BackgroundExcessive and inappropriate immune responses are the hallmark of several autoimmune disorders, including the inflammatory bowel diseases (IBD): Crohn’s disease (CD) and ulcerative colitis (UC). A complex etiology involving both environmental and genetic factors influences IBD pathogenesis. The role of microRNAs (miRNAs), noncoding RNAs involved in regulating numerous biological processes, to IBD pathology, in terms of initiation and progression, remains ill-defined. In the present study, we evaluated the relationship between colon, peripheral blood, and saliva whole miRNome expression in IBD patients and non-inflammatory bowel disease (non-IBD) controls to identify miRNAs that could discriminate CD from UC. Quantitative real-time PCR (qRT-PCR) was used to validate and assess miRNA expression.ResultsMicroarray analysis demonstrated that upwards of twenty six miRNAs were changed in CD and UC colon biopsies relative to the non-IBD controls. CD was associated with the differential expression of 10 miRNAs while UC was associated with 6 miRNAs in matched colon tissues. CD was associated with altered expression of 6 miRNAs while UC was associated with 9 miRNAs in whole blood. Expression of miR-101 in CD patients and miR-21, miR-31, miR-142-3p, and miR-142-5p in UC patients were altered in saliva.ConclusionsOur results suggest that there is specific miRNA expression patterns associated with UC versus CD in three separate tissue/body fluids (colon, blood, and saliva). Further, the aberrant miRNA expression profiles indicate that miRNAs may be contributory to IBD pathogenesis, or at least reflect the underlying inflammation. Scrutinizing miRNA expression in saliva and blood samples may be beneficial in monitoring or diagnosing disease in IBD patients. A panel of miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-146a, and miR-375) may be used as markers to identify and discriminate between CD and UC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0069-0) contains supplementary material, which is available to authorized users.

IL-10−/− mice, an animal model of Th1-mediated inflammatory bowel disease, were screened for the expression of 600 microRNAs (miRNAs) using colonic tissues and peripheral blood leukocytes (PBLs) from animals having either mild inflammation or severe intestinal inflammation. The development of colonic inflammation in IL-10−/− mice was accompanied by upregulation in the expression of ten miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-223, miR-326, miR-142-3p, miR-142-5p, miR-146a, and miR-155). Notably, the expression of all of these miRNAs plus miR-375 was elevated in PBLs of IL-10−/− mice at a time when colonic inflammation was minimal, suggesting that changes in specific miRNAs in circulating leukocytes may be harbingers of ensuing colonic pathology. In vitro exposure of colonic intraepithelial lymphocytes to IL-10 resulted in down-regulation of miR-19a, miR-21, miR-31, miR-101, miR-223, and miR-155. Interestingly, unlike IL-10−/− mice, changes in miRNAs in PBL of dextran sulfate sodium-treated mice were minimal, but were selectively elevated in the colon after pathology was severe. We further show that miR-223 is a negative regulator of the Roquin ubiquitin ligase, that Roquin curtails IL-17A synthesis, and that the 3′ UTR of Roquin is a target for miR-223, thus defining a molecular pathway by which IL-10 modulates IL-17-mediated inflammation. To identify additional miRNAs that may be involved in the regulation of Roquin, transcriptome analysis was done using cDNAs from HeLa cells transfected with 90 miRNA mimics. Twenty-six miRNAs were identified as potential negative regulators of Roquin, thus demonstrating functional complexity in gene expression regulation by miRNAs.

SummaryCostimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T ceils, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28-independent fashion since the mAb costimulates T cells from the CD28-deficient mice as well as wild-type mice. Expression doning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.T he generation of a T cell response has been shown to depend on two independent signals (1). The first signal for activation is mediated by the interaction of the antigenspecific TCR with processed peptides displayed by APCs. This specific signal must be accompanied by a second costimulatory signal(s) delivered by the APC to the T cells through cell-cell interactions or cytokines. The lack of costimulation prevents proliferation through the inhibition of autocrine lymphokine production. Moreover, in the absence of a second signal, some T cells are induced into a state of nonresponsiveness often called "anergy" (2).The nature of the costimulatory signal has recently been the subject of a large number of studies (3). CD28, which is expressed on a majority of T cells, is a receptor for the costimulatory molecules B7-1 and B7-2 (4). Binding of CD28, by either mAb or B7 family members, has been shown to costimulate antigen-specific T cell responses (5, 6), aUogeneic mixed lymphocyte reactions (7,8), and direct TCR-CD3 Anne I. Sperling (Bluestone Laboratory) and Jonathan M. Green (Thompson Laboratory) contributed equally to this work and should be considered co-first authors. activation through antibody-mediated stimulation (9). In addition, the blocking of CD28/1igand interactions substantially, but not completely, inhibits these immune responses (3).Recent studies, however, have shown that mice rendered CD28-deficient through mutations introduced by homologous recombination technology (CD28-/-), develop normal immune responses to viral infections in vivo, and reject allogeneic skin grafts (reference 10 and Green, J. M., C. B. Thompson, and J. A. Bluestone, unpublished data). The T cells from these mice can mount accessory cell-dependent responses to alloantigens, lectins, and anti-CD3 antibodies, although the respons...

Thyroid-stimulating hormone (TSH), a central neuroendocrine mediator of the hypothalamus-pituitary-thyroid axis, has been shown to affect various aspects of immunological development and function. To gain a better understanding of TSH involvement within the mammalian immune system, the expression and distribution of the TSH receptor (TSHr) has been studied by immunoprecipitation and by flow cytometric analyses. Using highly enriched populations of B cells, T cells, and dendritic cells, trace amounts of TSHr were precipitated from B cells and T cells, whereas high levels of TSHr were precipitated from the dendritic cell fraction. Flow cytometric analyses of TSHr expression on splenic and lymph node T cells revealed a major difference between those tissues in that only 2–3% of splenic T cells were TSHr+, whereas 10–20% of CD4+8− and CD4−8+ lymph node T cells expressed the TSHr, which was exclusively associated with CD45RBhigh cells and was not expressed during or after activation. The TSHr was not present on cells of the immune system during fetal or neonatal life. However, recombinant TSHβ was found to significantly enhance the phagocytic activity of dendritic cells from adult animals and to selectively augment IL-1β and IL-12 cytokine responses of dendritic cells following phagocytic activation. These findings identify a novel immune-endocrine bridge associated with professional APCs and naive T cells.

Murine small intestine intraepithelial lymphocytes (IELs) bear properties of both activated and nonactivated T cells, although the significance of that dichotomy remains unclear. In this study, we show that although IELs express CD69 in situ and ex vivo, and have cytotoxic activity ex vivo, most CD8+ IELs from normal mice are phenotypically similar to naive T cells in that they are CD45RBhigh, CD44low/int, and lack or have low levels of expression of CD25, Ly-6C, OX40, Fas ligand (FasL), and intracellular IFN-γ synthesis. Unlike CD8+ lymph node cells, IELs express high levels of the FasL gene, but do not express surface FasL until after CD3-mediated stimulation has occurred. Additionally, anti-CD3 stimulation of IELs in the presence of actinomycin-D did not inhibit FasL expression, suggesting that regulation FasL expression on IELs is controlled at least partially at the posttranscriptional level. Following CD3-mediated stimulation, IELs synthesize and secrete IFN-γ more rapidly and to greater levels than CD8+ lymph node cells, and they acquire the phenotype of fully activated effector cells as seen by an up-regulation of CD44, Ly-6C, OX40, FasL, and CD25 with the kinetics of memory T cells, with down-regulation of CD45RB expression. These findings indicate that contrary to previous interpretations, most small intestine IELs are not fully activated T cells, but rather that they are semiactivated T cells ready to shift to a fully activated state once a CD3-mediated signal has been received. These data also imply that under appropriate conditions it is possible for T cells to be sustained in a state of partial activation.

Neuroendocrine hormones of the hypothalamus-pituitary-thyroid axis can exert positive or negative immunoregulatory effects on intestinal lymphocytes. Small intestine epithelial cells were found to express receptors for thyrotropin-releasing hormone (TRH) and to be a primary source of intestine-derived thyroid-stimulating hormone (TSH). The gene for the TSH receptor (TSH-R) was expressed in intestinal T cells but not in epithelial cells, which suggested a hormone-mediated link between lymphoid and nonhematopoietic components of the intestine. Because mice with congenitally mutant TSH-R (hyt/hyt mice) have a selectively impaired intestinal T cell repertoire, TSH may be a key immunoregulatory mediator in the intestine.

It has been known for decades that the neuroendocrine system can both directly and indirectly influence the developmental and functional activity of the immune system. In contrast, far less is known about the extent to which the immune system collaborates in the regulation of endocrine activity. This is particularly true for immune-endocrine interactions of the hypothalamus-pituitary-thyroid axis. Although thyroid-stimulating hormone (TSH) can be produced by many types of extra-pituitary cells--including T cells, B cells, splenic dendritic cells, bone marrow hematopoietic cells, intestinal epithelial cells, and lymphocytes--the functional significance of those TSH pathways remains elusive and historically has been largely ignored from a research perspective. There is now, however, evidence linking cells of the immune system to the regulation of thyroid hormone activity in normal physiological conditions as well as during times of immunological stress. Although the mechanisms behind this are poorly understood, they appear to reflect a process of local intrathyroidal synthesis of TSH mediated by a population of bone marrow cells that traffic to the thyroid. This hitherto undescribed cell population has the potential to microregulate thyroid hormone secretion leading to critical alterations in metabolic activity independent of pituitary TSH output, and it has expansive implications for understanding mechanisms by which the immune system may act to modulate neuroendocrine function during times of host stress. In this article, the basic underpinnings of the hematopoietic-thyroid connection are described, and a model is presented in which the immune system participates in the regulation of thyroid hormone activity during acute infection.

Although cells of the immune system can produce thyroid stimulating hormone (TSH), the significance of that remains unclear. Using 5′ rapid amplification of cDNA ends, we show that mouse bone marrow cells produce a novel in-frame TSHβ splice variant generated from a portion of intron 4 with all of the coding region of exon 5 but none of exon 4. The TSHβ splice variant gene was expressed at low levels in the pituitary but at high levels in the bone marrow and the thyroid, and the protein was secreted from transfected CHO cells. Immunoprecipitation identified an 8 kDa product in lysates of CHO cells transfected with the novel TSHβ construct, and a 17 kDa product in lysates of CHO cells transfected with the native TSHβ construct. The splice variant TSHβ protein elicited a cAMP response from FRTL-5 thyroid follicular cells and a mouse alveolar macrophage cell line. Expression of the TSHβ splice variant but not the native form of TSHβ was significantly upregulated in the thyroid during systemic virus infection. These studies characterize the first functional splice variant of TSHβ, which may contribute to the metabolic regulation during immunological stress, and may offer an new perspective for understanding autoimmune thyroiditis.