Mechanisms of γδ T cell accumulation in visceral adipose tissue with aging.

γδ T cells are resident in visceral adipose tissue (VAT) where they show an age-associated increase in numbers and contribute to local and systemic chronic inflammation. However, regulation of this population and mechanisms for the age-dependent accumulation are not known. In this study, we identified a progressive trend of γδ T cell accumulation in VAT over the lifespan in mice and explored physiological mechanisms contributing to accumulation. Using isochronic parabiotic pairs of wild-type (WT) and T cell receptor delta knockout (TCRδ KO) mice at young and old age, we confirmed that VAT γδ T cells are predominately a tissue-resident population which is sustained in aging. Migration of peripheral γδ T cells into VAT was observed at less than 10%, with a decreasing trend by aging, suggesting a minor contribution of recruitment to γδ T cell accumulation with aging. Since tissue-resident T cell numbers are tightly regulated by a balance between proliferation and programmed cell death, we further explored these processes. Using in vivo EdU incorporation and the proliferation marker Ki67, we found that the absolute number of proliferating γδ T cells in VAT is significantly higher in the aged compared to young and middle-aged mice, despite a decline in the proportion of proliferating to non-proliferating cells by age. Analysis of apoptosis via caspase 3/7 activation revealed that VAT γδ T cells show reduced apoptosis starting at middle age and continuing into old age. Further, induction of apoptosis using pharmacological inhibitors of Bcl2 family proteins revealed that VAT γδ T cells at middle age are uniquely protected from apoptosis via a mechanism independent of traditional anti-apoptotic Bcl2-family proteins. Collectively, these data indicate that protection from apoptosis at middle age increases survival of tissue-resident γδ T cells resulting in an increased number of proliferative cells from middle age onward, and leading to the age-associated accumulation of γδ T cells in VAT. These findings are important to better understand how adipose tissue dysfunction and related changes in the immune profile contribute to inflammaging among the elderly.

Accumulation of γδ T cells in visceral fat with aging promotes chronic inflammation.

Adipose tissue dysfunction is strongly linked to the development of chronic inflammation and cardiometabolic disorders in aging. While much attention has been given to the role of resident adipose tissue immune cells in the disruption of homeostasis in obesity, age-specific effects remain understudied. Here, we identified and characterized a population of γδ T cells, which show unique age-dependent accumulation in the visceral adipose tissue (VAT) of both mice and humans. Diet-induced obesity likewise increased γδ T cell numbers; however, the effect was greater in the aged where the increase was independent of fat mass. γδ T cells in VAT express a tissue-resident memory T cell phenotype (CD44hiCD62LlowCD69+) and are predominantly IL-17A-producing cells. Transcriptome analyses of immunomagnetically purified γδ T cells identified significant age-associated differences in expression of genes related to inflammation, immune cell composition, and adipocyte differentiation, suggesting age-dependent qualitative changes in addition to the quantitative increase. Genetic deficiency of γδ T cells in old age improved the metabolic phenotype, characterized by increased respiratory exchange ratio, and lowered levels of IL-6 both systemically and locally in VAT. Decreased IL-6 was predominantly due to reduced production by non-immune stromal cells, primarily preadipocytes, and adipose-derived stem cells. Collectively, these findings suggest that an age-dependent increase of tissue-resident γδ T cells in VAT contributes to local and systemic chronic inflammation and metabolic dysfunction in aging.

Short-Term Dietary Restriction Rescues Mice From Lethal Abdominal Sepsis and Endotoxemia and Reduces the Inflammatory/Coagulant Potential of Adipose Tissue.

OBJECTIVES: Visceral adipose tissue is a major site for expression of proinflammatory and procoagulant genes during acute systemic inflammation. In this study, we tested whether the loss of fat mass by dietary restriction would remove the major source of these factors resulting in improved tolerance to sepsis and endotoxemia. DESIGN: Prospective, laboratory controlled experiments. SETTING: Aging and critical care research laboratory in a university hospital. SUBJECTS: Middle-aged (12-month old) male C57BL/6 mice. INTERVENTIONS: Mice were subjected to 40% dietary restriction for 3 weeks followed by induction of abdominal sepsis or endotoxemia by intraperitoneal injection with cecal slurry or lipopolysaccharide, respectively. MEASUREMENTS AND MAIN RESULTS: Compared with freely fed mice, dietary restricted mice exhibited dramatically improved survival (80% vs 0% after sepsis; p < 0.001 and 86% vs 12% after endotoxemia; p = 0.013) and significantly reduced visceral fat-derived messenger RNA expression of interleukin-6, thrombospondin-1, plasminogen activator inhibitor-1, and tissue factor, which positively correlated with fat mass. Plasma levels of interleukin-6 were significantly reduced by dietary restriction and correlated with adipose interleukin-6 messenger RNA levels and fat mass (p < 0.001; R = 0.64 and 0.89). In vitro culture of visceral fat explants from naive dietary restricted mice showed significantly reduced interleukin-6 secretion compared with that from freely fed mice in response to lipopolysaccharide. Analysis of major adipose immune cell populations by flow cytometry demonstrated that macrophages were the only cell population reduced by dietary restriction and that CD11c/CD206 (M2-type) and CD11c/CD206 (double negative) macrophages, in addition to T cells, are the major immune cell populations that produce interleukin-6 in middle-aged mice during systemic inflammation. CONCLUSIONS: Short-term dietary restriction drastically improved the survival outcome of middle-aged mice during both polymicrobial sepsis and sterile endotoxemia. Improved survival was accompanied by a significantly attenuated inflammatory response in adipose tissue, which is likely due to alterations of both fat mass quantity and qualitative changes, including a reduction in macrophage populations.

Lessons from mother: Long-term impact of antibodies in breast milk on the gut microbiota and intestinal immune system of breastfed offspring.

From birth to adulthood, the gut microbiota matures from a simple community dominated by a few major bacterial groups into a highly diverse ecosystem that provides both benefits and challenges to the host. Currently there is great interest in identifying environmental and host factors that shape the development of our gut microbiota. Breast milk is a rich source of maternal antibodies, which provide the first source of adaptive immunity in the newborn's intestinal tract. In this addendum, we summarize our recent data demonstrating that maternal antibodies in breast milk promote long-term intestinal homeostasis in suckling mice by regulating the gut microbiota and host gene expression. We also discuss important unanswered questions, future directions for research in this field, and implications for human health and disease.

Caspase-3 mediates the pathogenic effect of Yersinia pestis YopM in liver of C57BL/6 mice and contributes to YopM's function in spleen.

The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G(+), F4/80(+), and iNOS(+) cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.

Targeting the Wnt/ß-catenin signaling pathway in liver cancer stem cells and hepatocellular carcinoma cell lines with FH535.

Activation of the Wnt/ß-catenin pathway has been observed in at least 1/3 of hepatocellular carcinomas (HCC), and a significant number of these have mutations in the ß-catenin gene. Therefore, effective inhibition of this pathway could provide a novel method to treat HCC. The purposed of this study was to determine whether FH535, which was previously shown to block the ß-catenin pathway, could inhibit ß-catenin activation of target genes and inhibit proliferation of Liver Cancer Stem Cells (LCSC) and HCC cell lines. Using ß-catenin responsive reporter genes, our data indicates that FH535 can inhibit target gene activation by endogenous and exogenously expressed ß-catenin, including the constitutively active form of ß-catenin that contains a Serine37Alanine mutation. Our data also indicate that proliferation of LCSC and HCC lines is inhibited by FH535 in a dose-dependent manner, and that this correlates with a decrease in the percentage of cells in S phase. Finally, we also show that expression of two well-characterized targets of ß-catenin, Cyclin D1 and Survivin, is reduced by FH535. Taken together, this data indicates that FH535 has potential therapeutic value in treatment of liver cancer. Importantly, these results suggest that this therapy may be effective at several levels by targeting both HCC and LCSC.

Automated enumeration and viability measurement of canine stromal vascular fraction cells using fluorescence-based image cytometry method.

In recent years, the lipoaspirate collected from adipose tissue has been seen as a valuable source of adipose-derived mesenchymal stem cells for autologous cellular therapy. For multiple applications, adipose-derived mesenchymal stem cells are isolated from the stromal vascular fraction (SVF) of adipose tissue. Because the fresh stromal vascular fraction typically contains a heterogeneous mixture of cells, determining cell concentration and viability is a crucial step in preparing fraction samples for downstream processing. Due to a large amount of cellular debris contained in the SVF sample, as well as counting irregularities standard manual counting can lead to inconsistent results. Advancements in imaging and optics technologies have significantly improved the image-based cytometric analysis method. In this work, we validated the use of fluorescence-based image cytometry for SVF concentration and viability measurement, by comparing to standard flow cytometry and manual hemocytometer. The concentration and viability of freshly collected canine SVF samples are analyzed, and the results highly correlated between all three methods, which validated the image cytometry method for canine SVF analysis, and potentially for SVF from other species.

Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression.

Maintenance of intestinal homeostasis requires a healthy relationship between the commensal gut microbiota and the host immune system. Breast milk supplies the first source of antigen-specific immune protection in the gastrointestinal tract of suckling mammals, in the form of secretory IgA (SIgA). SIgA is transported across glandular and mucosal epithelial cells into external secretions by the polymeric Ig receptor (pIgR). Here, a breeding scheme with polymeric Ig receptor-sufficient and -deficient mice was used to study the effects of breast milk-derived SIgA on development of the gut microbiota and host intestinal immunity. Early exposure to maternal SIgA prevented the translocation of aerobic bacteria from the neonatal gut into draining lymph nodes, including the opportunistic pathogen Ochrobactrum anthropi. By the age of weaning, mice that received maternal SIgA in breast milk had a significantly different gut microbiota from mice that did not receive SIgA, and these differences were magnified when the mice reached adulthood. Early exposure to SIgA in breast milk resulted in a pattern of intestinal epithelial cell gene expression in adult mice that differed from that of mice that were not exposed to passive SIgA, including genes associated with intestinal inflammatory diseases in humans. Maternal SIgA was also found to ameliorate colonic damage caused by the epithelial-disrupting agent dextran sulfate sodium. These findings reveal unique mechanisms through which SIgA in breast milk may promote lifelong intestinal homeostasis, and provide additional evidence for the benefits of breastfeeding.

Regulation of the mucosal phenotype in dendritic cells by PPARγ: role of tissue microenvironment.

Mucosal DCs play a critical role in tissue homeostasis. Several stimuli can induce a mucosal phenotype; however, molecular pathways that regulate development of mucosal DC function are relatively unknown. This study sought to determine whether PPARγ contributes to the development of the "mucosal" phenotype in mouse DCs. Experiments demonstrated that PPARγ activation in BMDCs induced an immunosuppressive phenotype in which BMDCs had reduced expression of MHC class II and costimulatory molecules, increased IL-10 secretion, and reduced the ability to induce CD4 T cell proliferation. Activation of PPARγ enhanced the ability of BMDC to polarize CD4 T cells toward iTregs and to induce T cell expression of the mucosal homing receptor, CCR9. Activation of PPARγ increased the ability of BMDCs to induce T cell-independent IgA production in B cells. BMDCs from PPARγ(ΔDC) mice displayed enhanced expression of costimulatory molecules, enhanced proinflammatory cytokine production, and decreased IL-10 synthesis. Contrary to the inflammatory BMDC phenotype in vitro, PPARγ(ΔDC) mice showed no change in the frequency or phenotype of mDC in the colon. In contrast, mDCs in the lungs were increased significantly in PPARγ(ΔDC) mice. A modest increase in colitis severity was observed in DSS-treated PPARγ(ΔDC) mice compared with control. These results indicate that PPARγ activation induces a mucosal phenotype in mDCs and that loss of PPARγ promotes an inflammatory phenotype. However, the intestinal microenvironment in vivo can maintain the mucosal DC phenotype of via PPARγ-independent mechanisms.

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation.

BACKGROUND: Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). MATERIALS AND METHODS: Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. RESULTS: Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 µM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 µM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). CONCLUSIONS: LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation.

Bioactive lipids and cationic antimicrobial peptides as new potential regulators for trafficking of bone marrow-derived stem cells in patients with acute myocardial infarction.

Acute myocardial infarction (AMI) triggers mobilization of stem cells from bone marrow (BM) into peripheral blood (PB). Based on our observation that the bioactive sphingophospholipids, sphingosine-1 phosphate (S1P), and ceramide-1 phosphate (C1P) regulate trafficking of hematopoietic stem cells (HSCs), we explored whether they also direct trafficking of non-hematopoietic stem cells (non-HSCs). We detected a 3-6-fold increase in circulating CD34+, CD133+, and CXCR4+ lineage-negative (Lin-)/CD45- cells that are enriched in non-HSCs [including endothelial progenitors (EPCs) and very small embryonic-like stem cells (VSELs)] in PB from AMI patients (P<0.05 vs. controls). Concurrently, we measured a ∼3-fold increase in S1P and C1P levels in plasma from AMI patients. At the same time, plasma obtained at hospital admission and 6 h after AMI strongly chemoattracted human BM-derived CD34+/Lin- and CXCR4+/Lin- cells in Transwell chemotaxis assays. This effect of plasma was blunted after depletion of S1P level by charcoal stripping and was further inhibited by the specific S1P1 receptor antagonist such as W146 and VPC23019. We also noted that the expression of S1P receptor 1 (S1P1), which is dominant in naïve BM, is reduced after the exposure to S1P at concentrations similar to the plasma S1P levels in patients with AMI, thus influencing the role of S1P in homing to the injured myocardium. Therefore, we examined mechanisms, other than bioactive lipids, that may contribute to the homing of BM non-HSCs to the infarcted myocardium. Hypoxic cardiac tissue increases the expression of cathelicidin and ß-2 defensin, which could explain why PB cells isolated from patients with AMI migrated more efficiently to a low, yet physiological, gradient of stromal-derived factor-1 in Transwell migration assays. Together, these observations suggest that while elevated S1P and C1P levels early in the course of AMI may trigger mobilization of non-HSCs into PB, cathelicidin and ß-2 defensin could play an important role in their homing to damaged myocardium.

Inflammatory cytokine gene expression in mesenteric adipose tissue during acute experimental colitis.

BACKGROUND: Production of inflammatory cytokines by mesenteric adipose tissue (MAT) has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Animal models of colitis have demonstrated inflammatory changes within MAT, but it is unclear if these changes occur in isolation or as part of a systemic adipose tissue response. It is also unknown what cell types are responsible for cytokine production within MAT. The present study was designed to determine whether cytokine production by MAT during experimental colitis is depot-specific, and also to identify the source of cytokine production within MAT. METHODS: Experimental colitis was induced in 6-month-old C57BL/6 mice by administration of dextran sulfate sodium (2% in drinking water) for up to 5 days. The induction of cytokine mRNA within various adipose tissues, including mesenteric, epididymal, and subcutaneous, was analyzed by qRT-PCR. These adipose tissues were also examined for histological evidence of inflammation. The level of cytokine mRNA during acute colitis was compared between mature mesenteric adipocytes, mesenteric stromal vascular fraction (SVF), and mesenteric lymph nodes. RESULTS: During acute colitis, MAT exhibited an increased presence of infiltrating mononuclear cells and fibrotic structures, as well as decreased adipocyte size. The mRNA levels of TNF-α, IL-1ß, and IL-6 were significantly increased in MAT but not other adipose tissue depots. Within the MAT, induction of these cytokines was observed mainly in the SVF. CONCLUSIONS: Acute experimental colitis causes a strong site-specific inflammatory response within MAT, which is mediated by cells of the SVF, rather than mature adipocytes or mesenteric lymph nodes.

Toward a molecular pathogenic pathway for Yersinia pestis YopM.

YopM is one of the six "effector Yops" of the human-pathogenic Yersinia, but its mechanism has not been defined. After delivery to J774A.1 monocyte-like cells, YopM can rapidly bind and activate the serine/threonine kinases RSK1 and PRK2. However, in infected mice, effects of Y. pestis YopM have been seen only after 24-48 h post-infection (p.i.). To identify potential direct effects of YopM in-vivo we tested for effects of YopM at 1 h and 16-18 h p.i. in mice infected systemically with 10(6) bacteria. At 16 h p.i., there was a robust host response to both parent and ΔyopM-1 Y. pestis KIM5. Compared to cells from non-infected mice, CD11b(+) cells from spleens of infected mice produced more than 100-fold greater IFNγ. In the corresponding sera there were more than 100-fold greater amounts of IFNγ, G-CSF, and CXCL9, as well as more than 10-fold greater amounts of IL-6, CXCL10, and CXCL1. The only YopM-related differences were slightly lower CXCL10 and IL-6 in sera from mice infected 16 h with parent compared to ΔyopM-1 Y. pestis. Microarray analysis of the CD11b(+) cells did not identify consistent transcriptional differences of ≥4-fold at 18 h p.i. However, at 1 h p.i. mRNA for early growth response transcription factor 1 (Egr1) was decreased when YopM was present. Bone marrow-derived macrophages infected for 1 h also expressed lower Egr1 message when YopM was present. Infected J774A.1 cells showed greater expression of Egr1 at 1 h p.i. when YopM was present, but this pattern reversed at 3 h. At 6 h p.i., Cxcl10 mRNA was lower in parent-strain infected cells. We conclude that decreased Egr1 expression is a very early transcriptional effect of YopM and speculate that a pathway may exist from RSK1 through Egr1. These studies revealed novel early transcriptional effects of YopM but point to a time after 18 h of infection when critical transitional events lead to later major effects on cytokine gene transcription.

Multifactorial patterns of gene expression in colonic epithelial cells predict disease phenotypes in experimental colitis.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) is complex and the need to identify molecular biomarkers is critical. Epithelial cells play a central role in maintaining intestinal homeostasis. We previously identified five "signature" biomarkers in colonic epithelial cells (CEC) that are predictive of disease phenotype in Crohn's disease. Here we investigate the ability of CEC biomarkers to define the mechanism and severity of intestinal inflammation. METHODS: We analyzed the expression of RelA, A20, pIgR, tumor necrosis factor (TNF), and macrophage inflammatory protein (MIP)-2 in CEC of mice with dextran sodium sulfate (DSS) acute colitis or T-cell-mediated chronic colitis. Factor analysis was used to combine the five biomarkers into two multifactorial principal components (PCs). PC scores for individual mice were correlated with disease severity. RESULTS: For both colitis models, PC1 was strongly weighted toward RelA, A20, and pIgR, and PC2 was strongly weighted toward TNF and MIP-2, while the contributions of other biomarkers varied depending on the etiology of inflammation. Disease severity was correlated with elevated PC2 scores in DSS colitis and reduced PC1 scores in T-cell transfer colitis. Downregulation of pIgR was a common feature observed in both colitis models and was associated with altered cellular localization of pIgR and failure to transport IgA. CONCLUSIONS: A multifactorial analysis of epithelial gene expression may be more informative than examining single gene responses in IBD. These results provide insight into the homeostatic and proinflammatory functions of CEC in IBD pathogenesis and suggest that biomarker analysis could be useful for evaluating therapeutic options for IBD patients.

Bone marrow dendritic cell-mediated regulation of TLR and B cell receptor signaling in B cells.

Dendritic cells (DCs) play an essential role in regulation of immune responses. In the periphery, Ag presentation by DCs is critical for adaptive responses; for this reason, DCs are often targets of adjuvants that enhance vaccine responses. Activated mature DCs enhance B cell activation and differentiation by providing cytokines like BAFF and a proliferation-inducing ligand. However, the role of immature DCs in B cell tolerance is not well studied. Recently, mouse immature bone marrow-derived DCs (iBMDCs) have been shown to suppress anti-IgM-induced B cell activation. In this study, we tested the ability of mouse DCs to modulate B cell functions during TLR activation. We found that iBMDCs potently suppressed proliferation and differentiation of various B cell subsets on TLR stimulation. However, iBMDCs did not affect CD40-mediated B cell activation. Optimal suppression of B cell activation by iBMDCs required cell contact via the CD22 receptor on B cells. The B cell suppression was a property of iBMDCs or DCs resident in the bone marrow (BM), but not mature BM-derived DCs or DCs resident in the spleen. Presence of iBMDCs also enhanced the Ag-induced apoptotic response of BM B cells, suggesting that the suppressive effects of iBMDCs may have a role in B cell tolerance.

Adiponectin and plant-derived mammalian adiponectin homolog exert a protective effect in murine colitis.

BACKGROUND: Hypoadiponectinemia has been associated with states of chronic inflammation in humans. Mesenteric fat hypertrophy and low adiponectin have been described in patients with Crohn's disease. We investigated whether adiponectin and the plant-derived homolog, osmotin, are beneficial in a murine model of colitis. METHODS: C57BL/6 mice were injected (i.v.) with an adenoviral construct encoding the full-length murine adiponectin gene (AN+DSS) or a reporter-LacZ (Ctr and V+DSS groups) prior to DSS colitis protocol. In another experiment, mice with DSS colitis received either osmotin (Osm+DSS) or saline (DSS) via osmotic pumps. Disease progression and severity were evaluated using body weight, stool consistency, rectal bleeding, colon lengths, and histology. In vitro experiments were carried out in bone marrow-derived dendritic cells. RESULTS: Mice overexpressing adiponectin had lower expression of proinflammatory cytokines (TNF, IL-1ß), adipokines (angiotensin, osteopontin), and cellular stress and apoptosis markers. These mice had higher levels of IL-10, alternative macrophage marker, arginase 1, and leukoprotease inhibitor. The plant adiponectin homolog osmotin similarly improved colitis outcome and induced robust IL-10 secretion. LPS induced a state of adiponectin resistance in dendritic cells that was reversed by treatment with PPARγ agonist and retinoic acid. CONCLUSION: Adiponectin exerted protective effects during murine DSS colitis. It had a broad activity that encompassed cytokines, chemotactic factors as well as processes that assure cell viability during stressful conditions. Reducing adiponectin resistance or using plant-derived adiponectin homologs may become therapeutic options in inflammatory bowel disease.

Accumulation of CD4+ T cells in the colon of CsA-treated mice following myeloablative conditioning and bone marrow transplantation.

Syngeneic graft vs. host disease (SGVHD) was first described as a graft vs. host disease-like syndrome that developed in rats following syngeneic bone marrow transplantation (BMT) and cyclosporin A (CsA) treatment. SGVHD can be induced by reconstitution of lethally irradiated mice with syngeneic bone marrow cells followed by 21 days of treatment with the immunosuppressive agent CsA. Clinical symptoms of the disease appear 2-3 wk following cessation of CsA therapy, and disease-associated inflammation occurs primarily in the colon and liver. CD4(+) T cells have been shown to play an important role in the inflammatory response observed in the gut of SGVHD mice. Time-course studies revealed a significant increase in migration of CD4(+) T cells into the colon during CsA therapy, as well as significantly elevated mRNA levels of TNF-α, proinflammatory chemokines, and cell adhesion molecules in colonic tissue of CsA-treated animals compared with BMT controls, as early as day 14 post-BMT. Homing studies revealed a greater migration of labeled CD4(+) T cells into the gut of CsA-treated mice at day 21 post-BMT than control animals via CsA-induced upregulation of mucosal addressin cell adhesion molecule. This study demonstrates that, during the 21 days of immunosuppressive therapy, functional mechanisms are in place that result in increased homing of CD4(+) T effector cells to colons of CsA-treated mice.

Distinct CCR2(+) Gr1(+) cells control growth of the Yersinia pestis ΔyopM mutant in liver and spleen during systemic plague.

We are using a systemic plague model to identify the cells and pathways that are undermined by the virulence protein YopM of the plague bacterium Yersinia pestis. In this study, we pursued previous findings that Gr1(+) cells are required to selectively limit growth of ΔyopM Y. pestis and that CD11b(+) cells other than polymorphonuclear leukocytes (PMNs) are selectively lost in spleens infected with parent Y. pestis. When PMNs were ablated from mice, ΔyopM Y. pestis grew as well as the parent strain in liver but not in spleen, showing that these cells are critical for controlling growth of the mutant in liver but not spleen. In mice lacking expression of the chemokine receptor CCR2, wild-type growth was restored to ΔyopM Y. pestis in both organs. In spleen, the Gr1(+) cells differentially recruited by parent and ΔyopM Y. pestis infections were CCR2(+) Gr1(+) CD11b(+) CD11c(Lo-Int) MAC3(+) iNOS(+) (inducible nitric oxide synthase-positive) inflammatory dendritic cells (iDCs), and their recruitment to spleen from blood was blocked when YopM was present in the infecting strain. Consistent with influx of iDCs being affected by YopM in spleen, the growth defect of the ΔyopM mutant was relieved by the parent Y. pestis strain in a coinfection assay in which the parent strain could affect the fate of the mutant in trans. In a mouse model of bubonic plague, CCR2 also was shown to be required for ΔyopM Y. pestis to show wild-type growth in skin. The data imply that YopM's pathogenic effect indirectly undermines signaling through CCR2. We propose a model for how YopM exerts its different effects in liver and spleen.

Evidence of mobilization of pluripotent stem cells into peripheral blood of patients with myocardial ischemia.

OBJECTIVE: The ischemic myocardium releases multiple chemotactic factors responsible for the mobilization and recruitment of bone marrow-derived cells to injured myocardium. However, the mobilization of primitive pluripotent stem cells (PSCs) enriched in very small embryonic-like stem cells (VSELs) in various cardiac ischemic scenarios is not well understood. MATERIALS AND METHODS: Fifty-four ischemic heart disease patients, including subjects with stable angina, non-ST elevation myocardial infarction, and ST elevation myocardial infarction (STEMI) and 12 matched controls were enrolled. The absolute numbers of circulating stem/primitive cells in samples of peripheral blood (PB) were quantitated by ImageStream analysis and conventional flow cytometry. Gene expression of PSC (Oct-4 and Nanog), early cardiomyocyte (Nkx-2.5 and GATA-4), and endothelial (von Willebrand factor) markers was analyzed by real-time polymerase chain reaction. RESULTS: The absolute numbers of PSCs, stem cell populations enriched in VSELs, and hematopoietic stem cells present in PB were significantly higher in STEMI patients at presentation and declined over time. There was a corresponding increase in pluripotent, cardiac, and endothelial gene expression in unfractionated PB cells and sorted PB-derived primitive CD34(+) cells. The absolute numbers of circulating VSELs and hematopoietic stem cells in STEMI correlated negatively with patient age. CONCLUSIONS: Myocardial ischemia mobilizes primitive PSCs including pluripotent VSELs into the circulation. The peak of mobilization occurs within 12 hours in patients presenting with STEMI, which may represent a therapeutic window for future clinical applications. Reduced stem cell mobilization with advancing age could explain, in part, the observation that age is associated with poor prognosis in patients with myocardial infarction.

Association between chronic liver and colon inflammation during the development of murine syngeneic graft-versus-host disease.

The murine model of cyclosporine A (CsA)-induced syngeneic graft-versus-host disease (SGVHD) is a bone marrow (BM) transplantation model that develops chronic colon inflammation identical to other murine models of CD4(+) T cell-mediated colitis. Interestingly, SGVHD animals develop chronic liver lesions that are similar to the early peribiliary inflammatory stages of clinical chronic liver disease, which is frequently associated with inflammatory bowel disease (IBD). Therefore, studies were initiated to investigate the chronic liver inflammation that develops in the SGVHD model. To induce SGVHD, mice were lethally irradiated, reconstituted with syngeneic BM, and treated with CsA. All of the SGVHD animals that developed colitis also develop chronic liver inflammation. Liver samples from control and SGVHD animals were monitored for tissue pathology, RNA for inflammatory mediators, and phenotypic analysis and in vitro reactivity of the inflammatory infiltrate. Diseased animals developed lesions of intrahepatic and extrahepatic bile ducts. Elevated levels of mRNA for molecules associated with chronic liver inflammation, including mucosal cellular adhesion molecule -1, the chemokines CCL25, CCL28, CCR9, and T(H)1- and T(H)17-associated cytokines were observed in livers of SGVHD mice. CD4(+) T cells were localized to the peribiliary region of the livers of diseased animals, and an enhanced proliferative response of liver-associated mononuclear cells against colonic bacterial antigens was observed. The murine model of SGVHD colitis may be a valuable tool to study the entero-hepatic linkage between chronic colon inflammation and inflammatory liver disease.