Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics

Abstract

Background: Abdominal aortic aneurysm (AAA) is an asymptomatic chronic disease of the aorta and its evolution is unpredictable. Despite the existence of several pathological mechanisms contributing to the dilation of the human AAA wall, there is currently no specific therapy to prevent the fatal rupture of the aorta. Our objective was to identify novel mediators and/or biomarkers involved in the instability of the aortic wall that could help to prevent AAA progression.

Methods: Multiplexed quantitative proteomic analysis of human AAA and healthy aortic wall (medial and adventitial layers) was performed. Results were subsequently validated by western blot and immunohistochemistry, as well as by turbidimetry/ELISA of tissue-conditioned media. In addition, immunoglobulins A1 and A2 (IGA1 and IGA2) plasma levels were analyzed by turbidimetry in a pilot study [controls (n = 22) and AAA patients (n = 22)] and in a validation study with a 6-year follow-up [controls (n = 64) and AAA patients (n = 189)]. In vitro experiments were performed in THP-1-derived macrophages (basal or polarized to M1 or M2). Polymeric immunoglobulin receptor (PIGR) mRNA expression and secretion in macrophages were analyzed by Q-PCR and ELISA, respectively. Finally, the hematopoietic contribution of PIGR was assessed in experimental AAA (Ldlr^-/- mice fed an atherogenic diet and 1 μg/Kg/min angiotensin II infusion for 28 days) by bone marrow transplantation experiments.

Results: Functional analysis of biological pathways altered in human AAA wall revealed a significant upregulation of components of the adaptive immune response, including IGHA1 and IGHA2, as well as the IGA receptor, PIGR. In addition, IGA2, but not IGA1, plasma levels were significantly increased in a pilot study of AAA patients relative to controls (489 ± 38 vs 344 ± 36 mg/L, p < 0.01). This finding was further validated in a larger cohort, confirming the association of IGA2 with AAA presence independent of risk factors and treatments [OR = 2.140 (1.109-4.130), P < 0.05]. Furthermore, in the validation cohort, elevated IGA2 plasma levels were independently associated with AAA progression [HR = 1.941 (1.108-3.399), p < 0.05]. PIGR colocalized with macrophages in the AAA wall and, PIGR mRNA levels were increased following the differentiation of THP-1 monocytes into macrophages, as well as in M1-polarized THP-1 macrophages compared to M2 macrophages. Pigr deficiency in hematopoietic cells resulted in a significantly reduced AAA incidence (14 vs 57%) and decreased macrophage infiltration (3.5 ± 0.5 vs 5.6 ± 0.7%).

Conclusions: Increased IGA and PIGR is observed in the AAA wall. Pigr deficiency in hematopoietic cells decreases AAA progression, suggesting a therapeutic role for PIGR in AAA.

Keywords: Abdominal aortic aneurysm; Biomarkers; Immune response; Immunoglobulin A; Polymeric immunoglobulin receptor; Proteomics.

Fig. 1

Differentially regulated proteins in human AAA wall. Heat map depicting significant protein abundance changes (FDR < 5%) in the AAA media (A) and adventitial (B) layers compared to healthy control aortas. Increased (red) or decreased (blue) abundances are shown according to the indicated Zq scale. Correlation network obtained after functional analysis of the proteins differentially expressed in the medial and adventitial layer of human AAA aortas are displayed in the right panels. (C) Box plots of immunoglobulins increased in human AAA adventitial layer in comparison to healthy control aortas. Distribution of protein abundances (Zq values) are plotted. *p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001. IGHA1 immunoglobulin heavy constant A1, IGHA2 immunoglobulin heavy constant A2, IGHG1 immunoglobulin heavy constant G1, IGHG2 immunoglobulin heavy constant G2, IGHG3 immunoglobulin heavy constant G3, IGHG4 immunoglobulin heavy constant G4

Fig. 2

IGA1 and IGA2 levels are increased in human AAA wall. A Western blot and densitometric analysis of IGHA1 and IGHA2 after correction for GAPDH (loading control) in medial and adventitial layers of human AAA and healthy aortas (healthy media n = 14; AAA media n = 11; healthy adventitia n = 11; AAA adventitia n = 12). B Representative immunohistochemistry and quantification of IGA1 and IGA2 in serial aortic cross-sections from AAA and healthy aortas (n = 4 for both). C Quantification by immunoturbidimetry of IGA1 and IGA2 concentrations (mg/L) in the tissue-conditioned media of AAA and healthy aortas (healthy media n = 9; AAA media n = 9; healthy adventitia n = 9; AAA adventitia n = 9). Data represent means ± SEM. Mann–Whitney U test for healthy media vs. AAA media and healthy adventitia vs. AAA adventitia in (A) and (B). ***p < 0.001, ****p < 0.0001. Scale bars, 100 µm. A.U., arbitrary units. IGHA1 immunoglobulin heavy constant A1, IGHA2 immunoglobulin heavy constant A2, IGA1 Immunoglobulin A1 (including both heavy and light chains), IGA2 Immunoglobulin A2 (including both heavy and light chains)

Fig. 3

IGA2 plasma levels predict AAA evolution. A Total IGA, IGA1 and IGA2 concentration (mg/L) in plasma from AAA patients (n = 22) and controls (n = 22). B IGA2 concentration (mg/L) in plasma from AAA patients (n = 189) and controls (n = 64). (C) Cumulative Kaplan–Meier estimates with 95% confidence intervals for progression to surgical repair stratified by the lower and upper tertiles of IGA2 (mg/L) in AAA patients. Data represent means ± SEM. Mann–Whitney U test. ****p < 0.0001

Fig. 4

Polymeric immunoglobulin receptor levels are increased in human AAA wall. A Western blot and densitometric analysis of PIGR after correction for GAPDH (loading control) in medial and adventitial layers of human AAA and healthy aortas (healthy media n = 14; AAA media n = 11; healthy adventitia n = 11; AAA adventitia n = 12). B Quantification by ELISA of PIGR concentration (ng/mL) in the tissue-conditioned media of AAA and healthy aortas (healthy media n = 9; AAA media n = 9; healthy adventitia n = 9; AAA adventitia n = 9). C Representative immunohistochemistry and quantification of PIGR in serial aortic cross-sections from AAA and healthy aortas (n = 4 for both). Scale bars, 100 µm. D Representative double immunofluorescence staining of PIGR (red) and CD68 (green) in AAA wall. DAPI nuclear staining (blue) is shown. Scale bars, 50 μm. Data represent means ± SEM. Mann–Whitney U test for healthy media vs. AAA media and healthy adventitia vs. AAA adventitia in (A) and (B). **p < 0.01, ***p < 0.001. A.U., arbitrary units

Fig. 5

PIGR is expressed and released by human macrophages. A Relative PIGR mRNA expression of THP-1 monocytes vs. PMA-stimulated THP-1 macrophages (100 nM) at 24 and 48 h upon stimulation. PCR values normalized by GAPDH mRNA endogenous control are expressed as fold increases over basal condition at 24 or 48 h. B Quantification by ELISA of PIGR concentration (ng/mL) in the tissue-conditioned media of THP-1 monocytes and THP-1 macrophages at 24 and 48 h upon PMA stimulation. C Relative PIGR mRNA expression of non-polarized THP-1 macrophages (PMA 100 nM for 72 h, M0), polarized M1 macrophages (20 ng/mL IFN-γ + 100 ng/mL LPS, 24 h) and polarized M2 macrophages (20 ng/mL IL-4 + 20 ng/mL IL-13, 24 h). PCR values normalized by GAPDH mRNA endogenous control are expressed as fold increases over basal condition at 24 or 48 h. D Quantification by ELISA of PIGR concentration (ng/mL) in the tissue-conditioned media of non-polarized THP-1 macrophages (PMA 100 nM for 72 h, M0), polarized M1 macrophages (20 ng/mL IFN-γ + 100 ng/mL LPS, 48 h) and polarized M2 macrophages (20 ng/mL IL-4 + 20 ng/mL IL-13, 48 h). Data represent means ± SEM. Student's t-test. *p < 0.05, **p < 0.01, ****p < 0.0001. A.U., arbitrary units

Fig. 6

Pigr deficiency in the hematopoietic compartment decreases AAA development. A Experimental design and (B) incidence of AAA in Ldlr^−/− Pigr^+/+ (n = 14) and Ldlr^−/− Pigr^−/− (n = 14) chimeras at 28 days after subcutaneous AngII infusion via osmotic pumps. C Representative H&E staining and quantification of the maximal abdominal aortic diameter. D Representative photographs and quantification of CD68 immunostaining. Data represent means ± SEM. *p < 0.05, Mann–Whitney U test. Scale bars, 100 µm