A Comprehensive Exploration of Therapeutic Strategies in Inflammatory Bowel Diseases: Insights from Human and Animal Studies

Inflammatory bowel disease (IBD) is a collective term for a group of chronic inflammatory enteropathies which are characterized by intestinal inflammation and persistent or frequent gastrointestinal signs. This disease affects more than 3.5 million humans worldwide and presents some similarities between animal species, in particular, dogs and cats. Although the underlying mechanism that triggers the disease is not yet well understood, the evidence suggests a multifactorial etiology implicating genetic causes, environmental factors, microbiota imbalance, and mucosa immune defects, both in humans and in dogs and cats. Conventional immunomodulatory drug therapies, such as glucocorticoids or immunosuppressants, are related with numerous adverse effects that limit its long-term use, creating the need to develop new therapeutic strategies. Mesenchymal stromal cells (MSCs) emerge as a promising alternative that attenuates intestinal inflammation by modulating inflammatory cytokines in inflamed tissues, and also due to their pro-angiogenic, anti-apoptotic, anti-fibrotic, regenerative, anti-tumor, and anti-microbial potential. However, this therapeutic approach may have important limitations regarding the lack of studies, namely in veterinary medicine, lack of standardized protocols, and high economic cost. This review summarizes the main differences and similarities between human, canine, and feline IBD, as well as the potential treatment and future prospects of MSCs.


Introduction
Inflammatory bowel disease (IBD) is a collective term for a group of chronic inflammatory enteropathies, which are characterized by intestinal inflammation and persistent or regenerative therapies for IBD treatment are presented.Figure 2 illustrates the flowchart of study selection included in this article.Given the above, the goal of this study is to present a critical perspective, highlighting the key differences and similarities between human, canine, and feline IBD, as well as conventional therapy and innovative research, notably using MSCs and, in particular, adipose-derived mesenchymal stromal cells (ASCs).A scientific literature search was conducted using the primary electronic databases for scientific publication dissemination-PubMed, Scopus, and Web of Science-to compile and discuss the clinical trials using mesenchymal stem/stromal cells therapy for IBD treatment in human and companion animals.For this purpose, from all the articles published within the period between January 2000 to January 2024, referring to the association of the following keywords-dog, cat, human, mesenchymal stem/stromal cells and IBD-in their article title, abstract or keywords, data were extracted, analyzed, and discussed.All other types of articles were excluded, namely retrospective analyzes, preclinical studies in canine or feline models, studies with local infusion of MSCs instead IV infusion, treatment of perianal fistulas and studies published prior to 2000.Finally, a conclusion and future perspectives in regenerative therapies for IBD treatment are presented.Figure 2 illustrates the flowchart of study selection included in this article.

Inflammatory Bowel Disease 2.1. Etiopathogenesis
According to researchers, the etiology of the disease is likely triggered by a breakdown of immunologic tolerance to luminal antigens, such as commensal microorganisms and food components [5,21,22].A microbiota imbalance frequently results from the immune system's inability to discriminate between commensal bacteria and pathogenic bacteria [23,24].The intestinal mucosal immune barrier comprises gut-associated lymphoid tissue (GALT), secretory antibodies, and mesenteric lymph nodes that respond to toxins, antigens, and potentially dangerous pathogens [25].Despite exposure to numerous intraluminal microbial and dietary allergens and the presence of a high number of lymphoid cells, the intestine exhibits low physiological inflammation under normal circumstances.This is a result of the interaction between the epithelial mucosal barrier, innate immune system, and adaptive immune system [26].Figure 3  In humans and animals IBD, there is more permeability of the intestinal barrier due to improper tight junctions and adherent junction control.Immune cells may become more exposed to luminal antigens as a result of decreased barrier function [20,27].Intestinal Epithelial Cells (IECs) play an important role in regulating the immune response by expressing pattern-recognition receptors (PRRs) for microorganisms, such as Toll-like receptors (TLRs), and by producing a variety of humoral factors, including cytokines [26].The interaction of IECs, neutrophils, macrophages, dendritic cells (DCs), and eosinophils as well as their secreted products underlies the non-specific innate immune response, which promotes the differentiation of pathogen-associated molecular patterns [28].Depending on PRRs, one can determine whether the antigens are tolerated or are met with a reaction [20,29].Neutrophils are one of the first lines of cellular defense; they can remove infections by boosting inflammation through phagocytosis, producing reactive oxygen species (ROS) and by releasing intracellular chemicals known as neutrophil extracellular traps (NETs), promoting mucosal healing and inflammation reduction.Nonetheless, NETs also trigger epithelial cell death, and ROS also damage lipids and proteins in the mucosa barrier, altering their function [25].
The innate immune response culminates in the activation of dendritic cells, which function as a bridge between innate and adaptive immunity [30].Lymphocytes are the principal effector cells in the adaptive immune response; they are activated in the gut to destroy harmful antigens [28,31].Antigens are presented by dendritic cells to particular lymphocytes, such as naïve CD4+ T helper cells in secondary lymphoid organs (e.g., the mesenteric lymph node) [20,32].Depending on the profile of cytokines releases, T helper cells can differentiate into Th1, Th2, and Th17 cells and regulatory T cells (Tregs), which In humans and animals IBD, there is more permeability of the intestinal barrier due to improper tight junctions and adherent junction control.Immune cells may become more exposed to luminal antigens as a result of decreased barrier function [20,27].Intestinal Epithelial Cells (IECs) play an important role in regulating the immune response by expressing pattern-recognition receptors (PRRs) for microorganisms, such as Toll-like receptors (TLRs), and by producing a variety of humoral factors, including cytokines [26].The interaction of IECs, neutrophils, macrophages, dendritic cells (DCs), and eosinophils as well as their secreted products underlies the non-specific innate immune response, which promotes the differentiation of pathogen-associated molecular patterns [28].Depending on PRRs, one can determine whether the antigens are tolerated or are met with a reaction [20,29].Neutrophils are one of the first lines of cellular defense; they can remove infections by boosting inflammation through phagocytosis, producing reactive oxygen species (ROS) and by releasing intracellular chemicals known as neutrophil extracellular traps (NETs), promoting mucosal healing and inflammation reduction.Nonetheless, NETs also trigger epithelial cell death, and ROS also damage lipids and proteins in the mucosa barrier, altering their function [25].
The innate immune response culminates in the activation of dendritic cells, which function as a bridge between innate and adaptive immunity [30].Lymphocytes are the principal effector cells in the adaptive immune response; they are activated in the gut to destroy harmful antigens [28,31].Antigens are presented by dendritic cells to particular lymphocytes, such as naïve CD4+ T helper cells in secondary lymphoid organs (e.g., the mesenteric lymph node) [20,32].Depending on the profile of cytokines releases, T helper cells can differentiate into Th1, Th2, and Th17 cells and regulatory T cells (Tregs), which produce multiple cytokines with different actions (Figure 4) [28,32].After the antigens have been removed, lymphocytes are normally down-regulated to maintain intestinal homeostasis [26].In IBD, there is a continuous stimulation of these lymphocytes, resulting in chronic inflammation [8], and this disparity leads to the activation of macrophages and B cells, as well as the recruitment of circulating leukocytes into the gut [33].Some authors could not demonstrate a clear Th1 or Th2 cytokine expression in dogs and cats with CIE [4,15,28,34], and there is no indication of a Th17 signature [35].This finding in canine and feline CIE differs with human IBD, where patients with Crohn's disease had relevant Th1/Th17 (cell-mediated) cytokine polarization, and in ulcerative colitis, a significant Th2 (humoral) cytokine profile [4,36].
Biomedicines 2024, 12, x FOR PEER REVIEW 6 of 18 Th1/Th17 (cell-mediated) cytokine polarization, and in ulcerative colitis, a significant Th2 (humoral) cytokine profile [4,36].Genetic abnormalities in innate immune system have been linked to IBD susceptibility in both dogs and humans.Mutation in NOD2/CARD15 in humans, and TLR4 and TL5 in dogs, along with the presence of enteric microbiota, may result in increased proinflammatory cytokine production and decreased bacterial clearance, increasing chronic intestinal inflammation.In felines, the IBD pathophysiology has not been fully determined [15,37,38].

Conventional Therapies
Medical therapy is essential in the treatment of human IBD and aims to reduce inflammation.On the other hand, the majority of dogs and cats with CIE do not require any therapy other than dietary changes (e.g., novel protein source or protein hydrolysate used in hypoallergenic diets), antibiotic medication (e.g., metronidazole), or sulfasalazine/mesalamine (5-aminosalicylic acid-5-ASA) for canine colitis [2,6,15,39].Only a tiny percentage that do not respond to these therapies will require immunosuppressive treatment-dogs and cats with IBD [2].Various immunosuppressive medications are used alone or in combination to induce and then sustain remission [40].Glucocorticoids (prednisolone 1-2 mg/kg PO q12-24h or budesonide 1 mg/m2 PO q24h) and/or additional immunosuppressive drugs (cyclosporine or chlorambucil) are commonly used to treat dogs and cats with IBD, and azathioprine is also used in dogs [4,13,32,41].In dogs with PLE, if Genetic abnormalities in innate immune system have been linked to IBD susceptibility in both dogs and humans.Mutation in NOD2/CARD15 in humans, and TLR4 and TL5 in dogs, along with the presence of enteric microbiota, may result in increased proinflammatory cytokine production and decreased bacterial clearance, increasing chronic intestinal inflammation.In felines, the IBD pathophysiology has not been fully determined [15,37,38].

Conventional Therapies
Medical therapy is essential in the treatment of human IBD and aims to reduce inflammation.On the other hand, the majority of dogs and cats with CIE do not require any therapy other than dietary changes (e.g., novel protein source or protein hydrolysate used in hypoallergenic diets), antibiotic medication (e.g., metronidazole), or sulfasalazine/mesalamine (5-aminosalicylic acid-5-ASA) for canine colitis [2,6,15,39].Only a tiny percentage that do not respond to these therapies will require immunosuppressive treatment-dogs and cats with IBD [2].Various immunosuppressive medications are used alone or in combination to induce and then sustain remission [40].Glucocorticoids (prednisolone 1-2 mg/kg PO q12-24h or budesonide 1 mg/m 2 PO q24h) and/or additional immunosuppressive drugs (cyclosporine or chlorambucil) are commonly used to treat dogs and cats with IBD, and azathioprine is also used in dogs [4,13,32,41].In dogs with PLE, if there is no strong indication of an immune-mediated etiology, immunosuppressive therapy should not be a first option and should be carefully applied after a non-responsive nutrition modification trial [4,41,42].The use of supplemental medication such as pro-/prebiotics, gastroprotectors, and antiemetics may also benefit IBD patients [43][44][45].It is important to correct any possible imbalance, for example, Vitamin B12 deficiency and supportive care.For dogs with PLE that use prolonged corticosteroids, treatment with antiplatelet agents (e.g., clopidogrel) is indicated to prevent the thrombotic risk [4,29].
In human mild to moderately active IBD, oral 5-ASA (2-3 g/day) is the standard treatment, especially for colitis signs [46].5-ASA is an anti-inflammatory drug derived by sulfasalazine without sulfapyridine.In human patients with moderate to severe IBD or that did not respond to 5-ASA therapy in 2-4 weeks, corticosteroids are indicated for at least 6 to 8 weeks [47].Other immunosuppressive drugs such as azathioprine, methotrexate and cyclosporine can be used as an alternative treatment option for human IBD, similarly to companion animals [2,48].Antibiotherapy with metronidazole or ciprofloxacin is mostly used in cases of enterocutaneous and perianal fistulae [40,46,47].Finally, biological therapy including monoclonal antibodies targeting pro-inflammatory cytokine inhibitors (TNFα and IL-12/23), α4β7 anti-integrins, and more recently Janus Kinase (JAK) inhibitors, also are fundamental therapies for humans as these cytokines play an important role in the pathogenesis of IBD [47,49].Monoclonal antibodies would be also a potential treatment for canine and feline IBD but are currently not available in veterinary medicine, and the knowledge of the specific pathways addressed in companion animals IBD should be firstly well investigated [4,6].Table 1 summarizes the treatment steps to follow for humans, dogs, and cats with IBD.A small percentage of dogs with IRE do not respond well to medical therapy and are classified as having non-responsive enteropathy (NRE), which has a poor long-term prognosis and a high rate of euthanasia [50].Alternative immunomodulatory treatments, such as fecal microbiota transplantation (FMT) or stem cell therapy can be promising therapies and they need further studies [51,52].In humans, there is a stem cell therapy already licensed in the EU for the treatment of complicated perianal fistulas in adult patients with non-active or mildly active luminal Crohn's disease-Darvadstrocel (Alofsel®), comprising a suspension of expanded human allogeneic ASCs [53].

MSC Characterization
Stem cells can be classified based on their ability to differentiate and their source throughout the body in embryonic stem cells, adult stem cells, and also in induced pluripotent stem cells [5,[54][55][56][57]. Embryonic stem cells are totipotent cells; they can generate cells from all three germinal layers (endoderm, mesoderm, and ectoderm) as well as extraembryonic annexes (placenta and umbilical cord) [54,55,58].Adult stem cells are multipotent cells; they are only partly specialized and capable of producing a limited number of cell types from their own germinative layer [5,54,55].Finally, induced pluripotent stem cells are created by reprogramming adult cells into pluripotent cells in the laboratory [59][60][61].
Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are undifferentiated, non-hematopoietic cells with the ability to self-renew and found in a variety of adult or extraembryonic tissues [62][63][64].Due to their multipotency, they have the ability to differentiate under specific circumstances into a variety of cell types, including myocytes, osteoblasts, chondroblasts, tenocytes, cardiomyocytes, hepatocytes, neuronal cells, endothelial cells, and photoreceptors [5,64].MSCs play a critical function in the regulation of the immune system despite not meeting the prearranged requirements as immune cells [5,65].These cells are reported to be easy to isolate and feasible growth in in vitro culture and are able to secrete a wide range of pro-and anti-inflammatory factors, including prostaglandins, cytokines, and chemokines, which impact immune cells' activity.They have exceptional pleiotropic features, including the capacity to move toward injury sites via chemotaxis, angiogenesis, growth factor synthesis, and antifibrosis [57,62].
MCSs can be isolated from a wide variety of tissues, but are most commonly collected from adipose tissue (AT) and bone marrow (BM), since they are relatively simple sources for obtaining cells [61,62,66,67].For the collection of BM samples, surgery, general anesthesia, and a proper aseptic procedure are required, which is more invasive than obtaining AT samples [61,68].According to some researchers, MSCs obtained from AT have a stronger proliferative capacity than MSCs obtained from BM.However, there is still no agreement in the literature, and further differences between the cell's sources are detailed based on the donor's age or passage number of cells [61,69].
In culture, MSCs have a primarily fusiform shape and are capable of adhering to plastic [70].One of the MSCs' identifying characteristics is the expression of cell surface markers.The International Society for Cellular Therapy developed the minimum standards for classifying human MSCs in order to reach a wider consensus on their universal description.The capacity to stick to plastic under controlled culture conditions, cell surface expression of CD73, CD90, and CD105 (>95%), and the lack of the hematopoietic stem cell markers CD45, CD34, CD14 or CD11b, and CD79 are some of these requirements [71][72][73].Because of interspecies variations, it is known that human MSC markers could not be entirely compatible with those needed for canine and feline MSC identification [74].Some studies have shown that most canine and feline MSCs express cell surface markers CD44 and CD90 and lack the expression of hematopoietic marker CD34 [75,76].

MSCs' Mechanism of Action
Although the mechanism of action of therapeutic MSCs is still not fully known, it has attracted attention recently due to its growing use in immunological and regenerative medicine clinical trials on both human and animal patients [61,77,78].The mechanism of action most likely differs between species and are affected by a variety of factors such as culture conditions or an inflammatory environment [33].Adaptive immune cells (T and B lymphocytes) and innate immune cells (macrophages, dendritic cells, neutrophils, eosinophils, basophils, natural killer cells, and natural killer T cells) phenotype and function are both influenced by MSCs [77,[79][80][81].MSCs' anti-inflammatory, immunomodulatory, and immunosuppressive capabilities serve as the foundation for their therapeutic use.These cells can evolve into the desired cell type, allowing the wounded area to be healed [61,62].
Despite the fact that MSCs do not have intrinsic immunosuppressive capacity, this ability is developed when cells are triggered by a proinflammatory environment, with the production of cytokines such as INFγ, TNF-α, and interleukin-1 β [26,82].Additionally, they possess a remarkable capacity for immunomodulation through paracrine signaling, whereby they secrete a variety of molecules to nearby cells (cytokines, growth factors, and microvesicles that can carry a cargo of proteins and other bioactive molecules), or through cell-to-cell contact, which results in vascularization, cellular proliferation in damaged tissues, and a decrease in inflammation [5,83].
MSCs influence the innate immune system via three key mechanisms.Activating proinflammatory monocytes and macrophages is one of these methods.Macrophages can be change from M1 classic macrophages (with proinflammatory functions) in M2 anti-inflammatory macrophages in the presence of MSCs and their soluble factors such as IL-6, PGE 2 , TGF-β, and HGF [1,5,84,85].MSCs can also block monocyte development into mature DCs by producing soluble factors such IL-6, PGE2, TGF-β, and HGF [83,85].Finally, MSCs limit NK cell proliferation and cause polymorphonuclear cell and cytotoxic T cell apoptosis, which can be mediated by cell-to-cell contact or by soluble molecules such as PGE2 and IDO, but also TGF-β [5,26,84,86,87].
According to a number of studies, MSC infusion also influence the adaptive immune system by raising the frequency of regulatory T cells while decreasing the number of T cells that secrete inflammatory cytokines [63].The effect of MSCs on T cells appears to be proportional to the MSC/T cell ratio: a high MSC/T cell ratio inhibits T cell proliferation, whilst a low MSC/T cell ratio may promote T cell growth [33], which is an essential issue in IBD treatment.They also suppress B cell growth through cell-to-cell interaction and through a cell cycle arrest in the G0/G1 phase [61,84].
The therapy can be autologous or allogeneic, depending on whether the stem cells are derived from the patient or from another individual of the same species [61].Recent studies using xenogeneic stem cells from other species have yielded positive and safe outcomes.

Clinical Trials with MSCs
The immunomodulatory impact of MSC treatment has been documented in animal models and in humans during the last few years, with a considerable improvement in clinical presentation.Tables 2 and 3 summarize the clinical trials conducted to date on dogs and cats with MSC therapy for IBD.
IV-intravenous; ASCs-adipose-derived mesenchymal stromal cells; NLR-neutrophil-to-lymphocyte ratio; PLR-platelet-to-lymphocyte ratio; SII-systemic immune-inflammation index.Treatment with ACSs appears to be as effective in the treatment of feline IBD as prednisolone therapy.
MSCs as an alternative therapy for IBD are still a relatively new concept.However, in clinical studies on humans with inflammatory gastrointestinal and immunological disorders, this medication has proved to be efficacious and safe.Most studies in human IBD use BM-MSCs, and ASCs have been increasingly employed for the local treatment of perianal fistulas that result as a complication of CD [93][94][95][96].Table 4 summarizes published studies of intravenous MSC therapies in human IBD (CD and UC).

Discussion
CIE in dogs and cats is classified into many entities based on their response to medical therapy.In contrast, IBD in both animals and human patients is a condition that may requires immunosuppressive medications, biologic drugs and, in some cases, surgery [2].
Over the last decade, tremendous research efforts have resulted in a greater knowledge of the multifactorial disease complex of canine and feline IBD compared to humans.Despite the differences in classification of the disease, all species' etiopathogenesis culminate in a combination of genetic causes, environmental factors, microbiota imbalance, and mucosa immune defects.It is well recognized that the pathogenesis in humans and animals evolves an exacerbated reaction of lymphocytes to normal antigens, although the profile of released cytokines varies throughout species and even within investigations.
The majority of human research has used BM-MSCs, and in dogs and cats, ASCs.Adipose tissue is presently the most accessible and abundant source of MSCs, with a less painful collecting technique when compared to other sources.In Veterinary Medicine, adipose tissue is the most frequent source for stem cells obtainment when the MSC option is considered for IBD treatment.It is often obtained from animals undergoing routine surgery (e.g., ovariohysterectomies), where small quantities of adipose tissue are regularly discharged.Additionally, BM-MSCs are affected by age, and increasing age decreases cell number, proliferation, and differentiation capacities [109].ASCs, on the other hand, show more growth potential without losing their differentiation potential, resulting in massive cell numbers that are increasingly being used in cell therapy [110].
Studies carried out with MSCs in dogs and cats used doses between 2 and 4 million cells per kg/animal.Intravenous allogeneic cell administration was used in all studies, and in certain trials, MSC therapy was compared to conventional prednisolone therapy.ACSs safely increased life quality of dogs receiving or not receiving prednisolone concomitantly to stem cell therapy.In cats, Webb and Webb (2022) showed that therapy alone with prednisolone or therapy with MSCs appears to be equal effective for feline IBD therapy [91].Cats improved in clinical symptoms and scores when given ASCs or prednisolone, although the adverse effects of prolonged corticosteroid use are clearly severe.In human studies, dosages ranging from 1 to 3.5 million cells per kg/person were employed, and results have shown a safe and feasible therapy, with minimal or no side effects, possible with no association to stem cell administration.One study also demonstrated safety and efficacy in treatment with MSCs at doses of up to 10 million cells per kg/person [103].

Conclusions and Future Directions
Mesenchymal stromal cells appear to be a successful and safe therapy for IBD in humans, dogs, and cats.Future research is needed to draw more conclusions about these cell therapies, using more standardized techniques and larger patient cohorts.Other regenerative therapies have also been recently studied for IBD patients, such as stem cells' secretome, stem cells' extracellular vesicles, and organoids.MSCs' therapeutic effects occur through the production and release of trophic molecules, which is now referred to as the secretome [111].The MSC secretome is a complex combination of soluble products comprising a proteic soluble fraction (growth factors and cytokines) and a vesicular fraction (microvesicles and exosomes).Therapies without cell transplantation, such as the one offered by MSC's secretome, can offer key advantages over transplanting stem cells, although they are still poorly studied [111].
Exosomes are one type of extracellular vesicle that is now being researched as a potential therapy for IBD patients [112].Bilayer vesicular nanoparticles known as exosomes are released by cells as a result of environmental stimulation or self-activation and exhibit the same features as their parental cells.Exosomes produced from IECs have the ability to trigger immunoregulatory pathways to promote the maturation of macrophages with tolerogenic features, regulatory T cells (Treg), and DCs, as well as to maintain intestinal homeostasis [112].Extracellular vesicles produced from adipose tissue-derived MSCs have been shown to be effective against dextran sulfate sodium-induced colitis in mice.Nevertheless, the underlying processes have yet to be fully clarified [113].
Finally, organoids are sophisticated three-dimensional cell culture models created from stem cells that have a significantly greater ability for self-organization and reproduction of in vivo physiology than two-dimensional cell culture systems [114,115].Even though organoid systems may accurately mimic host biology, they may be restricted in research evaluating nutrients and drugs transport or host-microbiome interactions [116].Intestinal organoids have been used as a model to study the pathogenesis of IBD as they closely mimic the structure and behavior of the original organ.Ultimately, these organoids can be transplanted into IBD patients to repair epithelial damage and reverse all symptoms of the disease.This is expected to occur in the very near future.

Figure 1 .
Figure 1.Chronic inflammatory enteropathy classification in dogs and cats according to treatment response.

Figure 1 . 18 Figure 2 .
Figure 1.Chronic inflammatory enteropathy classification in dogs and cats according to treatment response.Biomedicines 2024, 12, x FOR PEER REVIEW 4 of 18

Figure 2 .
Figure 2. Flowchart of study selection.A total of 488 studies were analyzed.There were 18 studies that met the following criteria: clinical trials in humans, dogs, and cats with IBD treated with IV infusion of MSCs (Original figure created with BioRender.com).

18 Figure 3 .
Figure 3. Differences in IBD causes, risk factors, injury sites and clinical signs between human and companion animals (dogs and cats) (Original figure created with BioRender.com).

Figure 3 .
Figure 3. Differences in IBD causes, risk factors, injury sites and clinical signs between human and companion animals (dogs and cats).++-with special incidence.(Original figure created with BioRender.com).

Figure 4 .
Figure 4. Immune response mechanisms in the pathogenesis of IBD (Original figure created with BioRender.com).

Figure 4 .
Figure 4. Immune response mechanisms in the pathogenesis of IBD (Original figure created with BioRender.com).

Table 1 .
IBD treatment in humans, dogs, and cats.

Table 2 .
Clinical trials carried out with MCSs on canine IBD.

Table 3 .
Clinical trials carried out with MCSs on feline IBD.

Table 4 .
Clinical trials carried out with MCSs on human IBD.