Browsing by Author "Amado, Tiago"
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- Control of T cell effector functions by miRNAsPublication . Inácio, Daniel P.; Amado, Tiago; Silva-Santos, Bruno; Gomes, Anita QuintalThe differentiation of effector T cells is a tightly regulated process that relies on the selective expression of lineage-defining master regulators that orchestrate unique transcriptional programs, including the production of distinct sets of effector cytokines. miRNAs are post-transcriptional regulators that are now viewed as critical players in these gene expression networks and help defining cell identity and function. This review summarises the role of individual miRNAs in the regulation of the differentiation of effector T cell subsets, including CD4+ T helper cells, cytotoxic CD8+ T cells and innate-like NKT cells. Moreover, we refer to miRNAs that have been identified to affect simultaneously two or more effector T cell populations, impacting on the balance between effector T cells in vivo, thus constituting potential biomarkers or targets for therapies aiming at boosting immunity or controlling autoimmunity.
- Cross-regulation between cytokine and microRNA pathways in T cellsPublication . Amado, Tiago; Schmolka, Nina; Metwally, Hozaifa; Silva-Santos, Bruno; Gomes, Anita QuintalmicroRNA (miRNA) mediated regulation of protein expression has emerged as an important mechanism in T-cell physiology, from development and survival to activation, proliferation, and differentiation. One of the major classes of proteins involved in these processes are cytokines, which are both key input signals and major products of T-cell function. Here, we summarize the current data on the molecular cross-talk between cytokines and miRNAs: how cytokines regulate miRNA expression, and how specific miRNAs control cytokine production in T cells. We also describe the inflammatory consequences of deregulating the miRNA/cytokine axis in mice and humans. We believe this topical area will have key implications for immune modulation and treatment of autoimmune pathology.
- Dissecting the IFN-g versus IL-17-specific mRNAomes of effector gd T lymphocytesPublication . Inácio, Daniel; Pamplona, Ana; Amado, Tiago; Sobral, Daniel; Cunha, Carolina; Gomes, Anita Q.; Silva-Santos, BrunoThe ability of murine γδ T cells to rapidly produce the pro-inflammatory cytokines interleukin-17 (IL-17) or interferon-γ (IFN-γ) underlies their crucial roles in several pathophysiological contexts, from infection to cancer or autoimmunity. This functional capacity stems from a complex process of ‘developmental pre-programming’ in the thymus, after which a significant fraction of γδ T cells migrate to peripheral sites already committed to producing either IL-17 (gd17) or IFN-γ (gdIFN). While several studies have studied these gd T cell subtypes using surface markers that enrich for effector function, we still lack a characterization of the mRNA transcriptomes that specifically associate with IL-17 or IFN-g production by gd T cells. To overcome this limitation, in this study, we established a double reporter IL-17-GFP:IFN-γ-YFP mouse strain, which allowed us to isolate pure IL-17+, IFN-γ+, and the remaining IL-17-IFN-g-(DN) γδ T cell populations from the peripheral lymphoid organs to perform RNA sequencing and identify the subset-specific mRNAomes. Overall, we detected the expression of 12822 genes in gd T cells, with a significant number of genes being enriched in gd17 when compared with gdIFN and gdDN cells. Among these, 936 genes were differentially expressed between the three populations, with gd17 and gdIFN cells displaying the most distinct mRNAomes, which highlights their functional specialization, and gdIFN being more similar to DN than gd17 cells. A more detailed analysis of the top 30 differentially expressed genes among the most expressed genes by gd17 and gdIFN cells revealed that the majority of the signature genes increase their expression levels in the periphery upon their egress from the thymus, suggesting that these effector subsets only terminate their differentiation process at peripheral sites. Notably, gd17-associated signature genes are specifically expressed in this subset, unlike gdIFN signature genes, which are also often expressed by gdDN T cells, thus suggesting a developmental relationship between these two subpopulations. Collectively, our data allowed us to identify distinct mRNA signatures directly associated with cytokine expression in γδ T cells, several of which we are now further studying in disease models to identify potential new roles in pathophysiology.
- Dissecting the IFN-g versus IL-17-specific transcriptomes of effector gd T lymphocytes: a new role for signalling adaptor ThemisPublication . Inácio, Daniel; Amado, Tiago; Pamplona, Ana; Sobral, Daniel; Cunha, Carolina; Lesourne, Renaud; Gomes, Anita Q.; Silva-Santos, BrunoThe crucial role of murine γδ T cells in several (patho)physiological contexts stems from a complex process of ‘developmental preprogramming’ in the thymus, after which a significant fraction of γδ T cells populate peripheral sites already endowed with the capacity to secrete either IL-17 or IFN-γ1. However, despite the relevance of these γδ T cell effector subsets, we still lack knowledge on the transcriptomes that specifically associate with IL-17 or IFN-γ production. To address this, we established a double reporter IL-17-GFP:IFN-γ-YFP mouse strain, which allowed us to isolate pure peripheral IL-17-producing (γδ17) or IFN-γ-producing (γδIFN) γδ T cells to perform RNA-sequencing.
- Dissecting the role of microRNAs in effector versus regulatory CD4+ T cell differentiation during (auto)immune responses in vivoPublication . Cunha, Carolina; Romero, Paula Vargas; Pelicano, Catarina; Pais, Ana Teresa; Inácio, Daniel; Papotto, Pedro; Amado, Tiago; Gomes, Anita Q.; Santos-Silva, BrunoCD4+ T cells are key players in host defense against pathogens, but an incorrect balance between CD4+ T cell subsets, namely pro-inflammatory effector cells, including T helper 1 (Th)1 and Th17 cells (IFN-γ- and IL-17-producers, respectively), and anti-inflammatory regulatory cells (Treg; Foxp3+ subset), can lead to immune-mediated diseases. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. While individual miRNAs were shown to regulate the differentiation of specific CD4+ T cell populations, a holistic approach based on in vivo responses is missing and is critical to understanding how miRNA networks control this balance under physiological conditions. To address this, we have established a triple reporter mouse for Ifng, Il17, and Foxp3, and subject it to experimental autoimmune encephalomyelitis (EAE). We perform miRNA-seq analysis on Th1, Th17, and Treg cells isolated from the spleen (SPL) and lymph nodes (LNs) at the peak-plateau stage and found that 110 miRNAs are differentially expressed between effector and regulatory subsets. We further selected 8 candidate miRNAs that were specifically upregulated in one population versus the others. Both overexpression and inhibition studies showed that miR-126a limits IL-17+ expression in Th17 cells in vitro. Treatment with antagomiRs in vivo showed that silencing miR-122 increased the number of IL-17+ cells in the LNs and precipitated the onset of EAE, whereas inhibition of miR-1247 decreased the severity of the disease by reducing the number of IFN-γ+ cells, also in the LNs. Additionally, we identified IL-6 and TGF-β as the key cytokines upstream of miR-126a and miR-1247 expression, respectively. While both IL-6 and TGFβ also induce miR-122 expression, we found that IL-23 and IL-1β repress its expression. Interestingly, and given that IL-23 and IL-1β are critical to inducing Th17-mediated pathogenicity, we have consistently observed a pathogenic gene signature in CNS-derived Th17 cells when compared to peripheral Th17 cells with concomitantly decreased levels of miR-126a and miR-122. Overall, our results suggest that miR-126a and miR-122 regulate IL-17 expression and the pathogenic phenotype of Th17 cells to prevent excessive inflammation in the periphery while miR-1247 maintains the inflammatory phenotype of Th1 cells in an anti-inflammatory environment.
- Dissection of the IFN-γ versus IL-17-specific transcriptomes of γδ T cells: a new role for signaling adaptor ThemisPublication . Inácio, Daniel; Amado, Tiago; Pamplona, Ana; Sobral, Daniel; Cunha, Carolina; Lesourne, Renaud; Gomes, Anita Q.; Silva-Santos, BrunoThe crucial role of murine γδ T cells in several (patho)physiological contexts stems from a complex process of ‘developmental pre-programming’ in the thymus, after which a significant fraction of γδ T cells populate peripheral sites already endowed with the capacity to secrete either IL-17 or IFN-γ. However, despite the relevance of these γδ T cell effector subsets, we still lack knowledge on the transcriptomes that specifically associate with IL-17 or IFN-γ production. To address this, we established a double reporter IL-17-GFP:IFN-γ-YFP mouse strain, which allowed us to isolate pure peripheral IL-17-producing (γδ17) or IFN-γ-producing (γδIFN) γδ T cells to perform RNA-sequencing. This led to the identification of the distinct transcriptomes of γδ17 and γδIFN cells, which surprisingly diverged in 6337 differentially (over 1.5-fold) expressed genes. Pathway and gene ontology analyses indicated that γδ17 cells differ from γδIFN cells in their selective ability to sense and integrate external cues, whereas γδIFN stands out in replication, transcription, and translation processes. A detailed analysis of the top differentially expressed genes between γδ17 and γδIFN cells revealed that most of the signature genes of each subset increased their expression levels in the periphery (compared to the thymus), suggesting that γδ17 and γδIFN cells only terminate their differentiation process at peripheral sites. Among the top differentially expressed genes, we found Themis, a T cell-specific gene involved in the regulation of TCR signal strength, to be enriched in γδIFN cells. Importantly, we found that Themis deficiency leads to a dysregulated effector γδ T cell peripheral compartment at steady state, which upon infection with Plasmodium berguei ANKA sporozoites confers Themis-deficient mice full protection from experimental cerebral malaria, a γδIFN-dependent pathology. Accordingly, we observed a less activated and less proliferative γδIFN population in the peripheral lymph nodes of infected Themis-deficient mice compared to Themis-sufficient controls. This work demonstrates the relevance of the characterization of the γδIFN and γδ17 transcriptomes to uncover new players in the regulation of γδ T cell effector functions, which may open new avenues for their manipulation in disease settings.
- A key role for microRNAs in regulating IL-17 versus IFN-g production by gamma-delta T cellsPublication . Amado, Tiago; Schmolka, N.; Sobral, Daniel; Enguita, Francisco; Inácio, Daniel; Silva-Santos, Bruno; Gomes, Anita Q.γδ T cells are an important source of the pro-inflammatory cytokines IL-17 and IFN-γ under (patho)physiologic conditions. In the mouse, CD27+ γδ T cells are committed to IFN-γ expression, whereas their CD27- counterparts make IL-17 but are capable of co-expressing both cytokines under inflammatory conditions. We aim to characterize a novel layer of microRNA-mediated regulation of effector γδ T cell differentiation. First, by comparing the microRNA pools of the two CD27-based γδ T cell subsets, we found that miR-146a was selectively enriched in CD27- γδ T cells and restricted their IFN-γ production by targeting Nod1 mRNA. Next, to overcome the caveat of using surface markers, which do not allow isolation of pure populations of IL-17 or IFN-γ producing γδ T cells, we used a double reporter IL-17-GFP: IFNg-YFP mouse strain. Pure IL-17+ or IFN-γ+ γδ T cell populations were isolated from peripheral lymphoid organs and subjected to next-generation sequencing analysis of both microRNA and mRNA repertoires. This allowed us to identify, for the first time, miRNA and mRNA signatures directly associated with cytokine expression, rather than TCR Vγ usage of maturation markers. Furthermore, differentially expressed miRNAs and mRNAs were bioinformatically integrated into networks that allowed the identification of 6 miRNAs predicted to target key determinants of the IL-17 program; and 3 miRNA candidates for the IFN-γ program of γδ T cells. Ongoing molecular assays provide an unprecedented functional characterization of the impact of microRNAs on the identity and differentiation of effector γδ T cell subsets.
- A key role for microRNAs in regulating IL-17 versus IFN-γ production by γδ T cellsPublication . Inácio, Daniel; Amado, Tiago; Sobral, Daniel; Enguita, Francisco; Gomes, Anita Quintal; Silva-Santos, Brunoγδ T cells are key providers of proinflammatory cytokines in various contexts of (patho)physiology. They are preprogrammed in the thymus into distinct subsets producing either interleukin-17 (IL-17) or interferon-γ (IFN-γ), which segregate with CD27 expression. In the periphery, CD27−γδ T cells, which usually express IL-17, can be induced to coexpress IL-17 and IFN-γ. We have previously found that miR-146a was selectively enriched in these cells and restricted their IFN-γ production by targeting Nod1 mRNA. We aim at further dissecting microRNA-mediated regulation of effector γδ T cell differentiation independently of the use of surface markers, which do not allow the isolation of pure populations of IL-17+ or IFN-γ+ γδ T cells. Thus, we isolated these pure γδ T cell populations from peripheral lymphoid organs of a double reporter IL-17-GFP: IFN-γ-YFP mouse strain and subjected them to next-generation sequencing analysis of both microRNA and mRNA repertoires, which allowed us to identify miRNA and mRNA signatures directly associated with cytokine expression. Furthermore, differentially expressed miRNAs and mRNAs were bioinformatically integrated into networks that allowed the prediction of 6 and 3 miRNAs targeting key determinants of the IL-17 and IFN-γ programs of γδ T cells, respectively. Preliminary results, based on gain-of-function studies on fetal liver progenitor cells co-cultured with OP9-DL1 cells indicate that miR-326 and miR-450b may regulate γδ T cell development, inhibiting IFN-γ production. Further molecular assays are being performed on peripheral γδ T cells to provide a broader functional characterization of the impact of microRNAs on the identity and differentiation of effector γδ T cell subsets.
- A key role for microRNAs in the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Sobral, Daniel; Cunha, Carolina; Silva, Marta; Pamplona, Ana; Enguita, Francisco; Gomes, Anita Q.; Silva-Santos, BrunoThe ability of murine γδ T cells to rapidly produce the pro-inflammatory cytokines interleukin-17 (IL-17) or interferon-γ (IFN-γ) underlies their crucial roles in several (patho)physiological contexts. This capacity stems from a complex thymic process of ‘developmental pre-programming’, after which a large fraction of γδ T cells migrates to peripheral sites already committed to producing IL-17 or IFN-γ. We have previously found that miR-146a maintains the cell identity of peripheral IL-17-committed gδ T cells by inhibiting IFN-g production. To further address the role of microRNAs in γδ T cell differentiation, we isolated pure IL-17+ and IFN-γ+ γδ T cell populations from the peripheral lymphoid organs of a double reporter IL-17-GFP: IFN-γ-YFP mouse strain to perform small RNA-sequencing. This allowed us to identify distinct microRNA signatures associated with cytokine expression in γδ T cells, from which we selected ten microRNAs differentially expressed between IL-17+ and IFN-γ+ subsets to further characterize. We first analyzed the expression pattern of each candidate microRNA in γδ T cell subsets throughout mouse ontogeny and upon gain-of-function studies in in vitro γδ T cell cultures. Our results indicate that while some microRNAs regulate γδ T cell development in the thymus, other candidates modulate their peripheral effector functions. More specifically, using a miR-181a deficient mouse model, we have found that miR-181a, highly expressed in immature γδ T cell subsets in the thymus, shifts the IL-17/IFN-γ balance towards the IL-17-pathway in neonatal life, which is further maintained in the periphery during adult life. On the other hand, miR-7a-5p and miR-139-5p, overexpressed in peripheral IFN-g+ γδ T cells, regulate peripheral γδ T cell effector functions, either promoting functional plasticity or acting as an IFN-γ auto-repressor, respectively. Finally, miR-322-5p and miR-450b-3p, overexpressed in IL-17+ γδ T cells, may have therapeutic potential by modulating IFN-γ levels, which are critical in anti-tumoral and antiviral responses.
- A key role for microRNAs in the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Sobral, Daniel; Cunha, Carolina; Silva, Marta; Pamplona, Ana; Enguita, Francisco; Gomes, Anita Q.; Silva-Santos, BrunoThe ability of murine γδ T cells to rapidly produce the pro-inflammatory cytokines interleukin-17 (IL-17) or interferon-γ (IFN-γ) underlies their crucial roles in several (patho)physiological contexts. This capacity stems from a complex process of ‘developmental pre-programming’ in the thymus, after which a large fraction of γδ T cells migrate to peripheral sites already committed to producing either IL-17 or IFN-γ. To globally address the role of microRNAs in effector γδ T cell differentiation, we established a double reporter IL-17-GFP: IFN-γ-YFP mouse strain and isolated pure IL-17+ and IFN-γ+ γδ T cell populations from peripheral lymphoid organs to perform small RNA-sequencing. This allowed us to identify distinct microRNA signatures associated with cytokine expression in γδ T cells, from which we selected ten candidate microRNAs differentially expressed between IL-17+ and IFN-γ+ γδ T cells to functionally study further. Our results indicate that while some microRNAs, such as miR-128-3p and miR181a-5p, regulate γδ T cell development in the thymus, other candidates, including miR-7a-5p, miR-139-5p, miR-322-5p, and miR-450b-3p, modulate peripheral γδ T cell effector functions. Furthermore, using a miR-181a deficient mouse model, we have demonstrated that miR-181a, highly expressed in immature γδ T cell subsets in the thymus, shifts the in vivo IL-17/IFN-γ balance towards the IL-17 pathway in the neonatal thymus, which is further maintained in the periphery during adult life. These data demonstrate the impact of microRNAs on the development, differentiation, and functional identity of effector γδ T cell subsets, which may open new avenues for their manipulation in disease settings.
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