The results from the present study are consistent with an alteration of the collagen and TG2 immunofluorescence signal in the CD FBs compared to the CTR ones. These abnormalities could be involved in the decreased motility observed during the time-course experiments and in turn in the villous damage observed in CD.
The maintenance of a normal duodenal mucosal morphology results from a continuous interaction between the epithelium, the ECM represented by the basement membrane and the underlying FBs network, responsible for the secretion of collagen molecules and matrix stabilizing enzymes (TG2) . In CD the mucosal structure is deeply altered as proven by the typical “stigmata” of CD represented by mucosal atrophy, crypt hyperplasia and lymphocytic infiltration. Also the lamina propria, composed by type IV collagen and controlling the interface between the epithelium and the subepithelial compartment, is altered in CD [21, 22]. These aforementioned ECM alterations can be attributed to the secreted autoantibodies (anti-TG2 and anti-gliadin) interacting with the matrix proteins or alternatively to a primary defect of CD FBs which could abnormally express these proteins or enzymes . Moreover, FBs control the degradation of ECM through the secretion of lytic enzymes, as the metalloproteases (MMP), and their inhibitors [24–26]. Previous findings suggest that both these mechanisms can play a role in the CD progress. Schuppan et al. for example have demonstrated that ECM could act as a reservoir of autoantibodies, fuelling the mucosal inflammation and eventually modifying the pH and calcium concentrations, making the environment suitable for protein lysis and TG2 activation in terms of switching from isopeptide bonds to the deamidation using H2O as acyl acceptor [6, 23]. On the other hand, Verbeke et al. have reported a decreased immunofluorescence signal of type IV collagen and laminin with a “leaky” basement membrane . Accordingly, the increase of type I and IV collagen signals in FBs and the increase of TG2 activity in the medium could represent an attempt of CD FBs to restore the decreased collagen levels in the ECM of CD patients and to increase the formation of isopeptide bonds stabilizing the matrix fibrils. Although demonstrated only in vitro this scenario could also be present in the flattened CD mucosa; in other words, as primary cell cultures maintain the memory of their site of origin even after different passages, a restoration of the original in vivo scenario could be possible [27, 28]. To underline these mechanisms, the fibronectin levels did not differ between CD and healthy FBs, in line with the results obtained by Korhonen et al. who reported a comparable pattern both in CD and non-CD duodenal mucosa .
In the present study FBs resulted positive for αSMA, which is the most relevant marker of myofibroblasts (mFBs) and represents an intermediate state between FBs and smooth muscle cells . As observed in our experiments, mFBs display prominent cytoplasmatic actin microfilaments (stress fibers) and are connected to one another by adherent and gap junctions . In the gastrointestinal tract two kind of mFBs are usually recognized, the α-SMA positive and those negative, resident in the apical and basal parts of the villi, respectively . In detail, α-SMA positive FBs correspond to our in vitro population and are involved in both epithelial differentiation and ECM formation . More recently, using immunohistochemistry, some authors have demonstrated that α-SMA positive subepithelial mFBs constitutively express class II MHC molecules and are distinct from professional antigen presenting cells .
A further interesting finding from the present series, reported for the first time, is represented by the decreased motility of CD FBs compared with the control ones, as indicated by a 50% reduction of the displacement of CD FBs during the 72-hour movie recording. This finding refers to different biological aspects, as cell shape/dimension and migration are dynamic processes connected to the cellular fate (growth/death) and depending on the ECM composition and endocellular cytoskeleton. Thus, round shapen cells (circularity index = 1) appear as in stationary state while a Y-like shape as frequently observed in FBs cultures (circularity index < 1) characterizes those cells ready for migration . In fact, the “Y” shape is the most favorable for polarized migration, with a frontal adhesion point and a rear arc where active forces as given by the Laplace law on tension, prepare for the movement of the cell . The molecular mechanisms underlying cellular motility are largely unknown, even if the Rho family of GTPases seems to play a crucial role . Beyond the complex molecular processes connected to FB motility, several relevant clinical consequences have been reported. In particular, decreased FB motility has been reported in patients affected by diabetes mellitus (DM): for such patients a migratory defect represents a relevant impairment for wound healing, responsible for chronic skin ulcerations even requiring amputation . The discussed finding could represent a connection between CD and DM, which are supposed to present some common factors such as genetic background. Moreover, DM animal models are currently used to investigate particular aspects of CD as they are capable to develop duodenal atrophy when exposed to gluten feeding . It remains an interesting point for future investigation whether the observed alterations are common to other gastrointestinal autoimmune disorders (inflammatory bowel disease, autoimmune enteropathy) or they could be reverted in case of FBs derived from CD non atrophic mucosa, where a different FB population has been selected from duodenal micro-environment.