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. 2018 Sep 12:9:1936.
doi: 10.3389/fimmu.2018.01936. eCollection 2018.

Anti-LL37 Antibodies Are Present in Psoriatic Arthritis (PsA) Patients: New Biomarkers in PsA

Loredana Frasca  1 , Raffaella Palazzo  1 , Maria S Chimenti  2 , Stefano Alivernini  3   4 , Barbara Tolusso  3 , Laura Bui  5 , Elisabetta Botti  6 , Alessandro Giunta  6 , Luca Bianchi  6 , Luca Petricca  3 , Simone E Auteri  7 , Francesca Spadaro  8 , Giulia L Fonti  2 , Mario Falchi  9 , Antonella Evangelista  5 , Barbara Marinari  6 , Immacolata Pietraforte  10 , Francesca R Spinelli  7 , Tania Colasanti  7 , Cristiano Alessandri  7 , Fabrizio Conti  7 , Elisa Gremese  3   4 , Antonio Costanzo  11 , Guido Valesini  7 , Roberto Perricone  2 , Roberto Lande  1
Affiliations

Anti-LL37 Antibodies Are Present in Psoriatic Arthritis (PsA) Patients: New Biomarkers in PsA

Loredana Frasca et al. Front Immunol. .

Abstract

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis associated with psoriasis. A third of psoriatic patients develop PsA via unknown mechanisms. No reliable diagnostic markers are available for PsA, or prognostic biomarkers for PsA development in psoriasis. We previously uncovered a pro-inflammatory role for cathelicidin LL37 in lesional psoriasis skin. LL37 binds nucleic acids and stimulates plasmacytoid/myeloid dendritic cells (pDC, mDCs) to secrete type I interferon (IFN-I) and pro-inflammatory factors. LL37 becomes an autoantigen for psoriatic Th1-Th17/CD8 T cells. Anti-LL37 antibodies were detected in systemic lupus erythematosus, an autoimmune disease characterized by neutrophil-extracellular-traps release (NETosis) in target organs. LL37 can be substrate of irreversible post-translational modifications, citrullination or carbamylation, linked to neutrophil activity. Here we analyzed inflammatory factors, included LL37, in PsA and psoriasis plasma and PsA synovial fluids (SF)/biopsies. We show that LL37 (as a product of infiltrating neutrophils) and autoantibodies to LL37 are elevated in PsA, but not OA SF. Anti-LL37 antibodies correlate with clinical inflammatory markers. Anti-carbamylated/citrullinated-LL37 antibodies are present in PsA SF/plasma and, at lower extent, in psoriasis plasma, but not in controls. Plasma anti-carbamylated-LL37 antibodies correlate with PsA (DAS44) but not psoriasis (PASI) disease activity. Ectopic lymphoid structures, and deposition of immunoglobulin-(Ig)G-complexes (IC) co-localizing with infiltrating neutrophils, are observed in PsA and not OA synovial tissues (ST). Activated complement (C5a, C9), GM-CSF and IFN-I are up-regulated in PsA and not OA synovia and in PsA and psoriasis plasma but not in HD. C9 and GM-CSF levels in PsA SF correlate with clinical inflammatory markers and DAS44 (C9) and with anti-carbamylated/citrullinated-LL37 antibodies (GM-CSF and IFN-I). Thus, we uncover a role for LL37 as a novel PsA autoantibody target and correlation studies suggest participation of anti-LL37 antibodies to PsA pathogenesis. Notably, plasma antibodies to carbamylated-LL37, which correlate with DAS44, suggest their use as new disease activity markers. GM-CSF and complement C5a and C9 elevation may be responsible for autoantigens release by neutrophils and their modification, fueling inflammation and autoreactivity establishment. Finally, targeting GM-CSF, C5a, C9 can be beneficial in PsA.

Keywords: LL37; Psoriatic arthritis; autoantibodies complement; neutrophils; psoriasis.

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Figures

Figure 1
Figure 1
LL37 is expressed in synovial compartment of PsA. (A) LL37 was measured by ELISA in synovial fluids of PsA (N = 12) and control OA patients (N = 11), and LL37 levels are shown as median with Interquartile Range (IQR). P-value is calculated by two-tailed Mann-Whitney U test *p < 0.05. (B) Confocal microscopy images of synovial tissues of PsA and OA patients stained for myeloperoxidase (MPO; red), LL37 (gray) (original magnification 63x). For PsA, 1 representative staining of 7 patients is shown. For OA, 1 representative staining of 4 patients is shown.
Figure 2
Figure 2
Anti-LL37 antibodies are present in synovial fluids of PsA. Synovial fluids of PsA and OA (N = 11) patients were analyzed by ELISA for the presence of anti-LL37 (PsA, N = 19), anti-LL37cit (PsA, N = 21) and anti-LL37carb (PsA, N = 17). Antibody levels are shown as median with Interquartile Range (IQR). P-value is calculated by two-tailed Mann-Whitney U test, ***p < 0.0001. The mean+2 SD (standard deviation) of OA antibody reactivity to native LL37 or modified LL37 was used as cut-off (dotted line).
Figure 3
Figure 3
Anti-LL37 levels in SF PsA correlate with disease activity and inflammation markers. SF PsA levels of anti-LL37 Abs (N = 16) measured by ELISA as shown in Figure 2 were correlated with Disease Activity Score (DAS44), C reactive Protein (CRP), Eritrocyte Sedimentation Rate (ESR) and number of swollen joints. The correlation between the two variables was assessed by Spearman correlation coefficient.
Figure 4
Figure 4
Autoantibody reactivity to citrullinated and carbamylated LL37 in plasma of PsA and Pso patients. (A) Levels of anti-LL37cit (N = 29) and anti-LL37carb (N = 32) were measured in plasma of PsA, Pso (without PsA, N = 17) and healthy donors (HD, N = 14) by ELISA. Antibody levels are shown as median with Interquartile Range (IQR). P-value is calculated by two-tailed Mann-Whitney U test, *p < 0.05, ***p < 0.0001. The mean of HD antibody reactivity+2 SD was used as cut-off (dotted line). (B) Levels of PsA anti-LL37carb were correlated with Disease Activity Score (DAS44) by Spearman correlation coefficient and the significant P value by two-tailed Mann-Whitney U test.
Figure 5
Figure 5
IgG-immune complexes deposition and colocalization with neutrophil-derived LL37 in synovia of PsA. Confocal microscopy images of synovial tissues of two PsA patients stained for LL37 (red), IgG-IC (green) (original magnification, 63x). The dotted white line in PsA#2 indicates the inset of the corresponding picture of the bottom panel. Data are representative of 7 PsA patients.
Figure 6
Figure 6
Germinal Center-like structures in synovial tissues of PsA patients. IHC for CD3, CD20, CD21, CD23, Bcl6, and Ki67 (all DAB, brown) of synovial tissue from three early, naive to DMARDs treatment PsA patients (original magnification, 20x). The black arrows indicate a positive staining for CD21, CD23, Ki67, and Bcl6. The dotted black line indicates the insert of the corresponding picture at 40x magnification.
Figure 7
Figure 7
Complement C5a, C9, and GM-CSF in SF and plasma of PsA and Pso patients. (A) Content of C5a (N = 19), C9 (N = 19), and GM-CSF (N = 21) were measured by ELISA in SF of PsA and OA (N = 12). P-value is calculated by two-tailed Mann-Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.0001. (B) Plasma levels of C5a in PsA (N = 25) and Pso (without PsA, N = 14), PsA C9 (N = 18), and Pso (N = 25), PsA GM-CSF (N = 25) and Pso (N = 16) were assessed by ELISA. All data in (A) and (B) are shown as as median with Interquartile Range (IQR). P-value is calculated by two-tailed Mann-Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 8
Figure 8
GM-CSF correlates with complement and anti-LL37 antibodies in PsA. (A) Spearman correlation analysis was performed between levels of GM-CSF and anti-LL37carb in SF PsA (N = 17). (B) Spearman correlation analysis between GM-CSF and C5a, anti-LL37cit, and anti-LL37carb in plasma of PsA patients (N = 23).
Figure 9
Figure 9
Circulating C5a correlates with anti-LL37 antibodies and disease activity. Spearman correlation analysis was performed between levels of C5a with anti-LL37cit (N = 24), anti-LL37carb (N = 24) and DAS44 in plasma of PsA (N = 17).
Figure 10
Figure 10
C9 staining in synovial tissues of PsA. Confocal microscopy images of synovial tissues of two PsA and one OA patients stained for LL37 (red) and C9 (green) (original magnification, 63x). The dotted white line in PsA#2 indicates the inset of the corresponding picture of the lower panel. Data are representative of 7 PsA patients and 4 OA subjects.
Figure 11
Figure 11
Type I IFN in the synovial compartment of PsA. (A) IFNa was measured by ELISA in SF of PsA (N = 20) and OA (N = 12) subjects. Data are shown as median with Interquartile Range (IQR). P-value is calculated by two-tailed Mann-Whitney U test ***p < 0.001. (B) Confocal microscopy images of synovial tissues of one PsA and one OA patient stained for LL37 (red) and MxA (green) (original magnification, 63x). Data are representative of 7 PsA patients and 4 OA subjects.
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