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. 2014 Dec 23;9(12):e115474.
doi: 10.1371/journal.pone.0115474. eCollection 2014.

No evidence of pathogenic involvement of cathelicidins in patient cohorts and mouse models of lupus and arthritis

D Kienhöfer  1 , J Hahn  1 , I Schubert  1 , C Reinwald  1 , N Ipseiz  1 , S C Lang  1 , È Bosch Borràs  2 , K Amann  3 , C Sjöwall  4 , A E Barron  5 , A J Hueber  1 , B Agerberth  6 , G Schett  1 , M H Hoffmann  1
Affiliations

"VSports在线直播" No evidence of pathogenic involvement of cathelicidins in patient cohorts and mouse models of lupus and arthritis

D Kienhöfer (VSports app下载) et al. PLoS One. .

Abstract

Apart from their role in the immune defence against pathogens evidence of a role of antimicrobial peptides (AMPs) in autoimmune diseases has accumulated in the past years. The aim of this project was to examine the functional impact of the human cathelicidin LL-37 and the mouse cathelicidin-related AMP (CRAMP) on the pathogenesis of lupus and arthritis. Serum LL-37 and anti-LL-37 levels were measured by ELISA in healthy donors and patients with Systemic Lupus Erythematosus (SLE) and Rheumatoid arthritis (RA). Pristane-induced lupus was induced in female wild type (WT) and cathelicidin-deficient (CRAMP-/-) mice. Serum levels of anti-Sm/RNP, anti-dsDNA, and anti-histone were determined via ELISA, cytokines in sera and peritoneal lavages were measured via Multiplex. Expression of Interferon I stimulated genes (ISG) was determined by real-time PCR. Collagen-induced arthritis (CIA) was induced in male WT and CRAMP-/- mice and arthritis severity was visually scored and analysed histomorphometrically by OsteoMeasure software. Serum levels of anti-LL-37 were higher in SLE-patients compared to healthy donors or patients with RA. However, no correlation to markers of disease activity or organ involvement was observed. No significant differences of autoantibody or cytokine/chemokine levels, or of expression of ISGs were observed between WT and CRAMP-/- mice after pristane-injection. Furthermore, lung and kidney pathology did not differ in the absence of CRAMP. Incidence and severity of CIA and histological parameters (inflammation, cartilage degradation, and bone erosion) were not different in WT and CRAMP-/- mice. Although cathelicidins are upregulated in mouse models of lupus and arthritis, cathelicidin-deficiency did not persistently affect the diseases. Also in patients with SLE, autoantibodies against cathelicidins did not correlate with disease manifestation VSports手机版. Reactivity against cathelicidins in lupus and arthritis could thus be an epiphenomenon caused by extensive overexpression in blood and affected tissues. In addition, other cationic AMPs could functionally compensate for the deficiency of cathelicidins. .

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"V体育ios版" Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

"V体育安卓版" Figures

Figure 1
Figure 1. Autoantibodies against LL-37 in SLE are not linked to disease activity.
(A) In patients with SLE (n = 185) but not with RA (n = 92) or normal healthy donors (NHD, n = 74) serum autoAbs to LL-37 are detected. Shown are optical density (OD) indices, which are the ratios of the OD in the individual donor to the mean OD in NHD sera. One dot represents one patient, horizontal bars show the means. ***P<0.001, as determined by Kruskal-Wallis test with Dunn’s post-hoc test. Dashed line shows cutoff OD (1,93). (B) Sera of SLE patients (n = 21) were preincubated with saturating concentrations of LL-37 peptide before reactivity to plate-bound LL-37 was determined by ELISA. IgG reactivity to LL-37 can be blocked by preincubation with LL-37 peptide. ***P<0.001, as determined by Mann-Whitney U test. (C) Serum levels of LL-37 peptide are raised in a subgroup of SLE and RA patients. One dot represents one donor. Dashed line shows cutoff OD (2,77 ng/ml). (D) No correlation is found between SLE anti-LL-37 autoAbs and disease parameters of SLE. X-Y scatter plots of correlations between OD indices of anti-LL-37 autoAbs in SLE patients and SLEDAI (n = 163), anti-dsDNA autoAbs (n = 73), or serum levels of C-reactive protein (CRP, n = 74) or of IFNα (n = 28), respectively. Spearman coefficients (r) are depicted within the graphs. N.s., not significant. (E) Serum levels of LL-37 peptide are not linked to SLE disease activity. X-Y scatter plot of correlation between serum LL-37 levels and SLEDAI in 110 patients with SLE. Spearman coefficient (r) is depicted within the graph. N.s., not significant.
Figure 2
Figure 2. Longitudinal follow up of SLE patients does not uncover an association of anti-LL-37 autoAbs and disease activity.
Development over time of SLEDAI (dashed black line) and OD index of anti-LL-37 autoantibodies (red line) in 6 representative patients with SLE. A connection between anti-LL-37 and SLEDAI is not apparent.
Figure 3
Figure 3. Percentage of CRAMP-expressing cells is higher in pristane-injected animals.
Percentage of CRAMP+ cells in peritoneal lavage and peripheral blood of naïve mice and mice 7 days after pristane-injection. N = 5. *P<0.001, as determined by Student’s t-test.
Figure 4
Figure 4. Development of lupus autoAbs in C57BL/6 WT and CRAMP−/− mice.
CRAMP−/− and CRAMP+/+ mice develop similar levels of lupus autoantibodies after pristane-injection. Autoantibodies to dsDNA, Sm/RNP, histone, and CRAMP were analysed in sera of naïve and pristane-primed mice (n = 11–15) by ELISA and compared by ANOVA with Bonferroni post-hoc test. Red horizontal bars show means. No significant differences could be found except for autoantibodies to CRAMP. *P<0.05, ***P<0.001.
Figure 5
Figure 5. Cytokine/chemokine levels and expression of ISGs in sera and peritoneal lavages of naïve and pristane-injected mice are not significantly influenced by CRAMP-deficiency.
Scatter plots show individual measurements and means from naïve and pristane-injected mice of cytokine/chemokine concentrations in serum (A) or peritoneal lavage (B), or of expression of Interferon I-stimulated genes in serum (C), normalized to β-actin expression. One dot represents one mouse. No significant differences were found by ANOVA with Bonferroni post-hoc test.
Figure 6
Figure 6. Mild renal disease develops after pristane-injection in both WT andCRAMP−/− mice.
Representative immunofluorescence images showing predominantly mesangial IgG-deposits (A) and complement C3 deposits (B) in WT and CRAMP-deficient mice before and 6 months after pristane-injection. Scale bars, 50 µm (C) Representative PAS-stainings of kidneys from unchallenged and pristane-injected WT and CRAMP−/− mice showing mild mesangial cell and matrix expansion at day 180 in both groups. Scale bars, 50 µm (D) Quantification of proteinuria in CRAMP+/+ and CRAMP−/− mice before and 180 days after pristane-injection.
Figure 7
Figure 7. Transient alveolar haemorrhage develops after pristane-injection in both WT and CRAMP−/− mice.
(A) Classification of lung pathology in pristane-injected mice. Representative images and H&E-stainings of C57BL/6 mice two weeks after pristane-injection, with lungs showing no hemorrhage, partial, or full hemorrhage. Scale bars, 200 µm. (B) Time course of pristane-induced alveolar hemorrhage in WT and CRAMP−/− mice. Incidence and types of gross pathology at day 0, day 14, and day 180 after pristane-injection. (C) Low mortality after pristane-injection in both CRAMP+/+ (n = 14) and CRAMP−/− (n = 15) mice. No significant differences could be found by Logrank test.
Figure 8
Figure 8. Comparison of collagen-induced arthritis (CIA) in WT and CRAMP-deficient mice.
Incidence (A) of CIA and severity in affected mice (B). No significant differences could be found by comparing area under the curves of arthritic WT mice (n = 9) and CRAMP-deficient mice (n = 9) by Student’s t-test. Curves show means ± S.E.M. (C) Onset of CIA is not significantly different in CRAMP+/+ and CRAMP−/− mice. (D) Histomorphometric analysis of hind paws from WT and CRAMP−/− mice for synovial inflammation, cartilage damage, and bone erosion. Shown are scatter plots of individual measurements and means. One dot represents one mouse. No significant differences could be found by Student’s t-test. (E) Representative H&E stainings of joint sections from naïve mice and mice 37 days after collagen-injection. Left panels show sections from WT mice, right panels sections from CRAMP-deficient mice. Arrows indicate areas where inflammatory synovial tissue invades the subchondral space. Scale bars, 500 µm.
Figure 9
Figure 9. Complexes of cathelicidins and RNA or DNA induce low production of IFNα.
Bars show means and SEM of IFNα concentrations in supernatants from isolated human (A) or mouse (B) pDCs after incubation with free genomic DNA, free self-RNA, or DNA/RNA complexed to LL-37 (A) or (CRAMP). IFNα production from unstimulated pDCs or pDCs incubated with LL-37/CRAMP only was determined as a negative control, CpG2216 and R848 (Resiquimod) were used as a positive control for stimulation of TLR9 and TLR7/8, respectively. *p<0.05, as determined by Student’s t-test. N = 4–7.
Figure 10
Figure 10. Lupus antibodies induce agglomeration of mouse blood cells but no NETosis.
Representative fluorescence microscopy images and morphometry of murine blood cells incubated with various stimuli/controls and stained for DNA (DAPI, red) and neutrophil elastase (green). Incubation with PBS (A), IgG purified from naïve WT mice (B), IgG purified from PIL anti-CRAMP positive sera from WT mice (C), IgG purified from PIL anti-CRAMP-negative sera from CRAMP−/− mice (D), purified anti-CRAMP antibody (E), or PMA (F). The picture in (G) shows only NE-staining and reveals strictly cytoplasmatic expression of NE. Control stainings were performed using secondary FITC-conjugated Ab only (H). Scale bars, 100 µm. Neither incubation with IgG isolated from serum nor incubation with purified anti-CRAMP antibody induced NETosis, identifiable from extracellular washy DNA colocalizing with NE (as shown in F, white arrows). However, IgG from mice with PIL induced agglomeration of blood cells. (I) Quantification of the percentage of cells in aggregates >5x mean nuclear size and of cells having undergone NETosis. Bars show the mean and SD of 3 pictures for each treatment condition.
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