C57BL/6 (B6) and Balb/c mice were purchased from the Biological Resource Center ((BRC), Agency for Science, Technology, and Research (A^∗STAR), Singapore). C3^-/- (stock # 003641; on B6 background), C5ar1^-/- (stock #006845) and C3ar1^-/- mice (stock # 005712; both on Balb/c background) were from The Jackson Laboratory. Double C3ar1^-/- and C5ar1^-/- mice were generated by crossing C5ar1^-/- and C3ar1^-/- mice. All experiments were conducted with gender- and age-matched mice, and all mutants were backcrossed to B6 or Balb/c backgrounds for at least 10 generations. Animals were bred under specific pathogen-free conditions at the BRC (A^∗STAR, Singapore) and experiments were performed under the approval of the Institutional Animal Care and Use Committee (IACUC) in compliance with the Law and Guidelines for Animal Experiments of the BRC (A^∗STAR, Singapore).

Mice received an intraperitoneal (i.p.) injection of Brewer thioglycollate medium (3%) to increase macrophage yield and euthanized 3 days later. Peritoneal macrophages were collected by lavage of the peritoneal cavity with 10 ml fresh RPMI medium. Red blood cell lysis was performed using hypotonic ammonium chloride solution (0.084%) for 5 min at room temperature. Peritoneal macrophages were plated at 2 × 10⁵/well in 96-well plates, and stimulated with E. coli LPS (0.1 μg/ml) for 3 h with or without MSU (250 μg/ml) for an additional 4 h, in the presence of recombinant C3a (0.25 and 1.25 μg/ml) or C5a (1–10–50 ng/ml). After 24 h, cell culture supernatants were collected.

Human monocytic (THP1) cells were plated at 1 × 10⁵/well in 96-well plates and treated with PMA (200 nM) overnight to differentiate monocytes into macrophages. Cells were then washed and exposed to E. coli LPS (0.1 μg/ml) for 3 h with or without MSU (250 μg/ml) for an additional 4 h, in the presence of recombinant C5a (1–10–50 ng/ml). After 24 h, cell culture supernatants were collected.

Eight- to ten-week-old wild-type (WT), C3^-/-, C3ar^-/-, C5a^-/-, and C3ar^-/- C5ar^-/- mice were injected i.p. with 3 mg MSU crystals in 0.5 ml of saline. Control mice were injected with saline alone. After 6 h, mice were euthanized with CO₂ and peritoneal exudate cells were collected by lavage with cold medium. The resulting cell suspensions were centrifuged before red blood cell lysis using hypotonic ammonium chloride solution for 5 min at room temperature, then quantification of cell number. In some experiments, mice received linear C5a receptor antagonistic peptide (1 mg/kg of mouse weight, AnaSpec Inc) by i.p. injection 5 min prior to MSU treatment.

Total peritoneal cells were labeled with CD11b-APC (M1/70, eBioscience), Ly6C-PE-Cy7 (HK1.4, Biolegend), and Ly6G-PE (1A8, BD PharMingen) for 15 min at room temperature. Cells were then washed with PBS twice before analysis using a Fortessa flow cytometer (BD Biosciences). Data were analyzed using FlowJo (Treestar). Neutrophils were identified as CD11b⁺ Ly6G⁺ cells and monocytes as CD11b⁺ Ly6C⁺ Ly6G^- cells.

IL-1α and IL-1β were measured using DuoSet ELISA kits (R&D Systems), following the manufacturer’s instructions. C3a and C5a levels in peritoneal lavage fluids were quantified by a sandwich ELISA as previously described (Kandasamy et al., 2013). The following antibody pairs were used: rat anti-mouse C3a or C5a capture antibody (2 μg/ml, overnight, 4°C), and biotinylated rat-anti mouse C3a or C5a detection antibody (2 μg/ml, 1 h, room temperature), all from BD Pharmingen. The absorbance was read at 450 nm with the reference wavelength at 570 nm, using a Tecan M200 Infinite plate reader (Tecan).

Quantitative RT-PCR was performed using the following validated SYBR Green primers: Nlrp3, 5′-CCTCTAGCTTCTGCCGTGGTCTCT-3′ and 5′-CGAAGCAGCATTGATGGGACA-3′; Asc, 5′-CTGAGCAGCTGCAAACGACTAAA-3′ and 5′-CTTCTGTGACCCTGGCAATGAGT-3′; Casp1, 5′-CTGTCAGGGGCTCACTTTTCATTG-3′ and 5′-AATGTCCCGGGAAGAGGTAGAAAC-3′; Il1a, 5′-TCAGCACCACTTGGTAAATGAC-3′ and 5′-GTGTTTCTGGCAACTCCTTCAGC-3′; Il1b, 5′-GGTCAAAGGTTTGGAAGCAG-3′ and 5′-TGTGAAATGCCACCTTTTGA-3′; Gapdh, 5′-TCGTCCCGTAGACAAAATGG-3′ and 5′-TTGAGGTCAATGAAGGGGTC-3′. Amplification was performed using an Applied Biosystems 7500 Real-Time PCR System. The relative expression level of each gene was evaluated using the ΔΔC_t method. The difference between the C_t of the target gene and the C_t of the Gapdh housekeeping gene was normalized to the ΔC_t of the untreated condition.

Intracellular production of reactive oxygen species (ROS) was quantified using the H₂DCFDA fluorometric method: briefly, peritoneal macrophages were labeled with H₂DCF-DA (20 μM; BioChemika Fluka) for 30 min and then washed with PBS before 2 × 10⁵ cells per well were seeded into black 96-well plates. Cells were incubated with C5a (50 ng/ml) or without, for 5 h before fluorescence was measured at 10-min intervals for 100 min using the Infinite M200 plate reader (Tecan; excitation 485 nm, emission 538 nm). ROS levels are displayed as the percentage increase in ROS relative to untreated controls.

Statistical significance was assessed using unpaired two-tailed t tests. Data were analyzed using Prism 7 (GraphPad).

We first examined the interaction of the complement components C3a and C5a with macrophages in vitro. Mouse peritoneal macrophages were primed with LPS, and incubated with MSU crystals or not in the presence or absence of increasing concentrations of recombinant C3a or C5a. In the absence of MSU crystals, LPS alone, LPS and C3a, LPS and C5a did not induce the release of IL-1β and IL-1α (Figures 1A,B); while the addition of MSU crystals triggered the release of both IL-1α and IL-1β from macrophages (Figures 1A,B). This mechanism is consistent with the established dual activation model of the NLRP3 inflammasome, in that LPS is required to drive expression of the IL-1β precursor and NLRP3, while a second signal, such as MSU crystals, controls inflammasome formation and activation of caspase-1.

Macrophages co-stimulated with LPS/MSU crystals and increasing concentrations of C5a boosted IL-1β and IL-1α production in a dose-dependent manner (Figure 1B), whereas C3a had no such effect (Figure 1A). Similar results were obtained when the human monocytic THP1 cell line was used (Figure 1C). These data indicate that IL-1α/β release from peritoneal macrophages and human monocytic cells requires both LPS and MSU crystals, and that C5a, but not C3a, boosts this inflammasome-mediated release.

The mechanisms underlying complement-mediated recruitment of inflammatory leukocytes by MSU crystals in vivo are largely unknown. Therefore we first evaluated the ability of MSU crystals to activate the complement cascade in vivo by measuring amounts of C3a and C5a in the peritoneal fluid of mice following injection of MSU crystals, or vehicle only, into the peritoneal cavity of two commonly used mouse strains, C57Bl/6 and Balb/c. Two hours after MSU crystal injection C3a and C5a levels significantly increased in the lavage fluids from both C57Bl/6 (Figure 2A) and Balb/c mice (Figure 2B), relative to control animals, confirming that MSU crystals have the capacity to activate the complement cascade in vivo.

To evaluate the importance of the complement cascade in sterile inflammation induced by MSU crystals in vivo, we next assessed the infiltration of neutrophils and inflammatory monocytes into the peritoneal cavity of C3^-/- mice, which are unable to activate the complement cascade beyond the formation of the C3-convertase. C3^-/- and wild-type (WT) control mice were injected i.p. with MSU crystals or vehicle; after 6 h, cells were collected by peritoneal lavage, labeled for surface molecule expression and characterized by flow cytometry. WT animals receiving MSU crystals exhibited a robust inflammatory response, characterized by massive peritoneal infiltration of neutrophils and monocytes, compared to mice receiving vehicle (Figures 3A,B). MSU-elicited mobilization of neutrophils and monocytes in the peritoneal cavity of C3^-/- mice was significantly suppressed (Figures 3A,B), indicating that complement proteins play an important role in regulating neutrophil and monocyte recruitment in mice, thereby promoting gouty inflammation.

Active C3a and C5a are potent inflammatory mediators and chemoattractants for phagocytic cells, including neutrophils and monocytes. Early case reports found increased levels of C3a/C3adesArg in two patients with gout (Moxley and Ruddy, 1985; Jose et al., 1990); we therefore investigated whether C3a and C5a generated by MSU crystals in vivo are responsible for leukocyte infiltration. First, we induced MSU-elicited peritonitis in mice deficient in both C3a and C5a receptors (C3ar1^-/-C5ar1^-/-): significantly fewer neutrophils and monocytes were present in peritoneal lavage fluid from C3ar1^-/-C5ar1^-/- mice compared with WT controls (Figure 4A), indicating that anaphylatoxin-mediated signaling regulates leukocyte infiltration following MSU administration in vivo.

To refine our understanding of the roles played by C3a and C5a in MSU-elicited peritoneal neutrophil recruitment in vivo, mice deficient in C3aR alone, C5aR alone, and WT controls were injected i.p. with MSU crystals. Significantly fewer neutrophils were present in the peritoneal lavage fluid of C5ar1^-/- mice (Figure 4C), but not C3ar1^-/- mice (Figure 4B), compared to WT controls, indicating that C5aR, but not C3aR, signaling is crucial for neutrophil infiltration. We also observed a tendency toward a reduction of monocyte recruitment in C5ar^-/- mice compered to WT controls, but this difference does not reach statistical significance.

Human C5a receptor cyclic/linear antagonistic peptides have been used to suppress inflammation in experimental rodent models of inflammatory bowel disease, ischemia/reperfusion injury, and immune complex glomerulonephritis (Paczkowski et al., 1999; Short et al., 1999; March et al., 2004; Alexander et al., 2015). Therefore, we sought to determine whether a C5aR antagonist could also suppress C5a-mediated recruitment of neutrophils and monocytes in mice after MSU challenge. WT mice were treated i.p. with the C5aR antagonist or vehicle prior to MSU challenge; peritoneal lavage after 6 h revealed that mice pretreated with the C5aR antagonist peptide had significantly fewer neutrophils and monocytes in their peritoneal cavity compared to mice receiving vehicle alone (Figure 4D). Our results support a central role for C5a-mediated signaling in the MSU-elicited pathology caused largely by neutrophil infiltration in mice.

The importance of neutrophils and proinflammatory cytokines in MSU-induced inflammation led us to analyze, in more detail, the mechanisms underlying neutrophil recruitment induced by C5a. Recently, basic and clinical research studies have implicated IL-1β and its maturation by the NLRP3 inflammasome in the pathogenesis of gout (Martinon et al., 2006; So et al., 2007; Kono et al., 2010). IL-1β released from macrophages activates IL-1 receptors on epithelial cells and resident macrophages, leading to the release of pro-inflammatory cytokines and chemokines, which, in turn, recruit and activate leukocytes, amplifying the inflammatory positive-feedback loop (Kono et al., 2010). This inflammatory cascade is the major cause of gout. IL-1 receptor antagonists, such as IL-1RA or Anakinra, or anti-human IL-1β antibodies, suppress the acute neutrophil response to MSU challenge in mice (So et al., 2007; Schlesinger, 2012; Ottaviani et al., 2013; Goh et al., 2014), providing robust evidence that this pathway is major target for beneficial therapeutic interventions.

Because we previously demonstrated that C5a is crucial for leukocyte recruitment upon MSU challenge in vivo, we next sought to determine whether C5a regulates IL-1β production. IL-1β levels were measured in the peritoneal lavage fluid collected from WT and C3^-/- mice: IL-1β levels were significantly lower in C3^-/- mice compared with WT mice (Figure 5A). To assess the contribution of C3a and C5a signaling in IL-1β production induced by MSU crystals in vivo, IL-1β levels were also measured in peritoneal lavage of C3ar1^-/- (C3aR^-/-) or C5ar1^-/- (C5aR^-/-) mice: MSU administration induced abundant and comparable IL-1β production in WT and C3ar1^-/- mice, but far less so in C5ar1^-/- mice (Figure 5B). These results indicate that C5a generated by complement activation induced by MSU triggers IL-1β production in vivo, which in turn regulates neutrophil recruitment.

To gain insight into the molecular mechanisms by which C5a induces IL-1β in peritoneal macrophages, we first examined the possibility that C5aR signaling primes and/or activates the NLRP3 inflammasome. Peritoneal macrophages were exposed to LPS alone (a classical NLRP3 priming signal), C5a alone, or LPS in combination with C5a, for 4 h before quantification of the abundance of mRNA transcripts of the inflammasome components Nlrp3, Asc and Casp1, as well as Il1b and Il1a. While LPS robustly triggered Nlrp3, Il1b and Il1a transcription, C5a alone failed to do so (Figure 6A), and can therefore not be considered a priming stimulus.

Next we sought to investigate alternate mechanisms by which C5a boosts NLRP3 inflammasome activation. Since ROS have been shown to act as a primary inducer of NLRP3 inflammasome activity, we asked whether C5a, alike to MSU, triggers ROS production. We found that C5a exposure robustly increased ROS production in peritoneal macrophages (Figure 6B), suggesting that C5a is likely to increase NLRP3 activation at least in part via ROS production.