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Immune responses during lung infections must be tightly regulated in order to permit pathogen eradication while maintaining organ function. Although mechanisms involve complex networks of cytokines, the interferon (IFN) response has been shown to be an important driver of lung inflammation. Type I IFNs consist of a group of structurally similar cytokines that are produced during virus infection and are an integral part in regulating the immune response. However, in response to certain stimuli, type I IFNs have also been found to be central in the initiation of lung inflammatory responses by inducing the recruitment and activation of immune cells and thus may contribute to disease severity. Another cytokine that has been associated with chronic lung inflammation is the gp130 cytokine, Oncostatin M. Transient pulmonary overexpression of Oncostatin M by Adenovirus vector (AdOSM) induces lung inflammation biased toward Th2 cytokines associated with eosinophilia and alternatively activated (AA/M2) macrophage accumulation. Here we demonstrated that C57Bl/6 mice deficient of the type I interferon receptor (IFNAR1-/-), were less responsive against a suboptimal dose of AdOSM at day 7 post infection compared to AdOSM-treated wild-type. We observed a significant reduction in OSM mRNA and protein levels in AdOSM-treated IFNAR1-/- mice, compared to treated wild-type, which resulted in significant attenuation in OSM-induced inflammatory cell infiltration, epithelial hyperplasia, and alveolar wall thickening. Furthermore, IL-6 overexpression (as a comparator gp130 cytokine), induced lymphocyte accumulation in IFNAR1-/- mice, but at significantly lower levels than AdIL-6-treated wild-type. These results demonstrate that cross talk between IFNAR and gp130 cytokine signaling were required for maximal AdOSM- and AdIL-6-mediated pulmonary inflammation. We also observed that IFNAR1 deficiency directly and negatively regulated OSM-mediated responses in vitro. OSM-induced pSTAT3 levels were consistently lower in murine and human IFNAR1-deficient fibroblasts, compared to OSM-stimulated wild-type cells. This was associated with diminished OSM-induced IL-6 and MCP-1 production from IFNAR1-/- fibroblasts. Furthermore, we found that the combination of OSM and IFN-α led to increased IL-6 production from C57Bl/6 and BALB/c-derived Mouse Lung Fibroblasts (MLFs) then when either cytokine was used alone suggesting that these two cytokines can work in concert. Our findings are the first to suggest that IFNAR signaling participates in OSM-mediated responses in vitro and is required for maximal AdOSM-induced pulmonary inflammation in vivo. / Thesis / Master of Science (MSc)

Alterations to sphingolipid metabolism are associated with increased pulmonary inflammation, but the impact of inflammatory mediators, such as neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. NE is a protease associated CF lung disease progression, and can be found in up to micromolar concentrations in patient airways. While sphingolipids have been investigated in the context of CF, the focus has been on loss of cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we present a novel observation: oropharyngeal aspiration of NE increases airway ceramides in mice. Using a previously characterized mouse model of NE-induced inflammation, we demonstrate that NE increases de novo ceramide production, which is likely mediated via increased SPTLC2 levels. Inhibition of de novo sphingolipid synthesis using myriocin, an SPT inhibitor, decreases airway ceramide as well as the release of pro-inflammatory signaling molecules induced by NE. Furthermore, in a retrospective study of the sphingolipid content of CF sputum—the largest of its type in this patient cohort to date, we investigated the association between NE and sphingolipids. There were linear correlations between the concentration of active NE and ceramide, sphingomyelin, and monohexosylceramide moieties as well as sphingosine-1-phosphate. The presence of Methicillin-resistant Staphylococcus aureus (MRSA) positive culture and female gender both strengthened the association of NE and sphingolipids, but higher FEV1 % predicted weakened the association, and Pseudomonas aeruginosa had no effect on the association between NE and sphingolipids. These data suggest that NE may increase sphingolipids in CF airways as it did in our in vivo model, and that this association is stronger in patients that have worse lung function, are female, and whose lungs are colonized with MRSA. Modulating sphingolipid homeostasis could provide novel pharmacological approaches for alleviating pulmonary inflammation.

Rationnel : Le développement des techniques de reperfusion pulmonaire ex vivo (Ex vivo lung perfusion : EVLP) vise à réduire la pénurie d'organes disponibles pour la transplantation. Le monoxyde de carbone (CO) pourrait aider à la sélection des greffons en cours d'EVLP puisque sa production endogène est augmentée en situation d'ischémie reperfusion. Objectifs : Mettre au point une technique de mesure du CO exhalé (eCO) lors de l'EVLP et étudier ses variations en fonction des lésions d'ischémie reperfusion. Méthodes : À partir d'un modèle porcin d'EVLP et à l'aide d'un appareil de spectrométrie laser basé sur le principe d'une cavité résonnante (optical-feedback cavity-enhanced absorption spectroscopy : OF-CEAS), eCO était mesuré selon différents paramètres ventilatoires, en ambiances polluées par des gaz pauvres (< 0,015ppmv) ou riches (9ppmv) en CO et après perfusion d'un inhibiteur de l'hème oxygénase (SnPP). eCO était ensuite comparé après 30 min (J0) ou 24 h (J1) d'ischémie froide et sa valeur prédictive pour sélectionner des greffons était calculée.Résultats : La concentration d'eCO de poumons isolés était de 0,45 ± 0.19 ppmv. Les pics d'eCO se produisaient pendant la phase expiratoire. Ni les variations de fraction inspirée en oxygène, ni la pollution de l'air ambiant ne modifiaient eCO. Les concentrations d'eCO n'étaient pas différentes après perfusion de SnPP. eCO était plus élevé à J1 par rapport à J0 (1,35 ± 0,259 ppmv vs. 0,951 ± 0,313, p = 0,01) et était corrélé à un indice de perméabilité de la membrane alvéolo capillaire. La meilleure valeur seuil d'eCO déterminée à partir de la courbe ROC était 0,860 ppmv (sensibilité de 1 (0,31 – 1), spécificité de 0,44 (0,15 – 0,77), valeur prédictive positive de 0,37 (0,10 – 0,74), valeur prédictive négative de 1 (0,39 – 1)). Conclusions : La mesure d'eCO en condition d'EVLP est faisable. eCO est significativement augmenté lorsque les lésions d'ischémie reperfusion sont accrues mais eCO ne peut pas être utilisé de façon isolée pour sélectionner les greffons pulmonaires. / Rationale: Ex vivo lung perfusion (EVLP) is a promising technique to reduce the shortage of organs available for transplantation. Carbon monoxide (CO) might help in the selection of grafts during EVLP as its endogenous production is increased by ischemia reperfusion.Objectives: To develop a measurement technique of exhaled CO (eCO) during EVLP and to study its variations depending on ischemia reperfusion injuries.Methods: Using a pig model of EVLP and using a laser spectrometer technique based on the principle of a resonant cavity (optical feedback cavity-enhanced absorption spectroscopy-OF-CEAS), we measured eCO under different ventilatory parameters, various polluted environments with poor (<0,015ppmv) or rich (9ppmv) CO gas, and after infusion of an inhibitor of heme oxygenase (SnPP). We then compared eCO after 30 min (D0) or 24 h (D1) of cold ischemia and determined the predictive value of eCO to select lung grafts.Results: In isolated lungs, the concentration of eCO reached 0.45 ± 0.19 ppmv. eCO peaks during the expiratory phase. Neither variations of the fraction of inspired oxygen, nor the pollution of the ambient air altered eCO. eCO concentrations were not different after SnPP infusion. eCO was higher on day 1 compared to day 0 (1.35 ± 0.259 vs. 0.951 ± 0.313 ppmv, p = 0.01) and correlated with an index of the permeability of the alveolar capillary membrane. The best treshold value of eCO determined from the ROC curve was 0.860 ppmv (sensitivity 1 (0.31 to 1), specificity of 0.44 (0.15 to 0.77), positive predictive value of 0, 37 (0.10 to 0.74), negative predictive value of 1 (0.39 to 1)).Conclusions: Measurements of eCO during EVLP is feasible. eCO is significantly higher when ischemia-reperfusion injuries are increased. However, eCO can not be used in isolation to select lung grafts.

Le recrutement des neutrophiles qui caractérise l’inflammation observée lors de différentes pathologies pulmonaires conduit à la libération dans le milieu extracellulaire de protéases à sérine qui sont en partie responsables de la dégradation du tissu pulmonaire et/ou de la chronicité de l’inflammation. L’objectif initial de cette thèse était de développer une méthode de quantification de ces protéases, à partir des condensats d’air exhalé. En dépit de la sensibilité de la technique nous n’avons pas été en mesure de détecter des quantités significatives de protéases actives dans ces condensats. Ces résultats négatifs ont néanmoins permis de confirmer des hypothèses sur la distribution des protéases dans le milieu extracellulaire. La deuxième partie des travaux a été consacrée à la validation du modèle souris exposée à la fumée de cigarette comme modèle animal de bronchopneumopathie chronique obstructive. Nous avons purifié les trois protéases à sérine du neutrophile murin et avons construit des nouveaux substrats sensibles et spécifiques à partir des informations fournies par des études de modélisation moléculaire. Ces nouveaux outils permettent de valider l’utilisation du modèle souris pour comprendre le rôle des protéases à sérine dans la génération de peptides chimiotactiques au cours de la BPCO. / Neutrophils recruitment is a hallmark of the inflammation associated with different lung diseases. This recruitment leads to the release in the extracellular matrix of serine proteases that are responsible at least in part, of the degradation of the pulmonary tissue and/or of the chronicity of inflammation. The initial objective of this thesis was to develop a method of quantification of these proteases, based on the analysis of exhaled air condensate. But in spite of the sensitivity of the methods, we have not been able to detect any significant activity of neutrophil serine proteases in these condensates. This negative result however gave support a hypothesis we formulated on the extracellular biodistribution of proteases in lung secretions. The second part of this thesis was devoted to the validation of an animal model of chronic obstructive pulmonary disease, i.e. the mouse exposed to cigarette smoke. We have purified three murine serine neutrophil proteases and developed new sensitive and specific FRET substrates which were designed starting from molecular modeling studies. These new tools that validate the use of the mouse model of human COPD, will be of great help to understand the role of serine proteases for generating chemotactic peptides during this chronic disease.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2015-07-21T14:58:44Z No. of bitstreams: 1 Manoel Carneiro de Oliveira Junior.pdf: 4260915 bytes, checksum: ba3950686530a475258ace49a0675cdd (MD5) / Made available in DSpace on 2015-07-21T14:58:45Z (GMT). No. of bitstreams: 1 Manoel Carneiro de Oliveira Junior.pdf: 4260915 bytes, checksum: ba3950686530a475258ace49a0675cdd (MD5) Previous issue date: 2013-09-30 / Acute respiratory distress syndrome (ARDS) is a syndrome that presents high mortality rates, and the results of both insults pulmonary or extra-pulmonary (pneumonia or septic shock) are high, and is a disease characterized by respiratory insufficiency from the inflammatory response that leads to alteration of alveolar-capillary permeability, pulmonary edema and hypoxemia refractory to high flow oxygen. One of the most important mechanisms that determined the severity of this injury is the magnitude of the injury of alveolar epithelial barrier. The possibility of repairing epithelial at an early stage is the major determinant of recovery. Many of therapeutic modalities based on the attempt to decrease lung inflammation to minimize the initial injury and much of the inflammatory process occurs through activation of local and systemic cytokines such as TNF-α and IL-1β. A growing number of studies report that Low Level Laser Therapy (LLLT) have anti-inflammatory effects in models of LPS-induced pulmonary ARDS, however, so far, only the red spectrum lasers were studied. Therefore, this study aimed to investigate the role of infra red laser (830nm), 3J/cm2, 35mw, 80 seconds per point (03 points per application), in pulmonary inflammation, lung using LPS model (intratracheal) and also extrapulmonary (intraperitoneal) inducing ARDS. The laser application was performed directly in contact with the skin in the chest three points (corresponding to the end of the trachea - Section 01 right lung - point 02 and left lung - point 03), three times, beginning 01 hour after LPS administration. BALB / c mice (n = 40) were divided into control (n = 08; not administered LPS), IT (n = 07; intratracheal administered LPS (10 µg / mouse), IT + LLLT (n = 09; intratracheal LPS administered (10 µg / mouse) + LLLT), IP (n = 07; LPS administered intraperitoneal (100 µg / mouse), IP + LLLT (n = 09; administered intraperitoneal LPS (100 µg / mouse) + LLLT). Twenty-four hours after administration of LPS and Laser, animals were euthanized and the lungs removed for studies of pulmonary inflammation: Total cell count and differential, bronchoalveolar lavage (BAL), cytokines (IL-1beta, IL-6, IL-10, KC and TNF-α), BAL levels were also analyzed quantitatively the number of neutrophils in the lung parenchyma in lung tissue using histomorphometry techniques. Results showed that LLLT significantly reduced pulmonary and extra-pulmonary LPS induced in both configurations Experimental of ARDS, as evidenced by a reduction in the number of total cells and neutrophils in BAL, reduced levels of IL-1β, IL-6, KC, and TNF-α in BAL fluid as well as the number of neutrophils in the lung parenchyma. Therefore, we conclude that the 830nm infrared laser is effective in reducing pulmonary inflammation in both models pulmonary or extrapulmonary LPS-induced experimental ARDS. / A síndrome do desconforto respiratório agudo (SDRA) é uma síndrome que apresenta altas taxas de mortalidade, que pode ser resultante tanto de insultos pulmonares como extrapulmonares. A síndrome é caracterizada pela insuficiência respiratória proveniente da resposta inflamatória que cursa com alteração de permeabilidade alvéolo-capilar, edema e hipoxemia refratária aos altos fluxos de oxigênio. Um dos mais importantes mecanismos que determinam a severidade desta injúria é a magnitude da lesão da barreira epitélio alveolar. A possibilidade de reparação do epitélio em um estágio precoce é o maior determinante da recuperação. Muitas das modalidades terapêuticas baseiam-se na tentativa de diminuição da inflamação pulmonar para minimizar a lesão inicial, a qual se deve em grande parte ao processo inflamatório mediado pela ativação local e sistêmica por citocinas como TNF-α e IL-1β. Um número crescente de estudos relata que o laser de baixa intensidade apresenta efeitos antiinflamatórios em modelos de SDRA induzida por LPS e isquemia e reperfusão da artéria pulmonar. No entanto, até o momento, apenas lasers no espectro vermelho (650 – 655 nm) foram estudados. Portanto, o presente estudo tem como objetivo investigar o papel do laser de baixa intensidade (LBI), na faixa do infravermelho (830nm), 3J/cm2, 35mw, 80 segundos por ponto (03 pontos por aplicação), na inflamação pulmonar, usando um modelo de SDRA de origem pulmonar (LPS intratraqueal) e também extrapulmonar (LPS intraperitoneal). A aplicação do laser foi realizada diretamente em contato com a pele, em três pontos do tórax (correspondente ao final da traquéia - ponto 01, pulmão direito - ponto 02 e do pulmão esquerdo ponto 03), por três vezes, 01 hora após a administração de LPS. Camundongos BALB/c (n = 40) machos foram distribuídos em Controle (n = 08; não administrado com LPS), IT 10 (n = 07; LPS intratraqueal; 10 µg/camundongo), IT + Laser (n = 09; LPS intratraqueal; 10µg/camundongo + Laser), IP (n= 07; LPS intraperitoneal; 100µg/ camundongo), IP + Laser (n = 09; LPS intraperitoneal; 100 µg/camundongo + Laser). Os animais foram eutanaziados vinte e quatro horas após a administração de LPS. Foi avaliada a contagem de células totais e diferenciais no lavado bronco alveolar (LBA), os níveis de citocinas (IL-1β, IL-6, IL 10, KC e TNF-α), a densidade de neutrófilos no parênquima pulmonar. Os resultados demonstraram que o LBI significativamente reduziu o número de células totais e de neutrófilos no Lavado Bronco Alveolar (LBA), o número de neutrófilos no parênquima pulmonar, e os níveis de citocinas pró-inflamatórias no LBA tanto no modelo de SDRA pulmonar quanto extrapulmonar. Portanto, concluímos que o laser infravermelho 830nm é eficaz para reduzir a inflamação pulmonar, em ambos os modelos de SDRA intrapulmonar e extrapulmonar induzida por LPS.

Mechanical ventilation (MV) is widely used in spinal injury patients to compensate for respiratory muscle failure. MV is known to induce lung inflammation, while spinal cord injury (SCI) is known to contribute to local inflammatory response. Interaction between MV and SCI was evaluated in order to assess the impact it may have on the pulmonary inflammatory profile. Sprague Dawley rats were anesthetized for 24 h and randomized to receive either MV or not. The MV group included C4–C5 SCI, T10 SCI and uninjured animals. The nonventilated (NV) group included T10 SCI and uninjured animals. Inflammatory cytokine profile, inflammation related to the SCI level, and oxidative stress mediators were measured in the bronchoalveolar lavage (BAL). The cytokine profile in BAL of MV animals showed increased levels of TNF-α, IL-1β, IL-6 and a decrease in IL-10 (P = 0.007) compared to the NV group. SCI did not modify IL-6 and IL-10 levels either in the MV or the NV groups, but cervical injury induced a decrease in IL-1β levels in MV animals. Cervical injury also reduced MV-induced pulmonary oxidative stress responses by decreasing isoprostane levels while increasing heme oxygenase-1 level. The thoracic SCI in NV animals increased M-CSF expression and promoted antioxidant pulmonary responses with low isoprostane and high heme oxygenase-1 levels. SCI shows a positive impact on MV-induced pulmonary inflammation, modulating specific lung immune and oxidative stress responses. Inflammation induced by MV and SCI interact closely and may have strong clinical implications since effective treatment of ventilated SCI patients may amplify pulmonary biotrauma.

The receptor for advanced glycation end-products (RAGE) is a transmembrane cell surface protein of the immunoglobulin superfamily that acts as part of both the innate and adaptive immune system. RAGE is highly expressed in lung tissue and is therefore of interest in the pulmonary immune response. Specifically, RAGE mediates several cell-signaling responses such as inflammation and apoptosis. This work sought to elucidate the role of RAGE in the setting of chronic pulmonary irritation such as that found in long-term exposure to secondhand smoke (SHS). This irritation has several shared characteristics with lung diseases such as chronic obstructive pulmonary disease (COPD) and is therefore of value in discovering potential mechanistic targets for future therapeutic treatments for exacerbations of the disease. We validated the extracellular signal-regulated kinase (ERK) pathway as a downstream RAGE cascade to activate the cellular transcription factor NF-B and excluded the protein kinase B (AKT) pathway in chronic pulmonary inflammation. We also identified several proinflammatory cytokines mediated by RAGE in long-term SHS exposure; these included increased expression of TNF-, MIP-1, IL-13, and IFN, among others. Furthermore, we identified semisynthetic glycosaminoglycan ethers (SAGEs) as effective RAGE inhibitors in acute pulmonary inflammation and the improvement of lung function. RAGE is also implicated in many other diseases such as type II diabetes mellitus, Alzheimer’s disease, atherosclerosis, and obstetric complications. We investigated its postnatal expression in pups that experienced SHS-induced intrauterine growth restriction (IUGR) antenatally. Increased expression of RAGE correlated with concomitant decreased heart and kidney weights at 4 weeks of age. By 12 weeks of age, weights had improved to age-expected measurements, and detected RAGE protein levels had decreased. These results implicate a potential role for RAGE in disease pathologies of adults who experienced antenatal IUGR due to maternal SHS exposure during pregnancy. In addition to the RAGE signaling pathways, we also investigated the Gas6/AXL pathway in the lungs of pregnant preeclamptic rodents. Gas6 is a ligand for the transmembrane AXL receptor and has been found in increased levels in the serum of pregnant women with preeclampsia (PE). Previous studies in our lab demonstrated a rodent Gas6 model of PE. Using this model, we established that the maternal lung from Gas6-induced preeclamptic rats experienced increased AXL mRNA as well as higher total cell counts, protein, and inflammatory cytokines in bronchoalveolar lavage fluid (BALF). These findings established a connection between lung inflammation and the development of preeclampsia that was previously unknown.

Resistin-like molecule alpha (RELMα) is a secreted protein implicated in murine models of allergen-induced asthma, bleomycin-induced pulmonary fibrosis, and helminth infection. Transient pulmonary overexpression of Oncostatin M by Adenovirus vector (AdOSM) induces lung inflammation biased toward Th2 cytokines, eosinophil and alternatively activated (AA/M2) macrophage accumulation. In AdOSM-treated C57Bl/6 and BALB/c mice, we observed RELMα mRNA and protein markedly induced. RELMα is recognized as a marker of AA/M2 macrophages, and we observed by chromogenic in situ hybridization that RELMα mRNA co-expresses with the macrophage marker CD68, and RELMα mRNA was also highly induced in columnar airway epithelial cells upon AdOSM treatment. Assessing IL-6 as a comparator gp130 cytokine, AdIL-6 induced RELMα at significantly lower levels, however maximal induction of RELMα by AdOSM in C57Bl/6 mice required IL-6, assessed in IL-6–/– mice. Maximal induction of RELMα by AdOSM also required IL-33 in C57Bl/6 mice but not in BALB/c mice, assessed in IL-33–/– mice. We investigated functions of RELMα in response to OSM, in RELMα–/– mice. Inflammatory cell infiltration and Th2-associated cytokine responses were not altered in RELMα–/– in comparison to wildtype mice. However, RELMα-deficiency resulted in less accumulation of CD206+ AA/M2 macrophages, IFNγ+ Th1 cells in the lung, reduced induction of extracellular matrix gene mRNAs for COL1A1, COL3A1, MMP13, TIMP1, and reduced parenchymal alpha smooth muscle actin. RELMα–/– mice also showed less airway epithelial hyperplasia, increased epithelial cell damage/death (assessed morphologically) and increased LDH and soluble CK18 in response to AdOSM. Our findings suggest that RELMα does not modulate Th2 cytokines, but does participate in matrix deposition, airway remodelling mechanisms, and protection from inflammation-induced damage due to OSM-overexpression in lungs of C57Bl/6 mice. / Dissertation / Master of Science (MSc)