WT Bone Marrow Research Articles

ROCK1 Functions as a Suppressor of Inflammatory Cell Migration by Regulating PTEN Phosphorylation and Stability.

Abstract 240Neutrophils and macrophages are major cellular components of the innate immune response and are recruited rapidly in large numbers to sites of infection. The small family of Rho GTPases and its downstream effectors, Rho kinases (Rho-associated, coiled-coil containing protein kinase) have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases whose role in inflammation is not known. Here we show that deficiency of ROCK1 but not ROCK2 results in increased recruitment of macrophages (3.2 fold, n=8, *p<0.01) and neutrophils (3.4 fold, n=5 *p<0.05) compared to WT controls in an in vivo model of aseptic peritonitis. In vitro, deficiency of ROCK1 in bone marrow derived macrophages shows a significant increase in haptotactic transwell migration in response to M-CSF as well as MCP-1 on fibronectin as well as an increase in migration towards the wounded area in a wound healing assay compared to controls (∼3 fold, n=3, *p<0.005). Consistently, deficiency of ROCK1 in bone marrow derived neutrophils also shows a ∼2.63 fold increase in migration in response to fMLP compared to WT bone marrow derived neutrophils (BMNs) in a chemotactic migration assay. ROCK1 deficient macrophages also demonstrate a ∼2.5 fold increase in adhesion on fibronectin (n=3, *p<0.002). The enhanced migration and adhesion in ROCK1−/− macrophages was observed in spite of comparable expression of F4/80 (WT; 85.63% vs. ROCK1−/−; 88.68%, n=4), α4β1 and α5β1 integrins (WT; 67.49% & 88.2% vs. ROCK1−/−; 71.82 % & 87.09%, n=4), while no significant difference in the phagocytosis of sheep red blood cells was observed between WT and ROCK1−/− macrophages (Phagocytic index: WT; 98% vs. ROCK1−/− 97%, n=3, p>.05). Close examination of the cytoskeleton of ROCK1 deficient macrophages using confocal microscopy revealed more F-actin content on the entire cell surface compared to wildtype controls. Consistently, flow cytometric analysis using Alexa 488-phalloidin staining revealed abundance of F-actin in ROCK1−/− macrophages compared to WT controls (WT; 46.19% vs. ROCK1−/−; 65.23%, n=3, *p<0.05). Furthermore, immunofluorescence imaging of podosomes carried out using anti-vinculin antibody revealed more pronounced and increased podosomes in ROCK1 deficient macrophages compared to WT controls (n=3, *p<0.05). Biochemical analysis of ROCK1−/− macrophages revealed that the enhanced recruitment of ROCK1 deficient macrophages and neutrophils was apparent in spite of normal expression of ROCK2 in ROCK1−/− cells and a 60% reduction in overall ROCK activity. Interestingly, although both ROCK1 and ROCK2 co-immunoprecipitate with PTEN in response to cytokine induced stimulation, only ROCK1 appeared to be essential for PTEN phosphorylation, activation and stability. In the absence of ROCK1, PTEN phosphorylation, its activity and stability were significantly impaired in spite of the presence of ROCK2 (n=3, *p<0.05). Consequently, an increase in the activation of downstream targets of PTEN including AKT, GSK-3β and cyclinD1 was observed in ROCK1 deficient macrophages relative to controls (n=3). Taken together, these studies reveal a biochemical pathway involving ROCK1 and PTEN which is involved in the recruitment of macrophages and neutrophils during acute inflammation. Thus, ROCK1 likely functions as a physiologic regulator of PTEN whose function is to repress excessive recruitment of macrophages and neutrophils during acute inflammation. Disclosures:No relevant conflicts of interest to declare.

Angiopoietin-Like Protein 3 Supports the Activity of Mouse Hematopoietic Stem Cells in the Bone Marrow Niche.

Abstract 249The physiological role of Angiopoietin-like proteins (Angptls) in hematopoietic system is unknown. Here we showed that Angptl3 is expressed by both hematopoietic stem cells (HSCs) and bone marrow stromal cells. In particular, the expression of Angptl3 in the bone marrow stromal cells is significantly increased upon transplantation, suggesting that this protein may play an important role in the bone marrow under stress. We asked whether Angptl3 expression had a functional role in HSCs by utilizing mice ablated for Angptl3. Using Hoechst/pyronin Y staining and Brdu incorporation analysis, we found that HSCs in Angptl3-null mice exhibited significantly decreased quiescence compared to those in wild-type mice. To test the role of Angptl3 in the stress response of hematopoietic cells, we treated mice with 5-fluorouracil (5-FU), which is toxic to cycling cells and accelerates the entry of HSCs into the cell cycle. The survival of Angptl3-null mice was significantly lower than that of wild-type mice. To further identify the role of Angptl3 in stress response of HSCs, we examined whether Angptl3 affected DNA damage in HSCs upon transplantation. To this end, we transplanted WT bone marrow cells into lethally irradiated Angptl3 null recipients or WT mice. We found that HSCs in the Angptl3 null recipient mice had significantly increased gamma-H2AX foci compared to WT recipients, suggesting that Angptl3 protects HSCs from DNA damage. We further used the competitive reconstitution analysis to determine the roles of Angptl3 on HSC activity. Importantly, both Angptl3-null HSCs transplanted to wild-type recipients and wild-type HSCs transplanted to Angptl3-null recipients showed impaired repopulation. These results conclude that Angptl3 has both cell-autonomous and environmental effects that support the in vivo activity of HSCs.To identify the intracellular target of Angptl3 in HSCs, we performed DNA microarray and real-time RT-PCR analyses to compare the gene expression in HSCs isolated from WT and Angptl3 null mice. We found that Angptl3-null HSCs had increased expression of transcription factor Ikaros. Consistently, extrinsic treatment of HSCs by Angptl3 also suppressed the expression of Ikaros. Ikaros is a zinc finger transcription factor important for differentiation of lymphoid, myeloid, and erythroid cells, and its expression is low in multi-potent HSCs, but high in progenitors with lymphoid-myeloid potential. Since Angptl3 downregulates the expression of Ikaros in HSCs, we examined the effect of forced expression of Ikaros on HSC activities. Indeed, overexpression of Ikaros enhanced HSC cycling and DNA damage, and diminished their repopulation activity, indicating the downregulation of Ikaros by extrinsic Angptl3 maintains the stemness of HSCs.We studied the spatial relationship of Angptl3-expressing cells and the bone-marrow HSCs using immunohistochemical tools. We showed that 58.6% of Angptl3-producing cells were in contact with sinusoidal endothelial cells in bone marrow and that 60.8% of HSCs are adjacent to Angptl3-producing cells in the bone marrow. To directly test whether Angptl3-producing bone marrow stromal cells support HSC expansion, we co-cultured HSCs and CD45-SSEA4+ cells and used competitive reconstitution analysis to measure HSC activity. HSCs co-cultured with WT CD45-SSEA4+ cells had significantly increased repopulation relative to those co-cultured with Angptl3 null CD45-SSEA4+ cells (36% vs. 17%). This result demonstrated that bone marrow CD45-SSEA4+ cells support expansion of HSCs, and provided the functional evidence that Angptl3-producing stromal cells are a part of HSC niche in the bone marrow.Thus, Angptl3-producing cells are an important component of the HSC niche. Our experiments demonstrate a unique example of the extrinsic control of stemness by cell-autonomous effects from stem cells per se and by environmental effects from the niche cells. Disclosures:No relevant conflicts of interest to declare.

Abstract 5632: Cardiac Repair Following Myocardial Injury is Mediated by Notch1 Signaling in Mesenchymal Stem Cells

Background: Notch receptors regulate binary cell fate determination and are critical for embryonic development. In particular, Notch1 regulates the fate of cardiac progenitor cells and is an important mediator of vasculogenesis. However, it is little known whether Notch1 could contribute to the cardiac repair process following myocardial injury. Methods and Results: We generated mutant mice with cardiac-specific deletion of Notch1 ( α MHC-Cre/Notch1 flox/flox mice, C-N1−/−). After left descending artery (LAD) ligation, infarct size, cardiac dimension, and heart function were similar between C-N1−/− mice and control mice. However, global heterozygous Notch1 (N1+/−) mice exhibited larger infarct size, greater LV dimension, and worsening LV function compared to that of WT mice. These findings suggest that non-cardiac Notch1 contributes to cardiac repair. When the bone marrow (BM) of N1+/− mice was transplanted into WT mice, infarct size, and impaired heart function were greater, vascularity in the infarct border area was reduced, compared to WT mice transplanted with WT BM. Alternatively, WT BM transplantation (BMT) could rescue the increased myocardial injury observed in N1+/− mice. Using GFP labeled BM cell, we found more mesenchymal stem cells (MSCs) from WT BM could be recruited to the heart compared to that of N1+/− BM. Notch1 deficient also prevent the differentiation of BM-derived cells into vascular cells. In vitro, compared to WT MSCs, N1+/− MSCs showed less migration and proliferative capacity. When N1+/− MSCs were intramyocardially injected into the infarct heart of WT mice, infarct size was greater and blood vessel density was reduced compared that of WT MSCs. Indeed, N1+/− MSCs showed decreased survival after injection compared to WT MSCs. Furthermore, the expression of Notch1 target genes, Hey1 and HeyL, and cytokine receptors CSF3R and CXCR4 were reduced in N1+/− MSCs. This decrease was reversed by overexpression of Notch1 intracellular domain. Conclusions: These findings indicate that Notch signaling in MSC is critical for cardiac repair after myocardial infarction, and suggest that therapeutic strategies that increase Notch1 signaling in MSC could have clinical benefits in patients with ischemic heart disease.

Abstract 5459: Unexpected Acceleration of Atherosclerosis in Ldlr-null Mice Reconstituted With Group X Secretory Phospholipase A2-Deficient Bone Marrow

Group X (GX) secretory phospholipase A2 (sPLA2) has the most potent hydrolyzing activity toward phosphatidylcholine and elicits a marked release of arachidonic acid among several types of sPLA2. GX is expressed by vascular cells and inflammatory cells like macrophages and CD4+ T lymphocytes and is therefore believed to play a pathogenic role in atherosclerosis. We evaluated the effects of GX deficiency on systemic immune response and the development of atherosclerosis, a vascular disease sustained by lipid modifications and inflammation. GX KO mice showed increased spleen size confirmed by higher splenocyte number compared to C57BL6 controls (P&lt;0.01). LPS/IFN- γ -stimulated GX-deficient splenocytes produced more IL-12 than control splenocytes (3380 vs 1599 pg/ml, P&lt;0.05), with no difference in IL-10. Purified splenic CD4+ T cells from GX KO mice showed enhanced proliferation and production of the Th1 cytokine IFN- γ (5575 vs 1443 pg/ml, respectively, p&lt;0.05) compared with controls. GX-deficient blood mononuclear cells showed enhanced adhesion on activated HUVECs (8.8 vs 3.2 cell/field, P&lt;0.01) and on LDLr KO carotid arteries (4.2 vs 6.4 cell/mm, P&lt;0.01) ex vivo, compared to control cells. When challenged in vitro to test cell death susceptibility to a variety of apoptotic stimuli, bone marrow-derived GX-deficient macrophages displayed a significant increase of apoptosis compared to control cells. Finally, LDLr KO mice were lethally irradiated and reconstituted with either GX KO or wild type bone marrow. After 8 weeks of fat diet, mice reconstituted with GX KO bone marrow showed a ~100% increase of plaque size in the aortic sinus compared to mice with WT bone marrow (294 254 μ m2 vs 142 304 μ m2, respectively, P=0.001), despite similar plasma cholesterol level. This was associated with a more advanced plaque phenotype and marked accumulation of TUNEL-positive apoptotic cells (2.4 % vs 0.08 P&lt;0.001). Immunologic analysis of splenocytes and splenic CD4+ T cells from GX/LDLr chimeric mice showed increased immune cell activation and Th1-related cytokine production. In conclusion, GX sPLA2 deficiency increases innate and adaptive immune responses, enhances macrophage susceptibility to apoptosis and accelerates atherosclerosis in mice.

Abstract 5816: The Human Apoa-1 Transgene Suppresses Myeloid Proliferation and Accelerated Atherosclerosis in Ldlr Deficient Mice Reconstituted With ABC Transporter Deficient Bone Marrow

Overexpression or infusions of apoA-1 or HDL lead to reduced atherosclerosis. A major mechanism to explain this effect is thought to be cholesterol efflux from arterial wall macrophage foam cells via ABC transporters. In this study we have tested this hypothesis by transplanting bone marrow deficient in ABCA1 and ABCG1 into atherogenic diet-fed Ldlr deficient mice with or without expression of a human apoA-1 transgene. Unexpectedly, the apoA-1 transgene dramatically suppressed the accelerated atherosclerosis as well as leukocytosis, monocytosis and myeloid cell infiltration of organs in mice receiving double KO bone marrow. Double KO bone marrow showed a 5-fold expansion of hematopoietic stem cells, increased cycling of stem cells, increased myeloid progenitor cells and increased proliferation of bone marrow myeloid cells in response to growth factors. Double KO bone marrow myeloid cells showed increased association of ras with the plasma membrane possibly reflecting increased cholesterol content on the inner leaflet of the plasma membrane and increased ERK signaling in response to IL-3 or GM-CSF treatments. HDL promoted cholesterol efflux and decreased Ras membrane association and ERK signaling and inhibited proliferation of both WT and double KO bone marrow myeloid cells. The findings indicate a novel mechanism in which HDL may suppress atherosclerosis by decreasing myeloid cell proliferation and consequent monocytosis.

Abstract 5053: Deficiency of ASC (Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain) in Bone Marrow Cells Improved Myocardial Ischemia-Reperfusion Injury in Mice

Background: Inflammatory responses play a key role in the pathophysiology of myocardial ischemia-reperfusion (I/R) injury. ASC is an adaptor protein that forms inflammasome whose activation leads to caspase-1-dependent interleukin (IL)-1 β generation and subsequent inflammatory responses; however, the role of ASC in myocardial I/R injury remains to be determined. Methods and Results: ASC deficient (ASC −/− ) and wild-type (WT) mice were subjected to 30 min LAD occlusion, followed by reperfusion. ASC −/− mice showed improved LV dysfunction (%FS: 34.0% vs. 25.7% at 14 days p&lt;0.01), reduced infarct area/area at risk (IA/AAR: 18.7% vs. 28.6% at 48 h, p&lt;0.01), and scar formation (scar/LV area: 9.7% vs. 14.6% at 14 days, p&lt;0.01) after myocardial I/R. Immunostaining revealed decreased infiltration of macrophages (Mac3) and neutrophils (Gr-1), but not neovascularization (CD31), in the injured myocardium of the ASC −/− mice. Real-time RT-PCR and ELISA analyses demonstrated that the myocardial mRNA and protein expression of inflammatory cytokines, such as IL-1 β , IL-6, and MCP-1, after I/R were significantly decreased in the ASC −/− mice. Further, improved myocardial I/R injury was also observed in the caspase-1 −/− mice, compared with WT mice (IA/AAR: 20.2% vs. 28.6% at 48 h, p&lt;0.05). Since double immunostaining revealed that ASC was expressed in the infiltrated macrophages and neutrophils, bone marrow-transplanted (BMT) mice were prepared. The IA/AAR was significantly decreased in the BMT ASC−/−→WT mice, compared with BMT WT→WT (IA/AAR: 17.8% vs. 27.9% at 48 h, p&lt;0.01). Further, in vitro experiments showed that LPS-induced production of inflammatory cytokines such as IL-1 β , IL-6, and MCP-1 in the ASC −/− -bone marrow cells was significantly decreased compared, with that in the WT-bone marrow cells. Conclusion: ASC deficiency improved myocardial I/R injury via inflammatory cell infiltration and cytokine expression. These results suggest that ASC is a novel therapeutic target for myocardial I/R injury.

Transcellular biosynthesis of cysteinyl leukotrienes in vivo during mouse peritoneal inflammation

Leukotrienes (LTs) are lipid mediators of inflammation formed by enzymatic oxidation of arachidonic acid. One intriguing aspect of LT production is transcellular biosynthesis: cells expressing 5-lipoxygenase (5LO) form LTA(4) and transfer it to cells expressing LTA(4) hydrolase (LTA(4)H) or LTC(4) synthase (LTC(4)S) to produce LTB(4) or LTC(4). This process has been demonstrated in vivo for LTB(4), but not for cysteinyl LTs (cysLTs). We examined transcellular cysLT synthesis during zymosan-induced peritonitis, using bone marrow transplants with transgenic mice deficient in key enzymes of LT synthesis and analyzing all eicosanoids by liquid chromatography/tandem mass spectrometry. WT mice time-dependently produced LTB(4) and cysLTs (LTC(4), LTD(4), and LTE(4)). 5LO(-/-) mice were incapable of producing LTs. WT bone marrow cells restored this biosynthetic ability, but 5LO(-/-) bone marrow did not rescue LT synthesis in irradiated WT mice, demonstrating that bone marrow-derived cells are the ultimate source of all LTs in this model. Total levels of 5LO-derived products were comparable in LTA(4)H(-/-) and WT mice, but were reduced in LTC(4)S(-/-) animals. No differences in prostaglandin production were observed between these transgenic or chimeric mice. Bone marrow cells from LTA(4)H(-/-) or LTC(4)S(-/-) mice injected into 5LO(-/-) mice restored the ability to synthesize LTB(4) and cysLTs, providing unequivocal evidence of efficient transcellular biosynthesis of cysLTs. These results highlight the potential relevance of transcellular exchange of LTA(4) for the synthesis of LTs mediating biological activities during inflammatory events in vivo.

The roles of type I IFN receptor and neutrophils in regulating innate immunity to Leishmania parasites (133.26)

Abstract Type I IFNs exert diverse effector/regulatory functions in innate and adaptive immune responses to viruses and bacteria; however, their roles in parasitic infections are less clear. It has been reported that parasite-induced type I IFNs are critical for NO-dependent disease control, and that administration of IFN-β has a dose-dependent protective effect in L. major-infected mice. Surprisingly, we found that following infection with L. amazonensis parasites, IFNAR-/- mice developed significantly attenuated disease with low levels of cytokines and sera antigen-specific IgG. The marked reduction in tissue parasite loads and inflammatory responses even at 3 days of infection in IFNAR-/- mice suggests the possibility of neutrophil-mediated parasite clearance. First, although WT and IFNAR-/- mice were efficient in recruiting CD11b+F4/80+Gr1+ peritoneal neutrophils at 6 h post-injection of parasites, knockout mice displayed unique phenotypic changes in Gr1lowCD11bhigh macrophages and Gr1highCD11blow neutrophils at 24 h. Second, in addition to release more neutrophil elastase and myeloperoxidase, IFNAR-/- not STAT1-/- neutrophils had significantly higher rates of spontaneous and infection-induced apoptosis than did their WT controls. Third, while macrophages alone responded comparably to parasites, the interactions between macrophages and IFNAR-/- neutrophils (but not WT and STAT1-/- neutrophils), greatly enhanced parasite killing. Finally, adoptive transfer of WT bone marrow neutrophils into IFNAR-/- mice did enhance tissue parasite loads. This study suggests an important role for type I IFNs in regulating neutrophil turnover and Leishmania infection and provides new information on innate immunity to protozoan parasites.

Angiotensin II‐induced microvascular responses in mouse brain

Although angiotensin II (AngII)‐induced hypertension increases the risk for ischemic stroke, the mechanisms linking AngII to this cardiovascular disease remain poorly understood. We determined whether AngII (delivered over a 2 wk period with an Alzet pump) promotes leukocyte (LECA)‐ and platelet (PECA)‐endothelial cell adhesion in cerebral venules and/or alters blood brain barrier (BBB) permeability in C57Bl/6 (WT), AT1‐R−/− mice, and AT1‐R−/−‐&gt;WT bone marrow chimeras (BMC). Intravital video microscopy was used to monitor blood cell adhesion from a cranial window. AngII infusion induced dose‐dependent (400‐2000 ng/kg/min) increases in mean arterial pressure (MAP), LECA, and PECA. AT1‐R−/−, AT1‐R−/−‐&gt;WT BMC, and WT mice treated with Losartan (25 mg/kg/day) all exhibited no changes in MAP, LECA and PECA in response to AngII. BBB permeability, measured by Evans blue extravasation, was increased in response to AngII in WT and AT1‐R−/−‐&gt;WT BMC mice, but not in AT1‐R−/− mice and WT mice treated with Losartan. These findings implicate AT1‐receptors (AT1‐R) on blood cells in the inflammatory and pro‐thrombogenic phenotype induced during AngII‐induced hypertension. The accompanying BBB dysfunction appears to involve AT1‐R on endothelial cells, rather than blood cells. (Supported by HL26441)

AIRE regulates T-cell-independent B-cell responses through BAFF.

Autoimmune polyendocrine syndrome type I (APS I) results in multiple endocrine organ destruction and is caused by mutations in the autoimmune regulator gene (AIRE). APS I is characterized by circulating tissue-specific autoantibodies, and the presence of these antibodies is often predictive of organ destruction. The importance of AIRE in ensuring central tolerance by regulating the negative selection of autoreactive T cells has been shown clearly. However, in Aire(-/-) mice the phenotype (i.e., autoantibodies, liver infiltrates of B cells, splenomegaly, and marginal zone B-cell lymphoma) is predominantly B-cell mediated, suggesting an exaggerated activation of B cells. We have studied T-cell-independent B-cell responses in the absence of AIRE and found that Aire(-/-) mice have an increased response against T-cell-independent type II antigens. We linked this exaggerated response to the elevated serum levels of the B-cell-activating factor of the TNF family (BAFF) that were found both in APS I patients and in Aire(-/-) mice. Transfer of Aire(-/-) bone marrow into irradiated nude mice resulted in increased percentage of BAFF-expressing antigen-presenting cells compared with wt bone marrow, suggesting a T-cell-independent mechanism behind our findings. Furthermore, in vitro experiments showed that AIRE-deficient murine bone marrow-derived dendritic cells produced significantly more BAFF than wt cells when stimulated with IFN-gamma but not when stimulated with IL-10. Our results suggest a cell-intrinsic role for AIRE in peripheral dendritic cells by regulating IFN-gamma-receptor signaling and point toward complementary mechanisms by which AIRE is involved in maintaining tolerance.

Donor Bone Marrow Derived IL-17 Expressing Cells Exacerbate Chronic Graft-Versus-Host Disease in a Murine Bone Marrow Transplantation.

Th17 is a newly identified T cell lineage that secretes the proinflamatory cytokine IL-17. Th17 cells have been shown to play a crucial role in mediating autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), arthritis, and colitis. However, recent study showed that donor Th17 cell ameliorated acute graft-versus-host disease (GVHD) due to the downregulation of Th1 differentiation in a murine BMT model. The role of IL-17 on developing chronic GVHD, which is pathologically similar to autoimmue diseases, remains unanswered. To this end, we compared the development of chronic GVHD between the lethally irradiated mice (Balb/c, H-2d) given IL-17 knockout(IL- 17KO, C57BL/6 background, H-2b) bone marrow (BM) cells and wild type BM cells with low dose WT splenocytes to induce sublethal acute GVHD and chronic GVHD subsequently. Up to day 60 after BMT no significant differences in clinical symptoms of GVHD including body weight changes were observed between the mice co-injected with IL-17 KO BM cells plus WT splenocytes and WT BM cells plus WT splenocytes. After day 60 the mice receiving WT BM cells plus WT splenocytes experienced weight loss accompanied by skin inflammatory changes, while mice receiving IL-17KO BM plus WT splenocytes showed minimal signs of GVHD as well as mice receiving IL-17 KO BM or WT BM alone. Recovery of body weight on day 160 after BMT was statistically different between two groups (p=0.035). Taken together, IL-17 was exacerbation factor of chronic GVHD, but not acute GVHD. Next we further evaluated the percentage of Th17 cells derived from BM cells (not from infused splenocytes) in the spleen on day 160 after BMT. Percentages of IL-17 expressing cells in the mice receiving WT BM plus WT splenocytes in spleen were significantly higher than those of WT BM alone receiving mice in CD4 (p=0.03) subpopulations. In conclusion, Th17 cells critically involved in the pathogenesis of chronic GVHD. Neutralizing IL-17 would be potent strategy for preventing chronic GVHD. [Display omitted]

Absence of the Hemochromatosis Gene HFE Confers Protection Under Conditions of Stress Erythropoiesis.

A single mutation in the HFE gene (C282Y), a non-classical member of the MHC-I family, may lead to hereditary hemochromatosis (HH). Although the hallmark of HH is iron overload, several lines of evidence point to a distinct hematopoietic function for HFE:HH reticuloendothelial cells are iron deficient;reconstitution of Hfe-KO mice with bone marrow (BM) from wild-type (wt) animals leads to organ iron redistribution;erythropoietic abnormalities have been reported in HH patients, including altered red cell parameters, which have been associated with iron overload.In particular, however, the HFE promoter contains several GATA.1 responsive elements suggesting that it might be expressed in erythroid cells. Our goal was to characterize erythropoiesis in a mouse model of HH, discriminating between intrinsic roles for Hfe and iron overload in these process. Various parameters in Hfe-KO mice were compared to those in wt controls. Atomic absorption was used to measure organ iron content. Erythropoietic analysis was performed by complete blood cell counts and flow cytometry at 2, 5 and 12 months of life (N≥6 mice per group). Erythropoiesis was stressed by administration of phenylhydrazine, phlebotomy (N≥8 mice per group) or elimination of macrophages using clodronate liposomes (N≥4 mice per group). Hematological parameters were followed over time. Finally, lentiviral vectors were used to overexpress Hfe in the BM or liver of Hfe-KO animals (N≥3 mice per group), erythropoietic values being analyzed as described above.Our results indicate increased erythropoiesis at steady state in Hfe-KO animals, characterized by altered red blood cell (RBC) parameters. Analysis of erythroid populations in BM and spleen revealed an increase in the proportion of immature erythroid cells in these compartments of Hfe-KO mice, especially the spleen (9.4% ± 1.8% vs 5.1% ± 1.2% in wt mice, p≤0.05). Compared to normal mice, Hfe-KO mice recovered faster from anemia or hemolytic anemia induced, respectively, by phlebotomy and administration of phenylhydrazine. By day 8 their hemoglobin and hematocrit levels returned to steady state, while normalization required 10 days in wt mice. Preliminary results showed similar behavior in wt mice transplanted with Hfe-KO BM, compared to mice transplanted with wt BM. Since macrophages play an important role in erythropoiesis, we analyzed the erythropoietic behavior of wt and Hfe-KO mice after elimination of macrophages. Anemia induced by macrophage depletion was milder in Hfe-KO mice compared to wt mice (14.0 ± 0.4 g/dL vs 11.5 ± 0.1 g/dL), and characterized by a reduction in the hemoglobin content of RBC (MCH), with little change in the RBC count compared to steady state. In contrast anemia in wt mice was characterized by a reduction in both RBC the count and the MCH. To further investigate the correlation between Hfe, erythropoiesis and the potential role of iron overload in the erythropoietic alterations observed, we generated several lentiviral vectors to express Hfe in specific compartments, including the BM and liver. High levels of Hfe expression in BM led to a lethal anemia. On the other hand, low/normal level of expression led to reversion of the alterations in erythropoiesis at steady state, with no alteration in iron content or distribution compared to Hfe-KO control animals. Lentiviral Hfe gene transfer into the liver of Hfe-KO mice led to reduction of iron in the liver and some redistribution of this metal to the spleen. These mice showed the same erythropoietic alterations as Hfe-KO controls.

Abstract 3387: Hypoxia-induced Vascular Repair by Progenitor Cells Requires the Nadph Oxidase Nox2

Endothelial progenitor cells (EPC) promote vascular repair but the impact of systemic hypoxia on EPC function is unclear. Hypoxia is a strong stimulus for angiogenesis and affects reactive-oxygen species (ROS) production and signalling. We studied the role of hypoxia for EPC mobilization and vascular repair and the contribution of the NADPH oxidase Nox2, the most important source of ROS in endothelial cells, in this process. Murine EPCs were defined as linage negative and sca-1/flk-1 positive and measured by FACS from whole blood. Vascular repair was induced by the carotid artery wire injury model. Hypoxia (10%, 96hours) induced mobilisation of EPC and improved vascular repair after wire injury in wild-type mice but not in NADPH oxidase knockout mice (Nox2y/−). Hypoxia induces the formation of erythropoietin (EPO), which might contribute to EPC mobilization. EPO production in response to hypoxia however was enhanced in Nox2y/− as compared to WT mice. Importantly, EPO, which induced pronounced EPC mobilization in WT mice, failed to do so in Nox2−/− mice. These data suggest that EPO signalling is defective in Nox2y/− mice. Indeed, in EPCs from WT-mice, EPO induced ROS production and proliferation and both responses were missing in EPCs from Nox2y/− mice. Transplantation of bone marrow from Nox2y/− mice into WT mice resulted in an attenuated mobilization in response to hypoxia and EPO, whereas the transplantation of WT bone marrow into Nox2y/− mice restored the normal phenotype . This demonstrates that a failure of bone marrow stem cells to generate ROS in response to EPO underly the mobilization defect. We conclude that Nox2-derived ROS are required for EPO-signalling in stem cells and that hypoxia-induced EPC mobilisation and vascular repair requires the NADPH oxidase Nox2.

Innate Immunity to Pneumococcal Infection of the Central Nervous System Depends on Toll‐Like Receptor (TLR) 2 and TLR4

Recent studies have suggested that, in addition to Toll-like receptor (TLR) 2, other pattern recognition receptors mediate activation of the immune response after infection of the central nervous system (CNS) with Streptococcus pneumoniae (SP). Using a mouse meningitis model, we investigated the influence of TLR4 single deficiency (TLR4(-/-)), TLR2/TLR4 double deficiency (TLR2/4(-/-)), and TLR2/TLR4/TLR9 triple deficiency (TLR2/4/9(-/-)) on the immune response of the CNS to SP infection. To identify the cell populations that mediate the responses to SP, we generated TLR2/4(-/-)-wild-type (wt) bone marrow (BM) chimeras. Compared with infected wt mice, infected TLR2/4(-/-) and TLR2/4/9(-/-) mice had similar reductions in brain cytokine levels, pleocytosis, and cerebral pathologic findings, whereas no such effect was noted in infected TLR4(-/-) mice. The attenuated immune response was paralleled by an impaired host defense that resulted in worsening of disease. Analysis of the chimeric mice after infection showed that mere TLR2/4 deficiency, either of radioresistant cells or of transplanted BM-derived cells, was sufficient to mount a substantial cerebral immune response, such as that noted in wt mice. In murine SP meningitis, TLR2 and TLR4 expressed on radioresistant and transplanted BM-derived cells were major cellular sensors of invading SP inducing inflammatory responses.

CD40L is crucial for the development of autoimmunity induced by TLR7 overexpression

Antibodies against nuclear antigens secreted by autoreactive B cells are a hallmark of systemic autoimmunity. Failure to eliminate autoreactive B cells in the germinal center reaction has been reported in lupus disease. We have shown that control of TLR7 expression is essential to restrict B cell autoimmunity and DC expansion. However, the underlying mechanisms of TLR7‐induced autoimmunity still need to be scrutinized. We found increased spontaneous germinal center formation in Tg.TLR7 mice. B cells from these mice showed enhanced H2‐Ab1, stat1, T‐bet, AID expression and increased IgG1 and IgG2b immunoglobulin transcript. To test whether these B cell phenotypes required T cell help, we bred Tg.TLR7 with CD40L deficient mice. In the absence of CD40L, Tg.TLR7 mice did not develop spontaneous germinal center or any autoimmune phenotypes. Reconstitution experiments with an equal mix of Tg.TLR7.CD40L−/− and WT bone marrow showed that CD40L provided in trans can induced the disease. All of these results suggest that autoreactive B cell and autoimmune phenotype of Tg.TLR7 mice is CD40L‐dependent. Future experiments will determine whether T cells are needed to provide help through CD40L in this system.

Blocking TNF-α in mice reduces colorectal carcinogenesis associated with chronic colitis

The inflammatory bowel disease ulcerative colitis (UC) frequently progresses to colon cancer. To understand the mechanisms by which UC patients develop colon carcinomas, we used a mouse model of the disease whereby administration of azoxymethane (AOM) followed by repeated dextran sulfate sodium (DSS) ingestion causes severe colonic inflammation and the subsequent development of multiple tumors. We found that treating WT mice with AOM and DSS increased TNF-alpha expression and the number of infiltrating leukocytes expressing its major receptor, p55 (TNF-Rp55), in the lamina propria and submucosal regions of the colon. This was followed by the development of multiple colonic tumors. Mice lacking TNF-Rp55 and treated with AOM and DSS showed reduced mucosal damage, reduced infiltration of macrophages and neutrophils, and attenuated subsequent tumor formation. WT mice transplanted with TNF-Rp55-deficient bone marrow also developed significantly fewer tumors after AOM and DSS treatment than either WT mice or TNF-Rp55-deficient mice transplanted with WT bone marrow. Furthermore, administration of etanercept, a specific antagonist of TNF-alpha, to WT mice after treatment with AOM and DSS markedly reduced the number and size of tumors and reduced colonic infiltration by neutrophils and macrophages. These observations identify TNF-alpha as a crucial mediator of the initiation and progression of colitis-associated colon carcinogenesis and suggest that targeting TNF-alpha may be useful in treating colon cancer in individuals with UC.

NPM-ALK Induces Tumour Development in a Murine Model of Anaplastic Large Cell Lymphoma (ALCL) Independently of Bcl-3 Expression.

Anaplastic large cell lymphomas (ALCLs) define a subgroup of aggressive non-Hodgkin's lymphomas. In 40–60% of systemic ALCLs, a t(2;5) (p23;q35) translocation is found, which generates a fusion gene between nucleophosmin (NPM) and the receptor tyrosine kinase gene ALK (anaplastic lymphoma kinase). The NPM-ALK chimeric gene encodes a constitutively activated tyrosine kinase and is believed to initiate the process of lymphomagenesis. Since NPM-ALK has been shown to activate PI3-kinase and STAT3, proteins that are involved in apoptosis regulation, altered apoptosis might contribute to ALCL development. Bcl-3, which exerts an anti-apoptotic effect in B and T lymphocytes, has been reported to be up regulated in a subgroup of ALCLs. This raised the question whether Bcl-3 is required for tumour development induced by NPM-ALK. In a first set of experiments, the expression levels of Bcl-3 in NPM-ALK negative and NPM-ALK positive tumour cells were analysed. Bcl-3 expression was enhanced in NPM-ALK expressing cells compared to NPM-ALK negative cells or cells expressing a kinase dead NPM-ALK mutant. Next we used primary murine bone marrow cells from wt versus Bcl-3 −/− mice in order to clarify the contribution of Bcl-3 to NPM-ALK induced transformation. We employed a retroviral infection system utilizing a MCSV-based vector co-expressing NPM-ALK together with the enhanced green fluorescent protein (EGFP) via an internal ribosomal entry site to infect Bcl-3−/− and wild type (wt) bone marrow cells. Transformation of bone marrow cells was analysed by methylcellulose assay without cytokines. Transformation of NPM-ALK infected Bcl-3−/− bone marrow cells was comparable to NPM-ALK infected BL6 wt bone marrow cells. No colony formation was detectable after transfection of Bcl-3−/− bone marrow with empty vector as control. Finally, we utilized a murine transplantation model of ALCL. Lethally irradiated BL6 wt mice were transplanted with retrovirally NPM-ALK infected wild type or Bcl-3−/− bone marrow cells. As a control, BL6 wt mice received a transplant of Bcl-3−/− bone marrow cells infected with supernatant from viral producer cells transfected with empty vector. Mice transplanted with NPM-ALK infected wild type or Bcl-3−/− bone marrow cells rapidly died within a median survival time of 16 and 17 days respectively, whereas mice transplanted with Bcl-3 −/− bone marrow cells transfected with empty vector survived healthy for more than 300 days. Diseased mice macroscopically showed involvement of the spleen, predominantly. Histologically, spleens of diseased mice showed an extensive infiltration of ALK-positive tumour cells with proliferation of large histiocytic cells both in mice transplanted with NPM-ALK infected Bcl-3−/− and wild type bone marrow. In both groups, FACS-analysis revealed a high percentage of EGFP positive and thus NPM-ALK positive cells in bone marrow and spleen. In conclusion, NPM-ALK is able to transform bone marrow cells and to induce a lymphoma-like disease in the absence of Bcl-3. Bcl-3 is thus dispensable for ALCL development in a murine mouse model. Lack of Bcl-3 in the knockout mice may be compensated by the expression of other proteins. Therefore, Bcl-3 upregulation in a subgroup of human ALCLs may be not critical for lymphoma development.

Phospho- AKT Is Not Sufficient To Delay Neutrophil Apoptosis.

Neutrophils play an important role in the innate immune response and usually are the first cells to defend the organism against infections. The lifetime of neutrophils is tightly regulated and once the mature cells left the bone marrow most of them undergo apoptosis within 48 hours. The rate of neutrophil apoptosis can be modulated by a variety of cytokines, including granulocyte colony-stimulating factor (G-CSF). To investigate the involvement of the G-CSF receptor in the neutrophil susceptibility to apoptosis we utilized mice bearing truncation mutations on the G-CSF receptor (G-CSFR) and also the G:EpoR mutation, where the entire cytoplasmic (signaling) domain of the G-CSFR is replaced with that of the erythropoietin receptor (EpoR). We examined the role of AKT-mTOR pathway in neutrophil apoptosis. Bone marrow neutrophils from wild type (C57BL/6) and mutant mice where cultured in presence or absence of G-CSF and/or Rapamycin (a specific mTor inhibitor); the apoptosis and the level of phosphorylation of AKT and ERK were evaluated. Neutrophils extracted from G:EpoR mice do not respond to G-CSF treatment and undergo apoptosis within 24 hours, although WT neutrophils can be partially protected against apoptosis when treated with G-CSF. Interestingly, G-CSF treatment leads to activation of Akt and Erk kinases, even in G:EpoR neutrophils. Furthermore, the addition of Rapamycin had no effect in the apoptosis of WT neutrophils in presence or absence of G-CSF. To further elucidate the role of the AKT-mTOR pathway, Sca+Lin- WT bone marrow cells were utilized in an in vitro G-CSF dependent granulocytic differentiation assay. The experiments showed that the mTor pathway is important for the early stages of myeloid differentiation but has little influence in the late stages, and also in the life span of neutrophils. Interestingly, over expression of a constitutively active form of AKT (myr-AKT) in Sca+Lin- cells impaired proliferation even though the differentiation was not affected. To overcome this, a tetracycline response element (TRE) conditional expression system was utilized. These experiments showed that myr-AKT expression does not rescue neutrophils from apoptosis. In conclusion, our results indicate that after treatment with G-CSF; AKT and ERK are phosphorylated in WT and G:EpoR neutrophils, despite this, G-CSF does not suppress G:EpoR neutrophil apoptosis while WT neutrophils can have their apoptosis partially delayed. We also found that mTor is involved in the early stages of G-CSF dependent myeloid differentiation and its inhibition can partially arrest differentiation. Conversely, rapamycin does not affect neutrophil apoptosis, in presence or not of G-CSF. In concordance with these results overexpression of a constitutive active form of AKT, myr-AKT does not rescue neutrophils from apoptosis and it is also detrimental to early stages of granulocytic proliferation. These data strongly suggest that the AKT-mTor pathway while important in early myeloid differentiation and proliferation does not play a role in cytokine suppression of neutrophil apoptosis.

Systemic inflammation and end organ damage following trauma involves functional TLR4 signaling in both bone marrow-derived cells and parenchymal cells

Endogenous damage-associated molecular pattern (DAMP) molecules are released from cells during traumatic injury, allowing them to interact with pattern recognition receptors such as the toll-like receptors (TLRs) on other cells and subsequently, to stimulate inflammatory signaling. TLR4, in particular, plays a key role in systemic and remote organ responses to hemorrhagic shock (HS) and peripheral tissue injury in the form of bilateral femur fracture. TLR4 chimeric mice were generated to investigate the cell lineage in which functional TLR4 is needed to initiate the injury response to trauma. Chimeric mice were generated by adoptive bone marrow (BM) transfer, whereby donor marrow was given to an irradiated host using reciprocal combinations of TLR4 wild-type (WT; C3H/HeOuJ) and TLR4 mutant (Mu; C3H/HeJ) mice. After a period of engraftment, chimeric mice were then subjected to HS or bilateral femur fracture. Control groups, including TLR4-WT mice receiving WT BM and TLR4-Mu mice receiving Mu BM, responded to injury in a similar pattern to unaltered HeOuJ and HeJ mice, and protection was afforded to those mice lacking functional TLR4. In contrast, TLR4-WT mice receiving Mu BM and TLR4-Mu mice receiving WT BM demonstrated intermediate inflammatory and cellular damage profiles. These data demonstrate that functional TLR4 is required in BM-derived cells and parenchymal cells for an optimal inflammatory response to trauma.

P110γ and p110δ Phosphoinositide 3-Kinase Signaling Pathways Synergize to Control Development and Functions of Murine NK Cells

Phosphoinositide 3-kinases (PI-3Ks) are key enzymes for cell development, activation, and survival. Here we showed that PI-3K class IB and class IA catalytic subunits, p110gamma and p110delta, played a crucial role in the development and functions of murine NK cells. p110gamma deficiency and impairment of G protein-coupled receptor (GPRC) signaling prevented full NK cell maturation. Concomitant loss of p110gamma and p110delta exacerbated this defect, resulting in a very small population of NK cells with a highly immature phenotype in the bone marrow and periphery. Moreover, combined p110gamma and p110delta signals were required for cytotoxicity and activation of the kinase ERK during NK cell-target cell interaction. p110gamma played a major role in receptor-induced interferon-gamma (IFN-gamma) production through a pathway that involved the kinase ERK and 5-Lipoxigenase, which most likely generates lipid mediators activating GPRCs. Conversely, PI3Ks negatively regulated interleukin-12 (IL-12) and IL-18-induced IFN-gamma by modulating p38 kinase activation. Our data shed light on the multiple intersecting pathways through which PI3Ks control NK cell-mediated innate responses.