M1 State Research Articles

TMOD-25. UTILIZING PRIMARY MACROPHAGES AS AN IMPROVED SERUM-FREE IN VITRO MODEL OF TUMOR-ASSOCIATED MACROPHAGES IN GLIOBLASTOMA

Abstract BACKGROUND Tumor-associated macrophages (TAMs) account for up to 40% of tumor mass in glioblastoma and over 70% of the immune cells contained therein. TAMs influence glioblastoma (GBM) growth, invasion, and resistance to therapy. Unfortunately, in vitro macrophage models typically employ immortalized cell lines, such as THP-1, which require serum for their growth and maintenance, and are often stimulated with IL-4/13 to mimic TAM function in vitro. Unfortunately, THP-1-derived macrophages do not readily adopt the M2 state and may be poor TAM models. METHODS The buffy coat from peripheral blood was isolated from healthy donor blood and plated in RPMI 1640 supplemented with 10% human serum and 50ng/mL of M-CSF. 25 ng/mL of M-CSF was added on day 3. The macrophages were washed, lifted, and plated in serum-free X VIVO-15 supplemented with M-CSF. Afterwards macrophages were left in an M0 (resting) state, or polarized towards an M1(pro-inflammatory) by stimulating with LPS/IFNγ, M2 (anti-inflammatory) by polarizing with IL-4/13, or TAM state by co-culturing with GBM patient-derived xenolines. Macrophage polarization was characterized through cytokine arrays, invasion, angiogenesis, and phagocytosis assays, comparing them to M0, M1, and M2 macrophages. RESULTS M1-polarized THP-1 macrophages displayed an increase in M1-associated markers, such as CD80 and HLA-DR (p<0.0001); however, M2-polarized THP-1 macrophages did not display an increase in the M2-associated surface marker, CD206 (p = 0.6167). Interestingly, M1-polarized peripheral blood mononuclear cell (PBMC)-derived macrophages displayed increases in the M1-associated markers, CD80 and CD369 (p< 0.01). Additionally, M2-polarized PBMC-derived macrophages displayed increases in CD206 (p<0.0001). Interestingly, TAMs derived from either THP-1s or PBMCs did not closely align with either polarization state using cell-surface markers, dPCR, or cytokine arrays. CONCLUSIONS We have demonstrated a serum-free method of generating a TAM model by polarizing primary macrophages towards a GBM-supportive phenotype that differs molecularly and functionally from M0, M1, and M2 macrophages.

Hypoxic postconditioning modulates neuroprotective glial reactivity in a 3D cortical ischemic-hypoxic injury model

Stroke remains one of the major health challenges due to its high rates of mortality and long-term disability, necessitating the development of effective therapeutic treatment. This study aims to explore the neuroprotective effects of hypoxic postconditioning (HPC) using a cell-based 3D cortical ischemic-hypoxic injury model. Our model employs murine cells to investigate HPC-induced modulation of glial cell reactivity and intercommunication post-oxygen-glucose deprivation-reoxygenation (OGD-R) injury. We found that a single HPC session (1HPC) provided the most significant neuroprotection post-OGD-R compared to multiple intermittent hypoxic treatments, evidenced by improved spheroidal structure, enhanced cell survival and reduced apoptosis, optimal modulation of neuronal phenotypes, dampened ischemic responses, and augmented neurite outgrowth of spheroids. Furthermore, 1HPC suppressed both pro-inflammatory A1 and anti-inflammatory A2 astrocyte phenotypes despite the induction of astrocyte activation while reducing microglial activation with inhibited M1 and M2 reactive states. This was accompanied by a decrease in gene expression of the pro-inflammatory cytokines essential to microglia-astrocyte signaling, collectively suggesting a shift of glial cells away from their traditional reactive states for neuroprotection. This study highlights the potential of 1HPC as a novel therapeutic intervention for ischemic injury via the modulation of neuroprotective glial reactivity. Moreover, the 3D cortical ischemic-hypoxic injury model employed here holds enormous potential serving as a disease model to further elucidate the underlying mechanism of HPC, which can also extend to the applications in brain regeneration, drug development, and the modeling of neural diseases.

A Core-Brush Nanoplatform with Enhanced Lubrication and Anti-Inflammatory Properties for Osteoarthritis Treatment.

Osteoarthritis (OA) is recognized as a highly friction-related joint disease primarily associated with increased joint friction and inflammation due to pro-inflammatory M1-type macrophage infiltration in the articular cavity. Therefore, strategies to simultaneously increase lubrication and relieve inflammation to remodel the damaged articular microenvironment are of great significance for enhancing its treatment. Herein, a multifunctional core-brush nanoplatform composed of a ROS-scavenging polydopamine-coated SiO2 core and lubrication-enhancing zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) brush and loaded with the anti-inflammatory drug curcumin by a reactive oxygen species (ROS)-liable conjugation (named as SiO2@PP-Cur) is rationally designed. Benefiting from the grafted zwitterionic PMPC brush, a tenacious hydration layer with enhanced lubricity for reducing joint abrasions is developed. More importantly, based on the mono-iodoacetic acid-induced arthritis (MIA) rat model, intra-articular injection of SiO2@PP-Cur nanoplatform can effectively alleviate articular inflammation via promoting macrophage polarization from the pro-inflammatory M1 to anti-inflammatory M2 state by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and attenuating the degradation of cartilage matrix, resulting in the remodeling of the damaged microenvironment into a pro-regenerative microenvironment. As a result, SiO2@PP-Cur can considerably inhibit OA progression. Therefore, the work may provide a novel strategy for the development of an advanced core-brush nanoplatform for enhanced OA therapy.

Gamma Wave Stimulation Affects BACE1 Levels and Promotes the Transition of Microglia from M1 State to M2 State

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder and it presents with the hallmark neuropathological features of amyloid-beta (Aβ) plaques, tau tangles and active neuroinflammation. Beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a key mediator of increased Aβ, has been identified as the primary enzymatic source for production of this pathologic peptide while reactive microglial cells contribute to ongoing neurodegeneration perhaps through activation into their aggressive M1 state. The present study endeavors for the first time to address how gamma wave stimulation modulates BACE1 and microglial polarization (from M1 to beneficial M2) in different AD models. We hypothesize that gamma wave stimulation will reduce BACE1, decrease Aβ plaque burden and shift microglials towards M2 phenotype leading to reduced neuroinflammation and maintain cognitive function. This study will target mouse models of AD which have undergone gamma wave stimulation and its effects on BACE1 expression, microglial polarisation markers, Aβ plaque load and cognitive performance. Hypotheses the treatment will reduce BACE1 activity, decrease M1/M2 microglial ratio, improve cognitive performance and delay Aβ plaque accumulation These data suggest promising therapeutic strategies for neuroinflammation and amyloidogenesis in AD.

CTRP9 attenuates peripheral nerve injury-induced mechanical allodynia and thermal hyperalgesia through regulating spinal microglial polarization and neuroinflammation mediated by AdipoR1 in male mice

Peripheral nerve injury triggers rapid microglial activation, promoting M1 polarization within the spinal cord, which exacerbates the progression of neuropathic pain. C1q/TNF-related protein 9 (CTRP9), an adiponectin homolog, is known to suppress macrophage activation and exhibit anti-inflammatory properties through the activation of adiponectin receptor 1 (AdipoR1) in various disease contexts. Nevertheless, the involvement of CTRP9 in microglial polarization in the context of neuropathic pain is still unclear. Our study aimed to how CTRP9 influences spinal microglial polarization, neuroinflammation, and pain hypersensitivity, as well as the underlying mechanism, using a neuropathic pain model in male mice with spared nerve injury (SNI) of sciatic nerve. Our findings revealed SNI elevated the spinal CTRP9 and AdipoR1 levels in microglia. Furthermore, intrathecal administration of recombinant CTRP9 (rCTRP9) substantially weakened mechanical hypersensitivity and heat-related pain response triggered by SNI. On the other hand, rCTRP9 mediated a phenotypic switch in microglia, from the pro-inflammatory M1 state to the anti-inflammatory M2 state, by influencing the spinal AMPK/NF-κB mechanism in SNI mice. Additionally, treatment with AdipoR1 siRNA or an AMPK-specific antagonist both reversed the effects of CTRP9 on the phenotypic switching of spinal microglia and pain hypersensitivity. Collectively, these results indicate that CTRP9 ameliorates mechanical hypersensitivity and heat-related pain response, shifted the balance of microglia towards the anti-inflammatory M2 state, and suppresses neuroinflammatory responses by modulating the AMPK/NF-κB pathway, mediated by AdipoR1 activation, in mice with SNI.Graphical

Exploring the Role of Secondary Metabolites from Plants and Microbes as Modulators of Macrophage Differentiation.

Recent research has uncovered that secondary metabolites-biologically active compounds produced by plants, microbes, and other organisms-play a significant role in regulating the differentiation and function of macrophages. Macrophages, key components of the innate immune system, are crucial for a wide range of physiological processes, including immune response modulation, tissue homeostasis, and host defense against pathogens. This research delves into the mechanisms by which secondary metabolites influence macrophage differentiation signaling pathways, with a focus on how specific compounds affect macrophage polarization and functional phenotypes. Understanding these effects can open new avenues for developing therapeutic strategies that target macrophage-mediated immune responses. Secondary metabolites, such as nitrogen (N) and sulfur (S) containing compounds, terpenoids, and phenolic compounds from plants and microbes, can modulate macrophage differentiation by influencing cytokine production and activity. The activation of signaling pathways in macrophages involves multiple receptors and transcription factors, including IFN-γ receptor activation leading to STAT1 activation, TLR4 triggering IRF5, NFκB, and AP1, IL-4 receptor activation leading to STAT6 and IRF4 activation, PPARγ activation via the fatty acid receptor, TLR4 increasing CREB and C/EBP levels. The complex interplay between transcription factors and cytokines is crucial for maintaining the balance between the M1 and M2 states of macrophages. Despite these insights, further research is needed to unravel the specific molecular mechanisms involved and to identify promising secondary metabolites that could be translated into clinical applications.

On Calabi‐Yau Manifolds at Strong Topological String Coupling

AbstractIt was recently shown that integrating out M2 states on Calabi‐Yau manifolds captures non‐perturbative topological string physics in the free energy. In this note, It has been shown that the resulting expression manifests a certain duality symmetry: the free energy at strong string coupling is equal to the Calabi‐Yau period at weak string coupling. The duality yields the appropriate prescription for completing the integrating out in the ultraviolet.

Double-camouflaged tellurium nanoparticles for enhanced photothermal immunotherapy of tumor

The photothermal conversion properties of tellurium (Te) nanoparticles have been extensively investigated, rendering them a promising candidate for tumor photothermal therapy. However, there is still room for improvement in the development of efficient Te-based drug delivery systems. Here, Te nanoparticles are mineralized with bioactive molecules within attenuated Salmonella (S-Te), which are subsequently taken up by macrophages (RAW264.7) to construct a double-camouflaged delivery platform (RS-Te). Remarkably, RS-Te retains superior photothermal properties under near-infrared irradiation. The mineralization process eliminates bacterial proliferation potential, thereby mitigating the risk of excessive bacterial growth in vivo. Furthermore, the uptake of bacteria by macrophages not only polarizes them into M1 macrophages to induce an anti-tumor immune response but also circumvents any adverse effects caused by complex antigens on the bacterial surface. The results show that RS-Te can effectively accumulate and retain in tumors. RS-Te-mediated photothermal immunotherapy largely promotes the maturation of dendritic cells and priming of cytotoxic T cells induced by near-infrared laser irradiation. Moreover, RS-Te can switch the activation of macrophages from an immunosuppressive M2 phenotype to a more inflammatory M1 state. The double-camouflaged delivery system may offer highly efficient and safe cancer treatment.

DHA plays a protective role in cerebral ischemia–reperfusion injury by affecting macrophage/microglia type polarization

A close correlation exists between the macrophage/microglia(MΦ/MG) polarization states and the development of cerebral ischemia and reperfusion (I/R). Therefore it is of great significance to research on how to modulate the MΦ/MG states for improved patient outcomes. In particular, regulatory mechanisms involved in this process remain to be identified. Hereby, we aim to shed light on how docosahexaenoic acid (DHA) actively modulates the switch between M1 and M2 macrophage states by restraining the NACHT-LRR-PYD-containing protein three inflammasome (NALP3). We found that NALP3-positive cells were detected in clinical human cerebral infarction tissue samples and the mouse tMCAO model. In mice after DHA treatment, the number of NALP3-positive cells was significantly reduced, significantly decreasing infarct volume and improving the postoperative physical status of mice. NALP3-positive cells were found to be MΦ/MG after co-staining with CD11b. By extracting peritoneal macrophages, it was verified that DHA inhibited the activation of NALP3 and regulated the transformation of M1 and M2 cells, thereby reducing I/R injury.

Spatiotemporal Delivery of Dual Nanobodies by Engineered Probiotics to Reverse Tumor Immunosuppression via Targeting Tumor-Derived Exosomes.

The anti-PD-L1 and its bispecific antibodies have exhibited durable antitumor immunity but still elicit immunosuppression mainly caused by tumor-derived exosomes (TDEs), leading to difficulty in clinical transformation. Herein, engineered Escherichia coli Nissle 1917 (EcN) coexpressing anti-PD-L1 and anti-CD9 nanobodies (EcN-Nb) are developed and decorated with zinc-based metal-organic frameworks (MOFs) loaded with indocyanine green (ICG), to generate EcN-Nb-ZIF-8CHO-ICG (ENZC) for spatiotemporal lysis of bacteria for immunotherapy. The tumor-homing hybrid system can specifically release nanobodies in response to near-infrared (NIR) radiation, thereby targeting TDEs and changing their biological distribution, remodeling tumor-associated macrophages to M1 states, activating more effective and cytotoxic T lymphocytes, and finally, leading to the inhibition of tumor proliferation and metastasis. Altogether, the microfluidic-enabled MOF-modified engineered probiotics target TDEs and activate the antitumor immune response in a spatiotemporally manipulated manner, offering promising TDE-targeted immune therapy.

Quantitative proteomics reveals differential extracellular vesicle cargo from M1 and M2 monocyte-derived human macrophages.

Extracellular vesicles (EVs) mediate intercellular communication by carrying molecular cargo that facilitate diverse physiological processes. Macrophages, playing central roles in immune responses, release EVs that modulate various cellular functions. Given the distinct roles of M1 and M2 macrophage states, understanding the proteomic profiles of their EVs is important for elucidation of EV-mediated signalling and identifying potential biomarkers for diseases involving macrophage polarisation. We employed quantitative proteomics combined with bioinformatics to characterise the proteomic profile of EVs released by M1 and M2 monocyte-derived macrophages. We identified 1,731 proteins in M1/M2 EVs, 132 of which were significantly differentially between M1 and M2. Proteomic data, together with pathway analysis, found that M1/M2 macrophage EV cargo relate to cellular source, and may play roles in shaping immune responses, with M1 EV cargo associated with promotion of pro-inflammatory and antiviral functions, while M2 EV cargo associated with immune regulation and tissue repair. M1 EV cargo was associated with cytokine/chemokine signalling pathways, DNA damage, methylation, and oxidative stress. M2 EV cargo were associated with macrophage alternative-activation signalling pathways, antigen presentation, and lipid metabolism. We also report that macrophage EVs carry metallothioneins, and other related proteins involved in response to metals and oxidative stress.

Abstract B053: Tumor-associated macrophages diminishes IL1RAP-targeting CAR-T cell therapy efficacy in Ewing sarcoma

Abstract Ewing sarcoma (EwS) is a pediatric malignancy of soft tissue and bone with a poor prognosis, especially in its metastatic stages. We have previously identified IL1RAP as a promising target for immunotherapies like chimeric antigen receptor (CAR) T cell therapy against EwS. However, CAR-T cells, despite their success in hematological cancers, often show limited efficacy against solid tumors. In orthotopic xenograft models of Ewing sarcoma (EwS) we have observed that CD11b+ CD206+ macrophages, known as tumor-associated macrophages (TAMs), surround the primary tumor. Treatment with IL1RAP-targeting CAR-T cells specific to EwS results in T cells localizing with these macrophages and failing to infiltrate the primary tumor. This study aims to characterize the impact of TAMs on CAR-T cell efficacy in EwS. We hypothesized that TAMs, polarized by Ewing sarcoma cells, suppress IL1RAP CAR-T cell activity. To model these interactions, we co-cultured EwS cells, macrophages, and CAR-T cells, performing luciferin-based in vitro cytotoxicity assays. THP-1 monocytes were differentiated into macrophages and polarized into M1 or M2 states using cytokines (LPS + IFN-y, IL-4 + IL-13), or differentiated into TAMs by co-culture with EwS cell lines. In vitro cytotoxicity assays were conducted using luciferase-expressing EwS target cells, polarized THP-1 macrophages, and IL1RAP CAR-T effector cells, with luminescence indicating EwS cell survival. We found that M1 macrophages did not affect CAR-T cell targeting of EwS cells, whereas M2 macrophages and TAMs significantly decreased specific lysis (p < 0.001). Conditioned media from polarized macrophages had no effect on CAR-T cell performance against EwS, suggesting that physical proximity or direct contact with TAMs is necessary to suppress CAR-T cell function. Western blot analysis of polarized THP-1 macrophages revealed that M2-polarized macrophages upregulated IL1RAP expression compared to M1 macrophages. Ongoing experiments aim to determine whether elevated IL1RAP expression in M2 macrophages redirects IL1RAP CAR-T cells away from EwS cells or if M2 macrophages mediate general suppression of CAR-T cell activity. Additionally, we are investigating whether THP-1-derived TAMs and primary macrophages also upregulate IL1RAP expression when polarized and how this affects syngeneic CAR-T cell performance. Our findings indicate that TAMs can influence the efficacy of IL1RAP-targeting CAR-T cells against EwS. Further investigation into the specific mechanisms responsible for this suppressive effect could enhance CAR-T therapy for Ewing sarcoma and potentially other solid tumors. Citation Format: Yue Zhou (Joe) Huang, Melanie Rouleu, Poul Sorensen. Tumor-associated macrophages diminishes IL1RAP-targeting CAR-T cell therapy efficacy in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B053.

Investigating the pro-inflammatory differentiation of macrophages with bacterial ghosts in potential infection control.

In the complex realm of bacterial infections, particularly those caused by Staphylococcus aureus (S. aureus), macrophages play a pivotal role in orchestrating the immune response. During the initial stages of infection, the monocytes give rise to macrophages with a pro-inflammatory (M1 type) behaviour, engulfing and neutralizing the invading pathogens. However, under the sustained influence of S. aureus infection, monocytes can undergo a transition into an anti-inflammatory M2 state (pro-infection) rather than the M1 state (anti-infection), thereby compromising effective infection control. Therefore, it is necessary to develop a strategy that would preserve the pro-inflammatory functions of macrophages, in a safe and controlled manner. For this, we focused on harnessing the potential of S. aureus-derived ghost cells (GCs) which are non-live empty envelopes of bacterial cells, but with the antigenic determinants intact. Through a unique Lugol's-iodine treatment, we generated GCs and characterization of these GCs using gel electrophoresis, FTIR, flow cytometry, TEM, and SEM confirmed their structural integrity. Following this, we assessed the extend of cellular association of the GCs with RAW267.4 macrophages, and observed an immediate interaction between the two, as evident from the flowcytometry and microscopy studies. We then performed macrophage polarisation on a human monocyte-macrophage model cell line, THP-1. Our findings revealed that GCs effectively activated macrophages, and promoted a pro-inflammatory polarisation with the expression of M1 differentiation markers (CD86, TNFα, IL-1β, IL-6, IL-12) evaluated through both qPCR and ELISA. Interestingly an intermediary expression of M2 markers viz., CD206 and IL-10 was also observed, but was overruled by the enhanced expression of M1 markers at a later time point. Overall, our study introduces a novel approach utilizing GCs to guide naïve macrophages towards M1 subtypes, thereby potentiating immune responses during microbial infections. This innovative strategy can modulate macrophage function, ultimately improving outcomes in S. aureus infections and beyond.

Unveiling microglia heterogeneity in intracerebral hemorrhage

Microglia are important innate immune cells in the brain, and a rich diversity of subtypes has recently been discovered that expand beyond the traditional classification of traditional M1 (pro-inflammatory) and M2 (anti-inflammatory) classifications. Intracerebral hemorrhage (ICH) is a devastating form of stroke, and the understanding of its later-stage pathological mechanisms remains incomplete. In this study, through the analysis of single-cell transcripts from mice brains 14 days post-ICH, three disease-associated expression patterns of microglia were identified. These include a lipid metabolism and phagocytosis phenotype reminiscent of Disease-Associated Microglia (DAM) initially discovered in Alzheimer’s disease models, a phenotype associated with angiogenesis, and a relatively independent phenotype similar to the pro-inflammatory M1 state. These findings were further validated through immunofluorescence in both mouse and human specimens. In addition, analysis of single-cell transcripts from mice brains 3 days post-ICH suggested that microglia involved in lipid metabolism and phagocytosis likely emerge from early proliferating populations. Given the distinct origins and phenotypic characteristics of pro-inflammatory and reparative microglia, interventions targeting these cells hold the potential to modulate the delicate balance between injury and repair during the pathophysiological process of ICH, highlighting a pivotal direction for future therapeutic strategies.

Targeting Microglia in Neuroinflammation: H3 Receptor Antagonists as a Novel Therapeutic Approach for Alzheimer's Disease, Parkinson's Disease, and Autism Spectrum Disorder.

Histamine performs dual roles as an immune regulator and a neurotransmitter in the mammalian brain. The histaminergic system plays a vital role in the regulation of wakefulness, cognition, neuroinflammation, and neurogenesis that are substantially disrupted in various neurodegenerative and neurodevelopmental disorders. Histamine H3 receptor (H3R) antagonists and inverse agonists potentiate the endogenous release of brain histamine and have been shown to enhance cognitive abilities in animal models of several brain disorders. Microglial activation and subsequent neuroinflammation are implicated in impacting embryonic and adult neurogenesis, contributing to the development of Alzheimer's disease (AD), Parkinson's disease (PD), and autism spectrum disorder (ASD). Acknowledging the importance of microglia in both neuroinflammation and neurodevelopment, as well as their regulation by histamine, offers an intriguing therapeutic target for these disorders. The inhibition of brain H3Rs has been found to facilitate a shift from a proinflammatory M1 state to an anti-inflammatory M2 state, leading to a reduction in the activity of microglial cells. Also, pharmacological studies have demonstrated that H3R antagonists showed positive effects by reducing the proinflammatory biomarkers, suggesting their potential role in simultaneously modulating crucial brain neurotransmissions and signaling cascades such as the PI3K/AKT/GSK-3β pathway. In this review, we highlight the potential therapeutic role of the H3R antagonists in addressing the pathology and cognitive decline in brain disorders, e.g., AD, PD, and ASD, with an inflammatory component.

THE INFLUENCE OF ULTRA-WIDEBAND LOW-INTENSITY ULTRASOUND ON THE FUNCTIONAL ACTIVITY OF PERITONEAL MACROPHAGES IN VITRO

Studies have been conducted on the influence of various modes of ultrasound on the functional activity of peritoneal macrophages in nitro. It was found that the use of low-intensity broadband ultrasound (UMUS) significantly increases the cytotoxic and respiratory activity of macrophages compared to low-intensity pulsed ultrasound (LIPUS) and control. There was a significant increase in the level of endogenous peroxidase in the macrophage lysate, as well as a transition of macrophages from the M1 (pro-inflammatory) to M2 (anti-inflammatory) state when using UMUS.

Hyperbaric Oxygen Therapy as a Novel Approach to Modulating Macrophage Polarization for the Treatment of Glioblastoma.

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer with a poor prognosis despite current treatments. This is partially attributed to the immunosuppressive environment facilitated by tumor-associated macrophages, which predominantly underlie the tumor-promoting M2 phenotype. This study investigated the potential of hyperbaric oxygen (HBO) therapy, traditionally used to treat conditions such as decompression sickness, in modulating the macrophage phenotype toward the tumoricidal M1 state and disrupting the supportive tumor microenvironment. HBO has direct antiproliferative effects on tumor cells and reduces hypoxia, which may impair angiogenesis and tumor growth. This offers a novel approach to GBM treatment by targeting the role of the immune system within the tumor microenvironment. The effects of HBO on macrophage polarization and GBM cell viability and apoptosis were evaluated in this study. We detected that HBO promoted M1 macrophage cytokine expression while decreasing GBM cell viability and increasing apoptosis using GBM cell lines and THP-1-derived macrophage-conditioned media. These findings suggest that HBO therapy can shift macrophage polarization toward a tumoricidal M1 state. This can improve GBM cell survival and offers a potential therapeutic strategy. In conclusion, HBO can shift macrophages from a tumor-promoting M2 phenotype to a tumoricidal M1 phenotype in GBM. This can facilitate apoptosis and, in turn, improve treatment outcomes.

Critical role of CD206+ macrophages in promoting a cDC1-NK-CD8 T cell anti-tumor immune axis.

Tumor-associated macrophages (TAMs) are frequently categorized as being M1 or M2 polarized, even as substantial data challenges this binary modeling of macrophage cell state. One molecule consistently referenced as a delineator of a putative immunosuppressive M2 state is the surface protein CD206. We thus made a novel conditional CD206 (Mrc1) knock-in mouse to specifically visualize and/or deplete CD206+ M2-like TAMs and assess their correspondence with pro-tumoral immunity. Early, but not late depletion of CD206+ macrophages and monocytes (here, Mono/Macs) led to an indirect loss of a key anti-tumor network of NK cells, conventional type I dendritic cells (cDC1) and CD8 T cells. Among myeloid cells, we found that the CD206+ TAMs are the primary producers of CXCL9, and able to differentially attract activated CD8 T cells. In contrast, a population of stress-responsive TAMs (Hypoxic or Spp1+) and immature monocytes, which lack CD206 expression and become prominent following early depletion, expressed markedly diminished levels of CXCL9. Those NK and CD8 T cells which enter CD206-depleted tumors express vastly reduced levels of the corresponding receptor Cxcr3, the cDC1-attracting chemokine Xcl1 and cDC1 growth factor Flt3l transcripts. Consistent with the loss of this critical network, early CD206+ TAM depletion decreased tumor control by antigen specific CD8 T cells in mice. Likewise, in humans, the CD206Replete, but not the CD206Depleted Mono/Mac gene signature correlated robustly with CD8 T cell, NK cell and stimulatory cDC1 gene signatures and transcriptomic signatures skewed towards CD206Replete Mono/Macs associated with better survival. Together, these findings negate the unqualified classification of CD206+ M2-like macrophages as immunosuppressive by illuminating contexts for their role in organizing a critical tumor-reactive archetype of immunity.

TRPM2 channels are essential for regulation of cytokine production in lung interstitial macrophages.

Interstitial macrophages (IMs) are essential for organ homeostasis, inflammation, and autonomous immune response in lung tissues, which are achieved through polarization to a pro-inflammatory M1 and an M2 state for tissue repair. Their remote parenchymal localization and low counts, however, are limiting factors for their isolation and molecular characterization of their specific role during tissue inflammation. We isolated viable murine IMs in sufficient quantities by coculturing them with stromal cells and analyzed mRNA expression patterns of transient receptor potential (TRP) channels in naïve and M1 polarized IMs after application of lipopolysaccharide (LPS) and interferon γ. M-RNAs for the second member of the melastatin family of TRP channels, TRPM2, were upregulated in the M1 state and functional channels were identified by their characteristic currents induced by ADP-ribose, its specific activator. Most interestingly, cytokine production and secretion of interleukin-1α (IL-1α), IL-6 and tumor necrosis factor-α in M1 polarized but TRPM2-deficient IMs was significantly enhanced compared to WT cells. Activation of TRPM2 channels by ADP-ribose (ADPR) released from mitochondria by ROS-produced H2O2 significantly increases plasma membrane depolarization, which inhibits production of reactive oxygen species by NADPH oxidases and reduces cytokine production and secretion in a negative feedback loop. Therefore, TRPM2 channels are essential for the regulation of cytokine production in M1-polarized murine IMs. Specific activation of these channels may promote an anti-inflammatory phenotype and prevent a harmful cytokine storm often observed in COVID-19 patients.

Impact of exercise on uncoupling protein 1 and macrophage phenotype in mesenteric adipose tissue in an animal model of endometriosis

Endometriosis is a gynecological disorder characterized by growth of endometrial tissue outside the uterine cavity, with symptoms which may include chronic pelvic pain and infertility resulting in a decreased quality of life. Previous studies in our laboratory demonstrated that endometriosis animals have increased mesenteric adipose tissue (MAT), inflammation, and growth of implanted endometrial vesicles which are decreased by voluntary exercise. The levels of Uncoupling Protein 1 (UCP-1), a mitochondrial protein, are known to be increased by exercise in rodents, promoting thermogenesis and decreasing inflammation. Exercise can also increase expression of peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily. When activated PPARγ polarizes the macrophages into an anti-inflammatory M2 state, which can stimulate white adipose tissue beiging. We hypothesized that voluntary wheel running exercise can increase UCP-1 and M2 macrophages, decreasing MAT and endometrial vesicle size. Aim: To assess the impact of voluntary exercise on UCP-1 expression and macrophage phenotype in MAT of an animal model of endometriosis. Methods: Endometriosis was induced by implanting uterine tissue on the intestinal mesentery of female Sprague Dawley rats while control group only received sutures (SHAM). Exercise rats had access to a running wheel after surgery (ENDO-EX or SHAM-EX), while non-exercised animals didn’t (ENDO and SHAM; n=10-11/group). After sixty days, animals were sacrificed, and MAT was collected to determine CD68, UCP-1 and PPARγ expression by immunofluorescence staining. Results: Vesicles from ENDO-EX animals exhibited significantly decreased average weight (p<0.01) and smaller average total area (p<0.01) compared with the ENDO group. Endometriosis animals showed increased mesenteric adipose tissue when compared to sham group (p<0.01), which was reduced by voluntary exercise (p<0.001). Macrophage infiltration, UCP-1 and PPARγ expression increased with exercise. Conclusions: Increased UCP-1 and anti-inflammatory M2 macrophages following exercise may result in browning of MAT which can have a role in reducing the development of vesicles in the endometriosis model. These findings suggest that exercise may be a potential complementary therapeutic strategy for managing endometriosis symptoms. This work was supported by R15AT009915, R16GM149365, T32GM144896, PRI MD Summer Program and Porter Physiology Development Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.