Oxidation of polyunsaturated fatty acids contributes to different aspects of the inflammatory response due to the variety of products generated. Specifically, the oxidation of DHA produces the end-product, carboxyethylpyrrole (CEP), which forms a covalent adduct with proteins via an ϵ-amino group of lysines. Previously, we found that CEP formation is dramatically increased in inflamed tissue and CEP-modified albumin and fibrinogen became ligands for αDß2 (CD11d/CD18) and αMß2 (CD11b/CD18) integrins. In this study, we evaluated the effect of extracellular matrix (ECM) modification with CEP on the adhesive properties of M1-polarized macrophages, particularly during chronic inflammation. Using digested atherosclerotic lesions and in vitro oxidation assays, we demonstrated the ability of ECM proteins to form adducts with CEP, particularly, DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin, but not with collagen I. Using integrin αDß2-transfected HEK293 cells, WT, and αD-/- mouse M1- polarized macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading when compared with natural ECM ligands such as collagen IV, laminin, and fibrinogen. Integrin αDß2 is critical for M1 macrophage adhesion to CEP. Based on biolayer interferometry results, the isolated αD I-domain demonstrates markedly higher binding affinity to CEP compared to the “natural” αDß2 ligand fibrinogen. Finally, the presence of CEP-modified proteins in a 3D fibrin matrix significantly increased M1 macrophage retention. Therefore, CEP modification converts ECM proteins to αDß2- recognition ligands by changing a positively charged lysine to negatively charged CEP, which increases M1 macrophage adhesion to ECM and promotes macrophage retention during detrimental inflammation, autoimmunity, and chronic inflammation.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder and the third leading cause of mortality. In this thesis we performed a clustering analysis of four specific immune cells in the GSE136831 dataset, using the default recommended parameters of the Seurat package in R, and obtained 16 subclasses with various COPD and cell-type proportions. Clusters 3, 7 and 9 had more pronounced independence and were all composed of macrophage-dominated control samples. The results of the pseudo-time analysis based on Monocle 3 package in R showed three different patterns of cell evolution. All started with a high percentage of COPD states, one ended with a high rate of Control states, and the other two still finished with a high percentage of COPD states. The results of differentially expressed gene analysis corroborated the existence of finer clusters and provided support for their rational categorization based on the similar marker genes. The gene ontology (GO) enrichment analysis for cluster 0 and cluster 6 provided feedback on enriched biological process terms with significant and unique characteristics, which could help explore latent novel COPD treatment directions. Finally, some top-ranked potential pharmaceutical molecules were searched via the connectivity map (cMAP) database. / Graduate / 2023-08-12

Arginine transport is primarily mediated by the cationic amino acid transporters (CATs) in mammalian cells, but in aves the y+, b0,+ and B0,+ transport systems have also been observed. Arginine is the limiting catabolic substrate required for the production of nitric oxide (NO), a highly reactive compound that acts as a signaling molecule or killing compound. NO is synthesized by inducible nitric oxide synthase (iNOS) by macrophages for pathogen clearance. In mammals, CAT-2B is responsible for ARG import in the macrophage for NO synthesis, but the chicken CAT-2B isoform does not transport ARG. Therefore the objective of these studies was to identify the CAT(s) involved in mediating ARG uptake during a NO response in the chicken macrophage. Experiments were performed to measure: 1) ARG transporter mRNA and NO production from three sources of macrophages (HD11 cell line, n=6; primary 32d Cobb 500, n=8; Hyline W36, n=7) in response to Escherichia coli lipopolysaccharide (LPS); 2) the effect of CAT over-expression on NO production in response to LPS (HD11 cell line; n=8). In response to LPS iNOS mRNA abundance increased (P<0.05) 8.5-fold in the HD11 macrophages, 3.22-fold in broiler macrophages and 2.79-fold in layer macrophages. In all cells, CAT-1 was induced and CAT-2A increased (P<0.05) between 1.28 and 1.68-fold. CAT-2B was not detected at any time point or treatment condition. In the virally transformed chicken macrophage cell line (HD11) CAT-3 mRNA was induced, but in primary cells CAT-3 increased (P<0.05) 1.27-fold in broilers and 1.23-fold in layers. Transiently transfected chicken macrophages produce NO independent of LPS treatment by 6h, mock transfected controls did not respond by 6h. In the presence of LPS, CAT-1 transfected macrophages produced 50.0% more NO than mock transfected cells (P<0.05). CAT-2A and CAT-3 transfected macrophages produced only 17.6% and 72.1% of the total NO produced by controls (P<0.05). These results indicate that CAT-1 and CAT-3 are both sufficient to sustain ARG import for NO production in the chicken macrophage, but that CAT-1 produces a maximal response. These results also show that iNOS, despite its name, is constitutively present and can be activated by induction of CATs to import ARG.

No description available.

Muscular dystrophies are a heterogeneous group of genetic diseases that involve mutations in genes leading to progressive muscular weakness. Limb-Girdle Muscular Dystrophy 2B (LGMD2B) is a subset of muscular dystrophy caused by mutations in the DYSF gene, which encodes for dysferlin protein and has an incidence of 1/100,000-1/200,000 people, or 1/300 people of Libyan Jewish descent. Since there is no effective treatment that can cure or reverse effects of LGMD2B once diagnosed, our goal is to investigate and develop a protein therapy that mitigates effects of this disease in patients. Galectin-1 (Gal-1) is a small, soluble 14.5 kDa protein with a carbohydrate recognition domain capable of stabilizing the sarcolemma. The exact role that Gal-1 plays in myogenic cells is not fully understood, however, it is known that Gal-1 possesses anti-inflammatory properties and increases the terminal differentiation of committed myogenic cells. Our hypothesis is that Gal-1 treatment increases myogenic potential, improves membrane repair capability, and modulates the immune response in models of LGMD2B by stabilizing muscle integrity, leading to decreased disease manifestation. To test this hypothesis and assess the effect of Gal-1 treatment on myogenesis, anti-inflammatory modulation, and membrane repair, we designed, produced, and purified recombinant human galectin-1 (rHsGal-1) to be used in LGMD2B models. Our in vitro results indicate that after 2-3 days of treatment with 0.11μM rHsGal-1, A/J-/- myotubes enhance expression of myogenic late markers and increase in size and alignment. Additionally, after short-term treatment, rHsGal-1 improves membrane repair capability in a Ca2+ independent manner through an activated carbohydrate recognition domain (CRD) in in vitro and in vivo models of LGMD2B. We give evidence that rHsGal-1 upregulates anti-inflammatory cytokines, increases functional activity, and modulates the canonical NF-κB inflammatory pathway in dysferlin-deficient models by decreasing expression of TAK-1 and the p65 and p50 subunits in vitro and short-term in vivo treatment. Similar effects of the rHsGal-1 treatment were observed in patient-derived dysferlin-deficient human myotubes. Exploratory results show a potential decrease in muscle fat deposition in Bla/J mice. Furthermore, Gal-1 contributes to immune modulation by helping to initiate muscle regeneration by shifting M2 macrophage polarization. Together, our novel discoveries provide direct evidence that Gal-1 is a promising candidate to treat LGMD2B disease pathologies by improving expression of late-stage myogenic markers, improving membrane repair in vitro and short-term in vivo studies, promoting muscle regeneration through immune modulation, and reducing canonical NF-κB inflammation.

No description available.

No description available.

Antibody-recruiting molecules (ARMs) are therapeutic tools that simultaneously bind a hapten-specific serum antibody and a cancer cell surface protein, resulting in the activation and recruitment of an immune cell to the cancer surface. However, ARM efficacy is limited by the ability of ARMs to form a quaternary complex with the immune cell receptor, antibody, and cancer cell surface. The Rullo lab has previously developed and characterized a covalent ARM (cARM) that irreversibly links the ARM to the antibody and simplifies the quaternary binding equilibria. cARMs have shown a marked increase in both target immune recognition and therapeutic efficacy. However, cARM efficacy is still limited by the affinity of the antibody for the immune receptor. We aim to investigate how direct covalent engagement of the immune receptor and elimination the antibody-immune receptor binding equilibria impacts immune activation and therapeutic efficacy. This thesis focuses on the chemical programming of macrophages through direct covalent immune receptor engagement. We have developed and characterized covalent immune programmers (CIPs), which are molecules that contain a macrophage targeting domain and a tumour targeting domain. The macrophage targeting domain binds the activating receptor CD64 on the macrophage surface and contains a chemical warhead that covalently labels the receptor once bound. The tumour targeting domain can promote macrophage tumour engagement resulting in tumoricidal function. Flow cytometry experiments have shown that CIPS are able to bind Fc receptors specifically and effectively on the surface of macrophages. Further, CIPs were able to induce macrophage activation and induce target specific phagocytosis. These experiments have also shown that direct engagement of the receptor by the CIP is more effective than antibody-mediated engagement, suggesting that overall immune complex stability affects immune cell activation. Taken together, these concepts can be used to guide future immunotherapeutic design. / Thesis / Master of Science (MSc)

The use of the plasticizer bis(2-ethylhexyl)phthalate (DEHP) and other plasticizers in the manufacture of plastic products has been restricted due to adverse health outcomes such as obesity, metabolic syndrome, and asthma, for which inflammation has been described to be a driving factor. The emerging alternative plasticizer 1,2-cyclohexanedioic acid diisononyl ester (DINCH) still lacks information regarding its potential effects on the immune system. Here, we investigated the effects of DINCH and its naturally occurring metabolite monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH) on the innate immune response. Human THP-1 macrophages were exposed to 10 nM–10 μM DINCH or MINCH for 4 h, 16 h, and 24 h. To decipher the underlying mechanism of action, we applied an untargeted proteomic approach that revealed xenobiotic-induced activation of immune-related pathways such as the nuclear factor κB (NF-κB) signaling pathway. Key drivers were associated with oxidative stress, mitochondrial dysfunction, DNA damage repair, apoptosis, and autophagy. We verified increased reactive oxygen species (ROS) leading to cellular damage, NF-κB activation, and subsequent TNF and IL-1β release, even at low nM concentrations. Taken together, DINCH and MINCH induced cellular stress and pro-inflammatory effects in macrophages, which may lead to adverse health effects.

BACKGROUND: Distinguishing patients with axial spondyloarthritis (axial SpA) from patients with other causes of chronic back pain remains a challenge. The lack of reliable biomarkers contributes to the diagnostic delay in axial SpA. Recently, macrophage migration inhibitory factor (MIF) has been proposed as a candidate diagnostic and prognostic biomarker. MIF is a proinflammatory cytokine that was shown to be upregulated in several autoimmune diseases, including axial SpA. The putative role of CD8+ T cells in the disease process suggests further that serum markers of cytotoxicity might have value as serological biomarkers in axial SpA, and that subpopulations of cytotoxic lymphocytes might deserve attention as candidate cellular biomarkers. OBJECTIVE: The goal of this study was to compare serum levels of MIF and other candidate serum proteins in patients with axial SpA and controls, and to develop a flow cytometry panel to analyze cytotoxic lymphocyte cell subpopulations in these cohorts, including KIR+CD8+ T cells, Granzyme B+ CD8+ T cells, MAIT cells, and InEx cells. METHODS: Study subjects were recruited from the Brigham and Women’s Hospital Orthopedic and Arthritis Center. Four cohorts were compared: healthy controls (HC), patients with chronic low back pain (cLBP), axial SpA patients not on a biologic (axSpA/-), and axial SpA patients treated with a TNF inhibitor (axSpA/TNFi). Study subjects were matched for age, sex, and race, when possible. Serum was evaluated using the LEGENDplex Human CD8/NK panel (BioLegend) for thirteen markers including IL-17A, IL-6, TNF, granzyme B, and perforin. CRP and MIF were evaluated by DuoSet ELISA (R&D Systems). A high-dimensional flow cytometry panel was designed to evaluate 14 cell populations of interest. RESULTS: The severity of back pain in the cLBP controls and axSpA/- patients was comparable (BASDAI Q2 mean 5.0 +/- 1.9 vs. 5.0 +/- 3.0). axSpA/- patients had higher back pain, BASDAI and ASDAS scores than axSpA/TNFi patients consistent with higher disease activity in the biologic naïve group. Serum CRP values were significantly higher in axSpA/- patients compared with HC, cLBP controls, and axSpA/TNFi patients (P= 0.01, P=0.0029, P=0.004 respectively). Serum MIF levels were not statistically different between all four groups (P= 0.8069). Additionally, there were no statistically significant differences between the groups for any of the markers included in the LEGENDplex Human CD8/NK panel. A 32-color staining panel was developed to evaluate cytotoxic cell populations. CONCLUSION: In contrast to a previous study, we did not find differences in serum MIF levels between axial SpA patients and controls. Of the evaluated serum biomarkers, only CRP values correlated with active axial SpA. We have developed a promising flow cytometry panel that will help analyze subpopulations of cytotoxic cells. This ultimately could shed light on a candidate cellular biomarker. Our results underscore the need for more research into diagnostic biomarkers in axial SpA.