Hematopoietic stem and progenitors cells (HSPCs) are activated through TLR4 in vitro. However, it remains unclear whether in vivo TLR4 sensing by HSPCs occurs directly or via other cell intermediates. In this study, we examined the cellular mechanisms underlying murine hematopoietic stem cell (HSC) expansion and common lymphoid progenitor (CLP) depletion in a model of chronic low-dose LPS. Using adoptive-transfer approaches, we show that HSC and CLP sensitivity to chronic LPS depends on hematopoietic-derived, cell subset–autonomous TLR4. Like murine progenitors, human HSPCs are activated by TLR4 in vitro. Using humanized mice, a preclinical model relevant to human physiology, we show that persistent endotoxin increases the frequency of Ki-67+ HSCs and severely depletes CLPs and B precursors. Together, our findings show that murine HSPCs directly respond to endotoxin in vivo and that persistent LPS, a feature of several diseases of global health significance, impairs human lymphopoiesis.
Bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) express pattern recognition receptors, such as TLRs that detect microbial products, including bacterial endotoxin (1, 2). Although purified HSPCs can respond directly to the endotoxin LPS and other TLR ligands ex vivo (2, 3), the contribution of direct-sensing mechanisms by HSPCs in vivo is considered relatively minor (4). LPS-mediated emergency myelopoiesis is independent of hematopoietic-derived TLR4 and instead requires nonhematopoietic TLR4 (5). Likewise, HSPC expansion following polymicrobial sepsis is independent of the TLR4 signaling adaptors MyD88/TRIF (6). Emerging evidence reveals that persistent exposure to endotoxin is a feature of clinically significant conditions, including chronic infection, obesity, and HIV/AIDS (7–13). A murine model of chronic LPS exposure demonstrated significant changes to the BM compartment, including HSPC expansion and reduced lymphoid potential (1). Moreover, hematopoietic stem cells (HSCs) from these LPS-exposed mice had poor self-renewal following serial adoptive transfer, indicating functional impairments. The relative importance of direct versus indirect TLR4-sensing mechanisms in the chronic LPS setting remains unknown.
In this study, we examined the cell-mediated mechanisms underlying murine HSPC dysfunction following chronic low-dose LPS, including the tissue source (hematopoietic versus nonhematopoietic) and cellular site (direct versus indirect) of TLR4 signaling. We used an established model of persistent low-dose LPS exposure (1, 2, 9, 14) and adoptive-transfer strategies to determine the effects of chronic endotoxin on HSPC activity. We then assessed the biological consequences of persistent low-dose LPS to human hematopoiesis using a preclinical experimental model. Human HSPCs are activated by TLR ligands in vitro (15–17), but the in vivo impact has been understudied. Together, our findings demonstrate that murine HSPCs can directly sense chronic LPS in vivo and that chronic low-dose LPS perturbs human HSPCs and B-lineage progenitors.
Materials and Methods
C57BL/6, TLR4-deficient, and NSG (NOD scid γ, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were purchased from The Jackson Laboratory. Mice were bred or maintained in accordance with Institutional Animal Care and Use Committee policies at the University of Pittsburgh School of Medicine.
Human cord blood
Deidentified cord blood (CB) cells were obtained with approval from the University of Pittsburgh School of Medicine Institutional Review Board. Purified CD34+ CB cells were purchased from AllCells, or CB obtained from Loma Linda University was enriched for CD34+lin− cells by magnetic separation.
Murine reconstitution chimeras and humanized mice
Wild-type (WT) mice irradiated with 900 rad were subsequently engrafted with 2 × 106 WT or TLR4-deficient BM cells i.v. via tail vein. Donor and host were distinguished with CD45 alleles: WT (CD45.1/2 or CD45.1) and TLR4-knockout (KO) donor (CD45.2). At 12 wk postengraftment, mice were treated with 6 μg LPS or vehicle i.p. every other day for 4–6 wk. NSG mice irradiated with 200 rad were engrafted with 2 × 105 human (h) CD34+ CB cells infused i.v. via tail vein. At 16 wk postengraftment, mice were treated with LPS or vehicle for 4 wk as above. Mice were immediately sacrificed after the last LPS treatment.
Flow cytometry, BrdU labeling, and cell cycle analysis
Hematopoietic progenitors were isolated and stained for surface and intracellular markers, including Ki-67, as we reported previously (18).
For serum-free liquid culture assays, sorted HSCs, LKS−, or common lymphoid progenitor (CLP) subsets were cultured with X-VIVO 15 medium supplemented with rFlt3 ligand, stem cell factor, thrombopoietin, IL-7, or M-CSF, as we have done (1, 18). Sorted HSCs were cultured with 10 μg/ml LPS. At harvest at the time points indicated in each figure legend, cells were stained with Abs to B220, CD19, CD11b, Gr-1, or BrdU.
Statistical significance of differences between group means (p < 0.05) was established using the two-sample t test if comparisons were made between two independent groups or the paired t test if comparisons were made on paired samples, as indicated in each figure legend.
Results and Discussion
Hematopoietic-derived TLR4 directs HSC expansion and CLP reduction in a model of chronic LPS
TLR ligation can affect a wide variety of hematopoietic and nonhematopoietic cells. For example, activation of BM monocyte emigration by single, low-dose LPS exposure depends on TLR4+ BM reticular cells (19). To establish the relative importance of hematopoietic- versus nonhematopoietic-derived TLR4 in HSC expansion and CLP depletion following persistent LPS, we used reciprocal chimeras. We then examined the requirement for cell stage–autonomous TLR4 using mixed BM chimeras.
First, we generated chimeric mice by transferring TLR4KO BM into WT hosts (TLR4KO→WT) and vice versa (WT→TLR4KO), with donor and host distinguished by CD45 alleles. At 12 wk postengraftment, peripheral blood donor chimerism was 95% (data not shown), and chimeric and control mice were subsequently exposed to low-dose LPS. We found that chronic activation of multipotent HSPCs, as well as self-renewing longterm HSCs (LT-HSCs), depends on TLR4-expressing hematopoietic cells. Following persistent LPS exposure, LSK and LKS− cells from WT and WT→TLR4KO mice had a 20–40% increase in cellularity relative to PBS baseline for each cohort, whereas CLPs were reduced ∼50% relative to baseline (Fig. 1A, phenotypic details in Supplemental Fig. 1A, Supplemental Table I). In contrast, in TLR4KO and TLR4KO→WT mice, numbers of LSK, LKS−, and CLP subsets were statistically unchanged relative to baseline (p > 0.05), indicating that the effects of LPS on HSPC cellularity are independent of nonhematopoietic cell–mediated mechanisms. These findings suggest a major role for hematopoietic-derived TLR4 in the chronic model of LPS exposure and a comparatively minor role for nonhematopoietic-derived TLR4.
LKS− cells are enriched for myeloid precursors, whereas CLPs are enriched for lymphoid precursors. Consistent with our observations of increased numbers of LKS− cells and decreased CLPs in mice chronically exposed to LPS in vivo, peripheral blood from intact WT and WT→TLR4KO mice, but not TLR4KO and TLR4KO→WT mice, was myeloid skewed at the expense of lymphocytes in these animals (Fig. 1B). These findings were confirmed following examination of differentiation of each fraction in defined serum-free conditions ex vivo. LKS− cells sorted from LPS-exposed WT and WT→TLR4KO mice had a 3-fold increase in myeloid lineage production, as assessed using either the Mac-1 or Gr-1 differentiation markers, whereas no enhancement of myeloid outgrowth was detectable in LKS− cells derived from LPS-exposed TLR4KO and TLR4KO→WT chimeras (Fig. 1C, data not shown). CLPs exhibited a similar pattern of sensitivity, with LPS-mediated depletion apparent in WT and WT→TLR4KO mice but not in TLR4KO and TLR4KO→WT mice cohorts.
We examined whether LT-HSC numbers and proliferative activity also require hematopoietic-derived TLR4. LT-HSCs from LPS-exposed WT and WT→TLR4KO mice had a 30% increase in the frequency of proliferating BrdU+ cells and a 1.5–3-fold increase in cellularity relative to baseline for each group (Fig. 1D, 1E, phenotypic gating in Supplemental Fig. 1A). Similar to past studies showing increased basal proliferation of TLR4KO HSCs following adoptive transfer (20), we detected increased baseline proliferation of HSCs from PBS-treated TLR4KO→WT mice compared with PBS-treated WT or TLR4KO controls (Fig. 1D, left panel). This effect appears to be specific to the LT-HSC subset, because baseline short-term-HSC proliferation in these same animals was similar to controls (Fig. 1D, right panel). In contrast to TLR4-sufficient hematopoietic cells, which were elevated following chronic LPS, numbers and proliferation status of LT-HSCs from LPS-exposed TLR4KO and TLR4KO→WT mice were unchanged relative to vehicle controls (Fig. 1D, 1E). In these experiments, LT-HSCs were identified as CD150+CD48− LSK; exclusion of CD48 minimizes concerns about potential contamination of the gated HSC subset by CD150+ myeloid cells that may have upregulated Sca-1 during inflammation (1). Together, these studies with reciprocal chimeras emphasize a major role for hematopoietic-specific TLR4 in HSPC expansion and myeloid > lymphoid bias following persistent LPS.
HSPCs directly sense TLR4 ligand in vivo
Like CLPs (2), LT-HSCs bear surface TLR4 and are capable of directly responding to LPS in vitro. Fig. 2A depicts cell surface TLR4 expression on CD150+CD48− HSCs. The TLR4 coreceptor CD14 is also detectable (data not shown). We examined the ability of sort-purified LT-HSCs to proliferate in response to LPS, using a dose and time point that maximizes BrdU detection sensitivity and limits cell differentiation, thereby minimizing potential concerns about LPS effects on the immediate downstream progeny of cultured HSCs. Sort-purified LT-HSCs had a 50% increase in BrdU uptake following a 12-h LPS pulse (Fig. 2B). To empirically establish whether LPS acts directly on HSPCs in vivo, we generated mixed chimeras in which WT BM (CD45.1/2 or CD45.1) was mixed with TLR4KO BM (CD45.2) and engrafted into WT hosts (CD45.1 or CD45.1/2); data from two independent experiments using different allelic combinations were similar and are pooled. Because WT BM has a repopulation disadvantage against TLR4KO BM (20, 21), we used a 60:40 ratio (WT/KO) to ensure robust representation of both partners in the long-term chimeras. At 12 wk postreconstitution, chimerism was ∼50:50% (data not shown), and mice were then chronically exposed to LPS or PBS. Although numbers of TLR4-sufficient CLPs were reduced 50% in LPS- versus PBS-treated mice, cellularity was preserved in TLR4-deficient CLPs, demonstrating a requirement for cell-intrinsic TLR4 (Fig. 2C, left panel).
In the LT-HSC compartment, TLR4-sufficient HSC numbers increased 3-fold following LPS treatment, whereas TLR4-deficient HSC numbers were unchanged compared with baseline PBS for each group (Fig. 2C, right panels). Our findings in this low-dose LPS experimental model suggest that HSCs and CLPs directly sense TLR4 stimuli in vivo.
Persistent TLR4 stimulation perturbs human HSPCs in vivo
Human HSPCs respond to multiple TLR ligands, including TLR4, in vitro, but the in vivo implications are understudied, a major gap in our knowledge given that persistent TLR4 stimulation is a feature of multiple diseases with broad clinical impact (7, 9–11, 13). We transferred hCD34+ CB into NSG immune-deficient mice, rested animals for 4 mo to attain stable engraftment (28.0 ± 16.1% hCD45+ cells across 23 total mice engrafted by three independent CB donors), and then exposed animals to chronic LPS. Mirroring the myeloid skewing observed in intact WT mice following LPS exposure (Fig. 1), the relative proportion of hCD45+CD19+ B cells was diminished and the relative proportion of hCD45+CD33+ myeloid cells was enhanced following low-dose LPS treatment of humanized mice (Fig. 3A, left panel, phenotypic gating in Supplemental Fig. 1B). hCD45+ myeloid > B lymphoid skewing was also apparent in peripheral blood and spleen (Fig. 3B, data not shown). NK cells are capable of homeostatic expansion in lymphopenic environments, and it will be useful in future studies to determine whether hCD45+CD56+CD3− NK cellularity reflects de novo differentiation or homeostatic expansion of a small number of mature NK cells. Within the hCD45+ BM compartment, CLP, pro-B, and immature B subsets were virtually ablated, whereas granulocyte macrophage progenitor cells were increased in frequency, and megakaryocyte erythroid progenitor cells and common myeloid progenitor cells were comparable to controls (Fig. 3A, right panel). There was also a 2-fold increase in the frequency of CD34+lin− HSPCs positive for the Ki-67 proliferation Ag accompanied by myeloid > lymphoid skewing (Fig. 3C). Unlike the WT mouse model, in which total cellularity in blood and BM is relatively unaffected by LPS, humanized mice chronically exposed to LPS had a significant reduction in hCD45+ chimerism in both blood and BM compared with their PBS counterparts (Fig. 3D, data not shown). Although we note that xenogeneic studies should be interpreted with caution, collectively these data show that the xenotransplant model captures major aspects of chronic endotoxin exposure, including HSPC activation and disproportionate loss of the B lymphoid lineage. This approach may advance the evaluation of therapeutic strategies in a preclinical setting relevant to human physiology.
In summary, this study provides mechanistic insight into how persistent TLR4 ligand, a notable feature of diseases of global significance, including chronic infection, obesity, and HIV/AIDS, perturbs HSPC homeostasis. Our observations that cell-autonomous TLR4 is required for HSPC activation during chronic LPS exposure differ from observations in the acute LPS setting (4–6), thereby deepening our understanding of the distinct pathways of LPS detection in the acute versus chronic contexts. Recent studies demonstrate cooperation between cell-autonomous TLRs and the LPS-inducible inflammatory cytokine IFN-γ in enhancing TLR-mediated signals, as well as suppressing B cell fate (22). Increased levels of IFN-γ protein are detectable within BM of mice exposed to chronic LPS, suggesting a potential local source of this cytokine (data not shown). Moreover, that low-dose LPS disrupts human hematopoiesis in patterns appreciably similar to those observed in mouse highlights the potential impact of our findings with respect to human health.
We thank Dewayne Falkner for excellent cell sorting.
This work was supported by National Institutes of Health Grants AI079047 and AI105846 and the American Society of Hematology Bridge Grant Program (to L.B.); National Institutes of Health Grant P20 MD006988, Loma Linda University institutional grants from the Department of Pathology and Human Anatomy, the Department of Basic Sciences, the Center for Health Disparities and Molecular Medicine, and a grant to Promote Collaborative and Translational Research (to K.J.P.); and a Hillman Cancer Center support grant (to Y.D.). Y.D. was supported by National Institutes of Health Grant P30 CA4790413.
The online version of this article contains supplemental material.
Abbreviations used in this article:
common lymphoid progenitor
hematopoietic stem cell
hematopoietic stem and progenitor cell
The authors have no financial conflicts of interest.