RESUMEN
Intracellular pathogens that replicate in host myeloid cells have devised ways to inhibit the cell's killing machinery. Pyroptosis is one of the host strategies used to reduce the pathogen replicating niche and thereby control its expansion. The intracellular Leishmania parasites can survive and use neutrophils as a silent entry niche, favoring subsequent parasite dissemination into the host. Here, we show that Leishmania mexicana induces NLRP1- and caspase-1-dependent Gasdermin D (GSDMD)-mediated pyroptosis in neutrophils, a process critical to control the parasite-induced pathology. In the absence of GSDMD, we observe an increased number of infected dermal neutrophils two days post-infection. Using adoptive neutrophil transfer in neutropenic mice, we show that pyroptosis contributes to the regulation of the neutrophil niche early after infection. The critical role of neutrophil pyroptosis and its positive influence on the regulation of the disease outcome was further demonstrated following infection of mice with neutrophil-specific deletion of GSDMD. Thus, our study establishes neutrophil pyroptosis as a critical regulator of leishmaniasis pathology.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Leishmaniasis Cutánea , Neutrófilos , Proteínas de Unión a Fosfato , Piroptosis , Animales , Neutrófilos/metabolismo , Neutrófilos/inmunología , Proteínas de Unión a Fosfato/metabolismo , Ratones , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/metabolismo , Leishmaniasis Cutánea/parasitología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Leishmania mexicana/inmunología , GasderminasRESUMEN
Neutrophils are the first line of defence against invading pathogens. Although neutrophils are well-known professional killers, some pathogens including Leishmania (L.) parasites survive in neutrophils, using these cells to establish infection. Manipulation of neutrophil recruitment to the infection site is therefore of interest in this cutaneous disease. The c-MET tyrosine kinase receptor was shown to promote neutrophil migration to inflamed sites. Here, we investigated the importance of c-MET expression on neutrophils in their recruitment to the infection site and the role of c-Met expression in the pathology of leishmaniasis. Following infection with L. mexicana, mice with conditional deletion of c-MET in neutrophils controlled significantly better their lesion development and parasite burden compared to similarly infected wild type mice. Our data reveal a specific role for c-MET activation in Leishmania-induced neutrophil infiltration, a process correlating with their negative role in the pathology of the diseases. We further show that c-MET phosphorylation is observed in established cutaneous lesions. Exposure to L. mexicana upregulated c-Met expression predominantly in infected neutrophils and c-Met expression influenced ROS release by neutrophils. In addition, pharmacological inhibition of c-MET, administrated once the lesion is established, induced a significant decrease in lesion size associated with diminished infiltration of neutrophils. Both genetic ablation of c-MET in neutrophils and systemic inhibition of c-MET locally resulted in higher levels of CD4+T cells producing IFNγ, suggesting a crosstalk between neutrophils and these cells. Collectively, our data show that c-MET activation in neutrophils contributes to their recruitment following infection, and that L. mexicana induction of c-MET on neutrophils impacts the local pathology associated with this disease. Our results suggest a potential use for this inhibitor in the control of the cutaneous lesion during this parasitic infection.
Asunto(s)
Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/patología , Neutrófilos/inmunología , Proteínas Proto-Oncogénicas c-met/inmunología , Animales , Leishmaniasis Cutánea/metabolismo , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/inmunología , Proteínas Proto-Oncogénicas c-met/metabolismoRESUMEN
Injection of effector proteins to block host innate immune signaling is a common strategy used by many pathogenic organisms to establish an infection. For example, pathogenic Yersinia species inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signaling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1-caspase-8 death-inducing platform that confers antibacterial defense. While recent studies revealed that caspase-8 cleaves the pore-forming protein gasdermin D to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defense is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase-dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1ß release, which is critical for anti-Yersinia defense. During in vivo infection, IL-1ß neutralization increases bacterial burden in wild-type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signaling.
Asunto(s)
Inmunidad Innata , Macrófagos/metabolismo , Proteínas de Neoplasias/metabolismo , Neutrófilos/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Yersinia pseudotuberculosis/fisiología , Células 3T3 , Animales , Citocinas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Piroptosis , Infecciones por Yersinia pseudotuberculosis/inmunología , Infecciones por Yersinia pseudotuberculosis/microbiologíaRESUMEN
The skin microenvironment at the site of infection plays a role in the early events that determine protective T helper 1/type 1 immune responses during cutaneous leishmaniasis (CL) infection. During CL in nonhealing BALB/c mice, early interleukin-4 (IL-4) can instruct dendritic cells for protective Th1 immunity. Additionally, keratinocytes, which are the principal cell type in the skin epidermis, have been shown to secrete IL-4 early after Leishmania major infection. Here, we investigated whether IL-4/IL-13 signaling via the common IL-4 receptor alpha chain (IL-4Rα) on keratinocytes contributes to susceptibility during experimental CL. To address this, keratinocyte-specific IL-4Rα-deficient (KRT14cre IL-4Rα-/lox) mice on a BALB/c genetic background were generated by gene targeting and site-specific recombination (Cre/loxP) under the control of the keratinocyte-specific krt14 locus. Following high-dose infection with L. major IL-81 and LV39 promastigotes subcutaneously in the footpad, footpad swelling, parasite burden, IFN-γ/IL-4/IL-13 cytokine production, and type 1 and type 2 antibody responses were similar between KRT14cre IL-4Rα-/lox and littermate control IL-4Rα-/lox BALB/c mice. An intradermal infection with low-dose L. major IL-81 and LV39 promastigotes in the ear showed results in infected KRT14cre IL-4Rα-/lox BALB/c mice similar to those of littermate control IL-4Rα-/lox BALB/c mice, with the exception of a significant decrease observed in parasite burden only at the site of LV39 infection in the ear. Collectively, our results show that autocrine and paracrine signaling of IL-4/IL-13 through the IL-4Rα chain on keratinocytes does not influence the establishment of a nonhealing Th2 immune response in BALB/c mice during L. major infection.
Asunto(s)
Eliminación de Gen , Subunidad alfa del Receptor de Interleucina-4/genética , Queratinocitos/inmunología , Leishmaniasis Cutánea/inmunología , Transducción de Señal/inmunología , Animales , Comunicación Autocrina/inmunología , Linfocitos T CD4-Positivos , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/parasitología , Femenino , Interleucina-13/inmunología , Queratinocitos/parasitología , Leishmania major/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Comunicación Paracrina/inmunología , Células Th2/inmunologíaRESUMEN
Although Notch signaling plays important roles in lineage commitment and differentiation of multiple cell types including conventional T cells, nothing is currently known concerning Notch function in innate-like T cells. We have found that the homeostasis of several well-characterized populations of innate-like T cells including invariant NKT cells (iNKT), CD8ααTCRαß small intestinal intraepithelial lymphocytes, and innate memory phenotype CD8 T cells is controlled by Notch. Notch selectively regulates hepatic iNKT cell survival via tissue-restricted control of B cell lymphoma 2 and IL-7Rα expression. More generally, Notch regulation of innate-like T cell homeostasis involves both cell-intrinsic and -extrinsic mechanisms and relies upon context-dependent interactions with Notch ligand-expressing fibroblastic stromal cells. Collectively, using conditional ablation of Notch receptors on peripheral T cells or Notch ligands on putative fibroblastic stromal cells, we show that Notch signaling is indispensable for the homeostasis of three tissue-restricted populations of innate-like T cells: hepatic iNKT, CD8ααTCRαß small intestinal intraepithelial lymphocytes, and innate memory phenotype CD8 T cells, thus supporting a generalized role for Notch in innate T cell homeostasis.
Asunto(s)
Diferenciación Celular/inmunología , Homeostasis/inmunología , Receptores Notch/inmunología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Transgénicos , Receptores Notch/metabolismoRESUMEN
Infection of C57BL/6 mice with most Leishmania major strains results in a healing lesion and clearance of parasites from the skin. Infection of C57BL/6 mice with the L. major Seidman strain (LmSd), isolated from a patient with chronic lesions, despite eliciting a strong Th1 response, results in a nonhealing lesion, poor parasite clearance, and complete destruction of the ear dermis. We show here that in comparison to a healing strain, LmSd elicited early upregulation of IL-1ß mRNA and IL-1ß-producing dermal cells and prominent neutrophil recruitment to the infected skin. Mice deficient in Nlrp3, apoptosis-associated speck-like protein containing a caspase recruitment domain, or caspase-1/11, or lacking IL-1ß or IL-1 receptor signaling, developed healing lesions and cleared LmSd from the infection site. Mice resistant to LmSd had a stronger antigen-specific Th1 response. The possibility that IL-1ß might act through neutrophil recruitment to locally suppress immunity was supported by the healing observed in neutropenic Genista mice. Secretion of mature IL-1ß by LmSd-infected macrophages in vitro was dependent on activation of the Nlrp3 inflammasome and caspase-1. These data reveal that Nlrp3 inflammasome-dependent IL-1ß, associated with localized neutrophil recruitment, plays a crucial role in the development of a nonhealing form of cutaneous leishmaniasis in conventionally resistant mice.
Asunto(s)
Proteínas Portadoras/genética , Interleucina-1beta/genética , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Animales , Caspasa 1/genética , Caspasas/genética , Caspasas Iniciadoras , Modelos Animales de Enfermedad , Humanos , Interleucina-17/genética , Leishmania major/aislamiento & purificación , Leishmaniasis Cutánea/parasitología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , ARN Mensajero/biosíntesis , Receptores de Citocinas/genética , Receptores de Interleucina , Receptores Tipo I de Interleucina-1/genética , Células TH1/inmunologíaRESUMEN
The protozoan Leishmania mexicana parasite causes chronic non-healing cutaneous lesions in humans and mice with poor parasite control. The mechanisms preventing the development of a protective immune response against this parasite are unclear. Here we provide data demonstrating that parasite sequestration by neutrophils is responsible for disease progression in mice. Within hours of infection L. mexicana induced the local recruitment of neutrophils, which ingested parasites and formed extracellular traps without markedly impairing parasite survival. We further showed that the L. mexicana-induced recruitment of neutrophils impaired the early recruitment of dendritic cells at the site of infection as observed by intravital 2-photon microscopy and flow cytometry analysis. Indeed, infection of neutropenic Genista mice and of mice depleted of neutrophils at the onset of infection demonstrated a prominent role for neutrophils in this process. Furthermore, an increase in monocyte-derived dendritic cells was also observed in draining lymph nodes of neutropenic mice, correlating with subsequent increased frequency of IFNγ-secreting T helper cells, and better parasite control leading ultimately to complete healing of the lesion. Altogether, these findings show that L. mexicana exploits neutrophils to block the induction of a protective immune response and impairs the control of lesion development. Our data thus demonstrate an unanticipated negative role for these innate immune cells in host defense, suggesting that in certain forms of cutaneous leishmaniasis, regulating neutrophil recruitment could be a strategy to promote lesion healing.
Asunto(s)
Células Dendríticas/inmunología , Leishmania mexicana/inmunología , Leishmaniasis Cutánea/inmunología , Monocitos/inmunología , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/parasitología , Animales , Células Cultivadas , Enfermedad Crónica , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/parasitología , Citometría de Flujo , Leishmaniasis Cutánea/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Monocitos/parasitología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Adaptive immunity is initiated in T-cell zones of secondary lymphoid organs. These zones are organized in a rigid 3D network of fibroblastic reticular cells (FRCs) that are a rich cytokine source. In response to lymph-borne antigens, draining lymph nodes (LNs) expand several folds in size, but the fate and role of the FRC network during immune response is not fully understood. Here we show that T-cell responses are accompanied by the rapid activation and growth of FRCs, leading to an expanded but similarly organized network of T-zone FRCs that maintains its vital function for lymphocyte trafficking and survival. In addition, new FRC-rich environments were observed in the expanded medullary cords. FRCs are activated within hours after the onset of inflammation in the periphery. Surprisingly, FRC expansion depends mainly on trapping of naïve lymphocytes that is induced by both migratory and resident dendritic cells. Inflammatory signals are not required as homeostatic T-cell proliferation was sufficient to trigger FRC expansion. Activated lymphocytes are also dispensable for this process, but can enhance the later growth phase. Thus, this study documents the surprising plasticity as well as the complex regulation of FRC networks allowing the rapid LN hyperplasia that is critical for mounting efficient adaptive immunity.
Asunto(s)
Fibroblastos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Linfocitos/citología , Inmunidad Adaptativa , Animales , Movimiento Celular , Proliferación Celular , Células Dendríticas/citología , Fibroblastos/metabolismo , Homeostasis , Inflamación , Activación de Linfocitos , Linfotoxina-alfa/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente/métodos , Factor 88 de Diferenciación Mieloide/metabolismo , Células del Estroma/citología , Linfocitos T/citologíaRESUMEN
Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1ß stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Interleucina-1beta/inmunología , Activación de Linfocitos/inmunología , Linfocitos/inmunología , Transducción de Señal/inmunología , Animales , Presentación de Antígeno/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunidad Celular/inmunología , Interleucina-1beta/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C) on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs) from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1) response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2) response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response.
Asunto(s)
Moléculas de Adhesión Celular/inmunología , Células Dendríticas/inmunología , Inmunidad Celular , Inmunidad Innata , Inmunoglobulinas/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Animales , Células Dendríticas/patología , Femenino , Leishmaniasis Cutánea/patología , Ratones , Ratones Endogámicos BALB C , Piel/inmunología , Piel/parasitología , Piel/patología , Células TH1/inmunología , Células TH1/patología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1(-/-) mice are viable, fertile and show no altered hematopoietic compartment. In CD4(+) T cell- and dextran sodium sulfate-induced models of colitis, Trem1(-/-) mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1(-/-) mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1(-/-) mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1(-/-) mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1(+/+) controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.
Asunto(s)
Colitis/inmunología , Virus de la Influenza A/inmunología , Legionella pneumophila/inmunología , Enfermedad de los Legionarios/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Glicoproteínas de Membrana/deficiencia , Infecciones por Orthomyxoviridae/inmunología , Receptores Inmunológicos/deficiencia , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Colitis/terapia , Modelos Animales de Enfermedad , Enfermedad de los Legionarios/genética , Enfermedad de los Legionarios/patología , Enfermedad de los Legionarios/terapia , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/patología , Leishmaniasis Cutánea/terapia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Noqueados , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/terapia , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptor Activador Expresado en Células Mieloides 1RESUMEN
Leishmania major infection induces self-healing cutaneous lesions in C57BL/6 mice. Both IL-12 and IFN-γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3(-/-) ) mice (C57BL/6 background) that lack the major IL-12 producing and cross-presenting CD8α(+) and CD103(+) DC subsets. Batf3(-/-) mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3(-/-) mice secreted less IFN-γ, but more Th2- and Th17-type cytokines, mirrored by increased serum IgE and Leishmania-specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α(+) DCs isolated from lymph nodes of L. major-infected mice induced significantly more IFN-γ secretion by L. major-stimulated immune T cells than CD103(+) DCs. We next developed CD11c-diptheria toxin receptor: Batf3(-/-) mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf-3-dependent DCs from day 17 or wild-type mice depleted of cross-presenting DCs from 17-19 days after infection maintained significantly larger lesions similar to mice whose Batf-3-dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf-3-dependent DCs in protection against L. major.
Asunto(s)
Presentación de Antígeno , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Reactividad Cruzada , Células Dendríticas/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Proteínas Represoras/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/genética , Anticuerpos Antiprotozoarios/inmunología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Reactividad Cruzada/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Inmunoglobulina E/sangre , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Interferón gamma , Leishmania major/metabolismo , Leishmaniasis Cutánea/sangre , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Células Th2/inmunología , Células Th2/metabolismo , Células Th2/patologíaRESUMEN
Follicular helper T (TFH) cells are specialized in providing help for B cell differentiation and Ab secretion. Several positive and negative regulators of TFH cell differentiation have been described but their control is not fully understood. In this study, we show that Notch signaling in T cells is a major player in the development and function of TFH cells. T cell-specific gene ablation of Notch1 and Notch2 impaired differentiation of TFH cells in draining lymph nodes of mice immunized with T-dependent Ags or infected with parasites. Impaired TFH cell differentiation correlated with deficient germinal center development and the absence of high-affinity Abs. The impact of loss of Notch on TFH cell differentiation was largely independent of its effect on IL-4. These results show a previously unknown role for Notch in the regulation of TFH cell differentiation and function with implications for the control of this T cell population.
Asunto(s)
Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Receptores Notch/inmunología , Transducción de Señal/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Centro Germinal/citología , Centro Germinal/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+) T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+) T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4(+) T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre)) were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre) mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+) T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+) T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.
Asunto(s)
Interferón gamma/metabolismo , Leishmania major/fisiología , Leishmaniasis Cutánea/inmunología , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Células TH1/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Interacciones Huésped-Parásitos , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor Notch1/genética , Receptor Notch2/genética , Transducción de SeñalRESUMEN
Follicular Th (T(FH)) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5(high)PD-1(high) CD4(+) T(FH) cells that exhibit T(FH) features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal T(FH) cells fail to expand and to acquire a full-blown GC T(FH) phenotype, as reflected by a higher ratio of GC T(FH)/non-GC CD4(+) T cells in immunized adults than neonates (3.8 × 10(-3) versus 2.2 × 10(-3), p = 0.01). Following the adoptive transfer of naive adult OT-II CD4(+) T cells, OT-II T(FH) cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4(+) OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell-intrinsic factors. Postponing immunization to later in life increases the number of T(FH) cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC T(FH) and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the T(FH) cell development limits early life GC responses and that adjuvants/delivery systems supporting T(FH) differentiation may restore adultlike early life GC B cell responses.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Envejecimiento/inmunología , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Microambiente Celular/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Traslado Adoptivo , Animales , Animales Recién Nacidos , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Comunicación Celular/genética , Diferenciación Celular/genética , Senescencia Celular/inmunología , Islas de CpG/inmunología , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Inmunización , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Linfocitos T Colaboradores-Inductores/trasplante , Toxoide Tetánico/administración & dosificación , Toxoide Tetánico/inmunologíaRESUMEN
Cutaneous leishmaniasis affects 1 million people worldwide annually. Although conventional treatments primarily target the parasite, there is growing interest in host immune modulation. In this study, we investigated the impact of synthetic ß-carboline harmine (ACB1801), previously shown to be immunoregulatory in cancer, on the pathology caused by a drug-resistant Leishmania major strain causing persistent cutaneous lesions. Exposure to ACB1801 in vitro had a modest impact on parasite burden within host macrophages. Moreover, it significantly increased major histocompatibility complex II and costimulatory molecule expression on infected dendritic cells, suggesting an enhanced immune response. In vivo, ACB1801 monotherapy led to a substantial reduction in lesion development and parasite burden in infected C57BL/6 mice, comparable with efficacy of amphotericin B. Transcriptomics analysis further supported ACB1801 immunomodulatory effects, revealing an enrichment of TNF-α, IFN-γ, and major histocompatibility complex II antigen presentation signatures in the draining lymph nodes of treated mice. Flow cytometry analysis confirmed an increased frequency (1.5×) of protective CD4+IFN-γ+TNF-α+ T cells and a decreased frequency (2×) in suppressive IL-10+FoxP3- T cells at the site of infection and in draining lymph nodes. In addition, ACB1801 downregulated the aryl hydrocarbon receptor signaling, known to enhance immunosuppressive cytokines. Thus, these results suggest a potential use for ACB1801 alone or in combination therapy for cutaneous leishmaniasis.
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Leishmania major , Leishmaniasis Cutánea , Leishmaniasis , Humanos , Animales , Ratones , Harmina/farmacología , Harmina/uso terapéutico , Factor de Necrosis Tumoral alfa , Ratones Endogámicos C57BL , Inmunidad , Ratones Endogámicos BALB CRESUMEN
The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 µg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations.
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Antiprotozoarios , Resistencia a Medicamentos , Leishmaniasis Cutánea , Macrófagos , Antimoniato de Meglumina , Meglumina , Ratones Endogámicos BALB C , Compuestos Organometálicos , Insuficiencia del Tratamiento , Animales , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/parasitología , Antimoniato de Meglumina/uso terapéutico , Antimoniato de Meglumina/farmacología , Humanos , Antiprotozoarios/uso terapéutico , Antiprotozoarios/farmacología , Femenino , Meglumina/uso terapéutico , Meglumina/farmacología , Compuestos Organometálicos/uso terapéutico , Compuestos Organometálicos/farmacología , Ratones , Macrófagos/parasitología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Leishmania guyanensis/efectos de los fármacos , Adulto , Persona de Mediana Edad , Adulto Joven , Carga de Parásitos , AdolescenteRESUMEN
Drug resistance threatens the effective control of infections, including parasitic diseases such as leishmaniases. Neutrophils are essential players in antimicrobial control, but their role in drug-resistant infections is poorly understood. Here, we evaluated human neutrophil response to clinical parasite strains having distinct natural drug susceptibility. We found that Leishmania antimony drug resistance significantly altered the expression of neutrophil genes, some of them transcribed by specific neutrophil subsets. Infection with drug-resistant parasites increased the expression of detoxification pathways and reduced the production of cytokines. Among these, the chemokine CCL3 was predominantly impacted, which resulted in an impaired ability of neutrophils to attract myeloid cells. Moreover, decreased myeloid recruitment when CCL3 levels are reduced was confirmed by blocking CCL3 in a mouse model. Collectively, these findings reveal that the interplay between naturally drug-resistant parasites and neutrophils modulates the infected skin immune microenvironment, revealing a key role of neutrophils in drug resistance.
RESUMEN
Infection with Leishmania braziliensis causes cutaneous or mucocutaneous leishmaniasis in humans. Toll-like receptor 9 (TLR9) expression has been found in granulomas of lesions in L. braziliensis-infected individuals. L. braziliensis inoculation in mice induces very small lesions that are self-healing, whereas deficiency in the TLR adaptor molecule, MyD88, renders mice susceptible to infection. The TLR involved has not been identified, prompting us to investigate if TLR9 triggering by the parasite contributes to the strong resistance to infection observed in L. braziliensis-inoculated mice. The parasites activated wild-type (WT) dendritic cells (DCs) in vitro but not DCs derived from TLR9(-/-) mice. TLR9(-/-) mice inoculated with L. braziliensis exhibited a transient susceptibility characterized by increased lesion size and parasite burden compared to those of WT mice. Surprisingly, elevated levels of gamma interferon (IFN-γ) were measured at the site of infection and in draining lymph node T cells of TLR9(-/-) mice at the peak of susceptibility, suggesting that unlike observations in vitro, the parasite could induce DC activation leading to the development of Th1 cells in the absence of TLR9 expression. Taken together, these data show that TLR9 signaling is important for the early control of lesion development and parasite burden but is dispensable for the differentiation of Th1 cells secreting IFN-γ, and the high levels of this cytokine are not sufficient to control early parasite replication following L. braziliensis infection.
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Leishmania braziliensis , Leishmaniasis Cutánea/metabolismo , Receptor Toll-Like 9/fisiología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Leishmaniasis Cutánea/inmunología , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Células TH1/inmunología , Receptor Toll-Like 9/deficienciaRESUMEN
CD4⺠T helper cells are playing critical roles in host defense to pathogens and in the maintenance of immune homeostasis. Naïve CD4âºT cells, upon antigen-specific recognition, receive signals to differentiate into distinct effector T helper cell subsets characterized by their pattern of cytokine production and specific immune functions. A tight balance between these different subsets ensures proper control of the immune response. There is increasing evidence revealing an important role for Notch signaling in the regulation of CD4âºT helper cell differentiation or function in the periphery. However, the exact mechanisms involved remain unclear and appear contradictory. In this review, we summarize current knowledge and discuss recent advances in the field to reconcile different views on the role of Notch signaling in the differentiation of functional T helper subsets.