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High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/ß complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR-15a-16-1-KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.
Assuntos
Células da Medula Óssea/metabolismo , Macrófagos/metabolismo , MicroRNAs/fisiologia , Mieloma Múltiplo/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/genética , Mieloma Múltiplo/patologia , Microambiente TumoralRESUMO
Helminth parasites modulate immune responses in their host to prevent their elimination and to establish chronic infections. Our previous studies indicate that Taenia crassiceps-excreted/secreted antigens (TcES) downregulate inflammatory responses in rodent models of autoimmune diseases, by promoting the generation of alternatively activated-like macrophages (M2) in vivo. However, the molecular mechanisms triggered by TcES that modulate macrophage polarization and inflammatory response remain unclear. Here, we found that, while TcES reduced the production of inflammatory cytokines (IL-6, IL-12, and TNFα), they increased the release of IL-10 in LPS-induced bone marrow-derived macrophages (BMDM). However, TcES alone or in combination with LPS or IL-4 failed to increase the production of the canonical M1 or M2 markers in BMDM. To further define the anti-inflammatory effect of TcES in the response of LPS-stimulated macrophages, we performed transcriptomic array analyses of mRNA and microRNA to evaluate their levels. Although the addition of TcES to LPS-stimulated BMDM induced modest changes in the inflammatory mRNA profile, it induced the production of mRNAs associated with the activation of different receptors, phagocytosis, and M2-like phenotype. Moreover, we found that TcES induced upregulation of specific microRNAs, including miR-125a-5p, miR-762, and miR-484, which are predicted to target canonical inflammatory molecules and pathways in LPS-induced BMDM. These results suggest that TcES can modulate proinflammatory responses in macrophages by inducing regulatory posttranscriptional mechanisms and hence reduce detrimental outcomes in hosts running with inflammatory diseases.
Assuntos
Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , MicroRNAs/genética , Taenia/fisiologia , Animais , Biomarcadores , Citocinas/metabolismo , Feminino , Imunomodulação , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Teníase/genética , Teníase/imunologia , Teníase/metabolismo , Teníase/parasitologiaRESUMO
CXCR3, an X-linked gene, is subject to X chromosome inactivation (XCI), but it is unclear whether CXCR3 escapes XCI in immune cells. We determined whether CXCR3 escapes XCI in vivo, evaluated the contribution of allelic CXCR3 expression to the phenotypic properties of T cells during experimental infection with Leishmania, and examined the potential implications to sex differences in immune responses. We used a bicistronic CXCR3 dual-reporter mouse, with each CXCR3 allele linked to a green or red fluorescent reporter without affecting endogenous CXCR3 expression. Our results show that CXCR3 escapes XCI, biallelic CXCR3-expressing T cells produce more CXCR3 protein than monoallelic CXCR3-expressing cells, and biallelic CXCR3-expressing T cells produce more IFN-γ, IL-2, and CD69 compared with T cells that express CXCR3 from one allele during Leishmania mexicana infection. These results demonstrate that XCI escape by CXCR3 potentially contributes to the sex-associated bias observed during infection.
Assuntos
Receptores CXCR3/imunologia , Caracteres Sexuais , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Inativação do Cromossomo X/imunologia , Animais , Feminino , Infecções/imunologia , Masculino , Camundongos , Camundongos Mutantes , Receptores CXCR3/genéticaRESUMO
Daratumumab (Dara), a human immunoglobulin G1 kappa (IgG1κ) monoclonal anti-CD38 antibody, has been approved by the U.S. Food and Drug Administration for the treatment of relapsed multiple myeloma (MM) as a single agent as well as in combination with immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI). Although the scientific rationale behind the use of Dara in combination with IMiDs has been extensively explored, the molecular mechanisms underlying Dara-PI regimens have not yet been investigated. Here, we demonstrate that CD38 on the surface of MM cells is rapidly internalized after Dara treatment; we also show that Dara treatment impairs MM cell adhesion, an effect that can be rescued by using the endocytosis inhibitor Dynasore. Finally, we show that Dara potentiates bortezomib (BTZ) killing of MM cells in vitro and in vivo, independent of its function as an immune activator. In conclusion, our data show that Dara impairs MM cell adhesion, which results in an increased sensitivity of MM to proteasome inhibition.
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The complement system plays a critical role in immune responses against pathogens. However, its identity and regulation in the lung are not fully understood. This study aimed to explore the role of tripartite motif protein (TRIM) 72 in regulating complement receptor (CR) of the Ig superfamily (CRIg) in alveolar macrophage (AM) and innate immunity of the lung. Imaging, absorbance quantification, and flow cytometry were used to evaluate in vitro and in vivo AM phagocytosis with normal, or altered, TRIM72 expression. Pulldown, coimmunoprecipitation, and gradient binding assays were applied to examine TRIM72 and CRIg interaction. A pneumonia model was established by intratracheal injection of Pseudomonas aeruginosa. Mortality, lung bacterial burden, and cytokine levels in BAL fluid and lung tissues were examined. Our data show that TRIM72 inhibited CR-mediated phagocytosis, and release of TRIM72 inhibition led to increased AM phagocytosis. Biochemical assays identified CRIg as a binding partner of TRIM72, and TRIM72 inhibited formation of the CRIg-phagosome. Genetic ablation of TRIM72 led to improved pathogen clearance, reduced cytokine storm, and improved survival in murine models of severe pneumonia, specificity of which was confirmed by adoptive transfer of wild-type or TRIM72KO AMs to AM-depleted TRIM72KO mice. TRIM72 overexpression promoted bacteria-induced NF-κB activation in murine alveolar macrophage cells. Our data revealed a quiescent, noninflammatory bacterial clearance mechanism in the lung via AM CRIg, which is suppressed by TRIM72. In vivo data suggest that targeted suppression of TRIM72 in AM may be an effective measure to treat fatal pulmonary bacterial infections.
Assuntos
Proteínas de Transporte/metabolismo , Imunidade Inata/fisiologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Receptores de Complemento 3b/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Membrana , Camundongos Knockout , NF-kappa B/metabolismo , Fagocitose/fisiologia , Fagossomos/metabolismo , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/patologia , Proteínas com Motivo TripartidoRESUMO
Ly6Chi inflammatory monocytes (iMO) are critical for host defense against toxoplasmosis and malaria but their role in leishmaniasis is unclear. In this study, we report a detrimental role of Ly6Chi iMOs in visceral leishmaniasis (VL) caused by Leishmania donovani. We demonstrate that Ly6Chi iMOs are continuously recruited into the spleen and liver during L. donovani infection and they are preferential targets for the parasite. Using microarray-based gene expression profiling, we show that Ly6Chi iMOs isolated from the infected liver and spleen have distinct phenotypic and activation profiles. Furthermore, we demonstrate that blocking the recruitment of Ly6Chi iMOs into the liver and spleen during L. donovani infection using a CCR2 antagonist reduces the frequency of the pathogenic IFN-γ/IL10 dual producer CD4+ T cells in the spleen and leads to a significant reduction in parasite loads in the liver and spleen. Using STAT1-/- mice we show that STAT1 is critical for mediating the recruitment of Ly6Chi iMOs into organs during L. donovani infection, and adaptive transfer of wild type Ly6Chi iMOs into STAT1-/- recipients renders them susceptible to disease. Our findings reveal an unexpected pathogenic role for Ly6Chi iMOs in promoting parasite survival in VL and open the possibility of targeting this population for host-directed therapy during VL.
Assuntos
Inflamação/imunologia , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Fígado/imunologia , Monócitos/imunologia , Fator de Transcrição STAT1/metabolismo , Baço/imunologia , Animais , Antígenos Ly/metabolismo , Movimento Celular , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Fígado/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/parasitologia , Receptores CCR2/antagonistas & inibidores , Fator de Transcrição STAT1/genética , Baço/patologiaRESUMO
Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling which activates the expression of various genes related to apoptosis, inflammation, cell cycle and angiogenesis. Several experimental and clinical studies have investigated the role of STAT1 in primary tumor growth in breast cancer; however, its role in tumor metastasis remains to be determined. To determine the role of STAT1 in breast cancer metastasis, we analyzed growth and metastasis in WT or STAT1-/- mice orthotopically implanted with metastatic 4T1.2 cells. Primary tumor development was faster in STAT1-/- mice and these mice developed significantly bigger primary tumors and displayed more lung metastasis compared with WT counterparts. STAT1-/- mice showed elevated Ly6G+CD11b+ granulocytic MDSC infiltration in their primary tumors and spleens with concomitant upregulation of Mmp9 and Cxcl1 expression in tumors compared with WT counterparts. Blockade of IL-17A in primary tumor-bearing STAT1-/- mice suppressed accumulation of Ly6G+CD11b+ cells and markedly reduced lung metastasis. These data show that STAT1 is an important suppressor of primary breast tumor growth and metastasis. Importantly, we found anti-IL-17 treatment can rescue STAT1 deficient animals from developing exacerbated metastasis to the lungs which could be important for immunotherapies for immunocompromised breast cancer patients.
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The use of natural products as adjuvants has emerged as a promising approach for the development of effective vaccine formulations. Pentalinonsterol (PEN) is a recently isolated compound from the roots of Pentalinon andrieuxii and has been shown to possess antileishmanial activity against Leishmania spp. The objective of this study was to examine the immunomodulatory properties of PEN and evaluate its potential as an adjuvant. Macrophages and bone-marrow-derived dendritic cells (BMDCs) were stimulated with PEN and tested for gene expression, cytokine production, and their ability to activate T cells in vitro. PEN was also evaluated for its ability to generate antigen-specific Th1 and Th2 responses in vivo, following ovalbumin (OVA) immunization using PEN as an adjuvant. The results obtained demonstrate that PEN enhances the expression of NF-κB and AP1 transcription factors, promotes gene expression of Tnfα, Il6, Nos2, and Arg1, and upregulates MHCII, CD80, and CD86 in macrophages. PEN also enhanced IL-12 production in BMDCs and promoted BMDC-mediated production of IFN-γ by T cells. Further, mice immunized with OVA and PEN showed enhanced antigen-specific Th1 and Th2 cytokines in their splenocytes and lymph node cells, as well as increased levels of IgG1 and IgG2 in their sera. Taken together, this study demonstrates that PEN is a potent immunomodulatory compound and potentially can be used as an adjuvant for vaccine development against infectious diseases.
Assuntos
Adjuvantes Imunológicos/farmacologia , Apocynaceae/química , Citocinas/imunologia , Interleucina-12/imunologia , NF-kappa B/imunologia , Ovalbumina/imunologia , Esteróis/isolamento & purificação , Esteróis/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Adjuvantes Imunológicos/química , Animais , Citocinas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , NF-kappa B/metabolismo , Ovalbumina/química , Esteróis/química , Linfócitos T , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Helminths cause chronic infections and affect the immune response to unrelated inflammatory diseases. Although helminths have been used therapeutically to ameliorate inflammatory conditions, their anti-inflammatory properties are poorly understood. Alternatively activated macrophages (AAMÏs) have been suggested as the anti-inflammatory effector cells during helminth infections. Here, we define the origin of AAMÏs during infection with Taenia crassiceps, and their disease-modulating activity on the Experimental Autoimmune Encephalomyelitis (EAE). Our data show two distinct populations of AAMÏs, based on the expression of PD-L1 and PD-L2 molecules, resulting upon T. crassiceps infection. Adoptive transfer of Ly6C+ monocytes gave rise to PD-L1+/PD-L2+, but not PD-L1+/PD-L2- cells in T. crassiceps-infected mice, demonstrating that the PD-L1+/PD-L2+ subpopulation of AAMÏs originates from blood monocytes. Furthermore, adoptive transfer of PD-L1+/PD-L2+ AAMÏs into EAE induced mice reduced disease incidence, delayed disease onset, and diminished the clinical disability, indicating the critical role of these cells in the regulation of autoimmune disorders.
Assuntos
Transferência Adotiva/métodos , Antígenos Ly/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Ativação de Macrófagos , Células Precursoras de Monócitos e Macrófagos/imunologia , Taenia/imunologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Células Cultivadas , Encefalomielite Autoimune Experimental/terapia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismoRESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) is a major public health concern in Turkey and Sanliurfa represents the most endemic city in Turkey. Although children are most commonly affected by CL, detailed studies of pediatric CL in Turkey are lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this report we retrospectively evaluated clinical and epidemiological data of 8786 pediatric CL cases, and how children respond to antimonial therapy. CL was observed most frequently in children between 6-10 years old. Interestingly this group showed shorter duration of disease and smaller lesions compared to 0-5 year and 11-15 year old groups. Females were more affected in all groups. Lesion localization and types varied among groups, with 0-5 year old presenting head/neck and mucosal lesions, and more often suffered from recidivans type, this could be associated to the longest duration of the disease in this group. Eleven-15 year old group showed fewer lesions in the head/neck but more generalized lesions. Evaluation of treatment response revealed that intra-lesional treatment was preferred over intramuscular treatment. However, 0-5 year old received intramuscular treatment more often than the other groups. Furthermore, the majority of 0-5 year old group which received intra-lesional treatment did not received subsequent intra-lesional cycles, as did children in the range of 6-15 years old. CONCLUSIONS/SIGNIFICANCE: We report an increase in pediatric CL patients within the last four years. Analysis of pediatric CL patients by age revealed significant differences in CL progression. The data suggest that children between 0-5 years old responded better than other groups to intralesional treatment, since they received more often a single cycle of IL treatment, although follow up observation is required since they were more prone to develop recidivans. Eleven-15 year old patients comprise the largest percentage of patients receiving two or three cycles of intralesional treatment, suggesting that this group did not respond efficiently to intralesional treatment and highlighting the need for more effective therapeutic strategies against CL.
Assuntos
Leishmaniose Cutânea/epidemiologia , Adolescente , Distribuição por Idade , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Leishmaniose Cutânea/patologia , Masculino , Pediatria/estatística & dados numéricos , Estudos Retrospectivos , Turquia/epidemiologiaRESUMO
Ibrutinib, a BTK inhibitor, is currently used to treat various hematological malignancies. We evaluated whether ibrutinib treatment during development of murine bone marrow-derived dendritic cells (DCs) modulates their maturation and activation. Ibrutinib treatment increased the proportion of CD11c(+) DCs, upregulated the expression of MHC-II and CD80 and downregulated Ly6C expression by DCs. Additionally, ibrutinib treatment led to an increase in MHC-II(+), CD80(+) and CCR7(+) DCs but a decrease in CD86(+) DCs upon LPS stimulation. LPS/ibrutinib-treated DCs displayed increased IFNß and IL-10 synthesis and decreased IL-6, IL-12 and NO production compared to DCs stimulated with LPS alone. Finally, LPS/ibrutinib-treated DCs promoted higher rates of CD4(+) T cell proliferation and cytokine production compared to LPS only stimulated DCs. Taken together, our results indicate that ibrutinib enhances the maturation and activation of DCs to promote CD4(+) T cell activation which could be exploited for the development of DC-based cancer therapies.
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Oral cancer kills about 1 person every hour each day in the United States and is the sixth most prevalent cancer worldwide. The pro-inflammatory cytokine 'macrophage migration inhibitory factor' (MIF) has been shown to be expressed in oral cancer patients, yet its precise role in oral carcinogenesis is not clear. In this study, we examined the impact of global Mif deletion on the cellular and molecular process occurring during oral carcinogenesis using a well-established mouse model of oral cancer with the carcinogen 4-nitroquinoline-1-oxide (4NQO). C57BL/6 Wild-type (WT) and Mif knock-out mice were administered with 4NQO in drinking water for 16 weeks, then regular drinking water for 8 weeks. Mif knock-out mice displayed fewer oral tumor incidence and multiplicity, accompanied by a significant reduction in the expression of pro-inflammatory cytokines Il-1ß, Tnf-α, chemokines Cxcl1, Cxcl6 and Ccl3 and other molecular biomarkers of oral carcinogenesis Mmp1 and Ptgs2. Further, systemic accumulation of myeloid-derived tumor promoting immune cells was inhibited in Mif knock-out mice. Our results demonstrate that genetic Mif deletion reduces the incidence and severity of oral carcinogenesis, by inhibiting the expression of chronic pro-inflammatory immune mediators. Thus, targeting MIF is a promising strategy for the prevention or therapy of oral cancer.
Assuntos
Transformação Celular Neoplásica/genética , Fatores Inibidores da Migração de Macrófagos/genética , Neoplasias Bucais/etiologia , Neoplasias Bucais/metabolismo , Animais , Apoptose/genética , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos/metabolismo , Mediadores da Inflamação/metabolismo , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Neoplasias Bucais/patologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Invasividade Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Ibrutinib (PCI-32765) is an irreversible dual Btk/Itk inhibitor shown to be effective in treating several B cell malignancies. However, limited studies have been conducted to study the effect of this drug on myeloid cell function. Hence, we studied the effect of ibrutinib treatment on TLR-4 mediated activation of bone marrow derived dendritic cell culture (DCs). Upon ibrutinib treatment, LPS-treated DCs displayed lower synthesis of TNF-α and nitric oxide (NO) and higher induction of IL-6, TGF-ß, IL-10 and IL-18. While ibrutinib dampened MHC-II and CD86 expression on DCs, CD80 expression was upregulated. Further, ibrutinib-treated DCs promoted T cell proliferation and enhanced IL-17 production upon co-culture with nylon wool enriched T cells. Taken together, our results indicate that ibrutinib modulates TLR-4 mediated DC activation to promote an IL-17 response. We describe a novel mode of action for ibrutinib on DCs which should be explored to treat other forms of cancer besides B cell malignancies.
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Leishmania donovani is an intracellular parasite that infects professional phagocytes and causes visceral leishmaniasis (VL). The immune response during VL has been extensively studied in the context of T-helper (Th)1 and Th2 responses. Immunity against this parasite is dependent on IFN-γ production and subsequent macrophage activation, and the Th2 response promotes granuloma formation. The cytokine IL-17A is associated with neutrophilic inflammation. Depletion of neutrophils during experimental VL results in enhanced parasitic loads. Furthermore, although patients resistant to VL showed enhanced levels of IL-17A in circulation, little is known about the role of IL-17A during VL infection. Here, we used IL-17A-deficient mice and IL-17A reporter mice to address the role of IL-17A during VL. IL-17A(-/-) mice were highly resistant to VL infection, showing decreased parasites in the liver and spleen. This unexpected phenotype was associated with enhanced IFN-γ production by T cells and decreased accumulation of neutrophils and monocytes, resulting in reduced number of granulomas. We also found γδ T and Th17 cells as the main IL-17A(+) cells during VL infection. Our data reveal an unexpected role of IL-17A rendering susceptibility against L. donovani by regulating the IFN-γ response and promoting detrimental inflammation.
Assuntos
Interleucina-17/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Animais , Suscetibilidade a Doenças , Granuloma/imunologia , Granuloma/parasitologia , Interferon gama/imunologia , Leishmaniose Visceral/parasitologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/parasitologia , Neutrófilos/imunologia , Neutrófilos/parasitologia , Receptores de Interleucina-17/imunologia , Células Th1/imunologia , Células Th1/parasitologia , Células Th2/imunologia , Células Th2/parasitologiaRESUMO
Chronic inflammation of the intestinal mucosa is characteristic of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. Helminth parasites have developed immunomodulatory strategies that may impact the outcome of several inflammatory diseases. Therefore, we investigated whether Taenia crassiceps infection is able to decrease the inflammatory effects of dextran sulfate sodium- (DSS-) induced ulcerative colitis in BALB/c and C57BL/6 mice. Preinfection significantly reduced the manifestations of DSS-induced colitis, as weight loss and shortened colon length, and decreased the disease activity index independently of the genetic background of the mice. Taenia infection decreased systemic levels of proinflammatory cytokines while increasing levels of IL-4 and IL-10, and the inflammatory infiltrate into the colon was also markedly reduced. RT-PCR assays from colon showed that T. crassiceps-infected mice displayed increased expression of Arginase-1 but decreased expression of iNOS compared to DSS-treated uninfected mice. The percentages of T regulatory cells were not increased. The adoptive transfer of alternatively activated macrophages (AAMФs) from infected mice into mice with DSS-induced colitis reduced the severity of colon inflammation. Administration of indomethacin abrogated the anticolitic effect of Taenia. Thus, T. crassiceps infection limits the pathology of ulcerative colitis by suppressing inflammatory responses mechanistically associated with AAMФs and prostaglandins.
Assuntos
Colite Ulcerativa/parasitologia , Doença de Crohn/parasitologia , Inflamação/parasitologia , Prostaglandinas/biossíntese , Animais , Arginase , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/genética , Doença de Crohn/induzido quimicamente , Doença de Crohn/genética , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Óxido Nítrico Sintase Tipo II/biossíntese , Prostaglandinas/metabolismo , Taenia/patogenicidade , Teníase/complicações , Teníase/parasitologiaRESUMO
Host-pathogen interaction is an area of considerable interest. Intracellular parasites such as Leishmania reside inside phagocytes such as macrophages, dendritic cells and neutrophils. Macrophages can be activated by cytokines such as IFN-γ and Toll like receptor (TLR) agonists resulting in enhanced microbicidal activity. Leishmania parasites hijack the microbicidal function of macrophages, mainly by interfering with intracellular signaling initiated by IFN-γ and TLR ligands. Here we used transgenic Leishmania donovani parasites expressing the red fluorescent protein DsRed2 and imaging-flow cytometry technology to evaluate parasitic loads inside the macrophage in vitro. Further, this methodology enables us to visualize impairment in NFκB translocation to the nucleus in L. donovani infected macrophages. Additionally we show that uninfected bystander macrophages have a similar impairment in NFκB translocation as in L. donovani infected macrophages in response to the TLR4 agonist LPS. This evidence suggests a possible immunosuppressive role for infected macrophages in regulating the activation of uninfected bystander macrophages.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Leishmania donovani/fisiologia , Macrófagos/fisiologia , Macrófagos/parasitologia , Animais , Citometria de Fluxo/métodos , Citometria por Imagem/métodos , Interferon gama/metabolismo , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Ligantes , Proteínas Luminescentes/metabolismo , Ativação de Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo , Proteína Vermelha FluorescenteRESUMO
Three phenylpropanoid dimers (1-3) including two new metabolites were isolated from the extract of the twigs of Nectandra leucantha using antileishmanial bioassay-guided fractionation. The in vitro antiparasitic activity of the isolated compounds against Leishmania donovani parasites and mammalian cytotoxicity and immunomodulatory effects were evaluated. Compounds 1-3 were effective against the intracellular amastigotes within macrophages, with IC50 values of 26.7, 17.8, and 101.9 µM, respectively. The mammalian cytotoxicity, given by the 50% cytotoxic concentration (CC50), was evaluated against peritoneal macrophages. Compounds 1 and 3 were not toxic up to 290 µM, whereas compound 2 demonstrated a CC50 value of 111.2 µM. Compounds 1-3 also suppressed production of disease exacerbatory cytokines IL-6 and IL-10 but had minimal effect on nitric oxide production in L. donovani-infected macrophages, indicating that antileishmanial activity of these compounds is mediated via an NO-independent mechanism. Therefore, these new natural products could represent promising scaffolds for drug design studies for leishmaniasis.
Assuntos
Anisóis/isolamento & purificação , Anisóis/farmacologia , Antiprotozoários/isolamento & purificação , Antiprotozoários/farmacologia , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/farmacologia , Lauraceae/química , Leishmaniose/tratamento farmacológico , Fenilpropionatos/isolamento & purificação , Fenilpropionatos/farmacologia , Animais , Anisóis/química , Antiprotozoários/química , Brasil , Fatores Imunológicos/química , Concentração Inibidora 50 , Interleucina-10 , Interleucina-6 , Leishmania donovani/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estrutura Molecular , Óxido Nítrico/metabolismo , Fenilpropionatos/química , Caules de Planta/químicaRESUMO
C-type lectins are multifunctional sugar-binding molecules expressed on dendritic cells (DCs) and macrophages that internalize antigens for processing and presentation. Macrophage galactose-type lectin 1 (MGL1) recognizes glycoconjugates expressing Lewis X structures which contain galactose residues, and it is selectively expressed on immature DCs and macrophages. Helminth parasites contain large amounts of glycosylated components, which play a role in the immune regulation induced by such infections. Macrophages from MGL1(-/-) mice showed less binding ability toward parasite antigens than their wild-type (WT) counterparts. Exposure of WT macrophages to T. crassiceps antigens triggered tyrosine phosphorylation signaling activity, which was diminished in MGL1(-/-) macrophages. Following T. crassiceps infection, MGL1(-/-) mice failed to produce significant levels of inflammatory cytokines early in the infection compared to WT mice. In contrast, MGL1(-/-) mice developed a Th2-dominant immune response that was associated with significantly higher parasite loads, whereas WT mice were resistant. Flow cytometry and RT-PCR analyses showed overexpression of the mannose receptors, IL-4Rα, PDL2, arginase-1, Ym1, and RELM-α on MGL1(-/-) macrophages. These studies indicate that MGL1 is involved in T. crassiceps recognition and subsequent innate immune activation and resistance.
Assuntos
Antígenos de Helmintos/imunologia , Assialoglicoproteínas/metabolismo , Resistência à Doença/imunologia , Lectinas Tipo C/metabolismo , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Taenia/imunologia , Teníase/imunologia , Acetilgalactosamina/metabolismo , Animais , Assialoglicoproteínas/deficiência , Citocinas/biossíntese , Feminino , Galactose/metabolismo , Glicoconjugados/metabolismo , Imunidade , Espaço Intracelular/metabolismo , Cinética , Lectinas Tipo C/deficiência , Ativação de Macrófagos/imunologia , Proteínas de Membrana/deficiência , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Solubilidade , Teníase/parasitologiaRESUMO
Cutaneous leishmaniasis, caused mainly by Leishmania major, an obligate intracellular parasite, is a disfiguring disease characterized by large skin lesions and is transmitted by a sand fly vector. We previously showed that the chemokine receptor CXCR3 plays a critical role in mediating resistance to cutaneous leishmaniasis caused by Leishmania major. Furthermore, T cells from L. major-susceptible BALB/c but not L. major-resistant C57BL/6 mice fail to efficiently upregulate CXCR3 upon activation. We therefore examined whether transgenic expression of CXCR3 on T cells would enhance resistance to L. major infection in susceptible BALB/c mice. We generated BALB/c and C57BL/6 transgenic mice, which constitutively overexpressed CXCR3 under a CD2 promoter, and then examined the outcomes with L. major infection. Contrary to our hypothesis, transgenic expression of CXCR3 (CXCR3(Tg)) on T cells of BALB/c mice resulted in increased lesion sizes and parasite burdens compared to wild-type (WT) littermates after L. major infection. Restimulated lymph node cells from L. major-infected BALB/c-CXCR3(Tg) mice produced more interleukin-4 (IL-4) and IL-10 and less gamma interferon (IFN-γ). Cells in draining lymph nodes from BALB/c-CXCR3(Tg) mice showed enhanced Th2 and reduced Th1 cell accumulation associated with increased neutrophils and inflammatory monocytes. However, monocytes displayed an immature phenotype which correlated with increased parasite burdens. Interestingly, transgenic expression of CXCR3 on T cells did not impact the outcome of L. major infection in C57BL/6 mice, which mounted a predominantly Th1 response and spontaneously resolved their infection similar to WT littermates. Our findings demonstrate that transgenic expression of CXCR3 on T cells increases susceptibility of BALB/c mice to L. major.
Assuntos
Suscetibilidade a Doenças , Expressão Gênica , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Monócitos/imunologia , Receptores CXCR3/metabolismo , Células Th2/imunologia , Animais , Modelos Animais de Doenças , Feminino , Leishmaniose Cutânea/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Carga Parasitária , Receptores CXCR3/genética , Pele/parasitologia , Pele/patologiaRESUMO
The control of Trypanosoma cruzi infection is related to interferon-γ (IFN-γ) activation leading to intracellular clearance of parasites. The transcription factor signal transducer and activator of transcription 1 (STAT-1) is a key mediator of IFN-γ intracellular signalling and knockout of this protein leads to susceptibility to several intracellular microbes. To determine the role of STAT-1 in host susceptibility to T. cruzi infection we compared the survival, parasite loads and balance of IFN-γ and interleukin-10 (IL-10) responses between wild-type and STAT-1 knockout mice. We found that the lack of STAT-1 resulted in a more robust infection, leading to higher levels of blood and tissue parasites and markedly reduced survival. In addition, infected STAT-1 knockout mice had higher systemic levels of both IFN-γ and IL-10, suggesting that the absence of STAT-1 leads to a disequilibrium of pro-inflammatory and anti-inflammatory cytokines. Analysis of spleen cells indicates that CD4, CD8 cells generate IFN-γ and natural killer cells express IL-13 in STAT-1 knockout animals. The production of IL-17 is particularly enhanced in the absence STAT-1 expression but did not reduce mortality. Overall these results indicate that STAT-1 is important for the control of T. cruzi infection in mice.