Coeliac disease (CD) is a chronic inflammatory disease of the small bowel, triggered by dietary gluten, in genetically susceptible individuals [1]. The diagnosis of CD is based on the presence of characteristic lesions in small intestinal biopsy samples (intraepithelial lymphocytosis, elongation of the crypts and villous atrophy). However, since villous atrophy is not specific, CD diagnosis also requires a positive serology for anti-tissue-transglutaminase (atTG) antibodies or anti-endomysial antibodies (EMA), and it is confirmed by clinical and/or histological remission following gluten removal from diet [2]. The clinical spectrum of CD is very wide: patients can be asymptomatic (subclinical), present with trivial or apparently unrelated symptoms (non-classical CD), or be severely symptomatic, with frank malabsorption symptoms (classical CD) [3]. Moreover, CD patients might present with symptoms of associated autoimmune diseases, such as type 1 diabetes and autoimmune thyroiditis [4]. CD treatment is based solely on dietary intervention, the so-called gluten-free diet (GFD). With maintenance of a GFD, symptoms and serum coeliac antibodies gradually disappear, and healing of the intestinal damage typically occurs within 24 months of diet [1].

Despite the pathogenic interactions among genetic, immunological, and environmental factors in CD have been extensively investigated, little is known about intestinal stem cell (ISC) modulation and deregulation during the disease course and about the possible contribution of circulating bone marrow-derived stem cells to intestinal regeneration upon the commencement of a GFD. Aim of this study was to assess the circulating hematopoietic stem cells (HSC) traffic and putative ISC (CD133⁺/Lgr5⁺ crypt cell) density in active CD patients and to investigate their longitudinal modifications induced by GFD.

The intestinal tract has developed mechanisms to cope with the harsh mechanical and chemical conditions to which it is exposed, via a highly regulated process of self-renewal and turnover [7]. According to the “unifying hypothesis”, all the differentiated epithelial cells of the gut derive from a single ISC compartment, which resides at the crypt base. From their niche, ISC generate transit-amplifying cells that migrate upwards and progressively lose their proliferative capability and become fully differentiated villous epithelial cells. In 1974, two distinct theories were formulated regarding the identity and location of the ISC: the “+4 position model”, postulating that ISC correspond to slowly cycling label retaining cells (LRC) in +4 position within the crypt; and the “stem cell zone model”, that identified the ISC with small and rapidly proliferating crypt base columnar cells (CBC) [8]. Since the 1970s, several experimental models have supported the concept of ISC; such studies have led to the hypothesis of an ISC hierarchy, organized in two main compartments (quiescent LRC and actively cycling CBC), progressively recruited as a result of various degrees of damage, in order to ensure an effective crypt regeneration [9–11]. Among the candidate markers for ISC, CD133 and Lgr5 are particularly promising. The glycoprotein CD133 (prominin-1) is expressed in immature HSC, as well as in other stem and progenitor cells including neural and embryonic stem cells [12, 13]. CD133 has been used as a colon cancer stem cell marker [14] and has also been suggested as a reliable marker for ISC [15]. The leucine-rich repeat–containing G-protein–coupled receptor-5 (Lgr5) is one of the target genes of the Wnt-pathway and it has recently been shown to specifically label stem cells in the mouse small intestine [16, 17]. Interestingly, lineage-tracing studies showed that some Lgr5⁺ cells co-express CD133 and these CD133⁺ cells can generate the entire intestinal epithelium [14, 18].

Besides resident ISC, circulating multipotent stem cells of bone marrow (BM) origin represent another potential source for intestinal regeneration. Indeed, over the last years, several studies have demonstrated that BM-derived HSC may participate in gut repair at various levels: by giving rise to ISC, providing supporting elements within the ISC niche, and releasing soluble factors that promote healing through immune modulation, inhibition of apoptosis, and stimulation of resident ISC [19–21].

A better knowledge of ISC function and dysregulation in chronic inflammatory bowel diseases might help to understand the pathophysiology of such disorders and might also offer new insights into the development of stem cell-based therapies [11].

In particular, to the best of our knowledge, there is only one published study about the potential role of circulating BM-derived stem cells in CD, by Mastrandrea and coworkers, who found an increased traffic of circulating CD34⁺ HSC in active CD patients versus healthy controls [22]. The authors postulated that this phenomenon was related to the prevalence of apoptotic versus survival programs, a defect shared by chronic inflammatory diseases; in such conditions, HSC might represent a supplementary ISC source.

Indeed, it has been demonstrated that in active CD the status of chronic inflammation worsens the epithelial layer damage, thus causing activation of NF-κB, which leads to a vicious cycle of aberrant immune response, mucosal inflammation, increased mucosal permeability and impairment of the regenerative potential of the intestinal epithelium. Moreover, it has been shown that gliadin-derived cytotoxic peptides can induce oxidative stress, rearrangement of actin cytoskeleton, impairment of epithelial tight-junction assembly, and deregulation of the epithelial homeostasis and this might further impair the ISC functions [23–25].

Regarding ISC modulation and deregulation in CD, it has been observed that ISC differentiation towards Paneth cells (PC) and goblet cells may be disturbed in active CD. Rubio et al., found that PC in active CD are replaced by lysozyme-producing mucus cells and speculated about an ISC reprogramming, as an antimicrobial adaptation to signals generated by pathogenic duodenal bacteria [26]. Capuano et al., observed a down-regulation of Notch pathway and KLF4 signals in CD patients and a reduction of the number of goblet cells in small intestine of children with active CD and in those on a GFD, compared to controls [27].

Our results confirm an increased traffic of CD133⁺CD34⁺ HSC in the peripheral blood of active coeliac patients. Additionally, we observed for the first time that the putative ISC compartment, represented by of CD133⁺/Lgr5⁺ crypt epithelial cells, is depleted in active CD; this result is in line with the above-mentioned theory of impairment of the intestinal regenerative potential in CD. Interestingly, we found that circulating HSC traffic significantly increases in the first week of gluten-free diet, suggesting that BM-derived stem cells might participate in the enteric repair process at the very beginning of the dietary intervention, when the local ISC niche is depleted. Afterwards, the circulating HSC progressively decrease, together with the immune-mediated intestinal damage, and the local ISC compartment expands, leading to villous regeneration and clinical remission.

Given the retrospective nature of our study, it is not possible to determine whether and to what extent the HSCs are contributing directly to the repopulation of depleted ISCs. This hypothesis should be tested in animal models of CD, for instance upon sex-mismatched HSC transplantation [28].

Following a similar approach, BM contribution to the intestinal regeneration has been demonstrated in experimental models of inflammatory bowel disease (IBD); this evidence supported the rationale for the pharmacological mobilization of HSCs in the treatment of IBD [29, 30].

Finally, the dramatic increase in circulating HSC following initiation of GFD is worth speculation. We could hypothesize that the circulating BM-derived stem cells are migrating to sites of inflammation and being consumed in the lesions, and once the inflammation is reduced, their numbers increase in the circulation. Alternatively, it is possible that gluten removal induces the injured gut to release factors involved in HSC mobilization and homing, such as G-CSF and SDF-1 [31]. Experimental studies to assess intestinal gene expression modifications upon gluten removal would be of help to clarify the mechanisms underlying such HSC longitudinal modifications following GFD in CD.

The pathogenic interactions among genetic, immunological, and environmental factors in CD have been extensively investigated. On the contrary, little is known about ISC modulation and deregulation during the course of the disease. In the present study, we observed that the putative ISC compartment is depleted in active CD, likely due to a status of chronic inflammation, that leads to the impairment of the regenerative potential of the intestinal epithelium. Removing the inflammation-inducing factor (gluten) allowed for repopulation of the ISC compartment to occur. Circulating HSCs might actively contribute to this repopulation process, as suggested by the significant increase of HSC traffic at the very beginning of the dietary intervention. Afterwards, the circulating HSCs progressively decreased and the local ISC compartment expanded, leading to villous regeneration.

This study sheds some light on the dynamics of intestinal regeneration following GFD in coeliac patients and offers novel insights for the development of HSC-based treatments against CD. Further studies are required to dissect the molecular mechanisms underlying ISC impairment in active CD and their expansion upon gluten removal from the diet, and also to clarify the role of BM-derived stem cells in intestinal regeneration.