Homodimeric Granzyme A opsonizes Mycobacterium tuberculosis and inhibits its intracellular growth in human monocytes via TLR4 and CD14.

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete GzmA protective against intracellular Mtb growth. However, GzmA enzymatic activity is unnecessary for pathogen inhibition and the mechanisms of GzmA-mediated protection remain unknown. We show GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes and subsequent pathogen clearance within the phagolysosome. While monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind CD14 and TLR4. Without access to surface expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1, was associated with inhibitory activity. Further, GzmA colocalized with and was regulated by protein disulfide isomerase (PDI)A1, which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis and elimination of Mtb in human monocytes, and highlights PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

Quantitative cytofluorimetric analysis of mouse neutrophil extracellular traps.

The release of neutrophil extracellular traps (NETs) has been involved in numerous infectious and non-infectious diseases. Nevertheless, quantitative analysis of NETs in vivo has been challenging. Here, we present a protocol for NET quantification by flow cytometry in the bronchoalveolar lavage fluid (BALF) of mice upon pulmonary infection with S. aureus. We describe steps for bacteria growth and instillation and BALF recovery. We then detail staining to quantify the release of NETs and neutrophils recruited to the site of infection. For complete information on the generation and use of this protocol, please refer to Poli et al. (2021)1 and Poli et al. (2022).2.

Prioritized mass spectrometry increases the depth, sensitivity and data completeness of single-cell proteomics.

Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth of protein quantification, especially for proteins and modifications of biological interest. Here, to simultaneously advance all these aims, we developed prioritized Single-Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands of prioritized peptides across all single cells (thus increasing data completeness) while maximizing instrument time spent analyzing identifiable peptides, thus increasing proteome depth. These strategies increased the sensitivity, data completeness and proteome coverage over twofold. The gains enabled quantifying protein variation in untreated and lipopolysaccharide-treated primary macrophages. Within each condition, proteins covaried within functional sets, including phagosome maturation and proton transport, similarly across both treatment conditions. This covariation is coupled to phenotypic variability in endocytic activity. pSCoPE also enabled quantifying proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is freely available and widely applicable, especially for analyzing proteins of interest without sacrificing proteome coverage. Support for pSCoPE is available at http://scp.slavovlab.net/pSCoPE .

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia.

During gram-negative septicemia, interactions between platelets and neutrophils initiate a detrimental feedback loop that sustains neutrophil extracellular trap (NET) induction, disseminated intravascular coagulation, and inflammation. Understanding intracellular pathways that control platelet-neutrophil interactions is essential for identifying new therapeutic targets. Here, we found that thrombin signaling induced activation of the transcription factor NFAT in platelets. Using genetic and pharmacologic approaches, as well as iNFATuation, a newly developed mouse model in which NFAT activation can be abrogated in a cell-specific manner, we demonstrated that NFAT inhibition in activated murine and human platelets enhanced their activation and aggregation, as well as their interactions with neutrophils and NET induction. During gram-negative septicemia, NFAT inhibition in platelets promoted disease severity by increasing disseminated coagulation and NETosis. NFAT inhibition also partially restored coagulation ex vivo in patients with hypoactive platelets. Our results define non-transcriptional roles for NFAT that could be harnessed to address pressing clinical needs.

Zinc-dependent histone deacetylases drive neutrophil extracellular trap formation and potentiate local and systemic inflammation.

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) driven by viruses or bacteria, as well as in numerous immune-mediated disorders. Histone citrullination by the enzyme peptidylarginine deiminase 4 (PAD4) and the consequent decondensation of chromatin are hallmarks in the induction of NETs. Nevertheless, additional histone modifications that may govern NETosis are largely overlooked. Herein, we show that histone deacetylases (HDACs) play critical roles in driving NET formation in human and mouse neutrophils. HDACs belonging to the zinc-dependent lysine deacetylases family are necessary to deacetylate histone H3, thus allowing the activity of PAD4 and NETosis. Of note, HDAC inhibition in mice protects against microbial-induced pneumonia and septic shock, decreasing NETosis and inflammation. Collectively, our findings illustrate a new fundamental step that governs the release of NETs and points to HDAC inhibitors as therapeutic agents that may be used to protect against ARDS and sepsis.

Aged vasculature drives neutrophils mad.

Inflammaging drives age-related pathologies. In this issue of Immunity, Barkaway et al. illustrate how aged endothelial cells and mast cells promote reverse migration of neutrophils from inflamed tissue back into circulation, causing tissue damage at distal sites.

Dooming Phagocyte Responses: Inflammatory Effects of Endogenous Oxidized Phospholipids.

Endogenous oxidized phospholipids are produced during tissue stress and are responsible for sustaining inflammatory responses in immune as well as non-immune cells. Their local and systemic production and accumulation is associated with the etiology and progression of several inflammatory diseases, but the molecular mechanisms that underlie the biological activities of these oxidized phospholipids remain elusive. Increasing evidence highlights the ability of these stress mediators to modulate cellular metabolism and pro-inflammatory signaling in phagocytes, such as macrophages and dendritic cells, and to alter the activation and polarization of these cells. Because these immune cells serve a key role in maintaining tissue homeostasis and organ function, understanding how endogenous oxidized lipids reshape phagocyte biology and function is vital for designing clinical tools and interventions for preventing, slowing down, or resolving chronic inflammatory disorders that are driven by phagocyte dysfunction. Here, we discuss the metabolic and signaling processes elicited by endogenous oxidized lipids and outline new hypotheses and models to elucidate the impact of these lipids on phagocytes and inflammation.

Inositol 1,4,5-trisphosphate 3-kinase B promotes Ca2+ mobilization and the inflammatory activity of dendritic cells.

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca2+ mobilization. In DCs, Ca2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP3). Here, we showed that the IP3 receptor 3 (IP3R3) and ITPKB, a kinase that converts IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4), were both necessary for Ca2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP3R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca2+ mobilization through plasma membrane-localized IP3R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.

Endogenous oxidized phospholipids reprogram cellular metabolism and boost hyperinflammation.

Pathogen-associated molecular patterns (PAMPs) have the capacity to couple inflammatory gene expression to changes in macrophage metabolism, both of which influence subsequent inflammatory activities. Similar to their microbial counterparts, several self-encoded damage-associated molecular patterns (DAMPs) induce inflammatory gene expression. However, whether this symmetry in host responses between PAMPs and DAMPs extends to metabolic shifts is unclear. Here, we report that the self-encoded oxidized phospholipid oxPAPC alters the metabolism of macrophages exposed to lipopolysaccharide. While cells activated by lipopolysaccharide rely exclusively on glycolysis, macrophages exposed to oxPAPC also use mitochondrial respiration, feed the Krebs cycle with glutamine, and favor the accumulation of oxaloacetate in the cytoplasm. This metabolite potentiates interleukin-1ß production, resulting in hyperinflammation. Similar metabolic adaptions occur in vivo in hypercholesterolemic mice and human subjects. Drugs that interfere with oxPAPC-driven metabolic changes reduce atherosclerotic plaque formation in mice, thereby underscoring the importance of DAMP-mediated activities in pathophysiological conditions.

By Capturing Inflammatory Lipids Released from Dying Cells, the Receptor CD14 Induces Inflammasome-Dependent Phagocyte Hyperactivation.

A heterogeneous mixture of lipids called oxPAPC, derived from dying cells, can hyperactivate dendritic cells (DCs) but not macrophages. Hyperactive DCs are defined by their ability to release interleukin-1 (IL-1) while maintaining cell viability, endowing these cells with potent aptitude to stimulate adaptive immunity. Herein, we found that the bacterial lipopolysaccharide receptor CD14 captured extracellular oxPAPC and delivered these lipids into the cell to promote inflammasome-dependent DC hyperactivation. Notably, we identified two specific components within the oxPAPC mixture that hyperactivated macrophages, allowing these cells to release IL-1 for several days, by a CD14-dependent process. In murine models of sepsis, conditions that promoted cell hyperactivation resulted in inflammation but not lethality. Thus, multiple phagocytes are capable of hyperactivation in response to oxPAPC, with CD14 acting as the earliest regulator in this process, serving to capture and transport these lipids to promote inflammatory cell fate decisions.

Prolonged contact with dendritic cells turns lymph node-resident NK cells into anti-tumor effectors.

Natural killer (NK) cells are critical players against tumors. The outcome of anti-tumor vaccination protocols depends on the efficiency of NK-cell activation, and efforts are constantly made to manipulate them for immunotherapeutic approaches. Thus, a better understanding of NK-cell activation dynamics is needed. NK-cell interactions with accessory cells and trafficking between secondary lymphoid organs and tumoral tissues remain poorly characterized. Here, we show that upon triggering innate immunity with lipopolysaccharide (LPS), NK cells are transiently activated, leave the lymph node, and infiltrate the tumor, delaying its growth. Interestingly, NK cells are not actively recruited at the draining lymph node early after LPS administration, but continue their regular homeostatic turnover. Therefore, NK cells resident in the lymph node at the time of LPS administration become activated and exert anti-tumor functions. NK-cell activation correlates with the establishment of prolonged interactions with dendritic cells (DCs) in lymph nodes, as observed by two-photon microscopy. Close DC and NK-cell contacts are essential for the localized delivery of DC-derived IL-18 to NK cells, a strict requirement in NK-cell activation.

An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells.

Dendritic cells (DCs) use pattern recognition receptors to detect microorganisms and activate protective immunity. These cells and receptors are thought to operate in an all-or-nothing manner, existing in an immunologically active or inactive state. Here, we report that encounters with microbial products and self-encoded oxidized phospholipids (oxPAPC) induce an enhanced DC activation state, which we call "hyperactive." Hyperactive DCs induce potent adaptive immune responses and are elicited by caspase-11, an enzyme that binds oxPAPC and bacterial lipopolysaccharide (LPS). oxPAPC and LPS bind caspase-11 via distinct domains and elicit different inflammasome-dependent activities. Both lipids induce caspase-11-dependent interleukin-1 release, but only LPS induces pyroptosis. The cells and receptors of the innate immune system can therefore achieve different activation states, which may permit context-dependent responses to infection.

Toll-like receptor co-receptors as master regulators of the immune response.

Pattern recognition receptors (PRRs) are generally recognized as the initiators of all immune responses. PRRs bind molecular patterns associated with microorganisms or endogenous mediators released by stressed tissues. Upon ligand binding, PRRs induce the activation of an inflammatory process that ultimately leads to pathogen clearance or restoration of tissue homeostasis. PRRs govern these processes, regulating the activation of a complex network of transcription factors able to induce the appropriate immune response to a specific ligand. Toll-like-receptors (TLRs) are the first and best characterized PRR family, and for a long period of time they were believed to be autonomous proteins able to recognize and initiate all the immune response to a given stimulus. Recently this view was challenged by the discovery that so-called TLR co-receptors, such as CD14 and CD36, not only favor TLR-dependent signaling but can also transduce their own signal in a TLR-independent manner. Here we will discuss the capacity of TLR co-receptors to bind different microbial and endogenous ligands and to integrate TLR functions inducing specific signaling modules.

IL-15 cis presentation is required for optimal NK cell activation in lipopolysaccharide-mediated inflammatory conditions.

Natural killer (NK) cells have antitumor, antiviral, and antibacterial functions, and efforts are being made to manipulate them in immunotherapeutic approaches. However, their activation mechanisms remain poorly defined, particularly during bacterial infections. Here, we show that upon lipopolysaccharide or E. coli exposure, dendritic cells (DCs) produce three cytokines-interleukin 2 (IL-2), IL-18, and interferon ß (IFN-ß)-necessary and sufficient for NK cell activation. IFN-ß enhances NK cell activation by inducing IL-15 and IL-15 receptor α not only in DCs but, surprisingly, also in NK cells. This process allows the transfer of IL-15 on NK cell surface and its cis presentation. cis-presented NK cell-derived and trans-presented DC-derived IL-15 contribute equally to optimal NK cell activation.

CD14 and NFAT mediate lipopolysaccharide-induced skin edema formation in mice.

Inflammation is a multistep process triggered when innate immune cells - for example, DCs - sense a pathogen or injured cell or tissue. Edema formation is one of the first steps in the inflammatory response; it is fundamental for the local accumulation of inflammatory mediators. Injection of LPS into the skin provides a model for studying the mechanisms of inflammation and edema formation. While it is known that innate immune recognition of LPS leads to activation of numerous transcriptional activators, including nuclear factor of activated T cells (NFAT) isoforms, the molecular pathways that lead to edema formation have not been determined. As PGE2 regulates many proinflammatory processes, including swelling and pain, and it is induced by LPS, we hypothesized that PGE2 mediates the local generation of edema following LPS exposure. Here, we show that tissue-resident DCs are the main source of PGE2 and the main controllers of tissue edema formation in a mouse model of LPS-induced inflammation. LPS exposure induced expression of microsomal PGE synthase-1 (mPGES-1), a key enzyme in PGE2 biosynthesis. mPGES-1 activation, PGE2 production, and edema formation required CD14 (a component of the LPS receptor) and NFAT. Therefore, tissue edema formation induced by LPS is DC and CD14/NFAT dependent. Moreover, DCs can regulate free antigen arrival at the draining lymph nodes by controlling edema formation and interstitial fluid pressure in the presence of LPS. We therefore suggest that the CD14/NFAT/mPGES-1 pathway represents a possible target for antiinflammatory therapies.