Cdcs1 a major colitis susceptibility locus in mice; subcongenic analysis reveals genetic complexity.

BACKGROUND: The cytokine-deficiency-induced colitis susceptibility (Cdcs)1 locus is a major modifier of murine inflammatory bowel disease (IBD) and was originally identified in experimental crosses of interleukin-10-deficient (Il10(-/-)) mice. Congenic mice, in which this locus was reciprocally transferred between IBD-susceptible C3H/HeJBir-Il10(-/-) and resistant C57BL/6J-Il10(-/-) mice, revealed that this locus likely acts by inducing innate hypo- and adaptive hyperresponsiveness, associated with impaired NF-kappaB responses of macrophages. The aim of the present study was to dissect the complexity of Cdcs1 by further development and characterization of reciprocal Cdcs1 congenic strains and to identify potential candidate genes in the congenic interval. METHODS: In total, 15 reciprocal congenic strains were generated from Il10(-/-) mice of either C3H/HeJBir or C57BL/6J genetic backgrounds by 10 cycles of backcrossing. Colitis activity was monitored by histological grading. Candidate genes were identified by fine mapping of congenic intervals, sequencing, microarray analysis, and a high-throughput real-time reverse-transcription polymerase chain reaction (RT-PCR) approach using bone marrow-derived macrophages. RESULTS: Within the originally identified Cdcs1-interval, 3 independent regions were detected that likely contain susceptibility-determining genetic factors (Cdcs1.1, Cdcs1.2, and Cdcs1.3). Combining results of candidate gene approaches revealed Fcgr1, Cnn3, Larp7, and Alpk1 as highly attractive candidate genes with polymorphisms in coding or regulatory regions and expression differences between susceptible and resistant mouse strains. CONCLUSIONS: Subcongenic analysis of the major susceptibility locus Cdcs1 on mouse chromosome 3 revealed a complex genetic structure. Candidate gene approaches revealed attractive genes within the identified regions.

Cdcs1, a major colitogenic locus in mice, regulates innate and adaptive immune response to enteric bacterial antigens.

BACKGROUND & AIMS: The absence of interleukin 10, a key cytokine in gut homeostasis, causes severe colitis in C3H/HeJBir but not C57BL/6J mice. The major modifier for colitis was mapped on chromosome 3 and designated cytokine deficiency-induced colitis susceptibility 1 (Cdcs1). We developed reciprocal Cdcs1 congenic stocks on both interleukin 10-deficient backgrounds to identify the susceptibility gene and its function. METHODS: C3H/HeJBir congenic for the C57BL/6J-derived Cdcs1 allele and reciprocal C57BL/6J congenic for the C3H/HeJBir allele were analyzed for colitis development. Parental strains were compared by electrophoretic mobility shift assay to assess the candidacy of nuclear factor-kappaB p50 in the Cdcs1 interval. Functional differences were observed in innate and adaptive immune responses of parental and congenic stocks after bacterial ligand exposure in vitro (cytokine release from bone marrow-derived macrophage and dendritic cells) and in vivo (serum cytokines and primed CD4+ T cell proliferation). RESULTS: Cdcs1 was positioned within a minimum 7-megabase interval containing nuclear factor-kappaB p50. C3H/HeJBir colitis was significantly diminished by the C57BL/6J genome in this interval. Conversely, colitis in C57BL/6J was significantly exacerbated by the reciprocal C3H/HeJBir genome. C3H/HeJBir macrophages constitutively expressed higher nuclear factor-kappaB p50. Functional assays showed that C3H/HeJBir showed reduced innate responsiveness both in vivo and in vitro to bacterial ligands but showed increased CD4 T-cell responses compared with C57BL/6J. This differential responsiveness was controlled by the respective allele at Cdcs1. CONCLUSIONS: The colitogenic Cdcs1 allele impairs innate immunity to bacterial products and in turn skews the adaptive immune response toward compensatory hyperresponsiveness and chronic intestinal inflammation.

Hemoglobin in five genetically diverse Frankia strains.

Five strains of Frankia were selected to represent a wide range of genetic diversity and examined for presence of hemoglobin. All five strains produced hemoglobin when grown on media without (-N) or with (+N) combined nitrogen. This indicates that hemoglobin is common in Frankia and is not directly associated with nitrogen fixation. Frankia strain EAN1(pec) was examined in more detail. It showed greater hemoglobin concentration when grown at 2% O2 than at 20% O2 in the -N treatment but no effect of oxygen on hemoglobin concentration in the +N treatment. At both oxygen levels, it produced substantially more biomass in +N than in -N culture. It also produced significantly more biomass when the medium contained 0.2% CO2 than in the absence of CO2. The molecular mass of the hemoglobin as determined by size exclusion chromatography was 13.4 +/- 0.2 kDa (mean +/- SE, n = 3) and is consistent with that of a truncated hemoglobin. The hemoglobin had absorption spectra that were typical of a hemoglobin. The oxygen dissociation rate constants for the hemoglobin were 131.2 +/- 5.8 s(-1) for -N culture and 166 +/- 8.2 s(-1) for +N culture. These rapid rates are consistent with a function in facilitated diffusion of oxygen.

Hemoglobin in Frankia, a nitrogen-fixing actinomycete.

Frankia strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s(-1), and the oxygen association rate constant was 206 microM(-1) s(-1).