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TLR ligands and other allergen-nonspecific immunostimulatory molecules are ubiquitous in ambient air and have profound modulatory activities in animal models of allergic asthma. However, several of these molecules have been shown to promote exaggerated Th2-biased airway hypersensitivity responses (AHRs), whereas others attenuate the asthmatic phenotype. Therefore, it has proven difficult to extrapolate experimental results with purified molecules toward a more general understanding of the allergen-nonspecific immunomodulatory influence of living environments on the natural history of allergic asthma. These investigations determined how regular and intermittent airway exposures to an unpurified, but sterile house dust extract standard (HDEst) affected the OVA-specific AHR and immune status of previously Th2-sensitized mice. Low-dose daily and high-dose intermittent HDEst exposures modulated ongoing AHRs considerably, reducing eosinophil recruitment and methacholine responsiveness, while increasing neutrophilic inflammation. However, only daily airway delivery of low-dose HDEst attenuated OVA-specific Th2 cytokine production and Th2-biased AHRs to allergen challenge 1 mo later. Finally, whereas LPS mimicked many of the immunomodulatory characteristics of HDEst in this murine asthma model, daily airway HDEst delivery was highly effective in attenuating the AHR of OVA/alum-sensitized TLR4-deficient mice. Taken together, these investigations provide direct evidence that living environments contain allergen-nonspecific immunostimulatory molecules that influence the airway hypersensitivity status of allergen-sensitized mice by TLR4-dependent and independent mechanisms.

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.

Endotoxin and other immunostimulants ubiquitous in ambient air are potent mucosal adjuvants, yet only a minority of individuals develop aeroallergen hypersensitivities, whereas the majority develop tolerance. These investigations were performed to reconcile this paradox. During initial experiments, mice received a primary series of weekly intranasal OVA immunizations (1(0) vaccination). Selected mice also received intranasal sterile house dust extract (HDE) with each OVA vaccination, at a dose previously found to exert adjuvant activity. A third group of OVA-vaccinated mice received intranasal HDE on a daily basis, but at one seventh the adjuvant dose, beginning 1 week before the first and ending with the last 1(0) OVA vaccination. Mice were then left untreated for 4 weeks, and then received a secondary series of weekly intranasal OVA immunizations with adjuvant doses of HDE (2(0) sensitization). Three weeks later, OVA-specific airway challenges and immune responses were assessed. Analogous experiments were conducted with LPS. Mice receiving daily intranasal HDE or LPS during 1(0) OVA vaccination were highly resistant to 2(0) sensitization, whereas the mice in other experimental groups readily developed Th2-biased airway hypersensitivity. Tolerance was associated with poor OVA-specific CD4 cell proliferation and with local natural T-regulatory cell (Treg) expansion. Finally, Treg depletion by delivery of the anti-CD25 monoclonal antibody during 1(0) vaccination attenuated the tolerogenic effects of daily airway HDE exposures. These studies suggest that regular airway immunostimulant exposures selectively increase local Treg numbers and activity in an antigen-independent manner, thereby promoting the development of aeroallergen tolerance.