Case Report: Arterial Wall Inflammation in Atherosclerotic Cardiovascular Disease is Reduced by Olamkicept (sgp130Fc).

Inflammation is a strong driver of atherosclerotic cardiovascular disease (ASCVD). There is a large unmet need for therapies that prevent or reduce excessive inflammation while avoiding systemic immunosuppression. We showed previously that selective inhibition of pro-inflammatory interleukin-6 (IL-6) trans-signalling by the fusion protein olamkicept (sgp130Fc) prevented and reduced experimental murine atherosclerosis in low-density lipoprotein receptor-deficient (Ldlr -/-) mice on a high-fat, high-cholesterol diet independently of low-density lipoprotein (LDL) cholesterol metabolism. Therefore, we allowed compassionate use of olamkicept (600 mg intravenously biweekly for 10 weeks) in a patient with very-high-risk ASCVD. Despite optimal LDL cholesterol under maximum tolerated lipid-lowering treatment, the patient had a remaining very high risk for future cardiovascular events related to significant arterial wall inflammation with lipoprotein (a) [Lp(a)]-cholesterol as the main contributor. 18Fluorodeoxyglucose positron emission tomography/computed tomography (18FDG PET/CT) measurements were performed before and after the treatment period. Olamkicept reduced arterial wall inflammation in this patient without interfering with lipoprotein metabolism. No clinical or laboratory side effects were observed during or after treatment with olamkicept. Our findings in this patient matched the results from our mechanistic study in Ldlr -/- mice, which were extended by additional analyses on vascular inflammation. Olamkicept may be a promising option for treating ASCVD independently of LDL cholesterol metabolism. A Phase II trial of olamkicept in ASCVD is currently being prepared.

Selective inhibition of IL-6 trans-signaling by a miniaturized, optimized chimeric soluble gp130 inhibits TH17 cell expansion.

The cytokine interleukin-6 (IL-6) signals through three mechanisms called classic signaling, trans-signaling, and trans-presentation. IL-6 trans-signaling is distinctly mediated through a soluble form of its transmembrane receptor IL-6R (sIL-6R) and the coreceptor gp130 and is implicated in multiple autoimmune diseases. Although a soluble form of gp130 (sgp130) inhibits only IL-6 trans-signaling, it also blocks an analogous trans-signaling mechanism of IL-11 and its soluble receptor sIL-11R. Here, we report miniaturized chimeric soluble gp130 variants that efficiently trap IL-6:sIL-6R but not IL-11:sIL-11R complexes. We designed a novel IL-6 trans-signaling trap by fusing a miniaturized sgp130 variant to an IL-6:sIL-6R complex-binding nanobody and the Fc portion of immunoglobulin G (IgG). This trap, called cs-130Fc, exhibited improved inhibition of as well as increased selectivity for IL-6 trans-signaling compared to the conventional fusion protein sgp130Fc. We introduced affinity-enhancing mutations in cs-130Fc and sgp130Fc that further improved selectivity toward IL-6 trans-signaling. Moreover, cs-130Fc efficiently inhibited the expansion of T helper 17 (TH17) cells in cultures of mouse CD4+ T cells treated with IL-6:sIL-6R. Thus, these variants may provide or lead to the development of more precisely targeted therapeutics for inflammatory disorders associated with IL-6 trans-signaling.

Therapeutic Interleukin-6 Trans-signaling Inhibition by Olamkicept (sgp130Fc) in Patients With Active Inflammatory Bowel Disease.

BACKGROUND & AIMS: A large unmet therapeutic need exists in inflammatory bowel disease (IBD). Inhibition of interleukin (IL)-6 appears to be effective, but the therapeutic benefit of a complete IL6/IL6 receptor (IL6R) blockade is limited by profound immunosuppression. Evidence has emerged that chronic proinflammatory activity of IL6 is mainly mediated by trans-signaling via a complex of IL6 bound to soluble IL6R engaging the gp130 co-receptor without the need for membrane-bound IL6R. We have developed a decoy protein, sgp130Fc, that exclusively blocks IL6 proinflammatory trans-signaling and has shown efficacy in preclinical models of IBD, without signs of immunosuppression. METHODS: We present a 12-week, open-label, prospective phase 2a trial (FUTURE) in 16 patients with active IBD treated with the trans-signaling inhibitor olamkicept (sgp130Fc) to assess the molecular mechanisms, safety, and effectiveness of IL6 trans-signaling blockade in vivo. We performed in-depth molecular profiling at various timepoints before and after therapy induction to identify the mechanism of action of olamkicept. RESULTS: Olamkicept was well tolerated and induced clinical response in 44% and clinical remission in 19% of patients. Clinical effectiveness coincided with target inhibition (reduction of phosphorylated STAT3) and marked transcriptional changes in the inflamed mucosa. An olamkicept-specific transcriptional signature, distinguishable from remission signatures of anti-tumor necrosis factor (infliximab) or anti-integrin (vedolizumab) therapies was identified. CONCLUSIONS: Our data suggest that blockade of IL6 trans-signaling holds great promise for the therapy of IBD and should undergo full clinical development as a new immunoregulatory therapy for IBD. (EudraCT no., Nu 2016-000205-36).

Tocilizumab does not block interleukin-6 (IL-6) signaling in murine cells.

Tocilizumab is a humanized monoclonal antibody that is approved for the treatment of different human inflammatory diseases, including rheumatoid arthritis and cytokine release syndrome. Tocilizumab binds to the interleukin-6 receptor (IL-6R) and thereby blocks signaling of the pro-inflammatory cytokine IL-6. Initial studies and all authority assessment reports state that tocilizumab is effective in humans, but cannot bind to the murine or rat IL-6R and thus not block IL-6 signaling in the mouse. However, several recent studies described the use of tocilizumab in mice and reported biological effects that were attributed to IL-6 blockade. In this study, we investigate the capability of tocilizumab to block IL-6 signaling using different human and murine cell lines. Our results unequivocally confirm the original state of the art that tocilizumab blocks signaling via the human IL-6R, but does not block IL-6 signaling in murine cells.

Metabolic Functions of Gut Microbes Associate With Efficacy of Tumor Necrosis Factor Antagonists in Patients With Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Altered interactions between the mucosal immune system and intestinal microbiota contribute to pathogenesis of inflammatory bowel diseases (IBD). It is not clear how inhibitors of cytokines, such as antagonists of tumor necrosis factor (anti-TNF), affect the intestinal microbiome. We investigated the effects of anti-TNF agents on gut microbe community structure and function in a longitudinal 2-step study of patients with IBD. We correlated our findings with outcomes of treatment and investigated patterns of metabolites in fecal samples before and after anti-TNF therapy. METHODS: We performed a prospective study of 2 cohorts of patients in Germany; the discovery cohort comprised 12 patients with IBD, 17 patients with rheumatic disease, and 19 healthy individuals (controls); fecal samples were collected at baseline and 2, 6, and 30 weeks after induction of anti-TNF therapy. The validation cohort comprised 23 patients with IBD treated with anti-TNF or vedolizumab (anti-α4ß7 integrin) and 99 healthy controls; fecal samples were collected at baseline and at weeks 2, 6, and 14. Fecal microbiota were analyzed by V3-V4 16S ribosomal RNA gene amplicon sequencing. Clinical response and remission were determined by clinical disease activity scores. Metabolic network reconstruction and associated fecal metabolite level inference was performed in silico using the AGORA (Assembly of Gut Organisms through Reconstruction and Analysis) resource. Metabolomic analyses of fecal samples from a subset of patients were performed to validate metabolites associated with treatment outcomes. RESULTS: Anti-TNF therapy shifted the diversity of fecal microbiota in patients with IBD, but not with rheumatic disease, toward that of controls. Across timepoints, diversity indices did not vary significantly between patients with IBD who did or did not achieve clinical remission after therapy. In contrast, in silico modeling of metabolic interactions between gut microbes found metabolite exchange to be significantly reduced at baseline in fecal samples from patients with IBD and to be associated with later clinical remission. Predicted levels of butyrate and substrates involved in butyrate synthesis (ethanol or acetaldehyde) were significantly associated with clinical remission following anti-TNF therapy, verified by fecal metabolomic analyses. CONCLUSIONS: Metabolic network reconstruction and assessment of metabolic profiles of fecal samples might be used to identify patients with IBD likely to achieve clinical remission following anti-TNF therapy and increase our understanding of the heterogeneity of IBD.

The IL-6-neutralizing sIL-6R-sgp130 buffer system is disturbed in patients with type 2 diabetes.

Serum levels of interleukin-6 (IL-6) are increased in patients with type 2 diabetes (T2D). IL-6 exerts its pleiotropic effects via the IL-6 α-receptor (IL-6R), which exists in membrane-bound and soluble (sIL-6R) forms and activates cells via the ß-receptor glycoprotein 130 (gp130). The nonsynonymous single-nucleotide polymorphism (SNP) rs2228145 (Asp358Ala) within the IL6R locus is associated with T2D. The aim of this study was to determine whether sIL-6R in combination with soluble gp130 (sgp130) is able to form an IL-6-neutralizing buffer in healthy subjects and whether this is disturbed in T2D. We found that sIL-6R-sgp130 indeed forms an IL-6-neutralizing buffer in the serum of healthy humans, whose capacity is controlled by the SNP of the IL-6R. Circulating sIL-6R-sgp130 levels were lower in T2D subjects (P < 0.001), whereas IL-6 was high and inversely correlated with sIL-6R (r = -0.57, P < 0.001), indicating a severe disturbance of the buffer. This phenomenon is also observed in sex- and age-matched patients with both T2D and atherosclerosis but not in patients with atherosclerosis alone. In conclusion, sIL-6R and sgp130 serum levels were significantly lower in T2D patients compared with healthy subjects or atherosclerosis patients, although IL-6 levels were high. These data suggest that disturbance of the protective buffer may be closely associated with T2D pathophysiology.

Adjustment of triple shellac coating for precise release of bioactive substances with different physico-chemical properties in the ileocolonic region.

Formulations for the controlled release of substances in the human terminal ileum and colon are essential to target the gut microbiome and its interactions with the intestinal mucosa. In contrast to pharmaceutical enteric coatings, reliable food-grade alternatives are still scarce. Shellac coatings have been used for various active ingredients, but their stability is affected by the physicochemical properties of the encapsulated substances. It is well known, that shellac release can be modulated by an acidic subcoating. Here, we hypothesized that a triple shellac coating with an adjusted intermediate coating (acidic or alkaline) can be effectively used to counteract the differences in pH value of various encapsulated substances, allowing a precise targeting of the desired release pH value. First, the system was tested with riboflavin 5'-monophosphate sodium salt dihydrate (RMSD) as a characteristic model substance. Secondly, it was transferred to nicotinic acid (NA) and nicotinamide (NAM) as bioactive compounds with different physio-chemical properties: NAM, an alkaline crystalline and highly water-soluble substance, led to a premature release from conventional shellac microcapsules, whereas RMSD and NA with their medium solubility and neutral to acidic pH properties delayed the shellac dissolution. A precise modulation of the release profile of each substance was possible by the addition of different intermediate subcoatings: an acidic layer with citric acid counteracted the premature release of the alkaline and highly soluble NAM. In contrast, an alkaline sodium bicarbonate intermediate subcoating enhanced shellac swelling and delayed the release of NA and RMSD. In conclusion, the novel triple-layer shellac coating provides a much higher adaptability and reliability for nutritional formulations aiming at a targeted release in the ileocolonic region.

Mutations in Craniosynostosis Patients Cause Defective Interleukin-11 Receptor Maturation and Drive Craniosynostosis-like Disease in Mice.

Premature closure of the sutures that connect the cranial bones during development of the mammalian skull results in a phenotype called craniosynostosis. Recently, several craniosynostosis patients with missense mutations within the gene encoding the interleukin-11 receptor (IL-11R) have been described, but the underlying molecular mechanisms have remained elusive. IL-11 is a cytokine that has a crucial role in bone remodeling and activates cells via binding to the IL-11R. Here, we show that patient mutations prevented maturation of the IL-11R, resulting in endoplasmic reticulum retention and diminished cell surface appearance. Disruption of a conserved tryptophan-arginine zipper within the third domain of the IL-11R was the underlying cause of the defective maturation. IL-11 classic signaling via the membrane-bound receptor, but not IL-11 trans-signaling via the soluble receptor, was the crucial pathway for normal skull development in mice in vivo. Thus, the specific therapeutic inhibition of IL-11 trans-signaling does not interfere with skull development.

Soluble gp130 prevents interleukin-6 and interleukin-11 cluster signaling but not intracellular autocrine responses.

Interleukin-6 (IL-6) is a proinflammatory cytokine of the IL-6 family, members of which signal through a complex of a cytokine-specific receptor and the signal-transducing subunit gp130. The interaction of IL-6 with the membrane-bound IL-6 receptor (IL-6R) and gp130 stimulates "classic signaling," whereas the binding of IL-6 and a soluble version of the IL-6R to gp130 stimulates "trans-signaling." Alternatively, "cluster signaling" occurs when membrane-bound IL-6:IL-6R complexes on transmitter cells activate gp130 receptors on neighboring receiver cells. The soluble form of gp130 (sgp130) is a selective trans-signaling inhibitor, but it does not affect classic signaling. We demonstrated that the interaction of soluble gp130 with natural and synthetic membrane-bound IL-6:IL-6R complexes inhibited IL-6 cluster signaling. Similarly, IL-11 cluster signaling through the IL-11R to gp130 was also inhibited by soluble gp130. However, autocrine classic and trans-signaling was not inhibited by extracellular inhibitors such as sgp130 or gp130 antibodies. Together, our results suggest that autocrine IL-6 signaling may occur intracellularly.

Evaluation of interleukin-6 and its soluble receptor components sIL-6R and sgp130 as markers of inflammation in inflammatory bowel diseases.

PURPOSE: Interleukin-6 (IL-6) production and signalling are increased in the inflamed mucosa in inflammatory bowel diseases (IBD). As published serum levels of IL-6 and its soluble receptors sIL-6R and sgp130 in IBD are from small cohorts and partly contradictory, we systematically evaluated IL-6, sIL-6R and sgp130 levels as markers of disease activity in Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Consecutive adult outpatients with confirmed CD or UC were included, and their disease activity and medication were monitored. Serum from 212 CD patients (815 measurements) and 166 UC patients (514 measurements) was analysed, and 100 age-matched healthy blood donors were used as controls. RESULTS: IL-6 serum levels were significantly elevated in active versus inactive CD and UC, also compared with healthy controls. However, only a fraction of IBD patients showed increased serum IL-6. IL-6 levels ranged up to 32.7 ng/mL in active CD (> 5000-fold higher than in controls), but also up to 6.9 ng/mL in inactive CD. Increases in active UC (up to 195 pg/mL) and inactive UC (up to 27 pg/mL) were less pronounced. Associations between IL-6 serum levels and C-reactive protein concentrations as well as leukocyte and thrombocyte counts were observed. Median sIL-6R and sgp130 levels were only increased by up to 15%, which was considered of no diagnostic significance. CONCLUSIONS: Only a minority of IBD patients shows elevated IL-6 serum levels. However, in these patients, IL-6 is strongly associated with disease activity. Its soluble receptors sIL-6R and sgp130 do not appear useful as biomarkers in IBD.

Pharmacological inhibition of IL-6 trans-signaling improves compromised fracture healing after severe trauma.

Patients with multiple injuries frequently suffer bone fractures and are at high risk to develop fracture healing complications. Because of its key role both in systemic posttraumatic inflammation and fracture healing, the pleiotropic cytokine interleukin-6 (IL-6) may be involved in the pathomechanisms of trauma-induced compromised fracture healing. IL-6 signals are transmitted by two different mechanisms: classic signaling via the membrane-bound receptor (mIL-6R) and trans-signaling via its soluble form (sIL-6R). Herein, we investigated whether IL-6 classic and trans-signaling play different roles in bone regeneration after severe injury. Twelve-week-old C57BL/6J mice underwent combined femur osteotomy and thoracic trauma. To study the function of IL-6, either an anti-IL-6 antibody, which inhibits both IL-6 classic and trans-signaling, or a soluble glycoprotein 130 fusion protein (sgp130Fc), which selectively blocks trans-signaling, were injected 30 min and 48 h after surgery. Bone healing was assessed using cytokine analyses, flow cytometry, histology, micro-computed tomography, and biomechanical testing. Selective inhibition of IL-6 trans-signaling significantly improved the fracture healing outcome after combined injury, as confirmed by accelerated cartilage-to-bone transformation, enhanced bony bridging of the fracture gap and improved mechanical callus properties. In contrast, global IL-6 inhibition did not affect compromised fracture healing. These data suggest that classic signaling may mediate beneficial effects on bone repair after severe injury. Selective inhibition of IL-6 trans-signaling might have therapeutic potential to treat fracture healing complications in patients with concomitant injuries.

The balance of interleukin (IL)-6, IL-6·soluble IL-6 receptor (sIL-6R), and IL-6·sIL-6R·sgp130 complexes allows simultaneous classic and trans-signaling.

Interleukin (IL-)6 is the major pro-inflammatory cytokine within the IL-6 family. IL-6 signals via glycoprotein 130 (gp130) and the membrane-bound or soluble IL-6 receptor (IL-6R), referred to as classic or trans-signaling, respectively. Whereas inflammation triggers IL-6 expression, eventually rising to nanogram/ml serum levels, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130) are constitutively present in the upper nanogram/ml range. Calculations based on intermolecular affinities have suggested that systemic IL-6 is immediately trapped in IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes, indicating that sIL-6R and sgp130 constitute a buffer system that increases the serum half-life of IL-6 or restricts systemic IL-6 signaling. However, this scenario has not been experimentally validated. Here, we quantified IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes over a wide concentration range. The amounts of IL-6 used in this study reflect concentrations found during active inflammatory events. Our results indicated that most IL-6 is free and not complexed with sIL-6R or sgp130, indicating that the level of endogenous sgp130 in the bloodstream is not sufficient to block IL-6 trans-signaling via sIL-6R. Importantly, addition of the single-domain antibody VHH6, which specifically stabilizes IL-6·sIL-6R complexes but did not bind to IL-6 or sIL-6R alone, drove free IL-6 into IL-6·sIL-6R complexes and boosted trans-signaling but not classic signaling, demonstrating that endogenous sIL-6R has at least the potential to form complexes with IL-6. Our findings indicate that even though high concentrations of sIL-6R and sgp130 are present in human serum, the relative ratio of free IL-6 to IL-6·sIL-6R allows for simultaneous classic and trans-signaling.

Targeted Microbiome Intervention by Microencapsulated Delayed-Release Niacin Beneficially Affects Insulin Sensitivity in Humans.

OBJECTIVE: Gut microbiota represent a potential novel target for future prediabetes and type 2 diabetes therapies. In that respect, niacin has been shown to beneficially affect the host-microbiome interaction in rodent models. RESEARCH DESIGN AND METHODS: We characterized more than 500 human subjects with different metabolic phenotypes regarding their niacin (nicotinic acid [NA] and nicotinamide [NAM]) status and their gut microbiome. In addition, NA and NAM delayed-release microcapsules were engineered and examined in vitro and in vivo in two human intervention studies (bioavailability study and proof-of-concept/safety study). RESULTS: We found a reduced α-diversity and Bacteroidetes abundance in the microbiome of obese human subjects associated with a low dietary niacin intake. We therefore developed delayed-release microcapsules targeting the ileocolonic region to deliver increasing amounts of NA and NAM to the microbiome while preventing systemic resorption to avoid negative side effects (e.g., facial flushing). In vitro studies on these delayed-release microcapsules revealed stable conditions at pH 1.4, 4.5, and 6.8, followed by release of the compounds at pH 7.4, simulating the ileocolonic region. In humans in vivo, gut-targeted delayed-release NA but not NAM produced a significant increase in the abundance of Bacteroidetes. In the absence of systemic side effects, these favorable microbiome changes induced by microencapsulated delayed-release NA were associated with an improvement of biomarkers for systemic insulin sensitivity and metabolic inflammation. CONCLUSION: Targeted microbiome intervention by delayed-release NA might represent a future therapeutic option for prediabetes and type 2 diabetes.

Distinct Effects of IL-6 Classic and Trans-Signaling in Bone Fracture Healing.

Bone healing is a complex process with closely linked phases of inflammation, regeneration, and remodeling. IL-6 may crucially regulate this process; however, the underlying mechanisms are unclear. IL-6 signals are transmitted via the transmembrane glycoprotein 130 by two distinct mechanisms: classic signaling using the membrane-anchored IL-6 receptor and trans-signaling using its soluble form. Herein, we investigated the hypothesis that IL-6 classic and trans-signaling have different functions during bone healing. To investigate fracture healing, 12-week-old C57BL/6J mice underwent a femur osteotomy. To study the function of IL-6 during the inflammatory phase, either an anti-IL-6 antibody, which inhibits IL-6 classic and trans-signaling, or soluble glycoprotein 130 fusion protein, which selectively blocks trans-signaling, was injected after 30 minutes and 48 hours. To analyze IL-6 effects in the repair phase, compounds were injected from day 7 onwards. Global IL-6 inhibition in the early phase after fracture reduced systemic inflammation, the recruitment of immune cells, and bone regeneration, resulting in delayed fracture healing. Global IL-6 inhibition during the repair phase disturbed bone formation and remodeling. In contrast, inhibition of IL-6 trans-signaling exerted minor effects on the immune response and did not influence bone repair, suggesting that the classic pathway accounts for most of the effects observed after global IL-6 inhibition. Our results reveal that IL-6 classic signaling, but not IL-6 trans-signaling, is essential for bone repair.

Increased Tryptophan Metabolism Is Associated With Activity of Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Administration of tryptophan and some of its metabolites reduces the severity of colitis in mice, whereas removing tryptophan from the diet increases susceptibility to colitis. Transfer of the intestinal microbiome transfers the colitogenic phenotype from tryptophan starved animals to normally nourished mice. We aimed to systematically evaluate serum levels of tryptophan and its metabolites in patients with inflammatory bowel diseases (IBD), and study their association with clinical and serologic features. METHODS: We studied 535 consecutive patients with IBD (211 with ulcerative colitis [UC], 234 with Crohn's disease [CD]; 236 male), enrolled in Germany from August 2013 through April 2014 and followed until July 2016. Serum samples were collected from patients and 291 matched individuals without IBD (controls); levels of tryptophan were measured using high-performance liquid chromatography. Metabolites of tryptophan were measured in serum from 148 patients and 100 controls by mass spectrometry. We measured levels of interleukin 22 in serum from 28 patients by enzyme-linked immunosorbent assay. Paired stool and serum samples were collected from a subset of patients with active UC (n = 10) or CD (n = 8) to investigate associations between serum levels of tryptophan and composition of the fecal microbiota, analyzed by 16S ribosomal DNA amplicon sequencing. We used real-time polymerase chain reaction to measure levels of messenger RNAs in colonic biopsies from 60 patients with UC, 50 with CD, and 30 controls. We collected information on patients' disease activity scores, medications, laboratory assessments, and clinical examinations during recruitment and follow-up visits. RESULTS: Serum levels of tryptophan were significantly lower in patients with IBD than in controls (P = 5.3 × 10-6) with a stronger reduction in patients with CD (vs control; P = 1.1 × 10-10) than UC (vs control; P = 2.8 × 10-3). We found a negative correlation between serum levels of tryptophan and disease activity or levels of C-reactive protein. Levels of messenger RNAs encoding tryptophan 2,3-dioxygenase-2 and solute carrier family 6 member 19 (also called B0AT1) were significantly decreased in colonic biopsies from patients with IBD compared with controls, whereas level of messenger RNA encoding indoleamine 2,3-dioxygenase-1 was significantly increased. The composition of the fecal microbiota associated with serum levels of tryptophan. Analysis of tryptophan metabolites revealed activation of the kynurenine pathway, based on high levels of quinolinic acid, in patients with IBD compared with controls. Serum concentration of interleukin 22 associated with disease activity in patients with IBD; there was an inverse association between levels of interleukin 22 and serum levels of tryptophan. CONCLUSIONS: In an analysis of serum samples from more than 500 patients with IBD, we observed a negative correlation between serum levels of tryptophan and disease activity. Increased levels of tryptophan metabolites-especially of quinolinic acid-indicated a high activity of tryptophan degradation in patients with active IBD. Tryptophan deficiency could contribute to development of IBD or aggravate disease activity. Interventional clinical studies are needed to determine whether modification of intestinal tryptophan pathways affects the severity of IBD.

Efficacy of Sterile Fecal Filtrate Transfer for Treating Patients With Clostridium difficile Infection.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (CDI). However, transferring undefined living bacteria entails uncontrollable risks for infectious and metabolic or malignant diseases, particularly in immunocompromised patients. We investigated whether sterile fecal filtrates (containing bacterial debris, proteins, antimicrobial compounds, metabolic products, and oligonucleotides/DNA), rather than intact microorganisms, are effective in patients with CDI. METHODS: We performed a clinical case series to investigate the effects of fecal filtrate transfer (FFT) in 5 patients with symptomatic chronic-relapsing CDI at the Department of Internal Medicine I at the University Hospital Schleswig-Holstein (Kiel, Germany). Patients were followed up for at least 6 months and for up to 33 months. Stool was collected from 5 donors selected by the patients, and fully characterized according to FMT standards. Stool was sterile-filtered to remove small particles and bacteria; the filtrate was transferred to patients in a single administration via nasojejunal tube. Fecal samples were collected from patients before and at 1 week and 6 weeks after FFT. Microbiome, virome, and proteome profiles of donors and patients were compared. RESULTS: In all 5 patients, FFT restored normal stool habits and eliminated symptoms of CDI for a minimum period of 6 months. Proteome analyses of selected FFT filtrates showed no obvious protein candidates associated with therapeutic efficacy. 16S ribosomal RNA gene sequencing detected diverse bacterial DNA signatures in the filtrates. Analysis of virus-like particles from a filtrate found to reduce symptoms of CDI showed a complex signature of bacteriophages. Bacterial phylogeny and virome profile analyses of fecal samples from recipients indicated longitudinal changes in microbial and viral community structures after FFT. CONCLUSIONS: A preliminary investigation of 5 patients with CDI shows that transfer of sterile filtrates from donor stool (FFT), rather than fecal microbiota, can be sufficient to restore normal stool habits and eliminate symptoms. This finding indicates that bacterial components, metabolites, or bacteriophages mediate many of the effects of FMT, and that FFT might be an alternative approach, particularly for immunocompromised patients.

Different Soluble Forms of the Interleukin-6 Family Signal Transducer gp130 Fine-tune the Blockade of Interleukin-6 Trans-signaling.

Soluble forms of the IL-6 receptor (sIL-6R) bind to the cytokine IL-6 with similar affinity as the membrane-bound IL-6R. IL-6·sIL-6R complexes initiate IL-6 trans-signaling via activation of the ubiquitously expressed membrane-bound ß-receptor glycoprotein 130 (gp130). Inhibition of IL-6 trans-signaling has been shown to be favorable in numerous inflammatory diseases. Furthermore, different soluble forms of gp130 (sgp130) exist that, together with the sIL-6R, are thought to form a buffer for IL-6 in the blood. However, a functional role for the different sgp130 forms has not been described to date. Here we demonstrate that the metalloproteases ADAM10 and ADAM17 can produce sgp130 by ectodomain shedding of gp130, even though this mechanism only accounts for a minor proportion of sgp130 in the circulation. We further show that full-length sgp130 and the shorter forms sgp130-rheumatoid arthritis-associated peptide (RAPS) and sgp130-E10 are differentially expressed in a cell type- specific manner. Remarkably, full-length sgp130 is expressed by monocytes, but this expression is completely lost during differentiation into macrophages in vitro Using genetically engineered murine pre-B cells that secrete different forms of sgp130, we found that these secreted sgp130 proteins are able to prevent trans-signaling-driven cell proliferation of the secreting cells, whereas conditioned supernatant from these cells failed to block IL-6 trans-signaling in other cells. Thus, our data suggest that the different sgp130 forms are released from cells into their immediate surroundings and appear to form cell-associated gradients to modulate their own susceptibility for IL-6 trans-signaling.

Proteolytic Cleavage Governs Interleukin-11 Trans-signaling.

Interleukin (IL)-11 has been shown to be a crucial factor for intestinal tumorigenesis, lung carcinomas, and asthma. IL-11 is thought to exclusively mediate its biological functions through cell-type-specific expression of the membrane-bound IL-11 receptor (IL-11R). Here, we show that the metalloprotease ADAM10, but not ADAM17, can release the IL-11R ectodomain. Chimeric proteins of the IL-11R and the IL-6 receptor (IL-6R) revealed that a small juxtamembrane portion is responsible for this substrate specificity of ADAM17. Furthermore, we show that the serine proteases neutrophil elastase and proteinase 3 can also cleave the IL-11R. The resulting soluble IL-11R (sIL-11R) is biologically active and binds IL-11 to activate cells. This IL-11 trans-signaling pathway can be inhibited specifically by the anti-inflammatory therapeutic compound sgp130Fc. In conclusion, proteolysis of the IL-11R represents a molecular switch that controls the IL-11 trans-signaling pathway and widens the number of cells that can be activated by IL-11.