Kenneth Croitoru

NOD proteins: regulators of inflammation in health and disease

Entry of bacteria into host cells is an important virulence mechanism. Through peptidoglycan recognition, the nucleotide-binding oligomerization domain (NOD) proteins NOD1 and NOD2 enable detection of intracellular bacteria and promote their clearance through initiation of a pro-inflammatory transcriptional programme and other host defence pathways, including autophagy. Recent findings have expanded the scope of the cellular compartments monitored by NOD1 and NOD2 and have elucidated the signalling pathways that are triggered downstream of NOD activation. In vivo, NOD1 and NOD2 have complex roles, both during bacterial infection and at homeostasis. The association of alleles that encode constitutively active or constitutively inactive forms of NOD2 with different diseases highlights this complexity and indicates that a balanced level of NOD signalling is crucial for the maintenance of immune homeostasis.

Bacterial biogeography of the human digestive tract

We present bacterial biogeography as sampled from the human gastrointestinal tract of four healthy subjects. This study generated >32 million paired-end sequences of bacterial 16S rRNA genes (V3 region) representing >95,000 unique operational taxonomic units (OTUs; 97% similarity clusters), with >99% Goods coverage for all samples. The highest OTU richness and phylogenetic diversity was found in the mouth samples. The microbial communities of multiple biopsy sites within the colon were highly similar within individuals and largely distinct from those in stool. Within an individual, OTU overlap among broad site definitions (mouth, stomach/duodenum, colon and stool) ranged from 32–110 OTUs, 25 of which were common to all individuals and included OTUs affiliated with Faecalibacterium prasnitzii and the TM7 phylum. This first comprehensive characterization of the abundant and rare microflora found along the healthy human digestive tract represents essential groundwork to investigate further how the human microbiome relates to health and disease.

CD40 expression by human peripheral blood eosinophils.

In this study, we have investigated CD40 expression in human peripheral blood eosinophils and in human chronically inflamed nasal tissues, i.e., nasal polyps. We show by both reverse transcriptase-PCR and Northern blot analysis that eosinophils from allergic subjects express human CD40 mRNA. We also show that constitutive CD40 mRNA expression in eosinophils could be upregulated by exposure to IgA immune complexes and downregulated by IL-10 and the synthetic steroid budesonide. In addition, we demonstrate that eosinophils express CD40 protein by flow cytometry. Such expression is biologically functional as cross-linking CD40 with CD40 mAbs enhances eosinophil survival in a dose-dependent fashion; in addition, CD40 ligation stimulates eosinophils to release GM-CSF. CD40-mediated eosinophil survival was largely inhibited by an anti-GM-CSF neutralizing antibody suggesting GM-CSF involvement in the survival enhancing mechanism. CD40 mRNA was also detected in total RNA extracted from nasal polyp tissues but not in RNA isolated from normal nasal mucosa (inferior turbinate); by immunohistochemistry, we were able to detect immunoreactive CD40 protein in a variety of cell types in the polyp stroma, but primarily in eosinophils. These observations suggest previously unforeseen interactions between eosinophils and cells expressing the CD40 ligand and, thus, novel pathways by which eosinophils may contribute to the regulation of airway inflammation.

TH1/TH2,3 Imbalance due to Cytokine-Producing NK, γδ T and NK-γδ T Cells in Murine Pregnancy Decidua in Success or Failure of Pregnancy

PROBLEM: Recurrent spontaneous abortion in DBA/2‐mated CBA/J mice has been attributed to the production of Th1 cytokines (tumor necrosis factor [TNF]‐α and interferon [IFN]‐γ) by asialoGM1+ natural killer (NK) cells and Vγ1.1δ6.3+ T cells that infiltrate decidua by day 6.5, during the peri‐implantation period. Abortions can be prevented by a second population of Vγ1.1δ6.3 cells, which infiltrate on day 8.5 of gestation, and produce the Th2 cytokine interleukin (IL)‐10 and Th3 cytokine transforming growth factor (TGF)‐β2. In low abortion rate immunocompetent mice, most of the TGF‐β2 is derived from γδ T cells. However, TGF‐β2‐producing cells are present in the decidua of pregnant severe combined immune deficient (SCID) mice, which lack γδ T cells.
 METHODS: The cells in day 13.5 decidua of CBA×DBA/2 matings and SCID×SCID matings were identified using flow cytometry and combined surface staining for γδ and/or asialoGM1, and intracellular cytokine staining for TNF‐α, IFN‐γ, and TGF‐β2,3.
 RESULTS: TGF‐β2 and TNF‐α were found in asialoGM1+ NK cells in SCID mouse decidua. In CBA×DBA/2 mated mice, two major and one minor subsets of cytokine‐positive cells were identified: ‐γδ‐only T cells, double positive asialoGM1+γδ+ (NK‐γδ T) cells, and a small number of asialoGM1+γδ− NK‐only cells. The NK‐only and NK‐γδ T subsets showed a greater Th1/Th2,3 pattern of intracellular staining compared with the γδ‐only subset. In the CBA×DBA/2 and SCID×SCID systems, Th1/Th2,3 ratios could not predict actual observed abortion rates but did correlate with susceptibility to abortions (if exposed to an additional stimulus such as stress). The known effect of in vivo administration of anti‐asialoGM1 antibody on abortion rates within groups of mice exposed to such stresses could also be predicted.
 CONCLUSION: γδ+ cells in decidua (e.g. Vγ1+ cells which can recognize trophoblasts) differ based on the presence or absence of the NK marker‐asialo‐GM1. NK‐γδ T cells may be quite important in the Th1 response in early pregnancy that predisposes to abortions in CBA×DBA/2 matings, whereas γδ T‐only cells appear to be protective. In pregnant SCID mice, the TNF‐α+/TGF‐β2+ NK population is greatly expanded. An activating stimulus (such as stress or endotoxin) appears to be as important in triggering abortions, as is the Th1/Th2,3 ratio at the feto–maternal interface.

Defining the roles of perforin, Fas/FasL, and tumour necrosis factor α in T cell induced mucosal damage in the mouse intestine

Background and aims: Mucosal flattening and epithelial cell apoptosis are typical features of T cell induced inflammatory diseases of the bowel, such as coeliac disease and graft versus host disease. Mice injected with a T cell activating anti-CD3 antibody develop a severe diarrhoeal illness. We describe the histological features of this enteropathy and define the effector mechanisms involved in T cell induced mucosal injury in this in vivo model. Methods: Wild-type and genetically modified mice were injected with the anti-CD3 antibody 3C11 (50 μg). Changes in the murine intestine were characterised by light microscopy analysis and terminal uridine nick-end labelling (TUNEL) assay. The role of perforin, Fas/Fas ligand (FasL), tumour necrosis factor α (TNF-α), and interferon γ (IFN-γ) in T cell induced mucosal damage was assessed using selected immunodeficient mouse strains. Results: T cell activation caused severe damage, including small intestinal mucosal flattening and apoptosis of crypt epithelial cells. Mucosal damage was unaltered in anti-CD3 treated mice lacking IFN-γ, Fas, or TNF-α receptors. In mice lacking TNF-α receptors and Fas (TNF-R1×R2 lpr/lpr strain), enterocyte apoptosis was diminished but there was no significant reduction in tissue damage. Apoptosis and mucosal injury were significantly reduced in perforin knockout mice. Abrogation of both FasL and perforin (perforin KO×gld mice) further significantly reduced tissue damage and apoptotic bodies. Conclusions: T cell induced mucosal injury is mediated by the combined effect of multiple pathways but predominantly by perforin. The redundancy of the mechanisms of tissue damage will have significant impact on therapeutic strategies aimed at specific and targeted inhibition of inflammatory processes.

Regulation of obesity-related insulin resistance with gut anti-inflammatory agents.

Obesity has reached epidemic proportions, but little is known about its influence on the intestinal immune system. Here we show that the gut immune system is altered during high-fat diet (HFD) feeding and is a functional regulator of obesity-related insulin resistance (IR) that can be exploited therapeutically. Obesity induces a chronic phenotypic pro-inflammatory shift in bowel lamina propria immune cell populations. Reduction of the gut immune system, using beta7 integrin-deficient mice (Beta7(null)), decreases HFD-induced IR. Treatment of wild-type HFD C57BL/6 mice with the local gut anti-inflammatory, 5-aminosalicyclic acid (5-ASA), reverses bowel inflammation and improves metabolic parameters. These beneficial effects are dependent on adaptive and gut immunity and are associated with reduced gut permeability and endotoxemia, decreased visceral adipose tissue inflammation, and improved antigen-specific tolerance to luminal antigens. Thus, the mucosal immune system affects multiple pathways associated with systemic IR and represents a novel therapeutic target in this disease.

Compartmentalized transgene expression of granulocyte-macrophage colony- stimulating factor (GM-CSF) in mouse lung enhances allergic airways inflammation

To investigate the role of GM‐CSF in asthmatic airways inflammation, we have targeted GM‐CSF transgene to the airway cells in a mouse model of ovalbumin (OVA)‐induced allergic airways inflammation, a model in which there is marked induction of endogenous IL‐5 and IL‐4 but not GM‐CSF. Following intranasal delivery of a replication‐deficient adenoviral gene transfer vector (Ad), transgene expression was found localized primarily to the respiratory epithelial cells. Intranasal delivery of 0.03 × 109 plaque‐forming units (PFU) of AdGM‐CSF into naive BALB/c mice resulted in prolonged and compartmentalized release of GM‐CSF transgene protein with a peak concentration of ≈ 80 pg/ml detected in bronchoalveolar lavage fluid (BALF) at day 7, but little in serum. These levels of local GM‐CSF expression per se resulted in no eosinophilia and only a minimum of tissue inflammatory responses in the lung of naive mice, similar to those induced by the control vector. However, such GM‐CSF expression in the airways of OVA‐sensitized mice resulted in a much greater and sustained accumulation of various inflammatory cell types, most noticeably eosinophils, both in BALF and airway tissues for 15–21 days post‐OVA aerosol challenge, at which times airways inflammation had largely resolved in control mice. While the levels of IL‐5 and IL‐4 in BALF and the rate of eosinophil apoptosis were found similar between different treatments, there was an increased number of proliferative leucocytes in the lung receiving GM‐CSF gene transfer. Our results thus provide direct experimental evidence that GM‐CSF can significantly contribute to the development of allergic airways inflammation through potentiating and prolonging inflammatory infiltration induced by cytokines such as IL‐5 and IL‐4.

Association of host genome with intestinal microbial composition in a large healthy cohort

Intestinal microbiota is known to be important in health and disease. Its composition is influenced by both environmental and host factors. Few large-scale studies have evaluated the association between host genetic variation and the composition of microbiota. We recruited a cohort of 1,561 healthy individuals, of whom 270 belong in 123 families, and found that almost one-third of fecal bacterial taxa were heritable. In addition, we identified 58 SNPs associated with the relative abundance of 33 taxa in 1,098 discovery subjects. Among these, four loci were replicated in a second cohort of 463 subjects: rs62171178 (nearest gene UBR3) associated with Rikenellaceae, rs1394174 (CNTN6) associated with Faecalibacterium, rs59846192 (DMRTB1) associated with Lachnospira, and rs28473221 (SALL3) associated with Eubacterium. After correction for multiple testing, 6 of the 58 associations remained significant, one of which replicated. These results identify associations between specific genetic variants and the gut microbiome.

Preoperative biological therapy and short-term outcomes of abdominal surgery in patients with inflammatory bowel disease

Objective Previous investigations of short-term outcomes after preoperative exposure to biological therapy in inflammatory bowel disease (IBD) were conflicting. The authors aimed to assess postoperative outcomes in patients who underwent abdominal surgery with recent exposure to anti-tumour necrosis factor therapy. Design A retrospective case-control study with detailed matching was performed for subjects with IBD with and without exposure to biologics within 180 days of abdominal surgery. Postoperative outcomes were compared between the groups. Results 473 procedures were reviewed consisting of 195 patients with exposure to biologics and 278 matched controls. There were no significant differences in most postoperative outcomes such as: length of stay, fever (≥38.5°C), urinary tract infection, pneumonia, bacteraemia, readmission, reoperations and mortality. On univariate analysis, procedures on biologics had more wound infections compared with controls (19% vs 11%; p=0.008), but this was not significant in multivariate analysis. Concomitant therapy with biologics and thiopurines was associated with increased frequencies of urinary tract infections (p=0.0007) and wound infections (p=0.0045). Operations performed ≤14 days from last biologic dose had similar rates of infections and other outcomes when compared with those performed within 15–30 days or 31–180 days. Patients with detectable preoperative infliximab levels had similar rates of wound infection compared with those with undetectable levels (3/10 vs 0/9; p=0.21). Conclusion Preoperative treatment with TNF-α antagonists in patients with IBD is not associated with most early postoperative complications. A shorter time interval from last biological dose is not associated with increased postoperative complications. In most cases, surgery should not be delayed, and appropriate biological therapy may be continued perioperatively.

Expression of Dual TCR on DO11.10 T Cells Allows for Ovalbumin-Induced Oral Tolerance to Prevent T Cell-Mediated Colitis Directed against Unrelated Enteric Bacterial Antigens

The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4+ T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4+CD45RBhigh T cells developed colitis, and cotransferring DO11.10 CD45RBlowCD4+ T cells prevented CD4+CD45RBhigh T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4+ T cell subsets were associated with an increase in endogenous TCRα chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4+CD45RBhigh attenuated the colitis in association with increased TGF-β and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RBhigh T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.