Signal transducer and activator of transcription 4 (STAT4), but not IL-12 contributes to Pseudomonas aeruginosa-induced lung inflammation in mice

ARTICLE IN PRESS

Immunobiology 213 (2008) 469–479 www.elsevier.de/imbio

Signal transducer and activator of transcription 4 (STAT4), but not IL-12 contributes to Pseudomonas aeruginosa-induced lung inflammation in mice Rory O’Sullivana, Svetlana O. Carrigana, Jean S. Marshalla, Tong-Jun Lina,b, a

Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada B3K 6R8 Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3K 6R8

b

Received 14 April 2007; received in revised form 7 November 2007; accepted 19 November 2007

Abstract Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals and cystic fibrosis patients. This organism stimulates a complex inflammatory response in the lung, including production of various cytokines and chemokines. The specific contribution of these mediators in the host defense against this bacterium has yet to be fully characterized. Interleukin-12 (IL-12) is commonly known as a master regulator of innate and adaptive immunity. IL-12 induces its biological effects through its associated intracellular signaling molecule, the signal transducer and activator of transcription 4 (STAT4). To examine a specific role of IL-12 and STAT4 in P. aeruginosa lung infection in mice, STAT4-deficient (STAT4/) and IL-12 p40-deficient (IL-12 p40/) mice were infected with P. aeruginosa intranasally. Interestingly, STAT4/ mice, but not IL-12 p40/ mice after 24 h infection showed impaired production of the pro-inflammatory cytokines tumor necrosis factor, interleukin-1b, and macrophageinflammatory protein-2. However, neither STAT4 nor IL-12 p40 deficiency significantly affected INFg production or bacterial clearance compared to wild-type mice. Similarly, neutrophil recruitment was not affected in the STAT4/ and IL-12 p40/ mice. These results suggest that STAT4 contributes to P. aeruginosa-induced inflammation, but it is not essential for bacterial clearance. Although IL-12 is essential for the host defense against various pathogens, this cytokine is likely not a major player in the host response to P. aeruginosa lung infection. r 2007 Elsevier GmbH. All rights reserved. Keywords: STAT4; Pseudomonas aeruginosa; Inflammation; Lung; Cystic fibrosis

Introduction Pseudomonas aeruginosa is a ubiquitous, versatile Gram-negative bacillus which represents a major source of nosocomial infections (Lyczak et al., 2000). P. aeruginosa is commonly implicated in ventilatorassociated pneumonia, wound infection, bacteremia and Corresponding author at: Department of Pediatrics, IWK Health Center, Dalhousie University, 5850 University Avenue, Halifax, NS, Canada B3K 6R8. Tel.: +1 902 470 8834; fax: +1 902 470 7812. E-mail address: [email protected] (T.-J. Lin).

0171-2985/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2007.11.007

sepsis in burn victims, and ulcerative corneal keratitis (Lyczak et al., 2000). This organism is the major pathogen in the lung disease associated with cystic fibrosis (CF; Campana et al., 2004). The primary cell for the clearance of this bacterium is thought to be the neutrophil (Cripps et al., 1995). Infection by this organism is associated with a large influx of neutrophils and is characterized by the production of various inflammatory cytokines and chemokines such as TNF, IL-1b, and IL-8 (CXCL8, homologous to macrophage-inflammatory protein-2 (MIP-2/CXCL1) in mice) (Cripps et al., 1995). It has been postulated

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that P. aeruginosa-mediated lung injury is orchestrated by cytokines and chemokines. Altered Th1/Th2 cytokine balance can predispose the host to opportunistic infections. A predominant Th2type immune response has been observed in CF patients with P. aeruginosa lung infection (Moser et al., 2000; Hartl et al., 2006). A role of Th1 cytokines in P. aeruginosa lung infection is less clear. Interleukin-12 (IL-12) has been regarded as a ‘‘master’’ cytokine that mediates Th1 response. P. aeruginosa infection is associated with production of IL-12 (Murphey et al., 2004a) as well as IL-23 (Belladonna et al., 2006). However, a role of IL-12 in P. aeruginosa infection remains controversial. Suppressed IL-12 level has been associated with impaired clearance of P. aeruginosa, suggesting a protective role of IL-12 in the host defense against this pathogen (Murphey et al., 2004a). In contrast, others reported that decreased production of IL-12 is associated with better clearance of P. aeruginosa (Varma et al., 2005). Thus, a specific contribution of IL12 in the host defense against P. aeruginosa infection remains to be determined. IL-12 is a heterodimeric, 70-kDa cytokine with multiple established roles in innate and adaptive immunity (Trinchieri, 2003; Langrish et al., 2004). IL12 is composed of independently synthesized, covalently associated p40 and p35 subunits. The subunit p40 is shared by IL-23, a p40 and p19 heterodimer (Trinchieri, 2003; Langrish et al., 2004). These cytokines represent important physiological regulators in vivo for cellular response to microbial infections (Hunter, 2005). Animals deficient in IL-12/IL-23 p40 showed severely impaired resistance to certain bacterial, fungal, and mycobacterial pathogens (Decken et al., 1998; Lehmann et al., 2001; Cooper et al., 2002; Elkins et al., 2002), including pulmonary pathogens such as Klebsiella pneumoniae (Happel et al., 2003) and Mycobacterium tuberculosis (Khader et al., 2005). IL-12 exerts its effects at the cellular level by activating a signal transduction cascade known as the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathway (Watford et al., 2004). Common to many cell types, this pathway is initiated by IL-12 binding to a heterodimeric cell surface receptor (IL-12Rb1 and IL-12Rb2), leading to the activation of Jaks (Presky et al., 1996). Once activated, these kinases catalyze the phosphorylation of cytoplasmic STAT molecules, which dimerize and translocate into the nucleus. IL-12 signals almost exclusively through the STAT4 isoform, although low levels of other STAT activation have been observed (Watford et al., 2004). STAT dimers in the nucleus can act as transcription factors to promote the expression of a range of proinflammatory genes, including those that encode TNF and IL-1b (Imada and Leonard, 2000). STAT4 has been demonstrated as a critical mediator of early innate

immune responses against pulmonary Klebsiella infection (Deng et al., 2004). A role of STAT4 in the host defense against P. aeruginosa infection has not been reported previously. In this study, we used STAT4/ and IL-12 p40deficient (IL-12 p40/) mice to definitively determine a role of STAT4 and IL-12 in the host response to P. aeruginosa lung infection in vivo. We demonstrate that STAT4/ mice showed impaired production of the inflammatory mediators TNF, IL-1b, and MIP-2 in the airways. Despite the impaired ability to produce inflammatory cytokines/chemokine, STAT4-deficient (STAT4/) mice efficiently recruited neutrophils into the lung upon P. aeruginosa infection and effectively cleared this bacterium from the lung after 24 h lung infection. Surprisingly, IL-12 p40-deficient (IL-12 p40/  ) mice showed normal bacterial clearance, neutrophil infiltration, and cytokine and chemokine production in the airway following P. aeruginosa lung infection. These findings suggest that STAT4 contributes to P. aeruginosa-induced lung inflammation, but not neutrophil infiltration and bacterial clearance. IL-12 alone does not appear to contribute significantly to the host defense against P. aeruginosa lung infection.

Materials and methods Mice STAT4/ mice (BALB/c background) were purchased from the Jackson Laboratory (Bar Harbor, ME). STAT4/ mice were age-matched with wild-type BALB/c mice from Jackson Laboratory. IL-12 p40/ mice (C57BL/6 background, obtained from Jean Magram, Hoffmann-La Roche, Nutley, NJ) were bred in-house at the Carleton Animal Care Facility, Dalhousie University, Halifax, NS, Canada. Animals were bred under isolator conditions and placed in conventional housing for at least 4 weeks prior to experimentation. C57BL/6 mice (approximately 3–5 weeks old) were obtained from Charles River Laboratory (Wilmington, MA). All animals were given access to water and commercial chow throughout the course of the experiments. Animals were sacrificed by CO2 asphyxiation. Animal protocols were approved by the University Committee on Laboratory Animals, Dalhousie University, in accordance with the guidelines of the Canadian Council on Animal Care.

Lung infection with Pseudomonas aeruginosa and collection of lung and BALF The infection and tissue collection protocol used in this study was based on our previously described

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method (Power et al., 2004). P. aeruginosa strain 8821 (a gift from Dr. A. Chakrabarty, University of Illinois, Chicago, IL) is a mucoid strain isolated from a CF patient. Mice were infected with 1  107 or 1  109 P. aeruginosa intranasally. After 4 or 24 h, mice were sacrificed. Broncho-alveolar lavage fluid (BALF) was obtained by lavaging the lung with 3  1 ml PBS containing soybean trypsin inhibitor (100 mg/ml). The lung tissues were obtained for detection of cytokines, myeloperoxidase (MPO), and bacterial CFU counting (left lobes). Lung tissues were homogenized in 50 mM HEPES buffer (4 ml/mg lung) containing 100 mg/ml of soybean trypsin inhibitor. For counting bacterial CFU, 10 ml of the homogenate was plated on an agar dish and incubated for 24–48 h at 37 1C. The homogenate was centrifuged at 4 1C for 30 min at 14,000 rpm. The supernatant was stored at 80 1C for later cytokine analysis. The pellet was resuspended and homogenized in 0.5% cetyltrimethylammonium chloride (CTAC) (4 ml/mg lung) and centrifuged as described above. The clear extract was used for MPO assay. BALF (10 ml) was plated on an agar dish and incubated for 24–48 h for CFU counting. For detection of cytokines and MPO activity, BALF was centrifuged at 1500 rpm for 5 min at 4 1C. The supernatants were used for cytokine analysis. The pellets were resuspended in 1 ml NH4Cl (0.15 M) and spun as before to lyse red blood cells. The supernatants were discarded and the pellets were resuspended in 0.5% CTAC (250 ml/mice) and centrifuged and the clear extracts were used for MPO assay.

Myeloperoxidase (MPO) assay Seventy-five microliters of BALF and lung extracts was mixed with 75 ml of MPO substrate solution (3 mM 3,30 ,5,50 -tetramethyl-benzidine dihydrochloride, 120 mM resorcinol, 2.2 mM 3% H2O2) for 2 min in a 96-well plate. Reaction was terminated by addition of 150 ml of 2 M H2SO4, and optical density was measured at 450 nm.

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determined by assessing means with ANOVA and the Tukey–Kramer multiple comparisons test, or by using an unpaired t-test. Differences were considered significant at po0.05.

Results STAT4 but not IL-12 p40 deficiency leads to decreased production of inflammatory cytokine TNF and IL-1 To determine if IL-12 p40 deficiency or STAT4 deficiency affects P. aeruginosa-induced lung inflammation, we chose to determine levels of TNF, and IL-1b production. This is because inflammatory cytokine IL-1 and TNF together with chemokine MIP-2 influence neutrophil recruitment (Wagner and Roth, 2000). IL-1 and TNF are involved in P. aeruginosa lung infection as demonstrated in gene-deficient animals (Yu et al., 2000; Schultz et al., 2002). BALF or lung from P. aeruginosainfected animals was used to determine IL-1b and TNF production by ELISA. STAT4-deficient mice showed reduced IL-1b (Fig. 1A and B) and TNF (Fig. 2A and B) production in the BALF and lung when compared to control mice. In contrast, IL-12 p40 deficiency has no effect on P. aeruginosa-induced IL-1b (Fig. 1C and D) and TNF (Fig. 2C and D) production. IFNg levels in the BALF and lung homogenates from IL-12 p40/ and STAT4/ as well as their respective control mice were also determined. As shown in Table 1, no difference of IL-12 level was observed between IL-12 p40/, STAT4/, and their respective control mice. Infection with P. aeruginosa has little effect on IFNg production in wild-type, IL-12 p40/, or IFNg/ mice. In addition, level of IL-17A in IL-12 p40/ and the control mice was also determined. No significant difference of IL-17A levels was observed between the lung homogenates from IL-12 p40/ mice (no treatment: 169719 pg/ml; P. aeruginosa, 4 h: 163712 pg/ml; P. aeruginosa, 24 h: 170731 pg/ml) and those from control mice (no treatment: 172719 pg/ml; P. aeruginosa, 4 h: 171715 pg/ml; P. aeruginosa, 24 h: 185719 pg/ml).

Enzyme-linked immunosorbent assay (ELISA) Concentrations of TNF, IL-1b, MIP-2, IFNg, and IL17A were determined by ELISA, using DuoSet antibody pairs from R&D Systems (Minneapolis, MN) and a streptavidin alkaline phosphatase amplification system from Invitrogen Technologies (Carlsbad, CA).

Statistics Data are presented as mean7SEM of the indicated number of experiments. Statistical significance was

STAT4 but not IL-12 p40 deficiency leads to decreased production of neutrophil chemoattractant MIP-2 MIP-2 (homologous to human IL-8) is a potent neutrophil chemoattractant. To examine if IL-12 p40 or STAT4 deficiency affects production of MIP-2, BALF and lung tissues from IL-12 p40/ mice, STAT4/ mice, and their respective C57BL/6 or BALB/c control mice after P. aeruginosa infection for 4 or 24 h were collected for measuring MIP-2 protein level by ELISA.

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A

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Fig. 1. STAT4 deficiency but not IL-12 p40 deficiency leads to diminished IL-1b expression following P. aeruginosa lung infection. IL-12 p40/ mice or STAT4/ mice as well as their respective control mice were inoculated intranasally with P. aeruginosa (mucoid strain 8821, 1  109/mice, Psa). Mice that received saline were used as controls (NT). After 4 or 24 h, the BALF (A, C) and lung tissues (B, D) were collected for the determination of IL-1b by ELISA. Data are the mean7SE of 8–11 mice per group.

Significant production of MIP-2 in the BALF and lung after P. aeruginosa infection for 4 or 24 h was observed in IL-12 p40/ mice or STAT4/ mice and their control mice. Similar to IL-1b and TNF production, MIP-2 levels in both BALF and lung tissues from STAT4/ mice after P. aeruginosa infection for 24 h were lower (po0.05) compared to that of control animals (Fig. 3A and B). There was no significant difference of MIP-2 levels between IL-12 p40/ mice and their control mice (Fig. 3C and D), suggesting that IL-12 p40 deficiency has no effect on P. aeruginosainduced MIP-2 production in the lung. These results suggest that STAT4, but not IL-12 p40 is required for

the full production of MIP-2 following P. aeruginosa lung infection.

STAT4 or IL-12 p40 deficiency has no effect on neutrophil recruitment to the lungs of P. aeruginosa-infected mice Since neutrophils recruited in the lung play a major role in the clearance of P. aeruginosa, whether STAT4 or IL-12 affects neutrophil recruitment in response to P. aeruginosa lung infection was examined. IL-12 p40/ mice, STAT4/ mice, and their respective control mice

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Fig. 2. STAT4 deficiency but not IL-12 p40 deficiency leads to diminished TNF expression following P. aeruginosa lung infection. IL-12 p40/ mice or STAT4/ mice as well as their respective control mice were inoculated intranasally with P. aeruginosa (mucoid strain 8821, 1  109/mice, Psa). Mice that received saline were used as controls (NT). After 4 or 24 h, the BALF (A, C) and lung tissues (B, D) were collected for the determination of TNF by ELISA. Data are the mean7SE of 8–11 mice per group.

were infected intranasally with 1  109 CFU of P. aeruginosa for 4 or 24 h. BALF and lung samples were examined for MPO (a neutrophil marker) activity. Effective neutrophil recruitment was observed after P. aeruginosa infection for 24 h. MPO levels in BALF and lung tissues were similar between IL-12 p40/ mice and their control mice or between STAT4/ and their control animals (Fig. 4). These results suggest that in response to P. aeruginosa infection, STAT4/ mice and IL-12 p40/ mice effectively recruited neutrophils into their airways as their respective control animals did.

STAT4 or IL-12 p40 deficiency has no effect on the clearance of P. aeruginosa from the mice lung To examine if STAT4 or IL-12 has a role in bacterial clearance in vivo, IL-12 p40/ mice, STAT4/ mice, and their respective control mice were infected intranasally with 1  109 CFU of P. aeruginosa, and sacrificed at 24 h post-infection. Samples of BALF and lung tissue homogenate were plated onto agar for colony counting. No significant differences of CFU counts in BALF or lung tissues

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No effect of Pseudomonas aeruginosa infection on IFNg production in the lung

Table 1.

IFNg (pg/ml) in lung homogenates P. aeruginosa, 4h

P. aeruginosa, 24 h

No treatment

P. aeruginosa, 4h

P. aeruginosa, 24 h

75.8719.6 66.8716.9

69.1710.6 82.2726.2

56.2720.9 56.2721.4

43.9712.5 38.8713.8

42.8717.2 33.2716.9

29.6720.3 20.0717.3

106.1723.9 101.9719.4

90.8716.9 92.2717.8

87.0720.3 84.2721.9

50.6720.3 59.6729.1

35.9715.4 43.9719.0

27.0710.3 37.0711.2

No treatment IL-12+/+ mice IL-12 KO mice STAT4+/+ mice STAT4 KO mice

IFNg (pg/ml) in the BALF

IL-12 p40–/– (IL-12 KO) mice or STAT4 KO mice as well as their respective control mice were inoculated intranasally with P. aeruginosa (mucoid strain 8821, 1  109/mice). Mice that received saline were used as controls (no treatment). After 4 or 24 h, the BALF and lung tissues were collected for the determination of IFNg by ELISA. Data are the mean7SE of 8–11 mice per group.

C

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Fig. 3. STAT4 deficiency but not IL-12 p40 deficiency leads to diminished MIP-2 expression following P. aeruginosa lung infection. BALF (A, C) and lung tissues (B, D) were collected from IL-12 p40/ mice or STAT4/ mice as well as their respective control mice 4 or 24 h after intranasal administration with P. aeruginosa strain 8821 (1  109/mouse, Psa). Mice that received saline were used as controls (NT). MIP-2 levels were determined by ELISA. Data are the mean7SE of 8–11 mice per group.

were observed between IL-12 p40/ and their control mice or between STAT4/ and their control animals (Fig. 5). These results suggest that IL-12 p40/

mice and STAT4/ mice effectively cleared bacteria from their airways as their respective control animals did.

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Fig. 4. IL-12 p40 or STAT4 deficiency has no effect on neutrophil recruitment into the airways following P. aeruginosa lung infection. IL-12 p40/ mice or STAT4/ mice as well as their respective control mice were inoculated intranasally with P. aeruginosa (mucoid strain 8821, 1  109/mouse, Psa). Mice that received saline were used as controls (NT). After 4 or 24 h, the BALF (A, C) and lung tissues (B, D) were collected for the determination of myeloperoxidase (MPO) activities. Data are the mean7SE of 8–11 mice per group.

Discussion P. aeruginosa is a common cause of airway infection. P. aeruginosa lung infection is characterized by the influx of neutrophils and production of various inflammatory cytokines and chemokines. Although antibiotics will most likely continue to be a major component of treatment for P. aeruginosa lung infection, immunotherapy targeted at modulating the host immune response may provide an added advantage, particularly among patients who have impaired immunity or who are infected with resistant pathogens. Since imbalance of Th1 and Th2 cytokine production has been associated with P. aeruginosa infection (Moser et al., 2000), having a better understanding of the specific contribution of Th1/Th2 cytokines and their signaling pathways during

P. aeruginosa infection may ultimately facilitate the development of more specific and effective forms of immunotherapy. IL-12 is a major Th1 cytokine and has been associated with P. aeruginosa infection. Although IL-12 has been demonstrated to be essential for the host defense against several pathogens including K. pneumoniae (Happel et al., 2003) and M. tuberculosis (Khader et al., 2005), reports regarding the role of IL-12 in the host defense against P. aeruginosa infection, however, are inconsistent (Murphey et al., 2004a; Varma et al., 2005). In previous reports, evidence for a role of IL-12 is largely based on the association of IL-12 levels and host responses (Murphey et al., 2004a; Varma et al., 2005). Considering the significant role of IL-12 in innate and adaptive immunity in the lung, it is necessary to clarify

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BALF

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Fig. 5. IL-12 p40 or STAT4 deficiency has no effect on the clearance of P. aeruginosa from the mice lungs. STAT4/ mice (A, B) or IL-12 p40/ mice (C, D) as well as their respective control mice were challenged with P. aeruginosa (mucoid strain 8821, 1  109 CFU/mouse) intranasally. After 24 h, the BALF and the right lung were collected for colony counting. Data are the mean7SE of 8–11 mice per group.

its specific contribution in the immune response against P. aeruginosa. By using IL-12 p40-deficient mice, we demonstrated that this cytokine is not essential for the development of host defense against P. aeruginosa infection. It is important to note that IL-12 p40 subunit is shared by another cytokine known as IL-23 (Oppmann et al., 2000). IL-12 p40 and a subunit p19 constitute IL-23 (Oppmann et al., 2000). IL-12 and IL23 seem to have both overlapping and distinct functions (Oppmann et al., 2000). Our results suggest that both IL-12 and IL-23 are not required for host defense against P. aeruginosa. Since IL-12 primarily signals through STAT4, we used STAT4/ mice to further determine a role for IL-12 in P. aeruginosa infection. Similar to IL-12 p40/ mice, STAT4/ mice efficiently recruited neutrophils into the airways and effectively cleared P. aeruginosa from the lung. This finding is in contrast to those reports by others demonstrating that STAT4/ mice have impaired resistance to several intracellular pathogens including Babesia, Leishmania major, and Toxoplasma gondii infections (Stamm et al., 1999; Aguilar-Delfin et al., 2003; Bot et al., 2003) as well as extracellular bacteria K. pneumoniae (Deng et al.,

2004). STAT4 also appears to play a role in pulmonary immunity in a murine model of tuberculosis (Sugawara et al., 2003). These findings support the notion that mechanisms for the host defense against P. aeruginosa infection are distinct from those for other pathogens. It is also worthy of note that the STAT4/ mice in our study were bred on a BALB/c background, while the IL12 p40/ mice were bred on a C57BL/6 background. Although the difference in genetic background contributing to host resistance to P. aeruginosa has been reported (Sapru et al., 1999), both mice strains appear to mount an effective host response for the clearance of this bacterium in our study. Interestingly, in contrast to IL-12 p40/ mice, STAT4/ mice showed impaired production of MIP2, TNF, and IL-1b in both BALF and lung tissue when compared with wild-type mice. This result suggests that additional roles of STAT4 other than mediating IL-12 signaling may be required for the full development of inflammatory response to P. aeruginosa. STAT4 is also phosphorylated and activated in response to several immunoregulatory factors including urokinase-type plasminogen activator (uPA; Dumler et al., 1999), IFNa

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(Cho et al., 1996), IL-2 (Wang et al., 1999), and IL-17 (Subramaniam et al., 1999), in addition to IL-12. Indeed, many of these immunoregulatory factors have been associated with P. aeruginosa infection (Cripps et al., 1995; Gyetko et al., 2000; McAllister et al., 2005; Hartl et al., 2006). For example, deficiency of urokinase receptor, a uPA receptor, leads to impaired host defense against P. aeruginosa lung infection (Gyetko et al., 2000). The impaired MIP-2, TNF, and IL-1b production seen in STAT4/ mice suggests that STAT4regulated signaling pathway is needed for the full development of P. aeruginosa-induced inflammatory response in the airways. However, this reduction in cytokine/chemokine levels is either not substantial enough to influence bacterial clearance or is corrected by some other component of host response, since these animals showed normal neutrophil recruitment and bacterial clearance. IFNg is a major product following IL-12/STAT4 activation (Trinchieri, 2003) and has an essential role in resistance to many pathogenic bacteria, fungi, and intracellular parasites (Trinchieri, 2003). Level of IFNg was also examined in our model. No significant difference of IFNg level was observed in the lung from IL-12 p40/ mice, STAT4/ mice, or their respective control mice in both P. aeruginosa-infected or uninfected groups. Thus, P. aeruginosa infection had little effect on IFNg production. This result appears to be consistent with others demonstrating that administration of exogenous IFNg or IFNg deficiency had little effect on the bacterial clearance or animal survival following P. aeruginosa infection (Murphey et al., 2004b). This finding further supports the notion that the mechanism of host defense against P. aeruginosa is distinct from that utilized against other pathogens. In summary, we have demonstrated that IL-12 p40/ mice showed effective host response to P. aeruginosa lung infection, suggesting that IL-12 and IL-23 are likely not essential for the development of host response to this bacterium. In contrast, STAT4/ mice showed impaired MIP-2, TNF, and IL-1b production in the airways. However, these mice effectively recruited neutrophils and cleared P. aeruginosa from the lung. Thus, STAT4 is required for the regulation of P. aeruginosa-induced lung inflammation, but appears to be not essential for the host defense against this bacterium.

Acknowledgments The authors would like to thank Wei Chen and Fang Liu for their excellent technical assistance in the cytokine assays by ELISA. This work was supported by grants from the Canadian Institutes of Health Research (Grant MOP 81355), Canadian Cystic Fibro-

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sis Foundation (operating grant), and Izaak Walton Killam Health Center Establishment Grant to T.J.L. T.J.L. is supported by a New Investigator Award from the Canadian Institutes of Health Research and an Investigatorship from Izaak Walton Killam Health Center.

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