Apoptotic Cell-Directed Resolution of Lung Inflammation Requires Myeloid αv Integrin-Mediated Induction of Regulatory T Lymphocytes.

Intratracheal instillation of apoptotic cells enhances resolution of experimental lung inflammation by incompletely understood mechanisms. We report that this intervention induces functional regulatory T lymphocytes (Tregs) in mouse lung experimentally inflamed by intratracheal administration of lipopolysaccharide. Selective depletion demonstrated that Tregs were necessary for maximal apoptotic cell-directed enhancement of resolution, and adoptive transfer of additional Tregs was sufficient to promote resolution without administering apoptotic cells. After intratracheal instillation, labeled apoptotic cells were observed in most CD11c+CD103+ myeloid dendritic cells migrating to mediastinal draining lymph nodes and bearing migratory and immunoregulatory markers, including increased CCR7 and ß8 integrin (ITGB8) expression. In mice deleted for αv integrin in the myeloid line to reduce phagocytosis of dying cells by CD103+ dendritic cells, exogenous apoptotic cells failed to induce transforming growth factor-ß1 expression or Treg accumulation and failed to enhance resolution of lipopolysaccharide-induced lung inflammation. We conclude that in murine lung, myeloid phagocytes encountering apoptotic cells can deploy αv integrin-mediated mechanisms to induce Tregs and enhance resolution of acute inflammation.

ß8 Integrin Expression and Activation of TGF-ß by Intestinal Dendritic Cells Are Determined by Both Tissue Microenvironment and Cell Lineage.

Activation of TGF-ß by dendritic cells (DCs) expressing αvß8 integrin is essential for the generation of intestinal regulatory T cells (Tregs) that in turn promote tolerance to intestinal Ags. We have recently shown that αvß8 integrin is preferentially expressed by CD103(+) DCs and confers their ability to activate TGF-ß and generate Tregs. However, how these DCs become specialized for this vital function is unknown. In this study, we show that ß8 expression is controlled by a combination of factors that include DC lineage and signals derived from the tissue microenvironment and microbiota. Specifically, our data demonstrate that TGF-ß itself, along with retinoic acid and TLR signaling, drives expression of αvß8 in DCs. However, these signals only result in high levels of ß8 expression in cells of the cDC1 lineage, CD8α(+), or CD103(+)CD11b(-) DCs, and this is associated with epigenetic changes in the Itgb8 locus. Together, these data provide a key illustrative example of how microenvironmental factors and cell lineage drive the generation of regulatory αvß8-expressing DCs specialized for activation of TGF-ß to facilitate Treg generation.

A tolerogenic role for Toll-like receptor 9 is revealed by B-cell interaction with DNA complexes expressed on apoptotic cells.

Intracellular protein complexes containing nucleic acids are common targets of autoantibodies in many autoimmune diseases. Central tolerance to these antigens is incomplete, yet nucleosomal DNA is expressed on the surface of cells dying by apoptosis. It is commonly believed that autoimmunity is prevented by the rapid uptake of apoptotic cells (ACs) by neighbors or professional phagocytes to which they deliver anti-inflammatory signals. Self-reactive, innate-like B cells contact and are selected by intracellular antigens expressed on ACs; however, how self-tolerance is maintained is not well understood. Here we report that IL-10 production by B cells, stimulated by contact with ACs, results from the engagement of Toll-like receptor 9 (TLR9) within the B cell after recognition of DNA-containing complexes on the surface of ACs. Until now, TLR9 ligation has been considered an inflammatory signal, but we have confirmed a hitherto unexpected immunoregulatory role by demonstrating the absence of the protective effect of ACs during experimental autoimmune encephalitis (EAE) in TLR9-deficient mice. Human circulating CD27(+) B cells also respond to DNA-bearing ACs, but not to DNase-treated cells, by secreting IL-10. Chronic autoimmune disease may arise if this tolerance mechanism is not reimposed after episodes of inflammation, or if the regulatory B-cell response is subverted.

A blast from the past: clearance of apoptotic cells regulates immune responses.

Apoptosis, which is a programmed and physiological form of cell death, is known to shape the immune system by regulating populations of effector lymphocytes. However, the binding and ingestion of dying cells by monocytes/macrophages and dendritic cells can also influence immune responses markedly by enhancing or suppressing inflammation. Therefore, dead cells, which are a reflection of an organism's immediate past, can control its immunological future.

Genome analysis and CRISPR typing of Salmonella enterica serovar Virchow.

BACKGROUND: Salmonella enterica subsp. enterica serovar Virchow has been recognized as a significant health burden in Asia, Australia and Europe. In addition to its global distribution, S. Virchow is clinically significant due to the frequency at which it causes invasive infections and its association with outbreaks arising from food-borne transmission. Here, we examine the genome of an invasive isolate of S. Virchow SVQ1 (phage type 8) from an outbreak in southeast Queensland, Australia. In addition to identifying new potential genotyping targets that could be used for discriminating between S. Virchow strains in outbreak scenarios, we also aimed to carry out a comprehensive comparative analysis of the S. Virchow genomes. RESULTS: Genome comparisons between S. Virchow SVQ1 and S. Virchow SL491, a previously published strain, identified a high degree of genomic similarity between the two strains with fewer than 200 single nucleotide differences. Clustered Regularly Interspaced Palindromic Repeats (CRISPR) regions were identified as a highly variable region that could be used to discriminate between S. Virchow isolates. We amplified and sequenced the CRISPR regions of fifteen S. Virchow isolates collected from seven different outbreaks across Australia. We observed three allelic types of the CRISPR region from these isolates based on the presence/absence of the spacers and were able to discriminate S. Virchow phage type 8 isolates originating from different outbreaks. A comparison with 27 published Salmonella genomes found that the S. Virchow SVQ1 genome encodes 11 previously described Salmonella Pathogenicity Islands (SPI), as well as additional genomic islands including a remnant integrative conjugative element that is distinct from SPI-7. In addition, the S. Virchow genome possesses a novel prophage that encodes the Type III secretion system effector protein SopE, a key Salmonella virulence factor. The prophage shares very little similarity to the SopE prophages found in other Salmonella serovars suggesting an independent acquisition of sopE. CONCLUSIONS: The availability of this genome will serve as a genome template and facilitate further studies on understanding the virulence and global distribution of the S. Virchow serovar, as well as the development of genotyping methods for outbreak investigations.

11ß-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis.

11ß-Hydroxysteroid dehydrogenase type-1 (11ß-HSD1) converts inert cortisone into active cortisol, amplifying intracellular glucocorticoid action. 11ß-HSD1 deficiency improves cardiovascular risk factors in obesity but exacerbates acute inflammation. To determine the effects of 11ß-HSD1 deficiency on atherosclerosis and its inflammation, atherosclerosis-prone apolipoprotein E-knockout (ApoE-KO) mice were treated with a selective 11ß-HSD1 inhibitor or crossed with 11ß-HSD1-KO mice to generate double knockouts (DKOs) and challenged with an atherogenic Western diet. 11ß-HSD1 inhibition or deficiency attenuated atherosclerosis (74-76%) without deleterious effects on plaque structure. This occurred without affecting plasma lipids or glucose, suggesting independence from classical metabolic risk factors. KO plaques were not more inflamed and indeed had 36% less T-cell infiltration, associated with 38% reduced circulating monocyte chemoattractant protein-1 (MCP-1) and 36% lower lesional vascular cell adhesion molecule-1 (VCAM-1). Bone marrow (BM) cells are key to the atheroprotection, since transplantation of DKO BM to irradiated ApoE-KO mice reduced atherosclerosis by 51%. 11ß-HSD1-null macrophages show 76% enhanced cholesterol ester export. Thus, 11ß-HSD1 deficiency reduces atherosclerosis without exaggerated lesional inflammation independent of metabolic risk factors. Selective 11ß-HSD1 inhibitors promise novel antiatherosclerosis effects over and above their benefits for metabolic risk factors via effects on BM cells, plausibly macrophages.

Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis.

Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.

Preferential expression of integrin αvß8 promotes generation of regulatory T cells by mouse CD103+ dendritic cells.

BACKGROUND & AIMS: Immune responses in the intestine are controlled by regulatory T cells (Tregs), which prevent inflammation in response to commensal bacteria. A specific population of intestinal dendritic cells (DCs), marked by expression of CD103, generate Tregs more efficiently than other DC populations through mechanisms that involve retinoic acid and transforming growth factor (TGF)-ß. However, it is not clear how CD103(+) DCs are specialized for this function. We investigated the ability of CD103(+) DCs to promote Treg generation through activation of TGF-ß and the role of integrins with the αv subunit in this process. METHODS: Naïve T cells were cultured with purified DCs from mesenteric lymph nodes (MLNs) or intestines of wild-type and αv conditional knockout mice to assess generation of Tregs. Antigens were administered orally to mice, and antigen-specific generation of Tregs was measured in intestinal tissues. Expression of the integrin αv subunit was measured in purified subpopulations of DCs by quantitative polymerase chain reaction and immunoblot analyses. RESULTS: In vitro, CD103(+) DCs generated more Tregs in the presence of latent TGF-ß than other MLN DCs. Efficient generation of Tregs required expression of the integrin αv subunit by DCs; mice that lacked αv in immune cells did not convert naïve T cells to intestinal Tregs in response to oral antigen. CD103(+) DCs derived from the MLNs selectively expressed high levels of integrin αvß8 compared with other populations of DCs. CONCLUSIONS: Expression of αvß8 is required for CD103(+) DCs to become specialized and activate latent TGF-ß and generate Tregs during the induction of tolerance to intestinal antigens in mice.

Apoptotic cells induce CD103 expression and immunoregulatory function in myeloid dendritic cell precursors through integrin αv and TGF-ß activation.

In the mammalian gut CD103+ve myeloid DCs are known to suppress inflammation threatened by luminal bacteria, but stimuli driving DC precursor maturation towards this beneficial phenotype are incompletely understood. We isolated CD11+ve DCs from mesenteric lymph nodes (MLNs) of healthy mice; CD103+ve DCs were 8-24 fold more likely than CD103-ve DCs to exhibit extensive of prior phagocytosis of apoptotic intestinal epithelial cells. However, CD103+ve and CD103-ve MLN DCs exhibited similar ex vivo capacity to ingest apoptotic cells, indicating that apoptotic cells might drive immature DC maturation towards the CD103+ve phenotype. When cultured with apoptotic cells, myeloid DC precursors isolated from murine bone marrow and characterised as lineage-ve CD103-ve, displayed enhanced expression of CD103 and ß8 integrin and acquired increased capacity to induce T regulatory lymphocytes (Tregs) after 7d in vitro. However, DC precursors isolated from αv-tie2 mice lacking αv integrins in the myeloid line exhibited reduced binding of apoptotic cells and complete deficiency in the capacity of apoptotic cells and/or latent TGF-ß1 to enhance CD103 expression in culture, whereas active TGF-ß1 increased DC precursor CD103 expression irrespective of αv expression. Fluorescence microscopy revealed clustering of αv integrin chains and latent TGF-ß1 at points of contact between DC precursors and apoptotic cells. We conclude that myeloid DC precursors can deploy αv integrin to orchestrate binding of apoptotic cells, activation of latent TGF-ß1 and acquisition of the immunoregulatory CD103+ve ß8+ve DC phenotype. This implies that a hitherto unrecognised consequence of apoptotic cell interaction with myeloid phagocytes is programming that prevents inflammation.

The role and regulation of 11beta-hydroxysteroid dehydrogenase type 1 in the inflammatory response.

Cortisone, a glucocorticoid hormone, was first used to treat rheumatoid arthritis in humans in the late 1940s, for which Hench, Reichstein and Kendall were awarded a Nobel Prize in 1950 and which led to the discovery of the anti-inflammatory effects of glucocorticoids. To be effective, the intrinsically inert cortisone must be converted to the active glucocorticoid, cortisol, by the intracellular action of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Whilst orally administered cortisone is rapidly converted to the active hormone, cortisol, by first pass metabolism in the liver, recent work has highlighted an anti-inflammatory role for 11beta-HSD1 within specific tissues, including in leukocytes. Here, we review recent evidence pertaining to the anti-inflammatory role of 11beta-HSD1 and describe how inhibition of 11beta-HSD1, as widely proposed for treatment of metabolic disease, may impact upon inflammation. Finally, the mechanisms that regulate 11beta-HSD1 transcription will be discussed.

Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death.

Caspase-directed apoptosis usually fragments cells, releasing nonfunctional, prothrombogenic, membrane-bound apoptotic bodies marked for rapid engulfment by macrophages. Blood platelets are functional anucleate cells generated by specialized fragmentation of their progenitors, megakaryocytes (MKs), but committed to a constitutive caspase-independent death. Constitutive formation of the proplatelet-bearing MK was recently reported to be caspase-dependent, apparently involving mitochondrial release of cytochrome c, a known pro-apoptogenic factor. We extend those studies and report that activation of caspases in MKs, either constitutively or after Fas ligation, yields platelets that are functionally responsive and evade immediate phagocytic clearance, and retain mitochondrial transmembrane potential until constitutive platelet death ensues. Furthermore, the exclusion from the platelet progeny of caspase-9 present in the progenitor accounts for failure of mitochondrial release of cytochrome c to activate caspase-3 during platelet death. Thus, progenitor cell death by apoptosis can result in birth of multiple functional anucleate daughter cells.

Granulocyte macrophage-colony stimulating factor: A key modulator of renal mononuclear phagocyte plasticity.

Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) play key roles in the differentiation of macrophages and dendritic cells (DCs). We examined the effect of treatment with M-CSF-containing macrophage medium or GM-CSF-containing DC medium upon the phenotype of murine bone marrow-derived macrophages and DCs. Culture of macrophages for 5 days in DC medium reduced F4/80 expression and increased CD11c expression with cells effectively stimulating T cell proliferation in a mixed lymphocyte reaction. DC medium treatment of macrophages significantly reduced phagocytosis of both apoptotic cells and latex beads and strongly induced the expression of the chemokine receptor CCR7 known to be involved in DC trafficking to lymph nodes. Lysates of obstructed murine kidneys expressed both M-CSF and GM-CSF though M-CSF expression was dominant (M-CSF:GM-CSF ratio ∼30:1). However, combination treatment with both M-CSF and GM-CSF (ratio 30:1) indicated that small amounts of GM-CSF skewed macrophages towards a DC-like phenotype. To determine whether macrophage phenotype might be modulated in vivo we tracked CD45.1+ bone marrow-derived macrophages intravenously administered to CD45.2+ mice with unilateral ureteric obstruction. Flow cytometry of enzyme dissociated kidneys harvested 3 days later indicated CD11c and MHC Class II upregulation by adoptively transferred CD45.1+ cells with CD45.1+ cells evident in draining renal lymph nodes. Our data suggests that GM-CSF modulates mononuclear phagocyte plasticity, which likely promotes resolution of injury and healing in the injured kidney.

Exposure to the antimicrobial peptide LL-37 produces dendritic cells optimized for immunotherapy.

Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103+/CD141+ DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8+ (but not CD4+) T cells in vitro and in vivo. Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8+ T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols.

CD31 delays phagocyte membrane repolarization to promote efficient binding of apoptotic cells.

Homophilic ligation of CD31, a member of the Ig superfamily of adhesion receptors, promotes macrophage clearance of apoptotic leukocytes by a mechanism hitherto not described. In studying CD31-dependent regulation of beta1-integrin binding of fibronectin-coated Latex beads, we discovered a role for the voltage-gated potassium channel ether-à-go-go-related gene (ERG) as a downstream effector of CD31 signaling. ERG was identified by tandem mass spectrometry as a 140-kDa protein, which was selectively modified with biotin following the targeted delivery of a biotin-transfer reagent to CD31 using Fab fragments of an anti-CD31 mAb. Similar results were obtained with macrophages but not K562 cells, expressing a truncated cytoplasmic tail of CD31, which failed to regulate bead binding. Colocalization of CD31 with ERG was confirmed by immunofluorescence for K562 cells and macrophages. We now demonstrate that the resting membrane potential of macrophages is depolarized on contact with apoptotic cells and that CD31 inhibits the ERG current, which would otherwise function to repolarize. Sustained depolarization favored the firm binding of phagocytic targets, a prerequisite for efficient engulfment. Our results identify ERG as a downstream effector of CD31 in the regulation of integrin-dependent binding of apoptotic cells by macrophages.

Apoptosis and glomerulonephritis.

Glomerulonephritis (GN) is a form of autoimmunity in which apoptosis may be a double-edged sword. Resolution of GN can be promoted by apoptosis of infiltrating leucocytes and excess resident glomerular cells, leading to efficient anti-inflammatory clearance by macrophages and mesangial cells. However, unscheduled apoptosis in glomerular cells, especially epithelial cells ('podocytes') may drive progression of GN to hypocellular, nonfunctional scar. Defects in clearance of apoptotic cells may also have deleterious local effects, in addition to promoting autoimmunity itself. Nevertheless, there is strong promise for novel therapies based on new knowledge of apoptosis in GN, especially in regulation of leucocyte clearance from the inflamed glomerulus.

Comparison of multiplex PCR with conventional biochemical methods for the identification of Listeria spp. isolates from food and clinical samples in Queensland, Australia.

Listeria monocytogenes is an important foodborne pathogen with high mortality. L. monocytogenes and five other Listeria species can frequently be found in the same sample. To identify Listeria isolates found in foods to the species level, two multiplex PCRs were designed. The PCR and conventional biochemical methods were compared for the identification of 456 Listeria isolates collected from routine food quality monitoring schemes between June 2004 and February 2006 and for 62 L. monocytogenes isolates from patients between 1999 and 2005. The results showed that the PCR and biochemical methods had 100% agreement in Listeria identification. The distribution of Listeria species from foods was as follows: L. monocytogenes, 50.4%; L. innocua, 33.8%; L. welshimeri, 14.9%; L. seeligeri, 0.7%; L. grayi, 0.2%; and L. ivanovii, 0.0%. Additional analyses were performed to identify the major serotypes (1/2a, 1/2b, 1/2c, and 4b) and the three lineages of L. monocytogenes isolates from foods and patients, with 1/2a (69.6%) and 1/2b (21.7%) dominating the food isolates and 1/2b (54.8%) and 4b (30.7%) dominating the patient isolates. The lineage results showed that isolates of 1/2a and 1/2c belonged to lineage II and that isolates of 1/2b and 4b belonged to lineage I. The multiplex PCRs for Listeria identification that have been established provide an accurate and rapid method for food quality control. This study has provided the basic knowledge of distribution of Listeria species and L. monocytogenes serotypes in Queensland, Australia, which is useful for epidemiological investigations of listeriosis.

The death of human platelets during incubation in citrated plasma involves shedding of CD42b and aggregation of dead platelets.

The ability to readily identify dead platelets is invaluable to studies examining the means of their death, factors affecting their lifespan and their means of clearance by phagocytes. The aim of the present work was to develop a vital staining procedure for the rapid and objective discrimination of live from dead platelets that accrued in citrated platelet rich plasma (cPRP) incubated at 37 degrees C for several days. By transmission electron microscopy it was noted that platelet death was morphologically similar to necrosis and associated with aggregate formation. The vital dyes calcein-AM and FM 4-64 were found to robustly report the death of platelets and indicated that the aggregates which formed during incubation were populated exclusively by dead platelets. Additionally, platelet death was associated with the shedding of CD42b. Microscopic and cytometric analyses of incubated cPRP indicated that shedding of CD42b and aggregate formation by dead platelets were completely inhibited by the metalloproteinase inhibitor GM6001. Automated counting of platelets incubated in the presence of GM6001 revealed that death did not lead to a loss in cellularity. It is proposed that calcein-AM and FM4-64 are effective as vital stains for the reliable assessment of platelet viability and that platelet aggregation can occur by a novel mechanism dependent upon platelet death and metalloproteinase activity.