Carola Murphy

Natural killer cells protect mice from DSS-induced colitis by regulating neutrophil function via the NKG2A receptor

Natural killer (NK) cells are traditionally considered in the context of tumor surveillance and infection defense but their role in chronic inflammatory disorders such as inflammatory bowel disease is less clear. Here, we investigated the role of NK cells in dextran sodium sulfate (DSS)-induced colitis in mice. Depletion of NK cells impairs the survival of mice with colitis and is linked with dramatic increases in colonic damage, leukocyte infiltration, and pro-inflammatory profiles. Mice depleted of NK cells had increased numbers of neutrophils in colons and mesenteric lymph nodes, compared with control mice, in addition to acquiring a hyper-activation status. In vitro and in vivo studies demonstrate that NK cells downregulate pro-inflammatory functions of activated neutrophils, including reactive oxygen species and cytokine production, by direct cell-to-cell contact involving the NK cell–inhibitory receptor NKG2A. Our results indicate an immunoregulatory mechanism of action of NK cells attenuating DSS-induced colitis neutrophil-mediated inflammation and tissue injury via NKG2A-dependent mechanisms.

Natural Killer Cells Protect against Mucosal and Systemic Infection with the Enteric Pathogen Citrobacter rodentium

ABSTRACT Natural killer (NK) cells are traditionally considered in the context of tumor surveillance and viral defense, but their role in bacterial infections, particularly those caused by enteric pathogens, is less clear. C57BL/6 mice were orally gavaged with Citrobacter rodentium, a murine pathogen related to human diarrheagenic Escherichia coli. We used polyclonal anti-asialo GM1 antibody to actively deplete NK cells in vivo. Bioluminescent imaging and direct counts were used to follow infection. Flow cytometry and immunofluorescence microscopy were used to analyze immune responses. During C. rodentium infection, NK cells were recruited to mucosal tissues, where they expressed a diversity of immune-modulatory factors. Depletion of NK cells led to higher bacterial loads but less severe colonic inflammation, associated with reduced immune cell recruitment and lower cytokine levels. NK cell-depleted mice also developed disseminated systemic infection, unlike control infected mice. NK cells were also cytotoxic to C. rodentium in vitro.

Metabolic activity of probiotics - oxalate degradation.

Urinary tract stones are an important clinical problem in human and veterinary medicine. Hyperoxaluria is the single strongest promoter of kidney stone formation. The aims of the present study were to (a) evaluate oxalate degradation by a range of Bifidobacteria species and Lactobacillus species isolated from the canine and feline gastrointestinal tract in vitro and (b) to determine the impact of oxalate degradation by selected strains in vivo. The bacteria were grown in oxalate-containing media and their ability to degrade oxalate in vitro was determined using reverse-phased HPLC. Bifidobacteria species and Lactobacillus species that degraded oxalate in vitro and survived gastric transit were selected for further examination. The selected probiotics were fed to rats for 4 weeks. Urine was collected at weeks 0, 2 and 4 and oxalate levels determined by HPLC. In vitro degradation was detected for 11/18 of the Lactobacillus species. In contrast, the capacity to degrade oxalate was not detected for any of the 13 Bifidobacterium species tested. Lactobacillus animalis 223C, Lactobacillus murinus 1222, L. animalis 5323 and L. murinus 3133 were selected for further investigation in a rat model. Urinary oxalate levels were significantly reduced (p<0.05) in animals fed L. animalis 5323 and L. animalis 223C but were unaltered when fed L. murinus 1222, L. murinus 3133 or placebo. Probiotic organisms vary widely in their capacity to degrade oxalate. In vitro degradation does not uniformly translate to an impact in vivo. The results have therapeutic implications and may influence the choice of probiotic, particularly in the setting of enteric hyperoxaluria.

Use of bioluminescence imaging to track neutrophil migration and its inhibition in experimental colitis

Inflammatory bowel disease (IBD) is associated with neutrophil infiltration into the mucosa and crypt abscesses. The chemokine interleukin (IL)‐8 [murine homologues (KC) and macrophage inflammatory protein (MIP)‐2] and its receptor CXCR2 are required for neutrophil recruitment; thus, blocking this engagement is a potential therapeutic strategy. In the present study, we developed a preclinical model of neutrophil migration suitable for investigating the biology of and testing new drugs that target neutrophil trafficking. Peritoneal exudate neutrophils from transgenic β‐actin‐luciferase mice were isolated 12 h after intraperitoneal injection with thioglycollate, and were assessed phenotypically and functionally. Exudate cells were injected intravenously into recipients with dextran sodium sulphate (DSS)‐induced colitis followed by bioluminescence imaging of whole‐body and ex vivo organs at 2, 4 and 16–22 h post‐transfer. Anti‐KC antibody or an isotype control were administered at 20 µg/mouse 1 h before transfer, followed by whole‐body and organ imaging 4 h post‐transfer. The peritoneal exudate consisted of 80% neutrophils, 39% of which were CXCR2+. In vitro migration towards KC was inhibited by anti‐KC. Ex vivo bioluminescent imaging showed that neutrophil trafficking into the colon of DSS recipients was inhibited by anti‐KC 4 h post‐cell transfer. In conclusion, this study describes a new approach for investigating neutrophil trafficking that can be used in preclinical studies to evaluate potential inhibitors of neutrophil recruitment.

Local Bacteria Affect the Efficacy of Chemotherapeutic Drugs

In this study, the potential effects of bacteria on the efficacy of frequently used chemotherapies was examined. Bacteria and cancer cell lines were examined in vitro and in vivo for changes in the efficacy of cancer cell killing mediated by chemotherapeutic agents. Of 30 drugs examined in vitro, the efficacy of 10 was found to be significantly inhibited by certain bacteria, while the same bacteria improved the efficacy of six others. HPLC and mass spectrometry analyses of sample drugs (gemcitabine, fludarabine, cladribine, CB1954) demonstrated modification of drug chemical structure. The chemoresistance or increased cytotoxicity observed in vitro with sample drugs (gemcitabine and CB1954) was replicated in in vivo murine subcutaneous tumour models. These findings suggest that bacterial presence in the body due to systemic or local infection may influence tumour responses or off-target toxicity during chemotherapy.

The Sphingosine-1-Phosphate Analogue FTY720 Impairs Mucosal Immunity and Clearance of the Enteric Pathogen Citrobacter rodentium

ABSTRACT The sphingosine-1-phosphate (S1P) analogue FTY720 is therapeutically efficacious in multiple sclerosis and in the prevention of transplant rejection. It prevents the migration of lymphocytes to sites of pathology by trapping them within the peripheral lymph nodes, mesenteric lymph nodes (MLNs), and Peyers patches. However, evidence suggests that its clinical use may increase the risk of mucosal infections. We investigated the impact of FTY720 treatment on susceptibility to gastrointestinal infection with the mouse enteric pathogen Citrobacter rodentium. This attaching and effacing bacterium induces a transient bacterial colitis in immunocompetent mice that resembles human infection with pathogenic Escherichia coli. FTY720 treatment induced peripheral blood lymphopenia, trapped lymphocytes in the MLNs, and prevented the clearance of bacteria when mice were infected with luciferase-tagged C. rodentium. FTY720-treated C. rodentium-infected mice had enhanced colonic inflammation, with significantly higher colon mass, colon histopathology, and neutrophil infiltration than vehicle-infected animals. In addition, FTY720-treated infected mice had significantly lower numbers of colonic dendritic cells, macrophages, and T cells. Gene expression analysis demonstrated that FTY720-treated infected mice had an impaired innate immune response and a blunted mucosal adaptive immune response, including Th1 cytokines. The data demonstrate that the S1P analogue FTY720 adversely affects the immune response to and clearance of C. rodentium.

Technical Advance: Function and efficacy of an α4-integrin antagonist using bioluminescence imaging to detect leukocyte trafficking in murine experimental colitis

Leukocyte trafficking is a therapeutic target in IBD. The integrins α4β7 and α4β1 regulate leukocyte migration into tissues and lymphoid organs. Current strategies rely on biologics, such as mAb, to inhibit leukocyte recruitment. Here we show the in vivo therapeutic effects of a small molecule α4‐integrin antagonist (GSK223618A) in a leukocyte‐trafficking model and a murine model of colitis. Leukocytes isolated from MLNs of transgenic β‐actin‐luc+ mice were injected i.v. into recipients with DSS‐induced colitis. Recipient mice were orally gavaged with vehicle or an α4‐integrin antagonist 1 h pre‐adoptive transfer, followed by bioluminescence whole body and ex vivo organ imaging 4 h post‐transfer. To confirm its therapeutic effect, the α4‐integrin antagonist was given orally twice daily for 6 days to mice with DSS‐induced colitis, starting on Day 3. Clinical, macroscopic, and histological signs of inflammation were assessed and gene‐expression profiles analyzed. Using bioluminescence imaging, we tracked and quantified leukocyte migration to the inflamed gut and demonstrated its inhibition by a small molecule α4‐integrin antagonist. Additionally, the therapeutic effect of the antagonist was confirmed in DSS‐induced colitis in terms of clinical, macroscopic, and histological signs of inflammation. Gene expression analysis suggested enhancement of tissue healing in compound‐treated animals. Inhibition of leukocyte trafficking using small molecule integrin antagonists is a promising alternative to large molecule biologics. Furthermore, in vivo bioluminescence imaging is a valuable strategy for preclinical evaluation of potential therapeutics that target leukocyte trafficking in inflammatory diseases.

Activation of liver X receptor suppresses the production of the IL-12 family of cytokines by blocking nuclear translocation of NF-κBp50

There is now convincing evidence that liver X receptor (LXR) is an important modulator of the inflammatory response; however, its mechanism of action remains unclear. This study aimed to examine the effect of LXR on the IL-12 family of cytokines and examined the mechanism by which LXR exerted this effect. We first demonstrated that activation of murine-derived dendritic cells (DC) with a specific agonist to LXR enhanced expression of LXR following activation with LPS, suggesting a role in inflammation. Furthermore, we showed LXR expression to be increased in vivo in dextrane sulphate sodium-induced colitis. LXR activation also suppressed production of IL-12p40, IL-12p70, IL-27 and IL-23 in murine-derived DC following stimulation with LPS, and specifically targeted the p35, p40 and EBI3 subunits of the IL-12 cytokine family, which are under the control of the NF-κB subunit p50 (NF-κBp50). Finally, we demonstrated that LXR can associate with NF-κBp50 in DC and that LXR activation prevents translocation of the p50 subunit into the nucleus. In summary, our study indicates that LXR can specifically suppress the IL-12 family of cytokines though its association with NF-κBp50 and highlights its potential as a therapeutic target for chronic inflammatory diseases.

Shining a Light on Intestinal Traffic

Inflammatory bowel disease (IBD), encompassing Crohns disease and ulcerative colitis, is associated with enhanced leukocyte infiltration to the gut, which is directly linked to the clinical aspects of these disorders. Thus, leukocyte trafficking is a major target for IBD therapy. Past and emerging techniques to study leukocyte trafficking both in vitro and in vivo have expanded our knowledge of the leukocyte migration process and the role of inhibitors. Various strategies have been employed to target chemokine- and integrin-ligand interactions within the multistep adhesion cascade and the S1P/S1PR1 axis in leukocyte migration. Though there is an abundance of preclinical data demonstrating efficacy of leukocyte trafficking inhibitors, many have yet to be confirmed in clinical studies. Vigilance for toxicity and further research is required into this complex and emerging area of IBD therapy.

Intratumoural production of TNFα by bacteria mediates cancer therapy

Systemic administration of the highly potent anticancer therapeutic, tumour necrosis factor alpha (TNFα) induces high levels of toxicity and is responsible for serious side effects. Consequently, tumour targeting is required in order to confine this toxicity within the locality of the tumour. Bacteria have a natural capacity to grow within tumours and deliver therapeutic molecules in a controlled fashion. The non-pathogenic E. coli strain MG1655 was investigated as a tumour targeting system in order to produce TNFα specifically within murine tumours. In vivo bioluminescence imaging studies and ex vivo immunofluorescence analysis demonstrated rapid targeting dynamics and prolonged survival, replication and spread of this bacterial platform within tumours. An engineered TNFα producing construct deployed in mouse models via either intra-tumoural (i.t.) or intravenous (i.v.) administration facilitated robust TNFα production, as evidenced by ELISA of tumour extracts. Tumour growth was impeded in three subcutaneous murine tumour models (CT26 colon, RENCA renal, and TRAMP prostate) as evidenced by tumour volume and survival analyses. A pattern of pro-inflammatory cytokine induction was observed in tumours of treated mice vs. controls. Mice remained healthy throughout experiments. This study indicates the therapeutic efficacy and safety of TNFα expressing bacteria in vivo, highlighting the potential of non-pathogenic bacteria as a platform for restricting the activity of highly potent cancer agents to tumours.