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

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

N-Terminal Alms1 Protein Can Support Cilia Formation

<div><p>(A) Cotransfection of Alms1a siRNA-treated cells with a 5′ <i>Alms1</i> cDNA construct rescues primary cilia formation in mIMCD3 cells.</p><p>(B) Real-time PCR analysis of Alms1a siRNA and N-terminal Alms1-transfected cells. Upper panel: over-expression of the 5′ cDNA does not affect knockdown of endogenous <i>Alms1</i> mRNA with Alms1a siRNA. Lower panel: knockdown of endogenous <i>Alms1</i> mRNA does not affect overexpression of the 5′ cDNA. N, negative control siRNA; cDNA, 5′ <i>Alms1</i> cDNA; 1a, Alms1a siRNA.</p><p>(C) Stable expression of <i>Alms1</i> mRNA from the <i>Alms1^{L2131X/L2131X}</i> allele. Real-time PCR analysis of <i>Alms1</i> gene expression in an <i>Alms1^{L2131X/L2131X}</i> mouse and a wild-type littermate control.</p><p>(D) The N-terminal mouse Alms1 antibody detects Alms1 mutant protein at the ciliary basal body in primary kidney cells from the <i>Alms1^{L2131X/L2131X}</i> mouse. Shown are low and high magnifications of the ciliated cells. Arrowheads point out the base of cilia.</p><p>(E) Normal appearance of primary cilia in primary fibroblasts (MEF) and primary kidney cells (PKC) from the <i>Alms1^{L2131X/L2131X}</i> mouse strain.</p><p>(F) Inhibition of cilia formation in Alms1a siRNA-treated <i>Alms1^{L2131X/L2131X}</i> primary fibroblasts. Scale bars, 10 μm.</p></div

Kidney Abnormalities in <i>Alms1</i> Mutant Mice

<div><p>(A) H&E-stained kidney sections of a 6-mo-old <i>Alms1^{L2131X/L2131X}</i> mouse showing dilated cortex tubules compared with an age-matched wild-type control. Lack of kidney cilia is observed in some tubules in the cortex of <i>Alms1^{L2131X/L2131X}</i> kidney, whereas cilia in the medulla appear normal. H&E, hematoxylin-eosin; Acet, acetylated.</p><p>(B) Cortex cilia count comparison between <i>Alms1^{L2131X/L2131X}</i> and controls. 300–400 kidney nuclei were examined for each of six fields of <i>Alms1^{L2131X/L2131X}</i> and wild-type controls. The bar chart represents the average and standard deviations of cilia count per 100 kidney cortex epithelial cells from eight mice per group.</p><p>(C) In the cortex of <i>Alms1^{L2131X/L2131X}</i> kidney, cilia are lost selectively in LTA-labeled tubules but not in aquaporin-2-expressing tubules.</p><p>(D) Upper panel: clusters of Ki67-positive proliferating epithelial cells in the <i>Alms1^{L2131X/L2131X}</i> kidney, potentially lining the same convoluted tubule. Lower panel: TUNEL staining reveals apoptotic cells in <i>Alms1^{L2131X/L2131X}</i> kidneys but rarely in a wild-type control. Arrow, whole tubule cross sections were labeled by TUNEL, suggesting progression of nephropathy in <i>Alms1^{L2131X/L2131X}</i> mutant kidneys. WT, wild-type.</p><p>(E) Urinalysis of 3- to 6-mo-old <i>Alms1^{L2131X/L2131X}</i> mice and age-matched littermate controls. Urine from <i>Alms1^{L2131X/L2131X}</i> mice showed slight elevation of protein levels, <i>p</i> = 0.007. Scale bars, 50 μm.</p></div

Loss of Alms1 Does Not Affect Transcriptional Changes during Ciliogenesis but Causes Impairment in Flow-Induced Mechanosensation

<div><p>(A) Confocal microscopic analysis of cilia biogenesis in mIMCD3 cells. Short cilia can be detected at day 3 after transfection. mIMCD3 cells transfected with Alms1a siRNA have stunted cilia at days 3 and 5. Cells were stained with anti-acetylated tubulin (yellow, cilia) and TO-PRO-3 (red, nuclei).</p><p>(B) Suppression of Alms1 does not affect the upregulation of <i>Bbs4</i> and <i>Ttc10</i> during ciliogenesis. N, negative siRNA; 1a, Alms1a siRNA; 1b, Alms1b siRNA.</p><p>(C<b>)</b> Heat map representation of microarray analysis of mIMCD3 during ciliogenesis. 98 genes with the most dramatic changes in expression showed approximately equivalent expression changes in the presence of a scrambled siRNA control, or in the presence of specific siRNAs that decreased <i>Alms1</i> mRNA levels and blocked cilia formation.</p><p>(D) Stunted cilia formed in the presence of Alms1a siRNA (red) lack flow-induced Ca2+ influx in mIMCD3 cells, compared with a negative control siRNA (blue). Representative data are shown for cytosolic calcium change of individual cells in response to mechanical flow. Arrow points to the start of flow.</p></div