AAV2/8-humanFOXP3 gene therapy shows robust anti-atherosclerosis efficacy in LDLR-KO mice on high cholesterol diet
© Cao et al. 2015
Received: 12 March 2015
Accepted: 7 July 2015
Published: 18 July 2015
Inflammation is a key etiologic component in atherogenesis. Previously we demonstrated that adeno-associated virus (AAV) 2/8 gene delivery of Netrin1 inhibited atherosclerosis in the low density lipoprotein receptor knockout mice on high-cholesterol diet (LDLR-KO/HCD). One important finding from this study was that FOXP3 was strongly up-regulated in these Netrin1-treated animals, as FOXP3 is an anti-inflammatory gene, being the master transcription factor of regulatory T cells. These results suggested that the FOXP3 gene might potentially be used, itself, as an agent to limit atherosclerosis. To test this hypothesis AAV2/8 (AAV)/hFOXP3 or AAV/Neo (control) gene therapy virus were tail vein injected into the LDLR-KO/HCD animal model. It was found that hFOXP3 gene delivery was associated with significantly lower HCD-induced atherogenesis, as measured by larger aortic lumen cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-HCD-treated controls. Moreover these measurements taken from the hFOXP3/HCD-treated animals very closely matched those measurements taken from the normal diet (ND) control animals. These data strongly suggest that AAV/hFOXP3 delivery gave a robust anti-atherosclerosis therapeutic effect and further suggest that FOXP3 be examined more stringently as a therapeutic gene for clinical use.
Inflammation is now known to be a key regulatory process that is common denominator among several risk factors for atherosclerosis, in addition to accompanying and associated altered arterial biology [1, 2]. Furthermore, it appears that both the innate and adaptive arms of the immune system may also be involved in this overall inflammatory trend which is implicated in atherosclerosis [3–7]. We have carried out various therapeutic adeno-associated virus (AAV)-based gene therapy studies in an animal model of atherosclerosis (low-density lipoprotein receptor-knockout mouse on high cholesterol diet, LDLR-KO HCD), towards the specific goal of regulating the arterial immune cell infiltrate status with immuno-suppressive cytokines and leukocyte chemo-attractant/repellant chemokine genes, and thereby inhibiting atherosclerosis [8–15].
We recently published a study that demonstrated that AAV/Netrin1 systemic gene delivery was able to inhibit atherosclerosis in LDLR-KO mice on HCD . This was shown by high resolution ultrasound (HRUS) measurements of aortic lumen cross-sectional area, wall thickness, and systolic blood velocity. All of these measurements indicated that the Netrin1 gene delivery resulted in significantly lower atherosclerosis. However, upon analysis of the expression of various genes by Q-PCR we discovered that both Forkhead box P3’s (FOXP3) and CD25 expression were strongly up-regulated in the AAV/Netrin1-treated animals . Of course both FOXP3 and CD25 are hallmark markers of regulatory T cells (Treg). However, the exact mechanism by which FOXP3 and CD25 are up-regulated by Netrin1 in aortas challenged with HCD remains to be determined.
Of these two genes, FOXP3, in particular, the master transcription factor of regulatory T cells (Treg), is intriguing as a therapeutic gene as the Treg phenotype is tied to FOXP3 expression, and Treg affect both innate and adaptive immunity [3–7]. It is the induction of the FOXP3 gene which results in giving an immune suppressive function to Treg precursor cells, and the removal of expression of this same gene in mature Treg cells results in loss of Treg lineage identity and a marked reduction in immunosuppressive properties [16–19]. Here we characterize the effect of AAV-based human (h)FOXP3 gene delivery, by systemic tail vein injection, to inhibit atherosclerosis in the LDLR-KO/HCD animal model. In this study we use the human (h)FOXP3 transgene rather than the mouse (m)Foxp3 version as the hFOXP3 and mFOXP3 proteins are 86% homologous, and the use of the human version brings us potentially one step closer to clinical trials.
All experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Usage Committee of the Central Arkansas Veterans Healthcare System, Research and Development, at Little Rock. The project was funded by a Veterans Administration Merit Review grant to PLH.
AAV vector construction and virus generation
We directly addressed the hypothesis that hFOXP3 gene delivery can inhibit atherosclerosis by using AAV2/8 [AAV2 inverted terminal repeats (ITR) DNA combined with the AAV serotype 8 capsid] gene delivery. The human (h) FOXP3 cDNA was obtained from Open Biosystems and was ligated downstream from the cytomegalovirus immediate early promoter (CMVpr) within the gutted AAV vector dl3-97 to generate AAV/hFOXP3. The AAV/Neo vector has been described previously [8, 10–14]. AAV2/8 virus (AAV2 DNA in AAV8 virion) was produced using pDG8 helper and titered by dot blot analysis by standard methodologies [8, 10–14].
LDLR-KO mice (B6;129S7-Ldlr tm1Her /J) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Three groups of male mice, composed of ten animals each at 8 weeks old, were injected with AAV/Neo (positive control group), or AAV/hFOXP3 virus at a titer of 1 × 1010 e.g./ml via tail vein with 200 μL virus per mouse, two booster injections were followed at an interval of 5–6 days. High cholesterol diet (HCD) of 4% cholesterol and 10% cocoa butter diet (Harlan Teklad, Madison, Wis, USA) was provided from the first day of injection and maintained for the entire study period. Another group of mice fed with a ND was used as a negative control group. The normal background mouse chow was Harlan catalog #7012, and the HCD was #7012, 4% cholesterol/10% cocoa butter, custom formulated by Harlan. All experimental procedures conform to protocols approved by the Institutional Animal Care and Usage Committee of the Central Arkansas Veterans Health Care System at Little Rock.
Ultrasound imaging was carried out using a Vevo 770 High-Resolution Imaging system (Visualsonics, Toronto, Canada) with a RMV 707B transducer having a center frequency of 30 MHz. Animal preparation was done as described earlier . In brief, the mice were anesthetized using 1.5% isoflurane (Isothesia, Abbott Laboratories, Chicago, USA) with oxygen and laid supine out on a thermostatically heated platform. Abdominal hair was removed with a shaver and a chemical hair remover (Church & Dwight Co, Inc., NJ, USA). A pre-warmed transducing gel (Medline Industries, Inc., Mundelein, USA) was spread over the skin as a coupling medium for more accurate measurements. Two general levels of the vessel were visualized: thoracic region—below the aortic arches to the diaphragm and then the renal region—the upper abdominal region to the iliac bifurcation. Image acquisition was started on B-mode, where, a long axis view was used to visualize the length of the aorta. Then the scan head probe was turned 90° for a short-axis view to visualize the cross-sectional area of the aorta. Individual frames and cine loops (300 frames) were acquired at all levels of the aorta, and included both the long axis and short axis view and recorded at distances of 1 mm throughout the length of the aorta. Measurement of the flow velocity, orientation of the abdominal aorta by ultrasound, was accomplished by tilting the platform and the head of mouse down with the transducer probe towards the feet and tail of the mouse. This positioning resulted in the Doppler angle to be less than 60° for accurate measurements of blood flow velocity in the pulse-wave Doppler (PW) mode within abdominal aorta. Off-line measurements and data analysis was performed using the customized version of Vevo770 Analytical Software from both the longitudinal and transverse images. The complete imaging for each mouse lasted for about 25–30 min.
Measurement of plasma cholesterol
Total plasma cholesterol of AAV/Neo and AAV/FOXP3 mice were measured by VetScan VS2 (Abaxis, Union City, CA, USA) at the Veterans Animal Laboratory (VAMU).
Atherosclerotic lesion analysis by direct visualization
Whole dissected aortas were fixed in 10% buffered formalin, inspected under a dissecting microscope and any small pieces of adventitial fat that remained attached were removed very carefully without disturbing the aorta itself and the internal lipid accumulations/plaque. Unstained small animal aortas are normally translucent but show lipid deposition as white areas [21, 22]. Aortas were then photographed under natural light using a 10 megapixel digital camera (Nikon, Japan).
Observation of atherosclerosis by histology
Twenty weeks after first injection of virus and on HCD, mice were killed by CO2 exposure. Entire aortas, including the aortic arches, thoracic and abdominal aortas, were removed. The aorta was flushed with saline solution and fixed in 10% neutral buffered formalin (Sigma). After 24 h, the fixed tissue was used for paraffin embedding and sectioning for histological analysis. Finally representative sections were hematoxylin and eosin-stained.
Parameters were analyzed with statistics software SPSS 16.0 by nonparametric ANOVA test. If differences were detected between means, Newman–Keuls test was used for multiple comparisons. Difference were considered as significant if P < 0.05.
AAV vectors and animal treatments
Analysis of aortic structure
Visual inspection of aortas
Histologic views of representative aortas
Our earlier Netrin1 gene therapy study demonstrated that FOXP3 and CD25 were strongly overexpressed in aortas compared to controls , and that this overexpression was associated with significantly lower atherosclerosis. From these results we developed the hypothesis that the Treg phenotype, specifically induced by AAV/FOXP3 gene delivery, would show an inhibition of atherogenesis in our LDLR-KO/HCD model. This study has demonstrated that our hypothesis was correct and that CMVpr-FOXP3-gene delivery by AAV2/8 vector results in a robust protection of aortas from developing atherosclerosis in the LDLR-KO/HCD model. The protection afforded by the FOXP3-treated animals on HCD was shown by the finding that multiple aortic parameters were not statistically different from the ND control group. These data suggest that the FOXP3 gene gives a high level of efficacy against HCD-induced atherosclerosis in the well-established LDLR/KO-HCD animal model. None of our other therapeutic gene therapy in LDLR-KO/HCD mouse studies has given multiple aortic parameters which were statistically the same as the ND control group [8, 10–14]. This includes our “gold standard” IL10 gene, which has been utilized by at least three groups for inhibiting atherosclerosis [12, 15, 23–27].
Very likely the efficacious ability of FOXP3 lies in its role as the master transcription factor for the Treg development and phenotype. It is known that the loss of FOXP3 expression (or mutation FOXP3) results in increases in chronic autoimmunity . The phenotype of FOXP3 knockouts gives the “scurfy” phenotype in mice and in humans generates the X-linked autoimmunity–allergic dysregulation and immuno-dysregulation, X-linked syndromes [28, 29]. Tregs are also critical for lowering excess inflammation and giving tolerance to gut commensal microbes . However, if present in extreme excess, Tregs may allow for dysplastic and malignant cell growth and chronic infections through the governance of limited anti-tumor surveillance or limited anti-pathogenic organism immune responses [30–32].
As we age it is clear that inflammation increases and becomes a major threat to health [33–35]. Thus, the use of Tregs, the promotion of the Treg phenotype, is one possible approach to broadly limit this increasing inflammation, in the aged population. The use of AAV-based FOXP3 gene therapy, to promote the overall Treg phenotype, could be an effective therapeutic gene and agent against inflammation and diseases of the elderly. It is very important that the forced induction of the FOXP3 gene results in giving an immune suppressive function to Treg precursor cells, and that the removal of expression of this same gene in mature Treg cells results in loss of Treg lineage identity and a marked reduction in immunosuppressive properties [16, 19].
One possible mechanism for the AAV/hFOXP3-HCD animal group cholesterol levels being lower than the AAV/Neo-HCD group is the likelihood that Foxp3 is known to induce both transforming growth factor beta 1 (TGFβ1) and interleukin 10 (IL10). Both of these are known to be associated with lower cholesterol levels and both of TGFβ1 and IL10 are known to inhibit the development of atherosclerosis. Both inhibit macrophage trafficking into the arterial wall intima and thereby inhibit macrophage accumulation and foam cell formation. Additionally it has been reported by Klingenberg et al that Foxp3 expression is linked with lower cholesterol levels, as we see here . Thus, in summary, our data on FOXP3 gene delivery, and, in addition, the general literature on FOXP3 suggests that it will likely be a robust therapeutic gene for the treatment of atherosclerosis
Conceived and designed the experiments: PLH and MCI. Performed the experiments: MC HZ. Analyzed the data: PLH MCI MC HZ SAT MC LM JAF. Contributed reagents/materials/analysis tools: MC HZ PLH. Wrote the paper: PLH. Pre-submission manuscript review and correction: MCI MC SAT KDS LM JAF. All authors read and approved the final manuscript.
This study was funded by a VA Merit Review Grant to PLH.
Compliance with ethical guidelines
Competing interests The authors based in Little Rock, AR have no financial conflict of interest. Some of the authors based in Lubbock, TX are affiliated with Kiromic, LLC: MCI is the Chief Scientist and Founder of Kiromic, LLC; JAF is the Chief Medical Officer of Kiromic, LLC. Kiromic has filed a patent on this technology. However, Kiromic provided no funding for this project. This does not alter the authors’ adherence to Journal of Translational Medicine policies on sharing data and materials.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Libby P (2002) Inflammation in atherosclerosis. Nature 420(6917):868–874PubMedView ArticleGoogle Scholar
- Libby P, Ridker PM (2009) Hansson GK; Inflammation in atherosclerosis: from pathophysiology to practice. Leducq transatlantic network on atherothrombosis. J Am Coll Cardiol 54(23):2129–2138PubMed CentralPubMedView ArticleGoogle Scholar
- Hou X, Song J, Su J, Huang D, Gao W, Yan J et al (2015) CD4(+)Foxp3(+) Tregs protect against innate immune cell-mediated fulminant hepatitis in mice. Mol Immunol 63(2):420–427PubMedView ArticleGoogle Scholar
- Mahajan D, Wang Y, Qin X, Wang Y, Zheng G, Wang YM et al (2006) CD4 + CD25 + regulatory T cells protect against injury in an innate murine model of chronic kidney disease. J Am Soc Nephrol 17(10):2731–2741PubMedView ArticleGoogle Scholar
- Bacchetta R, Passerini L, Gambineri E, Dai M, Allan SE, Perroni L et al (2006) Defective regulatory and effector T cell functions in patients with FOXP3 mutations. J Clin Invest 116:1713–1722PubMed CentralPubMedView ArticleGoogle Scholar
- RoncaroloMG BattagliaM (2007) Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nat Rev Immunol 7:585–598View ArticleGoogle Scholar
- Quintana FJ, Iglesias AH, Farez MF, Caccamo M, Burns EJ, Kassam N et al (2010) Adaptive autoimmunity and Foxp3-based immunoregulation in zebrafish. PLoS One 5(3):e9478PubMed CentralPubMedView ArticleGoogle Scholar
- Zhu H, Cao M, Mirandola L, Figueroa JA, Cobos E, Chiriva-Internati M et al (2014) Comparison of efficacy of the disease-specific LOX1- and constitutive cytomegalovirus-promoters in expressing interleukin 10 through adeno-associated virus 2/8 delivery in atherosclerotic mice. PLoS One 9(4):e94665. doi:https://doi.org/10.1371/journal.pone.0094665 PubMed CentralPubMedView ArticleGoogle Scholar
- Hermonat PL (2014) Adeno-associated virus-based transgene delivery for treating progressive vascular diseases. Clon Transgen 3:e111View ArticleGoogle Scholar
- Zhu H, Cao M, Figueroa JA, Cobos E, Uretsky BF, Chiriva-Internati M et al (2014) AAV2/8-hSMAD3 gene delivery attenuates aortic atherogenesis, enhances Th2 response without fibrosis, in LDLR-KO mice on high cholesterol diet. J Trans Med 12(1):252View ArticleGoogle Scholar
- Zhu HQ, Cao M, Straub KD, Hermonat PL (2013) Systemic delivery of thiol-specific antioxidant hPRDX6 gene by AAV2/8 inhibits atherogenesis in LDLR KO mice on HCD. Gen Syndrom Gene Ther 4(135):2Google Scholar
- Cao M, Khan JA, Kang BY, Mehta JL, Hermonat PL (2012) Dual AAV/IL-10 plus STAT3 anti-inflammatory gene delivery lowers atherosclerosis in LDLR KO mice, but without increased benefit. Int J Vasc Med 2012:524235PubMed CentralPubMedGoogle Scholar
- Khan JA, Cao M, Kang B-Y, Liu Y, Mehta JL, Hermonat PL (2011) Systemic hNetrin-1 gene delivery by AAV8 alters leukocyte accumulation and atherogenesis in vivo. Gene Ther 18:437–444PubMedView ArticleGoogle Scholar
- Khan JA, Cao M, Kang BY, Liu Y, Mehta JL, Hermonat PL (2010) AAV/hSTAT3-gene delivery lowers aortic inflammatory cell infiltration in LDLR KO mice on high cholesterol. Atherosclerosis 213(1):59–66PubMedView ArticleGoogle Scholar
- Liu Y, Li D, Chen J, Xie J, Bandyopadhyay S, Zhang D (2006) Inhibition of atherogenesis in LDLR knockout mice by systemic delivery of adeno-associated virus type 2-hIL-10. Atherosclerosis 188:19–27PubMedView ArticleGoogle Scholar
- Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and function of CD4 + CD25 + regulatory T cells. Nat Immunol 4:330–336PubMedView ArticleGoogle Scholar
- Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057–1061PubMedView ArticleGoogle Scholar
- Ziegler SF (2006) FOXP3: of mice and men. Annu Rev Immunol 24:209–226PubMedView ArticleGoogle Scholar
- Williams LM, Rudensky AY (2007) Maitenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat Immunol 8:277–284PubMedView ArticleGoogle Scholar
- Martin-McNulty B, Vincelette J, Vergona R, Sullivan ME, Wang YX (2005) Noninvasive measurement of abdominal aortic aneurysms in intact mice by a high-frequency ultrasound imaging system. Ultrasound Med Biol 31(6):745–749PubMedView ArticleGoogle Scholar
- Daugherty A, Whitman SC (2003) Quantification of atherosclerosis in mice. Mol Biol 209(293–309):466Google Scholar
- Chen J, Tung CH, Mahmood U et al (2002) In vivo imaging of proteolytic activity in atherosclerosis. Circulation 105:2766–2771PubMedView ArticleGoogle Scholar
- Khan JA, Cao M, Kang BY, Liu Y, Mehta JL, Hermonat PL (2012) AAV/IL-10 plus STAT3 anti-inflammatory gene delivery lowers atherosclerosis in LDLR KO mice, but without increased benefit. Vasc Med, Int J, p 52435Google Scholar
- Chen S, Kapturczak MH, Wasserfall C, Glushakova OY, Campbell-Thompson M et al (2006) Interleukin 10 attenuates neointimal proliferation and inflammation in aortic allografts by a heme oxygenase-dependent pathway. Proc Natl Acad Sci 102:7251–7256View ArticleGoogle Scholar
- Yoshioka T, Okada T, Maeda Y, Ikeda U, Shimpo M et al (2004) Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice. Gene Ther 11:1772–1779PubMedView ArticleGoogle Scholar
- Sun J, Li X, Feng H, Gu H, Blair T, Li J et al (2011) Magnetic resonance imaging of bone marrow-cell mediated interleukin-10 gene therapy of atherosclerosis. PLoS One 6(9):e24529PubMed CentralPubMedView ArticleGoogle Scholar
- Han X, Kitamoto S, Wang H, Boisvert WA (2010) Interleukin-10 overexpression in macrophages suppresses atherosclerosis in hyperlipidemic mice. FASEB J 24:2869–2880PubMed CentralPubMedView ArticleGoogle Scholar
- Brunkow ME, Jeffery EW, Hjerrild KA, Paeper B, Clark LB, Yasayko SA et al (2001) Disruption of a new forkhead/wingedhelix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet 27:68–73PubMedView ArticleGoogle Scholar
- Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L et al (2001) The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 27:20–21PubMedView ArticleGoogle Scholar
- Littman DR, Rudensky A (2010) Th17 and regulatory T cells in mediating and restraining inflammation. Cell 140:845–858PubMedView ArticleGoogle Scholar
- Zou W (2006) Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 6:295–307PubMedView ArticleGoogle Scholar
- Workman CJ, Szymczak-Workman AL, Collison LW, Pillai MR, Vignali DA (2009) The development and function of regulatory T cells. Cell Mol Life Sci 66:2603–2622PubMed CentralPubMedView ArticleGoogle Scholar
- Chung HY, Kim HJ, Kim JW, Yu BP (2006) The inflammation hypothesis of aging. Annals NY Acad Sci 928:327–336View ArticleGoogle Scholar
- Kregel KC, Zhang HJ (2007) Anintegrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations. Am J Physiol Regul Comp Physiol 292:R18–R36View ArticleGoogle Scholar
- Chung HY, Cesari M, Anton S, Marzetti E, Giovannini S, Seo AY et al (2009) Leeuwenburgh. Molecular inflammation: Underpinnings of aging and age-related diseases. Aging Res Rev 8:18–30View ArticleGoogle Scholar
- Klingenberg R, Gerdes N, Badeau RM, Gistera A, Strothoff D, Ketelhuth DF et al (2013) Depletion of Foxp3 + regulatory T cells promotes hypercholesterolemia and atherosclerosis. J Clin Investig 123:1323–1334PubMed CentralPubMedView ArticleGoogle Scholar