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Local tissue acidosis affects anti-tumor immunity. In contrast, data on tissue pH levels in infected tissues and their impact on antimicrobial activity is sparse. In this study, we assessed the pH levels in cutaneous

With the increased interest in the microbiome research, gnotobiotic animals and techniques emerged again as valuable tools to investigate functional effects of host-microbe and microbe-microbe interactions. The increased demand for gnotobiotic experiments has resulted in the greater need for housing systems for short-term maintenance of gnotobiotic animals. During the last six years, the gnotobiotic facility of the Hannover Medical School has worked intensively with different housing systems for gnotobiotic animals. Here, we report our experience in handling, contamination incidence, and monitoring strategies that we apply for controlling gnotobiotic experiments. From our experience, the risk of introducing contaminants to animals housed in microisolator cages is higher than in isolators. However, with strict operating protocols, the contamination rate in these systems can be minimized. In addition to spore-forming bacteria and fungi from the environment, spore-forming bacteria from defined bacterial communities used in experiments represent the major risk for contamination of gnotobiotic experiments performed in microisolator cages. The presence/absence of contaminants in germ-free animals can be easily monitored by preparation of wet mounts and Gram staining of fecal samples. Contaminants in animals colonized with specific microorganisms need to be tracked with methods such as next-generation sequencing. However, when using PCR-based methods it is important to consider that relatively small amounts of bacterial DNA detected likely originates from food, bedding, or reagents and is not to be interpreted as true contamination.

CD83 is a maturation marker for dendritic cells. In the B cell lineage, CD83 is expressed especially on activated B cells and on light zone B cells during the germinal center (GC) reaction. The function of CD83 during GC responses is unclear. CD83−/− mice have a strong reduction of CD4+ T cells, which makes it difficult to analyze a functional role of CD83 on B cells during GC responses. Therefore, in the present study we generated a B cell–specific CD83 conditional knockout (CD83 B-cKO) model. CD83 B-cKO B cells show defective upregulation of MHC class II and CD86 expression and impaired proliferation after different stimuli. Analyses of GC responses after immunization with various Ags revealed a characteristic shift in dark zone and light zone B cell numbers, with an increase of B cells in the dark zone of CD83 B-cKO mice. This effect was not accompanied by alterations in the level of IgG immune responses or by major differences in affinity maturation. However, an enhanced IgE response was observed in CD83 B-cKO mice. Additionally, we observed a strong competitive disadvantage of CD83-cKO B cells in GC responses in mixed bone marrow chimeras. Furthermore, infection of mice with Borrelia burgdorferi revealed a defect in bacterial clearance of CD83 B-cKO mice with a shift toward a Th2 response, indicated by a strong increase in IgE titers. Taken together, our results show that CD83 is important for B cell activation and modulates GC composition and IgE Ab responses in vivo.

Analyzing human as well as animal microbiota composition has gained growing interest because structural components and metabolites of microorganisms fundamentally influence all aspects of host physiology. Originally dominated by culture-dependent methods for exploring these ecosystems, the development of molecular techniques such as high throughput sequencing has dramatically increased our knowledge. Because many studies of the microbiota are based on the bacterial 16S ribosomal RNA (rRNA) gene targets, they can, at least in principle, be compared to determine the role of the microbiome composition for developmental processes, host metabolism, and physiology as well as different diseases. In our review, we will summarize differences and pitfalls in current experimental protocols, including all steps from nucleic acid extraction to bioinformatical analysis which may produce variation that outweighs subtle biological differences. Future developments, such as integration of metabolomic, transcriptomic, and metagenomic data sets and standardization of the procedures, will be discussed.

Jun activation domain-binding protein 1 (JAB1) regulates ubiquitin-dependent protein degradation by deneddylation of cullin-based ubiquitin ligases and, therefore, plays a central role in regulating proliferation and apoptosis. Because these processes are decisive for B cell development, we investigated JAB1 functions in B cells by establishing a mouse strain with a B cell-specific JAB1 deletion. We show that JAB1 is essential for early B cell development, because the ablation of JAB1 expression blocks B cell development between the pro-B and pre-B cell stages. Furthermore, JAB1 deletion leads to aberrant expression of the apoptosis-triggering protein Fas ligand in pro-B cells. Concomitant B cell-specific overexpression of the antiapoptotic protein Bcl2 partially reverses the block in B cell development; rescued JAB1-deficient B cells reach the periphery and produce protective class-switched Abs after Borrelia burgdorferi infection. Interestingly, B cell-rescued mice exhibit no germinal centers but a striking extrafollicular plasma cell accumulation. In addition, JAB1 is essential for Bcl6 expression, a transcriptional repressor required for germinal center formation. These findings identify JAB1 as an important factor in checkpoint control during early B cell development, as well as in fate decisions in mature Ag-primed B cells.

HIF1A is a transcription factor that plays a central role for the adaptation to tissue hypoxia and for the inflammatory response of myeloid cells, including DCs. HIF1A is stabilized by hypoxia but also by TLR ligands under normoxic conditions. The underlying signaling events leading to the accumulation of HIF1A in the presence of oxygen are still poorly understood. Here, we show that in contrast to hypoxic stabilization of HIF1A, normoxic, TLR-mediated HIF1A accumulation in DCs follows a different pathway that predominantly requires MYD88-dependent NF-κB activity. The TLR-induced HIF1A controls a subset of proinflammatory genes that are insufficiently induced following hypoxia-mediated HIF1A induction. Thus, TLR activation and hypoxia stabilize HIF1A via distinct signaling pathways, resulting in differential HIF1A-dependent gene expression.

Dendritic cells (DC) play a key role in linking innate and adaptive immunity. In inflamed tissues, where DC become activated, oxygen tensions are usually low. Although hypoxia is increasingly recognized as an important determinant of cellular functions, the consequences of hypoxia and the role of one of the key players in hypoxic gene regulation, the transcription factor hypoxia inducible factor 1α (HIF-1α), are largely unknown. Thus, we investigated the effects of hypoxia and HIF-1α on murine DC activation and function in the presence or absence of an exogenous inflammatory stimulus. Hypoxia alone did not activate murine DC, but hypoxia combined with LPS led to marked increases in expression of costimulatory molecules, proinflammatory cytokine synthesis, and induction of allogeneic lymphocyte proliferation compared with LPS alone. This DC activation was accompanied by accumulation of HIF-1α protein levels, induction of glycolytic HIF target genes, and enhanced glycolytic activity. Using RNA interference techniques, knockdown of HIF-1α significantly reduced glucose use in DC, inhibited maturation, and led to an impaired capability to stimulate allogeneic T cells. Alltogether, our data indicate that HIF-1α and hypoxia play a crucial role for DC activation in inflammatory states, which is highly dependent on glycolysis even in the presence of oxygen.

Background Next-generation 16S ribosomal RNA gene sequencing is widely used to determine the relative composition of the mammalian gut microbiomes. However, in the absence of a reference, this does not reveal alterations in absolute abundance of specific operational taxonomic units if microbial loads vary across specimens. Results Here we suggest the spiking of exogenous bacteria into crude specimens to quantify ratios of absolute bacterial abundances. We use the 16S rDNA read counts of the spike-in bacteria to adjust the read counts of endogenous bacteria for changes in total microbial loads. Using a series of dilutions of pooled faecal samples from mice containing defined amounts of the spike-in bacteria Salinibacter ruber, Rhizobium radiobacter and Alicyclobacillus acidiphilus, we demonstrate that spike-in-based calibration to microbial loads allows accurate estimation of ratios of absolute endogenous bacteria abundances. Applied to stool specimens of patients undergoing allogeneic stem cell transplantation, we were able to determine changes in both relative and absolute abundances of various phyla, especially the genus Enterococcus, in response to antibiotic treatment and radio-chemotherapeutic conditioning. Conclusion Exogenous spike-in bacteria in gut microbiome studies enable estimation of ratios of absolute OTU abundances, providing novel insights into the structure and the dynamics of intestinal microbiomes. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0175-0) contains supplementary material, which is available to authorized users.

Analyzing human as well as animal microbiota composition has gained growing interest because structural components and metabolites of microorganisms fundamentally influence all aspects of host physiology. Originally dominated by culture-dependent methods for exploring these ecosystems, the development of molecular techniques such as high throughput sequencing has dramatically increased our knowledge. Because many studies of the microbiota are based on the bacterial 16S ribosomal RNA (rRNA) gene targets, they can, at least in principle, be compared to determine the role of the microbiome composition for developmental processes, host metabolism, and physiology as well as different diseases. In our review, we will summarize differences and pitfalls in current experimental protocols, including all steps from nucleic acid extraction to bioinformatical analysis which may produce variation that outweighs subtle biological differences. Future developments, such as integration of metabolomic, transcriptomic, and metagenomic data sets and standardization of the procedures, will be discussed.

Myeloid differentiation protein 88 (MyD88) is a general adaptor for the signaling cascade through receptors of the Toll/IL-1R family. When infected withLeishmania majorparasites, MyD88-deficient mice displayed a dramatically enhanced parasite burden in their tissues similar to that found in susceptible BALB/c mice. In contrast, MyD88 knockout mice did not develop ulcerating lesions despite a lack of interleukin-12 (IL-12) production and a predominant T helper 2 cell response. Blockade of IL-4 produced early (day 1) after infection restored a protective T helper 1 response in MyD88 knockout mice.

Background Staphylococcus aureus is a common pathogen among humans worldwide, with an increasing prevalence of multidrug resistance. The understanding of virulence factors inducing pathogenicity is still incomplete, and thus far the transfer of results from animal studies into successful clinical trials has been difficult. Methods In this study, we established an S. aureus infection model in mice engrafted with a human immune system, compared it with infected wild-type and nonhumanized mice, and investigated pathogenesis in these models. Results Staphylococcus aureus infection was aggravated in humanized mice, compared with wild-type or nonengrafted mice. The humanized mice displayed a significantly reduced survival percentage, increased weight loss, and a more-rapid increase in bacterial burden. In addition, S. aureus infection induced T-cell activation, apoptosis, and Fas receptor expression in humanized but not wild-type mice. Conclusions Our findings demonstrate the different pathogenetic mechanisms in wild-type and humanized mice and the possible benefit of including humanized mice in future studies involving S. aureus as a prior step to human clinical trials.

After infection with Borrelia burgdorferi, humans and mice under certain conditions develop arthritis. Initiation of inflammation is dependent on the migration of innate immune cells to the site of infection, controlled by interactions of a variety of adhesion molecules. In this study, we used the newly synthesized compound S18407, which is a prodrug of the active drug S16197, to analyze the functional importance of α4β1-dependent cell adhesion for the development of arthritis and for the antibacterial immune response. S16197 is shown to interfere specifically with the binding of α4β1 integrin to its ligands VCAM-1 and fibronectin in vitro. Treatment of B. burgdorferi-infected C3H/HeJ mice with the α4β1 antagonist significantly ameliorated the outcome of clinical arthritis and the influx of neutrophilic granulocytes into ankle joints. Furthermore, local mRNA up-regulation of the proinflammatory mediators IL-1, IL-6, and cyclooxygenase-2 was largely abolished. Neither the synthesis of spirochete-specific Igs nor the development of a Th1-dominated immune response was altered by the treatment. Importantly, the drug also did not interfere with Ab-mediated control of spirochete load in the tissues. These findings demonstrate that the pathogenesis, but not the protective immune response, in Lyme arthritis is dependent on the α4β1-mediated influx of inflammatory cells. The onset of inflammation can be successfully targeted by treatment with S18407.