Lipopolysaccharide-induced lung injury is independent of serum vitamin D concentration.

Vitamin D deficiency is increasing in incidence around the world. Vitamin D, a fat-soluble vitamin, has documented effects on the innate and adaptive immune system, including macrophage and T regulatory (Treg) cell function. Since Treg cells are important in acute lung injury resolution, we hypothesized that vitamin D deficiency increases the severity of injury and delays injury resolution in lipopolysaccharide (LPS) induced acute lung injury. Vitamin D deficient mice were generated, using C57BL/6 mice, through diet modification and limited exposure to ultraviolet light. At 8 weeks of age, vitamin D deficient and sufficient mice received 2.5 g/kg of LPS or saline intratracheal. At 1 day, 3 days and 10 days, mice were anesthetized and lung elastance measured. Mice were euthanized and bronchoalveolar lavage fluid, lungs and serum were collected. Ex vivo neutrophil chemotaxis was evaluated, using neutrophils from vitamin D sufficient and deficient mice exposed to the chemoattractants, KC/CXCL1 and C5a, and to bronchoalveolar lavage fluid from LPS-exposed mice. We found no difference in the degree of lung injury. Leukocytes were mildly decreased in the bronchoalveolar fluid of vitamin D deficient mice at 1 day. Ex-vivo, neutrophils from vitamin D deficient mice showed impaired chemotaxis to KC but not to C5a. Vitamin D deficiency modestly impairs neutrophil chemotaxis; however, it does not affect lung injury or its resolution in an LPS model of acute lung injury

Lung inflammation 1 day after either LPS instillation.

<p>Vitamin D-sufficient (VDS) or –deficient (VDD) treated with either LPS or PBS. N = 7 to 9 per group. (<b>A</b>) Bronchoalveolar lavage (BAL) fluid total cell count, p<0.05 comparing VDS and VDD mice after LPS instillation; (<b>B</b>) BAL fluid neutrophil count, p = 0.09 comparing VDS and VDD mice after LPS; (<b>C</b>) BAL fluid KC/CXCL1 concentration; (<b>D</b>) BAL fluid MIP2/CXCL2 concentration.</p

Lung injury and inflammation 3 days after LPS instillation.

<p>Vitamin D-sufficient (VDS) or –deficient (VDD) treated with either LPS or PBS. N = 8 per group. (<b>A</b>) Percent body weight change; (<b>B</b>) Lung elastance (H) over time following lung recruitment (mean ± SEM); (<b>C</b>) Left lung lobe weight (wt) normalized to day 0 body weight; (<b>D</b>) Total protein concentration of bronchoalveolar lavage (BAL) fluid; (<b>E</b>) IgM concentration of BAL fluid; (<b>F</b>) BAL fluid total cell count; (<b>G</b>) BAL neutrophil count.</p

Lung injury and inflammation 10 days after LPS instillation.

<p>Vitamin D-sufficient (VDS) or –deficient (VDD) were treated with either LPS or PBS. N = 8 to 10/group. (<b>A</b>) Percent body weight change, p<0.05 comparing VDS and VDD mice after LPS; (<b>B</b>) Lung elastance (H) over time following lung recruitment (mean ± SEM); (<b>C</b>) Left lung lobe weight (wt) normalized to day 0 body weight; (<b>D</b>) Total protein concentration of bronchoalveolar lavage (BAL) fluid; (<b>E</b>) IgM concentration of BAL fluid; (<b>F</b>) BAL fluid total cell count; (<b>G</b>) BAL neutrophil count.</p

Lung injury 1 day after LPS instillation.

<p>Vitamin D-sufficient (VDS) or –deficient (VDD) treated with either LPS or PBS. N = 7 to 9 per group. (<b>A</b>) Percent body weight change; (<b>B</b>) Lung elastance (H) over time following lung recruitment (mean ± SEM); (<b>C</b>) Left lung lobe weight (wt) normalized to day 0 body weight; (<b>D</b>) Total protein concentration of bronchoalveolar lavage (BAL) fluid; (<b>E</b>) IgM concentration of BAL fluid.</p

Neutrophil chemotaxis.

<p>Neutrophils were isolated from vitamin D-sufficient (VDS) or –deficient (VDD) mouse bone marrow for chemotaxis measurement and flow cytometry. (<b>A</b>) VDS neutrophil chemotaxis towards BAL fluid collected from VDS and VDD mice 1 day after LPS stimulation; Chemotaxis of neutrophils isolated from VDS and VDD mice towards varying concentrations of (<b>B</b>) KC/CXCL1 (N = 5 per group) and (<b>C</b>) complement C5a (N = 3 per group); (<b>D</b>) Percentage of CXCR2 positive cells in neutrophils isolated from bone marrow of VDS and VDD mice (N = 4 per group); (<b>E</b>) Expression of CXCR2 as determined by mean fluorescent intensity measured with flow cytometry of CXCR2+ cells from bone marrow of VDS and VDD mice (N = 4 per group).</p

Representative histology sections.

<p>400× magnfication of hematoxylin and eosin stained sections of right lung lobes collected from vitamin D sufficient (VDS) and deficient (VDD) mice after exposure to PBS or to LPS for varying lengths of time.</p