γδT Cells Are Prevalent in the Proximal Aorta and Drive Nascent Atherosclerotic Lesion Progression and Neutrophilia in Hypercholesterolemic Mice

<div><p>Unique innate immunity-linked γδT cells have been seen in early human artery lesions, but their role in lesion development has received little attention. Here we investigated whether γδT cells modulate atherogenesis in apolipoprotein E-deficient (ApoE KO) mice. We found that γδT cell numbers were markedly increased in the proximal aorta of ApoE-deficient <i>vs.</i> wild-type mice during early atherogenesis, particularly in the aortic root and arch, where they comprised most of the T cells and lesion progression is most rapid. γδT cells infiltrated intimal lesions in ApoE KO mice, but only the adventitia in wild-type mice, and were more prevalent than CD4⁺ T cells in early nascent lesions, as evaluated by <i>en face</i> confocal microscopy. These aortic γδT cells produced IL-17, but not IFN-γ, analyzed by <i>ex vivo</i> FACS. Furthermore, aortic arch lipid accumulation correlated strongly with abundance of IL-17-expressing splenic γδT cells in individual ApoE KO mice. To investigate the role of these γδT cells in early atherogenesis, we analyzed ApoE/γδT double knockout (DKO) compared to ApoE KO mice. We observed reduced early intimal lipid accumulation at sites of nascent lesion formation, both in chow-fed (by 40%) and Western diet-fed (by 44%) ApoE/γδT DKO mice. In addition, circulating neutrophils were drastically reduced in these DKO mice on Western diet, while expansion of inflammatory monocytes and splenic Th1 or Th17 lymphocytes was not affected. These data reveal, for the first time, a pathogenic role of γδT cells in early atherogenesis in ApoE KO mice, by mechanisms likely to involve their IL-17 production and induction of neutrophilia. Targeting γδT cells thus might offer therapeutic benefit in atherosclerosis or other inflammatory vascular diseases.</p></div

Endogenous moR-21 downregulates Txndc5 expression.

<p>(a) Txndc5 protein abundance in scrambled or moR-21 mimetic and scrambled or anti-moR-21 treated cells. Representative western blots are presented. Densitometry analysis for western blots is shown in the lower panel. Data are from at least 4 independent experiments and presented as fold change, compared with scrambled mimetic and scrambled anti-moR treated cells. (b) Upper panel: the moR-21 sponge was designed with bulged binding sites to avoid endonucleolytic cleavage by Ago2. Lower panel: the moR-21 sponge was constructed by inserting 7 copies of bulged moR-21 binding sites into the 3’UTR of a destabilized GFP reporter gene. (c) qRT-PCR analysis of Txndc5 mRNA in control or moR-21 sponge transfected HEK293 cells. Gapdh was used as an internal control for RT-qPCR. (d) immunoblot analysis of Txndc5 protein abundance in control or moR-21 sponge transfected HEK293 cells. Data are from 4 independent experiments. Values are mean ± SEM. The significance of differences between different treatments was determined by Student’s <i>t</i>-test. NS: non-significant, *: <i>P</i><0.05, **: <i>P</i><0.01, ***: <i>P</i><0.001.</p

γδT cell deficiency reduces aortic root lesion area in 24 wk-old chow-fed ApoE KO mice.

<p><b>A&D:</b> Aortic root lipid-rich lesions outlined in <i>green</i> (<i>en face</i> view) are significantly decreased in ApoE/γδT DKO mice (<i>p</i><0.0001). Lesion area was not affected by γδT cell deficiency in the aortic arch (<b>B&E</b>) or descending aorta (<b>C&F</b>).</p

Body and spleen weights, serum lipids and SAA levels in ApoE <i>vs.</i> ApoE/TCRδ DKO mice.

<p>Values are mean ± SE. *<i>P</i><0.02 <i>vs</i>. ApoE KO.</p><p>Aortic lesions in these mice are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109416#pone-0109416-g004" target="_blank">Fig. 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109416#pone-0109416-g005" target="_blank">5</a>.</p><p>Body and spleen weights, serum lipids and SAA levels in ApoE <i>vs.</i> ApoE/TCRδ DKO mice.</p

γδT cells are the dominant T cell subset in early lesions and associated adventitia of ApoE KO mice.

<p><i>En face</i> confocal images of aortic tissue from Western diet-fed ApoE KO mice stained with anti-CD3-FITC (<i>green</i>), anti-TCRγδ APC (<i>red</i>), and DAPI <i>(blue)</i>. γδT cells constitute the majority of CD3⁺ T cells in aortic root intima (<b><i>A</i></b>) and adventitia (<b><i>B</i></b>). γδT cells infiltrate the adventitia at intercostal artery branch points of the descending thoracic aorta (<b><i>C</i></b>). White scale bar = 10 µm. <b>D:</b> Quantitation: n = 3.</p

γδT cell deficiency reduces aortic arch lesion area in Western diet-fed ApoE KO mice.

<p>Mice were fed Western diet for 4 weeks and aortic root cross sections were stained with <b>A:</b> ORO and hematoxylin, or <b>B:</b> anti-CD68-Alexa Fluor 488, AlexaFluor 633 to visualize elastin, and DAPI. <b>C:</b> Aortic arch segments were stained with ORO <i>en face.</i><b>D&E:</b> Charts show total and macrophage-rich lesion areas in the aortic root. The aortic roots of 3 ApoE KO mice and 3 ApoE/γδT DKO mice were excluded from the analysis based on the lack of 3 complete cusps. <b>F:</b> Aortic arch lesions were significantly reduced in ApoE/γδT DKO mice (*<i>p</i><0.03).</p

MoR-21L plays a functional role in VSMC.

<p>(a) Location of moRs and miRs sequences on the predicted secondary structure surrounding the pre-miR-21 hairpin. The RNA structure prediction software mFold was used to predict pre-miR-21 secondary structure. (b) Increased expression of moR-21 and miR-21 in injured mouse carotid artery. Data are shown as mean ± SEM and are from 3 independent experiments. (c) Abundance of moR-21 and miR-21 in VSMC cultured under different conditions. VSMC were cultured in serum free medium (SFM) and medium containing 10% fetal bovine serum (FBS) or PDGF. BSA is the control for PDGF. Data are shown as mean ± SEM and are from 4 independent experiments. (d) Effects of over-expression of moR-21, miR-21, and scrambled mimetics on VSMC proliferation. VSMC were transfected with 20nM scrambled control, moR-21, and or miR-21 mimetics. Cell proliferation was measured at 0, 1, 2, and 3 days using Cell TiterGlo. Data are presented as relative proliferation, compared with scrambled mimetic-transfected cells on day 0. Data are shown as mean ± SEM and are from 5 independent experiments. <i>P</i> values were determined by one way repeated measure ANOVA. NS: nonsignificant, *: <i>P</i><0.05, ***: <i>P</i><0.001.</p

Endogenous moR-21 downregulates Txndc5 expression.

<p>(a) Txndc5 protein abundance in scrambled or moR-21 mimetic and scrambled or anti-moR-21 treated cells. Representative western blots are presented. Densitometry analysis for western blots is shown in the lower panel. Data are from at least 4 independent experiments and presented as fold change, compared with scrambled mimetic and scrambled anti-moR treated cells. (b) Upper panel: the moR-21 sponge was designed with bulged binding sites to avoid endonucleolytic cleavage by Ago2. Lower panel: the moR-21 sponge was constructed by inserting 7 copies of bulged moR-21 binding sites into the 3’UTR of a destabilized GFP reporter gene. (c) qRT-PCR analysis of Txndc5 mRNA in control or moR-21 sponge transfected HEK293 cells. Gapdh was used as an internal control for RT-qPCR. (d) immunoblot analysis of Txndc5 protein abundance in control or moR-21 sponge transfected HEK293 cells. Data are from 4 independent experiments. Values are mean ± SEM. The significance of differences between different treatments was determined by Student’s <i>t</i>-test. NS: non-significant, *: <i>P</i><0.05, **: <i>P</i><0.01, ***: <i>P</i><0.001.</p

Increased γδT cells in proximal aorta of ApoE KO vs. B6 mice.

<p>Single-cell suspensions from enzyme-digested aortas of 22 wk-old chow-fed mice were stained with anti-CD3-FITC, anti-CD45-PE and anti-TCRγδ-APC and analyzed by FACS. <b>A:</b> Gating strategy and representative FACS plots. <b>B:</b><i>Bar</i>s indicate means; <i>symbols</i> indicate the absolute number of aortic T cells in individual mice. γδT cells (CD3⁺TCRγδ⁺) were significantly increased in ApoE KO <i>vs.</i> B6 aorta (*<i>p</i><0.04). The absolute numbers of conventional αβT cells (CD3⁺TCRγδ⁻) were highly variable in individual aortas from ApoE KO mice and were not significantly different from the numbers in B6 mice.</p

γδT cell deficiency reduces neutrophilia and lymphopenia in ApoE KO mice.

<p>Blood leukocytes were stained with anti-Ly6G-FITC, anti-Ly6B(7/4)-PE, anti-CD11b-PERCP Cy5.5 and anti-Ly6C-APC. <b>A:</b> Representative FACS plots; X and Y axes are log fluorescence intensity, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109416#pone-0109416-g001" target="_blank">Figs 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109416#pone-0109416-g003" target="_blank">3</a>. Neutrophils are Ly6B<b>^hi</b> Ly6G<b>^hi</b>(<b><i>top</i></b>); inflammatory monocytes are Ly6G⁻, Ly6B<b>^hi</b>, CD11b⁺, Ly6C<b>^hi</b> (<b><i>bottom</i></b>). Blood neutrophils and Ly6C<b>^hi</b> monocytes are increased in ApoE KO (n = 14) <i>vs.</i> B6 (n = 6) mice; γδT cell deficiency (n = 9) reduces neutrophilia (<b>C</b>) but not Ly6C<b>^hi</b> monocyte levels (<b>D</b>). Circulating CD3⁺ T cells are decreased in ApoE KO, but not ApoE/γδT DKO, <i>vs.</i> B6 mice (<b>E</b>). γδT cells are decreased in ApoE KO <i>vs.</i> B6 mice, and absent in ApoE/γδT DKO mice. *p<0.05, **p<0.01 <i>vs.</i> B6; + p<0.05 <i>vs.</i> ApoE KO.</p