Complement Regulation in Human Tenocytes Under the Influence of Anaphylatoxin

International Journal of Molecular Sciences

Article Complement Regulation in Human Tenocytes under the Inﬂuence of Anaphylatoxin C5a

Sandeep Silawal 1, Benjamin Kohl 2 , Jingjian Shi 1 and Gundula Schulze-Tanzil 1,*

1 Institute of Anatomy and Cell Biology, Paracelsus Medical University, Nuremberg and Salzburg, General Hospital Nuremberg, Prof. Ernst Nathan Str. 1, 90419 Nuremberg, Germany; [email protected] (S.S.); [email protected] (J.S.) 2 Department of Traumatology and Reconstructive Surgery, Campus Benjamin Franklin, Charité—Universitätsmedizin Berlin, Corporate Member of Berlin Institute of Health, Freie Universität Berlin and Humboldt-Universität zu Berlin, Hindenburgdamm 30, 12203 Berlin, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-911-398-11-6772

Abstract: A central part of the complement system, the anaphylatoxin C5a was investigated in this study to learn its effects on tenocytes in respect to understanding the potential expression of other crucial complement factors and pro-inflammatory mediators involved in tendinopathy. Human hamstring tendon-derived tenocytes were treated with recombinant C5a protein in concentrations of 25 ng/mL and 100 ng/mL for 0.5 h (early phase), 4 h (intermediate phase), and 24 h (late phase). Tenocytes survival was assessed after 24 h stimulation by live-dead assay. The gene expression of complement-related factors C5aR, the complement regulatory proteins (CRPs) CD46, CD55, CD59, and of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 was monitored using qPCR. Tenocytes were immunolabeled for C5aR and CD55 proteins. TNFα production was monitored by ELISA. Tenocyte survival was not impaired through C5a stimulation. Interestingly, the gene expression of C5aR and that of the CRPs CD46 and CD59 was significantly Citation: Silawal, S.; Kohl, B.; Shi, J.; reduced in the intermediate and late phase, and that of TNFα only in an early phase, compared to Schulze-Tanzil, G. Complement α Regulation in Human Tenocytes the control group. ELISA analysis indicated a concomitant not significant trend of impaired TNF under the Influence of Anaphylatoxin protein synthesis at 4 h. However, there was also an early significant induction of CD55 and CD59 C5a. Int. J. Mol. Sci. 2021, 22, 3105. mediated by 25 ng/mL anaphylatoxin C5a. Hence, exposure of tenocytes to C5a obviously evokes a https://doi.org/10.3390/ijms22063105 time and concentration-dependent response in their expression of complement and pro-inflammatory factors. C5a, released in damaged tendons, might directly contribute to tenocyte activation and Academic Editor: Magali Cucchiarini thereby be involved in tendon healing and tendinopathy.

Received: 11 February 2021 Keywords: anaphylatoxin; C5a; complement system; complement regulation; tendinopathy; tenocytes Accepted: 15 March 2021 Published: 18 March 2021

Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in Tendinopathy has been defined as a chronic low grade inflammatory and degenerative published maps and institutional affil- iations. musculoskeletal disease [1,2]. Even though the etiology of this disease is still not clearly understood, there are various hypotheses that are frequently discussed. The collagen tears resulting from so-called micro traumata after repetitive mechanical overloading have been a long known classical explanation for the pain and functional impairment in tendinopathy [3]. Moreover, the inflammatory concept in the pathogenesis of tendinopathy has Copyright: © 2021 by the authors. regained a wider focus [2]. A low increase in pro-inflammatory cytokines such as tumor Licensee MDPI, Basel, Switzerland. necrosis factor (TNF)-α is associated with the pathological tendons and the affected teno- This article is an open access article cytes [4]. Tenocytes’ activity alters in response to any physiological and non-physiological distributed under the terms and mechanical loading, biochemical imbalance in their environment, or inflammation, leading conditions of the Creative Commons Attribution (CC BY) license (https:// to morphological changes and changes in their extracellular matrix synthesis [5–7]. In creativecommons.org/licenses/by/ addition, an increased presence of inflammatory cells such as macrophages, mast cells, 4.0/). and T-cells in pathological tendons was emphasized in comparison to healthy tendons

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7]. In addition, an increased presence of inflammatory cells such as macrophages, mast cells, and T-cells in pathological tendons was emphasized in comparison to healthy ten- Int. J. Mol. Sci. 2021, 22, 3105 dons in a systematic review [8]. These immune cells are actively involved2 of 13 in producing pro-inflammatory cytokines, such as TNFα, interleukin (IL)-6, IL-1β, etc., in addition to complement factors that can trigger the inflammation process [1,9]. A direct TNFα stimu- lationin a systematic of tenocytes review and [8]. Thesean indirect immune co cells-culture are actively with involvedleukocyte in producingsubpopulations, pro- peripheral α β bloodinflammatory mononuclear cytokines, cells such, and as TNF neutrophil, interleukins ha (IL)-6,ve been IL-1 studied, etc., in in addition our previous to com- in vitro study plement factors that can trigger the inflammation process [1,9]. A direct TNFα stimulation toof tenocytesunderstand and anthe indirect complement co-culture response with leukocyte in tenocytes subpopulations, [10]. peripheral blood mononuclearThe complement cells, and neutrophils system is have an important been studied first in our line previous of defensein vitro instudy our immune to system. Withunderstand more the than complement 30 different response proteins in tenocytes belonging [10]. to this system, its activation can be initi- atedThe through complement the classical, system is lectin an important, and spontaneous first line of defense hydrolysis in our immune pathway system.s [11]. The comple- With more than 30 different proteins belonging to this system, its activation can be initiated mentthrough protein the classical, activation lectin, andby spontaneouscleavage leads hydrolysis to commonly pathways [known11]. The complementeffects such as opsonization,protein inflammation activation by cleavage, and cell leads lysis to ( commonlyFigure 1).known effects such as opsonization, inflammation, and cell lysis (Figure1).

Figure 1. 1.Simplified Simplified scheme scheme of the of complementthe complement activation activation and regulation and regulation processes. C5aR:processes. C5a C5aR: C5a receptor, MAC: Membrane attack complex, MBL: mannose binding lectin. The image was self-made receptor, MAC: Membrane attack complex, MBL: mannose binding lectin. The image was self- by Sandeep Silawal. made by Sandeep Silawal. A common trunk protein complement factor C5 is fragmented through C5 convertase and otherA common extrinsic proteasestrunk protein such as complement cathepsin D into factor C5a, C5 a small is fragmented (9 kDa) but potentthrough so- C5 convertase andcalled other anaphylatoxin extrinsic and proteases C5b. C5b such attaches as tocathepsin the cell membrane. D into C5a, The C5ba small split fragment(9 kDa) but potent so- associates with other complement factors to generate a membrane attack complex (MAC, calledC5b–C9), anaphylatoxin leading to either and cellular C5b osmotic. C5b attach lysis ores other to the sublytic cell membrane. responses [12 The,13]. C5b C5a split fragment associatesbinds to the G-proteinwith other coupled complement membrane factors protein C5ato generate receptor (C5aR) a membrane or C5a receptor-like attack complex (MAC, C5b2 (C5L2),–C9) a, leading non-signaling to either C5abinding cellular protein, osmotic unfolding lysis or its other effects sublytic on the target responses cells. [12,13]. C5a bindsHereby, to C5a the triggers G-protein signal coupled cascades inm anembrane affected protein cell after itC5a attaches receptor to C5aR (C5aR) [11]. In-or C5a receptor- likeflammatory 2 (C5L2 effects), a non in various-signaling tissues C5a have binding been described protein, asunfolding a result of its the effects C5a/C5aR on the target cells. coupling such as their stimulatory effect on leukocytes, including chemotaxis of leukocytes, Herebysmooth muscle, C5a triggers contraction, signal or increase cascades in vascular in an permeability;affected cell hence, after C5a it attaches is known to C5aR [11]. In- flammatorybe a key player effects in the developmentin various tissues of inflammation have been and described sepsis [14,15 as]. a A result physiological of the C5a/C5aR cou- plingplasma such concentration as their of stimulatory 8.34 ± 2.05 ng/mL effect C5a on hasleukocytes been reported, including [16]. Interestingly, chemotaxis C5a of leukocytes, smoothand C3a havemuscle also contraction been associated, or withincrease tissue in regeneration vascular permeability; and repair [17]. hence, There are C5a is known to various mechanisms regulating complement effects for the protection of our own body becells. a key These player are exerted in the by so-calleddevelopment complement of inflammation regulatory proteins and sepsis (CRPs), [1 which4,15] are. A physiological plasmaavailable concentration in the system either of as8.34 soluble ± 2.05 factors ng/mL such C5a as C1-inhibitor, has been factorreported H, factor [16 H-like]. Interestingly, C5a andprotein C3a 1, clusterin,have also vitronectin, been associated or cell membrane-attached with tissue regeneration proteins, such and as repair CD46 (mem- [17]. There are var- iousbrane mechanisms co-factor), CD55 regulating (decay-accelerating complement factor), CD59effects (protectin), for the protection etc. [11]. Complement of our own body cells. regulatory proteins CD46 and CD55 regulate the complement cascade in the early cascade These are exerted by so-called complement regulatory proteins (CRPs), which are availa- level at which they attenuate the C3 convertase or accelerate its degradation preventing ble in the system either as soluble factors such as C1-inhibitor, factor H, factor H-like protein 1, clusterin, vitronectin, or cell membrane-attached proteins, such as CD46 (membrane co-factor), CD55 (decay-accelerating factor), CD59 (protectin), etc. [11]. Comple- ment regulatory proteins CD46 and CD55 regulate the complement cascade in the early

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cascade level at which they attenuate the C3 convertase or accelerate its degradation preventing the complement activation in the intermediate stage. CD59, on the other hand, hinders the association of membrane attack complex (MAC) as the terminal component of the complement cascade. Int. J. Mol. Sci. 2021, 22, 3105 TNFα and other pro-inflammatory mediators often accompany an increase3 of 13 in complement and can regulate each other in an inflammatory process [10,18,19]. Even though various cytokines have been discussed in the research field of tendon inflammation [20,21the], IL complement-1β, TNFα, activation IL-6, and in IL the-10 intermediate are considered stage. in CD59, various on the studies other hand, as the hinders key cytokines the association of membrane attack complex (MAC) as the terminal component of the in tendon diseases, as reported in a systematic review [9]. The complement system attracts complement cascade. increasingTNF attentionα and other in this pro-inflammatory tendon research mediators since oftencomplement accompany factors an increase and receptors in com- could be detectedplement in and tenocytes can regulate and each their other regulation in an inflammatory in-vitro in process response [10,18 to,19 physical]. Even though and biochemical influencesvarious cytokines [10,18,1 have9]. beenTherefore, discussed more in the research research fieldhas ofto tendon be performed inflammation to understand [20,21], the β α role ofIL-1 complement, TNF , IL-6, factors and IL-10 as possible are considered players in variousin the pathogenesis studies as the keyof tendinopathy cytokines in . tendon diseases, as reported in a systematic review [9]. The complement system attracts in- creasing attention in this tendon research since complement factors and receptors could be 2. Resultsdetected in tenocytes and their regulation in-vitro in response to physical and biochemical 2.1. Tenocyteinfluences Survive [10,18, 19C5a]. Therefore, Exposition more research has to be performed to understand the role of complement factors as possible players in the pathogenesis of tendinopathy. To assess whether C5a stimulation of tenocytes for 24 h (Figure 2A) impairs cell survival, 2.a Resultsvitality assay has been performed visualizing vital cells with green fluorescence and dead2.1. Tenocytecells with Survive red C5a fluorescence Exposition (Figure 2B). While areas covered with vital cells compared to thoseTo assess areas whether covered C5a in stimulation total by cells of tenocytes were measured for 24 h (Figure using2A) ImageJ impairs, no cell significant survival, a vitality assay has been performed visualizing vital cells with green fluorescence impairmentand dead in cellstenocytes with red vitality fluorescence in response (Figure2 toB). both While concentrations areas covered with of the vital anaphylatoxin cells C5a wascompared seen compared to those areas to covered the control in total bygroup cells were(Figure measured 2C). using ImageJ, no significant impairment in tenocytes vitality in response to both concentrations of the anaphylatoxin C5a was seen compared to the control group (Figure2C).

Figure 2. Schematic ﬁgure of the experimental setup (A). Tenocytes were stimulated with two Figure concentrations2. Schematic (25 figure ng/mL of and the 100 experimental ng/mL) of the setup recombinant (A). Tenocytes protein anaphylatoxin were stimulated C5a or remained with two concentrationsunstimulated (25 (control)ng/mL and for 24 100 h. (Bng) Representative/mL) of the recombinant images of live-dead protein staining anaphylatoxin of the tenocytes C5a or remainedafter unstimulated 24 h stimulation (control) with recombinant for 24 h protein. (B) Representative anaphylatoxin C5a. images Fluorescein of live diacetate-dead staining (green: of the tenocytesvital cells) after and 24 propidium h stimulation iodide (red:with dead recombinant cells) have beenprotein used anaphylatoxin for staining. Scale C5a. bar = Fluorescein 100 µm. diacetate(C )(green: Graphic vital presentation cells) and of tenocytes propidium vitality iodide after 24 (red: h stimulation dead cells) with have 25 ng/mL been (lightused blue) for staining. and Scale bar100 = ng/mL100 µm. (dark (C blue)) Graphic recombinant presentation C5a protein of intenocytes comparison vitality to the non-stimulatedafter 24 h stimulation control groups with 25 (grey). Number of living cells in relation to total cells (100%) measured in live-dead staining images ng/mL (light blue) and 100 ng/mL (dark blue) recombinant C5a protein in comparison to the of the tenocytes. Mean values and the standard errors of mean (SEM) are depicted. Shapiro–Wilk test non-stimulated control groups (grey). Number of living cells in relation to total cells (100%) was used to test the normal distribution of data. One sample t-test was performed in relation to the measuredtheoretical in live mean-dead of staining 100%. n = images 3 independent of the experiments tenocytes. were Mean performed values using and tenocytesthe standard derived errors of meanfrom (SEM) three are different depicted. donors. Shapiro–Wilk test was used to test the normal distribution of data. One sample t-test was performed in relation to the theoretical mean of 100%. n = 3 independent experiments were performed using tenocytes derived from three different donors.

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2.2. Tenocytes Show a Time and Concentration Dependent Regulation of C5aR, CRPs, TNFα, Int. J. Mol. Sci. 2021 22 , , 3105 and IL-6 Gene Expression 4 of 13 The C5aR gene expression was significantly reduced under both C5a concentrations at2.2. the Tenocytes intermedia Show ate Time phase and Concentrationtime point Dependentof analysis Regulation (4 h). This of C5aR, effect CRPs, could TNFα still, and be detected in theIL-6 tenocytes Gene Expression at the late phase of 24 h but only, when exposed to the higher concentration of 100The ng C5aR/mL geneC5a expression(Figure 3A was). Likewise, significantly the reduced transcriptional under both C5a activity concentrations of the gene encoding forat thecomplement intermediate regulatory phase time pointprotein of analysisCD46 also (4 h). showed This effect a significant could still be reduction detected at the inves- tigationin the tenocytes time point at the of late 24 phase h, but of 24only h but in only,response when to exposed the high to the concentration higher concen- of 100 ng/mL tration of 100 ng/mL C5a (Figure3A). Likewise, the transcriptional activity of the gene C5aencoding (Figure for 3B complement). In the early regulatory phase protein of C5a CD46 stimulation also showed (0.5 a significanth), a signi reductionficant increase in gene expressionat the investigation of complement time point regulatory of 24 h, but only proteins in response CD55 to and the highCD59 concentration in response of to the concen- tration100 ng/mL of 25 C5a ng (Figure/mL C5a3B). became In the early evident phase of(Figure C5a stimulation 3C,D), but (0.5 this h), aeffect significant was no longer de- tectableincrease inat genelater expression investigation of complement time points regulatory. proteins CD55 and CD59 in response to the concentration of 25 ng/mL C5a became evident (Figure3C,D), but this effect was no longerInterestingly, detectable at later the investigationgene expression time points. of CD59 decreased significantly at both later inves- tigationInterestingly, time points the gene (4 h expressionand 24 h) of, irrespectiv CD59 decreasede of significantlythe C5a concentrations at both later inves- (25 ng/mL or 100 ngtigation/mL) applied time points (Figure (4 h and 3D 24). h), irrespective of the C5a concentrations (25 ng/mL or 100 ng/mL) applied (Figure3D).

FigureFigure 3. 3. Tenocyte gene gene expression expression of C5aR of( AC5aR), CD46 (A(B),), CD46CD55 ((CB)) and, CD55CD59 (C(D)) and after CD59 0.5 h (n (=D 8),) after 0.5 h (n4 = h 8), (n =4 h 7), (n and = 7) 24, hand (n =24 6) h stimulation (n = 6) stimulation with 25 ng/mL with (light 25 ng blue)/mL and (light 100 blue) ng/mL and (dark 100 blue) ng/mL (dark recombinant C5a protein in comparison to the non-stimulated control groups (normalized to 1, blue) recombinant C5a protein in comparison to the non-stimulated control groups (normal- horizontal line). Mean values and the standard errors of mean are shown. Gene expression plotted ized to 1, horizontal line). Mean values and the standard errors of mean are shown. Gene ex- as relative changes with logarithmic scale of Y-axes. Rout outlier test Q = 1.0%. Shapiro Wilk and pressionKolmogorov–Smirnov plotted as relative normality changes tests were with performed logarithmic to prove scale the normal of Y-axe distributions. Rout ofoutlier data. Onetest Q = 1.0%. Shapirosample tWilk-test with and significance Kolmogorov in relation–Smirnov to control normality (*). * = p test≤ 0.05;s were ** = pperformed≤ 0.01. 6–8 independentto prove the normal distributionexperiments of with data human. One tenocytes sample derived t-test fromwith different significance donors in were relation performed. to control (*). * = p ≤ 0.05; ** = p ≤ 0.01. 6–8 independent experiments with human tenocytes derived from different donors were performed.The gene expression of the pro-inflammatory cytokine TNFα was significantly suppressed by 100 ng/mL C5a at the 0.5 h stimulation time (Figure4A). No further significant effect on TNFα gene expression was observed in later phases. IL-6 gene expression was The gene expression of the pro-inflammatory cytokine TNFα was significantly sup- not significantly affected by C5a stimulation (Figure4B). pressed by 100 ng/mL C5a at the 0.5 h stimulation time (Figure 4A). No further significant effect on TNFα gene expression was observed in later phases. IL-6 gene expression was not significantly affected by C5a stimulation (Figure 4B).

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Figure 4. Tenocyte gene expression of TNFα (A) and IL-6 (B) after 0.5 h, 4 h, and 24 h stimulation with 25 ng/mL (light blue) and 100 ng/mL (dark blue) recombinant C5a protein in comparison to Figurethe non 4.-stimulatedTenocyte gene control expression group ofs TNF(normalizedα (A) and toIL-6 1,( Bhorizontal) after 0.5 h,line 4 h,). andMean 24 values h stimulation and the Figure 4. Tenocyte gene expression of TNFα (A) and IL-6 (B) after 0.5 h, 4 h, and 24 h stimulation standardwith 25 error ng/mLs of (light mean blue) are andshown 100. ng/mL Control (darks have blue) been recombinant normalized C5a to protein 1. Gene in expression comparison plot- to with 25 ng/mL (light blue) and 100 ng/mL (dark blue) recombinant C5a protein in comparison tedthe as non-stimulatedrelative changes control with groups logarithmic (normalized scale of to Y 1,- horizontalaxes. Rout line). outlier Mean test values Q = 1.0%. and theShapiro standard Wilk to the non-stimulated control groups (normalized to 1, horizontal line). Mean values and the anderrors Kolmogorov of mean are–Smirnov shown. Controlsnormality have test beens were normalized performed to 1. to Gene prove expression the normal plotted distribution as relative of standard errors of mean are shown. Controls have been normalized to 1. Gene expression plot- data.changes One withsample logarithmic t-test with scale significance of Y-axes. Rout in relation outlier testto control Q = 1.0%. (*). Shapiro *** = p ≤ Wilk 0.001 and. n Kolmogorov–= 5 independ- ted as relative changes with logarithmic scale of Y-axes. Rout outlier test Q = 1.0%. Shapiro Wilk entSmirnov experiments normality with tests human were performedtenocytes from to prove different the normal donors distribution were performed. of data. One sample t-test andwith Kolmogorov signiﬁcance– inSmirnov relation tonormality control (*). test ***s = werep ≤ 0.001. performedn = 5 independent to prove the experiments normal distribution with human of data.tenocytes One sample from different t-test with donors significance were performed. in relation to control (*). *** = p ≤ 0.001. n = 5 independ- ent2.3. experimentsIntracellular with TNFα human Production tenocytes in Responsefrom different to C5a donors were performed. 2.3.An Intracellular intracellular TNFα concentrationProduction in Response of 4.19 to ± C5a2.78 pg TNFα protein per 1 µg total tenocyte protein2.3. IntracelAn was intracellularlular measured TNFα concentration Productionin the non in- ofstimulated Response 4.19 ± 2.78 to group pg C5a TNF atα 0.5protein h. No per significant 1 µg total tenocyte differences in theprotein TNFαAn wasintracellular protein measured concentrations concentration in the non-stimulated w ereof 4 .detected19 group± 2.78 at (pgFigure 0.5 TNFα h. No5) .protein signiﬁcantHowever, per differences a1 µgtrend total of in tenocyteprotein suppressionproteinthe TNF wasα protein couldmeasured concentrations be observed in the non in were- stimulatedthe detected early and group (Figure intermediate at5). 0.5 However, h. No pha significant as trendes of ofthe protein differences stimulation. in Thethesuppression TNFαdata in protein the could 24 be hconcentrations observedstimulation in the group w earlyere andshoweddetected intermediate no (Figure normal phases 5) .distribution However, of the stimulation. a; hencetrend, The ofthe protein inter- data in the 24 h stimulation group showed no normal distribution; hence, the interpretation pretationsuppression might could be speculativebe observed. in the early and intermediate phases of the stimulation. might be speculative. The data in the 24 h stimulation group showed no normal distribution; hence, the interpretation might be speculative.

Figure 5. ELISA analysis of intracellular TNFα after 0.5 h, 4 h, and 24 h stimulation with 25 ng/mL Figure(light blue)5. ELISA and 100analysis ng/mL of (dark intracellular blue) recombinant TNFα after C5a 0.5 protein h, 4 inh, comparisonand 24 h stimulation to the control with group 25 ng/mL (light(grey). blue) The and values 100 wereng/mL measured (dark blue) in pg recombinant per 1 µg of cellC5a lysateprotein total in comparison protein. Mean to valuesthe control and group (grey). The values were measured in pg per 1 µg of cell lysate total protein. Mean values and the Figurethe standard 5. ELISA errors analysis of mean of intracellular are shown. Controls TNFα after have 0.5 been h, 4 normalized h, and 24 toh stimulation 1. Rout outlier with test 25 ng/mL standard errors of mean are shown. Controls have been normalized to 1. Rout outlier test Q = 1.0%. (lightQ = 1.0%. blue) Shapiro and 100 Wilk ng/mL normality (dark blue) test was recombinant used to evaluate C5a protein the normal in comparison distribution to of the data. control One group Shapiro Wilk normality test was used to evaluate the normal distribution of data. One sample t-test (grey).sample Thet-test valueswas performed. were measured n = 3 independent in pg per 1 experiments µg of cell withlysate human total tenocytesprotein. Mean from different values and the was performed. n = 3 independent experiments with human tenocytes from different donors were standarddonors were error performed.s of mean are shown. Controls have been normalized to 1. Rout outlier test Q = 1.0%. performed. Shapiro Wilk normality test was used to evaluate the normal distribution of data. One sample t-test was performed. n = 3 independent experiments with human tenocytes from different donors were 2.4.performed. Immunolabeling of the Proteins C5aR and CD55 The protein expression levels of C5aR and CD55 were analyzed using immunostain- ing2.4. images Immunolabeling of these proteins.of the Proteins Despite C5aR not and reaching CD55 the level of significance, we could only observeThe a p trendrotein of expression a decrease level in boths of C5aR C5aR and and CD55 CD55 were protein analyzed expression using in immunostain- response to C5aing imagesstimulation of these compared proteins. to theDespite respective not reaching control thegroups level (Figure of significance,s 6 and 7). we C5aR could protein only observe a trend of a decrease in both C5aR and CD55 protein expression in response to C5a stimulation compared to the respective control groups (Figures 6 and 7). C5aR protein

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2.4. Immunolabeling of the Proteins C5aR and CD55 The protein expression levels of C5aR and CD55 were analyzed using immunostaining expressionimages of these of proteins.4 h stimulation Despite not with reaching 100 the ng level/mL of C5a significance, is not wenormal could onlyly distributed, and hence notobserve qualified a trend offor a decrease the interpretation in both C5aR and. Additional CD55 protein expressionF-actin staining in response demonstrated to the shape C5a stimulation compared to the respective control groups (Figures6 and7). C5aR protein andexpression morphology of 4 h stimulation of the with tenocytes 100 ng/mL to C5a better is not visualize normally distributed, the distribution and hence of the target protein C5aRnot qualified (Figure fors the 6 interpretation.and 7). Tenocytes Additional contained F-actin staining F-actin demonstrated filaments the shape as so-called stress fibers leadingand morphology to the offocal the tenocytes adhesion to better sites visualize of the thecells distribution with no of detectable the target protein difference evoked by the C5aR (Figures6 and7). Tenocytes contained F-actin filaments as so-called stress fibers treatmentleading to the with focal C5a. adhesion sites of the cells with no detectable difference evoked by the treatment with C5a.

FigureFigure 6.6.Tenocyte Tenocyt proteine protein expression expression of C5aR after of 4 andC5aR 24 hafter stimulation 4 and with 24 25 h ng/mL stimulation (light with 25 ng/mL (light blue) and 100 ng/mL (dark blue) recombinant C5a protein in comparison to the control group blue) and 100 ng/mL (dark blue) recombinant C5a protein in comparison to the control group (grey) (grey) (A). Fluorescence intensity indicating C5aR immunoreactivity was analyzed using ImageJ. (AMean). F valuesluorescence and the standard intensity errors indicating of mean areshown.Controls C5aR immunoreactivity have been normalized was to an 1. Routalyzed using ImageJ. Mean valuesoutlier testand Q the = 1.0%. standard Shapiro Wilk error normalitys of mean test was are used shown to test. Control the normals distributionhave been of normalized data. to 1. Rout outlier testOne Q sample = 1.0%.t-test Shapiro was performed Wilk in normality relation to control. test was (B) Representative used to test images the ofnormal tenocytes distribution of data. One sampleimmunolabeled t-test withwas C5aR performed speciﬁc antibodies. in relation Green to (Alexa control. 488) = ( C5aR,B) Representative blue (DAPI) = cell images nuclei, of tenocytes immuno- grey (Phalloidin Alexa 633) = actin cytoskeleton. Scale bar = 100 µm. (C) Isotype & negative controls labeledof the immunoﬂuorescence with C5aR specific staining. antibodies.n = 4 independent Green experiments (Alexa with488) human = C5aR, tenocytes blue from (DAPI) = cell nuclei, grey (Phalloidindifferent donors Alexa were performed.633) = actin cytoskeleton. Scale bar = 100 µm. (C) Isotype & negative controls of the immunofluorescence staining. n = 4 independent experiments with human tenocytes from different donors were performed.

Figure 7. Tenocyte protein expression of CD55 after 4 and 24 h stimulation with 25 ng/mL (light blue) and 100 ng/mL (dark blue) recombinant C5a protein in comparison to the control group (grey)

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expression of 4 h stimulation with 100 ng/mL C5a is not normally distributed, and hence not qualified for the interpretation. Additional F-actin staining demonstrated the shape and morphology of the tenocytes to better visualize the distribution of the target protein C5aR (Figures 6 and 7). Tenocytes contained F-actin filaments as so-called stress fibers leading to the focal adhesion sites of the cells with no detectable difference evoked by the treatment with C5a.

Figure 6. Tenocyte protein expression of C5aR after 4 and 24 h stimulation with 25 ng/mL (light blue) and 100 ng/mL (dark blue) recombinant C5a protein in comparison to the control group (grey) (A). Fluorescence intensity indicating C5aR immunoreactivity was analyzed using ImageJ. Mean values and the standard errors of mean are shown. Controls have been normalized to 1. Rout outlier test Q = 1.0%. Shapiro Wilk normality test was used to test the normal distribution of data. One sample t-test was performed in relation to control. (B) Representative images of tenocytes immunolabeled with C5aR specific antibodies. Green (Alexa 488) = C5aR, blue (DAPI) = cell nuclei, grey (Phalloidin Alexa 633) = actin cytoskeleton. Scale bar = 100 µm. (C) Isotype & negative controls of

Int. J. Mol. Sci. 2021, 22, 3105 the immunofluorescence staining. n = 4 independent experiments with7 of 13 human tenocytes from different donors were performed.

FigureFigure 7. 7.Tenocyte Tenocyte protein protein expression expression of CD55 after 4of and CD55 24 h stimulation after 4 and with 2524 ng/mL h stimulation (light with 25 ng/mL (light blue) and 100 ng/mL (dark blue) recombinant C5a protein in comparison to the control group (grey) blue)(A). Fluorescence and 100 ng intensity/mL indicating(dark blue) CD55 recombinant immunoreactivity C5a was analyzedprotein using in comparison ImageJ. Mean to the control group (grey) values and the standard errors of mean are shown. Controls have been normalized to 1. Rout outlier test Q = 1.0%. Shapiro Wilk normality test was used to test the normal distribution of data. One sample t-test was performed in relation to control. (B) Representative images of tenocytes immunolabeled with CD55 speciﬁc antibodies. Red (Cy3) = CD55, blue (DAPI) = cell nuclei, grey (Phalloidin Alexa 633) = actin cytoskeleton. Scale bar = 100 µm. (C) Isotype & negative controls of the immunoﬂuorescence staining. n = 4 independent experiments with tenocytes from different donors.

3. Discussion During the complement activation, the complement factors C3, C4, and C5 are split to generate the anaphylatoxins C3a, C4a, and C5a, respectively [11]. The aim of the study is to understand the effect of C5a, known as an inflammatory mediator, on human tenocytes with a particular focus on complement regulation and cytokines TNFα and IL-6. The results should help to hypothesize the putative role of C5a in tendinopathy. The C5 split fragment, C5a, is known to induce various inflammatory cell responses and has also been described as inducing apoptosis in adrenomedullary cells in an animal model of sepsis [15,22]. Additionally, a high concentration of 50 ng/mL recombinant C5a treatment of mouse kidney endothelial cells showed significantly higher rates of apoptosis than the lower concentrations or the control [23]. However, the vitality of human tenocytes exposed to concentrations of 25 ng/mL and 100 ng/mL C5a did not show significant changes; hence, cell vitality remained mainly unaffected. The selected concentration of 25 ng/mL C5a is between that used in the above-mentioned study [23] and the C5a amount measured in human plasma (8.34 ± 2.05 ng/mL) [16]. This displays the rather robust nature of the human tenocytes when exposed to these concentrations of anaphylatoxin C5a for the whole treatment time period. However, the tenocytes used in this study were derived from healthy tendons of young donors and one could hypothesize that cells from tendinopathic tendons might display a more sensitive response. The focus of our study is to understand the effect of C5a on tenocytes in regard to the inflammatory processes probably Int. J. Mol. Sci. 2021, 22, 3105 8 of 13

contributing to tendinopathy. The tenocytes react significantly in response to anaphylatoxin C5a with suppression of C5aR gene expression in the intermediate and late phases. No significant effects could be obtained in the early phase (Figure3A). The semi-quantitative evaluation of C5aR protein expression at 4 h displayed the gene expression pattern as a trend at a similar time point. The decrease of C5aR in gene and protein expression could suggest the self-regulation of the inflammatory effect in tenocytes representing a negative feedback loop. The CD46 gene expression of tenocytes was suppressed in a very late phase under 100 ng/mL C5a concentration (Figure3B). The suppression could also be observed after C3a stimulation in a previous study even in an earlier stage (i.e., in an intermediate phase) [18]. Under 25 ng/mL C5a stimulation, CD55 and CD59 gene expression of tenocytes was induced in a very early phase (Figure3C,D). This shows a possible self-protective capacity of the tenocytes in the early phase against the anaphylatoxin C5a. Interestingly, stimulation of tenocytes by another anaphylatoxin C3a more upstream localized in the complement cascade evoked a decrease in the CD55 gene expression in an intermediate phase [18]. Nonetheless, in our study, CD59 gene expression was reduced in both time periods and in response to both concentrations in the intermediate and the late phases (Figure3D). This displays an early response of tenocytes to C5a expressing the C5aR and CD59 genes, which probably recedes after 4 h to negative feedback. The pro-inflammatory cytokine TNFα is available at the site of inflammation, and blocking this agent is a clinical approach to treat various inflammatory diseases. It has been proven that tenocytes are also able to produce TNFα, and in response to TNFα, they react with an upregulation of the gene expression of pro-inflammatory (TNFα, IL-1β) and immunoregulatory (IL-6, IL-10) cytokines [20]. In addition, TNFα is known to be increased in the case of tendon pathology [4]. We know that C5aR gene expression is elevated in tenocytes in response to TNFα stimulation in an intermediate stage (4 h) of inflammation [10]. Conversely, in our present study, we could show that C5a suppresses the TNFα gene expression in these cells in an early phase of 0.5 h in comparison to the control group (Figure4A). This effect on TNF α could be determined in the intermediate phase of the stimulation (Figure5) at the protein level by ELISA analysis. In leukocytes, C5a stimulates the synthesis and release of pro-inflammatory cytokines such as TNFα, IL-1β, IL-6, etc. after 24 h [24,25]. However, C5a, as shown in a sepsis in vitro study, also decreased NFκB-dependent gene transcription of TNFα and impaired lipopolysaccharide-induced TNFα production in neutrophils [26]. The effect of TNFα suppression in tenocytes is a novel result. The suppression of both TNFα gene expression in the early phase, followed by a trend of impaired TNFα protein synthesis in the intermediate phase, reduced C5aR gene transcription in the intermediate and late phases, and early induction of CD55 and CD59 gene expression, could open the hypothesis that the tenocytes could have an anti- inflammatory self-regulatory mechanism against the inflammatory effect of anaphylatoxin C5a. TNFα alone can also trigger the IL-6 expression in the tenocytes and also a higher expression of IL-6 has been associated with tendinopathy [20,27]. Even though it is in our highest interest to understand the possible effect of anaphylatoxin C5a on the IL-6 synthesis focusing on tenocytes, in our study, no clear effect regarding IL-6 gene expression in tenocytes in response to this anaphylatoxin was detected (Figure4B). A previous study suggested that the immunoregulatory cytokine IL-6 is induced through TNFα in tenocytes, whereas the present study could not prove any regulation through C5a in the tenocytes [20]. However, one limitation of this study is that the number of independent experiments performed with different cell donors involved varied at the gene and protein expression analysis, which might be responsible for non-significant results at the protein level. The treatment of human tenocytes derived from non-inflamed hamstring tendons with anaphylatoxin C5a does not necessarily replicate typical micro-environmental conditions of tendinopathy. Interestingly, tenocytes exposed to C5a showed some anti-inflammatory responses, such as the early suppression of TNFα gene expression. Hence, future research of anaphylatoxin effects on tenocytes should include the presence of pro- or anti-inflammatory Int. J. Mol. Sci. 2021, 22, 3105 9 of 13

cytokines and cells from healthy and affected tendons of different disease stages to allow an even better understanding of the inﬂammatory aspects of tendinopathy.

4. Materials and Methods 4.1. Isolation of the Tenocytes and Tenocyte Culturing Tenocytes derived from human hamstring tendons collected from residual tissue after anterior cruciate ligament replacement surgeries were used in this project. The donors were exclusively males between 18 and 53 years old with an average age of 31.1 years old. Their use was approved by the Charité review board (Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany [Ethical approval number EA4-033-08]). Be- fore cutting the tissue into small pieces (1–4 mm), the paratenon of this tissue was carefully removed. The tissue was cultured in T25 culture ﬂasks (Sarstedt, Hildesheim, Germany) ◦ with a growth medium at 37 C and 5% CO2. The growth medium for tenocyte culturing consisted of Dulbecco’s MEM/Ham’s F-12 (1:1 mixture) (Merck KGaA, Darmstadt, Ger- many), 25 mg/mL ascorbic acid, 50 IU/mL streptomycin, 50 IU/mL penicillin, 2.5 µg/mL amphotericin B, and essential amino acids [all: Merck KGaA,]. A total of 10% fetal calf serum (FCS) was added to the growth medium for cell expansion purposes. The tenocytes emigrated from the explants after circa 7–10 days. To detach the cells from the ﬂask for expansion, 0.05% trypsin/1.0 mM ethylenediaminetetraacetic acid (EDTA) (Merck KGaA) was used. Cells at passages (P)4–7 were used for the experiments.

4.2. Stimulation of Tenocytes with Anaphylatoxin C5a Overall, 10,000 tenocytes/cm2 were cultivated for 24 h in 10% FCS containing growth medium and subsequently serum-starved for 1 h in starvation medium containing only 1% FCS before the stimulation experiment was performed. Simultaneously the C5a containing stimulation medium was prepared using 25 ng/mL and 100 ng/mL recombinant human C5a protein (Catalog Number: 2037-C5, R&D Systems, Minneapolis, MN, USA) in the starvation medium. Non-stimulated tenocytes were the control group (Figure2A).

4.3. Vitality Test of the Tenocytes After stimulation of the tenocytes cultivated on the poly-L-lysine (Merck KGaA) coated coverslips, the vitality staining of tenocytes was performed. The tenocytes were incubated in a mixture of phosphate-buffered saline (PBS) with fluorescein diacetate (final concentration = 15 µg/mL) and propidium iodide (final concentration = 1 µg/mL) (Thermo Fisher Scientific, Rockford, IL, USA). The green fluorescence representing the vital cells or red fluorescence indicating dead cells was visualized using a confocal laser scanning microscope (Leica TCS SPEII and DMi8, Wetzlar, Germany). The images of the tenocytes were analyzed using image processing software (ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA). Here, the area covered by living cells was calculated in relation to the total area colonized by cells (100%). Three independent experiments were included with three microscopic fields for each stimulation.

4.4. Measurement of TNFα Release by ELISA Human tenocyte monolayers were washed twice with PBS after completion of the experiment and were incubated with 300 µL lysis buffer (RIPA buffer, Thermo Fisher Scientiﬁc) supplemented with protease inhibitor cocktail tablets (Roche, Indianapolis, IN, USA) for 15 min on ice. The cells were additionally, mechanically lysed using cell scrapers. The lysed cells were diluted with PBS attaining a ﬁnal volume of 1 mL. The cell lysates were centrifuged for 15 min at 14,000× g. The acquired supernatants were transferred to another microcentrifuge tube and stored at −80 ◦C. The remaining cell debris was discarded. Roti®-Nanoquant kit (Carl Roth GmbH&Co.KG, Karlsruhe, Germany) was used to determine the protein concentration following the manufacturer’s protocol. Overall, 5 µg of the total protein was used in each sample to perform the ELISA assay. Int. J. Mol. Sci. 2021, 22, 3105 10 of 13

The Human TNFα Uncoated ELISA kit (Catalog Number 88-7346, Thermo Fisher Scientific) was used for the ELISA measurements. The 96-well plate provided with the kit was first coated with capture antibody and incubated at 4 ◦C overnight. On the next day, washing steps were performed with PBS containing 0.05 g/L Tween® 20 (Sigma-Aldrich Chemie GmbH, Munich, Germany). All of the following steps in the assay were executed according to the manufacturer’s instructions. Ultimately, 50 µL of 1 M H3PO4 (Sigma- Aldrich) into the wells terminated the reaction and the absorbance was measured at 450 nm using an ELISA plate reader (Infinite M200 Pro, Tecan, Männedorf, Switzerland).

4.5. Gene Expression Analysis 4.5.1. RNA Isolation and cDNA Synthesis After each stimulation time of 0.5 h, 4 h, and 24 h, cell culture supernatants were removed and the tenocytes were rinsed with PBS (Merck KGaA). After washing, the cells were lysed RNeasy lysis buffer (Qiagen GmbH, Hilden, Germany) containing one-hundredth of β-mercaptoethanol (Sigma-Aldrich Chemie GmbH). RNeasy mini kit (Qiagen GmbH) was used to extract the total RNA using the manufacturer’s instructions. RNA quantity and purity were subsequently analyzed using RNA 6000 Nano assay (Agilent Technolo- gies, Santa Clara, CA, USA). cDNA synthesis was performed with the QuantiTect Reverse Transcription Kit (Qiagen GmbH) in a Mastercycler (Eppendorf AG, Hamburg, Germany).

4.5.2. qPCR Real-time detection polymerase chain reaction (qPCR) analyses were performed in 20 µL reaction volume applying the derived cDNA. Specific primers for genes encoding complement components C5aR, CD46, CD55, CD59, and pro-inflammatory cytokines TNFα and IL-6 (Table1, Applied Biosystems (ABI), Foster City, USA) were used as genes of interest and the reference gene was hypoxanthine phosphoribosyl transferase (HPRT)-1 (Table1). HPRT1 served as the most stable reference gene tested before by comparing it with other reference genes including β-actin, TATA-binding protein, or glyceraldehyde 3-phosphate dehydrogenase. Together with TaqMan Gene Expression Master Mix solution (Thermo Fisher Scientific, Carlsbad, CA, USA), RNAse free water, respective primer, and 20 ng pro well aliquot of the derived cDNA were mixed to perform the TaqMan Gene Expression Assay. The assays were carried out in Real-Time-Cyclers (Opticon I, Bio-Rad Laboratories Inc., Hercules, CA, USA and Chromo4, Bio-Rad Laboratories, Hercules, CA, USA). As per the manufacturer´s instructions, the TaqMan analyses protocols describe the process as follows: the reaction mixture was heated at 50 ◦C for 2 min, the temperature rose to 95 ◦C for 10 min followed by the second stage. This stage runs in 40 cycles with 95 ◦C for 15 s to denaturate the DNA template and an annealing/ extending step at 60 ◦C for 1 min. Relative gene expression levels were normalized versus the reference gene. Finally, the calculation was performed using the 2-deltaCT method [28].

Table 1. Oligonucleotides used for qPCR analysis.

Primer Company Sequence Probe/Reference bp HPRT1 ABI * NM_000194.2 100 C5aR ABI * NM_001736.3 68 CD46 ABI * NM_172351.1 94 CD55 ABI * NM_000574.2 62 CD59 ABI * NM_203331.1 70 TNFα ABI * NM_000594.3 80 IL6 ABI * NM_000600.4 95 *: Sequence not provided by the company (ABI).

4.6. Immunoﬂuorescence Labeling of the Proteins C5aR and CD55 After 4 h and 24 h stimulation of the human tenocytes as described in Section 2.2, the cells were ﬁxated in 4% paraformaldehyde (PFA, Morphisto, Frankfurt am Main, Germany). Int. J. Mol. Sci. 2021, 22, 3105 11 of 13

After rinsing the cells in Tris-buffered saline (TBS) they were incubated in blocking buffer solution (5% protease-free donkey serum diluted in TBS with 0.1% Triton ×100) at room temperature (RT) for 20 min. The coverslips were rinsed thoroughly and incubated with the primary antibodies directed against C5aR1 and CD55 (for detailed information of antibodies and dyes, see Table2) in a humidifier chamber overnight at 4 ◦C. Once again, coverslips were washed thoroughly with TBS before incubation with donkey anti-mouse Alexa Fluor 488 and donkey-anti-goat Cyanine (Cy)3 (Table2), coupled with secondary antibodies for 60 min at RT. Cell nuclei were counterstained using 40,6-diamidino-2-phenylindole (DAPI) and the cytoskeleton with Phalloidin Alexa Fluor 633 (Table2). Negative controls were performed omitting the primary antibody during the staining procedure. Isotype controls were also performed using goat IgG isotype antibody and mouse IgG1 isotype antibody (Table2) as primary antibodies. Coverslips were washed three times with TBS before being covered with Fluoromount G (Southern Biotech, Birmingham, AL, USA). The images of cells with immunolabeled proteins (three microscopical fields) were subsequently prepared using confocal laser scanning microscopy (Leica). The cell fluorescence intensity of each immunolabeled protein was then measured by an image processing software (ImageJ) [29]. For a comparative analysis of cellular protein synthesis, corrected total cell fluorescence (CTCF) values were measured through which the overlapping background readings over the total cell covered area are deduced from the measured integrated fluorescence density [30,31]. CTCF = integrated fluorescence density—(area of selected cells X mean fluorescence of background readings).

Table 2. Antibodies and dyes used.

Specificity and Species Company Cat. No. Stock Concentration Used Dilution R&D systems, Goat anti-human CD55 (DAF) AF2009 200 µg/mL 1:20 Minneapolis, MN, USA GeneTex, Biozol, Mouse anti-human C5aR1 (CD88) GTX74845 1 mg/mL 1:20 Eching, Germany Thermo Fisher Scientific, Donkey anti-mouse-Alexa Fluor 488 A21202 2 mg/mL 1:200 Rockford, IL, USA Jackson Immuno Research, Donkey anti-goat-Cy3 705165147 1.5 mg/mL 1:200 Cambridgeshire, UK Thermo Fisher Scientific, Goat IgG isotype 02-6202 5 mg/mL 1:100 Rockford, IL, USA Thermo Fisher Scientific, Mouse IgG1 isotype 02-6102 5 mg/mL 1:100 Rockford, IL, USA Thermo Fisher Scientific, Phalloidin Alexa Fluor 633 A22284 300 µg/mL 1:1000 Rockford, IL, USA Roche Diagnostics GmbH, 40,6-diamidino-2-phenylindole (DAPI) 10236276001 1 µg/mL 1:100 Basel, Switzerland

4.7. Statistical Analysis GraphPad Prism (Version 8.1.4), GraphPad Software, San Diego, CA, USA was used for the statistical analysis. The normalized data were expressed as the mean and the standard error of mean (mean ± SEM). Shapiro Wilk normality test and/or Kolmogorov– Smirnov test normality tests were performed. The differences between experimental groups were considered signiﬁcant at p < 0.05 as determined by one sampled t-test. * = p ≤ 0.05; ** = p ≤ 0.01. The Rout test was performed to identify the outliers. The outliers were excluded from the statistics. Int. J. Mol. Sci. 2021, 22, 3105 12 of 13

5. Conclusions The presence of anaphylatoxin C5a can influence the expression of complement proteins and TNFα in tenocytes. Gene expression of the respective anaphylatoxin receptor C5aR was significantly reduced in 4 h (intermediate) and 24 h (late phase) and that of TNFα at 0.5 h (early phase). Moreover, early induction of CD55 and CD59 gene expression was found in response to the 25 ng/mL concentration of the anaphylatoxin C5a. All these data suggest an anti-inflammatory self-regulatory mechanism of the tenocytes against the inflammatory effect of anaphylatoxin C5a. However, lowered CRPs CD46 and CD59 gene expression in intermediate and late phases imply rather an inflammatory response by the tenocytes; hence, revealing a time and concentration-dependent reaction of the tenocytes to C5a. This study performed under non-inflammatory pre-conditions and void of additional external stressors allows for an understanding of the complement regulation and TNFα-related response of tenocytes directly exposed to this anaphylatoxin.

Author Contributions: Conceptualization, G.S.-T. and S.S.; methodology, S.S., G.S.-T. and B.K.; formal analysis, G.S.-T., B.K. and J.S.; investigation, S.S., B.K. and J.S.; resources, G.S.-T.; data curation, S.S., B.K.; writing—original draft preparation, S.S.; writing—review and editing, G.S.-T., B.K. and J.S.; visualization, S.S.; supervision, G.S.-T.; project administration, S.S., G.S.-T. and B.K. All authors have read and agreed to the published version of the manuscript. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by Charité Review Board (Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany [Ethical approval number EA4-033-08]). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Acknowledgments: We thank Marion Lemke and Sanjog Silawal for their technical assistance. We are very grateful for the support of Senat Krasnici and Thilo John for sample acquisition. We would also like to thank Nicola Daykin for proofreading this manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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