RESUMO
T cells play key protective but also pathogenic roles in COVID-19. We studied the expression of long non-coding RNAs (lncRNAs) in COVID-19 T-cell transcriptomes by integrating previously published single-cell RNA sequencing datasets. The long intergenic non-coding RNA MALAT1 was the most highly transcribed lncRNA in T cells, with Th1 cells demonstrating the lowest and CD8+ resident memory cells the highest MALAT1 expression, amongst CD4+ and CD8+ T-cells populations, respectively. We then identified gene signatures that covaried with MALAT1 in single T cells. A significantly higher number of transcripts correlated negatively with MALAT1 than those that correlated. Enriched functional annotations of the MALAT1- anti-correlating gene signature included processes associated with T-cell activation such as cell division, oxidative phosphorylation, and response to cytokine. The MALAT1 anti-correlating gene signature shared by both CD4+ and CD8+ T-cells marked dividing T cells in both the lung and blood of COVID-19 patients. Focussing on the tissue, we used an independent patient cohort of post-mortem COVID-19 lung samples and demonstrated that MALAT1 suppression was indeed a marker of MKI67+ proliferating CD8+ T cells. Our results reveal MALAT1 suppression and its associated gene signature are a hallmark of human proliferating T cells.
Assuntos
COVID-19 , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Regulação para Baixo , Proliferação de Células/genética , COVID-19/genética , Linfócitos T CD8-Positivos/metabolismoRESUMO
Despite extensive mapping of long noncoding RNAs in immune cells, their function in vivo remains poorly understood. In this study, we identify over 100 long noncoding RNAs that are differentially expressed within 24 h of Th1 cell activation. Among those, we show that suppression of Malat1 is a hallmark of CD4+ T cell activation, but its complete deletion results in more potent immune responses to infection. This is because Malat1-/- Th1 and Th2 cells express lower levels of the immunosuppressive cytokine IL-10. In vivo, the reduced CD4+ T cell IL-10 expression in Malat1-/- mice underpins enhanced immunity and pathogen clearance in experimental visceral leishmaniasis (Leishmania donovani) but more severe disease in a model of malaria (Plasmodium chabaudi chabaudi AS). Mechanistically, Malat1 regulates IL-10 through enhancing expression of Maf, a key transcriptional regulator of IL-10 Maf expression correlates with Malat1 in single Ag-specific Th cells from P. chabaudi chabaudi AS-infected mice and is downregulated in Malat1-/- Th1 and Th2 cells. The Malat1 RNA is responsible for these effects, as antisense oligonucleotide-mediated inhibition of Malat1 also suppresses Maf and IL-10 levels. Our results reveal that through promoting expression of the Maf/IL-10 axis in effector Th cells, Malat1 is a nonredundant regulator of mammalian immunity.
Assuntos
Interleucina-10/metabolismo , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Proteínas Proto-Oncogênicas c-maf/metabolismo , RNA Longo não Codificante/genética , Células Th1/imunologia , Células Th2/imunologia , Animais , Feminino , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Imunidade/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-maf/genética , Regulação para CimaRESUMO
Post-kala-azar dermal leishmaniasis (PKDL) is a chronic, stigmatizing skin condition occurring frequently after apparent clinical cure from visceral leishmaniasis. Given an urgent need for new treatments, we conducted a phase IIa safety and immunogenicity trial of ChAd63-KH vaccine in Sudanese patients with persistent PKDL. LEISH2a (ClinicalTrials.gov: NCT02894008) was an open-label three-phase clinical trial involving sixteen adult and eight adolescent patients with persistent PKDL (median duration, 30 months; range, 6-180 months). Patients received a single intramuscular vaccination of 1 × 1010 viral particles (v.p.; adults only) or 7.5 × 1010 v.p. (adults and adolescents), with primary (safety) and secondary (clinical response and immunogenicity) endpoints evaluated over 42-120 days follow-up. AmBisome was provided to patients with significant remaining disease at their last visit. ChAd63-KH vaccine showed minimal adverse reactions in PKDL patients and induced potent innate and cell-mediated immune responses measured by whole-blood transcriptomics and ELISpot. 7/23 patients (30.4%) monitored to study completion showed >90% clinical improvement, and 5/23 (21.7%) showed partial improvement. A logistic regression model applied to blood transcriptomic data identified immune modules predictive of patients with >90% clinical improvement. A randomized controlled trial to determine whether these clinical responses were vaccine-related and whether ChAd63-KH vaccine has clinical utility is underway.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Leishmania/imunologia , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Cutânea/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Adenovirus dos Símios/genética , Adolescente , Adulto , Criança , Feminino , Humanos , Injeções Intramusculares , Leishmania/isolamento & purificação , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Masculino , Prognóstico , Vacinas Sintéticas/imunologia , Adulto JovemRESUMO
Determining the mechanisms that distinguish protective immunity from pathological chronic inflammation remains a fundamental challenge. miR-132 has been shown to play largely immunoregulatory roles in immunity; however, its role in CD4+ T cell function is poorly understood. Here, we show that CD4+ T cells express high levels of miR-132 and that T cell activation leads to miR-132 up-regulation. The transcriptomic hallmark of splenic CD4+ T cells lacking the miR-132/212 cluster during chronic infection is an increase in mRNA levels of ribosomal protein (RP) genes. BTAF1, a co-factor of B-TFIID and novel miR-132/212-3p target, and p300 contribute towards miR-132/212-mediated regulation of RP transcription. Following infection with Leishmania donovani, miR-132-/- CD4+ T cells display enhanced expression of IL-10 and decreased IFNγ. This is associated with reduced hepatosplenomegaly and enhanced pathogen load. The enhanced IL-10 expression in miR-132-/- Th1 cells is recapitulated in vitro following treatment with phenylephrine, a drug reported to promote ribosome synthesis. Our results uncover that miR-132/212-mediated regulation of RP expression is critical for optimal CD4+ T cell activation and protective immunity against pathogens.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Ligação Proteica , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , Fator de Transcrição TFIID/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
BACKGROUND: An emerging problem in the treatment of breast cancer is the increasing incidence of metastases to the brain. Metastatic brain tumours are incurable and can cause epileptic seizures and cognitive impairment, so better understanding of this niche, and the cellular mechanisms, is urgently required. Microglia are the resident brain macrophage population, becoming "activated" by neuronal injury, eliciting an inflammatory response. Microglia promote proliferation, angiogenesis and invasion in brain tumours and metastases. However, the mechanisms underlying microglial involvement appear complex and better models are required to improve understanding of function. METHODS: Here, we sought to address this need by developing a model to study metastatic breast cancer cell-microglial interactions using intravital imaging combined with ex vivo electrophysiology. We implanted an optical window on the parietal bone to facilitate observation of cellular behaviour in situ in the outer cortex of heterozygous Cx3cr1GFP/+ mice. RESULTS: We detected GFP-expressing microglia in Cx3cr1GFP/+ mice up to 350 µm below the window without significant loss of resolution. When DsRed-expressing metastatic MDA-MB-231 breast cancer cells were implanted in Matrigel under the optical window, significant accumulation of activated microglia around invading tumour cells could be observed. This inflammatory response resulted in significant cortical disorganisation and aberrant spontaneously-occurring local field potential spike events around the metastatic site. CONCLUSIONS: These data suggest that peritumoral microglial activation and accumulation may play a critical role in local tissue changes underpinning aberrant cortical activity, which offers a possible mechanism for the disrupted cognitive performance and seizures seen in patients with metastatic breast cancer.
Assuntos
Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Microscopia Intravital/métodos , Microglia , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microambiente Tumoral/fisiologiaRESUMO
Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These findings provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously.
Assuntos
Células da Medula Óssea/citologia , Linfócitos T CD4-Positivos/citologia , Células-Tronco Hematopoéticas/citologia , Leishmania donovani/fisiologia , Leishmaniose Visceral/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células da Medula Óssea/metabolismo , Ciclo Celular , Proliferação de Células , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Fator de Necrose Tumoral alfa/genéticaRESUMO
Stromal cells and the immune functions that they regulate underpin multiple aspects of host defence, but the study of stromal cells as targets of infection and as regulators of anti-infective immunity is in its infancy and still limited to a few well-worked examples. In this review, the role of stromal cells at each sequential stage of infection is discussed, with examples drawn from across the spectrum of infectious agents, from prions to the parasitic helminths. Gaps in knowledge are identified, the challenges in studying stromal cell biology in the context of infection are highlighted, and the potential for stromal cell-targeted therapeutics is briefly discussed.
Assuntos
Infecções/patologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Adesão Celular , Humanos , Imunidade Inata , Infecções/imunologia , Células Estromais/imunologia , Células Estromais/patologiaRESUMO
Salmonella enterica serovar Typhi is a human-restricted Gram-negative bacterial pathogen responsible for causing an estimated 27 million cases of typhoid fever annually, leading to 217,000 deaths, and current vaccines do not offer full protection. The O-antigen side chain of the lipopolysaccharide is an immunodominant antigen, can define host-pathogen interactions, and is under consideration as a vaccine target for some Gram-negative species. The composition of the O-antigen can be modified by the activity of glycosyltransferase (gtr) operons acquired by horizontal gene transfer. Here we investigate the role of two gtr operons that we identified in the S Typhi genome. Strains were engineered to express specific gtr operons. Full chemical analysis of the O-antigens of these strains identified gtr-dependent glucosylation and acetylation. The glucosylated form of the O-antigen mediated enhanced survival in human serum and decreased complement binding. A single nucleotide deviation from an epigenetic phase variation signature sequence rendered the expression of this glucosylating gtr operon uniform in the population. In contrast, the expression of the acetylating gtrC gene is controlled by epigenetic phase variation. Acetylation did not affect serum survival, but phase variation can be an immune evasion mechanism, and thus, this modification may contribute to persistence in a host. In murine immunization studies, both O-antigen modifications were generally immunodominant. Our results emphasize that natural O-antigen modifications should be taken into consideration when assessing responses to vaccines, especially O-antigen-based vaccines, and that the Salmonellagtr repertoire may confound the protective efficacy of broad-ranging Salmonella lipopolysaccharide conjugate vaccines.
Assuntos
Anticorpos Antibacterianos/imunologia , Soros Imunes/imunologia , Antígenos O/imunologia , Salmonella typhi/imunologia , Animais , Anticorpos Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Modelos Animais de Doenças , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Soros Imunes/farmacologia , Imunização , Metilação , Camundongos , Antígenos O/metabolismo , Óperon , Salmonella typhi/classificação , Salmonella typhi/efeitos dos fármacos , Salmonella typhi/genética , Febre Tifoide/imunologia , Febre Tifoide/microbiologiaRESUMO
The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80(hi)CD11b(lo)CD11c(+) macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis.
Assuntos
Leishmania donovani/patogenicidade , Leishmaniose Visceral/patologia , Glicoproteínas de Membrana/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas Tirosina Quinases/metabolismo , Baço/irrigação sanguínea , Animais , Azepinas/farmacologia , Benzamidas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Linhagem Celular , Células Endoteliais/metabolismo , Feminino , Leishmaniose Visceral/parasitologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de Fator de Crescimento Neural/biossíntese , Transdução de Sinais/fisiologia , Baço/metabolismo , Esplenomegalia/parasitologia , Esplenomegalia/patologiaRESUMO
Natural killer (NK) cells play a well-recognized role in early pathogen containment and in shaping acquired cell-mediated immunity. However, indirect evidence in humans and experimental models has suggested that NK cells also play negative regulatory roles during chronic disease. To formally test this hypothesis, we employed a well-defined experimental model of visceral leishmaniasis. Our data demonstrated that NKp46(+)CD49b(+)CD3(-) NK cells were recruited to the spleen and into hepatic granulomas, where they inhibited host protective immunity in an interleukin-10 (IL-10)-dependent manner. Although IL-10 mRNA could be detected in activated NK cells 24 hr after infection, the inhibitory function of NK cells was only acquired later during infection, coincident with increased IL-10 mRNA stability and an enhanced capacity to secrete IL-10 protein. Our data support a growing body of literature that implicates NK cells as negative regulators of cell-mediated immunity and suggest that NK cells, like CD4(+) T helper 1 cells, may acquire immunoregulatory functions as a consequence of extensive activation.
Assuntos
Interleucina-10/genética , Células Matadoras Naturais/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Expressão Gênica , Granuloma/imunologia , Granuloma/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Leishmaniose Visceral/parasitologia , Fígado/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
We describe a two-photon microscopy-based method to evaluate the in vivo systemic transport of compounds. This method comprises imaging of the intact liver, kidney and intestine, the main organs responsible for uptake and elimination of xenobiotics and endogenous molecules. The image quality of the acquired movies was sufficient to distinguish subcellular structures like organelles and vesicles. Quantification of the movement of fluorescent dextran and fluorescent cholic acid derivatives in different organs and their sub-compartments over time revealed significant dynamic differences. Calculated half-lives were similar in the capillaries of all investigated organs but differed in the specific sub-compartments, such as parenchymal cells and bile canaliculi of the liver, glomeruli, proximal and distal tubules of the kidney and lymph vessels (lacteals) of the small intestine. Moreover, tools to image immune cells, which can influence transport processes in inflamed tissues, are described. This powerful approach provides new possibilities for the analysis of compound transport in multiple organs and can support physiologically based pharmacokinetic modeling, in order to obtain more precise predictions at the whole body scale.
Assuntos
Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Microscopia/métodos , Xenobióticos/análise , Xenobióticos/farmacocinética , Acetaminofen/farmacocinética , Acetaminofen/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Intestinos/efeitos dos fármacos , Rim/citologia , Rim/metabolismo , Células de Kupffer/efeitos dos fármacos , Fígado/citologia , Masculino , Camundongos Transgênicos , Gravação em VídeoRESUMO
BACKGROUND & AIMS: Kupffer cells (KCs), the resident tissue macrophages of the liver, play a crucial role in the clearance of pathogens and other particulate materials that reach the systemic circulation. Recent studies have identified KCs as a yolk sac-derived resident macrophage population that is replenished independently of monocytes in the steady state. Although it is now established that following local tissue injury, bone marrow derived monocytes may infiltrate the tissue and differentiate into macrophages, the extent to which newly differentiated macrophages functionally resemble the KCs they have replaced has not been extensively studied. METHODS: We studied the two populations of KCs using intravital microscopy, morphometric analysis and gene expression profiling. An ion homeostasis gene signature, including genes associated with scavenger receptor function and extracellular matrix deposition, allowed discrimination between these two KC sub-types. RESULTS: Bone marrow derived "KCs" accumulating as a result of genotoxic injury, resemble but are not identical to their yolk sac counterparts. Reflecting the differential expression of scavenger receptors, yolk sac-derived KCs were more effective at accumulating acetylated low density lipoprotein, whereas surprisingly, they were poorer than bone marrow-derived KCs when assessed for uptake of a range of bacterial pathogens. The two KC populations were almost indistinguishable in regard to i) response to lipopolysaccharide challenge, ii) phagocytosis of effete red blood cells and iii) their ability to contain infection and direct granuloma formation against Leishmania donovani, a KC-tropic intracellular parasite. CONCLUSIONS: Bone marrow-derived KCs differentiate locally to resemble yolk sac-derived KC in most but not all respects, with implications for models of infectious diseases, liver injury and bone marrow transplantation. In addition, the gene signature we describe adds to the tools available for distinguishing KC subpopulations based on their ontology. LAY SUMMARY: Liver macrophages play a major role in the control of infections in the liver and in the pathology associated with chronic liver diseases. It was recently shown that liver macrophages can have two different origins, however, the extent to which these populations are functionally distinct remains to be fully addressed. Our study demonstrates that whilst liver macrophages share many features in common, regardless of their origin, some subtle differences in function exist. DATA REPOSITORY: Gene expression data are available from the European Bioinformatics Institute ArrayExpress data repository (accession number E-MTAB-4954).
Assuntos
Medula Óssea , Humanos , Células de Kupffer , Fígado , Macrófagos , MonócitosRESUMO
Organ-specific immunity is a feature of many infectious diseases, including visceral leishmaniasis caused by Leishmania donovani. Experimental visceral leishmaniasis in genetically susceptible mice is characterized by an acute, resolving infection in the liver and chronic infection in the spleen. CD4+ T cell responses are critical for the establishment and maintenance of hepatic immunity in this disease model, but their role in chronically infected spleens remains unclear. In this study, we show that dendritic cells are critical for CD4+ T cell activation and expansion in all tissue sites examined. We found that FTY720-mediated blockade of T cell trafficking early in infection prevented Ag-specific CD4+ T cells from appearing in lymph nodes, but not the spleen and liver, suggesting that early CD4+ T cell priming does not occur in liver-draining lymph nodes. Extended treatment with FTY720 over the first month of infection increased parasite burdens, although this associated with blockade of lymphocyte egress from secondary lymphoid tissue, as well as with more generalized splenic lymphopenia. Importantly, we demonstrate that CD4+ T cells are required for the establishment and maintenance of antiparasitic immunity in the liver, as well as for immune surveillance and suppression of parasite outgrowth in chronically infected spleens. Finally, although early CD4+ T cell priming appeared to occur most effectively in the spleen, we unexpectedly revealed that protective CD4+ T cell-mediated hepatic immunity could be generated in the complete absence of all secondary lymphoid tissues.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Memória Imunológica , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Cloridrato de Fingolimode , Imunossupressores/farmacologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/parasitologia , Ativação Linfocitária/imunologia , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/imunologia , Tecido Linfoide/parasitologia , Camundongos , Camundongos Knockout , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/parasitologiaRESUMO
Many substances are hepatotoxic due to their ability to cause intrahepatic cholestasis. Therefore, there is a high demand for in vitro systems for the identification of cholestatic properties of new compounds. Primary hepatocytes cultivated in collagen sandwich cultures are known to establish bile canaliculi which enclose secreted biliary components. Cholestatic compounds are mainly known to inhibit bile excretion dynamics, but may also alter canalicular volume, or hepatocellular morphology. So far, techniques to assess time-resolved morphological changes of bile canaliculi in sandwich cultures are not available. In this study, we developed an automated system that quantifies dynamics of bile canaliculi recorded in conventional time-lapse image sequences. We validated the hepatocyte sandwich culture system as an appropriate model to study bile canaliculi in vitro by showing structural similarity measured as bile canaliculi length per hepatocyte to that observed in vivo. Moreover, bile canalicular excretion kinetics of CMFDA (5-chloromethylfluorescein diacetate) in sandwich cultures resembled closely the kinetics observed in vivo. The developed quantification technique enabled the quantification of dynamic changes in individual bile canaliculi. With this technique, we were able to clearly distinguish between sandwich cultures supplemented with dexamethasone and insulin from control cultures. In conclusion, the automated quantification system offers the possibility to systematically study the causal relationship between disturbed bile canalicular dynamics and cholestasis.
Assuntos
Canalículos Biliares/efeitos dos fármacos , Técnicas de Cultura de Células , Colágeno/química , Hepatócitos/efeitos dos fármacos , Animais , Canalículos Biliares/metabolismo , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Colestase Intra-Hepática/induzido quimicamente , Dexametasona/administração & dosagem , Fluoresceínas/farmacocinética , Hepatócitos/metabolismo , Insulina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
It is now recognized that innate immunity to intestinal microflora plays a significant role in mediating immune health, and modulation of microbial sensing may underpin the impact of plant natural products in the diet or when used as nutraceuticals. In this context, we have examined five classes of plant-derived flavonoids (flavonols, flavones, flavanones, catechins, and cyanidin) for their ability to regulate cytokine release induced by the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. We found that the flavonols selectively co-stimulated IL-1ß secretion but had no impact on the secretion of IL-6. Importantly, this costimulation of TLR2-induced cytokine secretion was dependent on regiospecific methylation of the flavonol scaffold with a rank order of quercetin-3,4'-dimethylether > quercetin-3-methylether > casticin. The mechanism underpinning this costimulation did not involve enhanced inflammasome activation. In contrast, the methylated flavonols enhanced IL-1ß gene expression through transcriptional regulation, involving mechanisms that operate downstream of the initial NF-κB and STAT1 activation events. These studies demonstrate an exquisite level of control of scaffold bioactivity by regiospecific methylation, with important implications for understanding how natural products affect innate immunity and for their development as novel immunomodulators for clinical use.
Assuntos
Flavonoides/química , Interleucina-1beta/biossíntese , Monócitos/metabolismo , Receptor 2 Toll-Like/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Cicloeximida/farmacologia , Sinergismo Farmacológico , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopeptídeos/farmacologia , Metilação/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Fosforilação/efeitos dos fármacos , Quercetina/análogos & derivados , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estereoisomerismo , Receptor 2 Toll-Like/agonistas , Transcrição Gênica/efeitos dos fármacosRESUMO
IL-10 is a critical regulatory cytokine involved in the pathogenesis of visceral leishmaniasis caused by Leishmania donovani and clinical and experimental data indicate that disease progression is associated with expanded numbers of CD4⺠IFNγ⺠T cells committed to IL-10 production. Here, combining conditional cell-specific depletion with adoptive transfer, we demonstrate that only conventional CD11c(hi) DCs that produce both IL-10 and IL-27 are capable of inducing IL-10-producing Th1 cells in vivo. In contrast, CD11c(hi) as well as CD11c(int/lo) cells isolated from infected mice were capable of reversing the host protective effect of diphtheria toxin-mediated CD11c⺠cell depletion. This was reflected by increased splenomegaly, inhibition of NO production and increased parasite burden. Thus during chronic infection, multiple CD11c⺠cell populations can actively suppress host resistance and enhance immunopathology, through mechanisms that do not necessarily involve IL-10-producing Th1 cells.
Assuntos
Antígeno CD11c/análise , Interleucina-10/biossíntese , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Células Th1/imunologia , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Toxina Diftérica , Progressão da Doença , Interleucina-17/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Baço/parasitologiaRESUMO
Experimental visceral leishmaniasis, caused by infection of mice with the protozoan parasite Leishmania donovani, is characterized by focal accumulation of inflammatory cells in the liver, forming discrete "granulomas" within which the parasite is eventually eliminated. To shed new light on fundamental aspects of granuloma formation and function, we have developed an in silico Petri net model that simulates hepatic granuloma development throughout the course of infection. The model was extensively validated by comparison with data derived from experimental studies in mice, and the model robustness was assessed by a sensitivity analysis. The model recapitulated the progression of disease as seen during experimental infection and also faithfully predicted many of the changes in cellular composition seen within granulomas over time. By conducting in silico experiments, we have identified a previously unappreciated level of inter-granuloma diversity in terms of the development of anti-leishmanial activity. Furthermore, by simulating the impact of IL-10 gene deficiency in a variety of lymphocyte and myeloid cell populations, our data suggest a dominant local regulatory role for IL-10 produced by infected Kupffer cells at the core of the granuloma.
Assuntos
Granuloma/imunologia , Interleucina-10/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Animais , Simulação por Computador , Modelos Animais de Doenças , Granuloma/parasitologia , Inflamação/imunologia , Inflamação/parasitologia , Interleucina-10/metabolismo , Células de Kupffer , Leishmaniose Visceral/parasitologia , Leucócitos , Fígado/imunologia , Fígado/parasitologia , Camundongos , Modelos Imunológicos , Carga ParasitáriaRESUMO
The leishmaniases are globally important parasitic diseases for which no human vaccines are currently available. To facilitate vaccine development, we conducted an open-label observational study to establish a controlled human infection model (CHIM) of sand fly-transmitted cutaneous leishmaniasis (CL) caused by Leishmania major. Between 24 January and 12 August 2022, we exposed 14 participants to L. major-infected Phlebotomus duboscqi. The primary objective was to demonstrate effectiveness of lesion development (take rate) and safety (absence of CL lesion at 12 months). Secondary and exploratory objectives included rate of lesion development, parasite load and analysis of local immune responses by immunohistology and spatial transcriptomics. Lesion development was terminated by therapeutic biopsy (between days 14 and 42 after bite) in ten participants with clinically compatible lesions, one of which was not confirmed by parasite detection. We estimated an overall take rate for CL development of 64% (9/14). Two of ten participants had one and one of ten participants had two lesion recurrences 4-8 months after biopsy that were treated successfully with cryotherapy. No severe or serious adverse events were recorded, but as expected, scarring due to a combination of CL and the biopsy procedure was evident. All participants were lesion free at >12-month follow-up. We provide the first comprehensive map of immune cell distribution and cytokine/chemokine expression in human CL lesions, revealing discrete immune niches. This CHIM offers opportunities for vaccine candidate selection based on human efficacy data and for a greater understanding of immune-mediated pathology. ClinicalTrials.gov identifier: NCT04512742 .
RESUMO
BACKGROUND: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with an airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. METHODS: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. FINDINGS: Forty patients (32M:8F, age: 22-98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. INTERPRETATION: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established. FUNDING: UK Research and Innovation/Medical Research Council through the UK Coronavirus Immunology Consortium, Barbour Foundation, General Sir John Monash Foundation, Newcastle University, JGW Patterson Foundation, Wellcome Trust.
Assuntos
COVID-19 , Lesão Pulmonar , Humanos , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , COVID-19/patologia , Lesão Pulmonar/patologia , Células Endoteliais , SARS-CoV-2 , Pulmão/patologiaRESUMO
LIGHT (TNFSF14) is a member of the TNF superfamily involved in inflammation and defence against infection. LIGHT signals via two cell-bound receptors; herpes virus entry mediator (HVEM) and lymphotoxin-beta receptor (LTßR). We found that LIGHT is critical for control of hepatic parasite growth in mice with visceral leishmaniasis (VL) caused by infection with the protozoan parasite Leishmania donovani. LIGHT-HVEM signalling is essential for early dendritic cell IL-12/IL-23p40 production, and the generation of IFNγ- and TNF-producing T cells that control hepatic infection. However, we also discovered that LIGHT-LTßR interactions suppress anti-parasitic immunity in the liver in the first 7 days of infection by mechanisms that restrict both CD4(+) T cell function and TNF-dependent microbicidal mechanisms. Thus, we have identified distinct roles for LIGHT in infection, and show that manipulation of interactions between LIGHT and its receptors may be used for therapeutic advantage.