Comparative analysis of cytokine profiles in autoinflammatory and autoimmune conditions.

BACKGROUND: Many disease states are characterized by a biased cytokine profile and determining the level of certain cytokines becomes an important diagnostic and research tool in clinical medicine. We hypothesized that, despite the highly dynamic nature, the profile of cytokines may be characteristic of disease. METHODS: The level of systemic cytokines was studied in an autoinflammatory condition, familial Mediterranean fever; in an autoimmune disease, systemic lupus erythematosus (SLE), and in healthy controls. RESULTS: Multivariate statistics with the use of seven variables clustered the patients and control subjects into the four well separated and distinct groups, corresponding to the SLE, FMF attack, FMF remission, and healthy states. CONCLUSIONS: The model suggested the existence of specific patterns in cytokine levels reflecting the healthy and specific disease states. These findings warrant further investigations to establish whether this approach may serve as a potential meta-biomarker for other inflammatory disorders.

Differential induction of total IgE by two Salmonella enterica serotypes.

The main goal of this study was to establish how the inflammation caused by infection with two different Salmonella enterica serotypes, S. Typhimurium and S. Enteritidis, may lead to the predisposition to allergy as measured by total IgE level in the blood. Infection by S. Typhimurium did not affect the systemic IgE concentration while in S. Enteritidis-infected patients there was a significant 3.5-fold increase. This effect was especially profound in patients >4 years old, with up to the 8-fold increase above the norm. The degree of dysbiosis in these two infections measured with the comparative counts of cultivated bacteria showed an inverse relationship with the IgE concentration. Earlier we reported the elevated level of IL-17 in patients infected by S. Enteritidis. In the current study a significant correlation was found between the concentrations of IL-17 and IgE suggesting a possible role played by this cytokine in triggering the production of IgE in response to S. Enteritidis infection.

Activated phenotype of circulating neutrophils in familial Mediterranean fever.

Familial Mediterranean fever (FMF) is autoinflammatory disorder, characterized by MEFV gene mutations and recurrent episodes of fever and serosal or synovial inflammation. Neutrophils are the predominant effector cells of acute inflammatory attacks in FMF; however pathogenic role and molecular phenotype of these cells remain largely unknown. To gain insight into the processes that contribute to the self-directed autoinflammation we characterized expression of a spectrum of genes involved in regulation of inflammation in unstimulated and LPS-activated neutrophils from FMF patients. Expression of 12 candidate immune genes encoding for inflammation-related molecules was assessed by quantitative RT-PCR in freshly isolated and LPS-stimulated peripheral polymorphonuclear neutrophils from fifteen FMF patients in attack-free period and ten healthy volunteers as controls. The relative expression was calculated using the second derivative method; the target gene expression was normalized to the expression of RPL32 gene. FMF neutrophils were characterized by up-regulated baseline gene expression of c-FOS (9.5-fold, p < 0.05), IL-8 (12-fold, p < 0.05), MMP9 (8-fold, p < 0.01), TLR2 (7-fold, p < 0.05) compared to the neutrophils from control subjects, a trend was also evident towards increased caspase-1 expression (3-fold, p = 0.09). Discriminant analysis clustered the patient and control subjects into two distinct groups (Wilks's lambda = 0.165, p = 0.042). Further, LPS-induced alterations of expression profiles were shared between FMF and healthy neutrophils, the profile consisting namely of up-regulated IL-1ß, TLR4, IL-8, and TNFAIP6 transcripts. Present study demonstrates distinct expression patterns of pre-activated neutrophils during attack-free period of FMF when compared to neutrophils from healthy controls. Furthermore, our data emphasize the importance of host-derived ligands in activation of FMF neutrophils.

Inflammatory Responses to Salmonella Infections Are Serotype-Specific.

The main purpose of this study was to investigate the profile of inflammatory response in patients with acute salmonellosis caused by two serotypes of Salmonella enterica, S. Enteritidis and S. Typhimurium, as well as in convalescent patients with previous acute disease caused by S. Enteritidis. Patients with acute disease showed significantly elevated levels of IL-1ß, IL-17, IL-10, and calprotectin compared to healthy control subjects. In convalescent patients, these markers were also significantly elevated, with the exception of IL-1ß. Multivariate statistical analyses with the use of these variables produced models with a good predictive accuracy resulting in excellent separation of the diseased and healthy cohorts studied. Overall, the results suggest that the profile of inflammatory response in this disease is determined, to a significant degree, by the serotype of Salmonella, and the profile of certain cytokines and calprotectin remains abnormal for a number of months following the acute disease stage.

Management of familial Mediterranean fever by colchicine does not normalize the altered profile of microbial long chain fatty acids in the human metabolome.

In our previous works we established that in an autoinflammatory condition, familial Mediterranean fever (FMF), the gut microbial diversity is specifically restructured, which also results in the altered profiles of microbial long chain fatty acids (LCFAs) present in the systemic metabolome. The mainstream management of the disease is based on oral administration of colchicine to suppress clinical signs and extend remission periods and our aim was to determine whether this therapy normalizes the microbial LCFA profiles in the metabolome as well. Unexpectedly, the treatment does not normalize these profiles. Moreover, it results in the formation of new distinct microbial LCFA clusters, which are well separated from the corresponding values in healthy controls and FMF patients without the therapy. We hypothesize that the therapy alters the proinflammatory network specific for the disease, with the concomitant changes in gut microbiota and the corresponding microbial LCFAs in the metabolome.

Profiles of Microbial Fatty Acids in the Human Metabolome are Disease-Specific.

The human gastrointestinal tract is inhabited by a diverse and dense symbiotic microbiota, the composition of which is the result of host-microbe co-evolution and co-adaptation. This tight integration creates intense cross-talk and signaling between the host and microbiota at the cellular and metabolic levels. In many genetic or infectious diseases the balance between host and microbiota may be compromised resulting in erroneous communication. Consequently, the composition of the human metabolome, which includes the gut metabolome, may be different in health and disease states in terms of microbial products and metabolites entering systemic circulation. To test this hypothesis, we measured the level of hydroxy, branched, cyclopropyl and unsaturated fatty acids, aldehydes, and phenyl derivatives in blood of patients with a hereditary autoinflammatory disorder, familial Mediterranean fever (FMF), and in patients with peptic ulceration (PU) resulting from Helicobacter pylori infection. Discriminant function analysis of a data matrix consisting of 94 cases as statistical units (37 FMF patients, 14 PU patients, and 43 healthy controls) and the concentration of 35 microbial products in the blood as statistical variables revealed a high accuracy of the proposed model (all cases were correctly classified). This suggests that the profile of microbial products and metabolites in the human metabolome is specific for a given disease and may potentially serve as a biomarker for disease.

Predominant role of host genetics in controlling the composition of gut microbiota.

BACKGROUND: The human gastrointestinal tract is inhabited by a very diverse symbiotic microbiota, the composition of which depends on host genetics and the environment. Several studies suggested that the host genetics may influence the composition of gut microbiota but no genes involved in host control were proposed. We investigated the effects of the wild type and mutated alleles of the gene, which encodes the protein called pyrin, one of the regulators of innate immunity, on the composition of gut commensal bacteria. Mutations in MEFV lead to the autoinflammatory disorder, familial Mediterranean fever (FMF, MIM249100), which is characterized by recurrent self-resolving attacks of fever and polyserositis, with no clinical signs of disease in remission. METHODOLOGY/PRINCIPAL FINDINGS: A total of 19 FMF patients and eight healthy individuals were genotyped for mutations in the MEFV gene and gut bacterial diversity was assessed by sequencing 16S rRNA gene libraries and FISH analysis. These analyses demonstrated significant changes in bacterial community structure in FMF characterized by depletion of total numbers of bacteria, loss of diversity, and major shifts in bacterial populations within the Bacteroidetes, Firmicutes and Proteobacteria phyla in attack. In remission with no clinical signs of disease, bacterial diversity values were comparable with control but still, the bacterial composition was substantially deviant from the norm. Discriminant function analyses of gut bacterial diversity revealed highly specific, well-separated and distinct grouping, which depended on the allele carrier status of the host. CONCLUSIONS/SIGNIFICANCE: This is the first report that clearly establishes the link between the host genotype and the corresponding shifts in the gut microbiota (the latter confirmed by two independent techniques). It suggests that the host genetics is a key factor in host-microbe interaction determining a specific profile of commensal microbiota in the human gut.

Elevated systemic antibodies towards commensal gut microbiota in autoinflammatory condition.

BACKGROUND: Familial Mediterranean fever (FMF) is an autoinflammatory condition, which is characterized by acute, self-limiting episodes of fever and serositis and chronic subclinical inflammation in remission. Here we investigated the consequence of this condition on the level of systemic antibodies directed towards common intestinal bacteria. METHODOLOGY/PRINCIPAL FINDINGS: The level of systemic antibodies towards the antigens of Bacteroides, Parabacteroides, Escherichia, Enteroccocus and Lactobaccilus was measured by ELISA in FMF patients at various stages of the disease and in healthy controls. The difference between remission and attack was not significant. IgG antibodies against the antigens of Bacteroides, Parabacteroides, Escherichia and Enteroccocus were significantly increased in FMF compared to control while IgA levels were not significantly affected. Western blot analyses demonstrated the IgG reactivity against multiple antigens of commensal bacteria in FMF. Serological expression cloning was performed to identify these antigens. No single dominant antigen was identified; the response was generalized and directed against a variety of proteins from Bacteroides, Parabacteroides, Escherichia, and other gut commensals. CONCLUSIONS/SIGNIFICANCE: This autoinflammatory syndrome is characterized by the increased systemic reactivity against commensal gut microbiota. This is probably the consequence of hypersensitivity of the inflammasome in FMF that triggers the inflammation and contributes to the excessive translocation of bacteria and bacterial antigens through the gut barrier.