NFATc1 mediates Toll-like receptor-independent innate immune responses during Trypanosoma cruzi infection.

Host defense against the intracellular protozoan parasite Trypanosoma cruzi depends on Toll-like receptor (TLR)-dependent innate immune responses. Recent studies also suggest the presence of TLR-independent responses to several microorganisms, such as viruses, bacteria, and fungi. However, the TLR-independent responses to protozoa remain unclear. Here, we demonstrate a novel TLR-independent innate response pathway to T. cruzi. Myd88(-/-)Trif(-/-) mice lacking TLR signaling showed normal T. cruzi-induced Th1 responses and maturation of dendritic cells (DCs), despite high sensitivity to the infection. IFN-gamma was normally induced in T. cruzi-infected Myd88(-/-)Trif(-/-) innate immune cells, and further was responsible for the TLR-independent Th1 responses and DC maturation after T. cruzi infection. T. cruzi infection induced elevation of the intracellular Ca(2+) level. Furthermore, T. cruzi-induced IFN-gamma expression was blocked by inhibition of Ca(2+) signaling. NFATc1, which plays a pivotal role in Ca(2+) signaling in lymphocytes, was activated in T. cruzi-infected Myd88(-/-)Trif(-/-) innate immune cells. T. cruzi-infected Nfatc1(-/-) fetal liver DCs were impaired in IFN-gamma production and DC maturation. These results demonstrate that NFATc1 mediates TLR-independent innate immune responses in T. cruzi infection.

Introduction

The host defense against invasion of intracellular pathogens relies on Th1 cell-derived IFN-γ that activates macrophages to kill the engulfed pathogens [1]. Toll-like receptor (TLR)-mediated recognition of pathogens has been established to induce activation of innate immune cells such as dendritic cells (DCs) and subsequent development of Th1 cells [2],[3]. However, recent evidence also indicates the presence of TLR-independent mechanisms for the recognition of microorganisms such as bacteria, viruses, and fungi [4],[5]. Accordingly, TLR-independent mechanisms for Th1 development have been demonstrated in several infectious models such as fungal and bacterial infections [6],[7]. However, TLR-independent recognition of protozoa remains unknown.

Trypanosoma cruzi is an intracellular protozoan parasite that causes Chagas' disease, a chronic disorder characterized by cardiomyopathy and malformation of the intestine [8]. Several components of T. cruzi have been shown to induce TLR-dependent activation of innate immunity and subsequent development of Th1 cells [9]–[14]. The absence of TLR-dependent activation of innate immunity results in high susceptibility to T. cruzi infection [15],[16] due to defective type I interferon (IFN)-mediated induction of the GTPase IFN-inducible p47 (IRG47) [17]. Invasion of infective metacyclic trypomastigotes of T. cruzi into host cells induces a close interaction between the parasites and the host, because T. cruzi utilize several host-derived factors in order to establish the infection. These include activation of Ca2+ signaling pathways and phosphatidylinositol-3 kinases [18]–[20]. However, it remains unclear how T. cruzi-mediated activation of host cytoplasmic signaling pathways is regulated and whether it is TLR-dependent or -independent.

In T cells, the nuclear factor of activated T cells (NFAT) family of transcription factors has been shown to mediate production of cytokines including IFN-γ [21],[22]. The NFAT family of proteins comprises four closely related members (NFATc1, NFATc2, NFATc3, and NFATc4) that are activated by Ca2+ signaling, and NFAT5 that is regulated by osmotic stress. The role of NFAT proteins in T cells has been well characterized [21],[22]. However, little is known about the role of NFAT proteins in innate immune responses, although some of the NFAT members are highly expressed and can modulate gene induction in macrophages (Mφ) [23],[24].

Here, we analyzed the mechanisms of TLR-independent activation of innate immunity during T. cruzi infection using T. cruzi-infected Myd88 mice. Our results demonstrate that NFATc1 mediates TLR-independent induction of IFN-γ in innate immune cells, leading to effective Th1 responses during T. cruzi infection.

Results

Normal Th1 response in T. cruzi-infected Myd88 −/− Trif −/− mice

Previously, we demonstrated that mice lacking both MyD88 and TRIF, in which TLR-dependent activation was abolished, are highly sensitive to infection with T. cruzi [17]. Because TLRs have been shown to control development of Th1 cells, we analyzed Th1 responses in T. cruzi-infected mice. Mice were intraperitoneally (i.p.) infected with T. cruzi trypomastigotes, and at 6 days of infection CD4+ T cells were isolated from the spleen and stimulated with anti-CD3 antibody (Ab) (Figure 1A). In T. cruzi-infected wild-type mice, there was considerable production of IFN-γ compared with that in non-infected control mice, indicating induction of potent Th1 responses. In Myd88 −/− and Myd88 −/− Trif −/− mice, IFN-γ production was similar to that in wild-type mice following T. cruzi infection. Next, we analyzed the antigen-specific Th1 response at 0, 4, 6, and 10 days after T. cruzi infection by stimulating CD4+ T cells with freeze-thawed T. cruzi in the presence of antigen presenting cells (APC) (Figure 1B). This stimulation induced marked production of IFN-γ at 6 and 10 days of the infection in wild-type mice. Even in CD4+ T cells derived from T. cruzi-infected Myd88 −/− and Myd88 −/− Trif −/− mice, antigen-specific production of IFN-γ was induced to levels similar to that of wild-type mice. Thus, the antigen-specific Th1 response was not impaired in Myd88 −/− and Myd88 −/− Trif −/− mice. We also analyzed IFN-γ production from CD4+ T cells by intracellular staining (Figure 1C, Figure S1A). The number of IFN-γ-producing CD4+ T cells was almost equally elevated in wild-type, Myd88 −/− and Myd88 −/− Trif −/− mice at 6 days (Figure 1C) as well as at 10 days (Figure S1A) after infection. Consistent with previous studies [25],[26], the number of IFN-γ producing CD8+ T cells and NK1.1+ natural killer cells was not increased at 10 days after T. cruzi infection (Figure S1B).

Figure 1

Th1 response and DC maturation in T. cruzi-infected Myd88 −/− Trif −/− mice.

(A, B) Splenic CD4+ T cells were isolated from wild-type, Myd88 −/− and Myd88 −/− Trif −/− mice after 6 days (A) or the indicated days (B) of T. cruzi infection or PBS injection, and stimulated with anti-CD3 antibody (A) or freeze-thawed T. cruzi in the presence of antigen presenting cells (B). After 24 h, supernatants were assayed for IFN-γ production by ELISA. Data are mean+s.d. of triplicate determination and a representative result of at least three independent experiments. In each experiment, two or three mice in each group were used. *: not detected. (C) Splenocytes were isolated from T. cruzi-infected or none-infected wild-type, Myd88 and Myd88 mice, and stimulated with 1 µg/ml ionomycin plus 50 ng/ml PMA. After surface staining with APC-conjugated anti-CD4 Ab, the cells were permeabilized and then stained with PE-conjugated anti-IFN-γ Ab, and analyzed by flow cytometry. The percentage of IFN-γ-producing CD4+ cells of individual mice is shown. (D)T. cruzi-infected or none infected bone marrow DCs of wild-type and Myd88 −/− Trif −/− were stained for expression of CD40, CD86, and I-Ab, and then analyzed by FACS.

Development of Th1 cells is critically controlled by DCs [27],[28]. In addition, stimulation of TLRs induces maturation of DCs [3]. Therefore, we analyzed expression of MHC class II and co-stimulatory molecules on T. cruzi-infected DCs. Bone marrow-derived DCs (BMDCs) were infected with T. cruzi trypomastigotes for 6 h, then were washed, cultured for 48 h, and analyzed for expression of MHC class II, CD40, and CD86 by flow cytometry (Figure 1D). T. cruzi infection resulted in enhanced expression of these molecules in wild-type BMDCs. Expression was also increased in BMDCs derived from Myd88 −/− Trif −/− mice after T. cruzi infection, indicating normal maturation of T. cruzi-infected DCs of Myd88 −/− Trif −/− mice. Thus, Th1 cell development and DC maturation were induced during T. cruzi infection even in the absence of TLR-dependent activation of innate immunity.

IFN-γ induction in T. cruzi-infected Myd88 −/− Trif −/− DCs and macrophages

T. cruzi infection induced maturation of DCs in the absence of TLR signaling. Therefore, we screened genes that were normally induced in T. cruzi-infected DCs of Myd88 −/− Trif −/− mice. BMDCs from wild-type, Myd88 −/− and Myd88 −/− Trif −/− mice were infected with T. cruzi trypomastigotes for 6 h, then mRNA was extracted and used for DNA microarray analysis. Approximately 80% of genes that were induced in T. cruzi-infected wild-type DCs (about 4000 genes) were MyD88-dependent, as the T. cruzi-mediated induction was reduced in Myd88 −/− DCs (Figure S2). Some of the genes that were normally induced in Myd88 −/− DCs, but not induced in Myd88 −/− Trif −/− DCs (MyD88/TRIF-dependent genes; 14% of genes that were induced in wild-type DCs) are known to be induced by type I IFNs. In addition, a majority of the small number of genes that were induced even in Myd88 −/− Trif −/− DCs (6%) were IFN-γ-inducible genes (Figure S3). In order to corroborate that IFN-γ-inducible genes are normally induced in T. cruzi-infected Myd88 −/− Trif −/− DCs, we analyzed mRNA expression of Ifng and IFN-γ-inducible genes, including Stat1, and Irgm, by real-time RT-PCR (Figure 2A). T. cruzi infection resulted in robust induction of Ifng, Stat1, and Irgm in wild-type, Myd88 −/−, Trif −/−, and Myd88 −/− Trif −/− DCs. T. cruzi-induced expression of Stat1 and Irgm in wild-type DCs was inhibited by addition of a de novo protein synthesis inhibitor, cycloheximide (CHX) (Figure 2B). In contrast, CHX did not inhibit T. cruzi-induced Ifng expression (Figure 2C). Next, in order to analyze whether the expression of the IFN-γ-inducible genes was secondary to induction of Ifng, we used BMDCs derived from Ifngr1 −/− mice in which the IFN-γ-mediated response was abolished (Figure 2D, E). In Ifngr1 −/− BMDCs, T. cruzi-mediated induction of Stat1 and Irgm was reduced, whereas induction of Ifng was unimpaired. These data indicate that Ifng was induced primarily in response to T. cruzi infection, and Stat1 and Irgm were induced secondary to Ifng induction. In peritoneal Mφ, similar patterns of T. cruzi-mediated gene expression were observed (Figure S4). Recently, the CD11clowB220+NK1.1+ subset of cells was identified as a natural killer (NK) cell subset with a high capacity for IFN-γ production in response to IL-12 or a TLR9 ligand [29]–[31]. To exclude the possibility of contamination of these cells in preparation of BMDCs or Mφ, we purified CD11chighB220−NK1.1− population from the spleen, and analyzed for IFN-γ expression (Figure S5A). CD11chighB220−NK1.1− cells showed very low levels of IFN-γ expression in response to IL-12/IL-18 stimulation compared with the NK cell subset (Figure S5B). However, these cells from wild-type and Myd88 mice expressed IFN-γ in response to T. cruzi infection (Figure 2F). Flow cytometric analysis further demonstrated that T. cruzi-infected CD11chigh splenic DCs expressed IFN-γ protein (Figure 2G). These findings indicate that T. cruzi infection induces IFN-γ production in DCs.

Figure 2

Expression of IFN-γ-inducible genes in T. cruzi-infected Myd88 DCs.

(A) Bone marrow DCs from wild-type, Myd88 −/−, Trif −/−, and Myd88 −/− Trif −/− mice were infected with T. cruzi for 3 or 6 h, and total RNA was isolated and mRNA expression of Stat1, Irgm and Ifng was quantified by real-time RT-PCR and normalized to the level of elongation factor-1α (EF1α). *: not detected (B, C) Bone marrow DCs from wild-type mice were pretreated with 1 µg/ml cycloheximide (CHX) for 5 min, then infected with T. cruzi for the indicated periods. Next, mRNA expression of Stat1, Irgm (B) and Ifng (C) was analyzed by real-time RT-PCR. *: not detected. Data are representative of three independent experiments. (D, E) Bone marrow DCs from wild-type and Ifngr1 −/− mice were infected with T. cruzi for 3 or 6 h. Next, mRNA expression of Stat1, Irgm (D) and Ifng (E) was analyzed. *: not detected (F) CD11chighB220−NK1.1− cells were isolated from the spleen of wild-type and Myd88 mice, and then T. cruzi-induced expression of Ifng was analyzed. Data are mean+s.d. of triplicate determination and a representative result of at least three independent experiments. (G) Splenocytes from wild-type mice were infected with T. cruzi for 18 h. After surface staining with APC-conjugated anti-CD11c Ab, the cells were permeabilized and then stained with PE-conjugated anti-IFN-γ Ab, and analyzed by flow cytometry. Representative results are shown from four independent experiments. The percentage of IFN-γ-producing CD11c+ cells of individual mice is shown.

IFN-γ-mediated DC maturation and Th1 responses in T. cruzi-infected Myd88 −/− Trif −/− mice

Next, we analyzed whether IFN-γ was involved in TLR-independent DC maturation and Th1 responses during T. cruzi infection. In BMDCs derived from Ifngr1 −/− mice, T. cruzi-induced enhancement of CD40, CD86, and MHC class II was partially reduced (Figure 3A). Furthermore, expression of these molecules was completely abolished in T. cruzi-infected DCs of Myd88 −/− Trif −/− Ifngr1 −/− mice. Enhanced expression of these molecules in response to exogenous IFN-γ was not observed in Ifngr1 −/− BMDCs (Figure S6A). These findings indicate that IFN-γ produced from T. cruzi-infected DCs mediated DC maturation. We also analyzed IFN-γ production from splenic CD4+ T cells of T. cruzi-infected mice (Figure 3B). In both Ifngr1 −/− and Myd88 −/− Trif −/− Ifngr1 −/− mice, T. cruzi antigen-dependent production of IFN-γ was severely reduced. Importance of IFN-γ production was further underscored by the finding that Ifngr1 −/− mice were more sensitive to T. cruzi infection than Myd88 −/− Trif −/− mice (Figure S6B). These results demonstrate that IFN-γ mediates TLR-independent DC maturation and Th1 development during T. cruzi infection.

Figure 3

IFN-γ dependent DC maturation and Th1 response in T. cruzi infection.

(A) Bone marrow DCs of wild-type, Ifngr1 −/−, and Myd88 −/− Trif −/− Ifngr1 −/− mice were infected with T. cruzi for 6 h, then washed and cultured for 48 h. The cells were analyzed for expression of CD40, CD86, and I-Ab using flow cytometry. Representative results are shown from three independent experiments. (B) Wild-type, Ifngr1 −/−, and Myd88 −/− Trif −/− Ifngr1 −/− mice were infected with T. cruzi. At six days after infection, CD4+ T cells were isolated from the spleen, and then stimulated with freeze-thawed T. cruzi in the presence of antigen presenting cells. After 24 h, supernatants were collected and assayed for IFN-γ production by ELISA. The values are the means+s.d. of four independent experiments each carried out in triplicate. #:P<0.05.

Importance of IL-12 in Th1 cell development has been established [32]. Indeed, IL-12p40-deficient mice were highly susceptible to T. cruzi infection with severely reduced Th1 responses ([33],[34] and Figure S7A). In addition, IL-12p40 concentration in the serum was decreased in T. cruzi-infected Ifngr1 mice (Figure S7B). In T. cruzi-infected Myd88 −/− Trif −/− mice, IL-12p40 production was severely reduced, but still induced [17], suggesting that IL-12 is produced via TLR-dependent and -independent pathways. Thus, IFN-γ, which is produced via the TLR-independent pathways, might induce IL-12p40 to activate T cells to fully differentiate into Th1 cells.

Involvement of Ca2+ signaling in IFN-γ induction in T. cruzi-infected Myd88 −/− Trif −/− cells

Next, we analyzed the molecular mechanisms for TLR-independent induction of IFN-γ after T. cruzi infection. In Myd88 −/− Trif −/− DCs, T. cruzi-induced phosphorylation of MAP kinases such as ERK, p38, and JNK, as well as degradation of IκBα was not observed at all (Figure S8A). In addition, T. cruzi infection did not induce DNA binding activity of NF-κB in Myd88 −/− Trif −/− DCs (Figure S8B). Thus, T. cruzi-mediated activation of NF-κB and MAP kinases was not induced in the absence of TLR signaling. Next, we stimulated DCs with T. cruzi trypomastigotes killed by repeated freeze-thaw steps. Live T. cruzi, but not killed parasites, induced Ifng expression (Figure 4A). Because many studies have demonstrated that T. cruzi utilize the host Ca2+ signaling to establish the infection [35], we assessed the intracellular Ca2+ concentration in T. cruzi-infected BMMφ using a fluorescent Ca2+ indicator Fluo-4 AM (Figure 4B, Figure S9A, B). T. cruzi infection led to rapid increase in intracellular Ca2+ level in both wild-type and Myd88 Mφ, which returned to the basal level after 18 min of the infection. Epimastigotes, which are not able to invade the host cells, did not induce the elevation of Ca2+ concentration in Mφ (Figure S9C). These results prompted us to examine whether Ca2+ mobilization induced by intracellular invasion of T. cruzi contributed to the TLR-independent Ifng induction. Accordingly, we treated wild-type and Myd88 −/− Trif −/− BMDCs with an intracellular Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), and infected with T. cruzi. In BAPTA-AM pre-treated DCs, T. cruzi-induced Ifng expression was severely reduced, although lipopolysaccharide (LPS)-induced response was not impaired (Figure 4C). In this condition, T. cruzi-induced elevation of intracellular Ca2+ concentration was severely reduced (Figure S10). In addition, stimulation with both phorbol myristate acetate (PMA)/Ca2+ ionophore or Ca2+ ionophore alone, which mimics Ca2+ signaling, induced expression of Ifng in both wild-type and Myd88 Mφ (Figure 4D). Taken together, these findings indicate that T. cruzi-dependent intracellular Ca2+ mobilization mediates TLR-independent Ifng induction.

Figure 4

Ca2+ signaling-dependent IFN-γ production in T. cruzi-infected DCs.

(A) Bone marrow DCs were stimulated with live T. cruzi or T. cruzi killed by repeated freeze-thaw steps for the indicated periods. Next, total RNA was isolated and analyzed for Ifng expression by real-time RT-PCR. The fold differences of each sample relative to EF1α are shown. *: not detected. (B) Bone marrow Mφ from wild-type and Myd88 mice were treated with Fluo-4AM for 30 min, and washed. Then, cells which were infected or non-infected with T. cruzi were analyzed by fluorescence microscopy at the indicated periods. Representative of three independent experiments. (C) Bone marrow DCs from wild-type and Myd88 −/− Trif −/− mice were pre-treated with BAPTA-AM (100 µM) for 30 min, and washed. Then, cells were infected with T. cruzi for 3 h. Expression of Ifng was analyzed by real-time RT PCR. Bone marrow DCs from wild-type mice were stimulated with LPS (100 ng/ml) for 3 h, and analyzed for expression of Il6 and Tnf. (D) Bone marrow Mφ from wild-type and Myd88 mice were stimulated with 5 µM Ca2+ ionophore plus 50 ng/ml PMA or 5 µM Ca2+ ionophore for the indicated periods, and analyzed for expression of Ifng. Data are mean+s.d., and representative one of three independent experiments. *: not detected.

NFATc1 activation in T. cruzi-infected Myd88 −/− Trif −/− cells

In the host cells, especially in T lymphocytes, Ca2+ mobilization induces activation of cytokine genes via calmodulin/calcineurin-dependent activation of the transcription factor NFAT. Therefore, we treated Myd88 −/− Trif −/− Mφ with FK506 to block calcineurin activation, and infected with T. cruzi. Treatment of FK506 resulted in a marked decrease in T. cruzi-induced expression of Ifng, despite normal LPS-induced response (Figure 5A). Among NFAT members, Nfatc1, Nfatc3, and Nfat5 mRNA were abundantly expressed in BMDCs (Figure S11). A previous study has shown that NFATc1 increased anti-CD3/anti-CD28-induced IFN-γ promoter activity in T cells [36]. Furthermore, it has been demonstrated that IFN-γ production was normal in NFATc3-deficient splenocytes [37]. In addition, NFAT5 has been shown to be activated by osmotic stress, but not by Ca2+ signaling [38]. Thus, we focused on NFATc1. In wild-type and Myd88 −/− Trif −/− BMMφ, T. cruzi trypomastigotes infection induced nuclear translocation of NFATc1 (Figure 5B). T. cruzi-induced nuclear translocation of NFATc1 was blocked by the pre-treatment with BAPTA-AM in wild-type and Myd88 −/− Trif −/− BMMφ (Figure S12A, B). These results indicate that NFATc1 is activated in response to T. cruzi infection in a TLR-independent manner. Next, we analyzed whether NFATc1 was involved in the T. cruzi-induced IFN-γ production. We obtained RAW264.7 macrophage clones expressing different levels of NFATc1 (Figure 5C). In NFATc1 expressing RAW264.7 cells, T. cruzi-induced expression of Ifng, Stat1, and Irgm was enhanced, and the extent of fold-induction correlated with the NFATc1 expression level (Figure 5D). T. cruzi-induced Ifng expression was severely reduced in the presence of FK506 (Figure 5E). These findings indicate the possible involvement of NFATc1 in mediating IFN-γ production in T. cruzi-infected innate immune cells.

Figure 5

T. cruzi-induced activation of NFATc1 in Myd88 −/− Trif −/− DCs and Mφ.

(A) Wild-type and Myd88 −/− Trif −/− bone marrow Mφ were infected with T. cruzi or stimulated with LPS for 6 h in the presence or absence of FK506 (0.5 µM or 2.5 µM). Next, total RNA was isolated and analyzed for Ifng, Tnf or Il6 expression by real-time RT-PCR. The fold differences of each sample relative to EF1α are shown. *: not detected. (B) Bone marrow Mφ from wild-type and Myd88 −/− Trif −/− mice were transfected with the NFATc1 expression plasmid. Cells were then infected with T. cruzi for 30 min., and stained with anti-NFATc1 antibody (red) and DAPI (blue). (C) RAW 264.7 cell clones (designated #3, #7, and #9) transfected with the NFATc1 expression plasmid were analyzed for expression of NFATc1 by immunoblot with antibodies specific for NFATc1 and β-actin. (D) RAW 264.7 cells expressing NFATc1 were infected with T. cruzi for 3 h. Next, total RNA was extracted and used for real-time RT-PCR analysis using primers specific for Ifng, Stat1, and Irgm. (E) RAW 264.7 cells expressing NFATc1 (clone #9) were infected with T. cruzi for 3 h in the presence or absence of FK506, and total RNA was extracted. Real-time RT-PCR analysis was performed using primers specific for Ifng. *: not detected. Data are mean+s.d., and a representative result of at least three independent experiments.

The NFAT family of transcription factors has been shown to interact with different transcription factors to effectively induce gene activation [22]. In the case of activation of the human IFNG promoter, T-bet (encoded by Tbx21) and NFAT have been shown to act synergistically on the promoter [39]. T-bet is a transcription factor that is essential for IFN-γ production in T cells and DCs [40]. Tbx21 has also been shown to be induced by IFN-γ in monocytes and DCs [41]. In accordance with those studies, Tbx21 was normally induced in T. cruzi-infected Myd88 −/− Trif −/− Mφ (Figure 6A). Expression of NFATc1 alone weakly activated the Ifng promoter in RAW264.7 Mφ, but introduction of both NFATc1 and T-bet synergistically activated the Ifng promoter in RAW264.7 Mφ (Figure 6B). These findings indicate that NFATc1 mediates activation of the Ifng promoter together with T-bet in innate immune cells, like the case in T cells.

Figure 6

NFATc1 and T-bet had a synergistic effect on activation of the Ifng promoter.

(A) Peritoneal Mφ from wild-type, Myd88 −/−, Trif −/−, and Myd88 −/− Trif −/− mice were infected with T. cruzi for 3 h, and analyzed for Tbx21 expression by real-time RT-PCR. Data are mean+s.d., and a representative result of at least three independent experiments. (B) RAW 264.7 cells were transiently transfected with either T-bet or NFATc1 expression vector, or both expression vectors together with the IFN-γ reporter plasmid. After 18 h of transfection, the cells were infected with T. cruzi for 18 h and activity of the reporter analyzed by luciferase assay. Data indicate mean+s.d., and a representative result of at least three independent experiments.

Impaired T. cruzi-induced responses in Nfatc1 −/− DCs

To determine the role of NFATc1 in T. cruzi-infected DCs, we investigated IFN-γ production and maturation in T. cruzi-infected Nfatc1 −/− DCs. Because mice lacking Nfatc1 are lethal before day14.5 of gestation [42], we obtained fetal liver-derived DCs (FLDCs). Fetal liver cells of both wild-type and Nfatc1 −/− embryos at day 12.5 of gestation differentiated into DCs expressing similar levels of CD11c in the presence of GM-CSF, Flt3 ligand and SCF (Figure S13). Moreover, LPS-induced maturation, as determined by enhanced surface expression of CD40, CD86, and MHC class II, was not impaired in Nfatc1 mice-derived cells (Figure S14A), indicating that development and LPS-induced maturation of Nfatc1 FLDCs was not compromised. T. cruzi-infected wild-type FLDCs expressed increased amounts of Ifng. In contrast, in FLDCs from Nfatc1 −/− embryos the T. cruzi-induced Ifng expression was impaired (Figure 7A). On the other hand, T. cruzi-induced Il6 and Tnf expression was observed normally in Nfatc1 −/− FLDCs (Figure 7B). In T. cruzi-infected Nfatc1 −/− FLDCs, expression of Il12b (encoding IL-12p40) was partially decreased (Figure S14B). Next, we analyzed T. cruzi-induced expression of MHC class II and co-stimulatory molecules on wild-type and Nfatc1 −/− FLDCs. In Nfatc1 −/− FLDCs, T. cruzi-mediated enhancement of CD40, CD86, and MHC class II was dramatically reduced (Figure 7C). The impaired surface expression of these molecules in Nfatc1 FLDCs was rescued by addition of exogenous IFN-γ (Figure 7C). These results indicate that NFATc1 mediates T. cruzi-induced IFN-γ production and maturation of DCs.

Figure 7

Defective T. cruzi-induced IFN-γ response in Nfatc1 −/− DCs.

(A, B) Wild-type and Nfatc1 −/− FLDCs were infected with T. cruzi for the indicated periods. Next, total RNA was extracted, and analyzed for expression of Ifng, Tnf and Il6. *: not detected. (C) Wild-type and Nfatc1 −/− FLDCs were infected with T. cruzi for 6 h, then washed and cultured for 24 h in the presence or absence of ρεχoμβιναντ IFN-γ. Surface expression of CD40, CD86, and I-Ab was analyzed by flow cytometry. Data are shown mean+s.d. of triplicate samples and a representative of four independent experiments.

Discussion

In the present study, we analyzed TLR-independent innate immune responses against the intracellular protozoan parasite T. cruzi. T. cruzi-infected Myd88 −/− Trif −/− mice displayed normal Th1 responses and normal DC maturation. A comprehensive analysis of gene expression profiles of T. cruzi-infected DCs identified IFN-γ as a TLR-independent gene which mediated DC maturation and Th1 responses even in the absence of TLR signaling. T. cruzi infection induced an increase in intracellular Ca2+ level in DCs and macrophages, which led to NFATc1 activation and IFN-γ induction in a TLR-independent manner. In Nfatc1 −/− DCs, T. cruzi-induced IFN-γ production and DC maturation was impaired. These findings demonstrate that NFATc1 is responsible for TLR-independent innate immune responses during T. cruzi infection.

The family of TLRs has been established to be critical for the innate recognition of T. cruzi [14]. TLR signaling pathways consist of two major components mediated by MyD88 and TRIF [43]. Myd88 −/− mice show a high susceptibility to T. cruzi infection [15],[16], while mice deficient in both MyD88 and TRIF are even more susceptible to T. cruzi infection [17]. These findings indicate that TLR-dependent recognition of T. cruzi is crucial to the host defense against the parasite. In this regard, TLR-dependent induction of IFN-β might be responsible for high susceptibility to T. cruzi infection in Myd88 −/− Trif −/− mice in spite of the normal Th1 responses [17].

A previous study showed the MyD88-dependent IFN-γ production in T. cruzi-infected mice [16]. However, surprisingly, we found that Myd88 mice exhibited normal Th1-dependent IFN-γ production. Discrepancy between both studies might be due to distinct experimental protocols. IFN-γ is known to facilitate IL-12 production. Indeed, IL-12p40-deficient mice were highly susceptible to T. cruzi infection with severely reduced Th1 responses [33],[34]. In T. cruzi-infected Myd88 −/− Trif −/− mice, IL-12p40 production was severely reduced, but still induced [17], suggesting that IL-12 is produced via TLR-dependent and -independent pathways. Considering that T. cruzi-infected Ifngr1 mice showed decreased level of serum IL-12p40 and that T. cruzi-infected Nfatc1 FLDCs exhibited reduced expression of IL-12p40, NFATc1-dependent IFN-γ production may facilitate IL-12p40 production. Alternatively, the direct involvement of NFATc1 in activation of IL-12p40 gene has been also shown [23]. Collectively, T. cruzi infection might cause not only the TLR-dependent IL-12p40 production, but also the NFATc1-mediated (TLR-independent) production of IFN-γ and IL-12p40, coordinating the host Th1 response.

IFN-γ was identified as a gene induced in T. cruzi-infected Myd88 −/− Trif −/− DCs. IFN-γ production by DCs was first demonstrated in IL-12-stimulated CD8α+ lymphoid DCs [44]. Subsequently, CD11clowB220+NK1.1+ cells were shown to produce high amounts of IFN-γ in response to IL-12 or a TLR9 ligand [45],[46]. These CD11clowB220+NK1.1+ cells have been shown to be a subset of NK cells [29]–[31]. Thus, IFN-γ production from DCs as well as Mφ is controversial [47],[48]. In order to exclude the possibility that our DC or Mφ preparations were contaminated by NK cell subsets, we isolated CD11chighB220−NK1.1− cells and analyzed IFN-γ production. These cells showed a severely reduced level of IL-12/IL-18-induced IFN-γ production compared with NK cells. In addition, IL-12/IL-18-induced IFN-γ expression was less than T. cruzi-induced expression in these cells (our unpublished data). In Nfatc1 −/− FLDCs, IL-12/IL-18-induced IFN-γ expression was not impaired (our unpublished data). These results indicate that T. cruzi-induced IFN-γ production in DCs is mediated by a pathway distinct from the IL-12 (or the TLR9 ligand)-induced one in NK subsets.

Protozoan parasites including T. cruzi require Ca2+ for their survival within the host cells [19]. In addition, T. cruzi evokes elevation of intracellular Ca2+ concentration in the host cells to establish the invasion [49],[50]. Our findings indicate that T. cruzi-induced activation of host Ca2+ signaling mediates IFN-γ production. In the host cells, the family of NFAT transcription factors, which is activated by calmodulin/calcineurin, is known to bridge Ca2+ to promote gene expression [51]. The role of NFAT proteins has been well characterized in T lymphocytes, and can induce activation of the Ifng gene [22],[52]. However, the role of NFAT proteins in innate immune cells remains unclear. Several reports indicate that NFAT proteins are activated in macrophages [24],[53]. In addition, cyclosporin A, which blocks calcineurin-dependent NFAT activation, has been shown to inhibit DC functions [54],[55]. In accordance with these reports, in the present study NFATc1 was activated in Myd88 Mφ in response to T. cruzi infection. Furthermore, analysis using FLDCs derived from Nfatc1 −/− embryos demonstrated that NFATc1 mediates T. cruzi-induced IFN-γ production and DC maturation. These findings establish a new signaling pathway mediating an innate immune response during T. cruzi infection. It is well established that the TLR-dependent pathway initiates innate immune responses against pathogens. In addition, in the case of invasion of protozoan parasites triggering activation of intracellular Ca2+ signaling, NFATc1 mediates the TLR-independent innate immune responses through induction of IFN-γ.

Bradykinin B2 receptor has been shown to mediate T. cruzi-dependent generation of inositol 1,4,5-trisphosphate, leading to elevated level of intracellular Ca2+ [56]. However, several other mechanisms that induce intracellular Ca2+ influx have been proposed in T. cruzi infection [18]. Identification of critical molecules that lead to NFATc1 activation during T. cruzi infection would be a future issue to be addressed. It would be also interesting in the future to analyze whether the NFAT pathway is involved in innate immune responses against other protozoan parasites such as Toxoplasma and Leishmania species.

In this study, we focused on DCs and Mφ, which initiate Th1 responses. However, in an in vivo condition, T. cruzi are expected to invade into several other types of cells than DCs and Mφ. Therefore, it is possible that the invasion of T. cruzi into cells of non-innate immune cell populations indirectly influences Th1 polarization in vivo. In the future, we should analyze whether the NFAT family of transcription factors is involved in these processes.

In summary, in the present study we revealed a new TLR-independent mechanism for the interaction between protozoan parasites and host innate immunity. Ca2+ is critical for both living organisms, and therefore the parasite utilizes host Ca2+ for its benefit. On the host side, Ca2+ signaling leads to activation of NFATc1 to eliminate the parasite. It would be interesting in the future to analyze the precise role of NFATc1 in protozoan parasite infection using innate immune cell-specific NFATc1-deficient mice.

Materials and Methods

Mice

All animal experiments were conducted in accordance with guidelines of the Animal Care and Use Committee of Osaka University and Kyushu University. Myd88 −/−, Trif −/−, Ifngr1 −/−, and Nfatc1 −/− mice were generated as previously described [17],[42]. Each mouse strain was backcrossed to C57BL/6 for at least five generations, and then used to generate double or triple-mutant mice.

Reagents

PE-conjugated anti-CD11c, PE-conjugated anti IFN-γ, APC-conjugated anti-CD11c, APC-conjugated anti-CD40, FITC-conjugated anti-NK1.1, FITC-conjugated anti-CD40, FITC-conjugated anti-CD86, FITC-conjugated anti-I-Ab and Pacific Blue-conjugated anti-B220 antibodies were purchased from BD Pharmingen. Anti-NFATc1 and anti-β actin antibodies were purchased from Santa Cruz. Ca2+ ionophore A23187 (C7522), PMA (P1585), and FK506 (F4679) were purchased from Sigma. Fluo-4 AM was purchased from Invitrogen. BAPTA-AM was from Calbiochem. Cycloheximide was from Nacalai tesque.

Preparation of macrophages (Mφ), dendritic cells (DCs), and antigen presenting cells (APC)

To isolate peritoneal Mφ, mice were i.p. injected with 2 ml of 4% thioglycolate medium (Sigma), and peritoneal exudate cells were isolated from the peritoneal cavity at three days post injection. The cells were incubated for 2 h, then washed three times with HBSS. The remaining adherent cells were used as peritoneal Mφ in experiments. To prepare bone marrow-derived DCs or Mφ, bone marrow cells were prepared from the femur and tibia, and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 µM 2-mercaptoethanol (2ME), and 10 ng/ml GM-CSF (Pepro Tech) or 30% L cell culture supernatant, respectively. After six days, the cells were used as bone marrow DCs or bone marrow Mφ in experiments. To prepare fetal liver-derived DCs (FLDCs), fetal liver (FL) were obtained from 12.5 days post-coitum murine embryos, and FL cells were dissociated by pipetting, passed through a nylon mesh, and then cultured in RPMI 1640 medium supplemented with 10% FBS, 100 µM 2ME, 20 ng/ml GM-CSF, 10 ng/ml Flt3 ligand (Pepro Tech), and 10 ng/ml SCF (Pepro Tech) as described [57]. After eight days, the floating cells were used as FLDCs in experiments. RAW 264.7 cells were transfected with the NFATc1 (pcDNA3) expression plasmid. The cells expressing NFATc1 were selected in the presence of 0.4 mg/ml G418 and cloned. Splenocytes from wild-type mice were irradiated (30 Gy) and used as APC.

Parasite and experimental infection

The trypomastigote stage of T. cruzi Tulahuen strain was maintained in vivo in Ifngr1 −/− mice by passages every other week or in vitro in LLC-MK2 cells by passages every four days. For in vitro experiments, 5×105 Mφ or DCs were infected with 1.5×106 trypomastigotes. For in vivo experiments, mice were i.p. injected with 6×101 trypomastigotes or PBS. Epimastigotes of Tulahuen strain were grown at 26°C in liver infusion tryptose liquid medium, supplemented with 2.5% hemoglobin and 10% fetal calf serum.

Real-time RT-PCR

Total RNA was isolated with TRIzol reagent (Invitrogen), and 1–2 µg of RNA was reverse transcribed using M-MLV reverse transcriptase (Promega) and random primers (Toyobo) after treatment with RQ1 DNase I (Promega). Real-time RT-PCR was performed on an ABI 7300 (Applied Biosystems) using the TaqMan Universal PCR Master Mix (Applied Biosystems). All data were normalized to the corresponding gene Eef1a1 encoding elongation factor-1α (EF-1α) expression, and the fold difference relative to the EF-1α was shown. Amplification conditions were: 50°C (2 min), 95°C (10 min), 40 cycles of 95°C (15 s), and 60°C (60 s). Primers of Tbx21, Stat1, Irgm, and Tnf were purchased from Assay on Demand (Applied Biosystems). Sequences for EF-1α, Il12b, Il6, and Ifng are as follows: EF-1α probe 5′-gcacctgagcagtgaagccagctgct-3′. forward primer 5′-gcaaaaacgacccaccaatg-3′. reverse primer 5′-ggcctggatggttcaggata-3′; Il6 probe 5′-ccttcttgggactgatgctggtgaca-3′. forward primer 5′-ctgcaagagacttccatccagtt-3′. reverse primer 5′-aagtagggaaggccgtggtt-3′; and Ifng probe 5′-gtcaccatccttttgccagttcctccag-3′. forward primer 5′-tcaagtggcatagatgtggaagaa-3′. reverse primer 5′-tggctctgcaggattttcatg-3′.

Intracellular cytokine staining

Splenic cells were isolated from T. cruzi-infected mice at the indicated time point and stimulated with PMA and ionomycin for 4 h in the presence of 10 µg/ml brefeldin A. In experiments to detect IFN-γ production from CD11c+ cells, splenic cells were infected with T. cruzi (1∶1) for 12 h, and further cultured for 6 h in the presence of 10 µg/ml brefeldin A. After staining of surface CD11c, CD4, CD8 or NK1.1, the cells were fixed with CytopermCytofix (BD Biosciences) for 20 min and incubated with PE-conjugated anti-IFN-γ Ab. Flow cytometric analysis was performed on FACSCantoII (BD Biosciences).

Measurement of cytokine production

For in vivo experiments, mice were i.p injected with T. cruzi, and CD4+ T cells were isolated from the spleen at the indicated days after the infection. 2.5×105 CD4+ T cells were stimulated with anti-CD3 Ab or freeze-thawed T. cruzi in the presence of 2.5×105 APC for 24 h. The culture supernatants were collected and diluted at 1∶5. ELISA was performed with anti-mouse IFN-γ Ab, avidin-HRP, and TMB solution purchased from eBioscience. Optical densities were determined at 450 nm wavelengths with reference at 570 nm. Levels of IFN- γ were calculated from the standard curve by using purified mouse IFN- γ purchased from eBioscience.

Flow cytometry

Bone marrow DCs or FLDCs were infected with T. cruzi for 6 h, washed and then cultured for 24 or 48 h. The T. cruzi-infected cells were stained with the combination of PE-conjugated anti-CD11c and the indicated antibodies at 4°C for 20 min, and washed. Flow cytometric analysis was performed on FACSCalibur or FACSCant II flow cytometer (BD Biosciences) and using FlowJo software (Tree Star). CD11chigh cells and NK cells were sorted using FACS Aria (BD Biosciences). The instrumental compensation was set in each experiment using single color, 2-color or 4-color stained samples.

Intracellular calcium measurements

This assay was performed as described [58]. In brief, bone marrow Mφ plated on glass-bottom dishes were incubated in serum-free RPMI 1640 supplemented with 2 µM Fluo-4 AM, the increase in fluorescent intensity of which indicates increased Ca2+ level, at 37°C for 30 min. The cells were then washed to remove the free extracellular dye, and were maintained in culture medium during the whole experiment. The analysis of changes of basal intracellular calcium concentrations in response to T. cruzi infection was performed using an IX71 fluorescence microscope (Olympus).

Immunofluorescence microscope

Bone marrow Mφ were transfected with pcDNA3-NFATc1 by nucleofection (mouse macrophage nucleofector kit; Amaxa). After 24 h, the cells were infected with T. cruzi for 30 min, washed with Tris-buffered saline (TBS), and then fixed with 3.7% formaldehyde in TBS for 15 min at room temperature. After permeabilization with 0.2% Triton X-100, cells were washed with TBS, incubated with anti-NFATc1 antibody in TBS containing 1% bovine serum albumin, then incubated with Alexa Fluor 594-conjugated goat anti-mouse immunoglobulin G (Molecular Probes). To stain the nucleus, cells were cultured with 0.5 mg/ml 4, 6-diamidino-2-phenylindole (DAPI; Wako). Stained cells were analyzed using an LSM510 confocal microscope (CarlZeiss).

Luciferase assay

RAW 264.7 cells were transfected with the indicated expression plasmids together with the reporter plasmid IFN-γ-Luc and the internal control plasmid phRG-TK by Nucleofection (Nucleofector Kit V; Amaxa). After 18 h, the cells were infected with T. cruzi for 18 h, and the luciferase activities of whole cell lysates were measured using the Dual-luciferase reporter assay system (Promega) and Lumat LD 9507 (Berthold).

Statistical analysis

Differences between control and experimental groups were evaluated by the Student's t-test.

IFN-γ production from lymphocytes after T. cruzi infection. (A) Splenocytes were isolated from wild-type, Myd88 and Myd88 mice at 10 days after T. cruzi infection, and stimulated with 1 µg/ml ionomycin plus 50 ng/ml PMA. After surface staining with APC-conjugated anti-CD4 Ab, the cells were permeabilized and then stained with PE-conjugated anti-IFN-γ Ab, and analyzed by flow cytometry. Representative results are shown from four independent experiments. The percentages of IFN-γ-producing CD4+ cells of individual mice are shown. (B) Splenocytes were isolated from wild-type and Myd88 mice at 10 days after T. cruzi infection, and stimulated with 1 µg/ml ionomycin plus 50 ng/ml PMA. After surface staining with FITC-conjugated anti-NK1.1 and CD8 Ab, cells were permeabilized and then stained with PE-conjugated anti-IFN-γ Ab, and analyzed by flow cytometry. Representative results are shown from two independent experiments. The percentages of IFN-γ-producing NK1.1+ or CD8+ cells of individual mice are shown.

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Microarray analysis of T. cruzi-infected DCs. Bone marrow DCs from wild-type, Myd88 and Myd88 mice were infected with T. cruzi for 6 h. Then, microarray analysis was performed using 5 µg of total RNA. Data are shown in fold-increase of T. cruzi-infected cells compared with non-infected cells. Red colored boxes indicate genes showing defective induction. Genes shown by yellow colored boxes indicate so-called IFN-α/β-inducible genes.

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TLR-independent expression of IFN-γ-inducible genes in T. cruzi-infected DCs. Bone marrow DCs from wild-type, Myd88 and Myd88 mice were infected with T. cruzi for 6 h. Then, microarray analysis was performed using 5 µg of total RNA. Data are shown in fold-increase of T. cruzi-infected cells compared with non-infected cells. Genes shown by yellow colored boxes have been reported as IFN-γ-inducible genes.

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Expression of IFN-γ-inducible genes in T. cruzi-infected peritoneal Mφ. (A, B, C) Peritoneal Mφ from wild-type, Myd88 , Trif, Myd88 and Ifngr1 mice were infected with T. cruzi for the indicated periods. Total RNA was extracted, and used for real-time RT-PCR analysis using primers specific for Ifng, Stat1 and Irgm. All data were normalized to the corresponding gene Eef1a1 encoding elongation factor-1α (EF1α) expression, and the fold difference relative to the EF1α was shown. Data are a representative of three independent experiments. *; not detected.

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Low level of IFN-γ expression in IL-12/IL-18 stimulated CD11chigh cells. (A) Splenic CD11chigh cells (CD11chigh B220− NK1.1−) and NK cells (CD11clow B220+ NK1.1+) were sorted by FACS Area (BD Bioscience). Numbers indicate percentages of CD11chigh NK1.1− and CD11chighB220− cells. (B) These cells were stimulated with 10 ng/ml IL-12 plus 10 ng/ml IL-18 for 6 h. Total RNA was isolated, and then Ifng mRNA expression was quantified by real-time RT-PCR and normalized to the level of EF1α. Data indicate mean+s.d. and a representative result of two independent experiments. *; not detected.

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IFN-γ-dependent DC maturation and host defense in T. cruzi infection. (A) Bone marrow DCs from wild-type and Ifngr1 mice were stimulated with 10 ng/ml murine IFN-γ for 48 h. IFNγ-stimulated DCs were stained with the combination of PE-conjugated anti-CD11c and the indicated antibodies at 4°C for 20 min, and washed. Flow cytometric analysis was performed on FACSCanto II (BD Biosciences). (B) Wild-type (n = 9), Myd88 (n = 5) and Ifngr1 (n = 11) mice were intraperitoneally infected with 1×104 T. cruzi. Serum numbers of trypomastigotes were monitored at the indicated times after infection. Note that many of Ifngr1 mice died before 15 days of the infection.

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IL-12 dependent Th1 response in T. cruzi infection. (A) Wild-type (n = 4) and Il12b (n = 4) mice were intraperitoneally infected with 60 T. cruzi. At 6 days after infection, CD4+ T cells were isolated from the spleen, and then stimulated with freeze-thawed T. cruzi in the presence of antigen presenting cells. After 24 h, supernatants were collected and assayed for IFN-γ production by ELISA. #:P<0.00066. *: not detected. (B) Wild-type (n = 2) and Ifngr1 (n = 2) mice were intraperitoneally infected with 60 T. cruzi. At 3 days postinfection with T. cruzi, concentrations of IL-12p40 in the sera from infected mice were quantified by ELISA. *: not detected.

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Impaired activation of MAP kinases and NF-κB in T. cruzi-infected Myd88 innate immune cells. (A) Bone marrow DCs were infected with T. cruzi for the indicated periods. Cell lysates were analyzed by Western blot analysis using antibodies specifically recognizing the indicated proteins. (B) Bone marrow DCs were infected with T. cruzi for the indicated periods. Nuclear extracts were subjected to EMSA using a radiolabeled oligonucleotide containing the murine κB site of the TNF promoter.

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T. cruzi-dependent increase on Ca2+ concentration in Mφ. (A) Cells showing bright fluorescence at 15 min after T. cruzi infection were counted. Average of numbers of cells with bright fluorescence (% in total cells counted) in twelve fields from three independent experiments (4 fields in each experiment) in ×400 magnification is shown. (B, C) Bone marrow Mφ from wild-type and Myd88 mice were incubated with Fluo-4AM for 30 min, then washed and infected with trypomastigotes or epimastigotes for the indicated periods. The cells were analyzed by IX71 fluorescence microscope (Olympus). Epimastigotes of Tulahuen strain were grown at 26°C in liver infusion tryptose liquid medium, supplemented with 2.5% hemoglobin and 10% fetal calf serum.

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Effect of Ca2+ chelator on T. cruzi-induced response in Mφ. (A) Peritoneal Mφ from wild-type mice were pre-incubated with 100 µM BAPTA-AM for 30 min in the presence of Fluo-4AM, then washed and infected or none-infected with T. cruzi for the indicated periods. Then, cells were analyzed by fluorescence microscopy. Representative of five independent experiments. (B) Cells showing bright fluorescence were counted at 10 min after T. cruzi infection, and average of total fifteen fields (from five independent experiments) is shown.

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Expression of NFAT family member in bone marrow DCs. Total RNA was isolated from bone marrow DCs of wild-type and Myd88. Microarray analysis was performed from 5 µg of total RNA. Levels of mRNA expression of NFAT members are shown as average difference.

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Ca2+-dependent nuclear translocation of NFATc1 in T. cruzi-infected Mφ. Bone marrow-derived macrophages from wild-type mice (A) and Myd88 mice (B) were transfected with the NFATc1 expression plasmid. Cells were treated with BAPTA-AM (100 µM) for 30 min and washed, and then infected with T. cruzi for 30 min. T. cruzi-infected cells were stained with anti-NFATc1 antibody (red) and DAPI (blue). The right panels show the percentage of nuclear translocated NFATc1. Average of number of cells with nuclear NFATc1 (% in total cells counted) in twelve fields from three independent experiments (four fields in each experiment) in ×400 magnification is shown.

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Generation of CD11c+ cells from Nfatc1 fetal liver cells. Fetal liver cells from 12.5 d.p.c. wild-type and Nfatc1 embryos were cultured with 20 ng/ml GM-CSF, 10 ng/ml Flt3 ligand, and 10 ng/ml SCF for 8 days. Expression of CD11c was analyzed and shown to be comparable between both genotypes. CD11c+ cells were enriched by MACS (Miltenyi Biotec) and used for experiments as FLDCs.

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Response of Nfatc1 FLDCs to LPS and T. cruzi. (A) Fetal liver DCs from 12.5 d.p.c. wild-type and Nfatc1 embryos were stimulated with 100 ng/ml LPS for 24 h. LPS-stimulated FLDCs were stained with the combination of PE-conjugated anti-CD11c and the indicated antibodies at 4°C for 20 min, and washed. Flow cytometric analysis was performed on FACSCalibur. (B) Fetal liver DCs from 12.5 p.d.c. wild-type and Nfatc1 embryos were infected with T. cruzi for the indicated periods. Total RNA was extracted, and used for real-time RT-PCR analysis using primers specific for Il12b. All data were normalized to the corresponding gene Eef1a1 encoding elongation factor-1α (EF1α) expression, and the fold difference relative to the Eef1α1 is shown.

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