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• W2149558843 abstract "Resident alveolar macrophages (rAMs) residing in the bronchoalveolar lumen of the airways play an important role in limiting excessive inflammatory responses in the respiratory tract. High phagocytic activity along with hyporesponsiveness to inflammatory insults and lack of autonomous IFN-β production are crucial assets in this regulatory function. Using a mouse model of asthma, we analyzed the fate of rAMs both during and after allergic bronchial inflammation. Although nearly indistinguishable phenotypically from naïve rAMs, postinflammation rAMs exhibited a strongly reduced basal phagocytic capacity, accompanied by a markedly increased inflammatory reactivity to Toll-like receptors TLR-3 (poly I:C), TLR-4 [lipopolysaccharide (LPS)], and TLR-7 (imiquimod). Importantly, after inflammation, rAMs exhibited a switch from an IFN-β-defective to an IFN-β-competent phenotype, thus indicating the occurrence of a new, inflammatory-released rAM population in the postallergic lung. Analysis of rAM turnover revealed a rapid disappearance of naïve rAMs after the onset of inflammation. This inflammation-induced rAM turnover is critical for the development of the hyperinflammatory rAM phenotype observed after clearance of bronchial inflammation. These data document a novel mechanism of innate imprinting in which noninfectious bronchial inflammation causes alveolar macrophages to acquire a highly modified innate reactivity. The resulting increase in secretion of inflammatory mediators on TLR stimulation implies a role for this phenomenon of innate imprinting in the increased sensitivity of postallergic lungs to inflammatory insults. Resident alveolar macrophages (rAMs) residing in the bronchoalveolar lumen of the airways play an important role in limiting excessive inflammatory responses in the respiratory tract. High phagocytic activity along with hyporesponsiveness to inflammatory insults and lack of autonomous IFN-β production are crucial assets in this regulatory function. Using a mouse model of asthma, we analyzed the fate of rAMs both during and after allergic bronchial inflammation. Although nearly indistinguishable phenotypically from naïve rAMs, postinflammation rAMs exhibited a strongly reduced basal phagocytic capacity, accompanied by a markedly increased inflammatory reactivity to Toll-like receptors TLR-3 (poly I:C), TLR-4 [lipopolysaccharide (LPS)], and TLR-7 (imiquimod). Importantly, after inflammation, rAMs exhibited a switch from an IFN-β-defective to an IFN-β-competent phenotype, thus indicating the occurrence of a new, inflammatory-released rAM population in the postallergic lung. Analysis of rAM turnover revealed a rapid disappearance of naïve rAMs after the onset of inflammation. This inflammation-induced rAM turnover is critical for the development of the hyperinflammatory rAM phenotype observed after clearance of bronchial inflammation. These data document a novel mechanism of innate imprinting in which noninfectious bronchial inflammation causes alveolar macrophages to acquire a highly modified innate reactivity. The resulting increase in secretion of inflammatory mediators on TLR stimulation implies a role for this phenomenon of innate imprinting in the increased sensitivity of postallergic lungs to inflammatory insults. The mucosal surfaces of the respiratory tract are continuously exposed to environmental antigens and must therefore restrain excessive inflammatory responses in order to fulfill their role in gaseous exchange and to prevent bystander tissue damage. Powerful mechanical and immunosuppressive mechanisms protect the lung against development of inappropriate immune reactivity and inflammation. When these defense mechanisms fail, chronic airway inflammatory diseases such as allergic asthma may develop.1Barnes P.J. Immunology of asthma and chronic obstructive pulmonary disease.Nat Rev Immunol. 2008; 8: 183-192Crossref PubMed Scopus (1044) Google Scholar In allergic asthma, the infiltration of the bronchial mucosa by leukocytes (mainly eosinophils), along with subepithelial fibrosis, goblet cell hyperplasia, and airway hyperresponsiveness, leads to reversible loss of lung function and in the long term to irreversible tissue remodeling.2Cohn L. Elias J.A. Chupp G.L. Asthma: mechanisms of disease persistence and progression.Annu Rev Immunol. 2004; 22: 789-815Crossref PubMed Scopus (707) Google Scholar Over several decades, immunosuppressive mechanisms have been identified that inhibit or limit the development of maladaptive pulmonary inflammatory responses. Allergen uptake and presentation by pulmonary plasmacytoid dendritic cells provides intrinsic protection against inflammatory responses to harmless antigen by skewing T-cell differentiation toward the tolerogenic CD4+ CD25+ regulatory T-cell phenotype.3de Heer H.J. Hammad H. Soullie T. Hijdra D. Vos N. Willart M.A. Hoogsteden H.C. Lambrecht B.N. Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen.J Exp Med. 2004; 200: 89-98Crossref PubMed Scopus (669) Google Scholar Immunomodulatory cytokines such as IL-10 and TGF-β are known to possess anti-inflammatory activities in the development of allergic asthma. In addition to their inhibitory effect on proinflammatory cytokine secretion and leukocyte maturation, IL-10 and TGF-β are commonly implicated in the generation of the inducible regulatory T-cell subsets Tr1 and Th3, respectively.4Auffray C. Sieweke M.H. Geissmann F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells.Annu Rev Immunol. 2009; 27: 669-692Crossref PubMed Scopus (1165) Google Scholar Resident alveolar macrophages (rAMs) are important sources of pulmonary IL-10 and TGF-β,5Lambrecht B.N. Alveolar macrophage in the driver's seat.Immunity. 2006; 24: 366-368Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar and have been shown to exert an immunosuppressive effect on T-lymphocytes and dendritic cells.6Ho A.S. Moore K.W. Interleukin-10 and its receptor.Ther Immunol. 1994; 1: 173-185PubMed Google Scholar Several studies have demonstrated a significant increase in allergic inflammation and T-cell reactivity in antigen-challenged lungs after depletion of rAMs.7Holt P.G. Oliver J. Bilyk N. McMenamin C. McMenamin P.G. Kraal G. Thepen T. Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages.J Exp Med. 1993; 177: 397-407Crossref PubMed Scopus (455) Google Scholar, 8Thepen T. Van Rooijen N. Kraal G. Alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice.J Exp Med. 1989; 170: 499-509Crossref PubMed Scopus (355) Google Scholar, 9Thepen T. McMenamin C. Girn B. Kraal G. Holt P.G. Regulation of IgE production in pre-sensitized animals: in vivo elimination of alveolar macrophages preferentially increases IgE responses to inhaled allergen.Clin Exp Allergy. 1992; 22: 1107-1114Crossref PubMed Scopus (83) Google Scholar The increased sensitivity of rAM-depleted lungs to antigen exposure observed in these studies was attributed to the loss of rAM-mediated suppression of dendritic cell maturation, function, and trafficking to mediastinal lymph nodes.10Stumbles P.A. Upham J.W. Holt P.G. Airway dendritic cells: co-ordinators of immunological homeostasis and immunity in the respiratory tract.APMIS. 2003; 111: 741-755Crossref PubMed Scopus (66) Google Scholar, 11Jakubzick C. Tacke F. Llodra J. van Rooijen N. Randolph G.J. Modulation of dendritic cell trafficking to and from the airways.J Immunol. 2006; 176: 3578-3584PubMed Google Scholar Along with the mucosal epithelia, rAMs are the first cells to contact inhaled infectious agents, as well as small-particulate debris and airborne allergens. After resolution of inflammation, the increased numbers and altered differentiation of long-lived antigen-reactive lymphocytes, a hallmark of the adaptive immune branch, stand in strong contrast to the return to basal levels for cell numbers and differentiation that are commonly reported for innate immune cells such as tissue macrophages. Recent reports, however, increasingly challenge the paradigm of innate cells retaining no memory of prior inflammatory insults.12Page K.R. Scott A.L. Manabe Y.C. The expanding realm of heterologous immunity: friend or foe?.Cell Microbiol. 2006; 8: 185-196Crossref PubMed Scopus (55) Google Scholar Murine rAMs have been reported to display a sustained desensitization to bacterial Toll-like receptor (TLR)-5 ligands after resolution of respiratory influenza infection.13Didierlaurent A. Goulding J. Patel S. Snelgrove R. Low L. Bebien M. Lawrence T. van Rijt L.S. Lambrecht B.N. Sirard J.C. Hussell T. Sustained desensitization to bacterial Toll-like receptor ligands after resolution of respiratory influenza infection.J Exp Med. 2008; 205: 323-329Crossref PubMed Scopus (299) Google Scholar In a mouse model of Sendai virus infection, a lasting effect on rAMs was observed, one that persisted after clearance of the virus. In this model of paramyxovirus-induced pathology, rAM activation persisted, resulting in a chronic lung condition with pathological features resembling asthma and chronic obstructive pulmonary disease.14Kim E.Y. Battaile J.T. Patel A.C. You Y. Agapov E. Grayson M.H. Benoit L.A. Byers D.E. Alevy Y. Tucker J. Swanson S. Tidwell R. Tyner J.W. Morton J.D. Castro M. Polineni D. Patterson G.A. Schwendener R.A. Allard J.D. Peltz G. Holtzman M.J. Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease.Nat Med. 2008; 14: 633-640Crossref PubMed Scopus (424) Google Scholar These observations indicate that infection may educate innate immune cells also, altering the way the cells respond to a subsequent inflammatory insult. This concept of innate imprinting has been documented in several mouse models of infection.12Page K.R. Scott A.L. Manabe Y.C. The expanding realm of heterologous immunity: friend or foe?.Cell Microbiol. 2006; 8: 185-196Crossref PubMed Scopus (55) Google Scholar Nonetheless, the extent to which innate imprinting also occurs after noninfectious, allergic inflammation and the nature of its functional outcome remain largely unknown. Here, we now provide evidence for a pronounced innate imprinting of rAMs as a consequence of allergic bronchial inflammation in mouse models for eosinophilic, Th2-biased mild to moderate asthma and for neutrophilic, Th1- and Th17-biased severe refractory asthma. The altered functional maturation of postinflammatory rAMs was evidenced by an enhanced responsiveness of the cells to TLR ligands and a newly acquired capacity to produce IFN-β, a type I IFN. Mechanistically, we provide evidence that the switch from a restrained to an unrestrained rAM inflammatory response is the consequence of allergic inflammation-induced rAM turnover, accompanied by the emergence after clearance of inflammation of a new population of secondary rAMs with increased reactivity to inflammatory insults. Female C57BL/6 mice, 6 to 8 weeks old, were purchased from Janvier (Le Genest-St-Isle, France). This wild-type strain expresses the CD45.2 alloantigen and served as recipient for the generation of CD45 chimeric mice. Female B6.SJL-Ptprca Pep3b/BoyJ mice, 12 weeks old, were purchased from Charles River International (Brussels, Belgium). This strain expresses the CD45.1 alloantigen and served as bone marrow donor for the generation of CD45 chimeric mice. Both mice strains were kept under specific pathogen-free conditions. All animal experiments were approved by the local ethics committee. For the allergic asthma model, C57BL/6 mice were immunized intraperitoneally with 20 μg of grade V chicken egg ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO), adsorbed on 1 mg aluminum hydroxide (alum; Sigma-Aldrich) in endotoxin-free PBS (Lonza, Walkersville, MD). To generate a model for a noneosinophilic severe refractory Th1-/Th17-mediated allergic bronchial inflammation, C57BL/6 mice were immunized subcutaneously with 20 μg of OVA in PBS emulsified in 75 μL complete Freund's adjuvant (CFA; Sigma-Aldrich). In both mouse models, OVA-sensitized mice were exposed to OVA aerosols, consisting of either 1% (allergic asthma model) or 0.1% (neutrophilic Th1-/Th17-mediated allergic inflammation model) of grade III OVA in PBS. Bone marrow cells were isolated under sterile conditions from the tibias and femurs of sex-matched CD45.1 donor mice. Briefly, tibias and femurs were flushed with sterile PBS and the cell suspension was filtered through 70-μm nylon mesh (BD Biosciences, San Diego, CA) to remove cell aggregates. Red blood cell lysis was performed before transplantation by incubation of the single-cell suspension in ACK lysing buffer (Lonza) for 3 minutes at room temperature. Recipient CD45.2 alloantigen-expressing C57BL/6 mice received 8 Gy of total body irradiation using a 5-MV photon beam of a linear accelerator (SL-75, Elekta, Crawley, UK). This radiation dose nearly completely depleted the bone marrow, but did not induce depletion of rAMs and memory T cells in previously OVA-alum-sensitized mice. A total of 8 × 106 CD45.1 donor bone marrow cells suspended in 250 μL sterile PBS were transplanted via lateral tail-vein injections into CD45.2 recipient mice. The drinking water of the CD45.2 recipient mice was supplemented with 0.2% neomycin trisulfate antibiotics (Sigma-Aldrich) from 5 days before until 14 days after the irradiation. Mice were anesthetized with 2,2,2-tribromomethanol (Avertin; Sigma-Aldrich), 2,5% in PBS. Bronchoalveolar lavage (BAL) was performed by making a small incision in the trachea, to allow passage of a lavage cannula. Lungs were flushed four times with 1 mL Ca2+-free and Mg2+-free Hank's balanced salt solution (HBSS; Invitrogen Life Technologies, Carlsbad, CA), supplemented with 0.05 mmol/L EDTA (ethylenediaminetetraacetic acid). Optionally, a prior lavage with 0.5 mL HBSS-EDTA was performed and BAL fluid (BALF) was isolated by centrifugation and collection of the supernatant. BALF cells were washed and resuspended in PBS for further use. Pre- and postinflammation rAMs isolated via BAL were cultured in complete culture medium: RPMI 1640 medium containing 1% heat-inactivated fetal calf serum, 25 mmol/L HEPES, 2 mmol/L l-glutamine, 1 mmol/L pyruvate, 100 U/mL penicillin/streptomycin (Invitrogen-Life Technologies), and 55 μmol/L 2-mercaptoethanol (Sigma-Aldrich). All cultures were enriched for macrophages by plastic adhesion for 1 hour at 37°C. Naïve and postinflammation rAMs were then stimulated for the indicated times with LPS (Escherichia coli 0111:B4; Sigma-Aldrich), polyriboinosinic:polyribocytidylic acid (poly I:C; InvivoGen, San Diego, CA), or imiquimod (InvivoGen) at 37°C. The expression of alveolar macrophage maturation markers was assessed on naïve and postinflammation rAMs by flow cytometry. Briefly, BALF cells were counted and suspended at a concentration of 106 cells/mL. High-affinity Fcγ receptors (FcγRs) were blocked by incubation with purified anti-mouse CD16/CD32 (Fc Block) for 15 minutes at 4°C and were stained with CD11c-allophycocyanin (APC), DEC-205-phycoerythrin (PE), F4/80-biotin, CD11b-PE, and/or CD115-PE for 1 hour at 4°C. Biotinylated F4/80 antibody was detected by an additional incubation step with streptavidin-PE for 20 minutes at 4°C. All antibodies and the streptavidin-PE were purchased from BD Biosciences. Autofluorescence was detected in the FL-1 channel. Turnover of naïve rAMs during the course of the bronchial inflammation elicited in both models was determined using CD45 chimeric mice. Naïve rAMs were identified by recipient-specific CD45.2 expression and by uptake of Fluoresbrite Yellow Green (YG) plain 1-μm latex microspheres (Polysciences, Warrington, PA), administered by the intrathecal route 48 hours before the first OVA aerosol exposure. Elicited cells were identified as microsphere− cells expressing donor CD45.1. Anti-mouse CD45.1-PE and CD45.2-PerCP-Cy5.5 antibodies (BD Biosciences) were used according to the manufacturer's instructions. Preincubation of the cells with Fc Block was used to prevent unwanted binding to Fc receptors. All samples were measured on a FACSCalibur flow cytometer (BD Biosciences) and were analyzed using CellQuest Pro software version 6.0 (BD Biosciences). BALF cell counts and cell-type composition was analyzed by flow cytometry. Cells preincubated with Fc Block were classified as monocytes (alveolar macrophages, elicited monocytes, and dendritic cells), neutrophils, eosinophils, or lymphocytes based on forward and side scatter gating and fluorescence intensities for anti-mouse CD3ε-Alexa488, B220-FITC, CCR3-PE, CD11c-APC, and I-Ab-biotin that was recognized by streptavidin-PerCP. All antibodies and streptavidin-PerCP were from BD Biosciences, except that CCR3-PE was from R&D Systems (Abingdon, UK). Additionally, the total number of BALF cells was calculated from the measured total cell count relative to the number of Flow-Count beads (Beckman Coulter, Brea, CA), of which a constant amount of was added to the sample. Total numbers of BALF cells were counted by use of a Bürker chamber (Marienfeld, Lauda-Königshofen, Germany). Trypan Blue was added to exclude dead cells. Differential cell counts obtained by flow cytometry were confirmed by morphological examination of cytospin preparations using a Shandon cytocentrifuge (TechGen, Zellik, Belgium) and stained with May-Grünwald-Giemsa stain (Sigma-Aldrich). The percentage of monocytes or macrophages, neutrophils, and eosinophils was determined by counting at least 400 cells. Both analyses were performed on an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with 4×, 10×, 20×, 40×, and 100× objectives. RNA isolation was performed using an RNeasy Plus mini kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. cDNA was synthesized using a SuperScript II reverse transcription reagent kit (Invitrogen-Life Technologies). Real-time quantitative PCR (qPCR) was performed on a LightCycler 480 system using a qPCR kit for SYBR Green I (both from Roche Molecular Systems, Pleasanton, CA). Real-time qPCR amplification was performed in triplicate reactions under the following conditions: a preincubation step at 95°C for 5 minutes, followed by 50 cycles at 95°C for 10 seconds and at 60°C for 30 seconds. The following primers were used, forward and reverse, respectively: murine Arg1, 5′-TGAACACGGCAGTGGCTTTA-3′ and 5′-GCATTCACAGTCACTTAGGTGGTTTA-3′; murine Inos, 5′-CAGCTGGGCTGTACAAACCTT-3′ and 5′-CATTGGAAGTGAAGCGTTTCG-3′; murine Usp18, 5′-AGCCCTCATGGTCTGGTTGGTT-3′ and 5′-GCACTCCGAGGCACTGTTATCC-3′; murine Ifit2, 5′-ATCTCTCCCTACTCTGCCCTCCTA-3′ and 5′-GCGTATAAATCAGCAATCCCTTCA-3′; murine Oas1 (Oas1a form), 5′-CCCTGGGCCCTTCCTGT-3′ and 5′-CCCGGGGGCACTTGTCT-3′; murine Ifi205, 5′-GGATAGAAGTTGTGGGGAGTGGC-3′ and 5′-CAGCCTTGGTGACCTTGACGA-3′; murine Il6, 5′-TAGTCCTTCCTACCCCATTTCC-3′ and 5′-TTGGTCCTTAGCCACTCCTTC-3′; murine Il12p35), 5′-AACCAGGGCCTTCTTTAG-3′ and 5′-GATCTGCCTGCCTTGGTCT-3′; and murine Rpl13a, 5′-CCTGCTGCTCTCAAGGTTGTT-3′ and 5′-TGGCTGTCACTGCCTGGTACTT-3′. Murine RPL13a mRNA was used as reference housekeeping gene for normalization. All primers were purchased from Invitrogen-Life Technologies. Uptake of YG+ microspheres by naïve and postinflammation rAMs was imaged with a confocal microscope (TCS SP5 system with an acousto-optical beam splitter; Leica, Wetzlar, Germany) using a 488-nm multiple argon laser line. Cytoplasm was stained with CellTracker Orange (Invitrogen-Life Technologies) and was excited with a 543-nm helium-neon laser. Nuclei were stained with 500 nmol/L DAPI (Invitrogen-Life Technologies) and excited with the 405-nm line of an UV diode laser. Stained cells were mounted in 1% N-propyl gallate in glycerol before image acquisition. Images were acquired using LAS AF software version 2.4.1, build 6384 (Leica) and were subsequently analyzed with Volocity software version 5.5 (PerkinElmer, Coventry, UK). Protein levels of mouse TNF-α, IL-6, IL-12p70, CXCL1, and CXCL2 in culture supernatant or BALF were quantified with a Bio-Plex suspension array system (Bio-Rad Laboratories, Hercules, CA) for simultaneous detection of cytokines, according to the manufacturer's protocol. The analytes were measured with a Bio-Plex protein array reader and Bio-Plex manager software version 5.0 (Bio-Rad Laboratories), using recombinant cytokine standards (all from Bio-Rad Laboratories). Culture supernatant levels of IFN-β were determined using a VeriKine mouse IFN-β ELISA kit (PBL Interferon Source, Piscataway, NJ) according to the manufacturer's protocol. Statistical analyses were performed using GraphPad Prism software version 5 (GraphPad Software, La Jolla, CA). Outlier statistics were used to choose between performing a one-way analysis of variance or Kruskal-Wallis nonparametric test, and Gaussian distribution of parameters was checked using a Kolmogorov-Smirnov test. Differences in means between each of two independent experimental groups were analyzed using an unpaired t-test or the nonparametric Mann-Whitney U-test at the 95% confidence interval. No statistical analysis was done for gene expression data, because these data involved pooled samples. We used a mouse model of allergic asthma in which a Th2-biased sensitization of C57BL/6 mice against the model allergen OVA is elicited by repeated intraperitoneal immunization using aluminum hydroxide as an adjuvant.3de Heer H.J. Hammad H. Soullie T. Hijdra D. Vos N. Willart M.A. Hoogsteden H.C. Lambrecht B.N. Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen.J Exp Med. 2004; 200: 89-98Crossref PubMed Scopus (669) Google Scholar Exposure of the sensitized mice to nebulized OVA then generated an eosinophilic airway inflammation mimicking the immunopathology of mild to moderate asthma (Figure 1A). The clearance of the allergic pulmonary inflammation was verified by harvesting BALF samples at different time points after the last of seven OVA challenges. This showed that the alveoli regained a new steady-state condition within 12 days. At this time point, absolute cell numbers had returned to basal levels (Figure 1A), and cytospin analysis showed that the cellular composition of the alveoli again was at 90% macrophages (Figure 1A). These macrophages form the new rAM population of postinflammation lungs and can therefore be considered postinflammation rAMs. In addition, at this time point Th2-associated inflammatory cytokines were no longer detectable in the BALF (data not shown). To determine to what extent the postinflammation rAM population exhibited a characteristic alveolar macrophage phenotype, we analyzed expression levels of alveolar macrophage markers by flow cytometry. Naïve and postinflammation rAM populations were isolated via BAL from naïve mice and OVA-challenged mice 15 days after the final OVA exposure. CD11c and DEC-205, hallmark surface markers of rAMs,15Gonzalez-Juarrero M. Shim T.S. Kipnis A. Junqueira-Kipnis A.P. Orme I.M. Dynamics of macrophage cell populations during murine pulmonary tuberculosis.J Immunol. 2003; 171: 3128-3135PubMed Google Scholar, 16Grundy M. Sentman C.L. GFP transgenic mice show dynamics of lung macrophages.Exp Cell Res. 2005; 310: 409-416Crossref PubMed Scopus (11) Google Scholar, 17Paine 3rd, R. Morris S.B. Jin H. Wilcoxen S.E. Phare S.M. Moore B.B. Coffey M.J. Toews G.B. Impaired functional activity of alveolar macrophages from GM-CSF-deficient mice.Am J Physiol Lung Cell Mol Physiol. 2001; 281: L1210-L1218PubMed Google Scholar, 18van Rijt L.S. Kuipers H. Vos N. Hijdra D. Hoogsteden H.C. Lambrecht B.N. A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma.J Immunol Methods. 2004; 288: 111-121Crossref PubMed Scopus (150) Google Scholar, 19Bilyk N. Holt P.G. The surface phenotypic characterization of lung macrophages in C3H/HeJ mice.Immunology. 1991; 74: 645-651PubMed Google Scholar, 20Bosio C.M. Dow S.W. Francisella tularensis induces aberrant activation of pulmonary dendritic cells.J Immunol. 2005; 175: 6792-6801PubMed Google Scholar were equally and uniformly expressed on both rAM populations (Figure 1B). High intrinsic fluorescence intensity, which is a general phenotypic characteristic of rAMs,21Vermaelen K. Pauwels R. Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights.Cytometry A. 2004; 61: 170-177Crossref PubMed Scopus (204) Google Scholar was present in both rAM populations. Also, F4/80, a broad macrophage marker,22Guth A.M. Janssen W.J. Bosio C.M. Crouch E.C. Henson P.M. Dow S.W. Lung environment determines unique phenotype of alveolar macrophages.Am J Physiol Lung Cell Mol Physiol. 2009; 296: L936-L946Crossref PubMed Scopus (162) Google Scholar was nearly equally and uniformly expressed on both cell populations. In contrast, naïve rAMs were uniformly negative for the expression of CD11b, whereas postinflammation rAMs expressed medium to high levels of CD11b. Furthermore, the monocyte marker CD115 (M-CSFR) and phagocytosis receptors FcγRIII (CD16/CD32) and SR-A (CD36) exhibited uniformly elevated expression levels at rAMs from postinflammation mice (Figure 1B; see also Supplemental Figure S1 at http://ajp.amjpathol.org). Thus, although postinflammation rAMs exhibit a characteristic alveolar macrophage marker profile (ie, autofluohigh CD11c+ DEC205+ F4/80+), they differ from naïve rAMs in the expression levels of macrophage/monocyte maturation and phagocytosis markers. Alveolar macrophages typically exhibit high phagocytic activity.23Gordon S.B. Read R.C. Macrophage defences against respiratory tract infections.Br Med Bull. 2002; 61: 45-61Crossref PubMed Scopus (190) Google Scholar To verify to what extent postinflammation rAMs had retained this functional trait, naïve and postinflammation rAMs were incubated ex vivo with fluorescent latex microspheres for up to 6 hours. Phagocytosis of the latex microspheres was assessed by confocal microscopy. The microspheres were readily engulfed by naïve rAMs, whereas microsphere uptake was strongly reduced in postinflammation rAMs at all time points assayed (Figure 1C). After 6 hours of incubation, nearly 85% of naïve rAMs were positive for uptake of microspheres, compared with only 10% of postinflammation rAMs. In addition, microsphere+ naïve rAMs consistently featured higher numbers of microspheres per cell, compared with microsphere+ postinflammation rAMs (Figure 1D). Reduced phagocytic activity is often observed in macrophages that have been alternatively differentiated.24Varin A. Mukhopadhyay S. Herbein G. Gordon S. Alternative activation of macrophages by IL-4 impairs phagocytosis of pathogens but potentiates microbial-induced signalling and cytokine secretion.Blood. 2010; 115: 353-362Crossref PubMed Scopus (138) Google Scholar These so-called M2 macrophages feature, in addition to a low phagocytic capacity, an arginine metabolism differing from M1 or classical differentiated macrophages by an increased arginase/iNOS expression ratio.25Martinez F.O. Helming L. Gordon S. Alternative activation of macrophages: an immunologic functional perspective.Annu Rev Immunol. 2009; 27: 451-483Crossref PubMed Scopus (2056) Google Scholar Analysis of Arg1 and Inos mRNA levels indeed confirmed this shift toward an M2 characteristic arginine metabolism. The expression of Arg-1, the prototypic M2 marker, was up to 50-fold higher in postinflammation rAMs, compared with naïve rAMs, whereas the expression of the M1 marker, Inos, scarcely differed between the two macrophage populations (Figure 1E). Next, to investigate to what extent TLR signaling in postinflammation rAMs is affected, we compared the response of pre- and postinflammation rAMs to ligation of the antibacterial Toll-like receptor TLR-4 and the antiviral Toll-like receptors TLR-3 and TLR-7. rAMs were isolated from naïve and postinflammation lungs and cultured ex vivo for 6 hours in the presence of E. coli LPS (0.1 μg/mL), poly I:C (10 μg/mL), or imiquimod (10 μg/mL). Production levels of inflammatory cytokines and chemokines were subsequently analyzed via a Bio-Plex suspension array system. Compared with naïve rAMs, postinflammation rAMs exhibited markedly increased protein levels for TNF-α, IL-6, IL-12p70, CXCL1 (KC), and CXCL2 (MIP-2) after stimulation with LPS and imiquimod (Figure 2). With poly I:C stimulation, however, these cytokines and chemokines remained undetectable or near basal levels (CXCL2) in the supernatant of both rAM cell cultures (Figure 2). rAMs differ from other tissue macrophages in their failure to autonomously produce IFN-β in response to TLR-3 and TLR-4 triggering.26Punturieri A. Alviani R.S. Polak T. Copper P. Sonstein J. Curtis J.L. Specific engagement of TLR4 or TLR3 does not lead to IFN-beta-mediated innate signal amplification and STAT1 phosphorylation in resident murine alveolar macrophages.J Immunol. 2004; 173: 1033-1042PubMed Google Scholar Strikingly, postinflammation rAMs switched from an IFN-β production-defective to an IFN-β production-competent phenotype after LPS and poly I:C stimulation, but failed to do so in response to imiquimod (Figure 3A). This difference was also confirmed at the level of autocrine IFN-β bioactivity, as apparent from the strongly increased transcript levels of Usp18, Ifit2, Oas1, and Ifi205 in LPS- and poly I:C-treated postinflammation rAMs (Figure 3B). The IFN-β biomarker function of these genes was confirmed by performing a similar analysis on the postinflammation rAMs from Ifnb KO mice,27Erlandsson L. Blumenthal R. Eloranta M.L. Engel H. Alm G. Weiss S. Leanderson T. Interferon-beta is required for interferon-alpha production in mouse fibroblast" @default.
• W2149558843 created "2016-06-24" @default.
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• W2149558843 date "2012-07-01" @default.
• W2149558843 modified "2023-09-29" @default.
• W2149558843 title "Innate Imprinting of Murine Resident Alveolar Macrophages by Allergic Bronchial Inflammation Causes a Switch from Hypoinflammatory to Hyperinflammatory Reactivity" @default.
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• W2149558843 doi "https://doi.org/10.1016/j.ajpath.2012.03.015" @default.
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