RESUMEN
Previously, we established that as a function of its mode of interaction with its ligand or cellular conditions such as membrane lipids, preexisting signaling intermediates activation status, a transmembrane receptor, as represented here with CD40, can induce counteractive cellular responses. Using CD40-binding peptides, recombinant mutated CD40-ligands, and an agonistic antibody, we have established the functional duality of CD40. CD40 builds up two constitutionally different signalosomes on lipid raft and non-raft membrane domains initiating two different signaling pathways. Although this initial signaling may be modified by the pre-existing signaling conditions downstream and may be subjected to feed-forward or negative signaling effects, the initial CD40-CD40L interaction plays a crucial role in the functional outcome of CD40. Herein, we have reviewed the influence of interaction between the CD40-CD40L evoking the functional duality of CD40 contingent upon different physiological states of the cells.
Asunto(s)
Antígenos CD40 , Ligando de CD40 , Humanos , Ligando de CD40/genética , Ligando de CD40/metabolismo , Antígenos CD40/metabolismo , Transducción de SeñalRESUMEN
NOD like receptors (NLR) are essential pathogen associated molecular pattern receptors of cytoplasmic origin. During several intracellular parasitic infections NLR played vital role for host protective immune response against the pathogen. Amongst various classes of NLR, NOD1 and NOD2 had been extensively studied and were found to be the most active member of the NLR family. Therefore, we wanted to study the role of NOD1/NOD2 during Leishmania donovani infection and the mechanism behind the utilization of this pathway as a therapeutic approach. Using the infected model of macrophage and BALB/c mice the expression of NOD1 and NOD2 were analysed. Our study showed that NOD2 but not NOD1 has been exploited during experimental VL, leading to the imbalance between Th-1/Th-2 cytokines profile. Over-expression of NOD2 and stimulation with its ligand muramyl dipeptide leads to successful clearance of parasite. During in vivo experiments we found that arabinosylated lipoarabinomannan helps in the restoration of NOD2 and with MDP in combination leads to effective clearance of parasite which rescued host protective immunity and comparatively more effective than Mw and MDP combination resulting in increase T cell response. Consequently, our study highlighted the significance of NOD2 during infection the immune-modulations of which can be used as a therapeutic target.
Asunto(s)
Inmunidad Innata/efectos de los fármacos , Factores Inmunológicos/farmacología , Mediadores de Inflamación/metabolismo , Leishmania donovani/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Lipopolisacáridos/farmacología , Proteína Adaptadora de Señalización NOD2/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Tripanocidas/farmacología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Leishmania donovani/inmunología , Leishmania donovani/patogenicidad , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/parasitología , Ratones Endogámicos BALB C , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Carga de Parásitos , Transducción de Señal/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/parasitología , Balance Th1 - Th2/efectos de los fármacosRESUMEN
Emergence of drug resistance during visceral leishmaniasis (VL) is a major obstacle imposed during successful therapy. An effective vaccine strategy against this disease is therefore necessary. Our present study exploited the SLA (soluble leishmanial antigen) and PGN (peptidoglycan) stimulated bone marrow-derived dendritic cells (DCs) as a suitable vaccine candidate during experimental VL. SLA-PGN-stimulated DCs showed a significant decrease in hepatic and splenic parasite burden, which were associated with increased production of nitric oxide and pro-inflammatory cytokines such as IL-12, IFN-γ and IL-17. Elevated level of IL-17 was accompanied with the generation of more Th17 cells. Further studies on DC provided the evidence that these SLA-PGN-stimulated DCs played an important role in providing necessary cytokines such as IL-6, IL-23 and TGF-ß for the generation of Th17 cells. Interestingly, inhibition of protein kinase C-ß (PKCß) in DCs led to decreased production of Th17 polarizing cytokines, causing reduction of the Th17 population size. Altogether, our finding highlighted the important role of DC-based PKCß in regulation of the function and generation of Th17 cells.
Asunto(s)
Antígenos de Protozoos/inmunología , Células Dendríticas/inmunología , Leishmania donovani/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/prevención & control , Peptidoglicano/inmunología , Células Th17/inmunología , Animales , Técnicas de Cocultivo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Inmunización , Mediadores de Inflamación/metabolismo , Leishmaniasis Visceral/parasitología , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa C beta/metabolismo , Células Th17/metabolismoRESUMEN
Leishmania donovani, a protozoan parasite, causes the disease visceral leishmanisis (VL), characterized by inappropriate CD8+ T-cell activation. Therefore, we examined whether the Toll-like Receptor 2 (TLR2) ligand Ara-LAM, a cell wall glycolipid from non-pathogenic Mycobacterium smegmatis, would restore CD8+ T-cell function during VL. We observed that by efficient upregulation of TLR2 signaling-mediated NF-κB translocation and MAPK signaling in CD8+ T-cells (CD25+CD28+IL-12R+IFN-γR+), Ara-LAM triggered signaling resulted in the activation of T-bet, which in turn, induced transcription favourable histone modification at the IFN-γ, perforin, granzyme-B promoter regions in CD8+ T-cells. Thus, we conclude that Ara-LAM induced efficient activation of effector CD8+ T-cells by upregulating the expression of IFN-γ, perforin and granzyme-B in an NF-κB and MAPK induced T-bet dependent manner in VL.
Asunto(s)
Linfocitos T CD8-positivos/parasitología , Leishmaniasis Visceral/inmunología , Proteínas de Dominio T Box/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Antígenos CD28/metabolismo , Proliferación Celular , Modelos Animales de Enfermedad , Granzimas/genética , Histonas/metabolismo , Interferón gamma/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Leishmania donovani , Leishmaniasis Visceral/sangre , Ligandos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Regiones Promotoras Genéticas , Receptores de Interferón/metabolismo , Receptores de Interleucina-12/metabolismo , Transducción de Señal , Regulación hacia Arriba , Receptor de Interferón gammaRESUMEN
CD40, as a single receptor that binds CD154 (CD40-ligand or CD40L), regulates counteractive effector functions such as production of pro- and anti-inflammatory cytokines. Therefore, we examined whether such dual messages are encrypted in CD40L. As such message encryption was never investigated, we hypothesized that mutation of certain amino acid residues should in principle enhance pro-inflammatory cytokine production whereas mutation of some others would enhance anti-inflammatory cytokine secretion. We mutated six such residues, which were previously showed to participate in CD40L function. Here, we report that the mutant CD154 129EâV was superior to the wild-type CD154 in killing of Leishmania donovani, induction of inducible nitric oxide synthase (iNOS) and production of IL-12 and relative phosphorylation of p38MAPK and ERK-1/2 in PBMC-derived macrophages. By contrast, 128SâV promoted L. donovani survival, reducing iNOS, but increasing IL-10 expression and predominant ERK-1/2 phosphorylation. The mutant 144GâV did not have significant effects. Other mutants (142EâV, 143KâA, 145YâF) mimicked the wild-type CD154. Molecular dynamics simulation suggested that these mutations induced differential conformational changes in the CD40-CD154 complex. Therefore, assortment of the contrasting messages encrypted in a given ligand performing counteractive functions presents a novel fundamental biological principle that can be used for devising various therapies.
Asunto(s)
Ligando de CD40/metabolismo , Antígenos CD40/química , Antígenos CD40/genética , Antígenos CD40/metabolismo , Ligando de CD40/química , Ligando de CD40/genética , Clonación Molecular , Citocinas/metabolismo , Expresión Génica , Humanos , Leishmania donovani/inmunología , Leishmania donovani/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Moleculares , Mutación , Nitritos/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Resonancia por Plasmón de Superficie , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs), a heterogeneous population of precursor cells, modulate protective immunity against visceral leishmaniasis by suppressing T cell functions. We observed that CD11b(+) Gr1(+) MDSCs, which initially expanded in soluble leishmanial antigen (SLA)-immunized mice and later diminished, suppressed proliferation of T cells isolated from SLA-immunized mice, but to a lesser extent than the case in naive mice. This lesser suppression of MDSCs accompanied the expression of F4/80 and the production of Cox-2, arginase I, nitric oxide, and PGE2. However, with SLA immunization, there was no difference in the expression of interleukin-2 (IL-2) or gamma interferon (IFN-γ) by T cells, in contrast to the case in nonimmunized mice, in which there is an influence. Glycyrrhizic acid (a triterpenoid compound)-mediated inhibition of Cox-2 in myeloid-derived suppressor cells influenced the capacity of T cells to proliferate and the expression of IL-2 and IFN-γ in Leishmania donovani-infected BALB/c mice. Further characterization confirmed that administration of glycyrrhizic acid to L. donovani-infected BALB/c mice results in an impairment of the generation of MDSCs and a reciprocal organ-specific proliferation of IFN-γ- and IL-10-expressing CD4(+) and CD8(+) T cells. Comprehensive knowledge on the Cox-2-mediated regulation of myeloid-derived suppressor cells might be involved in unlocking a new avenue for therapeutic interventions during visceral leishmaniasis.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Ácido Glicirrínico/farmacología , Factores Inmunológicos/farmacología , Leishmania donovani/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Células Mieloides/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Antígenos de Protozoos/administración & dosificación , Arginasa/genética , Arginasa/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/microbiología , Linfocitos T CD8-positivos/patología , Proliferación Celular , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Inmunización , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/inmunología , Leishmania donovani/patogenicidad , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/microbiología , Leishmaniasis Visceral/prevención & control , Ratones , Ratones Endogámicos BALB C , Células Mieloides/inmunología , Células Mieloides/microbiología , Células Mieloides/patología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/microbiología , Linfocitos T Reguladores/patologíaRESUMEN
Visceral leishmaniasis (VL), caused by a protozoan parasite Leishmania donovani, is still a threat to mankind due to treatment failure, drug resistance and coinfection with HIV. The limitations of first-line drugs have led to the development of new strategies to combat this dreaded disease. Recently, we have shown the immunomodulatory property of Ara-LAM, a TLR2 ligand, against leishmanial pathogenesis. In this study, we have extended our study to the effect of Ara-LAM on regulatory T cells in a murine model of VL. We observed that Ara-LAM-treated infected BALB/c mice showed a strong host-protective Th1 immune response due to reduced IL-10 and TGF-ß production, along with marked decrease in CD4(+) CD25(+) Foxp3(+) GITR(+) CTLA4(+) regulatory T cell (Treg) generation and activation. The reduction in Foxp3 expression was due to effective modulation of TGF-ß-induced SMAD signaling in Treg cells by Ara-LAM. Moreover, we demonstrated that Ara-LAM-induced IRF1 expression in the Treg cells, which negatively regulated foxp3 gene transcription, resulting in the reduced immunosuppressive activity of Treg cells. Interestingly, irf1 gene knockdown completely abrogated the effect of Ara-LAM on Treg cells. Thus, these findings provide detailed mechanistic insight into Ara-LAM-mediated modulation of Treg cells, which might be helpful in combating VL.
Asunto(s)
Factores de Transcripción Forkhead/análisis , Factores Inmunológicos/administración & dosificación , Factor 1 Regulador del Interferón/metabolismo , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Lipopolisacáridos/administración & dosificación , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD4/análisis , Antígeno CTLA-4/análisis , Modelos Animales de Enfermedad , Femenino , Proteína Relacionada con TNFR Inducida por Glucocorticoide/análisis , Interleucina-10/metabolismo , Subunidad alfa del Receptor de Interleucina-2/análisis , Leishmaniasis Visceral/patología , Masculino , Ratones Endogámicos BALB C , Subgrupos de Linfocitos T/química , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/química , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Visceral leishmaniasis (VL), which is endemic as a major infectious disease in the tropical and subtropical countries, is caused by a protozoan parasite Leishmania donovani. At present, restricted treatment options and lack of vaccines intensify the problem of controlling VL. Therefore, finding a novel immunoprophylactic or therapeutic principle is a pressing need. Here, we report that arabinosylated lipoarabinomannan (Ara-LAM), a TLR2-ligand isolated from Mycobacterium smegmatis, exhibits a strong immunomodulatory property that conferred protection against L. donovani infection. Although, Ara-LAM modulates TLR2 and MAPK signaling, it is not known whether Ara-LAM involves IFN-γ signaling for effective parasite clearance. Because, it is reported that IFN-γ signaling, a principle mediator of NO generation and macrophage and Tcell activation, is hampered during leishmanial pathogenesis. Ara-LAM increases IFN-γ receptor expression and potentiates IFN-γ receptor signaling through JAK-STAT pathway. Moreover, Ara-LAM reciprocally modulates IRF4 and IRF8 expression and reinstates anti-leishmanial Th1 response that eventuates in significantly reduced parasite load in spleen and liver of L. donovani-infected BALB/c mice. IFN-γRα silencing resulted in the suppression of these host-protective mechanisms affected by Ara-LAM. Thus, Ara-LAM-mediated restoration of IFN-γ responsiveness is a novel immuno-modulatory principle for protection against L. donovani susceptible host.
Asunto(s)
Arabinosa/metabolismo , Interacciones Huésped-Patógeno , Interferón gamma/inmunología , Leishmania donovani/fisiología , Leishmaniasis Visceral/inmunología , Lipopolisacáridos/metabolismo , Receptor de Interferón alfa y beta/metabolismo , Animales , Femenino , Macrófagos/inmunología , Masculino , Ratones Endogámicos BALB C , Factores de Transcripción/metabolismoRESUMEN
The antigen-presenting cellexpressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.