Vincent McDonald

Differential regulation of beta-defensin gene expression during Cryptosporidium parvum infection.

ABSTRACT Invasion of enterocytes by pathogenic microbes evokes both innate and adaptive immune responses, and microbial pathogens have developed strategies to overcome the initial host immune defense. β-Defensins are potentially important endogenous antibiotic-like effectors of innate immunity expressed by intestinal epithelia. In this study, the interplay between the enteric protozoan parasite Cryptosporidium parvum and host epithelial β-defensin expression was investigated. Using human and murine models of infection, we demonstrated that C. parvum infection differentially regulates β-defensin gene expression. Downregulation of murine β-defensin-1 mRNA and protein was observed in both in vitro and in vivo models of infection. Infection of the human colonic HT29 cell line with the parasite resulted in differential effects on various members of the defensin gene family. Partial reduction in human β-defensin-1 (hBD-1), induction of hBD-2, and no effect on hBD-3 gene expression was observed. Recombinant hBD-1 and hBD-2 peptides exhibited significant antimicrobial activity against C. parvum sporozoites in vitro. These findings demonstrate that C. parvum infection of enterocytes may affect the expression of various defensins in different ways and suggest that the overall outcome of the effect of antimicrobial peptides on early survival of the parasite may be complex.

Protection against the Early Acute Phase of Cryptosporidium parvum Infection Conferred by Interleukin-4-Induced Expression of T Helper 1 Cytokines

Immunity to Cryptosporidium parvum infection involves a T helper (Th) 1 response with interferon (IFN)- gamma and interleukin (IL)-12 activity, but the role of Th2 cytokines, such as IL-4, is unclear. Around the peak of infection, production of oocysts in IL-4-deficient and IL-4 receptor alpha -deficient neonatal BALB/c mice was greater than that in wild-type (wt) mice. Susceptibility to infection was increased or decreased, respectively, in wt mice treated with anti-IL-4 neutralizing antibodies or recombinant IL-4. Excretion of oocysts by IFN- gamma -deficient mice was unaffected by treatment with anti-IL-4, indicating that IL-4 stimulated IFN- gamma activity. Early during infection, wt mice had increased intestinal expression of IFN- gamma and IL-12 mRNA, compared with IL-4-deficient mice. Intestinal IL-4 was detected by Western blotting in wt mice 24 h after infection but not in uninfected control mice. These findings suggest that, early during C. parvum infection of BALB/c mice, there is production of IL-4 that promotes Th1-mediated immunity.

Interleukin-4 and transforming growth factor beta have opposing regulatory effects on gamma interferon-mediated inhibition of Cryptosporidium parvum reproduction.

ABSTRACT It was shown previously that enterocytes activated by gamma interferon (IFN-γ) are efficient effector cells in the killing of Cryptosporidium parvum. How this function is regulated is not clearly understood, but transforming growth factor β (TGF-β) and the Th2 regulatory cytokines may play a role. Using an in vitro cell culture system, we investigated how the key regulatory cytokines interleukin-4 (IL-4), IL-10, IL-13, and TGF-β might modulate the effect of IFN-γ in inducing resistance to infection in enterocyte cell lines. The results showed that TGF-β can abolish the inhibitory effect on C. parvum development and that neither IL-13 nor IL-10 influenced the action of IFN-γ. In contrast, IL-4 cooperated with low concentrations of IFN-γ (1 and 10 U/ml) to enhance parasite killing. One mechanism that appeared to be involved in the combined activity of IFN-γ and IL-4 was intracellular Fe2+ deprivation, but induction of nitric oxide production was not involved. In one cell line, the extents and durations of phosphorylation of STAT1, a transcription factor involved in IFN-γ signaling, were similar when cells were stimulated with IFN-γ alone and with IFN-γ and IL-4γ, suggesting that the cooperative effect of the cytokines was not related to STAT1 activation. The effects of the presence of TGF-β and IL-4 on IFN-γ function did not appear to involve any alteration in the level of expression of IFN-γ receptors.

The role of Cryptosporidium parvum-derived phospholipase in intestinal epithelial cell invasion.

In the Cryptosporidium parvum-infected intestinal epithelial cell, the parasite occupies an unusual extracytoplasmic location at the luminal surface, but how the invading zoites interact with the host cell to achieve this niche is poorly understood. This study examined the role of secretory phospholipase A2 (sPLA2), a known virulence factor for several pathogenic microorganisms, in establishing C. parvum intracellularly. Initially, it was established that there was sPLA2 activity in homogenates of C. parvum oocysts. C. parvum reproduction in two human enterocyte cell lines was significantly reduced by a specific PLA inhibitor, p-bromophenacylbromide, and by sheep anti-sPLA2 antibodies developed against PLA2 of bee (Apis mellifera) venom. Treatment of either C. parvum sporozoites or enterocytes with sPLA2 derived from cobra (Naja naja) venom before initiation of infection increased the numbers of intracellular parasites. Thus, C. parvum PLA2 may play an important part in establishing the parasite within the enterocyte.

Innate immune responses against Cryptosporidium parvum infection.

Cryptosporidium parvum infects intestinal epithelial cells and is commonly the parasite species involved in mammalian cryptosporidiosis, a major health problem for humans and neonatal livestock. In mice, immunologically mediated elimination of C. parvum requires CD4+ T cells and IFN‐γ. However, innate immune responses also have a significant protective role in both adult and neonatal mice. NK cells and IFN‐γ have been shown to be important components in immunity in T and B cell‐deficient mice, but IFN‐γ‐dependent resistance has also been demonstrated in alymphocytic mice. Epithelial cells may play a vital role in immunity as once infected these cells have increased expression of inflammatory chemokines and cytokines and demonstrate antimicrobial killing mechanisms, including production of NO and antimicrobial peptides. Toll‐like receptors facilitate the establishment of immunity in mice and are involved in the development of inflammatory responses of infected epithelial cells and also dendritic cells.

Roles for NK Cells and an NK Cell-Independent Source of Intestinal Gamma Interferon for Innate Immunity to Cryptosporidium parvum Infection

ABSTRACT A gamma interferon (IFN-γ)-dependent innate immune response operates against the intestinal parasite Cryptosporidium parvum in T- and B-cell-deficient SCID mice. Although NK cells are a major source of IFN-γ in innate immunity, their protective role against C. parvum has been unclear. The role of NK cells in innate immunity was investigated using Rag2−/− mice, which lack T and B cells, and Rag2−/− γc−/− mice, which, in addition, lack NK cells. Adult mice of both knockout lines developed progressive chronic infections; however, on most days the level of oocyst excretion was higher in Rag2−/− γc−/− mice and these animals developed morbidity and died, whereas within the same period the Rag2−/− mice appeared healthy. Neonatal mice of both mouse lines survived a rapid onset of infection that reached a higher intensity in Rag2−/− γc−/− mice. Significantly, similar levels of intestinal IFN-γ mRNA were expressed in Rag2−/− and Rag2−/− γc−/− mice. Also, infections in each mouse line were exacerbated by treatment with anti-IFN-γ neutralizing antibodies. These results support a protective role for NK cells and IFN-γ in innate immunity against C. parvum. In addition, the study implies that an intestinal cell type other than NK cells may be an important source of IFN-γ during infection and that NK cells may have an IFN-γ-independent protective role.

Cryptosporidium parvum Infection Rapidly Induces a Protective Innate Immune Response Involving Type I Interferon

Type II interferon (IFN), IFN-gamma, is important in innate immunity to the intestinal protozoan parasite Cryptosporidium species, which infects epithelial cells (enterocytes). This investigation is, to our knowledge, the first to characterize the role of type I IFN in innate immunity to this parasite. Pretreatment of human or murine enterocyte cell lines with IFN-alpha/beta inhibited parasite development, and we identified that a key mechanism of cytokine action was to prevent parasite invasion of enterocytes. IFN-alpha/beta was rapidly expressed by infected murine enterocytes and also by bone marrow-derived dendritic cells that were exposed to live parasites. Treatment of neonatal severe combined immunodeficiency mice with anti-IFN-alpha/beta neutralizing antibodies before infection increased oocyst reproduction, as measured at the peak of infection, and parasite numbers in gut epithelium were also increased 2 days after infection. The latter observation correlated with strong intestinal expression of both IFN-alpha and IFN-beta messenger RNA within 24 h after infection. Treatment with anti-IFN-alpha/beta, however, did not reduce early expression of IFN-gamma. These findings identify a novel early innate host response against Cryptosporidium parvum involving IFN-alpha/beta.

A potential role for interleukin-18 in inhibition of the development of Cryptosporidium parvum.

Accumulating evidence suggests that intestinal epithelial cells (IECs) constitutively express the immunoregulatory cytokine interleukin (IL)‐18. IECs also serve as the host cell for the intracellular parasitic protozoan Cryptosporidium parvum. In the present study, C. parvum infection of a human enterocyte cell‐line HCT‐8 resulted in increased expression of IL‐18 mRNA as measured by quantitative reverse transcription–polymerase chain reaction (RT‐PCR). IL‐18 protein was detected in control uninfected cells and following infection there was increased expression as measured by enzyme‐linked immunosorbent assay (ELISA). Gene expression revealed the presence of the IL‐18 receptor subunits not only in cell‐lines but also in freshly isolated IECs, suggesting that IL‐18‐mediated signalling events may contribute to epithelial host defence during infection. Recombinant IL‐18 inhibited intracellular development of the parasite in HCT‐8 and HT‐29 cells. Increased expression of bactericidal antibiotic peptides LL‐37 and α‐defensin 2 by IL‐18 in HCT‐8 and HT‐29 cells may represent one mode of action by which this pluripotent cytokine aids in limiting the development of intracellular pathogens such as C. parvum in the gastrointestinal tract.

Nitazoxanide for persistent diarrhoea in Zambian acquired immune deficiency syndrome patients: a randomized-controlled trial

Background:  Adults with acquired immune deficiency syndrome and persistent diarrhoea in Zambia have intestinal infection, predominantly protozoa.

Dysregulation of interferon-gamma-mediated signalling pathway in intestinal epithelial cells by Cryptosporidium parvum infection

The apicomplexan parasite Cryptosporidium parvum, the agent of cryptosporidiosis, primarily infects and reproduces in enterocytes. Interferon (IFN)‐γ is important for early control of the infection and acts directly on enterocytes to inhibit parasite development, although complete inhibition is not obtained. Addressing this latter observation, an investigation was made of the modulatory effect of C. parvum infection on IFN‐γ‐dependent enterocyte gene expression. Initial studies showed that IFN‐γ‐induced expression of indoleamine 2, 3 dioxygenase (IDO) mRNA and protein in CMT‐93 cells was abrogated by C. parvum infection. Infection also inhibited IDO expression by the human enterocyte cell lines HT29 and Caco‐2. Expression of IFN‐γ‐inducible genes important in the development of immune responses, including major histocompatibility complex class II and CIITA, was also inhibited by the parasite. Investigating a possible mechanism for these findings, it was shown that infection caused depletion of STAT1α protein, a key transcription factor in IFN‐γ signalling. These findings indicate C. parvum interferes with IFN‐γ‐dependent gene expression in enterocytes and suggest this activity could be a novel immuno‐evasive strategy employed by the parasite.