, but evidence for a prominent role of 62 the immune system is accumulating. The severity of Covid-19 pneumonia is associated with 63 lymphopenia and a cytokine release syndrome (CRS) (3), which contributes to the massive 64 migration of T cells into tissues, mainly the lung as revealed by accumulation of T cells 65 within lesions (4). 66

There is evidence that myeloid cells may be involved in the pathophysiology of 67 coronavirus infection, either directly, as a targets for the virus, or indirectly, as effectors of the 68 CRS (5) . Indeed, it is known from previous coronavirus outbreaks that macrophages are 69 susceptible to MERS-CoV and SARS-CoV-1 infection (6). Recently, macrophage and 70 monocyte accumulation in the alveolar lumen has been shown in a humanized mice model of 71 SARS-CoV-2 expressing human angiotensin-converting enzyme 2 (ACE2) (7). In addition, 72 SARS-CoV-2 nucleocapsid protein has been detected in lymph nodes and spleen-associated 73 CD169 + macrophages from Covid-19 patients (8). Finally, single cell RNA sequencing of 74 pulmonary tissue from Covid-19 patients revealed an expansion of interstitial macrophages 75 and monocyte-derived macrophages (MDM) but not of alveolar macrophages (9). However, 76 whether circulating monocytes and/or macrophages are targets of SARS-CoV-2 and whether 77 monocyte diversity is altered in Covid-19 patients require specific investigation since most 78 studies are based on this hypothesis. 79

Monocytes are innate hematopoietic cells that maintain vascular homeostasis and 80 ensure early responses to pathogens during acute infections. Three distinct human monocyte 81 subsets are described, based on the expression of CD14 and CD16 surface antigens: classical 82 CD14 + CD16monocytes, intermediate CD14 + CD16 + monocytes, and non-classical CD14 -83 CD16 + monocytes (10,11). Recently, it has been shown in murine models that classical 84 monocytes are the precursors of non-classical monocytes (12). There is evidence that 85 monocyte subsets exhibit a certain degree of functional specialization. During We show here that SARS-CoV-2 has the ability to infect human monocytes and 96 macrophages. SARS-CoV-2 infection stimulated the production of immunoregulatory 97 cytokines, interleukin (IL)-6 and IL-10 in both cell types and triggered in macrophages an 98 original transcriptional program enriched with M2-type genes. Macrophage polarization did 99 not account for permissivity to the virus since M1-and M2-polarized cells were similarly 100 infected by SARS-CoV-2. In Covid-19 patients, the numbers of classical, intermediate and 101

non-classical monocytes were decreased, irrespective of the level of severity. Their expression 102 of CD163, a molecule associated with the immunoregulatory phenotype, was significantly 103 higher than in healthy controls, whereas that of HLA-DR was decreased. Hence, SARS-CoV-104 2 drives circulating monocytes and macrophages, inducing immunoparalysis of the host for 105 the benefit of Covid-19 disease progression. 106

It has been shown that monocytes and macrophages express receptors for SARS-CoV-2 (24), 109 suggesting that the virus targets myeloid cells. We wondered whether SARS-CoV-2 was able 110 to infect human monocytes and macrophages. Monocytes, MDM and Vero cells were 111 incubated with SARS-CoV-2 strain IHU-MI3 (0.1 MOI) for 24 and 48 hours and infection 112 level was measured by RT-PCR and immunofluorescence. SARS-CoV-2 infected efficiently 113

Vero cells (Ct=18.69) after 24 hours, but a lytic process prevented the measurement of viral 114 replication, (Figure 1A ). Monocytes were also infected after 24 hours (Ct=22.44), but the 115 viral load remained constant thereafter (Ct=22.2) ( Figure 1A) . Similarly, macrophages were 116 efficiently infected with the SARS-CoV-2 strain IHU-MI3 after 24 (Ct=22.49) and 48 hours 117 (Ct=19.67). In contrast to Vero cells, monocytes and macrophages were not uniformly 118 infected, as observed by confocal microscopy ( Figure 1A , right panel). We next addressed 119 the ability of the IHU-MI3 strain of SARS-CoV-2 to induce the release of soluble mediators 120 from monocytes and MDMs. IL-1β, IL-6, IL-10, TNF-α, IFN-β and TGF-β1 levels were 121 measured in supernatants of monocytes or MDM stimulated with SARS-CoV-2 for 24 and 48 122 hours. IL-6, IL-10, and IL-1β levels were significantly increased in stimulated monocyte 123 supernatants as compared to unstimulated conditions after 24 hours ( Figure 1B ) and were 124 persistently increased after 48 hours ( Figure 1C ), whereas no difference was observed for 125 TNF-α. In MDM supernatants, levels of IL-6 and IL-10 were increased after 24 and 48 hours 126 ( Figure 1B and C) . TGF-β levels were significantly increased in supernatants from 127 monocytes and MDMs after 48 hours of stimulation. IFN-β was never detected in 128 supernatants from monocytes or macrophages stimulated by SARS-CoV-2 ( Figure 1B High-dimensional immune profiling by mass cytometry revealed immunosuppression 549

and dysfunction of immunity in COVID-19 patients. 

SARS-CoV-2 shedding and infectivity -Authors' reply. 429 The Lancet

Cytokine release syndrome in severe COVID-19

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Immunopathogenesis of coronavirus infections: implications 436 for SARS

Pathogenic human coronavirus infections: causes and 438 consequences of cytokine storm and immunopathology

The 441 pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

The Novel Severe Acute Respiratory Syndrome Coronavirus

Directly Decimates Human Spleens and Lymph Nodes. Infectious Diseases (except 446 HIV/AIDS

The 448 landscape of lung bronchoalveolar immune cells in COVID-19 revealed by single-cell 449 RNA sequencing

Nonclassical Monocytes in 451 Health and Disease

Identification and characterization of a 454 novel monocyte subpopulation in human peripheral blood

The fate and lifespan of human monocyte subsets in 457 steady state and systemic inflammation

Developmental and Functional Heterogeneity of 460

The myeloid cell compartment-cell by cell