Listening for rain: Principal component analysis and linear discriminant analysis for broadband acoustic rainfall detection.

Rain falling on the ocean creates acoustic signals. Ma and Nystuen [(2005). J. Atmos. Oceanic Technol. 22, 1225-1248] described an algorithm that compares three narrowband "discriminant" frequencies to detect rain. In 2022, Trucco, Bozzano, Fava, Pensieri, Verri, and Barla [(2022). IEEE J. Oceanic Eng. 47(1), 213-225] investigated rain detection algorithms that use broadband spectral data averaged over 1 h. This paper implements a rainfall detector that uses broadband acoustic data at 3-min time resolution. Principal Component Analysis (PCA) reduces the dimensionality of the broadband data. Rainfall is then detected via a Linear Discriminant Analysis (LDA) on the data's principal component projections. This PCA/LDA algorithm was trained and tested on 5 months of data recorded by hydrophones in a shallow noisy cove, where it was not feasible to average spectral data over 1 h. The PCA/LDA algorithm successfully detected 78 ± 5% of all rain events over 1 mm/h, and 73 ± 5% of all rain events over 0.1 mm/h, for a false alarm rate of ≈ 1% in both cases. By contrast, the Ma and Nystuen algorithm detected 32 ± 5% of the rain events over 1.0 mm/h when run on the same data, for a comparable false alarm rate.

Clearance of apoptotic neutrophils and resolution of inflammation.

The engulfment of apoptotic cells by phagocytes, a process referred to as efferocytosis, is essential for maintenance of normal tissue homeostasis and a prerequisite for the resolution of inflammation. Neutrophils are the predominant circulating white blood cell in humans, and contain an arsenal of toxic substances that kill and degrade microbes. Neutrophils are short-lived and spontaneously die by apoptosis. This review will highlight how the engulfment of apoptotic neutrophils by human phagocytes occurs, how heterogeneity of phagocyte populations influences efferocytosis signaling, and downstream consequences of efferocytosis. The efferocytosis of apoptotic neutrophils by macrophages promotes anti-inflammatory signaling, prevents neutrophil lysis, and dampens immune responses. Given the immunomodulatory properties of efferocytosis, understanding pathways that regulate and enhance efferocytosis could be harnessed to combat infection and chronic inflammatory conditions.

Lysis of human neutrophils by community-associated methicillin-resistant Staphylococcus aureus.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes infections associated with extensive tissue damage and necrosis. In vitro, human neutrophils fed CA-MRSA lyse by an unknown mechanism that is inhibited by necrostatin-1, an allosteric inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK-1). RIPK-1 figures prominently in necroptosis, a specific form of programmed cell death dependent on RIPK-1, RIPK-3, and the mixed-lineage kinase-like protein (MLKL). We previously reported that necrostatin-1 inhibits lysis of human neutrophils fed CA-MRSA and attributed the process to necroptosis. We now extend our studies to examine additional components in the programmed cell death pathway to test the hypothesis that neutrophils fed CA-MRSA undergo necroptosis. Lysis of neutrophils fed CA-MRSA was independent of tumor necrosis factor α, active RIPK-1, and MLKL, but dependent on active RIPK-3. Human neutrophils fed CA-MRSA lacked phosphorylated RIPK-1, as well as phosphorylated or oligomerized MLKL. Neutrophils fed CA-MRSA possessed cytoplasmic complexes that included inactive caspase 8, RIPK-1, and RIPK-3, and the composition of the complex remained stable over time. Together, these data suggest that neutrophils fed CA-MRSA underwent a novel form of lytic programmed cell death via a mechanism that required RIPK-3 activity, but not active RIPK-1 or MLKL, and therefore was distinct from necroptosis. Targeting the molecular pathways that culminate in lysis of neutrophils during CA-MRSA infection may serve as a novel therapeutic intervention to limit the associated tissue damage.

Phagocytosis of Staphylococcus aureus by human neutrophils prevents macrophage efferocytosis and induces programmed necrosis.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pose a significant threat to human health. Polymorphonuclear leukocytes (PMN) are the first responders during staphylococcal infection, but 15-50% of the initial ingested inoculum survives within the PMN phagosome and likely contributes directly or indirectly to disease pathogenesis. We hypothesize that surviving intracellular CA-MRSA undermine effective phagocyte-mediated defense by causing a decrease in macrophage uptake of PMN containing viable S. aureus and by promoting PMN lysis. In support of this hypothesis, PMN harboring viable CA-MRSA strain USA300 (PMN-SA) upregulated the "don't eat me" signal CD47, remained bound to the surface, and were inefficiently ingested by macrophages. In addition, coculture with PMN-SA altered the macrophage phenotype. Compared to macrophages fed USA300 alone, macrophages challenged with PMN-SA produced more IL-8 and less IL-1 receptor antagonist, TNF-α, activated caspase-1, and IL-1ß. Although they exhibited some features of apoptosis within 3 h following ingestion of S. aureus, including phosphatidylserine exposure and mitochondrial membrane depolarization, PMN-SA had sustained levels of proliferating cell nuclear Ag expression, absence of caspase activation, and underwent lysis within 6 h following phagocytosis. PMN lysis was dependent on receptor-interacting protein 1, suggesting that PMN-SA underwent programmed necrosis or necroptosis. These data are the first demonstration, to our knowledge, that bacteria can promote sustained expression of proliferating cell nuclear Ag and that human PMN undergo necroptosis. Together, these findings demonstrate that S. aureus surviving within PMN undermine the innate immune response and may provide insight into the pathogenesis of S. aureus disease.

In response to bacteria, neutrophils release extracellular vesicles capable of initiating thrombin generation through DNA-dependent and independent pathways.

Neutrophils release extracellular vesicles (EVs) and some subsets of neutrophil-derived EVs are procoagulant. In response to S. aureus, neutrophils produce EVs that associate electrostatically with neutrophil extracellular traps (NETs). DNA in NETs is procoagulant, but whether neutrophil EVs produced during bacterial challenge have similar activity is unknown. Given that EV activity is agonist- and cell-type dependent and coagulation contributes to sepsis, we hypothesized that sepsis-causing bacteria increase production of neutrophil-derived EVs, as well as EV-associated DNA, and intact EVs and DNA cause coagulation. We recovered EVs from neutrophils challenged with S. aureus (SA), S. epidermidis (SE), E. coli (EC), and P. aeruginosa (PA), and measured associated DNA and procoagulant activity. EVs from SA-challenged neutrophils (SA-EVs), which were previously characterized, displayed dose-dependent procoagulant activity as measured by thrombin generation (TG) in platelet-poor plasma. EV lysis and DNase treatment reduced TG by 90% and 37%, respectively. SE, EC, and PA also increased EV production and EV-associated extracellular DNA, and these EVs were also procoagulant. Compared to spontaneously released EVs, which demonstrated some ability to amplify Factor XII-dependent coagulation in the presence of an activator, only EVs produced in response to bacteria could initiate the pathway. SA-EVs and SE-EVs had more surface-associated DNA than EC-EVs and PA-EVs, and SA-EVs and SE-EVs contributed to initiation and amplification of TG in a DNA-dependent manner. However, DNA on EC- or PA-EVs played no role, suggesting that neutrophils release procoagulant EVs which can activate the coagulation cascade through both DNA-dependent and independent mechanisms.

Membrane-associated CD93 regulates leukocyte migration and C1q-hemolytic activity during murine peritonitis.

CD93 is emerging as a novel regulator of inflammation; however, its molecular function is unknown. CD93 exists as a membrane-associated glycoprotein on the surface of cells involved in the inflammatory cascade, including endothelial and myeloid cells. A soluble form (sCD93) is detectable in blood and is elevated with inflammation. In this study, we demonstrate heightened susceptibility to thioglycollate-induced peritonitis in CD93(-/-) mice. CD93(-/-) mice showed a 1.6-1.8-fold increase in leukocyte infiltration during thioglycollate-induced peritonitis between 3 and 24 h that returned to wild type levels by 96 h. Impaired vascular integrity in CD93(-/-) mice during peritonitis was demonstrated using fluorescence multiphoton intravital microscopy; however, no differences in cytokine or chemokine levels were detected with Luminex Multiplex or ELISA analysis. C1q-hemolytic activity in CD93(-/-) mice was decreased by 22% at time zero and by 46% 3 h after thioglycollate injection, suggesting a defect in the classical complement pathway. Leukocyte recruitment and C1q-hemolytic activity was restored to wild type levels when CD93 was expressed on either hematopoietic cells or nonhematopoietic cells in bone marrow chimeric mice. However, elevated levels of sCD93 in inflammatory fluid were observed only when CD93 was expressed on nonhematopoietic cells. Because cell-associated CD93 was sufficient to restore a normal inflammatory response, these data suggest that cell-associated CD93, and not sCD93, regulates leukocyte recruitment and complement activation during murine peritonitis.

Immune Targeting of Mycobacteria through Cell Surface Glycan Engineering.

Mycobacteria and other organisms in the order Mycobacteriales cause a range of significant human diseases, including tuberculosis, leprosy, diphtheria, Buruli ulcer, and non-tuberculous mycobacterial (NTM) disease. However, the intrinsic drug tolerance engendered by the mycobacterial cell envelope undermines conventional antibiotic treatment and contributes to acquired drug resistance. Motivated by the need to augment antibiotics with novel therapeutic approaches, we developed a strategy to specifically decorate mycobacterial cell surface glycans with antibody-recruiting molecules (ARMs), which flag bacteria for binding to human-endogenous antibodies that enhance macrophage effector functions. Mycobacterium-specific ARMs consisting of a trehalose targeting moiety and a dinitrophenyl hapten (Tre-DNPs) were synthesized and shown to specifically incorporate into outer-membrane glycolipids of Mycobacterium smegmatis via trehalose metabolism, enabling recruitment of anti-DNP antibodies to the mycobacterial cell surface. Phagocytosis of Tre-DNP-modified M. smegmatis by macrophages was significantly enhanced in the presence of anti-DNP antibodies, demonstrating proof-of-concept that our strategy can augment the host immune response. Because the metabolic pathways responsible for cell surface incorporation of Tre-DNPs are conserved in all Mycobacteriales organisms but absent from other bacteria and humans, the reported tools may be enlisted to interrogate host-pathogen interactions and develop immune-targeting strategies for diverse mycobacterial pathogens.

Piecing Complement Together with LEGO Bricks: Impacts on Interest, Confidence, and Learning in the Immunology Classroom.

Teaching and learning complex molecular cascades can often be challenging. In immunology, students struggle to visualize immunological processes, such as activation of the complement system, which involves three separate cascades leading to multiple effector functions. Offering learning activities that use tangible modeling can help students learn conceptually difficult content by fostering a visual understanding of concepts, as well as instill confidence and interest in the material. In this article, we describe a learning activity using LEGO bricks that demonstrates the activation of the classical, lectin, and alternative complement pathways and formation of the membrane attack complex. In both an introductory and advanced immunology course, we investigated the effect of the activity on student learning and subject confidence. Performance on examination questions about complement demonstrated that the LEGO activity improved learning in a naive student population (students in introductory immunology), but not in a previously informed student population (students in advanced immunology). In addition, self-reported confidence in the content was significantly higher in students who completed the LEGO activity in the advanced course, but not the introductory course, compared with those who did not do the activity. Students in both courses who did the activity had a positive perception of the activity, with a majority of students reporting that they enjoyed the activity and had more interest in the complement system.

Extracellular vesicles from A23187-treated neutrophils cause cGAS-STING-dependent IL-6 production by macrophages.

In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus, as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation.

Neutrophil-derived extracellular vesicles modulate the phenotype of naïve human neutrophils.

Neutrophils (PMN) regulate inflammation in many ways, including communication with other immune cells via extracellular vesicles (EVs). EVs released by human neutrophils activated with N-formylmethionyl-leucyl-phenylalanine (fMLF) (PMN-fMLF EVs) had an outside-out orientation and contained functionally important neutrophil plasma membrane proteins, including flavocytochrome b558, and enzymatically active granule proteins, elastase, and myeloperoxidase. Treatment of naïve PMN with PMN-fMLF EVs primed fMLF-stimulated NADPH oxidase activity, increased surface expression of the complement receptors CD11b/CD18 and CD35, the specific granule membrane protein CD66, and flavocytochrome b558 , and promoted phagocytosis of serum-opsonized Staphylococcus aureus. The primed oxidase activity reflected increased surface expression of flavocytochrome b558 and phosphorylation of SER345 in p47phox , two recognized mechanisms for oxidase priming. Taken together, these data demonstrate that stimulated PMN released EVs that altered the phenotype of naïve phagocytes by priming of the NADPH oxidase activity and augmenting phagocytosis, two responses that are integral to optimal PMN host defense.

Effect of extracellular vesicles from S. aureus-challenged human neutrophils on macrophages.

Staphylococcus aureus enhances neutrophil extracellular vesicle (EV) production. To investigate whether S. aureus viability influences EV biogenesis, EVs were isolated from human neutrophils incubated with viable bacteria (bEVs) or heat-killed bacteria (heat-killed EVs). Protein analysis, nanoparticle tracking and transmission electron microscopy showed comparable EV production between subsets, and both viable and nonviable bacteria were also detected in respective EV subsets. As anticipated, S. aureus, as well as bEVs with viable bacteria, were proinflammatory, and killing bacteria with gentamicin reduced cytokine production to baseline levels. Although heat-killed bacteria induced macrophage IL-6 production, heat-killed EVs did not. Additionally, we found that human and bacterial DNA associated with bEVs, but not heat-killed EVs, and that the DNA association could be partially decreased by disrupting electrostatic interactions. We investigated the potential for DNA isolated from EVs (EV-DNA) or EVs to cause inflammation. Although liposomal encapsulation of EV-DNA increased IL-6 production from baseline by 7.5-fold, treatment of bEVs with DNase I had no effect on IL-6 and IL-1ß production, suggesting that the DNA did not contribute to the inflammatory response. Filtered EVs, which lacked DNA and associated bacteria, exhibited less proinflammatory activity relative to bEVs, and enhanced macrophage expression of CD86 and HLA-DR. Ultimately, we show that bEVs isolated by differential centrifugation co-purify with bacteria and DNA, and studying their concerted activity and relative contribution to immune response is important to the study of host-pathogen interactions.

Detection and characterization of soluble CD93 released during inflammation.

BACKGROUND: CD93 is a cell surface glycoprotein that is required for efficient engulfment of apoptotic cells via an unknown mechanism. Recently, it was demonstrated that CD93 is proteolytically cleaved from the surface of activated human monocytes and neutrophils in response to inflammatory signals in vitro and that a soluble form of CD93 (sCD93) exists in human plasma. OBJECTIVE: The objective of this study was to examine the relationship among production of sCD93, inflammation, and engulfment of apoptotic cells. METHODS: Sterile peritonitis was induced in C57BL/6 mice, and lavage fluid was analyzed for presence of sCD93 by ELISA and Western blot. Cellular infiltrate was assessed by flow cytometry and analyzed for its capacity to shed CD93 in vitro. Peritoneal lavage fluid (PLF) was examined for its ability to regulate engulfment of apoptotic cells. RESULTS: There was an 8.9-fold increase in sCD93 following induction of peritonitis. Macrophages accounted for the majority of infiltrating leukocytes into the peritoneum when sCD93 levels were highest. Inflammatory peritoneal macrophages constitutively shed CD93 in vitro suggesting that this cell type contributes to elevated levels of sCD93 in vivo. Inflammatory PLF from wild-type mice containing elevated sCD93 significantly enhanced engulfment of apoptotic cells in vitro when compared to inflammatory fluid from CD93-deficient mice. CONCLUSIONS: These data demonstrate that inflammation triggers release of sCD93 in vivo, identify the inflammatory macrophage as a source of sCD93, and provide insight into the mechanism by which CD93 contributes to engulfment of apoptotic cells.

IFN-γ targets macrophage-mediated immune responses toward Staphylococcus aureus.

Infections, especially with Staphylococcus aureus (SA), commonly cause morbidity and mortality in patients with chronic granulomatous disease (CGD), a condition characterized by a defective phagocyte oxidase. IFN-γ reduces the frequency and consequences of infection in CGD by mechanisms that remain unknown. As IFN-γ promotes bacterial killing, efferocytosis of effete polymorphonuclear neutrophils (PMN), and cytokine production in macrophages-the same macrophage effector functions that are impaired in response to SA-we hypothesized that IFN-γ may reverse these defects and thereby, augment macrophage control of SA during infection. IFN-γ primed activation of the NADPH oxidase in a time-dependent manner, enhanced killing of ingested SA independent of any effects on phagocytosis, and increased binding of SA-laden neutrophils (PMN-SA) to macrophages. However, IFN-γ did not increase the percentage of apoptotic PMN or PMN-SA internalized by macrophages. Under conditions in which viable SA were eliminated, PMN-SA primed the inflammasome for subsequent activation by silica but did not induce IL-1ß production by macrophages. IFN-γ enhanced IL-6 production in response to SA or PMN-SA but did not increase inflammasome activation in response to either agonist. In summary, IFN-γ augmented direct killing of SA by macrophages, promoted engagement of PMN-SA, and enhanced macrophage-mediated cytokine responses that could collectively augment control of SA infection. Together, these findings support the hypothesis that IFN-γ improves responsiveness of macrophages to SA and provides insights into the mechanism of the clinical benefits of IFN-γ.

CD93: recent advances and implications in disease.

While it has been known for some time that CD93 regulates several processes involved in innate immunity and inflammation including phagocytosis and adhesion, the function of CD93 in disease progression is only now being elucidated. Recent in vivo studies in mice, and genome wide studies in mice and humans, have provided clues about its molecular function. Following a comprehensive review of CD93 expression patterns, this review will focus on recent findings over the last three years that address the putative function of CD93 in inflammation and innate immunity.

C1q and phagocytosis: the perfect complement to a good meal.

Complement component C1q is a member of a family of soluble proteins called defense collagens, which are important in host defense and apoptotic cell clearance. Failure to efficiently clear apoptotic cells in the absence of C1q is associated with autoimmunity. Here, we review the literature describing a central role for C1q in the enhancement of phagocyte function and focus specifically on C1q in apoptotic cell clearance. In addition, we highlight our recent findings demonstrating that C1q elicits a macrophage phenotype that is tailored specifically for clearance of apoptotic cells.

Macrophage response to apoptotic cells varies with the apoptotic trigger and is not altered by a deficiency in LRP expression.

Rapid engulfment of apoptotic cells in the absence of inflammation is required for maintenance of normal tissue homeostasis. The low-density lipoprotein receptor-related protein-1 (LRP/CD91) is a receptor mediating interactions between macrophages and apoptotic cells, but recent reports have challenged the requirement of this surface protein in this process. To explore the role of LRP in the recognition of apoptotic cells, target cells were generated with two distinct inducers of apoptotic cell death, etoposide and actinomycin-D. Jurkat T cells rendered apoptotic with etoposide exposed phosphatidylserine (PtdSer) and triggered engulfment by murine bone marrow-derived macrophages (BMDM), however they failed to suppress lipopolysaccharide-driven inflammatory cytokine secretion or, correspondingly, NF kappaB-dependent or TNFalpha promoter-driven transcriptional activity in transfected RAW264.7 macrophages. In contrast, induction of apoptosis in either Jurkat cells or HeLa epithelial cells with actinomycin-D resulted in diminution of proinflammatory signaling from RAW264.7 cells and BMDM. Treatment of actinomycin-treated Jurkat cells with Q-VD-OPh, an irreversible inhibitor of caspase activity, blocked apoptosis, as assessed by the inhibition of PtdSer exposure; however, the cells maintained anti-inflammatory activity. Anti-inflammatory signaling mediated by actinomycin-treated cells was not affected by a macrophage-specific deletion in LRP. Moreover, the presence of LRP on macrophages did not alter the efficiency of engulfment of apoptotic cells in vitro or in vivo. These data demonstrate that the method of induction of apoptosis of target cells influences subsequent macrophage responsiveness, and that LRP is not required for engulfment of apoptotic cells regardless of the method of induction.

Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis.

C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared with LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep RBC suboptimally opsonized with IgG or complement, compared with cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared with normal mouse serum, and the addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.