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1.
Cell ; 187(8): 1874-1888.e14, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38518773

RESUMO

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Pulmão , Polissacarídeos Bacterianos , Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Feminino , Masculino , Camundongos , Biofilmes , Escherichia coli/fisiologia , Hipotermia/metabolismo , Hipotermia/patologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/microbiologia , Pulmão/patologia , Pneumonia/microbiologia , Pneumonia/patologia , Pseudomonas aeruginosa/fisiologia , Células Receptoras Sensoriais , Polissacarídeos Bacterianos/metabolismo , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Nociceptores/metabolismo
2.
Nat Immunol ; 19(2): 192-201, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29335647

RESUMO

Pulmonary immunity requires tight regulation, as interstitial inflammation can compromise gas exchange and lead to respiratory failure. Here we found a greater number of aged CD11bhiL-selectinloCXCR4+ polymorphonuclear leukocytes (PMNs) in lung vasculature than in the peripheral circulation. Using pulmonary intravital microscopy, we observed lung PMNs physically interacting with B cells via ß2 integrins; this initiated neutrophil apoptosis, which led to macrophage-mediated clearance. Genetic deletion of B cells led to the accumulation of aged PMNs in the lungs without systemic inflammation, which caused pathological fibrotic interstitial lung disease that was attenuated by the adoptive transfer of B cells or depletion of PMNs. Thus, the lungs are an intermediary niche in the PMN lifecycle wherein aged PMNs are regulated by B cells, which restrains their potential to cause pulmonary pathology.


Assuntos
Linfócitos B/imunologia , Doenças Pulmonares Intersticiais/patologia , Neutrófilos/patologia , Fibrose Pulmonar/patologia , Animais , Doenças Pulmonares Intersticiais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibrose Pulmonar/imunologia
3.
Reprod Biol Endocrinol ; 21(1): 85, 2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37715251

RESUMO

Adhesion of the implanting blastocyst involves the interaction between integrin proteins expressed by trophoblast cells and components present in the basement membrane of the endometrial luminal epithelium. Although several factors regulating integrins and their adhesion to fibronectin are already known, we showed that Wnt signaling is involved in the regulation of blastocyst adhesion through the trafficking of integrins expressed by trophoblast cells. Localization of Itgα5ß1 by immunofluorescence and FN-binding assays were conducted on peri-implantation blastocysts treated with either Wnt5a or Wnt7a proteins. Both Wnt5a and Wnt7a induced a translocation of Itgα5ß1 at the surface of the blastocyst and an increase in FN-binding activity. We further demonstrated that uterine fluid is capable of inducing integrin translocation and this activity can be specifically inhibited by the Wnt inhibitor sFRP2. To identify the Wnt signaling pathway involved in this activity, blastocysts were incubated with inhibitors of either p38MAPK, PI3K pathway or CamKII prior to the addition of Wnts. Whereas inhibition of p38MAPK and PI3K had not effect, inhibition of CamKII reduced FN-binding activity induced by Wnts. Finally, we demonstrated that inhibition of Wnts by sFRP2 reduced the binding efficiency of the blastocyst to uterine epithelial cells. Our findings provide new insight into the mechanism that regulates integrin trafficking and FN-binding activity and identifies Wnts as a key player in blastocyst attachment to the uterine epithelium.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Fibronectinas , Animais , Camundongos , Feminino , Fosfatidilinositol 3-Quinases , Blastocisto , Via de Sinalização Wnt , Integrinas
4.
Dig Dis Sci ; 61(7): 1895-902, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27098414

RESUMO

BACKGROUND AND AIMS: The density of epithelial cell extrusion zones in the intestinal lining, also known as gap density (number of gaps/1000 epithelial cells counted), can be quantitated using probe-based confocal laser endomicroscopy (pCLE). Gap density has been reported to be higher than normal in both inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) patients. Epithelial cells destined for extrusion from the intestinal surface would stain positive for either activated caspase-1 or caspase-3 on mucosal biopsy samples. The aim of this study was to determine whether epithelial gap density on pCLE correlates with quantitative analysis of activated caspase staining of mucosal biopsy samples from patients. METHODS: We obtained pCLE images and biopsy samples of the terminal ileum during colonoscopies of healthy controls and patients with either IBD or IBS. The pCLE images and biopsy samples were blindly analyzed for gap density and for cells staining positive for activated caspases, respectively. The degree of correlation was determined using nonparametric statistical tests. RESULTS: The median results were 10 gaps/1000 cells counted for controls versus 33 gaps/1000 cells counted for chronic intestinal disorder patients (p = 0.02). Activated caspase staining showed 13 positive cells/1000 epithelial cells counted versus 26 positive cells/1000 epithelial cells counted, respectively (p = 0.02), thus showing a strong correlation with a Spearman's coefficient ρ of 0.61 (strong correlation for ρ = 0.4-0.75, p = 0.01). CONCLUSIONS: Intestinal epithelial gap density via pCLE correlated strongly with quantitative analysis of immunohistochemical staining of mucosal biopsy samples.


Assuntos
Células Epiteliais/fisiologia , Mucosa Gástrica/patologia , Imuno-Histoquímica , Microscopia Confocal , Adulto , Idoso , Caspases/metabolismo , Estudos de Coortes , Feminino , Humanos , Íleo/patologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Coloração e Rotulagem
5.
Nat Cell Biol ; 8(10): 1114-23, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16980957

RESUMO

Embryonic stem (ES) cells are pluripotent cells that can self-renew or differentiate into many cell types. A unique network of transcription factors and signalling molecules are essential for maintaining this capability. Here, we report that a spalt family member, Sall4, is required for the pluripotency of ES cells. Similarly to Oct4, a reduction in Sall4 levels in mouse ES cells results in respecification, under the appropriate culture conditions, of ES cells to the trophoblast lineage. Sall4 regulates transcription of Pou5f1 which encodes Oct4. Sall4 binds to the highly conserved regulatory region of the Pou5f1 distal enhancer and activates Pou5f1 expression in vivo and in vitro. Microinjection of Sall4 small interfering (si) RNA into mouse zygotes resulted in reduction of Sall4 and Oct4 mRNAs in preimplantation embryos and significant expansion of Cdx2 expression into the inner cell mass. These results demonstrate that Sall4 is a transcriptional activator of Pou5f1 and has a critical role in the maintenance of ES cell pluripotency by modulating Oct4 expression. The data also indicates that Sall4 is important for early embryonic cell-fate decisions.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Desenvolvimento Embrionário , Feminino , Camundongos , Dados de Sequência Molecular , Fator 3 de Transcrição de Octâmero/metabolismo , Gravidez , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , Transcrição Gênica
6.
Sci Transl Med ; 14(674): eabq6682, 2022 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-36475902

RESUMO

The lung naturally resists Aspergillus fumigatus (Af) in healthy individuals, but multiple conditions can disrupt this resistance, leading to lethal invasive infections. Core processes of natural resistance and its breakdown are undefined. We investigated three distinct conditions predisposing to lethal aspergillosis-severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection, influenza A viral pneumonia, and systemic corticosteroid use-in human patients and murine models. We found a conserved and essential coupling of innate B1a lymphocytes, Af-binding natural immunoglobulin G antibodies, and lung neutrophils. Failure of this axis concealed Af from neutrophils, allowing rapid fungal invasion and disease. Reconstituting the axis with immunoglobulin therapy reestablished resistance, thus representing a realistic pathway to repurpose currently available therapies. Together, we report a vital host resistance pathway that is responsible for protecting against life-threatening aspergillosis in the context of distinct susceptibilities.


Assuntos
COVID-19 , Neutrófilos , Humanos , Animais , Camundongos , SARS-CoV-2 , Esteroides/uso terapêutico
7.
J Exp Med ; 218(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34313733

RESUMO

Pulmonary innate immunity is required for host defense; however, excessive neutrophil inflammation can cause life-threatening acute lung injury. B lymphocytes can be regulatory, yet little is known about peripheral transitional IgM+ B cells in terms of regulatory properties. Using single-cell RNA sequencing, we discovered eight IgM+ B cell subsets with unique gene regulatory networks in the lung circulation dominated by transitional type 1 B and type 2 B (T2B) cells. Lung intravital confocal microscopy revealed that T2B cells marginate in the pulmonary capillaries via CD49e and require CXCL13 and CXCR5. During lung inflammation, marginated T2B cells dampened excessive neutrophil vascular inflammation via the specialized proresolving molecule lipoxin A4 (LXA4). Exogenous CXCL13 dampened excessive neutrophilic inflammation by increasing marginated B cells, and LXA4 recapitulated neutrophil regulation in B cell-deficient mice during inflammation and fungal pneumonia. Thus, the lung microvasculature is enriched in multiple IgM+ B cell subsets with marginating capillary T2B cells that dampen neutrophil responses.


Assuntos
Linfócitos B/patologia , Pulmão/patologia , Neutrófilos/patologia , Pneumonia/patologia , Animais , Aspergilose/microbiologia , Aspergilose/patologia , Linfócitos B/fisiologia , Capilares/patologia , Adesão Celular , Quimiocina CXCL13/metabolismo , Integrina alfa5/metabolismo , Microscopia Intravital , Lipoxinas/metabolismo , Pulmão/irrigação sanguínea , Pulmão/diagnóstico por imagem , Camundongos Mutantes , Pneumonia/diagnóstico por imagem , Receptores CXCR5/metabolismo , Análise de Célula Única
8.
Nat Commun ; 12(1): 1986, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790266

RESUMO

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia , Algoritmos , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Cromatografia Líquida , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Espectrometria de Massas , Fósforo-Oxigênio Liases/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Temperatura
9.
Microbiome ; 7(1): 1, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30606251

RESUMO

BACKGROUND: Inflammatory bowel diseases (IBD) are a group of complex and multifactorial disorders with unknown etiology. Chronic intestinal inflammation develops against resident intestinal bacteria in genetically susceptible hosts. We hypothesized that host intestinal immunoglobulin (Ig) G can be used to identify bacteria involved in IBD pathogenesis. RESULTS: IgG-bound and -unbound microorganisms were collected from 32 pediatric terminal ileum aspirate washes during colonoscopy [non-IBD (n = 10), Crohn disease (n = 15), and ulcerative colitis (n = 7)], and composition was assessed using the Illumina MiSeq platform. In vitro analysis of invasive capacity was evaluated by fluorescence in situ hybridization and gentamicin invasion assay; immune activation was measured by qPCR. Despite considerable inter-individual variations, IgG binding favored specific and unique mucosa-associated species in pediatric IBD patients. Burkholderia cepacia, Flavonifractor plautii, and Rumminococcus sp. demonstrated increased IgG binding, while Pseudomonas ST29 demonstrated reduced IgG binding, in IBD. In vitro validation confirmed that B. cepacia, F. plautii, and Rumminococcus display invasive potential while Pseudomonas protogens did not. CONCLUSION: Using IgG as a marker of pathobionts in larger patient cohorts to identify microbes and elucidate their role in IBD pathogenesis will potentially underpin new strategies to facilitate development of novel, targeted diagnostic, and therapeutic approaches. Interestingly, this method can be used beyond the scope of this manuscript to evaluate altered gut pathobionts in a number of diseases associated with altered microbiota including arthritis, obesity, diabetes mellitus, alcoholic liver disease, cirrhosis, metabolic syndrome, and carcinomas.


Assuntos
Bactérias/classificação , Imunoglobulina G/metabolismo , Doenças Inflamatórias Intestinais/cirurgia , Metagenômica/métodos , Adolescente , Bactérias/imunologia , Criança , Pré-Escolar , Colonoscopia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Intestinos/imunologia , Masculino , Filogenia
10.
Cell Host Microbe ; 23(1): 121-133.e4, 2018 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-29290576

RESUMO

Candida albicans bloodstream infection causes fungal septicaemia and death in over half of afflicted patients. Polymorphonuclear leukocytes (PMN) mediate defense against invasive candidiasis, but their role in protection versus tissue injury and sepsis is unclear. We observe PMN intravascular swarming and subsequent clustering in response to C. albicans yeast in a lethal septic mouse and human pulmonary circulation model. Live C. albicans sequester to the endothelium and are immediately captured by complement-dependent PMN chemotaxis, which is required for host survival. However, complement activation also leads to Leukotriene B4 (LTB4)-mediated intravascular PMN clustering and occlusion, resulting in capillaritis with pulmonary hemorrhage and hypoxemia. This clustering is unique to fungi and triggered by fungal cell wall components. PMN clustering is absent in mice lacking LTB4-receptor, and capillaritis is attenuated upon pharmacological LTB4 blockade without affecting phagocytosis. Therefore, therapeutically disrupting infection-induced capillaritis may limit organ injury without impairing host defense during fungal sepsis.


Assuntos
Arteriopatias Oclusivas/microbiologia , Candida albicans/imunologia , Candidíase/imunologia , Leucotrieno B4/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Sepse/imunologia , Animais , Arteriopatias Oclusivas/imunologia , Candidíase/microbiologia , Candidíase/patologia , Células Cultivadas , Feminino , Humanos , Pulmão/irrigação sanguínea , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sepse/microbiologia , Sepse/patologia
11.
PLoS One ; 8(12): e80656, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24312491

RESUMO

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1ß. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1ß to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3(-/-) and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1ß (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3(-/-) mice developed severe colitis; IL-1ß treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3(-/-) mice. In contrast, IL-1ß treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3(-/-) mice, and increased severity of disease in WT mice with IL-1ß treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3(-/-) compared to WT mice. IL-1ß treatments elevated macrophage infiltration into infected crypts in Nlrp3(-/-) mice, suggesting that IL-1ß may improve macrophage function, as exogenous administration of IL-1ß increased phagocytosis of C. rodentium by peritoneal Nlrp3(-/-) macrophages in vitro. As well, the exogenous administration of IL-1ß to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3(-/-) mice with IL-1ß seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1ß appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1ß improved bacterial clearance in Nlrp3(-/-) mice but increased tissue damage when given to WT mice.


Assuntos
Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Imunidade Inata , Interleucina-1beta/imunologia , Macrófagos/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/patologia , Feminino , Inflamassomos/genética , Inflamassomos/imunologia , Interleucina-1beta/genética , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR
12.
Inflamm Bowel Dis ; 19(5): 912-21, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23511029

RESUMO

BACKGROUND: Two distinct forms of intestinal epithelial cell (IEC) extrusion are described: 1 with preserved epithelial integrity and 1 that introduced breaches in the epithelial lining. In this study, we sought to determine the mechanism underlying the IEC extrusion that alters the permeability of the gut epithelium. METHODS: IEC extrusions in polarized T84 monolayer were induced with nigericin. Epithelial permeability was assessed with transepithelial electrical resistance and movements of latex microspheres and green fluorescent protein-transfected Escherichia coli across the monolayer. In vivo IEC extrusion was modulated in wild-type and a colitic (interleukin-10 knock-out) mouse model with caspase-1 activation and inhibition. Luminal aspirates and mucosal biopsies from control patients and patients with inflammatory bowel disease were analyzed for caspase-1 and caspase-3&7 activation. RESULTS: Caspase-1-induced IEC extrusion in T84 monolayers resulted in dose-dependent and time-dependent barrier dysfunction, reversible with caspase-1 inhibition. Moreover, the movements of microspheres and microbes across the treated epithelial monolayers were observed. Increased caspase-1-mediated IEC extrusion in interleukin-10 knock-out mice corresponded to enhanced permeation of dextran, microspheres, and translocation of E. coli compared with wild type. Caspase-1 inhibition in interleukin-10 knock-out mice resulted in a time-dependent reduction in cell extrusion and normalization of permeability to microspheres. Increased IEC extrusion in wild-type mice was induced with caspase-1 activation. In human luminal aspirates, the ratio of positively stained caspase-1 to caspase-3&7 cells were 1:1 and 2:1 in control patients and patients with inflammatory bowel disease, respectively; these observations were confirmed by cytochemical analysis of mucosal biopsies. CONCLUSIONS: IEC extrusion mediated by caspase-1 activation contributes to altered intestinal permeability in vitro and in vivo.


Assuntos
Permeabilidade da Membrana Celular , Extensões da Superfície Celular/patologia , Células Epiteliais/patologia , Doenças Inflamatórias Intestinais/patologia , Interleucina-10/fisiologia , Mucosa Intestinal/patologia , Animais , Apoptose , Western Blotting , Estudos de Casos e Controles , Caspase 1/metabolismo , Caspase 3/metabolismo , Diferenciação Celular , Proliferação de Células , Extensões da Superfície Celular/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Epiteliais/metabolismo , Imunofluorescência , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
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