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MiR-21 is strongly elevated in biopsies of pediatric patients with Eosinophilic Esophagitis (EoE)

MiR-21 is one of the most commonly dysregulated miRNA in a wide variety of cancers (Hatley et al., 2010; Jung and Calin, 2010). It is also reported as increased in heart diseases (Thum et al., 2008), lung fibrosis (Liu et al., 2010), type 1- diabetes (Ruan et al., 2011), T cell lymphoma (van der Fits et al., 2011), lupus (Garchow et al., 2011; Pan et al., 2010), and psoriasis (Meisgen et al., 2012; Zibert et al., 2010). Relevant to the role of miR-21 in promoting Th2 responses, miR-21 is up-regulated in three different mouse models of allergic inflammation (OVA, IL-13 transgenic and Aspergillus fumigatus) (Lu et al., 2009b). Further, we have also noted a strong induction of miR-21 in our airway inflammation studies, particularly for mice receiving Bcl6-deficient Tregs that developed severe Th2 inflammation. While inhibition of the Th1 cytokine IL-12 has been demonstrated as one mechanism for the association of miR-21 in Th2 diseases (Lu et al., 2011b; Lu et al., 2009b), our studies highlight a novel T cell intrinsic role for miR-21 in Th2 differentiation. Thus overall these studies suggest that miR-21 may not just be a passive marker up-regulated in Th2 diseases, but serves a functional role in augmenting Th2 responses via its role in myeloid cells and T cells. Thus, miR-21 may be part of a positive feedback loop amplifying the Th2 inflammatory responses.

MiRNAs are being used as diagnostic biomarkers for cancer and a number of autoimmune and inflammatory pathologies (O'Connell et al., 2012; Sonkoly and Pivarcsi, 2009; Tricoli and Jacobson, 2007). Thus, to pursue the idea of miR-21 as a novel biomarker for Th2-type inflammatory diseases, we wondered if miR-21 was up-regulated in human allergic diseases. Eosinophilic esophagitis (EoE) is an increasingly prevalent allergic disease where the esophagus becomes highly inflamed, frequently in response to food allergens (Liacouras et al., 2011; Noel and Rothenberg, 2005; Rothenberg, 2009). Further, EoE is a Th2-type inflammatory disease characterized by high eosinophil infiltration into the esophageal epithelium, with the hallmark Th2 cytokines implicated in the pathology. We therefore obtained esophageal biopsies from pediatric EoE patients (Subbarao et al., 2011). Our controls in the study were patients who had undergone esophagogastroduodenoscopy (EGD) for a variety of non-specific reasons but had normal histology of biopsies from the upper gastro-intestinal tract. MiR-21 was elevated an average of 50-fold in EoE biopsies over control biopsy levels, with statistical significance. In contrast, miR- 22 and miR-146b were not elevated in EoE biopsies (Figure 40A). To assess the specificity of miR-21 induction in Th2 diseases, we also tested biopsies from human patients with Crohn’s disease, a Th1-mediated disease (Dalal and Kwon, 2010). MiR-21 levels were unchanged in the biopsies of Crohn’s patients compared to controls, while miR-146b was undetected (Figure 40B). These results indicate that miR-21 is specifically elevated in human Th2 inflammatory diseases and could serve as a potential biomarker for Th2 conditions.

Figure 40: Increased miR-21 in the biopsies of pediatric patients with Eosinophilic Esophagitis (EoE).

(A) Quantitative PCR analysis of miR-21, miR-22 and miR-146b expression in esophageal biospsy RNA from healthy controls and eosinophilic esophagitis (EoE) patients, normalized using U6 as control. n = 18-20 subjects per group. (B) Quantitative PCR analysis of miR-21 expression in colon biopsy RNA from healthy controls and Crohn’s disease (CD) patients, normalized using U6 as control. n=2 controls, 7 CD patients. ***p<0.001 (two-tailed Student’s t-test) (error bars, s.e.m.)

A B m iR -2 1 (C o lo n ) Control CD patients 0.0 0.5 1.0 1.5 2.0 _NS

Circulating miR-21 can serve as a non-invasive biomarker for human Th2 inflammatory diseases

Circulating miRNAs are being used as non-invasive biomarkers in the cancer field (Cho, 2007). Since we noted increased circulating miR-21 in the sera of mice receiving Bcl6-deficient Tregs that correlated with a striking increase in miR-21 in the lungs and Th2 exacerbation, we tested sera of EoE patients. Indeed, consistent with the increase in miR-21 in the EoE biopsies, we noted an almost 30-fold increase in circulating miR-21 in the sera of EoE patients compared to controls (Figure 41). The other control miRs – 22 and 146b were not detected in the sera samples. This suggested that circulating miR-21 can be used as a non-invasive biomarker for Th2-type inflammation. Another report also noted increased miR-21 in the biopsies of EoE patients (Lu et al., 2012a; Lu et al., 2012b), although they noted only a 4-fold increase and reported little to no levels of circulating miR-21 in EoE (Lu et al., 2012b).

Figure 41: Increased miR-21 in the sera of pediatric patients with Eosinophilic Esophagitis (EoE).

Quantitative PCR analysis of miR-21 expression in serum RNA from healthy controls and eosinophilic esophagitis (EoE) patients, normalized using U6 as

Increased circulating miR-21 in the sera of asthmatic patients, however, miR-21 does not correlate with atopy

Some explanations for the differences between our results and the other

studies (Lu et al., 2012a; Lu et al., 2012b) assessing miR-21 levels in EoE are 1) possible differences in disease severity between patients in the two studies, 2)

differences in assay sensitivity in detecting miR-21 and 3) differences in control patients between the two studies. Notably, for the serum analysis, the Lu et al study compared circulating miR-21 in EoE patients to healthy atopic patients. In contrast, our controls in the EoE study were patients with normal histology of the upper GI tract who underwent EGD for non-specific reasons, but were not enriched for atopy. To better understand the relationship between atopy, allergic inflammatory disease and miR-21, we analyzed serum miR-21 levels in pediatric patients recruited into a study on the development of asthma. The patients were recruited as infants, based on a diagnosis of dermatitis, and subsequently characterized for atopic status and the development of asthma (Tepper et al., 2008). Atopic status was defined as the presence of specific IgE to at least 1 out of 10 allergens tested. We found that in a randomly selected set of 16 patients, tested at 5 years of age, serum miR-21 increased in the asthma patients by 4- fold, with a p value of 0.018 (Figure 42A). MiR-22 showed no increase in patients with asthma (Figure 42A). In our selected patients, 8 out of 16 patients were atopic, however atopy was randomly distributed between asthmatic and non- asthmatic patients. While asthmatic patients had on average a two-fold increase in serum IgE over non-asthmatic patients (130 IU/ml versus 60 IU/ml), the

difference in average IgE level was not significant (p=0.24). MiR-21 levels did not associate positively or negatively with atopy in this set of patients (Figure 42B). Thus, serum miR-21 is a novel biomarker for asthma, independent of atopy. Further research will be required to determine if serum miR-21 levels are a useful predictor of the development of asthma, EoE or other allergic inflammatory diseases.

Figure 42: Increased miR-21 in the sera of pediatric patients with asthma. Q (A) Quantitative PCR (QPCR) analysis of miR-21 and miR-22 expression in

serum samples prepared from 5 year old patients presenting at 1 year of age with dermatitis and then monitored for allergy symptoms and the development of asthma. Relative miR expression with U6 as control miR.

P (B) Pearson correlation analysis between IgE levels and miR21 levels (fold A B Serum miR-21 S er u m Ig E 0 2 4 6 8 0 100 200 300 400 500 _r2 _{= 0.03376} p = NS

Part IV: Role of Bcl6 in Treg lineage stability in the context of the Th2