High-resolution imaging of living gut mucosa: lymphocyte clusters beneath intestinal M cells are highly dynamic structures.

In the Peyer's patches of the small intestine, specialized epithelial cells, the membranous (M) cells, sample antigenic matter from the gut lumen and bring it into contact with cells of the immune system, which are then capable of initiating specific immune reactions. Using autofluorescence 2-photon (A2P) microscopy, we imaged living intestinal mucosa at a 0.5-µm resolution. We identified individual M cells without the aid of a marker and in vivo analyzed their sampling function over hours. Time-lapse recordings revealed that lymphocytes associated with M cells display a remarkable degree of motility with average speed rates of 8.2 µm/min, to form new M cell-associated lymphocyte clusters within less than 15 min. The lymphocytes drastically deform the M cells' cytoplasm and laterally move from one lymphocyte cluster to the next. This implies that the micro-compartment beneath M cells is a highly efficient container to bring potentially harmful antigens into contact with large numbers of immunocompetent cells. Our setup opens a new window for high-resolution 3D imaging of functional processes occurring in lymphoid and mucosal tissues.

Balancing intestinal and systemic inflammation through cell type-specific expression of the aryl hydrocarbon receptor repressor.

As a sensor of polyaromatic chemicals the aryl hydrocarbon receptor (AhR) exerts an important role in immune regulation besides its requirement for xenobiotic metabolism. Transcriptional activation of AhR target genes is counterregulated by the AhR repressor (AhRR) but the exact function of the AhRR in vivo is currently unknown. We here show that the AhRR is predominantly expressed in immune cells of the skin and intestine, different from other AhR target genes. Whereas AhRR antagonizes the anti-inflammatory function of the AhR in the context of systemic endotoxin shock, AhR and AhRR act in concert to dampen intestinal inflammation. Specifically, AhRR contributes to the maintenance of colonic intraepithelial lymphocytes and prevents excessive IL-1ß production and Th17/Tc17 differentiation. In contrast, the AhRR enhances IFN-γ-production by effector T cells in the inflamed gut. Our findings highlight the physiologic importance of cell-type specific balancing of AhR/AhRR expression in response to microbial, nutritional and other environmental stimuli.

Role of the C-type lectin receptors MCL and DCIR in experimental colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract. Though its exact etiology is still unclear, it is proposed that an imbalance in the intestinal homeostasis leads to a disturbed interaction between commensal microbiota and the mucosal immune system. Previous studies have shown that both innate and adaptive immunity are involved in an overwhelming colon inflammation, and thus contribute to the pathogenesis of IBD. In innate immunity, several pattern recognition receptors such as Toll-like receptors, NOD-like receptors or C-type lectin receptors (CLRs) are involved in IBD pathogenesis. Myeloid CLRs are mainly expressed by antigen-presenting cells and bind to glycan structures present on self or foreign antigens. The Macrophage-restricted C-type lectin (MCL) and the Dendritic cell immunoreceptor (DCIR) are two poorly characterized members of the CLR family. In this study, we investigated the role of MCL and DCIR in the pathogenesis of murine colitis. Both CLRs bound to intestinal microbiota to a different extent. They modulated the production of pro-inflammatory cytokines by antigen-presenting cells upon stimulation with heat-killed microbiota and impacted subsequent T cell responses. To analyze whether MCL and DCIR contribute to the pathogenesis of IBD, the dextran sulfate sodium (DSS) murine colitis model was employed. MCL-/- as well as DCIR-/- mice exhibited only a slightly increased severity of disease compared to wild-type mice indicating a limited role for MCL and DCIR in the regulation of intestinal immunity.

Transient ablation of regulatory T cells improves antitumor immunity in colitis-associated colon cancer.

Regulatory T cells (Treg) are supportive to cancer development in most tissues, but their role in colitis-associated colon cancer (CAC) remains unclear. In this study, we investigated the role of CD4(+)Foxp3(+) Treg in a mouse model of CAC and in patients with colon cancer. These Treg were increased strongly in number in a mouse model of CAC and in the peripheral blood of patients with colon cancer, exhibiting an activated phenotype as defined by elevated expression of GARP, CD103, CTLA-4, and IL10, along with an increased suppressive effect on the proliferation and Th1 cytokine expression of CD4(+)CD25(-) responder T cells ex vivo. Transient ablation of CD4(+)Foxp3(+) Treg during tumor development in the CAC model suppressed tumor outgrowth and distribution, accompanied by an increased number of CD8(+)IFNγ/granzyme B-producing effector T cells. Conversely, inactivation of IL10 in Treg did not elevate the antitumor response but instead further boosted tumor development. Our results establish a tumor-promoting function for Treg during CAC formation, but they also suggest that a selective, transient ablation of Treg can evoke antitumor responses, with implications for immunotherapeutic interventions in patients with CAC.

The C-Type Lectin Receptor SIGNR3 Binds to Fungi Present in Commensal Microbiota and Influences Immune Regulation in Experimental Colitis.

Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern-recognition receptors (PRRs) sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs) are PRRs recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 (SIGNR3) in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homolog of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze whether this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium-induced colitis model was employed. SIGNR3(-/-) mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3(-/-) mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.

Cellular-FLIP, Raji isoform (c-FLIP R) modulates cell death induction upon T-cell activation and infection.

Dysregulation of apoptosis caused by an imbalance of pro- and anti-apoptotic protein expression can lead to cancer, neurodegenerative, and autoimmune diseases. Cellular-FLIP (c-FLIP) proteins inhibit apoptosis directly at the death-inducing signaling complex of death receptors, such as CD95, and have been linked to apoptosis regulation during immune responses. While the isoforms c-FLIPL and c-FLIPS are well characterized, the function of c-FLIPR remains poorly understood. Here, we demonstrate the induction of endogenous murine c-FLIPR in activated lymphocytes for the first time. To analyze c-FLIPR function in vivo, we generated transgenic mice expressing murine c-FLIPR specifically in hematopoietic cells. As expected, lymphocytes from c-FLIPR transgenic mice were protected against CD95-induced apoptosis in vitro. In the steady state, transgenic mice had normal cell numbers and unaltered frequencies of B cells and T-cell subsets in lymphoid organs. However, when challenged with Listeria monocytogenes, c-FLIPR transgenic mice showed less liver necrosis and better bacterial clearance compared with infected wild-type mice. We conclude that c-FLIPR expression in hematopoietic cells supports an efficient immune response against bacterial infections.

Fermentable fiber ameliorates fermentable protein-induced changes in microbial ecology, but not the mucosal response, in the colon of piglets.

Dietary inclusion of fermentable carbohydrates (fCHO) is reported to reduce large intestinal formation of putatively toxic metabolites derived from fermentable proteins (fCP). However, the influence of diets high in fCP concentration on epithelial response and interaction with fCHO is still unclear. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO [14.5% crude protein (CP)/14.5% total dietary fiber (TDF)]; low fCP/high fCHO (14.8% CP/16.6% TDF); high fCP low fCHO (19.8% CP/14.5% TDF); and high fCP/high fCHO (20.1% CP/18.0% TDF) as dietary treatments. After 21-23 d, pigs were killed and colon digesta and tissue samples analyzed for indices of microbial ecology, tissue expression of genes for cell turnover, cytokines, mucus genes (MUC), and oxidative stress indices. Pig performance was unaffected by diet. fCP increased (P < 0.05) cell counts of clostridia in the Clostridium leptum group and total short and branched chain fatty acids, ammonia, putrescine, histamine, and spermidine concentrations, whereas high fCHO increased (P < 0.05) cell counts of clostridia in the C. leptum and C. coccoides groups, shifted the acetate to propionate ratio toward acetate (P < 0.05), and reduced ammonia and putrescine (P < 0.05). High dietary fCP increased (P < 0.05) expression of PCNA, IL1ß, IL10, TGFß, MUC1, MUC2, and MUC20, irrespective of fCHO concentration. The ratio of glutathione:glutathione disulfide was reduced (P < 0.05) by fCP and the expression of glutathione transferase was reduced by fCHO (P < 0.05). In conclusion, fermentable fiber ameliorates fermentable protein-induced changes in most measures of luminal microbial ecology but not the mucosal response in the large intestine of pigs.

Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy.

The mucosa of the gastrointestinal tract is a dynamic tissue composed of numerous cell types with complex cellular functions. Study of the vital intestinal mucosa has been hampered by lack of suitable model systems. We here present a novel animal model that enables highly resolved three-dimensional imaging of the vital murine intestine in anaesthetized mice. Using intravital autofluorescence 2-photon (A2P) microscopy we studied the choreographed interactions of enterocytes, goblet cells, enteroendocrine cells and brush cells with other cellular constituents of the small intestinal mucosa over several hours at a subcellular resolution and in three dimensions. Vigorously moving lymphoid cells and their interaction with constituent parts of the lamina propria were examined and quantitatively analyzed. Nuclear and lectin staining permitted simultaneous characterization of autofluorescence and admitted dyes and yielded additional spectral information that is crucial to the interpretation of the complex intestinal mucosa. This novel intravital approach provides detailed insights into the physiology of the small intestine and especially opens a new window for investigating cellular dynamics under nearly physiological conditions.

The CD40-CD40L pathway contributes to the proinflammatory function of intestinal epithelial cells in inflammatory bowel disease.

In inflammatory bowel diseases (IBD), intestinal epithelial cells (IECs) are involved in the outbalanced immune responses toward luminal antigens. However, the signals responsible for this proinflammatory capacity of IECs in IBD remain unclear. The CD40/CD40L interaction activates various pathways in immune and nonimmune cells related to inflammation and was shown to be critical for the development of IBD. Here we demonstrate CD40 expression within IECs during active IBD. Endoscopically obtained biopsies taken from Crohn's disease (n = 112) and ulcerative colitis patients (n = 67) consistently showed immunofluorescence staining for CD40 in IECs of inflamed ileal or colonic mucosa. In noninvolved mucosa during active disease, tissue obtained during Crohn's disease or ulcerative colitis in remission and biopsies from healthy controls (n = 38) IECs almost entirely lacked CD40 staining. Flow cytometry and RT-PCR analysis using different intestinal epithelial cell lines (HT29, SW480, and T84) showed IFN-gamma to effectively induce CD40 in IECs. Cells were virtually unresponsive to LPS or whole E. coli regarding CD40 expression. In addition, a moderate induction of CD40 was found in response to TNF-alpha, which exerted synergistical effects with IFN-gamma. CD40 ligation by CD40L-transfected murine fibroblasts or soluble CD40L increased the secretion of IL-8 in IFN-gamma pretreated HT29 cells. Our findings provide evidence for the epithelial expression and modulation of CD40 in IBD-affected mucosa and indicate its involvement in the proinflammatory function of IECs.

Multivesicular bodies in intestinal epithelial cells: responsible for MHC class II-restricted antigen processing and origin of exosomes.

In normal conditions intestinal epithelial cells (IECs) constitutively stimulate regulatory CD4(+) T cells. However, in Crohn's disease (CD), this major histocompatibility complex (MHC) class II-restricted antigen presentation results in stimulation of proinflammatory CD4(+) T cells. We hypothesized that these alternative functions might be mediated by differential sorting and processing of antigens into distinct MHC II-enriched compartments (MIICs). Accordingly, we analysed the endocytic pathways of lumenally applied ovalbumin (OVA) in IECs of the jejunum and ileum of wild-type (WT) and TNFDeltaARE/WT mice that develop a CD-resembling ileitis. Using quantitative reverse transcription polymerase chain reaction, we found that messenger RNA levels of interferon-gamma, tumour necrosis factor-alpha, interleukin-17 and interleukin-10 were significantly up-regulated in the inflamed ileum of TNFDeltaARE/WT mice, confirming CD-like inflammation. Fluorescence and immunoelectron microscopy revealed the presence of MHC II and invariant chain throughout the late endocytic compartments, with most molecules concentrated in the multivesicular bodies (MVB). OVA was targeted into MVB and, in contrast to other MIICs, accumulated in these structures within 120 min of exposure. The IEC-specific A33 antigen localized to internal vesicles of MVB and A33/class II-bearing exosomes were identified in intercellular spaces. Remarkably, the expression pattern of MHC II/invariant chain molecules and the trafficking of OVA were independent of mucosal inflammation and the specific region in the small intestine. MVB seem to be principally responsible for class II-associated antigen processing in IECs and to constitute the origin of MHC II-loaded exosomes. The distinctive functions of IECs in antigen presentation to CD4(+) T cells might arise as a result of differential processing within the MVB identified here.

Mechanisms of laser-induced dissection and transport of histologic specimens.

Rapid contact- and contamination-free procurement of histologic material for proteomic and genomic analysis can be achieved by laser microdissection of the sample of interest followed by laser-induced transport (laser pressure catapulting). The dynamics of laser microdissection and laser pressure catapulting of histologic samples of 80 mum diameter was investigated by means of time-resolved photography. The working mechanism of microdissection was found to be plasma-mediated ablation initiated by linear absorption. Catapulting was driven by plasma formation when tightly focused pulses were used, and by photothermal ablation at the bottom of the sample when defocused pulses producing laser spot diameters larger than 35 microm were used. With focused pulses, driving pressures of several hundred MPa accelerated the specimen to initial velocities of 100-300 m/s before they were rapidly slowed down by air friction. When the laser spot was increased to a size comparable to or larger than the sample diameter, both driving pressure and flight velocity decreased considerably. Based on a characterization of the thermal and optical properties of the histologic specimens and supporting materials used, we calculated the evolution of the heat distribution in the sample. Selected catapulted samples were examined by scanning electron microscopy or analyzed by real-time reverse-transcriptase polymerase chain reaction. We found that catapulting of dissected samples results in little collateral damage when the laser pulses are either tightly focused or when the laser spot size is comparable to the specimen size. By contrast, moderate defocusing with spot sizes up to one-third of the specimen diameter may involve significant heat and ultraviolet exposure. Potential side effects are maximal when samples are catapulted directly from a glass slide without a supporting polymer foil.

Validation of a novel ultra-short immunolabeling method for high-quality mRNA preservation in laser microdissection and real-time reverse transcriptase-polymerase chain reaction.

Laser microdissection allows isolation of tiny samples from tissue sections for analysis of gene expression by real-time quantitative polymerase chain reaction (PCR). Although immunohistochemical labeling is often required to identify target structures, it drastically degrades mRNA so that shortened protocols are needed. Here, we present a novel method that allows fluorescence double labeling to be performed in only one incubation of 5 minutes. Fab fragments directly coupled to fluorochromes are linked to primary antibodies before these complexes are applied to sections. We quantified the influences of fixatives, labeling solutions, and incubation time on the mRNA yield and compared our method with previously proposed protocols. While tissue components, ie, vimentin and Ki67 antigen, were sufficiently stained after only 5 minutes of incubation, the new method produced a minute loss of mRNA that did not significantly differ from that of untreated sections. In contrast, incubation times of 15 and 30 minutes reduced the mRNA yield by 99.8 to 99.9%. Furthermore, incubation periods longer than 5 minutes critically affected the ratio between the target and housekeeping genes tested by factors of up to 10.6. In conclusion, the novel method described here reduces mRNA loss and potential ratio shifts to a level that does not significantly differ from that of unlabeled samples.

Impact of formalin-fixation and paraffin-embedding on the ratio between mRNA copy numbers of differently expressed genes.

Several studies have shown that specific mRNA sequences can be successfully detected in formalin-fixed, paraffin-embedded tissues using reverse transcriptase-polymerase chain reaction (RT-PCR). Here, we test the hypothesis that gene expression levels can be accurately quantified in formalin-fixed, paraffin-embedded tissues by determining the ratio between the copy number of the mRNA molecule of interest and the mRNA copy number of a so-called housekeeping gene. The mRNA copy numbers of the variably expressed multiple drug resistance gene (MDR)-1 and four housekeeping genes (hypoxanthine phosphoribosyl-transferase-1, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and elongation factor-1a) were quantified by real-time-quantitative RT-PCR before and after formalin-fixation and paraffin-embedding of 576 tissue samples (heart, kidney, spleen, liver) from three beagle dogs. The results indicate that fixation and embedding drastically altered the ratios between the different mRNA copy numbers and that the relative expression levels of MDR-1 per any of the housekeeping genes were artificially increased or decreased up to more than tenfold. It would thus appear questionable to normalize quantitative expression data from fixed and embedded tissues by using housekeeping genes as reference. In contrast, tissue autolysis of up to 24 h and long-term storage of embedded tissues of up to 20 years had no additional effects.