RESUMO
Obesity is characterized by the expansion of the adipose tissue, usually accompanied by inflammation, with a prominent role of macrophages infiltrating the visceral adipose tissue (VAT). This chronic inflammation is a major driver of obesity-associated comorbidities. Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of various biological functions and the maintenance of the homeostasis of different tissues. In this study, we aimed to gain new insights into the expression and functional role of FHL2 in VAT in diet-induced obesity. We found enhanced FHL2 expression in the VAT of mice with Western-type diet (WTD)-induced obesity and obese humans and identified macrophages as the cellular source of enhanced FHL2 expression in VAT. In mice with FHL2 deficiency (FHL2KO), WTD feeding resulted in reduced body weight gain paralleled by enhanced energy expenditure and uncoupling protein 1 (UCP1) expression, indicative of activated thermogenesis. In human VAT, FHL2 was inversely correlated with UCP1 expression. Furthermore, macrophage infiltration and the expression of the chemokine MCP-1, a known promotor of macrophage accumulation, was significantly reduced in WTD-fed FHL2KO mice compared with wild-type (wt) littermates. While FHL2 depletion did not affect the differentiation or lipid metabolism of adipocytes in vitro, FHL2 depletion in macrophages resulted in reduced expressions of MCP-1 and the neuropeptide Y (NPY). Furthermore, WTD-fed FHL2KO mice showed reduced NPY expression in VAT compared with wt littermates, and NPY expression was enhanced in VAT resident macrophages of obese individuals. Stimulation with recombinant NPY induced not only UCP1 expression and lipid accumulation but also MCP-1 expression in adipocytes. Collectively, these findings indicate that FHL2 is a positive regulator of NPY and MCP-1 expression in macrophages and herewith closely linked to the mechanism of obesity-associated lipid accumulation and inflammation in VAT. Thus, FHL2 appears as a potential novel target to interfere with the macrophage-adipocyte crosstalk in VAT for treating obesity and related metabolic disorders.
Assuntos
Gordura Intra-Abdominal , Neuropeptídeo Y , Animais , Humanos , Camundongos , Tecido Adiposo/metabolismo , Dieta , Dieta Hiperlipídica , Inflamação/metabolismo , Gordura Intra-Abdominal/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Lipídeos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neuropeptídeo Y/metabolismo , Obesidade/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Microglial activation during neuroinflammation is crucial for coordinating the immune response against neuronal tissue, and the initial response of microglia determines the severity of neuro-inflammatory diseases. The CD83 molecule has been recently shown to modulate the activation status of dendritic cells and macrophages. Although the expression of CD83 is associated with early microglia activation in various disease settings, its functional relevance for microglial biology has been elusive. Here, we describe a thorough assessment of CD83 regulation in microglia and show that CD83 expression in murine microglia is not only associated with cellular activation but also with pro-resolving functions. Using single-cell RNA-sequencing, we reveal that conditional deletion of CD83 results in an over-activated state during neuroinflammation in the experimental autoimmune encephalomyelitis model. Subsequently, CD83-deficient microglia recruit more pathogenic immune cells to the central nervous system, deteriorating resolving mechanisms and exacerbating the disease. Thus, CD83 in murine microglia orchestrates cellular activation and, consequently, also the resolution of neuroinflammation.
Assuntos
Encefalomielite Autoimune Experimental , Camundongos , Animais , Microglia/metabolismo , Doenças Neuroinflamatórias , Sistema Nervoso Central/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Excessive macrophage (Mφ) activation results in chronic inflammatory responses or autoimmune diseases. Therefore, identification of novel immune checkpoints on Mφ, which contribute to resolution of inflammation, is crucial for the development of new therapeutic agents. Herein, we identify CD83 as a marker for IL-4 stimulated pro-resolving alternatively activated Mφ (AAM). Using a conditional KO mouse (cKO), we show that CD83 is important for the phenotype and function of pro-resolving Mφ. CD83-deletion in IL-4 stimulated Mφ results in decreased levels of inhibitory receptors, such as CD200R and MSR-1, which correlates with a reduced phagocytic capacity. In addition, CD83-deficient Mφ upon IL-4 stimulation, show an altered STAT-6 phosphorylation pattern, which is characterized by reduced pSTAT-6 levels and expression of the target gene Gata3. Concomitantly, functional studies in IL-4 stimulated CD83 KO Mφ reveal an increased production of pro-inflammatory mediators, such as TNF-α, IL-6, CXCL1 and G-CSF. Furthermore, we show that CD83-deficient Mφ have enhanced capacities to stimulate the proliferation of allo-reactive T cells, which was accompanied by reduced frequencies of Tregs. In addition, we show that CD83 expressed by Mφ is important to limit the inflammatory phase using a full-thickness excision wound healing model, since inflammatory transcripts (e.g. Cxcl1, Il6) were increased, whilst resolving transcripts (e.g. Ym1, Cd200r, Msr-1) were decreased in wounds at day 3 after wound infliction, which reflects the CD83 resolving function on Mφ also in vivo. Consequently, this enhanced inflammatory milieu led to an altered tissue reconstitution after wound infliction. Thus, our data provide evidence that CD83 acts as a gatekeeper for the phenotype and function of pro-resolving Mφ.
Assuntos
Proteínas de Checkpoint Imunológico , Interleucina-4 , Animais , Camundongos , Macrófagos , Fagócitos , InflamaçãoRESUMO
Historically, the eye has been considered as an organ free of lymphatic vessels. In recent years, however, it became evident, that lymphatic vessels or lymphatic-like vessels contribute to several ocular pathologies at various peri- and intraocular locations. The aim of this review is to outline the pathogenetic role of ocular lymphatics, the respective molecular mechanisms and to discuss current and future therapeutic options based thereon. We will give an overview on the vascular anatomy of the healthy ocular surface and the molecular mechanisms contributing to corneal (lymph)angiogenic privilege. In addition, we present (i) current insights into the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea triggered e.g. by inflammation or trauma, (ii) the role of lymphatic vessels in different ocular surface pathologies such as dry eye disease, corneal graft rejection, ocular graft versus host disease, allergy, and pterygium, (iii) the involvement of lymphatic vessels in ocular tumors and metastasis, and (iv) the novel role of the lymphatic-like structure of Schlemm's canal in glaucoma. Identification of the underlying molecular mechanisms and of novel modulators of lymphangiogenesis will contribute to the development of new therapeutic targets for the treatment of ocular diseases associated with pathological lymphangiogenesis in the future. The preclinical data presented here outline novel therapeutic concepts for promoting transplant survival, inhibiting metastasis of ocular tumors, reducing inflammation of the ocular surface, and treating glaucoma. Initial data from clinical trials suggest first success of novel treatment strategies to promote transplant survival based on pretransplant corneal lymphangioregression.
Assuntos
Transplante de Córnea , Glaucoma , Vasos Linfáticos , Neoplasias , Humanos , Vasos Linfáticos/patologia , Córnea , Linfangiogênese , Glaucoma/patologia , Inflamação/patologia , Neoplasias/patologiaRESUMO
Macrophages (MΦ) are remarkably plastic cells, which assume phenotypes in every shade between a pro-inflammatory classical activation, and anti-inflammatory or resolving activation. Therefore, elucidation of mechanisms involved in shaping MΦ plasticity and function is key to understand their role during immunological balance. The immune-modulating CD83 molecule is expressed on activated immune cells and various tissue resident MΦ, rendering it an interesting candidate for affecting MΦ biology. However, in-depth analyses of the precise kinetics and trafficking of CD83 within pro-inflammatory, LPS activated bone-marrow-derived MΦ have not been performed. In this study, we show that activation with LPS leads to a very fast and strong, but transient increase of CD83 expression on these cells. Its expression peaks within 2 h of stimulation and is thereby faster than the early activation antigen CD69. To trace the CD83 trafficking through MΦs, we employed multiple inhibitors, thereby revealing a de novo synthesis and transport of the protein to the cell surface followed by lysosomal degradation, all within 6 h. Moreover, we found a similar expression kinetic and trafficking in human monocyte derived MΦ. This places CD83 at a very early point of MΦ activation suggesting an important role in decisions regarding the subsequent cellular fate.
Assuntos
Lipopolissacarídeos , Macrófagos , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Diferenciação Celular , Membrana Celular , FenótipoRESUMO
Alterations in macrophage (Mφ) polarization, function, and metabolic signature can foster development of chronic diseases, such as autoimmunity or fibrotic tissue remodeling. Thus, identification of novel therapeutic agents that modulate human Mφ biology is crucial for treatment of such conditions. Herein, we demonstrate that the soluble CD83 (sCD83) protein induces pro-resolving features in human monocyte-derived Mφ biology. We show that sCD83 strikingly increases the expression of inhibitory molecules including ILT-2 (immunoglobulin-like transcript 2), ILT-4, ILT-5, and CD163, whereas activation markers, such as MHC-II and MSR-1, were significantly downregulated. This goes along with a decreased capacity to stimulate alloreactive T cells in mixed lymphocyte reaction (MLR) assays. Bulk RNA sequencing and pathway analyses revealed that sCD83 downregulates pathways associated with pro-inflammatory, classically activated Mφ (CAM) differentiation including HIF-1A, IL-6, and cytokine storm, whereas pathways related to alternative Mφ activation and liver X receptor were significantly induced. By using the LXR pathway antagonist GSK2033, we show that transcription of specific genes (e.g., PPARG, ABCA1, ABCG1, CD36) induced by sCD83 is dependent on LXR activation. In summary, we herein reveal for the first time mechanistic insights into the modulation of human Mφ biology by sCD83, which is a further crucial preclinical study for the establishment of sCD83 as a new therapeutical agent to treat inflammatory conditions.
Assuntos
Antígeno CD83 , Macrófagos , Linfócitos T , Humanos , Diferenciação Celular , FenótipoRESUMO
To facilitate the recovery process of chronic and hard-to-heal wounds novel pro-resolving treatment options are urgently needed. We investigated the pro-regenerative properties of soluble CD83 (sCD83) on cutaneous wound healing, where sCD83 accelerated wound healing not only after systemic but also after topical application, which is of high therapeutic interest. Cytokine profile analyses revealed an initial upregulation of inflammatory mediators such as TNFα and IL-1ß, followed by a switch towards pro-resolving factors, including YM-1 and IL-10, both expressed by tissue repair macrophages. These cells are known to mediate resolution of inflammation and stimulate wound healing processes by secretion of growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), which promote vascularization as well as fibroblast and keratinocyte differentiation. In conclusion, we have found strong wound healing capacities of sCD83 beyond the previously described role in transplantation and autoimmunity. This makes sCD83 a promising candidate for the treatment of chronic- and hard-to-heal wounds.
Assuntos
Interleucina-10 , Fator de Necrose Tumoral alfa , Fator de Crescimento Epidérmico , Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Macrófagos , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologiaRESUMO
Due to their ability to trigger strong immune responses, adenoviruses (HAdVs) in general and the serotype5 (HAdV-5) in particular are amongst the most popular viral vectors in research and clinical application. However, efficient transduction using HAdV-5 is predominantly achieved in coxsackie and adenovirus receptor (CAR)-positive cells. In the present study, we used the transduction enhancer LentiBOOST® comprising the polycationic Polybrene to overcome these limitations. Using LentiBOOST®/Polybrene, we yielded transduction rates higher than 50% in murine bone marrow-derived dendritic cells (BMDCs), while maintaining their cytokine expression profile and their capability to induce T-cell proliferation. In human dendritic cells (DCs), we increased the transduction rate from 22% in immature (i)DCs or 43% in mature (m)DCs to more than 80%, without inducing cytotoxicity. While expression of specific maturation markers was slightly upregulated using LentiBOOST®/Polybrene on iDCs, no effect on mDC phenotype or function was observed. Moreover, we achieved efficient HAdV5 transduction also in human monocytes and were able to subsequently differentiate them into proper iDCs and functional mDCs. In summary, we introduce LentiBOOST® comprising Polybrene as a highly potent adenoviral transduction agent for new in-vitro applications in a set of different immune cells in both mice and humans.
Assuntos
Adenovírus Humanos/genética , Células Dendríticas/virologia , Monócitos/virologia , Transdução Genética , Adenovírus Humanos/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Células Dendríticas/imunologia , Eletroporação , Vetores Genéticos , Brometo de Hexadimetrina , Especificidade de Hospedeiro , Humanos , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Fenótipo , Internalização do VírusRESUMO
Immune responses reflect a complex interplay of cellular and extracellular components which define the microenvironment of a tissue. Therefore, factors that locally influence the microenvironment and re-establish tolerance might be beneficial to mitigate immune-mediated reactions, including the rejection of a transplant. In this study, we demonstrate that pre-incubation of donor tissue with the immune modulator soluble CD83 (sCD83) significantly improves graft survival using a high-risk corneal transplantation model. The induction of tolerogenic mechanisms in graft recipients was achieved by a significant upregulation of Tgfb, Foxp3, Il27, and Il10 in the transplant and an increase of regulatory dendritic cells (DCs), macrophages (Mφ), and T cells (Tregs) in eye-draining lymph nodes. The presence of sCD83 during in vitro DC and Mφ generation directed these cells toward a tolerogenic phenotype leading to reduced proliferation-stimulating activity in MLRs. Mechanistically, sCD83 induced a tolerogenic Mφ and DC phenotype, which favors Treg induction and significantly increased transplant survival after adoptive cell transfer. Conclusively, pre-incubation of corneal grafts with sCD83 significantly prolongs graft survival by modulating recipient Mφ and DCs toward tolerance and thereby establishing a tolerogenic microenvironment. This functional strategy of donor graft pre-treatment paves the way for new therapeutic options in the field of transplantation.
Assuntos
Células Dendríticas , Sobrevivência de Enxerto , Tolerância Imunológica , Macrófagos , Linfócitos T ReguladoresRESUMO
PURPOSE: Limbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. METHODS: Human corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. RESULTS: LMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. CONCLUSION: These findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.
Assuntos
Epitélio Corneano , Limbo da Córnea , Animais , Células Cultivadas , Células Endoteliais , Células Epiteliais , Humanos , Melanócitos , Camundongos , Secretoma , Nicho de Células-Tronco , Células-TroncoRESUMO
ANP32B belongs to a family of evolutionary conserved acidic nuclear phosphoproteins (ANP32A-H). Family members have been described as multifunctional regulatory proteins and proto-oncogenic factors affecting embryonic development, cell proliferation, apoptosis, and gene expression at various levels. Involvement of ANP32B in multiple processes of cellular life is reflected by the previous finding that systemic gene knockout (KO) of Anp32b leads to embryonic lethality in mice. Here, we demonstrate that a conditional KO of Anp32b is well tolerated in adult animals. However, after immune activation splenocytes isolated from Anp32b KO mice showed a strong commitment towards Th17 immune responses. Therefore, we further analyzed the respective animals in vivo using an experimental autoimmune encephalomyelitis (EAE) model. Interestingly, an exacerbated clinical score was observed in the Anp32b KO mice. This was accompanied by the finding that animal-derived T lymphocytes were in a more activated state, and RNA sequencing analyses revealed hyperactivation of several T lymphocyte-associated immune modulatory pathways, attended by significant upregulation of Tfh cell numbers that altogether might explain the observed strong autoreactive processes. Therefore, Anp32b appears to fulfill a role in regulating adequate adaptive immune responses and, hence, may be involved in dysregulation of pathways leading to autoimmune disorders and/or immune deficiencies.
Assuntos
Imunidade Adaptativa/genética , Proteínas de Ciclo Celular/genética , Encefalomielite Autoimune Experimental/genética , Inflamação/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Imunidade Adaptativa/imunologia , Animais , Apoptose/genética , Proliferação de Células/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica/genética , Humanos , Fatores Imunológicos/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Células Th17/imunologiaRESUMO
Deficiency of CD83 in thymic epithelial cells (TECs) dramatically impairs thymic CD4 T cell selection. CD83 can exert cell-intrinsic and -extrinsic functions through discrete protein domains, but it remains unclear how CD83's capacity to operate through these alternative functional modules relates to its crucial role in TECs. In this study, using viral reconstitution of gene function in TECs, we found that CD83's transmembrane domain is necessary and sufficient for thymic CD4 T cell selection. Moreover, a ubiquitination-resistant MHCII variant restored CD4 T cell selection in Cd83(-/-) mice. Although during dendritic cell maturation CD83 is known to stabilize MHCII through opposing the ubiquitin ligase March1, regulation of March1 did not account for CD83's TEC-intrinsic role. Instead, we provide evidence that MHCII in cortical TECs (cTECs) is targeted by March8, an E3 ligase of as yet unknown physiological substrate specificity. Ablating March8 in Cd83(-/-) mice restored CD4 T cell development. Our results identify CD83-mediated MHCII stabilization through antagonism of March8 as a novel functional adaptation of cTECs for T cell selection. Furthermore, these findings suggest an intriguing division of labor between March1 and March8 in controlling inducible versus constitutive MHCII expression in hematopoietic antigen-presenting cells versus TECs.
Assuntos
Antígenos CD/fisiologia , Linfócitos T CD4-Positivos/imunologia , Células Epiteliais/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunoglobulinas/fisiologia , Glicoproteínas de Membrana/fisiologia , Timo/imunologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Células Dendríticas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ubiquitinação , Antígeno CD83RESUMO
To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B' core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8(+) T cells. Second, we generated the two-vector-based "modular promoter" system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B' core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/genética , Células Dendríticas/fisiologia , Vetores Genéticos , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Transgenes , Adenoviridae/genética , Antígenos CD/genética , Linfócitos T CD8-Positivos/imunologia , Citocinas/genética , Células Dendríticas/imunologia , Células HeLa , Fatores de Transcrição de Choque Térmico , Humanos , Imunoglobulinas/genética , Interleucina-12/genética , Antígeno MART-1/genética , Glicoproteínas de Membrana/genética , Proteína bcl-X/genética , Antígeno CD83RESUMO
The understanding of the immune response is right now at the center of biomedical research. There are growing expectations that immune-based interventions will in the midterm provide new, personalized, and targeted therapeutic options for many severe and highly prevalent diseases, from aggressive cancers to infectious and autoimmune diseases. To this end, immunology should surpass its current descriptive and phenomenological nature, and become quantitative, and thereby predictive.Immunology is an ideal field for deploying the tools, methodologies, and philosophy of systems biology, an approach that combines quantitative experimental data, computational biology, and mathematical modeling. This is because, from an organism-wide perspective, the immunity is a biological system of systems, a paradigmatic instance of a multi-scale system. At the molecular scale, the critical phenotypic responses of immune cells are governed by large biochemical networks, enriched in nested regulatory motifs such as feedback and feedforward loops. This network complexity confers them the ability of highly nonlinear behavior, including remarkable examples of homeostasis, ultra-sensitivity, hysteresis, and bistability. Moving from the cellular level, different immune cell populations communicate with each other by direct physical contact or receiving and secreting signaling molecules such as cytokines. Moreover, the interaction of the immune system with its potential targets (e.g., pathogens or tumor cells) is far from simple, as it involves a number of attack and counterattack mechanisms that ultimately constitute a tightly regulated multi-feedback loop system. From a more practical perspective, this leads to the consequence that today's immunologists are facing an ever-increasing challenge of integrating massive quantities from multi-platforms.In this chapter, we support the idea that the analysis of the immune system demands the use of systems-level approaches to ensure the success in the search for more effective and personalized immune-based therapies.
Assuntos
Alergia e Imunologia , Biologia Computacional , Medicina , Modelos Biológicos , Biologia de Sistemas , Animais , Biologia Computacional/métodos , Epitopos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade , Imunomodulação , Medicina/métodos , Transdução de Sinais , Biologia de Sistemas/métodosRESUMO
Dendritic cells (DC) and macrophages (MΦ) play a pivotal role in antimicrobial defense, in the regulation of immune responses, and in maintaining tissue homeostasis. The analysis of DC and MΦ function relies on primary cells albeit these cells are known to be difficult to transfect. This makes the use of small interfering RNA (siRNA) for targeted manipulation of gene expression by RNA interference difficult. In the following chapter, we provide a detailed protocol for the successful transfer of siRNA via electroporation into a defined population of mouse bone marrow-derived MΦ or DC that does not cause toxicity to the myeloid cells or nonspecific alterations of their biological functions. Factors that influence the transfection and knockdown rate will be highlighted.
Assuntos
Células da Medula Óssea/citologia , Células Dendríticas/metabolismo , Eletroporação/métodos , Macrófagos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Animais , Células Dendríticas/citologia , Técnicas de Silenciamento de Genes , Macrófagos/citologia , CamundongosRESUMO
Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, ß-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.
Assuntos
Antineoplásicos/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Toxina Diftérica/uso terapêutico , Interleucina-2/uso terapêutico , Melanoma/terapia , Neoplasias Cutâneas/terapia , Linfócitos T Reguladores/efeitos dos fármacos , Vacinas Anticâncer , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Terapia Combinada , Células Dendríticas/imunologia , Células Dendríticas/transplante , Citometria de Fluxo , Humanos , Tolerância Imunológica , Teste de Cultura Mista de Linfócitos , Melanoma/imunologia , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas Recombinantes de Fusão/uso terapêutico , Neoplasias Cutâneas/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
The demand for human monocyte-derived dendritic cells (moDCs), as well as for primary human B and T lymphocytes for immunological research purposes has been increased in recent years. Classically, these monocytes are isolated from blood, leukapheresis products or buffy coats of healthy donors by plastic adherence of peripheral blood mononuclear cells (PBMCs), followed by stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, while lymphocytes are usually isolated from the non-adherent fraction (NAF) by magnetic cell sorting. However, donor-blood is a limited resource and not every blood bank offers leukapheresis products or buffy coats for laboratory use. Additionally, a leukapheresis is very expensive and also the generation/isolation of cells is time- and cost-intensive. To overcome some of these obstacles, we evaluated if low-cost leukoreduction system chambers (LRSCs), which arise after routine donor plateletpheresis procedures, and are usually discarded, would be an alternative and appropriate source of PBMCs to generate moDCs and to isolate lymphocytes. By analyzing the number and phenotype of immature and mature dendritic cells (DCs), as well as of B and T lymphocytes derived from LRSCs, we found all cells to be of high quantity and quality. Further investigations on DCs comprising transwell migration assays, allogeneic mixed lymphocyte reactions (MLR), cytokine secretion assays, and cytotoxic T cell induction assays revealed high migratory, as well as stimulatory capacity of these cells. In addition, DCs and T cells were efficiently electroporated with mRNA and showed characteristic cytokine production after co-culture, demonstrating LRSCs as an efficient, valid, and economic source for generation of moDCs and lymphocytes for research purposes.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Células Dendríticas/citologia , Leucócitos Mononucleares/citologia , Plaquetoferese/métodos , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Criopreservação , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-4/metabolismoRESUMO
Modulation of immune responses is one of the main research aims in transplant immunology. In this study, we investigate the local immunomodulatory properties of soluble CD83 (sCD83) at the graft-host interface using the high-risk corneal transplantation model. In this model, which mimics the inflammatory status and the preexisting vascularization of high-risk patients undergoing corneal transplantation, allogeneic donor corneas are transplanted onto sCD83-treated recipient animals. This model allows the direct and precise application of the immune modulator at the transplantation side. Interestingly, sCD83 was able to prolong graft survival after systemic application as well as after topical application, which is therapeutically more relevant. The therapeutic effect was accompanied by an increase in the frequency of regulatory T cells and was mediated by the immune-regulatory enzyme IDO and TGF-ß. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-ß, a cytokine previously shown to be an essential mediator of IDO-dependent, long-term tolerance. These findings open new treatment avenues for local immune modulation after organ and tissue transplantation.
Assuntos
Antígenos CD/uso terapêutico , Transplante de Córnea , Facilitação Imunológica de Enxerto , Imunoglobulinas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Indolamina-Pirrol 2,3,-Dioxigenase/fisiologia , Glicoproteínas de Membrana/uso terapêutico , Linfócitos T Reguladores/efeitos dos fármacos , Tolerância ao Transplante/efeitos dos fármacos , Administração Oftálmica , Aloenxertos , Animais , Antígenos CD/administração & dosagem , Antígenos CD/imunologia , Células da Medula Óssea/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/análise , Sobrevivência de Enxerto , Imunoglobulinas/administração & dosagem , Imunoglobulinas/imunologia , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/biossíntese , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Injeções Intraperitoneais , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pré-Medicação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Solubilidade , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/administração & dosagem , Fator de Crescimento Transformador beta/fisiologia , Fator de Crescimento Transformador beta/uso terapêutico , Antígeno CD83RESUMO
Podocytes are essential to the structure and function of the glomerular filtration barrier; however, they also exhibit increased expression of MHC class II molecules under inflammatory conditions, and they remove Ig and immune complexes from the glomerular basement membrane (GBM). This finding suggests that podocytes may act as antigen-presenting cells, taking up and processing antigens to initiate specific T cell responses, similar to professional hematopoietic cells such as dendritic cells or macrophages. Here, MHC-antigen complexes expressed exclusively on podocytes of transgenic mice were sufficient to activate CD8+ T cells in vivo. In addition, deleting MHC class II exclusively on podocytes prevented the induction of experimental anti-GBM nephritis. Podocytes ingested soluble and particulate antigens, activated CD4+ T cells, and crosspresented exogenous antigen on MHC class I molecules to CD8+ T cells. In conclusion, podocytes participate in the antigen-specific activation of adaptive immune responses, providing a potential target for immunotherapies of inflammatory kidney diseases and transplant rejection.