[68Ga]Ga-NODAGA-TriGalactan, a low molecular weight tracer for the non-invasive imaging of the functional liver reserve.

BACKGROUND: Determination of the functional liver mass is important in a variety of clinical settings including liver surgery and transplantation. [99mTc]Tc-diethylenetriamine-pentaacetic acid galactosyl human serum albumin (99mTc-GSA) is a radiotracer targeting the asialoglycoprotein receptor (ASGR) and is routinely used in Japan for this purpose. Here we describe the development and evaluation of [68Ga]Ga-NODAGA-TriGalactan a low molecular weight PET-tracer targeting this structure. RESULTS: For synthesis TRIS as branching unit and NODAGA as chelator for labelling with [68Ga]Ga are included. Three galactose moieties are conjugated via a click chemistry approach resulting in the desired labelling precursor.68Ga-labelling could be accomplished in high radiochemical yield and purity. [68Ga]Ga-NODAGA-TriGalactan is very hydrophilic and revealed high plasma stability and low plasma protein binding. Fluorescence imaging showed binding on ASGR-positive organoids and the IC50-value was in the nanomolar range. Most importantly, both biodistribution as well as animal imaging studies using normal mice demonstrated high liver uptake with rapid elimination from all other organs leading to even higher liver-to-background ratios as found for 99mTc-GSA. CONCLUSION: [68Ga]Ga-NODAGA-TriGalactan shows high in vitro stability and selectively binds to the ASGR allowing imaging of the functional liver mass with high contrast. Thus, our first generation compound resulted already in an alternative to 99mTc-GSA for imaging the functional liver reserve and might allow the broader use of this imaging technique.

The breathtaking world of human respiratory in vitro models: Investigating lung diseases and infections in 3D models, organoids, and lung-on-chip.

The COVID-19 pandemic illustrated an urgent need for sophisticated, human tissue models to rapidly test and develop effective treatment options against this newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Thus, in particular, the last 3 years faced an extensive boost in respiratory and pulmonary model development. Nowadays, 3D models, organoids and lung-on-chip, respiratory models in perfusion, or precision-cut lung slices are used to study complex research questions in human primary cells. These models provide physiologically relevant systems for studying SARS-CoV-2 and, of course, other respiratory pathogens, but they are, too, suited for studying lung pathologies, such as CF, chronic obstructive pulmonary disease, or asthma, in more detail in terms of viral infection. With these models, the cornerstone has been laid for further advancing the organs by, for example, inclusion of several immune cell types or humoral immune components, combination with other organs in microfluidic organ-on-chip devices, standardization and harmonization of the devices for reliable and reproducible drug and vaccine testing in high throughput.

Salivary antibodies induced by BA.4/BA.5-convalescence or bivalent booster Immunoglobulin vaccination protect against novel SARS-COV-2 variants of concern.

Currently, SARS-CoV-2 Omicron BA.5 subvariants BF.7 and BQ.1.1 are rapidly emerging worldwide. To evaluate the SARS-CoV-2-neutralizing capacity of sera and saliva from triple vaccinated individuals, either boosted with an adapted bivalent COVID-19 vaccine or recovered from BA.4/BA.5 infection, we analyzed the sensitivity of replication-competent SARS-CoV-2 Omicron subvariants BA.4/5, BQ.1.1 and BF.7 to neutralization. Analysis of SARS-CoV-2-specific IgGs and IgAs showed increased serum IgG titers in the vaccinated group, while the serum and salivary IgA levels were comparable. Similar and efficient serum neutralization against the ancestral strain of SARS-CoV-2 and Omicron BA.4/BA.5 was detected in both cohorts, but critically reduced for BQ.1.1 and BF.7. In contrast, salivary neutralization against BA.4/BA.5 was increased in the convalescent compared to the vaccinated group, while salivary neutralizing capacity against BQ.1.1 and BF.7 was comparable in these groups. Further, personalized protective effects studied in a human 3D respiratory model revealed the importance of salivary protection against different Omicron subvariants. IMPORTANCE In BA.4/BA.5-convalescent versus vaccinated groups, salivary neutralization capacity increased against SARS-CoV-2 Omicron BA.4/BA.5. In contrast, it neutralized novel Omicron subvariants BQ.1.1 and BF.7 similarly. Salivary protection against various Omicron subvariants was even more evident when tested in a personalized approach using highly differentiated respiratory human 3D models.

Vaccination and Omicron BA.1/BA.2 Convalescence Enhance Systemic but Not Mucosal Immunity against BA.4/5.

Rising breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.4/5 led to the performance of various studies investigating systemic immunity and neutralizing antibodies in sera, but mucosal immunity remains understudied. In this cohort study, the humoral immune responses, including immunoglobulin levels and the presence of virus-neutralizing antibodies, of 92 vaccinated and/or BA.1/BA.2 convalescent individuals were investigated. Cohorts received two doses of ChAdOx1, BNT162b2, or mRNA-1273 and subsequent booster vaccination with either BNT162b2 or mRNA-1273, following BA.1/BA.2 infection. In addition, vaccinated and nonconvalescent or unvaccinated and BA.1 convalescent individuals were studied. Serum and saliva samples were used to determine SARS-CoV-2 spike-specific IgG and IgA titers and neutralizing activity against replication-competent SARS-CoV-2 wild-type virus and the Omicron BA.4/5 variant. Vaccinated/convalescent cohorts demonstrated strongest neutralization against BA.4/5, with 50% neutralization titer (NT50) values reaching 174.2; however, neutralization was reduced up to 11-fold, compared to wild-type virus. Both BA.1 convalescent and vaccinated nonconvalescent cohorts displayed the weakest neutralization against BA.4/5, with NT50 values being reduced to 4.6, accompanied by lower numbers of positive neutralizers. Additionally, salivary neutralization against wild-type virus was strongest in vaccinated and BA.2 convalescent subjects, but this elevated neutralization efficiency was lost when challenged with BA.4/5. Our data support the contention that current coronavirus disease 2019 (COVID-19) vaccines efficiently induce humoral immunity. However, antiviral effectiveness in serum and saliva is greatly reduced against novel variants of concern. These results suggest an adjustment of current vaccine strategies to an adapted or alternative vaccine delivery, such as mucosal booster vaccinations, which might establish enhanced or even sterilizing immunity against novel SARS-CoV-2 variants. IMPORTANCE Rising incidences of breakthrough infections caused by SARS-CoV-2 Omicron BA.4/5 have been observed. Although various studies were conducted investigating neutralizing antibodies in sera, mucosal immunity was barely evaluated. Here, we investigated mucosal immunity, since the presence of neutralizing antibodies at mucosal entry sites plays a fundamental role in disease limitation. We found strong induction of serum IgG/IgA, salivary IgA, and neutralization against SARS-CoV-2 wild-type virus in vaccinated/convalescent subjects but detected 10-fold reduced (albeit positive) serum neutralization against BA.4/5. Interestingly, vaccinated and BA.2 convalescent patients demonstrated the greatest serum neutralization against BA.4/5, but this advantageous neutralizing effect was not observed in the saliva. Our data support the contention that current COVID-19 vaccines are very efficient against severe/critical disease progression. Moreover, these results suggest an adjustment of the current vaccine strategy to adapted and alternative vaccine delivery, such as mucosal booster vaccinations, to establish robust sterilizing immunity against novel SARS-CoV-2 variants.

GlyPerA™ effectively shields airway epithelia from SARS-CoV-2 infection and inflammatory events.

New SARS-CoV-2 variants of concern (VOCs) and waning immunity illustrate that quick and easy-to-use agents are needed to prevent infection. To protect from viral transmission and subsequent inflammatory reactions, we applied GlyperA™, a novel antimicrobial formulation that can be used as mouth gargling solution or as nasal spray, to highly differentiated human airway epithelia prior infection with Omicron VOCs BA.1 and BA.2. This formulation fully protected polarized human epithelium cultured in air-liquid interphase (ALI) from SARS-CoV-2-mediated tissue destruction and infection upon single application up to two days post infection. Moreover, inflammatory reactions induced by the Omicron VOCs were significantly lowered in tissue equivalents either pre-treated with the GlyperA™ solution, or even when added simultaneously. Thus, the GlyperA™ formulation significantly shielded epithelial integrity, successfully blocked infection with Omicron and release of viral particles, and decreased intracellular complement C3 activation within human airway epithelial cell cultures. Crucially, our in vitro data imply that GlyperA™ may be a simple tool to prevent from SARS-CoV-2 infection independent on the circulating variant via both, mouth and nose.

Antiviral drugs block replication of highly immune-evasive Omicron subvariants ex vivo, but fail to reduce tissue inflammation.

The identification of the SARS-CoV-2 Omicron variants BA.4/BA.5, BF.7 and BQ.1.1 immediately raised concerns regarding the efficacy of currently used monoclonal antibody therapies. Here we examined the activity of monoclonal antibody therapies and antiviral drugs against clinical specimens for SARS-CoV-2 Omicron BA.4/BA.5, BF.7 and BQ.1.1 employing an immunofluorescence neutralization assay. Further we explored treatment of BA.4/BA.5 infections with efficient antiviral drugs and monoclonal antibodies in a 3D model of primary human bronchial epithelial cells. We found that the antiviral drugs Molnupiravir, Nirmatrelvir and Remdesivir efficiently inhibit BA.4/BA.5, BF.7 and BQ.1.1 replication. In contrast, only the monoclonal antibody Cilgavimab exerted an inhibitory effect, while Tixagevimab, Regdanvimab and Sotrovimab lost their efficacy against BA.4/BA.5. We found that only the prophylactic treatment with Cilgavimab impacted on tissue inflammation by reducing intracellular complement component 3 (C3) activation following BA.4/BA.5 infection in primary human airway epithelial grown in air-liquid-interphase, which was not the case when using antiviral drugs or Cilgavimab after establishment of infection. Of note, all tested monoclonal antibodies had no neutralizing activity during infection by BF.7 and BQ.1.1 variants. Our results suggest that despite a marked reduction of viral replication, potent antiviral drugs fail to reduce tissue levels of inflammatory compounds such as C3, which can still result in tissue destruction.

Antifibrotic Drug Nintedanib Inhibits CSF1R to Promote IL-4-associated Tissue Repair Macrophages.

Profibrotic and prohomeostatic macrophage phenotypes remain ill-defined, both in vivo and in vitro, impeding the successful development of drugs that reprogram macrophages as an attractive therapeutic approach to manage fibrotic disease. The goal of this study was to reveal profibrotic and prohomeostatic macrophage phenotypes that could guide the design of new therapeutic approaches targeting macrophages to treat fibrotic disease. This study used nintedanib, a broad kinase inhibitor approved for idiopathic pulmonary fibrosis, to dissect lung macrophage phenotypes during fibrosis-linked inflammation by combining in vivo and in vitro bulk and single-cell RNA-sequencing approaches. In the bleomycin model, nintedanib drove the expression of IL-4/IL-13-associated genes important for tissue regeneration and repair at early and late time points in lung macrophages. These findings were replicated in vitro in mouse primary bone marrow-derived macrophages exposed to IL-4/IL-13 and nintedanib. In addition, nintedanib promoted the expression of IL-4/IL-13 pathway genes in human macrophages in vitro. The molecular mechanism was connected to inhibition of the colony stimulating factor 1 (CSF1) receptor in both human and mouse macrophages. Moreover, nintedanib counterbalanced the effects of TNF on IL-4/IL-13 in macrophages to promote expression of IL-4/IL-13-regulated tissue repair genes in fibrotic contexts in vivo and in vitro. This study demonstrates that one of nintedanib's antifibrotic mechanisms is to increase IL-4 signaling in macrophages through inhibition of the CSF1 receptor, resulting in the promotion of tissue repair phenotypes.

Serum Neutralization Against SARS-CoV-2 Variants Is Heterogenic and Depends on Vaccination Regimen.

Omicron variants are still the dominant SARS-CoV-2 viruses worldwide, therefore determination of the level of protection from infection and severe disease is essential. Here, we investigated humoral and cellular immunity of individuals immunized by ChAdOx1, BNT162b2, and mRNA-1273 and our results show that IgG and neutralization titers wane over time. However, strongest neutralization against Omicron BA.1 and T-cell responses were detected in ChAdOx1 vaccinees 6 months after the second dose, while no long-lasting neutralization was shown against BA.2 in any cohort. Crucially, our investigation revealed that immunity against variants of concern is heterogenic and dependent on the immunization status.

ColdZyme® protects airway epithelia from infection with BA.4/5.

Vaccines against SARS-CoV-2 protect from critical or severe pathogenesis also against new variants of concern (VOCs) such as BA.4 and BA.5, but immediate interventions to avoid viral transmission and subsequent inflammatory reactions are needed. Here we applied the ColdZyme® medical device mouth spray to fully differentiated, polarized human epithelium cultured at an air-liquid interphase (ALI). We found using VOCs BA.1 and BA.4/5 that this device effectively blocked respiratory tissue infection. While infection with these VOCs resulted in intracellular complement activation, thus enhanced inflammation, and drop of transepithelial resistance, these phenomena were prevented by a single administration of this medical device. Thus, ColdZyme® mouth spray significantly shields epithelial integrity, hinders virus infection and blocks in a secondary effect intrinsic complement activation within airway cultures also in terms of the highly contagious VOCs BA.4/5. Crucially, our in vitro data suggest that ColdZyme® mouth spray may have an impact to protect against SARS-CoV-2 transmission, also in case of the Omicron BA.1, BA.4 and BA.5 variants.

Cilgavimab/Tixagevimab as alternative therapeutic approach for BA.2 infections.

Objectives: The identification of the SARS-CoV-2 Omicron variants BA.1 and BA.2 immediately raised concerns about the efficacy of currently used monoclonal antibody therapies. Here, we analyzed the activity of Sotrovimab and Regdanvimab, which are used in clinics for treatment of moderate to severe SARS-CoV-2 infections, and Cilgavimab/Tixagevimab, which are approved for prophylactic use, against BA.1 and BA.2 in a 3D model of primary human bronchial epithelial cells. Methods: Primary human airway epithelia (HAE) cells in a 3D tissue model were infected with clinical isolates of SARS-CoV-2 Delta, BA.1 or BA.2. To mimic the therapeutic use of mAbs, we added Regdanvimab, Sotrovimab or Cilgavimab/Tixagevimab 6 h after infection. In order to mirror the prophylactic use of Cilgavimab/Tixagevimab, we added this compound 6 h prior to infection to the fully differentiated, pseudostratified epithelia cultured in air-liquid interphase (ALI). Results: We observed that Sotrovimab, but not Regdanvimab, is active against BA.1; however, both antibodies lose their efficacy against BA.2. In contrast, we found that BA.2 was sensitive to neutralization by the approved prophylactic administration and the therapeutic use, which is not yet permitted, of Cilgavimab/Tixagevimab. Conclusion: Importantly, while the use of Tixagevimab/Cilgavimab is effective in controlling BA.2 but not BA.1 infection, monoclonal antibodies (mAbs) with efficacy against BA.1 are ineffective to reduce BA.2 virus replication in a human lung model. Our data may have implications on the variant specific clinical use of monoclonal antibodies.

Immune Responses Against SARS-CoV-2 WT and Delta Variant in Elderly BNT162b2 Vaccinees.

Background: Residents of nursing homes are one of the most vulnerable groups during the severe acute syndrome coronavirus 2 (SARS-CoV-2) pandemic. The aim of this study was to characterize cellular and humoral immune responses in >70-year-old participants before vaccination, after first and second vaccination with BNT162b2, in contrast to second-dose-vaccinated participants younger than 60 years. Methods: Peripheral blood mononuclear cells of 45 elderly and 40 younger vaccinees were analyzed by IFNγ ELISpot, specific immunoglobulin G antibody titers against SARS-CoV-2 spike protein, and neutralization abilities against SARS-CoV-2 wild-type (WT) and Delta variant (B.1.617.2). Results: Our results clearly demonstrate a significantly increased T cell response, IgG titers, and neutralization activities against SARS-CoV-2 WT and Delta between first and second vaccination with BNT162b2 in elderly vaccinees, thereby highlighting the importance of the second booster. Interestingly, similar cellular and humoral immune responses against SARS-CoV-2 WT and Delta were found after the second vaccine dose in the young and elderly groups. Conclusions: Our data demonstrate a full picture of cellular and humoral immune responses of BNT162b2-vaccinees in two age cohorts. In all vaccines, SARS-CoV-2 WT-specific antibodies with similar neutralizing activity were detected in all vaccinees. After the second vaccination, neutralization titers against SARS-CoV-2 Delta were impaired in both age groups compared with SARS-CoV-2 WT, thereby emphasizing the need for an additional booster to overcome rising variants of SARS-CoV-2.

A common framework of monocyte-derived macrophage activation.

Macrophages populate every organ during homeostasis and disease, displaying features of tissue imprinting and heterogeneous activation. The disconnected picture of macrophage biology that has emerged from these observations is a barrier for integration across models or with in vitro macrophage activation paradigms. We set out to contextualize macrophage heterogeneity across mouse tissues and inflammatory conditions, specifically aiming to define a common framework of macrophage activation. We built a predictive model with which we mapped the activation of macrophages across 12 tissues and 25 biological conditions, finding a notable commonality and finite number of transcriptional profiles, in particular among infiltrating macrophages, which we modeled as defined stages along four conserved activation paths. These activation paths include a "phagocytic" regulatory path, an "inflammatory" cytokine-producing path, an "oxidative stress" antimicrobial path, or a "remodeling" extracellular matrix deposition path. We verified this model with adoptive cell transfer experiments and identified transient RELMɑ expression as a feature of monocyte-derived macrophage tissue engraftment. We propose that this integrative approach of macrophage classification allows the establishment of a common predictive framework of monocyte-derived macrophage activation in inflammation and homeostasis.

Gene-selective transcription promotes the inhibition of tissue reparative macrophages by TNF.

Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn's Disease. Previously, we and others found that TNF blocks the emergence and function of alternative-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balance of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages, we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis, and signaling pathway deconvolution. We found that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way, dependent on JNK signaling via the type 1 TNF receptor on specific populations of alternative-activated macrophages. We further determined that JNK signaling has a profound and broad effect on activated macrophage gene expression. Our findings suggest that TNF's anti-M2 effects evolved to specifically modulate components of tissue and reparative M2 macrophages and TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor.

Nifedipine Potentiates Susceptibility of Salmonella Typhimurium to Different Classes of Antibiotics.

The calcium channel blocker nifedipine induces cellular iron export, thereby limiting the availability of the essential nutrient iron for intracellular pathogens, resulting in bacteriostatic activity. To study if nifedipine may exert a synergistic anti-microbial activity when combined with antibiotics, we used the mouse macrophage cell line RAW267.4, infected with the intracellular bacterium Salmonella Typhimurium, and exposed the cells to varying concentrations of nifedipine and/or ampicillin, azithromycin and ceftriaxone. We observed a significant additive effect of nifedipine in combination with various antibiotics, which was not observed when using Salmonella, with defects in iron uptake. Of interest, increasing intracellular iron levels increased the bacterial resistance to treatment with antibiotics or nifedipine or their combination. We further showed that nifedipine increases the expression of the siderophore-binding peptide lipocalin-2 and promotes iron storage within ferritin, where the metal is less accessible for bacteria. Our data provide evidence for an additive effect of nifedipine with conventional antibiotics against Salmonella, which is partly linked to reduced bacterial access to iron.

Cytokine-Mediated Regulation of ARG1 in Macrophages and Its Impact on the Control of Salmonella enterica Serovar Typhimurium Infection.

Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre+/-ARG1fl/fl and NRAMPG169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre+/-ARG1fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.

Ferritin H deficiency deteriorates cellular iron handling and worsens Salmonella typhimurium infection by triggering hyperinflammation.

Iron is an essential nutrient for mammals as well as for pathogens. Inflammation-driven changes in systemic and cellular iron homeostasis are central for host-mediated antimicrobial strategies. Here, we studied the role of the iron storage protein ferritin H (FTH) for the control of infections with the intracellular pathogen Salmonella enterica serovar Typhimurium by macrophages. Mice lacking FTH in the myeloid lineage (LysM-Cre+/+Fthfl/fl mice) displayed impaired iron storage capacities in the tissue leukocyte compartment, increased levels of labile iron in macrophages, and an accelerated macrophage-mediated iron turnover. While under steady-state conditions, LysM-Cre+/+Fth+/+ and LysM-Cre+/+Fthfl/fl animals showed comparable susceptibility to Salmonella infection, i.v. iron supplementation drastically shortened survival of LysM-Cre+/+Fthfl/fl mice. Mechanistically, these animals displayed increased bacterial burden, which contributed to uncontrolled triggering of NF-κB and inflammasome signaling and development of cytokine storm and death. Importantly, pharmacologic inhibition of the inflammasome and IL-1ß pathways reduced cytokine levels and mortality and partly restored infection control in iron-treated ferritin-deficient mice. These findings uncover incompletely characterized roles of ferritin and cellular iron turnover in myeloid cells in controlling bacterial spread and for modulating NF-κB and inflammasome-mediated cytokine activation, which may be of vital importance in iron-overloaded individuals suffering from severe infections and sepsis.

Lactate and IL6 define separable paths of inflammatory metabolic adaptation.

Lactate is an end point of Warburg-type metabolism found in inflammatory macrophages. Recently, lactate was shown to modify histones of lipopolysaccharide (LPS)-activated macrophages in a time-dependent way and promote the expression of genes linked to tissue repair, including arginase-1 (Arg1). We tested the interrelationships between histone lactylation (Kla) and tissue reparative gene expression and found that Kla was uncoupled from changes in gene expression linked to resolving M2 macrophage activation but correlated with Arg1 expression. LPS-induced Arg1 was instead dependent on autocrine-paracrine interleukin-6 (IL6) production, the IL6 receptor, and Stat3 signal transduction. We found that Kla increases as macrophages prepare to die under inflammatory stress, and Kla was absent in macrophages that cannot generate reactive nitrogen or have defects in diverse macrophage death pathways. Thus, Kla is a consequence rather than a cause of macrophage activation but occurs coincidently with an IL6- and Arg1-dependent metabolic rewiring under inflammatory duress.

Cutting Edge: TNF Is Essential for Mycobacteria-Induced MINCLE Expression, Macrophage Activation, and Th17 Adjuvanticity.

TNF blockade is a successful treatment for human autoimmune disorders like rheumatoid arthritis and inflammatory bowel disease yet increases susceptibility to tuberculosis and other infections. The C-type lectin receptors (CLR) MINCLE, MCL, and DECTIN-2 are expressed on myeloid cells and sense mycobacterial cell wall glycolipids. In this study, we show that TNF is sufficient to upregulate MINCLE, MCL, and DECTIN-2 in macrophages. TNF signaling through TNFR1 p55 was required for upregulation of these CLR and for cytokine secretion in macrophages stimulated with the MINCLE ligand trehalose-6,6-dibehenate or infected with Mycobacterium bovis bacillus Calmette-Guérin. The Th17 response to immunization with the MINCLE-dependent adjuvant trehalose-6,6-dibehenate was specifically abrogated in TNF-deficient mice and strongly attenuated by TNF blockade with etanercept. Together, interference with production or signaling of TNF antagonized the expression of DECTIN-2 family CLR, thwarting vaccine responses and possibly increasing infection risk.

A fully human anti-BMP6 antibody reduces the need for erythropoietin in rodent models of the anemia of chronic disease.

Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation, including anemia of chronic kidney disease. A black box warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in 2 different, well-established, and clinically relevant rodent models of ACD. In addition to counteracting hepcidin-driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared with either monotherapy in a qualitative and quantitative manner. Consequently, the combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic-free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.