L‐Citrulline Modulates Macrophage Polarization to an M2 Phenotype in a Model of Lipopolysaccharide‐Induced Lung Injury in Neonatal Rats

Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants resulting from inflammation and oxidative stress, leads to arrested pulmonary development and lifelong respiratory morbidities. Macrophages are the most abundant immune cells in the neonatal lung, orchestrating inflammation, maintaining homeostasis, and supporting tissue remodeling and repair. Macrophages can polarize into specific phenotypes in response to the micro-environment. M1 macrophages are pro-inflammatory and pathogen killing whereas, M2 macrophages are anti-inflammatory and support normal lung development. Existing therapies for BPD have inherent side effects. L-Citrulline (L-CIT), a non-essential amino acid, that is safe and well-tolerated by infants, reduces oxidative stress and lung inflammation. Thus, we hypothesize that L-CIT modulates macrophage polarization to an M2 phenotype in the presence of lipopolysaccharide (LPS) induced lung injury during early development.Sprague Dawley rat pups received saline (SAL) or L-CIT (2.5 g/kg) intraperitoneally during postnatal day (PND) 4-9. In addition, pups were treated with LPS (5mg/kg) or SAL intrapharyngeally during PND 5-8. Thus, we had 4 treatment groups, ie., SAL+SAL (controls), LPS+SAL, LPS+L-CIT and L-CIT+SAL. Pups were euthanized on PND 9. Bronchoalveolar Lavage Fluid (BALF): BALF was cytospinned, and cells were counted following hematoxylin and eosin staining. BALF supernatant was collected, and an arginase activity assay was performed for M2 marker expression. Lung homogenate: Lungs were dissected, homogenized and processed for western blotting of M2 (CD206) and M1 (Arg2) markers normalized to rat macrophage marker (F4/80).BALF analysis: The number of alveolar macrophages infiltrated in the BALF of animals treated with L-CIT+LPS (n=4) and those treated with SAL+LPS (n=4) were similar; both were significantly higher than control (p<.001; p<.0001, respectively). Arginase activity in the BALF was significantly higher in animals treated with L-CIT+LPS (p<.05; n= 4) compared to LPS alone (p<.05; n=4). Lung homogenate analysis: LPS+SAL treated animals had lower levels of protein expression for CD206 (M2 marker) (p<.05; n=4) compared to control animals. Whereas L-CIT+LPS treated animals had increased expression of CD206 (M2 marker) (p<.05; n=4) and lower expression of Arg2 (M1 marker) (p<.001; n=4) compared to control animals.In BPD pathogenesis, there is a major shift towards M1 polarization due to the presence of pro-inflammatory stimuli in the microenvironment, leading to arrested lung development. Our preliminary findings support a role for L-CIT in regulating macrophage polarization, favoring an anti-inflammatory M2 phenotype in the presence of inflammatory injury. This further suggests that L-CIT, a safe nutritional supplement, could reduce inflammatory lung injury during early development.