The focus of this research is to describe the production and characterization of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in insect cells, using Autographa californica buclear polyhedrosis virus (AcNPV) as an expression vector. All three forms of biological activity of hGM-CSF. Following N-glycanase treatment, the two glycosylated hGM-CSF proteins (15.5 and 16.5 KDa) which bound to Concanavalin A affinity column ran as a 14.5-15.5 KDa band on SDS-PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 KDa species. The N-terminal amino acid sequence of the recombinant hGM-CSF was identical to that of natural hGM-CSF deduced from cDNA. These results demonstrate that baculovirus-produced hGM-CSF could be N-glycosylated in Sf9 cells, the signal peptide of recombinant hGM-CSF could be recognized and cleaved by infected insect cells and the resultant molecule secreted into the medium.

Abstract Macrophages found in the tumor microenvironment play a crucial role in initiating an immunosuppressive tumor microenvironment that negatively impacts immunotherapy efficacy and aids tumor progression and metastasis. Constituting the most abundant immune cell in tumor microenvironment (TME), tumor associated macrophages (TAMs) have emerged as an attractive approach for anti-cancer therapy. However, two major challenges need to be overcome for successfully utilizing macrophages for immunotherapy. First, tumors repolarize the TAMs predominantly to M2 tumor-aiding phenotype by secreting various immunosuppressive cytokines. Second, cancer cells overexpress a membrane protein CD47 that interacts with signal-regulating protein alpha (SIRPalpha) expressed on macrophages. This crosstalk provides a downregulatory signal in the form of activation of SHP1/2 that inhibits cancer cell phagocytosis, and CD47, therefore, functions as a “don’t-eat-me” signal. We rationalized that these challenges can be overcome by engineering a nanoparticle system that can deliver a rationale combination of immunomodulatory agents to the TAMs that can both repolarize the M2 macrophages to M1 phenotype efficiently and concurrently block CD47-SIRPalpha interactions by inhibiting SHP2 signaling. Herein, we designed a lipid nanoparticle (LNP) system loaded with amphiphilic R848-cholesterol in its hydrophobic lipid bilayer, while SHP099 gets encapsulated in the hydrophilic core. Our previous studies have shown that the conjugation of cholesterol to the inhibitor stabilizes the lipid bilayer at a high inhibitor concentration. The LNPs showed high optimal drug loading, size, and stability. In vitro studies showed that the M2 macrophages treated with the LNPs system repolarized to M1 phenotype and expressed co-stimulatory molecules while having enhanced phagocytic potential. In vivo efficacy studies in 4T1 tumor-bearing mice showed that LNPs exhibit superior anti-tumor efficacy compared to other treatments. We evaluated the effect of MARCO-targeted LPNs by the conjugating anti-MARCO antibody on the LPN surface. However, no comparable difference in treatment efficacy was observed between the targeted MARCO-LNPs and the non-targeted LNPs. These results demonstrate that the MARCO targeting system designed in this study is largely ineffective in the targeted delivery of its drug cargo specifically to TAMs. Thus, the lipid nanoparticle-mediated co-delivery of a rational combination of TLR7/8 agonist and SHP2 inhibitor in the TAMs increases M2 to M1 repolarization and phagocytosis potential of macrophages. Recommended Citation Malik, V., Ramesh, A. and Kulkarni, A.A. (2021), TLR7/8 Agonist and SHP2 Inhibitor Loaded Nanoparticle Enhances Macrophage Immunotherapy Efficacy. Adv. Therap., 4: 2100086. https://doi.org/10.1002/adtp.202100086

IFNγ is an essential and pleiotropic activator of monocytes, but little is known about the effects IFNγ on cellular metabolism. Therefore, we sought to characterize and elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. First, we identified a critical role for IFNγ in the induction of immunoresponsive gene 1 (IRG1) and its product, itaconate. The immunometabolite, itaconate, has been reported to have antibacterial, anti-inflammatory and antioxidant activity. Irg1-/- mice, lacking itaconate, are highly susceptible and phenotypically similar to IFNγ knock out (GKO) mice upon infection with Mycobacterium tuberculosis. Therefore, we assessed the role of IRG1/itaconate in the context of non-tuberculous mycobacterial (NTM) infection, the most common type of infection in patients with immunodeficiencies caused by defects in IFNγ signaling. Our data suggest that impaired induction of itaconate in the context of mycobacterial infection may contribute to mycobacterial susceptibility and immune dysregulation in patients with defects in IFNγ signaling. Next, we evaluated the metabolic phenotype of IFNγ-stimulated human monocytes and found that IFNγ increased oxygen consumption rates (OCR), indicative of reactive oxygen species generation by both mitochondria and NADPH oxidase. Transcriptional profiling of human macrophages revealed that this oxidative phenotype was dependent on IFNγ-induced, nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for complete induction of the respiratory burst in IFNγ stimulated human monocytes.

Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity, myogenic potential, M2 macrophage polarization and decreases NF-κB inflammation in dysferlin-deficient models. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, and myoblast migration in dysferlin-deficient myotubes. Furthermore, rHsGal-1 showed an increased membrane repair capacity of dysferlin-deficient myotubes. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggests mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-l significantly reduces canonical NF-κB inflammation through TAK 1, P65, P50. Lastly we find 2.7 mg/kg in vivo rHsGal-1 treatment in BLA/J mice supports an M2 cyto-regenerative macrophage populations. Together our novel results reveal Gal-1 remediates disease pathologies in LGMD2B through changes in integral myogenic protein expression, mechanical membrane stabilization, immune modulation, and reducing canonical NF-κB inflammation.

The nervous system and immune system have broad and overlapping distributions in the body, and interactions of these ubiquitous systems are central to the field of neuroimmunology. Over the past two decades, there has been explosive growth in our understanding of neuroanatomical, cellular, and molecular mechanisms that mediate central modulation of immune functions through the autonomic nervous system. A major catalyst for growth in this field was the discovery that vagal nerve stimulation (VNS) caused a prominent attenuation of the systemic inflammatory response evoked by endotoxin in experimental animals. This effect was mediated by acetylcholine (ACh) stimulation of nicotinic receptors on splenic macrophages. Hence, the circuit was dubbed the “cholinergic anti-inflammatory pathway”. Subsequent work identified the α7 nicotinic ACh receptor (α7nAChR) as the crucial target for attenuation of pro-inflammatory cytokine release from macrophages and dendritic cells. Further investigation made the important discovery that cholinergic T cells within the spleen and not cholinergic nerve cells were the source of ACh that stimulated α7 receptors on splenic macrophages. Given the important role that inflammation plays in numerous disease processes, cholinergic anti-inflammatory mechanisms are under intensive investigation from a basic science perspective and in translational studies of animal models of diseases such as inflammatory bowel disease and rheumatoid arthritis. This basic work has already fostered several clinical trials examining the efficacy of VNS and cholinergic therapeutics in human inflammatory diseases. This review provides an overview of basic and translational aspects of the cholinergic anti-inflammatory response and relevant pharmacology of drugs acting at the α7nAChR.

Adipose tissue inflammation has an impact on liver health and it has been demonstrated that chronic alcohol consumption leads to the expression of pro-inflammatory markers in the adipose tissue. A thorough characterization of alcohol-induced adipose inflammation is lacking, and is important to understand in order to identify immune-related mechanisms that drive this phenomenon. Current therapeutic regimens for alcoholic liver disease are ineffective. It is critical to understand how other organs influence liver injury in this disease when developing novel and effective therapies in the future. Alcoholic liver disease exhibits a sexual dimorphism; women are more susceptible to liver injury than men and the same paradigm exists in rodent models. Here, I demonstrate that female mice have greater alcohol-induced adipose tissue inflammation than male mice, evidenced by greater expression of pro-inflammatory cytokines and cell markers. Further, female mice also exhibit higher expression of toll-like receptor genes in the adipose tissue, suggesting a potential role for the innate immune system in alcohol-induced adipose inflammation. Toll-like receptor 4 (TLR4) has been demonstrated to drive inflammation in both the liver and adipose tissue. I used both germline and conditional knockouts of Tlr4 to characterize alcohol-induced changes in the immune cell composition of adipose tissue. Alcohol increased the number of pro-inflammatory adipose tissue macrophages. This macrophage phenotype switching is partially dependent on TLR4; germline, but not myeloid-specific, Tlr4-deletion prevents macrophage phenotype switching. Overall, my work demonstrates that alcohol-induced adipose tissue inflammation is related to liver injury and that TLR4 contributes to adipose macrophage phenotype switching.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20983号 / 医博第4329号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 杉田 昌彦, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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