Human Immune Responses to Plasmodium falciparum infection: Molecular Evidence for a Suboptimal THαβ(TH9) and TH17 Bias over Ideal and Effective Traditional TH1 Immunity

Using microarray analysis, we showed up-regulation of Toll-like receptors 1,2,4,7,8, NF-[kappa][BETA], TNF-[alpha], p38-MAPK and MHC molecules in human peripheral blood mononuclear cells following infection with Plasmodium falciparum. We report herein further studies based on time-course microarray analysis with a focus on malaria-induced host immunity. Results show that in early malaria; selected immunity-related genes were up-regulated including alpha, beta, and gamma interferon related genes, as well as genes of IL-15, CD36, chemokines (CXCL10, CCL2, S100A8/9, CXCL9, and CXCL11), TRAIL and IgG Fc receptors. During acute febrile malaria, up-regulated genes included alpha, beta, and gamma interferon related genes, IL-8, IL-1[beta], IL-10 downstream genes, TGFB1, oncostatin-M, chemokines, IgG Fc receptors, ADCC signaling, complement-related genes, granzymes, NK cell killer/inhibitory receptors and Fas antigen. During remission, genes for NK receptors, immunoglobins, and granzymes/perforin were up-regulated. When viewed in terms of immunity type, malaria infection appeared to induce a mixed TH1 response, in which α and β interferon driven responses appear to predominate over the more classic IL-12 driven pathway. In addition, TH17 pathway also appears to be playing a significant role in the immunity to Plasmodium falciparum. Gene markers of TH17 (neutrophil-related genes, TGFB1 and IL-6 family (oncostatin-M)) and TH[alpha]/[beta] (IFN-[alpha]/[beta] and NK cytotoxicity and ADCC gene) immunity were up-regulated. Initiation of TH[alpha]/[beta] immune response was associated with an IFN-[alpha]/[beta] response which ultimately resulted in IL-10 and IFN-[gamma] achieved via a different pathway from the more classic IL-12 TH1 pattern. Based on these observations, we speculate that in Plasmodium falciparum infection, TH[alpha]/[beta] and TH17 immunity may predominate over the more traditional TH1 response.


Introduction
Host immunity may involve one or a combination of pathways. It has been suggested that TH1 immunity, initiated primarily by IL-12, may play an important immunological role in defense against viruses and intracellular bacteria/parasites, while TH2 responses, driven by IL4, have been shown to be more important against helminthes 1 . TH17 immunity is elicited against extracellular bacteria and this recently described host immunity pathway, is thought to be triggered by TGF-β working in tandem with IL-21 or IL-6 2 . Dendritic cells are important antigen processing cells and play an important role in stimulating both innate and adaptive immune responses to viruses, fungi and bacteria 3 . In these cells, IL-12 is up-regulated following infection with E. coli and Candida albicans, but not influenza virus. In contrast, interferon alpha/beta is upregulated after infection with influenza virus, but not E. coli and Candida albicans. These observations suggest that IL-12 and IFN-αβ may help drive different immune response maturation pathways 3 . It is not clear at this time as to which pathway may predominate in the host response to Plasmodium falciparum (P. falciparum) infection.
TH1 has been suggested to be the dominant and protective immune response against malaria both in rodents and humans [4][5] . Yet, the blood stage of P. falciparum can serve to immunosuppress the host's immune response to the liver stage of the parasite 6 .
Dendritic cell maturation is inhibited by P. falciparum infected RBCs 7 ; monocyte maturation is also suppressed by malaria pigments (hemozoin), and low serum IL-12 was reported in severe malaria [8][9] . These observations suggest that the IL-12 driven TH1 IFNg dominant response patterns seen following P. falciparum infection may not be functioning optimally; thus, against this background data; one may ask how the infected host is capable of mounting an effective TH1 response against the blood-borne stage of this parasite? An alternative pathway may be needed to achieve this host immune response.
To gain a better understanding of host immune response patterns associated P.
falciparum infection in humans, we carried out transcriptional profiling using microarray analysis of peripheral blood mononuclear cells (PBMCs) after P. falciparum infection.
Our earlier analysis showed an up-regulation of gene expression for Toll-like receptor signaling, NF-kB, TNF-, IFN-, IL-1, p38 MAPK, MHC class I & II molecules 10 . In this study, we further analyzed the data focusing on the specific type or types of host immunity induced by infection. Results indicated an up-regulation of TGF-β and the IL-6 family gene (oncostatin M), both of which induce TH17 immunity. We also observed an elevation in the expression of interferon gamma, NK cell cytotoxicity, antibodydependent cell cytotoxicity (ADCC), which are triggered by interferon alpha/beta (TH), instead of IL-12 (TH1). These results suggest multiple immune pathways may be activated following infection with P. falciparum. For the purposed of this thesis we have classified these additional pathways as: TH and TH17 immunity. The THαβ pathway is not generally recognized within the immunology community as a distinct T cell maturation pathway while the TH17 response pattern has only recently been recognized 11 . However, THαβ responses in which α and β interferons drive a succession of cellular process leading to mid-moderate IFN-γ production and NK cell and ADCC activation have been described in the host response to viruses but they have not been shown to play an important role in the maturation of the anti-malaria adaptive immune response.
To further clarify this point, traditionally TH1 leads to immunity against intracellular bacteria and protozoa; and THαβ against viral infections. Macrophage activation is characteristic of traditional TH1 immunity, while NK activation (natural cytotoxicity & ADCC) is more characteristic of TH immunity. Thus, malaria infection appears to be somewhat unique in that it appears to elicit a combined response reflective of both TH17 and THαβ host immunity.

Previous analysis
Subject recruitment, sample collection and preparation, and RNA purification were described previously in Ockenhouse and Hu's paper. Briefly, two groups of subjects were recruited for this study after Johns Hopkins University Institutional Review Board and US Army HSRRB ethical approvals were obtained. In the study cohort, 22 subjects, 20-45 years of age, were recruited from the Walter Reed Army Institute of Research (WRAIR). Subjects agreed to receive mosquito bites from laboratory-reared Anopheles stephensi infected by P. falciparum (3D7 strain). Once parasitemia was detected in the subjects's peripheral blood, these subjects received treatment with chloroquine. Blood samples were drawn during the un-infected baseline period (U) and again, when parasitemia was found for the first time (Early malaria) (E).
In the other study cohort, 15 adults were recruited from Cameroon, Africa. These subjects were 19-49 years of age with acute P. falciparum infection. All suffered from typical relapsing fever and blood smears showed parasitemia. These subjects received at least one week of anti-malarial drug treatment (Cotecxin). Blood drawing was performed during the acute febrile infection period (A) and one month later during the remission period (R). During the remission period, physical exams and blood smears were performed to ensure that malaria symptoms were no longer present and parasitemia was no longer detectable.
In the first study cohort, PBMCs were separated from whole blood samples by Ficoll-gradient at WRAIR. In the second study cohort, blood was collected in CPT tubes and PBMCs were isolated after centrifuge. A RNA stabilizing reagent-RNA later (Ambion, CA, USA) was added and samples were shipped to the USA on dry ice. Total RNA was extracted from both sets of samples using Trizol. The quality of sample RNA was estimated by spectrometry (OD>1.8) and gel electrophoresis.

Microarray preparation
Affymetrix U133A GeneChips (Affymetrix, Santa Clara, CA) were used in this study. The GeneChips contain 22,283 probe-sets, including 14,500 known wellcharacterized human genes and 18,400 transcripts. Before chip hybridization, a QIAGEN RNeasy cleanup kit was used to purify total RNA. Processing of templates for analysis on the Affymetrix U133A GeneChip was performed in accordance with methods described in the Affymetrix Technical Manual, Revision Three. Total RNA from the blood samples were hybridized into the arrays. Detailed cDNA preparation, in vitro transcription, staining, and scanning of Affymetrix U133A GeneChips were described previously in Chris Ockenhouse and Wanchung Hu's paper.

Data analysis
We used GeneSpring software and GeneSpring default normalization to perform one-way ANOVA tests, using un-infected samples as baseline. Significantly up-regulated genes were selected if false discovery rate (FDR) was <0.05 and fold change was >1.5x when compared to un-infected baseline. These genes were placed into three groups based on expression levels in different stages of the infection. In addition, Pearson's correlation analysis and Rank correlation analysis were performed to find the relationship between expression levels of genes after malaria infection. Pathway analysis was performed by using the Pathway Architect (Statagene Inc.) software to identify the specific immunological pathways involved.

Microarray accession numbers
The Affymetrix data sets can be accessed at http://www.ncbi.nlm.nih.gov/geo/ under the accession number GSE5418.

Results
A total of 2894 genes were differentially expressed out of the 22

TH1 immunity related gene up-regulation during malaria
After P. falciparum infection, many immune response-related genes of the TH1 immunological pathway were up-regulated (Table 1). Although interferon alpha/beta was not detected in this study, the major transcription factor of interferon alpha/beta synthesis, interferon-induced protein with tetratricopeptide repeat 4 (IFIT4), interferon-induced protein with tetratricopeptide repeat 1 (IFIT1), and interferon-induced protein with tetratricopeptide repeat 2 (IFIT2). The expression levels of the above genes were greater than 2-fold in early malaria, as compared to un-infection baseline. In acute febrile malaria, the expression levels of most of the alpha/beta interferon genes tended to decline and all returned to baseline levels of expression during the remission period (Table 1).
Interferon gamma and many interferon gamma-related genes were also up-regulated after malaria. These observations are consistent with a model in which malaria infection induces primarily a TH1 type immune response. These up-regulated genes included: interferon gamma, interferon inducible guanylate binding protein 1 (GBP1), interferon inducible guanylate binding protein 2 (GBP2), Janus kinase1 (JAK1), and transporter with antigen processing 1 (TAP1). The expression levels of GBP1, GBP2, and TAP1 ranged from 10.6 to 2.8 fold above baseline during early stage of malaria, and the expression level of GBP2 was greater than 2-fold change during acute febrile malaria.
These interferon gamma inducible genes all returned to baseline levels of expression during the remission periods of the disease. The up-regulation of a number of TH1 chemokines over the malaria disease course was also consistent with a TH1 model of anti-malaria immunity. Up-regulated TH1 related chemokine genes included: CXCL10, CCL3, CCL3 and CCR1. The expression levels of CXCL10 (5.3 fold) was highest during the early phase of malaria, while CCR3, and CCL4 were higher during febrile and recovery stages of the disease (Table 1).
There were many antibody-dependent cellular cytotoxicity (ADCC) and NK cell- Interleukin 15 can enhance NK cell differentiation and proliferation. ADCC is mainly mediated by NK cells, but it can also be mediated by macrophages. Fc receptor and ADCC signaling gene up-regulation is consistent with the ADCC machinery being turned on after P. falciparum infection. Among the above genes, gene expression levels were greater than 2-fold change (range 2 to 7 fold; see Table 1) in early malaria including: Fc gamma receptor 1A, Fc gamma receptor 3A, Fc epsilon receptor 1G, and TRAIL. Gene expression levels were greater than 2-fold change in acute febrile malaria including: Fc alpha receptor, Fc gamma receptor 3B, Fc epsilon receptor 1G, PI3K, MAP2K2, Arf6, KLRC3, KIR3DL2, and CEBP gamma (range 2-4 fold). Gene expression levels were greater than 2-fold change in the remission period, including: Fc gamma receptor 3B, Fc epsilon receptor 1G, PI3K, Arf6, KLRC3, and KIR3DL2 range 2-3 fold). Although ADCC signaling genes were not greater than 2-fold, they were higher than 1.5-fold change after malarial infection.
CTL related genes (CD8 T cells in Table 1) were only moderately up-regulated, during malaria Among these genes, granzyme B, CDC42, and Kras exhibited ≥ 2-fold change during acute febrile malaria. Granzyme B and Kras were > 2-fold change in the remission period.
Although macrophage/monocyte activation is traditionally considered to be a strong marker for classic TH1 type immunity, we surprisingly found that express levels of most macrophage activation genes were relatively unchanged or slightly suppressed after P. falciparum infection ( Table 2). These data support the hypothesis that there may be two pathways involved in the induction of TH1 types of immune responses: a TH interferon driven path and IL-12 driven path. (See Discussion Section). Most macrophage activation and proliferation related genes were un-changed. (Table 2) IL-12 is clearly the major cytokine needed to initiate a strong classic TH1 type immune response, and IL-12 expression levels remain unchanged over the course of this study (Table 2). This observation suggests that the induction of a TH1-type antimalarial immune response may be driven by cytokines other than IL-12. Like IL-12, other traditional TH1 cytokine: IL-3, M-CSF, and GM-CSF can also cause macrophage proliferation but these gene are also not up-regulated in response to infection with P. falciparum (Table2). In contrast, many macrophage inhibition-related genes were up-regulated after malaria including heme oxygenase 1 (HMOX1), JunB, and IRF1. JunB, PU.1 and IRF1 suppress macrophage proliferation. As indicated above, a macrophage differentiation inducer, MafB, is upregulated after malaria. Its antagonists, JunB and PU.1, were also up-regulated, so the effect of MafB could be suppressed. PU.1 up-regulation can drive monocytes to differentiate into dendritic cells instead of macrophages. Although macrophages can also express Fc receptor genes, we think these Fc receptor expression after malaria should mainly occur in NK cells. The Fc receptor signaling pathway (ADCC signaling pathway) in NK cell was all up-regulated (Table 1 ). Also, DAP12 up-regulation can suppress Fc receptor signaling in macrophages, so it is less likely that these up-regulated Fc receptors belong to macrophages. Although IL-10 itself was not up-regulated, many IL-10 downstream genes were up-regulated ( Figure 1). IL-10 can activate NK cells and deactivate macrophages. HMOX1 is the major downstream effector molecule to mediate IL-10 inhibition of macrophages. HMOX1 was up-regulated after malaria. HMOX1 generates CO molecule to mediate anti-inflammatory effects in macrophages, which is opposite to iNOS, which generates NO molecule to cause inflammatory effects.
Argininesuccinate synthetase (ASS) is the rate-limiting enzyme to synthesize arginine; the substrate of iNOS. iNOS is the central mediator representing macrophage classical activation; and un-changed ASS and iNOS express levels support the notion that macrophages are not being activated after malaria infection. In our study, macrophages were inhibited rather than activated after malaria. In summary, many elements of TH1 immunity were up-regulated following infection with P. falciparum, including: interferon alpha/beta inducible genes, interferon gamma inducible genes, NK cell related genes (NK cell cytotoxicity), ADCC related genes, TH1 chemokines, and CD8 T cell related genes.
However, macrophages appear to become deactivated and do not appear to proliferate after malarial infection.

TH17 immunity related gene up-regulation during malaria
A number of genes associated with the new described TH17 immunity pathway were up-regulated after infection with P. falciparum (see Table 3). Of these genes, IL-8 showed the greatest degree of up-regulation over the entire course of the disease cycle (early, febrile period and remission periods), with expression levels peaking during the febrile period of illness (8.5 fold increase; see Table 3). Other up-regulated genes included other TH17 related cytokines, IL1, TGF1, and oncostatin M [OSM], as well as TH17 related transcription factors, CEBP delta, and CEBP gamma. In addition, selected neutrophil-related genes were also up-regulated, including: neutrophil attracting chemokines, S100A9, CXCL2, and CXCL3, the neuterophil-related CD molecules, ICAM1, the NADPH oxidases gene NCF1, and acute reaction protein, PGE synthetase2.

TH2 immunity related genes unchanged during malaria
Most TH2 immunity related genes were remarkably unchanged over the course of the malaria disease cycle (Table 4). An extensive analysis of TH2 cytokines and chemokines, as well as Mast cell and eosinophil genes revealed that gene expression level were essentially unaffected by infection with P. falciparum; thus providing strong evidence that TH2-type immunity was not significantly initiated during malaria in this study. Expression levels for only a single gene involved in Mast cell activation, Fc epsilon receptor 1A surface receptor, was modified during the malaria disease cycle.
This gene was down-regulated during early malaria and mildly up-regulated during remission period.

Gene-to-gene relationship
We performed Pearson's correlation to analyze gene-to-gene relationship after malaria infection and found a number of relationships in our study subjects that were consistent with observations by other investigators in previous studies. We observed a strong negative Pearson's correlation between the expression levels of STAT1 and  (Table 5).
Rank correlation has also been done, but there were no significant results. Rank correlation is more strict than Pearson's correlation. If the data is normally distributed, Pearson's correlation analysis is more suitable than Rank correlation analysis.

Immunological pathways
Pathway analysis was conducted to explore immunological genetic circuitry  Figure 4 shows a sketch of the major pathways of host immunity that are felt to play a role in recovery from malaria based on the observations made in this study, as well as those made by other investigators in previous research. The changes in their expression levels during the early, acute, and remission stages of P. falciparum infection are summarized in Figure 4. As shown in Figure 5, the expression of genes related to TH2 immunity were unchanged following malaria infection; with most changes occurred in genes related to the TH17 and TH driven pathway of anti-malaria immunity.

Validation by plasma and PBMC protein expression
In our previous analysis, we have found out the concordance between transcription of

Discussion
In previous work, we showed that Toll-like receptor signaling genes, NFkB, TNFalpha, IFN gamma, IL-1 beta, p38 MAPK, and MHC I & II genes were significantly upregulated in acute febrile malaria in comparison with un-infected baseline 10 . In this current analysis, we identified more immunology genes, whose expression levels were significantly changed at different time-points over the course of the malaria disease cycle.
Based on these results, a clearer molecular picture of the range of host immune responses that develop following P. falciparum has started to emerge, which suggests that the THαβ variant pathway of original TH1 immunity and TH17 pathway may predominate over the more traditional IL-12 driven TH1 immunity pathway. However, it is important to Neutrophil overactivation in TH17 immunity is thought to play major role in the pathogenesis of cerebral malaria, acute renal failure, and ARDS [17][18][19] . Thus, malaria induced TH17 immunity could be related to both malaria-induced cerebral malaria, acute renal failure, and ARDS.
Evidence for dividing the more traditional TH1 immunity pathway into two subtypes (THαβ and TH1 immunity) comes primarily from both mouse and human models of malaria immunity. The major difference separating TH1 immunity and TH immunity are the effector cells involved. In the mouse model, interferon alpha/beta suppresses macrophage proliferation and neutrophiles and NK cells play a more prominent role, in contrast, IL-12 enhances macrophage proliferation [20][21] . In addition, interferon alpha/beta increases NK cell blastogenesis, so alpha/beta interferon enhances NK cell proliferation 22 . Thus, interferon alpha/beta and IL-12 mediated immunological events can be distinguished by the different effector cells they enhance, NK cells or macrophages.
Both IL-12 and interferon alpha/beta can induce CD4 T cells to secret interferon gamma. Interferon alpha/beta can induce CD4 T cells to produce both interferon gamma(+) and IL-10(+++). These interferon gamma and IL-10 secreting CD4 T cells were previously called a type 1 regulatory T cells because IL-10 has some antiinflammatory effect. However, if these types of T cells are purely immuno-regulator cells, it cannot explain why there is interferon gamma production from these CD4 T cells. IL-

is not a pure immunosuppressant because it can stimulate NK cells, B cells and CTLs.
In fact, the regulatory effect of these Tr1 cells could be due to cross-regulation between Tr1 cells and other T helper cells such as TH1, TH2, and TH17 cells. In a prior mouse study, interferon alpha/beta can substitute for IL-12 in the induction of interferon gamma production associated with the development of TH1 immunity 23 . In mice, interferon alpha/beta can also induce B cell isotype switching to produce IgG antibody 24 . IL-10 can stimulate B cell to produce IgG1 antibody and IFNg can stimulate B cells to produce IgG3 antibody. Interferon alpha/beta can also cause cross-priming of CD8 T cells 25 . Both interferon alpha/beta and IL-12 can serve as a third signal to facilitate clonal expansion of antigen specific CD8 T cells 26 . Again in mice, interferon alpha/beta can suppress the proliferation of IL-17 secreting CD4 T cells 2 . These observations in mice strongly suggest that interferon alpha/beta can induce an alternative TH1 immunity without the need of IL-12. In addition, interferon alpha/beta suppresses the induction of nitric oxide synthetase (iNOS) 27 . iNOS is the central effector molecule involved in the killing of ingested bacteria and parasites by macrophages. Activation of iNOS is a basic tenet of classic macrophage activation. iNOS activation has been shown to enhance the intracellular killing capability of macrophages by a 1000-fold 28 . iNOS can also be induced by interferon gamma through IL-12 mediated up-regulation. IL-10 can also serve as a strong macrophage de-activator. It can suppress macrophage proliferation, iNOS activation, and macrophage cytokine production. It can also suppress IL-12 production.
Thus, interferon alpha/beta and IL-12 should be seen as belonging to different inductive immunological pathways.
Mouse models of anti-malaria immunity indicate that interferon gamma and TH1 immunity play central roles in the developmental process 5 . Although administration of IL-12 can provide 100% protection against malaria parasite challenge, IL-12 plays a limited role in natural immunity against malaria in mice 4 . Active immunosuppression is well documented during malaria infection in mice 6 . IL-12 expression is down relegated in this model 29 . According to an IRF1 knockout study, mice can initiate TH1 immunity by bypassing the need for IL-12 production through the use of an alternate interferon alpha/beta driven pathway 30 . Based on recent microarray studies in malaria infected mice there is no evidence of IL-12 up-regulation following infection. Instead, interferon alpha/beta and its related genes were found to be significantly up-regulated 31-32 . Thus, providing further evidence at the gene level that interferon alpha/beta can substitute for IL-12 in the induction of malaria-specific interferon gamma TH1 type immune response after infection in rodents. However, the IFNαβ driven THαβ immunity only can produce mid-to-moderate IFNg, and IL-10, the main effector molecule in THαβ immunity, antagonizes the effect of IFNg upon macrophages. Because the ideal traditional TH1 immunity cannot be triggered in natural malarial infection in human to clear out the protozoa, malaria parasites can cause severe illness in human.
In human, there is also a substantial amount of evidence indicating that TH1 immunity can be induced by two pathways driven by αβ interferon or IL-12 33 .
Administration of IL-12 to human subjects suppresses NK cell proliferation 34 .
Macrophage activation serves to inhibit NK cell function; while NK cell activation serves to inhibit macrophage proliferation 20,35 . Given these observation it is reasonable to speculate that these two effector cells belong to different immunological pathways.In the present study, we found that a number of genes involved in the NK cell proliferation were up-regulated over the course of the malaria disease cycle (Table 1). In addition, as in the mouse model, genes associated with macrophage proliferation were not up-regulated following infection with P. falciparum. Increased NK cell populations have also been reported previously in acute malaria infection [36][37] . Thus, it appears that the interferon alpha/beta-NK cell pathway of THαβ immunity is preferentially enhanced after P. falciparum infection; while the IL-12 driven pathway appears unaffected. Other TH related cytokines also can cause NK cell activation or proliferation including IL-10 and IL-15, respectively.
In human, interferon alpha/beta has been shown to enhance NK cell cytotoxicity as well as antibody dependent cell cytotoxicity (ADCC) 38 , but IL-12 administration suppresses NK cell cytotoxicity and ADCC 39 . In addition, IL-12 induces NK cell to secret high levels of interferon gamma to activate macrophages instead of activating NK cell's cytotoxicity machinary 22 . In the current study, Fc receptors, alpha/beta interferon and ADCC related genes were up-regulated after malaria infection. Consistent with this model, patients with defects in NK cell production usually suffering from recurrent virus infections and have more complications following malaria infection 40 41 .
Further evidence that interferon alpha/beta can trigger TH1 immunity without the need for IL-12 comes from other human studies, in which, interferon alpha/beta suppressed STAT6 expression which is related to IL-4 induction and expression of TH2 immunity 42 . In our study interferon alpha/beta inducible genes were clearly up-regulated to induce THαβ immunity with ADCC; while TH2 related gene expression remained unchanged. In human models, interferon alpha/beta inhibits IL-12 production via an IL-10 dependent mechanism 43 . Thus, it seems reasonable to speculate that IL-12 and interferon alpha/beta do not belong to the same immunological pathway.
In human malarial infection, it has been shown that serum IL-12 levels are inversely correlated with malaria parasitemia 9 . The more severe the malaria is, the lower IL-12 expression levels become. The up-take of malarial pigment (Hemozoin) can downregulate IL-12 secretion in human monocytes 8  These molecules can actively suppress macrophage activation. When given alone and prior to malaria infection IL-12 can provide 100% protection against experimental Plasmodium infection through the activation of macrophages, which help to control the liver stage of infection 4 . However, based on the data collected during the course of the current study it also appears that infected individuals defend malaria through an alternate THαβ immunity, involving alpha/beta interferon and the activation of NK cells.
Interferon gamma plays a central role in immunity against malaria. However, the natural host immunity is suboptimal because IFNg is not the main effector in THαβ immunity. Although IL-10, a strong macrophage de-activator, was not up-regulated in this study, many downstream genes of IL-10 were up-regulated including heme oxygenase 1, and it suggests that IL-10 is actually up-regulated after malaria infection. In an ideal situation, TH1 immunity is basically immunity against intracellular bacteria/protozoa; THαβ immunity is basically immunity against viruses. Although, it appears that macrophages may not be activated in natural immunity against malaria, interferon gamma(+) and IL-10(+++) secreting CD4 T cells, cytotoxic CD8 T cells, IgG secreting B cells, and NK cells are up-regulated by interferon alpha/beta in malarial infection. In a clinical study in Gabon, interferon-γ and IL-10 secreting CD4 T cells were increasing and correspondent to the recovery of P. falciparum infection 46 . Pure TH1 interferon-γ producing T cells and pure TH2 IL-4 producing T cells were not changed during convalescence after malaria infection. Thus observation is consistent with a model, in which, interferon-γ and IL-10 secreting T cells play important roles in host immunity against malaria. In conclusion, it appears that the human immune response to P. falciparum is characterized by a suboptimal THα/β and TH17 bias which predominates over the more ideal and effective traditional TH1 responses driven by IL-12. Since treatment for malarial infection faces some difficulty like drug resistance, we strongly encourage to use IFNg(FDA approval drug) or IL-12 for malarial infection treatment to achieve the optimal host immunity to kill the intracellular protozoa. Red color means greater than 2-fold change; yellow color means 1.5-2-fold change; green color means no change blue color means down-regulation  3-B IL-4 centered pathway analysis. Network analysis was performed by using IL-4 as the central nodes. We used software to select IL-4 related genes from our genelist. Only 4 IL-4 related genes were selected.
3-C IL-12 centered pathway analysis. Network analysis was performed by using IL-12 as the central nodes. We used software to select IL-12 related genes from our genelist. Only 4 IL-12A/IL12B related genes were selected.