Background. Abnormalities affecting regulatory molecules and pentraxin (PTX) 3 have been suggested to contribute to development of lupus glomerulonephritis (LN). Characterization of novel pathogenic pathways could pave the way to targeted therapeutic approaches. Aims. To investigate the role and relevance of PTX3/anti-PTX3 related immunity in systemic lupus erythematosus (SLE) patients and in lupus murine models and to explore the in vivo effects of restoration of SERPINB3 levels. Methods. The overall project comprised four experimental phases. Three out of four experiments were carried out on murine models of SLE, either New Zealand Black/White (NZB/W F1) or Mrl/lpr mice, while one experiment was conducted in humans. In the first experiment, intraperitoneal administration of recombinant SERPINB3 (7.5 μg/0.1mL or 15 μg/0.1mL) or placebo (PBS 0.1 ml) was carried out in 40 NZB/W F1 mice divided into four groups of 10 mice each. Group 1 and 2 were treated before (preventive approach and group 3 and 4 after (therapeutic approach) development of proteinuria ≥100mg/dl. Two additional groups included 20 MRL/lpr mice which were prophylactically injected with SERPINB3 (10 mice, group 5) or PBS (10 mice, group 6). The second experiment involved 30 NZB/W F1 mice which underwent subcutaneous immunization with PTX3+alum (n=10), PBS+alum (n=10) or PBS alone (n=10) three times three weeks apart, starting before development of proteinuria. For both experiments, time of occurrence and levels of anti-dsDNA and anti-C1q antibodies, proteinuria and serum creatinine, overall- and proteinuria-free survival were assessed in mice followed up to natural death. Subanalysis regarding anti-PTX3 antibody levels and function were performed in the second experiment. The last experiment on mice involved 22 NZB/W F1 female littermates divided into two groups of 10 mice each and treated with the same approach described in the second experiment. Ten mice (5 from each group) were sacrificed at week 22 and the other 10 at 29 weeks. Histological and ultrastructural lesions were compared using optical microscopy, immunoelectron microscopy (IEM), immunofluorescence (IF) and confocal microscopy. Data on humans were retrieved enrolling 38 SLE patients (12 with biopsy-proven LN and 26 without LN) and 22 matched healthy donors (HD). Characterization and comparison of circulating levels of PTX3 specific (PTX3+) B cells between patients and controls was performed by flow-cytometry. The differences between groups for nonparametric continuous variables were analyzed using Mann-Whitney U test or Kruskal-Wallis’s ANOVA test when appropriate; proteinuria-free survival rate (proteinuria <300 mg/dl) and survival rates were evaluated by Kaplan-Meyer method using Mantel-Cox test for comparison. Chi-squared test was used for histological comparison. A p value<0.05 was considered statistically significant. Results. Experiments on mice showed positive results in terms of clinical effects deriving from restoration of SERPINB3 levels or immunization with PTX3. SERPINB3-administered mice displayed a milder LN, with lower and delayed occurrence of nephritogenic antibodies and milder proteinuria at several timepoints, as well as a prolonged survival versus PBS groups. Immunization with PTX3 evoked a vaccine-like response with occurrence of anti-PTX3 antibodies only in immunized mice and delayed and decreased levels of nephritogenic antibodies and proteinuria, resulting in a significantly longer disease-free and overall survival. Among mice sacrificed at given timepoints, notable differences were observed at IEM, IIF and confocal microscopy. PTX3-immunized mice displayed less or no electron-dense deposits (EDD) along the glomerular basement membrane and the mesangium, and remarkably decreased glomerular deposition of IgG, C1q and PTX3 compared to PBS-treated mice. Moreover, PTX3 was found inside the EDD at IEM and was shown to co-localized with nuclear material. LN patients displayed significantly lower levels of circulating PTX3+ B cells in comparison to SLE and HD, showing a persistent decrease among naïve and memory PTX3+ specific subsets. Conclusions. Clinical improvement of a lupus-like disease following restoration of SERPINB3 levels and immunization with PTX3 in lupus murine models suggests that very early abnormalities affecting molecules involved in tissue homeostasis, apoptosis and removal of apoptotic debris may induce further development of SLE and SLE-specific manifestations within an unpredictable lag time. PTX3 more strikingly emerged as a novel antigen in LN progression and PTX3/anti-PTX3 immunity appear to function as an early level of regulation which may fail in patients developing LN. Consistently, acquisition of a targeted anti-PTX3 immunity could hinder the progression from the preclinical to the clinical stages of disease. Altogether, these findings may add a piece of knowledge on the mechanisms supporting LN development and progression, and suggest that a targeted modulation of the native immunity could improve renal manifestations in selected patients. In the short term, dosage of serum anti-PTX3 antibodies may become a handful tool to help in stratifying lupus patients according to the risk of developing LN.  

Novel Pathogenic Pathways and Therapeutic Implications in Lupus Nephritis: The Emerging Role of PTX3-Related Immunity / Gatto, Mariele. - (2020 Nov 23).

Novel Pathogenic Pathways and Therapeutic Implications in Lupus Nephritis: The Emerging Role of PTX3-Related Immunity

Gatto, Mariele
2020

Abstract

Background. Abnormalities affecting regulatory molecules and pentraxin (PTX) 3 have been suggested to contribute to development of lupus glomerulonephritis (LN). Characterization of novel pathogenic pathways could pave the way to targeted therapeutic approaches. Aims. To investigate the role and relevance of PTX3/anti-PTX3 related immunity in systemic lupus erythematosus (SLE) patients and in lupus murine models and to explore the in vivo effects of restoration of SERPINB3 levels. Methods. The overall project comprised four experimental phases. Three out of four experiments were carried out on murine models of SLE, either New Zealand Black/White (NZB/W F1) or Mrl/lpr mice, while one experiment was conducted in humans. In the first experiment, intraperitoneal administration of recombinant SERPINB3 (7.5 μg/0.1mL or 15 μg/0.1mL) or placebo (PBS 0.1 ml) was carried out in 40 NZB/W F1 mice divided into four groups of 10 mice each. Group 1 and 2 were treated before (preventive approach and group 3 and 4 after (therapeutic approach) development of proteinuria ≥100mg/dl. Two additional groups included 20 MRL/lpr mice which were prophylactically injected with SERPINB3 (10 mice, group 5) or PBS (10 mice, group 6). The second experiment involved 30 NZB/W F1 mice which underwent subcutaneous immunization with PTX3+alum (n=10), PBS+alum (n=10) or PBS alone (n=10) three times three weeks apart, starting before development of proteinuria. For both experiments, time of occurrence and levels of anti-dsDNA and anti-C1q antibodies, proteinuria and serum creatinine, overall- and proteinuria-free survival were assessed in mice followed up to natural death. Subanalysis regarding anti-PTX3 antibody levels and function were performed in the second experiment. The last experiment on mice involved 22 NZB/W F1 female littermates divided into two groups of 10 mice each and treated with the same approach described in the second experiment. Ten mice (5 from each group) were sacrificed at week 22 and the other 10 at 29 weeks. Histological and ultrastructural lesions were compared using optical microscopy, immunoelectron microscopy (IEM), immunofluorescence (IF) and confocal microscopy. Data on humans were retrieved enrolling 38 SLE patients (12 with biopsy-proven LN and 26 without LN) and 22 matched healthy donors (HD). Characterization and comparison of circulating levels of PTX3 specific (PTX3+) B cells between patients and controls was performed by flow-cytometry. The differences between groups for nonparametric continuous variables were analyzed using Mann-Whitney U test or Kruskal-Wallis’s ANOVA test when appropriate; proteinuria-free survival rate (proteinuria <300 mg/dl) and survival rates were evaluated by Kaplan-Meyer method using Mantel-Cox test for comparison. Chi-squared test was used for histological comparison. A p value<0.05 was considered statistically significant. Results. Experiments on mice showed positive results in terms of clinical effects deriving from restoration of SERPINB3 levels or immunization with PTX3. SERPINB3-administered mice displayed a milder LN, with lower and delayed occurrence of nephritogenic antibodies and milder proteinuria at several timepoints, as well as a prolonged survival versus PBS groups. Immunization with PTX3 evoked a vaccine-like response with occurrence of anti-PTX3 antibodies only in immunized mice and delayed and decreased levels of nephritogenic antibodies and proteinuria, resulting in a significantly longer disease-free and overall survival. Among mice sacrificed at given timepoints, notable differences were observed at IEM, IIF and confocal microscopy. PTX3-immunized mice displayed less or no electron-dense deposits (EDD) along the glomerular basement membrane and the mesangium, and remarkably decreased glomerular deposition of IgG, C1q and PTX3 compared to PBS-treated mice. Moreover, PTX3 was found inside the EDD at IEM and was shown to co-localized with nuclear material. LN patients displayed significantly lower levels of circulating PTX3+ B cells in comparison to SLE and HD, showing a persistent decrease among naïve and memory PTX3+ specific subsets. Conclusions. Clinical improvement of a lupus-like disease following restoration of SERPINB3 levels and immunization with PTX3 in lupus murine models suggests that very early abnormalities affecting molecules involved in tissue homeostasis, apoptosis and removal of apoptotic debris may induce further development of SLE and SLE-specific manifestations within an unpredictable lag time. PTX3 more strikingly emerged as a novel antigen in LN progression and PTX3/anti-PTX3 immunity appear to function as an early level of regulation which may fail in patients developing LN. Consistently, acquisition of a targeted anti-PTX3 immunity could hinder the progression from the preclinical to the clinical stages of disease. Altogether, these findings may add a piece of knowledge on the mechanisms supporting LN development and progression, and suggest that a targeted modulation of the native immunity could improve renal manifestations in selected patients. In the short term, dosage of serum anti-PTX3 antibodies may become a handful tool to help in stratifying lupus patients according to the risk of developing LN.  
23-nov-2020
lupus nephritis, PTX3, electron microscopy, immunofluorescence, survival
Novel Pathogenic Pathways and Therapeutic Implications in Lupus Nephritis: The Emerging Role of PTX3-Related Immunity / Gatto, Mariele. - (2020 Nov 23).
File in questo prodotto:
File Dimensione Formato  
mariele_gatto_tesi.pdf

accesso aperto

Tipologia: Tesi di dottorato
Licenza: Non specificato
Dimensione 2.96 MB
Formato Adobe PDF
2.96 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3426251
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact