Gene polymorphisms in the NALP3 inflammasome are associated with interleukin-1 production and severe inflammation: relation to common inflammatory diseases?

OBJECTIVE: NALP3, ASC, and TUCAN are components of the NALP3 inflammasome, which triggers caspase 1-mediated interleukin-1beta (IL-1beta) release. Activating mutations in the gene encoding NALP3 (NLRP3) have recently been linked to familial periodic fever syndromes. We undertook this study to determine whether a patient with arthritis and antibiotic-resistant fever carried mutations in the genes encoding the NALP3 inflammasome. METHODS: Genetic analysis of NLRP3 and the gene encoding TUCAN (CARD-8) was performed on genomic DNA from the patient and from a population-based collection of DNA (806 subjects). For in vitro studies of IL-1beta production and caspase 1 activity, blood was obtained from the patient at different time points after administration of anakinra, an IL-1 receptor antagonist, as well as from 5 healthy age- and sex-matched control subjects. RESULTS: Mutation analysis of the patient's genes encoding NALP3, ASC, and TUCAN revealed variations in the NLRP3 (Q705K) and CARD-8 (C10X) genes. The allele frequencies of these single-nucleotide polymorphisms (SNPs) in the population were 6.5% and 34%, respectively. The elevated activity of caspase 1 and the high levels of IL-1beta measured in samples from the patient returned to normal levels after treatment with anakinra. CONCLUSION: Our results indicate that the patient's symptoms were due to elevated levels of IL-1beta, since treatment with anakinra effectively abolished the symptoms. The compound SNPs may explain the increased IL-1beta levels and inflammatory symptoms observed, but further studies are needed to reveal a functional relationship. The prevalence of the polymorphisms (4% of the population carry both SNPs) in the general population may suggest a genetic predisposition for common inflammatory disorders.     

Effect of platelet-rich plasma on rat Achilles tendon healing is related to microbiota

Background and purpose — In 3 papers in Acta Orthopaedica 10 years ago, we described that platelet-rich plasma (PRP) improves tendon healing in a rat Achilles transection model. Later, we found that microtrauma has similar effects, probably acting via inflammation. This raised the suspicion that the effect ascribed to growth factors within PRP could instead be due to unspecific influences on inflammation. While testing this hypothesis, we noted that the effect seemed to be related to the microbiota.

Material and methods — We tried to reproduce our old findings with local injection of PRP 6?h after tendon transection, followed by mechanical testing after 11 days. This failed. After fruitless variations in PRP production protocols, leukocyte concentration, and physical activity, we finally tried rats carrying potentially pathogenic bacteria. In all, 242 rats were used.

Results — In 4 consecutive experiments on pathogen-free rats, no effect of PRP on healing was found. In contrast, apparently healthy rats carrying Staphylococcus aureus showed increased strength of the healing tendon after PRP treatment. These rats had higher levels of cytotoxic T-cells in their spleens.

Interpretation — The failure to reproduce older experiments in clean rats was striking, and the difference in response between these and Staphylococcus-carrying rats suggests that the PRP effect is dependent on the immune status. PRP functions may be more complex than just the release of growth factors. Extrapolation from our previous findings with PRP to the situation in humans therefore becomes even more uncertain.     

Method of birth alters interferon-gamma and interleukin-12 production by cord blood mononuclear cells

Many uncertainties exist regarding the capability of cord blood mononuclear cells (CBMC) to produce cytokines. A number of conflicting reports led us to examine the effects of method of birth on CBMC production of interferon-gamma (IFN-γ), interleukin-4 (IL-4) and interleukin-12 (IL-12). While constitutive production of IL-4 was found in both vaginally and cesarean-delivered infants, constitutive IFN-γ or IL-12 production was found in neither. CBMC from vaginally delivered infants responded to stimulation with concanavalin A/phorbol 12-myristate 13-acetate (Con A/PMA), phytohemagglutinin (PHA), and lipopolysaccharide (LPS) with significantly higher levels of IFN-γ than CBMC from unlabored cesarean section (CS) infants. Production of IL-12 was increased in the vaginally delivered group in response to LPS and PHA but not to ConA/PMA. In contrast, mode of delivery was not associated with differences in IL-4 production. These results indicate that mode of delivery significantly alters the capability of CBMC to produce some cytokines and therefore should be taken into account in interpreting fetal/neonatal mononuclear cell function studies.     

Uropathogenic Escherichia coli triggers oxygen-dependent apoptosis in human neutrophils through the cooperative effect of type 1 fimbriae and lipopolysaccharide

Type 1 fimbriae are the most commonly expressed virulence factor on uropathogenic Escherichia coli. In addition to promoting avid bacterial adherence to the uroepithelium and enabling colonization, type 1 fimbriae recruit neutrophils to the urinary tract as an early inflammatory response. Using clinical isolates of type 1 fimbriated E. coli and an isogenic type 1 fimbria-negative mutant (CN1016) lacking the FimH adhesin, we investigated if these strains could modulate apoptosis in human neutrophils. We found that E. coli expressing type 1 fimbriae interacted with neutrophils in a mannose- and lipopolysaccharide (LPS)-dependent manner, leading to apoptosis which was triggered by the intracellular generation of reactive oxygen species. This induced neutrophil apoptosis was abolished by blocking FimH-mediated attachment, by inhibiting NADPH oxidase activation, or by neutralizing LPS. In contrast, CN1016, which did not adhere to or activate the respiratory burst of neutrophils, delayed the spontaneous apoptosis in an LPS-dependent manner. This delayed apoptosis could be mimicked by adding purified LPS and was also observed by using fimbriated bacteria in the presence of d-mannose. These results suggest that LPS is required for E. coli to exert both pro- and antiapoptotic effects on neutrophils and that the difference in LPS presentation (i.e., with or without fimbriae) determines the outcome. The present study showed that there is a fine-tuned balance between type 1 fimbria-induced and LPS-mediated delay of apoptosis in human neutrophils, in which altered fimbrial expression on uropathogenic E. coli determines the neutrophil survival and the subsequent inflammation during urinary tract infections.