Role Of Macrophages In Differentiation Research Articles

Vitamin A-Coupled Liposomes Carrying siRNA Against HSP47 Ameliorate Skin Fibrosis in Chronic Graft-Versus-Host Disease

Introduction: Chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation is characterized by multiple-organ fibrosis and often refractory to conventional immunosuppressive therapies, profoundly affecting quality of life of SCT survivors. Heat shock protein 47 (HSP47), a collagen specific molecular chaperone, plays a critical role in collagen synthesis in myofibroblasts. Vitamin A-coupled liposomes carrying HSP47 siRNA (VA-Lip HSP47) have been shown to deliver HSP47 siRNA to pathogenic myofibroblasts and ameliorate mouse liver cirrhosis. We tested if VA-Lip HSP47 could inhibit collagen synthesis in myofibroblasts and ameliorate skin fibrosis in chronic GVHD, using a well-established murine model of cutaneous chronic GVHD.Methods: VA-Lip HSP47 was provided by Nitto Denko Corporation (Osaka, Japan). BALB/c mice were exposed to a single dose of 6 Gy total body irradiation, followed by intravenous injection with 8 x 106 bone marrow cells plus 2.5 x 107 splenocytes from minor histocompatibility antigen mismatched B10.D2 or syngeneic BALB/c donors on day 0. Recipient mice were intravenously injected with 4.5 mg/kg VA-Lip HSP47 or vehicle thrice a week from day +1 to +41 after bone marrow transplantation (BMT). To deplete macrophages, a group of recipients were intraperitoneally injected with 0.5 mg/body of anti-CSF1 receptor monoclonal antibodies (aCSF1R) thrice a week from day +7 after BMT. Skin samples were harvested on day +42. For in vitro analysis, primary fibroblasts isolated from mouse skin were stimulated by 5 ng/ml TGF-b in the presence or absence of 50 nM of VA-Lip HSP47 or VA-Lip containing scrambled siRNA. HSP47 expressions were measured by quantitative PCR.Results:Masson’s trichrome staining of the skin sections demonstrated skin thickening with massive fibrosis of the dermis 42 days after BMT. Quantitative collagen assay confirmed collagen volume was significantly increased in the skin of allogeneic recipients compared to syngeneic controls. Immunofluorescent study showed myofibroblasts expressing HSP47 were accumulated in the dermis of allogeneic animals, while these cells were hardly detected in the skin of syngeneic controls. Macrophage-depletion with repeated administration of aCSF1R dramatically reduced HSP47+ myofibroblasts, suggesting a critical role of macrophages in differentiation of HSP47+ myofibroblasts. In vitro culture showed VA-Lip HSP47 knocked down HSP47 in mouse skin myofibroblasts, while VA-Lip carrying scrambled siRNA did not alter HSP47 expression (Figure 1). To confirm in vivo distribution of VA-Lip HSP47 specifically to the fibrotic skin lesion, mice with bleomycin-induced localized skin fibrosis were injected with VA-Lip HSP47 labeled with fluorescent dye. We confirmed VA-Lip HSP47 was distributed specifically to fibrotic skin, whereas no VA-Lip HSP47 particle was found in non-fibrotic skin of the same mouse. Next, recipients of allogeneic BMT were treated with VA-Lip HSP47 from day +1 to +41. VA-Lip HSP47 knocked down HSP47 expression in the skin of allogeneic mice, resulting in significantly reduced collagen-deposition and skin thickness (Figure 2). In contrast, VA-Lip HSP47 had no effects on collagen volumes in the skin of naïve mice. Finally, allogeneic recipients were injected with VA-Lip HSP47 thrice a week from day +21 to test if VA-Lip HSP47 could promote regression of established skin fibrosis in chronic GVHD. Skin thickness and collagen volume on day +42 were again reduced in allogeneic mice treated with VA-Lip HSP47 compared to allogeneic controls, indicating that VA-Lip HSP47 is a promising therapeutic agent for established skin fibrosis. Because VA-Lip HSP47 did not affect donor T-cell expansion in the spleen or systemic GVHD scores after allogeneic BMT, VA-Lip HSP47 ameliorated skin fibrosis independently of systemic immunosuppression.Conclusion: VA-Lip HSP47 abrogates HSP47 expression in pathogenic myofibroblasts in the skin and ameliorates skin fibrosis in chronic GVHD without affecting collagen synthesis in the normal skin. VA-Lip HSP47 is a safe and promising anti-fibrotic agent for skin fibrosis in chronic GVHD without inducing systemic immunosuppression. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Summary of the 2014 Alcohol and Immunology Research Interest Group (AIRIG) meeting

On November 21, 2014 the 19th annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at Loyola University Chicago Health Sciences Campus in Maywood, Illinois. The meeting focused broadly on inflammatory cell signaling responses in the context of alcohol and alcohol-use disorders, and was divided into four plenary sessions focusing on the gut and liver, lung infections, general systemic effects of alcohol, and neuro-inflammation. One common theme among many talks was the differential roles of macrophages following both chronic and acute alcohol intoxication. Macrophages were shown to play significant roles in regulating inflammation, oxidative stress, and viral infection following alcohol exposure in the liver, lungs, adipose tissue, and brain. Other work examined the role of alcohol on disease progression in a variety of pathologies including psoriasis, advanced stage lung disease, and cancer.

Establishment and characterization of macrophage cell line from thymus ofCatla catla(Hamilton, 1822)

A continuous cell line has been developed from thymus explants of Catla catla and the cells have been subcultured for 63 passages. The cells exhibited optimum growth at 30°C in L-15 medium containing 15% foetal bovine serum. The cultured cells engulfed yeast cells and fluorescent latex beads. These cells produced reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharide and phorbol esters. The culture supernatant from the cultured cells had lysozyme activity and these cells demonstrated Fc receptors. Almost all the cells were positive for alpha-naphthyl acetate esterase enzyme suggesting that the cells are of macrophage lineage and therefore, the cell line was designated as catla thymus macrophage (CTM) cell line. CTM cells formed aggregates around zoospores of Aphanomyces invadans, but were unable to inhibit the germination of spores. The karyotype analysis of CTM cells at 25th passage revealed a typical diploid model with 50 chromosomes per cell. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and cytochrome c oxidase subunit 1 confirmed that the CTM cell line originated from C. catla. This cell line should be useful for studying the role of macrophages in differentiation and maturation of thymocytes and can be a source of macrophage-specific enzymes and cytokines.

Differential Roles of Macrophages in Diverse Phases of Skin Repair

Influx of macrophages plays a crucial role in tissue repair. However, the precise function of macrophages during the healing response has remained a subject of debate due to their functional dichotomy as effectors of both tissue injury and repair. We tested the hypothesis that macrophages recruited during the diverse phases of skin repair after mechanical injury exert specific functions to restore tissue integrity. For this purpose, we developed a mouse model that allows conditional depletion of macrophages during the sequential stages of the repair response. Depletion of macrophages restricted to the early stage of the repair response (inflammatory phase) significantly reduced the formation of vascularized granulation tissue, impaired epithelialization, and resulted in minimized scar formation. In contrast, depletion of macrophages restricted to the consecutive mid-stage of the repair response (phase of tissue formation) resulted in severe hemorrhage in the wound tissue. Under these conditions, transition into the subsequent phase of tissue maturation and wound closure did not occur. Finally, macrophage depletion restricted to the late stage of repair (phase of tissue maturation) did not significantly impact the outcome of the repair response. These results demonstrate that macrophages exert distinct functions during the diverse phases of skin repair, which are crucial to control the natural sequence of repair events.