Soluble Signal Inhibitory Receptor on Leukocytes-1 Is Released from Activated Neutrophils by Proteinase 3 Cleavage

Abstract

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human granulocytes and monocytes that dampens antimicrobial functions. We previously showed that sputum neutrophils from infants with severe respiratory syncytial virus (RSV) bronchiolitis have decreased SIRL-1 surface expression compared with blood neutrophils and that SIRL-1 surface expression is rapidly lost from in vitro activated neutrophils. This led us to hypothesize that activated neutrophils lose SIRL-1 by ectodomain shedding. Here, we developed an ELISA and measured the concentration of soluble SIRL-1 (sSIRL-1) in patients with RSV bronchiolitis and hospitalized patients with COVID-19, which are both characterized by neutrophilic inflammation. In line with our hypothesis, sSIRL-1 concentration was increased in sputum compared with plasma of patients with RSV bronchiolitis and in serum of hospitalized patients with COVID-19 compared with control serum. In addition, we show that in vitro activated neutrophils release sSIRL-1 by proteolytic cleavage and that this diminishes the ability to inhibit neutrophilic reactive oxygen species production via SIRL-1. Finally, we found that SIRL-1 shedding is prevented by proteinase 3 inhibition and by extracellular adherence protein from Staphylococcus aureus. Notably, we recently showed that SIRL-1 is activated by PSMα3 from S. aureus, suggesting that S. aureus may counteract SIRL-1 shedding to benefit from preserved inhibitory function of SIRL-1. In conclusion, we report that SIRL-1 is released from activated neutrophils by proteinase 3 cleavage and that endogenous sSIRL-1 protein is present in vivo.

FIGURE 1.

Development of sSIRL-1 ELISA. (A) Schematic representation of SIRL-1 and its two potential soluble forms: VSTM1-v2 and sSIRL-1^ecto. (B and C) SDS-PAGE analysis of VSTM1-v2 and sSIRL-1^ecto, with or without pretreatment with PNGase F or 2-ME. Proteins were either stained directly by Coomassie blue (B) or transferred to a membrane and stained with SIRL-1 mAb clone 1A5 (C). Arrows indicate the position of VSTM1-v2 (v2), sSIRL-1^ecto (ecto), or PNGase F. (D) sSIRL-1^ecto, VSTM1-v2, and negative control sLAIR-1^ecto were titrated in a sSIRL-1 ELISA developed in-house (n ≥ 3; one representative example is shown). (E) sSIRL-1-negative heparin plasma samples (n = 5) were titrated and spiked with 70 pM sSIRL-1^ecto. sSIRL-1 concentration was measured by ELISA. Symbols represent the mean ± SD. The statistical difference between the spike and the recovery of sSIRL-1^ecto at different plasma dilutions was tested using a one-sample Wilcoxon test. WB, Western blot.

FIGURE 2.

sSIRL-1 is increased in patients with COVID-19 and patients with RSV bronchiolitis. sSIRL-1 was measured by ELISA in plasma, urine, and sputum. (A and B) sSIRL-1 concentration in plasma from 53 healthy donors, stratified per genotype of the rs612529 single-nucleotide polymorphism (T/T, n = 22; T/C, n = 17; C/C, n = 14). (C and D) sSIRL-1 concentration in serum of hospitalized patients with COVID-19 (n = 163) and control serum (n = 51). (E) sSIRL-1 concentration in heparin plasma or sputum of mechanically ventilated infants with severe RSV bronchiolitis or mechanically ventilated infants without infection (control individuals). Control plasma, n = 4; RSV plasma, n = 17; control sputum, n = 2; RSV sputum, n = 16. (F) sSIRL-1 concentration in paired plasma and urine samples of infants with severe RSV bronchiolitis (n = 8). (A, C, E, F) Each dot represents one donor, and the dark gray horizontal lines represent the mean. The shaded light gray area indicates the lower limit of detection (LLOD). Samples with undetectable sSIRL-1 were given a value of 0.5 × LLOD. Statistical differences were calculated using a Kruskal-Wallis test with Dunn’s correction (A, E), the Mann-Whitney test (C), or the Wilcoxon test (F). (B, D) The percentage of samples with detectable sSIRL-1. The error bars indicate the 95% confidence interval, calculated with Wilson-Brown. The statistical differences were calculated using Fisher’s exact test (B, D) in combination with Bonferroni correction for multiple testing (B). **p ≤ 0.01, ***p ≤ 0.001. ****p ≤ 0.0001.

FIGURE 3.

Activated neutrophils shed SIRL-1, which limits inhibition of ROS production via SIRL-1. Neutrophils were isolated from healthy donors and stimulated for 4 h at 37°C with curdlan (100 μg/ml) or TNF (100 ng/ml), with or without addition of a protease inhibitor cocktail. (A and B) SIRL-1 expression on neutrophils was analyzed by flow cytometry. Shown are representative histograms of the fluorescence intensity of cells stained with an SIRL-1 antibody (clone 1A5) (closed histogram) or an isotype control (open histogram) (A) and the quantification of the percentage of SIRL-1⁺ cells (B). Each symbol represents one donor (n = 6). (C) Supernatants from stimulated neutrophils were analyzed by sSIRL-1 ELISA. Each symbol represents one donor (n = 5). (D) Neutrophils were preincubated for 1 h with or without 50 ng/ml TNF, upon which neutrophils were added to 96-well plates that were coated with mAb anti-CD32 in combination with anti-SIRL-1 (clone 1A5) or an isotype control. ROS production was measured by Amplex red assay. Shown is the relative fluorescence intensity (RFU) at 60 min. The background signal in unstimulated samples was subtracted from all samples (n = 3). Statistical significance was determined using two-way ANOVA with Geisser-Greenhouse correction and Holm-Sidak’s multiple comparison test (B–D). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 4.

SIRL-1 is cleaved by PR3. (A, D, E) Neutrophils were isolated from healthy donors and stimulated for 2 h at 37°C with TNF (50 ng/ml), with or without addition of inhibitors against major protease classes (A; 10–40 µM) or a PR3 inhibitor (D and E; 10 µM). Cells were stained with an SIRL-1 antibody (clone 3F5) and analyzed by flow cytometry. (A) The bars indicate the percentage of SIRL-1⁺ cells (mean ± SD). Each symbol represents a donor (n = 3). (D) Representative histograms of the fluorescence intensity of cells stained with an SIRL-1 antibody and (E) the quantification of the percentage of SIRL-1⁺ cells (each symbol with connected line represents a donor) (n = 7). (B and C) PLB-985 cells with SIRL-1 overexpression were treated for 2 h with neutrophil elastase, cathepsin G, or PR3 (all 1 µM), followed by flow cytometric analysis (n = 4). Shown are representative histograms of the fluorescence intensity of cells stained with an SIRL-1 antibody (clone 3F5) or isotype control (B) and the quantification of the median fluorescence intensity (MFI), normalized to the MFI of untreated cells (C) (mean ± SD; each symbol represents one experiment; the dotted line represents the normalized MFI of the isotype control). (F) sSIRL-1^ecto and VSTM1-v2 were left untreated (−) or treated with buffer control (ctrl) or PR3 for 3 h at 25°C and analyzed by SDS-PAGE and Western blotting. The membrane was stained with an SIRL-1 antibody (clone 1A5). The arrows indicate the position of VSTM1-v2 or sSIRL-1^ecto. One representative experiment of n = 3 is shown. Statistical significance was determined using a mixed-effects model with Dunnett’s multiple comparisons test (A; TNF treatment alone was compared with TNF treatment with each of the protease inhibitors), one-way ANOVA with Dunnett’s multiple comparison test (C), or a mixed-effects model with Sidak’s multiple comparisons test (E), all with Geisser-Greenhouse correction. * p ≤ 0.05.

FIGURE 5.

S. aureus protein Eap inhibits SIRL-1 shedding. (A and B) Neutrophils were isolated from healthy donors and stimulated for 2 h at 37°C with TNF (50 ng/ml), with or without addition of 10–30 µg/ml Eap, EapH1, or EapH2 (indicated with H1 or H2, respectively). Cells were stained with a SIRL-1 mAb (clone 3F5) and analyzed by flow cytometry (n = 4–6). (A) The percentage of SIRL-1⁺ cells (mean ± SD). Each symbol represents a donor. (B) Representative histograms of the fluorescence intensity of cells stained with an SIRL-1 antibody. (C) PLB-985 cells with SIRL-1 overexpression were treated for 2 h with 1 µM PR3, with or without addition of Eap or EapH1 (30 µg/ml). SIRL-1 expression was analyzed by flow cytometry. The bars indicate the percentage of SIRL-1⁺ cells (mean ± SD). Each symbol represents an experiment (n = 3–4). Statistical significance was determined using a mixed-effects model with Dunnett’s multiple comparisons test (A) or Holm-Sidak’s multiple comparisons test (C), both with Geisser-Greenhouse correction. * p ≤ 0.05, ** p ≤ 0.01.