RESUMO
BACKGROUND: Type 2 innate lymphoid cells (ILC2s) play a pivotal role in type 2 asthma. CD226 is a costimulatory molecule involved in various inflammatory diseases. OBJECTIVE: We aimed to investigate CD226 expression and function within human and mouse ILC2s, and to assess the impact of targeting CD226 on ILC2-mediated airway hyperreactivity (AHR). METHODS: We administered IL-33 intranasally to wild-type mice, followed by treatment with anti-CD226 antibody or isotype control. Pulmonary ILC2s were sorted for ex vivo analyses through RNA sequencing and flow cytometry. Next, we evaluated the effects of CD226 on AHR and lung inflammation in wild-type and Rag2-/- mice. Additionally, we compared peripheral ILC2s from healthy donors and asthmatic patients to ascertain the role of CD226 in human ILC2s. RESULTS: Our findings demonstrated an inducible expression of CD226 in activated ILC2s, enhancing their cytokine secretion and effector functions. Mechanistically, CD226 alters intracellular metabolism and enhances PI3K/AKT and MAPK signal pathways. Blocking CD226 ameliorates ILC2-dependent AHR in IL-33 and Alternaria alternata-induced models. Interestingly, CD226 is expressed and inducible in human ILC2s, and its blocking reduces cytokine production. Finally, we showed that peripheral ILC2s in asthmatic patients exhibited elevated CD226 expression compared to healthy controls. CONCLUSION: Our findings underscore the potential of CD226 as a novel therapeutic target in ILC2s, presenting a promising avenue for ameliorating AHR and allergic asthma.
Assuntos
Antígenos de Diferenciação de Linfócitos T , Asma , Imunidade Inata , Linfócitos , Animais , Feminino , Humanos , Masculino , Camundongos , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Asma/imunologia , Interleucina-33/imunologia , Linfócitos/imunologia , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: Neutrophilic asthma is associated with disease severity and corticosteroid insensitivity. Novel therapies are required to manage this life-threatening asthma phenotype. Programmed cell death protein-1 (PD-1) is a key homeostatic modulator of the immune response for T-cell effector functions. OBJECTIVE: We sought to investigate the role of PD-1 in the regulation of acute neutrophilic inflammation in a murine model of airway hyperreactivity (AHR). METHODS: House dust mite was used to induce and compare neutrophilic AHR in wild-type and PD-1 knockout mice. Then, the therapeutic potential of a human PD-1 agonist was tested in a humanized mouse model in which the PD-1 extracellular domain is entirely humanized. Single-cell RNA sequencing and flow cytometry were mainly used to investigate molecular and cellular mechanisms. RESULTS: PD-1 was highly induced on pulmonary T cells in our inflammatory model. PD-1 deficiency was associated with an increased neutrophilic AHR and high recruitment of inflammatory cells to the lungs. Consistently, PD-1 agonist treatment dampened AHR, decreased neutrophil recruitment, and modulated cytokine production in a humanized PD-1 mouse model. Mechanistically, we demonstrated at the transcriptional and protein levels that the inhibitory effect of PD-1 agonist is associated with the reprogramming of pulmonary effector T cells that showed decreased number and activation. CONCLUSIONS: PD-1 agonist treatment is efficient in dampening neutrophilic AHR and lung inflammation in a preclinical humanized mouse model.
Assuntos
Asma , Receptor de Morte Celular Programada 1 , Humanos , Animais , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Pulmão , Células Th2 , Modelos Animais de DoençasRESUMO
BACKGROUND: Cannabinoids modulate the activation of immune cells and physiologic processes in the lungs. Group 2 innate lymphoid cells (ILC2s) are central players in type 2 asthma, but how cannabinoids modulate ILC2 activation remains to be elucidated. OBJECTIVE: Our goal was to investigate the effects of cannabinoids on ILC2s and their role in asthma. METHODS: A combination of cannabinoid receptor (CB)2 knockout (KO) mice, CB2 antagonist and agonist were used in the mouse models of IL-33, IL-25, and Alternaria alternata ILC2-dependent airway inflammation. RNA sequencing was performed to assess transcriptomic changes in ILC2s, and humanized mice were used to assess the role of CB2 signaling in human ILC2s. RESULTS: We provide evidence that CB2 signaling in ILC2s is important for the development of ILC2-driven airway inflammation in both mice and human. We showed that both naive and activated murine pulmonary ILC2s express CB2. CB2 signaling did not affect ILC2 homeostasis at steady state, but strikingly it stimulated ILC2 proliferation and function upon activation. As a result, ILC2s lacking CB2 induced lower lung inflammation, as we made similar observations using a CB2 antagonist. Conversely, CB2 agonism remarkably exacerbated ILC2-driven airway hyperreactivity and lung inflammation. Mechanistically, transcriptomic and protein analysis revealed that CB2 signaling induced cyclic adenosine monophosphate-response element binding protein (CREB) phosphorylation in ILC2s. Human ILC2s expressed CB2, as CB2 antagonism and agonism showed opposing effects on ILC2 effector function and development of airway hyperreactivity in humanized mice. CONCLUSION: Collectively, our results define CB2 signaling in ILC2s as an important modulator of airway inflammation.
Assuntos
Asma , Canabinoides , Pneumonia , Animais , Proliferação de Células , Citocinas , Humanos , Imunidade Inata , Inflamação , Interleucina-33 , Pulmão , Linfócitos , Camundongos , Camundongos Knockout , Receptor CB2 de Canabinoide , Receptores de CanabinoidesRESUMO
BACKGROUND: Type 2 innate lymphoid cells (ILC2s) are relevant players in type 2 asthma. They initiate eosinophil infiltration and airway hyperreactivity (AHR) through cytokine secretion. Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an inhibitory receptor considered to be an immune checkpoint in different inflammatory diseases. OBJECTIVE: Our aim here was to investigate the expression of LAIR-1 and assess its role in human and murine ILC2s. METHODS: Wild-type and LAIR-1 knockout mice were intranasally challenged with IL-33, and pulmonary ILC2s were sorted to perform an ex vivo comparative study based on RNA sequencing and flow cytometry. We next studied the impact of LAIR-1 deficiency on AHR and lung inflammation by using knockout mice and adoptive transfer experiments in Rag2-/-Il2rg-/- mice. Knockdown antisense strategies and humanized mice were used to assess the role of LAIR-1 in human ILC2s. RESULTS: We have demonstrated that LAIR-1 is inducible on activated ILC2s and downregulates cytokine secretion and effector function. LAIR-1 signaling in ILC2s was mediated via inhibitory pathways, including SHP1/PI3K/AKT, and LAIR-1 deficiency led to exacerbated ILC2-dependent AHR in IL-33 and Alternaria alternata models. In adoptive transfer experiments, we confirmed the LAIR-1-mediated regulation of ILC2s in vivo. Interestingly, LAIR-1 was expressed and inducible in human ILC2s, and knockdown approaches of Lair1 resulted in higher cytokine production. Finally, engagement of LAIR-1 by physiologic ligand C1q significantly reduced ILC2-dependent AHR in a humanized ILC2 murine model. CONCLUSION: Our results unravel a novel regulatory axis in ILC2s with the capacity to reduce allergic AHR and lung inflammation.
Assuntos
Alternariose/imunologia , Linfócitos/imunologia , Pneumonia/imunologia , Receptores Imunológicos/imunologia , Hipersensibilidade Respiratória/imunologia , Transferência Adotiva , Alternaria , Alternariose/fisiopatologia , Animais , Citocinas/imunologia , Feminino , Humanos , Imunidade Inata , Interleucina-33/farmacologia , Pulmão/imunologia , Pulmão/fisiopatologia , Transfusão de Linfócitos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/fisiopatologia , Receptores Imunológicos/genética , Hipersensibilidade Respiratória/fisiopatologiaRESUMO
BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are the dominant innate lymphoid cell population in the lungs at steady state, and their release of type 2 cytokines is a central driver in responding eosinophil infiltration and increased airway hyperreactivity. Our laboratory has identified a unique subset of ILC2s in the lungs that actively produce IL-10 (ILC210s). OBJECTIVE: Our aim was to characterize the effector functions of ILC210s in the development and pathology of allergic asthma. METHODS: IL-4-stimulated ILC210s were isolated to evaluate cytokine secretion, transcription factor signaling, metabolic dependence, and effector functions in vitro. ILC210s were also adoptively transferred into Rag2-/-γc-/- mice, which were then challenged with IL-33 and assessed for airway hyperreactivity and lung inflammation. RESULTS: We have determined that the transcription factors cMaf and Blimp-1 regulate IL-10 expression in ILC210s. Strikingly, our results demonstrate that ILC210s can utilize both autocrine and paracrine signaling to suppress proinflammatory ILC2 effector functions in vitro. Further, this subset dampens airway hyperreactivity and significantly reduces lung inflammation in vivo. Interestingly, ILC210s demonstrated a metabolic dependency on the glycolytic pathway for IL-10 production, shifting from the fatty acid oxidation pathway conventionally utilized for proinflammatory effector functions. CONCLUSION: These findings provide an important and previously unrecognized role of ILC210s in diseases associated with ILC2s such as allergic lung inflammation and asthma. They also provide new insights into the metabolism dependency of proinflammatory and anti-inflammatory ILC2 phenotypes.
Assuntos
Asma/imunologia , Hiper-Reatividade Brônquica/imunologia , Interleucina-10/imunologia , Linfócitos/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/imunologia , Proteínas Proto-Oncogênicas c-maf/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Neutrophils are essential during contact hypersensitivity (CHS), a common skin allergic disease. NF-E2-related factor-2 (Nrf2) is a key regulator of redox balance and skin homeostasis playing a protective role in CHS. In this study, we investigated Nrf2 role in neutrophil recruitment during the sensitization phase of CHS. Comparing wild-type and Nrf2 knockout mice, we demonstrated that Nrf2 regulated dinitrochlorobenzene-induced xenoinflammation, notably neutrophil recruitment to sensitized skin. Nrf2 protective role was associated with high expression of antioxidant genes (ho-1, gclc, nqo1 ) and decreased chemokine production (CCL2, CCL4, CCL11). Interestingly, skin sensitization induced CD36 upregulation in skin-resident macrophages. In vitro results confirmed that the transcription of cd36 gene in macrophages was dependent on Nrf2 and led to an improved capacity to phagocyte-damaged neutrophils by efferocytosis. Nrf2 emerges as a critical target in the sensitization phase of CHS regulating neutrophil recruitment and accumulation in the skin through antioxidant-dependent and -independent mechanisms.
Assuntos
Dermatite de Contato/imunologia , Regulação da Expressão Gênica/imunologia , Fator 2 Relacionado a NF-E2/imunologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Pele/imunologia , Animais , Antioxidantes , Quimiocinas/genética , Quimiocinas/imunologia , Dermatite de Contato/genética , Dermatite de Contato/patologia , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Neutrófilos/patologia , Pele/patologiaRESUMO
BACKGROUND & AIMS: Severe alcoholic hepatitis (SAH) is associated with a high risk of infection. The IL-33/ST2 pathway is involved in sepsis control but data regarding its role in alcohol-related liver disease (ALD) are lacking. We aimed to characterize the role of IL-33/ST2 in the polymorphonuclear neutrophils (PMNs) of patients with ALD and SAH. METHODS: Serum and circulating neutrophils were collected from patients with SAH, alcoholic cirrhosis and healthy controls. We quantified IL-33/ST2 pathway activity and CXCR2 at baseline and after exposure to IL-33. We also determined the migration capacity of PMNs. RESULTS: The decoy receptor of IL-33 (soluble ST2 [sST2]) was increased in SAH vs. cirrhosis and controls, demonstrating the defect in this pathway during ALD. The sST2 level was associated with response to treatment, 2-month survival, infection-free survival and probability of infection in SAH. Endotoxemia was weakly correlated with sST2. GRK2, a negative regulator of CXCR2, was overexpressed in PMNs of patients with SAH and cirrhosis and was decreased by IL-33. CXCR2 levels on PMNs were lower in SAH vs. cirrhosis and controls. Treatment with IL-33 partially restored CXCR2 expression in SAH and cirrhosis. PMN migration upon IL-8 was lower in patients with SAH and cirrhosis vs. controls. Treatment with IL-33 partially restored migration in those with SAH and cirrhosis. Interestingly, the migration capacity of PMNs and the response to IL-33 were enhanced in responders to corticosteroids (Lille <0.45) compared to non-responders. CONCLUSION: The IL33/ST2 pathway is defective in SAH and predicts outcome. This defect is associated with decreased CXCR2 expression on the surface of PMNs and lower migration capacity, which can be corrected by IL-33, especially in patients responding to steroids. These results suggest that IL-33 has therapeutic potential for SAH and its infectious complications. LAY SUMMARY: The neutrophils of patients with severe alcoholic hepatitis are associated with a defect in the IL-33/ST2 pathway. This defect is associated with lower migration capacities in neutrophils and a higher probability of getting infected. Administration of IL-33 to the neutrophils at least partly restores this defect and may be effective at reducing the risk of infection in patients with severe alcoholic hepatitis.
Assuntos
Movimento Celular/imunologia , Hepatite Alcoólica/sangue , Hepatite Alcoólica/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/sangue , Interleucina-33/sangue , Cirrose Hepática Alcoólica/sangue , Cirrose Hepática Alcoólica/imunologia , Neutrófilos/imunologia , Transdução de Sinais/imunologia , Adulto , Idoso , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Seguimentos , Humanos , Interleucina-33/farmacologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Prognóstico , Estudos Prospectivos , Receptores de Interleucina-8B/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Group 2 innate lymphoid cells (ILC2s) rapidly induce a type 2 inflammation in the lungs in response to allergens. Here, we focused on the role of iron, a critical nutritional trace element, on ILC2 function and asthma pathogenesis. We found that transferrin receptor 1 (TfR1) is rapidly up-regulated and functional during ILC2 activation in the lungs, and blocking transferrin uptake reduces ILC2 expansion and activation. Iron deprivation reprogrammed ILC2 metabolism, inducing a HIF-1α-driven up-regulation of glycolysis and inhibition of oxidative mitochondrial activity. Consequently, we observed that in vivo iron chelation or induction of hypoferremia reduced the development of airway hyperreactivity in experimental models of ILC2-driven allergic asthma. Human circulating ILC2s rapidly induced TfR1 during activation, whereas inhibition of iron uptake or iron deprivation reduced effector functions. Last, we found a negative relationship between circulating ILC2 TfR1 expression and airway function in cohorts of patients with asthma. Collectively, our studies define cellular iron as a critical regulator of ILC2 function.
Assuntos
Asma , Ferro , Linfócitos , Receptores da Transferrina , Receptores da Transferrina/metabolismo , Ferro/metabolismo , Animais , Linfócitos/metabolismo , Humanos , Asma/imunologia , Asma/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Imunidade Inata , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease of unknown etiology with a poor prognosis. It is a chronic and progressive disease that has a distinct radiological and pathological pattern from common interstitial pneumonia. The use of immunosuppressive medication was shown to be completely ineffective in clinical trials, resulting in years of neglect of the immune component. However, recent developments in fundamental and translational science demonstrate that immune cells play a significant regulatory role in IPF, and macrophages appear to be among the most crucial. These highly plastic cells generate multiple growth factors and mediators that highly affect the initiation and progression of IPF. In this review, we will provide an update on the role of macrophages in IPF through a systemic discussion of various regulatory mechanisms involving immune receptors, cytokines, metabolism, and epigenetics.
Assuntos
Fibrose Pulmonar Idiopática , Humanos , Fibrose Pulmonar Idiopática/genética , Cognição , Epigênese Genética , Macrófagos , Redes e Vias MetabólicasRESUMO
Ca2+ entry via Ca2+ release-activated Ca2+ (CRAC) channels is a predominant mechanism of intracellular Ca2+ elevation in immune cells. Here we show the immunoregulatory role of CRAC channel components Orai1 and Orai2 in Group 2 innate lymphoid cells (ILC2s), that play crucial roles in the induction of type 2 inflammation. We find that blocking or genetic ablation of Orai1 and Orai2 downregulates ILC2 effector function and cytokine production, consequently ameliorating the development of ILC2-mediated airway inflammation in multiple murine models. Mechanistically, ILC2 metabolic and mitochondrial homeostasis are inhibited and lead to the upregulation of reactive oxygen species production. We confirm our findings in human ILC2s, as blocking Orai1 and Orai2 prevents the development of airway hyperreactivity in humanized mice. Our findings have a broad impact on the basic understanding of Ca2+ signaling in ILC2 biology, providing potential insights into the development of therapies for the treatment of allergic and atopic inflammatory diseases.
Assuntos
Asma , Imunidade Inata , Camundongos , Humanos , Animais , Linfócitos , Homeostase , Inflamação , Proteína ORAI1/genéticaRESUMO
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are serious health problems that manifest as acute respiratory failure in response to different conditions, including viral respiratory infections. Recently, the inhibitory properties of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) were demonstrated in allergic and viral airway inflammation. In this study, we investigate the implication of LAIR-1 in ALI/ARDS and explore the underlying mechanisms. Polyinosinic:polycytidylic acid, a synthetic analog of double-stranded RNA, was used to mimic acute inflammation in viral infections. We demonstrate that LAIR-1 is predominantly expressed on macrophages and regulates their recruitment to the lungs as well as their activation in response to polyinosinic:polycytidylic acid. Interestingly, LAIR-1 deficiency increases neutrophil recruitment as well as lung resistance and permeability. In particular, we highlight the capacity of LAIR-1 to regulate the secretion of CXCL10, considered a key marker of macrophage overactivation in acute lung inflammation. We also reveal in COVID-19-induced lung inflammation that LAIR1 is upregulated on lung macrophages in correlation with relevant immune regulatory genes. Altogether, our findings demonstrate the implication of LAIR-1 in the pathogenesis of ALI/ARDS by means of the regulation of macrophages, thereby providing the basis of a novel therapeutic target.
Assuntos
Lesão Pulmonar Aguda , Pneumonia , Síndrome do Desconforto Respiratório , Humanos , Ativação de Macrófagos , Pulmão , Inflamação/patologia , Poli CRESUMO
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical disorders that mainly develop from viral respiratory infections, sepsis, and chest injury. Antigen-presenting cells play a pivotal role in propagating uncontrolled inflammation and injury through the excess secretion of pro-inflammatory cytokines and recruitment of immune cells. Autophagy, a homeostatic process that involves the degradation of cellular components, is involved in many processes including lung inflammation. Here, we use a polyinosinic-polycytidylic acid (poly(I:C))-induced lung injury mouse model to mimic viral-induced ALI/ARDS and show that disruption of autophagy in macrophages exacerbates lung inflammation and injury, whereas autophagy induction attenuates this process. Therefore, induction of autophagy in macrophages can be a promising therapeutic strategy in ALI/ARDS.
Assuntos
Lesão Pulmonar Aguda , Síndrome do Desconforto Respiratório , Animais , Camundongos , Células Apresentadoras de Antígenos , Macrófagos , Autofagia , Poli I-C/farmacologiaRESUMO
Kupffer cells play critical roles in both hepatitis B virus (HBV) persistence and clearance. Here, we provide a protocol for studying the interplay between Kupffer cells and HBV-positive hepatocytes ex vivo using mice as a model. This protocol includes hydrodynamic injection of HBV DNA into mouse hepatocytes, liver perfusion for isolating hepatocytes and Kupffer cells, and Seahorse metabolic analysis of Kupffer cells. This protocol allows the detailed analysis of how HBV-positive hepatocytes and Kupffer cells impact each other ex vivo. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).
Assuntos
Vírus da Hepatite B , Células de Kupffer , Animais , Vírus da Hepatite B/genética , Hepatócitos , Fígado , Camundongos , PerfusãoRESUMO
Regulatory T (Treg) cells are central to limit immune responses to allergens. Here we show that PD-L2 deficiency prevents the induction of tolerance to ovalbumin and control of airway hyperreactivity, in particular by limiting pTreg numbers and function. In vitro, PD-1/PD-L2 interactions increase iTreg numbers and stability. In mice lacking PD-L2 we find lower numbers of splenic pTregs at steady state, producing less IL-10 upon activation and with reduced suppressive activity. Remarkably, the numbers of splenic pTregs are restored by adoptively transferring PD-L2high dendritic cells to PD-L2KO mice. Functionally, activated pTregs lacking PD-L2 show lower Foxp3 expression, higher methylation of the Treg-Specific Demethylation Region (TSDR) and a decreased Tricarboxylic Acid (TCA) cycle associated with a defect in mitochondrial function and ATP production. Consequently, pyruvate treatment of PD-L2KO mice partially restores IL-10 production and airway tolerance. Together, our study highlights the importance of the PD-1/PD-L2 axis in the control of metabolic pathways regulating pTreg Foxp3 stability and suppressive functions, opening up avenues to further improve mucosal immunotherapy.
Assuntos
Fatores de Transcrição Forkhead , Proteína 2 Ligante de Morte Celular Programada 1 , Linfócitos T Reguladores , Animais , Fatores de Transcrição Forkhead/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos , Ovalbumina , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
There has been a global increase in rates of obesity with a parallel epidemic of non-alcoholic fatty liver disease (NAFLD). Autophagy is an essential mechanism involved in the degradation of cellular material and has an important function in the maintenance of liver homeostasis. Here, we explore the effect of Autophagy-related 5 (Atg5) deficiency in liver CD11c+ cells in mice fed HFD. When compared to control mice, Atg5-deficient CD11c+ mice exhibit increased glucose intolerance and decreased insulin sensitivity when fed HFD. This phenotype is associated with the development of NAFLD. We observe that IL-23 secretion is induced in hepatic CD11c+ myeloid cells following HFD feeding. We demonstrate that both therapeutic and preventative IL-23 blockade alleviates glucose intolerance, insulin resistance and protects against NAFLD development. This study provides insights into the function of autophagy and IL-23 production by hepatic CD11c+ cells in NAFLD pathogenesis and suggests potential therapeutic targets.
Assuntos
Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Animais , Autofagia , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina/genética , Interleucina-23/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.
Assuntos
Antígenos de Protozoários/imunologia , Medula Óssea/parasitologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Fígado/parasitologia , Baço/parasitologia , Imunidade Adaptativa , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/sangue , Medula Óssea/metabolismo , Feminino , Imunização , Interferon gama/metabolismo , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Baço/metabolismoRESUMO
The liver is unique in both its ability to maintain immune homeostasis and in its potential for immune tolerance following solid organ transplantation. Single-cell RNA sequencing (scRNA seq) is a powerful approach to generate highly dimensional transcriptome data to understand cellular phenotypes. However, when scRNA data is produced by different groups, with different data models, different standards, and samples processed in different ways, it can be challenging to draw meaningful conclusions from the aggregated data. The goal of this study was to establish a method to combine 'human liver' scRNA seq datasets by 1) characterizing the heterogeneity between studies and 2) using the meta-atlas to define the dominant phenotypes across immune cell subpopulations in healthy human liver. Publicly available scRNA seq data generated from liver samples obtained from a combined total of 17 patients and ~32,000 cells were analyzed. Liver-specific immune cells (CD45+) were extracted from each dataset, and immune cell subpopulations (myeloid cells, NK and T cells, plasma cells, and B cells) were examined using dimensionality reduction (UMAP), differential gene expression, and ingenuity pathway analysis. All datasets co-clustered, but cell proportions differed between studies. Gene expression correlation demonstrated similarity across all studies, and canonical pathways that differed between datasets were related to cell stress and oxidative phosphorylation rather than immune-related function. Next, a meta-atlas was generated via data integration and compared against PBMC data to define gene signatures for each hepatic immune subpopulation. This analysis defined key features of hepatic immune homeostasis, with decreased expression across immunologic pathways and enhancement of pathways involved with cell death. This method for meta-analysis of scRNA seq data provides a novel approach to broadly define the features of human liver immune homeostasis. Specific pathways and cellular phenotypes described in this human liver immune meta-atlas provide a critical reference point for further study of immune mediated disease processes within the liver.
Assuntos
Perfilação da Expressão Gênica , Homeostase , Fígado/imunologia , Fígado/metabolismo , Análise de Célula Única , Transcriptoma , Biomarcadores , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase/genética , Homeostase/imunologia , Humanos , Transdução de Sinais , Análise de Célula Única/métodosRESUMO
The prevalence of asthma and airway hyperreactivity (AHR) is increasing at an alarming rate. Group 2 innate lymphoid cells (ILC2s) are copious producers of type 2 cytokines, which leads to AHR and lung inflammation. Here, we show that mouse ILC2s express CD200 receptor (CD200R) and this expression is inducible. CD200R engagement inhibits activation, proliferation and type 2 cytokine production, indicating an immunoregulatory function for the CD200-CD200R axis on ILC2s. Furthermore, CD200R engagement inhibits both canonical and non-canonical NF-κB signaling pathways in activated ILC2s. Additionally, we demonstrate both preventative and therapeutic approaches utilizing CD200R engagement on ILC2s, which lead to improved airway resistance, dynamic compliance and eosinophilia. These results show CD200R is expressed on human ILC2s, and its engagement ameliorates AHR in humanized mouse models, emphasizing the translational applications for treatment of ILC2-related diseases such as allergic asthma.