Abstract BACKGROUND AND AIMS Renal fibrosis is a central feature of chronic kidney disease (CKD), and the severity of CKD correlates with the magnitude of renal fibrosis. Despite the causative role attributed to fibrosis in CKD progression, there is still no treatment available that directly targets renal fibrosis. Upon kidney injury, embryonic signalling pathways are activated, promoting the repair and regeneration of injured tissue. Maladaptive repair mechanisms have been associated with the development of renal fibrosis and sustained activation of Wnt/β-catenin signalling contributes to CKD progression. GPR124 (ADGRA2 gene), has been identified as a potential fibrotic mediator through its upregulation in pericytes during pericyte to myofibroblast transition. GPR124 has been identified to function as a co-receptor for Wnt7 mediating canonical Wnt signaling in endothelial cells. We observed elevated GPR124 expression in mouse kidneys after chronic ischaemia reperfusion injury (cIRI). To further explore the role of GPR124 in CKD progression, we have studied its correlation with disease severity in human NURTuRE kidney biopsies [1]. We have also looked at GPR124 therapeutic potential in mouse cIRI. Given its potential disease relevance, we set out to develop inhibitory GPR124 antibodies as novel therapeutics to treat renal fibrosis. METHOD Diagnostic FFPE kidney cortex biopsy samples from 497 NURTuRE patients were subjected to bulk RNA-Seq. Library preparation with Illumina Enrichment Tagmentation technology was followed by sequencing and alignment of paired-end reads to the reference genome GRCh38/ENSEMBL 97 and quantification. Monoclonal Anti-GPR124 antibodies were generated against the extracellular domain of human GPR124. Top candidates were selected by FACS binding and further characterized in a TCF/LEF luciferase assay for the inhibition of Wnt7 signaling. One hu/ms cross-reactive candidate was studied in a 14-day IRI mouse model to assess its anti-fibrotic effect in vivo. Mice were exposed to 30-min unilateral renal ischaemia, followed by 14 days recovery. Kidneys were studied for gene expression by RT-PCR. Animals were divided into sham (n = 5), control IRI (no antibody, n = 12) and treated groups (each n = 12). The antibody was administered before ischemia and during the recovery phase in two doses [50 mg/kg and 150 mg/kg]. RESULTS CONCLUSION NURTuRE provides us with a unique opportunity to assess relevance of a potential target candidate gene in kidney disease patients. The potential of GPR124 as a therapeutic target was highlighted by the upregulation of expression levels along disease severity and biological pathway analysis, where our data supports a potential role in extracellular matrix reorganization. The administration of GPR124 antibodies and inhibition of the GPR124-mediated Wnt signaling in vivo inhibited the development of ischaemia-induced fibrosis in the mouse kidney. The results strongly support a role for GPR124 in human kidney disease progression, through inhibition of fibrosis, and its potential as a future clinical target.

BACKGROUND & AIMS Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver macrophages. However, also hepatocytes, the parenchymal cells of the liver, possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct anti-viral mechanisms employed by hepatocytes. METHODS Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-κB signaling (IKKβΔHep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-α/β signaling-(IFNARΔHep), or interferon-α/β signaling in myeloid cells-(IFNARΔMyel) were infected. RESULTS Here, we demonstrate that LCMV activates NF-κB signaling in hepatocytes. LCMV-triggered NF-κB activation in hepatocytes did not depend on Kupffer cells or TNFR1- but rather on TLR-signaling. LCMV-infected IKKβΔHep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T-cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKKβ, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IKKβΔHep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IFNARΔHep, whereas IFNARΔMyel mice were able to control LCMV-infection. Hepatocytic NF-κB signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-α/β-mediated inhibition of HBV replication in vitro. CONCLUSIONS Together, these data show that hepatocyte-intrinsic NF-κB is a vital amplifier of interferon-α/β signaling pivotal for early, strong ISG responses, influx of immune cells and hepatic viral clearance.

Interferon γ (IFN-γ) was shown to be a macrophage activating factor already in 1984. Consistently, inborn errors of IFN-γ immunity underlie Mendelian Susceptibility to Mycobacterial Disease (MSMD). MSMD is characterized by genetic predisposition to disease caused by weakly virulent mycobacterial species. Paradoxically, macrophages from patients with MSMD were little tested. Here, we report a disease modeling platform for studying IFN-γ related pathologies using macrophages derived from patient specific induced pluripotent stem cells (iPSCs). We used iPSCs from patients with autosomal recessive complete- and partial IFN-γR2 deficiency, partial IFN-γR1 deficiency and complete STAT1 deficiency. Macrophages from all patient iPSCs showed normal morphology and IFN-γ-independent functionality like phagocytic uptake of bioparticles and internalization of cytokines. For the IFN-γ-dependent functionalities, we observed that the deficiencies played out at various stages of the IFN-γ pathway, with the complete IFN-γR2 and complete STAT1 deficient cells showing the most severe phenotypes, in terms of upregulation of surface markers and induction of downstream targets. Although iPSC-derived macrophages with partial IFN-γR1 and IFN-γR2 deficiency still showed residual induction of downstream targets, they did not reduce the mycobacterial growth when challenged with Bacillus Calmette–Guérin. Taken together, we report a disease modeling platform to study the role of macrophages in patients with inborn errors of IFN-γ immunity.

There are still knowledge gaps regarding the landscaping of value-based health care (VBHC). The aim was to review the Swiss VBHC market and assess knowledge and concerns around VBHC in the healthcare system. First, a secondary desk research (SDR) was performed to review the VBHC market, to map key stakeholders guiding VBHC in Switzerland and to develop key intelligence questions for the interviews. The SDR was performed on Google, Yahoo, Google Scholar and PUBMED. Second, eight in-depth interviews were conducted among persons in decision-making positions representing the academic, digital health, payer, industry and consultant sector. They were interviewed according to a structured questionnaire, which was pre-tested in three pilot interviews. The SDR revealed that in Switzerland the collection of standardized outcome measurements are limited to few institutions. Few initiatives are targeting hospitals to improve value for patients or assessing costs. As yet, no recognized outcome-based payment mechanism is available. According to the interviewees, the healthcare system is well funded and efficient, but VBHC could help to improve the system and reduce waste. In spite of this, VBHC is being explored in some areas in Switzerland but it is in its early stages. Main hurdles include low impetus to change, non-standardized patient pathway, no political commitment and inability to use AI in demonstrating value. In contrast, drivers for VBHC could include a highly effective health system, willingness of authorities to new concepts and trained professionals. The present work indicated that knowledge around VBHC and implementation of VBHC programs in the Swiss healthcare system is developing. In order to progress, behavioural change (quantity to quality) is key for the transition to VBHC. Additionally, evidence from real-world studies, pilot programs around VBHC components (such as patient related-outcome measures and costs) at hospitals and the usage of new technology to manage data are needed.

IFN-γ is an enigmatic cytokine that shows direct anti-viral effects, confers upregulation of MHC-II and other components relevant for antigen presentation, and that adjusts the composition and balance of complex cytokine responses. It is produced during immune responses by innate as well as adaptive immune cells and can critically affect the course and outcome of infectious diseases, autoimmunity, and cancer. To selectively analyze the function of innate immune cell-derived IFN-γ, we generated conditional IFN-γOFF mice, in which endogenous IFN-γ expression is disrupted by a loxP flanked gene trap cassette inserted into the first intron of the IFN-γ gene. IFN-γOFF mice were intercrossed with Ncr1-Cre or CD4-Cre mice that express Cre mainly in NK cells (IFN-γNcr1-ON mice) or T cells (IFN-γCD4-ON mice), respectively. Rosa26RFP reporter mice intercrossed with Ncr1-Cre mice showed selective RFP expression in more than 80% of the NK cells, while upon intercrossing with CD4-Cre mice abundant RFP expression was detected in T cells, but also to a minor extent in other immune cell subsets. Previous studies showed that IFN-γ expression is needed to promote survival of vaccinia virus (VACV) infection. Interestingly, during VACV infection of wild type and IFN-γCD4-ON mice two waves of serum IFN-γ were induced that peaked on day 1 and day 3/4 after infection. Similarly, VACV infected IFN-γNcr1-ON mice mounted two waves of IFN-γ responses, of which the first one was moderately and the second one profoundly reduced when compared with WT mice. Furthermore, IFN-γNcr1-ON as well as IFN-γCD4-ON mice survived VACV infection, whereas IFN-γOFF mice did not. As expected, ex vivo analysis of splenocytes derived from VACV infected IFN-γNcr1-ON mice showed IFN-γ expression in NK cells, but not T cells, whereas IFN-γOFF mice showed IFN-γ expression neither in NK cells nor T cells. VACV infected IFN-γNcr1-ON mice mounted normal cytokine responses, restored neutrophil accumulation, and showed normal myeloid cell distribution in blood and spleen. Additionally, in these mice normal MHC-II expression was detected on peripheral macrophages, whereas IFN-γOFF mice did not show MHC-II expression on such cells. In conclusion, upon VACV infection Ncr1 positive cells including NK cells mount two waves of early IFN-γ responses that are sufficient to promote the induction of protective anti-viral immunity.

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of interferon gamma (IFNγ) immunity and is characterized by severe infections by weakly virulent mycobacteria. Although IFNγ is the macrophage-activating factor, macrophages from these patients have never been studied. We demonstrate the generation of heterozygous and compound heterozygous (iMSMD-cohet) induced pluripotent stem cells (iPSCs) from a single chimeric patient, who suffered from complete autosomal recessive IFNγR1 deficiency and received bone-marrow transplantation. Loss of IFNγR1 expression had no influence on the macrophage differentiation potential of patient-specific iPSCs. In contrast, lack of IFNγR1 in iMSMD-cohet macrophages abolished IFNγ-dependent phosphorylation of STAT1 and induction of IFNγ-downstream targets such as IRF-1, SOCS-3, and IDO. As a consequence, iMSMD-cohet macrophages show impaired upregulation of HLA-DR and reduced intracellular killing of Bacillus Calmette-Guérin. We provide a disease-modeling platform that might be suited to investigate novel treatment options for MSMD and to gain insights into IFNγ signaling in macrophages.

Dendritic cells (DCs) are key directors of tolerogenic and immunogenic immune responses. During the steady state, DCs maintain T cell tolerance to self-antigens by multiple mechanisms including inducing anergy, deletion, and Treg activity. All of these mechanisms help to prevent autoimmune diseases or other hyperreactivities. Different DC subsets contribute to pathogen recognition by expression of different subsets of pattern recognition receptors, including Toll-like receptors or C-type lectins. In addition to the triggering of immune responses in infected hosts, most pathogens have evolved mechanisms for evasion of targeted responses. One such strategy is characterized by adopting the host's T cell tolerance mechanisms. Understanding these tolerogenic mechanisms is of utmost importance for therapeutic approaches to treat immune pathologies, tumors and infections. Transcriptional profiling has developed into a potent tool for DC subset identification. Here, we review and compile pathogen-induced tolerogenic transcriptional signatures from mRNA profiling data of currently available bacterial-or helminth-induced transcriptional signatures. We compare them with signatures of tolerogenic steady-state DC subtypes to identify common and divergent strategies of pathogen induced immune evasion. Candidate molecules are discussed in detail. Our analysis provides further insights into tolerogenic DC signatures and their exploitation by different pathogens.

Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 (IFNGR1 or IFNGR2) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that express Ifnγr1 either constitutively or myeloid specifically. Transduction of mouse Ifnγr1-/- HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity against Mycobacterium avium and Mycobacterium bovis Bacille Calmette-Guérin (BCG) in vitro. Transplantation of genetically corrected HSCs into Ifnγr1-/- mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients.

Chronic kidney disease (CKD) patients on dialysis often require Vitamin D receptor agonists (VDRAs) therapy for managing secondary hyperparathyroidism (SHPT). In a post-hoc analysis, the oral phosphate binder sucroferric oxyhydroxide (SFOH) had no apparent interaction with oral VDRAs. In contrast, the widely used oral phosphate binder sevelamer carbonate (SEV) showed potential interactions with oral VDRAs but no interactions with intravenous (IV) VDRAs. A cost minimization analysis (CMA) estimated the economic impact of using SFOH vs SEV in dialysis patients receiving VDRA agents in six European countries. The CMA assumed similar efficacy for SFOH and SEV, and that patients received either SFOH (1.5 g/day [3 tablets/day]) and oral VDRAs (0.28 μg/day) or SEV (6.4 g/day [8 tablets/day]) and IV VDRAs (1.84 μg/day or 200 IC/day). SFOH and SEV dosage was derived from two phase 3 trials (NCT01324128/NCT01464190). Drug acquisition costs were based on list ex-manufacturer prices. Costs for the administration of IV VDRAs and treatment of adverse events were not included. Annual treatment cost per patient for SFOH and (SEV) were: Austria €1,963 (€1,673), France €1,584 (€1,683), Germany €1,825 (€1,683), Italy €2,068 (€1,518), Switzerland €3,253 (€3,370); UK €2,872 (€3,270). Annual treatment costs per patient for oral and (IV) VDRAs were: Austria: €113 (€1,960), France: €88 (€2,649), Germany: €158 (€2,057), Italy: €32 (€1,791), Switzerland: €113 (€2,988) and UK: € 192 (€1,465). SFOH treatment with oral VDRAs resulted in annual cost savings per patient compared to SEV with IV VDRAs: Austria: €1,556, France: €2,661, Germany: €1,757, Italy: €1,208, Switzerland: €2,993, UK: € 1,671. The magnitude of the results was confirmed in sensitivity and scenario analyses. The CMA suggests that cost savings could be generated by using SFOH instead of SEV and, in consequence allowing SFOH patients to use oral VDRAs and avoid more costly IV VDRAs. Real-world data are needed to confirm these findings.