Interrupting an IFN-γ-dependent feedback loop in the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne

Abstract

Objectives: To study the molecular pathogenesis of PAPA (pyogenic arthritis, pyoderma gangrenosum and acne) syndrome, a debilitating hereditary autoinflammatory disease caused by dominant mutation in PSTPIP1.

Methods: Gene knock-out and knock-in mice were generated to develop an animal model. THP1 and retrovirally transduced U937 human myeloid leukaemia cell lines, peripheral blood mononuclear cells, small interfering RNA (siRNA) knock-down, site-directed mutagenesis, cytokine immunoassays, coimmunoprecipitation and immunoblotting were used to study inflammasome activation. Cytokine levels in the skin were evaluated by immunohistochemistry. Responsiveness to Janus kinase (JAK) inhibitors was evaluated ex vivo with peripheral blood mononuclear cells and in vivo in five treatment-refractory PAPA patients.

Results: The knock-in mouse model of PAPA did not recapitulate the human disease. In a human myeloid cell line model, PAPA-associated PSTPIP1 mutations activated the pyrin inflammasome, but not the NLRP3, NLRC4 or AIM2 inflammasomes. Pyrin inflammasome activation was independent of the canonical pathway of pyrin serine dephosphorylation and was blocked by the p.W232A PSTPIP1 mutation, which disrupts pyrin-PSTPIP1 interaction. IFN-γ priming of monocytes from PAPA patients led to IL-18 release in a pyrin-dependent manner. IFN-γ was abundant in the inflamed dermis of PAPA patients, but not patients with idiopathic pyoderma gangrenosum. Ex vivo JAK inhibitor treatment attenuated IFN-γ-mediated pyrin induction and IL-18 release. In 5/5 PAPA patients, the addition of JAK inhibitor therapy to IL-1 inhibition was associated with clinical improvement.

Conclusion: PAPA-associated PSTPIP1 mutations trigger a pyrin-IL-18-IFN-γ positive feedback loop that drives PAPA disease activity and is a target for JAK inhibition.

Keywords: Arthritis; Cytokines; Familial Mediterranean Fever; Inflammation.

Figure 1

Pstpip1 ^{A230T/ A230T} KI mice do not develop an autoinflammatory phenotype. (A) Strategy for generating Pstpip1 ^KO and Pstpip1 ^A230T mice. (B) Gross appearance of age-matched and gender-matched wild-type and homozygous Pstpip1 ^A230T/A230T KI mice. (C) Immunoblot analysis of LPS-primed BMDMs from WT, Mefv ^−/−, Pstpip1 ^−/− and Pstpip1 ^A230T/A230T mice with or without following ATP treatment to trigger the NLRP3 inflammasome as a positive control. (D) IL-1β measurements from LPS-primed BMDMs of WT, Pstpip1 ^A230T/A230T and Mefv^{− /−} mice with or without Clostridium botulinum C3 toxin for pyrin inflammasome activation. (E, F) IL-1β measurements of serum (E) and flow cytometry of peripheral blood cells for CD11b⁺ population (F) from Mefv ^B30.2/B30.2, Mefv ^B30.2/B30.2 Pstpip1 ^A230T/A230T, Mefv ^B30.2/B30.2 Pstpip1 ^A230T/+ and Pstpip1 ^A230T/A230T mice. P values were calculated with Mann-Whitney U test. BMDM, bone marrow-derived macrophages; LPS, lipopolysaccharide; n.s. not significant.

Figure 2

PAPA-associated PSTPIP1 mutations activate the pyrin inflammasome. (A) IL-1β measurements of cell culture supernatants from LPS-primed retroviral-transduced U937 cell lines expressing WT or PAPA-associated A230T and E250Q mutant PSTPIP1 proteins. (B) IL-1β measurements of cell culture supernatants from cell lines from (A) transiently transfected with negative control siRNA with no substantial sequence similarity to mouse or human gene sequences (siNC) or siRNA targeting the genes encoding pyrin, AIM2, NLRP3, NLRC4 or PSTPIP1, then primed with LPS. (C) IL-1β measurements of cell culture supernatants and immunoblot analysis of cell lysates from THP1-KO-NLRP3 cells transiently transfected with siNC or siRNA targeting the genes encoding pyrin or PSTPIP1, then treated with LPS with or without Clostridium difficile toxin A (TcdA). Results are from at least five independent experiments. P values were calculated with Mann-Whitney U test. LPS, lipopolysaccharide; n.s. not significant; n.t, no treatment; PAPA, pyogenic arthritis, pyoderma gangrenosum and acne.

Figure 3

PAPA-associated mutant PSTPIP1 activates the pyrin inflammasome through increased interaction. (A) IL-1β measurements of cell culture supernatants from LPS-primed retroviral-transduced U937 cell lines expressing WT or A230T or E250Q mutant PSTPIP1 proteins without (first three columns) or with secondary mutation, Y345F (second three columns) or W232A (last three columns). (B) Immunoblot analysis of pyrin-PSTPIP1 interaction in lysates of retroviral transduced U937 cells expressing myc-tagged WT or A230T, W232A or A230T/W232A mutant PSTPIP1 proteins assessed before (lysate) and after immunoprecipitation (IP) with antibody to myc or normal IgG. (C) IL-1β measurements of cell culture supernatants from LPS-primed retroviral-transduced U937 cell lines expressing full length WT or A230T or E250Q mutant PSTPIP1 (first three columns) or SH3 domain-truncated WT or mutant PSTPIP1 proteins (second three columns). All IL-1β measurements are from at least five independent experiments. The immunoprecipitation result is representative of three independent experiments. LPS, lipopolysaccharide; PAPA, pyogenic arthritis, pyoderma gangrenosum and acne.

Figure 4

IFN-γ is highly expressed on the skin of PAPA patients (dense dermal inflammatory infiltrate showed strong and diffuse staining of IFN-γ). The skin biopsies of normal skin from three healthy donors (Healthy Ctrl 1, 2 and 3), inflamed skin from two PAPA patients (PAPA pt. 1 at two different points in time, 3 months apart, marked as 1–1 and 1–2, and a second now-deceased PAPA patient with the E257K PSTPIP1 mutation who never received treatment with JAK inhibitors), and inflamed skin from two PAPA-like patients who were PSTPIP1-mutation negative (PAPA-like pt. 1 and 2) were stained with anti-human IFN-γ antibody (DAB, brown colour). Magnification×10; scale bar, 100 µm; representative images shown. JAK, Janus kinase; PAPA, pyogenic arthritis, pyoderma gangrenosum and acne.

Figure 5

IFN-γ induces pyrin expression and IL-18 secretion in myeloid cells with PAPA-associated PSTPIP1 mutations. (A) Immunoblot analysis of pyrin expression in cell lysates of U937 cells with or without IFN-γ treatment. (B) IL-18 measurements of cell culture supernatants from stable U937 cells expressing WT or A230T or E250Q mutant PSTPIP1 proteins with or without IFN-γ treatment. (C) Immunoblot analysis of pyrin expression in cell lysates of CD14⁺ monocytes from two healthy controls and two PAPA patients with or without IFN-γ treatment. (D) Bead-based multiplex analysis of IL-18, IL-6, TNFα and IL-1β levels in cell culture supernatants released by ex vivo cultured CD14⁺ monocytes from PAPA patients or healthy controls unprimed or primed with IFN-γ (n=4 each group). (E) IL-18 measurements of cell culture supernatants from CD14⁺ monocytes of PAPA patients transiently transfected with siNC or siMEFV then primed with IFN-γ. P values were calculated with Mann-Whitney U test. n.s. not significant; n.t. no treatment; PAPA, pyogenic arthritis, pyoderma gangrenosum and acne.

Figure 6

PAPA-associated mutations activate the pyrin inflammasome without affecting pyrin dephosphorylation. (A) Immunoblot analysis of pyrin-14-3-3ε interaction in lysates of stable U937 cells expressing myc-tagged WT or A230T, W232A or A230T/W232A mutant PSTPIP1 proteins assessed before (lysate) and after immunoprecipitation (IP) with antibody to myc or normal IgG. (B) Measurements of pyrin phosphorylation in cell lysates from stable U937 cells expressing myc-tagged WT, A230T or E250Q mutant PSTPIP1 proteins after IFN-γ treatment or IFN-γ followed by staurosporine treatment. Cell lysates were immunoblotted with antibodies against pyrin phospho-Ser242 and total pyrin. Band intensities of phosphorylated pyrin were quantified and normalised against total pyrin and plotted (top) from three independent immunoblot analyses (bottom, a representative immunoblot). (C) IL-18 measurements of cell culture supernatants from (B). IL-18 measurements were from at least five independent experiments. (D) Immunoblot analysis of pyrin phosphorylation and expression of PSTPIP1 and proIL-18 in cell lysates of CD14⁺ monocytes from two healthy controls and two PAPA patients after IFN-γ treatment or IFN-γ followed by staurosporine treatment. P values were calculated with the Mann-Whitney U test. PAPA, pyogenic arthritis, pyoderma gangrenosum and acne.

Figure 7

JAK inhibition suppresses PAPA syndrome-mediated inflammation. (A) IL-18 measurements of cell culture supernatants from stable U937 cells expressing WT or A230T mutant PSTPIP1 proteins after IFN-γ treatment and various doses of ruxolitinib or tofacitinib. (B) Immunoblot analysis of pyrin or proIL-18 expression in cell lysates from (A). (C) IL-18 measurements of cell culture supernatants from PBMCs of PAPA patients after IFN-γ treatment and various doses of ruxolitinib or tofacitinib. The IL-18 levels measured from cells treated with ruxolitinib or tofacitinib were normalised to the IL-18 levels measured from cells without ruxolitinib or tofacitinib treatment. (D) Immunoblot analysis of pyrin or PSTPIP1 expression in cell lysates from (C). (E) Daily prednisone doses of patients at baseline prior to starting a JAK inhibitor and at follow-up visits months later. (F) Several pyoderma gangrenosum skin lesions, characterised by poorly healing cutaneous ulcers with undermined edges, on the legs of patient 4 prior to starting ruxolitinib. (G) Six months after starting ruxolitinib, the cutaneous leg ulcers of patient 4 have healed with residual dermal scars. JAK, Janus kinase; PAPA, pyogenic arthritis, pyoderma gangrenosum and acne; PBMCs, peripheral blood mononuclear cell.