Lps induced TNF-α production by bronchoaveolar lavage-and lung cells is increased in TLR 9 deficient mice

Rationale: Toll-like receptors (TLRs) are proteins that recognize specific molecular patterns and have recently been implicated in the pathogenesis of chronic obstructive pulmonary diseases (COPD). Animal models that are used to study COPD involve for instance exposure to cigarette smoke (CS) or to lipopolysaccharide (LPS) which is known to stimulate TLR 4. Interestingly, CS induced cytokine production by macrophages is also mediated by TLR4 and TLR4 is implicated in CS induced pulmonary inflammation in a murine model of COPD (Karimi et al 2006, Maes et al 2006). Very recently we demonstrated that TLR 9 is involved in the CS-induced IL-8 production by human inflammatory cells (Mortaz et al, 2009). Now we want to confirm these data using TLR 9 KO mice. But before doing so, we wanted to exclude possible effects of LPS via TLR4 in these animals. Methods: Lungs of TLR 9-/- and wild type C57BL/6 (WT) mice were lavaged and bronchoalveolar lavage (BAL) fluid was examined for total cell number and differential cell number using diff quick staining. Lung and spleen tissue was isolated and frozen for PCR analysis. Total cell cultures derived from BAL (2∗10∧5 cells/200 μl), lung 8∗10∧5 cells/200 μl and spleen (2∗10∧5 cells/200 μl) were ex vivo stimulated with LPS (1000 ng/200μl). Tumor necrosis factor (TNF)-αlpha was measured in supernatant using a matched antibody cytoset from Arcus biologicals. Statistical analysis was performed in Prism. Results: Total BAL cells were 40% increased in TLR9-/- compared to WT mice. Differential cell count did not show PMN, eosinophil nor lymphocyte influx. The increase in cell number in TLR 9 -/- animals is caused by an increased number of bronchoalveolar macrophages. LPS increased the TNF-αlpha production of BAL-, lung- and spleen cells of WT-mice. Surprisingly, the TNF-αlpha levels were 42,6% increased in lung cell cultures from TLR 9 -/- animals compared to WT-mice (P