Chemokine receptor CCR1 disruption in bone marrow cells enhances atherosclerotic lesion development and inflammation in mice

Several chemokines or chemokine receptors are involved in atherogenesis. CCR1 is expressed by macrophages and lymphocytes, two major cell types involved in the progression of atherosclerosis, and binds to lesion-expressed ligands. We examined the direct role of the blood-borne chemokine receptor CCR1 in atherosclerosis by transplanting bone marrow cells from either CCR1+/+ or CCR1-/- mice into low-density lipoprotein-receptor (LDLr)-deficient mice. After exposure to an atherogenic diet for 8 weeks, no differences in fatty streak size or composition were detected between the 2 groups. After 12 weeks of atherogenic diet, however, an unexpected 70% increase in atherosclerotic lesion size in the thoracic aorta was detected in the CCR1-/- mice, accompanied by a 37% increase in the aortic sinus lesion area. CCR1-/- mice showed enhanced basal and concanavalin A-stimulated IFN-gamma production by spleen T cells and enhanced plaque inflammation. In conclusion, blood-borne CCR1 alters the immuno-inflammatory response in atherosclerosis and prevents excessive plaque growth and inflammation.     

The presence of leukocyte CC-chemokine receptor 2 in CCR2 knockout mice promotes atherogenesis

OBJECTIVE: To selectively determine the role of leukocyte CC-chemokine receptor 2 (CCR2) in atherogenesis. METHODS AND RESULTS: Bone marrow progenitor cells harvested from CCR2(+/+) mice were transplanted into irradiated CCR2(-/-) mice, representing the whole-body absence of CCR2 except in leukocytes. Transplantation of CCR2(-/-) bone marrow into CCR2(-/-) mice served as control. Eight weeks after bone marrow transplantation, the diet of regular chow was switched to a high-cholesterol diet for another 10 weeks in order to induce atherosclerosis. No significant differences in serum cholesterol and triglyceride levels were observed between the two groups. However, the mean cross-sectional aortic root lesion area of CCR2(+/+)-->CCR2(-/-) mice amounted up to 12.28+/-3.28x10(4) microm(2), compared with only 3.08+/-0.74 x 10(4) microm(2) observed in the CCR2(-/-)-->CCR2(-/-) group. Thus, the presence of CCR2 exclusively on leukocytes induces a fourfold increase in aortic lesion area. This extent of lesion development was comparable to C57Bl/6 mice receiving CCR2(+/+) bone marrow (10.08+/-3.30x10(4) microm(2)). CONCLUSION: These results point at a dominant role of leukocyte CCR2 in atherogenesis, implying that CCR2 from nonleukocyte sources, like endothelial cells or smooth muscle cells, is less critical in the initiation of atherosclerosis. Pharmacological inhibition of leukocyte CCR2 function might be a promising strategy to prevent atherosclerosis.     

Tumor necrosis factor-alpha increases circulating osteoclast precursor numbers by promoting their proliferation and differentiation in the bone marrow through up-regulation of c-Fms expression

Osteoclasts are essential cells for bone erosion in inflammatory arthritis and are derived from cells in the myeloid lineage. Recently, we reported that tumor necrosis factor-alpha (TNFalpha) increases the blood osteoclast precursor (OCP) numbers in arthritic patients and animals, which are reduced by anti-TNF therapy, implying that circulating OCPs may have an important role in the pathogenesis of erosive arthritis. The aim of this study is to investigate the mechanism by which TNFalpha induces this increase in OCP frequency. We found that TNFalpha stimulated cell division and conversion of CD11b+/Gr-1-/lo/c-Fms- to CD11b+/Gr-1-/lo/c-Fms+ cells, which was not blocked by neutralizing macrophage colony-stimulating factor (M-CSF) antibody. Ex vivo analysis of monocytes demonstrated the following: (i) blood CD11b+/Gr-1-/lo but not CD11b-/Gr-1- cells give rise to osteoclasts when they were cultured with receptor activator NF-kappaB ligand and M-CSF; and (ii) TNF-transgenic mice have a significant increase in blood CD11b+/Gr-1-/lo cells and bone marrow proliferating CD11b+/Gr-1-/lo cells. Administration of TNFalpha to wild type mice induced bone marrow CD11b+/Gr-1-/lo cell proliferation, which was associated with an increase in CD11b+/Gr-1-/lo OCPs in the circulation. Thus, TNFalpha directly stimulates bone marrow OCP genesis by enhancing c-Fms expression. This results in progenitor cell proliferation and differentiation in response to M-CSF, leading to an enlargement of the marrow OCP pool. Increased marrow OCPs subsequently egress to the circulation, forming a basis for elevated OCP frequency. Therefore, the first step of TNF-induced osteoclastogenesis is at the level of OCP genesis in the bone marrow, which represents another layer of regulation to control erosive disease.     

Kinetics of rebounding of lymphoid and myeloid cells in mouse peripheral blood, spleen and bone marrow after treatment with cyclophosphamide

Recently, we showed that post cyclophosphamide (CTX) microenvironment benefits the function of transferred T cells. Analysis of the kinetics of cellular recovery after CTX treatment showed that a single 4 mg/mouse CTX treatment decreased the absolute number of leukocytes in the peripheral blood (PBL) at days 3-15, and in the spleen and bone marrow (BM) at days 3-6. The absolute numbers of CD11c(+)CD11b(-) and CD11c(+)CD11b(+) dendritic cells (DCs), CD11b(+) and Ly6G(+) myeloid cells, T and B cells, CD4(+)CD25(+) T regulatory (T(reg)) cells, and NK1.1(+) cells also decreased. The cell numbers returned to control levels during the recovery phase. The absolute numbers of B cells remained low for 3 weeks. The numbers of DCs increased in PBL and spleen at day 9 but returned to control levels at day 15. These data indicate that CTX alters the cellular microenvironment in kinetics that might be precisely targeted to benefit the host.     

Differential role for cyclic AMP response element binding protein-1 in multiple stages of B cell development, differentiation, and survival

CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser(119-ala) phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed approximately 70% increase in pre-B1 (CD43(+)B220(+)CD24(+(int))) and approximately 60% decreased pre-BII (CD43(+)B220(+)CD24(++(high))) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed approximately 4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into RAG-2(-/-) mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43(+)B220(+)CD24(high) population that is normally absent in RAG-2(-/-) mice. In the periphery, the Tg mice exhibited decreased CD21(dim)CD23(high)IgM(+) follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells.     

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.     

Specific loss of toll-like receptor 2 on bone marrow derived cells decreases atherosclerosis in LDL receptor null mice

Innate immunity and, notably, Toll-like receptors (TLR), have an important role in atherogenesis. We have tested the hypothesis that the selective loss of TLR-2 by cells of bone marrow (BM) origin will protect low-density receptor-deficient (Ldlr (-/-)) mice from both early- and late-stage atherosclerosis. BM cells from Tlr2(+/+) and Tlr2(-/-) littermates were used to reconstitute lethally irradiated Ldlr(-/-) mice. Following a recovery period, mice were placed either on a diet containing 21% saturated fat - 0.15% cholesterol for 8?weeks to study early-stage atherosclerosis, or on a diet richer in cholesterol (1.5%) for 16?weeks to study late-stage atherosclerosis. Donor cell Tlr2 genotype did not alter serum cholesterol levels or lipoprotein profiles in recipient animals. After 8?weeks on the 0.15% cholesterol diet, deficiency of TLR-2 expression on cells of BM origin reduced atherosclerosis in the aortic root and the aortic arch in both genders of mice. In contrast, the BM recipients who received the 1.5% cholesterol diet for 16?weeks showed much larger lesions in the aortic root, and TLR-2 deficiency in BM cells failed to provide protection. Thus, TLR-2 expression in BM-derived cells contributes primarily to early stage atherosclerosis.     

C5aR expression in a novel GFP reporter gene knockin mouse: implications for the mechanism of action of C5aR signaling in T cell immunity

C5aR is a G protein-coupled receptor for the anaphylatoxin C5a and mediates many proinflammatory reactions. C5aR signaling also has been shown to regulate T cell immunity, but its sites and mechanism of action in this process remain uncertain. In this study, we created a GFP knockin mouse and used GFP as a surrogate marker to examine C5aR expression. GFP was knocked into the 3'-untranslated region of C5ar1 by gene targeting. We show that GFP is expressed highly on Gr-1(+)CD11b(+) cells in the blood, spleen, and bone marrow and moderately on CD11b(+)F4/80(+) circulating leukocytes and elicited peritoneal macrophages. No GFP is detected on resting or activated T lymphocytes or on splenic myeloid or plasmacytoid dendritic cells. In contrast, 5-25% cultured bone marrow-derived dendritic cells expressed GFP. Interestingly, GFP knockin prevented cell surface but not intracellular C5aR expression. We conclude that C5aR is unlikely to play an intrinsic role on murine T cells and primary dendritic cells. Instead, its effect on T cell immunity in vivo may involve CD11b(+)F4/80(+) or other C5aR-expressing leukocytes. Further, our data reveal a surprising role for the 3'-untranslated region of C5aR mRNA in regulating C5aR protein targeting to the plasma membrane.     

Orp8 Deficiency in Bone Marrow-Derived Cells Reduces Atherosclerotic Lesion Progression in LDL Receptor Knockout Mice

Introduction

Oxysterol binding protein Related Proteins (ORPs) mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow (BM) from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity.

Conclusions

Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.     

Participation of bone-marrow stem cells in the differentiation of mdx mice striated muscle

Two sets of experiments were carried out. The first one involved chimeric mice, obtained by intravenously injections of bone marrow derived cells taken from transgenic C57BL/6 mice, expressing GFP, to 5 Gy X-ray irradiated mdx or C57BL/6 mice. In 2 months M. quadriceps femoris of chimeric mice were destroyed by surgical clamp. Following the next 4-5 weeks, the same muscles were studied for the presence of GFP-positive striated muscle fibres. In the case of chimeric C57BL/6 mice GFP-positive striated muscle fibres were observed in 0.3 +/- 0.5 and in 0.2 +/- 0.3 % of destroyed muscle, and in lateral (control) muscle, consequently. In the case of chimeric mdx mice, positive results were observed in 1.7 +/- 0.4 and in 0.5 +/- 0.3 % of destroyed and control muscles, respectively. In the second set of experiments, the GFP-positive bone marrow cells were used for multiple intramuscular injections to M. quadriceps femoris of C57BL/6 or mdx mice in a dose of 2 x 10(5)-5 x 10(5) cells per mouse. Before injection, GFP-positive bone marrow cells were fractionated in a 63 % Percoll solution and then were exhausted from differentiated cells by magnetic manner using CD4, CD8, CD38, CD45R, CD119, Ly-6G, and F4/80 antibodies. After 2-3 weeks, as many as 0.15 +/- 0.40 and 0.1 +/- 0.2 % of GFP-positive muscle fibres were found in injected and control muscles of C57BL/6 mice, respectively. In the case of mdx mice, the frequency of GFP-positive striated muscle fibres was 2.0 +/- 0.8 and 1.2 +/- 0.6 % for injected and control muscles, respectively. A conclusion is made that bone marrow stem cells can take part in differentiation of mdx mouse muscles after their delivery by needle injections.     

Prevention of TGFβ induction attenuates angII-stimulated vascular biglycan and atherosclerosis in Ldlr−/− mice

Angiotensin II (angII) accelerates atherosclerosis, but the mechanisms are not fully understood. The aim of this study was to determine whether TGFβ is required for angII-induced atherosclerosis. Ldlr-null mice fed a normal chow diet were infused with angII or saline for 28 days. A single injection of TGFβ neutralizing antibody 1D11 (2 mg/kg) prevented angII-induction of TGFβ1 levels, and strikingly attenuated angII-induced accumulation of aortic biglycan content. To study atherosclerosis, mice were infused with angII or saline for 4 weeks, and then fed Western diet for a further 6 weeks. 1D11 had no effect on systolic blood pressure or plasma cholesterol; however, angII-infused mice that received 1D11 had reduced atherosclerotic lesion area by 30% (P < 0.05). Immunohistochemical analyses demonstrated that angII induced both lipid retention and accumulation of biglycan and perlecan which colocalized with apoB. 1D11 strikingly reduced the effect of angII on biglycan but not perlecan. 1D11 decreased total collagen content (P < 0.05) in the lesion area without changing plaque inflammation markers (CD68 and CD45). Thus, this study demonstrates that neutralization of TGFβ attenuated angII stimulation of biglycan accumulation and atherogenesis in mice, suggesting that TGFβ-mediated biglycan induction is one of the mechanisms underlying angII-promoted atherosclerosis.     

Distribution of cellular isoform of prion protein in T lymphocytes and bone marrow, analyzed by wild-type and prion protein gene-deficient mice

In this study, the authors investigated normal cellular prion protein (PrP(C)) expression on murine immune systems using prion protein gene-deficient mouse as negative control. Immunocytes expressing PrP(C) in adult and fetal mice were detected by flow cytometry with the monoclonal antibody against PrP(C), 6H4. Cells from thymus and bone marrow reacted positively with 6H4, while spleen cells, peritoneal cells, peripheral blood leukocytes, and intestinal intraepithelial lymphocytes were nonreactive. In thymus, PrP(C) was observed in CD4(-)CD8(-) double-negative thymocytes. PrP(C+) cells of double-negative thymocytes belonged to the CD3(-) subset, but not to the CD3(+) subset. Triple-negative PrP(C+) thymocytes expressed CD44 or CD25 antigens. Furthermore, PrP(C) was observed in c-kit(+) bone marrow cells. In fetuses, PrP(C+) cells were observed in the liver and thymus at day 16.0 and 15.0 of gestation, respectively. These results demonstrated that PrP(C) is expressed on immature immunocytes.     

Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr-/- mice expressing CETP

In addition to hepatic expression, cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are expressed by human macrophages. The combined actions of these proteins have profound effects on HDL structure and function. It is not known how these HDL changes influence atherosclerosis. To elucidate the role of leukocyte-derived HL on atherosclerosis in a background of CETP expression, we studied low density lipoprotein receptor-deficient mice expressing human CETP (CETPtgLDLr(-/-)) with a leukocyte-derived HL deficiency (HL(-/-) BM). HL(-/-) bone marrow (BM), CETPtgLDLr(-/-) mice were generated via bone marrow transplantation. Wild-type bone marrow was transplanted into CETPtgLDLr(-/-) mice to generate HL(+/+) BM, CETPtgLDLr(-/-) controls. The chimeras were fed a high-fat, high-cholesterol diet for 14 weeks to promote atherosclerosis. In female HL(-/-) BM, CETPtgLDLr(-/-) mice plasma HDL-cholesterol concentration during high-fat feeding was decreased 27% when compared with HL(+/+) BM, CETPtgLDLr(-/-) mice (P < 0.05), and this was associated with a 96% increase in en face aortic atherosclerosis (P < 0.05). In male CETPtgLDLr(-/-) mice, leukocyte-derived HL deficiency was associated with a 16% decrease in plasma HDL-cholesterol concentration and a 25% increase in aortic atherosclerosis. Thus, leukocyte-derived HL in CETPtgLDLr(-/-) mice has an atheroprotective role that may involve increased HDL levels.     

Bone Marrow Deficiency of MCPIP1 Results in Severe Multi-Organ Inflammation but Diminishes Atherogenesis in Hyperlipidemic Mice

Objective

MCPIP1 is a newly identified protein that profoundly impacts immunity and inflammation. We aim to test if MCPIP1 deficiency in hematopoietic cells results in systemic inflammation and accelerates atherogenesis in mice.

Approach and Results

After lethally irradiated, LDLR^−/− mice were transplanted with bone marrow cells from either wild-type or MCPIP1^−/− mice. These chimeric mice were fed a western-type diet for 7 weeks. We found that bone marrow MCPIP1^−/− mice displayed a phenotype similar to that of whole body MCPIP1^−/− mice, with severe systemic and multi-organ inflammation. However, MCPIP1^−/− bone marrow recipients developed >10-fold less atherosclerotic lesions in the proximal aorta than WT bone marrow recipients, and essentially no lesions in en face aorta. The diminishment in atherosclerosis in bone marrow MCPIP1^−/− mice may be partially attributed to the slight decrease in their plasma lipids. Flow cytometric analysis of splenocytes showed that bone marrow MCPIP1^−/− mice contained reduced numbers of T cells and B cells, but increased numbers of regulatory T cells, Th17 cells, CD11b+/Gr1+ cells and CD11b+/Ly6C^low cells. This overall anti-atherogenic leukocyte profile may also contribute to the reduced atherogenesis. We also examined the cholesterol efflux capability of MCPIP1 deficient macrophages, and found that MCPIP1deficiency increased cholesterol efflux to apoAI and HDL, due to increased protein levels of ABCA1 and ABCG1.

Conclusions

Hematopoietic deficiency of MCPIP1 resulted in severe systemic and multi-organ inflammation but paradoxically diminished atherogenesis in mice. The reduced atheroegensis may be explained by the decreased plasma cholesterol levels, the anti-atherogenic leukocyte profile, as well as enhanced cholesterol efflux capability. This study suggests that, while atherosclerosis is a chronic inflammatory disease, the mechanisms underlying atherogenesis-associated inflammation in arterial wall versus the inflammation in solid organs may be substantially different.     

Induction of central deletional T cell tolerance by gene therapy

Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag K(b) were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2K(b) resulted in lifelong expression of K(b) on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2K(b) transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with K(b) transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to K(b). Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.     

Genetically modified bone marrow-derived vehicle cells site specifically deliver an anti-inflammatory cytokine to inflamed interstitium of obstructive nephropathy

In this study, we used genetically modified bone marrow-derived CD11b(+)CD18(+) vehicle cells to deliver IL-1 receptor antagonist (IL-1ra) for treatment of inflamed renal interstitium in an animal model of unilateral ureteral obstruction (UUO). Vehicle cells that expressed the ICAM-1 ligands, CD11b and CD18, were obtained from bone marrow cells of DBA/2j mice and adenovirally transduced with the IL-1ra gene or glucocerebrosidase (GC) gene ex vivo. In kidneys treated to develop UUO, levels of ICAM-1, IL-1 beta, and IL-1R expression increased within 3 days compared with contralateral untreated kidneys in the same mice. Similarly, the macrophage infiltration in the cortical interstitium increased after 3 days in UUO kidneys, but not untreated kidneys. After UUO developed, DBA/2j mice were injected i.v. with either IL-1ra(+) vehicle cells (IL-1ra-treated mice) or GC(+) vehicle cells (GC-treated mice) at 24 h after UUO. Six days after the injection of these vehicle cells, marked increase of CD11b(+) IL-1ra(+) vehicle cells was observed in the ICAM-1-positive interstitium of UUO kidneys from IL-1ra-treated mice. In contrast, no CD11b(+) IL-1ra(+) cells appeared in ICAM-1-negative contralateral kidneys from these mice. Furthermore, the infiltration of macrophages (p < 0.001), expression of ICAM-1 (p < 0.005), and presence of alpha-smooth muscle actin (p = 0.005) in the interstitium of UUO kidneys were significantly decreased in IL-1ra-treated mice compared with GC-treated mice. These findings suggest that IL-1 may contribute to the development of renal interstitial injury and that our method can deliver a functioning gene encoding an antiinflammatory cytokine gene specifically at that site by interacting with local adhesion molecules.     

Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells, and an increase in granulocytes (CD11b(+)Ly6G(+)) in blood of miR155(-/-) transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the 'inflammatory' monocyte subset (CD11b(+)Ly6G(-)Ly6C(hi)) and reduces 'resident' monocytes (CD11b(+)Ly6G(-)Ly6C(low)) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155(-/-) transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.     

Expression of CD28 by bone marrow stromal cells and its involvement in B lymphopoiesis

Young mice lacking CD28 have normal numbers of peripheral B cells; however, abnormalities exist in the humoral immune response that may result from an intrinsic defect in the B cells. The goal of this study was to assess whether CD28 could be involved in the development of B cells. CD28 mRNA was detected preferentially in the fraction of bone marrow enriched for stromal cells. Flow cytometry and RT-PCR analysis demonstrated that CD28 was also expressed by primary-cultured stromal cells that supported B lymphopoiesis. Confocal microscopy revealed that in the presence of B-lineage cells, CD28 was localized at the contact interface between B cell precursors and stromal cells. In addition, CD80 was detected on 2-6% of freshly isolated pro- and pre-B cells, and IL-7 stimulation led to induction of CD86 on 15-20% of pro- and pre-B cells. We also observed that stromal cell-dependent production of B-lineage cells in vitro was greater on stromal cells that lacked CD28. Finally, the frequencies of B-lineage precursors in the marrow from young (4- to 8-wk-old) CD28(-/-) mice were similar to those in wild-type mice; however, older CD28(-/-) mice (15-19 mo old) exhibited a 30% decrease in pro-B cells and a 50% decrease in pre-B cells vs age-matched controls. Our results suggest that CD28 on bone marrow stromal cells participates in stromal-dependent regulation of B-lineage cells in the bone marrow. The localization of CD28 at the stromal cell:B cell precursor interface suggests that molecules important for T cell:B cell interactions in the periphery may also participate in stromal cell:B cell precursor interactions in the bone marrow.     

Genetic and immunological assessment of a bone marrow transplantation in a patient with a primary immune defect: leukocyte adhesion deficiency

Leukocyte adhesion deficiency (LAD) was suspected in a three weeks old girl from a family with an established history of LAD with a lack (less then 1%) of the beta 2 integrins CD 11a, b/CD 18 expression at the leukocytes surface, was engrafted with her mother HLA identical bone marrow at the age of 14 months. Repeated post transplantation (up to 22 months). Immunological assessments showed a good engraftment with 97% of the lymphocytes expressing CD11a/CD18. Cells proliferated normally in response to PHA and to Tetanus toxo?d after revaccination. The level of serum immunoglobulins was normal. Investigation of the CD18 intragenic polymorphic marker Avall before and after bone marrow transplantation (BMT) showed a transition from the Avall +/+ genotype to the mother's Avall +/- genotype. Similarly DNA fingerprints obtained with the patient genomic DNA, prepared from PBMC, prior and after transplantation, showed that the patient's DNA fingerprints pattern matched the mother's one. These findings are consistent with the good engraftment observed clinically. This study emphasizes the usefulness of the molecular techniques to evaluate the degree of chimerism in monitoring the outcome of bon marrow transplantation.