Functional Role of MicroRNAs in Thymocyte Development

The thymus, a central immune organ in humans and other mammals, is an important place for the development, differentiation, and maturation of immune cells. T lymphocytes, referred to as T cells, are derived from the early T-lineage precursor of the bone marrow or embryonic liver lymphoid stem cells, which develop and mature in the thymus and then migrate into peripheral immune organs or tissues to perform their biological functions [1-3]. During the development of the thymus, T cells can be divided into 3 stages based on the differential expression of CD4 and CD8 coreceptors (Fig. 1). The early T-cell phenotype consists of CD4^–CD8^– double-negative cells (DN), which mainly differentiate in the cortical region; subsequently, DN T cells differentiate into CD4⁺CD8⁺ double-positive cells (DP), begin to express T-cell receptor (TCR), and gradually migrate into the medulla. Furthermore, the MHC-restricted recognition ability is obtained by positive selection, and the tolerance to its own antigen is obtained by negative selection. Finally, these cells develop into mature T cells with only CD4 or CD8 single-positive (SP) expression, then migrate out of the thymus, and move to the peripheral lymphoid organs or tissues to settle and perform a corresponding immune function.

Studies have shown that microRNAs (miRNAs) are vital to many aspects of immunity, including various levels of T-cell immunity [4-9]. miRNAs are small, noncoding, single-strand ∼22 nucleotide (nt)-long RNAs generated from primary miRNAs (pri-miRNAs) containing a stem loop structure, which are processed in the nucleus by the enzymes Drosha and DiGeorge syndrome critical region 8 (DGCR8) into shorter pre-mi-RNAs [10]. The pre-miRNA is subsequently processed in the cytoplasm by Dicer, resulting in RNA duplex of -20–22 nt [11, 12]. The RNA duplex is incorporated into the RNA-induced silencing complex, where one of the strands undergoes degradation while the other forms the mature miRNA [13]. Mature miRNAs can bind to the 3′-untranslated region of complementary or partially complementary target gene mRNA and promote the degradation of target gene mRNA at posttranscriptional level or mediate its translation inhibition, thus exerting a wide range of physiological regulatory functions [14]. In humans, more than 1,000 miRNA family members have been reported to participate in important biological processes such as cell development, differentiation, proliferation, apoptosis, tumorigenesis, and -development. Accumulating evidence has shown that miRNAs play an important regulatory role in the development of thymocytes, indicating their importance in the development of the immune system.

Dynamic regulation of miRNA expression in sequential stages of thymocyte development is well documented [15]. An overall increase in miRNA levels at early stages of T-cell maturation (DP stage) with an increase in total cellular RNA content [15] suggests that its expression is associated with thymocyte development. Cobb et al. [16] further found that in the early stage of thymocyte development (DN to DP stage), conditional deletion of Dicer, a key enzyme required for miRNA maturation, would reduce the cellularity of DP thymocytes, which was driven mainly by reduced numbers and survival of αβ T cells, whereas the number of γδ T cells was not affected. Notably, the number of CD4 and CD8 SP T cells in the spleen and the total number of CD3⁺ T cells in the peripheral blood decreased obviously after conditional deletion of Dicer [16, 17]. In addition, individual miRNAs showed dynamic changes in different thymocyte subsets (DN, DP, and SP), and the increase in single miRNAs during thymocyte development was negatively correlated with the deletion of target genes [15, 18]. These studies indicated that specific miRNAs were involved in the development of thymocytes (Fig. 2).

In an miR-181a-overexpressing mouse model, the number of T cells in the peripheral circulation decreased significantly, especially the CD8⁺ T-cell number was reduced by 90% [19], suggesting that miR-181a may play an important role in the development of T cells. Functional analysis of miR-181a-1 and miR-181c by Liu et al. [20] showed that miR-181a-1 was ectopically expressed in thymic progenitor T cells and promoted the differentiation of CD4^–CD8^–DN to CD4⁺CD8⁺DP cells, while miR-181c did not have such a function, it might be that the unique pre-miRNA stem loop nt sequence of miR-181a-1 determined its functional specificity. Furthermore, Neilson et al. [15] found that miR-181a specifically increased in the CD4⁺CD8⁺DP stage of thymocyte development, and that miR-181a could inhibit the expression of B-cell lymphoma/leukemia 2 (Bcl-2) and CD69, for example, thereby affecting their coordination and participation in the positive selection process of CD4⁺CD8⁺DP T cells [21].

The sensitivity of TCR plays an important role in the positive and negative selection of T cells. Li et al. [22] confirmed that after the upregulation of miR-181a expression in mature T cells, the sensitivity of T cells to antigenic peptides would increase, and, meanwhile, the intracellular calcium ion outflow and cytokine IL-2 production would be enhanced; downregulation of miR-181a expression in immature T cells reduced T-cell sensitivity to antigenic peptides and impaired negative and positive selection of thymocyte development; in addition, the quantitative regulation of the sensitivity of miR-181a to T cells enabled mature T cells to recognize an antagonist (inhibitory antigen peptide) as an agonist. Li et al. [22] further found that miR-181a could increase the homeostasis of the phosphorylated intermediate by inhibiting the expression of tyrosine and serine phosphatase, thereby reducing the signal threshold of TCR and enhancing its -sensitivity. Subsequent analysis showed that miR-181a downregulated the expression of multiple phosphatase genes (such as SHP-2, PTPN22, DUSP5, and DUSP6). Importantly, the high expression of miR-181a was associated with the sensitivity of immature T cells, which suggested that miR-181a might act as an intrinsic “rheostat” for antigenic sensitivity during the development of T cells. Ebert et al. [23] also found that inhibition of miR-181a expression promoted T-cell responses to participate in the positive selection of its own peptides, leading to the maturation of T cells, while miR-181a could prevent the deletion of moderate affinity clone by regulating the threshold of the thymocyte TCR signal, ensuring the smooth progress of positive selection.

The miRNA expression profile of humans and mice showed that the level of miR-150 was upregulated during T-cell maturation [18, 24]. Zhou et al. [25] further discovered low expression of miR-150 in the CD4^–CD8^–DN stage of thymocytes, moderate expression in the CD4⁺CD8⁺DP stage and CD8⁺ T cells, while expression was high in CD4⁺ T cells – this dynamic change in miR-150 suggested that it might have a regulatory effect on thymocyte development. In miR-150 transgenic mice, overexpression of miR-150 blocked the development of murine thymocytes, especially the differentiation of DN3 to DN4, which ultimately led to a decrease in the number of CD4⁺ and CD8⁺ T cells [24]. In terms of the mechanism, the transcription factor c-myeloblastosis (c-Myb) was an important target molecule of miR-150, and its expression is downregulated when miR-150 was overexpressed in immature T cells [24]. In addition to c-Myb, NOTCH3 was also an important new target molecule of miR-150, which was the main regulatory factor of T-cell differentiation and can reduce the proliferation and survival of T cells [18]. These research works suggested that miR-150 was instrumental to T-cell maturation.

Other studies have shown that miR-150 is also involved in the development of different functional T-cell subsets. For example, the invariant nature killer T (iNKT) cells are a unique subset of T lymphocytes, which express a constant TCRVα14 and a partial marker of NK cells, CD161 (NK1.1), and NK cell receptors, NKR-P1C. Zheng et al. [26] demonstrated that miR-150 expression was upregulated during iNKT cell maturation and activation, and iNKT cell maturation in the thymus was impaired in the miR-150-knockout mouse model. Subsequently, they also confirmed that miR-150 knockout could change iNKT cell maturation and function by using adoptive cell transfer of bone marrow cells. Furthermore, Bezman et al. [27] studied miR-150 overexpression in mice and came to similar conclusions. The above studies indicate that miR-150 played important roles in the development and function of iNKT cells. However, whether it is also involved in the development of other T-cell subsets remains to be elucidated.

Under normal physiological conditions, miR-146a was expressed in various immune cells, including T cells [28]. It has been reported that overexpression of miR-146a in bone marrow lymphoid progenitor cells might impair the development of hematopoietic stem/progenitor cells, eventually resulting in a decreased number of CD4⁺ T cells in the peripheral blood [29]. In addition, Kirigin et al. [30] found that the expression level of miR-146a was different in thymocytes at different developmental stages of the thymus. Li et al. [31], using miR-146a overexpression in a mouse model, found that overexpression of miR-146a could promote T-cell proliferation and reduce T-cell numbers. More importantly, in this murine model, positive selection of thymocytes, a key process for central tolerance, was weakened, resulting in an increase in CD4⁺CD8⁺DP T cells and a decrease in CD4⁺SP and CD8⁺SP T cells. Moreover, the maturation process of CD8⁺SP T cells was weakened, which led to a more severe loss of CD8⁺SP T cells than CD4⁺SP T cells. The above studies showed that miR-146a can regulate thymocyte -development through the development of bone marrow lymphocyte progenitor cells and thymic cells. However, the related target molecular mechanism remains to be further studied.

MiR-17–92 is a cluster of 6 miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92–1). In the bone marrow, overexpression of miR-17–92 in lymphoid cells could lead to excessive cell proliferation, which may further affect the migration of lymphoid cells into the thymus, leading to dysplasia of thymocytes. During thymus development, Xiao et al. [32] used the mouse model that overexpressed miR-17–92 in the DN1 stage of thymocyte development and found that the number of peripheral T lymphocytes in this mouse model was significantly changed, especially CD4⁺ T cells, suggesting that miR-17–92 was involved in the thymocyte development process. Regelin et al. [33] further used miR-17–92^Δ/Δ mice and found that thymocytes lacking miR-17–92 had serious defects in early development, especially the differentiation process of DN to DP. These studies suggested that miR-17–92 played a crucial role in the regulation of thymocyte development and might be more related to the development of CD4⁺ T cells. However, its exact role and related mechanisms in various stages of thymocyte development remain unclear.

Natural regulatory T cells (nTreg) are CD4⁺ T cells produced by lymphocytes during thymus maturation, which express CD25 molecules. These cells are transported to peripheral lymphoid organs or tissues through the blood flow after thymus maturation and perform a corresponding immune function. Studies have found that miR-155 plays an important regulatory role in the development of nTreg cells [34-36]. In miR-155-knockout mice, the number of Treg cells in the thymus and peripheral lymphoid organs was decreased. Moreover, the proliferation capacity of Treg cells lacking miR-155 was also significantly weakened [34, 37]. IL-2 is a key factor in the development and survival of Treg cells [38-40]. Further studies have found that miR-155 can promote Treg cells in the thymus by enhancing the sensitivity of Treg to IL-2 and increasing signal transduction and activator of transcription 5 (STAT5) signaling pathways, thereby increasing the proliferation and survival of Treg cells in the thymus and periphery [37]. Interestingly, Kohlhaas et al. [34] also found that forkhead box protein 3 (Foxp3) can regulate the expression of miR-155 in nTreg cells. Given the importance of the STAT5 signaling pathway in Foxp3 expression [41], we hypothesized that there is a regulatory loop between miR-155, STAT5, and Foxp3 to regulate the development of nTreg cells. In addition, Sánchez-Díaz et al. [42] also found that the related pathway of C-type lectin could also regulate thymus development of Treg cells through the expression of miR-155, suggesting the complexity of the miR-155 molecule involved in thymic Treg development.

In addition, it was reported that miR-155 was involved in thymus development of iNKT cells. Burocchi et al. [43] used miR-155 overexpression in a mouse model and found that miR-155 overexpression could inhibit the development and maturation of iNKT cells in the thymus, showing that immature iNKT cells were largely retained in their second stage of thymus development (CD24^loCD44^hiNK1.1⁻), leading to a decrease in the number of iNKT cells migrating to the periphery. It was also found that the expression of 2 target molecules of miR-155, Ets1 and Itk, was downregulated during iNKT cell maturation in this murine model [43]. However, the exact regulatory mechanism remains to be elucidated.

In addition to the above miRNA family molecules, other miRNA members also participate in the regulation of thymocyte development (Table 1). For instance, miR-142 is an evolutionarily conserved miRNA that can be selectively expressed in hematopoietic tissue. Mildner et al. [44] showed that the deletion of miR-142 could affect cell homeostasis. Their further analysis revealed that miR-142 was required for thymocyte precursor development and T-cell maturation. The lack of miR-142 could lead to the abnormal development of stasis of the thymocyte precursor, and impair thymocyte proliferation and differentiation, which result in a large number of cells being retained in the DP stage. For other examples, the expression level of miR-125b in lymphatic stem cells was higher than that of myeloid and hematopoietic stem cells. This high expression promotes the development of lymphocyte lineages and participates in the survival of hematopoietic stem cells and the maintenance of lymphoid balance [45]. miR-205 could promote the development of T cells after stress by regulating forkhead box N1 and specific chemokines [46]. Moreover, miR-223 could further regulate the development of T cells by protooncogene control [47]. In addition, miR-133b regulated the differentiation of NKT17 cells in the thymus by regulating Th-POK expression and dendritic cell signaling [48]. Most recently, we used miR-126 knockdown in mice and found that miR-126 deficiency could affect the development of thymic CD4⁺SP cells through elevating its target molecule IRS-1 [49]. These studies further demonstrated the complexity of miRNA family molecules involved in thymocyte development.

In recent years, a series of studies have shown that miRNAs played an important regulatory role in thymocyte development, involving the development of lymphoid progenitor cells in the bone marrow, as well as DN/DP, and positive and negative selection of thymocytes. However, there are still many scientific issues to be further clarified. For example, what is the exact relationship among these different miRNA molecules, different stages of thymocyte development, and microenvironment in thymus? What about the related molecular mechanisms? The regulation mechanism of the expression of these miRNA molecules, especially the intrinsic link between the mRNAs involved in thymocyte development and clinical diseases, and how to carry out immunological biotherapy based on miRNA molecule-related clinical diseases? And so on. Further elucidation of these scientific issues will not only greatly enhance the understanding of the biological functions of miRNAs and the regulation mechanism of thymocyte development, but also be helpful in developing new strategies for immunotherapy against related clinical diseases.

This work was supported by the Program for High-Level Innovative Talents in Guizhou Province (QKH-RC-2016-4031), National Natural Science Foundation of China (31760258), Program for New Century Excellent Talents in University, Ministry of Education of China (NCET-12-0661), Program for Excellent Young Talents of Zunyi Medical University (15ZY-001), and the Project of Guizhou Provincial Department of Science and Technology (QKHJC-2018–1428).

The authors have no ethical conflicts to disclose.

All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Lin Hu designed and wrote the paper; Ling Mao and Shiming Liu wrote the paper; Juanjuan Zhao, Chao Chen, Mengmeng Guo, and Jie Yang designed the paper; Lin Xu, Wei Xu, and Zhixu He conceived, designed, and wrote the paper; and all authors reviewed the paper.

Edited by: H.-U. Simon, Bern.