MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2

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1 ARTICLE NUMBER: 164 DOI: 1.138/NMICROBIOL MERS coronvirus induces poptosis in kidney nd lung y upregulting Smd7 nd FGF2 Mn-Lung Yeung, Ynfeng Yo, Lilong Ji, Jsper F. W. Chn, Kwok-Hung Chn, Kwok-Fn Cheung, Honglin Chen, Vincent K. M. Poon, Aln K. L. Tsng, Kelvin K. W. To, Ming-Kwong Yiu, Jde L. L. Teng, Hin Chu, Jie Zhou, Qing Zhng, Wei Deng, Susnn K. P. Lu, Johnson Y. N. Lu, Ptrick C. Y. Woo, Tk-Mo Chn, Susn Yung, Bo-Jin Zheng, Dong-Yn Jin, Peter W. Mthieson, Chun Qin nd Kwok-Yung Yuen NATURE MICROBIOLOGY 1

DOI: 1.138/NMICROBIOL.216.4 DOI: NP Podocin Merge NP 5 µm γ-tuulin c Cell lystes d Conditioned Medi * p =.1 * p =.1 * p =.2 * p =.1 Virl lod (copy no. normlized to GAPDH) 1.6E+3 1.4E+3 1.2E+3 1.E+3 8.

Productive repliction in primry humn podocyte cell line.

2 DOI: 1.138/NMICROBIOL DOI: NP Podocin Merge NP 5 µm γ-tuulin c Cell lystes d Conditioned Medi * p =.1 * p =.1 * p =.2 * p =.1 Virl lod (copy no. normlized to GAPDH) 1.6E+3 1.4E+3 1.2E+3 1.E+3 8.E+2 6.E+2 4.E+2 * p =.1 Virl lod (copies/ml) 5.E+5 4.5E+5 4.E+5 3.5E+5 3.E+5 2.5E+5 2.E+5 1.5E+5 1.E+5 * p =.1 2.E+2 5E+4 hpi 24 hpi 48 hpi hpi 24 hpi 48 hpi Supplementry figure 1. Productive repliction in primry humn podocyte cell line. () Co-immunohistochemicl stining of NP (green) nd podocytespecific mrker, podocin (red) in primry humn podocyte cell line 1 24 h fter the chllenge of. () Western lot nlysis of NP in - nd -infected podocytes. γ-tuulin ws lso detected s loding control. Totl RNAs (c) nd the conditioned medi (d) of the -inoculted podocytes were hrvested for virl lod detection t the indicted time-points using RT-qPCR s previously descried. 2 The RNA from the cell lystes were normlized to the expression level of GAPDH mrna. Imges shown in nd re representtives of three independent experiments. Error rs shown in c nd d represent the men ± s.d. of three independent experiments. Sttisticl significnce ws evluted y Student s t-tests nd the rnges of p-vlues were indicted in c nd d. Smples from,, c nd d represent iologicl replictes. NATURE MICROBIOLOGY 1 2

1 -infected SARS-CoV-infected 1 2 3 4 Hours post-infection Hours

SARS-CoV-infected SARS-CoV NP 5 µm Apoptotic cells (%) DOI: 1.

Smd7-trnsfected TUNEL/DAPI 5 µm FGF2-trnsfected TUNEL/DAPI 5 µm

Apoptosis due to infection in kidney cells or NATURE

com/nturemicroiology 1 overexpression of Smd7 nd FGF2 in

() HK2 cells were infected y or SARS-CoV t M.O.I. = 9.

At different time-points (, 16, 28 nd 34 hpi), the infected

positively stined cells (red focl stin; ottom) over the totl

over tht of SARS-CoV were evluted y Student s t- tests nd the

Immunostinings of the virus NP (green; ottom) in ech infection

() HK2 cells were trnsfected with overexpression constructs of

ssys s descried ove. Different doses of cdna (,.25 nd.

5 μg with their vector ckone plsmids followed y trnsfection.

3 DOI: 1.138/NMICROBIOL ^ p =.1 Apoptotic cells (%) * p =.1 ψ p =.1 -infected SARS-CoV-infected Hours post-infection Hours post-infection TUNEL/DAPI -infected NP 5 µm TUNEL/DAPI SARS-CoV-infected SARS-CoV NP 5 µm Apoptotic cells (%) DOI: 1.138/NMICROBIOL μg Amount (µg) of Smd7/FGF2 constructs in.5 µg trnsfection complex Smd7-trnsfected FGF2-trnsfected Smd7-trnsfected TUNEL/DAPI 5 µm FGF2-trnsfected TUNEL/DAPI 5 µm Supplementry figure 2. Apoptosis due to infection in kidney cells or NATURE MICROBIOLOGY 1 overexpression of Smd7 nd FGF2 in uninfected kidney cells. () HK2 cells were infected y or SARS-CoV t M.O.I. = 9.6, seprtely. At different time-points (, 16, 28 nd 34 hpi), the infected cells were ssyed y terminl deoxynucleotidy trnsferse dutp nick end leling (TUNEL) ssys. The degree of poptosis (top) ws mesured y counting the numer of positively stined cells (red focl stin; ottom) over the totl numer of cells in the counted fields (lue nuclei counterstined y DAPI; ottom). Sttisticl nlyses of the percentge of poptotic cells induced y over tht of SARS-CoV were evluted y Student s t- tests nd the rnges of p-vlues were indicted on top of ech time-point. Immunostinings of the virus NP (green; ottom) in ech infection condition were included. () HK2 cells were trnsfected with overexpression constructs of Smd7 nd FGF2 followed y the detection of poptosis using TUNEL ssys s descried ove. Different doses of cdna (,.25 nd.5 μg) were first topped up to.5 μg with their vector ckone plsmids followed y trnsfection. The degree of poptosis (top) ws mesured y counting the numer of positively stined cells (red focl stin; ottom) over the totl numer of cells in the counted fields (lue nuclei counterstined y DAPI; ottom). Imges shown in nd re representtives of three independent experiments. Error rs shown in nd represent the men ± s.d. of three independent experiments. Sttisticl significnce ws evluted y Student s t-tests nd the rnges of p-vlues were indicted in nd. Smples from nd represent iologicl replictes. NATURE MICROBIOLOGY 31

4 DOI: DOI: 1.138/NMICROBIOL Smd7 mrna expression (fold) 5.E+3 4.5E+3 4.E+3 3.5E+3 3.E+3 2.5E+3 2.E+3 1.5E+3 1.E+3 5.E+2 * p =.1 * p =.1 SARS-CoV FGF2 mrna expression (fold) 5.E+3 4.5E+3 SARS-CoV 4.E+3 3.5E+3 3.E+3 2.5E+3 2.E+3 1.5E+3 1.E+3 5.E+2 infected infected BIRC3 mrna expression (fold) * p =.9 C M C M C: -infected M: -Infected Clu-3 HK2 c Clu-3 d 8.E+7 Clu-3 Virl lod (copy no. normlized to GAPDH) 4.E+7 3.5E+7 3.E+7 2.5E+7 DOI: 1.138/NMICROBIOL E+7 * p =.1 1.5E+7 1.E+7 5.E+6 BIRC3 Control Virl lod (copy no. normlized to GAPDH) 7.E+7 6.E+7 5.E+7 4.E+7 3.E+7 2.E+7 1.E+7 siclu sisod1 sicat sicontrol HK2 1.4E+9 HK2 Virl lod (copy no. normlized to GAPDH) 1.6E+9 1.4E+9 1.2E+9 1.E+9 8.E+8 6.E+8 4.E+8 1.2E+9 1.E+9 8.E+8 6.E+8 4.E+8 2.E+8 2.E+8 NATURE MICROBIOLOGY BIRC3 Control siclu sisod1 sicat sicontrol Supplementry figure 3. ltered host gene expressions in infected cells. () The reltive expression levels of Smd7 nd FGF2 in - nd SARS-CoV-infected Clu-3 cells were quntified y RT-qPCR s descried. 2 () The reltive expression levels of BIRC 3 mrna in -infected or -infected Clu-3 nd HK2 cells were mesured y RTqPCR. (c) The effect of overexpressed BIRC3 on repliction in Clu-3 (top) nd HK2 (ottom) were mesured y RT-qPCR. (d) Other deregulted genes not directly relted to Virl lod (copy no. normlized to GAPDH) poptosis, clusterin (CLU), superoxidse dimutse 1 (SOD1) nd ctlse (CAT) which expressions were ffected y infection s shown in Fig. 2c, were selected for investigting their effects on repliction. Their expressions were individully knocked down y sirna trnsfection followed y infection. The virl lods were mesured s descried ove. All vlues were normlized to the expression level of GAPDH mrna. Error rs shown in nd, c nd d represent the men ± s.d. of three independent experiments nd experiments done in triplicte, respectively. Sttisticl significnce ws evluted y Student s t-tests nd the rnges of p-vlues were indicted in, nd c. Smples from,, c nd d represent iologicl replictes. NATURE MICROBIOLOGY 1 4

5 DOI: 1.138/NMICROBIOL DOI: 1.E+7 DOI: 1.138/NMICROBIOL Virl titer (PFU/ml) 1.E+6 1.E+5 1.E+4 Clu-3 HK Antisense oligonucleotide (µg/ml) Inhiition of virl lod (%) * p =.1 * p =.1 M.O.I. = 9.6 M.O.I. =.96 sismd7 sicontrol Supplementry figure 4. The ntivirl ctivity of nti-smd7 oligonucleotide. () The virus plque forming units of -infected HK2 (drk dimond) nd Clu-3 cells (grey squre) were mesured t the indicted concentrtion of nti-smd7-treted cells. () HK2 cells trnsfected with sismd7 (left) or sicontrol (right) were infected with high (M.O.I. = 9.6; lck) or low (M.O.I. =.96) virl titer of. The virl lods were mesured y RT-qPCR s we previously descried 2. Error rs shown in nd represent the men ± s.d. of three independent experiments. Sttisticl significnce ws evluted y Student s t-tests nd the NATURE MICROBIOLOGY NATURE MICROBIOLOGY rnges of p-vlues were indicted in nd. Smples from nd represent iologicl replictes. NATURE MICROBIOLOGY 5 1

6 DOI: 1.138/NMICROBIOL DOI: RNA (copy no. normlized to GAPDH) Virl lod (copies/ml) 1.2E+7 1.E+7 8.E+6 6.E+6 4.E+6 2.E+6 8.E+7 7.E+7 6.E+7 5.E+7 4.E+7 3.E+7 2.E+7 Superntnt Cell lyste * p =.1 sicontrol sismd7 1.E+7 hpi 2 hpi 4 hpi 12 hpi RNA (copy no. normlized to GAPDH) DOI: 1.138/NMICROBIOL Negtive strnded RNA detection 9.E+6 8.E+6 7.E+6 6.E+6 5.E+6 4.E+6 3.E+6 2.E+6 1.E+6 sicontrol sismd7 hpi 2 hpi 4 hpi 12 hpi c sirna: sismd7 sicontrol hpi: C NP γ-tuulin Supplementry figure 5. Time-point nlysis of the knockdown effect of Smd7 on the repliction. () The RNAs from the mture viruses secreted to the conditioned medi (top) nd the mrna from the infected cell lystes (ottom) were collected from sismd7-trnsfected HK2 cells (grey). The virl lods were mesured y RTqPCR s we previously descried 2 nd then compred with tht of the sicontrol-trnsfected HK2 cells (lck). The red line indictes the virl lods from the superntnts of the sismd7- NATURE MICROBIOLOGY NATURE MICROBIOLOGY trnsfected HK2 cells t 12 hpi. () Negtive strnded RNA detection of the sismd7- nd sicontrol-trnsfected HK2 cells. Specific primer ws designed to reverse trnscrie the negtive strnded of the RNAs isolted from the infected cells t different time-points s descried in (). (c) Western lot nlysis of the NP from the sismd7- nd sicontrol-trnsfected HK2 cell lystes. Protein smples hrvested t different time-points of the infected cells (descried in ()) were resolved in sodium sulfte polycrylmide gel followed y the detection using nti- NP ntiodies. γ-tuulin ws detected s loding control. C = -infected HK2 smple extrcted from nother experiment ws included s positive control. Imge shown in c is representtive of experiments done in triplicte. Error rs shown in nd represent the men ± s.d. of experiments done in triplicte. Sttisticl significnce ws evluted y Student s t-tests nd the rnges of p-vlues were indicted in. Smples from, nd c represent iologicl replictes. NATURE MICROBIOLOGY 6 1

7 DOI: 1.138/NMICROBIOL DOI: DOI: 1.138/NMICROBIOL Virl lod (copy no.normlized to GAPDH) 3.E+9 2.5E+9 2.E+9 1.5E+9 1.E+9 5.E+8 * p =.1 NP γ-tuulin C A Virl lod (copy no.normlized to GAPDH) 1.6E+1 1.4E+1 1.2E+1 1.E+1 8.E+9 6.E+9 4.E+9 2.E+9 * p =.1 2 µm Etoposide * p =.1 NP γ-tuulin C I C I 5 µm 1 µm MDIVI Supplementry figure 6. The effect of poptosis on the repliction. () The virl lods (top) nd the levels of NP expression (ottom) were detected in the presence (A) or sence (C) of etoposide, n poptosis-inducer of the -infected cells t 24 hpi. () The virl lods (top) nd the levels of NP expression (ottom) were detected in the presence (I) or sence (C) of MDIVI, n poptosis-inhiitor of the -infected HK2 cells t 24 hpi. Imge shown in nd is representtive of experiments done in triplicte. Error rs shown in nd represent the men ± s.d. of experiments done in triplicte. NATURE NATURE MICROBIOLOGY MICROBIOLOGY Sttisticl significnce ws evluted y Student s t-tests nd the rnges of p-vlues were indicted in nd. Smples from nd represent iologicl replictes. NATURE MICROBIOLOGY 7 1

Rdiogrphic ltertions nd lung pthology of infected common mrmosets on dy 3

() Dorsl-ventrl nd lterl thorcic X-rys from MERS- CoV-inoculted common

() Gross pthology of the necropsied lungs from () showing mrked

8 DOI: 1.138/NMICROBIOL DOI: -inoculted Mock 4 mm -inoculted Mock 25 mm Supplementry figure 7. Rdiogrphic ltertions nd lung pthology of infected common mrmosets on dy 3 post-infection. () Dorsl-ventrl nd lterl thorcic X-rys from MERS- CoV-inoculted common mrmosets. () Gross pthology of the necropsied lungs from () showing mrked congestion nd consolidtion in oth lungs. The uninfected control X- rys nd gross pthology re shown djcent on the right side. Imges shown in nd re representtives of the six common mrmosets inoculted with. NATURE MICROBIOLOGY 1 8

3 4 3 µm FGF2 Smd7 3 µm 3 µm c 1 µm 1 µm Supplementry figure 8.

mrmosets (CM1 to CM6) nd n uninfected control.

kidney; P = MERS- NATURE MICROBIOLOGY NATURE MICROBIOLOGY www.nture.

cspse-3 (top), Smd7 (middle) nd FGF2 (ottom) were shown s descried in Fig. 4.

9 DOI: 1.138/NMICROBIOL DOI: GAPDH CM1 CM2 CM3 CM1 CM2 CM3 CM4 CM5 CM6 CM4 CM5 CM6 uninfected -NP P Cspse 3 DOI: 1.138/NMICROBIOL µm FGF2 Smd7 3 µm 3 µm c 1 µm 1 µm Supplementry figure 8. Detection of RNA nd ntigen from the lungs nd kidneys of -infected common mrmosets on dy 3 post-infection. () RT-qPCR for RNA from different prts of lungs nd kidneys of the -infected common mrmosets (CM1 to CM6) nd n uninfected control. = right upper loe; = left lower loe; = right lower loe; = left upper loe nd = kidney; P = MERS- NATURE MICROBIOLOGY NATURE MICROBIOLOGY CoV-infected HK2 smple extrcted from nother experiment ws included s positive control. () Immunohistochemicl stining of lungs from -infected common mrmosets for NP, cspse-3 (top), Smd7 (middle) nd FGF2 (ottom) were shown s descried in Fig. 4. (c) Histopthologicl chnges in lungs of -inoculted common mrmosets. Infected lung tissues showed cute ronchointerstitil pneumoni with influx of inflmmtory cells nd thickening of lveolr sept. Imges shown in, nd c re representtives of the six common mrmosets inoculted with. NATURE MICROBIOLOGY 9 1

10 DOI: 1.138/NMICROBIOL DOI: 1.8E+4 1.6E+4 1.4E+4 DOI: 1.138/NMICROBIOL E+4 Virl lod 1.E+4 8.E+3 6.E+3 4.E+3 2.E+3 mrna/gapdh expression (fold) (fold) Smd7 mrna/gapdh expression (fold) FGF2 1.6E+3 1.9E+3 1.4E+4 1.6E+4 dney tissues with vrious virl lod normlized with GAPDH Supplementry figure 9. Virl lods nd host gene expression in the kidney of common mrmosets inoculted with on dy 3 post-infection. Quntittive mesurement of RNA in the kidneys of -inoculted common mrmosets (top). The reltive expression levels of Smd7 (middle) nd FGF2 (ottom) of the corresponding smples were indicted. All vlues were normlized to GAPDH. Ech r, except lne 1 which is n NATURE NATURE MICROBIOLOGY MICROBIOLOGY uninfected control, represents kidney smple otined from common mrmoset inoculted with. NATURE MICROBIOLOGY 1 1

DOI: 1.138/NMICROBIOL.216.4 DOI: 1.138/NMICROBIOL.216.4 DOI: Norml Infected * * * * * * * 1 µm 1 µm Tuulr cells without elongted mitochondri (%) 9 8 7 6 5 4 3 2 1 Supplementry figure 1.

dneys from the -infected common mrmosets (CM1 to CM6) nd n uninfected control were fixed for the electron microscopic exmintion.

11 DOI: 1.138/NMICROBIOL DOI: 1.138/NMICROBIOL DOI: Norml Infected * * * * * * * 1 µm 1 µm Tuulr cells without elongted mitochondri (%) Supplementry figure 1. 2D electron microscopic exmintion of mitochondril frgmenttion in kidney tissues. dneys from the -infected common mrmosets (CM1 to CM6) nd n uninfected control were fixed for the electron microscopic exmintion. () A representtive picture shows the length of mitochondri of the infected injured tuulr cells (right). This is in contrst with tht of the norml uninfected control which showed undnt elongted mitochondri (sterisks = mitochondri with length > 2 μm). () Quntifiction of mitochondril frgmenttion. Mitochondril length ws mesured in individul tuulr cells to determine the percentge of cells tht showed filmentous mitochondri (length < 2 μm). Ech r, except lne 7 which is n uninfected control, represents kidney smple otined from common mrmoset inoculted with. NATURE MICROBIOLOGY NATURE NATURE MICROBIOLOGY 1 1

12 DOI: DOI: 1.138/NMICROBIOL Immunostin of uninfected common mrmoset kidneys FGF2 Cspse 3 3 µm Smd7 DOI: 1.138/NMICROBIOL µm 3 µm -NP 3 µm DPP4 3 µm ACE2 3 µm Histopthology of uninfected common mrmoset kidneys NATURE NATURE MICROBIOLOGY µm 1 µm Supplementry figure 11. Immunostin nd histopthologicl nlyses of the uninfected common mrmoset s kidney. () Immunohistochemicl stinings of cspse-3, Smd7, FGF2, NP, DPP4 nd ACE2 in kidneys of uninfected common mrmosets. Nuclei counterstined y DAPI were shown in lue color. () Hemtoxylin nd eosin stining of kidney sections of uninfected common mrmosets. Imges re representtives of n uninfected common mrmoset. NATURE MICROBIOLOGY

13 MICROBIOL DOI: 1.138/NMICROBIOL Rw dt for figure 1 -infected detection Lne Hours SARS-CoV-infected SARS-CoV detection infected GAPDH detection Lne Hours SARS-CoV-infected GAPDH detection Rw dt for figure 2 SARS-CoV SARS-CoV SARS-CoV SARS-CoV Smd7 NP γ-tuulin Cleved Cspse 3 SARS-CoV SARS-CoV FGF2 SARS-CoV NP Supplementry figure 12 NATURE MICROBIOLOGY 13

14 MICROBIOL DOI: 1.138/NMICROBIOL Rw dt for figure 3 nd Smd7 sismd7-1 sismd7-2 sicontrol Anti-FGF2 IgG Control sismd7-1 sismd7-2 sicontrol Cleved Cspse 3 Cleved Cspse 3 Anti-FGF2 IgG Control sismd7-1 sismd7-2 sicontrol γ-tuulin γ-tuulin Supplementry figure 12 (cont d) NATURE MICROBIOLOGY 14

138/N4 Rw dt for supplementry figure 1 NP

15 MICROBIOL DOI: 1.138/NMICROBIOL Rw dt for supplementry figure 1 NP γ-tuulin Supplementry figure 12 (cont d) NATURE MICROBIOLOGY 15

138/N4 Rw dt for supplementry figure 5c sirna: sismd7 sicontrol

16 MICROBIOL DOI: 1.138/NMICROBIOL Rw dt for supplementry figure 5c sirna: sismd7 sicontrol hpi: C NP sirna: sismd7 sicontrol hpi: C γ-tuulin Supplementry figure 12 (cont d) NATURE MICROBIOLOGY 16

17 MICROBIOL DOI: 1.138/NMICROBIOL Rw dt for supplementry figure 6 nd 2 µm Etoposide C A 2 µm Etoposide C A NP γ-tuulin MDIVI 5 µm 1 µm C I C I MDIVI 5 µm 1 µm C I C I γ-tuulin NP Supplementry figure 12 (cont d) NATURE MICROBIOLOGY 17

18 DOI: 1.138/NMICROBIOL DOI: 1.138/NMICROBIOL Rw dt for supplementry figure 8 CM1 CM2 CM3 CM1 CM2 CM3 GAPDH CM1 CM2 CM3 CM1 CM2 CM3 CM4 CM5 CM6 CM4 CM5 CM6 GAPDH CM4 CM5 CM6 CM4 CM5 CM6 P uninfected GAPDH P Supplementry figure 12 (cont d) NATURE MICROBIOLOGY

19 DOI: 1.138/NMICROBIOL DOI: 1.138/NMICROBIOL Supplementry References 1. Ni, L., Sleem, M. & Mthieson, P.W. Podocyte culture: tricks of the trde. Nephrology (Crlton) 17, (212). 2. Chn, J.F., et l. Differentil cell line susceptiility to the emerging novel humn etcoronvirus 2c EMC/212: implictions for disese pthogenesis nd clinicl mnifesttion. J Infect Dis 27, (213). NATURE MICROBIOLOGY 1 NATURE MICROBIOLOGY 191

20 DOI: DOI: 1.138/NMICROBIOL DOI: 1.138/NMICROBIOL Berlin Definition of cute respirtory distress syndrome (ARDS): 1) Timing: within 1 week of known clinicl insult or new or worsening respirtory symptoms. 2) Chest imging: ilterl opcities not fully explined y effusions, lor/lung collpse, or nodules. 3) Origin of edem: respirtory filure not fully explined y crdic filure or fluid overlod Tle S1. Clinicl score sheet for common mrmosets inoculted with. Score Generl Appernce Norml nd lert, moving without prompting Slow/ quiet, hunched, ut lert, interested, moving without prompting 5 Quieter, hunched,ut lert,moving needs lot of prompting 1 Loss of interest, lmost impossile to prompt to move, dull expression 15 flling sleep while wtched, little or no response to humn presence Skin nd Fur Norml Pilo erection/ unkept ppernce 5 Dischrges Orl/ nsl/ oculr 5 Respirtion Norml (6-1 pm 1 ) Incresed (1-12 pm) 5 Severely incresed (> 12 pm), lored, cough, open mouth rething 1 Dyspne, cynosis, fom 15 Food consumption Loss of ppetite 2 Anorexi 5 Totl 2 1 Breths per minute. 2 Euthnsi is indicted t clinicl score of 35. NATURE NATURE MICROBIOLOGY NATURE MICROBIOLOGY 1 2

Acute and gradual increases in BDNF concentration elicit distinct signaling and functions in neurons

Acute and gradual increases in BDNF concentration elicit distinct signaling and functions in neurons nd grdul increses in BDNF concentrtion elicit distinct signling nd functions in neurons Yunyun Ji,, Yun Lu, Feng Yng, Wnhu Shen, Tin Tze-Tsng Tng,, Linyin Feng, Shumin Dun, nd Bi Lu,.. - Grdul (normlized