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Plasmodium falciparum-infected erythrocytes and β-hematin induce partial maturation 1
of human dendritic cells and increase their migratory ability in response to lymphoid 2
chemokines. 3
4
Giusti Pablo (1) †
, Urban Britta C. (2, 3), Frascaroli Giada (4), Albrecht Letusa (5), Tinti Anna 5
(6), Troye-Blomberg Marita (1), Varani Stefania. (1)(7)(8). 6
7
(1)Department of Immunology, Wenner-Gren Institute, Stockholm University, Stockholm, 8
Sweden 9
(2) Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK 10
(3) KEMRI-Wellcome Trust Collaborative Research Programme, Centre for Geographical 11
Medicine, Kilifi, Kenya 12
(4) Institute for Virology, University of Ulm, Ulm, Germany 13
(5) Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, 14
Sweden 15
(6) Department of Biochemistry G. Moruzzi, University of Bologna, Bologna, Italy 16
(7) Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska 17
University Hospital, Huddinge, Sweden 18
(8) Department of Hematology and Clinical Oncology, “L. and A. Seragnoli”, University of 19
Bologna, Bologna, Italy. 20
21
Running title: dendritic cells, hemozoin, migration, malaria 22
23
†Corresponding author: 24
Pablo Giusti 25
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00649-10 IAI Accepts, published online ahead of print on 4 April 2011
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Department of Immunology 26
Wenner-Gren Institute 27
Svante Arrheniusväg 20c 28
Stockholm University 29
S-106 91 Stockholm 30
Sweden 31
Tel +46 (0)8164168 32
Fax + 47 (0)8 157356 33
E-mail: [email protected] 34
35
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Abstract 37
Acute and chronic Plasmodium falciparum infections alter the immune competence of the 38
host possibly through changes in dendritic cell functionality. Dendritic cells (DCs) are the 39
most potent activators of T cells and migration is integral to their function. Mature DCs 40
express lymphoid chemokine receptors (CCRs) which enables them to migrate to the lymph 41
nodes where they encounter naïve T cells. The present study aimed to investigate the impact 42
of the synthetic analog to malaria pigment hemozoin, i.e. β-hematin, or infected erythrocytes 43
(iRBCs) on the activation status of human monocyte-derived DCs and on their expression of 44
CCRs. 45
Human monocyte-derived DCs partially matured upon incubation with β-hematin as indicated 46
by an increased expression of CD80 and CD83. Both β-hematin and iRBCs provoked the 47
release of pro-inflammatory and anti-inflammatory cytokines such as IL-6, IL-10 and TNF-α, 48
but not IL-12, and induced up-regulation of the lymphoid chemokine receptor CXCR4, which 49
was coupled to an increased migration to lymphoid ligands. 50
Taken together, these results suggest that the partial and transient maturation of human 51
myeloid DCs upon stimulation with malaria-derived products and the increased IL-10, but 52
lack of IL-12 secretion, may lead to suboptimal activation of T cells. This may in turn lead to 53
impaired adaptive immune responses and therefore insufficient clearance of the parasites. 54
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Introduction 56
Plasmodium falciparum (Pf)-malaria is one of the most severe infectious diseases in the world 57
where as many as 3.3 billion people live at risk of infection and 247 million cases where 58
reported in 2006. The vast majority of victims are children below 5 years of age (49). 59
Immunity to malaria is developed only after repeated exposure and is not long lasting (18). 60
Several studies have demonstrated impairment of immune responses in Pf malaria infection 61
(46). 62
Previous studies indicate that an early pro-inflammatory cytokine-mediated 63
mechanism is crucial to control parasitemia and for the clearance of parasites. Excessive pro-64
inflammatory responses, however, can cause severe disease (31). The rapid production of 65
cytokines implies release either from pre-existing memory T-cells or cells of the innate 66
immune system, such as antigen-presenting cells (APCs) or natural killer (NK) cells. 67
Among APCs, dendritic cells (DCs) are the most potent in initiating, controlling and 68
regulating functionally distinct T-cell responses (2, 3). Three signals are required from a DC 69
to potently activate naive T cells and they consist of high levels of peptide-loaded human 70
leukocyte antigen (HLA) class II molecules, co-stimulatory molecules and cytokine secretion. 71
For proper stimulation of T cells, upon encountering a pathogen in the periphery, 72
DCs have to migrate to secondary lymphoid organs in order to encounter T cells (24). The 73
migratory capacities of DCs are guided by the expression of chemokine receptors (CCRs), 74
which in turn is regulated by DC maturation. In the periphery, the expression of CCR1 and 75
CCR5 enable DCs to detect inflammation and migrate towards the causative agent. Upon 76
maturation, those inflammatory CCR are down regulated and instead lymphoid CCRs such as 77
CXCR4 and CCR7, are up regulated, whose ligands are expressed in lymphatic tissues (2). 78
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The Pf parasite has been shown to affect DC responses through the action of the 79
malaria pigment hemozoin (Hz), the synthetic analog β-hematin or through adhesion of Pf 80
infected red blood cells (iRBCs). However, the data obtained so far are somewhat 81
contradictory. Coban et al. reported activation of a proportion of human monocyte-derived 82
DCs (MoDCs) upon Hz stimulation, as indicated by elevated expression of maturation 83
markers and IL-12 secretion (5). On the other hand, Hz-loaded human monocytes do not 84
differentiate into MoDCs, and Hz-loaded MoDCs exhibit an impaired maturation in response 85
to lipopolysaccharide (LPS), suggesting that Hz could have an inhibitory role in the 86
development of inflammatory DCs (36). 87
It has been shown that high-doses of mature iRBCs inhibit LPS-induced maturation 88
of human MoDCs and alter their immunostimulatory abilities (44). It was later suggested that 89
this effect could have been induced by apoptosis of DCs upon contact with high doses of 90
mature iRBCs (10). Another study has demonstrated that low doses of iRBCs induce semi-91
maturation of human MoDCs and release of TNF-α, IL-6 and IL-10 but not IL-12, whereas 92
free merozoites inhibited CD40L induced maturation of MoDCs (27). Furthermore, Pf 93
induces incomplete maturation of other DC subsets, such as plasmacytoid DCs (pDCs). 94
Parasite schizont extracts and iRBCs stimulate human pDCs to induce release of IFN-α but 95
not TNF-α to up regulate CCR7 and co-stimulatory molecules. Moreover, this effect was 96
reproduced in murine pDCs and shown to be dependent on TLR9 (29). 97
In vivo, the proportion of circulating DCs is reduced in children with Pf malaria (45). 98
Recent evidence shows that a minor blood-myeloid-DC population, the blood-dendritic-cell 99
antigen (BDCA)-3+ DCs, is expanded in children with severe malaria as compared to healthy 100
controls, which was associated with augmented IL-10 plasma levels and impaired DC 101
allostimulatory ability, indicating an immunomodulatory function for this APC subset (43). 102
Furthermore, analysis of the distribution of DCs in spleen of fatal Pf malaria cases showed 103
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that interdigitating DCs are reduced in the white pulp of the spleen whereas Prion-protein-104
expressing DCs are enriched in the red pulp and the marginal zone compared to fatal cases of 105
sepsis or controls (20). Therefore, despite contradictory in vitro results, analysis of DCs 106
during natural malaria infection suggests that a proportion of DCs are activated during 107
infection, and importantly exhibit an immunomodulatory phenotype as compared to classical 108
type-1 DC activation which leads to a Th1 skewed T-cell response. 109
In the present study, the phenotype and function of human MoDCs were evaluated 110
upon exposure to β-hematin, parasite lysates and iRBCs. As a measure of function the 111
expression of CCRs, maturation markers, cytokine release and the capacity of human MoDCs 112
to migrate towards chemoattractants and to activate allogeneic T cells were investigated. 113
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Materials and Methods 114
Dendritic cell cultures 115
Peripheral blood mononuclear cells (PBMCs) from anonymous blood donors (Karolinska 116
Hospital, Stockholm, Sweden) were isolated on a density gradient centrifugation using Ficoll-117
Paque (GE Healthcare, Uppsala, Sweden) and used for further cell purification. MoDCs were 118
generated by modification of a described method (32). Briefly, monocytes were isolated by 119
negative selection using MACS microbeads according to the manufacturer's instructions 120
(Monocyte Isolation Kit II, Miltenyi Biotech, Bergisch Gladbach, Germany). Purified 121
monocytes were resuspended in complete RPMI 1640 supplemented with 10% fetal calf 122
serum (FCS), 35 ng/ml IL-4, and 50 ng/ml GM-CSF (R&D Systems, Minneapolis, MN, 123
USA). Cell differentiation was monitored by light microscopy and flow cytometry. At day 5, 124
cells were harvested and 89 % ± 1 % of the cells exhibited a typical MoDC phenotype (CD14- 125
CD1a+). Parasite lysate, β-hematin or iRBCs were then added to differentiated MoDC 126
cultures. In order to induce maturation of MoDCs, 1 µM of prostaglandin E2 (PGE2) (R&D 127
Systems) and 50 ng/ml of TNF-α (R&D Systems) were added to the culture medium at day 5 128
if not stated otherwise. 129
Preparation of β-hematin 130
β-hematin, also called synthetic Hz is structurally similar to Hz formed naturally by parasites 131
(37, 40). It has been suggested that the manner of preparation of β-hematin from hemin could 132
affect the size and therefore effect of the crystals. Coban et al. showed recently that β-hematin 133
prepared from acid-catalysed reaction, had an optimal adjuvant effect compared to a 134
preparation based on an organic solution and we used this protocol with minor modifications 135
(4, 9, 41). Briefly, 75 mg of porcine hemin (Sigma-Aldrich, Steinheim, Netherlands) were 136
dissolved in 14.5 ml NaOH 0.1 M and incubated at 60°C. Within 10 min, 1.45 ml HCl (1M) 137
and 8.825 ml sodium acetate (12,8 M) were added and the solution was incubated for two 138
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hours at 60°C constantly stirring at a rate of ~150 rpm. The solution was then centrifuged at 139
15000 g for 13 min, and the pellet was resuspended in Tris-HCl buffer (100 mM, pH=8) 140
containing 2.5% SDS (KEBO Lab, Stockholm, Sweden) and incubated for 30 min 37°C to 141
dissolve heme aggregates. The solution was centrifuged at 15000 g for 13 min and the pellet 142
resuspended in pre-warmed NaHCO3 (100 mM, pH=9.2). After washing twice, Hz was dried 143
at 37°C. The resulting pellet was weighed and re-suspended to 10 mg/ml in sterile water. In 144
agreement with previous findings (8), the infrared (IR) spectrum of Hz showed intense bands 145
at 1710, 1660, 1299, 1279, 1209 cm-1
(Supplemental Figure 1). Two intense bands at 1660 146
and 1209 cm-1
were present in our preparation indicating a high grade of purity of the sample 147
(9). The endotoxin level in the preparation was below 0.05 endotoxin units (EU)/ml 148
(Bacteriology Lab, Sahlgrenska Hospital, Göteborg, Sweden) in our highest working 149
concentration. The highest working concentration used in this study was 20 µg of β-150
hematin/ml, which corresponds to 30µM and has been calculated to be equivalent to the 151
amount of hemozoin released into the blood stream at the burst of schizonts when 152
parasitaemia is 0.5% (12). 153
Parasite cultures 154
Pf parasite strains were cultured using human O+ RBCs (Karolinska Hospital, Stockholm, 155
Sweden) in RPMI 1640 medium containing 25mM Hepes, 2mM L-glutamine, 50 µg/ml 156
gentamycin, and 0.2% sodium bicarbonate and supplemented with 0,5% Albumax (Gibco™, 157
Paisley, UK). The F32 Tanzanian laboratory strain used in this study adheres to CD36 but not 158
to CD54 or chondroitin sulfate A (Albrecht L. personal communication). The parasites were 159
maintained in continuous cultures in candle jars as previously described (14). Parasite cultures 160
were set aside once a month and cultured separately without antibiotics for regular testing of 161
Mycoplasma contamination by the mycoplasma detection kit Mycoalert® (Lonza, Rockland 162
ME, USA). The parasite cultures were kept synchronous by treatment with 5% D-sorbitol in 163
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distilled water as previously described (17). In order to produce parasite lysates, late stage 164
parasites of the F32 strain were purified on 60% Percoll gradient centrifugation, sonicated as 165
previously described (42) and stored at -80°C until used. 166
Intact iRBCs were harvested from cultures of late parasite stages (schizonts and late 167
trophozoites) when the parasitemia reached approximately 10% and purified by using 168
magnetic separation as described (48). 169
Briefly, parasite cultures were pelleted and re-suspended in phosphate-buffered saline (PBS) 170
supplemented with 2% bovine serum albumin (BSA) and 2 mM EDTA. Parasites were added 171
to CS columns (Miltenyi Biotech) and incubated 10 min before extensively washing the 172
column. The column was then removed from the magnet and parasites were rinsed out with 173
PBS. The percentage of late stage iRBCs was determined by staining an aliquot of purified 174
late stage parasites with acridine orange and counting under UV-microscope. Uninfected 175
RBCs run in parallel under the same conditions were used as negative controls. Uninfected 176
and infected red blood cells (RBCs) were obtained from the same donors in each experiment. 177
178
MoDCs-parasites co-cultures 179
In order to prevent burst of the infected erythrocytes during co-culture, iRBCs were incubated 180
in aphidicolin which arrests the parasite cell cycle in the trophozoite stage (13). Briefly, 181
synchronous parasite cultures at ring stage (8-10% parasitemia) were cultured in 15 µg/ml of 182
aphidicolin (Sigma-Aldrich, St. Louis, USA) for maximum 24 hours. When the parasites had 183
reached the trophozoite stage the cultures were purified, including extensive washing to 184
remove any aphidicolin remnants, using magnetic separation as previously described (48). 185
Immature MoDCs were harvested and resuspended at 106 cells/ml in 24-well plates. Purified 186
trophozoites (90% ± 1% purity) were added at the ratios of iRBCs/DCs 10:1, and 100:1 or 187
uninfected RBCs were added at the high ratio. Based on the assumption that each iRBC 188
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contains 47 femtogram of Hz (15), DCs were in contact with 4.7µg/ml of Hz when incubated 189
with iRBCs at the ratio of 100:1 (iRBC:DCs). MoDCs and uninfected or infected RBCs were 190
co-cultured for 10 hours. TNF-α and PGE2 were added to separate DC cultures as a 191
maturation control. 192
193
Immunophenotyping of MoDCs 194
All antibodies used were mouse monoclonal antibodies either unconjugated or conjugated 195
with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Conjugated antibodies used for 196
cell-surface staining included those recognizing CD1a, CD14, CD80, CD83, CD86, HLA-DR, 197
(Pharmingen, San Diego, CA, USA). Unconjugated antibodies were CCR7, CXCR4 (R&D 198
Systems) that were made detectable using a secondary polyclonal goat anti-mouse PE 199
conjugated antibody (Dako Cytomation, DAKO A/S, Denmark). Data acquisition and analysis 200
were performed on a FACSCalibur flow cytometer (Beckton Dickinson, Mountain View, CA) 201
using BD CellQuest™Pro version 5.2.1 software. FACS data are presented here as mean 202
fluorescence intensity (MFI). 203
204
Migration assays 205
Migration of MoDCs was evaluated using a 48-well Boyden
chamber (Neuroprobe, 206
Pleasanton, CA, USA) with 5-µm-pore-size polycarbonate filters. Medium without FCS was 207
used as control of background migration. Cells were allowed to migrate for 1.5 hours in 208
response to either (CXCL)-12/stromal cell-derived factor (SDF)-1α or CCL19/Macrophage 209
inflammatory protein (MIP)-3β (PeproTech Inc., Rocky Hill, NJ, USA)
at a final 210
concentration of 100 ng/ml in RPMI 1640 medium containing 1% FCS. Thereafter MoDCs 211
adhering to the membrane were stained and counted. All samples were assayed in duplicates, 212
and the number of cells that migrated in five visual fields (original
magnification, x100) was 213
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determined for each well, as previously described (38). The net number of cells that migrated 214
was calculated by subtracting the number of cells that migrated in response to the medium
215
alone from the number of cells that migrated in response to chemokines. 216
217
Cytokine measurement in the supernatant of cell cultures 218
Supernatants from MoDC that were cultured in the presence or absence of parasite-derived 219
products were collected and subsequently stored at -70°C. Supernatants were analyzed for the 220
concentration of interferon (IFN)-γ, IL-1β, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α 221
and TNF-β using a Human Th1/Th2 11plex bead assay (Bender MedSystems, Vienna, 222
Austria) according to manufacturer’s instructions. Data acquisition and analysis were 223
performed by FACSCalibur and the FCAP Array software (Soft Flow, Hungary Ltd. 224
Hungary). 225
226
Mixed leukocyte reaction (MLR) 227
The ability of MoDCs stimulated with either β-hematin or iRBCs to induce T-cell 228
proliferation was assessed in an allogeneic MLR. As responder cells, we used allogeneic 229
PBMCs that were depleted of monocytes by CD14 negative selection (MACS microbeads, 230
Miltenyi Biotech) and then stained with carboxy-fluorescein diacetate succinimidyl ester 231
(CFSE) according to the manufacturer’s instructions (Invitrogen, Oregon, USA). Cells were 232
then resuspended in culture medium and 105 cells/well were added to a U-bottom 96-well 233
plate. MoDCs that were co-cultured with infected or uninfected erythrocytes for 10 hours as 234
described above, were isolated on a density gradient centrifugation using Ficoll-Paque (GE 235
Healthcare, Uppsala, Sweden), washed and resuspended in culture medium. MoDCs 236
stimulated with 20µg β-hematin were washed and resuspended in culture medium. Differently 237
stimulated MoDCs were then added to responder cells at MoDC to PBMC ratios of 1:10, 238
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1:30, 1:100 and 1:300. Cells were co-cultured for 5 days before being harvested and stained 239
for subsequent analysis. Monoclonal antibodies directed against CD3 and CD4 (Pharmingen, 240
San Diego, CA, USA) that were conjugated with peridinin-chlorophyll protein (PerCP) or 241
Allophycocyanin (APC) respectively were used to stain the cells for subsequent FACS 242
analysis. Data acquisition and analysis were performed on a FACSCalibur flow cytometer 243
(Beckton Dickinson, Mountain View, CA) using BD CellQuest™Pro version 5.2.1 software. 244
MoDC-stimulated CD4+CD3
+ cells that expressed less CFSE than CD4
+CD3
+ cells in 245
unstimulated PBMCs where gated as proliferating lymphocytes. 246
247
Statistical analysis 248
Data were analyzed using the Wilcoxon’s rank-sum test. Page’s trend test is a variant of the 249
nonparametric Friedman’s test and was used to analyze the dose response effects of β-hematin 250
on MoDCs by employing increasing doses of this compound. Comparisons were considered 251
statistically significant at p ≤ 0.05. 252 on August 2, 2019 by guest
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Results 253
β-hematin induces partial maturation of MoDCs and increases lymphoid CCR 254
expression 255
In order to evaluate the effect of β-hematin on the phenotype of MoDCs, cells were 256
stimulated with β-hematin or TNF-α/PGE2 overnight. MoDCs cultured in medium alone were 257
used as a negative control. As expected, cells that were stimulated with TNF-α/PGE2 showed 258
a statistically significant increased expression of CD80, CD83, HLA-DR, CCR7 and CXCR4 259
(Figure 1). 260
In order to examine what amount of β-hematin could affect MoDCs, we incubated 261
MoDCs with increasing doses of this compound. The results indicate a clear dose-response 262
relation for the activation markers CD80, CD83 and HLA-DR (Supplemental Figure 2) and 263
the most potent effect was observed for the highest dose of 20µg/ml. 264
After stimulation with β-hematin, we observed increased expression of CD80 on 265
MoDCs (p=0.046 and p=0.018, for 10 or 20 µg/ml β-hematin, respectively). Unstimulated 266
cells expressed low levels of CD83 and expression of CD83 on MoDCs increased 267
significantly in the presence of 10 µg/ml β-hematin (p=0.043) but not 20 µg/ml β-hematin. 268
TNF-α/PGE2 strongly increased the expression of HLA-DR and CCR7 molecules, whereas 269
the expression levels of these markers were only slightly, but not significantly, increased upon 270
β-hematin stimulation (Figure 1). Conversely, β-hematin significantly increased expression of 271
CXCR4 compared to unstimulated MoDCs when added at the concentration of 20 µg/ml 272
(p=0.028) which was comparable to the up-regulation of CXCR4 induced by TNF-α/PGE2 273
(Figure 1). A similar increase in CXCR4 expression on MoDCs was observed in the presence 274
of 10 µg/ml β-hematin although this was not significant (p=0.079). Thus, β-hematin induces 275
partial maturation of MoDCs and increases the expression of the lymphoid chemokine 276
receptor CXCR4 on the cell surface. 277
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To evaluate the kinetics of MoDC activation upon β-hematin or TNF-α/PGE2 278
stimulation, we performed a phenotypic analysis at 12, 36 and 60 hours after incubation with 279
these stimuli. Given our initial results, the kinetics of expression of activation markers on 280
MoDCs was analyzed only after stimulation with 20 µg/ml of β-hematin. At first, we 281
evaluated the data without taking into consideration the time points thus confirming the partial 282
activation described above. The baseline expression of CD80 was 56 +/- 3 MFI (mean–value 283
+/- standard error) and increased to 85+/- 4 MFI and to 96 +/- 4 MFI when cells were 284
stimulated with β-hematin or TNF-α/PGE2, respectively. For CD83 the background 285
expression was 21 +/- 4 MFI and increased to 35 +/- 6 MFI and to 68 +/- 6 MFI when cells 286
were stimulated with β-hematin or TNF-α/PGE2, respectively. HLA-DR expression increased 287
from 75+/- 3 to 91 +/- 2 MFI upon β-hematin stimulation and to 100 +/- 1 MFI using TNF-288
α/PGE2. 289
When we examined the kinetics of cell activation, increased expression of CD80 290
and HLA-DR on MoDCs was maintained after 60 hours of stimulation regardless of the type 291
of stimulus (data not shown). For CD83, maximum expression was observed 36 hours after 292
addition of the stimulus but expression levels dropped close to baseline over the 60 hour 293
period in response to β-hematin (Figure 2). Statistical analysis showed that the decrease in 294
CD83 expression on MoDCs was significant for unstimulated cells (p=0.021) and cells 295
stimulated with β-hematin between the 12 and 60 hour time points (p=0.043) (Figure 2). 296
When the cells were stimulated with TNF-α/PGE2 however, no statistically significant 297
difference was observed for CD83 expression levels (p=0.19) between the same time points. 298
Thus, the partial activation of MoDCs by β-hematin resulted in stably increased expression of 299
CD80 and HLA-DR, while expression of CD83 was transient as the levels were not 300
maintained after 60 hours of incubation. 301
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β-hematin impairs the up-regulation of CCR7 induced by maturation stimuli on MoDCs 303
In addition to the transient maturation of MoDCs with β-hematin, we investigated whether β-304
hematin could interfere with their maturation induced by TNF-α/PGE2. As shown in Figure 3, 305
addition of β-hematin to the cultures prior to TNF-α/PGE2 stimulation significantly impaired 306
the up-regulation of CCR7 (p=0.042 and p=0.017, for 10 µg and 20 µg of β-hematin, 307
respectively). No differences were observed for the expression levels of CD80, CD83, HLA-308
DR and CXCR4 on MoDCs matured with TNF-α/PGE2 in the presence or absence of β-309
hematin (data not shown). Thus, β-hematin selectively blocks CCR7 up-regulation induced by 310
TNF-α/PGE2 on MoDCs. 311
312
Contact with iRBCs induces up-regulation of the lymphoid chemokine receptor CXCR4 313
Next, we examined whether parasite lysates or intact iRBCs could induce transient maturation 314
of MoDCs. MoDCs from four donors were incubated for 18 hours with 15 or 75 µg/ml of 315
parasite lysate. The cells were subsequently harvested and stained for surface expression of 316
CD80, CD83, HLA-DR, CXCR4 and CCR7 and analyzed by FACS. No significant 317
differences were observed in the investigated markers upon contact with parasite lysates 318
compared to medium (data not shown). 319
To evaluate whether Pf antigens expressed on iRBCs could have an effect on 320
MoDCs, cells were incubated with aphidicolin-treated iRBCs, (F32 laboratory strain) or 321
uninfected RBCs, for 10 hours and then stained for CD83, HLA-DR, CCR7 and CXCR4. 322
Aphidicolin arrests parasite development at the trophozoite stage and therefore we were able 323
to analyse the effect of intact iRBCs on MoDC maturation independent of the effect of free 324
merozoites (12). 325
The expression levels of the investigated markers did not differ significantly 326
among MoDCs cultured in medium alone or with uninfected RBCs. In addition, no signs of 327
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maturation were observed by incubating MoDCs with low ratios of infected RBCs (1:1, 5:1 or 328
10:1, iRBC:MoDCs; data not shown). Nevertheless, we observed marginal up-regulation of 329
CD83, HLA-DR and CCR7 from 17+/-4 (mean–value +/- standard error), 81+/-5, 49+/-6 MFI 330
in the unstimulated cultures to MFIs of 24+/-5, 91+/-6 and 60+/-11, respectively, upon co-331
incubation with a high ratio of iRBCs to MoDCs (100:1) although the differences were not 332
statistically significant (Figure 4). Interestingly, the expression of CXCR4 was significantly 333
increased from 78+/-7 in unstimulated cells to 93+/-7 MFI when MoDCs were stimulated 334
with high doses of iRBCs (p=0.043) and to 106+/-11 when they were stimulated with TNF-335
α/PGE2 (p=0.028). 336
A separate experiment was performed to assess the impact of aphidicolin-treated 337
RBCs on MoDCs. No significant difference in the expression of the maturation markers 338
CD80, CD83 and HLA-DR was observed in MoDCs incubated with either untreated RBCs or 339
aphidicolin-treated RBCs (data not shown), indicating that aphidicolin does not affect 340
maturation of MoDCs. 341
Thus, high doses of iRBCs seem to induce marginal up-regulation of HLA DR 342
and co-stimulatory molecules on MoDCs but a significant increase in expression of the 343
lymphoid-chemokine receptor CXCR4. Importantly, intact parasitized erythrocytes are 344
required to induce this effect, since parasite lysates did not induce any maturation of DCs. 345
346
β-hematin-induced maturation of MoDCs is coupled to increased migratory capacity in 347
response to lymphoid chemokines 348
Upon maturation, MoDCs acquire the ability to migrate in response to lymphoid chemokines 349
by up-regulating the expression of CXCR4 and CCR7 molecules (47). Since we observed that 350
β-hematin induced significant up-regulation of CXCR4 and marginally increased levels of 351
CCR7, the capacity of β-hematin-treated MoDCs to migrate in response to lymphoid 352
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chemokines was assessed. 353
MoDCs treated with TNF-α/PGE2 exhibited a significantly increased migratory 354
capacity in response to the CXCR4 ligand CXCL12/SDF-1α (p=0.03) and the CCR7 ligand 355
CCL19/MIP-3β (p=0.04), when compared to untreated cells (Figure 5). Significantly 356
increased migration towards CXCL12/SDF-1α (p=0.03) as well as CCL19/MIP-3β (p=0.04) 357
was also observed in β-hematin-treated MoDCs compared to untreated cells indicating that 358
the increased expression of CXCR4 and CCR7 on β-hematin-treated MoDCs is functional. 359
360
MoDCs exhibit increased secretion of pro- and anti- inflammatory cytokines after 361
stimulation with β-hematin or high concentrations of iRBCs 362
To investigate whether malaria-derived stimuli could induce the production of soluble 363
mediators in MoDCs, the concentration of selected cytokines was measured in the cell-culture 364
supernatant obtained from β-hematin-, iRBC- and un-treated MoDCs. Baseline production of 365
all inflammatory cytokines in unstimulated MoDCs was negligible except for IL-8, as shown 366
in Figure 6a and 6b. In agreement with published data (21), the concentration of all examined 367
cytokines was unaffected upon stimulation with TNF-α/PGE2 (data not shown). In contrast, 368
MoDCs stimulated with 20 µg/ml of β-hematin released significantly higher levels of IL-6 369
(p=0.009) and IL-10 (p=0.021) as compared to unstimulated cells (Figure 6A). The secretion 370
of IL-6 was also increased when MoDCs were stimulated with 10 µg/ml of β-hematin 371
(p=0.05). We noted a slight increase in TNF-α secretion in β-hematin-stimulated cultures 372
compared to unstimulated controls, but this did not reach statistical significance (p=0.174). 373
No differences were found in IL-1β, IL-8 or IL-12p70 secretion between unstimulated 374
cultures and β-hematin-stimulated cultures. 375
Next, we analyzed cytokines release by MoDCs after stimulation with iRBCs. 376
At the low iRBCs:MoDCs (10:1) ratio no significant increment in the release of IL-1β, IL-6, 377
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IL-8, IL-10, IL-12p70 or TNF-α was observed (Figure 6B). However, using a high 378
iRBCs:MoDCs (100:1) ratio there was a significantly increased secretion of IL-1β (p=0.021), 379
IL-6 (p=0.021), IL-10 (p=0.021) and TNF-α (p=0.043) by MoDCs. Of note, the secretion of 380
IL-12p70 was very low upon iRBC stimulation in all donors examined. 381
When comparing the levels of cytokines secreted by DCs upon stimulation with β-382
hematin and iRBCs the same cytokines were elevated with both stimuli except for IL-1-β that 383
was not increased upon β-hematin stimulation. The levels of IL-6, IL-10 and TNF-α were 384
considerably higher when MoDCs were stimulated with iRBCs compared to β-hematin 385
stimulation. Thus, both β-hematin and iRBCs induce secretion of a number of pro- and anti-386
inflammatory cytokines in MoDCs but not IL-12. 387
388
iRBC-treated, but not β-hematin-treated MoDCs induce increased T-cell proliferation. 389
To investigate whether P.falciparum-stimulated MoDCs could induce T-cell proliferation at 390
early time points, we measured proliferation of allogeneic T cells in response to different 391
doses of DCs co-cultured with either 20µg of β-hematin or a high number of iRBCs (1:100). 392
MoDCs co-cultured with β-hematin induced T-cell proliferation comparable to that induced 393
by MoDCs that were unstimulated or co-cultured with uninfected RBCs in line with their only 394
partial phenotypical maturation (Figure 7). By contrast, MoDCs that had been co-cultured 395
with a high dose of intact iRBCs activated allogeneic T cells to a similar degree as TNF-396
α/PGE2 –treated MoDCs despite their partial phenotypic maturation, suggesting a difference 397
in the mode of action between MoDCs co-cultured with β-hematin and iRBCs. 398
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Discussion 399
In this study, we show that β-hematin-treatment of human MoDCs induces a significant up-400
regulation of CXCR4 as well as a slight up-regulation of CCR7. The increased levels of 401
expression of lymphoid CCRs on MoDCs upon β-hematin stimulation was coupled with 402
significantly increased migration towards the ligands for CXCR4 and CCR7, CXCL12/SDF-403
1α and CCL19/MIP-3β respectively. In addition, exposure of MoDCs to high doses of iRBCs 404
resulted in a similar pattern of up-regulation of these lymphoid CCR receptors. 405
Both CXCR4 and CCR7 have been found to be up-regulated in association with maturation of 406
MoDCs (33). Up-regulation of these receptors enables DCs to migrate to T-cell rich areas in 407
secondary lymphoid organs and screen massive numbers of naive T cells (31). Therefore, our 408
findings suggest that β-hematin increases migration of DCs to lymphoid organs. By contrast, 409
current evidence from rodent models of malaria shows that the time of interaction between T 410
cells and DCs is shortened, both in vitro and in vivo, when DCs had been exposed to Hz (26). 411
Together these results suggest that increased migration of DCs towards lymphoid-chemokine 412
gradients upon stimulation with Hz may be coupled to an improper DC-T cell interaction. 413
We also detected a significant up-regulation of some but not all co-stimulatory 414
markers when MoDCs were stimulated with β-hematin. It is unlikely that this effect is 415
mediated by TLR9 since recent evidence suggests that neither β-hematin (28) nor parasite-416
derived Hz (50) activate murine myeloid DCs via TLR9 and TLR9 is not expressed on human 417
myeloid DCs (35). Conversely, β-hematin-activation of myeloid DCs may act through the 418
Nalp3 inflammasome in accordance with previous findings in murine macrophages (7, 11). In 419
this context, it should be noted that our study was not designed to assess such aspects. 420
A kinetic study revealed that increased expression of CD80 and HLA-DR was 421
maintained on MoDCs after 60 hours of stimulation with β-hematin or TNF-α/PGE2. 422
However, although the expression of CD83 was maintained for 60 hours upon stimulation 423
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with TNF-α/PGE2, this was not true for MoDCs activated by β-hematin. Other studies have 424
shown that CD83 has a profound effect on DC maturation (16) and that modulation of CD83 425
can interfere with DC-T cell interactions (19). Thus, our data indicate that β-hematin -induced 426
DC maturation, reflected by CD83 expression, appears to be transient and not as potent as the 427
one induced by TNF-α/PGE2. 428
By contrast, exposure to iRBCs resulted only in up-regulation of chemokine 429
receptor expression whereas up-regulation of HLA-DR and co-stimulatory molecules was 430
only marginal and did not reach statistical significance. We (Urban and colleagues) and others 431
have shown that iRBCs alone did not induce up-regulation of HLA-DR and co-stimulatory 432
molecules on MoDCs (10, 44) but the expression levels of chemokine receptors where not 433
investigated. These results are therefore in agreement particularly when taking into account 434
that the marginal up-regulation of HLA-DR and CD83 we report here was observed after 10 435
hours of co-culture as opposed to the 48 - 68 hours co-culture of MoDCs and iRBCs in earlier 436
studies (10, 44). 437
In addition, we observed increased secretion of pro- and anti-inflammatory 438
cytokines by MoDCs incubated with Pf-derived stimuli within 24 hours of incubation and 439
increased immunostimulatory abilities of MoDCs after 10 hours co-culture with iRBCs, 440
whereas others found reduced allostimulation and no cytokine release by MoDCs upon 441
stimulation with iRBCs for a longer time (10). However, consistent in all reports is the lack of 442
IL-12 secretion by MoDCs stimulated with any Pf-derived product. 443
Of note, production of pro-inflammatory cytokines was much higher in MoDCs co-444
cultured with iRBCs compared to β-hematin suggesting that iRBCs contain additional factors 445
that drive secretion of inflammatory cytokines such as PfGPI (6). The high concentration of 446
IL-1β, IL-6 and TNF-α secreted within 10 hours by MoDCs co-cultured with iRBCs could 447
contribute to their ability to induce proliferation of allogeneic T cells. 448
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Together, our results suggest that parasite-products can induce partial and 449
transient phenotypic maturation of DCs at early time points but not IL-12 release. This is 450
coupled to increased DC immunostimulatory ability upon contact with intact iRBCs but not 451
with β-hematin. How these phenomena can influence adaptive immune responses to the 452
parasite can only be speculated. Partial and possibly transient DC maturation associated with 453
lack of IL-12 secretion and high levels of IL-10 are all phenomena that could support a 454
blunted adaptive immune response. 455
Importantly, we report here the sustained up-regulation of chemokine receptors 456
resulting in migration of MoDCs towards lymphokine gradients. These observations are in 457
line with findings by Millington et al (25, 26) who observed a significant decrease of mature 458
DCs in vivo by day 12 of mice infected with the parasite P. chabaudi chabaudi and no or only 459
marginal maturation of rodent DCs in response to iRBCs or Hz. Nevertheless, an increased 460
migration of myeloid DCs into the spleen was observed during P.chabaudii chabaudi 461
infection (39), and in particular migration from the marginal zone of the spleen into the CD4+
462
T-cell area within 5 days after parasites entered the bloodstream (20). In this context, DCs 463
induce activation of T cells but fail to sustain prolonged contact necessary for T cell 464
proliferation and differentiation (26). 465
Our in vitro observations of the effect of β-hematin and to a lesser extent iRBCs on 466
human DCs suggest that a similar mechanism may apply in humans; accumulation of malaria 467
pigment in myeloid DCs could induce migration of DCs into T cell areas of secondary 468
lymphoid organs due to increased expression of lymphoid chemokine receptors, followed by 469
increased stimulation of T-cell proliferation at early time points. However, these phenomena 470
are coupled with a lack of sustained up-regulation of CD83 and lack of IL-12 secretion, but 471
high levels of IL-10, which may result in altered T-cell differentiation. 472
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We also observed that β-hematin selectively impaired the up-regulation of CCR7 473
induced by TNF-α/PGE2 stimulation. Recent evidence indicates that, apart from chemotaxis, 474
CCR7 controls the cytoarchitecture, the migratory speed, and the maturation rate of DCs (34). 475
Therefore, impairing the CCR7 up-regulation on maturing MoDCs may be an effective tactic 476
for the malaria parasite to block DC functionality, thus hampering the initiation of the T-cell 477
responses, as seen in other parasitic infections (1). 478
It is well established that malaria parasites induce a large inflammatory response 479
during acute infection and a correlation has been found between high levels of certain 480
cytokines and severe complications during malaria infection (22, 23, 30). Our findings of 481
rapid release of pro- and anti-inflammatory mediators upon short-term stimulation with Pf-482
derived stimuli imply that myeloid DCs may make a significant contribution to the large 483
inflammatory response that is observed during acute Pf infection. 484
In conclusion, we found that contact of MoDCs with β-hematin or iRBCs induced 485
partial and transient maturation of these cells, provoked the release of pro- and anti-486
inflammatory cytokines other than IL-12 and up-regulated lymphoid CCR on the cell surface. 487
This was coupled to increased migration to lymphoid ligands when cells were stimulated with 488
β-hematin and increased proliferation of allogeneic T cells upon stimulation with iRBCs but 489
not β-hematin. 490
We therefore believe that myeloid DCs become activated at least early on during Pf 491
infection. However, the complex nature of the parasite may lead to transient and suboptimal 492
maturation process but increased migration to secondary lymphoid organs and subsequently 493
impaired activation of lymphocytes. Once careful studies with different parasite strains and 494
their products have established how DCs are affected by the parasite, it may become feasible 495
to manipulate DCs during malaria infection to induce more potent adaptive immunity. 496
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Acknowledgements 497
This work was supported by grants from the Swedish Agency for Research and Development 498
with Developing Countries (SIDA/SAREC, M.T-B. and S.V), as well as a grant within the 499
BioMalPar European Network of Excellence (LSHP-CT-2004-503578, M.T-B) and the 500
European Community's Seventh Framework Programme (FP7/2007-2013) under grant 501
agreement N° 242095. BCU is a Wellcome Trust Senior Fellow in Biomedical Science (grant 502
number 079082). 503
504
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Figure 1. β-hematin induces partial maturation of MoDCs and causes up-regulation of
lymphoid CCR expression.
MoDCs were incubated with or without β-hematin (10 or 20 µg/ml), and TNF-α/PGE2 were
added or not as maturation stimuli in designated wells. After 18 hours of incubation, cells
were harvested, stained for surface markers and analyzed by FACS. Y-axis reports mean
fluorescence intensity (MFI) for CD80, CD83, HLA-DR, CXCR4 and CCR7. The boxplots
illustrate the medians and the 25th and 75th quartile and the whiskers represent min and max
values for 5 donors. Data were analyzed using Wilcoxon’s rank sum test. *; P<0.05. **;
P<0.01.
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Figure 2. Kinetics of CD83 expression levels on MoDCs upon β-hematin stimulation.
MoDCs (1x106 cells/ml) were left unstimulated or were stimulated with -hematin (20
µg/ml) or the maturation stimuli (TNF-α/PGE2) and harvested after 12, 36 or 60 hours of
incubation. Cells were stained for the maturation marker CD83 (n=4 donors) and analyzed by
FACS. Data were analyzed using Wilcoxon’s rank sum test. *; P<0.05.
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Figure 3. β-hematin impairs up-regulation of CCR7 upon DC maturation.
MoDCs were incubated with or without β-hematin (10 or 20 µg/ml, n=5 and n=8 donors
examined, respectively) for 4 hours, and then treated with maturation stimuli (TNF-α/PGE2).
Cells were incubated for additional 18 hours, harvested, stained for surface markers and
analyzed by FACS. Y-axis indicates mean fluorescence intensity (MFI) for CCR7. The
boxplots illustrate the medians and the 25th and 75th quartiles and the whiskers represent the
min and max values. Data were analyzed using Wilcoxon’s rank sum test. *; P<0.05.
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Figure 4. Incubation with iRBCs induces up-regulation of CXCR4 expression.
MoDCs were co-cultured with uninfected RBCs (Ctrl RBC) or iRBCs at the ratio of 100:1
(RBCs/DCs). Maturation stimuli (TNF-α/PGE2) were added in separate wells as a positive
control for maturation. Cells were incubated for 10 hours, harvested, stained for surface
markers and analysed by FACS. The mean fluorescence intensity (MFI) of HLA-DR, CD83,
CXCR4 and CCR7 is presented in the figure. The boxplots illustrate the medians and the 25th
and 75th quartiles and the whiskers represent the min and max values of five donors. Data
were analyzed using Wilcoxon’s rank sum test. *; P<0.05.
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Figure 5. Partial maturation of MoDCs induced by β-hematin is coupled to increased
migration in response to lymphoid chemokines.
MoDCs were stimulated with β-hematin (20 µg/ml) or the maturation stimuli (TNF-α/PGE2)
and incubated for 18 hours. Cells were then harvested and seeded in the upper compartments
of a Boyden chamber and 100 ng/ml of CXCL12/SDF-1α or CCL19/MIP-3β were added to
the lower compartments. Culture medium alone was used as control for background
migration. Cells were incubated for 90 minutes to allow migration after which the cells that
had migrated to the lower compartment were counted in 5 representative fields for each well.
The net number of cells that migrated was calculated by subtracting the number of cells that
migrated in response to medium alone from the number of cells that migrated in response to
chemokines. The diagram represents mean values ± standard error of the mean of cells that
migrated in response to CXCL12/SDF-1α (n=6 donors) and to CCL19/MIP-3β (n=5 donors).
Data were analyzed using Wilcoxon’s rank sum test. *; P<0.05.
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Figure 6. Increased secretion of pro and anti- inflammatory cytokines by MoDCs upon
stimulation with high concentrations of β-hematin and iRBCs.
MoDC were left unstimulated or incubated with 10 or 20 µg/ml of β-hematin and cell culture
supernatants were subsequently analyzed for cytokine levels (A). MoDCs were incubated
with uninfected RBCs (Ctrl RBCs) or with a high (MoDCs+iRBCs 1:100) or a low
(MoDCs+iRBCs 1:10) concentration of iRBCs (B). Cell culture supernatants were collected
and subsequently analyzed for cytokine levels. Each graph illustrates the mean values and
standard error of the mean of cytokine concentration (n=4 donors). Data were analyzed using
Wilcoxon’s rank sum test. *; P<0.05.
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Figure 7. iRBC- but not β-hematin-treated MoDCs induce increased T cell proliferation.
MoDCs were co-cultured with medium alone (Ctrl DCs), 100:1 uninfected RBCs (Ctrl
RBCs), 20µg β-hematin (β-hematin), 100:1 infected erythrocytes (iRBC) or maturation
stimuli (TNF-α/PGE2). MoDCs where then put in co-cultures with CFSE-stained, allogeneic
PBMCs for 5 days. After incubation, the percentage of CD4+CD3
+ cells that had proliferated
were gated and are presented on the Y-axis while the different ratios of MoDC:T cells are
presented on the X-axis. The graph illustrates mean values of separate experiments from a
minimum of 6 donors. Error bars depict the standard error of the mean.
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