RESUMO
Antimicrobial peptides (AMPs) are a promising alternative to antibiotics in the fight against multi-drug resistant and immune system-evading bacterial infections. Protegrins are porcine cathelicidins which have been identified in porcine leukocytes. Protegrin-1 is the best characterized family member and has broad antibacterial activity by interacting and permeabilizing bacterial membranes. Many host defense peptides (HDPs) like LL-37 or chicken cathelicidin 2 (CATH-2) have also been shown to have protective biological functions during infections. In this regard, it is interesting to study if Protegrin-1 has the immune modulating potential to suppress unnecessary immune activation by neutralizing endotoxins or by influencing the macrophage functionality in addition to its direct antimicrobial properties. This study showed that Protegrin-1 neutralized lipopolysaccharide- (LPS) and bacteria-induced activation of RAW macrophages by binding and preventing LPS from cell surface attachment. Furthermore, the peptide treatment not only inhibited bacterial phagocytosis by murine and porcine macrophages but also interfered with cell surface and intracellular bacterial survival. Lastly, Protegrin-1 pre-treatment was shown to inhibit the amastigote survival in Leishmania infected macrophages. These experiments describe an extended potential of Protegrin-1's protective role during microbial infections and add to the research towards clinical application of cationic AMPs.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Catelicidinas , Lipopolissacarídeos , Macrófagos , Fagocitose , Animais , Camundongos , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fatores Imunológicos/farmacologia , Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Células RAW 264.7 , SuínosRESUMO
Vaccination is commonly used to protect dogs against leptospirosis, however, memory immune responses induced by canine Leptospira vaccines have not been studied. In the present study, antibody and T cell mediated responses were assessed in dogs before and 2 weeks after annual revaccination with a commercial tetravalent Leptospira vaccine containing serogroups Canicola and Australis. Vaccination significantly increased average log2 IgG titers from 6.50 to 8.41 in year 1, from 5.99 to 7.32 in year 2, from 5.32 to 8.32 in year 3 and from 5.32 to 7.82 in year 4. The CXCL-10 levels, induced by in vitro stimulation of PBMC with Canicola and Australis, respectively, significantly increased from 1039.05 pg/ml and 1037.38 pg/ml before vaccination to 2547.73 pg/ml and 2730.38 pg/ml after vaccination. IFN-γ levels increased from 85.60 pg/ml and 178.13 pg/ml before vaccination to 538.62 pg/ml and 210.97 pg/ml after vaccination. The percentage of proliferating CD4+ T cells in response to respective Leptospira strains significantly increased from 1.43 % and 1.25 % before vaccination to 24.11 % and 14.64 % after vaccination. Similar responses were also found in the CD8+ T cell subset. Vaccination also significantly enhanced the percentages of central memory CD4+ T cells from 12 % to 26.97 % and 27.65 %, central memory CD8+ T cells from 3 % to 9.47 % and 7.55 %, and effector CD8+ T cells from 3 % to 7.6 % and 6.42 %, as defined by the expression of CD45RA and CD62L, following stimulation with Canicola and Australis, respectively. Lastly, enhanced expression of the activation marker CD25 on T cells after vaccination was found. Together, our results show that next to IgG responses, also T cell responses are induced in dogs upon annual revaccination with a tetravalent Leptospira vaccine, potentially contributing to protection.
Assuntos
Doenças do Cão , Leptospira interrogans , Leptospira , Leptospirose , Cães , Animais , Vacinas Combinadas , Linfócitos T CD8-Positivos , Leucócitos Mononucleares , Vacinas Bacterianas , Doenças do Cão/prevenção & controle , Leptospirose/prevenção & controle , Leptospirose/veterinária , Vacinação/veterinária , Imunoglobulina GRESUMO
Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immunerelated diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.
Assuntos
Macrófagos , Monócitos , Animais , Cães , Diferenciação Celular , Macrófagos/metabolismo , Monócitos/metabolismo , FagocitoseRESUMO
Several reports demonstrated the susceptibility of domestic cats to SARS-CoV-2 infection. Here, we describe a thorough investigation of the immune responses in cats after experimental SARS-CoV-2 inoculation, along with the characterization of infection kinetics and pathological lesions. Specific pathogen-free domestic cats (n = 12) were intranasally inoculated with SARS-CoV-2 and subsequently sacrificed on DPI (days post-inoculation) 2, 4, 7 and 14. None of the infected cats developed clinical signs. Only mild histopathologic lung changes associated with virus antigen expression were observed mainly on DPI 4 and 7. Viral RNA was present until DPI 7, predominantly in nasal and throat swabs. The infectious virus could be isolated from the nose, trachea and lungs until DPI 7. In the swab samples, no biologically relevant SARS-CoV-2 mutations were observed over time. From DPI 7 onwards, all cats developed a humoral immune response. The cellular immune responses were limited to DPI 7. Cats showed an increase in CD8+ cells, and the subsequent RNA sequence analysis of CD4+ and CD8+ subsets revealed a prominent upregulation of antiviral and inflammatory genes on DPI 2. In conclusion, infected domestic cats developed a strong antiviral response and cleared the virus within the first week after infection without overt clinical signs and relevant virus mutations.
Assuntos
COVID-19 , Animais , Gatos , COVID-19/patologia , SARS-CoV-2 , Pulmão , Imunidade HumoralRESUMO
Mandatory potency testing of Leptospira vaccine batches relies partially on in vivo procedures, requiring large numbers of laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identified. We investigated innate immune responsiveness induced by inactivated L. interrogans serogroups Canicola and Icterohaemorrhagiae, and two bivalent, non-adjuvanted canine Leptospira vaccines containing the same serogroups. First, the transcriptome and proteome analysis of a canine monocyte/macrophage 030-D cell line stimulated with Leptospira strains, and vaccine B revealed more than 900 DEGs and 23 DEPs in common to these three stimuli. Second, comparison of responses induced by vaccine B and vaccine D revealed a large overlap in DEGs and DEPs as well, suggesting potential to identify biomarkers indicative of Leptospira vaccine quality. Because not many common DEPs were identified, we selected seven molecules from the identified DEGs, associated with pathways related to innate immunity, of which CXCL-10, IL-1ß, SAA, and complement C3 showed increased secretion upon stimulation with both Leptospira vaccines. These molecules could be interesting targets for development of biomarker-based assays for Leptospira vaccine quality control in the future. Additionally, this study contributes to the understanding of the mechanisms by which Leptospira vaccines induce innate immune responses in the dog.
Assuntos
Doenças do Cão , Leptospira , Leptospirose , Animais , Vacinas Bacterianas , Biomarcadores , Cães , Imunidade Inata , Leptospirose/prevenção & controle , Leptospirose/veterinária , Proteoma , Células Secretoras de Somatostatina , Transcriptoma , Vacinas CombinadasRESUMO
Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 co-inducer, we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin, which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line, also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that leucinostatin might be applied as a new co-inducer for Hsp70 expression.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Células Epiteliais/citologia , Proteínas de Choque Térmico HSP70/metabolismo , Epitélio Pigmentado da Retina/citologia , Estresse Fisiológico/efeitos dos fármacos , Animais , Células Cultivadas , CãesRESUMO
Tolerogenic dendritic cells (tolDCs) are a promising treatment modality for diseases caused by a breach in immune tolerance, such as rheumatoid arthritis. Current medication for these diseases is directed toward symptom suppression but no real cure is available yet. TolDC-based therapy aims to restore immune tolerance in an antigen-specific manner. Here we used a mouse model to address two major questions: (i) is a maturation stimulus needed for tolDC function in vitro and in vivo and is maturation required for functioning in experimental arthritis and (ii) can tolDCs modulate CD4+ T cell responses? To answer these questions, we compared matured and immature dexamethasone/vitamin D3-generated tolDCs in vitro. Subsequently, we co-transferred these tolDCs with naïve or effector CD4+ T cells to study the characteristics of transferred T cells after 3 days with flow cytometry and Luminex multiplex assays. In addition, we tested the suppressive capabilities of tolDCs in an experimental arthritis model. We found that tolDCs cannot only modulate naïve CD4+ T cell responses as shown by fewer proliferated and activated CD4+ T cells in vivo, but also effector CD4+ T cells. In addition, Treg (CD4+CD25+FoxP3+) expansions were seen in the proliferating cell population in the presence of tolDCs. Furthermore, we show that administered tolDCs are capable to inhibit arthritis in the proteoglycan-induced arthritis model. However, a maturation stimulus is needed for tolDCs to manifest this tolerizing function in an inflammatory environment. Our data will be instrumental for optimization of future tolDC therapies for autoimmune diseases.
Assuntos
Artrite Experimental/etiologia , Artrite Experimental/metabolismo , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Tolerância Imunológica , Animais , Artrite Experimental/patologia , Técnicas de Cocultura , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Imunomodulação , Imunofenotipagem , Ativação Linfocitária/imunologia , Masculino , Camundongos , Peptídeos/imunologia , Proteoglicanas/metabolismoRESUMO
Technologies that enable induction of therapeutic tolerance may revolutionize the treatment of autoimmune diseases by their supposed potential to induce drug-free and lasting disease remission. In combination with diagnostic tests that screen for individuals at risk, these approaches may offer chances to halt disease before serious damage in the tissues can occur. In fact, for healthy individuals at risk, this could lead to a preventive form of vaccination. For therapeutic tolerance to re-instate natural self-tolerance it seems essential to induce tolerance for the critical autoantigens involved in disease. However, for most autoimmune diseases such antigens are poorly defined. This is the case for both disease inciting autoantigens and antigens that become involved through epitope spreading. A possible source of surrogate auto-antigens expressed in tissues during inflammation are heat shock proteins (HSP) or stress proteins. In this mini-review we discuss unique characteristics of HSP which provide them with the capacity to inhibit inflammatory processes. Various studies have shown that epitopes of HSP60 and HSP70 molecules can function as vaccines to downregulate a variety of autoimmune inflammatory diseases. Currently, several research groups are developing cell therapies with the intention to reach therapeutic tolerance. In this review, in which we are proposing to ex vivo load tolerant dendritic cells with a Treg inducing HSP70 derived peptide called B29, we are discussing the chances to develop this as an autologous tolDC therapeutic tolerance therapy for rheumatoid arthritis.
Assuntos
Artrite Reumatoide/terapia , Autoantígenos/imunologia , Proteínas de Choque Térmico/imunologia , Tolerância Imunológica , Transferência Adotiva , Animais , Artrite Reumatoide/imunologia , Células Dendríticas/imunologia , Humanos , Linfócitos T Reguladores/imunologiaRESUMO
DC-specific ICAM3-grabbing non-integrin (DC-SIGN), which is expressed on DCs, can interact with a variety of pathogens such as HIV-1, hepatitis C, Ebola, cytomegalovirus, Dengue virus, Mycobacterium, Leishmania, and Candida albicans. We demonstrate that human milk can inhibit the DC-SIGN-mediated transfer of HIV-1 to CD4+ T lymphocytes as well as viral transfer by both immature and mature DCs. The inhibitory factor directly interacted with DC-SIGN and prevented the HIV-1 gp120 envelope protein from binding to the receptor. The human milk proteins lactoferrin, alpha-lactalbumin, lysozyme, beta-casein, and secretory leukocyte protease inhibitor did not bind DC-SIGN or demonstrate inhibition of viral transfer. The inhibitory effect could be fully alleviated with an Ab recognizing the Lewis X (LeX) sugar epitope, commonly found in human milk. LeX in polymeric form or conjugated to protein could mimic the inhibitory activity, whereas free LeX sugar epitopes could not. We reveal that a LeX motif present in human milk can bind to DC-SIGN and thereby prevent the capture and subsequent transfer of HIV-1 to CD4+ T lymphocytes. The presence of such a DC-SIGN-binding molecule in human milk may both influence antigenic presentation and interfere with pathogen transfer in breastfed infants.
Assuntos
Linfócitos T CD4-Positivos/virologia , Moléculas de Adesão Celular/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Lectinas Tipo C/metabolismo , Antígenos CD15/metabolismo , Leite Humano/imunologia , Leite Humano/metabolismo , Receptores de Superfície Celular/metabolismo , Trissacarídeos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Moléculas de Adesão Celular/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/virologia , Feminino , Infecções por HIV/transmissão , Humanos , Transmissão Vertical de Doenças Infecciosas , Lectinas Tipo C/fisiologia , Antígenos CD15/fisiologia , Ligação Proteica/imunologia , Receptores de Superfície Celular/fisiologia , Trissacarídeos/fisiologiaRESUMO
Most traditional vaccines are administered via the intramuscular route. Other routes of administration however, can induce equal or improved protective memory responses and might provide practical advantages such as needle-free immunization, dose sparing and induction of tissue-specific (mucosal) immunity. Here we explored the differences in immunological outcome after immunization with model antigens via two promising immunization routes (intradermal and intranasal) with or without the experimental adjuvant and TLR7/8-agonist R848. Because the adaptive immune response is largely determined by the local innate cells at the site of immunization, the effect of R848-adjuvation on local cellular recruitment, antigenic uptake by antigen-presenting cells and the initiation of the adaptive response were analyzed for the two routes of administration. We show a general immune-stimulating effect of R848 irrespective of the route of administration. This includes influx of neutrophils, macrophages and dendritic cells to the respective draining lymph nodes and an increase in antigen-positive antigen-presenting cells which leads for both intradermal and intranasal immunization to a mainly TH1 response. Furthermore, both intranasal and intradermal R848-adjuvated immunization induces a local shift in DC subsets; frequencies of CD11b+DC increase whereas CD103+DC decrease in relative abundance in the draining lymph node. In spite of these similarities, the outcome of immune responses differs for the respective immunization routes in both magnitude and cytokine profile. Via the intradermal route, the induced T-cell response is higher compared to that after intranasal immunization, which corresponds with the local higher uptake of antigen by antigen-presenting cells after intradermal immunization. Furthermore, R848-adjuvation enhances ex vivo IL-10 and IL-17 production after intranasal, but not intradermal, T-cell activation. Quite the opposite, intradermal immunization leads to a decrease in IL-10 production by the vaccine induced T-cells. This knowledge may lead to a more rational development of novel adjuvanted vaccines administered via non-traditional routes.
Assuntos
Adjuvantes Imunológicos , Imidazóis , Imunidade , Vacinação , Vacinas/imunologia , Administração Intranasal , Animais , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Vias de Administração de Medicamentos , Imunização , Injeções Intradérmicas , Camundongos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinação/métodos , Vacinas/administração & dosagemRESUMO
Probiotics and probiotic-related nutritional interventions have been described to have beneficial effects on immune homeostasis and gut health. In previous studies, Lactobacillus rhamnosus GG (LGG) soluble mediators (LSM) have been demonstrated to exert beneficial effects in preclinical models of allergic sensitization, bacterial infection, and intestinal barrier function. In the context of allergic diseases, differentiation of dendritic cells (DCs) and their interactions with T cell populations are crucial for driving tolerogenic responses. In this study, we set out to evaluate whether these LSM can modulate DC maturation and have an impact on prompting protective and/or tolerogenic T cell responses. Monocytes were isolated from PBMC of healthy blood donors and cultured in the presence of GM-CSF, IL-4, and LSM or unconditioned bacterial culture medium control (UCM) during 6 days to induce DC differentiation. Subsequently, these DCs were matured in the presence of TNF-α for 1 day and analyzed for their phenotype and ability to induce autologous T cell activation and differentiation to model recall antigens. After 7 days of co-culture, T cells were analyzed for activation and differentiation by flow cytometry of intracellular cytokines (IFN-γ, IL-2, IL-10, and IL-17A), activation markers (CD25), and Foxp3+ expression. LSM did not alter DC numbers or maturation status. However, these DCs did show improved capacity to induce a T cell response as shown by increased IL-2 and IFN-γ producing T cell populations upon stimulation with recall antigens. These enhanced recall responses coincided with enhanced Foxp3+ expression that was not observed when T cells were cultured in the presence of UCM-treated DCs. By contrast, the number of activated T cells (determined by CD25 expression) was only slightly increased. In conclusion, this study reveals that LSM can influence adaptive immune responses as shown by the modulation of DC functionality. These mechanisms might contribute to previous observed effects in animal models in vivo. Altogether, these results suggest that LSM may provide an alternative to live probiotics in case life bacteria may not be used because of health conditions, although further clinical testing is needed.
RESUMO
Dendritic cells (DCs) play a central role in balancing immune responses between tolerance induction and immune activation. Under steady state conditions DCs continuously sample antigens, leading to tolerance, whereas inflammatory conditions activate DCs, inducing immune activation. DCs express C-type lectin receptors (CLRs) for antigen capture and presentation, whereas Toll-like receptors (TLRs) are involved in pathogen recognition and DC activation. Recent data demonstrate that communication between TLRs and CLRs can affect the direction of immune responses. Several pathogens specifically target CLRs to subvert this communication to escape immune surveillance, either by inducing tolerance or skewing the protective immune responses.
Assuntos
Carboidratos/imunologia , Doenças Transmissíveis/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica , Lectinas Tipo C/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Animais , Apresentação de Antígeno/imunologia , Bactérias/imunologia , Bactérias/patogenicidade , Doenças Transmissíveis/patologia , Humanos , Imunidade , Schistosoma/imunologia , Schistosoma/patogenicidade , Receptores Toll-Like , Vírus/imunologia , Vírus/patogenicidadeRESUMO
Neutrophils are instrumental in innate immunity by mediating immediate removal of pathogens. Recent findings demonstrate that they are able to communicate with dendritic cells, which form a link between innate and adaptive immunity by stimulating long-term memory T cell responses. This communication occurs through the cell-surface receptors Mac-1 and carcinoembryonic antigen-related cellular adhesion molecule on neutrophils, which interact with C-type lectins such as dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN). Clearly, dendritic cells can prolong the lifespan of neutrophils, whereas neutrophils could direct dendritic cell-mediated T cell responses.
Assuntos
Comunicação Celular/imunologia , Células Dendríticas/imunologia , Neutrófilos/imunologia , Animais , Moléculas de Adesão Celular/imunologia , Humanos , Imunidade Ativa , Imunidade Inata , Inflamação/imunologia , Lectinas Tipo C/imunologia , Fígado/imunologia , Infiltração de Neutrófilos , Receptores de Superfície Celular/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Autoimmune and other chronic inflammatory diseases (AID) are prevalent diseases which can severely impact the quality of life of those that suffer from the disease. In most cases, the etiology of these conditions have remained unclear. Immune responses that take place e.g. during natural infection or after vaccination are often linked with the development or exacerbation of AID. It is highly debated if vaccines induce or aggravate AID and in particular adjuvants are mentioned as potential cause. Since vaccines are given on a large scale to healthy individuals but also to elderly and immunocompromised individuals, more research is warranted. Non-specific induction of naïve or memory autoreactive T cells via bystander activation is one of the proposed mechanisms of how vaccination might be involved in AID. During bystander activation, T cells unrelated to the antigen presented can be activated without (strong) T cell receptor (TCR) ligation, but via signals derived from the ongoing response directed against the vaccine-antigen or adjuvant at hand. In this study we have set up a TCR transgenic T cell transfer mouse model by which we were able to measure local bystander activation of transferred and labeled CD4+ T cells. Intramuscular injection with the highly immunogenic Complete Freund's Adjuvant (CFA) led to local in vivo proliferation and activation of intravenously transferred CD4+ T cells in the iliac lymph node. This local bystander activation was also observed after CFA prime and Incomplete Freund's Adjuvant (IFA) boost injection. Furthermore, we showed that an antigen specific response is sufficient for the induction of a bystander activation response and the general, immune stimulating effect of CFA or IFA does not appear to increase this effect. In other words, no evidence was obtained that adjuvation of antigen specific responses is essential for bystander activation.
Assuntos
Adjuvantes Imunológicos , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Adjuvante de Freund/imunologia , Inflamação/etiologia , Proteoglicanas/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos/efeitos adversos , Doenças Autoimunes/etiologia , Doenças Autoimunes/imunologia , Doença Crônica , Adjuvante de Freund/efeitos adversos , Humanos , Inflamação/imunologia , Ativação Linfocitária , Masculino , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteoglicanas/efeitos adversos , Vacinação/efeitos adversos , Vacinas/efeitos adversosRESUMO
The fundamental problem of autoimmune diseases is the failure of the immune system to downregulate its own potentially dangerous cells, which leads to destruction of tissue expressing the relevant autoantigens. Current immunosuppressive therapies offer relief but fail to restore the basic condition of self-tolerance. They do not induce long-term physiological regulation resulting in medication-free disease remissions. Heat shock proteins (HSPs) have shown to possess the capacity of inducing lasting protective immune responses in models of experimental autoimmune diseases. Especially mycobacterial HSP60 and HSP70 were shown to induce disease inhibitory IL-10-producing regulatory T cells in many different models. This in itself may seem enigmatic, since based on earlier studies, HSPs were also coined sometimes as pro-inflammatory damage-associated molecular patterns. First clinical trials with HSPs in rheumatoid arthritis and type I diabetes have also indicated their potential to restore tolerance in autoimmune diseases. Data obtained from the models have suggested three aspects of HSP as being critical for this tolerance promoting potential: 1. evolutionary conservation, 2. most frequent cytosolic/nuclear MHC class II natural ligand source, and 3. upregulation under (inflammatory) stress. The combination of these three aspects, which are each relatively unique for HSP, may provide an explanation for the enigmatic immune tolerance promoting potential of HSP.
RESUMO
Vaccines often contain adjuvants to strengthen the response to the vaccine antigen. However, their modes of action at the site of injection (SOI) are poorly understood. Therefore, we assessed the local effects of adjuvant on the innate immune system in mice. We investigated the safe, widely used adjuvants MF59 and aluminum hydroxide (alum), as well as trehalose-6,6'-dibehenate (TDB), Complete Freund's Adjuvant (CFA) and the Toll-Like-Receptor-ligands lipopolysaccharide (LPS) and Pam3CysSerLys4 (Pam3CSK4). We assessed muscle immune cell infiltration after adjuvant injection and observed 16h post immunization (hpi) an increased influx with CFA, MF59 and TDB, but not with alum, LPS or Pam3CSK4. An elevated influx with the latter three became visible only 72hpi. Contribution of granulocytes, macrophages and dendritic cells to the influx differed per adjuvant and in time. Adjuvants generally induced a local pro-inflammatory micro-milieu that was transient except for CFA and TDB. The gene expression of CXCL-1, CCL-2 and CCL-5, involved in recruitment of immune cells, varied per adjuvant and corresponded grossly with the observed influx of granulocytes and monocytes/macrophages. Muscles injected with CFA or MF59 (when co-injected with peptide) resulted in APC ex vivo capable to induce proliferation of peptide-specific T-cells. By adding in vitro an excess of peptide to the APC/T cell co-cultures, we observed an adjuvant-enhanced co-stimulation or antigen presentation by APC after CFA- but not MF59-injection. After TDB-injection this effect was observed only at 72hpi, but not 24hpi. Thus the cellular influx profile and the local cytokine and chemokine micro-milieu in the muscle were strongly influenced by the type of adjuvant. Additionally, the capacity of muscle APC to load and present antigen was affected by the adjuvant. These findings may assist the development of novel adjuvanted vaccines in a more rational manner.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Células Apresentadoras de Antígenos/imunologia , Imunidade Inata , Animais , Feminino , Injeções Intramusculares , Camundongos Endogâmicos BALB C , Músculos/imunologiaRESUMO
Regulatory T cells (Treg) function in the prevention of excessive inflammation and maintenance of immunological homeostasis. However, these cells may also interfere with resolution of infections or with immune reactions following vaccination. Effects of Treg on vaccine responses are nowadays investigated, but the impact of vaccination on Treg homeostasis is still largely unknown. This may be a relevant safety aspect, since loss of tolerance through reduced Treg may trigger autoimmunity. In exploratory clinical trials, healthy adults were vaccinated with an influenza subunit vaccine plus or minus the adjuvant MF59®, an adjuvanted hepatitis B subunit vaccine or a live attenuated yellow fever vaccine. Frequencies and phenotypes of resting (rTreg) and activated (aTreg) subpopulations of circulating CD4+ Treg were determined and compared to placebo immunization. Vaccination with influenza vaccines did not result in significant changes in Treg frequencies and phenotypes. Vaccination with the hepatitis B vaccine led to slightly increased frequencies of both rTreg and aTreg subpopulations and a decrease in expression of functionality marker CD39 on aTreg. The live attenuated vaccine resulted in a decrease in rTreg frequency, and an increase in expression of activation marker CD25 on both subpopulations, possibly indicating a conversion from resting to migratory aTreg due to vaccine virus replication. To study the more local effects of vaccination on Treg in lymphoid organs, we immunized mice and analyzed the CD4+ Treg frequency and phenotype in draining lymph nodes and spleen. Vaccination resulted in a transient local decrease in Treg frequency in lymph nodes, followed by a systemic Treg increase in the spleen. Taken together, we showed that vaccination with vaccines with an already established safe profile have only minimal impact on frequencies and characteristics of Treg over time. These findings may serve as a bench-mark of inter-individual variation of Treg frequencies and phenotypes following vaccination.
Assuntos
Linfócitos T Reguladores/efeitos dos fármacos , Vacinas Virais/farmacologia , Adulto , Animais , Feminino , Vacinas contra Hepatite B/imunologia , Vacinas contra Hepatite B/farmacologia , Humanos , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/farmacologia , Contagem de Linfócitos , Masculino , Camundongos , Fragmentos de Peptídeos , Protrombina , Linfócitos T Reguladores/imunologia , Vacinas/imunologia , Vacinas/farmacologia , Vacinas Virais/imunologia , Vacina contra Febre Amarela/imunologia , Vacina contra Febre Amarela/farmacologiaRESUMO
Disease suppressive T cell regulation may depend on cognate interactions of regulatory T cells with self-antigens that are abundantly expressed in the inflamed tissues. Heat shock proteins (HSPs) are by their nature upregulated in stressed cells and therefore abundantly present as potential targets for such regulation. HSP immunizations have led to inhibition of experimentally induced inflammatory conditions in various models. However, re-establishment of tolerance in the presence of an ongoing inflammatory process has remained challenging. Since tolerogenic DCs (tolDCs) have the combined capacity of mitigating antigen-specific inflammatory responses and of endowing T cells with regulatory potential, it seems attractive to combine the anti-inflammatory qualities of tolDCs with those of HSPs.