Increased Activation of M2 Macrophages in 1 Year‐Old Rat Lungs Following Neonatal Hyperoxia Exposure

Abstract

RationaleMacrophages are activated in response to insult. M1 macrophage polarization is associated with inflammation and tissue deterioration, whereas the M2 macrophage corresponds to wound repair and angiogenesis and has an anti‐inflammatory phenotype. Lipopolysaccharide (LPS) or interferon gamma (IFNγ) promote M1 differentiation and up‐regulate pro‐inflammatory cytokines like interleukin‐ (IL‐) 1β, IL‐6, and IL‐12 whereas M2 differentiation is regulated by IL‐4. Since hyperoxia exposure induces inflammation leading to the pathology seen in bronchopulmonary dysplasia (BPD) through induction of M1 macrophages, we tested the macrophage population in lung tissue of 1 year‐old rats. We postulated an increased activation of M2 macrophages in lungs of hyperoxia‐exposed rats, suggesting a continued wound healing response. Collagen levels were also assessed. We expected to see an increase in collagen levels of hyperoxic rats, suggesting that there is persistent phenotype after one year.MethodsNewborn rats were exposed to 85% hyperoxia (Hx) or normoxia (Nx) (21%) for 14 days and then kept at Nx conditions. At one year, the lung was lavaged with PBS, the left lung was inflated and fixed (4% formaldehyde). Cells were isolated using a 100 μm cell strainer, red blood cells were lysed (ammonium chloride), and the cells frozen in freezing medium. For flow cytometry, the cells were stained for CD45, CD24, CD163, CD80, CD86, CD204, and CD206. Analysis was done with an LSR‐2. Collagen levels were assessed using Masson's trichome‐stained lung sections. A blinded third party divded each image into grids and ranked each square on a scale of 0–3 (0=none, 3=severe) for fibrosis.ResultsAnalysis of CD80 and CD86 showed an increased proportion of the M1 macrophages in the Hx lung compared to the Nx lung (11.7% higher in Hx). The Hx M1 showed more of activation marker CD163 compared to the Nx M1 in the CD24− cell population (12% higher in Hx). Similarly, the proportion of CD204+ M2 was increased within the CD45+ cell population of Hx (6.3% higher in Hx). Comparing M1 to M2 macrophages, Hx exposure shifted the ratio of macrophages toward an M2 response. These findings were supported by the histologic assessment of collagen showing increased lung collagen in Hx compared to Nx rats (p=0.008).ConclusionOne year after neonatal Hx exposure, adult rat lungs demonstrate a macrophage population shift towards M2, suggesting a continued anti‐inflammatory response. Further, the increased level of collagen in Hx rats suggests that there is persistent damage after one year. The data indicates that humans born preterm with BPD may have long term alterations in inflammatory and repair pathways.Support or Funding InformationNHLBI, R01 HL115061‐03, Suppl (Eldridge) & NHLBI, R01 HL115061 (Eldridge)