key: cord-0698520-e6rwih22
authors: von Richthofen, Helen J.; Westerlaken, Geertje H.A.; Gollnast, Doron; Besteman, Sjanna; Delemarre, Eveline M.; Rodenburg, Karlijn; Moerer, Petra; Stapels, Daphne A.C.; Andiappan, Anand K.; Rötzschke, Olaf; Nierkens, Stefan; Leavis, Helen L.; Bont, Louis J.; Rooijakkers, Suzan H.M.; Meyaard, Linde
title: Soluble signal inhibitory receptor on leukocytes-1 is released from activated neutrophils by proteinase 3 cleavage
date: 2022-03-03
journal: bioRxiv
DOI: 10.1101/2022.03.03.482795
sha: cd995ddb29fe2b23090ecbbcf16a3ca2788af6a4
doc_id: 698520
cord_uid: e6rwih22

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human granulocytes and monocytes which 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 to 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 RSV bronchiolitis and hospitalized COVID-19 patients, which are both characterized by neutrophilic inflammation. In line with our hypothesis, sSIRL-1 concentration was increased in sputum compared to plasma of RSV bronchiolitis patients, and in serum of hospitalized COVID-19 patients compared to control serum. In addition, we show that in vitro activated neutrophils release sSIRL-1 by proteolytic cleavage, which can be prevented by proteinase 3 inhibition. Finally, we found that SIRL-1 shedding is prevented by extracellular adherence protein (Eap) from S. 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 are the first to report that SIRL-1 is released from activated neutrophils by proteinase 3 cleavage and that endogenous sSIRL-1 protein is present in vivo.

Immune inhibitory receptors (IIRs), also referred to as immune checkpoints, are pivotal in negative 40 regulation of immune cells (1, 2) . Although far less studied than the transmembrane form, many IIRs 41 also have a soluble form or homologue (3, 4) . Soluble IIRs can arise from ectodomain shedding of the 42 membrane-expressed receptor (5, 6), or can be a product of alternative splicing (7, 8) or a 43 homologous gene (9) . Soluble IIRs were found to be increased in the circulation of patients with 44 several forms of cancer (reviewed by (3)), sepsis (10), and . 45

Signal inhibitory receptor on leukocytes-1 (SIRL-1), encoded by the VSTM1 gene, is an IIR that is 46 expressed on human monocytes and granulocytes in peripheral blood (12) (13) (14) and lung (15). On 47 monocytes, but not granulocytes, SIRL-1 expression is associated with the single nucleotide 48 polymorphism (SNP) rs612529T/C (14, 15). SIRL-1 inhibits innate effector functions such as Fc 49

Receptor (FcR) induced production of reactive oxygen species (ROS) (14, 16, 17) and formation of 50 neutrophil extracellular traps (NET) (17, 18) . We recently showed that SIRL-1 recognizes amphipathic 51 α-helical peptides, including cathelicidin LL-37 and Staphylococcal phenol-soluble modulins (PSMs) 52 (19), as all well as several members of the S100 protein family (20), classifying SIRL-1 as an inhibitory 53 pattern recognition receptor (21) . 54

We previously showed that SIRL-1 surface expression on neutrophils and monocytes rapidly 55 decreases after activation in vitro (16) . Here, we hypothesized that activated neutrophils and 56 monocytes shed the ectodomain of SIRL-1, thereby releasing soluble SIRL-1 (sSIRL-1). In addition, it 57 has been described that VSTM1 encodes the splice variant VSTM1-v2, which lacks the exon that 58 encodes the transmembrane domain and is therefore predicted to give rise to a soluble form of . Even though these potential sources of sSIRL-1 have been reported, presence of endogenous 60 sSIRL-1 protein has not been demonstrated yet. In this study, we developed an ELISA to investigate 61 the presence and release mechanism of sSIRL-1 protein. 62

To determine PR3 cleavage, 20 µg/mL sSIRL-1 ecto or VSTM1-v2 were incubated with 2 µg/mL 133 PR3 in PBS with 0.5M NaCl and incubated 3h at 25°C, followed by SDS-PAGE and Western blot 134 analysis as described above, except that rabbit-anti-mouse IgG-HRP (DAKO; 1:10.000 diluted in TBS-T 135 with 1% BSA) was used as secondary antibody. 136 137 sSIRL-1 ELISA 138

To measure sSIRL-1 in cell supernatants, 96-wells flat-bottom MAXIsorp plates (Nunc) were coated 139 overnight at 4°C with capture mAb 1A5 (5 µg/mL in PBS, 50 µL/well). After washing with PBS 0.05% 140 (v/v) Tween-20, plates were blocked with 100 µL/well blocking buffer (1% (w/v) BSA, 3% (w/v) dry 141 milk in PBS). Next, plates were washed and incubated with undiluted cell supernatants and the 142 standard curve consisting of serially diluted sSIRL-1 ecto in PBS 1% BSA (50 µL/well), overnight at 4°C. 143

The following day, plates were washed and incubated with biotinylated mAb 3D3 for 1 hour at RT, 144 followed by washing and incubation with streptavidin poly-HRP (0.1 µg/mL; Sanquin) for 1 hour at 145

RT. Finally, plates were washed and incubated with 100 µL/well TMB-substrate (Biolegend/ITK). The 146

substrate reacted approximately 8 minutes, after which color development was stopped by adding 147 100 µL/well 1M H 2 SO 4 . Absorbance was measured at 450 nm on the CLARIOstar® (BMG Labtech). 148

Absorbance at 570 nm was used for background correction. All incubations, except coating of the 149 capture antibody, were done on a shaker. 150

The same procedure was used to measure sSIRL-1 in plasma, serum, urine or sputum 151 samples, with two exceptions: a different blocking buffer was used (3% (w/v) BSA in PBS), and 152 samples were pre-incubated 15 minutes on a shaker with 20 µg/mL HAMA blocking reagent 153 (Fitzgerald) before adding the samples to the ELISA plate, to prevent a-specific interactions. 154

We expressed the concentration of sSIRL-1 in pM rather than pg/mL, because endogenous 155 sSIRL-1 may have a different molecular weight than the recombinant sSIRL-1 ecto that was used for the 156 standard curve. 157 158

Neutrophils were isolated from peripheral blood using density gradient centrifugation on Ficoll (GE 160 Healthcare). The neutrophil pellet was incubated with ammonium chloride buffer to lyse the 161 erythrocytes. The remaining neutrophils were washed and suspended in RPMI 1640 containing 10% 162

(v/v) FCS and 1% (v/v) PS. Neutrophils were then stimulated with 50-100 ng/mL tumor necrosis factor 163 (TNF; Miltenyi) or 100 μg/mL of the Dectin-1 ligand Curdlan (Wako biochemical) in flatbottom plates 164

(Nunc) at 37°C, with or without addition of protease inhibitors. After 2 to 4 hours, neutrophils were 165 centrifuged 5 minutes at 500g to collect the supernatants. Cells were used for flow cytometry 166 analysis. Supernatants were centrifuged once more for 30 minutes at 25000g at 4°C to remove 167 cellular debris, followed by ELISA measurements. 168

The transduction and culture method of PLB-985 cells with SIRL-1 overexpression have been 171 previously described (16). Cells were treated 2h at 37°C with neutrophil elastase (Elastin Products 172 Company), cathepsin G (Biocentrum), or proteinase 3 (Elastin Products Company) (all 1 µM), followed 173 by flow cytometry analysis. 174 175

To determine SIRL-1 expression, cells were washed once with FACS buffer and stained with mAb 1A5 177 or 3F5 conjugated to AF647 or appropriate isotype controls (BD Biosciences) for 20 minutes at 4°C. For the ELISA, sSIRL-1 ecto was used as a standard, SIRL-1 mAb clone 1A5 as capture antibody 201 and SIRL-1 mAb clone 3D3 as detection antibody. In a competition assay, clone 1A5 and 3D3 did not 202 interfere with each other for binding to SIRL-1, and thus recognize different epitopes (Supplementary 203 Fig 1B) . The ELISA detected sSIRL-1 ecto and VSTM1-v2 equally well, with a lower limit of detection of 8 204 pM ( Fig 1D) . The ectodomain of the inhibitory receptor LAIR-1 (sLAIR-1 ecto ), which has 31% sequence 205 identity with sSIRL-1 ecto , was not detected in the ELISA, indicating specificity of the ELISA (Fig 1D) . We 206 spiked sSIRL-1 ecto into heparin plasmas that were sSIRL-1 negative in our ELISA. The spike was 207 recovered in all plasma dilutions tested (Fig 1E) , indicating that plasma is not interfering with the 208 sSIRL-1 measurement. Next, we spiked sSIRL-1 ecto into human pooled serum (HPS) and subjected it to 209 10 freeze thaw cycles, or incubation at 37°C, 56°C or 65°C for 30 minutes or 1 hour. sSIRL-1 210 concentration was stable in all freeze thaw cycles but decreased after 30 minutes incubation at 65 °C 211 (Supplementary Fig 1C) . Taken together, we developed a sensitive and specific assay to measure 212 sSIRL-1 protein concentration and showed that sSIRL-1 can be detected in presence of human plasma 213 components and is stable during multiple freeze-thaw cycles. 214 215

To investigate the presence of sSIRL-1 protein in vivo, we used the ELISA to measure sSIRL-1 in 217 plasma of healthy individuals, stratified per genotype of the rs612529T/C SNP. rs612529C associates 218 with decreased SIRL-1 expression on monocytes, but not on granulocytes (14, 15). sSIRL-1 was 219 detectable in only a small percentage of plasma samples from healthy individuals (8 out of 53, 15.1%) 220 (Fig 2A) . There was a tendency toward a lower percentage of samples with detectable sSIRL-1 in 221 individuals with a rs612529 C allele, but these differences were not statistically significant ( Fig 2B) . 222

To investigate the presence of sSIRL-1 in an inflammatory context, we measured sSIRL-1 in 223 patients with COVID-19 or RSV bronchiolitis, which are both characterized by excessive neutrophil 224 recruitment and activation (reviewed in (27, 28) . We detected sSIRL-1 in approximately 70% of sera 225 drawn from hospitalized adult COVID-19 patients, and the mean sSIRL-1 concentration was 226 significantly higher than in control serum (Fig 2C-D) . sSIRL-1 concentration in serum of COVID-19 227 patients was not affected by sex, age, nor the time since the start of symptoms or hospitalization 228 ( Supplementary Fig 2A, Fig 2C) . Serum samples in this cohort were collected at non-230 standardized time points, and we had restricted availability to clinical data, thereby limiting further 231 analysis on the correlation between sSIRL-1 concentration and disease activity. 232

We previously showed that RSV bronchiolitis patients have decreased SIRL-1 expression on 233 sputum neutrophils compared to peripheral blood neutrophils (17, 29), which is in line with our 234 hypothesis that activated neutrophils shed the ectodomain of SIRL-1. Indeed, in patients with severe 235 RSV bronchiolitis, we detected sSIRL-1 in 15 out of 16 sputa, and the mean sSIRL-1 concentration in 236 sputum was significantly increased compared to plasma ( Fig 2E) . We also detected sSIRL-1 in the 237 urine of the RSV patients, in similar concentrations as in plasma (Fig 2F) . In summary, we show that 238 sSIRL-1 concentration is mostly undetectable in healthy individuals, but increased in hospitalized 239 COVID-19 or severe RSV bronchiolitis patients. 240 241

To test if activated neutrophils indeed shed SIRL-1, we stimulated neutrophils from healthy controls 243 in vitro with TNF or curdlan, with or without addition of a broad-spectrum protease inhibitor cocktail. 244

We analyzed SIRL-1 expression on neutrophils by flow cytometry, and sSIRL-1 concentration in the 245 supernatant by ELISA. In agreement with our previous work (16), the percentage of SIRL-1 + 246 neutrophils decreased after activation (Fig 3A, B) . Concomitantly, we detected sSIRL-1 in the 247 supernatant ( Fig 3C) . Treatment with the protease inhibitor cocktail prevented this (Fig 3A-C) , 248

indicating that activated neutrophils release sSIRL-1 via proteolytic cleavage. 249 250

To examine which protease cleaves SIRL-1, we activated neutrophils with TNF in combination with 252 inhibitors against major classes of proteases: pepstatin A for aspartic proteases, E64 for cysteine 253 proteases, GM6001 for metalloproteases, leupeptin for serine and cysteine proteases, and aprotinin 254 for serine proteases. Treatment with 10 µM aprotinin resulted in a small but significant increase in 255 the percentage of SIRL-1-expressing cells after TNF stimulation, suggesting that SIRL-1 is shed by a 256 serine protease (Fig 4A) . Thus, we further investigated SIRL-1 cleavage by the serine proteases that 257 are predominantly secreted from activated neutrophils; cathepsin G, neutrophil elastase, and 258 proteinase 3 (PR3) (30). Treatment of SIRL-1-overexpressing PLB-985 cells with PR3 resulted in a 259 modest but consistent decrease in SIRL-1 expression, whereas cathepsin G and elastase did not affect 260 SIRL-1 expression on these cells (Fig 4B-C) . To confirm that PR3 cleaves SIRL-1, we activated 261 neutrophils with TNF in presence of a specific PR3 inhibitor. Indeed, the PR3 inhibitor partially 262

prevented the loss of SIRL-1 expression on TNF-activated neutrophils in all donors tested (Fig 4D-E) . 263

Finally, treatment of sSIRL-1 ecto and VSTM1-v2 with PR3 in a purified system resulted in proteolytic 264 cleavage as assessed by SDS-PAGE and Western Blot ( Fig 4F) . Thus, we show that PR3 cleaves SIRL-1. 265 266

SIRL-1 is activated by α-type phenol-soluble modulins (PSMs) from Staphylococci (19). S. aureus is 268 known for its large repertoire of secreted factors that can influence the host immune response, 269

including inhibitors against neutrophil proteases. Among these, S. aureus produces extracellular 270 adherence protein (Eap) and the homologues EapH1 and EapH2, which specifically inhibit neutrophil 271 serine proteases, including PR3 (24). We therefore questioned whether Eap can inhibit shedding of 272 SIRL-1 by neutrophils. Remarkably, treatment with Eap almost completely prevented the loss of 273 membrane-expressed SIRL-1 after TNF stimulation of neutrophils (Fig 5A, B) . A similar trend was seen 274 after treatment with EapH2, whereas treatment with EapH1 had no effect on membrane SIRL-1 275 expression. In PLB-985 cells, Eap and EapH1 both inhibited SIRL-1 cleavage by exogenous PR3 (Fig  276   5C ). Together, these data indicate that Eap can inhibit proteolytic cleavage of SIRL-1. 277

Here, we developed an ELISA to measure sSIRL-1 concentration (Fig 1) and are the first to show that 280 sSIRL-1 protein is present in vivo (Fig 2) . 281 282 sSIRL-1 concentration was increased in sputum of RSV bronchiolitis patients compared to plasma, 283 suggesting release of sSIRL-1 at the site of infection (Fig 2E) . sSIRL-1 was also detectable in sputum of 284 control infants, which can be explained by previous observations that sputum neutrophils of these 285 control patients are activated (17), possibly due to mechanical ventilation (31). From the site of 286 infection, sSIRL-1 may leak into the circulation, as there was a trend of increased sSIRL-1 in RSV 287 patient plasma compared to control plasma, although this comparison was limited by the low 288 number of control plasmas (Fig 2E) . Similarly, sSIRL-1 concentration was increased in serum of 289 hospitalized COVID-19 patients compared to control serum ( Fig 2C) . Lastly, sSIRL-1 was also 290 detectable in urine of RSV patients (Fig 2F) , indicating that, similar to soluble LAIR-1 (26), sSIRL-1 is 291 cleared by the kidneys. 292

Several studies have indicated a role of neutrophils in the pathophysiology of COVID-19 293 (reviewed in (28). Using a machine learning algorithm on a broad panel of inflammatory markers, 294 neutrophil-related markers predicted critical illness of COVID-19 patients most strongly (32). These 295 markers included resistin, lipocalin-2 and hepatocyte growth factor, which are all released by 296 degranulating neutrophils. Similarly, PR3 was highly present in sputum of COVID-19 patients (33), 297 and its concentration predicted COVID-19 disease severity (34). Due to restricted availability of data 298 and the collection of serum at non-standardized time points, we could not correlate sSIRL-1 299 concentration to COVID-19 disease progression, clinical parameters or inflammatory markers in our 300 cohort. Future studies will have to clarify if sSIRL-1 may serve as a biomarker to predict COVID-19 301 disease severity, or other diseases characterized by high neutrophil activation. 302

We found that SIRL-1 is cleaved from activated neutrophils by PR3 (Fig 3-4) . In addition to secretion 304 of soluble PR3, activated neutrophils can express PR3 on the plasma membrane on a subpopulation 305 of cells (35). Membrane-bound PR3 (mPR3) has been suggested to bind to the plasma membrane via 306 CD177, other membrane-expressed proteins, or directly to the lipid bilayer (36-38). We compared 307

shedding of SIRL-1 on FACS-sorted CD177 + versus CD177neutrophils and found that CD177 308 expression did not affect sSIRL-1 shedding (unpublished observations). Still, we showed that SIRL-1 309 surface expression decreased in a subpopulation of neutrophils after activation in vitro (Fig 3A, 4D , 310 5B), which may reflect cells with high mPR3 expression. On the other hand, SIRL-1 expression was 311 homogeneously low on neutrophils in sputum of RSV bronchiolitis patients (17, 29). Hence, the 312 relative contribution of soluble PR3 and mPR3 to SIRL-1 shedding remains to be determined. 313

Finally, we show that shedding of SIRL-1 by activated neutrophils was prevented by Eap, a 314 neutrophil serine protease inhibitor secreted by S. aureus (Fig 5) . The homologues EapH1 and EapH2 315 were less effective in preventing SIRL-1 shedding from activated neutrophils. In a previous study, 316 using short peptide substrates, EapH1 and EapH2 also had a lower capacity to inhibit PR3 than Eap 317 (Eap, K i = 0.23 nM; EapH1, K i = 1.0 nM; EapH2, K i = 21 nM) (24) (Fig 5A, B) . Alternatively, EapH1 and 318

EapH2 may differ in their ability to inhibit mPR3, as mPR3 has been shown to be more resistant to 319 PR3 inhibitors than soluble PR (39). The latter finding may also explain the partial effectiveness of the 320 PR3 inhibitor used in this study, and the differential abilities of leupeptin and aprotinin to inhibit 321 SIRL-1 shedding (Fig 4A, 5A) . 322

In addition to ectodomain shedding, sSIRL-1 may derive from the splice variant 40) . 324

controls (40). However, it remains to be determined whether endogenous VSTM1-v2 protein is 326 present in vivo. Both forms of sSIRL-1 were recognized by our ELISA (Fig 1D) , thus not allowing for 327 discrimination between these forms. Further research into VSTM1-v2 protein would benefit from the 328 development of an antibody that recognizes the intracellular tail of SIRL-1, which is present in 329 VSTM1-v2 but not in shed sSIRL-1. 330 331 Currently, we can only speculate on the function of SIRL-1 shedding. We previously argued that IIRs 332 that are constitutively expressed on a cell form a threshold to prevent unnecessary immune 333 activation. Some of these threshold receptors, so-called disinhibition receptors, are downregulated 334 after an activating stimulus surpasses the initial threshold, to facilitate subsequent cellular activation 335

(2). SIRL-1 is such an disinhibition receptor, based on its constitutive high expression on monocytes 336 and granulocytes in peripheral blood and lung (12, 14, 15) and downregulation during inflammation 337 (17, 29) . We thus propose that the function of SIRL-1 shedding is to rapidly remove SIRL-1 to facilitate 338 for a strong anti-microbial response, once the threshold for activation is surpassed. 339

Of course, the inhibitory function of SIRL-1 and hence the effect of SIRL-1 shedding also depends on 340 expression of its ligands. We found that SIRL-1 is activated by S100 proteins (20), cathelicidin LL-37, 341

and PSMs from Staphylococci (19). In inflammatory conditions with local tissue damage and DAMP 342 release, neutrophil released LL-37 and S100 proteins may act mostly on newly incoming neutrophils 343 with high SIRL-1 expression, to signal to these cells that no further immune activation is required. 344

PSMs may be beneficial for the host, for example by facilitating tolerance of resting neutrophils to 346 harmless Staphylococci such as S. epidermidis, but still allowing for full neutrophil activation once 347 SIRL-1 is shed in an inflammatory context. Interestingly, S. aureus, the most pathogenic member of 348 the Staphylococcus family, is unique in its secretion of Eap proteins (24). S. aureus may use Eap to 349 inhibit SIRL-1 shedding to benefit from the preserved inhibitory function of membrane-expressed 350 SIRL-1. Of particular interest is a recent study showing that S. aureus also requires Eap to prevent 

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Profiling of soluble immune 480 checkpoint proteins as potential non-invasive biomarkers in colorectal cancer and sepsis

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Signal Inhibitory 486 Receptor on Leukocytes-1 (SIRL-1) is a novel functional inhibitory immune receptor 487 expressed on human phagocytes

VSTM-v1, a potential myeloid 489 differentiation antigen that is downregulated in bone marrow cells from myeloid leukemia 490 patients

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Signal Inhibitory 499 Receptor on Leukocytes-1 is highly expressed on lung monocytes, but absent on 500 mononuclear phagocytes in skin and colon

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The Inflammatory Factors Associated with Disease Severity to Predict COVID-19 Progression

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Computational prediction 567 of the binding site of proteinase 3 to the plasma membrane

Characterization of the CD177 interaction 569 with the ANCA antigen proteinase 3

A hydrophobic patch on proteinase 3, 571 the target of autoantibodies in Wegener granulomatosis, mediates membrane binding via 572 NB1 receptors

New selective peptidyl di(chlorophenyl) phosphonate esters for 576 visualizing and blocking neutrophil proteinase 3 in human diseases

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