Essential role of local antibody distribution in mediating bone-resorbing effects.

The link between antibodies and bone mass is debated. Activated IgG, which interacts directly with Fc gamma receptors, stimulates osteoclastogenesis in vitro, and local injection in immune-activated mice leads to bone loss. Multiple myeloma patients with high serum IgG levels have induced osteoclast activation and display bone loss. In addition, bone loss has been linked to serum autoantibodies in autoimmune diseases, including anti-citrullinated protein antibodies (ACPA) in individuals with rheumatoid arthritis (RA). Whether serum IgG or autoantibodies regulate bone mass under healthy conditions is poorly studied. In elderly men, neither serum levels of polyclonal IgG nor autoantibody were associated with areal bone mineral density in the MrOS Sweden study. Repetitive systemic injections of high-dose polyclonal IgG complexes in mice did not exert any discernible impact on bone mineral density. However, repetitive local intra-articular injection of the same IgG complexes led to a localized reduction of trabecular bone density. These results indicate antibodies may only impact bone density when close to the bone, such as within the synovial joint.

Fc Gamma Receptors as Regulators of Bone Destruction in Inflammatory Arthritis.

Bone erosion is one of the primary features of inflammatory arthritis and is caused by excessive differentiation and activation of osteoclasts. Fc gamma receptors (FcγRs) have been implicated in osteoclastogenesis. Our recent studies demonstrate that joint-deposited lupus IgG inhibited RANKL-induced osteoclastogenesis. FcγRI is required for RANKL-induced osteoclastogenesis and lupus IgG-induced signaling transduction. We reviewed the results of studies that analyzed the association between FcγRs and bone erosion in inflammatory arthritis. The analysis revealed the dual roles of FcγRs in bone destruction in inflammatory arthritis. Thus, IgG/FcγR signaling molecules may serve as potential therapeutic targets against bone erosion.

Mood Disorder in Systemic Lupus Erythematosus Induced by Antiribosomal P Protein Antibodies Associated with Decreased Serum and Brain Tryptophan.

Antiribosomal P protein (anti-P) autoantibodies commonly develop in patients with systemic lupus erythematosus. We have previously established hybridoma clones producing anti-P mAbs. In this study, we explored the pathogenesis of behavioral disorders induced by anti-P Abs using these mAbs. New Zealand Black × New Zealand White F1, New Zealand White, C57BL/6, and BALB/c mice were treated with 1 mg of anti-P Abs once every 2 wk. The behavioral disorder was evaluated by the tail suspension test, forced swim test, and open field test. Following administration of anti-P Abs, New Zealand Black × New Zealand White F1 and C57BL/6 mice developed depressive behavior and showed increased anxiety with elevated serum TNF-α and IL-6 levels. Anti-P Abs were not deposited in the affected brain tissue; instead, this mood disorder was associated with lower serum and brain tryptophan concentrations. Tryptophan supplementation recovered serum tryptophan levels and prevented the behavioral disorder. TNF-α and IL-6 were essential for the decreased serum tryptophan and disease development, which were ameliorated by treatment with anti-TNF-α neutralizing Abs or dexamethasone. Peritoneal macrophages from C57BL/6 mice produced TNF-α, IL-6, and IDO-1 via interaction with anti-P Abs through activating FcγRs, which were required for disease development. IVIg, which has an immunosuppressive effect partly through the regulation of FcγR expression, also prevented the decrease in serum tryptophan and disease development. Furthermore, serum tryptophan concentrations were decreased in the sera of systemic lupus erythematosus patients with anti-P Abs, and lower tryptophan levels correlated with disease activity. Our study revealed some of the molecular mechanisms of mood disorder induced by anti-P Abs.

Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Targeting Pathomechanisms with Bruton Tyrosine Kinase Inhibitors.

A series of cases with rare thromboembolic incidents including cerebral sinus vein thrombosis (some of them fatal) and concomitant thrombocytopenia occurring shortly after vaccination with the coronavirus disease 2019 (COVID-19) vaccine AZD1222 (Vaxzevria) have caused significant concern and led to its temporary suspension in many countries. Immediate laboratory efforts in four of these patients have identified a tentative pathomechanism underlying this syndrome termed initially vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) and renamed recently vaccine-induced immune thrombotic thrombocytopenia (VITT). It encompasses the presence of platelet-activating antibodies to platelet factor-4/heparin complexes, possibly emulated by polyanionic constituents of AZD1222, and thus resembles heparin-induced thrombocytopenia (HIT). Because these immune complexes bind and activate platelets via Fcγ receptor IIA (FcγRIIA), high-dose intravenous immunoglobulin G has been suggested for treatment of VITT in addition to non-heparin anticoagulants. Here we propose inhibitors of Bruton tyrosine kinase (Btk) approved for B cell malignancies (e.g., ibrutinib) as another therapeutic option in VITT, as they are expected to pleiotropically target multiple pathways downstream of FcγRIIA-mediated Btk activation, for example, as demonstrated for the effective inhibition of platelet aggregation, dense granule secretion, P-selectin expression and platelet-neutrophil aggregate formation stimulated by FcγRIIA cross-linking. Moreover, C-type lectin-like receptor CLEC-2- and GPIb-mediated platelet activation, the interactions and activation of monocytes and the release of neutrophil extracellular traps, as encountered in HIT, could be attenuated by Btk inhibitors. As a paradigm for emergency repurposing of approved drugs in COVID-19, off-label use of Btk inhibitors in a low-dose range not affecting haemostatic functions could thus be considered a sufficiently safe option to treat VITT.

Yishen-tongbi decoction inhibits excessive activation of B cells by activating the FcγRIIb/Lyn/SHP-1 pathway and attenuates the inflammatory response in CIA rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Strong evidence supports that excessive activation of B cells plays a critical role in the pathogenesis of RA. Fc gamma receptor b (FcγRIIb) is the B cell inhibitory receptor and inhibits BCR (B cell receptor) signalling in part by selectively dephosphorylating CD19 which is considered a co-receptor for BCR and is essential for B cell activation. Our previous study demonstrated that a FcγRIIb I232T polymorphism presented a strong genetic link to RA and may lead to the excessive activation of B cells. Therefore, novel therapeutic strategies and drugs that can effectively inhibit the excessive activation of B cells by regulating the FcγRIIb are necessary for the treatment of RA. Therefore, we used Burkitt's lymphoma ST486 human B cells (lacking endogenous FcγRIIb) transfected with the 232Thr loss-of-function mutant to construct a FcγRIIb mutant cell line (ST486), and we demonstrated that YSTB treatment not only reduced proliferation and promoted apoptosis in ST486 cells but also did so in a dose-dependent manner. Furthermore, the intracellular Ca2+ flux of ST486 cells was decreased after treatment with YSTB, inhibiting the excessive activation of ST486 cells, and these effects correlated with the CD19/FcγRIIb-Lyn-SHP-1 pathways. Our data showed that YSTB treatment inhibited the expression of phosphorylated CD19 and upregulated the protein expression of FcγRIIb, Lyn, and SHP-1. Additionally, the CIA model was established to explore the anti-inflammatory and inhibitory effects of YSTB on bone destruction, and we found that YSTB decreased the paw oedema and arthritis index (AI) in CIA rats. It is worth mentioning that YSTB clearly decreased the AI earlier than methotrexate (MTX) (day 10 vs 16). Moreover, synovial hyperplasia, inflammatory cell infiltration and cartilage surface erosion in CIA rats were noticeably reduced after treatment with YSTB as evidenced by histopathological examination. Finally, we found that YSTB treatment suppressed bone erosion and joint space score (JNS) in CIA rats as evidenced by radiographic assessment. In summary, these data suggest that YSTB has great therapeutic potential for RA treatment.

Bovine IgG Prevents Experimental Infection With RSV and Facilitates Human T Cell Responses to RSV.

Respiratory syncytial virus (RSV) infections represent a major burden of disease in infants and are the second most prevalent cause of death worldwide. Human milk immunoglobulins provide protection against RSV. However, many infants depend on processed bovine milk-based nutrition, which lacks intact immunoglobulins. We investigated the potential of bovine antibodies to neutralize human RSV and facilitate-cell immune activation. We show cow's milk IgG (bIgG) and Intravenous Immunoglobulin (IVIG) have a similar RSV neutralization capacity, even though bIgG has a lower pre-F to post-F binding ratio compared to human IVIG, with the majority of bIgG binding to pre-F. RSV is better neutralized with human IVIG. Consequently, we enriched RSV specific T cells by culturing human PBMC with a mixture of RSV peptides, and used these T cells to study the effect of bIgG and IVIG on the activation of pre-F-pecific T cells. bIgG facilitated in vitro T cell activation in a similar manner as IVIG. Moreover, bIgG was able to mediate T cell activation and internalization of pathogens, which are prerequisites for inducing an adaptive viral response. Using in vivo mouse experiments, we showed that bIgG is able to bind the murine activating IgG Fc Receptors (FcγR), but not the inhibiting FcγRII. Intranasal administration of the monoclonal antibody palivizumab, but also of bIgG and IVIG prevented RSV infection in mice. The concentration of bIgG needed to prevent infection was ~5-fold higher compared to IVIG. In conclusion, the data presented here indicate that functionally active bIgG facilitates adaptive antiviral T cell responses and prevents RSV infection in vitro and in vivo.

Maternal Supplementation of Food Ingredient (Prebiotic) or Food Contaminant (Mycotoxin) Influences Mucosal Immune System in Piglets.

The early life period is crucial for the maturation of the intestinal barrier, its immune system, and a life-long beneficial host-microbiota interaction. The study aims to assess the impact of a beneficial dietary (short-chain fructooligosaccharides, scFOS) supplementation vs. a detrimental dietary environment (such as mycotoxin deoxynivalenol, DON) on offspring intestinal immune system developmental profiles. Sows were given scFOS-supplemented or DON-contaminated diets during the last 4 weeks of gestation, whereas force-feeding piglets with DON was performed during the first week of offspring life. Intestinal antigen-presenting cell (APC) subset frequency was analyzed by flow cytometry in the Peyer's patches and in lamina propria and the responsiveness of intestinal explants to toll-like receptor (TLR) ligands was performed using ELISA and qRT-PCR from post-natal day (PND) 10 until PND90. Perinatal exposure with scFOS did not affect the ontogenesis of APC. While it early induced inflammatory responses in piglets, scFOS further promoted the T regulatory response after TLR activation. Sow and piglet DON contamination decreased CD16+ MHCII+ APC at PND10 in lamina propria associated with IFNγ inflammation and impairment of Treg response. Our study demonstrated that maternal prebiotic supplementation and mycotoxin contamination can modulate the mucosal immune system responsiveness of offspring through different pathways.

Engineering of Fc Multimers as a Protein Therapy for Autoimmune Disease.

The success of Intravenous Immunoglobulin in treating autoimmune and inflammatory processes such as immune thrombocytopenia purpura and Kawasaki disease has led to renewed interest in developing recombinant molecules capable of recapitulating these therapeutic effects. The anti-inflammatory properties of IVIG are, in part, due to the Fc region of the IgG molecule, which interacts with activating or inhibitory Fcγ receptors (FcγRs), the neonatal Fc Receptor, non-canonical FcRs expressed by immune cells and complement proteins. In most cases, Fc interactions with these cognate receptors are dependent upon avidity-avidity which naturally occurs when polyclonal antibodies recognize unique antigens on a given target. The functional consequences of these avid interactions include antibody dependent cell-mediated cytotoxicity, antibody dependent cell phagocytosis, degranulation, direct killing, and/or complement activation-all of which are associated with long-term immunomodulatory effects. Many of these immunologic effects can be recapitulated using recombinant or non-recombinant approaches to induce Fc multimerization, affording the potential to develop a new class of therapeutics. In this review, we discuss the history of tolerance induction by immune complexes that has led to the therapeutic development of artificial Fc bearing immune aggregates and recombinant Fc multimers. The contribution of structure, aggregation and N-glycosylation to human IgG: FcγR interactions and the functional effect(s) of these interactions are reviewed. Understanding the mechanisms by which Fc multimers induce tolerance and attempts to engineer Fc multimers to target specific FcγRs and/or specific effector functions in autoimmune disorders is explored in detail.

Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling.

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

IL-4 controls activated neutrophil FcγR2b expression and migration into inflamed joints.

Neutrophils are the most abundant immune cells found in actively inflamed joints of patients with rheumatoid arthritis (RA), and most animal models for RA depend on neutrophils for the induction of joint inflammation. Exogenous IL-4 and IL-13 protect mice from antibody-mediated joint inflammation, although the mechanism is not understood. Neutrophils display a very strong basal expression of STAT6, which is responsible for signaling following exposure to IL-4 and IL-13. Still, the role of IL-4 and IL-13 in neutrophil biology has not been well studied. This can be explained by the low neutrophil surface expression of the IL-4 receptor α-chain (IL-4Rα), essential for IL-4- and IL-13-induced STAT6 signaling. Here we identify that colony stimulating factor 3 (CSF3), released during acute inflammation, mediates potent STAT3-dependent neutrophil IL-4Rα up-regulation during sterile inflammatory conditions. We further demonstrate that IL-4 limits neutrophil migration to inflamed joints, and that CSF3 combined with IL-4 or IL-13 results in a prominent neutrophil up-regulation of the inhibitory Fcγ receptor (FcγR2b). Taking these data together, we demonstrate that the IL-4 and CSF3 pathways are linked and play important roles in regulating proinflammatory neutrophil behavior.

Supplementation With the Sialic Acid Precursor N-Acetyl-D-Mannosamine Breaks the Link Between Obesity and Hypertension.

BACKGROUND: Obesity-related hypertension is a common disorder, and attempts to combat the underlying obesity are often unsuccessful. We previously revealed that mice globally deficient in the inhibitory immunoglobulin G (IgG) receptor FcγRIIB are protected from obesity-induced hypertension. However, how FcγRIIB participates is unknown. Studies were designed to determine if alterations in IgG contribute to the pathogenesis of obesity-induced hypertension. METHODS: Involvement of IgG was studied using IgG µ heavy chain-null mice deficient in mature B cells and by IgG transfer. Participation of FcγRIIB was interrogated in mice with global or endothelial cell-specific deletion of the receptor. Obesity was induced by high-fat diet (HFD), and blood pressure (BP) was measured by radiotelemetry or tail cuff. The relative sialylation of the Fc glycan on mouse IgG, which influences IgG activation of Fc receptors, was evaluated by Sambucus nigra lectin blotting. Effects of IgG on endothelial NO synthase were assessed in human aortic endothelial cells. IgG Fc glycan sialylation was interrogated in 3442 human participants by mass spectrometry, and the relationship between sialylation and BP was evaluated. Effects of normalizing IgG sialylation were determined in HFD-fed mice administered the sialic acid precursor N-acetyl-D-mannosamine (ManNAc). RESULTS: Mice deficient in B cells were protected from obesity-induced hypertension. Compared with IgG from control chow-fed mice, IgG from HFD-fed mice was hyposialylated, and it raised BP when transferred to recipients lacking IgG; the hypertensive response was absent if recipients were FcγRIIB-deficient. Neuraminidase-treated IgG lacking the Fc glycan terminal sialic acid also raised BP. In cultured endothelial cells, via FcγRIIB, IgG from HFD-fed mice and neuraminidase-treated IgG inhibited vascular endothelial growth factor activation of endothelial NO synthase by altering endothelial NO synthase phosphorylation. In humans, obesity was associated with lower IgG sialylation, and systolic BP was inversely related to IgG sialylation. Mice deficient in FcγRIIB in endothelium were protected from obesity-induced hypertension. Furthermore, in HFD-fed mice, ManNAc normalized IgG sialylation and prevented obesity-induced hypertension. CONCLUSIONS: Hyposialylated IgG and FcγRIIB in endothelium are critically involved in obesity-induced hypertension in mice, and supportive evidence was obtained in humans. Interventions targeting these mechanisms, such as ManNAc supplementation, may provide novel means to break the link between obesity and hypertension.

Selenium nanoparticles as new strategy to potentiate γδ T cell anti-tumor cytotoxicity through upregulation of tubulin-α acetylation.

Immune cell therapy presents a paradigm for the treatment of malignant tumors. Human Vγ9Vδ2 T cells, a subset of peripheral γδ T cells, have been shown to have promising anti-tumor activity. However, new methodology on how to achieve a stronger anti-tumor activity of Vγ9Vδ2 T cells is under continuous investigation. In this work, we used selenium nanoparticles (SeNPs) to strengthen the anti-tumor cytotoxicity of Vγ9Vδ2 T cells. We found SeNPs pretreated γδ T cells had significantly stronger cancer killing and tumor growth inhibition efficacy when compared with γδ T cells alone. Simultaneously, SeNPs pretreatment could significantly upregulate the expression of cytotoxicity related molecules including NKG2D, CD16, and IFN-γ, meanwhile, downregulate PD-1 expression of γδ T cells. Importantly, we observed that SeNPs promoted tubulin acetylation modification in γδ T cells through interaction between microtubule network and lysosomes since the latter is the primary resident station of SeNPs shown by confocal visualization. In conclusion, SeNPs could significantly potentiate anti-tumor cytotoxicity of Vγ9Vδ2 T cells, and both cytotoxicity related molecules and tubulin acetylation were involved in fine-tuning γδ T cell toxicity against cancer cells. Our present work demonstrated a new strategy for further enhancing anti-tumor cytotoxicity of human Vγ9Vδ2 T cells by using SeNPs-based nanotechnology, not gene modification, implicating SeNPs-based nanotechnology had a promising clinical perspective in the γδ T cell immunotherapy for malignant tumors.

Anti-HLA antibodies with complementary and synergistic interaction geometries promote classical complement activation on platelets.

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here, we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in vivo in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors.

A Novel, Fully Human Anti-fucosyl-GM1 Antibody Demonstrates Potent In Vitro and In Vivo Antitumor Activity in Preclinical Models of Small Cell Lung Cancer.

Purpose: The ganglioside fucosyl-GM1 (FucGM1) is a tumor-associated antigen expressed in a large percentage of human small cell lung cancer (SCLC) tumors, but absent in most normal adult tissues, making it a promising target in immuno-oncology. This study was undertaken to evaluate the preclinical efficacy of BMS-986012, a novel, nonfucosylated, fully human IgG1 antibody that binds specifically to FucGM1.Experimental Design: The antitumor activity of BMS-986012 was evaluated in in vitro assays using SCLC cells and in mouse xenograft and syngeneic tumor models, with and without chemotherapeutic agents and checkpoint inhibitors.Results: BMS-986012 showed a high binding affinity for FcγRIIIa (CD16), which resulted in enhanced antibody-dependent cellular cytotoxicity (ADCC) against FucGM1-expressing tumor cell lines. BMS-986012-mediated tumor cell killing was also observed in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) assays. In several mouse SCLC models, BMS-986012 demonstrated efficacy and was well tolerated. In the DMS79 xenograft model, tumor regression was achieved with BMS-986012 doses of 0.3 mg/kg and greater; antitumor activity was enhanced when BMS-986012 was combined with standard-of-care cisplatin or etoposide. In a syngeneic model, tumors derived from a genetically engineered model of SCLC were treated with BMS-986012 or anti-FucGM1 with a mouse IgG2a Fc and their responses evaluated; when BMS-986012 was combined with anti-PD-1 or anti-CD137 antibody, therapeutic responses significantly improved.Conclusions: Single-agent BMS-986012 demonstrated robust antitumor activity, with the addition of chemotherapeutic or immunomodulatory agents further inhibiting SCLC growth in the same models. These preclinical data supported evaluation of BMS-986012 in a phase I clinical trial of patients with relapsed, refractory SCLC. Clin Cancer Res; 24(20); 5178-89. ©2018 AACR.

Fcγ receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis.

BACKGROUND: Osteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA). METHODS: AIA was induced in knee joints of wild-type (WT), FcγRI,II,III-/-, and FcγRI,II,III,IV-/- mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR. RESULTS: FcγRI,II,III,IV-/- mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV-/- mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV-/- and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV-/- mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV-/- mice, AIA induction in knee joints of FcγRI,II,III-/- mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity. CONCLUSIONS: FcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint.

Human IgG subclass cross-species reactivity to mouse and cynomolgus monkey Fcγ receptors.

In therapeutic antibody discovery and early development, mice and cynomolgus monkey are used as animal models to assess toxicity, efficacy and other properties of candidate molecules. As more candidate antibodies are based on human immunoglobulin (IgG) subclasses, many strategies are pursued to simulate the human system in the test animal. However, translation rate from a successful preclinical trial to an approved drug is extremely low. This may partly be due to differences in interaction of human IgG based candidate molecules to endogenous Fcγ receptors of model animals in comparison to those of human Fcγ receptors. In this study, we compare binding characteristics of human IgG subclasses commonly used in drug development (IgG1, IgG2, IgG4) and their respective Fc silent versions (IgG1σ, IgG2σ, IgG4 PAA) to human, mouse, and cynomolgus monkey Fcγ receptors. To control interactions between Fab and Fc domains, the test IgGs all have the same variable region sequences. We found distinct variations of interaction of human IgG subclasses to model animal Fcγ receptors in comparison to their human counterparts. Particularly, cynomolgus monkey Fcγ receptors showed consistently tighter binding to human IgGs than human Fcγ receptors. Moreover, the presumably Fc silent human IgG4 PAA framework bound to cynomolgus monkey FcγRI with nanomolar affinity while only very weak binding was observed for the human FcγRI. Our results highlighted the need for a thorough in vitro affinity characterization of candidate IgGs against model animal Fcγ receptors and careful design of preclinical studies.

Identification of compounds that decrease numbers of Mycobacteria in human macrophages in the presence of serum amyloid P.

Mϕs are a heterogeneous population of cells and include classically activated Mϕs (M1) and alternatively activated Mϕs (M2). Mϕs can change from M1 to M2 and vice versa in response to environmental stimuli. Serum amyloid P (SAP) is a constitutive plasma protein that polarizes Mϕs to an M2 phenotype, and part of this effect is mediated through FcγRI receptors. In an effort to find ways to alter Mϕs phenotypes, we screened for compounds that can block the SAP-FcγRI interaction. From a screen of 3000 compounds, we found 12 compounds that reduced the ability of fluorescently labeled human SAP to bind cells expressing human FcγRI. Based on cell surface marker expression, 8 of the compounds inhibited the effect of SAP on skewing human Mϕs to an M2 phenotype and in the presence of SAP polarized Mϕs to an M1 phenotype. In diseases, such as tuberculosis, M1s are more effective at killing bacteria than M2s. SAP potentiated the numbers of the mycobacterial strains Mycobacterium smegmatis and Mycobacterium tuberculosis in Mϕs. When added along with SAP, 2 of the compounds reduced intracellular Mycobacterium numbers. Together, these results indicate that the blocking of SAP effects on Mϕs can skew these cells toward an M1 phenotype, and this may be useful in treating diseases, such as tuberculosis.

Bitter Melon Enhances Natural Killer-Mediated Toxicity against Head and Neck Cancer Cells.

Natural killer (NK) cells are one of the major components of innate immunity, with the ability to mediate antitumor activity. Understanding the role of NK-cell-mediated tumor killing in controlling of solid tumor growth is still in the developmental stage. We have shown recently that bitter melon extract (BME) modulates the regulatory T cell (Treg) population in head and neck squamous cell carcinoma (HNSCC). However, the role of BME in NK-cell modulation against HNSCC remains unknown. In this study, we investigated whether BME can enhance the NK-cell killing activity against HNSCC cells. Our results indicated that treatment of human NK-cell line (NK3.3) with BME enhances ability to kill HNSCC cells. BME increases granzyme B accumulation and translocation/accumulation of CD107a/LAMP1 in NK3.3 cells exposed to BME. Furthermore, an increase in cell surface expression of CD16 and NKp30 in BME-treated NK3.3 cells was observed when cocultured with HNSCC cells. Collectively, our results demonstrated for the first time that BME augments NK-cell-mediated HNSCC killing activity, implicating an immunomodulatory role of BME. Cancer Prev Res; 10(6); 337-44. ©2017 AACR.

Lifelong quercetin enrichment and cardioprotection in Mdx/Utrn+/- mice.

Duchenne Muscular Dystrophy (DMD) is associated with progressive cardiac pathology; however, the SIRT1/PGC1-α activator quercetin may cardioprotect dystrophic hearts. We tested the extent to which long-term 0.2% dietary quercetin enrichment attenuates dystrophic cardiopathology in Mdx/Utrn+/- mice. At 2 mo, Mdx/Utrn+/- mice were fed quercetin-enriched (Mdx/Utrn+/--Q) or control diet (Mdx/Utrn+/-) for 8 mo. Control C57BL/10 (C57) animals were fed a control diet for 10 mo. Cardiac function was quantified by MRI at 2 and 10 mo. Spontaneous physical activity was quantified during the last week of treatment. At 10 mo hearts were excised for histological and biochemical analysis. Quercetin feeding improved various physiological indexes of cardiac function in diseased animals. Mdx/Utrn+/--Q also engaged in more high-intensity physical activity than controls. Histological analyses of heart tissues revealed higher expression and colocalization of utrophin and α-sarcoglycan. Lower abundance of fibronectin, cardiac damage (Hematoxylin Eosin-Y), and MMP9 were observed in quercetin-fed vs. control Mdx/Utrn+/- mice. Quercetin evoked higher protein abundance of PGC-1α, cytochrome c, ETC complexes I-V, citrate synthase, SOD2, and GPX compared with control-fed Mdx/Utrn+/- Quercetin decreased abundance of inflammatory markers including NFκB, TGF-ß1, and F4/80 compared with Mdx/Utrn+/-; however, P-NFκB, P-IKBα, IKBα, CD64, and COX2 were similar between groups. Dietary quercetin enrichment improves cardiac function in aged Mdx/Utrn+/- mice and increases mitochondrial protein content and dystrophin glycoprotein complex formation. Histological analyses indicate a marked attenuation in pathological cardiac remodeling and indicate that long-term quercetin consumption benefits the dystrophic heart. NEW & NOTEWORTHY: The current investigation provides first-time evidence that quercetin provides physiological cardioprotection against dystrophic pathology and is associated with improved spontaneous physical activity. Secondary findings suggest that quercetin-dependent outcomes are in part due to PGC-1α pathway activation.

Characterization of the Syk-Dependent T Cell Signaling Response to an Altered Peptide.

Rheumatoid arthritis is an autoimmune disorder characterized by T cell dysregulation. We have shown that an altered peptide ligand (A9) activates T cells to use an alternate signaling pathway that is dependent on FcRγ and spleen tyrosine kinase, resulting in downregulation of inflammation. In the experiments described in this study, we have attempted to determine the molecular basis of this paradox. Three major Src family kinases found in T cells (Lck, Fyn, and Lyn) were tested for activation following stimulation by A9/I-Aq Unexpectedly we found they are not required for T cell functions induced by A9/I-Aq, nor are they required for APL stimulation of cytokines. On the other hand, the induction of the second messenger inositol trisphosphate and the mobilization of calcium are clearly triggered by the APL A9/I-Aq stimulation and are required for cytokine production, albeit the cytokines induced are different from those produced after activation of the canonical pathway. DBA/1 mice doubly deficient in IL-4 and IL-10 were used to confirm that these two cytokines are important for the APL-induced attenuation of arthritis. These studies provide a basis for exploring the effectiveness of analog peptides and the inhibitory T cells they induce as therapeutic tools for autoimmune arthritis.