Vitamin D deficiency and bacterial load in a murine model of sepsis-induced lung injury

Background We have previously shown that patients with acute lung injury are severely vitamin D deficient. Several studies have reported a high prevalence of vitamin D deficiency in critically ill patients with sepsis, associated with increased morbidity and mortality, but whether this association is cause or effect is unknown. Bacteraemic sepsis is more common in the winter months when serum vitamin D concentrations are lowest. The purpose of this study was to investigate the local and systemic effects of vitamin D deficiency in a murine model of sepsis-induced lung injury where we can predictably time the initiating insult. Methods We fed eight wild-type C57BL/6 mice a diet completely devoid of vitamin D for 6 weeks to induce severe vitamin D deficiency (serum concentration 9 nmol/L) and compared them with seven mice fed a vitamin D sufficient diet (42 nmol/L). We used caecal ligation and puncture (CLP) to establish sepsis. Animals were culled 16 h after CLP, and blood, peritoneal lavage fluid (PLF), and bronchoalveloar lavage fluid (BALF) were collected. Cell infiltrates were assessed by flow cytometry. Fluid protein concentrations were measured, and tissue protein permeability index was calculated as the ratio between fluid and serum protein. Bacterial load was evaluated as colony-forming units (CFU) per ml after 24 h incubation on appropriate media. Statistical analysis was done with Minitab 16 statistical software. Findings Vitamin D deficient mice had significantly increased bacterial load compared with dietary sufficient mice in BALF, blood, and PLF (BALF median 2·51 × 103 CFU per mL [IQR 0·65 × 103–1097 ×103] vs 0·51 ×103 [0·0–1·15 × 103], p=0·038; blood 66·1 × 103 [1·93 × 103–175 × 103] vs 0·56 × 103 [0·0–5·56 × 103], p=0·042; and PLF 336 × 103 [52 × 103–115 × 105] vs 8·22 × 103 [1·54 ×103–20·6 × 103], p=0·005). BALF protein permeability index was higher in deficient mice than in sufficient mice (median 3·3 [IQR 2·61–4·64] vs 1·8 [1·22–2·09], p=0·0003) but there was no difference in cell numbers recruited to the lung. PLF protein permeability index was also higher in the deficient group than in sufficient mice (46·8 [28·1–59·3] vs 27·2 [14·2–37·3], p=0·05), with an associated increase in neutrophils recruited to the peritoneum (p=0·04). Interpretation Vitamin D deficiency significantly increased the bacterial load systemically, locally, and within the lung in a murine model of peritonitis. This increase was associated with a rise in tissue permeability locally and within the lung. These data support pre-existing vitamin D deficiency as a determinant of the severity of bacteraemic sepsis and might account for some of the seasonal variations observed in the incidence of sepsis. Funding UK Medical Research Council