The results obtained for DMSO can be easily explained by the literature, as Smith, Gheux [ 38 ] showed that in co-culture models, the cytotoxicity of harmful compounds was reduced for the cells in the basolateral compartment. For CMC, the data obtained is harder to justify as no previous works addressed this topic, and the only similar work, by Pradhan, Mulenos [ 19 ], showed that the presence of cellulose, not Carboxymethyl Cellulose, in the apical compartment did not lead to a reduction in the viability of the Raji-B cells present in the basolateral compartment. A possible explanation for this behavior may be linked to a possible macromolecular crowding (MMC) effect, which has been described as the effect caused by the addition of polymeric materials to cell culture media, causing an excluded volume effect and leading to an increase in the deposition of cellular metabolites upon cell layer [ 39 ]. This behavior has been previously described for CMC regarding the increase of pro-collagen in skin cells [ 4 ]; and here, as cells are seeded on a permeable membrane, this may lead to the diffusion of cellular metabolites towards the basolateral compartment, thus causing the metabolism inhibition recorded.
When one considers the impact of the studied conditions upon the Hep G2 cells present in the basolateral compartment ( b), it is interesting to see that none of the studied conditions led to reductions in metabolic activity above the 30% threshold, defined by the ISO 10993-5:2009 standard as the cytotoxicity limit [ 37 ]. In fact, even for DMSO, which registered a steep drop in membrane integrity in the TEER data, the 30% metabolism inhibition threshold was not reached after 24 h, with an inhibition value of 17.8 ± 0.1% being observed. When analyzing the DMSO metabolism inhibition data obtained for the 6 h and the 24 h timepoints and crossing it with the TEER data, it was observed that the metabolism inhibition increased with the duration of the assay while TEER data was relatively stable from 2 h onwards. This may indicate that while the DMSO rapidly compromises membrane integrity, it does not permeate the membrane at the same rate, instead slowly permeating towards the basolateral compartment, thus leading to the increase in cytotoxicity that was observed. On the other hand, when assessing the data obtained for CMC, it can be observed that no statistically significant (p > 0.05) differences were found between sampling times, with a Hep G2 metabolism inhibition of ca. 10% being registered. When considering that CMC did not, apparently, alter apical membrane integrity and permeability, this data may indicate that CMC’s presence on the apical side leads to the production of some metabolite or stress that results in the viability loss registered. This is particularly evident when one considers that while CMC presented a TEER profile similar to that of the media control, it presented statistically significant (p < 0.05) metabolism reductions relative to the control conditions.
When considering CMC’s effect on intestinal barrier integrity, the data obtained ( a) showed no statistically significant (p > 0.05) variations in the TEER value relative to the control. In fact, CMC’s TEER values were very similar to those obtained for healthy membrane control (media only). On the other hand, for DMSO (damaged membrane), the reduction in TEER value was statistically significant (p < 0.05) from the onset of the assay, with an almost 70% reduction being observed within the first hour of the assay and no recovery being observed until the 24 h mark. As variations in TEER data can be directly linked with membrane health, integrity and permeability [ 35 , 36 ], the data obtained showed that CMC did not cause membrane rupture or irreversible permeability increases in the studied conditions. While no direct comparison of this data is possible, as no works regarding this topic were found, the results observed here are in line with the work of Pradhan, Mulenos [ 19 ], which has shown that cellulose does not cause any alterations to the barrier integrity of a Caco-2/HT29-MTX co-culture model.
When regarding the co-culture immunomodulation model, not only was cytokine production evaluated, but the membrane integrity and metabolic activity of the RAW cells in the basolateral compartment were accessed. For the last two, the results obtained showed that for the duration of the assay, CMC did not cause significant deleterious effects on membrane integrity ( a) or on the metabolism of the RAW cells present in the basolateral compartment ( b).
Interestingly, statistically significant (p < 0.05) differences were found between the controls and the conditions with CMC, as TEER values with lower variance were observed in the presence of CMC, even when the inflammatory stimulus was added to the system. These results are in line with those previously reported by Marescotti, Lo Sasso [40], which showed that LPS stimuli to the basolateral compartment of a Caco-2/HT29-MTX membrane did not cause any TEER decrease or increase in membrane permeation, with a possible explanation being a dysregulated LPS signaling through the down-regulation of the MD-2 and TLR4 receptors, as previously described by Abreu, Vora [41].
The results obtained regarding the immunomodulatory data can be divided in accordance with their origin: apical data obtained from the Caco-2/HT29-MTX membrane ( ) and basolateral data obtained from the RAW cells ( ). When considering the apical data, it is possible to see in that CMC’s presence led to a general increase in the secretion of pro-inflammatory cytokines with and without LPS stimulus.
Of the analyzed targets, IL-6 presented the highest promotions, with a curious pattern appearing as LPS presence did not lead to statistically significant (p > 0.05) differences, contrary to the statistically significant (p < 0.05) differences found between conditions with and without CMC. In fact, CMC’s effect on IL-6 production was so strong that, in its presence, this cytokine content was 692% and 642% (without and with LPS) above that of the basal control. This particular result is of concern, as IL-6 elicits an acute phase response and activates humoral and cellular responses via end-stage β-cell differentiation and T-cell activation, leading to the transition from an acute response to chronic inflammation [29]. Furthermore, IL-6-exacerbated secretion in intestinal epithelial cells (IEC) has been associated with intestinal inflammatory processes and shown to play a critical role in IBD and Chron’s disease [29]. When considering IL-8 and TNF-α values on the apical side, two different patterns of response were observed. For the first, it is interesting to note that CMC’s presence did not lead to statistically significant (p < 0.05) increases in this cytokine secretion relative to the basal and stimulation control. Furthermore, despite the lack of differences, CMC appeared to have a synergistic effect, with LPS as the highest relative production was registered in this condition. These IL-8 results are quite interesting as, in this particular co-culture model, the presence of increased levels of TNF-α in the basolateral compartment, as here observed, has been linked to the increased secretion of IL-8 in the apical compartment [16,42,43]. However, while this known interaction explains the statistically significant (p < 0.05) differences found between the basal control and the inflammation control, they do not explain the increases in both controls observed in the presence of CMC. Once again, this promotion found in the presence of CMC is worthy of record, as IL-8 is an early inflammatory marker due to its initiation of inflammatory cascades and mobilization of neutrophils and T lymphocytes, which will initiate the next steps in intestinal innate immune response, thus playing a key role in IBD [44,45]. Finally, analysis of the TNF-α data obtained showed that, once again, CMC’s presence led to statistically significant (p < 0.05) higher levels of this cytokine relative to the controls. For this cytokine, and as seen for IL-8, a synergistic effect could be observed between the inflammatory stimuli and CMC, with the highest relative production of TNF-α being observed in this condition. Considering that this cytokine is a known pro-inflammatory agent linked to the pathogenesis of IBD, chronic gut inflammation and the onset of Crohn’s disease in the intestine [46,47], the data reported here for CMC shows that this compound may pose a problem for gut health. Overall, the data reported for the apical compartment presents a picture that shows CMC as possessing a pro-inflammatory potential in the intestinal lumen, a behavior that has been attributed recently in the literature for CMC but for different reasons. In fact, in vivo works have depicted CMC as being responsible for the removal of the mucous layer that protects the gut mucosa, leading to the building of exacerbated microbial loads in the mucosa and, consequently, small bowel inflammation and IBD development [5,8,48]. If one considers the bibliographic data available and the data obtained here, the picture that emerges is that in addition to what has been published, CMC may also exert a pro-inflammatory effect upon the intestinal cell wall and, thus, it is likely that the reported deleterious effects of CMC are a result of both effects combined.
When considering the effects on the basal compartment ( ), the data obtained showed that of the thirteen targets analyzed, only five were detected in the multiplex assay, with three being pro-inflammatory (TNF-α, MCP-1 and IL-6) and the other two being anti-inflammatory.
For the first group, the standout result was registered for TNF-α, where a strong production was observed in the presence of the inflammatory stimulus, a behavior which has been previously described in the literature [16,42,43]. Interestingly, CMC’s presence in the apical compartment led to statistically significant (p < 0.05) lower values of TNF-α both in the presence and absence of LPS. These results are relevant as this cytokine has been widely associated with Crohn’s disease, gut inflammation, IBD and other pathologies, such as obesity, diabetes and colonic cancer. Thus, these reductions associated with CMC’s presence in the intestinal lumen may somewhat counteract the direct deleterious effects observed [46,47,49,50]. For the monocyte chemoattractant protein-1 (MCP-1), the results obtained showed, once again, a strong increase in the quantity secreted in the presence of the inflammatory stimulus. This result was in line with the literature, as LPS has been previously described as being responsible for the activation of the Toll-like receptor 4 (TLR-4) in RAW cells, leading, through the activation of various activation cascades, to the production of various pro-inflammatory cytokines and chemokines, among which is MCP-1 [51,52]. Similar to what was observed for TNF-α, CMC’s presence led to lower levels of this chemokine, with this difference being statistically significant (p < 0.05) in the absence of inflammatory stimulation. This is particularly interesting as CMC’s presence led to smaller quantities of MCP-1. Considering that this chemokine is essential in macrophage recruiting and its over-expression has been linked to tumor progression in the intestine, ulcerative colitis, IBD, obesity and diabetes, this under-expression in the presence of CMC may be of interest [53,54,55,56]. For IL-6, the first standout result is the lack of detected cytokines in the non-stimulated conditions, which by itself showed that CMC’s presence in the apical compartment did not have a pro-inflammatory effect in the basolateral side of the Transwell. Secondly, in the stimulated conditions, a significantly (p < 0.05) higher IL-6 value was observed in the basolateral compartment when CMC was present in the apical side of the system, showing that a possible synergistic effect between the inflammatory stimulus and some metabolites produced in these conditions may be occurring. When one considers that IL-6 production by macrophages in the gut has been linked to intestinal homeostasis and barrier function, IBD, colon cancer and colitis, this apparent stimuli of IL-6 production in the presence of CMC may be problematic [57,58,59,60].
When considering the data obtained for the two anti-inflammatory cytokines (IL-10 and IFN-β) detected, it is interesting to note the increases registered in the presence of CMC, with this augmented production being statistically significant (p < 0.05) for IFN-β. This behavior is even more relevant if one considers that IL-10 is associated with the dampening of intestinal inflammation, IBD and ulcerative colitis risk reduction, as this interferon limits the secretion of pro-inflammatory cytokines, deactivates macrophages and inhibits the secretion of Th1-related cytokines [61,62,63]. For IFN-β, the data obtained present some curiosities, as type I interferons are responsible for the inflammatory response to infections, which then modulate the cell cycle to suppress the infection and upregulate antigen presentation to innate immune cells, leading to the activation of T- and B-cells. Similarly, MCP-1 interferon secretion is stimulated through TLR activation by LPS, but curiously, this will also automatically regulate itself, as IFN-β production has been linked with an upregulation of cytochrome B beta chain expression. This will lead to the production of NADPH oxidase 2-derived H2O2, causing downregulation of TLR7 and culminating in the inhibition of IFN-β production [64,65,66,67]. While in this work, this downregulation is not on display, and the increased secretion in the presence of CMC in the apical side of the model may be of use in an in vivo setting. In fact, type I interferons play a critical role in the suppression of intestinal inflammation and IBD through the regulation of T regulatory (Treg) cell populations levels, with these cells maintaining intestinal homeostasis under the continuous challenge of microorganisms, their downregulation leading to accelerated colitis [68,69,70,71].