I n the immune response against viruses like influenza, NK cells play an important role1. NK cells express both

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1 OPEN SUBJECT AREAS: INFECTION MUCOSAL IMMUNOLOGY NK CELLS INFLUENZA VIRUS Differentil lung NK cell responses in vin influenz virus infected chickens correlte with pthogenicity Christine A. Jnsen 1, Eveline de Geus 1, Dphne A. vn Hrlem 1, Peter M. vn de Hr 1, Brndon Z. Löndt 2, Simon P. Grhm 2, Thoms W. Göel 3, Willem vn Eden 1, Shron M. Brookes 2 & Lonneke Vervelde 1 Received 26 April 213 Accepted 31 July 213 Pulished 21 August 213 Correspondence nd requests for mterils should e ddressed to C.A.J. (c..jnsen@uu. nl) Current ddress: The Roslin Institute nd Royl (Dick) School of Veterinry Studies, University of Edinurgh, Ester Bush, United Kingdom. 1 Deprtment of Infectious Diseses nd Immunology, Fculty of Veterinry Medicine, Utrecht University, Utrecht, the Netherlnds, 2 Deprtment of Virology, Animl Helth nd Veterinry Lortories Agency (AHVLA) Weyridge, Addlestone, Surrey, United Kingdom, 3 Institute for Animl Physiology, Deprtment of Veterinry Sciences, Ludwig Mximilins University Munich, Munich, Germny. Infection of chickens with low pthogenicity vin influenz (LPAI) virus results in mild clinicl signs while infection with highly pthogenic vin influenz (HPAI) viruses cuses deth of the irds within 36 8 hours. Since nturl killer (NK) cells hve een shown to ply n importnt role in influenz-specific immunity, we hypothesise tht NK cells re involved in this difference in pthogenicity. To investigte this, the role of chicken NK-cells in LPAI virus infection ws studied. Next ctivtion of lung NK cells upon HPAI virus infection ws nlysed. Infection with H9N2 LPAI virus resulted in the presence of virl RNA in the lungs which coincided with enhnced ctivtion of lung NK cells. The presence of HN1 viruses, mesured y detection of virl RNA, did not induce ctivtion of lung NK cells. This suggests tht decresed NK-cell ctivtion my e one of the mechnisms ssocited with the enhnced pthogenicity of HN1 viruses. I n the immune response ginst viruses like influenz, NK cells ply n importnt role1. NK cells express oth ctivting nd inhiitory receptors, nd the lnce etween these signls determines NK-cell ctivtion 2,3. The ctivting NK-cell receptor NKp6 is minly expressed on NK cells ut hs lso een reported on minor frction of NKT cells nd gmm delt T cells. NKp6 hs een demonstrted in different species including humns 6, monkeys 7, rodents 8, cttle 9, sheep 1 nd pigs 11. NKp6 nd NKp, nother memer of the fmily of nturl cytotoxicity receptors, ind virl hemgglutinin (HA) of vrious strins of influenz nd inding results in ctivtion of NK cells In vivo studies in mice hve shown tht NK cells 1 17 nd NKp6 18 re required for the clernce of influenz virus. In ptients with severe influenz infection, diminished frequencies of NK cells re oserved in the lood 19,2, nd pulmonry NK cells re lcking 21. This suggests n importnt role for NK cells in influenz-specific immunity. Wild qutic irds re the nturl reservoirs for influenz A viruses 22 which re le to infect oth humns nd nimls nd cuse sesonl epidemics of infectious respirtory disese in humns worldwide 22,23. These influenz viruses cn e chrcterized sed on the ntigenic properties of the virl surfce proteins HA nd neurminidse (NA) 2. In irds 16 HA sutypes nd 9 NA sutypes hve een descried 2. Avin influenz viruses re considered to e of either low pthogenicity or highly pthogenic, sed on the ility to induce clinicl disese nd/or deth in chickens 26. Infection with LPAI virus usully results in mild clinicl signs while infection with HPAI viruses induces systemic infection nd eventully deth of the host within 36 8 hours 27,28. Due to virl muttions these LPAI viruses my give rise to HPAI viruses 29. Some HPAI viruses cuse lethl infection in humns 3. Also LPAI viruses of the H7 nd H9 sutype hve een reported to infect humns This mkes vin influenz viruses potentil pndemic thret. The inding of the HA protein to NK cells, similr to the inding of the HA protein to receptors on the host cell, is dependent on silic cid residues on the NK-cell receptor. The inding of oth humn nd swine influenz viruses to 2,6-linked SA residues on humn NKp6 13 induces NKp6-medited killing. In contrst, HN1 HPAI viruses which prefer inding vi 2,3-SA residues ind to humn NKp6. The interction etween HN1 virus nd NKp6 is not le to induce NK-cell medited killing y itself. Killing of HN1 infected trgets is only oserved when oth NKp6 nd NKG2D re ctivted 3. This lck of NK-cell ctivtion upon the interction etween HN1 vin influenz viruses nd NKp6 itself my e property of these viruses which contriutes to SCIENTIFIC REPORTS 3 : 278 DOI: 1.138/srep278 1

2 their highly pthogenic nture. Alterntively, it my e cused y the fct tht the interctions etween vin HN1 virus nd the humn NKp6 through its 2,3-SA re insufficient to induce killing y NK cells. In the present study we hypothesise tht the lck of NK-cell ctivtion induced y HN1 viruses is property of these viruses, nd tht the diminished NK-cell ctivtion upon infection with highly pthogenic vin influenz virus is ssocited with enhnced pthogenicity. To investigte this, we performed infections in chickens, which cn e infected with oth LPAI viruses nd the dedly HPAI viruses. Studying NK-cell responses in chickens is chllenging due to the limited knowledge of non-mmmlin NK cells. Avin NK cells hve een descried s popultion of cells which express surfce CD8 homodimers, ut no T or B-cell specific ntigens 3. Furthermore, chicken NK cells hve een reported to express oth ctivting nd inhiitory receptors similr to wht hs een descried in humns 36,37. In recent study, we identified dditionl mrkers tht re expressed on chicken NK cells nd developed ssys to mesure NK-cell degrnultion nd killing 38. In the present study ctivtion of lung NK cells ws compred following infection with either LPAI or HPAI viruses. Since the role of chicken NK cells upon infection with AIV hs not een studied efore, we initilly studied NK-cell iology upon LPAI infection where we performed detiled kinetic study. With this knowledge we went on to study NK-cell ctivtion upon HPAI infection. The ltter ws limited y the timefrme of the infection (irds die within 36 8 hrs). Results Virl lod levels upon infection with LPAI virus. To investigte if infection with LPAI virus would ffect multiple lymphoid orgns, virl lod (RNA level) ws determined y qrt-pcr in lungs, lood, nd spleen etween nd 6 dys post infection. Virus ws detected in the lung t 1 dpi nd incresed virl lods were seen from 2 dpi onwrds (Fig. 1A). This increse continued until dpi, when virl lod peked t -Ct vlue of (men 6 SEM). At dpi, virl lod decresed nd ws only detectle in two irds t 6 dpi. A similr pttern ws oserved in PBMC (Fig. 1B). Virl lod in PBMC incresed up to dpi, ut ws lower thn tht in the lung (-Ct of ). At dpi virl lod declined nd ws undetectle in ll irds t 6 dpi. In spleen, the virl lod ws very low- the highest virl lod ws oserved t 3 dpi nd reched -Ct of (dt not shown). Chrcteriztion of vin NK cells in the lung. Avin NK cells hve een descried s popultion of cells which express surfce CD8 homodimers, ut no T or B-cell specific ntigens. These cells hve een reported in emryonic splenocytes, in the intestinl epithelium nd to lesser extend in PBMC nd spleen 3,39. Since lung NK cells hve not een studied efore in the chicken, we set out to investigte if lung NK cells cn e defined sed on the criteri used to descrie NK cells in these orgns. Therefore expression of B nd T-cell specific ntigens ws nlysed together with the expression of CD17 in lung cells of n uninfected ird. Surfce expression of CD17 ws detected on ctivted chicken NK cells, similr to wht hs een descried for mmmlin NK cells. Since surfce CD17 expression ws nlysed fter hours of culture without further stimultion using lung cells of n uninfected ird, the CD17 expression reflects the spontneous ctivtion of lung NK cells. As shown in Fig. 2, cells expressing surfce CD8 nd lcking T- or B-cell ntigens were oserved in lung. CD8 ws expressed on oth CD31 nd CD3- cells (Fig. 2A), while cells recognized y the B-cell mrker Bu-1 lck surfce expression of CD8 (Fig. 2B). CD3- cells lso lcked surfce expression of TCR1, TCR2 nd TCR3 (dt not shown). Anlysis of CD17 expression within these popultions of lung cells showed surfce CD17 expression on CD3- cells (Fig. 2C), nd cells expressing CD17 did not express B-cell ntigens (Fig. 2D). Within the CD3- popultion, cells expressing CD17 did not express CD8 (Fig. 2E). Thus, popultion of cells with surfce expression of CD8 tht lck T nd B cell ntigens ws oserved in the lung. Co-stining with mouse-nti-cd17 mas showed tht these cells tht lck B or T cell ntigens express CD17, nd tht CD17 expression ws ccompnied y loss of CD8 expression. Since B-cell mrker negtive cells were either CD31 or CD3- nd s CD17 is known to e expressed y CD3- NK cells nd CD31 cytotoxic T lymphocytes, we defined resting NK cells s CD3- CD81 cells (CD81 NK cells) nd ctivted NK cells s CD3- CD171 cells (ctivted NK cells). Frequencies of CD81 NK cells in the lung following infection with LPAI virus. To study if LPAI virus infection cuses chnge in the percentge of chicken NK cells in the lung, frequencies of CD3- nd CD81 NK cells isolted from the lungs were determined etween 1 6 dpi. Since we did not oserve ny difference etween the control irds t dy nd the 2 control irds t the different time points etween 1 nd 6 dpi, we hve comined these irds in one group of uninfected controls. The percentge of CD3- cells incresed until 3 dpi when it ws significntly higher compred to uninfected controls ( vs men 6 SEM, Fig. 3A). Figure 1 Virl lod fter infection with LPAI virus. Virl lod ws determined y qrt-pcr specific for the mtrix gene of the influenz virus. Reltive expression vlues were normlized ginst 28S rrna. Individul dt re shown for lung () nd PBMC () (n ; t 2 nd 3 dpi n 8), the r indictes the men. SCIENTIFIC REPORTS 3 : 278 DOI: 1.138/srep278 2

3 CD8α Bu-1 CD3 CD8α c d e CD17 CD17 CD17 CD3 Bu-1 CD8α Figure 2 Chrcteriztion of vin lung NK cells. Expression of B- nd T-cell specific ntigens on lung NK cells from n uninfected chicken ws nlysed y flow cytometry together with the expression of CD17. Since surfce CD17 expression ws nlysed fter hours of culture without further stimultion using lung cells of n uninfected ird, the CD17 expression reflects the spontneous ctivtion of lung NK cells. Cells were gted sed on forwrd-side sctter nd ded cells were excluded from the nlyses. Co-expression of CD3 nd CD8 () nd the B-cell mrker Bu-1 nd CD8 () ws nlysed ex vivo. Expression of CD17 within CD3 positive nd negtive popultions (c) Bu-1 positive nd negtive popultions (d) nd CD3 negtive CD8 positive nd negtive popultions (e) ws determined fter hour incution t 37uC. Popultions of cells showing NK-cell chrcteristics re indicted. Next, the percentge of CD81 NK cells were nlysed. In uninfected nimls, the frequency of CD81 NK cells ws % (Fig. 3B). At 3 dpi the percentge of CD81 NK cells in infected nimls incresed to %, pproximtely 2-fold higher thn the proportion of CD81 NK cells in uninfected controls. This increse in the percentge of CD81 NK cells ws mintined t nd dpi. At 6 dpi, the percentge of CD81 NK cells decresed to %, which ws similr to the percentge in uninfected controls. The proportion of CD81 NK cells in PBMC in uninfected controls (2. 6.7%) ws slightly higher compred to tht in lung ( %, Fig. 3D). During infection, the percentge CD81 NK cells vried, nd only t dpi the percentge of CD81 NK cells ws significntly higher compred to the uninfected controls (.2 6.8%, p,.). Different popultions of lung NK cells fter LPAI infection. The frequencies of NK cells in the lung fter LPAI virus infection were determined y flow cytometry using mrkers tht re known to e expressed on chicken NK cells; the chicken homologue of the humn NK-cell mrker CD6 nd the previously descried NK-cell mrkers 2E, 7C1 nd 28-. Expression of the NK-cell mrkers ws nlysed within the CD3- popultion. The percentge of CD61 NK cells incresed fter infection from % in uninfected controls to % t 1 dpi (p,., Fig. A). At dpi, the percentge CD61 NK cells temporrily dropped to levels lower thn in uninfected controls (.7 6.2, p,.). The frequency of 2E1 NK cells incresed continuously fter infection until 3 dpi ( %, p,. Fig. B). At dpi, the percentge of 2E1 NK cells decresed which resulted in levels similr to tht of uninfected controls t 6 dpi. In contrst to CD6 nd 2E, the percentge of 7C11 NK cells (. 6.1%) nd 28-1 NK cells (.2 6.%) in the lung ws rther low nd did not chnge fter infection (dt not shown). Interestingly, these mrkers were redily expressed on the supopultion of CD81 NK cells which re thought to represent popultion of resting NK cells. The percentge of 7C11 NK cells did not chnge during infection, while the percentge of 28-1 NK cells incresed during infection reching levels upto % ( % t dpi;.3 6.8% t 6 dpi; Fig. D). These differences in mrker positive cells imply the presence of different popultions of NK cells in the lung of chickens following LPAI virus infection. Infection with LPAI virus resulted in enhnced ctivtion of lung NK cells. To study the effect of LPAI virus infection on NK-cell ctivtion, cell surfce expression of CD17 ws nlysed. As virus detection in the spleen ws limited, NK-cell ctivtion induced y LPAI virus ws only determined for lung nd lood. Representtive exmples of CD17 expression on CD3- lung NK cells in uninfected controls nd t 1, 3 nd dpi show tht CD17 is only expressed t CD3- cell tht do not express CD8 (Fig. A)Immeditely fter infection, the proportion of ctivted lung NK cells temporrily incresed from % in uninfected controls to % t 1 dpi (p,.). At dpi the percentge of ctivted lung NK cells incresed gin to %. The dpi level ws greter thn the proportion of ctivted lung NK cells t 1 dpi (p,.) nd this high percentge of CD17 expressing cells continued until dpi. At 6 dpi, the frequency of ctivted lung NK cells diminished to % which ws similr to the level oserved in uninfected controls (Fig. B). SCIENTIFIC REPORTS 3 : 278 DOI: 1.138/srep278 3

4 %CD3- cells CD3- cells in lung % CD3-CD8α cells CD3 -CD8 α+ cells in lung dys post infection dys post infection c 8 CD3 -CD8 α+ cells in PBMC % CD3-CD8α+cells dys post infection Figure 3 Incresed frequencies of lung CD81 NK cells 3 dys post infection with LPAI virus. Frequencies of lung CD3- cells () lung CD81 NK cells () nd lood CD81 NK cells (c) were nlysed y flow cytometry. Men 6 SEM of five infected irds per dy nd seventeen uninfected irds (irds from dpi together with the uninfected irds from 1 6 dpi) re shown. Significnt differences compred to the uninfected controls were nlysed using Mnn-Whitney tests nd p,. is indicted y n sterisk. Although the percentge of ctivted NK cells in PBMC ws much lower compred to tht in lungs, similr pttern ws oserved in the chnges in expression erly post-infection (Fig. C). The percentge of ctivted NK cells incresed t 1 dpi from % in uninfected smples to % in infected irds (p,.). The percentge of ctivted NK cells then decresed gin to levels which were similr to uninfected controls. Gross pthology, nd virl RNA levels upon HN1 HPAI virus infection. Infection with LPAI virus resulted in incresed ctivtion of lung NK cells. The follow-up investigtion ws to determine the NK-cell response upon HPAI virus infection using the Eursin HN1 isoltes tytr nd tyeng91. The nlysis of NK-cell responses upon HPAI virus infection ws limited y sfety issues ssocited with working under Biosfety Continment Level 2 conditions. Therefore, ex vivo stining with NK-specific mas ws not included in these HPAI experiments. In contrst to the sence of lesions fter LPAI infection, HPAI infection induced gross pthology lesions tht were chrcteristic for AI virus infection (dt not shown). Birds inoculted with tytr HN1 HPAI virus displyed moderte splenomegly from 2 hpi, hyperplsi of the Burs of Fricius nd hyperemic lungs nd thymus. Petechi in cecl tonsils ws oserved occsionlly. Hyperemic lungs nd splenomegly ws lso oserved in chickens inoculted with tyeng91 HN1 HPAI virus t 2 hpi. Twelve hours fter infection with tytr, low virl lod levels were oserved in lungs of out of 6 nimls (-Ct of ; Fig. 6A). At 2 hpi, virus ws detected in lungs of ll nimls nd incresed to -Ct level of Twelve hours fter infection with tyeng91, virus ws detected in the lungs of ll nimls nd ws higher compred to tht oserved fter infection with tytr ( , p,., Fig. 6B) t the sme time point. At 2 hpi the virl lod ws similr to levels oserved 2 hpi with tytr ( ). In PBMC, virl lods were only detected t 2 hpi ( tytr, Fig. 6C; tyeng91, Fig. 6D). HN1 HPAI viruses do not induce ctivtion of lung NK cells. Similr to wht ws oserved fter LPAI infection, infection with the Eursin HPAI isolte tytr resulted in significnt increse in the percentge of CD3- cells t 8 hpi compred to uninfected controls (6.% versus.2% 6.3, p,., Fig. 7C). Similr results were oserved fter infection with the Eursin HPAI isolte tyeng91 (69.1% 6., p,., Fig. 7D). The increse in the percentge of CD3- cells ws only trnsient, t 2 hpi with either tytr or tyeng91 the percentge CD3- cells ws similr to the percentge in uninfected controls. Representtive exmples of CD17 expression on CD3- lung NK cells in uninfected controls nd chickens infected with tytr nd tyeng91 gin show tht CD17 is only expressed t CD3- cell tht do not express CD8 (Fig. 8A).After infection with tytr, the SCIENTIFIC REPORTS 3 : 278 DOI: 1.138/srep278

5 NK mrker expression within CD3- cells 1 8 %CD6+ cells %2E+ cells c 2 NK mrker expression within CD3-CD8α+ cells d 1 %7C1+ cells 1 1 %28-+ cells dys post infection dys post infection 6 Figure Different popultions of NK cells were oserved in the lung fter LPAI virus infection. Frequencies of NK cells in the lung fter LPAI virus infection were determined y flow cytometry using the NK-cell mrkers CD6 (), 2E (), 7C1 (c) nd 28- (d). The frequency of mrker positive cells within CD3- cells (A,B) nd CD3-CD81 (c,d) re shown. Men 6 SEM of five infected irds per dy nd seventeen uninfected irds (irds from dpi together with the uninfected irds from 1 6 dpi) re shown. Significnt differences compred to the uninfected controls were nlysed using Mnn- Whitney tests nd p,. is indicted y n sterisk. percentge of ctivted NK cells in the lung decresed drmticlly from % in uninfected controls compred to % t 8 hpi (p,.). This decrese in the frequency of ctivted NK cells continued t 12 nd 2 hpi (. 6.% nd. 6.7% respectively; Fig. 8B). Infection with tyeng91 lso resulted in rpid decrese of ctivted NK cells (6. 6.3% t 8 hpi, p,.) lthough the decrese ws not s strong when compred to tytr (Fig. 8C). At 2 hpi, the percentge of ctivted NK cells ws still significntly lower compred to hpi (.6 6.1% compred to %, p,.). In PBMC, the percentge of ctivted NK cells did not chnge upon infection with either tytr (Fig. 8D) or tyeng91 (Fig. 8E). Thus, infection with Eursin HPAI HN1 virus results in decresed ctivtion of lung NK cells. Discussion Nturl Killer cells nd the ctivting NK-cell receptor NKp6 hve een shown to ply n importnt role in influenz-specific immunity Since vin influenz viruses use distinct inding site on NKp6 compred to the humn nd swine influenz viruses nd inding is dependent on 2,3-SA rther thn 2,6-SA residues on the receptor 3, the lck of NK-cell cytotoxicity upon inding of vin HN1 virus to humn NKp6 itself in the sence of NKG2D crosslinking my e due to differences in receptor specificity. We proposed n lterntive mechnism nd hypothesised tht lck of NK-cell ctivtion upon inding to NKp6 itself is n intrinsic property of these HN1 HPAI viruses irrespective of the inding to 2,3- or 2,6- SA residues on the influenz specific NK-cell receptor NKp6. To investigte this, NK-cell responses fter infection were studied in chickens, which enled us to use system in which oth viruses nd NK cells re of vin origin. Studies on chicken NK cells hve een hmpered y the sence of specific mas for these cells. Originlly chicken NK cells were defined s CD3-CD81 cells 3. Similr results were oserved for lung NK cells, which cn e defined s T- nd B-cell mrker negtive, nd surfce CD8 positive. It is most likely tht this popultion of CD3-CD81 cells reflects resting NK cells, since NK-cell ctivtion results in the down regultion of the CD8 chin 38. This is gin confirmed y our oservtion tht CD17 is only expressed on cells tht do not express the CD8 chin.when studying the frequencies of CD3-CD81 NK cells in the lung upon LPAI infection, higher levels of CD3-CD81 cells in the lung compred to uninfected controls were oserved etween 3 nd dpi. Also the percentge of CD3- cells ws incresed t 3 dpi. This increse my imply n influx of NK cells in the lung, similr to wht hs een descried for mmmlin species. Since the solute numer of cells is not known, the increse in the percentge my lso reflect chnge in cell susets in the lung, rther thn n influx of NK cells. Immunohistochemistry is wrrnted to investigte the reson ehind the increse in CD3- nd CD3-CD81 cells in the lung. SCIENTIFIC REPORTS 3 : 278 DOI: 1.138/srep278

6 1 1.2 uninfected 1 dpi 3 dpi dpi CD17-APC %CD17+CD3- cells CD8α-PE c lung %CD17+CD3- cells PBMC dys post infection dys post infection Figure Infection with LPAI virus results in enhnced ctivtion of lung NK cells. To study possile differences in NK-cell ctivtion fter infection with A/chicken/United Ar Emirtes/99 H9N2 LPAI virus, cell surfce expression of CD17 ws nlysed y flow cytometry. Representtive FACS plots showing CD17 expression within lung CD3- cells from uninfected controls, nd infected nimls t 1, 3 nd dpi (). Men CD17 expression 6 SEM is shown for lung () nd PBMC (c) (n except for 2 dpi nd 3 dpi n 8). Significnt differences compred to the uninfected controls (irds from dpi together with the uninfected irds from 1 6 dpi) were nlysed using Mnn-Whitney tests nd p,. is indicted y n sterisk. Upon LPAI virus infection, diverse popultions of NK cells were oserved in the lung. The percentge of CD61NK cells rpidly incresed t 1 dpi, which my represent n influx of NK cells upon infection. The decrese in CD6 expression from 2 dpi onwrds possily reflects down regultion upon ctivtion similr to wht hs een descried for humn NK cells 1. Cler differences were oserved etween lung cells expressing the mrkers 2E, 7C1 nd 28-. Although ll mrkers hve een reported to e expressed on NK cells 38,39 nd the differences etween the mrkers suggest the existence of distinct popultions of NK cells, the ntigens recognized y these mrkers re not yet known. This complictes direct comprison etween NK-cell popultions in chickens nd in humns or mice. Infection with LPAI virus resulted in the enhnced ctivtion of lung NK cells s determined y mesuring cell surfce expression of CD17. This enhnced ctivtion ws oserved t 1 dpi nd gin t nd dpi. We propose the following model; fter infection, ctivtion of lung NK cells occurs. NK cells cn e ctivted directly vi the interction etween AIV nd NK cell receptor, or indirectly vi the production of cytokines like IL-12 nd IL-18 y infected mcrophges nd dendritic cells. Despite this ctivtion, virl lod continues to increse. As reflected y the increse in the percentge of CD3-CD81 cells t 3 dpi, we hypothesise tht resting NK cells enter the lung, susequently ecome ctivted (s shown y the second pek in ctivtion of lung NK cells t nd dpi). Upon the second increse in NK-cell ctivtion, decrese in virl lod is oserved. At 6 dpi, virl lod levels hve reduced drmticlly nd the frequencies of NK cells in the lung nd ctivtion of lung NK cells hve returned to levels oserved in uninfected controls. At 2 dpi nd 3 dpi, nlysis of ctivted lung NK cells showed two groups of irds: chickens with high percentge of ctivted NK cells or chickens with low percentge of ctivted NK cells. This cn e prtly explined y virl lod levels; t oth 2 nd 3 dpi two irds show lower virl lod compred to the rest of the group. Interestingly, the percentge ctivted NK cells in the uninfected controls differed etween the LPAI nd HPAI experiments. A possile explntion for this difference cn e the fct tht oth experiments were performed in different institutes nd therefore the chickens were housed in different niml fcilities, Since study in mice showed tht the environment hs strong impct on the priming of NK cells 2, housing under Biosfety Continment Level 2 conditions my result in less NK cell priming nd less spontneous degrnultion which is reflected y the lower percentge of ctivted NK cells in the lung. This fits with our previous oservtion tht uninfected SPF chickens tht were housed in isoltors lso show lower percentge of ctivted NK cells in the lung 3 compred to the lyer chickens tht were used in this study. In contrst to LPAI infection, infection with Eursin HN1 HPAI virus isoltes did not result in ctivtion of NK cells in the lung lthough similr mounts of virus were present. A decresed ctivtion of NK cells compred to uninfected controls ws found for two different HN1 HPAI viruses nd t multiple time points post infection. The decresed NK-cell ctivtion my hve severl cuses. Firstly, chicken NK cells express oth ctivting nd inhiitory NK-cell receptors 37,,, so it is possile tht chickens hve influenz-specific NK-cell receptors similr to NKp6. Although oth HPAI nd LPAI viruses ind through 2,3-SA residues, differences in inding etween HPAI nd LPAI viruses my still occur, for exmple due to differences in glycosyltion of HA 6 or to inding to dditionl NK receptors. The difference in inding my led to different lnce etween ctivting nd inhiiting receptors upon SCIENTIFIC REPORTS 3 : 278 DOI: 1.138/srep278 6

7 3 lung tytr 3 lung tyeng91 virl RNA levels (-Ct) 2 1 virl RNA levels (-Ct) 2 1 c PBMC tytr d PBMC tyeng91 virl RNA levels (-Ct) 2 1 virl RNA levels (-Ct) Figure 6 Virl lod levels in lung nd PBMC fter infection with HPAI virus. Virl lod ws determined y qrt-pcr specific for the mtrix gene of the influenz A virus. Reltive expression vlues were normlized ginst 28S rrna. Individul dt re shown for lung tissue (, ) nd PBMC (c, d) following infection with A/turkey/Turkey/1/2 (tytr) () or A/turkey/Englnd/-92/1991 (tyeng91) respectively (n 6). The r indictes the men. inding of LPAI or HPAI virus. Secondly, influenz virus hs een reported to e le to infect NK cells, which results in NK-cell poptosis nd reduced NK-cell cytotoxicity 7,8 nd the numer of NK cells is reduced in severely influenz infected ptients It my e possile tht HPAI viruses infect chicken NK cells, which could result in decresed NK-cell ctivtion. Thirdly, infection with HPAI virus is chrcterized y strong increse in virl lod nd cytokine storm in humns nd mcques 9,. Also in chickens strong induction of cytokines upon HPAI infection hs een reported, lthough the levels of cytokines induced seems to e dependent on the virl strin 1,2. The overwhelming mount of virus nd/or the presence of cytokines my produce n overstimultion of NK cells, resulting in NK-cell exhustion. Lstly, in this study we used H9N2 nd HN1 viruses which re oth endemic in poultry since their occurrence in the mid 9 s 3,. To formerly proof tht the lck of NK-cell ctivtion upon HN1 HPAI infection is n intrinsic 1 8 lung tytr 1 8 lung tyeng91 %CD3-cells 6 %CD3-cells Figure 7 Lymphocytes numers in the lungs fter infection with HPAI virus. Percentges of lung CD3- cells within the live gte upon infection with tytr () nd tyeng91 () were nlysed y flow cytometry. Men 6 SEM re shown (n 6). Significnt differences compred to the uninfected controls ( hpi group) were nlysed using Mnn-Whitney tests nd p,. is indicted y n sterisk. SCIENTIFIC REPORTS 3 : 278 DOI: 1.138/srep278 7

8 lung tytr lung tyeng91 8 hpi 12 hpi 8 hpi 12 hpi CD17-APC 2 CD8α-PE lung tytr c 2 lung tyeng91 %CD17+CD %CD17+CD3- cells d 2 PBMC tytr e PBMC tyeng91 %CD17+CD %CD17+CD3- cells Figure 8 Decresed ctivtion of lung NK cells fter infection with HPAI virus. Representtive FACS plots showing CD17 expression within lung CD3- cells from uninfected controls, nd irds infected with tytr nd tyeng91 t 8 nd12 hpi (). Possile differences in NK-cell ctivtion upon infection with the HN1 HPAI viruses tytr (, d) nd tyeng91 (c, e). Cell surfce expression of CD17 in CD3- cells ws nlysed y flow cytometry using the sme gting strtegy s in Figure 7. Men 6 SEM re shown for lung (, c) nd PBMC (d, e) (n 6). Significnt differences compred to the uninfected controls ( hpi group) were nlysed using Mnn-Whitney tests nd p,. is indicted y n sterisk. property of HPAI viruses in generl, experiments with n HPAI virus nd its respective precursors should e performed. In conclusion, we report enhnced ctivtion of lung NK cells fter infection with LPAI virus. Infection with HPAI virus results in decresed ctivtion of lung NK cells indicting tht decresed NK-cell ctivtion my e one of the mechnisms contriuting to the pthogenicity of HN1 HPAI viruses. The mechnism ehind this difference in ctivtion of lung NK cells upon LPAI nd HN1 HPAI infections remins to e elucidted. Methods Animls. One-dy old Lohmnn Brown chickens were otined from commercil reeder. Chickens were housed in groups nd fed d liitum on commercil feed. At the ge of 3 weeks, chickens were infected with either LPAI or HPAI virus. The LPAI H9N2 isolte A/chicken/United Ar Emirtes/99 (kindly provided y Intervet/ Merck Animl Helth, Boxmeer, The Netherlnds) ws diluted in sterile PBS to concentrtion of EID /ml nd chickens were inoculted intrnslly nd intrtrchelly (1 ml ech). For ech dy from nd 6 dpi, 2 uninfected nd infected irds (8 infected irds t 2 dpi nd 3 dpi) were killed y cervicl disloction nd lungs, spleen nd lood were collected. Pthology ws performed to identify lesions ssocited with AIV or immune ctivtion. In the nlyses results from uninfected irds t dpi re comined with the results from the uninfected irds t the other time points, since we did not oserve ny differences etween these irds. Together these irds re referred to s uninfected controls. Tissue smples from lung (prt L1), spleen nd PBMC were collected in TRIzol regent (Invitrogen, Bleiswijk, the Netherlnds), snp-frozen in liquid nitrogen nd stored in -8uC until RNA isoltion ws performed. To otin single cell suspension, lung tissue ws cut into smll pieces nd digested in RPMI contining 2 mg collgense A from Clostridium histolyticum nd mg DNAse I isolted from ovine pncres (Roche Applied Science, Almere, the Netherlnds) for 3 min t 37uC, nd homogenised using 7 mm cell striner (BD SCIENTIFIC REPORTS 3 : 278 DOI: 1.138/srep278 8

9 Biosciences, Frnklin Lkes, NJ, USA). Spleens were lso homogenised using 7 mm cell striner. Vile cells were isolted from lungs, spleen nd lood y Ficoll-Pque density grdient centrifugtion nd the totl numer of lymphocytes in the lung ws determined y counting the trypn lue negtive cells. Cells were resuspended in IMDM medium supplemented with 8% het inctivted FCS; 2% het inctivted chicken serum, 1 U/ml penicillin, 1 mg/ml streptomycin nd 2 mm glutmx ( NK medium ; Gico BRL, Pisly, United Kingdom) nd were used directly in the NK-cell ssys. Alterntively, chickens were infected with HN1 HPAI virus. Two different isoltes were used which disply different infection phenotypes in chickens: new Eursin linege [A/turkey/Turkey/1/2 (tytr)] nd n old Eursin linege [A/turkey/ Englnd/-92/1991 (tyeng91)] (B.Z. Löndt, personl communiction). Virus ws diluted in PBS to concentrtion of EID /.1 ml nd chickens were inoculted intrnslly nd introculrly ( ml ech). Birds were killed t, 8, 12 nd 2 (hpi) (6 irds per time point) nd lungs nd lood were collected. In the HPAI experiment, uninfected irds re the irds killed t hpi. Vile cells were isolted from these orgns s descried ove. All niml experiments involving LPAI virus were pproved y the Committee on Animl Experiments of the University of Utrecht (DEC 28.II.1.1) nd performed ccording the Dutch regultion on experimentl nimls. The experiments with the HN1 HPAI viruses were performed t the Animl Helth nd Veterinry Lortory Agencies ccording to the AHVLA committee for ethicl studies nd in ccordnce with the UK 1986 Animl Scientific Procedure Act nd AHVLA code of prctice for performnce of scientific studies using nimls (License numer 7/762). RNA isoltion nd quntittive rel time PCR. Frozen tissue smples were thwed nd lung nd spleen smples were homogenised (Retsch Mixer Mill 31, Fisher Scientific, Lndsmeer, The Netherlnds) in TRIzol (Invitrogen). PBMC smples were homogenised using syringe nd 21G needle. Totl RNA ws isolted ccording the TRIzol method using mnufcturer s instructions followed y DNAse tretment (RNse-Free DNse set, Qigen Benelux, Venlo, the Netherlnds). Purified RNA ws eluted in 3 ml RNAse free wter nd the RNA ws quntified y sornce mesurement t 28 nm. A mximum of ng RNA ws used for the cdna genertion using the iscript cdna synthesis kit (Biord lortories, Veenendl, the Netherlnds). Rel-time qrt-pcr ws performed to detect the mtrix gene of the influenz virus s previously descried 6. Briefly, conserved frgment of the influenz mtrix gene ws mplified using the primers M-Fw (9-CTTCTAACCGA GGTCGAAACGTA-39), M-Rev (9-CACTGGGCACGGTGAGC-39), nd the proe M(9-FAM-CTCAAAGCCGAGATCGCGCAGA-39-TAMRA) in comintion with the Tqmn Universl PCR mstermix (Applied Biosystems, Nieuwerkerk n de IJssel, The Netherlnds) on MyiQ Single color Reltime PCR Detection system (Biord). Reltive expression vlues were normlized ginst 28S rrna, s previously descried y Kiser et l 7. Men threshold cyle vlues (Ct) were determined sed on triplictes nd results re shown s -Ct vlues, which re clculted y sutrcting the experimentl Ct vlue from the mx Ct vlue. Flow cytometry. Chrcteriztion of vin NK cells in lung ws performed y flow cytometry using co-stinings with mouse-nti-chicken CD3 (CT3; IgG1,), mouse nti-chicken-cd8- PE mas (CT8, IgG1), mouse-nti-chicken-bu-1 (AV2, IgG1), mouse-nti-chicken TCR cd (TCR1, IgG1), mouse-nti-chicken TCR -V1 (TCR2, IgG1) nd mouse-nti-chicken TCR -V2 (TCR3, IgG1) on lung cells of n uninfected ird. All ntiodies were otined from Southern Biotec, Birminghm, AL, USA. Cells were stined with the ntiodies for 2 min t uc. Proportions of NK cells were nlysed y flow cytometry using mrkers tht re known to e expressed on chicken NK cells; the chicken homologue of the humn NK-cell mrker CD6 8 nd the previously descried NK-cell mrkers 2E, 7C1 38 nd Since some of the NK-cell mrkers re lso expressed on T cells 38, stining with NK-cell mrkers ws comined with nti-cd3 ma. Cells were stined with hyridom superntnts 38 for 3 min t uc, followed y got-nti-mouse IgG secondry A (Southern Biotec) for 2 min uc. Norml mouse serum ws used to lock non-specific inding followed y stining with nti- CD3-FITC nd nti- CD8-PE mas. CD17 expression ws nlysed y stining with mouse-nti-chcd17 iotinylted ma 38, followed y fluorochrome-lelled streptvidin secondry ntiody (BD Biosciences). Alterntively, n APC-conjugted nti-cd17 ntiody ws used. In ll experiments, stining with nti-cd17 ws comined with nti-cd3 nd nti- CD8 mas to exclude T cells from the nlyses. Prior to flow cytometry, 7-Amino-Actinomycin D (7AAD; BD Biosciences) or the live/dedh fixle violet cell stin (Invitrogen) ws used for exclusion of ded cells. At lest, cells in the live gte were cquired using FACS Cliur flowcytometer (BD Biosciences). Smples from the experiments with the HN1 HPAI viruses were cquired using MACSQuntH Anlyser (Miltenyi Biotec, Bergisch Gldch, Germny). Dt were nlysed using the softwre progrm FlowJO (Threestr Inc, Ashlnd, OR, USA) or FACS DIVA softwre (BD Biosciences). CD17 ssy. The CD17 ssy to study NK-cell ctivtion ws essentilly crried out s descried previously 38. Briefly, cells isolted from lung, spleen nd lood were resuspended in NK medium t concentrtion of cells/ml. Cells were cultured in the presence of 1 ml/ml Golgistop (BD Biosciences) nd nti-chcd17 ma during hours t 37uC, % CO 2. After incution, cells were wshed in PBS supplemented with.% BSA, nd stined with nti-chicken-cd3-fitc nd ntichicken-cd8-pe mas nd nlysed y flow cytometry. After stining cells from nimls infected with HPAI virus, cells were fixed using % prformldehyde (Merck, Drmstdt, Germny) for 1 minutes t room temperture. The cells were then wshed once in PBS supplemented with.% BSA nd flow cytometry ws performed s descried ove. Sttisticl nlyses. Non-prmetric sttisticl tests were used when the ssumption of normlly distriuted dt were not met. Differences etween the groups were nlysed using Mnn-Whitney U tests. A p-vlue of,. ws considered sttisticlly significnt. All sttisticl nlyses were performed using the softwre progrm SPSS 16. (SPSS Inc, Illinois, USA). 1. Trinchieri, G. Biology of nturl killer cells. Adv. Immunol. 7, (1989). 2. Cooper, M. A., Fehniger, T. A. & Cligiuri, M. A. The iology of humn nturl killer-cell susets. Trends Immunol. 22, (21). 3. Lnier, L. L. NK cell recognition. Annu. Rev. Immunol. 23, (2).. Yu, J. et l. NKp6 identifies n NKT cell suset susceptile to leukemic trnsformtion in mouse nd humn. J. Clin. Invest. 121, (211).. 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J. et l. Highly pthogenic (HN1) vin influenz induces n inflmmtory T helper type 1 cytokine response in the chicken. J. Interferon Cytokine Res. 31, 393 (211). 2. Reel, J. M. et l. Highly pthogenic or low pthogenic vin influenz virus sutype H7N1 infection in chicken lungs: smll differences in generl cute responses. Vet. Res. 2, 1 (211). 3. Guo, Y. J. et l. Chrcteriztion of the pthogenicity of memers of the newly estlished H9N2 influenz virus lineges in Asi. Virology 267, (2).. Xu, X., Suro, Cox, N. J. & Guo, Y. Genetic chrcteriztion of the pthogenic influenz A/Goose/Gungdong/1/96 (HN1) virus: similrity of its hemgglutinin gene to those of HN1 viruses from the 1997 outreks in Hong Kong. Virology 261, 1 19 (1999).. Reemers, S. S., vn Hrlem, D. A., Groot Koerkmp, M. J. & Vervelde, L. Differentil gene-expression nd host-response profiles ginst vin influenz virus within the chicken lung due to ntomy nd irflow. J. Gen. Virol. 9, (29). 6. vn der Goot, J. A. et l. Trnsmission of highly pthogenic vin influenz HN1 virus in Pekin ducks is significntly reduced y geneticlly distnt HN2 vccine. Virology 382, (28). 7. Kiser, P., Underwood, G. & Dvison, F. Differentil cytokine responses following Mrek s disese virus infection of chickens differing in resistnce to Mrek s disese. J. Virol. 77, (23). 8. Neulen, M. L. & Goel, T. W. Chicken CD6 defines NK cell susets in emryonic spleen nd lung. Dev. Comp. Immunol. 38, 1 1 (212). Acknowledgments This study ws finncilly supported y the Netherlnds Orgniztion for Scientific Reserch (NWO) Veni grnt , the EU sixth frmework progrm Flupth (grnt 22) nd Progrmme Impulse Veterinry Avin Influenz Reserch in The Netherlnds, Dutch Ministry of Agriculture, Nture nd Food Qulity. Animl nd lortory work performed t the AHVLA ws linked to BBSRC Reserch Grnt BB/E189/1. The funders hd no role in study design, dt collection nd nlysis, decision to pulish, or preprtion of the mnuscript. The uthors thnk Ildiko vn Rhijn for criticlly reding the mnuscript, Juthtip Kewchroen for technicl ssistnce with the LPAI virus experiments nd Bethny Nsh, John Ridgeon nd Michel Kelly for technicl ssistnce with the HPAI virus experiments, nd Prof. In H. Brown for ccess to collortive fcilities nd resources t AHVLA. The LPAI H9N2 isolte A/chicken/United Ar Emirtes/99 ws kindly provided y Intervet/Merck Animl Helth, Boxmeer, The Netherlnds. Author contriutions C.J. designed the study, generted dt of oth LPAI nd HPAI experiments, nlysed the dt nd wrote the mnuscript. E.d.G. ws involved in the design of the study, generted dt nd nlysed dt of the LPAI experiment. D.v.H. nd P.v.d.H. were involved in generting dt of the LPAI experiments s well s dt nlysis. B.L. ws involved in the design of the study, supplied the HPAI viruses, generted the HPAI dt nd wrote the mnuscript. S.G. supplied regents, nlysed the HPAI dt nd wrote the mnuscript. T.G. supplied mterils to nlyse NK cell frequencies nd function nd ws involved in writing the mnuscript. W.v.E. ws involved in dt nlysis nd writing the mnuscript. S.B. ws involved in the design of the study, supplied HPAI viruses nd wrote the mnuscript. L.V. generted dt of oth LPAI nd HPAI experiments, ws involved in dt nlysis nd wrote the mnuscript. Additionl informtion Competing finncil interests: The uthors declre no competing finncil interests. How to cite this rticle: Jnsen, C.A. et l. Differentil lung NK cell responses in vin influenz virus infected chickens correlte with pthogenicity. Sci. Rep. 3, 278; DOI:1.138/srep278 (213). This work is licensed under Cretive Commons Attriution 3. Unported license. To view copy of this license, visit SCIENTIFIC REPORTS 3 : 278 DOI: 1.138/srep278 1