key: cord-0761527-jyaig9lk
authors: Cérbulo-Vázquez, A.; García-Espinosa, M.; Briones-Garduño, J.C.; Arriaga-Pizano, L.; Ferat-Osorio, E.; Zavala-Barrios, B.; Cabrera-Rivera, G.L.; Miranda-Cruz, P.; García de la Rosa, M.T.; Prieto-Chávez, J.L.; Rivero-Arredondo, V.; Madera-Sandoval, R.L.; Cruz-Cruz, A.; Salazar-Rios, E.; Salazar-Rios, ME; Serrano-Molina, D; De Lira-Barraza, R. C.; Villanueva-Compean, A. H.; Esquivel-Pineda, A.; Ramirez-Montes de Oca, R.; Caldiño-Soto, F.; Ramírez-García, L.A.; Flores-Padilla, G.; Moreno-Álvarez, O.; Guerrero-Avendaño, GML; López-Macías, C.
title: The percentage of Monocytes CD39+ is higher in Pregnant COVID-19 than in Non-Pregnant COVID-19 patients
date: 2021-06-19
journal: bioRxiv
DOI: 10.1101/2021.06.18.449054
sha: a1969610de8db5560c8a8b9b3e4917301cf56417
doc_id: 761527
cord_uid: jyaig9lk

Current medical guidelines consider COVID-19 pregnant women a high-risk group. Physiological gestation down regulates the immunological response to maintain “maternal-fetal tolerance”; hence, a SARS-CoV-2 infection constitutes a potentially threatening condition to both the mother and the fetus. To establish the immune profile in pregnant COVID-19+ patients a cross-sectional study was conducted. Leukocyte immunophenotype, mononuclear leukocyte response to polyclonal stimulus and cytokine/chemokine serum concentration were analyzed in pregnant fifteen COVID-19+ and control groups (fifteen non-pregnant COVID-19+, and thirteen pregnant COVID-19-women). Pregnant COVID-19+ patients exhibit lower percentages of monocytes HLA-DR+ compared with control groups. Nevertheless, pregnant COVID-19+ women show a higher percentage of monocytes CD39+ than controls. Furthermore, a higher concentration of TNF-α, IL-6, MIP1b and IL-4 was observed within the pregnant COVID-19+ group. Our result shows that pregnant women express immunological characteristics that potentially mediate the immune response in COVID-19.

Analysis was performed using the Infinicyt™ Software 1.8. 168

Serum or cell culture supernatant was analyzed as follows; cytokines (IL-2, IL-4, IL-6, 170 IL-10, TNF-α, IFN-γ, and IL-17a) and chemokines (CXCL8/IL-8, CXCL10/IP-10, 171 CCL11/Eotaxin, CCL17/TARC, CCL2/MCP-1, CCL5/RANTES, CCL3/MIP-1a, 172 CXCL9/MIG, CXCL5/ENA-78, CCL20/ MIP-3a, CXCL1/GROa, CXCL11/I-TAC and 173 CCL4/MIP-1b) were determined using bead based immunoassays (CBA kit, Cat. 174 560484, BD PharMingen, San Diego, CA, USA; and LEGENDplex, Cat. 740003, 175 BioLegend, San Diego, CA, USA). Log-transformed data were used to construct 176 standard curves fitted to 10 discrete points using a 4-parameter logistic model. To assess the immune profile in pregnant women infected with SARS-CoV-2, we 188 analyzed; a) NP-COVID-19+, b) P-COVID-19+ and c) P-COVID-19-. Table 1 fibrinogen between the NP-COVID-19+ or P-COVID-19-groups vs. P- We did not observe any difference in the leukocyte count among groups, this is 195 consistent after phenotyping analysis by flow cytometry (Table S1 ). Furthermore, the 196 frequency of comorbidity (diabetes mellitus and systemic arterial hypertension) was 197 similar among the groups. On the other hand, some clinical characteristics display 198 differences, for example, heart rate was higher in P-COVID-19+ than in P- patients (p=0.048). Likewise, serum Lactate Dehydrogenase (LDH) concentration was 200 higher in P-COVID-19+ than in P-COVID-19-group. Nevertheless, patients in the P-201 COVID-19-group maintain higher oxygen saturation levels than in patients with or without pregnancy. Regarding serum D-dimer concentrations, higher 203 magnitudes were reported in P-COVID-19+ than in NP-COVID-19+. It is worthy of 204 highlighting that a SARS-CoV-2 infection does not increase the D-dimer concentration to the levels reported in physiological pregnancy. Similar gestational age, and number 206 of cases per trimester were analyzed in pregnant women. 207 208 

Cellular immune response is essential against SARS-CoV-2 infection. Using flow 223 cytometry, the proportion of leukocytes with an activated phenotype (HLA-DR+ or 224 CD69+) or inflammatory regulators (CD39+ or CD73+) was determined in peripheral 225 blood samples from the three groups by phenotyping, as described in the methods, and 226

were compared among each other. Figure 1 shows the lower proportion of monocytes 227 Along the same line, a lower percentage of monocytes HLA-DR+ was observed in NP-229 COVID-19+ than in P-COVID-19-group (p=0.0003), while NP-COVID-19+ and P-230 COVID-19+ show similar percentage of monocytes HLA-DR+. In contrast, the 231 percentage of CD4 or CD8 T lymphocytes that express HLA-DR or the early activation 232 marker (CD69) were similar among groups (Table S2) . 233

Regarding CD39, the percentage of monocytes CD39+ was lower in the NP-COVID-234 19+ than in the P-COVID-19+ group (p=0.004), a lower percentage was noticed in NP-235 COVID-19+ than in P-COVID-19-group; however, this difference was not statistically 236 significant (p = 0.663). A tendency in P-COVID-19+ women to express higher 237 percentage of monocytes CD39+ compared to non-infected gestating women (p=0.084) 238 was observed. CD39 expression was furthermore determine in B and T lymphocytes, 239 noticing a higher percentage of B CD39+ cells in P-COVID-19+ and P-COVID-19-240 compared to NP-COVID-19+ group, but these differences were not statistically 241 significant (p=0.127, p=0.487, respectively). On the other hand, T CD39+ cells are 242 higher in NP-COVID-19+ than in P-COVID-19+ or P-COVID-19-groups, although 243 these differences were not significant (p=0.201, p=0.057 respectively). With respect to 244 CD73, it was found that monocytes CD73+ reached a higher percentage in NP-COVID-245 

The proportion of lymphocytes and monocytes that synthesize cytokines IL-6, IFN-γ or 261 IL-1β was determined in P-COVID-19+ patients. Whole blood samples were cultivated 262 4 hours in absence or presence of polyclonal stimuli (LPS 250 ng/mL, PMA/Ion, 263 50ng/mL/1mg/mL) or human rIL-6 (100 ng/mL). Figure 2 shows the results of this 264 functional assay. The percentage of CD4 or CD8 T lymphocytes expressing IL-6 or 265 IFN-γ was less than 5% in the groups (Figure 2a, d, g, j) , and human rIL-6 did not 266 increase this percentage in CD4 or CD8 T lymphocytes (Figure 2b, e, h, k) . The 267 stimulus with PMA/Ion did increase the percentage of CD4 IL-6+ T lymphocytes in the 268 P-COVID-19+ group with respects to the P-COVID-19-group. However, this effect 269 was not seen in the CD8+ IL-6+ T lymphocytes (Figure 2c is only reached in CD8 IFN-γ+ T lymphocytes between the P-COVID-19+ and P-273 COVID-19-groups (p=0.019, Figure 2l) . 274

By the same token, the percentage of monocytes IL-1+ or IL-6+ is less than 10% in the 275 groups (Figure 2m, p) . The stimulus with human rIL-6 only increased the percentage of 276 monocytes IL-6+ in the P-COVID-19-group with respect to P-COVID-19+ (Figure 2q (Figure 3a, b, d, e) . Also, the TNF and IL-4 298 concentration is higher in P-COVID-19+ than in NP-COVID-19+ (Figure 3d, e) . Other 299 cytokine/chemokine show higher concentration in P-COVID-19+ than in P-COVID-19-300 , such as CXCL10 (IP10) and IL-2, although these do not reach a statistically significant 301 difference (Figure 3c and f, p = 0.456, and p=0.447, respectively) . Figure S1 shows 302 other cytokine/chemokine that show similar concentration between and among groups, 303 these includes CXCL8, CCL11, CCL17, CCL2, CCL5, CCL3, CXCL9, CXCL5, 304 CCL23, CXCL1, CXCL11, IL-17a, IFN-γ and IL-10. 305 306 307 

To know whether leukocytes from pregnant women with COVID-19 can be activated 314 by polyclonal confrontation and lead to cytokine/chemokine response, whole blood was 315 challenged; Figure 4 shows the concentration in supernatant. After 4 hours of culture 316 without stimulation (whole blood only), the concentration of TNF-α, IFN-γ, CCL3, 317 CCL4, IL-17a, CCL23, CXCL8 and IL-10 was similar among groups. Human rIL-6 318 induced a higher concentration of TNF-α, CCL3 and CCL4 (Figure 4e, f, d, h) , but only 319 TNF-α and CCL3 reached a difference statistically significant (Figure 4a, b) . Also, LPS 320 induced a higher concentration of TNF-α, CCL3, CCL4, CCL23 and CXCL8 in all 321 groups, these differences reached high significance (p<0.0001) when contrasted with 322 their respective pair without stimulus (in example; NP-COVID-19+ whole blood vs. P-323 COVID-19+ LPS). Finally, the stimulation with PMA/Ion induced a higher 324 concentration of CCL3, CCL4, IL-17a, CCL23, CXCL-8 and IL-10 in all groups rather 325 than in their respective pair without stimulus (in example; NP-COVID-19+ WB vs. P-326 COVID-19+ PMA/Ion). The PMA/Ion did not induce an increase IFN-γ response in all 327 groups, especially in NP-COVID-19+ group, however, the IFN-γ concentration was 328 higher in the pregnancy groups with and without COVID-19 compared to NP-COVID-329 19+ group, although no statistically significant difference was achieved (p> 0.9, p> 0.9 330 respectively). Figure S2 shows that the polyclonal stimulus did not induce an increase 331 response of CXCL10, CCL11, CCL17, CCL2, CCL5, CXCL9, CXCL5, CXCL1, 332 CXCL11, IL-10, IL-4 and IL-2. 

What immunological and hormonal protective factors lower the risk 487 of COVID-19 related deaths in pregnant women?

489 Maternal and Neonatal Morbidity and Mortality Among Pregnant Women With

Without COVID-19 Infection: The INTERCOVID Multinational Cohort Study

Update: Characteristics of Symptomatic Women of Reproductive Age with Laboratory-494

Confirmed SARS-CoV-2 Infection by Pregnancy Status -United States

497 Characteristics and Maternal and Birth Outcomes of Hospitalized Pregnant Women 498 with Laboratory-Confirmed COVID-19 -COVID-NET, 13 States

Immune 501 Regulation, Maternal Infection, Vaccination, and Pregnancy Outcome

Fetomaternal immune cross-talk and its consequences for 504 maternal and offspring's health

Clinical, laboratory and 507 imaging features of COVID-19: A systematic review and meta-analysis

Obesity a risk factor for increased COVID19 prevalence, severity and lethality 511 (Review)

COVID-19 and pregnancy: A review of 513 clinical characteristics, obstetric outcomes and vertical transmission

Outcome of coronavirus spectrum infections (SARS, MERS, COVID-19) during 517 pregnancy: a systematic review and meta-analysis

Laboratory Parameters in Detection of COVID-19 Patients with 521

Lung 524 pathology of fatal severe acute respiratory syndrome

Significant changes of 526 peripheral T lymphocyte subsets in patients with severe acute respiratory syndrome

Lymphopenia is 529 associated with severe coronavirus disease 2019 (COVID-19) infections: A systemic 530 review and meta-analysis

Chemokine up-534 regulation in SARS-coronavirus-infected, monocyte-derived human dendritic cells. 535 Blood

Temporal changes in 537 cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory 538 syndrome

Induction of IL-8 release in 540 lung cells via activator protein-1 by recombinant baculovirus displaying severe acute 541 respiratory syndrome-coronavirus spike proteins: identification of two functional 542 regions

Upregulation of the 544 chemokine (C-C motif) ligand 2 via a severe acute respiratory syndrome coronavirus 545 spike-ACE2 signaling pathway

Clinical features of patients 547 infected with 2019 novel coronavirus in Wuhan

SARS-CoV-2 infection: The role of cytokines in COVID-19 disease

Monocyte chemoattractant 553 protein-1 (MCP-1): an overview

Interleukin-1beta 555 causes pulmonary inflammation, emphysema, and airway remodeling in the adult 556 murine lung

Regulation of interferon-gamma during innate and 558 adaptive immune responses

COVID-19 infection among asymptomatic and symptomatic pregnant women: 561 Two weeks of confirmed presentations to an affiliated pair of

What Else Can CD39 Tell Us? Front Immunol

Coagulation in pregnancy

COVID-19 568 coagulopathy in pregnancy: Critical review, preliminary recommendations, and ISTH 569 registry-Communication from the ISTH SSC for Women's Health

D-572 dimer as an indicator of prognosis in SARS-CoV-2 infection: a systematic review

CT characteristics and 575 diagnostic value of COVID-19 in pregnancy

Clinical characteristics and laboratory results of 577 pregnant women with COVID-19 in Wuhan, China

Fluctuation in Normal Pregnancy: A Longitudinal Cohort Study of 4,117 Samples from 581 714 Healthy Danish Women

The increased expression of TREM-1 on 584 monocytes is associated with infectious and noninfectious inflammatory processes

Triggering receptor expressed on 588 myeloid cells-1 expression on monocytes is associated with inflammation but not with 589 infection in acute pancreatitis

Pregnant women infected with 592 pandemic H1N1pdm2009 influenza virus displayed overproduction of peripheral blood 593 CD69+ lymphocytes and increased levels of serum cytokines

CD39 and CD73 in immunity and 596 inflammation

Severe immunosuppression and not a cytokine storm characterizes COVID-19 599 infections

COVID-19-(P-COVID-19-). BMI; Body Mass Index. NLR

Wallis test

Pregnant-COVID-19-. Fisher's exact test. Significant p<0

Additionally, the frequency of symptoms was similar between NP-COVID-19 and P-375 COVID-19 (Table 2) , indicating a homogeneous clinical presentation, allowing the 376 identification of cellular and humoral characteristics in response against COVID-19 in 377 the presence or absence of pregnancy. 378Our study shows that the percentage of monocytes HLA-DR+ is lower in women with and without pregnancy than in P-COVID-19- (Fig 1) . Also, a lower 380 percentage has been observed in septic patients with critical condition or fatal outcome 381 (33, 34) , suggesting that this characteristic could be a helpful biomarker in The lower percentage of monocytes HLA-DR+ in COVID-19 could be the way to 383 downregulate the immune response by SARS-CoV-2 and to evade the immunity, or the 384 way that the immune response control activation and avoid over-stimulation. Our results 385 suggest that COVID-19 does not accentuate the low percentage of monocytes HLA-386 DR+, which would indicate that pregnancy does not limit the activation of monocytes in 387 peripheral blood upon a SARS-CoV-2 infection. More analysis is required to know the 388 biological significance of lower percentage of monocytes HLA-DR+ in Furthermore, a high percentage of lymphocytes CD69+ is reached in AH1N1 influenza, 390 another viral challenge that leads to unregulated inflammation in pregnant women (35). 391Likewise, we found a higher percentage of CD69+ cells in both CD4 and CD8 cells in 392 P-COVID-19+ than in NP-COVID-19+ and P-COVID-19-without reaching a statistical 393 significance (Table S2 ). This indicates a higher level of activation despite the multiple 394 mechanisms to ensure an immunotolerance during pregnancy. 395The expression of CD39 and CD73 on leukocytes may be an important mechanism for 396 resolving SARS-CoV-2 infection and COVID-19 disease. CD39 and CD73 are 397 ectoenzymes that sequentially metabolize ATP to adenosine (26, 36), thus controlling 398 inflammation through this alarmine, leading to an adenosine induced anti-inflammatory 399 response (26). Pregnant women with or without COVID-19 maintain higher percentages 400 of monocytes CD39+, and lower percentage of CD73+ than in NP-COVID-19+ (Fig 1) , 401suggesting that pregnant women control inflammation through monocytes 402 CD39+/CD73+. These could be a potential marker to monitor the evolution of COVID-40319. The function of CD39/CD73 is not limited to monocytes, however, our results 404 indicate that the percentage of B or T cells CD39+ or CD73+ is not significantly 405 modified by the effects of pregnancy or COVID-19 infection ( Figure 1 ). 406Activated leukocytes are a potential source of pro-inflammatory or regulatory cytokines 407 in peripheral blood of COVID-19 patients. We determined the percentage of leukocytes 408 IL-6+, IFN-γ+ or IL-1β+ after 4 hours of culture with or without polyclonal stimulation. 409Lymphocytes T CD4+ IL-6+ or IFN-γ+, CD8+IL-6+ or IFN-γ+ and monocytes IL-1β+ 410 or IL-6+ did not reach more than 5% of circulating cells, indicating a low baseline of 411 circulating cytokine producing leukocytes. After being stimulated with human rIL6, 412 there was no significant increase of IL-1β, IL-6 or IFN-γ producers in lymphocytes and 413 monocytes, indicating that the IL-6 in serum of COVID-19 patients could have a limited 414 stimulus to increase the synthesis of pro-inflammatory cytokines from circulating 415 leukocytes. Whole blood stimulation LPS or PMA/Ion increases the percentage of 416 lymphocytes T CD4+ and CD8+ and monocytes IFN-γ+ or IL-1β+ and IL-6+, 417indicating that these leukocytes are not anergic and retain the ability to synthesize 418 cytokines both in the presence and absence of pregnancy and COVID-19. However, 419 there is a trend to increase the percentage of monocytes IL-6+ and decrease the 420 percentage of lymphocytes CD4+IFN-γ+ and CD8+IFN-γ+ in patients with This suggests that both women with or without pregnancy develop quite similar 422 defenses to face COVID-19, increasing IL-6 and limiting IFN-γ response. 423The cytokine storm induced by COVID-19 could be greater in pregnant women, our 424 study shows that some cytokines (TNF-α, IL-6 and CCL3) reach a higher concentration 425 in serum of NP-COVID-19+ than in P-COVID-19+ patients, although it was only 426 significantly higher for TNF-α (Figure 3a) . Interestingly, we also found a highest 427 concentration of IL-4 in the P-COVID-19+ group with a statistically difference in NP- contributed with data acquisition, analysis and/or interpretation.