The author opted not to address eosinophil-associated gastrointestinal disorders (EGID) comprising eosinophilic oesophagitis, eosinophilic gastritis, eosinophilic enteritis and eosinophilic colitis as well as gastrointestinal manifestations of hypereosinophilic syndromes. These relatively rare conditions were extensively reviewed elsewhere[83,86,126,127]. The concluding part of this review is focused on two major colorectal diseases: IBD and CRC.
Eosinophils in the pathogenesis of IBD
IBD is represented mainly by two major conditions: ulcerative colitis (UC), which affects exclusively colonic mucosa, and Crohn’s disease (CD), a transmural asymmetrical inflammation that can involve the entire gastrointestinal tract. These diseases are highly prevalent all over the world[128-131]. Both UC and CD are chronic relapsing disorders with complex and not entirely understood mechanisms of development. IBD etiopathogenesis is, however, believed to include genetic predisposition, environmental/microbial impacts, intestinal barrier dysfunction and dysregulation of the mucosal immune system of the gut[6-10,132]. The latter two pathogenetic components can certainly be influenced by eosinophils, the abundance of which, correlating with disease severity, is well documented in the gut mucosa of patients with UC[133-140] and some CD cases[135,137,141,142]. The increased presence of eosinophils in the mucosa of these patients apparently results from an enhanced production of eotaxin 1 in the lamina propria[84,143-145] by colonocytes[84,136], macro-phages[84,145] or B lymphocytes. Furthermore, Manousou et al described an elevated expression of eotaxin receptor CCR3 in colonic biopsy samples from UC rather than CD patients. Although the authors interpreted this finding as a sign of the accumulation of CCR3-expressing T cells, it is likely that lamina propria eosinophils expressing the same receptor could be at least equally responsible for this UC-associated change. Conversely, serum eotaxin levels were reported to surge in active IBD[141,147,148], but the increase looked more pronounced in CD patients compared to UC cases[147,148]. The contrast between UC and CD becomes even more evident in view of different patterns of colonic eosinophil activation described by Lampinen et al[84,136,137,145,170,186,188] and indicating that in disease remission activated eosinophils persisted in the lamina propria of UC, but not CD patients[136,137].
The observed differences in colonic eosinophil presence and activity between the two IBD types are not surprising because it is traditionally accepted that CD pathogenesis, which has a stronger genetic component, is dominated by Th1 cytokine profile (combined with Th17 influences)[149-152], whereas Th2 immune response tends to characterise UC[150-153]. Given recent advances in the understanding of immune response types, involved cell populations and cytokine profiles[123,154,155], this straightforward division may now look simplistic, but the association of eosinophils with Th2 immunity is well proven. Therefore, their role in UC pathogenesis looks more evident and easier to explain.
Before further discussing the role of eosinophils in IBD development it will be useful to briefly address gut barrier-related aspects of disease onset. Although numerous factors admittedly contribute to IBD pathogenesis[6-8,156], the precise mechanism of disease initiation remains elusive. Multiple lines of evidence indicate that in most cases IBD can be triggered by an initial contact of the gut microbiota with the mucosal immune cells followed by the development of inadequate immune responses. It is probable that preconditions for this initial contact are associated with functional deficiencies of the protective barrier of the gut mucosa, and they are likely to differ between CD and UC. Indeed, the loose mucus layer of the small intestine may be easily penetrated by microorganisms that then directly contact the epithelium, particularly mucosal M-cells[102-104] of the ileum. Such events can be facilitated by α-defensin deficiency caused by Paneth cell dysfunction that is frequently observed in CD patients, including those genetically predisposed to the disease[157,158]. In contrast, M-cells in the normal colonic mucosa can hardly be reached by microbiota since two layers of mucus, especially the dense inner one[93-95], exclude any bacterial contact with the epithelium (Figure 2B). The protective role of the inner mucus layer is well illustrated by spontaneous colitis development in MUC2-deficient mice[159,160]. Bacterial penetration through the inner mucus layer was observed in UC patients, but it is not entirely clear what triggers the initial change of inner mucus layer properties. Mouse models suggest MUC2 secretion deficiency[159,160] or goblet cell depletion as probable causes, and goblet cell numbers in UC patients, indeed, tend to be reduced. Some authors believe that unresolved endoplasmic reticulum stress and the unfolded protein response are early events leading to goblet cell disfunction and mucus layer impairment in UC[164,165]. Altered eosinophil behaviour may well be associated with these phenomena, but this possibility remains to be investigated. Alternatively, the inner colonic mucus layer can be primarily damaged by mucus-degrading bacteria of the gut lumen. The latter process may potentially be modulated by dietary factors since it was experimentally demonstrated that dietary fibre deficiency leads to switching of the gut microbiota on using mucus glycoproteins as a nutrient source and eventual erosion of the mucus barrier. All the pathogenetic components discussed above may contribute to IBD initiation in humans, but further research in the area is obviously needed.
Whatever scenario causes IBD initiation, there is little doubt that bacterial antigen interaction with the gut-associated lymphoid tissue triggers complex cascades of inadequate immune responses leading to disease development. In these circum-stances, activated eosinophils present in the lamina propria predominantly start acting as effector cells, excessive protective response of which can cause serious damage to the host through several mechanisms. Activated eosinophils accumulating in the gut of IBD patients[133-142] have an extended lifespan, and their degranulation leads to a massive release of both cytotoxic granule proteins and pro-inflammatory cytokines. Gut epithelium is one of the key targets of cytotoxic eosinophil proteins as it was demonstrated that MBP alters colonic epithelium barrier function. Another mechanism of eosinophil contribution to colonic barrier dysfunction in UC involves muscarinic receptors expressed by these granulocytes. Wallon et al showed that cholinergic signals received by the muscarinic receptors caused corticotropin-releasing factor (CRF) production by eosinophils, and CRF induced degranulation of neighbouring mast cells that led to an increase in mucosal barrier permeability. Concerted action of eosinophils and mast cells may have multiple endpoints in IBD as the both types of cells are important sources of cytokines comprising TNFα that, intriguingly, induces M-cell appearance in the mouse colon during inflammation. Moreover, cooperation between eosinophils and mast cells was demonstrated in CD-associated fibrosis development at later stages of the disease. Inflammation intensity can also be aggravated by human gut eosinophils through blocking anti-inflammatory interleukin 22 (IL22) by overproducing IL22-binding protein in both CD and UC patients.
Interactions of gut eosinophils with other immune cells, especially lymphocytes, are very complex and poorly investigated in IBD patients. There is no room for discussing all of them here, but crosstalk between eosinophils and neutrophils deserves to be mentioned. Neutrophils are not present in the lamina propria in the normal conditions but are rapidly attracted there when inflammatory response develops (Figure 3). It is believed that chemokines, especially CXCL8, produced by gut epithelium in inflammation trigger neutrophil chemotaxis. Interestingly, eosinophil impact in this process is now becoming evident since they can synthesise CXCL8. Also, eosinophils were demonstrated to cooperate with colonic epithelium in producing a wider range of neutrophil chemoattractants. In conclusion of discussing the role of lamina propria eosinophils in IBD it needs to be noted that the presented facts are mostly related to human disease. There is a considerable body of additional information obtained in murine models which has been reviewed elsewhere.
Figure 3 Schematic representation of human colonic mucosa and overlaying mucus during inflammatory bowel disease (ulcerative colitis) flare-ups.
Rapid influx of neutrophils results in severe neutrophil infiltration of the lamina propria. Further massive transepithelial migration of neutrophils and other immune cells (especially eosinophils in ulcerative colitis) eliminates mucus layer structure, enables bacterial contact with the epithelium, causes epithelial cell death, ulcer formation and bleeding. Mucus infiltration with neutrophils and eosinophils is accompanied by abundant ETosis and release of both granule proteins and free eosinophil granules. Active inflammation also induces M-cell appearance in the epithelium overlaying lymphoid follicles. Cell images correspond to those used in Figure 2. Erythrocytes are presented by red circles. Small red dots correspond to free eosinophil granules.
Until recently, pathogenetic mechanisms of IBD were considered only at the level of events occurring within the gut wall. However, it is now becoming evident that gut mucus layer presents another, poorly investigated but potentially highly important, battlefield for innate immune responses. Although massive neutrophil influx to the mucosa, often leading to crypt abscess formation, is recognised as a hallmark feature of IBD, the importance and mechanisms of immune cell migration through mucosal epithelia remain poorly understood and insufficiently investigated[179,180]. Transepithelial neutrophil migration involving a chain of molecular events that include initial attachment to the basal surface of epithelial cells, movement through paracellular space (passing by desmosomes, adherens junctions and tight junctions) and eventual contact with the apical membrane of the epithelium is relatively well understood[179,180]. Moreover, it is known that following transmigration neutrophils release MMP9-rich microparticles that disrupt epithelial junctions facilitating further transmigration. Rapid eosinophil migration through tracheobronchial epithelium in experimental conditions was also reported, but remains unexplored in the gut. The process is, thus, well-defined, but the final destination of cells crossing the epithelial barrier remains obscure, being often indicated simply as “gut lumen”[179,180]. Neutrophil-associated markers of inflammation are, indeed, easily detectable in faeces of most patients with active UC and CD, as the popularity of faecal diagnostic tests, particularly stool calprotectin, proves[183,184]. Similarly, the presence of eosinophil markers in stool or colorectal perfusion fluid of IBD patients was repeatedly reported[184-188]. It is, however, apparent that any cells or biomolecules leaving gut epithelium surface should first enter mucus barrier already discussed above, and only its occasionally separated fragments can be incorporated into the faecal matter. The author of the present review previously hypothesised that colorectal mucus retains these highly informative cells and molecules and can be conveniently used for diagnosing colorectal diseases. Studies of our group convincingly demonstrated the abundance of inflammatory cells, predominantly neutrophils, in colorectal mucus collected from IBD patients either intrarectally[190,191] or non-invasively[192,193]. Common eosinophil presence in this material was also noted[191,193], especially in UC patients, and dramatically increased EDN levels were determined[191,194], typically with higher values in UC compared to CD. Detailed cytological analysis of these samples demonstrated that colorectal mucus from patients with active IBD commonly contains not only huge amounts of neutrophils, but also eosinophils, macrophages, erythrocytes as well as occasional plasma cells, lymphocytes and basophils. Most of these cells are viable and functionally active as our frequent observations of phagocytosis by neutrophils and macrophages indicate. Furthermore, it appears that signs of ETosis were also present in colorectal mucus from IBD patients. These findings allow hypothesising that gut mucus acts in IBD as a unique additional milieu, where immune responses expand from the mucosa. It is apparent that the abundance of active cells, especially granulocytes releasing contents of their granules, considerably loosens the inner mucus layer in the colon, thus making it both permeable for gut microbiota and facilitating further immune cell transmigration and movement through the mucus. These circumstances should favour antibacterial activity of the effector cells, but extensive collateral damage of host epithelium is highly likely. ETosis exerted by neutrophils, eosinophils and other immune cells[62-72] may be especially important in this context. Figure 3 reflects the author’s opinion on the extent of inflammatory process in the human gut.
Protection from invading microbiota appears to be the main biological aim of ETosis since histones enveloping released DNA are antibacterial, and the addition of DNA strands may increase mucus viscosity, thus mechanically compensating for MUC2 degradation. Further antibacterial action is provided by granule proteins of both neutrophils[196,197] and eosinophils (Table 1). However, ETosis in the mucus, especially combined with the release of intact eosinophil granules, can seriously damage enterocytes or colonocytes. Eosinophil-derived extracellular DNA traps have already been shown to injure airway epithelium in chronic obstructive pulmonary disease and chronic rhinosinusitis, where MBPs released from specific granules were especially toxic. This phenomenon is still poorly investigated in relation to UC and CD, but interest in IBD-associated ETosis is emerging. In addition to our results discussed above, NET presence has been demonstrated in biopsy samples from IBD patients[201-203], notably within crypt abscesses, i.e., beyond the mucosa. Although the significance of immune responses occurring within colorectal mucus remains to be elucidated, it is impossible to exclude that eosinophil-generated extracellular DNA threads, loaded with entrapped specific granules that release cytotoxic cationic proteins, can cause a continuing colonocyte damage leading to sustained ulceration. This so far unexplored mechanism may constitute an important pathogenetic factor in UC and, to some extent, in colonic CD. On the other hand, it should not be forgotten that transepithelial migration of immune cells is a major factor in mucosal disease resolution, as demonstrated for airway diseases[204,205]. Indeed, the presence of both inflammatory cells and biomarkers associated with them significantly decreased in colorectal mucus samples from successfully treated IBD patients. It is, therefore, probable that gradual distal movement of colorectal mucus creates favourable conditions for eliminating dead or obsolete immune cells from the surface of colonic epithelium if inflammation is successfully resolved.
The presented analysis of literature on eosinophil impact in IBD pathogenesis reveals that eosinophils are closely involved in this process through regulatory activities, interactions with other cells and tissues and effector functions that can often be excessive and damage the host. The impact of eosinophils appears to be especially important in altering the structure and protective functions of the mucosal barrier. The author also tried highlighting an interesting new research direction related to exploring poorly investigated immune responses occurring in the protective mucus layer of the gut. It is, however, apparent that our understanding of IBD pathogenesis, including eosinophil participation in it, remains fragmentary and needs further thorough investigation. For this reason, it would be premature to speculate on possible therapeutic interventions targeting eosinophils in IBD.
Eosinophils in the pathogenesis of CRC
CRC is one of the most frequent oncological conditions with estimated global figures of 1801000 new CRC cases and 861700 deaths due to this disease in 2018. Although there are many good reviews addressing various aspects of CRC pathogenesis[11-15], possible role of eosinophils in this process is usually overlooked despite the existence of reports deserving attention and briefly discussed below.
Eosinophil infiltration is often observed in malignancies, but for different tumours it was reported as either prognostically favourable or unfavourable[3,4,207-210]. Nevertheless, the presence of eosinophils in CRC patients is strongly linked with a decreased disease risk, better prognosis and extended patient survival. Indeed, elevated blood eosinophil counts were associated with a decreased CRC development risk as well as better prognosis[212-214]. Eosinophil infiltration of colorectal tumours is a common phenomenon, and higher numbers of infiltrating eosinophils detected both in the tumour tissue[215-217] and peritumourally[218-220] were repeatedly shown to be prognostically favourable. Despite these seemingly cogent findings, the quoted descriptive clinical studies could not provide any direct evidence of anti-cancer eosinophil action, and possible mechanisms of CRC growth inhibition by eosinophils remain poorly understood.
Tumour-associated inflammation is currently recognised among hallmarks of cancer. Eosinophil accumulation accompanying inflammation-related cancer cell death and proliferation in CRC is one of its components that probably reflects Th2 immune responses enhanced at the expense of Th1 immunity. Besides, eosinophils are likely to stimulate tissue remodelling and tumour-related angiogenesis[3,221]. The latter MBP-modulated effect may in theory promote tumour growth, however only non-cytotoxic MBP concentrations enhanced angiogenesis in vitro, and significant eosinophil infiltration in CRC is likely to produce high MBP concentrations. Ellyard et al argued that some components of Th2-driven inflammation in cancer can be associated with anti-tumour activity of CD4+ Th2 cells collaborating with tumour-infiltrating granulocytes, especially eosinophils that exert regulatory functions. In any case, it is now becoming clear that there are several mechanisms driving eosinophil attraction to tumours and defining their influence on malignant tissue. In particular, it was demonstrated in vitro that eosinophil chemotaxis could be induced by necrotic, but not viable cells of neoplastic intestinal epithelium. Experiments in a xenograft mouse model indicated that eosinophil infiltration developed rapidly, involved mostly tumour necrotic areas or capsule regions and was not associated with the presence of CD4+ T cells, thus suggesting that eosinophil chemotaxis depended on tumour-derived factors, such as damage-associated molecular pattern molecules (DAMPs) including the nuclear protein high mobility group box 1 (HMGB1). Notably, the presence of cell-free cytotoxic MBP was confined to the necrotic areas of tumours. There are also reports describing expression of eosinophil attractant ecalectin (variant of galectin-9) by human colorectal carcinoma cell lines and of eotaxin 1 in tumours resected from CRC patients. The latter phenomenon was recently investigated further in a mouse model by Hollande et al, who found that IL33 expressed by tumour cells induced eotaxin 1 production that led to eosinophil recruitment and degranulation-dependent suppression of tumour growth. Interestingly, eotaxin 1 concentration is negatively regulated by serine protease DPP4, and treatment with DPP4 inhibitor sitagliptin was shown to result in an increase in eotaxin 1 level. Sitagliptin is a drug already approved by the US FDA for hyperglycaemia treatment, and it may potentially be re-purposed as a new anti-tumour agent promoting tumoricidal action of eosinophils[228,229]. Coming back to IL33, its role as an influential modulator of early stages of eosinophil development, was noted in the beginning of this review, but it is also a potent eosinophil activator stimulating their degranulation. Moreover, it was reported that IL33-deficient mice had gut microbiota dysbiosis and were highly susceptible to both colitis and colitis-associated cancer, which could also be related to impaired eosinophil-driven responses. Hence, multiple parallel pathways are likely to be involved in generating eosinophil infiltration of colorectal tumours and direct killing of malignant cells, a phenomenon already proven experimentally. In vitro studies by French investigators assessing tumoricidal activity of eosinophils against Colo-205, a human colon carcinoma cell line, have shown that this effect was mediated by eosinophil-produced ECP, TNFα and proteolytic granzyme A. The same group later reported that eosinophil attachment to Colo-205 cells depended on interaction between adhesion molecules LFA-1 and ICAM-1 that was upregulated by IL18. Direct tumoricidal effect of tumour-infiltrating degranulating eosinophils was also observed in model experiments using genetically modified mice. It is, however, obvious that the tumoricidal action of eosinophils in vivo may involve multiple interactions with other immune cells. Notably, there is evidence that eosinophils can promote either Th2 or Th1 immune responses, depending on varying cytokine profiles[86,235]. Carretero et al have recently shown in experiments with xenograft-bearing mice that tumour-homing eosinophils secreted chemoattractants that guided CD8+ effector T cells to tumours, eventually causing tumour rejection. Thus, it is apparent that different immune response scenarios can be involved in anti-CRC action of eosinophils.
Completing this final section of the review, the author is tempted to briefly mention already discussed ETosis as another possible, but hitherto poorly investigated factor in CRC. One interesting link here is provided by recently published results implicating inflammation-induced ETosis in extracellular matrix remodelling and awakening of dormant cancer cells. As tumour-associated inflammation is a characteristic feature of CRC, and significantly increased extracellular trap formation in these tumours is now proven[238,239], it looks probable that previous reports of CRC-associated increase in the amount of DNA in stool[240,241] or on the surface of colorectal mucosa[190,242] at least partially reflected abundant ETosis occurring within mucus layers contacting tumour surface. In essence, cellular presence in the mucus overlaying colorectal tumour surface is quite similar to that depicted by Figure 3, the abundance of exfoliated malignant cells being the only major difference. Although today there is no published evidence of eosinophil contribution in CRC-related ETosis, this evidence is very likely to emerge soon. In contrast to anti-cancer effects of eosinophils in fully developed tumours, which were discussed above, it is impossible to exclude carcinogenicity of eosinophil-derived DNA traps loaded with highly cytotoxic released granules damaging colonic mucosa. Such a carcinogenic action could be involved at early stages of CRC development, especially in IBD-associated context[243,244]. Conversely, eosinophil-driven ETosis may prevent further tumour expansion at later stages of advanced tumour growth.
The presented analysis of literature establishes a link between eosinophil presence and favourable CRC prognosis, but functional versatility of these multifaceted cells may comprise both anti-cancer and tumour-promoting features. Only several experimental studies addressing eosinophil roles in cancer could be highlighted, however it already becomes transparent that alternative mechanisms involving both direct effector action of eosinophils and complex cooperation with other immune cells, especially T lymphocytes, can be engaged in different circumstances. This fascinating area still poses numerous unanswered questions requiring further intense investigation.