Plasticity of gene expression in the nervous system by exposure to environmental odorants that inhibit HDACs.

Eukaryotes respond to secreted metabolites from the microbiome. However, little is known about the effects of exposure to volatiles emitted by microbes or in the environment that we are exposed to over longer durations. Using Drosophila melanogaster, we evaluated a yeast-emitted volatile, diacetyl, found at high levels around fermenting fruits where they spend long periods of time. Exposure to the diacetyl molecules in headspace alters gene expression in the antenna. In vitro experiments demonstrated that diacetyl and structurally related volatiles inhibited conserved histone deacetylases (HDACs), increased histone-H3K9 acetylation in human cells, and caused changes in gene expression in both Drosophila and mice. Diacetyl crosses the blood-brain barrier and exposure caused modulation of gene expression in the mouse brain, therefore showing potential as a neuro-therapeutic. Using two separate disease models previously known to be responsive to HDAC inhibitors, we evaluated the physiological effects of volatile exposure. Diacetyl exposure halted proliferation of a neuroblastoma cell line in culture. Exposure to diacetyl vapors slowed progression of neurodegeneration in a Drosophila model for Huntington's disease. These changes strongly suggest that certain volatiles in the surroundings can have profound effects on histone acetylation, gene expression, and physiology in animals.

Hookworms dynamically respond to loss of Type 2 immune pressure.

The impact of the host immune environment on parasite transcription and fitness is currently unknown. It is widely held that hookworm infections have an immunomodulatory impact on the host, but whether the converse is true remains unclear. Immunity against adult-stage hookworms is largely mediated by Type 2 immune responses driven by the transcription factor Signal Transducer and Activator of Transcription 6 (STAT6). This study investigated whether serial passage of the rodent hookworm Nippostrongylus brasiliensis in STAT6-deficient mice (STAT6 KO) caused changes in parasites over time. After adaptation to STAT6 KO hosts, N. brasiliensis increased their reproductive output, feeding capacity, energy content, and body size. Using an improved N. brasiliensis genome, we found that these physiological changes corresponded with a dramatic shift in the transcriptional landscape, including increased expression of gene pathways associated with egg production, but a decrease in genes encoding neuropeptides, proteases, SCP/TAPS proteins, and transthyretin-like proteins; the latter three categories have been repeatedly observed in hookworm excreted/secreted proteins (ESPs) implicated in immunosuppression. Although transcriptional changes started to appear in the first generation of passage in STAT6 KO hosts for both immature and mature adult stages, downregulation of the genes putatively involved in immunosuppression was only observed after multiple generations in this immunodeficient environment. When STAT6 KO-adapted N. brasiliensis were reintroduced to a naive WT host after up to 26 generations, this progressive change in host-adaptation corresponded to increased production of inflammatory cytokines by the WT host. Surprisingly, however, this single exposure of STAT6 KO-adapted N. brasiliensis to WT hosts resulted in worms that were morphologically and transcriptionally indistinguishable from WT-adapted parasites. This work uncovers remarkable plasticity in the ability of hookworms to adapt to their hosts, which may present a general feature of parasitic nematodes.

Pleural macrophages translocate to the lung during infection to promote improved influenza outcomes.

Seasonal influenza results in 3 to 5 million cases of severe disease and 250,000 to 500,000 deaths annually. Macrophages have been implicated in both the resolution and progression of the disease, but the drivers of these outcomes are poorly understood. We probed mouse lung transcriptomic datasets using the Digital Cell Quantifier algorithm to predict immune cell subsets that correlated with mild or severe influenza A virus (IAV) infection outcomes. We identified a unique lung macrophage population that transcriptionally resembled small serosal cavity macrophages and whose presence correlated with mild disease. Until now, the study of serosal macrophage translocation in the context of viral infections has been neglected. Here, we show that pleural macrophages (PMs) migrate from the pleural cavity to the lung after infection with IAV. We found that the depletion of PMs increased morbidity and pulmonary inflammation. There were increased proinflammatory cytokines in the pleural cavity and an influx of neutrophils within the lung. Our results show that PMs are recruited to the lung during IAV infection and contribute to recovery from influenza. This study expands our knowledge of PM plasticity and identifies a source of lung macrophages independent of monocyte recruitment and local proliferation.

Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.

Health disparities in COVID-19: immune and vascular changes are linked to disease severity and persist in a high-risk population in Riverside County, California.

BACKGROUND: Health disparities in underserved communities, such as inadequate healthcare access, impact COVID-19 disease outcomes. These disparities are evident in Hispanic populations nationwide, with disproportionately high infection and mortality rates. Furthermore, infected individuals can develop long COVID with sustained impacts on quality of life. The goal of this study was to identify immune and endothelial factors that are associated with COVID-19 outcomes in Riverside County, a high-risk and predominantly Hispanic community, and investigate the long-term impacts of COVID-19 infection. METHODS: 112 participants in Riverside County, California, were recruited according to the following criteria: healthy control (n = 23), outpatients with moderate infection (outpatient, n = 33), ICU patients with severe infection (hospitalized, n = 33), and individuals recovered from moderate infection (n = 23). Differences in outcomes between Hispanic and non-Hispanic individuals and presence/absence of co-morbidities were evaluated. Circulating immune and vascular biomarkers were measured by ELISA, multiplex analyte assays, and flow cytometry. Follow-up assessments for long COVID, lung health, and immune and vascular changes were conducted after recovery (n = 23) including paired analyses of the same participants. RESULTS: Compared to uninfected controls, the severe infection group had a higher proportion of Hispanic individuals (n = 23, p = 0.012) than moderate infection (n = 8, p = 0.550). Disease severity was associated with changes in innate monocytes and neutrophils, lymphopenia, disrupted cytokine production (increased IL-8 and IP-10/CXCL10 but reduced IFNλ2/3 and IFNγ), and increased endothelial injury (myoglobin, VCAM-1). In the severe infection group, a machine learning model identified LCN2/NGAL, IL-6, and monocyte activation as parameters associated with fatality while anti-coagulant therapy was associated with survival. Recovery from moderate COVID infection resulted in long-term immune changes including increased monocytes/lymphocytes and decreased neutrophils and endothelial markers. This group had a lower proportion of co-morbidities (n = 8, p = 1.0) but still reported symptoms associated with long COVID despite recovered pulmonary function. CONCLUSION: This study indicates increased severity of COVID-19 infection in Hispanic individuals of Riverside County, California. Infection resulted in immunological and vascular changes and long COVID symptoms that were sustained for up to 11 months, however, lung volume and airflow resistance was recovered. Given the immune and behavioral impacts of long COVID, the potential for increased susceptibility to infections and decreased quality of life in high-risk populations warrants further investigation.

Diet high in linoleic acid dysregulates the intestinal endocannabinoid system and increases susceptibility to colitis in Mice.

Inflammatory bowel disease (IBD) is a multifactorial disease with increasing incidence in the U.S. suggesting that environmental factors, including diet, are involved. It has been suggested that excessive consumption of linoleic acid (LA, C18:2 omega-6), which must be obtained from the diet, may promote the development of IBD in humans. To demonstrate a causal link between LA and IBD, we show that a high fat diet (HFD) based on soybean oil (SO), which is comprised of ~55% LA, increases susceptibility to colitis in several models, including IBD-susceptible IL10 knockout mice. This effect was not observed with low-LA HFDs derived from genetically modified soybean oil or olive oil. The conventional SO HFD causes classical IBD symptoms including immune dysfunction, increased intestinal epithelial barrier permeability, and disruption of the balance of isoforms from the IBD susceptibility gene Hepatocyte Nuclear Factor 4α (HNF4α). The SO HFD causes gut dysbiosis, including increased abundance of an endogenous adherent invasive Escherichia coli (AIEC), which can use LA as a carbon source. Metabolomic analysis shows that in the mouse gut, even in the absence of bacteria, the presence of soybean oil increases levels of LA, oxylipins and prostaglandins. Many compounds in the endocannabinoid system, which are protective against IBD, are decreased by SO both in vivo and in vitro. These results indicate that a high LA diet increases susceptibility to colitis via microbial and host-initiated pathways involving alterations in the balance of bioactive metabolites of omega-6 and omega-3 polyunsaturated fatty acids, as well as HNF4α isoforms.

Sexual dimorphism in obesity is governed by RELMα regulation of adipose macrophages and eosinophils.

Obesity incidence is increasing worldwide with the urgent need to identify new therapeutics. Sex differences in immune cell activation drive obesity-mediated pathologies where males are more susceptible to obesity comorbidities and exacerbated inflammation. Here, we demonstrate that the macrophage-secreted protein RELMα critically protects females against high-fat diet (HFD)-induced obesity. Compared to male mice, serum RELMα levels were higher in both control and HFD-fed females and correlated with frequency of adipose macrophages and eosinophils. RELMα-deficient females gained more weight and had proinflammatory macrophage accumulation and eosinophil loss in the adipose stromal vascular fraction (SVF), while RELMα treatment or eosinophil transfer rescued this phenotype. Single-cell RNA-sequencing of the adipose SVF was performed and identified sex and RELMα-dependent changes. Genes involved in oxygen sensing and iron homeostasis, including hemoglobin and lncRNA Gm47283/Gm21887, correlated with increased obesity, while eosinophil chemotaxis and response to amyloid-beta were protective. Monocyte-to-macrophage transition was also dysregulated in RELMα-deficient animals. Collectively, these studies implicate a RELMα-macrophage-eosinophil axis in sex-specific protection against obesity and uncover new therapeutic targets for obesity.

Lipid-Sensing Receptor FFAR4 Modulates Pulmonary Epithelial Homeostasis following Immunogenic Exposures Independently of the FFAR4 Ligand Docosahexaenoic Acid (DHA).

The role of pulmonary free fatty acid receptor 4 (FFAR4) is not fully elucidated and we aimed to clarify the impact of FFAR4 on the pulmonary immune response and return to homeostasis. We employed a known high-risk human pulmonary immunogenic exposure to extracts of dust from swine confinement facilities (DE). WT and Ffar4-null mice were repetitively exposed to DE via intranasal instillation and supplemented with docosahexaenoic acid (DHA) by oral gavage. We sought to understand if previous findings of DHA-mediated attenuation of the DE-induced inflammatory response are FFAR4-dependent. We identified that DHA mediates anti-inflammatory effects independent of FFAR4 expression, and that DE-exposed mice lacking FFAR4 had reduced immune cells in the airways, epithelial dysplasia, and impaired pulmonary barrier integrity. Analysis of transcripts using an immunology gene expression panel revealed a role for FFAR4 in lungs related to innate immune initiation of inflammation, cytoprotection, and immune cell migration. Ultimately, the presence of FFAR4 in the lung may regulate cell survival and repair following immune injury, suggestive of potential therapeutic directions for pulmonary disease.

PTPN2 regulates bacterial clearance in a mouse model of enteropathogenic and enterohemorrhagic E. coli infection.

Macrophages intimately interact with intestinal epithelial cells, but the consequences of defective macrophage-epithelial cell interactions for protection against enteric pathogens are poorly understood. Here, we show that in mice with a deletion in protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in macrophages, infection with Citrobacter rodentium, a model of enteropathogenic and enterohemorrhagic E. coli infection in humans, promoted a strong type 1/IL-22-driven immune response, culminating in accelerated disease but also faster clearance of the pathogen. In contrast, deletion of PTPN2 specifically in epithelial cells rendered the epithelium unable to upregulate antimicrobial peptides and consequently resulted in a failure to eliminate the infection. The ability of PTPN2-deficient macrophages to induce faster recovery from C. rodentium was dependent on macrophage-intrinsic IL-22 production, which was highly increased in macrophages deficient in PTPN2. Our findings demonstrate the importance of macrophage-mediated factors, and especially macrophage-derived IL-22, for the induction of protective immune responses in the intestinal epithelium, and show that normal PTPN2 expression in the epithelium is crucial to allow for protection against enterohemorrhagic E. coli and other intestinal pathogens.

Sexual dimorphism in obesity is governed by RELMα regulation of adipose macrophages and eosinophils.

Obesity incidence is increasing worldwide with the urgent need to identify new therapeutics. Sex differences in immune cell activation drive obesity-mediated pathologies where males are more susceptible to obesity co-morbidities and exacerbated inflammation. Here, we demonstrate that the macrophage-secreted protein RELMα critically protects females against high fat diet-induced obesity. Compared to male mice, RELMα levels were elevated in both control and high fat dietfed females and correlated with adipose macrophages and eosinophils. RELMα-deficient females gained more weight and had pro-inflammatory macrophage accumulation and eosinophil loss, while both RELMα treatment and eosinophil transfer rescued this phenotype. Single cell RNA-sequencing of the adipose stromal vascular fraction was performed and identified sex and RELMα-dependent changes. Genes involved in oxygen sensing and iron homeostasis, including hemoglobin and lncRNA Gm47283, correlated with increased obesity, while eosinophil chemotaxis and response to amyloid-beta were protective. Monocyte-to-macrophage transition was also dysregulated in RELMα-deficient animals. Collectively, these studies implicate a RELMα-macrophage-eosinophil axis in sex-specific protection against obesity and uncover new therapeutic targets for obesity.

Myeloid- and epithelial-derived RELMα contribute to tissue repair following lung helminth infection.

Soil-transmitted helminth (STH) infections impact billions of individuals globally; however, there is a need to clarify the long-term impacts of these infections on pulmonary health owing to their transient migration and subsequent damage to the lungs. In mouse models of these infections using Nippostrongylus brasiliensis, lung pathology persists at later time points post single infection. These studies also indicate the persistent transcriptional expression of resistin-like molecule α (RELMα), an immunomodulatory protein induced in type 2 immunity and alternatively activated macrophages. Using constitutive and tamoxifen-inducible cell-specific RELMα knockout mouse strains, we identified that epithelial- and myeloid-derived RELMα protein remained elevated at 30 days post infection and altered the immune cell signature and gene expression in lung compartments. Histopathological assessment of alveolar damage revealed a role for RELMα in tissue repair, suggesting the importance of sustained RELMα expression for lung recovery from helminth infection. Acellular three-dimensional (3D) lung scaffolds were prepared from the lungs of wild-type (WT), RELMα KO-naive, or 30 days post N. brasiliensis-infected mice to assess their ability to support epithelial cell growth. N. brasiliensis infection significantly altered the scaffold and impaired epithelial cell growth and metabolic activity, especially in the RELMα KO scaffolds. These findings underscore a need to identify the long-term impacts of helminth infection on human pulmonary disease, particularly as alveolar destruction can develop into chronic obstructive pulmonary disease (COPD), which remains among the top global causes of death. Translation of these findings to human protein resistin, with sequence homology to RELMα therapeutic opportunities in lung repair.

Trickle infection with Heligmosomoides polygyrus results in decreased worm burdens but increased intestinal inflammation and scarring.

Introduction: Intestinal roundworms cause chronic debilitating disease in animals, including humans. Traditional experimental models of these types of infection use a large single-dose infection. However, in natural settings, hosts are exposed to parasites on a regular basis and when mice are exposed to frequent, smaller doses of Heligmosomoides polygyrus, the parasites are cleared more quickly. Whether this more effective host response has any negative consequences for the host is not known. Results: Using a trickle model of infection, we found that worm clearance was associated with known resistance-related host responses: increased granuloma and tuft cell numbers, increased levels of granuloma IgG and decreased intestinal transit time, as well as higher serum IgE levels. However, we found that the improved worm clearance was also associated with an inflammatory phenotype in and around the granuloma, increased smooth muscle hypertrophy/hyperplasia, and elevated levels of Adamts gene expression. Discussion: To our knowledge, we are the first to identify the involvement of this protein family of matrix metalloproteinases (MMPs) in host responses to helminth infections. Our results highlight the delicate balance between parasite clearance and host tissue damage, which both contribute to host pathology. When continually exposed to parasitic worms, improved clearance comes at a cost.

Resistin Concentration in Early Sepsis and All-Cause Mortality at a Safety-Net Hospital in Riverside County.

Background: Sepsis mortality has remained unchanged for greater than a decade, and early recognition continues to be the most important factor in mortality outcome. Plasma resistin concentration is increased in sepsis, but its mechanism and clinical relevance is unclear. As one function, resistin interacts with toll-like receptor 4 in competition with lipopolysaccharide, a main component of the gram-negative bacterial cell wall. It is not known if the type of infection leading to sepsis influences resistin production. The objective of this study was to investigate whether 1) early plasma resistin concentration can predict mortality, 2) elevated plasma resistin concentration is associated with clinical disease severity scores, such as SOFA, mSOFA and APACHE II, and 3) plasma resistin concentrations differ between gram negative versus other etiologies of sepsis. Methods: This was an exploratory study in the framework of a prospective observational design. Peripheral venous blood samples were obtained from subjects admitted to the intensive care unit at clinical recognition of sepsis (0 hour) and at 6 and 24 hours. Vasopressor utilization was not a requirement for inclusion. Plasma was analyzed for resistin concentration by ELISA. Cytokine concentrations including IL-6, IL-8, and IL-10 were determined by cytokine bead array. Cytokine data were evaluated against publicly available sepsis RNA expression datasets to compare protein versus RNA expression levels in predicting clinical disease state. Clinical data were collected from electronic health records for clinical severity index calculations and context for interpretation of resistin and cytokine concentrations. Subjects were followed up to 60 days, or until death, whichever came first. Statistical analysis was completed with R package and SPSS software. Results: Resistin levels were elevated in subjects admitted to the intensive care unit with sepsis. Four-hundred subjects were screened with 45 subjects included in the final analysis. Thirteen of 45 patients were non-survivors. Mortality within 60 days correlated with significantly higher resistin concentrations than in survivors. A resistin concentration of >126 ng/mL at clinical recognition of sepsis and >197 ng/mL within the first 24 hours were associated with mortality within 60 days with an area under the curve of 0.82 and 0.88, respectively. Most subjects with resistin concentration greater than these threshold values were deceased prior to 30 days. Resistin concentrations correlated with SOFA, mSOFA, and APACHE II scores in addition to having association with increases in inflammatory and sepsis biomarkers. These associations were validated with analysis of RNA expression datasets. Conclusion: Plasma resistin concentrations of >126 ng/mL at clinical recognition of sepsis and >197 ng/mL within the first 24 hours of clinical sepsis recognition are associated with all-cause mortality. Resistin concentration within this timeframe also has comparable mortality association to well-validated clinical severity indices of SOFA, mSOFA, and APACHE II scores.

CX3CR1-Expressing Myeloid Cells Regulate Host-Helminth Interaction and Lung Inflammation.

Many helminth life cycles, including hookworm, involve a mandatory lung phase, where myeloid and granulocyte subsets interact with the helminth and respond to infection-induced lung injury. To evaluate these innate subsets in Nippostrongylus brasiliensis infection, reporter mice for myeloid cells (CX3CR1GFP ) and granulocytes (PGRPdsRED ) are employed. Nippostrongylus infection induces lung infiltration of reporter cells, including CX3CR1+ myeloid cells and PGRP+ eosinophils. Strikingly, CX3CR1GFP/GFP mice, which are deficient in CX3CR1, are protected from Nippostrongylus infection with reduced weight loss, lung leukocyte infiltration, and worm burden compared to CX3CR1+/+ mice. This protective effect is specific for CX3CR1 as CCR2-deficient mice do not exhibit reduced worm burdens. Nippostrongylus co-culture with lung Ly6C+ monocytes or CD11c+ cells demonstrates that CX3CR1GFP/GFP monocytes secrete more pro-inflammatory cytokines and actively bind the parasites causing reduced motility. RNA sequencing of Ly6C+ or CD11c+ cells shows Nippostrongylus-induced gene expression changes, particularly in monocytes, associated with inflammation, chemotaxis, and extracellular matrix remodeling pathways. Analysis reveals cytotoxic and adhesion molecules as potential effectors against the parasite, such as Gzma and Gzmb, which are elevated in CX3CR1GFP/GFP monocytes. These studies validate a dual innate cell reporter for lung helminth infection and demonstrate that CX3CR1 impairs monocyte-helminth interaction.

Membrane Cholesterol Enrichment of Red Blood Cell-Derived Microparticles Results in Prolonged Circulation.

Particles fabricated from red blood cells (RBCs) can serve as vehicles for delivery of various biomedical cargos. Flipping of phosphatidylserine (PS) from the inner to the outer membrane leaflet normally occurs during the fabrication of such particles. PS externalization is a signal for phagocytic removal of the particles from circulation. Herein, we demonstrate that membrane cholesterol enrichment can mitigate the outward display of PS on microparticles engineered from RBCs. Our in-vitro results show that the phagocytic uptake of cholesterol-enriched particles by murine macrophages takes place at a lowered rate, resulting in reduced uptake as compared to RBC-derived particles without cholesterol enrichment. When administered via tail-vein injection into healthy mice, the percent of injected dose (ID) per gram of extracted blood for cholesterol-enriched particles was ∼1.5 and 1.8 times higher than the particles without cholesterol enrichment at 4 and 24 h, respectively. At 24 h, ∼43% ID/g of the particles without cholesterol enrichment was eliminated or metabolized while ∼94% ID/g of the cholesterol-enriched particles were still retained in the body. These results indicate that membrane cholesterol enrichment is an effective method to reduce PS externalization on the surface of RBC-derived particles and increase their longevity in circulation.

The interplay of helminthic neuropeptides and proteases in parasite survival and host immunomodulation.

Neuropeptides comprise a diverse and broad group of neurotransmitters in vertebrates and invertebrates, with critical roles in neuronal signal transduction. While their role in controlling learning and memory in the brains of mammals is known, their extra-synaptic function in infection and inflammation with effects on distinct tissues and immune cells is increasingly recognized. Helminth infections especially of the central nervous system (CNS), such as neurocysticercosis, induce neuropeptide production by both host and helminth, but their role in host-parasite interplay or host inflammatory response is unclear. Here, we review the neurobiology of helminths, and discuss recent studies on neuropeptide synthesis and function in the helminth as well as the host CNS and immune system. Neuropeptides are summarized according to structure and function, and we discuss the complex enzyme processing for mature neuropeptides, focusing on helminth enzymes as potential targets for novel anthelminthics. We next describe known immunomodulatory effects of mammalian neuropeptides discovered from mouse infection models and draw functional parallels with helminth neuropeptides. Last, we discuss the anti-microbial properties of neuropeptides, and how they may be involved in host-microbiota changes in helminth infection. Overall, a better understanding of the biology of helminth neuropeptides, and whether they affect infection outcomes could provide diagnostic and therapeutic opportunities for helminth infections.

Loss of protein tyrosine phosphatase non-receptor type 2 reduces IL-4-driven alternative macrophage activation.

Macrophages are a heterogeneous population of innate immune cells that are often divided into two major subsets: classically activated, typically pro-inflammatory (M1) macrophages that mediate host defense, and alternatively activated, tolerance-inducing (M2) macrophages that exert homeostatic and tissue-regenerative functions. Disturbed macrophage function/differentiation results either in inadequate, excessive immune activation or in a failure to induce efficient protective immune responses against pathogens. Loss-of-function variants in protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with chronic inflammatory disorders, but the effect of macrophage-intrinsic PTPN2 loss is still poorly understood. Here we report that PTPN2-deficient macrophages fail to acquire an alternatively activated/M2 phenotype. This was the consequence of reduced IL-6 receptor expression and a failure to induce IL-4 receptor in response to IL-6, resulting in an inability to respond to the key M2-inducing cytokine IL-4. Ultimately, failure to adequately respond to IL-6 and IL-4 resulted in increased levels of M1 macrophage marker expression in vitro and exacerbated lung inflammation upon infection with Nippostrongylus brasiliensis in vivo. These results demonstrate that PTPN2 loss interferes with the ability of macrophages to adequately respond to inflammatory stimuli and might explain the increased susceptibility of PTPN2 loss-of-function carriers to developing inflammatory diseases.

Dynamic changes in human single-cell transcriptional signatures during fatal sepsis.

Systemic infections, especially in patients with chronic diseases, may result in sepsis: an explosive, uncoordinated immune response that can lead to multisystem organ failure with a high mortality rate. Patients with similar clinical phenotypes or sepsis biomarker expression upon diagnosis may have different outcomes, suggesting that the dynamics of sepsis is critical in disease progression. A within-subject study of patients with Gram-negative bacterial sepsis with surviving and fatal outcomes was designed and single-cell transcriptomic analyses of peripheral blood mononuclear cells (PBMC) collected during the critical period between sepsis diagnosis and 6 h were performed. The single-cell observations in the study are consistent with trends from public datasets but also identify dynamic effects in individual cell subsets that change within hours. It is shown that platelet and erythroid precursor responses are drivers of fatal sepsis, with transcriptional signatures that are shared with severe COVID-19 disease. It is also shown that hypoxic stress is a driving factor in immune and metabolic dysfunction of monocytes and erythroid precursors. Last, the data support CD52 as a prognostic biomarker and therapeutic target for sepsis as its expression dynamically increases in lymphocytes and correlates with improved sepsis outcomes. In conclusion, this study describes the first single-cell study that analyzed short-term temporal changes in the immune cell populations and their characteristics in surviving or fatal sepsis. Tracking temporal expression changes in specific cell types could lead to more accurate predictions of sepsis outcomes and identify molecular biomarkers and pathways that could be therapeutically controlled to improve the sepsis trajectory toward better outcomes.

Lung Inflammatory Response to Environmental Dust Exposure in Mice Suggests a Link to Regional Respiratory Disease Risk.

PURPOSE: The Salton Sea, California's largest lake, is designated as an agricultural drainage reservoir. In recent years, the lake has experienced shrinkage due to reduced water sources, increasing levels of aerosolized dusts in surrounding regions. Communities surrounding the Salton Sea have increased asthma prevalence versus the rest of California; however, a connection between dust inhalation and lung health impacts has not been defined. METHODS: We used an established intranasal dust exposure murine model to study the lung inflammatory response following single or repetitive (7-day) exposure to extracts of dusts collected in regions surrounding the Salton Sea (SSDE), complemented with in vitro investigations assessing SSDE impacts on the airway epithelium. RESULTS: In these investigations, single or repetitive SSDE exposure induced significant lung inflammatory cytokine release concomitant with neutrophil influx. Repetitive SSDE exposure led to significant lung eosinophil recruitment and altered expression of genes associated with allergen-mediated immune response, including Clec4e. SSDE treatment of human bronchial epithelial cells (BEAS-2B) induced inflammatory cytokine production at 5- and 24-hours post-treatment. When BEAS-2B were exposed to protease activity-depleted SSDE (PDSSDE) or treated with SSDE in the context of protease-activated receptor-1 and -2 antagonism, inflammatory cytokine release was decreased. Furthermore, repetitive exposure to PDSSDE led to decreased neutrophil and eosinophilic influx and IL-6 release in mice compared to SSDE-challenged mice. CONCLUSION: These investigations demonstrate potent lung inflammatory responses and tissue remodeling in response to SSDE, in part due to environmental proteases found within the dusts. These studies provide the first evidence supporting a link between environmental dust exposure, protease-mediated immune activation, and respiratory disease in the Salton Sea region.

Macrophage-Regulatory T Cell Interactions Promote Type 2 Immune Homeostasis Through Resistin-Like Molecule α.

RELMα is a small, secreted protein expressed by type 2 cytokine-activated "M2" macrophages in helminth infection and allergy. At steady state and in response to type 2 cytokines, RELMα is highly expressed by peritoneal macrophages, however, its function in the serosal cavity is unclear. In this study, we generated RELMα TdTomato (Td) reporter/knockout (RαTd) mice and investigated RELMα function in IL-4 complex (IL-4c)-induced peritoneal inflammation. We first validated the RELMαTd/Td transgenic mice and showed that IL-4c injection led to the significant expansion of large peritoneal macrophages that expressed Td but not RELMα protein, while RELMα+/+ mice expressed RELMα and not Td. Functionally, RELMαTd/Td mice had increased IL-4 induced peritoneal macrophage responses and splenomegaly compared to RELMα+/+ mice. Gene expression analysis indicated that RELMαTd/Td peritoneal macrophages were more proliferative and activated than RELMα+/+ macrophages, with increased genes associated with T cell responses, growth factor and cytokine signaling, but decreased genes associated with differentiation and maintenance of myeloid cells. We tested the hypothesis that RαTd/Td macrophages drive aberrant T cell activation using peritoneal macrophage and T cell co-culture. There were no differences in CD4+ T cell effector responses when co-cultured with RELMα+/+ or RELMαTd/Td macrophages, however, RELMαTd/Td macrophages were impaired in their ability to sustain proliferation of FoxP3+ regulatory T cells (Treg). Supportive of the in vitro results, immunofluorescent staining of the spleens revealed significantly decreased FoxP3+ cells in the RELMαTd/Td spleens compared to RELMα+/+ spleens. Taken together, these studies identify a new RELMα regulatory pathway whereby RELMα-expressing macrophages directly sustain Treg proliferation to limit type 2 inflammatory responses.