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Comparative Study
. 2020 Oct 8;11(1):5086.
doi: 10.1038/s41467-020-18854-2.

Two distinct immunopathological profiles in autopsy lungs of COVID-19

Ronny Nienhold #  1 , Yari Ciani #  2 , Viktor H Koelzer #  3   4 , Alexandar Tzankov  5 , Jasmin D Haslbauer  5 , Thomas Menter  5 , Nathalie Schwab  1 , Maurice Henkel  1 , Angela Frank  1 , Veronika Zsikla  1 , Niels Willi  1 , Werner Kempf  6 , Thomas Hoyler  7 , Mattia Barbareschi  8 , Holger Moch  3 , Markus Tolnay  5 , Gieri Cathomas  1 , Francesca Demichelis  2   9 , Tobias Junt  7 , Kirsten D Mertz  10
Affiliations
Comparative Study

Two distinct immunopathological profiles in autopsy lungs of COVID-19

Ronny Nienhold et al. Nat Commun. .

Abstract

Coronavirus Disease 19 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has grown to a worldwide pandemic with substantial mortality. Immune mediated damage has been proposed as a pathogenic factor, but immune responses in lungs of COVID-19 patients remain poorly characterized. Here we show transcriptomic, histologic and cellular profiles of post mortem COVID-19 (n = 34 tissues from 16 patients) and normal lung tissues (n = 9 tissues from 6 patients). Two distinct immunopathological reaction patterns of lethal COVID-19 are identified. One pattern shows high local expression of interferon stimulated genes (ISGhigh) and cytokines, high viral loads and limited pulmonary damage, the other pattern shows severely damaged lungs, low ISGs (ISGlow), low viral loads and abundant infiltrating activated CD8+ T cells and macrophages. ISGhigh patients die significantly earlier after hospitalization than ISGlow patients. Our study may point to distinct stages of progression of COVID-19 lung disease and highlights the need for peripheral blood biomarkers that inform about patient lung status and guide treatment.

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Conflict of interest statement

V.H.K. has served as an invited speaker on behalf of Indica Labs. T.H. and T.J. are employees of Novartis. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. ISGhigh and ISGlow are two gene expression profiles in COVID-19 autopsy lungs.
a Heatmap showing K-means clustering of COVID-19 and normal lung samples based on expression levels of deregulated genes in COVID-19 versus normal lungs. b Comparison of upregulated and downregulated genes in lung samples from COVID-19 patients, normal lung samples, and samples from other infectious lung pathologies. c Principal component analysis (PCA) of COVID-19 and non-COVID-19 lung samples reveals segregation in two distinct groups based on diagnosis and viral load. d ISG signature expression in clusters 1 and 2 of COVID-19 lungs defines two profiles of COVID-19 autopsy lungs termed ISGhigh and ISGlow. Study patients with unambiguous sample segregation in either Cluster 1 or 2 were assigned the corresponding ISG activation label ISGhigh and ISGlow, respectively (n = 31 independent samples). e Hospitalization time in ISGhigh patients versus ISGlow patients (n = 14 independent samples). ISGhigh samples, red; ISGlow samples, blue. Box-plots elements indicate the median (center line), upper and lower quartiles (box limits). Whiskers extend to the most extreme value included in 1.5× interquartile range. Groups were compared using a two-sided Wilcoxon rank-sum test.
Fig. 2
Fig. 2. Co-infections in COVID-19 lungs identified by WGS metagenomics.
No differences in co-infections in ISGhigh and ISGlow COVID-19 lungs identified by WGS metagenomics. a Total number of reads generated for each sample. b Percentage of reads and c absolute numbers of reads not mapping to the human genome (GRCh37 hg19) (n = 34 independent samples). Box-plots elements indicate the median (center line), upper and lower quartiles (box limits). Whiskers extend to the most extreme value included in 1.5× interquartile range. d Bacterial and e viral co-infections across lung samples, WGS metagenomic analysis. Purple dots, numbers of reads sufficient for identification of non-human species. Samples are ordered by increasing the SARS-CoV-2 viral load in both the ISGlow and the ISGhigh group. Stacked bars, the relative abundance of the most common species. Gray bars represent frequent species, colored bars show pathogenic species. *One COVID-19 patient (C3) clustered in the normal control group. ISGhigh samples, red; ISGlow samples, blue.
Fig. 3
Fig. 3. Virological and cellular characteristics of the ISGhigh and ISGlow COVID-19 lung profiles.
a Correlation of viral load and ISG expression in COVID-19 lungs. Solid lines connect sample points from the same patient. The dotted line shows a regression for all samples, and the gray area delimits the 95% confidence intervals around it (Pearson’s correlation = 0.83, adjusted R-squared = 0.68, p-value = 1.66e−08). b Representative immunohistochemistry for SARS-CoV-2 on ISGhigh and ISGlow COVID-19 lung samples and controls. Size bar 100 μm. At least two different tissue blocks from different areas of the lungs were evaluated for each case. c Frequencies of immune cells on ISGhigh and ISGlow COVID-19 lung sections and controls. (n = 33 for CD3 and CD8, n = 32 for CD68, n = 30 for CD163). Box-plots elements indicate the median (center line), upper, and lower quartiles (box limits). Whiskers extend to the most extreme value included in 1.5× interquartile range. Groups were compared using a two-sided Wilcoxon rank-sum test. d Representative H&E stains and immunohistochemistry (CD3, CD8, CD68, CD163) of ISGhigh and ISGlow COVID-19 lungs and controls, size bar 500 μm. At least two different tissue blocks from different areas of the lungs were evaluated for each case. ISGhigh samples, red; ISGlow samples, blue.
Fig. 4
Fig. 4. Immune cell infiltrates on COVID-19 lung sections.
a Frequencies of immune cells on ISGhigh and ISGlow COVID-19 lung sections and controls (n = 33 for CD4 and CD20, n = 31 for CD123, n = 32 for CD8/PD1). Box-plots elements indicate the median (center line), upper and lower quartiles (box limits). Whiskers extend to the most extreme value included in 1.5× interquartile range. Groups were compared using a two-sided Wilcoxon rank-sum test. b Representative immunohistochemistry (CD4, CD20, CD123, CD8/PD1) of ISGhigh and ISGlow COVID-19 lungs and controls, size bar 500 μm. At least two different tissue blocks from different areas of the lungs were evaluated for each case. ISGhigh samples, red; ISGlow samples, blue.
Fig. 5
Fig. 5. Correlation of ISGhigh and ISGlow lung immunoprofiles with morphological changes.
a Expression of a cytokine signature (TNF, IL-1B, IL6, IFNA17, IFNB1, CCL2, CXCL9, CXCL10, CXCL11) in ISGhigh and ISGlow COVID-19 lung samples. This pro-inflammatory cytokine signature was significantly enriched in the ISGhigh subset (n = 31 independent samples). b Inverse correlation of viral load and activated CD8+ T cell signature (CD38, GZMA, GZMB, CCR5). Solid lines connect sample points from the same patient. The dotted line shows a regression for all the samples, and the gray area delimits the 95% Confidence Intervals around it (Pearson’s correlation = −0.5, adjusted R-squared = 0.22, p-value = 0.005). c Association of DAD stage with ISG expression (n = 31 independent samples). d Association of the pro-inflammatory cytokine signature with intra-alveolar hemorrhage (IAH) (n = 31 independent samples). e Pearson’s correlation of pro-inflammatory cytokines in the cytokine signature indicates the presence of co-regulated cytokines. f–i Association of cytokine signatures in ISGhigh and ISGlow COVID-19 lung samples with IAH. Association of: f Median IL6, TNF, IL1B expression. g Median IFNA17, IFNB1 expression. h Median CCL2 expression. i Median CXCL9/10/11 expression in ISGhigh and ISGlow COVID-19 lung samples versus IAH. Only the CXCL9/10/11 sub-signature was positively associated with IAH (n = 31 independent samples). j Association of CD68+ macrophage infiltrates with DAD (n = 27 independent samples). k Association of DAD stage with activated CD8+ T cell signature (n = 31 independent samples), l with CD8+ T cell counts (n = 29). m–p Association of cytokine signatures in ISGhigh and ISGlow COVID-19 lung samples with DAD stage. Association of: m Median IL6, TNF, IL1B expression. n Median IFNA17, IFNB1 expression. o Median CCL2 expression. p Median CXCL9/10/11 expression in ISGhigh and ISGlow COVID-19 lung tissue with DAD stage (n = 31 independent samples). ISGhigh samples, red; ISGlow samples, blue. All box-plots elements indicate the median (center line), upper and lower quartiles (box limits). Whiskers extend to the most extreme value included in 1.5× interquartile range. Groups were compared using a two-sided Wilcoxon rank-sum test.
Fig. 6
Fig. 6. Molecular characteristics of the ISGhigh and ISGlow COVID-19 lung profiles.
a Representative immunohistochemistry for p53 and Ki67. Size bar 100 μm. At least two different tissue blocks from different areas of the lungs were evaluated for each case. b Expression of C1QA and C1QB in ISGhigh and ISGlow lung samples (n = 31 independent samples). Box-plots elements indicate the median (center line), upper, and lower quartiles (box limits). Whiskers extend to the most extreme value included in 1.5× interquartile range. Groups were compared using a two-sided Wilcoxon rank-sum test. c Representative IHC stainings for complement activation products C5b-9 and C3d in ISGhigh, ISGlow COVID-19, and normal control lungs. Size bar 100 μm. At least two different tissue blocks from different areas of the lungs were evaluated for each case. ISGhigh samples, red; ISGlow samples, blue. d Schematic time course of COVID-19 lung disease based on lung autopsy findings. Early in the disease, an ISGhigh lung profile is observed, with high viral load, high expression of cytokines and ISGs, and sparse immune infiltrates. Late in the disease, an ISGlow lung profile is observed, with low viral load, low local expression of cytokines and ISGs, and strong infiltration of macrophages and lymphocytes. Patients who die early are not able to adequately control SARS-CoV-2, while patients who die late suffer from DAD and immunopathology. Infectious dose and individual predisposition to mount immune responses likely define whether or not a patient survives COVID-19. Green line: Relative viral loads, red line: Relative expression of lung ISGs and cytokines, blue line: pulmonary immune infiltrates and complement deposition. Dark gray area: lethal outcomes, arrows: individual variability.
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