No indications for overt innate immune suppression in critically ill COVID-19 patients

At the end of March 2020, there were in excess of 800.000 confirmed cases of coronavirus disease 2019 (COVID-19) worldwide. Several reports suggest that, in severe cases, COVID-19 may cause a hyperinflammatory 'cytokine storm'. However, unlike SARS-CoV infection, high levels of anti-inflammatory mediators have also been reported in COVID-19 patients. One study reported that 16% of COVID-19 patients who died developed secondary infection, which might indicate an immune-suppressed state. We explored kinetics of mHLA-DR expression, the most widely used marker of innate immune suppression in critically ill patients, in COVID-19 patients admitted to the ICU. Twenty-four confirmed COVID-19 patients were included, of which 75% was male and 79% had comorbidities. All patients were mechanically ventilated and exhibited large high levels of inflammatory parameters such as CRP and PCT. mHLA-DR expression levels were mostly within the normal range of 15000 - 45000 mAb/cell and showed no change over time. COVID-19 patients displayed notably higher mHLA-DR expression levels compared with bacterial septic shock patients. None of the COVID-19 patients developed a secondary infection. In conclusion, despite a pronounced inflammatory response, mHLA-DR expression kinetics indicate no overt innate immune suppression in COVID-19 patients. These data signify that innate immune suppression as a negative feedback mechanism following PAMP-induced inflammation appears not to be present in COVID-19.

Low monocytic (m)HLA-DR expression is the most widely used marker of innate immune suppression in critically ill patients. We recently showed that, in bacterial septic shock patients, low mHLA-DR expression is prevalent and associated with the development of secondary infections [1]. At the end of March 2020, there were in excess of 800.000 confirmed cases of coronavirus disease 2019 (COVID-19) worldwide, of whom more than 12.000 from the Netherlands. Several reports suggest that patients with severe COVID-19 may suffer from a hyperinflammatory "cytokine storm" [2,3]. However, unlike SARS-CoV infection, high levels of anti-inflammatory mediators (e.g. IL-10 and IL-4) have also been reported in COVID-19 [3].
Although there are few indications that secondary infections are common in COVID-19 patients, one study reported that 16% of COVID-19 patients who died developed secondary infections [4], which might indicate an immune-suppressed state. In the present work, we explored mHLA-DR expression kinetics in a cohort of 24 COVID-19 patients admitted to the Intensive Care Unit (ICU).
Between March 18 th -27 th , all COVID-19 patients admitted to our ICU were included in this prospective observational study. COVID-19 was confirmed by two positive RT-PCR tests for SARS-CoV-2 in throat swabs and by CT-scan findings. Fourteen patients were transferred from other ICUs. The median [IQR] ICU length of stay at the time of study inclusion was 3 [3][4] days. The study was carried out in accordance with the applicable rules concerning the review of research ethics committees and informed consent in the Netherlands. The ethics committee (CMO Arnhem-Nijmegen) board waived the need for informed consent because of the observational nature of the study and the non-invasiveness of blood withdrawal (all patients had an arterial canula in place, so no venapunctures were performed). All patients or legal representatives were informed about the study details and could abstain from participation. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 6, 2020. . https://doi.org/10.1101/2020.04.03.20049080 doi: medRxiv preprint Patient characteristics are listed in Table 1. In line with previous observations [3], the majority of patients was male and many had comorbidities. The median [IQR] time from onset of COVID-19 symptoms to ICU admission was 11 [8-13] days. All patients were mechanically ventilated and exhibited increases in inflammatory parameters ( Table 1)  In conclusion, despite a pronounced inflammatory response in COVID-19 patients, our preliminary results of mHLA-DR expression kinetics indicate no overt innate immune suppression. These findings are in accordance with a low incidence of secondary infections.
Therefore, innate immune suppression as a negative feedback mechanism following pathogenassociated molecular pattern-induced inflammation appears not to be present in COVID-19.
All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 6, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 6, 2020.
All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Ethics approval and consent to participate
The study was carried out in accordance with the applicable rules concerning the review of research ethics committees and informed consent in the Netherlands. The ethics committee (CMO Arnhem-Nijmegen) board waived the need for informed consent because of the observational nature of the study and the non-invasiveness of blood withdrawal (all patients had an arterial canula in place, so no venapunctures were performed). All patients or legal representatives were informed about the study details and could abstain from participation.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
The work was internally funded by the participating departments.  data obtained from bacterial septic shock patients using the same methodology, as recently published [1] (geometric mean ± 95% CI, please note that values obtained from septic shock patients at days 1-2 (n=203), 3-4 (n=205), and 6-8 (n=133) are plotted at day 1-3, 4-5, and 6-7, respectively). The dotted lines in panel A indicate the reference range in healthy subjects.
p-values next to the transparent grey line represent changes over time in COVID-19 patients calculated using mixed model analysis (on log transformed data for panels C and E).
Differences between COVID-19 and sepsis patients were analysed using unpaired t-tests on log-transformed data (p<0.0001 on all three timepoints).
All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.