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Abstract
MicroRNAs (miRNAs) are recently discovered regulators of gene expression, and early studies have indicated that they have a role in the regulation of haematopoiesis, the immune response and inflammation. They bind the 3’UTR of target mRNAs and mainly prevent translation of the protein product. Dysregulation of these molecules has been shown to be a hallmark of cancer and now investigators are examining their role in the pathogenesis of inflammatory diseases. miR-146 and miR-155 have been a particular focus for investigators, and these two miRNAs have been shown to be induced by proinflammatory stimuli such as interleukin 1, tumour necrosis factor α (TNFα) and Toll-like receptors (TLRs). They have also been detected in synovial fibroblasts and rheumatoid synovial tissue. Both have multiple targets, with miR-146 inhibiting TLR signalling and miR-155 regulating Th1 cells and also, interestingly, positively regulating mRNA for TNFα. The potential of miRNAs for improving our understanding of the pathogenesis of diseases such as rheumatoid arthritis, and for developing potentially new treatments for these diseases, is substantial.
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Dysregulated inflammation lies at the heart of rheumatoid arthritis (RA), and despite intense effort, how this disease is initiated and becomes chronic is still unclear. Increased expression of proinflammatory genes is a hallmark, and much effort has focused on the regulation of this process. This has largely been concerned with transcriptional control, with transcription factors such as NF-κB playing a particularly important role. More recently, however, post-transcriptional control of gene expression by micro-RNA (miRNA) has uncovered a new level of complexity in our understanding of gene expression in general, and we are beginning to determine the role of miRNA in immune and inflammatory responses.
The first miRNA was described when it was discovered that a small non-coding RNA, lin-4, controls developmental timing in C elegans by targeting the mRNA for a key developmental protein LIN-14.1 2 It was later discovered that an additional small RNA, let-7, also contributed to developmental control in worms,3 however at that stage it was thought that these cases of small RNA control were restricted to organisms such as C elegans in the context of development.4 However, three landmark papers published back to back in the journal Science in 2001 illustrated that these small RNA were not only representative of a wider class of small RNA molecules in nematodes but were also found in metazoans with descriptions in murine and human cells, where their sequences were highly conserved.5–7 These projects defined a new class of small non-coding RNA, termed miRNA, which consisted of 18–22 nucleotide RNA species processed from larger 60–70 nucleotide precursor RNA sequences which contain a hairpin secondary structure termed the pre-miRNA. The existence of over 326 miRNAs in humans has been confirmed,8 while some bioinformatic programs predict the exact number of small non-coding RNA genes to be over one thousand. The human genome annotation project RECODE claims that they are more widespread than protein coding genes, although this has to be confirmed experimentally.9 Figure 1 illustrates the biogenesis process of mature miRNA from primary transcripts, which involves two key RNase enzymes Drosha and Dicer; this is well reviewed elsewhere.10
Cloning and profiling of miRNA in mammals showed that unlike those previously discovered in C elegans whose expression was temporally regulated, the expression of particular miRNA was limited to particular cell types—for example, the expression of miR17–20 was detected in HeLa cells but undetectable in mouse kidney or frog ovary tissue.11 Additionally, miRNA expression profiles have been shown to become dysregulated in cancer, with many oncogenic translocations and breakpoint mutations occurring at miRNA loci, resulting in the altered expression of many miRNA.12 This implied that miRNAs play a role in controlling cell differentiation, cell type identity and proliferation. As the differentiation and maturation of many different cell types is a common feature of the immune response it thus seemed likely that miRNAs play a vital role in determining immune responses.
In this mini-review we will summarise how the study of miRNAs has enhanced our knowledge of the immune system and also highlight the role played by miRNAs in inflammatory diseases, notably RA.
MIRNAS AND THE CONTROL OF HAEMATOPOIESIS
The pathogenesis of RA and other inflammatory diseases involves multiple cell types including macrophages, dendritic cells, Th1 cells, B cells and mast cells. miRNAs have a determining role in haematopoiesis and therefore dysregulation of this process might be important to direct pathogenesis in inflammatory disease. Much work has been performed on analysing the expression profiles of miRNA across different cells of the haematopoietic system and also elucidating the function of these miRNAs. More recently, genetically modified mice, either knockout or knockin strains have been generated for particular miRNA whose profound phenotypes have illustrated the importance of miRNAs in controlling the immune response. Table 1 lists those miRNA whose expression has been shown to be crucial for the appropriate development of key cell types associated with inflammatory diseases.
One of the earliest expression profiling studies found that the expression of certain miRNA, miR-181, miR-223 and miR-142s was restricted to cells of the bone marrow, spleen and thymus. Of these, miR-181 was limited to B cells of the bone marrow and overexpression of this miR-181 in haematopoietic progenitor cells led to an increase in B-lineage cells. miR-223 expression was isolated to myeloid cells.13 Another early study profiled haematopoietic progenitor cells at different stages and found that in the differentiation of progenitor cells to mature cell types the expression of many miRNAs was downregulated. In this way particular miRNAs were thought to keep progenitor cells from differentiation. Examples discovered included the downregulation of miR-26a, miR-24 and miR-27a in mast cell differentiation alongside upregulation of miR-223, and the downregulation of miR-150 in the maturation of thymocytes to naïve T cells. Subsequently, in the differentiation to Th1 cells, miR-146 is decreased while miR-150 is again increased.
Subsequent studies outlined roles for miR-221 and miR-222 in erythropoiesis.14 They have been shown to target the c-kit mRNA. miR-223 was found to control granulopoiesis by targeting the transcription factor NFIA.15 The miR-17-5p-20a-106a cluster has also been shown to control monocytopoiesis.16
Another landmark study analysed the expression of miRNAs in haematopoietic progenitor stem cells and combined their data with mRNA expression data and miRNA:mRNA target predictions to build a picture of the way in which miRNAs control haematopoietic differentiation. They confirmed independently the findings from other studies, and the authors speculate that these miRNAs all function to block differentiation at different points by targeting specific mRNAs, thereby holding the cell in an undifferentiated state.17
MIRNAS AND T-CELL POLARITY
T cells play an important role in regulating immune responses and particularly, Th1 responses have been associated with the pathogenesis of RA. To assess the role of miRNAs in T-cell development, T cells deficient in the key miRNA processing enzyme Dicer were examined. Dicer deficiency resulted in aberrant helper T-cell differentiation and cytokine production. T cells could not be differentiated from CD8+ T cells in the absence of Dicer and although low in numbers, CD4+ T cells were generated which proliferated poorly upon stimulation and underwent increased apoptosis.18 19 A further study found that complementation of these cells with an miR-150 expression cassette restored correct T-cell differentiation but interestingly, blocked B-cell development.20 Examination of miRNA profiles of CD4+25+ regulatory T cells (Tregs) found that these cells displayed a unique profile compared with naïve T cells, including increased expression of particular miRNA such as miR-21, miR-146a miR-223, miR-214, miR-125a and miR-155, with decreased expression of miR-150 and miR-142-5p. Expression of most of these miRNAs could be induced by overexpression of the Treg-specific transcription factor FoxP3. This miRNA profile, however, was similar to the miRNA profile of active CD4+ helper T cells.21 Profiling of miRNAs in CD8+ cytotoxic T cells showed an increase in particular miRNA, including miR-21, in comparison with naïve T cells. The levels of this miRNA are reduced as these cells differentiate to memory T cells.22
The first miRNA-deficient animals generated, miR-155 −/− mice, displayed severe immunodeficiencies, particularly impaired B-cell responses and skewed Th2-helper T-cell responses.23 24 Expression profiling disclosed many possible target mRNA for miR-155 in the immune response, including transcription factors for example c-Maf, cytokines and signalling proteins. Interestingly, ablation of miR-155 was shown to result in decreased tumour necrosis factor α (TNFα) mRNA, indicating that miR-155 may somehow positively regulate TNFα production. More recently, it has emerged through the generation of genetically modified mice which contain mutated miR-155 binding sites in the activation-induced cytidine deaminase AID mRNA and other methods that miR-155 is a key regulator of B-cell maturation by control of the AID-mediated myc-IgH translocation process.25 26 Additionally, B-cell maturation is controlled by an additional miRNA, again highlighted by the generation of both loss-of-function and gain-of-function animals—miR-150. miR-150 is found specifically in mature, resting B and T lymphocytes.27 miR-150-deficient mice displayed expanded B-cell numbers and antibody production. The transcription factor target for miR-150, c-Myb is expressed in lymphocyte progenitors but absent in mature resting B cells.28 Conversely to miR-150, it is required for progression to the active B-cell state with the phenotypes of both miR-150-deficient and c-Myb-deficient animals displaying opposite phenotypes.28 Most recently, mice deficient in miR-223 have been generated which display expanded numbers of granulocyte progenitors that are hypermature and hypersensitive to activating stimuli.29 Thus miR-223 through targeting of the transcription factor Mef-2c has been shown to be an important negative regulator of granulopoiesis just as miR-150 serves to negatively regulate B-cell activation.
INDUCTION OF MIRNA BY INFLAMMATORY STIMULI
Apart from the important role of miRNA expression in determining cell fate, it became apparent that miRNA expression can be regulated dynamically in response to specific immune and inflammatory stimuli, as highlighted in fig 2. One of the first studies to highlight induction of a specific miRNA during a specific response was Kasashima et al, 2004,31 which profiled miRNAs in HL-60 cells differentiated into macrophages using phorbol esters. In particular, strong upregulation of miR-21 was seen. A more recent paper has defined the promoter region of the primary miR-21 transcript and indicated that its induction is dependent on the transcription factor AP-1 and may target the transcription factor NFIB.32 NFIB itself competes with AP-1 for a binding site in the predicted miR-21 promoter, and this targeting may function as positive feedback of miR-21 and possibly other AP-1-regulated genes. A paper examining the induction of miR-21 in myeloma cell lines found that its induction is also STAT3 dependent and can be induced by interleukin 6 (IL6) treatment.33
A study in 2006 profiled expression of miRNAs in the human monocyte cell line THP1 after treatment with the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS).30 In particular, strong upregulation of miR-146, miR-155 and miR-132 was seen. Induction of miR-146a was seen in response to treatment with many proinflammatory stimuli such as other TLR ligands (from bacterial but not viral sources) as well as cytokines such as IL1β, and its induction is apparently NF-κB dependent. The study highlighted two particular target mRNAs for miR-146a—the key TLR4 signalling proteins TRAF-6 and IRAK-1. A more recent study has shown through the use of antisense RNA that miR-146a functions to negatively regulate IL8 and RANTES production, although negative regulation of TRAF-6 and IRAK-1 was not seen.34 Thus it has been concluded that miR-146a represents a negative regulator of TLR signalling—highlighting new roles for miRNAs in signalling pathways.30 A second paper from the Baltimore group highlighted the induction of miR-155 in murine bone marrow derived macrophages in response to the TLR3 ligand poly-inositol-cytidine (poly(I:C)) and also interferon β. Its induction is rapid and large and dependent on the MAP kinase JNK. Furthermore its induction by interferons requires TNFα autocrine signalling.35
Induction of miR-155 by multiple TLR ligands has been confirmed23 24 36 37 and induction of miR-146a by the Epstein–Barr virus latent membrane protein LMP-1 has been demonstrated.38 39 Interestingly upon Epstein–Barr virus infection the upregulation of other miRNAs is seen, including miR-155 and miR-21.38
Induction of miRNAs in the lung after in vivo treatment with LPS has also been examined.40 Induction of miR-146a or miR-155 was not seen in this case, however the rapid induction of other miRNAs was observed, including miR-214, miR-21, miR-223 and miR-224. It is postulated that this induction of miRNAs may function to negatively regulate cytokine production in the lung.
Interestingly, an additional study has examined the potential of TLRs to downregulate miRNAs and consequently upregulate levels of target proteins. Intriguingly, infection of cholangiocytes with Cryptosporidium parvum or treatment with LPS alone leads to downregulation of the miRNA let-7i. A predicted target of let-7i is TLR4 itself and concurrently upregulation of the LPS receptor TLR4 protein is seen in these cells in a let-7i-dependent manner.41
MIRNA AND INFLAMMATORY DISEASES
Much of the focus on miRNAs has been on cancer with clear associations being observed for particular miRNAs, with some acting as oncogenes. For example, miR-155, miR-21 and miR-146 are all known to be upregulated in many types of cancer12 while expression of miR-15 and miR-16 is lost in some cancers and therefore these are said to act as tumour suppressor genes.42 More recently, roles for miRNAs in inflammatory diseases have been explored. These expression profiling studies are still in the early stages and are summarised in table 2. One particular study has outlined the role of a new miRNA in skin inflammation.43 miR-203 expression is limited to skin epithelia and, more specifically, to keratinocytes. It is found to be overexpressed in psoriasis. It is thought to bring about its proinflammatory effects through targeting of the mRNA for SOCS-3, a member of a key family of negative regulators of cytokine function.44 Therefore miR-203 can be said to be proinflammatory. In contrast, miR-146a, previously shown to be a negative regulator of cytokine production, is also found overexpressed in psoriasis. Other miRNAs upregulated include miR-21, in both psoriasis and atopic eczema and miR-17-5p. Interestingly, decreased levels of miR-125b are seen in both psoriasis and atopic eczema. miR-125b has previously been reported to be downregulated by TLR4 activation in cells.37 An additional study profiled miRNA expression in peripheral blood from patients with systemic lupus erythematosus and idiopathic thrombocytic purpura compared with healthy controls and found certain groups of miRNAs upregulated in both diseases whilst also identifying dysregulated miRNAs unique to each disease.45 miRNA expression profiles have also been studied in models of ischaemia.46
More recently, the expression of miRNAs in synovial fibroblasts treated with TNFα from patients with RA has been examined. Upregulation of miR-146a and miR-155 was seen. These two miRNAs are found at much higher levels in synovial fibroblasts from patients with RA than in controls or patients with osteoarthritis. The expression of miR-155 could be further enhanced by stimulation with a wide range of proinflammatory stimuli, including cytokines such as IL1β, TNFα and ligands for TLR2, TLR3 and TLR4. It was proposed that miR-155 might control the expression of matrix metalloproteinases 1 and 3 in these cells.47
Another study has analysed the expression of miR-146 in synovial tissue of patients with RA. They found much higher levels of both miR-146a and miR-146b in RA tissue than in control and osteoarthritic tissue. The expression of miR-146 could be induced in synovial tissue by treatment with IL1β and TNFα. In this case analysis showed that cells which expressed pre-miR-146 in synovial tissue were mainly macrophages but also some T and B lymphocytes.48
It is unsurprising, that these particular miRNAs are expressed at higher levels in rheumatoid synovial fibroblasts. TLRs and other proinflammatory stimuli have been implicated in the pathogenesis of RA for some time.49 50 This latest finding that TLR-dependent miRNAs are upregulated in these fibroblasts provides an extra line of evidence that activation of TLRs either by self or non-self signals may contribute to the initiation of RA. Interestingly, miR-155 has been shown to promote TNFα production.24 The promotion of TNFα production by miR-155 may be a key process in the pathogenesis of the disease. Another interesting point about the upregulation of these particular miRNAs is the observation that rheumatoid fibroblasts display similar characteristics to transformed cells.51 Perhaps it is the upregulation of these particular miRNAs, previously identified as oncogenes, which contributes to this phenotype. This highlights the point that miRNAs may provide the link between inflammation and cancer, as was suggested for miR-155. Figure 3 illustrates the upregulation of particular miRNAs in RA synovial fibroblasts.
CONCLUSIONS
Given that miRNAs are fundamental to the post-transcriptional control of gene expression, it is no surprise that functions for specific miRNAs in the immune and inflammatory response have been uncovered. Intense complexity can be expected, given the number of miRNAs in humans, and the multiple mRNAs they target. The best characterised so far for their immune effect are specific miRNAs associated with B-cell regulation, particularly miR-150 and miR-155, with miR-155 also having a role in Th2-cell development. For inflammation, miR-146 and miR-155 have been shown to be strongly induced in multiple cell types, including rheumatoid synovial fibroblasts, by proinflammatory stimuli. miR-146 regulates IRAK-1 and TRAF-6, signals for IL1 and TLRs. It may therefore be a key negative regulator of inflammation in RA. Clearly, many questions remain about miRNAs and inflammation in this emerging field. Given the multiplicity of miRNAs, how many might be ultimately implicated in the inflammatory response? What are the target genes for these miRNAs and how might they be implicated in disease pathogenesis? Are there single nucleotide polymorphisms in miRNA genes which might alter expression and link to disease? Might miRNA induction be important for inflammation-associated cancers? Finally, and perhaps most importantly, might these insights lead to new treatments for diseases such as RA? It is still too early to tell, but the prospect is extremely exciting.
REFERENCES
Footnotes
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Funding: Science Foundation Ireland, the Health Research Board of Ireland and EMBARK are gratefully acknowledged for providing financial support to the authors.
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Competing interests: None.