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(A) Changes in granulocyte (Gr1⁺) subpopulations in bone marrow cells (BMCs). (B) Changes in granulocyte (Gr1⁺) subpopulations in peripheral white blood cells (WBCs) following 24 h of bacteremia. n = 4 mice per group. *, P < 0.05 compared to control group of the same cell type; #, P < 0.05 compared to other groups of the same cell type.
Source publication
In response to severe bacterial infection, bone marrow hematopoietic activity shifts toward promoting granulopoiesis. The
underlying cell signaling mechanisms remain obscure. To study the role of Toll-like receptor 4 (TLR4)/stem cell antigen-1
(Sca-1) signaling in this process, bacteremia was induced in mice by intravenous injection of Escherichia...
Citations
... Septic infection was induced in mice as described previously with minor modifications (24)(25)(26)(27)29). Briefly, an intravenous (i.v.) injection (via the jugular or penile vein) of~1 × 10 7 to~1 × 10 8 colony-forming units (CFUs) of live or heat-inactivated Escherichia coli (E. coli strain E11775 from the American Type Culture Collection, Rockville, MD) in 100 ml of pyrogen-free saline/mouse was administered. ...
... Cell phenotype, intracellular expression of specificity protein 1 (SP1), and cell BrdU incorporation were determined with flow cytometry as previously described (24)(25)(26)(27)(28). Briefly, nucleated BMCs and PBMCs suspended in RPMI-1640 containing 2% BSA (1 × 10 6 cells in 100 ml medium) were added with a mixed panel of biotinylated anti-mouse lineage markers [10 µg/mL of each antibody against CD3e (clone 145-2C11), CD45R/B220 (clone RA3-6B2), CD11b (Mac-1, clone M1/70), TER-119 (clone TER-119)] and granulocyte differentiation antigen 1 (Gr-1 or Ly-6G/Ly-6C, clone RB6-8C5), or isotype control antibodies (clones A19-3, R35-95, A95-1) (BD Biosciences). ...
... These observations indicate that Sca-1 couples with Rac2 activation in signaling the EPC response. Cell surface crosslinking has been shown to promote Sca-1 signaling activity (25). The results of in vitro experiment via culturing marrow lin − c-kit + cells with or without LPS plus Sca-1 crosslinking antibodies for 24 h exhibited that Sca-1 crosslinking significantly enhanced LPS-induced upregulation of VEGFR2 mRNA expression by these marrow SPCs ( Figure 9C). ...
Early increase in the level of endothelial progenitor cells (EPCs) in the systemic circulation occurs in patients with septic infection/sepsis. The significance and underlying mechanisms of this response remain unclear. This study investigated the bone marrow EPC response in adult mice with septic infection induced by intravenous injection (i.v.) of Escherichia coli. For in vitro experiments, sorted marrow stem/progenitor cells (SPCs) including lineage(lin)⁻stem cell factor receptor (c-kit)⁺stem cell antigen-1 (Sca-1)⁻, lin⁻c-kit⁺, and lin⁻ cells were cultured with or without lipopolysaccharides (LPSs) and recombinant murine vascular endothelial growth factor (VEGF) in the absence and presence of anti-Sca-1 crosslinking antibodies. In a separate set of experiments, marrow lin⁻c-kit⁺ cells from green fluorescence protein (GFP)⁺ mice, i.v. challenged with heat-inactivated E. coli or saline for 24 h, were subcutaneously implanted in Matrigel plugs for 5 weeks. Marrow lin⁻c-kit⁺ cells from Sca-1 knockout (KO) mice challenged with heat-inactivated E. coli for 24 h were cultured in the Matrigel medium for 8 weeks. The marrow pool of EPCs bearing the lin⁻c-kit⁺Sca-1⁺VEGF receptor 2 (VEGFR2)⁺ (LKS VEGFR2⁺) and LKS CD133⁺VEGFR2⁺ surface markers expanded rapidly following septic infection, which was supported by both proliferative activation and phenotypic conversion of marrow stem/progenitor cells. Increase in marrow EPCs and their reprogramming for enhancing angiogenic activity correlated with cell-marked upregulation of Sca-1 expression. Sca-1 was coupled with Ras-related C3 botulinum toxin substrate 2 (Rac2) in signaling the marrow EPC response. Septic infection caused a substantial increase in plasma levels of IFN-γ, VEGF, G-CSF, and SDF-1. The early increase in circulating EPCs was accompanied by their active homing and incorporation into pulmonary microvasculature. These results demonstrate that the marrow EPC response is a critical component of the host defense system. Sca-1 signaling plays a pivotal role in the regulation of EPC response in mice with septic infection.
... The best characterized effect is increased differentiation of HSPCs into myeloid effector immune cells such as monocytes, macrophages, and granulocytes like neutrophils. [23][24][25][26][27][28] PAMP exposure causes HSPCs to secrete inflammatory cytokines (including IL-6, TNF-α, and IL-1β) when stimulated by TLR2 or TLR4 agonist in vitro, [26] and TLR4 stimulation causes persistent changes in HSPC chromatin accessibility at myeloid-associated gene enhancers that increases myeloid differentiation upon subsequent activation in vivo. [28] HPSCs also show increased cell division in response to TLR4 agonist in vitro, [23,29] or after β-glucan (a fungal PAMP) injection in vivo, [27] or after either Mycobacterium avium [30] or ...
... Escherichia coli [25] infection in vivo. In addition, systemic E. coli infection or dual stimulation of the PRRs TLR4 and the nucleotide-binding oligomerization domain containing (NOD) receptor NOD1 mobilizes BM hematopoietic stem cells (HSCs) to the spleen where they can differentiate after an infection. ...
Recent research highlights that inflammatory signaling pathways such as pattern recognition receptor (PRR) signaling and inflammatory cytokine signaling play an important role in both on‐demand hematopoiesis as well as steady‐state hematopoiesis. Knockout studies have demonstrated the necessity of several distinct pathways in these processes, but often lack information about the contribution of specific cell types to the phenotypes in question. Transplantation studies have increased the resolution to the level of specific cell types by testing the necessity of inflammatory pathways specifically in donor hematopoietic stem and progenitor cells (HSPCs) or in recipient niche cells. Here, we argue that for an integrated understanding of how these processes occur in vivo and to inform the development of therapies that modulate hematopoietic responses, we need studies that knockout inflammatory signaling receptors in a cell‐specific manner and compare the phenotypes caused by knockout in individual niche cells versus HSPCs.
... injection of heat-killed Escherichia coli. In both WT and D1 −/− D1L3 −/− mice, this treatment caused a major influx of neutrophils in the blood (Fig. S3 A) and broad induction of ICAM-1 and Sca-1 expression (Basit et al., 2006;Shi et al., 2013) 24 h later (Fig. S3, B and C). The induced neutrophilia was comparable with that observed after S. aureus infection (Fig. S3 D); furthermore, the LPS/E. ...
Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.
... The structure and function of TLR4 has been reviewed previously, and for this reason we only include a brief summary of its expression, namely ligand binding and signaling (Figure 1). TLR4 is expressed by hematopoietic stem cells as well as myeloid progenitors and mature neutrophils/monocytes/macrophages [9][10][11]. It is involved in the development of lymphoid cells [12], and it is expressed by various stromal cells [8]. ...
... The structure and function of TLR4 has been reviewed previously, and for this reason we only include a brief summary of its expression, namely ligand binding and signaling ( Figure 1). TLR4 is expressed by hematopoietic stem cells as well as myeloid progenitors and mature neutrophils/monocytes/macrophages [9][10][11]. It is involved in the development of lymphoid cells [12], and it is expressed by various stromal cells [8]. ...
Toll-like receptor 4 (TLR4) is a pattern-recognizing receptor that can bind exogenous and endogenous ligands. It is expressed by acute myeloid leukemia (AML) cells, several bone marrow stromal cells, and nonleukemic cells involved in inflammation. TLR4 can bind a wide range of endogenous ligands that are present in the bone marrow microenvironment. Furthermore, the TLR4-expressing nonleukemic bone marrow cells include various mesenchymal cells, endothelial cells, differentiated myeloid cells, and inflammatory/immunocompetent cells. Osteoblasts are important stem cell supporting cells localized to the stem cell niches, and they support the proliferation and survival of primary AML cells. These supporting effects are mediated by the bidirectional crosstalk between AML cells and supportive osteoblasts through the local cytokine network. Finally, TLR4 is also important for the defense against complicating infections in neutropenic patients, and it seems to be involved in the regulation of inflammatory and immunological reactions in patients treated with allogeneic stem cell transplantation. Thus, TLR4 has direct effects on primary AML cells, and it has indirect effects on the leukemic cells through modulation of their supporting neighboring bone marrow stromal cells (i.e., modulation of stem cell niches, regulation of angiogenesis). Furthermore, in allotransplant recipients TLR4 can modulate inflammatory and potentially antileukemic immune reactivity. The use of TLR4 targeting as an antileukemic treatment will therefore depend both on the biology of the AML cells, the biological context of the AML cells, aging effects reflected both in the AML and the stromal cells and the additional antileukemic treatment combined with HSP90 inhibition.
... Concomitantly, the bone marrow initiates the granulopoietic response to promote production of granulocytes through enhancing granulocyte lineage development at the expense of generating cells from other lineages (Ueda et al., 2005). Our recent investigations have demonstrated that the rapid activation of HSC proliferation along with reprogramming their commitment to granulocyte lineage differentiation is a critical step in the early stage of the granulopoietic response (Shi et al., 2013(Shi et al., , 2017(Shi et al., , 2018Zhang et al, 2008bZhang et al, , 2009). In the process of the granulopoietic response, the bone marrow accelerates granulocyte differentiation, shortens their marrow retention, and increases the release of these phagocytes into the systemic circulation (Sato et al., 1998;Shahbazian et al., 2004;Terashima et al., 1996). ...
... Live E. coli suspension in saline for i.v. injection in each experiment was freshly prepared as described previously (Shi et al., 2013(Shi et al., , 2017(Shi et al., , 2018. ...
... Flow cytometric analysis of cell phenotype, expression of SHH and Gli1, and incorporation of BrdU were performed as previously described (Shi et al., 2013(Shi et al., , 2017(Shi et al., , 2018Zhang et al., 2008b). Reagents used for labeling cells included a panel of biotinylated antibodies against mouse lineage markers [CD3e (clone 145-2C11), CD45R/B220 (clone RA3-6B2), CD11b (Mac-1, clone M1/70), TER-119 (clone TER-119), with or without Gr1 (granulocyte differentiation antigen 1, Ly-6G/Ly-6C, clone RB6-8C5) (BD Biosciences)]; biotinylated isotype control antibodies (clones A19-3, R35-95, and A95-1; BD Biosciences); fluorochrome-conjugated streptavidin (BD Biosciences); fluorochrome-conjugated antibodies against mouse c-kit (stem cell growth factor receptor or CD117, clone 2B8), Sca-1 (stem cell antigen-1 or Ly-6A/E, clone D7), Gr1 (Ly-6G, clone 1A8), CD34 (clone RAM34) (BD Biosciences), and F4/80 (clone BM8) (eBioscience, San Diego, CA); anti-human/mouse SHH (Clone E1, Santa Cruz Biotechnology, Inc., Dallas, TX); fluorochrome-conjugated polyclonal goat anti-mouse IgG (H + L; Life Technologies, Eugene, OR); fluorochrome-conjugated anti-human/anti-mouse Gli1 (Clone #388516, R&D Systems) and the isotype control antibody (Clone # 54447, R&D Systems); and BD BrdU Flow Kit (BD Biosciences). ...
Background
Activation of hematopoietic stem cells [HSCs, lineage(lin)⁻stem cell growth factor receptor (c‐kit)⁺stem cell antigen‐1(Sca‐1)⁺, or LKS cells in mice] is critical for initiating the granulopoietic response. This study determined the effect of alcohol exposure on sonic hedgehog (SHH) signaling in the regulation of HSC activation during bacteremia.
Methods
Acute alcohol intoxication was induced in mice by intraperitoneal (i.p.) injection of 20% alcohol (5 g alcohol/kg body weight). Control mice received i.p. saline. Thirty minutes later, mice were intravenously (i.v.) injected with Escherichia coli (E. coli, 1 to 5 × 10⁷ CFUs/mouse) or saline.
Results
SHH expression by lineage‐negative bone marrow cells (BMCs) was significantly increased 24 hours after E. coli infection. Extracellular signal‐regulated kinase 1/2 (ERK1/2)‐specificity protein 1 (Sp1) signaling promotes SHH expression. ERK1/2 was markedly activated in BMCs 8 hours following E. coli infection. Alcohol suppressed both the activation of ERK1/2 and up‐regulation of SHH expression following E. coli infection. E. coli infection up‐regulated GLI family zinc finger 1 (Gli1) gene expression by BMCs and increased Gli1 protein content in LKS cells. The extent of Gli1 expression was correlated with the activity of proliferation in LKS cells. Alcohol inhibited up‐regulation of Gli1 expression and activation of LKS cells in response to E. coli infection. Alcohol also interrupted the granulopoietic response to bacteremia.
Conclusion
These data show that alcohol disrupts SHH‐Gli1 signaling and HSC activation in the early stage of the granulopoietic response, which may serve as an important mechanism underlying the impairment of immune defense against bacterial infection in host excessively consuming alcohol.
... This triggers cell cycle entry of these cells through the activation of NFκB [50,51]. Moreover, activation of TLR4 upregulates Sca-1 expression on such precursors, that supports the development of granulocyte lineage [52,53]. Therefore, early neutrophil progenitors seem to be particular vulnerable for NAMPT depletion. ...
Granulocyte-colony stimulating factor (G-CSF)/nicotinamide phosphoribosyltransferase (NAMPT) signaling has been shown to be crucial for the modulation of neutrophil development and functionality. As this signaling pathway is significantly suppressed by type I interferons (IFNs), we aimed to study how the regulation of neutrophil differentiation and phenotype is altered in IFN-deficient mice during granulopoiesis. The composition of bone marrow granulocyte progenitors and their Nampt expression were assessed in bone marrow of type I IFN receptor knockout (Ifnar1-/-) mice and compared to wild-type animals. The impact of NAMPT inhibition on the proliferation, survival, and differentiation of murine bone marrow progenitors, as well as of murine 32D and human HL-60 neutrophil-like cell lines, was estimated. The progressive increase of Nampt expression during neutrophil progenitor maturation could be observed, and it was more prominent in IFN-deficient animals. Altered composition of bone marrow progenitors in these mice correlated with the dysregulation of apoptosis and altered differentiation of these cells. We observed that NAMPT is vitally important for survival of early progenitors, while at later stages it delays the differentiation of neutrophils, with moderate effect on their survival. This study shows that IFN-deficiency leads to the elevated NAMPT expression in the bone marrow, which in turn modulates neutrophil development and differentiation, even in the absence of tumor-derived stimuli.
... In vivo chronic treatment with LPS leads to HSC cycling and to myeloid differentiation with a consequent loss of their repopulating activity in transplantation experiments [49,50]. Indeed, the TLR4/Sca-1 axis contributes to granulopoiesis starting from HSC during bacterial infection or LPS treatment [51]. Notably, the time and the entity of the stimulation can influence cell lymphopoiesis as proved by chronic low-dose LPS perturbation in human HSC and B-lineage progenitors. ...
Toll-like receptors (TLRs) represent one of the bridges that regulate the cross-talk between the innate and adaptive immune systems. TLRs interact with molecules shared and preserved by the pathogens of origin but also with endogenous molecules (damage/danger-associated molecular patterns (DAMPs)) that derive from injured tissues. This is probably why TLRs have been found to be expressed on several kinds of stem/progenitor cells (SCs). In these cells, the role of TLRs in the regulation of the basal motility, proliferation, differentiation processes, self-renewal, and immunomodulation has been demonstrated. In this review, we analyze the many different functions that the TLRs assume in SCs, pointing out that they can have different effects, depending on the background and on the kind of ligands that they recognize. Moreover, we discuss the TLR involvement in the response of SC to specific tissue damage and in the reparative processes, as well as how the identification of molecules mediating the differential function of TLR signaling could be decisive for the development of new therapeutic strategies. Considering the available studies on TLRs in SCs, here we address the importance of TLRs in sensing an injury by stem/progenitor cells and in determining their behavior and reparative activity, which is dependent on the conditions. Therefore, it could be conceivable that SCs employed in therapy could be potentially exposed to TLR ligands, which might modulate their therapeutic potential in vivo . In this context, to modulate SC proliferation, survival, migration, and differentiation in the pathological environment, we need to better understand the mechanisms of action of TLRs on SCs and learn how to control these receptors and their downstream pathways in a precise way. In this manner, in the future, cell therapy could be improved and made safer.
... The most striking transcriptional response induced by EVI1 is upregulation of Sca-1, which is known to have an important role in the granulocytic response to bacteria via TLR4, the receptor for lipopolysaccharides (LPS) 53,54 ; PU.1 is known to play an important role in this 53 . Our data presented here indicate that upregulation of Sca-1 occurs via PU.1 action (Figs. ...
... The most striking transcriptional response induced by EVI1 is upregulation of Sca-1, which is known to have an important role in the granulocytic response to bacteria via TLR4, the receptor for lipopolysaccharides (LPS) 53,54 ; PU.1 is known to play an important role in this 53 . Our data presented here indicate that upregulation of Sca-1 occurs via PU.1 action (Figs. ...
Inv(3q26) and t(3:3)(q21;q26) are specific to poor-prognosis myeloid malignancies, and result in marked overexpression of EVI1, a zinc-finger transcription factor and myeloid-specific oncoprotein. Despite extensive study, the mechanism by which EVI1 contributes to myeloid malignancy remains unclear. Here we describe a new mouse model that mimics the transcriptional effects of 3q26 rearrangement. We show that EVI1 overexpression causes global distortion of hematopoiesis, with suppression of erythropoiesis and lymphopoiesis, and marked premalignant expansion of myelopoiesis that eventually results in leukemic transformation. We show that myeloid skewing is dependent on DNA binding by EVI1, which upregulates Spi1, encoding master myeloid regulator PU.1. We show that EVI1 binds to the −14 kb upstream regulatory element (−14kbURE) at Spi1; knockdown of Spi1 dampens the myeloid skewing. Furthermore, deletion of the −14kbURE at Spi1 abrogates the effects of EVI1 on hematopoietic stem cells. These findings support a novel mechanism of leukemogenesis through EVI1 overexpression.
... It is well known that TLR4/Sca-1 signaling plays an important role in the differentiation of hematopoietic precursor cells by promoting granulocyte lineage commitment [45]. Indeed, TLR4 is expressed by multipotent hematopoietic stem cells, and controls cell cycle entry of these cells. ...
Nicotinamide phosphoribosyltransferase (NAMPT) is the bottleneck enzyme of the NAD salvage pathway and thereby is a controller of intracellular NAD concentrations. It has been long known that the same enzyme can be secreted by a number of cell types and acts as a cytokine, although its receptor is at present unknown. Investigational compounds have been developed that target the enzymatic activity as well as the extracellular action (i.e. neutralizing antibodies). The present contribution reviews the evidence that links intracellular and extracellular NAMPT to myeloid biology, for example governing monocyte/macrophage differentiation, polarization and migration. Furthermore, it reviews the evidence that links this protein to some disorders in which myeloid cells have a prominent role (acute infarct, inflammatory bowel disease, acute lung injury and rheumatoid arthritis) and the data showing that inhibition of the enzymatic activity or the neutralization of the cytokine is beneficial in preclinical animal models.
... The homeostasis of HSC quiescence, self-renewal, proliferation, and differentiation secures maintaining the appropriate pool of HSCs while giving rise to all types of blood cells in the body. Our recent studies have revealed that primitive hematopoietic precursor cells in the adult bone marrow constitute a key component of the host immune defense system (2)(3)(4). During bacterial infection, marrow primitive hematopoietic precursor cells activate rapidly. ...
... Cell phenotype, cell membrane expression of SHH, and intracellular expression of specificity protein 1 (SP1) as well as Gli1 was determined with flow cytometry as previously described (2)(3)(4). Briefly, nucleated BMCs or WBCs suspended in RPMI-1640 containing 2% BSA (1 × 10 6 cells in 100 µl medium) were added with a mixed panel of biotinylated anti-mouse lin markers [10 µg/ml of each antibody against CD3e (clone 145-2C11), CD45R/B220 (clone RA3-6B2), CD11b (Mac-1, clone M1/70), TER-119 (clone TER-119)] with or without granulocyte differentiation antigen-1 (Gr-1 or Ly-6G/Ly-6C, clone RB6-8C5), or isotype control antibodies (clones A19-3, R35-95, A95-1) (BD Biosciences). ...
... The reduction in the number of Gli1 + LKS − cells in BMCs is likely caused by conversion of LKS − cells to cells no longer belonging to the LKS − phenotype. Our previous investigations have shown that re-expression of Sca-1 by LKS − cells leads to phenotypic conversion of LKS − cells to LKS cells, which play an important role in the rapid expansion of LKS cell pool in the bone marrow during the host response to serious bacterial infection (2,3,37). It is apparent that the phenotypic conversion of Gli1 + LKS − cells may also contribute to the marked increase in the number of Gli1 + LKS cells in BMCs following bacteremia in our current study. ...
Activation and reprogramming of hematopoietic stem/progenitor cells play a critical role in the granulopoietic response to bacterial infection. Our current study determined the significance of Sonic hedgehog (SHH) signaling in the regulation of hematopoietic precursor cell activity during the host defense response to systemic bacterial infection. Bacteremia was induced in male Balb/c mice via intravenous injection (i.v.) of Escherichia coli (5 × 10⁷ CFUs/mouse). Control mice received i.v. saline. SHH protein level in bone marrow cell (BMC) lysates was markedly increased at both 24 and 48 h of bacteremia. By contrast, the amount of soluble SHH ligand in marrow elutes was significantly reduced. These contrasting alterations suggested that SHH ligand release from BMCs was reduced and/or binding of soluble SHH ligand to BMCs was enhanced. At both 12 and 24 h of bacteremia, SHH mRNA expression by BMCs was significantly upregulated. This upregulation of SHH mRNA expression was followed by a marked increase in SHH protein expression in BMCs. Activation of the ERK1/2–SP1 pathway was involved in mediating the upregulation of SHH gene expression. The major cell type showing the enhancement of SHH expression in the bone marrow was lineage positive cells. Gli1 positioned downstream of the SHH receptor activation serves as a key component of the hedgehog (HH) pathway. Primitive hematopoietic precursor cells exhibited the highest level of baseline Gli1 expression, suggesting that they were active cells responding to SHH ligand stimulation. Along with the increased expression of SHH in the bone marrow, expression of Gli1 by marrow cells was significantly upregulated at both mRNA and protein levels following bacteremia. This enhancement of Gli1 expression was correlated with activation of hematopoietic stem/progenitor cell proliferation. Mice with Gli1 gene deletion showed attenuation in activation of marrow hematopoietic stem/progenitor cell proliferation and inhibition of increase in blood granulocytes following bacteremia. Our results indicate that SHH signaling is critically important in the regulation of hematopoietic stem/progenitor cell activation and reprogramming during the granulopoietic response to serious bacterial infection.
