Immunomodulation Of Macrophages Research Articles

Enzyme-responsive mannose-grafted magnetic nanoparticles for breast and liver cancer therapy and tumor-associated macrophage immunomodulation

ABSTRACT Background Chemo-immunotherapy modifies the tumor microenvironment to enhance the immune response and improve chemotherapy. This study introduces a dual-armed chemo-immunotherapy strategy combating breast tumor progression while re-polarizing Tumor-Associated Macrophage (TAM) using prodigiosin-loaded mannan-coated magnetic nanoparticles (PG@M-MNPs). Methods The physicochemical properties of one-step synthetized M-MNPs were analyzed, including X-ray diffraction, FTIR, DLS, VSM, TEM, zeta potential analysis, and drug loading content were carried out. Biocompatibility, cancer specificity, cellular uptake, and distribution of PG@M-MNPs were investigated using fluorescence and confocal laser scanning microscopy, and flow cytometry. Furthermore, the expression levels of IL-6 and ARG-1 after treatment with PG and PG@M-MNPs on M1 and M2 macrophage subsets were studied. Results The M-MNPs were successfully synthesized and characterized, demonstrating a size below 100 nm. The release kinetics of PG from M-MNPs showed sustained and controlled patterns, with enzyme-triggered release. Cytotoxicity assessments revealed an enhanced selectivity of PG@M-MNPs against cancer cells and minimal effects on normal cells. Additionally, immuno-modulatory activity demonstrates the potential of PG@M-MNPs to change the polarization dynamics of macrophages. Conclusion These findings highlight the potential of a targeted approach to breast cancer treatment, offering new avenues for improved therapeutic outcomes and patient survival.

Abstract 4987: TREM2 is a radiation-induced target for immunomodulation of tumor-associated macrophages in the prostate cancer tumor microenvironment

Abstract Background: The prostate cancer (PCa) tumor microenvironment (TME) is characterized by a predominance of myeloid cells that may have a key role in immunosuppression and disease progression. Stereotactic Body Radiation Therapy (SBRT) is utilized for treatment of localized and metastatic PCa and promotes inflammatory remodeling of the TME. We analyzed human PCa specimens at acute post-SBRT timepoints to identify radiation-induced targets in the PCa TME and used in vitro and in vivo models to test and validate our findings. Methods: We performed 10X single-cell RNA sequencing (scRNA-seq) on prostatectomy specimens from men with high-risk localized PCa; 12,988 cells from 4 patients that underwent pre-operative SBRT and 47,314 cells from 6 patients that underwent prostatectomy alone. THP-1 monocytes polarized into macrophage (Mɸ) states [M0, M1, M2 and tumor-associated macrophages (TAM), were used to characterize TREM2 expression and immune responses to radiation or conditioned media (CM) derived from irradiated PC3 cells. In vivo studies with C57BL/6 mice inoculated with MC38 flank tumors or PTEN-/- SPOPmut CHD1del mouse prostate organoid line were used to analyze intratumoral myeloid populations in response to SBRT (37.5Gy in 5 fractions). Results: scRNA-seq analysis (Cell Ranger 7.1.0 pipelines; Seurat v5 R package) revealed enrichment of a cluster of myeloid cells defined by a damage-associated Mɸ gene signature (TREM2, APOE, SPP1, LGALS3, CD9) in response to SBRT compared with non-irradiated specimens (35.6% vs 5.7%). TREM2 expression was significantly upregulated in THP-1 cells polarized in M2 (p<0.001) and TAM (p<0.001) conditions, compared to M1 conditions. Irradiation (20Gy x1) of THP-1 cells significantly increased TREM2 expression in M0 (p<0.01), M1 (p<0.001), and TAM (p<0.01) conditions. Proliferation of PC3 cells exposed to CM from irradiated TAMs was significantly increased (p<0.0001) compared to CM from non-irradiated TAM. Alternatively, CM derived from irradiated PC3 cells significantly increased (p<0.05) TREM2 expression in M0-polarized Mɸ. In vivo studies, in MC38 and organoid models, noted a significant reduction (p<0.0001) in tumor volume at day 7 post-SBRT. Multi-parametric flow cytometry demonstrated robust myeloid infiltration in response to SBRT dominated by CD11b+F4/80+ TAMs in irradiated tumors. Among irradiated tumors, TREM2 expression was markedly elevated in TAMs (organoid and MC38, p<0.0001). Conclusion: Preliminary data in human irradiated PCa and validation studies in preclinical models suggest TREM2 expression on myeloid cells is a radiation-induced target. Further studies are underway to understand the functional and immunological implications of SBRT-induced TREM2 expression on TAMs and whether this can be therapeutically targeted to enhance anti-tumor immunity in PCa. Citation Format: Janny A. Villa-Pulgarin, Un In Chan, Francesca Khani, Fabio Socciarelli, Jiansheng Wu, Ryan Carelli, Jeffrey Kraynak, Silvia C. Formenti, Luigi Marchionni, Massimo Loda, Himanshu Nagar, Ariel E. Marciscano, Christopher E. Barbieri. TREM2 is a radiation-induced target for immunomodulation of tumor-associated macrophages in the prostate cancer 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 4987.

Mesenchymal Stem Cell-Derived Exosomes Promote Recovery of The Facial Nerve Injury through Regulating Macrophage M1 and M2 Polarization by Targeting the P38 MAPK/NF-Κb Pathway.

Facial nerve (FN) injury seriously affects human social viability and causes a heavy economic and social burden. Although mesenchymal stem cell-derived exosomes (MSC-Exos) promise therapeutic benefits for injury repair, there has been no evaluation of the impact of MSC-Exos administration on FN repair. Herein, we explore the function of MSC-Exos in the immunomodulation of macrophages and their effects in repairing FN injury. An ultracentrifugation technique was used to separate exosomes from the MSC supernatant. Administrating MSC-Exos to SD rats via local injection after FN injury promoted axon regeneration and myelination and alleviated local and systemic inflammation. MSC-Exos facilitated M2 polarization and reduced the M1-M2 polarization ratio. miRNA sequencing of MSC-Exos and previous literature showed that the MAPK/NF-κb pathway was a downstream target of macrophage polarization. We confirmed this hypothesis both in vivo and in vitro. Our findings show that MSC-Exos are a potential candidate for treating FN injury because they may have superior benefits for FN injury recovery and can decrease inflammation by controlling the heterogeneity of macrophages, which is regulated by the p38 MAPK/NF-κb pathway.

Anticancer, Immunomodulatory, and Phytochemical Screening of Carthamus oxyacantha M.Bieb Growing in the North of Iraq.

Carthamus oxyacantha M.Bieb is a promising repository of active phytochemicals. These bioactive compounds work synergistically to promote the plant's antioxidant, anticancer, and immunomodulatory capabilities. The present study aimed to discover the potential immunomodulatory and cytotoxicity of different extracts of Carthamus oxycantha roots. Aqueous ethanol (70%), aqueous methanol (90%), ethyl acetate, and n-hexane extracts were tested against five cell lines (T47D, MDA-MB231, Caco-2, EMT6/P, and Vero). Among these extracts, ethyl acetate and n-hexane extracts showed significant activity in inhibiting the proliferation of cancerous cells because of the presence of several phytochemical compounds, including flavonoids, phenolics, and alkaloids. The n-hexane extract was the most potent extract against T47D and Caco-2 cell lines and had IC50 values of 0.067 mg/mL and 0.067 mg/mL, respectively. In comparison, ethyl acetate extract was active against T47D and MDAMB231, and IC50 values were 0.0179 mg/mL and 0.03 mg/mL, respectively. Both n-hexane and ethyl acetate extracts reduced tumor size (by 49.981% and 51.028%, respectively). Remarkably, Carthamus oxyacantha extracts decreased the average weight of the tumor cells in the in vivo model. The plant induced significant apoptotic activity by the activation of caspase-3, immunomodulation of macrophages, and triggering of pinocytosis. The implications of these intriguing findings demand additional research to broaden the scope of the understanding of this field, opening the doors to the possibilities of using Carthamus oxyacantha M.Bieb as an effective cancer treatment adjuvant in the future.

Immunomodulatory Activity and Its Mechanisms of Two Polysaccharides from Poria cocos.

Polyporaceae is an important fungal family that has been a source of natural products with a range of pharmaceutical activities in China. In our previous study, two polysaccharides, PCWPW and PCWPS, with significant antioxidant and antidepressant activity were obtained from Poria cocos. In this study, we evaluated their potential molecular mechanisms in the immunomodulation of macrophages. PCWPW and PCWPS were characterized by GC-MS analysis to contain 1,3-linked Glcp. ELISA assays results demonstrated that the secretion of TNF-α was significantly enhanced by PCWPW/PCWPS. RNA-seq data demonstrated that PCWPS treatment modulated the expression of immune-related genes in macrophages, which was further confirmed by RT-qPCR assays. The activation of TNF-α secretion was found to be mannose receptor (MR) dependent and suppressed by MR inhibitor pretreatment. Moreover, the amount of TNF-α cytokine secretion in PCWPW/PCWPS-induced RAW264.7 cells was decreased when pretreated with NF-κB or MAPK signaling pathway inhibitors. Collectively, our results suggested that PCWPW and PCWPS possessed immunomodulatory activity that regulates TNF-α expression through the NF-κB/MAPK signaling pathway by binding to mannose receptors. Therefore, PCWPW and PCWPS isolated from Poria cocos have potential as drug candidates for immune-related disease treatment.

Targeted modulation and enhancement of macrophages via sonodynamic therapy-driven cupferroptosis-like stress for implant-associated biofilm infections

As the vanguard against implant-associated infections (IAIs), macrophages facilitate the phagocytosis and pro-inflammatory polarization of planktonic and biofilm infections, but indirectly serve as a “Trojan horse” for intracellular bacterial infections. Effective drug delivery and macrophage immunomodulation to eliminate intracellular infections will contribute to the treatment of refractory IAIs and prevention of relapse. Herein, we developed an ultrasound-responsive copper-based nanoparticle targeting macrophages by encapsulating copper in a porphyrin metal-organic framework and surface-grafting D-Mannosamine to synthesize CuTCPP@MOF nanodots@ Mannosamine (CTMM) with macrophage-targeting functionality. Concurrent generation of hydroxyl radicals and singlet oxygen in biofilm microenvironment mediated by ultrasound disrupts the biofilm structure, achieving CTMM overload and interfering with bacterial biofilm growth and metabolism. By regulating macrophage oxidative stress metabolism, CTMM induced a cupferroptosis-like stress, characterized by concurrent cuproptosis and ferroptosis, to eliminate intracellular infections. Transcriptomic analysis verified that the cupferroptosis-like stress based on GPX4 axis modulation further promotes macrophage inflammatory activation. This CTMM+US-mediated bacterial clearance in biofilms and macrophage immunomodulation informs the "Trojan War" strategy to combat IAIs clinically.

Melatonin-pretreated human umbilical cord mesenchymal stem cells improved endometrium regeneration and fertility recovery through macrophage immunomodulation in rats with intrauterine adhesions†.

Intrauterine adhesions (IUA) are a common gynecological problem. Stem cell therapy has been widely used in the treatment of IUA. However, due to the complex and harsh microenvironment of the uterine cavity, the effectiveness of such therapy is greatly inhibited. This study aimed to investigate whether melatonin pretreatment enhances the efficacy of human umbilical cord mesenchymal stem cells (HucMSCs) in IUA treatment in rats. First, we explored the effect of melatonin on the biological activity of HucMSCs in vitro through a macrophage co-culture system, Cell Counting Kit 8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, immunofluorescence staining, and qRT-PCR. Subsequently, we established the IUA rat model and tracked the distribution of HucMSCs in this model. In addition, we observed the number of M1 and M2 macrophages through immunofluorescence staining and detected the levels of inflammatory cytokines. Four weeks after cell transplantation, HE, Masson, and immunohistochemical staining were performed. In vitro experiments showed that melatonin pretreatment of HucMSCs promoted proliferation, reduced apoptosis, up-regulated the stemness gene, and regulated macrophage polarization. In vivo, melatonin pretreatment caused more HucMSCs to remain in the uterine cavity. Melatonin-pretreated HucMSCs recruited more macrophages, regulated macrophage polarization, and reduced inflammation. Melatonin-pretreated HucMSCs relieved fibrosis, increased endometrium thickness, and up-regulated CD34, vimentin, proliferating cell nuclear antigen (PCNA), and alpha small muscle antigen (α-SMA) expression. Fertility tests showed that melatonin-pretreated HucMSCs increased the number of embryos. In summary, pretreatment with melatonin was beneficial for HucMSC treatment because it enhanced the cell's ability to recruit macrophages and regulate macrophage polarization, which led to the regeneration of the endometrium and improved pregnancy outcomes.

Immunomodulatory effects of seed peptide lunasin in RAW264.7 macrophages in obese microenvironments

In adiposity, immune cells infiltrate adipose tissues, especially macrophages, forming chronic inflammation. This study aimed to investigate the mechanism of lunasin regulating immune functions of RAW264.7 macrophages in obesity-related conditions. Lipopolysaccharide (LPS) triggered an acute inflammation, and adipocyte-conditioned medium (Ad-CM) and co-cultures of RAW264.7 macrophages and 3T3-L1 adipocytes were used to mimic obese microenvironments. Lunasin protected the vitality of RAW cells and suppressed leptin secretion in Ad-CM. In LPS, lunasin reduced 47% of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) staining, 28% of nitric oxide production, and 27% of tumor necrosis factor-α secretion in LPS-stimulated co-culture, while there were opposing effects in Ad-CM and co-culture without LPS. Moreover, LPS-stimulated migration was inhibited at 44% by lunasin, while Ad-CM-declined 49% of migration. Lunasin increased 21% and 42% of phagocytosis in LPS and Ad-CM challenges. Overall, the study first revealed that lunasin exerted immunomodulation in macrophages against LPS-stimulated inflammation but boosted immune functions in obesity-related microenvironments.

Extracellular Vesicles-Derived Hybrid Nanoplatforms for Amplified CD47 Blockade-Based Cancer Immunotherapy.

Immunomodulation of tumor-associated macrophages (TAMs) into tumor-inhibiting M1-like phenotype is a promising but challenging strategy. Cleverly, tumor cells overexpress CD47, a "don't eat me" signal that ligates with the signal regulatory protein alpha (SIRPα) on macrophages to escape phagocytosis. Thus, effective re-education of TAMs into the "eat me" type and blocking the CD47-SIRPα signaling play pivotal roles in tumor immunotherapy. Herein, it is reported that hybrid nanovesicles (hEL-RS17) derived from extracellular vesicles of M1 macrophages and decorated with RS17 peptide, an antitumor peptide that specifically binds to CD47 on tumor cells and blocks CD47-SIRPα signaling, can actively target tumor cells and remodel TAM phenotypes. Consequently, more M1-like TAMs infiltrate into tumor tissue to phagocytize more tumor cells due to CD47 blockade. By further co-encapsulating chemotherapeutic agent shikonin, photosensitizer IR820, and immunomodulator polymetformin in hEL-RS17, an enhanced antitumor effect is obtained due to the combinational treatment modality and close synergy among each component. Upon laser irradiation, the designed SPI@hEL-RS17 nanoparticles exert potent antitumor efficacy against both 4T1 breast tumor and B16F10 melanoma models, which not only suppresses primary tumor growth but also inhibits lung metastasis and prevents tumor recurrence, exhibiting great potential in boosting CD47 blockade-based antitumor immunotherapy.

100-OR: A Novel Macrophage-Regulation Drug for Treating Diabetic Foot Ulcer—A Phase III MRCT Study and Taiwan Experience

Macrophage regulation plays an essential role in Diabetic foot ulcer (DFU) healing. By recognizing the therapeutic potential in M1/M2-macrophage-regulation for DFUs, we aim to investigate the efficacy and safety of ON101, a topical new drug with a M1/M2-macrophage-regulating mechanism in phase 3 MRCT across the US, China, and Taiwan (NCT01898923). There are 236 eligible subjects with Wagner Grade 1 or 2 DFU(s), randomized 1:1 to receive ON101 or comparator for up to 16 weeks, followed by a 12-week follow-up. The incidence of complete healing was 60.7% by ON101 and 35.1% by the comparator (P=0.0001). The time to complete ulcer healing was faster in the ON101 group (P=0.002). Since the high levels of HbA1C, ulcer persistence, ulcer location, and amputation history as the risk factors leading to poor DFU outcomes, ON101 also showed demonstrated robust efficacy and safety in hard-to-heal ulcers. ON101, approved by Taiwan FDA in 2021, was shown with excellent therapeutic efficacy and safety in the treatment of DFUs. In a real-world scenario, case collation from 133 patients, ulcer severity with Wagner grade 1 to 4, after applying ON101, the average ulcer complete healing rate is 71%, no adverse events were related to ON101, which validates the drug safety of ON101. ON101 further supports its excellent ability to promote wound healing in DFU patients with various severities (Wagner grade 1 to 4). All those patients who applied ON101 alone or with other therapies reached ulcer healing under infection control and with necessary debridement. Macrophage-regulating drug achieved superior therapeutic efficacy in a large Phase 3 MRCT and has demonstrated excellent therapeutic efficacy in the real-world scenario where Wagner grade 3 and 4 DFUs reached ulcer healing when ON101 was combined with other therapies. This enlightens the future treatment in Wagner grade 1 to 4 DFUs by macrophage immunomodulation new drug as a promising solution. Disclosure S.Chen: Employee; Oneness Biotech Co., Ltd. S.Chu: None. C.Pu: None. J.Chen: Employee; Oneness Biotech Company. Y.Wu: Employee; Oneness Biotech Co., Ltd.

Polymers and Bioactive Compounds with a Macrophage Modulation Effect for the Rational Design of Hydrogels for Skin Regeneration.

The development of biomaterial platforms for dispensing reagents of interest such as antioxidants, growth factors or antibiotics based on functional hydrogels represents a biotechnological solution for many challenges that the biomedicine field is facing. In this context, in situ dosing of therapeutic components for dermatological injuries such as diabetic foot ulcers is a relatively novel strategy to improve the wound healing process. Hydrogels have shown more comfort for the treatment of wounds due to their smooth surface and moisture, as well as their structural affinity with tissues in comparison to hyperbaric oxygen therapy, ultrasound, and electromagnetic therapies, negative pressure wound therapy or skin grafts. Macrophages, one of the most important cells of the innate immune system, have been described as the key not only in relation to the host immune defense, but also in the progress of wound healing. Macrophage dysfunction in chronic wounds of diabetic patients leads to a perpetuating inflammatory environment and impairs tissue repair. Modulating the macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) could be a strategy for helping to improve chronic wound healing. In this regard, a new paradigm is found in the development of advanced biomaterials capable of inducing in situ macrophage polarization to offer an approach to wound care. Such an approach opens a new direction for the development of multifunctional materials in regenerative medicine. This paper surveys emerging hydrogel materials and bioactive compounds being investigated to induce the immunomodulation of macrophages. We propose four potential functional biomaterials for wound healing applications based on novel biomaterial/bioactive compound combination that are expected to show synergistic beneficial outcomes for the local differentiation of macrophages (M1-M2) as a therapeutic strategy for chronic wound healing improvement.

A 3D Bioprinted Nanoengineered Hydrogel with Photoactivated Drug Delivery for Tumor Apoptosis and Simultaneous Bone Regeneration via Macrophage Immunomodulation.

One of the significant challenges in bone tissue engineering (BTE) is the healing of traumatic tissue defects owing to the recruitment of local infection and delayed angiogenesis. Herein, a 3D printable multi-functional hydrogel composing polyphenolic carbon quantum dots (CQDs, 100 µg mL-1 ) and gelatin methacryloyl (GelMA, 12 wt%) is reported for robust angiogenesis, bone regeneration and anti-tumor therapy. The CQDs are synthesized from a plant-inspired bioactive molecule, 1, 3, 5-trihydroxybenzene. The 3D printed GelMA-CQDs hydrogels display typical shear-thinning behavior with excellent printability. The fabricated hydrogel displayed M2 polarization of macrophage (Raw 264.7) cells via enhancing anti-inflammatory genes (e.g., IL-4 and IL10), and induced angiogenesis and osteogenesis of human bone mesenchymal stem cells (hBMSCs). The bioprinted hBMSCs are able to produce vessel-like structures after 14 d of incubation. Furthermore, the 3D printed hydrogel scaffolds also show remarkable near infra-red (NIR) responsive properties under 808 nm NIR light (1.0W cm-2 ) irradiation with controlled release of antitumor drugs (≈49%) at pH 6.5, and thereby killing the osteosarcoma cells. Therefore, it is anticipated that the tissue regeneration and healing ability with therapeutic potential of the GelMA-CQDs scaffolds may provide a promising alternative for traumatic tissue regeneration via augmenting angiogenesis and accelerated immunomodulation.

Matrix stiffness-induced platelet activation determines immunomodulation of macrophages.

Although various bone defect repair materials have been used clinically, the influence of the material properties on bone repair and regeneration as well as the underlying mechanisms are not fully understood. We hypothesize that the material stiffness affects initial platelet activation during hemostasis phase, which in turn mediates subsequent osteoimmunomodulation of macrophages, finally determining clinical outcomes. To verify the hypothesis, the present work used polyacrylamide hydrogels with different stiffness (10, 70, and 260kPa) as model materials to investigate matrix stiffness induced platelet activation behavior and its mediation on osteoimmunomodulation of macrophages. The results showed that the matrix stiffness was positively related with activation degree of platelets. However, the extracts of platelets incubated on middle-stiff matrix polarized macrophages to pro-healing M2 phenotype when compared with that on soft and stiff matrixes. ELISA results showed when compared with that on soft and stiff matrixes, the platelets incubated on middle-stiff matrix released more TGF-β and PGE2, both of which could polarize macrophages to M2 phenotype. The M2 macrophages could promote angiogenesis of endothelial cells and osteogenesis of bone marrow mesenchymal stem cells, two important and coupled processes involved in bone repair and regeneration. These findings suggest bone repair materials with 70kPa stiffness can mediate proper platelet activation, which can polarize macrophages to pro-healing M2 phenotype, potentially contributing to bone repair and regeneration.

BODIPY-Based NO Probe for Macrophage-Targeted Immunotherapy Response Monitoring.

Precise and rapid detection of immune responses is critical for timely therapeutic regimen adjustment. Immunomodulation of tumor-associated macrophages (TAMs) from a protumorigenic phenotype (M2) to an antitumorigenic phenotype (M1) is crucial in macrophage-targeted immunotherapy. Herein, we developed a boron dipyrromethene (BODIPY)-based fluorescence probe BDP3 to detect the immune responses after immunotherapy by monitoring the nitric oxide (NO) released by M1 TAMs. With an aromatic primary monoamine structure and a p-methoxyanilin electron donor in the meso-position, BDP3 not only specifically activates stable and sensitive fluorescence by NO via a photoinduced electron transfer (PET) process but also achieves a long emission wavelength for efficient in vitro and in vivo imaging. Such NO-induced fluorescence signals of BDP3 are validated to correlate well with the phenotypes of TAMs detected in macrophage cell lines and tumor tissues. The distinct sensing effects toward two types of clinically used immunotherapeutic drugs further confirm the ability of BDP3 for specific monitoring of the M1/M2 switch in response to the macrophage-targeted immunotherapy. By virtue of good biocompatibility and appropriate tumor retention time, BDP3 could be a potential fluorescent probe for noninvasive evaluation of the immunotherapeutic efficacy of macrophage-targeted immunotherapy in living animals.

Novel HALO® image analysis to determine cell phenotype in porous precision-templated scaffolds.

Image analysis platforms have gained increasing popularity in the last decade for the ability to automate and conduct high-throughput, multiplex, and quantitative analyses of a broad range of pathological tissues. However, imaging tissues with unique morphology or tissues containing implanted biomaterial scaffolds remain a challenge. Using HALO®, an image analysis platform specialized in quantitative tissue analysis, we have developed a novel method to determine multiple cell phenotypes in porous precision-templated scaffolds (PTS). PTS with uniform spherical pores between 30 and 40 μm in diameter have previously exhibited a specific immunomodulation of macrophages toward a pro-healing phenotype and an overall diminished foreign body response (FBR) compared to PTS with larger or smaller pore sizes. However, signaling pathways orchestrating this pro-healing in 40 μm PTS remain unclear. Here, we use HALO® to phenotype PTS resident cells and found a decrease in pro-inflammatory CD86 and an increase in pro-healing CD206 expression in 40 μm PTS compared to 100 μm PTS. To understand the mechanisms that drive these outcomes, we investigated the role of myeloid-differentiation-primary-response gene 88 (MyD88) in regulating the pro-healing phenomenon observed only in 40 μm PTS. When subcutaneously implanted in MyD88KO mice, 40 μm PTS reduced the expression of CD206, and the scaffold resident cells displayed an average larger nuclear size compared to 40 μm PTS implanted in mice expressing MyD88. Overall, this study demonstrates a novel image analysis method for phenotyping cells within PTS and identifies MyD88 as a critical mediator in the pore-size-dependent regenerative healing and host immune response to PTS.

Superior protective effects of PGE2 priming mesenchymal stem cells against LPS-induced acute lung injury (ALI) through macrophage immunomodulation

BackgroundMesenchymal stem cells (MSCs) have demonstrated remarkable therapeutic promise for acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS). MSC secretomes contain various immunoregulatory mediators that modulate both innate and adaptive immune responses. Priming MSCs has been widely considered to boost their therapeutic efficacy for a variety of diseases. Prostaglandin E2 (PGE2) plays a vital role in physiological processes that mediate the regeneration of injured organs.MethodsThis work utilized PGE2 to prime MSCs and investigated their therapeutic potential in ALI models. MSCs were obtained from human placental tissue. MSCs were transduced with firefly luciferase (Fluc)/eGFP fusion protein for real-time monitoring of MSC migration. Comprehensive genomic analyses explored the therapeutic effects and molecular mechanisms of PGE2-primed MSCs in LPS-induced ALI models.ResultsOur results demonstrated that PGE2-MSCs effectively ameliorated lung injury and decreased total cell numbers, neutrophils, macrophages, and protein levels in bronchoalveolar lavage fluid (BALF). Meanwhile, treating ALI mice with PGE2-MSCs dramatically reduced histopathological changes and proinflammatory cytokines while increasing anti-inflammatory cytokines. Furthermore, our findings supported that PGE2 priming improved the therapeutic efficacy of MSCs through M2 macrophage polarization.ConclusionPGE2-MSC therapy significantly reduced the severity of LPS-induced ALI in mice by modulating macrophage polarization and cytokine production. This strategy boosts the therapeutic efficacy of MSCs in cell-based ALI therapy.

Photocurable fisetin silk fibroin hydrogel accelerates infected diabetic wounds healing through biofilm inhibition and macrophage immunomodulation

Infected diabetic wounds (IDW) are difficult to heal and lead to serious complications such as amputation. Inspired by natural elements, we developed an injectable photocurable hydrogel platform based on methacrylamide silk fibroin, encapsulated with ZnO NPs and plants derived fisetin, for accelerating IDW healing. The combined release of ZnO NPs and fisetin inhibited the biofilm formation of Staphylococcus aureus on the wound surface, while fisetin possessed the immunomodulatory function of promoting macrophage M2 polarization and the release of anti-inflammatory cytokines to promote tissue healing. Therefore, this natural hydrogel composite platform can be applied as a potential wound dressing with good biocompatibility.

Recombinant humanized collagen remodels endometrial immune microenvironment of chronic endometritis through macrophage immunomodulation.

Recently, evidence has suggested that chronic endometritis (CE) is a crucial factor associated with infertility and failure of assisted reproductive techniques, prompting concern in the reproductive field. Studies have shown that persistent infiltered immune cells stimulation result in the disturbance of endometrial immune microenvironment could lead to the infertility of CE patients finally. Conventional treatments are limited because they lack immune regulation, so it is urgent to develop a novel approach to treat CE and promote embryo implantation in patients with CE. Herein, we prepared recombinant humanized type III collagen (rhCol III) with high cell adhesion activity to regulate macrophages and repair the endometrium. In this study, M1 macrophages and M1 macrophages cultured medium and lipopolysaccharide (LPS) co-stimulated inflammatory endometrium stromal cells (ESCs) were established in vitro to mimic CE condition. rhCol III promoted M1 macrophages toward M2 phenotype, improved cell migration, viability and collagen components of inflammatory ESCs. Also, the inflammatory response of inflammatory ESCs was downregulated after rhCol III treatment. Subsequently, LPS was used for CE rat model and a 28-day observation was performed; inflammatory cells' infiltration, endometrium repair, extracellular matrix (ECM) remodeling and pregnancy outcomes were promoted after rhCol III endometrial infusion. In conclusion, rhCol III promoted (i) macrophage polarization toward M2 macrophages, (ii) pro-inflammatory cytokine production and anti-inflammatory cytokine reduction, (iii) ECM remodeling and (iv) fertility restoration. Meanwhile, rhCol III enhanced cell biological functions by interacting with discoidin domain receptors, regulated cell metabolism and reduced the inflammatory response through the inhibition of the NF-κB/YAP signaling pathway. Overall, the results illustrated the potential therapeutic prospects of rhCol III for CE treatment.

Biocompatibility and biodistribution of matrix-bound nanovesicles in vitro and in vivo.

Matrix-bound nanovesicles (MBV) are a distinct subtype of extracellular vesicles that are firmly embedded within biomaterials composed of extracellular matrix (ECM). MBV both store and transport a diverse, tissue specific portfolio of signaling molecules including proteins, miRNAs, and bioactive lipids. MBV function as a key mediator in ECM-mediated control of the local tissue microenvironment. One of the most important mechanisms by which MBV in ECM bioscaffolds support constructive tissue remodeling following injury is immunomodulation and, specifically, the promotion of an anti-inflammatory, pro-remodeling immune cell activation state. Recent in vivo studies have shown that isolated MBV have therapeutic efficacy in rodent models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV administered independent of the rest of the ECM, the in vitro and in vivo safety and biodistribution profile of MBV remain uncharacterized. The purpose of the present study was to thoroughly characterize the pre-clinical safety profile of MBV through a combination of in vitro cytotoxicity and MBV uptake studies and in vivo toxicity, immunotoxicity, and imaging studies. The results showed that MBV isolated from porcine urinary bladder are well-tolerated and are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive compared to the potent immunosuppressive drug cyclophosphamide. Furthermore, this safety profile was sustained across a wide range of MBV doses. STATEMENT OF SIGNIFICANCE: Matrix-bound nanovesicles (MBV) are a distinct subtype of bioactive extracellular vesicles that are embedded within biomaterials composed of extracellular matrix (ECM). Recent studies have shown therapeutic efficacy of MBV in models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV, the in vitro and in vivo biocompatibility and biodistribution profile of MBV remain uncharacterized. The results of the present study showed that MBV are a well-tolerated ECM-derived therapy that are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive. Collectively, these data highlight the translational feasibility of MBV therapeutics across a wide variety of clinical applications.

Disc regeneration by injectable fucoidan-methacrylated dextran hydrogels through mechanical transduction and macrophage immunomodulation

Modulating a favorable inflammatory microenvironment that facilitates the recovery of degenerated discs is a key strategy in the treatment of intervertebral disc (IVD) degeneration (IDD). More interestingly, well-mechanized tissue-engineered scaffolds have been proven in recent years to be capable of sensing mechanical transduction to enhance the proliferation and activation of nucleus pulposus cells (NPC) and have demonstrated an increased potential in the treatment and recovery of degenerative discs. Additionally, existing surgical procedures may not be suitable for IDD treatment, warranting the requirement of new regenerative therapies for the restoration of disc structure and function. In this study, a light-sensitive injectable polysaccharide composite hydrogel with excellent mechanical properties was prepared using dextrose methacrylate (DexMA) and fucoidan with inflammation-modulating properties. Through numerous in vivo experiments, it was shown that the co-culture of this composite hydrogel with interleukin-1β-stimulated NPCs was able to promote cell proliferation whilst preventing inflammation. Additionally, activation of the caveolin1-yes-associated protein (CAV1-YAP) mechanotransduction axis promoted extracellular matrix (ECM) metabolism and thus jointly promoted IVD regeneration. After injection into an IDD rat model, the composite hydrogel inhibited the local inflammatory response by inducing macrophage M2 polarization and gradually reducing the ECM degradation. In this study, we propose a fucoidan-DexMA composite hydrogel, which provides an attractive approach for IVD regeneration.