Mycoplasma Pneumonia During the Pathogenesis of Murine Responses

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Antigen-Pulsed Bone Marrow−Derived and Pulmonary Dendritic Cells Promote Th2 Cell Responses and Immunopathology in Lungs during the Pathogenesis of Murine This information is current as Mycoplasma Pneumonia of September 24, 2021. Nicole A. Dobbs, Xia Zhou, Mark Pulse, Lisa M. Hodge, Trenton R. Schoeb and Jerry W. Simecka J Immunol 2014; 193:1353-1363; Prepublished online 27 June 2014; Downloaded from doi: 10.4049/jimmunol.1301772 http://www.jimmunol.org/content/193/3/1353 http://www.jimmunol.org/ References This article cites 72 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/193/3/1353.full#ref-list-1

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antigen-Pulsed Bone Marrow–Derived and Pulmonary Dendritic Cells Promote Th2 Cell Responses and Immunopathology in Lungs during the Pathogenesis of Murine Mycoplasma Pneumonia

Nicole A. Dobbs,* Xia Zhou,† Mark Pulse,‡ Lisa M. Hodge,‡ Trenton R. Schoeb,x and Jerry W. Simecka‡

Mycoplasmas are a common cause of pneumonia in humans and animals, and attempts to create vaccines have not only failed to generate protective host responses, but they have exacerbated the disease. Mycoplasma pulmonis causes a chronic inﬂammatory

lung disease resulting from a persistent infection, similar to other mycoplasma respiratory diseases. Using this model, Th1 subsets Downloaded from promote resistance to mycoplasma disease and infection, whereas Th2 responses contribute to immunopathology. The purpose of the present study was to evaluate the capacity of cytokine-differentiated dendritic cell (DC) populations to inﬂuence the generation of protective and/or pathologic immune responses during M. pulmonis respiratory disease in BALB/c mice. We hypothesized that intratracheal inoculation of mycoplasma Ag–pulsed bone marrow–derived DCs could result in the generation of protective T cell responses during mycoplasma infection. However, intratracheal inoculation (priming) of mice with Ag-pulsed DCs resulted in

enhanced pathology in the recipient mice when challenged with mycoplasma. Inoculation of immunodeﬁcient SCID mice with Ag- http://www.jimmunol.org/ pulsed DCs demonstrated that this effect was dependent on lymphocyte responses. Similar results were observed when mice were primed with Ag-pulsed pulmonary, but not splenic, DCs. Lymphocytes generated in uninfected mice after the transfer of either Ag-pulsed bone marrow–derived DCs or pulmonary DCs were shown to be IL-13+ Th2 cells, known to be associated with immunopathology. Thus, resident pulmonary DCs most likely promote the development of immunopathology in mycoplasma disease through the generation of mycoplasma-speciﬁc Th2 responses. Vaccination strategies that disrupt or bypass this process could potentially result in a more effective vaccination. The Journal of Immunology, 2014, 193: 1353–1363.

ycoplasmas are underrecognized mucosal pathogens in problem in animals, causing a significant economic impact on humans with a profound impact on healthcare owing to livestock, poultry, and other food animals (9). Mycoplasma pul- by guest on September 24, 2021 M the number of urogential and respiratory diseases they monis causes a naturally occurring murine respiratory disease and cause (1–3). The common human respiratory pathogen Myco- is an excellent animal model of M. pneumoniae, allowing for the plasma pneumoniae is a frequent cause of community-acquired characterization of immune responses during the pathogenesis of pneumonia. It is responsible for up to 30% of all cases of pneu- mycoplasma respiratory disease (10). Both M. pulmonis and M. monia in the United States, in which 100,000 cases result in pneumoniae respiratory infections cause rhinitis, otitis media, hospitalization (4–7). Mycoplasma disease is also associated with laryngotracheitis, and bronchopneumonia. In terms of histopa- the exacerbation of other respiratory diseases, such as asthma (8). thology, both diseases are characterized by chronic inflammation, Additionally, mycoplasma respiratory infections are a major consisting of the accumulation of lymphocytes and macrophages along the respiratory airway (5, 11–14). Furthermore, several studies demonstrate that a component of mycoplasma respiratory *Division of Infectious Diseases, Department of Internal Medicine, University of disease is immunopathologic, with lymphocyte responses re- Texas Southwestern Medical Center, Dallas, TX 75390; †Department of Ophthalmol- sponsible for the severity of the inflammatory disease (15–18). ogy, Xiangya Hospital of Central South University, Changsha 410008, China; Recent studies have revealed that pulmonary T cell populations ‡Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107; and xDivision of Genomics, Department of are pivotal in determining the outcome of the mycoplasma in- Genetics, University of Alabama at Birmingham, Birmingham, AL 35294 fection. The depletion of Th cells results in less severe lung disease, Received for publication July 3, 2013. Accepted for publication May 22, 2014. demonstrating that a Th cell population mediates disease pathology This work was supported by Public Health Service Grants R01HL069431-01 and in the lung (19). Furthermore, additional studies indicate that Th2 R01AI042075 (to J.W.S.). The Flow Cytometry facility was supported by a shared cells are likely promote the development of immunopathology in instrument grant from the National Institutes of Health. mycoplasma disease (20, 21). However, adaptive immunity can Address correspondence and reprint requests to Dr. Jerry W. Simecka, Department of Cell Biology and Immunology, University of North Texas Health Science Center, still be beneficial by preventing dissemination of mycoplasma to 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. E-mail address: jerry. extrapulmonary tissues and conferring resistance to infection and [email protected] disease (18). Th1 cell responses appear to lead to resistance and Abbreviations used in this article: BMDC, bone marrow–derived DC; DC, dendritic dampen the inflammatory responses to infection (20). Addition- cell; FSL-1, fibroblast-stimulating lipopeptide-1; G4DC, bone marrow–derived DC + + grown in the presence of GM-CSF plus IL-4; GMDC, bone marrow–derived DC ally, CD8 T cells and CD25 regulatory T cells also reduce the grown in the presence of GM-CSF; LRN, lower respiratory lymph node; T10DC, severity of inflammatory disease (19) (A. Odeh and J.W. Simecka, bone marrow–derived DC grown in the presence of GM-CSF plus IL-10 and TGF- unpublished results). Therefore, Th cells, as well as other T cell b1; WT, wild-type. populations, have conflicting roles when it comes to protection Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 and pathology of mycoplasma disease, and the mechanisms that www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301772 1354 DCs PROMOTE MYCOPLASMA PNEUMONIA IMMUNOPATHOLOGY determine which arm of the immune response is activated are through the promotion of mycoplasma-specific Th2 responses. critical in the pathogenesis and outcome of mycoplasma respira- Vaccination strategies that disrupt or bypass this process could po- tory disease of the lower respiratory tract. tentially result in a more effective vaccination. Because of their central role in activation of T cell responses, APCs, that is, dendritic cells (DCs) and macrophages, may be Materials and Methods influential in the generation of harmful and/or beneficial pulmonary Mice immune responses, particularly with regard to the development of scid immune-mediated pathology or protection in mycoplasma pneu- BALB/cAnNHsd wild-type (WT) and SCID (BALB/cJHanHsd-Prkdc ) mice, tested to be virus- and mycoplasma-free, were obtained from Harlan monia (22–24). DCs are extremely potent APCs found in tissues Sprague Dawley (Indianapolis, IN). Mice were housed in sterile micro- and can activate both Th and cytotoxic T cells (25–31). However, isolator cages supplied with sterile bedding; food and water were provided studies suggest that the naive resident DCs in lungs are immature ad libitum. Female mice between 6 and 10 wk of age were used in the and are not as effective in Ag presentation (32, 33). In a recent study. All animal studies were reviewed and approved by the University of study, we demonstrated that in response to mycoplasma infec- North Texas Health Science Center Institutional Animal Care and Use Committee. tion pulmonary CD11c+ DCs, and not macrophages, were potent stimulators of mycoplasma-specific T cell responses in vitro (34). Bacterial growth conditions Furthermore, these DCs were colocalized with Th cells in the M. pulmonis UAB CT strain was used in all experiments. Stock cultures inflammatory lesions in the lungs of mycoplasma-infected mice. were grown as previously described (50). Stock cultures of mycoplasma Taken together, the in vitro and in vivo data suggest that the in- were stored at –80˚C and the average number of CFU for each batch was teraction of these cell types likely contributes to the immune determined from three to four aliquots that were thawed at different times. Downloaded from responses that impact disease pathogenesis. Although DCs could Intranasal infections in mice be supporting Th cell responses involved in resistance to disease For intranasal infections, a 1-ml aliquot of M. pulmonis stock was thawed or dampening inflammatory responses, DCs could also be in- and diluted in Hayflick’s medium to a concentration of 2 3 105 CFU/20 volved in the pathogenesis of immune-mediated inflammatory ml. Mice were anesthetized and infected with 20 ml diluted mycoplasma by lung disease, such as found with allergic and asthmatic disorders intranasal inoculation.

(35–38). Therefore, DCs are a major APC to investigate during http://www.jimmunol.org/ Mycoplasma Ag preparation mycoplasma respiratory disease, as they are likely to influence T cell responses critical to the outcome of infection at this time. Crude M. pulmonis membrane preparations used for pulsing of DCs for However, the impact of DC/T cell interactions in mycoplasma in vivo inoculations and in vitro stimulation of BMDCs were prepared by osmotic lysis as previously described (51). For in vitro Ag-specific pneumonia is not clear. responses, UV-irradiated mycoplasmas were used. These preparations The purpose of this study was to determine the impact of DC were shown in preliminary studies to stimulate cells similar to crude populations on T cell responses and mycoplasma disease patho- membrane Ag preparations, but they were easier and more reliable to genesis. Previous studies (39–44) have shown that using ex vivo– prepare. M. pulmonis was grown overnight at 37˚C in an orbital shaker in Hayflick’s medium. Then mycoplasma cells were pelleted by centrifuga- generated DCs can be pulsed with Ags as an approach to examine tion at 10,000 rpm for 20 min at 4˚C in a JA-20 rotor. The cell pellets were vaccination strategies against pathogens or tumor cells. Similar to resuspended in sterile PBS and placed into a sterile T-25 cell culture flask. by guest on September 24, 2021 those studies, we initially evaluated the capacity of cytokine dif- The mycoplasma was UV irradiated on a UV transilluminator (Fotodyne, ferentiated DC populations (45–49) to influence the generation of Hartland WI) for 20 min. A 100-ml sample of the UV-irradiated myco- protective and/or pathologic immune responses during mycoplasma preparation was placed onto mycoplasma plates and incubated for 7 d at 37˚C to assure there was no growth on the plates. plasma respiratory disease. Subpopulations of bone marrow–de- The protein concentrations of the Ag preparations were determined using rived DCs (BMDCs) were grown in the presence of GM-CSF only a Bradford protein assay (Bio-Rad, Hercules, CA) per the manufacturer’s (GMDCs), GM-CSF plus IL-4 (G4DCs), and GM-CSF plus IL-10 instructions. Aliquots were made and stored at 280˚C. and TGF-b1 (T10DCs). GM-CSF is required for the generation of Cultivation of BMDC subpopulations and purification DCs from bone marrow. The addition of IL-4 is typically used to generate higher numbers of more mature BMDCs in culture and Bone marrow cells were isolated from the tibiae and femora of healthy 6- to 8-wk-old female BALB/c mice (52, 53). RBCs were removed using ACK influences the differentiation of DCs (48, 49). Culture of BMDCs lysis buffer (54). The cells were suspended in complete RPMI culture in the presence of the regulatory cytokines TGF-b1 and IL-10 can medium (RPMI 1640 [HyClone Laboratories, Logan, UT], 10% heat- generate immature DCs or regulatory DCs (45–47). Because the inactivated FBS [HyClone Laboratories], 10 mM HEPES [Life Technol- pulmonary environment is already under the influence of these ogies, Grand Island, NY], 10 mM L-glutamine [Life Technologies], anti-inflammatory cytokines, the T10DCs may more closely mimic antibiotic/antimycotic solution [Life Technologies], and 50 mM 2-ME). Cells (107) were seeded into T-75 culture flasks in 20 ml complete RPMI the activity of pulmonary DCs in naive lungs (33). Using this culture medium. All BMDC groups were grown using the key DC growth approach, we hypothesized that intratracheal inoculation of mice factor GM-CSF (Invitrogen, Carlsbad, CA) at the concentration of 20 ng/ with mycoplasma Ag–pulsed BMDCs would prime mice, facili- ml added to the complete RPMI medium. tating the generation of protective or immunopathologic T cell To generate different BMDC populations, selected cytokines were added into the medium, as previously described (45–49). Three different BMDC responses against mycoplasma infection depending on the cyto- populations were derived in the presence of GM-CSF only (GMDCs), GM- kine-differentiated population used. In contrast, priming with Ag- CSF plus 20 ng/ml IL-4 (PeproTech, Rockhill, NJ) (G4DCs), or GM-CSF pulsed T10DCs enhanced the pathology observed in the recipient plus 20 ng/ml TGF-b1 (PeproTech) and 20 ng/ml IL-10 (PeproTech) mice when challenged with M. pulmonis, whereas priming with (T10DCs). BMDC populations were grown in their specific conditions for Ag-pulsed GMDCs or G4DCs had no effect. More severe disease 6 d at 37˚C in 5% CO2 adding 20 ml fresh medium every 2 d. After 6 d, the supernatant was retained. Cells were washed twice with complete RPMI was observed after priming with Ag-pulsed pulmonary (similar culture medium only (no cytokines or growth factors added). The common to T10DCs), but not splenic, resident tissue DCs. Lymphocytes DC surface marker CD11c was used to enrich each subpopulation for the generated in uninfected mice after intratracheal inoculation of BMDCs. This was accomplished using the paramagnetic beads conjugated + either Ag-pulsed T10DCs or pulmonary DCs were shown to be with anti-CD11c mouse Ab (N418 MicroBeads kit, Miltenyi Biotec, Auburn, CA) and sorted using the positive selection and magnetic columns Th2 cells, known to be associated with immunopathology. Thus, it on the autoMACS (Miltenyi Biotec) per the manufacturer’s instructions. appears that resident pulmonary DCs contribute to the develop- Purity of each BMDC population was $91% CD11c+ after the procedure ment of immunopathology in mycoplasma disease, most likely as determined by flow cytometry. The Journal of Immunology 1355

In vitro stimulation of BMDCs ries], 5% heat-inactivated FBS [HyClone Laboratories], 10 mM HEPES [Fisher Scientific], antibiotic/antimycotic solution [Life Technologies], 300 U/ml 3 5 + To study cytokine responses to various stimuli, 2 10 CD11c -purified Clostridium histolyticum type I collagenase [Worthington Biochemical, Free- BMDCs were seeded at 100 ml/well into 96-well round-bottom microtiter hold, NJ], and 50 U/ml DNase [Sigma-Aldrich, St. Louis, MO]). Tissues were plates in complete RPMI culture medium (containing no additional growth homogenized using a gentleMACS machine (Miltenyi Biotec) on the “lung factors or cytokines). Cells were stimulated with 100 ml of either myco- version 2.0” setting. After homogenization, a 0.25-ml sample was removed and plasma Ag preparation (5 mg/ml), ultrapure LPS (InvivoGen, San Diego, sonicated (Vibra-Cell sonicator; Sonics & Materials/Vibro Cell) for 1 min at 50 CA) (10 mg/ml), fibroblast-stimulating lipopeptide-1 (FSL-1) (InvivoGen), amplitudes without pulsing. After sonication, the tissue homogenate was serial a synthetic homolog of the MALP-2 agonist from M. fermentans (1 mg/ml), diluted (1:10) into Hayflick’s medium for determination of CFU numbers. or live M. pulmonis at a multiplicity of infection of 6 in a final volume of For cell isolation, the lung homogenates, after the sample used for CFU 200 ml/well culture medium. Cells cultured in medium only without the counts was removed, were incubated at 37˚C while rotating on a Nutator presence of stimuli served as the negative control. Cells were cultured at (Fisher Scientific) for 15–20 min. After incubation, the digestion mixture 37˚C and 5% CO2 for 24 h. After stimulation, the plates were centrifuged was passed through a 250-mm nylon mesh to remove undigested tissue. 3 at 200 g for 10 min at 4˚C, and then the cell culture supernatants were Cells were washed twice with RPMI wash medium and centrifuged at 200 3 g harvested and tested for cytokine production. for 10 min at 4˚C to pellet cells. Cells were then resuspended in 10 ml RPMI 1640 wash medium for further purification. Mononuclear cells were Cytokine ELISAs purified from cell suspension by density gradient centrifugation using IL-10 and IL-12p40 cytokines were measured with sandwich ELISAs. All Lympholyte-M (Cedarlane Laboratories, Burlington, NC) per the manu- ELISAs were performed using OptEIA mouse ELISA kits (BD Pharmin- facturer’s instructions. Cells were collected from the density gradient and gen, San Jose, CA) according to the manufacturer’s instructions with some washed twice with RPMI wash medium. Cells were passed through an modifications. To develop the assay, substrate solution (3,395,59-tetrame- additional 70-mm mesh filter and counted using a hemacytometer using thylbenzidine substrate; Moss, Pasadena, MD) was added to the wells. trypan blue exclusion of dead cells. Plates were read using a Synergy HT multimode microplate reader (Bio- Spleen and lower respiratory lymph node (LRN; mediastinal and hilar Downloaded from Tek Instruments, Winooski, VT) at an absorbance of 450 nm. The lowest lymph nodes) cells were isolated and individually crushed through a 250-mm concentration of the standard curves for IL-10 was 31.3 pg/ml and IL- nylon mesh into a petri dish containing 10 ml RPMI wash medium. Lym- 12p40 was 15.6 pg/ml. phocytes were washed twice with RPMI wash medium, followed by eryth- rocyte removal using ACK lysis buffer. Lymphocytes were counted with Intratracheal inoculation (priming) of mice with Ag-pulsed a hemacytometer using trypan blue exclusion of dead cells. BMDCs and tissue DCs Oxygen saturation

+ http://www.jimmunol.org/ CD11c -purified BMDCs or tissue-derived DCs were seeded into a 96-well The MouseOx pulse oximeter (Starr Life Sciences, Oakmont, PA) was used 3 5 flat-bottom plate at 2 10 cells/well with complete RPMI culture medium for noninvasive measurement of oxygen saturation in unaesthetized mice. (containing no growth factors or cytokines), just as stated in the stimulation The CollarClip sensor was placed around the necks of conscious mice and protocol. These DCs were cultured in the presence or absence of myco- the blood flow was measured through the carotid artery, providing cardiac ∼ plasma Ag (5 mg/ml). After overnight culture ( 12–16 h), the plates were and breathing reading scores. Each mouse was measured using the quick 3 centrifuged at 200 g for 10 min at 4˚C to pellet cells, and then each well averaging diagnostic window setting in the software. This setting takes an was washed three times with 200 ml complete RPMI culture medium average of 30 s of good readings on all variables, averaging 2–5 min to read (containing no growth factors or cytokines) to remove any unbound Ag. To a healthy mouse and averaging 20–30 min for a very sick mouse. Readings remove adherent DCs, 200 ml cold (4˚C) PBS was added to each well and measured on individual mice were taken every 2 d during a 14-d infection. the DCs were collected after thorough pipetting and combined into a tube.

DCs were counted by hematocytometer using trypan blue exclusion of dead Intracellular cytokine staining and flow cytometry by guest on September 24, 2021 cells, centrifuged at 200 3 g for 10 min at 4˚C, and resuspended at a concentration of 1 3 107 cells/ml in PBS. Mice were anesthetized with an i.p. The BD Cytofix/Cytoperm fixation/permeabilization kit (BD Pharmingen) was used for these procedures following the manufacturer’s instructions. injection of diluted ketamine-xylazine. When mice reached the surgical ∼ plane of anesthesia, 50 ml53 105 cells was inoculated intratracheally into Lymphocytes were stimulated overnight ( 12–16 h) in 96-well round- 6 m the lungs of anesthetized mice using a 1-ml syringe and 22-gauge, 1-inch bottom plates (10 cells/100 l) with or without UV-irradiated myco- m dosing needle with stainless steel ball at the end (Braintree Scientific, plasma Ag at 5 g/ml. Then, lymphocytes were additionally stimulated Braintree, MA). Control mice received 50 mlofPBSwithoutBMDCsor with 50 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (EMD unpulsed BMDCs. Animals were infected 10 d later. Millipore, Gibbstown, NJ) for 5 h before harvested. One hour after the addition of PMA and ionomycin to the lymphocytes, GolgiPlug, contain- Assessment of gross and histopathologic lesions ing brefeldin A (BD Pharmingen), was added to cultures at the recom- mended concentration 4 h prior to the harvest and staining. The cells were Lungs were removed, and two researchers examined each lobe for the incubated with a mixture of saturating amounts of the cell-surface Abs and presence of gross lesions. An estimated percentage of lesion damage was anti-CD16/CD32 to block Fc receptors. The cell surface markers used to determined visually for each lobe and recorded. The gross lesion scores for identify cells included PE-Cy7–labeled anti-CD3 mAb, Alexa Fluor 488– the lungs were calculated by multiplying the percentage of lesion damage by labeled anti-CD4 mAb, PerCP-labeled anti-CD8 mAb (all from BD the weight that each lobe contributes to the total lung weight (55). Pharmingen), and allophycocyanin-Cy7–labeled anti-CD44 mAb (Bio- Lungs were fixed in alcohol formalin (4% glacial acetic acid [Fisher Legend, San Diego, CA). Cells were incubated for 15 min at 4˚C protected Scientific, Pittsburgh, PA], 6% formaldehyde solution [Fisher Scientific], from light. To measure intracellular cytokines, cells were fixed and per- 40% deionized water, and 50% of 95% ethanol). Tissues were embedded in meabilized with Cytofix/Cytoperm solution provided in the kit, and the paraffin, sectioned at a thickness of 5 mm, and stained with H&E for light cells were incubated with saturating amounts of allophycocyanin-labeled microscopy. Each lung lobe was sectioned separately. Histology slides anti–IFN-g mAb and PE-labeled anti–IL-13 mAb (both from eBioscience, were randomly coded and subjectively scored at the University of Alabama San Diego, CA) diluted in 13 Perm/Wash buffer at 4˚C for 20 min and Birmingham for lesion severity (scale of 0–4) on the basis of the charac- protected from light. Cells were resuspended in 200 ml BD stabilizing teristic lesions of murine respiratory mycoplasmosis as described previ- fixative (BD Pharmingen) per well and protected from the light. Data were ously (56). Scores refer to 1) peribronchial and perivascular lymphoid acquired using a BD LSR II flow cytometer system and BD FACSDiva hyperplasia or infiltration (peribronchial infiltrate), 2) mixed neutrophilic version 5 software (BD Pharmingen). Lymphocyte gates and detector and histiocytic exudate in alveoli (alveolar exudate), 3) neutrophilic exu- voltages were set using unstained lymph node, lung, and spleen cells. The date in airway lumina (airway exudate), and 4) hyperplasia of airway flow cytometry data were further analyzed using FlowJo flow cytometry mucosal epithelium (epithelial) (56). A score for each lesion was weighted analysis software (Tree Star, Ashland, OR). The proportion of each target according to the percentage each lobe contributed to the total lung weight cell population was expressed as the percentage of the number of stained in arriving at a total lesion score for each set of lungs. cells. To determine the total number of specific lymphocyte populations, the percentages of stained cells, defined by the flow cytometry gates, were Mycoplasma numbers in lungs and mononuclear cell isolation multiplied by the total number of lymphocytes isolated from each tissue. The numbers of mycoplasma CFU in the lungs were determined as previ- CFSE labeling of DCs ously described but with a few modifications (19). Briefly, lung lobes were dissected and placed into gentleMACS (Miltenyi Biotec) C tubes containing Cells were washed twice with 10 ml PBS. Cells were suspended at 107/ml in 5 ml RPMI digest medium (10.4 g/l powder RPMI 1640 [HyClone Laborato- 1 ml PBS, and 1 ml 1 mM CSFE was added to stain the cells. Cells were 1356 DCs PROMOTE MYCOPLASMA PNEUMONIA IMMUNOPATHOLOGY incubated at 37˚C for 15 min. Cells were washed twice with PBS and then Priming of mice with Ag-pulsed GMDCs or T10DCs resulted in counted. The labeled cells were intratracheally inoculated, and 15 h later more severe respiratory disease. There was no difference in clinical the numbers of fluorescent cells recovered from LRNs and lungs were disease or weight loss between any of the PBS control or BMDC determined using flow cytometry. recipient mice within the first 6 d postinfection (Fig. 2A). All mice Isolation of pulmonary and splenic DCs exhibited no signs of clinical illness and were physically active As previously described (34), lung and splenic DCs were purified using and actively grooming demonstrated by the appearance of smooth paramagnetic bead-conjugated Ab and an autoMACS (Miltenyi Biotec) coats. By day 7 postinfection, mice given either Ag-pulsed GMDCs following the manufacturer’s instructions. To isolate DCs (CD11c+F4/802 or T10DCs had increased signs of clinical disease demonstrated cells) from spleens and lungs, mononuclear cells were first depleted of + + + by hunched posture, matted fur, immobility, and general malaise. F4/80 cells and CD11c cell were subsequently isolated. F4/80 cells were These groups showed increased weight loss as compared with the depleted using a two-step process. Mononuclear cells were incubated with biotinylated anti-mouse F4/80 Ab at a 1:40 dilution (Invitrogen/Caltag PBS control or G4DC recipient mice. Mice intratracheally inocu- Laboratories, Carlsbad, CA) followed by anti-biotin MicroBeads (Miltenyi lated with Ag-pulsed T10DCs lost significantly more weight by day Biotec). CD11c+ cell selection was done using CD11c (N418) MicroBeads 7 after mycoplasma infection than did any of the other BMDC , (Miltenyi Biotec). Using this approach, depletion of cells resulted in 2% recipient groups; however, the GMDCs had greater clinical illness residual F4/80+ cells remaining in the depleted cell population, whereas positive selection of cells resulted in $85% purity of CD11c+ cells. We were and weight loss compared with the mice given G4DCs or PBS. By unable to detect CD3+ cells in any of the APC populations. day 14 postinfection, mice given GMDCs or T10DCs still had significantly greater weight loss (21.4 and 21.9% weight loss, re- Statistical analyses spectively). Furthermore, the GMDC and T10DC recipient mice

Data were evaluated by ANOVA, followed by a Fisher protected least- had significantly higher gross lesion scores than those of the other Downloaded from square differences multigroup comparison. These analyses were per- groups of mice, consistent with clinical disease (Fig. 2B). Thus, formed using the StatView version 5.0.1 (SAS Institute, Cary, NC) computer priming naive mice with either Ag-pulsed GMDCs or T10DCs program. A p value #0.05 was considered statistically significant. resulted in increased severity of mycoplasma disease, whereas priming mice with G4DCs had no significant effect on disease se- Results verity.

IL-10 and IL-12 production differ between BMDC populations To determine whether priming of mice with Ag-pulsed DCs http://www.jimmunol.org/ grown in differing cytokine conditions affected host resistance to mycoplasma infection, the number of T cell populations can determine the outcome of many diseases, mycoplasma in the lungs of infected mice was determined at 14 d including mycoplasma, and DCs can influence the type of T cells postinfection. There was no significant difference in the number of generated in response to disease. BMDCs can be produced in po- mycoplasma CFU recovered from the lungs of any of the groups of larizing cytokine environments resulting in subclasses of BMDCs mice (Fig. 2C). Thus, the increase in disease severity was inde- that regulate T cell activation. We took advantage of this approach to pendent of the number mycoplasma in the lungs, suggesting that create different populations of BMDCs to test their ability to in- increased disease severity was due to greater immunopathology fluence the outcome of mycoplasma immunity. and not bacterial burden. To determine whether the BMDC subpopulations differed in the by guest on September 24, 2021 Acceleration of mycoplasma disease due to priming mice with cytokines they produced in response to mycoplasma and other BMDCs is Ag- and lymphocyte-dependent TLR-specific agonists, cells were stimulated with either viable mycoplasma, LPS (TLR4 agonist), or FSL-1 (a TLR2/TLR6 ag- The above studies demonstrated that intratracheal inoculation of onist) and the levels of IL-12p40 and IL-10 in culture supernatants naive mice with Ag-pulsed T10DCs or GMDCs resulted in in- were measured. GMDCs secreted significantly more IL-12p40 in creased disease severity after mycoplasma infection. We chose to response to live mycoplasma than did G4DCs or T10DCs (Fig. focus the remaining studies on T10DCs, as they showed the most 1A). A similar pattern was observed when the cells were stimu- consistent and profound effect on the outcome of mycoplasma- lated with either LPS or FSL-1. The T10DC group produced induced pathology and clinical disease in the previous studies. significantly more IL-10 than did G4DCs when stimulated with Additionally, normal lung DCs are typically immature and were mycoplasma and LPS (Fig. 1B). The GMDC group produced a predicted to be most similar to T10DCs (33). Histopathologic median amount of IL-10 under the same stimulatory conditions. examination at 14 d postinfection confirmed that inflammatory Overall, these results demonstrate that manipulation of BMDC lesions (exudate and lymphoid infiltration) are more severe in culture conditions resulted in their differentiation into DC sub- mice primed with Ag-pulsed T10DCs than in mice inoculated populations, and that these differentiated subpopulations were able with PBS (Fig. 3). Broth control mice that received Ag-pulsed to respond differently to mycoplasma and other stimuli. These T10DCs, but received broth instead of mycoplasma, were also results suggest that these cells may have the capability to influence evaluated. These mice did not have any lesion damage and looked the immune response in vivo. like uninfected mice (data not shown). It is possible that these responses were a result of the inoculation of DCs and not due to Priming of mice with Ag-pulsed BMDCs accelerates pulsing the cells with mycoplasma Ag. To examine this possi- mycoplasma disease bility, mice were intratracheally inoculated with either unpulsed To examine whether BMDC populations could influence myco- or Ag-pulsed T10DCs, and 10 d later mice were infected with plasma disease progression, CD11c+ BMDCs were isolated and M. pulmonis. The control group of mice received PBS. Clinical dis- pulsed overnight with mycoplasma Ag. Ag-pulsed cells suspended ease, weights, and arterial oxygen saturation were measured every in PBS were deposited intratracheally into the lungs of recipient 2 d for 14 d postinfection. naive mice. Control mice received PBS only intratracheally. Ten Priming mice with mycoplasma Ag-pulsed T10DCs increased days after adoptive transfer all mice were infected intranasally the severity of clinical signs of disease when compared with the with M. pulmonis. Clinical disease was monitored and body mice that received unpulsed DCs or PBS (control). Mice primed weights were recorded on days 0, 4, 7, 10, 12, and 14 postinfec- with Ag-pulsed DCs showed accelerated weight loss, but the tion. On day 14 postinfection, the lungs were scored for gross differences in weight loss between the groups became smaller by lesions, and numbers of mycoplasma CFU were determined. 14 d postinfection (Fig. 4A). There was a corresponding decrease The Journal of Immunology 1357

FIGURE 1. BMDC subpopulations display differing cytokine profiles after in vitro stimulation. Bone marrow DCs were derived in the presence of the following cytokines: GM-CSF alone (GMDCs, black bars), GM-CSF plus IL-4 (G4DCs, gray bars), and GM-CSF/TGF-b1 plus IL-10 (T10DCs, white bars). CD11c+ DCs were isolated and stimulated with LPS (TLR4 agonist), FSL-1 (TLR2/6 agonist), or viable M. pulmonis (M.p.) for ∼12–16 h (in the absence of cytokines). The levels of (A) IL-12p40 and (B) IL-10 in culture supernatants were subsequently measured. *p # 0.05 compared with similarly stimulated G4DCs; **p # 0.05 between the designated groups. The results are combined from three independent experiments; each sample was averaged from triplicates. Vertical bars and error bars represent the means 6 SEM. in arterial oxygen saturation. At 14 d postinfection, mycoplasma- vious findings measuring mycoplasma numbers at 14 d postinfec- infected mice primed with Ag-pulsed T10DCs had a significant tion. These results suggest that the acceleration and exacerbation of Downloaded from mean decrease in arterial oxygen saturation of 16.7% compared mycoplasma disease were dependent on the presence of Ag, even with a decrease of 4.6 and 2.5% in mice primed with unpulsed though there was some evidence of a slight nonspecific response DCs or PBS (Fig. 4B). Furthermore, the Ag-pulsed T10DC re- resulting from inoculation with unpulsed DCs and subsequent in- cipient mice had significantly higher gross lesion scores than any fection, and these differences were independent of the number of other groups of mice, consistent with clinical disease and oxygen mycoplasmas in the lung, suggesting that modulation of immune saturation (Fig. 4C). In an additional study, mice were primed with responses were responsible. http://www.jimmunol.org/ unpulsed T10DCs, with Ag-pulsed T10DCs, or with PBS, and the To determine whether the DC-mediated exacerbation of my- primed mice were infected with mycoplasma 10 d later. There was coplasma disease was lymphocyte-dependent, immunocompetent no difference in the number of mycoplasma recovered from lungs BALB/c WT and SCID BALB/c mice, lacking functional B and of mice 7 d postinfection (Fig. 4D), corresponding with the pre- T cells, were used to repeat the above experiments. Both SCID and by guest on September 24, 2021

FIGURE 2. Adoptive (intratracheal) transfer of Ag-pulsed GMDCs and T10DCs exacerbate mycoplasma disease. Bone marrow DCs were derived T10DCs in the presence of the following cytokines: GM-CSF alone (GMDCs), GM-CSF plus IL-4 (G4DCs), and GM-CSF/TGF-b1 plus IL-10 (T10DCs). Mycoplasma Ag–pulsed CD11c+ DCs were intratracheally deposited into the lungs of recipient mice. Control mice were inoculated with PBS. Ten days later, animals were intranasally infected with M. pulmonis.(A) Weight loss was recorded on days 4, 7, and 14 postinfection. By day 7 postinfection, both GMDC and T10DC recipient mice had greater weight loss than did the other groups of mice. Most T10DC recipient mice lost 10% or more of their original start weight by day 7 postinfection. The dotted line indicates 10% weight loss. (B) Gross lung lesions were scored on day 14 postinfection. The GMDC and T10DC recipient mice had greater lesion severity compared with other groups of mice. (C) There were no differences in CFU recovered from the lungs of mice 14 d postinfection. *p # 0.05 compared with control (PBS-inoculated) mice. The results are combined from six independent experiments (totals: n = 17 control, n = 20 GMDCs, n = 19 G4DCs, and n = 19 T10DCs). Vertical bars and error bars represent the means 6 SEM. 1358 DCs PROMOTE MYCOPLASMA PNEUMONIA IMMUNOPATHOLOGY

FIGURE 3. Histopathologic lung lesions were more severe in mice primed with Ag-pulsed T10DCs. Bone marrow DCs were derived in the presence of TGF-b1 plus IL-10 (T10DCs). Mycoplasma Ag–pulsed T10DCs (DC + Ag) were intratracheally inoculated into the lungs of recipient mice. Control mice were inoculated with PBS (control). Ten days later, animals were intranasally infected with M. pulmonis. Four- teen days later, lungs were removed and formalin fixed for histology. Lesion index scores refer to (A) neutrophilic exudate in airway lumina (airway exudate), (B) peribronchial and perivascular lymphoid hyperplasia or infiltration (peribronchial infiltrate) in lung or submucosal infiltrate in nasal passages, (C) hyperplasia of airway mucosal epithelium (epithelial), and (D) mixed neutrophilic and histiocytic exudate

in alveoli (alveolitis). Vertical bars and error bars represent Downloaded from means 6 SEM (totals: n = 7 control and n =10 DC + Ag). *p # 0.05 compared with control mice. http://www.jimmunol.org/

WT mice received either Ag-pulsed or unpulsed T10DCs and were lated overnight with UV-irradiated mycoplasma. Intracellular cy- subsequently infected 10 d later. Animals were monitored for 7 d tokine staining was performed to determine the number of Th cell postinfection because the most significant shift in disease occurs subsets present in these mice. Th1 cells were defined as CD3+CD4+ 1 wk into the infection. Similar to initial experiments, there was no CD44highIFN-g+IL-132, and Th2 cells were defined as CD3+CD4+ difference in clinical disease or weight loss between any of the CD44highIFN-g2IL-13+. groups at the early stages of infection (days 0–5) (data not shown). Mice that received mycoplasma Ag–pulsed T10DCs had a sig- However, as early as day 6, only the immunocompetent WT ani- nificant increase in the number of IL-13+ Th2 cells in the LRNs by guest on September 24, 2021 mals that had received the Ag-pulsed DCs showed the onset of when compared with both the PBS control mice and mice that clinical disease (hunched posture, matted fur, and malaise). The received unpulsed T10DCs (Fig. 7A). There was a significant SCID, PBS WT, and unpulsed T10DC mouse groups showed no increase in the number of Th2 cells in the animals that received signs of disease. On day 7, immunocompetent WT mice that re- unpulsed DCs, but not to the extent seen with the Ag-pulsed DC- ceived Ag-pulsed T10DCs lost significantly more weight than did inoculated mice (Fig. 7A). In contrast to IL-13+ Th2 cells, IFN-g+ any of the other groups (Fig. 5A). Consistent with increased Th1 cell numbers in the LRNs were not different between the clinical disease and weight loss, the immunocompetent Ag-pulsed different groups of mice (Fig. 7A). Similar results were found in T10DC recipient mice had significantly higher lesion scores than the lung lymphocytes, where there was an increase in the number did all other groups, including the immunodeficient SCID recip- of IL-13+ Th2 cells in mice intratracheally inoculated with Ag- ient mice (Fig. 5B). These data suggest that the exacerbation of pulsed DCs (Fig. 7B). mycoplasma respiratory disease, resulting from priming responses + with T10DCs, was not only dependent on Ag but also on the Priming with Ag-pulsed pulmonary, but not splenic, CD11c F4/802 DCs exacerbates mycoplasma respiratory disease presence of lymphocyte responses. The T10DCs used in the previous studies were cultured in anti- There is a significant increase in activated Th2 cells in inflammatory conditions, which may be similar to those found uninfected mice that receive Ag-pulsed T10DCs in the pulmonary environment (33). To determine whether resident Our previous studies demonstrate that Th2 responses contribute to pulmonary DCs have similar activity to the cultured BMDCs, the development of immunopathology in mycoplasma respiratory pulmonary and splenic CD11c+F4/802 DCs were isolated from disease (20). It is possible that intratracheal inoculation of DCs naive mice (34), pulsed overnight with mycoplasma Ag, and promotes the development of mycoplasma-specific Th2 responses, intratracheally inoculated into naive recipient mice. As done in the contributing to exacerbation of disease. Studies have shown that the previous studies, the Th cell subsets in lungs and LRNs of some draining lymph nodes are major sites of localization of fluorescently uninfected mice inoculated 10 d earlier with Ag-pulsed splenic labeled DCs after injection (57), and using this same approach, we and pulmonary DCs were determined. The rest of the mice were confirmed that CSFE-labeled T10DCs were located in the LRNs infected 10 d after inoculation of DCs, and 14 d postinfection and lungs 15 h after intratracheal inoculation, with ∼10% found in gross lung lesions were determined. LRNs (Fig. 6), but nothing was found in spleens. To determine Similar to what was seen in mice receiving Ag-pulsed T10DCs, whether inoculation of T10DCs activated Th cells, WT mice were mice inoculated with Ag-pulsed pulmonary DCs had a significant given Ag-pulsed or unpulsed T10DCs or PBS. Ten days after increase in the number of IL-13+ Th2 cells prior to infection in the adoptive transfer, uninfected mice were sacrificed lymphocytes LRNs (Fig. 8A). In contrast, there was no difference in either Th1 were isolated from the LRNs and lungs and subsequently stimu- or Th2 cell populations in mice given Ag-pulsed splenic DCs or The Journal of Immunology 1359 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Exacerbation of mycoplasma disease is Ag-dependent. Bone marrow DCs were derived in the presence of TGF-b1 plus IL-10 (T10DCs). Mycoplasma Ag–pulsed T10DCs (DC + Ag) or unpulsed T10DCs (DC) were intratracheally inoculated into the lungs of recipient mice. Control mice were inoculated with PBS (control). Ten days later, animals were intranasally infected with M. pulmonis.(A) Weight loss and (B) loss (decrease) in arterial oxygen saturation were recorded on days 4, 6, and 14 d postinfection. There was no difference in weight loss or arterial oxygen saturation at day 4 postinfection. At days 6 and 14 postinfection, there was a significant increase in the weight loss of mice receiving Ag-pulsed DCs, and by day 14 postinfection there was a decrease in arterial oxygen saturation of Ag-pulsed DC recipient mice as compared with mice receiving unpulsed DCs or PBS. (C) Mice were sacrificed at day 14 postinfection and lungs were scored for percentage of gross lesions. Lesion severity was significantly greater in animals that received Ag-pulsed DCs than in the other groups. (D) In additional studies, mice were sacrificed at day 7 postinfection, and the numbers of mycoplasma in lungs were determined. There was no significant difference in the numbers of mycoplasma recovered from the lungs of mice that received Ag-pulsed DCs than in the other groups. *p # 0.05 compared with control (PBS-inoculated) mice. The results are combined from two independent experiments (totals: n = 20 control, n = 18 DC + Ag, and n = 18 DC). Vertical bars and error bars represent the means 6 SEM.

PBS. Mice that received Ag-pulsed pulmonary DCs developed (58, 59), whereas some responses are protective against M. pul- more severe gross lesions than did mice that received Ag-pulsed monis infections (1, 20). Importantly, these responses are deter- splenic DCs or the PBS control animals (Fig. 8B). Overall these mined by the activation of different T cell populations, and the data indicate that resident pulmonary DCs can also promote im- mechanisms that initiate these responses are critical in the patho- munopathology associated with mycoplasma pneumonia, most genesis of mycoplasma respiratory disease of the lower respiratory likely through the generation of Th2-type cell responses. Inter- tract (19, 60). In a previous study (34), we found that DCs are the estingly, DC populations from nonpulmonary tissues (e.g., major APC population responsible for pulmonary T cell stimulation spleens) do not necessarily have the same capacity. in mycoplasma-infected mice and likely contribute to responses impacting disease pathogenesis. This idea is supported by results Discussion showing that DC populations from the lungs of infected mice were The generation of adaptive immunity is critical in determining the most capable of stimulating mycoplasma-speciﬁc T cell responses outcome of mycoplasma respiratory infection. It is clear that ele- in vitro and in vivo. However, it was not clear whether DCs were ments of the adaptive immune response contribute to the pathology promoting immunopathology or stimulating responses that could 1360 DCs PROMOTE MYCOPLASMA PNEUMONIA IMMUNOPATHOLOGY

FIGURE 5. Lymphocytes are required for the exacerbation mycoplasma disease. Bone marrow DCs were derived in the presence of TGF-b1 plus IL-10 (T10DCs). Ag-pulsed T10DCs (DC + Ag) or unpulsed T10DCs (DC) were intratracheally inoculated into the lungs of recipient immunocompetent (WT) or immunodeficient (SCID) mice. Control mice were inoculated with PBS (control). Ten days later, animals were intranasally infected with M. pulmonis.(A) Seven days postinfection, there was a significant increase in weight loss of immunocompetent mice that received Ag-pulsed DCs as compared with similarly treated SCID mice. There were also no significant differences in mice receiving unpulsed DCs or PBS (control). (B) Mice were sacrificed at day 7 postinfection and lungs were scored for percentage of gross lesions. Lesion severity was significantly greater in immunocompetent mice that received Ag- pulsed DC than in the other groups. *p # 0.05 compared with control (PBS-inoculated) mice. The results are combined from two independent experiments Downloaded from (total n $ 9). Vertical bars and error bars represent the means 6 SEM. either dampen harmful responses or control the infection. The pur- BMDC populations accelerated severity of mycoplasma disease. pose of this study was to determine the potential impact of DC This severity was indicated by higher weight losses, increased populations on mycoplasma disease pathogenesis and T cell gross lesions, and reduced oxygen saturation as compared with

responses. These results would not only increase our understanding mice that were not primed with Ag-pulsed DCs prior to infection. http://www.jimmunol.org/ of the immunopathogenic mechanisms of mycoplasma respiratory Furthermore, there were no changes in mycoplasma numbers re- disease and how they could exacerbate other respiratory diseases, but covered from lungs of infected mice due to priming with Ag- they may provide insights to develop effective vaccination strategies pulsed DCs. This is in contrast to other studies showing positive against mycoplasma diseases in humans and animals. effects of adoptive transfer of Ag-pulsed DCs on the host immune The results of the present study suggest that DCs contribute to response in other bacterial infection models using primary the development of the inflammatory lesions associated with BMDCs or JAWS II murine DC lines, against Helicobacter pylori mycoplasma respiratory diseases. Our previous study showed that and Chlamydia muridarum (61, 62), as well as for cancer therapy DC numbers increased in infected lungs and that they colocalized (reviewed in Ref. 63). However, DCs can exacerbate the severity in the inflammatory lesions with Th cells (34). Although it is of respiratory diseases, such as asthma hypersensitivity (64) and by guest on September 24, 2021 possible that the DCs are promoting responses to counter the se- airway reactivity associated with C. muridarum respiratory dis- verity of disease and/or resist infection, the present study found ease (65). Thus, DC populations can stimulate or support detri- that intratracheal instillation of Ag-pulsed pulmonary DC or mental immune responses involved in inflammatory lesions characteristic of mycoplasma pneumonia, and they do not readily help generate beneficial local immune responses that confer resistance to infection. It is however notable that in our studies not all DC populations promoted increased disease severity. Mice intratracheally primed with Ag-pulsed G4DCs developed disease similar to that of control mice. This is in contrast to Ag-pulsed BMDCs derived in the presence of GM-CSF alone (GMDCs) or GM-CSF combined with IL-10 plus TGF-b1 (T10DCs). Interestingly, culture of BMDCs in the presence of the regulatory cytokines TGF-b1 and IL-10 was reported to generate immature or regulatory DCs (45–47), which have similar features to DCs isolated from the lungs of naive mice (33), and in the present study, BMDCs derived in this cytokine environment were most consistent in priming mice for the accelerated development of inflammatory mycoplasma disease in mice postinfection. As suggested, the T10DCs may more closely mimic the activity of pulmonary DCs in naive lungs (33), and consistent FIGURE 6. Intratracheally inoculated DCs are located in lungs and with this idea, mice primed with Ag-pulsed lung DCs also de- LRNs of naive mice. Bone marrow DCs were derived in the presence of veloped more severe disease. However, this is not a characteristic TGF-b1 plus IL-10 (T10DCs). Mycoplasma Ag–pulsed T10DCs (DC + of all DC populations, as similar priming of mice using splenic Ag) were stained with the fluorescent dye CSFE and intratracheally in- DCs had no effect on mycoplasma disease, suggesting that pul- oculated into the lungs of recipient mice. Fifteen hours later, cells were monary DCs are more capable of initiating and/or supporting isolated from LRNs and from the lungs and spleens of the mice. The numbers of CSFE+ cells were determined using flow cytometry. As immunopathologic reactions responding to mycoplasma infection. a control, cells were also collected from mice intratracheally inoculated Overall, our results indicate that naive DCs in the lung promote with PBS, and no CSFE+ cells were found (data not shown). N.D., not the development of immune-mediated inflammatory lesions due to detected. The results are combined from two independent experiments mycoplasma infection, and it is likely that DCs found within the (total n $ 8). Vertical bars and error bars represent the means 6 SEM. inflammatory lesions contribute to disease severity (34). The Journal of Immunology 1361

FIGURE 7. Adoptive (intratracheal) transfer of Ag-pulsed DCs increases the number of IL-13+ Th2 cells but not IFN-g+ Th1 cells, in LRNs prior to infection. Bone marrow DCs were derived in the presence of TGF-b1 plus IL-10 (T10DCs). Mycoplasma Ag–pulsed T10DCs (DC + Ag) or unpulsed T10DCs (DC) were intratracheally inoculated into the lungs of recipient mice. Control mice were inoculated with PBS (control). Ten days later, lymphocytes were isolated from the (A) LRNs and the (B) lungs of the mice. These lymphocytes were stimulated overnight with UV-irradiated mycoplasma. The numbers of Th cells (CD3+CD4+CD82CD44high) expressing IFN-g (Th1, IL-132IFN-g+) or IL-13 (Th2, IL-13+IFN-g2) in each of the cell populations were determined. There was an increase in IL-13 producing speciﬁc Th2 cells in mice given Ag-pulsed DCs. There was also an increase in Th2 cells after inoculation with DCs alone, but at lower levels than mice given Ag-pulsed DCs. There were not changes in Th1 cells. *p # 0.05 in cell numbers as

compared with cells from control mice; **p # 0.05 between the designated groups. The results are combined from two independent experiments (total Downloaded from n = 6). Vertical bars and error bars represent the means 6 SEM.

The presentation of mycoplasma Ag by DCs within draining response to intratracheal adoptive transfer with either IL-10/TGF- lymph nodes and/or lungs leads to the activation of Th cells re- b1 DCs or lung DCs. Other studies further support a strong link sponsible for immunopathology associated with disease. DCs were between DCs and the propagation of Th2 cells, including the

found in both draining lymph nodes and lungs after adoptive expression of IL-13, in some lung diseases (64–66). In fact, our http://www.jimmunol.org/ transfer, which lead to exacerbation of mycoplasma lung disease. previous study (34) demonstrated that most of the DCs isolated Exacerbation of disease was dependent on lymphoid responses, as from lungs of mycoplasma infected mice were CD11b+CD8a2 priming of SCID mice, deficient in B and T cells, with Ag-pulsed conventional DCs. Other studies showed that CD11b+ and CD8a2 DCs had little to no disease, whereas similarly immunocompetent DCs can promote Th2 responses (67–69), which is consistent with mice showed increased disease severity. Additionally, Ag-pulsed a role for DCs in promoting in vivo Th2 responses during my- DCs generated a greater immune response than did DCs not ex- coplasma infection. Most importantly, several studies from our posed to mycoplasma Ag, and these results together demonstrate laboratory and ongoing studies using mycoplasma-specific Th cell that intratracheally inoculated DCs localized within the lymph lines demonstrate Th2 responses generated in response to myco- nodes and lungs, stimulating Ag-specific lymphocyte responses plasma are likely immunopathologic, whereas Th1 responses by guest on September 24, 2021 involved in the pathogenesis of inflammatory lesions associated confer resistance to disease (N. Dobbs, S. Bodhankar-Deo, and with mycoplasma pneumonia. Previous studies from our laboratory J.W. Simecka, unpublished results) (20). Thus, pulmonary DCs, as demonstrated that Th cells are critical in the development of in- well as IL-10/TGF-b DCs, stimulate Ag-specific IL-13+ Th2 flammatory lesions due to mycoplasma lung infection (19), and con- responses that likely accelerate the development of inflammatory sistent with a role of DCs in mediating inflammatory responses, responses after mycoplasma infection and could contribute to the activated Th cells were found in lower respiratory tract lymph exacerbation of asthma (8) and other respiratory diseases. nodes, as well as lungs, in response to adoptive transfer of Ag- Mycoplasmas are underrecognized pathogens, but they are pulsed DCs prior to infection. Interestingly, there was a preferen- known to cause 30% of all human pneumonia in the United States tial increase in activated Th cells producing IL-13, not IFN-g,in alone. Although mycoplasmas are susceptible to antibiotics, they

FIGURE 8. Pulmonary DCs, not splenic DCs, also exacerbate mycoplasma respiratory disease, possibly owing to the preferential increase of IL-13+ Th2 cells in the LRNs prior to infection. CD11c+F4/802 cells were isolated from the lungs (lung DC) or spleen (spleen DC) of naive mice, pulsed overnight with Mycoplasma Ag, and intratracheally instilled into the lungs of recipient mice. Control mice were inoculated with PBS. (A) Ten days later, lymphocytes were isolated from the LRNs of the mice. These lymphocytes were stimulated overnight with UV-irradiated mycoplasma. The numbers of Th cells (CD3+CD4+ CD82CD44high) expressing IFN-g (Th1, IL-132IFN-g+) or IL-13 (Th2, IL-13+IFN-g2) in each of the cell populations were determined. There was a significant increase in the numbers of specific Th2 cells in LRNs of mice inoculated with Ag-pulsed lung DCs, but not splenic DCs. (B) Additional groups of mice were intranasally infected with mycoplasma 10 d after inoculation with lung or splenic DCs. Mice were sacrificed at day 14 postinfection and lungs were scored for percentage of gross lesions. Lesion severity was significantly greater in mice that received Ag-pulsed lung DCs than in the other groups. *p # 0.05 compared with the other groups of mice. The results are combined from four independent experiments (n = 3/experiment). Vertical bars and error bars represent the means 6 SEM. 1362 DCs PROMOTE MYCOPLASMA PNEUMONIA IMMUNOPATHOLOGY still cause a significant impact on the health care industry as well as 3. Waites, K. B., J. W. Simecka, and D. F. Talkington. 2007. Pathogenesis of Mycoplasma pneumoniae infections:adaptive immunity, innate immunity, cell the agricultural industry. Therefore, a vaccine would be preferable biology, and virulence factors. In Community-Acquired Pneumonia. N. Suttorp, to reduce the amount of human and livestock infections. Attempts T. Welte, and R. Marre, eds. Birkhaüser, Basel, Switzerland, p. 183–199. have been made to develop a vaccine, but these have not only failed 4. Mogabgab, W. J. 1968. Mycoplasma pneumoniae and adenovirus respiratory illnesses in military and university personnel, 1959–1966. Am. Rev. Respir. Dis. to elicit protection but also in some cases exacerbated subsequent 97: 345–358. disease owing to mycoplasma infection. This makes it imperative to 5. Foy, H. M. 1993. Infections caused by Mycoplasma pneumoniae and possible understand the underlying cellular mechanisms involved in the carrier state in different populations of patients. Clin. Infect. Dis. 17(Suppl 1): S37–S46. generation of immunopathology or resistance to mycoplasma lung 6. Foy, H. M., G. E. Kenny, M. K. Cooney, and I. D. Allan. 1979. Long-term epide- infections. In our previous study (34), DCs were previously shown miology of infections with Mycoplasma pneumoniae. J. Infect. Dis. 139: 681–687. to be the major APC population responsible for T cell stimulation 7. Gray, G. C., L. B. Duffy, R. J. Paver, S. D. Putnam, R. J. Reynolds, and G. H. Cassell. 1997. Mycoplasma pneumoniae: a frequent cause of pneumonia in the lungs of mycoplasma-infected mice and were found to lo- among U.S. Marines in southern California. Mil. Med. 162: 524–526. calize in inflammatory lesions, interacting with Th cells. The 8. Martin, R. J., M. Kraft, H. W. Chu, E. A. Berns, and G. H. Cassell. 2001. A link present study further demonstrates that DCs in the lungs likely between chronic asthma and chronic infection. J. Allergy Clin. Immunol. 107: 595–601. stimulate immune responses leading to the development of in- 9. Simecka, J. W., J. K. Davis, M. K. Davidson, S. E. Ross, C. Stadtlander, and flammatory lesions characteristic of mycoplasma pneumonia. A G. H. Cassell. 1992. Mycoplasma diseases of animals. In Mycoplasmas. Mo- lecular Biology and Pathogenesis. J. Maniloff, R. N. McElhaney, L. R. Finch, likely scenario through which this occurs is that early after my- and J. B. Baseman, eds. American Society for Microbiology, Washington, DC, coplasma infection naive pulmonary DCs present Ag to T cells in p. 391–415. lower respiratory tract lymph nodes and promote a Th2 cell re- 10. Cassell, G. H. 1982. Derrick Edward Award Lecture. The pathogenic potential of mycoplasmas: Mycoplasma pulmonis as a model. Rev. Infect. Dis. 4(Suppl.): sponse. Mycoplasma-specific effector Th2 cells then migrate from S18–S34. Downloaded from lymph nodes into infected lung tissue, where resident DCs further 11. Dajani, A. S., W. A. Clyde, Jr., and F. W. Denny. 1965. Experimental infection stimulate these Th2 cells, resulting in inflammatory lesions. Im- with Mycoplasma pneumoniae (Eaton’s agent). J. Exp. Med. 121: 1071–1086. 12. Cassell, G. H., J. R. Lindsey, R. G. Overcash, and H. J. Baker. 1973. Murine portantly, these results further support the idea that vaccines and mycoplasma respiratory disease. Ann. N. Y. Acad. Sci. 225: 395–412. adjuvant combinations that avoid priming Th2 responses are 13. Cartner, S. C., J. W. Simecka, J. R. Lindsey, G. H. Cassell, and J. K. Davis. 1995. needed to minimize chances of exacerbation of mycoplasma dis- Chronic respiratory mycoplasmosis in C3H/HeN and C57BL/6N mice: lesion severity and antibody response. Infect. Immun. 63: 4138–4142. ease, as previously found (20). Because local immunization is 14. Yancey, A. L., H. L. Watson, S. C. Cartner, and J. W. Simecka. 2001. Gender is http://www.jimmunol.org/ optimal for generation of protective immunity (70), immunization a major factor in determining the severity of mycoplasma respiratory disease in mice. Infect. Immun. 69: 2865–2871. may require adjuvants that either enhance the activity of APCs, 15. Evenga˚rd, B., K. Sandstedt, G. Bo¨lske, R. Feinstein, I. Riesenfelt-Orn, and other than DCs, or alter the normal activity of DC populations in C. I. Smith. 1994. Intranasal inoculation of Mycoplasma pulmonis in mice with the respiratory tract. Adjuvants known to promote Th1 responses severe combined immunodeficiency (SCID) causes a wasting disease with grave arthritis. Clin. Exp. Immunol. 98: 388–394. through modification of APC activity would include selected 16. Keystone, E. C., D. Taylor-Robinson, M. F. Osborn, L. Ling, C. Pope, and TLR agonists, such as monophosphoryl lipid A or CpG- V. Fornasier. 1980. Effect of T-cell deficiency on the chronicity of arthritis in- oligodeoxynucleotide (71–76). In addition to stimuli to control duced in mice by Mycoplasma pulmonis. Infect. Immun. 27: 192–196. 17. Taylor, G., D. Taylor-Robinson, and G. W. Fernald. 1974. 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