Inhalation of ambient ozone alters populations of lung macrophages. However, the impact of altered lung macrophage populations on the pathobiology of ozone is poorly understood. We hypothesized that sub-populations of macrophages modulate the response to ozone. We exposed C57BL/6 mice to ozone (2 ppm × 3h) or filtered air. 24 h after the exposure, the lungs were harvested and digested and the cells underwent flow cytometry. Analysis revealed a novel macrophage subset present in ozone exposed mice, which were distinct from resident alveolar macrophages (AM) and identified by enhanced Gr-1+ expression (Gr-1 Macs). Further analysis identified that Gr-1+ Macs exhibited high expression of MARCO, CX3CR1, and NQO1. Gr-1+ Macs were present in the absence of CCR2, suggesting that they were not derived from a CCR2-dependent circulating intermediate. Using PKH26-PCL to label resident phagocytic cells, we demonstrated that Gr-1 Macs were derived from resident lung cells. This new subset was diminished in the absence of CX3CR1. Interestingly, CX3CR1-null mice exhibited enhanced responses to ozone, including increased airway hyperresponsiveness (AHR), exacerbated neutrophil influx, accumulation of 8-isoprostanes and protein carbonyls, and increased expression of cytokines (CXCL2, IL-1β, IL-6, CCL2, and TNF-α). Our results identify a novel subset of lung macrophages, which are derived from a resident intermediate, dependent upon CX3CR1, and appear to protect the host from the biological response to ozone.
This article is being retracted at the request of the authors because of concerns about the accuracy of the initial data from the animal physiology laboratory at Duke University. The authors re-exported source data from the animal ventilator (FlexiVent) and compared the output with the raw data originally received from the animal pulmonary physiology laboratory. The results from these initial comparisons suggested potential inconsistencies in the data, so the authors requested that an independent laboratory replicate the experiments of animal airway physiology presented in Figure 1 and Figure 4. The results of the replicated experiments validated the originally reported central role of mindin in airway hyper-responsiveness after exposure to either lipopolysaccharide (LPS) or ozone.Because the animal physiology laboratory at Duke University also analyzed cytokines, the authors had an independent laboratory replicate experiments to analyze the original role of cytokines reported in Figure 3 and Figure 6. In these replicate studies, which were limited in the number of animals and samples tested, the authors did not observe the same results and could not definitively determine whether or not the findings were valid.The inconsistent results led the authors to take a conservative approach, and they agreed to retract this paper. They regret any inconvenience to the scientific community.
Following an inquiry at Duke University, we have been informed that FlexiVent data provided to us by the animal pulmonary physiology laboratory may have been unreliable. The data in Figs. 1-4 are not affected. We have replicated the studies in Fig. 5A-F, and the new data support the originally published findings. We were unable to repeat the experiments in Fig. 5G; therefore, to maintain the accuracy of the scientific record, a corrected Fig. 5 and figure legend, representing the new data, are shown below. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600002 FIGURE 5. Ozone exposure in CX3CR1-null mice. C57BL/6 (WT) and CX3CR1 GFP/GFP (CX3CR1-null) mice were exposed to filtered air or 2 ppm of ozone for 3 h and then underwent analysis 12-24 h after exposure. A, Flow cytometric analysis of Gr-1 Macs in WT (open box) and CX3CR1-null mice (closed box) as a percentage of total cells at 24 h after exposure to filtered air (FA) or ozone. B, CX3CL1 protein expression was analyzed by ELISA in BAL from WT and CX3CR1-null mice 24 h after filtered air (open box) or ozone (closed box) exposure. C, AHR after increasing doses of methacholine in WT and CX3CR1-null mice 24 h after filtered air or ozone exposure. D, Total cells and neutrophils (PMNs) from BAL cell count differentials in WT (open box) and CX3CR1-null mice (closed box) 24 h after filtered air or ozone exposure. E, Analysis of total protein in BAL from WT (open box) and CX3CR1-null mice (closed box) 24 h after filtered air or ozone exposure. F, Analysis of cytokines by multiplex from concentrated BAL fluid in WT (open box) and CX3CR1-null mice (closed box) 12 h after filtered air or ozone exposure. Data for AHR are from n 5 7 WT FA, n 5 7 CX3CR1-null FA, n 5 12 WT O3, and n 5 14 CX3CR1-null O3 (# p , 0.05 for WT or CX3CR1-null O3 versus FA control, *p , 0.05 for WT versus CX3CR1-null O3 exposed, & p , 0.05 for WT versus CX3CR1-null FA exposed). Data for other experiments are from three to eight mice per group (WT-FA, WT-ozone, CX3CR1-null-FA, and CX3CR1-null-ozone).
Background: Our previous work demonstrated that the extracellular matrix protein mindin contributes to allergic airways disease. However, the role of mindin in nonallergic airways disease has not previously been explored.Objectives: We hypothesized that mindin would contribute to airways disease after inhalation of either lipopolysaccharide (LPS) or ozone.Methods: We exposed C57BL/6J and mindin-deficient (–/–) mice to aerosolized LPS (0.9 μg/m3 for 2.5 hr), saline, ozone (1 ppm for 3 hr), or filtered air (FA). All mice were evaluated 4 hr after LPS/saline exposure or 24 hr after ozone/FA exposure. We characterized the physiological and biological responses by analysis of airway hyperresponsiveness (AHR) with a computer-controlled small-animal ventilator (FlexiVent), inflammatory cellular recruitment, total protein in bronchoalveolar lavage fluid (BALF), proinflammatory cytokine profiling, and ex vivo bronchial ring studies.Results: After inhalation of LPS, mindin–/– mice demonstrated significantly reduced total cell and neutrophil recruitment into the airspace compared with their wild-type counterparts. Mindin–/– mice also exhibited reduced proinflammatory cytokine production and lower AHR to methacholine challenge by FlexiVent. After inhalation of ozone, mice had no detectible differences in cellular inflammation or total BALF protein dependent on mindin. However, mindin–/– mice were protected from increased proinflammatory cytokine production and AHR compared with their C57BL/6J counterparts. After ozone exposure, bronchial rings derived from mindin–/– mice demonstrated reduced constriction in response to carbachol.Conclusions: These data demonstrate that the extracellular matrix protein mindin modifies the airway response to both LPS and ozone. Our data support a conserved role of mindin in production of proinflammatory cytokines and the development of AHR in two divergent models of reactive airways disease, as well as a role of mindin in airway smooth muscle contractility after exposure to ozone.
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