Virus Infection Regulates Apoptosis During Influenza a Synthase Gene

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Transactivation of Inducible Nitric Oxide Synthase Gene by Kruppel-like Factor 6 Regulates Apoptosis during Influenza A Virus Infection This information is current as of October 2, 2021. Victoria Mgbemena, Jesus A. Segovia, Te-Hung Chang, Su-Yu Tsai, Garry T. Cole, Chiung-Yu Hung and Santanu Bose J Immunol published online 18 June 2012

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 18, 2012, doi:10.4049/jimmunol.1102742 The Journal of Immunology

Transactivation of Inducible Nitric Oxide Synthase Gene by Kruppel-like Factor 6 Regulates Apoptosis during Inﬂuenza A Virus Infection

Victoria Mgbemena,*,1 Jesus A. Segovia,*,1 Te-Hung Chang,* Su-Yu Tsai,* Garry T. Cole,†,‡ Chiung-Yu Hung,†,‡ and Santanu Bose*

Influenza A virus (flu) is a respiratory tract pathogen causing high morbidity and mortality among the human population. NO is a cellular mediator involved in tissue damage through its apoptosis of target cells and resulting enhancement of local inflammation. Inducible NO synthase (iNOS) is involved in the production of NO following infection. Although NO is a key player in the development of exaggerated lung disease during flu infection, the underlying mechanism, including the role of NO in apoptosis during infection, has not been reported. Similarly, the mechanism of iNOS gene induction during flu infection is not well defined in terms of the host transactivator(s) required for iNOS gene expression. In the current study, we identified Kruppel-like factor 6 Downloaded from (KLF6) as a critical transcription factor essential for iNOS gene expression during flu infection. We also underscored the requirement for iNOS in inducing apoptosis during infection. KLF6 gene silencing in human lung epithelial cells resulted in the drastic loss of NO production, iNOS promoter-specific luciferase activity, and expression of iNOS mRNA following flu infection. Chromatin immunoprecipitation assay revealed a direct interaction of KLF6 with iNOS promoter during in vitro and in vivo flu infection of human lung cells and mouse respiratory tract, respectively. A significant reduction in flu-mediated apoptosis was noted in KLF6-silenced cells, cells treated with iNOS inhibitor, and primary murine macrophages derived from http://www.jimmunol.org/ iNOS knockout mice. A similar reduction in apoptosis was noted in the lungs following intratracheal flu infection of iNOS knockout mice. The Journal of Immunology, 2012, 189: 000–000.

nfluenza A virus (flu) is an ssRNA virus that causes severe the infected host, it can also exaggerate the disease pathology respiratory diseases upon infection of the airway (1, 2). Flu associated with infection. This detrimental effect of NO is due I infection among high-risk individuals (e.g., elderly, immu- to its ability to launch a hyperinflammatory response, leading to nocompromised individuals) manifests itself in the progression of tissue damage (10). Two mechanisms are primarily responsible

an inflammatory disease state such as pneumonia (1–3). for inducing NO-mediated tissue damage: action of NO-induced by guest on October 2, 2021 NO (4) plays an important role in the innate immune response inflammatory mediators and apoptosis of NO-targeted cells. against pathogens (4–9). Although it confers a protective role in With regard to host defense against viruses, NO could act as a “double-edged sword.” Although functioning as an antiviral molecule, NO can contribute to theprogression/exaggeration of virus-induced disease (11). Flu infection results in NO pro- *Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229; †Department of Biology, duction as a result of induction of the inducible NO synthase The University of Texas, San Antonio, TX 78249; and ‡South Texas Center for (iNOS) gene (12). Although a specific transcription factor re- Emerging Infectious Diseases, The University of Texas, San Antonio, TX 78249 quired for iNOS gene expression during flu infection has not 1 V.M. and J.A.S. contributed equally to the work. been identified and characterized, one study suggested that NF- Received for publication September 21, 2011. Accepted for publication May 10, kB (an inflammatory transcription factor) may play a role in 2012. iNOS gene activation during flu infection (12). NO produced from This work was supported by National Institutes of Health Grants AI083387 (to S.B.) and AI071118 (to G.T.C.) and a grant from the Center for Innovation in Prevention the lungs during infection contributes to tissue damage and en- and Treatment of Airway Diseases (to S.B.). V.M. and J.A.S. were supported by hanced disease pathology associated with flu infection (13–15). National Institutes of Health/National Institute of Dental and Craniofacial Research NO-mediated apoptosis represents one of the key factors contrib- Grant DE14318 for the Craniofacial Oral-Biology Student Training in Academic Research program. The University of Texas Health Science Center at San Antonio uting to enhanced inflammation and tissue damage during nonin- FACS Core Facility was supported by Cancer Center Program Grant NIH P30 fectious respiratory diseases, such as asthma and chronic obstructive CA54174. pulmonary disease (10, 16). Although flu infection results in NO Address correspondence and reprint requests to Dr. Santanu Bose, Department of Microbiology and Immunology, The University of Texas Health Science Center at production, the contribution of NO in apoptosis during infection is San Antonio, 7703 Floyd Curl Drive, MC-7758, San Antonio, TX 78229. E-mail not known. Furthermore, with the exception of NF-kB, the role of address: [email protected] noninflammatory transcription factor(s) in the regulation of iNOS The online version of this article contains supplemental material. gene expression during respiratory virus infection (especially Abbreviations used in this article: BMDM, bone marrow-derived macrophage; ChIP, during flu infection) has not been examined. chromatin immunoprecipitation; flu, influenza A virus; iNOS, inducible NO synthase; In the current study, we identified triple zinc finger containing DNA KLF, Kruppel-like factor; KO, knockout; L-NIL, N6-(1-iminoethyl)-L-lysine dihydrochloride; PI, propidium iodide; poly-IC, polyinosinic-polycytidylic acid; shRNA, binding transcription factor Kruppel-like factor (KLF)6 (17, 18) as short hairpin RNA; UTHSCSA, University of Texas Health Science Center at San a transcription factor required for iNOS gene induction during flu Antonio; UV-flu, UV-irradiated flu; WT, wild-type. infection. We also demonstrated that induction of iNOS (leading to Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 production of NO) during flu infection contributes to apoptosis of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102742 2 ROLE OF KLF6 AND iNOS DURING INFLUENZA A VIRUS INFECTION both epithelial and immune cells (i.e., macrophages). The in vivo Viral infection of cells physiological relevance of the contribution of iNOS to apoptosis A549 cells were infected with purified flu in reduced serum and antibiotic- during flu infection was further documented using wild-type (WT) free Opti-MEM medium (Life Technologies). Following adsorption for and iNOS knockout (KO) mice. 1.5 h at 37˚C, cells were washed twice with serum-containing DMEM, and The KLF family of transcription factors controls a wide the infection was continued in the presence of serum-containing DMEM spectrum of biological and physiological processes, including cell for the specified times. BMDMs were grown in RPMI 1640 medium supplemented with 10% FBS, sodium pyruvate, L-glutamine, GM-CSF, growth, cell proliferation, and differentiation (17, 18). Although HEPESbuffer.BMDMswereinfectedwithpurifiedfluinserum KLF factors have been implicated in regulating normal cellular/ and antibiotic-free RPMI 1640 medium. Following adsorption for 1.5 h at tissue homeostasis, their role during infection has not been ex- 37˚C, cells were washed twice with serum-containing RPMI 1640, and the amined. In the current study, we identified KLF6 as a regulator infection was continued in the presence of serum-containing RPMI 1640 for the specified times. For some experiments, A549 cells were pretreated of iNOS gene expression during flu infection; furthermore, we with either water (vehicle) or N6-(1-iminoethyl)-L-lysine dihydrochloride demonstrated the requirement of iNOS expression for apoptosis (L-NIL) (200 mM; Cayman Chemical) for 2 h before infection. Infection during flu infection. was continued in the presence of L-NIL. NO quantification Materials and Methods NO production from infected cells was determined by measuring nitrite Virus and cell culture levels in the medium supernatant, as described previously (24). Medium supernatant collected from mock- and flu-infected cells were mixed with Influenza A [A/PR/8/34 (H1N1)] virus (flu) was grown in the allantoic Griess reagent for 10 min at room temperature. The absorbance (at 543 nm) cavities of 10-d-old embryonated eggs (19). Virus was purified by cen- was measured using a microplate reader. Diluted sodium nitrite was used to Downloaded from trifugation (two times) on discontinuous sucrose gradients (20). A549 cells derive a standard curve. were maintained in DMEM supplemented with 10% FBS, penicillin, streptomycin, and glutamine (21). Bone marrow-derived macrophages Luciferase assay (BMDMs) were obtained from femurs and tibias of WT and iNOS KO mice and were cultured for 6–8 d, as described earlier (19, 22). Cells were Luciferase assay was performed, as described previously (19, 25). A549 plated on six-well plates containing RPMI 1640, 10% FBS, 100 IU/ml cells were transfected (using Lipofectamine 2000 from Invitrogen) with penicillin, 100 mg/ml streptomycin, and 20 ng/ml GM-CSF. The flu titer pRL-null-Renilla luciferase and luciferase (firefly) reporter gene fused to human iNOS promoter (iNOS-luc). At 24 h posttransfection, cells were was monitored by plaque-assay analysis with MDCK cells. http://www.jimmunol.org/ infected with flu. At 12 and 24 h postinfection, cells were washed once with PBS and then lysed using passive lysis buffer (Promega). Luciferase Generation of stable short hairpin RNA-expressing A549 cells activity was measured using a Dual-Luciferase Reporter Assay System A549 cells transduced (in the presence of 5 mg/ml polybrene at a multi- (Promega), according to the manufacturer’s protocol. Transfection efficiency plicity of infection [MOI] of lentivirus) with either scrambled short hair- was normalized by measuring expression of Renilla luciferase. Luciferase pin RNA (shRNA) (control) or KLF6-specific shRNA-expressing lentivirus units were measured by standard methodology and are presented as fold (Santa Cruz Biotechnology, Santa Cruz, CA) were selected in the presence activation of luciferase activity. of puromycin dihydrochloride. Antibiotic-resistant cells were expanded to Apoptosis assay generate a population of stable cells expressing either scrambled shRNA or KLF6-specific shRNA. Flu-infected A549 cells and BMDMs were examined for apoptosis by annexin V labeling, using an annexin V/propidium iodide (PI) apoptosis by guest on October 2, 2021 RT-PCR detection kit (BioVision, Milpitas, CA) (26, 27). Total RNA was extracted from A549 cells using TRIzol reagent (Invitrogen). Flu infection of mice cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR was performed using 0.25 U Taq polymerase, Six- to eight-wk-old pathogen-free WT C57BL/6 and iNOS KO C57BL/6 m mice (The Jackson Laboratory) were anesthetized and inoculated (via 10 pmol each oligonucleotide primer, 1 mM MgCl2, and 100 M deoxy- 3 4 m nucleotide triphosphates in a final reaction volume of 25 ml. Following intratracheal route) with flu (5 10 PFU/mouse) in 100 l Opti-MEM amplification, the PCR products were analyzed on 1.5% agarose gel. Equal medium (Invitrogen) (19). Mock-infected animals were sham inoculated with 100 ml Opti-MEM. At 5 d postinfection, whole lung was collected. loading in each well was confirmed by analyzing the expression of the Lungs were used for in situ TUNEL analysis (with lung tissue sections) housekeeping gene GAPDH. The following primers were used to detect the indicated genes by RT-PCR: GAPDH forward, 59-GTCAGTGGTG- and Western blot analysis (with lung homogenate) with anti–caspase-3 Ab GACCTGACCT-39; GAPDH reverse, 59-AGGGGTCTACATGGCAACTG- (Cell Signaling Technology). Another set of WT mice was infected with 39; Human iNOS forward, 59-TCCGAGGCAAACAGCACATTCA-39; iNOS flu for 5 d, and the isolated lungs were used for tissue ChIP assay. The reverse, 59-GGGTTGGGGGTGTGGTGATGT-39; Human KLF6 forward, mouse experiments performed in the current study were reviewed and approved by The University of Texas Health Science Center at San Antonio 59-CTCTCAGCCTGGAAGCTTTTAGCCTAC-39;andKLF6 reverse, 59- (UTHSCSA) Institutional Animal Care and Use Committee. ACAGCTCCGAGGAACTTTCTCCCA-39. In situ TUNEL assay Chromatin immunoprecipitation assay TUNEL assay was performed to evaluate apoptotic cell death in the re- The standard chromatin immunoprecipitation (ChIP) assay protocol (23) spiratory tract. Formalin-fixed lungs from flu-infected WT and iNOS KO was followed for examining the binding of KLF6 to iNOS promoter in mice were used for in situ TUNEL assay using an ApopTag Peroxidase In cells and lung tissue. Following chromatin isolation from infected (at 1 Situ Apoptosis Detection Kit (Millipore, Billerica, MA). Light microscopy MOI) human A549 cells and mouse BMDMs (after cross-linking with was performed to capture digital images of the TUNEL-stained lung formaldehyde), it was immunoprecipitated at 4˚C with either human or sections. The digital images were used to count the number of TUNEL+ mouse specific anti-KLF6 Ab or isotype-matched control IgG (Santa Cruz cells in the lung sections using ImageJ software from the National Insti- Biotechnology). Human and mouse iNOS promoter-specific PCR primer tutes of Health (http://rsbweb.nih.gov/ij/), as described previously (28). For pairs (encompassing the consensus CACCC KLF6 binding site located in each analysis, an 858.95 3 646.74-mm area of TUNEL-stained lung section the proximal region of the iNOS promoter) were used to analyze the Ab- was scanned by ImageJ software. Gross apoptotic area was expressed as precipitated DNA. As a negative control, PCR primer pairs corresponding pixels/mm, and this value was used to calculate the percentage apoptotic to the iNOS promoter region that does not possess a consensus KLF6 area during each analysis. Three mice from each group were used to binding site were used. Primers used for the ChIP assay are shown in Table I. prepare lung sections (i.e., three flu-infected WT mice and three flu- Lung tissue derived from WT and iNOS KO mice was also used for ChIP infected iNOS KO mice). Data were collected from 27 areas (9 areas/ analysis, as described previously (23). To demonstrate that the amount of mouse) of the lung sections from each experimental group (i.e., 27 areas DNA used for immunoprecipitation (with either KLF6 Ab or control Ab) for flu-infected WT mice and 27 areas for flu-infected iNOS KO mice). was equivalent for each experiment, an equal portion of input DNA was The values obtained from 27 lung section areas of each experimental group analyzed for each sample. (WT and iNOS KO) were used for statistical analysis. The Journal of Immunology 3

Results transcripts were detected in flu-infected KLF6-silenced cells com- KLF6 is required for NO production pared with infected control cells (Fig. 1C). The loss of iNOS mRNAs in KLF6-silenced cells suggested that To study the role of KLF6 during infection, we used KLF6-specific KLF6 could positively transactivate the iNOS promoter. To test this shRNA to silence KLF6 expression in human lung epithelial A549 possibility, we used luciferase reporter gene fused to human iNOS cells. A549 cells are routinely used as model type II human alveolar promoter (iNOS-luc). Control and KLF6-silenced cells transfected epithelial cells, and alveolar cells are a major target of flu during with luciferase-expressing plasmids were infected with flu. Ex- productive infection of the human respiratory tract. A lentivirus- pression of the luciferase gene was monitored at 12 and 24 h mediated shRNA delivery system was used to generate stable postinfection. KLF6 directly regulates the iNOS promoter during A549 cells lacking KLF6 expression. Loss of KLF6 is evident in flu infection, because a significant reduction in luciferase activity stable cells expressing KLF6-specific shRNA (Fig. 1A). Control was observed in cells lacking KLF6 expression (Fig. 1D). These cells represent stable cells derived after transduction with scram- results showed that KLF6 is an important transcription factor bled shRNA expressing lentivirus. regulating iNOS gene expression during flu infection. Control and KLF6-silenced cells were subsequently used to examine whether KLF6 plays any role in NO production or iNOS Binding of KLF6 to iNOS promoter during infection of human gene expression during flu infection. To assess the requirement of lung epithelial cells KLF6 for NO production, control and KLF6-silenced cells were The role of KLF6 in the transactivation of the iNOS gene was further infected with flu. At 12 h postinfection, the medium supernatant was confirmed by performing a ChIP assay to investigate the direct collected to measure nitrite (the end product of NO) by Griess re- association of KLF6 with iNOS promoter during infection. Human agent. Griess assay revealed a significant reduction in NO production Downloaded from lung epithelial A549 cells were either mock infected or infected from KLF6-silenced cells compared with control cells (Fig. 1B). with flu for 6, 12, or 24 h. At various times postinfection, chromatin These results demonstrated that KLF6 is required for NO produc- from these cells was immunoprecipitated using either isotype- tion during flu infection. matched control IgG (negative control) or anti-KLF6 Ab. The KLF6 is required for iNOS gene expression KLF6–DNA binding complex was analyzed by PCR using primers To further evaluate the mechanism responsible for KLF6-dependent corresponding to the KLF6 binding site on the human iNOS pro- http://www.jimmunol.org/ NO production, we investigated whether KLF6 regulates iNOS gene moter (29) (Table I). A primer specific for the non-KLF6–binding expression. Total RNA was isolated from control and KLF6-silenced region on the human iNOS promoter element (an upstream region cells infected with flu for 12 h. The RNAwas used to determine iNOS in the human iNOS gene devoid of the KLF6-responsive region) mRNA levels by RT-PCR. iNOS gene expression is regulated by (Table I) was used as a negative control. Although KLF6 binding to KLF6 at the transcriptional level, because low levels of iNOS the human iNOS gene was not detected in mock-infected cells, flu infection led to the association of KLF6 with the human iNOS promoter (Fig. 2). Interestingly, maximal KLF6 binding was detected at 12 h postinfection, which corresponds to the time at which iNOS induction and NO production were observed during by guest on October 2, 2021 infection of A549 cells (Fig. 1). The specificity of KLF6 binding to the human iNOS promoter is borne out by the observation that immunoprecipitation with control IgG did not generate any amplified product. Moreover, amplification with primers that do not correspond to the KLF6 binding site on the human iNOS promoter did not yield any amplified product. These results demonstrate that, during flu infection, KLF6 directly transactivates human iNOS gene expression following its association with the human iNOS promoter. KLF6 regulates apoptosis during infection Our studies uncovered an important function for KLF6 during flu infection, because it is required for iNOS expression and subsequent NO production from infected cells. Previous studies showed that NO is a contributing factor for exaggerated lung disease (due to lung tissue damage) during flu infection (12–15). Because apoptosis could induce tissue damage, we next investigated whether the KLF6 gene is required for apoptosis during infection. Control FIGURE 1. KLF6 is required for NO production and iNOS gene ex- and KLF6-silenced cells were infected with flu (0.5 MOI) for pression. (A) RT-PCR analysis of KLF6 expression in stable A549 cells 48 h. Postinfection, the cells were used to determine apoptosis generated following transduction with scrambled shRNA (control) and by annexin V staining. Annexin V staining (which signifies early KLF6-specific shRNA (KLF6-silenced cells)-expressing lentivirus. (B)NO apoptosis) revealed a significant reduction in the apoptosis of production from mock- and flu-infected control and KLF6-silenced cells. infected KLF6-silenced cells compared with control cells (Fig. 3). NO was measured by monitoring nitrite production using Griess reagent. These results demonstrated that KLF6 could directly regulate im- 6 Each value represents the mean SD from three independent experiments portant cellular activity (i.e., programmed cell death or apoptosis) performed in triplicate. (C) RT-PCR analysis of iNOS expression in during flu infection. scrambled shRNA (control) and KLF6-specific shRNA-expressing stable A549 cells infected with flu. RT-PCR data are representative of three in- iNOS is required for apoptosis during infection of epithelial D dependent experiments. ( ) Activation of iNOS-luciferase reporter gene in cells and macrophages control and KLF6-silenced cells infected with flu for 12 or 24 h. The luciferase assay results are presented as mean 6 SD from three independent Based on our results showing the requirement of KLF6 for optimal experiments performed in triplicate. *p , 0.05, Student t test. apoptosis and transactivation of the iNOS gene by KLF6, we 4 ROLE OF KLF6 AND iNOS DURING INFLUENZA A VIRUS INFECTION 55.7 56.0 62.6 56.6 Temperature (˚C) Optimal Annealing 4666 1477 366 241 2 2 2 2 45 to 12 to 4417 to 1157 to 2 2 2 2 Product

FIGURE 2. Binding of KLF6 to human iNOS promoter during ﬂu infection. ChIP assay on A549 cells that were either mock infected or Downloaded from region of mouse iNOS promoter) region of mouse iNOS promoter) region of human iNOS promoter) region of human iNOS promoter) infected with ﬂu for 6, 12, or 24 h. Both anti-KLF6 Ab and isotype- 321 bp (encompassing 229 bp (encompassing matched control Ab (control Ab) were used. An upstream region in the 249 bp (encompassing 320 bp (encompassing human iNOS gene devoid of the KLF6 responsive region was probed as a negative control. ChIP data are representative of three independent experiments. input, Input DNA for each experiment. 9 9 9 9 9 9 9 9

speculated that iNOS (by generating and producing NO) may http://www.jimmunol.org/ contribute to apoptosis during flu infection. No study has evaluated the role of NO (and iNOS) in apoptosis during flu infection. The role of iNOS was examined by inhibiting iNOS enzymatic activity with the specific iNOS inhibitor L-NIL (30–33). A549 cells were treated with either vehicle (water) or L-NIL (200 mM) during flu (0.5 MOI) infection. A similar concentration of L-NIL (100–900 mM) was used previously to inhibit iNOS activity in lung epithelial cells (including A549 cells) (30–33). We (data not shown) and other investigators (30–33) did not observe any cytotoxicity in lung by guest on October 2, 2021 -GCTCTTGTTTCCCAGGTTAC-3 -AAAGTTGTGACCCTGGCAGC-3 -CCCAGTTTTGAAGTGACTACG-3 -GAGATGGCTCAGTTGGTAAAG-3 9 9 9 9 -CAGAGAGCTCCCTGCTGAGGAAA-3 5 5 -ACCAATAAGGAAACCGAGACACAG-3 9 5 5 -GGAGAACATTCAACCCCAGGTAATC-3 9 9 5 -GAGAGTTGTTTTTGCATAAAGGTCTC-3 5 9 5 5 Mouse iNOS reverse: Mouse iNOS forward: Mouse iNOS forward: Mouse iNOS forward: Human iNOS forward: Human iNOS forward: Human iNOS forward: Human iNOS reverse:

FIGURE 3. KLF6 expression is required for optimal apoptosis following flu infection. (A) Percentage of apoptotic (annexin V+) cells in flu-infected (48 h postinfection) control (expressing scrambled shRNA) and KLF6- silenced (expressing KLF6-specific shRNA) stable A549 cells. Annexin V staining quantified by FACS represents mean 6 SEM from five independent experiments performed in triplicate (B) The values in (A) were used to that does not bind to KLF6 (negative control) iNOS promoter iNOS promoter that does not bind to KLF6 (negative control) calculate the percentage of apoptosis in flu-infected control and KLF6- Encompassing the region in the human iNOS promoter Encompassing the KLF6 binding site at the human Encompassing the KLF6-binding site at the mouse Encompassing the region in the mouse iNOS promoter silenced cells. Apoptosis in flu-infected control cells was denoted as 100% , Table I. ChIP primers apoptosis. *p 0.05, two-tailed t test. The Journal of Immunology 5 epithelial cells treated with L-NIL (100–200 mM)for48h.At48h BMDMs were either mock infected or infected with flu for 6, 12, postinfection, the apoptosis status of infected cells was examined or 24 h. Chromatin from the BMDMs was immunoprecipitated by annexin V staining (annexin V+ and PI2 cells represent apoptotic using either an isotype-matched control IgG (negative control) or cells). Inhibition of iNOS activity markedly diminished the apo- anti-KLF6 Ab. A primer corresponding to the KLF6 binding site on ptosis of flu-infected cells (Fig. 4A, 4B). A nearly 80% reduction the mouse iNOS promoter was used to analyze the KLF6–DNA in apoptosis was observed in L-NIL–treated cells compared with binding complex by PCR. A primer specific for the non-KLF6– control cells. To our knowledge, these results uncovered, for the binding region on the mouse iNOS promoter (an upstream region first time, the role of NO in inducing apoptosis during flu infec- in the mouse iNOS gene) was used as a negative control. Although tion. NO specifically contributes to apoptosis during flu infection, KLF6 binding to the mouse iNOS gene was not observed in mock- because iNOS inhibition did not significantly alter the necrotic infected BMDMs, flu infection resulted in recruitment of KLF6 to status (annexin V+ and PI+ cells) of infected cells (Fig. 4C). the mouse iNOS promoter (Fig. 6). Interestingly, KLF6 associa- The role of iNOS in regulating apoptosis was further confirmed tion was detected early during infection (i.e., 6–12 h postinfec- using primary BMDMs isolated from iNOS KO mice. WT and tion), and the association of KLF6 with the iNOS promoter was iNOS KO BMDMs were infected with flu (0.5 MOI) for 48 h. lost at 24 h postinfection (Fig. 6). The time frame for KLF6 in- Postinfection, the apoptotic status was evaluated by annexin V teraction with the mouse iNOS promoter corresponded with NO staining. As shown in Fig. 5A and 5B, a significant decrease in production from flu-infected BMDMs, because optimal NO pro- apoptosis (36%) was observed in flu-infected iNOS KO BMDMs duction from flu-infected BMDMs was observed at 6–12 h post- compared with WT BMDMs. These results once again confirmed infection (data not shown). The specificity of KLF6 binding to that iNOS/NO plays a crucial role in apoptosis induction during the mouse iNOS promoter is borne out by the observation that flu infection. In contrast, the necrotic status of cells remained amplification with nonspecific primers (i.e., primers that do not Downloaded from unchanged in flu-infected WT BMDMs versus iNOS KO BMDMs correspond to the KLF6 binding site on the mouse iNOS pro- (Fig. 5C). moter) did not yield any amplified product. Moreover, immunoprecipitation with control IgG did not generate any amplified Binding of KLF6 to iNOS promoter during infection of primary product (data not shown). These results demonstrated that KLF6 macrophages interacts with the mouse iNOS promoter during flu infection of Experiments performed with WT and iNOS KO primary BMDMs primary macrophages. http://www.jimmunol.org/ revealed a role for iNOS in apoptosis during flu infection (Fig. 5). Because KLF6 regulates iNOS expression, we next investigated iNOS expression is essential for optimal apoptosis in the whether, similarly to lung epithelial cells, KLF6 also associates flu-infected respiratory tract with the iNOS promoter during flu infection of primary mouse The in vivo physiological role of iNOS was evaluated by infecting BMDMs. Previous studies mapped the KLF6 binding site in iNOS KO mice with flu. Previous studies showed that, compared the human iNOS gene to the proximal region (position 2164 to with WT mice, iNOS KO mice exhibit reduced lung disease se- 2168) (29). The consensus CACCC sequence proximal to the verity following flu infection (13–15). Interestingly, the reduced human iNOS gene was critical for KLF6 binding to the human lung disease was not due to diminished virus replication (infec- iNOS gene. We identified a similar proximal consensus CACCC tion), because flu burden in the lungs of WT and iNOS KO mice by guest on October 2, 2021 sequence (at position 291 to 295) in the mouse iNOS gene. was similar. In contrast with previous reports, our study revealed

FIGURE 4. iNOS activity is essential for apoptosis during flu infection of epithelial cells. (A) Percentage of annexin V+ cells (apoptotic cells) were detected by FACS analysis of flu-infected (48 h postinfection) A549 cells incubated with water (vehicle) or iNOS inhibitor L-NIL. Annexin V staining quantified by FACS represents mean 6 SEM from five independent experiments performed in triplicate. *p , 0.05, two-tailed t test. (B) The values in (A) were used to calculate the percentage of apoptosis in flu-infected cells (with or without L-NIL). Apoptosis in flu-infected vehicle-treated cells is denoted as 100% apoptosis. (C) Percentage of annexin V+ and PI+ cells (necrotic cells) were detected by FACS analysis of flu-infected (48 h postinfection) A549 cells incubated with water (vehicle) or iNOS inhibitor L-NIL. Annexin V + PI staining quantified by FACS represents mean 6 SEM from five independent experiments performed in triplicate. p = 0.12, two-tailed t test. (D) Representative FACS showing percentage of apoptotic (lower right quadrant, annexin V+ and PI2 cells) and necrotic (upper right quadrant, annexin V+ and PI+ cells) cells following flu infection of water- or L-NIL–treated cells. 6 ROLE OF KLF6 AND iNOS DURING INFLUENZA A VIRUS INFECTION

FIGURE 5. iNOS expression is required for apoptosis during flu infection of primary macrophages. (A) Percentage of annexin V+ cells (apoptotic cells) Downloaded from were detected by FACS analysis of flu-infected (48 h postinfection) WT and iNOS KO primary BMDMs. Annexin V staining quantified by FACS represents mean 6 SEM from four independent experiments performed in triplicate. *p , 0.05, two-tailed t test. (B) The values in (A) were used to calculate the percentage of apoptosis in flu-infected WT and iNOS KO BMDMs. Apoptosis in flu-infected WT BMDMs is denoted as 100% apoptosis. (C) Percentage of annexin V+ and PI+ cells (necrotic cells) was detected by FACS analysis of flu-infected (48 h postinfection) WT and iNOS KO BMDMs. Annexin V + PI staining quantified by FACS represents mean 6 SEM from four independent experiments performed in triplicate. p = 0.09, two-tailed t test. (D) Repre- sentative FACS showing the percentage of apoptotic (lower right quadrant, annexin V+ and PI2 cells) and necrotic (upper right quadrant, annexin V+ and PI+ cells) cells following flu infection of WT and iNOS BMDMs. http://www.jimmunol.org/ that flu replication (as assessed by flu hemagglutinin expression reduced inflammation (as deduced by cellularity of the airway) in in the lungs) in the airway is similar in WT and iNOS KO mice the lungs of infected iNOS KO animals (Supplemental Fig. 2). The (Supplemental Fig. 1). Because apoptosis contributes significantly TUNEL results were further confirmed by performing Western blot to disease severity, reduced apoptosis in flu-infected iNOS KO mice analysis with anti–caspase-3 Ab, which detects both the full-length may be one of the contributing factors responsible for diminished (35 kDa) and cleaved product (19 kDa) of caspase-3. Cleavage of airway damage in iNOS KO animals. To assess this possibility, WT procaspase-3 serves as a hallmark of apoptosis. Lung homogenates 3 4 and iNOS KO mice were inoculated with flu (5 10 PFU/mouse) prepared from flu-infected WT and iNOS KO mice were subjected by guest on October 2, 2021 via intratracheal route. At 5 d postinfection, the lungs were col- to Western blot analysis with caspase-3 Ab. Although cleaved lected, and in situ TUNEL assay with lung sections was conducted caspase-3 was abundant in the lungs of WT mice, a decrease in to evaluate the apoptotic status of the respiratory tract. Diminished cleaved caspase-3 product was noticed in iNOS KO lungs (Fig. 7C, apoptosis was observed in the lungs of iNOS KO animals com- 7D). These results showed that iNOS is a critical regulator of lung pared with WT mice (Fig. 7A). Scanning of the lung sections for apoptosis during flu infection. TUNEL+ cells (apoptotic cells) revealed significantly reduced KLF6 binds to the iNOS promoter during flu infection of mice apoptosis in iNOS KO mice (Fig. 7B). A representative TUNEL- stained lung section from flu-infected WT and iNOS KO mice is Our in vitro study showed that KLF6 is essential for iNOS gene shown in Supplemental Fig. 2. It is interesting to note that, con- expression by virtue of its binding to the iNOS promoter during sistent with a previous report (13), we also observed markedly flu infection of epithelial cells and macrophages (Figs. 2, 6). The in vivo physiological relevance of this observation was further confirmed by examining the interaction of KLF6 with the iNOS promoter in the lungs of mice infected with flu. Tissue ChIP assay was performed using lung harvested from flu-infected mice. Mice were infected with flu via intratracheal route. At 5 d postinfection, lungs were collected. Lungs isolated from mock-infected and flu- infected mice were processed for ChIP assay. Chromatin from the tissue was immunoprecipitated using either isotype-matched control IgG (negative control) or anti-KLF6 Ab. A primer corresponding to the KLF6 binding site on the mouse iNOS promoter was used to analyze the KLF6–DNA binding complex by PCR. A primer specific for the non-KLF6–binding region on the mouse iNOS promoter (an upstream region in the mouse iNOS gene) was used as a negative control. KLF6 binding to the mouse iNOS gene was not observed in mock-infected animals (Fig. 8). FIGURE 6. Association of KLF6 with mouse iNOS promoter during flu infection. ChIP assay on primary BMDMs that were either mock infected However, flu infection resulted in binding of KLF6 to the mouse or infected with flu for 6, 12, or 24 h. An upstream region in the mouse iNOS promoter (Fig. 8). The specificity of KLF6 binding to the iNOS gene devoid of the KLF6-responsive region was probed as a negative mouse iNOS promoter is evident by the observation that amplifica- control. The ChIP data are representative of three independent experi- tion with nonspecific primers (i.e., primers that do not correspond ments. input, Input DNA for each experiment. to the KLF6 binding site on the mouse iNOS promoter) did not The Journal of Immunology 7 Downloaded from

FIGURE 7. iNOS expression is required for optimal apoptosis in the lung of flu-infected mice. (A) For each experimental group, lung sections were prepared from three flu-infected WT mice and three flu-infected iNOS KO mice. The lung sections were used for TUNEL staining. ImageJ software was used to calculate TUNEL+ areas (representing apoptosis) in the lung sections, as detailed in Materials and Methods. The data are presented as the per- http://www.jimmunol.org/ centage apoptotic area. Each bar represents each infected animal, and the values represent the mean 6 SD for the percentage apoptotic area calculated from nine areas of the lung sections. (B) The values were compiled to calculate the percentage apoptotic area in flu-infected WT mice versus iNOS KO mice. The experiment was repeated twice in duplicate. *p = 0.0289, Student t test. (C) Lung homogenate prepared from flu-infected WT and iNOS KO mice was subjected to Western blot analysis with anti–caspase-3 Ab. The uncleaved full-length caspase-3 (35 kDa) and the cleaved caspase-3 product (19 kDa) are indicated by an arrow. (D) For each experimental group, lung homogenate was prepared from flu-infected WT (four mice) and iNOS KO (four mice) mice. Lung homogenate from each mouse (total n = 8 mice [n = 4 WT mice and n = 4 iNOS KO mice]) was subjected to Western blot analysis with anti–caspase- 3 Ab to detect the cleaved caspase-3 product (19 kDa), as shown in (C). The 19-kDa caspase-3 band from each infected mouse (total n = 8 mice) was quantified using Syngene GeneTools software, and the band density was plotted to show differences in the levels of cleaved caspase-3 product in flu- 2 2 infected WT versus iNOS KO mice. The band density is represented as adjusted volume intensity 3 mm (Adj Vol Int 3 mm ). The band density represents by guest on October 2, 2021 mean 6 SD from two independent experiments performed in duplicate. *p , 0.05, two-tailed t test. yield any amplified product. Moreover, immunoprecipitation with Discussion control IgG did not generate any amplified product (data not shown). iNOS gene expression is tightly regulated, because NO-mediated These results demonstrated the association of KLF6 with the mouse tissue damage could lead to enhanced disease phenotype (4–11). iNOS promoter during flu infection of the respiratory tract. Because of NO’s ability to induce apoptosis and enhance in- A schematic model depicting the role of KLF6 and iNOS is flammation (10, 11, 16), NO produced during virus infection shown in Fig. 9. significantly contributes to development of the disease state. These events culminate in an exacerbated disease condition as the result of tissue damage. Respiratory viruses, like influenza A virus, induce iNOS expression to generate NO, which contributes to enhanced disease pathophysiology associated with increased damage to the lung tissue (12–15). Although iNOS plays a critical role in determining the severity of the disease state following flu infection, it is not known whether iNOS/NO is required for apoptosis during flu infection or whether noninflammatory transcription factor(s) (with the exception of NF-kB, which is an inflammatory transcription factor) are required for iNOS gene expression during flu infection. Our studies uncovered two mechanisms associated with NO production during flu infection: we demonstrated that NO produced during infection plays an essential role in inducing apoptosis during infection, and we also identified a noninflamma- FIGURE 8. Binding of KLF6 to the mouse iNOS promoter during flu tory transcription factor (i.e., KLF6) as a transactivator of the infection of the mouse respiratory tract. Tissue ChIP assay was performed iNOS gene during infection. Based on our studies, we propose a using lung tissue isolated from mock-infected and flu-infected (5 d postinfection) mice. An upstream region in the mouse iNOS gene devoid of the model (Fig. 9) in which flu infection triggers binding of KLF6 to KLF6-responsive region was probed as a negative control. ChIP data are the iNOS promoter, leading to expression of iNOS gene/protein. representative of three independent experiments. input, Input DNA for NO generated by iNOS is a key contributing factor for induction each experiment. of the apoptotic pathway during infection. 8 ROLE OF KLF6 AND iNOS DURING INFLUENZA A VIRUS INFECTION

flu infection. In the current study, we uncovered the role for a noninflammatory transcription factor (i.e., KLF6) in controlling iNOS gene expression during flu infection. Our studies also illu- minated the role of the KLF family of transcription factors as an important regulator of gene expression associated with infection. NO production was dependent on infection with viable flu, because a significant reduction in NO levels was observed following infection with UV-irradiated flu (UV-flu) (Supplemental Fig. 4A). Interestingly, replication-incompetent flu (UV-flu) did not com- FIGURE 9. Schematic diagram depicting the role of KLF6 and iNOS pletely abrogate NO production, suggesting that viral components during flu infection. Flu infection stimulates binding of KLF6 to the iNOS promoter, resulting in transactivation of the iNOS gene. The enzymatic from input virus could trigger NO production; however, replication activity of iNOS generates NO, which is released from the cells during is required to sustain this process. The efficiency of UV inactivation infection. NO contributes to the infection process by inducing apoptosis. was evident from the lack of flu hemagglutinin expression in cells infected with UV-flu (Supplemental Fig. 4B). The KLF family of transcription factors controls a wide spectrum Apoptosis significantly contributes to pathogenesis and lung of biological and physiological processes, including cell growth, injury/disease pathology associated with flu infection (34–39). cell proliferation, and differentiation (17). The mammalian KLF Apoptosis of lung epithelial cells during infection with respiratory family consists of 17 DNA-binding proteins that possess three viruses, such as respiratory syncytial virus, results in their slough- zinc-fingered motifs. KLF transcription factors regulate the ing, and these cells aggregate with fibrin, mucin to form plugs function of various organ systems, including the hematological, Downloaded from associated with airway obstruction, a condition prevalent among digestive, cardiovascular, and respiratory systems; thus, they have respiratory syncytial virus-infected infants and children (40, 41). been implicated in the development/progression of diseases, such Apart from airway obstruction, a high degree of apoptosis of both as cancer, metabolic disorders, and cardiovascular and inflam- lung epithelial cells and immune cells (e.g., macrophages) during matory diseases (17). Among the KLF family, KLF6 is known respiratory virus infection results in delayed clearance of dead cells to regulate gene expression in various tissues by acting as either from the airway lumen, which culminates in the induction of a transactivator or a repressor of gene expression. KLF6- http://www.jimmunol.org/ necrosis and subsequent inflammation of the airway (42). A regulated genes include E-cadherin (48), TGF-b and TGF-b similar mechanism involving exaggerated apoptosis in the air- receptors (49), collagen (50), and urokinase plasminogen activator way may contribute to the development of severe lung damage (51). In addition, KLF6 was identified as a tumor-suppressor gene (including progression to a disease state such as pneumonia) associated mainly with prostate cancer (52). A role for KLF6 in during infection with highly virulent strains of flu (35–39). renal ischemia-reperfusion injury (53) and hepatic fibrosis (49, 50, Various mechanisms are involved in the induction of apoptosis 54) was reported. iNOS expression is also regulated by KLF6 following flu infection. Apoptosis during flu infection is mediated during diverse stress-related pathophysiological conditions, such by Siva-1 expression, extracellular calcium influx, Bax activation, as heat shock, serum starvation, and hypoxia (29). Although the by guest on October 2, 2021 and production of TRAIL (43–47). NO-induced apoptosis repre- human iNOS gene contains 10 consensus KLF6-binding motif sents one of the key factors contributing to enhanced inflammation “CAAAC,” the proximal 0.63-kb region of the iNOS gene is re- and tissue damage (10, 16). Moreover, NO production during in- quired for KLF6 binding to the iNOS gene for transactivation (29). fection with respiratory viruses, such as flu, results in exaggerated Another member of the KLF family, KLF4, also regulates iNOS disease pathology attributed to increased lung inflammation and and endothelial NO synthase expression during inflammation of the enhanced tissue damage (13–15). Although flu infection results in neuron and endothelium (55, 56). Although KLF factors have been NO production (12), the contribution of NO in apoptosis during implicated in regulating normal cellular/tissue homeostasis, their infection has not been evaluated. Our study showed that NO pro- role during infection was not known. In the current study, we duced during flu infection plays a critical role in inducing apoptosis. identified KLF6 as a regulator of iNOS gene expression during The contribution of NO to apoptosis in the airway was also vali- flu infection. dated in vivo using flu-infected WT and iNOS KO mice. Addi- Although the mechanism underlying iNOS gene transactivation tionally, we identified KLF6 as an essential transcription factor by KLF6 is not known, we surprisingly observed that polyinosinic- required for iNOS gene expression during flu infection. polycytidylic acid (poly-IC) (a TLR3 ligand)-mediated NO pro- The 16-kb iNOS gene is regulated by numerous transcription duction is dependent on KLF6 expression, because KLF6 silencing factors, such as NF-kB, STAT-1a, AP-1, Oct-1 (octamer factor), drastically inhibited NO production following poly-IC treatment T cell factor 4, STAT-3, NF-AT, and NF-IL6 (4). Among these (Supplemental Fig. 4C). Several studies reported that activated transcription factors, inflammatory transcription factors (e.g., AP- TLR3 (and poly-IC treatment) triggers iNOS-mediated NO re- 1, NF-kB, NF-AT, and STAT-1a) play an important role during lease (57–60), and our results suggested that KLF6 is required for infection and inflammatory response. NO is produced during flu TLR3-mediated iNOS induction. TLR3 is expressed in lung epi- infection, primarily as the result of iNOS induction (12–15). There thelial cells (and TLR3 is activated by poly-IC in these cells) has been no report that neuronal NOS is involved in NO genera- (61, 62); TLR3 activation during flu infection is not only required tion during infection. Thus, inhibition of neuronal NOS activity in for the proinflammatory response in lung epithelial cells, it plays A549 cells did not block NO generation following flu infection an important role in regulating flu pathogenesis in mice (61, 63–65). (Supplemental Fig. 3). The role of NF-kB in iNOS induction Thus, it is plausible that activation of the TLR3 pathway (resulting during flu infection has been suggested (12). It was also shown in activation of NF-kB and MAPK signaling) during flu infection that the iNOS gene is regulated by IRF-1 during flu infection (12). results in KLF6 binding to the iNOS promoter, leading to iNOS However, direct interaction/binding of NF-kB and IRF-1 to the gene expression. During this event, the iNOS gene-specific trans- iNOS promoter during flu infection has not been reported. More- activating property of KLF6 could be achieved by several over, with the exception of NF-kB, no other transcription factors mechanism(s): 1) Signal-specific phosphorylation of several KLFs have been implicated in transactivation of the iNOS gene during (e.g., KLF4) regulates their transactivating function. For example, The Journal of Immunology 9 phosphorylation of KLF4 by MAPK results in recruitment of innate host defense causes susceptibility to respiratory virus infections in cystic b b fibrosis. Immunity 18: 619–630. KLF4 to the TGF- R promoter and TGF- R gene expression 9. 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Sci. USA 101: 18141– (Department of Biochemistry, UTHSCSA) for assisting with the in vitro 18146. 25. Bose, S., N. Kar, R. Maitra, J. A. DiDonato, and A. K. Banerjee. 2003. Temporal neuronal NO synthase assay, and Dr. Benjamin J. Daniel and Karla M. activation of NF-kappaB regulates an interferon-independent innate antiviral Gorena from the UTHSCSA FACS facility for assistance. response against cytoplasmic RNA viruses. Proc. Natl. Acad. Sci. USA 100: 10890–10895. 26. Echchgadda, I., T. Chang, A. Sabbah, I. Bakri, Y. Ikeno, G. Hubbard, Disclosures B. Chatterjee, and S. Bose. 2011. Oncolytic targeting of androgen-sensitive The authors have no financial conflicts of interest. prostate tumor by the respiratory syncytial virus (RSV): consequences of deficient interferon-dependent antiviral defense. BMC Cancer 11: 43. 27. Echchgadda, I., S. Kota, I. DeLa Cruz, A. Sabbah, B. Chatterjee, and S. Bose. 2009. Anti-cancer oncolytic activity of respiratory syncytial virus. 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