RAGE and TLRs: relatives, friends or neighbours?

The innate immune system forms the first line of protection against infectious and non-infectious tissue injury. Cells of the innate immune system detect pathogen-associated molecular patterns or endogenous molecules released as a result of tissue injury or inflammation through various innate immune receptors, collectively termed pattern-recognition receptors. Members of the Toll-like receptor (TLR) family of pattern-recognition receptors have well established roles in the host immune response to infection, while the receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor predominantly involved in the recognition of endogenous molecules released in the context of infection, physiological stress or chronic inflammation. RAGE and TLRs share common ligands and signaling pathways, and accumulating evidence points towards their co-operative interaction in the host immune response. At present however, little is known about the mechanisms that result in TLR versus RAGE signalling or RAGE-TLR cross-talk in response to their shared ligands. Here we review what is known in relation to the physicochemical basis of ligand interactions between TLRs and RAGE, focusing on three shared ligands of these receptors: HMGB1, S100A8/A9 and LPS. Our aim is to discuss what is known about differential ligand interactions with RAGE and TLRs and to highlight important areas for further investigation so that we may better understand the role of these receptors and their relationship in host defense

Differential Effects of Allergen Challenge on Large and Small Airway Reactivity in Mice

<div><p>The relative contributions of large and small airways to hyperresponsiveness in asthma have yet to be fully assessed. This study used a mouse model of chronic allergic airways disease to induce inflammation and remodelling and determine whether <i>in vivo</i> hyperresponsiveness to methacholine is consistent with <i>in vitro</i> reactivity of trachea and small airways. Balb/C mice were sensitised (days 0, 14) and challenged (3 times/week, 6 weeks) with ovalbumin. Airway reactivity was compared with saline-challenged controls <i>in vivo</i> assessing whole lung resistance, and <i>in vitro</i> measuring the force of tracheal contraction and the magnitude/rate of small airway narrowing within lung slices. Increased airway inflammation, epithelial remodelling and fibrosis were evident following allergen challenge. <i>In vivo</i> hyperresponsiveness to methacholine was maintained in isolated trachea. In contrast, methacholine induced slower narrowing, with reduced potency in small airways compared to controls. <i>In vitro</i> incubation with IL-1/TNFα did not alter reactivity. The hyporesponsiveness to methacholine in small airways within lung slices following chronic ovalbumin challenge was unexpected, given hyperresponsiveness to the same agonist both <i>in vivo</i> and <i>in vitro</i> in tracheal preparations. This finding may reflect the altered interactions of small airways with surrounding parenchymal tissue after allergen challenge to oppose airway narrowing and closure.</p></div

Comparison of small airway responses to methacholine (MCh) in lung slices from saline (SAL)- and ovalbumin (OVA)-challenged mice.

<p>a) Representative images of MCh-induced airway narrowing. b) Representative traces showing the time course of changes in small airway lumen area. c) Average changes in % initial airway lumen area (SAL, OVA n = 9, 7). d) Contraction to 100 nM MCh over 5 min (SAL, OVA n = 4, 5). e) Lung slices cultured in the absence (open symbols) or presence (closed symbols) of IL-1α (10 ng/ml) and TNFα (50 ng/ml) for 48 h (paired tissues, SAL or OVA n = 4, 4) Data is expressed as % initial airway lumen area (mean ± SEM). *p<0.05 compared with appropriate controls.</p

Comparison of airway reactivity to methacholine (MCh) in saline- and ovalbumin-challenged mice (open circles and closed circles respectively).

<p>a) <i>In vivo</i> responses, measuring change in airway resistance (saline, ovalbumin n = 12, 8 respectively), b) <i>in vitro</i> responses in trachea, measuring change in force (saline, ovalbumin n = 18, 21) and c) <i>in vitro</i> responses in lung slices, measuring % decrease in small airway lumen area (saline, ovalbumin n = 9, 7). Data is expressed as mean ± S.E.M. *p<0.05 compared with saline controls.</p

Changes to airway wall morphology with chronic allergen challenge.

<p>Representative sections from mouse trachea (panels a, b) and lung (panels c–f). Following chronic challenge with saline (a, c, e) or ovalbumin (b, d, f), sections were stained with Masson’s trichrome stain for collagen (a, b, e, f) or Alcian blue-periodic acid Schiff to assess epithelial changes (c, d). Bar = 100 µm.</p

Bronchoalveolar Lavage Differential Cell Counts.

<p>Bronchoalveolar lavage (BAL) fluid was collected from saline- and ovalbumin (OVA)-challenged mice (n = 6/group) and assessed for total cellularity. Differential cell counts were made on cytospin preparations from each BAL fluid sample based on counts of 300 cells. Results are expressed as mean ± SEM. Statistical analysis of log-transformed data was conducted using unpaired t-tests.</p>***<p>p<0.001 compared with saline.</p

Comparison Of Methacholine Potency And Maximum In Trachea And Small Airways In Vitro.

<p>Airway reactivity to methacholine was assessed <i>in vitro</i> measuring increases in force in trachea and small airway narrowing in lung slices from saline- and ovalbumin (OVA)-challenged mice. Maximum responses and potency were obtained from fitted individual curves. Data is expressed as mean ± SEM.</p>*<p>p<0.05 compared with saline, unpaired t-test.</p

Comparison of small airway responses to methacholine (MCh) in lung slices from saline- and ovalbumin (OVA)-challenged mice under control and calcium-permeabilised conditions.

<p>Slices were treated with permeabilized to Ca²⁺ by simultaneous treatment with 20 mM caffeine and 50 µM ryanodine to open ryanodine receptors (RyR) in the sarcoplasmic reticulum of airway smooth muscle cells, depleting intracellular Ca²⁺ stores and clamping the intracellular Ca²⁺ concentration at extracellular levels. This abrogates subsequent caffeine contractions and MCh-induced Ca²⁺ oscillations, to permit the assessment of MCh responses due to RhoA/Rho kinase-mediated Ca²⁺ sensitisation alone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074101#pone.0074101-Bai1" target="_blank">[21]</a>. a) Representative trace showing response to 300 nM MCh under control and calcium-permeabilised conditions. b) Concentration-response to MCh (paired tissues, SAL or OVA n = 5, 3) expressed as % initial airway lumen area (mean ± SEM).</p