**4. MiRNAs and autoimmune diseases**

The emerging picture of a central role played by miRNAs in the onset, development and turning off of immune responses is strictly related to the findings of their involvement in autoimmune diseases. In some cases, the functions of specific miRNAs have been first elucidated in the disease and then in immune system physiology. The possibility to use murine models of autoimmunity allows investigators to study the selected miRNAs *in vivo* in order to understand how they facilitate or attenuate the disease. However, the identification of a specific miRNA in the mouse model does not mean a direct translation into human disease. Overall, disregulation of miRNAs observed in autoimmunity promote either activation of immune effectors and/or suppression of immune regulatory cells, thus contributing to disease development. In the following section, the contribution of miRNAs will be discussed according to the specific autoimmune disorder.

#### **5. Systemic Lupus Erythematosus (SLE)**

SLE is a chronic autoimmune disease with a complex pathogenesis, involving different organs [33]. Since systemic inflammation is the hallmark of the disease, deregulation of critical pro inflammatory pathways have been described [34]. Indeed, miRNAs deregulated in SLE target genes involved in the inflammatory responses

#### **5.1. miR146a**

210 Immunodeficiency

**3.2. Adaptive responses** 

are increased [10].

[30].

responses.

of miRNAs in turning on and off the inflammatory/innate responses. In this setting, miR21

Immune responses require an integration between the innate/inflammatory and adaptive arm. According to their specific functions, miRNAs represent a perfect set of molecules to finely regulate and coordinate also adaptive responses. miRNAs that are involved in developmental stages of hematopoiesis can show additional functions in differentiated immune cells. Indeed, miR181a, which is implicated in thymic selection, is able to strengthen TCR signaling and reinforce T cell activation upon antigen engagement [11]. This effect likely relies on phosphatase suppression and increase in ERK phosphorylation. A member of the same family, miR181b has been proposed as regulating CSR (class switch recombination) of B cells. CSR is induced by activation induced cytidine deaminase (AID) and is likely targeted by miR181b. Indeed, IgG switch promoted by LPS and IL-4 stimulation is impaired when levels of endogenous miR181b

The role of miRNAs in T cell responses can be also variable according to its endogenous levels and/or contemporary expression in antigen presenting cells (APCs). This might be the case of miR155. miR155 is encoded within the BIC region (B cell integration cluster), which is often involved in lymphomas. BIC deficient mice, which lacked miR155 production, did not show significant impairment in hematopoiesis. When immunization with different bacterial strains and subsequent challenge with the same pathogens were administered to BIC deficient mice, the animals died of infection. Indeed, immunizations did not translate into protective immunity as compared with wild type mice. The authors have shown that T cell activity was compromised because there was a shift towards Th2 phenotype due to downregulation of c-Maf, which is a transcription factor that drives Th2 cytokine secretion [29]. Furthermore, BIC deficient DCs failed to adequately activate T cell responses. In this model, B cells were not able to differentiate to plasmablasts and showed alterations in CSR (class switch recombination). This phenomenon may be due to miR155, that targets AID

Overall, these data indicate that miR155 has a pivotal role in sustaining adaptive immune

However, these data are partly in contrast with the study from Mao et al. [31], who showed that miR155 is upregulated upon TLR stimulation in murine bone marrow derived dendritic cells. Furthermore, transfection of murine epidermal DCs with miR155 coding plasmid increased its endogenous levels and attenuated T cells responses driven by DCs. These effects were reverted when a miR155 antisense sequence was co-transfected into epidermal DCs. The authors try to reconcile these conflicting data, explaining that endogenous levels may induce different effects of the same miRNA in different cell types. However, since

should turn off the response, by increasing IL-10 levels [28].

Type I IFN pathway is widely recognized as a primary deregulation of inflammatory responses in SLE pathogenesis [34]. IFN I pathway is elicited by TLR engagement. Among TLRs, TLR-7 contributes the most to this phenomenon. In 2009, Tang et al. [35] have shown that underexpression of miR146a is tightly related to the upregulation of type I IFN pathway. They analyzed 52 patients with SLE, 6 with Behcet'disease and 29 normal subjects and evaluated miR146a levels from PBMCs. Interestingly, miR146a was proportionally decreased according to disease state (no disease, inactive SLE and active SLE, with active SLE having the lowest levels). miR146a levels were inversely related to IFN score, which was calculated considering the expression of three representative

inducible genes. Finally, IFN pathway could be downregulated when overexpression of miR146a was attained in PBMCs taken from normal donors and SLE patients. The same group [36] has identified a genetic variant of the miR146a promoter region, that confers reduced binding affinity to the transcription factor ETS1, thus leading to reduced levels of miR146a and increased susceptibility to SLE. These data have been further confirmed by a genomic analysis, where a SLE associated polymorphic SNP variant, rs2431697, was found to be related to low expression levels of miR146a gene [37]. Overall, these findings suggest a pivotal role of miR146a in SLE susceptibility and development.

Emerging Role of MicroRNAs in the Pathophysiology of Immune System 213

inhibition. A similar activity of Dnmt1 suppression in CD4 T cells from SLE has been

RA is a systemic inflammatory disorder, primary involving synovial joints. The inflammatory milieu is the base for disease onset and progression. Several groups reported an increase of miR155 and 146a in synovial fibroblasts and PBMCs from RA patients [46, 47]. Interestingly, these miRNAs can be stimulated by inflammatory stimuli, though promoting opposite effects. miR155 sustains inflammation, while miR146a attenuates through TNFα suppression. In this setting, miR146a seems not able to promote its action. A possible explanation is that both miRNAs are elicited by the pro-inflammatory environment of RA, with miR155 enforcing inflammation, while miR146a should shut off it, but it is unable to

MS is an autoimmune disease of the central nervous system characterized by chronic inflammation, demyelination, and axonal damage. Demyelination is due to pro inflammatory T cells. Mireia Guerau-de-Arellano et al. [48] identified increased levels of miR128 and 27b in naive and miR340 in memory CD4 T cells from MS patients, favoring switch to Th1 phenotype. Gain-of-function experiments with these micro-RNAs enhanced the encephalitogenic potential of myelin-specific T cells in experimental autoimmune encephalomyelitis, while treatment with specific oligonucleotide miRNA inhibitors reverted to normal Th2 shift. These data further clarified the role of these miRNAs in MS pathogenesis and suggested a therapeutic strategy based on miR suppression by selected

The increasing evidences on the role of miRNAs in pathophysiology are radically changing the established paradigms of disease onset and development. However, we can assert that our undestanding of miRNA functions is still preliminary and further work is awaited to better define how these molecules integrate with known intracellular pathways. Indeed, we know that miRNAs exert a very finely tuned regulation of intracellular pathways. This effect is attained through a modulation of miR levels, that is very complex to study in simplified models both in vitro and in vivo. Indeed, the best method to study miRNAs is to over express or completely inhibit its expression, but it is unlikely that this method can perfectly mirror the real intracellular conditions. Immune responses represent an attracting system to explore miR functions, since they have to be tightly regulated. The data have shown that miRNA modulation is an efficient way to rapidly turn on and off immune responses, both preceding and integrating with the classical gene mastered pathways. Therefore, we believe that the study of miRNAs within

ascribed to miR21 and 148a [45]

**7. Multiple Sclerosis (MS)** 

exert its activity.

inhibitors.

**8. Conclusions** 

**6. Rheumathoid Arthritis (RA)** 

#### **5.2. miR125a, 126, 21 and 148a**

Autoimmune disorders are often characterized by disregulated expression of proinflammatory chemokines and its receptor that drive and sustain unchecked immune responses, favoring autoimmunity. This is the case for RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted), also known as CCL5, whose elevated levels are observed in the context of chronic systemic inflammations such as arthritis and nephritis. Renal damage is initiated by RANTES over-expression in mouse models of SLE [38].

Zhao et al. [39] have shown that miR125a levels are underexpressed in T cells of SLE patients, while its predicted target KLF13 (Kruppel like factor 13) was upregulated. KLF13 directly controls the expression of RANTES in T cells. Interestingly, prolonged mitogenic stimuli evoke miR125a upregulation in normal T cells, providing a negative feedback loop that controls chemokine expression and helps to turn off inflammatory responses. The deficiency of this mechanism in SLE patients provide further insights on the onset and progression of the disease.

DNA methylation is a relevant mechanism to regulate gene transcription in eukaryotic cells [40] and any perturbation of these pathways can have crucial impact in health and disease. T cells from SLE patients suffer of a global hypomethylation [41], which is related to disease activity. The reduction of DNA methylation depends upon the reduced levels of Dnmt1 (Dna methyl transferase-1), the key enzyme that transfers methyl groups to CpG islands. The paired analysis of CD4 T cells from normal donors and SLE patients revealed the presence of an upregulated miRNA in SLE-T cells, miR126, that was independent from costimulatory signals [42]. miR126 targets Dnmt1 and reduces its levels in SLE- CD4 T cells. miR126 downstream effects include hypomethylation of critical genes in autoimmune pathogenesis such as TNSFS7 and ITGAL, that encode CD70 and CD11a [43], respectively. Indeed, CD70 [44] is the cellular ligand for the tumor necrosis factor receptor family member CD27,and is required on activated T cells and B cells to stimulate the synthesis of IgG. CD11a, also known as lymphocyte function–associated antigen 1, belongs to the integrin family of cell surface receptors and can strengthen the adhesion of T lymphocytes to other immune cells. These events could be reverted by miR126 inhibition. A similar activity of Dnmt1 suppression in CD4 T cells from SLE has been ascribed to miR21 and 148a [45]
