Ccl19-ccr7–dependent reverse transendothelial migration of myeloid cells clears chlamydia muridarum from the arterial intima

Ccl19-ccr7–dependent reverse transendothelial migration of myeloid cells clears chlamydia muridarum from the arterial intima

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ABSTRACT Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic


cells. However, the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the


arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by


intimal myeloid cells. In mice infected with the intracellular pathogen _Chlamydia muridarum_, blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7–dependent RTM was


critical for the clearance of intimal _C. muridarum_. This process was inhibited by hypercholesterolemia. Thus, RTM protects the normal arterial intima, and compromised RTM during


atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions. Access through your institution Buy or subscribe This is a preview of subscription


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ATHEROSCLEROSIS Article Open access 11 January 2022 CHANGE HISTORY * _ 20 MARCH 2017 In the version of this article initially published, the label along the horizontal axis of the graph in


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Download references ACKNOWLEDGEMENTS We thank P. Lesnik (University of Pierre and Marie Curie) for CD11c–hBcl-2 mice; M.C. Nussenzweig (The Rockefeller University) for CD11c-eYFP mice; S.


Nunes de Vasconcelos (Toronto General Research Institute) for _Rag1_−/− mice; J. Gommerman (University of Toronto) for _Nos2_−/− mouse bones; and N. van Rooijen (Vrije Universiteit) for CLs


and PLs. Supported by the Canadian Institutes of Health Research (MOP-84446, MOP-106522 and MOP-89740 to M.I.C.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Toronto General Research


Institute, University Health Network, Toronto, Canada Mark Roufaiel, Allan Siu, Su-Ning Zhu, Andrew Lau, Hisham Ibrahim, Marwan Althagafi, Kelly Tai, Sharon J Hyduk, Kateryna O Cybulsky, 


Sherine Ensan, Angela Li, Rickvinder Besla, Henry M Becker, Haiyan Xiao, Clinton S Robbins, Jenny Jongstra-Bilen & Myron I Cybulsky * Department of Laboratory Medicine and Pathobiology,


University of Toronto, Toronto, Canada Mark Roufaiel, Allan Siu, Hisham Ibrahim, Marwan Althagafi, Rickvinder Besla, Henry M Becker, Clinton S Robbins, Jenny Jongstra-Bilen & Myron I


Cybulsky * Krembil Research Institute, University Health Network, Toronto, Canada Eric Gracey & Robert D Inman * Department of Immunology, University of Toronto, Toronto, Canada Eric


Gracey, Kelly Tai, Sherine Ensan, Angela Li, Henry M Becker, Robert D Inman, Clinton S Robbins, Jenny Jongstra-Bilen & Myron I Cybulsky * Department of Biochemistry, Center for Immunity


and Infection Lausanne, University of Lausanne, Switzerland Sanjiv A Luther * Department of Medicine, University of Toronto, Toronto, Canada Robert D Inman Authors * Mark Roufaiel View


author publications You can also search for this author inPubMed Google Scholar * Eric Gracey View author publications You can also search for this author inPubMed Google Scholar * Allan Siu


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publications You can also search for this author inPubMed Google Scholar * Haiyan Xiao View author publications You can also search for this author inPubMed Google Scholar * Sanjiv A Luther


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Clinton S Robbins View author publications You can also search for this author inPubMed Google Scholar * Jenny Jongstra-Bilen View author publications You can also search for this author


inPubMed Google Scholar * Myron I Cybulsky View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.R. designed and performed most of the


experiments, analyzed the data and contributed to writing the manuscript; E.G. and R.D.I. contributed to experiments with _C. muridarum_; A.S. and M.A. performed BrdU assays; S.-N.Z.


performed _ex vivo_ cannulation and perfusion of the aorta; A. Lau performed TUNEL staining and analysis; H.I. imaged live aortas _ex vivo_; K.T. performed qPCR analysis of mRNA in the


intima; S.J.H. and K.O.C. contributed to function-blocking antibody experiments; S.E., A. Li and R.B. contributed to flow-cytometry sorting studies; H.M.B. contributed to the experimental


design and editing; H.X. performed mouse husbandry; S.A.L. provided intellectual input and _Ccl19_−/− BM; C.S.R. provided intellectual input and contributed to flow-cytometry sorting


studies; J.J.-B. contributed to the experimental design, project supervision and writing of the manuscript; and M.I.C. provided overall project supervision, including oversight of


experiments and writing of the manuscript. CORRESPONDING AUTHOR Correspondence to Myron I Cybulsky. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial


interests. INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 QUANTIFICATION OF INTIMAL CD11C+ DCS. Representative composite _en face_ confocal microscopy images of the ascending


aortic arch lesser curvature from wild-type mice stained for CD11c (green) and nuclei (blue). Aortas were harvested 24 h after injection of PBS (upper panel) or LPS (lower panel). The


abundance of intimal CD11c+ cells is reduced in LPS-injected mice Scale bars, 400 μm. SUPPLEMENTARY FIGURE 2 DEPLETION OF CIRCULATING MONOCYTES VIA CLS AND APOPTOSIS OF CD11C+ DCS IN THE


INTIMA. (A) Representative flow cytometry plots of blood obtained from wild-type C57BL/6 mice 18 h after i.v. injection of PBS or clodronate liposomes (PL or CL, respectively). Classical


monocytes were identified as CD11bhighGr-1+ cells (circled). The CD11bhighGr-1high cells are neutrophils. Absolute numbers of classical monocytes per ml of blood is shown in the graph (mean


± s.e.m.; _n_ = 3; * _P_ < 0.05 (two-tailed Student’s T-test)). (B) Representative _en face_ confocal microscopy images of CD11c-DTR transgenic mouse intima 8 h after injection of DTx


(left panel), and wild-type C57BL/6 intima 12 h after LPS injection (right panel). DTx induced extensive apoptosis, which depleted CD11c+ cells, and served as a positive control for TUNEL


staining. Only occasional TUNEL+CD11c+ cells were found in the intima of LPS-injected mice (arrowhead). TUNEL+ nuclei (red) CD11c+ cells (green), nuclei (blue). Scale bars, 20 μm.


SUPPLEMENTARY FIGURE 3 ESTABLISHING THE EFFICACY OF FUNCTION-BLOCKING ANTIBODIES. (A) _In vitro_ CD8+ T cell chemotaxis assays. CD8+ T cells were purified from wild-type mouse lymph nodes. A


chemotactic gradient was established by adding recombinant murine CCL21 (100 nM) to the bottom wells of a chemotactic chamber. T cells in the top wells were incubated with function-blocking


antibodies to CCL21 or CCL19, or isotype-matched IgG. Three independent experiments were performed, each with triplicate wells. Values were normalized to the IgG-treated control group. mean


± s.e.m. ** _P_ < 0.01 (one-way ANOVA with Tukey’s comparison). (B) _In vivo_ adoptive transfer of UBC-GFP lymph node cells into wild-type and plt mice. One hour before the adoptive


transfer, mice were injected with function-blocking antibody to CCL19 or CCL21, or isotype-matched IgG. Two hours after adoptive transfer, blood, spleen, and lymph nodes were isolated, cell


suspensions were stained for CD3 and CD4 and analyzed by flow cytometry. Values represent GFP+CD4+ T cells (that were gated from CD3+ events in lymph nodes) relative to wild-type mice


injected with IgG. Data were normalized to blood and spleen values in order to control for variability in adoptive transfer efficiency. ** _P_ < 0.01, *** _P_ < 0.001 (one-way ANOVA


with Tukey’s comparison). Data are from three independent experiments (mean ± s.e.m. (A,B)). SUPPLEMENTARY FIGURE 4 DETERMINING THE DOSE OF _CHLAMYDIA_ IN INTRANASAL AND I.V. INOCULATION


MODELS. (A) Wild-type mice received an intranasal inoculation of _C. muridarum_. The dose, shown as inclusion forming units (IFU), is indicated. _C. muridarum_ 16s rRNA expression in blood


leukocytes was determined by qPCR 2.5 days after inoculation. Comparable infection of blood leukocytes is observed. The lower dose was selected for future studies. (B) Wild-type mice were


injected i.v. with the indicated dose of _C. muridarum_ and intimal CD11c+ cells per ascending aorta were quantified at 24 h. The highest dose was selected since it induces the greatest


decrease in intimal CD11c+ cells. (C) The maximal _Chlamydia_ infection in the intima was comparable after intranasal and i.v. injections. *** _P_ < 0.001 (two-tailed Student’s T-test


(A,C), one-way ANOVA with Tukey’s comparison (B)). Data are from three independent experiments (mean + s.e.m. (A-C)). SUPPLEMENTARY FIGURE 5 IL-1Β AND TNF INDUCE A DECREASE IN INTIMAL CD11C+


CELLS. (A, C) _In vivo_ assays: Cytokines were injected i.v. at the indicated doses and intimal CD11c+ cells per ascending aorta were quantified at 24 h. (B, D) _Ex vivo_ assays: Ascending


aortas were incubated with different concentrations of cytokines and intimal CD11c+ cells were determined at 12 h. ** _P_ < 0.01, *** _P_ < 0.001 (one-way ANOVA with Tukey’s comparison


(A,C), two-tailed Student’s T-test (B,D)). Data are from three independent experiments (mean + s.e.m. (A-D)). SUPPLEMENTARY FIGURE 6 DETECTION OF _C. MURIDARUM_ IN THE PLASMA AND BLOOD


LEUKOCYTES. (A) Wild-type C57BL/6 mice were injected with PBS liposomes (PL) or clodronate liposomes (CL) 12 h prior to _C. muridarum_ (107 EB i.v. per mouse). Blood was collected at 24 h


after _C. muridarum_ injection, cells were removed by centrifugation, and elementary bodies in the plasma were enumerated by assessing IFU in fibroblast cultures. mean ± s.e.m.; _n_ = 10 for


PL and _n_ =5 for CL; *** _P_ < 0.001 (two-tailed Student’s T-test). (B) Representative flow cytometry plots showing the sorting strategy for isolating blood B cells, T cells,


neutrophils, classical and non-classical monocytes. Blood samples were obtained from wild-type mice 24 h after i.v. injection of _C. muridarum_ and cells were stained for CD3, B220, Gr-1,


and CD11b. The CD3- B220- gate from the upper plot was analyzed for Gr-1 and CD11b in the lower plot. Cells were sorted directly into Trizol reagent for RNA isolation and detection of _C.


muridarum_ 16s rRNA. Plots are from one experiment representative of three experiments (_n_ = 6 mice). SUPPLEMENTARY FIGURE 7 RTM OF INTIMAL CD11C+ CELLS REMOVES _C. MURIDARUM_ FROM THE


ARTERIAL INTIMA. (A) Assessment of _C. muridarum_ EB clearance from the plasma of _Ccr7_-/- and wild-type (WT) mice after i.v. injection of _C. muridarum_ EBs (107 IFU per mouse). (B,C)


Quantification of _C. muridarum_ 16s rRNA by qPCR in blood leukocytes (B) and splenocytes (C) of wild-type and _Ccr7_-/- mice (key) after i.v. injection of _C. muridarum_ EBs.. Data are


expressed relative to values on day 1, set as 1. (D) Quantification of _C. muridarum_ 16s rRNA in BM-derived macrophages after infection _in vitro_ with _C. muridarum_ EBs.. Data are


expressed relative to 6 h values in wild-type cells. (E) Quantification of _C. muridarum_ 16s rRNA in the intima (left) and the number of intimal CD11c+ cells (right) in wild-type mice


injected i.v. with _C. muridarum_ EBs on day 0 and PTx or B-oligomer on day 3. Aortas were analyzed on days 3 and 4. (F) Assessment of systemic inflammatory response after _C. muridarum_


infection. Wild-type (dashed red) and _Ccr7_-/- (solid blue) mice were injected i.v. with _C. muridarum_ EBs on day 0 and daily blood samples were collected for serum analysis. Cytokines and


chemokines were analyzed using a LUMINEX MULTIPLEX ELISA. IL-3, IL-4, IL-17, and VEGF were not detectable in mouse serum. (G) Quantification of intimal CD11c+ cells in wild-type mice


reconstituted with wild-type (WT) or _Nos2_-/- BM (key). After engraftment, mice were injected i.v. with _C. muridarum_ EBs and intimal CD11c+ cells were quantified at the indicated time


points by confocal microscopy.. (H) Quantification of intimal CD11c+ cells in wild-type mice injected i.v. with _C. muridarum_ EBs and either iNOS inhibitor 1400W (10 mg/kg, i.v. 24 h prior


to tissue harvesting) or DMSO (carrier control). (I) Quantification of intimal CD3+ cells in wild-type mice at the indicated time points after i.v. injection of _C. muridarum_ EBs. (J )


Quantification of _C. muridarum_ 16s rRNA in the intima of CD11c-DTR mice on day 3 after i.v. injection of _C. muridarum_ EBs and depletion of intimal CD11c+ cells by injecting DTx on day 2.


Controls received PBS instead of DT. (K) Quantification of _C. muridarum_ 16s rRNA in the intima of CD11c-hBcl2 mice on days 3 and 4 after i.v. injection of _C. muridarum_ EBs. (L) PCR


analysis of mRNA in the aortic intima of wild-type (red) and _Ccr7_-/- (blue) mice at various times after i.v. injection of _C. muridarum_ EBs. Data are expressed relative to values on day


0, set as 1..* _P_ < 0.05, ** _P_ < 0.01, *** _P_ < 0.001 (one-way ANOVA (I) with Tukey’s post hoc test (C,D,E,G,H), two-way ANOVA (B) with Bonferroni’s post-hoc comparisons (F,L)),


or unpaired Student’s t-test (A,J,K)). Data are from three independent experiments (A-L; mean ± s.e.m.). SUPPLEMENTARY FIGURE 8 HYPERCHOLESTEROLEMIA INHIBITS RTM INDUCED BY TLR4 LIGANDS.


Quantification of intimal CD11c+ cells in _Ldlr_-/- mice (10 – 12 weeks old) that were fed either SCD or CRD for 1 week (key), then injected i.v. with PBS, LPS (100 μg) or poly(I:C) (150


μg), and aortas were harvested after 24 h. mean ± s.e.m.; _n_ = 5 mice per group; *** _P_ < 0.001 (one-way ANOVA (_P_ < 0.0001) with Tukey’s comparison (within each diet group);


two-way ANOVA (_P_ = 0.0001) with Bonferroni’s comparison (between diet groups)). Data are from three independent experiments. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES


Supplementary Figures 1–8 and Supplementary Table 1 (PDF 1043 kb) SOURCE DATA SOURCE DATA TO FIG. 1 SOURCE DATA TO FIG. 2 SOURCE DATA TO FIG. 3 SOURCE DATA TO FIG. 4 SOURCE DATA TO FIG. 5


SOURCE DATA TO FIG. 6 SOURCE DATA TO FIG. 7 SOURCE DATA TO FIG. 8 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Roufaiel, M., Gracey, E., Siu, A. _et


al._ CCL19-CCR7–dependent reverse transendothelial migration of myeloid cells clears _Chlamydia muridarum_ from the arterial intima. _Nat Immunol_ 17, 1263–1272 (2016).


https://doi.org/10.1038/ni.3564 Download citation * Received: 08 December 2015 * Accepted: 22 August 2016 * Published: 26 September 2016 * Issue Date: November 2016 * DOI:


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