Using house dust extracts to understand the immunostimulatory activities of living environments

Abstract

Laboratory and epidemiological studies have provided indirect but compelling evidence that toll-like receptor (TLR) signaling pathways play an important role in host responsiveness to ambient immunostimulatory factors. Nonetheless, direct evidence is limited. This paper will present our experience investigating the innate immunostimulatory activities of sterile house dust extracts (HDEs). In initial studies, bone marrow derived dendritic cells (BMDDCs) were cultured with HDEs, and cytokine production and co-stimulatory molecule expression were evaluated. In additional experiments, the TLR dependence of these responses was determined. HDEs induced concentration-dependent BMDDC activation. Moreover, the relative bioactivities of HDEs correlated with their endotoxin content. Finally, HDE-mediated responses were found to be partially dependent on TLR2, TLR4, and TLR9 and almost completely dependent on MyD88. These investigations provide the first direct evidence that TLR signaling pathways play a key role in innate responsiveness to non-infectious factors ubiquitous in living environments.

Fig. 1

HDEs induce BMDDC cytokine production. BALB/c BMDDCs were cultured at 1 × 10⁶ cells/ml with P-3-C (5 μg/ml), LPS (100 ng/ml), ISS (10 μg/ml), R848 (1 μg/ml), or HDEs (n = 15) for 24 h before supernatants were harvested for cytokine ELISA. HDE preparation and other methodological details for these experiments have been described previously. Presented results are reflective of 3 or more experiments (*P ≤ 0.05 versus unstimulated BMDDCs). For statistical analyses, results with individual HDEs were combined. (A) IL-6 and IL-12p40 production. (B) IL-12p70 production. HDEs were used at 1 mg/ml in these experiments.

Fig. 2

Associations between HDE endotoxin levels and bioactivities. BMDDCs were cultured with HDEs (0.1 mg/ml), as in Fig. 1 and HDE endotoxin levels were measured. Individual HDE endotoxin levels were then plotted against the cytokine responses induced by those HDEs (0.1 mg/ml). R = correlation coefficient.

Fig. 3

HDE induced BMDDC responses are TLR dependent. TLR2, TLR4, TLR9, MyD88 ko and WT (C157/B6) BMDDCs were cultured with purified TLR ligands or HDEs (n = 10) with high bioactivities, as in Fig. 1. HDE results are presented as means ± standard errors (* P ≤ 0.05 for WT versus KO BMDDCs). (A) WT versus TLR4 ko mice. (B) WT versus TLR2 ko mice. (C) WT versus TLR9 ko mice. (D) WT versus MyD88 ko mice.