SPA Promotes Atherosclerosis Through Mediating Macrophage Foam Cell Formation.

BACKGROUND: Atherosclerosis is a progressive inflammatory disease in which macrophage foam cells play a central role in disease pathogenesis. SPA (surfactant protein A) is a lipid-associating protein involved with regulating macrophage function in various inflammatory diseases. However, the role of SPA in atherosclerosis and macrophage foam cell formation has not been investigated. METHODS: SPA expression was assessed in healthy and atherosclerotic human coronary arteries and the brachiocephalic arteries of wild-type or ApoE-deficient mice fed high-fat diets for 4 weeks. Hypercholesteremic wild-type and SPA-deficient mice fed a high-fat diet for 6 weeks were investigated for atherosclerotic lesions in vivo. In vitro experiments using RAW264.7 macrophages, primary resident peritoneal macrophages extracted from wild-type or SPA-deficient mice, and human monocyte-derived macrophages from the peripheral blood of healthy donors determined the functional effects of SPA in macrophage foam cell formation. RESULTS: SPA expression was increased in atherosclerotic lesions in humans and ApoE-deficient mice and in response to a proatherosclerotic stimulus in vitro. SPA deficiency reduced the lipid profiles induced by hypercholesterolemia, attenuated atherosclerosis, and reduced the number of lesion-associated macrophage foam cells. In vitro studies revealed that SPA deficiency reduced intracellular cholesterol accumulation and macrophage foam cell formation. Mechanistically, SPA deficiency dramatically downregulated the expression of scavenger receptor CD36 (cluster of differentiation antigen 36) cellular and lesional expression. Importantly, SPA also increased CD36 expression in human monocyte-derived macrophages. CONCLUSIONS: Our results elucidate that SPA is a novel factor promoting atherosclerosis development. SPA enhances macrophage foam cell formation and atherosclerosis by increasing scavenger receptor CD36 expression, leading to increasing cellular OxLDL influx.

Multifaceted effects of Francisella tularensis on human neutrophil function and lifespan.

Francisella tularensis in an intracellular bacterial pathogen that causes a potentially lethal disease called tularemia. Studies performed nearly 100 years ago revealed that neutrophil accumulation in infected tissues correlates directly with the extent of necrotic damage during F. tularensis infection. However, the dynamics and details of bacteria-neutrophil interactions have only recently been studied in detail. Herein, we review current understanding regarding the mechanisms that recruit neutrophils to F. tularensis-infected lungs, opsonization and phagocytosis, evasion and inhibition of neutrophil defense mechanisms, as well as the ability of F. tularensis to prolong neutrophil lifespan. In addition, we discuss distinctive features of the bacterium, including its ability to act at a distance to alter overall neutrophil responsiveness to exogenous stimuli, and the evidence which suggests that macrophages and neutrophils play distinct roles in tularemia pathogenesis, such that macrophages are major vehicles for intracellular growth and dissemination, whereas neutrophils drive tissue destruction by dysregulation of the inflammatory response.

Bacterial lipoproteins and other factors released by Francisella tularensis modulate human neutrophil lifespan: Effects of a TLR1 SNP on apoptosis inhibition.

Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14 C] acetate-labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose-dependent neutrophil apoptosis inhibition via a TLR2-dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.

Cutting Edge: Helicobacter pylori Induces Nuclear Hypersegmentation and Subtype Differentiation of Human Neutrophils In Vitro.

Helicobacter pylori infects the human stomach and causes a spectrum of disease that includes gastritis, peptic ulcers, and gastric adenocarcinoma. A chronic, neutrophil-rich inflammatory response characterizes this infection. It is established that H. pylori stimulates neutrophil chemotaxis and a robust respiratory burst, but other aspects of this interaction are incompletely defined. We demonstrate here that H. pylori induces N1-like subtype differentiation of human neutrophils as indicated by profound nuclear hypersegmentation, a CD62Ldim, CD16bright, CD11bbright, CD66bbright, CD63bright surface phenotype, proinflammatory cytokine secretion, and cytotoxicity. Hypersegmentation requires direct neutrophil-H. pylori contact as well as transcription and both host and bacterial protein synthesis, but not urease, NapA, VacA, CagA, or CagT. The concept of neutrophil plasticity is new and, to our knowledge, these data are the first evidence that neutrophils can undergo subtype differentiation in vitro in response to bacterial pathogen infection. We hypothesize that these changes favor H. pylori persistence and disease.

Targeted inhibition of prostate cancer metastases with an RNA aptamer to prostate-specific membrane antigen.

Cell-targeted therapies (smart drugs), which selectively control cancer cell progression with limited toxicity to normal cells, have been developed to effectively treat some cancers. However, many cancers such as metastatic prostate cancer (PC) have yet to be treated with current smart drug technology. Here, we describe the thorough preclinical characterization of an RNA aptamer (A9g) that functions as a smart drug for PC by inhibiting the enzymatic activity of prostate-specific membrane antigen (PSMA). Treatment of PC cells with A9g results in reduced cell migration/invasion in culture and metastatic disease in vivo. Importantly, A9g is safe in vivo and is not immunogenic in human cells. Pharmacokinetic and biodistribution studies in mice confirm target specificity and absence of non-specific on/off-target effects. In conclusion, these studies provide new and important insights into the role of PSMA in driving carcinogenesis and demonstrate critical endpoints for the translation of a novel RNA smart drug for advanced stage PC.

Natural IgM mediates complement-dependent uptake of Francisella tularensis by human neutrophils via complement receptors 1 and 3 in nonimmune serum.

A fundamental step in the life cycle of Francisella tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum nor the receptors that mediate infection of neutrophils have been defined. In this study, human neutrophil uptake of GFP-expressing F. tularensis strains live vaccine strain and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components, we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis, whereas C5 was not. Second, we used purification and immunodepletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-Ag and capsule as prominent targets of these Abs on the bacterial surface. Finally, we demonstrate using receptor-blocking Abs that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-Ag polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3 opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner.

Francisella tularensis inhibits the intrinsic and extrinsic pathways to delay constitutive apoptosis and prolong human neutrophil lifespan.

Francisella tularensis is a facultative intracellular bacterium that infects many cell types, including neutrophils. We demonstrated previously that F. tularensis inhibits NADPH oxidase assembly and activity and then escapes the phagosome to the cytosol, but effects on other aspects of neutrophil function are unknown. Neutrophils are short-lived cells that undergo constitutive apoptosis, and phagocytosis typically accelerates this process. We now demonstrate that F. tularensis significantly inhibited neutrophil apoptosis as indicated by morphologic analysis as well as annexin V and TUNEL staining. Thus, ∼80% of infected neutrophils remained viable at 48 h compared with ∼50% of control cells, and ∼40% of neutrophils that ingested opsonized zymosan. In keeping with this finding, processing and activation of procaspases-8, -9, and -3 were markedly diminished and delayed. F. tularensis also significantly impaired apoptosis triggered by Fas crosslinking. Of note, these effects were dose dependent and could be conferred by either intracellular or extracellular live bacteria, but not by formalin-killed organisms or isolated LPS and capsule, and were not affected by disruption of wbtA2 or FTT1236/FTL0708-genes required for LPS O-antigen and capsule biosynthesis. In summary, we demonstrate that F. tularensis profoundly impairs constitutive neutrophil apoptosis via effects on the intrinsic and extrinsic pathways, and thereby define a new aspect of innate immune evasion by this organism. As defects in neutrophil turnover prevent resolution of inflammation, our findings also suggest a mechanism that may in part account for the neutrophil accumulation, granuloma formation, and severe tissue damage that characterizes lethal pneumonic tularemia.

Disruption of Francisella tularensis Schu S4 iglI, iglJ, and pdpC genes results in attenuation for growth in human macrophages and in vivo virulence in mice and reveals a unique phenotype for pdpC.

Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10(8) CFU iglJ- or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype.

The Francisella tularensis migR, trmE, and cphA genes contribute to F. tularensis pathogenicity island gene regulation and intracellular growth by modulation of the stress alarmone ppGpp.

The Francisella tularensis pathogenicity island (FPI) encodes many proteins that are required for virulence. Expression of these genes depends upon the FevR (PigR) regulator and its interactions with the MglA/SspA and RNA polymerase transcriptional complex. Experiments to identify how transcription of the FPI genes is activated have led to identification of mutations within the migR, trmE, and cphA genes that decrease FPI expression. Recent data demonstrated that the small alarmone ppGpp, produced by RelA and SpoT, is important for stabilizing MglA/SspA and FevR (PigR) interactions in Francisella. Production of ppGpp is commonly known to be activated by cellular and nutritional stress in bacteria, which indicates that cellular and nutritional stresses act as important signals for FPI activation. In this work, we demonstrate that mutations in migR, trmE, or cphA significantly reduce ppGpp accumulation. The reduction in ppGpp levels was similar for each of the mutants and correlated with a corresponding reduction in iglA reporter expression. In addition, we observed that there were differences in the ability of each of these mutants to replicate within various mammalian cells, indicating that the migR, trmE, and cphA genes are likely parts of different cellular stress response pathways in Francisella. These results also indicate that different nutritional and cellular stresses exist in different mammalian cells. This work provides new information to help understand how Francisella regulates its virulence genes in response to host cell environments, and it contributes to our growing knowledge of this highly successful bacterial pathogen.

Metabolic Reprogramming Mediates Delayed Apoptosis of Human Neutrophils Infected With Francisella tularensis.

Neutrophils (polymorphonuclear leukocytes, PMNs) have a distinctively short lifespan, and tight regulation of cell survival and death is imperative for their normal function. We demonstrated previously that Francisella tularensis extends human neutrophil lifespan, which elicits an impaired immune response characterized by neutrophil dysfunction. Herein, we extended these studies, including our transcriptional profiling data, and employed Seahorse extracellular flux analysis, gas chromatography-mass spectrometry metabolite analysis, flow cytometry and several other biochemical approaches to demonstrate that the delayed apoptosis observed in F. tularensis-infected neutrophils is mediated, in part, by metabolic reprogramming. Specifically, we show that F. tularensis-infected neutrophils exhibited a unique metabolic signature characterized by increased glycolysis, glycolytic flux and glucose uptake, downregulation of the pentose phosphate pathway, and complex glycogen dynamics. Glucose uptake and glycolysis were essential for cell longevity, although glucose-6-phosphate translocation into the endoplasmic reticulum was not, and we identify depletion of glycogen as a potential trigger of apoptosis onset. In keeping with this, we also demonstrate that ablation of apoptosis with the pan-caspase inhibitor Q-VD-OPh was sufficient to profoundly increase glycolysis and glycogen stores in the absence of infection. Taken together, our data significantly advance understanding of neutrophil immunometabolism and its capacity to regulate cell lifespan.

Neutrophil Survival Signaling During Francisella tularensis Infection.

Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover via constitutive apoptosis is essential for control of infection and resolution of inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen Francisella tularensis markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in F. tularensis-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of F. tularensis-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.

Helicobacter pylori-infected human neutrophils exhibit impaired chemotaxis and a uropod retraction defect.

Helicobacter pylori is a major human pathogen that colonizes the gastric mucosa and plays a causative role in development of peptic ulcers and gastric cancer. Neutrophils are heavily infected with this organism in vivo and play a prominent role in tissue destruction and disease. Recently, we demonstrated that H. pylori exploits neutrophil plasticity as part of its virulence strategy eliciting N1-like subtype differentiation that is notable for profound nuclear hypersegmentation. We undertook this study to test the hypothesis that hypersegmentation may enhance neutrophil migratory capacity. However, EZ-TAXIScan™ video imaging revealed a previously unappreciated and progressive chemotaxis defect that was apparent prior to hypersegmentation onset. Cell speed and directionality were significantly impaired to fMLF as well as C5a and IL-8. Infected cells oriented normally in chemotactic gradients, but speed and direction were impaired because of a uropod retraction defect that led to cell elongation, nuclear lobe trapping in the contracted rear and progressive narrowing of the leading edge. In contrast, chemotactic receptor abundance, adhesion, phagocytosis and other aspects of cell function were unchanged. At the molecular level, H. pylori phenocopied the effects of Blebbistatin as indicated by aberrant accumulation of F-actin and actin spikes at the uropod together with enhanced ROCKII-mediated phosphorylation of myosin IIA regulatory light chains at S19. At the same time, RhoA and ROCKII disappeared from the cell rear and accumulated at the leading edge whereas myosin IIA was enriched at both cell poles. These data suggest that H. pylori inhibits the dynamic changes in myosin IIA contractility and front-to-back polarity that are essential for chemotaxis. Taken together, our data advance understanding of PMN plasticity and H. pylori pathogenesis.

Francisella tularensis Schu S4 O-antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages.

Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.

Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes.

Flavocytochrome b(558), the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91(phox) (NOX2) and a smaller subunit, p22(phox). Although in neutrophils flavocytochrome b has been shown to localize to the plasma membrane and specific granules, little is known about its distribution in macrophages. Using immunofluorescent staining and live cell imaging of fluorescently tagged gp91(phox) and p22(phox), we demonstrate in a Chinese hamster ovary cell model system and in RAW 264.7 and primary murine bone marrow-derived macrophages that flavocytochrome b is found in the Rab11-positive recycling endocytic compartment, as well as in Rab5-positive early endosomes and plasma membrane. Additionally, we show that unassembled p22(phox) and gp91(phox) subunits localize to the endoplasmic reticulum, which redistribute to the cell surface and endosomal compartments following heterodimer formation. These studies show for the first time that flavocytochrome b localizes to intracellular compartments in macrophages that recycle to the plasma membrane, which may act as a reservoir to deliver flavocytochrome b to the cell surface and phagosome membranes.

Microtubules and Dynein Regulate Human Neutrophil Nuclear Volume and Hypersegmentation During H. pylori Infection.

Neutrophils (also called polymorphonuclear leukocytes, PMNs) are heterogeneous and can exhibit considerable phenotypic and functional plasticity. In keeping with this, we discovered previously that Helicobacter pylori infection induces N1-like subtype differentiation of human PMNs that is notable for profound nuclear hypersegmentation. Herein, we utilized biochemical approaches and confocal and super-resolution microscopy to gain insight into the underlying molecular mechanisms. Sensitivity to inhibition by nocodazole and taxol indicated that microtubule dynamics were required to induce and sustain hypersegmentation, and super-resolution Stimulated Emission Depletion (STED) imaging demonstrated that microtubules were significantly more abundant and longer in hypersegmented cells. Dynein activity was also required, and enrichment of this motor protein at the nuclear periphery was enhanced following H. pylori infection. In contrast, centrosome splitting did not occur, and lamin B receptor abundance and ER morphology were unchanged. Finally, analysis of STED image stacks using Imaris software revealed that nuclear volume increased markedly prior to the onset of hypersegmentation and that nuclear size was differentially modulated by nocodazole and taxol in the presence and absence of infection. Taken together, our data define a new mechanism of hypersegmentation that is mediated by microtubules and dynein and as such advance understanding of processes that regulate nuclear morphology.

Non-Randomized Trial of Dornase Alfa for Acute Respiratory Distress Syndrome Secondary to Covid-19.

Background: The most severe cases of Coronavirus-Disease-2019 (COVID-19) develop into Acute Respiratory Distress Syndrome (ARDS). It has been proposed that oxygenation may be inhibited by extracellular deoxyribonucleic acid (DNA) in the form of neutrophil extracellular traps (NETs). Dornase alfa (Pulmozyme, Genentech) is recombinant human deoxyribonuclease I that acts as a mucolytic by cleaving and degrading extracellular DNA. We performed a pilot study to evaluate the effects of dornase alfa in patients with ARDS secondary to COVID-19. Methods: We performed a pilot, non-randomized, case-controlled clinical trial of inhaled dornase for patients who developed ARDS secondary to COVID-19 pneumonia. Results: Improvement in arterial oxygen saturation to inhaled fraction of oxygen ratio (PaO2/FiO2) was noted in the treatment group compared to control at day 2 (95% CI, 2.96 to 95.66, P-value = 0.038), as well as in static lung compliance at days 3 through 5 (95% CI, 4.8 to 19.1 mL/cmH2O, 2.7 to 16.5 mL/cmH2O, and 5.3 to 19.2 mL/cmH2O, respectively). These effects were not sustained at 14 days. A reduction in bronchoalveolar lavage fluid (BALF) myeloperoxidase-DNA (DNA : MPO) complexes (95% CI, -14.7 to -1.32, P-value = 0.01) was observed after therapy with dornase alfa. Conclusion: Treatment with dornase alfa was associated with improved oxygenation and decreased DNA : MPO complexes in BALF. The positive effects, however, were limited to the time of drug delivery. These data suggest that degradation of extracellular DNA associated with NETs or other structures by inhaled dornase alfa can be beneficial. We propose a more extensive clinical trial is warranted. Clinical Trial Registration: ClinicalTrials.gov, Identifier: NCT04402970.

IL-5 mediates monocyte phenotype and pain outcomes in fibromyalgia.

ABSTRACT: Fibromyalgia (FM) is characterized by widespread chronic pain, fatigue, and somatic symptoms. The influence of phenotypic changes in monocytes on symptoms associated with FM is not fully understood. The primary aim of this study was to take a comprehensive whole-body to molecular approach in characterizing relationships between monocyte phenotype and FM symptoms in relevant clinical populations. Lipopolysaccharide-evoked and spontaneous secretion of IL-5 and other select cytokines from circulating monocytes was higher in women with FM compared to women without pain. In addition, greater secretion of IL-5 was significantly associated with pain and other clinically relevant psychological and somatic symptoms of FM. Furthermore, higher levels of pain and pain-related symptoms were associated with a lower percentage of intermediate monocytes (CD14++/CD16+) and a greater percentage of nonclassical monocytes (CD14+/CD16++) in women with FM. Based on findings from individuals with FM, we examined the role of IL-5, an atypical cytokine secreted from monocytes, in an animal model of widespread muscle pain. Results from the animal model show that IL-5 produces analgesia and polarizes monocytes toward an anti-inflammatory phenotype (CD206+). Taken together, our data suggest that monocyte phenotype and their cytokine profiles are associated with pain-related symptoms in individuals with FM. Furthermore, our data show that IL-5 has a potential role in analgesia in an animal model of FM. Thus, targeting anti-inflammatory cytokines such as IL-5 secreted by circulating leukocytes could serve as a promising intervention to control pain and other somatic symptoms associated with FM.

Mechanisms Driving Neutrophil-Induced T-cell Immunoparalysis in Ovarian Cancer.

T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.See related Spotlight by Cassatella, p. 725.