Tumor-assoziierte neutrophile Granulozyten während Pseudomonas aeruginosa-vermittelter Tumortherapie

Since the i.v. application of Pseudomonas aeruginosa lead to tumor clearance in WT mice, the quality of differentially primed CD4 and CD8 T cells to specifically act against tumor cells was investigated by adoptive transfer experiments in syngeneic Rag1-/- animals. Thereby, T cells isolated out of uninfected tumor-bearing WT mice allowed the complete rejection of new tumors, when a combination of such CD4 + CD8 T cells was injected into the recipient mice. Thereby it was demonstrated, that also uninfected tumor-bearing mice possess tumor-antigen specific T cells, which are nevertheless not able to reject established tumors. The adjuvant effect of the bacteria seemed to be necessary to enable tumor clearance in WT animals. However, when T cells were isolated out of WT mice, which had cleared CT26 tumors after infection with P. aeruginosa, the application of CD8 T cells alone prevented the development of tumors on modified Rag1 mice. By that, the development of a T cell memory and functional specificity was revealed. The impact of the bacterial infection on functional specializations of tumor-associated neutrophils (TANs) in the tumor microenvironment was furthermore investigated. Therefore, the expression of the extracellular phenotyping markers Icam, Fas, and CD62L, as well as the production of the cytokines TNF-alpha and active caspase 3, analyzed as intracellular phenotyping markers, was determined. The analyses revealed phenotype switches of the respective TAN population from N2 to N1 after the i.v. injection of P. aeruginosa into tumor-bearing WT- as well as Rag1-/- mice. The most and maximal significant changes of the percental number of TANs expressing a certain phenotype marker were observed three days post infection of tumor-bearing Rag1-/- mice. By the application of P. aeruginosa into tumor-bearing mice, tumor-supportive N2 TANs were obviously manipulated to switch into tumor-suppressing N1 TANs. Now, defined functions of N1 TANs can be investigated in future experiments and their onset in the present model probably induced by the bacteria-mediated tumor therapy.Da nach der i.v. Anwendung von Pseudomonas aeruginosa die Tumore auf den WT-Mäuse verschwanden, wurden unterschiedlich aktivierte CD4 und CD8 T-Zellen daraufhin untersucht, wie spezifisch sie gegen Tumorzellen agieren. Dafür wurden adoptive Transferexperimente in genidentischen Rag1-/- Mäuse durchgeführt. T-Zellen die aus unifizierten tumortragenden WT-Mäusen isoliert wurden ermöglichten eine komplette Abstoßung neuer Tumore, wenn eine Kombination aus solchen CD4 + CD8 T-Zellen in die Rezipienten-Mäuse injiziert wurde. Dadurch konnte gezeigt werden, dass auch uninfizierte tumortragende Mäuse Tumorantigen-spezifische T-Zellen besitzen. Diese waren allerdings nicht dazu fähig bereits ausgebildete Tumore zu beseitigen. Der adjuvante Effekt der Bakterien schien daher für eine solche Beseitigung von Tumoren in WT-Mäusen notwendig zu sein. Wenn dahingegen T-Zellen aus WT-Mäusen isoliert wurden, welche die Tumore nach der Infektion mit P. aeruginosa beseitigt hatten, war die Anwendung von ausschließlich CD8 T-Zellen ausreichend um die Entwicklung neuer Tumore in den modifizierten Rag1-Mäusen zu verhindern. Dadurch konnte das Bestehen von Gedächtnis-T-Zellen und deren funktionelle Spezifität bestätigt werden. Ebenfalls wurde der Einfluss der bakteriellen Infektion auf die funktionale Spezialisierung von Tumor-assoziierten Neutrophilen (TANs) im Tumormikromilieu untersucht. Dazu wurde die Expression der extrazellulären Phänotypisierungsmarker Icam, Fas und CD62L, sowie die Produktion der Zytokine TNF-alpha und aktive Caspase 3, die als intrazelluläre Phänotypisierungs-marker analysiert wurden, bestimmt. Die Analysen zeigten einen Phänotyp-Wechsel der jeweiligen TAN-Population von N2 zu N1 nach der i.v. Injektion von P. aeruginosa in tumortragenden WT- als auch Rag1-/--Mäusen an. Mittels der bakteriellen Injektion wurden Tumor-unterstützende N2 TANs offensichtlich dazu gebracht sich in Tumor-hemmende N1 TANs umzuwandeln. Nun können bestimmte Funktionen von N1 TANs in zukünftigen Experimenten untersucht und ihre Aktivierung wahrscheinlich mittels der Bakterien-vermittelten Tumortherapie im vorliegenden Model induziert werden

Composing a Tumor Specific Bacterial Promoter.

Systemically applied Salmonella enterica spp. have been shown to invade and colonize neoplastic tissues where it retards the growth of many tumors. This offers the possibility to use the bacteria as a vehicle for the tumor specific delivery of therapeutic molecules. Specificity of such delivery is solely depending on promoter sequences that control the production of a target molecule. We have established the functional structure of bacterial promoters that are transcriptionally active exclusively in tumor tissues after systemic application. We observed that the specific transcriptional activation is accomplished by a combination of a weak basal promoter and a strong FNR binding site. This represents a minimal set of control elements required for such activation. In natural promoters, additional DNA remodeling elements are found that alter the level of transcription quantitatively. Inefficiency of the basal promoter ensures the absence of transcription outside tumors. As a proof of concept, we compiled an artificial promoter sequence from individual motifs representing FNR and basal promoter and showed specific activation in a tumor microenvironment. Our results open possibilities for the generation of promoters with an adjusted level of expression of target proteins in particular for applications in bacterial tumor therapy

Identification and analysis steps of tumor specific promoters.

<p>A schematic overview. Two programs for basal promoter recognition, one based on sequence alignment kernel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.ref011" target="_blank">11</a>] and another on Hidden Markov Model (HMM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.ref012" target="_blank">12</a>] were applied to the TSP set (DNA sequences are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.s006" target="_blank">S1 Text</a>). As a negative control, a set of DNA fragments that do not initiate expression either in tumor or in spleen was selected (negative promoters, NP). Both programs recognize potential promoters in either dataset and the number of predictions greatly depends on user-defined threshold parameters. To evaluate the specificity of predictions, it was assumed that a basal promoter should be recognized in at least 75% of TSP and at most 50% of NPs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#sec014" target="_blank">Methods</a>). This will ensure the generality and specificity of the recognized feature.</p

Schematic representation of artificial promoters and their expression in tumor environment.

<p>Promoters were compiled by introduction of the respective elements taken from promoters P0.212, P1.6, P0.134 as well as consensus sequences into the template sequence. Additionally, regions around TSS were enriched for A/T to facilitate DNA melting. Expression values are relative to the expression of P0.212_1. Given are mean±SD. Only promoter P2.3 showed expression in tumor. No expression in tumor or spleen could be observed for the other promoters. Nucleotide sequences are presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.s003" target="_blank">S3 Fig</a>. Foot note: <b>I, II, III</b>—corresponding sequences were taken from promoters P0.212, P1.6, P0.134 respectively. <b>cons</b>—consensus sequences for the corresponding elements. <b>a, b</b>—modification of loci around putative start of transcription to facilitate DNA melting by random substitution of "C/G"s by "A/T"s.</p

Expression of promoters P1.6 and P0.212_1 in tumor, spleen and liver.

<p>Homogenates were analyzed via two color flow cytometry and plating to allow normalization. Given are mean and SD. Expression of P1.6 in spleen and liver can be considered negligible compared to expression in tumor. Therefore expression of promoter P1.6 was accepted as tumor specific.</p

Schematic representation of promoter structure.

<p>Left: binding motifs for factors TGIF, FNR and NagC are shown in green, yellow and brown, respectively. Basal promoter is shown as a directed arrow. Knockout of essential nucleotides within motifs were according to the literature and are represented by crossing lines. Mutation of non-essential nucleotides within motifs was random and is represented by dashed crossing lines. Nucleotides outside motifs were mutated randomly. Right: representative flow-cytometric analyses of GFP-expression in tumor and spleen. Each green point on the blots represents GFP expression levels of an individual bacterial cell. Displayed values of expression are relative to the expression values of the original promoter P0.212_1. All nucleotide substitutions are presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155338#pone.0155338.s002" target="_blank">S2 Fig</a>.</p

Induction of CD4(+) and CD8(+) anti-tumor effector T cell responses by bacteria mediated tumor therapy.

Facultative anaerobic bacteria like E. coli can colonize solid tumors often resulting in tumor growth retardation or even clearance. Little mechanistic knowledge is available for this phenomenon which is however crucial for optimization and further implementation in the clinic. Here, we show that intravenous injections with E. coli TOP10 can induce clearance of CT26 tumors in BALB/c mice. Importantly, re-challenging mice which had cleared tumors showed that clearance was due to a specific immune reaction. Accordingly, lymphopenic mice never showed tumor clearance after infection. Depletion experiments revealed that during induction phase, CD8(+) T cells are the sole effectors responsible for tumor clearance while in the memory phase CD8(+) and CD4(+) T cells were involved. This was confirmed by adoptive transfer. CD4(+) and CD8(+) T cells could reject newly set tumors while CD8(+) T cells could even reject established tumors. Detailed analysis of adoptively transferred CD4(+) T cells during tumor challenge revealed expression of granzyme B, FasL, TNF-α and IFN-γ in such T cells that might be involved in the anti-tumor activity. Our findings should pave the way for further optimization steps of this promising therapy

In Vivo Efficacy of Antimicrobials against Biofilm-Producing Pseudomonas aeruginosa.

Patients suffering from cystic fibrosis (CF) are commonly affected by chronic Pseudomonas aeruginosa biofilm infections. This is the main cause for the high disease severity. In this study, we demonstrate that P. aeruginosa is able to efficiently colonize murine solid tumors after intravenous injection and to form biofilms in this tissue. Biofilm formation was evident by electron microscopy. Such structures could not be observed with transposon mutants, which were defective in biofilm formation. Comparative transcriptional profiling of P. aeruginosa indicated physiological similarity of the bacteria in the murine tumor model and the CF lung. The efficacy of currently available antibiotics for treatment of P. aeruginosa-infected CF lungs, such as ciprofloxacin, colistin, and tobramycin, could be tested in the tumor model. We found that clinically recommended doses of these antibiotics were unable to eliminate wild-type P. aeruginosa PA14 while being effective against biofilm-defective mutants. However, colistin-tobramycin combination therapy significantly reduced the number of P. aeruginosa PA14 cells in tumors at lower concentrations. Hence, we present a versatile experimental system that is providing a platform to test approved and newly developed antibiofilm compounds

The Interferon-Induced Gene <i>Ifi27l2a</i> is Active in Lung Macrophages and Lymphocytes After Influenza A Infection but Deletion of <i>Ifi27l2a</i> in Mice Does Not Increase Susceptibility to Infection

<div><p>Interferons represent one of the first and essential host defense mechanisms after infection, and the activation of the IFN-pathway results in the transcriptional activation of hundreds of interferon-stimulated genes. The alpha-inducible protein 27 like 2A (<i>Ifi27l2a</i>) gene (human synonym: <i>ISG12</i>) is strongly up-regulated in the lung after influenza A infection in mice and has been shown in gene expression studies to be highly correlated to other activated genes. Therefore, we investigated the role of <i>Ifi27l2a</i> for the host defense to influenza A infections in more detail. RT-PCR analyses in non-infected mice demonstrated that <i>Ifi27l2a</i> was expressed in several tissues, including the lung. Detailed analyses of reporter gene expression in lungs from <i>Ifi27l2a-LacZ</i> mice revealed that <i>Ifi27l2a</i> was expressed in macrophages and lymphocytes but not in alveolar cells or bronchiolar epithelium cells. The number of macrophages and lymphocyte strongly increased in the lung after infection, but no significant increase in expression levels of the LacZ reporter gene was found within individual immune cells. Also, no reporter gene expression was found in bronchiolar epithelial cells, alveolar cells or infiltrating neutrophils after infection. Thus, up-regulation of <i>Ifi27l2a</i> in infected lungs is mainly due to the infiltration of macrophages and lymphocytes. Most surprisingly, deletion of <i>Ifi27l2a</i> in mouse knock-out lines did not result in increased susceptibility to infections with H1N1 or H7N7 influenza A virus compared to wild type C57BL/6N mice, suggesting a less important role of the gene for the host response to influenza infections than for bacterial infections.</p></div

Immunofluorescent analysis of LacZ reporter gene expression and cell-specific marker genes.

<p>Lungs of PR8 infected (2×10⁵ FFU) <i>Ifi27l2a^-/-</i> mice were prepared three days post infection and cryo-sections were co-stained with antibodies against Gr1 (A), CD11b (B) and LacZ (C). In the overlay picture, co-localization of LacZ and CD11b-stained macrophages was frequently observed (D). For CD11b/Gr1 double-positive neutrophils no co-immunostaining with LacZ could be detected.</p