Genome-Wide ENU Mutagenesis to Reveal Immune Regulators

AbstractA complete list of molecular components for immune system function is now available with the completion of the human and mouse genome sequences. However, identification and functional annotation of genes involved in immunological processes require a discovery methodology that can efficiently and broadly analyze the complex interplay of these components in vivo. Our recent experience indicates that genome-wide chemical mutagenesis in the mouse is an extremely powerful methodology for the identification of genes required for complex immunological processes

Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis

BACKGROUND: MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis. METHODS AND FINDINGS: By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p , 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1b, TNF-a, and IFN-c was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-c, TNF-a, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis. CONCLUSIONS: Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted. The Editors’ Summary of this article follows the references

A Role for Alström Syndrome Protein, Alms1, in Kidney Ciliogenesis and Cellular Quiescence

Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alström syndrome. Alström syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5′ fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alström syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alström syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alström syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia

Aberrant Mucin Assembly in Mice Causes Endoplasmic Reticulum Stress and Spontaneous Inflammation Resembling Ulcerative Colitis

Michael McGuckin and colleagues identify two mutations that cause aberrant mucin oligomerization in mice. The resulting phenotype, including endoplasmic reticulum stress, resembles clinical and pathologic features of human ulcerative colitis

Genome-wide ENU mutagenesis to reveal immune regulators

A complete list of molecular components for immune system function is now available with the completion of the human and mouse genome sequences. However, identification and functional annotation of genes involved in immunological processes require a discovery methodology that can efficiently and broadly analyze the complex interplay of these components in vivo. Our recent experience indicates that genome-wide chemical mutagenesis in the mouse is an extremely powerful methodology for the identification of genes required for complex immunological processes

A genomic view of immunology

The outstanding problems facing immunology are whole system issues: curing allergic and autoimmune disease and developing vaccines to stimulate stronger immune responses against pathogenic organisms and cancer. We hope that the human genome sequence wil

Contribution of the Box 1 and Box 2 motifs of cytokine receptors to Jak1 association and activation

Kinases of the Jak family (Jak1/2/3 and Tyk2) interact with the membrane proximal domain of different cytokine receptors and play a critical role in the activation of cytokine and growth factor signaling pathways. In this report we demonstrate that both the Box 1 and Box 2 motif collaborate in the association and activation of Jak1 by type I interferons. Mutational analysis of the β chain of type I interferon receptor (IFNαRβL/IFNAR2) revealed that Box 1 plays a more significant role in activation than in the association with Jak1 On the contrary, the Box 2 motif contributes more to the association with Jak1 than to kinase activation. Additionally, the study of the Jak1 binding sites on the IL2 receptor β (IL2Rβ), IFNγRα/IFNGR1, and IL10Rα/IL10R1 chains suggests that cytokine receptors have two different kinds of interaction with Jakl. One form of interaction involves the Box 1 and the previously described Box 2 motif, which we now designate as Box 2A, characterized by the VEVI and LEVL sequences present in IFNαRβL/IFNAR2 and IL2Rβ subunits, respectively. The second form of interaction requires a motif termed Box 2B, which is present in the IFNγRα/IFNGR1 (SILLPKS) and IL10Rα/IL10R1 (SVLLFKK) chains. Interestingly, Box 2B localizes close to the membrane region (8-10 amino acids from the membrane) similar to Box 1, whereas Box 2A is more distal (38-58 amino acids from the membrane)

Suppression of <i>Alms1</i> Expression Alters Primary Cilium Formation in Kidney Epithelial Cells

<div><p>(A) Elongated cilia, visualized with staining of acetylated tubulin (green), form normally in mIMCD3 cells after mock-transfection, transfection with a negative control siRNA, or transfection with two inactive siRNAs directed against Alms1 (Alms1c and Alms1d). Focal staining of acetylated tubulin without axoneme extension is seen after transfection with two active siRNAs targeting <i>Alms1</i> (Alms1a and Alms1b).</p><p>(B) Real-time PCR analysis with two mouse <i>Alms1</i> probes recognizing the junctions of exons 1 and 2 and exons 12 and 13, respectively: Alms1a and Alms1b siRNAs both cause 70%–80% knockdown of <i>Alms1</i> mRNA; no effect on <i>Alms1</i> mRNA was seen with the three siRNAs that were inactive in the ciliogenesis assay.</p><p>(C) Alms1a siRNA-treated cells lose endogenous Alms1 protein expression. Acet, acetylated. Scale bars, 10 μm.</p></div