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. 2022 Sep 26:13:912069.
doi: 10.3389/fimmu.2022.912069. eCollection 2022.

POP1 inhibits MSU-induced inflammasome activation and ameliorates gout

Lucia de Almeida  1 , Savita Devi  2 , Mohanalaxmi Indramohan  2 , Qi-Quan Huang  1 , Rojo A Ratsimandresy  2 , Richard M Pope  1 , Andrea Dorfleutner  2   3   4 , Christian Stehlik  2   3   4   5
Affiliations

POP1 inhibits MSU-induced inflammasome activation and ameliorates gout

Lucia de Almeida et al. Front Immunol. .

Abstract

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1β, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1β and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1β and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1β secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.

Keywords: caspase-1; gout; inflammasome; inflammation; macrophage; pyrin domain.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
POP1 ameliorates MSU-induced inflammation in mice. (A) In vivo imaging of MPO activity correlating to MSU-induced neutrophil infiltration into subcutaneous air pouches 6h after MSU crystal injection in wild type (WT), and POP1 transgenic (POP1TG) mice (left) and average radiance (right) presented as photons/sec/cm2/sr (n = 3 - 9 ± s.d.). (B) Subcutaneous air pouch exudates were analyzed for CD11b+Ly6G+ neutrophils by flow cytometry in mice injected with MSU crystals (n = 5 - 14 ± s.d.). (C) Representative example of a dot plot gating of CD11b and Ly6G stained cells from MSU crystal injected WT and POP1TG mice. (D) Representative H&E staining of subcutaneous air pouch lining of PBS or MSU crystal injected WT and POP1TG mice 6h after PBS or MSU crystal injection (left) showing the boxed area (top) enlarged (bottom), and quantification of subcutaneous air pouch lining presented as pixel determined at five different positions (right) (n = 4 ± s.d.). (E–G) Subcutaneous air pouch lavage exudates were analyzed for (E) IL-1β, (G) IL-6, TNF and CXCL1 by ELISA and for (F) ASC by immunoblot showing a representative example. (n = 5 - 9 ± s.d.). (H, I) Cleared synovial fluid obtained by ankle arthrocentesis from patients diagnosed with acute gout flares (P) using human plasma as a control (C) was analyzed for (H) IL-1β by ELISA and (I) ASC by immunoblot. (n = 6 ± s.d.). *p < 0.05.
Figure 2
Figure 2
POP1 ameliorates MSU-induced joint inflammation in mice. (A) In vivo imaging of MPO activity correlating to PBS (left ankle) or MSU (right ankle)-induced neutrophil infiltration into the hind limb ankle joint in wild type (WT) and POP1 transgenic (POP1TG) mice 24h after PBS or MSU crystal injection (left) and average radiance (right) presented as photons/sec/cm2/sr (n = 5 - 6 ± s.d.). (B) The ankle diameter was measured with a caliper before and 24h after MSU crystal injection into the hind limb ankle joint in WT and POP1TG mice (n = 11 - 12 ± s.d.). *p < 0.05.
Figure 3
Figure 3
POP1 inhibits inflammasome-dependent cytokine release in BMDM. (A, B) Wild type (WT) and POP1 transgenic (POP1TG) BMDM were primed with LPS and treated with MSU crystals as indicated and culture supernatants (SN) were analyzed for secreted (A) IL-1β and IL-18 and (B) IL-6 by ELISA (n = 3 ± s.d.). (C) Peritoneal macrophages were treated as above, and SN were analyzed for secreted IL-1β by ELISA (n = 3 ± s.d.). *p < 0.05.
Figure 4
Figure 4
POP1 inhibits MSU crystal-induced NLRP3 inflammasome assembly and caspase-1 activation. (A) Immunoprecipitation (IP) with immobilized anti-NLRP3 antibodies from either untreated or primed (Pam3CSK4) and activated (MSU crystals) GFP control and GFP-POP1 THP-1 cell lysates as indicated. Immunoblot of IPs and total cell lysates (TCL) for ASC, NLRP3, GFP and as a loading control tubulin. (B) Proximity ligation assay (PLA) between NLRP3 and caspase-1 in LPS primed and MSU crystal activated WT and POP1 transgenic (POP1TG) BMDM. DNA (blue), POP1 (green) and an arrow marks the PLA signal (red), showing a representative result (left) and quantification of PLA+ cells of three randomly selected fields of views (right). (C) LPS primed WT and POP1TG BMDM were activated with MSU crystals and culture supernatants (SN), and total cell lysates (TCL) analyzed by immunoblot for active caspase-1 p10, pro-caspase-1, ASC, HMGB1, mature IL-1β p17, pro-IL-1β, and tubulin as indicated, showing a representative result.
Figure 5
Figure 5
POP1 inhibits inflammasome-dependent cytokine release in human macrophages. (A, B) Control (Ctrl) and POP1 expressing THP-1 cells (POP1) were primed with LPS and treated with MSU crystals as indicated and culture supernatants (SN) were analyzed for secreted (A) IL-1β and IL-18 and (B) TNF by ELISA (n = 3 ± s.d.). (C, D) Ctrl shRNA and POP1 shRNA expressing THP-1 cells were primed and treated with MSU crystals as above and culture SN were analyzed for secreted (C) IL-1β and (D) TNF by ELISA (n = 3 ± s.d.). (E) LPS primed Control (Ctrl) THP-1 cells or THP-1 cells stably expressing POP1 or the indicated α helices of POP1 were activated with MSU crystals and culture supernatants were analyzed for secreted IL-1β by ELISA (n = 3 ± s.d.). *p < 0.05, ns, non specific.
Figure 6
Figure 6
Cell penetrating POP1 ameliorates gout. In vivo imaging of MPO activity correlating to MSU-induced neutrophil infiltration into subcutaneous air pouches 4h after MSU crystal injection in wild type mice injected with either TAT-GFP or TAT-POP1 30 minutes prior MSU crystal injection (left) and average radiance (right) presented as photons/sec/cm2/sr (n = 5 ± s.d.). *p < 0.05.
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