Jan Kehrmann

Impact of 5-aza-2′-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells

The epigenetic regulation of transcription factor genes is critical for T‐cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg‐specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5‐aza‐2′‐deoxycytidine and epigallocatechin‐3‐gallate on human CD4+ CD25− T cells for generating demethylation within FOXP3‐TSDR and inducing functional Treg cells. Gene expression, including lineage‐specifying transcription factors of the major T‐cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3‐TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5‐aza‐2′‐deoxycytidine induced FOXP3‐TSDR hypomethylation and expression of the Treg‐cell‐specific genes FOXP3 and LRRC32. Proliferation of 5‐aza‐2′‐deoxycytidine‐treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3‐TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin‐3‐gallate induced global DNA hypomethylation to a lesser extent than 5‐aza‐2′‐deoxycitidine, but no relevant hypomethylation within FOXP3‐TSDR or expression of Treg‐cell‐specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5‐aza‐2′‐deoxycitidine‐treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells.

Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry

During sepsis, a relative increase of regulatory T (Treg) cells has been reported. Its persistence is associated with lymphocyte anergy, immunoparalysis and a poor prognosis. Currently, an exact quantification of human Treg cells based on protein expression of marker molecules is ambiguous, as these molecules are expressed also by activated non-regulatory T cells. Furthermore, no firm criteria for flow cytometer gate settings exist so far. Recently, a specific DNA methylation pattern within FOXP3-TSDR has been reported that allows distinguishing Treg and non-regulatory T cells, independent of their activation status. Using this epigenetic marker, we established a single-tube real-time PCR based methylation assay (QAMA) for relative quantification of Treg cells. Validation was performed on defined ratios of methylated and unmethylated target sequence and on mixtures of Treg and non-regulatory T cells. DNA-methylation was measured in CD4+ T cells isolated from blood samples of 30 septic patients and 30 healthy subjects and compared with results of Treg cell quantification by flow cytometry based on CD4+ CD25hiCD127low measurement. In septic patients both methods showed an increased ratio of Treg cells to all CD4+ T cells. In healthy individuals, the results obtained by both methods were clearly positively correlated. However, the correlation between both methods in septic patients was only weak. We showed that quantification of Treg cells by QAMA detects CD4+ T cells with unmethylated FOXP3-TSDR, hidden in the CD25med/low fraction of flow cytometry. Given that unmethylated FOXP3-TSDR is the most specific feature of Treg cells to date, our assay precisely quantifies Treg cells, as it additionally detects those committed Treg cells, hidden in the CD25med/low fraction of CD4+ cells. Furthermore, QAMA is a reliable method, which is easier to standardize among laboratories and can thus improve reproducibility of Treg cell quantification.

Relevance of Foxp3 + regulatory T cells for early and late phases of murine sepsis

The role of Foxp3+ regulatory T (Treg) cells in the course of the early hyper‐inflammatory and subsequent hypo‐inflammatory phases of sepsis is ambiguous. Whereas Nrp1 expression has been reported to discriminate natural Treg cells from induced Treg cells, the Treg cell stability depends on the methylation status of foxp3‐TSDR. To specifically evaluate the role of Foxp3+ Treg cells in the early and late phases of sepsis, we induced sepsis by caecal ligation and puncture and subsequent Pseudomonas aeruginosa lung infection in a DEREG (DEpletion of REGulatory T cells) mouse model. We found an increase of Foxp3+ Treg cells to all CD4+ T cells during murine sepsis. Using a new methylation‐sensitive quantitative RT‐PCR method and deep amplicon sequencing, we demonstrated that natural (Nrp1+ Foxp3+) Treg cells and most induced (Nrp1− Foxp3+) Treg cells are stable and exhibit unmethylated foxp3‐TSDR, and that both Treg populations are functionally suppressive in healthy and septic mice. DEREG mice depleted of Foxp3+ Treg cells exhibit higher disease scores, mortality rates and interleukin‐6 expression levels than do non‐depleted DEREG mice in early‐phase sepsis, a finding indicating that Foxp3+ Treg cells limit the hyper‐inflammatory response and accelerate recovery. Treg cell depletion before secondary infection with P. aeruginosa 1 week after caecal ligation and puncture does not influence cytokine levels or the course of secondary infection. However, a moderate Treg cell recurrence, which we observed in DEREG mice during secondary infection, may interfere with these results. In summary, Treg cells contribute to a positive outcome after early‐phase sepsis, but the data do not support a significant role of Treg cells in immune paralysis during late‐phase sepsis.

Effects of green tea compound epigallocatechin-3-gallate against Stenotrophomonas maltophilia infection and biofilm.

We investigated the in vitro and in vivo activities of epigallocatechin-3-gallate (EGCg), a green tea component, against Stenotrophomonas maltophilia (Sm) isolates from cystic fibrosis (CF) patients. In vitro effects of EGCg and the antibiotic colistin (COL) on growth inhibition, survival, and also against young and mature biofilms of S. maltophilia were determined. Qualitative and quantitative changes on the biofilms were assessed by confocal laser scanning microscopy (CLSM). Further, in vivo effects of nebulized EGCg in C57BL/6 and Cftr mutant mice during acute Sm lung infection were evaluated. Subinhibitory concentrations of EGCg significantly reduced not only biofilm formation, but also the quantity of viable cells in young and mature biofilms. CLSM showed that EGCg-exposed biofilms exhibited either a change in total biofilm biovolume or an increase of the fraction of dead cells contained within the biofilm in a dose depended manner. Sm infected wild-type and Cftr mutant mice treated with 1,024 mg/L EGCg by inhalation exhibited significantly lower bacterial counts than those undergoing no treatment or treated with COL. EGCg displayed promising inhibitory and anti-biofilm properties against CF Sm isolates in vitro and significantly reduced Sm bacterial counts in an acute infection model with wild type and CF mice. This natural compound may represent a novel therapeutic agent against Sm infection in CF.

Performance of Vitek MS in identifying nontuberculous mycobacteria from MGIT liquid medium and Lowenstein-Jensen solid medium.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a fast and inexpensive method for bacterial identification. The aim of this study was to analyze the performance of Vitek MS in identifying 160 nontuberculous mycobacterial isolates of 24 species from Lowenstein-Jensen solid medium and BACTEC MGIT 960 liquid medium using a bead-based method. The system correctly identified 76.9% of the isolates (123 of 160) cultivated on solid medium and 76.9% (123 of 160) of positive liquid cultures. None of the isolates included in the study was misidentified. Although the overall performance of Vitek MS with the SARAMIS 4.12 database was comparable in identifying mycobacterial species grown on solid medium and in liquid medium, the identification rate varied notably between the various species analyzed, which currently limits the utility for identification in routine diagnostics for some species.

Principal component analysis of MALDI TOF MS mass spectra separates M. abscessus (sensu stricto) from M. massiliense isolates.

BackgroundThe discrimination of the members of the Mycobacterium abscessus complex is of clinical interest because one of the subspecies, M. massiliense, exhibits higher rates of response to antibiotic treatment for lung infection than do the other members of that complex. M. abscessus complex contains three subspecies that are laborious to identify; therefore, a routine diagnostic tool would be worthwhile.ResultsWe used principal component analysis, hierarchical cluster analysis, and single-peak analysis to examine peak lists derived from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) mass spectra of 50 clinical M. abscessus complex isolates, including 28 M. abscessus (sensu stricto), 19 M. massiliense, and 3 M. bolletii isolates grown in mycobacterium growth indicator tube liquid medium and prepared with a bead-based protocol. Principal component analysis but not hierarchical cluster analysis separated M. abscessus (sensu stricto) isolates and M. massiliense isolates into two clusters. Furthermore, single-peak analysis displayed 4 discriminating peaks that separated M. abscessus (sensu stricto) from M. massiliense isolates. M. bolletii isolates did not exhibit specific peaks but resembled the M. abscessus (sensu stricto) peak profile and also grouped within this principal component analysis cluster. Principal component analysis of all peak lists with the exclusion of the four discriminating peaks again separated M. abscessus (sensu stricto) from M. massiliense isolates, thus relativizing the importance of these peaks for subspecies identification.ConclusionsPrincipal component analysis of peak lists derived from MALDI TOF mass spectra is a robust and convenient method of discriminating M. massiliense isolates from the other members of the M. abscessus complex.

GenoType NTM-DR for identifying Mycobacterium abscessus subspecies and determining molecular resistance

ABSTRACT We studied the performance of a new line probe assay for identifying the subspecies and determining the macrolide and aminoglycoside resistance levels of 50 Mycobacterium abscessus isolates. Agreement of GenoType NTM-DR results with sequencing and phenotypic resistance results was 92% for subspecies identification and 98% for determining molecular and phenotypic resistance.

Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4+Foxp3+ regulatory T cells

Recently, we demonstrated elevated numbers of CD4+ Foxp3+ regulatory T (Treg) cells in Plasmodium yoelii‐infected mice contributing to the regulation of anti‐malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus‐derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3+ Treg cells expressing neuropilin‐1 (Nrp‐1) decreased at early time‐points during P. yoelii infection, whereas percentages of Helios+ Foxp3+ Treg cells remained unchanged. Both Foxp3+ Nrp‐1+ and Foxp3+ Nrp‐1− Treg cells from P. yoelii‐infected mice exhibited a similar T‐cell receptor Vβ chain usage and methylation pattern in the Treg‐specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3− T cells adoptively transferred to P. yoelii‐infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3+ Treg cells.

FOXP3 Expression in GARP-Transduced Helper T Cells Is Not Associated with FOXP3 TSDR Demethylation

Aim: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25hi FOXP3+ T (Treg) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (Th) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called Treg-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR. Methods: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. Results: Although GARP-transduced Th cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive Th cells. GARP-mediated FOXP3 upregulation in Th cells is not associated with Treg-specific demethylation of the FOXP3 TSDR. Conclusion: Although GARP-engineered Th cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards Treg cells in vitro, these cells do not completely mimic the epigenotype of natural Treg cells. Thus, concepts based on the genetic modification of Th cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application.

Diagnostic performance of blood culture bottles for vitreous culture compared to conventional microbiological cultures in patients with suspected endophthalmitis

The purpose of this investigation was to evaluate the performance of blood culture bottles in comparison to conventional microbiological culture techniques in detecting causative microorganisms of endophthalmitis and to determine their anti-infective susceptibility profiles. All consecutive cases with clinically suspected endophthalmitis in a university-based ophthalmology department between January 2009 and December 2016 were analysed in this retrospective comparative case series. Samples from 247 patients with suspected endophthalmitis underwent microbiological diagnostic work-up. All three culture methods were performed from 140 vitreous specimens. Vitreous fluid specimens were inoculated in blood culture bottles, aerobic and anaerobic broth solutions, and on solid media. Anti-infective susceptibility profiles were evaluated by semi-automated methods and/or gradient diffusion methods. Microorganisms were grown in 82 of 140 specimens for which all methods were performed (59%). Microorganisms were more frequently grown from blood culture bottles (55%) compared to broth solution (45%, p = 0.007) and solid media (33%, p < 0.0001). Considerable differences in the performance among culture media were detected for fungal pathogens. All grown fungi were detected by blood culture bottles (11 of 11, 100%). Broth solution recovered 64% and solid media 46% of grown fungi. No Gram-positive bacterium was resistant to vancomycin and all Gram-negative pathogens except for one isolate were susceptible to third-generation cephalosporins. In suspected endophthalmitis patients, blood culture bottles have a higher overall pathogen detection rate from vitreous fluid compared to conventional microbiological media, especially for fungi. The initial intravitreal antibiotic therapy with vancomycin plus third-generation cephalosporins appears to be an appropriate treatment approach for bacterial endophthalmitis.