IL-17 promotes differentiation of splenic LSK⁻ lymphoid progenitors into B cells following Plasmodium yoelii infection

Abstract

LSK⁻ (Lineage⁻Sca-1⁺c-kit⁻) cells are a lymphoid progenitor population that expands in the spleen and preferentially differentiates into mature B cells in response to Plasmodium yoelii infection in mice. Furthermore, LSK⁻ derived B cells can subsequently contribute to the ongoing immune response through the generation of parasite-specific antibody secreting cells, and germinal center and memory B cells. However, the factors that promote their differentiation into B cells in the spleen after infection are not defined. Here we show that LSK⁻ cells produce the cytokine IL-17 in response to Plasmodium infection. Using Il-17ra^−/− mice IL-17R signaling in cells other than LSK⁻ cells was found to support their differentiation into B cells. Moreover, primary splenic stromal cells grown in the presence of IL-17 enhanced the production of CXCL12, a chemokine associated with B-cell development in the bone marrow, by a population of IL-17RA–expressing podoplanin⁺CD31⁻ stromal cells, a profile associated with fibroblastic reticular cells. Subsequent blockade of CXCL12 in vitro reduced differentiation of LSK⁻ cells into B cells, supporting a direct role for this chemokine in this process. Immunofluorescence indicated that podoplanin⁺ stromal cells in the red pulp were the primary producers of CXCL12 after P. yoelii infection. Furthermore, podoplanin staining on stromal cells was more diffuse, and CXCL12 staining was dramatically reduced in Il-17ra^−/− mice after infection. Together these results identify a distinct pathway that supports lymphoid development in the spleen during acute Plasmodium infection.

Introduction

Previously, we identified an atypical lymphoid progenitor cell population in the spleen defined as Lin⁻Sca-1⁺c-Kit⁻ (LSK⁻) cells that differentiate into mature B cells in response to Plasmodium infection in mice. Furthermore, a proportion of LSK⁻ derived B cells were capable of differentiating into Plasmodium-specific antibody-secreting cells, and germinal center (GC) and memory B cells, suggesting their potential contribution to the humoral response against the parasite (1). However, the cells and factors that support differentiation of these splenic lymphoid progenitors into B cells after infection have not been defined.

Stromal cells are essential structural components of secondary lymphoid organs (SLOs), but they also interact with hematopoietic cells to promote or regulate adaptive immune responses. Their function can be modulated by cues from hematopoietic cells, other stromal cells, and the SLO environment (2). Also, stromal cells are essential for providing a niche within the bone marrow for the development of stem cells into various hematopoietic cell types under homeostatic conditions. In emergency conditions such as stress or infection hematopoietic stem and progenitor cells (HSPCs) are mobilized to the spleen from the bone marrow and take part in the process of extramedullary hematopoiesis (EMH), which coincides with a considerable expansion of red pulp cellularity (3–5). To what extent stromal cells in the splenic red pulp support EMH is unclear. However, a recent report indicated that stromal cells and endothelial cells contribute to the formation of an EMH niche associated with red pulp sinusoids in response to a number of stresses, including myeloablation, pregnancy, and blood loss. Both endothelial cells and stromal cells were found to express stem cell factor (SCF), while a subset of stromal cells was found to express CXCL12 (6).

The pro-inflammatory cytokine IL-17, which is produced by T cells, innate lymphoid cells and other cells, functions to promote the production of cytokines (G-CSF, IL-6, GM-CSF, SCF), chemokines (IL-8, CXCL1, CCL2), and prostaglandin E2 from cells such as endothelial cells, fibroblasts, macrophages, epithelial cells, keratinocytes and other stromal cell populations (7–9). Many of these cytokines and chemoattractants induced by IL-17 are important for stimulating granulopoiesis and recruitment of neutrophils to sites of inflammation. Systemic overexpression of IL-17 in vivo has also been shown to induce splenic EMH in mice, due to its ability to stimulate G-CSF and SCF production (8, 10). Also, IL-17 has been demonstrated to promote the formation of tertiary lymphoid tissues (TLTs) in such sites as the lung and brain, resulting in the accumulation of B cells, which form a follicle-like structure and T cells, which surround the B cells (11–14). The ability of IL-17 to promote TLT formation is due in part to its capacity to induce the production of CXCL12 by stromal cells (12–16). Based on its role in modulating stromal cells in non-lymphoid tissues, IL-17 may also actively influence the stromal cell compartment in the spleen to promote a niche for extramedullary lymphopoiesis.

In this report, we demonstrate that splenic LSK⁻ cells are the most abundant cell type that produces IL-17 at the peak of P. yoelii 17X infection. The absence of IL-17R signaling in the host, but not in LSK⁻ cells, led to a reduction in LSK⁻ cell differentiation into B cells, resulting in a decrease in germinal center B cells and antibody-secreting cells after infection. This result correlated with and contributed to an observed decrease in serum parasite-specific antibodies (Abs) and increased parasitemia in Il-17ra^−/− mice after P. yoelii 17X infection. In the absence of IL-17R signaling, splenic stromal cells produced less CXCL12 leading to impaired differentiation of LSK⁻ cells into B cells in vitro. Overall, these results identify a distinct pathway in which IL-17 production by splenic LSK⁻ cells serves to support their differentiation into B cells in response to acute Plasmodium infection.

Materials and Methods

Mice and infection

Female C57BL/6J and C57BL/6-Tg (UBC-GFP)30Scha/J (Ubc-GFP Tg) mice were purchased from The Jackson Laboratory, while male BALB/c mice were purchased from Harlan Laboratories. Il-17ra^{tm1a(KOMP)Wtsi} mice were generated by the knockout mouse project (UC Davis). Chimeric Il-17ra^{tm1a(KOMP)Wtsi} male mice were bred with female C57BL/6N (Charles River) mice. The F1 progeny Il-17ra^+/− mice were then backcrossed onto the C57BL/6N background. Back-crossing continued for ten generations to produce Il-17ra^−/− mice on a C57/BL/6N background, Il-17ra^−/− mice were then crossed with (UBC-GFP)30Scha/J mice to produce Il-17ra^−/− Ubc-GFP Tg mice. All mice were housed and bred in specific-pathogen-free facilities at the University of Arkansas for Medical Sciences in accordance with institutional guidelines. For infection with P. yoelii 17X, male BALB/c mice were infected with parasitized red blood cells (RBCs) derived from frozen stocks. Subsequently, 10⁵ parasitized erythrocytes derived from the passage were intraperitoneally injected into experimental female mice to establish infection. Parasitemia was evaluated by counting Giemsa (Harleco, Millipore) stained thin blood smears or by flow cytometry (17).

Flow cytometry and antibodies

Single cell suspension preparation and antibody labeling procedures are described elsewhere (1). For labeling stromal cells, the spleen was perfused with 0.2 mg/ml Liberase and 0.1 mg/ml DNase I (Roche) in RPMI 1640 media before cutting into small pieces and incubating at room temperature for 45 minutes on a rotating wheel; resulting cell suspension was passed through a 70-μm cell strainer to achieve a single cell suspension. Cells were then washed twice with RPMI 1640, followed by resuspension in complete RPMI (RPMI 1640 supplemented with 10% FBS, 1% non-essential amino acids, 1% sodium pyruvate, 1% L-glutamate, 1% penicillin-streptomycin, and 0.1% β-mercaptoethanol).

To prepare cells for flow cytometry 3 × 10⁶ splenocytes were incubated with Fc Block (10% 2.4G2 Fc Block, 0.5% normal rat IgG, and 0.5% normal mouse IgG) in FACS buffer (0.2% BSA and 0.2% 0.5M EDTA in 1× PBS) (10 min at 4°C). Surface staining was performed using appropriate dilutions of antibodies in FACS buffer (20 min at 4°C). For biotinylated antibodies, this step was followed by an addition of fluorochrome-conjugated streptavidin (SA) diluted appropriately in FACS buffer (10 min at 4°C). The antibodies IL-17RA, IgD, CD73, CD43, CD93, CD45.2, CD3e, CD11c, Ter-119, CD11b, CD5, NK1.1, CD8α, B220, CD4, CD38, c-kit, CD23, GL-7, CD90.2, CD21/35, and FoxP3 were purchased from eBioscience (San Diego, CA). Antibodies - Sca-1, γδ-TCR, Podoplanin, CD31, CD25, CXCR5, CD19, IgM, CD90.2, CD38, and fluorochrome-conjugated SA were purchased from Biolegend (San Diego, CA), while CD138, IL-17A, CD31, CXCR4, PD-1, CXCR5, and CD45 were purchased from BD Biosciences (San Jose, CA). For samples that did not require intracellular staining cells were fixed using a 4% paraformaldehyde (PFA) solution (Electron Microscopy Sciences).

For cytokine staining, splenocytes were incubated with PMA, Ionomycin and Brefeldin A (Sigma) (4 h at 37°C) before surface staining. Cells were fixed with 4%-PFA followed by permeabilization using 0.1% saponin diluted in FACS buffer and stained with antibodies diluted in this same buffer. Antibodies specific for IFN-γ, IL-10, and IL-17F were purchased from eBioscience, while the IL-17A antibody was purchased from BD Biosciences. Unconjugated anti-GFP rabbit monoclonal antibody and Alexa Fluor 488 conjugated goat anti-rabbit IgG were purchased from Thermo Fischer Scientific Inc. (Rockford, IL). For intracellular staining of transcription factors – FoxP3, AhR, and ROR-γt (eBioscience), a FoxP3 fixation/permeabilization kit (eBioscience) was used. Phenotypic analysis of the cell populations was attained by the acquisition of fluorochrome-labeled cells on an LSRII Fortessa flow cytometer (Becton Dickinson) and analysis using FlowJo software (version X, Tree Star). Fluorescence minus one (FMO) controls were used for setting the positive gates and indicating background staining for histogram plots.

Cell sorting

Procedure used for cell soring has been described previously (1). Briefly, LSK⁻ cells, fibroblast reticular cells (FRCs), follicular dendritic cells (FDCs), triple negative stromal cells (TNs) and blood endothelial cells (BECs) were harvested by sorting for Lin⁻CD45⁺Thy1.2⁻Sca-1⁺c-kit⁻ (LSK⁻), Lin⁻CD45⁻gp38⁺CD31⁻ (FRCs), Lin⁻CD45⁻gp38⁻CD31⁻CD35⁺ (FDCs), Lin⁻CD45⁻gp38⁻CD31⁻CD35⁻ (TNs), and Lin⁻CD45⁻gp38⁻CD31⁺ (BECs) populations.

Splenic stromal cell culture and in vitro lymphoid differentiation assay

Spleens isolated from 6–8 week old C57BL/6 mice were perfused with enzyme mix containing 0.2 mg/ml liberase (Roche) and 0.1 mg/ml DNAse I (ThermoFisher) and minced into small pieced using a scalpel. Following incubation with the enzyme mix for 45 min on a rotating wheel at room temperature the cell suspension was passed through a 70-μm cell strainer (Fisher). Resulting single cell suspension was then plated in a 100 mm cell culture plate (Corning) and incubated at 37°C in 5% CO₂ and non-adherent cells were washed off after four hours. Adherent cells were maintained in media (RPMI 1640 media supplemented with 20% FBS, 1% L-glutamate, 1% penicillin-streptomycin) for three weeks before purifying for CD45⁻gp38⁺CD31⁻ cells by sorting on an FACSAria II (Becton Dickinson). Recovered gp38⁺ stromal cells were kept in culture for an additional two weeks before using in lymphoid differentiation assays. For passage, splenic stromal cells were treated with 0.1% trypsin supplemented with 5 mM EDTA for five min at 37ºC. For lymphoid differentiation assays, splenic stromal cells were seeded, in 96-well flat-bottom, sterile tissue culture plates at a concentration of 10,000 cells per well and allowed to grow and adhere overnight in media. The following day the media was exchanged for complete RPMI. LSK⁻ cells from naïve mice were sorted (100 cells/well) directly onto the splenic stromal cell monolayer. The cells were cultured in complete RPMI medium with or without recombinant IL-17 (100 ng/ml) (eBioscience), for 7, or 14 d at 37ºC in 5% CO₂. The co-culture was analyzed by flow cytometry for the presence of CD19⁺B220⁺ B cells at the end of each time-point.

In vivo adoptive transfer

Splenic derived LSK⁻ cells were resuspended in endotoxin-free sterile PBS (Life Technologies) at a concentration of 1 × 10⁵ cells/ml, and 1 × 10⁴ cells were transferred per mouse into the venous sinus through the retro-orbital route for all recipients following isoflurane-induced anaesthetization.

ELISA

Recombinant P. yoelii 17X MSP-1₁₉ protein or whole parasite infected RBC lysate was used to coat high-binding Immulon HBX 4× plates (Bioexpress) overnight at 4°C. After blocking in 5% FCS in 1 × PBS, serially diluted serum was incubated on the plate followed by incubation with HRP-conjugated IgM or IgG Abs (Southern Biotech). SureBlue substrate (KPL) was added to detect the antigen-specific Abs. The wells were read at 450 nm on an FLUOstar Omega plate reader (BMG Labtech).

Immunofluorescence

Spleens were embedded in OCT compound (Electron Microscopy Sciences), frozen and sectioned at a thickness of 4 μm. Sections were then fixed in 75% methanol/25% acetone for 10 min at −20°C. After blocking, sections were incubated with appropriate dilutions of primary Abs (eBioscience) overnight at 4°C. Secondary anti-mouse AF488, anti-rabbit AF488, anti-rabbit Cy5, anti-rat DyLight649, or anti-hamster Cy3 (Jackson Immunoresearch) Abs were incubated on the slides for 2 h at 25°C the following day. Slides were mounted with Fluoroshield (Electron Microscopy Sciences) and subsequently imaged on an EVOS FL Auto 2 Imaging System (Life Technologies).

Quantitative RT-PCR

Splenic derived LSK⁻ cells and stromal cells sorted on an FACSAria II (Becton Dickinson) were subsequently resuspended in RNA lysis buffer (QIAGEN), and RNA was isolated using an RNeasy mini kit following the manufacturer’s protocol. The resulting RNA from each sample was treated with RNase-free DNase (Promega), followed by cDNA preparation using oligo-dT primers and SuperScript II Reverse Transcriptase (Life Technologies). For real-time qRT-PCR, cDNA from an equivalent number of cells was mixed with SYBR Green master mix (Bio-Rad) and appropriate primer sets; analysis was performed using a StepOne™ Real-Time PCR System and StepOne™ Software.

Statistical analysis

One-way analysis of variance (ANOVA) was used for comparing data obtained from a group of Plasmodium-infected mice, followed by Kruskal-Wallis test for multiple comparisons between the groups; whereas multiple t-tests were used for comparisons between two or more groups. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA).

Results

Expansion of pro-inflammatory IL-17–producing cells following Plasmodium infection

Infection with P. yoelii 17X induces severe anemia and acute changes in hematopoiesis in C57BL/6 mice (1, 4), which coincide with the induction of EMH in the spleen. Findings that corroborate with those seen in P. chabaudi AS (18, 19) and P. berghei (20) models of infection. Our previous data indicated that as part of the EMH response a lymphoid progenitor population defined as LSK⁻ cells expands in the spleen following acute infection with P. yoelii 17X. This lymphoid progenitor population was found to reach a peak in cell numbers at day 7 post-infection (p.i.) and taper off after peak parasitemia, around day 11 p.i. (1). Upon further evaluation, LSK⁻ cells are capable of producing the pro-inflammatory cytokine IL-17, but not IFN-γ, IL-17F or IL-10 after P. yoelii infection (Figure 1A–C, and data not shown). The frequency and number of IL-17–producing LSK⁻ cells peaks at day 7 p.i. (Figure 1A, B). Furthermore, at day 7 and 11 p.i. LSK⁻ cells are the major IL-17–producing cells in the spleen, making up 66% and 89% of the total IL-17⁺ cells at these time points (Figure 1D, E and Supplemental Figure 1). Only small expansions in the number of IL-17-producing CD4⁺, CD8⁺, and γδ TCR⁺ T cells, as well as NK cells, occur during infection compared to the number of IL-17⁺ cells seen in naïve mice (Figure 1E and Supplemental Figure 1).

Figure 1. Splenic LSK⁻ cells produce the cytokine IL-17 after P. yoelii 17X infection.

A. Representative flow plots and B. Total number of LSK⁻ cells producing IL-17 in the spleen of naïve or P. yoelii 17X infected C57BL/6 mice at days 0, 4, 7, 11 and 21 and 32 post-infection. ***p = 0.0012 (Kruskal-Wallis nonparametric ANOVA). C. Representative flow-plots showing the production of IL-17 and IFN-γ by LSK⁻ cells from WT mice at day 0 and 7 after P. yoelii 17X infection. The FMO control is shown to indicate background staining. D. Percentage and E. Total number of IL-17 producing immune cells in the spleen of C57BL/6 mice following acute P. yoelii infection at days 4, 7 and 11 post-infection. *p <0.01, **p <0.001, ***p =0.0001 (Two-way ANOVA). F. Representative histogram plots showing expression of ROR-γt, dot-plots indicating the median fluorescence intensity of ROR-γt expression and percentage of LSK⁻ cells expressing ROR-γt at day 0 and 7 following P. yoelii infection. FMO control is shown as grey filled histogram. G. Representative histogram plots showing expression of AhR, dot-plots indicating the median fluorescence intensity of AhR expression and percentage of LSK⁻ cells expressing AhR at day 0 and 7 following P. yoelii infection, FMO control shown as grey filled histogram. Data are representative of (A–C) or pooled from (D–E) three independent experiments, with n=3/4 mice/experiment. Error bars (B–G) S.E.M.

Next, LSK⁻ cells were assessed for expression of transcription factors associated with IL-17 production by T cells. While LSK⁻ cells from naïve spleens do not express RORγt, they are capable of expressing this key transcription factor involved in the commitment to the Th17 lineage (21) after infection (Figure 1F). Furthermore, a small percentage of LSK⁻ cells from naïve mice express the aryl hydrocarbon receptor (AhR) (Figure 1G), another transcription factor associated with the Th17 lineage (22, 23). Whereas only a marginal increase in the median fluorescent intensity for AhR expression occurs after infection, a higher frequency of LSK⁻ cells expresses this transcription factor (Figure 1G). Together this data indicate that LSK⁻ cells are the primary source of IL-17 in the spleen in response to acute P. yoelii infection and that this progenitor population expresses a number of transcription factors associated with IL-17 expression by CD4⁺ T cells.

Absence of IL-17R signaling diminishes the humoral response after Plasmodium infection

To determine if the IL-17 produced by LSK⁻ cells is necessary for promoting inflammation and/or development of these progenitor cells into B cells Il-17ra^−/− mice were infected with P. yoelii 17X. Following infection, no difference in parasitemia is observed between wild-type (WT) and Il-17ra^−/− mice over the first two weeks of infection (Figure 2A), indicating IL-17–mediated signaling has a minimal effect on the immune response during this stage of infection. Indeed, no difference in neutrophil or macrophage accumulation in the spleen occurs during this period between WT and Il-17ra^−/− mice, or mice treated with an anti-IL-17 blocking Ab (Supplemental Figure 2). Nor are any differences in Th1, Treg or Tfh cell numbers seen during this period (Supplemental Figure 2).

Figure 2. IL-17R deficiency leads to reductions in B cell populations after P. yoelii 17X infection.

A. Representative parasitemia curve showing the percentage of infected RBCs in WT (black dots) and Il-17ra^−/− (grey dots) mice. *p < 0.0005. B. P. yoelii 17X MSP-1₁₉–specific IgG measured in the serum at days 11, 17, and 36 p.i. by ELISA. *p <0.05, ***p <0.0001 (multiple t-test). Total number of C. antibody secreting cells (B220^intCD138⁺) *p ≤ 0.05, D. B cells (CD19⁺B220⁺) *p = 0.04; **p = 0.003; ***p <0.001, E. germinal center B cells (CD19⁺B220⁺CD38⁻GL-7⁺) *p = 0.0004, and F. memory B cells (CD19⁺B220⁺CD38⁺GL-7⁻CD73⁺) *p ≤ 0.05, G. Immature B cells (CD19⁺B220⁺CD93⁺) ***p < 0.005, *p < 0.05, and H. Newly formed B cells (CD19⁺B220⁺CD93⁻CD21/35⁻CD23⁻CD43⁺) *p = 0.03 (multiple t-test) in naïve and infected WT (black dots) and Il-17ra^−/− (grey dots) mice at days 0, 7, 11, 17 and 36 p.i. Data are pooled from three independent experiments, with n=3/4 mice/experiment. Error bars (A–J) S.E.M.

However, within the window of day 14 to 26 p.i., Il-17ra^−/− mice display a higher parasite burden compared to WT mice (Figure 2A). But, Il-17ra^−/− mice resolve the infection at the same time as WT mice, indicating that IL-17 plays a modest role in controlling Plasmodium infection. Resolution of P. yoelii infection involves the presence of B cells (24); therefore, the B-cell and Ab response were compared between WT and Il-17ra^−/− mice. The higher parasitemia in Il-17ra^−/− mice correlates with significantly lower merozoite surface protein-1 (MSP-1₁₉) IgG compared to WT mice at day 17 p.i. (Figure 2B). While no significant difference in antibody-secreting cells (B220^intCD138⁺) occurs in the spleen at this time (Figure 2C), a significant decrease in plasmablast numbers takes place in Il-17ra^−/− mice at day 11 p.i., which corresponds to the period just prior to peak parasitemia in WT mice. Moreover, a difference in parasite-specific IgG in the serum of WT and Il-17ra^−/− mice is already apparent at day 11 p.i., suggesting that IL-17 signaling is required before peak parasitemia to promote the production of antibody-secreting cells and control the infection. Furthermore, the large difference in serum MSP-1₁₉–specific IgG is temporary, as by day 36 p.i. the gap closes significantly between WT and Il-17ra^−/− mice (Figure 2B). Although IL-17 shows a moderate effect on controlling parasite burden in mice, there is a significant decrease in B cell numbers in the spleen of Il-17ra^−/− mice, throughout and after clearance of P. yoelii 17X infection (Figure 2D). Interestingly, the absence of IL-17RA results in a significant reduction of GC B cell numbers at day 36 p.i., which leads to a significant reduction in the number of antibody-secreting cells and memory B cells at this time (Figure 2C, E–F). Also, memory B cell numbers are significantly lower in Il-17ra^−/− mice at days 11 and 17 after infection (Figure 2F).

Furthermore, the loss of IL-17R signaling not only impacts mature B cell numbers but also reduces the number of immature B cells and newly formed B-lymphocytes (CD23⁻CD21/35⁻) compared to WT mice (Figure 2G, H). Importantly, the observed decreases in B cell numbers in Il-17ra^−/− mice are the direct result of the host response to P. yoelii 17X infection, as no significant differences in cell numbers for these B-cell populations occur in the spleens of naïve WT or Il-17ra^−/− mice (Figure 2C–H). Collectively, these data indicate that the absence of IL-17 mediated signaling affects several stages of B-cell development, which leads to an overall decline in B cell numbers and parasite-specific Ab production, resulting in a delay in parasite control in Il-17ra^−/− mice following P. yoelii infection.

Role of IL-17 on splenic LSK⁻ cell differentiation following Plasmodium infection

We previously showed that splenic LSK⁻ cells preferentially differentiate into mature B cells in response to P. yoelii infection, and a proportion of these progeny B cells are capable of producing parasite-specific Abs after activation (1). Therefore, the finding that LSK⁻ cells are the major producers of IL-17 at the peak of infection (Figure 1E), and the observed impact that the loss of IL-17R signaling has on the accumulation of B cell populations in the spleen, suggests a potential role for LSK⁻ cell-derived IL-17 in promoting their differentiation into B cells. Many different cell types express the IL-17RA subunit (25), including LSK⁻ cells (Supplemental Figure 3). However, the absence of IL-17R signaling does not impact LSK⁻ cell expansion (Supplemental Figure 3), further supporting a role for IL-17 in B cell differentiation and not LSK⁻ cell expansion. To test whether IL-17R signaling directly or indirectly influences the differentiation of LSK⁻ cells into B cells, splenic LSK⁻ cells from naïve Ubc-GFP Tg or Il-17ra^−/− Ubc-GFP Tg mice were transferred intravenously into WT or Il-17ra^−/− mice before infection of recipient mice with P. yoelii 17X (Figure 3A). At day 11 p.i. the spleens from recipient mice are examined for donor-derived (GFP⁺) cells. Irrespective of donor-cell background, recovery of GFP⁺ cells is significantly lower from Il-17ra^−/− recipients compared to WT recipients (Figure 3B).

Figure 3. Indirect IL-17R signaling is required to promote differentiation of LSK⁻ cells into B cells after infection.

A. Model showing the experimental design. Splenic LSK⁻ cells are sorted from naïve Ubc-GFP and Il-17ra^−/− Ubc-GFP Tg mice, and 5,000 cells are transferred i.v. into C57BL/6 and Il-17ra^−/− mice. Recipient mice are then infected i.p. with 10⁵ P. yoelii 17X pRBCs and the phenotype of the transferred cells is evaluated on day 11 p.i. Total number of GFP⁺ B. cells C. B cells, D. antibody secreting cells, E. germinal center B cells F. immature B cells and G. newly formed B cells recovered from WT (black dots) or IL-17ra^−/− (grey dots) recipient mice. *p ≤ 0.05, **p ≤0.01 (Kruskal-Wallis One way ANOVA). Data are representative of two independent experiments, with n=4/5 mice/experiment. Error bars (B–G) represent S.E.M.

Similarly, regardless of donor-cell background, recovery of GFP⁺ B cells is significantly lower from Il-17ra^−/− recipients compared to WT recipients (Figure 3C). This result translates into the identification of fewer antibody-secreting cells and GC B cells in Il-17ra^−/− recipients who received LSK⁻ cells from Ubc-GFP Tg or Il-17ra^−/− Ubc-GFP Tg mice. Whereas the total number of B cells, GC B cells or antibody-secreting cells show no significant difference in WT recipient mice that received either donor LSK⁻ cell phenotype (Figure 3D, E). Furthermore, the recovery of immature and newly formed B cells was less from Il-17ra^−/− mice compared to WT recipients (Figure 3F, G). These results suggest that IL-17 derived from LSK⁻ cells acts extrinsically on other cell types to promote development and differentiation of this progenitor population into B cells in response to P. yoelii 17X infection.

IL-17 promotes CXCL12 production by splenic stromal cells

Fibroblast-like stromal cells play a significant role in the development of lymphoid progenitor cells into B cells in the bone marrow (26, 27), and both immortalized bone marrow-derived OP9 stromal cells and primary splenic stromal cells can support the development of LSK⁻ cells into B cells (Supplemental Figure 3). Based on this information, it was of interest to determine if the addition of exogenous IL-17 could enhance the output of B cells in a co-culture assay. Since OP9 stromal cells lack IL-17RA expression (Supplemental Figure 3), primary stromal cells harvested from the spleen of naïve mice are cultured to generate a stable cell line. First, the expression of IL-17RA on the in vitro cultured stromal cell line was confirmed (Supplemental Figure 3). These stromal cells are then co-cultured with LSK⁻ cells isolated from naïve spleens in the presence or absence of IL-17. While no increase in B cell numbers occurs after one week of incubation with IL-17, a significant increase ensues after two weeks (Figure 4A). To corroborate the findings from the in vivo transfer experiments WT LSK⁻ cells are co-cultured with splenic stromal cells derived from WT or Il-17ra^−/− mice in the presence or absence of IL-17. In the absence of IL-17R signaling splenic stromal cells are not as efficient as WT stromal cells in supporting LSK⁻ cell differentiation into B cells. The addition of IL-17 only enhances B cell output in WT stromal cell cultures (Figure 4B), confirming that stromal cells can directly respond to IL-17 to promote B cell development from LSK⁻ cells.

Figure 4. IL-17 supports LSK⁻ cell differentiation into B cells in vitro.

A. Total number of B cells generated from co-cultures of splenic stromal cells and splenic LSK⁻ cells from naïve mice in media supplemented with or without IL-17 (100 ng/ml). The analysis is done at 7 or 14 days post-culture. *p <0.05 (two-way ANOVA). B. Total number of B cells recovered from co-culture of splenic LSK⁻ cells with splenic stromal cells isolated from WT (black bar) or Il-17ra^−/− mice (grey bar) in media supplemented with or without IL-17 at day 14 post-culture. *p <0.05, ***p <0.001 (two-way ANOVA). C. Splenic stromal cells are cultured in the presence (black bar) or absence (white bar) of IL-17 (100 ng/ml) for 14 days and mRNA expression of genes from splenic stromal cells are analyzed by quantitative RT-PCR and expressed relative to the expression of b-actin. *p <0.0005 (multiple t-test). D. CXCL12 production by splenic stromal cells is analyzed by ELISA, following incubation of stromal cells with or without IL-17 at days 7 or 14 post-culture. *p =0.0005 (two-way ANOVA). E. Representative histogram plots and scatter-dot-plots indicating the median fluorescence intensity of CXCR4 expression by splenic stromal cells at days 0 (solid line), 7 (dotted line), and 11 (dashed line) following P. yoelii 17X infection; FMO represented by grey filled histogram F. Total number of B cells recovered from the co-culture of splenic LSK⁻ cells and splenic stromal cells harvested from naïve mice in the presence or absence of IL-17 (100 ng/ml) and α-CXCL12 (10 μg/ml) at day 14 post-culture. **p <0.005, ***p =0.0005 (one way ANOVA). Data are representative of two independent experiments. Error bars (A–F) S.E.M.

These results indicate that IL-17 stimulation of stromal cells may promote the production of growth factors necessary for LSK⁻ cell differentiation. Therefore, splenic stromal cells incubated with or without IL-17 for 14 days were evaluated for expression of growth factors involved in B-cell development in the bone marrow and spleen (28–31) by quantitative RT-PCR to test this idea. Detection of transcripts for Il-6, Cxcl12, and Scf occurred in the stromal cells cultured in media alone. Following incubation with IL-17, only Cxcl12 saw an increase in gene expression. Also, transcripts for Baff, which are not detectable under media only conditions, are induced in response to IL-17 (Figure 4C).

Disruption of CXC12-CXCR4 interactions leads to impaired B cell differentiation by LSK⁻ cells

CXCL12 produced by stromal cells in the bone marrow plays a critical role in B cell lymphopoiesis (32–34). In response to CXCL12-abundant reticular (CAR) cells, the earliest B cell precursors migrate towards these cells to gain access to growth factors (35). Furthermore, CXCL12 plays an additional role in the retention of developing B cells (Pro-B cells, Pre-B cells) in the bone marrow, which allows them to complete their development into immature B cells (36, 37). Therefore, splenic stromal cells are cultured in the presence or absence of IL-17 for 7 or 14 days to determine if IL-17 can stimulate CXCL12 secretion by these cells. Splenic stromal cells are capable of secreting CXCL12 under media only conditions, and no increase in CXCL12 production happens at day 7 after addition of exogenous IL-17. However, by day 14 a measurable effect of IL-17 on CXCL12 production by the stromal cells is detected, resulting in a significant increase in production of this chemokine (Figure 4D).

CXCR4 is the receptor for CXCL12 and splenic LSK⁻ cells express this chemokine receptor (Figure 4E). Moreover, expression of CXCR4 on LSK⁻ cells is upregulated after P. yoelii infection (Figure 4E), suggesting that splenic LSK⁻ cells can respond to this chemokine produced by stromal cells. To test the importance of CXCL12 in the differentiation of LSK⁻ cells into B cells anti-CXCL12 blocking Ab is added to the co-culture assay. The addition of anti-CXCL12 reduces the positive impact that IL-17 has on B cell differentiation in this assay (Figure 4F). Collectively, these data indicate that CXCL12-CXCR4 interactions are important for LSK⁻ cell differentiation into B cells, a process that is enhanced indirectly by IL-17.

Loss of IL-17R signaling alters CXCL12 expression by splenic stromal cells in vivo

In lymph nodes, two particular types of stromal cells - fibroblast-reticular cells (FRCs; ER-TR7⁺gp38⁺CD31⁻) and integrin α7 pericytes (IAPs; ER-TR7⁺gp38⁻CD31⁻ITGA7⁺) have been shown to express the chemokine CXCL12 (2, 38). Although the characterization of stromal cells in lymph nodes is well defined, their architecture and organization in the spleen are not as well understood. However, a recent study showed CXCL12–producing stromal cells in the spleen are a subset of SCF-producing Tc21⁺ stromal cells that localize in the red pulp and expand during EMH, forming a niche for HSPCs in the spleen (6). The splenic stromal cell line utilized here in these studies has an FRC-like phenotype (CD45⁻Lin⁻gp38⁺CD31⁻) (Supplemental Figure 4), indicating that FRCs may be the population of stromal cells responsible for supporting LSK⁻ cell differentiation into B cells in vivo. Examination of stromal cells (CD45⁻Lin⁻) in the spleen of naïve and P. yoelii 17X infected WT and Il-17ra^−/− mice indicates that stromal cells expand after infection, but no difference in total numbers occurs between these mice (Supplemental Figure 4). A closer examination of different stromal cell populations in the spleen by flow cytometry, based on the endothelial cell marker CD31 and podoplanin (gp38) expression, reveals that FRCs (gp38⁺CD31⁻) are present at a lower frequency in naïve Il-17ra^−/− mice, compared to their naïve WT counterparts (Supplemental Figure 4). However, this difference did not translate to cellularity (Supplemental Figure 4). By day 9 p.i. there is a similar frequency of FRCs in the spleen of WT and Il-17ra^−/− mice. Furthermore, FRC (gp38⁺CD31⁻) and follicular dendritic cell (FDC; gp38⁻CD31⁻CD35⁺) populations expand equally after infection in WT and Il-17ra^−/− mice, while no expansion of blood endothelial cells ensues (BECs; gp38⁻CD31⁺) (Supplemental Figure 4).

Previous data suggest that IL-17 has a potential role in regulating protein production from stromal cells (11, 13–16). Therefore, gene expression for a number of hematopoietic growth factors and additional proteins that are produced by stromal cell populations in the LN (11, 14–16) were examined in stromal cells from P. yoelii infected WT and Il-17ra^−/− mice. Expression of Il-6 is significantly decreased in splenic stromal cells in the absence of IL-17R signaling, while Baff expression is unaltered (Figure 5A). Interestingly, while a significant reduction in Cxcl12 expression occurs in Il-17ra^−/− mice compared to WT mice Cxcl13 expression is increased in Il-17ra^−/− mice. FDCs are the primary producers of Cxcl13 within the B cell follicle, and this chemokine binds CXCR5, which is expressed by follicular B cells and follicular helper T cells to promote homing to the follicle in SLOs (39). These results indicate that IL-17R signaling can influence gene expression in stromal cells, and suggests that IL-17 can regulate the expression of chemokines that are necessary for the movement of hematopoietic cells, which may also include LSK⁻ cells, in the spleen.

Figure 5. Splenic stromal cells show population changes in Il-17ra^−/− mice after P. yoelii 17X infection.

A. RNA expression of Il-6, Baff, Cxcl12, and Cxcl13 by stromal cells (CD45⁻CD31⁻) from WT and Il-17ra^−/− mice at day 9 after P. yoelii 17X infection. *p <0.005 (multiple t-test). B. Representative flow-plots showing expression of IL-17RA by splenic stromal cells (CD45⁻Lin⁻) from naive mice (solid line), and mice infected with P. yoelii 17X at day 9 p.i. (dashed line); FMO control as grey filled histogram. C. RNA expression of Il17ra and Il17rc by FRCs (black), FDCs (grey), triple negative (TNs; gp38⁻CD31⁻CD21/35⁻) stromal cells (white), and endothelial cells (ECs; CD31⁺; checkered) from WT mice at day 9 following P. yoelii infection. *p <0.005, **p <0.0001 (two-way ANOVA). RNA expression of genes for D. growth factors and chemokines, E. functional proteins, and F. adhesion proteins by FRCs from WT and Il-17ra^−/− mice at day 9 following P. yoelii infection, *p <0.05, **p <0.001 (multiple t-test). G. Immunofluorescence analysis of spleen cross-sections from WT and Il-17ra^−/− mice infected with P. yoelii 17X for 9 days. ER-TR7 (Blue) labeling identifies mesenchymal stromal cells; podoplanin (Red) marks fibroblast-like stromal cells (FRCs) and CXCL12 is stained in green. Magnification 100X.

Upregulation of IL-17RA expression on splenic stromal cells ensues following P. yoelii 17X infection at day 9 p.i. (Figure 5B). While the expression of RNA for both IL-17R subunits occurs in various stromal cell populations, including FRCs, FDCs and triple negative stromal cells (TNs) that are comprised primarily of IAPs, FRCs show the highest level of expression for both subunits at the transcript level (Figure 5C).

Since FRCs are the dominant population found in the cultured stromal cell line (Supplemental Figure 4), and because they can produce CXCL12 in response to IL-17 stimulation, gene expression by FRCs isolated from infected WT and Il-17ra^−/− mice was examined. Amongst growth factors that have known roles in facilitating lymphopoiesis, a reduction in Cxcl12, Flt3l, Il-6, Scf, and Baff expression occurs in Il-17ra^−/− FRCs with the expression of Cxcl12, Flt3l, and Baff showing a significant decrease in the absence of IL-17 receptor signaling (Figure 5D). Amongst other growth factors evaluated, G-csf and Gm-csf, which are required for differentiation of neutrophils and monocytes respectfully, have reduced expression in Il-17ra^−/− FRCs. Analysis of FRC specific gene expression indicates that a significant decrease in transcripts for several genes including Cox1, Cox2, and Mmp9 occurs in the absence of IL-17R signaling (Figure 5E). To what degree if any a reduction in expression of these genes affects the ability of these reticular stromal cells to support lymphopoiesis is unclear at this time. Since contact-dependent communication is essential for hematopoietic development, particularly for the production of growth factors like CXCL12 (40, 41), gene expression for proteins that facilitate cell-to-cell contact were explored further. In particular, a reduction in RNA expression for Vcam-1 and gp38 occurs in Il-17ra^−/− FRCs (Figure 5F), suggesting a possible decline in an interaction of cells, including LSK⁻ cells, with FRCs.

Splenic sections from WT and Il-17ra^−/− mice were examined by immunofluorescence to determine if the loss of IL-17R signaling affects CXCL12 production by FRCs after infection. While no obvious differences in stromal cell architecture is apparent based on staining for the fibroblast marker ER-TR7, the use of podoplanin (gp38) to identify FRCs reveals a more diffuse staining pattern for this marker in the spleens of infected Il-17ra^−/− mice (Figure 5G). This finding correlates with the downregulation of gp38 gene expression seen in these cells. Also, staining for CXCL12 in WT sections overlaps primarily with that for podoplanin along the border of B-cell follicles, indicating that podoplanin⁺ cells are the major source of this chemokine in the spleen after infection. On the other hand, little to no CXCL12 staining is apparent in the splenic sections from Il-17ra^−/− mice, indicating that IL-17 can directly influence production of this chemokine by FRCs after infection. Overall this data suggests that following acute P. yoelii infection, IL-17 does not impact the expansion of splenic stromal cell populations, but instead modulates expression of chemoattractants, growth factors and adhesion proteins by stromal cells, which may serve to influence LSK⁻ cell differentiation into B cells.

LSK⁻ cells upregulate key adhesion molecules that facilitate tethering after infection

Chemokines are a major driving force behind the migration of hematopoietic cells and their development. Splenic LSK⁻ cells from naïve mice display robust expression of CXCR5, with a small proportion of the cells expressing CXCR4 (Figure 6A). However, after P. yoelii infection, LSK⁻ cells downregulate CXCR5 and reciprocally upregulate CXCR4 (Figure 6A–C). Although CXCR5⁺LSK⁻ cells expand after P. yoelii 17X infection (Figure 6D), CXCR4-expressing LSK⁻ cells show a greater than 100-fold expansion in the spleen (Figure 6E). These results suggest that a distinct migration of LSK⁻ cells from a CXCR5-sensitive niche to a CXCL12-abundant niche governed by CXCR4 occurs after infection.

Figure 6. Upregulation of CXCR4 and adhesion molecule expression by LSK⁻ cells after infection.

A. Representative flow plots showing expression of CXCR4 and CXCR5 on splenic LSK⁻ cells from naïve mice and mice infected with P. yoelii 17X at day 7 p.i. The percentage of B. CXCR5 and C. CXCR4 expressing LSK⁻ cells, and the total number of D. CXCR4⁺ and E. CXCR5⁺ LSK⁻ cells from the spleen of WT mice at days 0 and 7 after P. yoelii 17X infection. F. Representative flow-plots showing expression of CXCR4 and CD49d by LSK⁻ cells and G. the percentage of CD49d-expressing LSK⁻ cells from mice infected with P. yoelii 17X at days 0 and 7 p.i. The total number of H. CD49d⁺ I. CXCR4⁺ and J. CXCR5⁺ LSK⁻ cells from the spleens of WT (black dots) and Il-17ra^−/− mice (grey dots) at days 0, 7 and 17 following P. yoelii 17X infection. K. RNA expression of Itga4, Itgb1, Clec2, and Galectin8 by splenic LSK⁻ cells from naïve mice (white bar) and mice infected with P. yoelii 17X (black) at day 7 p.i. (day 7) *p <0.05, **p <0.001, ***p <0.0001 (multiple t-test). Data are representative of two independent experiments, with n=3/4 mice/experiment. Error bars (B–E, G–J) S.E.M.

VCAM-1 expression is a characteristic feature of reticular cells, and this adhesion molecule facilitates interaction with cells expressing integrin α4β1 (CD49d or VLA-4) (42). Interestingly, CXCR4⁺ LSK⁻ cells from naïve mice display integrin α4β1 expression on their surface (Figure 6F). Furthermore, an increase in CXCR4 expression by LSK⁻ cells correlates with an up-regulation of α4β1 expression following P. yoelii infection. (Figure 6F, G). However, IL-17R signaling does not have any impact on α4β1, CXCR4 or CXCR5 expression by LSK⁻ cells, as a similar number of LSK⁻ cells express these markers in WT and Il-17ra^−/− mice before and after infection (Figure 6H–J).

Recent studies have indicated that gp38 facilitates contact between FRCs and dendritic cells through interaction with Clec2 (2, 43). Additionally, Galectin-8 can also interact with gp38 (44). Based on this information the expression of transcripts for these two genes, as well as Itga4 and Itgb1, the genes for integrin α4β1, was confirmed on splenic LSK⁻ cells from naïve mice. However, a downregulation of Clec2 expression and a reciprocal upregulation of Galectin-8, Itga4, and Itgb1 expression occur after the infection (Figure 6K). This result indicates that Clec2 downregulation could happen upon interaction with gp38, or that infection with P. yoelii modulates expression of these ligands on FRCs, favoring Galectin-8 over Clec2 expression. Co-expression of CXCR4, Galectin-8, and α4β1 by LSK⁻ cells, as well as the findings that FRCs express CXCL12, gp38 and VCAM-1 after infection support the idea that FRCs serve as a likely candidate for interacting with and tethering these progenitor cells to them to promote their maturation in the spleen. Moreover, these data indicate that the absence of IL-17–mediated signaling results in the downregulation of key surface proteins on FRCs that could lead to an inhibition of cell-to-cell communication, thereby downregulating expression of chemokines and growth factors required for B-lymphocyte development.

Discussion

Several studies over the last two decades have documented the effects of infection and inflammation on hematopoiesis (45); particularly their role in favoring myelopoiesis and granulopoiesis over the production of lymphocytes (45–48). Plasmodium infection in mice is an example of a disease that induces such changes in hematopoiesis. Erythropoiesis is elicited in the spleen following Plasmodium infection to partially compensate for dys-erythropoiesis in the bone marrow (49, 50). Despite several reports indicating the noticeable effect of Plasmodium infection on EMH (1, 4, 18–20), the role of splenic stromal cells in the mobilization or differentiation of HSPCs has remained largely unexplored. We have previously shown that P. yoelii infection results in the expansion of a lymphoid LSK⁻ progenitor population in the spleen that differentiates into mature B cells that are activated in response to infection and can form a proportion of the Plasmodium-specific memory B cell and long-lived plasma cell pools (1). Herein we have extended these initial findings by showing that LSK⁻ cells produce the inflammatory cytokine IL-17 to promote CXCL12 production by splenic stromal cells. This chemoattractant subsequently supported the development of LSK⁻ cells into B cells, replicating the role that this chemokine plays in the development of progenitor cells into immature B cells in the bone marrow (32, 34, 35, 48, 51–54).

IL-17 plays a minimal role in attracting granulocytes to the spleen after P. yoelii infection, as evident from the similar number of neutrophils recruited to the spleen in WT and Il-17ra^−/− mice. Instead, the most prominent phenotype that emerges in infected Il-17ra^−/− mice involves the humoral response. Specifically, a significant decrease in parasite-specific IgG occurs during the resolution phase of the acute infection resulting in a higher parasite burden that peaks later and remains elevated longer in Il-17ra^−/− mice. One explanation for this phenotype is that LSK⁻ cell differentiation is impaired, resulting in a decline in progeny B cell numbers. Support for this idea comes from the finding that fewer immature and newly formed B cells are present in the spleen of Il-17ra^−/− mice after infection. Alternatively, the absence of IL-17R signaling could alter the activity of neutrophils recruited to the spleen after infection. A recent study has identified that IL-17 stimulated prostaglandin E2 production by stromal cells recruits BAFF-producing neutrophils to support B cell activation and survival (55). As we observed a decrease in expression of Cox2, which promotes prostaglandin synthesis, by FRCs from Il-17ra^−/− mice after infection, this could result in a decline in BAFF production by recruited neutrophils, leading to a subsequent decline in B cell numbers, specifically activated B cells undergoing plasmablast differentiation.

LSK⁻ cells are the major producer of IL-17 after Plasmodium infection. Therefore, we speculate that the phenotype observed in Il-17ra^−/− mice after P. yoelii infection is mainly due to the lack of IL-17 production by this cell type. This conclusion is based on the in vitro and in vivo data shown here that indicates that LSK⁻ cell-derived IL-17 promotes the differentiation of this lymphoid progenitor into B cells. However, we cannot rule out that localized production of IL-17 by the small number of other splenic hematopoietic cells that produce this cytokine after infection play a larger or supporting role in the development of the humoral response against Plasmodium.

As to how Il-17ra^−/− mice resolve the infection, previous reports have suggested that CLPs start repopulating the BM by day 11 after Plasmodium infection, and by the third week post-infection, CLPs are restored to their original cellularity (19). A restoration in normal B-cell lymphopoiesis in the bone marrow would alleviate the need for extramedullary B-cell lymphopoiesis and potentially a requirement for IL-17 in the spleen to support this process. Return to homeostatic lymphopoiesis may explain how in spite of a brief period of higher parasite burden, Il-17ra^−/− mice resolve the infection similar to WT mice. Alternatively, it is clear that germinal centers can form in Il-17ra^−/− mice, and therefore the emergence of higher affinity Abs, although at lower quantities, is sufficient to allow resolution of infection in these mice.

Transfer of LSK⁻ cells into recipient Il-17ra^−/− mice results in reduced expansion and differentiation of these progenitor cells into B cells following P. yoelii infection. The absence of IL-17RA on LSK⁻ cells shows a minimal effect on their ability to differentiate into B cells, indicating IL-17R mediated signaling is mostly important on neighboring cells. Although the potential contribution of various other IL-17R expressing cells cannot be ruled out; expression and robust up-regulation of IL-17RA on stromal cells after P. yoelii 17X infection, coupled with their ability to act as feeder cells for HSPC differentiation makes them a strong candidate for contributing to LSK⁻ cell differentiation. The impact of IL-17 on stromal cells, particularly in development and differentiation of B cells has been explored in many instances of infection (14), autoimmunity (12, 13, 15, 16) and tumor progression (56, 57). Various studies have indicated the pluripotent role of IL-17 in the induction of several chemokines and growth factors that play crucial parts in B cell development, suggesting an indirect relationship between IL-17 and B lymphopoiesis. On the other hand, there are reports that IL-17R-mediated signaling downregulates expression of CXCR4 on follicular T helper cells thereby triggering their migration away from the T-cell zone, to the T cell-B cell border, facilitating germinal center development (58). IL-17-mediated germinal center development is a major contributing factor to the production of autoantibodies in BXD2 mice (58–61). Interestingly, a significant decline in GC B cells numbers occurred in Il-17ra^−/− mice at day 36 p.i., which leads to a significant decrease in plasmablast and memory B cell numbers. These latter cell types are primarily derived from the germinal center reaction at this time. While this result suggests that IL-17 may impact the germinal center response, as seen in the BXD2 mice (58–61), its impact seems to be minimal as the difference in parasite-specific IgG diminishes over time. Also, no relapse in parasitemia occurs in Il-17ra^−/− mice at this time. Nor is a difference in protection noted when WT and Il-17ra^−/− mice are re-challenged with P. yoelii (data not shown). Future work is needed to determine the impact of IL-17 on the germinal center response in this model. Overall, these findings show a complicated relationship between IL-17 and B cells that depends on cross-talk among several factors.

In recent years, several studies have suggested an association of the pro-inflammatory cytokine IL-17 with lymphoid tissue development (11–16). IL-17 was shown to induce stromal cells to produce CXCL12 (14–16), and this chemokine was subsequently shown to be important for the development of tertiary lymphoid tissue (11, 12, 14). Also, CXCL12-producing stromal cells attract HSPCs to the stroma in both mouse (6) and human (62) spleens under conditions of EMH. CXCL12 also plays a crucial role in the progression of B cell progenitors through stages of development in the bone marrow. Expression of CXCL12 by IL-17–stimulated stromal cells in the spleen might indicate its potential role in supporting de novo B-cell development in this SLO. It is important to note that the absence of IL-17R signaling did not impede expansion of LSK⁻ cells in the spleen. Nor did it completely prevent differentiation of LSK⁻ cells into B cells in the adoptive transfer experiments, indicating redundancy exists in the components that contribute to the formation of B cells from this extramedullary B-cell lymphopoiesis pathway.

Even though our in vitro and in vivo data implicate FRCs (podoplanin⁺) as the splenic stromal cell population that directly responds to IL-17 further studies are required to determine if this is indeed the stromal cell population that LSK⁻ cells directly interact with in vivo to facilitate their differentiation into B cells. Furthermore, while this study has revealed a link between IL-17, and upregulation of CXCL12 and adhesion molecule expression by splenic stromal cells, additional factors that support B cell development from these progenitor cells remain to be defined. Our previous in vitro studies, suggest that B cell development from splenic LSK⁻ cells occur independently of Flt3L and IL-7 (1), and through additional in vitro studies BAFF was shown to have minimal to no impact on B cell development in these assays (data not shown). Overall, this report indicates a unique role that IL-17 plays in guiding splenic stromal cells to create a microenvironment fitting to support extramedullary lymphopoiesis during Plasmodium yoelii infection. Future work is also needed to verify if inflammation driven by pathogens that induce Th2 or Th17 responses cause a similar extramedullary B-cell lymphopoiesis process, and to determine if a similar pathway exists in humans.