Oral tolerance with heat shock protein 65 attenuates mycobacterium tuberculosis-inducedand high-fat-diet-driven atherosclerotic lesions
Dror Harats, MD* ,*,
Niva Yacov, BSc,
Boris Gilburd, PhD ,
Yehuda Shoenfeld, MD and
Jacob George, MD
* Institute of Lipid and Atherosclerosis Research, Sheba Medical Center, Tel Hashomer, Israel
Vascular Biogenics Ltd., Or-Yehuda, Israel
The Research Unit of Autoimmune Diseases, Sheba Medical Center, Sheba, Israel
Department of Cardiology, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel.
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Figure 1 Oral feeding with heat shock protein (HSP) 65 tolerizes immune responses to HSP65 in vivo. Low-density lipoprotein receptor deficient mice were immunized with HSP65 (10 µg/mouse) emulsified in incomplete Freunds adjuvant in the hind footpad. The mice were fed with either HSP65 or bovine serum albumin (BSA) before immunization as described in the Methods section. Lymphocytes obtained from draining inguinal lymph node cells of mice from both groups were cultured in the presence of different concentrations of HSP65 (A). Reactivity to HSP65 was determined by thymidine incorporation by lymphocytes and expressed as stimulation index. (B) Reactivity to HSP65 was evaluated in a similar manner in HSP65- or BSA-fed mice immunized with heat-killed preparation of Mycobacterium tuberculosis. *p < 0.01. Hatched bar = BSA-fed; solid bar = HSP65-fed.
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Figure 2 In vitro induction of tolerance to heat shock protein (HSP) 65 by co-culture of tolerized splenocytes with HSP65 responder lymph node cells. To assess in vitro tolerization, 105 splenocytes from HSP65 or bovine serum albumin (BSA)-fed (i.e., mice that were fed 5 times with HSP65, 50 µg/dose or BSA) were incubated in the presence of 104 lymph node cells from Mycobacterium tuberculosis immunized mice in the presence of different concentrations of HSP65. The figure represents average values obtained from three studies, each repeated twice. *p < 0.05. Hatched bar = BSA-fed splenocytes; solid bar = HSP-fed splenocytes.
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Figure 3 Induction of T helper 2 cytokines by orally tolerized mice. Lymph node cells were obtained from heat shock protein (HSP) 65 tolerized low-density lipoprotein receptor deficient mice immunized with HSP65 upon priming in vitro with 10 µg/ml HSP65 for 48 h. Interleukin-4 (IL-4) levels in the conditioned medium were assessed by a capture enzyme-linked immunosorbent assay kit as described in the Methods section. *p < 0.01.
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Figure 4 Oral tolerance with heat shock protein (HSP) 65 does not alter IgG isotype distribution. Sera was obtained from all mice tolerized with HSP65 and from non-tolerized mice from experiment 1 and 2. IgG anti-HSP65 isotypes were assessed in the first experiment (A), whereas IgG isotypes (total) were probed in the second experiment (B) employing a capture enzyme-linked immunosorbent assay. Hatched bar = HSP65; open bar = bovine serum albumin (BSA).
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Figure 5 Oral tolerance with heat shock protein (HSP) 65 attenuates atherogenesis in Mycobacterium tuberculosis and high-fat diet fed low-density lipoprotein receptor deficient mice (LDL-RD). The LDL-RD mice were fed with different doses of HSP65. One day after the last feeding, all mice were immunized with M. tuberculosis and euthanized 12 weeks afterwards. Lesion size was assessed after Oil red O staining of sections obtained at the level of the aortic sinus (A). *p < 0.01; **p < 0.001; ***p<0.0001. In the second experiment, LDL-RD mice were fed different concentrations HSP65, bovine serum albumin (BSA), or phosphate buffered saline (PBS) and fed a high fat diet for four weeks until euthanization. Atherosclerosis was assessed similar to the first experiment (B). *p < 0.01.
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Figure 6 Atherosclerosis in heat shock protein (HSP) 65 and bovine serum albumin (BSA) tolerized low-density lipoprotein receptor deficient (LDL-RD) mice. Representative Oil-red O stained section from BSA (A) and HSP65 (B) and fed LDL-RD mice.
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