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Keywords:

  • microsporidia;
  • encephalitozoon;
  • cytotoxic T cell;
  • CD8+ T cell;
  • persistence;
  • cell-mediated immunity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. General overview of the immune response to the microsporidia
  5. T cell response to the microsporidia and persistence
  6. Concluding remarks
  7. Acknowledgements
  8. References

The microsporidia are a diverse phylum of obligate intracellular parasites related to the fungi that cause significant and sometimes life-threatening disease in immune-compromised hosts, such as AIDS and organ transplant patients. More recently, their role in causing pathology in immune-competent populations has also been appreciated. Interestingly, in several instances, the microsporidia have been shown to persist in their hosts long term, causing at opposite ends of the spectrum either an intractable chronic diarrhea and wasting in patients with advanced-stage AIDS or asymptomatic shedding of spores in healthy populations. Much remains to be studied regarding the immune response to these pathogens, but it seems clear that CD8+ T cells are essential in clearing infection. However, in the infection models examined thus far, the role for CD4+ T cells is unclear at best. Here, we discuss the possible reasons and ramifications of what may be a weak primary CD4+ T cell response against Encephalitozoon cuniculi. Given the central role of the CD4+ T cell in other models of adaptive immunity, a better appreciation of its role in responding to microsporidia may provide insight into the survival strategies of these pathogens, which allow them to persist in hosts of varied immune status.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. General overview of the immune response to the microsporidia
  5. T cell response to the microsporidia and persistence
  6. Concluding remarks
  7. Acknowledgements
  8. References

The microsporidia are obligate intracellular parasites that were originally believed to be ‘primitive’ eukaryotes, but which have been recently reclassified as fungi (Table 1) (Weiss et al., 1999; Lee et al., 2010). Although initially regarded as rare, microsporidia are now believed to be common enteric pathogens that cause self-limited or asymptomatic infections in normal hosts. These organisms were first investigated in the late 19th century by Louis Pasteur and others studying pébrine, a disease of silkworms widespread in Europe at the time. The causative organism was later named Nosema bombycis (Naegeli, 1857) in a new order, forming the basis for the modern phylum Microsporidia. Now comprising over 1400 species in nearly 160 genera, many of medical, veterinary, and agricultural importance, their uniting and diagnostic feature is the internal polar tube that is extruded from the environmentally resistant spore upon germination and serves as a conduit for infection of the host cell (Fig. 1). Prior to the AIDS pandemic, these parasites received most of their attention as parasites of a variety of animals of agricultural importance (e.g. fish, fur-bearing animals, and beneficial and pestilent insects) and animals used in laboratory research (e.g. rabbits, rodents, and primates) (Wittner & Weiss, 1999; Wasson & Peper, 2000). Microsporidia have also been employed as biological control agents of insect pests, the most notable example being Antonospora locustae, a commercially available microsporidian insecticide for use against grasshoppers on rangelands (Henry & Oma, 1981). In the mid-twentieth century, there were also a few scattered reports in the literature of human infections, but their infrequency relegated these pathogens essentially to the status of medical curiosity.

image

Figure 1. Microsporidia spore in cell culture demonstrating extruded polar tube. Encephalitozoon hellem in cell culture (RK13 cells) stained with polyclonal rabbit antiserum to E. hellem followed by AlexaFluor 488 anti-rabbit IgG antibody (Xu & Weiss, 2005).

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Table 1. Characteristics of the microsporidia consistent with a fungal lineage
1Thymidylate synthase and dihydrofolate reductase are separate genes
2The small-subunit rRNA gene of microsporidia lacks a paromomycin-binding site, similar to the fungi
3The EF-1α sequence of the microsporidia has an insertion that is found only in fungi and animals, not in protozoa
4Microsporidia display similarities to the fungi during mitosis, for example closed mitosis, spindle pole bodies, and meiosis
5Microsporidia have chitin in their spore wall and store trehalose
6Analyses of many genes, for example glutamyl-tRNA synthetase, seryl-tRNA synthetase, vacuolar ATPase, TATA box-binding protein, seryl-tRNA synthetase, transcription initiation factor IIB, subunit A of vacuolar ATPase, and a GTP-binding protein and transcription factor IIB sequences, support a relationship between microsporidia and fungi
7Analysis of the E. cuniculi genome demonstrates that many of the E. cuniculi proteins are most similar to fungal homologs
8The presence in E. cuniculi of the principal enzymes for the synthesis and degradation of trehalose confirms that this disaccharide could be the major sugar reserve in microsporidia, as is seen in many fungi
9Analysis of glycosylation pathways and PTP 1 demonstrates that O-mannosylation as seen in fungi occurs in microsporidia
10Analysis of β-tubulin data that included additional species of microsporidia and more fungal phyla suggested that the Microsporidia were a sister group to the Zygomycota
11Microsporidian genomes harbor two syntenic ribosomal genes (RPL21 and RPS9) that are also syntenic throughout the fungi, but are not linked in other eukaryotic lineages
12An identified microsporidian sex-related locus is highly similar to the sex locus of the zygomycetes: both contain genes for a triose phosphate transporter (TPT), a high-mobility group (HMG) protein, and an RNA helicase

Since the mid-1980s, however, microsporidia have garnered increasing medical attention, especially in their capacity as opportunistic parasites of patients with AIDS (Cali, 1991; Weiss, 2001). In HIV-positive patients, the most common clinical manifestation of microsporidiosis is chronic diarrhea and wasting caused by enteric infection, but the spectrum of disease caused by these pathogens is broad and includes hepatitis, peritonitis, keratoconjunctivitis, sinusitis, bronchitis, pneumonia, cystitis, nephritis, myositis, encephalitis and other cerebral infections, and, rarely, urethritis, prostatic abscess, tongue ulcer, bone infection, and cutaneous infection (Wittner & Weiss, 1999; Franzen & Muller, 2001). Over 400 cases of human microspordiosis were documented by 1994 (Weber et al., 1994), and diagnostic surveys have revealed infection rates as high as 70% in HIV-infected populations (Weber et al., 1994; Wittner & Weiss, 1999). They have also caused infections in other immune-suppressed individuals, such as organ transplant recipients and patients undergoing chemotherapy (e.g. Metge et al., 2000; Sax et al., 1995; reviewed in Franzen & Muller, 2001).

The genome size of several different microsporidia has been determined and varies from 2.3 to 19.5 Mb (Wittner & Weiss, 1999), with Encephalitozoon cuniculi being 2.9 Mb (Katinka et al., 2001), Encephalitozoon hellem 2.5 Mb, and Encephalitozoon intestinalis 2.3 Mb (Corradi et al., 2010), which are among the smallest eukaryotic nuclear genomes identified. Data suggest that these organisms are diploid, but this has not been definitely proven. There are almost no introns in these compact genomes, the gene density is high, and proteins are shorter than the corresponding genes in Saccharomyces cervisiae. These small genomes demonstrate strategies for packing genetic information tightly into the genome as well as both streamlining and loss of metabolic pathways with increased host cell dependence (Keeling et al., 2010; Keeling & Corradi, 2011). There are currently no genetic systems for the manipulation of microsporidia, and thus one cannot use transfection techniques to study gene function, although our laboratory group is actively pursing the development of such techniques. In collaboration with Dr Patrick Keeling (University of British Columbia), Dr Saul Tzipori (Tufts University), Dr Elizabeth Didier (Tulane University), and the Broad Institute (MIT), we have recently completed the genomes of Enterocytozoon bieneusi, E. intestinalis, E. cuniculi (type 1, 2 and 3), E. hellem, and Vittaforma corneae, thus providing data that permit proteomic analysis across several microsporidia and permit studies on cloned antigens from these organisms (Corradi et al., 2010; Keeling et al., 2010). Genome data on these microsporidia are available at EuPathDB (http://microsporidiadb.org/micro/). In addition, we are completing the genome of Anncalia algerae, and genome annotation is in progress (Williams et al. 2008). Other groups have completed the genomes of several insect and invertebrate microsporidian pathogens: Nosema ceranae, Octosporea bayeri, and Vavria culicis floridensis, N. bombycis and the soil nematode pathogen Nematocida parisii (the majority of these genomes have been incorporated into MicrosporidiaDB) (Keeling et al., 2005; Corradi et al., 2008, 2009). These genomic data should facilitate new studies using these model systems to look at immune responses and host interactions among the microsporidia.

Microsporidia form characteristic unicellular spores that, for the human pathogenic microsporidia, range from 1.0 to 3.0 μm by 1.5 to 4.0 μm in size. The spore coat consists of an electron-dense, proteinaceous exospore, an electron-lucent endospore composed of chitin and protein, and an inner membrane or plasmalemma. Spores are resistant to environmental conditions, and this allows these organisms to persist in the environment, facilitating their transmission between hosts. These resistant spores may also allow these organisms to persist in infected tissues. A defining characteristic of all microsporidia is an extrusion apparatus consisting of a polar tube that is attached to the inside of the anterior end of the spore by an anchoring disk and coils around the sporoplasm in the spore. During germination, the polar tube rapidly everts forming a hollow tube that brings the sporoplasm into intimate contact with the host cell (Fig. 1). The polar tube provides a bridge to deliver the sporoplasm to the host cell. The mechanism by which the polar tube interacts with the host cell membrane is not known, but this may require the participation of the host cell. If a spore is phagocytosed by a host cell, germination will occur and the polar tube can pierce the phagocytic vacuole, delivering the sporoplasm into the host cell cytoplasm. The overall process of germination and formation of the polar tube inoculates the sporoplasm directly into a host cell. This germination is followed by merogony, during which the sporoplasm develops into meronts (the proliferative stage) that multiply, depending on the species, by either binary fission or multiple fission with the formation of multinucleate plasmodial forms. This stage is followed by sporogony, during which meront cell membranes thicken to form sporonts that, after subsequent division, give rise to sporoblasts that go on to form mature spores without additional multiplication. Once a host cell becomes distended with mature spores, the cell ruptures, releasing mature spores into the environment and completing its life cycle. The combination of multiplication during both merogony and sporogony results in a large number of spores being produced from a single infection and illustrates the enormous reproductive potential of these organisms.

Microsporidia are currently classified based on their ultrastructural features, including the size and morphology of the spores, number of coils of the polar tube, developmental life cycle, and host–parasite relationship (Wittner & Weiss, 1999). To date, at least 14 identified and two indeterminate species of Microsporidia have been found to infect humans, representing nine genera including Microsporidium, a taxon reserved for species of indeterminate assignment (Didier & Weiss, 2006). The following microsporidian phyla have been demonstrated to cause human infection (Table 2): Nosema (Nosema corneum renamed V. corneae (Silveira & Canning, 1995) and Nosema algerae reclassified initially as Brachiola algerae (Cali et al., 1998) and now as Anncaliia algerae (Franzen et al., 2006)), Pleistophora, Encephalitozoon, Enterocytozoon (Desportes et al., 1985), Septata (Cali et al., 1993) (reclassified as Encephalitozoon (Hartskeerl et al., 1995), Trachipleistophora (Field et al., 1996; Yachnis et al., 1996), Brachiola (Cali et al., 1998), Anncaliia (Franzen et al., 2006), Tubulinosema (Choudhary et al., 2011), and Microsporidium (Wittner & Weiss, 1999). Encephalitozoon hellem has been associated with superficial keratoconjunctivitis, sinusitis, respiratory disease, prostatic abscesses, and disseminated infection (Rastrelli et al., 1994). Encephalitozoon cuniculi has been associated with hepatitis, encephalitis, and disseminated disease (Orenstein et al., 1997; Sheth et al., 1997; Weber et al., 1997). Encephalitozoon (Septata) intestinalis has been associated with diarrhea, disseminated infection, and superficial keratoconjunctivitis (Cali et al., 1993; Visvesvara et al., 1995; Sheikh et al., 2000). Nosema, Vittaforma, and Microsporidium have been associated with stromal keratitis in immunocompetent hosts (Shadduck et al., 1990; Rastrelli et al., 1994; Sengupta et al., 2011). Pleistophora, Anncaliia, Tubulinosema, and Trachipleistophora have been associated with myositis (Chupp et al., 1993; Cali et al., 1996; Field et al., 1996; Cali & Takvorian, 2003; Choudhary et al., 2011). Trachipleistophora has been associated with encephalitis, keratitis, and disseminated disease (Field et al., 1996; Yachnis et al., 1996; Vavra et al., 1998). Enterocytozoon bieneusi, originally described in humans (Desportes et al., 1985), is associated with malabsorption, diarrhea, and cholangitis (Pol et al., 1993).

Table 2. Microsporidia identified as pathogenic to humans
Genus and speciesTypes of infectionAnimal reservoirsa
  1. a

    Animals in which organism has been found other than humans.

  2. b

    Cases reported in immunocompetent hosts.

  3. c

    Previously called Brachiola and Nosema.

  4. d

    Previously called Nosema.

Encephalitozoon
E. cuniculiHepatitis, peritonitis, encephalitisb, urethritis, prostatitis, nephritis, sinusitis, keratoconjunctivitis, cystitis, diarrheab, cellulitis, disseminated infectionMammals (rabbits, rodents, carnivores, primates)
E. hellemKeratoconjunctivitis, sinusitis, pneumonitis, nephritis, prostatitis, urethritis, cystitis, diarrhea, disseminated infectionPsittacine (parrots, lovebirds, budgerigars) and other birds (ostrich, chicken hummingbirds, finches)
E. intestinalisDiarrheab, intestinal perforation, cholangitis, nephritis, keratoconjunctivitisMammals (donkeys, dogs, pigs, cows, goats, primates)
Enterocytozoon
Enterocytozoon bieneusiDiarrheab, wasting syndrome, cholangitis, rhinitis, bronchitisMammals (pigs, primates, cows, dogs, cats) and birds (chickens)
Trachipleistophora
T. hominisMyositis, keratoconjunctivitis, sinusitisNone
T. anthropoptheraEncephalitis, disseminated infection, keratitisNone
Pleistophora
P. ronneafieiMyositisNone
Pleistophora sp.MyositisbFish
Anncaliiac
A. vesicularumMyositisNone
A. algeraeKeratoconjunctivitis, myositis, skin infectionMosquitoes
A. connoriDisseminated infection 
Nosema
N. ocularumKeratoconjunctivitisbNone
Vittaforma
Vittaforma corneaedKeratoconjunctivitisb, urinary tract infectionNone
Tubulinosema
Tubulinosema sp.MyositisNone (other species in insects)
Microsporidium
M. africanusCorneal ulcerbNone
M. ceylonesisCorneal ulcerbNone

Now widely acknowledged as opportunistic pathogens, evidence has emerged that the microsporidia cause disease in immune-competent individuals as well. Cases of microsporidiosis have been identified from all continents except Antarctica. Surveys of pathogens seen in stool samples in Africa, Asia, South America, and Central America have demonstrated that microsporidia are often found during careful stool examinations. Notable examples of microsporidiosis in immune-competent humans include gastrointestinal infections that have been discovered in travelers to and residents of underdeveloped countries, and ocular infections in contact lens wearers (Weber & Bryan, 1994; Desportes-Livage & Bylen, 1998). Indeed, the high seroprevalence of antimicrosporidian antibodies revealed by surveys of immune-competent individuals suggests that microsporidiosis in the general population may be common but self-limiting or asymptomatic. An early study (van Gool et al., 1997) revealed serum IgG specific for the polar tube of medically important genus Encephalitozoon in 8% and 5% of healthy Dutch blood donors and pregnant French women, respectively. A later investigation examined E. cuniculi polar tube-specific IgM, which is the first antibody secreted by plasma cells and perhaps more indicative of recent exposure, and found remarkably high positivity rates of 36% in healthy Japanese individuals of all ages and 59% in those aged 20 years or younger (Omura et al., 2007). A few studies have also addressed latent infections in healthy populations by staining for spores in stool samples. Spores shed in feces were detected in a study of healthy individuals in Cameroon, demonstrating that 68% were harboring microsporidia, with the highest prevalence in teenagers and the elderly and the highest parasite loads in children (Nkinin et al., 2007). Another study of HIV-negative, immune-competent patients in the Czech Republic with occupational risk exposure to animals found that 87% and 47% were shedding spores of Encephalitozoon spp. and E. bieneusi, respectively; the latter is the most common species causing diarrhea and wasting in patients with AIDS. Perhaps most interesting with regard to the present discussion is that spore shedding may continue even after the resolution of clinical gastrointestinal symptoms, as was observed in recent cases of travelers’ diarrhea (Wichro et al., 2005).

Despite the growing significance of the microsporidia, relatively little is known about environmental reservoirs for these pathogens, and modes of transmission to humans have not been explicitly documented. However, there is evidence that infections can occur by multiple routes (enumerated in Weiss, 2001) including water, respiratory, sexual, congenital, and zoonotic transmission, and in ocular infection by traumatic inoculation into the cornea. Viable infective spores of microsporidia are present in multiple body fluids (e.g. stool, urine, respiratory secretions) during infection, suggesting that person-to-person transmission can occur and that ocular infection may be transmitted by external autoinoculation because of contaminated fingers. It has been possible to transmit E. cuniculi via rectal infection in rabbits, suggesting the possibility of sexual transmission (Fuentealba et al., 1992). Encephalitozoon hellem has been demonstrated in the respiratory mucosa as well as in the prostate and urogenital tract of patients, raising the possibility of respiratory and sexual transmission in humans (Schwartz et al., 1994). Person-to-person transmission is supported by concurrent infections in cohabiting homosexual men (Wittner & Weiss, 1999). Although congenital transmission of E. cuniculi has been demonstrated in rabbits, mice, dogs, horses, alpaca, foxes, and squirrel monkeys, no such congenital transmission has been demonstrated in humans. Microsporidian spores are commonly found in surface water, and human pathogenic microsporidia have been found in municipal water supplies, tertiary sewage effluent, and ground water (Avery & Undeen, 1987; Dowd et al., 1998; Cotte et al., 1999). Encephalitozoon cuniculi spores remain viable for 6 days when in water and 4 weeks when dry at 22 °C, and N. bombycis spores may remain viable for 10 years in distilled water. It is likely that many of the microsporidia are waterborne pathogens. In addition, a recent report of a patient with rheumatoid arthritis receiving immunosuppressive therapy attributed a fatal case of myositis to Anncalia (Brachiola) algerae, a microsporidium initially identified from mosquitoes, underscoring the possibility of vectorborne transmission. Because of the probable risk of environmental transmission, the U.S. Environmental Protection Agency included these organisms on the two most recent Candidate Contaminant Lists CCL-1 and CCL-2 in 1998 and 2005, respectively; these actions identify the microsporidia as pathogens that may require regulation under the Safe Drinking Water Act. The ubiquity and hardiness of the spore ensure that much of the human population is subject to the possibility of infection.

Among the drugs tested in vitro and in vivo for the treatment of microsporidiosis, fumagillin and albendazole have demonstrated the most consistent activity and have been confirmed to have clinical efficacy in human microsporidian infections (Costa & Weiss, 2000). Albendazole binds to β-tubulin and is active against many microsporidia including all of the Encephalitozoonidae (E. hellem, E. cuniculi, E. intestinalis), and the sequence of the Encephalitozoon β-tubulin genes demonstrates an amino acid sequence associated with sensitivity to benzimidazoles (Li et al., 1996); however, the sequence of both Enterocytozoon (Akiyoshi et al., 2007) and Vittaforma (Franzen & Salzberger, 2008) demonstrate amino acids associated with albendazole resistance, which is consistent with the poor activity of albendazole in clinical cases of microsporidiosis caused by E. bieneusi. Fumagillin and its semi-synthetic analog TNP-470 have been found to have activity in vitro and in vivo against human pathogenic microsporidia including E. cuniculi, E. hellem, E. intestinalis, V. corneae, and E. bieneusi (Molina et al., 2002; Didier et al., 2006; Champion et al., 2010). In patients with immune suppression and intestinal microsporidiosis, such as patients with AIDS, discontinuation of therapy has often resulted in relapse of infection, which may be due to the persistence of the microsporidia in the host owing to an inadequate immune response as has been seen with other pathogens in this setting (Costa & Weiss, 2000). In ocular microsporidiosis such as keratoconjuctivitis, a similar relapse of infection has been seen, and persistence of microsporidian spores in conjunctiva scrapings has been documented (Sharma et al., 2011).

Given the frequency of latent infections in both immune-compromised and apparently immune-competent people, the question arises of what strategies the microsporidia have evolved to ensure persistence in their hosts. This is of practical significance considering the possibility of recrudescence should the host later become immune compromised. The possibility of this scenario occurring in humans is illustrated by a murine model of infection with E. cuniculi, where normal mice develop symptoms of disease followed by clinical recovery, but experience re-emergence of disease upon immune suppression with corticosteroids (Innes, 1970; Didier et al., 1994). Since the 1990s, over two dozen cases of microsporidiosis have been described in solid-organ and bone marrow transplant recipients (who usually must receive some form of immune suppressive therapy to ensure graft survival), some of which were life-threatening or resulted in fatal outcomes (reviewed in Galvàn et al., 2011). While in most cases it was unclear whether these infections originated from the donor or from latent infection of the recipient, the inability of either to have completely cleared the parasite despite initially displaying no clinical signs of infection suggests that the microsporidia are capable of modulating the immune response of the host in favor of their own survival. In this review, we discuss the mammalian T cell response to microsporidia with particular attention to aspects that may represent immune evasion strategies leading to persistence of these organisms.

General overview of the immune response to the microsporidia

  1. Top of page
  2. Abstract
  3. Introduction
  4. General overview of the immune response to the microsporidia
  5. T cell response to the microsporidia and persistence
  6. Concluding remarks
  7. Acknowledgements
  8. References

Infection with E. cuniculi in many mammals results in chronic infection with persistently high antibody titers and ongoing inflammation (e.g. persistent encephalitis in rabbits and chronic renal disease and congenital transmission in foxes). In immune-competent murine models of E. cuniculi infection, ascites develops and then clears; however, if corticosteroids are administered, the mice redevelop ascites, consistent with latent persistence of microsporidia in these animals (Didier, 1995). In SCID or athymic mice, infection with E. cuniculi results in death, with visceral dissemination of the organism and persistent ascites (Koudela et al., 1993). Adoptive transfer of sensitized syngeneic T-enriched spleen cells protects athymic or SCID mice against lethal E. cuniculi infection (Schmidt & Shadduck, 1984; Hermanek et al., 1993). Transfer of naive lymphocytes or hyperimmune serum failed to protect or prolong the survival of these mice. Although enhanced natural killer (NK) cell activity in E. cuniculi-infected mice has been reported, it does not seem to offer significant in vivo protection (Niederkorn et al., 1983).

Macrophages may have a role in the control of microsporidiosis. Cytokine-activated murine peritoneal macrophages can inhibit the replication of E. cuniculi in vitro (Didier & Shadduck, 1994). This inhibition may be mediated by nitric oxide, as studies demonstrate that inhibition of nitric oxide synthesis inhibited such killing (Didier, 1995; Didier et al., 2010). Mice deficient in inducible nitric oxide synthase, however, had no change in susceptibility to E. cuniculi infection (Khan & Moretto, 1999); therefore, nitric oxide is probably not the major mechanism for controlling dissemination of this organism in its host. Interferon-gamma (IFNγ) and lipopolysaccharide (LPS) treatment of macrophages infected with microsporidia induced a respiratory burst that controlled microsporidial replication (Didier & Shadduck, 1994; Franzen et al., 2005a; Fischer et al., 2008b; Didier et al., 2010); however, microsporidia are known to inhibit this respiratory burst in unstimulated macrophages (Monaghan et al., 2009). In human macrophages, Encephalitozoon sp. utilize Toll-like receptor ligand 2 (TLR2) for induction of immune responses and immune evasion (Fischer et al., 2008a). Macrophages may also be involved in the dissemination of infection. Spores have been shown to persist in phagosomes and escape macrophages by germination, occasionally penetrating into adjacent cells or tissue (Franzen et al., 2005b). In addition, infected macrophages produce chemokines that attract naïve monocytes that would be permissive to infection (Fischer et al., 2007).

Humoral immunity is not sufficient for protection against E. cuniculi infection, as adoptive transfer of immune B lymphocytes into athymic BALB/c (nu/nu) or SCID mice or passive transfer of hyperimmune serum into athymic mice does not protect these animals from death after infection (Schmidt & Shadduck, 1984; Hermanek et al., 1993). Nonetheless, during E. cuniculi infection, there is a strong antibody response to many components of this organism, and many of these antibodies are cross-reactive with other microsporidia. Maternal antibodies protect newborn rabbits from infection with E. cuniculi during the first 2 weeks of life (Bywater & Kellett, 1979). The in vitro infectivity of microsporidia is reduced by treatment with immune serum and complement (Schmidt & Shadduck, 1984), monoclonal antibody to the spore coat, or polyclonal antibodies to polar tube protein-1 (PTP1) (L.M. Weiss, unpublished data). IgM antibodies against PTP1 in normal human serum may play a role in preventing infection with E. cuniculi (Furuya et al., 2008). Overall, it is probable that antibodies play a role in limiting infection in the host, although they are clearly not sufficient to prevent mortality or to cure infection (Sak et al., 2006).

Interferon-gamma (IFNγ) and interleukin-12 (IL-12) are important for protective immunity against a number of intracellular viral, bacterial, and parasitic infections (Shtrichman & Samuel, 2001). Based on in vitro observations, it has been suggested that IFNγ plays an important role in the protective immunity against E. cuniculi infection. Both NK cells and γδ T cells, which are increased at early stages of infection, are likely important sources of IFNγ production (Khan & Moretto, 1999; Moretto et al., 2007). Studies with E. intestinalis and E. cuniculi have demonstrated that IFNγ knockout mice cannot clear infection (Achbarou et al., 1996). Treatment of E. cuniculi-infected mice with neutralizing antibody to IFNγ or IL-12 results in increased mortality (Khan & Moretto, 1999). The importance of IL-12 is illustrated by the fact that lethal infection with E. cuniculi also occurs in p40 knockout mice (which are unable to produce IL-12) (Khan et al., 1999). IFNγ production by dendritic cells (DCs) has been demonstrated to be important for priming the gut intraepithelial lymphocte response following oral infection with E. cuniculi (Moretto et al., 2007).

T cell response to the microsporidia and persistence

  1. Top of page
  2. Abstract
  3. Introduction
  4. General overview of the immune response to the microsporidia
  5. T cell response to the microsporidia and persistence
  6. Concluding remarks
  7. Acknowledgements
  8. References

The most significant manifestation of microsporidiosis, from the perspective of public health, is gastrointestinal tract infection in patients with AIDS. These organisms replicate in the epithelial lining of the small intestine, compromising villus architecture which leads to malabsorption followed by diarrhea. If infection is not eventually resolved, a wasting disease characteristic of advanced-stage AIDS results with often a fatal outcome. The importance of adaptive immunity in clearance of infection is best illustrated by the relationship between patient CD4+ cell count and outcome of microsporidian infection: AIDS patients with fewer than 50 CD4+ T cells per cubic milliliter of blood are especially prone to persistent diarrhea and weight loss, while HIV-negative patients and patients with AIDS whose T cell counts are restored via antiretroviral therapy are able to achieve resolution of disease (Goguel et al., 1997; Didier & Weiss, 2006). These observations are paralleled by experiments in laboratory mice, where animals severely immune suppressed either by genetic background (i.e. SCID mice with an absence of T- and B lymphocytes) or by pharmacologic agents (i.e. corticosteroids) develop a fatal ascites and dissemination of infection within the peritoneum, liver, and spleen (Innes, 1970; Schmidt & Shadduck, 1983; Koudela et al., 1993). As is the case with antiretroviral therapy in patients with AIDS, T cell reconstitution of SCID mice allows them to survive infection (Hermanek et al., 1993).

Using a mouse model of E. cuniculi infection in which animals are infected with spores intraperitoneally, the relative contributions of T cell subsets have been investigated. CD8+ T cells in particular have been shown to be critical in mediating protection, as CD8 gene knockout mice cannot survive infection. Cytokines secreted by this subset of T cells may play a role, but it is likely that their direct cytotoxicity is crucial as well, as evidenced by the lethal phenotype of perforin gene knockout mice upon infection (Khan et al., 1999). This requirement for CD8+ T cells makes sense because these are intracellular pathogens, and the only extracellular stage is the spore that may be hard to kill because of its thick coat and, even when engulfed, may be resistant to lysosomal enzymes.

In contrast to CD8+ T cells, CD4+ T cells are not required for protection against infection (Salat et al., 2006), and mice lacking CD4+ cells are able to survive high-dose infection (Khan et al., 1999). While unexpected in that CD8+ T cell responses in themselves typically require CD4+ T cell help (Bevan, 2004), it is not entirely surprising, as protective primary CD8+ T cell responses have been shown to develop even in the absence of CD4+ T cells to a variety of other infectious agents. Examples include the vaccinia (Binder & Kündig, 1991), influenza (Wu & Liu, 1994), and lymphocytic choriomeningitis viruses (Sun et al., 2004), the bacterium Listeria monocytogenes (Pope et al., 2001), the parasite Toxoplasma gondii (Combe et al., 2005), and the fungus Cryptococcus neoformans (Lindell et al., 2005). In other infectious disease and noninflammatory conditions where it is required, the nature of CD4+ T cell help presumably encompasses factors such as secreted IL-2 (Wilson & Livingstone, 2008), which promotes clonal expansion, and the activation of antigen-presenting cells (APCs) (Lu et al., 2000). The lack of a requirement for CD4+ T cells, which has been observed in certain infectious disease models, has been attributed to the presence of TLR ligands and other pathogen-associated molecular patterns that can directly stimulate APCs, or to pathogen recognition by other cell types which then release inflammatory mediators such as type I IFN or tumor necrosis factor. In the case of E. cuniculi, it has been suggested that cytokines produced by gamma delta T cells (γδ-T cells) are important in the priming of CD8+ T cells, as this T cell subset increases rapidly early in infection, γδ knockout mice exhibited poor CD8+ T cell responses, and CD8+ T cells isolated from these knockout animals failed to confer protection to naïve CD8 gene knockout mice (Moretto et al., 2001). In addition, innate recognition of Encephalitozoon spp. by TLR2 on macrophages induces an inflammatory response (Fischer et al., 2008a), which could help bypass the need for CD4+ T cells. IL-12 (Moretto et al., 2010) and IFNγ (Moretto et al., 2007) produced by DCs have also been shown to play a role in the priming of CD8+ T cells in this infection model. As additional investigative effort is directed toward events in the development of the vital CD8+ T cell response, our understanding of resistance to microsporidian infection will undoubtedly be enhanced. Nonetheless, given the central role of the helper T cell in adaptive immunity, the complete lack of apparent defects in protective immunity to E. cuniculi in CD4 gene knockout mice is striking: infected CD4 gene knockout mice do not suffer from increased parasite loads, nor are there defects in CD8+ T cell priming as there is no significant difference in the magnitude of the vital parasite-specific CD8+ T cell response (Moretto et al., 2000). Even if these helper T cells are not strictly required for CD8+ T cell activation, it is intriguing that their absence has no effect on the magnitude of this response, as they are the chief source of the IL-2 cytokine (Malek, 2008) that promotes T cell expansion.

If it is indeed true that CD4+ T cells play no clear role in protection, at least in the murine model thus examined, what would be the implication of this finding? Of course, the usual caveat applies that gene knockout mice maintained in the highly controlled and sanitized environment of the animal housing facility are at best an oversimplified model of human infections. It should be clear that in the long term, CD4+ T cells are realistically required to control infection, if only because the most severe manifestations of microsporidian infection are seen almost exclusively in patients with advanced-stage AIDS, whose CD4+ T cells are targeted and depleted by the human immunodeficiency virus. But even here, it is worthwhile to note that CD8+ T cell counts are also reduced in the advanced stages of AIDS (Schlumpberger et al., 1994), when microsporidiosis is likely to proceed to intractable chronic wasting disease. At the present time, owing to overwhelming usage of the CD4+ T cell count as correlate of HIV progression, there is not enough information to conclude whether microsporidiosis in patients with AIDS is more strongly correlated with the CD4+ or CD8+ T cell count. But it is relevant to note that microsporidiosis has been observed in the context of HIV and CD4+ T cell counts > 200 mm−3 (Sowerby et al., 1995), the upper threshold with which onset of opportunistic infections is commonly associated. Thus, it is reasonable to ask whether the activity of these T helper cells is relevant in responding to a microsporidian infection, or whether they are required only in the broad sense of maintaining an adaptive immune system.

It seems likely that there are CD4+ T cell responders to microsporidian infection, because antibodies to microsporidia are common worldwide, and B cells normally require help from follicular helper CD4+ T cells in the germinal center for productive antibody responses (Gatto & Brink, 2010). However, the contribution of this antibody response to protection is unclear. The few available studies on this subject indicate that host antibody to microsporidia probably plays at best an immunomodulatory role, as neither transfer of B lymphocytes from antigen-experienced animals (Enriquez, 1997) nor transfer of hyperimmune serum prevents lethal infection of athymic BALB/c mice (Schmidt & Shadduck, 1983), but can reduce the infectivity of spores in vitro (Schmidt & Shadduck, 1984). Moreover, it cannot be strictly assumed that CD4+ T cells are involved in the priming of the antibody response, as some classes of highly repetitive antigens found on microorganisms such as unusual polysaccharides or proteins composed of repeating motifs are able to stimulate antibody production in B cells by T cell-independent mechanisms (see Vos et al., 2000). While most of these studies have been on viruses and extracellular bacteria for which humoral immunity (e.g. in the form of neutralizing or opsonizing antibody) is generally much more effective, recent findings indicate that T-independent antibodies are also produced against the intracellular bacterial pathogen Ehrlichia muris (Racine et al., 2008). Thus, for the microsporidia that spend the vast majority of their lifestyle replicating inside a host cell, and whose highly glycosylated, repetitive amino acid motif–bearing polar tube and spore wall proteins (Xu & Weiss, 2005) are immunodominant in natural infections, it cannot be taken for granted that CD4+ T cells direct the humoral response; this is especially true in light of the fact that purified recombinant polar tube protein–pulsed DCs, generally considered the most potent kind of APC, were able to activate CD8+ but not CD4+ T cells (Moretto et al., 2010). Thus, at present, there is no clear evidence that microsporidia-specific CD4+ T cells are activated during peritoneal infection or that they contribute meaningfully to the protective response.

Given the central role of the CD4+ T cell in orchestrating adaptive immunity, the lack of evidence thus far for their appreciable role in protection against intraperitoneal infection is suggestive that this phenomenon may represent a means of immune evasion by these organisms, which can ensure their persistence in immune-competent and immune-compromised populations worldwide. The life cycle of these organisms itself may ensure that there exist only limited opportunities for antigen presentation to CD4+ T cells, which require antigen in the context of Major histocompatibility complex (MHC) class II. Although many species of microsporidia including Encephalitozoon infect macrophages, which as a professional APC expresses MHC II constitutively, the medically important species grow either in direct contact with the host cell cytoplasm [e.g. Enterocytozoon; Anncalia (Brachiola) algerae] or in a parasitophorous vacuole (e.g. Encephalitozoon) (Wittner & Weiss, 1999) that does not fuse with the lysosome (Weidner, 1975; Fasshauer et al., 2005). While there is indication that some spores may be taken up by phagocytosis rather than the classically envisioned invasion mechanism, which entails the parasite cytoplasm traveling through the extruded polar tube into a host cell (Couzinet et al., 2000), it seems that such spores either do not survive and proliferate (Orlik et al., 2010) or germinate within the phagosome and are propelled into the same or adjacent host cell cytoplasm by the long polar tube (Franzen et al., 2005b), much as they would have been via the classical invasion mechanism. Thus, it is unlikely that antigens derived from microsporidia replicating within the cell are presented by that same cell on MHC II via the classical endosome–lysosome pathway (see Burgdorf & Kurts, 2008), irrespective of the method of delivery of the pathogen to the host cell cytoplasm.

If microsporidia-derived antigens are not presented by the infected cell on MHC II to CD4+ T cells via the exogenous pathway, it is still possible that APCs may take up and present microbial antigen that becomes available when this infected cell dies or is lysed by a cytotoxic CD8+ T cell. Perhaps, this would partially account for the observation that depletion of both CD4+ and CD8+ T cells is required for lethality in a peroral model of E. cuniculi infection (Moretto et al., 2004), where gut mucosal DCs that continually sample luminal antigen abound (Milling et al., 2005). Interestingly, a subsequent study demonstrated that gut DCs indeed prime T cell immunity in perorally infected animals (Moretto et al., 2007), but again only the importance of CD8+ T cells was mechanistically affirmed. It has recently been demonstrated that TLR4 plays a major role in the activation of splenic DC during E. cuniculi infection, which results in the initiation of a protective immune response (Lawlor et al., 2010). The question, therefore, of whether there exists a microsporidia-specific CD4+ T cell response and whether it would play a clear role in protection remains. It is worthwhile to note that autophagy, which has been appreciated for more than a decade as a mode of MHC II presentation of endogenous, cytosolic antigen, is receiving increasing consideration as an avenue by which adaptive CD4+ T cell responses to intracellular pathogens may be achieved (see Crotzer & Blum, 2009). In the case of microsporidia, the entire parasitophorous vacuole could hypothetically be autophagocytosed, as has shown to be the case for T. gondii, another pathogen that resides in a nonfusogenic intracellular compartment (Andrade et al., 2006); alternatively, microsporidian proteins in the host cell cytoplasm could be targeted by microautophagy or chaperone-mediated autophagy mechanisms. Either of these could conceivably result in delivery to the endosomal/lysosomal compartment and subsequent presentation on MHC II (see Crotzer & Blum, 2009). This scenario would have important implications for the class II presentation of antigen by infected APCs, such as the macrophage, which plays a key role in the dissemination of infection to distal sites but is unlikely to present antigen via the traditional exogenous method.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. General overview of the immune response to the microsporidia
  5. T cell response to the microsporidia and persistence
  6. Concluding remarks
  7. Acknowledgements
  8. References

Based on the information available, the following hypothesis can be put forward (Fig. 2): E. cuniculi infection induces a strong CD8+ cytotoxic T lymphocyte (CTL) response, which restricts parasite growth by lysing the infected cells via a perforin-dependent mechanism. The induction of CD8+ CTLs is at least partially regulated by γδ T cells. This regulation may be dependent on the ability of γδ T cells to produce cytokines like IFN-γ. The CD8+ T cell subset plays a chief role in immunity to Encephalitozoon and appears to not require CD4+ help; in addition, clear evidence for the role of CD4+ T cells is lacking. It is important to note, however, that all of the animal studies conducted to date have examined only the primary response. Many recent immunologic studies indicate that while primary cytotoxic CD8+ T cell responses (particularly to microorganisms, which invariably elicit an inflammatory response via innate immunity) may operate independently of CD4+ T cells, CD4+ T cell help is required to generate and maintain a functional CD8+ memory T cell subset (Bevan, 2004). Thus, if there is indeed a paucity and/or dysfunction of microsporidia-specific CD4+ T cells elicited upon first encounter, it is possible that these repercussions may be better appreciated in a secondary challenge model of infection. The insights gained therein would have implications not only for patients with advanced-stage AIDS and other immune-suppressed patients who suffer from depressed CD4+ T cell counts, but also for apparently immune-competent individuals who shed spores asymptomatically after the resolution of primary disease. Moreover, a better appreciation for the role of the CD4+ T cell in microsporidiosis will obviously enhance our understanding of adaptive immunity to these pathogens and give us important clues to their long-term survival strategies.

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Figure 2. Role of CD8+ cytotoxic T cells in the immune response to microsporidia. CD8+ cytotoxic T cells have been shown to play a critical role in controlling infection in vivo, most likely by lysing infected host cells in a perforin-dependent manner. In contrast, CD4+ T cell help appears to be dispensable in the priming of CD8+ T cells, and the survival and parasite loads of infected mice lacking CD4+ T cells are not adversely impacted. Other immune cells including APCs (e.g. macrophages, DCs), which innately respond to infection with microsporidia, as well as NK cells and γδ T cells, may provide an early source of IFNγ by which CD8+ T cells may become primed to limit infection.

Adapted from Khan et al. (2001)

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References

  1. Top of page
  2. Abstract
  3. Introduction
  4. General overview of the immune response to the microsporidia
  5. T cell response to the microsporidia and persistence
  6. Concluding remarks
  7. Acknowledgements
  8. References