TISSUE-SPECIFIC STEM CELLS

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1 TISSUE-SPECIFIC STEM CELLS Direct Toll-Like Receptor-Mediated Stimulation of Hematopoietic Stem and Progenitor Cells Occurs In Vivo and Promotes Differentiation Toward Macrophages JAVIER MEGÍAS, a ALBERTO YÁÑEZ, b SILVIA MORIANO, b JOSÉ-ENRIQUE O CONNOR, a DANIEL GOZALBO, b MARÍA-LUISA GIL b a Laboratorio de Citomica, Unidad Mixta CIPF-UVEG, Centro de Investigacion Príncipe Felipe, Valencia, Spain; b Departamento de Microbiología y Ecología, Universitat de València, Burjassot, Spain Key Words. Toll-like receptors MyD88 Hematopoietic stem and progenitor cells Macrophages ABSTRACT As Toll-like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells (HSPCs), they may play a role in hematopoiesis in response to pathogens during infection. We show here that TLR2, TLR4, and TLR9 agonists (tripalmitoyl-s-glyceryl-l-cys-ser-(lys)4 [Pam3CSK4], lipopolysaccharide [LPS], and CpG oligodeoxynucleotide [ODN]) induce the in vitro differentiation of purified murine lineage negative cells (Lin 2 ) as well as HSPCs (identified as Lin 2 c-kit 1 Sca-1 1 IL-7Ra 2 [LKS] cells) toward macrophages (Mph), through a myeloid differentiation factor 88 (MyD88)-dependent pathway. In order to investigate the possible direct interaction of soluble microorganism-associated molecular patterns and TLRs on HSPCs in vivo, we designed a new experimental approach: purified Lin 2 and LKS cells from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into TLR2 2/2, Disclosure of potential conflicts of interest is found at the end of this article. TLR4 2/2, or MyD88 2/2 mice (CD45.2 alloantigen), which were then injected with soluble TLR ligands (Pam3CSK4, LPS, or ODN, respectively). As recipient mouse cells do not recognize the TLR ligands injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted cells were detected in the spleen and bone marrow of recipient mice, and in response to soluble TLR ligands, cells differentiated preferentially to Mph. These results show, for the first time, that HSPCs may be directly stimulated by TLR agonists in vivo, and that the engagement of these receptors induces differentiation toward Mph. Therefore, HSPCs may sense pathogen or pathogen-derived products directly during infection, inducing a rapid generation of cells of the innate immune system. STEM CELLS 2012;30: INTRODUCTION Toll-like receptors (TLRs) constitute a family of pattern-recognition receptors (PRRs) that recognize molecular signatures of microbial pathogens and function as sensors for infection. TLRs induce the activation of innate immune responses as well as the subsequent development of adaptive immune responses, as TLR signaling is an important component of dendritic cell (DC) maturation and activation. TLRs are type I membrane proteins characterized by an ectodomain responsible for recognition of microorganism-associated molecular patterns (MAMPs) and a cytoplasmic domain homologous to the cytoplasmic region of the interleukin (IL)-1 receptor (TIR-domain). Signal transduction starts with the recruitment of a set of intracellular TIR-domain-containing adaptors (myeloid differentiation factor 88 [MyD88], Toll-interleukin 1 receptor (TIR) domain containing adaptor protein [TIRAP], TIR-domaincontaining adapter-inducing interferon-b [TRIF] and TIR domain-containing adaptor-inducing IFN-b (TRIF)-related adaptor molecule [TRAM]) that interact with the cytoplasmic TIR domain of the TLRs. MyD88 is the universal adaptor molecule, shared by all TLRs except TLR3, that triggers expression of inflammatory cytokine and chemokine genes [1]. Recent findings suggest that TLRs may play a role in hematopoiesis during infection [2]. Murine hematopoietic stem cells (HSCs) and their progeny express TLRs, and upon in vitro exposure to soluble TLR2 and TLR4 ligands are stimulated to enter cell cycle and acquire lineage markers [3]. Signaling through TLR7/8 induces the differentiation of human bone marrow CD34 þ progenitor cells along the myeloid lineage [4], and the TLR1/2 agonist tripalmitoyl-s-glyceryl-l-cys-ser-(lys) 4 [Pam3CSK4] instructs commitment of human HSCs to a myeloid cell fate [5]. In addition, this newly described mechanism, which may represent a potential means for pathogen products to signal the rapid generation of innate immune cells, has been explored in some in vivo infectious models; production of DCs from murine lymphoid Author contributions: J.M.: performed experiments, interpreted data, and wrote the manuscript; A.Y.: designed research and interpreted data; S.M.: performed experiments; J.E.O.: interpreted data; D.G. and M.L.G.: designed research, interpreted data, and wrote the manuscript. Correspondence: María-Luisa Gil, Ph.D., Departamento de Microbiología y Ecología, Universitat de València, Edificio de Investigacion, C/ Dr. Moliner, 50, Burjassot, Valencia Spain. Telephone: ; Fax: ; m.luisa. gil@uv.es Received February 17, 2012; accepted for publication April 3, 2012; first published online in STEM CELLS EXPRESS April 17, VC AlphaMed Press /2012/$30.00/0 doi: /stem.1110 STEM CELLS 2012;30:

2 Megías, Ya~nez, Moriano et al precursors during herpes infection is TLR9 dependent [6], expansion of HSCs during vaccinia virus infection is MyD88 dependent [7], TLR-mediated signals play an essential role in monocyte expansion during systemic Listeria monocytogenes infection [8], and hematopoietic stem and progenitor cells (HSPCs) increase markedly following Mycobacterium tuberculosis infection in a TLR2/MyD88-dependent manner [9]. We have previously demonstrated that the fungal pathogen Candida albicans induces the proliferation of HSPCs and their differentiation toward the myeloid lineage in vitro. This response requires signaling through TLR2/MyD88 and gives rise to functional phagocytes that are able to internalize yeasts and secrete proinflammatory cytokines [10, 11]. Moreover, in an experimental model of invasive candidiasis in mice, we have shown that Lin c-kit þ Sca-1 þ IL-7Ra (LKS) cells are rapidly expanded and new monocyte-derived DCs (modcs) and inflammatory macrophages (Mph) are generated in the spleen in a TLR2-dependent manner [12]. All these results indicate that TLRs control hematopoiesis during infection and raise the question of whether this is due to the direct recognition of pathogens by progenitor cells and/ or to secondary effects due to pathophysiological changes during infection. To deal with this issue, we have performed a new experimental approach to investigate whether direct stimulation of HSPCs via TLRs occurs in vivo. Differentiation of transplanted wild-type CD45.1 HSPCs in TLR2 /, MyD88 /, or TLR4 / CD45.2 mice was determined following in vivo challenge with TLR ligands. Our results show, for the first time, that HSPCs are directly stimulated in vivo via TLRs. The in vivo differentiation of HSPCs in response to TLR2, TLR4, or TLR9 agonists is similar to the in vitro results, as in both cases, differentiation toward Mph is induced. These results indicate that HSPCs can sense pathogen products directly during infection to rapidly replenish the innate immune compartment and generate more of the mature cells needed to deal with the pathogen. MATERIALS AND METHODS Mice TLR2 /, MyD88 /, and TLR4 / mice (C57BL/6 background) provided by Dr. Shizuo Akira (Osaka University, Osaka, Japan) were bred and maintained at the animal production service facilities (University of Valencia); wild-type C57BL/6 mice (Harlan Iberica, Barcelona, Spain; were used as controls; wild-type CD45.1-positive allotype mice (B6.SJL- Ptprc a Pepc b /BoyCrl strain) were used as lineage negative (Lin ) progenitor cells and LKS cells transplant donors (Charles River Laboratories, Wilmington, MA; Mice of both sexes between 8 and 12 weeks old were used, and all assays involving mice were approved by the Institutional Animal Care and Use Committee (A , Universitat de València). Purification of Lin 2 and LKS Cells Lin and LKS cells were purified as previously described [10, 11]. Briefly, murine bone marrow was obtained by flushing the femurs and tibias; cells were depleted of lineage-positive cells by immunomagnetic cell sorting using MicroBeads (Miltenyi Biotec, Madrid, Spain; bone marrow cells were labeled with a cocktail of antibodies against a panel of lineage antigens (CD5, CD45R [B220], CD11b, Gr-1 [Ly-6G/C], 7-4, and Ter-119), and then cells were purified by negative selection according to the manufacturer s instructions (Lin progenitor cells). Purity of the sorted cells was assessed by labeling with anti-lin cocktail and by flow cytometry analysis, and no Lin þ cells were detected. LKS cells were purified from Lin cells by immunomagnetic positive selection using anti-sca-1 MicroBeads (Miltenyi Biotec). Purity of the sorted cells was assessed by labeling with phycoerythrin (PE)-labeled anti-c-kit monoclonal antibody (clone 3C1; Miltenyi Biotec) and PE-Cy7-labeled anti- IL-7Ra monoclonal antibody (clone SB/119; BD Pharmingen, San Jose, CA; A homogeneous LKS cell population was purified. In Vitro Assays Purified cells were immediately cultured in complete cell culture medium (RPMI 1640 medium supplemented with 2 mm L-glutamine, 50 lm 2-mercaptoethanol, 5% heat-inactivated fetal bovine serum (FBS), and 1% penicillin-streptomycin stock solution; Gibco, Barcelona, Spain; containing three cytokines: stem cell factor (SCF, 20 ng/ml), Flt-3 ligand (FL, 100 ng/ml) (Peprotech, Rocky Hill, NJ; peprotech.com), and IL-7 (10 ng/ml) (MBL, Woburn, MA; Lin progenitor cells were cultured in flatbottomed 24-well plates at a density of 400,000 cells per well in 0.5 ml, and LKS cells were cultured in round-bottomed 96-well plates at a density of 50,000 cells per well in 0.2 ml. Cells were challenged for 7 days with Pam3CSK4 (1 lg/ml), ultrapure Escherichia coli lipopolysaccharide (LPS) (1 lg/ml) or type A CpG oligodeoxynucleotide (ODN 1585) (10 lg/ml) (InvivoGen, San Diego, CA; Control cultures were performed without stimuli. At day 4, the culture medium was refreshed including both the cytokines and the stimuli. At day 7, cells were collected and analyzed by flow cytometry (see below). In Vivo Transplantation of CD45.1 Cells Murine bone marrow was extracted from CD45.1 mice and Lin progenitor cells or LKS cells were purified as described above. Approximately Lin cells in 100 ll of phosphate buffered saline (PBS) (purified from four mice) or LKS cells in 100 ll of PBS (purified from eight mice) were intravenously injected into one CD45.2 TLR2 /, one CD45.2 TLR4 /, or one CD45.2 MyD88 / knockout mouse. The transplanted mice were then injected with TLR ligands for 3 days. Each stimulated mouse received, by intravenous administration, three doses of TLR ligand at days 0, 1, and 2 after transplantation. TLR2 / mice were injected with 100 lg Pam3CSK4 per dose, TLR4 / mice with 100 lg LPS per dose, and MyD88 / mice with 100 lg ODN per dose (Fig. 3A). Each assay was performed with six Lin or LKS transplanted mice per group (TLR2 /, TLR4 /, or MyD88 / ); three mice within each group were challenged with TLR ligands and three mice were used as controls. Detection and Characterization of CD45.1 Transplanted Cells Each transplanted mouse was killed at day 3, and the spleen and bone marrow from femurs and tibias were removed aseptically. Total spleen cells were obtained by collagenase D treatment of the organ as previously described [13, 14]. Erythrocytes were lysed in ammonium chloride buffer. Fc receptors were blocked with FcR blocking reagent (Miltenyi Biotec), and cells were depleted of CD45.2 cells by immunomagnetic cell sorting using biotinylated anti-cd45.2 antibody and anti-biotin magnetic MicroBeads (both from Miltenyi Biotec). Recovered cells were microscopically counted, labeled with various combinations of antibodies, and analyzed by flow cytometry (see below). Antibodies and Flow Cytometry Analyses The following antibodies used in flow cytometry analyses were purchased from Miltenyi Biotec: cocktail of biotinylated anti-lineage antigens (CD5, CD45R [B220], CD11b, Gr-1 [Ly-6G/C], 7-4, and Ter-119), fluorescein isothiocyanate (FITC)-labeled anti-sca- 1 (clone D7), PE-labeled anti-c-kit (clone 3C1), FITC-labeled anti-cd45.1 (clone A20), allophycocyanin (APC)-labeled anti- CD11c (clone N418), and APC-labeled anti-mpdca-1 (clone JF05-1C2.4.1). The following antibodies were from ebioscience

3 1488 TLRs Directly Activate HSPCs In Vivo (San Diego, CA; PE-labeled anti- CD11b (clone M1/70) and PE-Cy7-labeled anti-f4/80 (clone BM8), or from BD Pharmingen: PE-Cy7-labeled anti-il-7ra (clone SB/119) and peridinin chlorophyll protein (PerCP)-Cy5.5- labeled anti-ly6c (clone AL-21). Flow cytometry analyses were performed on a FACSCanto cytometer (BD Biosciences, and the data were analyzed with FACSDiva and FlowJo software. Statistical Analysis Statistical differences were determined using one-way analysis of variance (ANOVA) followed by Dunnett s test for multiple comparisons and two-tailed Student s test for dual comparisons. Data are expressed as mean 6 SD. Significance was accepted at *, p <.05 and **, p <.01 level. RESULTS In Vitro Differentiation of Lin 2 and LKS Cells Is Altered by Exposure to TLR Ligands and Biased to Macrophage Production We investigated the in vitro effect of Pam3CSK4 (which only activates TLR2), LPS (which only activates TLR4), and ODN (which only activates TLR9) on differentiation of Lin progenitor cells. Control unchallenged cells from C57BL/6 mice were cultured for 7 days in complete cell culture medium containing SCF, FL, and IL-7 to guarantee the survival of stem cells and common myeloid and lymphoid progenitors; in these conditions, progenitors differentiate to a mixed population of cells (Fig. 1A, medium, Fig. 1B, white bars). These differentiated cells did not express the CD19 lymphoid marker (data not shown), indicating that in these conditions, B cells were not produced. Analysis of the expression of CD11b and CD11c allowed the identification of three different cell populations: (a) roughly 25% of cells were CD11b CD11c þ and also expressed mpdca-1 (data not shown), therefore corresponding to plasmacytoid DCs (pdcs), (b) 30% of cells were CD11b þ CD11c þ containing pre-dcs or classic DCs (cdcs, 12% of cells CD11b þ CD11c þ Ly6C F4/80 ) and modcs (5% of cells CD11b high CD11c þ Ly6C þ F4/80 þ ) and (c) 17% of cells were CD11b þ CD11c containing 5% Ly6C high monocytes (Mc, CD11b þ CD11c Ly6C þ F4/80 ). When Lin progenitor cells were cultured in the same conditions but in the presence of different TLR ligands, the differentiation pattern was completely different (Fig. 1A, Pam3CSK4,LPS,andODNandFig.1B,coloredbars).Thegeneration of DCs, both pdcs and cdcs, decreased dramatically, whereas a new major population of Mph (CD11b þ CD11c Ly6C þ F4/80 þ ) appeared. Although the response to TLR2, TLR4, and TLR9 ligands was qualitatively quite similar, the generation of Mph in response to Pam3CSK4 was higher than to LPSorODN.Asexpected,Lin progenitor cells from TLR2 / mice did not respond to Pam3CSK4, Lin progenitor cells from TLR4 / mice did not respond to LPS, and Lin progenitor cells from MyD88 / mice did not respond to Pam3CSK4 or ODN (Fig. 1B). Interestingly, Lin progenitor cells from MyD88 / mice did not respond to LPS, indicating that in these progenitor cells, the response to LPS was completely dependent on MyD88, although TLR4 can also signal through TRIF. It should be noted that the differentiation of Lin progenitor cells from knockout mice in the absence of TLR ligands was similar to the differentiation of cells from control mice (Fig. 1B). As the assayed Lin population contains a variety of transitional intermediates including HSCs, myeloid, and lymphoid committed progenitors, we next investigated whether stem cells and uncommitted progenitors would also be responsive to TLR ligands. LKS cells were purified and cultured in the same conditions as described above for Lin cells in presence or absence of TLR ligands (Fig. 2). In the absence of TLR ligands (medium), the LKS population from control (Fig. 2A, 2B) and knockout mice (Fig. 2B) similarly differentiated to a mixed population of cells. However, analysis of the expression of different markers on the culture cells showed that LKS cells generated fewer pdcs (10%) and more cdcs (34%) and Mc (16%) than the Lin population. When LKS cells were cultured in the presence of TLR ligands, the generation of pdcs and cdcs decreased and a new population of Mph appeared. Again, the TLR2 ligand induced the strongest production of Mph, and all responses were completely dependent on MyD88. Overall, these results clearly indicate that TLR2, TLR4, and TLR9 signaling via MyD88 biases the in vitro differentiation of Lin and LKS cells toward the production of Mph. Transplantation and Distribution of CD45.1 Lin 2 and LKS Cells in TLR2 2/2, TLR4 2/2, and MyD88 2/2 CD45.2 Mice: Effect of TLR Ligands The observed in vitro effect of TLR signaling on HSPCs may be of biological relevance in vivo. However, direct in vivo interaction between microbial pathogens, or their ligands, and TLRs on the HSPCs is difficult to demonstrate as HSPCs could respond to other stimuli generated when mature immune cells detect microbial products via their TLRs. In order to investigate the possible direct interaction of soluble MAMPs with TLRs on HSPCs in vivo, we designed a new experimental approach (Fig. 3A). We purified HSPCs (Lin or LKS cells) from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) and transplanted these cells into TLR2 /,TLR4 /,ormyd88 / mice (CD45.2 alloantigen). We then injected the mice with one daily dose of Pam3CSK4, LPS, or ODN, respectively, for 3 days. Using this experimental approach, the recipient mouse cells do not recognize the TLR ligand injected, so there should be no interference by cytokines or soluble mediators secreted by recipient cells. After 3 days, bone marrow and spleen cells were enriched for CD45.1 cells by depletion of CD45.2 cells and analyzed by multicolor fluorescence and flow cytometry. Analysis of the expression of CD45.1 allowed the detection of transplanted cells (Fig. 3B); approximately 3.3% of the Lin cells and 1.2% of the LKS cells detected in the spleen and 0.9% of the Lin cells and 0.7% of the LKS cells detected in the bone marrow were donor derived. A significant increase in CD45.1 cells was detected in the spleen of TLR2 / mice transplanted with both Lin and LKS cells and in the bone marrow of TLR2 / mice transplanted with Lin cells following Pam3CSK4 challenge (Fig. 3C). These results indicate that TLR2 signaling resulting from direct detection of Pam3CSK4 by the transplanted Lin /LKS cells induces their proliferation and/or improves their survival in vivo. Although LPS induced an increase in the percentage of CD45.1 cells in the spleens of TLR4 / mice transplanted with either Lin or LKS cells, this increase was not statistically significant. Similarly, treatment of MyD88 / mice with the TLR9 ligand ODN did not trigger any increase in the frequency of transplanted CD45.1 cells. Taken together, our data show that CD45.1 cells can be detected in the spleen and in the bone marrow 3 days after the transplantation, and that in these locations they are responsive to TLR2 agonists. Transplanted CD45.1 Lin 2 and LKS Cells Respond to TLR Ligands and Are Directed to Produce Mph Next, we analyzed the expression of different markers on the CD45.1 cells in order to determine whether the transplanted cells differentiated (Figs. 4, 5). Most of the CD45.1 cells in

4 Megías, Ya~nez, Moriano et al Figure 1. In vitro differentiation of Lin cells in response to TLR ligands. (A): Lin progenitor cells from C57BL/6 mice were cultured without stimuli (medium) or with Pam3CSK4 (1 lg/ml), LPS (1 lg/ml), or ODN (10 lg/ml) for 7 days, labeled with antibodies, and analyzed by flow cytometry. Cells were gated as pdcs (CD11b CD11c þ mpdca þ ), cdcs (pre-cdcs or cdcs: CD11b þ CD11c þ Ly6C F4/80 ), modcs (CD11b high CD11c þ Ly6C þ F4/80 þ ), Mc (CD11b þ CD11c Ly6C þ F4/80 ), and Mph (CD11b þ CD11c Ly6C þ F4/80 þ ). The indicated percentages refer to total analyzed cells. Ly6C versus F4/80 plots are subgated from the CD11b versus CD11c plot. At least 20,000 events were analyzed in each sample. Results shown are from one representative of three independent experiments. (B): The figure shows the percentages of pdcs, cdcs, and Mph generated from Lin progenitor cells from C57BL/6, TLR2 /, TLR4 /, and MyD88 / mice in the presence or absence of TLR ligands, analyzed following the same schedule as in (A). Data represent means 6 SD, from three experiments. **, p <.01 with respect to cells incubated with medium alone (medium) within each mouse type. Abbreviations: cdcs, classic dendritic cells; Lin, lineage negative cells; LPS, lipopolysaccharide; Mc, monocytes; modcs, monocyte-derived dendritic cells; Mph, macrophages; MyD88, myeloid differentiation factor 88; CpG ODN, oligodeoxynucleotide; pdcs, plasmacytoid dendritic cells; TLR, toll-like receptors.

5 1490 TLRs Directly Activate HSPCs In Vivo Figure 2. In vitro differentiation of LKS cells in response to TLR ligands. (A): LKS cells from C57BL/6 mice were cultured without stimuli (medium) or with Pam3CSK4 (1 lg/ml), LPS (1 lg/ml), or ODN (10 lg/ml) for 7 days, labeled with antibodies, and analyzed by flow cytometry. Cells were gated as pdcs (CD11b CD11c þ mpdca þ ), cdcs (pre-cdcs or cdcs: CD11b þ CD11c þ Ly6C F4/80 ), modcs (CD11b high CD11c þ Ly6C þ F4/80 þ ), Mc (CD11b þ CD11c Ly6C þ F4/80 ), and Mph (CD11b þ CD11c Ly6C þ F4/80 þ ). The indicated percentages refer to total analyzed cells. Ly6C versus F4/80 plots are subgated from the CD11b versus CD11c plot. At least 20,000 events were analyzed in each sample. Results shown are from one representative of three independent experiments. (B): The figure shows the percentages of pdcs, cdcs, and Mph generated from LKS cells from C57BL/6, TLR2 /, TLR4 /, and MyD88 / mice in the presence or absence of TLR ligands, analyzed following the same schedule as in (A). Data represent means 6 SD, from three experiments. *, p <.05, **, p <.01 with respect to cells incubated with medium alone (medium) within each mouse type. Abbreviations: cdcs, classic dendritic cells; LKS, Lin c-kit þ Sca-1 þ IL-7Ra ; LPS, lipopolysaccharide; Mc, monocytes; modcs, monocyte-derived dendritic cells; Mph, macrophages; MyD88, myeloid differentiation factor 88; CpGODN, oligodeoxynucleotide; pdcs, plasmacytoid dendritic cells; TLR, toll-like receptors. unstimulated mice did not express markers of stem cells (roughly, 1% 2% cells were c-kit þ Sca-1 þ IL-7Ra when LKS cells were transplanted and less than 1% when Lin cells were transplanted), indicating that after 3 days in vivo, both Lin and LKS cells had differentiated toward more mature cells. The cells did not express CD19 and B220 lymphoid markers either (data not shown), indicating that in these conditions, lymphopoiesis is not promoted. Finally, we analyzed the expression of myeloid markers and found that transplanted cells had differentiated toward this lineage, as significant percentages of CD11b þ cells were detected. Some cells expressed markers of mature pdcs, cdcs, or Mc, and the potential differentiation of transplanted cells was quite similar in TLR2 /, TLR4 /, or MyD88 / mice (Figs. 4, 5,

6 Megías, Ya~nez, Moriano et al Figure 3. Transplantation of CD45.1 Lin and LKS cells into CD45.2 mice and detection of CD45.1 cells in spleen and bone marrow. (A): Schematic protocol of cell transplantation (as described in Materials and Methods). (B): Three days after transplantation, donor-derived CD45.1 cells were detected in the spleen and bone marrow of CD45.2 control mice (unchallenged with TLR ligands). Dot plots show FSC against CD45.1 expression of the purified spleen and bone marrow cells. Percentage of recovered CD45.1 cells is calculated as follows: total number of recovered cells 100/(total number of transplanted cells), where total number of recovered cells ¼ % of CD45.1 cells determined by flow cytometry total number of purified cells from spleen or bone marrow/100. Indicated percentages are the mean 6 SD of nine mice. (C): Percentages of recovered CD45.1 cells from the spleen and bone marrow of TLR2 /, TLR4 /, and MyD88 / knockout mice transplanted with CD45.1 Lin progenitor cells or LKS cells and stimulated daily with 100 lg/day of Pam3CSK4, LPS, or ODN, respectively, for 3 days. Data represent means 6 SD, from three experiments. **, p <.01 with respect to CD45.1 cells recovered from transplanted unstimulated control mice. Abbreviations: FSC, forward scatter; HSPCs, hematopoietic stem and progenitor cells; Lin, lineage negative cells; LKS, Lin c-kit þ Sca-1 þ IL- 7Ra ; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88; CpGODN, oligodeoxynucleotide; TLR, toll-like receptors. unstimulated). However, after the injection of Pam3CSK4, LPS, and ODN, into the TLR2 /, TLR4 /, and MyD88 / mice, respectively, we detected the generation of a significant percentage of CD45.1 Mph (CD11b þ CD11c Ly6C þ F4/ 80 þ, 10% 20% CD45.1-positive cells) from both Lin and LKS cells (Figs. 4, 5). In response to Pam3CSK4 (TLR2 / mice), an increase in total CD11b þ cells, as well as Mph, was detected, indicating that transplanted cells proliferate and differentiate toward Mph. However, in response to LPS (TLR4 / mice) and to ODN (MyD88 / mice), the number of CD11b cells was not increased (for LKS transplanted cells, Fig. 5) and the increase in Mph correlated with a decrease in Mc (statistically significant in most cases, Figs. 4, 5). This result suggests that LPS and ODN induce less proliferation than Pam3CSK4, but that all stimuli induce differentiation to Mph, resulting in a decrease in the percentage of Mc. It should be noted that the percentage of pdcs generated from Lin cells was decreased both in the spleen and in the bone marrow of TLR2 /, in response to Pam3CSK4 (Fig. 4). Overall, the pattern of differentiation detected in vivo correlates with the in vitro assays, both in the percentages and the cell types that are generated in response to Pam3CSK4, LPS, and ODN. These results demonstrate that HSPCs, in the bone marrow and in the spleen, are directly stimulated by TLR2, TLR4, and TLR9 agonists, and that the engagement of these receptors gives rise preferentially to Mph. DISCUSSION Under physiological conditions, the process of HSC selfrenewal, as well as their conversion into lineage-committed progenitors, is tightly controlled to maintain daily blood cell production. Many cytokines and transcription factors finetune the proliferation of HSPCs and their differentiation into mature myeloid and lymphoid cells [15]. However, hematopoiesis can be dramatically altered during infections, which influence numbers and types of cells that are produced. During most bacterial, viral, and fungal infections, myelopoiesis becomes predominant with inhibition of other lineage (lymphoid and erythroid) development, and this is accompanied by alterations of the cellular composition and/or phenotype of bone marrow HSPCs [16, 17]. Additional perspective on hematopoiesis during infection has come from the discovery that murine and human HSPCs express functional TLRs, and that TLR signals provoke cell cycle entry and myeloid differentiation in vitro [3 5, 10 12]. HSPCs expansion and alterations in hematopoiesis during infection have been described in several models of bacterial, viral, and fungal infection, although the contribution of TLR signaling to this phenomenon is still a matter of discussion [16, 17].

7 1492 TLRs Directly Activate HSPCs In Vivo Figure 4. In vivo differentiation of CD45.1 Lin progenitor cells in response to TLR ligands. CD45.1 Lin progenitor cells were transplanted into TLR2 /, TLR4 /, and MyD88 / knockout mice and stimulated daily with 100 lg Pam3CSK4, LPS, or ODN, respectively, for 3 days. Afterward, recovered cells from the spleen and bone marrow of mice were labeled with antibodies and analyzed by flow cytometry. The CD45.1 population was gated and cells in this gate were identified as CD11b þ (total CD11b þ cells), pdcs (CD11b CD11c þ mpdca þ ), cdcs (pre-dcs or cdcs: CD11b þ CD11c þ Ly6C F4/80 ), Mc (CD11b þ CD11c Ly6C þ F4/80 ), and Mph (CD11b þ CD11c Ly6C þ F4/80 þ ). Percentages refer to the total analyzed CD45.1 population. At least 10,000 CD45.1 events were analyzed in each spleen sample and 2,000 in each bone marrow sample. Data represent means 6 SD, from three experiments. *, p <.05; **, p <.01 with respect to CD45.1 cells within each cell type recovered from transplanted nonstimulated mice. Abbreviations: cdcs, classic dendritic cells; Lin, lineage negative cells; LPS, lipopolysaccharide; Mc, monocytes; Mph, macrophages; MyD88, myeloid differentiation factor 88; CpGODN, oligodeoxynucleotide; pdcs, plasmacytoid dendritic cells; TLR, toll-like receptors. Although most of the infection models demonstrate that TLR-mediated signals play an essential role in the control of HSPC expansion [6 8, 12], other authors [18] have described that the expansion of HSPCs following bacterial infection occurs in the absence of TLR signaling. It is important to notice that the interpretation of in vivo results is difficult as TLR / / MyD88 / mice are more susceptible to most of the infections. Therefore, comparison of hematopoiesis between control and knockout mice during infection may reflect different tissue invasion by the microorganism, and consequently, differences in secretion of cytokines that can regulate hematopoiesis, a response to the pathogen that is also TLR/MyD88 mediated.

8 Megías, Ya~nez, Moriano et al Figure 5. In vivo differentiation of CD45.1 Lin c-kit þ Sca-1 þ IL-7Ra (LKS) cells in response to TLR ligands. CD45.1 LKS cells were transplanted into TLR2 /, TLR4 /, and MyD88 / knockout mice and stimulated daily with 100 lg Pam3CSK4, LPS, or ODN, respectively, for 3 days. Afterward, recovered cells from the spleen and bone marrow of mice were labeled with antibodies and analyzed by flow cytometry. The CD45.1 population was gated and cells in this gate were identified as CD11b þ (total CD11b þ cells), pdcs (CD11b CD11c þ mpdca þ ), cdcs (pre-dcs or cdcs: CD11b þ CD11c þ Ly6C F4/80 ), Mc (CD11b þ CD11c Ly6C þ F4/80 ), and Mph (CD11b þ CD11c Ly6C þ F4/ 80 þ ). Percentages refer to the total analyzed CD45.1 population. At least 2,000 CD45.1 events were analyzed in each spleen sample, and 2,000 in each bone marrow sample. Data represent means 6 SD, from three experiments. *, p <.05; **, p <.01 with respect to CD45.1 cells within each cell type recovered from transplanted unstimulated mice. Abbreviations: cdcs, classic dendritic cells; LPS, lipopolysaccharide; Mc, monocytes; Mph, macrophages; MyD88, myeloid differentiation factor 88; CpGODN, oligodeoxynucleotide; pdcs, plasmacytoid dendritic cells; TLR, toll-like receptors. Thus, in conventional in vivo models, the alterations in hematopoiesis and in the HSPC populations during infection can be explained by at least two mechanisms: (a) MAMPs may directly induce HSPCs proliferation and differentiation, as suggested by the in vitro results, or alternatively (b) the alterations could be caused by an indirect effect, due to pathophysiological changes during infection. These possibilities are not mutually exclusive, and both of them may involve TLR recognition of the pathogen. In this work, we deal with the issue of whether interaction of pathogens or the MAMPs released by them, directly on progenitor cells, can control hematopoiesis.

9 1494 TLRs Directly Activate HSPCs In Vivo Purified Lin cells (containing stem and all the progenitor cells) or the LKS population (containing stem and uncommitted progenitors) from the bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into TLR2 /, TLR4 /, or MyD88 / mice (CD45.2 alloantigen), which where then injected with Pam3CSK4, LPS, or ODN, respectively. Using this experimental approach, the cells of the recipient mice do not recognize the TLR agonist injected, and therefore any effects of cytokines or soluble mediators secreted by surrounding cells were excluded. Moreover, the recipient mice were not irradiated, in order to avoid an inflammatory environment that may generate artifact results. After 3 days, transplanted cells were detected in the spleen and in the bone marrow of mice. In these in vivo conditions and in the absence of TLR challenge, the HSPCs differentiated toward the myeloid lineage, as most of the cells had lost stem cell markers, did not express lymphoid markers, but most of them expressed myeloid markers. The differentiated cells expressed the markers of mature pdcs, cdcs, or Mc. This result is in accordance with Massberg et al. [19] who showed that migratory HSPCs give rise to myeloid cells in peripheral tissues. However, when each knockout mouse type was injected with the corresponding TLR agonist, the emergence of a new population of Mph was found. In this model, the differentiation of CD45.1 cells in response to the injected ligands can only be due to a direct ligand-tlr interaction on transplanted Lin or LKS cells and not to an indirect effect of TLR ligands on cells of the recipient knockout mice. The differentiation to Mph was similar in response to all TLR agonists assayed, although an increase in the total number of CD45.1- positive cells was found only in response to the TLR2 ligand. In addition, the increase in Mph was not accompanied by a decrease in Mc in response to the TLR2 ligand, further supporting the idea that Pam3CSK4 induces higher proliferation of CD45.1 cells than LPS or ODN. The stronger response to Pam3CSK4, as compared with LPS, is in accordance with the higher expression of TLR2 than TLR4 on long-term repopulating HSCs and LKS cells [3, 12]. The in vivo results clearly correlate with the in vitro assays, as TLR2, TLR4, and TLR9 agonists induce the differentiation of Lin or LKS cells toward Mph. Therefore, signaling through all the TLRs assayed induces the differentiation of HSPCs toward the same type of cell, although the response was stronger for TLR2 as compared with TLR4 and TLR9. In a previous work, we demonstrated that C. albicans drives the differentiation of Lin cells toward modcs in a TLR2/MyD88 and Dectin-1 (a PRR that recognizes the b-glucan of yeasts)-dependent manner in vitro [12]. Therefore, the response to C. albicans that is recognized simultaneously by both receptors [20] is different to the response to a specific TLR2 agonist, indicating that hematopoietic progenitor cells may integrate signals from different PRRs, and therefore the response may be pathogen specific. It should be noted that Dectin-1, in contrast to TLRs, is not expressed on the most primitive stem cells (side population), although the expression of this receptor is acquired by progenitors before their differentiation to mature cells, as Lin cells express significant levels of Dectin-1 [12]. The role of TLR2, TLR4, and TLR9 in the differentiation of Lin and LKS cells in vitro is in line with several previous reports showing that TLRs have a role in hematopoiesis during infection [3 12]. However, this is the first report showing that HSPCs use TLRs to directly sense pathogen-derived products in vivo and drive their differentiation toward myeloid lineage inducing the production of Mph. The ability of stem and progenitor cells to sense pathogen products may be protective, allowing rapid mobilization and generation of cells of the innate immune system, in a pathogen-specific manner. Moreover, in the case of local infections, migratory HSPCs that sense microbial danger signals in peripheral tissues can proliferate within that pathogen-challenged location and contribute to the supply of effector cells [21]. Therefore, TLR ligation directly activates HSPCs in vivo suggesting a mechanism by which pathogens may modulate hematopoiesis in real time. This points to a selection of innate immune populations during the infectious process, an idea that sits outside the current dogma of innate immune function, but which is gaining momentum in the literature. CONCLUSIONS Recent findings indicate that HSPCs respond to infection via multiple pathways. In this context, we have presented the first in vivo demonstration of direct HSPCs stimulation via TLRs. We showed here that TLR2, TLR4, and TLR9 agonists (Pam3CSK4, LPS, and ODN) induce the differentiation of purified murine Lin as well as HSPCs (identified as LKS cells) toward Mph both in vivo and in vitro. This new mechanism represents a potential means for the host to detect pathogen products and to signal the rapid generation of innate immune cells within the bone marrow or other tissues containing HSPCs. ACKNOWLEDGMENTS This work was supported by Grant SAF (Ministerio de Economía y Competitividad, Spain). Javier Megías and Alberto Ya~nez are recipients of fellowships from Fundacion Bancaja and Ministerio de Educacion y Ciencia, respectively. We are grateful to the SCSIE (Servicio Central de Soporte a la Investigacion Experimental), University of Valencia, for technical assistance. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest. REFERENCES 1 Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors. Nat Immunol 2010;11: King KY, Goodell MA. Inflammatory modulation of HSCs: Viewing the HSC as a foundation for the immune response. Nat Rev Immunol 2011;11: Nagai Y, Garrett K, Ohta S et al. Toll-like receptors on hematopoietic progenitor cells stimulate innate immune system replenishment. Immunity 2006;24: Sioud M, Fløisand Y, Forfang L et al. Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34þ progenitor cells along the myeloid lineage. 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