Organic barn dust inhibits surfactant protein D production through protein kinase-c alpha dependent increase of GPR116

Abstract

Prolonged exposure to organic barn dusts can lead to chronic inflammation and a broad range of lung problems over time, mediated by innate immune mechanisms. The immune surfactant or collectin surfactant protein D (SP-D) is a crucial multifunctional innate immune receptor. Little work to date has examined the effect of such collectins in response to organic dusts. We provide evidence here that agricultural organic dusts can inhibit mRNA and protein expression of SP-D in a human alveolar epithelial cell line, and an in vivo mouse model. This inhibition was not a result of lipopolysaccharide (LPS) or peptidoglycans, the two most commonly cited immune active components of these dusts. We further show that inhibition of the signaling molecule protein kinase C alpha (PKCα) can reverse this inhibition implicating it as a mechanism of SP-D inhibition. Examination of the SP-D regulatory receptor GPR116 showed that its mRNA expression was increased in response to dust and inhibited by blocking PKCα, implicating it as a means of inhibiting SP-D in the lungs in response to organic dusts. This reduction shows that organic barn dust can reduce lung SP-D, thus leaving workers potentially at risk for a host of pathogens.

Fig 1. Reduced expression of SP-D to HDE extracts in an in vivo mouse model system.

Mice exposed to HDE show slight increase in IL-6 and IL-8 and an influx of neutrophils (a). SP-A and SP-D protein in BAL (b) as well as SP-D mRNA (b) in lung tissue showed significant decreases with dust treatment. A549 cell line (c) treated for 24 hr with sterile barn hog barn dust (HDE) or grain dust extracts showed the same response in SP-D mRNA. Plethysmographic measurement of mice during exposure showed no significant change in breathing (PenH) (d). Bar graphs represent averages with error bars denoting SEM (N = 4 mice/group). Statistical significance denoted by asterisks (*p < 0.05, ***p < 0.001) as compared to respective media or saline treatment group.

Fig 2. SP-D is not inhibited by LPS or peptidoglycan, but by a heat labile fraction of barn dust.

A549 cells were exposed to HDE, LPS (100 EU/ml), peptidoglycan (10 μg/ml) or baked dust extract (5% v/v). Bar graphs represent averages with error bars denoting SEM (Cell cultures, N = 3 samples/group, repeated on three separate days). mRNA is given as fold change above media control levels. Statistical significance denoted by asterisks (**p < 0.01) as compared to respective media treatment group.

Fig 3

IL-6 and IL-8 response to barn dust is inhibited by SP-D. ELISA of A549 cells (a) or THP-1 cells (IL8 alone) (b) treated with SP-D (20 ng/ml) 1 hr prior to HDE exposure show a clear reduction in IL-8 expression 24 hr after HDE exposure. Bar graphs represent averages with error bars denoting SEM (Cell cultures, N = 3 samples/group, repeated on three separate days). mRNA is given as fold change above media control levels. Statistical significance denoted by asterisks (**p < 0.01) as compared to respective media treatment group.

Fig 4

PKCα, but not PKCɛ, is required for HDE-induced SP-D reduction (a). Inhibitors Gö6976 (PKCα) or Ro 31–8220 (PKCɛ were given 1 hr prior to treatment of cells with HDE for 24 hr. Bar graphs represent averages mRNA fold change above media control with error bars denoting SEM (Cell cultures, N = 3 samples/group, repeated on three separate days). (b) Inhibitor specificity of Gö6976 and Ro 31–8220 in PKCα and PKCɛ activity assays. Values given are in pmol/min/mg vs. media control. Statistical significance denoted by asterisks (*p < 0.05, ***p < 0.001) as compared to respective media treatment group.

Fig 5. GPR116 is increased by HDE exposure.

Treatment of A549 cells with HDE for 24 hr induced (a) a significant increase in GPR116 mRNA. Treatment of cells 1 hr prior HDE exposure with PKCα inhibitor Gö6976 was able to reverse this increase, but had no effect on unexposed cells. (b) mRNA from mice exposed to HDE for 24 hrs showed a similar increase. (c) Replacement of HDE with purified SP-D showed SP-D reduced GPR116 mRNA with no Gö6976 effect observed. Bar graphs represent averages with error bars denoting SEM (Cell cultures, N = 3 samples/group, repeated on three separate days. Mice, N = 4 mice/group). mRNA is given as fold change above media control levels. Statistical significance denoted by asterisks (*p < 0.05, **p < 0.01, ***p < 0.01) as compared to respective media treatment group.