Cytokine concentrations in stimulated splenocyte supernatant for mice receiving PBS or FL before and during sensitization to the nephritogenic antigen in the anti-GBM disease model.

<p>Data presented as mean±SEM, except for IL-10, presented as median (range) as data is non-parametric. IL-6, IL-12p70 and TNF were not detected.</p><p>Cytokine concentrations in stimulated splenocyte supernatant for mice receiving PBS or FL before and during sensitization to the nephritogenic antigen in the anti-GBM disease model.</p

FL therapy expands pDCs but increases delayed type hypersensitivity four days after sensitization to sheep globulin.

<p>(A) Experimental design. (B, C) Total spleen and LN cell number 4 and 10 days after sensitization, respectively. (D, E) Proportion of pDCs in the spleen and LN 4 and 10 days post-sensitization to sheep globulin respectively. (F, G) Footpad swelling in mice 4 and 10 days post-sensitization to sheep globulin, respectively. Black bars represent PBS treated mice. White bars represent FL treated mice. n = 4 per group. *P&lt;0.05, **P&lt;0.01.</p

FL administered before accelerated autologous phase anti-GBM disease did not protect against nephritis, but altered systemic immunity.

<p>(A) Proteinuria (dotted line represents measured level in non-nephritic WT mice, n = 4). (B) Serum urea (dotted line represents measured level in non-nephritic WT mice, n = 4). (C) Proportions of glomeruli with crescents and (D) glomerular segmental necrosis. (E) Representative images of glomeruli from PBS and FL treated mice taken at high power (400x, PAS stain). (F) Total spleen and LN cell number. (G) Proportion of CD11c+ cells from leukocytes in spleen and LN. (H) Proportion of pDCs in spleen and LN. (I) Serum mouse anti-sheep IgG antibody levels. Black bars represent PBS treated mice. White bars represent FL treated mice. OD, optical density. n = 6 per group. *P&lt;0.05.</p

FL administered throughout accelerated autologous phase anti-GBM disease reduced mouse survival.

<p>(A) Experimental design. (B) Survival following the induction of nephritis. Black bars represent PBS treated mice. White bars represent FL treated mice. n = 9 per group. *P&lt;0.05.</p

FL therapy throughout accelerated anti-GBM disease did not protect mice from nephritis.

<p>(A) Experimental design. (B) Proteinuria. (C) Percentage of glomerular crescents and (D) glomeruli with segmental necrosis. (E) Representative images of glomeruli from PBS and FL treated mice taken at high power (400x, PAS stain). (F–H) gcs, periglomerular and interstitial hpf CD11c+ immunofluorescence assessed by fluorescent microscopy of renal sections. (I) Representative images of CD11c+ immunofluorescent staining within the interstitial, glomerular and periglomerular regions. Black bars represent PBS treated mice. White bars represent FL treated mice. AU, arbitrary units; gcs, glomerular cross section; hpf, high-powered field. n = 7 per group. *P&lt;0.05.</p

The effects of 10 days of intraperitoneal FL on DC and Treg populations.

<p>(A) Experimental design, where FL or PBS was administered intraperitoneally to mice for daily for 10 days. (B) Total spleen and pooled LN cell number. (C) Representative FACS plots showing proportions of pDCs (CD11c+PDCA-1+ cells) in the spleen, gating on all CD11c+ leukocytes. (D) Proportions of CD11c+ cells in the spleen and LN. (E) Proportions of pDCs in the spleen and LN. (F) Representative FACS plots showing Tregs in the spleen, gating on lymphocytes, staining for CD4+ and foxp3+ cells. (G) Proportion of Tregs in the spleen and LN. Black bars represent PBS treated mice. White bars represent FL treated mice. n = 4 per group. *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001.</p

Cellular pro-inflammatory immune responses were heightened when FL was given during accelerated anti-GBM disease.

<p>(A) IL-17A+ spots and (B) IFNγ+ spots per 1 million stimulated splenocytes, measured by EliSpot. (C) Total spleen and LN cell number. (D, E) Proportion of CD11c+ cells and pDCs in the spleen and LN. (F) Representative FACS plots of splenocytes stained for CD11c and PDCA-1. (G) Proportion of activated T cells (CD4+CD25+/CD4+) in spleen and LN. (H) Representative FACS plots of pooled LN cells stained for CD4, CD25 and foxp3. (I, J) Proportions of Tregs (CD4+CD25+foxp3+/CD4+) and Teff (CD4+CD25+foxp3-/CD4+) in spleen and LN. Black bars represent PBS treated mice. White bars represent FL treated mice. n = 7 per group. *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001, ****P&lt;0.0001.</p

Cytokine concentrations in stimulated splenocyte supernatant for mice receiving PBS or FL for 10 days prior to commencement of the anti-GBM disease model.

<p>Data presented as mean±SEM, except for IL-10, presented as median (range) as data is non-parametric. TNF, IL-12p70 and IL-6 were not detectable.</p><p>Cytokine concentrations in stimulated splenocyte supernatant for mice receiving PBS or FL for 10 days prior to commencement of the anti-GBM disease model.</p

Intrarenal leukocytes in mice treated with PBS or FL throughout anti-GBM disease until day 9.

<p>c/gcs, cells per glomerular cross section; c/hpf, cells per high power field.</p><p>*P = 0.02.</p><p>Intrarenal leukocytes in mice treated with PBS or FL throughout anti-GBM disease until day 9.</p

Effects of FL given during sensitization, but not during the nephritic phase of injury.

<p>(A) Experimental design. (B) Serum urea (dotted line represents measured level in non-nephritic WT mice, n = 4). (C) Proteinuria (dotted line represents measured level in non-nephritic WT mice, n = 4). (D, E) Percentage of glomerular crescents and segmental necrosis. (F) Total spleen and LN cell number. (G) Proportion of Tregs in the spleen and LN. (H) Serum mouse anti-sheep IgG antibody concentrations. Black bars represent PBS treated mice. White bars represent FL treated mice. OD, optical density. n = 7 for PBS group and 9 for FL group. *P&lt;0.05, ***P&lt;0.001.</p