Abstract: The apicomplexan parasite Cryptosporidium is well-known for its capability to induce cryptosporidiosis, a severe diarrheal disease in human and animals. Cryptosporidium can also be a potential pathogen in human for cancer progression, particularly colorectal cancer. This review was designed to outline the information about the life cycle of the Cryptosporidium, the consequences of Cryptosporidium infection into the response mechanism in immune compromised host and finally the regulation of oncomiRNAs and tumor suppressor miRNAs upon Cryptosporidium infection. Host-Cryptosporidium interaction caused alteration of expression of a series of microRNAs or miRNAs as a result of controlling defense mechanism. Regulation of miRNAs in the infected cells may be identified as possible biomarkers in cancer progression. Upregulation of oncomicroRNAs or oncomiRNAs and the downregulation of tumor suppressor miRNAs in the host epithelial cells due to the Cryptosporidium infection may lead to cancer initiation on human.
INTRODUCTION
Cryptosporidium parvum (C. parvum) is a specie of genus Cryptosporidium, commonly known water borne and food borne parasite1-4. Even though the parasite is ubiquitously found in water, livestock and even in vegetables planted in wastewater, the harmful effect of Cryptosporidium is often being neglected5. It has been reported that Cryptosporidium can potentially cause cryptosporidiosis in human and animal6-8. Commonly, cryptosporidiosis is gastrointestinal abnormalities aroused after Cryptosporidium infection which is characterized by short-term or long-term diarrhea with abdominal pain9,10.
Apart from gastrointestinal or colorectal epithelial cell lines, the parasite was also observed to infect the epithelial cells of biliary and pulmonary area11,12. Cryptosporidium infection possesses a nature of minimal invasion and shows a critical innate immune response against infection to the host defense mechanism. Hence, there is a high possibility of alteration of gene expression in infected host cells13,14. Therefore, Cryptosporidium species infectivity is one of major concerns in today’s parasitological research.
Cryptosporidium belongs to the phylum apicomplexan which is particularly pathogenic in immune compromised patients. Several species of Apicomplexa are found to be responsible for introducing diseases in human15 because of their ability to introduce a complex mechanism in the host cells in order to maintain their maximum growth and perseverance16. Infection of Cryptosporidium on epithelial cells leads to activation of innate and adaptive immune response mechanism within the host cells. Cryptosporidium was found to interfere with the signaling pathways of cytokine production and apoptosis pathway along with other and change the host cell defense mechanism, thus alter the immune system15. Nuclear factor kappa B (NF-κB) signaling pathway is the first line defense mechanism of epithelial cells in host to encounter Cryptosporidium infection17,18. List of numerous genes, cytokines or chemokines are involved in the process of NF-κB activation including regulation of microRNAs19. Interestingly, NF-κB pathway is considered as a mastermind player in the metastasis, initiation and development of human carcinogenesis20.
The interrelation between cancer and Cryptosporidium was observed earlier, although, there was very little interest on that research initially. However, a couple of research work has been carried out later where evidence of the engagement of Cryptosporidium with colorectal patients has been established. The prevalence of Cryptosporidium was observed in immune compromised patients, where 12.6% of the 87 colorectal cancer patients’ faecal samples were Cryptosporidium positive21. An in vivo study on mice upon Cryptosporidium infection observed that infection leads to polyps and adenocarcinoma in the gastrointestinal tract; hence Cryptosporidium has been identified as a possible agent to trigger the colorectal neoplasia22,23.
Regulation of microRNAs has extensively been reported to be the controller of the onset and progression of human cancer24. Infection of Cryptosporidium to the human epithelial cell lines exhibits alteration of wide range of microRNAs expression profile. Several in vitro studies showed that Cryptosporidium infection on cell lines caused upregulation of oncomiRNAs and simultaneously downregulates tumor suppressor miRNAs3,7,25-42 (Table 1).
The rationale for this review is to understand subsequent mechanisms upon Cryptosporidium infection on epithelial cells. The elevated immune response causes the over expression of oncomiRNAs and simultaneously downregulation of tumor suppression miRNAs that enhanced the risk of initiation and progression of cancer. This review established the dysregulation miRNAs expression, one of the most important biomarkers of cancer regulation.
LIFE CYCLE OF CRYPTOSPORIDIUM
Cryptosporidium is referred to as a monoxenous organism that requires one host to complete life cycle. As a monoxenous organism the life cycle of the Cryptosporidium is completed within a host. Hosts apart, while remaining in the environment; the parasite exists as oocysts with thick outer wall which protect the organism in variable (high or low) temperature or such other environmental factors43. In desiccation the oocysts become inactivated, but the infective characteristic of the oocysts remain for several months even they pass through moist or cool climates44.
Usually the sporozoites are attached to the host epithelial cells. Once Cryptosporidium reach to the intestine, excystation of oocysts is triggered by different gastrointestinal conditions such as temperature, pH etc.45. Excystation of oocyst causes the release of four sporozoites out of the shell into the gastrointestinal tract. Subsequently the attachment of the sporozoites occurs to the host epithelial cells. A parasitophorous vacuole is formed at the ileocecal junction, because of the invasion of sporozoites. The sporozoites invade onto the ileocecal junction, which results the engulfment of the sporozoites by the epithelial cell and thus a parasitophorous vacuole is formed. Parasitophorous vacuole is not only a host of the Cryptosporidium, but also, the existence of a number of different protozoan parasites may occur within it, but interestingly, there is a unique structural feature named ‘feeder organelle’ is contained in Cryptosporidium, due to which the cytoplasm of the parasite is separated from the cell46.
| Table 1: | Regulation of miRNAs on Cryptosporidium-human epithelial cell line interaction and their associated pathway/targeted genes |
The shape of the sporozoite itself starts to be more spherical gradually once the feeder organelle develops, later on, a trophozoite is formed. By asexual reproduction cycle, the trophozoite changes to Type I meront may either develops into a Type II meront or the Type I meront may produce 6-8 merozoites47. The redeveloped merozoites immediately infect the host again and join into a recycling asexual reproduction cycle. Some, as usual tend to develop to Type II meront48.
On the other side, the Type II meronts start sexual production by releasing a set of four merozoites. Merozoites, which have released from Type II meronts are differentiated into macrogamonts. Also, after invasion the merozoites form microgamonts which further develop multi-nuclei to produce microgametes. Fertilization of the macrogamete is performed by the penetration of the free microgametes and thus a zygote is produced.
By the meiotic cell division process, four sporozoites are differentiated from one zygote. The sporozoites developed oocysts and the oocysts come out from the lumen. Two different types of oocysts namely, thin-walled oocysts and thick-wall oocysts of Cryptosporidium are produced inside lumen48. The oocysts with thin cell wall released sporozoites of Cryptosporidium directly into the lumen to infect onto the host cells. Thick-walled Cryptosporidium oocysts come out to be mixed into the faeces and have the capability to immediately infect the host cells or the faeces bring it into the environment48.
IMMUNE RESPONSE UPON CRYPTOSPORIDIUM INFECTION
The epithelial cell lines are activated both adaptive and innate immunity reactions to encounter Cryptosporidium infection18,49. Upon infection, host epithelial cells expressed several types of pattern recognition receptors (PRRs) including and nucleotide binding oligomerization domain-like receptors (NLRs) and Toll-like receptors (TLRs)19,50. Associated adaptor proteins were brought into the site by the expression of these receptors and thus trigger the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways51.
Innate immune reactions begin immediately after infection of C. parvum onto the epithelial cells. At the early stages, the inflammatory chemokines or cytokines are release due to inflammation caused by infection. Furthermore, the antimicrobial peptides are produced and the expression of adhesion molecules occurs52. Primarily the antimicrobial peptides (e.g., β-defensin 2 and nitric oxide) attempt to execute the Cryptosporidium or to inhibit its normal growth activity32. However, it is necessary to function PRRs to recognize pathogens and activate NF-κB signaling pathway in order to start up primary epithelial responses against infection17,18.
Innate immunity reactions triggered the adaptive immune responses by activating immune effector cells in the site of infection. Such cells include CD4+ and CD8+ lymphocytes, natural killer (NK) cells, macrophages, dendritic cells and innate lymphoid cells53,54.
Although both immunity reactions (innate and adaptive immunity) are activated upon infection, but Cryptosporidium itself developed some counter immune defense mechanism on the host cells, continues replication within the host and survive in the host. To note that, either the production of interferon gamma (IFN-γ) or IFN-γ signaling function in the epithelial cells are considered as essential factors to activate innate and adaptive immunity55. But upon Cryptosporidium infection, researchers observed a reduction of signaling transductor and activator of an essential transcription factor of IFN-γ signaling pathway, namely signaling transductor and activator of transcription1α (STAT1α)14. In the infected host cell, the depletion of transcription 1α (STAT1α) caused inhibition of transactivation of IFN-γ-dependent gene14. Additionally, C-C motif chemokine ligand 20 (CCL20), a cytokine responsible for the clearance of parasite was observed downregulated in the epithelial cells upon C. parvum infection28.
ELEVATED IMMUNE RESPONSE AND CANCER INITIATION
The first innate immune response against the Cryptosporidium infection is inflammation20, but repeated infection can trigger the formation of tumor by disabling the immune system that acts against the tumor cells. Besides, the induction of cell proliferation and instability of genetic process can be the results of inflammation. These abnormalities to the epithelial cells further result to oncogenic mutations20.
The defense mechanism starts with the activation of NF-κB pathway when Cryptosporidium infects on epithelial cells56. However, elevated NF-κB activation can be the potential regulator to produce colon cancer. Risk of colon cancer is high to inflammatory bowel disease (IBD) patients infected by Cryptosporidium due to the subsequent inflammation resulting from infection. Inflammation caused the secretion of tumor necrosis factor (TNF-α), interleukin (IL)-1, interleukin (IL)-17 and other pro-tumorigenic cytokines in order to induce NF-κB activity57. The downregulation of let-7 family and modulation of IL-6/STAT3 signaling in response to the inflammatory signals may lead to an uncontrolled proliferation of cells that results cancer initiation58.
CRYPTOSPORIDIUM INFECTION IN IMMUNOCOMPROMISED PATIENTS
Cryptosporidium invasion onto the human gastrointestinal epithelial cell lines results toll-like receptor (TLR)/NF-κB signaling activation. This activation leads to possible secretion and production of chemokines and cytokines including prostaglandin E2, interleukin (IL)-8 and interleukin (IL)-13, antimicrobial peptides and nitric oxide as a defensive mechanism to kill Cryptosporidium or inhibit its intensification59.
However, in immune compromised patients, there is insufficiency of T-cells (immune cells) containing a/b type T-cell receptor60. Thus, Cryptosporidium infection can alter the regulation of cellular mechanism and destroy complex regulatory network along with the regulation of miRNAs. Several studies confirmed that specific miRNA controlled different process of epithelial cells such as cellular differentiation regulation, cell death and proliferation, immunological response to microbial invasion, inflammatory responses, intracellular signaling pathways activation61-63, where interference of any of these processes supported the onset and progression of cancer in epithelial cell including colorectal cell.
For colorectal cancer, numerous molecular signaling pathways involved to suppress the T-cells and its subsets. Deactivation of immunity of CD4+ T cell is a result of canonical Wnt signaling in colorectal cell lines via suppression of IFN-γ and upregulation64 of IL-17a. Decreased CD4+ T cell count is also a detrimental factor in HIV patients since this results impairment in immune response mechanism65.
The ability of T cell, CD4+ is crucial in activation of T cell mediated NF-κB immune response mechanism66 and NF-κB signaling pathway is assumed to be an active player in initiation, development and metastasis in human cancer20. Since, the defense mechanism is compromised in cancer and HIV patients, the Cryptosporidium prevalence is high in such types of patients67.
ALTERATION OF MIRNAS UPON CRYPTOSPORIDIUM INFECTION
Cryptosporidium infection caused the activation TLR-4/NF-κB mediated signaling pathway and regulation of a number of miRNAs is associated to the pathway prior to infection. Cryptosporidium can modify the expression pattern of host epithelial cell line miRNAs by NF-κB p53 mediated signaling pathway activation25. The research identified miR-23b, miR-30b, miR-30c and miR-125b of cholangiocyte considerably upregulated along with a panel of miRNAs as a result of anti-microbial defense mechanism to counter the infection of C. parvum25. The study confirmed that, induced expression of selected miRNAs is due to transactivation of genes. Besides, a number of miRNAs of host biliary epithelial cells exhibit declined in expression17 including miR-98.
Upregulation of TLR4 due to the C. parvum infection result downregulation56 of let-7 miRNA. The subsequent inflammation produced by the C. parvum infection leads to an elevated expression of suppressors of inflammatory cytokine signaling (SOCS) and cytokine-inducible src homology 2-containing protein (CIS) and these alteration process is well maintained by downregulation of let-7 and miR-9836,42. The expression of NAD-dependent deacetylase sirtuin-1 (SIRT1) occurs due to the downregulation of let-7i, a miRNA precursor, in the H69 cell following infection68.
The C-C motif chemokine ligand 20 (CCL20), a cytokine responsible for the clearance of parasite was observed downregulated in epithelial expression upon the Cryptosporidium infection resulting from the downregulation28 of miR-21. When C. parvum was exposed to the cholangiocyte, reduction of miR-513 is observed that increase the expression of B7-H169. In vitro Cryptosporidium infection on biliary epithelial cell line showed downregulation of miR-221 of host cells and this downregulation of miR-221 is associated with the upregulation of intercellular adhesion molecule-1 (ICAM-1)38.
The miR-27b possesses a vital role in NF-κB signaling defense mechanism in infected host epithelial cell by targeting directly KH-type splicing regulatory protein (KSRP). The role of KSRP is to coordinate TLR4 mediated NF-κB signaling pathway. Hence, Cryptosporidium infection caused upregulation of miR-27b and this upregulation reduced the expression of KSRP in the epithelial cell line32.
Activation of NF-κB and histone deacetylases (HDACs) dependent defense mechanism in infected epithelial cells downregulates miR-424 and miR-503. Increase in expression of CX3CL1 is due to the downregulation of miRNA-424 and miRNA-50317.
CONCLUSION
The study established a relationship between the Cryptosporidium infection and regulation of miRNAs in the onset and progression of cancer. However, research on miRNAs regulation upon Cryptosporidium infection particularly in the in vitro gastrointestinal epithelial cells is inadequate. Therefore, further studies on miRNA and Cryptosporidium infection are vital to comprehend and reconfirm the mechanism of onset or progression of colorectal cancer by this parasite.
SIGNIFICANCE STATEMENT
This review discovered that there are significant dysregulation of oncomiRNAs and tumor suppressor miRNAs linked to the Cryptosporidium infection in the epithelial cells. Although the actual pathways for the dysregulation of tumor related with the miRNAs are needs to be understood, this review will be greatly beneficial for researchers to take as a reference for further studies. Current study proved that the development of cancer upon Cryptosporidium infection also causes the oncomiRNAs to be upregulated, while the tumor suppressor miRNAs are downregulated in the epithelial cell lines.
ACKNOWLEDGMENTS
This study is mainly supported by International Islamic University Malaysia, Publication Research Initiative Grant Scheme, P-RIGS18-037-0037.