通过使用双光子显微镜活体成像技术优化了K/B×N血清诱导的关节炎小鼠模型,并观察了嗜中性粒细胞的募集情况.皮下注射K/B×N血清可以快速诱导大量的嗜中性粒细胞募集到局部炎症关节.通过使用这种方法,发现阻断C5a活性会减少嗜中性粒细胞募集,说明C5a是关节炎期间嗜中性粒细胞募集的关键调节因子.因此,本试验将为补体相关生物制剂治疗类风湿关节炎提供新的靶点.

The infiltration of neutrophils and monocytes is a prominent feature of inflammatory diseases including human rheumatoid arthritis. Understanding how neutrophil recruitment is regulated during pathogenesis is crucial for developing anti-inflammatory therapies. We optimized the K/B×N serum-induced mouse arthritis model to study neutrophil trafficking dynamics in vivo using two-photon microscopy. Arthritogenic serum was injected subcutaneously into one hind footpad to induce a local arthritis with robust neutrophil recruitment. Using this approach, we showed that the depletion of monocytes with clodronate liposomes impaired neutrophil recruitment specifically at the transendothelial migration step. The depletion of CCR2(+) monocytes with the monoclonal antibody MC-21 reproduced these effects, implicating CCR2(+) monocytes as key regulators of neutrophil extravasation during arthritis initiation. However, monocyte depletion did not prevent neutrophil extravasation in response to bacterial challenge. These findings suggest that anti-inflammatory therapies targeting monocytes may act in part through antagonizing neutrophil extravasation at sites of aseptic inflammation.

Goblet cells in the small intestine act as passages delivering small antigens to tolerance-inducing dendritic cells in the lamina propria. How the balance between tolerance and immunity is maintained is an important question in immunology, and is of particular relevance to the small intestine, where innocuous antigens from the diet and potential pathogens are encountered simultaneously. McDole et al. show that goblet cells in the epithelium of the small intestine act as conduits through which small luminal antigens can be delivered to tolerance-inducing dendritic cells in the lamina propria, a layer of connective tissue beneath the epithelium. Through this mechanism, goblet cells could play a key part in promoting intestinal immune homeostasis. The intestinal immune system is exposed to a mixture of foreign antigens from diet, commensal flora and potential pathogens. Understanding how pathogen-specific immunity is elicited while avoiding inappropriate responses to the background of innocuous antigens is essential for understanding and treating intestinal infections and inflammatory diseases. The ingestion of protein antigen can induce oral tolerance, which is mediated in part by a subset of intestinal dendritic cells (DCs) that promote the development of regulatory T cells1. The lamina propria (LP) underlies the expansive single-cell absorptive villous epithelium and contains a large population of DCs (CD11c+ CD11b+ MHCII+ cells) comprised of two predominant subsets: CD103+ CX3CR1− DCs, which promote IgA production, imprint gut homing on lymphocytes and induce the development of regulatory T cells2,3,4,5,6,7,8,9, and CD103− CX3CR1+ DCs (with features of macrophages), which promote tumour necrosis factor-α (TNF-α) production, colitis, and the development of TH17 T cells5,6,7,10. However, the mechanisms by which different intestinal LP-DC subsets capture luminal antigens in vivo remains largely unexplored. Using a minimally disruptive in vivo imaging approach we show that in the steady state, small intestine goblet cells (GCs) function as passages delivering low molecular weight soluble antigens from the intestinal lumen to underlying CD103+ LP-DCs. The preferential delivery of antigens to DCs with tolerogenic properties implies a key role for this GC function in intestinal immune homeostasis.

Immune-mediated pulmonary diseases are a significant public health concern. Analysis of leukocyte behavior in the lung is essential for understanding cellular mechanisms that contribute to normal and diseased states. Here, we used two-photon imaging to study neutrophil extravasation from pulmonary vessels and subsequent interstitial migration. We found that the lungs contained a significant pool of tissue-resident neutrophils in the steady state. In response to inflammation produced by bacterial challenge or transplant-mediated, ischemia-reperfusion injury, neutrophils were rapidly recruited from the circulation and patrolled the interstitium and airspaces of the lung. Motile neutrophils often aggregated in dynamic clusters that formed and dispersed over tens of minutes. These clusters were associated with CD115+ F4/80+ Ly6C+ cells that had recently entered the lung. The depletion of blood monocytes with clodronate liposomes reduced neutrophil clustering in the lung, but acted by inhibiting neutrophil transendothelial migration upstream of interstitial migration. Our results suggest that a subset of monocytes serve as key regulators of neutrophil extravasation in the lung and may be an attractive target for the treatment of inflammatory pulmonary diseases.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by progressive inflammation with massive infiltration of leukocytes into the synovium of multiple joints. Although monocyte/macrophage infiltration is a prominent feature and correlated with disease severity, the exactly role of monocytes in the induction phase of joint inflammation is unknown. Using two-photon microscopy, we tested whether monocytes regulate the recruitment of neutrophils during K/B×N serum induced arthritis. We examined neutrophil trafficking dynamics in vivo in LysM-GFP mice using the established i.v. transfer model and a modified in which serum was injected subcutaneously (s.c.) into the footpad. The s.c. model had several advantages including: more synchronized cell trafficking behaviors, shorter disease course and focused inflammation in the affected limb. We found that clodronate-liposome depletion of monocytes impaired neutrophil recruitment in our model at two separate steps. First, transendothelial cell migration was inhibited significantly, leaving many neutrophils stranded along the endothelial surface. Moreover, the cells that managed to extravasate showed defective chemotaxis and localization to the joint. Our results suggest that monocytes serve as key mediators of neutrophil extravasation and may play a second role in controlling the local tissue distribution of neutrophils during arthritis. Key words: Arthritis, Monocyte, Neutrophil, two photon microscopy

BACKGROUND & AIMS: Helicobacter pylori infection increases gastric regulatory T cell (Treg) response, which may contribute to H pylori immune escape. We hypothesize that H pylori directs Treg skewing by way of dendritic cells (DCs) and thus inhibits interleukin-17(+) helper T cells (Th17) immunity. METHODS: Two-photon microscopy was used to locate DCs in gastric lamina propria of mice. The induction of Th17 and Treg responses by bacteria-pulsed murine bone marrow-derived DCs was analyzed by cytokine production and stimulation of T-cell proliferation. The effect of VacA, CagA, transforming growth factor-beta (TGF-beta), and IL-10 on Th17/Treg balance was assessed. The in vivo significance of Tregs on the H pylori-specific Th17 response and H pylori density was determined by using anti-CD25 neutralizing antibodies to deplete Tregs in mice. RESULTS: We showed that mucosal CD11c(+) DCs are located near the surface of normal gastric epithelium, and their number increased after H pylori infection. Study of the direct interaction of DCs with H pylori showed a Treg-skewed response. The Treg skewing was independent of H pylori VacA and CagA and dependent on TGF-beta and IL-10. In vivo Treg skewing by adoptive transfer of H pylori-pulsed DCs reduces the ratio of gastric IL-17/Foxp3 mRNA expressions. The depletion of CD25(+) Tregs results in early reduction of H pylori density, which is correlated with enhanced peripheral H pylori-specific Th17, but not Th1, response. CONCLUSIONS: Overall, our study indicates that H pylori alters the DC-polarized Th17/Treg balance toward a Treg-biased response, which suppresses the effective induction of H pylori-specific Th17 immunity.

Pathogen recognition by T cells is dependent on their exquisite specificity for self-major histocompatibility complex (MHC) molecules presenting a bound peptide. Although this specificity results from positive and negative selection of developing T cells in the thymus, the relative contribution of these two processes remains controversial. To address the relation between the selecting peptide-MHC complex and the specificity of mature T cells, we generated transgenic mice that express a single peptide-MHC class I complex. We demonstrate that positive selection of CD8 T cells in these mice results in an MHC-specific repertoire. Although selection on a single complex is peptide promiscuous, mature T cells are highly peptide specific. Thus, positive selection imparts MHC and peptide specificity on the peripheral CD8 T cell repertoire.

The nature of the peptides that drive positive selection of T cells in the thymus remains poorly defined. To examine the role of peptides during positive selection in vivo, we generated single‐chain‐trimer (SCT) transgenic mouse strains that express only the OVAp/Kb/β2m complex or the VSVp/Kb/β2m complex. Although the SCT transgene is expressed at levels that are comparable to Kb expression in B6 mice, very few CD8 T cells are selected by the SCT compared to B6 mice, a finding consistent with positive selection being peptide specific. Furthermore, the SCT selected CD8 T cells are highly reactive with Kb loaded with endogenous peptides, but are not reactive with Db. This demonstrates that endogenous Kb self peptides are effectively excluded by the covalently attached peptide, and that CD8 T cells preferentially react with the selecting class I molecule. A T cell clonal analysis revealed that SCT selected T cells were highly specific for Kb and were not more MHC cross‐reactive than T cells from B6 mice. These results suggest that CD8 T cells selected on a single peptide/MHC ligand are MHC specific and possess at least some peptide cross‐reactivity. The extent of this peptide cross reactivity is under investigation to define the relationship between the selecting and agonist peptides. In sum, these data strongly suggest that positive selection determines the peptide and MHC specificity of the CD8 T cell repertoire.

Although gene therapy has reduced manifestations of genetic diseases, immune responses can abrogate the effect. One approach to induce tolerance is to perform gene transfer in newborns when the immune system is immature. This has allowed a therapeutic response to be achieved for lysosomal storage diseases and hemophilia in mice. However, it is unclear if this approach would be effective in large animals with more-mature immune systems at birth, and it is unlikely that this will be effective in humans. Immune responses will likely pose a tremendous hurdle for patients with null mutations, and identification of a large animal model with a sufficiently mature immune system at birth should facilitate preclinical studies to identify ways to block an expected immune response in humans. Gene therapy experiments were performed in mucopolysaccharidosis I (MPS I) cats, which are deficient in the lysosomal enzyme α-L-iduronidase (IDUA) due to a 3 bp deletion. An amphotropic retroviral vector (RV) expressing the canine IDUA was injected IV into newborn cats at 10(9) or 10(10) TU/kg. The canine protein differs from the feline protein at 130 positions of 653 aa. Expression can be followed by analysis of enzyme activity in serum, as some of the enzyme produced by cells is secreted into blood. Cats achieved high serum activity that averaged 286+/−416 (SD; N=7) U/ml and 2355+/−2763 (SD) U/ml at 1 month after gene transfer for the low and high RV dose, respectively. However, all animals lost expression by 3 months, which was associated with the development of cytotoxic T lymphocytes (CTLs) that could kill autologous cIDUA-expressing fibroblasts, and with a marked decrease in RV RNA sequences in the liver. In contrast, antibodies to cIDUA did not develop. In an attempt to block a CTL response, two cats were treated with the immunosuppressive agent CTLA4- Ig for 2 weeks around the time of transduction with the high dose of RV. CTLA4-Ig binds to CD80 or CD86, and prevents them from stimulating an immune response by activating CD28 on the surface of lymphocytes. These cats achieved 187+/−161 U/ml of IDUA activity in serum, which has been stable for 10 months to date, and did not develop a CTL response These data demonstrate that CTLA4-Ig can prevent a CTL response after neonatal gene therapy. Factors that reduce the efficiency of the immune system in newborn mice and/or humans include reduced secretion of cytokines by lymphocytes, and reduced levels of the co-stimulatory partners CD40 ligand and CD40. In contrast, lymphocytes from newborn humans have normal levels of CD80/CD86 and CD28. We hypothesize that the CD80/CD86 interaction with CD28 may be of particular importance in the newborn period in cats, explaining why transient CTLA4-Ig is so effective at inducing tolerance. The cat appears to be an excellent pre-clinical model for neonatal gene therapy.

Dendritic cells (DC)-derived or tumor-derived exosomes are a population of nanometer sized membrane vesicles that can induce specific anti-tumor immunity. However, the immunogenic potential and efficiency of exosomes-based tumor vaccine are not satisfactory enough to achieve a curative effect in clinical trials. In this article we investigated whether IL-18 genetic modification of tumor cells can increase the efficacy of exosomes derived from IL-18 gene-modified tumor cells. We transfected carcinoembryonic antigen (CEA)-expressing tumor cells with a recombinant adenovirus encoding human IL-18 (AdhIL-18) and prepared the exosomes, Exo/IL-18, from IL-18 gene-modified tumor cells. We found that Exo/IL-18 naturally contain CEA and bioactive IL-18. Moreover, Exo-IL-18 are potent in chemoattracting DC and T cells, enhancing the proliferation and Th1 cytokine release of PBMC, and promoting the phenotypic and functional maturation of DC. Furthermore, Exo/IL-18-pulsed DC are quite potent to induce HLA-A*0201-restricted, CEA-specific CD8+ CTL from the PBMC of HLA-A*0201 CEA+ cancer patients in vitro. In almost all of these experiments, Exo/IL-18 show more potent functions than the conventionally prepared exosomes derived from parent tumor cells without IL-18 gene modification. Our findings suggest that Exo/IL-18 has more potent capability to induce specific anti-tumor immunity, and our strategy of IL-18 modification of exosomes is a feasible approach to develop exosomes-based tumor vaccines.

A novel member of mitochondrial carrier superfamily has been identified from human bone marrow stromal cells (BMSC) and designated as human BMSC-derived mitochondrial carrier protein (HuBMSC-MCP). It encodes a 321 amino-acid protein with three tandem related domains of about 100 amino acids. Each domain contains two hydrophobic stretches, which are thought to span the membrane as alpha-helices. Distant relationship analysis indicates that the protein is highly conserved between species from Caenorhabditis elegans to human. HuBMSC-MCP gene is mapped to chromosome 11p11. HuBMSC-MCP mRNA expression is detectable in various human tissues and cell lines. By confocal imaging, HuBMSC-MCP is localized to mitochondria and also detected in the pseudopodial protrusion of human breast adenocarcinoma MCF-7 cells. When transfected into dendritic cells (DC), HuBMSC-MCP could enhance DCs endocytotic capacity. Thus, HuBMSC-MCP is a phylogenetically conserved and widely expressed mitochondrial carrier protein which perhaps associates with mitochondrial oxidative phosphorylation.

OBJECTIVETo investigate the antitumor effect of combined adenovirus encoding E. coli cytosine deaminase (AdCD) and adenovirus encoding murine interleukin 2 (AdIL-2) on murine melanoma.METHODSC57BL/6 mice were inoculated s.c. with B16F10 melanoma cells and 3 days later received injections of AdCD and/or AdIL-2 at the site of tumor inoculation followed by administration of 5-flurocytosine (5FC) 300 mg/kg per day for 10 days.RESULTSMice receiving AdCD/5FC/AdIL2 therapy developed tumors more slowly and survived much longer when compared with mice treated with AdCD/5FC, AdIL2, AdlacZ/5FC, or PBS. Immunological analysis illustrated that combined treatment could enhance NK activity and CTL activity. Flow cytometry demonstrated that AdCD/5FC/AdIL2 therapy increased the expression of MHC-1 and CD80 molecules on freshly isolated tumor cells. The CD4+ and CD8+ T cell infiltration in the tumor increased significantly after the combined therapy.CONCLUSIONSOur data showed that combined transfer of CD suicide gene and IL-2 gene could inhibit the tumor growth more significantly. The increased specific and non-specific antitumor immunity might be responsible for the enhanced therapeutic effect.

In the present report, antitumor effect of combined transfer of suicide gene and cytokine gene was studied. Adenovirus engineered to express E. Coli. cytosine deaminase (AdCD) and/or adenovirus engineered to express murine granulocyte-macrophage colonystimulating factor (AdGMCSF) were used for the treatment of leukemia-bearing mice. The mice were inoculated s.c. with FBL-3 erythroleukemia cells and 3 days later received intratumoral injection of AdCD in the presence or absence of AdGMCSF followed by intraperitoneal 5-fluorocytosine (5FC) treatment. The results demonstrated that mice received combined therapy of AdCD/5FC and AdGMCSF developed tumors most slowly and survived much longer when compared with mice treated with AdCD/5FC alone, AdGMCSF alone, AdlacZ/5FC or PBS. Combined transfer of CD gene and GM-CSF gene achieved higher specific CTL activity than control therapies. Pathological examination illustrated that the tumor mass showed obvious necrosis and inflammatory cell infiltration in mice after combined therapy. The results demonstrated that combined transfer of suicide gene and cytokine gene could synergistically inhibit the growth of leukemia in mice and induce antitumor immunity of the host. The combination therapy might be a potential approach for cancer gene therapy.

The antitumor effect of the combined transfer of a suicide gene and a cytokine gene was evaluated in the present study. Adenoviruses expressing Escherichia coli cytosine deaminase (AdCD) and adenoviruses expressing murine interleukin-2 (AdIL-2) were utilized for the treatment of established tumors. The mice were inoculated s.c. with FBL-3 erythroleukemia cells and 3 days later received an intratumoral injection of AdCD in the presence or absence of AdIL-2 followed by intraperitoneal 5-fluorocytosine (5-FC) administration. The results demonstrated that tumor-bearing mice treated with AdCD/5-FC in combination with AdIL-2 showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdCD/5-FC, AdIL-2, adenovirus expressing β-galactosidase/5-FC or phosphate-buffered saline. The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD4+ and CD8+ T cells infiltrating the tumor after combined therapy. The splenic natural killer and cytotoxic T lymphocyte activities increased significantly in the mice after combined therapy with AdCD/5-FC/AdIL-2. Our results demonstrate that therapy combining a suicide gene and IL-2 gene can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently.

A recombinant vaccinia virus expressing marine granulocyte-macrophage colony-stimulating factor (VVGM-CSF) was tested for its antitumor activity. Murine pulmonary metastasis was established by injecting 2×105 B16F10 melanoma cells into the tail vein of C57BL/6 mice. Three days after B16F10 inoculation, WGM-CSF or VVTK, a thymidine kinase gene deficient control vaccinia virus, were injected intraperitoneally twice weekly for 2 weeks. Two weeks later, the mice were sacrificed and pulmonary metastasis foci counted. The results demonstrated that VVGM-CSF treatment significantly decreased the number of pulmonary metastasis and prolonged the survival time of tumor-bearing mice. Cytotoxic and phagocytic activities of the peritoneal macrophages were found to be markedly elevated hi mice treated with VVGM-CSF. Nitric oxide released from the macrophages was also found to be increased. These data, together with our other results, strongly demonstrated that continuous secretion of GM-CSF and activation of macrophages might partially explain the therapeutic effects of VVGM-CSF on murine pulmonary metastasis.