Inflammatory Bowel Diseases (IBDs) are characterized by chronic intestinal inflammation and fibrosis, the latter being the predominant denominator for long-term complications. Epithelial and mesenchymal 2D cultures are highly utilized in vitro models for the preclinical evaluation of anti-inflammatory and antifibrotic therapies. More recently, human intestinal organoids (HIOs), a new 3D in vitro model derived from pluripotent stem cells, have the advantage to closely resemble the architecture of the intestinal mucosa. However, the appropriate timing for the study of inflammatory and fibrotic responses, during HIO development, has not been adequately investigated. We developed HIOs from the human embryonic stem cell line, H1, and examined the expression of mesenchymal markers during their maturation process. We also investigated the effect of inflammatory stimuli on the expression of fibrotic and immunological mediators. Serial evaluation of the expression of mesenchymal and extracellular matrix (ECM) markers revealed that HIOs have an adequately developed mesenchymal component, which gradually declines through culture passages. Specifically, CD90, collagen type I, collagen type III, and fibronectin were highly expressed in early passages but gradually diminished in late passages. The proinflammatory cytokines IL-1α and TNF-α induced the mRNA expression of fibronectin, collagen types I and III, tissue factor (TF), and alpha-smooth muscle actin (α-SMA) primarily in early passages. Similarly, HIOs elicited strong mRNA and protein mesenchymal (CXCL10) and epithelial (CXCL1, CCL2, CXCL8, and CCL20) chemokine responses in early but not late passages. In contrast, the epithelial tight junction components, CLDN1 and JAMA, responded to inflammatory stimulation independently of the culture passage. Our findings indicate that this HIO model contains a functional mesenchymal component, during early passages, and underline the significance of the mesenchymal cells’ fitness in inflammatory and fibrotic responses. Therefore, we propose that this model is suitable for the study of epithelial-mesenchymal interactions in early passages when the mesenchymal component is active.

Bifidobacterium lactis, Lactobacillus acidophilus, Lactiplantibacillus plantarum and Saccharomyces boulardii are common probiotic supplements. Colonic subepithelial myofibroblasts (cSEMFs) are actively involved in mucosal wound healing and inflammation. cSEMFs, isolated from healthy individuals, were stimulated with 102 or 104 cfu/mL of these probiotic strains alone and in combination, and their effect on chemokine and wound healing factor expression was assessed by qRT-PCR, ELISA and Sircol Assay, and on cSEMFs migration, by Wound Healing Assay. These strains remained viable and altered cSEMFs’ inflammatory and wound healing behavior, depending on the strain and concentration. cSEMFs treated with a combination of the four probiotics had a moderate, but statistically significant, increase in the mRNA and/or protein expression of chemokines CXCL1, CXCL2, CXCL4, CXCL8, CXCL10, CCL2 and CCL5, and healing factors, collagen type I and III, fibronectin and tissue factor. In contrast, when each strain was administered alone, different effects were observed, with greater increase or decrease in chemokine and healing factor expression, which was balanced by the mixture. Overall, this study highlights that the use of multiple probiotic strains can potentially alert the gut mucosal immune system and promote wound healing, having a better effect on mucosal immunity than the use of single probiotics.

The probiotics Lactiplantibacillus plantarum UBLP-40, Lactobacillus rhamnosus UBLR-58 and Bifidobacterium longum UBBL-64 seem to promote wound healing when applied topically. Our aim was to investigate their effect on the mRNA expression of pro-inflammatory, healing and angiogenetic factors during the healing process of a standardized excisional wound model in rats. Rats subjected to six dorsal skin wounds were allocated to Control; L. plantarum; combined formula of L. rhamnosus plus B. longum; L. rhamnosus; and B. longum treatments, applied every two days, along with tissue collection. The pro-inflammatory, wound-healing, and angiogenetic factors of mRNA expression were assessed by qRT-PCR. We found that L. plantarum exerts a strong anti-inflammatory effect in relation to L. rhamnosus–B. longum, given alone or in combination; the combined regime of L. rhamnosus–B. longum, works better, greatly promoting the expression of healing and angiogenic factors than L. plantarum. When separately tested, L. rhamnosus was found to work better than B. longum in promoting the expression of healing factors, while B. longum seems stronger than L. rhamnosus in the expression of angiogenic factors. We, therefore, suggest that an ideal probiotic treatment should definitively contain more than one probiotic strain to speed up all three healing phases.

Idiopathic pulmonary fibrosis (IPF) is caused by progressive lung tissue impairment due to extended chronic fibrosis, and it has no known effective treatment. The use of conditioned media (CM) from an immortalized human adipose mesenchymal stem cell line could be a promising therapeutic strategy, as it can reduce both fibrotic and inflammatory responses. We aimed to investigate the anti-inflammatory and anti-fibrotic effect of CM on human pulmonary subepithelial myofibroblasts (hPSM) and on A549 pulmonary epithelial cells, treated with pro-inflammatory or pro-fibrotic mediators. CM inhibited the proinflammatory cytokine-induced mRNA and protein production of various chemokines in both hPSMs and A549 cells. It also downregulated the mRNA expression of IL-1α, but upregulated IL-1β and IL-6 mRNA production in both cell types. CM downregulated the pro-fibrotic-induced mRNA expression of collagen Type III and the migration rate of hPSMs, but upregulated fibronectin mRNA production and the total protein collagen secretion. CM’s direct effect on the chemotaxis and cell recruitment of immune-associated cells, and its indirect effect on fibrosis through the significant decrease in the migration capacity of hPSMs, makes it a plausible candidate for further development towards a therapeutic treatment for IPF.

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