IL-17A and TNF-α Exert Synergistic Effects on Expression of CXCL5 by Alveolar Type II Cells In Vivo and In Vitro

This information is current as Yuhong Liu, Junjie Mei, Linda Gonzales, Guang Yang, Ning of September 29, 2021. Dai, Ping Wang, Peggy Zhang, Michael Favara, Kenneth C. Malcolm, Susan Guttentag and G. Scott Worthen J Immunol published online 31 January 2011 http://www.jimmunol.org/content/early/2011/01/31/jimmun

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 31, 2011, doi:10.4049/jimmunol.1002016 The Journal of Immunology

IL-17A and TNF-a Exert Synergistic Effects on Expression of CXCL5 by Alveolar Type II Cells In Vivo and In Vitro

Yuhong Liu,*,1 Junjie Mei,*,1 Linda Gonzales,*,1 Guang Yang,* Ning Dai,* Ping Wang,* Peggy Zhang,* Michael Favara,* Kenneth C. Malcolm,† Susan Guttentag,*,‡ and G. Scott Worthen*,‡

CXCL5, a member of the CXC family of , contributes to neutrophil recruitment during lung inflammation, but its regulation is poorly understood. Because the T cell-derived IL-17A enhances host defense by triggering production of chemokines, particularly in combination with TNF-a, we hypothesized that IL-17A would enhance TNF-a–induced expression of CXCL5. Intratracheal coadministration of IL-17A and TNF-a in mice induced production of CXCL1, CXCL2, and CXCL5, which was associated with increased neutrophil influx in the lung at 8 and 24 h. The synergistic effects of TNF-a and IL17A were greatly attenuated in Cxcl52/2 mice at 24 h, but not 8 h, after exposure, a time when CXCL5 expression was at its peak in wild- Downloaded from type mice. Bone marrow chimeras produced using Cxcl52/2 donors and recipients demonstrated that lung-resident cells were the source of CXCL5. Using differentiated alveolar epithelial type II (ATII) cells derived from human fetal lung, we found that IL-17A enhanced TNF-a–induced CXCL5 transcription and stabilized TNF-a–induced CXCL5 transcripts. Whereas expression of CXCL5 required activation of NF-kB, IL-17A did not increase TNF-a–induced NF-kB activation. Apical costimulation of IL- 17A and TNF-a provoked apical secretion of CXCL5 by human ATII cells in a transwell system, whereas basolateral costimu- lation led to both apical and basolateral secretion of CXCL5. The observation that human ATII cells secrete CXCL5 in a polarized http://www.jimmunol.org/ fashion may represent a mechanism to recruit neutrophils in host defense in a fashion that discriminates the site of initial injury. The Journal of Immunology, 2011, 186: 000–000.

ung host defense requires both innate and adaptive im- signals in induction of ATII cell responses, and which mune responses acting in a highly coordinated manner. and chemokines are released, remains unclear. L Innate responses are initiated by microbial recognition via In addition to innate responses, adaptive immune responses, pattern recognition receptors. This recognition triggers the acti- characterized by specificity and memory and mediated primarily by vation of transcription factors, release of early response cytokines T and B lymphocytes, are important in host defense (11, 12). by guest on September 29, 2021 (particularly TNF-a) (1–3) and chemokines, recruitment of neu- Recent evidence suggests that a specific subset of CD4+ T cells trophils, and consequent clearance of microorganisms (4–6). Al- secreting IL-17 plays a nonredundant role in directing innate though alveolar macrophages (AMs) have been accepted as an responses (12). IL-17A is the prototype member of IL-17 family, important component of innate immunity in the lung, alveolar type which is associated with increased neutrophil influx through in- II (ATII) cells have recently been demonstrated to be an active duction of proinflammatory cytokines and chemokines during participant in the innate immune response. ATII cells participate bacterial infection (13–16). Which lung cell responds to IL-17A, in lung host defense not only by secreting surfactant lipids (7) and however, and the signals released that promote neutrophil accu- surfactant-associated (8) to block and opsonize bacteria mulation is not known. The best-known candidates, however, are and virus, but also by releasing chemokines to facilitate movement glutamic acid-leucine-arginine (ELR)+ CXC chemokines. of immune cells (9, 10). Despite these insights, the role of specific CXCL5 (known as LPS-induced CXC [LIX] in mice and epithelial cell-derived neutrophil-activating peptide-78 [ENA- 78] in human) is a unique member of ELR+ CXC family of chemokines, which are responsible for mediating neutrophil re- *Division of Neonatology, Children’s Hospital of Philadelphia, Philadelphia, PA cruitment into tissues during inflammation and infection (17). 19014; †Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206; and ‡University of Pennsylvania, Philadelphia, PA 19014 ENA-78 has been found to be involved in a variety of human 1Y.L., J.M., and L.G. contributed equally to this work. inflammatory diseases (18–27), such as chronic obstructive pul- monary disease, exacerbation of asthma and chronic obstructive Received for publication June 29, 2010. Accepted for publication December 23, 2010. pulmonary disease, acute coronary syndromes, diabetes mellitus, This work was supported by National Institutes of Health Grant HL068876. and inflammatory bowel diseases. Murine Cxcl5 was first cloned Address correspondence and reprint requests to Dr. G. Scott Worthen, Division of as a LPS response that could be attenuated by glucocorti- Neonatology, Abramson Research Center, Room #414C, Children’s Hospital of Phil- coids (28). In comparison with other CXC chemokines, specifi- adelphia, Philadelphia, PA19014. E-mail address: [email protected] cally CXCL1 (also referred to as keratinocyte chemoattractant Abbreviations used in this article: AM, alveolar macrophage; ATII, alveolar type II [KC]) and CXCL2 (also referred to as MIP-2), CXCL5 was shown cell; BALF, bronchoalveolar lavage fluid; BM, bone marrow; CHX, cycloheximide; DCI, dexamethasone plus 8-Br-cAMP plus isobutylmethylxanthine; ELR, glutamic to have distinct induction kinetics, tissue distribution, and sensi- acid-leucine-arginine; ENA-78, epithelial cell-derived neutrophil-activating peptide- tivity to glucocorticoids in an acute endotoxemia model (29). We 78; i.t., intratracheal; KC, keratinocyte chemoattractant; LIX, LPS-induced CXC have previously shown (10) that CXCL5 was expressed by ATII chemokine; qPCR, quantitative PCR; rm, recombinant mouse; WT, wild-type. cells through immunohistochemical analysis and primary rodent Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 ATII cell culture, in response to LPS stimulation, whereas KC and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002016 2 SYNERGISTIC EFFECTS OF IL-17A AND TNF-a ON CXCL5 EXPRESSION

MIP-2 are mainly expressed by alveolar macrophages. We further xylazene (10 mg/kg), and a midventral incision was performed. After generated Cxcl5-deficient mice and demonstrated the nonre- isolation of muscles, the trachea was exposed, and each mouse was in- 6 7 m dundant but opposing roles of CXCL5 in mediating neutrophil oculated with 10 or 10 CFU E. coli in 50 l in 0.9% saline. The mice were humanely sacrificed at 8 and 24 h postinfection, and BALF was col- influx to the lung in a localized LPS inhalation model and a severe lected. Escherichia coli pneumonia model (30). Upon LPS inhalation, CXCL5 expression in bronchoalveolar lavage fluid (BALF) was Intratracheal administration of recombinant mouse IL-17A a much more persistent than KC and MIP-2, which contributed to its and/or recombinant mouse TNF- dominant role in mediating neutrophil influx to the lung. Fur- C57BL/6 mice were divided into four groups as follows: 1) PBS alone; 2) thermore, in a model of severe E. coli pneumonia, CXCL5 release recombinant mouse (rm)IL-17A (625 ng/mouse) alone; 3) rmTNF-a (500 induced high levels of KC and MIP-2 in both BALF and plasma ng/mouse) alone; and 4) rmIL-17A plus rmTNF-a. All treatments were administered in a volume of 50 ml. Mice were euthanized at 4, 8, and 24 h. at least partially through inhibiting chemokine scavenging and 2/2 Cxcl5 mice were compared with C57BL/6 mice receiving rmIL-17A thereby modifying the inflammatory response. Although our group plus rmTNF-a at 8 and 24 h. has described CXCL5 as a product of ATII cells in mice and rats, and as a critical player in lung inflammation and pulmonary host Generation of chimeric mice by bone marrow reconstitution defense, whether human ATII cells respond similarly is unknown. The 6–8-wk-old recipient mice were lethally irradiated in two doses of 800 Furthermore, the stimuli that induce CXCL5 in the ATII cell are and 400 rad with an interval of 3 h. Bone marrow (BM) from donor mice poorly characterized. Because previous reports have indicated that was harvested from both the tibiae and femora. After lysis of red cells, ∼5 IL-17A in combination with TNF-a increased synergistically the million cells were i.v. injected into recipient mice after lethal irradiation. The BM reconstitution was performed in four groups of mice: 1) BM from expression of chemokines (16, 17), we hypothesized that IL-17A wild-type (WT) mice into WT mice; 2) BM from WT mice into Cxcl52/2 Downloaded from combined with TNF-a would exert synergistic effects on CXCL5 mice; 3) BM from Cxcl52/2 mice into WT mice; and 4) BM from Cxcl52/2 2 2 expression during lung inflammation. We further tested whether mice into Cxcl5 / mice. Eight weeks after irradiation, mice were used for 6 such synergistic effect would be manifested in primary human 10 E. coli i.t. inoculation. Analysis of blood genotypes after reconstitution indicated no admixture of recipient cells (data not shown). ATII cells, well known to be critical for development as well as normal homeostasis. Analysis of leukocytes in BALF

In this study, we first showed the expression of IL-17A, TNF-a, http://www.jimmunol.org/ At specified times after treatment, the mice were anesthetized and then and CXCL5 in severely inflamed mouse lungs. A synergistic ef- sacrificed.BALFwasconductedwith0.8-mlaliquotsofPBSina totalvolume fect on neutrophil influx was induced by intratracheal (i.t.) co- of 3.2 ml containing 100 mM diethylenetriamine pentaacetic acid. Leuko- administration of IL-17A and TNF-a, and this was associated with cytes from BALF were quantified using a hemacytometer and also processed increased expression of CXCL1 and CXCL5. In contrast to by cytospin. Slides were fixed and stained with Diff-quik staining kit (Sigma-Aldrich), and the neutrophil counts were calculated by multiplying CXCL1, CXCL5 played a dominant role in neutrophil influx neutrophil percentage by total number of leukocytes in the lavage. stimulated with IL-17A and TNF-a coinstillation at a later phase of influx. In differentiated primary human fetal ATII cells, we Cell culture demonstrated, for the first time to our knowledge, expression of Human fetal lung tissue (16–20 wk gestation) was obtained from Advanced CXCL5 by human ATII cells and confirmed the synergistic effect Bioscience Resources (Alameda, CA) under a protocol approved by by guest on September 29, 2021 of IL-17A and TNF-a on induction of CXCL5. The mechanism of Children’s Hospital of Philadelphia. Explants of the fetal lung were cul- this effect was due to both transcription and mRNA stability and tured 4 d in dexamethasone (10 nM) plus 8-Br-cAMP (0.1 mM) plus k isobutylmethylxanthine (0.1 mM) (DCI) at 37˚C in 5% CO2 incubator to required NF- B activation and new synthesis. We also induce a type II cell phenotype as described (32). A highly enriched showed, to our knowledge for the first time, a unique polarization population of type II cells was isolated as described (33) and cultured in both of stimulation and release of CXCL5 in ATII cells, which DCI to maintain the type II phenotype. may have an important role in acute lung injury during lung in- Nuclear run-on assay flammation and infection. For the measurement of gene transcription, nuclear run-on assay was performed following the protocol of Hildebrandt and colleagues (34). Materials and Methods Briefly, nuclei were isolated from human fetal ATII cells treated with or Reagents without TNF-a (10ng/ml) in the presence of IL-17 (25 ng/ml) for 24 h. Transcripts that were initiated in the cells were allowed to continue in the E. coli strain (ATCC 25922) was purchased from American Type Culture presence of NTPs for 15 min at 22˚C. RNA was extracted and reverse- Collection. Mouse rIL-17A (421-ML/CF), TNF-a (410-MT/CF), human a transcribed using the Superscript First-Strand System (Invitrogen) rIL-17A (317-IL), TNF- (210-TA), mouse CXCL5/LIX (DY443), and according to the manufacturer’s instructions. Real-time quantitative PCR human CXCL5/ENA-78 ELISA kit (DY254) were obtained from R&D k a (qPCR) was performed using TaqMan Univeral PCR Master Mix kit Systems. I B phosphorylation inhibitor BAY-11-7082 was obtained from (Applied Biosystems) with the TaqMan Assays (Applied Calbiochem. Stock solutions of the inhibitors were prepared in DMSO. Biosystems). The actinomycin D (A9415) was from Sigma-Aldrich. ENA-78 mRNA stability Mice a 2/2 2/2 Human alveolar type II cells were treated with TNF- (10 ng/ml) in the Generation of Cxcl5 mice has been described (30). Cxcl5 mice absence or presence of IL-17A (10 ng/ml) for 10 h. Cells were then washed (backcrossed with C57BL/6J mice for six generations) and their control and incubated with actinomycin D (5 mg/ml) to inhibit further transcrip- C57BL/6 mice were kept in specific pathogen-free conditions in the animal tion. Total RNA was extracted at 0, 4, 8, and 12 h, and CXCL5/ENA-78 facility of the Children’s Hospital of Philadelphia. All experimental pro- mRNA was quantified by qPCR. The percentages of the remaining mRNA cedures were in compliance with guidelines approved by the Institutional at different time points as compared with 0 h were calculated. Animal Care and Use Committee of the Children’s Hospital of Phila- delphia. Eight- to 12-wk-old and sex- and age-matched mice were used for Luciferase reporter assay experiments. Human ATII cells were seeded onto 96-well plates at a cell density of 5 3 4 E. coli pneumonia mouse model 10 cells/well in Waymouth’s medium (11220; Life Technologies) with DCI and incubated at 37˚C in 5% CO2 incubator. On day 4, the cells were The procedures for the E. coli pneumonia (ATCC 25922; American Type infected with adenovirus vectors that had NF-kB–driven firefly luciferase Culture Collection) mouse model have been previously described (31). element (3 3 105 PFU/well) and pRL-SV40-driven renilla luciferase el- Briefly, the mice were anesthetized with ketamine (100 mg/kg) and ement (3 3 105 PFU/well) (Vector Biolaboratories, Philadelphia, PA). The Journal of Immunology 3

FIGURE 1. Expression of IL-17A, TNF-a, and CXCL5 in mouse lung in response to E. coli i.t. chal- lenge. C57BL/6 mice were subjected to 107 CFU E. coli via i.t. inoculation; BALF was collected at differ- ent time points. The protein levels of IL-17A (A), TNF- a (B), and CXCL5 (C) in the BALF were measured by ELISA. Values are mean 6 SEM (n = 3–6). *p , 0.05 versus saline control group, **p , 0.01, ***p , 0.001.

Twenty-four hours postinfection, the cells were then treated with IL-17A, Results a TNF- , or both for 24 h in fresh medium. The cells were assayed for firefly Expression of IL-17A, TNF-a, and CXCL5 in mouse models of and renilla luciferase activities using Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was measured by the IVIS Imaging lung inflammation System (Series 100; Caliper Life Sciences). Data was normalized as in- To investigate the expression patterns of IL-17A, TNF-a, and structed. CXCL5 in pulmonary inflammation, we challenged C57BL/6 7 Human ATII cell monolayers on transwell filters mice with 10 CFU E. coli via i.t. inoculation. Mice exposed to E. coli challenge demonstrated increased IL-17A protein in BALF Human ATII cells were cultured on transwell filters (collagen-coated 8 h postinoculation that was sustained to 24 h (Fig. 1A). TNF-a Downloaded from transwell, 12-mm diameter, 0.4 mm pore size; Corning). The human ATII cells were seeded at 2.5 3 105 cells/cm2 in a volume of 500 ml/filter. protein level was markedly increased 8 h after E. coli inoculation In the lower compartment, 1.5 ml medium were added, thereby leveling and was decreased to 24 h (Fig. 1B). CXCL5, though increased at the height of the liquid levels to prevent hydrostatic pressure. Trans- 8 h, continued to increase through 24 h postinoculation (Fig. 1C). 2 epithelial resistance was measured was measured at .300 V/cm prior to Thus, expression of CXCL5 is associated with protein production stimulation of the cells. of IL-17A and TNF-a in BALF after E. coli challenge. http://www.jimmunol.org/ Multiplex 42-bead array assay Exogenous administration of IL-17A and TNF-a into the lung + The 96-well filter plate was prewetted with 200 ml assay buffer per well, exerts synergistic effect on the expressions of ELR CXC then the buffer was removed using a vacuum manifold. Each standard or chemokines and neutrophil influx m control or sample (25 l) was added into the appropriate wells. Assay a buffer was used for background. A total of 25 ml assay buffer was added to To further explore the role of IL-17A and TNF- in pulmonary the sample wells, and the same amount of cell culture medium was added inflammatory responses, we administered IL-17A and TNF-a, to the rest of the wells. The premixed beads (25 ml) were loaded into each alone or in combination, via i.t. instillation to C57BL/6 mice. Lung well and incubated overnight at 4˚C with shaking. The next day, the beads lavage was performed at 4, 8, and 24 h. IL-17A and TNF-a acted were washed two times by vacuum, and detection Abs were added. After m synergistically to promote neutrophil influx to the lung at 8 and incubation for 1 h at room temperature, 25 l streptavidin-PE was pipetted by guest on September 29, 2021 into each well. The beads were washed twice and then resuspended in 150 24h(Fig.2F). IL-17A and TNF-a together had a synergistic effect ml sheath fluid. The filter plate was run on Luminex 200. on CXCL5 expression at later time points (Fig. 2A). By comparison, the synergistic effect of IL-17A and TNF-a to increase CXCL1 Statistical analysis (Fig. 2B) and CXCL2 (Fig. 2C) was evident only at early time Data are presented as means 6 SEM. We used one-way ANOVA or two- points. The combination of two cytokines also synergistically way ANOVA to compare datasets. increased G-CSF expression (Fig. 2D) that was detectable at all

FIGURE 2. Exogenous administration of IL-17A and TNF-a into the lung exerts synergistic effect on expression of ELR+ CXC chemokines and neutrophil influx. C57BL/6 mice were i.t. instilled with PBS, IL-17A (625 ng/50 ml), TNF-a (500 ng/50 ml), or IL-17A + TNF-a ([625+500] ng/50 ml). Whole lung lavage was performed at 4, 8, and 24 h. The protein levels of CXCL5 (A), CXCL1 (B), CXCL2 (C), and G-CSF (D) in the BALF were measured by ELISA. The number of WBCs (E) and neutrophils (F) per milliliter of BALF were determined by light microscopy. Values are mean 6 SEM (n = 3–4). *p , 0.05 versus PBS, **p , 0.01, ***p , 0.001. 4 SYNERGISTIC EFFECTS OF IL-17A AND TNF-a ON CXCL5 EXPRESSION Downloaded from

FIGURE 3. CXCL5 plays a dominant role in mediating neutrophil influx to the lung induced by coinstillation of IL-17A + TNF-a. After coinstillation of IL-17A and TNF-a ([625+500] ng/50 ml) into the tracheas of both WT and Cxcl52/2 mice, whole lung lavage was performed at 8 and 24 h. The protein levels of CXCL5 (A), CXCL1 (B), CXCL2 (C), and G-CSF in the BALF of WT and Cxcl52/2 mice were measured by ELISA. The number of WBCs (D) and neutrophils (E) per ml of BALF were determined by light microscopy. Values are mean 6 SEM (n = 3–8). ***p , 0.001. N.D., not detected. http://www.jimmunol.org/ time points. None of the CXC chemokines, CXCL5, CXCL1, or 3A), CXCL1 (Fig. 3B) and CXCL2 (Fig. 3C) in the BALF were not CXCL2, were expressed in response to IL-17A alone, whereas the different from WT animals receiving IL-17A and TNF-a for 24 h. expression of CXCL5 and CXCL1 was increased by TNF-a The response to deletion of CXCL5 in the setting of similar stimulation alone at 24 and 4 h, respectively (Fig. 2A,2B). amounts of CXCL1 and CXCL2 at this time point indicates that CXCL5 plays a dominant role in neutropil influx to the lung in CXCL5 plays a dominant role in neutrophil influx after i.t. response to i.t. coadministration of IL-17A and TNF-a at this later administration of IL-17A and TNF-a by 24 h after exposure stage. Because significant enhancement of neutrophil recruitment (Fig. by guest on September 29, 2021 2F) upon coadministration of IL-17A and TNF-a was associated Pulmonary-resident cells are the primary source of CXCL5 with upregulated CXCL5 expression (Fig. 2A) in the mouse lung at upon E. coli challenge 8 and 24 h, we hypothesized that CXCL5 in the lung may play To determine which cells were responsible for CXCL5 production, a dominant role in neutrophil influx at later stages. To test this 2/2 we generated BM-reconstituted chimeric mice using WT and hypothesis, we coadministered IL-17A and TNF-a into Cxcl5 2 2 Cxcl5 / mice as both donors and recipients. After lethal irradi- and C57BL/6 WT mice. Although CXCL1 expression (Fig. 3B) 2/2 2 2 ation of both WT (denoted as W in the second letter) and Cxcl5 was higher in the Cxcl5 / mice than that of the WT mice after 8 h recipients (denoted as K in the second letter), these mice were of coadministration of IL-17A and TNF-a, there was no signifi- reconstituted with BM of healthy WT (denoted as W in the first cant difference in neutrophil influx (Fig. 3E) between the two 2/2 2 2 letter) or Cxcl5 donors (denoted as K in the first letter). After groups. In contrast, in the Cxcl5 / mice receiving IL-17A and challenge of these mice with i.t instillation of 106 CFU E. coli, TNF-a for 24 h, the total WBCs and neutrophils in the BALF only WT recipient mice showed detectable CXCL5 in BALF, in- were significantly reduced as compared with the WT mice (Fig. dicating that pulmonary-resident cells, not recruited hematopoietic 3D,3E). Despite the absence of CXCL5 in the targeted mice (Fig. cells or alveolar macrophages, express CXCL5 in the lung during lung inflammation (Fig. 4). Together with our previous findings that rat ATII cells expressed CXCL5 in vitro and in vivo upon LPS stimulation (10), these data suggest that ATII cells may be a major source of CXCL5 in the alveolar space during lung inflammation.

IL-17A and TNF-a act synergistically to induce CXCL5/ENA-78 expression in differentiated ATII cells derived from human fetal lung To determine whether human ATII cells express CXCL5 in response FIGURE 4. Lung-resident cells are the primary source of CXCL5 upon to TNF-a and IL-17A, epithelial cells isolated from second tri- E. coli challenge. The BM chimeric mice were i.t. challenged with 106 mester (16–20 wk gestation) human fetal lung and differentiated CFU E. coli. Eight hours after challenge, the BALF was collected and measured for CXCL5 level by ELISA. KK, BM from Cxcl52/2 donor mice in vitro to an ATII phenotype (33) were exposed to IL-17 and/or 2 2 2 2 a a into Cxcl5 / recipient mice; KW, BM from Cxcl5 / donor mice into TNF- for 24 h. Consistent with our in vivo data, TNF- alone WT recipient mice. Values are mean 6 SEM (n = 3–4). N.D, not detected; increased CXCL5/ENA-78 mRNA (Fig. 5A) and protein (Fig. 5B) WK, BM from WT donor mice into Cxcl5–/– recipient mice; WW, BM to a greater extent than did IL-17A alone in human ATII cells, but from WT donor mice into WT recipient mice. in both cases were dose dependent. The combination of IL-17A The Journal of Immunology 5 Downloaded from

FIGURE 5. IL-17A and TNF-a act synergistically to induce CXCL5/ENA-78 expression in differentiated ATII cells derived from human fetal lung. A, Human ATII cells were stimulated with IL-17A (10, 50 ng/ml) or TNF-a (2, 10 ng/ml) alone or in combination for 24 h. Total RNA was isolated, and CXCL5/ENA-78 mRNA level determined by qPCR and normalized to 18S rRNA (representative result from separate experiments with three donors). B, CXCL5/ENA-78 protein level in cell medium was measured by ELISA. Time course of CXCL5/ENA-78 mRNA (C) and protein (D) expression induced by http://www.jimmunol.org/ TNF-a and its augmentation by IL-17A. ATII cells were treated with TNF-a (10 ng/ml) or TNF-a (10 ng/ml) + IL-17A (10 ng/ml). Cell media and cell contents were collected at different time points. Values are mean 6 SEM. Data represent results from three independent donors. *p , 0.01 TNF-a + IL- 17A–treated cells versus TNF-a–treated cells, ***p , 0.001. and TNF-a induced a synergistic response on CXCL5/ENA-78 mechanisms. To investigate the mechanisms of the marked up- mRNA (Fig. 5A) and immunoreactive protein expression (Fig. 5B). regulation of CXCL5/ENA-78 expression upon IL-17A and TNF-a We then examined the time course of CXCL5/ENA-78 mRNA costimulation, we performed nuclear run-on assay in human ATII and protein expression in response to IL-17A and TNF-a compared cells treated with IL-17A (25 ng/ml) and/or TNF-a (10 ng/ml) for with TNF-a alone. TNF-a alone induced peak expression (still far 24 h to examine the transcription rate. Intact nuclei were isolated, by guest on September 29, 2021 below that of TNF-a plus IL-17A) of CXCL5/ENA-78 mRNA by and the RNA transcripts that had been initiated in vivo were 8–10 h, which then declined toward baseline by 24 h. The addi- allowed to complete in vitro. The relative levels of CXCL5/ENA- tion of IL-17A to TNF-a greatly enhanced CXCL5/ENA-78 78 transcription were normalized to that of GAPDH. As shown in mRNA expression, which peaked by 10 h and remained elevated Fig. 6A, IL-17A or TNF-a treatment increased novel CXCL5/ through 24 h (Fig. 5C). By contrast, CXCL5/ENA-78 protein was ENA-78 mRNA expression by ∼3-fold or 13-fold, respectively. not detected in the media in either case until 8 h and then in- A synergistic increase in CXCL5/ENA-78 transcription of ∼50 creased exponentially between 8 and 24 h, especially in response fold was observed in the presence of IL-17A plus TNF-a. to the combination of IL-17A and TNF-a (Fig. 5D). The modulation of mRNA stability has emerged as an important means of regulation for a number of proinflammatory (35). a IL-17A enhances TNF- –induced CXCL5/ENA-78 gene To understand whether this mechanism contributes to the syner- expression through both transcriptional and gistic effect of IL-17A and TNF-a on CXCL5/ENA-78 message, posttranscriptional mechanisms we tested stability of CXCL5/ENA-78 mRNA in human ATII cells Gene upregulation is commonly due to the induction of gene treated with TNF-a (10 ng/ml) in the presence or absence of IL- transcription, stabilization of mRNA, or the presence of both 17A (10 ng/ml) for 10 h. After incubation with actinomycin D,

FIGURE 6. IL-17A and TNF-a synergistically enhance CXCL5/ENA-78 mRNA transcription rate and stabilize CXCL5/ENA-78 mRNA in human fetal ATII cells. A, Nuclei were isolated from human fetal ATII cells treated with or without TNF-a (10 ng/ml) in the presence of IL-17A (25 ng/ml) for 24 h. Transcripts that were initiated in the cells were allowed to continue in vitro. The relative expression levels of novel CXCL5/ENA-78 mRNA was determined by qPCR and normalized to GAPDH. *p , 0.05 versus control, **p , 0.01. B, Human ATII cells were treated with TNF-a (10 ng/ml) or TNF-a + IL-17A (10 ng/ml) for 10 h. Cells were then incubated with actinomycin D to inhibit further transcription. CXCL5/ENA-78 mRNA expression over the next 12 h was quantified by qPCR and normalized to 18S rRNA. Values are mean 6 SEM. Data in A and B represent results for three independent donors. 6 SYNERGISTIC EFFECTS OF IL-17A AND TNF-a ON CXCL5 EXPRESSION

Cxcl5 promoter contains several NF-kB binding sites, the role of IL-17A in inducing NF-kB is controversial (35–37). To determine the relative effects of TNF-a and IL-17A on NF-kB activation, we transduced primary human ATII cells with recombinant adenoviral vectors containing an NF-kB–driven firefly luciferase reporter and a pRL-SV40–driven renilla luciferase reporter (the latter repre- senting a control for transduction efficiency). As seen in Fig. 8A, FIGURE 7. Inhibition of de novo protein synthesis abolishes the syn- a k a TNF- activated NF- B activity, whereas IL-17 did not. The ergistic effect of IL-17A and TNF- on CXCL5/ENA-78 mRNA expres- a sion. Human ATII cells were pretreated with CHX (5 mg/ml) for 1 h, and combination of TNF- and IL-17 did not induce synergistic ac- k then they were stimulated with IL-17A (25 ng/ml) and/or TNF-a (10 mg/ tivation of NF- B, suggesting the enhancement afforded by IL- ml). Ten hours later, cells were collected, and total RNA was isolated. 17 treatment was not through the NF-kB pathway. To further veri- qPCR were performed to quantify the relative expression of CXCL5/ENA- fy whether NF-kB signaling was involved in TNF-a–induced 78 mRNA normalized to 18S rRNA. *p , 0.05 versus control, ***p , CXCL5/ENA-78 expression, we pretreated human ATII cells with 0.001, &NS. Values are mean 6 SEM. Data represent results for three BAY-11-7082, an inhibitor of IkBa phosphorylation, for 30 min independent donors. and then stimulated them with IL-17A and/or TNF-a for 24 h. As seen in Fig. 8B, CXCL5/ENA-78 expression was greatly attenu- ated upon NF-kB inhibition in either stimulation with TNF-a RNA was extracted at various time points and CXCL5/ENA-78 alone or TNF-a plus IL-17A. These data demonstrated that TNF- measured using qPCR. In cells treated with TNF-a alone, CXCL5 a, but not IL-17A, activated NF- kB signaling, which was neces- Downloaded from t was determined to be 3.8 h, whereas in cells treated with both 1/2 sary, but not sufficient, for IL-17A enhancement of CXCL5/ENA- IL-17A and TNF-a, t was much longer (Fig. 6B). Therefore, 1/2 78 expression by human ATII cells. these data demonstrated that IL-17A enhances TNF-a–induced CXCL5/ENA-78 expression by effects on both increased tran- Human ATII cells secrete CXCL5/ENA-78 in a polarized and scription rates and prolonged stability of mRNA. regulated fashion

Inhibition of de novo protein synthesis abolishes the synergistic The alveolar lining layer has developed a highly organized cellular http://www.jimmunol.org/ a effect of IL-17A and TNF- on CXCL5/ENA-78 expression polarity: surfactant components are exclusively secreted to the To determine whether synthesis of an intermediate mediator (such apical sides; and fibrinogen is mainly secreted basolaterally (38). as a transcription factor or RNA-binding protein) might be in- To understand whether CXCL5/ENA-78 was secreted in a polar- volved in expression of CXCL5, we pretreated human ATII cells ized fashion in human ATII cells, a monolayer of cells were plated with 5 mg/ml cycloheximide (CHX), a protein synthesis inhibitor on transwell filters and stimulated with either IL-17A or TNF-a for 1 h prior to the stimulation with IL-17A and/or TNF-a for alone, the combination of both from either the upper or the lower 10 h. CXCL5/ENA-78 mRNA levels were determined by qPCR. compartments, or both compartments of the transwell (Fig. 9A). As shown in Fig. 7, the synergistic effect of IL-17A and TNF-a After 24 h stimulation, the CXCL5/ENA-78 levels in the super- on CXCL5/ENA-78 expression was abolished by treatment with natants from either the upper or lower chambers were measured by by guest on September 29, 2021 CHX (Fig. 7, black bars), even though CHX by itself increased ELISA. Apical stimulation by TNF-a provoked apical secretion of CXCL5/ENA-78 in control. Overall, these results indicate the ENA-78, whereas basolateral stimulation by TNF-a led to both synergistic effect of combined IL-17A and TNF-a on CXCL5/ apical and basolateral secretion of CXCL5/ENA-78 by human ENA-78 expression depends on new protein synthesis. ATII cells. This polarized response was also synergistically en- hanced by IL-17A costimulation (Fig. 9B). TNF-a, but not IL-17A, activates NF-kB signaling on CXCL5/ENA-78 expression in human ATII cells Effect of IL-17A and TNF-a on cytokine and chemokine NF-kB is a critical mediator of the cellular response to a variety of secretion in human ATII cells extracellular stimuli such as stress, cytokines, and bacteria. Al- Because coinstillation of IL-17A and TNF-a into murine lungs though TNF-a is well known to activate NF-kB activity, and the synergistically increased not only CXCL5, but also CXCL1, -2

FIGURE 8. TNF-a, but not IL-17A, activates NF- kB signaling on CXCL5/ENA-78 expression in human ATII cells. A, human ATII cells were transfected with adenovirus vectors that had NF-kB–driven firefly lu- ciferase element and pRL-SV40–driven renilla lucif- erase element. Twenty-four hours postinfection, the cells were then treated with IL-17A (25 ng/ml), TNF-a (10 ng/ml), or both for 24 h in fresh medium. The cells were assayed for firefly and renilla luciferase activities using Dual-Luciferase Reporter Assay System. Lucif- erase activities were measured by the IVIS Imaging. B and C, Human ATII cells were treated with or without phosphorylation IkBa inhibitor (BAY-11-7082) for 30 min and then stimulated with IL-17A and/or TNF-a for 24 h. The CXCL5/ENA-78 mRNA expression was measured by PCR (B) and protein levels were mea- sured by ELISA (C). Values are mean 6 SEM. Data represent results for three independent donors. ***p , 0.001 versus BAY-treated group. The Journal of Immunology 7

alveolus, based on its ability to secrete surfactants to prevent al- veolar collapse. More recently, a body of work indicates that ATII cells participate as well in host defense (7, 8, 40, 41). In this ar- ticle, we extend the function of ATII cells in inflammation by focusing on a unique member of the ELR+ CXC chemokine family, CXCL5. This chemokine, as we previously demonstrated, is mostly produced by AT II cells during lung inflammation (10) and plays a unique role in chemokine scavenging in blood and regulating neutrophil influx to the lung (30). IL-17 is a proinflammatory cytokine produced predominately by FIGURE 9. Human ATII cells secrete CXCL5/ENA-78 in a polarized Th17 cells. Although IL-17 is recognized to participate in host and regulated fashion. Human ATII cells seeded on transwell filters were defense and autoimmunity through the induction of proinflam- treated with PBS, IL-17A (25 ng/ml), TNF-a (10 ng/ml), or IL-17A+TNF- matory gene expression, including CXC chemokines, and subse- a from top or bottom chambers for 24 h. Supernatants were collected for quently leading recruitment of neutrophils (5), the molecular mech- measurement of CXCL5/ENA-78 protein. Values are mean 6 SEM. Data anisms by which this is achieved remain poorly defined. It has represent results for five independent donors. been reported that IL-17 treatment is a poor stimulus for gene ex- pression. However, IL-17 can cooperate with other cytokines, and G-CSF, we further explored whether IL-17A and TNF-a particularly TNF-a, to induce a synergistic response (42). We exerted a synergistic effect on the secretions of other cytokines developed a new model to test potential interactions between Downloaded from and chemokines in human ATII cells. We used multiplex bead cytokines. In this model, i.t. instillation of TNF-a produces a mild technology to measure the protein levels of a variety of cytokines inflammatory response, consistent with previous observations in and chemokines in media of human ATII cells that were treated mice (43). The coadministration of IL-17A, however, which to our with IL-17A and/or TNF-a for 24 h. As shown in Fig. 10, IL-17A knowledge has not previously been demonstrated, markedly in- alone induced only minimal cytokine and chemokine secretion, creased neutrophil accumulation. CXCL1 and -2 were upregulated a 2/2 but it synergized with TNF- in the induction of cytokine (IL-6, early, whereas CXCL5 was induced later. Cxcl5 mice showed http://www.jimmunol.org/ G-CSF, GM-CSF), and chemokine (IL-8, GRO, MCP-1, MIP-1b, markedly diminished neutrophil influx at later stages, but early MCP-3) expression (Fig. 10), suggesting that the synergism of neutrophil accumulation was unaffected. These data indicate TNF-a with IL-17A may play an important role in orchestrating ELR+ CXC chemokines, despite their similarities, may be re- pulmonary inflammatory responses through ATII cells. sponsible for different phases of the inflammatory response. Al- though the model itself is created through exogenous instillation, Discussion it represents conditions that occur in a variety of inflammatory During lung inflammation and infection, neutrophils generated in processes. the BM are quickly recruited to inflamed sites to form the first line To study mechanisms of synergism, we used human ATII cells of lung host defense. ELR+ CXC chemokines play an important in vitro as a model. We focused on human CXCL5/ENA-78 as by guest on September 29, 2021 role in directing this event and, at least in part, determine the a bona fide homolog of murine CXCL5/LIX for several reasons. nature and severity of the resulting inflammatory response. The Although human CXCL5/ENA-78 and human CXCL6/ GCP-2 are sources of these chemokines are less clear. Although traditionally similarly homologous with murine CXCL5/LIX, the genomic locus the AM has been considered to secrete most of the ELR+ CXC of mouse and human CXCL5 are nearly identical, whereas that of chemokines, we have shown murine AM do not express CXCL5 human CXCL6 is part of a duplicate region containing pseudo- during lung inflammation (10). In the present work, we suggest genes. Furthermore, murine CXCL5/LIX and human CXCL5/ that the ATII cell contributes to the lung inflammatory response ENA-78 share functional homology as well, as they both are ex- by virtue of its ability to secrete CXCL5 and a variety of other pressed in platelets (44) and ATII cells (30). chemokines in response to TNF-a and IL-17A, a frequently Using human ATII cells, we suggest that the mechanism by detected combination of cytokines. which IL-17A synergizes with TNF-a to induce CXCL5 is appar- The ATII cell has been described for many years, since the ently complex. IL-17A–induced synergism required new protein original work of Mason and Williams (39), as the defender of the synthesis, suggesting that synthesis of new transcription factor(s)

FIGURE 10. Effect of IL-17A and TNF-a on cytokine and chemokine secretion in human ATII cells. ATII cells were treated with IL-17A (25 ng/ml) and/ or TNF-a (10 ng/ml) for 24 h. Supernatants were collected for Luminex assay. Values are mean 6 SEM. Data represent results for three independent donors. **p , 0.01, ***p , 0.001. 8 SYNERGISTIC EFFECTS OF IL-17A AND TNF-a ON CXCL5 EXPRESSION

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drogen peroxide-induced loss of barrier function: the role of nitric oxide alternative nuclear factor-kappaB pathways: potential implications for autoim- http://www.jimmunol.org/ synthesis. J. Immunol. 169: 1474–1481. munity and rheumatoid arthritis. Arthritis Res. Ther. 10: 212. by guest on September 29, 2021