key: cord-0736440-9sm4wvyf
authors: Pontelli, Marjorie C; Castro, Italo A; Martins, Ronaldo B; Veras, Flávio P; Serra, Leonardo La; Nascimento, Daniele C; Cardoso, Ricardo S; Rosales, Roberta; Lima, Thais M; Souza, Juliano P; Caetité, Diego B; de Lima, Mikhael H F; Kawahisa, Juliana T; Giannini, Marcela C; Bonjorno, Letícia P; Lopes, Maria I F; Batah, Sabrina S; Siyuan, Li; Assad, Rodrigo L; Almeida, Sergio C L; Oliveira, Fabiola R; Benatti, Maíra N; Pontes, Lorena L F; Santana, Rodrigo C; Vilar, Fernando C; Martins, Maria A; Cunha, Thiago M; Calado, Rodrigo T; Alves-Filho, José C; Zamboni, Dario S; Fabro, Alexandre; Louzada-Junior, Paulo; Oliveira, Rene D R; Cunha, Fernando Q; Arruda, Eurico
title: Infection of human lymphomononuclear cells by SARS-CoV-2
date: 2020-08-07
journal: bioRxiv
DOI: 10.1101/2020.07.28.225912
sha: 7cb64b7fcc27189a2b53d8a998d1c818cfef58d8
doc_id: 736440
cord_uid: 9sm4wvyf

Although SARS-CoV-2 severe infection is associated with a hyperinflammatory state, lymphopenia is an immunological hallmark, and correlates with poor prognosis in COVID-19. However, it remains unknown if circulating human lymphocytes and monocytes are susceptible to SARS-CoV-2 infection. In this study, SARS-CoV-2 infection of human peripheral blood mononuclear cells (PBMCs) was investigated both in vitro and in vivo . We found that in vitro infection of whole PBMCs from healthy donors was productive of virus progeny. Results revealed that monocytes, as well as B and T lymphocytes, are susceptible to SARS-CoV-2 active infection and viral replication was indicated by detection of double-stranded RNA. Moreover, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from COVID-19 patients, and less frequently in CD4 (+) T lymphocytes. The rates of SARS-CoV-2-infected monocytes in PBMCs from COVID-19 patients increased over time from symptom onset. Additionally, SARS-CoV-2-positive monocytes and B and CD4+T lymphocytes were detected by immunohistochemistry in post mortem lung tissue. SARS-CoV-2 infection of blood circulating leukocytes in COVID-19 patients may have important implications for disease pathogenesis, immune dysfunction, and virus spread within the host.

CoV-2 antigen was found post mortem in the spleen and lymph nodes with 91 pathological signs of damage. In these organs, monocytes do contain viral 92 method. All experiments involving SARS-CoV-2 propagation were done in 158 biosafety level 3 laboratory. 159

160 In vitro infection of PBMCs. For these experiments, 10 6 PBMCs from 5 161 healthy donors were infected with SARS-CoV-2 (MOI=1) in RPMI with 0% FBS 162 at RT for 1 h under orbital agitation. Next, cells were pelleted at 300 × g, the 163

inoculum was washed and replaced by RPMI with 2% FBS and cells were 164 incubated at 37°C in 5% CO2. As controls, equivalent quantities of cells were 165 exposed to UV-inactivated SARS-CoV-2 and treated in the same way. We also 166 supernatants from in vitro assays. All real-time PCR assays were done on a 182

Step-One Plus thermocycler (Applied Biosystems, Foster City, CA, USA). Coverslips pre-treated with poly-lysine 0.1% (Sigma-Aldrich, cat. P8920) were 197 incubated with isolated PBMCs from patients or healthy donors at 37ºC, 20 198 minutes for cell adherence. After that, coverslip-containing cells were fixed with 199 4% paraformaldehyde (PFA) in PBS for 15 minutes, and then washed 3 times 200 with PBS. To detect viral antigens in cells, we used serum from a recovered 201 COVID-19 patient, which was first tested for specificity by immunofluorescence 202 in SARS-CoV-2 infected Vero CCL81 cells (Supplementary Fig 1B) . In 203 addition, for each experiment using the referred serum we included cells from 204 healthy donors or non-infected cells. As an isotype control of this serum, we 205 used a human serum collected in 2016. Biotin-conjugated anti-human IgG 206 (Sigma-Aldrich, cat. B-1140) was used as the secondary antibody, followed by 207 amplification with the TSA Cyanine 3 System (Perkin Elmer, NEL704A001KT), 208

following the manufacturer's protocol. To determine the phenotype of SARS-209 Treatment with trypsin for 60 min on ice after infection to remove surface-bound 242 viral particles was also included as a second control (Supplementary Fig 2) . 243 SARS-CoV-2 antibodies were detected with secondary anti-Mouse Alexa488. 244

Surface phosphatidylserine (PS) staining was carried out in whole blood using 245

ApoScreen AnnexinV-FITC apoptosis kit (SouthernBiotech cat 10010-02), 246 following manufacturer's guidelines. All data were acquired using a Verse or ab133335 ) and IL-6 (BD cat. 554400). After each round of staining, slides were 259 . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint scanned using a VS120 ScanScope (Olympus ) under 400x magnification. 260

Images were pseudocolored and overlaid in the first image of the preparation 261 counterstained with hematoxylin using ImageJ v1.50b (NIH, USA) and Adobe (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint

SARS-CoV-2 infection of human PBMCs is productive. Considering that 277 human lymphocyte and monocyte lineages are susceptible to SARS-CoV-2 278 infection in vitro, we sought to determine whether primary cultures of human 279

PBMCs could also be infected. Therefore, PBMCs from five healthy donors 280 were infected in vitro at a MOI=1. After 0, 6, 12, 24 and 48 hpi, supernatants 281 were harvested, and virus progeny was titrated. SARS-CoV-2 titers peaked 282 between 6 and 12 hpi, resulting in a 100-fold increase from the initial input, and 283 decreased steadily thereof (Fig 1A) . As expected, induction of general 284 intracellular alkalization by treatment with NH4Cl reduced progeny production by 285 approximately 10x (p=0.017). Interestingly, virus progeny production was not 286 entirely abolished by NH4Cl treatment, suggesting an entry pathway alternative 287 to endosomal acidification in PBMCs (Fig 1B) . 288

Even though expression of ACE2 is minimal in human PBMCs in general 289

[13, 14], we evaluated the viral production after blocking ACE2 and TMPRSS2. 290

Virus titers obtained after Camostat blockage of TMPRSS2 were not 291 significantly different from those obtained without the treatment (Fig 1C) , 292

suggesting that PBMC infection is not dependent on TMPRSS2. Conversely, 293 the blockage of ACE2 with anti-ACE2 antibody resulted in reduction, but not 294 abrogation of SARS-CoV-2 progeny production after 24 hpi (p=0.0216) (Fig  295   1C ), indicating that SARS-CoV-2 can infect human PBMCs independently of 296

Coronavirus replication entails the formation of abundant double-298 stranded RNAs (dsRNA) in the cytoplasm of infected cells, and thus its 299 intracellular detection is a reliable marker of viral replication. Therefore, infected 300

PBMCs were stained for SARS-CoV-2 and dsRNA and analyzed by confocal 301 microscopy. Most SARS-CoV-2-positive cells were also positive for dsRNA, and 302 rates of double-positive cells counted at 6 hours post-infection followed a 303 pattern that roughly matched the accumulation of progeny (Fig. 1D) . The 304 dsRNA staining was seen as clear puncta in SARS-CoV-2-infected cells, in a 305 pattern suggestive of virus factories. 306 307 . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint

vitro infection. To determine the susceptibility of circulating leukocytes to 309 SARS-CoV-2, PBMCs from five healthy donors were infected (MOI=1), and 310 analyzed the intracellular expression of SARS-CoV-2 antigens by flow 311 cytometry. After 24 hpi, SARS-CoV-2 was detected in all immunophenotyped 312 cells (Fig 2A) . Monocytes were the most susceptible cell type, showing 313 significant SARS-CoV-2 antigen staining (44.3%, p=0.039) (Fig 2B) . In addition 314 to monocytes, T CD4 + (14.2%, p=0.028), CD8 + (13.5%, p=0.019) and B 315 lymphocytes (7.58%) were also susceptible to SARS-2 infection (Fig 2C) . 316

Staining for SARS-CoV-2 was significantly reduced in cells treated with NH4Cl, 317

suggesting that acidification is important for in vitro infection of PBMCs. post-infection by analizing its binding to annexin V (Fig 3) . Despite the basal 326 annexin V staining (CD4 + mean 6.24%, CD8 + mean 12.36%) seen in non-327 infected cells (Fig 3A) , strong staining was observed both in live T CD4 + 328 (70.88%, p=0.0001) and CD8 + + lymphocytes (39.72%, p=0.0009) (Fig 3B) . 329

When cells were analyzed independently of Live/Dead staining, differences 330 were still significant and even increased for CD8 + (59.64%, p=0.0001) 331

( Supplementary Fig 4) , indicating that a considerable percentage of Annexin 

CoV-2. During April 7th to June 18 th , we enrolled 22 COVID-19 patients that 340

were admitted to the intensive care unit (ICU), presenting a moderate to severe 341 . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. analyzed PBMCs prepared from the whole blood of 22 patients and 11 healthy 345 donors by flow cytometry (Fig 4A) with staining for SARS-CoV-2 antigens. Cells 346 from COVID-19 patients showed significant expression of SARS-CoV-2 347 antigens (7.68%±1.56 p=0.008) in comparison with cells from healthy donors 348 (Fig 4B) . Interestingly, not all COVID-19 patients showed expressive staining 349

for SARS-CoV-2, and rates of SARS-CoV-2-positive cells ranged from 0.16 to 350 33.9% (Fig 4B) . Additionally, PMBCs from 15 COVID-19 patients were tested patients, with no discernible fluorescent signal seen in PBMCs from healthy 368 donors (Fig 4D) . Despite what was observed by FC experiments, some IF 369 staining was found in CD4 T lymphocytes, and after extensively screening, very 370 few T CD8 cells were found to be positive for IF (Supplementary Fig 5) . 371

Since the detection of SARS-CoV-2 in patients was found to be variable 372 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint frequencies were plotted on a heatmap for all cell immunophenotypes analyzed 376 (Fig 4E) . It became clear that rates of SARS-CoV-2-positive B lymphocytes 377 were high throughout the entire dataset. In contrast, rates of SARS-CoV-2-378 positive monocytes were higher after following time progression after symptoms 379 onset (Fig 4E) . Frequencies of SARS-CoV-2-positive cells correlated positively 380 with the length of time of COVID-19 progression after symptoms onset, 381 especially for inflammatory CD14 + CCR2 + monocytes (r=0.442 p=0.044) (Fig  382   4F) . 383

To confirm whether SARS-CoV-2 was actively replicating in PBMCs from 384 COVID-19 patients, we analyzed the presence of dsRNA in SARS-CoV-2-385 positive cells of different immunophenotypes by immunofluorescence and 386 confocal microscopy. Remarkably, dsRNA staining was found in most SARS-387

CoV-2-positive cell subsets, CD4 + T lymphocytes, B lymphocytes, and 388 monocytes (Fig 5) . Altogether, these data confirm that SARS-CoV-2 infects 389 circulating white blood cells from COVID-19 patients, and the frequencies of Fig 6) . Upon staining for SARS-CoV-2, slides were scanned, 402 the staining was erased, and re-stained sequentially for the surface antigens 403 CD4, CD20, and CD14. The serial immunolabelling indicated that CD4 + T 404 lymphocytes, B lymphocytes, and monocytes express SARS-CoV-2 antigens 405 (Fig 6) in the lungs of COVID-19 cases. Additionally, due to its well-known role 406 in lung tissue damage in COVID-19, IL-6-positive cells were also searched for 407 and, interestingly, several CD14 + monocytes expressing IL-6 were also positive 408 . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint for SARS-CoV-2 (Fig 6C-E) , indicating that inflammatory monocytes in lungs of 409 COVID-19 patients can also be infected with SARS-CoV-2. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint

The immunophenotyping of PBMCs infected in vitro with SARS-CoV-2 442 revealed that CD14 + , CD4 + , CD8 + and CD19 + cells were susceptible. Primary 443 human monocytes have been reported as susceptible to MERS-CoV and, more 444 recently, to SARS-CoV-2 [20, 21] . In contrast, another recent study did not 

and induce T-cell apoptosis through extrinsic and intrinsic pathways [20] . (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint

containing diverse immune cell types that bear close contact with SARS-CoV-2-511 infected lung cells, such as pneumocytes and alveolar macrophages [33] . In the 512 present study, we found CD4 + T and B lymphocytes and, importantly, also IL-6-513 expressing inflammatory monocytes positive for SARS-CoV-2 infiltrating the 514 lung tissue from fatal cases of COVID-19. Further studies will be required to 

The authors declare none. 

. CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint described for lymphocytes. Next, expression of CD14 and CD16 was used to 808 define circulating monocyte subpopulations. Expression of CCR2 by CD14 + and 809 CD14 + CD16 + cells was used to define inflammatory monocytes. Among every 810 defined subpopulation, expression of SARS-CoV-2 antigens was defined in 811 comparison with secondary antibody background and healthy donors staining 812

(flow plots and representative histograms). 813 814 Figure S4 . (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 7, 2020. ; https://doi.org/10.1101/2020.07.28.225912 doi: bioRxiv preprint

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MOI-1) and cultured for 48 h. (A) Overtime virus progeny 704 production from PBMCs infected with SARS-CoV-2

PBMCs were collected at each time point and titrated by TCID50. The small 706 symbols represent individual values (5 healthy donors) and error bars depict 707 standard deviation. (B) SARS-CoV-2 progeny titers in supernatants of

PBMCs at 24 and 48 hpi, with and without treatment with 20mM NH4Cl

Effects of blocking SARS-CoV-2 cell receptor ACE2 and TMPRSS2 on virus 710 progeny production

Camostat and virus progeny was titrated in supernatants at 24 hpi

Immunostaining for dsRNA in PBMCs cultured on poly-lysine -coated

coverslips 6h after SARS-CoV-2 infection. Cells were fixed

SARS-CoV-2 (red), dsRNA (cyan) and analyzed by confocal microscopy

Statistical analysis was performed using one-way or two-way ANOVA. Tukey's 716 or Holm-Sidak post-tests were applied when suitable