Long-Term Depletion of Conventional Dendritic Cells Cannot Be Maintained in an Atherosclerotic Zbtb46-DTR Mouse Model

<div><p>Background and aims</p><p>Increased evidence suggests a pro-atherogenic role for conventional dendritic cells (cDC). However, due to the lack of an exclusive marker for cDC, their exact contribution to atherosclerosis remains elusive. Recently, a unique transcription factor was described for cDC, namely <i>Zbtb46</i>, enabling us to selectively target this cell type in mice.</p><p>Methods</p><p>Low-density lipoprotein receptor-deficient (<i>Ldlr</i>^<i>-/-</i>) mice were transplanted with bone marrow from <i>Zbtb46</i>-diphtheria toxin receptor (DTR) transgenic mice following total body irradiation. <i>Zbtb46</i>-DTR→<i>Ldlr</i>^<i>-/-</i> chimeras were fed a Western-type diet for 18 weeks while cDC were depleted by administering diphtheria toxin (DT).</p><p>Results</p><p>Although we confirmed efficient direct induction of cDC death <i>in vitro</i> and <i>in vivo</i> upon DT treatment of <i>Zbtb46</i>-DTR mice, advanced atherosclerotic plaque size and composition was not altered. Surprisingly, however, analysis of <i>Zbtb46</i>-DTR→<i>Ldlr</i>^<i>-/-</i> chimeras showed that depletion of cDC was not sustained following 18 weeks of DT treatment. In contrast, high levels of anti-DT antibodies were detected.</p><p>Conclusions</p><p>Because of the observed generation of anti-DT antibodies and consequently the partial depletion of cDC, no clear decision can be taken on the role of cDC in atherosclerosis. Our results underline the unsuitability of <i>Zbtb46</i>-DTR→<i>Ldlr</i>^-/- mice for studying the involvement of cDC in maintaining the disease process of atherosclerosis, as well as of other chronic inflammatory diseases.</p></div

Chronic DT administration does not affect atherosclerotic plaque size and composition in <i>Zbtb46</i>-DTR→<i>Ldlr</i>^<i>-/-</i> mice.

<p>Representative images and quantification of (A) atherosclerotic lesion size (Oil Red O⁺ area), (B) vascular smooth muscle cells (α-SMA staining), (C) collagen (Sirius Red staining), (D) apoptosis (cleaved caspase-3 staining), and (E) CD11c⁺ cells in aortic root cryosections of control and DT-treated <i>Zbtb46</i>-DTR→<i>Ldlr</i>^<i>-/-</i> mice (n = 14–19); Univariate analyses were performed for plaque area and composition of aortic root sections. Data that failed the Levene's test of homogeneity of variances were mathematically transformed before statistical analysis was performed.</p

Weight and plasma characteristics from control and DT-treated <i>Zbtb46</i>-DTR→<i>Ldlr</i>^-/- mice.

<p>Weight and plasma characteristics from control and DT-treated <i>Zbtb46</i>-DTR→<i>Ldlr</i>^-/- mice.</p

Primer sequences for determination of chimerism.

<p>Primer sequences for determination of chimerism.</p

Insufficient cDC depletion is mediated by DT-specific humoral immunity, but independent of DTR resistance.

<p>(A) DT-specific IgG₁ antibodies were determined in plasma samples from control (n = 16) and DT-treated mice (n = 11) by ELISA and are shown as OD₄₅₀ values; Statistical significance was determined by means of a Student t test, ***<i>p</i><0.001; (B) Representative histogram of intracellular human DTR expression (anti-HB-EGF) assessed by flow cytometry of splenic cDC in <i>Ldlr</i>^-/- mice (dotted black), <i>Zbtb46</i>-DTR mice (blue) and <i>Zbtb46</i>-DTR→<i>Ldlr</i>^<i>-/-</i> mice (DT-treated, red) at the end of the study. An FMO sample is used as control (grey).</p

<i>In vivo</i> validation of the <i>Zbtb46</i>-DTR model to deplete cDC.

<p>(A,B) Representative dot plots (A) and flow cytometric analysis (B) of splenocytes of <i>Zbtb46</i>-DTR mice sacrificed 14h after a single injection with vehicle (control, n = 6) or 20ng DT/g.bw (n = 5); Differences between groups were analyzed by means of a Student t test, ***<i>p</i><0.001; (C) Quantification of cleaved caspase-3 positivity in spleens (average of 3 measurements per mouse, n = 2 mice per group); Differences between groups were analyzed by means of a Student t test, **<i>p</i><0.01; (D-F) Cleaved caspase-3 (red, arrow heads) and (D) CD68, (E) CD31, and (F) Ter119 staining of spleens from <i>Zbtb46</i>-DTR mice treated with vehicle (control) or DT (20ng/g.bw, 14h); (G) Double staining of spleens from control or DT-treated <i>Zbtb46</i>-DTR mice with cleaved caspase-3 (green) and CD11c (red). Arrow heads indicate apoptotic CD11c⁺ cells; Scale bar = 50μm.</p

Monoclonal antibodies used for flow cytometry.

<p>Monoclonal antibodies used for flow cytometry.</p

<i>In vitro</i> validation of the <i>Zbtb46</i>-DTR model to deplete cDC.

<p>(A,B) Representative CD11c/MHC class II contour plots (A) and quantification of flow cytometric analysis (B) of <i>in vitro</i> immature (iDC) and mature (mDC) BMDC from <i>Zbtb46</i>-DTR mice treated with 2000ng/ml DT for 24h or left untreated (control) (n = 3–4); (C) Quantification of <i>in vitro</i> cultures of BMDC from <i>Zbtb46</i>-DTR mice labeled with FITC Annexin-V for the detection of apoptotic cells after no treatment (control) or treatment with DT (2000ng/ml, 24h) (n = 3–4); Statistical analysis was performed by means of One-way ANOVA followed by a Tukey’s Multiple Comparison post-hoc test; *<i>p</i><0.05, **<i>p</i><0.01, significantly different from control iDC; ^§<i>p</i><0.05, ^§§<i>p</i><0.01, significantly different from control mDC.</p