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- Dissecting the role of microRNAs in effector versus regulatory CD4+ T cell differentiation during (auto)immune responses in vivoPublication . Cunha, Carolina; Romero, Paula Vargas; Inácio, Daniel; Pais, Ana Teresa; Pelicano, Catarina; Sobral, Daniel; Costa, Marina; Mensurado, Sofia; Sousa, Natacha Gonçalves; Papotto, Pedro; Enguita, Francisco; Gomes, Anita Q.; Silva-Santos, BrunoIntroduction: MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. They have been implicated in the regulation of the differentiation and function of CD4+ T cell subsets, key players in host defense against pathogens, but also responsible for immune-mediated diseases depending on the correct vs incorrect balance, respectively, between pro-inflammatory effector CD4+T cells, including the IFN-γ-producers T helper 1 (Th)1 and the IL-17-producers Th17 cells, and anti-inflammatory regulatory T cells (Treg). While individual miRNAs were found to regulate the differentiation of specific CD4+ T cell populations, an approach based on in vivo responses is still missing and is key to understanding how miRNA networks control this balance in pathophysiology. Methodology: We have established a triple reporter mouse for Ifng, Il17, and Foxp3, and subjected it to experimental autoimmune encephalomyelitis (EAE). We performed miRNA-seq analysis on Th1, Th17, and Treg cells isolated from the spleen and lymph nodes (LNs) at the peak plateau stage to identify miRNA candidates specifically expressed in one of the cell populations. We have in vivo modulated their expression levels using antagomiRs observed the course of EAE progression and characterised their upstream regulation in vitro in either Th1 or Th17 differentiation conditions. Results: The miRNA-seq data has allowed the identification of 110 miRNAs differentially expressed between effector (Th1 and Th17) and regulatory (Treg) subsets. From those, 9 were specifically upregulated in one population versus the others. In vivo miRNA modulation showed that silencing miR-122 precipitated the onset of EAE, whereas overexpressing miR-1247 decreased the severity of the disease. Cytokine-regulated miR-1247 and miR-122 expression levels are inversely associated with pathogenic signatures of Th1 and Th17 cells between lymphoid and central nervous systems. Discussion: Our results suggest that miR-122 and miR-1247 act as peripheral brakes to CD4+ T cell pathogenicity that are overruled in the inflamed target organ. These findings may have important implications for autoimmune diseases.
- 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.
- 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.
- Role of microRNAs on T cell differentiation during immune responses in vivoPublication . Cunha, Carolina; Romero, Paula Vargas; Pelicano, Catarina; Pais, Ana Teresa; Inácio, Daniel; Pappoto, Pedro; Amado, Tiago; Silva-Santos, Bruno; Gomes, Anita Q.CD4+ 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 TGF-β 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.
- 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 the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Silva, Marta; Sobral, Daniel; Cunha, Carolina; 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 the IL-17 or IFN-γ. We have previously found that one microRNA, miR-146a, maintains peripheral γδ T cell identity by inhibiting IFN-g production by the IL-17-committed CD27− gδ T cell subset. To further and more globally address the role of microRNAs in effector γδ T cell differentiation, we established a double reporter IL17-GFP:IFN-γ-YFP mouse strain and isolated pure IL-17+ and IFN-γ+ γδ T cell populations from the peripheral lymphoid organs to perform small RNA-sequencing. This allowed us to identify clearly 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 study further. We characterized the detailed expression pattern of each candidate microRNA in γδ T cell subsets throughout mouse ontogeny and upon gain-of-function studies in in vitro cultures of γδ T cells. 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. 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 in vivo 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 acting as an IFN-γ auto-repressor (miR-139-5p) or promoting functional plasticity (miR-7a-5p). Finally, miR-322-5p and miR-450b-3p, overexpressed in IL-17+ γδ T cells, may have therapeutic potential by modulating the production of IFNγ, whose levels are critical in anti-tumoral and anti-viral responses. These data demonstrate the impact of microRNAs on the differentiation and functional identity of effector γδ T cell subsets, which may open new avenues for their manipulation in disease settings.
- microRNAs are key regulators of the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Silva, Marta; Sobral, Daniel; Cunha, Carolina; Enguita, Francisco; Pamplona, Ana; 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 and non-redundant roles in several (patho)physiological contexts, such as tissue homeostasis, infection, autoimmunity and cancer. 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 IL-17 or IFN-γ, unlike their ab T cell counterparts1. So far, several miRNAs have been implied in the control of the differentiation and IFN-γ and IL-17 levels by ab Th1 and Th17 cells, respectively2. However, little is known about the action of these post-transcriptional regulators on γδ T cell differentiation. Schmolka et al. showed that miR-146a is selectively enriched in IL-17-biased CD27- γδ T cells and restricts their co-production of IFN-γ by targeting Nod1 mRNA, therefore regulating γδ T cell plasticity3. This isolated work illustrates the need of a more comprehensive study of the miRNA repertoires of γδ T cells and of the regulatory networks they take part in the control of IFN-γ and IL-17 production by these cells.
- A key role for microRNAs in the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Silva, Marta; Sobral, Daniel; Cunha, Carolina; Enguita, Francisco; Pamplona, Ana; 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 migrate 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 characterise. 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.