The microbiocidal activity of α-defensins was demonstrated in a cohort study of African adults, which showed that low Paneth cell α-defensin-gene expression was associated with a higher risk of diarrheal infections (15). Interestingly, although human α-defensins (HD) 5 and 6 are largely confined to the small intestine, they are also secreted in the colon of patients with ulcerative colitis (UC) due to the presence of metaplastic Paneth cells. This is thought to provide an alternative “on-demand” mechanism that provides antimicrobial expression and protection of the gut (14).

Apart from their microbiocidal role in the innate immune system, α-defensins are chemotactic for monocytes, dendritic, and T cells, thereby providing regulation of adaptive immunity via activation and recruitment of adaptive immune cells (4). Furthermore, α-defensins 1–4, known also as human neutrophil peptides (HNPs), enhance the expression of TNF-α and IL-1β in activated human monocytes and reduce the expression of vascular cell adhesion molecule (VCAM)-1 in human endothelial cells activated by TNF-α. This regulation of cytokine production and adhesion molecule expression indicates a potential role of HNPs in modulating inflammatory responses (16).

The antimicrobial function of β-defensins has also been well established. During health, the constitutive expression of human β-defensin (HBD) 1 by epithelial cells prevents microbial invasion by strengthening the intestinal mucosal barrier. The induction of HBD2, 3, and 4 during inflammation or infection may prevent further bacterial entry into an already compromised epithelium and contribute to antimicrobial defense during inflammation at this site (14). Like the α-defensins, β-defensins also exhibit chemotactic activity for immature dendritic cells (DCs) and memory T cells through the CC chemokine receptor type 6 (CCR6), thus promoting adaptive immune responses by recruiting these cells to the site of microbial invasion (17).

The expression of defensins in intestinal inflammation has been extensively studied, especially in the setting of inflammatory bowel disease (IBD) and its two main subsets, UC and Crohn’s disease (CD). There is a consensus in the literature that in UC, which involves superficial inflammation confined to the colonic mucosa, increased HBD2 and Paneth cell α-defensin expressions are characteristic (18). HBD2 levels were elevated in stool collected from children with active UC (median 356 ng/g, range 40–527) compared to healthy controls (median 13 ng/g, range 3–56; P = 0.0002) (19).

In colonic CD, there is attenuated induction of β-defensins as measured by mucosal mRNA (20). In ileal CD however, reduced Paneth cell α-defensin expression was observed in ileal biopsies (21). Kapel and colleagues (19) found only a three- to fourfold increase in fecal HBD2 levels in children with CD as opposed to the abovementioned >10-fold increase in fecal HBD2 levels for UC. This impaired induction of β-defensins in colonic CD has been attributed to low β-defensin-gene copy number (22), while other studies have suggested an association between CARD15/NOD2 mutations and HBD2 deficiency (23). The α-defensin deficiency in ileal CD has also been linked to NOD2 mutations (5). An important finding from the study by Kapel et al. (19) was a positive correlation between fecal calprotectin and HBD2, suggesting that fecal HBD2 is associated with intestinal inflammation.

Irritable bowel syndrome (IBS) is a very common functional bowel disorder in the absence of macroscopic and histologic inflammation, characterized by abdominal pain and altered bowel habits (24). According to the ROME III criteria, IBS can be subtyped into diarrhea predominant (IBS-D), constipation predominant (IBS-C), mixed diarrhea and constipation (IBS-M), and unsubtyped IBS (IBS-U) (25). There is mounting evidence that microbial dysbiosis is associated with IBS, with the implication of small intestinal bacterial overgrowth (SIBO) in its pathogenesis (26). This alteration in gut microbiota is hypothesized to lead to activation of the mucosal innate immune response (26), which has been supported by various studies. In particular, a study by Langhorst and co-workers comparing fecal HBD2 in patients with IBS, active UC, and healthy controls, found that fecal HBD2 was significantly elevated in the IBS group (mean ± SD: 76.0 ± 67.9 ng/g) in comparison to controls (29.9 ± 16.1 ng/g; P < 0.001), although to a lesser extent than patients with active UC (106.9 ± 91.5 ng/g). Their findings support the hypothesis of an activation of the mucosal innate defense in IBS toward low-grade mucosal inflammatory activity (24), which is in turn supported by studies that have found increased mast cells (27) and colonic lamina propria immune cells in patients with IBS (28).

Necrotizing enterocolitis (NEC) is one of the most common gastrointestinal emergencies in neonates (29). Its main risk factors are prematurity and low birthweight (29). The pathogenesis of NEC remains elusive but there is strong evidence that inappropriate bacterial colonization of the neonatal gut plays a role (30). This has been supported by various studies showing that probiotic supplementation in preterm neonates of very low birthweight (<1500 g) reduces the risk and mortality of NEC (31). Like IBS, this aberrant postnatal bacterial colonization may lead to an activation of the innate immune system of the gut.

A study by Jenke and colleagues aimed to assess this intestinal mucosal innate response via HBD2 expression in extremely low-birth-weight (ELBW) infants with NEC (32). They found that infants with moderate NEC had elevated fecal HBD2 concentrations before onset of symptoms, probably reflecting an adequate immune response (32). However, infants with severe NEC showed no increase in fecal HBD2 concentrations before and during the disease. This finding together with a lack of increase in fecal calprotectin concentration and normal villin expression, the latter of which is reduced with epithelial cell loss, suggests a specific deficiency of innate defense activation in ELBW infants rather than an impaired intestinal epithelial barrier, leading to a more severe course of NEC (32).

A study looking at cathelicidin expression in colonic mucosal biopsies of UC, CD, and healthy patients found a significantly higher expression in patients with UC when compared to those with CD (33). However, when inflamed and non-inflamed mucosa of patients with UC or CD was compared, no difference in expression was found. In addition, increased CD4 expression levels (a surrogate marker of infiltrating immune cells) in inflamed CD mucosa were not associated with increased cathelicidin expression (33). These findings suggest the dissociation between cathelicidin expression and inflammation. Other studies have supported this by showing that pro-inflammatory mediators do not upregulate cathelicidin expression, whether in vitro or in vivo (37, 38).

Regulation of cathelicidin expression is unclear. Cathelicidin expression was reported to be regulated by butyrate (a SCFA) through butyrate-induced differentiation of colonic epithelial cells (15, 37). However, Schauber and colleagues (33) showed that butyrate-enhanced colonocyte differentiation and butyrate-induced cathelicidin expression are regulated separately via distinct signaling pathways.

Shigellosis is a major cause of morbidity and mortality in developing countries (39). It is caused by infection with the highly contagious Shigella species, which invades the colonic mucosa causing inflammation that destroys the mucosal barrier (40). The clinical manifestations are the passage of bloody mucoid loose stools, abdominal cramps, rectal tenesmus, and fever (40).

Reduced cathelicidin levels have been observed in gut biopsies of patients with shigellosis (41). It has been suggested that this down-regulation is a strategy by pathogenic microbes to increase their virulence by circumventing host immune defenses. Adjunct therapy with butyrate during shigellosis resulted in enhanced expression of cathelicidin in rectal epithelia, prolonged cathelicidin secretion in stool, and early reduction in inflammation (40).

An increase in fecal lactoferrin levels occurs during intestinal inflammation (46) due to mucosal infiltration and degranulation of neutrophils, providing an additional source of lactoferrin to aid the mucosal innate response (45). Elevated fecal lactoferrin levels have been reported in IBD (47) with a sensitivity of 78%, and specificity of 90% in identifying inflammation in adults with chronic UC and CD (46). In addition, fecal lactoferrin showed good correlation to disease activity (endoscopic and histopathologic) and was 100% specific in ruling out IBS (46). Using an established cutoff point of 7.25 μg/mL for patients with IBD (48), similar findings of elevated fecal lactoferrin in pediatric patients with UC (1880 ± 565 μg/mL) and CD (1701 ± 382 μg/mL) compared to healthy controls (1.17 ± 0.47 μg/mL; P < 0.001) were observed (49). Fecal lactoferrin also correlated well with clinical activity indices and erythrocyte sedimentation rate (ESR) (49).

Osteoprotegerin is best known for its role in bone metabolism. It binds to RANKL and blocks its interaction with RANK, thereby inhibiting osteoclastogenesis (52). In addition, the RANK/RANKL/OPG system has a role in regulating intestinal inflammation by modulation of colonic DCs. In a murine model, exogenous OPG reduced DC survival, eliminating the antigen-presenting cell (APC) for colonic CD4⁺ T cells, thereby reducing inflammation (53).

TNF-related apoptosis-inducing ligand is a member of the TNF ligand superfamily that induces cellular apoptosis. The interaction of OPG with TRAIL during intestinal inflammation inhibits apoptosis of DCs and activated T cells, thereby perpetuating intestinal immune activation (54). This pro-inflammatory effect opposes the above findings of a potent anti-inflammatory effect of OPG when interacting with RANKL (53).

The role of OPG in intestinal inflammation was affirmed by a study of children with newly diagnosed CD that showed elevated serum and intestinal mucosal OPG levels (52). Importantly, fecal OPG was also raised in moderate/severe CD (6463 ± 8691 pg/mL) and mild CD (477 ± 848 pg/mL) when compared to healthy controls (63 ± 0.001 pg/mL; P < 0.0001). It was proposed that the excess circulating OPG was a result of increased mucosal OPG production due to inflammation. In addition, serum and fecal OPG decreased after treatment with exclusive enteral nutrition (EEN). This indicates that fecal OPG can be used as a marker of mucosal OPG expression and intestinal inflammatory severity in CD.

A recent study has provided further insight into the role of OPG in intestinal inflammation. Via in vitro methods, Nahidi and co-workers (55) found that OPG possesses pro-inflammatory properties via its induction of gut barrier dysfunction and secretion of pro-inflammatory cytokines. Their results also provide evidence that OPG, like TNF-α, exerts its pro-inflammatory effects by NF-κB activation.

Cryptosporidiosis is caused by infection with the waterborne protozoan parasite Cryptosporidium (56). It characteristically results in watery diarrhea (56) that is usually self-limited in immunocompetent individuals (57) but may be profuse and prolonged in immunocompromised patients (56).

The “disease-promoting” effect of OPG mediated by TRAIL, as discussed above, was further alluded to in an in vitro study of human ileal mucosal cells infected with Cryptosporidium (57). The results showed that treatment with TRAIL induced epithelial cell apoptosis and reduced parasite numbers. However, giving recombinant OPG blocked these therapeutic effects. Moreover, this study showed an early increase in OPG expression by the infected epithelial cells, suggesting that Cryptosporidium may upregulate OPG to protect against early apoptosis by TRAIL.

S100 proteins, especially calprotectin and S100A12, have been extensively studied in both the adult and pediatric IBD populations. Serum and mucosal levels of both of these biomarkers have been shown to be elevated in children with IBD (64).

Fecal calprotectin levels are also significantly elevated in children with IBD (median 1265 mg/kg) compared to children without IBD (median 30.5 mg/kg; P < 0.0001). A sensitivity of 100% and specificity of 67% was found for fecal calprotectin in identifying children with IBD (cutoff >50 mg/kg for IBD) (65). In addition, multiple studies have shown that fecal calprotectin correlates closely with endoscopic and histological grading of colonic inflammation in both UC and CD (66–68). A positive correlation between fecal calprotectin and clinical activity indices in both CD and UC has also been demonstrated (69) and therefore, fecal calprotectin has been proposed as a useful tool in monitoring disease activity in children with IBD (67).

Together, there is substantial evidence in the literature that fecal calprotectin is a sensitive marker of intestinal inflammation. Multiple studies have shown that fecal calprotectin can differentiate IBD from functional disorders like IBS (69, 70), with validation in children (67, 71). Based on a cutoff of 30 mg/kg, fecal calprotectin discriminated adults with active CD from those with IBS with 100% sensitivity and 97% specificity (72). However, it is not a disease-specific fecal marker and is also elevated in other gastrointestinal disorders like gastroenteritis (73) and colorectal cancer (CRC) (70), as well as during non-steroidal and non-inflammatory drug use (74).

Another potential aspect of fecal calprotectin is in predicting relapse in children with IBD. An elevated calprotectin level in stool was found to be associated with a 13-fold increased risk of relapse in adult IBD patients experiencing remission (75), with another study suggesting that fecal calprotectin is more accurate in predicting relapse in UC than CD (76).

S100A12 is also elevated in stool from children with active IBD (median 95.40 mg/kg; range 6.19–349.9 mg/kg) compared to healthy controls (median 0.69 mg/kg; range 0.39–17.73 mg/kg; P < 0.0001) (77). Moreover, it was found to have a sensitivity of 96% and specificity of 92% in distinguishing children with active IBD from healthy controls when a 10 mg/kg fecal S100A12 was used as a cutoff.

Cystic fibrosis (CF) is the most common life-shortening autosomal recessive disease in Caucasians, with an incidence of 1 in 2500 live births (78). There is evidence that CF predisposes to inflammatory changes not only in the respiratory system but also in the gastrointestinal tract (79).

In comparison to the abovementioned biomarkers, calprotectin has been more widely studied in CF. A study looking at children with CF found elevated fecal calprotectin levels and rectal nitric oxide production in majority of the subjects, indicating that intestinal inflammation is a major feature in CF (79). These values fell significantly after administration of the probiotic Lactobacillus GG, suggesting that the intestinal microbiota plays a role in CF intestinal inflammation (79). This was supported by Werlin and co-workers (78), who used wireless capsule endoscopy (WCE) and fecal calprotectin to investigate intestinal inflammation in children with CF. Fecal calprotectin was elevated only in pancreatic insufficient (PI) subjects, whereas WCE showed a high prevalence of small bowel injury in both PI and pancreatic sufficient (PS) children. The authors suggested that these findings support a “CF enteropathy” that is a primary feature of the CF phenotype and that its inflammatory component (as reflected by fecal calprotectin) changes with the degree of exocrine pancreatic impairment (78).

Nevertheless, it has been suggested that fecal sampling assessing intestinal inflammation in CF may potentially give false positive results due to the cross-reaction of ingested sputum proteins with intestinal inflammatory markers. However, this is likely a minor confounder to the significantly elevated fecal calprotectin detected in patients with CF (80).

In contrast, results from a recent study showed that unlike calprotectin, fecal S100A12 levels were not elevated in children with CF when compared to healthy controls (81).

In active IBD, there is increased intestinal epithelial cell turnover and rapid division (84). Hence, given the relationship of M2-PK to cell division, it has been postulated that fecal M2-PK concentrations are elevated in IBD patients (83). This has been supported by several studies.

Chung-Faye and co-workers (83) found fecal M2-PK to be significantly elevated in 81 adults with IBD and 7 with CRC when compared to 43 with IBS. Using a cutoff of 3.7 U/mL, fecal M2-PK had a sensitivity of 73% and specificity of 74% when used as a marker of organic gastrointestinal disease. Furthermore, M2-PK levels were greater in IBD patients with active compared to inactive disease. Their results also showed a high correlation between fecal M2-PK and calprotectin.

A pediatric study reported similar findings of significantly higher PK immunoreactivity in IBD patients (143.7 ± 24.6 U/g) when compared to healthy controls (1.2 ± 0.4 U/g; P < 0.00001) (82). Using the manufacturer recommended cutoff of 4 U/g, sensitivities of 94.3 and 100% were found for UC and CD, respectively. When a second cutoff of 5 U/g was used, false positives were reduced. However, specificity fell from 97.1% (cutoff of 4 U/g) to 94.3% (cutoff of 5 U/g) and sensitivity for CD fell to 94.1%. Regardless, the high sensitivity and specificity reflect the potential use of the fecal M2-PK test in pediatric IBD.

A more recent multicentre cohort study compared the ability of four fecal markers (calprotectin, lactoferrin, M2-PK, and S100A12) to predict the outcome in severe acute pediatric UC (88). Although all four markers reflected disease severity by their very elevated fecal values, only M2-PK had sufficient ability to predict corticosteroid treatment failure and the need for second-line therapy, presenting the potential for fecal M2-PK testing to be incorporated into clinical practice with further research. However, it was still inferior to the Pediatric UC activity index (PUCAI).

Two studies looking at ileal pouch-anal anastomosis (IPAA) in patients with UC and familial adenomatous polyposis (FAP) who underwent restorative proctocolectomy found that those with pouchitis had significantly higher fecal M2-PK levels (89, 90). Johnson and colleagues (90) found that fecal M2-PK could differentiate between non-inflamed and inflamed pouches with a sensitivity and specificity of 80 and 70.6%, respectively. Moreover, fecal M2-PK levels correlated significantly with disease activity indices, endoscopic and histological appearances, as well as the degree of neutrophilic infiltration (90). These findings were mirrored in the earlier study despite a smaller sample size (89).

Butyrate has anti-inflammatory effects that are mediated by the inhibition of NF-κB (i.e., inhibiting NF-κB nuclear translocation) in human colonic epithelial cells, therefore suppressing the gene transcription of pro-inflammatory cytokines. Histone deacetylase inhibition is the proposed mechanism behind this reduction in NF-κB translocation (93). The anti-inflammatory effects of butyrate have been demonstrated in UC, where butyrate enemas resulted in improved clinical disease activity and histological inflammation (94). The same anti-inflammatory effect was seen in CD, where butyrate reduced the expression of pro-inflammatory cytokines by intestinal biopsy specimens from CD patients (95).

Following the formation of an ileostomy, or after proximal colectomy for conditions such as IBD, where there is surgical diversion of the fecal stream (91), diversion colitis can develop due to the reduction in luminal butyrate levels (96). This results from the loss of butyrate as an energy source for colonocytes as well as a lack of its trophic effects on the colon (91). The anti-inflammatory effects of SCFAs were reiterated in a study which showed that SCFA-irrigation reversed the mucosal abnormalities in patients with diversion colitis (97).

Short-chain fatty acids have been found to have a role in diarrheal disorders, in accordance with their ability to stimulate fluid and electrolyte uptake. A study on cholera in children found that including rice starch in oral rehydration salts (ORS) resulted in faster clinical recovery (98). This was associated with striking increases in fecal bacterial concentrations and SCFA levels with time, indicating that this therapeutic effect might be mediated by SCFA enhancing sodium and water absorption and providing colonocytes with energy (98).

Short-chain fatty acids have also been implicated in the development of antibiotic-associated diarrhea (AAD). A study aimed at elucidating the pathogenesis of AAD found very low SCFA production in AAD patients versus controls (99). It is unclear whether AAD is secondary to impaired colonic fermentation resulting in decreased SCFA-stimulated sodium and water absorption, or the reduction in SCFA production is due to decreased colonic bacterial count (99).

It has been hypothesized that malabsorption of carbohydrates and to a lesser extent, protein, that occurs in CF associated with pancreatic insufficiency, can serve as substrates for fermentation by colonic microflora, thus leading to an increase in SCFA production and excretion in stool (100). Increased fecal SCFA output (50 ± 30 mmol/day) was found in patients with pancreatic insufficiency secondary to chronic pancreatitis unrelated to CF when compared to healthy individuals (10–20 mmol/day) (101). This finding of increased SCFA production in a maldigestive state similar to CF might indicate mechanisms other than pancreatic insufficiency in determining fecal SCFA output in CF patients. Notably, SCFA derivatives have been shown to correct the ΔF508-CFTR mutation in vitro, by correcting the inability of the ΔF508-CFTR protein to traffic to the cell surface membrane and by activating alternate chloride transport pathways (102).