Intranasal Administration of poly(I:C) and LPS in BALB/c Mice Induces Airway Hyperresponsiveness and Inflammation via Different Pathways

BACKGROUND: Bacterial and viral infections are known to promote airway hyperresponsiveness (AHR) in asthmatic patients. The mechanism behind this reaction is poorly understood, but pattern recognizing Toll-like receptors (TLRs) have recently been suggested to play a role. MATERIALS AND METHODS: To explore the relation between infection-induced airway inflammation and the development of AHR, poly(I:C) activating TLR3 and LPS triggering TLR4, were chosen to represent viral and bacterial induced interactions, respectively. Female BALB/c or MyD88-deficient C57BL/6 mice were treated intranasally with either poly(I:C), LPS or PBS (vehicle for the control group), once a day, during 4 consecutive days. RESULTS: When methacholine challenge was performed on day 5, BALB/c mice responded with an increase in airway resistance. The maximal resistance was higher in the poly(I:C) and LPS treated groups than among the controls, indicating development of AHR in response to repeated TLR activation. The proportion of lymphocytes in broncheoalveolar lavage fluid (BALF) increased after poly(I:C) treatment whereas LPS enhanced the amount of neutrophils. A similar cellular pattern was seen in lung tissue. Analysis of 21 inflammatory mediators in BALF revealed that the TLR response was receptor-specific. MyD88-deficient C57BL/6 mice responded to poly (I:C) with an influx of lymphocytes, whereas LPS caused no inflammation. CONCLUSION: In vivo activation of TLR3 and TLR4 in BALB/c mice both caused AHR in conjunction with a local inflammatory reaction. The AHR appeared to be identical regardless of which TLR that was activated, whereas the inflammation exhibited a receptor specific profile in terms of both recruited cells and inflammatory mediators. The inflammatory response caused by LPS appeared to be dependent on MyD88 pathway. Altogether the presented data indicate that the development of AHR and the induction of local inflammation might be the result of two parallel events, rather than one leading to another

Prostaglandin modulation of airway hyperresponsiveness and inflammation in murine models of asthma

Allergic asthma is an inflammatory disease of the airways and is characterised by chronic inflammation, reversible bronchoconstriction and airway hyperresponsiveness (AHR). The cyclooxygenases (COXs) are key enzymes in the biosynthesis of prostaglandins, which have diverse physiological and pathological functions, and are also important modulators of inflammatory responses in the lung. However, the role of COX-derived prostanoids in AHR and airway inflammation is still not fully understood. The overall aim of this Thesis was to define the role of COX-derived products in allergic airway responses in experimental mouse models of asthma. In the present work, lung physiology was measured using the flexiVent® system for direct measurements of respiratory system mechanics. Inflammation was assessed by measuring numbers of inflammatory cells in bronchoalveolar lavage (BAL) fluid as well as levels of inflammatory mediators in BAL fluid and lung tissue. Histological analysis of the lung tissue was also performed to assess possible structural changes. The aim of the first part of this Thesis was to develop an improved protocol for experimental studies of AHR and airway inflammation in sensitised and challenged mice, in order to optimise the delivery route and doses of allergen for antigen challenge. The result established that intranasal challenge of BALB/c mice caused a dose-dependent increase in AHR, whereas the BAL fluid cell response was augmented to the same extent by all doses of allergen. Intranasal challenge causes a more pronounced induction of both AHR and inflammation in sensitised BALB/c mice, compared to aerosol delivery of the same antigen. The second part of this Thesis aimed to define the function of the prostaglandins in the mouse models of allergic airway inflammation. The role of the prostaglandins was investigated both by using intervention with non-steroidal anti-inflammatory drugs (NSAIDs), as well as genetically modified mice where the synthesis of prostaglandin (PG) E2 had been disrupted by removal of one of the prostaglandin E synthases (mPGES-1). The findings support the concept that in general, COX products have a bronchoprotective function in the airways, i.e. local release of PGE2 modulates the airway smooth muscle reactivity and protects against excessive airway narrowing. Furthermore, the bronchoprotective effects in allergic airway inflammation are mainly due to PGE2, primarily generated by mPGES-1. The two isoenzymes, COX-1 and COX-2, also appeared to have separate functions, whereby COX-1 predominantly generated the prostaglandins recovered in BAL fluid and COX-2 activity was associated with the accumulation of inflammatory cells in BAL fluid. It is not clear, which prostaglandin mediated the COX-2 dependent effects on cellular inflammation. However, the observations suggest that neither mPGES-1 nor PGE2 are involved in the inhibitory function of COX-2 on cell recruitment in the allergic airway reaction. Taken together, the present Thesis supports the concept that AHR and the inflammatory response are distinct, and at least in part, uncoupled events. Furthermore, the findings raise awareness that BAL fluid inflammation is not a predictive surrogate marker of AHR. Finally, inhibition of PGE2 has been suggested as a target for anti-inflammatory therapies in humans, as PGE2 is a pro-inflammatory mediator and is involved in many inflammatory diseases. However, it is possible that inhibition of mPGES-1-derived PGE2 may have negative consequences in the airways

Effects of deep inspirations (DI) in healthy mice; () lung resistance (R) in mice given incremental doses of methacholine (MCH group), and () the effect of DI on lung compliance (C) presented as ΔC

Values are mean ± SEM, * < 0.05, ** < 0.01, *** < 0.001.<p><b>Copyright information:</b></p><p>Taken from "Different effects of deep inspirations on central and peripheral airways in healthy and allergen-challenged mice"</p><p>http://respiratory-research.com/content/9/1/23</p><p>Respiratory Research 2008;9(1):23-23.</p><p>Published online 28 Feb 2008</p><p>PMCID:PMC2291047.</p><p></p

Representative histological sections (hematoxylin and eosin stained) from healthy control animals in the PBS'98 group (picture A and B) and from animals having undergone a 98-day ovalbumin challenge protocol, the OVA'98 group (picture C and D)

Examination of sections from OVA'98 animals revealed a significant inflammation surrounding the airways and within the alveolar spaces. PBS'98 did not show any signs of inflammation.<p><b>Copyright information:</b></p><p>Taken from "Different effects of deep inspirations on central and peripheral airways in healthy and allergen-challenged mice"</p><p>http://respiratory-research.com/content/9/1/23</p><p>Respiratory Research 2008;9(1):23-23.</p><p>Published online 28 Feb 2008</p><p>PMCID:PMC2291047.</p><p></p

Poly(I:C) and LPS induced cell recruitment in BALF.

<p>(A, C) Absolute and (B, D) relative cell numbers in BALF in BALB/c mice (A, B) respectively MyD88deficient C57BL/6 mice (C, D) treated during 4 days with vehicle (PBS), poly(I:C) or LPS i.n.. Results are expressed as mean ± SEM. *p<0.05.</p

Poly(I:C) and LPS induced cytokine release in BALF.

<p>Levels of (A) IL-5, (B) IL-12, (C) MCP-1, (D) KC, (E) MIG and (F) VEGF in BALB/c mice treated during 4 days with vehicle (white), poly(I:C) (black) or LPS (gray) i.n. Results are expressed as mean ± SEM. PBS n = 10; poly I:C n = 12; LPS n = 13; ***p<0.001.</p

Poly(I:C) and LPS induced cell recruitment in lung tissue.

<p>Haematoxylin and eosin stained cells in BALB/c mice treated i.n. during 4 days with (A) vehicle, (B) poly(I:C) (mainly neutrophils) and (C) LPS (mainly lymphocytes). Magnification is ×200. (D) Semi quantitative grading score of cell infiltration in the lung.</p

LPS and poly(I:C) induced airway hyperresponsiveness.

<p>(A) Changes in lung resistance (R_L) and (B) compliance (C_L) in response to methacholine in BALB/c mice treated during 4 days with vehicle (PBS), poly(I:C) or LPS i.n.. Results are expressed as mean ± SEM. *p<0.05, ***p<0.001 vs. PBS.</p