key: cord-0686630-rw8oa757
authors: Takano, Tomohiro; Matsumura, Takayuki; Adachi, Yu; Terahara, Kazutaka; Moriyama, Saya; Onodera, Taishi; Nishiyama, Ayae; Kawana-Tachikawa, Ai; Miki, Shoji; Hosoya-Nakayama, Kaori; Nakamura-Hoshi, Midori; Seki, Sayuri; Tachikawa, Natsuo; Yoshimura, Yukihiro; Miyata, Nobuyuki; Horiuchi, Hiroshi; Sasaki, Hiroaki; Miyazaki, Kazuhito; Kinoshita, Noriko; Sudo, Tsutomu; Akiyama, Yutaro; Sato, Rubuna; Suzuki, Tadaki; Matano, Tetsuro; Takahashi, Yoshimasa
title: Myeloid cell dynamics correlating with clinical outcomes of severe COVID-19 in Japan
date: 2021-02-04
journal: Int Immunol
DOI: 10.1093/intimm/dxab005
sha: e48321f6b5cb542e7adf2bead9df707e3b5bc1d0
doc_id: 686630
cord_uid: rw8oa757

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19). However, data regarding myeloid cell expansion have been collected in Europe, where the mortality rate by COVID-19 is greater than those in other regions including Japan. Thus, characteristics of COVID-19-induced myeloid cell subsets remain largely unknown in the regions with low mortality rates. Here, we analyzed cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis, using blood samples from Japanese COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, but not other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Contrary to previous studies in Europe, this subset selectively expanded in survivors of severe cases and subsided before discharge, but such transient expansion was not observed in non-survivors in Japanese cohort. Analysis of plasma cytokine/chemokine levels revealed positive correlation of PMN-MDSC frequencies with interleukin 8 (IL-8) levels prior to the cell expansion, indicating the involvement of IL-8 on recruitment of PMN-MDSCs to peripheral blood following the onset of severe COVID-19. Thus, our data indicates that transient expansion of the PMN-MDSC subset results in improved clinical outcome. Thus, this myeloid cell subset may be a predictor of prognosis in cases of severe COVID-19 in Japan.

In December 2019, coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, Hubei Province, China. The virus spread rapidly, causing a global pandemic (1-5). Although most COVID-19 patients are asymptomatic or present with mild clinical symptoms that resemble seasonal coronavirus diseases, 19% of patients suffer from severe or critical disease with 2.3% mortality (6). The countermeasure for the pandemic is to protect those who are at greater risk of death from COVID-19, including the elderly and those with comorbidities, such as hypertension, diabetes, and cardiac/pulmonary diseases (6,7). Notably, the mortality rate is obviously lower in Asia than in US and Europe (8), but it remains unclear why the Asian population is resistant to COVID-19-related morbidity, although several hypotheses have been put forth.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that mediate immune suppression and are generated during a large array of pathogenic conditions ranging from cancer to obesity (9). In humans, MDSCs consist of at least three groups of cells, namely, early stage MDSCs (e-MDSCs), monocytic MDSCs (M-MDSCs), and polymorphonuclear MDSCs (PMN-MDSCs). These have been found in peripheral blood mononuclear cells (PBMCs), in addition to bone marrow and inflammatory tissues (9). Various infectious diseases have been reported to induce MDSCs, including bacterial, fungal, parasitic, and viral infections (10); however, their roles in disease pathogenesis are still unclear. Notably, individuals with the aforementioned risk factors for COVID-19 are prone to sustain increased frequencies of MDSCs (9,11). In addition, elevated levels of interleukin 6 (IL-6) and IL-8, well-known inducers of MDSCs (12- 14) , are observed in severe cases of COVID-19 (15, 16) .

A c c e p t e d M a n u s c r i p t 6 This information suggests a possible link between MDSCs and COVID-19. The expansion of MDSCs and MDSC-like cells has been repeatedly observed in severe COVID-19 patients by several research groups from European countries (17) (18) (19) (20) (21) . However, the previous results may not be directly applicable in other regions where the mortality rate is lower. In this study, we describe the transient but prominent expansion of the PMN-MDSC subset in survivors, but not in non-survivors, of severe COVID-19 in Japan. Our data suggest the beneficial role of PMN-MDSC subset, which potentially suppress excessive inflammation during recovery from severe COVID-19 in Japan.

This study protocol was approved by the National Institute of Infectious Diseases Ethic 

This study enrolled 40 patients with mild (n = 12), moderate I (n = 7), moderate II (n = 8), 

Blood samples were collected from the COVID-19 patients and healthy donors using a BD 

Plasma cytokines/chemokines were measured using a cytometric bead array kit (BD Biosciences) according to the manufacturer's instructions. Data were acquired using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FCAP Array Software Version 3.0 (BD Biosciences).

Data comprising the flow cytometric frequencies of cells were compared with two-way ANOVA with post-hoc Tukey's honest significant difference test. For plasma cytokine levels below the detection limit, the value was set to 0.01 pg/ml. Cytokine concentrations were compared with one-way ANOVA with post-hoc Tukey's honest significant difference test.

A c c e p t e d M a n u s c r i p t 9 Statistical significance was set at **p < 0.01, ***p < 0.001, and ****p < 0.0001. Spearman correlations between plasma cytokine concentrations and cell frequencies were identified in all specimens. Correlations with r > 0.4 or r < −0.4 and p < 0.01 were considered significant.

A simple regression line was shown for a significant correlation. GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses and graphical representations.

All scripts used in this manuscript are available upon reasonable request.

Three MDSC subsets (e-MDSCs, M-MDSCs, and PMN-MDSCs) in PBMCs were analyzed using flow cytometry (Fig. 1) (Fig. 3) . Compared to healthy controls, most cytokines/chemokines, except IL-8, were not significantly elevated in cohorts of this study.

We observed a significant and selective increase in IL-8 levels and PMN-MDSC subset from survivors of severe cases (Fig. 2 and 3) . These results imply a link between IL-8 and PMN-MDSC induction. In agreement with this, we found that IL-8 levels and PMN-MDSC frequency were positively correlated (Fig. 4) The current study still has a few limitations. 1) The sample size of this study was small due to a limited availability of patients. A recent cohort study in the United States revealed that male patients had higher plasma levels of innate immune cytokines including IL-8 along with more robust induction of non-classical monocytes (24). Therefore, the sex difference between the groups of severe-discharged (7 males and 1 female) and deceased (4 females and 1 male) patients might affect any events in induction of PMN-MDSCs (Supplementary Table 1 ). It is important to accumulate more data before generalizing the results in this study. 2) Frozen PBMCs were used in this study. We recognize that total cell numbers would be lowered by freezing and thawing process. However, in order to find the myeloid subsets that correlate with disease severity, it is acceptable to compare the frequency M a n u s c r i p t The frequencies of each MDSC subset relative to live PBMCs (7-AADwhole PBMCs) were analyzed for the first 3 weeks (day 0-21 after symptom onset). Data are represented as points indicating the data of independent samples and the mean (bar) ± SD of independent samples.

Healthy donors, n = 7, n' = 7. COVID-19 mild cases, n = 21, n' = 12; moderate I cases, n = 19, n' = 7; moderate II cases, n = 21, n' = 8; severe surviving cases, n = 12, n' = 7; severe fatal cases, n = 7, n' = 5 (n, samples; n', individuals). Two-way ANOVA and Tukey's posthoc test were performed for statistical analysis (***p < 0.001, ****p < 0.0001).

A c c e p t e d M a n u s c r i p t 19

Concentrations of plasma cytokines/chemokines were analyzed for the first 3 weeks (day 0-21 after symptom onset). Data are shown as points indicating the data of independent samples and the mean (bar) ± SD of independent samples. Healthy donors, n = 7, n' = 7. COVID-19 mild cases, n = 21, n' = 12; moderate I cases, n = 19, n' = 7; moderate II cases, n = 21, n' = 8;

severe surviving cases, n = 12, n' = 7; severe fatal cases, n = 7, n' = 5 (n, samples; n', individuals). One-way ANOVA and Tukey's post-hoc test were performed for statistical analysis (**p < 0.01, ***p < 0.001, ****p < 0.0001).

Correlations between plasma IL-8 concentrations and frequencies of e-MDSCs, M-MDSCs, or PMN-MDSCs relative to live PBMCs were analyzed. Healthy donors, n = 7, n' = 7;

COVID-19 mild cases, n = 27, n' = 12; moderate I cases, n = 24, n' = 7; moderate II cases, n

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We thank Michio Aiko, Rutaro Iwabuchi, Yasuko Tsunetsugu-Yokota, and Manabu Ato for technical support; and Ryoko Itami for secretarial assistance.

The authors declared no conflicts of interest.A c c e p t e d M a n u s c r i p t

A c c e p t e d M a n u s c r i p t