Pneumococcal Pneumonia Innate Immune Responses During Type I Alveolar Epithelial Cells Mount

Type I Alveolar Epithelial Cells Mount Innate Immune Responses during Pneumococcal Pneumonia

This information is current as Kazuko Yamamoto, Joseph D. Ferrari, Yuxia Cao, Maria I. of September 26, 2021. Ramirez, Matthew R. Jones, Lee J. Quinton and Joseph P. Mizgerd J Immunol 2012; 189:2450-2459; Prepublished online 27 July 2012;

doi: 10.4049/jimmunol.1200634 Downloaded from http://www.jimmunol.org/content/189/5/2450

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

Type I Alveolar Epithelial Cells Mount Innate Immune Responses during Pneumococcal Pneumonia

Kazuko Yamamoto,*,† Joseph D. Ferrari,* Yuxia Cao,* Maria I. Ramirez,* Matthew R. Jones,* Lee J. Quinton,* and Joseph P. Mizgerd*

Pneumonia results from bacteria in the alveoli. The alveolar epithelium consists of type II cells, which secrete surfactant and associated proteins, and type I cells, which constitute 95% of the surface area and meet anatomic and structural needs. Other than constitutively expressed surfactant proteins, it is unknown whether alveolar epithelial cells have distinct roles in innate immunity. Because innate immunity gene induction depends on NF-kB RelA (also known as p65) during pneumonia, we generated a murine model of RelA mutated throughout the alveolar epithelium. In response to LPS, only 2 of 84 cytokine transcripts (CCL20 and CXCL5) were blunted in lungs of mutants, suggesting that a very limited subset of immune mediators is selectively elaborated by the alveolar epithelium. Lung CCL20 induction required epithelial RelA regardless of stimulus, whereas lung CXCL5 expres- Downloaded from sion depended on RelA after instillation of LPS but not pneumococcus. RelA knockdown in vitro suggested that CXCL5 induction required RelA in type II cells but not type I cells. Sorted cell populations from mouse lungs revealed that CXCL5 was induced during pneumonia in type I cells, which did not require RelA. TLR2 and STING were also induced in type I cells, with RelA essential for TLR2 but not STING. To our knowledge, these data are the ﬁrst direct demonstration that type I cells, which constitute the majority of the alveolar surface, mount innate immune responses during bacterial infection. These are also, to our knowledge, the ﬁrst evidence for entirely RelA-independent pathways of innate immunity gene induction in any cell during http://www.jimmunol.org/ pneumonia. The Journal of Immunology, 2012, 189: 2450–2459.

he epithelium represents the first line of defense against the five NF-kB proteins, only p50 and RelA (also known as p65) pathogens in the lung. Epithelial cells provide a mechan- are readily detectable in lung nuclear fractions during acute pul- T ical barrier to prevent infection, and they can produce monary inflammation (7–9). p50 limits the expression of inflam- chemokines and cytokines that recruit and activate phagocytic cells matory cytokines and prevents lung injury during pneumonia (10, to eradicate organisms and infected cells (1, 2). The alveolar ep- 11). In contrast, RelA drives inflammatory responses by promot- ithelium consists of two main populations: alveolar type I (AT1) ing the expression of many cytokines, and the deletion of RelA and type II (AT2) epithelial cells. AT2 cells synthesize and secrete from all cells severely compromises antibacterial host defense (12, by guest on September 26, 2021 pulmonary surfactant, express chemokines and cytokines, and 13). Mice with a surfactant protein C (SPC)-driven dominant- participate in the innate immune response of the lung (3). Despite negative (dn)IkBa inhibitor of NF-kB have increased bacterial being the predominant cell in the alveolar space by number, AT2 burdens during pneumococcal pneumonia (12), suggesting that cells cover only 5% of the surface. The remaining 95% is covered NF-kB in AT2 cells contributes to host defense. These mice also by large attenuated AT1 cells (4–6). Although constituting so have decreased neutrophil recruitment and inflammatory cyto- much of the surface area of the lung, very little is known about any kines after LPS inhalation (14), indicating that NF-kBinAT2 potential contribution of AT1 cells to pulmonary innate immunity. cells participates in acute inflammatory responses. However, the Proinflammatory cytokines orchestrate innate immunity and are dnIkBa protein is not specific to distinct NF-kB proteins, and the mediated by multiple transcription factors, including NF-kB. Of efficacy of NF-kB inhibition by this approach is based on dy- namic stoichiometry, which has not been analyzed in these lung

*Pulmonary Center, Boston University School of Medicine, Boston, MA 02118; and cells. Importantly, neither these nor other studies to date have ex- †Department of Molecular Microbiology and Immunology, Nagasaki University amined roles of AT1 cells. The goal of the present study was to Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan evaluate unique roles of alveolar epithelial cells in innate immu- Received for publication February 23, 2012. Accepted for publication June 26, 2012. nity mediator expression elicited by bacterial stimuli in the lungs This work was supported by National Institutes of Health Grants HL068153 (to and to assess their dependence on NF-kB RelA. J.P.M.), HL079392 (to J.P.M.), HL104053 (to M.R.J.), HL092956 (to L.J.Q.), and HL083034 (to M.I.R.). K.Y. was supported by an American Lung Association Senior Research Fellowship. Materials and Methods The sequences presented in this article have been submitted to the National Center Mice for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih. flx/flx gov/geo/) under accession numbers GSE38398 and GSE38399). Rela mice (15) (provided by Dr. Roland Schmid, Technical University of Munich, Munich, Germany) were bred with SPC-reverse tetracycline Address correspondence and reprint requests to Dr. Joseph P. Mizgerd, Pulmonary tg/2 tg/tg Center, Boston University School of Medicine, 72 E. Concord Street, Boston, MA transactivator (rtTA) /(tetO)7CMV-Cre mice (16) (provided by 02118. E-mail address: [email protected] Dr. Jeffrey A. Whitsett, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH) to generate colonies of wild-type (WT) control mice Abbreviations used in this article: AT1, alveolar epithelial type I; AT2, alveolar 2/2 tg/tg flx/flx D/D tg/2 (SPC-rtTA /(tetO)7CMV-Cre /Rela )orRelA mice (SPC-rtTA / epithelial type II; dn, dominant-negative; PRR, pattern recognition receptor; qRT- tg/tg flx/flx (tetO)7CMV-Cre /Rela ) in which the Rela gene is selectively mutated PCR, quantitative RT-PCR; rtTA, reverse tetracycline transactivator; siRNA, small D/D interfering RNA; SPC, surfactant protein C; STING, stimulator of IFN gene; WT, in alveolar epithelial cells. Results obtained from RelA mice were wild-type. compared with sex-matched littermate controls. Doxycycline was provided in the chow (625 mg/kg, S-5086; Bio-Serv) to all mice throughout ges- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 tation and nursing to induce Cre-recombinase–mediated Rela mutation in www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200634 The Journal of Immunology 2451 the alveolar epithelium of the rtTA-transgenics, including both AT1 cells line [(24), obtained from Dr. Jeffrey A. Whitsett (Cincinnati Children’s and AT2 cells (17). Mice were not exposed to the doxycycline diet after Hospital Medical Center, Cincinnati, OH)] was cultured in a 1:1 mixture of weaning from their mothers at 3 wk age, preventing effects of confounding DMEM and F12 medium supplemented with 2% FBS, 100 U/ml penicillin, by doxycycline during experiments. At the time of experimentation, mice 100 mg/ml streptomycin, 5 mg/ml insulin, 10 mg/ml transferrin, 30 nM were 7–11 wk age. Experiments with nontransgenic mice were performed sodium selenite, 10 nM hydrocortisone, 10 nM b-estradiol, and 10 mM using C57BL/6 mice. All experimental protocols were approved by the HEPES. Cells were studied at ∼80% confluence. Cells were transfected Boston University Institutional Animal Care and Use Committee. by using 0.1 mM RelA-targeting small interfering RNA (siRNA; sense, 59-GCUCAAGAUCUGCCGAGUAUU-39,antisense,59-PUACUCGGC- Pneumonia AGAUCUUGAGCUU-39) or control nontargeting siRNA (D-001810-01-20), which did not complement known sequences in the mouse transcriptome Mice were anesthetized by i.p. injection of ketamine (50 mg/kg)/xylazine (both from Dharmacon). After 24 h, cells were washed once with PBS and (5 mg/kg). An angiocatheter was placed down the left bronchus, and mice received intratracheal instillations of 50 ml saline containing 106 CFU were stimulated after another 24 h culture with fresh media. Cells were stimulated for 6 h with 10 ng/ml recombinant murine TNF-a,10mg/ml Streptococcus pneumoniae serotype 3 (6303; American Type Culture m Collection) or 50 mg LPS (Escherichia coli O111:B4; Sigma-Aldrich) into LPS, or 5 g/ml BSA (all from Sigma-Aldrich). The cell pellets were the left lung lobe. These stimuli were chosen because they are relevant to collected after washing with PBS and resuspendedin1mlTRIzol(Invitrogen) human health, are each well characterized in murine models of pulmonary for RNA isolation. inflammation, and are to our knowledge the only two bacterial stimuli that Immunoblot assay have been studied in the context of epithelial NF-kB roles (12, 14, 18). Mice were euthanized by an aerosolized isoflurane overdose 15 h after Western blots were performed to determine siRNA-mediated knockdown S. pneumoniae instillation or 6 h after LPS instillation. of RelA in E10 and in MLE15 cells, as previously described (12). Abs for RelA (product no. 3034) and pan-actin (product no. 4968S) were purchased Bacterial clearance from Cell Signaling Technology. Downloaded from

Lungs were homogenized in sterile distilled H2O containing protease RNA puriﬁcation and analyses inhibitors (19). Homogenates were serially diluted, plated on 5% sheep blood agar plates using the drop plate method (20), and incubated over- Total RNA was isolated from mouse lung, alveolar epithelial cell lines, night at 37˚C. Colonies were counted to quantify CFU/lung. and isolated cells with an RNeasy Mini kit (Qiagen) or TRIzol reagent (Invitrogen) and an RNase-free DNase set (Qiagen). Quantitative RT-PCR Lung histology (qRT-PCR) was performed using the TaqMan RNA-to-CT one-step kit and the StepOnePlus real-time PCR systems (both from Applied Biosystems). Alveolar neutrophils were quantiﬁed by morphometric analyses of H&E- The primers and TaqMan probes for mouse CXCL5, TNF-a, IL-6, IL-1a, http://www.jimmunol.org/ stained sections of left (instilled) lung lobes, as described previously (17, IL-1b, CXCL1, CXCL2, TLR2, and TLR4 were previously published 21, 22). (12, 25, 26). The primers and TaqMan probe set were designed for mouse RelA interference in murine lung epithelial cell lines CCL20, G-CSF, and Ly6G using the CLC DNA Workbench software (CLC bio) with the following sequences: CCL20, forward, 59-CCTCAGCCTAA- The murine AT1-like cell line E10 [(23), obtained from Dr. Alvin Malkinson GAGTCAAGAAGA-39, reverse, 59-ACAAGTCCACTGGGACACAAA-39, (University of Colorado, Aurora, CO)], was cultured in CMRL-1066 probe, 59-ACACAGCCCAAGGAGGAAATGATCACAGC-39;G-CSF, medium containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, forward, 59-TTCCCCTGGTCACTGTCAGC-39,reverse,59-CACAG- and 100 mg/ml streptomycin. The murine AT2 cell-derived MLE15 cell CTTGTAGGTGGCACAC-39, probe, 59-ACCATCCCTGCCTCTGCC- by guest on September 26, 2021

FIGURE 1. A small subset of cytokines is decreased in the absence of alveolar epithelial RelA during LPS-induced pulmonary inflammation. (A) Lung inflammatory cytokine mRNA expression after 6 h intratracheal LPS in presence or absence of functional RelA in alveolar epithelial cells (SPC-rtTAwt/tg tg/tg F/F tetO-Cre RelA ). A total of 84 cytokine-related mRNA levels were determined using PCR array, and data for each group were expressed as Ct. Data shown are results from lung RNA pooled from six mice in each group. (B) Lung inflammatory cytokine mRNA expression (CXCL5 and CCL20) over a time course after intratracheal LPS instillation, measured in C57BL/6 mice using qRT-PCR (n = 3–5 in each time point, collected during two independent experiments). (C) Lung inflammatory cytokine mRNA expression after 0 or 6 h intratracheal LPS were measured by qRT-PCR in the presence and absence of functional RelA in alveolar epithelial cells. RelAD/D, transgenic mice with mutant RelA in alveolar epithelial cells (SPC-rtTAwt/tg tetO-Cretg/tg RelAF/F); WT, WT nontargeted mice (tetO-Cretg/tg RelAF/F, no Cre transgene). *p , 0.05 compared with WT mice (n = 3–4 mice/group in Ctrl, and n = 6 mice/group in LPS, collected during three independent experiments). 2452 IMMUNE RESPONSE OF ALVEOLAR EPITHELIAL CELLS AND PNEUMONIA

CCGAAG-39; Ly6G, forward, 59-TCCTGTGTGCTCATCCTTCTT-39, Isolation of AT1 cells from lung digests reverse, 59-TCCAGAGCAACGCAAAATCCAT-39,probe,59-TTCCTGCA- ACACAACTACCTGCCCCTT-39. TaqMan gene expression assays were After euthanasia, lungs were perfused with 10 ml HBSS via the right used for podoplanin (T1a) (Mm00494716_m1), caveolin-1 (Mm00483057_ ventricle through the pulmonary artery. One milliliter porcine elastase m1), Nkx2-1 (Mm00447558_ml), Sftpc (Mm00488144_m1), Sftpb (4.5U; Roche Diagnostics) was instilled through a tracheal cannula fol- (Mm00455681_ml), CC10 (Mm00442046_m1), PECAM-1 (Mm01242584_ lowed by 0.5 ml low melting agarose solution warmed to 45˚C. Lungs m1), p75 (Mm01309638_m1), and stimulator of IFN gene (STING; were immediately covered with ice for 2 min to gel the agarose and in- Mm01158117_m1; all from Applied Biosystems) mRNA analyses. For cubated in 2 ml elastase for 45 min at 37˚C. After this incubation, lung each sample, values were normalized to the content of 18S rRNA (27, 28). lobes were gently separated from the bronchi and minced in RPMI 1640 RT2 Profiler mouse inflammatory cytokines and receptors PCR array medium (Life Technologies) containing 50% FBS and 100 U/ml DNase I (PAMM-011; SABiosciences) was used for the multianalyte qRT-PCR (Qiagen). Cells in suspension were subsequently filtered through 100-, 70-, m analysis of mouse inflammatory cytokines. Results of the PCR arrays and 40- m nylon mesh. Cell suspensions from these lung digests were were deposited at National Center for Biotechnology Information Gene subjected to FACS. The following Abs and isotype controls were used: a Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), retrievable with PE-conjugated hamster anti-mouse podoplanin (T1 ), PE-conjugated ham- accession numbers GSE38398 and GSE38399. Efficacy of Rela gene rear- ster IgG (both hamster products from BioLegend), FITC-conjugated rat rangement was determined by examining sizes of amplification products anti-mouse CD45, FITC-conjugated rat IgG2b, rat anti-mouse CD16/CD32, using electrophoresis. For Rela RT-PCR, isolated RNA (1 mg) was reverse- and 7-aminoactinomycin D (all nonhamster products from BD Pharmin- transcribed using SuperScript VILO cDNA synthesis kit (Applied Biosystems), gen). Cell isolation was performed using a MoFlo cell sorter. Collected and PCR reactions were performed with GoTaq DNA polymerase (Promega). cells were stored in TRIzol (Invitrogen). Primers sequences for Rela were: forward, 59-ACAATAACCCCTTTCA- CGTTCCTA-39; reverse, 59-CCCAAGTCTTCATCAGCATCAA-39. Full- Immunofluorescence staining of sorted cells Rela length or rearranged products were indicated by amplification products Cells were stained for AT1 and AT2 markers using immunofluorescence. ∼ Downloaded from of 939 or 490 bp, respectively. Cytocentrifuged cell preparations sorted from lungs of C57BL/6 mice ELISA were fixed with 4% paraformaldehyde, blocked, and permeabilized. Slides were labeled with caveolin-1 or pro-SPC primary Abs followed ELISA kits (R&D Systems) for murine CXCL5 and CCL20 levels were by Alexa Fluor 488-conjugated donkey anti-rabbit IgG secondary Abs. used to measure their respective concentrations in lung homogenate or in Immunofluorescence images were taken using the AxioVision system supernatants of murine lung cell lines. (Zeiss). http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. During pneumococcal pneumonia, CCL20 but not CXCL5 requires alveolar epithelial RelA. (A) Lung inflammatory cytokine mRNA expression (CXCL5 and CCL20) over a time course after S. pneumoniae serotype 3 instillation, measured in C57BL/6 mice using qRT-PCR (n = 3 in each time point, collected during two independent experiments). (B) Lung inflammatory cytokine mRNA expression after 0 or 15 h intratracheal S. pneumoniae was measured by qRT-PCR in presence (WT) or absence of functional RelA (RelAD/D) in alveolar epithelial cells (n = 3–4 uninfected mice/group in Ctrl, and n = 5–6 infected mice/group in S. pneumoniae, collected during three independent experiments). (C) Lung inflammatory cytokine mRNA expression after 15 h intratracheal S. pneumoniae in presence (WT) or absence of functional RelA (RelAD/D) in alveolar epithelial cells. A total of 84 cytokine-related mRNA levels were determined using PCR array, and data for the each group were expressed as Ct. Data shown are results from lung RNA pooled from five to six mice in each group. (D) CXCL5 and CCL20 were measured by ELISA in lung homogenates harvested from uninfected mice and mice intratracheally infected with S. pneumoniae for 15 h in presence (WT) or absence (RelAD/D) of functional RelA in alveolar epithelial cells. *p , 0.05 compared with WT mice (n = 3 uninfected/group, and n = 3–8 infected/group, collected during two independent experiments). Sp, S. pneumoniae. The Journal of Immunology 2453

Statistical analyses To determine whether cytokines showed similar dependence on Statistical analyses were performed using GraphPad Prism software. Data RelA in alveolar epithelial cells in response to pneumococcus, qRT- were presented as means 6 SE. Real-time RT-PCR data were expressed as PCR was performed on lung mRNA extracts from mice with fold induction and therefore used geometric means 6 geometric SE used pneumococcal pneumonia. Whereas neither CCL20 nor CXCL5 as descriptive statistics. Statistical comparisons among groups for contin- was induced by 6 h, both were strongly induced by 15 h after uous variables were performed using a Student t test or a two-way ANOVA infection (Fig. 2A), consistent with prior publications demon- followed by Bonferroni post hoc analysis. A p value ,0.05 was considered significant. strating innate immunity gene induction responses to pneumo- cocci to involve slower kinetics compared with Gram-negative bacterial products (7, 12, 26, 33). As with LPS-induced inflam- Results mation, many genes induced in the lungs did not require RelA Phenotype of the alveolar epithelial RelA mutant mouse model specifically in alveolar epithelial cells (Fig. 2B). Also similar to We generated a mouse model devoid of functional RelA in alveolar LPS-induced inflammation, CCL20 induction did depend on RelA epithelial cells. Breeding was designed so that all offspring should in alveolar epithelial cells, being .90% diminished by the mu- be homozygous for tetO-Cre and floxed RelA, with half being tation of RelA in alveolar epithelial cells (Fig. 2B). Interestingly, negative and half positive for SPC-rtTA. When no doxycycline CXCL5 was induced despite RelA mutation in the alveolar epi- chow was included in the breeding cages, that ratio was observed. thelial cells 15 h after pneumococcal infection (Fig. 2B), which However, the inclusion of doxycycline chow during gestation and sharply contrasted with the results from lungs 6 h after LPS in- nursing resulted in a skewing from Hardy–Weinberg equilibrium stillation. A PCR array suggested similar cytokine expression (29, 30), with only 10% instead of 50% of the offspring being Downloaded from positive for the rtTA transgene and hence mutant for RelA in alveolar epithelial cells when genotyped at weaning (3 wk age). Furthermore, some but not all adult mice that survived RelA mutation via such a strategy displayed emphysematous-like en- larged alveolar air spaces. These results could indicate that RelA in epithelial cells is essential for preventing lethality during em- http://www.jimmunol.org/ bryonic and neonatal periods and for lung development postna- tally. However, these phenotypes more likely represent an artifact of the strategy, as evidenced by similar findings using the SPC- rtTA/tetO-Cre system in the absence of floxed genes (31, 32), with variable penetrance that depends on genetic background. The interpretation that these phenotypes resulted from the rtTA-based strategy rather than the RelA deletion is also supported by the fact that we observe neither phenotype in mice lacking RelA in all cells (12, 13). Although these phenotypes precluded extensive analyses by guest on September 26, 2021 of integrated responses in these mice, we were able to use them for a limited set of studies to interrogate cell-specific gene expression during acute pulmonary inflammation. Alveolar epithelial cell RelA is required for induction of selected cytokines The expression of lung inflammatory cytokines was evaluated using PCR array. To our surprise, during LPS-induced pulmonary inflammation, only 2 of 84 cytokines in the PCR array were decreased in the lungs of mice with RelA mutated in alveolar epithelial cells, CCL20 and CXCL5 (Fig. 1A). One gene, C-reactive protein, appeared to be increasedinmutantmiceinthisarray, but this was not followed up, as the mRNA levels were very low even in mutants (Fig. 1A). qRT-PCR confirmed that CCL20 and CXCL5 were significantly induced by LPS in the lungs, with CXCL5 peaking at 6–15 h and CCL20 peaking at 4–6 h (Fig. 1B). Induction of each was almost entirely abrogated by the mutation of RelA in alveolar epithelial cells (Fig. 1C). Seven other cytokines represented on the array (CXCL1, CXCL2, IL-1a, IL-1b,TNF-a, IL-6, and G-CSF) were measured by qRT-PCR, which confirmed that they were induced by LPS but were not FIGURE 3. Integrated immunity in mice with alveolar epithelial cell significantly affected by the mutation of RelA in alveolar epi- mutations in RelA. (A) Viable bacteria in the lung were quantified using thelial cells (Fig. 1C). That most cytokines examined were com- CFU assays after 15 h pneumococcal infection (n = 3–8 mice/group, B parable in WT and mutant lungs suggests that the strategy to collected during two independent experiments). ( ) Emigrated neutrophils were measured by quantifying neutrophils in alveolar airspaces using mor- target alveolar epithelial RelA did not have global or widespread phometry (n = 5–9 mice/group, collected during three independent experi- immunity consequences, but rather had very selective effects on a ments). (C) Neutrophil accumulation was evaluated by measuring Ly6G small subset of mediators. The strategy in these mice was designed mRNA in lung lysates after 6 h LPS challenge or 15 h pneumococcal to selectively target alveolar epithelial cells (16), suggesting AT2 infection, expressed as fold WT Ctrl (n = 3–4 mice/group in Ctrl, and or AT1 cells as potentially critical sources of the cytokines CCL20 n = 5–6 mice/group in each of LPS and in S. pneumoniae groups, and CXCL5 during LPS-induced pulmonary inflammation. collected over 3 independent experiments). Sp, S. pneumoniae. 2454 IMMUNE RESPONSE OF ALVEOLAR EPITHELIAL CELLS AND PNEUMONIA patterns in the lungs of mutant mice compared with WT after mRNA in the lung using qRT-PCR after either LPS or pneumo- pneumococcus infection (Fig. 2C). CXCL5 was unaffected by RelA coccus instillation (Fig. 3C). Thus, these data do not reveal roles mutation, CCL20 was diminished, and two additional transcripts for epithelial RelA in integrated innate immune responses. How- (for CXCR3 and IL-10Rb) were potentially blunted by epithelial ever, the limitations of this transgenic mouse model system, par- RelA mutation during pneumococcal pneumonia. Although these ticularly the perinatal lethality phenotype, restrict the numbers and results suggest additional new lines of inquiry, we chose to main- types of experiments that can be done using this model. A better tain focus on the epithelial-derived chemokines and pursue the understanding of the functional significance of epithelial RelA in suggestion that CCL20 is consistently dependent on epithelial integrated pulmonary immunity will require alternative and improved RelA whereas CXCL5 dependency appears to differ after LPS or models for cell-specific targeting of this transcription factor. pneumococcus. Importantly, the disparate roles for alveolar epi- Distinct cytokine expression patterns in alveolar epithelial cell thelial RelA in pneumococcal induction of CXCL5 and CCL20 lines in vitro were similarly observed when examined at the protein level using ELISA (Fig. 2D). These results suggest a heretofore unappre- To begin teasing apart specific roles of AT1 and AT2 cells in the ciated stimulus specificity relating to the role of alveolar epithelial expression of CCL20 and CXCL5, we turned to E10 and MLE15 RelA in CXCL5 induction, which could result from distinct cell cells, which are immortalized epithelial cell lines derived from sources (e.g., AT2 versus AT1 cells) and/or distinct molecular reg- mouse lungs. The E10 and MLE15 cell lines model many char- ulation (RelA-dependent versus -independent pathways) within the acteristics of AT1 and AT2 cells, respectively (12, 17, 24, 34). same cells. Consistent with prior studies suggesting that pneumococcus does

not activate NF-kB in alveolar epithelial cells (12), this stimulus Downloaded from Integrated immune responses failed to induce expression of CXCL5 or CCL20 from either cell Limitations of the animal model, discussed above, precluded ex- line (data not shown). After LPS or TNF-a stimulation, however, tensive analyses of integrated immune responses, but exploratory CXCL5 and CCL20 mRNA and protein levels were induced in studies were performed in an effort to reveal roles of epithelial both cell lines (Fig. 4C–F). Protein expression data suggest greater RelA. Bacterial burdens were measured 24 h after pneumococcal elaboration of CXCL5 by E10 and greater CCL20 by MLE15

infection. Neither WT nor mutant mice showed effective host (Fig. 4), supporting cell-specific cytokine profiles at the alveolar http://www.jimmunol.org/ defense during this infection, and there were no significant dif- surface. ferences between genotypes (Fig. 3A). In this transgenic model, We used an siRNA approach to identify the influence of RelA there were no significant effects of epithelial RelA mutation on on chemokine expression by these cell lines. RelA was successfully neutrophil recruitment in the lungs, measured by quantifying neu- knocked down in both cell lines (Fig. 4A, 4B). Induction of CCL20 trophils in the alveolar air spaces using morphometry after LPS required RelA following either stimulation and in both cell lines instillation (Fig. 3B) and by quantifying neutrophil-specific Ly6G (Fig. 4C, 4E). In contrast, RelA knockdown decreased CXCL5 by guest on September 26, 2021

FIGURE 4. CCL20 induction is consistently dependent on RelA, whereas CXCL5 induction is RelA-dependent in AT2-like cells (MLE15) but RelA-independent in AT1-like cells (E10). (A) Immunoblot shows that RelA was knocked down by siRNA in MLE15 cells. Data are representative of five independent experiments. (B) Immunoblot shows that RelA was knocked down by siRNA in E10 cells. Actin was used for loading control. Data are representative of four independent experiments. (C) CCL20 was measured by qRT-PCR of cell lysates and by ELISA of cell supernatants after 6 h stimulations (TNF-a and LPS) in MLE15 cells in which RelA was knocked down by siRNA. Results reflect data from five independent experiments. (D) CXCL5 was measured by qRT-PCR of cell lysates and by ELISA of cell supernatants after 6 h stimulations (TNF-a and LPS) in MLE15 cells in which RelA was knocked down by siRNA. Results reflect data from five independent experiments. (E) CCL20 was measured by qRT-PCR of cell lysates and by ELISA of cell supernatants after 6 h stimulations (TNF-a and LPS) in E10 cells in which RelA was knocked down by siRNA. Results reflect data from four independent experiments. (F) CXCL5 was measured by qRT-PCR of cell lysates and by ELISA of cell supernatants after 6 h stimulations (TNF-a and LPS) in E10 cells in which RelA was knocked down by siRNA. Results reflect data from four independent experiments. *p , 0.05 compared with control (nontargeted siRNA). The Journal of Immunology 2455 induction only in MLE15 cells (Fig. 4D, 4F). These data suggest ulation did not stain for caveolin-1 (Fig. 5C). Potential T1a+ cells the unexpected hypothesis that AT1 cells may be important sources in addition to AT1 cells may include lymphatic endothelial cells, of CXCL5, and they may be able to produce this chemokine in basal cells, and pleural cells (40, 41). PECAM-1 and p75, char- a RelA-independent manner. acteristic of lymphatic endothelial cells and basal cells, respectively, were less prominent in the T1a+ population compared with In vivo responses of primary AT1 cells during pneumonia the nonselected cells (Fig. 5B). Muc16, characteristic of pleural To elucidate distinct cytokine expression patterns from different cells but not other cells from the lung (42), was higher in the T1a+ epithelial cell types in vivo, AT1 cells were isolated from the lung population (data not shown), suggesting cells from the visceral usingAbagainstT1a, also known as podoplanin or RT140, a pleura as the other cells included with AT1 cells in these prepa- surface protein expressed by AT1 cells (35–39). In lung single- rations. We interpret these T1a+ cell pools as greatly enriched cell suspensions, the T1a+ cell population was clearly separated for AT1 cells and devoid of their most closely related and ap- from T1a2 cells (Fig. 5A). T1a+ cells express high podoplanin posed AT2 cells, although we recognize that they are not pure and caveolin-1, but not markers of other epithelial cells (SPC, AT1 cell populations. SPB, Nkx2-1, or CC10), suggesting successful separation of AT1 During LPS-induced pulmonary inflammation, both CXCL5 cells from AT2 cells and Clara cells (Fig. 5B). Immunofluores- and CCL20 were induced in cells other than AT1 cells and not cence of sorted cells confirmed that caveolin-1 staining, char- in the T1a+ cells (Fig. 5D, 5E). During pneumococcal pneu- acteristic of AT1 cells, was observed in the T1a+ population but monia, as after LPS, CCL20 was only induced in cells other than not the T1a2 population, whereas pro-SPC staining, characteristic AT1 cells (Fig. 5F). In contrast, CXCL5 was strongly induced in of AT2 cells, showed the opposite pattern (Fig. 5C). Complete the T1a+ AT1 cells during pneumococcal pneumonia (Fig. 5G). Downloaded from purity was not achieved, as a fraction of cells in the T1a+ pop- These results indicate that CXCL5 is induced in AT1 cells during http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. During pneumococcal pneumonia, CXCL5 is induced in AT1 cells. (A) AT1 cells were collected by sorting for surface expression of T1a within lung single-cell suspensions from C57BL/6 mice using flow cytometry. Results are representative of five mice collected during three independent experiments. (B) qRT-PCR of cell type marker expression among T1a+ cells and T1a2 cells reveals that the former are positive for AT1 markers (podoplanin and caveolin-1) whereas markers for AT2 (SPC, Nkx2-1) or other cells (CC10, PECAM-1, p75) were higher in the latter. Data are from five mice per group collected during three independent experiments. *p , 0.05 compared with T1a+ cells. (C) Immunofluorescence images of sorted CD452 T1a+ cells and CD452T1a2 cells demonstrates caveolin-1 staining (characteristic of type I cells) exclusively in the former, and pro-SPC staining (characteristic of type II cells) exclusively in the latter. Scale bars, 100 mm. Results are representative of four mice collected during two independent experiments. (D) CCL20 was primarily induced in T1a2 cells during LPS-induced pulmonary inflammation in C57BL/6 mice, measured using qRT-PCR (n = 3 mice/group in Ctrl, and n = 7 mice/group in LPS, collected during two independent experiments). (E) CXCL5 was primarily induced in T1a2 cells during LPS-induced pulmonary inflammation in C57BL/6 mice, measured using qRT-PCR (n = 3 mice/group in Ctrl, and n = 7 mice/group in LPS, collected during two independent experiments). (F) CCL20 was primarily induced in T1a2 cells during pneumococcal pneumonia in C57BL/6 mice, measured using qRT-PCR (n = 4 mice/group in Ctrl, and n = 9 mice/group in S. pneumoniae, collected during four independent experiments). (G) CXCL5 was primarily induced in T1a+ cells during pneumococcal pneumonia in C57BL/6 mice, measured using qRT-PCR, evidence that AT1 cells respond with innate immunity gene induction during pneumococcal pneumonia (n = 4 uninfected mice/group, and n = 9 infected mice/group, collected during four independent experiments). *p , 0.05 compared with uninfected control mice, †p , 0.05 compared with T1a+ cells. Sp, S. pneumoniae. 2456 IMMUNE RESPONSE OF ALVEOLAR EPITHELIAL CELLS AND PNEUMONIA pneumococcal pneumonia, which differs from LPS-induced in- AT1 cells express STING independent of RelA during flammation and which is to our knowledge the first direct evidence pneumococcal pneumonia of an innate immune response from type I alveolar epithelial cells. The discovery of innate immune responses in ATI cells raises the AT1 cells induce CXCL5 independent of RelA during question of whether these cells express distinct innate immunity- pneumococcal pneumonia initiating pattern recognition receptors (PRRs). We refined our cell sorting approaches to distinguish responses of T1a+ AT1 Collectively, the above data suggest the hypothesis that AT1 cells cells from both CD45+ leukocytes as well as cells not collected may be activated to express CXCL5 in a RelA-independent in either of those gates (including AT2 cells plus many others). fashion during pneumococcal pneumonia. To clarify RelA depen- The qRT-PCR of chemokines in these cell populations demon- dency of CXCL5 induction in AT1 cells, we applied our cell strated no induction of CCL20 or CXCL5 in leukocytes, rein- sorting approach to the lungs of mice with alveolar epithelial forcing the interpretations that CCL20 is induced selectively in mutations in RelA. Using an RT-PCR strategy to differentiate the AT2 cells whereas CXCL5 is induced in both AT2 and AT1 cells recombined Rela gene product from the floxed-but-not-recombined D D during pneumococcal pneumonia (Fig. 7A). Because they have version encoding WT RelA, we found that AT1 cells of RelA / been implicated in responses to pneumococcal pneumonia (43–46), mice expressed the mutant allele whereas the AT1 cells of WT the PRRs TLR2, TLR4, and STING were measured in these littermates did not (Fig. 6A). Leukocytes expressed WT RelA in sorted cell populations. TLR4 did not demonstrate much variability both cases (Fig. 6A). These results confirm effective transgene- between cell types and revealed little effect of infection (Fig. 7B). specific targeting of the Rela locus in alveolar epithelial cells, + TLR2 was much more highly expressed in leukocytes than epi- and they also add confidence to our interpretation of the T1a Downloaded from thelial cells, but it was induced in AT1 cells during infection (Fig. population as consisting of primarily AT1 cells with few non- 7B). Interestingly, STING was most highly expressed in T1a+ epithelial contaminants. To determine whether the AT1 cell in- cells compared with leukocytes or T1a2 cells, and STING also duction of CXCL5 during pneumococcal pneumonia required RelA, was significantly induced in AT1 cells during pneumonia (Fig. we compared CXCL5 expression from isolated T1a+ cells of WT D D 7B), supporting the concept that AT1 cells have roles in innate and RelA / mice. CXCL5 was significantly induced in T1a+ immunity. Because TLR2 and STING were induced in AT1 cells,

cells from both WT and mutant mice during pneumococcal pneu- http://www.jimmunol.org/ we endeavored to determine whether these cell-specific innate im- monia, with no significant differences due to genotype (Fig. 6B). mune responses were mediated by RelA. The expression of TLR2 These data demonstrate that the induction of CXCL5 in AT1 cells in T1a+ cells during pneumonia was entirely dependent on RelA during pneumococcal pneumonia does not require NF-kB RelA, (Fig. 7C). In contrast, STING was significantly induced in RelA- which is to our knowledge the first evidence of truly RelA- mutant AT1 cells, and this induction level did not differ from independent gene induction by any cell type during pneumonia. that observed in WT littermates (Fig. 7D). These results bolster the conclusion that AT1 cells respond to bacteria in the lungs, and they indicate that these cells use both RelA-dependent and -independent pathways to induce innate immunity genes. Similar by guest on September 26, 2021 to CXCL5, STING in AT1 cells is another innate immunity gene that can be induced independent of RelA during pneumonia.

Discussion These results demonstrate that NF-kB RelA in alveolar epithelial cells is essential for induction of selected cytokines in the lung, including CXCL5 and CCL20. The effects of alveolar epithelial mutation of RelA on cytokine expression were more modest than anticipated. Many cytokines were expressed similarly regardless of the epithelial cell mutation, including IL-1b,CXCL1,and CXCL2, which contrasts with prior publications in which the NF-kB pathway was targeted by overexpression of dnIkBa (14). This discrepancy may result from differences in the models, including distinct delivery systems for LPS or off-target effects of the SPC-driven dnIkBa transgene, or conceivably they may suggest possible roles for factors other than RelA being downstream of pathways inhibited by dnIkBa overexpression. Cytokines such FIGURE 6. AT1 cell induction of CXCL5 during pneumococcal pneu- as TNF-a and IL-1b more likely result from leukocytes than ep- monia is independent of RelA. (A) RelA is effectively targeted in AT1 cells ithelial cells (47, 48), suggesting that decreases observed in the by transgenesis. RT-PCR of WT and mutant RelA allele products in T1a+ SPC-dnIkBa transgenic mice may reflect indirect effects rather cells and CD45+ cells isolated from lung single-cell suspension of WT than direct expression by alveolar epithelial cells. In contrast to tg/tg F/F D/D (tetO-Cre RelA , no Cre transgene) or epithelial mutant RelA other cytokines, two chemokines were strongly diminished by wt/tg tg/tg F/F + (SPC-rtTA tetO-Cre RelA ) mice reveals that the T1a cells ex- RelA mutation in alveolar epithelial cells during LPS-induced press the mutant allele in targeted mice selectively. Results are represen- inflammation, that is, CCL20 and CXCL5. CCL20 is induced in tative of four mice collected during two independent experiments. (B) The human AT2 cells with LPS stimulation (49), but it can also be induction of CXCL5 in T1a+ cells does not require RelA. CXCL5 was measured by qRT-PCR in sorted T1a+ cells from uninfected mice or elaborated by other cells such as T lymphocytes (50). CCL20 during S. pneumoniae pneumonia, using WT or epithelial mutant RelAD/D has direct antimicrobial activities against Gram-negative bacteria mice (n = 3 mice/group in Ctrl, and n = 6 mice/group in S. pneumoniae, (50, 51), and it signals to dendritic cells and T cells in adaptive collected during three independent experiments). *p , 0.05 compared with immune responses (52). CXCL5 has been suggested to be an AT2 uninfected control mice. Sp, S. pneumoniae. cell-specific product based on immunohistochemistry (53), and it The Journal of Immunology 2457 Downloaded from http://www.jimmunol.org/

FIGURE 7. AT1 cell induction of TLR2 is dependent on RelA, whereas STING induction is RelA-independent. (A) Sorting strategy revised to differentiate gene expression from CD45+ leukocytes supports the concept that CCL20 and CXCL5 are exclusively epithelial in origin. The induction of CCL20 and CXCL5 was measured by qRT-PCR in T1a+ cells, T1a2 cells, and CD45+ cells collected from uninfected lungs and during S. pneumoniae pneumonia (n = 3 mice/group in Ctrl, and n = 6 mice/group in S. pneumoniae, collected during three independent experiments). (B) The expression of PRRs (TLR2, TLR4, and STING) was measured using qRT-PCR in T1a+ cells, T1a2 cells, and CD45+ cells collected from uninfected lungs and during S. pneumoniae pneumonia (n = 3 mice/group in Ctrl, and n = 6 mice/group in S. pneumoniae, collected during three independent experiments). *p , 0.05 compared with uninfected control mice, †p , 0.05 compared with T1a+ cells. (C) The induction of TLR2 in T1a+ cells, measured by qRT-PCR, was abrogated by RelA mutation during S. pneumoniae pneumonia, using lungs from WT (tetO-Cretg/tg RelAF/F, no Cre transgene) or epithelial mutant RelAD/D by guest on September 26, 2021 (SPC-rtTAwt/tg tetO-Cretg/tg RelAF/F) mice, with three mice per group in Ctrl and six mice per group in S. pneumoniae collected during three independent experiments. (D) STING was induced in T1a+ cells during S. pneumoniae pneumonia even when epithelial cells were deficient in RelA, using qRT-PCR to measure STING in lungs from WT or epithelial mutant RelAD/D mice (n = 3 mice/group in Ctrl, and n = 6 mice/group in S. pneumoniae, collected during independent experiments). *p , 0.05 compared with uninfected control mice, †p , 0.05 compared with WT mice. influences neutrophil influx to the lung after LPS inhalation and hybridization. There is little or no information about in vivo AT1 during E. coli pneumonia (54). Our results from targeted RelA responses to bacterial infection. The T1a-based sorting method mutation identify alveolar epithelial cells as essential sources of allows the study of primary AT1 cells collected from living lungs. selected innate immunity mediators important to lung host defense. The present studies reveal that AT1 cells respond in vivo during CCL20 induction was consistently abrogated by mutation of a bacterial infection of the lung. To our knowledge, this is the RelA in the alveolar epithelial cells. The AT2-like MLE15 cells first demonstration that AT1 cells induce innate immunity genes make much more CCL20 than do the AT1-like E10 cells, and this during pneumonia. The discovery that the cells constituting most induction is consistently dependent on RelA. The sorted cell of the alveolar surface respond to infection with the elaboration studies revealed that CCL20 is induced in cells other than AT1 of innate immunity mediators broadens our understanding of lung cells or leukocytes. Collectively, these three sets of results suggest cell biology and pulmonary immunity. that AT2 cells may be unique sources of CCL20 during acute pul- Our conclusion that AT1 cells have roles in innate immunity is monary inflammation, and this chemokine expression is uniformly further supported by the evidence that the PRRs TLR2 and STING dependent on RelA. are induced in AT1 cells during pneumococcal pneumonia, although In our study, CXCL5 was induced by LPS and pneumococcus TLR2 is more highly expressed by leukocytes. Interestingly, STING in T1a2 cells, which include AT2 cells, consistent with a prior is most highly expressed in AT1 cells at baseline and induced to study demonstrating CXCL5 induction in this cell type following the highest levels during infection in AT1 cells. STING mediates an intrapulmonary LPS challenge (53). Excitingly, our in vivo recognition pathways for pneumococcal DNA and drives the ex- studies revealed AT1 cells as a novel source of CXCL5 during pression of innate immunity mediators such as type I IFNs (43, 44). pneumococcal pneumonia. Previous studies have demonstrated Thus, our data suggest that AT1 cells during infection increase that purified bacterial products are capable of inducing chemo- their expression of both membrane and cytosolic receptors capable kines in primary AT1-like cells or cell lines under in vitro culture of responding to pneumococcus. conditions (55, 56), which we have here expanded and confirmed The significance of AT1 cell induction of innate immunity genes with studies of AT1-like E10 cultures. Owing to their anatomic (including but likely not limited to TLR2, STING, and CXCL5) is localizations and morphological characteristics, AT1 cells have unclear. Because the vast majority of the lung surface is AT1 cells, been difficult to study using immunohistochemistry or in situ the increased expression of PRRs and neutrophil chemokines 2458 IMMUNE RESPONSE OF ALVEOLAR EPITHELIAL CELLS AND PNEUMONIA may limit the spread of microbes throughout alveoli or across 10. Saccani, A., T. Schioppa, C. Porta, S. K. Biswas, M. Nebuloni, L. Vago, B. Bottazzi, M. P. Colombo, A. Mantovani, and A. Sica. 2006. p50 nuclear epithelial barriers. The fact that we did not see defects in in- factor-kB overexpression in tumor-associated macrophages inhibits M1 inflammation and host defense is certainly not evidence for a lack flammatory responses and antitumor resistance. Cancer Res. 66: 11432–11440. of a role; these particular challenges and the limitations of this 11.Mizgerd,J.P.,M.M.Lupa,M.S.Kogan,H.B.Warren,L.Kobzik,and G. P. Topulos. 2003. Nuclear factor-kB p50 limits inflammation and prevents transgenic mouse model (discussed above) may have been in- lung injury during Escherichia coli pneumonia. Am.J.Respir.Crit.CareMed. adequate to reveal functionally significant effects of AT1-derived 168: 810–817. genes. It is tempting to speculate that AT1 responses may be es- 12. Quinton, L. J., M. R. Jones, B. T. Simms, M. S. Kogan, B. E. Robson, S. J. Skerrett, and J. P. Mizgerd. 2007. 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