RESUMO
Epithelial surfaces form critical barriers to the outside world and are continuously renewed by adult stem cells1. Whereas dynamics of epithelial stem cells during homeostasis are increasingly well understood, how stem cells are redirected from a tissue-maintenance program to initiate repair after injury remains unclear. Here we examined infection by Heligmosomoides polygyrus, a co-evolved pathosymbiont of mice, to assess the epithelial response to disruption of the mucosal barrier. H. polygyrus disrupts tissue integrity by penetrating the duodenal mucosa, where it develops while surrounded by a multicellular granulomatous infiltrate2. Crypts overlying larvae-associated granulomas did not express intestinal stem cell markers, including Lgr53, in spite of continued epithelial proliferation. Granuloma-associated Lgr5- crypt epithelium activated an interferon-gamma (IFN-γ)-dependent transcriptional program, highlighted by Sca-1 expression, and IFN-γ-producing immune cells were found in granulomas. A similar epithelial response accompanied systemic activation of immune cells, intestinal irradiation, or ablation of Lgr5+ intestinal stem cells. When cultured in vitro, granuloma-associated crypt cells formed spheroids similar to those formed by fetal epithelium, and a sub-population of H. polygyrus-induced cells activated a fetal-like transcriptional program, demonstrating that adult intestinal tissues can repurpose aspects of fetal development. Therefore, re-initiation of the developmental program represents a fundamental mechanism by which the intestinal crypt can remodel itself to sustain function after injury.
Assuntos
Feto/citologia , Helmintos/fisiologia , Intestinos/citologia , Parasitos/fisiologia , Nicho de Células-Tronco , Células-Tronco/citologia , Animais , Antígenos Ly/biossíntese , Células Epiteliais/citologia , Feminino , Feto/metabolismo , Interferon gama/imunologia , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Nematospiroides dubius/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Infecções por Strongylida/parasitologiaRESUMO
Interleukin-22 (IL-22) plays a critical role in mucosal defense, although the molecular mechanisms that ensure IL-22 tissue distribution remain poorly understood. We show that the CXCL16-CXCR6 chemokine-chemokine receptor axis regulated group 3 innate lymphoid cell (ILC3) diversity and function. CXCL16 was constitutively expressed by CX3CR1(+) intestinal dendritic cells (DCs) and coexpressed with IL-23 after Citrobacter rodentium infection. Intestinal ILC3s expressed CXCR6 and its ablation generated a selective loss of the NKp46(+) ILC3 subset, a depletion of intestinal IL-22, and the inability to control C. rodentium infection. CD4(+) ILC3s were unaffected by CXCR6 deficiency and remained clustered within lymphoid follicles. In contrast, the lamina propria of Cxcr6(-/-) mice was devoid of ILC3s. The loss of ILC3-dependent IL-22 epithelial stimulation reduced antimicrobial peptide expression that explained the sensitivity of Cxcr6(-/-) mice to C. rodentium. Our results delineate a critical CXCL16-CXCR6 crosstalk that coordinates the intestinal topography of IL-22 secretion required for mucosal defense.
Assuntos
Quimiocina CXCL6/imunologia , Infecções por Enterobacteriaceae/imunologia , Interleucinas/imunologia , Mucosa/imunologia , Receptores CXCR/imunologia , Animais , Antígenos Ly/biossíntese , Linfócitos T CD4-Positivos/imunologia , Receptor 1 de Quimiocina CX3C , Quimiocina CXCL16 , Quimiocina CXCL6/biossíntese , Citrobacter rodentium/imunologia , Células Dendríticas/imunologia , Interleucina-23/biossíntese , Interleucinas/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor 1 Desencadeador da Citotoxicidade Natural/biossíntese , Receptores CXCR/biossíntese , Receptores CXCR/genética , Receptores CXCR6 , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/imunologia , Interleucina 22RESUMO
Within the thymus, two major thymic epithelial cell (TEC) subsets-cortical and medullary TECs-provide unique structural and functional niches for T cell development and establishment of central tolerance. Both lineages are believed to originate from a common progenitor cell, yet the cellular and molecular identity of these bipotent TEC progenitors/stem cells remains ill defined. Here we identify rare stromal cells in the murine adult thymus, which under low-attachment conditions formed spheres (termed "thymospheres"). These thymosphere-forming cells (TSFCs) displayed the stemness features of being slow cycling, self-renewing, and bipotent. TSFCs could be significantly enriched based on their distinct surface antigen phenotype. The FoxN1 transcription factor was dispensable for TSFCs maintenance in situ and for commitment to the medullary and cortical TEC lineages. In summary, this study presents the characterization of the adult thymic epithelial stem cells and demonstrates the dispensability of FoxN1 function for their stemness.
Assuntos
Células Epiteliais/citologia , Fatores de Transcrição Forkhead/genética , Células-Tronco/citologia , Linfócitos T/imunologia , Timo/citologia , Animais , Antígenos Ly/biossíntese , Antígenos de Neoplasias/biossíntese , Antígeno CD24/biossíntese , Moléculas de Adesão Celular/biossíntese , Diferenciação Celular/imunologia , Linhagem da Célula , Células Cultivadas , Pré-Escolar , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Lactente , Antígenos Comuns de Leucócito/biossíntese , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Técnicas de Cultura de Órgãos , Linfócitos T/citologiaRESUMO
While the functional plasticity of memory CD4(+) T cells has been studied extensively, less is known about this property in memory CD8(+) T cells. Here, we report the direct measurement of plasticity by paired daughter analysis of effector and memory OT-I CD8(+) T cells primed in vivo with ovalbumin. Naïve, effector, and memory OT-I cells were isolated and activated in single-cell culture; then, after the first division, their daughter cells were transferred to new cultures with and without IL-4; expression of IFN-γ and IL-4 mRNAs was measured 5 days later in the resultant subclones. Approximately 40% of clonogenic memory CD8(+) T cells were bipotential in this assay, giving rise to an IL-4(-) subclone in the absence of IL-4 and an IL-4(+) subclone in the presence of IL-4. The frequency of bipotential cells was lower among memory cells than naïve cells but markedly higher than among 8-day effectors. Separation based on high or low expression of CD62L, CD122, CD127, or Ly6C did not identify a phenotypic marker of the bipotential cells. Functional plasticity in memory CD8(+) T-cell populations can therefore reflect modulation at the level of a single memory cell and its progeny.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Plasticidade Celular/imunologia , Memória Imunológica/imunologia , Interleucina-4/farmacologia , Animais , Antígenos Ly/biossíntese , Biomarcadores/análise , Linhagem Celular , Interferon gama/biossíntese , Subunidade beta de Receptor de Interleucina-2/biossíntese , Interleucina-4/genética , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Selectina L/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/biossínteseRESUMO
Macrophages display phenotypic and functional heterogeneity dependent on the changing inflammatory microenvironment. Under some conditions, macrophages can acquire effector functions commonly associated with NK cells. In the current study, we investigated how the endogenous danger signal monosodium urate (MSU) crystals can alter macrophage functions. We report that naive, primary peritoneal macrophages rapidly upregulate the expression of the NK cell-surface marker NK1.1 in response to MSU crystals but not in response to LPS or other urate crystals. NK1.1 upregulation by macrophages was associated with mechanisms including phagocytosis of crystals, NLRP3 inflammasome activation, and autocrine proinflammatory cytokine signaling. Further analysis demonstrated that MSU crystal-activated macrophages exhibited NK cell-like cytotoxic activity against target cells in a perforin/granzyme B-dependent manner. Furthermore, analysis of tumor hemopoietic cell populations showed that effective, MSU-mediated antitumor activity required coadministration with Mycobacterium smegmatis to induce IL-1ß production and significant accumulation of monocytes and macrophages (but not granulocytes or dendritic cells) expressing elevated levels of NK1.1. Our findings provide evidence that MSU crystal-activated macrophages have the potential to develop tumoricidal NK cell-like functions that may be exploited to boost antitumor activity in vivo.
Assuntos
Antígenos Ly/biossíntese , Macrófagos/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/biossíntese , Neoplasias/terapia , Ácido Úrico/imunologia , Animais , Proteínas de Transporte/imunologia , Linhagem Celular Tumoral , Granzimas/metabolismo , Inflamassomos/imunologia , Interleucina-1beta/biossíntese , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Mycobacterium smegmatis/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Neoplasias/imunologia , Perforina/metabolismo , Fagocitose/imunologia , Regulação para Cima , Ácido Úrico/farmacologiaRESUMO
Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear. In this study, we used a renal bilateral ischemia-reperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b(+) cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies. Macrophage populations were isolated from kidney homogenates by fluorescence-activated cell sorting for whole genome microarray analysis. The CD11b(+)/Ly6C(high) population associated with the onset of renal injury and increase in proinflammatory cytokines, whereas the CD11b(+)/Ly6C(intermediate) population peaked during kidney repair. The CD11b(+)/Ly6C(low) population emerged with developing renal fibrosis. Principal component and hierarchical cluster analyses identified gene signatures unique to each population. The CD11b(+)/Ly6C(intermediate) population had a distinct phenotype of wound healing, confirmed by results of studies inhibiting the macrophage colony-stimulating factor 1 receptor,whereas the CD11b(+)/Ly6C(low) population had a profibrotic phenotype. All populations, including the CD11b(+)/Ly6C(high) population, carried differential inflammatory signatures. The expression of M2-specific markers was detected in both the CD11b(+)/Ly6C(intermediate) and CD11b(+)/Ly6C(low) populations, suggesting these in vivo populations do not fit into the traditional classifications defined by in vitro systems. Results of this study in a renal ischemia-reperfusion injury model allow phenotype and function to be assigned to CD11b(+)/Ly6C(+) monocyte/macrophage populations in the pathophysiology of disease after AKI.
Assuntos
Antígenos Ly/biossíntese , Rim/metabolismo , Macrófagos/classificação , Traumatismo por Reperfusão/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Traumatismo por Reperfusão/sangueRESUMO
Myeloid-derived suppressor cells (MDSCs) are a CD11b(+)Gr1(+) population in mice that can be separated into granulocytic (g-MDSC) and monocytic (m-MDSC) subtypes based on their expression of Ly6G and Ly6C. Both MDSC subtypes are potent suppressors of T cell immunity, and their contribution has been investigated in a plethora of diseases including renal cancer, renal transplant, and chronic kidney disease. Whether MDSCs contribute to the pathogenesis of acute kidney injury (AKI) remains unknown. Herein, using human C-reactive protein (CRP) transgenic (CRPtg) and CRP-deficient mice (CRP(-/-)) subjected to bilateral renal ischemia-reperfusion injury (IRI), we confirm our earlier finding that CRP exacerbates renal IRI and show for the first time that this effect is accompanied in CRPtg mice by a shift in the balance of kidney-infiltrating MDSCs toward a suppressive Ly6G(+)Ly6C(low) g-MDSC subtype. In CRPtg mice, direct depletion of g-MDSCs (using an anti-Gr1 monoclonal antibody) reduced the albuminuria caused by renal IRI, confirming they play a deleterious role. Remarkably, treatment of CRPtg mice with an antisense oligonucleotide that specifically blocks the human CRP acute-phase response also led to a reduction in renal g-MDSC numbers and improved albuminuria after renal IRI. Our study in CRPtg mice provides new evidence that MDSCs participate in the pathogenesis of renal IRI and shows that their pharmacological depletion is beneficial. If ongoing investigations confirm that CRP is an endogenous regulator of MDSCs in CRPtg mice, and if this action is recapitulated in humans, then targeting CRP or/and MDSCs might offer a new approach for the treatment of AKI.
Assuntos
Proteína C-Reativa/toxicidade , Nefropatias/induzido quimicamente , Células Mieloides/patologia , Traumatismo por Reperfusão/induzido quimicamente , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Antígenos Ly/biossíntese , Antígenos Ly/genética , Proteína C-Reativa/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Granulócitos/efeitos dos fármacos , Granulócitos/patologia , Humanos , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/patologia , Traumatismo por Reperfusão/patologia , Linfócitos TRESUMO
The role and origin of alveolar macrophages (AMs) in asthma are incompletely defined. We sought to clarify these issues in the context of acute allergic lung inflammation using house dust mite and OVA murine models. Use of liposomal clodronate to deplete resident AMs (rAMs) resulted in increased levels of inflammatory cytokines and eosinophil numbers in lavage fluid and augmented the histopathologic evidence of lung inflammation, suggesting a suppressive role for rAMs. Lung digests of asthmatic mice revealed an increased percentage of Ly6C(high)/CD11b(pos) inflammatory monocytes. Clodronate depletion of circulating monocytes, by contrast, resulted in an attenuation of allergic inflammation. A CD45.1/CD45.2 chimera model demonstrated that recruitment at least partially contributes to the AM pool in irradiated nonasthmatic mice, but its contribution was no greater in asthma. Ki-67 staining of AMs supported a role for local proliferation, which was increased in asthma. Our data demonstrate that rAMs dampen, whereas circulating monocytes promote, early events in allergic lung inflammation. Moreover, maintenance of the AM pool in the early stages of asthmatic inflammation depends on local proliferation, but not recruitment.
Assuntos
Asma/imunologia , Inflamação/imunologia , Macrófagos Alveolares/imunologia , Monócitos/imunologia , Alérgenos/imunologia , Alveolite Alérgica Extrínseca/imunologia , Animais , Antígenos Ly/biossíntese , Líquido da Lavagem Broncoalveolar/citologia , Antígeno CD11b/biossíntese , Proliferação de Células , Ácido Clodrônico/farmacologia , Citocinas/biossíntese , Modelos Animais de Doenças , Eosinófilos/imunologia , Antígenos Comuns de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Pneumonia/imunologia , Pyroglyphidae/imunologiaRESUMO
In the immunocompromised host, invasive infection with the fungal pathogen Candida albicans is associated with high morbidity and mortality. Sporadic cases in otherwise normal individuals are rare, and they are thought to be associated with genetic predisposition. Using a mouse model of systemic infection with C. albicans, we identified the SM/J mouse strain as unusually susceptible to infection. Genetic linkage studies in informative [C57BL/6JxSM/J]F2 mice identified a major locus on distal chromosome 15, given the appellation Carg5, that regulates C. albicans replication in SM/J mice. Cellular and molecular immunophenotyping experiments, as well as functional studies in purified cell populations from SM/J and C57BL/6J, and in [C57BL/6JxSM/J]F2 mice fixed for homozygous or heterozygous Carg5 alleles, indicate that Carg5-regulated susceptibility in SM/J is associated with a complex defect in the myeloid compartment of these mice. SM/J neutrophils express lower levels of Ly6G, and importantly, they show significantly reduced production of reactive oxygen species in response to stimulation with fMLF and PMA. Likewise, CD11b(+)Ly6G(-)Ly6C(hi) inflammatory monocytes were present at lower levels in the blood of infected SM/J, recruited less efficiently at the site of infection, and displayed blunted oxidative burst. Studies in F2 mice establish strong correlations between Carg5 alleles, Ly6G expression, production of serum CCL2 (MCP-1), and susceptibility to C. albicans. Genomic DNA sequencing of chromatin immunoprecipitated for myeloid proinflammatory transcription factors IRF1, IRF8, STAT1 and NF-κB, as well as RNA sequencing, were used to develop a "myeloid inflammatory score" and systematically analyze and prioritize potential candidate genes in the Carg5 interval.
Assuntos
Candidíase/genética , Candidíase/imunologia , Predisposição Genética para Doença , Animais , Antígenos Ly/biossíntese , Antígenos Ly/genética , Candidíase/microbiologia , Quimiocina CCL2/biossíntese , Quimiocina CCL2/sangue , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/microbiologia , Feminino , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Imunofenotipagem , Inflamação/genética , Inflamação/imunologia , Inflamação/microbiologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/microbiologia , Monócitos/patologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Neutrófilos/patologia , Cultura Primária de Células , Proteínas Repressoras/genética , Especificidade da Espécie , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/imunologiaRESUMO
Dendritic cells (DC) are critical for the initiation of immune responses; however, their role in priming IL-4-producing Th2 cells in vivo is not fully understood. We used a model of intradermal injection with fluorescent-labeled, nonviable larvae from the helminth parasite nonviable Nippostrongylus brasiliensis L3 larvae (Nb), a strong inducer of Th2 responses, together with IL-4-GFP reporter mice that enable a sensitive detection of IL-4 production to examine the contribution of DC to the priming of IL-4-producing CD4(+) T cells in vivo. We found that parasite material is taken up by two distinct DC populations in draining lymph nodes: a mostly CD11c(int)MHC class II (MHCII)(hi)CD11b(+)Ly6C(-) dermal DC population and a CD11c(hi)MHCII(int)CD11b(+)Ly6C(+) monocyte-derived DC population. After Nb treatment, these two DC populations appeared in the draining lymph nodes in comparable numbers and with similar kinetics; however, treatment with pertussis toxin blocked the migration of dermal DC and the priming of IL-4-producing T cells, but only partially affected monocyte-derived DC numbers. In line with this observation, transfer of OVA-loaded CD11c(int)MHCII(hi) DC from Nb-treated mice into naive hosts could sensitize OVA-specific CD4(+) T cells to IL-4 production, whereas transfer of CD11c(int)MHCII(hi) DC from naive mice, or CD11c(hi)MHCII(int) DC from Nb-treated or naive mice, induced CD4(+) T cell expansion but no IL-4 production. Phenotypic analysis of Nb-loaded CD11c(int)MHCII(hi) DC revealed expression of programmed death ligand 2, CD301b, IFN regulatory factor 4, and moderate upregulation of OX40 ligand. However, thymic stromal lymphopoietin and OX40 ligand were not required for Th2 priming. Thus, our data suggest that appropriate stimuli can induce DC to express the unique signals sufficient to direct CD4(+) T cells to Th2 differentiation.
Assuntos
Células Dendríticas/imunologia , Interleucina-4/biossíntese , Nippostrongylus/imunologia , Células Th2/imunologia , Animais , Antígenos Ly/biossíntese , Antígeno CD11c/biossíntese , Diferenciação Celular/imunologia , Citocinas/genética , Citocinas/imunologia , Proteínas de Fluorescência Verde , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/imunologia , Fatores Reguladores de Interferon/biossíntese , Interleucina-33 , Interleucina-4/imunologia , Interleucinas/imunologia , Larva/imunologia , Lectinas Tipo C/biossíntese , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligante OX40 , Proteína 2 Ligante de Morte Celular Programada 1/biossíntese , Fatores de Necrose Tumoral/biossíntese , Fatores de Necrose Tumoral/imunologia , Linfopoietina do Estroma do TimoRESUMO
The underlying mechanisms that lysosomal acid lipase (LAL) deficiency causes infiltration of myeloid-derived suppressor cells (MDSCs) in multiple organs and subsequent inflammation remain incompletely understood. Endothelial cells (ECs), lining the inner layer of blood vessels, constitute barriers regulating leukocytes transmigration to the site of inflammation. Therefore, we hypothesized that ECs are dysfunctional in LAL-deficient (lal(-/-)) mice. We found that Ly6G(+) cells transmigrated more efficiently across lal(-/-) ECs than wild-type (lal(+/+)) ECs, which were associated with increased levels of PECAM-1 and MCP-1 in lal(-/-) ECs. In addition, lal(-/-) ECs showed enhanced migration and proliferation, decreased apoptosis, but impaired tube formation and angiogenesis. lal(-/-) ECs also suppressed T cell proliferation in vitro. Interestingly, lal(-/-) Ly6G(+) cells promoted in vivo angiogenesis (including a tumor model), EC tube formation, and proliferation. Finally, the mammalian target of rapamycin (mTOR) pathway was activated in lal(-/-) ECs, and inhibition of mTOR reversed EC dysfunctions, including decreasing Ly6G(+) cell transmigration, delaying migration, and relieving suppression of T cell proliferation, which was mediated by decreasing production of reactive oxygen species. Our results indicate that LAL regulates EC functions through interaction with MDSCs and modulation of the mTOR pathway, which may provide a mechanistic basis for targeting MDSCs or mTOR to rejuvenate EC functions in LAL deficiency-related diseases.
Assuntos
Células Endoteliais/imunologia , Macrófagos/imunologia , Serina-Treonina Quinases TOR/imunologia , Migração Transendotelial e Transepitelial/imunologia , Doença de Wolman/imunologia , Animais , Antígenos Ly/biossíntese , Apoptose/imunologia , Células da Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Quimiocina CCL2/biossíntese , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Esterol Esterase/deficiência , Esterol Esterase/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Doença de Wolman/genética , Doença de WolmanRESUMO
To study gene functions specifically in NKp46+ cells we developed novel Cre mice allowing for conditional gene targeting in cells expressing Ncr1 (encoding NKp46). We generated transgenic Ncr1(greenCre) mice carrying an EGFPcre fusion under the control of a proximal Ncr1 promoter that faithfully directed EGFPcre expression to NKp46+ cells from lymphoid and nonlymphoid tissues. This approach allowed for direct detection of Cre-expressing NKp46+ cells via their GFP signature by flow cytometry and histology. Cre was functional as evidenced by the NKp46+ cell-specific expression of RFP in Ncr1(greenCre) Rosa-dtRFP reporter mice. We generated Ncr1(greenCre) Il2rg(fl/fl) mice that lack NKp46+ cells in an otherwise intact hematopoietic environment. Il2rg encodes the common gamma chain (γc ), which is an essential receptor subunit for cytokines (IL-2, -4, -7, -9, -15, and -21) that stimulate lymphocyte development and function. In Ncr1(greenCre) Il2rg(fl/fl) mice, NK cells are severely reduced and the few remaining NKp46+ cells escaping γc deletion failed to express GFP. Using this new NK-cell-deficient model, we demonstrate that the homeostasis of NKp46+ cells from all tissues (including the recently described intraepithelial ILC1 subset) requires Il2rg. Finally, Ncr1(greenCre) Il2rg(fl/fl) mice are unable to reject B16 lung metastases demonstrating the essential role of NKp46+ cells in antimelanoma immune responses.
Assuntos
Antígenos Ly/genética , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/patologia , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Animais , Antígenos Ly/biossíntese , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Fluorescência Verde/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Neoplasias Pulmonares/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 1 Desencadeador da Citotoxicidade Natural/biossínteseRESUMO
BACKGROUND: Japanese encephalitis (JE), a neuroinflammation caused by zoonotic JE virus, is the major cause of viral encephalitis worldwide and poses an increasing threat to global health and welfare. To date, however, there has been no report describing the regulation of JE progression using immunomodulatory tools for developing therapeutic strategies. We tested whether blocking the 4-1BB signaling pathway would regulate JE progression using murine JE model. METHODS: Infected wild-type and 4-1BB-knockout (KO) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, JEV-specific T cell, and type I/II IFN (IFN-I/II) innate responses were analyzed. RESULTS: Blocking the 4-1BB signaling pathway significantly increased resistance to JE and reduced viral burden in extraneural tissues and the CNS, rather than causing a detrimental effect. In addition, treatment with 4-1BB agonistic antibody exacerbated JE. Furthermore, JE amelioration and reduction of viral burden by blocking the 4-1BB signaling pathway were associated with an increased frequency of IFN-II-producing NK and CD4(+) Th1 cells as well as increased infiltration of mature Ly-6C(hi) monocytes in the inflamed CNS. More interestingly, DCs and macrophages derived from 4-1BB KO mice showed potent and rapid IFN-I innate immune responses upon JEV infection, which was coupled to strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7), and antiviral ISG genes (ISG49, ISG54, ISG56). Further, the ablation of 4-1BB signaling enhanced IFN-I innate responses in neuron cells, which likely regulated viral spread in the CNS. Finally, we confirmed that blocking the 4-1BB signaling pathway in myeloid cells derived from hematopoietic stem cells (HSCs) played a dominant role in ameliorating JE. In support of this finding, HSC-derived leukocytes played a dominant role in generating the IFN-I innate responses in the host. CONCLUSIONS: Blocking the 4-1BB signaling pathway ameliorates JE via divergent enhancement of IFN-II-producing NK and CD4(+) Th1 cells and mature Ly-6C(hi) monocyte infiltration, as well as an IFN-I innate response of myeloid-derived cells. Therefore, regulation of the 4-1BB signaling pathway with antibodies or inhibitors could be a valuable therapeutic strategy for the treatment of JE.
Assuntos
Antígenos Ly/biossíntese , Encefalite Japonesa/metabolismo , Interferon-alfa/biossíntese , Interferon beta/biossíntese , Monócitos/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/deficiência , Animais , Diferenciação Celular/fisiologia , Encefalite Japonesa/imunologia , Encefalite Japonesa/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Transdução de Sinais/fisiologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidoresRESUMO
RATIONALE: Autologous bone marrow-derived or cardiac-derived stem cell therapy for heart disease has demonstrated safety and efficacy in clinical trials, but functional improvements have been limited. Finding the optimal stem cell type best suited for cardiac regeneration is the key toward improving clinical outcomes. OBJECTIVE: To determine the mechanism by which novel bone-derived stem cells support the injured heart. METHODS AND RESULTS: Cortical bone-derived stem cells (CBSCs) and cardiac-derived stem cells were isolated from enhanced green fluorescent protein (EGFP+) transgenic mice and were shown to express c-kit and Sca-1 as well as 8 paracrine factors involved in cardioprotection, angiogenesis, and stem cell function. Wild-type C57BL/6 mice underwent sham operation (n=21) or myocardial infarction with injection of CBSCs (n=67), cardiac-derived stem cells (n=36), or saline (n=60). Cardiac function was monitored using echocardiography. Only 2/8 paracrine factors were detected in EGFP+ CBSCs in vivo (basic fibroblast growth factor and vascular endothelial growth factor), and this expression was associated with increased neovascularization of the infarct border zone. CBSC therapy improved survival, cardiac function, regional strain, attenuated remodeling, and decreased infarct size relative to cardiac-derived stem cells- or saline-treated myocardial infarction controls. By 6 weeks, EGFP+ cardiomyocytes, vascular smooth muscle, and endothelial cells could be identified in CBSC-treated, but not in cardiac-derived stem cells-treated, animals. EGFP+ CBSC-derived isolated myocytes were smaller and more frequently mononucleated, but were functionally indistinguishable from EGFP- myocytes. CONCLUSIONS: CBSCs improve survival, cardiac function, and attenuate remodeling through the following 2 mechanisms: (1) secretion of proangiogenic factors that stimulate endogenous neovascularization, and (2) differentiation into functional adult myocytes and vascular cells.
Assuntos
Osso e Ossos/citologia , Transdiferenciação Celular , Células Endoteliais/citologia , Células-Tronco Multipotentes/fisiologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Comunicação Parácrina/fisiologia , Proteínas Angiogênicas/biossíntese , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Antígenos Ly/biossíntese , Antígenos Ly/genética , Biomarcadores , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/transplante , Infarto do Miocárdio/patologia , Neovascularização Fisiológica/genética , Proteínas Proto-Oncogênicas c-kit/biossíntese , Proteínas Proto-Oncogênicas c-kit/genética , Remodelação Ventricular/fisiologiaRESUMO
MPYS (also known as STING, MITA, and TMEM173) is a type I IFN stimulator that is essential for host defense against DNA virus infection and appears important in defense against certain bacteria. The in vivo significance and mechanisms by which MPYS mediates host defense against nonviral pathogens are unknown. Using an MPYS-deficient mouse (Tmem173(
Assuntos
Antígenos Ly/biossíntese , Movimento Celular/imunologia , Listeriose/imunologia , Listeriose/patologia , Proteínas de Membrana/fisiologia , Monócitos/imunologia , Animais , Movimento Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Listeriose/genética , Fígado/imunologia , Fígado/microbiologia , Fígado/patologia , Proteínas de Membrana/deficiência , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Monócitos/microbiologia , Monócitos/patologia , Baço/imunologia , Baço/microbiologiaRESUMO
In this study, we explore the hypothesis that enhanced production of lactate by tumor cells, because of high glycolytic activity, results in inhibition of host immune response to tumor cells. Lactate dehydrogenase-A (LDH-A), responsible for conversion of pyruvate to lactate, is highly expressed in tumor cells. Lentiviral vector-mediated LDH-A short hairpin RNA knockdown Pan02 pancreatic cancer cells injected in C57BL/6 mice developed smaller tumors than mice injected with Pan02 cells. A decrease occurred in the frequency of myeloid-derived suppressor cells (MDSCs) in the spleens of mice carrying LDH-A-depleted tumors. NK cells from LDH-A-depleted tumors had improved cytolytic function. Exogenous lactate increased the frequency of MDSCs generated from mouse bone marrow cells with GM-CSF and IL-6 in vitro. Lactate pretreatment of NK cells in vitro inhibited cytolytic function of both human and mouse NK cells. This reduction of NK cytotoxic activity was accompanied by lower expression of perforin and granzyme in NK cells. The expression of NKp46 was decreased in lactate-treated NK cells. These studies strongly suggest that tumor-derived lactate inhibits NK cell function via direct inhibition of cytolytic function as well as indirectly by increasing the numbers of MDSCs that inhibit NK cytotoxicity. Depletion of glucose levels using a ketogenic diet to lower lactate production by glycolytic tumors resulted in smaller tumors, decreased MDSC frequency, and improved antitumor immune response. These studies provide evidence for an immunosuppressive role of tumor-derived lactate in inhibiting innate immune response against developing tumors via regulation of MDSC and NK cell activity.
Assuntos
Células Matadoras Naturais/imunologia , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Neoplasias/imunologia , Evasão Tumoral , Animais , Antígenos Ly/biossíntese , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Feminino , Glucose/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Granzimas/biossíntese , Humanos , Interleucina-6/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , L-Lactato Desidrogenase/genética , Lactato Desidrogenase 5 , Ácido Láctico/farmacologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/biossíntese , Neoplasias/genética , Neoplasias/metabolismo , Perforina/biossíntese , Interferência de RNA , RNA Interferente Pequeno , Baço/imunologiaRESUMO
Plasmacytoid dendritic cells (pDCs) play an important role in innate and adaptive immunity and were shown to be identical to previously described natural interferon (IFN)-α-producing cells. Here, we describe two functionally distinct pDC subpopulations that are characterized by the differential expression of stem cell antigen-1 (Sca-1; Ly-6A/E). Sca-1(-) pDCs are mainly found in the BM, appear first during development, show a higher proliferative activity, and represent the more precursor phenotype. Sca-1(+) pDCs are mostly located in secondary lymphoid organs and represent a later developmental stage. Sca-1(-) pDCs give rise to an Sca-1(+) subset upon activation or in response to endogenous type I IFN. Interestingly, in contrast to Sca-1(-) pDCs, Sca-1(+) pDCs are defective in IFN-α production upon endosomal TLR9 stimulation, whereas lysosomal signaling via TLR9 is functional in both subsets. Gene expression analysis revealed that osteopontin is strongly upregulated in Sca-1(-) pDCs. These data provide evidence for the molecular basis of the observed functional heterogeneity, as the intracellular isoform of osteopontin couples TLR9 signaling to IFN-α expression. Taken together, our results indicate that Sca-1(-) pDCs are an early developmental stage of pDCs with distinct innate functions representing the true murine natural IFN-α-producing cells.
Assuntos
Antígenos Ly/genética , Células Dendríticas/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/genética , Receptor Toll-Like 9/biossíntese , Animais , Antígenos Ly/biossíntese , Proliferação de Células , Células Dendríticas/imunologia , Feminino , Expressão Gênica , Interferon-alfa/biossíntese , Ativação Linfocitária/imunologia , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Osteopontina/biossíntese , Transdução de Sinais/imunologia , Regulação para CimaRESUMO
Invasive fungal infections by Candida albicans (Ca) are a frequent cause of lethal sepsis in intensive care unit patients. While a contribution of type I interferons (IFNs-I) in fungal sepsis remains unknown, these immunostimulatory cytokines mediate the lethal effects of endotoxemia and bacterial sepsis. Using a mouse model lacking a functional IFN-I receptor (Ifnar1â»/â»), we demonstrate a remarkable protection against invasive Ca infections. We discover a mechanism whereby IFN-I signaling controls the recruitment of inflammatory myeloid cells, including Ly6C(hi) monocytes and neutrophils, to infected kidneys by driving expression of the chemokines CCL2 and KC. Within kidneys, monocytes differentiate into inflammatory DCs but fail to functionally mature in Ifnar1â»/â» mice, as demonstrated by the impaired upregulation of the key activation markers PDCA1 and iNOS. The increased activity of inflammatory monocytes and neutrophils results in hyper-inflammation and lethal kidney pathology. Pharmacological diminution of monocytes and neutrophils by treating mice with pioglitazone, a synthetic agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ), strongly reduces renal immunopathology during Ca infection and improves mouse survival. Taken together, our data connect for the first time the sepsis-promoting functions of IFNs-I to the CCL2-mediated recruitment and the activation of inflammatory monocytes/DCs with high host-destructing potency. Moreover, our data demonstrate a therapeutic relevance of PPAR-γ agonists for microbial infectious diseases where inflammatory myeloid cells may contribute to fatal tissue damage.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Interferon Tipo I/metabolismo , Monócitos/imunologia , Neutrófilos/imunologia , Animais , Antígenos CD/biossíntese , Antígenos Ly/biossíntese , Candidemia/mortalidade , Candidíase/patologia , Quimiocina CCL2/biossíntese , Quimiocina CXCL1/biossíntese , Células Dendríticas/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Rim/imunologia , Rim/microbiologia , Masculino , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/biossíntese , PPAR gama/agonistas , Pioglitazona , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Transdução de Sinais/genética , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêuticoRESUMO
The transcription factor IFN regulatory factor (IRF)5 has been identified as a human systemic lupus erythematosus (SLE) susceptibility gene by numerous joint linkage and genome-wide association studies. Although IRF5 expression is significantly elevated in primary blood cells of SLE patients, it is not yet known how IRF5 contributes to SLE pathogenesis. Recent data from mouse models of lupus indicate a critical role for IRF5 in the production of pathogenic autoantibodies and the expression of Th2 cytokines and type I IFN. In the present study, we examined the mechanisms by which loss of Irf5 protects mice from pristane-induced lupus at early time points of disease development. We demonstrate that Irf5 is required for Ly6C(hi) monocyte trafficking to the peritoneal cavity, which is thought to be one of the initial key events leading to lupus pathogenesis in this model. Chemotaxis assays using peritoneal lavage from pristane-injected Irf5(+/+) and Irf5(-/-) littermates support an intrinsic defect in Irf5(-/-) monocytes. We found the expression of chemokine receptors CXCR4 and CCR2 to be dysregulated on Irf5(-/-) monocytes and less responsive to their respective ligands, CXCL12 and CCL2. Bone marrow reconstitution experiments further supported an intrinsic defect in Irf5(-/-) monocytes because Irf5(+/+) monocytes were preferentially recruited to the peritoneal cavity in response to pristane. Taken together, these findings demonstrate an intrinsic role for IRF5 in the response of monocytes to pristane and their recruitment to the primary site of inflammation that is thought to trigger lupus onset in this experimental model of SLE.
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Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Monócitos/imunologia , Monócitos/patologia , Terpenos/toxicidade , Animais , Antígenos Ly/biossíntese , Movimento Celular/imunologia , Células HEK293 , Humanos , Fatores Reguladores de Interferon/fisiologia , Lúpus Eritematoso Sistêmico/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Cavidade Peritoneal/patologiaRESUMO
Neuropathology in multiple sclerosis is closely linked to presence of macrophages in the CNS. Both M1 (inflammatory) and M2 (alternatively activated, noninflammatory) macrophages are found in the inflamed CNS and thought to differentiate from infiltrating monocytes. It is unclear whether the balance of M1 and M2 macrophages can be altered and whether this affects disease outcome. We show in this article that Ly6C(hi) inflammatory monocytes are the early and dominant infiltrating cells in the CNS during experimental autoimmune encephalomyelitis, a model for the acute phase of multiple sclerosis. Activation of invariant NKT (iNKT) cells reduced the frequency of Ly6C(hi) monocytes and increased the proportion of M2 macrophages in the CNS with associated improvement in neurologic impairment. In contrast, iNKT-deficient mice showed higher numbers of Ly6C(hi) monocytes, reduced M2, and much more severe disease. Adoptive transfer of M2-enriched cells to iNKT-deficient mice markedly improved neurologic impairment. In vitro and in vivo experiments showed that iNKT cells promote differentiation of monocytes to M2 macrophages in an IL-4 and CD1d-dependent process. These findings indicate that infiltrating Ly6C(hi) inflammatory monocytes are early players in acute neuroinflammation and that their frequency and differentiation can be influenced by activation of iNKT cells with resultant improvement in disease outcome.