RESUMO
While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity.
Assuntos
Antibacterianos/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Linezolida/uso terapêutico , Mitocôndrias/metabolismo , Peptídeos Cíclicos/uso terapêutico , Ribossomos/metabolismo , Células Th17/fisiologia , Animais , Autoimunidade/efeitos dos fármacos , Diferenciação Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Terapia de Alvo Molecular , Esclerose Múltipla/tratamento farmacológico , NAD/metabolismo , Fosforilação Oxidativa , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismoRESUMO
Segmented filamentous bacteria (SFB) are members of the commensal intestinal microbiome. They are known to contribute to the postnatal maturation of the gut immune system, but also to augment inflammatory conditions in chronic diseases such as Crohn's disease. Living primary tissue slices are ultrathin multicellular sections of the intestine and provide a unique opportunity to analyze tissue-specific immune responses ex vivo. This study aimed to investigate whether supplementation of the gut flora with SFB promotes T helper 17 (Th17) cell responses in primary intestinal tissue slices ex vivo. Primary tissue slices were prepared from the small intestine of healthy Taconic mice with SFB-positive and SFB-negative microbiomes and stimulated with anti-CD3/CD28 or Concanavalin A. SFB-positive and -negative mice exhibited distinct microbiome compositions and Th17 cell frequencies in the intestine and complex microbiota including SFB induced up to 15-fold increase in Th17 cell-associated mediators, serum amyloid A (SAA), and immunoglobulin A (IgA) responses ex vivo. This phenotype could be transmitted by co-housing of mice. Our findings highlight that changes in the gut microbiome can be observed in primary intestinal tissue slices ex vivo. This makes the system very attractive for disease modeling and assessment of new therapies.
Assuntos
Microbioma Gastrointestinal , Homeostase , Células Th17 , Animais , Células Th17/imunologia , Camundongos , Microbioma Gastrointestinal/imunologia , Homeostase/imunologia , Camundongos Endogâmicos C57BL , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologiaRESUMO
BACKGROUND: Aberrant extracellular matrix (ECM) deposition and remodelling is important in the disease pathogenesis of pulmonary fibrosis (PF). We characterised neoepitope biomarkers released by ECM turnover in lung tissue from bleomycin-treated rats and patients with PF and analysed the effects of two antifibrotic drugs: nintedanib and pirfenidone. METHODS: Precision-cut lung slices (PCLS) were prepared from bleomycin-treated rats or patients with PF. PCLS were incubated with nintedanib or pirfenidone for 48 h, and levels of neoepitope biomarkers of type I, III and VI collagen formation or degradation (PRO-C1, PRO-C3, PRO-C6 and C3M) as well as fibronectin (FBN-C) were assessed in the culture supernatants. RESULTS: In rat PCLS, incubation with nintedanib led to a reduction in C3M, reflecting type III collagen degradation. In patient PCLS, incubation with nintedanib reduced the levels of PRO-C3 and C3M, thus showing effects on both formation and degradation of type III collagen. Incubation with pirfenidone had a marginal effect on PRO-C3. There were no other notable effects of either nintedanib or pirfenidone on the other neoepitope biomarkers studied. CONCLUSIONS: This study demonstrated that nintedanib modulates neoepitope biomarkers of type III collagen turnover and indicated that C3M is a promising translational neoepitope biomarker of PF in terms of therapy assessment.
Assuntos
Fibrose Pulmonar Idiopática , Fibrose Pulmonar , Animais , Biomarcadores , Bleomicina/toxicidade , Colágeno Tipo III/metabolismo , Complemento C3/farmacologia , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/patologia , Indóis , Pulmão/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , RatosRESUMO
INTRODUCTION: Human respiratory syncytial virus (HRSV) is a common cause of respiratory tract infections (RTIs) globally and is one of the most fatal infectious diseases for infants in developing countries. Of those infected, 25%-40% aged ≤1 year develop severe lower RTIs leading to pneumonia and bronchiolitis, with ~10% requiring hospitalisation. Evidence also suggests that HRSV infection early in life is a major cause of adult asthma. There is no HRSV vaccine, and the only clinically approved treatment is immunoprophylaxis that is expensive and only moderately effective. New anti-HRSV therapeutic strategies are therefore urgently required. METHODS: It is now established that viruses require cellular ion channel functionality to infect cells. Here, we infected human lung epithelial cell lines and ex vivo human lung slices with HRSV in the presence of a defined panel of chloride (Cl-) channel modulators to investigate their role during the HRSV life-cycle. RESULTS: We demonstrate the requirement for TMEM16A, a calcium-activated Cl- channel, for HRSV infection. Time-of-addition assays revealed that the TMEM16A blockers inhibit HRSV at a postentry stage of the virus life-cycle, showing activity as a postexposure prophylaxis. Another important negative-sense RNA respiratory pathogen influenza virus was also inhibited by the TMEM16A-specific inhibitor T16Ainh-A01. DISCUSSION: These findings reveal TMEM16A as an exciting target for future host-directed antiviral therapeutics.
Assuntos
Anoctamina-1/farmacologia , Anticorpos Antivirais/imunologia , Proteínas de Neoplasias/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/imunologia , Células Cultivadas , Humanos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologiaRESUMO
BACKGROUND: Antiviral drugs such as rupintrivir may have an immune-modulatory effect in experimentally induced allergic asthma with subsequent RV infection. We infected lung slices of house-dust mite (HDM)-sensitized asthmatic mice ex vivo with human rhinovirus (RV) and investigated the effect of the antiviral drug rupintrivir on RV-induced cytokine response in lung tissue of HDM-sensitized mice ex vivo. METHODS: Mice were sensitized with HDM. Precision-cut lung slices (PCLS) were prepared from HDM-sensitized or non-sensitized mice. Lung slices were infected ex vivo with RV or RV together with rupintrivir. Modulation of immune responses was evaluated by cytokine secretion 48 h post infection. RESULTS: In vivo HDM sensitization resulted in a TH-2/TH-17-dominated cytokine response that persisted in PCLS ex vivo. RV infection of PCLS from non-sensitized mice resulted in the induction of an antiviral and pro-inflammatory immune response, as indicated by the secretion of IFN-α, IFN-ß, IFN-γ, TNF-α, MCP-1, IP-10, IL-10, and IL-17A. In contrast, PCLS from HDM-sensitized mice showed an attenuated antiviral response, but exaggerated IL-4, IL-6, and IL-10 secretion upon infection. Rupintrivir inhibited exaggerated pro-inflammatory cytokine IL-6 and TH-2 cytokine IL-4 in HDM-sensitized mice. CONCLUSIONS: In summary, this study demonstrates that treatment with rupintrivir influences virus-induced IL-4 and IL-6 cytokine release under experimental conditions ex vivo.
Assuntos
Interleucina-4/imunologia , Isoxazóis/farmacologia , Pulmão/imunologia , Pyroglyphidae/imunologia , Pirrolidinonas/farmacologia , Rhinovirus , Células Th2/imunologia , Animais , Antivirais/farmacologia , Citocinas/antagonistas & inibidores , Citocinas/imunologia , Feminino , Interleucina-4/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Cultura de Órgãos , Fenilalanina/análogos & derivados , Células Th2/efeitos dos fármacos , Valina/análogos & derivadosRESUMO
BACKGROUND: Factor XII (FXII) is a serine protease that is involved in activation of the intrinsic blood coagulation, the kallikrein-kinin system and the complement cascade. Although the binding of FXII to the cell surface has been demonstrated, the consequence of this event for proteolytic processing of membrane-anchored proteins has never been described. METHODS: The effect of FXII on the proteolytic processing of the low-density lipoprotein receptor-related protein 1 (LRP1) ectodomain was tested in human primary lung fibroblasts (hLF), alveolar macrophages (hAM) and in human precision cut lung slices (hPCLS). The identity of generated LRP1 fragments was confirmed by MALDI-TOF-MS. Activity of FXII and gelatinases was measured by S-2302 hydrolysis and zymography, respectively. RESULTS: Here, we demonstrate a new function of FXII, namely its ability to process LRP1 extracellular domain. Incubation of hLF, hAM, or hPCLS with FXII resulted in the accumulation of LRP1 ectodomain fragments in conditioned media. This effect was independent of metalloproteases and required FXII proteolytic activity. Binding of FXII to hLF surface induced its conversion to FXIIa and protected FXIIa against inactivation by a broad spectrum of serine protease inhibitors. Preincubation of hLF with collagenase I impaired FXII activation and, in consequence, LRP1 cleavage. FXII-triggered LRP1 processing was associated with the accumulation of gelatinases (MMP-2 and MMP-9) in conditioned media. CONCLUSIONS: FXII controls LRP1 levels and function at the plasma membrane by modulating processing of its ectodomain. GENERAL SIGNIFICANCE: FXII-dependent proteolytic processing of LRP1 may exacerbate extracellular proteolysis and thus promote pathological tissue remodeling.
Assuntos
Fator XII/farmacologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Gelatinases/metabolismo , Humanos , Domínios Proteicos , ProteóliseRESUMO
Multiple subsets of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent dendritic cells (DCs) control T-cell tolerance and immunity. In mice, Batf3-dependent CD103(+) DCs efficiently enter lymph nodes and cross-present antigens, rendering this conserved DC subset a promising target for tolerance induction or vaccination. However, only limited numbers of CD103(+) DCs can be isolated with current methods. Established bone marrow culture protocols efficiently generate monocyte-derived DCs or produce a mixture of FLT3L-dependent DC subsets. We show that CD103(+) DC development requires prolonged culture time and continuous action of both FLT3L and granulocyte macrophage colony-stimulating factor (GM-CSF), explained by a dual effect of GM-CSF on DC precursors and differentiating CD103(+) DCs. Accordingly, we established a novel method to generate large numbers of CD103(+) DCs (iCD103-DCs) with limited presence of other DC subsets. iCD103-DCs develop in a Batf3- and Irf8-dependent fashion, express a CD8α/CD103 DC gene signature, cross-present cell-associated antigens, and respond to TLR3 stimulation. Thus, iCD103-DCs reflect key features of tissue CD103(+) DCs. Importantly, iCD103-DCs express high levels of CCR7 upon maturation and migrate to lymph nodes more efficiently than classical monocyte-derived DCs. Finally, iCD103-DCs induce T cell-mediated protective immunity in vivo. Our study provides insights into CD103(+) DC development and function.
Assuntos
Antígenos CD/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Células Dendríticas/citologia , Células Dendríticas/imunologia , Cadeias alfa de Integrinas/imunologia , Proteínas Repressoras/imunologia , Animais , Antígenos CD/análise , Fatores de Transcrição de Zíper de Leucina Básica/análise , Diferenciação Celular , Células Cultivadas , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunidade Celular , Cadeias alfa de Integrinas/análise , Proteínas de Membrana/imunologia , Camundongos , Proteínas Repressoras/análise , Linfócitos T/imunologia , Receptor 3 Toll-Like/imunologiaRESUMO
Allergies are mainly characterized as an unrestrained Th2-biased immune response. Epidemiological data associate protection from allergic diseases with the exposure to certain infectious agents during early stages of life. Modulation of the immune response by pathogens has been considered to be a major factor influencing this protection. Recent evidence indicates that immunoregulatory mechanisms induced upon infection ameliorate allergic disorders. A longitudinal study has demonstrated reduced frequency and incidence of asthma in children who reported a prior infection with Salmonella. Experimental studies involving Salmonella enterica serovar Typhimurium-infected murine models have confirmed protection from induced allergic airway inflammation; however, the underlying cause leading to this amelioration remains incompletely defined. In this study, we aimed to delineate the regulatory function of Salmonella Typhimurium infection in the amelioration of allergic airway inflammation in mice. We observed a significant increase in CD11b+ Gr1+ myeloid cell populations in mice after infection with S. Typhimurium. Using in vitro and in vivo studies, we confirmed that these myeloid cells reduce airway inflammation by influencing Th2 cells. Further characterization showed that the CD11b+ Gr1+ myeloid cells exhibited their inhibitory effect by altering GATA-3 expression and interleukin-4 (IL-4) production by Th2 cells. These results indicate that the expansion of myeloid cells upon S. Typhimurium infection could potentially play a significant role in curtailing allergic airway inflammation. These findings signify the contribution of myeloid cells in preventing Th2-mediated diseases and suggest their possible application as therapeutics.
Assuntos
Antígeno CD11b/imunologia , Hipersensibilidade/imunologia , Inflamação/imunologia , Receptores de Quimiocinas/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Diferenciação Celular/imunologia , Fatores de Transcrição Forkhead/imunologia , Fator de Transcrição GATA3/imunologia , Hipersensibilidade/microbiologia , Inflamação/microbiologia , Interleucina-4/imunologia , Estudos Longitudinais , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/imunologia , Células Mieloides/microbiologia , Infecções por Salmonella/microbiologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/microbiologia , Células Th2/imunologia , Células Th2/microbiologiaRESUMO
Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8⺠T-cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC-SIGN (DC-specific-ICAM3-grabbing-nonintegrin/CD209) is a C-type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC-SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC-SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti-DC-SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti-DC-SIGN antibodies conjugated to OVA induced strong and persistent antigen-specific CD4⺠and CD8⺠T-cell responses, which efficiently protected from infection with OVA-expressing Listeria monocytogenes. Thus, we propose DC targeting via DC-SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/imunologia , Lectinas Tipo C/metabolismo , Listeria monocytogenes/imunologia , Receptores de Superfície Celular/metabolismo , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Antígeno CD11c/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Humanos , Imunidade Ativa , Imunidade Celular , Imunomodulação , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Listeria monocytogenes/genética , Camundongos , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Transgenes/genética , VacinaçãoRESUMO
The respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections associated with numerous hospitalizations. Recently, intramuscular (i.m.) vaccines against RSV have been approved for elderly and pregnant women. Noninvasive mucosal vaccination, e.g., by inhalation, offers an alternative against respiratory pathogens like RSV. Effective mucosal vaccines induce local immune responses, potentially resulting in the efficient and fast elimination of respiratory viruses after natural infection. To investigate this immune response to an RSV challenge, low-energy electron inactivated RSV (LEEI-RSV) was formulated with phosphatidylcholine-liposomes (PC-LEEI-RSV) or 1,2-dioleoyl-3-trimethylammonium-propane and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DD-LEEI-RSV) for vaccination of mice intranasally. As controls, LEEI-RSV and formalin-inactivated-RSV (FI-RSV) were used via i.m. vaccination. The RSV-specific immunogenicity of the different vaccines and their protective efficacy were analyzed. RSV-specific IgA antibodies and a statistically significant reduction in viral load upon challenge were detected in mucosal DD-LEEI-RSV-vaccinated animals. Alhydrogel-adjuvanted LEEI-RSV i.m. showed a Th2-bias with enhanced IgE, eosinophils, and lung histopathology comparable to FI-RSV. These effects were absent when applying the mucosal vaccines highlighting the potential of DD-LEEI-RSV as an RSV vaccine candidate and the improved performance of this mucosal vaccine candidate.
Assuntos
Anticorpos Antivirais , Imunidade nas Mucosas , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Células Th2 , Vacinas de Produtos Inativados , Animais , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/imunologia , Camundongos , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Feminino , Células Th2/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Imunização , Vírus Sincicial Respiratório Humano/imunologia , Vacinação/métodos , Vírus Sinciciais Respiratórios/imunologia , Carga Viral , Imunoglobulina A/imunologiaRESUMO
Rodent models of lipopolysaccharide (LPS)-induced pulmonary inflammation are used for anti-inflammatory drug testing. We aimed to characterize mice responses to aerosolized LPS alone or with intraperitoneal (i.p.) delivery of alpha1-antitrypsin (AAT). Balb/c mice were exposed to clean air or aerosolized LPS (0.21 mg/mL) for 10 min per day, for 3 d. One hour after each challenge, animals were treated i.p. with saline or with (4 mg/kg body weight) one of the AAT preparations: native (AAT), oxidized (oxAAT), recombinant (recAAT), or peptide of AAT (C-36). Experiments were terminated 6 h after the last dose of AATs. Transcriptome data of mice lungs exposed to clean air versus LPS revealed 656 differentially expressed genes and 155 significant gene ontology terms, including neutrophil migration and toll-like receptor signaling pathways. Concordantly, mice inhaling LPS showed higher bronchoalveolar lavage fluid neutrophil counts and levels of myeloperoxidase, inducible nitric oxide synthase, IL-1ß, TNFα, KC, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Plasma inflammatory markers did not increase. After i.p. application of AATs, about 1% to 2% of proteins reached the lungs but, except for GM-CSF, none of the proteins significantly influenced inflammatory markers. All AATs and C-36 significantly inhibited LPS-induced GM-CSF release. Surprisingly, only oxAAT decreased the expression of several LPS-induced inflammatory genes, such as Cxcl3, Cd14, Il1b, Nfkb1, and Nfkb2, in lung tissues. According to lung transcriptome data, oxAAT mostly affected genes related to transcriptional regulation while native AAT or recAAT affected genes of inflammatory pathways. Hence, we present a feasible mice model of local lung inflammation induced via aerosolized LPS that can be useful for systemic drug testing.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Pneumonia , alfa 1-Antitripsina , Animais , Humanos , Camundongos , Líquido da Lavagem Broncoalveolar , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Lipopolissacarídeos/efeitos adversos , Pulmão/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/tratamento farmacológico , alfa 1-Antitripsina/uso terapêuticoRESUMO
Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections in the elderly and in children, associated with pediatric hospitalizations. Recently, first vaccines have been approved for people over 60 years of age applied by intramuscular injection. However, a vaccination route via mucosal application holds great potential in the protection against respiratory pathogens like RSV. Mucosal vaccines induce local immune responses, resulting in a fast and efficient elimination of respiratory viruses after natural infection. Therefore, a low-energy electron irradiated RSV (LEEI-RSV) formulated with phosphatidylcholine-liposomes (PC-LEEI-RSV) was tested ex vivo in precision cut lung slices (PCLSs) for adverse effects. The immunogenicity and protective efficacy in vivo were analyzed in an RSV challenge model after intranasal vaccination using a homologous prime-boost immunization regimen. No side effects of PC-LEEI-RSV in PCLS and an efficient antibody induction in vivo could be observed. In contrast to unformulated LEEI-RSV, the mucosal vaccination of mice with PC formulated LEEI-RSV showed a statistically significant reduction in viral load after challenge. These results are a proof-of-principle for the use of LEEI-inactivated viruses formulated with liposomes to be administered intranasally to induce a mucosal immunity that could also be adapted for other respiratory viruses.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Humanos , Criança , Camundongos , Animais , Pessoa de Meia-Idade , Idoso , Lipossomos , Elétrons , Anticorpos Antivirais , Pulmão , Imunidade nas Mucosas , Modelos Animais de Doenças , Camundongos Endogâmicos BALB CRESUMO
Epithelial tissues represent vital interfaces between organisms and their environment. As they are constantly exposed to harmful pathogens, innocuous commensals, and environmental microbes, it is essential they sense and elicit appropriate responses toward these different types of microbes. Here, we demonstrate that the epithelial cytokine interleukin-36γ (IL-36γ) acts as a global discriminator of pathogenic and harmless microbes via cell damage and proteolytic activation. We show that intracellular pro-IL-36γ is upregulated by both fungal and bacterial epithelial microbes; yet, it is only liberated from cells, and subsequently processed to its mature, potent, proinflammatory form, by pathogen-mediated cell damage and pathogen-derived proteases. This work demonstrates that IL-36γ senses pathogen-induced cell damage and proteolytic activity and is a key initiator of immune responses and pathological inflammation within epithelial tissues. As an apically located epithelial proinflammatory cytokine, we therefore propose that IL-36γ is critical as the initial discriminator of harmless microbes and invasive pathogens within epithelial tissues.
Assuntos
Citocinas/metabolismo , Epitélio/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Interleucina-1/metabolismo , Animais , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
Suicide is a leading cause of death in the United States and is the second leading cause of death in the U.S. military. Previous research suggests that data obtained from social media networks may provide important clues for identifying at-risk individuals. To test this possibility, the social media profiles from 315 military personnel who died by suicide (n = 157) or other causes (n = 158) were coded for the presence of stressful life situations (i.e., triggers), somatic complaints or health issues (i.e., physical), maladaptive or avoidant coping strategies (i.e., behaviors), negative mood states (i.e., emotion), and/or negative cognitive appraisals (cognition). Content codes were subsequently analyzed using multilevel models from a dynamical systems perspective to identify temporal change processes characteristic of suicide death. Results identified temporal sequences unique to suicide, notably social media posts about triggers followed by more posts about cognitions, posts about cognitions followed by more posts about triggers, and posts about behaviors followed by fewer posts about cognitions. Results suggest that certain sequences in social media content may predict cause of death and provide an estimate of when a social media user is likely to die by suicide.
Assuntos
Militares , Mídias Sociais , Prevenção do Suicídio , Suicídio , Adaptação Psicológica , Adulto , Cognição , Feminino , Disparidades nos Níveis de Saúde , Humanos , Acontecimentos que Mudam a Vida , Masculino , Militares/psicologia , Militares/estatística & dados numéricos , Medição de Risco/métodos , Fatores de Risco , Rede Social , Estresse Psicológico/epidemiologia , Suicídio/psicologia , Suicídio/estatística & dados numéricos , Estados Unidos/epidemiologiaRESUMO
Macrophages are key cells of the innate immune system and act as tissue resident macrophages (TRMs) in the homeostasis of various tissues. Given their unique functions and therapeutic use as well as the feasibility to derive macrophages in vitro from hematopoietic stem cell (HSC) sources, we propose an "easy-to-use" immune cell spray (ICS) formulation to effectively deliver HSC-derived macrophages. To achieve this aim, we used classical pump spray devices to spray either the human myeloid cell line U937 or primary murine HSC-derived macrophages. For both cell types used, one puff could deliver cells with maintained morphology and functionality. Of note, cells tolerated the spraying process very well with a recovery of more than 90%. In addition, we used osmotic preconditioning to reduce the overall cell size of macrophages. While a 800 mosm hyperosmolar sucrose solution was able to reduce the cell size by 27%, we identified 600 mosm to be effective to reduce the cell size by 15% while maintaining macrophage morphology and functionality. Using an isolated perfused rat lung preparation, the combinatorial use of the ICS with preconditioned and genetically labeled U937 cells allowed the intra-pulmonary delivery of cells, thus paving the way for a new cell delivery platform.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Macrófagos/transplante , Monócitos/transplante , Animais , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/instrumentação , Estudos de Viabilidade , Células-Tronco Hematopoéticas/fisiologia , Humanos , Células K562 , Pulmão , Macrófagos/fisiologia , Camundongos , Monócitos/fisiologia , Osmose , Perfusão , Cultura Primária de Células/métodos , Ratos , Células U937RESUMO
The increasing number of severe infections with multi-drug-resistant pathogens worldwide highlights the need for alternative treatment options. Given the pivotal role of phagocytes and especially alveolar macrophages in pulmonary immunity, we introduce a new, cell-based treatment strategy to target bacterial airway infections. Here we show that the mass production of therapeutic phagocytes from induced pluripotent stem cells (iPSC) in industry-compatible, stirred-tank bioreactors is feasible. Bioreactor-derived iPSC-macrophages (iPSC-Mac) represent a highly pure population of CD45+CD11b+CD14+CD163+ cells, and share important phenotypic, functional and transcriptional hallmarks with professional phagocytes, however with a distinct transcriptome signature similar to primitive macrophages. Most importantly, bioreactor-derived iPSC-Mac rescue mice from Pseudomonas aeruginosa-mediated acute infections of the lower respiratory tract within 4-8 h post intra-pulmonary transplantation and reduce bacterial load. Generation of specific immune-cells from iPSC-sources in scalable stirred-tank bioreactors can extend the field of immunotherapy towards bacterial infections, and may allow for further innovative cell-based treatment strategies.
Assuntos
Infecções Bacterianas/prevenção & controle , Reatores Biológicos , Imunoterapia/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos/citologia , Infecções Respiratórias/prevenção & controle , Animais , Infecções Bacterianas/imunologia , Técnicas de Cultura de Células , Humanos , Macrófagos/fisiologia , Camundongos , Microscopia Eletrônica de Varredura , Pseudomonas aeruginosa/patogenicidade , Infecções Respiratórias/imunologiaRESUMO
Increased procoagulant activity in the alveolar compartment and uncontrolled inflammation are hallmarks of the acute respiratory distress syndrome (ARDS). Here, we investigated whether the contact phase system of coagulation is activated and may regulate inflammatory responses in human lungs. Components of the contact phase system were characterized in bronchoalveolar lavage fluids (BALF) from 54 ARDS patients and 43 controls, and their impact on cytokine/chemokine expression in human precision cut lung slices (PCLS) was assessed by a PCR array. Activation of the contact system, associated with high levels of coagulation factor XIIa (Hageman factor, FXIIa), plasma kallikrein and bradykinin, occurred rapidly in ARDS lungs after the onset of the disease and virtually normalized within one week from time of diagnosis. FXII levels in BALF were higher in ARDS non-survivors than survivors and were positively correlated with tumor necrosis factor (TNF)-α concentration. FXII induced the production and release of interleukin (IL)-8, IL-1ß, IL-6, leukemia inhibitory factor (LIF), CXCL5 and TNF-α in human PCLS in a kallikrein-kinin-independent manner. In conclusion, accumulation of FXII in ARDS lungs may contribute to the release of pro-inflammatory mediators and is associated with clinical outcome. FXII inhibition may thus offer a novel and promising therapeutic approach to antagonize overwhelming inflammatory responses in ARDS lungs without interfering with vital haemostasis.
Assuntos
Coagulação Sanguínea , Citocinas/metabolismo , Fator XII/metabolismo , Mediadores da Inflamação/metabolismo , Pulmão/metabolismo , Pneumonia/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Adulto , Líquido da Lavagem Broncoalveolar/química , Citocinas/genética , Feminino , Humanos , Pulmão/imunologia , Masculino , Pessoa de Meia-Idade , Pneumonia/sangue , Pneumonia/genética , Pneumonia/imunologia , Síndrome do Desconforto Respiratório/sangue , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/imunologia , Estudos Retrospectivos , Transdução de Sinais , Adulto JovemRESUMO
Probiotics can modulate the immune system, conferring beneficial effects on the host. Understanding how these microorganisms contribute to improve the health status is still a challenge. Previously, we have demonstrated that Enterococcus faecalis CECT7121 implants itself and persists in the murine gastrointestinal tract, and enhances and skews the profile of cytokines towards the Th1 phenotype in several biological models. Given the importance of dendritic cells (DCs) in the orchestration of immunity, the aim of this work was to elucidate the influence of E. faecalis CECT7121 on DCs and the outcome of the immune responses. In this work we show that E. faecalis CECT7121 induces a strong dose-dependent activation of DCs and secretion of high levels of IL-12, IL-6, TNFα, and IL-10. This stimulation is dependent on TLR signaling, and skews the activation of T cells towards the production of IFNγ. The influence of this activation in the establishment of Th responses in vivo shows the accumulation of specific IFNγ-producing cells. Our findings indicate that the activation exerted by E. faecalis CECT7121 on DCs and its consequence on the cellular adaptive immune response may have broad therapeutic implications in immunomodulation.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Enterococcus faecalis/imunologia , Imunomodulação/efeitos dos fármacos , Imunomodulação/imunologia , Interferon gama/imunologia , Probióticos/administração & dosagem , Animais , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Imunidade/efeitos dos fármacos , Imunidade/imunologia , Interleucina-10/imunologia , Interleucina-12/imunologia , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Therapeutic vaccinations against cancer are still largely ineffective. Major caveats are inefficient delivery of tumor antigens to dendritic cells (DCs) and excessive immune suppression by Foxp3+ regulatory T cells (Tregs), resulting in defective T cell priming and failure to induce tumor regression. To circumvent these problems we evaluated a novel combinatorial therapeutic strategy. We show that tumor antigen targeting to DC-SIGN in humanized hSIGN mice via glycans or specific antibodies induces superior T cell priming. Next, this targeted therapy was combined with transient Foxp3+ Treg depletion employing hSIGNxDEREG mice. While Treg depletion alone slightly delayed B16-OVA melanoma growth, only the combination therapy instigated long-term tumor regression in a substantial fraction of mice. This novel strategy resulted in optimal generation of antigen-specific activated CD8+ T cells which accumulated in regressing tumors. Notably, Treg depletion also allowed the local appearance of effector T cells specific for endogenous B16 antigens. This indicates that antitumor immune responses can be broadened by therapies aimed at controlling Tregs in tumor environments. Thus, transient inhibition of Treg-mediated immune suppression potentiates DC targeted antigen vaccination and tumor-specific immunity.