RESUMO
While conventional chimeric antigen-receptor (CAR)-T therapies have shown remarkable clinical activity in some settings, they can induce severe toxicities and are rarely curative. To address these challenges, we developed a controllable cell therapy where synthetic D-domain-containing proteins (soluble protein antigen-receptor X-linker [SparX]) bind one or more tumor antigens and mark those cells for elimination by genetically modified T cells (antigen-receptor complex [ARC]-T). The chimeric antigen receptor was engineered with a D-domain that specifically binds to the SparX protein via a unique TAG, derived from human alpha-fetoprotein. The interaction is mediated through an epitope on the TAG that is occluded in the native alpha-fetoprotein molecule. In vitro and in vivo data demonstrate that the activation and cytolytic activity of ARC-T cells is dependent on the dose of SparX protein and only occurs when ARC-T cells are engaged with SparX proteins bound to antigen-positive cells. ARC-T cell specificity was also redirected in vivo by changing SparX proteins that recognized different tumor antigens to combat inherent or acquired tumor heterogeneity. The ARC-SparX platform is designed to expand patient and physician access to cell therapy by controlling potential toxicities through SparX dosing regimens and enhancing tumor elimination through sequential or simultaneous administration of SparX proteins engineered to bind different tumor antigens.
Assuntos
Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Animais , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Imunoterapia Adotiva/métodos , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Neoplasias/terapia , Neoplasias/imunologia , Neoplasias/metabolismo , Ligação ProteicaRESUMO
Chimeric antigen receptor (CAR) T cell therapies have demonstrated impressive initial response rates in hematologic malignancies. However, relapse rates are significant, and robust efficacies in other indications, such as solid tumors, will likely require novel therapeutic strategies and CAR designs. To that end, we sought to develop simple, highly selective targeting domains (D domains) that could be incorporated into complex, multifunctional therapeutics. Herein, we describe the identification and characterization of D domains specific for CD123, a therapeutic target for hematologic malignancies, including acute myelogenous leukemia (AML). CARs comprised of these D domains mediate potent T cell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We describe a strategy of engineering less immunogenic D domains through the identification and removal of putative T cell epitopes and investigate the binding kinetics and affinity requirements of the resultant D domain CARs. Finally, we extended the utility of D domains by generating functional, bi-specific CARs comprised of a CD123-specific D domain and a CD19-specific scFv. The properties of D domains suggest that this class of targeting domain may facilitate the development of multi-functional CARs where conventional, scFv-based designs may be suboptimal.
Assuntos
Antineoplásicos/uso terapêutico , Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Domínios Proteicos/imunologia , Proteínas/uso terapêutico , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T/imunologia , Animais , Antígenos CD19/imunologia , Antineoplásicos/imunologia , Epitopos de Linfócito T/imunologia , Células HEK293 , Humanos , Células K562 , Leucemia Mieloide Aguda/terapia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ligação Proteica/imunologia , Proteínas/imunologia , Receptores de Antígenos Quiméricos/imunologia , Anticorpos de Cadeia Única/imunologia , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Treg cells can secrete latent TGF-ß1 (LTGF-ß1), but can also utilize an alternative pathway for transport and expression of LTGF-ß1 on the cell surface in which LTGF-ß1 is coupled to a distinct LTGF-ß binding protein termed glycoprotein A repetitions predominant (GARP)/LRRC32. The function of the GARP/LTGF-ß1 complex has remained elusive. Here, we examine in vivo the roles of GARP and TGF-ß1 in the induction of oral tolerance. When Foxp3(-) OT-II T cells were transferred to wild-type recipient mice followed by OVA feeding, the conversion of Foxp3(-) to Foxp3(+) OT-II cells was dependent on recipient Treg cells. Neutralization of IL-2 in the recipient mice also abrogated this conversion. The GARP/LTGF-ß1 complex on recipient Treg cells, but not dendritic cell-derived TGF-ß1, was required for efficient induction of Foxp3(+) T cells and for the suppression of delayed hypersensitivity. Expression of the integrin αvß8 by Treg cells (or T cells) in the recipients was dispensable for induction of Foxp3 expression. Transient depletion of the bacterial flora enhanced the development of oral tolerance by expanding Treg cells with enhanced expression of the GARP/LTGF-ß1 complex.
Assuntos
Tolerância Imunológica , Imunomodulação , Proteínas de Membrana/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Antígenos/imunologia , Biomarcadores , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Microbioma Gastrointestinal/imunologia , Expressão Gênica , Hipersensibilidade Tardia/genética , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/metabolismo , Tolerância Imunológica/genética , Imunofenotipagem , Integrinas/genética , Integrinas/metabolismo , Interleucina-2/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fenótipo , Ligação Proteica , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Activated T regulatory cells (Tregs) express latent TGF-ß1 on their cell surface bound to GARP. Although integrins have been implicated in mediating the release of active TGF-ß1 from the complex of latent TGF-ß1 and latent TGF-ß1 binding protein, their role in processing latent TGF-ß1 from the latent TGF-ß1/GARP complex is unclear. Mouse CD4(+)Foxp3(+) Treg, but not CD4(+)Foxp3(-) T cells, expressed integrin ß8 (Itgb8) as detected by quantitative RT-PCR. Itgb8 expression was a marker of thymically derived (t)Treg, because it could not be detected on Foxp3(+)Helios(-) Tregs or on Foxp3(+) T cells induced in vitro. Tregs from Itgb8 conditional knockouts exhibited normal suppressor function in vitro and in vivo in a model of colitis but failed to provide TGF-ß1 to drive Th17 or induced Treg differentiation in vitro. In addition, Itgb8 knockout Tregs expressed higher levels of latent TGF-ß1 on their cell surface consistent with defective processing. Thus, integrin αvß8 is a marker of tTregs and functions in a cell intrinsic manner in mediating the processing of latent TGF-ß1 from the latent TGF-ß1/GARP complex on the surface of tTregs.
Assuntos
Cadeias beta de Integrinas/imunologia , Integrinas/imunologia , Proteínas de Membrana/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Biomarcadores , Diferenciação Celular/imunologia , Colite/imunologia , Proteínas de Ligação a DNA/biossíntese , Fatores de Transcrição Forkhead/biossíntese , Proteínas de Fluorescência Verde/genética , Cadeias beta de Integrinas/biossíntese , Cadeias beta de Integrinas/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th17/imunologia , Fatores de Transcrição/biossíntese , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/imunologiaRESUMO
GARP/LRRC32 was defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-ß1. We find that GARP and latent TGF-ß1 are also found on mouse Tregs activated via TCR stimulation; however, in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus, and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-ß1 and TGF-ß1 loading into GARP and is independent of furin-mediated processing of pro-TGF-ß1 to latent TGF-ß1. Specific deletion of GARP in CD4(+) T cells results in lack of expression of latent TGF-ß1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of conventional T cells in vitro. Activated Tregs expressing GARP/latent TGF-ß1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17-producing cells and Tregs is caused preferentially by Tregs expressing the latent TGF-ß1/GARP complex on their cell surface rather than by secreted latent TGF-ß1.
Assuntos
Diferenciação Celular , Proteínas de Ligação a TGF-beta Latente/metabolismo , Proteínas de Membrana/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Fatores de Transcrição Forkhead/metabolismo , Furina/metabolismo , Regulação da Expressão Gênica , Proteínas de Ligação a TGF-beta Latente/genética , Proteínas de Membrana/genética , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Timo/imunologia , Timo/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Chimeric antigen receptor (CAR) T-cell therapies directed against B-cell maturation antigen (BCMA) have shown compelling clinical activity and manageable safety in subjects with relapsed and refractory multiple myeloma (RRMM). Prior reported CAR T cells have mostly used antibody fragments such as humanized or murine single-chain variable fragments or camelid heavy-chain antibody fragments as the antigen recognition motif. Herein, we describe the generation and preclinical evaluation of ddBCMA CAR, which uses a novel BCMA binding domain discovered from our D domain phage display libraries and incorporates a 4-1BB costimulatory motif and CD3-zeta T-cell activation domain. Preclinical in vitro studies of ddBCMA CAR T cells cocultured with BCMA-positive cell lines showed highly potent, dose-dependent measures of cytotoxicity, cytokine production, T-cell degranulation, and T-cell proliferation. In each assay, ddBCMA CAR performed as well as the BCMA-directed scFv-based C11D5.3 CAR. Furthermore, ddBCMA CAR T cells demonstrated in vivo tumor suppression in three disseminated BCMA-expressing tumor models in NSG-immunocompromised mice. On the basis of these promising preclinical data, CART-ddBCMA is being studied in a first-in-human phase I clinical study to assess the safety, pharmacokinetics, immunogenicity, efficacy, and duration of effect for patients with RRMM (NCT04155749).
Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Anticorpos de Cadeia Única , Animais , Antígeno de Maturação de Linfócitos B/metabolismo , Humanos , Imunoterapia Adotiva , Camundongos , Mieloma Múltiplo/patologia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/genética , Linfócitos TRESUMO
We previously described a population of regulatory macrophages that produced high levels of IL-10 and low levels of IL-12/23. We now describe and characterize the expression of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) by these macrophages. HB-EGF has previously been associated with a number of physiological and pathological conditions, including tumor growth and angiogenesis. The induction of HB-EGF in regulatory macrophages is due to new transcription and not to increased mRNA stability. The transcription factor Sp1 is a major factor in HB-EGF production, and knockdown of Sp1 substantially diminishes HB-EGF production. Sp1 was recruited to three sites within the first 2 kb of the HB-EGF promoter following stimulation, and the site located at -83/-54 was required for HB-EGF promoter activity. These regions of the promoter become more accessible to endonuclease activity following macrophage activation, and this accessibility was contingent on activation of the MAPK, ERK. We show that several experimental manipulations that give rise to regulatory macrophages also result in HB-EGF production. These observations indicate that in addition to the secretion of the anti-inflammatory cytokine IL-10, another novel characteristic of regulatory macrophages is the production of angiogenic HB-EGF.
Assuntos
Regulação da Expressão Gênica/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Macrófagos/imunologia , Animais , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/imunologia , Fator de Transcrição Sp1/metabolismo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Over the past three decades many techniques for expressing exogenous genes in a variety of cells and cell lines have been developed. Exogenous gene expression in macrophages has lagged behind that of other nonhematopioetic cells. There are many reasons for this, but most are due to technical difficulties associated with transfecting macrophages. As professional phagocytes, macrophages are endowed with many potent degradative enzymes that can disrupt nucleic acid integrity and make gene transfer into these cells an inefficient process. This is especially true of activated macrophages which undergo a dramatic change in their physiology following exposure to immune or inflammatory stimuli. Viral transduction of these cells has been hampered because macrophages are end-stage cells that generally do not divide; therefore, some of the vectors that depend on integration into a replicative genome have met with limited success. Furthermore, macrophages are quite responsive to "danger signals," and therefore several of the original viral vectors that were used for gene transfer induced potent anti-viral responses in these cells making these vectors inappropriate for gene delivery. Many of these difficulties have been largely overcome, and relatively high efficiency gene expression in primary human or murine macrophages is becoming more routine. In the present chapter we discuss some of the gene expression techniques that have met with success and review the advantages and disadvantages of each.
Assuntos
Técnicas Genéticas , Macrófagos/metabolismo , Adenoviridae/genética , Animais , Células da Medula Óssea/enzimologia , Núcleo Celular/genética , Eletroporação , Regulação Enzimológica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Lentivirus/genética , Macrófagos/enzimologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retroviridae/genética , Transdução Genética , TransfecçãoRESUMO
We generated three populations of macrophages (Mphi) in vitro and characterized each. Classically activated Mphi (Ca-Mphi) were primed with IFN-gamma and stimulated with LPS. Type II-activated Mphi (Mphi-II) were similarly primed but stimulated with LPS plus immune complexes. Alternatively activated Mphi (AA-Mphi) were primed overnight with IL-4. Here, we present a side-by-side comparison of the three cell types. We focus primarily on differences between Mphi-II and AA-Mphi, as both have been classified as M2 Mphi, distinct from Ca-Mphi. We show that Mphi-II more closely resemble Ca-Mphi than they are to AA-Mphi. Mphi-II and Ca-Mphi, but not AA-Mphi, produce high levels of NO and have low arginase activity. AA-Mphi express FIZZ1, whereas neither Mphi-II nor Ca-Mphi do. Mphi-II and Ca-Mphi express relatively high levels of CD86, whereas AA-Mphi are virtually devoid of this costimulatory molecule. Ca-Mphi and Mphi-II are efficient APC, whereas AA-Mphi fail to stimulate efficient T cell proliferation. The differences between Ca-Mphi and Mphi-II are more subtle. Ca-Mphi produce IL-12 and give rise to Th1 cells, whereas Mphi-II produce high levels of IL-10 and thus, give rise to Th2 cells secreting IL-4 and IL-10. Mphi-II express two markers that may be used to identify them in tissue. These are sphingosine kinase-1 and LIGHT (TNF superfamily 14). Thus, Ca-Mphi, Mphi-II, and AA-Mphi represent three populations of cells with different biological functions.
Assuntos
Citocinas/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Animais , Arginase/biossíntese , Arginase/imunologia , Antígeno B7-2/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Citocinas/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/classificação , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Fator de Crescimento Neural/biossíntese , Fator de Crescimento Neural/imunologia , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Proteínas/imunologia , Células Th1/imunologia , Células Th2/imunologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/biossíntese , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologiaRESUMO
The tumor microenvironment (TME) is established and maintained through complex interactions between tumor cells and host stromal elements. Therefore, therapies that target multiple cellular components of the tumor may be most effective. Sorafenib, a multi-kinase inhibitor, alters signaling pathways in both tumor cells and host stromal cells. Thus, we explored the potential immune-modulating effects of sorafenib in a murine HER-2-(neu) overexpressing breast tumor model alone and in combination with a HER-2 targeted granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting vaccine (3T3neuGM). In vitro, sorafenib inhibited the growth of HER-2 overexpressing NT2.5 tumor cells, inducing apoptosis. Sorafenib also interfered with ERK MAPK, p38 MAPK, and STAT3 signaling, as well as cyclin D expression, but did not affect HER-2 or AKT signaling. In vivo, single agent sorafenib disrupted the tumor-associated vasculature and induced tumor cell apoptosis, effectively inducing the regression of established NT2.5 tumors in immune competent FVB/N mice. Immune depletion studies demonstrated that both CD4+ and CD8+ T cells were required for tumor regression. Sorafenib treatment did not impact the rate of tumor clearance induced by vaccination with 3T3neuGM in tumor-bearing FVB/N mice relative to either sorafenib treatment or vaccination alone. In vivo studies further demonstrated that sorafenib enhanced the accumulation of both CD4+ and CD8+ T cells into the TME of vaccinated mice. Together, these findings suggest that GM-CSF-secreting cellular immunotherapy may be integrated with sorafenib without impairing vaccine-based immune responses.
Assuntos
Neoplasias da Mama/terapia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/transplante , Vacinas Anticâncer/imunologia , Imunoterapia Adotiva/métodos , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Receptor ErbB-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Terapia Combinada , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Humanos , Imunidade Celular , Camundongos , Camundongos Endogâmicos , Neoplasias Experimentais , Niacinamida/uso terapêutico , Receptor ErbB-2/imunologia , Transdução de Sinais/efeitos dos fármacos , Sorafenibe , Carga Tumoral , Microambiente TumoralRESUMO
Classical activating stimuli like LPS drive macrophages to secrete a battery of inflammatory cytokines, including interleukin (IL)-12/23, through Toll-like receptor (TLR) signaling. TLR activation in the presence of some factors, including prostaglandin E2 (PGE2), promotes an anti-inflammatory cytokine profile, with production of IL-10 and suppression of IL-12/23 secretion. Extracellular signal-regulated kinase (ERK) is a key regulator of macrophage IL-10 production. Since it inhibits ERK, we investigated the impact of Sorafenib on the cytokine profile of macrophages. In the presence of PGE2, Sorafenib restored the secretion of IL-12 and suppressed IL-10 production. Moreover, IL-12 secretion was enhanced by Sorafenib under conditions of TLR ligation alone. Furthermore, the impact of tumor culture supernatants, cholera toxin, and cAMP analogs (which suppress IL-12 secretion), was reversed by Sorafenib. Sorafenib inhibited the activation of the MAP kinase p38 and its downstream target mitogen and stress activated protein kinase (MSK), and partially inhibited protein kinase B (AKT) and its subsequent inactivation of the downstream target glycogen synthase kinase 3-ß (GSK3-ß). Interference with these pathways, which are pivotal in determining the balance of inflammatory versus anti-inflammatory cytokines, provides a potential mechanism by which Sorafenib can modulate the macrophage cytokine phenotype. These data raise the possibility that the use of Sorafenib as cancer therapy could potentially reverse the immunosuppressive cytokine profile of tumor-associated macrophages, rendering the tumor microenvironment more conducive to an anti-tumor immune response.
Assuntos
Benzenossulfonatos/farmacologia , Dinoprostona/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Piridinas/farmacologia , Animais , Linhagem Celular , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Interleucina-10/genética , Interleucina-12/genética , Camundongos , Niacinamida/análogos & derivados , Compostos de Fenilureia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Sorafenibe , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Macrophages display remarkable plasticity and can change their physiology in response to environmental cues. These changes can give rise to different populations of cells with distinct functions. In this Review we suggest a new grouping of macrophage populations based on three different homeostatic activities - host defence, wound healing and immune regulation. We propose that similarly to primary colours, these three basic macrophage populations can blend into various other 'shades' of activation. We characterize each population and provide examples of macrophages from specific disease states that have the characteristics of one or more of these populations.
Assuntos
Ativação de Macrófagos/fisiologia , Macrófagos/fisiologia , Animais , Citocinas/imunologia , Citocinas/fisiologia , Humanos , Imunidade Inata/imunologia , Imunidade Inata/fisiologia , Inflamação/imunologia , Cicatrização/imunologia , Cicatrização/fisiologiaRESUMO
NF-kappaB/Rel is a family of transcription factors whose activation has long been linked to the production of inflammatory cytokines. Here, we studied NF-kappaB signaling in the regulation of the anti-inflammatory cytokine, interleukin-10 (IL-10). We identified a role for a single NF-kappaB family member, NF-kappaB1 (p50), in promoting the transcription of IL-10. The NF-kappaB ciselement on IL-10 proximal promoter was located to -55/-46, where p50 can homodimerize and form a complex with the transcriptional co-activator CREB-binding protein to activate transcription. The other Rel family members appear to play a negligible role in IL-10 transcription. Mice lacking p50 were more susceptible to lethal endotoxemia, and macrophages taken from p50-/- mice exhibit skewed cytokine responses to lipopolysaccharide, characterized by decreased IL-10 and increased tumor necrosis factor and IL-12. Taken together, our studies demonstrate that NF-kappaB1 (p50) homodimers can be transcriptional activators of IL-10. The reciprocal regulation of pro- and anti-inflammatory cytokine production by NF-kappaB1 (p50) may provide potential new ways to manipulate the innate immune response.
Assuntos
Regulação da Expressão Gênica , Interleucina-10/metabolismo , Macrófagos/imunologia , Subunidade p50 de NF-kappa B/química , Subunidade p50 de NF-kappa B/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais/fisiologia , Animais , Sítios de Ligação , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Dimerização , Genes Reporter , Interleucina-10/genética , Lipopolissacarídeos/imunologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Subunidade p50 de NF-kappa B/genética , Regiões Promotoras Genéticas , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transcrição GênicaRESUMO
To gain insight into the molecular mechanism(s) whereby macrophages produce large amounts of IL-10, we analyzed IL-10 gene expression and temporally correlated it with modifications to chromatin associated with the IL-10 promoter. In resting cells, which make essentially no cytokines, the IL-10 promoter is associated with histones containing little or no detectable modifications. Macrophages stimulated in the presence of immune complexes begin to produce high levels of IL-10 pre-mRNA transcripts within minutes of stimulation. Coincident with this transcription was a rapid and dynamic phosphorylation of histone H3 at specific sites in the IL-10 promoter. Histone phosphorylation was closely followed by the binding of transcription factors to the IL-10 promoter. Blocking the activation of ERK prevented histone phosphorylation and transcription factor binding to the IL-10 promoter. In contrast to histone phosphorylation, the peak of histone acetylation at this promoter did not occur until after transcription had peaked. Inhibition of histone deactylase did not alter IL-10 gene expression, suggesting that phosphorylation but not acetylation was the proximal event responsible for IL-10 transcription. Our findings reveal a rapid and well-orchestrated series of events in which ERK activation causes a rapid and transient phosphorylation of histone H3 at specific regions of the IL-10 promoter, resulting in a transient exposure of the IL-10 promoter to the transcription factors that bind there. This exposure is essential for the efficient induction of IL-10 gene expression in macrophages. To our knowledge, this represents a unique way in which the expression of a cytokine gene is regulated in macrophages.