M2-like Phenotype Research Articles

The Mac Is Back: The Role of Macrophages in Human Healthy and Complicated Pregnancies.

Pregnancy is a fascinating immunological paradox: the semi-allogeneic fetus generally grows without any complications. In the placenta, fetal trophoblast cells come into contact with maternal immune cells. Inaccurate or inadequate adaptations of the maternal immune system could lead to problems with the functioning of the placenta. Macrophages are important for tissue homeostasis, cleanup, and the repair of damaged tissues. This is crucial for a rapidly developing organ such as the placenta. The consensus on macrophages at the maternal-fetal interface in pregnancy is that a major proportion have an anti-inflammatory, M2-like phenotype, that expresses scavenger receptors and is involved in tissue remodeling and the dampening of the immune reactions. Recent multidimensional analyses have contributed to a more detailed outlook on macrophages. The new view is that this lineage represents a highly diverse phenotype and is more prevalent than previously thought. Spatial-temporal in situ analyses during gestation have identified unique interactions of macrophages both with trophoblasts and with T cells at different trimesters of pregnancy. Here, we elaborate on the role of macrophages during early human pregnancy and at later gestation. Their possible effect is reviewed in the context of HLA incompatibility between mother and fetus, first in naturally conceived pregnancies, but foremost in pregnancies after oocyte donation. The potential functional consequences of macrophages for pregnancy-related immune reactions and the outcome in patients with recurrent pregnancy loss are also discussed.

Enhanced insulin-regulated phagocytic activities support extreme health span and longevity in multiple populations.

The immune system plays a central role in many processes of age-related disorders and it remains unclear if the innate immune system may play roles in shaping extreme longevity. By an integrated analysis with multiple bulk and single cell transcriptomic, so as DNA methylomic datasets of white blood cells, a previously unappreciated yet commonly activated status of the innate monocyte phagocytic activities is identified. Detailed analyses revealed that the life cycle of these monocytes is enhanced and primed to a M2-like macrophage phenotype. Functional characterization unexpectedly revealed an insulin-driven immunometabolic network which supports multiple aspects of phagocytosis. Such reprogramming is associated to a skewed trend of DNA demethylation at the promoter regions of multiple phagocytic genes, so as a direct transcriptional effect induced by nuclear-localized insulin receptor. Together, these highlighted that preservation of insulin sensitivity is a key to healthy lifespan and extended longevity, via boosting the function of innate immune system in advanced ages.

Blockade of the Immune Checkpoint CD47 by TTI-621 Potentiates the Response to Anti−PD-L1 in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is an incurable and cosmetically disfiguring disease associated with microenvironmental signals. We investigated the effects of CD47 and PD-L1 immune checkpoint blockades, as a strategy for targeting both innate and adaptive immunity. CIBERSORT analysis identified the immune-cell composition in the CTCL tumor microenvironment and the immune checkpoint expression profile for each immune-cell gene cluster from CTCL lesions. We investigated the relationship between MYC and CD47 and PD-L1 expression and found that MYC short hairpin RNA knockdown and MYC functional suppression by TTI-621 (SIRPαFc) and anti-PD-L1 (durvalumab) in CTCL cell lines reduced the expression of CD47 and PDL1 mRNA and protein as measured by qPCR and flow cytometry, respectively. Invitro, blockade of the CD47-SIRPα interaction with TTI-621 increased the phagocytic activity of macrophages against CTCL cells and enhanced CD8+ T-cell-mediated killing in a mixed leucocyte reaction. Moreover, TTI-621 synergized with anti-PD-L1 in macrophages reprogram to M1-like phenotypes and inhibited CTCL cell growth. These effects were mediated by cell death-related pathways, including apoptosis, autophagy, and necroptosis. Collectively, our findings show that CD47 and PD-L1 are critical regulators of immune surveillance in CTCL and that dual targeting of CD47 and PD-L1 will provide insight into tumor immunotherapy for CTCL.

Abstract P1-11-12: Soluble TNFα blockade enhances trastuzumab deruxtecan antitumor effect in HER2-positive breast cancer model

Abstract Background. Clinical trials have demonstrated that trastuzumab deruxtecan (T-DXd) provides a durable responses for patients with HER2-positive and HER2 low metastatic breast cancer (BC). With T-DXd treatment, approximately 50% of patients with HER2+ metastatic BC were still alive and progression-free at 24 months (DESTINY-Breast03). We previously shown that mucin 4 (MUC4) expression is an independent predictor of poor response to trastuzumab in HER2-positive BC patients. We also showed that MUC4 is upregulated by soluble TNFα (sTNFα) secreted by the tumor, which confers primary trastuzumab resistance since it hides trastuzumab epitope on the HER2 molecule, reducing its binding and diminishing its therapeutic effects. In preclinical models of de novo trastuzumab-resistant tumors, we proved that administration of the sTNFα blocking agent INB03 (DN) together with trastuzumab inhibited tumor growth and induced an innate immune response in the tumor microenvironment (TME). DN is a dominant-negative inhibitor of sTNFα that is not immunosuppressive because it does not affect transmembrane TNFα. Our goal is to study whether neutralizing sTNFα can improve T-DXd effects in a multiple HER2-targeted therapy-resistant model. Methods JIMT-1 is a HER2-positive BC cell line resistant to trastuzumab, pertuzumab and lapatinib, which expresses MUC4. JIMT-1 tumor-bearing nude mice were treated with (1) IgG 5 mg/kg, (2) T-DXd 5 mg/kg, (3) T-DXd 2.5 mg/kg, (4) T-DXd 1.25 mg/kg, (5) DN 10 mg/kg, (6) T-DXd 5 mg/kg +DN, (7) T-DXd 2.5 mg/kg +DN and (8) T-DXd 1.25 mg/kg +DN. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored regularly. Tumor-infiltrating macrophages, NK cells and myeloid-derived suppressor cells (MDSCs) were evaluated by immunofluorescence and flow cytometry. Results The dose-response curve of T-DXd exhibited tumor growth inhibitions of 83% (5 mg/kg), 61% (2.5 mg/kg) and 37% (1.25 mg/kg) vs IgG-treated tumors. DN alone had no antitumor effect. Combination of T-DXd with DN resulted in a reinforced antitumor effect, as the tumor growth inhibition escalated to 98% (T-DXd 5 mg/kg+ DN), 81% (T-DXd 2.5 mg/kg+DN) and 73% (T-DXd 1.25 mg/kg+DN). Moreover, we observed that addition of DN to T-DXd 1.25 and 5 mg/kg enhances the infiltration of resident macrophages (p < 0.05 and p<0.01, respectively) and promotes polarization to the M1-like phenotype (p < 0.05 and p<0.001, respectively). While T-DXd 2.5 and 5 mg/kg alone achieved a decrease in M2-like macrophages (p < 0.05), combination of T-DXd 1.25mg/kg+DN impaired M2-like polarization at similar levels of that observed with 2.5 and 5 mg/kg T-DXd (p < 0.05). Notably, the M1/M2 ratio escalated from 10.9% to 51.5% when DN was added to the lowest T-DXd dose (p < 0.01). In addition, T-DXd 2.5 and 5 mg/kg induce an increase in the proportion of NK cells in TME (p < 0.001 and p<0.05, respectively), which T-DXd 1.25 mg/kg+ DN treatment mimicked (p < 0.05). Although an increase in NK cell activation was observed with 1.25 and 2.5 mg/kg treatments (p < 0.05 and p<0.001, respectively), adding DN did not further improved this effect. Only the highest dose of T-DXd in combination with DN was able to increase NK cell degranulation (p < 0.05), compared to T-DXd alone. Finally, the percentage of MDSCs population decreases with the addition of DN to the T-DXd 2.5 and 5 mg/kg. Conclusions Our results suggest that sTNFα blockade is able to enhance T-DXd effect in a multiple HER2-targeted therapy resistant model. Notably, the administration of T-DXd 1.25 mg/kg+DN achieved a similar antitumor effect to T-DXd 5 mg/kg alone and also transforms the TME to an antitumor one with a reinforced immune response. This finding highlights that sTNFα and MUC4 expression are important variables in the response to T-DXd. Neutralization of sTNFα may open new therapeutic strategies for treatment of patients who present with MUC4 expression or have progression on T-DXd therapy. Citation Format: Sofia Bruni, Florencia Mauro, Cecilia Proietti, Rosalia Cordo-Russo, Mercogliano María Florencia, Roxana Schillaci. Soluble TNFα blockade enhances trastuzumab deruxtecan antitumor effect in HER2-positive breast cancer model [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 P1-11-12.

Blocking soluble TNFα sensitizes HER2-positive breast cancer to trastuzumab through MUC4 downregulation and subverts immunosuppression

BackgroundThe success of HER2-positive (HER2+) breast cancer treatment with trastuzumab, an antibody that targets HER2, relies on immune response. We demonstrated that TNFα induces mucin 4 (MUC4) expression, which shields...

Hepatocellular Carcinoma Cell-Derived Exosomal miR-21-5p Induces Macrophage M2 Polarization by Targeting RhoB

M2-like polarized tumor-associated macrophages (TAMs) are the major component of infiltrating immune cells in hepatocellular carcinoma (HCC), which have been proved to exhibit significant immunosuppressive and pro-tumoral effects. However, the underlying mechanism of the tumor microenvironment (TME) educating TAMs to express M2-like phenotypes is still not fully understood. Here, we report that HCC-derived exosomes are involved in intercellular communications and exhibit a greater capacity to mediate TAMs' phenotypic differentiation. In our study, HCC cell-derived exosomes were collected and used to treat THP-1 cells in vitro. Quantitative polymerase chain reaction (qPCR) results showed that the exosomes significantly promoted THP-1 macrophages to differentiate into M2-like macrophages, which have a high production of transforming growth factor-β (TGF-β) and interleukin (IL)-10. The analysis of bioinformatics indicated that exosomal miR-21-5p is closely related to TAM differentiation and is associated with unfavorable prognosis in HCC. Overexpressing miR-21-5p in human monocyte-derived leukemia (THP-1) cells induced down-regulation of IL-1β levels; however, it enhanced production of IL-10 and promoted the malignant growth of HCC cells in vitro. A reporter assay confirmed that miR-21-5p directly targeted Ras homolog family member B (RhoB) 3'-untranslatedregion (UTR) in THP-1 cells. Downregulated RhoB levels in THP-1 cells would weaken mitogen-activated protein kinase (MAPK) axis signaling pathways. Taken together, tumor-derived miR-21-5p promote the malignant advance of HCC, which mediated intercellular crosstalk between tumor cells and macrophages. Targeting M2-like TAMs and intercepting their associated signaling pathways would provide potentially specific and novel therapeutic approaches for HCC treatment.

Effective Reversal of Macrophage Polarization by Inhibitory Combinations Predicted by a Boolean Protein-Protein Interaction Model.

The function and polarization of macrophages has a significant impact on the outcome of many diseases. Targeting tumor-associated macrophages (TAMs) is among the greatest challenges to solve because of the low in vitro reproducibility of the heterogeneous tumor microenvironment (TME). To create a more comprehensive model and to understand the inner workings of the macrophage and its dependence on extracellular signals driving polarization, we propose an in silico approach. A Boolean control network was built based on systematic manual curation of the scientific literature to model the early response events of macrophages by connecting extracellular signals (input) with gene transcription (output). The network consists of 106 nodes, classified as 9 input, 75 inner and 22 output nodes, that are connected by 217 edges. The direction and polarity of edges were manually verified and only included in the model if the literature plainly supported these parameters. Single or combinatory inhibitions were simulated mimicking therapeutic interventions, and output patterns were analyzed to interpret changes in polarization and cell function. We show that inhibiting a single target is inadequate to modify an established polarization, and that in combination therapy, inhibiting numerous targets with individually small effects is frequently required. Our findings show the importance of JAK1, JAK3 and STAT6, and to a lesser extent STK4, Sp1 and Tyk2, in establishing an M1-like pro-inflammatory polarization, and NFAT5 in creating an anti-inflammatory M2-like phenotype. Here, we demonstrate a protein-protein interaction (PPI) network modeling the intracellular signalization driving macrophage polarization, offering the possibility of therapeutic repolarization and demonstrating evidence for multi-target methods.

Alpha fetoprotein promotes polarization of macrophages towards M2-like phenotype and inhibits macrophages to phagocytize hepatoma cells.

Alpha-fetoprotein(AFP) is a cancer biomarker for the diagnosis of hepatocellular carcinoma(HCC); however, its role in macrophage polarization and phagocytosis remains unclear. In the present study, we explored the correlation between AFP regulation of macrophage function and the possible regulatory mechanisms. Human mononuclear leukemia cells (THP-1) and monocytes from healthy donors were used to analyze the effect of AFP on the macrophages' phenotype and phagocytosis. THP-1 cells and healthy human donor-derived monocytes were polarized into M0 macrophages induced by phorbol ester (PMA), and M0 macrophages were polarized into M1 macrophages induced by lipopolysaccharide(LPS) and interferon-γ(IFN-γ). Interleukin-4(IL-4) and interleukin-13(IL-13) were used to induce M0 macrophage polarization into M2 macrophages. Tumor-derived AFP(tAFP) stimulated M0 macrophage polarization into M2 macrophages and inhibited M1 macrophages to phagocytize HCC cells. The role of AFP in promoting macrophage polarization into M2 macrophages and inhibiting the M1 macrophages to phagocytize HCC cells may be involved in activating the PI3K/Akt signaling pathway. AFP could also enhanced the migration ability of macrophages and inhibited the apoptosis of HCC cells when co-cultured with M1-like macrophages. AFP is a pivotal cytokine that inhibits macrophages to phagocytize HCC cells.

Convection-enhanced delivery of nanoencapsulated gene locoregionally yielding ErbB2/Her2-specific CAR-macrophages for brainstem glioma immunotherapy

Locoregional delivery of chimeric antigen receptor (CAR)-modified T (CAR-T) cells has emerged as a promising strategy for brain tumors. However, the complicated ex vivo cell manufacturing procedures and the rapid progression of the disease have limited its broader applications. Macrophages (MΦs) exhibit unique effector functions and a high degree of infiltration within the solid tumor microenvironment (TME), especially in the brain, where MΦs function as structural support, and the main immune effector cells of the CNS represent 5–12% of brain cells. Here, we report a synthetic universal DNA nanocarrier for in situ genetic editing of intratumoral MΦs with an ErbB2-specific CAR to direct their phagocytic activity towards tumors and subsequently initiate a locoregional antitumor immune response. Specifically, we demonstrated that when delivered locoregionally, the RP-182 peptide, located in the shell of a nanoparticle, targeted MΦs and reprogrammed M2-like tumor-associated macrophages (TAMs) to an antitumor M1-like phenotype. Subsequently, the CAR gene-laden DNA nanocomplex can be used to introduce ErbB2-targeted CAR, and the generated CAR-MΦs then act as “living” cures, thereby serially clearing the invasive tumor cells. Our work demonstrates a practical antitumor immunotherapy for brainstem gliomas (BSGs) that may be broadly applicable for patients suffering from other ErbB2-positive solid malignancies.

Translatome analyses by bio-orthogonal non-canonical amino acid labeling reveal that MR1-activated MAIT cells induce an M1 phenotype and antiviral programming in antigen-presenting monocytes.

MAIT cells are multifunctional innate-like effector cells recognizing bacterial-derived vitamin B metabolites presented by the non-polymorphic MHC class I related protein 1 (MR1). However, our understanding of MR1-mediated responses of MAIT cells upon their interaction with other immune cells is still incomplete. Here, we performed the first translatome study of primary human MAIT cells interacting with THP-1 monocytes in a bicellular system. We analyzed the interaction between MAIT and THP-1 cells in the presence of the activating 5-OP-RU or the inhibitory Ac-6-FP MR1-ligand. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) we were able to enrich selectively those proteins that were newly translated during MR1-dependent cellular interaction. Subsequently, newly translated proteins were measured cell-type-specifically by ultrasensitive proteomics to decipher the coinciding immune responses in both cell types. This strategy identified over 2,000 MAIT and 3,000 THP-1 active protein translations following MR1 ligand stimulations. Translation in both cell types was found to be increased by 5-OP-RU, which correlated with their conjugation frequency and CD3 polarization at MAIT cell immunological synapses in the presence of 5-OP-RU. In contrast, Ac-6-FP only regulated a few protein translations, including GSK3B, indicating an anergic phenotype. In addition to known effector responses, 5-OP-RU-induced protein translations uncovered type I and type II Interferon-driven protein expression profiles in both MAIT and THP-1 cells. Interestingly, the translatome of THP-1 cells suggested that activated MAIT cells can impact M1/M2 polarization in these cells. Indeed, gene and surface expression of CXCL10, IL-1β, CD80, and CD206 confirmed an M1-like phenotype of macrophages being induced in the presence of 5-OP-RU-activated MAIT cells. Furthermore, we validated that the Interferon-driven translatome was accompanied by the induction of an antiviral phenotype in THP-1 cells, which were found able to suppress viral replication following conjugation with MR1-activated MAIT cells. In conclusion, BONCAT translatomics extended our knowledge of MAIT cell immune responses at the protein level and discovered that MR1-activated MAIT cells are sufficient to induce M1 polarization and an anti-viral program of macrophages.

Key Role of Mesenchymal Stromal Cell Interaction with Macrophages in Promoting Repair of Lung Injury.

Lung macrophages (Mφs) are essential for pulmonary innate immunity and host defense due to their dynamic polarization and phenotype shifts. Mesenchymal stromal cells (MSCs) have secretory, immunomodulatory, and tissue-reparative properties and have shown promise in acute and chronic inflammatory lung diseases and in COVID-19. Many beneficial effects of MSCs are mediated through their interaction with resident alveolar and pulmonary interstitial Mφs. Bidirectional MSC-Mφ communication is achieved through direct contact, soluble factor secretion/activation, and organelle transfer. The lung microenvironment facilitates MSC secretion of factors that result in Mφ polarization towards an immunosuppressive M2-like phenotype for the restoration of tissue homeostasis. M2-like Mφ in turn can affect the MSC immune regulatory function in MSC engraftment and tissue reparatory effects. This review article highlights the mechanisms of crosstalk between MSCs and Mφs and the potential role of their interaction in lung repair in inflammatory lung diseases.

Immunomodulatory IL-23 receptor antagonist peptide nanocoatings for implant soft tissue healing.

Peri-implantitis, caused by an inflammatory response to pathogens, is the leading cause of dental implant failure. Poor soft tissue healing surrounding implants - caused by inadequate surface properties - leads to infection, inflammation, and dysregulated keratinocyte and macrophage function. One activated inflammatory response, active around peri-implantitis compared to healthy sites, is the IL-23/IL-17A cytokine axis. Implant surfaces can be synthesized with peptide nanocoatings to present immunomodulatory motifs to target peri-implant keratinocytes to control macrophage polarization and regulate inflammatory axises toward enhancing soft tissue healing. We synthesized an IL-23 receptor (IL-23R) noncompetitive antagonist peptide nanocoating using silanization and evaluated keratinocyte secretome changes and macrophage polarization (M1-like "pro-inflammatory" vs. M2-like "pro-regenerative"). IL-23R antagonist peptide nanocoatings were successfully synthesized on titanium, to model dental implant surfaces, and compared to nonfunctional nanocoatings and non-coated titanium. IL-23R antagonist nanocoatings significantly decreased keratinocyte IL-23, and downstream IL-17A, expression compared to controls. This peptide noncompetitive antagonistic function was demonstrated under lipopolysaccharide stimulation. Large scale changes in keratinocyte secretome content, toward a pro-regenerative milieu, were observed from keratinocytes cultured on the IL-23R antagonist nanocoatings compared to controls. Conditioned medium collected from keratinocytes cultured on the IL-23R antagonist nanocoatings polarized macrophages toward a M2-like phenotype, based on increased CD163 and CD206 expression and reduced iNOS expression, compared to controls. Our results support development of IL-23R noncompetitive antagonist nanocoatings to reduce the pro-inflammatory IL-23/17A pathway and augment macrophage polarization toward a pro-regenerative phenotype. Immunomodulatory implant surface engineering may promote soft tissue healing and thereby reduce rates of peri-implantitis.

Targeting Arg-1 and PD-L1 in M2-Tumor Associated Macrophages Impairs Juvenile Myelomonocytic Leukemia (JMML) Cell Proliferation and Migration

Background and Hypothesis: Tumor-associated macrophages (TAMs) are a key component of tumor-infiltrating immune cells. They are largely characterized into M1 or M2 types. TAMs express an anti-inflammatory M2-like phenotype, promote tumor progression. However, the role of M2-TAMs in driving disease pathogenesis in patients with Juvenile myelomonocytic leukemia (JMML), a rare form of pediatric leukemia driven to a large extent by mutations in the PTPN11 gene, which encodes the phosphatase SHP2 is unclear. We hypothesized that in JMML, inflammatory myeloid cells including neutrophils and M2-TAMs express higher levels of arginase-1 (Arg-1) and PD-L1, which may contribute to the local suppression of immune responses and support the development of JMML.
 Methods: To study how alterations in M1/M2 macrophages contribute to JMML development, we utilized a mouse model bearing Shp2E76K mutation (Ptpn11E76K/+) which manifests the cardinal features of human JMML. We hypothesized that Shp2E76K/+ mutations enhance the function of bone marrow derived macrophages (BMDMs), including M2-TAMs and contribute to T-cell suppression.
 Results: Our analysis of the bulk RNA-sequence data from 90 JMML patients showed increase in the expression of Arg-1 and PD-1. Furthermore, single cell RNA-seq analysis of macrophages from 4 JMML patients revealed higher expression of M2-macrophage markers/genes. Our results show that in M2-TAMs, Arg-1 and PD-L1 levels are elevated in BM and spleens of Shp2E76K/+ mice compared to WT. Moreover, M2-TAMs, Arg-1 and PD-L1 levels were also higher in BMDMs derived from Shp2E76K/+ mice compared to WT. The BMDMs from Shp2E76K/+ mice have greater proliferation and migration potential compared to WT BMDMs, which was significantly reduced by inhibiting the function of Arg-1 and PD-L1.
 Conclusion: Our results show that M2-TAMs, arginase-1, and PD-L1 create a pro-tumor microenvironment, which likely contributes to the growth of JMML cells. Inhibition of Arg-1 and PD-L1 is a novel therapeutic approach to treat patients with JMML.

Porous BCP ceramics with nanoscale whisker structure accelerate bone regeneration by regulating inflammatory response

Inflammation-induced by biomaterials is a critical event to determine the success and efficiency of tissue repair. Macrophages are a major population that participates the biomaterial induced inflammation. The response of macrophages depends on the characteristics of biomaterials, thus causing a cascade reaction in subsequent biological processes. In this study, porous biphase calcium phosphate (BCP) ceramics with the different surface structures were constructed to compare the effect of surface structure on bone generation potential, and further reveal the inflammation-involved mechanism. Our results demonstrated that macrophages on three ceramics showed distinct morphologies and spreading areas. The nanoscale whisker structure did induce more bone generation in the mice thigh muscle. The in vitro result revealed that nanoscale whisker structure could drive macrophage polarization towards M1-like phenotype, indicated by a higher expression of pro-inflammatory specific markers (iNOS and CCR7), and mass secretion of TNF-α. Further research indicated that additional TNF-α could promote the osteogenic differentiation of mesenchymal stem cells (MSCs). However, excess addition of TNF-α showed an opposite effect on the osteogenic differentiation of MSCs by initiating the NF-κB signaling pathway, which suppresses the osteogenesis process.

Interstitial Macrophages Lead Early Stages of Bleomycin-Induced Lung Fibrosis and Induce Fibroblasts Activation.

A progressive fibrosing phenotype is critical in several lung diseases. It is irreversible and associated with early patient mortality. Growing evidence has revealed pulmonary macrophages' role as modulators of the fibrotic processes. The proportion, phenotype, and function of alveolar (AM) and interstitial macrophages (IM) at the early stages of bleomycin-induced pulmonary fibrosis have not been clearly described. In this way, our study aimed to characterize these macrophage populations and investigate the effect on fibroblast activation. C57BL/6 mice were intratracheally injected with bleomycin and were sacrificed at day 3, 5, and 7 for the performance of flow cytometry and fluorescent-activated cell sorting analysis for protein and gene expression quantification. After bleomycin administration, the proportion of IM was significantly higher than that of AM, which showed a decay during the inflammatory phase, and peaked at day 7. At day 7 of the inflammatory phase, AM started shifting their phenotype from M1-like towards M2, while IM showed a M2-like phenotype. Conditioned medium derived from IM sorted at day 7 induced fibroblast activation and differentiation in myofibroblasts in vitro. Our findings indicate that IM are the largest macrophage population at the early stages of experimental pulmonary fibrosis and are secreted mediators able to activate fibroblasts, pointing to macrophage modulation as a potential therapeutic strategy to restrain progressive fibrosing lung disorders.

Expression of scavenger receptors CD163, CD204, and CD206 on macrophages in patients with pulmonary tuberculosis

The aim of the study was to evaluate the expression of scavenger receptors (CD163, CD204, CD206) on macrophages in patients with pulmonary tuberculosis, depending on the clinical form of the disease and sensitivity of the pathogen to anti-tuberculosis drugs.Materials and methods. 64 patients with pulmonary tuberculosis (TB) were examined: 26 patients with disseminated pulmonary tuberculosis (DTB) and 38 patients with infiltrative pulmonary tuberculosis (ITB). Of these, 42 patients secreted Mycobacterium tuberculosis (MBT) sensitive to basic antituberculosis drugs (ATBD), and 22 patients secreted MBT resistant to first-line anti-TB drugs. Material for the study was venous blood. To isolate monocytes from the whole blood in order to transform them into macrophages, Ficoll density gradient centrifugation with a density of 1.077 g / cm3 was used followed by immunomagnetic separation of CD14+ cells. Monocytes were cultured in the X-VIVO 10 medium with gentamicin and phenol red with the addition of macrophage colony-stimulating factor (M-CSF) (5 ng / ml) at a concentration of 1×106 cells / ml with stimulators: interleukin (IL)-4 (10 ng / ml) and interferon (IFN) γ (100 ng / ml). Immunophenotyping of macrophages was performed using monoclonal antibodies to CD163, CD204, and CD206 on the Beckman Coulter CytoFLEX LX Flow Cytometer. The analysis of the obtained data was carried out using the CytExpert 2.0 software. The results were analyzed using statistical methods.Results. Switching the phenotype of macrophages from the M1-like proinflammatory phenotype to M2-like antiinflammatory one contributes to the chronic course of pulmonary TB, dissemination, and persistence of infection. In the present study, we analyzed the features of the expression of CD163, CD204, and CD206 scavenger receptors on macrophages in patients with pulmonary TB. An increase in the number of macrophages carrying markers of the M2 subpopulation (CD163, CD204, and CD206) on their surface was noted, regardless of the clinical form of pulmonary TB and drug resistance of M. tuberculosis.Conclusion. Studying the mechanisms underlying M1 or M2 activation of macrophages is necessary for a deeper understanding of the immunopathogenesis of TB and the role of innate immunity cells in protecting the body from mycobacteria. The analysis of the expression of scavenger receptors CD163, CD204, and CD206 on macrophages allowed to conclude that, in pulmonary TB, especially in patients with drug resistant M. tuberculosis and infiltrative TB, regulatory mechanisms that suppress the activation of innate immunity are implemented together with polarization of macrophage differentiation towards the M2 phenotype. It may be the cause of immune deficiency induced by the pathogen.

An Engineered IFNγ-Antibody Fusion Protein with Improved Tumor-Homing Properties.

Interferon-gamma (IFNγ) is one of the central cytokines produced by the innate and adaptive immune systems. IFNγ directly favors tumor growth control by enhancing the immunogenicity of tumor cells, induces IP-10 secretion facilitating (CXCR3+) immune cell infiltration, and can prime macrophages to an M1-like phenotype inducing proinflammatory cytokine release. We had previously reported that the targeted delivery of IFNγ to neoplastic lesions may be limited by the trapping of IFNγ-based products by cognate receptors found in different organs. Here we describe a novel fusion protein consisting of the L19 antibody, specific to the alternatively spliced extra-domain B of fibronectin (EDB), fused to a variant of IFNγ with reduced affinity to its cognate receptor. The product (named L19-IFNγ KRG) selectively localized to tumors in mice, showed favorable pharmacokinetic profiles in monkeys and regained biological activity upon antigen binding. The fusion protein was investigated in two murine models of cancer, both as monotherapy and in combination with therapeutic modalities which are frequently used for cancer therapy. L19-IFNγ KRG induced tumor growth retardation and increased the intratumoral concentration of T cells and NK cells in combination with anti-PD-1.

Combination of hyaluronic acid conjugates with immunogenic cell death inducer and CpG for glioblastoma local chemo-immunotherapy elicits an immune response and induces long-term survival

The efficacy of standard glioblastoma (GBM) treatments has been limited due to the highly immunosuppressive tumor immune microenvironment, interpatient tumor heterogenicity and anatomical barriers, such as the blood brain barrier. In the present work, we hypothesized that a new local therapy based on the combination of doxorubicin (DOX) as an immunogenic cell death (ICD) inducer and CpG, a Toll-like receptor (TLR)-9 agonist, would act synergistically to eradicate GBM. DOX and CpG were first tested in an orthotopic GL261 GBM model showing enhanced survival. To improve the outcome with a reduced dose, we designed bioresponsive hyaluronic acid (HA)-drug conjugates for effective in situ chemoimmunotherapy. HA was derivatized with CpG. The new HA-CpG conjugate showed high efficacy in re-educating protumoral M2-like microglia into an antitumoral M1-like phenotype, inducing the expression of immune-stimulatory cytokines. DOX was also conjugated to HA. DOX conjugation increased ICD induction in GL261 cells. Finally, a combination of the conjugates was explored in an orthotopic GL261 GBM model. The local delivery of combined HA-DOX + HA-CpG into the tumor mass elicited antitumor CD8+ T cell responses in the brain tumor microenvironment and reduced the infiltration of M2-like tumor-associated macrophages and myeloid-derived suppressor cells. Importantly, the combination of HA-DOX and HA-CpG induced long-term survival in >66% of GBM-bearing animals than other treatments (no long-term survivor observed), demonstrating the benefits of conjugating synergistic drugs to HA nanocarrier. These results emphasize that HA-drug conjugates constitute an effective drug delivery platform for local chemoimmunotherapy against GBM and open new perspectives for the treatment of other brain cancers and brain metastasis.

Modulatory effects of mesenchymal stem cells on microglia in ischemic stroke.

Ischemic stroke accounts for 70-80% of all stroke cases. Immunity plays an important role in the pathophysiology of ischemic stroke. Microglia are the first line of defense in the central nervous system. Microglial functions are largely dependent on their pro-inflammatory (M1-like) or anti-inflammatory (M2-like) phenotype. Modulating neuroinflammation via targeting microglia polarization toward anti-inflammatory phenotype might be a novel treatment for ischemic stroke. Mesenchymal stem cells (MSC) and MSC-derived extracellular vesicles (MSC-EVs) have been demonstrated to modulate microglia activation and phenotype polarization. In this review, we summarize the physiological characteristics and functions of microglia in the healthy brain, the activation and polarization of microglia in stroke brain, the effects of MSC/MSC-EVs on the activation of MSC in vitro and in vivo, and possible underlying mechanisms, providing evidence for a possible novel therapeutics for the treatment of ischemic stroke.

Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice

Macrophages are a major population of immune cells in solid cancers, especially colorectal cancers. Tumor-associated macrophages (TAMs) are commonly divided into M1-like (tumor suppression) and M2-like (tumor promotion) phenotypes. Vasoactive intestinal peptide (VIP) is an immunoregulatory neuropeptide with a potent anti-inflammatory function. Inhibition of VIP signaling has been shown to increase CD8+ T cell proliferation and function in viral infection and lymphoma. However, the role of VIP in macrophage polarization and function in solid tumors remains unknown. Here, we demonstrated that conditioned medium from CT26 (CT26-CM) cells enhanced M2-related marker and VIP receptor (VPAC) gene expression in RAW264.7 macrophages. VIP hybrid, a VIP antagonist, enhanced M1-related genes but reduced Mrc1 gene expression and increased phagocytic ability in CT26-CM-treated RAW264.7 cells. In immunodeficient SCID mice, VIP antagonist alone or in combination with anti-PD-1 antibody attenuated CT26 tumor growth compared with the control. Analysis of tumor-infiltrating leukocytes found that VIP antagonist increased M1/M2 ratios and macrophage phagocytosis of CT26-GFP cells. Furthermore, Vipr2 gene silencing or VPAC2 activation affected the polarization of CT26-CM-treated RAW264.7 cells. In conclusion, the inhibition of VIP signaling enhanced M1 macrophage polarization and macrophage phagocytic function, resulting in tumor regression in a CT26 colon cancer model.