RESUMO
Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.
Assuntos
Anticorpos Neutralizantes , Receptor 1 de Quimiocina CX3C , Testes de Neutralização , Organoides , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Humanos , Vírus Sincicial Respiratório Humano/imunologia , Anticorpos Neutralizantes/imunologia , Organoides/metabolismo , Organoides/imunologia , Organoides/virologia , Organoides/citologia , Animais , Testes de Neutralização/métodos , Chlorocebus aethiops , Células Vero , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/imunologia , Anticorpos Antivirais/imunologia , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/metabolismo , Lactente , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/virologia , Anticorpos Monoclonais/imunologiaRESUMO
Peripheral monocyte-derived CX3C chemokine receptor 1 positive (CX3CR1+) cells play important roles in tissue homeostasis and gut repopulation. Increasing evidence also supports their role in immune repopulation of the brain parenchyma in response to systemic inflammation. Adoptive bone marrow transfer from CX3CR1 fluorescence reporter mice and high-resolution confocal microscopy was used to assess the time course of CX3CR1+ cell repopulation of steady-state and dextran sodium sulfate (DSS)-inflamed small intestine/colon and the brain over 4 weeks after irradiation. CX3CR1+ cell colonization and morphologic polarization into fully ramified cells occurred more rapidly in the small intestine than in the colon. For both organs, the crypt/mucosa was more densely populated than the serosa/muscularis layer, indicating preferential temporal and spatial occupancy. Repopulation of the brain was delayed compared with that of gut tissue, consistent with the immune privilege of this organ. However, DSS-induced colon injury accelerated the repopulation. Expression analyses confirmed increased chemokine levels and macrophage colonization within the small intestine/colon and the brain by DSS-induced injury. Early increases of transmembrane protein 119 and ionized calcium binding adaptor molecule 1 expression within the brain after colon injury suggest immune-priming effect of brain resident microglia in response to systemic inflammation. These findings identify temporal differences in immune repopulation of the gut and brain in response to inflammation and show that gut inflammation can impact immune responses within the brain.
Assuntos
Lesões Encefálicas/imunologia , Encéfalo/imunologia , Receptor 1 de Quimiocina CX3C/imunologia , Colite/imunologia , Mucosa Intestinal/imunologia , Monócitos/imunologia , Lesões Experimentais por Radiação/metabolismo , Animais , Encéfalo/patologia , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Receptor 1 de Quimiocina CX3C/genética , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Sulfato de Dextrana/toxicidade , Mucosa Intestinal/fisiologia , Camundongos , Camundongos Transgênicos , Monócitos/patologia , Lesões Experimentais por Radiação/genética , Lesões Experimentais por Radiação/patologiaRESUMO
T cells undergo rigorous selection in the thymus to ensure self-tolerance and prevent autoimmunity, with this process requiring innocuous self-antigens (Ags) to be presented to thymocytes. Self-Ags are either expressed by thymic stroma cells or transported to the thymus from the periphery by migratory dendritic cells (DCs); meanwhile, small blood-borne peptides can access the thymic parenchyma by diffusing across the vascular lining. Here we describe an additional pathway of thymic Ag acquisition that enables circulating antigenic macromolecules to access both murine and human thymi. This pathway depends on a subset of thymus-resident DCs, distinct from both parenchymal and circulating migratory DCs, that are positioned in immediate proximity to thymic microvessels where they extend cellular processes across the endothelial barrier into the blood stream. Transendothelial positioning of DCs depends on DC-expressed CX3CR1 and its endothelial ligand, CX3CL1, and disrupting this chemokine pathway prevents thymic acquisition of circulating proteins and compromises negative selection of Ag-reactive thymocytes. Thus, transendothelial DCs represent a mechanism by which the thymus can actively acquire blood-borne Ags to induce and maintain central tolerance.
Assuntos
Sangue/imunologia , Células Dendríticas/imunologia , Células Endoteliais/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Autoantígenos/imunologia , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/imunologia , Diferenciação Celular , Movimento Celular , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/imunologia , Células Dendríticas/citologia , Células Endoteliais/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Tolerância a Antígenos Próprios , Timócitos/citologia , Timo/citologiaRESUMO
Neuroinflammation has been put forward as a mechanism triggering axonal regrowth in the mammalian central nervous system (CNS), yet little is known about the underlying cellular and molecular players connecting these two processes. In this study, we provide evidence that MMP2 is an essential factor linking inflammation to axonal regeneration by using an in vivo mouse model of inflammation-induced axonal regeneration in the optic nerve. We show that infiltrating myeloid cells abundantly express MMP2 and that MMP2 deficiency results in reduced long-distance axonal regeneration. However, this phenotype can be rescued by restoring MMP2 expression in myeloid cells via a heterologous bone marrow transplantation. Furthermore, while MMP2 deficiency does not affect the number of infiltrating myeloid cells, it does determine the coordinated expression of pro- and anti-inflammatory molecules. Altogether, in addition to its role in axonal regeneration via resolution of the glial scar, here, we reveal a new mechanism via which MMP2 facilitates axonal regeneration, namely orchestrating the expression of pro- and anti-inflammatory molecules by infiltrating innate immune cells.
Assuntos
Axônios/imunologia , Transplante de Medula Óssea , Metaloproteinase 2 da Matriz/genética , Regeneração Nervosa/imunologia , Traumatismos do Nervo Óptico/imunologia , Nervo Óptico/imunologia , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Axônios/ultraestrutura , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/imunologia , Movimento Celular , Proteína GAP-43/genética , Proteína GAP-43/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Inflamação , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Metaloproteinase 2 da Matriz/deficiência , Metaloproteinase 2 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/citologia , Células Mieloides/imunologia , Regeneração Nervosa/genética , Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/patologia , Retina/imunologia , Retina/lesões , Retina/metabolismo , Transplante Heterólogo , Irradiação Corporal TotalRESUMO
Increasing evidence has demonstrated that oxidized low-density lipoproteins (oxLDL) and lipopolysaccharide (LPS) enhance accumulation of interleukin (IL)-1 beta-producing macrophages in atherosclerotic lesions. However, the potential synergistic effect of native LDL (nLDL) and LPS on the inflammatory ability and migration pattern of monocyte subpopulations remains elusive and is examined here. In vitro, whole blood cells from healthy donors (n = 20) were incubated with 100 µg/mL nLDL, 10 ng/mL LPS, or nLDL + LPS for 9 h. Flow cytometry assays revealed that nLDL significantly decreases the classical monocyte (CM) percentage and increases the non-classical monocyte (NCM) subset. While nLDL + LPS significantly increased the number of NCMs expressing IL-1 beta and the C-C chemokine receptor type 2 (CCR2), the amount of NCMs expressing the CX3C chemokine receptor 1 (CX3CR1) decreased. In vivo, patients (n = 85) with serum LDL-cholesterol (LDL-C) >100 mg/dL showed an increase in NCM, IL-1 beta, LPS-binding protein (LBP), and Castelli's atherogenic risk index as compared to controls (n = 65) with optimal LDL-C concentrations (≤100 mg/dL). This work demonstrates for the first time that nLDL acts in synergy with LPS to alter the balance of human monocyte subsets and their ability to produce inflammatory cytokines and chemokine receptors with prominent roles in atherogenesis.
Assuntos
Receptor 1 de Quimiocina CX3C/genética , LDL-Colesterol/farmacologia , Interleucina-1beta/genética , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Receptores CCR2/genética , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/imunologia , Adolescente , Adulto , Proteína C-Reativa/genética , Proteína C-Reativa/imunologia , Receptor 1 de Quimiocina CX3C/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/imunologia , HDL-Colesterol/sangue , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Expressão Gênica , Voluntários Saudáveis , Humanos , Interleucina-1beta/imunologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Pessoa de Meia-Idade , Monócitos/citologia , Monócitos/imunologia , Cultura Primária de Células , Receptores CCR2/imunologia , Triglicerídeos/sangueRESUMO
Attraction, retention, and differentiation of leukocytes to and within the kidney are governed by chemokines. The chemokine CX3CL1 (fractalkine) and its receptor CX3CR1 are exemplary in this regard as they are highly expressed and further upregulated in a range of kidney diseases. CX3CL1 is chiefly produced by renal endothelium and tubular epithelium, where it promotes leukocyte attraction. Recent data suggest that in addition to established soluble mediators, cellular interactions may enhance CX3CL1 expression. The receptor CX3CR1 is essential in myeloid phagocyte homing to the kidney at homeostasis, after acute cell depletion and in inflammation. CX3CR1 and its ligand are highly regulated in human kidney diseases such as IgA nephritis, systemic lupus erythematosus, and inflammatory conditions such as transplant rejection. A mechanistic role of CX3CR1 has been established in experimental models of nephrotoxic nephritis and renal candidiasis. It is debated in fibrosis. Recent publications demonstrate a role for CX3CR1+ myeloid cells in radio-contrast-agent and sepsis-induced kidney damage. Systemically, circulating CX3CR1+ monocytes reversibly increase in individuals with renal impairment and correlate with their cardiovascular risk. In this review, we discuss role and regulatory mechanisms of the CX3CL1-CX3CR1 axis in both localized and systemic effects of renal inflammation.
Assuntos
Receptor 1 de Quimiocina CX3C/imunologia , Quimiocina CX3CL1/imunologia , Nefropatias/imunologia , Rim/patologia , Animais , HumanosRESUMO
Respiratory syncytial virus (RSV) infection can cause bronchiolitis, pneumonia, morbidity, and some mortality, primarily in infants and the elderly, for which no vaccine is available. The RSV attachment (G) protein contains a central conserved domain (CCD) with a CX3C motif implicated in the induction of protective antibodies, thus vaccine candidates containing the G protein are of interest. This study determined if mutations in the G protein CCD would mediate immunogenicity while inducing G protein CX3C-CX3CR1 blocking antibodies. BALB/c mice were vaccinated with structurally-guided, rationally designed G proteins with CCD mutations. The results show that these G protein immunogens induce a substantial anti-G protein antibody response, and using serum IgG from the vaccinated mice, these antibodies are capable of blocking the RSV G protein CX3C-CX3CR1 binding while not interfering with CX3CL1, fractalkine.
Assuntos
Receptor 1 de Quimiocina CX3C/imunologia , Quimiocinas CX3C/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Antivirais/imunologia , Receptor 1 de Quimiocina CX3C/genética , Quimiocinas CX3C/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Domínios Proteicos , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/química , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/genética , Proteínas do Envelope Viral/químicaRESUMO
Efforts to improve the prognosis of steroid-resistant gut acute graft-versus-host-disease (SR-Gut-aGVHD) have suffered from poor understanding of its pathogenesis. Here we show that the pathogenesis of SR-Gut-aGVHD is associated with reduction of IFN-γ+ Th/Tc1 cells and preferential expansion of IL-17-IL-22+ Th/Tc22 cells. The IL-22 from Th/Tc22 cells causes dysbiosis in a Reg3γ-dependent manner. Transplantation of IFN-γ-deficient donor CD8+ T cells in the absence of CD4+ T cells produces a phenocopy of SR-Gut-aGVHD. IFN-γ deficiency in donor CD8+ T cells also leads to a PD-1-dependent depletion of intestinal protective CX3CR1hi mononuclear phagocytes (MNP), which also augments expansion of Tc22 cells. Supporting the dual regulation, simultaneous dysbiosis induction and depletion of CX3CR1hi MNP results in full-blown Gut-aGVHD. Our results thus provide insights into SR-Gut-aGVHD pathogenesis and suggest the potential efficacy of IL-22 antagonists and IFN-γ agonists in SR-Gut-aGVHD therapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Disbiose/imunologia , Doença Enxerto-Hospedeiro/imunologia , Interferon gama/imunologia , Interleucinas/imunologia , Fagócitos/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/imunologia , Modelos Animais de Doenças , Disbiose/genética , Disbiose/microbiologia , Disbiose/patologia , Microbioma Gastrointestinal/imunologia , Regulação da Expressão Gênica , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/microbiologia , Doença Enxerto-Hospedeiro/patologia , Interferon gama/deficiência , Interferon gama/genética , Interleucina-17/deficiência , Interleucina-17/genética , Interleucina-17/imunologia , Interleucinas/genética , Intestinos/imunologia , Intestinos/microbiologia , Intestinos/patologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas a Pancreatite/genética , Proteínas Associadas a Pancreatite/imunologia , Fagócitos/citologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais , Linfócitos T Auxiliares-Indutores , Linfócitos T Reguladores , Irradiação Corporal Total , Interleucina 22RESUMO
Human respiratory syncytial virus (RSV) is a major cause of serious respiratory tract infections in infants and the elderly. Recently it was shown that the RSV G glycoprotein mediates attachment to cells using CX3CR1 as a receptor, and that G-specific neutralizing antibodies can be detected using human airway epithelial (HAE) cell cultures. To investigate the contributions of G-specific antibodies to RSV neutralization, we performed HAE neutralization assays on sera from RSV G-immunized mice or RSV-infected infants. We confirmed that G-specific neutralization using serum from mice or humans could only be detected on HAE cultures. We also found that RSV G-specific antibodies in infants were either subgroup specific or cross-neutralizing. Altogether, our results suggest that G is an important target for generating neutralizing antibodies and would be beneficial to include in an RSV vaccine. Further, inclusion of G antigens from both RSV subgroups may enhance the vaccine cross protection potency.
Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Receptor 1 de Quimiocina CX3C/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Receptor 1 de Quimiocina CX3C/genética , Chlorocebus aethiops , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Expressão Gênica , Humanos , Soros Imunes/química , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Testes de Neutralização , Ligação Proteica , Receptores Virais/genética , Receptores Virais/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/patogenicidade , Células Vero , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/genéticaRESUMO
Acute kidney injury (AKI) to chronic kidney disease (CKD) progression has become a life-threatening disease. However, an effective therapeuticstrategyis still needed. The pathophysiology of AKI-to-CKD progression involves chronic inflammation and renal fibrosis driven by macrophage activation, which is physiologically dependent on colony-stimulating factor-1 receptor (CSF-1R) signaling. In this study, we modulated macrophage infiltration through oral administration of the CSF-1R inhibitor GW2580 in an ischemia-reperfusion (I/R)-induced AKI model to evaluate its therapeutic effects on preventing the progression of AKI to CKD. We found that GW2580 induced a significant reduction in the number of macrophages in I/R-injured kidneys and attenuated I/R-induced renal injury and subsequent interstitial fibrosis. By flow cytometry, we observed that the reduced macrophages were primarily Ly6C+ inflammatory macrophages in the GW2580-treated kidneys, while there was no significant difference in the number and percentage of Ly6C-CX3CR1+ macrophages. We further found that these reduced macrophages also demonstrated some characteristics of M2-like macrophages, which have been generally regarded as profibrotic subtypes in chronic inflammation. These results indicate the existence of phenotypic and functional crossover between Ly6C+ and M2-like macrophages in I/R kidneys, which induces AKI worsening to CKD. In conclusion, therapeutic GW2580 treatment alleviates acute renal injury and subsequent fibrosis by reducing Ly6C+ M2-like macrophage infiltration in ischemia-induced AKI.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Anisóis/farmacologia , Antígenos Ly/imunologia , Macrófagos/imunologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Traumatismo por Reperfusão/complicações , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/patologia , Animais , Anisóis/uso terapêutico , Antígenos Ly/efeitos dos fármacos , Antígenos Ly/metabolismo , Receptor 1 de Quimiocina CX3C/efeitos dos fármacos , Receptor 1 de Quimiocina CX3C/imunologia , Receptor 1 de Quimiocina CX3C/metabolismo , Modelos Animais de Doenças , Fibrose/tratamento farmacológico , Fibrose/etiologia , Fibrose/imunologia , Túbulos Renais/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/imunologiaRESUMO
In mice, monocytes (Mo) are conventionally described as CX3CR1low classical Mo (CMo) and CX3CR1high non-classical Mo (NCMo) based on the expression of EGFP in Cx3cr1+/EGFP mice and by analogy with human CX3CR1 expression. Although this terminology is widely used, it may not reflect the expression of CX3CR1 on Mo subsets. Using an unsupervised multiparametric analysis of blood Mo in steady state and after sterile peritonitis, we observed that CX3CR1 expression did not discriminate the CMo from the NCMo subsets. Our results highlight that despite being a reliable reporter to discriminate Mo subpopulations, EGFP level in Cx3cr1+/EGFP mice does not reflect CX3CR1 expression measured by a fluorescently-labeled CX3CL1 chemokine and a CX3CR1 specific antibody. In conclusion, authors should be cautious not to identify murine classical and non-classical Mo as CX3CR1low and CX3CR1high but rather use alternative markers such as the combination of Ly6C and CD43.
Assuntos
Receptor 1 de Quimiocina CX3C/imunologia , Monócitos/citologia , Monócitos/imunologia , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
HIV infection is associated with an increase in the proportion of activated CD8+ memory T cells (Tmem) that express CX3CR1, but how these cells are generated and maintained in vivo is unclear. We demonstrate that increased CX3CR1 expression on CD8+ Tmem in people living with HIV (PLWH) is dependent on coinfection with human CMV, and CX3CR1+CD8+ Tmem are enriched for a putatively immunosenescent CD57+CD28- phenotype. The cytokine IL-15 promotes the phenotype, survival, and proliferation of CX3CR1+CD57+CD8+ Tmem in vitro, whereas T cell receptor stimulation leads to their death. IL-15-driven survival is dependent on STAT5 and Bcl-2 activity, and IL-15-induced proliferation requires STAT5 and mTORC1. Thus, we identify mechanistic pathways that could explain how "inflammescent" CX3CR1+CD57+ CD8+ Tmem dominate the overall memory T cell pool in CMV-seropositive PLWH and that support reevaluation of immune senescence as a nonproliferative dead end.
Assuntos
Antígenos CD57/imunologia , Linfócitos T CD8-Positivos/imunologia , Receptor 1 de Quimiocina CX3C/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Memória Imunológica , Interleucina-15/imunologia , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Sobrevivência Celular/imunologia , Infecções por HIV/patologia , HumanosRESUMO
Although blockade of the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) immune checkpoint has revolutionized cancer treatment, how it works on tumor-infiltrating CD8+ T cells recognizing the same antigen at various differentiation stages remains elusive. Here, we found that the chemokine receptor CX3CR1 identified 3 distinct differentiation states of intratumor CD8+ T cell subsets. Adoptively transferred antigen-specific CX3CR1-CD8+ T cells generated phenotypically and functionally distinct CX3CR1int and CX3CR1hi subsets in the periphery. Notably, expression of coinhibitory receptors and T cell factor 1 (Tcf1) inversely correlated with the degree of T cell differentiation defined by CX3CR1. Despite lower expression of coinhibitory receptors and potent cytolytic activity, in vivo depletion of the CX3CR1hi subset did not alter the antitumor efficacy of adoptively transferred CD8+ T cells. Furthermore, differentiated CX3CR1int and CX3CR1hi subsets were impaired in their ability to undergo proliferation upon restimulation and had no impact on established tumors upon second adoptive transfer compared with the CX3CR1- subset that remained effective. Accordingly, anti-PD-L1 therapy preferentially rescued proliferation and cytokine production of the CX3CR1- subset and enhanced antitumor efficacy of adoptively transferred CD8+ T cells. These findings provide a better understanding of the phenotypic and functional heterogeneity of tumor-infiltrating CD8+ T cells and can be exploited to develop more effective immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Subpopulações de Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Transferência Adotiva , Animais , Receptor 1 de Quimiocina CX3C/imunologia , Humanos , CamundongosRESUMO
Intestinal mononuclear phagocytes (MPs) are composed of heterogeneous dendritic cell (DC) and macrophage subsets necessary for the initiation of immune response and control of inflammation. Although MPs in the normal intestine have been extensively studied, the heterogeneity and function of inflammatory MPs remain poorly defined. We performed phenotypical, transcriptional, and functional analyses of inflammatory MPs in infectious Salmonella colitis and identified CX3CR1+ MPs as the most prevalent inflammatory cell type. CX3CR1+ MPs were further divided into three distinct populations, namely, Nos2 +CX3CR1lo, Ccr7 +CX3CR1int (lymph migratory), and Cxcl13 +CX3CR1hi (mucosa resident), all of which were transcriptionally aligned with macrophages and derived from monocytes. In follow-up experiments in vivo, intestinal CX3CR1+ macrophages were superior to conventional DC1 (cDC1) and cDC2 in inducing Salmonella-specific mucosal IgA. We next examined spatial organization of the immune response induced by CX3CR1+ macrophage subsets and identified mucosa-resident Cxcl13 +CX3CR1hi macrophages as the antigen-presenting cells responsible for recruitment and activation of CD4+ T and B cells to the sites of Salmonella invasion, followed by tertiary lymphoid structure formation and the local pathogen-specific IgA response. Using mice we developed with a floxed Ccr7 allele, we showed that this local IgA response developed independently of migration of the Ccr7 +CX3CR1int population to the mesenteric lymph nodes and contributed to the total mucosal IgA response to infection. The differential activity of intestinal macrophage subsets in promoting mucosal IgA responses should be considered in the development of vaccines to prevent Salmonella infection and in the design of anti-inflammatory therapies aimed at modulating macrophage function in inflammatory bowel disease.
Assuntos
Receptor 1 de Quimiocina CX3C/imunologia , Imunoglobulina A/imunologia , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Estruturas Linfoides Terciárias/imunologia , Animais , Feminino , Microbioma Gastrointestinal/imunologia , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Salmonella enterica/imunologia , EstreptomicinaRESUMO
Tissue resident memory T cells (Trm) are critical for local protection against reinfection. The accumulation of T cells in the tissues requires a post-priming signal from TNFR superfamily members, referred to as signal 4. Glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18) signaling is important for this post-priming signal and for Trm formation during respiratory infection with influenza virus. As GITR signaling impacts both effector T cell accumulation and Trm formation, we asked if GITR differentially affects subsets of effector cells with different memory potential. Effector CD4+ T cells can be subdivided into 2 populations based on expression of lymphocyte antigen 6C (Ly6C), whereas effector CD8+ cells can be divided into 3 populations based on Ly6C and CX3CR1. The Ly6Chi and CX3CR1hi T cell populations represent the most differentiated effector T cells. Upon transfer, the Ly6Clo CD4+ effector T cells preferentially enter the lung parenchyma, compared to the Ly6Chi CD4+ T cells. We show that GITR had a similar effect on the accumulation of both the Ly6Chi and Ly6Clo CD4+ T cell subsets. In contrast, whereas GITR increased the accumulation of all three CD8+ T cell subsets defined by CX3CR1 and Ly6C expression, it had a more substantial effect on the least differentiated Ly6Clo CX3CR1lo subset. Moreover, GITR selectively up-regulated CXCR6 on the less differentiated CX3CR1lo CD8+ T cell subsets and induced a small but significant increase in CD127 selectively on the Ly6Clo CD4+ T cell subset. Thus, GITR contributes to accumulation of both differentiated effector cells as well as memory precursors, but with some differences between subsets.
Assuntos
Antígenos Ly/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteína Relacionada a TNFR Induzida por Glucocorticoide/genética , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Antígenos Ly/imunologia , Linfócitos T CD4-Positivos/classificação , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/classificação , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/imunologia , Diferenciação Celular , Movimento Celular , Feminino , Regulação da Expressão Gênica , Proteína Relacionada a TNFR Induzida por Glucocorticoide/deficiência , Proteína Relacionada a TNFR Induzida por Glucocorticoide/imunologia , Memória Imunológica , Imunofenotipagem , Vírus da Influenza A/crescimento & desenvolvimento , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Transdução de Sinais , Baço/imunologia , Baço/patologia , Baço/virologiaRESUMO
Postoperative ileus (POI) is triggered by an innate immune response in the muscularis externa (ME) and is accompanied by bacterial translocation. Bacteria can trigger an innate immune response via toll-like receptor (TLR) activation, but the latter's contribution to POI has been disproved for several TLRs, including TLR2 and TLR4. Herein we investigated the role of double-stranded RNA detection via TLR3 and TIR-domain-containing adapter-inducing interferon-ß (TRIF) signaling pathway in POI. POI was induced by small bowel intestinal manipulation in wt, TRIF-/-, TLR3-/-, type I interferon receptor-/- and interferon-ß reporter mice, all on C57BL/6 background, and POI severity was quantified by gene expression analysis, gastrointestinal transit and leukocyte extravasation into the ME. TRIF/TLR3 deficiency reduced postoperative ME inflammation and prevented POI. With bone marrow transplantation, RNA-sequencing, flow cytometry and immunohistochemistry we revealed a distinct TLR3-expressing radio-resistant MHCIIhiCX3CR1- IBA-1+ resident macrophage population within the deep myenteric plexus. TLR3 deficiency in these cells, but not in MHCIIhiCX3CR1+ macrophages, reduced cytokine expression in POI. While this might not be an exclusive macrophage-privileged pathway, the TLR3/TRIF axis contributes to proinflammatory cytokine production in MHCIIhiCX3CR1- IBA-1+ macrophages during POI. Deficiency in TLR3/TRIF protects mice from POI. These data suggest that TLR3 antagonism may prevent POI in humans.
Assuntos
Íleus/etiologia , Macrófagos/imunologia , Complicações Pós-Operatórias/etiologia , Receptor 3 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/imunologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Íleus/imunologia , Íleus/patologia , Imunidade Inata , Macrófagos/classificação , Macrófagos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plexo Mientérico/imunologia , Complicações Pós-Operatórias/imunologia , Complicações Pós-Operatórias/patologia , Tolerância a Radiação/imunologia , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/deficiência , Receptor 3 Toll-Like/genética , Quimeras de Transplante/imunologiaRESUMO
The transmembrane chemokine pathways CXCL16/CXCR6 and CX3CL1/CX3CR1 are strongly implicated in inflammation and angiogenesis. We investigated the involvement of these chemokine pathways and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of proliferative diabetic retinopathy (PDR). Vitreous samples from 32 PDR and 24 non-diabetic patients, epiretinal membranes from 18 patients with PDR, rat retinas, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to CXCL16-stimulated HRMECs was assessed. CXCL16, CX3CL1, ADAM10, ADAM17 and vascular endothelial growth factor (VEGF) levels were significantly increased in vitreous samples from PDR patients. The levels of CXCL16 were 417-fold higher than those of CX3CL1 in PDR vitreous samples. Significant positive correlations were found between the levels of VEGF and the levels of CXCL16, CX3CL1, ADAM10 and ADAM17. Significant positive correlations were detected between the numbers of blood vessels expressing CD31, reflecting the angiogenic activity of PDR epiretinal membranes, and the numbers of blood vessels and stromal cells expressing CXCL16, CXCR6, ADAM10 and ADAM17. CXCL16 induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB and VEGF in cultured Müller cells and tumor necrosis factor-α induced upregulation of soluble CXCL16 and ADAM17 in Müller cells. Treatment of HRMECs with CXCL16 resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and increased leukocyte adhesion to HRMECs. CXCL16 induced HRMEC proliferation, formation of sprouts from HRMEC spheroids and phosphorylation of ERK1/2. Intravitreal administration of CXCL16 in normal rats induced significant upregulation of the p65 subunit of NF-κB, VEGF and ICAM-1 in the retina. Our findings suggest that the chemokine axis CXCL16/CXCR6 and the processing metalloproteinases ADAM10 and ADAM17 might serve a role in the initiation and progression of PDR.
Assuntos
Proteína ADAM10/imunologia , Proteína ADAM17/imunologia , Secretases da Proteína Precursora do Amiloide/imunologia , Receptor 1 de Quimiocina CX3C/imunologia , Quimiocina CX3CL1/imunologia , Quimiocina CXCL16/imunologia , Retinopatia Diabética/imunologia , Proteínas de Membrana/imunologia , Animais , Retinopatia Diabética/patologia , Humanos , Masculino , RatosRESUMO
In the epididymis, prevention of autoimmune responses against spermatozoa and simultaneous protection against pathogens is important for male fertility. We have previously shown that mononuclear phagocytes (MPs) are located either in the epididymal interstitium or in close proximity to the epithelium. In the initial segments (IS), these 'intraepithelial' MPs extend slender luminal-reaching projections between epithelial cells. In this study, we performed an in-depth characterisation of MPs isolated from IS, caput-corpus and cauda epididymis of CX3CR1EGFP+/- mice that express EGFP in these cells. Flow cytometry analysis revealed region-specific subsets of MPs that express combinations of markers traditionally described in 'dendritic cells' or 'macrophages'. RNA sequencing identified distinct transcriptomic signatures in MPs from each region and revealed specific genes involved in inflammatory and anti-inflammatory responses, phagosomal activity and antigen processing and presentation. Functional fluorescent in vivo labelling assays showed that higher percentages of CX3CR1+ MPs that captured and processed antigens were detected in the IS compared to other regions. Confocal microscopy showed that in the IS, caput and corpus, circulatory antigens were internalised and processed by interstitial and intraepithelial MPs. However, in the cauda only interstitial MPs internalised and processed antigens, while intraepithelial MPs did not take up antigens, indicating that all antigens have been captured before they reached the epithelial lining. Cauda MPs may thus confer a stronger protection against blood-borne pathogens compared to proximal regions. By identifying immunoregulatory mechanisms in the epididymis, our study may lead to new therapies for male infertility and epididymitis and identify potential targets for immunocontraception.
Assuntos
Receptor 1 de Quimiocina CX3C/imunologia , Epididimo/imunologia , Fertilidade/genética , Fagócitos/imunologia , Espermatozoides/imunologia , Transcriptoma/imunologia , Animais , Apresentação de Antígeno , Antígenos CD/genética , Antígenos CD/imunologia , Autoantígenos/genética , Autoantígenos/imunologia , Receptor 1 de Quimiocina CX3C/deficiência , Receptor 1 de Quimiocina CX3C/genética , Comunicação Celular , Quimiocinas CC/genética , Quimiocinas CC/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Epididimo/citologia , Epididimo/metabolismo , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Masculino , Camundongos , Camundongos Knockout , Fagócitos/citologia , Fagócitos/metabolismo , Transporte Proteico , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Espermatozoides/citologia , Espermatozoides/metabolismoRESUMO
Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes and MHC-II and CD80 upregulation occurred upon GVHD induction. RNA sequencing-based analysis of purified microglia obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of Cx3cr1creER Tnffl/- mice reduced MHC-II expression and decreased CNS T cell infiltrates and VCAM-1+ endothelial cells. GVHD increased microglia TGF-ß-activated kinase-1 (TAK1) activation and NF-κB/p38 MAPK signaling. Selective Tak1 deletion in microglia using Cx3cr1creER Tak1fl/fl mice resulted in reduced TNF production and microglial MHC-II and improved neurocognitive activity. Pharmacological TAK1 inhibition reduced TNF production and MHC-II expression by microglia, Th1 and Th17 T cell infiltrates, and VCAM-1+ endothelial cells and improved neurocognitive activity, without blocking graft-versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF production of microglia in the CNS of GVHD patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as a mediator of CNS-GVHD, and provide a TAK1 inhibitor-based approach against GVHD-induced neurotoxicity.