The Emerging Role of B Lymphocytes in Cardiovascular Disease.

B cells are traditionally known for their ability to produce antibodies in the context of adaptive immune responses. However, over the last decade B cells have been increasingly recognized as modulators of both adaptive and innate immune responses, as well as players in an important role in the pathogenesis of a variety of human diseases. Here, after briefly summarizing our current understanding of B cell biology, we present a systematic review of the literature from both animal models and human studies that highlight the important role that B lymphocytes play in cardiac and vascular disease. While many aspects of B cell biology in the vasculature and, to an even greater extent, in the heart remain unclear, B cells are emerging as key regulators of cardiovascular adaptation to injury.

Mast Cells in Inflammation and Disease: Recent Progress and Ongoing Concerns.

Mast cells have existed long before the development of adaptive immunity, although they have been given different names. Thus, in the marine urochordate Styela plicata, they have been designated as test cells. However, based on their morphological characteristics (including prominent cytoplasmic granules) and mediator content (including heparin, histamine, and neutral proteases), test cells are thought to represent members of the lineage known in vertebrates as mast cells. So this lineage presumably had important functions that preceded the development of antibodies, including IgE. Yet mast cells are best known, in humans, as key sources of mediators responsible for acute allergic reactions, notably including anaphylaxis, a severe and potentially fatal IgE-dependent immediate hypersensitivity reaction to apparently harmless antigens, including many found in foods and medicines. In this review, we briefly describe the origins of tissue mast cells and outline evidence that these cells can have beneficial as well as detrimental functions, both innately and as participants in adaptive immune responses. We also discuss aspects of mast cell heterogeneity and comment on how the plasticity of this lineage may provide insight into its roles in health and disease. Finally, we consider some currently open questions that are yet unresolved.

Inflammasome-independent regulation of IL-1-family cytokines.

Induction, production, and release of proinflammatory cytokines are essential steps to establish an effective host defense. Cytokines of the interleukin-1 (IL-1) family induce inflammation and regulate T lymphocyte responses while also displaying homeostatic and metabolic activities. With the exception of the IL-1 receptor antagonist, all IL-1 family cytokines lack a signal peptide and require proteolytic processing into an active molecule. One such unique protease is caspase-1, which is activated by protein platforms called the inflammasomes. However, increasing evidence suggests that inflammasomes and caspase-1 are not the only mechanism for processing IL-1 cytokines. IL-1 cytokines are often released as precursors and require extracellular processing for activity. Here we review the inflammasome-independent enzymatic processes that are able to activate IL-1 cytokines, paying special attention to neutrophil-derived serine proteases, which subsequently induce inflammation and modulate host defense. The inflammasome-independent processing of IL-1 cytokines has important consequences for understanding inflammatory diseases, and it impacts the design of IL-1-based modulatory therapies.

Molecular mechanisms in genetically defined autoinflammatory diseases: disorders of amplified danger signaling.

Patients with autoinflammatory diseases present with noninfectious fever flares and systemic and/or disease-specific organ inflammation. Their excessive proinflammatory cytokine and chemokine responses can be life threatening and lead to organ damage over time. Studying such patients has revealed genetic defects that have helped unravel key innate immune pathways, including excessive IL-1 signaling, constitutive NF-κB activation, and, more recently, chronic type I IFN signaling. Discoveries of monogenic defects that lead to activation of proinflammatory cytokines have inspired the use of anticytokine-directed treatment approaches that have been life changing for many patients and have led to the approval of IL-1-blocking agents for a number of autoinflammatory conditions. In this review, we describe the genetically characterized autoinflammatory diseases, we summarize our understanding of the molecular pathways that drive clinical phenotypes and that continue to inspire the search for novel treatment targets, and we provide a conceptual framework for classification.

Regulatory T cells function in established systemic inflammation and reverse fatal autoimmunity.

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss of function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where a broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of rescued Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad-spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.

Does tissue imprinting restrict macrophage plasticity?

Macrophages have long been considered as particularly plastic cells. However, recent work combining fate mapping, single-cell transcriptomics and epigenetics has undermined the macrophage plasticity dogma. Here, we discuss recent studies that have carefully dissected the response of individual macrophage subsets to pulmonary insults and call for an adjustment of the macrophage plasticity concept. We hypothesize that prolonged tissue residency shuts down much of the plasticity of macrophages and propose that the restricted plasticity of resident macrophages has been favored by evolution to safeguard tissue homeostasis. Recruited monocytes are more plastic and their differentiation into resident macrophages during inflammation can result in a dual imprinting from both the ongoing inflammation and the macrophage niche. This results in inflammation-imprinted resident macrophages, and we speculate that rewired niche circuits could maintain this inflammatory state. We believe that this revisited plasticity model offers opportunities to reset the macrophage pool after a severe inflammatory episode.

Intracellular immune sensing promotes inflammation via gasdermin D-driven release of a lectin alarmin.

Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.

Alveolar macrophages are epigenetically altered after inflammation, leading to long-term lung immunoparalysis.

Sepsis and trauma cause inflammation and elevated susceptibility to hospital-acquired pneumonia. As phagocytosis by macrophages plays a critical role in the control of bacteria, we investigated the phagocytic activity of macrophages after resolution of inflammation. After resolution of primary pneumonia, murine alveolar macrophages (AMs) exhibited poor phagocytic capacity for several weeks. These paralyzed AMs developed from resident AMs that underwent an epigenetic program of tolerogenic training. Such adaptation was not induced by direct encounter of the pathogen but by secondary immunosuppressive signals established locally upon resolution of primary infection. Signal-regulatory protein α (SIRPα) played a critical role in the establishment of the microenvironment that induced tolerogenic training. In humans with systemic inflammation, AMs and also circulating monocytes still displayed alterations consistent with reprogramming six months after resolution of inflammation. Antibody blockade of SIRPα restored phagocytosis in monocytes of critically ill patients in vitro, which suggests a potential strategy to prevent hospital-acquired pneumonia.

SERPINB1-mediated checkpoint of inflammatory caspase activation.

Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation. While the reactive center loop of SERPINB1 inhibits neutrophil serine proteases, its carboxy-terminal CARD-binding motif restrained the activation of pro-caspase-1/-4/-5/-11. Consequently, knockdown or deletion of SERPINB1 prompted spontaneous activation of caspase-1/-4/-5/-11, release of the cytokine IL-1ß and pyroptosis, inducing elevated inflammation after non-hygienic co-housing with pet-store mice and enhanced sensitivity to lipopolysaccharide- or Acinetobacter baumannii-induced endotoxemia. Our results reveal that SERPINB1 acts as a vital gatekeeper of inflammation by restraining neutrophil serine proteases and inflammatory caspases in a genetically and functionally separable manner.

MTHFD2 is a metabolic checkpoint controlling effector and regulatory T cell fate and function.

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

Prenatal interleukin 6 elevation increases glutamatergic synapse density and disrupts hippocampal connectivity in offspring.

Early prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.

Resident macrophage-dependent immune cell scaffolds drive anti-bacterial defense in the peritoneal cavity.

Peritoneal immune cells reside unanchored within the peritoneal fluid in homeostasis. Here, we examined the mechanisms that control bacterial infection in the peritoneum using a mouse model of abdominal sepsis following intraperitoneal Escherichia coli infection. Whole-mount immunofluorescence and confocal microscopy of the peritoneal wall and omentum revealed that large peritoneal macrophages (LPMs) rapidly cleared bacteria and adhered to the mesothelium, forming multilayered cellular aggregates composed by sequentially recruited LPMs, B1 cells, neutrophils, and monocyte-derived cells (moCs). The formation of resident macrophage aggregates (resMφ-aggregates) required LPMs and thrombin-dependent fibrin polymerization. E. coli infection triggered LPM pyroptosis and release of inflammatory mediators. Resolution of these potentially inflammatory aggregates required LPM-mediated recruitment of moCs, which were essential for fibrinolysis-mediated resMφ-aggregate disaggregation and the prevention of peritoneal overt inflammation. Thus, resMφ-aggregates provide a physical scaffold that enables the efficient control of peritoneal infection, with implications for antimicrobial immunity in other body cavities, such as the pleural cavity or brain ventricles.

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.

Uncoupling of macrophage inflammation from self-renewal modulates host recovery from respiratory viral infection.

Tissue macrophages self-renew during homeostasis and produce inflammatory mediators upon microbial infection. We examined the relationship between proliferative and inflammatory properties of tissue macrophages by defining the impact of the Wnt/ß-catenin pathway, a central regulator of self-renewal, in alveolar macrophages (AMs). Activation of ß-catenin by Wnt ligand inhibited AM proliferation and stemness, but promoted inflammatory activity. In a murine influenza viral pneumonia model, ß-catenin-mediated AM inflammatory activity promoted acute host morbidity; in contrast, AM proliferation enabled repopulation of reparative AMs and tissue recovery following viral clearance. Mechanistically, Wnt treatment promoted ß-catenin-HIF-1α interaction and glycolysis-dependent inflammation while suppressing mitochondrial metabolism and thereby, AM proliferation. Differential HIF-1α activities distinguished proliferative and inflammatory AMs in vivo. This ß-catenin-HIF-1α axis was conserved in human AMs and enhanced HIF-1α expression associated with macrophage inflammation in COVID-19 patients. Thus, inflammatory and reparative activities of lung macrophages are regulated by ß-catenin-HIF-1α signaling, with implications for the treatment of severe respiratory diseases.

Notch4 signaling limits regulatory T-cell-mediated tissue repair and promotes severe lung inflammation in viral infections.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.

Citrulline depletion by ASS1 is required for proinflammatory macrophage activation and immune responses.

Citrulline can be converted into argininosuccinate by argininosuccinate synthetase (ASS1) in the urea cycle and the citrulline-nitric oxide cycle. However, the regulation and biological function of citrulline metabolism remain obscure in the immune system. Unexpectedly, we found that macrophage citrulline declines rapidly after interferon gamma (IFN-γ) and/or lipopolysaccharide (LPS) stimulation, which is required for efficient proinflammatory signaling activation. Mechanistically, IFN-γ and/or LPS stimulation promotes signal transducers and activators of transcription 1 (STAT1)-mediated ASS1 transcription and Janus kinase2 (JAK2)-mediated phosphorylation of ASS1 at tyrosine 87, thereby leading to citrulline depletion. Reciprocally, increased citrulline directly binds to JAK2 and inhibits JAK2-STAT1 signaling. Blockage of ASS1-mediated citrulline depletion suppresses the host defense against bacterial infection in vivo. We therefore define a central role for ASS1 in controlling inflammatory macrophage activation and antibacterial defense through depletion of cellular citrulline and, further, identify citrulline as an innate immune-signaling metabolite that engages a metabolic checkpoint for proinflammatory responses.

The signaling adaptor TRAF1 negatively regulates Toll-like receptor signaling and this underlies its role in rheumatic disease.

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis-associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex, SHARPIN, HOIP and HOIL-1, to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production, independently of tumor necrosis factor. Consistent with this, Traf1-/- mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling, providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP.

Evolution and Diversity of Immune Responses during Acute HIV Infection.

Understanding the earliest immune responses following HIV infection is critical to inform future vaccines and therapeutics. Here, we review recent prospective human studies in at-risk populations that have provided insight into immune responses during acute infection, including additional relevant data from non-human primate (NHP) studies. We discuss the timing, nature, and function of the diverse immune responses induced, the onset of immune dysfunction, and the effects of early anti-retroviral therapy administration. Treatment at onset of viremia mitigates peripheral T and B cell dysfunction, limits seroconversion, and enhances cellular antiviral immunity despite persistence of infection in lymphoid tissues. We highlight pertinent areas for future investigation, and how application of high-throughput technologies, alongside targeted NHP studies, may elucidate immune response features to target in novel preventions and cures.