The Phosphatase RosC from Streptomyces davaonensis is Used for Roseoflavin Biosynthesis and has Evolved to Largely Prevent Dephosphorylation of the Important Cofactor Riboflavin-5'-phosphate.

The antibiotic roseoflavin is a riboflavin (vitamin B2) analog. One step of the roseoflavin biosynthetic pathway is catalyzed by the phosphatase RosC, which dephosphorylates 8-demethyl-8-amino-riboflavin-5'-phosphate (AFP) to 8-demethyl-8-amino-riboflavin (AF). RosC also catalyzes the potentially cell-damaging dephosphorylation of the AFP analog riboflavin-5'-phosphate also called "flavin mononucleotide" (FMN), however, with a lower efficiency. We performed X-ray structural analyses and mutagenesis studies on RosC from Streptomyces davaonensis to understand binding of the flavin substrates, the distinction between AFP and FMN and the catalytic mechanism of this enzyme. This work is the first structural analysis of an AFP phosphatase. Each monomer of the RosC dimer consists of an α/ß-fold core, which is extended by three specific elongated strand-to-helix sections and a specific N-terminal helix. Altogether these segments envelope the flavin thereby forming a novel flavin-binding site. We propose that distinction between AFP and FMN is provided by substrate-induced rigidification of the four RosC specific supplementary segments mentioned above and by an interaction between the amino group at C8 of AFP and the ß-carboxylate of D166. This key amino acid is involved in binding the ring system of AFP and positioning its ribitol phosphate part. Accordingly, site-specific exchanges at D166 disturbed the active site geometry of the enzyme and drastically reduced the catalytic activity. Based on the structure of the catalytic core we constructed a whole series of RosC variants but a disturbing, FMN dephosphorylating "killer enzyme", was not generated.

Exposure to bacterial PAMPs before RSV infection exacerbates innate inflammation and disease via IL-1α and TNF-α.

Respiratory syncytial virus (RSV) can cause severe lower respiratory tract infections. Understanding why some individuals get more serious disease may help with diagnosis and treatment. One possible risk factor underlying severe disease is bacterial exposure before RSV infection. Bacterial exposure has been associated with increased respiratory viral-induced disease severity but the mechanism remains unknown. Respiratory bacterial infections or exposure to their pathogen associated molecular patterns (PAMPs) trigger innate immune inflammation, characterised by neutrophil and inflammatory monocyte recruitment and the production of inflammatory cytokines. We hypothesise that these changes to the lung environment alter the immune response and disease severity during subsequent RSV infection. To test this, we intranasally exposed mice to LPS, LTA or Acinetobacter baumannii (an airway bacterial pathogen) before RSV infection and observed an early induction of disease, measured by weight loss, at days 1-3 after infection. Neutrophils or inflammatory monocytes were not responsible for driving this exacerbated weight loss. Instead, exacerbated disease was associated with increased IL-1α and TNF-α, which orchestrated the recruitment of innate immune cells into the lung. This study shows that exposure to bacterial PAMPs prior to RSV infection increases the expression of IL-1α and TNF-α, which dysregulate the immune response resulting in exacerbated disease.

Both Classical and Non-Classical Monocytes Patrol Glomerular Capillaries and Promote Acute Glomerular Inflammation.

Monocyte patrolling of the vasculature has been ascribed primarily to the non-classical monocyte subset. However, a recent study of the glomerular microvasculature provided evidence that both classical and non-classical monocytes undergo periods of intravascular retention and migration. Despite this, whether these subsets contribute differentially to acute glomerular inflammation is unknown. This study used glomerular multiphoton intravital microscopy to investigate the capacity of classical and non-classical monocytes to patrol the glomerular microvasculature and promote acute, neutrophil-dependent glomerular inflammation. In imaging experiments in monocyte reporter Cx3cr1gfp/+ mice, co-staining with anti-Ly6B or anti-Ly6C revealed that both non-classical monocytes [CX3C chemokine receptor 1-green fluorescent protein positive (CX3CR1-GFP+)] and classical monocytes (CX3CR1-GFP+ and Ly6B+ or Ly6C+) underwent prolonged (>10 minutes) retention and migration in the glomerular microvasculature. On induction of acute glomerulonephritis, in these behaviors were increased in classical but not non-classical monocytes. Using non-classical monocyte-deficient Csf1rCreNr4a1fl/fl mice, or anti-CCR2 to deplete classical monocytes, the removal of either subset reduced neutrophil retention and activation in acutely inflamed glomeruli, while the depletion of both subsets, via anti-CCR2 treatment in Csf1rCreNr4a1fl/fl mice, led to further reductions in neutrophil activity. In contrast, in a model of CD4+ T cell-dependent glomerulonephritis, the depletion of either monocyte subset failed to alter neutrophil responses. These findings indicate that both classical and non-classical monocytes patrol the glomerular microvasculature and promote neutrophil responses in acutely inflamed glomeruli.

Bacterial vaginosis-driven changes in vaginal T cell phenotypes and their implications for HIV susceptibility.

Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome that is prevalent in reproductive-age women worldwide. Adverse outcomes associated with BV include an increased risk of sexually acquired Human Immunodeficiency Virus (HIV), yet the immunological mechanisms underlying this association are not well understood. To investigate BV driven changes to cervicovaginal tract (CVT) and circulating T cell phenotypes, participants with or without BV provided vaginal tract (VT) and ectocervical (CX) tissue biopsies and peripheral blood mononuclear cells (PBMC). Immunofluorescence analysis of genital mucosal tissues revealed a reduced density of CD3+CD4+CCR5+ cells in the VT lamina propria of individuals with compared to those without BV (median 243.8 cells/mm2 BV- vs 106.9 cells/mm2 BV+, p=0.043). High-parameter flow cytometry of VT biopsies revealed an increased frequency in individuals with compared to those without BV of dysfunctional CD39+ conventional CD4+ T cells (Tconv) (median frequency 15% BV- vs 30% BV+, padj=0.0331) and tissue-resident CD69+CD103+ Tconv (median frequency 24% BV- vs 38% BV+, padj=0.0061), previously reported to be implicated in HIV acquisition and replication. Our data suggests that BV elicits diverse and complex VT T cell alterations and expands on potential immunological mechanisms that may promote adverse outcomes including HIV susceptibility.

Helminth induced monocytosis conveys protection from respiratory syncytial virus infection in mice.

BACKGROUND: Respiratory syncytial virus (RSV) infection in infants is a major cause of viral bronchiolitis and hospitalisation. We have previously shown in a murine model that ongoing infection with the gut helminth Heligmosomoides polygyrus protects against RSV infection through type I interferon (IFN-I) dependent reduction of viral load. Yet, the cellular basis for this protection has remained elusive. Given that recruitment of mononuclear phagocytes to the lung is critical for early RSV infection control, we assessed their role in this coinfection model. METHODS: Mice were infected by oral gavage with H. polygyrus. Myeloid immune cell populations were assessed by flow cytometry in lung, blood and bone marrow throughout infection and after secondary infection with RSV. Monocyte numbers were depleted by anti-CCR2 antibody or increased by intravenous transfer of enriched monocytes. RESULTS: H. polygyrus infection induces bone marrow monopoiesis, increasing circulatory monocytes and lung mononuclear phagocytes in a IFN-I signalling dependent manner. This expansion causes enhanced lung mononuclear phagocyte counts early in RSV infection that may contribute to the reduction of RSV load. Depletion or supplementation of circulatory monocytes prior to RSV infection confirms that these are both necessary and sufficient for helminth induced antiviral protection. CONCLUSIONS: H. polygyrus infection induces systemic monocytosis contributing to elevated mononuclear phagocyte numbers in the lung. These cells are central to an anti-viral effect that reduces the peak viral load in RSV infection. Treatments to promote or modulate these cells may provide novel paths to control RSV infection in high risk individuals.

Mutation of the Plasmodium falciparum Flavokinase Confers Resistance to Roseoflavin and 8-Aminoriboflavin.

The riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilization. To determine their mechanism of action, we generated roseoflavin-resistant parasites by in vitro evolution. Relative to wild-type, these parasites were 4-fold resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Whole genome sequencing of the resistant parasites revealed a missense mutation leading to an amino acid change (L672H) in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene. The IC50 values for roseoflavin and 8-aminoriboflavin against the roseoflavin-resistant parasites created through in vitro evolution were indistinguishable from those against parasites in which the missense mutation was introduced into the native PfFK. We also generated two parasite lines episomally expressing GFP-tagged versions of either the wild-type or mutant forms of PfFK. We found that PfFK-GFP localizes to the parasite cytosol and that immunopurified PfFK-GFP phosphorylated riboflavin, roseoflavin, and 8-aminoriboflavin. The L672H mutation increased the KM for roseoflavin, explaining the resistance phenotype. Mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonized by increasing the extracellular concentration of riboflavin, consistent with it also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore, are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite proliferation by inhibiting riboflavin phosphorylation and via the generation of toxic flavin cofactor analogues.

Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia.

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE2) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4+/CD44+/CD62L+ and CD4+/CD44+/CD62L-/CD27+ T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation. This secondary immune activation drives local tissue-resident T cell development while limiting tissue injury.

Targeting TGFß-activated kinase-1 activation in microglia reduces CAR T immune effector cell-associated neurotoxicity syndrome.

Cancer immunotherapy with chimeric antigen receptor (CAR) T cells can cause immune effector cell-associated neurotoxicity syndrome (ICANS). However, the molecular mechanisms leading to ICANS are not well understood. Here we examined the role of microglia using mouse models and cohorts of individuals with ICANS. CD19-directed CAR (CAR19) T cell transfer in B cell lymphoma-bearing mice caused microglia activation and neurocognitive deficits. The TGFß-activated kinase-1 (TAK1)-NF-κB-p38 MAPK pathway was activated in microglia after CAR19 T cell transfer. Pharmacological TAK1 inhibition or genetic Tak1 deletion in microglia using Cx3cr1CreER:Tak1fl/fl mice resulted in reduced microglia activation and improved neurocognitive activity. TAK1 inhibition allowed for potent CAR19-induced antilymphoma effects. Individuals with ICANS exhibited microglia activation in vivo when studied by translocator protein positron emission tomography, and imaging mass cytometry revealed a shift from resting to activated microglia. In summary, we prove a role for microglia in ICANS pathophysiology, identify the TAK1-NF-κB-p38 MAPK axis as a pathogenic signaling pathway and provide a rationale to test TAK1 inhibition in a clinical trial for ICANS prevention after CAR19 T cell-based cancer immunotherapy.

Isolation, characterisation and description of the roseoflavin producer Streptomyces berlinensis sp. nov.

The Gram-positive bacteria Streptomyces davaonensis and Streptomyces cinnabarinus have been the only organisms known to produce roseoflavin, a riboflavin (vitamin B2) derived red antibiotic. Using a selective growth medium and a phenotypic screening, we were able to isolate a novel roseoflavin producer from a German soil sample. The isolation procedure was repeated twice, that is, the same strain could be isolated from the same location in Berlin 6 months and 12 months after its first isolation. Whole genome sequencing of the novel roseoflavin producer revealed an unusual chromosomal arrangement and the deposited genome sequence of the new isolate (G + C content of 71.47%) contains 897 genes per inverted terminal repeat, 6190 genes in the core and 107 genes located on an illegitimate terminal end. We identified the roseoflavin biosynthetic genes rosA, rosB and rosC and an unusually high number of riboflavin biosynthetic genes. Overexpression of rosA, rosB and rosC in Escherichia coli and enzyme assays confirmed their predicted functions in roseoflavin biosynthesis. A full taxonomic analysis revealed that the isolate represents a previously unknown Streptomyces species and we propose the name Streptomyces berlinensis sp. nov. for this roseoflavin producer.

Procoagulant platelets promote immune evasion in triple-negative breast cancer.

ABSTRACT: Triple-negative breast cancer (TNBC) is an aggressive tumor entity in which immune checkpoint (IC) molecules are primarily synthesized in the tumor environment. Here, we report that procoagulant platelets bear large amounts of such immunomodulatory factors and that the presence of these cellular blood components in TNBC relates to protumorigenic immune-cell activity and impaired survival. Mechanistically, tumor-released nucleic acids attract platelets to the aberrant tumor microvasculature, where they undergo procoagulant activation, thus delivering specific stimulatory and inhibitory IC molecules. This concomitantly promotes protumorigenic myeloid leukocyte responses and compromises antitumorigenic lymphocyte activity, ultimately supporting tumor growth. Interference with platelet-leukocyte interactions prevented immune cell misguidance and suppressed tumor progression, nearly as effective as systemic IC inhibition. Hence, our data uncover a self-sustaining mechanism of TNBC by using platelets to misdirect immune-cell responses. Targeting this irregular multicellular interplay may represent a novel immunotherapeutic strategy for TNBC without the adverse effects of systemic IC inhibition.

Repeated social defeat stress differently affects arthritis-associated hypersensitivity in male and female mice.

Chronic stress enhances the risk of neuropsychiatric disorders and contributes to the aggravation and chronicity of pain. The development of stress-associated diseases, including pain, is affected by individual vulnerability or resilience to stress, although the mechanisms remain elusive. We used the repeated social defeat stress model promoting susceptible and resilient phenotypes in male and female mice and induced knee mono-arthritis to investigate the impact of stress vulnerability on pain and immune system regulation. We analyzed different pain-related behaviors, measured blood cytokine and immune cell levels, and performed histological analyses at the knee joints and pain/stress-related brain areas. Stress susceptible male and female mice showed prolonged arthritis-associated hypersensitivity. Interestingly, hypersensitivity was exacerbated in male but not female mice. In males, stress promoted transiently increased neutrophils and Ly6Chigh monocytes, lasting longer in susceptible than resilient mice. While resilient male mice displayed persistently increased levels of the anti-inflammatory interleukin (IL)-10, susceptible mice showed increased levels of the pro-inflammatory IL-6 at the early- and IL-12 at the late arthritis stage. Although joint inflammation levels were comparable among groups, macrophage and neutrophil infiltration was higher in the synovium of susceptible mice. Notably, only susceptible male mice, but not females, presented microgliosis and monocyte infiltration in the prefrontal cortex at the late arthritis stage. Blood Ly6Chigh monocyte depletion during the early inflammatory phase abrogated late-stage hypersensitivity and the associated histological alterations in susceptible male mice. Thus, recruitment of blood Ly6Chigh monocytes during the early arthritis phase might be a key factor mediating the persistence of arthritis pain in susceptible male mice. Alternative neuro-immune pathways that remain to be explored might be involved in females.

Mutual regulation of CD4+ T cells and intravascular fibrin in infections.

Innate myeloid cells especially neutrophils and their extracellular traps are known to promote intravascular coagulation and thrombosis formation in infections and various other conditions. Innate myeloid cell-dependent fibrin formation can support systemic immunity while its dysregulation enhances the severity of infectious diseases. Less is known about the immune mechanisms preventing dysregulation of fibrin homeostasis in infection. During experimental systemic infections local fibrin deposits in the liver microcirculation cause rapid arrest of CD4+ T cells. Arrested T-helper cells mostly represent Th17 cells that partially originate from the small intestine. Intravascular fibrin deposits activate mouse and human CD4+ T cells which can be mediated by direct fibrin-CD4+ T-cell interactions. Activated CD4+ T cells suppress fibrin deposition and microvascular thrombosis by directly counteracting coagulation activation by neutrophils and classical monocytes. T-cell activation, which is initially triggered by IL-12p40- and MHC-II-dependent mechanisms, enhances intravascular fibrinolysis via LFA-1. Moreover, CD4+ T cells disfavor the association of the thrombin-activatable fibrinolysis inhibitor (TAFI) with fibrin whereby fibrin deposition is increased by TAFI in the absence but not in the presence of T cells. In human infections thrombosis development is inversely related to microvascular levels of CD4+ T cells. Thus, fibrin promotes LFA-1-dependent T-helper cell activation in infections which drives a negative feedback cycle that rapidly restricts intravascular fibrin and thrombosis development.

Radiotherapy Enhances Metastasis Through Immune Suppression by Inducing PD-L1 and MDSC in Distal Sites.

PURPOSE: Radiotherapy (RT) is a widely employed anticancer treatment. Emerging evidence suggests that RT can elicit both tumor-inhibiting and tumor-promoting immune effects. The purpose of this study is to investigate immune suppressive factors of radiotherapy. EXPERIMENTAL DESIGN: We used a heterologous two-tumor model in which adaptive concomitant immunity was eliminated. RESULTS: Through analysis of PD-L1 expression and myeloid-derived suppressor cells (MDSC) frequencies using patient peripheral blood mononuclear cells and murine two-tumor and metastasis models, we report that local irradiation can induce a systemic increase in MDSC, as well as PD-L1 expression on dendritic cells and myeloid cells, and thereby increase the potential for metastatic dissemination in distal, nonirradiated tissue. In a mouse model using two distinct tumors, we found that PD-L1 induction by ionizing radiation was dependent on elevated chemokine CXCL10 signaling. Inhibiting PD-L1 or MDSC can potentially abrogate RT-induced metastasis and improve clinical outcomes for patients receiving RT. CONCLUSIONS: Blockade of PD-L1/CXCL10 axis or MDSC infiltration during irradiation can enhance abscopal tumor control and reduce metastasis.

Clinically used broad-spectrum antibiotics compromise inflammatory monocyte-dependent antibacterial defense in the lung.

Hospital-acquired pneumonia (HAP) is associated with high mortality and costs, and frequently caused by multidrug-resistant (MDR) bacteria. Although prior antimicrobial therapy is a major risk factor for HAP, the underlying mechanism remains incompletely understood. Here, we demonstrate that antibiotic therapy in hospitalized patients is associated with decreased diversity of the gut microbiome and depletion of short-chain fatty acid (SCFA) producers. Infection experiments with mice transplanted with patient fecal material reveal that these antibiotic-induced microbiota perturbations impair pulmonary defense against MDR Klebsiella pneumoniae. This is dependent on inflammatory monocytes (IMs), whose fatty acid receptor (FFAR)2/3-controlled and phagolysosome-dependent antibacterial activity is compromized in mice transplanted with antibiotic-associated patient microbiota. Collectively, we characterize how clinically relevant antibiotics affect antimicrobial defense in the context of human microbiota, and reveal a critical impairment of IM´s antimicrobial activity. Our study provides additional arguments for the rational use of antibiotics and offers mechanistic insights for the development of novel prophylactic strategies to protect high-risk patients from HAP.

Expansion of Pathogenic Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis.

BACKGROUND: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1) or CTLA4 (cytotoxic T-lymphocyte-associated protein 4), have revolutionized cancer management but are associated with devastating immune-related adverse events including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. Although much has been learned about the role of T-cells in ICI myocarditis, little is understood about the identity, transcriptional diversity, and functions of infiltrating macrophages. METHODS: We used an established murine ICI myocarditis model (Ctla4+/-Pdcd1-/- mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization, molecular imaging, and antibody neutralization studies. RESULTS: We observed marked increases in CCR2 (C-C chemokine receptor type 2)+ monocyte-derived macrophages and CD8+ T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2+ subpopulation highly expressing Cxcl9 (chemokine [C-X-C motif] ligand 9), Cxcl10 (chemokine [C-X-C motif] ligand 10), Gbp2b (interferon-induced guanylate-binding protein 2b), and Fcgr4 (Fc receptor, IgG, low affinity IV) that originated from CCR2+ monocytes. It is important that a similar macrophage population expressing CXCL9, CXCL10, and CD16α (human homologue of mouse FcgR4) was expanded in patients with ICI myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9+Cxcl10+ macrophages via IFN-γ (interferon gamma) and CXCR3 (CXC chemokine receptor 3) signaling pathways. Depleting CD8+ T-cells or macrophages and blockade of IFN-γ signaling blunted the expansion of Cxcl9+Cxcl10+ macrophages in the heart and attenuated myocarditis, suggesting that this interaction was necessary for disease pathogenesis. CONCLUSIONS: These data demonstrate that ICI myocarditis is associated with the expansion of a specific population of IFN-γ-induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.

Myeloid Heterogeneity Mediates Acute Exacerbations of Pulmonary Fibrosis.

Epidemiological evidence indicates that exposure to particulate matter is linked to the development of idiopathic pulmonary fibrosis (IPF) and increases the incidence of acute exacerbations of IPF. In addition to accelerating the rate of lung function decline, exposure to fine particulate matter (particulate matter smaller than 2.5 µm [PM2.5]) is a risk factor for increased mortality in subjects with IPF. In this article, we show that exposure to PM2.5 mediates monocyte recruitment and fibrotic progression in mice with established fibrosis. In mice with established fibrosis, bronchoalveolar lavage cells showed monocyte/macrophage heterogeneity after exposure to PM2.5. These cells had a significant inflammatory and anti-inflammatory signature. The mixed heterogeneity of cells contributed to the proinflammatory and anti-inflammatory response. Although monocyte-derived macrophages were recruited to the lung in bleomycin-injured mice treated with PM2.5, recruitment of monocytes expressing Ly6Chi to the lung promoted progression of fibrosis, reduced lung aeration on computed tomography, and impacted lung compliance. Ly6Chi monocytes isolated from PM2.5-exposed fibrotic mice showed enhanced expression of proinflammatory markers compared with fibrotic mice exposed to vehicle. Moreover, IPF bronchoalveolar lavage cells treated ex vivo with PM2.5 showed an exaggerated inflammatory response. Targeting Ly6Chi monocyte recruitment inhibited fibrotic progression in mice. Moreover, the adoptive transfer of Ly6Chi monocytes exacerbated established fibrosis. These observations suggest that enhanced recruitment of Ly6Chi monocytes with a proinflammatory phenotype mediates acute exacerbations of pulmonary fibrosis, and targeting these cells may provide a potential novel therapeutic target to protect against acute exacerbations of IPF.

Tumor-infiltrating CCR2+ inflammatory monocytes counteract specific immunotherapy.

Tumor development and progression is shaped by the tumor microenvironment (TME), a heterogeneous assembly of infiltrating and resident host cells, their secreted mediators and intercellular matrix. In this context, tumors are infiltrated by various immune cells with either pro-tumoral or anti-tumoral functions. Recently, we published our non-invasive immunization platform DIVA suitable as a therapeutic vaccination method, further optimized by repeated application (DIVA2). In our present work, we revealed the therapeutic effect of DIVA2 in an MC38 tumor model and specifically focused on the mechanisms induced in the TME after immunization. DIVA2 resulted in transient tumor control followed by an immune evasion phase within three weeks after the initial tumor inoculation. High-dimensional flow cytometry analysis and single-cell mRNA-sequencing of tumor-infiltrating leukocytes revealed cytotoxic CD8+ T cells as key players in the immune control phase. In the immune evasion phase, inflammatory CCR2+ PDL-1+ monocytes with immunosuppressive properties were recruited into the tumor leading to suppression of DIVA2-induced tumor-reactive T cells. Depletion of CCR2+ cells with specific antibodies resulted in prolonged survival revealing CCR2+ monocytes as important for tumor immune escape in the TME. In summary, the present work provides a platform for generating a strong antigen-specific primary and memory T cell immune response using the optimized transcutaneous immunization method DIVA2. This enables protection against tumors by therapeutic immune control of solid tumors and highlights the immunosuppressive influence of tumor infiltrating CCR2+ monocytes that need to be inactivated in addition for successful cancer immunotherapy.

Contrasting roles of MERS-CoV and SARS-CoV-2 internal proteins in pathogenesis in mice.

Betacoronaviruses encode an internal (I) gene via an alternative reading frame within the nucleocapsid gene, called ORF8b for Middle-East respiratory syndrome coronavirus (MERS-CoV) and ORF9b for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Previous reports suggested that proteins 8b and 9b are involved in evading multiple innate immune signaling pathways. However, their roles in mediating pathogenesis in infected animals have not been determined. In this study, we abrogated the expression of protein 8b in MERS-CoV and protein 9b in SARS-CoV-2. Using mouse models of MERS-CoV and SARS-CoV-2 infection, we found that MERS-CoV lacking protein 8b expression was more virulent, while SARS-CoV-2 lacking protein 9b expression was attenuated compared with the respective wild-type viruses. Upon further analysis, we detected increased levels of type I interferon and enhanced infiltration of immune cells to the lungs of mice infected with MERS-CoV lacking protein 8b expression. These data suggest that the I protein of MERS-CoV plays a role in limiting pathogenesis while that of SARS-CoV-2 enhances disease severity. IMPORTANCE The function of betacoronavirus internal protein has been relatively understudied. The earliest report on the internal protein of mouse hepatitis virus suggested that the internal protein is a structural protein without significant functions in virus replication and virulence. However, the internal proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus, and SARS-CoV-2 have been shown to evade immune responses. Despite the reported functions of the internal protein in these highly pathogenic human coronaviruses, its role in mediating pathogenesis in experimentally infected animals has not been characterized. Our data indicated that despite the similar genomic location and expression strategy of these internal proteins, their effects on virulence are vastly different and virus specific, highlighting the complexity between host-virus interaction and disease outcome.

Randomized controlled trial investigating potential effects of relaxation on mitochondrial function in immune cells: A pilot experiment.

This study aimed to investigate the effect of a relaxation response induced by hypnosis on the mitochondrial energy production of immune cells compared to an everyday relaxing situation. Chronically stressed individuals (88% women) with at least moderate suggestibility were randomized to a hypnosis (20 min relaxation hypnosis; n = 20) or a control condition (20 min documentary; n = 22). Before and after intervention, peripheral blood was collected. The primary outcomes were mitochondrial respiration and density in immune cells measured by high-resolution respirometry and citrate synthase activity assays. As secondary outcome, perceived stress, anxiety, and depressive mood were assessed. The intervention led to no significant Group × Time effects on mitochondrial bioenergetic parameters but a significant Time effect (ηp2 = .09 -.10). Thus, there were no differences in the experimental conditions concerning the measured parameters of mitochondrial bioenergetics. Exploratory subanalyses indicated that stress, anxiety, and depressive mood were linked to lower mitochondrial respiration. Individuals with higher anxiety had less decrease in routine respiration over time than those with lower anxiety (ηp2 = .09). This study explores the effects of relaxation in the form of hypnosis compared to watching a video on the energy metabolism of immune cells. Relaxation, whether in targeted (hypnosis) or untargeted (documentary) form, affected mitochondrial respiration. Further research should focus on the long-term effects of relaxation on bioenergetics. The trial was retrospectively registered on 07/12/2021, DRKS00027356, https://drks.de/search/de/trial/DRKS00027356.