Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19

Abstract

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.

Keywords: COVID-19; Cellular immune response; Cytokines; Immunology; NF-kappaB.

Figure 1. Microglia exhibit distinct transcriptional responses in patients with COVID-19.

(A) Uniform manifold approximation and projection (UMAP) of 65,767 cells isolated from the frontal lobes of 8 patients postmortem. HSR, heat shock response; TCM, T central memory; TEM, T effector memory. (B) Relative abundance of microglial cell states as a percentage of total microglia. No significant differences are observed by diagnosis (q ≤ 0.05, Mann-Whitney). (C) Hierarchical clustering of mean marker gene expression by cell type and cell state by diagnosis. (D) MA plot of differentially expressed genes in total microglia in COVID-19 versus controls by pseudobulk differential expression analysis. Significantly upregulated genes are shown in red, and significantly downregulated genes are shown in blue (q < 0.05, Wald test). Genes shown in gray are not significantly differentially expressed. (E) Callouts of key markers of cell division and cell cycle arrest from D. All genes shown are significantly differentially expressed (q < 0.05, Wald test). (F) Gene set enrichment of Hallmark TNF-α Signaling Via NF-κB (M5890) from pseudobulk differential expression analysis (q = 5.96 × 10^–16, multilevel splitting Monte Carlo). (G) Median modular expression of Hallmark TNF-α Signaling Via NF-κB (M5890) by diagnosis. Points represent median expression in total microglia from each patient (q = 2.6 × 10^–2, Mann-Whitney). (H) Representative images of combined immunofluorescence and smFISH (RNAScope) from human frontal lobe tissue sections showing cell cycle–arrested, pro-inflammatory microglia in patients with COVID-19 relative to controls. Images are pseudocolored by channel as follows: DAPI: blue, IBA1: green, IL1B: red, CCL2: cyan, CDKN1A: magenta.

Figure 2. COVID-19 is distinguished from pneumonias of similar severity by expression of T cell and myeloid cell chemokines.

(A) Hierarchical clustering of 41 cytokines showing significant variability by diagnosis (q < 0.05, Kruskal-Wallis) from 187 BAL samples collected in the first 48 hours of intubation from 183 patients with an early BAL. (B) Hierarchical clustering of 25 cytokines showing significant variability by diagnosis (q < 0.05, Kruskal-Wallis) from 137 early plasma samples from 134 patients. (C) Expression of COVID-19–defining T lymphocyte and monocyte chemokines and key pro-inflammatory cytokines from 479 BAL samples collected throughout the duration of mechanical ventilation from 332 patients. (D) Expression of COVID-19–defining T lymphocyte and monocyte chemokines and key pro-inflammatory cytokines from 396 plasma samples collected throughout the duration of mechanical ventilation from 262 patients.

Figure 3. Cumulative but not peak exposure to pro-inflammatory cytokines is higher in patients with severe SARS-CoV-2 pneumonia compared with patients with severe pneumonia secondary to other pathogens.

(A) Hierarchical clustering of cumulative exposure to 44 BAL cytokines showing significant variability by diagnosis (q < 0.05, Kruskal-Wallis) from 327 patients estimated by geometric integration of the levels of 479 BAL samples collected throughout the duration of mechanical ventilation. LTAC, long-term acute care. (B) Hierarchical clustering of cumulative exposure to 51 plasma cytokines showing significant variability by diagnosis (q < 0.05, Kruskal-Wallis) from 258 patients estimated by geometric integration of the levels of 396 plasma samples collected throughout the duration of mechanical ventilation. (C) Cumulative expression of selected pro-inflammatory cytokines in BAL fluid from A. (D) Cumulative expression of selected pro-inflammatory cytokines in plasma from B. (E) Schematic for calculation of cumulative exposure for each cytokine assayed for each patient throughout the course of mechanical ventilation by geometric integration. BAL samples from 3 patients and plasma samples from 2 patients receiving long-term mechanical ventilation were excluded from these analyses.

Figure 4. Corticosteroid treatment is associated with reductions in T cell and myeloid cell chemokine expression predominantly in monocyte-derived alveolar macrophages.

(A) Box plots of cytokine expression for all BAL fluid and plasma cytokines exhibiting significantly altered expression (q < 0.05, Mann-Whitney) following corticosteroid treatment. Lightly shaded boxes represent cytokine expression values prior to corticosteroid treatment, and darkly shaded boxes represent expression values after corticosteroid treatment. (B) Paired comparisons of cytokine expression in BAL and plasma for all paired samples (paired Mann-Whitney). (C) Deconvolution of “bulk” cytokine expression in BAL fluid by scRNA-Seq of cells isolated from BAL fluid. Mean cytokine gene expression for each cell type detected in scRNA-Seq data (33) (black points) is overlaid on bulk cytokine expression by multiplexed cytokine array (filled bars) to identify cell type contributors to cytokine expression. MoAM, monocyte-derived alveolar macrophage; TRAM, tissue-resident alveolar macrophage; Treg, CD4⁺ regulatory T cell; iNKT, invariant natural killer T cell.