RESUMO
Chronic gut inflammatory diseases are associated with disruption of intestinal epithelial barriers and impaired mucosal immunity. HIV-1 (HIV) causes depletion of mucosal CD4+ T cells early in infection and disruption of gut epithelium, resulting in chronic inflammation and immunodeficiency. Although antiretroviral therapy (ART) is effective in suppressing viral replication, it is incapable of restoring the "leaky gut," which poses an impediment for HIV cure efforts. Strategies are needed for rapid repair of the epithelium to protect intestinal microenvironments and immunity in inflamed gut. Using an in vivo nonhuman primate intestinal loop model of HIV/AIDS, we identified the pathogenic mechanism underlying sustained disruption of gut epithelium and explored rapid repair of gut epithelium at the intersection of microbial metabolism. Molecular, immunological, and metabolomic analyses revealed marked loss of peroxisomal proliferator-activated receptor-α (PPARα) signaling, predominant impairment of mitochondrial function, and epithelial disruption both in vivo and in vitro. To elucidate pathways regulating intestinal epithelial integrity, we introduced probiotic Lactobacillus plantarum into Simian immunodeficiency virus (SIV)-inflamed intestinal lumen. Rapid recovery of the epithelium occurred within 5 h of L. plantarum administration, independent of mucosal CD4+ T cell recovery, and in the absence of ART. This intestinal barrier repair was driven by L. plantarum-induced PPARα activation and restoration of mitochondrial structure and fatty acid ß-oxidation. Our data highlight the critical role of PPARα at the intersection between microbial metabolism and epithelial repair in virally inflamed gut and as a potential mitochondrial target for restoring gut barriers in other infectious or gut inflammatory diseases.
Assuntos
Metabolismo Energético/fisiologia , Microbioma Gastrointestinal/fisiologia , Intestinos/imunologia , Intestinos/microbiologia , Mitocôndrias/metabolismo , PPAR alfa/metabolismo , Animais , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Epitélio/imunologia , Infecções por HIV , Humanos , Imunidade nas Mucosas , Interleucina-1beta/metabolismo , Intestinos/patologia , Lactobacillus plantarum/fisiologia , Macaca mulatta , Masculino , Metabolômica , Mitocôndrias/efeitos dos fármacos , Probióticos/administração & dosagem , Probióticos/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologiaRESUMO
Although anti-retroviral therapy (ART) is highly effective in suppressing HIV replication, it fails to eradicate the virus from HIV-infected individuals. Stable latent HIV reservoirs are rapidly established early after HIV infection. Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed. We report that ingenol-3-angelate (PEP005), the only active component in a previously FDA approved drug (PICATO) for the topical treatment of precancerous actinic keratosis, can effectively reactivate latent HIV in vitro and ex vivo with relatively low cellular toxicity. Biochemical analysis showed that PEP005 reactivated latent HIV through the induction of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB signaling pathway. Importantly, PEP005 alone was sufficient to induce expression of fully elongated and processed HIV RNAs in primary CD4+ T cells from HIV infected individuals receiving suppressive ART. Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone. Conversely, PEP005 suppressed HIV infection of primary CD4+ T cells through down-modulation of cell surface expression of HIV co-receptors. This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.
Assuntos
Azepinas/farmacologia , Diterpenos/farmacologia , Infecções por HIV/tratamento farmacológico , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triazóis/farmacologia , Latência Viral/efeitos dos fármacos , Azepinas/administração & dosagem , Diterpenos/administração & dosagem , HIV-1/efeitos dos fármacos , Humanos , Proteínas I-kappa B/farmacologia , Inibidor de NF-kappaB alfa , Fator B de Elongação Transcricional Positiva/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Triazóis/administração & dosagem , Ativação Viral/efeitos dos fármacosRESUMO
HIV causes rapid CD4+ T cell depletion in the gut mucosa, resulting in immune deficiency and defects in the intestinal epithelial barrier. Breakdown in gut barrier integrity is linked to chronic inflammation and disease progression. However, the early effects of HIV on the gut epithelium, prior to the CD4+ T cell depletion, are not known. Further, the impact of early viral infection on mucosal responses to pathogenic and commensal microbes has not been investigated. We utilized the SIV model of AIDS to assess the earliest host-virus interactions and mechanisms of inflammation and dysfunction in the gut, prior to CD4+ T cell depletion. An intestinal loop model was used to interrogate the effects of SIV infection on gut mucosal immune sensing and response to pathogens and commensal bacteria in vivo. At 2.5 days post-SIV infection, low viral loads were detected in peripheral blood and gut mucosa without CD4+ T cell loss. However, immunohistological analysis revealed the disruption of the gut epithelium manifested by decreased expression and mislocalization of tight junction proteins. Correlating with epithelial disruption was a significant induction of IL-1ß expression by Paneth cells, which were in close proximity to SIV-infected cells in the intestinal crypts. The IL-1ß response preceded the induction of the antiviral interferon response. Despite the disruption of the gut epithelium, no aberrant responses to pathogenic or commensal bacteria were observed. In fact, inoculation of commensal Lactobacillus plantarum in intestinal loops led to rapid anti-inflammatory response and epithelial tight junction repair in SIV infected macaques. Thus, intestinal Paneth cells are the earliest responders to viral infection and induce gut inflammation through IL-1ß signaling. Reversal of the IL-1ß induced gut epithelial damage by Lactobacillus plantarum suggests synergistic host-commensal interactions during early viral infection and identify these mechanisms as potential targets for therapeutic intervention.
Assuntos
Interleucina-1beta/biossíntese , Celulas de Paneth/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Animais , Imunofluorescência , Interações Hospedeiro-Parasita/imunologia , Imuno-Histoquímica , Interleucina-1beta/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/ultraestrutura , Mucosa Intestinal/virologia , Macaca mulatta , Masculino , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Celulas de Paneth/metabolismo , Celulas de Paneth/virologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/ultraestrutura , Carga ViralRESUMO
Circulating monocytes carrying human CMV (HCMV) migrate into tissues, where they differentiate into HCMV-infected resident macrophages that upon interaction with bacterial products may potentiate tissue inflammation. In this study, we investigated the mechanism by which HCMV promotes macrophage-orchestrated inflammation using a clinical isolate of HCMV (TR) and macrophages derived from primary human monocytes. HCMV infection of the macrophages, which was associated with viral DNA replication, significantly enhanced TNF-α, IL-6, and IL-8 gene expression and protein production in response to TLR4 ligand (LPS) stimulation compared with mock-infected LPS-stimulated macrophages during a 6-d in vitro infection. HCMV infection also potentiated TLR5 ligand-stimulated cytokine production. To elucidate the mechanism by which HCMV infection potentiated inducible macrophage responses, we show that infection by HCMV promoted the maintenance of surface CD14 and TLR4 and TLR5, which declined over time in mock-infected macrophages, and enhanced both the intracellular expression of adaptor protein MyD88 and the inducible phosphorylation of IκBα and NF-κB. These findings provide additional information toward elucidating the mechanism by which HCMV potentiates bacteria-induced NF-κB-mediated macrophage inflammatory responses, thereby enhancing organ inflammation in HCMV-infected tissues.
Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Macrófagos/imunologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/virologia , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/imunologia , Receptor 5 Toll-Like/imunologia , Replicação ViralRESUMO
The mucosa that lines the respiratory and gastrointestinal (GI) tracts is an important portal of entry for pathogens and provides the first line of innate immune defense against infections. Although an abundance of memory CD4(+) T cells at mucosal sites render them highly susceptible to HIV infection, the gut and not the lung experiences severe and sustained CD4(+) T cell depletion and tissue disruption. We hypothesized that distinct immune responses in the lung and gut during the primary and chronic stages of viral infection contribute to these differences. Using the SIV model of AIDS, we performed a comparative analysis of the molecular and cellular characteristics of host responses in the gut and lung. Our findings showed that both mucosal compartments harbor similar percentages of memory CD4(+) T cells and displayed comparable cytokine (IL-2, IFN-γ, and TNF-α) responses to mitogenic stimulations prior to infection. However, despite similar viral replication and CD4(+) T cell depletion during primary SIV infection, CD4(+) T cell restoration kinetics in the lung and gut diverged during acute viral infection. The CD4(+) T cells rebounded or were preserved in the lung mucosa during chronic viral infection, which correlated with heightened induction of type I IFN signaling molecules and innate viral restriction factors. In contrast, the lack of CD4(+) T cell restoration in the gut was associated with dampened immune responses and diminished expression of viral restriction factors. Thus, unique immune mechanisms contribute to the differential response and protection of pulmonary versus GI mucosa and can be leveraged to enhance mucosal recovery.
Assuntos
Citotoxicidade Imunológica/imunologia , Expressão Gênica/imunologia , Imunidade nas Mucosas/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Fator de Necrose Tumoral alfa/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica/genética , Modelos Animais de Doenças , Citometria de Fluxo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade nas Mucosas/genética , Memória Imunológica/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virologia , Macaca mulatta , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , Prognóstico , Recuperação de Função Fisiológica/genética , Recuperação de Função Fisiológica/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Transcriptoma/genética , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/genéticaAssuntos
Competência Clínica/estatística & dados numéricos , Dermatologia/educação , Dermatopatias/diagnóstico , Pigmentação da Pele/fisiologia , Estudantes de Medicina/estatística & dados numéricos , Dermatologia/estatística & dados numéricos , Erros de Diagnóstico/prevenção & controle , Erros de Diagnóstico/estatística & dados numéricos , Educação de Graduação em Medicina/estatística & dados numéricos , Humanos , Dermatopatias/fisiopatologiaRESUMO
As the "baby boomers" age, the percentage of the population over sixty-five years of age is increasing rapidly. Chronic disease management is an important component in the care of the elderly. The effects of aging on different organ systems are also pertinent; such as the weakening homeostatic response to injury in the older individuals. Mucosal surfaces have the largest combined surface area in the body and are the site of important host microbe interactions, especially in the gut which is prone to injury, both from local and systemic insult. This susceptibility has been known to increase with age. Therefore it is important to understand the interplay between aging, injury and recovery at the mucosal surface. Sex hormones play an important role in the maintenance of the mucosal barrier function as well as the mucosa associated immune function in both genders. Menopause in women is a defined time period in which major hormonal changes occur such as a decline in systemic estradiol levels. The differential levels of sex hormones contribute to the sexual dimorphism seen in response to injury at the mucosal surface, prior to and following menopause. Thus the effect of sex hormone and aging on mucosal mechanisms in response to injury is an important area of investigation.
Assuntos
Proteína C-Reativa/análise , Calcitonina/sangue , Leucemia-Linfoma Linfoblástico de Células T Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico por imagem , Biomarcadores/sangue , Criança , Diagnóstico Diferencial , Feminino , Humanos , Inflamação/sangue , Inflamação/diagnóstico por imagem , MasculinoRESUMO
OBJECTIVE: The human intestine harbors trillions of commensal microbes that live in homeostasis with the host immune system. Changes in the composition and complexity of gut microbial communities are seen in inflammatory bowel disease (IBD), indicating disruption in host-microbe interactions. Multiple factors including diet and inflammatory conditions alter the microbial complexity. The goal of this study was to develop an optimized methodology for fecal sample processing and to detect changes in the gut microbiota of patients with Crohn's disease receiving specialized diets. DESIGN: Fecal samples were obtained from patients with Crohn's disease in a pilot diet crossover trial comparing the effects of a specific carbohydrate diet (SCD) versus a low residue diet (LRD) on the composition and complexity of the gut microbiota and resolution of IBD symptoms. The gut microbiota composition was assessed using a high-density DNA microarray PhyloChip. RESULTS: DNA extraction from fecal samples using a column based method provided consistent results. The complexity of the gut microbiome was lower in IBD patients compared to healthy controls. An increased abundance of Bacteroides fragilis (B. fragilis) was observed in fecal samples from IBD positive patients. The temporal response of gut microbiome to the SCD resulted in an increased microbial diversity while the LRD diet was associated with reduced diversity of the microbial communities. CONCLUSION: Changes in the composition and complexity of the gut microbiome were identified in response to specialized carbohydrate diet. The SCD was associated with restructuring of the gut microbial communities.
RESUMO
BACKGROUND: Development of inflammatory bowel disease (IBD) involves the interplay of environmental and genetic factors with the host immune system. Mechanisms contributing to immune dysregulation in IBD are not fully defined. Development of novel therapeutic strategies is focused on controlling aberrant immune response in IBD. Current IBD therapy utilizes a combination of immunomodulators and biologics to suppress pro-inflammatory effectors of IBD. However, the role of immunomodulatory factors such as annexin A1 (ANXA1) is not well understood. The goal of this study was to examine the association between ANXA1 and IBD, and the effects of anti-TNF-α, Infliximab (IFX), therapy on ANXA1 expression. METHODS: ANXA1 and TNF-α transcript levels in PBMC were measured by RT PCR. Clinical follow up included the administration of serial ibdQs. ANXA1 expression in the gut mucosa was measured by IHC. Plasma ANXA1 levels were measured by ELISA. RESULTS: We found that the reduction in ANXA1 protein levels in plasma coincided with a decrease in the ANXA1 mRNA expression in peripheral blood of IBD patients. ANXA1 expression is upregulated during IFX therapy in patients with a successful intervention but not in clinical non-responders. The IFX therapy also modified the cellular immune activation in the peripheral blood of IBD patients. Decreased expression of ANXA1 was detected in the colonic mucosa of IBD patients with incomplete resolution of inflammation during continuous therapy, which correlated with increased levels of TNF-α transcripts. Gut mucosal epithelial barrier disruption was evident by increased plasma bacterial 16S levels. CONCLUSION: Loss of ANXA1 expression may support inflammation during IBD and can serve as a biomarker of disease progression. Changes in ANXA1 levels may be predictive of therapeutic efficacy.
Assuntos
Anexina A1/genética , Anexina A1/metabolismo , Doença de Crohn/metabolismo , Progressão da Doença , Regulação da Expressão Gênica , Adulto , Idoso , Anexina A1/sangue , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Doença de Crohn/sangue , Doença de Crohn/tratamento farmacológico , Doença de Crohn/imunologia , DNA Bacteriano/sangue , DNA Ribossômico/sangue , Feminino , Humanos , Inflamação/genética , Inflamação/metabolismo , Infliximab , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/genética , Adulto JovemRESUMO
BACKGROUND: Women and men have diverse responses to many infectious diseases. These differences are amplified following menopause. However, despite extensive information regarding the effects of sex hormones on immune cells, our knowledge is limited regarding the effects of sex and gender on the function of the mucosal immune system. Sex differences also manifest in the prevalence of gut associated inflammatory and autoimmune disorders, including Crohn's disease, ulcerative colitis and Celiac disease. It is thus hypothesized that a baseline sex-associated difference in immune activation may predispose women to inflammation-associated disease. METHODS: Peripheral blood samples and small intestinal biopsies were obtained from 34 healthy men and women. Immunophenotypic analysis of isolated lymphocytes was performed by flow cytometry. Oligonucleotide analysis was used to study the transcriptional profile in the gut mucosal microenvironment while real-time PCR analysis was utilized to identify differential gene expression in isolated CD4+ T cells. Transcriptional analysis was confirmed by protein expression levels for genes of interest using fluorescent immunohistochemistry. Data was analyzed using the GraphPad software package. RESULTS: Women had higher levels of immune activation and inflammation-associated gene expression in gut mucosal samples. CD4+ and CD8+ T cells had a significantly higher level of immune activation-associated phenotype in peripheral blood as well as in gut associated lymphoid tissue along with higher levels of proliferating T cells. CD4+ T cells that showed upregulation of IL1ß as well as the TH17 pathway-associated genes contributed a large part of the inflammatory profile. CONCLUSION: In this study, we demonstrated an upregulation in gene expression related to immune function in the gut microenvironment of women compared to men, in the absence of disease or pathology. Upon closer investigation, CD4+ T cell activation levels were higher in the LPLs in women than in men. Sex differences in the mucosal immune system may predispose women to inflammation-associated diseases that are exacerbated following menopause. Our study highlights the need for more detailed analysis of the effects of sex differences in immune responses at mucosal effector sites.
RESUMO
The antifungal metabolite 2,4-diacetylphloroglucinol plays a major role in the biocontrol capabilities of Pseudomonas fluorescens. The phloroglucinol biosynthetic locus of P. fluorescens F113 has been isolated previously. From nucleotide sequence data, a putative regulator gene (phlF) was identified upstream and divergently transcribed from the phlACBD phloroglucinol biosynthetic genes. PhlF shows similarity to various transcriptional repressors in the EMBL database and exhibits a helix-turn-helix motif in its amino acid sequence. phlF was cloned into an expression vector and the PhlF protein product was purified. Gel retardation experiments demonstrated PhlF to be a DNA-binding protein and showed that it binds to the phlA-phlF intergenic region. Introduction of phlF into P. fluorescens F113 in multiple copies resulted in repression of phloroglucinol production in this strain. This effect was mediated at the transcription level since the expression of a phloroglucinol biosynthetic gene fusion in this background was equally repressed. Furthermore, the inactivation of phlF results in derepression of phloroglucinol production in this strain.