Katrin Peter

Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease.

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 μmol/L to 11.8 ± 2.8 μmol/L in all post-transplant samples and to 3.5 ± 3 μmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.

Transcription and enhancer profiling in human monocyte subsets

Human blood monocytes comprise at least 3 subpopulations that differ in phenotype and function. Here, we present the first in-depth regulome analysis of human classical (CD14(++)CD16(-)), intermediate (CD14(+)CD16(+)), and nonclassical (CD14(dim)CD16(+)) monocytes. Cap analysis of gene expression adapted to Helicos single-molecule sequencing was used to map transcription start sites throughout the genome in all 3 subsets. In addition, global maps of H3K4me1 and H3K27ac deposition were generated for classical and nonclassical monocytes defining enhanceosomes of the 2 major subsets. We identified differential regulatory elements (including promoters and putative enhancers) that were associated with subset-specific motif signatures corresponding to different transcription factor activities and exemplarily validated novel downstream enhancer elements at the CD14 locus. In addition to known subset-specific features, pathway analysis revealed marked differences in metabolic gene signatures. Whereas classical monocytes expressed higher levels of genes involved in carbohydrate metabolism, priming them for anaerobic energy production, nonclassical monocytes expressed higher levels of oxidative pathway components and showed a higher mitochondrial routine activity. Our findings describe promoter/enhancer landscapes and provide novel insights into the specific biology of human monocyte subsets.

Metabolic Hallmarks of Tumor and Immune Cells in the Tumor Microenvironment

Cytotoxic T lymphocytes and NK cells play an important role in eliminating malignant tumor cells and the number and activity of tumor-infiltrating T cells represent a good marker for tumor prognosis. Based on these findings, immunotherapy, e.g., checkpoint blockade, has received considerable attention during the last couple of years. However, for the majority of patients, immune control of their tumors is gray theory as malignant cells use effective mechanisms to outsmart the immune system. Increasing evidence suggests that changes in tumor metabolism not only ensure an effective energy supply and generation of building blocks for tumor growth but also contribute to inhibition of the antitumor response. Immunosuppression in the tumor microenvironment is often based on the mutual metabolic requirements of immune cells and tumor cells. Cytotoxic T and NK cell activation leads to an increased demand for glucose and amino acids, a well-known feature shown by tumor cells. These close metabolic interdependencies result in metabolic competition, limiting the proliferation, and effector functions of tumor-specific immune cells. Moreover, not only nutrient restriction but also tumor-driven shifts in metabolite abundance and accumulation of metabolic waste products (e.g., lactate) lead to local immunosuppression, thereby facilitating tumor progression and metastasis. In this review, we describe the metabolic interplay between immune cells and tumor cells and discuss tumor cell metabolism as a target structure for cancer therapy. Metabolic (re)education of tumor cells is not only an approach to kill tumor cells directly but could overcome metabolic immunosuppression in the tumor microenvironment and thereby facilitate immunotherapy.

Tryptophan catabolism is associated with acute GVHD after human allogeneic stem cell transplantation and indicates activation of indoleamine 2,3-dioxygenase

Induction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway, acts as a potent immunoregulatory loop. To address its role in human allogeneic stem cell transplantation, we measured major tryptophan metabolites, such as quinolinic acid and kynurenine, in serial urine specimens from 51 patients by liquid chromatography-tandem mass spectrometry. Samples were collected between admission and day 90 after transplantation, and metabolite levels were correlated with early clinical events and outcome. In selected patients, IDO gene expression was assessed by quantitative RT-PCR in intestinal biopsies. Surviving patients had significantly lower metabolite levels on days 28, 42, and 90, respectively, compared with patients dying of GVHD and associated complications (n = 10). Kynurenine levels were directly correlated with severity and clinical course of GVHD: Mean urinary quinolinic acid levels were 4.5 ± 0.3 μmol/mmol creatinine in the absence of acute GVHD, 8.0 ± 1.1 μmol/mmol creatinine for GVHD grade 1 or 2, and 13.5 ± 2.7 μmol/mmol creatinine for GVHD grade 3 or 4 (P < .001), respectively. GVHD-dependent induction of IDO was further suggested by increased expression of IDO mRNA in intestinal biopsies from patients with severe GVHD. Our data indicate reactive release of kynurenines in GVHD-associated inflammation.

Lactic acid delays the inflammatory response of human monocytes

Lactic acid (LA) accumulates under inflammatory conditions, e.g. in wounds or tumors, and influences local immune cell functions. We previously noted inhibitory effects of LA on glycolysis and TNF secretion of human LPS-stimulated monocytes. Here, we globally analyze the influence of LA on gene expression during monocyte activation. To separate LA-specific from lactate- or pH-effects, monocytes were treated for one or four hours with LPS in the presence of physiological concentrations of LA, sodium lactate (NaL) or acidic pH. Analyses of global gene expression profiles revealed striking effects of LA during the early stimulation phase. Up-regulation of most LPS-induced genes was significantly delayed in the presence of LA, while this inhibitory effect was attenuated in acidified samples and not detected after incubation with NaL. LA targets included genes encoding for important monocyte effector proteins like cytokines (e.g. TNF and IL-23) or chemokines (e.g. CCL2 and CCL7). LA effects were validated for several targets by quantitative RT-PCR and/or ELISA. Further analysis of LPS-signaling pathways revealed that LA delayed the phosphorylation of protein kinase B (AKT) as well as the degradation of IκBα. Consistently, the LPS-induced nuclear accumulation of NFκB was also diminished in response to LA. These results indicate that the broad effect of LA on gene expression and function of human monocytes is at least partially caused by its interference with immediate signal transduction events after activation. This mechanism might contribute to monocyte suppression in the tumor environment.

Metabolic plasticity of human T cells: Preserved cytokine production under glucose deprivation or mitochondrial restriction, but 2-deoxy-glucose affects effector functions

The strong link between T‐cell metabolism and effector functions is well characterized in the murine system but hardly investigated in human T cells. Therefore, we analyzed glycolytic and mitochondrial activity in correlation to function in activated human CD4 and CD8 T cells. Glycolysis was barely detectable upon stimulation but accelerated beyond 24 h, whereas mitochondrial activity was elevated immediately in both T‐cell populations. Glucose deprivation or mitochondrial restriction reduced proliferation, had only a transient impact on “on‐blast formation” and no impact on viability, IFN‐γ, IL‐2, IL‐4, and IL‐10 production, whereas TNF was reduced. Similar results were obtained in bulk T cells and T‐cell subsets. Elevated respiration under glucose restriction demonstrated metabolic flexibility. Administration of the glycolytic inhibitor 2‐deoxy‐glucose suppressed both glycolysis and respiration and exerted a strong impact on cytokine production that persisted for IFN‐γ after removal of 2‐deoxy‐glucose. Taken together, glycolytic or mitochondrial restriction alone compromised proliferation of human T cells, but barely affected their effector functions. In contrast, effector functions were severely affected by 2‐deoxy‐glucose treatment.

Pioglitazone modulates tumor cell metabolism and proliferation in multicellular tumor spheroids

The anti-diabetic thiazolidinedione compound pioglitazone, a peroxisome proliferator-activated receptor-gamma agonist, and selective cyclooxygenase-2 inhibitors are clinically used in patients with advanced malignancies. Several previously published in vivo and in vitro studies showed growth inhibitory effects on different cancer cell lines. However, the underlying mechanisms are fairly unclear. Here, we analyzed the effects of pioglitazone in combination with other drugs in a three-dimensional multicellular tumor spheroid culture system (MCTS) generated from the two prostate carcinoma cell lines PC3 and LNCaP. As expected, pioglitazone also inhibited tumor cell proliferation in the MCTS system. Further studies revealed that pioglitazone lowered the pH of the culture medium, decreased oxygen consumption and increased lactate secretion in both tumor cell lines. Other glitazones, troglitazone and ciglitazone, had similar effects. The combination of pioglitazone with 2-deoxyglucose, a potent inhibitor of glycolysis, had an additive effect on the inhibition of cell proliferation and led to MCTS disintegration. Our data propose a new mechanism of growth inhibition by pioglitazone through modulation of the tumor cell metabolism.

Suppressive effects of tumor cell-derived 5′-deoxy-5′-methylthioadenosine on human T cells

ABSTRACT The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5′-deoxy-5′-methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTA-catabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAP-coding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting.

The association between acute graft-versus-host disease and antimicrobial peptide expression in the gastrointestinal tract after allogeneic stem cell transplantation

Intestinal microbiota disruption is associated with acute gastrointestinal (GI) Graft-versus-Host Disease (GvHD) and poor outcome after allogeneic stem cell transplantation (ASCT). Here, in a retrospective analysis of 200 patients undergoing ASCT at the Regensburg University Medical Center, we assessed the relative expression of Paneth cell antimicrobial peptides (AMPs), Human Defensins (HD) 5 and 6 and regenerating islet-derived 3α (Reg3α), in 292 human intestinal biopsies as well as Reg3α serum levels in relation to acute GI GvHD. In the absence of GI GvHD, the relative expression of Paneth cell AMPs was significantly higher in the small intestine (duodenum to ileum) than in the stomach and large intestine (cecum to rectum) for Reg3α (p≤0.001), HD5 (p≤0.002) and HD6 (p≤0.02). Acute stage 2–4 GI GvHD was associated with reduced expression of AMPs in the small intestine (p≤0.01) in comparison to stage 0–1 disease, accompanied by a decrease in Paneth cell count in case of severe acute GI GvHD (p<0.001). The opposite held true for the large intestine as we found stage 2–4 GI GvHD correlated with significantly higher expression of HD5, HD6, and Reg3α compared to mild or no acute GI GvHD (p≤0.002). Severe GI GvHD in both the lower and the upper GI tract also correlated with higher serum concentrations of Reg3α (p = 0.002). As indirect markers of intestinal microbiome diversity low levels of urinary 3-indoxyl sulfate levels were associated with severe stages of acute GI GvHD compared to mild stage or no acute GI GvHD (p = 0.05). In conclusion, acute GI GvHD correlates with intestinal expression of HD5, HD6 and Reg3α as well as Reg3α serum levels and is associated with intestinal dysbiosis.

Indoxyl 3-sulfate inhibits maturation and activation of human monocyte-derived dendritic cells

Indole is produced from l-tryptophan by commensal bacteria and further metabolized to indoxyl 3-sulfate (I3S) in the liver. Physiologic concentrations of I3S are related to a lower risk to develop graft versus host disease in allogeneic stem cell transplanted patients pointing towards an immunoregulatory function of I3S. Here we investigated the impact of I3S on the maturation of human monocyte-derived dendritic cells (DCs). Even pathophysiologic concentrations of I3S did not affect viability of mature DCs, but I3S decreased the expression of co-stimulatory molecules such as CD80 and CD86 on mature DCs. Furthermore, I3S inhibited IL-12 and IL-6 secretion by mature DCs while IL-10 was significantly upregulated. Co-culture of I3S-treated mature DCs with allogeneic T cells revealed no alteration in T cell proliferation. However, interferon gamma and TNF production of T cells was suppressed. As I3S exerted no direct effect on T cells, the defect in T cell activation was mediated by I3S-treated mature DCs. Our study suggests an anti-inflammatory and tolerizing effect of I3S on human DCs.