Negative feedback regulation of IL-32 production by iNOS activation in response to dsRNA or influenza virus infection

Negative feedback regulation of IL-32 production byiNOS activation in response to dsRNA or influenzavirus infection

Wei Li, Fang Yang, Yan Liu, Rui Gong, Li Liu, Yong Feng, Pan Hu,

Wei Sun, Qian Hao, Lei Kang, Jianguo Wu and Ying Zhu

The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan,

P. R. China

iNOS plays an important role in mediating inflammation. In this study, we found that iNOS-

derived NO was increased 2.4-fold in the serum samples of 101 patients infected with influenza

A virus in comparison with samples of 105 healthy individuals. In A549 human lung epithelial

cells, infection with influenza A virus or stimulation with poly(I:C)1IFN-c resulted in increased

mRNA and protein levels of both IL-32 and iNOS, with subsequent release of NO. Over-

expression of IL-32 resulted in upregulated iNOS expression with subsequent NO production.

Knock down of IL-32 by IL-32-specific siRNA resulted in the inhibition of dsRNA-induced

expression of iNOS and NO release, indicating that IL-32 is an upstream regulatory factor of

dsRNA-triggered iNOS production. Surprisingly, over-expression of iNOS resulted in the

reduction of IL-32 expression, and suppression of iNOS by the selective iNOS inhibitor

S-methylisothiourea sulfate stimulated IL-32 expression, indicating that a negative feedback

mechanism operates between the iNOS/NO and IL-32 systems. These findings suggest that

influenza A virus infection activates IL-32 and iNOS expression by a heretofore unrecognized

complex mechanism, in which the two pro-inflammatory factors regulate each other, involving

positive and negative feedback regulatory loops.

Key words: Gene regulation . IL-32 . Inducible nitric oxide synthase . Inflammation .

Influenza virus

Introduction

In a recent study we found that influenza virus (IV) infection in

patients is associated with increased serum levels of the inflammatory

cytokine IL-32 and the COX-2-induced prostaglandin PGE2. In vitro,

IV infection of A549 lung epithelial cells resulted in release of IL-32

and activation of COX-2 expression [1]. As a proxy of virus infection

we used stimulation of the cells with dsRNA1IFN-g, a combination

previously shown to act synergistically when administered intra-

tracheally [2] and in mouse peritoneal macrophages in vitro [3]. We

demonstrated that production of IL-32 depends on prior COX-2

activation, but also that IL-32 can exert negative feedback control

over activation of COX-2 [1].

In the present study we have pursued the analysis of these

inflammatory pathways by testing for activation of the iNOS gene

and subsequent production of NO. NO is synthesized from L-arginine

by NOS in numerous mammalian cells and tissues and plays a critical

role in signal modulation during immune responses, chronic

inflammation and carcinogenesis. Three isoforms of NOS have been

identified until now: two constitutively expressed and calcium-

dependent isoforms endothelial NOS and neuronal NOS; one indu-

cible and calcium-independent isoform (iNOS), which catalyzes the

production of high amount of NO in response to diverse stimuli [4].Increased IL-32 and iNOS expression stimulated by viral infec-

tions has been reported in previous investigations [1, 5, 6]. However,

the function of IL-32 in the pro-inflammatory network is still unclear.

Since IL-32 and iNOS are obligatory mediators of inflammation, the

SHORT COMMUNICATION

Correspondence: Professor Ying Zhue-mail: [email protected]

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2009. 39: 1019–1024 DOI 10.1002/eji.200838885 Immunity to infection 1019

question arises as to whether there is a relationship between the two

proteins or they act as independent effectors of host inflammatory

response to viral infection. Our results showed that influenza A virus

infection or dsRNA treatment activates IL-32 and iNOS expression by

a heretofore unrecognized mechanism, in which influenza A virus

stimulates iNOS expression through IL-32, and iNOS or NO exerts

feedback inhibition on IL-32 production.

Results and discussion

IL-32 and iNOS activation in response to influenza Avirus infection and dsRNA

We first determined the effects of influenza A virus infection and

poly(I:C)1IFN-g treatment on the expression level of IL-32, iNOS

and iNOS-derived NO release by clinical data analysis and cell

culture experiments. The serum levels of IL-32 and NO were

significantly higher in patients with influenza A virus infection

than in the healthy individuals (means7SEM for IL-32,

182.7743.2 versus 114.5728.9 pg/mL; for NO, 12.373.4 versus

5.271.1mM, respectively, po0.001). Human lung epithelial cells

(A549) were infected with influenza A virus or treated with

poly(I:C)1IFN-g. The culture supernatants and cell lysates were

harvested at 0, 6, 12, 24 and 48 h after infection or induction.

IL-32 production (Fig. 1A) and NO accumulation (Fig. 1B) in

culture supernatants were measured. IL-32, iNOS mRNA (Fig. 1C)

and protein (Fig. 1D) expression levels in cell lysates were

examined by RT-PCR and Western blot analysis, respectively. Both

influenza A virus infection and dsRNA treatment activated the

expression of IL-32 and iNOS in a time-dependent manner.

To identify the viral components responsible for iNOS expression,

we tested cells for activation of the iNOS promoter following trans-

fection with each of all ten genes of IV. For comparison with dsRNA

parallel cultures were similarly tested following treatment with

poly(I:C) or poly(I:C)1IFN-g. Results showed that poly(I:C),

poly(I:C)1IFN-g and NS1 are the most important factors in the

induction of human iNOS promoter activities in both 293T

(Fig. 1E) and A549 cells (Fig. 1F), suggesting that dsRNA and NS1

are key viral components involved in virus-triggered iNOS expression

during IV infection. Although both components merit thorough

analysis, for the present study we focused only on the effect of dsRNA

in the regulation of IL-32 and iNOS expression.

dsRNA stimulates iNOS through the IL-32 pathway

The effects of IL-32 on the activation of human iNOS promoter

were determined. A549 cells were cotransfected with the reporter

plasmid phiNOS-Luc and Flag2A-IL-32 into four cell lines. Results

from luciferase activity assays (Fig. 2A) showed that the level of

iNOS promoter activity was increased by IL-32 over-expression in

A549, Jurkat, U937 and HEK 293T cells.

To determine the effects of IL-32 on the activation of iNOS

mRNA and NO production, A549 cells were transfected with

different amount of Flag2A-IL-32. Results from RT-PCR

using iNOS-specific, or b-actin-specific primers showed that

the levels of iNOS mRNA were increased as the amount of

Figure 1. Induction of IL-32 and iNOS in A549 or 293T cells in response toinfluenza A virus infection or treatment with dsRNA. Time-dependentaccumulation of IL-32 (A) and NO (B) in the supernatant fluids of A549-cellcultures in response to influenza A virus infection (MOI 5 1) or treatmentwith poly(I:C) (50mg/mL)1IFN-g (150U/mL). (C) Time-dependent accumu-lation of IL-32 and iNOS mRNA in cell lysates by RT-PCR analysis. (D) IL-32and iNOS protein by Western blot analysis. A549 cells harvested atindicated time points after influenza A virus infection (MOI 5 1) or after48 h treatment with poly(I:C)1IFN-g. (E and F) Screening of viral proteinsand dsRNA as inducers of iNOS: 293T cells (E) or A549 cell cultures (F)were cotransfected with reporter plasmid phiNOS-Luc, and pRL-TK alongwith each viral gene construct or the control vector, or co-treated withpoly(I:C)1IFN-g.48 h post transfection luciferase activities were measuredand normalized versus renilla luciferase activities measured. Data showmean7SE (n 5 3) and are representative of three independent assays(�po0.05). Student’s t test was used to determine statistical significance.

Eur. J. Immunol. 2009. 39: 1019–1024Wei Li et al.1020


Flag2A-IL-32 increased, but the levels of b-actin mRNA remained

relatively constant (Fig. 2B). Furthermore, NO release in culture

supernatants was stimulated by IL-32 over-expression in a dose-

dependent manner (Fig. 2C).

To confirm these results, A549 cells were transfected with

IL-32-specific siRNA along with reporter plasmid phiNOS-Luc,

followed by treatment with poly(I:C)1IFN-g. Transfection with a

plasmid expressing an irrelevant siRNA was used as control.

Results from luciferase activity assay showed that the level of

iNOS promoter activity was decreased by knocking down

IL-32 (Fig. 2D). Furthermore, NO accumulation in supernatants

of cultures stimulated by poly(I:C)1IFN-g was suppressed

by IL-32-specific siRNA (Fig. 2E). These data suggest that

IL-32 is an upstream regulatory factor of dsRNA-triggered iNOS

production.

Feedback inhibition of IV induced IL-32 expression byiNOS-derived NO

To test the possibility that iNOS or NO, produced following

viral infection, reciprocally affects the activity of the IL-32

promoter, we cotransfected cells with the reporter plasmid

pIL-32-Luc and pcDNA3.1-hiNOS. Results from luciferase

activity assays showed that the level of IL-32 promoter

activity was decreased by iNOS over-expression (Fig. 3A). Similar

results were observed in four cell lines: A549, Jurkat, U937 and

293T.

To determine the possible regulatory effect of iNOS or NO

on IL-32 mRNA expression, A549 cells were transfected with

different amounts of pcDNA3.1-hiNOS and treated with poly(I:C)

1IFN-g as the inducer. Results from RT-PCR using IL-32-specific or

Figure 2. dsRNA induces iNOS in an IL-32-dependent manner. (A) Reporter plasmid phiNOS-Luc and pRL-TK were cotransfected along withFlag2A-IL-32 or Flag2A into cultures of A549, Jurkat, U937 or 293T cells. Luciferase activities were measured after 48 h. (B and C) A549 cells weretransfected with different amounts of Flag2A-IL-32 plasmid. RT-PCR for iNOS and b-actin (internal control) in cell lysates (B), measurements for NOin culture supernatants (C) were performed after 48 h. (D) A549-cell cultures were cotransfected with reporter plasmid phiNOS-Luc and pRL-TKalong with siRNA-IL-32 or siRNA-control and treated with poly(I:C) (50 mg/mL)1IFN-g (150 U/mL) for 48 h. Luciferase activities were then measured.(E) A549 cells were transfected by siRNA-IL-32 or siRNA-control and stimulated with poly(I:C)1IFN-g for 48 h. Time-dependent release of NO inculture supernatants was measured. Data show mean7SE (n 5 3 or 4) and are representative of three independent assays (�po0.05). Student’s t testwas used to determine statistical significance.

Eur. J. Immunol. 2009. 39: 1019–1024 Immunity to infection 1021


b-actin-specific primers showed that, with increasing amounts of

pcDNA3.1-hiNOS, the levels of IL-32 mRNA decreased whereas the

levels of b-actin mRNA remained relatively constant (Fig. 3B).

To confirm these results, we treated A549 cells with poly(I:C)

1IFN-g in the presence or absence of the selective iNOS inhibitor

S-methylisothiourea sulfate (SMT). IL-32 production in super-

natants of cultures stimulated with poly(I:C)1IFN-g was

enhanced by SMT (Fig. 3C). Furthermore, A549 cells were

transfected by reporter plasmid pIL-32-Luc and infected by

influenza A virus in the presence or absence of different

concentrations of SMT. Results from luciferase activity assays

showed that SMT augmented the level of IL-32 promoter activity

in a dose-dependent manner (Fig. 3D). Moreover, production of

IL-32 protein in culture supernatants was enhanced by SMT in a

dose-dependent manner (Fig. 3E).

Our data, obtained from observations on influenza infection

in patients as well as from experiments on cultures infected with

the virus or exposed to dsRNA, indicate that IL-32 plays an

important role in the inflammatory reactions occurring during IV

infections or in other conditions that otherwise involve exposure

to dsRNA. Our study identifies a positive induction cascade,

virus/dsRNA-IL-32-iNOS-NO, that controls itself through a

negative feedback loop iNOS-NO-IL-32.

On the basis of this study and our previous work [1],

we propose a hypothetical model (Fig. 3F) according to which

influenza A virus or dsRNA triggers production of COX-2 and

PGE2, IL-32, iNOS and NO resulting in a host inflammatory

response. The model calls for cross-talk among the three genes

involved, by which (i) COX-2 upregulates IL-32 production; and

conversely, IL-32 attenuates COX-2 expression and reduces COX-

2-derived PGE2 synthesis; (ii) IL-32 upregulates iNOS expression,

and NO production; and conversely, iNOS or NO suppress IL-32

production. Accordingly, mitigated PGE2 production is due to

IL-32-mediated decreased expression of COX-2.

Concluding remarks

This novel notion that virus infection and dsRNA treatment

activate COX-2, IL-32 and iNOS expression by a mechanism in

which these three pro-inflammatory factors regulate each other in

an order of COX-2/IL-32/iNOS, involving positive regulations

and negative feedbacks, expands our understanding of relevant

highly pathophysiological processes caused by influenza A virus

and should also help to develop novel therapeutic strategies

aimed at controlling airway inflammation.

Materials and methods

Patients

After informed consent, venous blood was drawn from 101 adult

patients who were seropositive for influenza A (56 males, 45

females, aged 38.9713.6 years) and 105 healthy individuals (60

males, 45 females, aged 37.2711.1 years) who were seronega-

tive. The collection of blood samples for research was approved

by the Institutional Review Board of the College of Life Sciences,

Figure 3. Feedback inhibition by NO of IV induced IL-32 expression anda hypothetical inflammatory network model. (A) Reporter plasmidpIL-32-Luc and pRL-TK were cotransfected along with pcDNA3.1-hiNOSor control vector (pcDNA3.1) into A549, Jurkat, U937- or 293T-cellcultures. Luciferase activity was measured 48 h post transfection.(B) A549 cells were transfected with different amounts of pcDNA3.1-hiNOS plasmid and stimulated with poly(I:C) (50mg/mL)1IFN-g(150 U/mL) for 48 h. Levels of IL-32 mRNA in cell lysates were measuredby RT-PCR analysis. (C) A549 cells were stimulated with poly(I:C)1IFN-gin the presence or absence of 100mM SMT for 48 h. Production of IL-32 inculture supernatants was measured at indicated time points. (D) A549-cell cultures were cotransfected with reporter plasmid pIL-32-Luc andpRL-TK and infected with influenza A virus (MOI 5 1) with or withoutSMT at indicated concentrations. Luciferase activities were measured48 h post transfection. (E) A549 cells were infected with influenza Avirus (MOI 5 1) with or without SMT at indicated concentrations.Production of IL-32 in culture supernatants were measured 48 h postinfection. Data show mean7SE (n 5 3) (�po0.05). Student’s t test wasused to determine statistical significance. (F) A hypothetical model forregulation of pro-inflammatory factors COX-2, IL-32 and iNOS expres-sion in response to influenza A virus infection or dsRNA treatment.Influenza A virus or dsRNA triggers production of COX-2/PGE2, IL-32,iNOS/NO and resulting in subsequent host inflammatory responses.The cross-talk among the three genes involved are described by which(i) COX-2 upregulates IL-32 production; and conversely, IL-32 attenuatesCOX-2 expression and reduces COX-2-derived PGE2 synthesis; (ii) IL-32upregulates iNOS expression, and NO production; and conversely, iNOSor NO suppress IL-32 production.



Wuhan University in accordance with guidelines for the protec-

tion of human subjects.

Plasmids, antibodies and inhibitors

pcDNA3.1-hiNOS containing the human iNOS coding region was

a gift from Dr. Shapiro Richard A (University of Pittsburgh, USA).

The luciferase reporter vector (pGL3) containing an IL-32

promoter region (�746/125) pIL-32-Luc, pGL3 containing an

iNOS promoter region (�8.3 K/190) phiNOS-Luc, renilla inter-

nal control vector pRL-TK (Promega), IL-32 expression vector

Flag2A-IL-32, expression constructs containing ten IV genes and

IL-32-specific siRNA expression plasmid siRNA-IL32 were docu-

mented in our previous studies [1, 7, 8].

SMT (Alexis Biochemicals, Grunberg, Germany), synthetic

poly(I:C) (Sigma, St. Louis, MO, USA), and recombinant human

IFN-g (Peprotech, London, UK) were dissolved in PBS and used at a

final concentration of 100mM, 50mg/mL, 150 U/mL, respectively.

Virus and cell culture

IV strain A/chicken/Hubei/327/2004 (H5N1) was used as

described previously [1, 9]. Human Embryonic Kidney cells

(HEK 293T) were cultured in DMEM, human lung epithelial cells

(A549) were cultured in F12K medium, human T-cell lympho-

blast-like cells (Jurkat) and human leukemic monocyte

lymphoma cells (U937) were cultured in RPMI 1640 medium.

Transient transfection and luciferase reporter geneassays

Cells were plated at density of 4.0�105 cells per 24-well or

6-well plate and grown to confluence reaching about 80%

at the time of transfection. The plasmids were co-transfected into

cells using Lipofectamine 2000 reagent (Invitrogen). Poly(I:C),

IFN-g and SMT were added into the culture media immediately

after transfection. Twenty-four hours post transfection, cells

were serum-starved for another 24 h before being harvested.

Luciferase activities were then measured and renilla luciferase

activities were determined as internal control for transfection

efficiency as previously described [1, 10]. Assay results were

expressed as RLU (relative luciferase activity unit) or as LUC

(luciferase activity).

RT-PCR and Western blot analysis

Total RNA extraction, reverse transcription and detection primers

for IL-32, iNOS and b-actin were described previously [1, 8].

Protein extracts were prepared and quantified using protein

assay kit (Bio-Rad). Western blot analysis was performed using

antibodies against IL-32 and iNOS and sample loading was

normalized by using antibody to b-actin. Immunoblots were visua-

lized with the ECL detection system (Pierce, Rockford, IL, USA).

Measurement of NO release and ELISA for IL-32

NO was determined by mixing 50 mL of culture medium with

50 mL of Greiss reagent (Promega) as described previously [8].

IL-32 production in culture supernatants was measured by ELISA

as described previously [1].

Statistical analysis

All experiments were carried out on triplicate or quadruplicate

cell cultures. Each set of experiments was repeated at least three

times with similar results, and representative ones are shown.

The clinical data were analyzed by Mann–Whitney U test and the

experimental results by Student’s t test. Differences were

considered statistically significant at a value of pr0.05.

Acknowledgements: We thank Hubei provincial Center for Disease

Control and Prevention (Hubei CDC) for the generous assistance in

collecting serum samples from patients seropositive to influenza A

antigen and healthy individuals in this study. This work was

supported by research grants from the National Natural Science

Foundation of China (No. 30570066), the Major State Basic Research

Development Program of China (‘‘973’’ project No. 2007CB512803

and No. 2009CB522506), Hubei Provincial Science Foundation for

Distinguished Youth Scholar to Y.Z. and the Ph.D. Program

Foundation of Ministry of Education of China (No. 20050486012).

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: IV: influenza virus � SMT: S-methylisothiourea sulfate

Full correspondence: Professor Ying Zhu, The State Key Laboratory of

Virology, College of Life Sciences, Wuhan University, Wuhan 430072,

P. R. China

Fax: 186-27-68754592

e-mail: [email protected]

Additional correspondence: Professor Jianguo Wu, The State Key

loboratory of Virology, College of Life Sciences Wuhan University,

Wuhan 430072,

P. R. China

Fax: 186-27-68754979

e-mail: [email protected]

Received: 5/9/2008

Revised: 17/12/2008

Accepted: 21/1/2009



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Negative feedback regulation of IL-32 production by iNOS activation in response to dsRNA or influenza virus infection