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- microRNA-122 and microRNA-1247 regulate the pathogenic phenotype of effector CD4+ T cell subsets in (auto)immune responses in vivoPublication . Cunha, Carolina; Romero, Paula Vargas; Pelicano, Catarina; Pais, Ana Teresa; Inácio, Daniel; Papotto, Pedro; Sobral, Daniel; Amado, Tiago; Gomes, Anita Q.; Silva-Santos, BrunoAutoimmune diseases are often associated with an imbalance 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 Foxp3+ regulatory cells (Treg). The differentiation of these distinct CD4+ T cell subsets is known to be regulated by microRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression at the post-transcriptional level. While various individual miRNAs have been implicated in this process, a holistic approach focused on in vivo immune responses is missing to better understand how miRNA networks shape the CD4+ T cell compartment in pathophysiology. To address this biological question, we established a triple reporter mouse for Ifng, Il17, and Foxp3, and subjected it to experimental autoimmune encephalomyelitis (EAE), a widely used rodent model of Multiple Sclerosis (MS). We performed miRNA-seq analysis on Th1, Th17, and Treg cells isolated from the spleen and lymph nodes (LNs) at the peak-plateau stage of EAE, and found 110 miRNAs to be differentially expressed between the effector and regulatory subsets. From there, we studied the functional role of 5 candidate miRNAs as they were specifically upregulated in one population versus the others. In vivo miRNA modulation showed that silencing miR-122 (upregulated in Th17 cells) increased the frequency of IL-17A+ cells in the LNs and precipitated the onset of EAE, whereas upregulation of miR-1247 (highly expressed in the Th1 subset) decreased the severity of the disease reducing the number of IFN-γ+ cells in the LNs. We further found that both IL-6 and TGF-β induce miR-122 expression, whereas IL-23 and IL-1β repress its expression. Given that IL-23 and IL-1β are critical to induce Th17-mediated pathogenicity, our data suggests that miR-122 is expressed in a non-pathogenic context. Interestingly, we have observed a pathogenic gene signature in CNS-derived Th17 cells (when compared to peripheral Th17 cells) with concomitantly decreased levels of miR-122, suggesting that miR-122 may regulate Th17 pathogenicity. Similarly, we observed that once Th1 cells infiltrate the CNS, their levels of miR-1247 decrease, and they produce higher levels of IFN-γ. Furthermore, as we found that this miRNA is induced by the anti-inflammatory cytokines IL-10 and TGF-β, we propose that miR-1247 constitutes an auto-regulatory mechanism of Th1 cells in the periphery, which is disrupted upon CNS infiltration. Overall, our results suggest that miR-122 and miR-1247 control the pathogenic phenotype of effector Th17 and Th1 cells, respectively, during CNS autoimmunity. These findings may have important implications for autoimmune diseases, which we are now assessing in samples from MS patients.
- 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.
- 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 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 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.