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The Journal of Immunology, 2007, 178, 5753 -5761
Copyright © 2007 by The American Association of Immunologists, Inc.
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Protection from Direct Cerebral Cryptococcus Infection by Interferon-{gamma}-Dependent Activation of Microglial Cells1

Qing Zhou*, Ruth A. Gault{dagger}, Thomas R. Kozel{dagger} and William J. Murphy2,{dagger}

* Division of Blood and Marrow Transplantation, Cancer Center and Department of Pediatrics, MMC 109, University of Minnesota, Minneapolis, MN 55455; and {dagger} Department of Microbiology and Immunology, University of Nevada, Reno, Reno, NV 89557


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The brain represents a significant barrier for protective immune responses in both infectious disease and cancer. We have recently demonstrated that immunotherapy with anti-CD40 and IL-2 can protect mice against disseminated Cryptococcus infection. We now applied this immunotherapy using a direct cerebral cryptococcosis model to study direct effects in the brain. Administration of anti-CD40 and IL-2 significantly prolonged the survival time of mice infected intracerebrally with Cryptococcus neoformans. The protection was correlated with activation of microglial cells indicated by the up-regulation of MHC II expression on brain CD45lowCD11b+ cells. CD4+ T cells were not required for either the microglial cell activation or anticryptococcal efficacy induced by this immunotherapy. Experiments with IFN-{gamma} knockout mice and IFN-{gamma}R knockout mice demonstrated that IFN-{gamma} was critical for both microglial cell activation and the anticryptococcal efficacy induced by anti-CD40/IL-2. Interestingly, while peripheral IFN-{gamma} production and microglial cell activation were observed early after treatment, negligible IFN-{gamma} was detected locally in the brain. These studies indicate that immunotherapy using anti-CD40 and IL-2 can augment host immunity directly in the brain against C. neoformans infection and that IFN-{gamma} is essential for this effect.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The brain and the CNS represent formidable barriers for successful immunotherapy in infectious disease and cancer. Cryptococcus neoformans is a type of encapsulated yeast that causes the most common life-threatening AIDS-related fungal infection (1). The major target organ of C. neoformans infection is the CNS, with meningoencephalitis and meningitis being the most common clinical presentations of cryptococcosis progression. Immune responses in the CNS are particularly limited by the blood brain barrier (BBB),3 a layer of tightly packed capillary endothelial cells that serves to control the passage of molecules to the brain and so protect the brain integrity.

Microglial cells are one of the few residential immune cell types found in the brain (2). The critical importance of microglial cells has only begun to be recognized in immune responses to brain injury (3, 4, 5, 6), infections (7, 8, 9, 10, 11), tumor (2, 12, 13, 14), and autoimmune disease (11, 15, 16, 17). At a resting stage, microglial cells possess a phenotype with low expression of the leukocyte common Ag CD45, positive expression of the beta2 integrin CD11b, and very low or negative expression of MHC II. Upon activation, microglial cells become phagocytic macrophage-like cells and up-regulate the expression of CD45 and MHC II as well as other stimulatory molecules (18, 19, 20). Several studies have provided the evidence for beneficial aspects of microglial cell activation in response to CNS infections (7, 8, 9). More importantly, MHC II-positive perivascular microglial cells are reported to be critical for host resistance to C. neoformans (21).

CD40/CD40 ligand interaction has been demonstrated to be an important connection between innate and adaptive immunity (22, 23). CD40, a member of the TNFR superfamily, is found on APCs. CD40 is also found on microglial cells, especially at the activated stage (24, 25). A recent study has shown that CD40 and CD40L interaction is essential for full microglial cell activation (24).

We have recently shown a therapeutic effect against systemic cryptococcosis by using CD40 stimulation with an agonist mAb in combination with IL-2 (26). This antifungal effect was correlated with the reduced dissemination of C. neoformans as indicated by the decreased fungal burdens observed in the various organs of mice, including the brain. However, in this model where Cryptococcus was given i.v. it was unclear whether anti-CD40/IL-2 treatment was promoting systemic immune responses that prevented the yeast from entering the brain or was enhancing the resistance locally in the brain. Hence, we applied anti-CD40 and IL-2 immunotherapy in a murine model of direct cerebral Cryptococcus infection to study the effect of systemic anti-CD40/IL-2 administration on brain resistance. Our results demonstrated that CD40 stimulation with IL-2 significantly prolonged the survival time of mice previously infected by the cerebral route with C. neoformans. This therapeutic effect of anti-CD40/IL-2 was associated with enhanced microglial cell activation as indicated by the up-regulation of MHC II expression, and IFN-{gamma} was critical for the efficacy of this immunotherapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 mice were obtained from Charles River Laboratories and the National Cancer Institute (Frederick, MD). B6.129S7-Ifngtm1Ts/J (IFN-{gamma} knockout (KO)), B6.129S7-Ifngr1tm1Agt/J (IFN-{gamma}R KO), and C57BL/6J wild-type (WT) control mice were purchased from The Jackson Laboratory. All mice were maintained in an animal facility at the University of Nevada, Reno, NV, and all studies were approved by the university’s Institutional Animal Care and Use Committee. All mice were between 8 and 16 wk of age.

Murine model of cerebral cryptococcosis

Serotype A strain C. neoformans CN6 was used. C. neoformans was cultured in 5 ml of Sabouraud (SAB) glucose broth (BD Biosciences) for 6–8 h in a shaking incubator at 30°C and 250 rpm before the transfer of 5–6 million cells to 70 ml of SAB glucose broth in a 250-ml flask. C. neoformans was then cultured overnight in a shaker at 30°C and 100 rpm before being harvested, washed, counted, and diluted in Dulbecco’s PBS (DPBS) (Mediatech). Mice were infected with C. neoformans (20–30 yeast cells) via the intracerebral (i.c.) route as described previously (27). Briefly, mice were anesthetized with isoflurane vapor. A 50-µl inoculum of C. neoformans or sterile DPBS was injected at a point 5–6 mm posterior to the eyes on the midline. Mice recovered fully from the i.c. injection after 30 min. The viability of the inoculum was determined by quantitative culturing on SAB dextrose agar plates (BD Biosciences). Viability was >95%. Infected mice were observed for morbidity, primarily brain edema and lethargy with partial paralysis. Morbid mice were euthanized by CO2 based on 20% weight loss and clinical signs of meningitis including hydrocephalus, unbalanced movement, or paralysis.

Anti-CD40 and IL-2 treatment in mice

Recombinant human IL-2 (TECIN (Teceleukin); Roche) was provided by the National Cancer Institute (Frederick, MD). Agonist rat anti-mouse CD40 (clone FGK115B-3, a subclone of FGK115 that was a gift from Dr. B. Blazar, University of Minnesota, Minneapolis, MN), was produced as demonstrated previously (26). Protein/IgG quantification was performed by spectrophotometry, and Ab content was determined by rat IgG ELISA. The endotoxin level of the purified Ab was 1.77 endotoxin units/mg Ab as determined by quantitative Limulus amoebocyte lysate assay (QCL-1000; BioWhittaker). Purified rat IgG was purchased from Jackson ImmunoResearch Laboratories.

Mice were treated with anti-CD40/IL-2 18–20 h after C. neoformans infection for all of the studies. The regimen is consistent with a previous study using a dissemination model with minor modifications (26). Agonist anti-CD40 or isotype control rat IgG (Jackson ImmunoResearch Laboratories) was administered i.p. once a day for 4 days (50 µg/dose). IL-2 was given at 500,000 IU i.p. twice a day twice a week for a total of eight injections. DPBS (control for IL-2; 0.2 ml/dose) was given i.p. on the same schedule as IL-2. Six to eight mice per group were used in survival studies and each survival study was repeated 2–3 times.

Organ C. neoformans colony forming assay

Five days into the anti-CD40 and IL-2 treatment, mice were euthanized by CO2 and multiple organs including the brain, lungs, livers, and kidneys were collected. Organ samples were prepared in sterile water with a homogenizer and were appropriately diluted. The diluted samples (100 µl) were then plated on SAB plates and cultured at 30°C for two days. C. neoformans colonies were counted, calculated, and presented as CFU per organ.

In vivo CD4+ T cell depletion studies

CD4+ T cells were depleted with an anti-CD4 Ab (clone GK1.5, a gift from Dr. G. B. Huffnagle, University of Michigan, Ann Arbor, MI). Mice were injected i.p. with anti-CD4 Ab (300 µg/dose) or isotype control rat IgG (Jackson ImmunoResearch Laboratories) starting 1 day before C. neoformans infection and every 5 days for a total of three injections during the full course of anti-CD40 and IL-2 treatments. Cell depletion efficiency (>90%) was determined by flow cytometric analysis of splenocytes.

Brain mononuclear cell preparation

Three mice per group were sacrificed 5 or 11 days after C. neoformans infection and the brains from each group were harvested and minced. Brain leukocytes were isolated by gradient separation using 40 and 60% Percoll (28). After centrifugation at 1600 rpm at room temperature for 45 min, the middle layer was carefully removed and the cells were washed with staining buffer containing 1% FBS and 1% penicillin/streptomycin in DPBS and resuspended in blocking buffer containing 1% human AB serum, 1% FBS, and 1% penicillin/streptomycin in DPBS for labeling.

Flow cytometric analysis

For flow cytometric analysis, three mice per group were sacrificed 5 or 11 days after C. neoformans infection. Mononuclear cells from the brains were prepared as described above. For identification of various leukocyte populations, brain leukocyte suspensions were labeled for 25 min at 4°C in the dark with the following Abs: FITC-conjugated rat anti-mouse CD11b, FITC-conjugated rat anti-mouse CD3, PE-conjugated rat anti-mouse CD4, PE-conjugated rat anti-mouse CD8a, PE-Cy 5-conjugated rat anti-mouse CD45 (BD Pharmingen), PE-conjugated rat anti-mouse IgG2b, and/or PE-conjugated rat anti-mouse I-A/I-E (eBioscience). Cells were then washed with staining buffer. All results were obtained using a FACScan flow cytometer (Becton Dickinson). Forward and side scatter settings were gated to exclude debris. Approximately 10,000 cells were analyzed for each determination. Nonspecific binding was corrected with isotype-matched controls.

RNase protection assay

For the determination of cytokine mRNA levels in the brains, three mice per group were sacrificed 5 or 11 days after the injection of C. neoformans and the brain samples were collected. RNA samples were extracted by traditional phenol/chloroform extraction as demonstrated previously (29). RNA Samples were then assayed with a multiprobe RNase protection assay system (R&D Systems) according to the manufacturer’s instructions. The multiprobe template sets mCK-2b and mCK-3b were used. Mouse mRNAs detected in mCK-2b template sets include those of IL-12p35, IL-12p40, IL-10, IL-1{alpha}, IL-1beta, IL-1R{alpha}, IL-18, IL-6, IFN-{gamma}, MIF, L32, and GAPDH; mRNAs detected in mCK3b template sets include those of TNF-beta, LT-beta, TNF-{alpha}, IL-6, IFN-{gamma}, IFN-beta, TGF-beta1, TGF-beta2, TGF-beta3, MIF, L32, and GAPDH. The housekeeping genes L32 and GAPDH were used to standardize the result of each mRNA sample.

Statistics

Survival data were plotted by using the Kaplan-Meier method and analyzed ith the log-rank test. A comparison of organ CFU was made with a two-way ANOVA test. Comparisons of MHC II expression on microglial cells was made with a one-way ANOVA test. Comparisons of brain cytokine mRNA expression and T cell infiltration in the brains were made with an unpaired Student’s t test. A-value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Anti-CD40 and IL-2 immunotherapy enhances the host resistance to cerebral cryptococcosis

We have previously shown that CD40 stimulation using the agonist mAb ({alpha}-CD40) together with IL-2 can augment an anticryptococcal response in a murine disseminated disease model (26). However, the effects of anti-CD40/IL-2 treatment on cerebral resistance were unclear. To address this question, we applied this immunotherapeutic regimen in a direct cerebral infection model where C. neoformans was directly injected into the brains of mice. In this model, a markedly lower challenge inoculum (20–30 cells/dose, compared with 105 cells/dose used in systemic cryptococcosis model) of C. neoformans was used because of extremely aggressive infection in the brain by the yeast. The C. neoformans infection was primarily confined locally in the brain at the early stage (day 5; Fig. 1B, p < 0.05). Dissemination of the yeast to other organs including the lungs, liver, and kidneys was observed at the time of the death of mice, ~10–15 days postinfection due to CNS pathology (data not shown). One day following C. neoformans infection, rat IgG/PBS control, anti-CD40 alone, IL-2 alone, or anti-CD40/IL-2 was administered systemically. The dosage of anti-CD40/IL-2 used was similar to the regimen used to elicit antifungal effects in our previously reported study of a disseminated cryptococcosis model (26). Remarkably, similar to the results observed with the disseminated cryptococcosis model, treatment with neither anti-CD40 nor IL-2 alone failed to alter the course of cerebral C. neoformans infection, whereas anti-CD40 in combination with IL-2 significantly prolonged the survival time of mice infected with Cryptococcus via the i.c. route (Fig. 1A, p < 0.005). This increase in survival was correlated with a significant reduction in fungal burdens in the brains of anti-CD40/IL-2-treated mice on day 5 (Fig. 1B, p < 0.05). This indicated that systemic treatment with anti-CD40 and IL-2 is capable of promoting host resistance directly in the brains of mice infected by the i.c. route with C. neoformans.


Figure 1
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  		FIGURE 1. Anti-CD40 and IL-2 immunotherapy provided antifungal effects in mice previously infected i.c. with C. neoformans. One day after infection (20 yeast cells/dose), C57BL/6 mice (n = 7 mice/group) were treated with IL-2 alone ({blacktriangleup}), anti-CD40 alone ({blacktriangledown}), anti-CD40 and IL-2 ({square}), or control rat IgG and PBS ({blacksquare}). Anti-CD40 or isotype control rat IgGs were administered at 50 µg/dose i.p. once a day for 4 days. IL-2 was administered at 500,000 IU/dose i.p. twice a day twice a week for a total of eight injections. DPBS (vehicle control for IL-2; 0.2 ml/dose) was given at the same schedule as IL-2. A, The combination of anti-CD40 ({alpha}-CD40) and IL-2 ({square}) significantly prolonged the survival time of mice compared with rat IgG/PBS control ({square} vs {blacksquare}, p < 0.005), IL-2 alone ({square} vs {blacktriangleup}, p < 0.005), or anti-CD40 alone ({square} vs {blacktriangledown}, p < 0.05). Survival analysis was plotted according to the Kaplan-Meier method. Statistical differences were determined with the log-rank test. B, Five days after infection C. neoformans burdens in the brains of mice were significantly higher than those in other organs (p < 0.001). There was no C. neoformans detected in the liver by CFU assay on day 5. The combination of anti-CD40 and IL-2 significantly reduced fungal burdens in the organs of treated mice compared with mice treated with rat IgG/PBS (p < 0.005), IL-2 alone (p < 0.005), or anti-CD40 alone (p < 0.05) on day 5. Statistical differences were determined with two-way ANOVA.

 
Anti-CD40 and IL-2 enhanced MHC class II expression on microglial cells

We then determined the mechanism underlying the increase in resistance after anti-CD40 and IL-2 immunotherapy by assessing the status of cells within the brain. To determine whether microglial cells were affected by the systemic administration of anti-CD40/IL-2, brain mononuclear cells were isolated on day 5 and labeled for microglial cell surface marker CD45, CD11b, and MHC class II, which is a marker for microglial cell activation (24, 30). Cells were gated based on CD45low and CD11b expression, and MHC II expression was measured. Neither anti-CD40 nor IL-2 alone induced MHC II expression on microglial cells. However, anti-CD40 in combination with IL-2 significantly up-regulated MHC II expression compared with rat IgG/PBS, anti-CD40 alone, or IL-2 alone controls (Fig. 2, p < 0.001), and this was correlated with protection. Increased MHC II expression was also observed at a later time point of anti-CD40/IL-2 treatment (day 11; data not shown).


Figure 2
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  		FIGURE 2. Anti-CD40 and IL-2 up-regulated MHC class II expression on microglial cells. Five days after C. neoformans i.c. infection, three mice per group were sacrificed. Brain samples were taken and leukocytes were isolated and labeled with specific Abs to surface molecules. Flow cytometric analysis was used to assess MHC class II molecule expression. Flow histogram graphs of MHC class II expression on CD45lowCD11b+ cells were shown (A–D). Isotype control histograms are depicted shaded and the specific MHC II are shown as open histograms. Statistical differences are shown in E. Only the combination of anti-CD40 ({alpha}-CD40) and IL-2 enhanced MHC class II expression on microglial cells, and this enhancement was significant compared with rat IgG/PBS, IL-2 alone, and anti-CD40 alone treatment. Statistical differences were determined by one-way ANOVA with post hoc comparisons using a Tukey test. p < 0.05 is considered significant.

 
To verify whether cryptococcal stimulation was required for this effect on microglial cells, mice were injected i.c. with PBS instead of C. neoformans and then treated with anti-CD40/IL-2. Significantly increased MHC II expression on CD45lowCD11b+ cells was also found in the brains of anti-CD40- and IL-2-treated mock infected mice (Fig. 3, p < 0.001) compared with rat IgG/PBS controls. These results indicate that anti-CD40 and IL-2 immunotherapy can rapidly augment microglial cell activation independently of the presence of C. neoformans.


Figure 3
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  		FIGURE 3. MHC class II up-regulation induced by anti-CD40 and IL-2 was independent of Ag stimulation. C57BL/6 mice were injected with either C. neoformans or PBS i.c. before anti-CD40 and IL-2 treatment. Five days after C. neoformans or PBS i.c. injection, three mice per group were sacrificed for brain leukocyte determination. Flow cytometric analysis was used to assess MHC class II molecule expression. Flow histogram graphs of MHC class II expression on CD45lowCD11b+ cells were shown (A–D). A bar graph along with statistical differences is shown in E. Anti-CD40 ({alpha}-CD40) and IL-2 significantly enhanced MHC class II expression on microglial cells either in the presence or absence of C. neoformans. Statistical differences were determined by one-way ANOVA with post hoc comparisons using a Tukey test. p < 0.05 is considered significant. Representative data from one of two experiments are shown.

 
Peripheral CD4+ T cells are not necessary for the protection provided by anti-CD40 and IL-2 immunotherapy in the brain

C. neoformans may disseminate to extrapulmonary sites, particularly the brain, when the host immune system is suppressed with profound CD4+ T cell deficiency during AIDS. Therefore, applying several immunotherapies for cryptococcosis in clinical settings is not optimal given a potential dependence on CD4+ T cells for efficacy (31, 32, 33). To study the anticryptococcal efficacy of anti-CD40/IL-2 immunotherapy in a clinically relevant condition, CD4+ T cells in mice were previously depleted by a specific anti-CD4 Ab 1 day before C. neoformans infection and during the full course of anti-CD40/IL-2 treatment. Anti-CD40 and IL-2 treatment significantly prolonged the survival time of CD4+ T cell-depleted mice previously infected i.c. with C. neoformans (Fig. 4, p < 0.0002; CD4-depleted rat IgG/PBS vs CD4 depleted anti-CD40/IL-2) as well as the WT mice (Fig. 4, p < 0.005; WT {alpha}-CD40/IL-2 vs WT rat IgG/PBS). Prior CD4+ T cell depletion also did not alter the outcome of MHC II up-regulation on microglial cells (Fig. 5, p < 0.001; CD4-depleted rat IgG/PBS vs CD4-depleted anti-CD40/IL-2), further demonstrating the importance of the activation of this cell type in correlation with the protection. Although CD4+ T cell-depleted mice tended to succumb at an earlier time to cerebral cryptococcosis as compared with WT controls, the difference was not significant (p = 0.09). These results suggest that CD4+ T cells are not required for microglial cell activation or for the anticryptococcal efficacy of anti-CD40 and IL-2 immunotherapy in cerebral cryptococcosis.


Figure 4
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  		FIGURE 4. CD4+ T cells are dispensable for the antifungal effect of anti-CD40 and IL-2 treatment in the brain. C57BL/6 mice (n = 8 mice/group) received either isotype control rat IgG or anti-CD4 ({alpha}-CD40) Ab starting 1 day prior to C. neoformans injection and every 4 days during the anti-CD40 and IL-2 treatment. The combination of anti-CD40 and IL-2 treatments significantly prolonged the survival time of CD4+ T cell-depleted (dep) mice with cerebral cryptococcosis ({triangleup} vs {blacktriangleup}, p < 0.0002), as well as WT mice ({square} vs {blacksquare}, p < 0.005). Survival analysis was plotted according to the Kaplan-Meier method, and statistical differences were determined with the log-rank test. Representative data from one of two similar experiments are shown.

 

Figure 5
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  		FIGURE 5. CD4+ T cells were not required for MHC class II up-regulation induced by anti-CD40 and IL-2 in the brain. C57BL/6 mice were received either isotype control rat IgG or anti-CD4 Ab starting 1 day prior to C. neoformans injection and every 4 days during the anti-CD40 and IL-2 treatment. Five days after C. neoformans i.c. injection, three mice per group were sacrificed for brain leukocyte determination. Flow cytometric analysis was used to assess MHC class II molecule expression. Flow histogram graphs of MHC class II expression on CD45lowCD11b+ cells are shown (A–D). A bar graph along with statistical differences is shown in E. Anti-CD40 ({alpha}CD40) and IL-2 significantly increased MHC class II expression on microglial cells in CD4+ T cell-depleted (dep) mice (p < 0.001) as well as WT controls (p < 0.001). Statistical differences were determined by one-way ANOVA with post hoc comparisons using a Tukey test. p < 0.05 is considered significant. Representative data from one of two experiments were shown.

 
IFN-{gamma} signaling is required for the protection induced by anti-CD40 and IL-2

IFN-{gamma} is a multifunctional Th1 cytokine involved in brain inflammation and the pathogenesis of various brain diseases (34, 35) and has been shown to participate in the immune response in the brain through activating microglial cells (36, 37). IFN-{gamma} has been demonstrated to enhance the anticryptococcal activity of murine microglial cells in vitro by inducing MHC class II expression and NO production (37, 38, 39). To determine whether IFN-{gamma} was an important mediator of anti-CD40 and IL-2 immunotherapy in the brain, the efficacy of anti-CD40/IL-2 treatment in IFN-{gamma} KO (GKO) mice were compared with WT mice. Our results showed that the absence of IFN-{gamma} completely abrogated the protection induced by anti-CD40 and IL-2 immunotherapy for cerebral cryptococcosis as compared with the prolonged survival time of treated WT mice (Fig. 6A, p < 0.005). Importantly, studies of brain leukocytes by flow cytometric analysis revealed that in the absence of IFN-{gamma}, anti-CD40 and IL-2 treatment resulted in a complete failure in inducing MHC class II expression on microglial cells (Fig. 7, A–E), indicating the correlation between the activation of microglial cells and the protection in the brain.


Figure 6
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  		FIGURE 6. Requirement for IFN-{gamma} and IFN-{gamma}R in the antifungal effects of anti-CD40 and IL-2 treatment. A, C57BL/6J WT and GKO mice (n = 8 mice/group) were infected with C. neoformans (30 cells/dose) 1 day before treatment with either rat IgG/PBS control or anti-CD40 ({alpha}-CD40) and IL-2. The combination of anti-CD40 and IL-2 failed to protect GKO mice from cerebral cryptococcosis ({triangleup} and {blacktriangleup}), whereas anti-CD40 and IL-2 significantly prolonged the survival time of WT mice ({square} vs {blacksquare}, p < 0.005). B, Requirement for IFN-{gamma}R in the antifungal effects of anti-CD40 and IL-2 treatment. C57BL/6J WT and IFN-{gamma}R KO mice (n = 8 mice/group) were infected with C. neoformans (20 cells/dose) followed by treatment with either rat IgG/PBS control or anti-CD40 and IL-2. The combination of anti-CD40 and IL-2 failed to prolong the survival time of the IFN-{gamma}R KO mice previously infected with C. neoformans i.c. ({triangleup} and {blacktriangleup}), while anti-CD40 and IL-2 significantly prolonged the survival time of WT mice ({square} vs {blacksquare}, p < 0.005). Survival analysis was plotted according to the Kaplan-Meier method, and statistical differences were determined with the log-rank test.

 

Figure 7
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  		FIGURE 7. IFN-{gamma} and IFN-{gamma}R were required for MHC class II up-regulation induced by anti-CD40 and IL-2 in the brain. C57BL/6J WT mice or GKO mice were infected with C. neoformans (30 cells/dose) i.c. and treated with either rat IgG/PBS or anti-CD40/IL-2 1 day after the infection. Five days after the C. neoformans i.c. injection, three mice per group were sacrificed for brain leukocyte detection. Flow cytometric analysis was used to assess MHC class II molecule expression. Flow histogram graphs of MHC class II expression on CD45lowCD11b+ cells are shown (A–D). A bar graph and statistical differences are shown in E. Absence of IFN-{gamma} totally abrogated the anti-CD40 ({alpha}-CD40)/IL-2 induced MHC class II expression, while MHC class II expression was significantly up-regulated in WT treated mice (p < 0.01). F, C57BL/6J WT mice or IFN-{gamma}R KO mice were infected with C. neoformans (20 cells/dose) i.c. and treated with either rat IgG/PBS or anti-CD40/IL-2. Five days after C. neoformans i.c. injection, three mice per group were sacrificed for brain leukocyte detection. The absence of the IFN-{gamma}R totally abrogated the anti-CD40/IL-2-induced MHC class II expression, while MHC class II expression was significantly up-regulated in WT treated mice (p < 0.01). Statistical differences were determined by one-way ANOVA with post hoc comparisons with Tukey test. p < 0.05 is considered significant. Representative data from one of two experiments are shown.

 
A recent study with IFN-{gamma}R KO mice demonstrated that IFN-{gamma}R is also required for inducing a protection against C. neoformans in the lungs of mice (40). To examine whether IFN-{gamma}R is necessary for anticryptococcal efficacy of anti-CD40 and IL-2 in the brain, IFN-{gamma}R KO mice were infected with C. neoformans and treated with anti-CD40/IL-2. Similar to the responses observed with GKO mice, anti-CD40/IL-2 failed to protect IFN-{gamma}R KO mice from cerebral cryptococcosis (Fig. 6B). Moreover, the absence of IFN-{gamma}R abrogated the induction of MHC II expression on microglial cells (Fig. 7F). Thus, IFN-{gamma} is critical for the anticryptococcal efficacy of anti-CD40/IL-2 in the brain, as well as the activation of microglial cells.

IFN-{gamma} mRNA level was elevated in the brains of mice at a later time point (day 11) but not early (day 5) and was correlated with increased T cell infiltration into the brain

In the brain, endogenous IFN-{gamma} can be produced by activated T and NK cells in response to proinflammatory signals such as IL-12 secretion by APCs or activated macrophages and microglial cells (10, 41). IFN-{gamma} also has the ability to cross the BBB and remain functional to facilitate brain immune cells in the inflammation process (42). To investigate whether IFN-{gamma} was being produced locally in the brain by anti-CD40 and IL-2 immunotherapy at the time when the up-regulation of MHC II on microglial cells was observed (days 5 and 11), mice were sacrificed 5 or 11 days after C. neoformans infection and brain samples as well as spleen samples were collected. mRNAs were isolated and a RNase protection assay was used to determine cytokine mRNA production in both organs. Brain mononuclear cells were also isolated at the same time points to assess T cell infiltration in the brains. The results demonstrated no difference in IFN-{gamma} mRNA level in the brains by anti-CD40/IL-2 treatment by day 5 (Fig. 8A). This result was correlated with little T cell infiltration in the brain at this time point (Fig. 8, E and F). However, anti-CD40/IL-2 treatment induced significant increases in IFN-{gamma} mRNA levels in the spleens of treated mice, indicating systemic IFN-{gamma} production by this treatment on day 5 (Fig. 8B, p < 0.05). This induction of systemic IFN-{gamma} production was confirmed by elevated serum IFN-{gamma} levels observed 5 days into the treatment that were similar to the levels previously observed on day 11 (26) (Fig. 8D). These results demonstrated that the IFN-{gamma}-dependent activation of microglial cells at an early time point (day 5) is associated with increased peripheral IFN-{gamma} induction, but not in the brain. At a later time point of anti-CD40/IL-2 treatment (day 11) significantly elevated IFN-{gamma} mRNA levels in the brains was observed (Fig. 8C, p < 0.05). This result was correlated with significant increase in T lymphocyte infiltration to the brains induced by anti-CD40/IL-2 (Fig. 8, G and H, p < 0.05). These results further indicate that anti-CD40/IL-2 treatment could promote IFN-{gamma} local production through increasing T cell infiltration in the brain at later time points.


Figure 8
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  		FIGURE 8. IFN-{gamma} mRNA production was increased locally in the brain by infiltrating T cells induced by anti-CD40 ({alpha}-CD40)/IL-2 on day 11; however, it was not changed at an early time point (day 5). C57BL/6 mice were infected with C. neoformans (15 cells/dose) and treated with either control rat IgG/PBS or anti-CD40/IL-2. Five or 11 days after C. neoformans infection, three mice per group were sacrificed. Brain and spleen samples were prepared. mRNA was isolated and a RNase protection assay was used to determine multiple cytokine mRNA levels in the brains (A and C) and spleens (B). Data were standardized with housekeeping gene L32 and presented as a mRNA:L32 ratio. At the same time point (day 5 and Day 11), brain mononuclear cells were also isolated and flow cytometric analysis was used to determine T lymphocyte infiltration in the brains. A, IFN-{gamma} mRNA level was not changed by anti-CD40/IL-2 treatment in the brains of mice infected with C. neoformans i.c. on day 5. B, Anti-CD40 and IL-2 increased IFN-{gamma} mRNA expression in the spleens of mice (p < 0.05) on day 5. C, the IFN-{gamma} mRNA level was elevated by anti-CD40/IL-2 treatment in the brains of mice on day 11. D, Serum IFN-{gamma} levels were determined by cytokine ELISA 5 and 11 days after infection. Serum IFN-{gamma} levels were significantly increased by anti-CD40/IL-2 on days 5 and 11 (p < 0.01). E–H, Little CD4+ (E) and CD8+ T cell (F) infiltration was found in the brains at an early time point (day 5). A significant increase in the CD4+ (G) and CD8+ T cell populations (H) were found in the brains of mice induced by anti-CD40/IL-2 later on day 11. Statistical differences were determined by an unpaired Student t test. p < 0.05 is considered significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The CNS represents a significant barrier to immune and pharmacologic intervention in a variety of disease states ranging from infectious disease to cancer. C. neoformans infection, which causes meningoencephalitis and meningitis, is one of the four most frequent CNS-involving conditions leading to the morbidity and mortality of AIDS patients (43). Antifungal therapies currently available to treat cerebral cryptococcosis are limited and inefficient (44), with extremely high cost and high risk for relapse if a secondary prophylaxis is not applied (43, 45). Although immunotherapy perhaps offers a potential venue for cryptococcosis treatment, it was unclear whether beneficial effects within the brain could result with systemic administration. Our previous studies have demonstrated that anti-CD40 and IL-2 immunotherapy protects mice from disseminated cryptococcosis. Our results showed that anti-CD40 and IL-2 significantly prolonged the survival time of mice infected by the i.c. route with C. neoformans and that this protective effect was correlated with the up-regulation of MHC class II molecule on microglial cells. IFN-{gamma} was critical for the efficacy of anti-CD40/IL-2 immunotherapy within the brain, although the data would indicate that peripheral and not local production of IFN-{gamma} was responsible for early microglial cell activation.

Unlike many immunotherapeutic regimens for treating cryptococcosis, CD4+ T cells were not required for the anticryptococcal efficacy of anti-CD40 and IL-2. CD4+ T cells are critical in restricting C. neoformans growth and preventing the dissemination of the yeast to extrapulmonary sites, including the brain (46). CD4+ T cells can also play an important role in host immune defense against C. neoformans in the brain by interacting with MHC II+ microglial cells in an Ag-specific context and activating their fungicidal activity by cytokine secretion (21). However, in this study the absence of CD4+ T cells did not make the mice more susceptible to C. neoformans i.c. infection or alter the anticryptococcal activity of anti-CD40 and IL-2 immunotherapy, indicating that other IFN-{gamma}-producing immune cell types, such as CD8+ T cells and NK cells, may be able to activate microglial cells.

Microglial cell activation was correlated with the prolonged survival time induced by anti-CD40 and IL-2 immunotherapy in mice infected i.c. with C. neoformans, indicating the importance of this cell type in brain immunity responding to the infections and immunotherapies. MHC II+ microglial cells have been shown to be an important element in host resistance to cerebral cryptococcosis (21). Activated microglial cells have been demonstrated to engulf C. neoformans and function as APCs by up-regulating MHC class II expression as well as other costimulatory molecules such as CD80, CD86, and CD40 (47) through which they are able to interact with T cells and gain enhanced microbial resistance (47, 48, 49). Therefore, the marked up-regulation of MHC class II expression on microglial cells induced by anti-CD40 and IL-2 treatment in this study indicated that these cells were activated. Activated microglial cells are also primary effector cells in the brain that produce NO and inflammatory cytokines such as TNF-{alpha}, IL-6, and IL-12 in response to various stimuli (50, 51, 52, 53). Little proinflammatory cytokine mRNA expression in the brain, including TNF-{alpha}, IL-12, IL-1, and IL-18, was detected by day 5 after C. neoformans i.c. infection even with anti-CD40/IL-2 treatment, even though the activation of microglial cells was detected. During the later course in the treatment significantly increased T lymphocyte infiltration in the brains was detected and these cells may contribute to local IFN-{gamma} production in the brain. Hence, at these later time points (i.e., day 11) the pathology in the brains of the directly infected mice is significant.

Consistent with studies conducted in a murine model of systemic cryptococcosis, IFN-{gamma} is also necessary for the efficacy of the anticryptococcal effect of anti-CD40/IL-2 in the brain. IFN-{gamma} is able to cross the BBB and persist in the brain at very low concentrations (42), and the biological effects of IFN-{gamma} are largely restrained by immunosuppressive factors in the brain such as TGF-beta and IL-10 (54). IFN-{gamma} as single regimen has shown a limited therapeutic effect on cryptococcosis (55, 56), and the amount of IFN-{gamma} given systemically is limited by the severe toxicity observed (57). The benefit of anti-CD40/IL-2 treatment was to stimulate immune cells to produce IFN-{gamma} at a physiologically effective level without this toxicity. Anti-CD40 single treatment could also increase IFN-{gamma} production to an extent of about half the amount induced by anti-CD40 in combination with IL-2 (data not shown); however anti-CD40 alone failed to up-regulate MHC class II expression on microglial cells or protect mice from cerebral cryptococcosis. These results suggests that there may be a threshold of IFN-{gamma} required for microglial cell activation or that IL-2 may be needed to facilitate the passage of IFN-{gamma} across the BBB through IL-2-induced capillary leakage. This also indicates that, although critical, IFN-{gamma} may not be the only immunomodulatory factor that is responsible for the anticryptococcal efficacy of anti-CD40 and IL-2 immunotherapy but is clearly required. This therapeutic effect of anti-CD40/IL-2 treatment has been shown in different strains of inbred mice and is not limited to the results presented in this study (data not shown). Determining the effects of other important mediators such as IL-12 and TNF-{alpha} is also of interest.

The anticryptococcal effect of IFN-{gamma} is likely achieved indirectly through the activation of immune cells to display fungicidal activity. IFN-{gamma} is a pivotal stimulator for the expression of MHC II on microglial cells, which are necessary in host resistance to C. neoformans infection (21). Another possibility is IFN-{gamma} is required to alter the BBB, which allows anti-CD40 to enter the brain and activate microglial cells. Although mice eventually died of C. neoformans outgrowth in the brain, these results demonstrate that immunotherapy with anti-CD40 and IL-2 can have significant effects on immune function and resistance in the brain. These results also have implications in the application of this immunotherapeutic regimen in circumstances where cancer is present in the CNS.


   Acknowledgments
 
We thank Dr. Bruce R. Blazar (University of Minnesota Hospital and Cancer Center, Minneapolis, MN) for the gift of the FGK115 hybridoma. We thank Weihong Ma, Danice E. C. Wilkins, and Dr. Doug Redelman for generous help.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Public Health Service Grant CA095572-04. Back

2 Address correspondence and reprint requests to William J. Murphy, University of Nevada, Department of Microbiology, Applied Research Facility, Room 342, Mail Stop 199, Reno, NV 89577. E-mail address: wmurphy{at}medicine.nevada.edu Back

3 Abbreviations used in this paper: BBB, blood brain barrier; DPBS, Dulbecco’s PBS; KO, knockout; GKO, IFN-{gamma} KO; i.c., intracerebral; SAB, Sabouraud; WT, wild type. Back

Received for publication October 30, 2006. Accepted for publication February 1, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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