Systemic inflammation in acute intermittent porphyria: a case-control study

Abstract

This study aimed to examine whether acute intermittent porphyria (AIP) is associated with systemic inflammation and whether the inflammation correlates with disease activity. A case-control study with 50 AIP cases and age-, sex- and place of residence-matched controls was performed. Plasma cytokines, insulin and C-peptide were analysed after an overnight fast using multiplex assay. Long pentraxin-3 (PTX3) and complement activation products (C3bc and TCC) were analysed using enzyme-linked immunosorbent assay (ELISA). Urine porphobilinogen ratio (U-PBG, µmol/mmol creatinine), haematological and biochemical tests were performed using routine methods. Questionnaires were used to register AIP symptoms, medication and other diseases. All 27 cytokines, chemokines and growth factors investigated were increased significantly in symptomatic AIP cases compared with controls (P < 0·0004). Hierarchical cluster analyses revealed a cluster with high visfatin levels and several highly expressed cytokines including interleukin (IL)-17, suggesting a T helper type 17 (Th17) inflammatory response in a group of AIP cases. C3bc (P = 0·002) and serum immunoglobulin (Ig)G levels (P = 0·03) were increased significantly in cases with AIP. The U-PBG ratio correlated positively with PTX3 (r = 0·38, P = 0·006), and with terminal complement complex (TCC) levels (r = 0·33, P = 0·02). PTX3 was a significant predictor of the biochemical disease activity marker U-PBG in AIP cases after adjustment for potential confounders in multiple linear regression analyses (P = 0·032). Prealbumin, C-peptide, insulin and kidney function were all decreased in the symptomatic AIP cases, but not in the asymptomatic cases. These results indicate that AIP is associated with systemic inflammation. Decreased C-peptide levels in symptomatic AIP cases indicate that reduced insulin release is associated with enhanced disease activity and reduced kidney function.

Keywords: chemokines; complement; cytokines; human; inflammation.

© 2016 British Society for Immunology.

Figures

Figure 1

Increased levels of cytokines and growth factors in asymptomatic (Asympt.) and symptomatic (Sympt.) acute intermittent porphyria (AIP) cases compared with matched controls (Ctrl). (a) Increased levels of tumour necrosis factor (TNF), (b) interleukin (IL)‐1β and (c) IL‐17, (d) interferon (IFN)‐γ and the growth factors (e) basic fibroblast growth factor (FGF basic), and (f) vascular endothelial growth factor (VEGF), (g) granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and (h) platelet‐derived growth factor (PDGF)‐BB in asymptomatic and symptomatic AIP cases compared with their respective matched controls (Ctrl). The concentration of cytokines and chemokines were measured using a multiplex cytokine assay. The results are expressed as pg/ml. The results from the age‐ and sex‐matched controls (n = 50, including 35 controls for the symptomatic cases) and the cases with AIP (n = 50, 35 symptomatic) are shown as scatter‐plots with the median. The horizontal dotted gridline indicate each cytokine's upper reference value (97·5 percentile) in 42 healthy controls. The data were analysed using the Wilcoxon matched‐pairs signed‐rank test.

Figure 2

Hierarchical cluster analysis of cytokines in acute intermittent porphyria (AIP) cases and matched controls. The cytokine levels in each AIP case and matched controls were divided on the median levels of each cytokine in the 50 matched controls. The matrix results were then transformed using natural logarithm. The ln expression levels of individual cytokines are represented by a colour scale, with high values in red, intermediate levels in white and low cytokine levels in blue. The cluster analysis was performed using r with Euclidian distance and complete clustering. The main clusters of AIP cases and controls are named (a) and (b), and the subclusters are named (a1), (a2), (b1) and (b2) on the dendrogram. The cytokines clustered into three clusters named 1, 2 and 3. The mean ln expressions of all the cytokines levels are given as box‐plots below the dendrogram. The age in years, gender, U‐porphobilinogen (U‐PBG) and U‐ALA ratio expressed as µmol/mmol creatinine, pre‐albumin (g/l) and the relative estimated glomerular filtration rate (eGFR) Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) (ml/min/1·73 m2) based on cystatin C levels, are shown as a bar graphs below the heatmap for each AIP case and control. Asymptomatic (open circles) and symptomatic (filled circles) AIP cases are indicated below the heatmap, those with the R167W mutation are indicated with underscored symbols.

Figure 3

Correlation matrix of porphyrins and precursors, biomarkers of inflammation and kidney function. The data in the left lower part are Spearman's correlation coefficients, r, in the acute intermittent porphyria group (n = 50), r ≥ 0·28 and r ≤ −0·28 (blue colour). The pairwise correlation between the different variables is depicted. The corresponding significant P‐values (P < 0·05) are indicated by red colour in the upper right part. The following variables were included: urine porphobilinogen ratio (PBG); urine 5‐aminolevulinic acid ratio (ALA); uroporphyrin (Urop.); total porphyrins (Total p.); serum cystatin C (Cyst. C); plasma long pentraxin 3 (PTX3); interleukin (IL)‐7 and ‐9; chemokine ligand (CCL) 3, 4 and 11; serum immunoglobulin A (IgA); blood (B) monocyte count (Monoc.); serum C3bc/C3 ratio (C3bc/C3); the plasma levels of the terminal complement complex (TCC); complement C3 and C4.

Figure 4

Prealbumin levels, kidney function, C‐peptide, insulin and glucose/insulin ratio, visfatin and porphyrin precursor levels in asymptomatic (Asympt.) and symptomatic (Sympt.) acute intermittent porphyria (AIP) cases compared with controls (Ctrl). (a) Serum prealbumin levels (g/l), (b) relative eGFR (estimated glomerular filtration rate) Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) (ml/min/1·73 m2), (c) plasma C‐peptide (pg/ml), (d) plasma insulin (pg/ml), (e) glucose/insulin ratio (mmol/l/pg/ml), (f) plasma visfatin levels (pg/ml), (g) urine delta‐amino levulinic acid (ALA, µmol/mmol creatinine), (h) urine porphobilinogen (PBG, µmol/mmol creatinine), in asymptomatic (Asympt.) and symptomatic (Sympt.) acute intermittent porphyria cases compared with their respective matched controls (Ctrl). Prealbumin, cystatin C, C‐peptide, insulin and visfatin were analysed using immunoassays. Glucose, ALA and PBG were analysed using standard biochemical assays. The results from the age‐ and sex‐matched controls (n = 50; of these 35 were controls for the symptomatic cases) and the cases with AIP (n = 50, 35 symptomatic) are shown as scatter‐plots with the median. The paired data were analysed using the Wilcoxon's matched‐pairs signed‐rank test. The asymptomatic and symptomatic AIP cases were compared using the Mann–Whitney U‐test.

Figure 5

Possible pathways from increased ALA, PBG and porphyrins to increased cytokines. Increased levels of delta‐aminolevulinic acid (ALA), porphobilinogen (PBG) and porphyrins from the liver of acute intermittent porphyria (AIP) patients may cause tissue damage in several tissues, including the nervous system, the liver itself, pancreas and kidneys. Reduced prealbumin indicates inflammation in the liver, and reduced eGFR (estimated glomerular filtration rate) is due to kidney damage. Reduced insulin and C‐peptide secretion from the pancreas may be due to reduced hormone secretion or organ damage. Organ damage may activate complement measured as increased levels of the terminal complement complex (TCC) and C5a, which activates cells to release cytokines, interleukins and growth factors. Another pathway of cytokine release may possibly be the release of damage‐associated molecular patterns (DAMPS) from damaged cells including uric acid crystals in gout due to reduced kidney function which activate directly or indirectly immune cells to release cytokines, interleukins and growth factors. Whether enhanced levels of ALA, PBG and porphyrins may act as DAMPS themselves remains to be elucidated. Other inflammatory stimuli due to smoking and dietary factors, etc. may also activate cells to release cytokines.