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Keywords:

  • inflammation;
  • lymphocytes;
  • major histocompatibility complex;
  • malaria

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II+CD11c non-T, non-B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II+CD11c non-T, non-B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag-2−/− mice with adoptively transferred normal spleen cells indicated that these cells were non-lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II+CD11c non-T, non-B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin-6 in response to infected red blood cells, but had only a limited ability to activate antigen-specific CD4+ T cells. This study revealed a novel interaction between MHC II+CD11c non-lymphoid cells and lymphoid cells in the accumulations of these non-lymphoid cells in the spleen during infection with P. yoelii.

List of Abbreviations
7-AAD

7-aminoactinomycin D

APC

antigen-presenting cell

CD

cluster of differentiation

CFSE

carboxyfluorescein succinimidyl ester

DC

dendritic cell

FcR

Fc receptor

FITC

fluorescein isothiocyanate

IL

interleukin

iNOS

inducible nitric oxide

iRBC

infected red blood cell

L. monocytogenes

Listeria monocytogenes

mAb

monoclonal antibody

MFI

mean fluorescence intensity

MHC

major histocompatibility complex

NK

natural killer

OVA

ovalbumin

P.

Plasmodium

PDCA-1

plasmacytoid dendritic cell antigen-1

RBC

red blood cell

TCR

T cell receptor

Protective immune responses against the blood stage of malarial infection require antibody and CD4+ T cell immune responses [1]. Presentation of antigens to T cells by APCs initiates activation of adaptive immunity. The most critical APCs that activate naïve CD4+ T cells are DCs that capture parasite-derived antigens and thus activate specific T cells that recognize them [2, 3]. DCs are heterogeneous and include both several conventional DC subsets and plasmacytoid DCs. Conventional DCs, highly specialized APCs that can activate naïve T cells, are characterized by their strong expression of MHC II and CD11c. In addition to these DCs that are present during the steady state, infection or inflammation induces some other DC subsets [4, 5]. Infection with L. monocytogenes induces recruitment of a monocyte-derived DC subset (TipDC) that can produce TNF-α and iNOS in the spleen and mediates innate immune defense against the pathogen [6]. DCs with regulatory functions have also been described. CD11clowCD45RBhigh DCs produce large amounts of IL-10 and are capable of suppressing T cell responses and inducing differentiation of Type 1 regulatory T cells [7].

Modulation of the function of DCs during Plasmodium infection has been the subject of several investigations [8]. RBCs that are infected with P. falciparum adhere to DCs and inhibit their maturation, reducing activation of specific T cell immune responses [9]. With progress of the blood stage of infection, maturation of DCs and their ability to activate adaptive immune responses are inhibited and their ability to secrete IL-12/IL-10 in response to Toll-like receptor signaling is reversed [10-12]. Studies of DC subsets have indicated that during P. yoelii infection regulatory DCs become the most prevalent DC population. These cells preferentially induce IL-10-producing CD4+ T cells and inhibit excessive immune responses during systemic infectious diseases [13]. In a model of P. chabaudi infection, researchers demonstrated that CD8+ DCs are the major DC population during the early phase of infection, whereas CD8 DCs play a major role in the later phase of infection and promote IL-4- or IL-10-producing CD4+ T cells [14].

The spleen is the major organ involved in generating protective immune responses during malarial infection [15]. Splenectomy of mice immune to P. vinckei vinckei showed the critical role played by the spleen [16]. The mice lost their protective immunity after splenectomy because of depletion of CD4+ T cells. Splenomegaly is a prominent symptom of malaria. The size of the spleen dramatically increases during Plasmodium infection because of influx and expansion of immune cells together with hematopoiesis. The microarchitecture of the spleen is also altered during malarial infection [17, 18]. However, the mechanisms by which protective immunity is generated in the spleen during infection are not clearly understood.

Given the significant changes in splenic cellular composition and activation of immune cells by systemic inflammation that accompany Plasmodium infection, we postulated that the non-DC population may function as APCs during infection with Plasmodium species. Because expression of MHC II is obligatory for activating CD4+ T cells, we investigated MHC II+CD11c non-T, non-B cells, which accumulate in the spleen during P. yoelii infection. Our study demonstrated that the population of MHC II+ cells changes during infection and that MHC II+CD11c non-T, non-B cells become more numerous by approximately 10 days after infection. Although these cells are of non-lymphoid lineage, their increase in the spleen depends on the presence of lymphoid cells. These cells produce TNF-α and IL-6; however, their ability to activate specific CD4+ T cells is limited.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Mice and Plasmodium yoelii infection

Rag-2−/− mice were provided by Dr. Y. Yoshikai (Kyushu University, Fukuoka, Japan) [19], and OT-II transgenic mice expressing the TCR specific for OVA323–339/I-Ab by Dr. H. Kosaka (Osaka University, Osaka, Japan) [20]. These mice were maintained in the Laboratory Animal Center for Animal Research at Nagasaki University and were used at the age of 8–14 weeks. C57BL/6 (B6) mice were purchased from SLC (Hamamatsu, Japan). All animal experiments were conducted according to the Guidelines of the Laboratory Animal Center for Biomedical Research at Nagasaki University. For adoptive transfer, Rag-2−/− mice were administered spleen cells (5 × 107) from B6.Ly5.1 mice i.v. via the tail vein. Mice were infected with P. yoelii 17XNL (P. yoelii) by i.p. injection of 1 × 104 iRBCs. The degree of parasitemia was monitored by microscopic examination of standard blood films.

Cell isolation and flow cytometry

Mouse spleens were cut into small fragments and incubated with Hank's balanced salt solution containing collagenase (400 U/mL, Wako) for 45 min at 37°C. Bone marrow cells were collected from mouse femurs by flushing with medium. After lysing RBCs with Gey's solution, the FcRs were blocked with anti-FcR mAb (2.4G2, 10 µg/mL) for 15 min at 4°C and the splenocytes stained with fluorochrome-conjugated mAbs specific for CD3 (145-2C11), CD19 (1D3), CD11c (N418), MHC II (M5/114), CD45R (RA3-6B2), CD45.1 (A20), CD80 (16-10A1), CD86 (GL-1), CD138 (281-2), IgM (11/41), IgD (11-26c), IgG1 (RMG1-1), IgG2a/2b (R2-40), Ly6C (AL-21), Ly6G (1A8), CD11b (M1/70), F4/80 (BM8), NK1.1 (PK136) and their isotype controls (all from e-Bioscience, San Diego, CA, USA) or with allophycocyanin-anti-PDCA-1 (Miltenyi Biotec, Gladbach, Germany). 7-AAD was used to gate out dead cells and flow cytometry performed using FACS Canto II (BD Bioscience, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

To purify subpopulations of MHC II+ cells, FcRs were blocked with anti-FcR mAbs and splenocytes stained with PECy7-anti-CD3, PECy7-anti-CD19, PE-anti-CD11c, and FITC-anti-MHC II and biotin-anti-IgM mAbs plus APC-streptavidin, then labeled with anti-Cy7 Microbeads (Miltenyi Biotec). CD3+ and CD19+ cells were depleted using AutoMACS (Miltenyi Biotec). 7-AAD was added to exclude dead cells and MHC II+CD11chiCD3CD19 (DCs), MHC II+CD11cCD3CD19IgM+, and MHC II+CD11cCD3CD19IgM populations were sorted using a FACS Aria II (BD Biosciences). For morphological analysis, sorted cells were centrifuged using Cytospin (Block Scientific, Bohemia, NY, USA) and stained with Diff-Quik (Sysmex, Kobe, Japan).

Cytokine assays

Infected red blood cells from the blood of infected mice (parasitemia, 30–50%) were purified (> 95%) by centrifugation in 74% Percoll density gradient as described previously [21]. MHC II+CD11chiCD3CD19, MHC II+CD11cCD3CD19IgM+, and MHC II+CD11cCD3CD19IgM cells were purified by cell sorting as described. Cells (1 × 105) were cultured in the presence of iRBC or RBC (4 × 106) in a final volume of 200 µL for 16 hr and the concentrations of cytokines in the supernatant determined by a sandwich ELISA as described previously [22].

Antigen-presentation assay

OT-II mice were immunized i.p. with OVA (200 µg) in complete Freund's adjuvant. After 5 days, CD4+ T cells were prepared from the spleens of OT-II mice using a CD4+ T cell isolation kit (Milteny Biotech, CD4+ T cells; 87.5%) and labeled with 15 µM CFSE (Invitrogen, Carlsbad, CA, USA) for 10 min. MHC II+CD11chiCD3CD19 DCs and MHC II+CD11cCD3CD19 cells were prepared by cell sorting and pulsed with OVA323–339 (10 µg/mL) or with OVA (1 mg/mL) for 3 hr. OT-II (1 × 105) and MHC II+CD3CD19 cells (1 × 104) were cocultured for 3 days and cell divisions assessed on the basis of diminution of CFSE dye using a FACS Canto II. The supernatant was collected after 2 days of culture to measure the concentrations of IL-2. ELISA was performed as described previously [22].

Statistical analysis

The statistical significance of differences was determined by two-sided Student's t-test using GraphPad PRISM 5 software. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Increase in MHC II+ cells during the course of infection with Plasmodium yoelii

After excluding T and B cells with CD3 and CD19 markers, MHC class II+ cells were examined using spleen cells from B6 mice infected with P. yoelii. Splenic CD3CD19 cells were stained for CD11c and MHC II, and MHC II+ cells divided into three subpopulations based on the degree of CD11c expression, namely CD11chi, CD11cint and CD11c cells (Fig. 1a). In MHC II+CD3CD19 cells, the degree of MHC II expression was greater in CD11chi cells (MFI: uninfected, 6199; infection day 8, 3279) than in CD11cint (MFI: uninfected, 2884; day 8, 2219) or CD11c (MFI: uninfected, 2638; day 8, 1295) cells. MHC II+CD11chiCD3CD19 cells included conventional DCs and constituted the major population of MHC II+ cells prior to infection. MHC II+CD11cintCD3CD19 cells included plasmacytoid DCs and regulatory DCs [7]. After day 5 post-infection, the numbers of both MHC II+CD11chiCD3CD19 and MHC II+CD11cintCD3CD19 cells decreased steadily (Fig. 1b). In contrast, the number of MHC II+CD11cCD3CD19 cells increased until day 9 post-infection in parallel with the degree of parasitemia (Fig. 1c). However, the number of these cells decreased after day 11 post-infection despite continuing increase in parasitemia. These MHC II+CD11cCD3CD19 cells were next stained for CD138, a plasma cell marker, and Igs (Fig. 2a). To our surprise, 23% and 59% of this population expressed IgD and IgM in uninfected mice, respectively, and 6% and 44% expressed IgD and IgM in the infected mice, respectively. IgM+ B cells in the CD3CD19MHC II+ population in the infected mice were mostly IgDB220 and were distinct from those in uninfected mice (Fig. 2b). The morphology of each population was examined (Fig. 2c). CD11chi DCs and MHC II+CD11cCD3CD19IgM+ cells from the infected mice were homogeneous in size and staining patterns. However, MHC II+CD11cCD3CD19IgM cells were heterogeneous in size and may have included multiple cell types. The proportion of these MHC II+CD11cCD3CD19IgM cells in the peripheral blood and bone marrow were also examined (Fig. 2d). These cells increased in spleen, blood and bone marrow on days 6 and 8 post-infection, suggesting that greater numbers of them were being generated in the bone marrow. Since it became clear that the CD3CD19MHC II+ population contained B cells, these IgM+ cells were excluded from further study, and we thereafter focused on MHC II+CD11cCD3CD19IgM cells.

image

Figure 1. Numbers of MHC II+CD11cCD3CD19 cells increase in the spleens of B6 mice during infection with P. yoelii. (a) Spleen cells from mice infected with P. yoelii for 0–15 days were stained with mAbs. Gating for CD3CD19 spleen cells (histogram) and expression of CD11c and MHC II on CD3CD19 gated cells are shown. The number in each box indicates the proportion of cells within the CD3CD19 population. (b) Spleen cells from mice infected with P. yoelii for 0–15 days. The number of MHC II+CD11chi, MHC II+CD11cint and MHC II+CD11c cells within the CD3CD19 gate in each mouse was calculated by multiplying the total number of spleen cells by the proportion of each subset. (c) The severity of parasitemia on various days post-infection in an individual mouse is shown. The bars indicate the mean value on each day.

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image

Figure 2. There are IgM+ cells among the MHC II+CD11cCD3CD19 cells of infected mice. (a) B6 mice were infected with P. yoelii and spleen cells from a pair of infected (bold line) and uninfected (dotted line) mice 8 days post-infection analyzed by flow cytometry. Cells were stained for CD3, CD19, CD11c, MHC II and surface Ig or with isotype control (gray shadow). Flow cytometry profiles of the marker expression of CD11chi (top line), CD11cint (middle line) and CD11c (bottom line) populations after gating for MHC II+CD3CD19 cells are shown. (b) The flow cytometric profiles of MHC II+CD11cCD3CD19 gated populations after staining for CD3, CD19, CD11c, MHC II, B220 and IgD or IgM are shown. Representative data of four similar results are shown. (c) MHC II+CD11chiCD3CD19 (DC), MHC II+CD11cCD3CD19IgM+ and IgM cells were sort-purified from the B6 mice infected with P. yoelii (day 9 post-infection, parasitemia, 37.7%). The cells were placed on a glass slide using a cytospin and stained with Diff-Quick (Sysmex, Kobe, Japan). (d) Cells were collected from spleen, blood and bone marrow of mice prior to (0) and 6 (parasitemia 5.0 ± 1.3%) or 8 (parasitemia 16.4 ± 6.9%) days post-infection with P. yoelii. The proportion of MHC II+CD11cCD3CD19IgM cells of the total live cells was determined using flow cytometry. Bars indicate the mean values.

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The phenotypes of each MHC II+CD3CD19IgM subset were examined next (Fig. 3a). MHC II+CD3CD19IgMCD11chi cells are conventional DCs. Most of this population expressed CD11b, F4/80 and the costimulatory molecules CD80 and CD86. During P. yoelii infection, the proportion of cells expressing F4/80 was reduced, whereas that of cells expressing Ly6C was increased. Additionally, expression of CD40, CD80 and CD86 was increased. MHC II+CD11cintCD3CD19IgM cells, most of which expressed Ly6C, CD11b, CD80 and CD86, were a minor population in uninfected mice. This population may have contained several distinct subsets, including pDCs that express B220 and PDCA-1. Some cells in this group expressed NK1.1, suggesting that this group included NK DCs or interferon-producing killer DCs [23]. After 8 days post-infection, MHC II+CD11cintCD3CD19IgM cells that expressed B220 and PDCA-1 had almost disappeared. Expression of their costimulatory molecules was upregulated. MHC II+CD11cCD3CD19IgM cells, which may have contained several different cell types including those expressing B220, Ly6G, Ly6C, NK1.1, CD11b, and F4/80 were a minor population in uninfected mice, as were IgD+ B cells. Eight days post-infection, the number of these cells increased, whereas those expressing B220, Ly6G, IgD, NK1.1, and F4/80 had almost disappeared. Thus, this population of MHC II+CD11cCD3CD19IgM cells in infected mice was distinct from those in uninfected mice and lacked expression of many cell type specific markers. Approximately 41% of this population expressed Ly6C and most appeared to express PDCA-1 to a moderate degree.

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Figure 3. Increase of MHC II+CD3CD19IgM cells in the spleen requires lymphoid cells. (a, b). B6 (wild type) and Rag-2−/− mice were infected with P. yoelii (8 days post-infection B6, parasitemia 29.0%; Rag-2−/−, parasitemia 12.4%). Spleen cells from a pair of infected (bold line) and uninfected (dotted line) mice were stained with CD3, CD19, CD11c, MHC II, IgM, and with the indicated mAbs or isotype control (gray shadow). (a) Flow cytometry profiles of the marker expression on CD11chi, CD11cint and CD11c populations from B6 mice after gating for MHC II+CD3CD19IgM cells are shown. (b) Flow cytometry profiles of CD11c and MHC II expression on CD3CD19IgM gated spleen cells are shown. The numbers indicate the proportions of each subset (c) The number of CD11chi, CD11cint and CD11c cells within MHC II+CD3CD19 IgM gates in each mouse was calculated by multiplying the total number of spleen cells by the proportions of each subset. A summary of these experiments is shown. Student's t-test, *P < 0.05. The degree of parasitemia: B6 mice, 28.3 ± 3.8%; Rag-2−/− mice, 37.4 ± 21.9%. WT, wild type.

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Origin of MHC II+CD11cCD3CD19IgM cells

To examine whether MHC II+CD11cCD3CD19IgM cells increase during P. yoelii infection in the absence of B and T cells, we infected Rag-2−/− mice with P. yoelii (Fig. 3b). After infection with P. yoelii, splenocytes from Rag-2−/− mice exhibited striking differences from those of wild-type mice. Infected Rag-2−/− mice (5.6 ± 0.8 × 107; parasitemia, 37.4 ± 21.9%) had more spleen cells than uninfected Rag-2−/− mice (1.1 ± 0.4 × 107). However, the numbers of MHC II+CD11c−iCD3CD19IgM cells did not increase in Rag-2−/− mice (Fig. 3c), suggesting that lymphoid cells are involved in the increase in this population during infection with P. yoelii.

Because lymphoid cells were required for the accumulation of MHC II+CD11cCD3CD19IgM cells during infection with P. yoelii, the following two possibilities were considered: (1) these cells were derived from the lymphoid lineage; or (2) they were of myeloid lineage and became MHC II+CD11cIgM cells under the influence of lymphocytes during infection. To examine these possibilities, Rag-2−/− mice (CD45.2+) were adoptively infused with splenocytes, which contain lymphoid cells, from B6.Ly5.1 (CD45.1+) mice. These mice were maintained for 3 weeks to allow homeostatic proliferation of the donor cells and were then infected with P. yoelii [24]. Eight days post-infection, accumulation of MHC II+CD11cCD3CD19IgM cells was separately examined in CD45.1+ and CD45.1 populations (Fig. 4). The number of MHC II+CD11cCD3CD19IgM cells did not significantly increase in the donor CD45.1+ population; however, the number in the host CD45.2+ population did significantly increase, suggesting that the majority of MHC II+CD11cCD3CD19IgM cells that are derived from the myeloid lineage accumulate in the spleens of P. yoelii-infected mice mainly have a non-lymphoid lineage. Thus, it was concluded that MHC II+CD11cCD3CD19IgM cells that are derived from the myeloid lineage accumulate in the spleens of P. yoelii-infected mice under the influence of lymphocytes.

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Figure 4. The origin of the MHC II+CD3CD19IgM cells that increased in the spleen during P. yoelii infection. Rag-2−/− mice (CD45.2+) were adoptively transferred with spleen cells (5 × 107) from B6.Ly5.1 mice (CD45.1+) and infected or uninfected with P. yoelii. On day 8 post-infection spleen cells were harvested and stained for CD3, CD19, IgM, CD45.1, CD11c and MHC II. (a) The CD11c and MHC II profiles of CD3CD19IgMCD45.1+ and CD3CD19IgMCD45.1 gated populations are shown. The proportion of CD45.1+ cells was 36.4 ± 8.8% and 46.9 ± 11.8% in uninfected and infected mice, respectively. (b) The number of MHC II+CD11cCD3CD19IgM cells in CD45.1 (host) and CD45.1+ (donor) gates was calculated by multiplying the total number of spleen cells by the proportions of each subset. Student's t-test, *, P < 0.05. The degree of parasitemia was 27.6 ± 26.0%.

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Function of MHC II+CD11c non-lymphoid cells that accumulate in the spleen

The functional capacities of MHC-II+CD11c non-lymphoid cells that accumulate in the spleen as a defense mechanism against P. yoelii infection were examined. First, purified populations of MHC II+CD11cCD3CD19IgM cells were incubated with iRBCs and production of TNF-α, IL-6 and IL-12 evaluated (Fig. 5). Conventional DCs from uninfected mice were used as positive controls. In response to iRBC, MHC II+CD11cCD3CD19IgM cells from infected mice produced TNF-α and IL-6, but not IL-12. Production of IL-10 was undetectable (data not shown). Second, the ability of these cells to present antigens to CD4+ T cells was evaluated by using OT-II OVA-specific TCR transgenic mice (Fig. 6). OT-II mice were immunized with OVA to enrich memory/effector type OT-II cells that are sensitive to the antigen presentation of OVA. MHC II+ subpopulations isolated from the spleens of infected and uninfected mice were pulsed with OVA323–339 or OVA and cocultured with OT-II cells. OT-II cell proliferation was assessed on the basis of diminution in CFSE and the amount of IL-2 production, which was determined by ELISA. MHC II+CD11chi DCs from both uninfected and infected mice efficiently stimulated proliferation of, and IL-2 production by, OT-II cells. MHC-II+CD11cCD3CD19 cells from uninfected and infected mice were able to stimulate proliferation of OT-II cells in the presence of the OVA323–339 peptide, but not in the presence of OVA, and minimal IL-2 production was stimulated in OT-II cells. Based on these findings, it was concluded that MHC-II+CD11c non-lymphoid cells from infected mice can produce inflammatory cytokines in response to iRBC to a similar degree as DCs, but have only a limited ability to activate antigen-specific CD4+ T cells.

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Figure 5. MHC II+CD11cCD3CD19IgM cells produce TNF-α and IL-6 in response to iRBCs. After enrichment of CD3CD19 cells using AutoMACS, MHC II+CD11c+CD3CD19 (DC), MHC II+CD11cIgMCD3CD19 and MHC II+CD11cIgM+CD3CD19 cells were purified by cell sorting from spleen cells of uninfected and infected mice. Cells were cultured in the absence (open bar) or presence of RBCs (gray bar) or iRBCs (black bar) for 16 hr. Production of TNF-α IL-6 and IL-12 were determined by ELISA in triplicate wells. The results are expressed as mean ± SD. Representative data of three similar experiments are shown.

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Figure 6. MHC II+CD11c cells that increase in the spleen during P. yoelii infection are poor APCs. MHC II+CD11c+CD3CD19 (DC, CD11chi) and MHC II+CD11cCD3CD19 (CD11c) spleen cells were sort-purified from uninfected and P. yoelii-infected mice on day 8 post-infection. These cells were pulsed with medium alone, OVA323–339 or OVA for 3 hr and cocultured with CFSE-labeled CD4+ T cells that had been purified from OVA-immunized OT-II (CD45.1) mice. (a) The cells were stained with APC-anti-CD4 and PECy7-anti-CD45.1 mAbs, and CFSE profiles of OT-II cells determined using flow cytometry. (b) After culture for 2 days in the presence of medium alone, OVA323–339 or OVA, the concentrations of IL-2 in the culture supernatant were determined using ELISA and expressed as mean ± SD. Representative data of three similar experiments are shown.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

During infection with malarial parasites, dramatic changes in the cellular composition in the spleen occur. We studied subsets of MHC II+ non-lymphoid cells during infection with P. yoelii. To exclude T and B cells, we focused our study on MHC II+CD3CD19 cells in the spleen and divided them into three groups on the basis of their degree of expression of CD11c. The numbers of MHC II+CD11chiCD3CD19 and MHC II+CD11cintCD3CD19 cells in the spleen increased approximately 5 days post-infection, and then generally reduced until approximately 10 days post-infection with P. yoelii. In contrast, the number of MHC II+CD11cCD3CD19 cells increased steadily during the first 5–10 days post-infection. We saw increases in these cells not only in the spleen, but also in blood and bone marrow, suggesting that some of these splenic cells are derived from bone marrow. Despite our initial plan to exclude T and B cells, further analysis revealed that, although the cells lacked the B cell markers CD19 and B220 as well as plasma cell marker CD138, this population included IgM+IgD B cells that increased in number during infection with P. yoelii. Thus, we focused our study on MHC II+CD11cCD3CD19IgM cells. In uninfected mice, few MHC II+CD11cCD3CD19IgM cells, which were heterogeneous populations expressing a variety of surface markers, were present. After infection with P. yoelii, the number of MHC II+CD11cCD3CD19IgM cells increased in the spleen and most did not express cell type-specific markers apart from PDCA-1 and Ly6C (∼41%). We also observed increased numbers of these cells in the spleens of mice infected with P. bergei ANKA (data not shown). During infection with P. chabaudi, Ly6C+ monocytes are reportedly generated in the bone marrow in a C–C chemokine receptor type 2-dependent manner and migrate to the spleen; these cells produce proinflammatory cytokines in response to the malarial antigen and express small amounts of MHC II, but they are poor APCs [25]. Although the MHC II+CD11cCD3CD19IgM cells that we identified are functionally similar to these Ly6C+ monocytes, there are some phenotypical differences. Their Ly6C+ monocytes express CD11b and CD11c while ours do not express these markers and only ∼41% express Ly6C.

To confirm that this increased population truly consisted of non-lymphoid cells, we used Rag-2−/− mice, which lack T and B cells. However, to our surprise, these cells did not increase in the spleens of Rag-2−/− mice during P. yoelii infection. This finding raised two possibilities: either the MHC II+CD11cCD3CD19IgM cells that increase during infection are lymphoid cells or their accumulation in the spleen depends on lymphoid cells. To examine these possibilities, we used Rag-2−/− mice containing B6 splenocytes. Our results suggested that although most accumulating MHC II+CD11cCD3CD19IgM cells are derived from non-lymphoid cells, their accumulation in the spleen is dependent on lymphoid cells. Accumulation of this population may require multiple steps, including their generation in the bone marrow, exit to the peripheral circulation, and migration to the splenic tissue. During P. yoelii infection, lymphocytes are activated and they may produce cytokines, which are required for the generation or migration of these cells into the spleen.

We observed a moderate degree of PDCA-1 expression in the MHC II+CD11cCD3CD19IgM population during P. yoelii infection. Although PDCA-1 is reportedly a marker of plasmacytoid DCs [26], recent studies have revealed that this marker is also expressed on a subpopulation of B cells [27-29]. Although PDCA-1+ B cells are a minor population in naïve mice, a large proportion of B lineage cells express PDCA-1 after infection with influenza virus or L. monocytogenes, or under generalized autoimmune conditions such as MRL-lpr. Upon activation, PDCA-1+ B cells can secrete type I IFNs and the immunosuppressive enzyme indoleamine 2,3-dioxygenase [28]. This suggests that secretion of IFN-α by PDCA-1+ B cells during infection with L. monocytogenes contributes to innate immune responses against bacterial infection [29]. Thus, it is likely that induction of PDCA-1 on MHC II+CD11cCD3CD19IgM cells is due to their activation during malarial infection, rather than expansion of a particular cell subset that expresses PDCA-1.

Functionally, the MHC II+CD11cCD3CD19IgM cells were able to produce TNF-α and IL-6 in response to iRBCs, suggesting that they may contribute to the inflammatory response to P. yoelii infection. Their production of IL-10 in response to iRBC was not detectable (data not shown). Although these cells expressed MHC II, they were unable to present protein antigens and activate T cells. Thus, MHC II+CD11cCD3CD19 cells are similar to Ly6C+ monocytes, which express MHC II weakly and are unlikely to function as APCs in vivo [25]. Our study confirmed that CD11c+ DCs are major APCs in the spleen during P. yoelii infection. Lymphocytes that are activated by these DCs produce cytokines, which may be required for the accumulation of MHC II+CD11c non-lymphoid cells in the spleen. These non-lymphoid cells produce proinflammatory cytokines such as TNF-α and IL-6 in response to parasitized RBCs and promote immune responses that may inhibit the growth of parasites, as suggested by previous studies [25].

During the blood stage of infection with malarial parasites, the battle between the parasites and the immune system primarily occurs in the spleen. Induction of effective immune responses in the spleen is required to develop effective immune defenses against invading parasites. Our study clarified the interaction between MHC II+CD11c non-lymphoid cells and lymphoid cells in the accumulation of these non-lymphoid cells in the spleen. A further understanding of the varieties of cell types in the spleen and their interactions will help to explain the mechanisms underlying modulation of immune responses during infection with malarial parasites and will be important for developing an effective vaccine against this critical infectious disease.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

We thank Drs H. Kosaka (Osaka University, Osaka, Japan) and Y. Yoshikai (Kyushu University, Fukuoka, Japan) for providing mice and M. Masumoto (Nagasaki University, Nagasaki, Japan) for cell sorting. This study was supported by the Global COE Program at Nagasaki University and by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture to K.Y.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  • 1
    Good M.F., Xu H., Wykes M., Engwerda C.R. (2005) Development and regulation of cell-mediated immune responses to the blood stages of malaria: implications for vaccine research. Annu Rev Immunol 23: 6999.
  • 2
    Steinman R.M., Banchereau J. (2007) Taking dendritic cells into medicine. Nature 449: 41926.
  • 3
    Shortman K., Naik S.H. (2007) Steady-state and inflammatory dendritic-cell development. Nat Rev Immunol 7: 1930.
  • 4
    Geissmann F., Manz M.G., Jung S., Sieweke M.H., Merad M., Ley K. (2010) Development of monocytes, macrophages, and dendritic cells. Science 327: 65661.
  • 5
    Dominguez P.M., Ardavin C. (2010) Differentiation and function of mouse monocyte-derived dendritic cells in steady state and inflammation. Immunol Rev 234: 90104.
  • 6
    Serbina N.V., Salazar-Mather T.P., Biron C.A., Kuziel W.A., Pamer E.G. (2003) TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection. Immunity 19: 5970.
  • 7
    Wakkach A., Fournier N., Brun V., Breittmayer J.P., Cottrez F., Groux H. (2003) Characterization of dendritic cells that induce tolerance and Tr 1 cell differentiation in vivo. Immunity 18: 60517.
  • 8
    Wykes M.N., Good M.F. (2008) What really happens to dendritic cells during malaria? Nat Rev Microbiol 6: 86470.
  • 9
    Urban B.C., Ferguson D.J., Pain A., Willcox N., Plebanski M., Austyn J.M., Roberts D.J. (1999) Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400: 737.
  • 10
    Ocana-Morgner C., Mota M.M., Rodriguez A. (2003) Malaria blood stage suppression of liver stage immunity by dendritic cells. J Exp Med 197: 14351.
  • 11
    Perry J.A., Olver C.S., Burnett R.C., Avery A.C. (2005) Cutting edge: the acquisition of TLR tolerance during malaria infection impacts T cell activation. J Immunol 174: 59215.
  • 12
    Wilson N.S., Behrens G.M., Lundie R.J., Smith C.M., Waithman J., Young L., Forehan S.P., Mount A., Steptoe R.J., Shortman K.D., De Koning-Ward T.F., Belz G.T., Carbone F.R., Crabb B.S., Heath W.R., Villadangos J.A. (2006) Systemic activation of dendritic cells by toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity. Nat Immunol 7: 16572.
  • 13
    Wong K.A., Rodriguez A. (2008) Plasmodium infection and endotoxic shock induce the expansion of regulatory dendritic cells. J Immunol 180: 71626.
  • 14
    Sponaas A.M., Cadman E.T., Voisine C., Harrison V., Boonstra A., O'Garra A., Langhorne J. (2006) Malaria infection changes the ability of splenic dendritic cell populations to stimulate antigen-specific T cells. J Exp Med 203: 142733.
  • 15
    Engwerda C.R., Beattie L., Amante F.H. (2005) The importance of the spleen in malaria. Trends Parasitol 21: 7580.
  • 16
    Kumar S., Good M.F., Dontfraid F., Vinetz J.M., Miller L.H. (1989) Interdependence of CD4+ T cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development. J Immunol 143: 201723.
  • 17
    Achtman A.H., Khan M., Maclennan I.C., Langhorne J. (2003) Plasmodium chabaudi chabaudi infection in mice induces strong B cell responses and striking but temporary changes in splenic cell distribution. J Immunol 171: 31724.
  • 18
    Beattie L., Engwerda C.R., Wykes M., Good M.F. (2006) CD8+ T lymphocyte-mediated loss of marginal metallophilic macrophages following infection with Plasmodium chabaudi chabaudi AS. J Immunol 177: 251826.
  • 19
    Shinkai Y., Rathbun G., Lam K.P., Oltz E.M., Stewart V., Mendelsohn M., Charron J., Datta M., Young F., Stall A.M., Alt F.W. (1992) RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68: 85567.
  • 20
    Barnden M.J., Allison J., Heath W.R., Carbone F.R. (1998) Defective TCR expression in transgenic mice constructed using cDNA-based α- and β-chain genes under the control of heterologous regulatory elements. Immunol Cell Biol 76: 3440.
  • 21
    Ing R., Segura M., Thawani N., Tam M., Stevenson M.M. (2006) Interaction of mouse dendritic cells and malaria-infected erythrocytes: uptake, maturation, and antigen presentation. J Immunol 176: 44150.
  • 22
    Miyakoda M., Kimura D., Yuda M., Chinzei Y., Shibata Y., Honma K., Yui K. (2008) Malaria-specific and nonspecific activation of CD8+ T Cells during blood stage of Plasmodium berghei infection. J Immunol 181: 14208.
  • 23
    Chan C.W., Crafton E., Fan H.N., Flook J., Yoshimura K., Skarica M., Brockstedt D., Dubensky T.W., Stins M.F., Lanier L.L., Pardoll D.M., Housseau F. (2006) Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity. Nat Med 12: 20713.
  • 24
    Goldrath A.W., Bogatzki L.Y., Bevan M.J. (2000) Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation. J Exp Med 192: 55764.
  • 25
    Sponaas A.M., Freitas Do Rosario A.P., Voisine C., Mastelic B., Thompson J., Koernig S., Jarra W., Renia L., Mauduit M., Potocnik A.J., Langhorne J. (2009) Migrating monocytes recruited to the spleen play an important role in control of blood stage malaria. Blood 114: 552231.
  • 26
    Krug A., French A.R., Barchet W., Fischer J.A., Dzionek A., Pingel J.T., Orihuela M.M., Akira S., Yokoyama W.M., Colonna M. (2004) TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21: 10719.
  • 27
    Blasius A.L., Giurisato E., Cella M., Schreiber R.D., Shaw A.S., Colonna M. (2006) Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation. J Immunol 177: 32605.
  • 28
    Vinay D.S., Kim C.H., Chang K.H., Kwon B.S. (2010) PDCA expression by B lymphocytes reveals important functional attributes. J Immunol 184: 80715.
  • 29
    Bao Y., Han Y., Chen Z., Xu S., Cao, X. (2010) IFN-α-producing PDCA-1+ Siglec-H B cells mediate innate immune defense by activating NK cells. Eur J Immunol 41: 65768.