COVID‐19 rapidly increases MDSCs and prolongs innate immune dysfunctions

We used unsupervised immunophenotyping of blood leukocytes and measured cytokine production by innate immune cell exposed to LPS and R848. We show that COVID‐19 induces a rapid, transient upregulation of myeloid‐derived suppressor cells (MDSCs) accompanied by a rapid, sustained (up to 3 months) hyporesponsiveness of dendritic cells and monocytes. Blood MDSCs may represent biomarkers and targets for intervention strategies in COVID‐19 patients.


COVID-19 rapidly increases MDSCs and prolongs innate immune dysfunctions
Inflammatory and danger signals stimulate hematopoiesis and the generation of myeloid-derived suppressor cells (MDSCs) that suppress innate and adaptive immune responses [1]. High levels of blood MDSCs are associated with nosocomial infections, morbidity, and mortality in critically ill patients with sepsis [2]. Severe COVID-19 is characterized by exuberant inflammation, leading to a cascade of immune-related manifestations. Lymphopenia and impaired immune effector cell functions contribute to COVID-19 pathogenesis and increase the risk of secondary infections and death [3]. While increased expression of MDSCs has been reported in COVID-19 patients [4][5][6][7], scare studies performed longterm, longitudinal analyses in recovered patients.
COVID-19 patients had higher leukocyte counts and longer hospital stay than moderate COVID-19 patients (Supporting Information Table S1). Ten age-and sexmatched healthy individuals were used as controls.
At study inclusion, PMN-MDSCs and M-MDSCs counts were 10-fold and fourfold higher in severe than in moderate COVID-19 patients (Fig. 1A). Other cell populations were similar in severe and moderate COVID-19 patients. PMN-MDSCs and M-MDSCs levels correlated with each other (ρ = 0.43; p = 0.03). PMN-MDSCs inversely correlated with lymphocyte counts (Fig. 1B). A similar, not statistically significant, inverse correlation was detected between MDSCs and CD4 + and CD8 + T cells and T regulatory cells (Fig.  S2). Since the levels of M-MDSCs in blood, but not in the airways, correlated with COVID-19 severity [5], the quantification of MDSCs in peripheral blood may represent an interesting biomarker of COVID-19.
Thirty-three cytokines/chemokines/ growth factors (measured using a 49plex assay) were detected in the serum of patients (Fig. 1C) (Fig. 1C). Interestingly, EGF, HGF, PDGF-BB, and VGEF have been shown to expand and chemo-attract MDSCs, and IL-1β and IL-7 to stimulate myelopoiesis and sustain the expansion and T-cell suppressing activity of MDSCs [1,2]. Thus, the inflammatory milieu in COVID-19 patients contains mediators that promote the generation and activity of MDSCs.
Whole blood was stimulated with LPS and R848. Intracellular cytokine staining followed by flow cytometry analysis was used to quantify the proportion of monocytes and DCs producing TNF and IL-6 ( Fig. 2). In healthy controls, 0.02/4.3% of monocytes produced TNF/IL-6 at baseline, 24/17% in response to LPS, and 79/46% in response to R848, respectively. The percentage of blood monocytes producing TNF and IL-6 in response to LPS and R848 was 1.3-to 4.9-fold lower in COVID-19 patients. The reduction was more striking in severe than in moderate COVID-19 patients. The impaired response of monocytes persisted 3 months (Fig. 2Aand B). In healthy controls, 0.6% of DCs produced TNF/IL-6 at baseline, 38/36% in response to LPS, and 68/58% in response to R848. TNF and IL-6 positive DCs were 2.1-to 5.1-fold lower in COVID-19 patients, more impaired in severe than in moderate COVID-19 patients. Impaired cytokine response persisted 3 months (Fig. 2C and D).
Upon stimulation with LPS and R848, 17 of 24 and 13 of 24 of cytokines were detected at lower concentrations in blood from patients than healthy controls (Fig. 2E). Interestingly, six of 24 and seven of 24 of the cytokines were detected at lower concentrations in patients analyzed after 3 months, implying prolonged immunological defects. Patients with moderate and severe COVID-19 were similarly affected.
MDSCs represented 10%-15% of blood leukocytes, peaked in severe COVID-19, and were associated with cytokine levels, lymphocytopenia, worse outcome, and impaired cytokine production by monocytes and DCs. Three months after inclusion, leukocyte counts were back to normal. Yet, blood, monocytes, and DCs still displayed reduced cytokine production, revealing long-term immune disturbances. Likewise, MDSCs were normalized while cellular abnormalities were uncovered several weeks after SARS-CoV-2 infection [9]. Whether MDSCs play a role in persistent immune dysfunctions is unknown, but would involve long-lasting imprinting independent from MDSCs elevated counts. The suppressive activity of MDSCs might vary over time, as reported during sepsis [2]. Overall, failure to restore immune homeostasis in COVID-19 patients may be a driver of long-COVID, increasing the risk of infections. Long COVID is reminiscent of the postsepsis syndrome characterized by immunosuppression associated with persistent low-grade inflammation [10].
Our work has limitations. The number of patients was small, which may have limited the detection of differences or correlations. Additional markers could be used to trace MDSCs. However, we elected to minimize analytical variations by labeling whole blood quickly after drawing and analyzing flow cytometry data by unsupervised clustering. Finally, we have not assessed the immunosuppressive capacity of MDSCs. Yet, MDSCs of COVID-19 patients were shown to inhibit the prolif-eration and cytokine production by T cells [4][5][6].
To conclude, our data suggest that MDSCs in peripheral blood represent biomarkers to stratify COVID-19 patients. Targeting MDSCs and/or immune dysfunctions might proof useful to counterbalance immunosuppression, reduce nosocomial and long-term infections, and decrease late mortality in severe COVID-19 patients.

Acknowledgements:
This work was supported by the Swiss National Science Foundation (CRSII3_147662S and 310030_207418), European Union (676129), Société Académique Vaudoise (Switzerland), and Porphyrogenis Foundation (Switzerland). We thank Profs. Vollenweider and Waeber (Lausanne University Hospital, Switzerland) for their contribution. Open access funding provided by Universite de Lausanne.

Conflict of interest:
The authors declare no commercial or financial conflict of interest.

Data availability statement:
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.