IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation.

The molecular mechanisms driving specific regulation of neutrophils are not completely understood to date. In order to characterize fundamental granulocyte features on protein level, we analyzed changes in proteome composition as reaction to stress from cell activation processes. For this purpose, we isolated primary granulocytes from equine whole blood through density gradient centrifugation followed by sodium chloride lysis and stimulated cells for 30 min with interleukin-8 (IL8) due to its role as a chemotactic factor for neutrophils. We additionally used phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), which are primarily associated to neutrophil extracellular trap formation and release of reactive oxygen species. From mass spectrometry analysis, we identified a total of 2,032 proteins describing the whole granulocyte proteome, including 245 proteins (12% of identified proteome) newly associated to in vivo expression in primary equine granulocytes (hypothetical proteins). We also found distinct and different changes in protein abundance (ratio ≥ 2) after short stimulation of cells with various stimuli, pointing to rapid and differentiated reaction pattern. IL8 stimulation resulted in increased protein abundance of 58 proteins (3% of proteome), whereas PMA induced changed protein abundance of 207 (10 % of proteome) and LPS of 46 proteins (2% of proteome). Enrichment analyses clearly showed fundamental differences between stimuli, with primary association of IL8 stimulation to processes in immune response, receptor signaling and signal transduction. Top enrichment for PMA on the other hand pointed to vesicle mediated transport and exocytosis. Stimulation with LPS did not result in any significant enrichment. Although we detected 43% overlap of enrichment categories for IL8 and PMA stimulation, indicating that activation of neutrophils with different stimuli partly induces some similar biological processes and pathways, hierarchical clustering showed clear differences in distribution and biological relevance of clusters between the chosen stimuli. Our studies provide novel information on the granulocyte proteome and offer insights into early, differentiated granulocyte reaction to stimuli, which contribute to a better understanding of molecular mechanisms involved in activation and recruitment of neutrophils, through inflammatory stimuli.

The molecular mechanisms driving specific regulation of neutrophils are not completely understood to date. In order to characterize fundamental granulocyte features on protein level, we analyzed changes in proteome composition as reaction to stress from cell activation processes. For this purpose, we isolated primary granulocytes from equine whole blood through density gradient centrifugation followed by sodium chloride lysis and stimulated cells for 30 min with interleukin-8 (IL8) due to its role as a chemotactic factor for neutrophils. We additionally used phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), which are primarily associated to neutrophil extracellular trap formation and release of reactive oxygen species. From mass spectrometry analysis, we identified a total of 2,032 proteins describing the whole granulocyte proteome, including 245 proteins (12% of identified proteome) newly associated to in vivo expression in primary equine granulocytes (hypothetical proteins). We also found distinct and different changes in protein abundance (ratio ≥ 2) after short stimulation of cells with various stimuli, pointing to rapid and differentiated reaction pattern. IL8 stimulation resulted in increased protein abundance of 58 proteins (3% of proteome), whereas PMA induced changed protein abundance of 207 (10 % of proteome) and LPS of 46 proteins (2% of proteome). Enrichment analyses clearly showed fundamental differences between stimuli, with primary association of IL8 stimulation to processes in immune response, receptor signaling and signal transduction. Top enrichment for PMA on the other hand pointed to vesicle mediated transport and exocytosis. Stimulation with LPS did not result in any significant enrichment. Although we detected 43% overlap of enrichment categories for IL8 and PMA stimulation, indicating that activation of neutrophils with different stimuli partly induces some similar biological processes and pathways, hierarchical clustering showed clear differences in distribution and biological relevance of clusters between the chosen stimuli. Our studies provide novel information on the granulocyte proteome and offer insights into early, differentiated granulocyte reaction to stimuli, which contribute to a better understanding of molecular mechanisms involved in activation and recruitment of neutrophils, through inflammatory stimuli.

INTRODUCTION
Granulocytes have initially been labeled as short-lived, terminally differentiated cells, driving innate immune response through phagocytosis, degranulation, ROS release and, as described more recently, NETosis (1,2). However, today, neutrophil diversity and plasticity, with many different subpopulations and finely tuned functional features are evident (3)(4)(5)(6)(7)(8). Still relatively little is known about specific, differentiated regulation mechanisms in early granulocyte activation involved in subsequent innate immune responses. For this reason, we investigated fundamental granulocyte features by analyzing changes in proteome composition as reaction to cell activation and allocating these changes to different biological processes and pathways in an equine model. In cells from the adaptive immune system, we previously found major differences in regulation of lymphocyte protein expression in autoimmune disease (9)(10)(11)(12). Moreover, we detected differences in the granulocyte proteome, with Talin1 as a key player in disease pathogenesis, indicating a role of the innate immune system in lymphocyte-driven autoimmune disease (13,14). In retrospect, the granulocytes analyzed in these studies most likely represent the subpopulation of low density neutrophils (LDN), which were recently discovered (15). In present study, LDN were excluded from analysis, due to granulocyte isolation protocol. Here, we were especially interested in the impact of initial activation on downstream innate immune response and the pathways switched on in course of activation-induced cell stress in order to provide fundamental knowledge on granulocyte activation mechanisms.

Sample Processing
The blood used in this study originated from three resident horses of the LMU equine clinic (aged 12, 20, and 21; kept in straw-embedded stalls with daily access to paddocks), which are at the student's disposal for supervised ultrasoundand health assessment training. Health status was assessed by standard clinical routine examinations. No experimental procedures were performed on these horses. Venous whole blood was collected in tubes supplemented with 25.000 I.U. heparin. After rough sedimentation of erythrocytes, PMN were isolated from plasma by density gradient centrifugation (RT, 290 × g, 25 min, brake off) using Ficoll-Paque PLUS separating solution (GE Life Sciences, Freiburg, Germany). Cells were washed gently (4 C, 400 × g, 10 min) in cold PBS (DPBS devoid of CaCl 2 and MgCl 2 ; Gibco/ThermoFisher Scientific, Germany) and remaining erythrocytes were removed by sodium chloride lysis (lysis in 0.2% NaCl, after 30 s addition of equal part 1.6% NaCl to restore isotonicity). Cells were washed (4 • C, 400 × g, 10 min) and resuspended in PBS with 0.2% Glucose. From each animal used in the experiment, we prepared aliquot portions of 6 × 10 5 cells/500 µl. These cell aliquots were separately stimulated with recombinant equine interleukin-8 (IL8; Kingfisher Biotec; 1 ng/ml), phorbol 12-myristate 13acetate (PMA; Sigma-Aldrich/Merck, Darmstadt, Germany; 5 µg/ml) and lipopolysaccharide (LPS; Sigma-Aldrich/Merck, Darmstadt, Germany; 5 µg/ml) for 30 min in a CO 2 incubator (37 • C, 5% CO 2 ). Untreated medium control (mc) was incubated under the same conditions but without stimulating agent. After stimulation, each of the stimulated and mc aliquots was topped up to 1 ml with PBS with 0.2% Glucose and pelleted (4 • C, 2,300 × g, 10 min). All Samples were stored at −20 • C. Shortly before mass spectrometry analysis, cells were thawed and lysed in urea buffer (8 M urea in 0.1 M Tris/HCl pH 8.5), and protein amount was determined with Bradford protein assay (16).

Mass Spectrometry Analysis
From each sample, 10 µg total protein was digested with LysC and trypsin by filter-aided sample preparation (FASP) as previously described (17). Acidified eluted peptides were analyzed in the data-dependent mode on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) online coupled to a UItimate 3000 RSLC nano-HPLC (Dionex). Samples were automatically injected and loaded onto the C18 trap column, eluted after 5 min and separated on the C18 analytical column (75 µm ID × 15 cm, Acclaim PepMAP 100 C18. 100 Å/size, LC Packings, Thermo Fisher Scientific, Bremen, Germany) by a 90 min non-linear acetonitrile gradient at a flow rate of 250 nl/min. MS spectra were recorded at a resolution of 60,000. After each MS1 cycle, the 10 most abundant peptide ions were selected for fragmentation.

Data Processing
Label-free quantitative analysis was performed using Progenesis QI software (version 2.5, Non-linear Dynamics, Waters, Newcastle upon Tyne, U.K.) as described (18,19), with raw MS spectral files imported, followed by automatic peak picking and retention time alignment and normalization of total peak intensities across all samples to minimize loading differences. All MS/MS spectra were exported from Progenesis QI software as Mascot generic files (mgf) and searched against Ensembl Horse protein database (version 3.0, http://www.ensembl.org) for peptide identification with Mascot (version 2.5.1). Search parameters used were 10 ppm peptide mass tolerance, 20 mmu fragment mass tolerance, one missed cleavage allowed, carbamidomethylation as fixed modification and methionine oxidation as well as deamidation of asparagine and glutamine as variable modifications. Mascot integrated decoy database search was set to a false discovery rate (FDR) of 1% when searching was performed on the concatenated mgf files with a percolator ion score cut-off of 13 and an appropriate significance threshold p. Identifications were re-imported into Progenesis QI and redundancies grouped following the rules of parsimony.

Data Analysis
Differential protein abundance was determined by comparison of the mean normalized peptide abundance from the extracted ion chromatograms. Proteins were considered differentially expressed at stimulating agent/mc ratio ≥ 2.0. Bioinformatic analysis was performed on human orthologs of gene names from differentially expressed equine proteins with open source software ShinyGO v0.60: http://bioinformatics.sdstate.edu/go60/ (20) with the following settings: search species human, Pvalue cutoff (FDR) 0.05, number of most significant terms to show 30. P-value for enrichment analysis was calculated via hypergeometric distribution, followed by correction using FDR. Venn diagram was made with open source tool: http:// bioinformatics.psb.ugent.be/webtools/Venn/.

Two Thousand Thirty-Two Proteins Describing the Granulocyte Proteome
Using mass spectrometry analysis, we identified the equine whole granulocyte proteome, comprising a total of 2,032 proteins. Among the identifications, we found 245 proteins (hypothetical proteins) which have not been associated to the in vivo protein expression repertoire of equine granulocytes so far (Supplemental Table 1). These proteins represent 12% of the total granulocyte proteome identified here.

Short Stimulation Time of Only 30 min Results in Rapid and Differentiated Reactions of Cells
After stimulation with three different stimulating agents, we found distinct changes in granulocyte protein abundance compared to medium controls (ratio cut-off ≥ 2). In detail, cells stimulated with LPS showed higher expression levels of 46 proteins (2% of proteome), whereas PMA induced increased protein abundance of 207 proteins (10% of proteome). IL8 stimulation resulted in increased protein expression levels of 58 proteins (3% of proteome) (Supplemental Table 2). All of these differentially abundant proteins summed up to a total of 252, from which only 15 showed higher expression levels in all three stimulating agent groups (Figure 1, Table 1). Analysis of differentially expressed proteins per stimulation group revealed 12 unique proteins from LPS and 174 from PMA stimulated cells as well as 22 proteins with unique appearance in cells stimulated with IL8 ( Figure 1, Table 1).

Reaction of Innate Immune Cells to Different Stimuli Are Respectively Clustered in Three Distinct Networks
In order to understand the association of the differentially expressed proteins to biological processes and their known role in granulocyte activation pathways, we analyzed the data from the 15 proteins present in all groups (Figure 1, Table 1, Supplemental Figure 1, Supplemental Table 3) as well as LPS, PMA, and IL8 groups with open source software ShinyGO. LPS stimulation data did not result in any significant enrichment and clustering of the differentially expressed proteins in these cells. Therefore, we looked into GO category assignment for these proteins and found eight high level categories mainly connected to cell metabolism, intracellular transport and response to stress (Supplemental Table 4). Data from IL8 and PMA stimulated cells, however, revealed three distinct clusters. Comparison of these enrichment category clusters showed a 43% overlap between IL8 and PMA stimulation groups, with neutrophil activation and cellular catabolic processes as the two major shared functional categories (Figure 3, Table 3). The unique clusters for each stimulant, however, showed a clear difference in reaction of cells to stimuli: PMA stimulated innate immune cells reacted with processes involved in intracellular transportation processes, whereas IL8 stimulated cells showed involvement in signal transduction pathways (Figure 3, Table 3).

Unique Reaction of Cells to IL8 Stimulation Associates to Receptor Signaling, Signal Transduction, and Immune Response
For more detailed analysis, we subsequently focused on proteins which were differentially abundant in either IL8 or PMA stimulated cells and therefore described as unique for respective stimulus. Hierarchical clustering of enrichment analysis data from unique proteins expressed after PMA stimulation pointed to primary involvement in vesicle-mediated and intracellular transport as well as exocytosis on the one and metabolic processes on the other hand ( Figure 2, Table 2). Results from IL8 stimulated cells showed primary association of uniquely expressed proteins to receptor signaling, signal transduction, and immune response with top enrichment for Fc-epsilon receptor and Tumor necrosis factor (TNF) mediated signaling pathways (Figure 2, Table 2). Also, enrichment of PI 3kinase activity pointed to processes in cytoskeleton dynamics (Figure 2, Table 2). Interestingly, the protein proteasome 26S subunit, ATPase 6 (PSMC6), was allocated to the majority of functional enrichment categories from the IL8 stimulation group ( Table 2).

DISCUSSION
Knowledge about the molecular mechanisms involved in specific granulocyte activation and subsequent choice of pathways depending on different stressors is still incomplete to date. Moreover, in the past, granulocytes have frequently been underestimated in their ability to execute distinct heterogenic reactions rather than uniform response cascades to any, mainly pathogen-induced stimulus. The past decade has yielded more details on granulocyte heterogeneity and function, not only for processes in the innate immune system but also for regulatory involvement in adaptive immune responses (4,21). Nevertheless, there are still many signaling processes in granulocyte activation, which need clarification. To gain deeper insight into these processes and to find possible downstream reaction differences between initiating stimuli, we performed a short stimulation assay with freshly obtained, primary equine granulocytes. PMA and LPS, were used as universal stimuli. PMA induces exocytosis, ROS release and NET formation through direct activation of protein kinase C (PKC) and subsequent signal transduction cascade (22). LPS triggers similar responses by binding to TLR4 on neutrophils (23). For specific activation of granulocytes, we used IL8. This cytokine is expressed by a variety of cells, such as monocytes, fibroblasts and endothelial cells and acts as a potent chemoattractant for granulocytes, inducing neutrophil recruitment and chemotaxis via chemokine receptors CXCR1 and CXCR2 (24). Granulocyte activation and identification of resulting differences in behavior, gene regulation and protein expression have previously been performed in other uncommon models such as cattle (25) and pigs (26). Predominantly, studies focus on human granulocytes, however, most of these studies concentrate on one particular morphologic (granules, membrane proteins) (27)(28)(29) or functional (NET formation) (30, 31) feature of granulocytes. Few studies describe stimulatory experiments and their effect on the whole granulocyte proteome (32,33).
Compared to these studies, we chose a very short stimulation time in order to detect early proteome changes with possibly transient character. Also, we did not separate the proteome via 2D prior to mass spectrometry analysis. From our initial proteomics experiment, we unraveled the equine granulocyte proteome, detecting 2,032 proteins (Supplemental Table 1). Similar proteomic based studies have been performed on human granulocytes (34), granulocytes from other species such as cattle (35) and rats (36), as well as neutrophil-associated BALF proteins in horses (37). However, to our knowledge, the full equine neutrophil proteome has not been described to date. Interestingly, 12% of the identified proteins in our study were classified as "hypothetical proteins, " whose existence is predicted, but experimental evidence for in vivo expression is lacking. With our studies, we could confirm actual in vivo expression of these proteins, associating them to primary granulocyte proteome in horses. We chose equine granulocytes to conduct our experiments, because the equine and human immune system share a wide range of similarities both in granulocyte-lymphocyte ratio, composition and function (38)(39)(40). Furthermore, the horse is prone to allergies and autoimmune diseases, which are similarly found in humans (41)(42)(43)(44)(45) and adaptive as well as innate immune cells from horses have proven to be valuable tools for studying human diseases (37,42,44,46). Despite certain differences between human and horse neutrophils (47,48), the horse is still a very promising model, especially for processes and diseases which are not adequately addressed by rodent models. However, more investigations are needed to determine its exact and true translational value, which we provide a basis for with our studies.
Among all identified proteins, we found a total of 252 differentially abundant proteins after cell stimulation with different stimuli (Supplemental Table 2). Fifteen of these proteins showed higher expression levels in all three stimulating agent groups (Figure 1, Table 1, Supplemental Figure 1,    Proteins used were uniquely expressed in each stimulation group. Table 3), indicating onset of some mutual reactions to the different stimuli. A larger number of unique proteins with differential expression per stimulant, however, pointed to predominantly differentiated reactions to the different stimuli (Figure 1, Table 1). Further assessment of all differentially abundant proteins from PMA and IL8 samples with ShinyGO enrichment analysis revealed 57% unique network clustering for each stimulant, respectively (Figure 3, Table 3). This shows the ability of granulocytes to distinguish between stimuli and regulate specific pathways in response to selective cell-stressors, although partial immune response is executed independent of stimulation type. Subsequent analysis of solely those proteins that changed abundance uniquely after either IL8 or PMA stimulation highlighted their association to stimulantcharacteristic reactions, such as exocytosis and degranulation after PMA stimulation (22), and cytoskeleton dynamics after stimulation with IL8 (49, 50) (Figure 2, Table 2). IL8 stimulation yielded the identification of proteasome 26S subunit, ATPase 6 (PSMC6), which showed higher abundance unique to this stimulant (IL8/mc ratio 2.1; p < 0.001). PSMC6 is an ATP-dependent proteolytic complex responsible for ubiquitin-dependent protein degradation (51,52), which is an important regulator of the majority of cellular activity and homeostasis (53). Divergent levels of proteasome activity have a strong impact on disease pathogenesis of several diseases and are used as drug targets in disease treatment (51,(54)(55)(56). Thus, the higher abundance of PSMC6 in IL8 stimulated cells might indicate activation of the proteasome in granulocytes with functional importance in downstream regulation of immune response to stress. Subsequent analysis of proteomic data from IL8 stimulated cells revealed that PSMC6 was present in the majority of functional enrichment clusters from biological processes, including the top enriched functional categories tumor necrosis factor (TNF) mediated signaling and Fc-epsilon receptor pathways ( Table 2). These two pathways are essential for signal transduction in cells, with a wide functional variety of downstream responses such as apoptosis but also immune and inflammatory responses as well as cell survival, activation and differentiation (57,58). Interestingly, occurrence of Fc receptors on granulocytes have initially been described as a marker of neutrophil heterogeneity rather than a necessity for optimal neutrophil aggregation and adhesion (59). Especially Fc-epsilon receptor signaling is only present in neutrophils under certain conditions and their exact role is still   (58). Furthermore, Fc receptors are unlikely to mediate PMA-induced cell activation (59), which is consistent with our findings on PMA-stimulated granulocytes, where we found no allocation of uniquely expressed proteins to Fc receptor signaling pathways (Figures 2, 3, Tables 2, 3).

Supplemental
Our findings undermine the ongoing appreciation of granulocyte function toward finely tuned, heterogeneous, specific reactions of more than one subpopulation of neutrophils (4,7,8,(60)(61)(62)(63). Furthermore, our data shows that a stimulation time of only 30 min is sufficient to initiate substantial and specific changes in granulocyte proteome as reaction to individual stimulating agents (Figure 1, Table 1, Supplemental Table 2). These rapid changes most likely occur due to gene induction of early responding genes but may also be the result of posttranslational modifications mediated by proteins that are activated early in neutrophil responses to stimuli, such as phosphatidylinositol 3-kinase (PI 3-kinase). Interestingly, PI 3kinase activity appears as functional category from enrichment analysis of our IL8 data (Figure 2A, Table 2), supporting the involvement of PI 3-kinase in IL8-induced protein changes. With its ability to phosphorylate molecules acting as second messengers and thereby switch on downstream intracellular signaling (64), and its involvement in neutrophil chemokinesis and phagosome formation (49,50,65), PI 3-kinase merits further investigations in future functional studies. No matter the origin of the changed granulocyte protein repertoire described in our data, it gives insight into early onset of granulocyte activation on protein level, which may be useful to modulate granulocyte mediated pathological processes in future functional experiments. However, more experiments are needed, not only for determination of minimal stimulation times triggering regulation of protein expression levels in granulocytes, but also for analysis of expression kinetics in course of longer stimulation assays. From other comprehensive studies on equine neutrophils we know, that neutrophil extracellular traps (NETs) readily occur in response to adequate stimuli (66) as opposed to cells from other animal models (67). Our protein data however, lack association to this process (Figure 2,  Table 2). We assume that the expression differences of proteins associated to NET formation occur after longer stimulation time, as recently described (66). Therefore, increasing the stimulation time in these assays could address protein repertoire changes associated to NET-relevant biological processes such as DNA decondensation, histone citrullination, and related signal transduction. Also, we would expect more prominent clustering of IL8 induced protein changes to cytoskeleton dynamics involved in chemotaxis and phagosome formation, as functional answers of cells to stimuli fluctuate over time (68). Keeping in mind the dynamic character of protein expression patterns in course of cell activation, our data put a spotlight merely on the first reaction to stimuli. This is a very interesting time point in our opinion, because it shows the initiating functional answers in activated cells, which are potentially accessible to experimental modulation. Adding proteomic data from more stimulation times would give a more precise insight into dynamic whole-cell proteome changes throughout the activation process of granulocytes, similar to previous analysis of pre-determined cytokines and degranulation markers by kinetic flow cytometry (69), which needs to be addressed in future studies.

CONCLUSION
With our data we provide a fundamental study on activation of primary granulocytes and regulation of downstream immune response by showing that different stimuli provoke divergent and rapid downstream responses through regulation of protein expression in these cells. These expression differences show involvement in various different pathways and biological processes which, among some similarities, differ between stimuli and support knowledge on heterogeneity of granulocytes and their highly selective response to stimuli. The presented data may therefore act as a guide for further, in-depth research on granulocyte response patterns and behavior in health and disease.

DATA AVAILABILITY STATEMENT
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (70) partner repository with the dataset identifier PXD013648.

AUTHOR CONTRIBUTIONS
CD conceived and designed the experiments. RD and SH performed the experiments. RD, SH, MW, and CD analyzed the data. RD wrote the manuscript. All authors critically read the manuscript and approved the final version to be published.

FUNDING
This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG DE 719/4-3 (to CD).