Type 1 Immunity Provides Both Optimal Mucosal and Systemic Protection against a Mucosally Invasive, Intracellular Pathogen
http://www.100md.com
感染与免疫杂志 2005年第8期
Department of Internal Medicine, Saint Louis University Health Sciences Center, 3635 Vista Avenue, St. Louis, Missouri 63110
ABSTRACT
It has been hypothesized that optimal vaccine immunity against mucosally invasive, intracellular pathogens may require the induction of different types of immune responses in mucosal and systemic lymphoid tissues. Mucosal type 2/3 responses (producing interleukin-4 [IL-4], IL-6 and/or transforming growth factor ) could be necessary for optimal induction of protective secretory immunoglobulin A responses. On the other hand, systemic type 1 responses (including gamma interferon [IFN-], tumor necrosis factor alpha, and optimal cytotoxic T-cell responses) are likely to be critical for protection against the disseminated intracellular replication that occurs after mucosal invasion. Despite these predictions, we recently found that vaccines inducing highly polarized type 1 immunity in both mucosal and systemic tissues provided optimal mucosal and systemic protection against the protozoan pathogen Trypanosoma cruzi. To further address this important question in a second model system, we now have studied the capacity of knockout mice to develop protective immune memory. T. cruzi infection followed by nifurtimox treatment rescue was used to immunize CD4, CD8, 2-microglobulin, inducible nitric oxide synthase (iNOS), IL-12, IFN-, and IL-4 knockout mice. Despite the previously demonstrated importance of CD4+ T cells, CD8+ T cells, and nitric oxide for T. cruzi immunity, CD4, CD8, and iNOS knockout mice developed mucosal and systemic protective immunity. However, IL-12, IFN-, and 2-microglobulin-deficient mice failed to develop mucosal or systemic protection. In contrast, IL-4 knockout mice developed maximal levels of both mucosal and systemic immune protection. These results strongly confirm our earlier conclusion from studies with polarizing vaccination protocols that type 1 immunity provides optimal mucosal and systemic protection against a mucosally invasive, intracellular pathogen.
INTRODUCTION
Chagas disease is a major cause of death in endemic areas, with an estimated 20 million people infected throughout Latin America. Although some cases of human infection are caused by contaminated blood transfusions, most transmission occurs from contact with Trypanosoma cruzi-infected reduviid excreta. T. cruzi insect-derived metacyclic trypomastigotes (IMT) can infect humans through breaks in the skin (e.g., through the reduviid bite site) or via mucosal routes (e.g., conjunctival exposure and ingestion). The relative significance of each route for human infection is currently unknown. In any case, induction of both mucosal and systemic parasite-specific immunity is an important goal for T. cruzi vaccine development, and detailed analyses of the immune subsets required for mucosal and systemic protection are needed.
Combined mucosal and systemic immunity could maximize protection against many chronic diseases caused by mucosally invasive, intracellular pathogens (e.g., T. cruzi, Mycobacterium tuberculosis, and human immunodeficiency virus). However, no vaccines available for human use are known to induce both optimal mucosal and systemic protection concurrently. Mucosal and systemic protection could require different immune responses. T cells producing interleukin-4 (IL-4), IL-5, and IL-10 (type 2 phenotype), or producing high levels of transforming growth factor (TGF-; type 3 phenotype), induce secretory immunoglobulin A responses protective against mucosal infection (12, 36). On the other hand, T cells producing gamma interferon (IFN-), tumor necrosis factor alpha, and IL-2 (type 1 phenotype) are clearly protective against systemic intracellular replication of many human pathogens (1, 23). Type 1 and type 2/3 responses have reciprocal inhibitory activities presenting a significant obstacle for development of vaccines designed to induce differential T-cell responses in mucosal and systemic immune compartments (3, 4, 7, 9, 10). Therefore, it is of critical importance for the field of vaccine immunobiology to define the specific mucosal and systemic responses protective against mucosally invasive, intracellular pathogens and to study the interactions between protective mucosal and systemic responses.
The identification of key cytokines required for generation of different T-cell subsets has made it possible to induce highly polarized antigen-specific type 1 and type 2 responses. IL-12 and IL-4 are critical for induction of type 1 and type 2 responses, respectively (23). Kumar et al. reported that in vitro-generated CD4+ Th1 but not Th2 cells could adoptively transfer protection against systemic T. cruzi challenges (18). We demonstrated that recombinant IL-12 and anti-IL-4 induced type 1-biased responses in vivo that were highly protective against normally lethal T. cruzi systemic challenges (15, 27). IL-4 plus anti-IFN- induced type 2 polarized responses that failed to protect against systemic T. cruzi challenges (15). More recently, we extended these observations showing that despite the prediction that an ideal vaccine should induce optimal mucosal type 2 immunity protective against initial invasion in the relevant mucosal lymphoid tissue, type 1 polarized responses induced by intranasal vaccination were optimally protective against both mucosal and systemic T. cruzi challenges (13).
We now use a second model system to examine the mucosal and systemic protective effects of T. cruzi-specific type 1 and type 2 responses. An infection/nifurtimox rescue protocol was employed to induce T. cruzi-specific "immune memory" in CD4, CD8, 2-microglobulin, inducible nitric oxide synthase (iNOS), IL-12, IFN-, and IL-4 knockout mice. The data demonstrate that type 1 responses are absolutely essential for both optimal mucosal and systemic protection.
MATERIALS AND METHODS
Mice. Six- to 8-week-old mice were used in these experiments. Wild-type C57BL6/J and the following knockout mice in the same genetic backgound were purchased from The Jackson Laboratory (Bar Harbor, ME): iNOS (–/–), IL-4 (–/–), 2-microglobulin (–/–), IFN- (–/–), and IL-12 p40 (–/–). Wild-type BALB/c mice were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). CD4 (–/–) and CD8 (–/–) knockout mice in the BALB/c genetic background were provided courtesy of T. Mak (Amgen, Ontario, Canada). Mice were housed under pathogen-free conditions throughout these studies.
Parasites and challenges. Blood-form trypomastigotes (BFT) and IMT from the Tulahuen strain of Trypanosoma cruzi were prepared as previously described (13). BFT were prepared from highly parasitemic BALB/c heparinized blood, enumerated by microscopic hemocytometer counts, and diluted with phosphate-buffered saline to the desired concentration. One hundred to 1,000 BFT were injected intraperitoneally for primary infections, and 1,000 to 20,000 BFT were injected subcutaneously at the base of the tail for subsequent infections. IMT were prepared from the collected excreta of T. cruzi-infected reduviid insects (Dipetalogaster maximus), enumerated by direct hemocytometer count, and fed orally to mice. These mice were fasted for 4 h and were given 0.5 ml of 1.5% sodium bicarbonate in Hanks' buffer intragastrically 15 min prior to oral delivery of 1,000 to 10,000 IMT parasites.
Infection/nifurtimox treatment protocols for induction of immune memory. For studies of systemic immunity, wild-type and knockout mice were infected intraperitoneally with 100 BFT. Seven days after infection, treatment with the trypanocidal drug nifurtimox (100 mg/kg/day intragastrically, Monday through Friday) was begun and continued for 4 weeks. Nifurtimox was obtained from John Becher at the Centers for Disease Control Drug Service, Atlanta, GA. One month after cessation of nifurtimox, mice were reinfected with 5,000 BFT intraperitoneally, followed by 4 more weeks of nifurtimox treatment beginning 1 week after the second parasite injection. To study mucosal immunity, a similar protocol was followed, substituting oral delivery of 1,000 to 10,000 IMT, instead of intraperitoneal BFT, for each infection/nifurtimox treatment cycle.
Assessment of protective immunity. Four to six weeks after the second cycle of T. cruzi infection/nifurtimox treatment was finished, nave and "memory immune" knockout mice were challenged without further nifurtimox treatment. To assess systemic protection, mice were challenged subcutaneously with 5,000 BFT (a standard lethal dose in wild-type mice) and followed for survival over >3 months. For assessment of mucosal memory immunity, mice were challenged with 1,000 to 10,000 IMT orally and studied 11 to 14 days later for the levels of T. cruzi replication detectable in gastric mucosal tissues—previously shown to be the site of initial mucosal invasion after oral IMT challenge (14)—and in the gastric draining lymph nodes. Total gastric DNA was extracted and studied by real-time PCR with primers specific for a T. cruzi-specific gene target as described previously (27). Briefly, 200 ng of total gastric DNA was added to SYBR green PCRs with primers specific for a 65-bp fragment of the T. cruzi gene coding for cruzipain. Samples were then run in an ABI Prism 7700 sequence detection system, and quantities in terms of T. cruzi molecular equivalents per 200 ng gastric DNA were calculated based on standard curves generated with DNA collected from a known number of T. cruzi epimastigotes. Draining gastric lymph nodes were harvested and passed through a wire mesh to obtain mononuclear cell suspensions. These cells were washed and then plated in two- to fivefold serial dilutions in medium (LDNT+) optimized for the outgrowth of T. cruzi epimastigotes from infected cells. These plates were inspected microscopically every 2 weeks for parasite outgrowth. The number of parasites per million lymph node cells can be calculated by dividing 1 million by the minimal number of cells plated that were associated with the outgrowth of parasites as described previously (14, 27).
Flow cytometry. Lymphocyte populations were stained with a combination of anti-CD8-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), and anti-CD3-peridinin-chlorophyll-protein C complex (PerCP; Pharmingen, San Diego, CA) and studied with a FACScaliber flow cytometer.
RESULTS
Development of T. cruzi memory immune model. Many of the relevant immune knockout mice are known to be highly susceptible to primary T. cruzi infection, generally succumbing to overwhelming infection within a few weeks after even very-low-level parasite challenges. In order to study memory immune responses we needed to develop a treatment rescue protocol that would prevent death in these immunodeficient mice during primary infection. Shown in Fig. 1 is the infection/nifurtimox treatment protocol used to generate T. cruzi-specific immune memory in wild-type and immunodeficient mice. Mice were challenged mucosally with IMT or systemically with BFT. Beginning 1 week after challenge, nifurtimox was administered intragastrically on a daily basis (Monday through Friday) for at least 1 month. After stopping nifurtimox, mice were observed for at least 1 more month to allow the effects of nifurtimox to wear off and be sure that recrudescence of overwhelming parasitemia did not occur. This cycle of infection, followed by nifurtimox treatment, followed by observation without further nifurtimox for at least 1 more month, was repeated before testing whether immune memory protective against mucosal and systemic T. cruzi challenges had developed in these mice. Control nave mice received parallel treatments with nifurtimox without the first two parasite challenges to control for any possible positive or negative effects of nifurtimox on immunity. Using this protocol we were able to generate memory immune animals for study in most knockout strains despite infection with virulent T. cruzi. To prove that survival in knockout strains was not due to spontaneous reversion to wild type, combinations of flow cytometric and cytokine secretion studies were completed postchallenge. Shown in Fig. 2 are representative flow cytometry histograms demonstrating the absence of CD4+ and CD8+ T cells in CD4 and CD8 knockout mice, respectively, surviving the T. cruzi infection/nifurtimox rescue protocol.
Molecular requirements for memory immune systemic protection. Table 1 presents the overall results from our new investigations of the immunologic requirements for protective systemic immunity. As described above, control mice had received 2 cycles of 1-month treatments with nifurtimox without preceding T. cruzi infection before investigating their ability to survive a primary BFT challenge. Memory immune mice had been immunized with 2 cycles of infection followed by nifurtimox rescue, prior to assessment of their ability to survive a tertiary challenge without further nifurtimox treatment. CD4, CD8, and iNOS knockout mice were able to develop robust immune memory protective against systemic parasite challenges despite previous publications documenting their enhanced susceptibility to primary T. cruzi infection (16, 25, 26). Surviving CD4, CD8, and iNOS knockout mice were challenged systemically again 2 months later with 10-fold-higher BFT doses, and these mice survived for at least another 2-month period of observation. These results demonstrate compensatory mechanisms for these defects in memory immune responses. On the other hand, even with 6 to 8 weeks of nifurtimox treatment, we were unable to rescue IL-12, IFN-, or 2-microglobulin knockout mice from death after primary T. cruzi BFT challenges. Therefore, the cytokine mainly responsible for generation of type 1 immunity (IL-12), the major effector cytokine associated with type 1 immunity (IFN-), and the downstream type 1 effector functions restricted by class I molecules coexpressed with 2-microglobulin are all absolutely essential for the development of immune memory protective against systemic T. cruzi infection. In contrast, the major generating and effector cytokine associated with type 2 responses, IL-4, was not necessary for the development of systemic protection. These results confirm numerous other reports demonstrating that type 1 immune responses are critical for systemic protection against intracellular pathogens.
Molecular requirements for mucosal protection. We developed an oral T. cruzi challenge model that mimics 1 of the natural mucosal routes of infection in humans (14). T. cruzi IMT invade via the gastric epithelium after oral ingestion, which drains to lymphoid tissue present within the lesser curvature of the stomach. Mucosal parasite replication can be studied in gastric epithelial and draining lymphoid tissue after oral challenge using a T. cruzi-specific real-time PCR assay and a quantitative parasite culture technique (14, 27). After harvesting total gastric DNA 10 to 14 days after oral challenge, molecular equivalents of T. cruzi present in these samples are quantified by real-time PCR. Single-cell suspensions of the gastric draining lymph node cells are prepared and plated under limiting dilution parasite culture conditions to quantitate viable T. cruzi organsims. In three previous publications (13, 14, 27), as well as throughout the experiments described here, similar results have been obtained with these real-time PCR and quantitative parasite culture techniques. Figure 3 presents comparative results obtained by real-time PCR and quantitative parasite culture in studies of wild-type and CD4 knockout mice.
Table 2 presents the overall real-time PCR results obtained in our mucosal immune studies. The oral IMT challenge is less virulent than systemic BFT challenge, and all immunodeficient mice could be rescued from overwhelming parasitemia after oral IMT challenge by nifurtimox treatment. This allowed us to directly examine whether mice with these genetic defects, including mice deficient in key components of type 1 immunity, could develop mucosal immune memory. CD4, CD8, and iNOS knockout mice were able to develop protective mucosal immunity not significantly different from that detected in wild-type mice, further demonstrating compensatory mechanisms for these immune defects. However, similar to the systemic immune studies reported above, and consistent with our previous type 1- and type 2-biasing vaccine work (13), IL-12, IFN-, and 2-microglobulin knockout mice failed to develop detectable protection against T. cruzi mucosal challenge. Therefore, we have clearly shown that the cytokine mainly responsible for generation of type 1 immunity (IL-12), the major effector cytokine associated with type 1 immunity (IFN-), and the downstream type 1 effector functions restricted by class I molecules coexpressed with 2-microglobulin are all absolutely essential for the development of both T. cruzi mucosal and systemic protective immune memory. In contrast, the major generating and effector cytokine for type 2 responses, IL-4, was not necessary for the development of immune memory protective against either mucosal or systemic challenge. Figure 4 graphically illustrates that IL-4 knockout mice, but not IFN- knockout mice, could develop protective mucosal immunity.
DISCUSSION
Because CD4+ Th2 cells (secreting IL-4, IL-5, and IL-6) generally are required for the optimal induction of secretory immunoglobulin A responses previously shown to be involved in mucosal protection (20, 21, 36), we were surprised to find in our recently published studies that intranasal vaccines inducing highly polarized type 1 responses without detectable vaccine-induced IL-4 responses were optimally protective against T. cruzi mucosal challenges (13). Therefore, we were interested in studying the importance of type 1 and type 2 immune responses for mucosal immunity in a second T. cruzi model system. Our previous work involved studies of the differential protective effects of type 1 and type 2 immune responses induced preinfection. To further address this important question in a second model system, we now have studied the capacity of knockout mice to develop protective immune memory. T. cruzi infection followed by nifurtimox treatment rescue was used to immunize CD4, CD8, 2-microglobulin, iNOS, IL-12, IFN-, and IL-4 knockout mice. CD4, CD8, and iNOS knockout mice developed compensatory mechanisms protective against mucosal and systemic T. cruzi infection. However, IL-12-, IFN--, and 2-microglobulin-deficient mice failed to develop any detectable mucosal or systemic protection. In contrast, IL-4 knockout mice had no deficiency in their ability to develop either mucosal or systemic protective immune memory. These results strongly confirm our earlier conclusion from studies with polarizing vaccination protocols that type 1 immunity provides optimal mucosal and systemic protection against a mucosally invasive, intracellular pathogen.
We have previously shown in SCID adoptive transfer experiments that CD4+ Th1 cells were required for transfer of the ability to develop primary immunity protective against systemic T. cruzi challenge (15). On the other hand, in the same publication we demonstrated that CD4+ T cells were not required for the transfer of protective systemic effector functions that had already developed in memory immune mice. These results suggest that CD4+ Th1 cells act primarily as helper cells for the development of protective effector mechanisms, but do not function directly as critical immune effector cells during T. cruzi infection. Based on these results, we predicted that CD4 knockout mice would be deficient in helper activity and would not be capable of developing protective systemic immune memory in our infection/nifurtimox rescue protocol. However, we found this not to be the case. As shown in Tables 1 and 2, CD4 knockout mice immunized with 2 infection/nifurtimox rescue cycles developed significant immune memory protective against further mucosal and systemic T. cruzi challenges. Surviving memory immune CD4 knockout mice were sacrificed 2 to 3 months after the final T. cruzi challenge, and their splenocytes were studied by flow cytometry to confirm the absence of CD4+ T cells (Fig. 2). Therefore, although in the normal wild-type genetic background it appears that CD4+ Th1 cells are required for the development of protective immune memory, mice lacking CD4+ T cells are capable of developing protective immunity by some alternative mechanism(s). Recent work has demonstrated that nonconventional subsets of T cells including (i) CD1-restricted T-cell receptor /+, CD4 CD8 double-negative T cells (24, 33), (ii) T cell receptor /+ T cells (17, 35), and (iii) NK T cells (6, 28) can develop potent type 1 immune phenotypes, and perhaps one or more of these subsets may provide alternative helper functions for the development of protective immune memory in the CD4 knockout mice studied with our infection/nifurtimox rescue model. Learning precisely what these alternative mechanisms are and how to induce them may result in the development of new vaccination strategies useful in patients with functional depletion of CD4+ T cells (e.g., AIDS patients).
The presence of CD8+ cytotoxic T lymphocytes (CTL) capable of recognizing and lysing T. cruzi-infected cells has been shown to correlate with protective immunity (25, 29, 30), and vaccines that induce T. cruzi-specific CD8+ CTL responses often are extremely effective at inducing protective immunity (8, 34). Our results are consistent with these previous results but also present new information. We initially tried infection/nifurtimox rescue protocols delaying treatment until 2 weeks after T. cruzi challenge. CD8 knockout mice challenged with as few as 100 BFT died usually before nifuritmox was begun or shortly thereafter when nifurtimox treatment was delayed for this long after infection (data not shown). These results illustrate the importance of CD8+ CTL responses for the immune clearance of primary infection. However, after we revised our protocol to begin nifurtimox earlier, 1 week after infection, we found that some CD8 knockout mice could be rescued after low doses of BFT challenge. Treatment of these mice for 1 month with nifurtimox resulted in long-term survival of these animals; recurrence of parasitemia was not seen after cessation of nifurtimox treatment. Their survival allowed us to study whether CD8+ CTL are necessary for the development of protective immune memory. Despite their extreme susceptibility to primary T. cruzi systemic challenges, CD8 knockout mice rescued by the early nifurtimox treatment were able to develop systemic immunity protective against repeated BFT challenges (Table 1 and Results). In addition, using the less virulent oral challenges with T. cruzi IMT, we were more easily able to generate memory immune mice in the CD8 knockout background that were found to develop protective immunity against further oral IMT challenges (Table 2), providing additional support for the conclusion that CD8+ T cells are not absolutely required for the development of protective T. cruzi memory immunity. Surviving memory immune CD8 knockout mice were sacrificed 2 to 3 months after the final T. cruzi challenge, and their splenocytes were studied by flow cytometry to confirm the absence of CD8+ T cells (Fig. 2). The fact that 2-microglobulin-deficient mice were found to be much more susceptible to T. cruzi infection, being unable to develop any protective immune memory, suggests that they lack other important effector functions in addition to defects in CD8+ CTL. T cells stimulated by other nonpolymorphic class I-like molecules (e.g., CD1) also require 2-microglobulin expression and may provide complementary cytolytic effector functions important for protective T. cruzi immunity. Identifying these alternative mechanisms of protective effector function will be important for future vaccine development.
Nitric oxide is a known trypanocidal agent capable of killing both intracellular and extracellular T. cruzi. Many groups, including ours, have shown that type 1 cytokine responses can induce nitric oxide production leading to inhibition of T. cruzi growth within infected macrophages (11, 15, 22, 32). However, immune memory induced by our infection/nifurtimox rescue protocol could protect against both mucosal and systemic T. cruzi challenges even in iNOS knockout mice (Tables 1 and 2). These results demonstrate that other microbicidal functions such as superoxide derivatives (2, 19) and/or peroxynitrite (5, 19, 31) can provide compensatory trypanocidal activity effective for protective immune memory in mice lacking the inducible nitric oxide synthase gene. Measurements of these other microbicidal functions could provide important surrogate markers for studies of protective immunity.
In summary, we have shown that the murine immune system is capable of compensating for multiple deficiencies of key immunologic functions important for T-cell memory. Elucidating the compensatory mechanisms will have important implications for the development of vaccines and/or immunotherapies for use in patients with certain immunodeficiencies. In addition, combined with our previous studies of type 1 and type 2 polarizing vaccination protocols, the current results conclusively demonstrate the critical importance of type 1 immunity for both mucosal and systemic protection against T. cruzi infection. These results have important practical implications suggesting that vaccines designed to induce concurrent mucosal and systemic protection against mucosally invasive, intracellular pathogens can focus on a more feasible goal of inducing homologous type 1 immune responses in both mucosal and systemic tissues, rather than differential induction of type 2 and type 1 responses in mucosal and systemic tissues, respectively. However, to date this question has only been addressed in our T. cruzi gastric invasion model. T. cruzi can invade mucosal surfaces after conjunctival challenges as well. In addition, several other important human pathogens establish chronic intracellular infections after invasion through other mucosal surfaces (e.g., human immunodeficiency virus and Mycobacterium tuberculosis). Future work will need to test the more general hypothesis that type 1 immunity will provide optimal protection of other mucosal surfaces against invasion by T. cruzi and other important human intracellular pathogens.
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ABSTRACT
It has been hypothesized that optimal vaccine immunity against mucosally invasive, intracellular pathogens may require the induction of different types of immune responses in mucosal and systemic lymphoid tissues. Mucosal type 2/3 responses (producing interleukin-4 [IL-4], IL-6 and/or transforming growth factor ) could be necessary for optimal induction of protective secretory immunoglobulin A responses. On the other hand, systemic type 1 responses (including gamma interferon [IFN-], tumor necrosis factor alpha, and optimal cytotoxic T-cell responses) are likely to be critical for protection against the disseminated intracellular replication that occurs after mucosal invasion. Despite these predictions, we recently found that vaccines inducing highly polarized type 1 immunity in both mucosal and systemic tissues provided optimal mucosal and systemic protection against the protozoan pathogen Trypanosoma cruzi. To further address this important question in a second model system, we now have studied the capacity of knockout mice to develop protective immune memory. T. cruzi infection followed by nifurtimox treatment rescue was used to immunize CD4, CD8, 2-microglobulin, inducible nitric oxide synthase (iNOS), IL-12, IFN-, and IL-4 knockout mice. Despite the previously demonstrated importance of CD4+ T cells, CD8+ T cells, and nitric oxide for T. cruzi immunity, CD4, CD8, and iNOS knockout mice developed mucosal and systemic protective immunity. However, IL-12, IFN-, and 2-microglobulin-deficient mice failed to develop mucosal or systemic protection. In contrast, IL-4 knockout mice developed maximal levels of both mucosal and systemic immune protection. These results strongly confirm our earlier conclusion from studies with polarizing vaccination protocols that type 1 immunity provides optimal mucosal and systemic protection against a mucosally invasive, intracellular pathogen.
INTRODUCTION
Chagas disease is a major cause of death in endemic areas, with an estimated 20 million people infected throughout Latin America. Although some cases of human infection are caused by contaminated blood transfusions, most transmission occurs from contact with Trypanosoma cruzi-infected reduviid excreta. T. cruzi insect-derived metacyclic trypomastigotes (IMT) can infect humans through breaks in the skin (e.g., through the reduviid bite site) or via mucosal routes (e.g., conjunctival exposure and ingestion). The relative significance of each route for human infection is currently unknown. In any case, induction of both mucosal and systemic parasite-specific immunity is an important goal for T. cruzi vaccine development, and detailed analyses of the immune subsets required for mucosal and systemic protection are needed.
Combined mucosal and systemic immunity could maximize protection against many chronic diseases caused by mucosally invasive, intracellular pathogens (e.g., T. cruzi, Mycobacterium tuberculosis, and human immunodeficiency virus). However, no vaccines available for human use are known to induce both optimal mucosal and systemic protection concurrently. Mucosal and systemic protection could require different immune responses. T cells producing interleukin-4 (IL-4), IL-5, and IL-10 (type 2 phenotype), or producing high levels of transforming growth factor (TGF-; type 3 phenotype), induce secretory immunoglobulin A responses protective against mucosal infection (12, 36). On the other hand, T cells producing gamma interferon (IFN-), tumor necrosis factor alpha, and IL-2 (type 1 phenotype) are clearly protective against systemic intracellular replication of many human pathogens (1, 23). Type 1 and type 2/3 responses have reciprocal inhibitory activities presenting a significant obstacle for development of vaccines designed to induce differential T-cell responses in mucosal and systemic immune compartments (3, 4, 7, 9, 10). Therefore, it is of critical importance for the field of vaccine immunobiology to define the specific mucosal and systemic responses protective against mucosally invasive, intracellular pathogens and to study the interactions between protective mucosal and systemic responses.
The identification of key cytokines required for generation of different T-cell subsets has made it possible to induce highly polarized antigen-specific type 1 and type 2 responses. IL-12 and IL-4 are critical for induction of type 1 and type 2 responses, respectively (23). Kumar et al. reported that in vitro-generated CD4+ Th1 but not Th2 cells could adoptively transfer protection against systemic T. cruzi challenges (18). We demonstrated that recombinant IL-12 and anti-IL-4 induced type 1-biased responses in vivo that were highly protective against normally lethal T. cruzi systemic challenges (15, 27). IL-4 plus anti-IFN- induced type 2 polarized responses that failed to protect against systemic T. cruzi challenges (15). More recently, we extended these observations showing that despite the prediction that an ideal vaccine should induce optimal mucosal type 2 immunity protective against initial invasion in the relevant mucosal lymphoid tissue, type 1 polarized responses induced by intranasal vaccination were optimally protective against both mucosal and systemic T. cruzi challenges (13).
We now use a second model system to examine the mucosal and systemic protective effects of T. cruzi-specific type 1 and type 2 responses. An infection/nifurtimox rescue protocol was employed to induce T. cruzi-specific "immune memory" in CD4, CD8, 2-microglobulin, inducible nitric oxide synthase (iNOS), IL-12, IFN-, and IL-4 knockout mice. The data demonstrate that type 1 responses are absolutely essential for both optimal mucosal and systemic protection.
MATERIALS AND METHODS
Mice. Six- to 8-week-old mice were used in these experiments. Wild-type C57BL6/J and the following knockout mice in the same genetic backgound were purchased from The Jackson Laboratory (Bar Harbor, ME): iNOS (–/–), IL-4 (–/–), 2-microglobulin (–/–), IFN- (–/–), and IL-12 p40 (–/–). Wild-type BALB/c mice were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). CD4 (–/–) and CD8 (–/–) knockout mice in the BALB/c genetic background were provided courtesy of T. Mak (Amgen, Ontario, Canada). Mice were housed under pathogen-free conditions throughout these studies.
Parasites and challenges. Blood-form trypomastigotes (BFT) and IMT from the Tulahuen strain of Trypanosoma cruzi were prepared as previously described (13). BFT were prepared from highly parasitemic BALB/c heparinized blood, enumerated by microscopic hemocytometer counts, and diluted with phosphate-buffered saline to the desired concentration. One hundred to 1,000 BFT were injected intraperitoneally for primary infections, and 1,000 to 20,000 BFT were injected subcutaneously at the base of the tail for subsequent infections. IMT were prepared from the collected excreta of T. cruzi-infected reduviid insects (Dipetalogaster maximus), enumerated by direct hemocytometer count, and fed orally to mice. These mice were fasted for 4 h and were given 0.5 ml of 1.5% sodium bicarbonate in Hanks' buffer intragastrically 15 min prior to oral delivery of 1,000 to 10,000 IMT parasites.
Infection/nifurtimox treatment protocols for induction of immune memory. For studies of systemic immunity, wild-type and knockout mice were infected intraperitoneally with 100 BFT. Seven days after infection, treatment with the trypanocidal drug nifurtimox (100 mg/kg/day intragastrically, Monday through Friday) was begun and continued for 4 weeks. Nifurtimox was obtained from John Becher at the Centers for Disease Control Drug Service, Atlanta, GA. One month after cessation of nifurtimox, mice were reinfected with 5,000 BFT intraperitoneally, followed by 4 more weeks of nifurtimox treatment beginning 1 week after the second parasite injection. To study mucosal immunity, a similar protocol was followed, substituting oral delivery of 1,000 to 10,000 IMT, instead of intraperitoneal BFT, for each infection/nifurtimox treatment cycle.
Assessment of protective immunity. Four to six weeks after the second cycle of T. cruzi infection/nifurtimox treatment was finished, nave and "memory immune" knockout mice were challenged without further nifurtimox treatment. To assess systemic protection, mice were challenged subcutaneously with 5,000 BFT (a standard lethal dose in wild-type mice) and followed for survival over >3 months. For assessment of mucosal memory immunity, mice were challenged with 1,000 to 10,000 IMT orally and studied 11 to 14 days later for the levels of T. cruzi replication detectable in gastric mucosal tissues—previously shown to be the site of initial mucosal invasion after oral IMT challenge (14)—and in the gastric draining lymph nodes. Total gastric DNA was extracted and studied by real-time PCR with primers specific for a T. cruzi-specific gene target as described previously (27). Briefly, 200 ng of total gastric DNA was added to SYBR green PCRs with primers specific for a 65-bp fragment of the T. cruzi gene coding for cruzipain. Samples were then run in an ABI Prism 7700 sequence detection system, and quantities in terms of T. cruzi molecular equivalents per 200 ng gastric DNA were calculated based on standard curves generated with DNA collected from a known number of T. cruzi epimastigotes. Draining gastric lymph nodes were harvested and passed through a wire mesh to obtain mononuclear cell suspensions. These cells were washed and then plated in two- to fivefold serial dilutions in medium (LDNT+) optimized for the outgrowth of T. cruzi epimastigotes from infected cells. These plates were inspected microscopically every 2 weeks for parasite outgrowth. The number of parasites per million lymph node cells can be calculated by dividing 1 million by the minimal number of cells plated that were associated with the outgrowth of parasites as described previously (14, 27).
Flow cytometry. Lymphocyte populations were stained with a combination of anti-CD8-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), and anti-CD3-peridinin-chlorophyll-protein C complex (PerCP; Pharmingen, San Diego, CA) and studied with a FACScaliber flow cytometer.
RESULTS
Development of T. cruzi memory immune model. Many of the relevant immune knockout mice are known to be highly susceptible to primary T. cruzi infection, generally succumbing to overwhelming infection within a few weeks after even very-low-level parasite challenges. In order to study memory immune responses we needed to develop a treatment rescue protocol that would prevent death in these immunodeficient mice during primary infection. Shown in Fig. 1 is the infection/nifurtimox treatment protocol used to generate T. cruzi-specific immune memory in wild-type and immunodeficient mice. Mice were challenged mucosally with IMT or systemically with BFT. Beginning 1 week after challenge, nifurtimox was administered intragastrically on a daily basis (Monday through Friday) for at least 1 month. After stopping nifurtimox, mice were observed for at least 1 more month to allow the effects of nifurtimox to wear off and be sure that recrudescence of overwhelming parasitemia did not occur. This cycle of infection, followed by nifurtimox treatment, followed by observation without further nifurtimox for at least 1 more month, was repeated before testing whether immune memory protective against mucosal and systemic T. cruzi challenges had developed in these mice. Control nave mice received parallel treatments with nifurtimox without the first two parasite challenges to control for any possible positive or negative effects of nifurtimox on immunity. Using this protocol we were able to generate memory immune animals for study in most knockout strains despite infection with virulent T. cruzi. To prove that survival in knockout strains was not due to spontaneous reversion to wild type, combinations of flow cytometric and cytokine secretion studies were completed postchallenge. Shown in Fig. 2 are representative flow cytometry histograms demonstrating the absence of CD4+ and CD8+ T cells in CD4 and CD8 knockout mice, respectively, surviving the T. cruzi infection/nifurtimox rescue protocol.
Molecular requirements for memory immune systemic protection. Table 1 presents the overall results from our new investigations of the immunologic requirements for protective systemic immunity. As described above, control mice had received 2 cycles of 1-month treatments with nifurtimox without preceding T. cruzi infection before investigating their ability to survive a primary BFT challenge. Memory immune mice had been immunized with 2 cycles of infection followed by nifurtimox rescue, prior to assessment of their ability to survive a tertiary challenge without further nifurtimox treatment. CD4, CD8, and iNOS knockout mice were able to develop robust immune memory protective against systemic parasite challenges despite previous publications documenting their enhanced susceptibility to primary T. cruzi infection (16, 25, 26). Surviving CD4, CD8, and iNOS knockout mice were challenged systemically again 2 months later with 10-fold-higher BFT doses, and these mice survived for at least another 2-month period of observation. These results demonstrate compensatory mechanisms for these defects in memory immune responses. On the other hand, even with 6 to 8 weeks of nifurtimox treatment, we were unable to rescue IL-12, IFN-, or 2-microglobulin knockout mice from death after primary T. cruzi BFT challenges. Therefore, the cytokine mainly responsible for generation of type 1 immunity (IL-12), the major effector cytokine associated with type 1 immunity (IFN-), and the downstream type 1 effector functions restricted by class I molecules coexpressed with 2-microglobulin are all absolutely essential for the development of immune memory protective against systemic T. cruzi infection. In contrast, the major generating and effector cytokine associated with type 2 responses, IL-4, was not necessary for the development of systemic protection. These results confirm numerous other reports demonstrating that type 1 immune responses are critical for systemic protection against intracellular pathogens.
Molecular requirements for mucosal protection. We developed an oral T. cruzi challenge model that mimics 1 of the natural mucosal routes of infection in humans (14). T. cruzi IMT invade via the gastric epithelium after oral ingestion, which drains to lymphoid tissue present within the lesser curvature of the stomach. Mucosal parasite replication can be studied in gastric epithelial and draining lymphoid tissue after oral challenge using a T. cruzi-specific real-time PCR assay and a quantitative parasite culture technique (14, 27). After harvesting total gastric DNA 10 to 14 days after oral challenge, molecular equivalents of T. cruzi present in these samples are quantified by real-time PCR. Single-cell suspensions of the gastric draining lymph node cells are prepared and plated under limiting dilution parasite culture conditions to quantitate viable T. cruzi organsims. In three previous publications (13, 14, 27), as well as throughout the experiments described here, similar results have been obtained with these real-time PCR and quantitative parasite culture techniques. Figure 3 presents comparative results obtained by real-time PCR and quantitative parasite culture in studies of wild-type and CD4 knockout mice.
Table 2 presents the overall real-time PCR results obtained in our mucosal immune studies. The oral IMT challenge is less virulent than systemic BFT challenge, and all immunodeficient mice could be rescued from overwhelming parasitemia after oral IMT challenge by nifurtimox treatment. This allowed us to directly examine whether mice with these genetic defects, including mice deficient in key components of type 1 immunity, could develop mucosal immune memory. CD4, CD8, and iNOS knockout mice were able to develop protective mucosal immunity not significantly different from that detected in wild-type mice, further demonstrating compensatory mechanisms for these immune defects. However, similar to the systemic immune studies reported above, and consistent with our previous type 1- and type 2-biasing vaccine work (13), IL-12, IFN-, and 2-microglobulin knockout mice failed to develop detectable protection against T. cruzi mucosal challenge. Therefore, we have clearly shown that the cytokine mainly responsible for generation of type 1 immunity (IL-12), the major effector cytokine associated with type 1 immunity (IFN-), and the downstream type 1 effector functions restricted by class I molecules coexpressed with 2-microglobulin are all absolutely essential for the development of both T. cruzi mucosal and systemic protective immune memory. In contrast, the major generating and effector cytokine for type 2 responses, IL-4, was not necessary for the development of immune memory protective against either mucosal or systemic challenge. Figure 4 graphically illustrates that IL-4 knockout mice, but not IFN- knockout mice, could develop protective mucosal immunity.
DISCUSSION
Because CD4+ Th2 cells (secreting IL-4, IL-5, and IL-6) generally are required for the optimal induction of secretory immunoglobulin A responses previously shown to be involved in mucosal protection (20, 21, 36), we were surprised to find in our recently published studies that intranasal vaccines inducing highly polarized type 1 responses without detectable vaccine-induced IL-4 responses were optimally protective against T. cruzi mucosal challenges (13). Therefore, we were interested in studying the importance of type 1 and type 2 immune responses for mucosal immunity in a second T. cruzi model system. Our previous work involved studies of the differential protective effects of type 1 and type 2 immune responses induced preinfection. To further address this important question in a second model system, we now have studied the capacity of knockout mice to develop protective immune memory. T. cruzi infection followed by nifurtimox treatment rescue was used to immunize CD4, CD8, 2-microglobulin, iNOS, IL-12, IFN-, and IL-4 knockout mice. CD4, CD8, and iNOS knockout mice developed compensatory mechanisms protective against mucosal and systemic T. cruzi infection. However, IL-12-, IFN--, and 2-microglobulin-deficient mice failed to develop any detectable mucosal or systemic protection. In contrast, IL-4 knockout mice had no deficiency in their ability to develop either mucosal or systemic protective immune memory. These results strongly confirm our earlier conclusion from studies with polarizing vaccination protocols that type 1 immunity provides optimal mucosal and systemic protection against a mucosally invasive, intracellular pathogen.
We have previously shown in SCID adoptive transfer experiments that CD4+ Th1 cells were required for transfer of the ability to develop primary immunity protective against systemic T. cruzi challenge (15). On the other hand, in the same publication we demonstrated that CD4+ T cells were not required for the transfer of protective systemic effector functions that had already developed in memory immune mice. These results suggest that CD4+ Th1 cells act primarily as helper cells for the development of protective effector mechanisms, but do not function directly as critical immune effector cells during T. cruzi infection. Based on these results, we predicted that CD4 knockout mice would be deficient in helper activity and would not be capable of developing protective systemic immune memory in our infection/nifurtimox rescue protocol. However, we found this not to be the case. As shown in Tables 1 and 2, CD4 knockout mice immunized with 2 infection/nifurtimox rescue cycles developed significant immune memory protective against further mucosal and systemic T. cruzi challenges. Surviving memory immune CD4 knockout mice were sacrificed 2 to 3 months after the final T. cruzi challenge, and their splenocytes were studied by flow cytometry to confirm the absence of CD4+ T cells (Fig. 2). Therefore, although in the normal wild-type genetic background it appears that CD4+ Th1 cells are required for the development of protective immune memory, mice lacking CD4+ T cells are capable of developing protective immunity by some alternative mechanism(s). Recent work has demonstrated that nonconventional subsets of T cells including (i) CD1-restricted T-cell receptor /+, CD4 CD8 double-negative T cells (24, 33), (ii) T cell receptor /+ T cells (17, 35), and (iii) NK T cells (6, 28) can develop potent type 1 immune phenotypes, and perhaps one or more of these subsets may provide alternative helper functions for the development of protective immune memory in the CD4 knockout mice studied with our infection/nifurtimox rescue model. Learning precisely what these alternative mechanisms are and how to induce them may result in the development of new vaccination strategies useful in patients with functional depletion of CD4+ T cells (e.g., AIDS patients).
The presence of CD8+ cytotoxic T lymphocytes (CTL) capable of recognizing and lysing T. cruzi-infected cells has been shown to correlate with protective immunity (25, 29, 30), and vaccines that induce T. cruzi-specific CD8+ CTL responses often are extremely effective at inducing protective immunity (8, 34). Our results are consistent with these previous results but also present new information. We initially tried infection/nifurtimox rescue protocols delaying treatment until 2 weeks after T. cruzi challenge. CD8 knockout mice challenged with as few as 100 BFT died usually before nifuritmox was begun or shortly thereafter when nifurtimox treatment was delayed for this long after infection (data not shown). These results illustrate the importance of CD8+ CTL responses for the immune clearance of primary infection. However, after we revised our protocol to begin nifurtimox earlier, 1 week after infection, we found that some CD8 knockout mice could be rescued after low doses of BFT challenge. Treatment of these mice for 1 month with nifurtimox resulted in long-term survival of these animals; recurrence of parasitemia was not seen after cessation of nifurtimox treatment. Their survival allowed us to study whether CD8+ CTL are necessary for the development of protective immune memory. Despite their extreme susceptibility to primary T. cruzi systemic challenges, CD8 knockout mice rescued by the early nifurtimox treatment were able to develop systemic immunity protective against repeated BFT challenges (Table 1 and Results). In addition, using the less virulent oral challenges with T. cruzi IMT, we were more easily able to generate memory immune mice in the CD8 knockout background that were found to develop protective immunity against further oral IMT challenges (Table 2), providing additional support for the conclusion that CD8+ T cells are not absolutely required for the development of protective T. cruzi memory immunity. Surviving memory immune CD8 knockout mice were sacrificed 2 to 3 months after the final T. cruzi challenge, and their splenocytes were studied by flow cytometry to confirm the absence of CD8+ T cells (Fig. 2). The fact that 2-microglobulin-deficient mice were found to be much more susceptible to T. cruzi infection, being unable to develop any protective immune memory, suggests that they lack other important effector functions in addition to defects in CD8+ CTL. T cells stimulated by other nonpolymorphic class I-like molecules (e.g., CD1) also require 2-microglobulin expression and may provide complementary cytolytic effector functions important for protective T. cruzi immunity. Identifying these alternative mechanisms of protective effector function will be important for future vaccine development.
Nitric oxide is a known trypanocidal agent capable of killing both intracellular and extracellular T. cruzi. Many groups, including ours, have shown that type 1 cytokine responses can induce nitric oxide production leading to inhibition of T. cruzi growth within infected macrophages (11, 15, 22, 32). However, immune memory induced by our infection/nifurtimox rescue protocol could protect against both mucosal and systemic T. cruzi challenges even in iNOS knockout mice (Tables 1 and 2). These results demonstrate that other microbicidal functions such as superoxide derivatives (2, 19) and/or peroxynitrite (5, 19, 31) can provide compensatory trypanocidal activity effective for protective immune memory in mice lacking the inducible nitric oxide synthase gene. Measurements of these other microbicidal functions could provide important surrogate markers for studies of protective immunity.
In summary, we have shown that the murine immune system is capable of compensating for multiple deficiencies of key immunologic functions important for T-cell memory. Elucidating the compensatory mechanisms will have important implications for the development of vaccines and/or immunotherapies for use in patients with certain immunodeficiencies. In addition, combined with our previous studies of type 1 and type 2 polarizing vaccination protocols, the current results conclusively demonstrate the critical importance of type 1 immunity for both mucosal and systemic protection against T. cruzi infection. These results have important practical implications suggesting that vaccines designed to induce concurrent mucosal and systemic protection against mucosally invasive, intracellular pathogens can focus on a more feasible goal of inducing homologous type 1 immune responses in both mucosal and systemic tissues, rather than differential induction of type 2 and type 1 responses in mucosal and systemic tissues, respectively. However, to date this question has only been addressed in our T. cruzi gastric invasion model. T. cruzi can invade mucosal surfaces after conjunctival challenges as well. In addition, several other important human pathogens establish chronic intracellular infections after invasion through other mucosal surfaces (e.g., human immunodeficiency virus and Mycobacterium tuberculosis). Future work will need to test the more general hypothesis that type 1 immunity will provide optimal protection of other mucosal surfaces against invasion by T. cruzi and other important human intracellular pathogens.
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