Pro-inflammatory M1 Macrophages Research Articles

Reprogramming Tumor-Associated Macrophage Using Nanocarriers: New Perspectives to Halt Cancer Progression.

Cancer remains a significant challenge for public healthcare systems worldwide. Within the realm of cancer treatment, considerable attention is focused on understanding the tumor microenvironment (TME)-the complex network of non-cancerous elements surrounding the tumor. Among the cells in TME, tumor-associated macrophages (TAMs) play a central role, traditionally categorized as pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. Within the TME, M2-like TAMs can create a protective environment conducive to tumor growth and progression. These TAMs secrete a range of factors and molecules that facilitate tumor angiogenesis, increased vascular permeability, chemoresistance, and metastasis. In response to this challenge, efforts are underway to develop adjuvant therapy options aimed at reprogramming TAMs from the M2 to the anti-tumor M1 phenotype. Such reprogramming holds promise for suppressing tumor growth, alleviating chemoresistance, and impeding metastasis. Nanotechnology has enabled the development of nanoformulations that may soon offer healthcare providers the tools to achieve targeted drug delivery, controlled drug release within the TME for TAM reprogramming and reduce drug-related adverse events. In this review, we have synthesized the latest data on TAM polarization in response to TME factors, highlighted the pathological effects of TAMs, and provided insights into existing nanotechnologies aimed at TAM reprogramming and depletion.

A cross-linked macropore hydrogel based on M1 macrophage lysate and alginate regulates tumor-associated macrophages for the treatment of melanoma

Pro-inflammatory M1 macrophages possess the ability to change the immunosuppressive tumor microenvironment by releasing various inflammatory factors simultaneously, which can effectively inhibit tumor progression and relapse. Promoting macrophage polarization towards M1 may be an effective way to treat Melanoma. However, the risk of cytokine storm caused by the proliferation and excessive activation of M1 macrophages greatly limits it as a biosafety therapeutic strategy in anti-tumor immunotherapy. Therefore, how to engineer natural M1 macrophage to a biocompatible biomaterial that maintains the duration time of tumor suppressive property duration time still remains a huge challenge. To achieve this goal, we developed an injectable macroporous hydrogel (M1LMHA) using natural M1 macrophage lysates and alginate as raw materials. M1LMHA had excellent biocompatibility, adjustable degradation rate and could sustainably release varieties of natural inflammatory factors, such as tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and interleukin-12 (IL-12), etc. M1LMHA could repolarize anti-inflammatory M2 macrophages to M1 macrophages by the synergistic effect of released tiny inflammatory factors via the NF-κB pathway. This study supported that M1LMHA might be an effective and safe tool to activate tumor-associated immune cells, improving the efficiency of anti-tumor immunotherapy.

Biologic Adjuvants to Rotator Cuff Repairs Induce Anti-Inflammatory Macrophage 2 Polarization and Reduce Inflammatory Macrophage 1 Polarization In Vitro

PurposeTo examine the effect of various biologic adjuvants on the polarization of macrophages in an in vitro model for rotator cuff tears. MethodsTissue was harvested from 6 patients undergoing arthroscopic rotator cuff repair. An in vitro model of the supraspinatus and subacromial bursa was created and treated with control, platelet rich plasma (PRP), autologous activated serum (AAS), or a combination of PRP+AAS. The effect of treatment on macrophage polarization between M1 pro-inflammatory macrophages or M2 anti-inflammatory macrophages was measured using gene expression, protein expression, flow cytometry and nitric oxide (NO) production. ResultsTendon and bursa treated with PRP, AAS and PRP+AAS significantly decreased the gene expression of M1 markers IL-12 and TNF-a, while significantly increasing the expression of M2 markers Arginase, IL-10 and TGF-b (p<0.05) compared to treatment with control. ELISA analysis of protein production demonstrated that compared to control, co-culture treated with PRP, AAS and PRP+AAS significantly decreased markers of M1-macrophages (IL-6, IL-12, and TNF-a), while significantly increasing the expression of markers of M2-macrophages (Arginase, IL-10, and TGF-b) (p<0.05). Flow cytometry analysis of surface markers demonstrated that compared to control, tendon and bursa treated with PRP, AAS and PRP+AAS significantly decreased markers of M1-macrophages (CD80, CD86, CD64, CD16), while significantly increasing the expression of markers of M2-macrophages (CD163 and CD206) (p<0.05). Treatment of the co-culture with PRP, AAS and PRP+AAS consistently demonstrated a decrease in NO production (p<0.05) compared to control. AAS and PRP+AAS demonstrated an increased macrophage shift to M2 compared to PRP alone, whereas there was not as uniform of a shift when comparing PRP+AAS to AAS alone. ConclusionsIn an in vitro model of rotator cuff tears, the treatment of supraspinatus tendon and subacromial bursa with PRP, AAS and PRP+AAS demonstrated an increase in markers of anti-inflammatory M2-macrophages and a concomitant decrease in markers of pro-inflammatory M1-macrophages. AAS and PRP+AAS contributed to a large shift to macrophage polarization to the anti-inflammatory M2 compared to PRP.

Probiotics Mitigate Exercise Fatigue, Memory Deficits, Depression and Neuro/inflammation in a Mouse Model of Gulf War Illness

We have previously found learning/memory, exercise intolerance, metabolic disturbance, gut dysbiosis and deregulation of neuroinflammatory genes, suggesting a possible microbiota-gut-brain interaction in GWI pathology (Kozlova et al., 2022a PMID: 34710444; 2022b PMID: 34801513). Here, we tested the hypothesis that probiotic treatment (P) prevents GWI symptoms. Adult male C57Bl/6N mice were separated into 4 groups (n=16/group): GW receiving pyridostigmine bromide (8.7 mg/kg, PB, po), permethrin (1.3 mg/kg, PER, top), DEET (33%, top); CON/S (vehicle plus stress); CON/S+P and GW+P. Probiotic groups received a gut-protective probiotic (P) cocktail of L. reuteri, L. rhamnosus, L. casei, B. longum (po, 108 CFU/mL, 3 times/wk) for 2 wk prior to and during GW agent treatment and until sacrifice 5 months later. Fecal RT-qPCR showed colonization of all strains except L. rhamnosus after 6-7 doses when compared to saline sham. Colonization after GW agent exposure was strain-specific and varied with time post-exposure. On an exercise endurance (EE) test GW (but not GW+P) mice tired faster relative to CON/S at PT56 (p&lt;0.05) but not at PT150. Repeat testing on the passive avoidance apparatus (15 wk after previous test) showed that GW but not GW+P mice displayed reduced latency to enter aversive chamber on day 1 of acquisition trials, indicating deficient memory (p&lt;0.05). Depressive-like behavior (mean percent time spent mobile) on tail suspension test (TST) showed less time spent mobile for GW (p&lt;0.05) but not GW+P mice than controls at PT50 (but not PT150). Probiotics protected against the GW-induced rise in liver C-reactive protein (CRP) (p&lt;0.05) and brain interleukin-6 (IL-6) (p&lt;0.05). Immunofluorescence probing in the hippocampal CA1 and dentate gyrus showed significantly greater counts of Iba-1 positive cells in GW (but not GW+P) vs CON/S (p&lt;0.05). NeuN immunostaining on the same sections showed abnormal neuronal phenotype indicative of reorganized RNA in GW but not GW+P) vs CON/S (p&lt;0.05). Multi-gene profiling of innate and adaptive immunity genes in brain homogenates (prefrontal cortex, hypothalamus, hippocampus) identified GW agent-induced deregulation (&gt;1.5-fold; p&lt;.05) of pro-inflammatory genes, i.e., upregulation of C5ar1 and Cxcl10 and downregulation of Tlr4; changes prevented by probiotic treatment. In the gut, M1 proinflammatory macrophages appeared to be more abundant in GW (but not GW+P) vs CON/S (p&lt;0.05). We used 16S next generation sequencing to examine probiotic treatment effects on gut microbial communities. In combination, our findings indicate that GW mice can be used to model multi-symptom illness and support previous evidence that the underlying pathophysiology in GWI likely involve systemic, gut and neuroinflammation and bacterial dysbiosis. Importantly, we show for the first time that probiotic treatment protects against some GWI symptom domains. Further study is needed to uncover pathophysiological pathways through which the microbiota-gut-brain axis impacts gut and brain health and toxicant-related pathology. DoD grant GW-180072 to M.C.C. 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.

Vascular Endothelial Mitochondrial Oxidative Stress in Response to Placental Macrophages from Preeclamptic Pregnancies

Preeclampsia (PE), a hypertensive disorder of pregnancy characterized by maternal hypertension, fetal growth restriction (FGR), and chronic immune activation and inflammation, is a major cause of maternal and fetal morbidity and mortality worldwide. Prior clinical studies demonstrate that proinflammatory M1 macrophages are increased in women with PE. We do not understand the role that M1 macrophage polarization may play in PE pathophysiology. We set out to begin characterization of macrophages in patient samples obtained from the UMMC biobank and their ability to induce mitochondrial oxidative stress (ROS) in human umbilical vein endothelial cells (HUVECs). After obtaining informed consent, whole blood and placental tissues were collected from normal pregnant (n=5) and preeclamptic women (n=8) who delivered in the labor and delivery unit UMMC. Lymphocytes were isolated from blood on a Ficoll-Histopaque gradient and analyzed by flow cytometry for total and M1 macrophages. Total macrophages were isolated from digested placental tissue by magnetic bead separation using the Human Macrophage Isolation Kit (Miltenyi). Isolated macrophages were co-cultured with HUVECs overnight. After removal of culture inserts containing macrophages, HUVECs were serum-starved and incubated with MitoSox Red, collected, and analyzed on a flow cytometer. Total circulating macrophages were increased in placentas from PE women vs normal pregnant patients (10.8% PMBCs vs. 3.5% PMBCs, respectively, p&lt;0.05). Furthermore, macrophage polarization to M1 macrophages was significantly higher in women with PE (15.02% of total macrophages) compared to NP women (6.23% of total macrophages, p&lt;0.05 vs. PE). We also determined that mitochondrial ROS production was significantly elevated in HUVEC cells co-cultured with macrophages from PE pregnancies (15.37 A.U.) when compared to HUVECs co-cultured with normal pregnant control macrophages (7.788 A.U., p&lt;0.05 vs. PE). Future studies will include enrollment of additional participants and determination of endothelial activation by measurement of adhesion molecules, growth factors, extracellular matrix proteins, and cytokines in HUVECs and conditioned media via Western Blot, ELISA, and multiplex analysis. These data show that PE placental macrophages induce mitochondrial ROS production in endothelial cells and may play a role to directly induce vascular dysfunction contributing to hypertension during a preeclamptic pregnancy. This work was supported by National Institutes of Health Grants T32HL105324 to C. A. Shields, and R01HL151407 to D. C. Cornelius. 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.

Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation

HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.

CD226 promotes renal fibrosis by regulating macrophage activation and migration.

It has been found that CD226 plays an important role in regulating macrophage function, but its expression and function in macrophages during renal fibrogenesis have not been studied. Our data demonstrated that CD226 expression in macrophages was obviously upregulated in the unilateral ureteral obstruction model, while CD226 deficiency attenuated collagen deposition in renal interstitium along with fewer M1 within renal cortex and renal medulla and a lower level of proinflammatory factors compared to that of control littermates. Further studies demonstrated that Cd226-/- bone marrow-derived macrophages transferring could significantly reduce the tubular injury, collagen deposition, and proinflammatory cytokine secretion compared with that of Cd226+/+ bone marrow-derived macrophages transferring in the unilateral ureteral obstruction model. Mechanistic investigations revealed that CD226 promoted proinflammatory M1 macrophage accumulation in the kidney via suppressing KLF4 expression in macrophages. Therefore, our results uncovered a pathogenic role of CD226 during the development of chronic kidney disease by promoting monocyte infiltration from peripheral blood into the kidney and enhancing macrophage activation toward the inflammatory phenotype by suppressing KLF4 expression.

Galectin-3 absence alters lymphocytes populations dynamics behavior and promotes functional recovery after spinal cord injury in mice

Spinal cord injury (SCI) results from various mechanisms that damage the nervous tissue and the blood-brain barrier, leading to sensory and motor function loss below the injury site. Unfortunately, current therapeutic approaches for SCI have limited efficacy in improving patients outcomes. Galectin-3, a protein whose expression increases after SCI, influences the neuroinflammatory response by favoring pro-inflammatory M1 macrophages and microglia, while inhibiting pro-regenerative M2 macrophages and microglia, which are crucial for inflammation resolution and tissue regeneration. Previous studies with Galectin-3 knock-out mice demonstrated enhanced motor recovery after SCI. The M1/M2 balance is strongly influenced by the predominant lymphocytic profiles (Th1, Th2, T Reg, Th17) and cytokines and chemokines released at the lesion site. The present study aimed to investigate how the absence of galectin-3 impacts the adaptive immune system cell population dynamics in various lymphoid spaces following a low thoracic spinal cord compression injury (T9-T10) using a 30 g vascular clip for one minute. It also aimed to assess its influence on the functional outcome in wild-type (WT)and Galectin-3 knock-out (GALNEG) mice. Histological analysis with hematoxylin-eosin and Luxol Fast Blue staining revealed that WT and GALNEG animals exhibit similar spinal cord morphology. The absence of galectin-3 does not affect the common neuroanatomy shared between the groups prompting us to analyze outcomes between both groups. Following our crush model, both groups lost motor and sensory functions below the lesion level. During a 42-day period, GALNEG mice demonstrated superior locomotor recovery in the Basso Mouse Scale (BMS) gait analysis and enhanced motor coordination performance in the ladder rung walk test (LRW) compared to WT mice. GALNEG mice also exhibited better sensory recovery, and their electrophysiological parameters suggested a higher number of functional axons with faster nerve conduction. Seven days after injury, flow cytometry of thymus, spleen, and blood revealed an increased number of T Reg and Th2 cells, accompanied by a decrease in Th1 and Th17 cells in GALNEG mice. Immunohistochemistry conducted on the same day exhibited an increased number of Th2 and T Reg cells around the GALNEG's spinal cord lesion site. At 42-day dpi immunohistochemistry analyses displayed reduced astrogliosis and greater axon preservation in GALNEG's spinal cord seem as a reduction of GFAP immunostaining and an increase in NFH immunostaining, respectively. In conclusion, GALNEG mice exhibited better functional recovery attributed to the milder pro-inflammatory influence, compensated by a higher quantity of T Reg and Th2 cells. These findings suggest that galectin-3 plays a crucial role in the immune response after spinal cord injury and could be a potential target for clinical therapeutic interventions.

Shift from Pro- to Anti-Inflammatory Phase in Pelvic Floor Muscles at Postpartum Matches Histological Signs of Regeneration in Multiparous Rabbits.

Background and Objectives: Pelvic floor muscles (PFM) play a core role in defecation and micturition. Weakening of PFM underlies urogynecological disorders such as pelvic organ prolapse and stress urinary incontinence. Vaginal delivery damages PFM. Muscle trauma implies an inflammatory response mediated by myeloid cells, essential for subsequent recovery. Molecular signaling characterizing the pro-inflammatory phase shifts M1 macrophages to M2 macrophages, which modulate muscle repair. The present study aimed to evaluate histological characteristics and the presence of M1 and M2 macrophages in bulbospongiosus (Bsm) and pubococcygeus muscles (Pcm). Materials and Methods: Muscles from young nulliparous (N) and multiparous rabbits on postpartum days three (M3) and twenty (M20) were excised and histologically processed to measure the myofiber cross-sectional area (CSA) and count the centralized myonuclei in hematoxylin-eosinstained sections. Using immunohistochemistry, M1 and M2 macrophages were estimated in muscle sections. Kruskal-Wallis or one-way ANOVA testing, followed by post hoc tests, were conducted to identify significant differences (p < 0.05). Results: The myofiber CSA of both the Bsm and Pcm of the M3 group were more extensive than those of the N and M20 groups. Centralized myonuclei estimated in sections from both muscles of M20 rabbits were higher than those of N rabbits. Such histological outcomes matched significant increases in HLA-DR immunostaining in M3 rabbits with the CD206 immunostaining in muscle sections from M20 rabbits. Conclusions: A shift from the pro- to anti-inflammatory phase in the bulbospongiosus and pubococcygeus muscles of multiparous rabbits matches with centralized myonuclei, suggesting the ongoing regeneration of muscles.

Left Ventricular Remodelling Associated with the Transient Elevated [68Ga]Ga-Pentixafor Activity in the Remote Myocardium Following Acute Myocardial Infarction.

Previous studies have initially reported accompanying elevated 2-deoxy-2[18F]fluoro-D-glucose ([18F]F-FDG) inflammatory activity in the remote area and its prognostic value after acute myocardial infarction (AMI). Non-invasive characterization of the accompanying inflammation in the remote myocardium may be of potency in guiding future targeted theranostics. [68Ga]Ga-Pentixafor targeting chemokine receptor 4 (CXCR4) on the surface of inflammatory cells is currently one of the promising inflammatory imaging agents. In this study, we sought to focus on the longitudinal evolution of [68Ga]Ga-Pentixafor activities in the remote myocardium following AMI and its association with cardiac function. Twelve AMI rats and six Sham rats serially underwent [68Ga]Ga-Pentixafor imaging at pre-operation, and 5, 7, 14 days post-operation. Maximum and mean standard uptake value (SUV) and target-to-background ratio (TBR) were assessed to indicate the uptake intensity. Gated [18F]F-FDG imaging and immunofluorescent staining were performed to obtain cardiac function and responses of pro-inflammatory and reparative macrophages, respectively. The uptake of [68Ga]Ga-Pentixafor in the infarcted myocardium peaked at day 5 (all P = 0.003), retained at day 7 (all P = 0.011), and recovered at day 14 after AMI (P > 0.05), paralleling with the rise-fall pro-inflammatory M1 macrophages (P < 0.05). Correlated with the peak activity in the infarct territory, [68Ga]Ga-Pentixafor uptake in the remote myocardium on day 5 early after AMI significantly increased (AMI vs. Sham: SUVmean, SUVmax, and TBRmean: all P < 0.05), and strongly correlated with contemporaneous EDV and/or ESV (SUVmean and TBRmean: both P < 0.05). The transitory remote activity recovered as of day 7 post-AMI (AMI vs. Sham: P > 0.05). Corresponding with the peaked [68Ga]Ga-Pentixafor activity in the infarcted myocardium, the activity in the remote region elevated accordingly and led to contemporaneous left ventricular remodelling early after AMI. Further studies are warranted to clarify its clinical application potential.

ROS-responsive sprayable hydrogel as ROS scavenger and GATA6+ macrophages trap for the prevention of postoperative abdominal adhesions

Postoperative abdominal adhesions are a common clinical problem after surgery and can cause many serious complications. Current most commonly used antiadhesion products are less effective due to their short residence time and focus primary on barrier function. Herein, we developed a sprayable hydrogel barrier (sHA-ADH/OHA-E) with self-regulated drug release based on ROS levels at the trauma site, to serve as a smart inflammatory microenvironment modulator and GATA6+ macrophages trap for non-adherent recovery from abdominal surgery. Sulfonated hyaluronic acid (HA) conjugates modified with adipic dihydrazide (sHA-ADH), and oxidized HA conjugates grafted with epigallocatechin-3-gallate (EGCG) via ROS-cleavable boronate bonds (OHA-E) were synthesized. sHA-ADH/OHA-E hydrogel was facilely fabricated within 5 s after simply mixing sHA-ADH and OHA-E through forming dynamic covalent acylhydrazones. With good biocompatibility, appropriate mechanical strength, tunable shear-thinning, self-healing, asymmetric adhesion, and reasonable in vivo retention time, sHA-ADH/OHA-E hydrogel meets the requirements of a perfect physical barrier. Intriguingly, sulfonic acid groups endowed the hydrogel with satisfactory anti-fibroblast and macrophage attachment capability, and were demonstrated for the first time to act as polyanion traps to prevent GATA6+ macrophages aggregation. Importantly, EGCG could be intelligently released by ROS triggering to alleviate oxidative stress and promote proinflammatory M1 macrophage polarize to antiinflammatory M2 phenotype. Further, the fibrinolytic system balance was restored to reduce fibrosis. Thanks to the above advantages, the sHA-ADH/OHA-E hydrogel exhibited excellent anti-adhesion effects in a rat sidewall defect–cecum abrasion model and is expected to be a promising and clinically translatable antiadhesion barrier.

Predominance of M2 macrophages in organized thrombi in chronic thromboembolic pulmonary hypertension patients.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a debilitating disease characterized by thrombotic occlusion of pulmonary arteries and vasculopathy, leading to increased pulmonary vascular resistance and progressive right-sided heart failure. Thrombotic lesions in CTEPH contain CD68+ macrophages, and increasing evidence supports their role in disease pathogenesis. Macrophages are classically divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which are involved in wound healing and tissue repair. Currently, the phenotype of macrophages and their localization within thrombotic lesions of CTEPH are largely unknown. In our study, we subclassified thrombotic lesions of CTEPH patients into developing fresh thrombi (FT) and organized thrombi (OT), based on the degree of fibrosis and remodeling. We used multiplex immunofluorescence histology to identify immune cell infiltrates in thrombotic lesions of CPTEH patients. Utilizing software-assisted cell detection and quantification, increased proportions of macrophages were observed in immune cell infiltrates of OT lesions, compared with FT. Strikingly, the proportions with a CD206+INOS- M2 phenotype were significantly higher in OT than in FT, which mainly contained unpolarized macrophages. Taken together, we observed a shift from unpolarized macrophages in FT toward an expanded population of M2 macrophages in OT, indicating a dynamic role of macrophages during CTEPH pathogenesis.

Zinc- and Fluoride-Releasing Bioactive Glass as a Novel Bone Substitute

Bioglass 45S5, a silica-based glass, has pioneered a new field of biomaterials. Bioglass 45S5 promotes mineralization through calcium ion release and is widely used in the dental field, including toothpaste formulations. However, the use of Bioglass 45S5 for bone grafting is limited owing to the induction of inflammation, as well as reduced degradation and ion release. Phosphate-based glasses exhibit higher solubility and ion release than silica-based glass. Given that these glasses can be synthesized at low temperatures (approximately 1,000°C), they can easily be doped with various metal oxides to confer therapeutic properties. Herein, we fabricated zinc- and fluoride-doped phosphate-based glass (multicomponent phosphate [MP] bioactive glass) and further doped aluminum oxide into the MP glass (4% Al-MP glass) to overcome the striking solubility of phosphate-based glass. Increased amounts of zinc and fluoride ions were detected in water containing the MP glass. Doping of aluminum oxide into the MP glass suppressed the striking dissolution in water, with 4% Al-MP glass exhibiting the highest stability in water. Compared with Bioglass 45S5, 4% Al-MP glass in water had a notably reduced particle size, supporting the abundant ion release of 4% Al-MP glass. Compared with Bioglass 45S5, 4% Al-MP glass enhanced the osteogenesis of mouse bone marrow–derived mesenchymal stem cells. Mouse macrophages cultured with 4% Al-MP glass displayed enhanced induction of anti-inflammatory M2 macrophages and reduced proinflammatory M1 macrophages, indicating M2 polarization. Upon implanting 4% Al-MP glass or Bioglass 45S5 in a mouse calvarial defect, 4% Al-MP glass promoted significant bone regeneration when compared with Bioglass 45S5. Hence, we successfully fabricated zinc- and fluoride-releasing bioactive glasses with improved osteogenic and anti-inflammatory properties, which could serve as a promising biomaterial for bone regeneration.

Influence of physico-chemical properties of two lipoxin emulsion-loaded hydrogels on pre-polarized macrophages: a comparative analysis.

Inflammation, a crucial defense mechanism, must be rigorously regulated to prevent the onset of chronic inflammation and subsequent tissue damage. Specialized pro resolving mediators (SPMs) such as lipoxin A4 (LXA4) have demonstrated their ability to facilitate the resolution of inflammation by orchestrating a transition of M1 pro-inflammatory macrophages towards an anti-inflammatory M2 phenotype. However, the hydrophobic and chemically labile nature of LXA4 necessitates the development of a delivery system capable of preserving its integrity for clinical applications. In this study, two types of emulsion were formulated using different homogenization processes:mechanical overhead stirrer (MEB for blank Emulsion and MELX for LXA4 loaded-Emulsion) or Luer-lock syringes (SEB for blank Emulsion and SELX for LXA4 loaded-Emulsion)). Following characterization, including size and droplet morphology assessment by microscopy, the encapsulation efficiency (EE) was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To exert control over LXA4 release, these emulsions were embedded within silanized hyaluronic acid hydrogels. A comprehensive evaluation, encompassing gel time, swelling, and degradation profiles under acidic, basic, and neutral conditions, preceded the assessment of LXA4 cumulative release using LC-MS/MS. Physicochemical results indicate that H-MELX (Mechanical overhead stirrer LXA4 Emulsion loaded-Hydrogel) exhibits superior efficiency over H-SELX (Luer-lock syringes LXA4 Emulsion loaded-Hydrogel). While both formulations stimulated pro-inflammatory cytokine secretion and promoted a pro-inflammatory macrophage phenotype, LXA4 emulsion-loaded hydrogels displayed a diminished pro-inflammatory activity compared to blank emulsion-loaded hydrogels. These findings highlight the biological efficacy of LXA4 within both systems, with H-SELX outperforming H-MELX in terms of efficiency. To the best of our knowledge, this is the first successful demonstration of the biological efficacy of LXA4 emulsion-loaded hydrogel systems on macrophage polarization. These versatile H-MELX and H-SELX formulations can be customized to enhance their biological activity making them promising tools to promote the resolution of inflammation in diverse clinical applications.

Biotechnological Effects of Advanced Smart-Bile Acid Cyclodextrin-Based Nanogels for Ear Delivery and Treatment of Hearing Loss.

Inner ear delivery requires safe and effective drug delivery vehicles incorporating high-viscosity formulations with permeation enhancers. This study designs novel thermoresponsive-smart polymer-bile acid and cyclodextrin-based nanogels for inner ear delivery. Nanogels are examined for their rheological and physical properties. The biocompatibility studies will be assessed on auditory and macrophage cell lines by investigating the impact of nanogels on cellular viability, mitochondrial respiration, glycolysis, intracellular oxidative stress, inflammatory profile, and macrophage polarization. Novel ther nanogels based on bile acid and beta-cyclodextrin show preserved porous nanogels' inner structure, exhibit non-Newtonian, shear-thinning fluid behavior, have fast gelation at 37°C and minimal albumin adsorption on the surface. The nanogels have minimal impact on cellular viability, mitochondrial respiration, glycolysis, intracellular oxidative stress, and inflammatory profile of the auditory cell line House Ear Institute-Organ of Corti 1 after 24h incubation. Nanogel exposure of 24h to macrophage cell line RAW264.7 leads to decreased viability, mitochondrial dysfunction, and increased intracellular ROS and inflammatory cytokines. However, polarization changes from M2 anti-inflammatory to M1 pro-inflammatory macrophages are minimal, and inflammatory products of RAW264.7 macrophages do not overly disrupt the survivability of HEI-OC1 cells. Based on these results, thermoresponsive bile acid and cyclodextrin nanogels can be potential drug delivery vehicles for inner ear drug delivery.

Abstract 2180: Modulation of immune infiltration patterns in inflammation-associated colorectal cancer through rice bran-based dietary strategies

Abstract The correlation between gut microbiota dysbiosis and inflammation-associated colorectal cancer (CRC) has generated considerable interest in dietary interventions targeting immune modulation. Our prior study demonstrated the colon cancer protective effects of rice bran (RB) and ex vivo Bifidobacterium longum fermented rice bran (FRB) diet intervention against azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC in gut microbiota-intact specific pathogen-free mice (SPF). RB showed higher levels of protection compared to FRB against CRC, as evidenced by diminished neoplastic lesion size and restored colonic epithelial integrity. The major objective of the present study was to leverage cutting-edge multispectral imaging technique for simultaneous detection of multiple immune cells markers and for a comprehensive analysis of distinct immune landscapes. We also performed spatial correlations within the tumor microenvironment of AOM/DSS-SPF mice undergoing RB and FRB dietary interventions. The primary focus was on T cell subtypes including cytotoxic T cells, helper T cells, regulatory T (Treg) cells, natural killer (NK) cells, NKT cells, B cells and macrophages (MΦ) subtypes (M1 MΦ, and M2 MΦ) to uncover the intricacies of the immune landscape and the interplay for CRC prevention through dietary intervention. The results indicated that RB intervention for 15 weeks led to reductions in overall T cell subtypes in the colonic tissue, particularly Treg cells, which are associated with protecting tumors against immune surveillance in CRC. On the other hand, FRB intervention also resulted in reductions in overall T cell subtypes but exhibited higher infiltration of NK cells and proinflammatory M1 macrophages in the colonic tissue after 15 weeks. While an increased presence of NK cells might be beneficial in countering CRC, the infiltration of M1 macrophages could potentially lessen the impact of FRB compared to RB intervention in CRC protection. The outcomes suggest that the better protective efficacy of non-fermented RB diets is linked to the modulation of Treg cell infiltration, whereas the efficacy of the FRB diet is to some extent limited by the presence of M1 macrophages. Overall, this study provides valuable insights into the complex immune landscape influenced by RB and FRB interventions in a murine model of inflammation-induced CRC, and a novel mechanism for the immunomodulatory effects of dietary RB interactions with gut microbiota. In the future, observing immune modulation throughout diet intervention would enhance our understanding of the role played by these immune cells. Citation Format: Robin Kumar, Lakshmi Sai Pratyusha Bugata, Md. Imtiazul Kabir, Rajesh Agarwal, Elizabeth P. Ryan, Komal Raina. Modulation of immune infiltration patterns in inflammation-associated colorectal cancer through rice bran-based dietary strategies [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 2180.

Abstract 1468: Low exposures to amphibole or serpentine asbestos in germline Bap1-mutant mice induce mesotheliomacharacterized by an immunosuppressive tumor microenvironment

Abstract The purpose of this study was to determine if minimal exposures to amphibole (crocidolite) and serpentine (chrysotile) asbestos fibers significantly increase the incidence and rate of malignant mesothelioma (MM) onset in mice with a germline heterozygous Bap1 mutation, and to assess the effect of the resulting gene × environment interactions on the tumor immune microenvironment (TIME). The studies were carried out by chronically injecting intraperitoneally Bap1+/- and Bap1+/+ (wild-type, WT) mice with minimal doses of asbestos (in some experiments, as low as 0.0125 mg x 8 injections; total, 0.1 mg/mouse) and then monitoring the mice for tumor formation for up to 90 weeks. Pro-inflammatory and pro-tumorigenic factors in the TIME were evaluated by immunohistochemistry (IHC) and immunofluorescence (IF) for immune cell markers. With either type of asbestos and at each dose tested, MMs occurred at a significantly higher rate and earlier onset time in Bap1-mutant mice than in WT littermates. MMs from mice injected with chrysotile, but less so with crocidolite, had widespread adhesions in the peritoneal cavity owing to an overwhelming immune response, particularly at the higher dose used. IHC staining demonstrated an increased number of immune cells in granulomas and MMs. In particular, pro-inflammatory M1 macrophages were found mainly in granulomas, whereas pro-tumorigenic M2 macrophages were predominant in MMs. IF staining revealed that the relative number of M2 macrophages was strikingly greater in chrysotile-induced than crocidolite-induced MMs, suggesting that chrysotile induces a more profound immune response. MMs from Bap1-mutant mice showed upregulation of both CD39/CD73-adenosine and Ccl2/Ccr2 pathways, which collectively are known to mechanistically create favorable conditions for neoplastic transformation, invasion, and the development of an immunosuppressive TIME, in part by attracting M2 macrophages. This implies that chemoprevention and immunotherapy strategies targeting these pro-tumorigenic immune pathways could be beneficial in BAP1 mutation carriers. In conclusion, we provide evidence that low-level exposures to either chrysotile or crocidolite in germline Bap1 heterozygous mice result in a markedly increased risk of MM characterized by an immunosuppressive tumor microenvironment. Drawing parallels to humans, these experimental findings suggest that exposure of BAP1 mutation carriers to even minimal doses of different forms of asbestos may be associated with significantly enhanced susceptibility to MM. Citation Format: Yuwaraj Kadariya, Eleonora Sementino, Maggie Ruan, Mitchell Cheung, Andres J. Klein-Szanto, Kathy Cai, Joseph R. Testa. Low exposures to amphibole or serpentine asbestos in germline Bap1-mutant mice induce mesotheliomacharacterized by an immunosuppressive tumor microenvironment [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 1468.

Ischemia and reperfusion-injured liver-derived exosomes elicit acute lung injury through miR-122-5p regulated alveolar macrophage polarization

Acute lung injury (ALI) is a common postoperative complication, particularly in pediatric patients after liver transplantation. Hepatic ischemia–reperfusion (HIR) increases the release of exosomes (IR-Exos) in peripheral circulation. However, the role of IR-Exos in the pathogenesis of ALI induced by HIR remains unclear. Here, we explored the role of exosomes derived from the HIR-injured liver in ALI development. Intravenous injection of IR-Exos caused lung inflammation in naive rats, whereas pretreatment with an inhibitor of exosomal secretion (GW4869) attenuated HIR-related lung injury. In vivo and in vitro results show that IR-Exos promoted proinflammatory responses and M1 macrophage polarization. Furthermore, miRNA profiling of serum identified miR-122-5p as the exosomal miRNA with the highest increase in young rats with HIR compared with controls. Additionally, IR-Exos transferred miR-122-5p to macrophages and promoted proinflammatory responses and M1 phenotype polarization by targeting suppressor of cytokine signaling protein 1(SOCS-1)/nuclear factor (NF)-κB. Importantly, the pathological role of exosomal miR-122-5p in initiating lung inflammation was reversed by inhibition of miR-122-5p. Clinically, high levels of miR-122-5p were found in serum and correlated to the severity of lung injury in pediatric living-donor liver transplant recipients with ALI. Taken together, our findings reveal that IR-Exos transfer liver-specific miR-122-5p to alveolar macrophages and elicit ALI by inducing M1 macrophage polarization via the SOCS-1/NF-κB signaling pathway.

Narrow-band UVB radiation triggers diverse changes in the gene expression and induces the accumulation of M1 macrophages in human skin

BackgroundThe underlying molecular mechanisms that determine the biological effects of UVB radiation exposure on human skin are still only partially comprehended. ObjectivesOur goal is to examine the human skin transcriptome and related molecular mechanisms following a single exposure to UVB in the morning versus evening. MethodsWe exposed 20 volunteer females to four-fold standard erythema doses (SED4) of narrow-band UVB (309–313 nm) in the morning or evening and studied skin transcriptome 24 h after the exposure. We performed enrichment analyses of gene pathways, predicted changes in skin cell composition using cellular deconvolution, and correlated cell proportions with gene expression. ResultsIn the skin transcriptome, UVB exposure yielded 1384 differentially expressed genes (DEGs) in the morning and 1295 DEGs in the evening, of which the most statistically significant DEGs enhanced proteasome and spliceosome pathways. Unexposed control samples showed difference by 321 DEGs in the morning vs evening, which was related to differences in genes associated with the circadian rhythm. After the UVB exposure, the fraction of proinflammatory M1 macrophages was significantly increased at both timepoints, and this increase was positively correlated with pathways on Myc targets and mTORC1 signaling. In the evening, the skin clinical erythema was more severe and had stronger positive correlation with the number of M1 macrophages than in the morning after UVB exposure. The fractions of myeloid and plasmacytoid dendritic cells and CD8 T cells were significantly decreased in the morning but not in the evening. ConclusionsNB-UVB-exposure causes changes in skin transcriptome, inhibiting cell division, and promoting proteasome activity and repair responses, both in the morning and in the evening. Inflammatory M1 macrophages may drive the UV-induced skin responses by exacerbating inflammation and erythema. These findings highlight how the same UVB exposure influences skin responses differently in morning versus evening and presents a possible explanation to the differences in gene expression in the skin after UVB irradiation at these two timepoints.

A chronic pro-inflammatory environment contributes to the physiopathology of actinic lentigines

Actinic lentigines (AL) or age spots, are skin hyperpigmented lesions associated with age and chronic sun exposure. To better understand the physiopathology of AL, we have characterized the inflammation response in AL of European and Japanese volunteers. Gene expression profile showed that in both populations, 10% of the modulated genes in AL versus adjacent non lesional skin (NL), i.e. 31 genes, are associated with inflammation/immune process. A pro-inflammatory environment in AL is strongly suggested by the activation of the arachidonic acid cascade and the plasmin pathway leading to prostaglandin production, along with the decrease of anti-inflammatory cytokines and the identification of inflammatory upstream regulators. Furthermore, in line with the over-expression of genes associated with the recruitment and activation of immune cells, immunostaining on skin sections revealed a significant infiltration of CD68+ macrophages and CD4+ T-cells in the dermis of AL. Strikingly, investigation of infiltrated macrophage subsets evidenced a significant increase of pro-inflammatory CD80+/CD68+ M1 macrophages in AL compared to NL. In conclusion, a chronic inflammation, sustained by pro-inflammatory mediators and infiltration of immune cells, particularly pro-inflammatory M1 macrophages, takes place in AL. This pro-inflammatory loop should be thus broken to normalize skin and improve the efficacy of age spot treatment.