CD40-mediated immune cell activation enhances response to anti-PD-1 in murine intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: While cholangiocarcinomas (CCAs) commonly express programmed cell death 1 (PD-1) and its ligand (PD-L1), they respond poorly to immune checkpoint inhibitors (ICIs). We aimed to determine whether stimulating antigen-presenting cells, including macrophages and dendritic cells, using a CD40 agonist could improve this response. METHODS: We compared treatment responses in subcutaneous, orthotopic, and 2 plasmid-based murine intrahepatic CCA (iCCA) models. Mice were treated for 4 weeks with weekly IgG control, a CD40 agonistic antibody, anti-PD-1, or the combination of both (anti-CD40/PD-1). Flow cytometric (FACS) analysis of lymphocytes and myeloid cell populations (including activation status) was performed. We used dendritic cell knockout mice, and macrophage, CD4+ and CD8+ T cell depletion models to identify effector cells. Anti-CD40/PD-1 was combined with chemotherapy (gemcitabine/cisplatin) to test for improved therapeutic efficacy. RESULTS: In all 4 models, anti-PD-1 alone was minimally efficacious. Mice exhibited a moderate response to CD40 agonist monotherapy. Combination anti-CD40/PD-1 therapy led to a significantly greater reduction in tumor burden. FACS demonstrated increased number and activation of CD4+ and CD8+ T cells, natural killer cells, and myeloid cells in tumor and non-tumor liver tissue of tumor-bearing mice treated with anti-CD40/PD-1. Depletion of macrophages, dendritic cells, CD4+ T cells, or CD8+ T cells abrogated treatment efficacy. Combining anti-CD40/PD-1 with gemcitabine/cisplatin resulted in a significant survival benefit compared to gemcitabine/cisplatin alone. CONCLUSION: CD40-mediated activation of macrophages and dendritic cells in iCCA significantly enhances response to anti-PD-1 therapy. This regimen may enhance the efficacy of first-line chemotherapy. LAY SUMMARY: Checkpoint inhibition, a common form of immune therapy, is generally ineffective for the treatment of cholangiocarcinoma. These tumors suppress the infiltration and function of surrounding immune cells. Stimulating immune cells such as macrophages and dendritic cells via the CD40 receptor activates downstream immune cells and enhances the response to checkpoint inhibitors.

Behind the scenes of vitamin D binding protein: more than vitamin D binding.

Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein.

Integrated Transcriptomics Establish Macrophage Polarization Signatures and have Potential Applications for Clinical Health and Disease.

Growing evidence defines macrophages (Mφ) as plastic cells with wide-ranging states of activation and expression of different markers that are time and location dependent. Distinct from the simple M1/M2 dichotomy initially proposed, extensive diversity of macrophage phenotypes have been extensively demonstrated as characteristic features of monocyte-macrophage differentiation, highlighting the difficulty of defining complex profiles by a limited number of genes. Since the description of macrophage activation is currently contentious and confusing, the generation of a simple and reliable framework to categorize major Mφ phenotypes in the context of complex clinical conditions would be extremely relevant to unravel different roles played by these cells in pathophysiological scenarios. In the current study, we integrated transcriptome data using bioinformatics tools to generate two macrophage molecular signatures. We validated our signatures in in vitro experiments and in clinical samples. More importantly, we were able to attribute prognostic and predictive values to components of our signatures. Our study provides a framework to guide the interrogation of macrophage phenotypes in the context of health and disease. The approach described here could be used to propose new biomarkers for diagnosis in diverse clinical settings including dengue infections, asthma and sepsis resolution.

Macrophage Activation Mechanisms in Human Monocytic Cell Line-derived Macrophages.

BACKGROUND: Although the mechanisms of macrophage activation are important for cancer immunotherapy, they are poorly understood. Recently, easy and robust assay systems for assessing the macrophage-activating factor (MAF) using monocytic cell line-derived macrophages were established. MATERIALS AND METHODS: Gene-expression profiles of U937- and THP-1-derived macrophages were compared using gene expression microarray analysis and their responses against several MAFs were examined by in vitro experiments. RESULTS: Activated states of these macrophages could not be assigned to a specific sub-type but showed, however, different unique characteristics. CONCLUSION: The unique of monocytic cell line-derived macrophages could provide clues to understand the activation mechanism of macrophages and, therefore, help to develop effective cancer immunotherapy with MAFs.

Degalactosylated/Desialylated Bovine Colostrum Induces Macrophage Phagocytic Activity Independently of Inflammatory Cytokine Production.

BACKGROUND/AIM: Colostrum contains antibodies, such as immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM), and, therefore, has potent immunomodulating activity. In particular, IgA has an O-linked sugar chain similar to that in the group-specific component (Gc) protein, a precursor of the Gc protein-derived macrophage-activating factor (GcMAF). In the present study, we investigated the macrophage-activating effects of degalactosylated/desialylated bovine colostrum. RESULTS: We detected the positive band in degalactosylated/ desialylated bovine colostrum by western blotting using Helix pomatia agglutinin lectin. We also found that degalactosylated/ desialylated bovine colostrum could significantly enhance the phagocytic activity of mouse peritoneal macrophages in vitro and of intestinal macrophages in vivo. Besides, degalactosylated/desialylated bovine colostrum did not mediate the production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). CONCLUSION: Similar to the use of GcMAF, degalactosylated/desialylated bovine colostrum can be used as a potential macrophage activator for various immunotherapies.

Effect of Edwardsiella tarda immunization on systemic immune response, mucosal immune response and protection in catla (Catla catla).

The effect of immunization on systemic and cutaneous mucosal immune responses of fish and their possible relation with protection has not been fully assessed. In this study, healthy catla (Catla catla) were immunized against Edwardsiella tarda using two antigenic preparations namely, whole cell bacterin (B) and bacterin mixed with Freund's complete adjuvant in a 1:1 (v/v) ratio (B+A) followed by a booster dose after 3 weeks of first injection. Different systemic and cutaneous mucosal immune responses were measured at weekly interval upto 8th week post vaccination (pv). Fish were challenged 8 weeks pv with live E. tarda to study vaccine induced protection. The result showed that although there were strong systemic as well as mucosal immune responses, particularly after booster dose, the challenge produced low to moderate protection in terms of relative percent survival (RPS). The maximum RPS (50 %) was recorded in the adjuvanted bacterin group after 8 weeks pv. Low to moderate protection after challenge, which may be attributed to the intracellular nature of E. tarda and/or use of crude antigenic preparation, accounts for new strategy to be developed for immunization programme against such intracellular pathogen. The results collectively suggest possible involvement of systemic as well as mucosal immune responses in inducing protective immunity in catla.

Molecular cloning and function characterization of a new macrophage-activating protein from Tremella fuciformis.

Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1ß, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway.

Leptin on peritoneal macrophages of patients with endometriosis.

PROBLEM: The expression of cyclooxygenase (COX)-2 is considered as a marker of macrophage activation and has been implicated in the development of endometriosis. Leptin is an immunomodulator, which may also affect the development of endometriosis. However, how leptin contributes to these pathological processes has not been completely understood. The aim of this study was to investigate the effects of leptin on peritoneal macrophages and its relationship with endometriosis. METHODS OF STUDY: Peritoneal fluid from 60 women of reproductive age was obtained while they underwent laparoscopy. Forty patients had endometriosis and 20 patients did not have endometriosis. The concentration of leptin in the peritoneal fluid and prostaglandin F(2alpha) levels was measured by ELISA, and the other protein expression using Western blot when peritoneal macrophages were stimulated with leptin. RESULTS: Concentration of leptin in peritoneal fluid was increased in patients with endometriosis compared with disease-free normal control. Functional leptin receptor was present in peritoneal macrophages. Treatment of peritoneal macrophages with leptin induced COX-2 expression. Production of prostaglandin F(2alpha) by peritoneal macrophages was increased after leptin stimulation in women with endometriosis. CONCLUSION: Elevated concentration of leptin in peritoneal fluid may contribute to the pathological process of endometriosis through activation of peritoneal macrophages.

Interferon-gamma: a historical perspective.

This article reviews the main lines of thinking and exploration that have led to our current conception of the role of IFN-gamma in immune defense and autoimmunity. In 1965 the first report appeared describing production of an interferon-like virus inhibitor in cultured human leukocytes following exposure to the mitogen phytohemagglutinin. In the early 1970s the active principle became recognized as being distinct from classical virus-induced interferons, leading to its designation as immune interferon or Type II interferon, and eventually IFN-gamma. Up to that point interest in the factor had come almost exclusively from virologists, in particular those among them who were believers in interferon. Evidence first coming forward in the 1980s that IFN-gamma is indistinguishable from macrophage-activating factor (MAF), then a prototype lymphokine, was the signal for immunologists at large to become interested. Today IFN-gamma ranks among the most important endogenous regulators of immune responses.

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Mercury chloride effects on the function and cellular integrity of sea bass (Dicentrarchus labrax) head kidney macrophages.

The objective of this work was to study mercury chloride effects on the function and integrity of sea bass (Dicentrarchus labrax) head kidney macrophages (S-HKM), and to evaluate the response of HgCl2-exposed cells to macrophage activating factor(s) (MAF) produced by sea bass head kidney leukocytes. There was considerable variability in the effects of HgCl2 on the production of reactive oxygen species (ROS) by S-HKM. When incubated with HgCl2, cells from five out of nine fish tested showed a decrease in ROS production as compared to cells incubated with medium alone. In those cultures, MAF addition prevented the mercury chloride-induced decrease in ROS production. In other S-HKM cultures isolated from different fish, mercury chloride abrogated the up-regulating effect of MAF on the respiratory burst. MAF activation of the phagocytic activity of S-HKM was also impaired by HgCl2 addition. Mercury chloride induced apoptosis in S-HKM cultures and MAF addition prevented this effect.

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide.

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.

Compatibility of the totally replaced hip. Reduction of wear by amorphous diamond coating.

Particulate wear debris in totally replaced hips causes adverse local host reactions. The extreme form of such a reaction, aggressive granulomatosis, was found to be a distinct condition and different from simple aseptic loosening. Reactive and adaptive tissues around the totally replaced hip were made of proliferation of local fibroblast like cells and activated macrophages. Methylmethacrylate and high-molecular-weight polyethylene were shown to be essentially immunologically inert implant materials, but in small particulate form functioned as cellular irritants initiating local biological reactions leading to loosening of the implants. Chromium-cobalt-molybdenum is the most popular metallic implant material; it is hard and tough, and the bearings of this metal are partially self-polishing. In total hip implants, prerequisites for longevity of the replaced hip are good biocompatibility of the materials and sufficient tribological properties of the bearings. The third key issue is that the bearing must minimize frictional shear at the prosthetic bone-implant interface to be compatible with long-term survival. Some of the approaches to meet these demands are alumina-on-alumina and metal-on-metal designs, as well as the use of highly crosslinked polyethylene for the acetabular component. In order to avoid the wear-based deleterious properties of the conventional total hip prosthesis materials or coatings, the present work included biological and tribological testing of amorphous diamond. Previous experiments had demonstrated that a high adhesion of tetrahedral amorphous carbon coatings to a substrate can be achieved by using mixing layers or interlayers. Amorphous diamond was found to be biologically inert, and simulator testing indicated excellent wear properties for conventional total hip prostheses, in which either the ball or both bearing surfaces were coated with hydrogen-free tetrahedral amorphous diamond films. Simulator testing with such total hip prostheses showed no measurable wear or detectable delamination after 15,000,000 test cycles corresponding to 15 years of clinical use. The present work clearly shows that wear is one of the basic problems with totally replaced hips. Diamond coating of the bearing surfaces appears to be an attractive solution to improve longevity of the totally replaced hip.

Structurally well-defined macrophage activating factor derived from vitamin D3-binding protein has a potent adjuvant activity for immunization.

Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice.

A defect in inducible beta-galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.

Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso-Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso-Pc efficiently activated macrophages whereas lyso-Pc-treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso-Pc-inducible beta-galactosidase of B lymphocytes. Lyso-Pc-inducible beta-galactosidase of B lymphocytes was found to be defective in mi mutant mice.

Differential expression of IL-1 beta, TNF-alpha, IL-6, and IL-8 in human monocytes in response to lipopolysaccharides from different microbes.

Macrophages respond to bacterial lipopolysaccharides (LPS) and activate several host defense functions through production of mediators. However, it is not clear whether the degree of macrophage responsiveness to different sources of LPS is equivalent to or varies with the source of LPS. Therefore, in this report, we examined the extent of the human monocyte response to LPS derived from two oral pathogens, Actinobacillus actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg). Additionally, due to its well-established ability to activate monocytes, we used LPS from Escherichia coli (Ec). Human monocytes, when activated with a specific source of LPS, exhibited rapid expression of mRNA for IL-1 beta, TNF-alpha, and IL-8, which was followed by IL-6, as measured by RNA-PCR. Moreover, the expression of mRNA for these cytokines was followed by cytokine synthesis. Monocytes from the same subject, when activated with LPS from Pg, Aa, or Ec expressed quantitatively different levels of mRNA and proteins for all four cytokines. A given LPS induced either high or low expression of the battery of cytokines tested, indicating that the expression of these pro-inflammatory cytokines may be regulated by a single or a cluster of gene(s). However, no apparent differences in the time course of mRNA expression for these cytokines were observed in response to any of the LPS tested. Furthermore, the relative ability of the different sources of LPS to induce mRNA for cytokines varied throughout a wide range of LPS concentrations. This suggests that differences exist in the sensitivity of monocytes to a specific LPS, rather than in the kinetics of the secretory process itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant chicken interferon: a potent antiviral agent that lacks intrinsic macrophage activating factor activity.

Crude preparations of chicken interferon (ChIFN) from various sources contain both antiviral and macrophage-activating factor (MAF) activity. Previous serological data indicated that unlike mammals, birds might express only a single type of IFN in response to viruses and mitogens that exhibits both activities. We have now expressed a complementary DNA for virus-induced ChIFN in transfected COS cells and in Escherichia coli. Purified recombinant ChIFN is a powerful antiviral agent and has high Mx promoter-inducing activity. However, as the sole agent, recombinant ChIFN lacks MAF activity: it does not induce the secretion of nitric oxide in primary monocyte-derived chicken macrophages. A neutralizing antiserum prepared against cloned ChIFN blocks most of the antiviral and Mx promoter-inducing activity present in preparations of natural ChIFN, but does not inhibit the MAF activity. These results demonstrate that chicken cells can be induced to secrete a novel cytokine which probably represents the avian homolog of mammalian IFN-gamma.

Roles of beta-galactosidase of B lymphocytes and sialidase of T lymphocytes in inflammation-primed activation of macrophages.

The outer surface of mouse B lymphocytes carries constitutive and inducible beta-galactosidase isozymes. A brief (30 min) treatment of B lymphocytes with lysophosphatidylcholine (lyso-Pc) immediately induced an approximate 3-fold higher beta-galactosidase activity than the constitutive isozyme of untreated B lymphocytes. Thus, the lyso-Pc-inducible isozyme is not a de novo enzyme. Outer surface of mouse T lymphocytes carries constitutive (non-Neu-1) and inducible (Neu-1) sialidase isozymes. The lyso-Pc-inducible beta-galactosidase of B lymphocytes and the Neu-1 sialidase of T lymphocytes were required for conversion of vitamin D3-binding protein (Gc protein) to a potent macrophage activating factor. This enzymatic generation of the macrophage activating factor was mediated via enzyme-associated receptors.