A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease

Crohn's disease is a chronic inflammatory disorder of the gastrointestinal tract, which is thought to result from the effect of environmental factors in a genetically predisposed host. A gene location in the pericentromeric region of chromosome 16, IBD1, that contributes to susceptibility to Crohn's disease has been established through multiple linkage studies(1-6), but the specific gene(s) has not been identified. NOD2, a gene that encodes a protein with homology to plant disease resistance gene products is located in the peak region of linkage on chromosome 16 (ref. 7). Here we show, by using the transmission disequilibium test and case-control analysis, that a frameshift mutation caused by a cytosine insertion, 3020insC, which is expected to encode a truncated NOD2 protein, is associated with Crohn's disease. Wild-type NOD2 activates nuclear factor NF-kappaB, making it responsive to bacterial lipopolysaccharides; however, this induction was deficient in mutant NOD2. These results implicate NOD2 in susceptibility to Crohn's disease, and suggest a link between an innate immune response to bacterial components and development of disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62856/1/411603a0.pd

Proteasome subunit deficiency influences the innate immune response to Streptococcus pneumoniae

Proteasomen, die die proteolytisch aktiven Untereinheiten LMP7, LMP2 und MECL1 inkorporieren, nennt man Immunoproteasomen. Während einer Immunreaktion führen diese regulierende sowie modulierende Funktionenaus. Sie sind konstitutiv exprimiert in Zellen hämatopoetischen Ursprungs, ein Bestandteil des angeborenen Immunsystems, die die erste Angriffsfront gegen pathogene Mikroorganismen ausbilden. Um die Bedeutung des Immunoproteasoms für die angeborene Immunantwort bei einer Streptococcus pneumoniae Infektion auf zu zeigen, charakterisierten wir den Krankheitsverlauf einer bakteriellen Pneumonie und analysierten lokale aber auch systemische Immunreaktionen in LMP7 ko Mäusen mit Hilfe eines S. pneumoniae Infektionsmodels. Die hier generierten Daten zeigten einen fortgeschrittenen Krankheitsverlauf in LMP7 ko Mäuse, der in einer systemischen inflammatorischen Immunreaktion endete und sich in klinischen Parametern, wie physiologische Kondition, spezifische diagnostische Marker und Immunsuppression, andeutete. Der Zustand der Sepsis entwickelte sich vermutlich aufgrund einer erhöhten bakteriellen Last im Blut und führte zu einer vorzeitigen Mortalität infizierter LMP7 ko Tiere. Obwohl die Fähigkeit von LMP7 ko Leukozyten ex vivo Bakterien zu eliminieren nicht beeinträchtigt war, zeigten LMP7 ko Mäuse in vivo eine verminderte Genexpression immunmodulierender Moleküle, wie Pentraxine, Fikoline und Kollektine. Diese Moleküle fördern die Aufnahme, Elimination und Degradation pathogener Mikroorganismen. Die reduzierte Expression opsonierender Moleküle wurde begleitet von einer veränderten proteasomalen Zusammensetzung in murinen Makrophagen und Lebergewebe. Zusammengefasst lässt sich sagen, dass diese Ergebnisse eine bisher unbekannte Rolle von Immunoproteasomalen Untereinheiten bei der Regulierung der angeborenen Immunantwort auf extrazelluläre bakterielle Infektionen unterstreichen.Immunoproteasomes, harboring the active site subunits LMP7, LMP2, and MECL1 exert protective, regulatory or modulating functions during infection-induced immune responses. Immunoproteasomes are constitutively expressed in hematopoietic derived cells, constituting the first line of defense against invading pathogens. To clarify the impact of immunoproteasomes on the innate immune response against Streptococcus pneumoniae, we characterized the progression of disease and analyzed the local as well as systemic innate immune response in LMP7 ko mice by using a S. pneumoniae infection model. Data showed that mice deficient in LMP7 suffered from a more severe case of pneumonia which ended in a systemic inflammatory response indicated by aggravated clinical signs, diagnostic parameters, and immune suppression. The systemic inflammatory response probably established in consequence of an increased bacteremia and resulted in early mortality. Although, bacterial killing efficiency of LMP7 ko leukocytes was unaffected ex vivo, LMP7 ko mice exhibited a reduction in the transcription of genes encoding immune modulating molecules such as pentraxins, ficolins, and collectins, which facilitate opsonophagocytosis. The reduced expression of opsonins was accompanied by an affected subunit composition of proteasomes in murine macrophages and liver. In summary these results highlight an unsuspected role for immuno-subunits in modulating the innate immune response to extracellular bacterial infections

Proteasome β5i Subunit Deficiency Affects Opsonin Synthesis and Aggravates Pneumococcal Pneumonia.

Immunoproteasomes, harboring the active site subunits β5i/LMP7, β1i/LMP2, and β2i/MECL1 exert protective, regulatory or modulating functions during infection-induced immune responses. Immunoproteasomes are constitutively expressed in hematopoietic derived cells, constituting the first line of defense against invading pathogens. To clarify the impact of immunoproteasomes on the innate immune response against Streptococcus pneumoniae, we characterized the progression of disease and analyzed the systemic immune response in β5i/LMP7-/- mice. Our data show that β5i/LMP7 deficiency, which affected the subunit composition of proteasomes in murine macrophages and liver, was accompanied by reduced transcription of genes encoding immune modulating molecules such as pentraxins, ficolins, and collectins. The diminished opsonin expression suggested an impaired humoral immune response against invading pneumococci resulting in an aggravated systemic dissemination of S. pneumoniae in β5i/LMP7-/- mice. The impaired bacterial elimination in β5i/LMP7-/- mice was accompanied by an aggravated course of pneumonia with early mortality as a consequence of critical illness during the late phase of disease. In summary our results highlight an unsuspected role for immuno-subunits in modulating the innate immune response to extracellular bacterial infections

Efficiency of bacterial killing is unaltered in β5i/LMP7^-/- leukocytes.

<p>(A) Analysis of phagocytosis of fluorochrome-labelled particles by BMM, determined by flow cytometry (each group with n = 3). (B) Analysis of the intracellular early phase of killing and (C) late phase of killing of D39Δcps by BMM. Therefore, BMM were co-cultivated with D39Δcps MOI 10, cells were lysed, and cfu/ml were determined (each group with n = 3). (D) Analysis of NO secretion by WT and β5i/LMP7^-/- BMM, stimulated either with LPS (1 μg/ml) or heat-inactivated D39Δcps lysate (MOI 50). NO concentrations were photometrically determined in the supernatant by Griess-Reagent (each group with n = 7; statistical analysis by student´s t-test. * p<0.05).</p

β5i/LMP7^-/- mice suffer more than WT mice from severe pneumonia.

<p>(A) Monitoring of body weight 24 h and 48 h after transnasal application of 5×10⁶ CFU PN36/mouse (each group with n = 12–29). (B) Analysis of mRNA expression of the acute phase protein SAA in liver after transnasal application of 5×10⁶ CFU PN36/mouse, performed by RT and Real Time PCR (each group with n = 3–14) (statistical analysis by Mann Whitney U Test. *p<0.05; **p<0.01; ***p<0.001). (C) Assessment of the approximate survival rate for up to 10 days upon transnasal application of 7.5x10⁴ CFU PN36/mouse (each group with n = 17).</p

β5i/LMP7 deficiency as well as proteasome inhibition enhances AP-1 activation in stimulated macrophages.

<p>(A-B) Immuno-blot on phosphorylated and non-phosphorylated members of AP-1 and the NF-κB activating pathway in WT and β5i/LMP7^-/- BMM, stimulated <i>in vitro</i> for 0 min, 30 min, and 240 min with (A) LPS (1 μg/ml) or (B) D39Δcps (MOI 10) (each group with n = 3). (C) Immuno-blot on phosphorylated and non-phosphorylated cJun in RAW 264.7 cells, pretreated with DMSO or epoxomicin (250 nM) for 2 h, and subsequently stimulated with LPS (1 μg/ml) or D39Δcps (MOI 10) for 0 h, 2 h, 4 h, and 6 h. (D) Immuno-blot on catalytic β-subunits of the proteasome in RAW 264.7 cells incubated for 2 h with DMSO or epoxomicin (250 nM). Molecular shift indicates inhibitor binding.</p

β5i/LMP7^-/- mice exhibit a diminished expression of opsonizing molecules in lung, liver and macrophages.

<p>(A) Gene expression analysis of lungs, 48 h after transnasal application of 5×10⁶ CFU PN36/mouse, performed by RT and Real Time PCR (each group with n = 5–6; statistical analysis by Mann Whitney U Test. *p<0.05). (B) Gene expression analysis of BMM co-cultivated with D39Δcps (MOI 10) for 4 h, performed by RT and Real Time PCR (each group with n = 4, illustrated experiment is representative for 4 individual replicates; showing mean± SD; statistical analysis by student’s t-test **p<0.01). (C) Gene expression analysis of liver 48 h after transnasal application of 5×10⁶ CFU PN36/mouse, performed by RT and Real Time PCR (each group with n = 12–14; statistical analysis by Mann Whitney U Test. *p<0.05; **p<0.01).</p

Both immuno- and standard subunits of the proteasome are constitutively expressed in leukocytes and liver.

<p>(A) Analysis of proteasome catalytic β-subunits expression on protein level in liver, 48 h after transnasal application of 5×10⁶ CFU PN36/mouse, performed by immuno-blot. (B) Analysis of proteasome catalytic β-subunits expression on protein level in BMM performed by immuno-blot (each group with n = 3). (C) 2D gel electrophoresis of purified 20S proteasomes, isolated from WT BMM. Proteasome subunits were visualized by coomassie stain. (D) Determination of β-subunits, incorporated in BMM 20S, detected by immuno-blot.</p

Systemic levels of chemokines increase after infection, but do not differ between WT and β5i/LMP7^-/- mice.

<p>Determination of chemokines in plasma, obtained 48 h after transnasal application of 5×10⁶ CFU PN36/mouse, and measured by cytokine multiplex assay (each group with n = 5–7).</p

β5i/LMP7 deficiency has no apoptotic or cytotoxic effect upon infection.

<p><b>(A)</b> Analysis of the activity of caspase 3/7 in cell lysate and LDH in supernatant of stimulated BMM. They were co-cultivated with D39Δcps (MOI 10) for 4 h, or treated with 10 μM MG132 for 24 h as positive control (each group with n = 4). <b>(B)</b> Analysis of potential liver damage, 48 h post infection, by measuring ALAT activity in serum of mice transnasally infected with 5×10⁶ CFU <i>PN36</i>/mouse (each group with n = 5–6).</p