Cryptosporidium
5.5 Adaptive immunity
As important as innate immunity is in the initial stages of infection, adap- tive immunity is required to clear the parasites completely. Experimentally, infections in nude and severe combined immunodeficiency (SCID) mice can- not be cleared by the immune system and are chronic. T cells are particularly important in providing protection against cryptosporidial infection in mice. In- nate T cells such␥␦T cells are found in abundance in the gut mucosa. In cryp- tosporidiosis, they contribute to resistance to infection; neonates deficient in these cells are more susceptible than control mice. However, it is the adaptive
␣T cells that are necessary for control of infection, as shown by the fact that adult mice lacking␣T cells develop chronic infections.
5.5.1 T cells: CD4+ cells
CD4+ cells proliferate and produce IFN-␥ in response to infection. CD4+
lymphocytes are necessary for the resolution of infection. In general, patients with CD4+ counts greater than 200 cells/mm3tend to have less severe disease than those with less than 50 cells/mm3. Individuals with a depleted CD4+
T cell number due to HIV infection (see Chapter 19) have more severe and potentially life-threatening disease, demonstrating the importance of T cells in resistance and recovery from infection. Mice rendered deficient in CD4+ T cells by monoclonal antibody treatment have markedly decreased immunity, while adoptive transfer of CD4+ T cells to T cell-deficient mice can greatly reduce infection levels.
Intestinal epithelial lymphocytes (IELs) are nonconventional lymphocytes located among the epithelial cells in the lumen. These are important effec- tor cells in C. parvum infections, and a proportion of them are CD4+ cells.
Adoptive transfer of CD4+ IELs cells in SCID mice have been shown to reduce parasite load and give better protection against Cryptosporidium infections than adoptive transfer of CD8+ IELs, despite the fact that the majority (≈85 per cent) of IELs in mice are CD8+. This is consistent with infections of mice that are deficient in MHC molecules; MHC-II deficient mice that lack func- tional CD4+ are more susceptible than are MHC-I deficient mice that lack CD8+ T cells.
5.5.2 T cells: CD8+ cells
Although not as protective as CD4+ T cells, CD8+ T cells also play an im- portant role in response to infection. Increased numbers are observed in an- imals that have recovered from challenge infection, and adoptively trans- ferred CD8+ cell populations decrease infection, albeit not as markedly as CD4+ cells. The mechanism by which CD8+ T cells confer protection is not entirely clear, but it may be via the production IFN-␥ early in infec- tion. CD8+ T cells have been shown to produce IFN-␥ when stimulated with the cryptosporidial-specific antigen gp15 ex vivo. Additionally, they may act through cytotoxicity, as antigen-sensitised CD8+ T cells can reduce the parasite load in infected intestinal epithelial cell cultures by lysing infected intestinal epithelial cells.
5.5.3 Cytokines
5.5.3.1 IFN-␥
In humans, increased amounts of IFN-␥ are generated in response to cryp- tosporidial specific antigen after prior exposure. Treatment with an antibody to remove IFN-␥ enhances infection in mice and SCID mice have shown that resistance to C. parvum infection is IFN-␥-dependent. This evidence suggests that IFN-␥ plays an important role in innate, as well as adaptive, immunity. More severe infection in neonatal BALB/c mice developed upon
IFN-γ TNF-α
IECs
IELs
IL-4 IL-5 Th2 Cytokines Immunoregulatory
cytokines IL-10 TGF-β
?
Th1 Cytokines CD4+
Th1 NK cell
CD8+
Tc
IL-18 LL-37 β-defensin 2
IL-12 APC
IL-18
?
?
IL-15 IFN-γ
IFN-γ
Figure 5.2 The putative role of cytokines inCryptosporidiuminfection.The intestinal immune response to Cryptosporidium is largely Th1 in nature, driven by the cytokine IFN-␥(orange dots). The production of immunoregulatory or Th2 cytokines are hypothesised to dampen Th1 responses in the intestine. The purple dashed lines depict hypothetical or unknown sources or roles of cytokines.
Abbreviations: IEC, intestinal epithelial cell; IEL, intestinal epithelial lymphocyte; IFN, interferon; IL, interleukin; TGF, transforming growth factor; TNF, tumour necrosis factor.
administration of monoclonal antibodies to remove CD4+ T cells and IFN-
␥, when compared to antibody treatment-mediated removal of CD4+ T cells or CD8+ T cells, strongly suggesting that IFN-␥ might be produced in Cryp- tosporidium-infected mice by non-T cells such as NK cells.
Several different mechanisms of resistance mediated by the cytokine have been proposed. A common mechanism of action is through the induction of iNOS synthesis of NO in infection (Chapter 1). However, IFN-␥does not seem to me- diate expression of iNOS by the intestinal epithelium or synthesis of NO in re- sponse to C. parvuminfectionin vivo. InCryptosporidium-infected cells ex- posed to exogenous IFN-␥, depletion of intracellular iron may be a possible mechanism of action responsible for inhibition ofC. parvumgrowth. Alterna- tively, it is possible that IFN-␥activation of TNF-␣expression via up-regulation of its transcription factor NF-could impact onCryptosporidiuminfection (Figure 5.2).
5.5.3.2 TNF-␣
While TNF-␣is increased duringC. parvuminfection, and may enhance other immune responses, the lack of this cytokine does not appear to impact signifi- cantly on infection or enteric symptoms. Although treatment with recombinant TNF-␣can reduce parasite numbers in infected epithelial cells, neutralisation
of TNF-␣by antibody treatment in mice has no effect on infection, and TNF-␣ deficient mice are no more susceptible to infection than immunologically in- tact mice.
5.5.3.3 IL-12
IL-12 is a key inducer of IFN-␥, critical in resistance and protection of other Api- complexan parasites. Incryptosporidiosis, treatment of both immunocompe- tent and immunodeficient mice with IL-12 before infection prevented or greatly reduced the severity of infection, and was attributed to a decrease in IFN-␥
reduction. IL-12 deficient mice are susceptible to infection, but they generate some IFN-␥and are able to recover from infection. Infected calves produced IL-12 in response to infection, but treatment with recombinant IL-12 does not provide protection from challenge inoculation withC. parvumoocysts. IL-12 may have other roles besides induction of IFN-␥, as IL-12p40 gene knockout mice treated with IFN-␥-neutralising antibody excreted over eightfold higher oocyst numbers than IFN-␥knockout mice.
5.5.3.4 IL-18
IL-18 is a pluripotent cytokine involved in innate and adaptive immune mech- anisms, and it is produced by epithelial cells and a number of different im- mune cells. It is up-regulated in response toC. parvuminfectionin vitroand in mice. Recombinant IL-18 can inhibit intracellular development of the parasite in HCT-8 and HT-29 cell lines, possibly by increasing expression of bacterici- dal antibiotic peptides LL-37 and␣-defensin 2 (see Figure 5.2). IL-18 deficient mice are susceptible to infection, supporting a protective role for this cytokine in cryptosporidial infection.
Although IL-18 is well known to act in conjunction with IL-12 as an IFN-␥in- ducer, treatment of IL-12 deficient mice with recombinant IL-18 decreasedC.
parvumload markedly demonstrating a protective role for this cytokine alone.
Additionally, treatment of IFN-␥deficient mice with neutralising anti-IL-18 an- tibodies resulted in an increased parasite excretion, suggesting an additional role for IL-18 besides induction of IFN-␥. Potentially, this role could be through the induction of Th2 cytokines, since increased gene expressions of IL-4 and IL-13 were observed in splenocytes of anti-IL-18-treated mice deficient in the production of IL-12 and IFN-␥.
5.5.3.5 Th2 cytokines
Th2 cytokines such as IL-4 and IL-5 have been detected following infection.
These cytokines probably play a role in effective control ofC. parvuminfection by suppressing the production of Th1 cytokines (see Figure 5.2) although, para- doxically, it is also possible that IL-4 may enhance the Th1 response by stimulat- ing IFN-␥production. Although one study has demonstrated that IL-4 deficient mice shed oocysts for longer than immunologically intact mice, the majority of studies have failed to demonstrate a critical role for IL-4 in cryptosporidial infection. In human volunteers, IL-4 expression was associated with prior sen- sitisation toCryptosporidiuminfection, but it did not correlate with symptoms or oocyst production.
5.5.3.6 Immunoregulatory cytokines
IL-10 and TGF-are important immunoregulators in the intestinal tract, and they inhibit synthesis of pro-inflammatory cytokines. IL-10 increases in both HIV-infected and malnourished individuals infected withCryptosporidium, as well as in infected calves and in some mouse models of cryptosporidial infec- tion. TGF-also has been shown to increase in both animals and humans after infection. This cytokine is important in the repair of epithelial cells and can down-regulate Th1 cytokines such as IFN-␥ activityin vitro, as well as inhibit C. parvumdevelopment.
5.5.4 Antibody response
In the general population, the seropositivity rate in humans is high and re- ported to be anywhere from 25 to ⬎60 per cent, depending on the location and population being surveyed. While antibody responses (specifically im- munoglobulin (IgG and IgA)) are mounted against parasite antigens following primary infection, the role of these antibodies during recovery appears limited.
There is some evidence that humoral responses play a role, albeit modest, in protection from reinfection and, indeed, individuals with antibody deficiencies such as X-linked hyper-IgM and IgA deficiencies are more susceptible toCryp- tosporidiuminfection. On the contrary, high titres of parasite IgG and IgA can be found in HIV-infected individuals, who remain susceptible to infection with Cryptosporidium.
Antibodies are directed against several immunodominant antigens and these can block invasionin vitroand reduce parasite loads in mice. In calves, oocyst shedding and parasite load was reduced by passive transfer of colostrum from cows immunised with P23 antigen ofC. parvum, suggesting that partial protec- tion may be achieved if local antibody concentrations are generated and main- tained. However, antibodies do not seem to be essential, at least in mice; B cell deficient mice are no more susceptible to infection than immunologically in- tact control mice.