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Targeting the α4β7-MAdCAM-1 axis with vedolizumab (VDZ) is a front-line therapeutic paradigm in ulcerative colitis (UC). However, mechanism(s) of action (MOA) of VDZ remain relatively undefined. Here, we examined three distinct cohorts of patients with UC (n=83, n=60, and n=21), to determine the effect of VDZ on the mucosal and peripheral immune system. Transcriptomic studies with protein level validation were used to study drug MOA using conventional and transgenic murine models. We found a significant decrease in colonic and ileal naïve B and T cells and circulating gut-homing plasmablasts (β7+) in VDZ-treated patients, pointing to gut-associated lymphoid tissue (GALT) targeting by VDZ. Murine Peyer’s patches (PP) demonstrated a significant loss cellularity associated with reduction in follicular B cells, including a unique population of epithelium-associated B cells, following anti-α4β7 antibody (mAb) administration. Photoconvertible (KikGR) mice unequivocally demonstrated impaired cellular entry into PPs in anti-α4β7 mAb treated mice. In VDZ-treated, but not anti-tumor necrosis factor-treated UC patients, lymphoid aggregate size was significantly reduced in treatment responders compared to non-responders, with an independent validation cohort further confirming these data. GALT targeting represents a novel MOA of α4β7-targeted therapies, with major implications for this therapeutic paradigm in UC, and for the development of new therapeutic strategies.

Two resident macrophage subsets reside in peritoneal fluid. Macrophages also reside within mesothelial membranes lining the peritoneal cavity, but they remain poorly characterized. Here, we identified two macrophage populations (LYVE1hi MHC IIlo-hi CX3CR1gfplo/− and LYVE1lo/− MHC IIhi CX3CR1gfphi subsets) in the mesenteric and parietal mesothelial linings of the peritoneum. These macrophages resembled LYVE1+ macrophages within surface membranes of numerous organs. Fate-mapping approaches and analysis of newborn mice showed that LYVE1hi macrophages predominantly originated from embryonic-derived progenitors and were controlled by CSF1 made by Wt1+ stromal cells. Their gene expression profile closely overlapped with ovarian tumor-associated macrophages previously described in the omentum. Indeed, syngeneic epithelial ovarian tumor growth was strongly reduced following in vivo ablation of LYVE1hi macrophages, including in mice that received omentectomy to dissociate the role from omental macrophages. These data reveal that the peritoneal compartment contains at least four resident macrophage populations and that LYVE1hi mesothelial macrophages drive tumor growth independently of the omentum.

Clotting factors made by resident peritoneal macrophages promote microbial trapping in fluid microenvironments where microbes might otherwise escape., Macrophages resident in different organs express distinct genes, but understanding how this diversity fits into tissue-specific features is limited. Here, we show that selective expression of coagulation factor V (FV) by resident peritoneal macrophages in mice promotes bacterial clearance in the peritoneal cavity and serves to facilitate the well-known but poorly understood “macrophage disappearance reaction.” Intravital imaging revealed that resident macrophages were nonadherent in peritoneal fluid during homeostasis. Bacterial entry into the peritoneum acutely induced macrophage adherence and associated bacterial phagocytosis. However, optimal control of bacterial expansion in the peritoneum also required expression of FV by the macrophages to form local clots that effectively brought macrophages and bacteria in proximity and out of the fluid phase. Thus, acute cellular adhesion and resident macrophage–induced coagulation operate independently and cooperatively to meet the challenges of a unique, open tissue environment. These events collectively account for the macrophage disappearance reaction in the peritoneal cavity.

This cohort study investigates the association of sex hormones with disease severity and inflammatory cytokines in patients with COVID-19., Key Points Question Are circulating sex hormones associated with disease severity in patients with COVID-19? Findings In a cohort study of 152 patients with COVID-19, including 143 patients who were hospitalized, testosterone concentrations at presentation and on day 3 were inversely associated with disease severity and circulating inflammatory cytokine concentrations in men but not in women. Transcriptional profiling of circulating mononuclear cells revealed upregulation of hormone signaling pathways in patients requiring intensive care vs those with milder disease. Meaning These findings suggest that low testosterone concentrations may play a mechanistic role in worse outcomes observed in men with COVID-19, underscoring the need for clinical trials to test this hypothesis., Importance Male sex is a risk factor for developing severe COVID-19 illness. It is not known whether sex hormones contribute to this predisposition. Objective To investigate the association of concentrations of serum testosterone, estradiol, and insulinlike growth factor 1 (IGF-1, concentrations of which are regulated by sex hormone signaling) with COVID-19 severity. Design, Setting, and Participants This prospective cohort study was conducted using serum samples collected from consecutive patients who presented from March through May 2020 to the Barnes Jewish Hospital in St Louis, Missouri, with COVID-19 (diagnosed using nasopharyngeal swabs). Exposures Testosterone, estradiol, and IGF-1 concentrations were measured at the time of presentation (ie, day 0) and at days 3, 7, 14, and 28 after admission (if the patient remained hospitalized). Main Outcomes and Measures Baseline hormone concentrations were compared among patients who had severe COVID-19 vs those with milder COVID-19 illness. RNA sequencing was performed on circulating mononuclear cells to understand the mechanistic association of altered circulating hormone concentrations with cellular signaling pathways. Results Among 152 patients (90 [59.2%] men; 62 [40.8%] women; mean [SD] age, 63 [16] years), 143 patients (94.1%) were hospitalized. Among 66 men with severe COVID-19, median [interquartile range] testosterone concentrations were lower at day 0 (53 [18 to 114] ng/dL vs 151 [95 to 217] ng/dL; P = .01) and day 3 (19 [6 to 68] ng/dL vs 111 [49 to 274] ng/dL; P = .006) compared with 24 men with milder disease. Testosterone concentrations were inversely associated with concentrations of interleukin 6 (β = −0.43; 95% CI, −0.52 to −0.17; P

Tertiary lymphoid organs often arise during chronic inflammation, including in inflammatory bowel diseases like ileitis. The impact of TLOs on disease progression or immunity and how they integrate with or influence the surrounding tissue microenvironment remain unclear. In human Crohn's disease, we earlier observed that TLOs were positioned along mesenteric collecting lymphatic vessels (CLVs) that govern lymph outflow from the intestine. Here we show in the TNFΔARE mouse model of Crohn’s-like ileitis that TLOs form at valve regions of CLVs, supporting lymphangiogenic outgrowths. These TLOs compress CLVs, preventing forward lymph flow and immune cell egress to draining lymph nodes, while fostering lymph leak and backflow. Lymph transport through TLOs was restored if TNF neutralization was begun early. However, robustly developed TLOs resisted regression and restoration of flow. Thus, TLOs can profoundly remodel communication between tissues and their draining lymph nodes by strongly suppressing dissemination of cells and molecules.

Summary Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn’s disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn’s disease.

Pioneer reports of Crohn’s disease (CD) suggested that impaired lymphatic flow might drive its pathogenesis but remains unsettled. Nodules of tertiary lymphoid organs (TLO) are found in association with collecting lymphatic vessels (CLVs) of the mesentery that normally conducts lymph outflow from the intestine. Whether TLOs affect lymph transport is unknown. In the TNFΔARE mouse model of Crohn’s-like ileitis, TLOs are found in valves regions. Using lymphatic reporters and photoconversion to study cell trafficking from the intestine, our findings indicated that TLOs halts immune cells traveling from the inflamed ileum to the lymph node, effectively trapping DCs, B, and T cells, and impacting the development of microbe tolerogenic regulatory T cells. Lymphatic transport defects were intrinsic to the CLVs because the soluble fluorescent tracer’s passage through TLO was also blocked. Lymph blockage promoted retrograde lymphatic flow returning towards the gut wall due to incapable valves. Moreover, significant lymph leakage was found, specifically at the TLOs. Neutralizing anti-TNF mAb treatment into TNFΔARE mice is ineffective in eliminating TLOs or restoring lymphatic trafficking when administered in female mice with advanced disease. In males, the therapy was able to restore forward flow to the lymph node. However, even in the presence of TNF inhibition, both sexes demonstrated TLO lymph leakage. Thus, mesenteric TLOs that form during chronic ileitis drive broadly impaired lymph transit of molecules and cells from the intestine that is only partially reversible by neutralizing the cytokine cascade underlying the disease and establish a perennial tissue alteration.

The concept that impaired lymphatic flow during the progression of Crohn’s disease, a major form of inflammatory bowel disease, emerged decades ago, but remains unresolved. In Crohn’s disease, tertiary lymphoid organs (TLOs) are associated with collecting lymphatic vessels (CLVs) of the mesentery that govern lymph outflow from the intestine. Whether TLOs affect lymph transport is unknown. In the TNFDARE mouse model of Crohn’s-like ileitis, TLOs formed at valve regions which supported lymphangiogenic outgrowths to the TLOs. Photoconversion approaches to study cell trafficking indicated that TLOs received immune cells traveling from the intestine but scarcely allowed their egress, effectively trapping DCs, B, and T cells. Using adoptive transfer of microbiota-dependent TCR we found reduced overall Tregs differentiation and their presence shifted from the lymph nodes to TLOs, altering intestinal-induced responses. Strikingly, although anti-TNF therapy dampens gut inflammation, TLO are refractory the therapy. Moreover, passage of soluble fluorescent tracer through TLO-rich CLVs was also impeded, indicating transport defects were intrinsic to the CLVs. Indeed, lymph flow diverted at TLO to neighboring CLV branches, where dysfunctional valves allowed lymph return to the gut wall, accompanied by lymph leakage at the TLOs. Culturing lymphatic endothelial cells with TNF demonstrate that the cytokine alone can reduce key genes found in lymphatic valves. These data indicate that TNF disrupts lymphatic valve maintenance pathways, promoting formation of mesenteric TLOs that broadly impair lymph transit of molecules and cells from the intestine, impairing immune surveillance of the mucosal barrier, altering homeostasis.

Background Short gut syndrome (SGS) following massive small bowel resection (SBR) is a major cause of pediatric mortality and morbidity secondary to nutritional deficiencies and the sequelae of chronic total parenteral nutrition use, including liver steatosis. Despite the importance of lymphatic vasculature in fat absorption, lymphatic response after SBR has not been studied. We hypothesize that lymphatic vessel integrity is compromised in SGS, potentially contributing to the development of impaired lipid transport leading to liver steatosis and metabolic disease. Methods Mice underwent 50% proximal SBR or sham operations. Imaging of lymphatic vasculature in the lamina propria and mesentery was compared between sham and SBR Prox1 ERCre-Rosa26LSLTdTomato mice. mRNA expression levels of lymphangiogenic markers were performed in C57BL/6J mice. Results Lymphatic vasculature was significantly altered after SBR. Mesenteric lymphatic collecting vessels developed new branching structures and lacked normal valves at branch points, while total mucosal lymphatic capillary area in the distal ileum decreased compared to both sham and intraoperative controls. Intestinal Vegfr3 expression also increased significantly in resected mice. Conclusions Intestinal lymphatics, in both the lamina propria and mesentery, dramatically remodel following SBR. This remodeling may affect lymphatic flow and function, potentially contributing to morbidities and nutritional deficiencies associated with SGS.

Background and aims: AVX-470 is an orally administered, bovine-derived, anti-tumour necrosis factor (TNF) antibody with local activity in the gastrointestinal tract. In the first-in-human clinical trial of AVX-470 in active ulcerative colitis, we evaluated inflammatory biomarkers in colon tissue as measures of disease activity and early response to treatment. Methods: Thirty-six patients received active drug (AVX-470 at 0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Colon biopsy samples were collected from 5 regions of colon at baseline and week 4. Tissue inflammatory biomarkers were evaluated by immunohistochemistry and quantitative reverse transcription–polymerase chain reaction (qRT-PCR), epithelial cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and bovine immunoglobulin by immunohistochemistry and mass spectrometry. Endoscopic activity (Ulcerative Colitis Endoscopic Index of Severity [UCEIS]) at colonoscopy was assessed in each colonic region by a central reader. Results: Bovine immunoglobulin was observed in mucosal tissue before and after dosing in lamina propria and submucosal layers of biopsy tissue. Baseline levels of TNF, myeloperoxidase (MPO), CD68 and interleukin (IL)-1β and, to a lesser extent, IL-6 mRNA were 2- to 3-fold higher in distal vs proximal colon tissue, corresponding to the 2- to 3-fold differences in baseline severities of endoscopic scores. Reductions of >10-fold in TNF and, to lesser extents, in MPO and epithelial cell apoptosis were observed in proximal and distal colon biopsies after 4 weeks of AVX-470 3.5g/day treatment. Reductions in TNF scores were correlated with changes in MPO and CD3 immunohistochemistry scores. Conclusions: These results are consistent with anti-TNF activity of orally administered AVX-470 in colon mucosal tissue in ulcerative colitis patients and demonstrate the utility of tissue biomarkers in assessing disease and treatment response in early clinical studies. Clinical Trial Registration Number: This trial was registered with Clinicaltrials.gov as study [NCT01759056][1] and with EudraCT as study 2012-004859-27. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01759056&atom=%2Feccojc%2Fearly%2F2016%2F02%2F22%2Fecco-jcc.jjw026.atom

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, which is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF). We developed and characterized AVX-470, a novel polyclonal antibody specific for human TNF. We evaluated the oral activity of AVX-470m, a surrogate antibody specific for murine TNF, in several well-accepted mouse models of IBD.AVX-470 and AVX-470m were isolated from the colostrum of dairy cows that had been immunized with TNF. The potency, specificity, and affinity of both AVX-470 and AVX-470m were evaluated in vitro and compared with infliximab. AVX-470m was orally administered to mice either before or after induction of colitis, and activity was measured by endoscopy, histopathology, immunohistochemistry, and quantitative measurement of messenger RNA levels. Colitis was induced using either 2,4,6-trinitrobenzene sulfonate or dextran sodium sulfate.AVX-470 and AVX-470m were shown to be functionally comparable in vitro. Moreover, the specificity, neutralizing potency, and affinity of AVX-470 were comparable with infliximab. Orally administered AVX-470m effectively reduced disease severity in several mouse models of IBD. Activity was comparable with that of oral prednisolone or parenteral etanercept. The antibody penetrated the colonic mucosa and inhibited TNF-driven mucosal inflammation with minimal systemic exposure.AVX-470 is a novel polyclonal anti-TNF antibody with an in vitro activity profile comparable to that of infliximab. Oral administration of a surrogate antibody specific for mouse TNF is effective in treating mouse models of IBD, delivering the anti-TNF to the site of inflammation with minimal systemic exposure.

Background and aims: AVX-470 is an oral, polyclonal bovine-derived anti-tumour necrosis factor (TNF) antibody in development for treatment of inflammatory bowel disease (IBD). AVX-470 neutralizes TNF locally in the gastrointestinal tract, minimizing systemic exposure. This was a double-blind, placebo-controlled, first-in-human trial designed to assess the safety, pharmacokinetics, immunogenicity and preliminary efficacy of 4 weeks of AVX-470 in patients with active ulcerative colitis (UC). Methods: Thirty-seven patients with active UC were randomized and 36 received AVX-470 (0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Endoscopic activity was assessed by colonoscopy pre- and post-treatment. The primary endpoint was safety. Secondary endpoints included pharmacokinetics and immunogenicity. Clinical and endoscopic response and remission were assessed as exploratory endpoints. Results: Thirty-three (92%) patients completed treatment and follow-up. The incidence of adverse events was similar across treatment groups and no allergic reactions or opportunistic infections were reported. AVX-470 therapy did not induce human anti-bovine antibodies (HABA). Bovine immunoglobulin (Ig) with TNF binding capacity was detected in stool, while bovine Ig levels in serum were low. Across all AVX-470 doses, 25.9% of patients achieved clinical response compared with 11.1% on placebo, with greatest improvements in the 3.5g/day group associated with proximal colon endoscopic improvement and reductions in serum CRP and IL-6. Conclusions: AVX-470 was safe and well tolerated in this first-in-human trial in UC, with efficacy trends for clinical, endoscopic and biomarker endpoints in the highest dose group (3.5g/day). Results suggest benefit of an orally delivered locally active agent in moderate to severe UC. Clinical Trial Registration Number: This trial was registered with Clinicaltrials.gov as study [NCT01759056][1] and with EudraCT as study 2012-004859-27. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01759056&atom=%2Feccojc%2Fearly%2F2016%2F03%2F17%2Fecco-jcc.jjw036.atom