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Autoimmune enteropathy (AIE) is a rare syndrome characterized by chronic diarrhea, malabsorption, and severe villous atrophy, generally associated with autoimmune comorbidities and circulating anti-enterocyte antibodies.1, 2 Most cases of AIE occur in the context of an X-linked genetic syndrome known as IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked syndrome), a disease affecting male infants, and secondary to mutations in the coding region or in regulatory elements of Foxp3 gene.3, 4 Foxp3, also known as scurfin, is a forkhead transcription factor essential for the development and function of CD4+CD25+ regulatory T cells (Tregs), a subset of T lymphocytes that play a fundamental role in the regulation of T-cell activation and immune homeostasis.5 Consistently, IPEX patients display a profound defect in Tregs and a severe reduction of FoxP3 expression in most tissues.6 Nonetheless, in the last few years, non-IPEX adult-onset forms of AIE are being increasingly reported in both genders.7 These patients exhibit reduced expression of Foxp3 and reduced suppressive function of CD4+CD25+ Tregs,8 raising the possibility that defective autosomal genes could also lead to a deficit of Foxp3 expression and function. These findings have led to postulate that an unrestricted activation of gut-associated lymphoid tissue, due to an impairment in Tregs quantity and/or function, may be responsible for the intestinal inflammation and villous atrophy which dominate the pathological picture of the syndrome.1 Indeed, immunohistochemistry studies have documented a dense mixed T-cell infiltrate (CD4+ and CD8+) in the duodenal mucosa of patients with active AIE, which could be associated with a prominent intraepithelial lymphocytosis in a significant proportion of cases.9 This is consistent with the long-known role of activated T cells and T-cell-derived cytokines in the pathogenesis of enteropathy and villous atrophy in humans,10 and substantiates the clinical success of T-cell-directed therapies in both IPEX and non-IPEX AIEs. Indeed, even if clinical manifestations of AIE usually respond to corticosteroids, steroid-refractory cases are common, requiring rescue therapy with additional immunomodulators, such as azathioprine, cyclosporine, and tacrolimus, while bone marrow transplantation has been employed with some success in severe, drug-refractory cases.2 Despite these interventions, AIE is still affected by a high rate of complications and mortality, and novel therapeutic approaches to control the syndrome are urgently needed. In order to discover novel targets for selective and more effective drugs, it is thus highly relevant to obtain insights into characteristics of infiltrating intestinal inflammatory cells during active AIE. In this context, existing knowledge regarding AIE pathophysiology mostly derive from studies conducted on peripheral blood cells or histological sections,6, 9, 11 while very little is known regarding phenotypic and functional characteristics of inflammatory cells that infiltrate the intestinal mucosa. Here, we report for the first time a detailed phenotypic and functional analysis of intraepithelial (IELs) and lamina propria (LP) T lymphocytes that infiltrate the small intestinal mucosa in a severe case of non-IPEX adult-onset AIE, following their variations during drug-induced disease remission. The appearance of unconventional IELs that co-produced IFN-γ and IL-17 was observed in the duodenal mucosa, which was largely absent in healthy controls and patients with celiac or Crohn’s disease, and which relieved upon pharmacological treatment. This was associated with a failure of LP CD4+CD25low Treg cells to control IL-17 induction in IELs. Intriguingly, activated Foxp3lowTregs in AIE upregulated membrane-bound transforming growth factor-β (TGF-β), and TGF-β strongly enhanced IL-17 induction in CD8+IELs. These results shed new light on the role of Foxp3+Tregs and TGF-β in the regulation of intestinal homeostasis in vivo. For immunofluorescence, paraffin sections from AIE biopsies (PRE and POST) and uninflamed duodenal biopsies as control were submitted to deparaffinization in xylene and hydration through a series of decreasing alcohol concentrations. Heat-induced antigen retrieval was obtained using Diva Decloaker solution (Biocare Medical, Concord, CA) at 120 °C for 3 min in the Decloaker chamber. After blocking and permeabilization with PBS-BSA 1%-NP40 1% for 30 min, tissues were incubated with rabbit polyclonal anti-CD8 antibody (1:100 ab4055, Abcam, Cambridge, UK) and goat polyclonal anti-phosphorylated SMAD2/3 antibody (1:50 sc-11769, Santa Cruz Biotechnology, Santa Cruz, CA) overnight. The sections were then washed and incubated with a donkey anti-rabbit Alexa 488 (1:1,000, Life Technologies, Carlsbad, CA) and a donkey anti-goat Alexa 568 (1:1,000, Life Technologies) for 45 min at room temperature. The negative control was performed omitting the primary antibodies and incubating tissues only with secondary antibodies for 45 min at room temperature. Tissues were then counterstained with DAPI and slides were mounted with Dako-Fluorescent mounting Media (Dako, Carpinteria, CA). Slides were observed using a Leica SP5 confocal microscope with a 63 × N.A. 1.4 PL APO objective. We next evaluated the effector cytokine profile of CD4+ and CD8+ T cells extracted from the LP (Figure 3c) and from the intraepithelial compartment (Figure 3d). We focused our attention on the expression of IFN-γ and IL-17, since T helper cells that co-produce these cytokines are associated with intestinal inflammation.15, 16, 17 We also assessed the production of IL-22 and GM-CSF, two additional key cytokines regulating intestinal homeostasis.18, 19 We observed increased levels of IL-17 production by LP CD4+lymphocytes in active AIE that was largely reverted upon therapy-induced resolution of inflammation (Figure 3e, upper panel). Conversely, no major differences were found between AIE and controls in the proportion of CD4+T cells producing IFN-γ (Figure 3e), GM-CSF or IL-22 (Supplementary Figure 1 online). LP CD8+ T cells expressed very high levels of IFN-γ in all cases, but IL-17-producing and IL-17/IFN-γ co-producing CD8+ T cells were rare (Figure 3e lower panel). Noteworthy, a dramatic increase of IL-17-producing lymphocytes was found in the intraepithelial compartment. (Figure 3f). As shown, IL-17-producing CD4+ T cells were more strongly increased in the intraepithelial compartment as compared to the LP, and IL-17/IFN-γ co-producing cells were also more frequent (Figure 3f). Moreover, in active AIE also CD8+ IELs produced very high levels of IL-17, the large majority of these IL-17+CD8+ IELs also co-producing IFN-γ (Figure 3f, lower panel). In marked contrast, in healthy controls IL-17/IFN-γ co-producing CD8+ IELs were nearly undetectable (Figure 3f). Furthermore, steroid and azathioprine treatments strongly reduced IL-17 production in particular in the intraepithelial compartment. To exclude that enhanced IL-17 expression was merely resulting from the ongoing mucosal inflammation, ileal biopsies from a group of patients with active Crohn’s disease were analyzed. As shown in Supplementary Figure 2 online, enhanced percentages of IL-17-producing CD4+ and CD8+ T cells were found both in the LP and in the intraepithelium compartment of active Crohn’s disease patients as compared to uninflamed controls as expected. However, while the fraction of IL-17-producing T cells in Crohn’s disease and AIE were similar in the LP, IL-17+ IELs were much more frequent in AIE, in particular in the CD8 compartment. These data suggest that AIE is characterized by a selective increase of IL-17-producing LP CD4+ T cells, and by the massive appearance of unconventional IL-17 and IFN-γ co-producing CD4+ and CD8+ IELs. In particular IL-17-producing CD8+ IELs are largely absent in the healthy duodenum and rare in the ileum of Crohn’s disease patients, and disappear upon therapy-induced resolution of inflammation. The efficient cytokine-dependent induction of IL-17 in IELs prompted us to define the contribution of individual cytokines to this unexpected cellular plasticity. We therefore measured IFN-γ and IL-17 production in response to TCR stimulation and Th17-promoting cytokines in CD4+ and CD8+ IELs and analyzed their modulation in the selective absence of either IL-2317 or TGF-β123 (Figure 4c). The absence of TGF-β1 induced a strong and significant reduction of IL-17 production in CD8+ IELs, but had only a weak and not significant effect on IL-17 production by CD4+ IELs (Figure 4c, left panel). Furthermore, the effect of TGF-β1 was specific for IL-17, since IFN-γ production did not change significantly. Conversely, the absence of IL-23 had only a rather weak inhibitory effect on IL-17 induction in both CD4+ and CD8+ IELs (Figure 4c, right panel). We conclude that TCR-activated IELs efficiently acquire IL-17-producing capacities without losing IFN-γ production in the presence of Th17-promoting cytokines. Intriguingly, TGF-β strongly enhanced the generation of IL-17 and IFN-γ co-producing CD8+ IELs, but had no relevant effect on CD4+ IELs. CD25+ Tregs are unable to produce pro-inflammatory cytokines, but unlike helper T cells they upregulate membrane-bound TGF-β in its latent form (LAP, Latency Associated Protein), which is presented by the Treg-specific surface receptor GARP (glycoprotein A repetitions predominant).25 Notably, TGF-β signaling in mouse effector T cells is required for the inhibition of experimental colitis by Foxp3+Tregs.26 We wondered if the dysfunctional Foxp3lowTregs in AIE could enhance IL-17 production of CD8+IELs via TGF-β, and to this end we compared the capacity of CD4+CD25+IL-7Rlow Tregs to upregulate GARP and LAP surface expression following TCR activation (Figure 5c). Foxp3lowTregs upregulated GARP and LAP similar to Tregs from healthy controls, indicating that the induction of membrane-bound TGF-β production was not inhibited in Foxp3lowTregs in AIE. Finally, to address whether CD8+ IELs also received signals from TGF-β in AIE in vivo, we stained duodenal AIE sections for CD8 and phosphorylated Smad2/3, (p-SMAD2/3), since the latter are phosphorylated and thus activated upon TGF-β receptor (TGF-β R) engagement. As shown in Figure 5d, we observed a higher number of CD8+ cells (green cells) in active AIE (PRE) in situ compared to healthy controls that decreased upon therapy as expected. Moreover, p-Smad2/3 positive cells (red) were present both in the steady state and in AIE before and after treatment. Moreover, some CD8+ lymphocytes were positive for p-Smad2/3 in all cases, suggesting that they were exposed to and activated by TGF-β in vivo both in the healthy gut and in AIE. In conclusion, while normal duodenal Tregs suppress IL-17 production by IELs, Foxp3lowTregs in AIE, not only failed to suppress, but even enhanced IL-17 production in CD8+ IELs. This paradoxic finding could be explained by the fact that Foxp3lowTregs efficiently upregulated membrane-bound TGF-β, which in turn could enhance IL-17 production by CD8+ IELs. Consistent with this scenario, TGF-β signaling in CD8+IELs could be detected in situ in active AIE, demonstrating that bioactive TGF-β was present under this inflammatory condition. AIE is a fascinating disease from an immunological perspective, as insights into its pathogenesis may lead to valuable information regarding mechanisms of tolerance and in vivo regulation of immune responses in the human gut. Specifically, the recognition that most AIE cases are secondary to reduced numbers and functions of Foxp3+ Tregs7 makes this rare disease a model to study a partial defect of Tregs in the modulation of intestinal immune homeostasis. However, most studies on AIE pathogenesis focused on the reduced numbers and functions of Tregs in peripheral blood rather than analyzing functional or phenotypic changes in regulatory and effector lymphocytes in the intestine. Nevertheless, such information would be of relevance in designing novel anti-inflammatory drugs and therapeutic approaches for this rare condition. The here analyzed AIE patient had severe duodenal inflammation, an elevated titer of anti-enterocyte antibodies and reduced Foxp3 expression in CD25+ Tregs; moreover γ/δ lymphoma, IPEX as well as CD were ruled out, indicating that she was actually affected by AIE.6, 8 Intestinal homeostasis requires not only Foxp3+Tregs but also IL-10,29 that can be provided by both Foxp3+ and Foxp3− CD4+ Tregs in mice.14, 30, 31, 32 Consistently, defects in the IL-10/IL-10R pathway induces severe early-onset colitis in humans.33 We detected relatively high IL-10 production by CD4+ T cells in active AIE (PRE) that diminished upon treatment-induced remission, indicating that defective Foxp3 expression in Tregs but not impaired IL-10 production is the underlying defect in AIE. Consistent with previous reports,34 we found that the small intestinal mucosa was massively infiltrated with T cells during active AIE, with a preponderance of CD8+ over CD4+ T cells in the LP. In addition, we observed a very selective upregulation of IL-17 in LP CD4+T cells. These alterations were largely reverted upon treatment-induced mucosal healing. IL-17 is important for epithelial barrier function and has anti-microbial as well as pro-inflammatory activities, and the upregulation of IL-17 might be an unsuccessful attempt by the intestinal immune system to re-establish gut homeostasis in AIE. Indeed, while anti-IFN-γ antibodies had a beneficial effect in IBD patients, anti-IL-17A was unexpectedly detrimental.35 Nevertheless, mucosal Th17 cells and in particular those co-producing IL-17 and IFN-γ or GM-CSF could also drive chronic intestinal inflammation, and might thus also play a pathogenic role.15, 19, 36 A second major change in active AIE that was reverted upon mucosal healing was the abundant IL-17 production by IELs, in particular IL-17 and IFN-γ-co-producing CD8+IELs. Importantly, IL-17-producing CD8+ IELs were rare in other inflammatory diseases of the small intestine, such as CD and ileal Crohn’s disease. CD8+IELs have been implicated in multiple pathological processes of the small intestine, including the progression of inflammatory bowel diseases,37 as well as in the pathogenesis of CD by directly targeting intestinal epithelial cells and causing villous atrophy.38, 39 In addition, IFN-γ/IL-17 co-producing CD8+ T cells have been described in other immunological disorders of the intestine, including active CD40 and microscopic colitis.41 Interestingly, recent evidence in mice suggests that IELs may be under the direct control of intestinal Tregs, as an experimental reduction in numbers and functionality of Tregs leads to a severe form of intestinal inflammation characterized by infiltration of the intraepithelial compartment by IL-17-secreting CD8+ T cells.42 Consistently, we found that the induction of IL-17-producing CD8+IELs in humans was inhibited by Tregs in control patients, but not in AIE. IL-17 and IFN-γ co-producing T cells can be generated from IL-17-producing CD4+ T cells with cytokines that induce IFN-γ, such as IL-1β and IL-23 or IL-12.20, 43 In contrast, we showed here that IFN-γ-producing CD8+IELs could acquire IL-17 production without losing IFN-γ production in response to a cocktail of IL-17-promoting cytokines, i.e., IL-1β, IL-6, IL-23 and TGF-β1. IELs are thus not terminally differentiated cells but are highly plastic,43 and could change their cytokine profile in response to locally uncontrolled cytokine production. Pro-inflammatory cytokines in the LP are probably derived from myeloid cells, and interestingly we observed increased frequencies of CD103+ myeloid cells in active AIE. Indeed, intestinal CD103+DC produce pro-inflammatory cytokines and induce Th17 differentiation in mice.44 An important and unexpected finding of this study was that IL-17 production by IELs and LP T cells had largely different requirements.45 Thus, while LP CD4+ T cells rapidly upregulated IL-17 upon TCR stimulation in the absence of exogenous cytokines, LP CD8+ T cells poorly upregulated IL-17 production even under optimal Th17 conditions, consistent with their low IL-17 production in active AIE. Intriguingly, the anti-inflammatory cytokine TGF-β, which strongly inhibits the induction of IFN-γ production in naïve CD4+ T cells19, 44 and restrains colitis,26 enhanced IL-17 production in CD8+IELs, but had no inhibitory effect on IFN-γ. This enhancing effect of TGF-β was specific for CD8+ IELs, because TGF-β had no enhancing effect on CD4+IELs. Thus, pro-inflammatory cytokines were sufficient to induce IL-17 in TCR-activated CD4+IELs, possibly because they contain pre-committed Th17 cell precursors46, 47 which do not require TGF-β to upregulate IL-17.46, 48 Finally, our data suggests that a relevant source of intestinal TGF-β, which promotes IL-17 induction in CD8+ IELs in AIE, might be the defective Foxp3lowTregs themselves. Indeed, latent TGF-β was efficiently and selectively upregulated on Foxp3lowTregs in AIE, and the latter further enhanced IL-17 production selectively by CD8+IELs. Thus, the defective Foxp3lowTregs might enhance IL-17 production by CD8+IELs via TGF-β Moreover, some duodenal CD8+T cells contained phosphorylated Smad2/3, demonstrating that they received signals from the TGF-βR also under inflammatory conditions in vivo and suggesting that bioactive TGF-β was available. Indeed, it was previously shown in mice that the presence of apoptotic cells and pathogen-associated molecular patterns in the inflamed intestine is a physiological situation that leads to the induction of both TGF-β and pro-inflammatory cytokines.49 Overall these findings indicate that TGF-β plays an important role not only in the steady state, but also under inflammatory conditions. In conclusion, our data shows that in AIE the quantitative deficit of Foxp3 expression in intestinal CD4+CD25+Tregs is associated with a selectively dysregulated IL-17 production in particular by CD8+ lymphocytes in the epithelium, which is reverted upon successful pharmacological treatment. The enhanced IL-17 production by both CD4+ and CD8+IELs in AIE suggests that human Foxp3+Tregs have a non-redundant role to control IL-17 production by IELs. Moreover, TGF-β, possibly derived from defective Tregs in AIE, could be selectively important for IL-17 production by CD8+IELs. This data broadens our understanding on the role of Foxp3+Tregs and TGF-β in intestinal immune homeostasis, and raises the question if mongersen, an efficient treatment of Crohn’s disease50 that enhances TGF-β responsiveness, is also an appropriate treatment for AIE.  Guarantor of the article: Flavio Caprioli, MD, PhD. Specific author contributions: Designed, performed and analyzed experiments and wrote the paper: M.P.; designed experiments and wrote the paper: J.G.; designed and supervised the study and experiments and wrote the paper: F.C.; collected and analyzed histological data: U.G.; critically reviewed the manuscript for important intellectual content: A.M., S.T., G.N., P.L., G.E., M.P., L.E., S.A., D.C.; All Authors approved the final draft submitted. Financial support: This work was supported by the Cariplo Foundation and the Italian Ministry of health (Giovani Ricercatori GR-2011- 02352001). Potential competing interests: None. Supplementary Information accompanies this paper on the Clinical and Translational Gastroenterology website

Uncontrolled IL-17 Production by Intraepithelial Lymphocytes in a Case of non-IPEX Autoimmune Enteropathy