The Fulani ethnic group from West Africa is relatively better protected against Plasmodium falciparum malaria as compared to other sympatric ethnic groups, such as the Dogon. However, the mechanisms behind this lower susceptibility to malaria are largely unknown, particularly those concerning innate immunity. Antigen-presenting cells (APCs), and in particular dendritic cells (DCs) are important components of the innate and adaptive immune systems. Therefore, in this study we investigated whether APCs obtained from Fulani and Dogon children exhibited differences in terms of activation status and toll-like receptor (TLR) responses during malaria infection. Lower frequency and increased activation was observed in circulating plasmacytoid DCs and BDCA-3+ myeloid DCs of infected Fulani as compared to their uninfected counterparts. Conversely, a higher frequency and reduced activation was observed in the same DC subsets obtained from peripheral blood of P. falciparum-infected Dogon children as compared to their uninfected peers. Moreover, infected individuals of both ethnic groups exhibited higher percentages of both classical and inflammatory monocytes that were less activated as compared to their non-infected counterparts. In line with APC impairment during malaria infection, TLR4, TLR7 and TLR9 responses were strongly inhibited by P. falciparum infection in Dogon children, while no such TLR inhibition was observed in the Fulani children. Strikingly, the TLR-induced IFN-γ release was completely abolished in the Dogon undergoing infection while no difference was seen within infected and non-infected Fulani. Thus, P. falciparum infection is associated with altered activation status of important APC subsets and strongly inhibited TLR responses in peripheral blood of Dogon children. In contrast, P. falciparum induces DC activation and does not affect the innate response to specific TLR ligands in Fulani children. These findings suggest that DCs and TLR signalling may be of importance for the protective immunity against malaria observed in the Fulani.
Citation: Arama C, Giusti P, Boström S, Dara V, Traore B, Dolo A, et al. (2011) Interethnic Differences in Antigen-Presenting Cell Activation and TLR Responses in Malian Children during Plasmodium falciparum Malaria. PLoS ONE6(3): e18319. https://doi.org/10.1371/journal.pone.0018319
Editor: Francesco Dieli, University of Palermo, Italy
Received: November 14, 2010; Accepted: March 3, 2011; Published: March 31, 2011
Copyright: © 2011 Arama et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Swedish Agency for Research and Development with Developing Countries, http://www.sida.se (Sida/SAREC SWE 1995-117), the European and Developing Countries Clinical Partnership's grant, http://www.edctp.org (EDCTP CG-TA 05 40204_003), as well as a grant within the BioMalPar European Network of Excellence, http://www.biomalpar.org (LSHP-CT-2004-503578) and from the European Community's Seventh Framework Programme, http://www.evimalar.org/ (FP7/2007-2013) under grant agreement N°242095. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Half of the world's population remains at risk of contracting malaria. During 2008 malaria was estimated to be responsible for 767.000 deaths in Africa alone, mostly in children below the age of 5 . Previous studies performed in a rural area in Mali have shown different susceptibility to Plasmodium falciparum (P. falciparum) infection between two ethnic groups; the Fulani and the Dogon. These populations live under similar social, cultural and geographic conditions and are exposed to identical malaria pressure . However, the Fulani show less clinical symptoms of malaria, and parasites are less frequently detected in their blood , . In addition, they exhibit higher titres of P. falciparum-specific IgG subclasses , and IgM antibodies , . Recent evidence shows lower numbers of regulatory T cells  and more potent early IFN-γ responses against the parasite  in Fulani as compared to other sympatric ethnic groups, suggesting that these immunological phenomena can lead to more robust anti-malarial immunity. Nevertheless, little is known about the functionality of innate immunity in Fulani and sympatric ethnic groups and how this branch of immunity influences susceptibility to malaria infection.
Innate immunity has two important functions in the defence against the parasite. First, it acts to inhibit rapid parasite growth thereby limiting the onset of malaria pathology. Second, it is crucial for the development of parasite-specific adaptive immunity . Antigen-presenting cells (APCs), and in particular dendritic cells (DCs) are important components of the innate and adaptive branches of the immune system. In humans, two main populations of DCs have been described in peripheral blood; the CD11c−, CD123+, blood dendritic cell antigen (BDCA)-2+ plasmacytoid DCs (pDCs) and the CD11c+, CD123− myeloid DCs (mDCs). The latter can be further divided into three subtypes; the BDCA-1+ DCs, the BDCA-3+ DCs and the CD16+ DCs –.
It has been shown that monocyte/macrophage- and mDC functions are impaired in vitro by the malaria parasite or by products derived from infection –. Another study has shown that HLA-DR levels are decreased on circulating DCs of P. falciparum-infected children as compared to healthy controls . In addition, higher numbers of circulating BDCA-3+ mDCs have been detected in the blood of severely infected children combined with reduced immunostimulatory ability. This indicates that malaria may impair mDC functionality and suggests that the BDCA-3+ DC subset may play a crucial role in the immune response against the parasite in humans , .
P. falciparum also modulates pDC responses in vitro by inducing increased secretion of interferon (IFN)-α . Daily injection of this cytokine protects against severe malaria caused by P. berghei in mice  and higher plasma levels of IFN-α have been associated with milder P. falciparum malaria episodes in humans . However, while mDCs are crucial in triggering an effective T-cell response during malaria infection, pDCs are not required for the induction of malaria parasite-specific responses as recently shown in murine models of experimental infection , .
Evidence demonstrates a fundamental role for monocytes in activation of NK cells  and in differentiation of regulatory T cells  upon P. falciparum stimulation in vitro. Nevertheless, the role of this cell subset during the infection in the natural host is only partially understood. Monocytes can trigger adaptive immune responses and contribute to parasite clearance through antibody-dependent cellular inhibition and phagocytosis . Two major monocyte subpopulations have been described ; one classical (CD14+CD16−) and one inflammatory (CD14+CD16+) ; the latter has been found to be increased during acute uncomplicated malaria , . A recent study reported that the levels of CD16/Fcγ receptor IIIA on CD16+ monocytes were higher in children with severe P. falciparum anaemia, which could enhance erythrophagocytosis and TNF-α production in this cell subset .
APCs interact with pathogens through specialized receptors termed pattern-recognition receptors (PRRs) that have evolved to detect danger signals such as pathogen-associated molecular patterns (PAMPs) . A well-studied family of PRRs are the toll-like receptors (TLRs) , which are expressed differently in different APC populations. The monocyte and mDC subsets express mainly TLR1 through TLR6, while TLR7 and TLR9 are expressed in pDCs , . Recent evidence suggests that TLRs are central mediators of pro-inflammatory responses during malaria –. For example, it is known that Plasmodium DNA bound to either malaria pigment  or protein components , activates the TLR9 pathway and that P. falciparum glycosylphosphatidylinositol (GPI) activates TLR2 . However, the role of TLRs in the development of immunity to malaria and in the pathology of malaria is not well understood.
In this study, we evaluated the activation status of different DC and monocyte subpopulations in peripheral blood of Fulani and Dogon children with or without P. falciparum malaria infection in an endemic area of Mali. In addition, the impact of P. falciparum on the activation of APCs was examined by stimulating peripheral blood mononuclear cells (PBMCs) with specific TLR ligands. LPS was employed to activate TLR4 on monocytes and mDC subsets while imiquimod and CpG-A ODN were used to selectively stimulate TLR7 and TLR9, respectively, which are expressed in pDCs , , .
Study subject characteristics
The mean age of the Dogon children was 6.36 years for uninfected and 6.70 years for the infected ones. In the Fulani children, the mean age was 6.44 years for uninfected and 4.50 years for the infected ones. The infected Fulani were younger than the infected Dogon (Table 1). During P. falciparum infection, the mean temperature was significantly higher in the infected Dogon (37.74°C) as compared to infected Fulani (36.71°C, p≤0.05). The mean parasite density was 18758 [150-122000] asexual parasites/µl for Fulani and 13845 [575-48625] asexual parasites/µl for the Dogon.
Interethnic and intraethnic differences in the frequency and immunophenotype of blood DCs during P. falciparum infection
Circulating DCs are defined as leukocyte lineage negative and HLA-DR positive mononuclear cells . Blood DCs were divided into four different subsets by employing monoclonal antibodies against the surface markers BDCA-1, BDCA-2, BDCA-3 and CD16 after gating lineage negative PBMCs. The activation status of each subset was assessed by measuring HLA-DR and CD86 levels.
The infected Dogon exhibited significantly higher frequency of circulating BDCA-2+ (p = 0.034) and BDCA-3+ (p = 0.016) DCs than their uninfected peers (Figure 1A). Conversely, the infected Fulani had a slightly reduced frequency of BDCA-2+ (p = 0.060) and BDCA-3+ (p = 0.038) DCs as compared to their uninfected peers. When comparing only the infected children from each ethnic group, the percentage of circulating BDCA-2+ and BDCA-3+ DCs was significantly lower in Fulani as compared to Dogon (p = 0.04 and p = 0.01, respectively). No interethnic differences were observed between the uninfected children from each group. A similar trend was observed when evaluating absolute numbers of BDCA-2+ and BDCA-3+ DCs/ml of blood although differences were not significant (data not shown). The percentage and absolute numbers of circulating CD16+ and BDCA-1+ DCs did not show any inter- or intra-ethnic difference (Figure 1A and data not shown).
PBMCs from the study participants were stained with monoclonal antibodies for subsequent flow cytometric analysis using a FACSCalibur. The samples were categorised as uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 13) and infected Fulani (n = 10). An increase was observed of BDCA2+ and BDCA3+ cells in the circulation of infected Dogon whereas the opposite was seen for the Fulani (Figure 1A). The expression of activation marker HLA-DR (Figure 1B) and CD86 (Figure 1C) was lower in BDCA2+ and BDCA3+ cells of the Dogon when undergoing infection while it was increased in the Fulani. It is known that DCs travel to the lymph nodes upon activation. Therefore, it is possible that the lower numbers seen in the infected Fulani is a consequence of activation. The boxplots illustrate the medians and the 25th and 75th quartile and the whiskers represent the 10% and 90% percentiles. Data were analyzed by Mann-Whitney rank sum test. *; p≤0.05. **;p<0.01. ***; p<0.001.
When examining DC-activation status, we observed that expression levels of the activation marker HLA-DR was significantly lower on all examined DC subsets in the infected Dogon as compared to uninfected peers (Figure 1B). In addition, the Dogon children undergoing infection had a decreased expression of CD86 on BDCA-2+ DCs (Figure 1C) as compared to their uninfected peers (p = 0.0003). Conversely, the infected Fulani children exhibited significantly increased levels of HLA-DR on pDCs (p = 0.009) and slightly increased levels of HLA-DR on BDCA-3+ mDCs (p = 0.057) as compared to uninfected peers. In addition, expression levels of HLA-DR were reduced on CD16+ mDCs of both Dogon and Fulani children undergoing infection as compared to their uninfected peers.
When comparing children undergoing infection from each ethnic group, the Fulani exhibited higher levels of HLA-DR and CD86 on the BDCA-1+, BDCA-2+ and BDCA-3+ DCs but not on CD16+ DCs as compared to the infected Dogon children. Thus, interethnic differences were observed in terms of blood DC activation status upon malaria and all circulating DC subpopulations excluding CD16+ DCs were significantly more mature in Fulani than in Dogon children during P. falciparum infection. In order to evaluate if these differences could be dependent on parasite burdens, we correlated levels of parasitaemia with HLA-DR expression levels on the affected DC subpopulations from each study participant. No significant correlation was observed in Dogon and Fulani children undergoing P. falciparum infection. For BDCA-1+ DCs the results of the correlation tests were Rho = −0.38 (p = 0.106) for Dogon children and Rho = 0.20 (p = 0.72) for Fulani children. For BDCA-2+ DCs the results were Rho = −0.16 (p = 0.49) for Dogon children and Rho = 0.20 (p = 0.72) for Fulani children. For BDCA-3+ DCs the results were Rho = −0.28 (p = 0.22) for infected Dogon children and Rho = −0.40 (p = 0.48) for infected Fulani children. This suggests that the differences observed in the DC subpopulations could not be correlated to the differences in parasite burden between the populations.
Interethnic and intraethnic differences in monocyte frequency and activation status during P. falciparum infection
Given the importance of monocytes in inflammation and clearance of parasites, we investigated the classical (CD14+CD16−) and inflammatory (CD14+CD16+) monocytic subsets during P. falciparum infection in the children participating in the study.
A significantly increased proportion of both classical (p = 0.02) and inflammatory (p = 0.02) monocytes was observed in Dogon children undergoing infection as compared to their uninfected peers. A similar trend, although not significant, was found between infected and non-infected Fulani (Figure 2A).
PBMCs were stained with monoclonal antibodies and subsequently analyzed using FACSCalibur. The samples were categorized as uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 13) and infected Fulani (n = 10). An increase of circulating monocytes was observed for both groups although only statistically significant for the Dogon (Figure 2A). There was a decreased expression of HLA-DR (Figure 2B) in classical monocytes for both ethnic groups. The levels of HLA-DR were also suppressed in the inflammatory monocytes although not statistically significant for the Dogon. The expression of CD86 (Figure 2C) was decreased on both monocytic subsets although only statistically significant for the Dogon classical monocytes from both Dogon and Fulani. Although the levels of activation markers seem similarly regulated in both groups the classical monocytes of the Fulani exhibit a pattern of higher activation when undergoing infection than the Dogon when infected. The boxplots illustrate the medians and the 25th and 75th quartile and the whiskers represent the 10% and 90% percentiles. Data were analyzed by Mann-Whitney rank sum test. *; p≤0.05. ***; p<0.001.
When examining the activation status of circulating monocytes, a significantly decreased expression of HLA-DR was observed on classical monocytes from both Dogon and Fulani groups undergoing infection as compared to their uninfected peers (p = 0.0001 and p = 0.004, respectively; Figure 2B). A decreased expression of HLA-DR was also observed in inflammatory monocytes of Fulani (p = 0.02) but not of Dogon children (p = 0.41) undergoing infection as compared to their uninfected peers. Finally, in infected Dogon children the expression of the co-stimulatory marker CD86 was also significantly impaired on classical and inflammatory monocytes as compared to uninfected peers (Figure 2C). When comparing children undergoing infection from each ethnic group, the Fulani exhibited higher levels of HLA-DR and CD86 on classical but not on inflammatory monocytes as compared to Dogon children. Thus, interethnic differences were observed in classical monocytes during malaria infection and these cells were significantly more activated in infected Fulani than in infected Dogon children.
P. falciparum infection impairs cytokine responses upon TLR stimulation in Dogon but not in Fulani children
The ability of APCs to secrete cytokines upon TLR stimulation was investigated by stimulating PBMCs with ligands for TLR4, TLR7 and TLR9, i.e. LPS, imiquimod and CpG-A, respectively.
In Dogon, the TLR4 pathway was strongly impaired during P. falciparum infection, as shown by inhibited production of IL-1β, IL-6, IL-10, TNF-α and IFN-γ by PBMCs upon LPS stimulation (Figure 3). Similarly, release of IL-1β, IL-6, IL-10, TNF-α, IFN-α and IFN-γ was inhibited in infected Dogon as compared to uninfected peers when PBMCs were treated with a TLR9 ligand. Also TLR7 responses were impaired upon P. falciparum infection in Dogon children and IFN-α and TNF-α levels were lower in PBMCs stimulated with imiquimod in infected Dogon as compared to their uninfected peers.
PBMCs (1 million/ml) were stimulated or not with LPS (100 ng/ml), CpG-A (3 µg/ml) or imiquimod (1 µg/ml). After 16 hours, supernatants were collected and analysed for the presence of cytokines. The samples were categorized as uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 14) and infected Fulani (n = 23). The levels of IL-1β, IL-6, IL-10, TNF-α and INF-γ were severely supressed in the infected Dogon as compared to their uninfected peers when the cells had been stimulated with either LPS or CpGA. As a result, the levels of the infected Fulani were higher than infected Dogon for all cytokines except for IFN-α when stimulated with LPS and except for IL-1β and IL-10 when stimulated with CpG. Similarly, when stimulated with imiquimod cells of the infected Fulani secreted higher levels of IL-6, IFN-α, TNF-α and INF-γ than the infected Dogon due to the suppressed secretion of Dogon when undergoing infection. Data presented as box plots illustrate the medians and the 25th and 75th quartile and the whiskers represent the 10% and 90% percentiles. Data were analyzed using Mann-Whitney rank sum test. *; p≤0.05. **; p<0.01. ***; p<0.001.
Conversely, no differences were observed in cytokine release between PBMCs obtained from infected and uninfected Fulani upon TLR4, TLR7 and TLR9 stimulation (Figure 3). As compared to infected Dogon children, the infected Fulani secreted significantly higher levels of IL-1β, IL-6, IL-10, TNF-α and IFN-γ upon TLR4 stimulation and higher levels of IL-6, IFN-α, TNF-α and IFN-γ when PBMCs were stimulated with TLR7 or TLR9 ligands.
Taken together, these results indicate that P. falciparum infection impaired TLR4-, TLR7-, and TLR9-induced cytokine responses in the Dogon children, while such responses were unaffected in the Fulani children. As a consequence, the levels of most cytokines that were released upon TLR stimulation were higher in Fulani than Dogon children undergoing P. falciparum infection.
To further analyse the clinical relevance of the cytokines secreted by the PBMCs from Dogon and Fulani children when exposed to P. falciparum infection, the ratios of TNF-α/IL10 and IFN-γ/IL-10 were calculated. The TNF-α/IL10 ratio was significantly decreased upon P. falciparum infection in Dogon children when PBMCs were stimulated with TLR7 (p = 0.02) and TLR9 (p = 0.01) ligands, indicating an impaired pro-inflammatory activity in these specific TLR pathways upon P. falciparum infection in this ethnic group (Figure 4A). Conversely, the TNF-α/IL10 ratio was slightly increased upon P. falciparum infection in Dogon children when PBMCs were stimulated with LPS (p = 0.08). This might indicate that the TLR4 signalling pathway may be less affected than the TLR7 and TLR9 signalling in Dogon undergoing infection. This ratio was not affected in infected Fulani as compared to uninfected peers upon TLR4, TLR7 and TLR9 stimulation. When comparing children undergoing infection from each ethnic group, the TNF-α/IL-10 ratio was higher in Fulani than Dogon children when cells were stimulated with TLR9 ligands (p = 0.04), suggesting a significantly higher proinflammatory activity induced by TLR9 ligation in Fulani than Dogon children undergoing P. falciparum infection. Similarly, the ratio of IFN-γ/IL-10 was decreased in the infected Dogon children as compared to their uninfected peers upon stimulation with TLR4 (p<0.0001) and TLR9 (p<0.0001; Figure 4B). When comparing PBMCs obtained from children undergoing infection from each ethnic group, the IFN-γ/IL-10 ratio was significantly higher in Fulani than in Dogon upon stimulation with TLR4 and TLR9 ligands (p = 0.002 and p = 0.0051; respectively). The same patterns were observed for cells stimulated with TLR7 although the data did not reach statistical significance. Thus, these data confirm that, during P. falciparum malaria infection, PBMCs from Fulani children exhibit a higher pro-inflammatory profile upon TLR stimulation as compared to the Dogon children.
PBMCs (1 million/ml) were stimulated or not with LPS (100 ng/ml), CpG-A (3 µg/ml) or imiquimod (1 µg/ml). After 16 hours, supernatants were collected, cytokine levels were analyzed and the TNF-α/IL-10 (Figure 4A) and IFN-γ/IL-10 (Figure 4B) ratios were calculated. The samples from 77 children were categorized as uninfected Dogon (n = 20), infected Dogon (n = 20), uninfected Fulani (n = 23) and infected Fulani (n = 14). The TNF-α/IL-10 ratio (Figure 4A) was significantly lower in the infected Dogon as compared to their uninfected peers when stimulated with CpG or imiquimod. This resulted in significantly higher TNF-α/IL-10 ratio of infected Fulani as compared to the infected Dogon. Due to the severe suppression of IFN-γ the IFN-γ/IL-10 ratios (Figure 4B) were severely suppressed in the LPS and CpG stimulated cells of the Dogon and therefore higher in the infected Fulani than in the infected Dogon. The boxplots illustrate the medians and the 25th and 75th quartile and the whiskers represent the 10% and 90% percentiles. Data were analyzed using Mann-Whitney rank sum test. *; p≤0.05. **; p<0.01. ***; p<0.001.
In this study, we investigated certain aspects of the innate immunity to malaria in children from two different ethnic groups that live in a malaria endemic area in Mali and exhibit different susceptibility to P. falciparum infection. The impact of ethnicity on allele and genotype frequency for innate soluble factors, such as IL-4 and IL-10, has been previously established in asymptomatic individuals belonging to Dogon and Fulani's groups in Mali . However, functional studies focusing on the role of P. falciparum in affecting innate responses in these two ethnic groups are lacking.
Our findings indicate that P. falciparum infection inhibited the expression of HLA-DR in mDCs and pDCs in Dogon children. These results are in line with previous findings showing reduced expression of HLA-DR on mDCs of Kenyan children during acute malaria . HLA class II expression on DCs is fundamental for presenting antigens to T cells and inducing their activation . Higher numbers of circulating BDCA-3+ mDCs have been detected in blood of severely infected children . Similarly, we observed that the frequency of this mDC subset was increased in peripheral blood of Dogon children undergoing malaria infection as compared to uninfected peers. It has also been previously shown that APCs from children with severe malaria exhibit lower immunostimulatory abilities than those of healthy children . Thus, the reduced expression of HLA-DR on the surface of DCs in Dogon children undergoing malaria infection may indicate impaired DC functionality, which in turn, may contribute to impaired cell-mediated responses.
Conversely, higher activation levels were observed in blood pDCs and BDCA-3+ mDCs of infected Fulani as compared to uninfected children belonging to the same ethnic group. It is known that DC activation is coupled to increased cell migration to lymphoid organs . Therefore, activation of these DC subsets may trigger their migration to the spleen and other lymphoid organs and thereby account for the lower frequency of pDCs, and BDCA-3+ mDCs observed in the peripheral blood of infected Fulani. However, the absolute numbers of circulating DCs were only slightly affected by malaria infection in Fulani. Therefore, the reduced frequency of these DC subsets in infected subjects may also be explained by a relative increase in some other leukocyte population. In fact, it is known that the Fulani have a higher spleen rate than other sympatric ethnic groups , , ,  that could be coupled to an increased number of T- and B-cell subsets in circulation. Thus, it is possible that circulating BDCA-3+ mDCs and pDCs become activated and migrate to secondary lymphoid organs such as the spleen to a higher extent in the Fulani than in the Dogon during malaria infection.
Recent evidence suggests that BDCA-3+ DCs in humans exhibit superior capacity in stimulating cytotoxic T-cell (CTL) responses. These cells may be the counterpart of murine CD8-α+ DCs , , which are known to cross-present parasite antigens and mediate effective CTL responses against blood stage P. berghei in a murine model of malaria . In addition, pDCs are known to play a crucial role in triggering humoral immunity against viruses , . Thus, the specific activation of these DC subsets observed in the Fulani, but not in the Dogon, during P. falciparum infection may contribute to more robust anti-malarial cellular IFN-γ responses  and antibody responses , , ,  that have been observed in the Fulani as compared to other sympatric ethnic groups.
The frequencies of both classical and inflammatory monocytes were increased both in Dogon and Fulani children upon P. falciparum infection. These results are in accordance with previous findings in Thai-Burmese patients where patients with acute malaria exhibited a higher frequency of inflammatory monocytes in peripheral blood . We also found that P. falciparum infection impaired monocyte immunophenotypes, as reflected by the reduced expression of activation markers on classical and inflammatory monocytes in both Fulani and Dogon children, suggesting that P. falciparum alters the functionality of these cells in both ethnic groups. However, interethnic differences were observed during malaria infection as classical monocytes were significantly more activated in infected Fulani than in infected Dogon children.
To further evaluate the innate response in the Fulani and Dogon children during malaria infection, we investigated cytokine release by PBMCs after stimulation with specific TLR ligands. Cytokines are crucial in the pathogenesis of malaria and the balance between pro- and anti- inflammatory cytokines plays a central role in the clinical outcome of this infection , . In this context, we report that, in Dogon children, TLR4, TLR7 and TLR9 pathways were impaired by P. falciparum infection while such TLR inhibition was not observed in the Fulani children. This is in line with the impairment of DC phenotype that was observed in Dogon but not Fulani children upon malaria infection. In humans, TLR7 and TLR9 are mainly expressed in pDCs and it has been established that TLR9 is involved in recognition of malarial products , , . It is also known that TLR7 and TLR9 signalling mediates robust induction of type I IFNs, increased cross presentation of antigens and strong CTL responses , . In our study, impairment has been specifically observed in the pro-inflammatory activity of these two TLR pathways upon P. falciparum infection in Dogon children, but not in the Fulani ethnic group.
In general, the importance of TLRs in malaria is underlined by the fact that TLR4 polymorphisms can predispose to severe malaria  and that polymorphisms in both TLR4 and TLR9 may play a role in the severity of pregnancy associated malaria . Contradictory findings however, have been reported about the effect of malaria infection on TLR signalling. Enhanced pro-inflammatory cytokine production has been shown in response to TLR1/2, TLR4, TLR7/8 and TLR9 stimulation in PBMCs obtained from volunteers undergoing experimental malaria  or patients with natural infection , while others have suggested that malaria has a suppressive effect on TLR signalling, possibly through cross-tolerance following recognition of Plasmodium antigens by TLRs . In partial agreement with the latter study, we observed that P. falciparum infection suppresses TLR responses in Dogon children while no effects were observed in the Fulani children. There may be several reasons for the discrepancies seen in these studies. Firstly, both the studies by McCall et al. and Franklin et al. were performed in adults while, in our study, only children were included. Secondly, these studies were performed in malaria naïve adults at the earliest stages of P. falciparum infection  and in an area with low transmission of malaria , respectively. Conversely, our study area is mesoendemic with high transmission during rainy seasons. As this work was performed at the end of the rainy season, the children included in our study should have experienced at least 2 episodes of P. falciparum infection during the same season as estimated by previous observations in the same area (Arama C. unpublished results). Thus, malaria infection may have a biphasic influence on the innate immune system, initially inducing TLR priming and then, in case of frequent exposure to the parasite, tolerance will ensue. Interestingly, such a suppressive phase was observed in Dogon but not in the children belonging to the Fulani ethnic group.
Moreover, we observed higher release of IFN-γ by PBMCs of infected Fulani children as compared to infected Dogon upon TLR stimulation. This is in line with recent findings indicating that PBMCs from Fulani specifically stimulated with P. falciparum in vitro release higher amount of IFN-γ than Dogon . Studies in both humans and mice indicate that the magnitude of an early IFN-γ response is a crucial determinant of the outcome of malaria infection , . Our findings suggest that the impaired innate IFN-γ responses upon TLR stimulation in infected Dogon as compared to infected Fulani may be one of the underlying causes for the relative susceptibility to malaria in the Dogon ethnic group as compared to the Fulani.
It is possible that APC dysfunction could be dependent on the levels of parasitaemia in an infected individual. This suggests that the APC alteration observed in Dogon individuals upon P. falciparum infection merely depends on differences in parasitaemias in this ethnic group as compared to Fulani. However, we did not observe any significant correlation between parasitaemia and HLA-DR expression levels on distinct APC subsets in infected children. Therefore, differences in levels of parasitaemia do not appear to account for the APC dysfunction observed in Dogon children as compared to Fulani in our cohort.
In conclusion, in this study we found that P. falciparum infection impairs the phenotype of blood DCs and alters TLR responses of PBMCs from Dogon but not Fulani children. In particular, malaria infection induces differential innate IFN-γ release in the two ethnic groups. These findings suggest that activation of certain DC subsets and additional mechanisms triggering cytokine release by APCs through TLR activation may represent immunological correlates of protection against P. falciparum malaria. However, the prospective evaluation of a larger cohort of patients is required to confirm our results and to analyze the potential occurrence of distinct TLR polymorphisms in these two ethnic groups. The understanding of innate immune responses to P. falciparum in naturally exposed children may yield important insights in the development of immunity to malaria with particular regard to rational vaccine development.
Materials and Methods
Ethical approval was obtained from the institutional review boards of the University of Bamako, Mali (N°08_64/FMPOS) and by the Swedish ethical research committee (03-536). Before the inclusion of each child in the study, the community leaders' permission and individual consent form were obtained from the parents of each child.
The study was conducted in a rural area of the Dogon valley of Mali where malaria is mesoendemic with intense transmission during the rainy season. The study area and the study population have been described in details elsewhere . Children between 2 and 10 years of age belonging to either the Fulani or the Dogon ethnic group were included in the study. Forty children from the Dogon population and 37 from the Fulani were recruited. The study included healthy children and infected children that were classified as uncomplicated malaria. A thick blood smear was performed in all subjects included in the study. The slides were stained in 3% Giemsa and examined for the presence of P. falciparum parasites. Presence of malaria infection was defined as a positive thick smear with or without any malaria symptom. Among the Dogon, 20 children were undergoing malaria infection and 20 children were healthy, while among Fulani 14 children were suffering from malaria and 23 were uninfected. Unfortunately not all slides could be recovered from the field study so we only have parasitaemias for eight of the Fulani and nineteen of the Dogon children.
Blood collection and cell preparation
Venous whole blood samples were collected from healthy and infected children of both ethnicities. From each volunteer, 6 ml of peripheral blood was collected in heparin tubes (BD Vacutainer® Plasma Tube, Franklin Lakes, NJ, USA) and were transported to the Malaria Research and Training Centre Laboratory in Bandiagara, north-east of Mali, for further experiments. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) and used for APC immunophenotyping and TLR stimulation.
Purified PBMCs were fixed and frozen immediately after purification as previously described . Briefly, cells were resuspended in PBS to a concentration of 2 million cells/ml and treated with DNAse (Sigma-Aldrich®, Steinheim, Germany) for 5 minutes at 37°C at a concentration of 666 units/ml. Cells were then washed and resuspended in 3 ml of prewarmed (37°C) 4% paraformaldehyde (PFA) (Sigma-Aldrich®, Steinheim, Germany), incubated for 5 minutes and then washed in cold PBS +1% foetal bovine serum (FBS, Gibco, Paisley, UK) Finally, cells were resuspended in 1 ml PBS +10% DMSO and frozen at −80°C. For staining, cells were thawed and resuspended in PBS 5 mM EDTA 2% FBS. FcR blocking reagent and mouse monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE), peridinin chlorophyll protein (PerCP) or allophycocyanin were added. Antibodies used for cell-surface staining included those recognizing BDCA-1 (clone AD5-8E7), BDCA-2 (clone AC144), BDCA-3 (clone AD5-14H12), CD3 (BW264/56), CD14 (clone TUK4), CD19 (clone LT19), and mouse isotype controls IgG1 and IgG2a (Miltenyi Biotec, GmbH, Germany) and CD16 (clone B73.1), CD56 (clone NCAM16.2), CD86 (clone 2331 FUN-1), HLA-DR (clone L243 G46-6; Pharmingen, San Diego, CA, USA). The cells were finally resuspended in PBS 1% PFA, acquired and analysed using a FACSCalibur (BD Biosciences, San Diego, CA, USA) and the BD CellQuest™Pro version 5.2.1 software.
PBMCs stimulation with TLR ligands
PBMCs were resuspended in RPMI supplemented with 10% FCS, 200 mM L-Glutamine, 100 µg/ml streptomycin and 100 U/ml penicillin to a final concentration of 1 million cells/ml. Two hundred µl of cell suspension were distributed into 5 different wells of 96-well flat bottom plate for cell cultures (Sarstedt Inc. Newton, NC, USA). Cells were stimulated with LPS (Sigma-Aldrich® Steinheim, Germany) (100 ng/ml), CpG-A (Metabion, Martinsried, Germany) (3 µg/ml), imiquimod (InvivoGen, San Diego, USA) (1 µg/ml) and unstimulated control. The plates were incubated for 16 hrs at 37°C + 5% CO2 and then the cells and supernatants were collected and stored at −80°C for subsequent cytokine analysis.
Determination of cytokines in supernatants by cytometric bead array
Levels of IL-1β, IL-6, IL-8, IL-10, IL-12 (p70) and TNF-α in supernatants obtained from stimulated PBMCs were determined by cytometric-bead array (CBA) technology (BD Biosciences, San Diego, CA, USA) and the fluorescent signals were detected by Flow Cytometry according to the manufacturer's recommendation. This sensitive technique allows detection by flow cytometry of multiple cytokines in small quantities of samples. All samples were diluted 1∶10 in Assay Diluent (BD Biosciences, San Diego, CA, USA). Data were acquired with fluorescent activated cell sorter (FACS) Calibur (BD Biosciences, San Diego, CA, USA) and analyzed using CellQuest and FCAP Array software. The lower detection limit for the various cytokines was 4.8, 4.7, 3.2, 3.8, 3.7 and 4.9 pg/ml for IL-1β, IL-6, IL-8, IL-10, IL-12 (p70) and TNF-α, respectively.
Determination of cytokine levels in supernatants by ELISA
Levels of IFN-α and IFN-γ were measured by conventional sandwich enzyme-linked immunosorbent assay (ELISA) kits using pairs of capture and detection antibodies (Mabtech, Nacka, Sweden). All samples were tested in duplicates according to the manufacturer's recommendation. The optical densities were measured at 405 nm in an ELISA plate reader (VmaxTM Kinetic Microplate Reader, Menlo Park, CA, USA). The assay sensitivity was 2 pg/ml for IFN-α and 7 pg/ml for IFN-γ, respectively.
Statistical significance was determined using non-parametric tests. A Kruskal-Wallis test was employed to analyze the results when more than two groups were involved. The Mann-Whitney U-test was employed to evaluate significance between two unpaired groups when the Kruskal-Wallis test showed a significant difference between the four groups. Linear regression analysis (Spearman rank correlation test) was employed when analysing possible correlations. For all tests p≤0.05 was considered significant and no correction was made for using multiple tests. The data were analyzed using the StatView software.
We sincerely thank the participants and the residents of Manteourou and surrounding villages in Mali for their ongoing support of this study.
Conceived and designed the experiments: CA PG SB VD BT AD OD SV MT-B. Analyzed the data: CA PG SV. Contributed reagents/materials/analysis tools: MT-B SV BT AD OD. Wrote the paper: CA PG SV MT-B. Provided medical care for study participants and collected clinical data at the field site: CA VD AD. Processed biological specimens and performed clinical assays: CA PG SB.
- 1. World Health Organization (2009) World malaria report 2009. World Health Organization2009World malaria report 2009
- 2. Dolo A, Modiano D, Maiga B, Daou M, Dolo G, et al. (2005) Difference in susceptibility to malaria between two sympatric ethnic groups in Mali. Am J Trop Med Hyg 72(3): 243–248.A. DoloD. ModianoB. MaigaM. DaouG. Dolo2005Difference in susceptibility to malaria between two sympatric ethnic groups in Mali.Am J Trop Med Hyg723243248
- 3. Israelsson E, Vafa M, Maiga B, Lysen A, Iriemenam NC, et al. (2008) Differences in fcgamma receptor IIa genotypes and IgG subclass pattern of anti-malarial antibodies between sympatric ethnic groups in Mali. Malar J 7: 175.E. IsraelssonM. VafaB. MaigaA. LysenNC Iriemenam2008Differences in fcgamma receptor IIa genotypes and IgG subclass pattern of anti-malarial antibodies between sympatric ethnic groups in Mali.Malar J7175
- 4. Bolad A, Farouk SE, Israelsson E, Dolo A, Doumbo OK, et al. (2005) Distinct interethnic differences in immunoglobulin G class/subclass and immunoglobulin M antibody responses to malaria antigens but not in immunoglobulin G responses to nonmalarial antigens in sympatric tribes living in west Africa. Scand J Immunol 61(4): 380–386.A. BoladSE FaroukE. IsraelssonA. DoloOK Doumbo2005Distinct interethnic differences in immunoglobulin G class/subclass and immunoglobulin M antibody responses to malaria antigens but not in immunoglobulin G responses to nonmalarial antigens in sympatric tribes living in west Africa.Scand J Immunol614380386
- 5. Farouk SE, Dolo A, Bereczky S, Kouriba B, Maiga B, et al. (2005) Different antibody- and cytokine-mediated responses to Plasmodium falciparum parasite in two sympatric ethnic tribes living in Mali. Microbes Infect 7(1): 110–117.SE FaroukA. DoloS. BereczkyB. KouribaB. Maiga2005Different antibody- and cytokine-mediated responses to Plasmodium falciparum parasite in two sympatric ethnic tribes living in Mali.Microbes Infect71110117
- 6. Torcia MG, Santarlasci V, Cosmi L, Clemente A, Maggi L, et al. (2008) Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria. Proc Natl Acad Sci U S A 105(2): 646–651.MG TorciaV. SantarlasciL. CosmiA. ClementeL. Maggi2008Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria.Proc Natl Acad Sci U S A1052646651
- 7. McCall MB, Hopman J, Daou M, Maiga B, Dara V, et al. (2010) Early interferon-gamma response against Plasmodium falciparum correlates with interethnic differences in susceptibility to parasitemia between sympatric Fulani and Dogon in Mali. J Infect Dis 201(1): 142–152.MB McCallJ. HopmanM. DaouB. MaigaV. Dara2010Early interferon-gamma response against Plasmodium falciparum correlates with interethnic differences in susceptibility to parasitemia between sympatric Fulani and Dogon in Mali.J Infect Dis2011142152
- 8. Stevenson MM, Riley EM (2004) Innate immunity to malaria. Nat Rev Immunol 4(3): 169–180.MM StevensonEM Riley2004Innate immunity to malaria.Nat Rev Immunol43169180
- 9. Piccioli D, Tavarini S, Borgogni E, Steri V, Nuti S, et al. (2007) Functional specialization of human circulating CD16 and CD1c myeloid dendritic-cell subsets. Blood 109(12): 5371–5379.D. PiccioliS. TavariniE. BorgogniV. SteriS. Nuti2007Functional specialization of human circulating CD16 and CD1c myeloid dendritic-cell subsets.Blood1091253715379
- 10. Lindstedt M, Lundberg K, Borrebaeck CA (2005) Gene family clustering identifies functionally associated subsets of human in vivo blood and tonsillar dendritic cells. J Immunol 175(8): 4839–4846.M. LindstedtK. LundbergCA Borrebaeck2005Gene family clustering identifies functionally associated subsets of human in vivo blood and tonsillar dendritic cells.J Immunol175848394846
- 11. Jongbloed SL, Kassianos AJ, McDonald KJ, Clark GJ, Ju X, et al. (2010) Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens. J Exp Med 207(6): 1247–1260.SL JongbloedAJ KassianosKJ McDonaldGJ ClarkX. Ju2010Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens.J Exp Med207612471260
- 12. Urban BC, Ferguson DJ, Pain A, Willcox N, Plebanski M, et al. (1999) Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400(6739): 73–77.BC UrbanDJ FergusonA. PainN. WillcoxM. Plebanski1999Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells.Nature40067397377
- 13. Urban BC, Roberts DJ (2002) Malaria, monocytes, macrophages and myeloid dendritic cells: Sticking of infected erythrocytes switches off host cells. Curr Opin Immunol 14(4): 458–465.BC UrbanDJ Roberts2002Malaria, monocytes, macrophages and myeloid dendritic cells: Sticking of infected erythrocytes switches off host cells.Curr Opin Immunol144458465
- 14. Skorokhod OA, Alessio M, Mordmuller B, Arese P, Schwarzer E (2004) Hemozoin (malarial pigment) inhibits differentiation and maturation of human monocyte-derived dendritic cells: A peroxisome proliferator-activated receptor-gamma-mediated effect. J Immunol 173(6): 4066–4074.OA SkorokhodM. AlessioB. MordmullerP. AreseE. Schwarzer2004Hemozoin (malarial pigment) inhibits differentiation and maturation of human monocyte-derived dendritic cells: A peroxisome proliferator-activated receptor-gamma-mediated effect.J Immunol173640664074
- 15. Urban BC, Mwangi T, Ross A, Kinyanjui S, Mosobo M, et al. (2001) Peripheral blood dendritic cells in children with acute Plasmodium falciparum malaria. Blood 98(9): 2859–2861.BC UrbanT. MwangiA. RossS. KinyanjuiM. Mosobo2001Peripheral blood dendritic cells in children with acute Plasmodium falciparum malaria.Blood98928592861
- 16. Urban BC, Cordery D, Shafi MJ, Bull PC, Newbold CI, et al. (2006) The frequency of BDCA3-positive dendritic cells is increased in the peripheral circulation of Kenyan children with severe malaria. Infect Immun 74(12): 6700–6706.BC UrbanD. CorderyMJ ShafiPC BullCI Newbold2006The frequency of BDCA3-positive dendritic cells is increased in the peripheral circulation of Kenyan children with severe malaria.Infect Immun741267006706
- 17. Todryk SM, Urban BC (2008) Dendritic cells in Plasmodium infection. Future Microbiol 3: 279–286.SM TodrykBC Urban2008Dendritic cells in Plasmodium infection.Future Microbiol3279286
- 18. Pichyangkul S, Yongvanitchit K, Kum-arb U, Hemmi H, Akira S, et al. (2004) Malaria blood stage parasites activate human plasmacytoid dendritic cells and murine dendritic cells through a toll-like receptor 9-dependent pathway. J Immunol 172(8): 4926–4933.S. PichyangkulK. YongvanitchitU. Kum-arbH. HemmiS. Akira2004Malaria blood stage parasites activate human plasmacytoid dendritic cells and murine dendritic cells through a toll-like receptor 9-dependent pathway.J Immunol172849264933
- 19. Vigario AM, Belnoue E, Gruner AC, Mauduit M, Kayibanda M, et al. (2007) Recombinant human IFN-alpha inhibits cerebral malaria and reduces parasite burden in mice. J Immunol 178(10): 6416–6425.AM VigarioE. BelnoueAC GrunerM. MauduitM. Kayibanda2007Recombinant human IFN-alpha inhibits cerebral malaria and reduces parasite burden in mice.J Immunol1781064166425
- 20. Luty AJ, Perkins DJ, Lell B, Schmidt-Ott R, Lehman LG, et al. (2000) Low interleukin-12 activity in severe Plasmodium falciparum malaria. Infect Immun 68(7): 3909–3915.AJ LutyDJ PerkinsB. LellR. Schmidt-OttLG Lehman2000Low interleukin-12 activity in severe Plasmodium falciparum malaria.Infect Immun68739093915
- 21. deWalick S, Amante FH, McSweeney KA, Randall LM, Stanley AC, et al. (2007) Cutting edge: Conventional dendritic cells are the critical APC required for the induction of experimental cerebral malaria. J Immunol 178(10): 6033–6037.S. deWalickFH AmanteKA McSweeneyLM RandallAC Stanley2007Cutting edge: Conventional dendritic cells are the critical APC required for the induction of experimental cerebral malaria.J Immunol1781060336037
- 22. Voisine C, Mastelic B, Sponaas AM, Langhorne J (2010) Classical CD11c+ dendritic cells, not plasmacytoid dendritic cells, induce T cell responses to Plasmodium chabaudi malaria. Int J Parasitol 40(6): 711–719.C. VoisineB. MastelicAM SponaasJ. Langhorne2010Classical CD11c+ dendritic cells, not plasmacytoid dendritic cells, induce T cell responses to Plasmodium chabaudi malaria.Int J Parasitol406711719
- 23. Newman KC, Korbel DS, Hafalla JC, Riley EM (2006) Cross-talk with myeloid accessory cells regulates human natural killer cell interferon-gamma responses to malaria. PLoS Pathog 2(12): e118.KC NewmanDS KorbelJC HafallaEM Riley2006Cross-talk with myeloid accessory cells regulates human natural killer cell interferon-gamma responses to malaria.PLoS Pathog212e118
- 24. Scholzen A, Mittag D, Rogerson SJ, Cooke BM, Plebanski M (2009) Plasmodium falciparum-mediated induction of human CD25Foxp3 CD4 T cells is independent of direct TCR stimulation and requires IL-2, IL-10 and TGFbeta. PLoS Pathog 5(8): e1000543.A. ScholzenD. MittagSJ RogersonBM CookeM. Plebanski2009Plasmodium falciparum-mediated induction of human CD25Foxp3 CD4 T cells is independent of direct TCR stimulation and requires IL-2, IL-10 and TGFbeta.PLoS Pathog58e1000543
- 25. Bouharoun-Tayoun H, Druilhe P (1992) Plasmodium falciparum malaria: Evidence for an isotype imbalance which may be responsible for delayed acquisition of protective immunity. Infect Immun 60(4): 1473–1481.H. Bouharoun-TayounP. Druilhe1992Plasmodium falciparum malaria: Evidence for an isotype imbalance which may be responsible for delayed acquisition of protective immunity.Infect Immun60414731481
- 26. Passlick B, Flieger D, Ziegler-Heitbrock HW (1989) Identification and characterization of a novel monocyte subpopulation in human peripheral blood. Blood 74(7): 2527–2534.B. PasslickD. FliegerHW Ziegler-Heitbrock1989Identification and characterization of a novel monocyte subpopulation in human peripheral blood.Blood74725272534
- 27. Ziegler-Heitbrock L (2007) The CD14+ CD16+ blood monocytes: Their role in infection and inflammation. J Leukoc Biol 81(3): 584–592.L. Ziegler-Heitbrock2007The CD14+ CD16+ blood monocytes: Their role in infection and inflammation.J Leukoc Biol813584592
- 28. Chimma P, Roussilhon C, Sratongno P, Ruangveerayuth R, Pattanapanyasat K, et al. (2009) A distinct peripheral blood monocyte phenotype is associated with parasite inhibitory activity in acute uncomplicated Plasmodium falciparum malaria. PLoS Pathog 5(10): e1000631.P. ChimmaC. RoussilhonP. SratongnoR. RuangveerayuthK. Pattanapanyasat2009A distinct peripheral blood monocyte phenotype is associated with parasite inhibitory activity in acute uncomplicated Plasmodium falciparum malaria.PLoS Pathog510e1000631
- 29. Jaworowski A, Kamwendo DD, Ellery P, Sonza S, Mwapasa V, et al. (2007) CD16+ monocyte subset preferentially harbors HIV-1 and is expanded in pregnant Malawian women with Plasmodium falciparum malaria and HIV-1 infection. J Infect Dis 196(1): 38–42.A. JaworowskiDD KamwendoP. ElleryS. SonzaV. Mwapasa2007CD16+ monocyte subset preferentially harbors HIV-1 and is expanded in pregnant Malawian women with Plasmodium falciparum malaria and HIV-1 infection.J Infect Dis19613842
- 30. Ogonda LA, Orago AS, Otieno MF, Adhiambo C, Otieno W, et al. (2010) The levels of CD16/Fc gamma receptor IIIA on CD14+ CD16+ monocytes are higher in children with severe Plasmodium falciparum anemia than in children with cerebral or uncomplicated malaria. Infect Immun 78(5): 2173–2181.LA OgondaAS OragoMF OtienoC. AdhiamboW. Otieno2010The levels of CD16/Fc gamma receptor IIIA on CD14+ CD16+ monocytes are higher in children with severe Plasmodium falciparum anemia than in children with cerebral or uncomplicated malaria.Infect Immun78521732181
- 31. Jin MS, Lee JO (2008) Structures of the toll-like receptor family and its ligand complexes. Immunity 29(2): 182–191.MS JinJO Lee2008Structures of the toll-like receptor family and its ligand complexes.Immunity292182191
- 32. Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: Update on toll-like receptors. Nat Immunol 11(5): 373–384.T. KawaiS. Akira2010The role of pattern-recognition receptors in innate immunity: Update on toll-like receptors.Nat Immunol115373384
- 33. Kadowaki N, Ho S, Antonenko S, Malefyt RW, Kastelein RA, et al. (2001) Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med 194(6): 863–869.N. KadowakiS. HoS. AntonenkoRW MalefytRA Kastelein2001Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.J Exp Med1946863869
- 34. Muzio M, Bosisio D, Polentarutti N, D'amico G, Stoppacciaro A, et al. (2000) Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: Selective expression of TLR3 in dendritic cells. J Immunol 164(11): 5998–6004.M. MuzioD. BosisioN. PolentaruttiG. D'amicoA. Stoppacciaro2000Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: Selective expression of TLR3 in dendritic cells.J Immunol1641159986004
- 35. Coban C, Ishii KJ, Uematsu S, Arisue N, Sato S, et al. (2007) Pathological role of toll-like receptor signaling in cerebral malaria. Int Immunol 19(1): 67–79.C. CobanKJ IshiiS. UematsuN. ArisueS. Sato2007Pathological role of toll-like receptor signaling in cerebral malaria.Int Immunol1916779
- 36. Franklin BS, Rodrigues SO, Antonelli LR, Oliveira RV, Goncalves AM, et al. (2007) MyD88-dependent activation of dendritic cells and CD4(+) T lymphocytes mediates symptoms, but is not required for the immunological control of parasites during rodent malaria. Microbes Infect 9(7): 881–890.BS FranklinSO RodriguesLR AntonelliRV OliveiraAM Goncalves2007MyD88-dependent activation of dendritic cells and CD4(+) T lymphocytes mediates symptoms, but is not required for the immunological control of parasites during rodent malaria.Microbes Infect97881890
- 37. Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, et al. (2007) Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to toll-like receptor 9. Proc Natl Acad Sci U S A 104(6): 1919–1924.P. ParrocheFN LauwN. GoutagnyE. LatzBG Monks2007Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to toll-like receptor 9.Proc Natl Acad Sci U S A104619191924
- 38. Wu X, Gowda NM, Kumar S, Gowda DC (2010) Protein-DNA complex is the exclusive malaria parasite component that activates dendritic cells and triggers innate immune responses. J Immunol 184(8): 4338–4348.X. WuNM GowdaS. KumarDC Gowda2010Protein-DNA complex is the exclusive malaria parasite component that activates dendritic cells and triggers innate immune responses.J Immunol184843384348
- 39. Krishnegowda G, Hajjar AM, Zhu J, Douglass EJ, Uematsu S, et al. (2005) Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: Cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 280(9): 8606–8616.G. KrishnegowdaAM HajjarJ. ZhuEJ DouglassS. Uematsu2005Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: Cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity.J Biol Chem280986068616
- 40. Krug A, Rothenfusser S, Hornung V, Jahrsdorfer B, Blackwell S, et al. (2001) Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol 31(7): 2154–2163.A. KrugS. RothenfusserV. HornungB. JahrsdorferS. Blackwell2001Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells.Eur J Immunol31721542163
- 41. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, et al. (2000) Immunobiology of dendritic cells. Annu Rev Immunol 18: 767–811.J. BanchereauF. BriereC. CauxJ. DavoustS. Lebecque2000Immunobiology of dendritic cells.Annu Rev Immunol18767811
- 42. Vafa M, Maiga B, Berzins K, Hayano M, Bereczky S, et al. (2007) Associations between the IL-4 -590 T allele and Plasmodium falciparum infection prevalence in asymptomatic Fulani of Mali. Microbes Infect 9(9): 1043–1048.M. VafaB. MaigaK. BerzinsM. HayanoS. Bereczky2007Associations between the IL-4 -590 T allele and Plasmodium falciparum infection prevalence in asymptomatic Fulani of Mali.Microbes Infect9910431048
- 43. Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392(6673): 245–252.J. BanchereauRM Steinman1998Dendritic cells and the control of immunity.Nature3926673245252
- 44. Vafa M, Israelsson E, Maiga B, Dolo A, Doumbo OK, et al. (2009) Relationship between immunoglobulin isotype response to Plasmodium falciparum blood stage antigens and parasitological indexes as well as splenomegaly in sympatric ethnic groups living in Mali. Acta Trop 109(1): 12–16.M. VafaE. IsraelssonB. MaigaA. DoloOK Doumbo2009Relationship between immunoglobulin isotype response to Plasmodium falciparum blood stage antigens and parasitological indexes as well as splenomegaly in sympatric ethnic groups living in Mali.Acta Trop10911216
- 45. Bereczky S, Dolo A, Maiga B, Hayano M, Granath F, et al. (2006) Spleen enlargement and genetic diversity of Plasmodium falciparum infection in two ethnic groups with different malaria susceptibility in Mali, west Africa. Trans R Soc Trop Med Hyg 100(3): 248–257.S. BereczkyA. DoloB. MaigaM. HayanoF. Granath2006Spleen enlargement and genetic diversity of Plasmodium falciparum infection in two ethnic groups with different malaria susceptibility in Mali, west Africa.Trans R Soc Trop Med Hyg1003248257
- 46. Poulin LF, Salio M, Griessinger E, Anjos-Afonso F, Craciun L, et al. (2010) Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8alpha+ dendritic cells. J Exp Med 207(6): 1261–1271.LF PoulinM. SalioE. GriessingerF. Anjos-AfonsoL. Craciun2010Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8alpha+ dendritic cells.J Exp Med207612611271
- 47. Lundie RJ, de Koning-Ward TF, Davey GM, Nie CQ, Hansen DS, et al. (2008) Blood-stage Plasmodium infection induces CD8+ T lymphocytes to parasite-expressed antigens, largely regulated by CD8alpha+ dendritic cells. Proc Natl Acad Sci U S A 105(38): 14509–14514.RJ LundieTF de Koning-WardGM DaveyCQ NieDS Hansen2008Blood-stage Plasmodium infection induces CD8+ T lymphocytes to parasite-expressed antigens, largely regulated by CD8alpha+ dendritic cells.Proc Natl Acad Sci U S A105381450914514
- 48. Jego G, Palucka AK, Blanck JP, Chalouni C, Pascual V, et al. (2003) Plasmacytoid dendritic cells induce plasma cell differentiation through type I interferon and interleukin 6. Immunity 19(2): 225–234.G. JegoAK PaluckaJP BlanckC. ChalouniV. Pascual2003Plasmacytoid dendritic cells induce plasma cell differentiation through type I interferon and interleukin 6.Immunity192225234
- 49. Varani S, Cederarv M, Feld S, Tammik C, Frascaroli G, et al. (2007) Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells. J Immunol 179(11): 7767–7776.S. VaraniM. CederarvS. FeldC. TammikG. Frascaroli2007Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells.J Immunol1791177677776
- 50. Luoni G, Verra F, Arca B, Sirima BS, Troye-Blomberg M, et al. (2001) Antimalarial antibody levels and IL4 polymorphism in the Fulani of west Africa. Genes Immun 2(7): 411–414.G. LuoniF. VerraB. ArcaBS SirimaM. Troye-Blomberg2001Antimalarial antibody levels and IL4 polymorphism in the Fulani of west Africa.Genes Immun27411414
- 51. Clark IA, Budd AC, Alleva LM, Cowden WB (2006) Human malarial disease: A consequence of inflammatory cytokine release. Malar J 5: 85.IA ClarkAC BuddLM AllevaWB Cowden2006Human malarial disease: A consequence of inflammatory cytokine release.Malar J585
- 52. Walther M, Woodruff J, Edele F, Jeffries D, Tongren JE, et al. (2006) Innate immune responses to human malaria: Heterogeneous cytokine responses to blood-stage Plasmodium falciparum correlate with parasitological and clinical outcomes. J Immunol 177(8): 5736–5745.M. WaltherJ. WoodruffF. EdeleD. JeffriesJE Tongren2006Innate immune responses to human malaria: Heterogeneous cytokine responses to blood-stage Plasmodium falciparum correlate with parasitological and clinical outcomes.J Immunol177857365745
- 53. Coban C, Ishii KJ, Kawai T, Hemmi H, Sato S, et al. (2005) Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin. J Exp Med 201(1): 19–25.C. CobanKJ IshiiT. KawaiH. HemmiS. Sato2005Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin.J Exp Med20111925
- 54. Liu YJ (2005) IPC: Professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol 23: 275–306.YJ Liu2005IPC: Professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors.Annu Rev Immunol23275306
- 55. Pulendran B, Ahmed R (2006) Translating innate immunity into immunological memory: Implications for vaccine development. Cell 124(4): 849–863.B. PulendranR. Ahmed2006Translating innate immunity into immunological memory: Implications for vaccine development.Cell1244849863
- 56. Mockenhaupt FP, Cramer JP, Hamann L, Stegemann MS, Eckert J, et al. (2006) Toll-like receptor (TLR) polymorphisms in African children: Common TLR-4 variants predispose to severe malaria. Proc Natl Acad Sci U S A 103(1): 177–182.FP MockenhauptJP CramerL. HamannMS StegemannJ. Eckert2006Toll-like receptor (TLR) polymorphisms in African children: Common TLR-4 variants predispose to severe malaria.Proc Natl Acad Sci U S A1031177182
- 57. Mockenhaupt FP, Hamann L, von Gaertner C, Bedu-Addo G, von Kleinsorgen C, et al. (2006) Common polymorphisms of toll-like receptors 4 and 9 are associated with the clinical manifestation of malaria during pregnancy. J Infect Dis 194(2): 184–188.FP MockenhauptL. HamannC. von GaertnerG. Bedu-AddoC. von Kleinsorgen2006Common polymorphisms of toll-like receptors 4 and 9 are associated with the clinical manifestation of malaria during pregnancy.J Infect Dis1942184188
- 58. McCall MB, Netea MG, Hermsen CC, Jansen T, Jacobs L, et al. (2007) Plasmodium falciparum infection causes proinflammatory priming of human TLR responses. J Immunol 179(1): 162–171.MB McCallMG NeteaCC HermsenT. JansenL. Jacobs2007Plasmodium falciparum infection causes proinflammatory priming of human TLR responses.J Immunol1791162171
- 59. Franklin BS, Parroche P, Ataide MA, Lauw F, Ropert C, et al. (2009) Malaria primes the innate immune response due to interferon-gamma induced enhancement of toll-like receptor expression and function. Proc Natl Acad Sci U S A 106(14): 5789–5794.BS FranklinP. ParrocheMA AtaideF. LauwC. Ropert2009Malaria primes the innate immune response due to interferon-gamma induced enhancement of toll-like receptor expression and function.Proc Natl Acad Sci U S A1061457895794
- 60. Boutlis CS, Yeo TW, Anstey NM (2006) Malaria tolerance–for whom the cell tolls? Trends Parasitol 22(8): 371–377.CS BoutlisTW YeoNM Anstey2006Malaria tolerance–for whom the cell tolls?Trends Parasitol228371377
- 61. Hay SI, Okiro EA, Gething PW, Patil AP, Tatem AJ, et al. (2010) Estimating the global clinical burden of Plasmodium falciparum malaria in 2007. PLoS Med 7(6): e1000290.SI HayEA OkiroPW GethingAP PatilAJ Tatem2010Estimating the global clinical burden of Plasmodium falciparum malaria in 2007.PLoS Med76e1000290
- 62. De Souza JB, Williamson KH, Otani T, Playfair JH (1997) Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria. Infect Immun 65(5): 1593–1598.JB De SouzaKH WilliamsonT. OtaniJH Playfair1997Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria.Infect Immun65515931598
- 63. Pinto LA, Trivett MT, Wallace D, Higgins J, Baseler M, et al. (2005) Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry. Cytometry B Clin Cytom 63(1): 47–55.LA PintoMT TrivettD. WallaceJ. HigginsM. Baseler2005Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry.Cytometry B Clin Cytom6314755