MicroRNAs are small, noncoding RNAs that function as posttranscriptional modulators of gene expression by binding target mRNAs and inhibiting translation. They are therefore crucial regulators of several biological as well as immunological events. Recently, miR-511-3p has been implicated in the development and differentiation of APCs, such as dendritic cells (DCs), and regulating several human diseases. Interestingly, miR-511-3p is embedded within the human MRC1 gene that encodes the mannose receptor. In this study, we sought to examine the impact of miR-511-3p up-or downregulation on human DC surface phenotype, cytokine profile, immunogenicity (using IDO activity as a surrogate), and downstream T cell polarization. Using gene silencing and a selection of microRNA mimics, we could successfully suppress or induce the expression of miR-511-3p in DCs. Consequently, we show for the first time, to our knowledge, that inhibition and/or overexpression of miR-511-3p has opposing effects on the expression levels of two key C-type lectin receptors, namely the mannose receptor and DC-specific ICAM 3 nonintegrin at protein and mRNA levels, thereby affecting C-type lectin receptor-induced modulation of IDO activity in DCs. Furthermore, we show that downregulation of miR-511-3p drives an anti-inflammatory DC response characterized by IL-10 production. Interestingly, the miR-511-3p low DCs also promoted IL-4 secretion and suppressed IL-17 in cocultures with autologous T cells. Together, our data highlight the potential role of miR-511 in regulating DC function and downstream events leading to Th polarization and immune modulation.
The peroxisome proliferator activated receptor gamma (PPARγ) is a ligand activated transcription factor expressed in dendritic cells (DCs), where it exerts anti-inflammatory responses against TLR4-induced inflammation. Recently, microRNA-511 (miR-511) has also emerged as a key player in controlling TLR4-mediated signalling, and in regulating the function of DCs. Interestingly, PPARγ has been previously highlighted as a putative target of miR-511 activity; however the link between miR-511 and PPARγ and its influence on human DC function within the context of LPS-induced inflammatory responses is unknown. Using a selection of miR-511-3p-specific inhibitors and mimics, we demonstrate for the first time that up or downregulation of miR-511-3p inversely correlates with PPARγ mRNA levels and transcriptional activity following treatment with PPARγ synthetic agonist rosiglitazone (RSG), in the presence or absence of LPS. Additionally, we show that PPARγ activation with RSG modulates LPS-induced DC activation and downregulates pro-inflammatory cytokine production following downregulation of miR-511-3p. Lastly, PPARγ activation was shown to suppress LPS-mediated induction of indoleamine 2,3-dioxygenase (IDO) activity in DCs, most likely due to changes in miR-511-3p expression. These data suggest that PPARγ-induced modulation of DC phenotype and function is influenced by miR-511-3p expression, which may serve as a potential therapeutic target against inflammatory diseases.
New strategies for immune modulation have shown real promise in regenerative medicine as well as the fight against autoimmune diseases, allergies, and cancer. Dendritic cells (DCs) are gatekeepers of the immune system and their ability in shaping the adaptive immune responses makes DCs ideal targets for immune modulation. Carbohydrates are abundant in different biological systems and are known to modulate DC phenotype and function. However, how simple monosaccharides instruct DC function is less well understood. In this study, we used a combinatorial array of immobilized monosaccharides to investigate how they modulate DC phenotype and function and crucially the impact of such changes on downstream adaptive immune responses. Our data show that a selection of monosaccharides significantly suppress lipopolysaccharide-induced DC activation as evidenced by a reduction in CD40 expression, IL-12 production, and indoleamine 2,3-dioxygenase activity, while inducing a significant increase in IL-10 production. These changes are indicative of the induction of an anti-inflammatory or regulatory phenotype in DCs, which was further confirmed in DC–T cell co-cultures where DCs cultured on the ‘regulatory’ monosaccharide-coated surfaces were shown to induce naïve T cell polarization toward regulatory phenotype. Our data also highlighted a selection of monosaccharides that are able to promote mixed Treg and Th17 cell differentiation, a T cell phenotype expected to be highly immune suppressive. These data show the potential immunomodulatory effects of immobilized monosaccharides in priming DCs and skewing T cell differentiation toward an immune-regulatory phenotype. The ability to fine-tune immune responses using these simple carbohydrate combinations (e.g. as coatings for existing materials) can be utilized as novel tools for immune modulation with potential applications in regenerative medicine, implantable medical devices, and wound healing where reduction of inflammatory responses and maintaining immune homeostasis are desirable.
The use of T cells is increasing both in healthcare and in research yet the preservation methodologies for longer periods of times are yet to be optimized. In order to overcome these issues, we have optimized a protocol in sample handling and preservation of T cells in order to perform a successful donor homologous co-culture with DCs and preserve these cells for subsequent testing. This method will help in saving time and effort as well as the ease of use for experiments requiring use of T cells in mono or co-cultures. Handling and preservation of T cells using our methodology showed stability and viability of these cells in co-cultures. Data showed viability of > 93% before and after liquid nitrogen preservation. Moreover, preserved cells had no unspecific activation which can be seen in unchanged expression of the T cell activation marker CD25. T cell proliferation profile showed that preserved T cells used in DC-T cell co-cultures (LPS stimulated DCs) had the ability to interact and proliferate indicating potency of these cells. This provides evidence of the efficiency of our handling and preservation methodology in maintaining cell viability and stability. Preserving donor T cells would facilitate reuse of these cells in donor homologous co-cultures reducing inconvenience of multiple donations of fresh blood and provides accessibility of the same population of T cells for experiments that requires repetition, commercial availability of the cells or for preservation of cells for clinical therapies such as chimeric antigen receptor T cells.
The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor expressed in dendritic cells (DCs), where it exerts anti-inflammatory responses against TLR4-induced inflammation. Recently, microRNA-511 (miR-511) has also emerged as a key player in controlling TLR4-mediated signalling and in regulating the function of DCs. Interestingly, PPARγ has been previously highlighted as a putative target of miR-511 activity; however, the link between miR-511 and PPARγ and its influence on human DC function within the context of LPS-induced inflammatory responses is unknown. Using a selection of miR-511-3p-specific inhibitors and mimics, we demonstrate for the first time that knockdown or overexpression of miR-511-3p inversely correlates with PPARγ mRNA levels and affects its transcriptional activity following treatment with rosiglitazone (RSG; PPARγ agonist), in the presence or absence of LPS. Additionally, we show that PPARγ-mediated suppression of DC activation and pro-inflammatory cytokine production in miR-511-3p knockdown DCs is abrogated following overexpression of miR-511-3p. Lastly, PPARγ activation suppressed LPS-mediated induction of indoleamine 2,3-dioxygenase (IDO) activity in DCs, most likely due to changes in miR-511-3p expression. Our data thus suggests that PPARγ-induced modulation of DC phenotype and function is influenced by miR-511-3p expression, which may serve as a potential therapeutic target against inflammatory diseases.
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