RESUMEN
No licensed vaccine exists against visceral leishmaniasis (VL), a disease caused by the Leishmania donovani parasite. We have previously reported both macrophages and dendritic cells play important role in the protection induced by a live attenuated centrin gene-deleted L. donovani (LdCen-/- ) parasite vaccine. The role of neutrophils in orchestrating the initial innate response to pathogens is widely recognized. To investigate the early interaction of LdCen-/- with neutrophils, we immunized mice intradermally in the ear pinna with LdCen-/- Compared with LdWT infection, LdCen-/- parasites induced higher recruitment of neutrophils to the ear dermis and ear draining lymph nodes (dLN) as early as 6-18 h after immunization, which were predominantly proinflammatory in nature. Neutrophils from ear dLN of LdCen-/- -immunized mice exhibited heightened expression of costimulatory molecules and attenuated expression of coinhibitory molecules necessary for higher T cell activation. Further phenotypic characterization revealed heterogeneous neutrophil populations containing Nα and Nß subtypes in the ear dLN. Of the two, the parasitized Nα subset from LdCen-/- -immunized mice exhibited much stronger Ag-specific CD4+ T cell proliferation ex vivo. Adoptive transfer of neutrophils bearing LdCen-/- parasites induced an increased Th1 response in naive mice. Importantly, neutrophil depletion significantly abrogated Ag-specific CD4+ T cell proliferation in LdCen-/- -immunized mice and impaired protection against virulent challenge. Conversely, replenishing of neutrophils significantly restored the LdCen-/- -induced host-protective response. These results suggest that neutrophils are indispensable for protective immunity induced by LdCen-/- parasite vaccine.
Asunto(s)
Leishmania donovani/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/prevención & control , Activación de Linfocitos , Infiltración Neutrófila , Neutrófilos/inmunología , Células TH1/inmunología , Animales , Femenino , Leishmania donovani/genética , Vacunas contra la Leishmaniasis/genética , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/inmunología , Ratones , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
No vaccine exists against visceral leishmaniasis. To develop effective vaccines, we have previously reported protective role of live attenuated centrin gene-deleted Leishmania donovani (LdCen-/- ) parasites through induction of Th1 type immune response in mice, hamsters, and dogs. In this study, we specifically explored the role of Th17 cells in LdCen-/- -induced host protection in mice. Our results showed that compared with wild-type L. donovani infection, LdCen-/- parasites induce significantly higher expression of Th17 differentiation cytokines in splenic dendritic cells. There was also induction of IL-17 and its promoting cytokines in total splenocytes and in both CD4 and CD8 T cells following immunization with LdCen-/- Upon challenge with wild-type parasites, IL-17 and its differentiating cytokines were significantly higher in LdCen-/- -immunized mice compared with nonimmunized mice that resulted in parasite control. Alongside IL-17 induction, we observed induction of IFN-γ-producing Th1 cells as reported earlier. However, Th17 cells are generated before Th1 cells. Neutralization of either IL-17 or IFN-γ abrogated LdCen-/- -induced host protection further confirming the essential role of Th17 along with Th1 cytokines in host protection. Treatment with recombinant IL-23, which is required for stabilization and maintenance of IL-17, heightened Th17, and Tc17 responses in immunized mice splenocytes. In contrast, Th17 response was absent in immunized IL-23R-/- mice that failed to induce protection upon virulent Leishmania challenge suggesting that IL-23 plays an essential role in IL-17-mediated protection by LdCen-/- parasites. This study unveiled the role of IL-23-dependent IL-17 induction in LdCen-/- parasite-induced immunity and subsequent protection against visceral leishmaniasis.
Asunto(s)
Interleucina-17/metabolismo , Interleucina-23/metabolismo , Leishmania donovani/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Animales Modificados Genéticamente , Femenino , Humanos , Leishmania donovani/genética , Vacunas contra la Leishmaniasis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Protozoarias/genética , Receptores de Interleucina/genética , Células TH1/parasitología , Células Th17/parasitología , Vacunas Atenuadas/inmunologíaRESUMEN
The clinical outcome of Leishmania pathogenesis ranges from active skin lesions to fatal visceral dissemination and severely impaired T cell immunity. It is well established that a strong Th1 immune response is protective against cutaneous forms of the disease, however a mixed Th1/Th2 response is most commonly observed against visceral infections as evident from previous studies. Aside from Th1/Th2 cytokines, the pro-inflammatory IL-17 cytokine family plays an important role in the clearance of intracellular pathogens. In Leishmania induced skin lesions, IL-17 produced by Th17 cells is shown to exacerbate the disease, suggesting a role in pathogenesis. However, a protective role for IL-17 is indicated by the expansion of IL-17 producing cells in vaccine-induced immunity. In human visceral leishmaniasis (VL) it has been demonstrated that IL-17 and IL-22 are associated with protection against re-exposure to Leishmania, which further suggests the involvement of IL-17 in vaccine induced protective immunity. Although there is no vaccine against any form of leishmaniasis, the development of genetically modified live attenuated parasites as vaccine candidates prove to be promising, as they successfully induce a robust protective immune response in various animal models. However, the role of IL-17 producing cells and Th17 cells in response to these vaccine candidates remains unexplored. In this article, we review the role of IL-17 in Leishmania pathogenesis and the potential impact on vaccine induced immunity, with a special focus on live attenuated Leishmania parasites.
Asunto(s)
Interleucina-17/metabolismo , Leishmania/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis/inmunología , Células Th17/inmunología , Animales , Humanos , Inmunidad Celular , Balance Th1 - Th2 , Vacunas AtenuadasRESUMEN
Visceral leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that genetically modified Leishmania donovani parasites, here described as live attenuated parasites, induce a host protective adaptive immune response in various animal models. In this study, we demonstrate an innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice both in vitro and in vivo. In vitro infection of macrophages with live attenuated parasites (compared to that with wild-type [WT] L. donovani parasites) induced significantly higher production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-12 [IL-12], gamma interferon [IFN-γ], and IL-6), chemokines (monocyte chemoattractant protein 1/CCL-2, macrophage inflammatory protein 1α/CCL-3, and IP-10), reactive oxygen species (ROS), and nitric oxide, while concomitantly reducing anti-inflammatory cytokine IL-10 and arginase-1 activities, suggesting a dominant classically activated/M1 macrophage response. The classically activated response in turn helps in presenting antigen to T cells, as observed with robust CD4(+) T cell activation in vitro. Similarly, parasitized splenic macrophages from live attenuated parasite-infected mice also demonstrated induction of an M1 macrophage phenotype, indicated by upregulation of IL-1ß, TNF-α, IL-12, and inducible nitric oxide synthase 2 and downregulation of genes associated with the M2 phenotype, i.e., the IL-10, YM1, Arg-1, and MRC-1 genes, compared to WT L. donovani-infected mice. Furthermore, an ex vivo antigen presentation assay showed macrophages from live attenuated parasite-infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by ovalbumin-specific CD4(+) T cells, resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasites promotes a state of classical activation (M1 dominant) in macrophages that leads to the generation of protective Th1 responses in BALB/c mice.
Asunto(s)
Inmunidad Innata , Leishmania donovani/genética , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Macrófagos/inmunología , Células TH1/inmunología , Inmunidad Adaptativa , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Quimiocina CCL3/genética , Quimiocina CCL3/inmunología , Femenino , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/parasitología , Ratones , Ratones Endogámicos BALB CRESUMEN
OBJECTIVE: Primary Sjögren's syndrome (SS) is characterized by autoimmune activation and loss of function in secretory epithelia. The present study was undertaken to investigate and characterize changes in the epithelia associated with the loss of gland function in primary SS. METHODS: To identify changes in epithelial gene expression, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with primary SS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. The effect of bone morphogenetic protein 6 (BMP-6) on salivary gland function was tested using adeno-associated virus-mediated gene transfer to the salivary glands of C57BL/6 mice. RESULTS: A significant increase in expression of BMP-6 was observed in RNA isolated from SS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector-treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with SS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. CONCLUSION: In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in primary SS, the present results suggest that BMP-6-induced salivary and lacrimal gland dysfunction in primary SS is independent of the autoantibodies and immune activation associated with the disease.
Asunto(s)
Proteína Morfogenética Ósea 6/metabolismo , Aparato Lagrimal/metabolismo , Glándulas Salivales/metabolismo , Síndrome de Sjögren/metabolismo , Animales , Autoanticuerpos/metabolismo , Proteína Morfogenética Ósea 6/genética , Femenino , Técnicas de Transferencia de Gen , Humanos , Aparato Lagrimal/inmunología , Aparato Lagrimal/fisiopatología , Ratones , Ratones Endogámicos C57BL , Glándulas Salivales/inmunología , Glándulas Salivales/fisiopatología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/fisiopatología , Xerostomía/inmunología , Xerostomía/metabolismo , Xerostomía/fisiopatologíaRESUMEN
No human vaccine is available for visceral leishmaniasis (VL). Live attenuated centrin gene-deleted L. donovani (LdCen-/-) parasite vaccine has been shown to induce robust innate immunity and provide protection in animal models. Toll-like receptors (TLRs) are expressed in innate immune cells and are essential for the early stages of Leishmania infection. Among TLRs, TLR-9 signaling has been reported to induce host protection during Leishmania infection. Importantly, TLR-9 ligands have been used as immune enhancers for non-live vaccination strategies against leishmaniasis. However, the function of TLR-9 in the generation of a protective immune response in live attenuated Leishmania vaccines remains unknown. In this study, we investigated the function of TLR-9 during LdCen-/- infection and found that it increased the expression of TLR-9 on DCs and macrophages from ear-draining lymph nodes and spleen. The increase in TLR-9 expression resulted in changes in downstream signaling in DCs mediated through signaling protein myeloid differentiation primary response 88 (MyD88), resulting in activation and nuclear translocation of nuclear factor-κB (NF-κB). This process resulted in an increase in the DC's proinflammatory response, activation, and DC-mediated CD4+T cell proliferation. Further, LdCen-/- immunization in TLR-9-/- mice resulted in a significant loss of protective immunity. Thus, LdCen-/- vaccine naturally activates the TLR-9 signaling pathway to elicit protective immunity against virulent L. donovani challenge.
RESUMEN
Vaccination with live attenuated Leishmania parasites such as centrin deleted Leishmania donovani (LdCen-/-) against visceral leishmaniasis has been reported extensively. The protection induced by LdCen-/- parasites was mediated by both CD4+ and CD8+ T cells. While the host immune mediators of protection are known, parasite determinants that affect the CD4+ and CD8+ T cell populations remain unknown. Parasite encoded inflammatory cytokine MIF has been shown to modulate the T cell differentiation characteristics by altering the inflammation induced apoptosis during contraction phase in experimental infections with Leishmania or Plasmodium. Neutralization of parasite encoded MIF either by antibodies or gene deletion conferred protection in Plasmodium and Leishmania studies. We investigated if the immunogenicity and protection induced by LdCen-/- parasites is affected by deleting MIF genes from this vaccine strain. Our results showed that LdCen-/-MIF-/- immunized group presented higher percentage of CD4+ and CD8+ central memory T cells, increased CD8+ T cell proliferation after challenge compared to LdCen-/- immunization. LdCen-/-MIF-/- immunized group presented elevated production of IFN-γ+ and TNF-α+ CD4+ T cells concomitant with a reduced parasite load in spleen and liver compared to LdCen-/-group following challenge with L. infantum. Our results demonstrate the role of parasite induced factors involved in protection and long-term immunity of vaccines against VL.
Asunto(s)
Leishmania donovani , Vacunas contra la Leishmaniasis , Leishmaniasis Visceral , Parásitos , Animales , Ratones , Linfocitos T CD8-positivos , Leishmaniasis Visceral/parasitología , Leishmania donovani/genética , Linfocitos T CD4-Positivos , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Neutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo. METHODOLOGY/FINDINGS: LdCen-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4+T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen-/- parasites by DCs. Neutrophil depletion in LdCen-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4+Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4+ T cells in vivo. CONCLUSIONS: Collectively, LdCen-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.
Asunto(s)
Leishmania donovani , Vacunas contra la Leishmaniasis , Leishmaniasis Visceral , Animales , Comunicación Celular , Células Dendríticas , Leishmania donovani/fisiología , Leishmaniasis Visceral/parasitología , Ratones , Neutrófilos , Vacunas AtenuadasRESUMEN
Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen-/-) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen-/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We showed that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver as well as reduced mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the disease promoting cytokines IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-γ resulting in higher IFN-γ/IL-10 and IFN-γ/IL4 ratios compared to non-immunized animals. LmexCen-/- immunization also resulted in long-lasting protection against L. donovani infection. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against the Old World visceral leishmaniasis.
RESUMEN
Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene-deleted parasite strain (LmCen-/- ) that induced protection against homologous and heterologous challenges. We demonstrated that the protection is mediated by IFN (Interferon) γ-secreting CD4+ T-effector cells and multifunctional T cells, which is analogous to leishmanization. In addition, in a leishmanization model, skin tissue-resident memory T (TRM) cells were also shown to be crucial for host protection. In this study, we evaluated the generation and function of skin TRM cells following immunization with LmCen-/- parasites and compared those with leishmanization. We show that immunization with LmCen-/- generated skin CD4+ TRM cells and is supported by the induction of cytokines and chemokines essential for their production and survival similar to leishmanization. Following challenge with wild-type L. major, TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice. Furthermore, upon challenge, CD4+ TRM cells induce higher levels of IFNγ and Granzyme B in the immunized and leishmanized mice than in non-immunized mice. Taken together, our studies demonstrate that the genetically modified live attenuated LmCen-/- vaccine generates functional CD4+ skin TRM cells, similar to leishmanization, that may play a crucial role in host protection along with effector T cells as shown in our previous study.
Asunto(s)
Leishmania major , Vacunas contra la Leishmaniasis , Parásitos , Animales , Inmunidad , Interferón gamma , Vacunas contra la Leishmaniasis/genética , Células T de Memoria , Ratones , Piel , Combinación Trimetoprim y SulfametoxazolRESUMEN
Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen-/-) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen-/- parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen-/- immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen-/- infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen-/- immunized hamsters. This study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.
Asunto(s)
Eliminación de Gen , Genes Protozoarios , Leishmania donovani/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/prevención & control , Leishmania donovani/genética , Leishmaniasis Visceral/inmunología , Proteínas Protozoarias , Vacunas Atenuadas/inmunologíaRESUMEN
Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Leishmania major/genética , Leishmania major/patogenicidad , Vacunas Atenuadas/uso terapéutico , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Dexametasona/farmacología , Femenino , Citometría de Flujo , Edición Génica , Ingeniería Genética , Humanos , Terapia de Inmunosupresión , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Psychodidae/parasitología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
No vaccine exists against visceral leishmaniasis. Toward developing vaccines against VL, we have reported previously on the immunogenicity of live attenuated LdCen-/- parasites in animal models. Immunization with LdCen-/- parasites has been shown to induce durable protective immunity in pre-clinical animal models. Although the innate immune responses favoring a Th1 type immunity are produced following LdCen-/- immunization, the molecular determinants of such responses remain unknown. To identify early biomarkers of immunogenicity associated with live attenuated parasitic vaccines, we infected macrophages derived from healthy human blood donors with LdCen-/- or LdWT parasites ex vivo and compared the early gene expression profiles. In addition to altered expression of immune related genes, we identified several microRNAs that regulate important cytokine genes, significantly altered in LdCen-/- infection compared to LdWT infection. Importantly, we found that LdCen-/- infection suppresses the expression of microRNA-21 (miR-21) in human macrophages, which negatively regulates IL12, compared to LdWT infection. In murine DC experiments, LdCen-/- infection showed a reduced miR-21 expression with a concomitant induction of IL12. Silencing of miR-21 using specific inhibitors resulted in an augmented induction of IL12 in LdWT infected BMDCs, illustrating the role of miR-21 in LdWT mediated suppression of IL12. Further, exosomes isolated from LdCen-/- infected DCs contained significantly reduced levels of miR-21 compared to LdWT infection, that promoted proliferation of CD4+ T cells in vitro. Similar miR-21 mediated IL12 regulation was also observed in ex vivo human macrophage infection experiments indicating that miR-21 plays a role in early IL12 mediated immunity. Our studies demonstrate that LdCen-/- infection suppresses miR-21 expression, enables IL12 mediated induction of adaptive immunity including proliferation of antigen experienced CD4+ T cells and development of a Th1 immunity, and suggest that miR-21 could be an important biomarker for LdCen-/- vaccine immunity in human clinical trials. One Sentence Summary: Role of miR-21 in vaccine induced immunity.
Asunto(s)
Inmunidad/inmunología , Leishmania donovani/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/inmunología , Macrófagos/inmunología , MicroARNs/inmunología , Animales , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Humanos , Inmunización/métodos , Leishmania donovani/genética , Leishmania donovani/fisiología , Vacunas contra la Leishmaniasis/administración & dosificación , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/prevención & control , Macrófagos/parasitología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , MicroARNs/genética , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/parasitología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
The protozoan parasite Leishmania has evolved several strategies to undermine host defense mechanisms by inducing Th2-type adaptive immunity and suppressing effector functions of Th1 phenotype. In our earlier studies, using centrin gene-deleted Leishmania (LdCen-/-) parasites as an immunogen, we have shown induction of an effective Th1-type immunity and robust memory responses that mediate protection against virulent challenge. However, role of inhibitory signals in Leishmania vaccine induced immunity in general, and LdCen-/- in particular has not been studied. Herein, we report that immunization with LdCen-/- parasites produces more functional Th1-type CD4+ T cells via downregulation of CD200-CD200R immune inhibitory axis compared to wild-type infection. We found that expression of CD200 and CD200R was significantly reduced in LdCen-/- infection compared to wild-type infection. Diminished CD200-CD200R signaling in LdCen-/- infection enabled proliferation of CD4+ T cells and resulted in the induction of pro-inflammatory cytokines and suppression of anti-inflammatory response. The effects of diminished CD200-CD200R signaling by LdCen-/- were most evident in the suppression of IL-10-producing CD4+ T cells that helped enhance more Th1 cytokine producing and multi-functional T cells compared to wild-type infection. In vivo blocking of CD200 expression with anti-CD200 treatment in wild-type infected mice limited Th2 response as indicated by reduction of IL-10-producing Tr1 cells and reduced parasite burden. On the other hand, treatment with anti-CD200 improved the LdCen-/- vaccine-induced multifunctional response and reduction in splenic parasite load upon challenge. Taken together, these studies demonstrate the role of CD200-CD200R signals in the protection induced by LdCen-/- parasites.
Asunto(s)
Antígenos de Protozoos/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Leishmania donovani/fisiología , Leishmaniasis Visceral/inmunología , Células TH1/inmunología , Animales , Antígenos CD/metabolismo , Antígenos de Protozoos/genética , Antígenos CD4/metabolismo , Proteínas de Unión al Calcio/genética , Diferenciación Celular , Proteínas Cromosómicas no Histona/genética , Regulación hacia Abajo , Femenino , Técnicas de Inactivación de Genes , Humanos , Inmunofenotipificación , Activación de Linfocitos , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microorganismos Modificados Genéticamente , Transducción de SeñalRESUMEN
Leishmania donovani parasites are the cause of visceral leishmaniasis and are transmitted by bites from phlebotomine sand flies. A prominent feature of vector-transmitted Leishmania is the persistence of neutrophils at bite sites, where they protect captured parasites, leading to enhanced disease. Here, we demonstrate that gut microbes from the sand fly are egested into host skin alongside Leishmania parasites. The egested microbes trigger the inflammasome, leading to a rapid production of interleukin-1ß (IL-1ß), which sustains neutrophil infiltration. Reducing midgut microbiota by pretreatment of Leishmania-infected sand flies with antibiotics or neutralizing the effect of IL-1ß in bitten mice abrogates neutrophil recruitment. These early events are associated with impairment of parasite visceralization, indicating that both gut microbiota and IL-1ß are important for the establishment of Leishmania infections. Considering that arthropods harbor a rich microbiota, its potential egestion after bites may be a shared mechanism that contributes to severity of vector-borne disease.
Asunto(s)
Microbioma Gastrointestinal/inmunología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/transmisión , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Psychodidae/parasitología , Animales , Antiparasitarios/farmacología , Cricetinae , Femenino , Mordeduras y Picaduras de Insectos/parasitología , Insectos Vectores/parasitología , Leishmania donovani/efectos de los fármacos , Leishmaniasis Visceral/parasitología , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/efectos de los fármacos , Infiltración Neutrófila/inmunología , Neutrófilos/inmunologíaRESUMEN
Currently, there is no vaccine against visceral leishmaniasis (VL). Toward developing an effective vaccine, we have reported extensively on the immunogenicity of live attenuated LdCentrin-/- mutants in naive animal models. In VL endemic areas, asymptomatic carriers outnumber symptomatic cases of VL and are considered to be a reservoir of infection. Vaccination of asymptomatic cases represents a viable strategy to eliminate VL. Immunological correlates of protection thus derived might have limited applicability in conditions where the immunized host has prior exposure to virulent infection. To examine whether LdCen-/- parasites can induce protective immunity in experimental hosts that have low-level parasitemia from a previous exposure mimicking an asymptomatic condition, we infected C57Bl/6 mice with wild-type Leishmania donovani parasites expressing LLO epitope (LdWT LLO 103, i.v.). After 3 weeks, the mice with low levels of parasitemia were immunized with LdCen-/- parasites expressing 2W epitope (LdCen-/-2W 3 × 106 i.v.) to characterize the immune responses in the same host. Antigen experienced CD4+ T cells from the asymptomatic (LdWT LLO infected) LdCen-/-2W immunized, and other control groups were enriched using LLO- and 2W-specific tetramers, followed by Flow cytometric analysis. Our analysis showed that comparable CD4+ T cell proliferation and CD4+ memory T cell responses (TCM) represented by CD62Lhi, CCR7+, and IL-7R+ T cell populations were induced with LdCen-/-2W in both asymptomatic and naive animals that received LdCen-/- immunization. Upon restimulation with peptide, TCM cells differentiated into effector T cells and there was no significant difference in the recall response in animals with asymptomatic infection. Following virulent challenge, comparable reduction in splenic parasite burden was observed in both asymptomatic and naive LdCen-/- immunized animals concomitant with the development of multifunctional CD4+ and CD8+ T cells. Further, LdCen-/-2W immunization resulted in complete clearance of the preexisting asymptomatic infection (LdWT LLO ). Our results demonstrate that LdCen-/-2W immunization could be efficacious for use in asymptomatic VL individuals. Further, immunization with LdCen-/- could help in reducing the parasite burden in the asymptomatic cases and aid in controlling the VL in endemic areas.
RESUMEN
No licensed human vaccines are currently available against leishmaniasis. Several anti-leishmanial vaccines are currently undergoing testing, including genetically modified live-attenuated parasite vaccines. Studies with live attenuated Leishmania vaccines such as centrin deleted Leishmania donovani parasites (LdCen -/-) showed protective immunity in animal models. Such studies typically examined the biomarkers of protective immunity however the biomarkers of attenuation in the parasite preparations have not received adequate attention. As several candidate vaccines enter clinical trials, a more complete product characterization to enable maintenance of product quality will help meet regulatory requirements. Towards this goal, we have determined the complete genome sequence of LdCen -/- and its parent strain Ld1S-2D (LdWT) and characterized the LdCen -/- vaccine strain using bioinformatics tools. Results showed that the LdCen -/- parasites, in addition to loss of the centrin gene, have additional deletions ranging from 350 bp to 6900 bp in non-contiguous loci on several chromosomes, most commonly in untranslated regions. We have experimentally verified a subset of these adventitious deletions that had no impact on the attenuation of the LdCen -/- parasites. Our results identified hitherto unknown features of attenuation of virulence that could be used as markers of product quality in production lots and highlight the importance of product characterization in parasitic vaccines.
Asunto(s)
Leishmania donovani/genética , Vacunas Atenuadas/genética , Secuenciación Completa del Genoma/métodos , Biología Computacional/métodos , Marcadores Genéticos , Genoma de Protozoos , Humanos , Vacunas Antiprotozoos/genética , Eliminación de SecuenciaRESUMEN
No licensed human vaccines are currently available against any parasitic disease including leishmaniasis. Several antileishmanial vaccine formulations have been tested in various animal models, including genetically modified live-attenuated parasite vaccines. Experimental infection studies have shown that Leishmania parasites utilize a broad range of strategies to undermine effector properties of host phagocytic cells, i.e., dendritic cells (DCs) and macrophages (MΦ). Furthermore, Leishmania parasites have evolved strategies to actively inhibit TH1 polarizing functions of DCs and to condition the infected MΦ toward anti-inflammatory/alternative/M2 phenotype. The altered phenotype of phagocytic cells is characterized by decreased production of antimicrobial reactive oxygen, nitrogen molecules, and pro-inflammatory cytokines, such as IFN-γ, IL-12, and TNF-α. These early events limit the activation of TH1-effector cells and set the stage for pathogenesis. Furthermore, this early control of innate immunity by the virulent parasites results in substantial alteration in the adaptive immunity characterized by reduced proliferation of CD4(+) and CD8(+) T cells and TH2-biased immunity that results in production of anti-inflammatory cytokines, such as TGF-ß, and IL-10. More recent studies have also documented the induction of coinhibitory ligands, such as CTLA-4, PD-L1, CD200, and Tim-3, that induce exhaustion and/or non-proliferation in antigen-experienced T cells. Most of these studies focus on viral infections in chronic phase, thus limiting the direct application of these results to parasitic infections and much less to parasitic vaccines. However, these studies suggest that vaccine-induced protective immunity can be modulated using strategies that enhance the costimulation that might reduce the threshold necessary for T cell activation and conversely by strategies that reduce or block inhibitory molecules, such as PD-L1 and CD200. In this review, we will focus on the polarization of antigen-presenting cells and subsequent role of costimulatory and coinhibitory molecules in mediating vaccine-induced immunity using live-attenuated Leishmania parasites as specific examples.
RESUMEN
Live-attenuated parasite vaccines are being explored as potential vaccine candidates since other approaches of vaccination have not produced an effective vaccine so far. In order for live-attenuated parasite vaccines to be tested in preclinical studies and possibly in clinical studies, the safety and immunogenicity of these organisms must be rigorously evaluated. Here we describe methods to test persistence in the immunized host and immunogenicity, and to identify biomarkers of vaccine safety and efficacy with particular reference to genetically attenuated Leishmania parasites.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ingeniería Genética , Leishmania/inmunología , Vacunas Antiprotozoos/efectos adversos , Vacunas Antiprotozoos/inmunología , Seguridad , Animales , Biomarcadores/metabolismo , Membrana Celular/parasitología , Femenino , Inmunidad Innata , Macrófagos/citología , Ratones , Vacunas Antiprotozoos/genética , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani There are no vaccines and available drugs against leishmaniasis are toxic. Immunomodulators that specifically boost the anti-microbial activities of the immune cells could alleviate several of these limitations. Therefore, finding novel immunomodulators for VL therapy is a pressing need. This study is aimed to evaluate the immunomodulatory role of leptin, an adipocyte-derived hormone capable of regulating the immune response, in L. donovani-infected mice. We observed that recombinant leptin treatment reduced splenic parasite burden compared with non-treated infected normal mice. Decrease in parasite burden correlated with an induction of innate immune response in antigen-presenting cells that showed an increase in nitric oxide, enhanced pro-inflammatory cytokine (interferon gamma [IFNγ], interleukin12 [IL]12, and IL1ß) response in the splenocytes, indicating host-protecting Th1 response mediated by leptin. Moreover, in infected normal mice, leptin treatment induced IFNγ production from both CD4(+) and CD8(+) T cells, compared with non-treated infected mice. Alternatively, leptin-deficient (Ob/Ob) mice had higher splenic and liver parasite burden compared with the infected normal mice. However, leptin treatment failed to reduce the splenic parasite burden and improve a host-protective cytokine response in these mice. In addition, in contrast to dendritic cells (DCs) from a normal mouse, Ob/Ob mouse-derived DCs showed a defect in the induction of innate immune response on Leishmania infection that could not be reversed by leptin treatment. Therefore, our findings reveal that leptin has a differential immunomodulatory effect in controlling VL in normal and Ob/Ob mice.