[Show abstract][Hide abstract] ABSTRACT: Coxiella burnetii, the causative agent of Q fever, is recognized by TLR2. TLR10 can act as an inhibitory receptor on TLR2-derived immune responses. Therefore, we investigated the role of TLR10 on C. burnetii-induced cytokine production and assessed whether genetic polymorphisms in TLR10 influences the development of chronic Q fever. HEK293 cells, transfected with TLR2, TLR10 or TLR2/TLR10, and human peripheral blood mononuclear cells (PBMCs) in the presence of anti-TLR10, were stimulated with C. burnetii. In both assays, the absence of TLR10 resulted in increased cytokine responses after C. burnetii stimulation. In addition, the effect of single nucleotide polymorphisms (SNPs) in TLR10 was examined in healthy volunteers whose PBMCs were stimulated with C. burnetii Nine Mile or the Dutch outbreak isolate C. burnetii 3262. Individuals bearing SNPs in TLR10 displayed increased cytokine production upon C. burnetii 3262 stimulation. Furthermore, 139 chronic Q fever patients and 220 controls were genotyped for TLR10 N241H, I775V and I369L. None of these polymorphisms were associated with increased susceptibility to chronic Q fever. In conclusion, TLR10 has an inhibitory effect on in vitro cytokine production by C. burnetii, but the presence of TLR10 polymorphisms does not lead to an increased risk of developing chronic Q fever.
[Show abstract][Hide abstract] ABSTRACT: Major abscesses and diabetic foot infections (DFIs) are predominant subtypes of complicated skin and skin structure infections (cSSSIs), and are mainly caused by Staphylococcus aureus and β-hemolytic streptococci. This study evaluates the potential benefit of direct pathogen-specific real-time polymerase chain reaction (PCR) assays in the identification of causative organisms of cSSSIs. One-hundred and fifty major abscess and 128 DFI biopsy samples were collected and microbial DNA was extracted by using the Universal Microbe Detection kit for tissue samples. Pathogen-specific PCRs were developed for S. aureus and its virulence factor Panton-Valentine leukocidin (PVL), Streptococcus pyogenes, S. agalactiae, S. dysgalactiae, and the S. anginosus group. Identification by pathogen-specific PCRs was compared to routine culture and both methods were considered as the gold standard for determination of the sensitivity and specificity of each assay. Direct real-time PCR assays of biopsy samples resulted in a 34 % higher detection of S. aureus, 37 % higher detection of S. pyogenes, 18 % higher detection of S. agalactiae, 4 % higher detection of S. dysgalactiae subspecies equisimilis, and 7 % higher detection of the S. anginosus group, compared to routine bacterial culture. The presence of PVL was mainly confined to S. aureus isolated from major abscess but not DFI biopsy samples. In conclusion, our pathogen-specific real-time PCR assays had a higher yield than culture methods and could be an additional method for the detection of relevant causative pathogens in biopsies.
European Journal of Clinical Microbiology 07/2015; 34(9). DOI:10.1007/s10096-015-2428-4 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Toll-like receptors (TLRs) play a central role in the innate immune response to complicated skin and skin structure infections (cSSSIs), with TLR10 being the first family member known to have an inhibitory function. This study assessed the role of TLR10 in recognition of cSSSI-related pathogens and whether genetic variation in TLR10 influences susceptibility to cSSSIs.
Human peripheral blood mononuclear cells (PBMCs) preincubated with anti-TLR10 antibody and HEK-293 cells overexpressing TLRs were exposed to cSSSI pathogens, and cytokine secretion was determined by enzyme-linked immunosorbent assay. A total of 318 patients with cSSSI and 328 healthy controls were genotyped for 4 nonsynonymous single-nucleotide polymorphisms in TLR10, and functional consequences of the TLR10 SNPs were assessed via in vitro stimulation assays.
PBMC stimulation with cSSSI pathogens in the presence of TLR10 neutralizing antibody significantly increased interleukin 6 secretion. Overexpression of TLR10 completely abrogated TLR2-induced interleukin 8 secretion by HEK-293 cells in response to cSSSI pathogens. Three polymorphisms in TLR10, I775L, I369L, and N241H, were associated with reduced susceptibility to cSSSIs. The presence of the TLR10 alleles 775L, 369L, or 241H increased interleukin 6 secretion by PBMCs in response to cSSSI pathogens.
TLR10 is a modulatory receptor of innate immune responses to cSSSI-related pathogens, and genetic variants in TLR10 are associated with protection against cSSSIs.
The Journal of Infectious Diseases 04/2015; 212(9). DOI:10.1093/infdis/jiv229 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Complicated skin and skin structure infections (cSSSIs) are caused by Gram-positive and Gram-negative, aerobic and anaerobic pathogens, with a polymicrobial aetiology being frequent. Recognition of invading pathogens by the immune system results in the production of pro- and anti-inflammatory cytokines, which are extremely important for intercellular communication and control of infection. This study assessed whether genetic variation in genes encoding cytokines influences the susceptibility to cSSSIs. For the association study, 318 patients with cSSSI and 328 healthy controls were genotyped for single nucleotide polymorphisms (SNPs) in cytokine genes IL1A, IL1B, IL1RN, TNF, IL10, IL17A, IL17F and IFNG. For immunological validation, peripheral blood mononuclear cells (PBMCs) from 74 healthy individuals, genotyped for SNPs of interest, were stimulated with Staphylococcus aureus or Escherichia coli and corresponding cytokine levels were determined by enzyme-linked immunosorbent assay (ELISA). Polymorphisms IL6 rs1800797, TNF rs1800629, IL10 rs1800871, IL17A rs8193036 and IFNG rs2069705 influenced susceptibility to cSSSIs. No differences in cytokine responses, stratified for genotype, were detected after PBMC stimulation. No association with cSSSIs was observed for polymorphisms IL1A rs17561 and rs1800587, IL1B rs16944 and rs1143627, IL1RN rs4251961, TNF rs361525, IL10 rs1800896, IL17A rs2275913 and IL17F rs763780. In conclusion, polymorphisms in IL6, TNF, IL10, IL17A and IFNG are associated with susceptibility to cSSSIs.
European Journal of Clinical Microbiology 07/2014; 33(12). DOI:10.1007/s10096-014-2201-0 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Complicated skin and skin structure infections (cSSSIs) are characterized by infections with gram-positive or gram-negative aerobic or anaerobic bacteria, as well as by a polymicrobial etiology. These invading microorganisms are recognized by pattern-recognition receptors (PRRs) of the innate immune system. This study assessed whether genetic variation in genes encoding PRRs influences the susceptibility to cSSSIs.
A total of 318 patients with cSSSI and 328 healthy controls were genotyped for 9 nonsynonymous single-nucleotide polymorphisms (SNPs) in PRR genes coding for Toll-like receptors (TLRs) 1, 2, 4, and 6; NOD-like receptor 2; and the signaling adaptor molecule TIRAP. Associations between susceptibility to cSSSIs and a SNP were investigated by means of logistic regression models. In an additional cohort of 74 healthy individuals in whom the same SNPs were genotyped, peripheral blood mononuclear cells (PBMCs) were obtained and stimulated with Staphylococcus aureus. Interleukin 6 concentrations were determined in supernatants by enzyme-linked immunosorbent assay to determine the correlation between genotypes and levels of IL-6 secretion.
In the genetic association analysis, polymorphisms in TLR1 (S248N and R80T), TLR2 (P631H), and TLR6 (P249S) were associated with an increased susceptibility to cSSSIs. No association with susceptibility to cSSSIs was observed for polymorphisms TLR2 (R753Q), TLR4 (D299G and T399I), NOD2 (P268S), and TIRAP (S180L). In the functional analysis, individuals bearing the TLR1 248N or 80T allele showed lower IL-6 secretion upon stimulation with S. aureus.
Polymorphisms in TLR1, TLR2, and TLR6 are associated with increased susceptibility to cSSSIs. For TLR1, impaired proinflammatory cytokine production due to the polymorphism is most likely the mechanism mediating this effect.
The Journal of Infectious Diseases 02/2014; 210(2). DOI:10.1093/infdis/jiu080 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacteroides fragilis, an intestinal flora commensal microorganism, is frequently isolated from abscesses and soft tissue infections. This study aimed to identify pattern recognition receptors (PRRs) involved in B. fragilis recognition and to characterize the induced cytokine profile. Human PBMCs were stimulated with heat-killed B. fragilis and cytokine levels were determined by ELISA. Roles of individual PRRs were assessed using specific blockers of receptor signaling pathways and PBMCs carrying single nucleotide polymorphisms of PRR genes. Cell lines expressing human TLR2 or TLR4 were employed to assess TLR-specificity of B. fragilis. TLR1, TLR2 and NOD2 were the main PRRs responsible for recognition of B. fragilis, while TLR4, TLR6, NOD1 and Dectin-1 were not involved. B. fragilis induced strong IL-6 and IL-8, moderate IL-1β and TNF-α, and poor IL-10, IL-17, IL-23 and IFN-γ production. This study identifies the receptor pathways of the innate immune response to B. fragilis, and thus provides new insights in the host defense against B. fragilis.
[Show abstract][Hide abstract] ABSTRACT: Upon the invasion of the host by microorganisms, innate immunity is triggered through pathogen recognition by pattern recognition
receptors (PRRs). Toll-like receptors (TLRs) are the best-studied class of PRRs, and they recognize specific pathogen-associated
molecular patterns (PAMPs) from various microorganisms. A large number of studies have shown that genetic variation in TLRs
may influence susceptibility to infections. We assessed the genetic variation of TLR2, which encodes one of the most important TLRs, in various populations around the globe and correlated it with changes in
the function of the molecule. The three best-known nonsynonymous TLR2 polymorphisms (1892C>A, 2029C>T, and 2258G>A) were assessed in different populations from the main continental masses: Romanians,
Vlax-Roma, Dutch (European populations), Han Chinese (East Asia), Dogon, Fulani (Africa), and Trio Indians (America). The
2029C>T polymorphism was absent in both European and non-European populations, with the exception of the Vlax-Roma, suggesting
that this polymorphism most likely arose in Indo-Aryan people after migration into South Asia. The 1892C>A polymorphism that
was found exclusively in European populations, but not in Asian, African, or American volunteers, probably occurred in proto-Indo-Europeans.
Interestingly, 2258G>A was present only in Europeans, including Vlax-Roma, but at a very low frequency. The differential pattern
of the TLR2 polymorphisms in various populations may explain some of the differences in susceptibility to infections between these populations.
Infection and immunity 02/2012; 80(5):1917-22. DOI:10.1128/IAI.00121-12 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin-22 (IL-22) is a mediator in antimicrobial responses and inflammatory autoimmune diseases. Although IL-22 and its receptor, IL-22R, have been identified in the synovium of rheumatoid arthritis patients, the source of IL-22 and its contribution to disease pathogenicity remain to be established. This study was undertaken to investigate the regulation of IL-22 by Th17 cells in vitro and to evaluate the potential for IL-22 depletion in an experimental arthritis model using mice deficient in the IL-1 receptor antagonist (IL-1Ra-/-).
Naive murine T cells were cultured under conditions leading to polarization of the cells into subsets of Th1, Th2, induced Treg, and Th17. Cytokines were measured in the culture supernatants, and the cells were analyzed by fluorescence-activated cell sorting. Tissue samples from the inflamed ankle synovium of IL-1Ra-/- mice were isolated, and messenger RNA levels of marker genes were quantified. IL-1Ra-/- mice were treated with neutralizing anti-IL-22 antibodies. Synovial cells were isolated from the inflamed tissue and sorted into fractions for analysis of cytokine production.
In vitro tests showed that Th17 cells produced high levels of IL-22 after stimulation with IL-1 or IL-23. Interestingly, a synergistic increase in the production of IL-22 was observed after combining IL-1 and IL-23. In vivo, IL-1Ra-/- mice displayed a progressive erosive arthritis, characterized by up-regulation of IL-17 in mildly and severely inflamed tissue, whereas the levels of IL-22 and IL-22R were increased only in severely inflamed synovia. Anti-IL-22 treatment of IL-1Ra-/- mice significantly reduced the inflammation and bone erosion. Analysis of isolated single cells from the inflamed synovia revealed that IL-22 was mainly produced by IL-17-expressing T cells.
These findings suggest that IL-22 plays an important role in IL-1-driven chronic joint destruction.
[Show abstract][Hide abstract] ABSTRACT: Background: Bacteroids fragilis and Candida albicans are both part of the commensal intestinal flora. When B. fragilis spreads to normally sterile parts of the body it is a potent inducer of abscess formation. These abscesses are often polymicrobial and synergistic effects in promoting larger abscesses and bacterial persistence have been observed for bacterial co-infections. In contrast, the presence of fungi in abscesses and the effect of fungal and microbial co-infections on the host immune response has been poorly studied. The aim of this study was to assess the modulatory effect of B. Fragilis on the C. albicans induced cytokine profile. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were stimulated with heat- killed B. fragilis (107/ml), heat- killed C. albicans (105/
ml), or the combination and cytokine levels were determined in supernatants by
Results: Both B. fragilis (107/ml) and C. albicans (105/ml) are potent inducers of IL-8
and IL-6, with a moderate IL-1b and TNFa production, while induction of IL-23, IFNc
and IL-10 is low. In contrast to B. fragilis, C. albicans is a potent inducer of IL-17. Coincubation
of Bacteroides fragilis and C. albicans resulted in a significant decrease of IL-
17 secretion by PBMCs, whereas co-incubation had an additive effect on most other
cytokines. B. fragilis inhibited IL-17 production even if added to the cells two hours
after stimulation with C. albicans. B. fragilis induced these effects through Toll-like
receptor 2 (TLR2), and the TLR2 stimulus Pam3Cys had similar inhibitory effects on
C. albicans-induced IL-17 secretion. Conclusion: B. fragilis inhibits C. albicans induced IL-17 secretion through TLR2-mediated signalling. This finding may have important consequences for the patho-physiology of bacterial-fungal mixed abscesses, as well as during co-colonization of the intestinal mucosa with these two microorganisms.