Eduardo Martínez-Naves

Paneth cells as a site of origin for intestinal inflammation

The recognition of autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn’s disease. Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity, causes Paneth cell dysfunction. As Atg16l1HM mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells, the human orthologue of which harbours rare inflammatory bowel disease risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis. Unresolved ER stress is a common feature of inflammatory bowel disease epithelium, and several genetic risk factors of Crohn’s disease affect Paneth cells. Here we show that impairment in either UPR (Xbp1ΔIEC) or autophagy function (Atg16l1ΔIEC or Atg7ΔIEC) in intestinal epithelial cells results in each other’s compensatory engagement, and severe spontaneous Crohn’s-disease-like transmural ileitis if both mechanisms are compromised. Xbp1ΔIEC mice show autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased intestinal epithelial cell death, inositol requiring enzyme 1α (IRE1α)-regulated NF-κB activation and tumour-necrosis factor signalling, which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity, and augmentation of autophagy in intestinal epithelial cells ameliorates ER stress-induced intestinal inflammation and eases NF-κB overactivation and intestinal epithelial cell death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth-cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal Crohn’s disease as a specific disorder of Paneth cells.

Molecular typing of HLA-B27 alleles

HLA-B27 represents a family of closely related antigens. Six alleles which differ in a limited number of nucleotide substitutions have been described (B*2701—B*2706). These changes are clustered in α1 and α2 domains. Polymerase chain reaction strategies were designed to amplify specific regions of class I exons 2 and 3. Amplified sequences were tested with eight sequence-specific oligonucleotides to distinguish all B27 subtypes. We also subtyped B27 in 50 healthy Spanish individuals using this procedure. The B*2705 subtype is over-represented in our population (96%). The remaining 4% carried the B*2702 allele. This finding is in agreement with the frequencies described by other techniques (cytotoxic T lymphocytes and isoeletric-focusing) for Caucasian populations. Class I oligotyping is a poorly developed field with significant potential applications. This procedure of genotyping B27 alleles is a reliable method which can be used in transplantation and B27-associated disease studies.

Endoplasmic reticulum stress: implications for inflammatory bowel disease pathogenesis

Purpose of review To provide an overview of the emerging role of cellular stress responses in inflammatory bowel disease (IBD). Recent findings The unfolded protein response (UPR) is a primitive cellular pathway that is engaged when responding to endoplasmic reticulum stress and regulates autophagy. Highly secretory cells such as Paneth cells and goblet cells in the intestines are particularly susceptible to endoplasmic reticulum stress and are exceedingly dependent upon a properly functioning UPR to maintain cellular viability and homeostasis. Primary genetic abnormalities within the components of the UPR (e.g. XBP1, ARG2, ORMDL3), genes that encode proteins reliant upon a robust secretory pathway (e.g. MUC2, HLAB27) and environmental factors that create disturbances in the UPR (e.g. microbial products and inflammatory cytokines) are important factors in the primary development and/or perpetuation of intestinal inflammation. Summary Endoplasmic reticulum stress is an important new pathway involved in the development of intestinal inflammation associated with IBD and likely other intestinal inflammatory disorders.

Immature Human Dendritic Cells Infected with Leishmania infantum Are Resistant to NK-Mediated Cytolysis but Are Efficiently Recognized by NKT Cells

Dendritic cells (DC) play an important role in innate and adaptive immunity, interacting with T cells, NK, and NKT cells. A critical step in the interaction of the parasitic protozoa Leishmania with their host is the evasion of both innate and adaptive immunity, producing a long-lasting chronic infection. There is growing evidence that these parasites can modify the Ag-presenting and immunoregulatory functions of DCs. The cells and mechanisms involved in innate immune response against Leishmania are still poorly understood. In this study, we investigated how Leishmania infantum infection affects DC interactions with NK and invariant NKT (iNKTs) cells in humans. We found that infected immature DCs (iDCs) do not up-regulate HLA class I molecules. Despite this, iDCs become resistant to killing mediated by autologous NK cells due to the up-regulation of HLA-E expression, which protects target cells from NK-mediated lysis through interaction with the inhibitory receptor CD94/NKG2A. Furthermore, iDCs infected with L. infantum up-regulate CD1d cell surface expression and consequently can be efficiently recognized and killed by iNKT cells that produce IFN-γ. These data suggest that L. infantum could be able to evade NK recognition; in contrast, iNKTs may play an important role in the immune response against Leishmania.

The germline repertoire of T cell receptor β-chain genes in multiple sclerosis patients from Spain

Recently several reports have described contradictory results after studying the association between restriction fragments length polymorphisms (RFLP) of T cell receptor (TcR) beta-chain genes and multiple sclerosis (MS). We studied the allelic, genotypic and haplotypic distribution of RFLPs of TcR beta chain gene segments C beta, V beta 8 and V beta 11 in 97 unrelated multiple sclerosis (MS) patients, 11 with chronic progressive MS and 86 with relapsing/remitting (R/R) MS. We found the distribution of the TcR haplotypes defined by the alleles of the three loci studied in the MS patients was significantly different from that found in control individuals. The distribution of TcR haplotypes in R/R MS patients was also different from that observed in controls. Our data suggest that the TcR beta chain gene complex contains one or more genes involved in genetic susceptibility to develop MS.

DNA polymorphisms and linkage relationship of the human complement component C6, C7, and C9 genes

In this report we describe the linkage between genes encoding human complement componentsC6,C7, andC9. Polymorphisms have been described at the DNA level for theC7 andC9 genes. We have studied 20 individuals by Southern blot analysis with fourC6 cDNA subclones to detect restriction fragment length polymorphisms (RFLPs). We have found a Taq I polymorphism defined by two alleles of 8.0 (C6 H) and 6.0 (C6 L) kilobases (kb). RFLP segregation for theC6, C7, andC9 loci in informative families allowed us to estimate the maximum Lod scores at a recombination fraction of Φ=0.0 (C6–C7), Φ=0.0 (C7–C9), and Φ=0.0 (C6–C9). Significant linkage disequilibrium was found betweenC6 andC7 and betweenC7 andC9 loci in directly determined haplotypes of unrelated parents. Data from this study show that the genes encoding the human terminal complement componentsC6, C7, andC9 define a cluster in the short arm of chromosome 5. We propose that the clusters involving theC8A andC8B and theC6, C7, andC9 genes be referred to as MACI and MACH, respectively.

Expression of Human CD1d Molecules Protects Target Cells from NK Cell-Mediated Cytolysis

The cytotoxic activity of NK cells can be inhibited by classical and nonclassical MHC molecules. The CD1 system is formed by a family of glycoproteins that are related to classical MHC. CD1a, b, and c molecules present lipids or glycolipids to T cells and are involved in defense against microbial infections, especially mycobacteria. It has been shown recently that these molecules can inhibit target cell lysis by human NK cells. It has also been shown that mouse CD1d molecules can protect cells from NK cell-mediated cytotoxicity. In the present study, we describe how human CD1d, orthologous to murine CD1 molecules, can inhibit NK cell-mediated cytolysis. We have expressed CD1d in the HLA class I-deficient cell lines L721.221 and C1R. The inhibitory effect is observed when effector NK cells from different donors are used, as well as in different cell lines with NK activity. The inhibitory effect was reversed by incubating the target cells with a mAb specific for human CD1d. Incubation of target cells with the ligands for CD1d, α-galactosylceramide (α-GalCer), and β-GalCer abolishes the protective effect of CD1d in our in vitro killing assays. Staining the effector cells using CD1d tetramers loaded with α-GalCer was negative, suggesting that the putative inhibitory receptor does not recognize CD1d molecules loaded with α-GalCer.

Mesoporous Silicon Microparticles Enhance MHC Class I Cross-Antigen Presentation by Human Dendritic Cells

The mesoporous silicon microparticles (MSMPs) are excellent vehicles for releasing molecules inside the cell. The aim of this work was to use MSMPs to deliver viral specific MHC class I restricted epitopes into human antigen presenting cells (monocyte derived dendritic cells, MDDCs) to facilitate their capture, processing, and presentation to CD8+ (cytotoxic) T lymphocytes. We show for the first time that MSMPs vehiculation of antigenic peptides enhances their MHC class I presentation by human MDDCs to CD8 T lymphocytes.

Structural characterization of two CD1A allelic variants

CD1 molecules are specialized in presenting lipidic antigens to T lymphocytes. They are structurally and evolutionary related to MHC molecules and show very limited polymorphism. We have previously described and partially characterized a new human CD1A allele differing from the wild type CD1A by a substitution of Cysteine by Tryptophan at position 52 in the alpha1 domain of the CD1A molecule. The frequency of this allele varies from 10% in individuals of Caucasian origin to 56% in Chinese people. The aim of the present work was to structurally characterize this CD1A allele. To do this we have cloned and sequenced the full-length cDNA encoding the new CD1A allele. The cDNA sequence of this allele encodes a protein differing the wild type in two amino acids at positions 14 (Threonine versus Isoleucine) and 52 (Cysteine versus Tryptophan). The cDNAs encoding both wild type and mutant CD1A were cloned in the expression vector pSRalphaNeo and transfected into C1R and L721.221 cells. Cell surface expression of the protein products in transfected cell lines were analyzed by flow cytometry and immunoprecipitation using CD1a-specific monoclonal antibodies. Our results indicate that both allelic products are efficiently expressed on the cell surface.

Intestinal epithelial cell endoplasmic reticulum stress promotes MULT1 up-regulation and NKG2D-mediated inflammation

Endoplasmic reticulum (ER) stress is commonly observed in intestinal epithelial cells (IECs) and can, if excessive, cause spontaneous intestinal inflammation as shown by mice with IEC-specific deletion of X-box–binding protein 1 (Xbp1), an unfolded protein response–related transcription factor. In this study, Xbp1 deletion in the epithelium (Xbp1&Dgr;IEC) is shown to cause increased expression of natural killer group 2 member D (NKG2D) ligand (NKG2DL) mouse UL16-binding protein (ULBP)–like transcript 1 and its human orthologue cytomegalovirus ULBP via ER stress–related transcription factor C/EBP homology protein. Increased NKG2DL expression on mouse IECs is associated with increased numbers of intraepithelial NKG2D-expressing group 1 innate lymphoid cells (ILCs; NK cells or ILC1). Blockade of NKG2D suppresses cytolysis against ER-stressed epithelial cells in vitro and spontaneous enteritis in vivo. Pharmacological depletion of NK1.1+ cells also significantly improved enteritis, whereas enteritis was not ameliorated in Recombinase activating gene 1−/−;Xbp1&Dgr;IEC mice. These experiments reveal innate immune sensing of ER stress in IECs as an important mechanism of intestinal inflammation.