MUCOSAL EPITHELIAL CELLS ARE CRITICAL PLAYERS IN THE HOST'S INNATE IMMUNITY TO CRYPTOSPORIDIUM INFECTION

In addition to providing a natural barrier that limits infection, the gastrointestinal epithelium also plays a critical role in the initial recognition of parasites and the triggering of adaptive immunity. Epithelial cells are equipped with several defence mechanisms to guard against infection by pathogens. Recent studies indicate that epithelial cells express a variety of pathogen pattern recognition receptors (PRRs), such as the Toll-like receptors (TLRs) and nucleotide binding and oligomerization domain-like receptors (NLRs), which recognize pathogens or pathogen-associated molecular patterns (PAMPs) (Kawai and Akira, Reference Kawai and Akira2010). TLRs recognize microbes on the cell surface and in endosomes, whereas NLRs sense microbial molecules in the cytosol. Upon specific pathogen recognition, these receptors recruit adaptor proteins and activate downstream signalling cascades that regulate the activity of nuclear factor kappa-B (NF-κB), mitogen-activated protein kinases (MAPK), or caspase-dependent signalling pathways (Kawai and Akira, Reference Kawai and Akira2010). This activation induces the expression of several adhesion molecules, inflammatory mediators (e.g. cytokines/chemokines), and antimicrobial peptides, initiating innate epithelial immune responses against pathogen infection (Fig. 1).

Fig. 1. TLR4/NF-κB signalling pathway and TLR4/MAPK signalling pathway in gastrointestinal epithelium. Epithelial cells express a variety of pathogen pattern recognition receptors (PRRs), such as TLRs, which recognize PAMPs. TLRs recognize microbes on the cell surface. Upon specific pathogen recognition, these receptors recruit adaptor proteins and activate downstream signalling cascades that regulate the activity of nuclear factor kappa-B (NF-κB), mitogen-activated protein kinases (MAPK), or caspase-dependent signalling pathways. This activation induces the expression of several adhesion molecules, inflammatory mediators (for example, cytokines/chemokines), and antimicrobial peptides, initiating innate epithelial immune responses against pathogen infection.

Activation of the NF-κB pathway through TLRs/NLRs is a common response in many epithelial cells in response to microbial infection. The NF-κB family of transcription factors consists of five members: p50, p52, p65 (RelA), c-Rel and RelB. In most cells, NF-κB exists in a latent state in the cytoplasm bound to inhibitory κBs that mask its nuclear localization signal (Hayden and Ghosh, Reference Hayden and Ghosh2008). Activation of NF-κB causes it to move into the nucleus and regulate the expression of a number of host genes, including miRNAs (Zhou et al. Reference Zhou, Hu, Liu, Gong, Drescher and Chen2009, Reference Zhou, O'Hara and Chen2011). The transcription activation domain (TAD) necessary for the positive regulation of gene expression is present only in p65, c-Rel and RelB. Thus, promoter binding of p65, c-Rel and RelB usually is associated with gene transactivation. Due to their lack of TADs, p50 and p52 usually repress gene transcription, unless they are associated with a TAD-containing NF-κB family member or another protein capable of coactivator recruitment (Poppelmann et al. Reference Poppelmann, Klimmek, Strozyk, Voss, Schwarz and Kulms2005).

After entry into host epithelial cells, Cryptosporidium spp. resides within a unique intracellular but extracytoplasmic niche, separating the parasite from direct interaction with other cell types. Therefore, Cryptosporidium spp. has been classified as a ‘minimally invasive’ mucosal pathogen (Chen et al. Reference Chen, Keithly, Paya and LaRusso2002). Because of the ‘minimally invasive’ nature of Cryptosporidium infection, mucosal epithelial cells are critical to the host's anti-Cryptosporidium immunity. Indeed, a recent study with newborn mice showed similar acute patterns of C. parvum infection in C57BL/6 wild-type and T and B cell-deficient Rag2^−/− mice (Korbel et al. Reference Korbel, Barakat, Di Santo and McDonald2011). Upon Cryptosporidium infection, epithelial cells quickly initiate a series of innate immune reactions, including production of antimicrobial peptides (e.g. β-defensins and LL-37) and release of inflammatory cytokines/chemokines, such as interleukin-8 (IL-8) (Borad and Ward, Reference Borad and Ward2010; Petry et al. Reference Petry, Jakobi and Tessema2010; McDonald et al. Reference McDonald, Korbel, Barakat, Choudhry and Petry2013). Production and secretion of antimicrobial peptides can kill Cryptosporidium or inhibit parasite growth. These cytokines/chemokines of epithelial cell origin can mobilize and activate immune effector cells (e.g. lymphocytes, macrophages and neutrophils) to the infection sites (Blikslager et al. Reference Blikslager, Moeser, Gookin, Jones and Odle2007). Recent evidence implies that the release of epithelial exosomes may be an additional element of epithelial anti-Cryptosporidium defence (Hu et al. Reference Hu, Gong, Roth, Huang, Ward, Zhu, Larusso, Hanson and Chen2013).

NON-CODING RNAS ARE EMERGING REGULATORS IN HOST ANTIMICROBIAL IMMUNITY AND DEFENCE

It has become increasingly apparent that the non-protein-coding portion of the genome is of crucial functional importance for normal development and physiology, as well as for disease (Mercer et al. Reference Mercer, Dinger and Mattick2009). This non-protein-coding portion reflects these RNA transcripts that do not encode a protein product, so-called ncRNAs. ncRNA genes include highly abundant and functionally important RNAs, including transfer RNA (tRNA), ribosomal RNA (rRNA), snRNAs, miRNAs, siRNAs, piRNAs, and lincRNAs (Fig. 2). Among these ncRNAs, miRNAs and lincRNAs have received much attention as newly identified families of regulators in animal and plant cells. miRNAs comprise a large family of ∼21-nucleotide-long RNAs that can bind to the 3′-untranslated region (3′UTR) of mRNAs based on perfect or nearly perfect complementarity. LincRNAs are thought to be longer than 200 nucleotides and have little or no protein-coding capacity. So far, a total of 1048 mature miRNAs and almost 3300 lincRNAs have been identified in humans (Kozomara and Griffiths-Jones, Reference Kozomara and Griffiths-Jones2011; Schonrock et al. Reference Schonrock, Harvey and Mattick2012). Of note, some viruses and parasite species also express miRNAs (Kincaid and Sullivan, Reference Kincaid and Sullivan2012). However, it appears that Cryptosporidium spp. does not have the siRNA machinery and, thus, might not express miRNA molecules (Abrahamsen et al. Reference Abrahamsen, Templeton, Enomoto, Abrahante, Zhu, Lancto, Deng, Liu, Widmer, Tzipori, Buck, Xu, Bankier, Dear, Konfortov, Spriggs, Iyer, Anantharaman, Aravind and Kapur2004). Whether Cryptosporidium spp. expresses lincRNAs has not been investigated.

Fig. 2. Type of human non-coding RNAs. Almost 70% of the human genome is transcribed, and only 2% encode for proteins. ncRNAs are loosely grouped into two major classes based on transcript size: small ncRNAs and lincRNAs.

Accumulating data indicate that miRNAs and lincRNAs are an essential part of the complex regulatory networks that control various cellular processes, including differentiation and fate of cells, as well as immune responses in epithelial and immune cells (Zhou et al. Reference Zhou, O'Hara and Chen2011; Carpenter et al. Reference Carpenter, Aiello, Atianand, Ricci, Gandhi, Hall, Byron, Monks, Henry-Bezy, Lawrence, O'Neill, Moore, Caffrey and Fitzgerald2013). miRNAs are predicted to promote fine-tuning of post-transcriptional regulation to >60% of mammalian protein-coding mRNAs (Liu and Olson, Reference Liu and Olson2010), and have emerged as key post-transcriptional regulators of gene expression. LincRNAs can function in cis, recruiting protein complexes to their site of transcription, thus creating a locus-specific address (Chaumeil et al. Reference Chaumeil, Le Baccon, Wutz and Heard2006), and also in trans to regulate distantly located genes (Huarte et al. Reference Huarte, Guttman, Feldser, Garber, Koziol, Kenzelmann-Broz, Khalil, Zuk, Amit, Rabani, Attardi, Regev, Lander, Jacks and Rinn2010). The recent discovery of lincRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2), which mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests a role for lincRNAs in managing chromatin states in a gene-specific fashion (Rinn et al. Reference Rinn, Kertesz, Wang, Squazzo, Xu, Brugmann, Goodnough, Helms, Farnham, Segal and Chang2007). LincRNAs can be induced in innate immune cells and may act as key regulators of the inflammatory response (Guttman et al. Reference Guttman, Amit, Garber, French, Lin, Feldser, Huarte, Zuk, Carey, Cassady, Cabili, Jaenisch, Mikkelsen, Jacks, Hacohen, Bernstein, Kellis, Regev, Rinn and Lander2009; Carpenter et al. Reference Carpenter, Aiello, Atianand, Ricci, Gandhi, Hall, Byron, Monks, Henry-Bezy, Lawrence, O'Neill, Moore, Caffrey and Fitzgerald2013). Pathologically, lincRNAs have been associated with human inflammatory diseases and malignant and neurological disorders (Huarte et al. Reference Huarte, Guttman, Feldser, Garber, Koziol, Kenzelmann-Broz, Khalil, Zuk, Amit, Rabani, Attardi, Regev, Lander, Jacks and Rinn2010; Carpenter et al. Reference Carpenter, Aiello, Atianand, Ricci, Gandhi, Hall, Byron, Monks, Henry-Bezy, Lawrence, O'Neill, Moore, Caffrey and Fitzgerald2013).

ALTERATIONS IN NCRNA GENE EXPRESSION IN EPITHELIAL CELLS FOLLOWING C. PARVUM INFECTION

miRNAs are initially transcribed as primary transcripts known as pri-miRNAs by RNA polymerase II (RNA pol II) and cropped into ∼70- to 100-nucleotide-long hairpin precursors (termed pre-miRNAs) in the nucleus by the RNAse III Drosha (Lee et al. Reference Lee, Kim, Han, Yeom, Lee, Baek and Kim2004). Pre-miRNAs are actively transported by exportin-5 to the cytoplasm, where they are cleaved by the enzyme Dicer to form mature miRNAs. This cleavage event gives rise to a double-stranded ∼22 nt product composed of the mature miRNA guide strand and the miRNA* passenger strand. The mature miRNA is then loaded into the RNA-induced silencing complex (RISC), while the passenger strand is degraded. The RISC identifies target mRNAs by base-pair complementarity, resulting in mRNA cleavage and/or translational suppression (Winter et al. Reference Winter, Jung, Keller, Gregory and Diederichs2009). The majority of miRNA genes are located in intergenic regions or in antisense orientation to annotated genes, indicating that they form independent transcription units. Approximately 50% of human miRNA genes are expressed from non-protein-coding transcripts. Other miRNAs are located within introns of annotated genes, which may be transcribed as part of their ‘host genes’ (Saini et al. Reference Saini, Griffiths-Jones and Enright2007).

Recent studies indicate that transcription of miRNA genes in epithelial cells infected by C. parvum can be elaborately controlled through various regulatory mechanisms, including transactivation and transrepression by nuclear transcription factors associated with the downstream signalling pathways of TLR/NLRs; in particular, by the NF-κB pathway (Chen et al. Reference Chen, O'Hara, Nelson, Splinter, Small, Tietz, Limper and LaRusso2005). Using an in vitro model of human biliary cryptosporidiosis, a significant alteration in miRNA expression profile has been identified in infected host epithelial cells following infection in vitro (Zhou et al. Reference Zhou, Hu, Liu, Gong, Drescher and Chen2009). The up-regulated miRNAs include miR-23b, miR-30b, miR-30c, miR-125b, miR-15b, miR-16, miR-27b, miR-24 and miR-21; downregulated miRNAs include let-7i, miR-513, miR-98, and miR-214 (Fig. 3). Further exploration has identified that transcription of a subset of miRNA genes is regulated through NF-κB activation in human biliary epithelial cells in response to C. parvum infection (Table 1). Specifically, inhibition of NF-κB activation by SC-514, an IKK2 inhibitor, blocked C. parvum-induced up-regulation of a subset of pri-miRNAs, including pri-miR-125b-1, pri-miR-21, pri-miR-23b-27b-24-1 and pri-miR-30b. Moreover, direct binding of the NF-κB p65 subunit to the promoter elements of mir-125b-1, mir-21, mir-23b-27b-24-1 and mir-30b genes was identified by chromatin immunoprecipitation analysis and confirmed by the luciferase reporter assay using the constructs covering the potential promoter elements of these miRNA genes. The data demonstrated that mir-125b-1, mir-21, mir-30b and mir-23b-27b-24-1 cluster genes were transactivated through promoter binding of the NF-κB p65 subunit following C. parvum infection. In contrast, C. parvum transactivated mir-30c and mir-16 genes in biliary epithelial cells in a p65-independent manner (Zhou et al. Reference Zhou, Hu, Liu, Gong, Drescher and Chen2009).

Fig. 3. The miRNA expression profile in human epithelial cells in response to C. parvum infection. The left panel shows a heat-map of miRNAs that showed changes in expression in H69 cells following C. parvum infection. The horizontal axis indicates samples of non-infected cells (n = 3; Control-1, -2, and -3) and cells after exposure to live C. parvum for 12 h (n = 3, C. parvum-1, -2, and -3). The right panel shows expression of miRNAs in H69 cells following C. parvum infection. Cellular levels of miRNAs were presented as the log2 (Hy5/Hy3) ratios that passed the filtering criteria variation across the samples. P values are from the t’ test. hsa = Homo sapiens (Zhou et al. Reference Zhou, Hu, Liu, Gong, Drescher and Chen2009).

Table 1. Validated targets of miRNAs relevant to innate immunity following C. parvum infection

Abbreviations: C. parvum, Cryptosporidium parvum; HDAC, Histone deacetylase; C/EBPβ, CCAAT/enhancer-binding protein beta; TLR4, Toll-like receptor 4; SNAP23, snaptosome-associated protein of 23 KD; CIS, cytokine-inducible Src homology 2; SOCS4, suppressors of cytokine signalling 4; iNOS, inducible nitric oxide synthase; B7-H1, B7 homologue 1; CX3CL1, chemokine (C-X3-C motif) ligand 1; KSRP, the KH-type splicing regulatory protein; ICAM-1, Intercellular cell adhesion molecule 1; ND, not determined.

In addition to the up-regulation of some miRNAs, NF-κB signalling may also be involved in the down-regulation of miRNA genes in response to C. parvum infection. Transcription of the let-7i gene in biliary epithelial cells in response to C. parvum infection has been reported to be suppressed through promoter binding by the NF-κB subunit p50 along with CCAAT/enhancer-binding protein beta (C/EBPβ) (O'Hara et al. Reference O'Hara, Splinter, Gajdos, Trussoni, Fernandez-Zapico, Chen and LaRusso2010). Furthermore, a recent study showed that C. parvum infection suppressed transcription of the mir-424-503 gene in an NF-κB-dependent manner. Increased promoter recruitment of NF-κB p50 associated with the mir-424-503 gene was observed in infected epithelial cells (Zhou et al. Reference Zhou, Gong, Chen, Miller, Eischeid and Chen2013).

ANTIMICROBIAL MOLECULES FROM EPITHELIAL CELLS CONTROLLED BY MIRNAS FOLLOWING C. PARVUM INFECTION

Classic epithelial cell-derived antimicrobial peptides, such as β-defensin 2 and LL-37, have been shown to have anti-cryptosporidial activity in vitro (Chen et al. Reference Chen, O'Hara, Nelson, Splinter, Small, Tietz, Limper and LaRusso2005; Dommett et al. Reference Dommett, Zilbauer, George and Bajaj-Elliott2005) and may play a role in mucosal anti-C. parvum defence in vivo. Nitric oxide (NO) has been shown to play an important role in epithelial defence against C. parvum in some models (Leitch and He, Reference Leitch and He1994; Gookin et al. Reference Gookin, Chiang, Allen, Armstrong, Stauffer, Finnegan and Murtaugh2006; Nordone and Gookin, Reference Nordone and Gookin2010), while other models have failed to show similar effects (Hayward et al. Reference Hayward, Chmura and Cosyns2000; Lean et al. Reference Lean, McDonald, Bajaj-Elliott, Pollok, Farthing and McDonald2003).

NO production in epithelial cells is mainly regulated by inducible nitric oxide synthase (iNOS) (Pautz et al. Reference Pautz, Art, Hahn, Nowag, Voss and Kleinert2010). NO production in epithelial cells after C. parvum infection is increased through NF-κB-dependent mechanisms, with the involvement of miRNA-mediated stabilization of iNOS mRNA (Zhou et al. Reference Zhou, Gong, Eischeid and Chen2012). Specifically, the RNA stability of iNOS mRNA is enhanced in host epithelial cells following C. parvum infection. Increased stability of iNOS mRNA facilitates the protein synthesis of iNOS, promoting NO production in infected cells. Underlying mechanisms of iNOS mRNA stabilization are associated with the suppression of the KH-type splicing regulatory protein (KSRP), an RNA-binding protein that interacts with the mRNAs that have AU-rich elements (AREs) in their 3′UTR. Binding of these ARE-containing mRNAs by KSRP causes mRNA degradation; thus, KSRP is a key mediator of mRNA decay (Linker et al. Reference Linker, Pautz, Fechir, Hubrich, Greeve and Kleinert2005; Winzen et al. Reference Winzen, Thakur, Dittrich-Breiholz, Shah, Redich, Dhamija, Kracht and Holtmann2007). iNOS mRNA is one of the mRNA molecules regulated by KSRP; there are several AREs within the 3′UTR of iNOS mRNA (Linker et al. Reference Linker, Pautz, Fechir, Hubrich, Greeve and Kleinert2005). Interestingly, KSRP is a target for miR-27b and, thus, transactivation of the mir-23b-27b-24-1 gene in C. parvum-infected epithelial cells through the NF-κB pathway causes translational suppression of KSRP (Zhou et al. Reference Zhou, Hu, Liu, Gong, Drescher and Chen2009, Reference Zhou, Gong, Eischeid and Chen2012). Therefore, NO production in epithelial cells in response to C. parvum infection involves miR-27b-mediated stabilization of iNOS mRNA. Indeed, functional manipulation of KSRP or miR-27b caused reciprocal alterations in iNOS mRNA stability and NO production in infected cells. Forced expression of KSRP and inhibition of miR-27b resulted in an increased burden of C. parvum infection (Zhou et al. Reference Zhou, Gong, Eischeid and Chen2012) (Table 1; Fig. 4).

Fig. 4. Post-transcriptional gene suppression mediated by miRNAs is involved in the fine-tuning of epithelial anti-Cryptosporidium defence. Cryptosporidium parvum infection alters expression profile of miRNA genes in infected epithelial cells. A panel of miRNA genes is transactivated or transsuppressed through the activation of the NF-κB pathway. These NF-κB-responsive miRNAs may modulate epithelial anti-parasite responses at every step of the innate immune network, including feedback regulation to TLR/NF-κB signalling, post-transcriptional regulation of many NF-κB-regulated immune or inflammatory genes, release of epithelial exosomes and production of antimicrobial effector molecules.

It is still unclear whether synthesis of other antimicrobial molecules is regulated by miRNAs. The 3′UTRs of human β-defensin 2 and LL-37 mRNAs are relatively short (105 nt for β-defensin 2 and 67 nt for LL-37) and, therefore, may not be directly targeted by miRNAs. Secretion of mucins in the gastrointestinal tract is an important element of mucosal defence and could protect the host against infection by extracellular stages of C. parvum. Additional epithelial cell-derived molecules, such as serum amyloid A 3 (Saa3) and regenerating islet-derived 3 gamma (Reg3g), have also recently been demonstrated to display antimicrobial activity (Reigstad et al. Reference Reigstad, Lunden, Felin and Backhed2009; Choi et al. Reference Choi, McAleer, Zheng, Pociask, Kaplan, Qin, Reinhart and Kolls2013). Their involvement in Cryptosporidium infection of gastrointestinal epithelium and potential association with miRNA-mediated post-transcriptional suppression has yet to be investigated.

MIRNAS IN PRODUCTION OF CYTOKINES/CHEMOKINES FROM EPITHELIAL CELLS AND MUCOSAL INFILTRATION DURING C. PARVUM INFECTION

Release of inflammatory cytokines and chemokines from epithelial cells following C. parvum infection will trigger adaptive immunity and attract inflammatory infiltration, including NK cells, macrophages and lymphocytes. Based on bioinformatics analysis, approximately 29% of cytokine/chemokine mRNAs have potential target sites for miRNAs (Asirvatham et al. Reference Asirvatham, Gregorie, Hu, Magner and Tomasi2008). Human gastrointestinal epithelial cell lines increase the production of IL-8, chemokine (C-X3-C motif) ligand 1 (CX3CL1, also known as fractalkine), chemokine (C-X-C motif) ligand 1 (CXCL1), prostaglandin E2 (PGE₂), and C-X-C motif chemokine 10 (CXCL10) following C. parvum infection. PGE₂ is catalysed by cyclooxygenase-2 (Cox-2). Notably, many of the mRNAs of these cytokines/chemokines, such as IL-8 and Cox-2, contain AREs in their 3′UTRs (Winzen et al. Reference Winzen, Thakur, Dittrich-Breiholz, Shah, Redich, Dhamija, Kracht and Holtmann2007). As discussed above, binding of the ARE-containing mRNAs by KSRP causes mRNA degradation, and KSRP is a target of miR-27b. Therefore, translational suppression of KSRP through induction of miR-27b in infected cells should influence the synthesis of these ARE-associated cytokines/chemokines. Indeed, stabilization of IL8 and Cox-2 mRNAs was confirmed to be associated with suppression of KSRP and miR-27b induction in cells following C. parvum infection (Zhou et al. Reference Zhou, Gong, Eischeid and Chen2012) (Table 1; Fig. 4).

mRNAs of other cytokines and chemokines may directly be targeted by C. parvum-responsive miRNAs in infected epithelial cells. CX3CL1 is a unique member of the CX3C family, and it binds only to and is the unique ligand of its receptor, CX3CR1 (Stievano et al. Reference Stievano, Piovan and Amadori2004). CX3CL1 is known to function as an adhesion molecule to interact with immune cells that express CX3CR1, including CD4⁺ and CD8⁺ T-cells, NK cells and monocytes (Chakravorty et al. Reference Chakravorty, Cockwell, Girdlestone, Brooks and Savage2002). Increased expression and release of CX3CL1 was demonstrated in cultured human biliary epithelial cells after C. parvum infection (Zhou et al. Reference Zhou, Gong, Chen, Miller, Eischeid and Chen2013). Such induction of CX3CL1 in infected cells depends, at least partially, on the transrepression of the mir-424-503 gene. Both miR-424 and miR-503 can bind to the 3′UTR of CX3CL1 and cause translational suppression. Consequently, transrepression of the mir-424-503 gene in infected cells may facilitate the synthesis of CX3CL1 protein and promote infiltration of CX3CR1⁺ immune cells to infection sites (Zhou et al. Reference Zhou, Gong, Chen, Miller, Eischeid and Chen2013) (Table 1; Fig. 4).

Besides cytokine/chemokine release, C. parvum infection also induces expression of adhesion and costimulatory molecules in host epithelial cells (Zhou et al. Reference Zhou, O'Hara and Chen2011). Intercellular adhesion molecule 1 (ICAM-1) is well known for its importance in stabilizing cell-cell interactions and is critical for the firm arrest and transmigration of leucocytes out of blood vessels into tissues (Muller, Reference Muller2009). Upregulation of ICAM-1 transcription and protein expression was detected in human gastrointestinal epithelial cells following C. parvum infection at both the transcriptional and posttranscriptional levels in vitro (Gong et al. Reference Gong, Hu, Zhou, Liu, Feng, Soukup and Chen2011). At the posttranscriptional level, miR-221 regulates ICAM-1 translation through targeting the ICAM-1 3′UTR (Gong et al. Reference Gong, Hu, Zhou, Liu, Feng, Soukup and Chen2011). Significant down-regulation of miR-221 has been identified in cultured human biliary epithelial cells following C. parvum infection. Functionally, down-regulation of miR-221 results in the relief of miR-221-mediated translational suppression, and, thus, facilitates ICAM-1 synthesis in epithelial cells and inflammatory infiltration of leucocytes during C. parvum infection (Gong et al. Reference Gong, Hu, Zhou, Liu, Feng, Soukup and Chen2011) (Table 1; Fig. 4). Moreover, mucosal epithelial cells can express several co-stimulatory molecules, thus modulating the function of infiltrated immune cells at the mucosal site during infection. B7-H1 is a key member of the B7 family of costimulatory molecules important in regulation of immune response, in particular T cell homoeostasis (Dong and Chen, Reference Dong and Chen2006). Expression of B7-H1 by epithelial cells in response to C. parvum infection is tightly regulated to ensure an appropriate antimicrobial immune response. Many researchers have indicated that B7-H1 protein is usually undetectable, although expression of B7-H1 mRNA is common in many cells, suggesting post-transcriptional suppression. B7-H1 is a target of miR-513; miR-513 targeting may account for the absence of B7-H1 protein in cells under a non-stimulation condition (Gong et al. Reference Gong, Zhou, Hu, Liu, Sosnowska, Drescher, Dong and Chen2010). In addition, down-regulation of miR-513 is required for up-regulation of B7H1 protein levels in human biliary epithelial cells following C. parvum infection, suggesting a role of miR-513 in regulating inflammatory responses through targeting of B7-H1 (Gong et al. Reference Gong, Zhou, Hu, Liu, Sosnowska, Drescher, Dong and Chen2010) (Table 1; Fig. 4).

RELEASE OF EPITHELIAL CELL-DERIVED EXOSOMES: ANTI-CRYPTOSPORIDIUM ACTIVITY AND RELEASE REGULATION

Exosomes represent a specific subtype of secreted membrane vesicles that are ∼30–100 nm in size and are formed inside secreting cells in endosomal compartments called multi-vesicular bodies (MVBs) (Théry, Reference Théry2011). Exosomes are produced by a variety of cells, including reticulocytes, epithelial cells, neurons and tumour cells (Théry, Reference Théry2011). Exosomal vesicles shuttle a wide variety of bioactive molecules, such as proteins, lipids, mRNAs and miRNAs, and thereby traffic molecules from the cytoplasm and membranes of one cell to other cells or extracellular spaces (Smalheiser, Reference Smalheiser2007; Valadi et al. Reference Valadi, Ekstrom, Bossios, Sjostrand, Lee and Lötvall2007). There is increasing evidence that exosomes play an important role in normal physiological processes, development, viral infection and other human diseases (Yu et al. Reference Yu, Harris and Levine2006; Théry, Reference Théry2011). We recently demonstrated that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by C. parvum (Hu et al. Reference Hu, Gong, Roth, Huang, Ward, Zhu, Larusso, Hanson and Chen2013). Intriguingly, released exosomes contain antimicrobial peptides with anti-C. parvum activity, including β-defensin-2 and LL-37. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. A direct binding of exosomes to the parasite surface was observed in cell cultures after exposure to freshly excysted C. parvum sporozoites by scanning and transmission EM. These parasites directly bound by exosomes showed a decrease in viability, suggesting the anti-C. parvum activity of exosomes at physiological conditions (Hu et al. Reference Hu, Gong, Roth, Huang, Ward, Zhu, Larusso, Hanson and Chen2013). Of note, the life cycle of C. parvum, both in vitro and in vivo, has extracellular stages (i.e. sporozoites, merozoites and microgametocytes), and they are likely vulnerable to exosomal binding/targeting (Table 1; Fig. 4).

Interestingly, release of exosomes from infected epithelium following C. parvum infection involves a miRNA-mediated exocytic mechanism (Hu et al. Reference Hu, Gong, Roth, Huang, Ward, Zhu, Larusso, Hanson and Chen2013). Secretion of exosomes is regulated by various stimuli, including the activation of P2X receptor by ATP on monocytes and neutrophils, thrombin receptor on platelets, and TLR4 by LPS on dendritic cells (Bhatnagar and Schorey, Reference Bhatnagar and Schorey2007). Formation of exosomes within MVBs and targeting of tranmembrane proteins involve a complex intracellular sorting network, including the endosomal sorting complex required for transport (ESCRT) machinery (van Niel et al. Reference van Niel, Porto-Carreiro, Simoes and Raposo2006). Fusion of MVBs with plasma membrane is an exocytic process that requires the association of v-SNAREs (from the vesicles) and t-SNAREs (at the membrane) to form a ternary SNARE (SNAP receptor) complex. The SNARE complex brings the two membranes in opposition, a necessary step in overcoming the energy barrier required for membrane fusion (Südhof and Rothman, Reference Südhof and Rothman2009). Cryptosporidium parvum-stimulated release of exosomes involves TLR4/IKK2 activation and the SNAP23-associated vesicular exocytic process (Hu et al. Reference Hu, Gong, Roth, Huang, Ward, Zhu, Larusso, Hanson and Chen2013). Whereas a basal level of exosomal luminal release exists in cultured biliary epithelial monolayers and in the murine biliary tract, a TLR4-dependent increase in luminal release of epithelial exosomes was detected following C. parvum infection. Activation of TLR4 signalling increases SNAP23 expression and enhances phosphorylation of SNAP23 in infected cells. SNAP23 is a target of the let-7 family of miRNAs. Since TLR4 signalling mediates transrepression of the let-7 miRNA genes in C. parvum-infected epithelial cells (Hu et al. Reference Hu, Gong, Roth, Huang, Ward, Zhu, Larusso, Hanson and Chen2013), release of let-7-mediated SNAP23 translational repression facilitates SNAP23 protein synthesis in infected cells, promoting exosomal luminal release from infected epithelium (Hu et al. Reference Hu, Gong, Roth, Huang, Ward, Zhu, Larusso, Hanson and Chen2013) (Table 1; Fig. 4).

In addition, more recent studies have shown that miRNAs are also important components of exosomes. Intriguingly, exosome-shuttled miRNA molecules can be delivered to other cell types through exosomal uptake (Valadi et al. Reference Valadi, Ekstrom, Bossios, Sjostrand, Lee and Lötvall2007). Given the importance of miRNAs in epithelial innate immune responses following C. parvum infection, it would be interesting to determine whether exosomes from epithelial cells also carry miRNAs and thus modulate epithelial-immune cell interactions and epithelial anti-C. parvum defence, via exosomal delivery of miRNAs. Because Cryptosporidium spp. does not have the siRNA machinery, delivery of exosomal-shuttled miRNAs to the parasite may not directly influence parasite biology. Nevertheless, these miRNAs shuttled in epithelial cell-derived exosomes released to the basolateral domain during C. parvum infection may modulate host anti-C. parvum immunity, a process that has been demonstrated in the intestinal epithelium during other mucosal infections (Mallegol et al. Reference Mallegol, Van Niel, Lebreton, Lepelletier, Candalh, Dugave, Heath, Raposo, Cerf-Bensussan and Heyman2007). Given the evidence that exosomes from both immune and non-immune cells positively and negatively modulate the immune response (Robbins and Morelli, Reference Robbins and Morelli2014), the role for basolateral exosomes from epithelial cells in host anti-C. parvum immunity needs further experimental elucidation.

MIRNAS AND FEEDBACK REGULATION OF EPITHELIAL ANTI-C. PARVUM IMMUNE RESPONSES

To carry out a fine-tuning of immune responses in response to infection, epithelial cells have developed multiple strategies for the feedback regulation of intracellular signalling pathways. Several endogenous proteins have recently been identified to counter-regulate intracellular signalling cascades and promote resolution of inflammation, such as Toll-interacting protein and A20 to the TLR and NF-κB signalling (Hayden and Ghosh, Reference Hayden and Ghosh2008). The cytokine-inducible Src homology 2 protein (CIS) and suppressors of cytokine signalling (SOCS) proteins are a family of intracellular molecules that have emerged as key physiological regulators of cytokine responses in many cell types (Yoshimura et al. Reference Yoshimura, Naka and Kubo2007). The best-characterized SOCS family members are CIS and SOCS1-3, which function in a classical, negative-feedback loop and inhibit cytokine signalling by interacting with JAK/STAT signalling cascades (Mansell et al. Reference Mansell, Smith, Doyle, Gray, Fenner, Crack, Nicholson, Hilton, O'Neill and Hertzog2006; Yoshimura et al. Reference Yoshimura, Naka and Kubo2007).

These effector molecules of various intracellular signalling cascades can be targets of miRNAs. Targets of miR-146 include IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) (Taganov et al. Reference Taganov, Boldin, Chang and Baltimore2006). MyD88 has been identified as a novel target of miR-155 (Tang et al. Reference Tang, Xiao, Liu, Li, Zhu, Li, Xie, Zhuang, Zou and Mao2010). Therefore, up-regulation of miR-146 and miR-155 has been implicated as a negative feedback to inhibit TLR/NF-κB signalling in cells following microbial challenge (Taganov et al. Reference Taganov, Boldin, Chang and Baltimore2006; Tang et al. Reference Tang, Xiao, Liu, Li, Zhu, Li, Xie, Zhuang, Zou and Mao2010). However, expression of miR-146 and miR-155 remains unchanged in biliary epithelial cells following C. parvum infection (Zhou et al. Reference Zhou, Hu, Liu, Gong, Drescher and Chen2009). On the other hand, miRNAs down-regulated in cells following C. parvum infection may exert a positive feedback regulation to epithelial cell immune response. Specifically, TLR4 is a target of let-7; thus, suppression of let-7 in cells following C. parvum infection contributes to an enhanced level of TLR4 protein in infected cells, facilitating epithelial antimicrobial defence (Chen et al. Reference Chen, Splinter, O'Hara and LaRusso2007) (Table 1; Fig. 4). In addition, it has been reported that miR-98 and let-7 target CIS (Hu et al. Reference Hu, Zhou, Liu, Gong, Eischeid, Dittman and Chen2009). Cryptosporidium parvum infection induces CIS expression in infected epithelial cells through TLR/NF-κB-suppressed expression of miR-98 and let-7 (Hu et al. Reference Hu, Zhou, Liu, Gong and Chen2010). Induction of CIS expression enhances IκB degradation, promoting NF-κB activation (Hu et al. Reference Hu, Zhou, Liu, Gong and Chen2010) (Table 1; Fig. 4).

CONCLUDING REMARKS

While we have begun to recognize the critical role for ncRNAs in mucosal epithelial cells in the innate immunity against Cryptosporidium infection, our current understanding has been limited to miRNAs. A potential role for other ncRNAs, in particular lincRNAs, is yet to be investigated. The interactions of ncRNAs and their targets are complex. It has been proposed that a single miRNA can repress hundreds of target transcripts, and multiple miRNAs may target the same transcript. New technologies will help to identify ncRNA targeting globally, such as cross-linking-argonaute/RNA immunoprecipitation, proteomic approaches and high-throughput sequencing assays (Ørom and Lund, Reference Ørom and Lund2010; Leung et al. Reference Leung, Young, Bhutkar, Zheng, Bosson, Nielsen and Sharp2011). The development of ncRNA knockouts could greatly advance our understanding of ncRNA-mediated anti-C. parvum immune responses in vivo. ncRNAs may also be critical players in other pathogenic aspects of C. parvum infection, such as the lasting effects in intestinal epithelium. Unravelling the regulatory functions of ncRNAs in antiparasite immunity is still in its infancy, but will likely yield new insights into our understanding of the immunobiology and immunopathology of cryptosporidiosis. Given the importance of adaptive immunity to clear infection, the role for ncRNAs in adaptive immunity may be critical for anti-Cryptosporidium immunity, an under-studied area of Cryptosporidium research.