As reviewed by Parija and Jeremiah, Blastocystis spp. were first described by Brittan and Swayne during the 1849 cholera epidemic in London. To this day, the taxonomic classification of this organism remains controversial in the scientific community (35). In the early 1900s, Alexeieff and Emile Brumpt classified Blastocystis as a harmless saprophytic yeast of the intestinal tract. However, in 1967, Zierdt et al. reclassified the organism as a protist based on its morphology and phenotypic properties (36). Thus, after various observations over the decades, Blastocystis is currently classified as an anaerobic, non-invasive protist that colonizes the mammalian intestinal tract and belongs to the Stramenopiles, a clade of protists that group with algae, diatoms, and water molds (37). It possesses a high genetic diversity and a vast host range, including livestock and humans (38). Epidemiological estimates suggest that more than 1 billion people worldwide are colonized with Blastocystis spp., varying at 0.5%–23.1% in developed countries versus 22.1%–100% in resource-limited countries (39–41). Blastocystis spp. is currently comprised of 28 subtypes (STs), 12 of which are reported to colonize the GIT of humans (ST1–ST8, ST10, ST12, ST14, and ST16), but among them, only ST1–ST4 account for more than 90% of the human infections reported globally, whereas ST5–ST8, ST10, ST12, ST14, and ST16 are rarely found in human stool (42–46). Blastocystis has been detected in the human enteric microbiota in both healthy and disease conditions by different methods such as real-time PCR, amplicon-based NGS, and metagenomics. Its presence within the gut microbiota is associated with a shift in bacterial composition (47). The association between Blastocystis and the shift in microbial diversity and composition, as well as its effects on the host response remain to be determined. Although reports of this parasite are becoming more common in human gut studies, the direct Blastocystis mucosal immune response in the murine model is still under-explored.

Whether Blastocystis is pathogenic under some circumstances, for example, during host immune suppression, remains unclear, largely because screening for the presence of other known microorganisms such as viruses, protozoa, or bacteria has not been systematically performed (48, 49). Some studies have described a positive correlation between Blastocystis colonization and conditions such as irritable bowel syndrome or inflammatory bowel disease in humans (50, 51), whereas the high prevalence of this protist observed in healthy human guts has fueled a separate debate on whether this parasite is non-pathogenic and a commensal of the human GIT. Indeed, a sample cohort of healthy humans in Ireland identified that 56% of individuals were positive for Blastocystis by 18S rRNA PCR (52).

In vitro models suggest that Blastocystis can regulate host immune responses. Colonic epithelial cells exposed to Blastocystis Nand II strain released IL-8 and GM-CSF cytokines (53), possibly triggered by Blastocystis cysteine proteases, which are released by the zoonotic isolate WR1 (ST4) and activate the NF-κB transcription factor (54). In addition, Blastocystis ST7 (B isolate) can evade the immune response by downregulating inducible nitric oxide synthase (iNOS) (55) and degrading human secretory immunoglobulin A (IgA) (56).

Iguchi et al. determined that Blastocystis RN94-9 strain (a subtype 4 isolate from a laboratory rat) is not inconspicuous. Although rats experimentally infected with ST4 RN94-9 cysts largely failed to display pathogenic markers during colonization, such as weight loss, diarrhea symptoms, mucosal epithelium lesions, or inflammatory cell infiltrates (57), they did, however, induce type 1 proinflammatory cytokines such as IFN-γ, IL-12, and TNF-α and cause a mild goblet cell hyperplasia that was associated with an increase in the production of neutral mucins (57). Goblet cells are essential cells for maintaining gastrointestinal homeostasis. They synthesize neutral and acidic mucins to produce and sustain the enteric mucus layer, an innate barrier against pathogens (58). Alterations in the neutral and acidic mucin ratio have also been detected in rats infected by the parasitic nematode Nippostrongylus brasiliensis (59). While this imbalance in the production of neutral versus acidic mucins is thought to change mucus layer charge, whether it influences parasite-mucosa attachment is less clear.

In addition to the activation of a Th1 cytokine signature, Blastocystis modulates the in vivo Th17 cytokine immune response. Th17 immunity is strongly correlated with the clearance of extracellular parasites, inflammation, and auto-immune diseases (60). IL-17, IL-23, and IL-22 cytokines have a prominent role in Th17 subset activation (61). BALB/C mice infected with Blastocystis spp. developed significant increases in their IL-17 and IL-23 levels in the intestinal mucosa (62). In summary, Blastocystis appears to modulate the enteric host immune response by inducing specific Th1/Th17 cytokine production. Hence, colonization by Blastocystis may generate a level of bystander inflammation that could conceivably protect mice from pathogenic infections or make them more susceptible to certain auto-immune conditions. Therefore, studies to understand the kinetics, level of, and persistence of cytokine induction during Blastocystis colonization are needed. In addition, the data thus far investigating Blastocystis colonization in vivo certainly argue for the necessity to screen for the presence of commensal parasites in the gut in experimental workflows in order to gain a better understanding of their role in gastrointestinal disease models.

Tritrichomonas spp. comprise a complex group of neglected gut-dwelling protists that have similar morphology but possess extant diversity. Studies within this group of parasites are limited, largely because of a lack of high-quality reference genomes or methods for axenic culture. Closely related species such as Tritrichomonas muris, Tritrichomonas musculus, and Tritrichomonas rainier have been discovered recently as common commensal protists that establish persistent, long-lived infections that impact the microbiome and mucosal immune homeostasis within the intestinal tract of wild and laboratory mice (63). Colonization of mice with these protists shows little to no evidence of epithelium lesion, blood cell infiltration, or other detectable gut-related disease symptoms, e.g., diarrhea, weight loss, or lethargy. Colonized mice do experience, however, a mild goblet cell hyperplasia and display significant changes in their mucosal immunity, which protect them from GIT bacterial infections but increase their susceptibility to inflammatory bowel-like diseases (64, 65). These studies certainly highlight the importance of screening the microbiota of laboratory mice for Tritrichomonas commensal infection.

In the healthy gut under homeostatic conditions, immune sentinels and taste-chemosensory cells called tuft cells are commonly found in low numbers within the epithelia of the small and large intestine in mice. However, in response to various parasitic infections, tuft cells are known to expand exponentially. For example, enteric non-pathogenic commensal protists, including Tritrichomonas spp., and pathogens such as nematodes (e.g., N. brasiliensis, Heligmosomoides polygyrus, and Trichinella spiralis) and trematodes (e.g., Echinostoma caproni) cause tuft cell activation and expansion, triggering mainly type 2 host immunity involving ILC2s (66–68).

Howitt et al. first described markedly different intestinal tuft cell numbers between specific pathogen-free BIH mice bred “in-house” versus BIH mice purchased from the Jackson Laboratory (JAX). When cecal contents from BIH mice were fed to the JAX mice, intestinal tuft cells proliferated to a level identified in the bred “in-house” BIH mice, and the transmissible component responsible for the phenotypic change was a single-celled protist that they referred to as T. muris (65). Two other contemporaneous studies likewise concluded that this and a related gut-dwelling commensal protist referred to as T. musculus alter immune cell homeostasis within the gut microenvironment, conferring the protection of colonized mice from mucosal bacterial infections at the expense of increasing their susceptibility to colitis and colorectal tumors (60, 67).

The Howitt study showed that shortly after T. muris colonization, tuft cells release the cytokine IL-25 and promote ILC2 activation and the secretion of IL-13, resulting in goblet cell hyperplasia and skewing the immune potential of the gut toward a Th2 response (65). Two years later, Nadjsombati et al. showed that the related parabasalid T. rainier also induced tuft cell expansion, ILC2 activation, and goblet cell hyperplasia in the small intestine of laboratory mice, similar to T. muris (69). Their work established that succinate secreted by T. rainier is a pivotal ligand sensed by the succinate receptor (SUCNR1) expressed on tuft cells and that this was sufficient to initiate a Th2-biased immune response in the small intestine (69). Succinate is a metabolite produced by hydrogenosomes, a mitochondria-like organelle that synthesizes ATP in anaerobic protists, including Tritrichomonas spp (70). This remarkable finding revealed for the first time that a metabolite unique to a eukaryotic microbe was able to directly activate type 2 immune cell responses via a tuft cell-ILC2 sensing and activation circuit (19).

Paradoxically enteric Tritrichomonas spp. infection also activates Th1 and Th17 immune cells and their signature cytokines during mouse colonization, in addition to initiating Th2-biased immune responses in the small intestine. In the large intestine specifically, T. muris stimulates a Th1-biased pro-inflammatory response, increasing IL-12/IL-23p40 and IFN-γ proteins to levels that exacerbate colitis in Rag1^−/− mice post-adoptive T-cell transfer (71). Chudnovskiy et al. also demonstrated a CD45⁺ hematopoietic cell expansion and the secretion of high levels of IgA in the large intestine of mice colonized with T. musculus (T.mu), a T. muris-related species (64). Like T. muris, T.mu also induced mild goblet cell hyperplasia and an exacerbated inflammatory response in colitis and tumorigenesis models. Within the cecal epithelium, high levels of IL-18 were released, the result of activating an ASC-dependent inflammasome, and this was important for the induction of colonic Th1 and Th17 immune responses in T.mu-colonized mice (64). The altered inflammatory state driven by the IL-18 release within the colonic epithelium was sufficient to protect mice from Salmonella typhimurium enterocolitis and established that the presence of T.mu significantly increased anti-bacterial defenses within the enteric mucosa (64). Recent work has identified that T.mu activates both the NLRP1B and NLRP3 inflammasomes and also causes a dramatic increase in luminal extracellular ATP levels (72). T.mu colonization likewise triggered IL-13 by ILC2s in the mucosa, as previously reported by Howitt et al. using T. muris (65). It also altered host glucose homeostasis by increasing gluconeogenesis, as well as increasing free choline production in the gut of infected hosts. The parasite metabolite that triggered this pathway is unknown, but there is evidence for a succinate-independent process (73). Altogether, the data described here emphasize the importance of investigating commensal protists in the gut microbiota in order to devise new therapeutic interventions to control inflammatory, pathogenic, and/or metabolic diseases.

T. gondii is an intracellular protozoan parasite capable of infecting essentially any warm-blooded animal. It is highly prevalent worldwide, with about 1/3 of the human population chronically infected with T. gondii parasites (74). The parasite is a leading cause of infectious blindness and is considered a life-threatening disease among the immunocompromised. It is also capable of causing abortion or birth defects during congenital infection (75, 76). Toxoplasma transmission occurs by oral ingestion of infectious tissue cysts or oocysts; hence, intestinal mucosal immunity constitutes the first line of defense and is one of the most important barriers against T. gondii (13, 77, 78). In the small intestine, T. gondii differentiates into tachyzoites, the rapidly replicating form of the parasite, which ultimately disseminates infection beyond the intestinal mucosa, colonizing other tissues, such as skeletal muscle and the central nervous system, before it differentiates into the bradyzoite form, which establishes latent infection in the form of tissue cysts (13). When in the gut, Toxoplasma alters the mucosal homeostatic balance and induces a cascade of immunological events involving components of both innate and adaptive immunity, characterized by a polarized Th1 immune response that induces high levels of IFN-γ and a collapse of Tregs (13, 79). The heightened inflammatory state has been shown to cause a Crohn’s disease-like enteritis, the result of an IFN-γ-mediated acute ileitis that occurs in some inbred mice (77, 80–83). Type 1 immunity is normally associated with host protection and is necessary for T. gondii clearance. However, in some Toxoplasma murine infection models, a dysregulated Th1 response can occur, which causes irreversible pathological alterations and inflammatory responses that promote severe tissue damage and host death (82–84). Here, we describe the mucosal-associated immune cell types that are induced during T. gondii infection in the gut with reference to well-described parasite factors that regulate the development of the T. gondii-associated mucosal immune response.

Gut epithelial and lamina propria cells are key players in host defense against Toxoplasma infection in the intestine. The gut epithelium is an important physical barrier and functions as the first line of innate defense against the parasite. In response to oral Toxoplasma infection, the gut epithelium releases alarmins IL-18 and IL-33 (both IL-1 family members), which synergize with IL-12 to promote protective IFN-γ-mediated immune responses that regulate T. gondii infection within the ileum. The release of IL-18 is caspase-1/11, ASC, and inflammasome sensor NLRP3- and NLRP1b-dependent (85). IL-18 plays a pivotal role in limiting parasite replication, as mice deficient in IL-18 or IL-18R experience 20–100-fold higher parasite loads and die acutely, compared to infected wild-type mice with the same genetic background (85, 86). It has also been reported that T. gondii actively crosses the gut epithelial barrier by invading, replicating, and lysing epithelial cells (78, 87). This process ruptures the gut epithelium and releases the damage-associated molecular pattern (DAMP) cytokine IL-33, which also synergizes with IL-12 to promote ILC1 production of IFN-γ and CCR2-dependent recruitment of inflammatory monocytes required for resistance to T. gondii (88). T. gondii tachyzoites are also released into the lamina propria compartment. Such collateral damage facilitates the translocation of bacteria across the gut epithelium, which exacerbates the inflammatory response and causes serious gut pathology (78, 89–92).

The presence of IFN-γ driven by T. gondii infection is also responsible for the rapid loss of Paneth cells within the host mucosa and results in a myriad of pleiotropic effects. Paneth cells are epithelial cells that secrete a suite of antimicrobial proteins and peptides in order to sustain mucosal homeostasis (15). In the presence of Toxoplasma, alterations in both membrane and mitochondrial integrity occur in Paneth, cells which activate an mTORC1-dependent cell death pathway (93–95). The loss of Paneth cells is also partially associated with the activation of TLR11 signaling in the gut (93). Likewise, IELs, another epithelial cell type comprised mainly of CD8α T cells, play a critical role in the initiation of tissue damage and immunopathology in response to Toxoplasma infection. The progression of pathological inflammation and necrosis is associated with the expansion of CCR2^hiCD103⁺ IELs in the epithelium, which are principally induced by the presence of the CCL2 chemokine. Interestingly, CCR2^−/− mice experience diminished intestinal inflammation but had higher mortality rates, due largely to a failure to regulate Toxoplasma growth (96). Hence, IELs perform a critical function. Failure to regulate them precisely represents a double-edged sword: IELs secrete anti-inflammatory mediators, like TGF-β, to regulate mucosal inflammation in the GIT but can also expand to produce high levels of IFN-γ, which drives the immunopathology in response to Toxoplasma infection (77).

After crossing the epithelial barrier, parasites reach the LP, where immune cells are abundantly present. In the LP, innate immune cells, including inflammatory monocytes, dendritic cells, and NK cells, are critical players that both initiate and regulate the immune response against T. gondii infection (76). The myeloid compartment, specifically macrophages and dendritic cells, is responsible for sensing parasite antigens (pathogen-associated molecular patterns (PAMPs)) and rapidly respond by secreting IL-12, one of the major cytokines released in response to Toxoplasma infection. IL-12 principally drives the marked IFN-γ production by NK and T cells (97, 98). Among innate immune cells of non-myeloid origin, the role of ILCs during T. gondii infection is not well defined, so it remains unclear precisely how they contribute to the induction or regulation of mucosal immunity against Toxoplasma. Although it is known that ILC1 cells can secrete IFN-γ, the contribution of this cell type to the total aggregated IFN-γ levels during Toxoplasma infection is likely less relevant than that of CD4⁺ T cells. Indeed, López-Yglesias et al. demonstrated that in the absence of ILC1, Tbx21^−/− mice were still able to generate a robust IFN-γ response driven by CD4⁺ T cells (94). Of note, Wagage et al. suggested that RORyt⁺ ILC3 cells limit T-cell responses and pathology during T. gondii infection; however, they showed that ILC3 frequency is significantly decreased in the presence of the parasite (99). More studies on ILCs are required to fully understand their role in oral T. gondii infection models. Likewise, the role of neutrophils, another innate immune cell of myeloid origin, has not been systematically studied, so its role in contributing to mucosal immunity during Toxoplasma infection is not clear. Neutrophils rapidly migrate into the lamina propria during acute infection and can be detected within 3 days post-oral inoculation of T. gondii cysts (100, 101). Coombes et al. reported that tachyzoites preferentially infect infiltrating neutrophils, which function as reservoirs for T. gondii dissemination to other host tissues (102). Further studies are necessary to define the role of neutrophils during Toxoplasma intestinal infection.

One of the most important and well-studied cell types responding to T. gondii infection is DCs. These cells are the main source of IL-12 production and are central for the activation of T cells during both intraperitoneal and oral T. gondii infection. Several studies previously demonstrated that T. gondii proteins can directly activate Toll-like receptors (TLRs) to induce IL-12 production by DCs and macrophages. These include Micronemes 1 and 4 (TLR2/TLR4 agonists) and Profilin (TLR11/TLR12 agonists) (103–107). Although most of these studies were carried out using intraperitoneal infection models, it is known that profilin and TLR11 interactions also activate the intestinal innate immune response during oral infection with T. gondii. Benson et al. showed that mice deficient in TRL11 expression are more susceptible to Toxoplasma infection, the result of a marked reduction in IL-12 secretion and a partial defect in the generation of Th1 cells (108). In addition, recent studies have reported that IL-12—released during Toxoplasma infection—arises mainly from uninfected lamina propria DCs (CD11b⁻CD103⁺) rather than infected ones (109–111). It has been suggested that these cells respond to soluble parasite factors secreted prior to parasite invasion or parasite proteins that have been actively injected into DCs by T. gondii (109–111). Other parasite factors that regulate mucosal immunity include the Type II dense granule protein 15 (GRA15_II), which activates NF-κB within innate immune infected cells and results in elevated IL-12 production, lower parasite burden, and less intestinal inflammation. This event was reported to occur only when the Type I rhoptry kinase 16 (ROP16_I), another parasite factor that is involved in STAT3, STAT5, and STAT6 activation, was heterologously expressed within T. gondii Type II strains (112, 113). Hence, the combined expression of GRA15_II and ROP16_I in a Type II background strain conferred host resistance to acute oral infection by T. gondii. Finally, GRA24 is also released into the host cell cytoplasm to induce IL-12 production by triggering the activation of host mitogen-activated protein kinase p38 (p38 MAPK), which subsequently results in the transcription of the IL-12 gene (114–117). Additionally, different studies reported that there is a large CCR2-dependent influx of inflammatory monocytes into the lamina propria compartment during Toxoplasma infection, and IFN-γ-activated inflammatory monocytes are associated with the control of parasite replication and host resistance (111, 118). These cells are capable of producing IL-12 in the gut during infection, and this helps in the recruitment of protective Th1 cells in a CXCR3-dependent manner (119). Moreover, it is also reported that inflammatory monocytes are critical for T. gondii clearance by IFN-γ-dependent activation of iNOS/NOS2, the release of NO, and the activation of IRGs (immunity-related GTPases), which destroy the parasitophorous vacuole membrane (PVM) to limit parasite replication (92, 120, 121).

As mentioned before, T. gondii infection induces a very strong and polarized Th1 immune response, associated with a high frequency of IFN-γ-producing CD4⁺ T cells in murine models. Although Th1 immunity is required to control parasite replication, by inducing such a strong inflammatory response, in some mouse genetic backgrounds, it also drives a severe, and often lethal, intestinal immunopathology (81, 93, 122). López-Yglesias et al. demonstrated that even in the absence of T-bet, the transcriptional factor signature for Th1 cells, the IFN-γ-producing CD4⁺ T-cell response, is still markedly increased in Tbx21^−/− mice, suggesting that the immunopathogenesis during infection can be driven by a T-bet-independent pathway (94). Additionally, the T. gondii-Th1-induced tissue damage in the intestinal mucosa is associated with the collapse of resident Tregs and a subsequent reduction of IL-10 levels during infection (79). This phenomenon is associated with the conversion of Tregs into Tbet⁺ IFN-γ-producing cells and favors the development of a Th1-driven Crohn’s disease-like immunopathology (79, 123). Finally, Rachinel et al. suggested that the acute ileitis driven by T. gondii is associated with the expression of the parasite immunodominant antigen SAG1 (surface antigen 1), which elicits a CD4⁺ T cell-dependent lethal inflammatory process in the oral B6 mouse model (124). Although Th1 responses dominate during T. gondii infection, a few studies suggest that IL-17A- and IL-22-secreting Th17 cells also play an important role in maintaining persistent levels of antimicrobial peptide production, given that mice deficient in class I-restricted T cell-associated molecule (CRTAM) expression on T cells failed to control T. gondii-driven immunopathology and microbial translocation (125, 126).

Among all the intestinal pathogenic protozoa parasites that will be discussed in this review, it is notable that the area of study regarding T. gondii biology has evolved dramatically during the past few years. In addition, advances in gene editing tools and experimental models are continually contributing to our understanding of important host–parasite interactions that impact host immune responses against T. gondii. However, the role of many parasite factors and their interaction impacting the development of host intestinal immunity remains unknown. Ultimately, further studies are required to comprehensively elucidate the underpinnings of the protective immune response that can be elicited to revert immunopathology and confer a host-protective effect by reducing or preventing T. gondii-driven acute ileitis.

The intestinal extracellular protozoan parasite G. intestinalis (syn. Giardia duodenalis and Giardia lamblia) is highly prevalent worldwide and can infect all mammals, including humans, domestic animals, and wildlife. Giardiasis is considered one of the most prevalent enteric diseases caused by a protozoan parasite, with estimates ranging from 2%–5% to 20%–30% of Giardia-infected people in high and low-middle-income countries, respectively. Approximately 280 million new cases are documented every year worldwide (127–129). Immunocompetent individuals typically resolve the infection spontaneously within a few weeks after exposure, and some people never develop any symptoms (130, 131). However, susceptible individuals often exhibit a myriad of different gastrointestinal symptoms, including abdominal cramps, nausea, and diarrhea (132). These clinical symptoms are frequently associated with the development of malabsorption syndrome and with children’s development and growth impairment, especially in chronic and recurrent Giardia infections (133–135). Recent data suggest that giardiasis is one of the four main contributors to stunting in children (135–137). Transmission of Giardia occurs through the oral–fecal route with the ingestion of parasite cysts present in contaminated water or food (138), highlighting the importance of the intestinal mucosal immune response triggered by Giardia infection, which will be discussed here.

Within the gastrointestinal environment, Giardia cysts release trophozoites, the parasite form that adheres to the epithelial monolayer to colonize the small intestine, especially the duodenum. Trophozoites attach to the microvilli in the epithelium to replicate and complete their life cycle, but this alters mucosal homeostasis and induces several host responses. In mice, Giardia infection is mainly characterized by a protective Th17 immune response, with high levels of IgA secretion, and the presence of IL-17 is associated with parasite clearance (139, 140).

The intestinal epithelium functions as a physical barrier to protect from invading pathogens reaching the deeper layers of the mucosa. Although Giardia trophozoites do not invade the mucosa or submucosa layers in immunocompetent organisms, many studies suggest that Giardia trophozoites attach to the intestinal epithelial cells (IECs) and rupture the epithelial barrier by disrupting tight junction proteins within the IECs, such as claudins and occludin, which increases intestinal permeability, facilitating the entry and spread of enteric pathogens (141–143). In addition to functioning as a physical barrier to avoid parasite invasion in the lamina propria, the intestinal epithelial layer is responsible for producing various chemicals, such as nitric oxide (NO), and antimicrobial peptides that inhibit Giardia replication in the epithelium (144). However, Giardia-produced arginine deiminase (ADI) is thought to reduce the availability of arginine in the gut, which negatively affects NO production by enterocytes (144). In addition, Stadelmann et al. reported that arginine consumption by Giardia is also associated with decreased proliferation of intestinal epithelial cells during infection (145). Epithelial Paneth cells produce and release α-defensins, an AMP that, in combination with NO from nitric oxide synthase (NOS2), acts to control Giardia burden and eliminate infection (146). Giardia infection also upregulates mucus production during intestinal infection (147–149).

Giardia infection results in the rupture of the epithelial barrier, and this event is associated with the release of chemokines that recruit immune cells required for the protective response against Giardia. The epithelial disruption also promotes microbial translocation and the release of parasite antigens within the lamina propria, which induces the activation of recruited and resident myeloid cells (140, 150–152). However, studies regarding the specific role of macrophages and dendritic cells during Giardia infection in mice are sparse in the literature. It is known that DCs are one of the main sources of IL-6, and in the absence of this cytokine, IL-6 knockout mice fail to control Giardia replication during in vivo infections (153–155). It is reported that Giardia can induce a cytokine profile that is less inflammatory, and it seems that the reduction of proinflammatory cytokines is mediated by the activation of TLR2 signaling in DCs and macrophages (156, 157). Paradoxically, infection in mice that lack TLR2 is associated with reduced parasite burden and less Giardia-associated pathology (156). Further studies are necessary to understand the specific role of myeloid cells during Giardia in vivo infection.

Giardia induces the recruitment of mast cells into the small intestine lamina propria, and at the site of infection, these cells degranulate and release histamines and protease-1 (MMCP-1) (158, 159). Li et al. described that MMCP-1 interacts with cholecystokinin (CCK), and this interaction results in more intestinal contractility, which is thought to be associated with cramps, one of the most common symptoms noted by infected people (128, 159). The literature suggests that mast cells are recruited via activation of the complement lectin pathway since mice lacking mannose-binding lectin 2 (MBL2) and complement factor 3a receptor (C3aR) expression are impaired in mast cell recruitment during Giardia infection (160). Additionally, the expression of MBL2 in the intestinal mucosa is dependent on Giardia-induced IL-17 (147).

As mentioned before, Giardia infection induces the differentiation and expansion of IL-17-producing CD4⁺ T cells, which confer a host-protective effect by limiting parasite replication. It is reported that the control of infection is dependent on T cells but independent of Th1 and Th2 immunity (139, 140, 161). However, as is the case for many other pathogens, a protective immune response that limits the Giardia burden may also generate significant pathology. Known sequelae associated with Giardia infection in mice are accelerated intestinal transit contributing to parasite-induced diarrhea, muscle hypercontractility, mast cell activation, and cramping (158, 159, 162, 163). Lastly, activated CD8⁺ T effector cells (CD44^hi and CD69^hi) mediate tissue damage and microvillous shortening, which is responsible for disaccharidase enzyme (sucrase and lactase) deficiency and malabsorption of electrolytes, nutrients, and water (156, 164). The mechanism of CD8⁺ T-cell activation during Giardia infection, however, remains unclear but is thought to be influenced by the intestinal microbiota (156).

While there has been significant progress in understanding the host–parasite interaction and the immune response induced by Giardia, further studies are needed to determine which parasite factors specifically trigger innate immune activation, as well as the signals implicated in Th17 activation within the intestinal mucosa, and how CD8⁺ T cells become activated to induce pathology. Finally, new studies are required to better understand the nature of the protective immune response induced by Giardia, which is associated with a reduced risk of developing a severe diarrheal disease in children, and whether Giardia parasitism may be relevant or harmful in the context of a co-infection with another enteric pathogen.

Cryptosporidium spp. are epicellular protozoan parasites that colonize the gastrointestinal tract of mammals. The species C. parvum and Cryptosporidium hominis cause the majority of human infections globally (165, 166). In immunocompetent individuals, cryptosporidiosis is usually a self-limiting infection, with mild (abdominal pain and diarrhea) or absent symptoms, but this parasite can cause serious diseases in immunodeficient patients leading to severe, life-threatening diarrhea (166, 167). The enteric disease is also the second leading cause of severe diarrheal illness in children under 5 years old, with an estimated 60,000 deaths per year worldwide (168). In addition, it is responsible for affecting the growth and development of children. The limited availability of therapeutic treatments and the total lack of a vaccine represent a challenge for disease prevention (168). Parasite transmission occurs through the oral–fecal route upon the ingestion of infectious oocysts (169). Among the three pathogenic protozoa discussed in this review, Cryptosporidium infection and its associated mucosal immunity are much less explored by the scientific community, mainly because of a lack of experimental tools, including the ability to readily cultivate the parasite life cycle in the laboratory and the limited availability of animal models. Nevertheless, recent advances adapting Cryptosporidium to mice are facilitating new insight and permitting investigation of the intestinal mucosal immune response during in vivo infection.

Within the intestinal lumen, released sporozoites adhere to the epithelial plasmalemma to form a parasitophorous vacuole, a compartment isolated from both the lumen and host cell cytoplasm where parasites replicate and promote colonization of the small intestine. Cryptosporidiosis is characterized by crypt hyperplasia, villous atrophy, and diffuse shortening or loss of brush border microvilli, which results in malabsorption, dehydration, and diarrhea (166, 167). Cryptosporidium initiates an inflammatory process by activating epithelial cells (IECs) and inducing the secretion of “alarmin” cytokines (IL-8, TNF-α, and IL-1β) and chemokines (CCL2, CCL5, CXCL1, CXCL8, CXCL9, CXCL10, etc.) (170, 171). This event triggers the recruitment of effector immune cells to the site of infection and inhibits parasite adhesion to the mucosal epithelium (169–172). Various other experimental studies have concluded that production of β-defensin antimicrobial peptides, as well as cytokines, chemokines, and prostaglandin E2 by IECs, occurs via activation of the TLR2/TLR4/NF-κB signaling pathway during Cryptosporidium infection (171, 173, 174). Moreover, NF-κB activation induces the expression of anti-apoptotic factors that prolongs the life of IECs, promoting parasite replication and the propagation of the infection (175, 176).

The secretion of chemokines by Cryptosporidium-activated IECs is responsible for the recruitment of inflammatory monocytes, macrophages, and NK cells to the site of infection, and the presence of local IL-18 and IL-12 induces a synergistic activation of macrophages and NK cells to secrete high levels of IFN-γ in infected neonatal mice (170, 177). Depletion of NK cells was associated with an exacerbated infection in neonatal mice (178). Furthermore, Choudhry et al. reported that the neutralization of IL-18 during Cryptosporidium infection, after in vivo treatment with anti-IL-18 antibodies, resulted in decreased IFN-γ expression in Rag2^−/−γc^−/− mice (177). Different studies using transgenic mice have shown that deficiencies in either Th1 cytokines, such as IL-12, IL-18, and IFN-γ, or inflammasome components (caspase-1) are associated with a more susceptible or sometimes lethal phenotype to Cryptosporidium infection (179–183). Additionally, a recent study has demonstrated that Cryptosporidium is recognized by the inflammasome sensor NOD-like receptor family pyrin domain containing 6 (NLRP6), which activates enterocytes to release bioactive IL-18 and is required for parasite control (183). Finally, the synergistic secretion of IL-18 and IL-12 by activated enterocytes stimulates ILCs to produce IFN-γ, and this event promotes parasite control by enterocytes (184). Thus, the combination of IL-12, IL-18, and IFN-γ as well as inflammasome activation seem to be associated with a protective mechanism against infection.

Given the fact that IFN-γ suppresses Cryptosporidium infection and controls parasite replication, it has been described that CD4⁺ T cells are also essential for parasite elimination and the establishment of an effective immune response following infection (166). Many studies have reported that neutralization of IFN-γ in Rag2^−/− or SCID mice was associated with increased Cryptosporidium burden, persistent diarrhea, and progress of intestinal pathology, sometimes leading to mouse death (179, 185–189). Intriguingly, McDonald et al. showed evidence that during early Cryptosporidium infection, mice secreted intestinal IL-4. They showed that susceptibility to infection was increased and associated with higher production of oocysts when mice were treated with anti-IL-4 antibodies. Their data suggest that early IL-4 can promote a protective, Th1-mediated mucosal immune response that inhibits parasite development (190). The concomitant neutralization of IL-4 and IL-5 was also shown to increase oocyst shedding in infected mice (191). Lastly, the role of CD8⁺ T cells and B cells during Cryptosporidium infection is less clear. Although it has been reported that they expand during infection, whether they are essential for the clearance of infection remains enigmatic (192, 193). Additionally, while parasite-specific IgM and IgG levels have been shown to increase during infection, they do not prevent the host from secondary infection, although specific antibodies are known to reduce oocyst shedding in a reinfection event (194, 195).

Despite the difficulties of conducting experimental studies in the Cryptosporidium field, knowledge is quickly evolving given the development of new technologies and lab strategies to improve parasite culture, life cycle, and experimental models. These new techniques are allowing previously unanswered questions about the biology of Cryptosporidium parasitism to be explored, including how parasite proteins can be delivered directly to the cytosol of infected host cells (196). Still, many questions remain unanswered, such as what parasite factors are recognized by the host cells that initiate the innate mucosal immune response or what extracellular or intracellular receptors are involved in parasite recognition.