Cold-induced urticarial autoinflammatory syndrome related to factor XII activation

Abstract

Hereditary autoinflammatory diseases are caused by gene mutations of the innate immune pathway, e.g. nucleotide receptor protein 3 (NLRP3). Here, we report a four-generation family with cold-induced urticarial rash, arthralgia, chills, headache and malaise associated with an autosomal-dominant inheritance. Genetic studies identify a substitution mutation in gene F12 (T859A, resulting in p.W268R) which encodes coagulation factor XII (FXII). Functional analysis reveals enhanced autocatalytic cleavage of the mutated protein and spontaneous FXII activation in patient plasma and in supernatant of transfected HEK293 cells expressing recombinant W268R-mutated proteins. Furthermore, we observe reduced plasma prekallikrein, cleaved high molecular weight kininogen and elevated plasma bradykinin. Neutrophils are identified as a local source of FXII. Interleukin-1β (IL-1β) is upregulated in lesional skin and mononuclear donor cells exposed to recombinant mutant proteins. Treatment with icatibant (bradykinin-B2-antagonist) or anakinra (interleukin-1-antagonist) reduces disease activity in patients. In conclusion, our findings provide a link between contact system activation and cytokine-mediated inflammation.

Fig. 1. Phenotype and genotype in patients with FXII-associated cold autoinflammatory syndrome (FACAS).

a Urticarial rash of the face of a 9-year-old girl and the leg of her 35-year-old mother. b Skin histopathology reveals dermal edema (H.E. yellow arrow) and perivascular infiltrates composed of macrophages (CD163) and few intravascular neutrophils (myeloperoxidase [MPO], yellow arrow). Original magnification ×400  (n = 2 technical replicates). c Family pedigree over four generations identifying affected (shaded black) and unaffected (unshaded) individuals. Exome sequenced individuals are underlined. Where conducted, the results of Sanger sequencing of p.W286R are shown to top left of each individual whereby “+” denotes heterozygote for variant and wild-type, and “−” denotes healthy reference. d Representative sequencing chromatogram of the heterozygous genotype in an affected individual (top) and chromatogram of healthy individual from the same region (bottom).

Fig. 2. FXII W268R results in fragmentation and spontaneous activation of FXII.

a FXII fragmentation in FACAS plasma samples and FXII W268R mutant proteins. Citrate plasma of affected (#1, #2, #5) and non-affected (#3, #4, #6) family members, recombinant wild-type and W268R FXII expressed in HEK293 cells and plasma from an FXII-HAE patient was separated by SDS-PAGE (reducing blot) and immunoblotted for FXII. Red arrows at 50 kDa indicate heavy chain of cleaved FXII in the plasma of affected patients and recombinant W268R FXII (rFXII W268R), which is barely visible in non-affected and hereditary angioedema (HAE) III plasma or wild-type FXII (rFXII wt). Recombinant W268R and wild-type FXII were expressed in HEK293 cells. Black arrows point to rFXII W268R fragments observed by mass spectrometry in c, n = 5 technical replicates. b Immunoblot (reduced conditions) of recombinant mutant proteins (in the presence or absence of surface activator kaolin) and controls (rFXII wt and purified αFXIIa). In addition to 50 kDa (heavy chain, HC) and 25 kDa (light chain, LC) bands, a further band appears at 60 kDa (II) that is absent in rFXII wt and αFXIIa. Black arrows point to rFXII W268R fragments observed by mass spectrometry in c, n = 3 technical replicates. c Schematic presentation of FXII W268R procession. Red arrows point to mutation at position W268R. Proteolytic sites are indicated by blue arrows. Cleavage after position 353 results in FXIIa W268R HC (50 kDa) and FXIIa W268R LC (25 kDa) fragments. Additional cleavage results in a truncated FXII W268R fragment (60 kDa) cleaved at position 447. d FXIIa activity was measured by a chromogenic assay demonstrating considerable spontaneous FXIIa activity in plasma of affected individuals (n = 3), but less in healthy donors (n = 3 healthy family members). e Spontaneous FXIIa activity was also observed in culture supernatants from HEK293 cells expressing W268R, but not wild-type FXII. Graphs show individual data points and the mean ± s.e.m. Statistical significance is indicated by unpaired Student’s t test with Welch’s correction (d, e). Source data are provided as a source data file.

Fig. 3. FXII W268R activates the kallikrein–kinin pathway.

a Citrate plasma of affected (n = 3) and healthy family members (n = 3) was immunoblotted for prekallikrein (PK) showing substantial cleavage of PK (black arrow) to kallikrein (red arrow), n = 2 technical replicates. This corresponds to reduced PK activity (b) and plasma levels (c) in affected individuals (n = 3 affected vs. n = 3 healthy). d FXIIa-C1-inhibitor (FXIIa-C1-INH) and e) PK-C1-INH complexes in patient plasma (n = 3 affected vs. n = 3 healthy). f Citrate plasma of affected (n = 3) and healthy family members (n = 3) was immunoblotted for high-molecular-weight kininogen (HMWK) showing its complete degradation (black arrow) to cleaved HMWK (cHMWK) (red arrow), n = 2 technical replicates. Correspondingly, increased cHMWK activity (g) and bradykinin plasma levels (h) (n = 3 affected vs. n = 3 healthy; n = 2 affected vs. n = 7 healthy and n = 2 HAE I/II) are detectable in the plasma of affected patients. PK and cHMWK activity were measured by chromogenic assays, PK and bradykinin plasma levels were assessed by ELISA. Graphs show individual data points and the mean ± s.e.m. Statistical significance is indicated by unpaired Student’s t test with Welch’s correction (b, c, d, e, g) or one-way ANOVA with Sidak’s multiple comparisons test (h). Source data are provided as a source data file.

Fig. 4. FXII is expressed by skin and blood neutrophils in FACAS.

a Representative image from serial sections of the lesional skin of FACAS patient ca. 1 h after cold exposure. FXII immunoreactivity is restricted to neutrophils at perivascular sites and within blood vessels (white arrows), n = 2 technical replicates. b Exemplary images from cytospins isolated from the peripheral blood of FACAS patients and healthy control subjects. Only single FACAS neutrophils (0.72%), but not healthy control neutrophils (0%), present with double-positive cytoplasmatic staining for FXII and MPO. Original magnification ×400, n = 3 technical replicates. c Peripheral blood mononuclear cells were isolated from healthy control subjects by density-gradient centrifugation and stimulated with increasing doses of bradykinin. IL-8 secretion was assessed by ELISA. Pooled data of n = 3 different donors, n = 2 technical replicates. The graph shows individual data points and the mean ± s.e.m. Statistical significance is indicated by one-way ANOVA with Sidak’s multiple comparisons. Source data are provided as a source data file.

Fig. 5. FXII W268R induces and increases IL-1β secretion and expression.

a–c Peripheral blood mononuclear cells were isolated from healthy control subjects by density-gradient centrifugation. Interleukin-1β (IL-1β) secretion requires a two-step process: (i) transcriptional upregulation of pro-IL-1β via toll-like receptors and (ii) conversion of pro-IL-1β into its active form by inflammasome activation. Cells were pre-stimulated with toll-like receptor agonist lipopolysaccharide (LPS), and nucleotide binding like receptor protein 3 (NLRP3) inflammasome activation was induced by the danger molecule ATP (LPS + ATP = blue symbols). Red symbols denote LPS-stimulated cells only, and green symbols represent ATP-activated cells without priming by LPS. Experiments were performed in the presence of FXII-depleted plasma (control) and addition of recombinant FXII wild-type (rFXII wt) and W268R (rFXII W268R) protein (a) or rFXII W268R-S544A (b). Experiments in (c) were conducted with purified FXII, FXIIa, and different doses of the small peptide-based inhibitor PPACK. Black arrows indicate priming effect by the mutant rFXII W268R (a) and the active site incapacitated rFXII W268R-S544A (b), which was absent in rFXII wt and control. Also, the presence of FXIIa (with or without PPACK) did not induce changes in IL-1β release (c). IL-1β secretion was assessed by ELISA. Pooled data of n = 3 different (a) or n = 2 (b, c) experiments. Bar graphs show individual data points and the mean ± s.e.m. Statistical significance in is indicated by one-way ANOVA (a, c) with Tukey’s multiple comparisons test for ATP between the groups control vs. rFXII W268R and rFXII wt vs. rFXII W268R (a) or unpaired Student’s t test (b). d, e Representative image from serial sections of the lesional skin from FACAS patient following cold exposure exhibits IL-1β immunoreactivity. IL-1β expression is pronounced around blood vessels (yellow arrows) and co-localizes with neutrophils (d) and macrophages (e) (white arrows). Original magnification ×400, n = 2 technical replicates. Source data are provided as a source data file.

Fig. 6. The bradykinin B2 receptor antagonist icatibant resolves FACAS signs and symptoms.

During winter with continuous skin symptoms, the index patient administered icatibant 30 mg. a Urticarial rash completely resolved within 90 min post icatibant application (from the left to right). b Total and individual symptoms reported and assessed daily over a time period of 8 days by the patient using a standardized daily health assessment form covering the main symptoms rash, fatigue, headache, arthralgia, and fever/chills. The total score ranges from 0 = no symptoms to 50 = maximum of symptoms. Each subscore can range from 0 = no symptoms to 10 = maximum of symptoms. The subscores do not show fever/chills as these symptoms were not present at that time. Icatibant was given on 4 consecutive days (red arrows).

Fig. 7. Proposed pathomechanism underlying FXII W268R mutation in FACAS.

Liver-derived and pre-activated FXII induces plasma prekallikrein cleavage, HMWK cleavage leading to constantly raised bradykinin (BK) levels in the patient plasma (1) and increased permeability of the vasculature (3). BK may also induce IL-1β release from monocytes and tissue macrophages (Mɸ) leading to the autoinflammatory phenotype observed in FACAS patients (2), (5). IL-1β may further induce BK accumulation by upregulation of its receptors and downregulation of its inactivating enzyme ACE (4). In the skin, BK activates Mɸ to produce IL-8 and IL-1β, recruiting neutrophils into the tissue (5). These neutrophils may act as a source of auto-activated FXIIa in the skin, which leads to further contact system activation and BK generation (6). Under normal conditions, C1-Inhibitor can control BK generation, however, upon cold exposure inhibitory capacity may drop leading to a local increase in BK. Activation of neutrophils may occur via a paracrine feedback loop involving plasma kallikrein and HMWK. In addition, BK may activate skin mast cells to release heparin and polyphosphates (7), which supports further activation of FXII.