Objective. In gout, incompletely defined molecular factors alter recognition of dormant articular and bursal monosodium urate monohydrate (MSU) crystal deposits, thereby inducing self-limiting bouts of characteristically severe neutrophilic inflammation. To define primary determinants of cellular recognition, uptake, and inflammatory responses to MSU crystals, we conducted a study to test the role of Toll-like receptor 2 (TLR-2), TLR-4, and the cytosolic TLR adapter protein myeloid differentiation factor 88 (MyD88), which are centrally involved in innate immune recognition of microbial pathogens.Methods. We isolated bone marrow-derived macrophages (BMDMs) in TLR-2 ؊/؊ , TLR-4 ؊/؊ , MyD88 ؊/؊ , and congenic wild-type mice, and assessed phagocytosis and cytokine expression in response to endotoxin-free MSU crystals under serum-free conditions. MSU crystals also were injected into mouse synovium-like subcutaneous air pouches.Results. TLR-2 ؊/؊ , TLR-4 ؊/؊ , and MyD88 ؊/؊ BMDMs demonstrated impaired uptake of MSU crystals in vitro. MSU crystal-induced production of interleukin-1␤ (IL-1␤), tumor necrosis factor ␣, keratinocyte-derived cytokine/growth-related oncogene ␣, and transforming growth factor ␤1 also were significantly suppressed in TLR-2 ؊/؊ and TLR-4 ؊/؊ BMDMs and were blunted in MyD88 ؊/؊ BMDMs in vitro. Neutrophil influx and local induction of IL-1␤ in subcutaneous air pouches were suppressed 6 hours after injection of MSU crystals in TLR-2 ؊/؊ and TLR-4 ؊/؊ mice and were attenuated in MyD88 ؊/؊ mice.Conclusion. The murine host requires TLR-2, TLR-4, and MyD88 for macrophage activation and development of full-blown neutrophilic, air pouch inflammation in response to MSU crystals. Our findings implicate innate immune cellular recognition of naked MSU crystals by specific TLRs as a major factor in determining the inflammatory potential of MSU crystal deposits and the course of gouty arthritis.

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Phagocyte ingestion of monosodium urate (MSU) crystals can induce proinflammatory responses and trigger acute gouty inflammation. Alternatively, the uptake of MSU crystals by mature macrophages can be noninflammatory and promote resolution of gouty inflammation. Macrophage activation by extracellular MSU crystals involves apparent recognition and ingestion mediated by TLR2 and TLR4, with subsequent intracellular recognition linked to caspase-1 activation and IL-1β processing driven by the NACHT-LRR-PYD-containing protein-3 inflammasome. In this study, we examined the potential role in gouty inflammation of CD14, a phagocyte-expressed pattern recognition receptor that functionally interacts with both TLR2 and TLR4. MSU crystals, but not latex beads, directly bound recombinant soluble (s) CD14 in vitro. CD14−/− bone marrow-derived macrophages (BMDMs) demonstrated unimpaired phagocytosis of MSU crystals but reduced p38 phosphorylation and ∼90% less IL-1β and CXCL1 release. Attenuated MSU crystal-induced IL-1β release in CD14−/− BMDMs was mediated by decreased pro-IL-1β protein expression and additionally by decreased caspase-1 activation and IL-1β processing consistent with diminished NACHT-LRR-PYD-containing protein-3 inflammasome activation. Coating of MSU crystals with sCD14, but not sTLR2 or sTLR4, restored IL-1β and CXCL1 production in CD14−/− BMDMs in vitro. Gain of function of CD14 directly enhanced TLR4-mediated signaling in response to MSU crystals in transfected Chinese hamster ovary cells in vitro. Last, MSU crystal-induced leukocyte influx at 6 h was reduced by∼75%, and local induction of IL-1β decreased by >80% in CD14−/− mouse s.c. air pouches in vivo. We conclude that engagement of CD14 is a central determinant of the inflammatory potential of MSU crystals.

Ten strains of an unknown Campylobacter species were isolated from the livers of chickens with spotty liver disease in Australia. The strains were Gram-stain-negative, microaerobic, catalase- and oxidase-positive and urease-negative. Unlike most other species of the genus Campylobacter, most of the tested strains of this novel species hydrolysed hippurate and half of them could not reduce nitrate. All strains showed resistance, or intermediate resistance, to nalidixic acid and most of them were resistant to cephalothin. Examination of negatively stained cells under transmission electron microscopy revealed that they were S-shaped, with bipolar unsheathed flagella. Phylogenetic analyses based on the 16S rRNA gene and the heat shock protein 60 (hsp60) gene sequences indicated that the strains formed a robust clade that was clearly distinct from recognized Campylobacter species. Unusually, they had a DNA G+C content of 27.9 mol%, lower than any previously described Campylobacter species, and they showed less than 84 % average nucleotide identity to the nearest sequenced species. Taken together, these data indicate that the strains belong to a novel Campylobacter species, for which the name Campylobacter hepaticus sp. nov. is proposed. The type strain is HV10T (=NCTC 13823T=CIP 111092T).

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