Inflammatory Bowel Disease

How similar are human and animal digestive systems?

 

This is not an exhaustive list of species-specific differences, nor can one be made given their unknown full extent, but rather an example of how these differences impact the face, construct, predictive, and intrinsic validity of animal models.

 

Not all species-specific differences can be accounted for in animal models, as there are hundreds of them, their relevance for inflammatory bowel diseases (IBD) is unclear, and their interaction with other organ systems in the animal model makes it difficult to predict how they would have behaved within the human system.

 

Although gastrointestinal tracts in humans and species commonly used in animal experimentation are grossly composed of similar organs, they also present with specificities in anatomy and physiology that reflect distinct evolutionary paths, dietary needs, feeding patterns and digestive strategies.

Species-specific differences in intestinal structure, organisation, histology and function lead to variations in microbiome/mucus/bile acids composition, surface areas interactions, absorption of orally administered compounds, susceptibility to IBD and response to treatments.

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Species-specific differences in gastrointestinal anatomy and physiology

In humans, the duodenum-produced hormone motilin triggers periodic contractions to clear residual food particles and prevent bacterial overgrowth. IBD is associated with decreased gut motility, contributing to microbial dysbiosis and inflammation.

However, rodents, who lack the gene encoding motilin, have different gut motility patterns compared to humans, making it difficult to gather human-relevant insights on mechanisms that underly pathological alterations of gut motility Kitazawa & Kaiya, Front. Endocrinol., May 2019.

Furthermore, segmental contractions in the rodent colon differ from the peristaltic waves observed in humans, impacting how external triggering factors (environmental compounds, drugs) interact with the gut mucosa and the gut microbiome.

 

In contrast to humans, mice and rats feed almost incessantly and mostly during the night, leading to lower pH in the rodent stomach compared to humans Nagpal et al., Front. Microbiol., Nov 2018, McConnell et al., J. Pharm. & Pharmac., Jan 2008. Variations in intestinal pH can influence the solubility and absorption of certain drugs and/or favor the presence of metabolizing enzymes and microbiota that are not typical for the human gastrointestinal (GI) tract, resulting in inter-species differences in drug metabolism and composition of the gut microbiome Kohl et al., J. Exp. Zool., Apr 2013.

 

The human mucosal surface contains circular folds that increase the surface area and provide a niche for mucus-associated bacteria, while the mouse mucosal surface is smooth, which likely also contributes to differences in microbiome composition.

In non-human primates, who have a more easily digestible diet than humans, the small intestine is generally shorter compared to humans, potentially reducing the exposure time of the intestinal lining to toxins/pathogens and limiting the extent of microbiome interactions, dysbiosis, and intestinal inflammation.

 

Moreover, there are several prominent species-specific differences in location, distribution, density, secretion levels and protein sequences of mucin-producing goblet cells and the antimicrobial compounds-secreting Paneth cells, affecting immune responses, intestinal inflammation and microbiota composition Timmermans et al., Cell, Aug 2024, Yamaguchi et al., J. Immuno., Sep 2002. These inter-species differences, at tissue, cellular and molecular level, represent an additional barrier to successful translation of animal studies to CD and UC patients. 

 

 

Species-specific differences in gut microbiome

Qualitative and quantitative inter-species differences in GI microbiome have a major impact on the activity of drug-metabolizing enzymes, on intestinal pH, on gut inflammation, and on integrity of the gut barrier, thereby affecting predictive value of animal models of IBD.

 

The study of the microbiome has historically relied on mouse studies, based on coarse comparisons at higher taxonomy hierarchy level with a poorly characterized mouse GI microbiota.

However, metagenomics analysis of gut microbiome at lower taxonomy level have since shown major inter-species differences in GI microbiome composition, quantity, and function between humans and other species Hugenholtz & de Vos, Cell. Mol. Life Sci., Nov 2017, Kieser et al., Plos, Mar 2022, Fan et al., Curr. Microbio., Mar 2023, De Jonge et al., Front Microbiol., Jun 2022.

Notably, the Faecalibacterium genus, that plays a protective role in IBD patients, has a high relative abundance of over 10%, whereas it is hardly detected in mice. Faecalibacterium is decreased in patients with IBD, especially in ileal CD Sokol et al., IBD, Feb 2009.

Within the same phylum Firmicutes, the genus of Roseburia, that is altered in IBD compared to healthy individuals, has a relative abundance of 5-10% in humans versus 1-5% in mice.

The Bifidobacterium genus, of which alterations were equally observed in IBD, has a relative abundance of 1-5% in humans and 0.01-1% in mice.

 

Initiatives were taken to humanize mice with human microbiota and to inter-cross inbred strains. However, it was found that the GI microbiome in mice varied immensely depending on the housing conditions, even more so than on the genetic background Hugenholtz & de Vos, Cell. Mol. Life Sci., Nov 2017.

Previously, DNA sequencing of fecal samples of mouse strains of diverse genetic, provider, housing and diet backgrounds and comparison to the human gastrointestinal genome has demonstrated that only 4% of mouse GI microbial genes were shared with human GI microbial genes Xiao et al., Nature Biotech., Sep 2015.

 

Rats tend to have a higher abundance of the phylum Bacteroidetes compared to mice. Mice, on the other hand, have a higher abundance of the family Muribaculaceae within the phylum Bacteroidetes Nagpal et al., Front. Microbiol., Nov 2018.

Although the gut microbiota in humans was closer to NHP than to mice and rats, there are still significant differences between the two species.

For example, African green monkeys show an opposite microbial taxa and genes responses to a typical high-protein, high-fat, low-fiber Western diet compared to humans, suggesting that NHP are not a good model for studying the effect of the human gut microbiota on host metabolism and microbiome-associated human diseases Amato et al., Microbiome, Nov 2015.

 

The so-called human microbiota-associated (HMA) mouse models do not replicate patients' microbiome Arrieta et al., Cell Host Micr., May 2016, possibly as a result of adaptations of microbes to the mouse intestinal environment.

Human-based research has shown that the gut microbiome intervenes in shaping both the innate and adaptive immune system from infancy through adulthood through human host-specific interactions between human host-specific immune system and human host-specific microbiota.

Therefore, it is not surprising that HMA mice have a low immune maturation with reduced numbers of adaptive and innate intestinal immune cells when compared with mice that harbor a murine microbiota.

Since the engrafted human microbiota in mice does not recapitulate the dysbiosis nor the immune responses characteristic of IBD, the use of HMA animals to model IBD is not likely to be of benefit to patients. 

 

 

Species-specific differences in feeding patterns

Species-specific nutritional needs, physiology and behavioral patterns affect dietary balance, the microbiome composition, and susceptibility to IBD.

 

Because of their higher energy demands, small animals need to have a short retention time of foods. To maintain a population of gut miocrobiota all while allowing food particles to pass on rapidly, rodents partly recycle their microbiota through proximate colon folds that act as mucus traps and enable transport of microbiome and mucus back to the colon Bjornhag et al., Zoosyst. & Evol., Apr 2008.

The need to recycle nutrients is also reflected in the practice of coprophagy characteristic of rodents and several other species (dogs, horses, some NHP). Coprophagy is not considered as normal behaviour in humans, therefore extrapolation of dietary effects on intestinal inflammation in species that practice coprophagy to humans can produce inaccurate interpretations Jiminez et al., Gut Pathogens, Nov 2015.

 

 

Species-specific differences in xenobiotics metabolizing enzymes

Certain drugs, such as non-steroidal anti-inflammatory drugs (NSAID), were found to exacerbate intestinal inflammation in patients Sostres et al., Arth. Res. & Th., Jul 2013.

It is believed that NSAID exerts this effect by inhibiting cyclooxygenase COX1 enzymes, responsible for producing prostaglandins that stimulate the protective mucus layer production. Because of inter-species differences in activity of COX1/COX2 enzymes, as well as eventual prostaglandin-independent compensatory mechanisms, the toxic effects of NSAID were not captured in animal testing.

In addition, gene polymorphisms for the main NSAID metabolizing enzyme, CYP2C, were associated with inter-individual differences in metabolic clearance, plasma elimination half-life, plasma concentrations of NSAID McEvoy et al., Front. Pharmacol., Jun 2021.

 

There are significant inter-species differences in expression, activity, and substrate specificity of gastrointestinal phase I (CYP) and phase II (UDP-glucuronosyltransferases - UGT, sulfotransferases - SULT) enzymes that metabolize xenobiotics Turpeinen et al., Xenob., Sep 2008,  Maksyumiuk et al., PLOS One, Jan 2024, Windmill et al., Tox. Sci., Mar 2000.

 

Moreover, animal testing does not recapitulate human-relevant inter-individual variations in metabolism of xenobiotics, such as in gene polymorphisms, and expression levels of individual CYP enzymes Paine et al., Drug Met. & Disp., May 2006.

 

 

Species-specific differences in xenobiotics transporters

Substrate specificity, tissue distribution, relative abundance, and functional activity of drug transporters varies both between rodents, dogs and monkeys, and between tested species and humans Tucker et al., Bioch. Pharmac., Jan 2012, Chu et al., QxMD, Mar 2013, further compromising the predictive value of animal testing.

 

 

Species-specific differences in stress response capacity

Significant inter-species differences in organism's ability to detect, respond to, and adapt to stressors contributes to poor predictivity of animal testing.

This also means that modelling environmental triggers (pollution, chemicals, drugs) of IBD in animals is likely to produce different outcomes in humans.

 

Xenobiotics are detected by xenobiotic receptors, also known as xenosensors, that control the expression of genes encoding metabolizing enzymes and transporters by acting as transcription factors. Some of the key xenosensors expressed in human intestine and liver include the aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR).

Xenosensors show marked inter-species differences in their activation by xenobiotics Mackowiak et al., Drug Met. & Disp., Sep 2018.

For example, rifampicin and SR12813 are potent agonists for human PXR but not for mouse PXR, leading to the induction of P450 enzymes followed by increased metabolism and clearance of co-administered drugs.

 

The response to toxicants commonly involves activation of protective nuclear factor erythroid 2-related factor 2 (Nrf2) pathways, and subsequently, the activation of expression of genes encoding ROS-neutralizing antioxidant enzymes like glutathione S-transferases (GST) and superoxide dismutase (SOD).

In the context of IBD, by activating the expression of antioxidant and cytoprotective genes, a robust Nrf2 response could help mitigate the damage from chronic inflammation.

However, inter-species differences in Nrf2-mediated baseline level, threshold of activation and robustness of response, likely contribute to differences in phenotype and pathophysiology between animal models of IBD and IBD patients Russomanno et al., Tox. Sci., Aug 2023, Donato et al., Arch. Tox., Feb 2022.

 

 

Species-specific differences in metabolic rates

Species-specific differences in metabolic rates might eight increase susceptibility to IBD in experimental animals through an increased production of ROS or, on the contrary, decrease it through a better nutrient absorption and metabolism of xenobiotics Toutain et al., Comp. Vet. Pharmac., Jan 2010.

 

Metabolic rates of mammals are adapted to their body sizes, physiology, diet, behaviour, environment and activity levels Pettersen et al., J. Exper. Biol., Jan 2018. For instance, in comparison to humans, mice, rats and rabbits have higher metabolic rates, while pigs and sheep have lower metabolic rates Kleiber, J. Theor. Bio., Sep 1975.

Human metabolism also differs significantly from that of other primates. In spite of their higher physical activity levels, non-human primates have lower metabolic rates, presumably due to higher energy demands associated with the human brain Yegian et al., PNAS, Nov 2024.

 

 

Species-specific differences in mucins

Mucins play several key roles in maintaining GI health by providing lubrication for food and stool progression, by participating in activation/inhibition of immune signaling pathways, and by protecting the intestinal epithelium from toxins/pathogens.

In IBD, defects in mucin production and function can lead to a compromised barrier, exacerbating inflammation and disease progression Theodoratou et al., Nature Rev. Gastro. & Hepat., Jun 2014.

 

The role of mucins has been extensively studied in animals, however, such experiments are highly invasive, technically challenging, and not directly translatable to humans.

 

The main mucin types found in the intestines and in the stomach, Muc2, Muc5b, and Muc5ac, overlap between humans, rats, and mice. Nonetheless, there are major species-specific differences in mucins' composition, glycosylation patterns, expression levels, and functional roles Hugenholtz & de Vos, Cell. Mol. Life Sci., Nov 2017.

Changes in glycosylation patterns can impact the protective mucus layer in the gut. For instance, decreased glycosylation has been observed in the intestinal mucus of IBD patients, which might lead to increased bacterial contact with the epithelium, potentially triggering inflammation.

Dysregulation in mucin synthesis and secretion can compromise the mucosal barrier, making it easier for pathogens to invade and cause inflammation.

Inter-species differences in functional roles might make certain mucins more critical in maintaining gut barrier integrity in one species compared to another.

Species-specific differences in mucus-associated GI microbiome can affect mucus adhesion, degradation, and overall layer thickness.

In addition, differences in the immune systems of humans and rodents can affect how mucins modulate immune responses and intestinal inflammation.

 

 

Species-specific differences in bile acids

Bile acids (BA) act as signaling molecules that interact with the farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor 1 (GPBAR1) that are highly expressed in the ileum and the colon.

FXR is believed to have a protective effect on IBD, by playing a role in maintaining intestinal barrier integrity, by controlling the expression of genes involved in inflammatory responses and by suppressing the production of pro-inflammatory cytokines and chemokines. Its exact role remains, nonetheless, to be elucidated in a human-based system, since animal studies on FXR have shown inconsistent and unreliable results Andersen et al., Cell, Nov 2021.

 

Bile acid malabsorption in the ileum is frequently observed in IBD patients, particularly those with CD, and can lead to diarrhea, abdominal pain and bloating Vitek, IBD, Feb 2015.

 

The gut microbiota can modulate the BA pool size and composition through conversion of primary BA to secondary BA, and amino acids conjugation.

Reciprocally, BAs were found to shape the gut microbiota through regulation of growth and survival of specific bacterial species.

Patients with IBD develop an altered BA composition characterized by an increase in primary BA and a decrease in secondary BA, probably as a consequence of gut dysbiosis Xu et al., Med., Jun 2024. 

  

The BA pool compositions in rodents are very different in humans.

In mice and rats, cholic acid (CA), chenodeoxycholic acid (CDCA), and ursodeoxycholic acid (UDCA), are converted to hydrophilic α-muricholic acid (α-MCA) and β-MCA, reducing BA-induced epithelial damage and influencing interactions with the gut microbiota.

Muricholic acids are less common in humans than in rodents, because the liver enzyme responsible for their synthesis, CYP2C70, is rodent-specific Takahashi et al., J. Lip. Res., Dec 2016.

 

Furthermore, rodents’ liver and gut microbiome can hydroxylate LCA into the less toxic hyodeoxycholic acid (HDCA), and this metabolic pathway is more active in rodents than in humans, contributing to inter-species differences in BA toxicity Lin et al., Drug Metab. Dis., Mar 2020.

In humans, BA toxicity exacerbates epithelial damage and intestinal permeability characteristic of IBD, whereas this process is less prominent in rodents.

 

In humans, most primary BA are glycine conjugated, while in rodents, the majority of primary BAs are taurine conjugated Xue et al., Cells, Oct 2021.

Rodent MCAs and taurine-conjugated primary BA are more hydrophilic compared to their human BA counterparts, which makes rodent BA less toxic, that is less likely to disrupt the phospholipid bilayer of cell membranes.

 

in contrast to humans, in which CDCA is the most potent endogenous FXR agonist, the taurine-conjugated α/βMCA exert antagonistic FXR activity in rodents, which explains higher synthesis rates of BA in rodents compared to humans Zhang et al., J. Clin. Med., Dec 2021.

These species-specific differences have repercussions at several levels of BA metabolism and signaling pathways, likely contributing to failures of translation of findings in animal studies to humans.

 

 

Species-specific differences in gene expression regulation

Non-coding RNA, including microRNA (miRNA) and long non-coding RNA (lncRNA), plays a key role in gene expression regulation.

The various lncRNA have been found to act through a variety of regulatory mechanisms, including epigenetic (by recruiting epigenetic modifiers to specific genomic regions), post-transcriptional (by influencing the stability, translation, or degradation of mRNA) and through other noncoding RNA regulators (by sequestering miRNA and preventing them from binding to their target mRNA).

 

Excessive immunological response and inflammation, characteristic of IBD, could be aided by dysregulation or insufficient miRNA-mediated repression Preda & Istratescu, UC Et., Sep 2022.

Since lncRNA can influence the expression of genes that maintain the epithelial barrier and regulate the immune reactivity, dysregulation of specific lncRNA can increase intestinal permeability and exacerbate inflammation.

However, in spite of presence of conserved regions, sequences and expression profiles of lncRNA were found to vary significantly between species Sarropoulos et al., Nature, Jun 2019.

 

 

Species-specific differences in immune system

Innate and adaptive immunity responses play a crucial role in the pathogenesis of IBD.

 

Extensive differences between humans and mice were demonstrated in the structure of innate and adaptive immunity, including in balance of leukocyte subsets, defensins, Toll receptors, inducible NO synthase, the NK inhibitory receptor families Ly49 and KIR, FcR, Ig subsets, the B cell (BLNK, Btk, and λ5) and T cell (ZAP70 and common γ-chain) signaling pathway components, Thy-1, γδ T cells, cytokines and cytokine receptors, Th1/Th2 differentiation, costimulatory molecule expression and function, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression Mestas & Hughes, J Immunol, Mar 2004.

 

In addition, it was shown that transcriptional responses to acute inflammatory stresses of different etiologies in mouse models do not correlate with human acute inflammatory diseases Seok et al., PNAS, Jan 2013. 

 

Major species-specific differences were also found in transcriptional regulation, chromatin state and higher order chromatin organization. Notably, cis-regulatory sequences next to immune-system-related genes have shown the most divergence, as they have apparently undergone more rapid evolution than others Yue et al., Nature, Nov 2014.

 

It was suggested that using animal models with a humanized immune system might improve translatability to humans, however, such an approach would face persistent, insurmountable challenges: the role of the human immune system in IBD is complex and not fully understood, the equivalence of humanized animals to the human immune system was never demonstrated by objective measures Davis, Cell, Dec 2008, and the cross-talk between the human immune system and the rest of human organ systems cannot be recapitulated in animals.

 

Additionally, another aspect that is particularly relevant for IBD and that cannot be recapitulated in humanized animal models, is the development and adaptation of the immune system in response to the environment, including the microbiome, diet? and housing. This missing aspect in animal models is crucial, since as twin studies show, the human immune system is more shaped by the environment than by genes Brodin et al., Cell, Jan 2015.