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* The Molecular Immunology Laboratory, The Queensland Institute of Medical Research, The Bancroft Centre, Brisbane, Queensland, Australia;
Department of Pathogenic Biology and Immunology, Shihezi University School of Medicine, Shihezi, Xinjiang Autonomous Region, People’s Republic of China; and
Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand
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Abstract |
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. Because TNF- is known to mature DCs, we used TNF-KO mice to determine the role of this cytokine in the loss of DC function. In the TNF-KO mice, phenotype, Ag presentation, and IL-12 secretion by DCs were restored to normal following both lethal infections. This study shows that the systemic production of TNF- contributes to poor DC function during lethal infections. These studies may explain, at least in part, immunosuppression that is associated with malaria. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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An investigation into the subtype of CD11c+ DCs that mediates immunity to P. chabaudi found that both CD8+ and CD8– DCs presented malarial peptides during infection and could induce IFN-
production by CD4 T cells (8). However, only CD8– DCs isolated at the acute phase of infection stimulated Ag-specific T cell proliferation and the production of IL-4 and IL-10 by malaria Ag-specific transgenic T cells (8). Moreover, a comparison of peptide-pulsed CD8– and CD8+ DCs found that both DC subsets have the ability to prime and boost CD8 T cell responses to P. berghei and are involved in the activation of memory CD8 T cells (9). Another study has reported that DCs from early in a P. chabaudi infection secrete IL-12 and stimulate an IFN--dominated T cell response (10). However, as the malaria infection progresses, DCs become refractory to the production of TLR-mediated IL-12 and TNF-, while increasing their ability to produce IL-10 and retaining their capacity for activation of naive T cells (10).
A recent study found that DCs selectively phagocytose pRBCs and present pRBC-derived Ags to CD4+ T cells, thereby promoting the development of protective Th1-dependent immune responses to P. chabaudi blood-stage malaria infection (11). In contrast, another study found that DCs exposed to P. berghei pRBC were selectively deficient in priming CD8+, but not CD4+ T cells, which resulted in decreases in both proliferation and cytokine production (4). These DCs are also unable to cross-present OVA to OTI T cells (5). Similarly, it has been shown that P. yoelii blood stage infections also induce DCs to suppress CD8+ T cell responses in natural malaria infections (3).
These differences noted in DC function are difficult to reconcile. A possible explanation is the different species and strains of parasites that were investigated by the different groups. In our study, we investigated DC function following lethal P. berghei or P. vinckei infections and compared this to DCs from nonlethal P. chabaudi or P. yoelii 17XNL infections, to determine whether the species of parasite affected DC function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Specific pathogen-free, 6- to 8-wk-old, female C57BL/6J mice were obtained from the Animal Resources Centre (Murdoch, Western Australia). Specific pathogen-free, 6- to 8-wk-old, female TNF-knockout (KO) mice (on a C57BL/6 background, >10 backcrosses to C57BL/6) were provided by Dr. C. Engwerda (Queesnland Institute of Medical Research, Brisbane, Australia) and OTII mice (on a C57BL/6 background, >10 backcrosses to C57BL/6) were provided by Dr. A. Baz (Queesnland Institute of Medical Research, Brisbane, Australia). The OTII mice have a transgenic TCR specific for OVA on CD4+ T cells. These studies have been reviewed and approved by the Queesnland Institute of Medical Research Animal Ethics Committee.
Cell culture
Cells were washed and cultured in Iscove’s DMEM (without phenol red) with 5% FCS (Invitrogen Life Technologies), 2 mM glutamine (Invitrogen Life Technologies), 25 mM 2–2-ME, and 45 µg/ml penicillin/streptomycin (Invitrogen Life Technologies). The absence of phenol red from the culture medium significantly reduced the autofluorescence shown by cells.
Isolation of splenic DCs
Spleens from 8- to 10-wk-old naive or infected C57BL/6 mice were digested with collagenase D and DNase (Boehringer Mannheim, U.K (12, 13).), RBCs were lysed with Geys solution and DCs were isolated using anti-CD11c MACS beads according to the manufacturer’s instructions (Miltenyi Biotec). Isolated DCs were always labeled with anti-CD11c-PE for FACS analysis to confirm purity. An additional column was undertaken if purity was lower than 95%. Multiple columns were always used to give purity of >98%.
Infection of mice
To define changes to DCs in infected mice, cohorts of mice were infected i.v. with 104 P. berghei, 104 P. vinckei, 105 P. yoelii 17XNL or 105 P. chabaudi parasitized red cells, and spleens taken after 7 days for flow cytometry analysis as preliminary studies found that changes to the DCs were not clear before this time with all strains of parasite. Different size inocula were used so that parasitemias would become patent on approximately the same day. DCs were isolated from cohorts of 3–6 infected and naive mice.
Flow cytometry of DCs
To minimize nonspecific labeling, DCs were preincubated with either purified rat Ig or 5% rat serum in 5% BSA/PBS for 20 min before cell labeling. In some cases, mAb specific for Fc receptors (2.4G2) was used to confirm absence of Fc-mediated binding. The mAbs used were directly conjugated with FITC, PE, or allophycocyanin and were titrated on whole spleen cells before use. DCs were routinely analyzed for MHC class II (M5/115.15.2), CD80 (16-10A1), CD86 (GL1), and CD11c (HL3), expression using reagents purchased from BD Pharmingen. Samples were analyzed on a FACSCalibur, gating on viable cells, using CellQuest software (Version 3.3). Viability was assessed by trypan blue or labeling with 7-actinomycin D. Approximately 104 to 2 x 105 cells from each sample were analyzed.
Assay for DC endocytic properties
To assess the uptake of latex particles, spleen cell suspensions or isolated DCs were incubated for 1 h at 37°C with either 1 µm FITC-labeled or unlabelled latex beads in Iscoves DMEM with 5% FCS, washed in cold 0.02 M EDTA in PBS for 5 min, washed three times in ice-cold medium, fixed with 2% paraformaldehyde, and assessed by flow cytometry.
Cytokines
The levels of cytokines in the serum were measured using the Becton Dickinson bead array kits according to the manufacturer’s instructions.
IL-12 secretion
DCs were isolated and cultured on Multiscreen-HA sterile plates previously coated with 10 µg/ml anti-IL-12 (p70) Ab (BD Pharmingen) overnight, washed, and unbound sites blocked with 5% FCS. Two x 105 cells were added to each well in medium alone or with 1.0 µM phosphorothioate modified CpG oligonucleotide, 1668 (5' TCC ATG ACG TTC CTG ATG CT 3') (14) with 10 µg LPS (Escherichia coli; Sigma-Aldrich) (14). After overnight culture at 37°C, plates were washed with 0.05% Tween 20 PBS and incubated with 0.5 µg/ml biotin-anti-IL-12 (BD Pharmingen) followed by alkaline phosphatase-labeled-streptavidin (BD Pharmingen). NBT/BCIP tablets (Sigma-Aldrich) were used to visualize spots. The IL-12 ELISPOT for the TNF-KO experiments were done with a Becton Dickson IL-12p70 set.
DC-allogenic T cell mixed lymphocyte cultures
DCs isolated from the spleens of naive or infected mice as described above were titrated and cultured in triplicates with 2.5 x 105 naive T cells, isolated from BALB/c or OTII mice. The OTII cultures were supplemented with OVA. The T cells were isolated using either MACS anti-Thy 1 beads (Miltenyi Biotec) or R&D Systems mouse T cell enrichment kit. Both methods gave >98% purity. After 3 days, the culture wells were pulsed with [3H]thymidine and radioactive uptake was measured.
Statistics
Error bars shown are means ± SE of means. The p values were calculated using the Mann-Whitney nonparametric t test, with a two-sided tail, based on pooled data from two to four replicate experiments. In studies with smaller sample groups, the nonparametric t test with a two-sided tail, based on pooled data from replicate experiments, was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Cohorts of mice were infected with either P. chabaudi, P. yoelii 17XNL, P. berghei, or P. vinckei and the parasitemia monitored for 48 days, or until mice became ill and had to be sacrificed (Fig. 1A). Because mice given lethal infections usually die after 8–10 days, the maturation of CD11c+ DCs following all infections was assessed by the level of expression of MHC class II, CD80, and CD86 after 7 days compared with DCs from naive mice. All preparations were analyzed at the same time. The DCs from mice infected with P. berghei or P. vinckei had very mature DCs with 10–15-fold higher levels of MHC class II (Fig. 1, B and C) compared with DCs from P. yoelii 17XNL-infected, P. chabaudi-infected, or naive mice. DCs were then assessed 2, 4, and 7 days after infection with P. vinckei (Fig. 1D) or P. berghei (Fig. 1E). With both species of parasite, DCs were similar to naive mice until day 4 and then matured by day 7. Preliminary studies found that changes to DCs were only consistent after 6 days.
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Because inflammation has been associated with malaria, we assessed levels of inflammatory cytokines TNF- and IL-12 in the serum 4 and 7 days after infection with lethal P. berghei or P. vinckei and compared these to naive mice or mice infected with P. yoelii 17XNL (Fig. 2). Mice infected with P. vinckei (p < 0.0004) or P. berghei (p < 0.032) showed 
8–10-fold increases in levels of systemic TNF- after 7 days compared with serum from the same naive mice before infection. Mice infected with P. yoelii did not have increased levels of systemic TNF- whereas mice infected with P. chabaudi doubled the level of cytokine (p > 0.05; Fig. 2A). In contrast, mice infected with P. vinckei or P. berghei did not have detectable levels of serum IL-12, which was found in the serum of mice with nonlethal P yoelii and P. chabaudi infections (Fig. 2B). Subsequent studies focused on P. yoelii 17XNL infections to represent the nonlethal infections as it reached higher levels of parasitemia than P. chabaudi, similar to the lethal infections.
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DCs endocytose Ag, process the Ag into peptides, and present the peptides to T cells. We thus investigated whether very mature DCs seen during lethal rodent malaria were capable of endocytosing 1-µm latex particles. DCs were isolated from naive mice and 4 and 7 days after infection with P. yoelii, P. berghei, or P. vinckei and incubated with the latex beads (Fig. 3). Within 4 days of the infection with both lethal species of parasite, DCs had lost some of their capacity to endocytose latex beads and after 7 days of infection, the capacity was reduced in 65–85% of cells. DCs from P. yoelii infections also lost some of their capacity for endocytosis, but only after 7 days and not to the same level as DCs from P. berghei (p < 0.0012) or P. vinckei (p < 0.0002) infected mice. The loss in endocytic capacity preceded increases in systemic TNF- levels and DC maturation.
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mediates changes in DCs during lethal infections
Because TNF- induces the maturation of DCs (15), the hypermature DCs seen following P. berghei or P. vinckei infections could have been a result of the high levels of systemic TNF- found following lethal infections (Fig. 2). This hypothesis was supported by the observation that TNF- levels peaked by day 7 (Fig. 2) and this correlated with the hypermaturation of DCs at the same time (Fig. 1). As such, TNF-KO mice were used to determine the role of this cytokine in DC maturation and function.
Cohorts of C57BLl/6 (wild type, WT) or TNF-KO mice were infected with P. yoelii 17XNL, P. berghei, or P. vinckei, and after 7 days the DCs were isolated from their spleens. The absolute numbers of CD11c+ DCs isolated from either WT or TNF-KO mouse infected with P. yoelii 17XNL, P. berghei, or P. vinckei were not significantly (p > 0.05) different (Fig. 4A). Mice infected with P. yoelii 17XNL, did however have 3–5-fold more DCs compared with naive mice or mice infected with P. berghei or P. vinckei.
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had no effect on DCs from mice infected with P. yoelii 17XNL. These data were confirmed by blocking TNF with an anti-TNF Ab (data not shown).
We then investigated whether the phenotypic change in DCs mediated by TNF- affected the priming of naive T cells by DCs. Cohorts of WT or TNF-KO mice were infected with P. berghei or P. vinckei, and then CD11c+ DCs were isolated and cultured with syngeneic T cells from OVA-specific TCR transgenic (OTII) mice in the presence of OVA (Fig. 4B). CD11c+ DCs from WT mice infected with P. berghei (Fig. 4C) were very poor at initiating proliferation of OTII T cells, but DCs from TNF-KO-infected mice were as functional as DCs from naive mice (Fig. 4C). When WT mice were infected with P. vinckei, higher numbers of CD11c+ DCs were inhibitory for T cell priming (Fig. 4C). In contrast, DCs from TNF-KO mice infected with P. vinckei were as efficient as DCs from naive mice in priming naive OTII T cells (Fig. 4C).
Because DCs are a significant source of IL-12, this function of DCs was also examined. CD11c+ DC populations were isolated from infected and naive mice and p70 IL-12 production assessed using an ELISPOT assay (Fig. 4D). Following infection of WT mice with P. vinckei (p < 0.0005) or P. berghei (p < 0.024), the number of DCs secreting IL-12 were low compared with naive mice, but levels were significantly increased in TNF-KO mice to levels equivalent to naive mice but not mice infected with P. yoelii 17XNL or P. chabaudi (Fig. 4D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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. Because TNF matures DCs, we infected TNF-KO mice and found that DC maturation and function were normal. This study showed that in at least two lethal infections, the production of TNF- during infection mediates the "hypermaturation" of DCs and thus blocks DC function. We have thus identified a mechanism by which DC function is compromised. A recent study found that the cross-presentation of viral Ags by DCs to CD8 T cells was impaired during P. berghei infections (5) and we now show the reason for the changes to DC function.
TNF- is associated with pathology of malaria, especially with acute respiratory distress syndrome and cerebral malaria (17, 18). Studies in humans infected with P. falciparum have associated severe disease with higher levels of serum TNF- (19). We found that high levels of serum TNF- associated with lethal but not nonlethal infections, and that a high level of the cytokine compromised DC function. We also found that the restoration of DC function by removal of TNF-, did not improve the survival of mice (data not shown) indicating that the loss of DC function is not the only factor to contribute to lethal disease. T cell-dependent TNF- is a critical component in both parasite killing and disease promotion (20). Experiments with lethal P. berghei and blocking Abs have demonstrated that all T cell mediated antiparasitic immunity and all T cell-mediated weight loss were TNF-dependent. Blocking TNF- in mice that received parasite-specific T cells prolonged the survival of the mice (20). However, another study found that treatment of susceptible A/J mice with recombinant IL-12 significantly decreased the peak parasitemias and improved survival from P. chabaudi infections while the simultaneous depletion of TNF- and IFN- in the IL-12-treated mice resulted in 100% mortality (21). Taken together, these studies indicate that IL-12 regulates the development of resistance to nonlethal P. chabaudi via a CD4+ Th1 response, which involves the TNF- (20, 21). We now show that compared with P. chabaudi or P. yoelii infections, lethal infections are associated with high levels of serum TNF and that in the absence of TNF, DC function was improved. Moreover, IL-12 was not measurable in the serum of mice with lethal infections but was in abundance in serum of mice infected with either P. chabaudi or P. yoelii. A possible explanation is that the high levels of TNF induce maturation of DCs, which make them refractile to stimulation for IL-12 secretion.
Finally, although we found that the high levels of systemic TNF- increased the maturation of DCs with a concomitant loss of DC function, our studies did not identify the source of TNF. Studies have found that when mouse peritoneal macrophages were incubated with erythrocytes infected with nonlethal or lethal variants of Plasmodium yoelii or with P. berghei, in the presence of polymyxin B to exclude the effects of any contaminating endotoxin, the macrophages secreted very high levels of TNF- (22). Subsequent work found that heat-stable soluble products of rodent malarial parasites activated macrophages to secrete TNF- into the serum in a dose related amounts (23). Thus, lethal parasites that reach high parasitemias rapidly may secrete higher levels of the heat-stable soluble products and as such activate macrophages to secrete high levels of systemic TNF-. In contrast, nonlethal P. yoelii infections take much longer to reach high parasitemias and immunity has a chance to develop as noted by the detection of systemic IL-12.
In conclusion, although the secretion of TNF- has a significant role in mediating survival in a nonlethal P. chabaudi infection (21), this cytokine mediates disease in lethal models (20). In this study, we have identified a factor that directly inhibits DC function which may help in understanding malaria pathogenesis.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by the National Health and Medical Research Council. S.J. was supported by the China Scholarship Council. 
2 Address correspondence and reprint requests to Dr. Michael F. Good, Queensland Institute of Medical Research, Bancroft Centre, 300 Herston Road, Brisbane, Queensland, Australia. E-mail address: Michael.Good{at}qimr.edu.au
3 Abbreviations used in this paper: DC, dendritic cell; KO, knockout; WT, wild type.
Received for publication March 27, 2007. Accepted for publication July 13, 2007.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and TNF- and occurs via a nitric oxide-dependent mechanism. J. Immunol. 155: 2545-2556. [Abstract]This article has been cited by other articles:
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  R. Ing and M. M. Stevenson Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-Stage Plasmodium chabaudi AS Infection in Mice Infect. Immun., February 1, 2009; 77(2): 770 - 782. [Abstract] [Full Text] [PDF]
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E. T. Cadman, A. Y. Abdallah, C. Voisine, A.-M. Sponaas, P. Corran, T. Lamb, D. Brown, F. Ndungu, and J. Langhorne Alterations of Splenic Architecture in Malaria Are Induced Independently of Toll-Like Receptors 2, 4, and 9 or MyD88 and May Affect Antibody Affinity Infect. Immun., September 1, 2008; 76(9): 3924 - 3931. [Abstract] [Full Text] [PDF]
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 K. A. Wong and A. Rodriguez Plasmodium Infection and Endotoxic Shock Induce the Expansion of Regulatory Dendritic Cells J. Immunol., January 15, 2008; 180(2): 716 - 726. [Abstract] [Full Text] [PDF]
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