RESUMO
We investigated mechanisms by which TLR9 signaling promoted the development of the protective response to Cryptococcus neoformans in mice with cryptococcal pneumonia. The afferent (week 1) and efferent (week 3) phase immune parameters were analyzed in the infected wild-type (TLR9(+/+)) and TLR-deficient (TLR9(-/-)) mice. TLR9 deletion diminished 1) accumulation and activation of CD11b(+) dendritic cells (DCs), 2) the induction of IFN-γ and CCR2 chemokines CCL7, CCL12, but not CCL2, at week 1, and 3) pulmonary accumulation and activation of the major effector cells CD4(+) and CD8(+) T cells, CD11b(+) lung DCs, and exudate macrophages at week 3. The significance of CCL7 induction downstream of TLR9 signaling was investigated by determining whether CCL7 reconstitution would improve immunological parameters in C. neoformans-infected TLR9(-/-) mice. Early reconstitution with CCL7 1) improved accumulation and activation of CD11b(+) DCs at week 1, 2) restored early IFN-γ production in the lungs, and 3) restored the accumulation of major effector cell subsets. CCL7 administration abolished the difference in lung fungal burdens between TLR9(+/+) and TLR9(-/-) mice at week 3; however, significant reduction of fungal burdens between PBS- and CCL7-treated mice has not been observed, suggesting that additional mechanism(s) apart from early CCL7 induction contribute to optimal fungal clearance in TLR9(+/+) mice. Collectively, we show that TLR9 signaling during the afferent phase contributes to the development of protective immunity by promoting the early induction of CCL7 and IFN-γ and the subsequent early recruitment and activation of DCs and additional effector cells in mice with cryptococcal pneumonia.
Assuntos
Quimiocina CCL7/imunologia , Criptococose/imunologia , Cryptococcus neoformans , Pulmão/imunologia , Pneumonia/imunologia , Animais , Antígeno CD11b/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Comunicação Celular/imunologia , Contagem de Colônia Microbiana , Criptococose/complicações , Criptococose/microbiologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Interferon gama/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Quimioatraentes de Monócitos/imunologia , Pneumonia/complicações , Pneumonia/microbiologia , Transdução de Sinais , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologiaRESUMO
Previous research in our laboratory has demonstrated that repeated intranasal exposure to Aspergillus fumigatus conidia in C57BL/6 mice results in a chronic pulmonary inflammatory response that reaches its maximal level after four challenges. The inflammatory response is characterized by eosinophilia, goblet cell metaplasia, and T helper T(H)2 cytokine production, which is accompanied by sustained interleukin-17 (IL-17) expression that persists even after the T(H)2 response has begun to resolve. T(H)17 cells could develop in mice deficient in gamma interferon (IFN-γ), IL-4, or IL-10. In the lungs of IL-17 knockout mice repeatedly challenged with A. fumigatus conidia, inflammation was attenuated (with the most significant decrease occurring in eosinophils), conidial clearance was enhanced, and the early transient peak of CD4(+) CD25(+) FoxP3(+) cells blunted. IL-17 appeared to play only a minor role in eosinophil differentiation in the bone marrow but a central role in eosinophil extravasation from the blood into the lungs. These observations point to an expanded role for IL-17 in driving T(H)2-type inflammation to repeated inhalation of fungal conidia.
Assuntos
Aspergillus fumigatus/imunologia , Aspergillus fumigatus/patogenicidade , Interleucina-17/imunologia , Aspergilose Pulmonar/imunologia , Eosinofilia Pulmonar/imunologia , Esporos Fúngicos/imunologia , Animais , Antígenos CD4/biossíntese , Eosinófilos/imunologia , Fatores de Transcrição Forkhead/biossíntese , Interferon gama/imunologia , Interleucina-10/imunologia , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Interleucina-4/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia , Células Th17/imunologia , Células Th2/imunologiaRESUMO
The composition of the lung microbiome contributes to both health and disease, including obstructive lung disease. Because it has been estimated that over 70% of the bacterial species on body surfaces cannot be cultured by currently available techniques, traditional culture techniques are no longer the gold standard for microbial investigation. Advanced techniques that identify bacterial sequences, including the 16S ribosomal RNA gene, have provided new insights into the depth and breadth of microbiota present both in the diseased and normal lung. In asthma, the composition of the microbiome of the lung and gut during early childhood development may play a key role in the development of asthma, while specific airway microbiota are associated with chronic asthma in adults. Early bacterial stimulation appears to reduce asthma susceptibility by helping the immune system develop lifelong tolerance to innocuous antigens. By contrast, perturbations in the microbiome from antibiotic use may increase the risk for asthma development. In chronic obstructive pulmonary disease, bacterial colonisation has been associated with a chronic bronchitic phenotype, increased risk of exacerbations, and accelerated loss of lung function. In cystic fibrosis, studies utilising culture-independent methods have identified associations between decreased bacterial community diversity and reduced lung function; colonisation with Pseudomonas aeruginosa has been associated with the presence of certain CFTR mutations. Genomic analysis of the lung microbiome is a young field, but has the potential to define the relationship between lung microbiome composition and disease course. Whether we can manipulate bacterial communities to improve clinical outcomes remains to be seen.
Assuntos
Fibrose Cística/microbiologia , Pneumopatias Obstrutivas/microbiologia , Pulmão/microbiologia , Metagenoma , Mucosa Respiratória/imunologia , Humanos , Pulmão/patologia , FumarRESUMO
Clearance of pulmonary infection with the fungal pathogen Cryptococcus neoformans is associated with the accumulation and activation of lung macrophages. However, the phenotype of these macrophages and the mechanisms contributing to their accumulation are not well-defined. In this study, we used an established murine model of cryptococcal lung infection and flow cytometric analysis to identify alveolar macrophages (AMs) and the recently described exudate macrophages (ExMs). Exudate macrophages are distinguished from AMs by their strong expression of CD11b and major histocompatibility complex class II and modest expression of costimulatory molecules. Exudate macrophages substantially outnumber AMs during the effector phase of the immune response; and accumulation of ExMs, but not AMs, was chemokine receptor 2 (CCR2) dependent and attributable to the recruitment and subsequent differentiation of Ly-6C(high) monocytes originating from the bone marrow and possibly the spleen. Peak ExM accumulation in wild-type (CCR2(+/+)) mice coincided with maximal lung expression of mRNA for inducible nitric oxide synthase and correlated with the known onset of cryptococcal clearance in this strain of mice. Exudate macrophages purified from infected lungs displayed a classically activated effector phenotype characterized by cryptococcal-enhanced production of inducible nitric oxide synthase and tumor necrosis factor α. Cryptococcal killing by bone marrow-derived ExMs was CCR2 independent and superior to that of AMs. We conclude that clearance of cryptococcal lung infection requires the CCR2-mediated massive accumulation of fungicidal ExMs derived from circulating Ly-6C(high) monocytes.
Assuntos
Criptococose/imunologia , Cryptococcus neoformans/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Pneumonia/imunologia , Receptores CCR2/imunologia , Animais , Antígenos Ly/imunologia , Antígenos CD11/imunologia , Modelos Animais de Doenças , Exsudatos e Transudatos/imunologia , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Camundongos Mutantes , Pneumonia/microbiologia , Receptores CCR2/genéticaRESUMO
Little is known about the dynamics of early ecological succession during experimental conventionalization of the gastrointestinal (GI) tract; thus, we measured changes in bacterial communities over time, at two different mucosal sites (cecum and jejunum), with germfree C57BL/6 mice as the recipients of cecal contents (input community) from a C57BL/6 donor mouse. Bacterial communities were monitored using pyrosequencing of 16S rRNA gene amplicon libraries from the cecum and jejunum and analyzed by a variety of ecological metrics. Bacterial communities, at day 1 postconventionalization, in the cecum and jejunum had lower diversity and were distinct from the input community (dominated by either Escherichia or Bacteroides). However, by days 7 and 21, the recipient communities had become significantly diverse and the cecal communities resembled those of the donor and donor littermates, confirming that transfer of cecal contents results in reassembly of the community in the cecum 7 to 21 days later. However, bacterial communities in the recipient jejunum displayed significant structural heterogeneity compared to each other or the donor inoculum or the donor littermates, suggesting that the bacterial community of the jejunum is more dynamic during the first 21 days of conventionalization. This report demonstrates that (i) mature input communities do not simply reassemble at mucosal sites during conventionalization (they first transform into a "pioneering" community and over time take on the appearance, in membership and structure, of the original input community) and (ii) the specific mucosal environment plays a role in shaping the community.
Assuntos
Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Ceco/microbiologia , Ecossistema , Vida Livre de Germes , Jejuno/microbiologia , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Bacteroides/genética , Bacteroides/crescimento & desenvolvimento , Bacteroides/isolamento & purificação , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/genética , Fezes/microbiologia , Genes de RNAr , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Proteobactérias/genética , Proteobactérias/crescimento & desenvolvimento , Proteobactérias/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodosRESUMO
RATIONALE: Bronchoalveolar lavage fluid (BAL) from human lung allografts demonstrates the presence of a multipotent mesenchymal stromal cell population. However, the clinical relevance of this novel cellular component of BAL and its association with bronchiolitis obliterans syndrome (BOS), a disease marked by progressive airflow limitation secondary to fibrotic obliteration of the small airways, remains to be determined. OBJECTIVES: In this study we investigate the association of number of mesenchymal stromal cells in BAL with development of BOS in human lung transplant recipients. METHODS: Mesenchymal colony-forming units (CFUs) were quantitated in a cohort of 405 BAL samples obtained from 162 lung transplant recipients. Poisson generalized estimating equations were used to determine the predictors of BAL mesenchymal CFU count. MEASUREMENTS AND MAIN RESULTS: Higher CFU counts were noted early post-transplantation; time from transplant to BAL of greater than 3 months predicted 0.4-fold lower CFU counts (P = 0.0001). BOS diagnosis less than or equal to 365 days before BAL was associated with a 2.11-fold higher CFU count (P = 0.02). There were 2.62- and 2.70-fold higher CFU counts noted in the presence of histologic diagnosis of bronchiolitis obliterans (P = 0.05) and organizing pneumonia (0.0003), respectively. In BAL samples obtained from BOS-free patients greater than 6 months post-transplantation (n = 173), higher mesenchymal CFU counts (≥10) significantly predicted BOS onset in both univariate (hazard ratio, 5.61; 95% CI, 3.03-10.38; P < 0.0001) and multivariate (hazard ratio, 5.02; 95% CI, 2.40-10.51; P < 0.0001) Cox regression analysis. CONCLUSIONS: Measurement of mesenchymal CFUs in the BAL provides predictive information regarding future BOS onset.
Assuntos
Bronquiolite Obliterante/etiologia , Líquido da Lavagem Broncoalveolar/citologia , Transplante de Pulmão/efeitos adversos , Células-Tronco Mesenquimais/fisiologia , Adulto , Idoso , Biomarcadores , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Distribuição de Poisson , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Estatísticas não Paramétricas , Células-Tronco/citologia , Adulto JovemRESUMO
Multipotent mesenchymal progenitor cells, termed "mesenchymal stem cells" (MSCs), have been demonstrated to reside in human adult lungs. However, there is little information regarding the associations of these local mesenchymal progenitors with other resident somatic cells and their potential for therapeutic use. Here we provide in vivo and in vitro evidence for the ability of human adult lung-resident MSCs (LR-MSCs) to interact with the local epithelial cells. The in vivo retention and localization of human LR-MSCs in an alveolar microenvironment was investigated by placing PKH-26 or DsRed lentivirus-labeled human LR-MSCs in the lungs of immunodeficient (SCID) mice. At 3 weeks after intratracheal administration, 19.3 ± 3.21% of LR-MSCs were recovered, compared with 3.47 ± 0.51% of control fibroblasts, as determined by flow cytometry. LR-MSCs were found to persist in murine lungs for up to 6 months and demonstrated preferential localization to the corners of the alveoli in close proximity to type II alveolar epithelial cells, the progenitor cells of the alveolar epithelium. In vitro, LR-MSCs established gap junction communications with lung alveolar and bronchial epithelial cells and demonstrated an ability to secrete keratinocyte growth factor, an important modulator of epithelial cell proliferation and differentiation. Gap junction communications were also demonstrable between LR-MSCs and resident murine cells in vivo. This study demonstrates, for the first time, an ability of tissue-specific MSCs to engraft in their organ of origin and establishes a pathway of bidirectional interaction between these mesenchymal progenitors and adult somatic epithelial cells in the lung.
Assuntos
Células Epiteliais Alveolares/metabolismo , Comunicação Celular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Alvéolos Pulmonares/cirurgia , Animais , Separação Celular/métodos , Rastreamento de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Fator 7 de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Junções Comunicantes/metabolismo , Vetores Genéticos , Sobrevivência de Enxerto , Humanos , Lentivirus/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos , Camundongos SCID , Compostos Orgânicos/metabolismo , Alvéolos Pulmonares/metabolismo , Fatores de Tempo , Transfecção , Proteína Vermelha FluorescenteRESUMO
Aspergillus fumigatus, a ubiquitous airborne fungus, can cause invasive infection in immunocompromised individuals but also triggers allergic bronchopulmonary aspergillosis in a subset of otherwise healthy individuals repeatedly exposed to the organism. This study addresses a critical gap in our understanding of the immunoregulation in response to repeated exposure to A. fumigatus conidia. C57BL/6 mice were challenged intranasally with A. fumigatus conidia weekly, and leukocyte composition, activation, and cytokine production were examined after two, four, and eight challenges. Approximately 99% of A. fumigatus conidia were cleared within 24 h after inoculation, and repeated exposure to A. fumigatus conidia did not result in hyphal growth or accumulation of conidia with time. After 2 challenges, there was an early influx of neutrophils and regulatory T (T(reg)) cells into the lungs but minimal inflammation. Repeated exposure promoted sustained expansion of the draining lymph nodes, while the influx of eosinophils and other myeloid cells into the lungs peaked after four exposures and then decreased despite continued A. fumigatus challenges. Goblet cell metaplasia and low-level fibrosis were evident during the response. Repeated exposure to A. fumigatus conidia induced T cell activation in the lungs and the codevelopment by four exposures of T(H)1, T(H)2, and T(H)17 responses in the lungs, which were maintained through eight exposures. Changes in CD4 T cell polarization or T(reg) numbers did not account for the reduction in myeloid cell numbers later in the response, suggesting a non-T-cell regulatory pathway involved in dampening inflammation during repeated exposure to A. fumigatus conidia.
Assuntos
Aspergillus fumigatus/imunologia , Aspergilose Pulmonar/imunologia , Células Th1/fisiologia , Células Th17/fisiologia , Células Th2/fisiologia , Animais , Linfócitos T CD4-Positivos , Inflamação/imunologia , Inflamação/patologia , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Esporos Fúngicos/imunologia , Fatores de TempoRESUMO
The immune response to Cryptococcus neoformans following pulmonary infection of C57BL/6 wild-type (WT) mice results in the development of persistent infection with characteristics of allergic bronchopulmonary mycosis (ABPM). To further clarify the role of Th1/Th2 polarizing cytokines in this model, we performed kinetic analysis of cytokine responses and compared cytokine profiles, pathologies, and macrophage (Mac) polarization status in C. neoformans-infected WT, interleukin-4-deficient (IL-4(-/-)), and gamma interferon-deficient (IFN-γ(-/-)) C57BL/6 mice. Results show that cytokine expression in the infected WT mice is not permanently Th2 biased but changes dynamically over time. Using multiple Mac activation markers, we further demonstrate that IL-4 and IFN-γ regulate the polarization state of Macs in this model. A higher IL-4/IFN-γ ratio leads to the development of alternatively activated Macs (aaMacs), whereas a higher IFN-γ/IL-4 ratio leads to the generation of classically activated Macs (caMacs). WT mice that coexpress IL-4 and IFN-γ during fungal infection concurrently display both types of Mac polarization markers. Concurrent stimulation of Macs with IFN-γ and IL-4 results in an upregulation of both sets of markers within the same cells, i.e., formation of an intermediate aaMac/caMac phenotype. These cells express both inducible nitric oxide synthase (important for clearance) and arginase (associated with chronic/progressive infection). Together, our data demonstrate that the interplay between Th1 and Th2 cytokines supports chronic infection, chronic inflammation, and the development of ABPM pathology in C. neoformans-infected lungs. This cytokine interplay modulates Mac differentiation, including generation of an intermediate caMac/aaMac phenotype, which in turn may support chronic "steady-state" fungal infection and the resultant ABPM pathology.
Assuntos
Criptococose/imunologia , Citocinas/imunologia , Pneumopatias Fúngicas/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Animais , Criptococose/patologia , Cryptococcus neoformans/imunologia , Feminino , Imunofluorescência , Imuno-Histoquímica , Interleucina-4/deficiência , Pneumopatias Fúngicas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Although γherpesvirus infections are associated with enhanced lung fibrosis in both clinical and animal studies, there is limited understanding about fibrotic effects of γherpesviruses on cell types present in the lung, particularly during latent infection. Wild-type mice were intranasally infected with a murine γherpesvirus (γHV-68) or mock-infected with saline. Twenty-eight days postinfection (dpi), â¼14 days following clearance of the lytic infection, alveolar macrophages (AMs), mesenchymal cells, and CD19-enriched cell populations from the lung and spleen express M(3) and/or glycoprotein B (gB) viral mRNA and harbor viral genome. AMs from infected mice express more transforming growth factor (TGF)-ß(1), CCL2, CCL12, TNF-α, and IFN-γ than AMs from mock-infected mice. Mesenchymal cells express more total TGF-ß(1), CCL12, and TNF-α than mesenchymal cells from mock-infected mice. Lung and spleen CD19-enriched cells express more total TGF-ß(1) 28 dpi compared with controls. The CD19-negative fraction of the spleen overexpresses TGF-ß(1) and harbors viral genome, but this likely represents infection of monocytes. Purified T cells from the lung harbor almost no viral genome. Purified T cells overexpress IL-10 but not TGF-ß(1). Intracellular cytokine staining demonstrated that lung T cells at 28 dpi produce IFN-γ but not IL-4. Thus infection with a murine γherpesvirus is sufficient to upregulate profibrotic and proinflammatory factors in a variety of lung resident and circulating cell types 28 dpi. Our results provide new information about possible contributions of these cells to fibrogenesis in the lungs of individuals harboring a γherpesvirus infection and may help explain why γHV-68 infection can augment or exacerbate fibrotic responses in mice.
Assuntos
Citocinas/biossíntese , Infecções por Herpesviridae/imunologia , Fibrose Pulmonar/etiologia , Rhadinovirus/patogenicidade , Infecções Tumorais por Vírus/imunologia , Animais , Sequência de Bases , Quimiocina CCL2/biossíntese , Primers do DNA/genética , DNA Viral/genética , Modelos Animais de Doenças , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Pulmão/imunologia , Pulmão/virologia , Ativação de Macrófagos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Masculino , Mesoderma/imunologia , Mesoderma/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quimioatraentes de Monócitos/biossíntese , Fibrose Pulmonar/genética , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/virologia , Baço/imunologia , Baço/virologia , Linfócitos T/imunologia , Linfócitos T/virologia , Fator de Crescimento Transformador beta1/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia , Carga ViralRESUMO
Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study, we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First, CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes, including macrophages, CD4+ T cells, and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40, systemic cryptococcal dissemination was increased, and mice died of central nervous system infection. Second, CD40 promoted pathological changes in the airways, including intraluminal mucus production and subepithelial collagen deposition, but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively, these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some, but not all, elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung.
Assuntos
Antígenos CD40/imunologia , Criptococose , Cryptococcus neoformans/fisiologia , Pneumopatias Fúngicas , Pulmão , Animais , Antígenos CD40/genética , Células Cultivadas , Infecções Fúngicas do Sistema Nervoso Central/imunologia , Infecções Fúngicas do Sistema Nervoso Central/microbiologia , Criptococose/imunologia , Criptococose/microbiologia , Criptococose/patologia , Cryptococcus neoformans/imunologia , Interferon gama/imunologia , Leucócitos/citologia , Leucócitos/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Pneumopatias Fúngicas/imunologia , Pneumopatias Fúngicas/microbiologia , Pneumopatias Fúngicas/patologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
To determine whether TLR9 signaling contributes to the development of the adaptive immune response to cryptococcal infection, wild-type (TLR9+/+) and TLR9 knockout (TLR9-/-) BALB/c mice were infected intratracheally with 10(4) C. neoformans 52D. We evaluated 1) organ microbial burdens, 2) pulmonary leukocyte recruitment, 3) pulmonary and systemic cytokine induction, and 4) macrophage activation profiles. TLR9 deletion did not affect pulmonary growth during the innate phase, but profoundly impaired pulmonary clearance during the adaptive phase of the immune response (a 1000-fold difference at week 6). The impaired clearance in TLR9-/- mice was associated with: 1) significantly reduced CD4(+), CD8+ T cell, and CD19+ B cell recruitment into the lungs; 2) defects in Th polarization indicated by altered cytokine responses in the lungs, lymphonodes, and spleen; and 3) diminished macrophage accumulation and altered activation profile, including robust up-regulation of Arg1 and FIZZ1 (indicators of alternative activation) and diminished induction of inducible nitric oxide synthase (an indicator of classical activation). Histological analysis revealed defects in granuloma formation and increased numbers of intracellular yeast residing within macrophages in the lungs of TLR9-/- mice. We conclude that TLR9 signaling plays an important role in the development of robust protective immunity, proper recruitment and function of effector cells (lymphocytes and macrophages), and, ultimately, effective cryptococcal clearance from the infected lungs.
Assuntos
Imunidade Adaptativa/imunologia , Criptococose/imunologia , Cryptococcus neoformans/imunologia , Pneumopatias , Transdução de Sinais/imunologia , Receptor Toll-Like 9/metabolismo , Animais , Cryptococcus neoformans/patogenicidade , Citocinas/imunologia , Feminino , Humanos , Pneumopatias/imunologia , Pneumopatias/microbiologia , Pneumopatias/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Receptor Toll-Like 9/genéticaRESUMO
To investigate the role of CD11c(+) cells in endotoxin-induced acute lung injury, wild-type or CD11c-diphtheria toxin receptor transgenic mice were treated with intraperitoneal diphtheria toxin (5 ng/g b.wt.) in the presence or absence of intratracheal lipopolysaccharide (51 microg). Lipopolysaccharide treatment resulted in 100% mortality in CD11c-depleted animals but not in control animals. Analysis of local lung tissue revealed no differences in acute lung injury severity; however, analysis of distal tissues revealed severe damage and necrosis to multiple organs (liver, spleen, and kidneys) in CD11c-diphtheria toxin receptor mice but not in wild-type mice. In addition, dramatic increases in systemic levels of liver enzymes (alanine aminotransferase, 657 U/L, aspartate aminotransferase, 1401 U/L), blood urea (53 mg/dl), and 8-iso-prostaglandin F(2alpha), a marker of oxidative stress (350 pg/ml), were observed. These data demonstrate that CD11c(+) cells play a critical role in protecting the organs from systemic injury caused by a pulmonary endotoxin challenge.
Assuntos
Lesão Pulmonar Aguda/complicações , Lesão Pulmonar Aguda/patologia , Antígeno CD11c/metabolismo , Insuficiência de Múltiplos Órgãos/complicações , Insuficiência de Múltiplos Órgãos/patologia , Lesão Pulmonar Aguda/sangue , Lesão Pulmonar Aguda/enzimologia , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Biomarcadores/metabolismo , Nitrogênio da Ureia Sanguínea , Capilares/efeitos dos fármacos , Capilares/patologia , Citocinas/genética , Citocinas/metabolismo , Toxina Diftérica/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência de Múltiplos Órgãos/sangue , Insuficiência de Múltiplos Órgãos/enzimologia , Necrose , Estresse Oxidativo/efeitos dos fármacos , Alvéolos Pulmonares/irrigação sanguínea , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Solubilidade/efeitos dos fármacos , Análise de SobrevidaRESUMO
Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans is associated with the CCR2-mediated accumulation of lung dendritic cells (DC) and the development of a T1 adaptive immune response. The objective of this study was to identify the circulating DC precursor(s) responsible for this large increase in lung DC numbers. An established murine model was used to evaluate putative DC precursors in the blood, bone marrow, and lungs of CCR2(+/+) mice and CCR2(-/-) mice throughout a time course following infection with C. neoformans. Results demonstrate that numbers of Ly-6C(high) monocytes increased in parallel in the peripheral blood and lungs of CCR(+/+) mice, whereas CD11c(+) MHC class II(+) pre-DC were 10-fold less prevalent in the peripheral blood and did not differ between the two strains. Accumulation of Ly-6C(high) monocytes correlated with a substantial increase in the numbers of CD11b(+) DC in the lungs of infected CCR2(+/+) mice. Comparative phenotypic analysis of lung cells recovered in vivo suggests that Ly-6C(high) monocytes differentiate into CD11b(+) DC in the lung; differentiation is associated with up-regulation of costimulatory molecules and decreased Ly-6C expression. Furthermore, in vitro experiments confirmed that Ly-6C(high) monocytes differentiate into CD11b(+) DC. Accumulation of Ly-6C(high) monocytes and CD11b(+) DC was not attributable to their proliferation in situ. We conclude that the CCR2-mediated accumulation of CD11b(+) DC in the lungs of Cryptococcus-infected mice is primarily attributable to the continuous recruitment and differentiation of Ly-6C(high) monocytes.
Assuntos
Antígenos Ly/biossíntese , Antígeno CD11b/biossíntese , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Pneumopatias Fúngicas/imunologia , Pulmão/imunologia , Monócitos/imunologia , Receptores CCR2/fisiologia , Animais , Antígenos Ly/fisiologia , Contagem de Células , Diferenciação Celular/genética , Movimento Celular/genética , Proliferação de Células , Criptococose/imunologia , Criptococose/metabolismo , Criptococose/patologia , Cryptococcus neoformans/imunologia , Citocinese/genética , Citocinese/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Feminino , Pulmão/metabolismo , Pulmão/patologia , Pneumopatias Fúngicas/microbiologia , Pneumopatias Fúngicas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/metabolismo , Monócitos/patologia , Receptores CCR2/biossíntese , Receptores CCR2/deficiência , Células-Tronco/imunologia , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
RATIONALE: No effective treatment exists for idiopathic pulmonary fibrosis, and its pathogenesis remains unclear. Accumulating evidence implicates herpesviruses as cofactors (either initiating or exacerbating agents) of fibrotic lung disease, but a role for latent herpesvirus infection has not been studied. OBJECTIVES: To develop a murine model to determine whether latent herpesvirus infection can augment fibrotic responses and to gain insight into potential mechanisms of enhanced fibrogenesis. METHODS: Mice were infected with murine gammaherpesvirus 14 to 70 days before a fibrotic challenge with fluorescein isothiocyanate or bleomycin so that the virus was latent at the time of fibrotic challenge. Measurements were made after viral infection alone or after the establishment of fibrosis. MEASUREMENTS AND MAIN RESULTS: gammaHerpesvirus is latent by 14 days post infection, and infection 14 to 70 days before fibrotic challenge augmented fibrosis. Fibrotic augmentation was not dependent on reactivation of the latent virus to a lytic state. Total cell numbers and fibrocyte numbers were increased in the lungs of latently infected mice administered fibrotic challenge compared with mock-infected mice that received fibrotic challenge. Latent infection up-regulates expression of proinflammatory chemokines, transforming growth factor-beta1, and cysteinyl leukotrienes in alveolar epithelial cells. CONCLUSIONS: Latent gammaherpesvirus infection augments subsequent fibrotic responses in mice. Enhanced fibrosis is associated with the induction of profibrotic factors and the recruitment of fibrocytes. Our data complement existing human and animal data supporting the hypothesis that gammaherpesviruses can serve as initiating cofactors in the pathogenesis of pulmonary fibrosis.
Assuntos
Infecções por Herpesviridae/complicações , Fibrose Pulmonar Idiopática/complicações , Animais , Quimiocina CCL2/sangue , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Gammaherpesvirinae/fisiologia , Fibrose Pulmonar Idiopática/virologia , Pulmão/imunologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quimioatraentes de Monócitos/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/sangue , Ativação Viral/fisiologia , Latência Viral/fisiologiaRESUMO
RATIONALE: Idiopathic pulmonary fibrosis (IPF) and other idiopathic interstitial pneumonias (IIPs) have similar clinical and radiographic features, but their histopathology, response to therapy, and natural history differ. A surgical lung biopsy is often required to distinguish between these entities. OBJECTIVES: We sought to determine if clinical variables could predict a histopathologic diagnosis of IPF in patients without honeycomb change on high-resolution computed tomography (HRCT). METHODS: Data from 97 patients with biopsy-proven IPF and 38 patients with other IIPs were examined. Logistic regression models were built to identify the clinical variables that predict histopathologic diagnosis of IPF. MEASUREMENTS AND MAIN RESULTS: Increasing age and average total HRCT interstitial score on HRCT scan of the chest may predict a biopsy confirmation of IPF. Sex, pulmonary function, presence of desaturation, or distance walked during a 6-minute walk test did not help discriminate pulmonary fibrosis from other IIPs. CONCLUSIONS: Clinical data may be used to predict a diagnosis of IPF over other IIPs. Validation of these data with a prospective study is needed.
Assuntos
Fibrose Pulmonar Idiopática/diagnóstico , Fatores Etários , Biópsia , Diagnóstico Diferencial , Teste de Esforço/métodos , Teste de Esforço/estatística & dados numéricos , Feminino , Humanos , Pneumonias Intersticiais Idiopáticas/diagnóstico , Fibrose Pulmonar Idiopática/patologia , Pulmão/diagnóstico por imagem , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Testes de Função Respiratória/métodos , Testes de Função Respiratória/estatística & dados numéricos , Estudos Retrospectivos , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios X/métodosRESUMO
The origin and turnover of connective tissue cells in adult human organs, including the lung, are not well understood. Here, studies of cells derived from human lung allografts demonstrate the presence of a multipotent mesenchymal cell population, which is locally resident in the human adult lung and has extended life span in vivo. Examination of plastic-adherent cell populations in bronchoalveolar lavage samples obtained from 76 human lung transplant recipients revealed clonal proliferation of fibroblast-like cells in 62% (106 of 172) of samples. Immunophenotyping of these isolated cells demonstrated expression of vimentin and prolyl-4-hydroxylase, indicating a mesenchymal phenotype. Multiparametric flow cytometric analyses revealed expression of cell-surface proteins, CD73, CD90, and CD105, commonly found on mesenchymal stem cells (MSCs). Hematopoietic lineage markers CD14, CD34, and CD45 were absent. Multipotency of these cells was demonstrated by their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. Cytogenetic analysis of cells from 7 sex-mismatched lung transplant recipients harvested up to 11 years after transplant revealed that 97.2% +/- 2.1% expressed the sex genotype of the donor. The presence of MSCs of donor sex identity in lung allografts even years after transplantation provides what we believe to be the first evidence for connective tissue cell progenitors that reside locally within a postnatal, nonhematopoietic organ.
Assuntos
Transplante de Pulmão/fisiologia , Células-Tronco Mesenquimais/citologia , Adulto , Antígenos CD/análise , Líquido da Lavagem Broncoalveolar/citologia , Células do Tecido Conjuntivo/citologia , Células do Tecido Conjuntivo/fisiologia , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Pneumopatias/classificação , Pneumopatias/cirurgia , Masculino , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Transplante HomólogoRESUMO
Urease, a major virulence factor for Cryptococcus neoformans, promotes lethal meningitis/encephalitis in mice. The effect of urease within the lung, the primary site of most invasive fungal infections, is unknown. An established model of murine infection that utilizes either urease-producing (wt and ure1::URE1) or urease-deficient (ure1) strains (H99) of C. neoformans was used to characterize fungal clearance and the resultant immune response evoked by these strains within the lung. Results indicate that mice infected with urease-producing strains of C. neoformans demonstrate a 100-fold increase in fungal burden beginning 2 weeks post-infection (as compared with mice infected with urease-deficient organisms). Infection with urease-producing C. neoformans was associated with a highly polarized T2 immune response as evidenced by increases in the following: 1) pulmonary eosinophils, 2) serum IgE levels, 3) T2 cytokines (interleukin-4, -13, and -4 to interferon-gamma ratio), and 4) alternatively activated macrophages. Furthermore, the percentage and total numbers of immature dendritic cells within the lung-associated lymph nodes was markedly increased in mice infected with urease-producing C. neoformans. Collectively, these data define cryptococcal urease as a pulmonary virulence factor that promotes immature dendritic cell accumulation and a potent, yet non-protective, T2 immune response. These findings provide new insights into mechanisms by which microbial factors contribute to the immunopathology associated with invasive fungal disease.
Assuntos
Cryptococcus/enzimologia , Células Dendríticas/imunologia , Urease/farmacologia , Animais , Ensaio de Unidades Formadoras de Colônias , Criptococose/imunologia , Cryptococcus neoformans/enzimologia , Células Dendríticas/efeitos dos fármacos , Proteínas Fúngicas/farmacologia , Humanos , Hospedeiro Imunocomprometido , Leucócitos/patologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Urease/deficiência , Urease/genéticaRESUMO
The present study dissected the role of a Th2 bias in pathogenesis of Cryptococcus neoformans H99 infection by comparing inhalational H99 infections in wild-type BALB/c and IL-4/IL-13 double knockout mice. H99-infected wild-type mice showed all major hallmarks of Th2 but not Th1/Th17 immunity in the lungs and lung-associated lymph nodes. In contrast, the IL-4/13(-/-) mice developed robust hallmarks of Th1 and Th17 but not Th2 polarization. The IL-4/IL-13 deletion prevented pulmonary eosinophilia, goblet cell metaplasia in the airways and resulted in elevated serum IgE, and a switch from alternative to classical activation of macrophages. The development of a robust Th1/Th17 response and classical activation of macrophages resulted in significant containment of H99 in the lungs of IL-4/13(-/-) mice compared with unopposed growth of H99 in the lungs of wild-type mice. However, IL-4/13(-/-) mice showed only 1-week longer survival compared with wild-type mice. The comparison of brain and spleen cryptococcal loads at weeks 2, 3, and 4 postinfection revealed that the systemic dissemination in IL-4/13(-/-) mice occurred with an approximate 1-week delay but subsequently progressed with similar rate as in the wild-type mice. Furthermore, wild-type and IL-4/13(-/-) mice developed equivalently severe meningitis/encephalitis at the time of death. These data indicate that the Th2 immune bias is a crucial mechanism for pulmonary virulence of H99, whereas other mechanisms are largely responsible for its central nervous system tropism and systemic dissemination.
Assuntos
Criptococose/imunologia , Cryptococcus neoformans/patogenicidade , Interleucina-17/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Animais , Quimiotaxia de Leucócito , Criptococose/patologia , Cryptococcus neoformans/imunologia , Progressão da Doença , Interleucina-13/deficiência , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-4/deficiência , Interleucina-4/genética , Interleucina-4/imunologia , Ativação de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th2/imunologia , VirulênciaRESUMO
Development of allograft rejection continues to be the major determinant of morbidity and mortality postlung transplantation. We have recently demonstrated that a population of donor-derived mesenchymal stem cells is present in human lung allografts and can be isolated and expanded ex vivo. In this study, we investigated the impact of lung resident mesenchymal stem cells (LR-MSCs), derived from allografts of human lung transplant recipients, on T cell activation in vitro. Similar to bone marrow-derived MSCs, LR-MSCs did not express MHC II or the costimulatory molecules CD80 or CD86. In vitro, LR-MSCs profoundly suppressed the proliferative capacity of T cells in response to a mitogenic or an allogeneic stimulus. The immunosuppressive function of LR-MSCs was also noted in the absence of direct cell contact, indicating that LR-MSCs mediated their effect predominantly via a soluble mediator. LR-MSCs isolated from lung transplant recipients demonstrated PGE(2) secretion at baseline (385 +/- 375 pg/ml), which increased in response to IL-1beta (1149 +/- 1081 pg/ml). The addition of PG synthesis inhibitors (indomethacin and NS-398) substantially abrogated LR-MSC-mediated immunosuppression, indicating that PGE(2) may be one of the major soluble mediators impacting T cell activity. This is the first report to demonstrate that human tissue-derived MSCs isolated from an allogeneic environment have the potential to mediate immunological responses in vitro.