Abstract-Heme oxygenase (HO-1) is the rate-limiting enzyme in the catabolism of heme, which leads to the generation of biliverdin, iron, and carbon monoxide. It has been shown to have important antioxidant and antiinflammatory properties that result in a vascular antiatherogenic effect. To determine whether HO-1 expression in macrophages constitutes a significant component of the protective role in atherosclerosis, we evaluated the effect of decreased or absent HO-1 expression in peritoneal macrophages on oxidative stress and inflammation in vitro, and the effect of complete deficiency of HO-1 expression in macrophages in atherosclerotic lesion formation in vivo. We found that compared with HO-1 ϩ/ϩ controls, peritoneal macrophages from HO-1 Ϫ/Ϫ and HO-1 ϩ/Ϫ mice exhibited (1) increased reactive oxygen species (ROS) generation, (2) increased proinflammatory cytokines such as monocyte chemotactic protein 1 (MCP-1) and interleukin 6 (IL-6), and (3) increased foam cell formation when treated with oxLDL, attributable in part to increased expression of scavenger receptor A (SR-A). Bone marrow transplantation experiments performed in lethally irradiated LDL-R null female mice, reconstituted with bone marrow from HO-1 Ϫ/Ϫ versus HO-1 ϩ/ϩ mice, revealed that HO-1 Ϫ/Ϫ reconstituted animals exhibited atherosclerotic lesions with a greater macrophage content as evaluated by immunohistochemistry and planimetric assessment. We conclude that HO-1 expression in macrophages constitutes an important component of the antiatherogenic effect by increasing antioxidant protection and decreasing the inflammatory component of atherosclerotic lesions.
Induction of heme oxygenase-1 (HO-1) is protective in tissue injury in models of allograft rejection and vascular inflammation through either prevention of oxidative damage or via immunomodulatory effects. To examine the specific role of HO-1 in modulating the immune response, we examined the differences in immune phenotype between HO-1 knockout (HO-1(-/-)) and wild-type (HO-1(+/+)) mice. Consistent with previous findings, marked splenomegaly and fibrosis were observed in HO-1(-/-) mice. The lymph nodes of HO-1-deficient mice demonstrated a relative paucity of CD3- and B220-positive cells, but no such abnormalities were observed in the thymus. Flow cytometric analysis of isolated splenocytes demonstrated no differences in the proportions of T lymphocytes, B lymphocytes or monocytes/macrophages between the HO-1(-/-) and HO-1(+/+) mice. Significantly higher baseline serum IgM levels were observed in HO-1(-/-) versus HO-1(+/+) mice. Under mitogen stimulation with either lipopolysaccharide or anti-CD3/anti-CD28, HO-1(-/-) splenocytes secreted disproportionately higher levels of pro-inflammatory Th1 cytokines as compared to those from HO-1(+/+) mice. These findings demonstrate significant differences in the immune phenotype between the HO-1(-/-) and the HO-1(+/+) mice. The absence of HO-1 correlates with a Th1-weighted shift in cytokine responses suggesting a general pro-inflammatory tendency associated with HO-1 deficiency.
The development of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice provides for their use as a model of human type 1 diabetes. To test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (rAAV) vectors containing murine cDNAs for immunomodulatory cytokines IL-4 or IL-10. Skeletal muscle transduction of female NOD mice with IL-10, but not IL-4, completely abrogated diabetes. rAAV-IL-10 transduction attenuated the production of insulin autoantibodies, quantitatively reduced pancreatic insulitis, maintained islet insulin content, and altered splenocyte cytokine responses to mitogenic stimulation. The beneficial effects were host specific, as adoptive transfer of splenocytes from rAAV IL-10-treated animals rapidly imparted diabetes in naive hosts, and the cells contained no protective immunomodulatory capacity, as defined through adoptive cotransfer analyses. These results indicate the utility for rAAV, a vector with advantages for therapeutic gene delivery, to transfer immunoregulatory cytokines capable of preventing type 1 diabetes. In addition, these studies provide foundational support for the concept of using immunoregulatory agents delivered by rAAV to modulate a variety of disorders associated with deleterious immune responses, including allergic reactions, transplantation rejection, immunodeficiencies, and autoimmune disorders.
Historically, heme oxygenase-1 (HO-1) has been viewed as a cytoprotective protein, ameliorating the effects of inflammatory cellular damage. The demonstration, however, of a beneficial role for HO-1 in allograft protection from acute and chronic rejection, 1-3 strongly suggests an important function of this enzyme in both innate and adaptive immune responses. In the original description of a mouse model of HO-1 deficiency, Poss and Tonegawa 4 noted an age-related overgrowth of the CD4 ϩ T-cell population, suggesting impaired regulation of T-cell proliferation. Our previous work assessing immune function in HO-1 Ϫ/Ϫ mice supported this notion because it showed a predominance of Th1-type cytokine secretion [eg, interleukin (IL)-1, interferon-␥, tumor necrosis factor-␣, IL-6] from splenocytes after polyclonal stimulation of T cells, implying that HO-1 activity is important in modulation of lymphocyte activation.5 This is of particular interest given reports that HO-1 is constitutively expressed in the CD4 ϩ CD25 ϩ subset of Treg cells, 6 and that HO-1 levels increase even further after T-cell stimulation.7 Furthermore, the report by Song and colleagues 8 demonstrated that carbon monoxide (CO), a byproduct of HO activity, has a very strong inhibitory effect on CD3-activated T-cell proliferation. Previously, we proposed a hypothetical model for the immunomodulatory effects of HO-1 in the maintenance of peripheral tolerance based on then available evidence that CO produced in regulatory T (Treg) cells may be an integral component of the suppression of T-cell activation by Treg cells in the presence of effector T (Teff) cells and antigen-presenting cells (APCs). 9The purpose of the present study was to analyze the role of HO-1 in Treg-mediated suppression. As a first step, we performed a phenotypic analysis of lymphocytes obtained from the peripheral lymphoid organs of HO-1 Ϫ/Ϫ mice for potential abnormalities. In the second step, we explored the functional significance of these findings in a series of T-cell proliferation/suppression assays. Finally, we examined the possibility that HO-1 exSupported by the National Institutes of Health (grants K08 AI 57362 to M.H.K., and R01 DK 075532 and R01 HL068157 to A.A).
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