RESUMO
The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.
Assuntos
Encéfalo/citologia , Linfócitos T CD4-Positivos/metabolismo , Feto/citologia , Microglia/citologia , Microglia/metabolismo , Sinapses/metabolismo , Adulto , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Escala de Avaliação Comportamental , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Criança , Feminino , Feto/embriologia , Humanos , Lectinas Tipo C/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neurogênese/genética , Parabiose , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Análise de Célula Única , Baço/citologia , Baço/metabolismo , Sinapses/imunologia , TranscriptomaRESUMO
The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.
Assuntos
Astrócitos , Produtos Biológicos , Animais , Encéfalo , Humanos , Interleucina-2/genética , Interleucinas , Camundongos , Doenças Neuroinflamatórias , Linfócitos T ReguladoresRESUMO
Rationale: Sensitization to Fel d 1 (Felis domesticus allergen 1) contributes to persistent allergic rhinitis and asthma. Existing treatment options for cat allergy, including allergen immunotherapy, are only moderately effective, and allergen immunotherapy has limited use because of safety concerns. Objectives: To explore the relationship among the pharmacokinetic, clinical, and immunological effects of anti-Fel d 1 monoclonal antibodies (REGN1908-1909) in patients after treatment. Methods: Patients received REGN1908-1909 (n = 36) or a placebo (n = 37) in a phase 1b study. Fel d 1-induced basophil and IgE-facilitated allergen binding responses were evaluated at baseline and Days 8, 29, and 85. Cytokine and chemokine concentrations in nasal fluids were measured, and REGN1908-1909 inhibition of allergen-IgE binding in patient serum was evaluated. Measurements and Main Results: Peak serum drug concentrations were concordant with maximal observed clinical response. The anti-Fel d 1 IgE/cat dander IgE ratio in pretreatment serum correlated with Total Nasal Symptom Score improvement. The allergen-neutralizing capacity of REGN1908-1909 was observed in serum and nasal fluid and was detected in an inhibition assay. Type 2 cytokines (IL-4, IL-5, and IL-13) and chemokines (CCL17/TARC, CCL5/RANTES [regulated upon activation, normal T-cell expressed and secreted]) in nasal fluid were inhibited in REGN1908-1909-treated patients compared with placebo (P < 0.05 for all); IL-13 and IL-5 concentrations correlated with Total Nasal Symptom Score improvement. Ex vivo assays demonstrated that REGN1908 and REGN1909 combined were more potent than each alone for inhibiting FcεRI- and FcεRII (CD23)-mediated allergic responses and subsequent T-cell activation. Conclusions: A single, passive-dose administration of Fel d 1-neutralizing IgG antibodies improved nasal symptoms in cat-allergic patients and was underscored by suppression of FcεRI-, FcεRII-, and T-helper cell type 2-mediated allergic responses. Clinical trial registered with www.clinicaltrials.gov (NCT02127801).
Assuntos
Alérgenos/efeitos adversos , Antialérgicos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Gatos , Glicoproteínas/efeitos adversos , Fatores Imunológicos/uso terapêutico , Rinite Alérgica/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antialérgicos/administração & dosagem , Anticorpos Monoclonais/imunologia , Método Duplo-Cego , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Imunoterapia , Masculino , Pessoa de Meia-Idade , Efeito Placebo , Rinite Alérgica/etiologia , Rinite Alérgica/imunologiaRESUMO
BACKGROUND: Grass pollen-specific immunotherapy involves immunomodulation of allergen-specific TH2 responses and induction of IL-10+ and/or TGF-ß+CD4+CD25+ regulatory T cells (induced Treg cells). IL-35+CD4+CD25+ forkhead box protein 3-negative T (IL-35-inducible regulatory T [iTR35]) cells have been reported as a novel subset of induced Treg cells with modulatory characteristics. OBJECTIVE: We sought to investigate mechanisms underlying the induction and maintenance of immunologic tolerance induced by IL-35 and iTR35 cells. METHODS: The biological effects of IL-35 were assessed on group 2 innate lymphoid cells (ILC2s); dendritic cells primed with thymic stromal lymphopoietin, IL-25, and IL-33; and B and TH2 cells by using flow cytometry and quantitative RT-PCR. Grass pollen-driven TH2 cell proliferation and cytokine production were measured by using tritiated thymidine and Luminex MagPix, respectively. iTR35 cells were quantified in patients with grass pollen allergy (seasonal allergic rhinitis [SAR] group, n = 16), sublingual immunotherapy (SLIT)-treated patients (SLIT group, n = 16), and nonatopic control subjects (NACs; NAC group, n = 16). RESULTS: The SAR group had increased proportions of ILC2s (P = .002) and IL-5+ cells (P = .042), IL-13+ cells (P = .042), and IL-5+IL-13+ ILC2s (P = .003) compared with NACs. IL-35 inhibited IL-5 and IL-13 production by ILC2s in the presence of IL-25 or IL-33 (P = .031) and allergen-driven TH2 cytokines by effector T cells. IL-35 inhibited CD40 ligand-, IL-4-, and IL-21-mediated IgE production by B cells (P = .015), allergen-driven T-cell proliferation (P = .001), and TH2 cytokine production mediated by primed dendritic cells. iTR35 cells suppressed TH2 cell proliferation and cytokine production. In addition, allergen-driven IL-35 levels and iTR35 cell counts were increased in patients receiving SLIT (all, P < .001) and NACs (all, P < .001) compared with patients with SAR. CONCLUSION: IL-35 and iTR35 cells are potential novel immune regulators induced by SLIT. The clinical relevance of SLIT can be underscored by restoration of protective iTR35 cells.
Assuntos
Alérgenos/imunologia , Interleucinas/imunologia , Linfócitos/imunologia , Poaceae/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/terapia , Imunoterapia Sublingual , Adulto , Feminino , Humanos , Tolerância Imunológica , Masculino , Pessoa de Meia-Idade , Rinite Alérgica Sazonal/imunologia , Adulto JovemRESUMO
BACKGROUND: A 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide (LPP) immunotherapy for rhinoconjunctivitis with or without asthma over 4 physician visits is safe, well tolerated, and effective. OBJECTIVE: We sought to investigate immunologic mechanisms of LPP immunotherapy in a subset of patients who participated in a phase III, multicenter, randomized, double-blind, placebo-controlled trial (clinical.govNCT02560948). METHODS: Participants were randomized to receive LPP (n = 21) or placebo (n = 11) for 3 weeks over 4 visits. Grass pollen-induced basophil, T-cell, and B-cell responses were evaluated before treatment (visit [V] 2), at the end of treatment (V6), and after the pollen season (V8). RESULTS: Combined symptom and rescue medication scores (CSMS) were lower during the peak pollen season (-35.1%, P = .03) and throughout the pollen season (-53.7%, P = .03) in the LPP-treated group compared with those in the placebo-treated group. Proportions of CD63+ and CD203cbrightCRTH2+ basophils were decreased following LPP treatment at V6 (10 ng/mL, P < .0001) and V8 (10 ng/mL, P < .001) compared to V2. No change in the placebo-treated group was observed. Blunting of seasonal increases in levels of grass pollen-specific IgE was observed in LPP-treated but not placebo-treated group. LPP immunotherapy, but not placebo, was associated with a reduction in proportions of IL-4+ TH2 (V6, P = .02), IL-4+ (V6, P = .003; V8, P = .004), and IL-21+ (V6, P = .003; V8, P = .002) follicular helper T cells. Induction of FoxP3+, follicular regulatory T, and IL-10+ regulatory B cells were observed at V6 (all P < .05) and V8 (all P < .05) in LPP-treated group. Induction of regulatory B cells was associated with allergen-neutralizing IgG4-blocking antibodies. CONCLUSION: For the first time, we demonstrate that the immunologic mechanisms of LPP immunotherapy are underscored by immune modulation in the T- and B-cell compartments, which is necessary for its effect.
Assuntos
Alérgenos/imunologia , Asma/terapia , Conjuntivite/terapia , Lolium/imunologia , Peptídeos/uso terapêutico , Pólen/imunologia , Rinite Alérgica Sazonal/terapia , Adulto , Asma/imunologia , Linfócitos B Reguladores/imunologia , Conjuntivite/imunologia , Dessensibilização Imunológica , Método Duplo-Cego , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Peptídeos/imunologia , Rinite Alérgica Sazonal/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Adulto JovemRESUMO
BACKGROUND: Grass pollen subcutaneous immunotherapy (SCIT) is associated with induction of serum IgG4-associated inhibitory antibodies that prevent IgE-facilitated allergen binding to B cells. OBJECTIVE: We sought to determine whether SCIT induces nasal allergen-specific IgG4 antibodies with inhibitory activity that correlates closely with clinical response. METHODS: In a cross-sectional controlled study, nasal fluid and sera were collected during the grass pollen season from 10 SCIT-treated patients, 13 untreated allergic patients (with seasonal allergic rhinitis [SAR]), and 12 nonatopic control subjects. Nasal and serum IgE and IgG4 levels to Phleum pratense components were measured by using the Immuno Solid Allergen Chip microarray. Inhibitory activity was measured by IgE-facilitated allergen binding assay. IL-10+ regulatory B cells were quantified in peripheral blood by using flow cytometry. RESULTS: Nasal and serum Phl p 1- and Phl p 5-specific IgE levels were increased in patients with SAR compared to nonatopic control subjects (all, P < .001) and SCIT-treated patients (nasal, P < .001; serum Phl p 5, P = .073). Nasal IgG4 levels were increased in the SCIT group compared to those in the SAR group (P < .001) during the pollen season compared to out of season. IgG-associated inhibitory activity in nasal fluid and serum was significantly increased in the SCIT group compared to that in the SAR (both, P < .01). The magnitude of the inhibitory activity was 93% (P < .001) in nasal fluid compared to 66% (P < .001) in serum and was reversed after depletion of IgG. Both nasal fluid (r = -0.69, P = .0005) and serum (r = -0.552, P = .0095) blocking activity correlated with global symptom improvement. IL-10+ regulatory B cells were increased in season compared to out of season in the SCIT group (P < .01). CONCLUSION: For the first time, we show that nasal IgG4-associated inhibitory activity correlates closely with the clinical response to allergen immunotherapy in patients with allergic rhinitis with or without asthma.
Assuntos
Alérgenos/imunologia , Anticorpos Neutralizantes/imunologia , Dessensibilização Imunológica , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Mucosa Nasal/imunologia , Phleum/imunologia , Pólen/imunologia , Adulto , Linfócitos B Reguladores/imunologia , Biomarcadores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/terapiaRESUMO
Interaction between the complement system and carbon nanotubes (CNTs) can modify their intended biomedical applications. Pristine and derivatised CNTs can activate complement primarily via the classical pathway which enhances uptake of CNTs and suppresses pro-inflammatory response by immune cells. Here, we report that the interaction of C1q, the classical pathway recognition molecule, with CNTs involves charge pattern and classical pathway activation that is partly inhibited by factor H, a complement regulator. C1q and its globular modules, but not factor H, enhanced uptake of CNTs by macrophages and modulated the pro-inflammatory immune response. Thus, soluble complement factors can interact differentially with CNTs and alter the immune response even without complement activation. Coating CNTs with recombinant C1q globular heads offers a novel way of controlling classical pathway activation in nanotherapeutics. Surprisingly, the globular heads also enhance clearance by phagocytes and down-regulate inflammation, suggesting unexpected complexity in receptor interaction. FROM THE CLINICAL EDITOR: Carbon nanotubes (CNTs) maybe useful in the clinical setting as targeting drug carriers. However, it is also well known that they can interact and activate the complement system, which may have a negative impact on the applicability of CNTs. In this study, the authors functionalized multi-walled CNT (MWNT), and investigated the interaction with the complement pathway. These studies are important so as to gain further understanding of the underlying mechanism in preparation for future use of CNTs in the clinical setting.
Assuntos
Complemento C1q/imunologia , Fator H do Complemento/imunologia , Imunidade Inata , Macrófagos/imunologia , Nanotubos de Carbono/efeitos adversos , Linhagem Celular , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Ativação do Complemento , Proteínas do Sistema Complemento , Humanos , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestrutura , FagocitoseRESUMO
Interleukin 2 (IL-2) is a key homeostatic cytokine, with therapeutic applications in both immunogenic and tolerogenic immune modulation. Clinical use has been hampered by pleiotropic functionality and widespread receptor expression, with unexpected adverse events. Here, we developed a novel mouse strain to divert IL-2 production, allowing identification of contextual outcomes. Network analysis identified priority access for Tregs and a competitive fitness cost of IL-2 production among both Tregs and conventional CD4 T cells. CD8 T and NK cells, by contrast, exhibited a preference for autocrine IL-2 production. IL-2 sourced from dendritic cells amplified Tregs, whereas IL-2 produced by B cells induced two context-dependent circuits: dramatic expansion of CD8+ Tregs and ILC2 cells, the latter driving a downstream, IL-5-mediated, eosinophilic circuit. The source-specific effects demonstrate the contextual influence of IL-2 function and potentially explain adverse effects observed during clinical trials. Targeted IL-2 production therefore has the potential to amplify or quench particular circuits in the IL-2 network, based on clinical desirability.
Assuntos
Interleucina-2 , Células Matadoras Naturais , Linfócitos T Reguladores , Animais , Imunidade Inata , Interleucina-2/biossíntese , Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
SAMHD1, a human host factor found in myeloid cells which restricts HIV-1 replication. It depletes the dNTPs pool for viral cDNA syntheses, thus preventing the viral replication in the cells. The viral accessory protein, Vpx, exists only in SIVmac/HIV-2 particles. Vpx in SIVmac can induce proteosomal degradation of SAMHD1, which then leads to a decrease in the cytoplasmic dNTP pool. The protein-protein interaction between Vpx and SAMHD1 and its consequences are still unclear. Methods: In this study, we cloned, for the first time, Vpx gene from a HIV-2 infected patient and found up to 30% sequence variation compared to known HIV-2 strains. We then analyzed the role of SAMHD1 protein expression in transfected THP-1 and U937 cells by transfecting with the Vpx gene derived from SIVmac, HIV-2 from the NIH sample as well as HIV-2 from a Saudi patient. We found that Vpx gene expression led to reduced levels of intracellular SAMHD1. When the supernatants of the transfected cell lines were examined for secreted cytokines, chemokines and growth factors, Vpx expression seemed to be suppressive of pro-inflammatory response, and skewed the immune response towards an anti-inflammatory response. These results suggest that Vpx can act at two levels: clearance of intracellular restriction factor and suppression of cytokine storm: both aimed at long-term latency and host-pathogen stand-off, suggesting that Vpx is likely to be a potential therapeutic target.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-2/fisiologia , Interações Hospedeiro-Patógeno , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Citocinas/metabolismo , Suscetibilidade a Doenças , Regulação Viral da Expressão Gênica , Humanos , Imunofenotipagem , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Ligação Proteica , Análise de Sequência de DNA , Proteínas Virais Reguladoras e Acessórias/genética , Latência ViralRESUMO
Syncytiotrophoblast derived Extracellular Vesicles (STBEV) from normal pregnancy (NP) have previously been shown to interact with circulating monocytes and B cells and induce pro-inflammatory cytokine release. Early-onset preeclampsia (EOPE) is associated with an exacerbated inflammatory response, yet there is little data regarding late-onset PE (LOPE) and immune function. Here, using a macrophage/monocyte cell line THP-1, we investigated the inflammatory potential of STBEV, comprising medium/large-STBEV (>200nm) and small-STBEV (<200nm), isolated from LOPE (n=6) and normal (NP) (n=6) placentae via dual-lobe ex-vivo placental perfusion and differential centrifugation. THP-1 cells bound and internalised STBEV isolated from NP and LOPE placentae, as revealed by flow cytometry, confocal microscopy, and ELISA. STBEV-treated THP-1 cells were examined for cytokine gene expression by RT-qPCR and the cell culture media examined for secreted cytokines/chemokines. As expected, NP medium/large-STBEV significantly upregulated the transcriptional expression of TNF-α, IL-10, IL-6, IL-12, IL-8 and TGF-ß compared to PE medium/large-STBEV. However, there was no significant difference in the small STBEV population between the two groups, although in general, NP small STBEVs slightly upregulated the same cytokines. In contrast, LOPE STBEV (medium and large) did not induce pro-inflammatory responses by differentiated THP-1 macrophages. This decreased effect of LOPE STBEV was echoed in cytokine/chemokine release. Our results appear to suggest that STBEV from LOPE placentae do not have a major immune-modulatory effect on macrophages. In contrast, NP STBEV caused THP-1 cells to release pro-inflammatory cytokines. Thus, syncytiotrophoblast extracellular vesicles from LOPE dampen immune functions of THP-1 macrophages, suggesting an alternative mechanism leading to the pro-inflammatory environment observed in LOPE.
Assuntos
Vesículas Extracelulares/fisiologia , Macrófagos/imunologia , Placenta/imunologia , Pré-Eclâmpsia/imunologia , Trofoblastos/ultraestrutura , Adulto , Citocinas/biossíntese , Citocinas/genética , Feminino , Citometria de Fluxo , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Gravidez , Células THP-1RESUMO
Surfactant protein D (SP-D) is expressed in the mucosal secretion of the lung and contributes to the innate host defense against a variety of pathogens, including influenza A virus (IAV). SP-D can inhibit hemagglutination and infectivity of IAV, in addition to reducing neuraminidase (NA) activity via its carbohydrate recognition domain (CRD) binding to carbohydrate patterns (N-linked mannosylated) on NA and hemagglutinin (HA) of IAV. Here, we demonstrate that a recombinant fragment of human SP-D (rfhSP-D), containing homotrimeric neck and CRD regions, acts as an entry inhibitor of IAV and downregulates M1 expression considerably in A549 cells challenged with IAV of H1N1 and H3N2 subtypes at 2 h treatment. In addition, rfhSP-D downregulated mRNA levels of TNF-α, IFN-α, IFN-ß, IL-6, and RANTES, particularly during the initial stage of IAV infection of A549 cell line. rfhSP-D also interfered with IAV infection of Madin Darby canine kidney (MDCK) cells through HA binding. Furthermore, rfhSP-D was found to reduce luciferase reporter activity in MDCK cells transduced with H1+N1 pseudotyped lentiviral particles, where 50% of reduction was observed with 10 µg/ml rfhSP-D, suggestive of a critical role of rfhSP-D as an entry inhibitor against IAV infectivity. Multiplex cytokine array revealed that rfhSP-D treatment of IAV challenged A549 cells led to a dramatic suppression of key pro-inflammatory cytokines and chemokines. In the case of pH1N1, TNF-α, IFN-α, IL-10, IL-12 (p40), VEGF, GM-CSF, and eotaxin were considerably suppressed by rfhSP-D treatment at 24 h. However, these suppressive effects on IL-10, VEGF, eotaxin and IL-12 (p40) were not so evident in the case of H3N2 subtype, with the exception of TNF-α, IFN-α, and GM-CSF. These data seem to suggest that the extent of immunomodulatory effect of SP-D on host cells can vary considerably in a IAV subtype-specific manner. Thus, rfhSP-D treatment can downregulate pro-inflammatory milieu encouraged by IAV that otherwise causes aberrant inflammatory cell recruitment leading to cell death and lung damage.
Assuntos
Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteína D Associada a Surfactante Pulmonar/imunologia , Células A549 , Animais , Aves , Citocinas/imunologia , Cães , Hemaglutininas/imunologia , Humanos , Células Madin Darby de Rim Canino , Peptídeos/genética , Peptídeos/imunologia , Proteína D Associada a Surfactante Pulmonar/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Bovine conglutinin, the first animal collectin to be discovered, is structurally very similar to Surfactant Protein D (SP-D). SP-D is known to interact with Mycobacterium tuberculosis, and the closely-related M. bovis, the causative agent of bovine tuberculosis. We speculated that due to the overall similarities between conglutinin and SP-D, conglutinin is likely to have a protective influence in bovine tuberculosis. We set out to investigate the role of conglutinin in host-pathogen interaction during mycobacterial infection. We show here that a recombinant truncated form of conglutinin (rfBC), composed of the neck and C-type lectin domains, binds specifically and in a dose-dependent manner to the model organism Mycobacterium bovis BCG. rfBC showed a significant direct bacteriostatic effect on the growth of M. bovis BCG in culture. In addition, rfBC inhibited the uptake of M. bovis BCG by THP-1 macrophages (human monocyte lineage cell line) and suppressed the subsequent pro-inflammatory response. Conglutinin is well-known as a binder of the complement activation product, iC3b. rfBC was also able to inhibit the uptake of complement-coated M. bovis BCG by THP-1 macrophages, whilst modulating the pro-inflammatory response. It is likely that rfBC inhibits the phagocytosis of mycobacteria by two distinct mechanisms: firstly, rfBC interferes with mannose receptor-mediated uptake by masking lipoarabinomannan (LAM) on the mycobacterial surface. Secondly, since conglutinin binds iC3b, it can interfere with complement receptor-mediated uptake via CR3 and CR4, by masking interactions with iC3b deposited on the mycobacterial surface. rfBC was also able to modulate the downstream pro-inflammatory response in THP-1 cells, which is important for mobilizing the adaptive immune response, facilitating containment of mycobacterial infection. In conclusion, we show that conglutinin possesses complement-dependent and complement-independent anti-mycobacterial activities, interfering with both known mechanisms of mycobacterial uptake by macrophages. As mycobacteria are specialized intracellular pathogens, conglutinin may inhibit M. bovis and M. tuberculosis from establishing an intracellular niche within macrophages, and thus, negatively affect the long-term survival of the pathogen in the host.
Assuntos
Colectinas/imunologia , Proteínas do Sistema Complemento/imunologia , Mycobacterium bovis/imunologia , Soroglobulinas/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Animais , Biomarcadores , Bovinos , Colectinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose/imunologia , Soroglobulinas/metabolismo , Células THP-1 , Tuberculose Bovina/metabolismoRESUMO
Development of nanoparticles as tissue-specific drug delivery platforms can be considerably influenced by the complement system because of their inherent pro-inflammatory and tumorigenic consequences. The complement activation pathways, and its recognition subcomponents, can modulate clearance of the nanoparticles and subsequent inflammatory response and thus alter the intended translational applications. Here, we report, for the first time, that human properdin, an upregulator of the complement alternative pathway, can opsonize functionalized carbon nanotubes (CNTs) via its thrombospondin type I repeat (TSR) 4 and 5. Binding of properdin and TSR4+5 is likely to involve charge pattern/polarity recognition of the CNT surface since both carboxymethyl cellulose-coated carbon nanotubes (CMC-CNT) and oxidized (Ox-CNT) bound these proteins well. Properdin enhanced the uptake of CMC-CNTs by a macrophage cell line, THP-1, mounting a robust pro-inflammatory immune response, as revealed by qRT-PCR, multiplex cytokine array, and NF-κB nuclear translocation analyses. Properdin can be locally synthesized by immune cells in an inflammatory microenvironment, and thus, its interaction with nanoparticles is of considerable importance. In addition, recombinant TSR4+5 coated on the CMC-CNTs inhibited complement consumption by CMC-CNTs, suggesting that nanoparticle decoration with TSR4+5, can be potentially used as a complement inhibitor in a number of pathological contexts arising due to exaggerated complement activation.
Assuntos
Proteínas ADAMTS/imunologia , Macrófagos/imunologia , Nanotubos de Carbono/química , Properdina/imunologia , Proteínas ADAMTS/genética , Carboximetilcelulose Sódica/química , Ativação do Complemento , Citocinas/genética , Células HEK293 , Humanos , Inflamação/imunologia , Properdina/genética , Ligação Proteica , Células THP-1RESUMO
Mycobacterium tuberculosis can proficiently enter macrophages and diminish complement activation on its cell surface. Within macrophages, the mycobacterium can suppress macrophage apoptosis and survive within the intracellular environment. Previously, we have shown that complement regulatory proteins such as factor H may interfere with pathogen-macrophage interactions during tuberculosis infection. In this study, we show that Mycobacterium bovis BCG binds properdin, an upregulator of the complement alternative pathway. TSR4+5, a recombinant form of thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an inhibitor of the alternative pathway, was also able to bind to M. bovis BCG. Properdin and TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Quantitative real-time PCR revealed elevated pro-inflammatory responses (TNF-α, IL-1ß, and IL-6) in the presence of properdin or TSR4+5, which gradually decreased over 6 h. Correspondingly, anti-inflammatory responses (IL-10 and TGF-ß) showed suppressed levels of expression in the presence of properdin, which gradually increased over 6 h. Multiplex cytokine array analysis also revealed that properdin and TSR4+5 significantly enhanced the pro-inflammatory response (TNF-α, IL-1ß, and IL-1α) at 24 h, which declined at 48 h, whereas the anti-inflammatory response (IL-10) was suppressed. Our results suggest that properdin may interfere with mycobacterial entry into macrophages via TSR4 and TSR5, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. This study offers novel insights into the non-complement related functions of properdin during host-pathogen interactions in tuberculosis.
Assuntos
Macrófagos/fisiologia , Mycobacterium bovis/fisiologia , Properdina/fisiologia , Trombospondinas/fisiologia , Citocinas/genética , Humanos , Células THP-1RESUMO
Complement system homeostasis is important for host self-protection and anti-microbial immune surveillance, and recent research indicates roles in tissue development and remodelling. Complement also appears to have several points of interaction with the blood coagulation system. Deficiency and altered function due to gene mutations and polymorphisms in complement effectors and regulators, including Factor H, have been associated with familial and sporadic autoimmune inflammatory - thrombotic disorders, in which autoantibodies play a part. These include systemic lupus erythematosus, rheumatoid arthritis, atypical haemolytic uremic syndrome, anti-phospholipid syndrome and age-related macular degeneration. Such diseases are generally complex - multigenic and heterogeneous in their symptoms and predisposition/susceptibility. They usually need to be triggered by vascular trauma, drugs or infection and non-complement genetic factors also play a part. Underlying events seem to include decline in peripheral regulatory T cells, dendritic cell, and B cell tolerance, associated with alterations in lymphoid organ microenvironment. Factor H is an abundant protein, synthesised in many cell types, and its reported binding to many different ligands, even if not of high affinity, may influence a large number of molecular interactions, together with the accepted role of Factor H within the complement system. Factor H is involved in mesenchymal stem cell mediated tolerance and also contributes to self-tolerance by augmenting iC3b production and opsonisation of apoptotic cells for their silent dendritic cell engulfment via complement receptor CR3, which mediates anti-inflammatory-tolerogenic effects in the apoptotic cell context. There may be co-operation with other phagocytic receptors, such as complement C1q receptors, and the Tim glycoprotein family, which specifically bind phosphatidylserine expressed on the apoptotic cell surface. Factor H is able to discriminate between self and nonself surfaces for self-protection and anti-microbe defence. Factor H, particularly as an abundant platelet protein, may also modulate blood coagulation, having an anti-thrombotic role. Here, we review a number of interaction pathways in coagulation and in immunity, together with associated diseases, and indicate where Factor H may be expected to exert an influence, based on reports of the diversity of ligands for Factor H.
Assuntos
Doenças Autoimunes/imunologia , Fator H do Complemento/imunologia , Inflamação/imunologia , Trombose/imunologia , Animais , HumanosRESUMO
Allergen immunotherapy (AIT) is an immune modulating treatment for allergic diseases. Although highly effective, some patients do not respond to the treatment. To date there are no surrogate biomarkers that are predictive of the clinical response to AIT. More and more is known about the underlying immunological mechanism involved in AIT. Through modulation of both innate and adaptive immune responses, involving reduced ILC2 and enhanced Treg and Breg induction and functionality, along with induction of IgG4 antibody production which have the capacity to inhibit both allergen-induced basophil responsiveness and CD23-mediated IgE-facilitated allergen presentation, the result is an immune skewing towards a more balanced Type I response. So far, however there is not a clear correlation with the observed immunological changes and predictive correlates of clinical efficacy. The most promising biomarker of successful AIT is IgE-FAB as a reflection of functional IgG4. Cellular responses and cytokine analysis gives a great deal of insight into the mechanisms of AIT but may not represent useful or indeed reliable biomarkers in a clinical setting. There is a need for more research for confirmation and interpretation of the possible association with biomarkers and clinical response to AIT.
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Carbon nanotubes (CNTs) are increasingly being developed for use in biomedical applications, including drug delivery. One of the most promising applications under evaluation is in treating pulmonary diseases such as tuberculosis. Once inhaled or administered, the nanoparticles are likely to be recognised by innate immune molecules in the lungs such as hydrophilic pulmonary surfactant proteins. Here, we set out to examine the interaction between surfactant protein D (SP-D), a key lung pattern recognition molecule and CNTs, and possible downstream effects on the immune response via macrophages. We show here that a recombinant form of human SP-D (rhSP-D) bound to oxidised and carboxymethyl cellulose (CMC) coated CNTs via its C-type lectin domain and enhanced phagocytosis by U937 and THP-1 macrophages/monocytic cell lines, together with an increased pro-inflammatory response, suggesting that sequestration of SP-D by CNTs in the lungs can trigger an unwanted and damaging immune response. We also observed that functionalised CNTs, opsonised with rhSP-D, continued to activate complement via the classical pathway, suggesting that C1q, which is the recognition sub-component of the classical pathway, and SP-D have distinct pattern recognition sites on the CNTs. Consistent with our earlier reports, complement deposition on the rhSP-D opsonised CNTs led to dampening of the pro-inflammatory immune response by THP-1 macrophages, as evident from qPCR, cytokine array and NF-κB nuclear translocation analyses. This study highlights the importance of understanding the interplay between innate immune humoral factors including complement in devising nanoparticle based drug delivery strategies.
Assuntos
Macrófagos/citologia , Nanotubos de Carbono/química , Fagocitose , Proteína D Associada a Surfactante Pulmonar/química , Complemento C1q/química , Via Clássica do Complemento , Citocinas/metabolismo , Humanos , Proteínas Recombinantes/química , Células THP-1 , Fatores de Transcrição/metabolismo , Transcriptoma , Células U937RESUMO
Dendritic cells (DCs) are the most potent antigen-presenting cells capable of priming naïve T-cells. Its C-type lectin receptor, DC-SIGN, regulates a wide range of immune functions. Along with its role in HIV-1 pathogenesis through complement opsonization of the virus, DC-SIGN has recently emerged as an adaptor for complement protein C1q on the surface of immature DCs via a trimeric complex involving gC1qR, a receptor for the globular domain of C1q. Here, we have examined the nature of interaction between C1q and DC-SIGN in terms of domain localization, and implications of C1q-DC-SIGN-gC1qR complex formation on HIV-1 transmission. We first expressed and purified recombinant extracellular domains of DC-SIGN and its homologue DC-SIGNR as tetramers comprising of the entire extra cellular domain including the α-helical neck region and monomers comprising of the carbohydrate recognition domain only. Direct binding studies revealed that both DC-SIGN and DC-SIGNR were able to bind independently to the recombinant globular head modules ghA, ghB, and ghC, with ghB being the preferential binder. C1q appeared to interact with DC-SIGN or DC-SIGNR in a manner similar to IgG. Mutational analysis using single amino acid substitutions within the globular head modules showed that TyrB175 and LysB136 were critical for the C1q-DC-SIGN/DC-SIGNR interaction. Competitive studies revealed that gC1qR and ghB shared overlapping binding sites on DC-SIGN, implying that HIV-1 transmission by DCs could be modulated due to the interplay of gC1qR-C1q with DC-SIGN. Since C1q, gC1qR, and DC-SIGN can individually bind HIV-1, we examined how C1q and gC1qR modulated HIV-1-DC-SIGN interaction in an infection assay. Here, we report, for the first time, that C1q suppressed DC-SIGN-mediated transfer of HIV-1 to activated pooled peripheral blood mononuclear cells, although the globular head modules did not. The protective effect of C1q was negated by the addition of gC1qR. In fact, gC1qR enhanced DC-SIGN-mediated HIV-1 transfer, suggesting its role in HIV-1 pathogenesis. Our results highlight the consequences of multiple innate immune pattern recognition molecules forming a complex that can modify their functions in a way, which may be advantageous for the pathogen.
RESUMO
Surfactant proteins SP-A and SP-D are pattern recognition innate immune molecules that belong to the C-type lectin family. In lungs, they play an important role in the clearance of pathogens and control of inflammation. SP-A and SP-D are also expressed in the female reproductive tract where they play an important role in pregnancy and parturition. However, the role of SP-A and SP-D expressed at the feto-maternal interface (decidua) remains unclear. Here, we have examined the expression of SP-A and SP-D in the murine decidua at 17.5 (pre-parturition) and 19.5dpc (near parturition) and their effect on lipopolysaccharide (LPS)-treated decidual macrophages. SP-A and SP-D were localized to stromal cells in the murine decidua at 17.5 and 19.5dpc in addition to cells lining the maternal spiral artery. Purified pre-parturition decidual cells were challenged with LPS with and without SP-A or SP-D, and expression of F4/80 and TNF-α were measured by flow cytometry. On their own, SP-A or SP-D did not affect the percentage of F4/80 positive cells while they suppressed the percentage of TNF-α positive cells. However, simultaneous addition of SP-A or SP-D, together with LPS, reduced TNF-α secreting F4/80 positive cells. It is likely that exogenous administration of SP-A and SP-D in decidua can potentially control infection and inflammation mediators during spontaneous term labor and infection-induced preterm labor. Thus, the presence of SP-A and SP-D in the murine decidua is likely to play a protective role against intrauterine infection during pregnancy.