Figure 1.
Decreased Gr1low monocyte counts in IL-17 receptor A deficient mice.
(A,B) Peripheral blood total white cell (A) and monocyte (B) counts were assessed by an automated analyzer in wild type (wt) and Interleukin-17 receptor A-deficient (Il17ra-/-) mice (n = 9–12). (C–E) Gr1high and Gr1low monocyte subgroups were analyzed by flow cytometry after gating for CD11b+CD115+ events (example in C, D: proportion of Gr1high monocytes and E: absolute concentrations in wt and Il17ra-/- mice, n = 9–12, t-tests).
Figure 2.
Monocyte characterization in mixed chimeric wt/IL-17 receptor A deficient mice.
Monocyte analysis in mixed bone marrow chimeric wt(CD45.1)/Il17ra-/-(CD45.2) mice (recipients: CD45.2 wt mice). (A) Monocytes were gated by CD115 and IL-17 receptor expression was assessed as control of complete chimerism. (B) Gr1high and Gr1low monocytes in mixed chimeric mice were analyzed for the intensity of Il17ra expression (MFI, n = 16 from 3 independent transplantations, Bonferroni after One-way-ANOVA). (C) Gr1high monocytes were sorted from bone-marrow and expression of IL-17 receptor subunits analyzed by qPCR (means of n = 2 independent cell sorts).
Figure 3.
Absence of IL-17 receptor A reduces monocyte counts in a cell specific manner.
Leukocyte analysis in mixed bone marrow chimeric wt (CD45.1)/Il17ra-/- (CD45.2) mice. (A) The proportion of Il17ra-/- cells among blood lymphocytes (Ly), granulocytes (PMN) and monocytes (mono). (B) Absolute monocyte concentrations for each genotype. (C,D) The proportion of Gr1high (C, n = 20 mice from 3 independent transplantations) and Ly6Chigh monocytes (D, n = 6) was significantly higher among Il17ra-/- than wt cells. (E) Absolute blood monocyte concentrations (Bonferroni after One-way-ANOVA, n = 20 mice from 3 independent transplantations). (F) Proportion of blood monocytes staining as dead cells (n = 16 from 3 independent transplantations, Bonferroni after One-way-ANOVA)).
Figure 4.
Homeostatic tissue macrophages are altered without IL-17 receptor.
(A–D) Monocytes, macrophages and dendritic cells from mixed bone marrow chimeric wt/Il17ra-/- mice were analyzed by flow cytometry after enzymatic digestion of lungs (examples in A, statistical analysis in B), in spleen (C) and peritoneal cavity (D) (n = 8 and 2 independent transplantations, paired t-tests).
Figure 5.
Il17ra-/-Gr1low monocyte development in the bone marrow.
(A,B) Bone marrow progenitors were defined among lineage negative cells as hematopoietic stem cells (HSC, CD117+Sca+), myeloid progenitors (MP, CD117+Sca−), common myeloid progenitors (CMP, CD34+Fcγr− among MP) and granulocyte macrophage progenitors (GMP, CD34+Fcγr+ among MP). To assess the role of the IL-17 receptor on a cell specific level, the proportion of wt and Il17ra-/- cells at each stage was analyzed in mixed bone marrow chimeric mice (B, n = 10 from 3 independent transplantations). (C,D) Similarly bone marrow CD11b+CD115+ monocytes were analyzed for genotype (C) and Gr1high proportion (D) in wt and Il17ra-/- cells (n = 10 from 3 independent transplantations, paired t-test).
Figure 6.
Lack maintenance of Il17ra-/-Gr1low monocyte population after normal initial recovery.
(A-D) Mixed bone marrow chimeric wt/Il17ra-/- mice underwent monocyte depletion by liposomal clodronate. During recovery, monocyte genotype (A), proportion of Gr1high and Gr1low cells among the monocytes of each genotype (B) and absolute blood concentrations of Gr1high (C) and Gr1low monocytes (D) of each genotype were measured (n = 8, 2 independent transplantations, Bonferroni after two-way-ANOVA).
Figure 7.
Kinetics of labeled Gr1high and Gr1low monocytes in the presence and absence of Il17ra-/-.
(A,B) Monocytes in mixed chimeric wt/Il17ra-/- mice were labeled with fluorescent latex beads (A). The proportion of labeled CD11b+CD115+ cells among each monocyte subgroup (Gr1high and Gr1low monocytes) within each genotype was assessed over time (B, n = 4-5, 2 independent transplantations, Bonferroni after ANOVA). (C) Dividing cells in mixed bone marrow chimeric wt/Il17ra-/- mice labeled with a single BrdU injection and peripheral blood monocytes were analyzed for BrdU incorporation at the indicated timepoints. In every animal, the proportion of labeled cells among Gr1high and Gr1low monocytes of either genotype was assessed over time (n = 4, * indicates sign. differences between wt and Il17ra-/- Gr1low monocytes at the indicated timepoints, # indicates a sign. change over time within wt (black bars) or Il17ra-/- (white bars) Gr1low monocytes, analysis with Bonferroni after ANOVA). (D) Proliferation of bone marrow Gr1high and Gr1low monocytes of both genotypes was assessed 20 h after BrdU injection (n = 7 from 2 independent transplantations, Bonferroni after ANOVA).
Figure 8.
In peritonitis, Il17ra-/- monocyte recruitment is sustained, but macrophage phenotype changed.
(A,B) Peritonitis was induced in mixed bone marrow chimeric wt/Il17ra-/- mice. Peripheral blood (at start and end of the experiment) and peritoneal neutrophils (PMN, CD11b+CD115−Gr1high, A) and monocytes (CD11b+CD115+, B) were analyzed by flow cytometry after 10 h (n = 4, Bonferroni after ANOVA). (C) At day 3, intraperitoneal myeloid CD11b+ cells were analyzed and compared to peripheral blood monocytes at start and end of the experiment (n = 4–6, 2 independent transplantations, Bonferroni after ANOVA). (D) CD11c expression on wt and Il17ra-/- myeloid (CD11b+) cells was compared in lung, spleen, resting and inflamed peritoneal cavity of mixed bone marrow chimeric wt/Il17ra-/- mice (n = 4–8, paired t-tests).
Figure 9.
Reduced renal macrophage generation in response to urethral obstruction in the absence of IL-17 receptor.
Renal fibrosis was induced by unilateral ureteral obstruction (UUO) in mixed wt/Il17ra-/- bone marrow chimeric mice. Myeloid cell accumulation in the obstructed kidney was assessed for wt and Il17ra-/- cells in identical environments (A,B) Total CD11b+ myeloid cell infiltration to the kidney (A, ctrl = contralateral kidney, UUO = obstructed kidney) and their genotype (B, #indicates significance level against expected 50% wt ratio, *between UUO and ctrl) was determined. (C,D) For assessment of differentiation, renal myeloid cell CD11c, F4/80 (C) and Gr1 (D) surface marker expression was tested (n = 6 from 2 independent transplantations, paired t-tests). (E) Renal myeloid cell proliferation was assessed 24 h after a single injection of BrdU (n = 4).
Figure 10.
Decreased renal fibrosis in the absence of IL-17 receptor on myeloid cells.
Unilateral ureteral obstruction was performed in wt and Il17ra-/- bone marrow chimeric mice. (A–C) Histologic assessment of fibrosis was conducted after Masson's trichrome (A), Sirius red (B, birefringent sirius red positive fibrotic tissue area analyzed from n = 5/group from 2 independent transplantations, 40× original magnification) and αSMA- immunofluorescent staining (C, n = 7 from 2 independent transplantations, scale bar = 100 µm) on day 7. (D) mRNA expression of collagen I, fibrinonectin and CTGF was assessed in obstructed kidneys by quantitative PCR (day seven, n = 3, t-tests).