Mature Macrophages Research Articles

DOP28 Aconitate decarboxylase 1 (ACOD1) represents a crucial regulator of monocyte recruitment, survival and proinflammatory status in intestinal inflammation

Abstract Background Aconitate decarboxylase 1 (ACOD1; also known as IRG-1) is the ubiquitous source of itaconate, which dampens inflammasome activation by preventing HIF1a production of IL-1b and prevents TNF production through NRF2 activation. We reasoned that expression of ACOD1 is an inhibitory pathway within monocytes to curtail their inflammatory function, which could be manipulated in IBD therapy. Methods We performed single-cell RNA sequencing of monocytes from colonoscopic biopsies of treatment naïve IBD patients, as well as complementary analysis of DSS treated C57BL/6J (WT) mice, using single-cell protein-mRNA (Primeflow) technologies for validation. To determine the role of ACOD1 in colitis, Acod1–/– mice were administered 2% DSS in drinking water for 6 days (acute colitis) or 4 days followed by 14 day recovery period (inflammation resolution) and assessed the colon myeloid compartment by multi-parameter flow cytometry. This was complemented with competitive bone marrow chimeric mice to assess the intrinsic role of ACOD1 controlling monocyte survival and proinflammatory status. Transcriptional profiling (Nanostring) was used to determine the molecular pathways influenced by ACOD1. Results We found that ACOD1/Acod1 represented an evolutionarily conserved of feature of inflammation-associated monocytes, with expression present in mucosal but not blood monocytes. Acod1–/– mice demonstrated increased susceptibility to acute intestinal injury with DSS compared to co-housed WT controls, characterised by increased weight loss and colon shortening with significant accumulation of colonic neutrophils and CD64+Ly6C+ MHC-II+/– monocytes. Consistent with Acod1 limiting inflammasome activation, Acod1–/– monocytes demonstrated significantly greater IL-1b producing capabilities than WT controls. To assess Acod1 deficient and sufficient haemopoietic cells in in the same environment, we generated competitive bone marrow chimeric mice. Acod1 deficiency conferred a significant advantage to monocyte recruitment to the colon, characterised by a 10fold increase in Ly6C+ MHC-II–colon monocytes in colitis. Transcriptional profiling of WT and Acod1–/– colonic monocytes revealed significantly greater expression of molecules required for monocyte extravasation, including Sell (CD62L), Icam1, Ccr2 and Itgal (CD11a). In addition, expression of genes indicative of macrophage maturation such as Itgax (CD11c), Cd163 and Cx3cr1 were lower in the context of Acod1 deficiency, suggesting that normal maturation of these cells may be perturbed by loss of Acod1. Conclusion ACOD1/Acod1 represents an evolutionarily conserved, monocyte specific immuno-metabolic pathway that acts to limit monocyte recruitment, fate and pro-inflammatory function within the inflamed intestine.

Lack of iron, zinc, and vitamins as a contributor to the etiology of atopic diseases.

Micronutritional deficiencies are common in atopic children suffering from atopic dermatitis, food allergy, rhinitis, and asthma. A lack of iron, in particular, may impact immune activation with prolonged deficiencies of iron, zinc, vitamin A, and vitamin D associated with a Th2 signature, maturation of macrophages and dendritic cells (DCs), and the generation of IgE antibodies. In contrast, the sufficiency of these micronutrients establishes immune resilience, promotion of regulatory cells, and tolerance induction. As micronutritional deficiencies mimic an infection, the body's innate response is to limit access to these nutrients and also impede their dietary uptake. Here, we summarize our current understanding of the physiological function of iron, zinc, and vitamins A and D in relation to immune cells and the clinical consequences of deficiencies in these important nutrients, especially in the perinatal period. Improved dietary uptake of iron is achieved by vitamin C, vitamin A, and whey compounds, whereas zinc bioavailability improves through citrates and proteins. The addition of oil is essential for the dietary uptake of beta-carotene and vitamin D. As for vitamin D, the major source comes via sun exposure and only a small amount is consumed via diet, which should be factored into clinical nutritional studies. We summarize the prevalence of micronutritional deficiencies of iron, zinc, and vitamins in the pediatric population as well as nutritional intervention studies on atopic diseases with whole food, food components, and micronutrients. Dietary uptake via the lymphatic route seems promising and is associated with a lower atopy risk and symptom amelioration. This review provides useful information for clinical studies and concludes/emphasizes that a healthy, varied diet containing dairy products, fish, nuts, fruits, and vegetables as well as supplementing foods or supplementation with micronutrients as needed is essential to combat the atopic march.

Impact of the flame retardant 2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) in THP-1 macrophage-like cell function via small extracellular vesicles.

2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the most widespread environmental brominated flame-retardant congeners which has also been detected in animal and human tissues. Several studies have reported the effects of PBDEs on different health issues, including neurobehavioral and developmental disorders, reproductive health, and alterations of thyroid function. Much less is known about its immunotoxicity. The aim of our study was to investigate the effects that treatment of THP-1 macrophage-like cells with PBDE-47 could have on the content of small extracellular vesicles' (sEVs) microRNA (miRNA) cargo and their downstream effects on bystander macrophages. To achieve this, we purified sEVs from PBDE-47 treated M(LPS) THP-1 macrophage-like cells (sEVsPBDE+LPS) by means of ultra-centrifugation and characterized their miRNA cargo by microarray analysis detecting the modulation of 18 miRNAs. Furthermore, resting THP-1 derived M(0) macrophage-like cells were cultured with sEVsPBDE+LPS, showing that the treatment reshaped the miRNA profiles of 12 intracellular miRNAs. This dataset was studied in silico, identifying the biological pathways affected by these target genes. This analysis identified 12 pathways all involved in the maturation and polarization of macrophages. Therefore, to evaluate whether sEVsPBDE+LPS can have some immunomodulatory activity, naïve M(0) THP-1 macrophage-like cells cultured with purified sEVsPBDE+LPS were studied for IL-6, TNF-α and TGF-β mRNAs expression and immune stained with the HLA-DR, CD80, CCR7, CD38 and CD209 antigens and analyzed by flow cytometry. This analysis showed that the PBDE-47 treatment does not induce the expression of specific M1 and M2 cytokine markers of differentiation and may have impaired the ability to make immunological synapses and present antigens, down-regulating the expression of HLA-DR and CD209 antigens. Overall, our study supports the model that perturbation of miRNA cargo by PBDE-47 treatment contributes to the rewiring of cellular regulatory pathways capable of inducing perturbation of differentiation markers on naïve resting M(0) THP-1 macrophage-like cells.

Multinucleation resets human macrophages for specialized functions at the expense of their identity

Macrophages undergo plasma membrane fusion and cell multinucleation to form multinucleated giant cells (MGCs) such as osteoclasts in bone, Langhans giant cells (LGCs) as part of granulomas or foreign‐body giant cells (FBGCs) in reaction to exogenous material. How multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrate comparative transcriptomics with functional assays in purified mature mononuclear and multinucleated human osteoclasts, LGCs and FBGCs. Strikingly, in all three types of MGCs, multinucleation causes a pronounced downregulation of macrophage identity. We show enhanced lysosome‐mediated intracellular iron homeostasis promoting MGC formation. The transition from mononuclear to multinuclear state is accompanied by cell specialization specific to each polykaryon. Enhanced phagocytic and mitochondrial function associate with FBGCs and osteoclasts, respectively. Moreover, human LGCs preferentially express B7‐H3 (CD276) and can form granuloma‐like clusters in vitro, suggesting that their multinucleation potentiates T cell activation. These findings demonstrate how cell–cell fusion and multinucleation reset human macrophage identity as part of an advanced maturation step that confers MGC‐specific functionality.

Type I interferon signaling facilitates resolution of acute liver injury by priming macrophage polarization.

Due to their broad functional plasticity, myeloid cells contribute to both liver injury and recovery during acetaminophen overdose-induced acute liver injury (APAP-ALI). A comprehensive understanding of cellular diversity and intercellular crosstalk is essential to elucidate the mechanisms and to develop therapeutic strategies for APAP-ALI treatment. Here, we identified the function of IFN-I in the myeloid compartment during APAP-ALI. Utilizing single-cell RNA sequencing, we characterized the cellular atlas and dynamic progression of liver CD11b+ cells post APAP-ALI in WT and STAT2 T403A mice, which was further validated by immunofluorescence staining, bulk RNA-seq, and functional experiments in vitro and in vivo. We identified IFN-I-dependent transcriptional programs in a three-way communication pathway that involved IFN-I synthesis in intermediate restorative macrophages, leading to CSF-1 production in aging neutrophils that ultimately enabled Trem2+ restorative macrophage maturation, contributing to efficient liver repair. Overall, we uncovered the heterogeneity of hepatic myeloid cells in APAP-ALI at single-cell resolution and the therapeutic potential of IFN-I in the treatment of APAP-ALI.

Immunomodulatory Effects of Non-Thermal Plasma in a Model for Latent HIV-1 Infection: Implications for an HIV-1-Specific Immunotherapy.

In people living with HIV-1 (PLWH), antiretroviral therapy (ART) eventually becomes necessary to suppress the emergence of human immunodeficiency virus type 1 (HIV-1) replication from latent reservoirs because HIV-1-specific immune responses in PLWH are suboptimal. Immunotherapies that enhance anti-HIV-1 immune responses for better control of virus reemergence from latent reservoirs are postulated to offer ART-free control of HIV-1. Toward the goal of developing an HIV-1-specific immunotherapy based on non-thermal plasma (NTP), the early immunological responses to NTP-exposed latently infected T lymphocytes were examined. Application of NTP to the J-Lat T-lymphocyte cell line (clones 10.6 and 15.4) stimulated monocyte recruitment and macrophage maturation, which are key steps in initiation of an immune response. In contrast, CD8+ T lymphocytes in a mixed lymphocyte reaction assay were not stimulated by the presence of NTP-exposed J-Lat cells. Furthermore, co-culture of NTP-exposed J-Lat cells with mature phagocytes did not modulate their antigen presentation to primary CD8+ T lymphocytes (cross-presentation). However, reactivation from latency was stimulated in a clone-specific manner by NTP. Overall, these studies, which demonstrated that ex vivo application of NTP to latently infected lymphocytes can stimulate key immune cell responses, advance the development of an NTP-based immunotherapy that will provide ART-free control of HIV-1 reactivation in PLWH.

Macrophage colony-stimulating factor potentially induces recruitment and maturation of macrophages in recurrent pituitary neuroendocrine tumors.

Although pituitary neuroendocrine tumors (PitNETs) are usually benign, some are highly invasive and recurrent. Recurrent PitNETs are often treatment-resistant and there is currently no effective evidence-based treatment. Tumor-associated macrophages (TAMs) promote tumor growth in many cancers, but the effect of TAMs on PitNETs remains unclear. This study investigated the role of TAMs in the incidence of recurrent PitNETs. Immunohistochemical analysis revealed that the densities of CD163- and CD204-positive TAMs tended to increase in recurrent PitNETs. Compared with TAMs in primary lesions, those in recurrent lesions were enlarged. To clarify the cell-cell interactions between TAMs and PitNETs, in vitro experiments were performed using a mouse PitNET cell line AtT20 and the mouse macrophage cell line J774. Several cytokines related to macrophage chemotaxis and differentiation, such as M-CSF, were elevated significantly by stimulation with macrophage conditioned medium. When M-CSF immunohistochemistry analysis was performed using human PitNET samples, M-CSF expression increased significantly in recurrent lesions compared with primary lesions. Although no M-CSF receptor (M-CSFR) expression was observed in tumor cells of primary and recurrent PitNETs, flow cytometric analysis revealed that the mouse PitNET cell line expressed M-CSFR. Cellular proliferation in mouse PitNETs was inhibited by high concentrations of M-CSFR inhibitors, suggesting that cell-to-cell communication between PitNETs and macrophages induces M-CSF expression, which in turn enhances TAM chemotaxis and maturation in the tumor microenvironment. Blocking the M-CSFR signaling pathway might be a novel therapeutic adjuvant in treating recurrent PitNETs.

Nanoparticle delivery of CD40 siRNA suppresses alloimmune responses by inhibiting activation and differentiation of DCs and macrophages.

CD40 is an important costimulatory molecule expressed on antigen-presenting cells (APCs) and plays a critical role for APC activation, offering a promising therapeutic target for preventing allograft rejection. Here, we developed a biodegradable nanoparticle small interfering RNA delivery system (siCD40/NPs) to effectively deliver CD40 siRNA (siCD40) into hematopoietic stem cells (HSCs), myeloid progenitors, and mature dendritic cells (DCs) and macrophages. Injection of siCD40/NPs not only down-regulated CD40 expression in DCs and macrophages but also inhibited the differentiation of HSCs and/or myeloid progenitors into functional DCs and macrophages. Furthermore, siCD40/NPs treatment significantly prolonged allograft survival in mouse models of skin allotransplantation. In addition to reiteration of the role of CD40 in APC activation, our findings highlight a previously unappreciated role of CD40 in DC and macrophage differentiation from their progenitors. Furthermore, our results support the effectiveness of siCD40/NPs in suppressing alloimmune responses, providing a potential means of facilitating tolerance induction and preventing allotransplant rejection.

Bcl6 drives stem-like memory macrophages differentiation to foster tumor progression

Cancer development is a long-lasting process during which macrophages play a pivotal role. However, how macrophages maintain their cellular identity, persistence, expanding and pro-tumor property during malignant progression remains elusive. Inspired by the recent report of the activation of stem cell-like self-renewal mechanism in mature macrophages, we postulate that intra-tumoral macrophages might be trained to assume stem-like properties and memory-like activity favoring cancer development. Herein we demonstrated that tumor infiltrating macrophages rapidly converted into the CD11b+F4/80+Ly6C−Bcl6+ phenotype, and adopted stem cell-like properties involving expression of stemness-related genes, long-term persistence and self-renewing. Importantly, Bcl6+ macrophages stably maintained cell identity, gene signature, metabolic profile, and pro-tumor property even after long-term culture in tumor-free medium, which were hence termed stem cell-like memory macrophages (SMMs). Mechanistically, we showed that transcriptional factor Bcl6 co-opted the demethylase Tet2 and the deacetylase SIRT1 to confer the epigenetic imprinting and mitochondrial metabolic traits to SMMs, bolstering the stability and longevity of trained immunity in tumor-associated macrophages (TAMs). Furthermore, tumor-derived redHMGB1 was identified as the priming signal, which, through TLR4 and mTOR/AKT pathway, induced Bcl6-driven program underpinning SMMs generation. Collectively, our study uncovers a distinct macrophage population with a hybrid of stem cell and memory cell properties, and unveils a regulatory mechanism that integrates transcriptional, epigenetic and metabolic pathways to promote long-lasting pro-tumor immunity.

Dietary-Polysaccharide-Modified Fish-Oil-Based Double Emulsion as a Functional Colloidal Formulation for Oral Drug Delivery.

Oral delivery is the most convenient drug administration route. However, oral delivery of peptides is extremely challenging due to the physical and chemical barriers within the gastrointestinal tract. Polysaccharides are often utilized as polymeric biomaterials in drug delivery. Among these, dietary polysaccharides extracted from okra, yam, and spirulina have been reported to stimulate innate immunity with well-known nutritional benefits. In this study, we developed a dietary-polysaccharide-modified fish-oil-based emulsion for oral co-delivery of a hydrophilic PD-L1 blocking peptide and the hydrophobic small molecule simvastatin. The optimal emulsion was nano-sized and exhibited a negative surface charge, high drug encapsulation efficiency of over 97%, low viscosity, and sustained drug release manner. The formulation could significantly increase the uptake of peptides by intestinal Caco-2 cells, which demonstrated the great potential of the formulation for promoting the oral absorption of peptides. Additionally, these dietary polysaccharides could promote dendritic cell maturation and cytokine expression in macrophages, demonstrating that these nutraceutical polysaccharides had dual roles of functioning as promising colloidal delivery systems and as potential immune modulators or adjuvants. Thus, this food-based colloidal delivery system shows promise for the oral delivery of peptide drugs and lays a great platform for future applications in immunotherapy.

GABA regulates IL-1β production in macrophages.

Neurotransmitters have been well documented to determine immune cell fates; however, whether and how γ-amino butyric acid (GABA) shapes the function of innate immune cells is still obscure. Here, we demonstrate that GABA orchestrates macrophage maturation and inflammation. GABA treatment during macrophage maturation inhibits interleukin (IL)-1β production from inflammatory macrophages. Mechanistically, GABA enhances succinate-flavin adenine dinucleotide (FAD)-lysine specific demethylase1 (LSD1) signaling to regulate histone demethylation of Bcl2l11 and Dusp2, reducing formation of the NLRP3-ASC-Caspase-1 complex. The GABA-succinate axis reduces succinylation of mitochondrial proteins to promote oxidative phosphorylation (OXPHOS). We also find that GABA alleviates lipopolysaccharides (LPS)-induced sepsis as well as high-fat-diet-induced obesity in mice. Our study shows that GABA regulates pro-inflammatory macrophage responses associated with metabolic reprogramming and protein succinylation, suggesting a strategy for treating macrophage-related inflammatory diseases.

Mesenchymal stem cells alleviate systemic sclerosis by inhibiting the recruitment of pathogenic macrophages

Systemic sclerosis (SSc) is a recalcitrant autoimmune disease for which there is no cure. Mesenchymal stem cell (MSC)-based treatment has emerged as a promising therapeutic option for several autoimmune diseases. Previously, we found that the immunoregulatory potential of MSCs can be greatly enhanced by IFN-γ and TNF-α. Here, we found that IFN-γ- and TNF-α-pretreated MSCs significantly alleviated skin fibrosis in a bleomycin (BLM)-induced SSc model. Macrophages were found to be the predominant profibrotic immune cell population in the pathogenesis of SSc. The accumulation of macrophages was significantly decreased by MSC treatment. Importantly, MSCs primarily reduced the population of maturing macrophages with high CCR2 expression by inhibiting the generation of CCL2 from fibroblasts and macrophages. This finding may help to improve MSC-based clinical treatments for SSc patients.

Manganese suppresses the development of oral leukoplakia by activating the immune response.

Manganese ion (Mn2+ ) is reported to promote the antitumor immune response by activating the cGAS-STING pathway, but it is unknown whether Mn2+ can prevent the malignant transformation of precancerous lesions. The effects of Mn2+ in treating oral leukoplakia (OLK) were explored in this work. Peripheral blood Mn analysis of the patients was performed using inductively coupled plasma atomic emission spectroscopy (ICP-AES). A coculture model of dendritic cells (DCs)/macrophages, CD8+ T cells, and dysplastic oral keratinocytes (DOKs) was employed to analyze the role and mechanism of Mn2+ in a simulated OLK immune microenvironment. Western blot, RT-PCR, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and lactate dehydrogenase (LDH) assays were adopted to detect the mechanism of Mn2+ in this model. 4-nitroquinoline oxide (4NQO)-induced OLK mice were used to assess the role of Mn2+ in suppressing OLK progression, and a novel Mn2+ -loaded guanosine-tannic acid hydrogel (G-TA@Mn2+ hydrogel) was fabricated and evaluated for its advantages in OLK therapy. The content of Mn in patients' peripheral blood was negatively related to the progression of OLK. Mn2+ promoted the maturation and antigen presentation of DCs and macrophages and enhanced the activation of CD8+ T cells in the coculture model, resulting in effective killing of DOKs. Mechanistic analysis found that Mn2+ enhanced the anti-OLK immune response by activating the cGAS-STING pathway. Moreover, Mn2+ suppressed the development of 4NQO-induced carcinogenesis in the mouse model. In addition, the G-TA@Mn2+ hydrogel had better anti-OLK effects. Mn2+ enhanced the anti-OLK immune response by activating the cGAS-STING pathway, and the G-TA@Mn2+ hydrogel is a potential novel therapeutic approach for OLK treatment.

Isocitrate Dehydrogenase 2 Mutation Drives Bone Marrow Macrophage Dysfunction without a Complete Block in Hematopoietic Differentiation

Myelodysplastic syndromes (MDS) represent a broad category of myeloid malignancies characterized by myeloid skewed hematopoiesis, blood cytopenias and increased risk of transformation to leukemia. While not common, isocitrate dehydrogenase 2 (IDH2) mutations are represented in clonal hematopoiesis and in MDS, are found in patients with higher International Prognosis Scoring System (IPSS) disease scores and confer a higher risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite which disrupts hematopoiesis. However, the mechanisms that induces clonal progression and transformation remains incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutation on mature myeloid cell functions and the bone marrow microenvironment (BMME) remains to be elucidated. Using RAW264.7 cells, a monocyte/macrophage-like murine cell line, we retrovirally infected cells with human wt IDH2 or the R172K IDH2 mutant, a common IDH2 mutation. After confirming a 30-fold increase of 2HG (p<.005) and observing evidence of hypermethylation in the IDH2 mutant RAW cells, we tested several key functions of bone marrow macrophages: ability to engulf apoptotic cells (efferocytosis), erythropoietic support, and cytokine production. After labeled apoptotic neutrophils were fed as efferocytic targets to RAW cells at a 10:1 ratio for 6 hours, we observed a 10% decrease of efferocytosis in IDH2 mutants (p < 0.05). Next, we cocultured RAW cells with primary murine late-stage erythroid progenitors and quantified their capacity to form colonies (CFU-E) in the presence of erythropoietin. Compared to wt, IDH2 mutant RAW cells inhibited CFU-E formation by 25% (p < 0.0075). Cytokine array on cell culture supernatant from mutant RAW cells had elevated proinflammatory chemokines/cytokines (CCL5, CXCL10, TNF-a) and decreased cytokines related to inflammation resolution and efferocytosis (GDF-15, Gas6, Endostatin, CCL2). To test these findings further in vivo we generated a murine bone marrow transplant model with retrovirally infected hematopoietic stem cells (HSC: lineage-, c-Kit+, Sca-1+) containing the human wt or R172K IDH2 mutation with an mCherry reporter and transplanted into CD45.1 congenic mice (10 mutant, 10 wt, 1:1 male:female). Mutant IDH2 HSCs demonstrated better engraftment (85-90%) compared to wt (70-80%) and also had higher levels of mCherry positivity within CD45.2+ cells (80-90% mutant vs 40-60% in wt). Longitudinal blood draws showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes in IDH2 mutants by 16 weeks post-transplant. In contrast to current literature, IDH2 mutant cells did not exhibit a complete block in myeloid cell differentiation, as the mutant clone gave rise to mature monocytes, macrophages, and neutrophils. We also harvested bone marrow at 16 weeks post transplantation. Compared to wt IDH2, the mutant IDH2 bone marrow cells exhibited myeloid skewing hematopoiesis and a 3-fold increase in bone marrow monocytes (CD45+, Ly6G-, Ly6C++, F4/80+, p < 0.00001). In the IDH2 mutant bone marrow cells, myeloid progenitors were also expanded: common myeloid progenitors (CMPs: 4-fold increase p < 0.001, CD45+, lineage-, c-Kit+, Sca-1-, CD34+, CD16/32-), and granulocytic monocytic precursors (GMPs: 3-fold increase p < 0.0001, CD45+, c-Kit+, Sca-1-, CD34+, CD16/32+). We also observed a previously unreported loss of long-term hematopoietic stem cells (LT-HSC: 13.5-fold reduction, p < 0.00001, CD45+, lineage-, c-Kit+, Sca-1+, Flt3-, CD48-, CD150+). When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow derived macrophages fed normal apoptotic neutrophils at a 10:1 ratio for 3 or 24 hours. In summary, we identify defects in efferocytic function and cytokine production in monocytes and macrophages derived from IDH2 mutant hematopoietic stem and progenitor cells. We have previously shown that defects in the efferocytic function of bone marrow macrophages induce increases in inflammatory signals and contribute to skewing of hematopoiesis. Since these macrophage dysfunctions have not previously been attributed to IDH2 single mutation, they represent a potential novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and transformation to leukemia.

Targeted Genomic Insertion of Cars in iPSC-Derived Macrophages Leads to Improved Expression and Anti-Tumor Activity

Expression of chimeric antigen receptors (CARs) provides an important strategy to improve the anti-tumor activity of immune cells. T cells equipped with CARs specific to tumor antigens have been very effective for treatment of B cell malignancies. However, CAR-T cell-mediated treatment had limited success in solid tumors. Since macrophages are capable of infiltrating into tumor microenvironment, detecting phagocytic targets and engulfing them, as well as stimulating other endogenous immune cells, macrophages engineered with CAR constructs have been gaining interest for cancer immunotherapy. We have successfully generated CAR-expressing induced pluripotent stem cells (iPSCs) through either PiggyBac transposon-mediated transfection or lentiviral transduction. Subsequently, the CAR-expressing iPSCs were differentiated into macrophages (iPSC-CARMACs). The CAR expression in undifferentiated iPSCs were high and stable, but gradually downregulated during the differentiation into macrophages. Due to the loss of CAR expression, these iPSC-CARMACs only modestly improved phagocytic activity compared to wild-type iPSC-derived macrophages (iPSC-MACs) in vitro. Additionally, we did not find a significant benefit for in vivo anti-tumor activity of the iPSC-CARMACs compared to iPSC-MACs. We hypothesized the deficiency of anti-tumor activity might be due to the loss of CAR-expression that can become silenced after random integration into the genome. Therefore, to test this hypothesis as well as improve and stabilize the CAR expression, we engineered human iPSCs using CRISPR-Cas9 mediated CAR insertion into AAVS1 locus, a known safe harbor locus that is less prone to genetic silencing. We engineered a series of CAR constructs containing the same anti-Mesothelin (Meso) extracellular single-chain antibody variable fragment, combined with different intracellular signaling domains including Bai1, MegF10, MerTK and CD3ζ. To determine the phagocytic activity of the anti-Meso CARs expressing these different intracellular domains, we initially introduced CAR constructs into THP-1 cells. We tested these CAR-expressing THP-1-derived macrophages in phagocytosis assay with Meso-expressing A1847 ovarian cancer cells. We found Bai1 and MegF10 triggered higher levels of engulfment of tumor cells than other CAR constructs either by themselves or when combined with anti-CD47 antibody (Figure A). Given the potential anti-tumor activity of anti-Meso-Bai1 and anti-Meso-MegF10 CARs, we next generated anti-Meso-Bai1 and anti-Meso-MegF10 CAR-expressing iPSC-CARMACs by targeting at AAVS1 locus to improve CAR expression compared to the previous iPSC-CARMACs made by transposon transfection or lentiviral transduction. We successfully obtained iPSCs expressing anti-Meso-Bai1 CAR integrated into AAVS1 site (AAVS1-Meso-Bai1-iPSCs). We have differentiated the AAVS1-Meso-Bai1-iPSCs to macrophages (Meso-Bai1-CARMACs) and demonstrated Meso-Bai1-CARMACs significantly improved phagocytic activity compared to iPSC-MACs with or without the addition of anti-CD47 antibody (Figure B). Notably, CAR expression was stably maintained at a high level on the mature macrophages generated from the AAVS1-Meso-Bai1 iPSCs compared to those either from transposon or lentiviral vector mediated CAR expressing iPSCs (Figure C). We isolated two homozygous clones of AAVS1-Meso-Bai1 iPSCs that have even stronger CAR expression than bulk population of AAVS1-Meso-Bai1 iPSCs. The differentiation of these single clones, as well as the evaluation of their in vitro activity are in progress. We are also testing the in vivo function of these new AAVS1-iPSC-CARMACs in an ovarian tumor model. Together, these studies demonstrate that we can successfully produce CAR-expressing iPSC derived macrophages, which exhibit improved anti-tumor activity compared to wild-type iPSC derived macrophages. CAR expression can be best maintained on macrophages by AAVS1-locus targeting insertion. Importantly, these studies also demonstrate that strategies used to genetically modify iPSC-derived cells may need to be modified or optimized depending on which mature cell type of interest is being produced. That is, while randomly integrated CARs seem to work well in iPSC-derived NK cells and T cells, this targeted insertion approach is likely needed for derivation of more functional iPSC-derived CAR-macrophages. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterization of immunomodulatory responses induced by manuka honey.

Manuka honey (MH) is known for its wound-healing, anti-microbial, anti-oxidant and anti-tumor properties. However, there is conflicting evidence regarding the role of MH in inflammatory responses, with some studies highlighting its pro-inflammatory capacity and others showing that it has a predominantly anti-inflammatory activity. The current study is aimed at characterizing the immunomodulatory capacity of MH using both in vitro and in vivo approaches, focusing on the underlying mechanisms. Treatment of RAW 264.7 macrophages with 1% MH (w/v) resulted in a significant increase in the gene expression (~26-fold) and secretion (~27-fold) of tumor necrosis factor-alpha (TNF-α). Similarly, an increase was observed in the gene expression of other inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS), as well as the chemokines; (C-X-C motif) ligand 2 (CXCL2) and (C-C) motif ligand 2 (CCL2). Using an in vivo model, intraperitoneal (i.p.) administration of MH in C57BL/6 mice elicited a peritoneal response characterized by a significant expansion in the number of peritoneal exudate cells (PECs), which was mainly due to a 35-fold increase in the recruitment of neutrophils. Importantly, this response was evident in toll-like receptor 4 (TLR4)-defective C3H/HeJ mice, indicating that the observed stimulatory effect occurs independently of TLR4 and unlikely to be mediated by any lipopolysaccharide (LPS) contaminant. MH administration also led to changes in the phenotypic expression and functional maturation of peritoneal macrophages, as evidenced by a shift towards the CD11blo F4/80lo phenotype and an increase in the expression of major histocompatibility complex (MHC) class II proteins. In contrast, the MH-initiated peritoneal response was largely abrogated in mice deficient in myeloid differentiation primary response 88 (MyD88) protein, a critical adaptor of most TLR signaling pathways. Thus, the current findings help to characterize the immunostimulatory properties of MH and their dependence on TLR signaling, and highlight the potential utility of MH as an immunomodulatory agent in a variety of disorders.

Early treatment with anti-α4β7 antibody facilitates increased gut macrophage maturity in SIV-infected rhesus macaques.

Despite advances in combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to experience gastrointestinal dysfunction. Infusions of anti-α4β7 monoclonal antibodies (mAbs) have been proposed to increase virologic control during simian immunodeficiency virus (SIV) infection in macaques with mixed results. Recent evidences suggested that therapeutic efficacy of vedolizumab (a humanized anti-α4β7 mAb), during inflammatory bowel diseases depends on microbiome composition, myeloid cell differentiation, and macrophage phenotype. We tested this hypothesis in SIV-infected, anti-α4β7 mAb-treated macaques and provide flow cytometric and microscopic evidence that anti-α4β7 administered to SIV-infected macaques increases the maturity of macrophage phenotypes typically lost in the small intestines during SIV disease progression. Further, this increase in mature macrophage phenotype was associated with tissue viral loads. These phenotypes were also associated with dysbiosis markers in the gut previously identified as predictors of HIV replication and immune activation in PLWH. These findings provide a novel model of anti-α4β7 efficacy offering new avenues for targeting pathogenic mucosal immune response during HIV/SIV infection.

Unbalanced Arginine pathway and altered maturation of pleural macrophages in Th2-deficient mice during Litomosoides sigmodontis filarial infection.

Filarial parasites are tissue dwelling worms transmitted by hematophagous vectors. Understanding the mechanisms regulating microfilariae (the parasite offspring) development is a prerequisite for controlling transmission in filarial infections. Th2 immune responses are key for building efficient anti-parasite responses but have been shown to also lead to detrimental tissue damage in the presence of microfilariae. Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity was therefore used to characterize pleuropulmonary pathology and associated immune responses in wild-type and Th2 deficient mice. Wild-type and Th2-deficient mice (Il-4rα-/-/Il-5-/- ) were infected with L. sigmodontis and parasite outcome was analyzed during the patent phase (when microfilariae are in the general circulation). Pleuropulmonary manifestations were investigated and pleural and bronchoalveolar cells were characterized by RNA analysis, imaging and/or flow cytometry focusing on macrophages. Il-4rα-/-/Il-5-/- mice were hypermicrofilaremic and showed an enhanced filarial survival but also displayed a drastic reduction of microfilaria-driven pleural cavity pathologies. In parallel, pleural macrophages from Il-4rα-/-/Il-5-/- mice lacked expression of prototypical alternative activation markers RELMα and Chil3 and showed an altered balance of some markers of the arginine metabolic pathway. In addition, monocytes-derived F4/80intermediate macrophages from infected Il-4rα-/-/Il-5-/- mice failed to mature into resident F4/80high large macrophages. Altogether these data emphasize that the presence of both microfilariae and IL-4R/IL-5 signaling are critical in the development of the pathology and in the phenotype of macrophages. In Il-4rα-/-/Il-5-/- mice, the balance is in favor of parasite development while limiting the pathology associated with the host immune response.

Regulation of the macrophage-hepatic stellate cell interaction by targeting macrophage peroxisome proliferator-activated receptor gamma to prevent non-alcoholic steatohepatitis progression in mice.

Macrophages display remarkable plasticity and can interact with surrounding cells to affect hepatic immunity and tissue remodelling during the progression of liver diseases. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a critical role in macrophage maturation, polarization and metabolism. In this study, we investigated the role of PPARγ in macrophage-hepatic stellate cell (HSC) interaction during non-alcoholic steatohepatitis (NASH) development. Wild-type, Ppargfl/fl and PpargΔLyz2 mice were fed a methionine- and choline-deficient (MCD) diet to induce NASH. Depletion of macrophages was performed using an injection of gadolinium chloride intraperitoneally. PPARγ-overexpressing or PPARγ-knockout macrophages were stimulated with saturated fatty acid (SFA) and cocultured with HSCs in a conditioned medium or the transwell coculture system. Depletion of macrophages inhibited HSC activation and ameliorated NASH progression in MCD diet-fed mice. Coculturing HSCs with macrophages or culturing HSCs in a macrophage-conditioned medium-facilitated HSC activation, and this effect was magnified when macrophages were metabolically activated by SFA. Moreover, the absence of PPARγ in macrophages enhanced metabolic activation, promoting the migration and activation of HSCs through IL-1β and CCL2. In contrast, overexpression of PPARγ in macrophages obtained the opposite effects. In vivo, macrophage-specific PPARγ knockout affected the phenotype of hepatic macrophages and HSCs, involving the MAPK and NLRP3/caspase-1/IL-1β signalling pathways. Infiltrating hepatic monocyte-derived macrophages became the predominant macrophages in NASH liver, especially in PpargΔLyz2 mice, paralleling with aggravated inflammation and fibrosis. Regulating macrophage PPARγ affected the metabolic activation of macrophages and their interaction with HSCs. Macrophage-specific PPARγ may be an attractive therapeutic target for protecting against NASH-associated inflammation and fibrosis.

Dielectrophoretic characterization of macrophage phenotypes.

Different macrophage phenotypes play important roles in diverse biological processes and diseases. In this study, we have characterized the dielectrophoretic responses of human monocytes and macrophage phenotypes: nonactivated (M0), pro-inflammatory (M1), and pro-healing (M2a). Dielectrophoretic responses of cells change as a function of frequency of the applied electric field. We measured the crossover frequency at which cells transition from negative to positive dielectrophoresis (DEP) or vice versa using interdigitated electrodes. For these characterization experiments, we also developed a new low-conductivity media formulation that retained 100% of the initial viability for 1h. Human THP1 monocytes showed a distinguishable DEP response from mature macrophages. M1 macrophages also showed a distinct DEP response compared to M0 and M2a macrophages. No clear distinction could be drawn between M0 and M2a. The median values of the crossover frequencies of monocytes, M0, M1, and M2a were 38, 21, 11, and 23kHz, respectively. Membrane capacitances of these cells were calculated consequently, and the values were 0.0111, 0.0128, 0.0244, and 0.0117F/m2 for monocytes, M0, M1, and M2a, respectively. These results show how bioelectric properties are influenced by changes in macrophage phenotype.