Published online before print February 5, 2008
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* Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, and
Internal Medicine, Department of Pathology, Diagnostic and Veterinary Clinic, University of Perugia, Perugia, Italy
1Correspondence: Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06126 Perugia, Italy. E-mail: vecchiar{at}unipg.it
ABSTRACT
In this study, we analyzed the possibility that Indinavir (IDV), a well-known protease inhibitor (PI) used in highly active antiretroviral therapy, could affect immune response against the opportunistic fungus Cryptococcus neoformans. In particular, the quality of dendritic cell (DC) response was analyzed. The results reported here show that IDV treatment induces an expansion of DC with CD8
phenotype in spleens of infected hosts. Splenic CD11c+ DC expressed elevated costimulatory molecules such as CD40 and CD80, showed an increased expression of mRNA for proinflammatory cytokines, and secreted abundant IL-12. Integration of all aforementioned regulatory effects results in development of an efficient, T cell-protective response that reflects a consistent reduction in fungus colonization at a cerebral level. These results could help to elucidate the immunoregulatory activity of PI and point out the beneficial effects of IDV in regulating DC functions and antifungal activity. Therefore, although new PI are being introduced in the clinical setting, nevertheless, given its low cost and proven efficacy, IDV could still be considered a potential key compound in the treatment of HIV in resource-limited settings.
Key Words: protease inhibitors • Cryptococcus neoformans • APC • immunoregulation • costimulatory molecules
INTRODUCTION
Cryptococcus neoformans is a ubiquitous, encapsulated yeast that is responsible for morbidity and mortality in patients with impaired cell-mediated immunity [1 ], especially in those with AIDS [2 ]. In immunocompetent individuals, primary lung infections with C. neoformans usually resolve without therapy, but in immunocompromised individuals, C. neoformans can disseminate to the CNS and cause potentially fatal infections. Amphotericin B and fluconazole represent the gold standard therapy for cryptococcosis, but this approach has important clinical limitations including toxic side-effects and for fluconazole, development of resistance after prolonged therapy [3 ]. Thus, the search for new, powerful drugs against cryptococcosis remains a crucial issue. The treatment of HIV-positive patients with highly active antiretroviral therapy (HAART), a combination treatment including a cocktail of viral RT and protease inhibitors (PI), has proven successful in delaying the onset of AIDS [4 ]. HAART leads to a remarkable improvement in the immune status of HIV-positive patients with a significant increase in the number of CD4+ T cells [5 ]. These patients also show a greatly reduced incidence of opportunistic infections such as cryptococcosis [6 , 7 ], an effect attributed to the direct activity of PI on microbial pathogens [8 9 10 ]. Indinavir (IDV) has been approved for the treatment of HIV, but the introduction of new PI with enhanced safety has made this drug no longer an obvious choice. However, low dose of IDV coupled with Ritonavir has been found efficacious and tolerable. Therefore, given its low cost and proven efficacy, IDV remains a key compound in the treatment of HIV in resource-limited settings [11 ].
In a previous paper, we described the direct, inhibitory effects of IDV on selected virulence factors of C. neoformans [12 ]. Furthermore, the beneficial effects of IDV in quenching the deleterious effects exerted by C. neoformans on immunity were also demonstrated [13 ].
Dendritic cells (DC) are APC specialized in regulating T cell immunity [14
]. In their immature state, DC reside in peripheral tissues, where they survey for incoming pathogens. An encounter with pathogens leads to DC activation and migration to secondary lymphoid organs, where they trigger a specific T cell response. In particular, CD11c+/CD8+ DC trigger the development of Th1-type cells, whereas CD11c+/CD8– DC induce a Th2-type response [15
]. DC have been shown to be important for the generation of a protective, cell-mediated response against C. neoformans in mice [16
]. The aim of this study was to evaluate if pretreatment of mice with IDV can influence the immune response to C. neoformans systemic infection and in particular, if it can regulate DC activation.
MATERIALS AND METHODS
Reagents and media
IDV was obtained from Antonio Cassone (Istituto Superiore di Sanità, Rome, Italy). RPMI 1640 with L-glutamine added and FCS were obtained from Gibco-BRL (Paisley, Scotland). All reagents and media were negative for endotoxin, as assessed by Limulus amebocyte lysate assay (Sigma Chemical Co., St. Louis, MO, USA).
Microorganisms
A thinly encapsulated strain of C. neoformans var neoformans serotype A (No. 6995=NIH 37) was obtained from Central Bureau voor Schimmel cultures (Delft, The Netherlands). The cultures were maintained by serial passages on Sabouraud agar (BioMèrieux, Lyon, France). The cells were harvested by suspending a single colony in saline, washed twice, counted in a hemocytometer, and adjusted to the desired concentration. Heating at 60°C for 30 min was used to kill C. neoformans cells.
Mice and infection
Female, 8- to 10-week-old, inbred BALB/c mice were obtained from Harlan Nossan Laboratories (Milan, Italy) and housed at the Animal Facilities of the University of Perugia (Italy). Procedures involving animals and their care were conducted in conformity with national and international laws and policies. Mice were given 10 or 25 µmol/0.2 ml IDV i.p. on Days –3, –2, and –1 and were subsequently infected i.v. with C. neoformans cells (5x107/0.5 ml, Day 0). Infected animals were monitored for organ clearance. Quantification of fungal growth at different times after infection was assessed by plating serial dilutions of brain homogenates onto Sabouraud agar. The experiments were repeated three to five times by using four animals/experimental group.
Cell separation and counts
Spleens from mice untreated or treated with IDV (25 µmol/0.2 ml) and infected or not with C. neoformans were recovered 5 days postinfection and homogenized in 3 ml sterile saline. Cells were centrifuged, resuspended in PBS, and counted by hemocytometer. DC or CD3+ T cells were isolated from splenocytes using CD11c (N418) or CD90 (Thy1.2) mAb-conjugated MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively, and purified by magnetic separation according to the manufacturer’s instructions. Purity of separated cells was >90%. Absolute numbers of purified splenic CD11c+ DC were calculated on the basis of spleen cellularity of each group. Possible contamination of splenic CD11c+ DC with other cell types was excluded by flow cytometry analysis (see below).
Flow cytometry analysis
For analysis of percentage of CD11c-positive cells, unfractionated spleen cells (106) from mice untreated or treated with IDV (25 µmol/0.2 ml) and infected or not with C. neoformans were fixed in 4% formalin for 5 min at room temperature, washed, and incubated for 40 min on ice with FITC-labeled mAb to CD11c (1 µl/106 cells; hamster IgG, Miltenyi Biotec). After incubation, cells were washed twice with fluorescence buffer (FB; PBS containing 0.5% BSA and 0.4% sodium azide), resuspended in FB, and analyzed using a FACScan flow cytofluorometer (BD Biosciences, Franklin Lakes, NJ, USA). In selected experiments, a morphological examination of purified splenic CD11c+ DC was performed to identify low, midlow, and high DC subsets [17
]. For evaluation of costimulatory molecule expression, splenocytes were incubated with FITC-labeled mAb to CD11c and with R-phycoerythrin (RPE)-labeled mAb to CD8 (1 µl/106 cells; rat IgG2a, Miltenyi Biotec), CD40 (0.25 µg/106 cells; rat IgG2a), or CD80 (0.25 µg/106 cells; hamster IgG, all from BioLegend, San Diego, CA, USA). After incubation, cells were washed twice with FB, resuspended in FB, and analyzed by two-color flow cytometry. In selected experiments, expression of CD8 was evaluated on CD11chigh DC. To check possible contamination of splenic CD11c+ DC with other cell types, splenocytes (106) from mice treated as above were incubated with FITC-labeled mAb to CD11c (1 µl/106 cells; hamster IgG, Miltenyi Biotec) and with RPE-labeled mAb to PDCA-1 (0.25 µg/106 cells; rat IgG2b, eBioscience Inc., San Diego, CA, USA), NK cells (1 µl/106 cells; mouse IgG2a), CD4 (1 µl/106 cells; rat IgG2b), CD8 (1 µl/106 cells; rat IgG2b), or CD11b (1 µl/106 cells; mouse IgG2b, all from ImmunoTools GmbH, Germany). After incubation, cells were washed twice with FB, resuspended in FB, and analyzed by two-color flow cytometry. Control staining of cells with irrelevant antibodies was used to obtain background fluorescence values. Data are expressed as percentage of positive cells or mean fluorescence intensity (MFI).
Intracellular staining
For evaluation of percentage of CD11c+/IL-12p70+ cells, unfractionated spleen cells (106) from mice untreated or treated with IDV (25 µmol/0.2 ml) and infected or not with C. neoformans were fixed with 4% formalin for 5 min at room temperature, washed, and incubated for 40 min on ice with RPE-labeled mAb to CD11c (0.25 µg/106 cells; hamster IgG, BioLegend). After incubation, cells were washed twice with FB and permeabilized for 5 min with 0.1% saponin, washed with 0.1% saponin, and incubated for 20 min on ice with biotin-labeled polyclonal antibody to IL-12 (5 µl/tube, Cedarlane Laboratories, Ontario, Canada). After incubation, cells were washed with 0.1% saponin and incubated for 20 min on ice with FITC-conjugated anti-biotin antibody (1 µl/106 cells; mouse IgG1, Miltenyi Biotec). After incubation, cells were washed with 0.1% saponin, resuspended with FB, and analyzed using a FACScan flow cytofluorometer. In selected experiments, unfractionated spleen cells (108) from mice untreated or treated with IDV (25 µmol/0.2 ml) and infected or not with C. neoformans were stimulated in vitro for 18 h with heat-inactivated C. neoformans (E:T=1:2). After incubation, intracellular staining of IL-12p70 was performed, as described above, on purified splenic CD11c+ DC, and intracellular staining of IFN-
and IL-2 was performed using RPE-labeled anti-IFN- (2.5 µg/ml; mouse IgG1, BD PharMingen, Franklin Lakes, NJ, USA) or RPE-labeled anti-IL-2 (dilution, 1:10; rat IgG2b, Miltenyi Biotec) mAb on purified splenic CD3+ T cells. After incubation and washing, cells were analyzed using a FACScan flow cytofluorometer. Control staining of cells with irrelevant antibodies was used to obtain background fluorescence values.
Cytokine production
Blood serum from mice, untreated or treated with IDV (25 µmol/0.2 ml) and infected or not with C. neoformans, was tested for IL-12p70 levels by a specific ELISA assay (Biosource, Camarillo, CA, USA). Unfractionated spleen cells (10x106/ml) from mice treated as described above, were cocultured for 18 h with heat-inactivated C. neoformans (E:T=1:2), and the supernatants were tested for IL-12p70, IFN-, and IL-2 levels by specific ELISA assays (Biosource). Cytokine titers were calculated by reference to standard curves, constructed with known amounts of recombinant cytokines.
RNA extraction and RT-PCR analysis
Purified splenic DC (106) from mice untreated or treated with IDV (25 µmol/0.2 ml) and infected or not with C. neoformans were lysed using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA was extracted. The RT reaction was performed using Moloney murine leukemia virus RT, as described in the manufacturer’s instructions (Invitrogen), and PCR amplification of IL-12p40, TNF-, and GADPH genes was performed using specific primers (Table 1
). RT-PCR products were visualized by electrophoresis through 1.5% agarose gels in 1% Tris-acetate-EDTA containing ethidium bromide (0.05 µg/ml).
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Table 1. Primer Sequences Used for Quantitative RT-PCR
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, and GADPH clones for use as standards were prepared by PCR from cDNA derived from purified splenic DC as described above and cloned into p-Drive vector (Qiagen S.p.A., Milano, Italy). These were verified by sequence analysis. Plasmid DNA was diluted tenfold, and the starting concentration for the dilution series was 108 gene copies/µl.
Real-time RT-PCR
For each target gene, primers were selected using "Beacon Designer" software (Bio-Rad). All primers are listed in Table 1
. Real-time RT-PCR (quantitative RT-PCR) was performed in a 96-well PCR plate using SYBR Green (all from Bio-Rad). For real-time PCR reaction, 100 ng/µl reverse-transcribed RNA was used, and cDNA was normalized according to GADPH as an internal control gene. Amplification conditions were the same for all cytokines and GADPH mRNAs assayed: 3 min at 95°C, 40 cycles of 10 s at 95°C, and 30 s at 62°C. For the Melt Curve, the amplification condition was 1 min at 50°C and 90 cycles of 10 s at 50°C. The experiments were performed on an "iCycler IQ Multicolor Real-time PCR Detection System" (Bio-Rad).
Histological analysis
For histology, 10 days after infection, brains were excised and fixed immediately in formalin. Sections (1.5 µm) of paraffin-embedded tissues were stained using the periodic acid-Schiff (PAS) procedure, which detects neutral and acid polysaccharides. On PAS-stained sections, cryptococcal blastospores stain red [18
].
Statistical analysis
Statistical analysis was performed with the Primer of Biostatistics software program. Data are reported as the mean ± SE from three to five separate experiments. Data were evaluated by one-way ANOVA. Post-hoc comparisons were performed with Bonferroni’s test. A value of P < 0.05 was taken as significant.
RESULTS
We previously demonstrated that IDV affects selected virulence factors of C. neoformans and that IDV-treated C. neoformans is more susceptible to killing by natural phagocytes [12
]. As a result of the importance of DC in the induction of a specific antifungal immune response, we questioned whether IDV could affect selected DC functions. Indeed, DC play a critical role in determining the type and intensity of T cell responses [15
, 19
]; thus, we exploited the possibility that IDV could affect DC phenotype during C. neoformans infection. To this end, splenic DC were recovered from mice treated with IDV (25 µmol/0.2 ml) for 3 consecutive days before systemic challenge with C. neoformans and analyzed by cytofluorimetric analysis 5 days after infection. We first determined the cellularity of the spleens in mice from different experimental groups. As reported in Figure 1
, spleen cellularity was similar in all determinations performed (Fig. 1A)
. However, the treatment with IDV increased the percentage as well as the total number of splenic CD11c+ DC in challenged mice. This increase was ascribed to CD11clow and CD11chigh subsets (Fig. 1B
and 1C)
. The gating strategy of results reported in Figure 1B
1C
1D
, is shown in Figure 1E
. Similar results were obtained when purified splenic CD11c+ DC were used (Fig. 1F)
. Given that CD11chigh DC are known to contain CD8+ and CD8–, which drive the development of distinct Th cells [20
], we analyzed CD8 expression on the two CD11c+ DC subsets. The results indicate a significantly higher expression of CD8 on CD11chigh (Fig. 1D)
but not on CD11clow DC (not shown). Contamination with different cell types was excluded by flow cytometry analysis, as described in Materials and Methods. Moreover, IDV induces a moderate but significant increase of splenic DC percentage as well as improvement of the percentage of DC positive for CD8, CD40, and CD80 (Fig. 2A
) in C. neoformans-infected mice. However, IDV is incapable of regulating these molecules in uninfected mice (Fig. 2A)
. The gating strategy of results reported in Figure 2A
is shown in Figure 2B
.
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Figure 1. Effect of IDV on spleen cellularity and splenic DC phenotype. Spleens were recovered 5 days postinfection with C. neoformans, and total number of splenocytes was determined by hemocytometer counts (A). The percentage of splenic DC was evaluated after incubation of cells with labeled mAb to CD11c (FITC; B). Absolute numbers of purified splenic DC recovered after magnetic separation were calculated on the basis of spleen cellularity (C). Double-positive CD8+/CD11chigh cells were determined on unfractionated splenocytes; data are expressed as MFI (D). NS, Untreated mice; IDV (25 µmol/0.2 ml, Days –3, –2, –1); Cn, C. neoformans (5x107/mouse, Day 0). Data reported are mean ± SE from three separate experiments; *, P < 0.05 (IDV+Cn vs. Cn). (E) The gating strategy for analysis of low (R1) and high (R2) positive CD11c+ DC in unfractionated splenocytes is reported. (F) Purified splenic CD11c+ DC have been gated in three populations as follows: R1 CD11clow DC, R2 CD11cmid low DC, and R3 CD11chigh DC. SSC-H, Side-scatter height; FSC-H, forward-scatter height; FL1-H, fluorescence 1 height.
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Figure 2. CD8, CD40, and CD80 expression on splenic DC of mice treated with IDV and challenged with C. neoformans. Splenocytes were recovered 5 days postinfection and treated with mAb to CD11c (FITC) or CD11c plus CD8, CD40, or CD80 (all RPE; A) and analyzed using a FACScan flow cytofluorometer. Values represent the percentage of positive cells. Data reported are mean ± SE from three separate experiments; *, P < 0.05 (IDV+Cn vs. Cn). (B) The gating strategy is reported.
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+ DC secrete IL-12 and induce a Th1 response, and CD8– DC do not secrete IL-12 and induce a Th2 response [20
]. To evaluate the possible effects of DC on the ensuing T cell immunity, the kinetics of IL-12 production was analyzed in DC from mice treated or untreated with IDV and challenged with C. neoformans. Data reported in Figure 3A
show that when assayed 5 and 11 days postinfection, unfractionated spleen cells from IDV-treated, C. neoformans-infected mice produce significantly higher levels of IL-12p70 with respect to IDV-untreated, C. neoformans-infected mice. Thus, the observed high-level production of IL-12p70 is apparently a result of DC. Indeed, as reported in Figure 3B
(top and middle panels), 5 days after infection, spleens from mice treated with IDV show a significant increase in the frequency of IL-12p70+/CD11c+ cells with respect to spleens from IDV-untreated mice. Similar results were obtained with splenic CD11c+ DC purified from splenocytes cultured for 18 h with heat-inactivated C. neoformans (Fig. 3B
, bottom panel). Given that IL-12 plays a critical role in cross-talk with T cells, promoting a protective response against cryptococcosis [21
], IL-12 was determined in serum of mice treated or not with IDV and infected with C. neoformans. The results, reported in Figure 3C
, show that up-regulation of IL-12p70 production is evident as early as 1 day postinfection and persists after 5 and 11 days. The IDV-mediated activation of DC was analyzed further by determining IL-12p40 and TNF- transcripts in purified splenic DC 5 days after fungal challenge. This time-point was chosen on the basis of previous results showing an up-regulation of costimulatory molecules and IL-12p70 production. The results reported in Figure 4
show that there is a significant increase in IL-12p40 (Fig. 4A
and 4B)
and TNF- (Fig. 4A
and 4C)
expression in DC from IDV-treated mice. It is noteworthy that the up-regulation mediated by IDV was appreciable only in challenged mice and not in normal mice (Fig. 4)
.
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Figure 3. IL-12p70 determination in culture supernatant of splenocytes, in splenic DC, or in serum of mice treated with IDV and challenged with C. neoformans. IL-12p70 levels were determined at different times after infection by specific ELISA assay (A and C). Percentage of positive DC for intracellular IL-12p70 in unfractionated splenocytes is shown as a histogram (B, top panel) and dot-plot (B, middle panel); percentage of purified splenic CD11c+ DC positive for intracellular IL-12p70 is shown as a histogram (B, bottom panel). In histograms, data are reported as mean ± SE from five separate experiments; *, P < 0.05 (IDV+Cn vs. Cn).
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Figure 4. Expression levels of IL-12p40 and TNF- genes on purified splenic CD11c+ DC of mice treated with IDV and challenged with C. neoformans. The test was performed 5 days after infection. Total RNA was isolated and analyzed for mRNA expression with RT-PCR (A). Transcript copy numbers were determined by quantitative RT-PCR using cDNA as a template. Copy numbers were normalized against the copy number of the GADPH gene (B and C). Results shown are from one representative experiment out of three with similar results.
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and IL-2. The results, reported in Figure 5
, show that the production of IFN- by unfractionated splenocytes from IDV-treated, C. neoformans-infected mice was increased 5 days postinfection, and this effect persisted until Day 11 (Fig. 5A)
. Similar results were obtained when IL-2 production was evaluated (Fig. 5B)
. In selected experiments, 5 days after C. neoformans infection, mice were killed, spleens were removed, and unfractionated splenocytes were cultured for 18 h with heat-inactivated C. neoformans. CD3+T cells were subsequently purified, and intracellular staining showed that in IDV-pretreated mice, IFN- (Fig. 5C)
and IL-2 (Fig. 5D)
production was significantly increased.
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Figure 5. Kinetics of IFN- and IL-2 production from splenocytes of mice treated with IDV and challenged with C. neoformans. Cells were cultured in vitro for 18 h in the presence of heat-inactivated C. neoformans (E:T=1:2), and supernatants were tested for IFN- (A) and IL-2 (B) levels by specific ELISA assays. In selected experiments, 5 days after C. neoformans infection, unfractionated splenocytes were cultured as above, and CD3+ T cells were subsequently purified, and intracellular staining for IFN- (C) and IL-2 (D) was evaluated. Data reported are mean ± SE from three separate experiments; *, P < 0.05 (IDV+Cn vs. Cn).
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Figure 6. Determination of fungal clearance in brains from mice treated with IDV and challenged with C. neoformans. CFU recovery from the brain was determined 3, 5, 10, 15, and 20 days after fungal infection; IDV (25 or 10 µmol/0.2 ml, Days –3, –2, –1). Data reported are mean ± SE from five separate experiments; *, P < 0.05 (IDV-treated vs. Cn).
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Figure 7. Histological analysis of brains from mice treated with IDV. Mice were treated with IDV (10 µmol/0.2 ml, Days –3, –2, –1) and subsequently infected with C. neoformans. Ten days after infection, animals were killed, brains were excised, and brain sections were stained with the PAS. (A and B) Brain sections from mice infected with C. neoformans. (C and D) Brain sections from mice treated with IDV and infected with C. neoformans. Original magnification, x2.5 (A and C); x40 (B and D). Images shown are from one representative experiment out of three with similar results.
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In previous studies, we demonstrated that IDV attenuates the virulence of C. neoformans by interfering with fungal urease and protease production and capsule formation [12 ]. The IDV effects on C. neoformans virulence were also evaluated in an in vivo system. In fact, C. neoformans pre-exposed to IDV maintained the attenuated virulence as demonstrated by easy clearance of the fungus from host [13 ]. In this study, we investigated the possible immunoregulatory effects of IDV in murine cryptococcosis. In particular, we studied the influence of IDV treatment on biological functions of DC during the course of infection. Several studies have so far been devoted to clarify the mechanism of the partial immune reconstitution observed in AIDS patients undergoing PI treatment [22 , 23 ], but data about immunoregulatory activity of PI are scarce and mostly obtained in in vitro systems. Recently, an effect of IDV on the increase in phagocytic activity by monocyte-derived macrophages has been described [24 ]. Indeed, the identification of immunoregulatory effects of IDV could also help to elucidate the PI mechanisms in the observed reconstitution of immunological response, which occurs even when the virological response fails [25 ].
The results reported here indicate that IDV treatment in infected animals induces an expansion of splenic CD11c+ DC; induces increased expression of CD8 on CD11chigh DC; induces an increment of CD40 and CD80 double-positive CD11c+ DC; induces increased mRNA expression and secretion of proinflammatory cytokines by splenic DC; induces an increase in IL-12 production by splenic DC; fosters a Th cell-protective response by promoting IFN- and IL-2 production by T cells; impairs C. neoformans growth in vivo, particularly, at cerebral level.
It has been reported that treatment with PI, in combination with at least two nucleoside RT inhibitors (HAART), is effective in suppressing viral load with consequent restoration of immune response, ascribed predominantly to recovery of CD4+ T cells [26
27
28
]. Moreover, some data exist about HAART immunoregulation. Indeed, it has been observed that in HIV-uninfected subjects that received HAART prophylaxis including IDV, no change in total leukocyte and CD4+ T cell count was observed; however, a suppression of proinflammatory cytokines as well as IFN- secretion was manifested in mononuclear cells [29
]. We did not observe a significant regulation of proinflammatory cytokine secretion in uninfected mice treated with IDV; this apparent discrepancy could be a result of the different experimental setting. In addition, we observed that IDV is able to stimulate secretion of cytokines such as IL-12 and TNF- only in infected mice, suggesting that in our experimental system, IDV is unable "per se" to influence the immune response, even at the transcriptional level, as demonstrated by similar levels of cytokine mRNA expression in untreated and IDV-treated mice.
Moreover, the significant increase in splenic DC during C. neoformans infection in IDV-treated mice could be a result of IDV-mediated inhibition of DC migration to the periphery. This is consistent with early studies evidencing that IDV down-regulates DC-specific, ICAM-grabbing nonintegrin expression, which in turn, inhibits DC interaction with ICAM-2, thereby down-regulating transendothelial migration of DC [30 ]. Another important effect mediated by IDV is the inhibition of Kaposi’s sarcoma through blockage of several pathways involved in tumor growth/invasion [31 ].
Our results extend the comprehension of the IDV-mediated effects on host immune response, and in particular, we report that IDV increases the percentage of CD8+/CD11c+ DC and the expression of CD8 on CD11chigh DC, suggesting an induction of their maturation. Given that CD8+/CD11c+ DC in murine spleen is involved in inducing a Th1-type response [32
] and that the development of Th1 is considered critical in combating fungal infection [33
], the increase of splenic CD8+/CD11c+ DC, even if moderate in absolute number, could implement a protective immune response. The mechanism involved in the observed phenomenon is obscure, and further studies are necessary to clarify this issue. Previously, it has been observed that IDV inhibits DC maturation by impairing the expression of CD40 and CD80, but it is unable to affect CD86 expression. The same study also reported that IDV has only a limited effect on DC-induced, allogenic T cell proliferation [34
]. Differently from previous results, we did not observe inhibition of constitutive expression of CD40 and CD80 on splenic DC after administration of IDV; however, previous observations are referred to an in vitro system and to DC undergoing maturation. Noteworthy, we observed a decrease in CD40 and CD80 expression in splenic DC of infected mice 5 days postinfection. This inhibition was reverted in IDV-treated mice, suggesting that IDV could protect splenic DC from multiple immunosuppressive effects, engendered by capsular material of C. neoformans [35
].
The increase in IL-12 production by splenocytes 5 days postinfection is mainly ascribed to splenic DC. However, an increase in IL-12 was observed in serum of IDV-treated animals as early as 1 day postinfection. The fact that the effects of IDV on splenic DC were observed late after infection could be a result of activation of DC in a secondary phase of immune response. This implies that an early activation is conducted by other immunological districts or other immune cells, as demonstrated by early detection of IL-12 in serum and late recovery of IL-12 in splenic DC.
The beneficial effect of IDV in accomplishment of antifungal defense is supported by its capacity to increase the production of IFN- and IL-2 by splenocytes. In particular, intracellular staining for IFN- and IL-2, performed in purified splenic T cells from IDV-treated and infected mice, showed a moderate but significant increase of both cytokines. Indeed, even a moderate enhancement of IFN- or IL-2 has been considered relevant in other experimental systems [36
, 37
]. It is worthy to mention that the presence of IFN- and IL-2 has been correlated with protective responses against C. neoformans infection [38
, 39
]. As a matter of fact, clear evidence that IDV positively affects immune response to C. neoformans is documented by the drastic reduction in CFU observed in the brain, the target organ in systemic cryptococcosis, and confirmed by examination of histological samples in which significant reduction in cerebral tissue lesions was evident 10 days after infection.
Collectively, our results show that in a healthy host, IDV treatment does not affect the biological function of immune cells; conversely, under stress conditions, such as infection, the inhibition of some cellular proteases could possibly be beneficial in inducing, driving, or prolonging selected, protective immune functions.
ACKNOWLEDGEMENTS
This work was supported by a grant from the National Research Program on AIDS, contract no. 50G.38. The authors are grateful to Gabriella F. Mansi for editorial assistance.
Received July 11, 2007; revised January 8, 2008; accepted January 9, 2008.
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