The Journal of Immunology

CXCL1 Regulates Pulmonary Host Defense to Klebsiella Infection via CXCL2, CXCL5, NF-kB, and MAPKs

Shanshan Cai,*,† Sanjay Batra,*,† Sergio A. Lira,‡ Jay K. Kolls,x,{ and Samithamby Jeyaseelan*,†,‖

Pulmonary bacterial infections are a leading cause of death. Since the introduction of antibiotics, multidrug-resistant Klebsiella pneumoniae became an escalating threat. Therefore, development of methods to augment antibacterial defense is warranted. recruitment is critical to clear bacteria, and neutrophil migration in the lung requires the production of ELR+ CXC . Although lung-specific CXCL1/keratinocyte cell-derived (KC) transgene expression causes neutrophil- mediated clearance of K. pneumoniae, the mechanisms underlying KC-mediated host defense against K. pneumoniae have not been explored. In this study, we delineated the host defense functions of KC during pulmonary K. pneumoniae infection using KC2/2 mice. Our findings demonstrate that KC is important for expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine, and activation of NF-kB and MAPKs in the lung. Furthermore, KC derived from both hematopoietic and resident cells contrib- utes to host defense against K. pneumoniae. Neutrophil depletion in mice before K. pneumoniae infection reveals no differences in the production of MIP-2 and LPS-induced CXC chemokine or activation of NF-kB and MAPKs in the lung. Using murine bone marrow-derived and alveolar , we confirmed KC-mediated upregulation of MIP-2 and activation of NF-kB and MAPKs on K. pneumoniae infection. Moreover, neutralizing KC in bone marrow-derived macrophages before K. pneumoniae challenge decreases bacteria-induced production of KC and MIP-2, and activation of NF-kB and MAPKs. These findings reveal the importance of KC produced by hematopoietic and resident cells in regulating pulmonary host defense against a bacterial pathogen via the activation of transcription factors and MAPKs, as well as the expression of cell adhesion molecules and other neutrophil chemoattractants. The Journal of Immunology, 2010, 185: 6214–6225.

espite the development of newer generation antibiotics, contributing to neutrophil influx is a prerequisite to designing im- the morbidity and mortality associated with bacterial proved treatment or prevention strategies, or both, to attenuate ex- D pneumonia is still extensive (1–3). In addition, clinical cessive neutrophil-mediated lung parenchymal damage. management of patients infected with antibiotic-resistant bacterial Bacterial lung infection is characterized by excessive capillary strains, including Klebsiella pneumoniae, has been difficult. Innate leakage, activation of transcription factors, upregulation of cell host defense mediated through is pivotal to controlling adhesion molecules, expression of proinflammatory mediators, and bacterial replication in the host. However, neutrophil influx acts as accumulation of neutrophils in the alveolar spaces with diffuse a double-edged sword: an insufficient neutrophil recruitment can damage of alveolar epithelial and endothelial cells (7–9). The lead to life-threatening infection, whereas an extreme accumulation production of chemokines is a critical step leading to the re- of neutrophils can result in excessive lung injury, such as acute lung cruitment of neutrophils to organs during microbial insult (7–9). injury/adult respiratory distress syndrome (4–6). Therefore, the These chemokines are classified into four groups based on the ar- ideal therapeutic approach to target neutrophils would be to atten- rangement of a cysteine motif positioned near the N terminus: C, uate their destructive potential whereas maintaining their antibac- CC, CXC, and CX3C. These groups are further characterized by the terial defense function. However, understanding the mechanisms position of the ELR motif (glutamic acid-leucine-arginine) before the CXC sequence. These ELR+ CXC chemokines are powerful *Laboratory of Lung Biology, Department of Pathobiological Sciences and †Center neutrophil chemoattractants, and seven of this group have been for Experimental Infectious Disease Research, Louisiana State University, Baton identified in humans: IL-8; neutrophil-activating peptide 2; growth- Rouge, LA 70803; ‡Immunology Institute, Mount Sinai School of Medicine, New York, NY; and xDepartment of Genetics, {Department of Pediatrics, and ‖Department related oncogene-a,-b, and -g; -derived neutrophil- of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA activating peptide 78; and granulocyte chemotactic protein 2. Of 70112 these, IL-8 is the most potent neutrophil chemoattractant in hu- Received for publication December 1, 2009. Accepted for publication September 10, mans. Although a homolog of human IL-8 has not been identi- 2010. fied, CXCL1/keratinocyte cell-derived chemokine (KC) (10, 11), This work was supported by a Scientist Award from the Flight Attendant Medical Research Institute (YCSA-062466) and the National Institutes of Health (Grants R01 CXCL2/MIP-2 (12, 13), and CXCL5/LPS-induced CXC chemo- HL-091958 and R01 HL-091958S1 via the American Recovery and Reinvestment kine (LIX) (14, 15) have been found to be potent neutrophil che- Act to S.J.). moattractants in mice. Address correspondence and reprint requests to Dr. Samithamby Jeyaseelan, Depart- KC has been shown to be essential in inducing neutrophil re- ment of Pathobiological Sciences, Louisiana State University, Skip Bertman Drive, Baton Rouge, LA 70803. E-mail address: [email protected] cruitment to the lungs because 1) inhibition of KC using a blocking Abbreviations used in this paper: AM, alveolar ; BALF, bronchoalveolar Ab resulted in attenuation of neutrophil migration to the airspaces lavage fluid; BMDM, bone marrow-derived macrophage; i.t., intratracheal; KC, after Escherichia coli LPS challenge in a rat model (10, 11), and keratinocyte cell-derived chemokine; LIX, LPS-induced CXC chemokine; MOI, 2) transgenic KC mice, which constitutively expressed KC within multiplicity of infection; MPO, myeloperoxidase. the lungs, have more neutrophil influx and less bacterial burden in Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 the organs after challenge with K. pneumoniae (16). However, the www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903843 The Journal of Immunology 6215 signaling cascades associated with neutrophil-mediated bacterial kB and MAPKs. Moreover, our findings suggest that KC mediates clearance in the lungs have yet to be explored. With the advent of pulmonary host defense in an autocrine and paracrine manner. deletion technology in mice, it is possible to determine the mechanisms of these chemokines in pulmonary host defense. In Materials and Methods this study, we sought to determine the mechanisms underlying Animals KC-mediated pulmonary defense using mice with a targeted dis- KC gene-deficient mice (KC2/2) (17) were backcrossed 10 times with ruption in KC. Our results reveal that KC derived from both he- C57BL/6; therefore, C57BL/6 mice were used as control mice (KC+/+). All matopoietic and resident cells regulate host defense in the lung via animal experiments were conducted using a protocol approved by the expression of other neutrophil chemokines and activation of NF- Louisiana State University Animal Welfare Committee. Female mice 8–10

FIGURE 1. Mortality in KC2/2 mice infected with K. pneumoniae. A, Enhanced mortality in CXCL1/KC2/2 mice after i.t. K. pneumoniae challenge. Mice were i.t. infected with 103 CFU K. pneumoniae/mouse, and survival was assessed up to 15 d. Data are from two separate experiments (n = 10 mice/ group). Significance between groups was examined by Wilcoxon rank sign test; asterisk indicates the difference between KC2/2 and KC+/+ mice (pp , 0.05). B and C, Impaired bacterial clearance in the lungs and spleens in KC2/2 mice challenged i.t. with K. pneumoniae. The CFUs were examined in homogenates obtained from the lungs and spleens at 24 and 48 h post-K. pneumoniae challenge (n = 5/group from two independent experiments; pp , 0.05; significant differences between KC2/2 and KC+/+ mice). D and E,KC2/2 mice have reduced cellular/neutrophil accumulation in the airspaces (BALF) after K. pneumoniae (103 CFU/mouse) inoculation. F, Attenuated neutrophil influx in the lung parenchyma, as measured by MPO activity, in KC2/2 mice after K. pneumoniae infection. D–F, n = 6/group from two separate experiments; pp , 0.05; significant differences between KC2/2 and KC+/+ mice. 6216 CXCL1/KC IN BACTERIAL PNEUMONIA wk old, ranging from 20 to 25 g in weight, were used in our experiments. at a 1:1000 dilution, whereas Abs to total p38 and GAPDH were added to the Mice were maintained under specific pathogen-free conditions and kept on concentration of 1:5000. Immunostaining was performed using an appro- a 12:12 h light/dark cycle with free access to food and water. priate secondary Ab at a dilution of 1:2000 and developed with ECL plus Western blot detection system (Thermo-Fisher, Piscataway, NJ). K. pneumoniae inoculation NF-kB activation We used serotype 2 of K. pneumoniae strain (ATCC 43816; American Type Culture Collection, Manassas, VA) because this strain induces sub- An ELISA-based NF-kB DNA binding assay was performed in the lungs stantial inflammation in mice at 103 CFU/animal (18). In brief, the bacteria according to the manufacturer’s protocol (Active Motif, Carlsbad, CA) to were grown for 6 h to midlogarithmic phase at 37˚C in tryptic soy broth detect the activation of the p65 subunit of NF-kB as described earlier (18). while shaking at 225 rpm, harvested by centrifugation, and washed twice in Nuclear extract was prepared using the Nuclear Extraction Kit from Active sterile isotonic saline. The bacteria were resuspended in saline at a con- Motif. In brief, a total of 7.5 mg nuclear extract from each lung sample centration of 103 CFU/50 ml/mouse for in vivo experiments. The mice were post-K. pneumoniae challenge was added to the NF-kB–specific oligonu- anesthetized with ketamine (100 mg/kg)/xylazine (10 mg/kg) mixture, and cleotide coated 96-well plate and incubated for 1 h at room temperature. a midventral incision was performed. After isolation of muscles, the tra- After washing the plate three times, a primary Ab specific for NF-kB/p65 chea was exposed and inoculated with 103 CFU K. pneumoniae. A 50-ml was added and incubated for 1 h at room temperature. After three washings aliquot of a serially diluted suspension of the initial inocula was plated onto to remove excess primary Ab, an anti-HRP conjugate was added to the tryptic soy agar and MacConkey plates for validation of CFUs. plate, and the color development was monitored at the OD of 450 nm. Survival studies Generation of KC chimeras Mice were inoculated intratracheal (i.t.) with 103 CFU K. pneumoniae in 50 Donor and recipient mice between 6–8 wk old were used to generate chi- ml of 0.9% saline, and survival was examined up to 15 d. meras, as described in our earlier publication (19). Recipient mice were gamma-irradiated in two 525-rad doses between 3 h. Bone marrow cells Determination of lung and spleen bacterial CFUs (8 3 106/mouse) were injected into the tail vein of the irradiated recipients, The lungs and spleens of mice were weighed and homogenized in 1 ml of 0.9% saline using a tissue homogenizer. The solid tissue was allowed to sediment for 10 min at room temperature, and the supernatants were serially diluted. Twenty-microliter aliquots of each sample were plated on tryptic soy agar and MacConkey agar plates, and bacterial colonies were enu- merated after incubation at 37˚C. Bronchoalveolar lavage fluid collection At the designated time points, the animals were euthanized and exsangui- nated by cardiac puncture. The trachea was cannulated with a 20-gauge catheter, and bronchoalveolar lavage fluid (BALF) was collected as described earlier (18–21). A total of 3.0 ml BALF was retrieved from each mouse, and 140 ml BALF was centrifuged and placed on glass cytospin slides, which were then stained by Diff-Quick reagents (Fisher Scientific, Chicago, IL) to enumerate leukocyte subtypes. Two milliliters of the undiluted BALF was centrifuged, passed via a 0.22-mm filter, and used immediately or stored at 280˚C for future use. Myeloperoxidase assay Neutrophil migration to the lung can be assessed by myeloperoxidase (MPO) activity in the lungs, which was determined as previously described (18–21). In brief, the lungs were weighed, homogenized, centrifuged, and the pellet was resuspended in 50 mM potassium phosphate buffer, pH 6.0 (supplemented with 0.5% hexadecyltrimethylammonium bromide). Sam- ples were then sonicated, incubated at 60˚C for 2 h, and assayed for MPO activity in a hydrogen peroxide/O-dianisidine buffer at 460 nm. The MPO activity was calculated between 0 and 90 s. The lung samples were used for MPO activity within 3 wk postharvest. and chemokine determination Cytokine and chemokine levels were measured in BALF, lung homoge- nates, culture supernatants obtained from bone marrow-derived macro- phages (BMDMs), and alveolar macrophages (AMs) using a specific sand- wich ELISA as described in earlier publications. The minimum detection limit is 8 pg/ml cytokine protein (18–21).

Western blotting of lung homogenates The harvested lungs were homogenized in 1 ml PBS buffer containing 0.1% Triton X-100, and complete protease and phosphatase inhibitor mixture (Roche, Indianapolis, IN) using a TissueLyser II (Qiagen, Valencia, CA) for 30 s in chilled conditions and centrifuged at maximum speed in a micro- centrifuge at 4˚C for 20 min to remove debris. The resulting supernatant fluids were used for Western blotting. To ensure equal loading of protein FIGURE 2. K. pneumoniae-induced cytokine/chemokine production in onto the gel, we performed a Bradford protein assay (Bio-Rad, Hercules, KC2/2 mice. A–C, KC-dependent signaling regulates the expression of CA). The lung homogenates were analyzed on 8–15% Tris-glycine gels. The MIP-2 and LIX in the lungs post-K. pneumoniae inoculation. Mice were resolved proteins were transferred onto a polyvinylidene difluoride mem- K. pneumoniae brane using standard protocols. The specific Abs to phospho-p44/42 MAPK infected with , BALF was collected, and MIP-2, LIX, and (Erk1/2) (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), phos- TNF-a concentrations were measured at 24 and 48 h postinfection. A total pho–stress-activated protein kinase/JNK (Thr183/Tyr185), phospho-Ik ki- of 4–6 animals were used at each time point in each group. Significant 2 2 nase (IKK)a/b (Ser176/180), phospho–NF-kB/p65 (Ser536), phospho–Ik- differences between KC / and control (KC+/+) mice are indicated by Ba (Ser32), IKKb, NF-kB/p65, IkBa, VCAM-1, and ICAM-1 were added asterisks. pp , 0.05. The Journal of Immunology 6217 and mice were maintained on 0.2% neomycin sulfate for the first 3 wk. The polyacrylamide gels. Proteins were transferred onto polyvinylidene di- reconstituted mice were used for experiments 6 wk after the transplantation. fluoride membranes and were detected using the indicated Ab specific for We found that greater than 84% of blood leukocytes were derived from activated/phosphorylated NF-kB or total MAPKs, such as JNK, ERK, and donor mice at the time the mice were used for experiments (6–8 wk after p38. The Western blots were quantitated against GAPDH and expressed as transplantation). Irradiated mice that were not transplanted with donor cells fold increase in response to K. pneumoniae after background (unstimulated) died between days 18 and 22 after transplantation (data not shown). subtraction. In addition, culture media obtained from macrophages at 18 h after K. pneumoniae infection were used for cytokine/chemokine deter- Neutrophil depletion mination by ELISA. The protocol used to deplete neutrophils (GR1+) in mice has been described AM culture in our earlier reports (21) with modifications. A total of 50 mg anti-GR1+ mAb (sodium azide-free and low LPS content; RB6-8C5 from BD Phar- AMs were isolated as described in earlier publications (22, 23). Positive mingen, San Diego, CA) in 50 ml was administrated i.p. at 12 and 2 h before selection by CD11b MicroBeads (Stem Cell Technologies, Vancouver, bacterial infection. In control experiments, 50 mg of nonspecific isotype- British Columbia, Canada) was applied to isolate AMs from BAL cells. matched control mAb in equal volumes was administrated at the same time Pooled BAL cells were resuspended in RoboSep buffer, and CD11b PE points before bacterial infection. To validate the efficiency of neutrophil labeling reagent was mixed with cell suspension, followed by incubation at depletion by the Ab, we obtained differential WBC counts in blood every 12 room temperature for 15 min. EasySep PE selection mixture was thereafter h up to 3 d, and #2% neutrophils were found up to 3 d after depletion. added to the cell suspension, and the suspension was further incubated for 15 min at room temperature. EasySep Magnetic nanoparticles were lastly + BMDM culture mixed with the cell suspension, followed by magnetic separation of CD11b cells. Cells were washed with PBS, centrifuged at 1200 rpm for 10 min, and BMDMs were differentiated using DMEM containing 10% FBS and resuspended in DMEM containing 5% FBS. Cells were infected with M-CSF. BM cells were differentiated in culture dishes by supplementing K. pneumoniae (MOI of 1) and harvested at designated time points using DMEM with M-CSFon days 3 and 5 for 7 d. Cells were thenstimulated forthe urea/CHAPS/Tris buffer. Samples were centrifuged and supernatants were indicated times with K. pneumoniae (multiplicity of infection [MOI] of 1) used for Western blotting. In addition, culture media were collected at 18 h and processed in urea/CHAPS/Tris buffer before being separated on SDS postinfection and were used for cytokine/chemokines determination. Fi-

FIGURE 3. Activation of K. pneumoniae-mediated NF-kB and MAPKs, and expression of ICAM-1 and VCAM-1 in lung tissues of KC2/2 mice. A, KC- mediated regulation of IKK (an upstream kinase of NF-kB) and NF-kB/p65 and IkBa in the lungs of KC2/2 mice post-K. pneumoniae infection. The blots are representative of three independent experiments with similar results. B, Reduced activation of the NF-kB/p65 subunit of NF-kB as detected by p65 DNA binding in the nuclear extracts of murine lungs at 24 and 48 h after K. pneumoniae infection (n = 3–4 mice/group at a time point.). C, Attenuated upregulation of MAPKs and cell adhesion molecules (VCAM-1 and ICAM-1) in the lungs of KC2/2 mice after K. pneumoniae inoculation. Results are representative of three separate experiments with identical results. Values that are significantly different between KC2/2 and KC+/+ are indicated by asterisks (pp , 0.05). D, Densitometric analysis of Western blots from three independent experiments to quantify the protein levels of IKK, NF-kB, and phospho-IKK, –NF-kB, -IkBa and -MAPKs, as well as ICAM-1, VCAM-1, and total p38 post-K. pneumoniae infection. The results obtained were normalized against GAPDH and expressed as mean 6 SE. The difference between the KC2/2 and KC+/+ animals was determined and expressed as pp , 0.05. 6218 CXCL1/KC IN BACTERIAL PNEUMONIA nally, the total number of live cells and the percentage of purified AMs followed by Bonferroni’s post hoc analysis for multiple comparisons. All were determined by utilizing trypan blue exclusion and cytospin prepara- statistical calculations were performed using InStat software and GraphPad tions stained with Wright-Giemsa, respectively. Percentage recovery of Prism 4.0. Differences were considered statistically significant at p , 0.05 AMs was calculated as the ratio of AMs to the total macrophages isolated when compared with control. Survival curves were compared by Wilcoxon from the BALF. Using this technique, we obtained ∼85–90% AMs. rank sign test. Statistical analysis Results Data are expressed as mean 6 SE. The intensity of immunoreactive bands KC2/2 mice are highly susceptible to K. pneumoniae infection was determined using a Gel Digitizing Software (UN-SCAN-IT gel) from Silk Scientific (Orem City, UT). Data were analyzed by one-way ANOVA, To assess the requirement of KC in host defense against K. pneu- moniae, we infected KC2/2 and KC+/+ animals with K. pneumoniae (103 CFU/mouse) through the i.t. route, and survival was monitored for up to 15 d. We observed decreased survival in the KC2/2 group, and only 14.3% of mice survived to day 15 (Fig. 1A). In contrast, 75% of K. pneumoniae-infected KC+/+ mice survived up to 15 d after K. pneumoniae infection (Fig. 1A).

FIGURE 4. Role of bone marrow and non-bone marrow cells in host defense against K. pneumoniae. KC produced from bone marrow and non- bone marrow/resident cells after K. pneumoniae infection is essential for host defense. Bone marrow chimeras were generated by lethal irradiation of KC2/2 and KC+/+ mice and reconstituted with bone marrow cells via tail FIGURE 5. K. pneumoniae-induced cytokine/chemokine production in injection. A, Bacterial CFU in the lungs at 48 h post-K. pneumoniae in- lungs of neutrophil-depleted KC+/+ and KC2/2mice. A–D, KC derived from fection. B and C, Total WBC and neutrophil counts in BALF at 48 h post- neutrophils does not contribute to MIP-2 and LIX production. Mice were K. pneumoniae infection. D, KC levels in BALF at 48 h after K. pneumoniae pretreatedwithanti-Gr1orcontrolmAbat12and2hbeforeinfectionwith challenge. A total of 5–7 mice/group were used in 2 independent experi- 103 CFU K. pneumoniae. Cytokine/chemokines levels were determined at 24 ments, and statistical significance was expressed as pp , 0.05. and 48 h post-K. pneumoniae infection (n = 6–8 animals/group; pp , 0.05). The Journal of Immunology 6219

KC2/2 mice have impaired clearance of K. pneumoniae observed decreased activity in the lungs of KC2/2 mice as com- To determine whether the enhanced mortality in KC2/2 mice to K. pared with their WT counterparts (Fig. 1F), demonstrating at- pneumoniae was due to an impaired ability to limit bacterial growth tenuated neutrophil recruitment to the lung parenchyma. in the lungs and/or bacterial dissemination to the bloodstream, we 2 2 KC deficiency results in decreased MIP-2 and LIX production infected KC / and their control mice (KC+/+) with 103 CFU K. in response to K. pneumoniae pneumoniae, and assessed bacterial numbers in the lung and spleen at 24 and 48 h. As compared with KC+/+ (C57BL/6) mice, KC2/2 Because the impairment in K. pneumoniae-induced neutrophil 2 2 animals had a greater bacterial burden in the lungs at 48 h and more accumulation observed in KC / mice could reflect a decrease in bacterial dissemination at 24 h (Fig. 1B,1C). the levels of /chemokines, we quantified the expression of TNF-a, MIP-2, and LIX in BALF at 24 and 48 h after K. KC deficiency impairs inflammatory cell recruitment to the pneumoniae challenge. The levels of MIP-2 were found to be lung after K. pneumoniae challenge attenuated in KC2/2 mice at both 24 and 48 h after K. pneumoniae To assess the myeloid cells recruitment to the lungs of KC2/2 and infection, whereas the levels of LIX were found to be decreased at KC+/+ mice post-K. pneumoniae infection, we i.t. infected both 24 h postinfection (Fig. 2A,2B). However, TNF-a levels in BALF 2 2 groups of mice with K. pneumoniae and then enumerated the were not different between KC / and KC+/+ mice (Fig. 2C). recruited cells. In KC2/2 mice, pulmonary total WBC and neu- k trophil recruitment was attenuated at 24 and 48 h after K. pneu- K. pneumoniae-induced KC regulates NF- B activation in the moniae challenge. In control (saline-challenged) groups, however, lung no neutrophil influx in the lungs was observed in both KC2/2 and Previous reports have demonstrated that NF-kB is a major regula- KC+/+ mice (Fig. 1D,1E). We also measured MPO activity in tor of cytokines and chemokines, we next examined whether KC the lung parenchyma of infected KC2/2 and KC+/+ mice, and modulates NF-kB activation post-K. pneumoniae challenge. To

FIGURE 6. Activation of K. pneumoniae-induced NF-kB and MAPKs in lung tissues of neutrophil-depleted KC+/+ and KC2/2 mice. A, Wild type and KC2/2 mice challenged with 103 CFU i.t. were euthanized at 24 and 48 h postinfection, and lungs were used for Western blotting. The membranes were probed with activated NF-kB and MAPK Abs. B, Densitometric analysis of Western blots from three independently performed experiments to quantify the protein levels of IKK, NF-kB, ICAM-1, VCAM-1, and phospho-IKK, –NF-kB, and -MAPKs after K. pneumoniae challenge. The data obtained from these experiments were normalized against GAPDH and expressed as mean 6 SE from two independent experiments. The significance between the KC2/2 and KC+/+ mice was indicated as pp , 0.05. 6220 CXCL1/KC IN BACTERIAL PNEUMONIA elucidate NF-kB activation, we first determined the effect of K. wild type counterparts, although this decrease was significant only pneumoniae-induced KC on the phosphorylation of IKK, the up- at a later time point (48 h) (Fig. 3B,3D). stream kinase of NF-kB and Ik-Ba. Our data revealed activation of K. pneumoniae-induced MAPK activation is impaired in IKKa/b by KC. In a similar manner, we observed an increase 2 2 KC / mice in NF-kB /p65 phosphorylation at Ser536 at 48 h postinfection. Although we also observed a modest decrease in the expression of Decreased neutrophil accumulation in the lungs in response to K. 2/2 IKKb and NF-kB/p65 proteins in KC2/2 mice after K. pneumoniae pneumoniae in KC mice could be attributed to reduced acti- infection, these changes were not significant as compared with the vation of MAPKs, which can then regulate other transcription posttranslational modifications (phosphorylation) of NF-kB (Fig. factors besides NF-kB, such as AP-1 and STAT-1 (24, 25). 3A–D). In addition, we repeatedly could not detect phosphorylated Therefore, we examined the activation of JNK, ERK, and p38 2/2 +/+ IKKa/b or NF-kB/p65 in the control (saline-challenged) KC2/2 kinases in the lungs of KC and KC mice after K. pneumo- and KC+/+ animals. Moreover, our time-course experiments niae infection. Activation of JNK was attenuated both at 24 and 48 revealed a decrease in the phosphorylation of IkBa (Ser32) in the h after K. pneumoniae infection, whereas the activation of ERK lungs of KC2/2 mice starting from 24 h posttreatment and persisted was reduced only at 48 h (Fig. 3C,3D). In addition, activation of 2/2 up to 48 h post-K. pneumoniae infection. Concomitantly, we also p38 was attenuated in the lungs of KC mice at 24 h post- observed an increased accumulation of IkBa protein in KC2/2 infection (Fig. 3C,3D). mice at 48 h post-K. pneumoniae infection (Fig. 3A–D). To confirm decreased activation of NF-kB /p65 in KC2/2 mice after K. KC contributes to K. pneumoniae-induced ICAM-1 and pneumoniae infection, we determined p65 DNA binding using the VCAM-1 expression nuclear extracts from the lungs. Our findings revealed a decrease in Successful neutrophil recruitment to the lung in response to infec- NF-kB/p65 DNA binding in KC2/2 mice as compared with their tion requires the efficient upregulation of cell adhesion molecules

FIGURE 7. Activation of NF-kB and MAPKs in BMDMs of KC2/2 mice infected with K. pneumoniae. A, Decreased activation of NF-kB and MAPKs in BMDMs obtained from KC2/2 mice post-K. pneumoniae infection. Activation of IKK, NF-kB, and MAPKs was detected at 24 and 48 h after K. pneumoniae stimulation. The blot is a representative of three independent experiments with identical results. B, Densitometric analysis of IKK, NF-kB, and MAPK activation in BMDMs up to 60 min after stimulation with K. pneumoniae (MOI of 1). Data expressed as mean 6 SE of three blots from three mice in each group (pp , 0.05). C–E, Attenuated production of MIP-2 in BMDMs obtained from KC2/2 mice post-K. pneumoniae infection. BMDMs were stimulated with K. pneumoniae at an MOI of 1, and supernatants collected at 18 h postinfection were used to determine MIP-2, KC, and TNF-a release. The data were obtained from three independent experiments. The Journal of Immunology 6221 on vascular endothelium and leukocytes. We therefore examined the KC+/+ irradiated mice reconstituted with KC2/2 bone marrow did expression of ICAM-1 and VCAM-1 in lung homogenates after not show differences in lung CFU, neutrophil influx, or KC levels K. pneumoniae infection. In KC2/2 mice, the expression of both after K. pneumoniae infection (Fig. 3A–D) As expected, neutro- ICAM-1 and VCAM-1 was attenuated in the lung at 48 h after phil recruitment to the lung or KC production was not observed in K. pneumoniae infection relative to the KC+/+ mice (Fig. 3C,3D). reconstituted mice after saline challenge (data not shown). Bone marrow cell and non-bone marrow cell-derived KC Neutrophils do not contribute to KC production in the lungs contributes to host defense after pulmonary K. pneumoniae after K. pneumoniae challenge infection We observed substantial neutrophil recruitment to the lung at 48 h We next studied the relative importance of bone marrow (radio- post-K. pneumoniae infection (Fig. 1C–E). Therefore, we depleted sensitive) versus resident/nonhematopoietic (radioresistant) cell- neutrophils from KC+/+ and KC2/2 mice to assess their role in the derived KC in host defense using bone marrow chimeras. Lung production of cytokines/chemokines, as well as activation of NF- CFUs in response to K. pneumoniae infection were increased in kB and MAPKs in the lungs. To deplete neutrophils, we treated irradiated KC2/2 reconstituted with KC+/+ bone marrow (Fig. 4A). mice with anti–Gr-1 (RB6-8C5) Ab at 12 and 2 h before K. pneumoniae-induced total leukocyte and neutrophil influx, as K. pneumoniae challenge, whereas isotype-matched Ab was used well as KC production, was attenuated in irradiated KC2/2 as a control. Neutrophil depletion did not substantially alter the reconstituted with KC2/2 bone marrow (Fig. 4B–D). As compared differences in MIP-2, LIX, and TNF-a levels between KC+/+ and with irradiated KC+/+ mice reconstituted with KC+/+ bone marrow, KC2/2 mice after K. pneumoniae infection (Fig. 5). In a similar irradiated KC2/2 mice reconstituted with KC+/+ bone marrow or manner, activation of NF-kB and MAPKs, and expression of

FIGURE 8. Effect of KC neutralization on the production of cytokines/chemokines in BMDMs after K. pneumoniae challenge. A–C, BMDMs from KC+/+ mice produce KC, MIP-2, and TNF-a during differentiation in the absence of K. pneumoniae infection on day 7. Supernatants were collected at 1, 3, 5, and 7 d culture, and cytokine/chemokine levels were assessed by sandwich ELISA. Data are representative of two independent experiments using three mice per group (pp , 0.05). D–F, Blocking KC using Ab-attenuated KC and MIP-2 but not TNF-a production in BMDMs. BMDMs were pretreated with KC neutralizing Ab 2 h before K. pneumoniae infection (MOI of 1). Supernatants were collected 2, 4, and 8 h after K. pneumoniae challenge, and cytokine/ chemokines levels were measured by sandwich ELISA. Data are pooled from two independent experiments using five mice per group, and significant difference was expressed as pp , 0.05. 6222 CXCL1/KC IN BACTERIAL PNEUMONIA

VCAM-1 and ICAM-1 was not different between KC+/+ and KC and MIP-2 production and activation of NF-kB and MAPKs KC2/2 mice postinfection (Fig. 6). were attenuated in AMs obtained from KC2/2 mice as compared 2 2 with cells obtained from their littermate control mice (Fig. 10). BMDMs obtained from KC / mice have attenuated activation of NF-kB and MAPKs in response to K. pneumoniae We next assessed whether KC deficiency has any effect on acti- Discussion vation of NF-kB and MAPKs in myeloid cells. In particular, we Community-acquired pneumonia caused by K. pneumoniae is have used macrophages because these cells are known to mediate a leading cause of death due to extensive lung damage (26). Unlike host defense. When KC2/2 macrophages were stimulated with many other Gram-negative bacterial pathogens, K. pneumoniae can K. pneumoniae, reduced activation of NF-kB, as well as JNK, induce disseminated infection even with a relatively low dose. ERK, and p38, was observed (Fig. 7A,7B). We observed attenu- Although initial treatment of community-acquired pneumonia is ated MIP-2 production in macrophages obtained from KC2/2 empirical antibiotic coverage, increasing numbers of immuno- mice, although TNF-a levels between KC2/2 and KC+/+ mice compromised patients, a growing proportion of elderly individuals, were not different after K. pneumoniae infection (Fig. 7C–E). To and the emergence of antibiotic-resistant K. pneumoniae makes this examine the basal levels of cytokines/chemokines in BMDMs, we treatment difficult. Therefore, modulation of the host immune re- measured the protein levels of MIP-2, KC, and TNF-a. Our results sponse itself is an attractive option for clinical management, par- also show that BMDMs release KC, MIP-2, and TNF-a during ticularly among patients with immune system impairment or differentiation on day 7 in the absence of K. pneumoniae infection, infected with antibiotic-resistant bacteria. Development of more although these levels are much less than in the presence of effective agents requires a better understanding of the mechanisms K. pneumoniae infection (Fig. 8A–C). by which innate defense against bacteria is regulated. Successful host defense against bacteria involves the brisk re- KC mediates expression of cytokine/chemokines and activation cruitment of neutrophils because they phagocytose and eliminate k of NF- B and MAPKs in an autocrine and paracrine manner microbes. Neutrophil migration to the lungs involves the expression To test the hypothesis that KC-induced production of cytokine/ of chemokines. Earlier reports have demonstrated that KC, MIP-2, chemokines and activation of NF-kB and MAPKs involves auto- and LIX are important for neutrophil accumulation in the lungs (11, crine and paracrine responses in BMDMs, we used a KC neutral- 13–15). These investigations have primarily been performed using izing Ab. As demonstrated in Figs. 8 and 9, blocking KC reduced blocking Abs. Subsequently, several KC transgenic mouse strains release of KC and MIP-2, but not TNF-a (Fig. 8D,8E), and acti- have been used to demonstrate the importance of KC in neutrophil vation of NF-kB and MAPKs (Fig. 9). We used murine AMs to influx in the lungs, although the transgene expression was ex- confirm the findings obtained from BMDMs, and demonstrated that pressed in all tissues, including the lung (27, 28). Therefore,

FIGURE 9. Effect of KC blocking on activation of NF-kB and MAPKs in BMDMs challenged with K. pneumoniae. A, KC blocking decreased activation of NF-kB and MAPKs in BMDMs obtained from KC+/+ (C57BL/6) mice after pulmonary K. pneumoniae infection. Activation of NF-kB and MAPKs was detected at various time points after stimulation. The blot is representative of four blots with identical results. B and C, Densitometric analysis of IKK, NF- kB, IkBa, and MAPK activation in BMDMs at 2, 4, and 8 h poststimulation with an MOI of 1. Data expressed as mean 6 SE of three independent experiments in each group (pp , 0.05). The Journal of Immunology 6223

FIGURE 10. Effect of KC disruption on cytokine/chemokine production and activation of NF-kB and MAPKs in AMs after K. pneumoniae infection. A–C, Decreased production of MIP-2 in AMs obtained from KC2/2 mice to K. pneumoniae challenge. Alveolar macrophages were stimulated with K. pneumoniae at an MOI of 1, and supernatants collected at 18 h postinfection were used to determine MIP-2, TNF-a, and KC levels. The data were obtained from two independent experiments using four mice in total. D, Reduced activation of NF-kB and MAPKs in AMs obtained from KC2/2 mice after K. pneumoniae infection. Activation of IKK, NF-kB, and MAPKs was detected at various time points after K. pneumoniae stimulation (MOI of 1). The blot is a representative of three experiments with identical results. E, Densitometric analysis of IKK, NF-kB, and MAPK activation in BMDMs up to 8 h after K. pneumoniae stimulation with an MOI of 1. Data expressed as mean 6 SE of three blots from three mice in each group from two independent experiments (pp , 0.05). a lung-specific KC transgene-expressing mouse was later de- support that KC not only has a direct role but also has an indirect veloped to demonstrate the importance of KC in K. pneumoniae- role via upregulating other proinflammatory mediators in the lungs induced neutrophil recruitment and bacterial clearance. An im- to augment host defense during bacterial infections. portant challenge that remains is the determination of the Neutrophil infiltration into mucosal epithelia is a hallmark of KC-mediated signaling mechanisms in the lungs during pulmo- bacterial pneumonia, and this involves a multistep paradigm. nary bacterial infection. In this report, we have used a loss-of- Neutrophils in the bloodstream adhere to the vascular endothelium function model using mice deficient in KC to delineate the and migrate across the endothelium to reach sites of inflammation mechanisms associated with K. pneumoniae-induced neutrophil- in various organs, including the lungs (29). This process is complex mediated bacterial clearance in the lungs. This model provided and involves multiple adhesion molecules from different families an opportunity to address the mechanisms related to KC-induced, that are expressed on the surface of endothelial cells and leuko- neutrophil-mediated host defense in the lungs against bacterial cytes in response to different cytokines. These endothelial proteins pathogens, including K. pneumoniae. Using this murine model, we recognize specific leukocyte glycoproteins and facilitate their re- confirmed the previous observations (27, 28) that KC is essential versible adherence to the vessel wall, known as tethering. This is for survival and the clearance of K. pneumoniae from the host. followed by firm attachment of neutrophils to the endothelium and A role for chemokines to amplify the inflammation in the lungs eventual extravasation through diapedesis. In this investigation, has been documented in an investigation using KC transgenic mice we identified that K. pneumoniae regulates the expression of (28). That report provided evidence that KC regulates the ex- VCAM-1 and ICAM-1 after K. pneumoniae infection, and this is pression of MIP-2 in the lungs in KC transgenic mice infected one of the KC-mediated defense mechanisms involved in neu- with K. pneumoniae, whereas KC does not regulate TNF-a, IFN- trophil accumulation and bacterial clearance in the lungs. g, IL-12, and IL-10 (28). Our data show that KC also regulates A central feature of inflammation is the activation of tran- other chemokines, such as LIX, in response to K. pneumoniae scription factors. Among these, NF-kB is well studied and infection. These observations reveal that KC can augment becomes activated in response to stress, cytokines, free radicals, neutrophil-mediated lung inflammation and host defense against UV irradiation, oxidized low-density lipoprotein, and bacterial or bacteria via the upregulation of MIP-2 and LIX. These findings viral Ags. NF-kB activation has been reported to contribute to 6224 CXCL1/KC IN BACTERIAL PNEUMONIA

A key finding of our investigation is that depletion of neutrophils resulted in no substantial change in the production ofKC inthe lungs. These results are consistent with the fact that several other cell types are involved in the initiation of KC production during pulmonary infectious challenge. However, Kp-induced production of TNF-a was substantially reduced in the lungs of neutrophil-depleted KC+/+ and KC2/2 mice, whereas Kp-induced TNF-a levels were not different between KC+/+ and KC2/2 mice. Our observations suggest that neutrophils may be the key source for TNF-a, a cyto- kine that is not regulated by KC-dependent signaling cascade(s). We used macrophages in our in vitro system because this cell type is a vital mediator of host defense in the lungs and found that mac- rophages modulate lung inflammation on K. pneumoniae infection via KC-mediated activation of NF-kB and MAPKs. Our data also show that KC regulates MIP-2 in macrophages on K. pneumoniae infection. Because KC controls the production of MIP-2 from macrophages and LIX from alveolar epithelial type II cells in the lung, it is likely that KC-mediates both autocrine and paracrine effects on K. pneumoniae infection. In this regard, we have ob- served expression of KC and its receptor (CXCR2) in large and small airway cells, alveolar type II cells, macrophages, and neu- FIGURE 11. Schematic depicting the functions of KC leading to neu- trophils (data not shown). trophil accumulation after K. pneumoniae infection. KC activates of NF- 2/2 k KC mice have been used in a few prior studies: 1) KC/low- B and MAPK cause upregulation of chemokines, such as KC, MIP-2, 2/2 LIX, and adhesion molecules, including VCAM-1 and ICAM-1. In turn, density lipoprotein receptor mice exhibit protective effects 2/2 these events ultimately cause neutrophil recruitment to clear the bacterial against the development of atherosclerosis (36), and 2) KC infection from the lungs. mice also show protection against dextran sulfate sodium-induced colitis (17). We took advantage of the gene-deficient mice to dem- neutrophil accumulation in the lungs by bacteria via multiple onstrate the host defense mechanisms induced by KC after K. mechanisms, including upregulation of cell adhesion molecules pneumoniae infection. The findings from this study unveil that (VCAM-1 and ICAM-1). In this regard, studies demonstrate that multiple signaling cascades involving transcription factors, kina- endogenous NF-kB protects the host by preventing excessive in- ses, and the expression of proinflammatory mediators are altered flammation and injury during E. coli (30) and pneumococcal in Gram-negative pneumonia via KC (Fig. 11, proposed model). pneumonia (31). Moreover, NF-kB is shown to be essential for This investigation offers the first step, to our knowledge, into the antibacterial host defense (30, 31). Our findings from this study biology of KC in mediating pulmonary antibacterial defense on support an important role for KC in late NF-kB activation (48 h) bacterial infection. in the lungs after K. pneumoniae infection. In addition, our find- ings also suggest the involvement of other transcription factors Acknowledgments such as AP-1/STAT-1 because MAPKs are known to regulate AP-1 We thank Nades Palaniyar (University of Toronto, Toronto, Ontario, Can- and STAT-1 (32), and our results reveal that KC mediates activation ada) for helpful discussions and Rachel Zemans and Ken Malcolm (National of all three MAPKs at a late time point (48 h). Jewish Health, Denver, CO), Dan Chisenhall, Pete Mottram (Louisiana The relative contribution of myeloid cells (macrophages and State University), and Lung Biology laboratory members, and Gayathriy neutrophils) versus resident tissue cells in neutrophil accumulation Balamayooran and Himanshu Raje for critical reading of the manuscript. into the lung from the bloodstream has not been clear. 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