Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota.

Reversible genomic DNA inversions control the expression of numerous gut bacterial molecules, but how this impacts disease remains uncertain. By analyzing metagenomic samples from inflammatory bowel disease (IBD) cohorts, we identified multiple invertible regions where a particular orientation correlated with disease. These include the promoter of polysaccharide A (PSA) of Bacteroides fragilis, which induces regulatory T cells (Tregs) and ameliorates experimental colitis. The PSA promoter was mostly oriented "OFF" in IBD patients, which correlated with increased B. fragilis-associated bacteriophages. Similarly, in mice colonized with a healthy human microbiota and B. fragilis, induction of colitis caused a decline of PSA in the "ON" orientation that reversed as inflammation resolved. Monocolonization of mice with B. fragilis revealed that bacteriophage infection increased the frequency of PSA in the "OFF" orientation, causing reduced PSA expression and decreased Treg cells. Altogether, we reveal dynamic bacterial phase variations driven by bacteriophages and host inflammation, signifying bacterial functional plasticity during disease.

A personalized network framework reveals predictive axis of anti-TNF response across diseases.

Personalized treatment of complex diseases has been mostly predicated on biomarker identification of one drug-disease combination at a time. Here, we use a computational approach termed Disruption Networks to generate a data type, contextualized by cell-centered individual-level networks, that captures biology otherwise overlooked when performing standard statistics. This data type extends beyond the "feature level space", to the "relations space", by quantifying individual-level breaking or rewiring of cross-feature relations. Applying Disruption Networks to dissect high-dimensional blood data, we discover and validate that the RAC1-PAK1 axis is predictive of anti-TNF response in inflammatory bowel disease. Intermediate monocytes, which correlate with the inflammatory state, play a key role in the RAC1-PAK1 responses, supporting their modulation as a therapeutic target. This axis also predicts response in rheumatoid arthritis, validated in three public cohorts. Our findings support blood-based drug response diagnostics across immune-mediated diseases, implicating common mechanisms of non-response.

Antibiotic use differentially affects the risk of anti-drug antibody formation during anti-TNFα therapy in inflammatory bowel disease patients: a report from the epi-IIRN.

OBJECTIVE: Anti-drug antibodies (ADA) to anti-tumour necrosis factor (anti-TNF) therapy drive treatment loss of response. An association between intestinal microbial composition and response to anti-TNF therapy was noted. We therefore aimed to assess the implications of antibiotic treatments on ADA formation in patients with inflammatory bowel disease (IBD). DESIGN: We analysed data from the epi-IIRN (epidemiology group of the Israeli IBD research nucleus), a nationwide registry of all patients with IBD in Israel. We included all patients treated with anti-TNF who had available ADA levels. Survival analysis with drug use as time varying covariates were used to assess the association between antibiotic use and ADA development. Next, specific pathogen and germ-free C57BL mice were treated with respective antibiotics and challenged with infliximab. ADA were assessed after 14 days. RESULTS: Among 1946 eligible patients, with a median follow-up of 651 days from initiation of therapy, 363 had positive ADA. Cox proportional hazard model demonstrated an increased risk of ADA development in patients who used cephalosporins (HR=1.97, 95% CI 1.58 to 2.44), or penicillins with ß-lactamase inhibitors (penicillin-BLI, HR=1.4, 95% CI 1.13 to 1.74), whereas a reduced risk was noted in patients treated with macrolides (HR=0.38, 95% CI 0.16 to 0.86) or fluoroquinolones (HR=0.20, 95% CI 0.12 to 0.35). In mice exposed to infliximab, significantly increased ADA production was observed in cephalosporin as compared with macrolide pretreated mice. Germ-free mice produced no ADA. CONCLUSION: ADA production is associated with the microbial composition. The risk of ADA development during anti-TNF therapy can possibly be reduced by avoidance of cephalosporins and penicillin-BLIs, or by treatment with fluoroquinolones or macrolides.

Differential Serum-intestinal Dynamics of Infliximab and Adalimumab in Inflammatory Bowel Disease Patients.

BACKGROUND AND AIMS: Therapeutic drug monitoring is used to guide anti-tumour necrosis factor [TNF] therapy. However, the associations between serum drug levels [SDL], TNF-bound, and free anti-TNF in the target tissue are incompletely defined. We aimed to assess the interactions between these parameters in inflammatory bowel disease [IBD] patients. METHODS: ENZYME-LINKED IMMUNOSORBENT: assays [ELISA assays] were used to detect free drug and TNF-drug complexes in intestinal tissues. Concurrent SDL, anti-drug antibodies [ADA], pharmacotherapy, clinical response, endoscopic appearance, and histological severity were determined. Comparisons between anti-TNFs and paired inflamed/non-inflamed tissue were performed. Variables were correlated and potential interactions detected using multivariate analysis. RESULTS: A total of 95 biopsies taken from 49 anti-TNF treated IBD patients [26 receiving infliximab and 23 adalimumab] were studied. Free drug levels were higher in inflamed compared with non-inflamed paired specimens. Tissue free-drug and TNF-drug complexes levels were higher in adalimumab-treated patients. In adalimumab-treated patients, SDL were correlated with free drug, but not TNF-drug complex levels, in both inflamed and non-inflamed segments. In infliximab-treated patients, higher SDL were associated with the presence of tissue free drug in both inflamed and non-inflamed segments, whereas TNF-drug complexes were mostly detected in non-inflamed but not in inflamed tissue. In the presence of ADA, neither free drug nor TNF-infliximab complexes were measured in the tissue. Tissue levels did not correlate well with clinical, endoscopic, or histological scores. CONCLUSIONS: SDL correlated with tissue free drug levels; however, different dynamics were observed for TNF-drug complex levels. Infliximab and adalimumab tissue drug dynamics differ. Better understanding of these interactions may allow future therapeutic optimisation.

Infliximab-Tumor Necrosis Factor Complexes Elicit Formation of Anti-Drug Antibodies.

BACKGROUND & AIMS: Some patients develop anti-drug antibodies (ADAs), which reduce the efficacy of infliximab, a monoclonal antibody against tumor necrosis factor (TNF), in the treatment of immune-mediated diseases, including inflammatory bowel diseases. ADAs arise inconsistently, and it is not clear what factors determine their formation. We investigated features of the immune system, the infliximab antibody, and its complex with TNF that might contribute to ADA generation. METHODS: C57BL/6 mice were given injections of infliximab and recombinant human TNF or infliximab F(ab')2 fragments. Blood samples were collected every 2-3 days for 2 weeks and weekly thereafter for up to 6 weeks; infliximab-TNF complexes and ADAs were measured by enzyme-linked immunosorbent assay (ELISA). Intestinal biopsy and blood samples were obtained from patients having endoscopy who had received infliximab therapy for inflammatory bowel diseases; infliximab-TNF complexes were measured with ELISA. Infliximab-specific plasma cells were detected in patient tissue samples by using mass cytometry. We studied activation of innate immune cells in peripheral blood mononuclear cells (PBMCs) from healthy donors incubated with infliximab or infliximab-TNF complexes; toll-like receptors (TLRs) were blocked with antibodies, endocytosis was blocked with the inhibitor PitStop2, and cytokine expression was measured by real-time polymerase chain reaction and ELISAs. Uptake of infliximab and infliximab-TNF complexes by THP-1 cells was measured with confocal microscopy. RESULTS: Mice given increasing doses of infliximab produced increasing levels of ADAs. Blood samples from mice given injections of human TNF and infliximab contained infliximab-TNF complexes; complex formation was associated with ADA formation with an area under the curve of 0.944 (95% confidence interval, 0.851-1.000; P = .003). Intestinal tissues from patients, but not blood samples, contained infliximab-TNF complexes and infliximab-specific plasma cells. Incubation of PBMCs with infliximab-TNF complexes resulted in a 4.74-fold increase in level of interleukin (IL) 1ß (IL1B) messenger RNA (P for comparison = .005), increased IL1B protein secretion, and a 2.69-fold increase in the expression of TNF messenger RNA (P for comparison = 0.013) compared with control PBMCs. Infliximab reduced only IL1B and TNF expression. Antibodies against TLR2 or TLR4 did not block the increases in IL1B or TNF expression, but endocytosis was required. THP-1 cells endocytosed higher levels of infliximab-TNF complexes than infliximab alone. CONCLUSIONS: In mice, we found ADA formation to increase with dose of infliximab given and concentration of infliximab-TNF complexes detected in blood. Based on studies of human intestinal tissues and blood samples, we propose that infliximab-TNF complexes formed in the intestine are endocytosed by and activate innate immune cells, which increase expression of IL1B and TNF and production of antibodies against the drug complex. It is therefore important to optimize the infliximab dose to a level that is effective but does not activate an innate immune response against the drug-TNF complex.

Cell-centred meta-analysis reveals baseline predictors of anti-TNFα non-response in biopsy and blood of patients with IBD.

OBJECTIVE: Although anti-tumour necrosis factor alpha (anti-TNFα) therapies represent a major breakthrough in IBD therapy, their cost-benefit ratio is hampered by an overall 30% non-response rate, adverse side effects and high costs. Thus, finding predictive biomarkers of non-response prior to commencing anti-TNFα therapy is of high value. DESIGN: We analysed publicly available whole-genome expression profiles of colon biopsies obtained from multiple cohorts of patients with IBD using a combined computational deconvolution-meta-analysis paradigm which allows to estimate immune cell contribution to the measured expression and capture differential regulatory programmes otherwise masked due to variation in cellular composition. Insights from this in silico approach were experimentally validated in biopsies and blood samples of three independent test cohorts. RESULTS: We found the proportion of plasma cells as a robust pretreatment biomarker of non-response to therapy, which we validated in two independent cohorts of immune-stained colon biopsies, where a plasma cellular score from inflamed biopsies was predictive of non-response with an area under the curve (AUC) of 82%. Meta-analysis of the cell proportion-adjusted gene expression data suggested that an increase in inflammatory macrophages in anti-TNFα non-responding individuals is associated with the upregulation of the triggering receptor expressed on myeloid cells 1 (TREM-1) and chemokine receptor type 2 (CCR2)-chemokine ligand 7 (CCL7) -axes. Blood gene expression analysis of an independent cohort, identified TREM-1 downregulation in non-responders at baseline, which was predictive of response with an AUC of 94%. CONCLUSIONS: Our study proposes two clinically feasible assays, one in biopsy and one in blood, for predicting non-response to anti-TNFα therapy prior to initiation of treatment. Moreover, it suggests that mechanism-driven novel drugs for non-responders should be developed.

Microprocessor Recruitment to Elongating RNA Polymerase II Is Required for Differential Expression of MicroRNAs.

The cellular abundance of mature microRNAs (miRNAs) is dictated by the efficiency of nuclear processing of primary miRNA transcripts (pri-miRNAs) into pre-miRNA intermediates. The Microprocessor complex of Drosha and DGCR8 carries this out, but it has been unclear what controls Microprocessor's differential processing of various pri-miRNAs. Here, we show that Drosophila DGCR8 (Pasha) directly associates with the C-terminal domain of the RNA polymerase II elongation complex when it is phosphorylated by the Cdk9 kinase (pTEFb). When association is blocked by loss of Cdk9 activity, a global change in pri-miRNA processing is detected. Processing of pri-miRNAs with a UGU sequence motif in their apical junction domain increases, while processing of pri-miRNAs lacking this motif decreases. Therefore, phosphorylation of RNA polymerase II recruits Microprocessor for co-transcriptional processing of non-UGU pri-miRNAs that would otherwise be poorly processed. In contrast, UGU-positive pri-miRNAs are robustly processed by Microprocessor independent of RNA polymerase association.

Anti-infliximab Antibodies with Neutralizing Capacity in Patients with Inflammatory Bowel Disease: Distinct Clinical Implications Revealed by a Novel Assay.

BACKGROUND: About 60% of infliximab (IFX)-treated patients develop antidrug antibodies (ADA), although their clinical significance remains disputed. The aim of this study was to develop an assay for assessing ADA-neutralizing potential, and clinical significance. METHODS: An immune assay was devised in which the inhibition of IFX binding to plated-tumor necrosis factor in the presence of patient sera or controls, was assessed and defined as IFX-tumor necrosis factor binding reduction ratio (ITBR). The assay was compared to a bioassay in which tumor necrosis factor-α-induced interleukin-8 secretion from HT-29 cells was assessed after addition of IFX to ADA-containing sera or control sera. RESULTS: Both assays detected neutralizing antibodies in 39 of 44 ADA-positive sera. The median ITBR was 3.66 (mean 4.9 ± 3.2) in 29 ADA-positive patients with loss of response (LOR), and 1.3 (mean 1.9 ± 1.3) in 15 patients without LOR (P = 0.001). ADA titers in both groups were similar (median 9.5 and 10.2 µg/mL, respectively P = 0.74). Using an ITBR of 1.65, the sensitivity for LOR detection was 86.2% and the specificity was 66.7%. (positive predictive value 83%; negative predictive value 71.4%; P = 0.001). When early ADA-IFX-sera from IFX-treated patients with or without subsequent LOR were compared, the median ITBRs were 1.1 and 0.57, respectively (P = 0.028). CONCLUSIONS: Detection of neutralizing antibody activity was superior to antibody quantization by enzyme-linked immunosorbent assay with respect to correlation with clinical LOR, and for prediction of subsequent LOR. These findings may assist in optimizing infliximab therapy in patients with inflammatory bowel disease.

A Systematic Genetic Screen to Dissect the MicroRNA Pathway in Drosophila.

A central goal of microRNA biology is to elucidate the genetic program of miRNA function and regulation. However, relatively few of the effectors that execute miRNA repression have been identified. Because such genes may function in many developmental processes, mutations in them are expected to be pleiotropic and thus are discarded in most standard genetic screens. Here, we describe a systematic screen designed to identify all Drosophila genes in ∼40% of the genome that function in the miRNA pathway. To identify potentially pleiotropic genes, the screen analyzed clones of homozygous mutant cells in heterozygous animals. We identified 45 mutations representing 24 genes, and we molecularly characterized 9 genes. These include 4 previously known genes that encode core components of the miRNA pathway, including Drosha, Pasha, Dicer-1, and Ago1. The rest are new genes that function through chromatin remodeling, signaling, and mRNA decapping. The results suggest genetic screens that use clonal analysis can elucidate the miRNA program and that ∼100 genes are required to execute the miRNA program.

Silencing by small RNAs is linked to endosomal trafficking.

Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

Reconstitution of expression of H-2K region-encoded murine MHC class I glycoproteins in MHC class I-deficient B16BL6 melanoma cells affects the expression and function of antigen-processing machinery.

We have recently reported that reconstitution of expression of major histocompatibility complex (MHC) class I glycoproteins in MHC-deficient and highly metastatic B16BL6 melanoma cells attenuates their malignant capacities by modulation of compartmentalization and functions of cell membrane receptors for growth factors [Assa-Kunik E, et al. J Immunol 2003;171:2945-52]. Our present study provides evidence that re-expression of an H-2K MHC class I-encoding gene in these cells also augments the expression of the Tap-2 peptide transporter and the inducible proteasome subunits, i.e. Lmp-2, Lmp-7 and Lmp-10. Up-regulation of inducible proteasome subunits was also followed by a significant changed in the proteolytic activity of the proteasome complex. We suggest that, in addition to providing a framework for proper presentation of antigenic peptides, MHC class I glycoproteins may regulate the immune response by modulating the expression and function of other genes, whose products are essential for proper antigen processing and presentation.

Attenuation of the Fas-L independent B16BL6 melanoma lymphocidic capacity by H-2K class I molecules.

We have previously reported that the capacity of highly malignant B16BL6 murine melanoma cells to induce cell death in naive syngeneic lymphocytes stems from the absence of major histocompatibility complex (MHC) class I glycoproteins in these melanoma cells. Our present study provides evidence that the above-mentioned lymphocidic activities of B16BL6 cells are selectively attenuated when the expression of H-2K (but not H-2D or H-2L) MHC class I glycoproteins is reconstituted in these cells. The induction of apoptosis in naive lymphocytes by H-2K-deficient melanoma cells does not involve the Fas ligand (Fas-L)/FAS signaling module, as demonstrated by employing lymphocytes derived from Fas-L(gld)- or Fas(lpr)-deficient mice in co-culture experiments. Furthermore, these tumor cells fail to induce Fas-L-mediated fratricide in co-cultured lymphocytes and do not express Fas-L either when grown alone or co-cultured with lymphocytes. These findings explain the previously widely reported selective down-regulation of certain MHC class I-encoded glycoproteins (H-2K, bur not H-2D or H-2L) during tumor progression. Namely, the initiation of an effective immune response against H-2K-deficient cells could be abrogated at very early steps, as the result of the induction of Fas-L/Fas-independent cell death among naive lymphoid cells.

Alterations in the expression of MHC class I glycoproteins by B16BL6 melanoma cells modulate insulin receptor-regulated signal transduction and augment [correction of augments] resistance to apoptosis.

In a variety of malignancies, the immune-escape phenotype is associated, in part, with the inability of tumor cells to properly present their Ags to CTLs due to a deranged expression of MHC class I glycoproteins. However, these molecules were found to possess broader nonimmune functions, including participation in signal transduction and regulation of proliferation, differentiation, and sensitivity to apoptosis-inducing factors; processes, which are characteristically impaired during malignant transformation. We investigated whether the deranged expression of MHC class I expression by tumor cells could affect proper receptor-mediated signal transduction and accentuate their malignant phenotype. The malignant and H-2K murine MHC class I-deficient B16BL6 melanoma cells were characterized by an attenuated capacity to bind insulin due to the retention of corresponding receptor in intracellular stores. The restoration of H-2K expression in these cells, which abrogated their capacity to form tumors in mice, enhanced membrane translocation of the receptor, presumably, by modulating its glycosylation. The addition of insulin to H-2K-expressing melanoma cells cultured in serum-free conditions precluded apoptotic death by up-regulating the activity of protein kinase B (PKB)/Akt. In contrast, the deficiency for H-2K characteristic to the malignant clones was associated with a constitutive high activity of PKB/Akt, which rendered them resistant to apoptosis, induced by deprivation of serum-derived growth factors. The possibility to correct the regulation of PKB/Akt activity by restoration of H-2K expression in B16BL6 melanoma cells may be considered as an attractive approach for cancer therapy, since an aberrant activation of this enzyme is characteristic to resistant malignancies.