This study shows that IL-17A production is a characteristic feature of active CD. At diagnosis children with CD had highly elevated levels of IL-17A mRNA in their jejunal mucosa that returned to background levels on a gluten-free diet. Moreover, IL-17A production was at least partly due to a reaction against gluten because ex vivo
challenge of biopsies of treated symptom-free CD patients with gluten peptides increased IL-17A mRNA levels. These findings confirm the previous studies suggesting a role for IL-17A in CD, i.e. demonstrating IL-17A expression in active CD, IL-17A responses to ex vivo
challenge with gliadin peptides and gliadin specific Th17 cells in CD patients 
. We found that ex vivo
challenge of intestinal biopsies from CD patients with gluten peptides caused increased IL-17A levels in most samples while only a few responded with concomitant increase in the IFN-γ level. One explanation for this somewhat unexpected observation could be that the kinetics for induction of IL-17A is faster than for IFN-γ.
In the present study we take the analysis of active, untreated CD further by performing immunohistochemical analysis combined with morphometry of the small intestinal mucosa for cells expressing the IL-17A protein. A significantly increased frequency of IL-17A+
cells, particularly in the epithelium, was found in active CD compared to controls. These results are in line with a recent study by Lahdenperä et al. 
in which IL-17A+
cells were demonstrated in the small intestinal LP with an increased average frequency in active CD. In their study, however, the epithelium was not analyzed.
New findings are that both Th17 and Tc17 cells contribute significantly to the IL-17A response in the inflamed small intestinal mucosa of patients with active CD and that IL-17A mRNA levels in the mucosa correlate with the mRNA levels of IFN-γ and Foxp3 mRNAs but not with the level of IL-10 mRNA. Tc17 cells, which are CD8+
cells, are mainly present in the epithelium and we show that they are responsible for most of the IL-17A activity in this compartment. In active CD, IL-17A producing IELs, i.e. the Tc17 cells, were more than 6 times as abundant as in IELs of controls and was ≈10 times higher than in LPLs in patients with untreated CD. Furthermore, only a 1.5 fold increase of IL-17A producing LPLs was seen in active CD compared to controls. IL-17A produced by the Tc17 cells in the epithelium may contribute significantly to the antimicrobial defense by recruitment of immune cells to infected sites and by up-regulation of anti-microbial peptides 
. That CD has traits of an antibacterial reaction is in concert with our previous finding that levels of the α-defensins, HD-5 and HD-6, as well as lysozyme are increased in the small intestinal mucosa in active CD 
. Generally, Tc17 cells are found in the intestinal mucosa and in the lung and shown to have functional plasticity, low cytolytic function, involvement in the immune response to viruses and in the disease process of certain autoimmune diseases like psoriasis 
. In fact, Tc17 cells are expanded in psoriasis skin lesions 
. We demonstrate that CD8+
IELs in the inflamed mucosa in active CD produce both IL-17A and IFN-γ (this study and 
). Taken together with the findings that Tc17 cells can show functional plasticity, simultaneously produce IL-17A and IFN-γ and convert into Tc1 cells 
, we hypothesize, as depicted in , that the Tc17 cells undergo functional plasticity during the disease process in untreated CD developing into the characteristic CD8+
IELs producing large amounts of IFN-γ and finally develop into the hyperstimulated cytotoxic T lymphocytes with lost specificity control as shown by Meresse et al. 
. Our results furthermore suggest a parallel activation of Th17/Tc17 cells and Tregs. Mouse studies have shown that pro-inflammatory autoimmune Th17 cells are controlled in the small intestine partly by conversion to Tregs 
. In CD this control seems to be insufficient and it is even possible that Tregs convert into Th17 cells 
. One explanation for the fact that high levels of IL-17A occur in the presence of elevated levels of down-regulatory cytokines like IL-10 in active CD might be that the Tc17 cells are resistant to inhibition by Tregs (), as was shown to be the case for Tc17 cells in psoriatic lesions 
Hypothetical scenario of the immune situation in the small intestinal mucosa during the inflammatory reaction.
The CD associated bacteria investigated in this study influenced the IL-17A expression in the small intestinal mucosa of CD patients in two ways. Firstly they were capable of inducing an IL-17A response on their own suggesting that the increased IL-17A response seen in active CD in part could be directed against the CD associated bacteria and secondly, addition of these bacteria during ex vivo
challenge with gluten digest influenced the IL-17A response, either suppressing or enhancing it depending on whether the patient had a strong response to gluten digest alone or not. Therefore we believe that the CD associated bacteria play an important role in determining the magnitude of the IL-17A response. An IL-17A response to the CD associated bacteria might contribute to break the tolerance to gluten with the consequence that the individual develops CD. We used a mixture of CD associated bacteria for challenge of the biopsies. The mixture included L. umeaense
, a spore-forming, segmented filamentous bacterium that seems to adhere to small intestinal epithelium and hence shows great resemblance to the SFB (segmented, filamentous bacteria belonging to the genus Candidatus
) that proved of importance for development of the gut immune system in mice and in particular the development of mucosal Th17 cells 
. Recent studies have shown that L. umeaense
and SFB are different species but are indeed related and both belong to the Lachnospiraceae
. It is possible that more than one species of adherent bacteria is capable of driving development of Th17 cells or that L. umeaense
is the counterpart in humans to SFB in mice. An additional, not mutually exclusive, possibility is that L. umeaense
bacteria stimulate Tregs to IL-10 production and/or conversion of Th17 cells to Tregs. It has been shown that a mixture of 46 indigenous intestinal bacterial strains of the genus Clostridium
induces Tregs in mouse colon 
. Several of the bacterial strains belonged to the XIVa cluster of Clostridium
that is closely related to the new genus Lachoanaerobaculum
When the mixture of the CD associated bacteria was added during challenge with gluten peptides two different patterns in IL-17A response emerged compared to the response to challenge with gluten peptides alone, i.e. enhancement or suppression. Interestingly it turned out that those patients who showed a suppressed IL-17A response when CD associated bacteria were included were all children born during the Swedish CD epidemic. In contrast, children born after the epidemic all showed an enhancement of the IL-17A response. Our previous study on the microbiota in the small intestine showed that adherent rod-shaped bacteria detectable by scanning electron microscopy were most frequent in young CD patients during the epidemic 
. The fact that it 10 years later was possible to isolate CD associated rod-shaped bacteria from biopsies of patients born during the epidemic suggests that the patients acquired an unfavorable composition of their small intestinal microbiota at that time and that dysbiosis of the microbiota has prevailed since then 
. Thus, it is possible that the different outcomes of adding CD associated bacteria are dependent on differences in the resident microbiota of the patients. The suppressed IL-17A response in patients born during the epidemic could mean that they have developed Tregs directed against CD associated bacteria present in their resident microbiota or that they have become impaired in their capacity to generate an adequate anti-bacterial response. Notably, the response to challenge with gluten peptides alone was significantly higher in these children than in those born after the epidemic suggesting that they are strongly gluten reactive and that the level of the anti-gluten response is enhanced by the components in the resident microbiota. Other groups have also suggested that intestinal dysbiosis may be a contributing factor for CD 
. In accordance with this notion, two recent studies report differences between the fecal microbiota of infants genetically predisposed for developing CD and those who are not 
. A link between dysbiosis of human gut microbiota with outgrowth of potential pathogenic bacteria and other immune disorders has previously been suggested. Of particular interest in relation to CD are the reports showing that patients with inflammatory bowel disease have a composition of their gut microbiota that differs from healthy individuals 
The enhanced IL-17A response seen in biopsies from CD patients born after the epidemic upon challenge with a combination of gluten peptides and a mixture of CD associated bacteria differed from the IL-17A response seen in fresh biopsies from CD patients with active disease. Thus, CD4+ cells, Th17 cells, totally dominated as the cellular source of IL-17A in the challenged biopsies, both within the epithelium and in the LP. This could be due to slower kinetics for raising a Tc17 cell response than for a Th17 cell response in the epithelium or that an epithelial reaction dominated by Tc17 cells is a feature of the immune situation in the mucosa of patients presenting with the disease in whom the Tc17 cells are converted into IFN-γ producing Tc1 cells as the disease progresses ().
A limitation of our study is that ex vivo
challenge experiments require fresh biopsies from symptom-free CD patients on a gluten-free diet. Biopsies included in this study were all collected based on clinical requirements for the diagnostic procedure 
. Collection of a second biopsy after treatment with gluten-free diet is therefore performed only in a fraction of the patients and hence only few biopsies from treated CD patients are available for research and consequently the number of biopsies subjected to ex vivo
challenge in this study is limited.
Collectively, the data presented here suggest that the IL-17A producing cells play a major role in the pathogenesis of CD, that both gluten and CD associated bacteria provoke an IL-17A response in the intestinal mucosa of CD patients and that the magnitude of the adverse IL-17A reaction to gluten is markedly influenced by the composition of the resident microbiota and the amount of CD associated bacteria present. The results are in line with the hypothesis that circumstances causing disturbances when the resident microbiota of the small intestine is established can lead to long-lasting dysbiosis with increased amounts of one or several of the CD associated bacterial species we have identified. These changes in microbiota may influence the magnitudes of IL-17A responses to gluten and be risk factors for contraction of CD in predisposed children. Possibly some of the contradictive results on IL-17A responses in CD in the literature 
can be explained by the degree of dysbiosis in the individual’s gut microbiota and the presence or absence of the CD associated bacteria.