Cytokine signatures of end organ injury in COVID-19

Abstract

Increasing evidence has shown that Coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunologic response. We aimed to assess the differences in inflammatory cytokines in COVID-19 patients compared to contemporaneously hospitalized controls and then analyze the relationship between these cytokines and the development of Acute Respiratory Distress Syndrome (ARDS), Acute Kidney Injury (AKI) and mortality. In this cohort study of hospitalized patients, done between March third, 2020 and April first, 2020 at a quaternary referral center in New York City we included adult hospitalized patients with COVID-19 and negative controls. Serum specimens were obtained on the first, second, and third hospital day and cytokines were measured by Luminex. Autopsies of nine cohort patients were examined. We identified 90 COVID-19 patients and 51 controls. Analysis of 48 inflammatory cytokines revealed upregulation of macrophage induced chemokines, T-cell related interleukines and stromal cell producing cytokines in COVID-19 patients compared to the controls. Moreover, distinctive cytokine signatures predicted the development of ARDS, AKI and mortality in COVID-19 patients. Specifically, macrophage-associated cytokines predicted ARDS, T cell immunity related cytokines predicted AKI and mortality was associated with cytokines of activated immune pathways, of which IL-13 was universally correlated with ARDS, AKI and mortality. Histopathological examination of the autopsies showed diffuse alveolar damage with significant mononuclear inflammatory cell infiltration. Additionally, the kidneys demonstrated glomerular sclerosis, tubulointerstitial lymphocyte infiltration and cortical and medullary atrophy. These patterns of cytokine expression offer insight into the pathogenesis of COVID-19 disease, its severity, and subsequent lung and kidney injury suggesting more targeted treatment strategies.

Figure 1

Cytokine expression of COVID19 and control patients. The curved line of the violin box plots show the density of day 1 of hospital admission cytokine expression levels. The horizontal line in the inner box plot represents the median and interquartile range. Each dot represents a subject (COVID19, n = 90; Control, n = 51). Significance of comparisons were determined by an unadjusted linear regression models using log-scaled cytokines and robust standard errors. P-values after adjustment for multiple comparisons accompany the respective comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Cytokine expression of inflammatory cytokines in mild and severe COVID19 patients. The curved line of the violin box plots show the density of day 1 of hospital admission the cytokine expression levels. The horizontal line in the inner box plot represents the median and interquartile range. Each dot represents a subject (COVID-19, Mild = 56; Severe, n = 34). Significance of comparisons were determined by an unadjusted linear regression models using log-scaled cytokines and robust standard errors. p-values after adjustment for multiple comparisons accompany the respective comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Cytokine and clinical laboratory correlations of COVID-19 and control patients. Correlation heatmap of 39 cytokines from patient serum comparing cytokine concentrations at day 1 of hospital admission with first clinical laboratory parameters obtained in the first 72 h of admission. Correlation heatmaps are stratified by COVID-19 patients and controls. Only significant correlations (p < 0.05) after adjustment for multiple comparisons are presented with a Spearman's correlation coefficient value. The Spearman's correlation coefficient is visualized by color intensity. INR International Normalized Ratio, LDH lactate dehydrogenase.

Figure 4

Cytokine and SOFA score correlations between positive and negative COVID-19 patients. Correlation matrix of 39 cytokines from patient serum comparing cytokine concentrations at day 1 of hospital admission with SOFA scores. Correlation heatmaps are stratified by COVID-19 patients and controls. Only significant correlations (p < 0.05) after adjustment for multiple comparisons are presented with a Spearman's correlation coefficient value. The Spearman's correlation coefficient is visualized by color intensity. SOFA Sequential Organ Failure Assessment.

Figure 5

Associations between cytokine expression levels and clinical outcomes within COVID19 patients. (a) Forest plots representing the estimates of association for day 1 of hospital admission cytokine expression with clinical outcomes of mortality, need for intubation (ARDS), and development of acute kidney injury (AKI) among COVID-19 patients. Each box shows the estimated Hazard Ratio (HR) and each whisker represents the 95% Confidence Interval (CI) of the HR. Cox Proportional Hazard (PH) models with robust standard errors were used to compute all estimates with time 0 as day 1 of hospital admission. (b) Venn diagram showing 23 cytokines significantly associated (p < 0.05) with clinical outcomes such as mortality, ARDS, and development of AKI after pvalue adjustment for multiple comparisons.

Figure 6

Histopathology findings of lung and kidney in COVID19 patients and controls. Representative H&E and TUNEL staining in (a) lung and (b) kidney tisuses from patients with COVI19 (COVID-19, n = 9) and controls (Control, n = 5). Black arrows indicate mononuclear inflammatory cells. Lung injury was assessed on a scale of 0–2 for each of the following criteria: (i) alveolar polymorphonuclear neutrophils, (ii) chronic alveolar inflammation/macrophages, (iii) acute alveolar wall Inflammation, (iv) chronic alveolar wall inflammation, (v) hyaline membranes, (vi) Type 2 hyperplasia only, (vii) Type 2 hyperplasia with fibroblasts, and (viii) organizing pneumonia and squamous metaplasia. The final injury score was derived from the following calculation: Score = I + ii + iii + iv + v + vi + vii + viii. G indicates glomerulus. Banff Score: g, glomerulitis; i, interstitial inflammation; ptc, peritubular capillaritis; ct, tubular atrophy; ci, interstitial fibrosis; cv, vascular fibrous intimal thickening; ti, total inflammation. *p < 0.05, **p < 0.01.