Human Macrophages Preferentially Infiltrate the Superficial Adipose Tissue.

Human abdominal subcutaneous adipose tissue consists of two individual layers—the superficial adipose tissue (SAT) and deep adipose tissue (DAT)—separated by the Scarpa’s fascia. The present study focuses on the analysis of morphological and immunological differences of primary adipocytes, adipose-derived stem cells (ASC), and tissue-infiltrating immune cells found in SAT and DAT. Adipocytes and stromal vascular fraction (SVF) cells were isolated from human SAT and DAT specimens and phenotypically characterized by in vitro assays. Ex vivo analysis of infiltrating immune cells was performed by flow cytometry. Primary adipocytes from SAT are larger in size but did not significantly differ in cytokine levels of LEPTIN, ADIPOQ, RBP4, CHEMERIN, DEFB1, VISFATIN, MCP1, or MSCF. ASC isolated from SAT proliferated faster and exhibited a higher differentiation potential than those isolated from DAT. Flow cytometry analysis indicated no specific differences in the relative numbers of ASC, epithelial progenitor cells (EPC), or CD3⁺ T-cells, but showed higher numbers of tissue-infiltrating macrophages in SAT compared to DAT. Our findings suggest that ASC isolated from SAT have a higher regenerative potential than DAT-ASC. Moreover, spatial proximity to skin microbiota might promote macrophage infiltration in SAT.

Preadipocytes in human granulation tissue: role in wound healing and response to macrophage polarization.

BACKGROUND: Chronic non-healing wounds pose a global health challenge. Under optimized conditions, skin wounds heal by the formation of scar tissue. However, deregulated cell activation leads to persistent inflammation and the formation of granulation tissue, a type of premature scar tissue without epithelialization. Regenerative cells from the wound periphery contribute to the healing process, but little is known about their cellular fate in an inflammatory, macrophage-dominated wound microenvironment. METHODS: We examined CD45-/CD31-/CD34+ preadipocytes and CD68+ macrophages in human granulation tissue from pressure ulcers (n=6) using immunofluorescence, immunohistochemistry, and flow cytometry. In vitro, we studied macrophage-preadipocyte interactions using primary human adipose-derived stem cells (ASCs) exposed to conditioned medium harvested from IFNG/LPS (M1)- or IL4/IL13 (M2)-activated macrophages. Macrophages were derived from THP1 cells or CD14+ monocytes. In addition to confocal microscopy and flow cytometry, ASCs were analyzed for metabolic (OXPHOS, glycolysis), morphological (cytoskeleton), and mitochondrial (ATP production, membrane potential) changes. Angiogenic properties of ASCs were determined by HUVEC-based angiogenesis assay. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR. RESULTS: CD45-/CD31-/CD34+ preadipocytes were observed with a prevalence of up to 1.5% of total viable cells in human granulation tissue. Immunofluorescence staining suggested a spatial proximity of these cells to CD68+ macrophages in vivo. In vitro, ASCs exposed to M1, but not to M2 macrophage secretome showed a pro-fibrotic response characterized by stress fiber formation, elevated alpha smooth muscle actin (SMA), and increased expression of integrins ITGA5 and ITGAV. Macrophage-secreted IL1B and TGFB1 mediated this response via the PI3K/AKT and p38-MAPK pathways. In addition, ASCs exposed to M1-inflammatory stress demonstrated reduced migration, switched to a glycolysis-dominated metabolism with reduced ATP production, and increased levels of inflammatory cytokines such as IL1B, IL8, and MCP1. Notably, M1 but not M2 macrophages enhanced the angiogenic potential of ASCs. CONCLUSION: Preadipocyte fate in wound tissue is influenced by macrophage polarization. Pro-inflammatory M1 macrophages induce a pro-fibrotic response in ASCs through IL1B and TGFB1 signaling, while anti-inflammatory M2 macrophages have limited effects. These findings shed light on cellular interactions in chronic wounds and provide important information for the potential therapeutic use of ASCs in human wound healing.

Oxidant therapy improves adipogenic differentiation of adipose-derived stem cells in human wound healing.

BACKGROUND: Adipose-derived stem cells (ASC) and adipocytes are involved in numerous physiological and pathophysiological conditions, which have been extensively described in subcutaneous and visceral fat depots over the past two decades. However, much less is known about ASC and adipocytes outside classical fat tissue depots and their necessity in tissue remodeling after injury. Therefore, we investigated the etiology of adipocytes in human granulation tissue and define their possible role wound healing. METHODS: Identification of human wound tissue adipocytes was determined by immunohistochemical staining of granulation tissue sections from patients undergoing surgical debridement. Stromal cell fractions from granulation tissue and subcutaneous fat tissue were generated by collagenase type II-based protocols. Pro- and anti-inflammatory wound bed conditions were mimicked by THP1- and CD14+ monocyte-derived macrophage models in vitro. Effects of macrophage secretome on ASC differentiation and metabolism were determined by immunoblotting, flow cytometry, and microscopy assessing early and late adipocyte differentiation states. Functional rescuing experiments were conducted by lentiviral transduction of wildtype PPARG, IL1RA, and N-chlorotaurine (NCT) treatment. RESULTS: Single and clustered adipocyte populations were detected in 11 out of 13 granulation tissue specimens and single-cell suspensions from granulation tissue showed adipogenic differentiation potential. Pro-inflammatory signaling by IFNG/LPS-stimulated macrophages (M (IFNG/LPS)) inhibited the maturation of lipid droplets in differentiated ASC. In contrast, anti-inflammatory IL4/IL13-activated macrophages (M (IL4/IL13)) revealed minor effects on adipocyte development. The M (IFNG/LPS)-induced phenotype was associated with a switch from endogenous fatty acid synthesis to glycolysis-dominated cell metabolism and increased pro-inflammatory cytokine production. Impaired adipogenesis was associated with increased, but seemingly non-functional, CEBPB levels, which failed to induce downstream PPARG and CEBPA. Neither transgenic PPARG overexpression, nor inhibition of IL1B was sufficient to rescue the anti-adipogenic effects induced by IFNG/LPS-activated macrophages. Instead, macrophage co-treatment during stimulation with NCT, a mild oxidant produced by activated granulocytes present in human wounds in vivo, significantly attenuated the anti-adipogenic effects. CONCLUSIONS: In conclusion, the appearance of adipocytes in wound tissue indicates a prevailing anti-inflammatory environment that could be promoted by NCT treatment and may be associated with improved healing outcomes.

Repression of the BH3-only molecule PMAIP1/Noxa impairs glucocorticoid sensitivity of acute lymphoblastic leukemia cells.

Glucocorticoid (GC)-induced apoptosis plays a major role in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Members of the BCL2 family of pro- and anti-apoptotic proteins are regulated by GC, but to what extent these regulations contribute to GC-induced cell death and resistance development is poorly understood. Using primary lymphoblasts from ALL children during systemic GC monotherapy and related cell lines, we have previously shown that the response of the BCL2 rheostat to GC was dominated by induction of the pro-apoptotic BH3-only molecules BMF and BCL2L11/Bim, but we also observed an unexpected significant repression of the pro-apoptotic BCL2 protein PMAIP1/Noxa. Here, we report that GC represses Noxa mRNA levels and also interferes with its protein stability in a proteasome-dependent manner. Prevention of GC-mediated Noxa repression by conditional expression of transgenic Noxa changed the kinetics of GC-induced apoptosis to resemble cell death induced by BimEL alone. Hence, GC appear to activate functionally relevant pro- as well as anti-apoptotic pathways in ALL cells. Interfering with the anti-apoptotic component of the GC response might contribute to improved therapeutic approaches and circumvention of resistance to this therapy.

Immunophenotypic characterization of human T cells after in vitro exposure to different silicone breast implant surfaces.

The most common complication of silicone breast implants is capsular contracture (massive scar formation around the implant). We postulate that capsular contracture is always a sequel to inflammatory processes, with both innate and adaptive immune mechanisms participating. In general, fibroblasts and macrophages have been used as cell types to evaluate in vitro the biocompatibility of breast implant surfaces. Moreover, also T cells have been found at the implant site at the initial stage of fibrous capsule formation. However, only few studies have addressed the influence of surfaces with different textures on T-cell responses. The aim of the present study was to investigate the immune response of human peripheral blood mononuclear cells (PBMC) to commercially available silicone breast implants in vitro. PBMC from healthy female blood donors were cultured on each silicone surface for 4 days. Proliferation and phenotype of cultured cells were assessed by flow cytometry. Cytokine levels were determined by multiplex and real-time assay. We found that silicone surfaces do not induce T-cell proliferation, nor do they extensively alter the proportion of T cell subsets (CD4, CD8, naïve, effector memory). Interestingly, cytokine profiling identified matrix specific differences, especially for IL-6 and TNF-α on certain surface topographies that could lead to increased fibrosis.