key: cord-1033096-slbfft33 authors: Gomez Rial, J.; Curras Tuala, M. J.; Rivero Calle, I.; Gomez Carballa, A.; Cebey Lopez, M.; Rodriguez Tenreiro, C.; Dacosta Urbieta, A.; Rivero Velasco, C.; Rodriguez Nunez, N.; Trastoy Pena, R.; Rodriguez Garcia, J.; Salas, A.; Martinon Torres, F. title: Increased serum levels of sCD14 and sCD163 indicate a preponderant role for monocytes in COVID-19 immunopathology date: 2020-06-04 journal: nan DOI: 10.1101/2020.06.02.20120295 sha: 2ed18e33f71a7afe6f7f78dd1882d6d17ba85cbc doc_id: 1033096 cord_uid: slbfft33 Background. Emerging evidence indicates a potential role for monocyte in COVID-19 immunopathology. We investigated two soluble markers of monocyte activation, sCD14 and sCD163, in covid19 patients with the aim of characterizing their potential role in monocyte-macrophage disease immunopathology. To the best of our knowledge, this is the first study of its kind. Methods. Fifty-nine SARS-Cov-2 positive hospitalized patients, classified according to ICU or non-ICU admission requirement, were prospectively recruited and analyzed by ELISA for levels of sCD14 and sCD163, along with other laboratory parameters, and compared to a healthy control group. Results. sCD14 and sCD163 levels were significantly higher among COVID-19 patients, independently of ICU admission requirement, compared to the control group. We found a significant correlation between sCD14 levels and other inflammatory markers, particularly Interleukin-6, in the non-ICU patients group. sCD163 showed a moderate positive correlation with the time at sampling from admission, increasing its value over time, independently of severity group. Conclusions. Monocyte-macrophage activation markers are increased and correlate with other inflammatory markers in SARS-Cov-2 infection, in association to hospital admission. These data suggest a potentially preponderant role for monocyte-macrophage activation in the development of immunopathology of covid19 patients. Background. Emerging evidence indicates a potential role for monocyte in COVID-19 immunopathology. We investigated two soluble markers of monocyte activation, sCD14 and sCD163, in covid19 patients with the aim of characterizing their potential role in monocyte-macrophage disease immunopathology. To the best of our knowledge, this is the first study of its kind. Methods. Fifty-nine SARS-Cov-2 positive hospitalized patients, classified according to ICU or non-ICU admission requirement, were prospectively recruited and analyzed by ELISA for levels of sCD14 and sCD163, along with other laboratory parameters, and compared to a healthy control group. Results. sCD14 and sCD163 levels were significantly higher among COVID-19 patients, independently of ICU admission requirement, compared to the control group. We found a significant correlation between sCD14 levels and other inflammatory markers, particularly Interleukin-6, in the non-ICU patients' group. sCD163 showed a moderate positive correlation with the time at sampling from admission, increasing its value over time, independently of severity group. Emerging evidence from SARS-Cov-2 infected patients suggests a key role for monocyte-macrophage in the immunopathology of COVID-19 infection, with a predominant monocyte-derived macrophage infiltration observed in severely damaged lungs [1] , and morphological and inflammation-related changes in peripheral blood monocytes that correlate with the patients' outcome [2] An overexuberant inflammatory immune response with production of a cytokine storm and T-cell immunosuppression are the main hallmarks of severity in these patients [3] . This clinical course resembles viral-associated hemophagocytic syndrome (VASH), a rare severe complication of various viral infections mediated by proinflammatory cytokines, resulting in multiorgan failure and death [4] . A chronic expansion of inflammatory monocytes and over-activation of macrophages have been extensively described in this syndrome [5; 6; 7] . VAHS has been identifies as a major contributor to death of patients in past pandemics outbreaks [8] including previous SARS and MERS outbreaks [9] and is currently suggested for SARS-Cov-2 outbreak. [10] CD14 and CD163 are both myeloid differentiation markers found primarily on monocytes and macrophages, and detection of soluble release of both in plasma is considered a good biomarker of monocyte-macrophage activation [11; 12] . Elevated plasma levels of soluble CD14 (sCD14) are associated to poor prognosis in VIH-infected patients, are a strong predictor of morbidity and mortality [13; 14] , and associated with diminished CD4+-T cell restoration [15] . In addition, soluble CD163 (sCD163) plasma levels are a good proxy for monocyte expansion and disease progression during HIV infection [16] . In measles infection, a leading cause of death associated with increased susceptibility to secondary infections and immunosuppression, sCD14 and sCD163 levels were found to be significantly higher, indicating an important and persistent monocytemacrophage activation [17] . We hypothesized that monocytes/macrophages may be an important component of immunopathology associated to SARS-Cov-2 infection. In this paper, we analyze plasma levels of soluble monocyte activation markers in COVID-19 patients and their correlation with severity and other inflammatory markers. 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 June 4, 2020. . https://doi.org/10.1101/2020.06.02.20120295 doi: medRxiv preprint We recruited 59 patients with confirmed PCR-positive diagnostic for SARS-Cov-2 infection, classified according ICU admission requirement (n=22 patients), or non-ICU requirement (n=37), and age-matched healthy individuals (n=20) as control group. Demographic data, main medication treatment and routine lab clinical parameters including inflammatory biomarkers were collected for all infected patients. Leftover sera samples from routine analytical control were employed for the research analysis, after obtaining corresponding informed consent. Time elapsed from hospital admission to sample extraction was also recorded. To determine levels of soluble monocyte activation markers in serum specimens, appropriate sandwich ELISA (Quantikine, R&D systems, United Kingdom) were used following manufacturer indications. Briefly, diluted sera samples were incubated for 3 hours at room temperature in the corresponding microplate strips coated with capture antibody. After incubation, strips were properly washed and incubated with the corresponding Human Antibody conjugate for 1 hour. After washing, reactions were revealed and optical density at 450 nm was determined in a microplate reader. Concentration levels were interpolated from the standard curve using a four-parameter logistic (4-PL) curve-fit in Prism8 GraphPad software. Final values were corrected applying the corresponding dilution factor employed. Data are expressed as median and interquartile range. All statistical analyses were performed using the statistical package R. Mann-Whitney tests were used for comparison between ICU and non-ICU groups versus healthy controls. Pearson's correlation coefficients were used to quantify the association between sCD14 and sCD163 concentration and other lab parameters in non-ICU patients. Data outliers, falling outside the 1.5 interquartile range, were excluded from the 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 June 4, 2020. . https://doi.org/10.1101/2020.06.02.20120295 doi: medRxiv preprint statistical analysis. The nominal significance level considered was 0.05. Bonferroni adjustment was used to account for multiple testing. Patients in the ICU group showed significant differences in several clinical laboratory parameters when compared to non-ICU group: lymphocytes, ferritin, D-dimer, Lactate Dehydrogenase (LDH), Procalcitonin (PCT), Interleukin-6 (IL-6). The absolute value for circulating monocytes did not show significant differences between groups. However, these values may have been distorted by the use of tocilizumab, an IL-6 blocking drug extensively employed in the ICU group which interferes with monocyte function. Age and time elapsed from admission to sample extraction did not show differences between groups. Values are summarized in Table 1 . Median levels for sCD14 in sera from ICU patients were 2444.0 (95%CI: 1914.0-3251.0) ng/ml, compared to 2613.0 (95%CI: 2266.0-2991.0) ng/ml in non-ICU patients. The healthy control group median value was 1788.0 (95%CI: 1615.0-1917.0) ng/ml. We observed significant differences for values from infected patients relative to control group (P-value<0.0001), however no significant differences were observed between ICU and non-ICU groups. Median levels for sCD163 in sera from ICU patients were 911.5 (95%CI: 624.7-1167.0) ng/ml, and 910.4 (95%CI: 733.1-1088.0) ng/ml in non-ICU patients. The healthy control group value was 495.6 (95%CI: 332.5-600.7) ng/ml. In the same way as with sCD14, we observed significant differences for values from infected patients compared to control group (P-value<00001), but no differences between ICU and non-ICU infected patients. Values are summarized in Table 2 and Figure 1 . We assessed correlation between sCD14 and sCD163 levels and time elapsed from hospital admission to sample extraction (Figure 2) . We found a significant positive correlation between sCD163 levels and time elapsed (r 2 =0.3246, P-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 June 4, 2020. (Figure 3) . Particularly, IL-6 also shows a significant positive correlation with sCD14 (r 2 =0.6034, P-value=0.0003) (Figure 4 ). We analyzed possible age-dependence of sCD14 and sCD163 levels. Values did not show association between these biomarker levels and the age of patients. Our results show, for the first time, increased levels of sCD14 and sCD163 in sera from SARS-Cov-2 infected patients admitted to hospital. We did not observe any differences between ICU or non-ICU patients, probably due to the interference on monocyte function produced by the use of tocilizumab and/or corticoid treatment in ICU patients as previously demonstrated [18; 19] . However, levels of sCD14 showed a strong correlation with clinical laboratory parameters, including acute phase reactants (ferritin, LDH, C-reactive protein, procalcitonin) and a strong correlation with IL-6 levels in the non-ICU patient group, where no tocilizumab and/or corticoids treatments were used. Furthermore, sCD163 levels 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 June 4, 2020. . https://doi.org/10.1101/2020.06.02.20120295 doi: medRxiv preprint showed a correlation with the time elapsed from hospital admission to sample extraction, increasing its value over time of hospital admission, suggesting a potential indicator of progression of disease. Monocytes and macrophages constitute a key component of immune responses against viruses, acting as bridge between innate and adaptive immunity [20] . Activation of macrophages has been demonstrated to be pivotal in the pathogenesis of the immunosuppression associated to several viral infections (VIH, measles), where expansion of specific subsets of monocytes and macrophages in peripheral blood are observed, and considered to be drivers of immunopathogenesis [21] . Our results support the hypothesis of a preponderant role for monocytes in SARS-Cov-2 immunopathology, associated to an over exuberant immune response. Increased levels of monocyte-macrophage activation markers and the correlation with other inflammatory biomarkers (particularly IL-6), indicate a close relationship between monocyte activation and immunopathology in these patients. Inflammatory markers are closely related to severity in COVID-19 pathology [22] and selective blockade of IL-6 has been demonstrated to be a good therapeutic strategy in COVID-19 pathology [23] . Our results thus suggest that monocyte-macrophage activation can act as driver cells of the cytokine storm and immunopathology associated to severe clinical course of COVID-19 patients. Further, monitorization of monocyte activity trough these soluble activation markers and/or follow-up of circulating inflammatory monocytes in peripheral blood, could be useful to assess disease progression in the same way as in other viral infections [16] . In addition, our results identify monocyte-macrophage as a good target for the design of therapeutic intervention using drugs that inhibit monocytemacrophage activation and differentiation. In this sense, anti-GM CSF inhibitor drugs, currently under clinical trials for rheumatic and other auto-inflammatory diseases, might provide satisfactory results in COVID-19 patients. Other drugs targeting monocyte and/or macrophage could also be useful in COVID-19, as in other inflammatory diseases [24] . The strategy of inhibiting monocyte differentiation has proved useful in avoiding cytokine storm syndrome after CAR-T cell immunotherapy [25] , suggesting a possible therapeutic application to COVID-19 immunopathology [26] The present study has several limitations, 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 June 4, 2020. . https://doi.org/10.1101/2020.06.02.20120295 doi: medRxiv preprint including the relatively low numbers of patients tested and the interference of tocilizumab and corticoids in ICU patients' results. However, these preliminary results are strongly suggestive of an important implication of monocytemacrophage in COVID-19 immunopathology, as highlighted by the correlations found between these biomarker levels and inflammatory parameters. Further studies using broader series are needed to confirm our findings. In summary, our data underscore the preponderant role of monocyte and macrophage immune response in COVID-19 immunopathology and provide pointers for future interventions in drug strategies and monitoring plans for these patients. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential We want to acknowledge the effort of all first-line healthcare workers of the patients included in this study. 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 June 4, 2020. . https://doi.org/10.1101/2020.06.02.20120295 doi: medRxiv preprint [26] J. Gomez-Rial, and F. Martinon-Torres, A strategy targeting monocytemacrophage differentiation to avoid pulmonary complications in sars-cov2 infection. Clinical Immunology in press (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. The copyright holder for this preprint this version posted June 4, 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. The copyright holder for this preprint this version posted June 4, 2020. . https://doi.org/10.1101/2020.06.02.20120295 doi: medRxiv preprint Table 2 . Concentration (ng/ml) of serum levels of sCD14 and sCD163 in patients from ICU and non-ICU groups, and healthy controls. Data are represented as median and interquartile range. 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 June 4, 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. The copyright holder for this preprint this version posted June 4, 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 June 4, 2020. 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