Proteomic Analyses in COVID-19-Associated Secondary Hemophagocytic Lymphohistiocytosis

Abstract

Context: COVID-19 has been associated with features of a cytokine storm syndrome with some patients sharing features with the hyperinflammatory disorder, secondary hemophagocytic lymphohistiocytosis (sHLH).

Hypothesis: We hypothesized that proteins associated with sHLH from other causes will be associated with COVID-sHLH and that subjects with fatal COVID-sHLH would have defects in immune-related pathways.

Methods and models: We identified two cohorts of adult patients presenting with COVID-19 at two tertiary care hospitals in Seattle, Washington in 2020 and 2021. In this observational study, we assessed clinical laboratory values and plasma proteomics. Subjects identified as having sHLH (ferritin > 1000 plus cytopenias in two or more lineages [WBC < 5000 odds ratio [OR] ANC (absolute neutrophil count) < 1000, hemoglobin < 9 or hematocrit < 27, platelets < 100,000], and elevated transaminases [either AST (aspartate aminotransferase) or ALT (alanine aminotransferase) > 30] OR subjects with a ferritin > 3000) were compared with those with COVID-19 without sHLH. We identified 264 patients with COVID-19 of whom 24 met our sHLH definition. Eight patients who died of COVID-sHLH underwent genomic sequencing to identify variants in immune-related genes.

Results: Nine percent of enrolled COVID-19 subjects met our defined criteria for sHLH (n = 24/264). Using broad serum proteomic approaches (O-link and SomaScan), we identified three proteins increased in subjects with COVID-19-associated sHLH (soluble PD-L1 [sPD-L1], tumor necrosis factor-R1, and interleukin [IL]-18BP, p < 0.05 for O-link and false discovery rate < 0.05 for SomaScan), supporting a role for proteins previously associated with other forms of sHLH (IL-18BP and soluble tumor necrosis factor receptor 1). We also identified candidate proteins and pathways associated with COVID-sHLH, including sPD-L1 and the syntaxin pathway. We detected pathogenic variants in DOCK8 and TMPRSS15 in deceased individuals with COVID-sHLH, further suggesting that alterations in immune-related processes may contribute to hyperinflammation and fatal outcomes in COVID-19.

Interpretations and conclusions: Proteins increased in COVID-19-associated sHLH, such as sPD-L1, and pathways, such as the syntaxin pathway, suggest important roles for the immune response in driving sHLH in the context of COVID-19.

Figure 1.

Flowchart of subject inclusion. A, Cohort 1. B, Cohort 2. MSD = Meso Scale Discovery, sHLH = secondary hemophagocytic lymphohistiocytosis.

Figure 2.

Soluble PD-L1 (sPD-L1) is elevated in two independent cohorts of COVID-secondary hemophagocytic lymphohistiocytosis (sHLH) subjects. (A) Plasma samples (n = 82) from cohort 1 were analyzed on the O-link platform and PD-L1 displayed as normalized protein expression (NPX) values. Open circles: no sHLH (n = 75), red squares: with sHLH (n = 7). (B) Plasma samples from cohort 2 were analyzed using the SomaScan platform (n = 101) and PD-L1 was displayed as normalized relative fluorescent unit (RFU). Open circles: no sHLH (n = 91); red squares: with sHLH (n = 10). (C) Plasma samples from cohort 2 were analyzed for sPD-L1 (n = 181) using an Meso Scale Discovery (MSD) assay. Open circles: no sHLH, red squares (n = 164): with sHLH (n = 17). Bars indicate the median and interquartile range. * p < 0.05, ** p < 0.01 by two-tailed Mann-Whitney U test.

Figure 3.

Syntaxin and interleukin (IL)-1 pathway proteins and coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) are differentially expressed in COVID-secondary hemophagocytic lymphohistiocytosis (sHLH) subjects. A, Plasma samples from cohort 2 were analyzed using the SomaScan platform (n = 101; no sHLH n = 91; sHLH n = 10). Volcano plot displays differentially expressed proteins between subjects with sHLH vs. those without sHLH adjusted for age, sex, and body mass index. False discovery rate (FDR) < 0.05. B, Plasma levels of CHCHD10 from cohort 2 displayed as normalized relative fluorescent units (RFU) from SomaScan data presented in (A). (C) Normalized read counts of transcripts from RNA sequencing of CD14+ monocytes from subjects without sHLH (n = 22) or subjects with sHLH (n = 3) from cohort 1. Open circles: no sHLH, red squares: with sHLH. Bars indicate the median and interquartile range. *** p < 0.001 by two-tailed Mann-Whitney U test. STX = syntaxin protein.