RESUMEN
We previously demonstrated that MCP-1 is important for E. coli-induced neutrophil migration to the lungs. However, E. coli neither disseminates nor induces death in mice. Furthermore, the cell types and the host defense mechanisms that contribute to MCP-1-dependent neutrophil trafficking have not been defined. In this study, we sought to explore the cell types and the mechanisms associated with Klebsiella pneumoniae-mediated MCP-1-dependent neutrophil influx. MCP-1(-/-) mice are more susceptible to pulmonary K. pneumoniae infection and show higher bacterial burden in the lungs and dissemination. MCP-1(-/-) mice also display attenuated neutrophil influx, cytokine/chemokine production, and activation of NF-κB and MAPKs following intratracheal K. pneumoniae infection. rMCP-1 treatment in MCP-1(-/-) mice following K. pneumoniae infection rescued impairment in survival, bacterial clearance, and neutrophil accumulation in the lung. Neutrophil numbers in the blood of MCP-1(-/-) mice were associated with G-CSF concentrations in bronchoalveolar lavage fluid and blood. Bone marrow or resident cell-derived MCP-1 contributed to bacterial clearance, neutrophil accumulation, and cytokine/chemokine production in the lungs following infection. Furthermore, exogenous MCP-1 dose dependently increased neutrophil counts and G-CSF concentrations in the blood. Intriguingly, administration of intratracheal rG-CSF to MCP-1(-/-) mice after K. pneumoniae infection rescued survival, bacterial clearance and dissemination, and neutrophil influx in MCP-1(-/-) mice. Collectively, these novel findings unveil an unrecognized role of MCP-1 in neutrophil-mediated host immunity during K. pneumoniae pneumonia and illustrate that G-CSF could be used to rescue impairment in host immunity in individuals with absent or malfunctional MCP-1.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/sangre , Factor Estimulante de Colonias de Granulocitos/fisiología , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/terapia , Infiltración Neutrófila/inmunología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/terapia , Animales , Modelos Animales de Enfermedad , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Infecciones por Klebsiella/sangre , Klebsiella pneumoniae/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/genética , Neumonía Bacteriana/sangreRESUMEN
Conventional dendritic cells (DCs) are essential mediators of antitumor immunity. As a result, cancers have developed poorly understood mechanisms to render DCs dysfunctional within the tumor microenvironment (TME). After identification of CD63 as a specific surface marker, we demonstrate that mature regulatory DCs (mregDCs) migrate to tumor-draining lymph node tissues and suppress DC antigen cross-presentation in trans while promoting T helper 2 and regulatory T cell differentiation. Transcriptional and metabolic studies showed that mregDC functionality is dependent on the mevalonate biosynthetic pathway and its master transcription factor, SREBP2. We found that melanoma-derived lactate activates SREBP2 in tumor DCs and drives conventional DC transformation into mregDCs via homeostatic or tolerogenic maturation. DC-specific genetic silencing and pharmacologic inhibition of SREBP2 promoted antitumor CD8+ T cell activation and suppressed melanoma progression. CD63+ mregDCs were found to reside within the lymph nodes of several preclinical tumor models and in the sentinel lymph nodes of patients with melanoma. Collectively, this work suggests that a tumor lactate-stimulated SREBP2-dependent program promotes CD63+ mregDC development and function while serving as a promising therapeutic target for overcoming immune tolerance in the TME.
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Células Dendríticas , Ácido Láctico , Melanoma , Transducción de Señal , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , Células Dendríticas/inmunología , Progresión de la Enfermedad , Tolerancia Inmunológica/inmunología , Ácido Láctico/metabolismo , Melanoma/inmunología , Melanoma/patología , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones Endogámicos C57BL , Transducción de Señal/inmunología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Therapeutic resistance to immune checkpoint blockade has been commonly linked to the process of mesenchymal transformation (MT) and remains a prevalent obstacle across many cancer types. An improved mechanistic understanding for MT-mediated immune evasion promises to lead to more effective combination therapeutic regimens. Herein, we identify the Hedgehog transcription factor, Gli2, as a key node of tumor-mediated immune evasion and immunotherapy resistance during MT. Mechanistic studies reveal that Gli2 generates an immunotolerant tumor microenvironment through the upregulation of Wnt ligand production and increased prostaglandin synthesis. This pathway drives the recruitment, viability, and function of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) while also impairing type I conventional dendritic cell, CD8 + T cell, and NK cell functionality. Pharmacologic EP2/EP4 prostaglandin receptor inhibition and Wnt ligand inhibition each reverses a subset of these effects, while preventing primary and adaptive resistance to anti-PD-1 immunotherapy, respectively. A transcriptional Gli2 signature correlates with resistance to anti-PD-1 immunotherapy in stage IV melanoma patients, providing a translational roadmap to direct combination immunotherapeutics in the clinic. SIGNIFICANCE: Gli2-induced EMT promotes immune evasion and immunotherapeutic resistance via coordinated prostaglandin and Wnt signaling.
RESUMEN
Bacterial pneumonia remains a significant cause of mortality in the United States. The innate immune response is the first line of defense against invading bacteria. Neutrophil recruitment to the lungs is the first step in a multistep sequence leading to bacterial clearance. Ligand interaction with pattern-recognizing receptors (PRRs) leads to chemokine production, which drives neutrophils to the site of infection. Although we demonstrated that RIP2 is important for host defense in the lungs against Escherichia coli, the individual roles of NOD1 and NOD2 in pulmonary defense have not been addressed. Here, we explored the role of NOD2 in neutrophil-mediated host defense against an extracellular pathogen, E. coli. We found enhanced bacterial burden and reduced neutrophil and cytokine/chemokine levels in the lungs of NOD2â»/â» mice following E. coli infection. Furthermore, we observed reduced activation of NF-κB and mitogen-activated protein kinases (MAPKs) in the lungs of NOD2â»/â» mice upon E. coli challenge. Moreover, NOD2â»/â» neutrophils show impaired intracellular bacterial killing. Using NOD2/RIP2â»/â» mice, we observed bacterial burden and neutrophil accumulation in the lungs similar to those seen with NOD2â»/â» mice. In addition, bone marrow-derived macrophages obtained from NOD2/RIP2â»/â» mice demonstrate a reduction in activation of NF-κB and MAPKs similar to that seen with NOD2â»/â» mice in response to E. coli. These findings unveil a previously unrecognized role of the NOD2-RIP2 axis for host defense against extracellular Gram-negative bacteria. This pathway may represent a novel target for the treatment of lung infection/inflammation.
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Infecciones por Escherichia coli/inmunología , Pulmón/inmunología , Proteína Adaptadora de Señalización NOD2/inmunología , Neumonía Bacteriana/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transducción de Señal , Animales , Carga Bacteriana , Citocinas/metabolismo , Femenino , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Neutrófilos/microbiología , Proteína Adaptadora de Señalización NOD2/deficiencia , Neumonía Bacteriana/microbiología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficienciaRESUMEN
The tumor-intrinsic NOD-, LRR- and pyrin domain-containing protein-3 (NLRP3) inflammasome-heat shock protein 70 (HSP70) signaling axis is triggered by CD8+ T cell cytotoxicity and contributes to the development of adaptive resistance to anti-programmed cell death protein 1 (PD-1) immunotherapy by recruiting granulocytic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into the tumor microenvironment. Here, we demonstrate that the tumor NLRP3-HSP70 axis also drives the accumulation of PMN-MDSCs into distant lung tissues in a manner that depends on lung epithelial cell Toll-like receptor 4 (TLR4) signaling, establishing a premetastatic niche that supports disease hyperprogression in response to anti-PD-1 immunotherapy. Lung epithelial HSP70-TLR4 signaling induces the downstream Wnt5a-dependent release of granulocyte colony-stimulating factor (G-CSF) and C-X-C motif chemokine ligand 5 (CXCL5), thus promoting myeloid granulopoiesis and recruitment of PMN-MDSCs into pulmonary tissues. Treatment with anti-PD-1 immunotherapy enhanced the activation of this pathway through immunologic pressure and drove disease progression in the setting of Nlrp3 amplification. Genetic and pharmacologic inhibition of NLRP3 and HSP70 blocked PMN-MDSC accumulation in the lung in response to anti-PD-1 therapy and suppressed metastatic progression in preclinical models of melanoma and breast cancer. Elevated baseline concentrations of plasma HSP70 and evidence of NLRP3 signaling activity in tumor tissue specimens correlated with the development of disease hyperprogression and inferior survival in patients with stage IV melanoma undergoing anti-PD-1 immunotherapy. Together, this work describes a pathogenic mechanism underlying the phenomenon of disease hyperprogression in melanoma and offers candidate targets and markers capable of improving the management of patients with melanoma.
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Melanoma , Receptor Toll-Like 4 , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Inmunoterapia , Melanoma/patología , Progresión de la Enfermedad , Microambiente TumoralRESUMEN
The tumor-intrinsic NOD-like receptor family, pyrin-domain-containing-3 (NLRP3) inflammasome, plays an important role in regulating immunosuppressive myeloid cell populations in the tumor microenvironment (TME). While prior studies have described the activation of this inflammasome in driving pro-tumorigenic mechanisms, emerging data is now revealing the tumor NLRP3 inflammasome and the downstream release of heat shock protein-70 (HSP70) to regulate anti-tumor immunity and contribute to the development of adaptive resistance to anti-PD-1 immunotherapy. Genetic alterations that influence the activity of the NLRP3 signaling axis are likely to impact T cell-mediated tumor cell killing and may indicate which tumors rely on this pathway for immune escape. These studies suggest that the NLRP3 inflammasome and its secreted product, HSP70, represent promising pharmacologic targets for manipulating innate immune cell populations in the TME while enhancing responses to anti-PD-1 immunotherapy. Additional studies are needed to better understand tumor-specific regulatory mechanisms of NLRP3 to enable the development of tumor-selective pharmacologic strategies capable of augmenting responses to checkpoint inhibitor immunotherapy while minimizing unwanted off-target effects. The execution of upcoming clinical trials investigating this strategy to overcome anti-PD-1 resistance promises to provide novel insight into the role of this pathway in immuno-oncology.
RESUMEN
Although anti-PD-1 therapy has improved clinical outcomes for select patients with advanced cancer, many patients exhibit either primary or adaptive resistance to checkpoint inhibitor immunotherapy. The role of the tumor stroma in the development of these mechanisms of resistance to checkpoint inhibitors remains unclear. We demonstrated that pharmacologic inhibition of the TGFß signaling pathway synergistically enhanced the efficacy of anti-CTLA-4 immunotherapy but failed to augment anti-PD-1/PD-L1 responses in an autochthonous model of BRAFV600E melanoma. Additional mechanistic studies revealed that TGFß pathway inhibition promoted the proliferative expansion of stromal fibroblasts, thereby facilitating MMP-9-dependent cleavage of PD-L1 surface expression, leading to anti-PD-1 resistance in this model. Further work demonstrated that melanomas escaping anti-PD-1 therapy exhibited a mesenchymal phenotype associated with enhanced TGFß signaling activity. Delayed TGFß inhibitor therapy, following anti-PD-1 escape, better served to control further disease progression and was superior to a continuous combination of anti-PD-1 and TGFß inhibition. This work illustrates that formulating immunotherapy combination regimens to enhance the efficacy of checkpoint blockade requires an in-depth understanding of the impact of these agents on the tumor microenvironment. These data indicated that stromal fibroblast MMP-9 may desensitize tumors to anti-PD-1 and suggests that TGFß inhibition may generate greater immunologic efficacy when administered following the development of acquired anti-PD-1 resistance.See related Spotlight on p. 1444.
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Resistencia a Antineoplásicos/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Resistencia a Antineoplásicos/fisiología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Inmunoterapia/métodos , Masculino , Metaloproteinasa 9 de la Matriz/inmunología , Melanoma/metabolismo , Melanoma/patología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas B-raf/genética , Pirazoles/farmacología , Quinolinas/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.
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Modelos Animales de Enfermedad , Infecciones por Haemophilus , Haemophilus influenzae , Neumonía/microbiología , Animales , RatonesRESUMEN
Deficiency in the E3 ubiquitin ligase Itch causes a skin-scratching phenotype in mice. We found that there was increased phosphorylation and activation of the mitogen-activated protein kinase p38α in spontaneous and experimentally induced skin lesions of Itch-deficient (Itch-/-) mice. Itch bound directly to the TGF-ß-activated kinase 1-binding protein 1 (Tab1) through a conserved PPXY motif and inhibited the activation of p38α. Knockdown of Tab1 by short hairpin RNA attenuated the prolonged p38α phosphorylation exhibited by Itch-/- cells. Similarly, reconstitution of Itch-/- cells with wild-type Itch, but not the ligase-deficient Itch-C830A mutant, inhibited the phosphorylation and activation of p38α. Compared to the skin of wild-type mice, the skin of Itch-/- mice contained increased amounts of the mRNAs of proinflammatory cytokines, including tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-1ß, IL-11, and IL-19. Inhibition of p38 or blocking the interaction between p38α and Tab1 with a cell-permeable peptide substantially attenuated skin inflammation in Itch-/- mice. These findings provide insight into how Itch-mediated regulatory mechanisms prevent chronic skin inflammation, which could be exploited therapeutically.