Sheena M. Cruickshank

Colonic dendritic cells, intestinal inflammation, and T cell-mediated bone destruction are modulated by recombinant osteoprotegerin.

Autoimmune associated bone disease and intestinal inflammation are closely linked with deregulation and hyperactivation of autoreactive CD4 T cells. How these T cells are activated and mediate disease is not clear. Here we show that in the Interleukin 2-deficient mouse model of autoimmunity spontaneous osteopenia and colitis are caused by increased production of the ligand for receptor activator of NFkappaB (RANKL). RANKL acting via its receptor, receptor activator of NFkappaB (RANK), increases bone turnover and promotes intestinal dendritic cell (DC) survival in vivo. Modulation of RANKL-RANK interactions with exogenous recombinant osteoprotegerin (Fc-OPG) reverses skeletal abnormalities and reduces colitis by decreasing colonic DC numbers. This study identifies a common causal link between bone disease and intestinal inflammation and establishes the importance of DC in mediating colonic inflammation in vivo.

Expression and cytokine regulation of immune recognition elements by normal human biliary epithelial and established liver cell lines in vitro

BACKGROUND/AIMS Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines. METHODS Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence. RESULTS All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2. CONCLUSIONS These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

AIM2 and NLRC4 inflammasomes contribute with ASC to acute brain injury independently of NLRP3

Significance Cerebral ischemia (CI; stroke, brain injury, vascular dementia, neonatal hypoxia, and many other conditions) affects people at all stages of life. Of the many diseases associated with CI, stroke alone causes up to 10% of deaths worldwide and is a leading cause of disability; yet treatment options are extremely limited, so this represents an area of massive unmet clinical need. Inflammation involving the cytokine interleukin-1 is a major contributor to cell death in the ischemic brain. Inflammation can be regulated by large protein complexes called inflammasomes. Here we show that the NLRC4 (NLR family, CARD domain containing 4) and AIM2 (absent in melanoma 2) inflammasomes, but not the NLRP3 (NLR family, pyrin domain containing 3) inflammasome, contribute to inflammation and injury in an ischemic brain and are thus potential therapeutic targets for these devastating diseases. Inflammation that contributes to acute cerebrovascular disease is driven by the proinflammatory cytokine interleukin-1 and is known to exacerbate resulting injury. The activity of interleukin-1 is regulated by multimolecular protein complexes called inflammasomes. There are multiple potential inflammasomes activated in diverse diseases, yet the nature of the inflammasomes involved in brain injury is currently unknown. Here, using a rodent model of stroke, we show that the NLRC4 (NLR family, CARD domain containing 4) and AIM2 (absent in melanoma 2) inflammasomes contribute to brain injury. We also show that acute ischemic brain injury is regulated by mechanisms that require ASC (apoptosis-associated speck-like protein containing a CARD), a common adaptor protein for several inflammasomes, and that the NLRP3 (NLR family, pyrin domain containing 3) inflammasome is not involved in this process. These discoveries identify the NLRC4 and AIM2 inflammasomes as potential therapeutic targets for stroke and provide new insights into how the inflammatory response is regulated after an acute injury to the brain.

Local Arginase 1 Activity Is Required for Cutaneous Wound Healing

Chronic nonhealing wounds in the elderly population are associated with a prolonged and excessive inflammatory response, which is widely hypothesized to impede healing. Previous studies have linked alterations in local L-arginine metabolism, principally mediated by the enzymes arginase (Arg) and inducible nitric oxide synthase (iNOS), to pathological wound healing. Over subsequent years, interest in Arg/iNOS has focused on the classical versus alternatively activated (M1/M2) macrophage paradigm. Although the role of iNOS during healing has been studied, Arg contribution to healing remains unclear. Here, we report that Arg is dynamically regulated during acute wound healing. Pharmacological inhibition of local Arg activity directly perturbed healing, as did Tie2-cre-mediated deletion of Arg1, revealing the importance of Arg1 during healing. Inhibition or depletion of Arg did not alter alternatively activated macrophage numbers but instead was associated with increased inflammation, including increased influx of iNOS+ cells and defects in matrix deposition. Finally, we reveal that in preclinical murine models reduced Arg expression directly correlates with delayed healing, and as such may represent an important future therapeutic target.

Functional characterization of T cells in abdominal aortic aneurysms

Abdominal aortic aneurysms (AAA) exhibit features of a chronic inflammatory disorder. The functional attributes of the T cells in AAA tissue are unclear, with little quantitative or functional data. Using a novel, non‐enzymatic method to isolate viable cells from AAA tissue, functional properties of AAA T cells were investigated for the first time. Composition and phenotype of AAA T cells was determined by flow cytometry and verified by immunohistochemistry. Tissue mononuclear cells (MNCs) were cultured in the presence of T‐cell mitogens, and cell cycle analysis and cytokine production assessed. Typical cell yield was 4·5 × 106 cells per gram of AAA tissue. The majority (58·1 ± 5·3%) of haematopoietic (CD45+) cells recovered were CD3+ T cells, B cells comprised 41·1 ± 5·7%, natural killer cells 7·3 ± 2·5%, and macrophages 2%. Freshly isolated T cells were in resting (G1) state, with 25% expressing the activation‐associated cell surface antigens major histocompatibility complex II and CD25. When stimulated in vitro, a significant proportion entered S and G2 phase of the cell cycle, up‐regulated CD25, and secreted tumour necrosis factor‐α, interferon‐γ, interleukin (IL)‐5 and IL‐6. Despite patient differences, the composition of the AAA inflammatory infiltrate was remarkably consistent, and when re‐stimulated ex‐vivo T cells produced a stereotypical cytokine response, consistent with the hypothesis that AAA T cells can promote tissue inflammation by secretion of proinflammatory cytokines, and in addition provide signals for B‐cell help.

Transforming growth factor‐β promotes ‘death by neglect’ in post‐activated human T cells

Transforming growth factor‐β (TGF‐β) is central to the wound repair processes that follow local trauma and inflammation. In order to mimic the early events of wound‐healing, we studied the effects of TGF‐β on mitogen‐stimulated peripheral blood cells. TGF‐β added at the initiation of mitogenesis did not significantly alter T‐cell activation, proliferation, CD45 isoform switching, or activation‐induced cell death. By contrast, TGF‐β added 72 hr post‐activation (or later) enhanced the cumulative increase in apoptotic T cells. TGF‐β had no effect on mitogen‐induced up‐regulation of Fas (CD95) or Fas ligand and did not enhance killing of the Fas‐sensitive Jurkat cell line by activated T cells. Furthermore, TGF‐β had no direct effect on levels of mRNA for members of the bcl family (bcl‐X, bfl‐1, bik, bak, bax, bcl‐2 and mcl‐1). These findings suggest that TGF‐β does not directly induce apoptosis via the Fas system or by direct effects on bcl proteins. However, interleukin‐2, which can ‘rescue’ lymphocytes from spontaneous apoptosis due to cytokine deprivation, abolished the pro‐apoptotic effects of TGF‐β on post‐activated T cells, thus demonstrating that TGF‐β increases the cytokine‐dependence of T cells for survival. We propose a novel role for TGF‐β in the suppression of inflammation by promoting the elimination of post‐activated T cells once the initiating stimulus has been resolved.

Colonic epithelial cell mediated suppression of CD4 T cell activation

Background and aims: As the first point of contact with enteric antigens, intestinal epithelial cells (IEC) may be key in regulating mucosal immune responses. We determined therefore if murine colonic epithelial cells (CEC) have tolerogenic or activating effects on CD4 T cells. Methods: Using a novel CEC, macrophages, and CD4 T cell coculture system, mitogen and antigen specific responses of naïve and antigen primed CD4 T cells were assessed. Results: Although a proportion of CEC express the costimulatory molecules B7.1, B7.2, CD40, and CD54, they were unable to promote mitogen or antigen driven activation of CD4 T cells, even in the presence of exogenous costimulatory signals. CD4 T cells cocultured with CEC were CD25lo and CD45RBlo and remained in the G1 phase of the cell cycle. CEC were also able to prevent CD4 T cell activation by professional antigen presenting cells. CEC mediated suppression of T cell activation was cell contact dependent and transforming growth factor β independent. Conclusions: These observations suggest that CEC contribute to the maintenance of T cell tolerance in the gut by preventing inappropriate activation of CD4 T cells.

Rapid dendritic cell mobilization to the large intestinal epithelium is associated with resistance to Trichuris muris infection.

The large intestine is a major site of infection and disease, yet little is known about how immunity is initiated within this site and the role of dendritic cells (DCs) in this process. We used the well-established model of Trichuris muris infection to investigate the innate response of colonic DCs in mice that are inherently resistant or susceptible to infection. One day postinfection, there was a significant increase in the number of immature colonic DCs in resistant but not susceptible mice. This increase was sustained at day 7 postinfection in resistant mice when the majority of the DCs were mature. There was no increase in DC numbers in susceptible mice until day 13 postinfection. In resistant mice, most colonic DCs were located in or adjacent to the epithelium postinfection. There were also marked differences in the expression of colonic epithelial chemokines in resistant mice and susceptible mice. Resistant mice had significantly increased levels of epithelium-derived CCL2, CCL3, CCL5, and CCL20 compared with susceptible mice. Furthermore, administering neutralizing CCL5 and CCL20 Abs to resistant mice prevented DC recruitment. This study provides clear evidence of differences in the kinetics of DC responses in hosts inherently resistant and susceptible to infection. DC responses in the colon correlate with resistance to infection. Differences in the production of DC chemotactic chemokines by colonic epithelial cells in response to infection in resistant vs susceptible mice may explain the different kinetics of the DC response.

Increased natural killer cell activity in patients with an abdominal aortic aneurysm

Natural killer (NK) cells have an emerging role in the development of chronic disease and in the direction and maintenance of inflammatory responses. Abdominal aortic aneurysms (AAA) is a chronic inflammatory disorder of unknown aetiology. The aim was to investigate whether NK cells showed altered function in patients with an AAA.

Arginase-1-expressing macrophages are dispensable for resistance to infection with the gastrointestinal helminth Trichuris muris

Alternatively activated macrophages (AAMs) have key roles in the immune response to a variety of gastrointestinal helminths such as Heligmosomoides bakeri and Nippostrongylus brasiliensis. In addition, AAMs have been implicated in the resolution of infection‐induced pathology in Schistosoma mansoni infection. AAMs exert their activity in part via the enzyme arginase‐1 (Arg1), which hydrolyses l‐arginine into urea and ornithine, and can supply precursor substrate for proline and polyamine production. Trichuris muris is a worm that resides in the large intestine with resistance being characterized by a Th2 T‐cell response, which drives alternatively activated macrophage production in the local environment of the infection. To investigate the role of AAMs in T. muris infection, we used independent genetic and pharmacologic models of arginase deficiency. In acute infection and Th2‐dominated immunity, arginase‐deficient models expelled worms normally. Macrophage‐Arg1‐deficient mice showed cytokine and antibody levels comparable to wild‐type animals in acute and chronic infection. We also found no role for AAMs and Arg1 in infection‐induced pathology in the response to T. muris in either chronic (Th1 dominated) or acute (Th2 dominated) infections. Our data demonstrate that, unlike other gastrointestinal helminths, Arg1 expression in AAMs is not essential for resistance to T. muris in effective resolution of helminth‐induced inflammation.