Decreased microbiome diversity in the HIV small airway epithelium.

BACKGROUND: Persons living with human immunodeficiency virus (PLWH) face an increased burden of chronic obstructive pulmonary disease (COPD). Repeated pulmonary infections, antibiotic exposures, and immunosuppression may contribute to an altered small airway epithelium (SAE) microbiome. METHODS: SAE cells were collected from 28 PLWH and 48 HIV- controls through bronchoscopic cytologic brushings. DNA extracted from SAE cells was subjected to 16S rRNA amplification and sequencing. Comparisons of alpha and beta diversity between HIV+ and HIV- groups were performed and key operational taxonomic units (OTUs) distinguishing the two groups were identified using the Boruta feature selection after Random Forest Analysis. RESULTS: PLWH demonstrated significantly reduced Shannon diversity compared with HIV- volunteers (1.82 ± 0.10 vs. 2.20 ± 0.073, p = 0.0024). This was primarily driven by a reduction in bacterial richness (23.29 ± 2.75 for PLWH and 46.04 ± 3.716 for HIV-, p < 0.0001). Phyla distribution was significantly altered among PLWH, with an increase in relative abundance of Proteobacteria (p = 0.0003) and a decrease in Bacteroidetes (p = 0.0068) and Firmicutes (p = 0.0002). Six discriminative OTUs were found to distinguish PLWH from HIV- volunteers, aligning to Veillonellaceae, Fusobacterium, Verrucomicrobiaceae, Prevotella, Veillonella, and Campylobacter. CONCLUSIONS: Compared to HIV- controls, PLWH's SAE microbiome is marked by reduced bacterial diversity and richness with significant differences in community composition.

Integrative Genomics of Emphysema-Associated Genes Reveals Potential Disease Biomarkers.

Chronic obstructive pulmonary disease is the third leading cause of death worldwide. Gene expression profiling across multiple regions of the same lung identified genes significantly related to emphysema. We sought to determine whether the lung and epithelial expression of 127 emphysema-related genes was also related to lung function in independent cohorts, and whether any of these genes could be used as biomarkers in the peripheral blood of patients with chronic obstructive pulmonary disease. To that end, we examined whether the expression levels of these genes were under genetic control in lung tissue (n = 1,111). We then determined whether the mRNA levels of these genes in lung tissue (n = 727), small airway epithelial cells (n = 238), and peripheral blood (n = 620) were significantly related to lung function measurements. The expression of 63 of the 127 genes (50%) was under genetic control in lung tissue. The lung and epithelial mRNA expression of a subset of the emphysema-associated genes, including ASRGL1, LPHN2, and EDNRB, was strongly associated with lung function. In peripheral blood, the expression of 40 genes was significantly associated with lung function. Twenty-nine of these genes (73%) were also associated with lung function in lung tissue, but with the opposite direction of effect for 24 of the 29 genes, including those involved in hypoxia and B cell-related responses. The integrative genomics approach uncovered a significant overlap of emphysema genes associations with lung function between lung and blood with opposite directions between the two. These results support the use of peripheral blood to detect disease biomarkers.

Azithromycin and risk of COPD exacerbations in patients with and without Helicobacter pylori.

BACKGROUND: Helicobacter pylori (HP) infection is associated with reduced lung function and systemic inflammation in chronic obstructive pulmonary disease (COPD) patients. Azithromycin (AZ) is active against HP and reduces the risk of COPD exacerbation. We determined whether HP infection status modifies the effects of AZ in COPD patients. METHODS: Plasma samples from 1018 subjects with COPD who participated in the Macrolide Azithromycin (MACRO) in COPD Study were used to determine the HP infection status at baseline and 12 months of follow-up using a serologic assay. Based on HP infection status and randomization to either AZ or placebo (PL), the subjects were divided into 4 groups: HP+/AZ, HP-/AZ, HP+/PL, and HP-/PL. Time to first exacerbation was compared across the 4 groups using Kaplan-Meier survival analysis and a Cox proportional hazards model. The rates of exacerbation were compared using both the Kruskal-Wallis test and negative binomial analysis. Blood biomarkers at enrolment and at follow-up visits 3, 12, and 13 (1 month after treatment was stopped) months were measured. RESULTS: One hundred eighty one (17.8%) patients were seropositive to HP. Non-Caucasian participants were nearly three times more likely to be HP seropositive than Caucasian participants (37.4% vs 13.6%; p < 0.001). The median time to first exacerbation was significantly different across the four groups (p = 0.001) with the longest time in the HP+/AZ group (11.2 months, 95% CI; 8.4-12.5+) followed by the HP-/AZ group (8.0 months, 95% CI; 6.7-9.7). Hazard ratio (HR) for exacerbations was lowest in the HP+/AZ group after adjustment for age, sex, smoking status, ethnicity, history of peptic ulcer, dyspnea, previous hospital admission, GOLD grade of severity, and forced vital capacity (HR, 0.612; 95% CI, 0.442-0.846 vs HR, 0.789; 95% CI, 0.663-0.938 in the HP-/AZ group). Circulating levels of soluble tumor necrosis factor receptor-75 were reduced only in the HP+/AZ group after 3 months of AZ treatment (-0.87 ± 0.31 µg/L; p = 0.002); levels returned to baseline after discontinuing AZ. CONCLUSIONS: AZ is effective in preventing COPD exacerbations in patients with HP seropositivity, possibly by modulating TNF pathways related to HP infection.

Host Response to the Lung Microbiome in Chronic Obstructive Pulmonary Disease.

RATIONALE: The relatively sparse but diverse microbiome in human lungs may become less diverse in chronic obstructive pulmonary disease (COPD). This article examines the relationship of this microbiome to emphysematous tissue destruction, number of terminal bronchioles, infiltrating inflammatory cells, and host gene expression. METHODS: Culture-independent pyrosequencing microbiome analysis was used to examine the V3-V5 regions of bacterial 16S ribosomal DNA in 40 samples of lung from 5 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 4) and 28 samples from 4 donors (controls). A second protocol based on the V1-V3 regions was used to verify the bacterial microbiome results. Within lung tissue samples the microbiome was compared with results of micro-computed tomography, infiltrating inflammatory cells measured by quantitative histology, and host gene expression. MEASUREMENTS AND MAIN RESULTS: Ten operational taxonomic units (OTUs) was found sufficient to discriminate between control and GOLD stage 4 lung tissue, which included known pathogens such as Haemophilus influenzae. We also observed a decline in microbial diversity that was associated with emphysematous destruction, remodeling of the bronchiolar and alveolar tissue, and the infiltration of the tissue by CD4(+) T cells. Specific OTUs were also associated with neutrophils, eosinophils, and B-cell infiltration (P < 0.05). The expression profiles of 859 genes and 235 genes were associated with either enrichment or reductions of Firmicutes and Proteobacteria, respectively, at a false discovery rate cutoff of less than 0.1. CONCLUSIONS: These results support the hypothesis that there is a host immune response to microorganisms within the lung microbiome that appears to contribute to the pathogenesis of COPD.

The bronchial epithelial cell bacterial microbiome and host response in patients infected with human immunodeficiency virus.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is an important comorbidity in patients living with human immunodeficiency virus (HIV). Previous bacterial microbiome studies have shown increased abundance of specific bacterium, like Tropheryma whipplei, and no overall community differences. However, the host response to the lung microbiome is unknown in patients infected with HIV. METHODS: Two bronchial brush samples were obtained from 21 HIV-infected patients. One brush was used for bacterial microbiome analysis using the Illumina MiSeqTM platform, while the other was used to evaluate gene expression patterns of the host using the Affymetrix Human Gene ST 2.0 array. Weighted gene co-expression network analysis was used to determine the relationship between the bacterial microbiome and host gene expression response. RESULTS: The Shannon Diversity was inversely related to only one gene expression module (p = 0.02); whereas evenness correlated with five different modules (p ≤ 0.05). After FDR correction only the Firmicutes phylum was significantly correlated with any modules (FDR < 0.05). These modules were enriched for cilia, transcription regulation, and immune response. Specific operational taxonomic units (OTUs), such as OTU4 (Pasteurellaceae), were able to distinguish HIV patients with and without COPD and severe emphysema. CONCLUSION: These data support the hypothesis that the bacterial microbiome in HIV lungs is associated with specific host immune responses. Whether or not these responses are also seen in non-HIV infected individuals needs to be addressed in future studies.

The relationship between Helicobacter pylori seropositivity and COPD.

RATIONALE: Chronic systemic infections such as those with Helicobacter pylori (H. pylori) may contribute to the evolution and progression of chronic obstructive pulmonary disease (COPD). Using data from the Lung Health Study (LHS), we determined the relationship of H. pylori infection with the severity and progression of COPD. METHODS: Using an immunoassay, we measured H. pylori immunoglobulin G (IgG) antibody titres in serum samples of 4765 patients with mild-to-moderate COPD. We then determined their relationship with the individual's FEV1 and the rate of decline in FEV1 and mortality over 11 years using multiple regression analysis. RESULTS: Approximately 18% of the patients were seropositive to H. pylori and these individuals demonstrated lower FEV1 (L) values at every study visit compared with individuals who were seronegative for H. pylori (p value=0.00012). However, patients with seropositivity to H. pylori were on average 0.012 m shorter than those with seronegativity (p value=0.0015). The significant relationship between FEV1 and H. pylori seropositivity disappeared when FEV1 per cent predicted (FEV1pp) was used (p value=0.45). H. pylori seropositive individuals had greater circulating C reactive protein (CRP) levels compared with H. pylori seronegative individuals (p value=0.012), and had increased risk of cardiovascular mortality (relative risk 1.61, p=0.05). CONCLUSIONS: H. pylori infection was associated with reduced lung function that is most likely due to the effect of the bacterium on lung growth earlier in life. It is also associated with systemic inflammation and increased risk of cardiovascular mortality in patients with COPD. TRIAL REGISTRATION NUMBERS: NCT00000568 and NCT00000569.

Atypical activation of the unfolded protein response in cystic fibrosis airway cells contributes to p38 MAPK-mediated innate immune responses.

Inflammatory lung disease is the major cause of morbidity and mortality in cystic fibrosis (CF); understanding what produces dysregulated innate immune responses in CF cells will be pivotal in guiding the development of novel anti-inflammatory therapies. To elucidate the molecular mechanisms that mediate exaggerated inflammation in CF following TLR signaling, we profiled global gene expression in immortalized human CF and non-CF airway cells at baseline and after microbial stimulation. Using complementary analysis methods, we observed a signature of increased stress levels in CF cells, specifically characterized by endoplasmic reticulum (ER) stress, the unfolded protein response (UPR), and MAPK signaling. Analysis of ER stress responses revealed an atypical induction of the UPR, characterized by the lack of induction of the PERK-eIF2α pathway in three complementary model systems: immortalized CF airway cells, fresh CF blood cells, and CF lung tissue. This atypical pattern of UPR activation was associated with the hyperinflammatory phenotype in CF cells, as deliberate induction of the PERK-eIF2α pathway with salubrinal attenuated the inflammatory response to both flagellin and Pseudomonas aeruginosa. IL-6 production triggered by ER stress and microbial stimulation were both dependent on p38 MAPK activity, suggesting a molecular link between both signaling events. These data indicate that atypical UPR activation fails to resolve the ER stress in CF and sensitizes the innate immune system to respond more vigorously to microbial challenge. Strategies to restore ER homeostasis and normalize the UPR activation profile may represent a novel therapeutic approach to minimize lung-damaging inflammation in CF.

Respiratory viral detection and small airway inflammation in lung tissue of patients with stable, mild COPD.

BACKGROUND: Viral respiratory tract infections are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). In lung tissue specimens from patients with stable, mild COPD and from control smokers without airflow obstruction, we determined the prevalence and load of nucleic acid from common respiratory viruses and concomitant inflammation of small airways measuring less than 2-mm in diameter. METHODS: Frozen lung tissue obtained from patients with stable, mild COPD (n = 20) and control subjects (n = 20) underwent real-time quantitative PCR (qPCR) for 13 respiratory viruses, and quantitative histology for inflammation of small airways. The two groups were compared for viral prevalence and load, and airway inflammation. The relationship between viral load and airway inflammatory cells was also analyzed. RESULTS: Viral nucleic acid were detected in lung tissue of 18/40 (45.0%) of the individuals studied and included seven co-infections that were characterized by a "dominant virus" contributing to most of the total measured viral load. Lung tissue of COPD patients had a significantly higher prevalence of viral nucleic acid (particularly influenza A virus), and increased inflammation of small airways by macrophages and neutrophils versus controls. In qPCR-positive individuals, linear regression analysis showed a direct correlation between viral load and airway neutrophils, and between influenza A virus load and airway macrophages. CONCLUSION: The lung tissue of patients with stable, mild COPD has a higher prevalence and load of respiratory viruses versus non-obstructed control subjects, and increased inflammation of small airways. Respiratory viruses may represent potential targets in COPD patient management.

Single-Cell Analysis Identifies NOTCH3-Mediated Interactions between Stromal Cells That Promote Microenvironment Remodeling and Invasion in Lung Adenocarcinoma.

Cancer immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD); however, a significant proportion of patients do not respond. Recent transcriptomic studies to understand determinants of immunotherapy response have pinpointed stromal-mediated resistance mechanisms. To gain a better understanding of stromal biology at the cellular and molecular level in LUAD, we performed single-cell RNA sequencing of 256,379 cells, including 13,857 mesenchymal cells, from 9 treatment-naïve patients. Among the mesenchymal cell subsets, FAP+PDPN+ cancer-associated fibroblasts (CAF) and ACTA2+MCAM+ pericytes were enriched in tumors and differentiated from lung-resident fibroblasts. Imaging mass cytometry revealed that both subsets were topographically adjacent to the perivascular niche and had close spatial interactions with endothelial cells (EC). Modeling of ligand and receptor interactomes between mesenchymal and ECs identified that NOTCH signaling drives these cell-to-cell interactions in tumors, with pericytes and CAFs as the signal receivers and arterial and PLVAPhigh immature neovascular ECs as the signal senders. Either pharmacologically blocking NOTCH signaling or genetically depleting NOTCH3 levels in mesenchymal cells significantly reduced collagen production and suppressed cell invasion. Bulk RNA sequencing data demonstrated that NOTCH3 expression correlated with poor survival in stroma-rich patients and that a T cell-inflamed gene signature only predicted survival in patients with low NOTCH3. Collectively, this study provides valuable insights into the role of NOTCH3 in regulating tumor stroma biology, warranting further studies to elucidate the clinical implications of targeting NOTCH3 signaling. SIGNIFICANCE: NOTCH3 signaling activates tumor-associated mesenchymal cells, increases collagen production, and augments cell invasion in lung adenocarcinoma, suggesting its critical role in remodeling tumor stroma.

The lung tissue microbiome in chronic obstructive pulmonary disease.

RATIONALE: Based on surface brushings and bronchoalveolar lavage fluid, Hilty and coworkers demonstrated microbiomes in the human lung characteristic of asthma and chronic obstructive pulmonary disease (COPD), which have now been confirmed by others. OBJECTIVES: To extend these findings to human lung tissue samples. METHODS: DNA from lung tissue samples was obtained from nonsmokers (n = 8); smokers without COPD (n = 8); patients with very severe COPD (Global Initiative for COPD [GOLD] 4) (n = 8); and patients with cystic fibrosis (CF) (n = 8). The latter served as a positive control, with sterile water as a negative control. All bacterial community analyses were based on polymerase chain reaction amplifying 16S rRNA gene fragments. Total bacterial populations were measured by quantitative polymerase chain reaction and bacterial community composition was assessed by terminal restriction fragment length polymorphism analysis and pyrotag sequencing. MEASUREMENT AND MAIN RESULTS: Total bacterial populations within lung tissue were small (20-1,252 bacterial cells per 1,000 human cells) but greater in all four sample groups versus the negative control group (P < 0.001). Terminal restriction fragment length polymorphism analysis and sequencing distinguished three distinct bacterial community compositions: one common to the nonsmoker and smoker groups, a second to the GOLD 4 group, and the third to the CF-positive control group. Pyrotag sequencing identified greater than 1,400 unique bacterial sequences and showed an increase in the Firmicutes phylum in GOLD 4 patients versus all other groups (P < 0.003) attributable to an increase in the Lactobacillus genus (P < 0.0007). CONCLUSIONS: There is a detectable bacterial community within human lung tissue that changes in patients with very severe COPD.

Cancer-Associated Fibroblasts Promote Immunosuppression by Inducing ROS-Generating Monocytic MDSCs in Lung Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

The Impact of DNA Polymerase and Number of Rounds of Amplification in PCR on 16S rRNA Gene Sequence Data.

PCR amplification of 16S rRNA genes is a critical yet underappreciated step in the generation of sequence data to describe the taxonomic composition of microbial communities. Numerous factors in the design of PCR can impact the sequencing error rate, the abundance of chimeric sequences, and the degree to which the fragments in the product represent their abundance in the original sample (i.e., bias). We compared the performance of high fidelity polymerases and various numbers of rounds of amplification when amplifying a mock community and human stool samples. Although it was impossible to derive specific recommendations, we did observe general trends. Namely, using a polymerase with the highest possible fidelity and minimizing the number of rounds of PCR reduced the sequencing error rate, fraction of chimeric sequences, and bias. Evidence of bias at the sequence level was subtle and could not be ascribed to the fragments' fraction of bases that were guanines or cytosines. When analyzing mock community data, the amount that the community deviated from the expected composition increased with the number of rounds of PCR. This bias was inconsistent for human stool samples. Overall, the results underscore the difficulty of comparing sequence data that are generated by different PCR protocols. However, the results indicate that the variation in human stool samples is generally larger than that introduced by the choice of polymerase or number of rounds of PCR.IMPORTANCE A steep decline in sequencing costs drove an explosion in studies characterizing microbial communities from diverse environments. Although a significant amount of effort has gone into understanding the error profiles of DNA sequencers, little has been done to understand the downstream effects of the PCR amplification protocol. We quantified the effects of the choice of polymerase and number of PCR cycles on the quality of downstream data. We found that these choices can have a profound impact on the way that a microbial community is represented in the sequence data. The effects are relatively small compared to the variation in human stool samples; however, care should be taken to use polymerases with the highest possible fidelity and to minimize the number of rounds of PCR. These results also underscore that it is not possible to directly compare sequence data generated under different PCR conditions.

Past, Present, and Future Research on the Lung Microbiome in Inflammatory Airway Disease.

COPD, asthma, and cystic fibrosis (CF) are obstructive lung diseases with distinct pathophysiologies and clinical phenotypes. In this paper, we highlight recent advances in our understanding of relationships between clinical phenotypes, host inflammatory response, and lung microbiota in these diseases. Although COPD, asthma, and CF largely have distinct lung microbiota and inflammatory profiles, certain commonalities exist. In all three of these lung diseases, and in healthy persons, anaerobic taxa that are typically associated with oral microbiota (eg, Prevotella species, Veillonella species) are present in the airways and associated with increased host inflammatory response. Similarly, across all three diseases, members of the Proteobacteria phylum are associated with more advanced disease. Finally, we highlight challenges in translating these findings into advances in clinical care, including continued knowledge gaps regarding the causal relationships between host inflammatory response, lung microbiota, medication effects, and clinical phenotypes.

Fecal Short-Chain Fatty Acids Are Not Predictive of Colonic Tumor Status and Cannot Be Predicted Based on Bacterial Community Structure.

Colonic bacterial populations are thought to have a role in the development of colorectal cancer with some protecting against inflammation and others exacerbating inflammation. Short-chain fatty acids (SCFAs) have been shown to have anti-inflammatory properties and are produced in large quantities by colonic bacteria that produce SCFAs by fermenting fiber. We assessed whether there was an association between fecal SCFA concentrations and the presence of colonic adenomas or carcinomas in a cohort of individuals using 16S rRNA gene and metagenomic shotgun sequence data. We measured the fecal concentrations of acetate, propionate, and butyrate within the cohort and found that there were no significant associations between SCFA concentration and tumor status. When we incorporated these concentrations into random forest classification models trained to differentiate between people with healthy colons and those with adenomas or carcinomas, we found that they did not significantly improve the ability of 16S rRNA gene or metagenomic gene sequence-based models to classify individuals. Finally, we generated random forest regression models trained to predict the concentration of each SCFA based on 16S rRNA gene or metagenomic gene sequence data from the same samples. These models performed poorly and were able to explain at most 14% of the observed variation in the SCFA concentrations. These results support the broader epidemiological data that questions the value of fiber consumption for reducing the risks of colorectal cancer. Although other bacterial metabolites may serve as biomarkers to detect adenomas or carcinomas, fecal SCFA concentrations have limited predictive power.IMPORTANCE Considering that colorectal cancer is the third leading cancer-related cause of death within the United States, it is important to detect colorectal tumors early and to prevent the formation of tumors. Short-chain fatty acids (SCFAs) are often used as a surrogate for measuring gut health and for being anticarcinogenic because of their anti-inflammatory properties. We evaluated the fecal SCFA concentrations of a cohort of individuals with different colonic tumor burdens who were previously analyzed to identify microbiome-based biomarkers of tumors. We were unable to find an association between SCFA concentration and tumor burden or use SCFAs to improve our microbiome-based models of classifying people based on their tumor status. Furthermore, we were unable to find an association between the fecal community structure and SCFA concentrations. Our results indicate that the association between fecal SCFAs, the gut microbiome, and tumor burden is weak.

Leveraging Existing 16S rRNA Gene Surveys To Identify Reproducible Biomarkers in Individuals with Colorectal Tumors.

An increasing body of literature suggests that both individual and collections of bacteria are associated with the progression of colorectal cancer. As the number of studies investigating these associations increases and the number of subjects in each study increases, a meta-analysis to identify the associations that are the most predictive of disease progression is warranted. We analyzed previously published 16S rRNA gene sequencing data collected from feces and colon tissue. We quantified the odds ratios (ORs) for individual bacterial taxa that were associated with an individual having tumors relative to a normal colon. Among the fecal samples, there were no taxa that had significant ORs associated with adenoma and there were 8 taxa with significant ORs associated with carcinoma. Similarly, among the tissue samples, there were no taxa that had a significant OR associated with adenoma and there were 3 taxa with significant ORs associated with carcinoma. Among the significant ORs, the association between individual taxa and tumor diagnosis was equal to or below 7.11. Because individual taxa had limited association with tumor diagnosis, we trained Random Forest classification models using only the taxa that had significant ORs, using the entire collection of taxa found in each study, and using operational taxonomic units defined based on a 97% similarity threshold. All training approaches yielded similar classification success as measured using the area under the curve. The ability to correctly classify individuals with adenomas was poor, and the ability to classify individuals with carcinomas was considerably better using sequences from feces or tissue.IMPORTANCE Colorectal cancer is a significant and growing health problem in which animal models and epidemiological data suggest that the colonic microbiota have a role in tumorigenesis. These observations indicate that the colonic microbiota is a reservoir of biomarkers that may improve our ability to detect colonic tumors using noninvasive approaches. This meta-analysis identifies and validates a set of 8 bacterial taxa that can be used within a Random Forest modeling framework to differentiate individuals as having normal colons or carcinomas. When models trained using one data set were tested on other data sets, the models performed well. These results lend support to the use of fecal biomarkers for the detection of tumors. Furthermore, these biomarkers are plausible candidates for further mechanistic studies into the role of the gut microbiota in tumorigenesis.

Normalization of the microbiota in patients after treatment for colonic lesions.

BACKGROUND: Colorectal cancer is a worldwide health problem. Despite growing evidence that members of the gut microbiota can drive tumorigenesis, little is known about what happens to it after treatment for an adenoma or carcinoma. This study tested the hypothesis that treatment for adenoma or carcinoma alters the abundance of bacterial populations associated with disease to those associated with a normal colon. We tested this hypothesis by sequencing the 16S rRNA genes in the feces of 67 individuals before and after treatment for adenoma (N = 22), advanced adenoma (N = 19), and carcinoma (N = 26). RESULTS: There were small changes to the bacterial community associated with adenoma or advanced adenoma and large changes associated with carcinoma. The communities from patients with carcinomas changed significantly more than those with adenoma following treatment (P value < 0.001). Although treatment was associated with intrapersonal changes, the change in the abundance of individual OTUs in response to treatment was not consistent within diagnosis groups (P value > 0.05). Because the distribution of OTUs across patients and diagnosis groups was irregular, we used the random forest machine learning algorithm to identify groups of OTUs that could be used to classify pre and post-treatment samples for each of the diagnosis groups. Although the adenoma and carcinoma models could reliably differentiate between the pre- and post-treatment samples (P value < 0.001), the advanced-adenoma model could not (P value = 0.61). Furthermore, there was little overlap between the OTUs that were indicative of each treatment. To determine whether individuals who underwent treatment were more likely to have OTUs associated with normal colons we used a larger cohort that contained individuals with normal colons and those with adenomas, advanced adenomas, and carcinomas. We again built random forest models and measured the change in the positive probability of having one of the three diagnoses to assess whether the post-treatment samples received the same classification as the pre-treatment samples. Samples from patients who had carcinomas changed toward a microbial milieu that resembles the normal colon after treatment (P value < 0.001). Finally, we were unable to detect any significant differences in the microbiota of individuals treated with surgery alone and those treated with chemotherapy or chemotherapy and radiation (P value > 0.05). CONCLUSIONS: By better understanding the response of the microbiota to treatment for adenomas and carcinomas, it is likely that biomarkers will eventually be validated that can be used to quantify the risk of recurrence and the likelihood of survival. Although it was difficult to identify significant differences between pre- and post-treatment samples from patients with adenoma and advanced adenoma, this was not the case for carcinomas. Not only were there large changes in pre- versus post-treatment samples for those with carcinoma, but also these changes were toward a more normal microbiota.

The cellular and molecular determinants of emphysematous destruction in COPD.

The introduction of microCT has made it possible to show that the terminal bronchioles are narrowed and destroyed before the onset of emphysematous destruction in COPD. This report extends those observations to the cellular and molecular level in the centrilobular phenotype of emphysematous destruction in lungs donated by persons with very severe COPD (n = 4) treated by lung transplantation with unused donor lungs (n = 4) serving as controls. These lung specimens provided companion samples to those previously examined by microCT (n = 61) that we examined using quantitative histology (n = 61) and gene expression profiling (n = 48). The histological analysis showed that remodeling and destruction of the bronchiolar and alveolar tissue is associated with macrophage, CD4, CD8, and B cell infiltration with increased formation of tertiary lymphoid organs. Moreover, gene set enrichment analysis showed that genes known to be expressed by natural killer (NK), lymphoid tissue inducer (LTi), and innate lymphoid cell 1 (ILC1) cells, but not ILC2 or ILC3 cells, were enriched in the expression profiles associated with CD4, CD8, and B cell infiltration. Based on these findings, we postulate that the centrilobular phenotype of emphysematous destruction COPD is driven by a Th1 response activated by infiltrating ILC1, NK, and LTi cells.