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

content, access via your institution ACCESS OPTIONS Access through your institution Subscribe to this journal Receive 12 print issues and online access $209.00 per year only $17.42 per issue

Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL

ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS IDENTIFICATION OF A NON-CANONICAL

CHEMOKINE-RECEPTOR PATHWAY SUPPRESSING REGULATORY T CELLS TO DRIVE ATHEROSCLEROSIS Article Open access 22 January 2024 LIMITED PROLIFERATION CAPACITY OF AORTIC INTIMA RESIDENT MACROPHAGES

REQUIRES MONOCYTE RECRUITMENT FOR ATHEROSCLEROTIC PLAQUE PROGRESSION Article 07 September 2020 C-TYPE LECTIN RECEPTOR CLEC4A2 PROMOTES TISSUE ADAPTATION OF MACROPHAGES AND PROTECTS AGAINST

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

Figure 1a ('Dose (mg)') is incorrect. The correct label is 'Dose (μg)'. The error has been corrected in the HTML and PDF versions of the article. _ REFERENCES * Cybulsky,

M.I., Cheong, C. & Robbins, C.S. Macrophages and dendritic cells: partners in atherogenesis. _Circ. Res._ 118, 637–652 (2016). CAS  PubMed  Google Scholar  * Millonig, G. et al. Network

of vascular-associated dendritic cells in intima of healthy young individuals. _Arterioscler. Thromb. Vasc. Biol._ 21, 503–508 (2001). CAS  PubMed  Google Scholar  * Jongstra-Bilen, J. et

al. Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis. _J. Exp. Med._ 203, 2073–2083 (2006). CAS  PubMed  PubMed Central  Google

Scholar  * Choi, J.H. et al. Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves. _J. Exp. Med._ 206, 497–505 (2009). CAS  PubMed  PubMed Central  Google

Scholar  * Paulson, K.E. et al. Resident intimal dendritic cells accumulate lipid and contribute to the initiation of atherosclerosis. _Circ. Res._ 106, 383–390 (2010). CAS  PubMed  Google

Scholar  * Kawai, T. & Akira, S. The roles of TLRs, RLRs and NLRs in pathogen recognition. _Int. Immunol._ 21, 317–337 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Soloff, A.C.

& Barratt-Boyes, S.M. Enemy at the gates: dendritic cells and immunity to mucosal pathogens. _Cell Res._ 20, 872–885 (2010). CAS  PubMed  Google Scholar  * Martel, C. et al. Lymphatic

vasculature mediates macrophage reverse cholesterol transport in mice. _J. Clin. Invest._ 123, 1571–1579 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Huttenlocher, A. &

Poznansky, M.C. Reverse leukocyte migration can be attractive or repulsive. _Trends Cell Biol._ 18, 298–306 (2008). CAS  PubMed  PubMed Central  Google Scholar  * Xu, X. & Ge, Q.

Maturation and migration of murine CD4 single positive thymocytes and thymic emigrants. _Comput. Struct. Biotechnol. J._ 9, e201403003 (2014). PubMed  PubMed Central  Google Scholar  *

Serbina, N.V. & Pamer, E.G. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2. _Nat. Immunol._ 7, 311–317 (2006). CAS 

PubMed  Google Scholar  * Fujimura, N. et al. CCR2 inhibition sequesters multiple subsets of leukocytes in the bone marrow. _Sci. Rep._ 5, 11664 (2015). CAS  PubMed  PubMed Central  Google

Scholar  * Condeelis, J. & Pollard, J.W. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. _Cell_ 124, 263–266 (2006). CAS  PubMed  Google Scholar  *

Woodfin, A. et al. The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils _in vivo_. _Nat. Immunol._ 12, 761–769 (2011). CAS  PubMed  PubMed

Central  Google Scholar  * Campbell, L.A. & Kuo, C.C. _Chlamydia pneumoniae_--an infectious risk factor for atherosclerosis? _Nat. Rev. Microbiol._ 2, 23–32 (2004). CAS  PubMed  Google

Scholar  * Grayston, J.T. Infections caused by _Chlamydia pneumoniae_ strain TWAR. _Clin. Infect. Dis._ 15, 757–761 (1992). CAS  PubMed  Google Scholar  * Grayston, J.T. & Campbell, L.A.

The role of _Chlamydia pneumoniae_ in atherosclerosis. _Clin. Infect. Dis._ 28, 993–994 (1999). CAS  PubMed  Google Scholar  * Filardo, S., Di Pietro, M., Farcomeni, A., Schiavoni, G. &

Sessa, R. _Chlamydia pneumoniae_-mediated inflammation in atherosclerosis: a meta-analysis. _Mediators Inflamm._ 2015, 378658 (2015). PubMed  PubMed Central  Google Scholar  * Zimmer, S.,

Grebe, A. & Latz, E. Danger signaling in atherosclerosis. _Circ. Res._ 116, 323–340 (2015). CAS  PubMed  Google Scholar  * Moazed, T.C., Kuo, C.C., Grayston, J.T. & Campbell, L.A.

Evidence of systemic dissemination of _Chlamydia pneumoniae_ via macrophages in the mouse. _J. Infect. Dis._ 177, 1322–1325 (1998). CAS  PubMed  Google Scholar  * Gieffers, J. et al.

_Chlamydia pneumoniae_ infection in circulating human monocytes is refractory to antibiotic treatment. _Circulation_ 103, 351–356 (2001). CAS  PubMed  Google Scholar  * Evani, S.J. &

Ramasubramanian, A.K. Biophysical regulation of _Chlamydia pneumoniae_-infected monocyte recruitment to atherosclerotic foci. _Sci. Rep._ 6, 19058 (2016). CAS  PubMed  PubMed Central  Google

Scholar  * Kuo, C.C. et al. _Chlamydia pneumoniae_ (TWAR) in coronary arteries of young adults (15-34 years old). _Proc. Natl. Acad. Sci. USA_ 92, 6911–6914 (1995). CAS  PubMed  PubMed

Central  Google Scholar  * Kuo, C. & Campbell, L.A. Detection of _Chlamydia pneumoniae_ in arterial tissues. _J. Infect. Dis._ 181 (Suppl. 3), S432–S436 (2000). CAS  PubMed  Google

Scholar  * Belland, R.J., Ouellette, S.P., Gieffers, J. & Byrne, G.I. _Chlamydia pneumoniae_ and atherosclerosis. _Cell. Microbiol._ 6, 117–127 (2004). CAS  PubMed  Google Scholar  *

Zhu, S.N., Chen, M., Jongstra-Bilen, J. & Cybulsky, M.I. GM-CSF regulates intimal cell proliferation in nascent atherosclerotic lesions. _J. Exp. Med._ 206, 2141–2149 (2009). CAS  PubMed

  PubMed Central  Google Scholar  * Gautier, E.L. et al. Enhanced dendritic cell survival attenuates lipopolysaccharide-induced immunosuppression and increases resistance to lethal endotoxic

shock. _J. Immunol._ 180, 6941–6946 (2008). CAS  PubMed  Google Scholar  * Förster, R., Davalos-Misslitz, A.C. & Rot, A. CCR7 and its ligands: balancing immunity and tolerance. _Nat.

Rev. Immunol._ 8, 362–371 (2008). PubMed  Google Scholar  * Nibbs, R.J. & Graham, G.J. Immune regulation by atypical chemokine receptors. _Nat. Rev. Immunol._ 13, 815–829 (2013). PubMed

  Google Scholar  * Gracey, E. et al. Pulmonary _Chlamydia muridarum_ challenge activates lung interstitial macrophages which correlate with IFN-γ production and infection control in mice.

_Eur. J. Immunol._ 45, 3417–3430 (2015). CAS  PubMed  Google Scholar  * Rajaram, K. & Nelson, D.E. _Chlamydia muridarum_ infection of macrophages elicits bactericidal nitric oxide

production via reactive oxygen species and cathepsin B. _Infect. Immun._ 83, 3164–3175 (2015). CAS  PubMed  PubMed Central  Google Scholar  * de Bruijn, M.F. et al. High-level expression of

the ER-MP58 antigen on mouse bone marrow hematopoietic progenitor cells marks commitment to the myeloid lineage. _Eur. J. Immunol._ 26, 2850–2858 (1996). CAS  PubMed  Google Scholar  * Choi,

J.H. et al. Flt3 signaling-dependent dendritic cells protect against atherosclerosis. _Immunity_ 35, 819–831 (2011). CAS  PubMed  Google Scholar  * Link, A. et al. Fibroblastic reticular

cells in lymph nodes regulate the homeostasis of naive T cells. _Nat. Immunol._ 8, 1255–1265 (2007). CAS  PubMed  Google Scholar  * Poznansky, M.C. et al. Thymocyte emigration is mediated by

active movement away from stroma-derived factors. _J. Clin. Invest._ 109, 1101–1110 (2002). CAS  PubMed  PubMed Central  Google Scholar  * Lee, J.Y., Buzney, C.D., Poznansky, M.C. &

Sackstein, R. Dynamic alterations in chemokine gradients induce transendothelial shuttling of human T cells under physiologic shear conditions. _J. Leukoc. Biol._ 86, 1285–1294 (2009). CAS 

PubMed  PubMed Central  Google Scholar  * Worbs, T., Mempel, T.R., Bölter, J., von Andrian, U.H. & Förster, R. CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo.

_J. Exp. Med._ 204, 489–495 (2007). CAS  PubMed  PubMed Central  Google Scholar  * Matloubian, M. et al. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P

receptor 1. _Nature_ 427, 355–360 (2004). CAS  PubMed  Google Scholar  * Zachariah, M.A. & Cyster, J.G. Neural crest-derived pericytes promote egress of mature thymocytes at the

corticomedullary junction. _Science_ 328, 1129–1135 (2010). CAS  PubMed  PubMed Central  Google Scholar  * Randolph, G.J., Beaulieu, S., Lebecque, S., Steinman, R.M. & Muller, W.A.

Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. _Science_ 282, 480–483 (1998). CAS  PubMed  Google Scholar  * Weiss, G. & Schaible, U.E.

Macrophage defense mechanisms against intracellular bacteria. _Immunol. Rev._ 264, 182–203 (2015). CAS  PubMed  PubMed Central  Google Scholar  * van Gils, J.M. et al. The neuroimmune

guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques. _Nat. Immunol._ 13, 136–143 (2012). CAS  PubMed  PubMed Central  Google Scholar  *

Angeli, V. et al. Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobilization. _Immunity_ 21, 561–574 (2004). CAS  PubMed  Google Scholar  * Park,

Y.M., Febbraio, M. & Silverstein, R.L. CD36 modulates migration of mouse and human macrophages in response to oxidized LDL and may contribute to macrophage trapping in the arterial

intima. _J. Clin. Invest._ 119, 136–145 (2009). CAS  PubMed  Google Scholar  * Llodrá, J. et al. Emigration of monocyte-derived cells from atherosclerotic lesions characterizes regressive,

but not progressive, plaques. _Proc. Natl. Acad. Sci. USA_ 101, 11779–11784 (2004). PubMed  PubMed Central  Google Scholar  * Keiper, T. et al. The role of junctional adhesion molecule-C

(JAM-C) in oxidized LDL-mediated leukocyte recruitment. _FASEB J._ 19, 2078–2080 (2005). CAS  PubMed  Google Scholar  * Moore, K.J., Sheedy, F.J. & Fisher, E.A. Macrophages in

atherosclerosis: a dynamic balance. _Nat. Rev. Immunol._ 13, 709–721 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Daley, J.M., Thomay, A.A., Connolly, M.D., Reichner, J.S. &

Albina, J.E. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. _J. Leukoc. Biol._ 83, 64–70 (2008). CAS  PubMed  Google Scholar  * Ensan, S. et al. Self-renewing

resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth. _Nat. Immunol._ 17, 159–168 (2016). CAS  PubMed  Google Scholar 

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

View author publications You can also search for this author inPubMed Google Scholar * Su-Ning Zhu View author publications You can also search for this author inPubMed Google Scholar *

Andrew Lau View author publications You can also search for this author inPubMed Google Scholar * Hisham Ibrahim View author publications You can also search for this author inPubMed Google

Scholar * Marwan Althagafi View author publications You can also search for this author inPubMed Google Scholar * Kelly Tai View author publications You can also search for this author

inPubMed Google Scholar * Sharon J Hyduk View author publications You can also search for this author inPubMed Google Scholar * Kateryna O Cybulsky View author publications You can also

search for this author inPubMed Google Scholar * Sherine Ensan View author publications You can also search for this author inPubMed Google Scholar * Angela Li View author publications You

can also search for this author inPubMed Google Scholar * Rickvinder Besla View author publications You can also search for this author inPubMed Google Scholar * Henry M Becker View author

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

View author publications You can also search for this author inPubMed Google Scholar * Robert D Inman View author publications You can also search for this author inPubMed Google Scholar *

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:

https://doi.org/10.1038/ni.3564 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently

available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative