SARS-CoV-2 ORF8 Mediates Signals in Macrophages and Monocytes through MyD88 Independently of the IL-17 Receptor

Abstract

SARS-CoV-2 has caused an estimated 7 million deaths worldwide to date. A secreted SARS-CoV-2 accessory protein, known as open reading frame 8 (ORF8), elicits inflammatory pulmonary cytokine responses and is associated with disease severity in COVID-19 patients. Recent reports proposed that ORF8 mediates downstream signals in macrophages and monocytes through the IL-17 receptor complex (IL-17RA, IL-17RC). However, generally IL-17 signals are found to be restricted to the nonhematopoietic compartment, thought to be due to rate-limiting expression of IL-17RC. Accordingly, we revisited the capacity of IL-17 and ORF8 to induce cytokine gene expression in mouse and human macrophages and monocytes. In SARS-CoV-2-infected human and mouse lungs, IL17RC mRNA was undetectable in monocyte/macrophage populations. In cultured mouse and human monocytes and macrophages, ORF8 but not IL-17 led to elevated expression of target cytokines. ORF8-induced signaling was fully preserved in the presence of anti-IL-17RA/RC neutralizing Abs and in Il17ra-/- cells. ORF8 signaling was also operative in Il1r1-/- bone marrow-derived macrophages. However, the TLR/IL-1R family adaptor MyD88, which is dispensable for IL-17R signaling, was required for ORF8 activity yet MyD88 is not required for IL-17 signaling. Thus, we conclude that ORF8 transduces inflammatory signaling in monocytes and macrophages via MyD88 independently of the IL-17R.

Figure 1.. Mouse macrophages do not respond to IL-17.

BMDMs from (A) WT mice or (B) RAW 264.7 cells were treated with IL-17 (200 ng/mL) or E. coli LPS (1 μg/mL) for 3 or 6 h. Expression of the indicated genes was quantified by qPCR normalized to Gapdh. Data are presented as fold-change relative to unstimulated ± SEM of 3–4 independent experiments. Each symbol indicates an individual mouse (n=4) or biological replicates (n=3). (C) Histograms of CD11b, CD11c and CD45 staining on ST2, RAW 264.7 and BMDM cells is shown. Significance was assessed by 1-way ANOVA with Bonferroni’s or Kruskal-Wallis with Dunn’s multiple comparisons tests. Data is representative of 2 independent experiments.

Figure 2.. ORF8 but not IL-17 activates monocytes/macrophages.

Primary BMDMs derived from (A) C57Bl/6 mice, (B) RAW 264.7 cells or (C) human monocytes derived from PBMCs were stimulated with IL-17 (200 ng/mL), His-ORF8 (1 μg/mL) or LPS (1 μg/mL) for 3 h. Expression of the indicated genes was quantified by qPCR normalized to Gapdh. Data are presented as fold-change relative to unstimulated ± SEM of 3–5 independent experiments. Each symbol indicates one mouse (n=5), biological replicates (n= 3) or human donor sample (n=5). (D) Human KCs were treated with IL-17 (50–100 ng/mL) or ORF8 (1 μg/mL) for 3 h. Expression of indicated genes was quantified by qPCR normalized to GAPDH or RPLP0. Data are fold-change relative to unstimulated ± SEM. Each symbol represents a biological replicate from two independent experiments. Significance was assessed by 1-way ANOVA with Bonferroni’s or Kruskal-Wallis with Dunn’s multiple comparisons tests.

Figure 3.. IL-17 receptor subunit expression in SARS-CoV-2-infected humans and mouse lung.

(A) UMAP of scRNA-seq data from lungs of SARS-Cov-2-infected K18-hACE2 transgenic mice (USA-WA1/2020 strain, 4 d) showing murine Il17ra, IL17rc and viral Orf8 in monocyte/macrophage and fibroblast clusters. (B) Severe COVID19 patient BALF IL17RA, IL17RC and viral ORF8 in monocyte/macrophage and fibroblast clusters. Each point is an individual cell; colors are based on marker annotation. Gray indicates that gene of interest was below threshold in the marker set. Cutoff: Log2Norm > 1 (log- transformed and normalized). Data are from published datasets, see Methods.

Figure 4.. ORF8 signals are MyD88-dependent.

(A) BMDMs from the indicated mice were treated with IL-17 (200 ng/mL), ORF8 (1 μg/mL) or LPS (1 μg/mL ) for 3 h. Expression of the indicated genes was quantified by qPCR normalized to Gapdh. Data are presented as fold-change relative to unstimulated ± SEM. Each symbol indicates one mouse (n=5–9) and are from 2 independent experiments. (B) RAW 264.7 cells were pre-treated with control IgG, IL-17RA or IL-17RC Abs at 5 μg/mL for 1 h and stimulated with IL-17 (50 ng/mL), ORF8 (1 μg/mL) or LPS (1 μg/mL) for 3 h. Expression of indicated genes was quantified by qPCR normalized to Gapdh. Data are as fold-change relative to untreated, and show mean (± SEM). Each symbol represents a biological replicate (n=4) and data are from 2 independent experiments. Significance was assessed by 2-way ANOVA with Bonferroni’s test.

Figure 5.. ORF8 signals are MyD88-dependent, but do not require IL-1R1.

BMDMs from the indicated mice were treated with IL-17 (200 ng/mL) or ORF8 (1 μg/mL) for 3 h. Expression of the indicated genes was quantified by qPCR normalized to Gapdh. Data are presented as fold-change relative to unstimulated ± SEM. Each symbol indicates experimental replicates from 2 mice performed once. Significance was assessed by 2-way ANOVA with Bonferroni’s test.

Figure 6.. ORF8-induced gene expression is not a result of endotoxin contamination.

RAW 264.7 cells were pre-treated with polymyxin B (30 μg/mL) or vehicle for 1 h and then stimulated with ORF8 (1 μg/mL) or LPS at the indicated concentrations for 3 h. Expression of the indicated genes was quantified by qPCR normalized to Gapdh. Data are presented as fold-change relative to unstimulated, and show mean (± SEM). Each symbol represents one biological replicate from 2 independent experiments. Significance was assessed by 2-way ANOVA with Bonferroni’s test.