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Exhaled breath condensate (EBC) is routinely collected and analyzed in breath research. Because it contains aerosol droplets, EBC samples from SARS-CoV-2 infected individuals harbor the virus and pose the threat of infectious exposure. We report for the first time a safe and consistent method to fully inactivate SARS-CoV-2 in EBC samples and make EBC samples safe for processing and analysis. EBC samples containing infectious SARS-CoV-2 were treated with several concentrations of acetonitrile. The most commonly used 10% acetonitrile treatment for EBC processing failed to completely inactivate the virus in samples and viable virus was detected by the assay of SARS-CoV-2 infection of Vero E6 cells in a biosafety level 3 laboratory. Treatment with either 50% or 90% acetonitrile was effective to completely inactivate the virus, resulting in safe, non-infectious EBC samples that can be used for metabolomic analysis. Our study provides SARS-CoV-2 inactivation protocol for the collection and processing of EBC samples in the clinical setting and for advancing to metabolic assessments in health and disease.
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COVID-19 , SARS-CoV-2 , Testes Respiratórios , Expiração , Humanos , MetabolômicaRESUMO
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.
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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
Enteric defensins likely play a key role in the management of the human microbiome throughout development. The functional and mechanistic diversity of defensins is much greater than was initially thought. Defensin expression and overall Paneth cell physiology likely plays a key role in the development of colitis and other inflammatory or dysbiotic diseases of the gut. As our understanding of enteric defensins grows, their potential as tools of clinical intervention becomes more apparent. In this review, we focus on the function and activity of Paneth Cell defensins and highlight their role in disease.
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HIV-1 disease progression is paradoxically characterized by systemic chronic immune activation and gut mucosal immune dysfunction, which is not fully defined. Annexin A1 (ANXA1), an inflammation modulator, is a potential link between systemic inflammation and gut immune dysfunction during the simian immunodeficiency virus (SIV) infection. Gene expression of ANXA1 and cytokines were assessed in therapy-naïve rhesus macaques during early and chronic stages of SIV infection and compared with SIV-negative controls. ANXA1 expression was suppressed in the gut but systemically increased during early infection. Conversely, ANXA1 expression increased in both compartments during chronic infection. ANXA1 expression in peripheral blood was positively correlated with HLA-DR+CD4+ and CD8+ T-cell frequencies, and negatively associated with the expression of pro-inflammatory cytokines and CCR5. In contrast, the gut mucosa presented an anergic cytokine profile in relation to ANXA1 expression. In vitro stimulations with ANXA1 peptide resulted in decreased inflammatory response in PBMC but increased activation of gut lymphocytes. Our findings suggest that ANXA1 signaling is dysfunctional in SIV infection, and may contribute to chronic inflammation in periphery and with immune dysfunction in the gut mucosa. Thus, ANXA1 signaling may be a novel therapeutic target for the resolution of immune dysfunction in HIV infection.
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Anexina A1/biossíntese , Trato Gastrointestinal/patologia , Imunidade nas Mucosas , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Citocinas/biossíntese , Perfilação da Expressão Gênica , Macaca mulatta , Vírus da Imunodeficiência Símia/crescimento & desenvolvimentoRESUMO
This study investigated the effects of HIV-1 infection and antiretroviral therapy (ART) on the expression of intestinal drug efflux transporters, i.e., P-glycoprotein (Pgp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP), and metabolic enzymes, such as cytochrome P450s (CYPs), in the human upper intestinal tract. Intestinal biopsy specimens were obtained from HIV-negative healthy volunteers, ART-naive HIV-positive (HIV(+)) subjects, and HIV(+) subjects receiving ART (10 in each group). Intestinal tissue expression of drug transporters and metabolic enzymes was examined by microarray, real-time quantitative reverse transcription-PCR (qPCR), and immunohistochemistry analyses. Microarray analysis demonstrated significantly lower expression of CYP3A4 and ABCC2/MRP2 in the HIV(+) ART-naive group than in uninfected subjects. qPCR analysis confirmed significantly lower expression of ABCC2/MRP2 in ART-naive subjects than in the control group, while CYP3A4 and ABCG2/BCRP showed a trend toward decreased expression. Protein expression of MRP2 and BCRP was also significantly lower in the HIV(+) naive group than in the control group and was partially restored to baseline levels in HIV(+) subjects receiving ART. In contrast, gene and protein expression of ABCB1/Pgp was significantly increased in HIV(+) subjects on ART relative to HIV(+) ART-naive subjects. These data demonstrate that the expression of drug-metabolizing enzymes and efflux transporters is significantly altered in therapy-naive HIV(+) subjects and in those receiving ART. Since CYP3A4, Pgp, MRPs, and BCRP metabolize or transport many antiretroviral drugs, their altered expression with HIV infection may negatively impact drug pharmacokinetics in HIV(+) subjects. This has clinical implications when using data from healthy volunteers to guide ART.
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Infecções por HIV/enzimologia , Infecções por HIV/metabolismo , HIV-1/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Fármacos Anti-HIV/farmacologia , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
BACKGROUND: The impact of HIV infection on pattern recognition receptor (PRR) expression in gut-associated lymphoid tissue and its association with dysbiosis is not well understood. METHODS: PRR and cytokine gene expression were examined in mesenteric lymph nodes (mLN) of rhesus macaques during acute and chronic (untreated and early antiretroviral (ART) treated) infections. Gene expression was correlated with microbial abundance in the gut and immune activation. RESULTS: PRR expression rapidly increases during acute infection and is significantly decreased in chronic infection. Early ART maintains elevated PRR expression. Correlation analysis revealed three distinct groups of bacterial taxa that were associated with gene expression changes in infection. CONCLUSIONS: PRR and cytokine gene expression in the gut-draining mLN are rapidly modulated in response to viral infection and are correlated with gut dysbiosis. These data suggest that the dysregulation of PRR and related cytokine expression may contribute to chronic immune activation in SIV infection.
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Antirretrovirais/farmacologia , Citocinas/genética , Microbioma Gastrointestinal , Regulação da Expressão Gênica , Receptores de Reconhecimento de Padrão/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Doença Aguda , Animais , Doença Crônica , Citocinas/metabolismo , Linfonodos/imunologia , Linfonodos/virologia , Receptores de Reconhecimento de Padrão/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologiaRESUMO
Immunization of macaques with attenuated simian immunodeficiency virus (SIV) with deletions in nef (SIVΔnef) is shown to elicit protective immunity to infection by pathogenic SIV, yet the mechanisms that orchestrate protection and prevent pathogenesis remains unknown. We utilized whole-genome transcriptional profiling to reveal molecular signatures of protective immunity in circulating CD8+ T cells of rhesus macaques vaccinated with SIVmac239Δnef and challenged with pathogenic SIVmac251. Our findings suggest that protective immunity to pathogenic SIV infection induced by SIVmac239∆nef is associated with balanced induction of T cell activation and immunoregulatory mechanisms and dampened activation of interferon-induced signaling pathways and cytolytic enzyme production as compared with pathogenic SIVmac251 infection of unvaccinated controls. We provide evidence that protective immunity to SIVmac251 correlates with induction of biomarkers of T cell activation, differentiation, signaling, and adhesion that were down regulated in unvaccinated controls. The study highlights potential immunomodulatory networks associated with protective immunity against the virus.
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Vírus da Imunodeficiência Símia/fisiologia , Animais , Linfócitos T CD8-Positivos/fisiologia , Feminino , Regulação Viral da Expressão Gênica/fisiologia , Macaca mulatta , Receptores de Superfície Celular , Vacinas contra a SAIDS/imunologia , Transcriptoma , Carga ViralRESUMO
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.
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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
OBJECTIVE: Although HAART effectively suppresses viral replication, it fails to eradicate latent viral reservoirs. The 'shock and kill' strategy involves the activation of HIV from latent reservoirs and targeting them for eradication. Our goal was to develop new approaches for activating HIV from latent reservoirs. DESIGN: We investigated capacity of Ingenol B (IngB), a newly modified derivative of Ingenol ester that was originally isolated from a Brazilian plant in Amazon, for its capacity and mechanisms of HIV reactivation. METHODS: Reactivation of HIV-1 by IngB was evaluated in J-Lat A1 cell culture model of HIV latency as well as in purified primary CD4 T cells from long-term HAART-treated virologically-suppressed HIV-infected individuals. The underlining molecular mechanisms of viral reactivation were investigated using flow cytometry, RT-qPCR and chromatin immunoprecipitation. RESULTS: IngB is highly effective in reactivating HIV in J-Lat A1 cells with relatively low cellular toxicity. It is also able to reactivate latent HIV in purified CD4 T cells from HAART-treated HIV-positive individuals ex vivo. Our data show that IngB may reactivate HIV expression by both activating protein kinase C (PKC)δ-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and directly inducing NF-κB protein expression. Importantly, IngB has a synergistic effect with JQ1, a BET bromodomain inhibitor, in latent HIV reactivation. CONCLUSIONS: IngB is a new promising compound to activate latent HIV reservoirs. Our data suggest that formulating novel derivatives from Ingenol esters may be an innovative approach to develop new lead compounds to reactivate latent HIV.
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Linfócitos T CD4-Positivos/efeitos dos fármacos , Diterpenos/metabolismo , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Adulto , Idoso , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Imunoprecipitação da Cromatina , Citometria de Fluxo , HIV-1/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Provírus/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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.
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UNLABELLED: Epithelial barrier dysfunction during human immunodeficiency virus (HIV) infection has largely been attributed to the rapid and severe depletion of CD4(+) T cells in the gastrointestinal (GI) tract. Although it is known that changes in mucosal gene expression contribute to intestinal enteropathy, the role of small noncoding RNAs, specifically microRNA (miRNA), has not been investigated. Using the simian immunodeficiency virus (SIV)-infected nonhuman primate model of HIV pathogenesis, we investigated the effect of viral infection on miRNA expression in intestinal mucosa. SIV infection led to a striking decrease in the expression of mucosal miRNA compared to that in uninfected controls. This decrease coincided with an increase in 5'-3'-exoribonuclease 2 protein and alterations in DICER1 and Argonaute 2 expression. Targets of depleted miRNA belonged to molecular pathways involved in epithelial proliferation, differentiation, and immune response. Decreased expression of several miRNA involved in maintaining epithelial homeostasis in the gut was localized to the proliferative crypt region of the intestinal epithelium. Our findings suggest that SIV-induced decreased expression of miRNA involved in epithelial homeostasis, disrupted expression of miRNA biogenesis machinery, and increased expression of XRN2 are involved in the development of epithelial barrier dysfunction and gastroenteropathy. IMPORTANCE: MicroRNA (miRNA) regulate the development and function of intestinal epithelial cells, and many viruses disrupt normal host miRNA expression. In this study, we demonstrate that SIV and HIV disrupt expression of miRNA in the small intestine during infection. The depletion of several key miRNA is localized to the proliferative crypt region of the gut epithelium. These miRNA are known to control expression of genes involved in inflammation, cell death, and epithelial maturation. Our data indicate that this disruption might be caused by altered expression of miRNA biogenesis machinery during infection. These findings suggest that the disruption of miRNA in the small intestine likely plays a role in intestinal enteropathy during HIV infection.
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HIV , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatologia , Infecções por Lentivirus/metabolismo , MicroRNAs/metabolismo , Vírus da Imunodeficiência Símia , Adulto , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Biologia Computacional , Densitometria , Citometria de Fluxo , Humanos , Mucosa Intestinal/imunologia , Microdissecção e Captura a Laser , Infecções por Lentivirus/fisiopatologia , Macaca mulatta , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Carga 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.
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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/genéticaRESUMO
A majority of individuals infected with human immunodeficiency virus (HIV) have inadequate access to antiretroviral therapy and ultimately develop debilitating oral infections that often correlate with disease progression. Due to the impracticalities of conducting host-microbe systems-based studies in HIV infected patients, we have evaluated the potential of simian immunodeficiency virus (SIV) infected rhesus macaques to serve as a non-human primate model for oral manifestations of HIV disease. We present the first description of the rhesus macaque oral microbiota and show that a mixture of human commensal bacteria and "macaque versions" of human commensals colonize the tongue dorsum and dental plaque. Our findings indicate that SIV infection results in chronic activation of antiviral and inflammatory responses in the tongue mucosa that may collectively lead to repression of epithelial development and impact the microbiome. In addition, we show that dysbiosis of the lingual microbiome in SIV infection is characterized by outgrowth of Gemella morbillorum that may result from impaired macrophage function. Finally, we provide evidence that the increased capacity of opportunistic pathogens (e.g. E. coli) to colonize the microbiome is associated with reduced production of antimicrobial peptides.
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Disbiose , Perfilação da Expressão Gênica , Metagenoma , Microbiota , Boca/microbiologia , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Vírus da Imunodeficiência Símia , Animais , Análise por Conglomerados , Regulação da Expressão Gênica , Interferon gama/metabolismo , Macaca mulatta , Masculino , Mucosa Bucal/imunologia , Mucosa Bucal/metabolismo , Mucosa Bucal/microbiologia , Mucosa Bucal/patologia , Filogenia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologiaRESUMO
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.
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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
Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.
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Células Apresentadoras de Antígenos/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Lactobacillus/imunologia , Adulto , Idoso , Antígenos CD36/metabolismo , Doença Crônica , Estudos de Coortes , Células Dendríticas/metabolismo , Feminino , Infecções por HIV/enzimologia , Infecções por HIV/microbiologia , Humanos , Imunidade/imunologia , Inflamação/sangue , Inflamação/imunologia , Inflamação/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Fosforilação , Receptores Imunológicos/metabolismo , Receptor 2 Toll-Like/metabolismo , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
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.
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BACKGROUND: Opportunistic oral infections can be found in over 80% of HIV + patients, often causing debilitating lesions that also contribute to deterioration in nutritional health. Although appreciation for the role that the microbiota is likely to play in the initiation and/or enhancement of oral infections has grown considerably in recent years, little is known about the impact of HIV infection on host-microbe interactions within the oral cavity. In the current study, we characterize modulations in the bacterial composition of the lingual microbiome in patients with treated and untreated HIV infection. Bacterial species profiles were elucidated by microarray assay and compared between untreated HIV infected patients, HIV infected patients receiving antiretroviral therapy, and healthy HIV negative controls. The relationship between clinical parameters (viral burden and CD4+ T cell depletion) and the loss or gain of bacterial species was evaluated in each HIV patient group. RESULTS: In untreated HIV infection, elevated viremia was associated with significantly higher proportions of potentially pathogenic Veillonella, Prevotella, Megasphaera, and Campylobacter species in the lingual microbiome than observed in healthy controls. The upsurge in the prevalence of potential pathogens was juxtaposed by diminished representation of commensal Streptococcus and Veillonella species. Colonization of Neisseria flavescens was lower in the lingual microbiome of HIV infected patients receiving antiretroviral therapy than in uninfected controls. CONCLUSIONS: Our findings provide novel insights into the potential impact of HIV infection and antiretroviral therapy on the community structure of the oral microbiome, and implicate potential mechanisms that may increase the capacity of non-commensal species to gain a stronger foothold.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Bactérias/classificação , Infecções Bacterianas/microbiologia , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Metagenoma , Língua/microbiologia , Adulto , Idoso , Bactérias/genética , Feminino , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-IdadeRESUMO
Interruption of suppressive highly active antiretroviral therapy (HAART) in HIV-infected patients leads to increased HIV replication and viral rebound in peripheral blood. Effects of therapy interruption on gut-associated lymphoid tissue (GALT) have not been well investigated. We evaluated longitudinal changes in viral replication and emergence of viral variants in the context of T cell homeostasis and gene expression in GALT of three HIV-positive patients who initiated HAART during primary HIV infection but opted to interrupt therapy thereafter. Longitudinal viral sequence analysis revealed that a stable proviral reservoir was established in GALT during primary HIV infection that persisted through early HAART and post-therapy interruption. Proviral variants in GALT and peripheral blood mononuclear cells (PBMCs) displayed low levels of genomic diversity at all times. A rapid increase in viral loads with a modest decline of CD4(+) T cells in peripheral blood was observed, while gut mucosal CD4(+) T cell loss was severe following HAART interruption. This was accompanied by increased mucosal gene expression regulating interferon (IFN)-mediated antiviral responses and immune activation, a profile similar to those found in HAART-naive HIV-infected patients. Sequence analysis of rebound virus suggested that GALT was not the major contributor to the postinterruption plasma viremia nor were GALT HIV reservoirs rapidly replaced by HIV rebound variants. Our data suggest an early establishment and persistence of viral reservoirs in GALT with minimal diversity. Early detection of and therapy for HIV infection may be beneficial in controlling viral evolution and limiting establishment of diverse viral reservoirs in the mucosal compartment.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV/isolamento & purificação , Mucosa Intestinal/virologia , Adulto , Análise por Conglomerados , HIV/classificação , HIV/genética , Humanos , Leucócitos Mononucleares/virologia , Masculino , Filogenia , Plasma/virologia , Polimorfismo Genético , Provírus/classificação , Provírus/genética , Provírus/isolamento & purificação , Viremia , Suspensão de TratamentoRESUMO
Gastrointestinal mucosa is an early target of HIV and a site of viral replication and severe CD4(+) T cell depletion. However, effects of HIV infection on gut mucosal innate immune defense have not been fully investigated. Intestinal Paneth cell-derived α-defensins constitute an integral part of the gut mucosal innate defense against microbial pathogens. Using the SIV-infected rhesus macaque model of AIDS, we examined the level of expression of rhesus enteric α-defensins (REDs) in the jejunal mucosa of rhesus macaques during all stages of SIV infection using real-time PCR, in situ hybridization, and immunohistochemistry. An increased expression of RED mRNAs was found in PC at the base of the crypts in jejunum at all stages of SIV infection as compared with uninfected controls. This increase correlated with active viral replication in gut-associated lymphoid tissue. Loss of RED protein accumulation in PC was seen in animals with simian AIDS. This was associated with the loss of secretory granules in PC, suggesting an increase in degranulation during advanced SIV disease. The α-defensin-mediated innate mucosal immunity was maintained in PC throughout the course of SIV infection despite the mucosal CD4(+) T cell depletion. The loss of RED protein accumulation and secretion was associated with an increased incidence of opportunistic enteric infections and disease progression. Our findings suggest that local innate immune defense exerted by PC-derived defensins contributes to the protection of gut mucosa from opportunistic infections during the course of SIV infection.