<p>Macrophages are an important key effector cell in the immune system which can practically be found in every tissue. Macrophages have for a long time been considered a population of cells only responsible for pro-inflammatory responses and anti-microbial activities. But over the past decade, many have come to realize the amazing plasticity of macrophages in response to different stimulations. The anti-microbial and pro-inflammatory macrophage is known as classically activated macrophages but newly discovered phenotypes have been revealed named wound-healing macrophages and regulatory macrophages. Through systematic screening we have identified an inducible macrophage activation state which has both wound-healing and regulatory capabilities activated by the novel cytokine combination IL-4/IL-10 with or without TGF-β.</p>

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<p>Obesity due to a high-fat diet is characterized by accumulation of inflammatory macrophages in tissues, leading to chronic low-grade systemic inflammation. Obese individuals also exhibit impaired tissue healing. With a high-fat feeding, cells are exposed to the elevated levels of dietary fatty acids (FAs), and such a change of microenvironment may alter their properties. Stem cells are cells capable of multipotent differentiation, and this potential allows them to play a promising role in healing and regenerative medicine. However, the effect of obesity, particularly various types of dietary FAs, on the function of stem cells remains largely unknown. Furthermore, obesity is a primary risk factor of osteoarthritis (OA), a disease of entire of joint involving degradation of cartilage, synovitis, and subchondral bone changes. Yet, the mechanisms linking obesity and OA are not fully understood. Furthermore, although macrophages are well recognized for their inflammatory role in obesity, little is known regarding functionality of these cells in regulating the effect of obesity on OA. This dissertation develops fundamental stem cell isolation and culture techniques, and utilizes animal models to investigate (1) the influences of high-fat diet induced-obesity on function of adult stem cells, (2) examine the effect of obesity and dietary FAs on OA, and (3) evaluate the role of macrophages in obesity-associated OA by depleting macrophages using a transgenic mouse model.</p><p>A variety of adult stem cell populations including bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs), and infrapatellar-derived stem cells (IFP cells) were successfully isolated from lean and obese mice and expanded in vitro. Obese stem cells demonstrated altered multilineage differentiation potential and distinct immunophenotypes as compared to lean stem cells. Furthermore, FA treatment of lean stem cells significantly changed their multipotency but did not completely recapitulate the properties of obese stem cells.</p><p>Supplementation of &#969;-3 polyunsaturated fatty acids (PUFAs) in a high-fat diet was capable to mitigate injury-induced OA and decrease serum inflammatory cytokine levels. &#969;-3 PUFAs also significantly enhanced wound repair, while saturated FAs and &#969;-6 PUFAs act as a detrimental factor in OA, synovitis, and wound healing. Spontaneous locomotion of the mice was independent of OA development. Furthermore, using mathematical models and weight-matched mice, we found that OA was significantly associated with dietary FA content but not with body weight and mouse activity. These results suggest that metabolic factor plays a more significant role in obesity-associated OA than mechanical factor. </p><p>Despite their temporary improved metabolic parameters and reduced osteophyte formation, obese mice receiving short-term, systemic macrophage depletion did not mitigate cartilage degeneration following joint injury. Instead, macrophage depletion significantly enhanced joint synovitis in the surgery-operated joint. Macrophage-depleted mice also exhibited up-regulated expression of inflammatory cytokines in synovial fluid. These findings indicate that despite their recognized pro-inflammatory role, macrophages are vital in regulating the homeostasis of immune cells in the joint following injury. </p><p>Taken together, this research further elucidates the relationships among obesity, stem cells, and OA. The results from our study may provide a framework to develop stem cell therapy for obese patients and intervention program for obese OA patients in the future.</p> / Dissertation

Human macrophages are one of the main targets for HIV-1 infection, despite their moderately low surface expression levels of the main HIV-1 receptor, CD4. The site of HIV-1 fusion can occur at the surface or following uptake through an endosomal pathway and it might be anticipated that the site would affect the progress of HIV-1 through the cell to the nucleus. Previous pharmacological studies provide one line of evidence for an endosomal entry route which is dependent on Detergent Resistant Membranes (DRMs). However, these findings need confirmation using a genetic approach, as small molecules may have multiple non-specific effects. For this study, a novel genetic approach was developed to manipulate sub-cellular CD4 distribution and investigate whether it determines the HIV-1 entry pathway in macrophages. This was achieved by transducing human embryonic stem cells (hESC) with lentiviral vectors and differentiating these cells into homogeneous genetically modified macrophages. This cellular system by-passes the challenges posed by the refractoriness to direct genetic manipulation of heterogeneous primary macrophages. Firstly, as proof of principle, a short hairpin RNA targeting CD4 was expressed in hESC-macrophages, resulting in knockdown of CD4 and, as anticipated, strong inhibition of HIV-1 infection. Secondly, expression of LCK in hESC-macrophages effectively tethered CD4 at the cell surface, and sequestered HIV-1 into an unproductive pathway, presumably through surface fusion, rather than progressing successfully to the nucleus. Thirdly, endogenous CD4 was substituted with CD4 mutants designed to be excluded from DRMs, which resulted in reduced successful HIV-1 entry versus substituted control CD4. The results support the model in which the productive entry pathway of HIV-1 in macrophages occurs via fusion after a raft-dependent endocytic uptake pathway, and requires CD4 localization to lipid rafts.

The kinetics and mechanism of accumulation for liposomal ciprofloxacin (Lipo-CPFX) into the rat alveolar macrophage NR8383 cells were studied in vitro, in comparison to unformulated ciprofloxacin (CPFX). Upon incubation with CPFX or Lipo-CPFX, cellular drug accumulation was determined from the cell lysates or efflux was from the extracellular media by fluorescence-HPLC. The accumulation for Lipo-CPFX reached the asymptotic values at ≥ 2 hours, which was a result of uptake and efflux. The uptake appeared to be due to liposomes, mediated via cellular energy-independent mechanism like lipid fusion. In contrast, the efflux appeared to be due to ciprofloxacin, partly cellular energy-dependent, and involve probenecid-sensitive multidrug resistance proteins (MRPs). Overall, Lipo-CPFX enabled greater drug accumulation into the NR8383 cells than CPFX. This logically suggests a greater potential to treat respiratory infections especially caused by bacteria resistant to phagocytic killing.

The aim of this study was to investigate macrophage interactions with electrospun scaffolds and quantify the expression of vital angiogenic growth factors in vitro. This study will further help in evaluating the potential of these electrospun constructs as vascular grafts for tissue repair and regeneration in situ. Human peripheral blood macrophages were seeded in serum free media on electrospun (10 mm) discs of polydioxanone (PDO), elastin and PDO:elastin blends (50:50, 70:30 and 90:10). The growth factor secretion was analyzed by ELISA. Macrophages produced high levels of vascular endothelial growth factor (VEGF) and acidic fibroblast growth factor (aFGF). Transforming growth factor beta-1 (TGF-β1) secretion was relatively low and there was negligible production of basic fibroblast growth factor (bFGF). Histology revealed direct correlation between cell infiltration into scaffolds and the PDO concentration. There was greater macrophage infiltration through fibrous networks of the PDO and 90:10 scaffolds. Therefore, it can be anticipated that these scaffolds will support tissue regeneration and angiogenesis.

Therapeutic ultrasound (TUS) is a treatment modality that is used to accelerate tissue healing. TUS is thought to affect cellular processes of tissue healing, especially those that occur in the inflammatory and early proliferative phases. TUS can be applied using various parameter selections including intensity, wavelength, duty cycle and treatment duration and no clear consensus exists on optimal parameters for healing enhancement. Macrophages are important mediators of inflammation and their actions are critical to normal progression into the proliferative phase of healing. They complete many functions during these periods of tissue healing, among those being release of cytokines and growth factors. These paracrine factors affect other inflammatory cells, resident cells of the healing tissue, including fibroblasts and endothelial cells that are necessary for restoration of damaged tissue. The hypothesis of this investigation is that TUS enhances early healing, in part, through stimulation of macrophage release of paracrine factors involved in coordination of the cellular aspects of tissue healing and that specific levels of TUS are most stimulatory for macrophages. This study examined macrophage release of interleukin-1beta (IL-1Beta), vascular endothelial growth factor (VEGF), transforming growth factor-Beta 1 (TGF-B1) and fibroblast mitogens, in response to varied levels of TUS. Fibroblasts incubated up to 48-hours in media conditioned by TUS-stimulated macrophages were not induced to proliferate regardless of the parameters sets of TUS applied. TUS (1 MHz, 400mW/cm2 SATA, 20% duty cycle, 10-minute exposure) induced macrophage release of VEGF and IL-1Beta within 10-minutes post-TUS, without any additional release being stimulated at 1-hour post-insonation. No other combination of TUS parameters studied induced release of IL-1Beta and VEGF. TUS did not induce release of TGF-Beta 1 at either time point post-TUS. VEGF and IL-1Beta release occurred in conjunction with lactate dehydrogenase (LDH) release from treated macrophages, indicating non-specific cell membrane permeabilization was involved in the cellular response. For IL-1Beta, TUS-stimulated release was inhibited at lower exposure temperatures. Inhibition of TUS-induced release at lower temperatures indicates that a cellular metabolic process, most likely exocytosis, was also stimulated by TUS. Based on these results, it appears that TUS exposure at 1 MHz, 400mW/cm2 SATA, 20% duty cycle induces non-specific and cell-mediated release of secretory proteins. Thus, enhanced release of cytokines and growth factors from macrophages is a possible mechanism by which TUS enhances tissue healing.

Macrophages (MФ) and mast cells are important cell types in the context of tissue remodeling and regeneration. Mast cells participate in the early stages of wound healing and modulate the acute inflammatory responses to biomaterials. Mast cells can secrete a myriad of different cytokines by the process of degranulation; the process of regulated secretion in which preformed contents stored in their granules are rapidly released by exocytosis. Some of these cytokines such as IL-4, IL-13 and TNF-α can modulate the MФ phenotype. Macrophages (MΦ) are innate immune cells, crucial for tissue homeostasis, presentation of foreign and self-antigens following infection/injury, pathogen clearance, inflammation resolution, angiogenesis, and wound healing. MΦ display plasticity and can acquire pro-inflammatory (M1) or angiogenic/wound healing (M2) phenotypes depending upon the environmental stimuli. The phenotypic profile of MФ as M1 or M2 following exposure to the biomaterial can dictate the downstream processes of tissue remodeling and angiogenesis. An analysis of how these two cell types interact with electrospun biomaterials and how different properties of an electrospun biomaterial impacts the MΦ phenotype is the focus of this thesis. Mast cells synthesize several potent angiogenic factors and can also stimulate fibroblasts, endothelial cells and macrophages. An understanding of how they participate in wound healing and angiogenesis is important to further our knowledge about in situ vascular prosthetic regeneration. The adhesion, proliferation and cytokine secretion of bone marrow derived murine mast cells (BMMC) on electrospun polydioxanone (PDO), polycaprolactone (PCL) and silk scaffolds, as well as tissue culture plastic (TCP) has been investigated in the presence or absence of IL-3, SCF, IgE and IgE with a crosslinking antigen, dinitrophenol-conjugated albumin (DNP). It was previously believed that only activated BMMCs exhibit adhesion and cytokine secretion. However, this study shows non-activated BMMC adhesion to electrospun scaffolds. Silk scaffold was not found to be conducive for mast cell adhesion and cytokine secretion. Activation by IgE and DNP significantly enhanced mast cell adhesion, proliferation, migration and secretion of TNF-α, MIP-1α and IL-13. This indicates that mast cells might play a role in MФ polarization (M1/M2), biomaterial integration into the host tissue, regeneration, and possibly angiogenesis. In the next study, bone marrow derived murine macrophages (BMMΦs, 106 cells) were seeded on TCP (24 well plate) and PDO scaffolds (15 mm discs) electrospun from varying polymer concentrations (60, 100, and 140 mg/ml). Scaffold evaluation showed that large polymer concentrations led to larger fiber diameters, which in turn led to larger pore-sizes and porosity but a smaller surface area to volume ratio. After 24 hrs of culture, the cell lysates were analyzed for Arginase (Arg1) and inducible nitric oxide synthase (iNOS) expression by western blot and cell culture supernatants were analyzed for Nitric oxide (NO2-), Tumor Necrosis Factor – alpha (TNF-α), Interleukin-6 (IL-6), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor – beta1 (TGF-β1) and basic fibroblast growth factor (bFGF) levels. The results indicated a correlation between Arg1 expression and increasing fiber/pore-size, indicating that the larger fiber/pore-sizes polarize towards a M2 phenotype. Also, the expression of iNOS was downregulated on the larger fiber/pore-size. The levels of NO2- were significantly higher on the lower fiber/pore-sizes indicating an M1 phenotype. The levels of VEGF, TGF-β1 and bFGF increased with increasing fiber/pore-sizes. The results showed higher Arg1 expression in M2s on the 60 mg/ml scaffold created by the air-flow impedance method compared to the 60 mg/ml scaffold created on the solid mandrel created by traditional electrospinning. The Arg1 expression was reduced on the compressed 140 mg/ml PDO scaffold compared to the normal 140 mg/ml scaffold. This result indicates that pore-size might be playing a greater role compared to fiber diameter in BMMФ phenotype modulation. In order to assess the angiogenic potential of BMMΦs cultured on PDO scaffolds, a 3D angiogenesis bead assay was performed using conditioned media from the BMMΦ:PDO interaction. The results of the 3D angiogenesis bead assay showed that the conditioned media from BMMΦs of M0 and M2 phenotypes cultured on the 140 mg/ml PDO scaffold induced larger sprouting and higher percentage density of sprouts when compared to the 60 mg/ml PDO scaffold and TCP. To investigate the signaling mechanism involved in this phenotypic switch, BMMΦs were isolated from the bone marrow of MyD88 knockout (KO) mice (Jackson Laboratories) and cultured on PDO (60 and 140 mg/ml) scaffolds (106 /disc) and TCP for 24 hrs and their Arg1 and iNOS expression was analyzed by western blot. The expression of Arg1 and iNOS was severely impaired on the BMMΦs from MyD88-/- mice cultured on the 140 mg/ml scaffold when compared to the 60 mg/ml PDO scaffold and TCP. This result indicates that scaffolds with different fiber/pore-sizes signal differently. A subcutaneous mouse model (described in Chapter 6) was used to evaluate the angiogenic and regenerative potential of PDO scaffolds in vivo. The DIVAA assay showed statistically higher FITC-dextran signal intensity for the 140 mg/ml scaffold compared to the 60 mg/ml scaffold indicating greater angiogenic response in the 140 mg/ml tube. However, problems of high background were observed in this assay with the use of electrospun PDO. The observed high background was probably due to the formation of complexes between dextran and adsorbed plasma proteins on the surface of the PDO. More studies are needed to optimize this assay for use with biomaterials such as PDO. H&E staining of the harvested PDO tubes (60 mg/ml and 140 mg/ml) was also performed. The cross-sections of these tubes showed greater cell recruitment and infiltration into the fibrous structures of the 140 mg/ml tube compared to the 60 mg/ml tube. This result corroborates the in vitro result of BMMФ infiltrating deeper into the structures of the 140 mg/ml scaffold compared to the 60 mg/ml scaffold. The scaffolds were also analyzed by immunostaining for iNOS (indicative of M1 phenotype of MФs). The results showed statistically higher ratios of iNOS positive:negative areas on the 60 mg/ml scaffold compared to the 140 mg/ml scaffold. Overall, these studies indicate that 140 mg/ml scaffold supports greater cell recruitment and cell infiltration in vivo but a smaller ratio of iNOS positive:negative areas compared to the 60 mg/ml scaffold, which supports a predominately M1 MФ phenotype. The studies indicate that varying properties of PDO can alter both the phenotype and function of BMMΦs in vitro and in vivo. We have also shown that the 140 mg/ml scaffold signal BMMΦs through MyD88-dependent mechanisms. A complete understanding of the way materials signal would allow us to control or modulate undesirable immune reactions to biomaterials in vivo. These studies would also help engineer biomaterials that promote angiogenesis and regeneration.

1 Abstract Vitamin D3 is important for keeping the right concetration of Ca2+ in plasma. Therefore it is essential for proper bone growth and development. Nevertheless, vitamin D3 has also a number of immunomodulating effects. Our thesis has been targeted on evaluation and comparison of vitamin D3 influence on expression of chosen surface markers (CD14, CD54, HLA-DR, CD16, CD36 and CD163) with THP-1 cells and monocytes gained from human peripheral blood. Other aims have been analysing the vitamin D3 influence on longevity of THP-1 cells and measuring the soluble CD14 and IL-8 production with THP-1 cells under the vitamin D3 influence. The cells have been stimulated with five different concentrations of vitamin D3 for the time 24, 48 and 72 hours. Higher used concetrations of vitamin D3, i.e. 100 nM and 1000 nM have increased the expression of CD14 with THP-1 cells in the time 48 and 72 hours of the stimulation time. With the monocytes from peripheral human blood the increase of the CD14 expression hasn't been remarkable from the physiological point of view. Together with the vitamin D3 concentration increase the sCD14 production with THP­1 cells was considerably higher. The sCD14 was the highest in the time 72 hours after the stimulation with the highest used vitamin D3 concetration. The IL-8 quantity with...

Monocytes and macrophages are important components of the innate immune response. These mononuclear phagocytes form a heterogeneous cell population, of which phenotype and functions can be modified under the influence of different signals coming from the surrounding microenvironment. The aim of this work was to modulate the phenotype of these cells by a variety of stimulants and to compare the changes induced on the model of THP-1 monocytic cell line and on the human peripheral blood monocytes. Surface marker expression was analyzed by flow cytometry. Further on, IL-8 production was evaluated by Luminex assay and the concentration of soluble calprotectin was assessed by ELISA. The most significant changes in surface marker expression were induced by exposure to IFNγ. This cytokine increased the expression of CD54, CD14 and HLA-DR on the surface of THP-1 cell line. Higher concentrations of IFNγ promoted higher apoptotic rate and augmented calprotectin expression and production in THP-1 cell line. On the surface of monocytes, IFNγ stimulation resulted only in the upregulation of CD54 expression. IL-4 increased the expression of CD36 by THP-1 cell line and inhibited the expression of CD163 by human monocytes. LPS stimulation caused the suppression of HLA-DR activation in monocytes and enhanced IL-8...