Induces Macrophage Apoptosis Research Articles

Therapeutic Insulin Analogue Concentrations at Infusion Sites Enhanced the Pro-Inflammatory Response and Apoptosis in an In Vitro Macrophage-Material Interaction Model.

Continuous subcutaneous insulin infusion for Type 1 diabetes relies upon insulin infusion sets (IIS) to reliably deliver insulin to a subcutaneous depot, where it is absorbed into systemic circulation. However, IIS are plagued by short wear times and high failure rates, due in part to inconsistent insulin absorption that can arise over time. While emerging evidence suggests that the local inflammatory response to the IIS cannula may impact both wear times and unreliable insulin adsorption, the mechanisms are poorly understood. Here, we investigated the effects of local infused insulin concentrations on the biomaterial host response to better understand the underlying factors that limit the IIS performance. We first modeled the insulin concentration for a constant basal infusion rate to select a relevant insulin concentration range of 0.1-10 U/mL within the infusion site. We then examined the influence of a commercial insulin analogue (Humulin-N) using an in vitro macrophage-material model, which uses adsorbed fibroblast lysate (containing damage-associated molecular patterns) to activate macrophages and recapitulates macrophage responses on implanted biomaterials. RAW-Blue macrophages cultured on lysate-adsorbed surfaces had increased nuclear factor-κB (NF-κB) and activating protein 1 (AP-1) activity and intracellular reactive oxygen species (ROS) accumulation compared to control surfaces. Humulin-N concentration (0.5-10 U/mL) enhanced the NF-κB/AP-1 activity and ROS accumulation in macrophages on lysate-adsorbed surfaces. However, Humulin-N had no effect on NF-κB/AP-1 or ROS in the absence of the inflammatory stimulus. Additionally, high insulin concentrations arising from therapeutic doses induced macrophage apoptosis with and without adsorbed lysate. This study contributes to emerging evidence that infused insulin affects the tissue response to IIS.

Liposomal sodium clodronate mitigates radiation-induced lung injury through macrophage depletion

Radiation-induced lung injury (RILI) is a severe complication arising from thoracic tumor radiotherapy, which constrains the possibility of increasing radiation dosage. Current RILI therapies provide only limited relief and may result in undesirable side effects. Therefore, there is an urgent demand for effective and low-toxicity treatments for RILI. Macrophages play a pivotal role in RILI, promoting inflammation in the initial stages and facilitating fibrosis in the later stages. Sodium clodronate, a bisphosphonate, can induce macrophage apoptosis when encapsulated in liposomes. In this study, we explored the potential of liposomal sodium clodronate (LC) as a specific agent for depleting macrophages to alleviate acute RILI. We assessed the impact of LC on macrophage consumption both in vitro and in vivo. In a mouse model of acute RILI, LC treatment group led to a reduction in alveolar macrophage counts, mitigated lung injury severity, and lowered levels of pro-inflammatory cytokines in both plasma and bronchoalveolar lavage fluid. Additionally, we further elucidated the specific effects and mechanism of LC on macrophages in vitro. Alveolar macrophages MHS cells were subjected to varying concentrations of LC (0, 50, 100, 200 μg/ml), and the results demonstrated its dose-dependent inhibition of cell proliferation and induction of apoptosis. Moreover, LC decreased the secretion of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Conditioned media from LC-treated macrophages protected alveolar epithelial cells MLE-12 from radiation-induced damage, as demonstrated by reduced apoptosis and DNA damage. These findings imply that LC-mediated macrophage depletion may present a promising therapeutic strategy for alleviating radiation-induced lung injury.

Study on the mechanism of glucocorticoid receptor mitochondrial translocation and glucocorticoid-induced apoptosis in macrophages

Objective Our research aimed to investigate the therapeutic effects of Tubastatin-A, a glucocorticoid receptor (GR) mitochondrial translocation inhibitor, and mitoquinone (MitoQ), an antioxidant, on attenuating dexamethasone (DEX)-induced macrophage apoptosis. Methods We treated RAW264.7 macrophages with different combinations of DEX and either Tubastatin-A or MitoQ. Parameters such as mitochondrial GR translocation, mitochondrial reactive oxygen species levels, mitochondrial membrane potential, mitochondrial permeability transition pore opening, cytochrome C efflux to the cytosol, and apoptosis were subsequently evaluated in the different treatment groups via qRT–PCR, western blotting, and immunofluorescence assays. Results DEX intervention increased the translocation of GRs into the mitochondria, while reducing the expression of the mitochondrial gene MT-CO1 and the activity of mitochondrial respiratory chain complex IV in macrophages. In addition, DEX administration increased mtROS levels, mitochondrial permeability transition pore opening, and mitochondrial cytochrome C release in macrophages, which promoted their apoptosis. We found that Tubastatin-A inhibited mitochondrial GR translocation and reversed the DEX-induced increase in GR levels within the mitochondria. Furthermore, Tubastatin-A mitigated various mitochondrial changes induced by DEX, including reducing the efflux of mitochondrial cytochrome C and inhibiting macrophage apoptosis. Similarly, MitoQ exerted its effects on macrophage apoptosis by reducing mtROS levels through the mitochondrial pathway. Conclusions The DEX-mediated translocation of GR into mitochondria disrupts the mitochondrial function of macrophages, which induces their apoptosis. By inhibiting mitochondrial translocation of GR and reducing mtROS levels, Tubastatin-A and MitoQ can effectively attenuate macrophage apoptosis, which has clinical implications for reducing the notable side effects associated with glucocorticoid use.

C-terminal region of Rv1039c (PPE15) protein of Mycobacterium tuberculosis targets host mitochondria to induce macrophage apoptosis.

Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic mycobacterium. Some of these proteins are known to play role in virulence and immune response modulation, but many are still uncharacterized. This study investigated the role of C-terminal region of Rv1039c (PPE15) in inducing mitochondrial perturbations and macrophage apoptosis. Our in-silico studies revealed the disordered, coiled, and hydrophobic C-terminal region in Rv1039c has similarity with C-terminal of mitochondria-targeting pro-apoptotic host proteins. Wild type Rv1039c and C-terminal deleted Rv1039c (Rv1039c-/-Cterm) recombinant proteins were purified and their M. smegmatis knock-in strains were constructed which were used for in-vitro experiments. Confocal microscopy showed localization of Rv1039c to mitochondria of PMA-differentiated THP1 macrophages; and reduced mitochondrial membrane depolarization and production of mitochondrial superoxides were observed in response to Rv1039c-/-Cterm in comparison to full-length Rv1039c. The C-terminal region of Rv1039c was found to activate caspases 3, 7 and 9 along with upregulated expression of pro-apoptotic genes like Bax and Bim. Rv1039c-/-Cterm also reduced the Cytochrome-C release from the mitochondria and the expression of AnnexinV/PI positive and TUNEL positive cells as compared to Rv1039c. Additionally, Rv1039c was observed to upregulate the TLR4-NF-κB-TNF-α signalling whereas the same was downregulated in response to Rv1039c-/-Cterm. These findings suggested that the C-terminal region of Rv1039c is a molecular mimic of pro-apoptotic host proteins which induce mitochondria-dependent macrophage apoptosis and evoke host immune response. These observations enhance our understanding about the role of PE/PPE proteins at host-pathogen interface.

Current research on the interaction between Helicobacter pylori and macrophages.

Helicobacter pylori (H. pylori) is a gram-negative bacteria with a worldwide infection rate of 50%, known to induce gastritis, ulcers and gastric cancer. The interplay between H. pylori and immune cells within the gastric mucosa is pivotal in the pathogenesis of H. pylori-related disease. Following H. pylori infection, there is an observed increase in gastric mucosal macrophages, which are associated with the progression of gastritis. H. pylori elicits macrophage polarization, releases cytokines, reactive oxygen species (ROS) and nitric oxide (NO) to promote inflammatory response and eliminate H. pylori. Meanwhile, H. pylori has developed mechanisms to evade the host immune response in order to maintain the persistent infection, including interference with macrophage phagocytosis and antigen presentation, as well as induction of macrophage apoptosis. Consequently, the interaction between H. pylori and macrophages can significantly impact the progression, pathogenesis, and resolution of H. pylori infection. Moreover, macrophages are emerging as potential therapeutic targets for H. pylori-associated gastritis. Therefore, elucidating the involvement of macrophages in H. pylori infection may provide novel insights into the pathogenesis, progression, and management of H. pylori-related disease.

SAM68 directs STING signaling to apoptosis in macrophages

DNA is a danger signal sensed by cGAS to engage signaling through STING to activate innate immune functions. The best-studied downstream responses to STING activation include expression of type I interferon and inflammatory genes, but STING also activates other pathways, including apoptosis. Here, we report that STING-dependent induction of apoptosis in macrophages occurs through the intrinsic mitochondrial pathway and is mediated via IRF3 but acts independently of gene transcription. By intersecting four mass spectrometry datasets, we identify SAM68 as crucial for the induction of apoptosis downstream of STING activation. SAM68 is essential for the full activation of apoptosis. Still, it is not required for STING-mediated activation of IFN expression or activation of NF-κB. Mechanistic studies reveal that protein trafficking is required and involves SAM68 recruitment to STING upon activation, with the two proteins associating at the Golgi or a post-Golgi compartment. Collectively, our work identifies SAM68 as a STING-interacting protein enabling induction of apoptosis through this DNA-activated innate immune pathway.

Effect and Mechanism of Mycobacterium avium MAV-5183 on Apoptosis of Mouse Ana-1 Macrophages.

To investigate the effects and mechanisms of Mycobacterium avium MAV-5183 protein on apoptosis in mouse Ana-1 macrophages. A pET-21a-MAV-5183 recombinant plasmid was constructed. The recombinant MAV-5183 protein was cloned, expressed, purified, and identified using an anti-His-tagged antibody. Rabbits were immunized to obtain antiserum, and its potency and immunoreactivity were assessed through WB. Mouse Ana-1 macrophages were incubated with varying concentrations of MAV-5183 protein. Flow cytometry, following ANNEXIN V-FITC/PI double staining, detected apoptosis. Western Blot analysis was conducted to identify apoptosis-related molecules Caspase-9/8/3 and vesicle-related molecules ASC, NLRP3, and Cleaved-casp1. ELISA measured TNF-α and IL-6 levels in the culture supernatant. LDH activity and ROS levels were analyzed separately. RT-qPCR measured mRNA levels of Caspase-9/8/3, ASC, NLRP3, Caspase-1, IL-1β, Bax, MAPK-p38, Bcl-2, TNF-α, and IL-6. MAV-5183 protein was successfully cloned, purified, and identified. In in vitro studies on Ana-1 macrophages, MAV-5183 protein increased the expression of Caspase-9/8/3, ASC, NLRP3 (P < 0.01), induced ROS secretion (P < 0.05), and promoted inflammatory cytokine secretion (TNF-α, IL-6, P < 0.0001); however, it did not significantly affect LDH (P > 0.05). MAV-5183 also induced apoptosis in Ana-1 macrophages (P < 0.05). RT-qPCR results indicated a significant increase in mRNA expression of Caspase-9/8/3, ASC, NLRP3, TNF-α, IL-6, MAPK-p38, and pro-apoptotic factor Bax (P < 0.01), with no significant effect on Bcl-2 and IL-1β mRNA (P > 0.05). The data indicate that MAV-5183 induces macrophage apoptosis through a caspase-dependent pathway and promotes inflammatory cytokine secretion via ROS.

Microplastics induced apoptosis in macrophages by promoting ROS generation and altering metabolic profiles

The ubiquitous presence of Microplastics (MPs) in various environments documented in recent years has recently raised significant concerns about their toxic effects. While macrophages serve as the first line of defense against toxic substances and pathogens, the impact and mechanisms of microplastics on these immune cells remain unclear. This study aims to explore whether MPs induce macrophage apoptosis through the promotion of reactive oxygen species (ROS) generation and alterations in metabolic profiles. The viability of RAW264.7 cells decreased as the concentration of 0.5 µm or 5 µm MPs ranged from 0.2 to 1.5 mg/mL, with a more pronounced effect observed in the 0.5 µm MPs group. Zebrafish exposed to 0.5 µm or 5 µm MPs at a concentration of 0.5 mg/mL exhibited decreased macrophage abundance and increased apoptosis, accompanied by alterations in the expression of inflammatory and apoptosis-related genes. While 0.5 µm MPs were observed to enter macrophages, 5 µm MPs only adhered to the cell membrane surface. Both particle sizes induced ROS generation and disrupted cellular metabolism in RAW264.7 cells. Notably, macrophages exhibited a more pronounced response to 0.5 µm MPs, characterized by heightened ROS generation, increased secretion of pro-inflammatory mediators, and a significant decrease in sphingolipid metabolism. These findings suggest that the adverse effects on macrophages are greater with 0.5 µm MPs compared to 5 µm MPs, possibly attributed to particle size effects. This study contributes additional evidence on the impact of MPs on human immune cells.

NK-derived exosome miR-1249-3p inhibits Mycobacterium tuberculosis survival in macrophages by targeting SKOR1

Murine Natural Killer cells were cultivated in vitro to isolate NK-derived exosomes. Subsequent quantification via qPCR confirmed enrichment of miR-1249-3p. Ana-1 murine macrophages were cultured in vitro and subsequently inoculated with Mycobacterium tuberculosis (MTB) strain H37Rv. NK-exo and NK-exo miR-1249-3p were separately applied to the infection model, followed by immunological assays conducted post-48-hour co-culture. Western blot analyses corroborated that NK-exo exhibited exosomal marker proteins Granzyme A (GzmA), Granzyme B (GzmB), and Perforin (PFN), alongside a notable enrichment of miR-1249-3p. Functionally, NK-exo augmented the expression levels of Caspase-9,-8, and -3, as well as PARP, while attenuating the expression of NLRP3, ASC, and Cleaved-Caspase-1. Furthermore, qPCR demonstrated an up-regulation of Caspase-9, -8, and -3, along with pro-apoptotic factors Bax and Bid, and a concomitant down-regulation of the anti-apoptotic factor Bcl-2. The expression levels of inflammatory markers ASC, NLRP3, Cleaved-Caspase-1, and IL-1β were concomitantly decreased. ELISA findings indicated diminished levels of TNF-α and ROS secretion. NK-exo miR-1249-3p specifically targeted and attenuated the expression of SKOR-1, engendering up-regulation of apoptosis-associated proteins and down-regulation of inflammation-related proteins, consequently affecting cellular fate.Our empirical evidence substantiates that NK-exo induces macrophage apoptosis, thereby mitigating MTB survival. Furthermore, NK-exo miR-1249-3p directly targets and inhibits SKOR-1 expression, leading to macrophage apoptosis and consequently hampering the proliferation of MTB. The data implicate the potential therapeutic relevance of NK-exo and miR-1249-3p in managing drug-resistant tuberculosis.

HgCl2 exposure mediates pyroptosis of HD11 cells and promotes M1 polarization and the release of inflammatory factors through ROS/Nrf2/NLRP3

Mercury (Hg) is a serious metal environmental pollutant. HgCl2 exposure causes pyroptosis. When macrophages are severely stimulated, they often undergo M1 polarization and release inflammatory factors. However, the mechanisms by which mercuric chloride exposure induces macrophage apoptosis, M1 polarization, and inflammatory factors remain unclear. HD11 cells were exposed to different concentrations of Hg chloride (180, 210 and 240 nM HgCl2). The results showed that mercury chloride exposure up-regulated ROS, C-Nrf2 and its downstream factors (NQO1 and HO-1), and down-regulated N-Nrf2. In addition, the expressions of focal death-related indicators (Caspase-1, NLRP3, GSDMD, etc.), M1 polarization marker CD86 and inflammatory factors (TNF-α, IL-1β) increased, and the above changes were related to mercury. Oxidative stress inhibitor (NAC) can block ROS/ NrF2-mediated oxidative stress, inhibit mercury-induced pyroptosis and M1 polarization, and effectively reduce the release of inflammatory factors. The addition of Vx-765 to inhibit pyroptosis can effectively alleviate M1 polarization of HD11 cells and reduce the expression of inflammatory factors. HgCl2 mediates pyroptosis of HD11 cells by regulating ROS/Nrf2/NLRP3, promoting M1 polarization and the release of inflammatory factors.

Salmonella Typhimurium secondary infection induced macrophages apoptosis NO-dependent during Mycobacterium neoaurum infection

Mycobacterium neoaurum (M. neoaurum) and Salmonella Typhimurium (S. Typhimurium) are important zoonotic pathogens, and both are intracellular bacteria, which can induce cellular immunity. Coinfection is prevalent worldwide, even more prevalent than single infection. This study aimed to investigate the effect of M. neoaurum/S. Typhimurium coinfection on the percentage property of C57BL/6 mice regulatory T cells (Tregs) and the immune activity of RAW264.7 cells. The secretion of TNF-α, IFN-γ, IL-1β, IL-12 and iNOS in RAW264.7 cells was determined by ELISA. The expression of CD40+ , CD80+ and CD86+ costimulatory molecules on the surface of macrophages was analyzed by flow cytometry. A Nitric oxide (NO) assay was used to detect the production of NO in RAW264.7 cells. The apoptosis of RAW264.7 cells was detected by flow cytometry. The results showed that macrophage expressed a large number of cytokines and surface costimulatory molecules to enhance immune activity. S. Typhimurium secondary infection significantly increased the expression of iNOS and generation of NO, and M. neoaurum/S. Typhimurium-induced apoptosis was NO-dependent. Our data provide a theoretical basis for secondary infections by other pathogens after Nontuberculous Mycobacterium (NTM) infection, and lay a foundation for further research.

Statement of Retraction: A novel Fas-binding outer membrane protein and lipopolysaccharide of Leptospira interrogans induce macrophage apoptosis through the Fas/FasL-caspase-8/-3 pathway

Statement of Retraction: A novel Fas-binding outer membrane protein and lipopolysaccharide of Leptospira interrogans induce macrophage apoptosis through the Fas/FasL-caspase-8/-3 pathway

TWIST1 rescue calcium overload and apoptosis induced by inflammatory microenvironment in S. aureus-induced osteomyelitis.

Currently, there is no effective therapy for Staphylococcus aureus-induced osteomyelitis. It is widely recognized that the inflammatory microenvironment around abscess plays an essential role in protracting the course of S. aureus-induced osteomyelitis. In this study, we found TWIST1 was highly expressed in macrophages around abscesses but less related to local S. aureus in the later stages of Staphylococcus aureus-infected osteomyelitis. Mouse bone marrow macrophages show apoptosis and elevated TWIST1 expression when treated with the inflammatory medium. Knockdown of TWIST1 induced macrophage apoptosis, impaired the bacteria phagocytosis/killing abilities, and promoted cell apoptosis markers expression in inflammatory microenvironment stimulation. Furthermore, inflammatory microenvironments were responsible for inducing calcium overload in macrophage mitochondrial while calcium overload inhibition significantly rescued macrophage apoptosis, bacteria phagocytosis/killing abilities and improved the mice's antimicrobial ability. Our findings indicated that TWIST1 is a crucial molecule that protects macrophages from calcium overload induced by inflammatory microenvironments.

Chlamydia psittaci inclusion membrane protein CPSIT_0842 induces macrophage apoptosis through MAPK/ERK-mediated autophagy.

Chlamydia psittaci is a multi-host zoonotic pathogen, which mainly infects poultry and inflicts an appreciable economic burden on the livestock farming industry. C. psittaci inclusion membrane proteins are uniquely positioned at the host-pathogen interface and are important virulence proteins. We have previously confirmed that Incs regulate host cell survival to help Chlamydia sp. evade host-cell-mediated defense mechanisms. However, the role of the Inc, CPSIT_0842, in the regulation of cell death following the establishment of persistent C. psittaci infection remains unknown. This study explored the effect of CPSIT_0842 on the crosstalk between the autophagic and apoptotic pathways in macrophages. Results showed that CPSIT_0842 initiated autophagy and blocked autophagic flux in human macrophages, as indicated by autophagy-related protein LC3-II, Beclin-1, and p62 upregulation, autophagosome accumulation, and lysosomal protein LAMP1 diminution. We also showed that the disruption of autophagic flux had a regulatory effect on CPSIT_0842-induced apoptosis. Moreover, the suppression of autophagy initiation by 3-methyladenine attenuated CPSIT_0842-induced apoptosis. By contrast, the induction of autophagic flux by rapamycin did not significantly affect CPSIT_0842-induced apoptosis. Taken together, these findings demonstrate that CPSIT_0842 induced macrophage apoptosis by initiating incomplete autophagy through the MAPK/ERK/mTOR signaling pathway, which may be instrumental to the ability of C. psittaci to evade the host innate immune response and establish persistent infection. The improved understanding of the autophagic and cell death pathways triggered upon bacterial inclusion will likely help in the development of novel treatment strategies for chlamydia infection.

MicroRNA-202-5p-dependent inhibition of Bcl-2 contributes to macrophage apoptosis and atherosclerotic plaque formation

BackgroundThe identification of microRNA (miRNA)-related molecular mechanisms has advanced the development of new therapeutics for atherosclerosis (AS). The roles of miR-202-5p- in the pathogenic mechanisms of AS have not been explored. MethodsMacrophages were transfected with a series of miR-202-5p mimic/inhibitor, and then assessed for changes in viability, apoptosis, and secretion of inflammatory cytokines. The regulatory mechanism of miR-202-5p was explored through dual-luciferase reporter gene assay. A mouse model of AS was developed in ApoE-/- mice fed with high-fat diet to examine the in vivo effects of miR-202-5p on atherosclerotic plaque formation, collagen synthesis, and fiber cap thickness. ResultsElevated miR-202-5p was found in atherosclerotic plaque tissues of the mice. miR-202-5p was able to induce macrophage apoptosis and release of pro-inflammatory factors. Besides, miR-202-5p limited Bcl-2 expression and elevated the levels of Bax, cleaved caspase-3, and cleaved caspase-9. Bcl-2 was concluded as a target gene of miR-202-5p. The pro-apoptotic effect of miR-202-5p on macrophages was achieved via limiting Bcl-2. In the mouse AS model, restoration of miR-202-5p stimulated atherosclerotic plaque formation, but reduced collagen synthesis and fiber cap thickness. ConclusionThese data collectively suggest a pro-apoptotic action of miR-202-5p in macrophages that contributes to atherosclerotic plaque formation.

Abstract PD2-05: CSF-1R antibody targeting therapy with combined metronomic chemotherapy enhances a B and T cell response for the treatment of metastatic triple negative breast cancer

Abstract Background: Increased macrophage infiltration is associated with the poorest outcome following neo-adjuvant chemotherapy in patients with triple negative breast cancer (TNBC). We have extensively characterized three different preclinical syngeneic claudin-low Trp53-/- tumor models (T11, T12 and 2151R), that have high Tumor Associated Macrophage (TAM) infiltration. These “claudin low” models closely phenocopy the EMT/TAM subtype observed in patients. Treatment using conventional chemotherapy, Cyclophosphamide (CTX) in combination with Pexidartinib (PXB, PLX3397) an anti CSF-1R small molecule inhibitor yielded a durable response in two of the models, T12 and 2151R (Singh et al. Cancer Res. 2022). However, due to potential liver toxicity and dual targeting of c-kit and FLT-3, PXB is not moving forward into the clinic for TNBC. Accordingly, we asked if SNDX-ms6352 a specific, high affinity monoclonal antibody targeting CSF-1R in combination with CTX might provide an alternative approach for treating primary TNBC as well as established lung metastases. Methods: Tumor chunks were transplanted into the mammary fat pad to generate primary tumors. Single cells from primary T12 mammary tumors were introduced via tail vein (TV) injection to obtain lung metastases. After fifteen days for primary tumors and twelve days following TV injection, respectively, the mice were randomized into treatment groups and administered four weekly treatments of IgG, CTX, SNDX-ms6352 or CTX+SNDX-ms6352. Primary tumors, lungs and mammary glands were harvested and fixed overnight in 4% paraformaldehyde then placed in 70% EtOH for paraffin embedding and stained for H&amp;E, immunohistochemistry (IHC), immunofluorescence (IF) and imaging mass cytometry (IMC). Results: Following, four weekly treatments of CTX+SNDX-ms6453 we observed a significantly decreased primary tumor volume as compared to IgG, CTX and SNDX-ms6352 alone. Mice that fully responded to the treatment were re-challenged with tumor cells in the contralateral mammary gland to determine if there was a long-term immune memory response. We observed a fifty percent tumor re-challenge rejection in both T12 and 2151R. Increased expression of CD20 B and CD8 T cells was observed within the mammary gland of the complete responders, this was accompanied by macrophage depletion in the SNDX-ms6352 and combination treated mice. SNDX-ms6352 induced macrophage apoptosis as evidenced by the activation of cleaved caspase 3. For lung metastases TV injected mice were administered BrdU to identify micro-metastatic lesions. Following four weekly combination treatments of CTX+/-SNDX-ms6352 we observed a decreased lung metastatic burden and increased overall survival as compared to IgG, CTX and SNDX-ms6352 alone. Interestingly we also observed increased CD8 T-cell infiltration, but only in the combination treated mice following 28 and 56 days post treatment. Conclusion: These results suggest that targeting macrophages enhances the immunostimulatory effect of low dose cyclophosphamide in treating not only primary tumors, but also established lung metastasis via the activation of the tumor immune microenvironment. Supported by CA148761 and T32 CA203690 grants. Citation Format: Diego Pedroza, Weiguo Wu, Paul Porter, Xiang Zhang, Jeffrey Rosen. CSF-1R antibody targeting therapy with combined metronomic chemotherapy enhances a B and T cell response for the treatment of metastatic triple negative breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-05.

DUSP1 mediates BCG induced apoptosis and inflammatory response in THP-1 cells via MAPKs/NF-κB signaling pathway

Tuberculosis (TB) is a zoonotic infectious disease caused by Mycobacterium tuberculosis (Mtb). Apoptosis and necrosis caused by the interaction between the host and the pathogen, as well as the host’s inflammatory response, play an important role in the pathogenesis of TB. Dual-specificity phosphatase 1 (DUSP1) plays a vital role in regulating the host immune responses. However, the role of DUSP1 in the regulation of THP-1 macrophage apoptosis induced by attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection remains unclear. In the present study, we report that infection with BCG significantly induces macrophage apoptosis and induces the production of DUSP1, TNF-α and IL-1β. DUSP1 knockdown significantly inhibited BCG-induced macrophage apoptosis and activation of MAPKs/NF-κB signaling pathway. In addition, DUSP1 knockdown suppressed BCG-induced inflammation in vivo. Taken together, this study demonstrates that DUSP1, as a regulator of MAPKs/NF-κB signaling pathway, plays a novel role in BCG-induced macrophage apoptosis and inflammatory response.

Clinical isolates of Mycobacterium tuberculosis with different genotypes exhibit distinct host macrophage responses in vitro.

Introduction. Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, can survive as an intracellular parasite after entering macrophages via phagocytosis. M.tb strains are genotypically distinct and engage in diverse pathogen-host interactions, with different host immune responses triggered by different M.tb strains. Importantly, differences in intracellular accumulation and triggering of host macrophage responses during early infection stages are key determinants that shape the final outcomes of host innate immune responses to different M.tb strains.Hypothesis/Gap Statement. Clinical M.tb strains with different genotypes elicit different host innate immune responses in vitro.Aim. This work aimed to compare host innate immune responses elicited by genotypically diverse, clinically derived M.tb strains in vitro.Methodology. RAW264.7 cells were infected with three lineage 2 and lineage 4 clinically derived M.tb strains and strain H37Rv. Strains were evaluated for differences in intracellular growth, induction of macrophage apoptosis, and induction of expression of proinflammatory cytokines and associated pattern recognition receptors.Results. Highly variable cytokine profiles were observed subsequent to RAW264.7 cell infection with the different strains. The Beijing genotype strain, a modern Beijing strain belonging to lineage 2, induced milder host proinflammatory responses and less apoptosis and exhibited greater intracellular growth as compared to the other strains. Moreover, mRNA expression levels of iNOS in Beijing and MANU2 genotype strains exceeded corresponding levels obtained for the T1 genotype strain. Meanwhile, mRNA expression levels of toll-like receptor (TLR)-encoding genes TLR2 and TLR7 in macrophages infected with the Beijing genotype strain were higher than corresponding levels observed in MANU2 genotype strain-infected macrophages.Conclusion. The higher intracellular survival rate and lower level of host cell apoptosis associated with macrophage infection with the Beijing genotype strain indicated greater virulence of this strain relative to that of the other strains. Furthermore, in vitro immune responses induced by the Beijing genotype strain were unique in that this strain induced a weaker inflammatory response than was induced by T1 or MANU2 genotype strains. Nevertheless, additional evidence is needed to confirm that Beijing genotype strains possess greater virulence than strains with other genotypes.

Programmed prodrug breaking the feedback regulation of P-selectin in plaque inflammation for atherosclerotic therapy

Inflammation is the main driver of the aggravation of arteriosclerosis, and the complex inflammatory response in plaque is usually the result of the interaction of various cells and cytokines. Therefore, it is difficult to comprehensively regulate the inflammatory process of arteriosclerosis by intervening a single target, resulting in the poor effect of existing treatment method. Based on our clinical findings that P-selectin stably and highly expressed in patients’ plaque endothelial cells, the programmed prodrug, low molecular weight heparin-indomethacin nanoparticles (LI NPs), were established as anti-inflammatory agent to multiphase inhibit arteriosclerosis by cascade interference of P-selectin. Structurally, LI NPs was obtained by simple esterification of low molecular weight heparin and indomethacin without any additives, guaranteeing the biocompatibility and applicability of LI NPs. Functionally, LI NPs could interfere with P-selectin in the inflammatory process, such as inhibiting macrophage adhesion, reducing the secretion of inflammatory factors, and inducing macrophage apoptosis. In the arteriosclerosis mice model, LI NPs significantly reduced the plaque area and showed satisfactory curative effect, which is related to the intervention of the multiphase inflammation between endothelial cells and macrophages. In conclusion, the programmed prodrug LI NPs offered a promising approach for the clinical therapy of arteriosclerosis.

Soluble egg antigen of Schistosoma japonicum induces macrophage apoptosis in mice

To investigate the dynamic changes of macrophage numbers and apoptosis during Schistosoma japonicum infection, and to investigate the possible mechanisms of macrophage apoptosis induced by S. japonicum soluble egg antigen (SEA). C57BL/6 mice at ages of 6~8 weeks were randomly divided into 4 groups, including three experimental groups and a normal control group. Each mouse in the experimental groups was infected with (12 ± 1) cercariae of S. japonicum via the abdominal skin, and all mice in an experimental group were sacrificed 3, 5, 8 weeks post-infection, respectively, while mice in the control group were not infected with S. japonicum cercariae and sacrificed on the day of S. japonicum infection in the experimental group. Mouse liver specimens and peritoneal exudation cells were sampled in each group, and the dynamic changes of macrophage numbers and apoptosis were detected. Mouse peritoneal macrophages were isolated, purified and treated with S. japonicum SEA, PBS and ovalbumin (OVA) in vitro, and the macrophage apoptosis was detected using flow cytometry. The mRNA and protein expression of BCL-2 protein family members were determined in macrophages using real-time quantitative PCR (qP-CR) and Western blotting assays, and the activation of caspase 3 was determined using flow cytometry and Western blotting. In addition, macrophages were in vitro treated with S. japonicum SEA in presence of a caspase inhibitor, H2O2 or N-acetyl-L-cysteine, and the apoptosis of macrophages was detected using flow cytometry. The total macrophage numbers continued to increase in mouse liver [(0.873 ± 0.106) × 106, (2.737 ± 0.460) × 106 and (3.107 ± 0.367) × 106 cells, respectively; F = 81.900, P < 0.01] and peritoneal specimens [(5.282 ± 1.136) × 105, (7.500 ± 1.200) × 105 and (12.800 ± 0.800) × 105 cells, respectively; F = 55.720, P < 0.01] 3, 5 and 8 weeks post-infection with S. japonicum, and the numbers of apoptotic macrophages also continued to increase in mouse liver [(0.092 ± 0.018) × 106, (0.186 ± 0.025) × 106 and (0.173 ± 0.0270) × 106 cells; F = 57.780, P < 0.01] and peritoneal specimens [(0.335 ± 0.022) × 105, (0.771 ± 0.099) × 105 and (1.094 ± 0.051) × 105 cells; F = 49.460, P < 0.01] 3, 5 and 8 weeks post-infection with S. japonicum. The apoptotic rate of SEA-treated macrophages [(24.330 ± 0.784)%] was significantly higher than that of PBS-[(18.500 ± 1.077)%] and OVA-treated macrophages [(18.900 ± 1.350)%] (both P values < 0.01). There were no significant differences in the mRNA or protein expression of Bcl-2 [Bcl - 2 mRNA expression: (1.662 ± 0.943) vs. (1.000 ± 0.000), t = 1.215, P > 0.05; BCL protein expression: (0.068 ± 0.004) vs. (0.070 ± 0.005), t = 0.699, P > 0.05], Bax [Bax mRNA expression: (0.711 ± 0.200) vs. (1.000 ± 0.000), t = 2.507, P > 0.05; BAX protein expression: (0.089 ± 0.005) vs. (0.097 ± 0.003), t = 2.232, P > 0.05] and Bak [Bak mRNA expression: (1.255 ± 0.049) vs. (1.00 ± 0.00), t = 0.897, P > 0.05; BAK protein expression: (0.439 ± 0.048) vs. (0.571 ± 0.091), t = 2.231, P > 0.05] between in SEA- and PBS-treated macrophages. S. japonicum SEA induced macrophage apoptosis in the presence of a caspase inhibitor (F = 0.411, P > 0.05); however, SEA failed to induce macrophage apoptosis in the presence of H2O2 or NAC (F = 11.880 and 9.897, both P values < 0.05). S. japonicum SEA may induce macrophage apoptosis through promoting reactive oxygen species expression during S. japonicum infections in mice.