Periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) is a disorder of innate immunity and Th1 activation responsive to IL-1 blockade

Abstract

The syndrome of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) is the most common periodic fever disease in children. However, the pathogenesis is unknown. Using a systems biology approach we analyzed blood samples from PFAPA patients whose genetic testing excluded hereditary periodic fevers (HPFs), and from healthy children and pediatric HPF patients. Gene expression profiling could clearly distinguish PFAPA flares from asymptomatic intervals, HPF flares, and healthy controls. During PFAPA attacks, complement (C1QB, C2, SERPING1), IL-1-related (IL-1B, IL-1RN, CASP1, IL18RAP), and IFN-induced (AIM2, IP-10/CXCL10) genes were significantly overexpressed, but T cell-associated transcripts (CD3, CD8B) were down-regulated. On the protein level, PFAPA flares were accompanied by significantly increased serum levels of chemokines for activated T lymphocytes (IP-10/CXCL10, MIG/CXCL9), G-CSF, and proinflammatory cytokines (IL-18, IL-6). PFAPA flares also manifested a relative lymphopenia. Activated CD4(+)/CD25(+) T-lymphocyte counts correlated negatively with serum concentrations of IP-10/CXCL10, whereas CD4(+)/HLA-DR(+) T lymphocyte counts correlated positively with serum concentrations of the counterregulatory IL-1 receptor antagonist. Based on the evidence for IL-1β activation in PFAPA flares, we treated five PFAPA patients with a recombinant IL-1 receptor antagonist. All patients showed a prompt clinical and IP-10/CXCL10 response. Our data suggest an environmentally triggered activation of complement and IL-1β/-18 during PFAPA flares, with induction of Th1-chemokines and subsequent retention of activated T cells in peripheral tissues. IL-1 inhibition may thus be beneficial for treatment of PFAPA attacks, with IP-10/CXCL10 serving as a potential biomarker.

Fig. 1.

Study population and procedures. (A) Demographic and clinical characteristics of 21 patients with PFAPA syndrome. IQR, interquartile range. (B) Schematic of the overall study population and number of patients analyzed in each different assay or experiment. Definitions of classic and stringent PFAPA criteria are provided in Materials and Methods. Infectious serology comprised antistreptolysin O, anti-DNaseB, antibody titers against HSV 1 and 2, EBV, and CMV. ANA, anti-nuclear antibody; ND, not done; PAPA, pyogenic arthritis, pyoderma gangrenosum, and acne syndrome.

Fig. 2.

Distinct whole-blood gene expression profiles in PFAPA syndrome. Microarray analysis of messenger RNA extracted from whole blood of six PFAPA patients during and between episodes, six healthy controls and six hereditary periodic fever patients during flares [two FMF (M694V/E148Q and M694del in MEFV), one TRAPS (C52G in TNFRSF1A), three CAPS (two G569R, and L632F in NLRP3/CIAS1)]. (A) Principal component analysis of differentially expressed genes, with each dot representing one subject. (B–F) Hierarchical clustering of differentially expressed genes in the four study groups, with genes selected and listed according to their intensity of expression and canonical pathways, respectively. Gene symbols are depicted in the left column. At least twofold and statistically significant differences of probe set expression between active and inactive PFAPA disease states are shown (Wilcoxon signed-rank test P < 0.007, FDR 2%). Asterisks in the right column indicate at least 1.5-fold significantly changed transcript levels when comparing flares of PFAPA with flares of hereditary autoinflammatory diseases (Mann-Whitney U test P < 0.03, FDR 20%). Expression values were normalized per gene (row) to the mean of the healthy control group; each column represents one individual. As shown on the color bars, red indicates relative up-regulation, blue relative down-regulation.

Fig. 3.

Distinct inflammatory protein measures in PFAPA patients. Values of (A) serum cytokines and chemokines and (B) CRP in PFAPA patients during and between flares, healthy controls, and HPF patients during flare. Results for IL-1β, IL-2, IL-4, IL-5, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, Eotaxin, FGF basic, GM-CSF, IFN-γ, MCP-1, MIP-1α, PDGF BB, RANTES (regulated upon activation, normal T cell-expressed and -secreted), TNF-α, and VEGF did not show any statistically significant differences in any of the comparisons and therefore are not shown. NS, not significant.

Fig. 4.

Clinical and laboratory response of PFAPA patients treated with recombinant IL-1R antagonist (anakinra). Subcutaneous injection of five PFAPA patients with one dose of the recombinant IL-1R antagonist (anakinra) each, between 21 and 48 h following the onset of flare; one patient (patient 4) received an additional dose of recombinant IL-1R antagonist (anakinra) 24 h after the first dose. (A) Summary of demographic and clinical data of the five PFAPA patients; *Patient 5 received daily cimetidine but continued to have classic flares. (B) Fever curves before and after treatment; red arrows indicate injection of recombinant IL-1R antagonist (anakinra), black arrows indicate administration of ibuprofen, gray arrow indicates administration of acetaminophen. (C) Laboratory values and cytokine/chemokine measurements in sera before and after injection of recombinant IL-1R (anakinra).