Martina Dorsch

Adaptation of Solitary Intestinal Lymphoid Tissue in Response to Microbiota and Chemokine Receptor CCR7 Signaling

Besides Peyer’s patches, solitary intestinal lymphoid tissue (SILT) provides a structural platform to efficiently initiate immune responses in the murine small intestine. SILT consists of dynamic lymphoid aggregates that are heterogeneous in size and composition, ranging from small clusters of mostly lineage-negative cells known as cryptopatches to larger isolated lymphoid follicles rich in B cells. In this study, we report that in chemokine receptor CCR7-deficient mice SILT is enlarged, although unchanged in frequency and cellular composition compared with wild-type mice. This phenotype is conferred by bone marrow-derived cells and is independent of the presence of intestinal bacteria. Remarkably, particularly small-sized SILT predominates in germfree wild-type mice. Colonization of wild-type mice with commensal bacteria provokes an adjustment of the spectrum of SILT to that observed under specific pathogen-free conditions by the conversion of pre-existing lymphoid structures into larger-sized SILT. In conclusion, our findings establish that intestinal microbes influence the manifestation of gut-associated lymphoid tissues and identify CCR7 signaling as an endogeneous factor that controls this process.

Anti-inflammatory and immunomodulatory properties of α1-antitrypsin without inhibition of elastase

The rationale of α1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastase-deficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-α (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-α, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1β gene expression, were observed in lungs of AAT-deficient patients treated with AAT therapy compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastase-deficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60–80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40- to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition.

Selective blockade of interleukin-6 trans-signaling improves survival in a murine polymicrobial sepsis model*

Objective: The pleiotropic cytokine interleukin (IL)-6 seems to play a pivotal role in sepsis, but contradictory findings in animal models impede a rationale for therapies directed against IL-6. IL-6 signals by two mechanisms via the ubiquitous transmembrane glycoprotein 130 (gp130): “classic” signaling using membrane-bound IL-6 receptor (IL-6R) and trans-signaling using soluble IL-6R (sIL-6R). Trans-signaling is selectively inhibited by soluble gp130 (sgp130). The aim of this study was to systematically compare complete blockade of IL-6 signaling (using a neutralizing anti-IL-6 antibody) and selective blockade of IL-6 trans-signaling (using a fusion protein of sgp130 and the crystallizable fragment of immunoglobulin G1, sgp130Fc) in a standardized cecal ligation and puncture (CLP) sepsis model. Design: Animal study. Setting: Animal laboratory. Subjects: C57BL/6J mice. Interventions: We performed a 96-hr dose-response study and a 24-hr study to investigate short-term mechanisms. In the 96-hr study, CLP was performed in 120 randomized mice (20 mice received vehicle, 10 mice per dose group). Mice were treated with equimolar doses of sgp130Fc (0.01/0.1/1/10 mg/kg) or anti-IL-6 (0.008/0.08/0.8/8 mg/kg) 24 hrs before CLP. Two additional groups received 0.5 mg/kg sgp130Fc 24 hrs before or 1 mg/kg sgp130Fc 24 hrs after CLP. Survival and activity scores were obtained daily until 96 hrs after CLP. In the 24-hr study, mice were randomized into four groups with 10 animals each (sham/vehicle, CLP/vehicle, CLP/anti-IL-6 [0.8 mg/kg], and CLP/sgp130Fc [1 mg/kg]) and killed after 24 hrs. Measurements and Main Results: In contrast to anti-IL-6, pretreatment with sgp130Fc significantly and dose-dependently increased survival from 45% to 100%. In addition, 1 mg/kg sgp130Fc administered 24 hrs after CLP increased survival from 45% to 80%. Mechanistically, sgp130Fc efficacy was reflected by complete prevention of epithelial cell apoptosis in the jejunum after CLP, which was not achieved with anti-IL-6. Conclusion: Selective inhibition of IL-6 trans-signaling by sgp130Fc has considerable potential for the treatment of sepsis and related disorders.

Chemokine Receptor CXCR5 Supports Solitary Intestinal Lymphoid Tissue Formation, B Cell Homing, and Induction of Intestinal IgA Responses

Solitary intestinal lymphoid tissue (SILT) comprises a spectrum of phenotypically diverse lymphoid aggregates interspersed throughout the small intestinal mucosa. Manifestations of SILT range from tiny lymphoid aggregates almost void of mature lymphocytes to large structures dominated by B cells. Large SILT phenotypically resemble a single Peyer’s patch follicle, suggesting that SILT might contribute to intestinal humoral immune responses. In this study, we track the fate of individual SILT in vivo over time and analyze SILT formation and function in chemokine receptor CXCR5-deficient mice. We show that, in analogy to Peyer’s patches, formation of SILT is invariantly determined during ontogeny and depends on CXCR5. Young CXCR5-deficient mice completely lack SILT, suggesting that CXCR5 is essential for SILT formation during regular postnatal development. However, microbiota and other external stimuli can induce the formation of aberrant SILT distinguished by impaired development of B cell follicles in CXCR5-deficient mice. Small intestinal transplantation and bone marrow transplantation reveal that defect follicle formation is due to impaired B cell homing. Moreover, oral immunization with cholera toxin or infection with noninvasive Salmonella fail to induce efficient humoral immune responses in CXCR5-deficient mice. Bone marrow transplantation of CXCR5-deficient recipients with wild-type bone marrow rescued B cell follicle formation in SILT but failed to restore full humoral immune responses. These results reveal an essential role of CXCR5 in Peyer’s patch and SILT development and function and indicate that SILT do not fully compensate for the lack of Peyer’s patches in T cell-dependent humoral immune responses.

A high specificity and affinity interaction with serum albumin stimulates an anion conductance in malaria-infected erythrocytes.

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl- and lactate outward currents with an EC50 of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

Experimental Bronchial Asthma – The Strength of the Species Rat

No single animal species reflects the complete range of human respiratory anatomy, physiology and - often not mentioned - age-related changes. The rat was the first experimental asthma model, but was overtaken in numbers by murine models many years ago. Data will be compiled to document that the rat model still has an important role in the research of bronchial asthma and other lung diseases. In pharmaceutical research for new drugs the rat model is still indispensable. Here specific aspects will be highlighted where the rat offers advantages in comparison to other species: the anatomical feature of the proprietary bronchial circulation, genetics and proteomics, lung function and finally economical considerations.

Role of Energy Sensor TlpD of Helicobacter pylori in Gerbil Colonization and Genome Analyses after Adaptation in the Gerbil

ABSTRACT Helicobacter pylori maintains colonization in its human host using a limited set of taxis sensors. TlpD is a proposed energy taxis sensor of H. pylori and dominant under environmental conditions of low bacterial energy yield. We studied the impact of H. pylori TlpD on colonization in vivo using a gerbil infection model which closely mimics the gastric physiology of humans. A gerbil-adapted H. pylori strain, HP87 P7, showed energy-dependent behavior, while its isogenic tlpD mutant lost it. A TlpD-complemented strain regained the wild-type phenotype. Infection of gerbils with the complemented strain demonstrated that TlpD is important for persistent infection in the antrum and corpus and suggested a role of TlpD in horizontal navigation and persistent corpus colonization. As a part of the full characterization of the model and to gain insight into the genetic basis of H. pylori adaptation to the gerbil, we determined the complete genome sequences of the gerbil-adapted strain HP87 P7, two HP87 P7 tlpD mutants before and after gerbil passage, and the original human isolate, HP87. The integrity of the genome, including that of a functional cag pathogenicity island, was maintained after gerbil adaptation. Genetic and phenotypic differences between the strains were observed. Major differences between the gerbil-adapted strain and the human isolate emerged, including evidence of recent recombination. Passage of the tlpD mutant through the gerbil selected for gain-of-function variation in a fucosyltransferase gene, futC (HP0093). In conclusion, a gerbil-adapted H. pylori strain with a stable genome has helped to establish that TlpD has important functions for persistent colonization in the stomach.

Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat

BackgroundEngineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain.ResultsAfter microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat.ConclusionThe Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.

Orthotopic transplantation of rat ovaries as a tool for strain rescue

Spontaneous mutations in inbred strains provide valuable resources for new disease models. Unfortunately, these mutations may affect reproduction, which require considerable efforts in breeding management. We transplanted ovaries of such mutant rat strains orthotopically into ovariectomized premature coisogenic recipients. A reproductive cycle was established in each of the recipients within 5 to 6 weeks after transplantation. Moreover, one-third became pregnant and had litters an average of 3 months after transplantation. These experiments demonstrate that orthotopic transplantation of ovaries can be used in the management of subfertile rat colonies.

Longitudinal MRI contrast enhanced monitoring of early tumour development with manganese chloride (MnCl2) and superparamagnetic iron oxide nanoparticles (SPIOs) in a CT1258 based in vivo model of prostate cancer

BackgroundCell lines represent a key tool in cancer research allowing the generation of neoplasias which resemble initial tumours in in-vivo animal models. The characterisation of early tumour development is of major interest in order to evaluate the efficacy of therapeutic agents. Magnetic resonance imaging (MRI) based in-vivo characterisation allows visualisation and characterisation of tumour development in early stages prior to manual palpation. Contrast agents for MRI such as superparamagnetic iron oxide nanoparticles (SPIOs) and manganese chloride (MnCl2) represent powerful tools for the in-vivo characterisation of early stage tumours. In this experimental study, we labelled prostate cancer cells with MnCl2 or SPIOs in vitro and used 1 T MRI for tracing labelled cells in-vitro and 7 T MRI for tracking in an in-vivo animal model.MethodsLabelling of prostate cancer cells CT1258 was established in-vitro with MnCl2 and SPIOs. In-vitro detection of labelled cells in an agar phantom was carried out through 1 T MRI while in-vivo detection was performed using 7 T MRI after subcutaneous (s.c.) injection of labelled cells into NOD-Scid mice (n = 20). The animals were scanned in regular intervals until euthanization. The respective tumour volumes were analysed and corresponding tumour masses were subjected to histologic examination.ResultsMnCl2in-vitro labelling resulted in no significant metabolic effects on proliferation and cell vitality. In-vitro detection-limit accounted 105 cells for MnCl2 as well as for SPIOs labelling. In-vivo 7 T MRI scans allowed detection of 103 and 104 cells. In-vivo MnCl2 labelled cells were detectable from days 4–16 while SPIO labelling allowed detection until 4 days after s.c. injection. MnCl2 labelled cells were highly tumourigenic in NOD-Scid mice and the tumour volume development was characterised in a time dependent manner. The amount of injected cells correlated with tumour size development and disease progression. Histological analysis of the induced tumour masses demonstrated characteristic morphologies of prostate adenocarcinoma.ConclusionsTo the best of our knowledge, this is the first study reporting direct in-vitro MnCl2 labelling and 7 T based in-vivo MRI tracing of cancer cells in a model of prostate cancer. MnCl2 labelling was found to be suitable for in-vivo tracing allowing long detection periods. The labelled cells kept their highly tumourigenic potential in-vivo. Tumour volume development was visualised prior to manual palpation allowing tumour characterisation in early stages of the disease.