Autoantibodies against chemokines post-SARS-CoV-2 infection correlate with disease course.

Infection with severe acute respiratory syndrome coronavirus 2 associates with diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse coronavirus disease 2019 (COVID-19) outcomes. Here we discovered that antibodies against specific chemokines were omnipresent post-COVID-19, were associated with favorable disease outcome and negatively correlated with the development of long COVID at 1 yr post-infection. Chemokine antibodies were also present in HIV-1 infection and autoimmune disorders, but they targeted different chemokines compared with COVID-19. Monoclonal antibodies derived from COVID-19 convalescents that bound to the chemokine N-loop impaired cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising chemokine antibodies may modulate the inflammatory response and thus bear therapeutic potential.

Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein.

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization, and, similar to fp.006 and hr2.016, protects mice expressing human angiotensin-converting enzyme 2 against infection when present as a bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

The thioredoxin system: Balancing redox responses in immune cells and tumors.

The thioredoxin (TRX) system is an important contributor to cellular redox balance and regulates cell growth, apoptosis, gene expression, and antioxidant defense in nearly all living cells. Oxidative stress, the imbalance between reactive oxygen species (ROS) and antioxidants, can lead to cell death and tissue damage, thereby contributing to aging and to the development of several diseases, including cardiovascular and allergic diseases, diabetes, and neurological disorders. Targeting its activity is also considered as a promising strategy in the treatment of cancer. Over the past years, immunologists have established an essential function of TRX for activation, proliferation, and responses in T cells, B cells, and macrophages. Upon activation, immune cells rearrange their redox system and activate the TRX pathway to promote proliferation through sustainment of nucleotide biosynthesis, and to support inflammatory responses in myeloid cells by allowing NF-κB and NLRP3 inflammasome responses. Consequently, targeting the TRX system may therapeutically be exploited to inhibit immune responses in inflammatory conditions. In this review, we summarize recent insights revealing key roles of the TRX pathway in immune cells in health and disease, and lessons learnt for cancer therapy.

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2.

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants. One sentence summary: Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course.

Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 infection and autoimmune disorders, but they target different chemokines than those in COVID-19. Monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential. One-Sentence Summary: Naturally arising anti-chemokine antibodies associate with favorable COVID-19 and predict lack of long COVID.

Peroxisomes Are Critical for the Development and Maintenance of B1 and Marginal Zone B Cells but Dispensable for Follicular B Cells and T Cells.

Antioxidant systems maintain cellular redox (oxidation-reduction) homeostasis. In contrast with other key redox pathways, such as the thioredoxin system, glutathione, and NF-E2-related factor 2 (Nrf2), little is known about the function of the redox-sensitive organelle "peroxisome" in immune cells. In this study, we show that the absence of peroxisomes in conditional Pex5-deficient mice strikingly results in impaired homeostatic maintenance of innate-like B cells, namely, B1 and marginal zone B cells, which translates into a defective Ab response to Streptococcus pneumoniae Surprisingly, however, follicular B2 cell development, homeostatic maintenance, germinal center reactions, Ab production, class switching, and B cell memory formation were unaffected in Pex5-deficient animals. Similarly, T cell development and responses to viral infections also remained unaltered in the absence of Pex5 Thus, this study highlights the differential requirement of peroxisomes in distinct lymphocyte subtypes and may provide a rationale for specifically targeting peroxisomal metabolism in innate-like B cells in certain forms of B cell malignancies involving B1 cells.

Differential sensitivity of inflammatory macrophages and alternatively activated macrophages to ferroptosis.

Acumulation of oxidized membrane lipids ultimately results in ferroptotic cell death, which can be prevented by the selenoenzyme glutathione peroxidase 4 (Gpx4). In vivo conditions promoting ferroptosis and susceptible cell types are still poorly defined. In this study, we analyzed the conditional deletion of Gpx4 in mice specifically in the myeloid cell lineages. Surprisingly, development and maintenance of LysM+ macrophages and neutrophils, as well as CD11c+ monocyte-derived macrophages and dendritic cells were unaffected in the absence of Gpx4. Gpx4-deficient macrophages mounted an unaltered proinflammatory cytokine response including IL-1ß production following stimulation with TLR ligands and activation of several inflammasomes. Accordingly, Gpx4fl/fl LysM-cre mice were protected from bacterial and protozoan infections. Despite having the capacity to differentiate to alternatively activated macrophages (AAM), these cells lacking Gpx4 triggered ferroptosis both in vitro and in vivo following IL-4 overexpression and nematode infection. Exposure to nitric oxide restored viability of Gpx4-deficient AAM, while inhibition of iNOS in proinflammatory macrophages had no effect. These data together suggest that activation cues of tissue macrophages determine sensitivity to lipid peroxidation and ferroptotic cell death.

Redox regulation of immunometabolism.

Metabolic pathways and redox reactions are at the core of life. In the past decade(s), numerous discoveries have shed light on how metabolic pathways determine the cellular fate and function of lymphoid and myeloid cells, giving rise to an area of research referred to as immunometabolism. Upon activation, however, immune cells not only engage specific metabolic pathways but also rearrange their oxidation-reduction (redox) system, which in turn supports metabolic reprogramming. In fact, studies addressing the redox metabolism of immune cells are an emerging field in immunology. Here, we summarize recent insights revealing the role of reactive oxygen species (ROS) and the differential requirement of the main cellular antioxidant pathways, including the components of the thioredoxin (TRX) and glutathione (GSH) pathways, as well as their transcriptional regulator NF-E2-related factor 2 (NRF2), for proliferation, survival and function of T cells, B cells and macrophages.

The thioredoxin-1 inhibitor Txnip restrains effector T-cell and germinal center B-cell expansion.

Thioredoxin-1 (Trx1) is a vital component for cellular redox homeostasis. In T cells, Trx1 donates electrons for the de novo synthesis of deoxyribonucleotides to allow rapid cell proliferation. The Trx-interacting protein (Txnip) binds to the reduced Trx1 and inhibits its activity. However, the role of Txnip in adaptive immunity in vivo is unknown. Here, we show that absence of Txnip increased proliferation of effector T cells and GC B-cell responses in response to lymphocytic choriomeningitis virus and Qß virus-like particles, respectively, but did not affect development and homeostasis of T and B cells. While downregulation of Txnip and concomitant upregulation of Trx1 is critical for rapid T-cell expansion upon viral infection, re-expression of Txnip and consequently inhibition of Trx1 is important to restrain late T-cell expansion. Importantly, we demonstrated that T-cell receptor (TCR) engagement but not CD28 costimulation is critically required for Txnip downregulation. Thus, this study further uncovers positive and negative control of lymphocyte proliferation by the Trx1 system.

Electrophilic Nrf2 activators and itaconate inhibit inflammation at low dose and promote IL-1ß production and inflammatory apoptosis at high dose.

Controlling inflammation is critical for preventing many diseases including cancer, autoimmune disorders and hypersensitivity reactions. NF-E2-related factor 2 (Nrf2) is a key transcription factor that controls the cellular antioxidant and cytoprotective response. Moreover, Nrf2 has been implicated in the regulation of inflammatory processes, although the ultimate mechanism by which this is achieved is unknown. Here, we investigated mechanisms of inflammation and cell death pathways induced by a variety of Nrf2 activators including dimethyl fumarate (DMF) and the endogenous metabolite itaconate. We found that exposure of bone marrow-derived dendritic cells (BMDCs) to low concentrations of a variety of electrophilic Nrf2 activators including itaconate prior to Toll-like receptor (TLR) stimulation inhibits transcription of pro-inflammatory cytokines (such as interleukin [IL]-12 and IL-1ß) by activation of Nrf2. By contrast, high doses of these electrophilic compounds after TLR activation promote inflammatory apoptosis and caspase-8-dependent IL-1ß processing and release independently of Nrf2. Interestingly, tert-butylhydroquinone (tBHQ), a non-electrophilic Nrf2-activator, failed to induce IL-1ß production. These results have important implications for clinical application of electrophilic compounds.

Endothelial Lactate Controls Muscle Regeneration from Ischemia by Inducing M2-like Macrophage Polarization.

Endothelial cell (EC)-derived signals contribute to organ regeneration, but angiocrine metabolic communication is not described. We found that EC-specific loss of the glycolytic regulator pfkfb3 reduced ischemic hindlimb revascularization and impaired muscle regeneration. This was caused by the reduced ability of macrophages to adopt a proangiogenic and proregenerative M2-like phenotype. Mechanistically, loss of pfkfb3 reduced lactate secretion by ECs and lowered lactate levels in the ischemic muscle. Addition of lactate to pfkfb3-deficient ECs restored M2-like polarization in an MCT1-dependent fashion. Lactate shuttling by ECs enabled macrophages to promote proliferation and fusion of muscle progenitors. Moreover, VEGF production by lactate-polarized macrophages was increased, resulting in a positive feedback loop that further stimulated angiogenesis. Finally, increasing lactate levels during ischemia rescued macrophage polarization and improved muscle reperfusion and regeneration, whereas macrophage-specific mct1 deletion prevented M2-like polarization. In summary, ECs exploit glycolysis for angiocrine lactate shuttling to steer muscle regeneration from ischemia.

Cyclopentenone Prostaglandins and Structurally Related Oxidized Lipid Species Instigate and Share Distinct Pro- and Anti-inflammatory Pathways.

Oxidized lipids play a critical role in a variety of diseases with two faces: pro- and anti-inflammatory. The molecular mechanisms of this Janus-faced activity remain largely unknown. Here, we have identified that cyclopentenone-containing prostaglandins such as 15d-PGJ2 and structurally related oxidized phospholipid species possess a dual and opposing bioactivity in inflammation, depending on their concentration. Exposure of dendritic cells (DCs)/macrophages to low concentrations of such lipids before Toll-like receptor (TLR) stimulation instigates an anti-inflammatory response mediated by nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent inhibition of nuclear factor κB (NF-κB) activation and downstream targets. By contrast, high concentrations of such lipids upon TLR activation of DCs/macrophages result in inflammatory apoptosis characterized by mitochondrial depolarization and caspase-8-mediated interleukin (IL)-1ß maturation independently of Nrf2 and the classical inflammasome pathway. These results uncover unexpected pro- and anti-inflammatory activities of physiologically relevant lipid species generated by enzymatic and non-enzymatic oxidation dependent on their concentration, a phenomenon known as hormesis.

Thioredoxin-1 distinctly promotes NF-κB target DNA binding and NLRP3 inflammasome activation independently of Txnip.

Antioxidant systems, such as the thioredoxin-1 (Trx1) pathway, ensure cellular redox homeostasis. However, how such systems regulate development and function of myeloid cells is barely understood. Here we show that in contrast to its critical role in T cells, the murine Trx1 system is dispensable for steady-state myeloid-cell hematopoiesis due to their capacity to tap the glutathione/glutaredoxin pathway for DNA biosynthesis. However, the Trx1 pathway instrumentally enables nuclear NF-κB DNA-binding and thereby pro-inflammatory responses in monocytes and dendritic cells. Moreover, independent of this activity, Trx1 is critical for NLRP3 inflammasome activation and IL-1ß production in macrophages by detoxifying excessive ROS levels. Notably, we exclude the involvement of the Trx1 inhibitor Txnip as a redox-sensitive ligand of NLRP3 as previously proposed. Together, this study suggests that targeting Trx1 may be exploited to treat inflammatory diseases.

B1 and Marginal Zone B Cells but Not Follicular B2 Cells Require Gpx4 to Prevent Lipid Peroxidation and Ferroptosis.

Aerobic organisms need to maintain cellular redox homeostasis. Glutathione peroxidase-4 (Gpx4) has the unique ability to protect cells against lipid peroxidation. Here, we show that Gpx4 is absolutely required to prevent ferroptosis during development, maintenance, and responses of innate-like B cells, namely, the B1 and marginal zone (MZ) B cells. In contrast, Gpx4 is dispensable for the development, germinal center reactions, and antibody responses of follicular B2 cells. Mechanistically, we show increased lipid metabolism and sensitivity to lipid peroxidation and ferroptosis in B1 and MZ B cells compared to follicular B2 cells, consistent with the requirement of Gpx4 in innate-like B cells. This high sensitivity to ferroptosis of innate-like B cells may be used to therapeutically target Gpx4 in certain forms of B cell malignancies involving B1 cells.

The thioredoxin-1 and glutathione/glutaredoxin-1 systems redundantly fuel murine B-cell development and responses.

Antioxidant systems maintain cellular redox homeostasis. The thioredoxin-1 (Trx1) and the glutathione (GSH)/glutaredoxin-1 (Grx1) systems are key players in preserving cytosolic redox balance. In fact, T lymphocytes critically rely on reducing equivalents from the Trx1 system for DNA biosynthesis during metabolic reprogramming upon activation. We here show that the Trx1 system is also indispensable for development and functionality of marginal zone (MZ) B cells and B1 cells in mice. In contrast, development of conventional B cells, follicular B-cell homeostasis, germinal center reactions, and antibody responses are redundantly sustained by both antioxidant pathways. Proliferating B2 cells lacking Txnrd1 have increased glutathione (GSH) levels and upregulated cytosolic Grx1, which is barely detectable in expanding thymocytes. These results suggest that the redox capacity driving proliferation is more robust and flexible in B cells than in T cells, which may have profound implications for the therapy of B and T-cell neoplasms.

The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation.

The thioredoxin-1 (Trx1) system is an important contributor to cellular redox balance and is a sensor of energy and glucose metabolism. Here we show critical c-Myc-dependent activation of the Trx1 system during thymocyte and peripheral T-cell proliferation, but repression during T-cell quiescence. Deletion of thioredoxin reductase-1 (Txnrd1) prevents expansion the CD4-CD8- thymocyte population, whereas Txnrd1 deletion in CD4+CD8+ thymocytes does not affect further maturation and peripheral homeostasis of αßT cells. However, Txnrd1 is critical for expansion of the activated T-cell population during viral and parasite infection. Metabolomics show that TrxR1 is essential for the last step of nucleotide biosynthesis by donating reducing equivalents to ribonucleotide reductase. Impaired availability of 2'-deoxyribonucleotides induces the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal function of the Trx1 system in metabolic reprogramming of thymic and peripheral T cells and provide a rationale for targeting Txnrd1 in T-cell leukemia.

Synthesis and Structure-Activity Relationship Studies of Anti-Inflammatory Epoxyisoprostane Analogues.

The lactone derivative of the epoxyisoprostane EC is a highly effective inhibitor of the secretion of the proinflammatory cytokine IL-6. Herein, a modular synthesis of analogues is described, allowing flexible variations of the cyclic side chain of the parent lactone. A structure-activity relationship study identified a lactam analogue that retains the high activity. Furthermore, the exocyclic allylic alcohol was shown to be crucial for the observed effect.

Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level.

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus-to our knowledge-identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories "cell wall/membrane biogenesis" and "defense mechanisms" were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.