Yanira Riffo-Vasquez

Role of sex hormones in allergic inflammation in mice

Background A number of clinical studies have documented both a pro‐ and anti‐inflammatory role for sex hormones in the context of lung inflammation and worsening of asthma.

Role of cytokines and chemokines in bronchial hyperresponsiveness and airway inflammation.

Over the last decade there has been an intense interest in the potential role of cytokines and chemokines as important mediators in various atopic diseases, including asthma and the mechanisms by which these mediators regulate airway inflammation and bronchial hyperresponsiveness. This research effort has recently culminated in the publication of clinical studies that have assessed the role of interleukin (IL)-4 [Borish et al., Am J Respir Crit Care Med 160, 1816-1823 (1999)], IL-5 [Leckie et al., Lancet 356, 2144-2148 (2000)], and IL-12 [Bryan et al., Lancet 356, 2149-2153 (2000)] in allergic asthma, and the results have been disappointing. This is not surprising given the pleiotropic role cytokines play in the allergic response confirmed by numerous animal studies providing evidence of functional redundancy. The alternative view is that our current concepts in asthma pathogenesis need significant revision. This review will summarise the evidence for the role of cytokines and chemokines in various aspects of asthma pathophysiology; namely, bronchial hyperresponsiveness, eosinophil recruitment to the airways, mucus secretion, and airway remodelling.

TRPV1 Deletion Enhances Local Inflammation and Accelerates the Onset of Systemic Inflammatory Response Syndrome

The transient receptor potential vanilloid 1 (TRPV1) is primarily localized to sensory nerve fibers and is associated with the stimulation of pain and inflammation. TRPV1 knockout (TRPV1KO) mice show enhanced LPS-induced sepsis compared with wild type (WT). This implies that TRPV1 may have a key modulatory role in increasing the beneficial and reducing the harmful components in sepsis. We investigated immune and inflammatory mechanisms in a cecal ligation and puncture (CLP) model of sepsis over 24 h. CLP TRPV1KO mice exhibited significant hypothermia, hypotension, and organ dysfunction compared with CLP WT mice. Analysis of the inflammatory responses at the site of initial infection (peritoneal cavity) revealed that CLP TRPV1KO mice exhibited: 1) decreased mononuclear cell integrity associated with apoptosis, 2) decreased macrophage tachykinin NK1-dependent phagocytosis, 3) substantially decreased levels of nitrite (indicative of NO) and reactive oxygen species, 4) increased cytokine levels, and 5) decreased bacteria clearance when compared with CLP WT mice. Therefore, TRPV1 deletion is associated with impaired macrophage-associated defense mechanisms. Thus, TRPV1 acts to protect against the damaging impact of sepsis and may influence the transition from local to a systemic inflammatory state.

Effect of Mycobacterium tuberculosis chaperonins on bronchial eosinophilia and hyper-responsiveness in a murine model of allergic inflammation

Background Epidemiological evidence suggests that infection with Mycobacterium tuberculosis protects children against asthma. Several laboratories have shown that, in mouse models of allergic inflammation, administration of the whole live tuberculosis vaccine, Mycobacterium bovis bacillus Calmette–Guerin (BCG), prevents ovalbumin (OVA)‐induced pulmonary eosinophilia.

Comparison of the Moonlighting Actions of the Two Highly Homologous Chaperonin 60 Proteins of Mycobacterium tuberculosis

ABSTRACT Evidence is emerging that the two chaperonin (Cpn) 60 proteins of Mycobacterium tuberculosis, Cpn60.1 and Cpn60.2, have moonlighting actions that may contribute to the pathology of tuberculosis. We studied the release of Cpn60.1 from M. tuberculosis and infected macrophagelike cells and compared recombinant Cpn60.1 and Cpn60.2 in a range of cell-based assays to determine how similar the actions of these highly homologous proteins are. We now establish that Cpns are similar as follows: (i) Cpn60.1, as it has been shown for Cpn60.2, is released by M. tuberculosis in culture, and Cpn60.1 is furthermore released when the bacterium is in quiescent, but not activated, macrophagelike cells, and (ii) both proteins only showed a partial requirement for MyD88 for the induction of proinflammatory cytokine production compared to lipopolysaccharide. However, we also found major differences in the cellular action of Cpns. (i) Cpn60.2 proved to be a more potent stimulator of whole blood leukocytes than Cpn60.1 and was the only one to induce tumor necrosis factor alpha synthesis. (ii) Cpn60.1 bound to ca. 90% of circulating monocytes compared to Cpn60.2, which bound <50% of these cells. Both chaperonins bound to different cell surface receptors, while monocyte activation by both proteins was completely abrogated in TLR4−/− mice, although Cpn60.2 also showed significant requirement for TLR2. Finally, an isogenic mutant lacking cpn60.1, but containing intact cpn60.2, was severely inhibited in generating multinucleate giant cells in an in vitro human granuloma assay. These results clearly show that, despite significant sequence homology, M. tuberculosis Cpn60 proteins interact in distinct ways with human or murine macrophages.

Quantitative assessment of nanoparticle surface hydrophobicity and its influence on pulmonary biocompatibility.

To date, the role of nanoparticle surface hydrophobicity has not been investigated quantitatively in relation to pulmonary biocompatibility. A panel of nanoparticles spanning three different biomaterial types, pegylated lipid nanocapsules, polyvinyl acetate (PVAc) and polystyrene nanoparticles, were characterized for size, surface charge, and stability in biofluids. Surface hydrophobicity of five nanoparticles (50-150nm) was quantified using hydrophobic interaction chromatography (HIC) and classified using a purpose-developed hydrophobicity scale: the HIC index, range from 0.00 (hydrophilic) to 1.00 (hydrophobic). This enabled the relationship between the nanomaterial HIC index value and acute lung inflammation after pulmonary administration to mice to be investigated. The nanomaterials with low HIC index values (between 0.50 and 0.64) elicited little or no inflammation at low (22cm(2)) or high (220cm(2)) nanoparticle surface area doses per animal, whereas equivalent surface area doses of the two nanoparticles with high HIC index values (0.88-0.96) induced neutrophil infiltration, elevation of pro-inflammatory cytokines and adverse histopathology findings. In summary, a HIC index is reported that provides a versatile, discriminatory, and widely available measure of nanoparticle surface hydrophobicity. The avoidance of high (HIC index>~0.8) surface hydrophobicity appears to be important for the design of safe nanomedicines for inhalation therapy.

Cytokines in airway inflammation

With over 50 potential asthma mediators, cytokines are the latest group of substances which have been investigated for their potential role in this disease. The use of murine models of allergic inflammation has facilitated the investigation of the role of individual cytokines in this response. The use of targeted gene disruption, overexpression of genes and monoclonal antibodies directed against cytokines have allowed a detailed examination of the role cytokines play in IgE production, eosinophil recruitment and bronchial hyperresponsiveness, which are the characteristic features of the asthma phenotype. Despite the introduction of this new methodology, conflicting reports relating to the role of cytokines in allergic inflammation, highlight the complexity of allergic inflammation and challenge the notion that a single cytokine can explain the asthma phenotype.

Pulmonary Inflammation Impacts on CYP1A1- Mediated Respiratory Tract DNA Damage Induced by the Carcinogenic Air Pollutant Benzo(a)pyrene

Pulmonary inflammation can contribute to the development of lung cancer in humans. We investigated whether pulmonary inflammation alters the genotoxicity of polycyclic aromatic hydrocarbons (PAHs) in the lungs of mice and what mechanisms are involved. To model nonallergic acute inflammation, mice were exposed intranasally to lipopolysaccharide (LPS; 20 µg/mouse) and then instilled intratracheally with benzo[a]pyrene (BaP; 0.5 mg/mouse). BaP-DNA adduct levels, measured by 32P-postlabeling analysis, were approximately 3-fold higher in the lungs of LPS/BaP-treated mice than in mice treated with BaP alone. Pulmonary Cyp1a1 enzyme activity was decreased in LPS/BaP-treated mice relative to BaP-treated mice suggesting that pulmonary inflammation impacted on BaP-induced Cyp1a1 activity in the lung. Our results showed that Cyp1a1 appears to be important for BaP detoxification in vivo and that the decrease of pulmonary Cyp1a1 activity in LPS/BaP-treated mice results in a decrease of pulmonary BaP detoxification, thereby enhancing BaP genotoxicity (ie, DNA adduct formation) in the lung. Because less BaP was detoxified by Cyp1a1 in the lungs of LPS/BaP-treated mice, more BaP circulated via the blood to extrapulmonary tissues relative to mice treated with BaP only. Indeed, we observed higher BaP-DNA adduct levels in livers of LPS/BaP-treated mice compared with BaP-treated mice. Our results indicate that pulmonary inflammation could be a critical determinant in the induction of genotoxicity in the lung by PAHs like BaP. Cyp1a1 appears to be involved in both BaP bioactivation and detoxification although the contribution of other enzymes to BaP-DNA adduct formation in lung and liver under inflammatory conditions remains to be explored.

A role for MC3R in modulating lung inflammation

In this study we set out to ascertain whether melanocortin peptides could be potential therapeutic agents in allergic and non-allergic models of lung inflammation by identifying the receptor(s) involved using a molecular, genetic and pharmacological approach. Western blot analyses revealed expression of the melanocortin receptor (MCR) type 1 and 3 on alveolar macrophages from wild-type mice. Alveolar macrophage incubation, with the selective MC3R agonist [D-TRP(8)]-gamma-MSH and pan-agonist alpha-MSH but not the selective MC1R agonist MS05, led to an increase in cAMP in wild-type macrophages. This increase occurred also in macrophages taken from recessive yellow (e/e; bearing a mutant and inactive MC1R) mice but not from MC3R-null mice. In an allergic model of inflammation, the pan-agonist alpha-MSH and selective MC3R agonist [D-TRP(8)]-gamma-MSH displayed significant attenuation of both eosinophil and lymphocyte accumulation but not IL-5 levels in wild-type and recessive yellow e/e mice. However in MC3R-null mice, alpha-MSH failed to cause a significant inhibition in these parameters, highlighting a preferential role for MC3R in mediating the anti-inflammatory effects of melanocortins in this model. Utilising a non-allergic model of LPS-induced lung neutrophilia, the pan-agonist alpha-MSH and selective MC3R agonist [D-TRP(8)]-gamma-MSH displayed significant attenuation of neutrophil accumulation and inhibition of TNF-alpha release. Thus, this study highlights that melanocortin peptides inhibit leukocyte accumulation in a model of allergic and non-allergic inflammation and this protective effect is associated with activation of the MC3R. The inhibition of leukocyte accumulation is via inhibition of TNF-alpha in the non-allergic model of inflammation but not IL-5 in the allergic model. These data have highlighted the potential for selective MC3R agonists as novel anti-inflammatory therapeutics in lung inflammation.

P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of inflammation

The small GTPase Rac is required for neutrophil recruitment during inflammation, but its guanine-nucleotide exchange factor (GEF) activators seem dispensable for this process, which led us to investigate the possibility of cooperation between Rac-GEF families. Thioglycollate-induced neutrophil recruitment into the peritoneum was more severely impaired in P-Rex1(-/-) Vav1(-/-) (P1V1) or P-Rex1(-/-) Vav3(-/-) (P1V3) mice than in P-Rex null or Vav null mice, suggesting cooperation between P-Rex and Vav Rac-GEFs in this process. Neutrophil transmigration and airway infiltration were all but lost in P1V1 and P1V3 mice during lipopolysaccharide (LPS)-induced pulmonary inflammation, with altered intercellular adhesion molecule 1-dependent slow neutrophil rolling and strongly reduced L- and E-selectin-dependent adhesion in airway postcapillary venules. Analysis of adhesion molecule expression, neutrophil adhesion, spreading, and migration suggested that these defects were only partially neutrophil-intrinsic and were not obviously involving vascular endothelial cells. Instead, P1V1 and P1V3 platelets recapitulated the impairment of LPS-induced intravascular neutrophil adhesion and recruitment, showing P-Rex and Vav expression in platelets to be crucial. Similarly, during ovalbumin-induced allergic inflammation, pulmonary recruitment of P1V1 and P1V3 eosinophils, monocytes, and lymphocytes was compromised in a platelet-dependent manner, and airway inflammation was essentially abolished, resulting in improved airway responsiveness. Therefore, platelet P-Rex and Vav family Rac-GEFs play important proinflammatory roles in leukocyte recruitment.