1. Volume 20, Issue 8, Pages 1830-1843 (August 2017)
Triggering of NOD2 Receptor Converts Inflammatory Ly6Chigh into Ly6Clow Monocytes with Patrolling Properties 
Anne-Julie Lessard, Manon LeBel, Benoit Egarnes, Paul Préfontaine, Peter Thériault, Arnaud Droit, Alexandre Brunet, Serge Rivest, Jean Gosselin 
Cell Reports 
Volume 20, Issue 8, Pages (August 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 20, 1830-1843DOI: (10.1016/j.celrep.2017.08.009)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
MDP Treatment Promotes the Conversion of Inflammatory Ly6Chigh Monocytes into a Ly6Clow Patrolling Subset
Wild-type, Nr4a1−/−, and Nod2−/− mice (n = 3–6 mice/group) were treated daily with vehicle (saline 0.9%, i.v.) or MDP (10 mg/kg, i.v.), and blood was collected at indicated times after MDP treatment.
(A) Absolute count of blood total monocytes following treatment with vehicle (full lines) or MDP (dashed lines) as measured by flow cytometry.
(B) Flow cytometry dot plots for blood Ly6Chigh, Ly6Cinter, and Ly6Clow monocyte number from wild-type, Nr4a1−/−, and Nod2−/− mice treated with vehicle or MDP (48 hr).
(C) Kinetics of blood Ly6Chigh, Ly6Cinter, and Ly6Clow monocyte number following daily treatment with vehicle (full lines) or MDP (dashed lines) as measured by flow cytometry.
Data are presented as mean ± SEM of three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ (unpaired t test).
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4. Figure 2
MDP Treatment Accelerates the Re-emergence of Ly6Clow Blood Monocytes following Clodronate Administration
(A) Experimental design of clodronate liposomes (Clo-lipo) and MDP administration. Wild-type mice (n = 3–6 mice/group) were injected with PBS-liposomes (PBS-lipo) as control or Clo-lipo (i.v.) in order to deplete blood monocytes. MDP was injected daily (i.v.) 24 hr following clodronate administration and animals were sacrificed at indicated times.
(B) Flow cytometry analysis of blood Ly6Chigh, Ly6Cinter, and Ly6Clow monocyte subsets in naive, PBS-lipo-, or Clo-lipo-injected mice following vehicle (saline) or MDP treatment. Animals were sacrificed at indicated times post-treatment, and absolute counts of re-emerging monocyte subsets were determined by flow cytometry.
(C) Absolute values of total, Ly6Chigh, and Ly6Clow blood monocytes in clodronate (Clo-lipo)-injected mice treated daily with vehicle or MDP. Animals were sacrificed at indicated times following Clo-lipo administration. In naive mice, absolute count of total monocytes was 191, ± 10,125.5, Ly6Chigh monocytes was 106, ± 7,524.75, and Ly6Clow monocytes was 64, ± 4,
(D) Experimental design of DiO-labeled liposomes and MDP treatment. DiO-labeled liposomes (DiO-lipo) or PBS-liposomes (control) were injected intravenously in wild-type mice 48 hr following Clo-lipo administration in order to label newly synthesized Ly6Chigh monocytes. Mice were treated daily with vehicle or MDP (i.v.) and sacrificed at indicated times.
(E) Flow cytometry of Ly6Chigh, Ly6Cinter, and Ly6Clow monocyte subsets at indicated times following vehicle or MDP treatments after gating on all monocytes (top) or DiO-labeled monocytes (bottom). Data are presented as mean ± SEM of three independent experiments.
(F) Flow cytometry analysis of CD45.1+ bone marrow-derived monocyte subsets before proceeding to engraftment. Grafted CD45.1 bone marrow monocytes were mostly Ly6Chigh cells (94.8%).
(G) Flow cytometry analysis of CD45.1+ and CD45.2+ cells in blood of CD45.2 mice adoptively transferred with CD45.1 BM cells.
(H) Total bone marrow cells (35 × 106 cells) were adoptively transferred (i.v.) into CD45.2 recipient mice. Animals were treated with vehicle or MDP (i.v.) 2 hr after adoptive transfer. Blood samples were collected at indicated time and analyzed by flow cytometry for the levels of Ly6Clow and Ly6Chigh CD45.1+ monocytes. Data are representative of three independent experiments with one mouse/experiment. ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ (two-way ANOVA), MDP-treated compared with vehicle-treated animals.
(I) Sorted Ly6Chigh CD45+ bone marrow-derived monocytes (2.5 × 106 cells) were adoptively transferred into CD45.2 recipient mice. Animals were treated with MDP (i.v.), and blood was collected 1 hr following treatment for flow cytometry analysis. Data are representative of two experiments performed with one mouse/experiment.
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5. Figure 3
MDP Treatment Increases Ly6Clow Monocytes in Bone Marrow and Spleen of Mice
(A and C) Flow cytometry gating strategies of total, Ly6Chigh, and Ly6Clow monocytes in (A) bone marrow and (C) spleens of wild-type mice. Bone marrow monocytes are gated as Live+Lin−CD117−CD115+CD11b+ and spleen monocytes as Live+Lin−CD11b+CD11c−. Monocyte subsets were analyzed according to their Ly6C expression in vehicle (control) or MDP (i.v.) treated mice (n = 5) and are presented as absolute number ± SEM of two independent experiments.
(B and D) Frequencies of Ly6Clow and Ly6Chigh monocytes are shown in (B) bone marrow cells and (D) spleen cells of mice treated with vehicle or MDP (i.v.) at indicated times.
(E) Gating strategies of CD142+ CD16− (P1, blue), CD14+ CD16+ (P2, red), and CD14± CD162+ (P3, green) monocyte subsets in freshly isolated, non-stimulated (cell cultures), or MDP-treated human mononuclear cells for 18 hr.
(F) Frequencies (%) of blood CD142+ CD16− (P1) and CD14± CD162+ (P3) monocyte subsets from freshly isolated, non-stimulated, or MDP treated human mononuclear cells for 18 hr.
Dot plots show the distribution and mean ± SEM (horizontal lines) of monocytes subset frequencies of three independent experiments. Each symbol represents an independent individual. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ (unpaired t test).
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6. Figure 4
MDP Treatment Induces Crawling of Differentiated Ly6Chigh Monocytes
(A and B) Intravital imaging of monocytes in the dermis (ear) of Cx3cr1+/gfp mice treated with (A) vehicle or MDP (i.v., 72 hr post-treatment) and (B) Cx3cr1+/gfp mice injected with clodronate-liposomes (Clo-lipo) 24 hr prior to vehicle or MDP treatments. Images were recorded at indicated time following Clo-lipo administration. The scale bar represents 20 μm. See also Movies S1, S2, S3, and S4.
(C and D) Flow cytometry analysis of Ly6Chigh, Ly6Cinter, and Ly6Clow blood monocytes from corresponding Cx3cr1+/gfp in vivo imaged mice. Data are presented as absolute numbers of monocyte subsets.
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7. Figure 5
Homing of Converted Monocytes in MDP-Treated Mice following Thioglycollate-Induced Peritonitis
(A) Intravital imaging of peritoneal microvessels of Cx3cr1+/gfp mice treated daily with vehicle (top) or MDP (i.v.) (bottom) prior to intraperitoneal injection of thioglycollate. Images were recorded at indicated times following thioglycollate administration. The scale bar represents 20 μm. See also Movies S5, S6, S7, S8, and S9.
(B) Numbers of Ly6Clow and Ly6Chigh monocytes were assessed by flow cytometry in peritoneal lavages performed on in vivo imaged Cx3cr1+/gfp mice at indicated times following thioglycollate administration.
(C) Intravital imaging of peritoneal microvessels of Cx3cr1+/gfp mice treated daily with vehicle or MDP (i.v.) prior to intraperitoneal injection of thioglycollate. Animals were injected with 2.5 μg (i.v.) of CCR2 PE antibody prior to imaging. Images were recorded at 5 hr following thioglycollate administration. The scale bar represents 20 μm.
(D) Levels of IL-6, TNF-α, IL-10, and TGF-β1 were determined in peritoneal lavages of in vivo imaged mice at indicated times following thioglycollate administration. No significant levels of IL-6, TNF-α, IL-10, and TGF-β1 were detected in MDP treated mice (data not shown).
Data are presented as mean ± SEM of two independent experiments (n = 3 mice/group). ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ (two-way ANOVA followed by Bonferroni test in B and Mann-Whitney test in D).
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8. Figure 6
MDP Treatment Increases Levels of Ly6Clow Monocytes and Reduces Inflammation in LPS-Treated Mice
(A and B) Flow cytometry analysis (A) and absolute counts (B) of Ly6Chigh, Ly6Cinter, and Ly6Clow monocytes in blood (left) and spleen (right) of wild-type mice treated daily with MDP (48 hr). Mice were injected with LPS (1 μg) 24 hr following the last vehicle or MDP injection. Animals were sacrificed 48 hr after LPS administration.
(C) Levels of TNF-α and IL-6 were determined in sera (left) and spleens (right) of mice.
Data are presented as mean ± SEM of two independent experiments (n = 4 mice/group). ∗p ≤ 0.05 (Mann-Whitney test) compared with indicated groups.
Cell Reports  , DOI: ( /j.celrep )
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9. Figure 7
Impact of MDP Treatment on Gene Expression in Ly6Chigh and Ly6Clow Monocyte Subsets
(A) Venn diagram illustrating the overlap of the differentially expressed genes (p < 0.05 after Benjamini-Hochberg correction for multiple testing) in Ly6Chigh and Ly6Clow blood monocytes of MDP-treated mice (18 hr). The number in each section of the diagram represents the number of genes modulated by MDP treatment.
(B) Heatmap displaying the log2 fold change in expression of 51 genes regulated in monocyte subsets following MDP treatment. Key genes are presented in red. Data are representative of two or more independent experiments.
Cell Reports  , DOI: ( /j.celrep )
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