Macrophage Populations Research Articles

Infection history imprints prolonged changes to the epigenome, transcriptome and function of Kupffer cells

Liver macrophages fulfill various homeostatic functions and represent an essential line of defense against pathogenic insults. However, it remains unclear whether a history of infectious disease in the liver leads to long-term alterations to the liver macrophage compartment. We utilized a curable model of parasitic infection invoked by the protozoan parasite Trypanosoma brucei brucei to investigate whether infection history can durably reshape hepatic macrophage identity and function. Employing a combination of fate mapping, single-cell CITE-sequencing, single-nuclei multiome analysis, epigenomic analysis, and functional assays, we studied the alterations to the liver macrophage compartment during and after the resolution of infection. We show that T.brucei brucei infection alters the composition of liver-resident macrophages, leading to the infiltration of monocytes that differentiate into various infection-associated macrophage populations with divergent transcriptomic profiles. Whereas infection-associated macrophages disappear post-resolution of infection, monocyte-derived macrophages engraft in the liver, assume a Kupffer cell (KC)-like profile and co-exist with embryonic KCs in the long-term. Remarkably, the prior exposure to infection imprinted an altered transcriptional program on post-resolution KCs that was underpinned by an epigenetic remodeling of KC chromatin landscapes and a shift in KC ontogeny, along with transcriptional and epigenetic alterations in their niche cells. This reprogramming altered KC functions and was associated with increased resilience to a subsequent bacterial infection. Our study demonstrates that a prior exposure to a parasitic infection induces trained immunity in KCs, reshaping their identity and function in the long-term. Although the liver is frequently affected during infections, and despite housing a major population of resident macrophages known as Kupffer cells (KCs), it is currently unclear whether infections can durably alter KCs and their niche cells. Our study provides a comprehensive investigation into the long-term impact of a prior, cured parasitic infection, unveiling long-lasting ontogenic, epigenetic, transcriptomic and functional changes to KCs as well as KC niche cells, which may contribute to KC remodeling. Our data suggest that infection history may continuously reprogram KCs throughout life with potential implications for subsequent disease susceptibility in the liver, influencing preventive and therapeutic approaches.

Simultaneous regulation of AGE/RAGE signaling and MMP-9 expression by an immunomodulating hydrogel accelerates healing in diabetic wounds

PurposeIn chronic hyperglycemia, the advanced glycation end product (AGE) interacts with its receptor (RAGE) and contributes to impaired wound healing by inducing oxidative stress, generating dysfunctional macrophages, and prolonging the inflammatory response. Additionally, uncontrolled levels of proteases, including metallomatrix protease-9 (MMP-9), in the diabetic wound bed degrade the extracellular matrix (ECM) and biological cues that augment healing. A multifunctional antimicrobial hydrogel (Immuno-gel) containing RAGE and MMP-9 inhibitors can regulate the wound microenvironment and promote scar-free healing. ResultsImmuno-gel was characterized and the wound healing efficacy was determined in vitro cell culture and in vivo diabetic Wistar rat wound model using ELISA, Western blot, and Immunofluorescence staining. The Immuno-gel exhibited a highly porous morphology with excellent in vitro cytocompatibility. AGE-stimulated macrophages treated with the Immuno-gel released higher levels of pro-healing cytokines in vitro. In the hydrogel-wound interface of diabetic Wistar rats, Immuno-gel treatment significantly reduced MMP-9 and NF-κB expression and enhanced pro-healing (M2) macrophage population and pro-healing cytokines. ConclusionAltogether, this study suggests that Immuno-gel simultaneously attenuates macrophage dysfunction through the inhibition of AGE/RAGE signaling and reduces MMP-9 overexpression, both of which favor scar-free healing. The combinatorial treatment with RAGE and MMP-9 inhibitors via Immuno-gel simultaneously modulates the diabetic wound microenvironment, making it a promising novel treatment to accelerate diabetic wound healing.

Controlling redox state by edaravone at transplantation site enhances bone regeneration

In cell-based bone augmentation, transplanted cell dysfunction and apoptosis can occur due to oxidative stress caused by the overproduction of reactive oxygen species (ROS). Edaravone (EDA) is a potent free radical scavenger with potential medical applications. This study aimed to investigate the effect of controlling oxidative stress on bone regeneration using EDA. Bone marrow-derived cells were collected from 4-week-old rats, and EDA effects on cell viability and osteogenic differentiation were evaluated. Collagen gels containing PKH26-prelabeled cells were implanted into the calvarial defects of 12-week-old rats, followed by daily subcutaneous injections of normal saline or 500 μM EDA for 4 d. Bone formation was examined using micro-computed tomography and histological staining. Immunofluorescence staining was performed for markers of oxidative stress, macrophages, osteogenesis, and angiogenesis. EDA suppressed ROS production and hydrogen peroxide-induced apoptosis, recovering cell viability and osteoblast differentiation. EDA treatment in vivo increased new bone formation. EDA induced the transition of the macrophage population toward the M2 phenotype. The EDA group also exhibited stronger immunofluorescence for vascular endothelial growth factor and CD31. In addition, more PKH26-positive and PKH26-osteocalcin-double-positive cells were observed in the EDA group, indicating that transplanted cell survival was prolonged, and they differentiated into bone-forming cells. This could be attributed to oxidative stress suppression at the transplantation site by EDA. Collectively, local administration using EDA facilitates bone regeneration by improving the local environment and angiogenesis, prolonging survival, and enhancing the osteogenic capabilities of transplanted cells.

Detection of early-stage NASH using non-invasive hyperpolarized 13C metabolic imaging

Non-alcoholic steatohepatitis (NASH) is characterized from its early stages by a profound remodeling of the liver microenvironment, encompassing changes in the composition and activities of multiple cell types and associated gene expression patterns. Hyperpolarized (HP) 13C MRI provides a unique view of the metabolic microenvironment, with potential relevance for early diagnosis of liver disease. Previous studies have detected changes in HP 13C pyruvate to lactate conversion, catalyzed by lactate dehydrogenase (LDH), with experimental liver injury. HP ∝\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\propto $$\\end{document}-ketobutyrate (∝\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\propto $$\\end{document} KB) is a close molecular analog of pyruvate with modified specificity for LDH isoforms, specifically attenuated activity with their LDHA-expressed subunits that dominate liver parenchyma. Building on recent results with pyruvate, we investigated HP ∝\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\propto $$\\end{document} KB in methionine-choline deficient (MCD) diet as a model of early-stage NASH. Similarity of results between this new agent and pyruvate (~ 50% drop in cytoplasmic reducing capacity), interpreted together with gene expression data from the model, suggests that changes are mediated through broad effects on intermediary metabolism. Plausible mechanisms are depletion of the lactate pool by upregulation of gluconeogenesis (GNG) and pentose phosphate pathway (PPP) flux, and a possible shift toward increased lactate oxidation. These changes may reflect high levels of oxidative stress and/or shifting macrophage populations in NASH.

Monitoring melanoma patients on treatment reveals a distinct macrophage population driving targeted therapy resistance

Resistance to targeted therapy remains a major clinical challenge in melanoma. To uncover resistance mechanisms, we perform single-cell RNA sequencing on fine-needle aspirates from resistant and responding tumors of patients undergoing BRAFi/MEKi treatment. Among the genes most prominently expressed in resistant tumors is POSTN, predicted to signal to a macrophage population associated with targeted therapy resistance (TTR). Accordingly, tumors from patients with fast disease progression after therapy exhibit high POSTN expression levels and high numbers of TTR macrophages. POSTN polarizes human macrophages toward a TTR phenotype and promotes resistance to targeted therapy in a melanoma mouse model, which is associated with a phenotype change in intratumoral macrophages. Finally, polarized TTR macrophages directly protect human melanoma cells from MEKi-induced killing via CD44 receptor expression on melanoma cells. Thus, interfering with the protective activity of TTR macrophages may offer a strategy to overcome resistance to targeted therapy in melanoma.

Airway basal stem cells are necessary for the maintenance of functional intraepithelial airway macrophages.

Adult stem cells play a crucial role in tissue homeostasis and repair through multiple mechanisms. In addition to being able to replace aged or damaged cells, stem cells provide signals that contribute to the maintenance and function of neighboring cells. In the lung, airway basal stem cells also produce cytokines and chemokines in response to inhaled irritants, allergens, and pathogens, which affect specific immune cell populations and shape the nature of the immune response. However, direct cell-to-cell signaling through contact between airway basal stem cells and immune cells has not been demonstrated. Recently, a unique population of intraepithelial airway macrophages (IAMs) has been identified in the murine trachea. Here, we demonstrate that IAMs require Notch signaling from airway basal stem cells for maintenance of their differentiated state and function. Furthermore, we demonstrate that Notch signaling between airway basal stem cells and IAMs is required for antigen-induced allergic inflammation only in the trachea where the basal stem cells are located whereas allergic responses in distal lung tissues are preserved consistent with a local circuit linking stem cells to proximate immune cells. Finally, we demonstrate that IAM-like cells are present in human conducting airways and that these cells display Notch activation, mirroring their murine counterparts. Since diverse lung stem cells have recently been identified and localized to specific anatomic niches along the proximodistal axis of the respiratory tree, we hypothesize that the direct functional coupling of local stem cell-mediated regeneration and immune responses permits a compartmentalized inflammatory response.

Cholinergic macrophages promote the resolution of peritoneal inflammation

The non-neural cholinergic system plays a critical role in regulating immune equilibrium and tissue homeostasis. While the expression of choline acetyltransferase (ChAT), the enzyme catalyzing acetylcholine biosynthesis, has been well documented in lymphocytes, its role in the myeloid compartment is less understood. Here, we identify a significant population of macrophages (Mϕs) expressing ChAT and synthesizing acetylcholine in the resolution phase of acute peritonitis. Using Chat-GFP reporter mice, we observed marked upregulation of ChAT in monocyte-derived small peritoneal Mϕs (SmPMs) in response to Toll-like receptor agonists and bacterial infections. These SmPMs, phenotypically and transcriptionally distinct from tissue-resident large peritoneal macrophages, up-regulated ChAT expression through a MyD88-dependent pathway involving MAPK signaling. Notably, this process was attenuated by the TRIF-dependent TLR signaling pathway, and our tests with a range of neurotransmitters and cytokines failed to induce a similar response. Functionally, Chat deficiency in Mϕs led to significantly decreased peritoneal acetylcholine levels, reduced efferocytosis of apoptotic neutrophils, and a delayed resolution of peritonitis, which were reversible with exogenous ACh supplementation. Intriguingly, despite B lymphocytes being a notable ChAT-expressing population within the peritoneal cavity, Chat deletion in B cells did not significantly alter the resolution process. Collectively, these findings underscore the crucial role of Mϕ-derived acetylcholine in the resolution of inflammation and highlight the importance of the non-neuronal cholinergic system in immune regulation.

Relationship of microvascular density and tumor-associated macrophages with orofacial squamous cell carcinoma progression

The current study aimed to investigate the characteristics of tumor-associated macrophages (TAMs) and their association with microvascular density (MVD) in tumor progression in different grades of orofacial squamous cell carcinoma (OSCC) in the Pakistani population. This prospective study included 234 patients with oral cancer reported at different hospitals in Pakistan diagnosed with OSCC. Tumors were graded on the Anneroth grading system and the association between the frequency of TAMs and MVD was examined in vivo. The macrophages visible through immunohistochemistry for CD68 and the microvessels observed through immunohistochemistry for CD34 were manually counted in 3 high-power fields. The CD68 and CD34 counts were significantly lower in well-differentiated squamous cell carcinoma compared to poorly differentiated squamous cell carcinoma. Linear regression analysis revealed a positive correlation between the area percentage of CD68 immunoreactivity and the grade of the tumor (r = 0.776). Vice versa, a positive correlation also existed between the area percentage of CD34 immunoreactivity and the grade of the tumor (r = 0.690). Pearson correlation revealed a positive association between the TAMs and MVD (r = 0.680; P < .001). There was an increased population of tumor-associated macrophages and tumor angiogenesis with the increasing grade of orofacial squamous cell carcinoma. (Oral Surg Oral Med Oral Pathol Oral Radiol YEAR;VOL:page range).

THP-1 Monocytic Cells Are Polarized to More Antitumorigenic Macrophages by Serial Treatment with Phorbol-12-Myristate-13-Acetate and PD98059.

Background and Objectives: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in regulating macrophage polarization towards an antitumorigenic (M1) phenotype rather than a protumorigenic (M2) one in various experimental models. Here, we evaluated the effect of PD98059, a mitogen-activated protein kinase kinase MAPKK MEK1-linked pathway inhibitor, on the differentiation and polarization of THP-1 monocytes in response to phorbol-12-myristate-13-acetate (PMA) under various culture conditions for tumor microenvironmental application. Materials and Methods: Differentiation and polarization of THP-1 were analyzed by flow cytometry and RT-PCR. Polarized THP-1 subsets with different treatment were compared by motility, phagocytosis, and so on. Results: Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). CD11bhigh THP-1 cells treated with PMA and PMA-post-PD were categorized into M0 (HLA-DRlow and CD206low), M1 (HLA-DRhigh and CD206low), and M2 (HLA-DRlow and CD206high), resulting in an increased population of M1 macrophages. The transcription levels of markers of macrophage differentiation and polarization confirmed the increased M1 polarization of THP-1 cells with post-PD treatment rather than with PMA-only treatment. The motility and cytotoxicity of THP-1 cells with post-PD treatment were higher than THP-1 cells with PMA, suggesting that post-PD treatment enhanced the anti-tumorigenicity of THP-1 cells. Confocal microscopy and flow cytometry showed the effect of post-PD treatment on phagocytosis by THP-1 cells. Conclusions: We have developed an experimental model of macrophage polarization with THP-1 cells which will be useful for further studies related to the tumor microenvironment.

Gut Dysbiosis Drives Inflammatory Bowel Disease Through the CCL4L2-VSIR Axis in Glycogen Storage Disease.

Patients with glycogen storage disease type Ib (GSD-Ib) frequently have inflammatory bowel disease (IBD). however, the underlying etiology remains unclear. Herein, this study finds that digestive symptoms are commonly observed in patients with GSD-Ib, presenting as single or multiple scattered deep round ulcers, inflammatory pseudo-polyps, obstructions, and strictures, which differ substantially from those in typical IBD. Distinct microbiota profiling and single-cell clustering of colonic mucosae in patients with GSD are conducted. Heterogeneous oral pathogenic enteric outgrowth induced by GSD is a potent inducer of gut microbiota immaturity and colonic macrophage accumulation. Specifically, a unique population of macrophages with high CCL4L2 expression is identified in response to pathogenic bacteria in the intestine. Hyper-activation of the CCL4L2-VSIR axis leads to increased expression of AGR2 and ZG16 in epithelial cells, which mediates the unique progression of IBD in GSD-Ib. Collectively, the microbiota-driven pathomechanism of IBD is demonstrated in GSD-Ib and revealed the active role of the CCL4L2-VSIR axis in the interaction between the microbiota and colonic mucosal immunity. Thus, targeting gut dysbiosis and/or the CCL4L2-VISR axis may represent a potential therapy for GSD-associated IBD.

HGG-31. GENOMIC CORRELATIONS WITH HEMORRHAGE RISK IN PEDIATRIC PATIENTS WITH DIFFUSE HEMISPHERIC GLIOMA

Abstract BACKGROUND Pediatric diffuse hemispheric glioma (DHG) is a histone-mutated (H3.3G34R/V) high-grade glioma with poor prognosis. Clinical observation and previous reports have identified that a subset of patients present with tumor-associated hemorrhage. Here, we present our findings from radiological review of these patients and determine genomic risk factors predictive for hemorrhage risk in this cohort. METHODS Data was abstracted through the Children’s Brain Tumor Network (CBTN) and EGAS00001004301. Transcriptomic and genomic analyses were completed in R 4.3.1 using edgeR, msigdbr, and xCell. Presence of blood products was determined on preoperative MRI by a board certified pediatric neuroradiologist. RESULTS 48 samples were available across cohorts with transcriptomic or genomic data. 10 samples (21%) had pre-operative imaging and transcriptomic data available. Initial analysis determined that 6 samples (60%) had acute/chronic hemorrhage based on radiological review. Samples with hemorrhage had increased levels of VEGFA (LFC: 3.45, p=2E-07) and CA9 (LFC: 5.97, p=8E-05) expression on the transcriptomic level suggesting that patients with high expression of these markers (AngioHi) had increased risk of hemorrhage compared to patients with low expression of these markers (AngioLo). AngioHi patients had notably higher levels of IL8 (LFC: 3.56, p=0.01) and increased macrophage populations. Genomic information was available for 15 samples (31%, 7 AngioHi, 8 AngioLo). PTEN alterations (n=4, 57%), FBXW7 alterations (n= 2, 29%), or PDGFRa driver alterations (n= 3, 43%) were found in the AngioHi cohort while none of these alterations were identified in the AngioLo cohort. Notably, at least one of these mutations was seen in all cases in the AngioHi cohort. CONCLUSIONS These findings identify potential mutational alterations predictive for tumor-associated hemorrhage in pediatric DHG. Current work is being completed to further characterize how these mutations impact vascular remodeling for patients with DHG and may offer targeted therapeutic opportunities.

The hepatocyte epidermal growth factor receptor (EGFR) pathway regulates the cellular interactome within the liver fibrotic niche.

Liver fibrosis is the consequence of chronic liver injury in the presence of an inflammatory component. Although the main executors of this activation are known, the mechanisms that lead to the inflammatory process that mediates the production of pro-fibrotic factors are not well characterized. Epidermal growth factor receptor (EGFR) signaling in hepatocytes is essential for the regenerative processes of the liver; however, its potential role in regulating the fibrotic niche is not yet clear. Our group generated a mouse model that expresses an inactive truncated form of the EGFR specifically in hepatocytes (ΔEGFR mice). Here, we have analyzed the response of WT and ΔEGFR mice to chronic treatment with carbon tetrachloride (CCl4), which induces a pro-inflammatory and fibrotic process in the liver. The results indicated that the hallmarks of liver fibrosis were attenuated in CCl4-treated ΔEGFR mice when compared with CCl4-treated WT mice, coinciding with a faster resolution of the fibrotic process and ameliorated damage. The absence of EGFR activity in hepatocytes induced changes in the pattern of immune cells in the liver, with a notable increase in the population of M2 macrophages, more related to fibrosis resolution, as well as in the population of lymphocytes related to eradication of the damage. Transcriptome analysis of hepatocytes, and secretome studies of extracellular media from in vitro experiments, allowed us to elucidate the specific molecular mechanisms regulated by EGFR that mediate hepatocyte production of both pro-fibrotic and pro-inflammatory mediators; these have consequences for the deposition of extracellular matrix proteins, as well as for the immune microenvironment. Overall, our study uncovered novel mechanistic insights regarding EGFR kinase-dependent actions in hepatocytes that reveal its key role in chronic liver damage. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Tropoelastin modulates systemic and local tissue responses to enhance wound healing

Wound healing is facilitated by biomaterials-based grafts and substantially impacted by orchestrated inflammatory responses that are essential to the normal repair process. Tropoelastin (TE) based materials are known to shorten the period for wound repair but the mechanism of anti-inflammatory performance is not known. To explore this, we compared the performance of the gold standard Integra Dermal Regeneration Template (Integra), polyglycerol sebacate (PGS), and TE blended with PGS, in a murine full-thickness cutaneous wound healing study. Systemically, blending with TE favorably increased the F4/80+ macrophage population by day 7 in the spleen and contemporaneously induced elevated plasma levels of anti-inflammatory IL-10. In contrast, the PGS graft without TE prompted prolonged inflammation, as evidenced by splenomegaly and greater splenic granulocyte and monocyte fractions at day 14. Locally, the inclusion of TE in the graft led to increased anti-inflammatory M2 macrophages and CD4+T cells at the wound site, and a rise in Foxp3+ regulatory T cells in the wound bed by day 7. We conclude that the TE-incorporated skin graft delivers a pro-healing environment by modulating systemic and local tissue responses. Statement of significanceTropoelastin (TE) has shown significant benefits in promoting the repair and regeneration of damaged human tissues. In this study, we show that TE promotes an anti-inflammatory environment that facilitates cutaneous wound healing. In a mouse model, we find that inserting a TE-containing material into a full-thickness wound results in defined, pro-healing local and systemic tissue responses. These findings advance our understanding of TE's restorative value in tissue engineering and regenerative medicine, and pave the way for clinical applications.

A mouse model of TB-associated lung fibrosis reveals persistent inflammatory macrophage populations during treatment.

Post-TB lung disease (PTLD) causes a significant burden of global disease. Fibrosis is a central component of many clinical features of PTLD. To date, we have a limited understanding of the mechanisms of TB-associated fibrosis and how these mechanisms are similar to or dissimilar from other fibrotic lung pathologies. We have adapted a mouse model of TB infection to facilitate the mechanistic study of TB-associated lung fibrosis. We find that the morphologies of fibrosis that develop in the mouse model are similar to the morphologies of fibrosis observed in human tissue samples. Using Second Harmonic Generation (SHG) microscopy, we are able to quantify a major component of fibrosis, fibrillar collagen, over time and with treatment. Inflammatory macrophage subpopulations persist during treatment; matrix remodeling enzymes and inflammatory gene signatures remain elevated. Our mouse model suggests that there is a therapeutic window during which adjunctive therapies could change matrix remodeling or inflammatory drivers of tissue pathology to improve functional outcomes after treatment for TB infection.

The Aging Lacrimal Gland of Female C57BL/6J Mice Exhibits Multinucleate Macrophage Infiltration Associated With Lipid Dysregulation.

Loss of function of the lacrimal gland (LG), which produces the aqueous tear film, is implicated in age-related dry eye. To better understand this deterioration, we evaluated changes in lipid metabolism and inflammation in LGs from an aging model. LG sections from female C57BL/6J mice of different ages (young, 2-3 months; intermediate, 10-14 months; old, ≥24 months) were stained with Oil Red-O or Toluidine blue to detect lipids. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blotting of LG lysates determined differences in the expression of genes and proteins related to lipid metabolism. A photobleaching protocol to quench age-related autofluorescence was used in LG sections to evaluate changes in immunofluorescence associated with NPC1, NPC2, CTSL, and macrophages (F4/80, CD11b) with age using confocal fluorescence microscopy. Old LGs showed increased lipids prominent in basal aggregates in acinar cells and in extra-acinar sites. LG gene expression of Npc1, Npc2, Lipa, and Mcoln2, encoding proteins involved in lipid metabolism, was increased with age. NPC1 was also significantly increased in old LGs by western blotting. In photobleached LG sections, confocal fluorescence microscopy imaging of NPC1, NPC2, and CTSL immunofluorescence showed age-associated enrichment in macrophages labeled to detect F4/80. Although mononuclear macrophages were detectable in LG at all ages, this novel multinucleate macrophage population containing NPC1, NPC2, and CTSL and enriched in F4/80 and some CD11b was increased with age at extra-acinar sites. Lipid-metabolizing proteins enriched in F4/80-positive multinucleated macrophages are increased in old LGs adjacent to sites of lipid deposition in acini.

Neutrophils and galectin-3 defend mice from lethal bacterial infection and humans from acute respiratory failure

Respiratory infection by Pseudomonas aeruginosa, common in hospitalized immunocompromised and immunocompetent ventilated patients, can be life-threatening because of antibiotic resistance. This raises the question of whether the host’s immune system can be educated to combat this bacterium. Here we show that prior exposure to a single low dose of lipopolysaccharide (LPS) protects mice from a lethal infection by P. aeruginosa. LPS exposure trained the innate immune system by promoting expansion of neutrophil and interstitial macrophage populations distinguishable from other immune cells with enrichment of gene sets for phagocytosis- and cell-killing-associated genes. The cell-killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs and a prognostically favorable hypoinflammatory plasma biomarker subphenotype. Taken together, our study provides impetus for harnessing the potential of galectin-3-expressing neutrophils to protect from lethal infections and respiratory failure.

Stimulus-response signaling dynamics characterize macrophage polarization states

The functional state of cells is dependent on their microenvironmental context. Prior studies described how polarizing cytokines alter macrophage transcriptomes and epigenomes. Here, we characterized the functional responses of 6 differentially polarized macrophage populations by measuring the dynamics of transcription factor nuclear factor κB (NF-κB) in response to 8 stimuli. The resulting dataset of single-cell NF-κB trajectories was analyzed by three approaches: (1) machine learning on time-series data revealed losses of stimulus distinguishability with polarization, reflecting canalized effector functions. (2) Informative trajectory features driving stimulus distinguishability ("signaling codons") were identified and used for mapping a cell state landscape that could then locate macrophages conditioned by an unrelated condition. (3) Kinetic parameters, inferred using a mechanistic NF-κB network model, provided an alternative mapping of cell states and correctly predicted biochemical findings. Together, this work demonstrates that a single analyte's dynamic trajectories may distinguish the functional states of single cells and molecular network states underlying them. A record of this paper's transparent peer review process is included in the supplemental information.

The rationale of reprogramming TREM2+ macrophages for overcoming the resistance to immune checkpoint inhibitors in pancreatic adenocarcinoma.

e14649 Background: Pancreatic ductal adenocarcinoma (PDAC) resists to immune checkpoint inhibitors as single agents. TAMs expressing the triggering receptor expressed on myeloid cells (TREM2) protein were found to be a dominant macrophage population in PDAC; targeting TREM2 has been found to enhance the efficacy of immune checkpoint inhibitor-based therapies in preclinical studies of other malignancies. Here, we studied the role of targeting TREM2 in PDAC by testing the combination of non-depleting anti-TREM2 antibody and anti-PD-1 antibody in a mouse model of PDAC. We also examined the relationship between TREM2 expression and effector T cells in surgically resected human PDAC collected from a previous clinical trial treated with the neoadjuvant combination therapy of nivolumab (anti-PD-1 antibody), urelumab (anti-CD137 agonist antibody), and the pancreatic cancer GVAX vaccine. Methods: C57BL/6 mice were orthotopically implanted with KPC tumors. Anti-TREM2 was injected intraperitoneally (i.p.) once a week and anti-PD-1 was injected i.p. twice a week. Tumor volume was measured by ultrasound. TREM2 was stained on slides which were previously obtained from surgically resected PDACs following the neoadjuvant combination treatment of nivolumab, urelumab, and GVAX and stained for multiple immune cell markers with a multiplex staining-striping immunohistochemistry (IHC) technique. Results: Tumor growth post-tumor implantation was found to be significantly decreased by the anti-PD-1 and anti-TREM2 combination compared with anti-PD-1 alone, anti-TREM2 alone, or control treatment (p &lt; 0.0001). Survival of tumor-bearing mice was significantly prolonged by the anti-PD-1 and anti-TREM2 combination when compared to anti-PD-1(p &lt; 0.05), anti-TREM2(p &lt; 0.05), and control (p &lt; 0.01). Multiplex IHC analysis of human PDACs following neoadjuvant immunotherapy showed a higher density of granzyme B+CD8+ effector T cells correlated with higher density of TREM2+ cells (p = 0.0357). Conclusions: Our study demonstrates the anti-tumor efficacy of anti-TREM2 antibody in combination with an immune checkpoint inhibitor in mouse PDAC. However, the analysis of human PDAC following neoadjuvant immunotherapy suggests that TREM2 on TAMs may be activated following the activation of effector T cells. Taken together, our results suggest that TREM2+ TAMs should be reprogrammed instead of being depleted in the design of anti-TREM2-based therapy for PDAC.

ScRNA-seq reveals transcriptional dynamics of Encephalitozoon intestinalis parasites in human macrophages.

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Dissemination to other organ systems is also observed, and is potentially facilitated by macrophages. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the host and parasite during infection. While a small population of infected macrophages mount a response, most remain transcriptionally unchanged, suggesting that the majority of parasites may avoid host detection. The parasite transcriptome reveals large transcriptional changes throughout the life cycle, providing a blueprint for parasite development. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication and dissemination. Together, our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program.

Macrophage transcriptome changes in response to cardiac injuries are determined by the type of lesion rather than their ontogenetic nature

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): CRC1425 - DFG Introduction Resident macrophages account for 5% of the cells in the healthy heart. In response to cardiac injury, monocytes infiltrate and differentiate into recruited macrophages complementing the original resident macrophage population, and together orchestrate cardiac remodeling. We aim to identify the extent to which macrophage origin, tissue location and type of cardiac injury determine macrophage phenotypes in ischemic and non-ischemic cardiac injuries over time. Results and methods We use a reporter mouse line CX3CR1Yfp CreER/+:R26tdT/+ to visualize and quantify fluxes of resident and recruited macrophages and their respective localizations within the heart following ischemia and reperfusion (I/R) injury and pressure overload after transversal aortic constriction (TAC), respectively. Interestingly, macrophage numbers peaked during the first week post-surgery in both the local/acute and the global/continuous injury models. Initially, recruited macrophages outnumbered the resident macrophage pool within the infarct area but ultimately reached a near 1:1 equilibrium at 4 weeks post I/R injury. In the remote myocardium the 1:1 equilibrium established already during the first week. In TAC-injured hearts, the 1:1 ratio of recruited and resident hearts also established within the first week post-surgery and persisted up to 8 weeks of follow-up even as cardiac function deteriorated. In the infarct, recruited macrophages showed increased cytokine synthesis, defensive response, and cell adhesion pathways during the first week after injury. Many inflammatory pathways remained activated up to day 28 post MI despite simultaneous activation of the anti-inflammatory response. Formerly resident macrophages and their progeny also mounted an immune response throughout the healing process. Unexpectedly, by day 28 post MI when a stable scar had formed, resident and recruited macrophage population transcriptomes converted onto a common, pro-inflammatory steady state phenotype, that differed from their originally divergent gene expression profiles in the healthy heart by over one thousand genes. In the remote zone, however, resident macrophages maintained cellular homeostasis function, unlike recruited macrophages that retained an inflammatory program. Following TAC surgery, transcriptional profiles remained differentially regulated at all time points tested, where recruited subset induced immune response and resident macrophages upregulated tissue remodeling genes. Conclusion Ischemic and non-ischemic cardiac injuries induce a transient peak of monocyte recruitment and macrophage differentiation in and around cardiac lesions which lead to a permanent integration of recruited monocyte-derived macrophages into the pool of tissue resident macrophages – albeit at different paces. The type of injury and macrophage localization within the changing tissue microenvironment determine partial or complete override of ontogenic cell programs.