Sepsis-trained macrophages promote antitumoral tissue-resident T cells.

Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity.

FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.

ß-Glucan Reverses the Epigenetic State of LPS-Induced Immunological Tolerance.

Innate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). We apply an integrated epigenomic approach to characterize the molecular events involved in LPS-induced tolerance in a time-dependent manner. Mechanistically, LPS-treated monocytes fail to accumulate active histone marks at promoter and enhancers of genes in the lipid metabolism and phagocytic pathways. Transcriptional inactivity in response to a second LPS exposure in tolerized macrophages is accompanied by failure to deposit active histone marks at promoters of tolerized genes. In contrast, ß-glucan partially reverses the LPS-induced tolerance in vitro. Importantly, ex vivo ß-glucan treatment of monocytes from volunteers with experimental endotoxemia re-instates their capacity for cytokine production. Tolerance is reversed at the level of distal element histone modification and transcriptional reactivation of otherwise unresponsive genes. VIDEO ABSTRACT.

Sepsis expands a CD39+ plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity.

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions.

Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.

Broad defects in the energy metabolism of leukocytes underlie immunoparalysis in sepsis.

The acute phase of sepsis is characterized by a strong inflammatory reaction. At later stages in some patients, immunoparalysis may be encountered, which is associated with a poor outcome. By transcriptional and metabolic profiling of human patients with sepsis, we found that a shift from oxidative phosphorylation to aerobic glycolysis was an important component of initial activation of host defense. Blocking metabolic pathways with metformin diminished cytokine production and increased mortality in systemic fungal infection in mice. In contrast, in leukocytes rendered tolerant by exposure to lipopolysaccharide or after isolation from patients with sepsis and immunoparalysis, a generalized metabolic defect at the level of both glycolysis and oxidative metabolism was apparent, which was restored after recovery of the patients. Finally, the immunometabolic defects in humans were partially restored by therapy with recombinant interferon-γ, which suggested that metabolic processes might represent a therapeutic target in sepsis.

The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin.

Ancient organisms have a combined coagulation and immune system, and although links between inflammation and hemostasis exist in mammals, they are indirect and slower to act. Here we investigated direct links between mammalian immune and coagulation systems by examining cytokine proproteins for potential thrombin protease consensus sites. We found that interleukin (IL)-1α is directly activated by thrombin. Thrombin cleaved pro-IL-1α at a site perfectly conserved across disparate species, indicating functional importance. Surface pro-IL-1α on macrophages and activated platelets was cleaved and activated by thrombin, while tissue factor, a potent thrombin activator, colocalized with pro-IL-1α in the epidermis. Mice bearing a mutation in the IL-1α thrombin cleavage site (R114Q) exhibited defects in efficient wound healing and rapid thrombopoiesis after acute platelet loss. Thrombin-cleaved IL-1α was detected in humans during sepsis, pointing to the relevance of this pathway for normal physiology and the pathogenesis of inflammatory and thrombotic diseases.

The ATP-Binding Cassette Gene ABCF1 Functions as an E2 Ubiquitin-Conjugating Enzyme Controlling Macrophage Polarization to Dampen Lethal Septic Shock.

Sepsis is a bi-phasic inflammatory disease that threatens approximately 30 million lives and claims over 14 million annually, yet little is known regarding the molecular switches and pathways that regulate this disease. Here, we have described ABCF1, an ATP-Binding Cassette (ABC) family member protein, which possesses an E2 ubiquitin enzyme activity, through which it controls the Lipopolysaccharide (LPS)- Toll-like Receptor-4 (TLR4) mediated gram-negative insult by targeting key proteins for K63-polyubiquitination. Ubiquitination by ABCF1 shifts the inflammatory profile from an early phase MyD88-dependent to a late phase TRIF-dependent signaling pathway, thereby regulating TLR4 endocytosis and modulating macrophage polarization from M1 to M2 phase. Physiologically, ABCF1 regulates the shift from the inflammatory phase of sepsis to the endotoxin tolerance phase, and modulates cytokine storm and interferon-ß (IFN-ß)-dependent production by the immunotherapeutic mediator, SIRT1. Consequently, ABCF1 controls sepsis induced mortality by repressing hypotension-induced renal circulatory dysfunction.

The kinase Jnk2 promotes stress-induced mitophagy by targeting the small mitochondrial form of the tumor suppressor ARF for degradation.

Mitophagy is essential for cellular homeostasis, but how mitophagy is regulated is largely unknown. Here we found that the kinase Jnk2 was required for stress-induced mitophagy. Jnk2 promoted ubiquitination and proteasomal degradation of the small mitochondrial form of the tumor suppressor ARF (smARF). Loss of Jnk2 led to the accumulation of smARF, which induced excessive autophagy that resulted in lysosomal degradation of the mitophagy adaptor p62 at steady state. Depletion of p62 prevented Jnk2-deficient cells from mounting mitophagy upon stress. Jnk2-deficient mice displayed defective mitophagy, which resulted in tissue damage under hypoxic stress, as well as hyperactivation of inflammasomes and increased mortality in sepsis. Our findings define a unique mechanism of maintaining immunological homeostasis that protects the host from tissue damage and mortality.

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity.

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2(D57N) mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.

The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis.

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

A computational approach to developing a multi-epitope vaccine for combating Pseudomonas aeruginosa-induced pneumonia and sepsis.

Pseudomonas aeruginosa is a complex nosocomial infectious agent responsible for numerous illnesses, with its growing resistance variations complicating treatment development. Studies have emphasized the importance of virulence factors OprE and OprF in pathogenesis, highlighting their potential as vaccine candidates. In this study, B-cell, MHC-I, and MHC-II epitopes were identified, and molecular linkers were active to join these epitopes with an appropriate adjuvant to construct a vaccine. Computational tools were employed to forecast the tertiary framework, characteristics, and also to confirm the vaccine's composition. The potency was weighed through population coverage analysis and immune simulation. This project aims to create a multi-epitope vaccine to reduce P. aeruginosa-related illness and mortality using immunoinformatics resources. The ultimate complex has been determined to be stable, soluble, antigenic, and non-allergenic upon inspection of its physicochemical and immunological properties. Additionally, the protein exhibited acidic and hydrophilic characteristics. The Ramachandran plot, ProSA-web, ERRAT, and Verify3D were employed to ensure the final model's authenticity once the protein's three-dimensional structure had been established and refined. The vaccine model showed a significant binding score and stability when interacting with MHC receptors. Population coverage analysis indicated a global coverage rate of 83.40%, with the USA having the highest coverage rate, exceeding 90%. Moreover, the vaccine sequence underwent codon optimization before being cloned into the Escherichia coli plasmid vector pET-28a (+) at the EcoRI and EcoRV restriction sites. Our research has developed a vaccine against P. aeruginosa that has strong binding affinity and worldwide coverage, offering an acceptable way to mitigate nosocomial infections.

Understanding Immunosuppression after Sepsis.

Following a severe primary infection or trauma, the risk of developing pneumonia increases due to acquired immune defects collectively known as sepsis-induced immunosuppression. In this issue of Immunity, Roquilly et al. (2017) show that dendritic cells and macrophages developing in the lung after the resolution of a severe infection acquire tolerogenic properties that contribute to persistent immunosuppression and susceptibility to secondary infections.

Local Modulation of Antigen-Presenting Cell Development after Resolution of Pneumonia Induces Long-Term Susceptibility to Secondary Infections.

Lung infections cause prolonged immune alterations and elevated susceptibility to secondary pneumonia. We found that, after resolution of primary viral or bacterial pneumonia, dendritic cells (DC), and macrophages exhibited poor antigen-presentation capacity and secretion of immunogenic cytokines. Development of these "paralyzed" DCs and macrophages depended on the immunosuppressive microenvironment established upon resolution of primary infection, which involved regulatory T (Treg) cells and the cytokine TGF-ß. Paralyzed DCs secreted TGF-ß and induced local Treg cell accumulation. They also expressed lower amounts of IRF4, a transcription factor associated with increased antigen-presentation capacity, and higher amounts of Blimp1, a transcription factor associated with tolerogenic functions, than DCs present during primary infection. Blimp1 expression in DC of humans suffering sepsis or trauma correlated with severity and complicated outcomes. Our findings describe mechanisms underlying sepsis- and trauma-induced immunosuppression, reveal prognostic markers of susceptibility to secondary infections and identify potential targets for therapeutic intervention.

Deletion of Tmem268 in mice suppresses anti-infectious immune responses by downregulating CD11b signaling.

Transmembrane protein 268 (TMEM268) is a novel, tumor growth-related protein first reported by our laboratory. It interacts with the integrin subunit ß4 (ITGB4) and plays a positive role in the regulation of the ITGB4/PLEC signaling pathway. Here, we investigated the effects and mechanism of TMEM268 in anti-infectious immune response in mice. Tmem268 knockout in mice aggravated cecal ligation and puncture-induced sepsis, as evidenced by higher bacterial burden in various tissues and organs, congestion, and apoptosis. Moreover, Tmem268 deficiency in mice inhibited phagocyte adhesion and migration, thus decreasing phagocyte infiltration at the site of infection and complement-dependent phagocytosis. Further findings indicated that TMEM268 interacts with CD11b and inhibits its degradation via the endosome-lysosome pathway. Our results reveal a positive regulatory role of TMEM268 in ß2 integrin-associated anti-infectious immune responses and signify the potential value of targeting the TMEM268-CD11b signaling axis for the maintenance of immune homeostasis and immunotherapy for sepsis and related immune disorders.

Sepsis Impairs IFN-γ Production in CD8 T Cells through Changes in Local Chromatin Landscape.

Sepsis is a complex condition of inflammatory and immune dysregulation, triggered by severe infection. In survivors, chronic inflammation and immune dysregulation linger, facilitating the emergence of infections. CD8 dysfunction contributes to immunosuppression in sepsis survivors. We devised an animal model that enabled us to identify and analyze CD8-intrinsic defects induced by sepsis. We adoptively transferred CD45.1 CD8 OT-I T cells into CD45.2 congenic mice and subjected them to cecal ligature and puncture, to induce abdominal sepsis. One month later, we isolated the transferred CD8 cells. Surface marker expression confirmed they had not been activated through the TCR. CD8 OT-I T cells isolated from septic (or sham-operated) mice were transferred to second recipients, which were challenged with OVA-expressing Listeria monocytogenes. We compared effector capacities between OT-I cells exposed to sepsis and control cells. Naive mice that received OT-I cells exposed to sepsis had higher bacterial burden and a shorter survival when challenged with OVA-expressing L. monocytogenes. OT-I cells isolated from septic mice produced less IFN-γ but had conserved activation, expansion potential, and cytotoxic function. We observed lower transcript levels of IFN-γ and of the long noncoding RNA Ifng-as1, a local regulator of the epigenetic landscape, in cells exposed to sepsis. Accordingly, local abundance of a histone modification characteristic of active promoter regions was reduced in sepsis-exposed CD8 T cells. Our results identify a mechanism through which inflammation in the context of sepsis affects CD8 T cell function intrinsically.

Decoding the potential role of regulatory T cells in sepsis-induced immunosuppression.

Sepsis, a multiorgan dysfunction with high incidence and mortality, is caused by an imbalanced host-to-infection immune response. Organ-support therapy improves the early survival rate of sepsis patients. In the long term, those who survive the "cytokine storm" and its secondary damage usually show higher susceptibility to secondary infections and sepsis-induced immunosuppression, in which regulatory T cells (Tregs) are evidenced to play an essential role. However, the potential role and mechanism of Tregs in sepsis-induced immunosuppression remains elusive. In this review, we elucidate the role of different functional subpopulations of Tregs during sepsis and then review the mechanism of sepsis-induced immunosuppression from the aspects of regulatory characteristics, epigenetic modification, and immunometabolism of Tregs. Thoroughly understanding how Tregs impact the immune system during sepsis may shed light on preclinical research and help improve the translational value of sepsis immunotherapy.

Sepsis shapes the human γδ TCR repertoire in an age- and pathogen-dependent manner.

Sepsis affects 25 million children per year globally, leading to 2.9 million deaths and substantial disability in survivors. Extensive characterization of interactions between the host and bacteria in children is required to design novel preventive and therapeutic strategies tailored to this age group. Vγ9Vδ2 T cells are the first T cells generated in humans. These cells are defined by the expression of Vγ9Vδ2 T-cell receptors (TCRs, using the TRGV9 and TRDV2 gene segments), which react strongly against the prototypical bacterial phosphoantigen HMBPP. We investigated this reactivity by analyzing the TCR δ (TRD) repertoire in the blood of 76 children (0-16 years) with blood culture-proven bacterial sepsis caused by HMBPP-positive Escherichia coli or by HMBPP-negative Staphylococcus aureus or by HMBPP-negative Streptococcus pneumoniae. Strikingly, we found that S. aureus, and to a lesser extent E. coli but not S. pneumoniae, shaped the TRDV2 repertoire in young children (<2 years) but not in older children or adults. This dichotomy was due to the selective expansion of a fetal TRDV2 repertoire. Thus, young children possess fetal-derived Vγ9Vδ2 T cells that are highly responsive toward specific bacterial pathogens.

Differentiation of Type 17 Mucosal-Associated Invariant T Cells in Circulation Contributes to the Severity of Sepsis.

Mucosal-associated invariant T (MAIT) cells are essential in defending against infection. Sepsis is a systemic inflammatory response to infection and a leading cause of death. The relationship between the overall competency of the host immune response and disease severity is not fully elucidated. This study identified a higher proportion of circulating MAIT17 with expression of IL-17A and retinoic acid receptor-related orphan receptor γt in patients with sepsis. The proportion of MAIT17 was correlated with the severity of sepsis. Single-cell RNA-sequencing analysis revealed an enhanced expression of lactate dehydrogenase A (LDHA) in MAIT17 in patients with sepsis. Cell-culture experiments demonstrated that phosphoinositide 3-kinase-LDHA signaling was required for retinoic acid receptor-related orphan receptor γt expression in MAIT17. Finally, the elevated levels of plasma IL-18 promoted the differentiation of circulating MAIT17 cells in sepsis. In summary, this study reveals a new role of circulating MAIT17 in promoting sepsis severity and suggests the phosphoinositide 3-kinase-LDHA signaling as a driving force in MAIT17 responses.

A combinatorial F box protein directed pathway controls TRAF adaptor stability to regulate inflammation.

Uncontrolled activation of tumor necrosis factor receptor-associated factor (TRAF) proteins may result in profound tissue injury by linking surface signals to cytokine release. Here we show that a ubiquitin E3 ligase component, Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by destabilizing a sentinel TRAF inhibitor, Fbxl2. Fbxo3 and TRAF protein in circulation positively correlated with cytokine responses in subjects with sepsis, and we identified a polymorphism in human Fbxo3, with one variant being hypofunctional. A small-molecule inhibitor targeting Fbxo3 was sufficient to lessen severity of cytokine-driven inflammation in several mouse disease models. These studies identified a pathway of innate immunity that may be useful to detect subjects with altered immune responses during critical illness or provide a basis for therapeutic intervention targeting TRAF protein abundance.