RESOURCE https://doi.org/10.1038/s43587-021-00067-x Cellular and plasma proteomic determinants of COVID-19 and non-COVID-19 pulmonary diseases relative to healthy aging Laura Arthur1,8, Ekaterina Esaulova 1,8, Denis A. Mogilenko 1, Petr Tsurinov1,2, Samantha Burdess1, Anwesha Laha1, Rachel Presti 3, Brian Goetz4, Mark A. Watson1, Charles W. Goss5, Christina A. Gurnett6, Philip A. Mudd 7, Courtney Beers4, Jane A. O’Halloran3 and Maxim N. Artyomov1 ✉ We examine the cellular and soluble determinants of coronavirus disease 2019 (COVID-19) relative to aging by performing mass cytometry in parallel with clinical blood testing and plasma proteomic profiling of ~4,700 proteins from 71 individuals with pul- monary disease and 148 healthy donors (25–80 years old). Distinct cell populations were associated with age (GZMK+CD8+ T cells and CD25low CD4+ T cells) and with COVID-19 (TBET−EOMES− CD4+ T cells, HLA-DR+CD38+ CD8+ T cells and CD27+CD38+ B cells). A unique population of TBET+EOMES+ CD4+ T cells was associated with individuals with COVID-19 who experienced moderate, rather than severe or lethal, disease. Disease severity correlated with blood creatinine and urea nitrogen levels. Proteomics revealed a major impact of age on the disease-associated plasma signatures and highlighted the divergent contri- bution of hepatocyte and muscle secretomes to COVID-19 plasma proteins. Aging plasma was enriched in matrisome proteins and heart/aorta smooth muscle cell-specific proteins. These findings reveal age-specific and disease-specific changes associ- ated with COVID-19, and potential soluble mediators of the physiological impact of COVID-19. OVID-19 is caused by the novel severe acute respiratory syn- cell composition changes significantly with age, as does the environ- drome coronavirus 2 (SARS-CoV-2), a pandemic virus that ment, for example, the plasma proteome20. Therefore, understanding rapidly spread worldwide, killing over two million individu- the COVID-19-driven immune response in the context of the aging C 1 als as of February 2021 (World Health Organization ). Most indi- immune system is critically important in determining why patho- viduals infected by SARS-CoV-2 are asymptomatic or have mild gens like SARS-CoV-2 more frequently initiate a severe clinical pre- to moderate clinical symptoms2. However, a notable portion of sentation in older individuals. However, a typical study design for infected individuals develop severe symptoms, including high fever, immunophenotyping peripheral blood mononuclear cells (PBMCs) shortness of breath and muscle pain. The most severe cases of infec- from COVID-19 includes only a comparison between middle-aged tion progress to acute respiratory distress syndrome, multiorgan healthy or recovered individuals and patients with COVID-19 who failure and death. COVID-19 severity has been associated with lym- are typically 60 years and older13,21,22. phopenia3–5, elevated C-reactive protein6 and increased proinflam- In this study, we use clinical blood testing, mass cytometry matory cytokines such as interleukin (IL)-1β7,8, IL-6 (refs. 9–12), IL-8 and unbiased proteomics profiling of ~4,700 proteins to examine (ref. 10) and tumor necrosis factor (TNF)9,10, indicating an ongoing the phenotypic characteristics of plasma and PBMCs in nonobese systemic immune response. Several recent studies have character- individuals with respiratory distress with or without laboratory- ized the altered composition of the immune cells in patients with confirmed infection by SARS-CoV-2 (71 individuals) and compare COVID-19 compared to healthy or recovered patients13–15. In these these cohorts to samples from age-stratified healthy nonobese indi- studies, it remains unclear which emerging features are specific viduals (148 individuals from 25 to 80 years old). to COVID-19 and how many observations are shared with other inflammatory pathologies. Results Compared to other respiratory infections, COVID-19 has sev- Study design and clinical cohorts. First, we considered individuals eral unique features. The risk of progression to severe disease and who presented with respiratory illness symptoms and had a phy- mortality is greater in individuals with comorbidities like obesity, sician-ordered SARS-CoV-2 test performed at the Barnes Jewish hypertension and diabetes16. Most strikingly, COVID-19 is charac- Hospital between 26 March 2020 and 28 August 2020 (Washington terized by a profound age-associated susceptibility; individuals over University 350 (WU350) cohort). Based on nasopharyngeal test- 65 years old have the highest infection fatality rate and account for ing by PCR with reverse transcription (RT–PCR), participants more than 70% of COVID-19 deaths17–19. It is known that immune were defined as SARS-CoV-2 positive (CV; 140 females and 173 1Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, MO, USA. 2JetBrains Research, Saint Petersburg, Russia. 3Department of Medicine, Washington University School of Medicine, Saint Louis, MO, USA. 4Siteman Cancer Center, Washington University School of Medicine, Saint Louis, MO, USA. 5Division of Biostatistics, Washington University School of Medicine, Saint Louis, MO, USA. 6Department of Neurology, Washington University School of Medicine, Saint Louis, MO, USA. 7Department of Emergency Medicine, Washington University School of Medicine, Saint Louis, MO, USA. 8These authors contributed equally: Laura Arthur, Ekaterina Esaulova. ✉e-mail: [email protected] NatURE AGING | VOL 1 | JUNE 2021 | 535–549 | www.nature.com/nataging 535 RESOURCE NATURE AGING a WU350 cohort, 411 people SARS-CoV-2- SARS-CoV-2- negative positive Donors used for the analyses NCV Clinical blood panels and differentials BMI < 33* CV day 0–3 after 75 ) ER visit Immunostaining and ears 50 CyTOF of PBMCs 0255075 100 ABF300 healthy cohort, 156 people Age (y 25 Plasma proteomics by A 0 SomaScan B BMI < 30 C Non-smoker D E A (44)B (28)C (20)D (29)E (35) Healthy NCV (27) CV_severe (21) 02550 75 100 CV_moderateCV_deceased (18) (14) Age (years) b ) −1 30 l ) 1.5 µ ) ) −1 l 2.0 −1 −1 l l µ ) 3 µ µ 20 −1 l 20 1.0 µ 1.5 2 1.0 10 10 0.5 1 WBC (K 0.5 Monocyte (K Neutrophil (K 0 Lymphocyte (K 0 0 0 0 Imm. granulocytes (K A B C D E A B C D E A B C D E A B C D E NCV NCV NCV NCV NCV CV_severe CV_severe CV_severe CV_severe CV_severe CV_moderateCV_deceased CV_moderateCV_deceased CV_moderateCV_deceased CV_moderateCV_deceased CV_moderate CV_deceased c ) 25 ) 500 6 −1 l −1 µ 15 ) 20 400 −1 l µ 4 15 10 300 10 200 2 RDW (%) 5 RBC (M 5 100 Hemoglobin (g dl 0 0 0 Platelet count (K 0 A B C D E A B C D E A B C D E A B C D E NCV NCV NCV NCV CV_severe CV_severe CV_severe CV_severe CV_moderateCV_deceased CV_moderate CV_moderate CV_moderateCV_deceased d ) 125 −1 10.0 ) ) −1 ) −1 100 −1 6 4 7.5 75 5.0 4 2 50 2.5 2 25 Albumin (g dl Calcium (mg dl Creatinine (mg dl 0 0 0 0 Urea nitrogen (mg dl A B C D E A B C D E A B C D E A B C D E NCV NCV NCV NCV CV_severe CV_severe CV_severe CV_severe CV_moderateCV_deceased CV_moderateCV_deceased CV_moderateCV_deceased CV_moderateCV_deceased Fig. 1 | Study outline and clinical characterization of healthy and COVID-19/non-COVID-19 cohorts. Blood panels were performed for the following cohorts: A (25–34 years), n = 36; B (35–44 years), n = 21; C (45–54 years), n = 16; D (55–65 years), n = 24; E (>65 years), n = 25; CV, COVID-19 (32–91 years, 70.8 mean, 11.2 s.d.), n = 53; NCV, non-COVID-19 (32–87 years, 52.8 mean, 17 s.d.), n = 17. See Extended Data Fig. 1 for statistics related to b–d. a, Study outline. An asterisk represents four patients who had a BMI < 33. b–d, Selected WBC differentials (b); RBC, hemoglobin and platelet differentials (c); and clinical blood values (d) for cohorts A–E and CV/NCV cohorts. The lower and upper hinges of all box plots represent the 25th and 75th percentiles. Horizontal bars show the median value. Whiskers extend to values that are no further than 1.5 times the IQR from either the upper or the lower hinge. RDW, RBC distribution width; ER, emergency room. males) or SARS-Cov-2 negative (NCV; 98 females and 40 males; butions proportional to those of the nonobese individuals (53 CV Fig. 1a). The population was heterogeneous for body mass index individuals: median BMI, 25.5; interquartile range (IQR), 21.9–28.4; (BMI), where nearly half of individuals were moderately or severely 27 NCV individuals: median BMI, 27.3; IQR, 25.6–29.8; Extended obese (BMI > 33; Extended Data Fig. 1a). Given that obesity is a Data Fig. 1b,c). We cannot conclusively rule out SARS-CoV-2 infec- recognized risk factor for severe COVID-19 (ref. 16) and known to tion in participants with negative SARS-CoV-2 tests because the strongly impact immune and proteomic homeostasis23, we chose to false-negative rate of the nasopharyngeal RT–PCR test is reported minimize these confounding factors in our analysis and excluded to be 0.018–0.33 (ref. 24); however, 13 of 27 NCV individuals were participants with moderate and severe obesity. Our selected CV and retested, and none of the retests was positive for SARS-CoV-2, and NCV cohorts consisted of 80 individuals, with age and sex distri- none of the 27 individuals had a subsequent hospital readmission. 536 NatURE AGING | VOL 1 | JUNE 2021 | 535–549 | www.nature.com/nataging NATURE AGING RESOURCE The most common diagnoses at discharge were pneumonia and concentration, indicative of liver health, did not decrease with chronic obstructive pulmonary disease (Supplementary Table 1).