RESUMO
Introduction: Atopic diseases have been steadily increasing over the past decades and effective disease-modifying treatment options are urgently needed. These studies introduce a novel synthetic Toll-like receptor 4 (TLR4) agonist, INI-2004, with remarkable efficacy as a therapeutic intranasal treatment for seasonal allergic rhinitis. Methods: Using a murine airway allergic sensitization model, the impact of INI-2004 on allergic responses was assessed. Results: One or two intranasal doses of INI-2004 significantly reduced airway resistance, eosinophil influx, and Th2 cytokine production - providing strong evidence of allergic desensitization. Further investigations revealed that a liposomal formulation of INI-2004 exhibited better safety and efficacy profiles compared to aqueous formulations. Importantly, the liposomal formulation demonstrated a 1000-fold increase in the maximum tolerated intravenous dose in pigs. Pre-clinical GLP toxicology studies in rats and pigs confirmed the safety of liposomal INI-2004, supporting its selection for human clinical trials. Discussion: These findings lay the groundwork for the ongoing clinical evaluation of INI-2004 in allergic rhinitis as a stand-alone therapy for individuals poly-sensitized to multiple seasonal allergens. The study underscores the significance of innovative immunotherapy approaches in reshaping the landscape of allergic rhinitis management.
Assuntos
Administração Intranasal , Modelos Animais de Doenças , Receptor 4 Toll-Like , Animais , Receptor 4 Toll-Like/agonistas , Camundongos , Suínos , Feminino , Lipossomos , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/tratamento farmacológico , Alérgenos/imunologia , Alérgenos/administração & dosagem , Dessensibilização Imunológica/métodos , Ratos , Citocinas/metabolismo , Camundongos Endogâmicos BALB CRESUMO
Antigenically divergent H7N9 viruses pose a potential threat to public health, with the poor immunogenicity of candidate H7N9 vaccines demonstrated in clinical trials underscoring the urgent need for more-effective H7N9 vaccines. In the present study, mice were immunized with various doses of a suspended-MDCK-cell-derived inactivated H7N9 vaccine, which was based on a low-pathogenic H7N9 virus, to assess cross-reactive immunity and cross-protection against antigenically divergent H7N9 viruses. We found that the CRX-527 adjuvant, a synthetic TLR4 agonist, significantly enhanced the humoral immune responses of the suspended-MDCK-cell-derived H7N9 vaccine, with significant antigen-sparing and immune-enhancing effects, including robust virus-specific IgG, hemagglutination-inhibiting (HI), neuraminidase-inhibiting (NI), and virus-neutralizing (VN) antibody responses, which are crucial for protection against influenza virus infection. Moreover, the CRX-527-adjuvanted H7N9 vaccine also elicited cross-protective immunity and cross-protection against a highly pathogenic H7N9 virus with a single vaccination. Notably, NI and VN antibodies might play an important role in cross-protection against lethal influenza virus infections. This study showed that a synthetic TLR4 agonist adjuvant has a potent immunopotentiating effect, which might be considered worth further development as a means of increasing vaccine effectiveness.
Assuntos
Anticorpos Antivirais , Imunidade Humoral , Subtipo H7N9 do Vírus da Influenza A , Vacinas contra Influenza , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae , Receptor 4 Toll-Like , Vacinas de Produtos Inativados , Animais , Subtipo H7N9 do Vírus da Influenza A/imunologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Camundongos , Anticorpos Antivirais/imunologia , Cães , Células Madin Darby de Rim Canino , Vacinas de Produtos Inativados/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Feminino , Anticorpos Neutralizantes/imunologia , Proteção Cruzada/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Adjuvantes de Vacinas , Imunoglobulina G/imunologia , Imunoglobulina G/sangueRESUMO
Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and ß-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice. The results indicated a synergistic effect of MPLA and Rha, two immunostimulators that function via interacting with toll-like receptor 4 and recruiting endogenous anti-Rha antibodies, respectively. All the tested MPLA-Rha conjugates exhibited potent adjuvant activities to promote antibody production against both protein and carbohydrate antigens. Overall, MPLA-α-Rha exhibited better activities than MPLA-ß-Rha, and 6'-linked conjugates were slightly better than 1-linked ones. Particularly, MPLA-1-α-Rha and MPLA-6'-α-Rha were the most effective adjuvants in promoting IgG antibody responses against protein antigen keyhole limpet hemocyanin and carbohydrate antigen sTn, respectively.
Assuntos
Lipídeo A , Ramnose , Lipídeo A/análogos & derivados , Lipídeo A/química , Lipídeo A/farmacologia , Lipídeo A/imunologia , Animais , Ramnose/química , Ramnose/imunologia , Ramnose/farmacologia , Camundongos , Adjuvantes de Vacinas/química , Adjuvantes de Vacinas/farmacologia , Feminino , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/síntese química , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Hemocianinas/química , Hemocianinas/imunologiaRESUMO
Agonists of Toll like receptors (TLRs) have attracted interest as adjuvants and immune modulators. A crystal structure of TLR4/MD2 with E. coli LPS indicates that the fatty acid at C-2 of the lipid A component of LPS induces dimerization of two TLR4-MD2 complexes, which in turn initiates cell signaling leading to the production of (pro)inflammatory cytokines. To probe the importance of the (R)-3-hydroxymyristate at C-2 of lipid A, a range of bis- and mono-phosphoryl lipid A derivatives with different modifications at C-2 were prepared by a strategy in which 2-methylnaphthyl ethers were employed as permanent protecting group that could be readily removed by catalytic hydrogenation. The C-2 amine was protected as 9-fluorenylmethyloxycarbamate, which at a later stage could be removed to give a free amine that was modified by different fatty acids. LPS and the synthetic lipid As induced the same cytokines, however, large differences in activity were observed. A compound having a hexanoyl moiety at C-2 still showed agonistic properties, but further shortening to a butanoyl abolished activity. The modifications had a larger influence on monophosphoryl lipid As. The lipid As having a butanoyl moiety at C-2 could selectively antagonize TRIF associated cytokines induced by LPS or lipid A.
Assuntos
Citocinas , Lipídeo A , Lipopolissacarídeos , Lipídeo A/química , Lipídeo A/farmacologia , Lipídeo A/análogos & derivados , Lipídeo A/síntese química , Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/química , Humanos , Antígeno 96 de Linfócito/metabolismo , Antígeno 96 de Linfócito/química , Desenho de Fármacos , Relação Estrutura-Atividade , Transdução de Sinais/efeitos dos fármacosRESUMO
In this work, we present the potential of Fourier transform infrared (FTIR) microspectroscopy to compare on whole cells, in an unbiased and untargeted way, the capacity of bacterial lipopolysaccharide (LPS) and two rationally designed molecules (FP20 and FP20Rha) to activate molecular circuits of innate immunity. These compounds are important drug hits in the development of vaccine adjuvants and tumor immunotherapeutics. The biological assays indicated that FP20Rha was more potent than FP20 in inducing cytokine production in cells and in stimulating IgG antibody production post-vaccination in mice. Accordingly, the overall significant IR spectral changes induced by the treatment with LPS and FP20Rha were similar, lipids and glycans signals being the most diagnostic, while the effect of the less potent molecule FP20 on cells resulted to be closer to control untreated cells. We propose here the use of FTIR spectroscopy supported by artificial intelligence (AI) to achieve a more holistic understanding of the cell response to new drug candidates while screening them in cells.
Assuntos
Lipopolissacarídeos , Aprendizado de Máquina , Receptor 4 Toll-Like , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Animais , Espectroscopia de Infravermelho com Transformada de Fourier , Camundongos , Lipopolissacarídeos/farmacologia , Humanos , Desenho de Fármacos , Células RAW 264.7RESUMO
INTRODUCTION: Hepatitis C is a liver disease with high chronicity, the cause of cirrhosis and hepatocarcinoma. The main obstacle to controlling hepatitis C is the lack of vaccines. The aim of the work was to compare the immunogenic activity of nonstructural recombinant proteins NS3, NS4 and NS5B of hepatitis C virus (HCV) as components of a subunit candidate vaccine and to analyze the adjuvant properties of two available commercial drugs, polymuramil and pyrogenalum. MATERIALS AND METHODS: BALB/c, DBA/2J and C57BL/6 mice were immunized with nonstructural proteins without adjuvants or with polymuramyl (NOD1 and NOD2 agonist) and pyrogenalum (TLR-4 agonist). The activity of antibodies was determined in ELISA, the cellular response - by antigen-specific lymphocyte proliferation and by production of IFN-γ in vitro. RESULTS: Recombinant proteins showed different immunogenicity. NS4 induced antibodies more efficiently than NS3 and NS5B. Significant differences were found in the immune response of three inbred lines mice: the level of IFN-γ in BALB/c and DBA/2J mice induced by NS5B protein was 30 times higher than in C57Bl/6 mice. In contrast, the induction of antibodies in BALB/c mice was lower than in C57Bl/6 and DBA/2J. Polymuramil did not increase the humoral response to NS5B and enhanced the cellular response only in C57BL/6 mice. The combined use of polymuramil with pyrogenalum significantly increased both the humoral and cellular response of mice to all recombinant HCV proteins. CONCLUSION: Different immunogenic properties and different functions of recombinant non-structural HCV proteins indicate the feasibility of their combined inclusion in subunit vaccines. It was established for the first time that immunization with HCV proteins with a complex adjuvant (polymuramyl + pyrogenalum) has a synergistic effect, significantly exceeding the effect of each of them separately.
Assuntos
Hepatite C , Receptor 4 Toll-Like , Vacinas de DNA , Vacinas contra Hepatite Viral , Animais , Camundongos , Adjuvantes Imunológicos/farmacologia , Hepacivirus , Imunidade Celular , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas Recombinantes , Receptor 4 Toll-Like/agonistas , Vacinas de DNA/farmacologia , Vacinas contra Hepatite Viral/farmacologia , Proteínas não Estruturais ViraisRESUMO
Importance: Metastatic soft tissue sarcomas (STSs) have limited systemic therapy options, and immunomodulation has not yet meaningfully improved outcomes. Intratumoral (IT) injection of the toll-like receptor 4 (TLR4) agonist glycopyranosyl lipid A in stable-emulsion formulation (GLA-SE) has been studied as immunotherapy in other contexts. Objective: To evaluate the safety, efficacy, and immunomodulatory effects of IT GLA-SE with concurrent radiotherapy in patients with metastatic STS with injectable lesions. Design, Setting, and Participants: This phase 1 nonrandomized controlled trial of patients with STS was performed at a single academic sarcoma specialty center from November 17, 2014, to March 16, 2016. Data analysis was performed from August 2016 to September 2022. Interventions: Two doses of IT GLA-SE (5 µg and 10 µg for 8 weekly doses) were tested for safety in combination with concurrent radiotherapy of the injected lesion. Main Outcomes and Measures: Primary end points were safety and tolerability. Secondary and exploratory end points included local response rates as well as measurement of antitumor immunity with immunohistochemistry and T-cell receptor (TCR) sequencing of tumor-infiltrating and circulating lymphocytes. Results: Twelve patients (median [range] age, 65 [34-78] years; 8 [67%] female) were treated across the 2 dose cohorts. Intratumoral GLA-SE was well tolerated, with only 1 patient (8%) experiencing a grade 2 adverse event. All patients achieved local control of the injected lesion after 8 doses, with 1 patient having complete regression (mean regression, -25%; range, -100% to 4%). In patients with durable local response, there were detectable increases in tumor-infiltrating lymphocytes. In 1 patient (target lesion -39% at 259 days of follow-up), TCR sequencing revealed expansion of preexisting and de novo clonotypes, with convergence of numerous rearrangements coding for the same binding sequence (suggestive of clonal convergence to antitumor targets). Single-cell sequencing identified these same expanded TCR clones in peripheral blood after treatment; these T cells had markedly enhanced Tbet expression, suggesting TH1 phenotype. Conclusions and Relevance: In this nonrandomized controlled trial, IT GLA-SE with concurrent radiotherapy was well tolerated and provided more durable local control than radiotherapy alone. Patients with durable local response demonstrated enhanced IT T-cell clonal expansion, with matched expansion of these clonotypes in the circulation. Additional studies evaluating synergism of IT GLA-SE and radiotherapy with systemic immune modulation are warranted. Trial Registration: ClinicalTrials.gov Identifier: NCT02180698.
Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Humanos , Feminino , Idoso , Masculino , Receptor 4 Toll-Like/agonistas , Linfócitos T , Neoplasias de Tecidos Moles/tratamento farmacológico , Neoplasias de Tecidos Moles/radioterapia , Sarcoma/tratamento farmacológico , Sarcoma/radioterapia , Receptores de Antígenos de Linfócitos TRESUMO
Consumption of diets high in fat has been linked to the development of obesity and related metabolic complications. Such associations originate from the enhanced, chronic, low-grade inflammation mediated by macrophages in response to translocated bacteria, bacterial products, or dietary constituents such as fatty acids (FAs). Nucleotide-binding Oligomerization Domain 2 (NOD2) senses muramyl dipeptide (MDP), a component of bacterial peptidoglycan. The inability to sense peptidoglycan through NOD2 has been demonstrated to lead to dysbiosis, increased bacterial translocation, inflammation and metabolic dysfunction. Currently, it is unknown how consumption of HFDs with different FA compositions might influence NOD2-dependent responses. In this study, we subjected WT mice to a control diet or to HFDs comprised of various ratios of unsaturated to saturated fats and determined the macrophage response to TLR4 and NOD2 agonists. A HFD with equal ratios of saturated and unsaturated fats enhanced subsequent responsiveness of macrophages to LPS but not to MDP. However, a high-unsaturated fat diet (HUFD) or a high-saturated fat diet (HSFD) both decreased the responsiveness to NOD2 agonists compared to that observed in control diet (CD) fed mice. These data suggest that dietary fatty acid composition can influence the subsequent macrophage responsiveness to bacterial products.
Assuntos
Gorduras na Dieta , Macrófagos , Proteína Adaptadora de Sinalização NOD2 , Receptor 4 Toll-Like , Animais , Camundongos , Acetilmuramil-Alanil-Isoglutamina , Dieta Hiperlipídica , Gorduras na Dieta/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Peptidoglicano/metabolismo , Receptor 4 Toll-Like/agonistasRESUMO
Heavy-ion radiation received during radiotherapy as well as the heavy-ion radiation received during space flight are equally considered harmful. Our previous study showed that TLR4 low toxic agonist, monophosphoryl lipid A (MPLA), alleviated radiation injury resulting from exposure to low-LET radiation. However, the role and mechanism of MPLA in heavy-ion-radiation injury are unclear. This study aimed to investigate the role of MPLA on radiation damage. Our data showed that MPLA treatment alleviated the heavy-ion-induced damage to microstructure and the spleen and testis indexes. The number of karyocytes in the bone marrow from the MPLA-treated group was higher than that in the irradiated group. Meanwhile, western blotting analysis of intestine proteins showed that pro-apoptotic proteins (cleaved-caspase3 and Bax) were downregulated while anti-apoptotic proteins (Bcl-2) were upregulated in the MPLA-treated group. Our in vitro study demonstrated that MPLA significantly improved cell proliferation and inhibited cell apoptosis after irradiation. Moreover, immunofluorescence staining and quantification of nucleic γ-H2AX and 53BP1 foci also suggested that MPLA significantly attenuated cellular DNA damage repair. Collectively, the above evidence supports the potential ability of MPLA to protect against heavy-ion-radiation injury by inhibiting apoptosis and alleviating DNA damage in vivo and vitro, which could be a promising medical countermeasure for the prevention of heavy-ion-radiation injury.
Assuntos
Lesões por Radiação , Receptor 4 Toll-Like , Humanos , Masculino , Apoptose/efeitos da radiação , Dano ao DNA , Reparo do DNA , Receptor 4 Toll-Like/agonistasRESUMO
Bacterial lipopolysaccharides (LPS) are potent innate immunostimulants targeting the Toll-like receptor 4 (TLR4), an attractive and validated target for immunostimulation in cancer therapy. Although LPS possess anti-tumor activity, toxicity issues prevent their systemic administration at effective doses in humans. We first demonstrated that LPS formulated in liposomes preserved a potent antitumor activity per se upon systemic administration in syngeneic models, and significantly enhance the antitumor activity of the anti-CD20 antibody rituximab in mice xenografted with the human RL lymphoma model. Liposomal encapsulation also allowed a 2-fold reduction in the induction of pro-inflammatory cytokines by LPS. Mice receiving an intravenous administration demonstrated a significant increase of neutrophils, monocytes and macrophages at the tumor site as well as an increase of macrophages in spleen. Further, we chemically detoxified LPS to obtain MP-LPS that was associated with a 200-fold decrease in the induction of proinflammatory cytokines. When encapsulated in a clinically approved liposomal formulation, toxicity, notably pyrogenicity (10-fold), was limited while the antitumor activity and immunoadjuvant effect were maintained. This improved tolerance profile of liposomal MP-LPS was associated with the preferential activation of the TLR4-TRIF pathway. Finally, in vitro studies demonstrated that stimulation with encapsulated MP-LPS reversed the polarization of M2 macrophages towards an M1 phenotype, and a phase 1 trial in healthy dogs validated its tolerance upon systemic administration up to very high doses (10µg/kg). Altogether, our results demonstrate the strong therapeutic potential of MPLPS formulated in liposomes as a systemically active anticancer agent, supporting its evaluation in patients with cancer.
Assuntos
Adjuvantes Imunológicos , Lipopolissacarídeos , Receptor 4 Toll-Like , Animais , Cães , Humanos , Camundongos , Citocinas , Lipossomos , Receptor 4 Toll-Like/agonistasRESUMO
Acute kidney injury (AKI) is common in surgical and critically ill patients. This study examined whether pretreatment with a novel Toll-like receptor 4 agonist attenuated ischemia-reperfusion injury (IRI)-induced AKI (IRI-AKI). We performed a blinded, randomized-controlled study in mice pretreated with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD), a synthetic Toll-like receptor 4 agonist. Two cohorts of male BALB/c mice received intravenous vehicle or PHAD (2, 20, or 200 µg) at 48 and 24 h before unilateral renal pedicle clamping and simultaneous contralateral nephrectomy. A separate cohort of mice received intravenous vehicle or 200 µg PHAD followed by bilateral IRI-AKI. Mice were monitored for evidence of kidney injury for 3 days postreperfusion. Kidney function was assessed by serum blood urea nitrogen and creatinine measurements. Kidney tubular injury was assessed by semiquantitative analysis of tubular morphology on periodic acid-Schiff (PAS)-stained kidney sections and by kidney mRNA quantification of injury [neutrophil gelatinase-associated lipocalin (Ngal), kidney injury molecule-1 (Kim-1), and heme oxygenase-1 (Ho-1)] and inflammation [interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (Tnf-α)] using quantitative RT-PCR. Immunohistochemistry was used to quantify proximal tubular cell injury and renal macrophages by quantifying the areas stained with Kim-1 and F4/80 antibodies, respectively, and TUNEL staining to detect the apoptotic nuclei. PHAD pretreatment yielded dose-dependent kidney function preservation after unilateral IRI-AKI. Histological injury, apoptosis, Kim-1 staining, and Ngal mRNA were lower in PHAD-treated mice and IL-1ß mRNA was higher in PHAD-treated mice. Similar pretreatment protection was noted with 200 mg PHAD after bilateral IRI-AKI, with significantly reduced Kim-1 immunostaining in the outer medulla of mice treated with PHAD after bilateral IRI-AKI. In conclusion, PHAD pretreatment leads to dose-dependent protection from renal injury after unilateral and bilateral IRI-AKI in mice.NEW & NOTEWORTHY Pretreatment with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide; a novel synthetic Toll-like receptor 4 agonist, preserves kidney function during ischemia-reperfusion injury-induced acute kidney injury.
Assuntos
Injúria Renal Aguda , Traumatismo por Reperfusão , Receptor 4 Toll-Like , Animais , Masculino , Camundongos , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Injúria Renal Aguda/prevenção & controle , Rim/patologia , Lipocalina-2 , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , RNA Mensageiro , Receptor 4 Toll-Like/agonistasRESUMO
A full nonclinical safety package was performed to support the clinical use of SPA14, a novel liposome-based vaccine adjuvant containing the synthetic toll-like receptor 4 agonist E6020 and saponin QS21. E6020 and QS21 were tested negative for their potential genotoxic effects in Ames, micronucleus, or mouse-lymphoma TK (thymidine kinase) assay. To evaluate the potential local and systemic effects of SPA14, two toxicity studies were performed in rabbits. In the first dose range finding toxicity study, rabbits received two intramuscular injections of SPA14 at increasing doses of E6020 combined with two antigens, a control (saline), the two antigens alone, or the antigens adjuvanted with a liposome-based adjuvant AS01B. No systemic toxicity was detected, supporting the dose of 5 µg of E6020 for the subsequent pivotal study. In the second repeated dose toxicity study, rabbits received four intramuscular injections of SPA14 alone, a control (saline), SPA14 combined with two antigens, the two antigens alone, or the antigens combined with AF03 adjuvant, which is a squalene-based emulsion. SPA14 alone or in combination with the antigens was well tolerated and did not cause any systemic toxicity. Finally, two safety pharmacology studies were conducted to assess potential cardiovascular and respiratory effects of E6020 and SPA14 in conscious telemetered cynomolgus monkeys and beagle dogs, respectively. One subcutaneous injection of E6020 in monkeys and one intramuscular injection of SPA14 in dogs had no consequences on respiratory and cardiovascular functions. Altogether these results support the clinical development of SPA14.
Assuntos
Adjuvantes de Vacinas , Receptor 4 Toll-Like , Camundongos , Animais , Coelhos , Cães , Receptor 4 Toll-Like/agonistas , Lipossomos , Adjuvantes Imunológicos/farmacologiaRESUMO
BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are a multipotent cell population of clinical interest because of their ability to migrate to injury and tumor sites, where they may participate in tissue repair and modulation of immune response. Although the processes regulating MSC function are incompletely understood, it has been shown that stimulation of Toll-like receptors (TLRs) can alter MSC activity. More specifically, it has been reported that human bone marrow-derived MSCs can be "polarized" by TLR priming into contrasting immunomodulatory functions, with opposite (supportive or suppressive) roles in tumor progression and inflammation. Adipose-derived MSCs (ASCs) represent a promising alternative MSC subpopulation for therapeutic development because of their relative ease of isolation and higher abundance compared with their bone marrow-derived counterparts; however, the polarization of ASCs remains unreported. METHODS: In this study, we evaluated the phenotypic and functional consequences of short-term, low-level stimulation of ASCs with TLR3 and TLR4 agonists. RESULTS: In these assays, we identified transient gene expression changes resembling the reported pro-inflammatory and anti-inflammatory MSC phenotypes. Furthermore, these priming strategies led to changes in the functional properties of ASCs, affecting their ability to migrate and modulate immune-mediated responses to prostate cancer cells in vitro. CONCLUSIONS: TLR3 stimulation significantly decreased ASC migration, and TLR4 stimulation increased ASC immune-mediated killing potential against prostate cancer cells.
Assuntos
Células-Tronco Mesenquimais , Neoplasias da Próstata , Humanos , Masculino , Diferenciação Celular , Células Cultivadas , Células-Tronco Mesenquimais/fisiologia , Obesidade , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Neoplasias da Próstata/metabolismo , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismoRESUMO
A phase I trial (NCT03447314; 204686) evaluated the safety and efficacy of GSK1795091, a Toll-like receptor 4 (TLR4) agonist, in combination with immunotherapy (GSK3174998 [anti-OX40 monoclonal antibody], GSK3359609 [anti-ICOS monoclonal antibody], or pembrolizumab) in patients with solid tumors. The primary endpoint was safety; other endpoints included efficacy, pharmacokinetics, and pharmacodynamics (PD). Manufacturing of GSK1795091 formulation was modified during the trial to streamline production and administration, resulting in reduced PD (cytokine) activity. Fifty-four patients received GSK1795091 with a combination partner; 32 received only the modified GSK1795091 formulation, 15 received only the original formulation, and seven switched mid-study from the original to the modified formulation. Despite the modified formulation demonstrating higher systemic GSK1795091 exposure compared with the original formulation, the transient, dose-dependent elevations in cytokine and chemokine concentrations were no longer observed (e.g., IP-10, IL10, IL1-RA). Most patients (51/54; 94%) experienced ≥1 treatment-emergent adverse event (TEAE) during the study. Safety profiles were similar between formulations, but a higher incidence of TEAEs associated with immune responses (chills, fatigue, pyrexia, nausea, and vomiting) were observed with the original formulation. No conclusions can be made regarding GSK1795091 anti-tumor activity due to the limited data collected. Manufacturing changes were hypothesized to have caused the change in biological activity in this study. Structural characterization revealed GSK1795091 aggregate size in the modified formulation to be twice that in the original formulation, suggesting a negative correlation between GSK1795091 aggregate size and PD activity. This may have important clinical implications for future development of structurally similar compounds.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias , Humanos , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Citocinas , Lipídeo A/uso terapêutico , Neoplasias/tratamento farmacológico , Receptor 4 Toll-Like/agonistas , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversosRESUMO
Toll-like receptor (TLR) agonists can act as immune stimulants alone or as part of alum or oil formulations. Humoral and cellular immune responses were utilized to assess quantitative and qualitative immune response enhancement by TLR agonists using recombinant protective antigen (rPA) of B. anthracis as a model antigen. To rPA, combined with aluminum hydroxide (Alhydrogel; Al(OH)3) or squalene (AddaVax™), was added one of 7 TLR agonists: TLR2 agonist Pam3CysSK4 (PamS), TLR3 agonist double stranded polyinosinic:polycytidylic acid (PolyIC), TLR4 agonists Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), TLR7-8 agonists 3M-052 or Resiquimod (Resiq), or TLR9 agonist CPG 7909 (CPG). CD-1 or BALB/c mice received two intraperitoneal or intramuscular immunizations 14 days apart, followed by serum or spleen sampling 14 days later. All TLR agonists except PamS induced high levels of B. anthracis lethal toxin-neutralizing antibodies and immunoglobulin G (IgG) anti-PA. Some responses were >100-fold higher than those without a TLR agonist, and IP delivery (0.5 mL) induced higher TLR-mediated antibody response increases compared to IM delivery (0.05 mL). TLR7-8 and TLR9 agonists induced profound shifts of IgG anti-PA response to IgG2a or IgG2b. Compared to the 14-day immunization schedule, use of a shortened immunization schedule of only 7 days between prime and boost found that TLR9 agonist CPG in a squalene formulation maintained higher interferon-γ-positive cells than TLR4 agonist GLA. Variability in antibody responses was lower in BALB/c mice than CD-1 mice but antibody responses were higher in CD-1 mice. Lower serum 50% effective concentration (EC50) values were found for rPA-agonist formulations and squalene formulations compared to Al(OH)3 formulations. Lower EC50 values also were associated with low frequency detection of linear peptide epitopes. In summary, TLR agonists elicited cellular immune responses and markedly boosted humoral responses.
Assuntos
Bacillus anthracis , Adjuvantes Imunológicos , Hidróxido de Alumínio , Animais , Antígenos , Imunoglobulina G , Camundongos , Camundongos Endogâmicos BALB C , Esqualeno , Receptor 2 Toll-Like , Receptor 4 Toll-Like/agonistas , Receptor 7 Toll-Like/agonistas , Receptor Toll-Like 9/agonistasRESUMO
The glucocorticoid-induced tumor necrosis factor receptor (GITR) agonistic antibody (DTA-1) has been proved to elicit robust immune response in various kinds of tumors. However, only a few of the HCC patients could benefit from it, and the mechanism of DTA-1 resistance remains unknown. Here, we measured GITR expression in different immunocytes in HCC microenvironment, and we observed that tumor-infiltrating regulatory T cells (Ti-Tregs) significantly expressed GITR, which were associated with poor prognosis. Meanwhile, we analyzed the variation of tumor-infiltrating immune components and associated inflammation response after DTA-1 treatment in orthotopic liver cancer model of mice. Surprisingly, DTA-1 treatment reduced the infiltration of Tregs but failed to activate CD8+ T cells and elicit antitumor efficacy. In particular, DTA-1 treatment enforced alternative M2 polarization of macrophage, and macrophage depletion could enhance DTA-1-mediated antitumor efficacy in HCC. Mechanistically, macrophage M2 polarization attributed to the IL-4 elevation induced by Th2 immune activation in the treatment of DTA-1, resulting in DTA-1 resistance. Furthermore, Toll-like receptor 4 (TLR4) agonist could diminish the macrophage (M2) polarization and reverse the M2-mediated DTA-1 resistance, eliciting robust antitumor effect in HCC. Our finding demonstrated that the TLR4 agonist synergized with DTA-1 was a potential strategy for HCC treatment.
Assuntos
Carcinoma Hepatocelular , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Neoplasias Hepáticas , Receptor 4 Toll-Like , Animais , Linfócitos T CD8-Positivos , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Proteína Relacionada a TNFR Induzida por Glucocorticoide/agonistas , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores , Receptor 4 Toll-Like/agonistas , Microambiente TumoralRESUMO
Despite success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability remain. Molecular adjuvants targeting pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulates various PRRs, including toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors. We hypothesized that targeting PRRs using molecular adjuvants on nanoparticles (NPs) along with a stabilized spike protein antigen could stimulate broad and efficient immune responses. Adjuvants targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer NPs were combined with the S1 subunit of spike protein and assessed in vitro with isogeneic mixed lymphocyte reactions (isoMLRs). For in vivo studies, the adjuvant-NPs were combined with stabilized spike protein or spike-conjugated NPs and assessed using a two-dose intranasal or intramuscular vaccination model in mice. Combination adjuvant-NPs simultaneously targeting TLR and RIG-I receptors (MPLA+PUUC, CpG+PUUC, and R848+PUUC) differentially induced T cell proliferation and increased proinflammatory cytokine secretion by APCs in vitro. When delivered intranasally, MPLA+PUUC NPs enhanced CD4+CD44+ activated memory T cell responses against spike protein in the lungs while MPLA NPs increased anti-spike IgA in the bronchoalveolar (BAL) fluid and IgG in the blood. Following intramuscular delivery, PUUC NPs induced strong humoral immune responses, characterized by increases in anti-spike IgG in the blood and germinal center B cell populations (GL7+ and BCL6+ B cells) in the draining lymph nodes (dLNs). MPLA+PUUC NPs further boosted spike protein-neutralizing antibody titers and T follicular helper cell populations in the dLNs. These results suggest that protein subunit vaccines with particle-delivered molecular adjuvants targeting TLR4 and RIG-I could lead to robust and unique route-specific adaptive immune responses against SARS-CoV-2.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Proteína DEAD-box 58 , Nanopartículas , Receptores Imunológicos , Receptor 4 Toll-Like , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Sistemas de Liberação de Medicamentos , Imunidade Humoral , Imunoglobulina G , Camundongos , Nanopartículas/química , Receptores Imunológicos/agonistas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Receptor 4 Toll-Like/agonistasRESUMO
Microglia have diverse physiological and pathological functions. However, the transcriptional mechanisms remain elusive. Here we sought new transcription factors relevant to microglial functions from the microglial transcriptome of stressed mice and evaluated their roles in primary microglia. TLR2 and TLR4 agonists increased Rel, Atf3, and Cebpb and decreased Hhex in primary microglia as repeated social defeat stress. Although Hhex was not studied in microglia, TLR2 and TLR4 agonists decreased Hhex, and Hhex overexpression attenuated TLR4-increased expression of inflammation-related genes. These findings suggest that Hhex negatively regulates inflammation-related genes in microglia and that TLR2/4 activation reduces Hhex, facilitating TLR4-mediated neuroinflammation.
Assuntos
Proteínas de Homeodomínio , Microglia , Fatores de Transcrição , Animais , Proteínas de Homeodomínio/metabolismo , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
TLR4 is a key pattern recognition receptor that can sense pathogen- and danger- associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro-inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species-specific, and barely predictable structure-activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan-based lipid A-mimicking molecules that can activate human and murine TLR4-mediated signaling with picomolar affinity. Exploiting crystal structure - based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible ß(1â6)-linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non-reducing ßGlcN(1â1')ßGlcN scaffold. Excellent stereoselectivity in a challenging ß,ß-1,1' glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of ß,ß-1,1'-linked diglucosamine-derived glycolipids entailing multiple long-chain (R)-3- acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D-molecular shape and conformational rigidity of unnatural ß,ß-1,1'-linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4-mediated signaling in a species-independent manner.
Assuntos
Lipídeo A , Receptor 4 Toll-Like , Adjuvantes Imunológicos/química , Animais , Dissacarídeos/química , Humanos , Imunoterapia , Lipídeo A/química , Camundongos , Estudos Prospectivos , Receptor 4 Toll-Like/agonistasRESUMO
In this study, we have identified Interferon-stimulated genes (ISGs), especially IFIT1, 2 and 3, as target genes of propionate-induced signalling in the human epithelial cell line A549, the monocytic cell line THP-1 as well as in primary, human peripheral blood-derived macrophages (PBMs). Induction of the IFIT gene family by propionate negatively regulates TLR-induced signalling. Propionate stimulation results in downregulation of pro-inflammatory cytokine and chemokine expression as well as MHC class II expression upon TLR1/2 and TLR4 re-stimulation in A549 and THP-1 cells as well as in PBMs, demonstrating that propionate-induced signalling is involved in the induction of TLR cross-tolerance. Signalling pathway analysis clearly demonstrates that propionate-induced IFIT expression is mediated by FFAR2 in a Gαq/11 signalling pathway-dependent manner. Furthermore, propionate-induced IFIT expression is dependent on IFN type I and/or type III-mediated signalling since pre-treatment of A549 cells with Ruxolitinib, a specific JAK1/2 tyrosine kinase inhibitor, prior to stimulation with propionate, inhibited the upregulation of IFIT1 expression. The hypo-responsiveness towards TLR1/2 and TLR4 agonists seems to be mediated by different members of the IFIT gene family in a cell type-specific manner. Collectively, our data indicate that propionate-induced signalling controls pro-inflammatory responses by activation of IFN type I and/or type III-induced and IFIT-mediated counter-regulatory mechanisms in order to protect against exacerbating inflammatory reactions.