PTX3 Regulation of Inflammation, Hemostatic Response, Tissue Repair, and Resolution of Fibrosis Favors a Role in Limiting Idiopathic Pulmonary Fibrosis

PTX3 is a soluble pattern recognition molecule (PRM) belonging to the humoral innate immune system, rapidly produced at inflammatory sites by phagocytes and stromal cells in response to infection or tissue injury. PTX3 interacts with microbial moieties and selected pathogens, with molecules of the complement and hemostatic systems, and with extracellular matrix (ECM) components. In wound sites, PTX3 interacts with fibrin and plasminogen and favors a timely removal of fibrin-rich ECM for an efficient tissue repair. Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown origin, associated with excessive ECM deposition affecting tissue architecture, with irreversible loss of lung function and impact on the patient’s life quality. Maccarinelli et al. recently demonstrated a protective role of PTX3 using the bleomycin (BLM)-induced experimental model of lung fibrosis, in line with the reported role of PTX3 in tissue repair. However, the mechanisms and therapeutic potential of PTX3 in IPF remained to be investigated. Herein, we provide new insights on the possible role of PTX3 in the development of IPF and BLM-induced lung fibrosis. In mice, PTX3-deficiency was associated with worsening of the disease and with impaired fibrin removal and subsequently increased collagen deposition. In IPF patients, microarray data indicated a down-regulation of PTX3 expression, thus suggesting a potential rational underlying the development of disease. Therefore, we provide new insights for considering PTX3 as a possible target molecule underlying therapeutic intervention in IPF

iNOS Activity Modulates Inflammation, Angiogenesis, and Tissue Fibrosis in Polyether-Polyurethane Synthetic Implants

There is considerable interest in implantation techniques and scaffolds for tissue engineering and, for safety and biocompatibility reasons, inflammation, angiogenesis, and fibrosis need to be determined. The contribution of inducible nitric oxide synthase (iNOS) in the regulation of the foreign body reaction induced by subcutaneous implantation of a synthetic matrix was never investigated. Here, we examined the role of iNOS in angiogenesis, inflammation, and collagen deposition induced by polyether-polyurethane synthetic implants, using mice with targeted disruption of the iNOS gene (iNOS−/−) and wild-type (WT) mice. The hemoglobin content and number of vessels were decreased in the implants of iNOS−/− mice compared to WT mice 14 days after implantation. VEGF levels were also reduced in the implants of iNOS−/− mice. In contrast, the iNOS−/− implants exhibited an increased neutrophil and macrophage infiltration. However, no alterations were observed in levels of CXCL1 and CCL2, chemokines related to neutrophil and macrophage migration, respectively. Furthermore, the implants of iNOS−/− mice showed boosted collagen deposition. These data suggest that iNOS activity controls inflammation, angiogenesis, and fibrogenesis in polyether-polyurethane synthetic implants and that lack of iNOS expression increases foreign body reaction to implants in mice

Role of the chemokines CCL3/MIP-1α and CCL5/RANTES in sponge-induced inflammatory angiogenesis in mice

Barcelos, Luciola S Coelho, Amanda M Russo, Remo C Guabiraba, Rodrigo Souza, Adriano L S Bruno-Lima, Guilherme Jr Proudfoot, Amanda E I Andrade, Silvia P Teixeira, Mauro M Microvasc Res. 2009 Sep;78(2):148-54. doi: 10.1016/j.mvr.2009.04.009. Epub 2009 May 8.; International audience; OBJECTIVE: We examined the potential contribution of CCL3 and CCL5 to inflammatory angiogenesis in mice. METHODS: Polyester-polyurethane sponges were implanted in mice and blood vessel counting and hemoglobin, myeloperoxidase and N-acetylglucosaminidase measurements used as indexes for vascularization, neutrophil and macrophage accumulation, respectively. RESULTS: CCL3 and CCL5 were expressed throughout the observation period. Exogenous CCL3 enhanced angiogenesis in WT, but angiogenesis proceeded normally in CCL3(-/-) mice, suggesting that endogenous CCL3 is not critical for sponge-induced angiogenesis in mice. CCL5 expression was detected at day 1, but levels significantly increased thereafter. Exogenous CCL5 reduced angiogenesis in WT mice possible via CCR5 as CCL5 was without an effect in CCR5(-/-) mice. Treatment of WT with the CCR1/CCR5 antagonist, Met-RANTES, prevented neutrophil and macrophage accumulation, but enhanced sponge vascularization. CONCLUSION: Thus, endogenous CCL3 appears not to play a role in driving sponge-induced inflammatory angiogenesis in mice. The effects of CCL5 were anti-angiogenic and appeared to be mediated via activation of CCR5

The atypical chemokine receptor ACKR2 is protective against sepsis

Sepsis is a systemic inflammatory response as a result of uncontrolled infections. Neutrophils are the first cells to reach the primary sites of infection and chemokines play a key role in recruiting neutrophils. However, in sepsis chemokines could also contribute to neutrophil infiltration to vital organs leading to multiple organ failure. ACKR2 is an atypical chemokine receptor, which can remove and degrade inflammatory CC chemokines. The role of ACK2 in sepsis is unknown. Using a model of cecal ligation and puncture (CLP), we demonstrate here that ACKR2 deficient (−/−) mice exhibited a significant reduction in the survival rate compared to similarly treated wild type (WT) mice. However, neutrophil migration to the peritoneal cavity and bacterial load were similar between WT and ACKR2−/− mice during CLP. In contrast, ACKR2−/− mice showed increased neutrophil infiltration and elevated CC chemokine levels in the lung, kidney and heart compared to the WT mice. In addition, ACKR2−/− mice also showed more severe lesions in the lung and kidney than those in the WT mice. Consistent with these results, WT mice under non-severe sepsis (90% survival) had higher expression of ACKR2 in these organs than mice under severe sepsis (no survival). Finally, the lungs from septic patients showed increased number of ACKR2+ cells compared to those of non-septic patients. Our data indicate that ACKR2 may have a protective role during sepsis, and the absence of ACKR2 leads to exacerbated chemokine accumulation, neutrophil infiltration and damage to vital organs

Eosinophil-Associated Innate IL-17 Response Promotes Aspergillus fumigatus Lung Pathology

Aspergillus fumigatus is a common widespread microorganism with environmental, biological and clinical relevance. After inhalation, swollen conidia can germinate, colonize and invade pulmonary tissues. Eosinophils have been described as key cells in A. fumigatus lung infection. However, their specific role in protecting or damaging lung tissue as well as their relatioship among different A. fumigatus strains is poorly understood. Previously, it has been reported that eosinophils are able to produce IL-17 and mediate an innate response that protected mice from infection using Af293 and CEA10 strains. Here, we have developed a set of new experiments with the CEA17-derived A1163 strain of A. fumigatus. Using ΔdblGATA1 mice, we demonstrate that eosinophils produce IL-17 and are involved in control of neutrophil, macrophage and lymphocyte recruitment. We found that eosinophils also induce high levels of cytokines and chemokines, generating an intense inflammatory process. Eosinophils are responsible for increased pulmonary dysfunction and elevated lethality rates in mice. Curiously, fungal burden was not affected. To address the role of IL-17 signaling, pharmacological inhibition of this mediator in the airways with anti-IL-17 antibody was able to reduce inflammation in the airways and protect infected mice. In conclusion, our results demonstrate that eosinophils control IL-17-mediated response and contribute to lung pathology after A. fumigatus infection. Therefore, eosinophils may represent a potential target for controlling exacerbated inflammation and prevent tissue damage during this fungal infection

IgG Induced by Vaccination With Ascaris suum Extracts Is Protective Against Infection

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p < 0.001), CUT 59% (p < 0.001), and ExAD 51% (p < 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity

Efeito da administração do antagonista de receptor CXCR2 no modelo de fibrose pulmonar induzida por bleomicina

Exportado OPUSMade available in DSpace on 2019-08-13T16:03:13Z (GMT). No. of bitstreams: 1 disserta__o_russo_rc_2005.pdf: 2998267 bytes, checksum: 9e14a1f8288398f32dccd43b2426d295 (MD5) Previous issue date: 12A fibrose pulmonar é uma doença caracterizada pela deposição intersticial progressiva de colágeno, que acarreta alterações na arquitetura normal pulmonar e perda da função, podendo levar ao óbito. As inflamações agudas pulmonares e a sua cronificação estão associadas a fenômenos fibróticos, sendo responsáveis por seu desencadeamento. A inflamação que precede a instalação da fibrose pulmonar é caracterizada pelo influxo de células inflamatórias, culminando na liberação de mediadores que perpetuam o dano inicial. Desta forma, é provável que a inibição da resposta inflamatória seja capaz de diminuir a deposição de colágeno intersticial. O modelo de fibrose pulmonar induzido por bleomicina é caracterizado por um intenso influxo de neutrófilos, concomitantemente com uma produção de citocinas, níveis elevados das quimiocinas CXCL1-3/KC e CXCL1-2/MIP-2, e posterior deposição de colágeno no parênquima pulmonar.No presente trabalho, estudamos os efeitos da administração do DF2162, um antagonista de receptores de quimiocinas CXCR2, no modelo experimental de fibrose pulmonar induzida por bleomicina em camundongos. Nossos dados mostram que a administração da dose de 6 mg/kg de DF2162, duas vezes ao dia, inibiu de forma significativa os picos de influxo neutrofílicos após administração de 0,125 U de bleomicina por via intra-traqueal. Entretanto, DF2162 não alterou os níveis das citocinas de caráter modulatório tais como IFN, IL-10 e VEGF, importantes para o processo inflamatório. As quimiocinas CXCL1-3/KC, CXCL1-2/MIP-2, CCL2/JE, CCL3/MIP-1 e CXCL10/IP-10 quantificadas também não apresentaram mudanças no seu perfil de produção, com exceção para CCL5/RANTES, que foi inibida, e CXCL9/MIG, que apresentou níveis elevados numa fase mais inicial, pelo tratamento com este antagonista. Além disso, foram verificadas alterações patológicas que revelaram uma inflamação menos severa e uma menor de deposição de colágeno intersticial no pulmão dos animais tratados com DF2162. Entretanto, apesar de uma melhora em todos os aspectos inflamatórios estudados, os animais que receberam DF2162 apresentaram um índice de letalidade de 66,6%; enquanto que no grupo controle foi observado a morte de apenas 25% dos animais.Estes dados em conjunto sugerem que o receptor CXCR2 exerça papel importante na regulação do processo inflamatório e fibrose pulmonar induzida por bleomicina. Apesar disso, o incremento na taxa de letalidade no grupo de animais que receberam tratamento com DF2162 após instilação de bleomicina provavelmente não está relacionado ao processo fibrótico em si, mas depende de outros fatores que serão investigados em estudos posteriores.Pulmonary fibrosis is a disease characterized by progressive interstitial collagen deposition, which causes changes in the normal lung architecture and loss of function, which could lead to death. Acute pulmonary inflammatory processes and their chronification are associated with fibrotic phenomena acting as triggering events. The inflammation that precedes the emergence of pulmonary fibrosis is characterized by an increased cell influx, which culminates in the liberation of inflammatory mediators, which perpetuate the initial lesion. Therefore it is likely that the inhibition of the inflammatory response might be able to decrease the interstitial collagen deposition. The bleomycin-induced pulmonary fibrosis model is characterized by intense neutrophil influx concomitant with cytokine production, high levels of chemokines CXCL1-3/KC and CXCL1-2/MIP-2, followed by collagen deposition on the pulmonary parenchyma. In this study, we analyzed the effects of DF2162 administration, which is a CXCR2 chemokine receptor antagonist on bleomycin-induced pulmonary fibrosis model in mice. Our results show that the administration of 6 mg/kg of DF2162 twice a day significantly inhibited the neutrophilic influx peaks caused by intra-tracheal instillation of 0,125 U of bleomycin. However, DF2162 did not promote changes in the levels of modulatory cytokines such as IFN, IL-10 e VEGF, which are important for the inflammatory process. We did not observe changes in the levels of chemokines CXCL1-3/KC, CXCL1-2/MIP-2, CCL2/JE, CCL3/MIP-1 and CXCL10/IP-10 with the exception of CCL5/RANTES, whose production was inhibited and CXCL9/MIG, whose levels incresased during the early phase of DF2162 treatment. Furthermore, we observed pathological changes revealing less severe and reduced interstitial collagen deposition in the lungs of DF2162-treated animals. In spite of the observed improvement in all inflammatory aspects studied, the animals receiving DF2162 had a higher mortality (66.6%) than the animals in the control group, which showed only 25% lethality.These data suggest that the CXCR2 receptor exerts an important role in the regulation of the inflammatory process and pulmonary fibrosis induced by bleomycin. Notwithstanding, the increment in letality in the group of mice that received DF2162 treatment after bleomycin instillation is likely not associated with the fibrotic process itself, but depends on factors which shall be later studied

Intervenção farmacológica no sistema quimiocinas: possíveis alvos para o tratamento da fibrose pulmonar

Exportado OPUSMade available in DSpace on 2019-08-13T13:54:52Z (GMT). No. of bitstreams: 1 tese_russo_rc_2009.pdf: 65950198 bytes, checksum: 9a272665e4752113dba3f51ad3587da1 (MD5) Previous issue date: 21A fibrose pulmonar é uma doença crônica respiratória letal cujos sinais clínicos incluem uma piora da dispnéia, perda progressiva do volume pulmonar e troca gasosa anormal. A fibrose pode ser desencadeada por fenômenos inflamatórios pulmonares que geralmente precedem a deposição de colágeno no parênquima pulmonar. Inicialmente, o uso de corticosteróides foi utilizado como estratégia para seu tratamento, com finalidade de reduzir a inflamação pulmonar e também a fibroplasia. Porém, o aparecimento de efeitos adversos durante o período de tratamento limitaram sua aplicação terapêutica. Dados clínicos têm revelado uma estreita correlação entre a fibrose pulmonar, os níveis de quimiocinas circulantes ou no lavado bronco-alveolar, e o perfil leucocitário encontrado. Diante deste cenário, as quimiocinas se tornam importantes alvos para o tratamento da fibrose pulmonar, uma vez que são responsáveis por guiarem as respostas inflamatória e fibrogênica.Esta tese de doutorado reúne dados sobre o estudo de novos fármacos capazes de inibir a fibrose pulmonar induzida de maneira experimental em camundongos e revela diferentes formas de intervir no sistema quimiocina durante processos inflamatórios e fibrogênicos pulmonares. Os alvos utilizados para estudo foram: (i) o receptor de quimiocina CXCR2, (ii) a quimiocina CCL3 e (iii) o elemento de sinalização comum dos receptores de quimiocinas, a PI3K. A administração oral do antagonista alostérico não-competitivo de receptores CXCR2 (DF2162) no modelo de fibrose pulmonar induzida por bleomicina foi responsável pelo efeito protetor observado nos animais quando tratados de maneira preventiva (do dia 0 ao 16) ou terapêutica (do dia 8 ao 16). A redução da fibrose pulmonar nos animais tratados com DF2162 pareceu ser causada pelos efeitos inibitórios sobre a angiogênese, podendo até ser de natureza dual, por inibição da transmigração neutrofílica para as vias aéreas. Através do uso da proteína ligadora de quimiocinas-CCL3 (Evasin-1), isolada e clonada de glândulas salivares do carrapato Rhipicephalus sanguineus, estudamos os efeitos inibitórios de CCL3 durante a inflamação pulmonar e fibrose induzida por bleomicina. O tratamento preventivo (do dia 0 ao 25) e terapêutico (do dia 8 ao 25) com Evasin-1 causou a redução da fibrose pulmonar em camundongos, associada a um menor infiltrado leucocitário e redução da produção de citocinas pro-fibrogênicas. Por último, para avaliar a participação de um elemento comum da via de sinalização intracelular utilizada pelos receptores de quimiocinas, a PI3K, no desenvolvimento da fibrose pulmonar, animais deficientes para a isoforma da PI3K foram desafiados com bleomicina. A deficiência para PI3K causou atenuação da fibrose e aumento na sobrevida dos animais, além de apresentarem redução no influxo leucocitário nas vias aéreas. A inibição da PI3K também gerou modificações funcionais em células endoteliais e fibroblastos in vitro, sugerindo que PI3K participa do processo de angiogênese e na secreção de colágeno por fibroblastos. Em conjunto, estes dados demonstram uma possível modulação do sistema quimiocinas no contexto de patologias inflamatórias pulmonares e oferecem novos fármacos como candidatos para o tratamento da fibrose pulmonar. Futuros ensaios clínicos poderão validar seu uso terapêutico.Pulmonary fibrosis is a chronic respiratory lethal disease whose clinical signs include a worsening of dyspnea, progressive loss of lung volume and abnormal gas exchange. Fibrosis may be triggered by a series of inflammatory lung condition that usually precedes the deposition of collagen in the lung parenchyma. Initially, the use of corticosteroids was used as a strategy for the treatment of pulmonary fibrosis in order to reduce the pulmonary inflammation as well fibroplasia. However, the appearance of adverse effects during the treatment period limited their therapeutic application. Clinical data have shown a close correlation between pulmonary fibrosis, levels of circulating chemokines or boncho-alveolar lavage, and white blood cell counts. In this scenario, the chemokines become important targets for the treatment of pulmonary fibrosis, since they are responsible for guiding the inflammatory and fibrogenic responses.This thesis presents a compilation study of new drugs that may inhibit lung fibrosis induced experimentally in mice and reveals different ways of intervening in the chemokine system during pulmonary inflammatory and fibrogenic process. The targets used for the study were: (i) the chemokine receptor CXCR2, (ii) the chemokine CCL3 and (iii) the common element of signaling pathway of chemokine receptors, the PI3K. The oral administration of the allosteric antagonist non-competitive of CXCR2 receptor (DF2162) in bleomycin-induced pulmonary fibrosis mice model, was responsible for the protective effect observed in animals when treated in a preventively (from day 0 to 16) or therapeutic (from day 8 to 16) schedule. The reduction of pulmonary fibrosis in those animals treated with DF2162 appeared to be caused by inhibitory effects on angiogenesis, and may even be of dual nature, via inhibition of neutrophil transmigration into the airways. Through the use of the chemokine binding proteins-CCL3 (Evasin-1), first isolated and cloned from salivary glands of Rhipicephalus sanguineus, we studied the inhibitory effects of CCL3 during bleomycin-induced pulmonary inflammation and fibrosis. Preventive (from day 0 to 25) and therapeutic (from day 8 to 25) treatments with Evasin-1 caused a reduction of pulmonary fibrosis in mice, associated with a lower leukocyte infiltration and reduced production of pro-fibrogenic cytokines. Finally, to evaluate the importance of a common element of intracellular signaling pathway used by chemokine receptors, the PI3K, in the development of pulmonary fibrosis, deficient animals for the isoform of PI3K were challenged with bleomycin. PI3K deficiency led to attenuation of fibrosis and increased animal survival, besides a reduction in leukocyte influx into the airways. The inhibition of PI3K also caused functional changes in endothelial cells and fibroblasts in vitro, suggesting that PI3K participates in the process of angiogenesis and the secretion of collagen by fibroblasts. Together, these data demonstrate a potential modulation of the chemokine system in inflammatory lung diseases context and provide new drug candidates for the pulmonary fibrosis treatment. Future clinical trials may validate their therapeutic use

Pharmacological blockade of protease-Activated Receptor 2 improves airway remodeling and lung inflammation in experimental allergic asthma

Protease-activated receptors (PARs) are metabotropic G-protein-coupled receptors that are activated via proteolytic cleavage of a specific sequence of amino acids in their N-terminal region. PAR2 has been implicated in mediating allergic airway inflammation. This study aims to study the effect of PAR2 antagonist ENMD1068in lung inflammation and airway remodeling in experimental asthma. Allergic lung inflammation was induced in sensitized BALB/c mice through intranasal instillations of ovalbumin (OVA), and mice were pretreated with ENMD1068 1 hour before each OVA challenge. Bronchoalveolar lavage fluid (BALF) was collected, and the lungs were removed at different time intervals after OVA challenge to analyze inflammation, airway remodeling and airway hyperresponsiveness. Ovalbumin promoted leukocyte infiltration into BALF in a PAR2-dependent manner. ENMD1068 impaired eosinophil peroxidase (EPO) and myeloperoxidase (MPO) activity in the lung parenchyma into BALF and reduced the loss of dynamic pulmonary compliance, lung resistance in response to methacholine, mucus production, collagen deposition and chemokine (C-C motif) ligand 5 expression compared to those in OVA-challenged mice. We propose that proteases released after an allergen challenge may be crucial to the development of allergic asthma in mice, and PAR2 blockade may be useful as a new pharmacological approach for the treatment of airway allergic diseases

iNOS Activity Modulates Inflammation, Angiogenesis, and Tissue Fibrosis in Polyether-Polyurethane Synthetic Implants

There is considerable interest in implantation techniques and scaffolds for tissue engineering and, for safety and biocompatibility reasons, inflammation, angiogenesis, and fibrosis need to be determined. The contribution of inducible nitric oxide synthase (iNOS) in the regulation of the foreign body reaction induced by subcutaneous implantation of a synthetic matrix was never investigated. Here, we examined the role of iNOS in angiogenesis, inflammation, and collagen deposition induced by polyether-polyurethane synthetic implants, using mice with targeted disruption of the iNOS gene (iNOS−/−) and wild-type (WT) mice. The hemoglobin content and number of vessels were decreased in the implants of iNOS−/− mice compared to WT mice 14 days after implantation. VEGF levels were also reduced in the implants of iNOS−/− mice. In contrast, the iNOS−/− implants exhibited an increased neutrophil and macrophage infiltration. However, no alterations were observed in levels of CXCL1 and CCL2, chemokines related to neutrophil and macrophage migration, respectively. Furthermore, the implants of iNOS−/− mice showed boosted collagen deposition. These data suggest that iNOS activity controls inflammation, angiogenesis, and fibrogenesis in polyether-polyurethane synthetic implants and that lack of iNOS expression increases foreign body reaction to implants in mice