Interleukin-22 drives nitric oxide-dependent DNA damage and dysplasia in a murine model of colitis-associated cancer.

The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh infection induces DNA damage in this model and whether this depends on NO has not been determined. Here we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22-dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process.

Helicobacter hepaticus infection in mice: models for understanding lower bowel inflammation and cancer.

Pioneering work in the 1990s first linked a novel microaerobic bacterium, Helicobacter hepaticus, with chronic active hepatitis and inflammatory bowel disease in several murine models. Targeted H. hepaticus infection experiments subsequently demonstrated its ability to induce colitis, colorectal cancer, and extraintestinal diseases in a number of mouse strains with defects in immune function and/or regulation. H. hepaticus is now widely utilized as a model system to dissect how intestinal microbiota interact with the host to produce both inflammatory and tolerogenic responses. This model has been used to make important advances in understanding factors that regulate both acquired and innate immune response within the intestine. Further, it has been an effective tool to help define the function of regulatory T cells, including their ability to directly inhibit the innate inflammatory response to gut microbiota. The complete genomic sequence of H. hepaticus has advanced the identification of several virulence factors and aided in the elucidation of H. hepaticus pathogenesis. Delineating targets of H. hepaticus virulence factors could facilitate novel approaches to treating microbially induced lower bowel inflammatory diseases.

Acute appendicitis is characterized by a uniform and highly selective pattern of inflammatory gene expression.

Acute appendicitis (AA) is the most common life-threatening surgical emergency in pediatrics. To characterize the nature of the inflammatory response in AA, gene expression profiles were generated. We found remarkable uniformity in the genes that were differentially expressed between patients with appendicitis and control groups. Sixty-four probe sets were differentially expressed in samples from patients with both severe and mild appendicitis compared to control samples, and within this group we were able to identify four dominant clusters. Interestingly, expression levels of interleukin (IL)-8 significantly correlated with histologic score, and expression of IL-8 protein was observed within both neutrophils and mononuclear cells by immunohistochemistry, suggesting a possible role in the etiology of appendicitis. Although there was some overlap between genes reported to be differentially expressed in Crohn's disease (CD) and those observed in AA, differential expression of genes involved in interferon responses that characterize CD was not observed.

Targeted mutation of TNF receptor I rescues the RelA-deficient mouse and reveals a critical role for NF-kappa B in leukocyte recruitment.

NF-kappaB binding sites are present in the promoter regions of many acute phase and inflammatory response genes, suggesting that NF-kappaB plays an important role in the initiation of innate immune responses. However, targeted mutations of the various NF-kappaB family members have yet to identify members responsible for this critical role. RelA-deficient mice die on embryonic day 15 from TNF-alpha-induced liver degeneration. To investigate the importance of RelA in innate immunity, we genetically suppressed this embryonic lethality by breeding the RelA deficiency onto a TNFR type 1 (TNFR1)-deficient background. TNFR1/RelA-deficient mice were born healthy, but were susceptible to bacterial infections and bacteremia and died within a few weeks after birth. Hemopoiesis was intact in TNFR1/RelA-deficient newborns, but neutrophil emigration to alveoli during LPS-induced pneumonia was severely reduced relative to that in wild-type or TNFR1-deficient mice. In contrast, radiation chimeras reconstituted with RelA or TNFR1/RelA-deficient hemopoietic cells were healthy and demonstrated no defect in neutrophil emigration during LPS-induced pneumonia. Analysis of RNA harvested from the lungs of mice 4 h after LPS insufflation revealed that the induction of several genes important for neutrophil recruitment to the lung was significantly reduced in TNFR1/RelA-deficient mice relative to that in wild-type or TNFR1-deficient mice. These results suggest that TNFR1-independent activation of RelA is essential in cells of nonhemopoietic origin during the initiation of an innate immune response.

Mechanisms of granulocytosis in the absence of CD18.

Genetic deficiency in CD18 leads to disease characterized by myeloid hyperplasia, including profound granulocytosis and splenomegaly. Myeloid hyperplasia could directly result from the disruption of CD18 functions essential to granulopoiesis or basal leukocyte trafficking. Alternatively, myeloid hyperplasia could be reactive in nature, due to disruption of essential roles of CD18 in leukocyte responses to microbial challenge. To distinguish between these mechanisms, the hematopoietic systems of lethally irradiated wild-type (WT) mice were reconstituted with either WT fetal liver cells or CD18-deficient fetal liver cells, or an equal mixture of both types of cells. Granulocytosis and splenomegaly developed in mice that received CD18-deficient fetal liver cells. Splenomegaly was prevented and granulocytosis was inhibited by more than 95% in mice that had received both CD18-deficient and WT fetal liver cells, suggesting that myeloid hyperplasia was largely reactive in nature. Consistent with this postulate, the circulating life spans in the blood and the fraction of neutrophils that incorporated BrdU in the bone marrow were not increased for CD18-deficient neutrophils compared with the WT. However, these animals did develop mild granulocytosis compared with mice reconstituted with WT cells alone, and a higher percentage of CD18-deficient leukocytes were neutrophils compared with the WT leukocytes. These observations suggest that the granulocytosis observed in the absence of CD18 occurs through at least 2 mechanisms: one that is dramatically improved by the presence of WT cells, likely reactive in nature, and a second that is independent of the WT hematopoietic cells, involving an alteration in the lineage distribution of blood leukocytes.

Typhlocolitis in NF-kappa B-deficient mice.

Activation of inflammatory gene expression by the transcription factor NF-kappaB is a central pathway in many inflammatory disorders, including colitis. Increased NF-kappaB activity has been linked with development of colitis in humans and animal models, thus it was unexpected when NF-kappaB-deficient mice developed spontaneous typhlocolitis. To further characterize this finding, we induced typhlocolitis in rederived NF-kappaB-deficient mice using intragastric infection with Helicobacter hepaticus. At 6 wk postinfection (PI), severe colitis with increased type 1 cytokine expression was seen in infected mice that lacked the p50 subunit of NF-kappaB and were also heterozygous for the p65 subunit of NF-kappaB(p50(-/-)p65(+/-)). Mice lacking the p50 subunit alone (p50(-/-)) were less severely affected, and wild-type mice and p65(+/-) mice were unaffected. T cell development in NF-kappaB-deficient mice was normal. These data indicate that p50 and p65 subunits of NF-kappaB have an unexpected role in inhibiting the development of colitis.

Nuclear factor kappa B is required for the development of marginal zone B lymphocytes.

Although immunoglobulin (Ig)M(hi)IgD(lo/-)CD21(hi) marginal zone B cells represent a significant proportion of naive peripheral splenic B lymphocytes, few of the genes that regulate their development have been identified. This subset of peripheral B cells fails to emerge in mice that lack nuclear factor (NF)-kappa Bp50. Less drastic reductions in marginal zone B cell numbers are also seen in the spleens of recombination activating gene (Rag)-2(-/-) mice reconstituted with NF-kappa Bp65(-/-) fetal liver cells and in c-Rel(-/-) mice. In contrast, steady-state levels of IgD(hi) splenic follicular B cells are not significantly reduced in the absence of NF-kappa Bp50, NF-kappa Bp65, or c-Rel. Reconstitution of B cells in Rag-2(-/-) mice with a mixture of p50(-/-)/p65(-/-) fetal liver cells and Rag-2(-/-) bone marrow cells revealed that the generation of marginal zone B cells requires the expression of NF-kappa B in developing B cells, as opposed to supporting cells.

Effects of CD18 deficiency on the emigration of murine neutrophils during pneumonia.

We hypothesized that CD18 deficiency would impair the ability of neutrophils to emigrate from pulmonary blood vessels during certain pneumonias. To directly compare the abilities of wild-type (WT) and CD18-deficient neutrophils to emigrate, mice with both types of leukocytes in their blood were generated by reconstituting the hemopoietic systems of lethally irradiated C57BL/6 mice with mixtures of fetal liver cells from WT and CD18-deficient mice. Percentages of CD18-deficient neutrophils in the circulating and emigrated pools were compared during experimental pneumonias. Similar percentages were observed in the blood and bronchoalveolar lavage fluid 6 or 24 h after intratracheal instillation of Streptococcus pneumoniae, demonstrating that no site on the CD18 molecule was required for either its adhesive or its signaling functions during neutrophil emigration. However, 6 h after instillation of Escherichia coli LPS or Pseudomonas aeruginosa, the percentage of CD18-deficient neutrophils in the bronchoalveolar lavage fluid was only about one-fourth of that observed in the blood. This difference persisted for at least 24 h after instillation of E. coli LPS. Thus, neutrophil emigration elicited by the Gram-negative stimuli E. coli LPS or P. aeruginosa was compromised by deficiency of CD18. These data, based on comparing WT and gene-targeted CD18-deficient neutrophils within the same animals, provide evidence for molecular pathways regulating neutrophil emigration, which could not be appreciated in previous studies with pharmacological blockade or genetic deficiency of CD18.

The p65 subunit of NF-kappa B is redundant with p50 during B cell proliferative responses, and is required for germline CH transcription and class switching to IgG3.

B cells lacking individual NF-kappa B/Rel family members exhibit defects in activation programs. We generated small resting B cells lacking p65 or p50 alone, or lacking both p50 and p65, then evaluated the ability of these cells to proliferate, secrete Ig, and undergo Ig class switching. B cells lacking p65 proliferated well in response to all stimuli tested. However, these cells demonstrated an isolated defect in switching to IgG3, which was associated with a decrease in gamma 3 germline CH gene expression. Whereas, previously reported, B cells lacking p50 alone had a severe proliferative defect in response to LPS, a moderate defect in response to CD40 ligand (CD40L), and normal proliferation to Ag receptor cross-linking using dextran-conjugated anti-IgD Abs (alpha delta-dex), B cells lacking both p50 and p65 exhibited severely impaired proliferation in response to LPS, alpha delta-dex, and CD40L. This defect could be overcome by simultaneous administration of alpha delta-dex and CD40L. In response to this latter combination of stimuli, B cells lacking both p50 and p65 secreted Ig and underwent isotype switching to IgG1 as efficiently as B cells lacking p50 alone. These data demonstrate a role for the p65 subunit of NF-kappa B in germline CH gene expression as well as functional redundancy between p50 and p65 during proliferative responses.

Failure of lymphopoiesis after adoptive transfer of NF-kappaB-deficient fetal liver cells.

Mice deficient in the p65 subunit of NF-kappaB die during fetal development. Introduction of p50/p65-deficient fetal liver cells into lethally irradiated hosts resulted in a severe deficit of fetal liver-derived lymphocytes and their immediate precursors but an overabundance of fetal liver-derived granulocytes. Surprisingly, simultaneous transplantation of wild-type bone marrow cells rescued the production of p50/p65-deficient lymphocytes. Expression of immunoglobulin K light chains on these rescued NF-kappaB-deficient B lymphocytes was normal. These results suggest that while p50 and p65 do not regulate the maturation of pre-B cells, NF-kappaB mediates the development or survival of an early lymphocyte precursor through regulation of an extracellular factor.

The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine.

The 44-amino-acid E5 transforming protein of bovine papillomavirus can induce growth transformation of cultured rodent fibroblast cell lines. Previous studies revealed that efficient transformation of mouse C127 cells by the E5 protein required a central core of hydrophobic amino acids and several specific carboxyl-terminal amino acids. Although a randomly derived sequence of hydrophobic amino acids could functionally replace the wild-type hydrophobic core, most such sequences could not. We show here that the conserved glutamine at position 17 in the hydrophobic domain is also important for transformation and that insertion of the glutamine can rescue the transforming activity of many but not all otherwise defective mutants containing random hydrophobic sequences. However, a class of mutants was identified that transform efficiently even in the absence of glutamine, demonstrating that the presence of this amino acid is not absolutely required for efficient transformation. E5 proteins containing the glutamine appear to display increased homodimer formation compared with mutant proteins lacking the glutamine, but this amino acid has no apparent effect on protein stability.

Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16.

To examine the biological properties of the bovine papillomavirus type 1 (BPV) and human papillomavirus type 16 (HPV16) E5 genes, each was cloned separately into a retroviral expression vector and helper-free recombinant viruses were generated in packaging cell lines. The BPV E5 retroviruses efficiently caused morphologic and tumorigenic transformation of cultured lines of murine fibroblasts, whereas the HPV16 E5 viruses were inactive in these assays. In contrast, infection of the p117 established line of murine epidermal keratinocytes with either the BPV or the HPV16 E5 retrovirus resulted in the generation of tumorigenic cells. Pam212 murine keratinocytes were also transformed to tumorigenicity by the HPV16 E5 gene but not by the gene carrying a frameshift mutation. These results establish that the HPV16 E5 gene is a transforming gene in cells related to its normal host epithelial cells.

High fat feeding increases brown fat GDP binding in lean but not obese Zucker rats.

The effects of high fat feeding on brown fat thermogenesis in rodents are controversial. In this study, we examined the effects of 8 d of high fat feeding on brown fat mitochondrial GDP binding (an in vitro index of thermogenic activity) in lean (Fa/?) and obese (fa/fa) Zucker rats. Six-week-old female rats were fed a defined low fat control diet (9.5% of energy from fat) only during the dark cycle (1200-2400 h), and food intake was measured daily at 1200, 1600, 2300, and 2400 h for 7 d (the control period). For the next 8 d, half of the lean and obese rats were fed a high fat diet (65% of energy from fat), and the others remained on the low fat control diet. Each rat was fed the amount of energy equivalent to its average energy intake during the 7-d control period. Rats were killed at 0800-1000 h. In the lean rats, high fat feeding increased GDP binding. This increased binding in the lean rats appeared to reflect levels of dietary fat and carbohydrate and was independent of caloric intake. The blunted GDP binding of the obese rats failed to respond to dietary changes.

Genetic evidence that acute morphologic transformation, induction of cellular DNA synthesis, and focus formation are mediated by a single activity of the bovine papillomavirus E5 protein.

The bovine papillomavirus (BPV) type 1 E5 gene encodes a 44-amino-acid protein that can stably transform cultured rodent cells when expressed in the absence of all other viral genes. We have previously constructed a BPV-simian virus 40 recombinant virus (Pava-1) which efficiently expresses the BPV type 1 E5 gene in infected cells (J. Settleman and D. DiMaio, Proc. Natl. Acad. Sci. USA 85:9007-9011, 1988). Within 48 h of Pava-1 infection, the vast majority of mouse C127 cells underwent a dramatic morphologic transformation which was accompanied by cell proliferation. Infection of C127 cells made quiescent by contact inhibition and serum starvation caused a great induction of cellular DNA synthesis. These morphologic and mitogenic responses were proportional to the virus multiplicity of infection. Mutational analysis indicated that the E5 gene is both necessary and sufficient for these activities. Analysis of a variety of E5 missense mutants revealed a strong correlation between their phenotypes in the acute transformation assays following infection and in the stable focus-forming assay following transfection. Most of the defective mutants expressed normal levels of E5 protein following infection, indicating that their defective phenotypes are not due to the synthesis of an unstable protein. The failure to genetically resolve these E5 activities suggests that the ability of the E5 protein to cause acute morphologic transformation and reentry into the cell cycle may be intimately related to its ability to cause stable cell transformation and that these functions are probably mediated by a single biochemical activity of the E5 protein.

Transforming activity of a 16-amino-acid segment of the bovine papillomavirus E5 protein linked to random sequences of hydrophobic amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the smallest transforming protein yet described. Previous results from our laboratory indicate that a hydrophobic core and specific carboxyl-terminal amino acids are required for the E5 protein to exert its transforming function. In this study, additional substitution mutations were generated in the E5 gene to determine the minimal amino acid sequence requirements for focus formation in mouse C127 cells. In most cases examined, substitution of the hydrophobic middle third of the E5 protein with unrelated hydrophobic sequences severely inhibited transforming activity. However, we have identified one hydrophobic amino acid sequence apparently unrelated to the wild-type one that can replace the middle third of the wild-type E5 protein without affecting the ability of the protein to stably transform cells or interact with cell membranes. Furthermore, a mutant E5 protein in which only the carboxyl-terminal 16 amino acids of the protein have been derived from E5 sequences retains transforming activity. Since several residues in the carboxyl-terminal portion of the E5 protein can be freely substituted with different amino acids (B. H. Horwitz, A. L. Burkhardt, R. Schlegel, and D. DiMaio, Mol. Cell. Biol. 8:4071-4078, 1988), the results reported here imply that much of the specific information necessary for cell transformation can be supplied by a subset of the carboxyl-terminal 16 amino acids of this protein.

44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the E5 gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the E5 protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the E5 protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.

Bovine papillomavirus E2 gene regulates expression of the viral E5 transforming gene.

We have performed transient-expression experiments with CV1 monkey kidney cells to investigate the role of the bovine papillomavirus type 1 (BPV1) E2 gene in the regulation of the E5 transforming gene. Direct analysis of the 7-kilodalton open reading frame (ORF) E5 protein and measurements of the expression of an E5-chloramphenicol acetyltransferase fusion protein indicate that the efficient expression of ORF E5 requires the full-length E2 gene, which can be supplied in trans. The viral long control region is required in cis for this response to ORF E2, and it acts in a position- and orientation-independent fashion characteristic of a transcriptional enhancer. Deletion analysis suggests that the P2443 promoter is required for efficient expression of the E5 gene. The E2 repressor activity encoded in the 3' end of the E2 gene inhibits the expression of ORF E5. These effects define a major BPV1 regulatory circuit and appear to explain the transformation behavior of a variety of BPV1 mutants.

Mutational analysis of open reading frame E4 of bovine papillomavirus type 1.

Open reading frame (ORF) E4 is a 353-base-pair ORF of bovine papillomavirus type 1. To determine the biological activities of this ORF in mouse C127 cells, we analyzed the effects of two constructed mutations which are predicted to prevent synthesis of ORF E4 proteins while leaving the amino acid sequence encoded by the overlapping ORF E2 unchanged. Neither mutation interfered with the abilities of the mutants to efficiently induce focus formation, induce growth in soft agarose, or transactivate an inducible bovine papillomavirus type 1 enhancer. Also, neither mutation prevented establishment of the viral DNA as an extrachromosomal plasmid in transformed cells. These results suggest that ORF E4 proteins are not required for these biological activities, and they are consistent with the observation of others (J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986) that the ORF E4 protein of a human papillomavirus is associated with late gene expression during papilloma formation.