Teske Schoffelen

Cryptococcus gattii induces a cytokine pattern that is distinct from other cryptococcal species.

Understanding more about the hosts immune response to different Cryptococcus spp. will provide additional insight into the pathogenesis of cryptocococcis. We hypothesized that the ability of C. gattii to cause disease in immunocompetent humans depends on a distinct innate cytokine response of the host to this emerging pathogen. In the current study we assessed the cytokine profile of human peripheral blood mononuclear cells (PBMCs) of healthy individuals, after in vitro stimulation with 40 different well-defined heat-killed isolates of C. gattii, C. neoformans and several hybrid strains. In addition, we investigated the involvement of TLR2, TLR4 and TLR9 in the pro-inflammatory cytokine response to C. gattii. Isolates of C. gattii induced higher concentrations of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 and the Th17/22 cytokine IL-17 and IL-22 compared to C. neoformans var neoformans and C. neoformans var grubii. In addition, clinical C. gattii isolates induced higher amounts of cytokines than environmental isolates. This difference was not observed in C. neoformans var. grubii isolates. Furthermore, we demonstrated a likely contribution of TLR4 and TLR9, but no role for TLR2, in the hosts cytokine response to C. gattii. In conclusion, clinical heat-killed C. gattii isolates induced a more pronounced inflammatory response compared to other Cryptococcus species and non-clinical C. gattii. This is dependent on TLR4 and TLR9 as cellular receptors.

Specific Interferon-γ detection for the diagnosis of previous Q fever

BACKGROUND Current practice for diagnosis of Q fever, caused by the intracellular pathogen Coxiella burnetii, relies mainly on serology and, in prevaccination assessment, on skin tests (STs), which both have drawbacks. In this study, C. burnetii-specific interferon γ (IFN-γ) production was used as a new diagnostic tool for previous Q fever, circumventing most of these drawbacks. Our aim was to compare this test to serology and ST. METHODS One thousand five hundred twenty-five individuals from an endemic area with a risk for chronic Q fever were enrolled. IFN-γ production was measured after in vitro stimulation of whole blood with C. burnetii antigens. Various formats using different C. burnetii antigens were tested. Serology and ST were performed in all individuals. RESULTS In all assay formats, C. burnetii-specific IFN-γ production was higher (P < .0001) in seropositive or ST-positive subjects than in seronegative and ST-negative subjects. Whole blood incubated for 24 hours with C. burnetii Nine Mile showed optimal performance. After excluding subjects with equivocal serology and/or borderline ST results, IFN-γ production was 449 ± 82 pg/mL in the positive individuals (n = 219) but only 21 ± 3 pg/mL in negative subjects (n = 908). Using Bayesian analysis, sensitivity and specificity (87.0% and 90.2%, respectively) were similar to the combination of serology and ST (83.0% and 95.6%, respectively). Agreement with the combination of serology and ST was moderate (84% concordance; κ = 0.542). CONCLUSIONS Specific IFN-γ detection is a novel diagnostic assay for previous C. burnetii infection and shows similar performance and practical advantages over serology and ST. Future studies to investigate the clinical value in practice are warranted.

Limited humoral and cellular responses to Q fever vaccination in older adults with risk factors for chronic Q fever

OBJECTIVES In the Netherlands, people at risk for chronic Q fever were vaccinated against Coxiella burnetii with the inactivated whole cell vaccine Q-vax®. We aimed to measure the immune responses to C. burnetii six and twelve months after vaccination in this relevant population. METHODS In 260 vaccinees, antibody responses were assessed by immunofluorescence assay (IFA), complement fixation test and ELISA. The cellular immune responses were assessed by measuring C. burnetii-specific interferon (IFN)-γ production in blood. Serological results of 200 individuals with past Q fever were used for comparison. RESULTS At six months, 46% of vaccinees showed low IFA antibody titres and 67% had a positive IFN-γ assay; At twelve months, both were 60%. In contrast, individuals with a past Q fever were seropositive in 99.5% at six and twelve months, with relatively higher IFA titres. Interestingly, vaccinees with positive IFN-γ assay pre-vaccination, showed a higher seroconversion rate than IFN-γ negative vaccinees: 74% vs. 41% (p < 0.001). CONCLUSIONS The immune response after Q-vax® vaccination is lower and restricted to a smaller proportion than found after past Q fever and than previously described after vaccination, suggesting decreased vaccine immunogenicity in this high-risk population. A positive IFN-γ assay before vaccination in seronegative vaccinees likely points to pre-existing immunity resulting in boosting by vaccination.

A combination of interferon-gamma and interleukin-2 production by Coxiella burnetii-stimulated circulating cells discriminates between chronic Q fever and past Q fever.

Infection with Coxiella burnetii may lead to life-threatening chronic Q fever endocarditis or vascular infections, which are often difficult to diagnose. The present study aims to investigate whether measurement of in-vitro interferon-gamma (IFN-γ) production, a key cytokine in the immune response against C. burnetii, differentiates chronic from a past cleared infection, and whether measurement of other cytokines would improve the discriminative power. First, C. burnetii-specific IFN-γ production was measured in whole blood of 28 definite chronic Q fever patients and compared with 135 individuals with past Q fever (seropositive controls) and 908 seronegative controls. IFN-γ production was significantly higher in chronic Q fever patients than in controls, but with overlapping values between patients and seropositives. Secondly, the production of a series of other cytokines was measured in a subset of patients and controls, which showed that interleukin (IL)-2 production was significantly lower in patients than in seropositive controls. Subsequently, measuring IL-2 in all patients and all controls with substantial IFN-γ production showed that an IFN-γ/IL-2 ratio >11 had a sensitivity and specificity of 79% and 96%, respectively, to diagnose chronic Q fever. This indicates that a high IFN-γ/IL-2 ratio is highly suggestive for chronic Q fever. In an additional group of 25 individuals with persistent high anti-Coxiella phase I IgG titres without definite chronic infection, all but six showed an IFN-γ/IL-2 ratio <11. In conclusion, these findings hold promise for the often difficult diagnostic work-up of Q fever and the IFN-γ/IL-2 ratio may be used as an additional diagnostic marker.

Recognition of Coxiella burnetii by Toll-like Receptors and Nucleotide-Binding Oligomerization Domain–like Receptors

BACKGROUND Infection with Coxiella burnetii can lead to acute and chronic Q fever. Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6, nucleotide-binding oligomerization domain receptor 1 (NOD1), NOD2, and the mitogen-activated protein kinases are central in the innate immune response against microorganisms, but little is known about their role in the recognition of C. burnetii in humans. METHODS Human peripheral blood mononuclear cells (PBMCs) were stimulated with C. burnetii Nine Mile and the Dutch outbreak isolate C. burnetii 3262. TLRs were inhibited using specific antibodies or antagonists. Additionally, the influence of human polymorphisms in TLRs and Nod-like receptors (NLRs) on C. burnetii-induced cytokine production was assessed. RESULTS Inhibition of TLR2, p38, JNK, and ERK led to decreased cytokine responses in C. burnetii-stimulated human PBMCs. Humans with polymorphisms in TLR1 and NOD2 had reduced cytokine production, compared with humans with wild-type genotypes, after stimulation. Interestingly, polymorphisms in TLR6 led to decreased cytokine production after C. burnetii 3262 stimulation but not after C. burnetii Nine Mile stimulation. CONCLUSIONS The TLR1/TLR2 heterodimer and NOD2 are important recognition receptors for the induction of cytokine responses against C. burnetii in humans. Furthermore, an interesting finding was the divergent recognition of C. burnetii Nine Mile and C. burnetii 3262.

Adverse events and association with age, sex and immunological parameters of Q fever vaccination in patients at risk for chronic Q fever in the Netherlands 2011

BACKGROUND Following a large Q fever outbreak in the Netherlands, patients at risk for chronic Q fever received a whole-cell Q fever vaccine. Sensitized people were excluded based on pre-vaccination screening with skin test (ST) and serology. An investigational IFN-γ-production assay was added. No previous experience existed for Q fever vaccination in this patient risk-group with predefined cardiac valvular anomalies or aortic aneurysm/prosthesis and many co-morbidities. We studied the adverse events (AE) and their association with patient characteristics and immunological parameters. METHODS AE registration covered the week after skin test and 90 days following vaccination, with the use of diaries, interviews and spontaneous reports. Serious (S)AE were assessed immediately to ensure safety. We coded AE according to reported severity. Univariate and multivariate analysis addressed associations. RESULTS Pre-vaccination screening led to exclusion of 182 patients with positive serology and 207 patients with positive skin test-reading. The skin test did not lead to any causally related SAE. Subsequent vaccination of 1370 patients did not reveal unexpected AE; however, 80% of vaccinees reported local AE (in 26% of these pronounced or extensive). The two causally related SAE (0.1%) both concerned a persistent subcutaneous injection site mass. AE were more frequent in women, younger patients, and those without immunosuppressive co-morbidity/medication. The occurrence of local AE after skin test was associated with pre-vaccination positive serology and high IFN-γ production. This was also true for local AE following vaccination, with a strong association with local AE after skin test as well. The proportion of vaccinees with positive serology and positive IFN-γ values 6 months after vaccination was higher in those with local AE after skin test or after vaccination (non-significant, probably due to small numbers). CONCLUSION Q fever vaccination was safe but reactogenic in this high-risk patient-group. Rates of local AE were higher in women, younger age groups and in those with positive immunological parameters. Vaccinees with local AE after skin test or after vaccination appear to have more pronounced post-vaccination immune responses.

Coxiella burnetii infection (Q fever) in rheumatoid arthritis patients with and without anti-TNFα therapy

Q fever is a zoonosis caused by the intracellular bacterium Coxiella burnetii. The Netherlands experienced a major Q fever outbreak between 2007 and 2010, with an estimate of more than 40 000 infected individuals.1 Initial infection is asymptomatic in over 50% of the infected individuals or causes a mostly self-limiting febrile disease.2 However, chronic Q fever may develop months to years after initial infection. This serious, life-threatening condition presents mostly as endocarditis or infection of an aortic aneurysm or vascular prosthesis, and is accompanied by high IgG antibody titres against phase I C burnetii .3 Individuals most at risk for chronic Q fever are those with pre-existing valvulopathy, vascular aneurysm or prosthesis and yet undefined types of immune suppression.4 ,5 Tumour necrosis factor-α (TNFα) plays an important role in the defence against intracellular bacteria such as C burnetii . In vitro studies show that TNFα is involved in internalisation and intracellular killing of C burnetii in monocytes.6 ,7 In addition, C burnetii- infected TNFα knockout mice develop early bacteraemia and severe …

Genetic Variation in Pattern Recognition Receptors and Adaptor Proteins Associated With Development of Chronic Q Fever

BACKGROUND Q fever is an infection caused by Coxiella burnetii. Persistent infection (chronic Q fever) develops in 1%-5% of patients. We hypothesize that inefficient recognition of C. burnetii and/or activation of host-defense in individuals carrying genetic variants in pattern recognition receptors or adaptors would result in an increased likelihood to develop chronic Q fever. METHODS Twenty-four single-nucleotide polymorphisms in genes encoding Toll-like receptors, nucleotide-binding oligomerization domain-like receptor-2, αvβ3 integrin, CR3, and adaptors myeloid differentiation primary response protein 88 (MyD88), and Toll interleukin 1 receptor domain-containing adaptor protein (TIRAP) were genotyped in 139 patients with chronic Q fever and in 220 controls with cardiovascular risk-factors and previous exposure to C. burnetii. Associations between these single-nucleotide polymorphisms and chronic Q fever were assessed by means of univariate logistic regression models. Cytokine production in whole-blood stimulation assays was correlated with relevant genotypes. RESULTS Polymorphisms in TLR1 (R80T), NOD2 (1007fsX1), and MYD88 (-938C>A) were associated with chronic Q fever. No association was observed for polymorphisms in TLR2, TLR4, TLR6, TLR8, ITGAV, ITGB3, ITGAM, and TIRAP. No correction for multiple testing was performed because only genes with a known role in initial recognition of C. burnetii were included. In the whole-blood assays, individuals carrying the TLR1 80R-allele showed increased interleukin 10 production with C. burnetii exposure. CONCLUSIONS Polymorphisms in TLR1 (R80T), NOD2 (L1007fsX1), and MYD88 (-938C>A) are associated with predisposition to development of chronic Q fever. For TLR1, increased interleukin 10 responses to C. burnetii in individuals carrying the risk allele may contribute to the increased risk of chronic Q fever.

Specific in vitro interferon-gamma and IL-2 production as biomarkers during treatment of chronic Q fever.

Background: Antibiotic treatment of chronic Q fever is cumbersome and of long duration. To monitor treatment, there is a need for alternative biomarkers. Coxiella burnetii-specific interferon (IFN)-γ and interleukin (IL)-2 production reflect the type of effector and memory T-cell response. In chronic Q fever, C. burnetii-specific IFN-γ production is higher and IL-2 production is lower than in individuals with past Q fever. Here we explore whether C. burnetii-specific IFN-γ and IL-2 production correlate to treatment response. Methods: We studied the longitudinal C. burnetii-specific IFN-γ/IL-2 ratio in fifteen proven chronic Q fever patients. All patients were followed for at least 18 months during antibiotic treatment. Treatment was considered successful when clinical recovery was observed, a positive PCR for C. burnetii DNA in blood became persistently negative, anti-phase I IgG showed a fourfold decrease or more, and imaging techniques showed disappearance of infectious foci. Results: Overall, the IFN-γ/IL-2 ratio declined when patients experienced a successful treatment outcome. When treatment failed, IFN-γ/IL-2 ratios did not significantly decrease. The median (±IQR) slope of the longitudinal IFN-γ/IL-2 ratio with successful treatment was -2.10 (-7.02 to -0.06), and -0.15 (-1.13 to 0.25) with unsuccessful treatment (P = 0.19). Q fever endocarditis patients had higher IFN-γ/IL-2 ratios than patients with endovascular infections. Conclusion: We propose that the IFN-γ/IL-2 ratio can be used as an additional biomarker for monitoring chronic Q fever treatment, with declining ratios being indicative of successful treatment.

Early cytokine and antibody responses against Coxiella burnetii in aerosol infection of BALB/c mice.

Coxiella burnetii, a Gram-negative intracellular bacterium, can give rise to Q fever in humans and is transmitted mainly by inhalation of infected aerosols from animal reservoirs. Serology is commonly used to diagnose Q fever, but the early cellular immune response-i.e., C. burnetii-specific interferon γ (IFN-γ) production in response to antigen challenge-might be an additional diagnostic. Detection of IFN-γ responses has been used to identify past and chronic Q fever infections, but the IFN-γ response in acute Q fever has not been described. By challenging immunocompetent BALB/c mice with aerosols containing phase I C. burnetii, the timing and extent of IFN-γ recall responses were evaluated in an acute C. burnetii infection. Other cytokines were also measured in an effort to identify other potential diagnostic markers. The data show that after initial expansion of bacteria first in lungs and then in other tissues, the infection was cleared from day 10 onwards as reflected by the decreasing number of bacteria. The antigen-induced IFN-γ production by splenocytes coincided with emergence of IgM phase II antibodies at day 10 postinfection and preceded appearance of IgG antibodies. This was accompanied by the production of proinflammatory cytokines including interleukin (IL) 6, keratinocyte-derived cytokine, and IFN-γ-induced protein 10, followed by monocyte chemotactic protein 1, but not by IL-1β and tumor necrosis factor α, and only very low production of the anti-inflammatory cytokine IL-10. These data suggest that analysis of antigen-specific IFN-γ responses could be a useful tool for diagnosis of acute Q fever. Moreover, the current model of C. burnetii infection could be used to give new insights into immunological factors that predispose to development of persistent infection.