Neutrophilextrace llular traps in the intestinalm ucosa of Eim eria-infected anim als

Tamara Muñoz-Caro,Liliana Machado Ribeiro da Silva,Zaída Rentería-Solis,Anja Taubert,Carlos HermosillaInstitute of Parasitology,Justus Liebig University Giessen,Giessen,Germany



Neutrophilextrace llular traps in the intestinalm ucosa of Eim eria-infected anim als

Tamara Muñoz-Caro#,Liliana Machado Ribeiro da Silva#,Zaída Rentería-Solis,Anja Taubert,
Carlos Hermosilla*
Institute of Parasitology,Justus Liebig University Giessen,Giessen,Germany

Original article http://dx.doi.org/10.1016/j.apjtb.2016.01.001

ARTICLE INFO

Article history:

Rec

eived in revised form 27Nov, 2nd revised form 30 Nov 2015

Accepted 28 Dec 2015

Availableonline8 Jan 2016

Eimeria bovis

Eimeria arloingi

Neutrophil extracellular traps

Apicomplexa

Coccidiosis

ABSTRACT

Ob jective:To investigate the presence of neutrophil extracellular traps(NETs)in vivo by analysing intestinalsections from experimentally Eimeria bovis-and naturally Eimeria arloingi-infected animals.

M ethods:Intestinal samples of Eimeria arloingi-and Eimeria bovis-infected animals were analysed by using immunohistochem ical and fl uorescence approach by using monoclonal antibodies.

Resu lts:Classical NET components were con fi rmed by co-localization of extracellular DNA being decorated w ith neutrophil elastase and histones in Eimeria-infected tissue samples.Here,extrusion of NETs was exclusively detected in intestinal polymorphonuclear neutrophils in fi ltrating Eimeria-infected sites.In vivo NETs were either found in close proxim ity or in direct contact to different Eimeria stages suggesting a stage-independent process.NETs were also found w ithin the gut lumen driven by polymorphonuclear neutrophils thatwere contacting released oocysts.

Conclusions:We postulate that NETsm ight play an important role in innate defence reactions in coccidiosis therefore signi fi cantly altering the outcome of infection.

1.Introduction

Coccidiosis isa protozoan disease caused by differentspecies of the genus Eimeria which causes considerable animal health problems and econom ic losses in the ruminant industry worldw ide due to severe clinical enteritis and/or typhlocolitis[1–7]. Ruminant Eimeria infections w ith pathogenic species,such as Eimeria bovis(E.bovis)in cattle or Eimeria arloingi (E.arloingi)in goats,commonly induce clinical disease only in young animals,since homologous reinfections generally areunder immunological control[8].However,relatively little is known on early host innate immune reactions against Eimeria infections contributing to protection of animals through the interaction w ith cells of the cellular adaptive immune response [9–11].In this context,polymorphonuclear neutrophils(PMN) play a key role since they are themost abundant cells in the blood and the fi rst ones to be recruited to the site of infection [12–14].PMN own several effectormechanisms to combat and eventually kill pathogens,such as phagocytosis,reactive oxygen species production,the release of antim icrobial peptides/proteins and the formation of neutrophil extracellular traps(NETs)[12,14,15].NETs are generally released after PMN cell death and are primarily situated in the extracellular space [16].The formation of NETs(NETosis)is a NADPH oxidase (NOX)-dependent mechanism[15,17–22],which leads to the extrusion of a m ixture of nuclear and cytoplasm ic granule contents leading to the formation of DNA-rich web-like structuresbeing decorated w ith histones(H1,H2A/H2B,H3,H4)and granular effector molecules,such as neutrophil elastase(NE), lactoferrin,pentraxin,myeloperoxidase(MPO)and others [14,16,19].Unlike NOX-dependent NETosis,NOX-independent NETosis is accompanied by a substantially lower level of ERK activation and rather moderate level of Akt activation,whereas the activation of p38 is sim ilar in both pathways[23]. Irrespective of NOX-dependency,pathogens may either be immobilized w ithin sticky DNA fi bres or be killed via the local high concentration of effector molecules.Interestingly,Yipp etal.recently demonstrated that PMN,which undergo NETosis w ithout cell lysis are still viable and retain their ability to phagocytisebacteria[24].In agreementw ith these fi ndings,PMN also seem to be able to release NETs of m itochondrial origin which are of smaller size than the ones originating from classical NETosis[25].So far,NET formation was described to be induced by different protozoan parasites in vitro,such as Plasmodium falciparum[26],Leishmania spp.[27],E.bovis [22,28],Toxoplasma gondii(T.gondii)[29–31],E.arloingi[5], Besnoitia besnoiti(B.besnoiti)[20],and Cryptosporidium parvum[32].In addition,monocyte-derived extracellular traps (ETs)have recently been reported to be formed in response to tachyzoites of B.besnoiti and T.gondii in vitro[29–31].Recent analyses on Eimeria-induced NETosis con fi rmed its dependency on NOX,NE and MPO activities[5,22,28].M ore detailed investigations on molecular mechanisms of E.bovistriggered NETosis have demonstrated that this cell death pathway is CD11b-,ERK1/2-,p38-,m itogen-activated protein kinase-and Ca++-dependent[22].

There isa vastamountof data on the in vivo role of NETs in various bacterial infections[12,33],in metabolic[34,35], reproductive[36,37]and autoimmune disorders[38–40],and in cancer progression[41,42].However,in vivo data on NETs regarding parasitic diseases are scarce.The fi rst evidence of parasite-induced NETs in vivo came from Plasmodium falciparum-infected children[26].Detailed analyses of cutaneous Leishmania lesions from human patients in Brazil also proved the in vivo existence of Leishmania-triggered NETs as demonstrated by the simultaneous presence of extracellular DNA and histones[27].Abi Abdallah et al.provided fi rst indications on the in vivo relevance of NETs against T.gondii in amurinemodel of infection[30].

The aim of the currentstudywas to show in vivo evidence on NETosis in response to Eimeria infections.Typical NET structures were found in gut tissue sections of both E.bovis-and E.arloingi-infected animals indicating that this effectormechanism naturally occurs during primary Eimeria infections. However,the actual ef fi cacy of this effectormechanism in vivo remains to be elucidated in Eimeria-infected animals.

2.M aterials and m ethods

2.1.Intestinal samples of E.arloingi-and E.bovis infected animals

A two-month-old Serpentina goat kid of the province of A lentejo,Portugal,which died due to a severe natural E.arloingi infection served asdonor for intestinalsamples[5].In the case of E.bovis,intestinal gut samples originating from experimentally E.bovis-(strain H)infected calves,which were published before[8],were used.Caprine and bovine intestinal gut samples(jejunum,ileum,caecum,colon)were w ithdrawn for immediate fi xation[4%formaldehyde in phosphatebuffered saline(PBS),24 h]and embedded in paraf fi n according to procedures described by S¨uhwold etal.[8].Then 3–5μm cross-sections of formalin-fi xed tissues were deparaf fi nized according to standard histological procedures.Thereafter,the samples were exposed to descendant concentrations of isopropanol(90%,80%,70%,and 50%,3 m in each)and rehydrated in distilled water(3 m in).The samples were incubated in haematoxylin solution(Sigma–A ldrich)for 90 s,then washed 5 times in bi-distilled water and placed for 5m in in tap water.A fterwards,the sampleswerewashed in bi-distilled water again,stained w ith eosin staining solution(Sigma–A ldrich,30 s) and washed again tw ice in bi-distilled water.Finally,the samples were dehydrated in ascending isopropanol concentrations (70%,80%and 90%,30 s each),incubated tw ice in isopropanol (100%,2 m in)and tw ice in xylol(100%,2 m in).Finally,all samples were mounted w ith Pertex™(Leica Biosystems)for further investigations.

2.2.Immunohistochemical detection of NETs

For the immunohistochem ical detection of NETs,paraf fi nfi xed sections were deparaf fi nized as previously described.For antigen-demasking,a heating treatmentwas performed.Therefore,slides were cooked in a steamer in 10 mmol/L Tris base (Sigma–Aldrich)and 1 mmol/L ethylene diam ine tetraacetic acid solution(pH 9.0)(Sigma–A ldrich),for 15m in for caprine samples and 30min forbovine samples.Thereafter,the samples were allowed to cool down for 20m in at room temperature and then washed thrice in PBS for 2 m in.To inhibit endogenous peroxidase activity the sections were exposed to 1%H2O2(Sigma–Aldrich,30m in,room temperature),then washed thrice in PBS(2 m in).Unspeci fi c protein binding was excluded by treatment w ith 1%bovine serum album in(BSA)(Sigma–A ldrich)and 0.1%sodium azide in PBS(Sigma–A ldrich)for 30 m in at room temperature.A fterwards,the samples were incubated in primary antibody solution[anti-histone H3(D1H2) XP®rabbitmonoclonal antibody,No.4499(Cell Signaling); overnight,4°C,1:100 dilution in blocking solution].The samples were washed thrice in PBS and exposed to the secondary antibody[goat anti-rabbit immunoglobulin G(H+L) secondary antibody,horseradish peroxidase conjugate(Life Technologies);1:50 in PBS,1 h,room temperature].For signal development the samples were exposed to 3.3′-diam inobenzidine(Sigma–A ldrich,125μg/m L,10 m in,room temperature)and then washed thricew ith PBS.Counterstaining was performed in haematoxylin staining solution(Sigma–A ldrich,1:5 in distilled water,90 s).Thereafter the sampleswere washed(5 m in,distilled water)and dehydrated in ascending isopropanol concentrations(50%,70%,80%and 90%,30 m in each),isopropanol 100%(2×2 m in)and xylene(100%, 2×2 m in).The samples were mounted in Pertex™(Leica Biosystems).In order to test for unspeci fi c NET formation a rat ileum tissue section was equally processed in parallel.Visualization was achieved and documented by using an inverted Olympus BX51®m icroscopeequippedw ith a digital cameraand an analySIS®software(Olympus).

2.3.Fluorescence-based detection of NETs

Fluorescence-based detection of NETs was performed according to von Ko¨ckritz-Blickwede et al.w ith some slight modi fi cations[17].Brie fl y,the samples were deparaf fi nized in xylene(Fisher Scienti fi c,3×10 m in),100%alcohol(Fisher Scienti fi c,2×5 m in),95%alcohol(2×5 m in)and 70% alcohol(2×5 m in).Thereafter,the sampleswere washed w ithPBS(3×10 dips)and heated in a m icrowave(2×5 m in in citrate buffer,pH 6.0,Dako,S2369).A fterwards,the samples were cooled for 20 min at room temperature,washed thrice w ith PBS and blocked w ith 2%BSA–PBS+foetal calf serum (Sigma–A ldrich,45 m in,room temperature).The samples were then exposed to primary antibody solution(rabbit antihuman NE,1:500;AB68672,Abcam,3 h,4°C,hum idity chamber,2%BSA–PBS).To avoid drying-out,the cross sections were covered w ith para fi lm.Then the samples were washed four times w ith PBS and incubated in secondary antibody solution(Invitrogen,A lexa Fluor®488 conjugated goat anti-rabbit antibodies,1:500,30 m in,room temperature,hum idity chamber,covered w ith para fi lm).A fter four washings w ith PBS,the samples were mounted either in ProlongGold®w ith 4′,6-diam idino-2-phenylindole(DAPI)staining or in ProlongGold®after staining w ith Sytox Orange®(Invitrogen, 1:1000,5m in,room temperature,in the dark).The visualization of extracellular DNA and NE-positive signals was achieved using an inverted Olympus IX81®fl uorescencem icroscope.

3.Resu lts

Haematoxylin-eosin-stained sections of E.bovis-and E.arloingi-infected intestinal tissue samples showed a strong leukocytic mucosal in fi ltration,mainly composed of PMN, monocytes and eosinophils,into parasitised areas of the jejunum,ileum and caecum/colon.Some mucosal leucocytes were found in direct contact w ith the surface of infected host cells carrying different Eimeria stages such as oocysts (Figures 1A and 2C),macrogamonts(Figures 1B,C and 2A,B) and also at the periphery of developing macromeronts (Figure2D).These features demonstrate that these immune cells are capable to effectively transm igrate into affected intestinal mucosa in vivo.Accordingly,thehistopathology ofboth Eimeria infections exhibited a dramatic damage due to a high parasitic load alongside w ith a striking epithelial destruction and detachment(dysentery).PMN were even found w ithin the intestinal lumen in close contact w ith extracellular E.bovis oocysts(Figure 1D).

Figure 1.Haematoxylin-eosin staining of E.bovis-infected intestinal tissue.A:Intestinal leucocyte contacting E.bovis oocysts(arrow).B and C:Intestinal leucocyte contacting E.bovis macrogamonts(arrow s).D:Intestinal leucocyte contacting an E.bovis oocyst in lumen(arrow).Oo:Oocysts;Ma: Macrogamonts.Scale bars=20μm.

Figure 2.Haematoxylin-eosin staining of E.arloingi-infected intestinal tissue.A and B:Leucocyte in fi ltration contacting E.arloingi macrogamontstages (arrows);C:Leucocyte in fi ltration contacting E.arloingi oocysts stages (arrows);D:E.arloingi macromeront being surrounded by leucocyte in fi ltration(arrow s).Mm:Macromeront;Oo:Oocysts;M a:M acrogamonts. Scale bars=20μm.

Figure3.Co-localization of extracellular DNA and histones in E.arloingitriggered NET structures in infected intestinal tissue.Intestinal tissue(jejunum)sections from E.arloingi-infected animals were used for immuno fl uorescence analysis in order to identify NETs by (monoclonal)antibody-based detection ofhistones(H1,H2A,H2B,H3 and H4,in green).DNA was stainedw ith Sytox orange(in red).A:Anti-histone staining of H1,H2A,H2B,H3 and H4;B:Sytox orange staining of DNA; C:Overlay of A and B.W hite arrow s indicate NET structures being extruded from PMN.Scale bars=20μm.

In addition,the co-localization ofmucosalextracellular DNA w ith histones(H1,H2A,H2B,H3,H4)(Figure 3)and NE (Figure 4)in Eimeria-induced NETs corroborated the classical characteristics of NETs in vivo.Furthermore,sections from the jejunum revealed a strong in fl ux of PMN into Eimeria-infected areas with some of them releasing NETs as seen by the colocalization of H3 and extracellular nucleic acids derived from dead PMN(Figure 5),making this feature distinguishable from non-NET-releasing PMN which retain their typical cellular morphology.According to this,a recent study supports the use of immunostaining w ith citrullinated histone-3 antibodies to identify NETs in tissue sections show ing that nuclear NETs extensions display orientations in different planes,in contrast to the ones observed in nuclear crush smears[43].In both Eimeria species infections,single PMN were found releasing H3-positive NET structures in close proxim ity to Eimeria stages(Figure6B). Diffused aswell as small NET typeswere extruded by caprine PMN in fi ltrating mucosal areas of E.arloingi replication. Overall,in vivo NET-associated results clearly con fi rm previousin vitro data on E.bovis-[10–22]and E.arloingi-triggered NET release[5],and their role as novel effectormechanism against these apicomplexan parasites.

Figure 4.Co-localization of extracellular DNA and NE in E.arloingitriggered NET structures in infected intestinal tissue.Intestinal tissue(jejunum)sections from E.arloingi infected animals were used for immuno fl uorescence analysis in order to identify NETs by (polyclonal)antibody-based detection ofNE(in green)in combinationw ith DAPI staining(in blue)to identify nuclear and extracellular DNA.A: DAPI-stained DNA;B:NE staining;C:Overlay of A and B.W hite arrow s indicate NET structures.Scale bars=20μm.

Figure 5.Histone detection in E.bovis-(A)and E.arloingi-infected(B) intestinal samp les.NETs were identi fi ed by combining haematoxylin staining(in blue)w ith the(monoclonal)antibody-based detection ofhistone H3[Cell Signaling,1:100(in brown)].Red arrows indicate PMN releasing NET structures;yellow arrow shows inactive PMN.Scale bars=20μm.

Figure 6.Histone detection in E.arloingi-infected intestinal samples.A:PMN contacting E.arloingi macrogamont(arrow);B:PMN releasing NETs in close proxim ity to macrogamonts of E.arloingi(arrow).NETs were identi fi ed by combining haematoxylin staining(in blue)w ith the (monoclonal)antibody-based detection of histone H3[Cell Signaling, 1:100(in brown)].Ma:Macromeront.Scale bars=20μm.

4.Discussion

Early innate leucocyte-mediated reactionsagainstbovine and caprine Eimeria parasites have scarcely been investigated in the past,although the fi rst encounter between parasites and innate immune cells should be decisive for the subsequentoutcome of infection[5,22].PMN appear to play a pivotal role in rum inant Eimeria-triggered early host innate defence in vivo since this leucocyte population was identi fi ed in parasitized intestine of E.bovis-[44],Eimeria ninakohlyakimovae-[45]and E.arloingi-infected animals[5].Detailed molecular investigations have revealed that PMN do not only interact directly w ith viable E.bovis stages and antigens,but also serve as an early source of immunomodulatory molecules, such as chemokine(C–C motif)ligand 3 and tumour necrosis factorα[10],which supportmonocyte/macrophage in fi ltration and activation[46].PMN were also shown to adhere to E.bovis-infected endothelium under physiological fl ow conditions[47],and their phagocytic and oxidative burst activities were found enhanced in response to sporozoites of E.bovis in vitro and ex vivo[10].Furthermore,E.bovis-and E.arloingi-triggered NETosiswas reported as additional PMN effectormechanism in vitro[5,22,28].

The current in vivo data indicate NETosis as a generally occurring effector mechanism against Eimeria parasites.Colocalization studies on intestinal extracellular DNA being decorated w ith both histones and NE con fi rmed the presence of NETs in Eimeria-infected mucosa.Here,different patterns of NETswereobserved as particularly seen in NE-positive staining which showed rather diffuse than spread form of NETs.Differential types of NETs have already been described in Haemonchus contortus-triggered NETosis[48].Interestingly,in vivo NET release occurred irrespective of the Eimeria species and was also independent of the parasitic stages,i.e.merozoites I and oocysts,as previously demonstrated elsewhere[5,22].In agreement,different E.bovis and E.arloingi stages(i.e. sporozoites,merozoites I and oocysts)were previously identi fi ed as potent NET inducers in vitro[5,22,28].Moreover, it was demonstrated that Eimeria-induced NETosis is neither stage-,species-nor host-speci fi c process[22].The evidence of NET release in vivo in close proxim ity to parasitized areas containing intracellular Eimeria stages as well as NETs sticking to extracellular oocysts in the lumen of the intestine suggest NETosis as early host effector mechanism as previously postulated elsewhere[5,32].

Sim ilar in vivo NET-related studies have been performed in otherapicomplexan parasitessuch as T.gondii[49].Here,in vivo NETswere con fi rmed by using immunohistochem istry analysis in T.gondii-infectedm ice lung tissue observing theextracellular DNA release co-localized w ith MPO molecules[30]. Nonetheless,in this former in vivo murine study neither direct contact of T.gondii-tachyzoites w ith NETs nor NETsentrapped parasites were demonstrated[30].These in vivo results coincide well w ith our fi ndings where hardly any parasites were found entrapped by NETs.Taking into account that in vivo immunohistochemistry NET-related analyses of rum inant Eimeria-infected gut tissue sections m ight be a disadvantage due to the large size of the animals,itm ight beeasier in the future to obtain evidence of parasites entrapped in NETs in Eimeria-infected rodentmodels,such as Eimeria falciformis or Eimeria vermiformis[50].

Referring to oocyst-induced NETosis,itappears noteworthy thatin the caseof E.arloingi oocysts(which areequipped w ith a m icropyle),a blockage of sporozoite release by NETs was postulated[5].In agreement,oocyst-induced NETosis was also reported for Cryptosporidium parvum where these stages were almost completely covered by NET structures[32].Besides the interference w ith the E.arloingi excystation process,NETs were also released towards unsporulated oocysts in the gut lumen.However,so far it remains to be elucidated whether they are affected or even destroyed by the local high concentrations of antim icrobial peptides/proteases,such as NE, MPO,pentraxin,lactoferrin and gelatinase[14].

Notonly PMN butalso eosinophilsandmonocyteshavebeen reported to play a crucial role in E.bovis-,Eimeria ninakohlyakimovae-and E.arloingi-induced coccidiosis[5,44–46]. Interestingly,ETs have recently also been reported to be released by other immune cells than PMN[29].Thus,ETs can also be generated by macrophages[51,52],eosinophils[53,54], mast cells[55,56],basophils[57,58]and monocytes[21,31]. Independent of the leucocyte type,all ETs contain a vast amount of potent antimicrobial components and thus are able to interact w ith trapped pathogens[39].Referring to parasitedriven formation of ETs,monocyte-derived ETs have recently been reported to be formed after exposure to B.besnoiti and T.gondii tachyzoites leading to parasite entrapment[21,31].It is noteworthy that Taubert etal.also reported enhancedmonocytic activities throughout experimental E.bovis infection although the detection of formation of ETswas not part of the study[46].

Regarding potential detrimental effects of NETs on Eimeria sp.,extra-and intra-cellular stages have to be considered differently.Extracellular stages of Eimeria sp.,such as sporozoites ormerozoites in search of an adequate host cell,are unlikely to be killed by NETs,butwere proven to be immobilized and hampered from host cell invasion[5,22,28].However, intracellular stages can hardly be attacked by NET structures. Nevertheless,the function of NETsmay here be attributed to other leucocyte recruitment(e.g.macrophages,cytotoxic CD8+cells)to the pathogen's site to deliver more effective parasitocidal actions.A lternatively,the local high concentration of NET-related antim icrobial molecules m ight additionally damage the cellmembraneof infected cells,thereby exposing parasitic stages directly to NETs.Consistently to this assumption,in heavily Eimeria-infected mucosa,NETs were often observed sticking to epithelial host cells carrying intracellular stages.Actually the fi rstever published dataon parasiteinduced NETs also reported in vivo NETs entrapping Plasmodium falciparum-infected host cells(erythrocytes)w ithin blood vessels[26].Given that E.bovis-infected host cells express parasite-derived antigens(EbHSAg)on their surface membrane[59],these molecules might be recognized by PMN-derived pathogen recognition receptors,such as Toll-like receptors.In this context,we demonstrated the presenceofmRNA transcripts of TLR1,TLR2,TLR4,TLR6,TLR7 and TLR10 genes in bovine PMN[60],and further characterized their pivotal role in the activation process of PMN after speci fi c TLR-ligand binding[61].In the human system there is some evidence on TLR4-dependent platelet–neutrophil interactions leading to the formation of NETs in plasma from severely septic patients[62]. Overall,future functional experiments have to clarify whether NETs may exhibit any detrimental effect on intracellular stages of Eimeria sp.

Con fl ict of interest statement

We declare thatwe have no con fl ict of interest.

Acknow ledgm ents

We would like to thank Gabriele Fuchs-Moll(Laboratory Section of Experimental Surgery,Justus Liebig University Giessen,Germany)for her excellent technical assistance in immunohistochem istry analysis.We are also deeply thankful to the Portuguese Association of Serpentina Goat Farmers for supplying samples and technical support during fi eld studies. W e would like to thank Luísa Rosendo Fialho(Department of Veterinary Medicine,University of Evora,Portugal),Christin Ritter(Institute of Parasitology,Justus Liebig University Giessen,Germany)and Ricardo Hartley(Laboratory of Cryobiology and Analysis of Spermatic Functionality,Institute of Animal Science,Faculty of Veterinary Sciences,Austral University of Chile)for their excellent technical support.This work was supported and fi nanced by the German Research Foundation (DFG;Grant No.TA 219/4-1).

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17 Nov 2015

*Corresponding author:Prof.Dr.Dr.habil.CarlosHermosilla,DVM,DipEVPC, Visiting Professor(UACH).Institute of Parasitology,BFS,Justus Liebig University Giessen,Schubertstr.81,Giessen,Germany.

Tel:+49 641 99 38461

Fax:+49 641 99 38469

E-mail:Carlos.R.Hermosilla@vetmed.uni-giessen.de

All animal proceduresw ere performed according to the Justus Liebig University Animal Care Ethic Comm ittee and in accordance to the current European Animal W elfare Legislation:ART13TFEU.

Foundation Pro ject:Supported by the German Research Foundation(DFG; Grant No.TA 219/4-1).

Peer review under responsibility of Hainan M edical University.The journal implements double-blind peer review practiced by specially invited international editorial board members.

#These authors contributed equally to thiswork.2221-1691/Copyright©2016 Hainan Medical University.Production and hosting by Elsevier B.V.This is an open accessarticle under the CC BY-NC-ND license(http:// creativecommons.org/licenses/by-nc-nd/4.0/).