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Acute respiratory virus infections can have profound and long-term effects on lung function that persist even after the acute responses have fully resolved. In this study, we examined gene expression by RNA sequencing in the lung tissue of wild-type BALB/c mice that were recovering from a sublethal infection with the pneumonia virus of mice (PVM), a natural rodent pathogen of the same virus family and genus as the human respiratory syncytial virus. We compared these responses to gene expression in PVM-infected mice treated with Lactobacillus plantarum, an immunobiotic agent that limits inflammation and averts the negative clinical sequelae typically observed in response to acute infection with this pathogen. Our findings revealed prominent differential expression of inflammation-associated genes as well as numerous genes and gene families implicated in mitosis and cell-cycle regulation, including cyclins, cyclin-dependent kinases, cell division cycle genes, E2F transcription factors, kinesins, centromere proteins, and aurora kinases, among others. Of particular note was the differential expression of the cell division cycle gene Cdc20b, which was previously identified as critical for the ex vivo differentiation of multi-ciliated cells. Collectively, these findings provided us with substantial insight into post-viral repair processes and broadened our understanding of the mechanisms underlying Lactobacillus-mediated protection.
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Despite an ongoing focus on the role of diet in health and disease, we have only a limited understanding of these concepts at the cellular and molecular levels. While obesity has been clearly recognized as contributing to metabolic syndrome and the pathogenesis of adult asthma, recent evidence has linked high sugar intake alone to an increased risk of developing asthma in childhood. In this study, we examined the impact of diet in a mouse model of allergic airways inflammation with a specific focus on eosinophils. As anticipated, male C57BL/6 mice gained weight on a high-calorie, high-fat diet. However, mice also gained weight on an isocaloric high-sucrose diet. Elevated levels of leptin were detected in the serum and airways of mice maintained on the high-fat, but not the high-sucrose diets. We found that diet alone had no impact on eosinophil numbers in the airways at baseline or their recruitment in response to allergen (Alternaria alternata) challenge in either wild-type or leptin-deficient ob/ob mice. However, both bronchoalveolar lavage fluid and eosinophils isolated from lung tissue of allergen-challenged mice exhibited profound diet-dependent differences in cytokine content. Similarly, while all wild-type mice responded to allergen challenge with significant increases in methacholine-dependent total airway resistance (Rrs), airway resistance in mice maintained on the isocaloric high-sucrose (but not the high-calorie/high-fat) diet significantly exceeded that of mice maintained on the basic diet. In summary, our findings revealed that mice maintained on an isocaloric high-sucrose diet responded to allergen challenge with significant changes in both BAL and eosinophil cytokine content together with significant increases in Rrs. These results provide a model for further exploration of the unique risks associated with a high-sugar diet and its impact on allergen-associated respiratory dysfunction.
Assuntos
Alérgenos/toxicidade , Asma/patologia , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Eosinófilos/imunologia , Pneumonia/complicações , Sacarose/toxicidade , Animais , Asma/etiologia , Asma/metabolismo , Líquido da Lavagem Broncoalveolar/química , Eosinófilos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/toxicidade , Edulcorantes/toxicidadeRESUMO
Respiratory virus infections can have long-term effects on lung function that persist even after the acute responses have resolved. Numerous studies have linked severe early childhood infection with respiratory syncytial virus (RSV) to the development of wheezing and asthma, although the underlying mechanisms connecting these observations remain unclear. Here, we examine airway hyperresponsiveness (AHR) that develops in wild-type mice after recovery from symptomatic but sublethal infection with the natural rodent pathogen, pneumonia virus of mice (PVM). We found that BALB/c mice respond to a limited inoculum of PVM with significant but reversible weight loss accompanied by virus replication, acute inflammation, and neutrophil recruitment to the airways. At day 21 post-inoculation, virus was no longer detected in the airways and the acute inflammatory response had largely resolved. However, and in contrast to most earlier studies using the PVM infection model, all mice survived the initial infection and all went on to develop serum anti-PVM IgG antibodies. Furthermore, using both invasive plethysmography and precision-cut lung slices, we found that these mice exhibited significant airway hyperresponsiveness at day 21 post-inoculation that persisted through day 45. Taken together, our findings extend an important and versatile respiratory virus infection model that can now be used to explore the role of virions and virion clearance as well as virus-induced inflammatory mediators and their signaling pathways in the development and persistence of post-viral AHR and lung dysfunction.
Assuntos
Vírus da Pneumonia Murina/imunologia , Infecções por Pneumovirus/complicações , Infecções por Pneumovirus/veterinária , Hipersensibilidade Respiratória/etiologia , Animais , Anticorpos Antivirais/imunologia , Humanos , Pulmão/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Pneumonia Murina/fisiologia , Infecções por Pneumovirus/imunologia , Infecções por Pneumovirus/virologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/fisiologiaRESUMO
The unprecedented impact of the coronavirus disease 2019 (COVID-19) pandemic has resulted in global challenges to our health-care systems and our economic security. As such, there has been significant research into all aspects of the disease, including diagnostic biomarkers, associated risk factors, and strategies that might be used for its treatment and prevention. Toward this end, eosinopenia has been identified as one of many factors that might facilitate the diagnosis and prognosis of severe COVID-19. However, this finding is neither definitive nor pathognomonic for COVID-19. While eosinophil-associated conditions have been misdiagnosed as COVID-19 and others are among its reported complications, patients with pre-existing eosinophil-associated disorders (e.g., asthma, eosinophilic gastrointestinal disorders) do not appear to be at increased risk for severe disease; interestingly, several recent studies suggest that a diagnosis of asthma may be associated with some degree of protection. Finally, although vaccine-associated aberrant inflammatory responses, including eosinophil accumulation in the respiratory tract, were observed in preclinical immunization studies targeting the related SARS-CoV and MERS-CoV pathogens, no similar complications have been reported clinically in response to the widespread dissemination of either of the two encapsulated mRNA-based vaccines for COVID-19.
Assuntos
COVID-19 , Eosinófilos , Vacinas contra COVID-19 , Humanos , Prognóstico , SARS-CoV-2 , VacinaçãoRESUMO
The eosinophil is an enigmatic cell with a continuing ability to fascinate. A considerable history of research endeavor on eosinophil biology stretches from the present time back to the nineteenth century. Perhaps one of the most fascinating aspects of the eosinophil is how accumulating knowledge has changed the perception of its function from passive bystander, modulator of inflammation, to potent effector cell loaded with histotoxic substances through to more recent recognition that it can act as both a positive and negative regulator of complex events in both innate and adaptive immunity. This book consists of chapters written by experts in the field of eosinophil biology that provide comprehensive clearly written protocols for techniques designed to underpin research into the function of the eosinophil in health and disease.
Assuntos
Eosinófilos/metabolismo , Eosinófilos/fisiologia , Biologia Molecular/métodos , Imunidade Adaptativa , Proteínas Granulares de Eosinófilos , Humanos , InflamaçãoRESUMO
Human eosinophilic leukocytes are found in peripheral blood and tissues at homeostasis and at elevated levels in atopic disorders. As inbred strains of mice (Mus musculus) are currently the models of choice for the study of disease mechanisms in vivo, a full understanding of mouse eosinophils is critical for interpretation of experimental findings. Toward this end, several years ago we presented a protocol for generating mouse eosinophils in tissue culture from unselected bone marrow progenitors (Dyer et al., J Immunol 181: 4004-4009, 2008). This method has been implemented widely and has proven to be effective for generating phenotypically normal eosinophils from numerous mouse strains and genotypes. Here we provide a detailed version of this protocol, along with suggestions and notes for its careful execution. We have also included several protocol variations and suggestions for improvements.
Assuntos
Técnicas de Cultura de Células/métodos , Eosinófilos/citologia , Células-Tronco Mesenquimais/citologia , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Diferenciação Celular , Eosinófilos/metabolismo , Eosinófilos/fisiologia , Interleucina-5/metabolismo , Contagem de Leucócitos , Camundongos , Células-TroncoRESUMO
Flow cytometry is a critical tool that can be employed to detect unique cells and to isolate cells from tissues based on their antigen profiles. While mouse eosinophils can be readily detected by one or more distinct antigen profiles, many of these strategies do not result in accurate eosinophil counts. We present here our basic protocol, which permits quantitative detection of eosinophils and isolation of eosinophils from bone marrow, spleen, and lung tissue of allergen-challenged wild-type and unchallenged IL5 transgenic mice. With small protocol variations, eosinophils can be isolated from small intestines and muscle tissue, the latter from infiltrates characteristic of muscular dystrophy (mdx) mice.
Assuntos
Separação Celular/métodos , Eosinófilos/citologia , Citometria de Fluxo/métodos , Alérgenos/imunologia , Animais , Sangue/metabolismo , Células Sanguíneas/citologia , Medula Óssea/imunologia , Células da Medula Óssea/citologia , Eosinófilos/metabolismo , Eosinófilos/fisiologia , Feminino , Separação Imunomagnética/métodos , Contagem de Leucócitos/métodos , Pulmão/citologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Receptores de IgG/imunologia , Baço/citologia , Baço/imunologiaRESUMO
No longer regarded simply as end-stage cytotoxic effectors, eosinophils are now recognized as complex cells with unique phenotypes that develop in response stimuli in the local microenvironment. In our previous study, we documented eosinophil infiltration in damaged muscle characteristic of dystrophin-deficient (mdx) mice that model Duchenne muscular dystrophy. Specifically, we found that eosinophils did not promote the generation of muscle lesions, as these persisted in eosinophil-deficient mdx.PHIL mice. To obtain additional insight into these findings, we performed RNA sequencing of eosinophils isolated from muscle tissue of mdx, IL5tg, and mdx.IL5tg mice. We observed profound up-regulation of classical effector proteins (major basic protein-1, eosinophil peroxidase, and eosinophil-associated ribonucleases) in eosinophils isolated from lesion-free muscle from IL5tg mice. By contrast, we observed significant up-regulation of tissue remodeling genes, including proteases, extracellular matrix components, collagen, and skeletal muscle precursors, as well as the immunomodulatory receptor, Trem2, in eosinophils isolated from skeletal muscle tissue from the dystrophin-deficient mdx mice. Although the anti-inflammatory properties of Trem2 have been described in the monocyte/macrophage lineage, no previous studies have documented its expression in eosinophils. We found that Trem2 was critical for full growth and differentiation of bone marrow-derived eosinophil cultures and full expression of TLR4. Immunoreactive Trem2 was also detected on human peripheral blood eosinophils at levels that correlated with donor body mass index and total leukocyte count. Taken together, our findings provide important insight into the immunomodulatory and remodeling capacity of mouse eosinophils and the flexibility of their gene expression profiles in vivo.
Assuntos
Eosinófilos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Animais , Sangue/metabolismo , Contagem de Células , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos Knockout , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Receptor 4 Toll-Like/metabolismoRESUMO
Virus-induced inflammation plays a critical role in determining the clinical outcome of an acute respiratory virus infection. We have shown previously that the administration of immunobiotic Lactobacillus plantarum (Lp) directly to the respiratory tract prevents lethal inflammatory responses to subsequent infection with a mouse respiratory virus pathogen. While Lp-mediated protective responses involve non-redundant contributions of both Toll-like receptor 2 (TLR2) and NOD2, the cellular basis of these findings remains unclear. Here, we address the impact of Lp and its capacity to suppress inflammation in virus-infected respiratory epithelial cells in two cell culture models. We found that both MLE-12 cells and polarized mouse tracheal epithelial cells (mTECs) were susceptible to infection with Influenza A and released proinflammatory cytokines, including CCL2, CCL5, CXCL1, and CXCL10, in response to replicating virus. MLE-12 cells express NOD2 (81 ± 6.3%) and TLR2 (19 ± 4%), respond to Lp, and are TLR2-specific, but not NOD2-specific, biochemical agonists. By contrast, we found that mTECs express NOD2 (81 ± 17%) but minimal TLR2 (0.93 ± 0.58%); nonetheless, mTECs respond to Lp and the TLR2 agonist, Pam2CSK4, but not NOD2 agonists or the bifunctional TLR2-NOD2 agonist, CL-429. Although MLE-12 cells and mTECS were both activated by Lp, little to no cytokine suppression was observed in response to Lp followed by virus infection via a protocol that replicated experimental conditions that were effective in vivo. Further study and a more complex approach may be required to reveal critical factors that suppress virus-induced inflammatory responses.
Assuntos
Reações Cruzadas/imunologia , Inflamação/etiologia , Lactobacillus plantarum/fisiologia , Probióticos , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Imunofenotipagem , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Receptores de Reconhecimento de Padrão/metabolismo , Mucosa Respiratória/microbiologia , Mucosa Respiratória/patologia , Receptor 2 Toll-Like/metabolismo , Viroses/complicações , Viroses/virologia , Redução de PesoRESUMO
Chronic inhalation of fungi and fungal components has been linked to the development of respiratory disorders, although their role with respect to the pathogenesis of acute respiratory virus infection remains unclear. Here, we evaluate inflammatory pathology induced by repetitive administration of a filtrate of the ubiquitous fungus, Alternaria alternata, and its impact on susceptibility to infection with influenza A. We showed previously that A. alternata at the nasal mucosae resulted in increased susceptibility to an otherwise sublethal inoculum of influenza A in wild-type mice. Here we demonstrate that A. alternata-induced potentiation of influenza A infection was not dependent on fungal serine protease or ribonuclease activity. Repetitive challenge with A. alternata prior to virus infection resulted proinflammatory cytokines, neutrophil recruitment, and loss of alveolar macrophages to a degree that substantially exceeded that observed in response to influenza A infection alone. Concomitant administration of immunomodulatory Lactobacillus plantarum, a strategy shown previously to limit virus-induced inflammation in the airways, blocked the exaggerated lethal response. These observations promote an improved understanding of severe influenza infection with potential clinical relevance for individuals subjected to continuous exposure to molds and fungi.
Assuntos
Alternaria , Alternariose/imunologia , Vírus da Influenza A , Macrófagos Alveolares/imunologia , Infecções por Orthomyxoviridae/fisiopatologia , Alternaria/metabolismo , Alternariose/patologia , Alternariose/fisiopatologia , Animais , Bactérias/crescimento & desenvolvimento , Líquido da Lavagem Broncoalveolar/microbiologia , Citocinas/metabolismo , Suscetibilidade a Doenças , Feminino , Inflamação , Lactobacillus plantarum/fisiologia , Pulmão/imunologia , Masculino , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Ribonucleases/metabolismo , Serina Proteases/metabolismo , Índice de Gravidade de DoençaRESUMO
Eosinophils and their secretory mediators play an important role in the pathogenesis of infectious and inflammatory disorders. Although eosinophils are largely evolutionally conserved, their physiologic functions are not well understood. Given the availability of new eosinophil-targeted depletion therapies, there has been a renewed interest in understanding eosinophil biology as these strategies may result in secondary disorders when applied over long periods of time. Recent data suggest that eosinophils are not only involved in immunological effector functions but also carry out tissue protective and immunoregulatory functions that actively contribute to the maintenance of homeostasis. Prolonged eosinophil depletion may therefore result in the development of secondary disorders. Here, we review recent literature pointing to important roles for eosinophils in promoting immune defense, antibody production, activation of adipose tissue, and tissue remodeling and fibrosis. We also reflect on patient data from clinical trials that feature anti-eosinophil therapeutics.
Assuntos
Eosinófilos/imunologia , Síndrome Hipereosinofílica/imunologia , Inflamação/imunologia , Animais , Formação de Anticorpos , Humanos , Imunidade Celular , Imunomodulação , Interleucina-5 , CicatrizaçãoRESUMO
Eosinophils have broad and extensive immunomodulatory capacity; recent studies have focused on the roles of distinct eosinophil subsets in specific tissue microenvironments. Ly6G is a GPI-linked leukocyte surface Ag understood primarily as a marker of mouse neutrophils, although its full function is not known. Here, we show that Ly6G/Gr1, detected by mAbs 1A8 (anti-Ly6G) and RB6-8C5 (anti-Gr1), is detected prominently on a significant fraction of eosinophils from mouse bone marrow and bone marrow-derived culture, with fractions expressing this Ag increasing in IL-5-enriched microenvironments. Among our findings, we identified SiglecF+ Gr1+ eosinophils in bone marrow from naïve, allergen-challenged and IL-5 transgenic mice; SiglecF+ Gr1+ eosinophils were also prominent ex vivo in bone marrow-derived eosinophils (bmEos) in IL-5-enriched culture. Reducing the IL-5 concentration 20-fold had no impact on the rate of generation of SiglecF+ bmEos but did result in a marked increase in the Gr1- fraction (from 17.4 ± 2% to 30 ± 2.3%, ***P < 0.005). Reducing the IL-5 concentration also enhanced chemotaxis; SiglecF+ Gr1- bmEos were considerably more responsive to eotaxin-1 than were their SiglecF+ Gr1+ counterparts. These results suggest that (i) IL-5 regulates the expression of Ly6G/Gr1, either directly or indirectly, in cells of the eosinophil lineage, (ii) eosinophils generated in response to high concentrations of IL-5 can be distinguished from those generated under homeostatic conditions by expression of the Ly6G/Gr1 cell surface Ag, and (iii) expression of Ly6G/Gr1 may have an impact on function, directly or indirectly, including the potential to undergo chemotaxis in response to eotaxin-1.
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Antígenos Ly/metabolismo , Antígenos de Superfície/metabolismo , Eosinófilos/citologia , Eosinófilos/metabolismo , Interleucina-5/metabolismo , Animais , Antígenos de Diferenciação Mielomonocítica/metabolismo , Células da Medula Óssea/citologia , Sobrevivência Celular , Quimiocina CCL11/sangue , Quimiotaxia de Leucócito , Homeostase , Camundongos , Fenótipo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
[This corrects the article DOI: 10.1038/s42003-019-0647-4.].
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The systemic capillary leak syndrome (SCLS, Clarkson disease) is a disorder of unknown etiology characterized by recurrent episodes of vascular leakage of proteins and fluids into peripheral tissues, resulting in whole-body edema and hypotensive shock. The pathologic mechanisms and genetic basis for SCLS remain elusive. Here we identify an inbred mouse strain, SJL, which recapitulates cardinal features of SCLS, including susceptibility to histamine- and infection-triggered vascular leak. We named this trait "Histamine hypersensitivity" (Hhs/Hhs) and mapped it to Chromosome 6. Hhs is syntenic to the genomic locus most strongly associated with SCLS in humans (3p25.3), revealing that the predisposition to develop vascular hyperpermeability has a strong genetic component conserved between humans and mice and providing a naturally occurring animal model for SCLS. Genetic analysis of Hhs may reveal orthologous candidate genes that contribute not only to SCLS, but also to normal and dysregulated mechanisms underlying vascular barrier function more generally.
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Síndrome de Vazamento Capilar/genética , Animais , Síndrome de Vazamento Capilar/etiologia , Síndrome de Vazamento Capilar/fisiopatologia , Permeabilidade Capilar/genética , Permeabilidade Capilar/fisiologia , Mapeamento Cromossômico , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Histamina/fisiologia , Humanos , Vírus da Influenza A Subtipo H3N2 , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos , Infecções por Orthomyxoviridae/complicações , Pele/irrigação sanguínea , Especificidade da Espécie , SinteniaRESUMO
The hallmark features of allergic asthma are type 2 (eosinophilic) inflammation and airways hyperresponsiveness (AHR). Although these features often comanifest in mouse lungs in vivo, we demonstrate in this study that the serine protease Alp1 from the ubiquitous mold and allergen, Aspergillus fumigatus, can induce AHR in mice unable to generate eosinophilic inflammation. Strikingly, Alp1 induced AHR in mice devoid of protease-activated receptor 2/F2 trypsin-like receptor 1 (PAR2/F2RL1), a receptor expressed in lung epithelium that is critical for allergic responses to protease-containing allergens. Instead, using precision-cut lung slices and human airway smooth muscle cells, we demonstrate that Alp1 directly increased contractile force. Taken together, these findings suggest that Alp1 induces bronchoconstriction through mechanisms that are largely independent of allergic inflammation and point to a new target for direct intervention of fungal-associated asthma.
Assuntos
Aspergillus fumigatus/imunologia , Asma/imunologia , Asma/microbiologia , Proteínas Fúngicas/imunologia , Serina Endopeptidases/imunologia , Alérgenos/imunologia , Animais , Aspergillus fumigatus/enzimologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Broncoconstrição/efeitos dos fármacos , Broncoconstrição/imunologia , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Inflamação/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/imunologia , Receptor PAR-2/genética , Receptor PAR-2/imunologiaRESUMO
Eosinophilic leukocytes develop in the bone marrow and migrate from peripheral blood to tissues, where they maintain homeostasis and promote dysfunction via release of preformed immunomodulatory mediators. In this study, we explore human eosinophil heterogeneity with a specific focus on naturally occurring variations in cytokine content. We found that human eosinophil-associated cytokines varied on a continuum from minimally (coefficient of variation [CV] ≤ 50%) to moderately variable (50% < CV ≤ 90%). Within the moderately variable group, we detected immunoreactive IL-27 (953 ± 504 pg/mg lysate), a mediator not previously associated with human eosinophils. However, our major finding was the distinct and profound variability of eosinophil-associated IL-16 (CV = 103%). Interestingly, eosinophil IL-16 content correlated directly with body mass index (R 2 = 0.60, ***p < 0.0001) in one donor subset. We found no direct correlation between eosinophil IL-16 content and donor age, sex, total leukocytes, lymphocytes, or eosinophils (cells per microliter), nor was there any relationship between IL-16 content and the characterized -295T/C IL-16 promoter polymorphism. Likewise, although eosinophil IL-1ß, IL-1α, and IL-6 levels correlated with one another, there was no direct association between any of these cytokines and eosinophil IL-16 content. Finally, a moderate increase in total dietary fat resulted in a 2.7-fold reduction in eosinophil IL-16 content among C57BL/6-IL5tg mice. Overall, these results suggest that relationships between energy metabolism, eosinophils, and IL-16 content are not direct or straightforward. Nonetheless, given our current understanding of the connections between asthma and obesity, these findings suggest important eosinophil-focused directions for further exploration.
Assuntos
Citocinas/imunologia , Eosinófilos/imunologia , Interleucina-16/imunologia , Adulto , Idoso , Animais , Asma/imunologia , Medula Óssea/imunologia , Feminino , Humanos , Contagem de Leucócitos/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Adulto JovemRESUMO
Eosinophils are present in muscle lesions associated with Duchenne muscular dystrophy and dystrophin-deficient mdx mice that phenocopy this disorder. Although it has been hypothesized that eosinophils promote characteristic inflammatory muscle damage, this has not been fully examined. In this study, we generated mice with the dystrophin mutation introduced into PHIL, a strain with a transgene that directs lineage-specific eosinophil ablation. We also explored the impact of eosinophil overabundance on dystrophinopathy by introducing the dystrophin mutation into IL-5 transgenic mice. We evaluated the degree of eosinophil infiltration in association with myofiber size distribution, centralized nuclei, serum creatine kinase, and quantitative histopathology scores. Among our findings, eosinophils were prominent in the quadriceps muscles of 4-wk-old male mdx mice but no profound differences were observed in the quantitative measures of muscle damage when comparing mdx versus mdx.PHIL versus mdx.IL5tg mice, despite dramatic differences in eosinophil infiltration (CD45+CD11c-Gr1-MHC class IIloSiglecF+ eosinophils at 1.2 ± 0.34% versus <0.1% versus 20 ± 7.6% of total cells, respectively). Further evaluation revealed elevated levels of eosinophil chemoatttractants eotaxin-1 and RANTES in the muscle tissue of all three dystrophin-deficient strains; eotaxin-1 concentration in muscle correlated inversely with age. Cytokines IL-4 and IL-1R antagonist were also detected in association with eosinophils in muscle. Taken together, our findings challenge the long-held perception of eosinophils as cytotoxic in dystrophin-deficient muscle; we show clearly that eosinophil infiltration is not a driving force behind acute muscle damage in the mdx mouse strain. Ongoing studies will focus on the functional properties of eosinophils in this unique microenvironment.
Assuntos
Eosinófilos/imunologia , Distrofia Muscular de Duchenne/imunologia , Animais , Modelos Animais de Doenças , Distrofina/imunologia , Feminino , Interleucina-4/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/imunologia , Receptores de Interleucina-1/imunologiaRESUMO
Eosinophil peroxidase (EPX) is a major constituent of the large cytoplasmic granules of both human and mouse eosinophilic leukocytes. Human EPX deficiency is a rare, autosomal-recessive disorder limited to the eosinophil lineage. Our intent was to explore the impact of EPX gene deletion on eosinophil content, structure, and function. In response to repetitive intranasal challenge with a filtrate of the allergen, Alternaria alternata, we found significantly fewer eosinophils peripherally and in the respiratory tracts of EPX-/- mice compared to wild-type controls; furthermore, both the major population (Gr1-/lo ) and the smaller population of Gr1hi eosinophils from EPX-/- mice displayed lower median fluorescence intensities (MFIs) for Siglec F. Quantitative evaluation of transmission electron micrographs of lung eosinophils confirmed the relative reduction in granule outer matrix volume in cells from the EPX-/- mice, a finding analogous to that observed in human EPX deficiency. Despite the reduced size of the granule matrix, the cytokine content of eosinophils isolated from allergen-challenged EPX-/- and wild-type mice were largely comparable to one another, although the EPX-/- eosinophils contained reduced concentrations of IL-3. Other distinguishing features of lung eosinophils from allergen-challenged EPX-/- mice included a reduced fraction of surface TLR4+ cells and reduced MFI for NOD1. Interestingly, the EPX gene deletion had no impact on eosinophil-mediated clearance of gram-negative Haemophilus influenzae from the airways. As such, although no clinical findings have been associated with human EPX deficiency, our findings suggest that further evaluation for alterations in eosinophil structure and function may be warranted.
Assuntos
Peroxidase de Eosinófilo/deficiência , Eosinófilos/enzimologia , Eosinófilos/patologia , Pulmão/enzimologia , Pulmão/fisiologia , Alérgenos/imunologia , Alternaria/fisiologia , Animais , Antígenos de Diferenciação Mielomonocítica/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Eosinófilos/ultraestrutura , Haemophilus influenzae/fisiologia , Humanos , Interleucina-3/metabolismo , Pulmão/microbiologia , Camundongos Endogâmicos C57BL , Proteína Adaptadora de Sinalização NOD1/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Receptor 4 Toll-Like/metabolismoRESUMO
Severe respiratory virus infections feature robust local host responses that contribute to disease severity. Immunomodulatory strategies that limit virus-induced inflammation may be of critical importance, notably in the absence of antiviral vaccines. In this study, we examined the role of the pleiotropic cytokine IL-6 in acute infection with pneumonia virus of mice (PVM), a natural rodent pathogen that is related to respiratory syncytial virus and that generates local inflammation as a feature of severe infection. In contrast to Influenza A, PVM is substantially less lethal in IL-6 -/- mice than it is in wild-type, a finding associated with diminished neutrophil recruitment and reduced fluid accumulation in lung tissue. Ly6Chi proinflammatory monocytes are recruited in response to PVM via a CCR2-dependent mechanism, but they are not a major source of IL-6 nor do they contribute to lethal sequelae of infection. By contrast, alveolar macrophages are readily infected with PVM in vivo; ablation of alveolar macrophages results in prolonged survival in association with a reduction in virus-induced IL-6. Finally, as shown previously, administration of immunobiotic Lactobacillus plantarum to the respiratory tracts of PVM-infected mice promoted survival in association with diminished levels of IL-6. We demonstrated in this study that IL-6 suppression is a critical feature of the protective mechanism; PVM-infected IL-6 -/- mice responded to low doses of L. plantarum, and administration of IL-6 overcame L. plantarum-mediated protection in PVM-infected wild-type mice. Taken together, these results connect the actions of IL-6 to PVM pathogenesis and suggest cytokine blockade as a potential therapeutic modality in severe infection.
Assuntos
Interleucina-6/imunologia , Vírus da Pneumonia Murina/imunologia , Infecções por Pneumovirus/imunologia , Animais , Inflamação , Interleucina-6/farmacologia , Lactobacillus plantarum/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Probióticos/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Sistema Respiratório/imunologia , Sistema Respiratório/virologiaRESUMO
Neutrophils are white blood cells that are mobilized to damaged tissues and to sites of pathogen invasion, providing the first line of host defense. Chemokines displayed on the surface of blood vessels promote migration of neutrophils to these sites, and tissue- and pathogen-derived chemoattractant signals, including N-formylmethionylleucylphenylalanine (fMLP), elicit further migration to sites of infection. Although nearly all chemoattractant receptors use heterotrimeric G proteins to transmit signals, many of the mechanisms lying downstream of chemoattractant receptors that either promote or limit neutrophil motility are incompletely defined. Here, we show that regulator of G protein signaling 5 (RGS5), a protein that modulates G protein activity, is expressed in both human and murine neutrophils. We detected significantly more neutrophils in the airways of Rgs5-/- mice than WT counterparts following acute respiratory virus infection and in the peritoneum in response to injection of thioglycollate, a biochemical proinflammatory stimulus. RGS5-deficient neutrophils responded with increased chemotaxis elicited by the chemokines CXC motif chemokine ligand 1 (CXCL1), CXCL2, and CXCL12 but not fMLP. Moreover, adhesion of these cells was increased in the presence of both CXCL2 and fMLP. In summary, our results indicate that RGS5 deficiency increases chemotaxis and adhesion, leading to more efficient neutrophil mobilization to inflamed tissues in mice. These findings suggest that RGS5 expression and activity in neutrophils determine their migrational patterns in the complex microenvironments characteristic of inflamed tissues.