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.

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.

Anti-IL-23p19 therapy inhibits the adoptive transfer of syngeneic graft-versus-host disease.

Syngeneic graft-versus-host disease (SGVHD), a chronic inflammatory disease, develops following irradiation, syngeneic bone marrow transplantation (BMT) and treatment with the immunosuppressive agent cyclosporine A (CsA). We have shown that TH1 and TH17 cytokine responses are increased during the development of SGVHD. The current study was designed to further investigate the involvement of TH17 immunity in SGVHD-associated colitis. IL-23 is a TH17 cytokine responsible for maintaining the effector functions of TH17 cells. The administration of anti-mouse IL-23p19 was shown to significantly reduce the clinical symptoms of primary and secondary SGVHD-associated colitis resulting in a significant reduction in both TH1 and TH17 associated cytokine expression. These results demonstrate that the TH17-associated cytokine, IL-23, may prove to be a beneficial therapeutic target in the treatment of chronic colon inflammation.

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.

A gut feeling about murine syngeneic GVHD.

Murine syngeneic graft-versus-host disease (SGVHD) results in chronic colon and liver inflammation following syngeneic bone marrow transplantation (BMT) and treatment with the calcineurin inhibitor, cyclosporine A (CsA). SGVHD was initially thought to arise as a result of an autoreactive immune response, but more recently it has been shown that enhanced antimicrobial responses develop in SGVHD mice. Consequently, we performed studies to analyze the role of the microbiota in the development of murine SGVHD. Treatment with broad-spectrum antibiotics eliminated disease-associated inflammatory immune responses and pathology, linking the role of the microbiota and microbial-specific immunity to the development of murine SGVHD. In a broader context, these results bring into question the role that anti-microbial immune responses play in post-transplant immune pathologies that develop following allogeneic stem cell transplantation and use of calcineurin inhibitors.

Murine syngeneic graft-versus-host disease is responsive to broad-spectrum antibiotic therapy.

Murine syngeneic graft-versus-host disease (SGVHD) initiates colon and liver inflammation following lethal irradiation, reconstitution with syngeneic bone marrow transplantation, and therapy with the immunosuppressive agent cyclosporine A. Previous studies have demonstrated that the inducible disease is mediated by CD4(+) T cells with increased reactivity of peripheral and liver-associated lymphocytes against intestinal microbial Ags. In the current report, studies were performed to analyze the specificity of the CD4(+) T cell response of T cells isolated from diseased animals and to determine the in vivo role of the microbiota to the development of SGVHD. Increased major histocompatibility Ag (MHC) class II-restricted responsiveness of SGVHD CD4(+) T cells against microbial Ags isolated from the ceca of normal animals was observed. The enhanced proliferative response was observed in the CD62L(-) memory population of CD4(+) T cells. To determine the role of the bacterial microbiota in the development of murine SGVHD, control and CsA-treated bone marrow transplantation animals were treated with broad-spectrum antibiotics (metronidazole, ciprofloxacin) after transplantation. Cyclosporine A-treated animals that were given antibiotic therapy failed to develop clinical symptoms and pathological lesions in the target tissues characteristic of SGVHD. Furthermore, the reduction in intestinal bacteria resulted in the elimination of the enhanced antimicrobial CD4(+) T cell response and significantly reduced levels of the inflammatory cytokines, IFN-γ, IL-17, and TNF-α. The elimination of the disease-associated inflammatory immune responses and pathology by treatment with broad-spectrum antibiotics definitively links the role of the microbiota and microbial-specific immunity to the development of murine SGVHD.

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.

Development of a T(H)17 immune response during the induction of murine syngeneic graft-versus-host disease.

Syngeneic graft-versus-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow (BM) and treatment with a 21 day course of the immunosuppressive agent cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2-3 weeks post-CsA with inflammation occurring in the colon and liver. Previously we have demonstrated that CD4(+) T cells and a T helper cell type 1 cytokine response (T(H)1) are involved in the development of SGVHD associated intestinal inflammation. Studies have recently discovered an additional T cell lineage that produces IL-17 and is termed T(H)17. It has been suggested that inflammatory bowel disease is a result of a T(H)17 response rather than a T(H)1 response. This study was designed to investigate T(H)17 involvement in SGVHD-associated colitis. Following induction of SGVHD, the levels of T(H)17 and T(H)1 cytokine mRNA and protein were measured in control and SGVHD animals. In vivo cytokine neutralization was performed to determine the role of the prototypic T(H)17 cytokine, IL-17, in the disease process. We found that during CsA-induced murine SGVHD there was an increase in both T(H)17 and T(H)1 mRNA and cytokines within the colons of diseased mice. The administration of an anti-mouse IL-17A mAb did not alter the course of disease. However, neutralization of IL-17A resulted in an increased production of IL-17F, a related family member, with an overlapping range of effector activities. These results demonstrate that in the pathophysiology of SGVHD, there is a redundancy in the T(H)17 effector molecules that mediate the development of SGVHD.

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.

IL-10 promotes resistance to apoptosis and metastatic potential in lung tumor cell lines.

Treatment of non-small cell lung carcinoma (NSCLC) remains at a disappointingly low success rate. Not only is metastatic spread common in NSCLC, but therapeutic success decreases dramatically once metastases are present. Understanding factors which contribute to poor prognosis in NSCLC is critical for development of more successful therapeutic approaches. Interleukin-10 (IL-10) expression has been shown in several studies to correlate with a poorer prognosis in NSCLC; however, the mechanisms by which IL-10 affects lung tumor growth and metastases are unclear. The goal of this study was to evaluate the effects of tumor-derived IL-10 on the growth and metastasis of lung cancer cells in a murine model. Lewis lung carcinoma cells were stably transfected with the chicken ovalbumin gene (cOVA) as a model tumor antigen (LL43 tumor cells) and subsequently transfected with the murine IL-10 gene (LL43-10 tumor cells). Subcutaneous growth of the LL43 tumor cells was not affected by expression of IL-10. However, LL43-10 tumors had a fourfold increase in tumor microvessel density, as indicated by CD31 staining. Metastatic potential was also increased in IL-10-expressing lung tumor cells, leading to a greater number of tumor cells in lymph nodes draining the primary tumor site. Finally, exposure of Lewis lung tumor cells in vitro to exogenous IL-10 dramatically increased their resistance to UV-induced apoptosis. These results indicate that a primary effect of IL-10 on lung cancer cells may be to increase their metastatic potential by promoting angiogenesis and resistance to apoptosis.

Gr1+ cells control growth of YopM-negative yersinia pestis during systemic plague.

YopM, a protein toxin of Yersinia pestis, is necessary for virulence in a mouse model of systemic plague. We previously reported YopM-dependent natural killer (NK) cell depletion from blood and spleen samples of infected mice. However, in this study we found that infection with Y. pestis KIM5 (YopM(+)) caused depletion of NK cells in the spleen, but not in the liver, and antibody-mediated ablation of NK cells had no effect on bacterial growth. There was no YopM-associated effect on the percentage of dendritic cells (DCs) or polymorphonuclear leukocytes (PMNs) in the early stage of infection; however, there was a YopM-associated effect on PMN integrity and on the influx of monocytes into the spleen. Ablation of Gr1(+) cells caused loss of the growth defect of YopM(-) Y. pestis in both the liver and spleen. In contrast, ablation of macrophages/DCs inhibited growth of both parent and mutant bacteria, accompanied by significantly fewer lesion sites in the liver. These results point toward PMNs and inflammatory monocytes as major cell types that control growth of YopM(-) Y. pestis. Infection with fully virulent Y. pestis CO92 and a YopM(-) derivative by intradermal and intranasal routes showed that the absence of YopM significantly increased the 50% lethal dose only in the intradermal model, suggesting a role for YopM in bubonic plague, in which acute inflammation occurs soon after infection.

Inhibition of interleukin-10 signaling in lung dendritic cells by toll-like receptor 4 ligands.

The homeostatic microenvironment in lung is immunosuppressive and interleukin-10 (IL-10) helps maintain this microenvironment. Despite constitutive production of IL-10 in normal lung, macrophages (MØs) and dentritic cells (DCs) remain capable of responding to microorganisms, suggesting that these innate immune cells have a mechanism to override the immunosuppressive effects of IL-10. Prior studies by the authors revealed that Toll-like receptor (TLR) ligands inhibit IL-10 receptor signaling in alveolar macrophages (AMØs), thereby obviating the immunosuppressive activity of IL-10. This report compares the immunologic phenotypes of AMØs and lung DCs and their ability to respond to IL-10 following exposure to microbial stimuli. IL-10 was constitutively produced by normal lung epithelium and exposure to lipopolysaccharide (LPS) in vivo increased the expression of IL-10 during the first 24 hours. AMØs constitutively produced IL-10 mRNA, whereas both AMØs and LDCs constitutively expressed IL-12 mRNA. AMØs and LDCs, as well as bone marrow-derived MØs and DCs, had reduced capacity to activate STAT3 in response to IL-10 if pretreated with LPS. Inhibition was not associated with decreased expression of IL-10 receptor (IL-10R) and was dependent on the MyD88 signaling pathway. These results demonstrate a common underlying regulatory mechanism in both DCs and MØs by which microbial stimuli can override the immunosuppressive effect of constitutive IL-10 production in the lung.

Suppression of experimental colitis in mice by CD11c+ dendritic cells.

BACKGROUND: The innate immune system serves a critical role in homeostasis of the gastrointestinal (GI) tract. Both macrophages (MØs) and dendritic cells (DCs) have been shown to have pathogenic roles in animal models of inflammatory bowel disease. However, studies by several labs have established that resident MØs and DCs within the normal GI tract maintain an immunosuppressive phenotype compared to that seen in other peripheral sites. Recent studies by our lab demonstrated that the depletion of both MØs and DCs before the initiation of dextran sodium sulfate (DSS)-induced colitis resulted in exacerbation of disease, partly caused by increased neutrophil influx. METHODS/RESULTS: In this current report, DSS-induced colitis was shown to be significantly more severe when DCs were selectively depleted in mice as indicated by changes in weight loss, stool consistency, rectal bleeding, and histopathology. In contrast to enhanced colitis in MØ/DC-depleted mice, which was associated with increased neutrophil influx, increased colitis in DC-depleted mice was not associated with an increase in neutrophils in the colon, as shown by CXCL1 chemokine levels and myeloperoxidase (MPO) activity. However, increased IL-6 gene and protein expression in colon tissues correlated positively with increased colitis severity in DC-depleted mice compared to colitis in DC-intact mice. CONCLUSIONS: This study demonstrates that resident DCs can suppress the severity of acute DSS colitis and that regulation of IL-6 production may contribute to DC-mediated control of intestinal inflammation.

Adoptive transfer of murine syngeneic graft-vs.-host disease by CD4+ T cells.

Syngeneic graft-vs.-host disease (SGVHD) develops in rodents following the treatment of lethally irradiated, bone marrow (BM) reconstituted animals with a short course of the immunosuppressive agent cyclosporine A (CsA). Using an in vivo depletion approach, we recently demonstrated that CD4(+), but not CD8(+), T cells participated in inducing SGVHD. Studies were therefore undertaken to adoptively transfer SGVHD into lethally irradiated, syngeneic BM reconstituted secondary recipients. Whole T cell populations as well as purified CD4(+)T cells isolated from SGVHD, but not normal or transplant control, animals mediated the transfer of SGVHD into secondary recipients. These cells have an apparent specificity for enteric bacterial antigens. The pathologic process that developed was identical to that observed in the animals with de novo SGVHD after syngeneic BMT and CsA therapy. It was shown that a radiation-sensitive mechanism prevented the transfer of SGVHD into normal, nonirradiated secondary recipients. The ability to reproducibly transfer SGVHD into secondary recipients will enhance our ability to study regulatory mechanisms that are altered during CsA therapy and permit the development of murine CsA-induced SGVHD.

Induction of murine syngeneic graft-versus-host disease by cells of recipient origin.

BACKGROUND: Syngeneic graft-versus-host disease (SGVHD) develops after lethal irradiation, reconstitution with syngeneic bone marrow (BM), and treatment with a 21-day course of the immunosuppressant cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2-3 weeks after CsA treatment, with inflammation in the colon and liver. It has been demonstrated that CD4+ T cells and a T helper cell type 1 cytokine response (Th1) are involved in the development of SGVHD associated intestinal inflammation. The immune response associated with SGVHD is thought to be the result of the reconstitution of the recipient immune system with the syngeneic donor BM. However, definitive studies have not addressed this issue experimentally. METHODS: To determine the origin of the effector cells that participate in SGVHD, C3H/HeN recipient mice were lethally irradiated and transplanted with BM from normal immunocompetent mice or from immunodeficient, severe combined immune deficient, or Rag-2-/- animals. RESULTS: CsA-treated animals, but not control animals, developed inflammation characteristic of SGVHD in the colon and liver regardless of the source of the donor marrow. Furthermore, immunologically, all CsA treated animals responded similarly with increased production of inflammatory cytokines and an increase in activated CD4+ T cells in the periphery and colon relative to controls. CONCLUSION: These results demonstrate that after lethal irradiation and in the absence of donor T cells, T cells of recipient origin can expand and mediate the induction of CsA-induced SGVHD.

Suppression of experimental colitis by intestinal mononuclear phagocytes.

The contribution of innate immunity to inflammatory bowel disease (IBD) remains an area of intense interest. Macrophages (MØ) and dendritic cells (DC) are considered important factors in regulating the onset of IBD. The goal of this study was to determine if intestinal mononuclear phagocytes (iMNP) serve a pathological or protective role in dextran sulfate sodium (DSS)-induced colitis in mice. Using a conditional MØ/DC depletion transgenic mouse line--MØ Fas-induced apoptosis--to systemically deplete iMNP, DSS colitis histopathology was shown to be more severe in MØ/DC-depleted compared with MØ/DC-intact mice. Similarly, localized iMNP depletion by clodronate-encapsulated liposomes into C57BL/6, BALB/c, and CB.17/SCID mice also increased DSS colitis severity, as indicated by increased histopathology, weight loss, rectal bleeding, decreased stool consistency, and colon length compared with MØ/DC-intact, DSS-treated mice. Histology revealed that iMNP depletion during DSS treatment led to increased neutrophilic inflammation, increased epithelial injury, and enhanced mucin depletion from Goblet cells. iMNP depletion did not further elevate DSS-induced expression of TNF-alpha and IFN-gamma mRNA but significantly increased expression of CXCL1 chemokine mRNA. Myeloperoxidase activity was increased in colons of MØ/DC-depleted, DSS-treated mice, compared with DSS alone, coincident with increased neutrophil infiltration in diseased colons. Neutrophil depletion combined with MØ/DC depletion prevented the increase in DSS colitis severity compared with MØ/DC depletion alone. This study demonstrates that iMNP can serve a protective role during development of acute colitis and that protection is associated with MØ/DC-mediated down-regulation of neutrophil infiltration.

A critical role for the inflammatory response in a mouse model of preneoplastic progression.

The tumor microenvironment, which includes inflammatory cells, vasculature, extracellular matrix, and fibroblasts, is a critical mediator of neoplastic progression and metastasis. Using an inducible transgenic mouse model of preneoplastic progression in the mammary gland, we discovered that activation of inducible fibroblast growth factor receptor-1 (iFGFR1) in the mammary epithelium rapidly increased the expression of several genes involved in the inflammatory response. Further analysis revealed that iFGFR1 activation induced recruitment of macrophages to the epithelium and continued association with the alveolar hyperplasias that developed following long-term activation. Studies using HC-11 mammary epithelial cells showed that iFGFR1-induced expression of the macrophage chemoattractant osteopontin was required for macrophage recruitment in vitro. Finally, conditional depletion of macrophages inhibited iFGFR1-mediated epithelial cell proliferation and lateral budding. These findings show that inflammatory cells, specifically macrophages, are critical for mediating early events in an inducible transgenic mouse model of preneoplastic progression.

Development of peritoneal adhesions in macrophage depleted mice.

BACKGROUND: We present a new mouse model for the study of peritoneal adhesions using macrophage Fas-induced apoptosis (Mafia) transgenic mice expressing a Fas-FKBP construct under control of the murine c-fms promoter. Mafia mice allow systemic macrophage depletion by dimerization of Fas with a synthetic dimerizer, AP20187. Results demonstrate that macrophage depletion in Mafia mice induces peritoneal adhesion formation when the peritoneal cavity is also exposed to an irritant. The Mafia mouse model presents a reproducible, non-surgical approach for research in adhesion formation and prevention. MATERIALS AND METHODS: Mafia mice were treated with AP20187 using an intravenous (i.v.) or intraperitoneal (i.p.) injection. Control groups included mock-treated Mafia mice and both AP20187 and mock-treated wild type mice. Seven days after treatment, mice were observed for the presence of adhesions. RESULTS: After i.p. injection with AP20187, 76% of Mafia mice developed adhesions whereas none of the mock-treated Mafia or wild-type mice developed adhesions, and only one AP20187-treated wild-type mouse (5.8%) developed a mild adhesion. Mafia mice treated with AP20187 i.v. exhibited macrophage depletion not significantly different than i.p. treated mice, but did not develop adhesions. In contrast, Mafia mice treated with AP20187 i.v. developed adhesions when diluent was also injected into the peritoneal cavity, whereas i.p diluent alone had no effect. CONCLUSION: Macrophage depletion, combined with a peritoneal irritant, results in peritoneal adhesion formation in transgenic Mafia mice. Macrophages appear to play a protective role in the development and/or repair of peritoneal adhesions.