Summarized by: Egbert Simon and Rami Rafeh, Fall 2007

LAY SUMMARY

There is evidence that Hematopoietic Stem Cells (HSCs) exhibit the unique ability to self-renew.  However, little is known about the mechanisms associated with promoting this ability during the asymmetric division of HSCs.  This paper examines the role of an immune regulator known as Interleukin-10 (IL-10) on HSC self-renewal using animal models divided into two groups that differed in genotype.  The genotypic difference in the two groups was used in order to identify the origin of the cells being observed as either being derived from the donor or from the recipient (Kang et al, 2007).  Also used in this study was an animal model that lacks the gene for IL-10, known as IL-10 knockout (KO) mice.

In comparing normal hematopoietic systems to those lacking IL-10, it was found that IL-10 KO mice had slightly lower bone marrow cells in comparison to the wild type (WT) mice.  Also, there was a noticeable decrease in red blood cells in IL-10 KO mice.  When testing the ability of the bone marrow cells (BMCs) to sustain long-term cultures, it was noted that IL-10 KO mice had a 3-fold decrease in colony formation over a 4 week period (Kang et al, 2007).  The repopulating capacity of HSCs in bone marrow was shown to be significantly lower in IL-10 KO mice than in WT mice.  Irradiated WT mice which received arrested BMCs originating from donor WT mice, were cultured on engineered stromal cells enabling them to secrete higher levels of IL-10 than normal.  This showed a higher level of reconstitution unlike the recipients that received BMCs cultured on normal stromal cells.  To mimic stressed conditions in the bone marrow that induce an immune response, WT mice received radiation causing an increase in IL-10 production compared to non-irradiated WT mice.  Notably, there was a higher level of IL-10 production for a period of 8 days following radiation within the osteoblasts that line the endosteal surface of the trabecular region in the irradiated WT mice (Kang et al, 2007). When taken together, these findings suggest that Interleukin-10 not only plays a role in regulating the body’s immune response, but may also be involved HSC self-renewal activity.

SCIENTIFIC SUMMARY

Understanding hematopoiesis and the regenerative abilities of the bone marrow has been the focus of a great deal of research.  Hematopoietic Stem Cells (HSC) have the ability to reconstitute all the cellular components and blood lineages.  This paper examines the role of Interleukin-10 (IL-10), and it’s affect on HSC self-renewal.  IL-10 has been observed as an immune cytokine that has inhibitory effects, but it is also suggested that its role in HSC self renewal is understated.  The following review looks into the paper’s findings.

The investigators used two types of mice as receipt or donors in their experimentation; Ly5.1 or Ly5.2.  IL-10 knockout (KO) mice were used as a model of an IL-10 disrupted system.  The investigators harvested the hematopoietic progenitors prior to injection of 5-fluorouracil (5-FU), which was used in order to eliminate all bone marrow cell lines.  The investigators then sorted for Lin-Sca-1+c-kit+ cells (LSK), and later purified for side population cells (SP).

In their first experiment, the investigators used the LSK cells in stroma-free culture to quantitatively measure the amount of colonies formed with or without the addition of IL-10.  They were able to achieve this by using a retroviral vector to infect MSCs via centrifuge, sort by green fluorescent protein (GFP+) and then culture the concentration in a 96-hour ELISA assay.  The next experiment involved plating an in vitro assay with erythropoietin (EPO) and other factors.  The investigators were able to measure stromal cells and their ability to form long-term colony forming cells.  In their third experiment, the investigators irradiated recipient mice and injected donor cells along with the recipient’s own helper cells.  They were able to measure repopulation ability of the transplanted donor cells into the recipient by using flow cytometry against surface antigens of the bone marrow cells or blood cells expressing donor origin leukocytes.  By staining for myeloid cells and antibodies towards lymphoid engraftment, the investigators were able to measure the number of HSCs in the competitive repopulating units (CRU) assay.  Finally, the last experiment described in the paper was setup in order to measure IL-10 receptor expression using a cytokine assay and immunohistochemistry analysis of bone marrow cells.  They were able to do this by incubating antibodies against the alpha subunit of the IL-10 receptor.  Concentrations of tumor necrosis factor-alpha (TNFa) and interferon-gamma (IFNγ) were measured in culture and the investigators were able to detect IL-10 production in normal and irradiated BM.

The results of the experiments showed that IL-10 KO mice had less HSC production as compared to wildtype, with the mature cell population left unaffected.  It is suggested that the progenitors were intact while the HSC level was affected.  Since it was shown that you can decrease the number of primitive HSC populations, the investigators tested if they can in turn increase HSC production ex vivo versus normal stromal cells.  Here, it was demonstrated that regenerative abilities were found with IL-10 outside the microenvironment of the bone marrow (BM).  By examining TNFa and IFNg, the investigators were able to induce stress on the microenvironment to mimic the immune response of the body.  It was shown that IL-10 could act as an important ligand towards self renewal during a stress-induced environment, without inhibiting TNFa or IFNg secretion.  Upon examination, it was demonstrated that SP cells expressed IL-10 receptors, which increased production of IL-10 in the lining of the trabecular regions of the stressed bone marrow as compared to normal production in the steady state marrow.  Altogether, this shows that IL-10 may act by preserving HSC via self renewal in the bone marrow.

Expansion of primitive HSCs can be very useful in the reconstitution of blood lineages in the bone marrow.  This paper suggests that IL-10’s role in the self renewal process is underestimated and should not solely be viewed as a cytokine involved in immune system modulation.  The investigators are not suggesting that IL-10 is the only cytokine involved in self-renewal but instead believe that more attention should be focused in understanding the factors involved in the marrow milieu, in particular, regeneration.  It is hoped that one day, ex vivo expansion of these lineages is possible and the reason why research is needed to understand the regulatory factors that are involved in HSC preservation and reconstitution.