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. 2016 Mar;27(3):792-803.
doi: 10.1681/ASN.2015010009. Epub 2015 Jul 9.

Ly6Chigh Monocytes Protect against Kidney Damage during Sepsis via a CX3CR1-Dependent Adhesion Mechanism

Benjamin G Chousterman  1 , Alexandre Boissonnas  2 , Lucie Poupel  3 , Camille Baudesson de Chanville  3 , Julien Adam  4 , Nahid Tabibzadeh  5 , Fabrice Licata  3 , Anne-Claire Lukaszewicz  6 , Amélie Lombès  3 , Philippe Deterre  3 , Didier Payen  6 , Christophe Combadière  2
Affiliations

Ly6Chigh Monocytes Protect against Kidney Damage during Sepsis via a CX3CR1-Dependent Adhesion Mechanism

Benjamin G Chousterman et al. J Am Soc Nephrol. 2016 Mar.

Abstract

Monocytes have a crucial role in both proinflammatory and anti-inflammatory phenomena occurring during sepsis. Monocyte recruitment and activation are orchestrated by the chemokine receptors CX3CR1 and CCR2 and their cognate ligands. However, little is known about the roles of these cells and chemokines during the acute phase of inflammation in sepsis. Using intravital microscopy in a murine model of polymicrobial sepsis, we showed that inflammatory Ly6C(high) monocytes infiltrated kidneys, exhibited altered motility, and adhered strongly to the renal vascular wall in a chemokine receptor CX3CR1-dependent manner. Adoptive transfer of Cx3cr1-proficient monocyte-enriched bone marrow cells into septic Cx3cr1-depleted mice prevented kidney damage and promoted mouse survival. Modulation of CX3CR1 activation in septic mice controlled monocyte adhesion, regulated proinflammatory and anti-inflammatory cytokine expression, and was associated with the extent of kidney lesions such that the number of lesions decreased when CX3CR1 activity increased. Consistent with these results, the pro-adhesive I249 CX3CR1 allele in humans was associated with a lower incidence of AKI in patients with sepsis. These data show that inflammatory monocytes have a protective effect during sepsis via a CX3CR1-dependent adhesion mechanism. This receptor might be a new therapeutic target for kidney injury during sepsis.

Keywords: chemokine receptor; immunology; kidney dysfunction.

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Figures

Figure 1.
Figure 1.
Ly6Chigh monocytes exhibit increased adhesion to the renal endothelium during sepsis. Two-photon laser scanning microscopic images (left) with volume rendering (right) of ECFP+ (cyan squares) and GFP+ (green squares) cells in kidneys of MacBlue×Cx3cr1gfp/+ mice 6 hours after (A) sham or (B) CLP operations. CFP signals are in blue, GFP signals are in green, and autofluorescent renal tubules are in red. Overlay of flow cytometric surface marker expression gated on GFP+ (green histograms) and ECFP+ cells (cyan histograms) in kidneys are shown for each condition. Mean percentages±SD of gated cells are indicated (n=6 mice per group out of three independent experiments). Background staining (gray histograms) gated on nonfluorescent cells is represented. (C) Dot plots represent Ly6C and CX3CR1-GFP expression, gated on blood CD11b+ECFP+ cells 6 hours after sham or CLP operations. Mean percentages±SD of gated cells are indicated (n=6 mice per group out of three independent experiments). (D) Time series two-photon laser scanning microscopic images of kidney cortex of MacBlue×Cx3cr1gfp/+ mice 6 hours after CLP. Examples of circulating (blue squares), crawling (green squares), and adhering monocytes (purple squares) are presented. ECFP signals are in cyan, GFP signals are in green, renal tubules are autofluorescent, and blood vessels are visualized by 2 MDa rhodamine-dextran. (E) Relative frequency of the three behaviors. Bars represent mean±SEM (n=4 sham and n=3 control from independent experiments; ANOVA with Bonferroni adjustment for multiple comparisons were used; ***P<0.001). (F) Dwell time and contact duration with renal endothelium of ECFP+ monocytes. Black bars indicate means. (n=4 sham and n=4 control from independent experiments; Mann-Whitney test were used; ***P<0.001). (G) Radar chart representation shows ECFP+ cell dynamic signatures in sham-operated (green) and CLP-operated (red) mice. Mean values are presented within the 95% confidence interval of the measured value scale for each parameter. Data represent a pool of cells from (n=4 sham and n=4 control from independent experiments; Mann-Whitney test were used; *** P<0.001). (See also Supplemental Figure 1 and Supplemental Movies 1–4).
Figure 2.
Figure 2.
CX3CR1 promotes Ly6Chigh monocyte adhesion and prevents renal damage during sepsis. (A) Photomicrographs and (B) quantification of kidney histologic lesions 24 hours after CLP in WT (blue) and Cx3cr1−/− mice (red). Bars represent mean±SD (n=10 WT, n=8 Cx3cr1−/−, from at least two repeated experiments; ANOVA with Bonferroni adjustment was used; ***P<0.001). (C) Urea and creatinine measurements in plasma of CLP-operated WT and Cx3cr1−/− mice bars represent mean±SEM (n=10 WT sham, 6 Cx3cr1−/− sham, 15 WT, and 15 Cx3cr1−/− CLP, from at least two repeated experiments; ANOVA with Bonferroni adjustment was used; *P<0.05). (D) Number of Ly6Chigh monocytes in bone marrow, blood, and kidney of WT (blue) and Cx3cr1−/− (red) mice. Bars represent mean±SD (n=10 WT sham, 5 WT CLP at 6 hours, 12 WT CLP at 24 hours, and 6 Cx3cr1−/− sham, CLP at 6 hours, and CLP at 24 hours, from at least two repeated experiments; ANOVA with Bonferroni adjustment was used; *P<0.05). (E) Two-photon laser scanning microscopic images with overlay of monocyte migratory tracks (pink) in kidney cortex of MacBlue×Cx3cr1gfp/+ and MacBlue×Cx3cr1gfp/gfp mice, 6 hours after CLP. ECFP signals are in cyan, GFP signals are in green, renal tubules are autofluorescent, and blood vessels are visualized by 2 MDa rhodamine-dextran. (F) Relative frequency of the three monocyte behaviors in MacBlue×Cx3cr1gfp/+ and MacBluexC×3cr1gfp/gfp mice. Bars represent mean±SEM (n=3 MacBlue×Cx3cr1gfp/+; n=4 MacBluexC×3cr1gfp/gfp from independent experiments; ANOVA with Bonferroni adjustment was used; **P<0.01). (G) Dwell time and contact duration with the renal endothelium of ECFP+Cx3cr1gfp/+ and ECFP+Cx3cr1gfp/gfp monocytes. Black bars indicate means. (H) Radar chart representation shows ECFP+ cell dynamic signatures in CLP-operated MacBlue×Cx3cr1gfp/+ (red) and MacBlue×Cx3cr1gfp/gfp (blue) mice. Mean values are presented within the 95% confidence interval of the measured value scale for each parameter (for all two-photon experiments, n=3 MacBlue×Cx3cr1gfp/+; n=4 MacBluexC×3cr1gfp/gfp from independent experiments; Mann-Whitney test for multiple comparisons was used; ***P<0.001). (See also Supplemental Figure 2 and Supplemental Movie 5).
Figure 3.
Figure 3.
Bone marrow–derived monocytes protect against damage to kidney tissue during sepsis. (A) Quantification of kidney histologic lesions 24 hours after CLP in WT, Ccr2−/− and Ccr2−/− mice with adoptive transfer of WT bone marrow monocytes before surgery. Bars represent mean±SD (n=10 WT, 11 Ccr2−/−, 9 WT in Ccr2−/−; data from at least two repeated experiments; ANOVA with Bonferroni adjustment was used; ****P<0.0001). (B) Survival of CLP-operated Cx3cr1−/− mice after adoptive transfer of WT (gray line) or Cx3cr1−/− bone marrow monocytes (black line) (n=7 per group out of three independent experiments; survival curves were compared with a log-rank test; *P<0.05). (C) Quantification of kidney histologic lesions 24 hours after CLP in Cx3cr1−/− mice with adoptive transfer of WT (gray) or Cx3cr1−/− (black) bone marrow monocytes before surgery (n=9 per group from at least two repeated experiments; ANOVA with Bonferroni adjustment was used; **** P<0.0001).
Figure 4.
Figure 4.
CX3CR1 activation controls Ly6Chigh monocyte adherence and the outcome of CLP-mediated sepsis. (A) Two-photon laser scanning microscopic images with overlay of monocyte migratory tracks (pink) in kidney cortex of MacBlue×Cx3cr1gfp/+ mice treated with CX3CL1 or F1, 6 hours after CLP. ECFP signals are in cyan, GFP signals are in green, and renal tubules are autofluorescent. (B) Relative frequency of the three behaviors and (C) dwell time and contact duration in MacBlue×Cx3cr1gfp/+ untreated (red), treated with CX3CL1 (blue), or treated with F1 (purple). Bars represent mean±SEM (n=3 mice per group from independent experiments; ANOVA with Bonferroni adjustment was used; **P<0.01; ***P<0.001). (D) Radar chart representation shows ECFP+ cell dynamic signatures in the different experimental conditions. Mean values are presented within the 95% confidence interval of the measured value scale for each parameter. For all two-photon experiments (n=3 mice per group from independent experiments; Mann-Whitney test was used; *P<0.05; ***P<0.001). (E) Quantification of kidney histologic lesions 24 hours after CLP in control, CX3CL1, and F1-treated mice. Bars represent mean±SD (n=10 control, 12 F1, and 9 CX3CL1, from at least two independent experiments; ANOVA with Bonferroni adjustment was used; *P<0.05; **P<0.01; ***P<0.001). (See also Supplemental Movies 6 and 7.)
Figure 5.
Figure 5.
CX3CR1 blockade increases kidney inflammation and reduces IL-1ra production by Ly6Chigh monocyte during sepsis. (A) Intracellular production of IL-1ra was evaluated by intracellular cytokine staining gated on CD11b+ Ly6Gneg NK1.1neg Ly6Chigh cells from sham-operated (green) and CLP-operated mice (red) 6 hours after surgery. Gray histogram represents isotype staining. (B) Mean fluorescence intensity of IL-1ra intracellular staining was compared between sham-operated (green) and CLP-operated mice (red) treated or not treated with F1 (purple), 6 hours after surgery (n=6 sham, F1, and 12 WT CLP from two independent experiments; ANOVA t test was used; *P<0.05; ***P<0.001). Kidneys from CLP-operated mice treated with PBS (control) or F1 were extracted 6 hours after CLP and were evaluated by quantitative PCR for (C) TNF-α production, (D) TGF-β production, and (E) IL1ra production. Results are represented as fold increase of CLP-operated mice (n=6 in each groups from two independent experiments; t test was performed; P value are indicated).
Figure 6.
Figure 6.
The proadhesive CX3CR1 I249 allele is associated with a reduced incidence of AKI in septic patients. PBMCs from individuals with CX3CR1 VV (black) or VI (gray) genotypes were assayed for adhesion with (A) CX3CL1-expressing HEK (n=9–10 individuals) or (B) with adherent smooth muscle cells (SMCs) treated with TNF-α and IFN-γ (n=5 individuals in each group). Bars indicate mean±SEM. A t test was used; *P<0.05. (C) V249I polymorphism distribution and incidence of AKI in patients with VV− (black bar) or VI/II− (gray bar) polymorphisms (n=239; chi-squared test was used; and 95% confidence intervals of the estimate are presented).
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References

    1. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM, Sibbald WJ, The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine : Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 101: 1644–1655, 1992 - PubMed
    1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care. Crit Care Med 29: 1303–1310, 2001 - PubMed
    1. Vincent JL, Sakr Y, Sprung CL, Ranieri VM, Reinhart K, Gerlach H, Moreno R, Carlet J, Le Gall JR, Payen D, Sepsis Occurrence in Acutely Ill Patients Investigators : Sepsis in European intensive care units: Results of the SOAP study. Crit Care Med 34: 344–353, 2006 - PubMed
    1. Angus DC, van der Poll T: Severe sepsis and septic shock. N Engl J Med 369: 840–851, 2013 - PubMed
    1. Cohen J: The immunopathogenesis of sepsis. Nature 420: 885–891, 2002 - PubMed

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