Jeremy S. Schaefer

Selective Upregulation of microRNA Expression in Peripheral Blood Leukocytes in IL-10 −/− Mice Precedes Expression in the Colon

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 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 10 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 downregulation 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 selectively elevated in the colon after pathology was severe. We further show that miR-223 is a negative regulator of the Roquin ubiquitin ligase, Roquin curtails IL-17A synthesis, and the 3′ untranslated region 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.

MicroRNA signatures differentiate Crohn’s disease from ulcerative colitis

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.

Bone marrow cells produce a novel TSHβ splice variant that is upregulated in the thyroid following systemic virus 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 (RACE), we show that mouse bone marrow (BM) 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 BM and the thyroid, and the protein was secreted from transfected Chinese hamster ovary (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 (AM) 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 a new perspective for understanding autoimmune thyroiditis.

ICOS promotes IL‐17 synthesis in colonic intraepithelial lymphocytes in IL‐10−/− mice

In the absence of IL‐10, colonic inflammation ensues, which is characterized by high levels of IL‐17. Here, we demonstrate a direct correlation between ICOS expression and IL‐17 production in cIELs. IL‐10−/− mice had increased numbers of cIELs and greater colon weight. Although the CD69 early activation antigen was expressed on cIELs from normal and IL‐10−/− mice, ICOS was expressed only on cIELs from IL‐10−/− mice. IL‐17‐producing cells in IL‐10−/− mice consisted of CD4+ and CD8+ cIELs; however, CD4+ cells were the predominant IL‐17‐producing cell population. Culture of cIELs from IL‐10−/− mice with IL‐23 resulted in an increase in ICOS and IL‐17 expression, whereas IL‐10 suppressed expression of ICOS and IL‐17. This occurred in primary cultures and recall stimulation experiments. The ICOS ligand B7RP‐1 was up‐regulated on colonic epithelial cells and on a population of large granular leukocytes during inflammation. Culture of cIELs with B7RP‐1+ DCs enhanced IL‐17A production from normal cIELs but failed to do so using cIELs from ICOS−/− mice. In vivo treatment of IL‐10−/− mice with antibody to ICOS resulted in a significant reduction in colonic pathology. These findings implicate ICOS as an activational signal of Th17 cells during chronic intestinal inflammation, and they suggest that under some conditions, control of ICOS expression may help to suppress chronic intestinal inflammation.

A novel thyroid stimulating hormone β-subunit isoform in human pituitary, peripheral blood leukocytes, and thyroid

Thyroid stimulating hormone (TSH) is produced by the anterior pituitary and is used to regulate thyroid hormone output, which in turn controls metabolic activity. Currently, the pituitary is believed to be the only source of TSH used by the thyroid. Recent studies in mice from our laboratory have identified a TSHbeta isoform that is expressed in the pituitary, in peripheral blood leukocytes (PBL), and in the thyroid. To determine whether a human TSHbeta splice variant exists that is analogous to the mouse TSHbeta splice variant, and whether the pattern of expression of the splice variant is similar to that observed in mice, PCR amplification of RNAs from pituitary, thyroid, PBL, and bone marrow was done by reverse-transcriptase PCR and quantitative realtime PCR. Human pituitary expressed a TSHbeta isoform that is analogous to the mouse TSHbeta splice variant, consisting of a 27 nucleotide portion of intron 2 and all of exon 3, coding for 71.2% of the native human TSHbeta polypeptide. Of particular interest, the TSHbeta splice variant was expressed at significantly higher levels than the native form or TSHbeta in PBL and the thyroid. The TSHalpha gene also was expressed in the pituitary, thyroid, and PBL, but not the BM, suggesting that the TSHbeta polypeptide in the thyroid and PBL may exist as a dimer with TSHalpha. These findings identify an unknown splice variant of human TSHbeta. They also have implications for immune-endocrine interactions in the thyroid and for understanding autoimmune thyroid disease from a new perspective.

Immunological regulation of metabolism—a novel quintessential role for the immune system in health and disease

The hypothalamus‐pituitary‐thyroid (HPT) axis is an integrated hormone network that is essential for maintaining metabolic homeostasis. It has long been known that thyroid stimulating hormone (TSH), a central component of the HPT axis, can be made by cells of the immune system; however, the role of immune system TSH remains enigmatic and most studies have viewed it as a cytokine used to regulate immune function. Recent studies now indicate that immune system‐derived TSH, in particular, a splice variant of TSHβ that is preferentially made by cells of the immune system, is produced by a subset of hematopoietic cells that traffic to the thyroid. On the basis of these and other findings, we propose the novel hypothesis that the immune system is an active participant in the regulation of basal metabolism. We further speculate that this process plays a critical role during acute and chronic infections and that it contributes to a wide range of chronic inflammatory conditions with links to thyroid dysregulation. This hypothesis, which is amenable to empirical analysis, defines a previously unknown role for the immune system in health and disease, and it provides a dynamic connection between immune‐endocrine interactions at the organismic level.—Schaefer, J. S., Klein, J. R. Immunological regulation of metabolism—a novel quintessential role for the immune system in health and disease. FASEB J. 25, 29–34 (2011). www.fasebj.org

Differential Cytokine Patterns in Mouse Macrophages and Gingival Fibroblasts After Stimulation With Porphyromonas gingivalis or Escherichia coli Lipopolysaccharide

BACKGROUND A major cause of chronic inflammatory periodontal disease is Porphyromonas gingivalis, a non-motile, Gram-negative, rod-shaped, anaerobic bacterium. Within gingival tissue, both macrophages and fibroblasts participate in the immune response to foreign entities by releasing cytokines and expressing molecules to recruit and activate lymphocytes. However, the contribution of gingival macrophages and fibroblasts to the immune response to P. gingivalis infection is not fully known. METHODS The AMJ2-C8 cell line (AM cells), a mouse alveolar macrophage cell line, and ESK-1 cells, a mouse gingival fibroblast cell line made in our laboratory, were treated with lipopolysaccharide (LPS) from either P. gingivalis or Escherichia coli. The expression of immune response molecules was quantified by real-time polymerase chain reaction and enzyme-linked immunoassay. RESULTS AM and ESK-1 cells responded differently to P. gingivalis and E. coli LPS stimulation. The ESK-1 gingival fibroblast cell line was more responsive to E. coli LPS stimulation as seen by elevated levels of interleukin (IL)-6, inducible nitric oxide, and monocyte chemotactic protein-1 expression relative to stimulation by P. gingivalis LPS. Conversely, the AM macrophage cell line was more responsive to P. gingivalis LPS stimulation, particularly for interleukin IL-1β, IL-6, and monocyte chemotactic protein-1, relative to stimulation by E. coli LPS. CONCLUSION These findings demonstrate that E. coli LPS induces a stronger cytokine and chemokine response in gingival fibroblasts, whereas P. gingivalis LPS induces a stronger response in macrophages.

Small intestine inflammation in Roquin-mutant and Roquin-deficient mice.

Roquin, an E3 ubiquitin ligase that localizes to cytosolic RNA granules, is involved in regulating mRNA stability and translation. Mice that have a M199R mutation in the Roquin protein (referred to as sanroque or Roquinsan/san mice) develop autoimmune pathologies, although the extent to which these occur in the intestinal mucosa has not been determined. Here, we demonstrate that Roquinsan/san mice reproducibly develop intestinal inflammation in the small intestine but not the colon. Similarly, mice generated in our laboratory in which the Roquin gene was disrupted by insertion of a gene trap cassette (Roquingt/gt mice) had small intestinal inflammation that mimicked that of Roquinsan/san mice. MLN cells in Roquinsan/san mice consisted of activated proliferating T cells, and had increased numbers of CD44hi CD62Llo KLRG1+ short-lived effector cells. Proportionally more small intestinal intraepithelial lymphocytes in Roquinsan/san mice expressed the ICOS T cell activation marker. Of particular interest, small intestinal lamina propria lymphocytes in Roquinsan/san mice consisted of a high proportion of Gr-1+ T cells that included IL-17A+ cells and CD8+ IFN-γ+ cells. Extensive cytokine dysregulation resulting in both over-expression and under-expression of chemotactic cytokines occurred in the ileum of Roquinsan/san mice, the region most prone to the development of inflammation. These findings demonstrate that chronic inflammation ensues in the intestine following Roquin alteration either as a consequence of protein mutation or gene disruption, and they have implications for understanding how small intestinal inflammation is perpetuated in Crohns disease (CD). Due to the paucity of animal models of CD-like pathophysiology in the small intestine, and because the primary gene/protein defects of the Roquin animal systems used here are well-defined, it will be possible to further elucidate the underlying genetic and molecular mechanisms that drive the disease process.

MicroRNAs: how many in inflammatory bowel disease?

Purpose of review MicroRNAs (miRNAs), small noncoding RNA molecules of approximately 22 nucleotides, have emerged as critical mediators of gene expression. As the dysregulation of gene expression can have far reaching impact on health and disease, miRNAs are being examined as potent new mediators of disease as either biomarkers or potential therapeutic targets. The purpose of this review is to evaluate the contribution of miRNAs to inflammatory bowel disease (IBD) pathophysiology. Recent findings Recent studies have evaluated the expression of miRNAs in tissue and body fluid specimens from patients with the main subtypes of IBD – Crohns disease and ulcerative colitis. Unique miRNA expression patterns that may distinguish IBD subtypes have been uncovered. Summary Significant progress has been made in illuminating the complex interactive networks of miRNAs and gene targets in IBD. The potential use of miRNAs as disease biomarkers or therapeutics shows promise. However, there are still significant hurdles to overcome before miRNA-based therapeutics and diagnostics will be of clinical utility.

A role for IL-10 in the transcriptional regulation of Roquin-1

Roquin-1, a RING finger E3 ubiquitin ligase, functions as a modulator of inflammation; however, nothing is known about how Rc3h1 expression is regulated. Here, we describe an opposing relationship between Roquin-1 and the IL-17 proinflammatory cytokine by demonstrating that enforced expression of Rc3h1 restricts Il17a expression, and that exposure of T cells to IL-10, a cytokine with immunosuppressive activity, increases Rc3h1 expression. Luciferase reporter assays conducted using eight transcription factor plasmids (STAT1, STAT3, STAT5, GATA2, c-Rel, IKZF1, IKZF2, and IKZF3) demonstrated that STAT1, STAT3, GATA2, and c-Rel increased Rc3h1 promoter activity, whereas IKZF2 decreased activity. Gene expression of those five transcription factors increased in T cells exposed to IL-10. Transcription factor-specific siRNAs suppressed the IL-10 effect on Rc3h1 transcription. These findings identify a role for IL-10 in regulating Rc3h1 transcription, and they have implications for understanding how Roquin-1 controls the immune response.