Macrophage Effector Functions Research Articles

HIF-1α regulates mitochondrial function in bone marrow-derived macrophages, but not in tissue-resident alveolar macrophages.

HIF-1α plays a critical role in shaping macrophage phenotype and effector function. We have previously shown that tissue-resident alveolar macrophages (TR-AMs) have extremely low glycolytic capacity at steady-state, but can shift toward glycolysis under hypoxic conditions. Here, using inducible HIF-1α knockout (Hif1a -/- ) TR-AMs and bone marrow-derived macrophages (BMDMs) and show that TR-AM HIF-1α is required for the glycolytic shift under prolyl hydroxylase inhibition, but is dispensable at steady-state for inflammatory effector function. In contrast, HIF-1α deletion in BMDMs led to diminished glycolytic capacity at steady-state and reduced inflammatory capacity, but higher mitochondrial function. Gene set enrichment analysis revealed enhanced c-Myc transcriptional activity in Hif1a -/- BMDMs, and upregulation of gene pathways related to ribosomal biogenesis and cellular proliferation. The findings highlight the heterogeneity of HIF-1α function in distinct macrophage populations and provide new insight into how HIF-1α regulates gene expression, inflammation, and metabolism in macrophages.

Abstract Tu012: Innate Immune Pathway cGAS-STING in Macrophage Function in Atherosclerosis

Macrophages are key cells in atherosclerotic cardiovascular disease. Macrophages within a single plaque are heterogenous: some are lipid-laden “foam cells” with varying ability to recycle lipoproteins or clear dead cells while others secrete traditional “pro-inflammatory” signals. Determining what signals bias macrophages to one phenotype versus another could identify a therapeutic target. Our lab showed that after acute myocardial infarction, macrophages are skewed to a reparative phenotype by loss of cGAS-STING signaling. cGAS-STING are a pattern recognition receptor-adaptor pair that are activated by double stranded DNA in a sequence-independent manner. Global deletion of Sting and Cgas have been shown to protect against atherosclerosis in mice. We hypothesize that hematopoietic-specific loss of Cgas will reduce plaque burden by biasing macrophages to a less inflammatory phenotype and upregulating pro-resolving functions. To test this, B6.129S7- Ldlr tm1Her /J ( Ldlr -/-) mice (female, age 8-12 weeks, 9 per group) underwent total body irradiation followed by bone marrow transplant from C57BL6/N- Cgas -/- ( Cgas -/-) or wildtype donor mice (female, age 6-8 weeks). After bone marrow reconstitution, recipient mice were started on Western diet. Mice will continue Western diet for 12 weeks (through April 24, 2024). Then, aortas, peripheral blood, spleen, and bone marrow will be collected for further analysis including plaque burden, fibrous cap thickness, and macrophage phenotype. We will also perform in vitro studies using primary macrophages to determine how Cgas -/- affects macrophage effector functions critical for atherosclerosis. To date, mice that received Cgas -/- or wildtype donor bone marrow had similar baseline cell count, triglyceride and total cholesterol levels. Bone marrow derived macrophages (BMDM) from Cgas -/- mice have increased expression of fibronectin. Thus, at baseline Cgas -/- BMDM express high levels of a marker associated with pro-reparative macrophages. More studies are needed to characterize this phenotype and determine the consequence for in vivo atherosclerosis.

Abstract 1599: Metabolism as a key regulator of macrophage phenotype in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related deaths in the US, with a five-year survival rate of 12%. Barriers to treatment include the highly fibrotic tumor microenvironment (TME), the high interstitial pressure which acts to collapse the blood vessels, and the influx of immunosuppressive immune cells. Within this immune milieu, we find that the most abundant are tumor-associated macrophages (TAMs). These TAMs display a phenotype similar to anti-inflammatory (M2-like) macrophages. Despite TAMs exerting an immunosuppressive function within the TME, there is not a complete overlap with the characteristics associated with M2 macrophages. Interestingly, TAMs still display many markers associated with the pro-inflammatory (M1-like) macrophages. This dichotomy suggests there may be a level of plasticity within the macrophage compartment that can be exploited to lead to better tumor control. Previous studies have shown a connection between arginine metabolism and macrophage effector function along the M1/M2 axis. In macrophages, arginine can be broken down in two ways: via iNOS into nitric oxide, which aids in effector function, or via Arginase 1 (Arg1) into ornithine and later proline, which is an essential component of the extracellular matrix (ECM). This study seeks to elucidate how metabolic perturbations influence macrophage function and phenotype. Here we observed that changes in oxygen availability and glucose abundance shifted macrophage expression of iNOS and Arg1. In vivo we found that conditionally knocking out Arg1 in the myeloid compartment leads to lower fibrosis when inducing inflammation in the pancreas. Furthermore, macrophages polarized with PDAC cancer-conditioned media display a sharp increase in Arg1 expression as compared to unpolarized macrophages, as well as increased resistance to both glycolytic inhibitors and mitochondrial inhibitors. This preliminary data demonstrates an important link between macrophage metabolism and function in the context of pancreatic disease. Citation Format: Cecily Anaraki, Christopher Halbrook. Metabolism as a key regulator of macrophage phenotype in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1599.

Immunometabolic Maladaptations to the Tumor Microenvironment.

Tumors consist of cancer cells and a wide range of tissue resident and infiltrating cell types. Tumor metabolism, however, has largely been studied on whole tumors or cancer cells and the metabolism of infiltrating immune cells remains poorly understood. It is now clear from a range of analyses and metabolite rescue studies that metabolic adaptations to the tumor microenvironment (TME) directly impede T-cell and macrophage effector functions. The drivers of metabolic adaptation to the TME and metabolic immune suppression include depletion of essential nutrients, accumulation of waste products or immune suppression metabolites, and metabolic signaling through altered posttranslational modifications. Each infiltrating immune cell subset differs, however, with specific metabolic requirements and adaptations that can be maladaptive for antitumor immunity. Here, we review T-cell and macrophage adaptation and metabolic immune suppression in solid tumors. Ultimately, understanding and addressing these challenges will improve cancer immunotherapy and adoptive chimeric antigen receptor T-cell therapies.

Differential impact of high-salt levels in vitro and in vivo on macrophage core functions

The consumption of processed food is on the rise leading to huge intake of excess dietary salt, which strongly correlates with development of hypertension, often leading to cardiovascular diseases such as stroke and heart attack, as well as activation of the immune system. The effect of salt on macrophages is especially interesting as they are able to sense high sodium levels in tissues leading to transcriptional changes. In the skin, macrophages were shown to influence lymphatic vessel growth which, in turn, enables the transport of excess salt and thereby prevents the development of high blood pressure. Furthermore, salt storage in the skin has been linked to the onset of pro-inflammatory effector functions of macrophages in pathogen defence. However, there is only little known about the mechanisms which are involved in changing macrophage function to salt exposure. Here, we characterize the response of macrophages to excess salt both in vitro and in vivo. Our results validate and strengthen the notion that macrophages exhibit chemotactic migration in response to salt gradients in vitro. Furthermore, we demonstrate a reduction in phagocytosis and efferocytosis following acute salt challenge in vitro. While acute exposure to a high-salt diet in vivo has a less pronounced impact on macrophage core functions such as phagocytosis, our data indicate that prolonged salt challenge may exert a distinct effect on the function of macrophages. These findings suggest a potential role for excessive salt sensing by macrophages in the manifestation of diseases related to high-salt diets and explicitly highlight the need for in vivo work to decipher the physiologically relevant impact of excess salt on tissue and cell function.

A254 CANAGLIFLOZIN EXACERBATES CHEMICALLY INDUCED COLITIS WHEN COMBINED WITH A HIGH SUGAR DIET

Abstract Background Consumption of high-sugar diets exacerbates inflammation and hinders the healing process in animal models of acute colitis. Canagliflozin (CANA) is a sodium glucose cotransporter-2 (SGLT2) inhibitor primarily used for the management of type 2 diabetes and cardiovascular disease. CANA has demonstrated the ability to suppress human T cell and macrophage effector function and to improve inflammation in rat colitis models. Based on these findings, we hypothesized that CANA would mitigate the deleterious effects of a high sugar diet in a DSS colitis model through its effects on immune effector cells. Aims The aim of this study was to examine the effects of a high sugar diet and CANA administration on colonic inflammation in an animal model of acute colitis. Methods Adult wild-type male and female 129/SvEv mice were placed on standard chow or a high-sugar diet (HSD) containing 50% sucrose (AIN76A) ± CANA (10mg/kg) gavaged daily (n=4-6 for each group). Following three days on the respective diet, mice received 1.8-2% dextran sodium sulfate (DSS) in their drinking water for 5 days, followed by water alone for 4 days. Disease Activity Index (DAI) was computed daily based on changes in animal weight, stool consistency, and blood in stool. Colonic tissues were evaluated for histological changes, weight-to-length ratio, and cytokine levels using the MesoScale discovery platform. Results Mice on the HSD had increased severity of colitis compared with chow fed mice as evidenced by enhanced DAI, increased weight loss, and increased levels of colonic TNFα (pampersand:003C0.02). Response to CANA treatment was both sex and diet dependent. Contrary to our hypothesis, CANA treatment increased severity of colitis in male mice on the HSD as evidenced by increased DAI and delayed healing with increased enterocyte injury and increased levels of lamina propria neutrophils and lymphocytes. CANA treatment did not exacerbate disease severity in females on the HSD but did delay healing as evidenced by increased lamina propria neutrophils and lymphocytes and worse enterocyte injury. These effects were not observed in mice that were fed standard chow diets with CANA. Conclusions A high-sugar diet significantly exacerbates chemically induced colitis and delays healing. This effect is intensified when combined with CANA, especially in male mice. These findings indicate that effects of SGLT2 inhibitors on colonic inflammation can be modulated by both dietary intake and sex. Funding Agencies CCC

Macrophage N-glycan processing inhibits antibody-dependent cellular phagocytosis.

Factors regulating macrophage effector function represent potential targets to optimize the efficacy of antibody-mediated therapies. Macrophages are myeloid cells capable of engulfing and destroying diseased or damaged target cells. Antibodies binding to the target cell surface can engage macrophage Fc gamma receptors (FcγRs) to elicit antibody-dependent cellular phagocytosis (ADCP), a process that contributes to treatments mediated by anti-tumor antibodies. Conversely, macrophage ADCP of apoptotic T cells is also linked to tolerance in the tumor environment. Here we evaluated the role of asparagine(N)-linked glycans in the function of macrophages derived from primary human monocytes. Macrophages treated with kifunensine, an inhibitor of N-glycan processing, exhibited greater target binding and ADCP of antibody-coated target cells. Kifunensine treatment increased ADCP of both rituximab-coated Raji B cells and trastuzumab-coated SKBR3 cells. ADCP required FcγRs; inhibiting CD64 / FcγRI led to the greatest reduction, followed by CD32 / FcγRII and then CD16 / FcγRIII in most donors. Kifunensine treatment also increased the antibody-binding affinity of CD16. Differences in the abundance of phosphorylated immune receptors, including Siglec-9, CD32a, and LAIR-1 correlated with the increased ADCP. These results demonstrate that N-glycan processing regulates macrophage effector function.

Phagocyte population in the inflammatory pattern of the bronchi of asthma patients regulated by interleukin-17A and interferon-gamma during the airway response to a cold stimulus

Introduction. Based on the common effector functions of polymorphonuclear neutrophils and macrophages as phagocytic cells, their role in the formation of an acute reaction of the respiratory tract to a cold stimulus in patients with asthma is of concern.Aim. The study of the concentration of phagocytes, IL-17A and IFN-γ in the inflammatory pattern of the bronchi of asthma patients depending on the airway reaction to a cold stimulus.Materials and methods. 129 patients with asthma were examined. The design of the study included questioning patients using a validated questionnaire Asthma Control Test (ACT, Quality Metric Inc., 2002), collection of induced and spontaneously produced sputum, exhaled breath condensate (EBC), bronchoprovocation test with a 3-minute isocapnic hyperventilation with cold (-20ºС) air (IHCA) with an assessment of the airway response (ΔFEV1) by spirometry.Results. Group 1 (n=55) included individuals with ΔFEV1 -10% and below, group 2 (n=74) – with ΔFEV1 above -10%: -15 (-21; -11) and -3.7 (-6.1; -0.38)%, respectively (p=0.0002). According to the level of ACT (17 [13; 21.5] and 19 [14; 22] points) and indicators of lung function (FEV1 [93.0±2.4 and 97.1±2.4%] and FEF25-75 [63.5±3.5 and 72.0±3.7%]), the patients had no significant intergroup differences. The pattern of bronchial inflammation in group 1 was mixed (neutrophils ≥40%), in group 2 – eosinophilic. In response to the IHCA test, the number of neutrophils significantly increased in the sputum of patients in group 1, the number of macrophages and the number of structurally intact epithelial cells decreased, in proportion to this, the level of IFN-γ and IFN-γ-inducible protein IP-10 (CXCL10) increased in the EBC in relation to patients of the 2nd group. A direct relationship was found between baseline concentrations of IP-10 and IFN-γ (Rs=0.7; p<0.01) in EBC.Conclusion. The airway response to a cold stimulus of patients with asthma is accompanied by functional activation of phagocytic cells with an escalation of neutrophilic inflammation and a decrease in the number of macrophages infiltrating the bronchi associated with an increase in the concentration of IFN-γ, which stimulates the processes of respiratory burst and triggers cell destruction and cytolysis.

Development of a novel in vitro model to study the modulatory role of the respiratory complex I in macrophage effector functions.

Increasing evidence demonstrate that the electron transfer chain plays a critical role in controlling the effector functions of macrophages. In this work, we have generated a Ndufs4-/- murine macrophage cell lines. The Ndufs4 gene, which encodes a supernumerary subunit of complex I, is a mutational hotspot in Leigh syndrome patients. Ndufs4-/- macrophages showed decreased complex I activity, altered complex I assembly, and lower levels of maximal respiration and ATP production. These mitochondrial respiration alterations were associated with a shift towards a pro-inflammatory cytokine profile after lipopolysaccharide challenge and improved ability to phagocytose Gram-negative bacteria.

BATF2 enhances proinflammatory cytokine responses in macrophages and improves early host defense against pulmonary Klebsiella pneumoniae infection.

Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a transcription factor that is emerging as an important regulator of the innate immune system. BATF2 is among the top upregulated genes in human alveolar macrophages treated with LPS, but the signaling pathways that induce BATF2 expression in response to Gram-negative stimuli are incompletely understood. In addition, the role of BATF2 in the host response to pulmonary infection with a Gram-negative pathogen like Klebsiella pneumoniae (Kp) is not known. We show that induction of Batf2 gene expression in macrophages in response to Kp in vitro requires TRIF and type I interferon (IFN) signaling, but not MyD88 signaling. Analysis of the impact of BATF2 deficiency on macrophage effector functions in vitro showed that BATF2 does not directly impact macrophage phagocytic uptake and intracellular killing of Kp. However, BATF2 markedly enhanced macrophage proinflammatory gene expression and Kp-induced cytokine responses. In vivo, Batf2 gene expression was elevated in lung tissue of wild-type (WT) mice 24 h after pulmonary Kp infection, and Kp-infected BATF2-deficient (Batf2-/-) mice displayed an increase in bacterial burden in the lung, spleen, and liver compared with WT mice. WT and Batf2-/- mice showed similar recruitment of leukocytes following infection, but in line with in vitro observations, proinflammatory cytokine levels in the alveolar space were reduced in Batf2-/- mice. Altogether, these results suggest that BATF2 enhances proinflammatory cytokine responses in macrophages in response to Kp and contributes to the early host defense against pulmonary Kp infection.NEW & NOTEWORTHY This study investigates the signaling pathways that mediate induction of BATF2 expression downstream of TLR4 and also the impact of BATF2 on the host defense against pulmonary Kp infection. We demonstrate that Kp-induced upregulation of BATF2 in macrophages requires TRIF and type I IFN signaling. We also show that BATF2 enhances Kp-induced macrophage cytokine responses and that BATF2 contributes to the early host defense against pulmonary Kp infection.

Modulation of Macrophage Redox and Apoptotic Processes to Leishmania infantum during Coinfection with the Tick-Borne Bacteria Borrelia burgdorferi.

Canine leishmaniosis (CanL) is a zoonotic disease caused by protozoan Leishmania infantum. Dogs with CanL are often coinfected with tick-borne bacterial pathogens, including Borrelia burgdorferi in the United States. These coinfections have been causally associated with hastened disease progression and mortality. However, the specific cellular mechanisms of how coinfections affect microbicidal responses against L. infantum are unknown. We hypothesized that B. burgdorferi coinfection impacts host macrophage effector functions, prompting L. infantum intracellular survival. In vitro experiments demonstrated that exposure to B. burgdorferi spirochetes significantly increased L. infantum parasite burden and pro-inflammatory responses in DH82 canine macrophage cells. Induction of cell death and generation of mitochondrial ROS were significantly decreased in coinfected DH82 cells compared to uninfected and L. infantum-infected cells. Ex vivo stimulation of PBMCs from L. infantum-seronegative and -seropositive subclinical dogs with spirochetes and/or total Leishmania antigens promoted limited induction of IFNγ. Coexposure significantly induced expression of pro-inflammatory cytokines and chemokines associated with Th17 differentiation and neutrophilic and monocytic recruitment in PBMCs from L. infantum-seropositive dogs. Excessive pro-inflammatory responses have previously been shown to cause CanL pathology. This work supports effective tick prevention and risk management of coinfections as critical strategies to prevent and control L. infantum progression in dogs.

Loss of stearoyl-CoA desaturase 2 disrupts inflammatory response in macrophages.

Macrophages are innate immune cells that patrol tissues and are the first responders to detect infection. They orchestrate the host immune response in eliminating invading pathogens and the subsequent transition from inflammation to tissue repair. Macrophage dysfunction contributes to age-related pathologies, including low-grade inflammation in advanced age that is termed "inflammaging." Our laboratory has previously identified that macrophage expression of a fatty acid desaturase, stearoyl-CoA desaturase 2 (SCD2), declines with age. Herein, we delineate the precise cellular effects of SCD2 deficiency in murine macrophages. We found that deletion of Scd2 from macrophages dysregulated basal and bacterial lipopolysaccharide (LPS)-stimulated transcription of numerous inflammation-associated genes. Specifically, deletion of Scd2 from macrophages decreased basal and LPS-induced expression of Il1b transcript that corresponded to decreased production of precursor IL1B protein and release of mature IL1B. Furthermore, we identified disruptions in autophagy and depletion of unsaturated cardiolipins in SCD2-deficient macrophages. To assess the functional relevance of SCD2 in the macrophage response to infection, we challenged SCD2-deficient macrophages with uropathogenic Escherichia coli and found that there was impaired clearance of intracellular bacteria. This increased burden of intracellular bacteria was accompanied by increased release of pro-inflammatory cytokines IL6 and TNF but decreased IL1B. Taken together, these results indicate that macrophage expression of Scd2 is necessary for maintaining the macrophage response to inflammatory stimuli. This link between fatty acid metabolism and fundamental macrophage effector functions may potentially be relevant to diverse age-related pathologies. IMPORTANCE Macrophages are immune cells that respond to infection, but their dysfunction is implicated in many age-related diseases. Recent evidence showed that macrophage expression of a fatty acid enzyme, stearoyl-CoA desaturase 2, declines in aged organisms. In this work, we characterize the effects when stearoyl-CoA desaturase 2 is deficient in macrophages. We identify aspects of the macrophage inflammatory response to infection that may be affected when expression of a key fatty acid enzyme is decreased, and these findings may provide cellular insight into how macrophages contribute to age-related diseases.

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.

Differential effect of lactate on synovial fibroblast and macrophage effector functions.

The synovial membrane is the main site of inflammation in rheumatoid arthritis (RA). Here several subsets of fibroblasts and macrophages, with distinct effector functions, have been recently identified. The RA synovium is hypoxic and acidic, with increased levels of lactate as a result of inflammation. We investigated how lactate regulates fibroblast and macrophage movement, IL-6 secretion and metabolism via specific lactate transporters. Synovial tissues were taken from patients undergoing joint replacement surgery and fulfilling the 2010 ACR/EULAR RA criteria. Patients with no evidence of degenerative or inflammatory disease were used as control. Expression of the lactate transporters SLC16A1 and SLC16A3 on fibroblasts and macrophages was assessed by immunofluorescence staining and confocal microscopy. To test the effect of lactate in vitro we used RA synovial fibroblasts and monocyte-derived macrophages. Migration was assessed via scratch test assays or using trans-well inserts. Metabolic pathways were analysed by Seahorse analyser. IL-6 secretion was determined by ELISA. Bioinformatic analysis was performed on publicly available single cell and bulk RNA sequencing datasets. We show that: i) SLC16A1 and SLC16A3 which regulate lactate intake and export respectively, are both expressed in RA synovial tissue and are upregulated upon inflammation. SLC16A3 is more highly expressed by macrophages, while SLC16A1 was expressed by both cell types. ii) This expression is maintained in distinct synovial compartments at mRNA and protein level. iii) Lactate, at the concentration found in RA joints (10 mM), has opposite effects on the effector functions of these two cell types. In fibroblasts, lactate promotes cell migration, IL-6 production and increases glycolysis. In contrast macrophages respond to increases in lactate by reducing glycolysis, migration, and IL-6 secretion. In this study, we provide the first evidence of distinct functions of fibroblasts and macrophages in presence of high lactate levels, opening new insights in understanding the pathogenesis of RA and offering novel potential therapeutic targets.

Genetically Engineered Artificial Exosome-Constructed Hydrogel for Ovarian Cancer Therapy.

Owing to the insidious onset of ovarian cancer, most patients are in the advanced stage with extensive peritoneal metastasis when they are diagnosed. Treatment of peritoneal metastasis from advanced ovarian cancer remains a significant challenge. Inspired by the massive macrophages in the peritoneal environment, here, we reported an artificial exosome-based peritoneal-localized hydrogel to domesticate peritoneal macrophages as the therapeutic target for realizing potent ovarian cancer therapy, where artificial exosomes derived from genetically sialic-acid-binding Ig-like lectin 10 (Siglec-10)-engineered M1-type macrophages were chemically designed as gelator. Upon triggering immunogenicity with X-ray radiation, our hydrogel encapsulating efferocytosis inhibitor MRX-2843 enabled a cascade regulation to orchestrate polarization, efferocytosis, and phagocytosis of peritoneal macrophages for realizing robust phagocytosis of tumor cells and powerful antigen presentation, offering a potent approach for ovarian cancer therapy via bridging the innate effector function of macrophages with their adaptive immune response. Moreover, our hydrogel is also applicable for potent treatment of inherent CD24-overexpressed triple-negative breast cancer, providing an emerging therapeutic regimen for the most lethal malignancies in women.

Neutrophils show distinct patterns of activation in the context of infection with different pathogens

Abstract Alternatively activated neutrophils (N2) enhance lung anti-helminth trained macrophage effector function in response to infection with the helminth Nippostrongylus. brasiliensis (Nb) (Fei Chen, et al., NI, 2014, CR, 2022). However, potential differences in activation patterns of neutrophils with different pathogens is not yet well studied. We report here lung neutrophils bear unique gene expression profiles at 2 days after infection with Nb, fungi (Aspergillosis fumigatus (Af)), and bacteria (Staphylococcus aureus (SA). AF and SA triggered genes associated with cell death and autophagy but differed greatly in metabolic activity. In contrast, Nb induced gene expression patterns characteristic of type 2 immunity, xenobiotic pathways, and cell proliferation. Further analysis revealed increased edu incorporation, KI-67 and IL-13 expression in neutrophils in the lung but not in the blood and bone marrow. Furthermore, the proliferating neutrophil subset predominantly expressed IL-13 and was IL-4R dependent. To directly examine neutrophils proliferating in the lung after Nb infection, Nb-primed neutrophils (CD45.2) were transferred to CD45.1 Nb inoculated recipients. At day 10 after inoculation, donor neutrophils were readily detected and were proliferating as measured by ki-67 staining and edu incorporation. These studies suggest that neutrophils exhibit distinct activation states following infection with different pathogens. Unlike neutrophils typically associated with microbial infection, where rapid turnover from cell death typically occurs, neutrophils activated following Nb infection persist and undergo cell cycling within the remodeled lung tissue microenvironment.

Abstract 6370: Cell-based luminescent reporter bioassays for immunotherapies targeting macrophage effector functions

Abstract Macrophages play a key role in the elimination of cancer via phagocytosis and presentation of tumor antigens to T cells. Antibody-dependent cellular phagocytosis (ADCP), mediated by macrophages and other myeloid cells, is an important mechanism-of-action (MoA) for antibody-based cancer immunotherapies. Therapeutic strategies that enhance ADCP and other macrophage effector functions can augment direct tumor destruction and enable anti-tumor immunity. ADCP is initiated by binding of antibody Fc domains to Fc gamma receptors (FcgRs), and FcgR crosslinking results in signal transduction that drives inflammatory cytokine production and phagocytosis. Simultaneously, macrophage effector functions are regulated by immune checkpoints. Blockade of these immune checkpoints (e.g., SIRPa, ILT4), or agonists of activation pathways (e.g., TLR receptors) can enhance macrophage effector functions. Current assays to measure macrophage function are tedious, low throughput, and highly variable. Here, we present a suite of cell-based luminescent reporter bioassays to measure macrophage effector functions: 1) A monocytic cell background was used to measure ADCP via endogenous expression of multiple FcgRs and integration of a FcgR-activation dependent luminescent reporter. 2) Direct detection of cellular phagocytosis can be measured using the HiBiT split luciferase system. 3) Modulation of macrophage-expressed immune checkpoints can be determined using cell-based reporter bioassays for SIRPa/CD47 and ILT4/HLA-G. 4) Activation of TLRs 1, 2, 4, 5 and 6 can be measured using a TLR reporter bioassay. These bioassays are robust, easy-to-use, and are pre-qualified according to ICH guidelines. Together, these novel reporter bioassays provide a robust, high-throughput toolbox to facilitate discovery and development of immunotherapies targeting macrophage effector functions. Citation Format: Jonathan Mitchell, Julia Gilden, Jim Hartnett, Pete Stecha, Gopal B. Krishnan, Frank Fan, Mei Cong, Jamison Grailer. Cell-based luminescent reporter bioassays for immunotherapies targeting macrophage effector functions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6370.

Abstract 2517: Optimizing breast cancer therapy by inhibiting the adenosine receptor and oxygen consumption

Abstract Immune checkpoint blockade has shown remarkable promise in melanoma and other tumor types. However, a large proportion of patients do not respond or develop acquired resistance. Tumors can activate multiple checkpoints and immunosuppressive pathways to evade immune surveillance, which are likely a cause of treatment failure. Therefore, inhibition of multiple checkpoints may be necessary for maximal efficacy of immunotherapies. The adenosine A2 receptor (A2aR), was shown to function as an immune checkpoint. Its blockade inhibited tumor growth and metastases and synergized with other checkpoint inhibitors. We propose that using AZD4635, a potent and selective A2aR antagonist, will enhance the therapeutic efficacy of anti-PD-1/PD-L1. In addition, we propose that blocking tumor oxygen consumption using deferiprone (DFP), phenformin (Phen) and metformin (Met) will further enhance A2aR and PD-1/PD-L1 blockade efficacy. We evaluated the effects of MET, Phen, DFP, and AZD4635 on tumor cells, T cell, and macrophage proliferation and effector function in vitro. IC50 measurements showed that T cells are the most sensitive to inhibition by these drugs, while 4T1 tumor cells appear to be the least sensitive. Naïve mouse T cells were activated with anti-CD3 and anti-CD28 coated dynabeads in the presence of titrating doses of all four drugs for 72 hours then analyzed by flow cytometry for proliferation and activation status. AZD4635 and DFP increased the expression of the activation marker CD25 (IL2Ra) on CD8 T cells. In addition, AZD4635 also increased expression of Granzyme B on both CD4 and CD8 T cells. Met appears to have little to no effect on T cell proliferation activation. We utilized the Seahorse XF Mito Stress Test assay to measure the mitochondrial respiratory activity of T cells in the presence these drugs. T cells were activated with dynabeads for 72 hours and then incubated with titrating doses of the drugs overnight before running on the Seahorse assay. DFP and Phen had the strongest effect on Maximal Respiration and Spare Respiratory Capacity at dilutions of 1uM followed by 16uM. A similar, albeit less profound effect was observed in groups treated with Met and AZD4635. Lastly, we obtained and generated two mouse breast cancer cell lines (4T1 and E0771) bearing HIF-1 reporter systems. We treated 4T1-HRE and E0771 cells with increasing doses of cobalt chloride (CoCl2) to chemically mimic hypoxia by inducing HIF1a expression. Both 4T1-HRE and E0771-HRE demonstrated increased luciferase activity that correlated with increasing doses of CoCl2 in vitro. Future experiments will focus on characterizing the adenosine pathway in vivo in 4T1-HRE and E0771-HRE tumors and examine how drugs that target the adenosine A2AaR receptor (AZD4635), and oxygen consumption (DFP, Phen and Met) influence tumor oxygen consumption in vivo as well as the activation states of immune cells in the tumor microenvironment. Citation Format: Sadna Budhu, Mayuresh Mane, Mamadou A. Bah, Juan Zurita, Inna Serganova, Soe Min, Anais Assouvie, Jedd D. Wolchok, Jason Koutcher, Vladimir Ponomarev, Taha Merghoub. Optimizing breast cancer therapy by inhibiting the adenosine receptor and oxygen consumption [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2517.

Whole-Transcriptome Analysis Highlights Adenylyl Cyclase Toxins-Derived Modulation of NF-κB and ERK1/2 Pathways in Macrophages.

Edema toxin (ET), one of the main toxic factors of Bacillus anthracis (B. anthracis), is a kind of potent adenylate cyclase (AC). B. anthracis has adapted to resist macrophage microbicidal mechanisms in part by secreting ET. To date, there is limited information on the pathogenic mechanisms used by ET to manipulate macrophage function, especially at the transcriptome level. We used RNA sequencing to study transcriptional changes in RAW264.7 cells treated with ET. We aimed to identify molecular events associated with the establishment of infection and followed changes in cellular proteins. Our results indicate that ET inhibited TNF-α expression in the RAW264.7 mouse macrophage cell line by activating the cAMP/PKA pathway. ET challenge of macrophages induced a differential expression of genes that participate in multiple macrophage effector functions such as cytokine production, cell adhesion, and the inflammatory response. Furthermore, ET influenced the expression of components of the ERK1/2, as well as the NF-αB signaling pathways. We also showed that ET treatments inhibit the phosphorylation of the ERK1/2 protein. ET also attenuated NF-αB subunit p65 phosphorylation and transcriptional activity of NF-αB via the cAMP/PKA pathway in macrophages. Since the observed modulatory effects were characteristic only of the bacterial exotoxin ET, we propose this may be a mechanism used by B. anthracis to manipulate macrophages and establish systemic infection.

Glutamine Metabolism Supports the Functional Activity of Immune Cells against Aspergillus fumigatus.

The reprogramming of cellular metabolism of immune cells is an essential process in the regulation of antifungal immune responses. In particular, glucose metabolism has been shown to be required for protective immunity against infection with Aspergillus fumigatus. However, given the intricate cross talk between multiple metabolic networks and signals, it is likely that cellular metabolic pathways other than glycolysis are also relevant during fungal infection. In this study, we demonstrate that glutamine metabolism is required for the activation of macrophage effector functions against A. fumigatus. Glutamine metabolism was found to be upregulated early after fungal infection and glutamine depletion or the pharmacological inhibition of enzymes involved in its metabolism impaired phagocytosis and the production of both proinflammatory and T-cell-derived cytokines. In an in vivo model, inhibition of glutaminase increased susceptibility to experimental aspergillosis, as revealed by the increased fungal burden and inflammatory pathology, and the defective cytokine production in the lungs. Moreover, genetic variants in glutamine metabolism genes were found to regulate cytokine production in response to A. fumigatus stimulation. Taken together, our results demonstrate that glutamine metabolism represents an important component of the immunometabolic response of macrophages against A. fumigatus both in vitro and in vivo. IMPORTANCE The fungal pathogen Aspergillus fumigatus can cause severe and life-threatening forms of infection in immunocompromised patients. The reprogramming of cellular metabolism is essential for innate immune cells to mount effective antifungal responses. In this study, we report the pivotal contribution of glutaminolysis to the host defense against A. fumigatus. Glutamine metabolism was essential both in vitro as well as in in vivo models of infection, and genetic variants in human glutamine metabolism genes regulated cytokine production in response to fungal stimulation. This work highlights the relevance of glutaminolysis to the pathogenesis of aspergillosis and supports a role for interindividual genetic variation influencing glutamine metabolism in susceptibility to infection.