The effect of intestinal flora metabolites on macrophage polarization.

Intestinal flora metabolites played a crucial role in immunomodulation by influencing host immune responses through various pathways. Macrophages, as a type of innate immune cell, were essential in chemotaxis, phagocytosis, inflammatory responses, and microbial elimination. Different macrophage phenotypes had distinct biological functions, regulated by diverse factors and mechanisms. Advances in intestinal flora sequencing and metabolomics have enhanced understanding of how intestinal flora metabolites affect macrophage phenotypes and functions. These metabolites had varying effects on macrophage polarization and different mechanisms of influence. This study summarized the impact of gut microbiota metabolites on macrophage phenotype and function, along with the underlying mechanisms associated with different metabolites produced by intestinal flora.

Zhiqiao Gancao decoction regulated JAK2/STAT3/ macrophage M1 polarization to ameliorate intervertebral disc degeneration.

Background: Zhiqiao Gancao decoction (ZQGCD) was created by Professor Gong Zhengfeng, a renowned Chinese medicine expert. Clinical studies have shown its efficacy in alleviating pain and enhancing lumbar function in intervertebral disc degeneration (IDD) patients. However, the precise mechanism of ZQGCD in treating IDD remains unclear. Methods: The active components of ZQGCD were identified using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A rat model of intervertebral disc degeneration was established, and rats in each group received ZQGCD for three weeks. Assessment parameters included hyperalgesia status, observation of intervertebral disc tissue degeneration and macrophage infiltration, and analysis of JAK2/STAT3 pathway protein expression in the intervertebral disc. Primary macrophage M1 polarization was induced using LPS, with cells treated using the JAK2 inhibitor (AZD1480) and ZQGCD to evaluate macrophage polarization, cellular supernatant inflammatory factors, and JAK2/STAT3 pathway expression. Macrophage supernatant served as a conditioned medium to observe its effects on the proliferation of nucleus pulposus cells (NPCs) and the expression of collagen II and MMP3 proteins. Results: A total of 81 active components were identified in ZQGCD. Following ZQGCD treatment, infiltrating macrophages in intervertebral disc tissues of model rats decreased, the content of M1 macrophages decreased, while the content of M2 macrophages increased, the expression of proinflammatory factors and pain-inducing factors in serum decreased, and the expression of substance P in intervertebral disc tissue decreased. Consequently, the intervertebral disc degeneration and hyperalgesia of rats were improved. In vitro studies revealed that LPS induced M1 macrophage polarization. By inhibiting the JAK2/STAT3 pathway, both JAK2 inhibitors and ZQGCD effectively suppressed M1 polarization, resulting in decreased levels of IL-1ß, IL-6, TNF-α, and various other inflammatory factors. Consequently, this inhibition led to a delay in the degeneration of NPCs. Conclusion: There is macrophage infiltration in the intervertebral disc tissue of IDD rats, and JAK2/STAT3 pathway is activated, macrophages are polarized to M1 type, resulting in inflammatory microenvironment, leading to intervertebral disc degeneration and hyperalgesia. ZQGCD exhibited a delaying effect on IDD and improved hyperalgesia by inhibiting the JAK2/STAT3/macrophage M1 polarization pathway.

Role and mechanism of IRF9 in promoting the progression of rheumatoid arthritis by regulating macrophage polarization via PSMA5.

Aim: To explore the mechanisms of IRF9 in the progression of rheumatoid arthritis(RA), and the effects of IRF9 on M1/M2 polarization. Methods: RA dataset (GSE55457) was downloaded from GEO. Correlation analysis between IRF9 and its downstream target protein PSMA5 was performed using bioinformatics analysis. The M1/M2 cell ratio of peripheral blood mononuclear cells which from 20 healthy specimen and 40 RA patients was determined. The expression of IRF9 and PSMA5 was detected using qPCR and Western blot. Then, knockdown IRF9 in RAW264.7 cell line (sh-IRF9 RAW264.7) was constructed. The effect of sh-IRF9 RAW264.7 on RA was explored by constructing a CIA mouse model. Results: IRF9 is upregulated in RA and is of good early screening effect. The results of pathway analysis showed that IRF9 targets and regulates the PSMA5 signaling pathway. IRF9 and PSMA5 were significantly elevated in RA patients, M1/M2 ratio was also increased. The effects of IRF9 on RAW264.7 macrophages were deeply explored in vitro, revealing that knockdown of IRF9 suppressed PSMA5, M1/M2 ratio and the secretion of pro-inflammatory factor in RAW264.7. In mouse in vivo experiments, sh-IRF9 RAW264.7 cells were found to modulate RA by downregulating PSMA5, modulating the M1/M2 ratio through enhancing the anti-inflammatory factor, and suppressing the pro-inflammatory factor. Conclusion: IRF9 promoted the progression of RA via regulating macrophage polarization through PSMA5.

Tirofiban prevents the effects of SARS-CoV-2 spike protein on macrophage activation and endothelial cell death.

SARS-CoV-2 viral-derived particles have been proposed to have a causal role in tissue inflammation. Macrophage is the culprit cell in the pathogenesis of destructive inflammatory response to the SARS-CoV-2 virus. We investigated whether the spike protein might play a role in perturbing the physiological process of resolution of inflammation. Using an in vitro model of M2 polarized macrophages, we found that recombinant spike protein produced typical M1 morphological features in these alternative differentiated cells. In the presence of spike, M2-macrophages lose their elongated morphology, become rounded and acquire a strong capability to stimulate lymphocyte activation and proliferation. Moreover, in M2 macrophages, spike activated the signal transducer and activator-1 (STAT1) the pivotal mediator of pro-inflammatory macrophages. We observed STAT1 activation also in endothelial cells cultured with recombinant spike, accompanied by Bax upregulation and cell death. Blockade of beta3 integrin with the RGD mimetic tirofiban reverted the spike-induced costimulatory effects on M2 macrophages. Also, tirofiban counteracted STAT1 and Bax activation in endothelial cells cultured with spike and reduced endothelial cell death. In conclusion, we found that some proinflammatory effects of the spike protein can involve the integrin pathway and provide elements supporting use of RGD mimetics against SARS-Cov-2.

Advances in understanding the immunity of the brain and its borders: Focus on brain macrophages.

A recent study outlines the phenotypes of brain border region macrophages in developing, normal and glioblastoma-affected brains. For the first time, the authors show in-vivo turnover of human brain border macrophages. The findings have implications for the understanding of brain border immunity and potential macrophage targeting therapies. KEYPOINTS: Human border region macrophages are distinct from microglia. These distinct phenotypes are established early during embryonal development - Brain border macrophages are partially replaced by bone marrow-derived myeloid cells. The transcriptional phenotypes of glioblastoma-associated macrophage are determined by the anatomical region.

Identification and validation of Rab GTPases RAB13 as biomarkers for peritoneal metastasis and immune cell infiltration in colorectal cancer patients.

Background: As one of the most common cancer, colorectal cancer (CRC) is with high morbidity and mortality. Peritoneal metastasis (PM) is a fatal state of CRC, and few patients may benefit from traditional therapies. There is a complex interaction between PM and immune cell infiltration. Therefore, we aimed to determine biomarkers associated with colorectal cancer peritoneal metastasis (CRCPM) and their relationship with immune cell infiltration. Methods: By informatic analysis, differently expressed genes (DEGs) were selected and hub genes were screened out. RAB13, one of the hub genes, was identificated from public databases and validated in CRC tissues. The ESTIMATE, CEBERSORT and TIMER algorithms were applied to analyze the correlation between RAB13 and immune infiltration in CRC. RAB13's expression in different cells were analyzed at the single-cell level in scRNA-Seq. The Gene Set Enrichment Analysis (GSEA) was performed for RAB13 enrichment and further confirmed. Using oncoPredict algorithm, RAB13's impact on drug sensitivity was evaluated. Results: High RAB13 expression was identified in public databases and led to a poor prognosis. RAB13 was found to be positively correlated with the macrophages and other immune cells infiltration and from scRNA-Seq, RAB13 was found to be located in CRC cells and macrophages. GSEA revealed that high RAB13 expression enriched in a various of biological signaling, and oncoPredict algorithm showed that RAB13 expression was correlated with paclitaxel sensitivity. Conclusion: Our study indicated clinical role of RAB13 in CRC-PM, suggesting its potential as a therapeutic target in the future.

Improving the efficacy of cancer immunotherapy by host-defence caerin 1.1 and 1.9 peptides.

Cancer remains a major global health challenge. Immunotherapy has revolutionized the management of cancer, yet only a limited number of patients respond to such treatments. This is largely attributed to the immunosuppressive tumor microenvironment, which diminishes the effectiveness of immunotherapy. Recent studies have underscored the potential of naturally derived caerin 1 peptides, particularly caerin 1.1 and caerin 1.9, which exhibit strong antitumor effects and enhance the efficacy of immunotherapies in animal models. This review encapsulates the current research aimed at augmenting the effectiveness of immunotherapy, focusing on the role of caerin 1.1 and caerin 1.9 in boosting immunotherapeutic outcomes, elucidating possible mechanisms, and discussing their limitations and challenges.

Nanosensor-Enabled Detection and Identification of Intracellular Bacterial Infections in Macrophages.

Opportunistic bacterial pathogens can evade the immune response by residing and reproducing within host immune cells, including macrophages. These intracellular infections provide reservoirs for pathogens that enhance the progression of infections and inhibit therapeutic strategies. Current sensing strategies for intracellular infections generally use immunosensing of specific biomarkers on the cell surface or polymerase chain reaction (PCR) of the corresponding nucleic acids, making detection difficult, time-consuming, and challenging to generalize. Intracellular infections can induce changes in macrophage glycosylation, providing a potential strategy for signature-based detection of intracellular infections. We report here the detection of bacterial infection in macrophages using a boronic acid (BA)-based pH-responsive polymer sensor array engineered to distinguish mammalian cell phenotypes by their cell surface glycosylation signatures. The sensor was able to discriminate between different infecting bacteria in minutes, providing a promising tool for diagnostic and screening applications.

Osteogenic Protection against Fine Dust with Erucic Acid-Induced Exosomes.

Fine dust causes various disorders, including cardiovascular, neurological, renal, reproductive, motor, systemic, respiratory, and cancerous diseases. Therefore, it is essential to study functional materials to prevent these issues. This study investigated the beneficial effects of erucic acid against fine dust using methods such as miRNA profiling, quantitative PCR, flow cytometry, ELISA, and Alizarin O staining. Erucic acid effectively suppresses inflammation and upregulates osteogenic activators in fibroblasts exposed to fine dust. Additionally, erucic acid-induced exosomes (EIEs) strongly counteract the negative effects of fine dust on osteocytic differentiation and inflammation. Despite fine dust exposure, EIEs promoted osteocytic differentiation in adipose-derived stem cells (ASCs) and enhanced osteogenesis and phagocytosis in macrophages. The significant upregulation of RunX2 and BMP7 by EIEs indicates its strong role in osteocytic differentiation and protection against the effects of fine dust. EIEs also boosts immune activity and acts as an osteogenic trigger for macrophages. MicroRNA profiling revealed that EIEs dramatically upregulated miRNAs, including hsa-miRNA-1301-3p, hsa-miRNA-1908-5p, hsa-miRNA-423-5p, and hsa-miRNA-122-5p, which are associated with osteogenic differentiation and immunity. Therefore, EIEs show potential as biomaterials to prevent environment-borne diseases.

Phagocytosis Checkpoints in Glioblastoma: CD47 and Beyond.

Glioblastoma multiforme (GBM) is one of the deadliest human cancers with very limited treatment options available. The malignant behavior of GBM is manifested in a tumor which is highly invasive, resistant to standard cytotoxic chemotherapy, and strongly immunosuppressive. Immune checkpoint inhibitors have recently been introduced in the clinic and have yielded promising results in certain cancers. GBM, however, is largely refractory to these treatments. The immune checkpoint CD47 has recently gained attention as a potential target for intervention as it conveys a "don't eat me" signal to tumor-associated macrophages (TAMs) via the inhibitory SIRP alpha protein. In preclinical models, the administration of anti-CD47 monoclonal antibodies has shown impressive results with GBM and other tumor models. Several well-characterized oncogenic pathways have recently been shown to regulate CD47 expression in GBM cells and glioma stem cells (GSCs) including Epidermal Growth Factor Receptor (EGFR) beta catenin. Other macrophage pathways involved in regulating phagocytosis including TREM2 and glycan binding proteins are discussed as well. Finally, chimeric antigen receptor macrophages (CAR-Ms) could be leveraged for greatly enhancing the phagocytosis of GBM and repolarization of the microenvironment in general. Here, we comprehensively review the mechanisms that regulate the macrophage phagocytosis of GBM cells.

Signalling Pathways of Inflammation and Cancer in Human Mononuclear Cells: Effect of Nanoparticle Air Pollutants.

Fine inhalable particulate matter (PM) triggers an inflammatory response in the airways and activates mononuclear cells, mediators of tissue homeostasis, and tumour-promoting inflammation. We have assessed ex vivo responses of human monocytes and monocyte-derived macrophages to standardised air pollutants: carbon black, urban dust, and nanoparticulate carbon black, focusing on their pro-inflammatory and DNA-damaging properties. None of the PM (100 µg/mL/24 h) was significantly toxic to the cells, aside from inducing oxidative stress, fractional DNA damage, and inhibiting phagocytosis. TNFα was only slightly increased. PM nanoparticles increase the expression and activate DNA-damage-related histone H2A.X as well as pro-inflammatory NF-κB. We have shown that the urban dust stimulates the pathway of DNA damage/repair via the selective post-translational phosphorylation of H2A.X while nanoparticulate carbon black increases inflammation via activation of NF-κB. Moreover, the inflammatory response to lipopolysaccharide was significantly stronger in macrophages pre-exposed to urban dust or nanoparticulate carbon black. Our data show that airborne nanoparticles induce PM-specific, epigenetic alterations in the subsets of cultured mononuclear cells, which may be quantified using binary fluorescence scatterplots. Such changes intercede with inflammatory signalling and highlight important molecular and cell-specific epigenetic mechanisms of tumour-promoting inflammation.

The complex role of macrophages in pancreatic cancer tumor microenvironment: a review on cancer progression and potential therapeutic targets.

Pancreatic cancer (PC) is one of the deadliest cancers worldwide with low survival rates and poor outcomes. The treatment landscape for PC is fraught with obstacles, including drug resistance, lack of effective targeted therapies and the immunosuppressive tumor microenvironment (TME). The resistance of PC to existing immunotherapies highlights the need for innovative approaches, with the TME emerging as a promising therapeutic target. The recent advancements in understanding the role of macrophages, this context highlight their significant impact on tumor development and progression. There are two important types of macrophages: M1 and M2, which play critical roles in the TME. Therapeutics strategies including, depletion of tumor-associated macrophages (TAMs), reprogramming TAMs to promote anti-tumor activity, and targeting macrophage recruitment can lead to promising outcomes. Targeting macrophage-related pathways may offer novel strategies for modulating immune responses, inhibiting angiogenesis, and overcoming resistance to chemotherapy in PC treatment.

CD5L up-regulates the TGF-ß signaling pathway and promotes renal fibrosis.

Renal fibrosis is the common final pathway of progressive renal diseases, in which the macrophages play an important role. ELISA was used to detect CD5 antigen-like (CD5L) in serum samples from end-stage renal disease (ESRD), as well as in mice serum with unilateral ureteral occlusion (UUO). Recombinant CD5L was injected into UUO mice to assess renal injury, fibrosis, and macrophage infiltration. The expression of CD5L was significantly upregulated in the serum of patients with ESRD and UUO mice. Histological analysis showed that rCD5L-treated UUO mice had more severe renal injury and fibrosis. Furthermore, rCD5L promoted the phenotypic transfer of monocytes from Ly6Chigh to LyC6low. RCD5L promoted TGF-ß signaling pathway activation by promoting Smad2/3 phosphorylation. We used Co-IP to identify HSPA5 interact with CD5L on cell membrane could inhibit the formation of the Cripto/HSPA5 complex, and promote the activation of the TGF-ß signaling pathway. The CD5L antibody could reduce the degree of renal fibrosis in UUO mice.

Emodin in-situ delivery with Pluronic F-127 hydrogel for myocardial infarction treatment: Enhancing efficacy and reducing hepatotoxicity.

AIMS: This study evaluates the therapeutic potential of emodin in enhancing the anti-inflammatory phenotype of macrophages, proposing a novel treatment strategy for myocardial infarction (MI). Our objective is to overcome the challenge of myocardial repair post-MI by developing an innovative in-situ myocardial drug delivery system that reduces associated hepatotoxicity. MATERIALS AND METHODS: Through network pharmacology, it was identified that emodin primarily treats MI through anti-inflammatory actions. We investigated the influence of emodin on macrophage polarization using cellular assays and examined its therapeutic impacts and hepatotoxicity in animal models across various doses. A novel in-situ drug delivery system was devised using Pluronic F-127, a thermosensitive hydrogel, to enhance solubility and enable localized delivery to the myocardium. KEY FINDINGS: In vitro studies confirmed that emodin effectively induces macrophage polarization toward an anti-inflammatory phenotype. In vivo analyses demonstrated a dose-dependent therapeutic effect on the myocardium, although higher doses led to significant hepatotoxicity. The innovative drug delivery system increased emodin's solubility, facilitated precise myocardial targeting, and markedly reduced systemic exposure and liver toxicity. SIGNIFICANCE: This study introduces an advanced approach to treating MI by leveraging the natural anti-inflammatory properties of emodin combined with drug delivery technology. This strategy not only enhances the clinical feasibility of emodin for MI treatment but also represents a significant advancement in therapeutic methods. It focuses on increasing the drug concentration in the myocardium while minimizing the systemic side effects of the drug.

Human liver sinusoidal endothelial cells support the development of functional human pluripotent stem cell-derived Kupffer cells.

In mice, the first liver-resident macrophages, known as Kupffer cells (KCs), are thought to derive from yolk sac (YS) hematopoietic progenitors that are specified prior to the emergence of the hematopoietic stem cell (HSC). To investigate human KC development, we recapitulated YS-like hematopoiesis from human pluripotent stem cells (hPSCs) and transplanted derivative macrophage progenitors into NSG mice previously humanized with hPSC-liver sinusoidal endothelial cells (LSECs). We demonstrate that hPSC-LSECs facilitate stable hPSC-YS-macrophage engraftment for at least 7 weeks. Single-cell RNA sequencing (scRNA-seq) of engrafted YS-macrophages revealed a homogeneous MARCO-expressing KC gene signature and low expression of monocyte-like macrophage genes. In contrast, human cord blood (CB)-derived macrophage progenitors generated grafts that contain multiple hematopoietic lineages in addition to KCs. Functional analyses showed that the engrafted KCs actively perform phagocytosis and erythrophagocytosis in vivo. Taken together, these findings demonstrate that it is possible to generate human KCs from hPSC-derived, YS-like progenitors.

CD36 restricts lipid-associated macrophages accumulation in white adipose tissues during atherogenesis.

Visceral white adipose tissues (WAT) regulate systemic lipid metabolism and inflammation. Dysfunctional WAT drive chronic inflammation and facilitate atherosclerosis. Adipose tissue-associated macrophages (ATM) are the predominant immune cells in WAT, but their heterogeneity and phenotypes are poorly defined during atherogenesis. The scavenger receptor CD36 mediates ATM crosstalk with other adipose tissue cells, driving chronic inflammation. Here, we combined the single-cell RNA sequencing technique with cell metabolic and functional assays on major WAT ATM subpopulations using a diet-induced atherosclerosis mouse model (Apoe-null). We also examined the role of CD36 using Apoe/Cd36 double-null mice. Based on transcriptomics data and differential gene expression analysis, we identified a previously undefined group of ATM displaying low viability and high lipid metabolism and labeled them as "unhealthy macrophages". Their phenotypes suggest a subpopulation of ATM under lipid stress. We also identified lipid-associated macrophages (LAM), which were previously described in obesity. Interestingly, LAM increased 8.4-fold in Apoe/Cd36 double-null mice on an atherogenic diet, but not in Apoe-null mice. The increase in LAM was accompanied by more ATM lipid uptake, reduced adipocyte hypertrophy, and less inflammation. In conclusion, CD36 mediates a delicate balance between lipid metabolism and inflammation in visceral adipose tissues. Under atherogenic conditions, CD36 deficiency reduces inflammation and increases lipid metabolism in WAT by promoting LAM accumulation.

Inhibition of insulin-like growth factors increases production of CXCL9/10 by macrophages and fibroblasts and facilitates CD8+ cytotoxic T cell recruitment to pancreatic tumours.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with an urgent unmet clinical need for new therapies. Using a combination of in vitro assays and in vivo preclinical models we demonstrate that therapeutic inhibition of the IGF signalling axis promotes the accumulation of CD8+ cytotoxic T cells within the tumour microenvironment of PDAC tumours. Mechanistically, we show that IGF blockade promotes macrophage and fibroblast production of the chemokines CXCL9 and CXCL10 to facilitate CD8+ T cell recruitment and trafficking towards the PDAC tumour. Exploring this pathway further, we show that IGF inhibition leads to increased STAT1 transcriptional activity, correlating with a downregulation of the AKT/STAT3 signalling axis, in turn promoting Cxcl9 and Cxcl10 gene transcription. Using patient derived tumour explants, we also demonstrate that our findings translate into the human setting. PDAC tumours are frequently described as "immunologically cold", therefore bolstering CD8+ T cell recruitment to PDAC tumours through IGF inhibition may serve to improve the efficacy of immune checkpoint inhibitors which rely on the presence of CD8+ T cells in tumours.

Lipid-associated macrophages' promotion of fibrosis resolution during MASH regression requires TREM2.

While macrophage heterogeneity during metabolic dysfunction-associated steatohepatitis (MASH) has been described, the fate of these macrophages during MASH regression is poorly understood. Comparing macrophage heterogeneity during MASH progression vs regression, we identified specific macrophage subpopulations that are critical for MASH/fibrosis resolution. We elucidated the restorative pathways and gene signatures that define regression-associated macrophages and establish the importance of TREM2+ macrophages during MASH regression. Liver-resident Kupffer cells are lost during MASH and are replaced by four distinct monocyte-derived macrophage subpopulations. Trem2 is expressed in two macrophage subpopulations: i) monocyte-derived macrophages occupying the Kupffer cell niche (MoKC) and ii) lipid-associated macrophages (LAM). In regression livers, no new transcriptionally distinct macrophage subpopulation emerged. However, the relative macrophage composition changed during regression compared to MASH. While MoKC was the major macrophage subpopulation during MASH, they decreased during regression. LAM was the dominant macrophage subtype during MASH regression and maintained Trem2 expression. Both MoKC and LAM were enriched in disease-resolving pathways. Absence of TREM2 restricted the emergence of LAMs and formation of hepatic crown-like structures. TREM2+ macrophages are functionally important not only for restricting MASH-fibrosis progression but also for effective regression of inflammation and fibrosis. TREM2+ macrophages are superior collagen degraders. Lack of TREM2+ macrophages also prevented elimination of hepatic steatosis and inactivation of HSC during regression, indicating their significance in metabolic coordination with other cell types in the liver. TREM2 imparts this protective effect through multifactorial mechanisms, including improved phagocytosis, lipid handling, and collagen degradation.

Macrophages in vascular disease: Roles of mitochondria and metabolic mechanisms.

Macrophages are a dynamic cell type of the immune system implicated in the pathophysiology of vascular diseases and are a major contributor to pathological inflammation. Excessive macrophage accumulation, activation, and polarization is observed in aortic aneurysm (AA), atherosclerosis, and pulmonary arterial hypertension. In general, macrophages become activated and polarized to a pro-inflammatory phenotype, which dramatically changes cell behavior to become pro-inflammatory and infiltrative. These cell types become cumbersome and fail to be cleared by normal mechanisms such as autophagy. The result is a hyper-inflammatory environment causing the recruitment of adjacent cells and circulating immune cells to further augment the inflammatory response. In AA, this leads to excessive ECM degradation and chemokine secretion, ultimately causing macrophages to dominate the immune cell landscape in the aortic wall. In atherosclerosis, monocytes are recruited to the vascular wall, where they polarize to the pro-inflammatory phenotype and induce inflammatory pathway activation. This leads to the development of foam cells, which significantly contribute to neointima and necrotic core formation in atherosclerotic plaques. Pro-inflammatory macrophages, which affect other vascular diseases, present with fragmented mitochondria and corresponding metabolic dysfunction. Targeting macrophage mitochondrial dynamics has proved to be an exciting potential therapeutic approach to combat vascular disease. This review will summarize mitochondrial and metabolic mechanisms of macrophage activation, polarization, and accumulation in vascular diseases.

Prognostic and immunotherapeutic significances of M2 macrophage-related genes signature in lung cancer.

Objective: We aimed to investigate the immunological significance of M2 macrophage-related genes in lung cancer (LC) patients, specifically focusing on constructing a risk score to predict patient prognosis and response to immunotherapy. Methods: We developed a novel risk score by identifying and incorporating 12 M2 macrophage-related genes. The risk score was calculated by multiplying the expression levels of risk genes by their respective coefficients. Through comprehensive enrichment analysis, we explored the potential functions distinguishing high- and low-risk groups. Moreover, we examined the relationship between patients in different risk groups and immune infiltration as well as their response to immunotherapy. The single-cell RNA sequencing data were acquired to ascertain the spatial pattern of RNF130 expression. The expression of RNF130 was examined using TCGA datasets and verified by HPA. The qRT-PCR was employed to examine RNF130 expression in LC cells. Finally, in vitro experiments were carried out to validate the expression and function of RNF130. Results: Our results indicated that the risk score constructed from 12 M2 macrophage-related genes was an independent prognostic factor. Patients in the high-risk group had a significantly worse prognosis compared to those in the low-risk group. Functional enrichment analysis showed a significant relationship between the risk score and immunity. Furthermore, we explored immune infiltration in different risk groups using seven immune algorithms. The results demonstrated a negative correlation between high-risk group patients and immune infiltration of B cells, CD4+ cells, and CD8+ cells. We further validated these findings using an immunotherapy response database, which revealed that high-risk patients were more likely to exhibit immune evasion and might have poorer immunotherapy outcomes. Additionally, drug sensitivity analysis indicated that patients in the high-risk group were more sensitive to certain chemotherapeutic and targeted drugs than those in the low-risk group. Single-cell analysis indicated that macrophages were the primary site of RNF130 distribution. The results from the TCGA and HPA database demonstrated a trend toward a low expression of RNF130 in LC. Finally, in vitro experiments further validated the expression and function of RNF130 in LC cells. Conclusions: The high-risk group constructed with M2 macrophage-related genes in LC was closely associated with poor prognosis, low immune cell infiltration, and poorer response to immunotherapy. This risk score can help differentiate and predict the prognosis and immune status of LC patients, thereby aiding in the development of precise and personalized immunotherapy strategies.