A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells.

Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings.

A Monocyte-Orchestrated IFN-I-to-IL-4 Cytokine Axis Instigates Protumoral Macrophages and Thwarts Poly(I:C) Therapy.

Type I IFNs (IFN-I) are important for tumor immune surveillance and contribute to the therapeutic responses for numerous treatment regimens. Nevertheless, certain protumoral activities by IFN-I have been increasingly recognized. Indeed, our recent work showed that systemic poly(I:C)/IFN treatment can undesirably trigger high arginase (ARG1) expression within the tumor-associated monocyte/macrophage compartment. Using a line of CRISPR-generated Arg1-YFP reporter knock-in mice, we have determined that a subset of tumor-associated macrophages represent the major Arg1-expressing cell type following poly(I:C)/IFN stimulation. More detailed analyses from in vitro and in vivo models demonstrate a surprising IFN-to-IL-4 cytokine axis in transitional monocytes, which can subsequently stimulate IL-4 target genes, including Arg1, in macrophages. Intriguingly, IFN stimulation of transitional monocytes yielded concurrent M2 (YFP+)- and M1 (YFP-)-skewed macrophage subsets, correlated with an inhibitory crosstalk between IFN-I and IL-4. Genetic abrogation of IL-4 signaling in mice diminished poly(I:C)/IFN-induced ARG1 in tumors, leading to enhanced activation of CD8+ T cells and an improved therapeutic effect. The present work uncovered a monocyte-orchestrated macrophage phenotype conversion mechanism that may have broad implications.

Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.

Melanoma-associated fibroblasts impair CD8+ T cell function and modify expression of immune checkpoint regulators via increased arginase activity.

This study shows that melanoma-associated fibroblasts (MAFs) suppress cytotoxic T lymphocyte (CTL) activity and reveals a pivotal role played by arginase in this phenomenon. MAFs and normal dermal fibroblasts (DFs) were isolated from surgically resected melanomas and identified as Melan-A-/gp100-/FAP+ cells. CTLs of healthy blood donors were activated in the presence of MAF- and DF-conditioned media (CM). Markers of successful CTL activation, cytotoxic degranulation, killing activity and immune checkpoint regulation were evaluated by flow cytometry, ELISPOT, and redirected killing assays. Soluble mediators responsible for MAF-mediated effects were identified by ELISA, flow cytometry, inhibitor assays, and knock-in experiments. In the presence of MAF-CM, activated/non-naïve CTLs displayed dysregulated ERK1/2 and NF-κB signaling, impeded CD69 and granzyme B production, impaired killing activity, and upregulated expression of the negative immune checkpoint receptors TIGIT and BTLA. Compared to DFs, MAFs displayed increased amounts of VISTA and HVEM, a known ligand of BTLA on T cells, increased L-arginase activity and CXCL12 release. Transgenic arginase over-expression further increased, while selective arginase inhibition neutralized MAF-induced TIGIT and BTLA expression on CTLs. Our data indicate that MAF interfere with intracellular CTL signaling via soluble mediators leading to CTL anergy and modify immune checkpoint receptor availability via L-arginine depletion.

The pro-apoptotic ARTS protein induces neutrophil apoptosis, efferocytosis, and macrophage reprogramming to promote resolution of inflammation.

ARTS (Sept4_i2) is a pro-apoptotic protein and a product of the Sept4 gene. ARTS acts upstream of mitochondria to initiate caspase activation. ARTS induces apoptosis by specifically binding XIAP and allowing de-repression of active caspases required for Mitochondrial Outer Membrane Permeabilzation (MOMP). Moreover, ARTS promotes apoptosis by inducing ubiquitin-mediated degradation of both major anti-apoptotic proteins XIAP and Bcl-2. In the resolution phase of inflammation, the infiltrating leukocytes, which execute the acute innate response, undergo apoptosis and are subsequently cleared by phagocytic macrophages (i.e. efferocytosis). In this course, macrophages undergo reprogramming from inflammatory, to anti-inflammatory, and eventually to resolving macrophages that leave the injury sites. Since engulfment of apoptotic leukocytes is a key signaling step in macrophage reprogramming and resolution of inflammation, we hypothesized that a failed apoptosis in leukocytes in vivo would result in an impaired resolution process. To test this hypothesis, we utilized the Sept4/ARTS-/- mice, which exhibit resistance to apoptosis in many cell types. During zymosan A-induced peritonitis, Sept4/ARTS-/- mice exhibited impaired resolution of inflammation, characterized by reduced neutrophil apoptosis, macrophage efferocytosis and expression of pro-resolving mediators. This was associated with increased pro-inflammatory cytokines and reduced anti-inflammatory cytokines, secreted by resolution-phase macrophages. Moreover, ARTS overexpression in leukocytes in vitro promoted an anti-inflammatory behavior. Overall, our results suggest that ARTS is a key master-regulator necessary for neutrophil apoptosis, macrophage efferocytosis and reprogramming to the pro-resolving phenotype during the resolution of inflammation.

Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens.

Toll-like receptor (TLR) signaling in macrophages is required for antipathogen responses, including the biosynthesis of nitric oxide from arginine, and is essential for immunity to Mycobacterium tuberculosis, Toxoplasma gondii and other intracellular pathogens. Here we report a 'loophole' in the TLR pathway that is advantageous to these pathogens. Intracellular pathogens induced expression of the arginine hydrolytic enzyme arginase 1 (Arg1) in mouse macrophages through the TLR pathway. In contrast to diseases dominated by T helper type 2 responses in which Arg1 expression is greatly increased by interleukin 4 and 13 signaling through the transcription factor STAT6, TLR-mediated Arg1 induction was independent of the STAT6 pathway. Specific elimination of Arg1 in macrophages favored host survival during T. gondii infection and decreased lung bacterial load during tuberculosis infection.

Myeloid-derived suppressor cells shift Th17/Treg ratio and promote systemic lupus erythematosus progression through arginase-1/miR-322-5p/TGF-ß pathway.

Immune cells play important roles in systemic lupus erythematosus (SLE). We previously found that myeloid-derived suppressor cell (MDSC)-derived arginase-1 (Arg-1) promoted Th17 cell differentiation in SLE. In the present study, we performed RNA-chip to identify the microRNA regulation network between MDSCs and Th17 cells. miR-542-5p in humans, as the homologous gene of miR-322-5p in mice was significantly up-regulated in the Th17+MDSC group compared with Th17 cells cultured alone and down-regulated in the Th17+MDSC+Arg-1 inhibitor group compared with the Th17+MDSC group. We further evaluated the miR-322-5p and Th17/Treg balance in mice and found that the proportions of both Th17 cells and Tregs were elevated and that miR-322-5p overexpression activated the transforming growth factor-ß pathway. Moreover, although miR-322-5p expression was higher in SLE mice, it decreased after treatment with an Arg-1 inhibitor. The proportion of Th17 cells and Th17/Treg ratio correlated with miR-322-5p levels. In conclusion, MDSC-derived Arg-1 and mmu-miR-322-5p not only promote Th17 cell and Treg differentiation, but also shift the Th17/Treg ratio in SLE. The Arg-1/miR-322-5p axis may serve as a novel treatment target for SLE.

Interactions between macrophages and helminths.

Macrophages, the major population of tissue-resident mononuclear phagocytes, contribute significantly to the immune response during helminth infection. Alternatively activated macrophages (AAM) are induced early in the anti-helminth response following tissue insult and parasite recognition, amplifying the early type 2 immune cascade initiated by epithelial cells and ILC2s, and subsequently driving parasite expulsion. AAM also contribute to functional alterations in tissues infiltrated with helminth larvae, mediating both tissue repair and inflammation. Their activation is amplified and occurs more rapidly following reinfection, where they can play a dual role in trapping tissue migratory larvae and preventing or resolving the associated inflammation and damage. In this review, we will address both the known and emerging roles of tissue macrophages during helminth infection, in addition to considering both outstanding research questions and new therapeutic strategies.

Myeloid-Derived Suppressor Cells Contribute to Susceptibility to Trypanosoma congolense Infection by Suppressing CD4+ T Cell Proliferation and IFN-γ Production.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of bone marrow-derived myeloid cells that have immune-suppressive activities. These cells have been reported to suppress T cell immunity against tumors as well as in some parasitic and bacterial infections. However, their role during Trypanosoma congolense infection has not been studied. Given that immunosuppression is a hallmark of African trypanosomiasis, we investigated the role of MDSCs in immunity to T. congolense infection. We found increased numbers of MDSCs in the spleen and liver of infected mice, which correlated with increased parasitemia. Depletion of MDSCs significantly increased the percentage of proliferating and IFN-γ-producing CD4+ T cells from the spleen of T. congolense-infected mice. Furthermore, MDSCs from T. congolense-infected mice directly suppressed CD4+ T cell proliferation in a coculture setting. This suppressive effect was abolished by the arginase-1 inhibitor, Nω-hydroxy-nor-l-arginine (nor-NOHA), indicating that MDSCs suppress CD4+ T cell proliferation and function in an arginase-1-dependent manner. Indeed, depletion of MDSCs during infection led to control of the first wave of parasitemia and prolonged survival of infected mice. This was also associated with increased CD4+ T cell proliferation and IFN-γ production. Taken together, our findings identify an important role of MDSCs in the pathogenesis of experimental T. congolense infection via suppression of T cell proliferative and effector cytokine responses in an arginase-1-dependent manner.

CD71+ Erythroid Suppressor Cells Promote Fetomaternal Tolerance through Arginase-2 and PDL-1.

Survival of the allogeneic pregnancy depends on the maintenance of immune tolerance to paternal alloantigens at the fetomaternal interface. Multiple localized mechanisms contribute to the fetal evasion from the mother's immune rejection as the fetus is exposed to a wide range of stimulatory substances such as maternal alloantigens, microbes and amniotic fluids. In this article, we demonstrate that CD71+ erythroid cells are expanded at the fetomaternal interface and in the periphery during pregnancy in both humans and mice. These cells exhibit immunosuppressive properties, and their abundance is associated with a Th2 skewed immune response, as their depletion results in a proinflammatory immune response at the fetomaternal interface. In addition to their function in suppressing proinflammatory responses in vitro, maternal CD71+ erythroid cells inhibit an aggressive allogeneic response directed against the fetus such as reduction in TNF-α and IFN-γ production through arginase-2 activity and PD-1/programmed death ligand-1 (PDL-1) interactions. Their depletion leads to the failure of gestation due to the immunological rejection of the fetus. Similarly, fetal liver CD71+ erythroid cells exhibit immunosuppressive activity. Therefore, immunosuppression mediated by CD71+ erythroid cells on both sides (mother/fetus) is crucial for fetomaternal tolerance. Thus, our results reveal a previously unappreciated role for CD71+ erythroid cells in pregnancy and indicate that these cells mediate homeostatic immunosuppressive/immunoregulatory responses during pregnancy.

IL-35Ig-expressing dendritic cells induce tolerance via Arginase 1.

The cytokine interleukin IL-35 is known to exert strong immunosuppressive functions. Indoleamine 2,3-dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL-35 and the activity of those enzymes. We transfected a single chain IL-35Ig gene construct in murine splenic DCs (DC35 ) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL-35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC35 , and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed-type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC35 was associated with the detection of CD25+ CD39+ , rather than Foxp3+ , regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC35 appears to be an early event in IL-35Ig-mediated immunosuppression.

Flow cytometric analysis of the leukocyte landscape during bleomycin-induced lung injury and fibrosis in the rat.

Bleomycin-induced lung injury and fibrosis is a well-described model to investigate lung inflammatory and remodeling mechanisms. Rat models are clinically relevant and are also widely used, but rat bronchoalveolar lavage (BAL) cells are not fully characterized with flow cytometry due to the limited availability of antibodies for this species. We optimized a comprehensive time-dependent flow cytometric analysis of cells after bleomycin challenge, confirming previous studies in other species and correlating them to histological staining, cytokine profiling, and collagen accumulation analysis in rat lungs. For this purpose, we describe a novel panel of rat surface markers and a strategy to identify and follow BAL cells over time. By combining surface markers in rat alveolar cells (CD45+), granulocytes and other myeloid cells, monocytes and macrophages can be identified by the expression of CD11b/c. Moreover, different activation states of macrophages (CD163+) can be observed: steady state (CD86-MHC-IIlow), activation during inflammation (CD86+,MHC-IIhigh), activation during remodeling (CD86+MHC-IIlow), and a population of newly recruited monocytes (CD163-α-granulocyte-). Hydroxyproline measured as marker of collagen content in lung tissue showed positive correlation with the reparative phase (CD163- cells and tissue inhibitor of metalloproteinases (TIMP) and IL-10 increase). In conclusion, after a very early granulocytic recruitment, inflammation in rat lungs is observed by activated macrophages, and high release of IL-6 and fibrotic remodeling is characterized by recovery of the macrophage population together with TIMP, IL-10, and IL-18 production. Recruited monocytes and a second peak of granulocytes appear in the transitioning phase, correlating with immunostaining of arginase-1 in the tissue, revealing the importance of events leading the changes from injury to aberrant repair.

Immune off-target effects of Brentuximab Vedotin in relapsed/refractory Hodgkin Lymphoma.

Hodgkin Lymphoma (HL) is associated with deep microenvironment re-shaping and myeloid dysfunction. Given that only limited data are available regarding the role of Brentuximab Vedotin (BV) as single agent in transplant-naive relapsed/refractory (R/R) patients and its off-target effects on immune system, we evaluated the amount of regulatory T-cells (T-regs), myeloid-derived suppressor cells (MDSC) subpopulations, and their functional marker, serum arginase-1 (s-Arg-1), in peripheral blood of 15 consecutive R/R HL patients. After a median of four BV cycles, the overall response rate (complete response + partial response) was 47%, with 4 (27%) complete metabolic remissions. BV reduced the absolute number of three MDSC subtypes and s-Arg-1 levels. Patients with baseline s-Arg-1 ≥200 ng/ml had inferior progression-free survival at 36 months compared to those with low s-Arg-1. T-regs dysfunction was recovered by BV: absolute T-regs count was increased after treatment with BV, independently of metabolic response achieved, with a significant reduction of CD30+ T-regs. Our data disclose off-target effects of BV in the microenvironment that could explain its deep and durable clinical efficacy.

Arginase-1-based vaccination against the tumor microenvironment: the identification of an optimal T-cell epitope.

L-arginine depletion by regulatory cells and cancer cells expressing arginase-1 (Arg-1) is a vital contributor to the immunosuppressive tumor microenvironment in patients with cancer. We have recently described the existence of pro-inflammatory effector T cells that recognize Arg-1. Hence, Arg-1-specific self-reactive T cells are a naturally occurring part of the memory T-cell repertoire of the human immune system. Here, we further characterize a highly immunogenic epitope from Arg-1. We describe frequent T-cell-based immune responses against this epitope in patients with cancer, as well as in healthy donors. Furthermore, we show that Arg-1-specific T cells expand in response to the TH2 cytokine interleukin (IL)-4 without any specific stimulation. Arg-1-specific memory TH1 cells that respond to increased IL-4 concentration may, therefore, drive the immune response back into the TH1 pathway. Arg-1-specific T cells thus appear to have an important function in immune regulation. Because Arg-1 plays an important role in the immunosuppressive microenvironment in most cancers, an immune modulatory vaccination approach can readily be employed to tilt the balance away from immune suppression in these settings.

Immunometabolic phenotype of BV-2 microglia cells upon murine cytomegalovirus infection.

Microglia are resident brain macrophages with key roles in development and brain homeostasis. Cytomegalovirus (CMV) readily infects microglia cells, even as a possible primary target of infection in development. Effects of CMV infection on a cellular level in microglia are still unclear; therefore, the aim of this research was to assess the immunometabolic changes of BV-2 microglia cells following the murine cytomegalovirus (MCMV) infection. In light of that aim, we established an in vitro model of ramified BV-2 microglia (BV-2∅FCS, inducible nitric oxide synthase (iNOSlow), arginase-1 (Arg-1high), mannose receptor CD206high, and hypoxia-inducible factor 1α (HIF-1αlow)) to better replicate the in vivo conditions by removing FCS from the cultivation media, while the cells cultivated in 10% FCS DMEM displayed an ameboid morphology (BV-2FCS high, iNOShigh, Arg-1low, CD206low, and HIF-1αhigh). Experiments were performed using both ramified and ameboid microglia, and both of them were permissive to productive viral infection. Our results indicate that MCMV significantly alters the immunometabolic phenotypic properties of BV-2 microglia cells through the manipulation of iNOS and Arg-1 expression patterns, along with an induction of a glycolytic shift in the infected cell cultures.

The AP-1 Transcription Factor c-Jun Promotes Arthritis by Regulating Cyclooxygenase-2 and Arginase-1 Expression in Macrophages.

Activation of proinflammatory macrophages is associated with the inflammatory state of rheumatoid arthritis. Their polarization and activation are controlled by transcription factors such as NF-κB and the AP-1 transcription factor member c-Fos. Surprisingly, little is known about the role of the AP-1 transcription factor c-Jun in macrophage activation. In this study, we show that mRNA and protein levels of c-Jun are increased in macrophages following pro- or anti-inflammatory stimulations. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment cluster analyses of microarray data using wild-type and c-Jun-deleted macrophages highlight the central function of c-Jun in macrophages, in particular for immune responses, IL production, and hypoxia pathways. Mice deficient for c-Jun in macrophages show an amelioration of inflammation and bone destruction in the serum-induced arthritis model. In vivo and in vitro gene profiling, together with chromatin immunoprecipitation analysis of macrophages, revealed direct activation of the proinflammatory factor cyclooxygenase-2 and indirect inhibition of the anti-inflammatory factor arginase-1 by c-Jun. Thus, c-Jun regulates the activation state of macrophages and promotes arthritis via differentially regulating cyclooxygenase-2 and arginase-1 levels.

Recombinant human arginase I elicited immunosuppression in activated macrophages through inhibiting autophagy.

Arginase I has been documented to impair T cell function and attenuate cellular immunity, however, there is little evidence to reveal the effect of arginase I on macrophage function. Recently, recombinant human arginase I (rhArg) has been developed for cancer therapy and is in clinical trial for hepatocellular carcinoma, whereas the potential immunosuppression induced by rhArg limited its therapeutic efficacy. To improve the clinical outcome of rhArg, addressing the immune suppression appears to be particularly important. In this study, we found that rhArg attenuated macrophage functions, including inhibiting macrophage cell proliferation, nitric oxide (NO) and reactive oxygen species (ROS) production, cytokine secretion, MHC-II surface expression, and phagocytosis, thereby inducing immunosuppression in lipopolysaccharides (LPS)/interferon-γ (IFN-γ)-activated macrophages. Notably, we observed that rhArg downregulated autophagy in activated macrophages. Moreover, application of trehalose (an autophagy inducer) significantly restored the impaired immune function in activated macrophages, suggesting the essential role of autophagy in rhArg-induced immunosuppression. To further illustrate the effect of autophagy in immunosuppression, we then observed the effect of 3-MA (an autophagy inhibitor) on the immune function of macrophages. As expected, inhibiting autophagy by 3-MA attenuated immune functions in activated macrophages. Collectively, this study elucidated that rhArg induced immunosuppression in activated macrophages via inhibiting autophagy, providing potential strategy to ameliorate the immune suppression which is of great significance to cancer therapy and facilitating the development of rhArg as a potential therapy for malignant carcinomas.

L-Arginine Inhibited Inflammatory Response and Oxidative Stress Induced by Lipopolysaccharide via Arginase-1 Signaling in IPEC-J2 Cells.

This study aimed to explore the effect of L-arginine on lipopolysaccharide (LPS)-induced inflammatory response and oxidative stress in IPEC-2 cells. We found that the expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), cluster of differentiation 14 (CD14), nuclear factor-kappaBp65 (NF-κBp65), chemokine-8 (IL-8), tumor necrosis factor (TNF-α) and chemokine-6 (IL-6) mRNA were significantly increased by LPS. Exposure to LPS induced oxidative stress as reactive oxygen species (ROS) and malonaldehyde (MDA) production were increased while glutathione peroxidase (GSH-Px) were decreased in LPS-treated cells compared to those in the control. LPS administration also effectively induced cell growth inhibition through induction of G0/G1 cell cycle arrest. However, compared with the LPS group, cells co-treatment with L-arginine effectively increased cell viability and promoted the cell cycle into the S phase; L-arginine exhibited an anti-inflammatory effect in alleviating inflammation induced by LPS by reducing the abundance of TLR4, MyD88, CD14, NF-κBp65, and IL-8 transcripts. Cells treated with LPS+L-arginine significantly enhanced the content of GSH-Px, while they decreased the production of ROS and MDA compared with the LPS group. Furthermore, L-arginine increased the activity of arginase-1 (Arg-1), while Arg-1 inhibitor abolished the protection of arginine against LPS-induced inflammation and oxidative stress. Taken together, these results suggested that L-arginine exerted its anti-inflammatory and antioxidant effects to protect IPEC-J2 cells from inflammatory response and oxidative stress challenged by LPS at least partly via the Arg-1 signaling pathway.

Deficient arginase II expression without alteration in arginase I expression attenuated experimental autoimmune encephalomyelitis in mice.

In the past there have been a multitude of studies that ardently support the role of arginase II (Arg II) in vascular and endothelial disorders; however, the regulation and function of Arg II in autoimmune diseases has thus far remained unclear. Here we report that a global Arg II null mutation in mice suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. During EAE, both Arg I and Arg II were induced in spinal cords, but only Arg II was induced in spleens and splenic dendritic cells (DCs). DC activation by lipopolysaccharide (LPS), CD40L or TLR8 agonist significantly enhanced Arg II expression without affecting Arg I expression. Conversely, DC differentiating cytokines [IL-4 and granulocyte macrophage-colony-stimulating factor (GM-CSF)] yielded opposite effects. In addition, Arg I and Arg II were regulated differentially during Th1 and Th17 cell polarization. Arg II deficiency in mice delayed EAE onset, ameliorated clinical symptoms and reduced myelin loss, accompanied by a remarkable reduction in the EAE-induced spinal cord expression of Th17 cell markers (IL-17 and RORγt). The abundance of Th17 cells and IL-23+ cells in relevant draining lymph nodes was significantly reduced in Arg II knockout mice. In activated DCs, Arg II deficiency significantly suppressed the expression of Th17-differentiating cytokines IL-23 and IL-6. Interestingly, Arg II deficiency did not lead to any compensatory increase in Arg I expression in vivo and in vitro. In conclusion, Arg II was identified as a factor promoting EAE likely via an Arg I-independent mechanism. Arg II may promote EAE by enhancing DC production of Th17-differentiating cytokines. Specific inhibition of Arg II could be a potential therapy for multiple sclerosis.

Upregulation of PD-L1 by SPP1 mediates macrophage polarization and facilitates immune escape in lung adenocarcinoma.

Tumor-associated macrophages (TAMs) polarization represents a key regulatory process of tumor progression. However, the underlying mechanisms are unclear. This study aimed to investigate the relationship between secreted phosphoprotein 1 (SPP1) and TAMs in lung adenocarcinoma cells. THP-1 monocytes were differentiated into macrophages using PMA. PMA-treated THP-1 cells were co-cultured with human A549 cells culture supernatant. SPP1 expression in TAMs isolated from lung adenocarcinoma tissues and PMA-treated THP-1 cells were measured. Macrophage polarization was identified by flow cytometric analysis. Cell migration and apoptosis were assessed by Transwell migration assays and flow cytometric analysis, respectively. SPP1 is highly expressed in tumor tissues and TAMs isolated from patients with an advanced TNM stage, and also in PMA-treated THP-1 cells. Co-culture with A549 cells strongly induced SPP-1 expression as well as M2 polarization of THP-1 cells, but it had little effect on short hairpin SPP1 (shSPP1)-transfected THP-1 cells. Interestingly, programmed death ligand 1 (PD-L1), a critical regulator of M2 polarization, was downregulated in SPP1 knockdown THP-1 cells. Inhibition of PD-L1 induced a greater decline of the M2 markers IL-10 and Arg-1 but an increase in the M1 markers IL-12 and TNF-α. In addition, SPP1 knockdown in THP-1 cells can mitigate migration but promote apoptosis of A549 cells, and PD-L1 inhibition can further enhance this effect. THP-1 cells co-cultured with A549 cells attenuated CD4+ T-cell activation, whereas SPP1 inhibition restored T-cell activation. These results highlight the importance of SPP1 in mediating macrophage polarization and lung cancer evasion, suggesting a potential therapeutic target for lung cancer.