LPS binds to TLR4 to elicit cellular inflammatory responses. By administering a low dose of LPS daily for 30 days, we intended to mimic inflammatory conditions similar to those occurring during chronic pathogenic infection or autoimmunity. Indeed, we detected dramatic induction of a number of inflammatory cytokines such as IL-6, KC, TNFα and IL-10 on Day 15 of the treatment, but cytokine levels returned to normal at the time when HSC were analyzed. It should be noted that the analysis on Day 15 serves as a snap shot of the inflammatory responses triggered by LPS. Expression of other cytokines tested may also be affected, but the induction follows a different kinetics. Furthermore, LPS may stimulate the production of additional cytokines which were not included in our assay. Whether this burst of inflammatory cytokines has any adverse effects on stem cells remains to be tested. However, several such cytokines are known to have direct effects on the properties of HSC. For example, IL-6 and IL-10 have stimulatory effects on HSC self-renewal whereas TNFα exerts inhibitory effects 
. Apart from the roles of inflammatory cytokines, HSCs are also found to express TLR4 and, thus, have the potential to directly bind LPS, which initiates downstream signaling within HSC 
. Our current data cannot distinguish the HSC-intrinsic from cytokine-mediated effects on HSC maintenance after LPS treatment, although it is likely that both mechanisms are at play.
Regardless the mechanisms by which LPS exerts its deleterious effect on HSC activity, we have shown that chronic LPS treatment profoundly impairs the repopulating potential of wild type HSC. This defect is accompanied by a significant increase in the number of phenotypic HSC and a reduction in the percentage of label-retaining cells, which could be interpreted to mean that an increased turnover of LT-HSC occurred. Since the viability of HSC was unchanged when assayed after the 30-day LPS treatment, the increased turnover is mostly likely due to accelerated proliferation. Although BrdU labeling used in the label-retaining assay has been thought to disturb the quiescence of HSC 
, data from examination of Ki67 levels in HSC by intracellular staining also suggested that LPS treatment directs the cells out of the G0 state. LT-HSCs are normally present in a quiescent state residing in their niches in the bone marrow 
. Loss of quiescence is believed to be detrimental to stem cell integrity and eventually leads to stem cell exhaustion 
. Therefore, increased HSC cycling stimulated by LPS could compromise the ability of HSC to reconstitute the bone marrow of transplant recipients. At the dose of 1 µg of LPS per mouse, although we detected a slight myeloid skewing in treated animals, we did not find the same effect when treated HSCs were transplanted into primary or secondary recipients, which was observed when mice were treated with 6 µg of LPS 
. Perhaps, the threshold is higher for upsetting the balance between lymphoid and myeloid potential of HSCs.
We also found that LPS treatment in vivo stimulated Id1 expression in Lin−
LSK cells, a population containing HSC as well as other progenitors. It is of note that incubation of LSK with LPS in vitro could not stimulate Id1 expression, which suggests that up-regulation of Id1 by LPS is likely mediated by inflammatory cytokines. More importantly, LPS injection into Id1 deficient mice did not appear to have any significant effects on HSC function and properties compared to control Id1 deficient mice, even though it led to skewing of lymphoid versus myeloid differentiation. This raises a possibility that the effects of LPS might be at least partially mediated by Id1 expression. Admittedly, Id1 deficiency itself markedly diminishes the long-term repopulating potential of HSC 
. We thought that loss of Id1 results in elevated E protein activities and pre-mature differentiation of LT-HSC into downstream lineages, thus exhausting the LT-HSC pool. Incidentally, one report did not reveal any HSC intrinsic defect in Id1 deficient mice using a less stringent measurement 
. In the current study, we found that LPS administration not only failed to further reduce but also somewhat elevated the engraftment capacity of Id1 deficient HSC. This apparent rescuing effect by LPS could be due to the induction of other Id proteins as we have found Id2 expression could be modestly induce by LPS (). The effects of Id1 deficiency under normal and inflammatory conditions underscore the importance of maintaining an appropriate level of Id proteins for the wellbeing of HSC.
There are several mechanisms that could act separately or coordinately to render Id1 deficient mice insensitive to LPS-mediated effects. First, ablation of Id1 blunted the cytokine storm triggered by LPS. For example, Id1 deficient mice produced significantly lower levels of TNFα and IL-10 than wild type mice on Day 15 of LPS injection (). The cell types predominantly contributing to the differences in systemic cytokine production in wild type and Id1−/−
mice are currently unknown although dendritic cells and macrophages are known to express Id1. Likewise, whether the difference in the levels of these cytokines contributes to the distinct responses of LPS between wild type and Id1 deficient HSC remains to be verified. Second, the LPS insensitivity of Id1−/−
mice could be HSC intrinsic. Id proteins are inhibitors of E proteins which play an important part in maintaining stem cell quiescence 
. Disruption of the E protein genes, E47, led to a hyperproliferative phenotype of HSC and decreased long-term repopulating potential 
. These are reminiscent of the situations after LPS treatment of wild type mice, in which Id1 may be up-regulated and E protein function is inhibited. Finally, Id proteins are also expressed in many of the cell types that make up the bone marrow niche, including osteoblasts, endothelial cells, mesenchymal stem cells and macrophages 
. LPS could alter the behavior of these cells by increasing Id1 levels and, thus, create unfavorable environments for HSC to live in. Taken together, the intimate relationships between Id1 expression and inflammation, as well as HSC and their niches, make Id1 one of the plausible mediators of the effects on HSCs during inflammation, which can be elicited by LPS or other pathogenic agents.
LT-HSCs can be subdivided into dormant and activated populations, and hematopoietic stresses such as bone marrow injury or mobilization with G-CSF can activate dormant HSCs to self-renew 
. Undoubtedly, a balance between the numbers of dormant and activated HSCs is crucial for the maintenance of a healthy pool of stem cells 
. Up-regulation of Id1 by inflammatory stimuli could lead to disruption of HSC quiescence and increases in proliferation. Repeated occurrence of such scenarios could deplete the dormant fraction of HSC, which would be detrimental for the longevity of HSC. Parenthetically, it remains to be determined if periodical stimulation of Id1 expression throughout life is beneficial for rejuvenating HSC. After all, Id1 is necessary for maintaining steady-state levels of LT-HSC.