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SHABNAM HASHEMI ET AL.

ing infection. Apoptosis is a form of programmed cell regulation of necrosis (14). This study aimed to in- death by receptor activation and is a conserved vestigate the effects of mix-infection with IBV and process. It is an important part of the host’s innate APEC on activation of host apoptosis on the chicken immune response, which helps in early control of trachea and identifying genes involved in apoptosis the various infections (2). One of the strategies that by RNA-seq. supports intracellular survival and persistence of the viruses, bacteria, and parasites is inhibited host cell by apoptosis and the reorganization of cellular recep- MATERIALS AND METHODS tors, or by the activation of apoptotic proteins such as caspases. Many pathogenic bacteria and viruses Virus and bacterial strains. Avian infectious can repress apoptosis during infection by encoding bronchitis virus variant 2 from the virus collection proteins to escape from immune attack, or viruses and avian pathogenic E. coli serotype O78: K80 from can promote apoptotic death of the host cells (3). the University of Tehran culture collection was used There are four apoptotic pathways consist of: the in this study. internal mitochondrial pathway (the intrinsic path- way), the death receptor-mediated external apoptotic Chicken challenge and tissue collection. Forty pathway (the extrinsic pathway), the endoplasmic re- SPF (specific pathogen-free) chickens were divided ticulum stress pathway, and the B-granzyme-medi- equally into two groups: one experimental group and ated apoptosis pathway. The protein molecules that one control group. All groups were kept in separate were involved in the apoptosis mechanisms are Cys- isolators. At 12 days of age, each chicken was inocu- 4 teinyl aspartate specific proteins (Caspase), adapter lated via oculonasal inoculation of 100 µl of 10 EID50 proteins, B-cell lymphoma-2 (Bcl2), an inhibitor of infective doses of IBV, and 72 hours later were inocu- apoptosis proteins (IAPs) (4). lated via the intratracheal route of 100 µl of 108 CFU/ Regulation of intrinsic apoptosis processes is by the ml infective dose of APEC, and control groups were BCL-2 and caspase families of proteins (5, 6). inoculated with 100 μl PBS. Chickens were eutha- Caspases can be classified into three groups: apopto- nized at 3 days post-injection (dpi), and the tracheal sis initiator (CASP2, CASP8, CASP9, and CASP10), organs were harvested. A piece of trachea was added inflammatory CASPs (CASP1, CASP4, CASP15, directly to the RNAlater solution (Thermo Fisher Sci- CASP11, and CASP12), and apoptosis executioner entific) and then stored at -80°C. A piece of trachea (CASP3, CASP6, and CASP7) (7). The inhibitors of samples from the challenge group and control group apoptosis proteins (IAP) can regulate the activity of were included in the qPCR validation of the RNA- caspases. Cathepsin D can also regulate apoptosis (8). seq results for IBV detection and culture for APEC's Other factors for regulating immune responses, colony count. apoptosis, and cell survival, are the mitogen-acti- vated protein kinases (MAPKs) families and NFKB RNA isolation. According to the manufacturer's in- transcription factors (9). struction, total RNA from samples with the adequate Three MAPK families have been characterized: virus and bacterial load was extracted from samples Jun kinase (JNK/SAPK), extracellular signal-regu- of control and challenged groups using TRIzol re- lated kinase (ERK), and p38 MAPK (10). Caspase-8 agent (Thermo Fisher Scientific). On-column DNA is a cell-extrinsic apoptotic signal that can cause digestion was performed using RNase-free DNase transduction by death receptors (DRs), DR4, DR5, (SinaClone) to remove contaminating genomic DNA. and Fas into a death-inducing signaling complex RNA samples quantified using an Agilent Bioanalyz- (DISC) (11, 12). er 2100. These total RNAs were subjected to the Bei- One of the molecules that are transducer meta- jing Genome Institute (BGI) (Shenzhen, China) for bolic and mitogenic signals to promote cell growth, RNA-seq and transcriptome analysis. migration, proliferation, and apoptosis are phospho- inositide 3-kinases (PI3Ks) family and PTPs (13). Illumina library construction, RNA sequenc- Also, receptor-interacting protein kinase (RIPK) 1 ing, and analysis. Illumina HiSeq 2500 platform, and RIPK3 that can be induced by tumor necrosis paired-end, and strand-specific sequencing pro- factor (TNF) signaling, are essential kinases for the duced an average read length of ~150 bp. Acquired

18 IRAN. J. MICROBIOL. Volume 13 Number 1 (February 2021) 17-22 http://ijm.tums.ac.ir TRANSCRIPTOME ANALYSIS OF APOPTOSIS GENES

data were processed to obtain raw reads, which Table 1. KEGG pathway analysis for apoptotic genes. Path- were then extracted for quality control, trimming, way analysis enrichment using DEGs between control and read mapping, and identified differentially ex- the group of inoculated with IBV and APEC. FDR < 0.05 pressed genes (DEGs) using CLC Biomedical soft- was used as a threshold to select significant KEGG path- ware (Trial version) that were aligned using refer- ways. The KEGG ID was gga04210. ence genome (Gallus Gallus reference) extracted of

NCBI. For DEGs analysis, after normalized data by Genes Fold change Genes Fold change

CLC Biomedical software and statistical analysis value value by Beggarly test, DEGs were extracted by compar- TRADD DIV RIPK1 16 ing gene expression levels between the infected and BCL2A1 DIV DAB2IP 14 control groups. For visualization and discovery anal- MAPK1 553 PTPN13 13 ysis of DEGs to perform functional annotation, GO APAF1 228 TNFSF10 13

(Gene Ontology) analysis was performed by Enrichr TRAF2 199 PIK3R2 13

(http://amp.pharm.mssm.edu/Enrichr), then, terms PIK3CA 133 CTSC 11 with corrected FDR<0.05 were considered signifi- CASP3 107 BID 10 cantly enriched in differentially expressed genes. Ob- CASP7 65 CFLAR 10 tained DEGs were analyzed for pathway enrichment RAF1 52 CASP2 9 analysis based on the KEGG (Kyoto Encyclopedia of CTSD 48 FADD 9 Genes and Genomes) database (FDR<0.05) to explore ITPR3 40 MAP3K14 8 the various biological processes. The apoptosis path- PIK3CD 26 NFKB1 8 way was selected among the obtained pathways, and EIF2AK3 16 PIK3CB 8 the genes group was collected, then genes were en- ATM 16 ITPR2 3 riched in the STRING database for predicted protein interactions.

RESULTS

Functional analysis of apoptosis genes. After the analysis of DEGs by comparing extracted genes be- tween the infected group and the control group, this was identified that all of the apoptosis genes were up-regulated. The list of apoptotic genes is in Table 1 from most fold change value to least, respective- ly. The more fold change value was for TRADD and BCL2A1 genes that TRADD is one of the proteins associated with the extrinsic pathway, and BCL2A1 is a member in the intrinsic pathway, and the less fold change value was for MAP3K14, NFKB1, PIK3CB, and ITPR2 genes (Table 1). The genes were enriched in the STRING database for predicted protein interactions. The apoptotic genes contained 28 nodes associated with 79 interac- tions (edges) (Fig. 1). Fig. 1. STRING analysis of the relationship between differ- entially expressed genes (FDR < 0.05) related to the apop-

totic pathways in up-regulated genes in the mixture infection DISCUSSION group with IBV and APEC. The network nodes represent the proteins encoded by the DEGs, and the edges represent pro- This novel experiment characterized the type of tein-protein interactions. apoptotic pathways in the chicken trachea mix-in-

http://ijm.tums.ac.ir IRAN. J. MICROBIOL. Volume 13 Number 1 (February 2021) 17-22 19 SHABNAM HASHEMI ET AL.

fected with IBV (variant 2) and APEC (O78: K80) in ously. the early phase of infection by survey transcriptome Between these two pathways, most of our study changes in the chicken trachea with focuses on the genes were for the intrinsic pathway. However, we use of the strand-specific NGS method. know that the molecules in one pathway can influ- Apoptosis is a conserved process for early con- ence the other, and two pathways are linked (28). A trol of the various infections that is consists of group of negative regulators of both caspases and cell many pathways. The cells can sense the presence death is an inhibitor of apoptosis proteins (IAP) (29). of pathogen proteins and eliminate them by induc- In our study, anti-apoptotic protein CFLAR, RIPK1, ing apoptosis (15). In this study, we found a total of and TRAF2 were found. CFLAR and TRAF2 sup- investigated twenty-eight apoptotic genes (Table 1) pressed CASP8 activation (30). RIPK1 kinase activi- were up-regulated, and they consisted of six path- ty and RIPK3 have both caspase-dependent and inde- ways related to cell death, and some regulator and pendent mechanisms that contribute to TNF induced apoptosis inhibitors. In the extrinsic pathway by apoptosis. In addition to the above, two regulators, transduction of cell-extrinsic apoptotic signals by CTSD (Cathepsin D) and CTSC, were found in our the death receptors (DRs), the caspase-8 recruiting study, too, that they both are an important regulator into a death-inducing signaling complex (DISC) and of apoptotic pathways in cells and autophagy (31, 32). induce a conformational change in the receptor that The endoplasmic reticulum stress pathway was leads to recruitment of TNFR1-associated death another apoptotic pathway that was found in our domain protein (TRADD) or Fas-associated death study. Three ER-resident transmembrane proteins domain protein (FADD) that they lead to apoptosis ERN1 (endoplasmic reticulum to nucleus signaling (16). In this study, the expression of TRADD was 1), ATF6 (activating transcription factor 6), and EI- higher than FAAD. This study also demonstrated the F2AK3 (eukaryotic translation initiation factor 2-α PTPN13 gene, which is an enzyme member of the kinase 3) are the stress sensors of ER, among them protein tyrosine phosphatase (PTP) family, that can EIF2AK3-EIF2S1 or the ERN1-MAPK8/9/10 path- interact and dephosphorylate the Fas receptor, which way is a crucial mediator of ER stress-induced au- suggested this is the role of this protein in Fas-medi- tophagy (33, 34), that EIF2AK3 was found in our ated programmed cell death (17). In previous studies, study. The previous study showed that in mammal PLCB2, TRAF2, JAK1, CBLB, FOS, SOCS4, TN- Vero cells, when infected with IBV, the endoplasmic FRSF1A, CASP8, and TRADD in E. coli infection reticulum stress pathway was reported. Also, IBV were shown (18, 19). was found to induce ER stress in infected cells at a In the intrinsic pathway, the release of the pro-apop- late stage of infection (35), while this study showed totic proteins including cytochrome c, activates the that infection with a mix of IBV and APEC induced BCL-2 family members Bax and Bak results in this form of apoptosis in trachea cells in the early MOMP (mitochondrial outer membrane permeabili- phase of infection. zation), from the inter-membrane space into the cyto- Also, ITPR2 and ITPR3 were found in this study. sol (20, 21). Cytochrome c bind to APAF1 and form- Apoptosis is one Ca2+-mediated event influenced by ing the apoptosome and activating caspase-9, and this ITPR isoforms such as ITPR3 and ITPR2 (36, 37). molecule cleave and activate caspase-3 and caspase-7 Cell death by NFKB was another pathway in our (22). In present research showed BCL2A1 and BID. study. NFKB consists of five subunits: RelB, p65 BID is the proapoptotic Bcl2 family member that can (RelA), p50 (NFKB1), c-Rel, and p52 (NFKB2) that migrate to mitochondria by protease-cleaved (Trun- they are homo-and hetero-dimmers. The most well- cated BID (tBID)) (23), and APAF1 is related to this known NFKB dimer is p50: p65 (38), that they are pathway. Current research found ATM that ATM is generally thought to be transcriptional repressors a substrate for caspase 3 in vitro. ATM can prevent by bounding to DNA (39). In our study, NFKB was DNA repair and DNA damage signaling by cleavage shown to be one of the subunits of NFKB. during apoptosis and generate kinase-inactive (24). Raf-1 is one of the upstream component in the The presence of caspase-1, caspase-9, caspase-18, MAPK that can activate MEK (MAP kinase kinase) Bcl-2L1, BCL2-antagonist/killer 1, Mcl-1, caspase-3, and then activates MAP kinase by phosphorylation Bax, and Bak in IBV infection (25, 26) and the pres- (42). Subsequently other downstream kinases can be ence of caspase 3/7 in APEC (27) was shown previ- activated by MAP kinase (43).

20 IRAN. J. MICROBIOL. Volume 13 Number 1 (February 2021) 17-22 http://ijm.tums.ac.ir TRANSCRIPTOME ANALYSIS OF APOPTOSIS GENES

Previously MAPK8 was shown to be the main points. Cell 2004; 116: 205-219. pathway for TNFSF10-induced autophagy (44). 6. Galluzzi L, Vitale I, Abrams J, Alnemri E, Baehrecke E, Blagosklonny M, et al. Molecular definitions of cell Our study demonstrated that MAPK14, MAPK1, death subroutines: recommendations of the Nomencla- TNFSF10, and RAF-1, also exist in this pathway. ture Committee on Cell Death 2012. Cell Death Differ Other protein groups that contribute to cell death 2012; 19: 107-120. are PI3K complexes. In our study PI3KCA was found, 7. Zhang Y, Zhong X, Chen Y, Liu S, Wu G, Liu Y. As- PI3KCB, PI3KCD, and PIK3R2. Class I PI3Ks form sociation between CASP-8 gene polymorphisms and a functional heterodimer composed of two subunits cancer risk in some Asian population based on a HuGE consist of catalytic (110 kDa (p110)) and regulatory review and meta-analysis. Genet Mol Res 2013; 12: (85 kDa (p85)). The two regulatory subunits are p85β 6466-6476. (PIK3R2) and p85α (45). The catalytic p110 subunit 8. Deiss LP, Galinka H, Berissi H, Cohen O, Kimchi A. has four isoforms consist of PI3KCA, PI3KCG, Cathepsin D protease mediates programmed cell death PI3KCB, and PI3KCD. PI3K activates protein kinase induced by interferon-gamma, Fas/APO-1 and TNF-al- B (PKB), which activates the mTOR pathway (46). pha. EMBO J 1996; 15: 3861-3870. The previous study showed PIK3CD and PIK3R1 in 9. Concetti J, Wilson CL. NFKB1 and cancer: friend or foe? Cells 2018; 7: 133. APEC infection, too (19). 10. Zhang W, Liu HT. MAPK signal pathways in the regu- The pathways responsible for causing cell death, lation of cell proliferation in mammalian cells. Cell Res in this review were the extrinsic pathway, intrinsic 2002; 12: 9-18. pathway, endoplasmic reticulum stress pathway, 11. Ashkenazi A, Dixit VM. Apoptosis control by death and MAPK signaling pathway, and cell death by NFkB decoy receptors. Curr Opin Cell Biol 1999; 11: 255- and activates mTOR pathway. Although many apop- 260. totic and anti-apoptotic proteins are involved in dif- 12. Thorburn A. Death receptor-induced cell killing. Cell ferent apoptotic pathways, research is still required Signal 2004; 16: 139-144. in this field from a deeper perspective and the ther- 13. Engelman JA, Luo J, Cantley LC. The evolution of apeutic side. phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 2006; 7: 606-619. 14. Dondelinger Y, Aguileta M, Goossens V, Dubuisson C,

Grootjans S, Dejardin E, et al. RIPK3 contributes to ACKNOWLEDGEMENTS TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase in- The authors gratefully thank Iran National Science hibition. Cell Death Differ 2013; 20: 1381-1392. Foundation for supporting this study by granting this 15. Everett H, McFadden G. Apoptosis: an innate immune project (95836652). response to virus infection. Trends Microbiol 1999; 7: 160-165.

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