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Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and the third leading cause for cancer-related death worldwide. The tumor is difficult-to-treat due to its inherent resistance to chemotherapy. Antistromal therapy is a novel therapeutic approach, targeting cancer-associated fibroblasts (CAF) in the tumor microenvironment. CAF-derived microfibrillar-associated protein 5 (MFAP-5) is identified as a novel target for antistromal therapy of HCC with high translational relevance. Biocompatible polypept(o)ide-based polyion complex micelles (PICMs) constructed with a triblock copolymer composed of a cationic poly(l-lysine) complexing anti-MFAP-5 siRNA (siMFAP-5) via electrostatic interaction, a poly(γ-benzyl-l-glutamate) block loading cationic amphiphilic drug desloratatine (DES) via π-π interaction as endosomal escape enhancer and polysarcosine poly(N-methylglycine) for introducing stealth properties, are generated for siRNA delivery. Intravenous injection of siMFAP-5/DES PICMs significantly reduces the hepatic tumor burden in a syngeneic implantation model of HCC, with a superior MFAP-5 knockdown effect over siMFAP-5 PICMs or lipid nanoparticles. Transcriptome and histological analysis reveal that MFAP-5 knockdown inhibited CAF-related tumor vascularization, suggesting the anti-angiogenic effect of RNA interference therapy. In conclusion, multicompartment PICMs combining siMFAP-5 and DES in a single polypept(o)ide micelle induce a specific knockdown of MFAP-5 and demonstrate a potent antitumor efficacy (80% reduced tumor burden vs untreated control) in a clinically relevant HCC model.
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Hyperactive FMS-like receptor tyrosine kinase-3 mutants with internal tandem duplications (FLT3-ITD) are frequent driver mutations of aggressive acute myeloid leukemia (AML). Inhibitors of FLT3 produce promising results in rationally designed cotreatment schemes. Since FLT3-ITD modulates DNA replication and DNA repair, valid anti-leukemia strategies could rely on a combined inhibition of FLT3-ITD and regulators of cell cycle progression and DNA integrity. These include the WEE1 kinase which controls cell cycle progression, nucleotide synthesis, and DNA replication origin firing. We investigated how pharmacological inhibition of FLT3 and WEE1 affected the survival and genomic integrity of AML cell lines and primary AML cells. We reveal that promising clinical grade and preclinical inhibitors of FLT3 and WEE1 synergistically trigger apoptosis in leukemic cells that express FLT3-ITD. An accumulation of single and double strand DNA damage precedes this process. Mass spectrometry-based proteomic analyses show that FLT3-ITD and WEE1 sustain the expression of the ribonucleotide reductase subunit RRM2, which provides dNTPs for DNA replication. Unlike their strong pro-apoptotic effects on leukemia cells with FLT3-ITD, inhibitors of FLT3 and WEE1 do not damage healthy human blood cells and murine hematopoietic stem cells. Thus, pharmacological inhibition of FLT3-ITD and WEE1 might become an improved, rationally designed therapeutic option.
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Apoptose , Proteínas de Ciclo Celular , Dano ao DNA , Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Proteínas Tirosina Quinases , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Animais , Pirazóis , PirimidinonasRESUMO
KH-type splicing regulatory protein (KSRP) is a single-stranded nucleic acid-binding protein with multiple functions. It is known to bind AU-rich motifs within the 3'-untranslated region of mRNA species, which in many cases encode dynamically regulated proteins like cytokines. In the present study, we investigated the role of KSRP for the immunophenotype of macrophages using bone marrow-derived macrophages (BMDM) from wild-type (WT) and KSRP-/- mice. RNA sequencing revealed that KSRP-/- BMDM displayed significantly higher mRNA expression levels of genes involved in inflammatory and immune responses, particularly type I interferon responses, following LPS stimulation. In line, time kinetics studies revealed increased levels of interferon-γ (IFN-γ), interleukin (IL)-1ß and IL-6 mRNA in KSRP-/- macrophages after 6 h subsequent to LPS stimulation as compared to WT cultures. At the protein level, KSRP-/- BMDM displayed higher levels of these cytokines after overnight stimulation. Matching results were observed for primary peritoneal macrophages of KSRP-/- mice. These showed higher IL-6, tumor necrosis factor-α (TNF-α), C-X-C motif chemokine 1 (CXCL1) and CC-chemokine ligand 5 (CCL5) protein levels in response to LPS stimulation than the WT controls. As macrophages play a key role in sepsis, the in vivo relevance of KSRP deficiency for cytokine/chemokine production was analyzed in an acute inflammation model. In agreement with our in vitro findings, KSRP-deficient animals showed higher cytokine production upon LPS administration in comparison to WT mice. Taken together, these findings demonstrate that KSRP constitutes an important negative regulator of cytokine expression in macrophages.
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Proteínas de Transporte , Interleucina-6 , Animais , Camundongos , Interleucina-6/genética , Lipopolissacarídeos , Macrófagos , Citocinas , Regiões 3' não TraduzidasRESUMO
Mammalian cells replicate ~ 3 × 109 base pairs per cell cycle. One of the key molecules that slows down the cell cycle and prevents excessive DNA damage upon DNA replication stress is the checkpoint kinase ataxia-telangiectasia-and-RAD3-related (ATR). Proteolysis-targeting-chimeras (PROTACs) are an innovative pharmacological invention to molecularly dissect, biologically understand, and therapeutically assess catalytic and non-catalytic functions of enzymes. This work defines the first-in-class ATR PROTAC, Abd110/Ramotac-1. It is derived from the ATR inhibitor VE-821 and recruits the E3 ubiquitin-ligase component cereblon to ATR. Abd110 eliminates ATR rapidly in human leukemic cells. This mechanism provokes DNA replication catastrophe and augments anti-leukemic effects of the clinically used ribonucleotide reductase-2 inhibitor hydroxyurea. Moreover, Abd110 is more effective than VE-821 against human primary leukemic cells but spares normal primary immune cells. CRISPR-Cas9 screens show that ATR is a dependency factor in 116 myeloid and lymphoid leukemia cells. Treatment of wild-type but not of cereblon knockout cells with Abd110 stalls their proliferation which verifies that ATR elimination is the primary mechanism of Abd110. Altogether, our findings demonstrate specific anti-leukemic effects of an ATR PROTAC.
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Proteínas Mutadas de Ataxia Telangiectasia , Proliferação de Células , Replicação do DNA , Leucemia , Humanos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Replicação do DNA/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Leucemia/patologia , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Leucemia/genética , Linhagem Celular Tumoral , Proteólise/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Hidroxiureia/farmacologia , Hidroxiureia/análogos & derivados , Dano ao DNA/efeitos dos fármacosRESUMO
Integrin receptors are heterodimeric surface receptors that play multiple roles regarding cell-cell communication, signaling, and migration. The four members of the ß2 integrin subfamily are composed of an alternative α (CD11a-d) subunit, which determines the specific receptor properties, and a constant ß (CD18) subunit. This review aims to present insight into the multiple immunological roles of integrin receptors, with a focus on ß2 integrins that are specifically expressed by leukocytes. The pathophysiological role of ß2 integrins is confirmed by the drastic phenotype of patients suffering from leukocyte adhesion deficiencies, most often resulting in severe recurrent infections and, at the same time, a predisposition for autoimmune diseases. So far, studies on the role of ß2 integrins in vivo employed mice with a constitutive knockout of all ß2 integrins or either family member, respectively, which complicated the differentiation between the direct and indirect effects of ß2 integrin deficiency for distinct cell types. The recent generation and characterization of transgenic mice with a cell-type-specific knockdown of ß2 integrins by our group has enabled the dissection of cell-specific roles of ß2 integrins. Further, integrin receptors have been recognized as target receptors for the treatment of inflammatory diseases as well as tumor therapy. However, whereas both agonistic and antagonistic agents yielded beneficial effects in animal models, the success of clinical trials was limited in most cases and was associated with unwanted side effects. This unfavorable outcome is most probably related to the systemic effects of the used compounds on all leukocytes, thereby emphasizing the need to develop formulations that target distinct types of leukocytes to modulate ß2 integrin activity for therapeutic applications.
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Integrinas , Síndrome da Aderência Leucocítica Deficitária , Humanos , Animais , Camundongos , Antígenos CD18/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Leucócitos/metabolismo , Diferenciação CelularRESUMO
Messenger RNA (mRNA) is a powerful tool for nucleic acid-based therapies and vaccination, but efficient and specific delivery to target tissues remains a significant challenge. In this study, we demonstrate lipoamino xenopeptide carriers as components of highly efficient mRNA LNPs. These lipo-xenopeptides are defined as 2D sequences in different 3D topologies (bundles or different U-shapes). The polar artificial amino acid tetraethylene pentamino succinic acid (Stp) and various lipophilic tertiary lipoamino fatty acids (LAFs) act as ionizable amphiphilic units, connected in different ratios via bisamidated lysines as branching units. A series of more lipophilic LAF4-Stp1 carriers with bundle topology is especially well suited for efficient encapsulation of mRNA into LNPs, facilitated cellular uptake and strongly enhanced endosomal escape. These LNPs display improved, faster transfection kinetics compared to standard LNP formulations, with high potency in a variety of tumor cell lines (including N2a neuroblastoma, HepG2 and Huh7 hepatocellular, and HeLa cervical carcinoma cells), J774A.1 macrophages, and DC2.4 dendritic cells. High transfection levels were obtained even in the presence of serum at very low sub-microgram mRNA doses. Upon intravenous application of only 3 µg mRNA per mouse, in vivo mRNA expression is found with a high selectivity for dendritic cells and macrophages, resulting in a particularly high overall preferred expression in the spleen.
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Nanopartículas , Baço , Camundongos , Animais , Baço/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nanopartículas/química , Lipídeos/química , Transfecção , Macrófagos/metabolismo , Células Dendríticas/metabolismo , RNA Interferente Pequeno , Lipossomos/metabolismoRESUMO
Deeper knowledge about the role of the tumor microenvironment (TME) in cancer development and progression has resulted in new strategies such as gene-based cancer immunotherapy. Whereas some approaches focus on the expression of tumoricidal genes within the TME, DNA-based vaccines are intended to be expressed in antigen-presenting cells (e.g., dendritic cells, DCs) in secondary lymphoid organs, which in turn induce anti-tumor T cell responses. Besides effective delivery systems and the requirement of appropriate adjuvants, DNA vaccines themselves need to be optimized regarding efficacy and selectivity. In this work, the concept of DC-focused transcriptional targeting was tested by applying a plasmid encoding for the luciferase reporter gene under the control of a derivative of the human fascin1 gene promoter (pFscnLuc), comprising the proximal core promoter fused to the normally more distantly located DC enhancer region. DC-focused activity of this reporter construct was confirmed in cell culture in comparison to a standard reporter vector encoding for luciferase under the control of the strong ubiquitously active cytomegalovirus promoter and enhancer (pCMVLuc). Both plasmids were also compared upon intravenous administration in mice. The organ- and cell type-specific expression profile of pFscnLuc versus pCMVLuc demonstrated favorable activity especially in the spleen as a central immune organ and within the spleen in DCs.
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Neoplasias , Humanos , Camundongos , Animais , Regiões Promotoras Genéticas , Genes Reporter , Neoplasias/metabolismo , Células Dendríticas , Luciferases/metabolismo , Microambiente TumoralRESUMO
Background: Systemic inflammation with elevated inflammatory cytokines is a hallmark in patients with cirrhosis and the main driver of decompensation. There is insufficient data on whether inflammatory cytokine levels differ between hepatic and jugular veins, which may have implications for further immunological studies. Methods: Blood from the hepatic and jugular veins of 40 patients with cirrhosis was collected during hepatic venous pressure gradient (HVPG) measurements. Serum levels of 13 inflammatory cytokines (IL-1ß, Int-α2, Int-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33) were quantified by cytometric bead array. Results: Cytokine levels of IFN-α2, IFN-γ, TNF-α, IL-6, IL-8, IL-10, IL-17A, IL-18, IL-23, and IL-33 were significantly elevated in patients with decompensated cirrhosis compared to patients with compensated cirrhosis. When comparing patients with clinically significant portal hypertension (CSPH, HVPG ≥ 10 mmHg) to patients without CSPH, there were significantly enhanced serum levels of IL-6 and IL-18 in the former group. There was no significant difference between cytokine serum levels between blood obtained from the jugular versus hepatic veins. Even in subgroup analyses stratified for an early cirrhosis stage (Child-Pugh (CP) A) or more decompensated stages (CP B/C), cytokine levels were similar. Conclusion: Cytokine levels increase with decompensation and increasing portal hypertension in patients with cirrhosis. There is no relevant difference in cytokine levels between hepatic and jugular blood in patients with cirrhosis.
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Hipertensão Portal , Interleucina-10 , Humanos , Interleucina-18 , Interleucina-17 , Interleucina-33 , Citocinas , Fator de Necrose Tumoral alfa , Veias Jugulares , Interleucina-6 , Interleucina-8 , Cirrose Hepática , Interleucina-23RESUMO
The design of functional polymers coupled with stimuli-triggered drug release mechanisms is a promising achievement to overcome various biological barriers. pH trigger methods yield significant potential for controlled targeting and release of therapeutics due to their simplicity and relevance, especially upon cell internalization. Here, we introduce reactive polymers that conjugate primary or secondary amines and release potential drugs under acidic conditions. For that purpose, we introduced methacrylamide-based monomers with pendant 2-propionic-3-methylmaleic anhydride groups. Such groups allow the conjugation of primary and secondary amines but are resistant to radical polymerization conditions. We, therefore, polymerized 2-propionic-3-methylmaleic anhydride amide-based methacrylates via reversible addition-fragmentation chain transfer (RAFT) polymerization. Their amine-reactive anhydrides could sequentially be derivatized by primary or secondary amines into hydrophilic polymers. Acidic pH-triggered drug release from the polymeric systems was fine-tuned by comparing different amines. Thereby, the conjugation of primary amines led to the formation of irreversible imide bonds in dimethyl sulfoxide, while secondary amines could quantitatively be released upon acidification. In vitro, this installed pH-responsiveness can contribute to an effective release of conjugated immune stimulatory drugs under endosomal pH conditions. Interestingly, the amine-modified polymers generally showed no toxicity and a high cellular uptake. Furthermore, secondary amine-modified immune stimulatory drugs conjugated to the polymers yielded better receptor activity and immune cell maturation than their primary amine derivatives due to their pH-sensitive drug release mechanism. Consequently, 2-propionic-3-methylmaleic anhydride-based polymers can be considered as a versatile platform for pH-triggered delivery of various (immuno)drugs, thus enabling new strategies in macromolecule-assisted immunotherapy.
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Anidridos Citracônicos , Polímeros , Polímeros/química , Aminas/química , Anidridos , Concentração de Íons de HidrogênioRESUMO
Aging attenuates the overall responsiveness of the immune system to eradicate pathogens. The increased production of pro-inflammatory cytokines by innate immune cells under basal conditions, termed inflammaging, contributes to impaired innate immune responsiveness towards pathogen-mediated stimulation and limits antigen-presenting activity. Adaptive immune responses are attenuated as well due to lowered numbers of naïve lymphocytes and their impaired responsiveness towards antigen-specific stimulation. Additionally, the numbers of immunoregulatory cell types, comprising regulatory T cells and myeloid-derived suppressor cells, that inhibit the activity of innate and adaptive immune cells are elevated. This review aims to summarize our knowledge on the cellular and molecular causes of immunosenescence while also taking into account senescence effects that constitute immune evasion mechanisms in the case of chronic viral infections and cancer. For tumor therapy numerous nanoformulated drugs have been developed to overcome poor solubility of compounds and to enable cell-directed delivery in order to restore immune functions, e.g., by addressing dysregulated signaling pathways. Further, nanovaccines which efficiently address antigen-presenting cells to mount sustained anti-tumor immune responses have been clinically evaluated. Further, senolytics that selectively deplete senescent cells are being tested in a number of clinical trials. Here we discuss the potential use of such drugs to improve anti-aging therapy.
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Imunossenescência , Humanos , Imunossenescência/fisiologia , Inflamação/metabolismo , Envelhecimento/fisiologia , Citocinas/metabolismo , Sistema Imunitário/metabolismoRESUMO
Hepatocytes exert pivotal roles in metabolism, protein synthesis and detoxification. Non-parenchymal liver cells (NPCs), largely comprising macrophages, dendritic cells, hepatic stellate cells and liver sinusoidal cells (LSECs), serve to induce immunological tolerance. Therefore, the liver is an important target for therapeutic approaches, in case of both (inflammatory) metabolic diseases and immunological disorders. This review aims to summarize current preclinical nanodrug-based approaches for the treatment of liver disorders. So far, nano-vaccines that aim to induce hepatitis virus-specific immune responses and nanoformulated adjuvants to overcome the default tolerogenic state of liver NPCs for the treatment of chronic hepatitis have been tested. Moreover, liver cancer may be treated using nanodrugs which specifically target and kill tumor cells. Alternatively, nanodrugs may target and reprogram or deplete immunosuppressive cells of the tumor microenvironment, such as tumor-associated macrophages. Here, combination therapies have been demonstrated to yield synergistic effects. In the case of autoimmune hepatitis and other inflammatory liver diseases, anti-inflammatory agents can be encapsulated into nanoparticles to dampen inflammatory processes specifically in the liver. Finally, the tolerance-promoting activity especially of LSECs has been exploited to induce antigen-specific tolerance for the treatment of allergic and autoimmune diseases.
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Hepatite , Neoplasias Hepáticas , Humanos , Fígado/patologia , Hepatócitos , Hepatite/patologia , Células Estreladas do Fígado , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Microambiente TumoralRESUMO
Linear polyglycerol is known as a highly hydrophilic and biocompatible polymer that is currently considered for numerous medical applications. Derived from this well-known structure, the synthesis of highly biocompatible, thermoresponsive polyether copolymers via statistical anionic ring-opening copolymerization of ethyl glycidyl ether (EGE) and ethoxy ethyl glycidyl ether (EEGE) is described. Subsequent deprotection of the acetal groups of EEGE yields copolymers of linear glycerol (linG) and EGE, P(linG-co-EGE). These copolymers showed monomodal and narrow molecular weight distributions with dispersities D ≤ 1.07. The microstructure was investigated via in situ1H NMR kinetics experiments, revealing reactivity ratios of rEEGE = 1.787 ± 0.007 and rEGE = 0.560 ± 0.002, showing a slightly favored incorporation of EEGE over EGE. Due to the deliberate incorporation of rather hydrophobic EGE units into the water soluble linPG, tunable thermoresponsive behavior is achieved with cloud point temperatures Tcp between 9.0-71.4 °C. Besides the commonly utilized method turbidimetry, temperature-dependent 1H NMR measurements were used for more accurate and reproducible results. The change of the hydrodynamic radii rH of the copolymers and their aggregates upon reaching Tcp was investigated via DOSY NMR spectroscopy. To explore possible biomedical applications, as an example, the cell viability and immunology of an exemplary P(linG-co-EGE) copolymer sample was investigated. Since both, cell viability and immunology are comparable to the gold standard PEG, the herein presented copolymers show high potential as biocompatible and thermoresponsive alternatives to PEG for biomedical applications.
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The cysteine protease cathepsin S (CatS) is overexpressed in many tumors. It is known to be involved in tumor progression as well as antigen processing in antigen-presenting cells (APC). Recent evidence suggests that silencing CatS improves the anti-tumor immune response in several cancers. Therefore, CatS is an interesting target to modulate the immune response in these diseases. Here, we present a series of covalent-reversible CatS inhibitors based on the α-fluorovinylsulfone and -sulfonate warheads. We optimized two lead structures by molecular docking approaches, resulting in 22 final compounds which were evaluated in fluorometric enzyme assays for CatS inhibition and for selectivity towards the off-targets CatB and CatL. The most potent inhibitor in the series has subnanomolar affinity (Ki =0.08â nM) and more than 100,000-fold selectivity towards cathepsins B and L. These new reversible and non-cytotoxic inhibitors could serve as interesting leads to develop new immunomodulators in cancer therapy.
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Catepsinas , Neoplasias , Humanos , Simulação de Acoplamento Molecular , Catepsinas/química , Catepsina L , Catepsina B , Fatores Imunológicos , Neoplasias/tratamento farmacológicoRESUMO
The approval of immune-checkpoint inhibitors (CPI) and mitogen activated protein kinase inhibitors (MAPKi) in recent years significantly improved the treatment management and survival of patients with advanced malignant melanoma. CPI aim to counter-act receptor-mediated inhibitory effects of tumor cells and immunomodulatory cell types on effector T cells, whereas MAPKi are intended to inhibit tumor cell survival. In agreement with these complementary modes of action preclinical data indicated that the combined application of CPI and MAPKi or their optimal sequencing might provide additional clinical benefit. In this review the rationale and preclinical evidence that support the combined application of MAPKi and CPI either in concurrent or consecutive regimens are presented. Further, we will discuss the results from clinical trials investigating the sequential or combined application of MAPKi and CPI for advanced melanoma patients and their implications for clinical practice. Finally, we outline mechanisms of MAPKi and CPI cross-resistance which limit the efficacy of currently available treatments, as well as combination regimens.
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Melanoma , Neoplasias Cutâneas , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/patologia , Neoplasias Cutâneas/tratamento farmacológico , Imunoterapia/métodos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/uso terapêuticoRESUMO
Regulatory T cells (Treg) are essential for the maintenance of peripheral tolerance. Treg dysfunction results in diverse inflammatory and autoimmune diseases with life-threatening consequences. ß2-integrins (CD11a-d/CD18) play important roles in the migration of leukocytes into inflamed tissues and cell signaling. Of all ß2-integrins, T cells, including Treg, only express CD11a/CD18, termed lymphocyte function-associated antigen 1 (LFA-1), on their surface. In humans, loss-of-function mutations in the common subunit CD18 result in leukocyte adhesion deficiency type-1 (LAD-1). Clinical symptoms vary depending on the extent of residual ß2-integrin function, and patients may experience leukocytosis and recurrent infections. Some patients can develop autoimmune diseases, but the immune processes underlying the paradoxical situation of immune deficiency and autoimmunity have been scarcely investigated. To understand this complex phenotype, different transgenic mouse strains with a constitutive knockout of ß2-integrins have been established. However, since a constitutive knockout affects all leukocytes and may limit the validity of studies focusing on their cell type-specific role, we established a Treg-specific CD18-floxed mouse strain. This mini-review aims to delineate the role of LFA-1 for the induction, maintenance, and regulatory function of Treg in vitro and in vivo as deduced from observations using the various ß2-integrin-deficient mouse models.
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Doenças Autoimunes , Antígeno-1 Associado à Função Linfocitária , Humanos , Camundongos , Animais , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos T Reguladores/metabolismo , Camundongos Transgênicos , Antígenos CD18/genética , Antígenos CD18/metabolismo , Diferenciação Celular/genéticaRESUMO
Leukocyte adhesion deficiency type 1 (LAD-1) is a rare disease resulting from mutations in the gene encoding for the common ß-chain of the ß2-integrin family (CD18). The most prominent clinical symptoms are profound leukocytosis and high susceptibility to infections. Patients with LAD-1 are prone to develop autoimmune diseases, but the molecular and cellular mechanisms that result in coexisting immunodeficiency and autoimmunity are still unresolved. CD4+FOXP3+ Treg are known for their essential role in preventing autoimmunity. To understand the role of Treg in LAD-1 development and manifestation of autoimmunity, we generated mice specifically lacking CD18 on Treg (CD18Foxp3), resulting in defective LFA-1 expression. Here, we demonstrate a crucial role of LFA-1 on Treg to maintain immune homeostasis by modifying T cell-DC interactions and CD4+ T cell activation. Treg-specific CD18 deletion did not impair Treg migration into extralymphatic organs, but it resulted in shorter interactions of Treg with DC. In vivo, CD18Foxp3 mice developed spontaneous hyperplasia in lymphatic organs and diffuse inflammation of the skin and in multiple internal organs. Thus, LFA-1 on Treg is required for the maintenance of immune homeostasis.
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Doenças Autoimunes , Autoimunidade , Camundongos , Animais , Antígeno-1 Associado à Função Linfocitária/genética , Linfócitos T Reguladores , Doenças Autoimunes/genéticaRESUMO
Transcutaneous immunization (TCI) utilizing the TLR7 agonist imiquimod (IMQ-TCI) induces T cell-driven protective immunity upon application onto intact skin. In our present work, we combine the anti-psoriatic agent dithranol with IMQ-TCI to boost vaccination efficacy (Dithranol/IMQ-based transcutaneous vaccination (DIVA)). Using ovalbumin-derived peptides as model antigens in mice, DIVA induced superior cytolytic CD8+ T cells and CD4+ T cells with a TH1 cytokine profile in the priming as well as in the memory phase. Regarding the underlying mechanisms, dithranol induced an oxidant-dependent, monocyte-attracting inflammatory milieu in the skin boosting TLR7-dependent activation of dendritic cells and macrophages leading to superior T cell priming and protective immunity in vaccinia virus infection. In conclusion, we introduce the non-invasive vaccination method DIVA to induce strong primary and memory T cell responses upon a single local treatment. This work provides relevant insights in cutaneous vaccination approaches, paving the way for clinical development in humans.
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BACKGROUND: Protease-activated receptor 2 (PAR2) signaling controls skin barrier function and inflammation, but the roles of immune cells and PAR2-activating proteases in cutaneous diseases are poorly understood. OBJECTIVE: To dissect PAR2 signaling contributions to skin inflammation with new genetic and pharmacological tools. METHODS/RESULTS: We found markedly increased numbers of PAR2+ infiltrating myeloid cells in skin lesions of allergic contact dermatitis (ACD) patients and in the skin of contact hypersensitivity (CHS) in mice, a murine ACD model for T cell-mediated allergic skin inflammation. Cell type-specific deletion of PAR2 in myeloid immune cells as well as mutation-induced complete PAR2 cleavage insensitivity significantly reduced skin inflammation and hapten-specific Tc1/Th1 cell response. Pharmacological approaches identified individual proteases involved in PAR2 cleavage and demonstrated a pivotal role of tissue factor (TF) and coagulation factor Xa (FXa) as upstream activators of PAR2 in both the induction and effector phase of CHS. PAR2 mutant mouse strains with differential cleavage sensitivity for FXa versus skin epithelial cell-expressed proteases furthermore uncovered a time-dependent regulation of CHS development with an important function of FXa-induced PAR2 activation during the late phase of skin inflammation. CONCLUSIONS: Myeloid cells and the TF-FXa-PAR2 axis are key mediators and potential therapeutic targets in inflammatory skin diseases.