Marijo Parcina

Tumour-derived prostaglandin E2 and transforming growth factor-β synergize to inhibit plasmacytoid dendritic cell-derived interferon-α

In previous studies we reported that plasmacytoid dendritic cells (PDC) infiltrating head and neck cancer tissue are functionally impaired, but the molecular basis for the functional deficiency remained unclear. Here we demonstrate that tumour‐derived prostaglandin E2 (PGE2) and transforming growth factor‐β (TGF‐β) increase interleukin‐8 (IL‐8) but synergistically inhibit interferon‐α (IFN‐α) and tumour necrosis factor (TNF) production of Toll‐like receptor 7 (TLR7)‐ and Toll‐like receptor 9 (TLR9)‐stimulated PDC. The inhibitory effect of PGE2 could be mimicked by the induction of cyclic AMP (cAMP) and by inhibitors of cyclooxygenase. The contribution of tumour‐derived TGF‐β was confirmed by the TGF‐β antagonist SB‐431542. Suppression of tumour‐derived PGE2 and TGF‐β restored TLR‐induced IFN‐α production of PDC. Additionally, PGE2‐ and TGF‐β‐treated PDC display a ‘tolerogenic’ phenotype because of a downregulation of CD40 accompanied by an upregulation of CD86. Finally, in TLR‐stimulated PDC, PGE2 and TGF‐β reduce the CCR7 : CXCR4 ratio, suggesting that PDC are impaired in their ability to migrate to tumour‐draining lymph nodes but are retained in stromal cell‐derived factor 1 (SDF‐1)‐expressing tissues. Based on these data, cyclooxygenase inhibitors and TGF‐β antagonists may improve TLR7‐ and TLR9‐based tumour immunotherapy.

Staphylococcus aureus-Induced Plasmacytoid Dendritic Cell Activation Is Based on an IgG-Mediated Memory Response

Type I IFNs represent a major antimicrobial defense mechanism due to their property of enhancing immune responses by priming both innate and adaptive immune cells. Plasmacytoid dendritic cells (pDC) are the major source of type I IFN in the human body and represent innate immune cells involved in first-line defense against invading pathogens. Although pDC activation has been extensively studied upon stimulation with synthetic TLR ligands, viruses, and intracellular bacteria, there is only scarce information on extracellular bacteria. In this study we show that the triggering of human pDC-derived IFN-α secretion by Staphylococcus aureus is independent of TLR2 and specific for coagulase-positive staphylococci. Specificity of the pDC response to S. aureus is independent of the bacterial virulence factors protein A and α-toxin but is mediated by Ag-specific IgG and CD32. S. aureus-induced pDC activation can be blocked by inhibitory DNA oligonucleotides and chloroquine, suggesting that engagement of TLR7/9 by bacterial nucleic acids after CD32-mediated uptake of these compounds may play a central role in this process. Altogether, we propose that in marked contrast to nonselective TLR2-dependent activation of most innate immune cells, pDC activation by S. aureus represents an Ag-specific memory response since it requires the presence of class-switched immunoglobulins.

Pathogen-Triggered Activation of Plasmacytoid Dendritic Cells Induces IL-10–Producing B Cells in Response to Staphylococcus aureus

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell–independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the VH3+ BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A–bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scidPrkdc/γc−/− mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell–derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell–mediated immune tolerance.

Impact of Rifaximin on the Frequency and Characteristics of Spontaneous Bacterial Peritonitis in Patients with Liver Cirrhosis and Ascites

Background Rifaximin is a non-absorbable antibiotic used to prevent relapses of hepatic encephalopathy which may also be a candidate for prophylaxis of spontaneous bacterial peritonitis (SBP). Aim To detect the impact of rifaximin on the occurrence and characteristics of SBP. Methods We prospectively studied all hospitalized patients that underwent a diagnostic paracentesis in our department from March 2012 to April 2013 for SBP and recorded all clinical data including type of SBP prophylaxis, prior use of rifaximin, concomitant complications of cirrhosis, as well as laboratory results and bacteriological findings. Patients were divided into the following three groups: no antibiotic prophylaxis, prophylaxis with rifaximin or with systemically absorbed antibiotic prophylaxis. Results Our study cohort comprised 152 patients with advanced liver cirrhosis, 32 of whom developed SBP during the study period. As expected, our study groups differed regarding a history of hepatic encephalopathy and SBP before inclusion into the study. None of the 17 patients on systemic antibiotic prophylaxis developed SBP while 8/27 patients on rifaximin and 24/108 without prophylaxis had SBP (p = 0.02 and p = 0.04 versus systemic antibiotics, respectively). In general, episodes of SBP were similar for patients treated with rifaximin and those without any prophylaxis. However, Escherichia coli and enterococci were dominant in the ascites of patients without any prophylaxis, while mostly klebsiella species were recovered from the ascites samples in the rifaximin group. Conclusion Rifaximin pretreatment did not lead to a reduction of SBP occurrence in hospitalized patients with advanced liver disease. However, the bacterial species causing SBP were changed by rifaximin.

Induction of Type I IFN Is a Physiological Immune Reaction to Apoptotic Cell-Derived Membrane Microparticles

Membrane microparticles (MMP) released from apoptotic cells deliver signals that secure the anti-inflammatory response beyond the nearest proximity of the apoptotic cell. Plasmacytoid dendritic cells (pDC) are sentinels prepared to detect cellular processes that endanger the organism. They play a key role in the regulation of both pro- and anti-inflammatory immune responses. Based on the assumption that pDC could participate in the initiation of the anti-inflammatory response to apoptotic cells, we investigated the effects of apoptotic cell-derived MMP on human pDC. The results obtained in our experiments confirmed that MMP released from apoptotic cells trigger IFN-α secretion from human pDC. They further suggest that pDC activation results from sensing of DNA contained in MMP. MMP-DNA displays a particularly strong stimulatory activity compared with MMP-RNA and other sources of DNA. Inhibition of MMP-induced IFN-α secretion by cytochalasin D, chloroquine, and an inhibitory G-rich oligodeoxynucleotide identify TLR9 as the receptor for MMP-DNA. In marked contrast to the pDC response in autoimmune patients, in healthy subjects MMP-mediated stimulation of pDC-derived IFN-α was found to be independent of FcγRIIA (CD32A). Based on our findings, we conclude that induction of pDC-derived IFN-α by MMP is a physiological event; future investigations are necessary to elucidate whether pDC activation promotes inflammation or propagates tolerance in the context of apoptotic cell clearance.

Analysis of Transmission of MRSA and ESBL-E among Pigs and Farm Personnel.

Livestock-associated bacteria with resistance to two or more antibiotic drug classes have heightened our awareness for the consequences of antibiotic consumption and spread of resistant bacterial strains in the veterinary field. In this study we assessed the prevalence of concomitant colonization with livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) and enterobacteriaceae expressing extended-spectrum betalactamases (ESBL-E) in farms at the German-Dutch border region. Nasal colonization of pigs with MRSA (113/547 (20.7%)) was less frequent than rectal colonization with ESBL-E (163/540 (30.2%)). On the individual farm level MRSA correlated with ESBL-E recovery. The data further provide information on prevalence at different stages of pig production, including abattoirs, as well as in air samples and humans living and working on the farms. Notably, MRSA was detected in stable air samples of 34 out of 35 pig farms, highlighting air as an important MRSA transmission reservoir. The majority of MRSA isolates, including those from humans, displayed tetracycline resistance and spa types t011 and t034 characteristic for LA-MRSA, demonstrating transmission from pigs to humans. ESBL-E positive air samples were detected on 6 out of 35 farms but no pig-to-human transmission was found. Detection of ESBL-E, e.g. mostly Escherichia coli with CTX-M-type ESBL, was limited to these six farms. Molecular typing revealed transmission of ESBL-E within the pig compartments; however, related strains were also found on unrelated farms. Although our data suggest that acquisition of MRSA and ESBL-E might occur among pigs in the abattoirs, MRSA and ESBL-E were not detected on the carcasses. Altogether, our data define stable air (MRSA), pig compartments (ESBL-E) and abattoir waiting areas (MRSA and ESBL-E) as major hot spots for transmission of MRSA and/or ESBL-E along the pig production chain.

Purified apoptotic bodies stimulate plasmacytoid dendritic cells to produce IFN-alpha

A characteristic feature of many autoimmune diseases like systemic lupus erythematodes (SLE) is the development of autoantibodies against various cytoplasmatic or nuclear antigens. The mechanisms leading to this autoantibody formation are not completely understood to date. Nevertheless several publications released during the last years point out that a dysregulation of apoptosis or an ineffective clearance of apoptotic cells contribute to the development of autoimmunity and to the formation of autoantibodies [1–3]. Our group has shown an increased rate of apoptosis in patients suffering from SLE. We observed an even stronger increase of apoptosis in SLE patients suffering from an active infectious disease (Figure 1) [4]. This data support the hypothesis that a dysregulation of apoptosis contributes to the development of autoimmunity in SLE patients. Moreover, an upregulation of IFN-alpha and IFNalpha related genes has been described in serum and cells from SLE patients [5–7]. To date, it is not known whether these two phenomena (increased apoptosis, IFN-alpha production) are linked. Some authors postulate that DNA containing immune complexes or ribonucleoprotein immune complexes can trigger the secretion of IFN-alpha in a TLR-9 or TLR-7 dependent manner [8,9]. Plasmacytoid dendritic cells (pDC) are the main producers of Type I interferons in peripheral blood. pDCs are a rare and unique population of cells, which were initially recognized for their role in antiviral immune responses. To date, it is generally accepted that these cells are involved in several immunological processes. pDCs can support B-cell antibody production, induce and regulate T-cell responses and they can influence cell trafficking [10,11]. In this context, we are interested whether apoptotic bodies isolated from dying lymphocytes are able to stimulate pDCs cells to produce IFN-alpha. Apoptotic bodies were isolated from activated lymphocytes after induction of apoptotis by UV-B irradiation. After co-incubation of apoptotic bodies with enriched pDCs, obtained from healthy normal donors, an enzyme linked immunosorbent assay was performed to quantitfy IFN alpha in the cellular supernatants. We observed an increased secretion of IFN-alpha after co-incubation of apoptotic blebs with pDCs (unpublished data, Figure 2). Taken together, these data indicate that a dysregulation of apoptosis and a disturbed clearance of apoptotic material and a dysregulation of apoptosis result in an accumulation of apoptotic bodies in blood and other tissues of autoimmune patients.

Identification of agr-positive methicillin-resistant Staphylococcus aureus harbouring the class A mec complex by MALDI-TOF mass spectrometry

A small peptide called PSM-mec is encoded on the type II, III and VIII SCCmec cassettes present in the genomes of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains. This peptide is excreted by agr-positive strains, which represent about 89% of the strains of our collection and can be identified by the presence of delta toxin in mass spectrometry. The presence of the peptide in the MALDI-TOF MS spectra of whole cells was proved by a knock-down experiment employing a clone that expressed antisense RNA to psm-mec. Furthermore, evaluation of a collection of clinical agr-positive MRSA and MSSA isolates and type strains showed that, using a detection window of m/z 2411-2419, the PSM-mec is detected by mass spectrometry of whole cells with a sensitivity of 0.95 and a specificity of 1, thereby enabling rapid identification of a subgroup of MRSA with a method that is used during routine identification procedures.

Poke weed mitogen requires Toll-like receptor ligands for proliferative activity in human and murine B lymphocytes.

Poke weed mitogen (PWM), a lectin purified from Phytolacca americana is frequently used as a B cell-specific stimulus to trigger proliferation and immunoglobulin secretion. In the present study we investigated the mechanisms underlying the B cell stimulatory capacity of PWM. Strikingly, we observed that highly purified PWM preparations failed to induce B cell proliferation. By contrast, commercially available PWM preparations with B cell activity contained Toll-like receptor (TLR) ligands such as TLR2-active lipoproteins, lipopolysaccharide and DNA of bacterial origin. We show that these microbial substances contribute to the stimulatory activity of PWM. Additional experimental data highlight the capacity of PWM to enable B cell activation by immunostimulatory DNA. Based on these findings we propose that the lectin sensitizes B cells for TLR stimulation as described for B cell receptor ligation and that B cell mitogenicity of PWM preparations results from synergistic activity of the poke weed lectin and microbial TLR ligands present in the PWM preparations.

Heterogeneity of Host TLR2 Stimulation by Staphylocoocus aureus Isolates

High lipoprotein expression and potent activation of host Toll-like receptor-2 (TLR2) are characteristic features of the staphylococcal species. Expression of TLR2 in the host is important for clearance of Staphylococcus aureus infection and host survival. Thus, we hypothesized that bacterial regulation of its intrinsic TLR2-stimulatory capacity could represent a means for immune evasion or host adaptation. We, therefore, compared clinical S. aureus isolates in regards to their TLR2 activation potential and assessed the bacterial factors that modulate TLR2-mediated recognition. S. aureus isolates displayed considerable variability in TLR2-activity with low to absent TLR2-activity in 64% of the isolates tested (68/106). Notably, strain-specific TLR2-activity was independent of the strain origin, e.g. no differences were found between strains isolated from respiratory specimen from cystic fibrosis patients or those isolated from invasive disease specimen. TLR2-activity correlated with protein A expression but not with the agr status. Capsule expression and small colony variant formation had a negative impact on TLR2-activity but any disruption of cell wall integrity enhanced TLR2 activation. Altogether, heterogeneity in host TLR2-activity reflects differences in metabolic activity and cell wall synthesis and/or remodeling.