Epithelial TLR4 Signaling Activates DUOX2 to Induce Microbiota-Driven Tumorigenesis.

BACKGROUND & AIMS: Chronic colonic inflammation leads to dysplasia and cancer in patients with inflammatory bowel disease. We have described the critical role of innate immune signaling via Toll-like receptor 4 (TLR4) in the pathogenesis of dysplasia and cancer. In the current study, we interrogate the intersection of TLR4 signaling, epithelial redox activity, and the microbiota in colitis-associated neoplasia. METHODS: Inflammatory bowel disease and colorectal cancer data sets were analyzed for expression of TLR4, dual oxidase 2 (DUOX2), and NADPH oxidase 1 (NOX1). Epithelial production of hydrogen peroxide (H2O2) was analyzed in murine colonic epithelial cells and colonoid cultures. Colorectal cancer models were carried out in villin-TLR4 mice, carrying a constitutively active form of TLR4, their littermates, and villin-TLR4 mice backcrossed to DUOXA-knockout mice. The role of the TLR4-shaped microbiota in tumor development was tested in wild-type germ-free mice. RESULTS: Activation of epithelial TLR4 was associated with up-regulation of DUOX2 and NOX1 in inflammatory bowel disease and colorectal cancer. DUOX2 was exquisitely dependent on TLR4 signaling and mediated the production of epithelial H2O2. Epithelial H2O2 was significantly increased in villin-TLR4 mice; TLR4-dependent tumorigenesis required the presence of DUOX2 and a microbiota. Mucosa-associated microbiota transferred from villin-TLR4 mice to wild-type germ-free mice caused increased H2O2 production and tumorigenesis. CONCLUSIONS: Increased TLR4 signaling in colitis drives expression of DUOX2 and epithelial production of H2O2. The local milieu imprints the mucosal microbiota and imbues it with pathogenic properties demonstrated by enhanced epithelial reactive oxygen species and increased development of colitis-associated tumors. The inter-relationship between epithelial reactive oxygen species and tumor-promoting microbiota requires a 2-pronged strategy to reduce the risk of dysplasia in colitis patients.

Morphine tolerance is attenuated in germfree mice and reversed by probiotics, implicating the role of gut microbiome.

Prolonged exposure to opioids results in analgesic tolerance, drug overdose, and death. The mechanism underlying morphine analgesic tolerance still remains unresolved. We show that morphine analgesic tolerance was significantly attenuated in germfree (GF) and in pan-antibiotic-treated mice. Reconstitution of GF mice with naïve fecal microbiota reinstated morphine analgesic tolerance. We further demonstrated that tolerance was associated with microbial dysbiosis with selective depletion in Bifidobacteria and Lactobacillaeae. Probiotics, enriched with these bacterial communities, attenuated analgesic tolerance in morphine-treated mice. These results suggest that probiotic therapy during morphine administration may be a promising, safe, and inexpensive treatment to prolong morphine's efficacy and attenuate analgesic tolerance. We hypothesize a vicious cycle of chronic morphine tolerance: morphine-induced gut dysbiosis leads to gut barrier disruption and bacterial translocation, initiating local gut inflammation through TLR2/4 activation, resulting in the activation of proinflammatory cytokines, which drives morphine tolerance.

Low-Fat, High-Fiber Diet Reduces Markers of Inflammation and Dysbiosis and Improves Quality of Life in Patients With Ulcerative Colitis.

BACKGROUND & AIMS: A high-fat diet has been associated with an increased risk of ulcerative colitis (UC). We studied the effects of a low-fat, high-fiber diet (LFD) vs an improved standard American diet (iSAD, included higher quantities of fruits, vegetables, and fiber than a typical SAD). We collected data on quality of life, markers of inflammation, and fecal markers of intestinal dysbiosis in patients with UC. METHODS: We analyzed data from a parallel-group, cross-over study of 17 patients with UC in remission or with mild disease (with a flare within the past 18 mo), from February 25, 2015, through September 11, 2018. Participants were assigned randomly to 2 groups and received a LFD (10% of calories from fat) or an iSAD (35%-40% of calories from fat) for the first 4-week period, followed by a 2-week washout period, and then switched to the other diet for 4 weeks. All diets were catered and delivered to patients' homes, and each participant served as her or his own control. Serum and stool samples were collected at baseline and week 4 of each diet and analyzed for markers of inflammation. We performed 16s ribosomal RNA sequencing and untargeted and targeted metabolomic analyses on stool samples. The primary outcome was quality of life, which was measured by the short inflammatory bowel disease (IBD) questionnaire at baseline and week 4 of the diets. Secondary outcomes included changes in the Short-Form 36 health survey, partial Mayo score, markers of inflammation, microbiome and metabolome analysis, and adherence to the diet. RESULTS: Participants' baseline diets were unhealthier than either study diet. All patients remained in remission throughout the study period. Compared with baseline, the iSAD and LFD each increased quality of life, based on the short IBD questionnaire and Short-Form 36 health survey scores (baseline short IBD questionnaire score, 4.98; iSAD, 5.55; LFD, 5.77; baseline vs iSAD, P = .02; baseline vs LFD, P = .001). Serum amyloid A decreased significantly from 7.99 mg/L at baseline to 4.50 mg/L after LFD (P = .02), but did not decrease significantly compared with iSAD (7.20 mg/L; iSAD vs LFD, P = .07). The serum level of C-reactive protein decreased numerically from 3.23 mg/L at baseline to 2.51 mg/L after LFD (P = .07). The relative abundance of Actinobacteria in fecal samples decreased from 13.69% at baseline to 7.82% after LFD (P = .017), whereas the relative abundance of Bacteroidetes increased from 14.6% at baseline to 24.02% on LFD (P = .015). The relative abundance of Faecalibacterium prausnitzii was higher after 4 weeks on the LFD (7.20%) compared with iSAD (5.37%; P = .04). Fecal levels of acetate (an anti-inflammatory metabolite) increased from a relative abundance of 40.37 at baseline to 42.52 on the iSAD and 53.98 on the LFD (baseline vs LFD, P = .05; iSAD vs LFD, P = .09). The fecal level of tryptophan decreased from a relative abundance of 1.33 at baseline to 1.08 on the iSAD (P = .43), but increased to a relative abundance of 2.27 on the LFD (baseline vs LFD, P = .04; iSAD vs LFD, P = .08); fecal levels of lauric acid decreased after LFD (baseline, 203.4; iSAD, 381.4; LFD, 29.91; baseline vs LFD, P = .04; iSAD vs LFD, P = .02). CONCLUSIONS: In a cross-over study of patients with UC in remission, we found that a catered LFD or iSAD were each well tolerated and increased quality of life. However, the LFD decreased markers of inflammation and reduced intestinal dysbiosis in fecal samples. Dietary interventions therefore might benefit patients with UC in remission. ClinicalTrials.gov no: NCT04147598.

Conservation of pathogenic TCR homology across class II restrictions in anti-ribonucleoprotein autoimmunity: extended efficacy of T cell vaccine therapy.

T cells have been shown to mediate aspects of anti-ribonucleoprotein (RNP) autoimmunity, and are a potential target of therapy in lupus and related diseases. In this study, we assessed the relevance of a conserved class of anti-RNP T cells to autoimmune disease expression and therapy. Our data show that anti-RNP T cell selection induced a limited set of homologous CDR3 motifs at high frequency. Homologous CDR3 motifs have been reported in other autoimmune diseases. Vaccination with irradiated anti-RNP (but not anti-tetanus toxoid) CD4(+) cells induced remission of anti-RNP-associated nephritis in ≥ 80% of treated mice, even with donor/recipient MHC class II mismatch, and in both induced and spontaneous autoimmunity. Vaccine responder sera inhibited anti-70k T cell proliferation and bound hybridomas expressing the conserved CDR3 motifs. Our data indicate that a limited set of TCR CDR3 motifs may be important for the pathogenesis of anti-RNP lupus and other autoimmune diseases. The ability to target a consistent set of pathogenic T cells between individuals and across class II restrictions may allow for the more practical development of a standardized anti-RNP T cell vaccine preparation useful for multiple patients.

Generation of functional, antigen-specific CD8+ human T cells from cord blood stem cells using exogenous Notch and tetramer-TCR signaling.

In vitro differentiation of mouse and human stem cells into early T cells has been successfully demonstrated using artificial Notch signaling systems. However, generation of mature, antigen-specific, functional T cells, directly from human stem cells has remained elusive, except when using stromal coculture of stem cells retrovirally transfected with antigen-specific T cell receptors (TCRs). Here we show that human umbilical cord blood (UCB)-derived CD34+CD38-/low hematopoietic stem cells can be successfully differentiated into functional, antigen-specific cytotoxic CD8+ T cells without direct stromal coculture or retroviral TCR transfection. Surface-immobilized Notch ligands (DLL1) and stromal cell conditioned medium successfully induced the development of CD1a+CD7+ and CD4+CD8+ early T cells. These cells, upon continued culture with cytomegalovirus (CMV) or influenza-A virus M1 (GIL) epitope-loaded human leukocyte antigen (HLA)-A*0201 tetramers, resulted in the generation of a polyclonal population of CMV-specific or GIL-specific CD8+ T cells, respectively. Upon further activation with antigen-loaded target cells, these antigen-specific, stem cell-derived T cells exhibited cytolytic functionality, specifically CD107a surface mobilization, interferon gamma (IFNg) production, and Granzyme B secretion. Such scalable, in vitro generation of functional, antigen-specific T cells from human stem cells could eventually provide a readily available cell source for adoptive transfer immunotherapies and also allow better understanding of human T cell development.

Functional redundancy of MyD88-dependent signaling pathways in a murine model of histidyl-transfer RNA synthetase-induced myositis.

We have previously shown that i.m. administration of bacterially expressed murine histidyl-tRNA synthetase (HRS) triggers florid muscle inflammation (relative to appropriate control proteins) in various congenic strains of mice. Because severe disease develops even in the absence of adaptive immune responses to HRS, we sought to identify innate immune signaling components contributing to our model of HRS-induced myositis. In vitro stimulation assays demonstrated HRS-mediated activation of HEK293 cells transfected with either TLR2 or TLR4, revealing an excitatory capacity exceeding that of other bacterially expressed fusion proteins. Corresponding to this apparent functional redundancy of TLR signaling pathways, HRS immunization of B6.TLR2(-/-) and B6.TLR4(-/-) single-knockout mice yielded significant lymphocytic infiltration of muscle tissue comparable to that produced in C57BL/6 wild-type mice. In contrast, concomitant elimination of TLR2 and TLR4 signaling in B6.TLR2(-/-).TLR4(-/-) double-knockout mice markedly reduced the severity of HRS-induced muscle inflammation. Complementary subfragment analysis demonstrated that aa 60-90 of HRS were absolutely required for in vitro as well as in vivo signaling via these MyD88-dependent TLR pathways--effects mediated, in part, through preferential binding of exogenous ligands capable of activating specific TLRs. Collectively, these experiments indicate that multiple MyD88-dependent signaling cascades contribute to this model of HRS-induced myositis, underscoring the antigenic versatility of HRS and confirming the importance of innate immunity in this system.

Metaproteomics reveals diet-induced changes in gut microbiome function according to Crohn's disease location.

BACKGROUND: Crohn's disease (CD) is characterized by chronic intestinal inflammation. Diet is a key modifiable factor influencing the gut microbiome (GM) and a risk factor for CD. However, the impact of diet modulation on GM function in CD patients is understudied. Herein, we evaluated the effect of a high-fiber, low-fat diet (the Mi-IBD diet) on GM function in CD patients. All participants were instructed to follow the Mi-IBD diet for 8 weeks. One group of CD patients received one-time diet counseling only (Gr1); catered food was supplied for the other three groups, including CD patients (Gr2) and dyads of CD patients and healthy household controls (HHCs) residing within the same household (Gr3-HHC dyads). Stool samples were collected at baseline, week 8, and week 36, and analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: At baseline, the metaproteomic profiles of CD patients and HHCs differed. The Mi-IBD diet significantly increased carbohydrate and iron transport and metabolism. The predicted microbial composition underlying the metaproteomic changes differed between patients with ileal only disease (ICD) or colonic involvement: ICD was characterized by decreased Faecalibacterium abundance. Even on the Mi-IBD diet, the CD patient metaproteome displayed significant underrepresentation of carbohydrate and purine/pyrimidine synthesis pathways compared to that of HHCs. Human immune-related proteins were upregulated in CD patients compared to HHCs. CONCLUSIONS: The Mi-IBD diet changed the microbial function of CD patients and enhanced carbohydrate metabolism. Our metaproteomic results highlight functional differences in the microbiome according to disease location. Notably, our dietary intervention yielded the most benefit for CD patients with colonic involvement compared to ileal-only disease. Video Abstract.

Intestinal Epithelial Inactivity of Dual Oxidase 2 Results in Microbiome-Mediated Metabolic Syndrome.

BACKGROUND & AIMS: Metabolic syndrome (MetS) is characterized by obesity, glucose intolerance, and hepatic steatosis. Alterations in the gut microbiome play important roles in the development of MetS. However, the mechanisms by which this occurs are poorly understood. Dual oxidase 2 (DUOX2) is an antimicrobial reduced nicotinamide adenine dinucleotide phosphate oxidase expressed in the gut epithelium. Here, we posit that epithelial DUOX2 activity provides a mechanistic link between the gut microbiome and the development of MetS. METHODS: Mice carrying an intestinal epithelial-specific deletion of dual oxidase maturation factor 1/2 (DA IEC-KO), and wild-type littermates were fed a standard diet and killed at 24 weeks. Metabolic alterations were determined by glucose tolerance, lipid tests, and body and organ weight measurements. DUOX2 activity was determined by Amplex Red. Intestinal permeability was determined by fluorescein isothiocyanate-dextran, microbial translocation assessments, and portal vein lipopolysaccharide measurements. Metagenomic analysis of the stool microbiome was performed. The role of the microbiome was assessed in antibiotic-treated mice. RESULTS: DA IEC-KO males showed increased body and organ weights accompanied by glucose intolerance and increased plasma lipid and liver enzyme levels, and increased adiposity in the liver and adipose tissue. Expression of F4/80, CD68, uncoupling protein 1, carbohydrate response element binding protein, leptin, and adiponectin was altered in the liver and adipose tissue of DA IEC-KO males. DA IEC-KO males produced less epithelial H2O2, had altered relative abundance of Akkermansiaceae and Lachnospiraceae in stool, and showed increased portal vein lipopolysaccharides and intestinal permeability. Females were protected from barrier defects and MetS, despite producing less H2O2. Antibiotic depletion abrogated all MetS phenotypes observed. CONCLUSIONS: Intestinal epithelial inactivity of DUOX2 promotes MetS in a microbiome-dependent manner.

Combining Pentoxifylline With Vedolizumab for Crohn's Disease: Results of a Randomised, Placebo-controlled Pilot Study.

BACKGROUND AND AIMS: The efficacy of current biologics may be limited by targeting only one pathway. Pentoxifylline [PTX] interferes with tumour necrosis factor [TNF] gene expression. We performed a randomised, placebo-controlled pilot study to determine if PTX plus vedolizumab [VDZ] in patients with Crohn's disease [CD] is safe and improves response compared with VDZ monotherapy. METHODS: Thirty adult patients with active CD were randomised to VDZ/PTX or VDZ/placebo and followed for 24 weeks. Endoscopic activity and inflammatory cytokines were measured at baseline and Week 24. Descriptive statistics were used to determine estimates of effect. RESULTS: Demographics were similar but baseline disease activity was higher in the VDZ/PTX group. There was no difference in clinical remission at Week 14 (60.0% vs 66.67%, odds ratio [OR] 0.76, 95% confidence interval [CI] 0.16, 3.51) or steroid-free clinical remission at Week 24 in patients receiving VDZ/PTX. Improved clinical response was noted in the VDZ/PTX group at Weeks 6, 14, and 24 [Week 6: 20% vs 6.67%, Week 14: 26.67% vs 6.67%, Week 24: 40% vs 20%]. The rate of endoscopic remission was similar between the groups [40% vs 33.33%], with a greater mean decrease in Simple Endoscopic Score-CD [SES-CD] and C-reactive protein [CRP] with VDZ/PTX [SES-CD -3.17 vs -0.15, CRP -5.56 vs 0.46]. An increase in serum TNF-α concentration was observed with VDZ/placebo group; PTX mitigated this effect. No serious adverse events occurred. CONCLUSIONS: VDZ/PTX did not provide benefit over VDZ monotherapy in clinical or endoscopic remission but appeared to improve clinical response and was safe. These data should inform a fully powered study.

Transcriptional Behavior of Regulatory T Cells Predicts IBD Patient Responses to Vedolizumab Therapy.

BACKGROUND: Inflammatory bowel disease (IBD) involves chronic T cell-mediated inflammatory responses. Vedolizumab (VDZ), a monoclonal antibody against α4ß7 integrin, inhibits lymphocyte extravasation into intestinal mucosae and is effective in ulcerative colitis (UC) and Crohn's disease (CD). AIM: We sought to identify immune cell phenotypic and gene expression signatures that related to response to VDZ. METHODS: Peripheral blood (PBMC) and lamina propria mononuclear cells (LPMCs) were analyzed by flow cytometry and Cytofkit. Sorted CD4 + memory (Tmem) or regulatory T (Treg) cells from PBMC and LPMC were analyzed by RNA sequencing (RNA-seq). Clinical response (≥2-point drop in partial Mayo scores [UC] or Harvey-Bradshaw index [CD]) was assessed 14 to 22 weeks after VDZ initiation. Machine-learning models were used to infer combinatorial traits that predicted response to VDZ. RESULTS: Seventy-one patients were enrolled: 37 received VDZ and 21 patients remained on VDZ >2 years. Fourteen of 37 patients (38%; 8 UC, 6 CD) responded to VDZ. Immune cell phenotypes and CD4 + Tmem and Treg transcriptional behaviors were most divergent between the ileum and colon, irrespective of IBD subtype or inflammation status. Vedolizumab treatment had the greatest impact on Treg metabolic pathways, and response was associated with increased expression of genes involved in oxidative phosphorylation. The strongest clinical predictor of VDZ efficacy was concurrent use of thiopurines. Mucosal tissues offered the greatest number of response-predictive biomarkers, whereas PBMC Treg-expressed genes were the best predictors in combinatorial models of response. CONCLUSIONS: Mucosal and peripheral blood immune cell phenotypes and transcriptional profiles can inform VDZ efficacy and inform new opportunities for combination therapies.

Vedolizumab (VDZ) is effective in the treatment of IBD. Immunophenotyping and RNAseq of T cells were used to inform its mechanism of action. Changes in T regulatory cells in the periphery and mucosa have the greatest relationship to VDZ response.

Lamina Propria Phagocyte Profiling Reveals Targetable Signaling Pathways in Refractory Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Lamina propria phagocytes are key mediators of inflammatory bowel disease (IBD). We aimed to understand the transcriptomic and functional differences in these cells based on location, disease type, inflammation state, and medication use in patients with IBD. METHODS: Phagocytic immune cells in the lamina propria, as defined by the marker CD11b, were isolated from 54 unique patients (n = 111 gut mucosal biopsies). We performed flow cytometry for cell phenotyping (n = 30) and RNA sequencing with differential gene expression analysis (n = 58). We further cultured these cells in vitro and exposed them to janus kinase inhibitors to measure cytokine output (n = 27). Finally, we matched patient genomic data to our RNA sequencing data to perform candidate gene expression quantitative trait locus analysis (n = 34). RESULTS: We found distinct differences in gene expression between CD11b+ cells from the colon vs ileum, as well as in different inflammatory states and, to a lesser degree, IBD types (Crohn's disease or ulcerative colitis). These genes mapped to targetable immune pathways and metabolic and cancer pathways. We further explored the janus kinase-signal transducer and activator of transcription pathway, which was upregulated across many comparisons including in biopsies from anti-tumor necrosis factor refractory patients. We found that isolated CD11b+ cells treated with janus kinase inhibitors had decreased secretion of cytokines tumor necrosis factora and interleukin-8 (P ≤ .05). We also found 3 genetic variants acting as expression quantitative trait loci (P ≤ .1) within our CD11b+ data set. CONCLUSIONS: Lamina propria phagocytes from IBD mucosa provide pathogenetic clues on the nature of treatment refractoriness and inform new targets for therapy.

T cell vaccination therapy in an induced model of anti-RNP autoimmune glomerulonephritis.

To establish the relevance of targeting disease-associated T cells in anti-RNP-associated glomerulonephritis, mice developing nephritis following immunization with U1-70-kd small nuclear ribonucleoprotein (snRNP) were treated with a single dose of irradiated antigen-selected T cell vaccine. T cell receptor usage in nephritic kidneys revealed oligoclonal use of T Cell Receptor V Beta (TRBV) genes as previously found in spleens and lungs of immunized mice with pulmonary disease. The CDR3 regions from T cell isolates showed sequence homology to those in humans with anti-RNP autoimmunity. Following T cell vaccination, urinalysis returned to normal in 5/7 treated mice (71% response rate) whereas all mock-treated mice continued to have an active urinary sediment (Fisher's Exact p=0.02). An oligoclonal population of T cells homologous to those identified in humans with anti-RNP autoimmunity is implicated in disease pathogenesis, and T cell vaccination is associated with a high rate of clinical improvement in established nephritis.

Microbial Signatures and Innate Immune Gene Expression in Lamina Propria Phagocytes of Inflammatory Bowel Disease Patients.

BACKGROUND & AIMS: The interaction between intestinal microbiota and the immune system plays a vital role in inflammatory bowel disease (IBD). Although numerous deep-sequencing studies have suggested dysbiosis in IBD, identifying specific bacteria from the stool or mucosa that are responsible for disease susceptibility or severity has remained a challenge. Lamina propria phagocytes ideally are localized to interact with bacteria that are in close proximity to, or have invaded, the tissue. Thus, we examined the microbial populations associated with the lamina propria phagocytes in 20 Crohn's disease and 12 ulcerative colitis patients. Specifically, we aimed to address whether the phagocyte-associated microbiota differed from the mucosa-associated microbiota and whether this varied based on IBD type or the state of inflammation. METHODS: 16S ribosomal RNA gene sequencing and innate immune gene expression profiling was done on CD11b+ lamina propria phagocytes isolated from the biopsies obtained from IBD patients. RESULTS: Phagocyte-associated microbiota was enriched in bacterial species belonging to phylum Proteobacteria, whereas species belonging to phylum Bacteroidetes were enriched in the mucosal microbiota of IBD patients. Disease type was the most influential factor in driving differences in the microbiota of both the mucosa and the lamina propria phagocytes, irrespective of inflammation state o`r anatomic location. Crohn's disease and ulcerative colitis specimens showed similar patterns of increased inflammatory gene expression in phagocytes isolated from inflamed areas compared with those isolated from uninflamed regions. CONCLUSIONS: This pilot study shows the feasibility of using lamina propria phagocytes to characterize the microbiota in IBD patients. The approach used in this study can narrow the spectrum of potentially dysbiotic bacterial populations and clinically relevant gene expression signatures in IBD patients.

Expression of SARS-CoV-2 Entry Molecules ACE2 and TMPRSS2 in the Gut of Patients With IBD.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have intestinal inflammation and are treated with immune-modulating medications. In the face of the coronavirus disease-19 pandemic, we do not know whether patients with IBD will be more susceptible to infection or disease. We hypothesized that the viral entry molecules angiotensin I converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are expressed in the intestine. We further hypothesized that their expression could be affected by inflammation or medication usage. METHODS: We examined the expression of Ace2 and Tmprss2 by quantitative polymerase chain reacion in animal models of IBD. Publicly available data from organoids and mucosal biopsies from patients with IBD were examined for expression of ACE2 and TMPRSS2. We conducted RNA sequencing for CD11b-enriched cells and peripheral and lamina propria T-cells from well-annotated patient samples. RESULTS: ACE2 and TMPRSS2 were abundantly expressed in the ileum and colon and had high expression in intestinal epithelial cells. In animal models, inflammation led to downregulation of epithelial Ace2. Expression of ACE2 and TMPRSS2 was not increased in samples from patients with compared with those of control patients. In CD11b-enriched cells but not T-cells, the level of expression of ACE2 and TMPRSS2 in the mucosa was comparable to other functional mucosal genes and was not affected by inflammation. Anti-tumor necrosis factor drugs, vedolizumab, ustekinumab, and steroids were linked to significantly lower expression of ACE2 in CD11b-enriched cells. CONCLUSIONS: The viral entry molecules ACE2 and TMPRSS2 are expressed in the ileum and colon. Patients with IBD do not have higher expression during inflammation; medical therapy is associated with lower levels of ACE2. These data provide reassurance for patients with IBD.

Immune profiling of human tumors identifies CD73 as a combinatorial target in glioblastoma.

Immune checkpoint therapy with anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of many solid tumors. However, the clinical efficacy of immune checkpoint therapy is limited to a subset of patients with specific tumor types1,2. Multiple clinical trials with combinatorial immune checkpoint strategies are ongoing; however, the mechanistic rationale for tumor-specific targeting of immune checkpoints is elusive. To garner an insight into tumor-specific immunomodulatory targets, we analyzed 94 patients representing five different cancer types, including those that respond relatively well to immune checkpoint therapy and those that do not, such as glioblastoma multiforme, prostate cancer and colorectal cancer. Through mass cytometry and single-cell RNA sequencing, we identified a unique population of CD73hi macrophages in glioblastoma multiforme that persists after anti-PD-1 treatment. To test if targeting CD73 would be important for a successful combination strategy in glioblastoma multiforme, we performed reverse translational studies using CD73-/- mice. We found that the absence of CD73 improved survival in a murine model of glioblastoma multiforme treated with anti-CTLA-4 and anti-PD-1. Our data identified CD73 as a specific immunotherapeutic target to improve antitumor immune responses to immune checkpoint therapy in glioblastoma multiforme and demonstrate that comprehensive human and reverse translational studies can be used for rational design of combinatorial immune checkpoint strategies.

Fluid supplementation accelerates epithelial repair during chemical colitis.

The dextran sulfate sodium (DSS) model of colitis is a common animal model of inflammatory bowel disease that causes pain and distress. In this study, we aimed to determine whether fluid supplementation can be used as a welfare-based intervention to minimize animal suffering. C57Bl/6 females undergoing acute colitis by administration of 3% DSS in drinking water were supplemented with 1 mL intraperitoneal injections of NaCl and compared to non-supplemented control mice. Mouse behavior and locomotive activity were assessed on days 5-6 after DSS initiation by means of tail suspension, novel object recognition and open field activity tests. Mice were euthanized after either the acute (day 7) or the recovery phase (day 12) of colitis and inflammation, epithelial proliferation, and differentiation were assessed by means of histology, immunohistochemistry, quantitative PCR, and western blot. We found that fluid-supplemented mice had reduced signs of colitis with no alterations in behavior or locomotive activity. Furthermore, we observed an accelerated epithelial repair response after fluid hydration during the acute phase of colitis, characterized by increased crypt proliferation, activation of ERK1/2, and modulation of TGF-ß1 expression. Consistent with these findings, fluid-supplemented mice had increased numbers of goblet cells, upregulated expression of differentiation markers for absorptive enterocytes, and reduced inflammation during the recovery phase. Our results show that fluid hydration does not reduce stress in DSS-treated mice but alters colitis evolution by reducing clinical signs and accelerating epithelial repair. These results argue against the routine use of fluid supplementation in DSS-treated mice.

Intestinal Epithelial Cells Respond to Chronic Inflammation and Dysbiosis by Synthesizing H2O2.

The microbes in the gastrointestinal tract are separated from the host by a single layer of intestinal epithelial cells (IECs) that plays pivotal roles in maintaining homeostasis by absorbing nutrients and providing a physical and immunological barrier to potential pathogens. Preservation of homeostasis requires the crosstalk between the epithelium and the microbial environment. One epithelial-driven innate immune mechanism that participates in host-microbe communication involves the release of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), toward the lumen. Phagocytes produce high amounts of ROS which is critical for microbicidal functions; the functional contribution of epithelial ROS, however, has been hindered by the lack of methodologies to reliably quantify extracellular release of ROS. Here, we used a modified Amplex Red assay to investigate the inflammatory and microbial regulation of IEC-generated H2O2 and the potential role of Duox2, a NADPH oxidase that is an important source of H2O2. We found that colonoids respond to interferon-γ and flagellin by enhancing production of H2O2 in a Duox2-mediated fashion. To extend these findings, we analyzed ex vivo production of H2O2 by IECs after acute and chronic inflammation, as well as after exposure to dysbiotic microbiota. While acute inflammation did not induce a significant increase in epithelial-driven H2O2, chronic inflammation caused IECs to release higher levels of H2O2. Furthermore, colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased H2O2 production compared with healthy controls. Collectively, these data suggest that IECs are capable of H2O2 production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of H2O2 by IECs as a read-out of innate defense by the mucosa.

Metastatic Melanoma Patient Had a Complete Response with Clonal Expansion after Whole Brain Radiation and PD-1 Blockade.

We report here on a patient with metastatic melanoma who had extensive brain metastases. After being treated with the sequential combination of whole brain radiation therapy followed by the PD-1-inhibitory antibody, pembrolizumab, the patient had a durable complete response. Retrospective laboratory studies of T cells revealed that, after treatment with anti-PD-1 commenced, effector CD8+ T cells in the blood expanded and the ratio of CD8+:Treg T cells increased. A CD8+ T-cell clone present in the initial brain metastases was expanded in the blood after anti-PD-1 treatment, which suggested an antitumor role for this clone. Immunohistochemical analysis confirmed the presence of CD8+ T cells and low PD-L1 expression in the brain metastases before immunotherapy initiation. This sequence of therapy may provide an option for melanoma patients with unresponsive brain metastases. Cancer Immunol Res; 5(2); 100-5. ©2017 AACR.

Voltage-dependent calcium channels in young and old human red cells.

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of alpha1 subunit of high-voltage activated Ca channels (pan alpha1) and against conserved residues of alpha1C and alpha1E subunits. No specific reaction was detected with antibodies against conserved residues of alpha1A, alpha1B, or alpha1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan alpha1 or anti-alpha1E blots, whereas two bands (200 and 230 kDa) were detected by anti-alpha1C exposure. Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl + 5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15-50% reduction of 45Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Qand N-type blockers also reduced Ca influx to various extents (25-60%). Old cells were generally insensitive to L-type but not to non-L-type blockers. Raising external K to about 70-80 mM reduced by 50-100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.

Linneg Sca-1high CD49fhigh prostate cancer cells derived from the Hi-Myc mouse model are tumor-initiating cells with basal-epithelial characteristics and differentiation potential in vitro and in vivo.

A cell line was established from ventral prostate (VP) tumors of one-year-old Hi-Myc mice. These cells, called HMVP2 cells, are LinnegSca-1highCD49fhigh with high CD44 and CD29 expression and express CK14, Sca-1 and CD49f (but not CK8), suggesting basal-epithelial characteristics. Furthermore, HMVP2 cells form spheroids and both the cells and spheroids produce tumors in syngeneic mice. After four days of culture, HMVP2 spheroids underwent a gradual transition from LinnegSca-1highCD49fhigh expression to LinnegSca-1lowCD49flow while a subpopulation of the cells retained the original LinnegSca-1highCD49fhigh expression pattern. Additional cell subpopulations expressing Lin positive markers were also present suggesting further differentiation of HMVP2 spheroids. Two additional highly tumorigenic cell lines (HMVP2A1 and HMVP2A2) were isolated from HMVP2 cells after subsequent tumor formation in FVB/N mice. Concurrently, we also established cell lines from the VP of 6 months old Hi-Myc mice (named as HMVP1) and FVB/N mice (called NMVP) having less aggressive growth properties compared to the other three cell lines. AR expression was reduced in HMVP2 cells compared to NMVP and HMVP1 cells and almost absent in HMVP2A1 and HMVP2A2 cells. These cell lines will provide valuable tools for further mechanistic studies as well as preclinical studies to evaluate preventive and/or therapeutic agents for prostate cancer.