Multivariate inference of pathway activity in host immunity and response to therapeutics

Developing a quantitative view of how biological pathways are regulated in response to environmental factors is central for understanding of disease phenotypes. We present a computational framework, named Multivariate Inference of Pathway Activity (MIPA), which quantifies degree of activity induced in a biological pathway by computing five distinct measures from transcriptomic profiles of its member genes. Statistical significance of inferred activity is examined using multiple independent self-contained tests followed by a competitive analysis. The method incorporates a new algorithm to identify a subset of genes that may regulate the extent of activity induced in a pathway. We present an in-depth evaluation of specificity, robustness, and reproducibility of our method. We benchmarked MIPA's false positive rate at less than 1%. Using transcriptomic profiles representing distinct physiological and disease states, we illustrate applicability of our method in (i) identifying gene–gene interactions in autophagy-dependent response to Salmonella infection, (ii) uncovering gene–environment interactions in host response to bacterial and viral pathogens and (iii) identifying driver genes and processes that contribute to wound healing and response to anti-TNFα therapy. We provide relevant experimental validation that corroborates the accuracy and advantage of our method

ATG5 regulates plasma cell differentiation

Autophagy is a conserved homeostatic process in which cytoplasmic contents are degraded and recycled. Two ubiquitin-like conjugation pathways are required for the generation of autophagosomes, and ATG5 is necessary for both of these processes. Studies of mice deficient in ATG5 reveal a key role for autophagy in T lymphocyte function, as well as in B cell development and B-1a B cell maintenance. However, the role of autophagy genes in B cell function and antibody production has not been described. Using mice in which Atg5 is conditionally deleted in B lymphocytes, we showed here that this autophagy gene is essential for plasma cell homeostasis. In the absence of B cell ATG5 expression, antibody responses were significantly diminished during antigen-specific immunization, parasitic infection and mucosal inflammation. Atg5-deficient B cells maintained the ability to produce immunoglobulin and undergo class-switch recombination, yet had impaired SDC1 expression, significantly decreased antibody secretion in response to toll-like receptor ligands, and an inability to upregulate plasma cell transcription factors. These results build upon previous data demonstrating a role for ATG5 in early B cell development, illustrating its importance in late B cell activation and subsequent plasma cell differentiation

Functional profiling of the gut microbiome in disease-associated inflammation

The microbial residents of the human gut are a major factor in the development and lifelong maintenance of health. The gut microbiota differs to a large degree from person to person and has an important influence on health and disease due to its interaction with the human immune system. Its overall composition and microbial ecology have been implicated in many autoimmune diseases, and it represents a particularly important area for translational research as a new target for diagnostics and therapeutics in complex inflammatory conditions. Determining the biomolecular mechanisms by which altered microbial communities contribute to human disease will be an important outcome of current functional studies of the human microbiome. In this review, we discuss functional profiling of the human microbiome using metagenomic and metatranscriptomic approaches, focusing on the implications for inflammatory conditions such as inflammatory bowel disease and rheumatoid arthritis. Common themes in gut microbial ecology have emerged among these diverse diseases, but they have not yet been linked to targetable mechanisms such as microbial gene and genome composition, pathway and transcript activity, and metabolism. Combining these microbial activities with host gene, transcript and metabolic information will be necessary to understand how and why these complex interacting systems are altered in disease-associated inflammation

Proteins encoded in genomic regions associated with immune-mediated disease physically interact and suggest underlying biology

Genome-wide association studies have uncovered hundreds of DNA changes associated with complex disease. The ultimate promise of these studies is the understanding of disease biology; this goal, however, is not easily achieved because each disease has yielded numerous associations, each one pointing to a region of the genome, rather than a specific causal mutation. Presumably, the causal variants affect components of common molecular processes, and a first step in understanding the disease biology perturbed in patients is to identify connections among regions associated to disease. Since it has been reported in numerous Mendelian diseases that protein products of causal genes tend to physically bind each other, we chose to approach this problem using known protein–protein interactions to test whether any of the products of genes in five complex trait-associated loci bind each other. We applied several permutation methods and find robustly significant connectivity within four of the traits. In Crohn's disease and rheumatoid arthritis, we are able to show that these genes are co-expressed and that other proteins emerging in the network are enriched for association to disease. These findings suggest that, for the complex traits studied here, associated loci contain variants that affect common molecular processes, rather than distinct mechanisms specific to each association.Massachusetts Institute of Technology (MIT IDEA2 Program)Harvard University. Biological and Biomedical Sciences ProgramEunice Kennedy Shriver National Institute of Child Health and Human Development (U.S.) (NICHD RO1 grant HD055150-03)National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) (K08 NIH-NIAMS career development award (AR055688))National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (DK083756)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (DK086502)Denmark. Forskningsradet for Sundhed og SygdomCenter for the Study of Inflammatory Bowel Diseas

An Image-Based Genetic Assay Identifies Genes in T1D Susceptibility Loci Controlling Cellular Antiviral Immunity in Mouse

The pathogenesis of complex diseases, such as type 1 diabetes (T1D), derives from interactions between host genetics and environmental factors. Previous studies have suggested that viral infection plays a significant role in initiation of T1D in genetically predisposed individuals. T1D susceptibility loci may therefore be enriched in previously uncharacterized genes functioning in antiviral defense pathways. To identify genes involved in antiviral immunity, we performed an image-based high-throughput genetic screen using short hairpin RNAs (shRNAs) against 161 genes within T1D susceptibility loci. RAW 264.7 cells transduced with shRNAs were infected with GFP-expressing herpes simplex virus type 1 (HSV-1) and fluorescent microscopy was performed to assess the viral infectivity by fluorescence reporter activity. Of the 14 candidates identified with high confidence, two candidates were selected for further investigation, Il27 and Tagap. Administration of recombinant IL-27 during viral infection was found to act synergistically with interferon gamma (IFN-γ) to activate expression of type I IFNs and proinflammatory cytokines, and to enhance the activities of interferon regulatory factor 3 (IRF3). Consistent with a role in antiviral immunity, Tagap-deficient macrophages demonstrated increased viral replication, reduced expression of proinflammatory chemokines and cytokines, and decreased production of IFN-β. Taken together, our unbiased loss-of-function genetic screen identifies genes that play a role in host antiviral immunity and delineates roles for IL-27 and Tagap in the production of antiviral cytokines

A noncanonical autophagy pathway restricts Toxoplasma gondii growth in a strain-specific manner in IFN-γ-activated human cells

ABSTRACT A core set of autophagy proteins is required for gamma interferon (IFN-γ)-mediated clearance of Toxoplasma gondii in the mouse because of their control of several downstream effectors, including immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs). However, these effectors are absent (i.e., IRGs) from or nonessential (i.e., GBPs) in IFN-γ-activated human cells, raising the question of how these cells control parasite replication. Here, we define a novel role for ubiquitination and recruitment of autophagy adaptors in the strain-specific control of T. gondii replication in IFN-γ-activated human cells. Vacuoles containing susceptible strains of T. gondii became ubiquitinated, recruited the adaptors p62 and NDP52, and were decorated with LC3. Parasites within LC3-positive vacuoles became enclosed in multiple layers of host membranes, resulting in stunting of parasite replication. However, LC3-positive T. gondii-containing vacuoles did not fuse with endosomes and lysosomes, indicating that this process is fundamentally different from xenophagy, a form of autophagy involved in the control of intracellular bacterial pathogens. Genetic knockout of ATG16L or ATG7 reverted the membrane encapsulation and restored parasite replication, indicating that core autophagy proteins involved in LC3 conjugation are important in the control of parasite growth. Despite a role for the core autophagy machinery in this process, upstream activation through Beclin 1 was not sufficient to enhance the ubiquitination of T. gondii-containing vacuoles, suggesting a lack of reliance on canonical autophagy. These findings demonstrate a new mechanism for IFN-γ-dependent control of T. gondii in human cells that depends on ubiquitination and core autophagy proteins that mediate membrane engulfment and restricted growth

Early life environment and natural history of inflammatory bowel diseases

Background: Early life exposures may modify risk of inflammatory bowel diseases (IBD; Crohn’s disease (CD), ulcerative colitis (UC)). However, the relationship between early life exposures and natural history of IBD has not been previously examined. Methods: This single center study included patients with CD or UC recruited in a prospective IBD registry. Enrolled patients completed a detailed environmental questionnaire that assessed various early life environmental exposures. Our primary outcome was requirement for disease-related surgery in CD and UC. Logistic regression models defined independent effect of early life exposures, adjusting for potential confounders. Results: Our study included 333 CD and 270 UC patients. Just over half were female with a median age at diagnosis of 25 years. One-third of the cohort had history of bowel surgery (31%) and nearly half had used at least one biologic agent (47%). Among those with CD, being breastfed was associated with reduced risk of CD-related surgery (34% vs. 55%), while childhood cigarette smoke exposure was associated with increased risk. On multivariate analysis, history of being breastfed (odds ratio (OR) 0.21, 95% confidence interval [CI] 0.09–0.46) and cigarette smoke exposure as a child (OR 2.17, 95% CI 1.10–4.29) remained independently associated with surgery. None of the early life variables influenced disease phenotype or outcome in UC. Conclusion: A history of being breastfed was associated with a decreased risk while childhood cigarette smoke exposure was associated with an increased risk of surgery in patients with CD. Further investigation to examine biological mechanisms is warranted. Electronic supplementary material The online version of this article (doi:10.1186/s12876-014-0216-8) contains supplementary material, which is available to authorized users

GSDMD is critical for autoinflammatory pathology in a mouse model of Familial Mediterranean Fever

Pyroptosis is an inflammasome-induced lytic cell death mode, the physiological role of which in chronic inflammatory diseases is unknown. Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory disease worldwide, affecting an estimated 150,000 patients. The disease is caused by missense mutations in Mefv that activate the Pyrin inflammasome, but the pathophysiologic mechanisms driving autoinflammation in FMF are incompletely understood. Here, we show that Clostridium difficile infection of FMF knock-in macrophages that express a chimeric FMF-associated Mefv(V726A) Pyrin elicited pyroptosis and gasdermin D (GSDMD)-mediated interleukin (IL)-1 beta secretion. Importantly, in vivo GSDMD deletion abolished spontaneous autoinflammatory disease. GSDMD-deficient FMF knock-in mice were fully protected from the runted growth, anemia, systemic inflammatory cytokine production, neutrophilia, and tissue damage that characterize this autoinflammatory disease model. Overall, this work identifies pyroptosis as a critical mechanism of IL-1 beta-dependent autoinflammation in FMF and highlights GSDMD inhibition as a potential antiinflammatory strategy in inflammasome-driven diseases

Sub-clinical detection of gut microbial biomarkers of obesity and type 2 diabetes

Background: Obesity and type 2 diabetes (T2D) are linked both with host genetics and with environmental factors, including dysbioses of the gut microbiota. However, it is unclear whether these microbial changes precede disease onset. Twin cohorts present a unique genetically-controlled opportunity to study the relationships between lifestyle factors and the microbiome. In particular, we hypothesized that family-independent changes in microbial composition and metabolic function during the sub-clinical state of T2D could be either causal or early biomarkers of progression. Methods: We collected fecal samples and clinical metadata from 20 monozygotic Korean twins at up to two time points, resulting in 36 stool shotgun metagenomes. While the participants were neither obese nor diabetic, they spanned the entire range of healthy to near-clinical values and thus enabled the study of microbial associations during sub-clinical disease while accounting for genetic background. Results: We found changes both in composition and in function of the sub-clinical gut microbiome, including a decrease in Akkermansia muciniphila suggesting a role prior to the onset of disease, and functional changes reflecting a response to oxidative stress comparable to that previously observed in chronic T2D and inflammatory bowel diseases. Finally, our unique study design allowed us to examine the strain similarity between twins, and we found that twins demonstrate strain-level differences in composition despite species-level similarities. Conclusions: These changes in the microbiome might be used for the early diagnosis of an inflamed gut and T2D prior to clinical onset of the disease and will help to advance toward microbial interventions. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0271-6) contains supplementary material, which is available to authorized users