M1 Infiltration Research Articles

Macrophage determines immnophenotype and predicts anti-PD-L1 response of urothelial cancer: Results from phase II clinical trial.

e15093 Background: Immune-checkpoint blockades (ICBs) are revolutionizing treatment of advanced or metastatic urothelial cancer (mUC) whereas the promising response merely occurred in a subset of patients. To dig into underlying determinants, we investigated the comprehensive tumor microenvironment (TME) of 348 tumor samples derived from a large phase II trial (IMvigor210: NCT#02108652). Methods: Based on the 348 pre-treatment samples, the LASSO-Bootstraping algorithm were applied to figure out the optimal predictive biomarker. Assessment of immune-cell proportions, pathway activity, and tumor purity used CIBERSORT algorithm, ssGSEA methodology, and ESTIMATE algorithm, respectively. Unsupervised clustering methods were applied to identify TME infiltrating patterns. Moreover, the predictive value of M1 infiltration in mUC were systematically explored, as well as its correlation to integrative molecular subtypes, tumor intrinsic mutation and metabolic pathways. An additional TCGA dataset was applied to validate the results. Results: Bootstrapping and ROC curve analysis indicated that M1 was non-inferior to TMB in predicting ICBs response (M1: AUC = 0.706; TMB: AUC = 0.728; Delong test: P = 0.333), but surpassed more-than-10,000 counterparts covering TME-infiltrating cells, metabolic pathways, tumor intrinsic pathways and hallmarks of cancer. Kaplan-Meier survival analysis (M1: P < 0.0001, Hazard Ratio = 0.25, 95% CI: 0.14 − 0.44) supported its promising prognostic value. Moreover, study into TME patterns demonstrated its role in determining immunophenotypes (AUC = 0.785). M1 frequency is positively correlated with CD8+ T cells fraction (P = 7e-04), whereas the Treg cell, neutrophil and monocyte infiltration which refer to suppressive immune microenvironment were negatively correlated (P = 0.0066, P = 7e-04, P = 2e-04, respectively). Furthermore, significant correlation was observed between M1 and tumor intrinsic characteristics, including TMB, neoantigen and PD-L1 level of tumor (P = 3.83e-5, P = 1.69e-7, and P = 0.0018, respectively). The TCGA data reproductively supported the majority of aforementioned results. Conclusions: Our study lend statistical power to the notion that Macrophage independently contribute to anti-tumor immune function in the context of ICB therapy. Nonetheless, these findings provide important insight into the role of M1-Macrophage, macrophage polarization and innate immune machinery in therapeutic responses to ICB. Clinical trial information: NCT02951767 .

Increased Vascular Adhesion Protein 1 (VAP-1) Levels are Associated with Alternative M2 Macrophage Activation and Poor Prognosis for Human Gliomas.

Glioma is characterized by a high heterogeneity in the brain tumor. Abundant tumor-associated macrophages (TAMs) exist as neoplastic tissues, implicating tumor plasticity and thus leading to therapeutic challenges. Vascular adhesion protein (VAP-1) potentially serves as a mediator for TAM immunity in tumor milieu. We previously demonstrated that VAP-1 could contribute to tumor malignancy, but its characteristics in TAM immunity of glioma progression are still unclear. This study explored the association of VAP-1 expression with TAM distribution as well as the resulting clinical significance and prognostic value in human gliomas. An in-depth analysis of AOC3 (VAP-1) gene expression was performed using 695 glioma samples derived from the cancer genome atlas (TCGA)-lower grade glioma and glioblastoma (GBMLGG) cohort. Bioinformatic analysis confirmed that VAP-1 expression is associated with poor prognosis of glioma patients (p = 0.0283). VAP-1 and TAM biomarkers (CD68, iNOS, and CD163) were evaluated by immunohistochemistry in 108 gliomas from Kaohsiung Medical University Hospital. VAP-1+ was expressed in 56 (51.85%) cases and this phenotype revealed a significant association with overall survival in Kaplan–Meier analysis (p < 0.0001). Immunohistochemical double staining showed that VAP-1 immunoreactivity was present around CD163+ M2 infiltration location, including aggressive lesions and neighboring neovasculature. We demonstrated that high VAP-1 expression levels positively correlated with CD163+ M2 activation and coexpression of these two proteins was associated with worse survival in gliomas (p < 0.0001). Multivariate analysis indicated that VAP-1 alone and co-expressed with CD163 were the significantly independent indicators (both p < 0.0001). Furthermore, VAP-1/CD163 coexpression exhibited excellent diagnostic accuracy in gliomas (AUC = 0.8008). In conclusion, VAP-1 and TAM CD163 M2 coexpression was found in glioma tissues belonging to a highly malignant subgroup that was associated with poor prognosis. These results implied VAP-1 abundance is closely linked to alternative M2 activation during glioma progression. From the aforementioned data, a reasonable inference is that VAP-1 combined with targeting M2 immunity might be an effective therapeutic target for human gliomas.

Heat stress ameliorates tenotomy-induced inflammation in muscle-specific response via regulation of macrophage subtypes.

During disuse-induced muscle atrophy, macrophages play a significant role in inflammatory responses that occur with muscle degeneration and repair. Heat treatment has been shown to alleviate muscle atrophy; however, the effect of heat on inflammatory responses following tenotomy has not been evaluated. This study examined the effects of heat stress on proinflammatory (M1-like) and anti-inflammatory (M2-like) macrophage populations. Also, cytokine protein expression in oxidative soleus and glycolytic plantaris muscles following Achilles tendon transection (tenotomy) was analyzed. Male Wistar rats were assigned into control, control plus heat stress, tenotomy, and tenotomy plus heat stress groups. Tenotomy was performed for 8 (TEN8) and 14 (TEN14) days to induce muscle inflammation. Heat treatments, 30 min at 40.5-41.5°C, were given 24 h before and 1-6 consecutive days after tenotomy (TEN8 group) or every other day (TEN14 group). Tenotomy induced muscle necrosis, extensive infiltration of M1- (CD68+), and M2- (CD163+) like macrophages and increased tumor necrosis factor-α (TNFα) but not interleukin-10 (IL-10) protein expression. Heat stress caused a reduction in necrotic fibers, M1-like macrophage invasion, and TNFα protein expression in tenotomized soleus muscle. Additionally, heat stress enhanced M2-like macrophage accumulation during the 14 days following tenotomy in soleus muscle but did not affect IL-10 protein level. Our results indicate that heat stress can limit tenotomy-induced inflammatory responses through modulation of macrophage subtypes and TNFα protein expression, preferentially in oxidative muscle. These findings shed light on the ability of heat stress as a therapeutic strategy to manipulate macrophages for optimal inflammation during muscle atrophy.NEW & NOTEWORTHY We investigated differential effects of heat stress on modulating inflammation following 8 and 14 days of tenotomy in soleus and plantaris muscles. Heat exposure could reduce necrosis, suppress pro-inflammatory macrophage infiltration, and diminish TNFα protein expression in tenotomized muscle, which preferentially occurred in soleus muscle. Additionally, heat stress enhanced anti-inflammatory macrophages in soleus muscle in the 14-day study period. Neither tenotomy nor heat stress had an impact on IL-10 protein expression in either muscle examined.

Tumor associated macrophages and its nanovesicles: Immunological insights in cerebral glioma

Purpose: The immune system has a key role in glioma progression, especially the tumor associated macrophages (TAMs). In-vivo, we aimed to study the total TAMs and differential M1 and M2 TAM infiltration in low grade (LGG) versus high grade gliomas (HGG). Also, we investigated the implication of total TAMs and differential M1 and M2 TAMs infiltration on glioma progression. In-vitro, we studied the effect of soluble factors present in nanovesicles (NV) released from M1 TAMs on the fate of glioma cells.Methods: In-vivo, we performed immunohistochemistry using iNOS and CD163 (markers for M1 and M2 respectively). In-vitro, we polarized the human monocytes U937 cell line into M1, we isolated the NV from the M1-conditioned medium (CM) by centrifugation and filtration; then, the protein content of the NV was quantified by the protein assay. We added M1-NV on U251 glioma cells and we studied the cellular activation of glioma cells using the MTT assay. To assess the apoptosis of U251, we used the flow-cytometry. Apoptotic cells were identified by annexin V and Propidium Iodide (markers for early and late apoptosis respectively).Results: in-vivo, there is an M1/M2 imbalance in early stages of glioma which is associated with earlier progression to high malignancy. Also, the higher M2 infiltration, the earlier is the progression. In-vitro, M1-NV had a more potent anti-tumor effect compared to its corresponding CM. We assume that our experimental results can be a future treatment for the cerebral glioma.

Macrophage correlates with immunophenotype and predicts anti-PD-L1 response of urothelial cancer.

Immune-checkpoint blockades (ICBs) have been routinely implemented to treat metastatic urothelial cancer (mUC), whereas robust biomarkers are urgently warranted. Herein, we explored latent promising biomarkers based on 348 pretreatment mUC samples from IMvigor210.Methods: The genome, transcriptome, immunome, and metabolome were systemically analyzed using the external TCGA dataset for validation. Kaplan-Meier and ROC curve analyses were performed to estimate the predictive capacity of M1-macrophage infiltration. Chi-square/Spearman/Mann Whitney U test are used to determine its correlation to genetic, biochemical, and clinicopathological parameters.Results: M1 frequency is a robust biomarker for predicting the prognosis and response to ICBs, which is non-inferior to tumor mutation burden (TMB) or tumor neoantigen burden (TNB), and exceeds CD8 T cells, T cell inflamed gene expression profile (GEP), and PD-L1 expression. Moreover, M1 infiltration is associated with immune phenotypes (AUC = 0.785) and is negatively correlated with immune exclusion. Additionally, transcriptomic analysis showed immune activation in the high-M1 subgroup, whereas it showed steroid and drug metabolism reprograming in the M1-deficient subset, which characterized the limited sensitivity to ICB therapy. Notably, investigation of the corresponding intrinsic genomic profiles highlighted the significance of TP53 and FGFR alterations.Conclusions: M1 infiltration is a robust biomarker for immunotherapeutic response and immunophenotype determination in an mUC setting. Innate immunity activation involving macrophage polarization remodeling and anti-FGFR mutations may be promising strategies for synergy with anti-PD-L1 treatments and may help prolong the clinical survival of patients with mUC.

Gentiopicroside (GENT) protects against sepsis induced by lipopolysaccharide (LPS) through the NF-κB signaling pathway.

Sepsis is a high-mortality disease without effective therapeutic options. The hyperactivation of the monocyte-macrophage system, especially M1 macrophages, triggers the onset of septic shock. Gentiopicroside (GENT), the main active component in the traditional Chinese medicinal herb Radix Gentianae, has been shown to have anti-inflammatory properties. Nevertheless, this anti-inflammatory effect has not been fully elucidated. In vitro, we stimulated primary bone marrow-derived macrophages (BMMs) or peritoneal elucidated macrophages (PEMs) by lipopolysaccharide (LPS) and interferon (IFN)-γ and pre-treated with GENT and we tested the cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF) α production by enzyme linked immunosorbent assay (ELISA) or real-time quantitative PCR (qPCR). Further, we determined the NF-κB-mediated inflammatory pathway such as IKKα/β and p65 phosphorylation by Western blot. Then we detected the p65 nuclear localization by immunofluorescent staining. Moreover, NF-κB inhibitor and p65-targeted siRNAs were further used to validate the anti-inflammatory mechanism of GENT. In vivo, GENT (50 mg/kg) was administered intragastrically before and after LPS (40 mg/kg) injection. The death time were recorded and the serum levels of IL-1β, IL-6 and TNFα were tested by ELISA, and the IL-1β, IL-6 and TNFα mRNA expression in the lung were test by qPCR and the M1 infiltration in the lung were determined by F4/80 and INOS immunofluorescent staining. In vitro, we observed that GENT reduced the inflammatory cytokine production of BMMs stimulated by (LPS)/IFN-γ and ameliorated the phosphorylation of IKKα/β and p65, the degradation of IκBα, and the translocation of p65 into the nucleus. We did not find GENT has any effect on MAPK signaling under LPS/IFN-γ stimulation. NF-κB inhibitor and p65 siRNAs eliminated the inhibition effect of GENT. In vivo, we observed GENT prevented mice from dying in the LPS-induced shock model and decreased the serum levels of IL-1β and IL-6, the mRNA expression of IL-1β, IL-6 and TNFα in lung tissue, and the amount of M1 macrophage infiltration in the lung. GENT prevented LPS/IFN-γ-induced inflammatory cytokine production by macrophages through the NF-κB signaling pathway in vitro and protected against the endotoxin shock induced by LPS in vivo.

Paclitaxel-loaded biodegradable ROS-sensitive nanoparticles for cancer therapy.

BackgroundReactive oxygen species (ROS), such as hydrogen peroxide and superoxide, trigger biodegradation of polymer-based nanoparticles (NPs) bearing pinacol-type boronic ester groups. These NPs may selectively release their cargo, in this case paclitaxel (PTX), at the high levels of ROS present in the intracellular environment of inflamed tissues and most tumors.PurposeThe main objective was to determine anti-tumor efficacy of PTX-loaded ROS-sensitive NPs and to examine whether macrophage infiltration had any impact on treatment efficacy.MethodsNPs were synthesized and their characteristics in the presence of H2O2 were demonstrated. Both confocal microscopy as well as flow cytometry approaches were used to determine degradation of ROS-sensitive NPs. HeLa cells were cultured in vitro and used to establish tumor xenografts in nude mice. In vivo experiments were performed to understand toxicity, biodistribution and anti-tumor efficacy of the NPs. Moreover, we performed immunohistochemistry on tumor sections to study infiltration of M1 and M2 subsets of macrophages.ResultsWe demonstrated that PTX delivered in NPs containing a ROS-sensitive polymer exhibits a better anti-tumor efficacy than PTX in NPs containing ROS-non-sensitive polymer, free PTX or Abraxane® (nab-PTX). The biodistribution revealed that ROS-sensitive NPs exhibit retention in liver, spleen and lungs, suggesting a potential to target cancer metastasizing to these organs. Finally, we demonstrated a correlation between infiltrated macrophage subsets and treatment efficacy, possibly contributing to the efficient anti-tumor effects.ConclusionTreatment with ROS-sensitive NPs containing PTX gave an improved therapeutic effect in HeLa xenografts than their counterpart, free PTX or nab-PTX. Our data revealed a correlation between macrophage infiltration and efficiency of the different antitumor treatments, as the most effective NPs resulted in the highest infiltration of the anti-tumorigenic M1 macrophages.

SAT-149 BIOMARKERS OF MACROPHAGE POLARIZATION ASSOCIATE ETIOLOGIC CAUSES AND ACTIVITY OF INTRINSIC ACUTE KIDNEY INJURIES

Macrophage polarization is a major contributing factor in acute kidney injury (AKI). We aim to check the value of urine sCD163 and urine sediment macrophage subtypes in differentiating etiologic causes of various intrinsic renal AKI in a large cohort. A total of 205 patients with renal intrinsic acute kidney injuries verified by renal biopsy were enrolled. Uninary sCD163 was quantified using enzyme-linked immunosorbent assay, macrophage subtypes in urine sediments and in renal biopsy samples were determined by immunostaining. Compared to healthy controls (0 pg/ml) and AKI due to interstitial or tubular injuries (median 55.1 pg/ml), the level of urinary sCD163 was markedly higher in glomerulopathy, especially in diffuse proliferative glomerulonephritis (median 285.8 pg/ml, P<0.001) and significantly correlated with the proportion of cellular crescents formation (P=0.043). Urine sediment analysis of macrophage M1/M2 ratio could differentiate acute tubulointerstitial nephritis (M1/M2>2.35, with sensitivity of 98% and specificity of 100%) from crescentic glomerulonephritis (M1/M2<0.27, with sensitivity of 100% and specificity of 69%). Urinary sCD163 levels and urine M2 subtype positively correlated with infiltrated M2 macrophage in glomeruli, whereas urine M1 subtype positively correlated with infiltrated M1 macrophage in interstitium.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Markers of macrophage polarization associate etiologic causes and activity of intrinsic AKI, which could be viewed as the noninvasive “liquid biopsy” in the presence of various intrinsic kidney diseases.

Urine macrophages reflect kidney macrophage content during acute tubular interstitial and glomerular injury

Macrophage polarization is a major contributing factor in acute kidney injury (AKI). We aim to determine its biomarker value in differentiating etiologic causes of various intrinsic renal AKI. A total of 205 patients with renal intrinsic AKI were enrolled. Urinary sCD163 was quantified and macrophage subtypes in urine and in renal biopsy were determined. Compared to healthy controls and AKI due to interstitial or tubular injuries (0 pg/μmol), urinary sCD163 was markedly higher in glomerulopathy, especially in diffuse proliferative glomerulonephritis (275.5 pg/μmol) and significantly correlated with cellular crescent formation. Urine sediment analysis of M1/M2 ratio could differentiate acute tubulointerstitial nephritis (M1/M2 > 2.35) from crescentic glomerulonephritis (M1/M2 < 0.27). Urinary sCD163 levels and M2 subtype positively correlated with infiltrated M2 in the glomeruli, whereas urine M1 positively correlated with infiltrated M1 in the interstitium. Of note, urinary sCD163 showed better diagnositic performance in differentiating disease etiologies compared to tradiational urinary biomarkers of AKI (NGAL and KIM-1) and markers of myeloid cells (CD11b) and pan macrophages (CD68). Thus markers of macrophage polarization could be viewed as the noninvasive “liquid biopsy” in the presence of various intrinsic kidney diseases.

566-P: Mild Increase of Proinflammatory Macrophages Correlates with Reduction of Nerve Fiber Density in Sural Nerve of Human Type 2 Diabetic Subjects

The dominant infiltration of pro-inflammatory M1 macrophages (M1) compared to anti-inflammatory M2 macrophages (M2) exerts insulin resistance in peripheral tissues of type 2 diabetes (T2DM). This inflammatory reaction is also associated with the pathogenesis and development of diabetic complications including neuropathy (DPN). It is still unclarified, however, regarding macrophage infiltration in human DPN. The aim of this study is to clarify the significance of macrophage infiltration in sural nerve (SN) in subjects with T2DM. SN of autopsy cases was dissected from 2014 to 2016. Eleven cases (Male 6, Female 5) of non-T2DM (nDM) and 11 cases (Male 3, Female 8) of T2DM (T2D) were subjected to the evaluation. Double immunohistochemistry was performed in cross section of SN for CD68 (Pan-macrophage) and CD206 (M2). M1 was defined as CD68+ and CD206- and M2 was as CD206+, respectively. Epon-embedded semithin section was used for morphometrical assessment of SN fibers. In frozen SN, mRNA expression was evaluated by qPCR. Average age and BMI were comparable between 2 groups. HbA1c was significantly greater in T2D (mean±SE) (7.2±0.5%) than nDM (5.5±0.2%) (p&amp;lt;0.05). Pathological evaluation showed 2.4 times M1 in T2D more than that in nDM (p&amp;lt;0.05). On the other hand, M2 was comparable between 2 groups. Expression of iNOS for M1 in T2D was 1.8 times increased compared to that of nDM (p&amp;lt;0.05), whilst, that of CD163 for M2 was comparable between two groups. Correlation analysis between macrophage infiltration and clinical parameters disclosed that the M1 infiltration proportionally correlated with the value of HbA1c (p&amp;lt;0.05), whereas there was no correlation between M2 infiltration and HbA1c. SN fiber density inversely correlated with M1 population (p&amp;lt;0.05). Collectively, mild, but significant pro-inflammatory condition is associated with SN fiber loss, suggesting the pathogenic role of M1 in DPN. Disclosure H. Mizukami: None. S. Osonoi: None. K. Takahashi: None. K. Kudo: None. C. Itabashi: None. S. Yagihashi: None.

Macrophage phenotypes and Gas6/Axl signaling in apical lesions

Background/purposeMacrophages participate in the periapical inflammation with pro-inflammatory M1 cells and anti-inflammatory M2 cells. Gas6/Axl signal is the responsible pathway for the activation of M1 and polarization of M2. The aim of this study was to compare the number of CD16+ M1 cells, CD206+ M2 cells, and Gas6/Axl expression between apical granulomas and radicular cysts. Materials and methodsTwenty-four cases of granuloma and twenty of cysts were submitted to immunohistochemistry using anti-CD16 and anti-CD206 antibodies for determining M1 and M2 macrophages and investigating the cells with positive Gas6 and Axl expression. ResultsThere were more numerous of M1 macrophages in radicular cysts (175.9 ± 87.7) compared to apical granuloma (116.6 ± 55.8), and M2 macrophages was higher in cysts (204.0 ± 97.6) than granuloma (152.9 ± 64.6). The level of Gas6/Axl expression were similar. There was a significant different in M1 macrophage (P = 0.014) between two diagnosis. In patients with or without root resorption, the number of M1 were 194.6 ± 57.2 compared with 139.1 ± 79.6. The number of M2 were 241.7 ± 81.4 and 164.6 ± 77.1. The expression of Axl was stronger in root resorption patients (191.1 ± 43.6), but the tendency in Gas6 expression was similar. Significant differences were noted in high M2 infiltration and Axl positive lesions. ConclusionIt appears that macrophages associated with significantly higher numbers in radicular cysts than apical granuloma. Meanwhile, macrophages and Axl receptor was intensively expressed in patients with root resorption, related to severe inflammation.

Tubulointerstitial Infiltration of M2 Macrophages in Henoch-Schönlein Purpura Nephritis Indicates the Presence of Glomerular Crescents and Bad Clinical Parameters.

Henoch-Schönlein purpura (HSP) is the most common systemic vasculitis in children, and renal involvement (HSP nephritis, HSPN) is a severe manifestation. HSPN is histologically classified by the International Study of Kidney Disease in Children (ISKDC) based on mesangial hypercellularity and the extent of glomerular crescents. Macrophages, categorized as M1 or M2, frequently infiltrate in various glomerular and tubulointerstitial diseases and infiltration of specific subtypes is associated with disease progression. Therefore, to identify whether infiltration of M1 or M2 macrophages has clinical significance, we quantified the subtypes of macrophages in 49 HSPN specimens and correlated the counts with histologic features and clinical parameters. Higher tubulointerstitial M2 counts were associated with chronic renal failure (CRF), ISKDC classes III-IV, and crescents (P<0.001, 0.002, 0.001). Glomerular M2 counts were significantly related to ISKDC classes III-IV and crescents (area under curve, AUC 0.804, 0.833). Tubulointerstitial M2 counts were associated with CRF, ISKDC classes III-IV, and crescents (AUC 0.872, 0.778, 0.830). Tubulointerstitial M2 counts also revealed higher AUC than tubulointerstitial M1 counts for CRF (P=0.036) and ISKDC classes III-IV (P=0.047). Glomerular M2 counts revealed higher AUC than glomerular M1 counts for ISKDC classes III–IV (P=0.024). Tubulointerstitial M2 counts were the most powerful parameter for CRF (AUC 0.872) and revealed even higher AUC than ISKDC classification (AUC 0.716) with borderline significance (P=0.086) for CRF. In summary, tubulointerstitial M2 counts were a superior parameter to tubulointerstitial M1 counts and even to ISKDC classification indicating the presence of CRF.

Periostin Links Skin Inflammation to Melanoma Progression in Humans and Mice.

It is widely accepted that chronic inflammation initiates and promotes carcinogenesis and tumor progression in various cell types. However, this paradigm has not been comprehensively investigated in melanoma. To investigate the effects of chronic inflammation on the progression of melanoma, we established a murine inflammatory skin model and investigated the relationship between skin inflammation and melanoma progression. In a murine model, B16F10 melanoma cells in inflamed skin grew significantly more rapidly than cells in control skin. The stromal expression of periostin was upregulated in inflamed skin, and significantly more CD163+ M2 macrophages were recruited to the melanomas in inflamed skin. We then immunohistologically examined the expression of stromal periostin and the infiltration of CD163+ M2 macrophages in human acral lentiginous melanomas (n = 94) and analyzed the statistical associations with clinicopathological variables. In human melanomas, high periostin expression and a large number of infiltrated M2 macrophages were significantly correlated with poor prognosis. Furthermore, we confirmed that periostin promotes the proliferation of murine and human melanoma cells in vitro. Our findings indicate that periostin and M2 macrophages play a critical role in melanoma progression and prognosis in both humans and mice, indicating that periostin is a potential target for treating progressive melanoma.

Infiltrated M2 tumour-associated macrophages in the stroma promote metastasis and poor survival in oesophageal squamous cell carcinoma.

Although M2 tumour-associated macrophages (M2 TAMs) have been shown to be associated with the progression and metastasis of breast cancer, their role in oesophageal squamous cell carcinoma (ESCC) remains less well understood. Therefore, to understand the clinicopathological significance of infiltrated M2 TAMs in ESCC, statistical analysis was performed after immunohistochemical evaluation of CD163 expression, a well-accepted surface marker of M2 TAMs in ESCC. To gain insight into the effect of M2 TAMs, ESCC cell lines Eca109 and KYSE150 cells were co-cultured with M2 TAMs artificially induced from THP-1 cells. The variations in the proliferation, migration and invasion were assessed using the MTT, wound-healing and Transwell assays, respectively. The variation in the typical biomarkers of the epithelial-mesenchymal transition (EMT) was evaluated using western blotting. Infiltrated M2 TAMs were confirmed to predominate in the stroma of ESCC relative to normal controls. Moreover, it turned out that M2 TAMs were shown to promote the migration and invasion of ESCC cells but not proliferation. Furthermore, M2 TAMs were observed to induce EMT in ESCC cells. Together, our results showed that infiltrated M2 TAMs in the stroma is a feature accompanying ESCC metastasis and that M2 TAMs can promote the migration and invasion, but not proliferation, of ESCC cells, thereby inducing EMT. Thus, M2 TAMs could be an alternative therapeutic target in ESCC.

Molecular Subtypes of Urothelial Bladder Cancer: Results from a Meta-cohort Analysis of 2411 Tumors

BackgroundPrevious molecular subtyping for bladder carcinoma (BLCA) involved <450 samples, with diverse classifications. ObjectiveTo identify molecular subtypes by curating a large BLCA dataset. Design, setting, and participantsGene expression publicly available were combined and reanalyzed. The dataset contained 2411 unique tumors encompassing non-muscle-invasive (NMIBC) and muscle-invasive BLCA (MIBC). Subtypes were reproduced on The Cancer Genome Atlas, UROMOL, and IMvigor210. InterventionSubtypes were assigned by gene expression. Outcome measurements and statistical analysisKaplan-Meier analyses were performed for subtype-clinical outcome correlations; Chi-square/Fisher exact tests were used for subtype-clinicopathological parameters associations. Results and limitationsWe identified six molecular subtypes with different overall survival (OS) and molecular features. Subtype Neural-like (median OS, 87 mo) is prevalent in MIBC and characterized by high WNT/β-catenin signaling. HER2-like (107.7 mo) is distributed evenly across NMIBC and MIBC, with higher ERBB2 amplification and signaling. Papillary-like (>135 mo), an NMIBC subtype enriched in urothelial differentiation genes, shows a high frequency of actionable FGFR3 mutations, amplifications, and FGFR3-TACC3 fusion. Luminal-like (91.7 mo), predominantly NMIBC, has higher MAPK signaling and more KRAS and KMT2C/D mutations than other subtypes. Mesenchymal-like (MES; 86.6 mo) and Squamous-cell carcinoma-like (SCC; 20.6 mo) are predominant in MIBC. MES is high in AXL signaling, whereas SCC has elevated PD1, CTLA4 signaling, and macrophage M2 infiltration. About 20% of NMIBCs show MIBC subtype traits and a lower 5-yr OS rate than Papillary-like NMIBC (81% vs 96%). The main limitations of our study are the incomplete clinical annotation, and the analyses were based on transcriptome subset due to comparisons across gene expression quantification technologies. ConclusionsBLCA can be stratified into six molecular subtypes. NMIBC, with a high risk of progression, displays the molecular features of MIBC. Patient summaryBiomarkers are urgently needed to guide patient treatment selection and avoid unnecessary toxicities in those who fail to respond. We believe molecular subtyping is a promising way to tailor disease management for those who will benefit most.

Treatment with TNF-α inhibitor rectifies M1 macrophage polarization from blood CD14+ monocytes in patients with psoriasis independent of STAT1 and IRF-1 activation

BackgroundPsoriasis is a systemic inflammatory disease with dramatic responses to TNF-α inhibitors. TNF-α is mainly produced by macrophages. However, how macrophage polarization contributes to psoriasis remains unknown. ObjectiveWe aimed to investigate the molecular mechanisms of macrophage polarization in psoriasis. Methods8 patients with moderate to severe psoriasis (Male/Female: 4/4, average age: 47.9 years old) and 8 healthy controls (Male/Female: 4/4, average age: 49.3 years old) were recruited. Their peripheral CD14+ monocytes were isolated with magnetic beads and then were differentiated into macrophages. The differential macrophage polarization was compared among normal controls, psoriatic patients before and after TNF-α inhibitors. The U937 cells were used to investigate the mechanisms by which TNF-α altered the macrophage polarization. ResultsThe ratio of M1 to M2a macrophage polarization was higher in psoriatic patients comparing with that in controls. The decreasing M1/M2a ratio was parallel to decreasing PASI severity score after adalimumab treatment. Consistently, TNF-α blockage decreased M1/M2a ratio in U937 cells. The induction of STAT1 and IRF-1 in polarized U937 M1 cells was inhibited by TNF-α inhibitor. However, STAT1 and/or IRF-1 interference could not resume M1 polarization. In skin, the increased M1 and M2 infiltration in lesions returned to baseline after successful treatment with TNF-α inhibitor. ConclusionsIncreased M1 polarization is associated with higher disease severity in psoriasis, resuming to baseline after successful treatment by TNF-α inhibitors. TNF-α blockage inhibits M1 polarization through STAT1- and IRF-1-independent pathways. Macrophage polarization may contribute to disease progression in psoriasis.

Distribution of M1 and M2 macrophages in tumor islets and stroma in relation to prognosis of non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) remains the most common cause of cancer related death worldwide. Tumor-infiltrating macrophages are believed to play an important role in growth, progression, and metastasis of tumors. In NSCLC, the role of macrophages remains controversial; therefore, we aimed to evaluate the distribution of macrophages (M1 and M2) in tumor islets and stroma and to analyze their relations to patients’ survival.MethodsLung tissue specimens from 80 NSCLC patients who underwent surgical resection for NSCLC (pathological stage I-III) and 16 control group subjects who underwent surgery because of recurrent spontaneous pneumothorax were analyzed. Immunohistochemical double staining of CD68/iNOS (markers for M1 macrophages) and CD68/CD163 (markers for M2 macrophages) was performed and evaluated in a blinded manner. The numbers of M1 and M2 macrophages in tumor islets and stroma were counted manually.ResultsPredominant infiltration of M1 and M2 macrophages was observed in the tumor stroma compared with the tumor islets. M2 macrophages predominated over M1 macrophages in the tumor tissue. Tumor islets-infiltrating M1 macrophages and the number of total tumor-infiltrating M2 macrophages were independent predictors of patients survival: high infiltration of M1 macrophages in tumor islets was associated with increased overall survival in NSCLC (P < 0.05); high infiltration of total M2 macrophages in tumor (islets and stroma) was associated with reduced overall survival in NSCLC (P < 0.05).ConclusionsThis study demonstrated that high infiltration of M1 macrophages in the tumor islets and low infiltration of total tumor-infiltrating M2 macrophages were associated with improved NSCLC patients’ survival.Trial registrationClinicalTrials.gov NCT01955343, registered on September 27, 2013

Infiltration of M1, but not M2, macrophages is impaired after unilateral ureter obstruction in Nrf2-deficient mice

Chronic inflammation can be a major driver of the failure of a variety of organs, including chronic kidney disease (CKD). The NLR family pyrin domain-containing 3 (NLRP3) inflammasome has been shown to play a pivotal role in inflammation in a mouse kidney disease model. Nuclear factor erythroid 2-related factor 2 (Nrf2), the master transcription factor for anti-oxidant responses, has also been implicated in inflammasome activation under physiological conditions. However, the mechanism underlying inflammasome activation in CKD remains elusive. Here, we show that the loss of Nrf2 suppresses fibrosis and inflammation in a unilateral ureter obstruction (UUO) model of CKD in mice. We consistently observed decreased expression of inflammation-related genes NLRP3 and IL-1β in Nrf2-deficient kidneys after UUO. Increased infiltration of M1, but not M2, macrophages appears to mediate the suppression of UUO-induced CKD symptoms. Furthermore, we found that activation of the NLRP3 inflammasome is attenuated in Nrf2-deficient bone marrow–derived macrophages. These results demonstrate that Nrf2-related inflammasome activation can promote CKD symptoms via infiltration of M1 macrophages. Thus, we have identified the Nrf2 pathway as a promising therapeutic target for CKD.

M1 Macrophage–Induced Endothelial-to-Mesenchymal Transition Promotes Infantile Hemangioma Regression

Infantile hemangiomas are benign tumors of vascular endothelial cells (ECs), characterized by three distinct stages: proliferating phase, involuting phase, and involuted phase. The mechanisms that trigger involution of hemangioma into fibro-fatty tissue remain unknown. We report a novel mechanism by which M1-polarized macrophages induce endothelial-to-mesenchymal transition (EndMT) and promote hemangioma regression. M1- but not M2-polarized macrophages induced EndMT in ECs. Tumor necrosis factor-α and, to a lesser extent, IL-1β and interferon-γ were the most potent cytokines produced by the M1 macrophages that induce invitro EndMT. Western blot analysis and gene expression profiling showed that ECs treated with M1 macrophages, tumor necrosis factor-α, or IL-1β decreased the expression of endothelial markers, whereas mesenchymal markers increased concomitantly. Immunohistochemical staining of patient samples revealed that a significant perivascular infiltration of M1, but not M2, macrophages coincides with endothelial expression of the critical EndMT transcription factors Snail/Slug in involuting hemangiomas. Most strikingly, M1 macrophage-treated ECs isolated from patient hemangiomas (HemECs) but not untreated HemECs readily differentiated into adipocytes on adipogenic induction. Thus, invitro EndMT and adipogenesis of HemECs have, in part, recapitulated the natural history of hemangioma regression. In conclusion, our findings indicate that EndMT induced by M1 macrophages promotes infantile hemangioma regression and may lead to novel therapeutic treatments for this vascular tumor.

Regulatory T cells and skeletal muscle regeneration.

Skeletal muscle regeneration results from the activation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Inflammatory and immune cells have a crucial role in the regeneration process. Acute muscle injury causes an immediate transient wave of neutrophils followed by a more persistent infiltration of M1 (proinflammatory) and M2 (anti-inflammatory/proregenerative) macrophages. New studies show that injured muscle is also infiltrated by a specialized population of regulatory T (Treg) cells, which control both the inflammatory response, by promoting the M1-to-M2 switch, and the activation of satellite cells. Treg cells accumulate in injured muscle in response to specific cytokines, such as IL-33, and promote muscle growth by releasing growth factors, such as amphiregulin. Muscle repair during aging is impaired due to reduced number of Treg cells and can be enhanced by IL-33 supplementation. Migration of Treg cells could also contribute to explain the effect of heterochronic parabiosis, whereby muscle regeneration of aged mice can be improved by a parabiotically linked young partners. In mdx dystrophin-deficient mice, a model of human Duchenne muscular dystrophy, muscle injury, and inflammation is mitigated by expansion of the Treg-cell population but exacerbated by Treg-cell depletion. These findings support the notion that immunological mechanisms are not only essential in the response to pathogenic microbes and tumor cells but also have a wider homeostatic role in tissue repair, and open new perspectives for boosting muscle growth in chronic muscle disease and during aging.