| MicroRNAs (miRNAs) are a new class of small RNA molecules that play an important role in post-transcriptional gene regulation. Bioinformatic analysis has estimated that the mammalian miRNA repertoire may directly regulate up to30%of all protein-encoding genes. In recent years, rapidly accumulating evidence has demonstrated that miRNAs are key players in diverse physiological and pathophysiological processes. Therefore, it is not surprising that miRNAs act as indispensable fine tuners for the regulation of endogenous gene expression in immune homeostasis.Among the functionally evaluated miRNAs, miR-146a was the first to be reported as a key factor modulating the innate immune response. Upon LPS stimulation and virus infection, miR-146a acts as a negative feedback regulator and restrains the Toll-like receptor signaling pathways by targeting IRAK1/2and TRAF6in various innate cells such as monocytes, lung epithelial alveolar cells, and microglial cells. In the adoptive immune system, miR-146a has previously been reported to target FADD to modulate activation-induced cell death and IL-2expression in Jurkat T cells. Ablation of miR-146a in mice leads to increased numbers of Treg cells but impairs their suppressor function because of unrestrained function of STAT1. These studies suggest that miR-146a functions as a robust "brake" during the inflammation and adoptive immune responses.miRNA expression profiling has shown that miR-146a levels are dynamically regulated during the physiological and pathological immune responses. In comparison to naive B or T lymphocytes, miR-146a is much more abundant in various differentiated lymphocyte subsets such as Tfh, Treg, and germinal center (GC) B cells. In addition, our previous data showed that miR-146a expression levels were significantly elevated in the CD4+T cells of rheumatoid arthritis patients and that this elevation was closely associated with the serum TNF-a level. This expression pattern is not restricted to rheumatoid arthritis, and similar results have been noted in patients with cancers or autoimmune disorders, including Sjogren’s syndrome and psoriasis. miR-146a accumulation in these inflammatory or autoimmune conditions cannot explain or be explained by its immunosuppressive nature in innate cells.To investigate the potential pathological roles of accumulated miR-146a in vivo, we developed a transgenic mouse strain constitutively expressing miR-146a. When there was a3-8fold increase in miR-146a expression, which is reminiscent of that found in patients, the transgenic mice spontaneously developed immune symptoms remarkably similar to the manifestations of autoimmune lymphoproliferative syndrome (ALPS). Further cellular and molecular analyses suggested that Fas suppression by miR-146a represents a major etiological factor for these pathological manifestations.To investigate the pathogenic roles of miR-146a, concentrated lentivirus was microinjected, as described previously, into the perivitelline space of single-cell embryos to generate mouse strains carrying the mmu-miR-146a transgene. Four of seven (57%) founder animals expressed GFP at detectable levels by FACS. The founders were backcrossed with BALB/c mice for more than9generations. Since the genomic DNA fragment encoding miR-146a was preceded by a ubiquitin promoter and GFP in the vector FUGW-miR146a, a convenient approach was established for screening out transgenic mice with higher miR-146a expressions by detecting the GFP expression level in PBMCs. The expression levels of miR-146a were quantified by real-time PCR. The data showed3-8fold forced expression in transgenic mice, and the levels varied among hearts, livers, spleens, lungs, kidneys, and lymph nodes. The expression level of miR-146a coincidently matched that in patients with autoimmune disorders. Theoretically, in addition to miR-146a, our transgenic construct could produce mature miR-146a*. Although some star strand of miRNAs could be stable, our data indicated that this is not the case for miR-146a*. We measured the expression of miR-146a*and miR-146a using qRT-PCR (Taqman platform). Normalized to an endogenous control, snoRNA-135, we found that there are at least1700folds difference in concentration between miR-146a and miR-146a. At such low level of concentration, it is unlikely miR-146a*can make any contribution to the phenotype observed in our TG mice. Our approach for generating transgenic mice with high miRNA expression is feasible for studying miR-146a function in immune disorders and may therefore be applied to extensive investigation of miRNA functions in vivo.Starting at the age of3weeks, miR-146a transgenic mice exhibited greatly enlarged spleens and lymph nodes. Especially in the lymph nodes, the cellularity was3-fold higher in transgenic animals at week3, peaked to8-fold at week4, and was maintained at4-fold from week6. Except for monocyte counts, the hematological analysis showed no significant differences in white and red blood cell counts, red blood cell width distribution, mean corpuscular volume, lymphocyte count, hematocrit, hemoglobin level, or platelet production between the wild-type and transgenic mice at the age of8weeks (n=10). In addition, the histopathological analysis also showed inflammation infiltrates in the lungs and livers of transgenic mice at the age of8weeks.We examined various subsets of lymphocytes in the lymph organs of miR-146a transgenic mice. Thymocyte development in transgenic mice was largely normal. While there was no significant change in the frequency of lymphocyte subsets compared to that in wild-type mice, the numbers of CD4+and CD8+T cells were increased greatly due to the dramatic increase in cellularity. Previous studies have suggested that miR-146a is involved in T cell activation and Treg cell function. In miR-146a transgenic mice, a slight decrease in Treg cell frequency was observed. The spontaneous T cell activation was assessed with surface markers, including CD25, CD69, CD44, and CD62L. No obvious activation was observed for either CD4+or CD8+T cells, which suggested that the reduced frequency of Treg cells was not sufficient to induce T cell activation. Moreover, in our in vitro assays, T cell survival in transgenic mice was also found to be normal. An important observation, however, was an abnormal increase in both the cell number and frequency of CD3+CD4+CD-TCRαβ+double-negative (DN) T cells in the spleens of miR-146a transgenic mice at the age of8to52weeks.The levels of inflammatory proteins in mouse sera were examined using the Milliplex assay. Although no significant changes were observed in the cytokines and chemokines tested, higher concentrations of total serum immunoglobulin were observed in miR-146a transgenic mice; that is, young adult transgenic mice (at the age of8weeks) had3-to6-fold higher serum concentrations of total IgG1, IgG2b, IgG2a, and IgG3. In aged mice (at the age of52weeks), the elevation in IgGl and IgG2a levels was retained, although at reduced levels, and no difference was detected in IgM or IgA levels in both8-and52-week-old mice.Consistent with elevated IgG levels, further analysis showed that the B220+B lymphocyte population in lymph nodes was considerably increased. Furthermore, a similar proportion of B cell subsets was observed between wild-type and transgenic mice, including mature B (B220+AA4.1-) and immature B (B220+AA4.1+), newly formed or activated B (B220+IgM+CD21lowCD23low), and follicular B cells (B220+IgM+CD21int CD23high). B cell development in the bone marrow was also normal in transgenic mice. However, with PNA staining, we observed that multiple GC-like structures developed spontaneously in the lymph nodes of transgenic mice, as early as8weeks after birth. Using the confocal immunofluorescence analysis, the similar results with germinal centers were observed in peripheral lymph nodes of transgenic mice at the age of8weeks. Further analysis at the cellular level showed that there was no significant difference in the percentage of Tfh cells between transgenic and wild-type mice. However, in both young adult and aged mice, the fractions and numbers of GC B cells significantly increased in peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), Peyer’s patches (PPs), and spleens (SP) of transgenic mice.In summary, starting at a young age, miR-146a transgenic mice developed enlarged spleens and lymph nodes, inflammatory infiltrations in the livers and lungs, increased frequency and numbers of T and B cells, elevated fractions of double-negative T cells, higher serum IgG levels, and accumulation of GC B cells. These pathological phenotypes remarkably coincided with clinical manifestations of ALPS.Dysfunction of Fas, which leads to interference with the Fas-mediated apoptosis pathway, is generally regarded as a pathogenesis factor in most individuals with ALPS. Therefore, the Fas expression in various lymphocyte populations (CD3+CD4+T, CD3+CD8+T, CD3+CD4-CD8TCRap+DN T, B220+PNA+CD38low non-GCB, and B220+PNA-CD38high GC B cells) was examined using FACS. As shown previously, the GC B cells were the only cells among these cell populations that had a higher surface density of Fas. Remarkably, the surface Fas level was downregulated in the B220+PNA+CD38low population of GC B cells from transgenic mice. This downregulation pattern suggests that miR-146a acts as a rheostat rather than a binary off-switch to decrease the Fas expression in GC B cells. The bioinformatics analysis also indicated that Fas could be a direct target of miR-146a. We tested this hypothesis with a dual luciferase assay. In agreement with a previous report, our results showed that miR-146a overexpression reduced the luciferase activity when cotransfected with plasmids containing the3’-UTR of Fas (from the mouse, rat, or human) in293FT cells, indicating that Fas is the direct target of miR-146a. To demonstrate that the suppression of Fas by miR-146a was specifically achieved during the GC formation, rather than originating from the B cell development process, we mixed non-GC B cells isolated from miR-146a transgenic or wild-type mice with wild-type CD4+T cells, followed by adoptive transfer into SCID mice. Seven days after transfer, the Fas expression levels were examined. A significantly higher amount (27-42%) of GC B cells were accumulated in transferred B cells from transgenic mice, compared to those (4-16%) from wild-type animals. Among the GC B cell population on both the third and seventh day after adoptive transfer, there was a significant reduction in the Fas expression on the surface of GC B cells originating from the non-GC B cells of donor transgenic mice. However, no significant alteration of Fas was observed in GC B cells from transgenic mice on either the third or seventh day after adoptive transfer. From the third to the seventh day after adoptive transfer, a slight increase of Fas expression was observed in GC B cells of wild-type mice, in agreement with a previous report that the expression of Fas was increased during the GC formation. Moreover, these data indicated that miR-146a may be intrinsically required for downregulating Fas during the GC formation.To further elucidate the mechanism underlying miR-146a-mediated B cell dysfunction, naive B cells were sorted from wild-type and transgenic mice, and the gene expression profiles were investigated using the cDNA microarray approach. Microarray data were used to assess miR-146a targets with the Sylamer method, which was designed to match the complementation between the seed region of miR-146a and the3’-UTR of downregulated mRNAs. The analysis showed that the elements capable of pairing to the seed region of miR-146a were significantly enriched in transgenic B cells. The previously validated miR-146a targets, such as TRAF6, IRAK1, and CXCR4, were identified in our microarray results. The functional bioinformatics analysis also suggested that the targets of miR-146a are enriched in the NF-κB, AP-1, IRF, and STAT signaling pathways. In agreement with the notion that miR-146a targets the NF-κB pathway in a negative feedback manner to regulate macrophage proliferation in response to antigens or mitogens, B cells from miR-146a transgenic mice proliferated to a lesser extent than those from wild-type mice that were stimulated with LPS.Alternatively, the expansion of naive B cells in our transgenic mouse model could be explained by lymphocyte homeostasis. To test this possibility, B cells isolated from wild-type and transgenic mice were adoptively transferred to6-to8-week-old recipient SCID mice. Using an EdU-incorporation assay, we found that B cells from transgenic mice showed hyper-homeostatic proliferation at day7after transfer. Furthermore, adoptive transfer of CD4+T cells was also performed to investigate T cell expansion in transgenic mice. Surprisingly, transferring CD4+T cells alone from transgenic mice showed a proliferation rate similar to that for wild-type mice. A previous report showed that ablation of Fas specifically in GC B cells impaired the homeostasis of both T and B lymphocytes. Therefore, a combination of T and B cells from the lymph nodes of transgenic mice was adoptively transferred, and homeostasis proliferation of various subsets gated as B220+, CD4+, and CD8+was analyzed. Interestingly, T cells from wild-type mice showed hyper-homeostatic proliferation when transferred together with miR-146a transgenic B cells. These findings suggested that the sustained expression of miR-146a in B cells is the major factor leading to the enhanced homeostatic expansion of B cells and T cells, which may be an etiologic factor for ALPS-like pathogenesis.We generated miR-146a transgenic mice that spontaneously develop immunological disorders that resemble the major clinical manifestations of ALPS. The majority of ALPS patients exhibit chronic and non-malignant lymphadenopathy and splenomegaly with significantly increased numbers of double-negative T cells and higher serum IgG levels at an early age. In addition, ALPS symptoms are typically worse in children but can be relieved in adults to some degree. In miR-146a transgenic mice, enlarged spleens and lymph nodes were observed at a very early age (3weeks), and the difference peaked around the fourth week after birth.. Elevated levels of double-negative T cell were also observed in the spleens of all young transgenic mice. The serum concentrations of IgG1, IgG2b, IgG2a, and IgG3were3-6fold higher in transgenic mice at early ages, and the total serum immunoglobulin levels at the age of52weeks almost returned to normal levels, except for those of IgG1and IgG2a. Infiltrative lung lesions and liver dysfunction have been found among4-5%of ALPS patients; similarly, we observed lung and liver infiltration with inflammatory cells in miR-146transgenic mice at a much higher incidence rate. Throughout the life, ALPS patients are susceptible to Hodgkin and non-Hodgkin lymphoma; strikingly, we also detected a high incidence of lymphoma development in aged miR-146a transgenic mice.However, there are some significant divergences between our model and ALPS patients. For example, higher serum autoantibody levels are observed among25%of ALPS patients; despite the accumulation of GC B cells, the levels of anti-dsDNA and anti-ANA in miR-146a transgenic mice were undetectable. In addition, during the progress of ALPS in patients, IL-10significantly increases in circulation and lymphoid tissues. We examined a large panel of cytokines and chemokines in the sera from transgenic mice and did not find any significant change. These data imply that overexpression of a single miRNA, miR-146a, in vivo may predominately promote lymphocyte proliferation without fully breaking the threshold for immunological tolerance. This is consistent with the current notion that the full manifestation of ALPS in human requires accumulation of genetic defects.Typical ALPS patients inherit genetic defects in the apoptosis pathway, which leads to breakdown of lymphocyte homeostasis and normal immunological tolerance. The major susceptible genes identified include Fas, caspase10, and Fas ligands. Previous reports have shown that Fas mutations in ALPS patients are accompanied by the reduction in Fas protein expression. In this study, we demonstrated that enhanced miR-146a expression results in the downregulation of Fas in GC B cells and eventually leads to the development of major ALPS symptoms. The critical etiologic factor for disease development in our model appears to be the overexpression of miR-146a in naive B cells. Our adaptive transfer experiments clearly showed that transgenic naive B cells are hyperproliferative, capable of promoting homeostatic proliferation of T cells, and prone to differentiating into GC B cells with reduced surface Fas levels. These findings agree with previous studies showing that the ablation of Fas, specifically in GC B cells but not in T cells, can unbalance the lymphocyte homeostasis and lead to hyper-lymphoproliferation. Moreover, downregulated Fas in GC B cells may also promote the survival of activated mature T cells and increase the possiblity to lose CD8or CD4coreceptor expression on activated mature T cells, thereby leading to the accumulation of DN T cells. In addition, recent studies showed that chronic active Epstein-Barr virus (EBV)-infected patients exhibit clinical ALPS manifestations, including increased double-negative T cell levels, hepatosplenomegaly, and lymphadenopathy. Interestingly, EBV infection can dramatically induce miR-146a expression in EBV-susceptible B cells. Taken together, these findings support the notion that miR-146a may be associated with the development of ALPS and may provide a novel explanation for the pathogenesis of ALPS, especially in cases accompanied with virus infection and chronic inflammation. High levels of miR-146a are induced in patients with autoimmune disorders, including rheumatoid arthritis, Sjogren’s syndrome, and psoriasis, which is in agreement with our observations in transgenic mice. On the other hand, the current conventional wisdom, with its roots in data from miR-146a-knockout mice, characterizes miR-146a as an immune suppressor. Ablation of miR-146a in mice results in a spontaneous autoimmune disorder characterized by elevated levels of serum autoantibodies against dsDNA, abnormal activation of T cells, and increased numbers but impaired suppressor function of Treg cells. However, we do not deem these nonreciprocal phenotypes between transgenic and knockout mice to be necessarily contradictory. Instead, with LPS stimulation, B cells from miR-146a transgenic mice indeed had lower proliferation rates, which is reciprocal to data from miR-146a-ablated mice. Moreover, TRAF6, IRAK1, and CXCR4were identified as the major targets in miR-146a-deficient macrophages, and the expression levels of these genes were significantly dampened in our transgenic naive B cells. Furthermore, the increased miR-146a expression in mice also reduced the frequency of Treg cells, although not to a degree that could result in conventional T cell activation. Nevertheless, our data demonstrate a novel functional role of miR-146a in facilitating lymphocyte homeostasis proliferation. We hypothesize that the autoimmunity that develops in miR-146a-deficient mice reflects its suppressive effects on the NF-B pathway, with a dominant function inside innate immune cells, while the ALPS-like symptoms manifested in miR-146a transgenic mice illustrate its nature in promoting the homeostatic expansion of B cells. |
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