Supplementary Figure 1. Overall experimental design including the rationale behind specific details. Supplementary Figure 2. Confirmation of the ASF strains and B. fibrisolvens by PCR. Supplementary Figure 3. Formulations of the 3 diets provided to gnotobiotic mice in these experiments. Supplementary Figure 4. A combination of a high-fiber diet and a butyrate-producing bacterium (Butyrivibrio fibrisolvens) yields higher levels of butyrate in the lumen of the colon than either factor in isolation. Supplementary Figure 5. Detection of b-catenin mutations in tumor cells. Supplementary Figure 6. Analysis of b catenin nuclear localization in control and experimental tumors. Supplementary Figure 7. Characterization of squamous cell tumors. Supplementary Figure 8. Analysis of wild-type and mutant B. fibrisolvens strains. Supplementary Figure 9. Expression of Warburg effect markers. Supplementary Figure 10. Butyrate oxidation and uptake in colorectal tumors and normal colonic tissues. Supplementary Figure 11. Representative IHC images showing cleaved caspase 3 positive cells (arrows) in normal colonic tissue and tumors from mice in control and experimental conditions. Supplementary Figure 12. Flow cytometric analysis of immune cell populations from BALB/c mice colonized with the 4 ASF and B. fibrisolvens in gnotobiotic isolators. Supplementary Figure 13. Serum and colonic cytokine levels (pg/mL) from BALB/c mice colonized with the 4 ASF and B. fibrisolvens in gnotobiotic isolators based on Luminex assays. Supplementary Figure 14. Minimal inflammation based on histologic assessment of H&E-stained sections of colorectal tumor sections from 7-10 mice of each treatment group 5 months after exposure to the 5 AOM/3 DSS regimen. Supplementary Figure 15. Control experiment demonstrating that inflammation plays a minor role in colorectal tumorigenesis in the gnotobiotic model where BALB/c mice were polyassociated with 4 ASF plus B. fibrisolvens and then treated with AOM/DSS. Supplementary Figure 16. Relative Il18 mRNA levels normalized to Gapdh levels in normal colonic tissue and tumors based on RT-qPCR experiments. Supplementary Table 1. Luminal Concentrations (μM) of Other Two Major SCFAs (Propionate and Acetate). Supplementary Table 2. Clinical Information Regarding Human Adenocarcinomas. Supplementary Table 3. Statistical tests used for each figure panel.

Variation in immune homeostasis, the state in which the immune system is maintained in the absence of stimulation, is highly variable across populations. This variation is attributed to both genetic and environmental factors. However, the identity and function of specific regulators have been difficult to identify in humans. We evaluated homeostatic antibody levels in the serum of the Collaborative Cross (CC) mouse genetic reference population. We found heritable variation in all antibody isotypes and subtypes measured. We identified 4 quantitative trait loci (QTL) associated with 3 IgG subtypes: IgG1, IgG2b, and IgG2c. While 3 of these QTL map to genome regions of known immunological significance (major histocompatibility and immunoglobulin heavy chain locus), Qih1 (associated with variation in IgG1) mapped to a novel locus on Chromosome 18. We further associated this locus with B cell proportions in the spleen and identify Methyl-CpG binding domain protein 1 under this locus as a novel regulator of homeostatic IgG1 levels in the serum and marginal zone B cells (MZB) in the spleen, consistent with a role in MZB differentiation to antibody secreting cells.

Abstract Immune homeostasis is the state that the immune system maintains in the absence of insult. Perturbation of immune homeostasis can impact autoimmunity/allergy and adversely affect infectious responses. To date, much of the analysis of immune homeostasis has focused on systemic immunity, but it is also likely to be important in an organ specific manner. Since the lungs are a major site of environmental exposure and infection, we used the Collaborative Cross (CC) mouse genetic reference population to study the genetic regulation of the breadth of baseline immune cell populations in the lung and identify loci regulating these cells at homeostasis. We found that all 54 immune cell phenotypes measured showed strong genetic variation in cell type abundances. We identified 28 quantitative trait loci associated with variation in 24 immune cell populations or the relationship between cell populations. Further, we identified significant associations between 20 of these loci and responses to either influenza A virus (IAV) or Severe acute respiratory syndrome associated coronavirus (SARS-CoV) disease in the same strains of mice. Notably, a locus mapped for variation in Ly6C+ monocyte abundance was associated with SARS-CoV weight loss, as well as titer at days 2 and 4 post-infection. This locus is also associated with influenza-induced disease, as measured by weight loss post-infection. Our analysis highlights the strong genetic control of immune homeostasis, and the key role that immune homeostasis plays in contributing to downstream infectious responses. In particular, our analysis suggests that the abundance of a variety of lung leukocyte populations prior to infection could serve as predictors of immune responses to respiratory viruses.

SummaryVariation in immune homeostasis, immune system stability, in organ systems such as the lungs is likely to shape the host response to infection at these exposed tissues. We evaluated immune homeostasis in immune cell populations in the lungs of the Collaborative Cross (CC) mouse genetic reference population. We found vast heritable variation in leukocyte populations with the frequency of many of these cell types showing distinct patterns relative to classic inbred strains C57BL/6J and BALB/cJ. We identified 28 quantitative trait loci (QTL) associated with variation in baseline lung immune cell populations, including several loci that broadly regulate the abundance of immune populations from distinct developmental lineages, and found that many of these loci have predictive value for influenza disease outcomes, demonstrating that genetic determinants of homeostatic immunity in the lungs regulate susceptibility to virus-induced disease. All told, we highlight the need to assess diverse mouse strains in understanding immune homeostasis and resulting immune responses.

Abstract Immune homeostasis is the state where the immune system maintains stability in the absence of insult. Much of the analysis of immune homeostasis has focused on systemic immunity, but it is also likely to be important in an organ specific manner. There is evidence that homeostatic immunity can affect subsequent responses to infection or vaccination. Since the lungs are a major site of infection, we used the Collaborative Cross (CC) mouse genetic reference population to study the genetic regulation of the breadth of baseline immune cell populations in the lung and identify loci regulating these cells at the steady state. We found that all immune cell populations measured showed strong genetic (i.e. strain-specific) variation in cell type abundances. We identified 12 quantitative trait loci (QTL) associated with variation in 12 immune cell populations or the relationships between cell populations. Given the role of various immune cells in the lungs during respiratory virus pathogenesis, we asked whether any of the mapped QTL correlated with influenza A virus (IAV) or Severe acute respiratory syndrome associated coronavirus (SARS-CoV) disease following infection in the same strains of mice. Notably, a locus we mapped for baseline abundance of CD8+ T cells in the lungs was associated with peak weight loss following IAV infection. Additionally, a locus mapped for variation in Ly6C+ monocyte/macrophage abundance was associated with SARS-CoV titer at days 2 and 4 post-infection. These data suggest that abundance of lung leukocyte populations prior to infection could serve as predictors of immune responses to respiratory viruses.

Host genetic factors play a fundamental role in regulating humoral immunity to viral infection, including influenza A virus (IAV). Here, we utilize the Collaborative Cross (CC), a mouse genetic reference population, to study genetic regulation of variation in antibody response following IAV infection. CC mice show significant heritable variation in the magnitude, kinetics, and composition of IAV-specific antibody response. We map 23 genetic loci associated with this variation. Analysis of a subset of these loci finds that they broadly affect the antibody response to IAV as well as other viruses. Candidate genes are identified based on predicted variant consequences and haplotype-specific expression patterns, and several show overlap with genes identified in human mapping studies. These findings demonstrate that the host antibody response to IAV infection is under complex genetic control and highlight the utility of the CC in modeling and identifying genetic factors with translational relevance to human health and disease.

Influenza A virus (IAV) is a respiratory pathogen that causes substantial morbidity and mortality during both seasonal and pandemic outbreaks. Infection outcomes in unexposed populations are affected by host genetics, but the host genetic architecture is not well understood. Here, we obtain a broad view of how heritable factors affect a mouse model of response to IAV infection using an 8 × 8 diallel of the eight inbred founder strains of the Collaborative Cross (CC). Expanding on a prior statistical framework for modeling treatment response in diallels, we explore how a range of heritable effects modify acute host response to IAV through 4 d postinfection. Heritable effects in aggregate explained ∼57% of the variance in IAV-induced weight loss. Much of this was attributable to a pattern of additive effects that became more prominent through day 4 postinfection and was consistent with previous reports of antiinfluenza myxovirus resistance 1 (Mx1) polymorphisms segregating between these strains; these additive effects largely recapitulated haplotype effects observed at the Mx1 locus in a previous study of the incipient CC, and are also replicated here in a CC recombinant intercross population. Genetic dominance of protective Mx1 haplotypes was observed to differ by subspecies of origin: relative to the domesticus null Mx1 allele, musculus acts dominantly whereas castaneus acts additively. After controlling for Mx1, heritable effects, though less distinct, accounted for ∼34% of the phenotypic variance. Implications for future mapping studies are discussed.

The complement system is a critical part of host defense to many bacterial, viral, and fungal infections. It works alongside pattern recognition receptors to stimulate host defense systems in advance of activation of the adaptive immune response. In this study, we directly test the role of complement in SARS-CoV pathogenesis using a mouse model and show that respiratory disease is significantly reduced in the absence of complement even though viral load is unchanged. Complement-deficient mice have reduced neutrophilia in their lungs and reduced systemic inflammation, consistent with the observation that SARS-CoV pathogenesis is an immune-driven disease. These data suggest that inhibition of complement signaling might be an effective treatment option following coronavirus infection.

Host immunity plays a key role in protection against infection, however, there is significant individual-to-individual variation in the induction and quality of immune response that is driven in part by genetic factors. A growing body of evidence suggests that variation in immune status prior to specific insult may influence subsequent response to vaccination or pathogen challenge. Therefore, understanding the variation in immune status prior to specific insult is necessary for assessing variability in immune response to vaccination or pathogen challenge. The Collaborative Cross (CC) mouse population is a genetically diverse population that was designed to mimic the genetic and phenotypic diversity that is observed in humans and allows us to investigate mechanistically the contribution of genetics to complex phenotypes. Consequently, we asked whether there was variation in antibody levels in 57 CC strains prior to pathogen challenge and if that variation was associated with specific genetic factors. To assess this, we measured antibody levels for IgM, total IgG, IgG1, IgG2a, IgG2b, and IgG2c by ELISA and mapped quantitative trait loci (QTL) associated with variation in antibody concentrations. We observed substantial variation in all antibody isotypes measured. We also identified a suggestive (p=0.1) QTL on chromosome 18. Allele effects show that the C57Bl/6J allele at the locus of interest is linked to higher levels of total IgG. Subsequent analysis of genes under the QTL identified Methyl-CpG binding domain protein-1 (Mbd1) as the most promising candidate gene associated with total IgG variation. Ongoing studies are evaluating the role of Mbd1 in the regulation of total IgG production.

ABSTRACT Zoonotic viruses circulate as swarms in animal reservoirs and can emerge into human populations, causing epidemics that adversely affect public health. Portable, safe, and effective vaccine platforms are needed in the context of these outbreak and emergence situations. In this work, we report the generation and characterization of an alphavirus replicon vaccine platform based on a non-select agent, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain 3526 (VRP 3526). Using both noroviruses and coronaviruses as model systems, we demonstrate the utility of the VRP 3526 platform in the generation of recombinant proteins, production of virus-like particles, and in vivo efficacy as a vaccine against emergent viruses. Importantly, packaging under biosafety level 2 (BSL2) conditions distinguishes VRP 3526 from previously reported alphavirus platforms and makes this approach accessible to the majority of laboratories around the world. In addition, improved outcomes in the vulnerable aged models as well as against heterologous challenge suggest improved efficacy compared to that of previously attenuated VRP approaches. Taking these results together, the VRP 3526 platform represents a safe and highly portable system that can be rapidly deployed under BSL2 conditions for generation of candidate vaccines against emerging microbial pathogens. IMPORTANCE While VEE virus replicon particles provide a robust, established platform for antigen expression and vaccination, its utility has been limited by the requirement for high-containment-level facilities for production and packaging. In this work, we utilize an attenuated vaccine strain capable of use at lower biocontainment level but retaining the capacity of the wild-type replicon particle. Importantly, the new replicon platform provides equal protection for aged mice and following heterologous challenge, which distinguishes it from other attenuated replicon platforms. Together, the new system represents a highly portable, safe system for use in the context of disease emergence.

Mannose binding lectin (MBL) generally plays a protective role during viral infection, yet MBL-mediated complement activation promotes Ross River virus (RRV)-induced inflammatory tissue destruction, contributing to arthritis and myositis. As MBL binds to carbohydrates, we hypothesized that N-linked glycans on the RRV envelope glycoproteins act as ligands for MBL. Using a panel of RRV mutants lacking the envelope N-linked glycans, we found that MBL deposition onto infected cells was dependent on the E2 glycans. Moreover, the glycan-deficient viruses exhibited reduced disease and tissue damage in a mouse model of RRV-induced myositis compared to wild-type RRV, despite similar viral load and inflammatory infiltrates within the skeletal muscle. Instead, the reduced disease induced by glycan-deficient viruses was linked to decreased MBL deposition and complement activation within inflamed tissues. These results demonstrate that the viral N-linked glycans promote MBL deposition and complement activation onto RRV-infected cells, contributing to the development of RRV-induced myositis.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4+ T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4+ T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease.

ABSTRACT Chikungunya virus (CHIKV) is an alphavirus responsible for causing epidemic outbreaks of polyarthralgia in humans. Because CHIKV is initially introduced via the skin, where γδ T cells are prevalent, we evaluated the response of these cells to CHIKV infection. CHIKV infection led to a significant increase in γδ T cells in the infected foot and draining lymph node that was associated with the production of proinflammatory cytokines and chemokines in C57BL/6J mice. γδ T cell−/− mice demonstrated exacerbated CHIKV disease characterized by less weight gain and greater foot swelling than occurred in wild-type mice, as well as a transient increase in monocytes and altered cytokine/chemokine expression in the foot. Histologically, γδ T cell−/− mice had increased inflammation-mediated oxidative damage in the ipsilateral foot and ankle joint compared to wild-type mice which was independent of differences in CHIKV replication. These results suggest that γδ T cells play a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage. IMPORTANCE Recent epidemics, including the 2004 to 2007 outbreak and the spread of CHIKV to naive populations in the Caribbean and Central and South America with resultant cases imported into the United States, have highlighted the capacity of CHIKV to cause explosive epidemics where the virus can spread to millions of people and rapidly move into new areas. These studies identified γδ T cells as important to both recruitment of key inflammatory cell populations and dampening the tissue injury due to oxidative stress. Given the importance of these cells in the early response to CHIKV, this information may inform the development of CHIKV vaccines and therapeutics.

New systems genetics approaches are needed to rapidly identify host genes and genetic networks that regulate complex disease outcomes. Using genetically diverse animals from incipient lines of the Collaborative Cross mouse panel, we demonstrate a greatly expanded range of phenotypes relative to classical mouse models of SARS-CoV infection including lung pathology, weight loss and viral titer. Genetic mapping revealed several loci contributing to differential disease responses, including an 8.5Mb locus associated with vascular cuffing on chromosome 3 that contained 23 genes and 13 noncoding RNAs. Integrating phenotypic and genetic data narrowed this region to a single gene, Trim55, an E3 ubiquitin ligase with a role in muscle fiber maintenance. Lung pathology and transcriptomic data from mice genetically deficient in Trim55 were used to validate its role in SARS-CoV-induced vascular cuffing and inflammation. These data establish the Collaborative Cross platform as a powerful genetic resource for uncovering genetic contributions of complex traits in microbial disease severity, inflammation and virus replication in models of outbred populations.

ABSTRACT Toll-like receptors (TLRs) are sensors that recognize molecular patterns from viruses, bacteria, and fungi to initiate innate immune responses to invading pathogens. The emergence of highly pathogenic coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) is a concern for global public health, as there is a lack of efficacious vaccine platforms and antiviral therapeutic strategies. Previously, it was shown that MyD88, an adaptor protein necessary for signaling by multiple TLRs, is a required component of the innate immune response to mouse-adapted SARS-CoV infection in vivo. Here, we demonstrate that TLR3−/−, TLR4−/−, and TRAM−/− mice are more susceptible to SARS-CoV than wild-type mice but experience only transient weight loss with no mortality in response to infection. In contrast, mice deficient in the TLR3/TLR4 adaptor TRIF are highly susceptible to SARS-CoV infection, showing increased weight loss, mortality, reduced lung function, increased lung pathology, and higher viral titers. Distinct alterations in inflammation were present in TRIF−/− mice infected with SARS-CoV, including excess infiltration of neutrophils and inflammatory cell types that correlate with increased pathology of other known causes of acute respiratory distress syndrome (ARDS), including influenza virus infections. Aberrant proinflammatory cytokine, chemokine, and interferon-stimulated gene (ISG) signaling programs were also noted following infection of TRIF−/− mice that were similar to those seen in human patients with poor disease outcome following SARS-CoV or MERS-CoV infection. These findings highlight the importance of TLR adaptor signaling in generating a balanced protective innate immune response to highly pathogenic coronavirus infections. IMPORTANCE Toll-like receptors are a family of sensor proteins that enable the immune system to differentiate between “self” and “non-self.” Agonists and antagonists of TLRs have been proposed to have utility as vaccine adjuvants or antiviral compounds. In the last 15 years, the emergence of highly pathogenic coronaviruses SARS-CoV and MERS-CoV has caused significant disease accompanied by high mortality rates in human populations, but no approved therapeutic treatments or vaccines currently exist. Here, we demonstrate that TLR signaling through the TRIF adaptor protein protects mice from lethal SARS-CoV disease. Our findings indicate that a balanced immune response operating through both TRIF-driven and MyD88-driven pathways likely provides the most effective host cell intrinsic antiviral defense responses to severe SARS-CoV disease, while removal of either branch of TLR signaling causes lethal SARS-CoV disease in our mouse model. These data should inform the design and use of TLR agonists and antagonists in coronavirus-specific vaccine and antiviral strategies.

ABSTRACT Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. IMPORTANCE The 2012 outbreak of MERS-CoV raises the specter of another global epidemic, similar to the 2003 SARS-CoV epidemic. MERS-CoV is related to BtCoV HKU5 in target regions that are essential for drug and vaccine testing. Because no small animal model exists to evaluate MERS-CoV pathogenesis or to test vaccines, we constructed a recombinant BtCoV HKU5 that expressed a region of the SARS-CoV spike (S) glycoprotein, thereby allowing the recombinant virus to grow in cell culture and in mice. We show that this recombinant virus targets airway epithelial cells and causes disease in aged mice. We use this platform to (i) identify a broad-spectrum antiviral that can potentially inhibit viruses closely related to MERS-CoV, (ii) demonstrate the absence of increased eosinophilic immune pathology for MERS-CoV N protein-based vaccines, and (iii) mouse adapt this virus to identify viral genetic determinants of cross-species transmission and virulence. This study holds significance as a strategy to control newly emerging viruses.

Abstract It is controversial whether dietary fiber protects against colorectal cancer because of conflicting results from human epidemiologic studies. These studies have been complicated by the participants’ genetic heterogeneity and differences in the composition of microbiota within their gastrointestinal tracts. To eliminate these confounding variables, we utilized a gnotobiotic mouse model of colorectal cancer. Our experiments were designed to investigate the function of butyrate because it is a short-chain fatty acid produced by bacterial fermentation of fiber in the colon at high (mM) levels and has potent energetic and epigenetic properties in host colonocytes. Here, we report that fiber did, in fact, have a chemoprotective effect but in a microbiota- and butyrate-dependent manner. The incidence, number, size, and histopathologic progression of AOM/DSS-induced colorectal tumors were significantly diminished when BALB/c mice were provided a high-fiber diet only if they were colonized with defined microbiota that included a butyrate-producing bacteria. This chemoprotective effect was attenuated when mice were colonized with the same microbiota except that the wild-type butyrate producer was replaced by a mutant strain with a 0.8-kb deletion in the butyryl-CoA synthesis operon. To confirm that butyrate is a causal factor, the chemoprotective effect was recapitulated in mice without any butyrate-producing bacteria if they were provided a butyrate-fortified diet. Our data support a general mechanism that includes microbial fermentation of fiber rather than fiber exclusively speeding colonic transit to minimize the exposure of colonocytes to ingested carcinogens. Our data also support a molecular mechanism that is metaboloepigenetic. Normal colonocytes utilize butyrate as their preferred energy source, whereas cancerous colonocytes rely on glucose because of the Warburg effect. Due to this metabolic difference, butyrate accumulated in tumors (as measured by LC-MS) and functioned as an HDAC inhibitor to increase histone acetylation levels and apoptosis. To support the applicability of this model to human cancer, we demonstrate that butyrate also accumulates at higher levels in human colorectal tumors than in normal colonic tissue, and this is associated with higher levels of histone acetylation in tumors. These results link diet and microbiota to a common metabolite that influences epigenetics and cancer predisposition. Citation Format: Dallas Donohoe, Darcy Holley, Leonard Collins, Stephanie Montgomery, Alan Whitmore, Andrew Hillhouse, Kaitlin Curry, Sarah Renner, Alicia Greenwalt, Elizabeth Ryan, Virginia Godfrey, Mark Heise, Deborah Threadgill, James Swenberg, David Threadgill, Scott Bultman. Dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY04-02. doi:10.1158/1538-7445.AM2014-SY04-02

Genetic variation between diverse mouse species is well-characterized, yet existing knowledge of the mouse transcriptome comes largely from one mouse strain (C57BL/6J). As such, it is unlikely to reflect the transcriptional complexity of the mouse species. Gene transcription is dynamic and condition-specific; therefore, to better understand the mouse transcriptional response to respiratory virus infection, we infected the eight founder strains of the Collaborative Cross with either influenza A virus or severe acute respiratory syndrome coronavirus and sequenced lung RNA samples at 2 and 4 days after infection. We found numerous instances of transcripts that were not present in the C57BL/6J reference annotation, indicating that a nontrivial proportion of the mouse genome is transcribed but poorly annotated. Of these novel transcripts, 2150 could be aligned to human or rat genomes, but not to existing mouse genomes, suggesting functionally conserved sequences not yet recorded in mouse genomes. We also found that respiratory virus infection induced differential expression of 4287 splicing junctions, resulting in strain-specific isoform expression. Of these, 59 were influenced by strain-specific mutations within 2 base pairs of key intron-exon boundaries, suggesting cis-regulated expression. Our results reveal the complexity of the transcriptional response to viral infection, previously undocumented genomic elements, and extensive diversity in the response across mouse strains. These findings identify hitherto unexplored transcriptional patterns and undocumented transcripts in genetically diverse mice. Host genetic variation drives the complexity and diversity of the host response by eliciting starkly different transcriptional profiles in response to a viral infection.

Abstract Whether dietary fiber protects against colorectal cancer is controversial because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We used gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as a histone deacetylase (HDAC) inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared with normal colonic tissues. Significance: These results, which link diet and microbiota to a tumor-suppressive metabolite, provide insight into conflicting epidemiologic findings and suggest that probiotic/prebiotic strategies can modulate an endogenous HDAC inhibitor for anticancer chemoprevention without the adverse effects associated with synthetic HDAC inhibitors used in chemotherapy. Cancer Discov; 4(12); 1387–97. ©2014 AACR. See related commentary by Sebastián and Mostoslavsky, p. 1368 This article is highlighted in the In This Issue feature, p. 1355