TYPE Original Research

PUBLISHED 03 April 2023

DOI 10.3389/fimmu.2023.1151780

Elevated circulating
OPEN ACCESS monocytes and monocyte
EDITED BY
William Tolbert,
Henry M Jackson Foundation for the
activation in COVID-19
Advancement of Military Medicine (HJF),
United States convalescent individuals
REVIEWED BY
Anuradha Rajamanickam,
International Centers for Excellence in Juwon Park 1,2*, Logan S. Dean 1,2, Boonyanudh Jiyarom 1,
Research (ICER), India
Shetty Ravi Dyavar,
Louie Mar Gangcuangco 1,3, Parthav Shah 3,4, Thomas Awamura 2,
Adicet Bio, Inc, United States Lauren L. Ching 2, Vivek R. Nerurkar 2, Dominic C. Chow 3,
*CORRESPONDENCE Fritzie Igno 3, Cecilia M. Shikuma 1,2,3 and Gehan Devendra 3,5*
Juwon Park
[email protected] 1
Hawaii Center for AIDS, John A. Burns School of Medicine, University of Hawai’i at Manoa, Honolulu,
Gehan Devendra HI, United States, 2Department of Tropical Medicine, Medical Microbiology, and Pharmacology, John A.
[email protected] Burns School Medicine, University of Hawai’i at Manoa, Honolulu, HI, United States, 3Department of
Medicine, John A. Burns School of Medicine, University of Hawai’i at Manoa, Honolulu, HI, United States,
SPECIALTY SECTION 4
John A. Burns School of Medicine, University of Hawai'i at Manoa, Honolulu, HI, United States,
This article was submitted to 5
Department of Pulmonary and Critical Care, Queen’s Medical Center, Honolulu, HI, United States
Viral Immunology,
a section of the journal
Frontiers in Immunology

RECEIVED 26 January 2023

ACCEPTED 21 March 2023 Background: Monocytes and macrophages play a pivotal role in inflammation
PUBLISHED 03 April 2023 during acute SARS-CoV-2 infection. However, their contribution to the
CITATION development of post-acute sequelae of SARS-CoV-2 infection (PASC) are not
Park J, Dean LS, Jiyarom B, fully elucidated.
Gangcuangco LM, Shah P, Awamura T,
Ching LL, Nerurkar VR, Chow DC, Igno F,
Shikuma CM and Devendra G (2023) Methods: A cross-sectional study was conducted comparing plasma cytokine
Elevated circulating monocytes and and monocyte levels among three groups: participants with pulmonary PASC
monocyte activation in COVID-19
(PPASC) with a reduced predicted diffusing capacity for carbon monoxide
convalescent individuals.
Front. Immunol. 14:1151780. [DLCOc, <80%; (PG)]; fully recovered from SARS-CoV-2 with no residual
doi: 10.3389/fimmu.2023.1151780 symptoms (recovered group, RG); and negative for SARS-CoV-2 (negative
COPYRIGHT group, NG). The expressions of cytokines were measured in plasma of study
© 2023 Park, Dean, Jiyarom, Gangcuangco,
cohort by Luminex assay. The percentages and numbers of monocyte subsets
Shah, Awamura, Ching, Nerurkar, Chow,
Igno, Shikuma and Devendra. This is an (classical, intermediate, and non-classical monocytes) and monocyte activation
open-access article distributed under the (defined by CD169 expression) were analyzed using flow cytometry analysis of
terms of the Creative Commons Attribution
License (CC BY). The use, distribution or peripheral blood mononuclear cells.
reproduction in other forums is permitted,
provided the original author(s) and the Results: Plasma IL-1Ra levels were elevated but FGF levels were reduced in PG
copyright owner(s) are credited and that
the original publication in this journal is
compared to NG. Circulating monocytes and three subsets were significantly higher
cited, in accordance with accepted in PG and RG compared to NG. PG and RG exhibited higher levels of CD169+
academic practice. No use, distribution or monocyte counts and higher CD169 expression was detected in intermediate and
reproduction is permitted which does not
comply with these terms. non-classical monocytes from RG and PG than that found in NG. Further correlation
analysis with CD169+ monocyte subsets revealed that CD169+ intermediate
monocytes negatively correlated with DLCOc%, and CD169+ non-classical
monocytes positively correlated with IL-1a, IL-1b, MIP-1a, Eotaxin, and IFN-g.

Conclusion: This study present evidence that COVID convalescents exhibit

monocyte alteration beyond the acute COVID-19 infection period even in
convalescents with no residual symptoms. Further, the results suggest that
monocyte alteration and increased activated monocyte subsets may impact

Frontiers in Immunology 01 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

pulmonary function in COVID-19 convalescents. This observation will aid in

understanding the immunopathologic feature of pulmonary PASC development,
resolution, and subsequent therapeutic interventions.

KEYWORDS

SARS-CoV-2, Long-COVID, post-acute sequalae of SARS-CoV-2 infection, pulmonary

sequelae, monocytes, CD169

Introduction monocyte response in individuals with pulmonary PASC (PPASC)

remain unclear. Given that blood monocytes provide a window into
It is estimated that one-third of patients infected with severe the systemic immune response, reflecting the risk of potential
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who complications after recovery from acute infection, it is important
develop coronavirus-19 (1) continue to experience residual to characterize these monocyte populations to gain insight into the
symptoms, collectively referred to as ‘Long-COVID’ or ‘post- role that monocyte dysregulation plays in PPASC.
acute sequelae of SARS-CoV-2 infection’ (PASC) (2). PASC In this study, we analyzed circulating monocytes and plasma
symptoms are highly heterogeneous with a wide range of cytokine expression in COVID-19 convalescents, comparing them
presentations, including fatigue, dyspnea, sleep disorders, anxiety, to uninfected individuals. We further assessed the relationship
and loss of memory and/or concentration. Among individuals with between these parameters and quantitative measures of lung
PASC, pulmonary complications, such as persistent dyspnea and function. We found that COVID-19 convalescents, regardless of
chronic cough are common (3, 4). The pathophysiology of COVID- residual pulmonary symptoms, displayed increased monocyte levels
19 is complex and appears to involve multiple inflammatory and and had an activated phenotype, defined as CD169 + cells.
immunological pathways (5). Studies have shown that COVID-19 Moreover, the percentages and numbers of CD169+ monocyte
patients display high systemic levels of cytokines and profound subsets were associated with the diffusing capacity of the lungs for
immune cell dysregulation that correlates with disease severity carbon monoxide (DLCOc)% and proinflammatory cytokines,
(6, 7). suggesting that alterations in monocyte subset activation may
Monocytes and macrophages are essential immune cells contribute to the development of chronic lung sequelae in
involved in host immunity and tissue homeostasis (8–10). These individuals after resolution of COVID-19 infection.
cells also possess inflammatory (11) and tissue-repairing capabilities
and thus actively participate in all phases of the inflammatory
response. Monocytes can be activated by infection and/or Methods
inflammatory conditions, leading to differentiation and
polarization into macrophages with pro-inflammatory phenotypes Study cohort and selection of participants
(9, 12, 13). High monocyte count and activated monocyte
phenotype have been linked to various pathological conditions This cross-sectional study investigated PASC complications
(13–17). During SARS-CoV-2 infection, elevated peripheral among individuals living in Hawaii. Participants with PASC were
monocyte levels and altered phenotype were observed in patients recruited from the Post-COVID Recovery and Care Clinic of an
(14, 15, 18). Analysis of circulating monocytes has shown to predict academic tertiary care hospital (Queen’s Medical Center, Honolulu,
disease severity and mortality in COVID-19 (19, 20). A Hawaii) between September 2020 and Mar 2021, prior to the
comprehensive analysis of immune cells revealed long-term detection of Omicron variants in Hawaii. Participants were
perturbations of innate and adaptive immune populations that grouped into the following: A) individuals who reported
persisted at least 6 months after SARS-CoV-2 infection (21). persistent pulmonary symptoms (dyspnea, fatigue, cough, or
COVID-19 convalescents with prolonged symptoms displayed shortness of breath) beyond 30 days after COVID-19 infection
highly activated myeloid cells, lacked naïve T and B cells, and with reduced diffusion capacity for carbon monoxide (corrected for
exhibited elevated type I and III interferon levels (22). A recent hemoglobin-DLCOc, <80%) by pulmonary function test
study found that intermediate (CD14+CD16+) and non-classical (pulmonary PASC group [PG]; n=11); B) individuals who have
monocytes (CD14loCD16+) were significantly elevated in PASC fully recovered from SARS-CoV-2 infection with no residual
patients. Furthermore, SARS-CoV-2 S1 protein was detected in symptoms >30 days after acute infection (recovered group [RG];
non-classical monocytes, but not classical and intermediate n=10); and C) individuals confirmed to have not contracted
monocytes in PASC patients, suggesting that non-classical COVID-19 using negative SARS-CoV-2 antibody test (negative
monocytes may contribute to inflammation in PASC (23). group [NG], n=10). The PG and RG groups had documented
Although our understanding of innate immunity underlying the positive SARS-CoV-2 by polymerase chain reaction (PCR) and a
pathophysiology of PASC is evolving, a detailed understanding of replicated SARS-CoV-2 IgG antibody test. The study was approved

Frontiers in Immunology 02 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

by the Queens Medical Center Institutional Review Committee with by addition of Human TruStain FcX (BioLegend, San Diego, CA,
the University of Hawaii IRB ceding authority (24: RA-2020-053). 1:200) in hanks balanced salt solution (HBSS) supplemented with
1% bovine serum albumin (BSA) (flow buffer) at room temperature
(RT) for 15 minutes. Subsequently, cells were stained with the
Pulmonary function tests titrated fluorophore conjugated extracellular antibodies; CD45-
BV711 (BD Biosciences, East Rutherford, NJ), CD11b-PE-Cy-7
Pulmonary function testing (PFT) was performed on (BioLegend, San Diego, CA), CD14-BV605 (BioLegend, San
individuals PPASC. All PG participants underwent PFTs (Vyaire) Diego, CA), CD16-BV421 (BioLegend, San Diego, CA), CD169-
with the measurements of forced vital capacity (FVC), forced APC (Biolegend, San Diego, CA) at RT for 30 minutes and then
expiratory volume in 1 second (FEV1), total lung capacity (TLC), washed twice with ice-cold flow buffer. Samples were then washed
and DLCOc% interpreted in accordance with European Respiratory twice with ice-cold flow buffer and resuspended to 800 ɱL of flow
Society (ERS)/American Thoracic Society (ATS) guidelines (24). buffer for acquisition. Samples were acquired with identical voltage
settings on a LSR Fortessa (BD Sciences, East Rutherford, NJ) with
approximately 1.0x109 events collected per sample. 10 uL of
Plasma and peripheral blood mononuclear AccuCheck Counting Beads (Life Technologies, Carlsbad, CA)
cells isolation were added prior to acquisition for calculation of absolute cell
counts. Compensation beads (Invitrogen, Waltham, MA) were
Whole blood was collected from study participants in ethylene prepared for accurate compensation controls. Data was analyzed
diamine tetra-acetic acid (EDTA) tubes (BD, Vacutainer) by using FlowJo (Treestar, Ashland, OR) software and absolute cell
venipuncture and processed based on a well-established method counts were determined according to manufacturer protocols.
(25). In brief, whole blood was centrifuged, plasma removed and
cryopreserved at −80°C until downstream analysis. Remaining
venous blood was diluted with an equal volume of phosphate Statistics
buffered saline (PBS) and layered on top of Ficoll-Paque Plus (GE
Healthcare Biosciences, Piscataway, NJ) following the A cross-sectional study comparing PG, RG, and NG
manufacturer’s protocol. PBMC were separated by centrifugation participants was undertaken. Flow cytometry results between the
at 400 × g for 30 minutes at room temperature (RT). PBMC were groups were compared using Mann Whitney-U test. Patient
collected from the buffy coat, red blood cells lysed, and then washed characteristics between groups were compared using Chi-squared
twice in PBS supplemented with 1% fetal bovine serum (FBS). Cells test, Fisher’s exact test, or Mann Whitney-U test, as appropriate.
were then counted, viability determined, and cryopreserved until Correlation between monocyte subsets, CD169+ monocyte subsets,
further analysis. and inflammatory markers were analyzed using Spearman
correlation. P-value < 0.05 was considered statistically significant
for all tests. Statistical analyses were performed using IBM SPSS
Multiplex cytokine analysis version 28 (Chicago, IL) and GraphPad Prism 9. (Graph Pad, San
Diego, CA).
Plasma was thawed and prepared following the manufacturer’s
guidelines for each kit. All samples were run in duplicate in a single
plate per panel. Biomarkers to assess inflammation (IFN-g, IL-13, Results
IL-1a, IL-8, IL-1b, IL-1Ra, TNF-a), leukocyte chemotaxis (Eotaxin,
MCP-1, MIP-3a, MIP-1a, RANTES/CCL5), and tissue remodeling/ Study cohort description
fibrosis (PDGF-AA, PDGF-AA/BB, FGF, VEGF, TGF-a) were
measured using the R&D System ™ Human XL Cytokine The median age of the participants was 53, 54, and 55 years for
Discovery Premixed Kit. Data were acquired on a MAGPIX® PG (n=11), RG (n=10), and NG (n=10), respectively. 18.2% of
Instrument (Luminex Corporation, Austin, TX). Data analysis individuals with PPASC had respiratory history (asthma or COPD).
was done using GraphPad Prism 9. Net median fluorescent Higher rates of hospital admission were seen among PG compared
intensity (MFI) was calculated (MFI value minus background with RG (36% vs 0%, P = 0.015). Among the hospitalized patients
value) and the average net MFI of duplicate samples (n=4), one patient was admitted to the medical intensive care unit
was determined. (ICU) due to increasing oxygen demand and eventual mechanical
ventilatory support. No significant differences in pre-existing
conditions, body mass index (BMI), and smoking prevalence
Flow cytometric analysis between PG and RG were seen. Overall, 68.8% of the participants
had received a SARS-CoV-2 vaccine at the time of enrollment.
Cryopreserved PBMC were thawed and washed twice in PBS. Individuals with PPASC experienced prolonged pulmonary
Typically, cells were incubated with LIVE/DEAD™ Fixable Yellow complications including, dyspnea, fatigue, cough, and shortness of
Dead Cell Stain (Invitrogen, 1:1000) at 4°C for 30 minutes, followed breath; 45.5% of them had at least two pulmonary symptoms. Those

Frontiers in Immunology 03 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

with PPASC reported symptoms lasting for a median duration of six COVID-19 convalescents display elevated
months from post COVID-19 infection. All individuals from RG circulating monocytes
resolved symptoms within 4 weeks after disease onset and reported
no symptoms at the time of sample collection. The baseline To determine the impact of long-term consequence of COVID-
participant characteristics are displayed in Table 1. 19 on blood monocytes and monocyte alteration in PPASC, PBMC
cells isolated from NG (n=10), RG (n=10), and PG (n=11) cohorts
were analyzed by flow cytometry. A representative gating strategy
Soluble biomarker levels in for identifying monocytes within the PBMC fractions from three
COVID-19 convalescents groups was shown in Figure 2A. Monocyte subsets; classical
(CD14+ CD16-), intermediate (CD14+ CD16+), or non-classical
To examine blood biomarkers associated with PPASC, we (CD14lo CD16+) were defined by CD14 and CD16 surface levels
assessed 17 analytes in the plasma of participants from the NG, within the monocytes (Figure 2A). We found both the percentages
RG, and PG by Luminex assay and compared the plasma and numbers of total circulating monocytes were higher in the
concentration of analytes among the groups. Analytes included COVID-19 convalescents (PG and RG) than NG (Figures 2B, C). In
cytokines associated with “cytokine storm”; IL-1a, IL-1b, IL-1Ra, comparison to NG, both the percentage and number of classical and
IL-8, IL-13, and TNF-a. “leukocyte chemotaxis”; Eotaxin, MCP-1, intermediate monocytes were significantly increased in PG and RG
MIP-3a, MIP-1a, Fractalkine, IP-10, MCP-3, and, RANTES.), “tissue (Figures 3A, B, D, E). Also, the percentage and number of non-
remodeling”; PDGF-AA, PDGF-AA/BB, FGF, VEGF, and TGF-a. classical monocytes were significantly increased in PG, but only
The IL-1Ra was elevated (2.2 fold higher) in the PG compared to NG, non-classical monocyte numbers were increased in RG, compared
and its’ level trended higher in PG as compared to RG (Figure 1A). to NG (Figures 3C, F). However, we did not observe any significant
PDGF-AA, PDGF-AB, and TGF-a were decreased in the RG, difference between PG and RG in three monocyte subsets
compared to the NG, and their levels tended to be lower in the PG percentages and numbers (Figure 3). Altogether, these
(Figures 1B–D). FGF remained significantly lower in the RG and PG observations suggest that circulating monocyte levels remain
compared to NG (Figure 1E). There was no difference observed in 13 elevated for several months after SARS-CoV-2 infection, even
analytes among three groups (Supplementary Figure 1). convalescents who have no residual symptom.

TABLE 1 Characteristics of study participants.

PG RG NG P-value
(N=11) (N=10) (N=10)
Age (years) 53 [44, 58] 54 [46.2, 59.2] 55 [44, 58.2] 0.953

Male Gender 8 (72.7%) 6 (60%) 6 (60%) 0.778

Months post-COVID infection 6 [4, 12] 5 [1.5, 9.5]* – 0.515

2
Body Mass Index (kg/m ) 30.8 [24.3, 35.1] 26.9 [22.0, 34.6] No data 0.676

Received vaccination for COVID-19 9 (81.8%) 5 (50%) 8 (88.9%)* 0.116

Hospital admission (n, %)** 4 (36.3%) 0 0 0.015

Race

White/Caucasian 3 (27.3%) 1 (10%) 5 (50%) 0.160

Asian 4 (36.4%) 4 (40%) 5 (50%)

Native Hawaiian/Pacific Islander 3 (27.3%) 2 (20%) 0

Other 1 (9.0%) 3 (30%) 0

Co-morbidities

Asthma 1 (9.1%) 1 (10%) 1 (10%) 0.997

COPD 1 (9.1%) 0 0 0.391

Hypertension 3 (27.3%) 2 (20%) 1 (10%) 0.605

Diabetes 2 (18.2%) 1 (10%) 1 (10%) 0.809

Smoking history (past/current) 4 (36.4%) 2 (20%) 0 0.109

Current alcohol use 3 (27.3%) 5 (50%) 0 0.038

P-value calculated using Kruskal-Wallis test, Chi-square test, or Fisher’s exact test.
*1 missing data.
**One patient was admitted to the medical ICU for mechanical ventilatory support.
Bold indicates statistical significance.

Frontiers in Immunology 04 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

A B C

D E

FIGURE 1
Plasma cytokine levels among groups. (A) IL-1Ra, (B) PDGF-AA, (C) PDGF-AB/BB, (D) TGF-a, and (E) FGF levels were measured in plasma collected
from NG, RG, and PG. The data are represented as the box and scatter plots with each circle representing a single individual. P values were
calculated using the Mann–Whitney U-test. *p < 0.05, **p < 0.01, ns, non-significant.

CD169+ monocytes in monocytes (Figure 4D). Significant increases in CD169 +

COVID-19 convalescents percentages were observed in intermediate and non-classical
monocytes, only between RG and NG, with no difference between
CD169, a type I interferon-inducible receptor, is expressed on PG and NG, or PG and RG observed (Figures 4E–G). These data
monocytes and macrophages (26–28). CD169+ monocytes and indicate that circulating monocytes from COVID-19 convalescents
macrophages have been thought to be important players in remain increased and display a higher CD169 expression.
inflammatory response of inflammatory and autoimmune diseases
(29–31). Monocytes from COVID-19 patients had increased CD169
levels during acute SARS-CoV2 infection and monocyte CD169 was The relationship between CD169+
identified as a biomarker in early COVID-19 infection (27). To monocytes and lung function in PPASC
further investigate difference in monocyte activation among the
groups, we analyzed CD169 expression in monocytes. When PG participants had PFTs performed during their period of
monocytes were stratified based on CD169 expression, the prolonged respiratory symptoms. 18.2% of participants reported pre-
percentage of CD169+ monocytes were significantly higher in the existing pulmonary conditions prior to infection with SARS-CoV-2
PG and RG than in the NG (Figure 4A). Also, CD169+ monocyte (Table 1). We explored the relationship between the percentages and
numbers were significantly increased in the PG and RG, compared counts of CD169+ monocyte with DLCOc%. We found negative
to NG (Figure 4B). The MFI of CD169 on classical monocytes did correlations between CD169+ monocytes and CD169+ intermediate
not differ among the groups. However, CD169 MFI of intermediate monocytes percentages and CD169+ intermediate monocyte counts
and non-classical monocytes in PG and RG was significantly higher (r=-0.758; P=0.009, r=-0.71; P=0.02, r=-0.69; P=0.051, respectively)
than those in NG (Figure 4C). Interestingly, when the percentage of and DLCOc% in PG (Figures 5A–C). Taken together, these results
CD169+ cells was examined in the three groups in each respective suggested that elevated CD169 expression in monocytes may serve as
monocyte population, no difference was observed in classical biomarker for determining lung function in PPASC.

Frontiers in Immunology 05 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

B C

FIGURE 2
Comparison of circulating monocyte levels among the groups. Representative flow cytometry gating strategy for identification of monocytes and
monocyte subsets in PBMC from NG, RG, and PG groups. Lymphocytes and monocytes were selected using CD45+ followed by gating for CD11b+
cells. (A1) non-classical (CD14lo/CD16+), (A2) intermediate (CD14+/CD16+, (A3) Classical (CD14+/CD16-), and (B) Total monocyte percentages as a
proportion of total identified CD45+ cells in NG, RG, and PG groups. (C) Total monocyte counts in NG, RG, and PG groups. Mann-Whitney-U Test *p
< 0.05, **p < 0.01, ***p < 0.001, ns, non-significant.

Monocyte relationship with systemic levels monocyte counts, while MCP-1 was associated with the CD169+
of cytokines in PPASC percentage of intermediate monocytes. IL-1a and IL-1b were
positively correlated with the percentage of CD169+ non-classical
In order to determine whether the changes in monocytes monocytes. IL-1a, MIP-1a, IFN-g, Eotaxin, and PDFG-AA were
correlated with cytokine expression in PPASC, we performed positively correlated with CD169+ non-classical monocyte counts
spearman rank correlation to assess associations between cytokines (Table 2 and Figure 6). Altogether, these results suggested that
and monocyte subset counts and percentages. In Table 2, monocyte monocyte subsets and the cells with CD169 upregulation were
parameters (monocyte subsets and CD169+ total monocyte and associated with a proinflammatory cytokine environment in PPASC.
monocyte subsets) showed a positive correlation with cytokines
(VEGF, IL-8, IL-1a, IL-1b, PDGF-AA, PDGF-AB/BB, Eotaxin, Discussion
MIP-1a, MCP-1, and IFN-g). VEGF levels were positively
associated with CD169+ total monocyte counts and non-classical In this study, we observed that COVID-19 convalescents with
monocyte percentages. The percentage of intermediate monocytes pulmonary PASC displayed altered circulating monocyte levels and
was positively associated with IL-1a, IL-1b, MIP-1a, PDGF-AA, and activation, which may last several months after infection.
PDGF-AB/BB cytokines, but negatively associated with FGF. Eotaxin, Interestingly, monocyte alterations were also observed in
MIP-1a, and VEGF were positively associated with intermediate individuals whose symptoms had resolved completely. These

Frontiers in Immunology 06 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

A B C

D E F

FIGURE 3
Comparison of circulating monocyte subsets among the groups. (A) Classical monocyte percentage, (B) Intermediate monocyte percentage, (C) Non-Classical
monocyte percentage, as a proportion of total identified CD45+ cells in NG, RG, and PG groups. (D) Classical monocyte counts, (E) Intermediate monocyte
counts, and (F) Non-Classical monocyte in NG, RG, and PG groups. Mann-Whitney-U Test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-
significant.

findings highlight that COVID-19 convalescents exhibit monocyte likely to have impairment of lung function (32, 33). Also, pulmonary
dysregulation beyond the resolution of initial infection. symptoms were presented without impairment of lung function or
Monocytes have begun to emerge as key cellular modulators of cardiopulmonary exercise test among COVID-19 survivors (33). Also,
COVID-19 pathophysiology. During acute SARS-CoV-2 infection, monitoring of PFTs in severe COVID-19 survivors with lung
monocytes are dysregulated, exhibiting aberrant functions and abnormalities post discharge demonstrated significant pulmonary
mediation of the cytokine storm associated with severe COVID- sequelae (34, 35). These study demonstrates that hospitalized
19. Zhou et al. (11) found elevated levels of CD14+ CD16 + COVID-19 survivors were more likely to have persistent pulmonary
monocytes in ICU COVID-19 patients, compared to non-ICU PASC symptoms compared with those without hospitalization.
patients and healthy controls. Indeed, granulocyte-macrophage- Hospitalized COVID-19 survivors tended to have reduced DLCOc%
colony stimulating factor (GM-CSF) and IL-6 expressing CD14+ (61.98%) compared to non-hospitalized COVID-19 survivors
monocytes were significantly increased in the ICU patients. Few (66.89%). A correlation analysis of CD169+ monocyte subsets and
studies have detailed the immune profiles in individuals with PASC DLCOc% suggested that alteration of activated monocyte subsets may
despite accumulating evidence indicating that substantial impact pulmonary function in COVID-19 convalescents. Nonetheless,
perturbation of the innate immune system is presents in PASC. we cannot exclude the possibility that this association may reflect other
However, it is still largely unknown if immune cell perturbations residual symptoms because a nonnegligible proportion of PPASC
contribute to the immunopathology of PPASC. individuals also experienced various other symptoms.
This is the first study, to our knowledge, to evaluate the The cytokine profile revealed no changes in major inflammatory
associations of monocyte levels with lung function in a community- cytokines between NG, RG, and PG. Similar observations in COVID-
based cohort. In this study, we selected individuals with PPASC 19 convalescents have been reported in another cross-sectional study.
through a primary questionnaire and secondary evaluation of IL-1a, IL-1b, IL-8, IFN-g, VEGF-A, and TNF-a had returned to
pulmonary function. This approach clarified the presence of normal levels 6 months after recovery, but IL-1Ra was still elevated in
pulmonary symptoms and may not discern individuals who had COVID-19 convalescents, compared to healthy controls (20). Another
their symptoms overestimated based on the questionnaire. Studies study showed that COVID-19 convalescents at 4 months post
demonstrate that pulmonary symptoms were present regardless of infection had higher levels of IFN-b, IFN-l1, CXCL9, CXCL10, IL-
initial COVID severity, but patients with severe COVID-19 were more 8, and sTIM-3, regardless of symptoms, compared to uninfected

Frontiers in Immunology 07 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

B C D

E F G

FIGURE 4
Characterization of circulating CD169+ monocytes in NG, RG, and PG groups. Representative histogram and dot plot of CD169+/CD14+ monocytes
in NG, RG, and PG groups. Percentage of total CD169+ monocytes from total CD14+ monocytes is shown above the gate (B). Total CD169+
monocyte percentage from CD45+ cells, (C) CD169+ monocyte, (D) MFI of CD169 on monocyte subsets in NG, RG, and PG. The percentage of
CD169+ cells identified in (E) classical monocytes, (F) intermediate monocytes, and (G) non-classical monocytes within NG, RG, and PG groups.
Mann-Whitney-U Test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant.

A B C

FIGURE 5
Spearman correlation between DLCOc% and (A) percentage of CD169+ total monocytes, (B) percentage of CD169+ intermediate monocytes, and
(C) CD169+ intermediate monocyte count in PG.

Frontiers in Immunology 08 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

TABLE 2 Spearman correlations of circulating molecules and monocyte populations in PG.

Parameter Analyte R-value P-value

Total CD169+ Monocyte Count VEGF 0.84 0.002

MIP-1a 0.611 0.05

IL-1a 0.709 0.017

IL-1b 0.8 0.005

Intermediate Monocyte % of CD45+ Cells
PDGF-AA 0.764 0.009

PDGF-AB/BB 0.782 0.006

FGF -0.627 0.044

Eotaxin 0.627 0.044

Intermediate Monocyte Count MIP-1a 0.63 0.042

VEGF 0.736 0.013

IL-8 0.747 0.04

Non-classical Monocyte % of CD45+ cells
MCP-1 -0.709 0.018

Non-classical Monocyte Count VEGF 0.836 0.002

CD169+ % of Intermediate Monocytes MCP-1 0.691 0.022

IL-1a 0.618 0.047

CD169+ Non-classical Monocyte % of CD45+ cells
IL-1b 0.745 0.011

IFN-g 0.636 0.04

Eotaxin 0.655 0.034

+
CD169 Non-classical Monocyte Count PDGF-AA 0.618 0.048

IL-1 a 0.68 0.024

MIP-1a 0.783 0.006

controls. IFN-b and IFN-l1 still remained elevated in COVID-19 various studies alters the detectable cytokine profiles. Another
convalescents with PASC but others’ expressions were reduced at possibility is that COVID-19 convalescents are not classified into
month 8, compared to month 4 (22). Queiroz et al. (36) showed that symptomatic and asymptomatic. In this case, less systemic
IL-2, TNF-a, and IL-17 levels were higher in COVID-19 inflammation is represented, but the respiratory environment
convalescents than acute COVID-19 patients. Also, individuals with may show distinct proinflammatory conditions in individuals
PASC had higher levels of IL-17 and IL-2, but lower levels IL-10, IL-6, with PPASC. Further analysis of cellular composition and
and IL-4 levels, compared individuals without sequelae. However, cytokines in blood and BALF from individuals with PPASC over
there was no significant difference in IL-6 levels between post- a longitudinal period is required to understand the dynamic
COVID-19 individuals with and without sequelae (36). Oher studies features of respiratory and systemic immunity in PPASC during
investigating immune features of COVID-19 convalescent trends disease progression and resolution.
observed elevated levels of IL-6 and IL-1b (20, 37, 38) in individuals While no difference in major inflammatory cytokines between
with PASC. Notably, increased IL-1b, IL-6, and TNF levels were the three groups was observed in our study, correlations of CD169+
reported in association with PASC development in a large-scale cohort monocyte subsets with cytokines suggested that specific activated
study (38). Interestingly, published scRNA-sequencing datasets monocyte subsets produce high levels of proinflammatory cytokines
generated from severe COVID-19 patients demonstrate increased in PPASC. Correlations of D-dimer with CD169+ non-classical
transcript reads of IL-1b and TNF-a from bronchoalveolar lavage monocytes observed herein were corroborated by the findings of
fluid (BALF) macrophages (38), supporting their hypothesis that Pandori et al. (39) in a cohort of individuals hospitalized for
proinflammatory reprogramming of lung macrophages and/or COVID-19 (39). Interestingly, their cohort did not display the
precursor monocytes may drive prolonged and exacerbated increases in total monocyte populations in their hospitalized
PASC symptomology. group but displayed decreases in non-classical monocyte
Some discrepancies in the reported cytokine levels from PASC percentages and steady levels of classical and intermediate
studies continue to generate questions regarding the importance of monocyte percentages from total CD45+ cells in participants
a heterogeneous multisystemic condition. One might speculate the hospitalized for COVID-19 up to 90 days following admission.
varying windows of sample collection post-infection between These trends suggest that decreased monocyte proportions are

Frontiers in Immunology 09 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

A B

C D

E F

FIGURE 6
CD169+ non-classical monocytes were associated with IL-1a, IL-1b, MIP-1a, Eotaxin, and IFNg in PG. Spearman correlation between the percentage
of non-classical CD169+ monocytes and (A) IL-1a and (B) IL-1b. Spearman correlation between CD169+ non-classical monocyte count and (C) MIP-
1a, (D) Eotaxin, (E) IL-1a, and (F) IFNg.

present during hospitalization from COVID-19, but COVID-19 pathogenesis and what the consequences of viral reservoirs in non-
convalescents demonstrated elevated monocyte levels, potentially in classical monocytes are in PASC persistence. Further studies for
a dysregulated nature (20, 22, 23). monitoring monocyte and cytokine perturbations and viral reservoirs
A recent publication analyzed soluble factors related to over time in a larger cohort warrants further investigation to identify
monocyte/macrophage dysregulation and SARS‐CoV‐2 spike (S1) a suitable biomarker for PPASC prognosis prediction and prognosis.
protein in COVID-19 convalescents (40). They demonstrated that Limitations of this study include a small sample size and a lack
prolonged perturbations of IL-5 and IL-17F levels were observed in of initial COVID-19 severity and medical history in comparison
individuals with sequelae. Also, these individuals showed few groups (NG and RG). Recent studies have demonstrated that female
significant correlations among tested cytokines, but this association sex, age, and smoking are risk factors for PASC (41–43). However,
was not evident in individuals without sequelae. Furthermore, higher the majority of our PPASC individuals were male (72.7%) and
circulating S1 levels were detected in individuals with sequelae, older, thus we did not have younger participants to stratify our
compared to individuals without sequelae. In line with their analyses by age. Likely due to a small sample size, there were no
findings, persistent S1 protein were found significantly increased in differences in the level of monocytes or immunological parameters
non-classical monocytes in individuals with PASC up to 15 months between males and females. Sample size was based on feasibility,
post-infection (23), indicating the presence of replicating viral rather than an objective estimation step driven by a hypothesis and
reservoirs in PASC. Further questions are raised as to whether our sample size was not adequately powered for multivariable
monocyte subsets represent a key inflammatory driver of PPASC analyses. Therefore, we cannot be certain if main risk factors for

Frontiers in Immunology 10 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

severe and PASC includes, age, sex, smoking, and comorbidities collection. LG analyzed data and assisted in generating table. LC,
contribute to monocyte dysregulation in COVID-19 convalescents. VN, DC, and CS contributed to subject recruitment and sample
Our clinical data was also limited by chart review and/or patient collection. JP, LD, LG, and TA wrote the draft of the manuscript.
recall. From our chart review of the PPASC patients (N=11), one PS, VN, DC, FI, CS, and GD revised the manuscript. All the authors
patient received a combination of remdesivir, dexamethasone, and assisted in editing, provided critical review, and approved the final
convalescent plasma; one patient received remdesivir and version of the submission. All authors contributed to the article and
dexamethasone. Some patients could not recall whether they approved the submitted version.
received any specific interventions for their acute COVID-19
episode. The length of post COVID-19 infection was variable, 1
to 10 months post-infection. Variability in time of sample collection Funding
may have influenced monocyte population characteristics. Due to
the exploratory nature, the limited sample size, and variable This work was supported by Myra W. and Jean Kent Angus
sampling time, we acknowledge that a large sample size would Foundation, NIH/NIMHD (U54MD007601), NIH/NHBLI
have provided increased statistical power and more informed (K12HL143960), the University of Washington/Fred Hutch Center
conclusions of monocyte roles in PPASC. In addition, a for AIDS Research, an NIH-funded program under award number
longitudinal evaluation of monocytes dynamics and the P30AI027757, the Molecular and Cellular Immunology Core through
phenotypic changes after COVID-19 infection should be carried the funding of the Centers of Biomedical Research Excellence (COBRE)
out to determine whether monocytes dysfunction is associated with program (P30GM114737), and the NIH/ NIMHD Minority Health
the clinical outcome of respiratory failure. It would also be of Research Training (MHRT) program (T37MD008636).
clinical interest to track the perturbations of monocyte populations
in relation to acute infection, COVID-19 disease context, and then
into PPASC development or recovery.
Acknowledgments
In summary, our data indicate that systemic monocyte alteration
Authors thank the staff of the Hawaii Center for AIDS for
continues within COVID-19 convalescents with pulmonary
participant recruitment, blood collection, and sample processing for
symptoms, which is also found in COVID-19 convalescents with
the study during a particular challenging period. Dr. Chris Farrar
no residual symptoms. Also, COVID-19 convalescents exhibit
and Dr. Alexandra Gurary at the Flow Cytometry Core, John A.
activated monocyte phenotypes, denoted by CD169 expression, and
Burns School of Medicine, University of Hawaii at Manoa who
this activated phenotype is associated with poor lung function and
provided technical guidance for flow cytometry experiments.
increased proinflammatory cytokines. The drivers of PPASC
Authors are also grateful to the study participants.
pathogenesis require further investigation, but possibilities include
high circulating monocyte levels, increased CD169+ intermediate and
non-classical monocytes, and IL-1Ra expression. These observations Conflict of interest
will aid in informing the ongoing decipherment of the
immunopathology that contributes to PPASC development, The authors declare that the research was conducted in the
COVID-19 recovery, and subsequent therapeutic interventions. absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.

Data availability statement

Publisher’s note
The raw data supporting the conclusions of this article will be
All claims expressed in this article are solely those of the authors
made available by the authors upon request.
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
Ethics statement reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
The study was approved by the Queens Medical Center endorsed by the publisher.
Institutional Review Committee with the University of Hawaii IRB
ceding authority (24: RA-2020-053). The patients/participants
provided their written informed consent to participate in this study. Supplementary material
The Supplementary Material for this article can be found online at:
Author contributions https://www.frontiersin.org/articles/10.3389/fimmu.2023.1151780/
full#supplementary-material
JP supervised and designed the experimental approaches. LD
conducted and analyzed flow cytometry data. BJ collected and SUPPLEMENTARY FIGURE 1
Levels of plasma cytokine in NG, RG, and PG. (A) No differences in Eotaxin,
processed human blood samples and conducted the Luminex MCP-1, MIP-3a, MIP-1a, IFNg, IL-13, IL-1b, TNFa, VEGF, RANTES/CCL5, (B) IL-
experiments and data analysis. GD analyzed PFT and clinical data 8 and IL-1a.

Frontiers in Immunology 11 frontiersin.org

Park et al. 10.3389/fimmu.2023.1151780

References
1. Seery V, Raiden SC, Algieri SC, Grisolia NA, Filippo D, De Carli N, et al. Blood 24. Stanojevic S, Kaminsky DA, Miller MR, Thompson B, Aliverti A, Barjaktarevic I,
neutrophils from children with covid-19 exhibit both inflammatory and anti- et al. Ers/Ats technical standard on interpretive strategies for routine lung function
inflammatory markers. EBioMedicine (2021) 67:103357. doi: 10.1016/ tests. Eur Respir J (2022) 60(1):2101499. doi: 10.1183/13993003.01499-2021
j.ebiom.2021.103357 25. SahBandar IN, Ndhlovu LC, Saiki K, Kohorn LB, Peterson MM, D'Antoni ML,
2. Groff D, Sun A, Ssentongo AE, Ba DM, Parsons N, Poudel GR, et al. Short-term et al. Relationship between circulating inflammatory monocytes and cardiovascular
and long-term rates of postacute sequelae of sars-Cov-2 infection: A systematic review. disease measures of carotid intimal thickness. J Atheroscler Thromb (2020) 27(5):441–8.
JAMA Netw Open (2021) 4(10):e2128568. doi: 10.1001/jamanetworkopen.2021.28568 doi: 10.5551/jat.49791
3. Jiang DH, Roy DJ, Gu BJ, Hassett LC, McCoy RG. Postacute sequelae of severe 26. Affandi AJ, Olesek K, Grabowska J, Nijen Twilhaar MK, Rodriguez E, Saris A,
acute respiratory syndrome coronavirus 2 infection: A state-of-the-Art review. JACC et al. Cd169 defines activated Cd14(+) monocytes with enhanced Cd8(+) T cell
Basic Transl Sci (2021) 6(9):796–811. doi: 10.1016/j.jacbts.2021.07.002 activation capacity. Front Immunol (2021) 12:697840. doi: 10.3389/fimmu.2021.697840
4. Montani D, Savale L, Noel N, Meyrignac O, Colle R, Gasnier M, et al. Post-acute 27. Doehn JM, Tabeling C, Biesen R, Saccomanno J, Madlung E, Pappe E, et al.
covid-19 syndrome. Eur Respir Rev (2022) 31:210185. doi: 10.1183/16000617.0185-2021 Cd169/Siglec1 is expressed on circulating monocytes in covid-19 and expression levels
5. Merad M, Blish CA, Sallusto F, Iwasaki A. The immunology and are associated with disease severity. Infection (2021) 49(4):757–62. doi: 10.1007/s15010-
immunopathology of covid-19. Science (2022) 375(6585):1122–7. doi: 10.1126/ 021-01606-9
science.abm8108 28. Hartnell A, Steel J, Turley H, Jones M, Jackson DG, Crocker PR.
6. Terpos E, Ntanasis-Stathopoulos I, Elalamy I, Kastritis E, Sergentanis TN, Politou Characterization of human sialoadhesin, a sialic acid binding receptor expressed by
M, et al. Hematological findings and complications of covid-19. Am J Hematol (2020) resident and inflammatory macrophage populations. Blood (2001) 97(1):288–96.
95(7):834–47. doi: 10.1002/ajh.25829 doi: 10.1182/blood.v97.1.288

7. Giamarellos-Bourboulis EJ, Netea MG, Rovina N, Akinosoglou K, Antoniadou A, 29. York MR, Nagai T, Mangini AJ, Lemaire R, van Seventer JM, Lafyatis R. A
Antonakos N, et al. Complex immune dysregulation in covid-19 patients with severe macrophage marker, siglec-1, is increased on circulating monocytes in patients with
respiratory failure. Cell Host Microbe (2020) 27(6):992–1000.e3. doi: 10.1016/ systemic sclerosis and induced by type I interferons and toll-like receptor agonists.
j.chom.2020.04.009 Arthritis Rheum (2007) 56(3):1010–20. doi: 10.1002/art.22382

8. Bassler K, Schulte-Schrepping J, Warnat-Herresthal S, Aschenbrenner AC, 30. Biesen R, Demir C, Barkhudarova F, Grun JR, Steinbrich-Zollner M, Backhaus
Schultze JL. The myeloid cell compartment-cell by cell. Annu Rev Immunol (2019) M, et al. Sialic acid-binding ig-like lectin 1 expression in inflammatory and resident
37:269–93. doi: 10.1146/annurev-immunol-042718-041728 monocytes is a potential biomarker for monitoring disease activity and success of
therapy in systemic lupus erythematosus. Arthritis Rheum (2008) 58(4):1136–45.
9. Guilliams M, Mildner A, Yona S. Developmental and functional heterogeneity of doi: 10.1002/art.23404
monocytes. Immunity (2018) 49(4):595–613. doi: 10.1016/j.immuni.2018.10.005
31. Xiong YS, Cheng Y, Lin QS, Wu AL, Yu J, Li C, et al. Increased expression of
10. Knoll R, Schultze JL, Schulte-Schrepping J. Monocytes and macrophages in siglec-1 on peripheral blood monocytes and its role in mononuclear cell reactivity to
covid-19. Front Immunol (2021) 12:720109. doi: 10.3389/fimmu.2021.720109 autoantigen in rheumatoid arthritis. Rheumatol (Oxford) (2014) 53(2):250–9.
11. Zhou Y, Fu B, Zheng X, Wang D, Zhao C, Qi Y, et al. Pathogenic T-cells and doi: 10.1093/rheumatology/ket342
inflammatory monocytes incite inflammatory storms in severe covid-19 patients. Natl 32. Darawshy F, Abu Rmeileh A, Kuint R, Padawer D, Karim K, Fridlender Z, et al.
Sci Rev (2020) 7(6):998–1002. doi: 10.1093/nsr/nwaa041 Residual symptoms, lung function, and imaging findings in patients recovering from
12. Schulte-Schrepping J, Reusch N, Paclik D, Bassler K, Schlickeiser S, Zhang B, sars-Cov-2 infection. Intern Emerg Med (2022) 17(5):1491–501. doi: 10.1007/s11739-
et al. Severe covid-19 is marked by a dysregulated myeloid cell compartment. Cell 022-02950-w
(2020) 182(6):1419–40.e23. doi: 10.1016/j.cell.2020.08.001 33. Abdallah SJ, Voduc N, Corrales-Medina VF, McGuinty M, Pratt A, Chopra A,
13. Liao M, Liu Y, Yuan J, Wen Y, Xu G, Zhao J, et al. Single-cell landscape of et al. Symptoms, pulmonary function, and functional capacity four months after covid-
bronchoalveolar immune cells in patients with covid-19. Nat Med (2020) 26(6):842–4. 19. Ann Am Thorac Soc (2021) 18(11):1912–7. doi: 10.1513/AnnalsATS.202012-
doi: 10.1038/s41591-020-0901-9 1489RL
14. Liu N, Jiang C, Cai P, Shen Z, Sun W, Xu H, et al. Single-cell analysis of covid-19, 34. Smet J, Stylemans D, Hanon S, Ilsen B, Verbanck S, Vanderhelst E. Clinical
sepsis, and hiv infection reveals hyperinflammatory and immunosuppressive signatures status and lung function 10 weeks after severe sars-Cov-2 infection. Respir Med (2021)
in monocytes. Cell Rep (2021) 37(1):109793. doi: 10.1016/j.celrep.2021.109793 176:106276. doi: 10.1016/j.rmed.2020.106276
15. Mann ER, Menon M, Knight SB, Konkel JE, Jagger C, Shaw TN, et al. 35. Daher A, Balfanz P, Cornelissen C, Muller A, Bergs I, Marx N, et al. Follow up of
Longitudinal immune profiling reveals key myeloid signatures associated with covid- patients with severe coronavirus disease 2019 (Covid-19): Pulmonary and
19. Sci Immunol (2020) 5(51). doi: 10.1126/sciimmunol.abd6197 extrapulmonary disease sequelae. Respir Med (2020) 174:106197. doi: 10.1016/
j.rmed.2020.106197
16. Kreuter M, Lee JS, Tzouvelekis A, Oldham JM, Molyneaux PL, Weycker D, et al.
Monocyte count as a prognostic biomarker in patients with idiopathic pulmonary 36. Queiroz MAF, Neves P, Lima SS, Lopes JDC, Torres M, Vallinoto I, et al.
fibrosis. Am J Respir Crit Care Med (2021) 204(1):74–81. doi: 10.1164/rccm.202003- Cytokine profiles associated with acute covid-19 and long covid-19 syndrome. Front
0669OC Cell Infect Microbiol (2022) 12:922422. doi: 10.3389/fcimb.2022.922422
17. Scott MKD, Quinn K, Li Q, Carroll R, Warsinske H, Vallania F, et al. Increased 37. Pan Y, Jiang X, Yang L, Chen L, Zeng X, Liu G, et al. Sars-Cov-2-Specific
monocyte count as a cellular biomarker for poor outcomes in fibrotic diseases: A immune response in covid-19 convalescent individuals. Signal Transduct Target Ther
retrospective, multicentre cohort study. Lancet Respir Med (2019) 7(6):497–508. (2021) 6(1):256. doi: 10.1038/s41392-021-00686-1
doi: 10.1016/S2213-2600(18)30508-3 38. Schultheiss C, Willscher E, Paschold L, Gottschick C, Klee B, Henkes SS, et al.
18. Szabo PA, Dogra P, Gray JI, Wells SB, Connors TJ, Weisberg SP, et al. The il-1beta, il-6, and tnf cytokine triad is associated with post-acute sequelae of covid-
Longitudinal profiling of respiratory and systemic immune responses reveals myeloid 19. Cell Rep Med (2022) 3(6):100663. doi: 10.1016/j.xcrm.2022.100663
cell-driven lung inflammation in severe covid-19. Immunity (2021) 54(4):797–814.e6. 39. Pandori WJ, Padgett LE, Alimadadi A, Gutierrez NA, Araujo DJ, Huh CJ, et al.
doi: 10.1016/j.immuni.2021.03.005 Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a
19. Marais C, Claude C, Semaan N, Charbel R, Barreault S, Travert B, et al. Myeloid novel subset of Cd9(+) monocytes associated with covid-19 hospitalization. J Leukoc
phenotypes in severe covid-19 predict secondary infection and mortality: A pilot study. Biol (2022) 112(5):1053–63. doi: 10.1002/JLB.4COVA0122-076R
Ann Intensive Care (2021) 11(1):111. doi: 10.1186/s13613-021-00896-4
40. Schultheiss C, Willscher E, Paschold L, Gottschick C, Klee B, Bosurgi L, et al.
20. Utrero-Rico A, Gonzalez-Cuadrado C, Chivite-Lacaba M, Cabrera-Marante O, Liquid biomarkers of macrophage dysregulation and circulating spike protein illustrate
Laguna-Goya R, Almendro-Vazquez P, et al. Alterations in circulating monocytes the biological heterogeneity in patients with post-acute sequelae of covid-19. J Med
predict covid-19 severity and include chromatin modifications still detectable six Virol (2023) 95(1):e28364. doi: 10.1002/jmv.28364
months after recovery. Biomedicines (2021) 9(9). doi: 10.3390/biomedicines9091253
41. Afanasieva OI, Filatova AY, Arefieva TI, Klesareva EA, Tyurina AV,
21. Ryan FJ, Hope CM, Masavuli MG, Lynn MA, Mekonnen ZA, Yeow AEL, et al. Radyukhina NV, et al. The association of lipoprotein(a) and circulating monocyte
Long-term perturbation of the peripheral immune system months after sars-Cov-2 subsets with severe coronary atherosclerosis. J Cardiovasc Dev Dis (2021) 8(6).
infection. BMC Med (2022) 20(1):26. doi: 10.1186/s12916-021-02228-6 doi: 10.3390/jcdd8060063
22. Phetsouphanh C, Darley DR, Wilson DB, Howe A, Munier CML, Patel SK, et al.
Immunological dysfunction persists for 8 months following initial mild-to-Moderate sars- 42. Bai F, Tomasoni D, Falcinella C, Barbanotti D, Castoldi R, Mule G, et al. Female
Cov-2 infection. Nat Immunol (2022) 23(2):210–6. doi: 10.1038/s41590-021-01113-x gender is associated with long covid syndrome: A prospective cohort study. Clin
Microbiol Infect (2022) 28(4):611.e9–e16. doi: 10.1016/j.cmi.2021.11.002
23. Patterson BK, Francisco EB, Yogendra R, Long E, Pise A, Rodrigues H, et al.
Persistence of sars cov-2 S1 protein in Cd16+ monocytes in post-acute sequelae of 43. Subramanian A, Nirantharakumar K, Hughes S, Myles P, Williams T, Gokhale
covid-19 (Pasc) up to 15 months post-infection. Front Immunol (2021) 12:746021. KM, et al. Symptoms and risk factors for long covid in non-hospitalized adults. Nat
doi: 10.3389/fimmu.2021.746021 Med (2022) 28(8):1706–14. doi: 10.1038/s41591-022-01909-w

Frontiers in Immunology 12 frontiersin.org