Dermal Fibroblasts Promote Alternative Macrophage Activation Improving Impaired Wound Healing.

Tight control of inflammation is required for tissue repair and wound healing and depends on alternative polarization of macrophages as checkpoint for inflammatory resolution. Its perturbations lead to impaired regeneration. Administration of cells/cell factors capable of reversing inflammation and rescuing alternative polarization could be promising for treating inflammatory diseases. We show that human dermal fibroblasts (dFb) are ideal candidates for such a task by demonstrating a new function of these cells, which is modulating macrophage polarization. Coculture of dFb with human monocytes in vitro or injection of dFb into mice with thioglycollate-induced peritonitis favors alternative macrophage activation and reduces inflammation by releasing tumor necrosis factor-inducible gene 6 protein and Cox-2 products. Silencing these factors in dFb abolishes the reported effects, demonstrating their importance for immunomodulation. Importantly, in a model of delayed wound healing due to prolonged inflammation (db/db mice), administration of dFb improves defective tissue repair with augmentation of alternative macrophage polarization and inflammation resolution. Human dFb are low immunogenic cells, easy to obtain, and can be expanded extensively in vitro conserving their immunomodulatory capacity; this, together with our findings, suggests that dFb might represent an alternative for cell-based therapies of conditions characterized by excessive inflammation and delayed tissue repair.

Artificial extracellular matrices composed of collagen I and high-sulfated hyaluronan promote phenotypic and functional modulation of human pro-inflammatory M1 macrophages.

The sequential phases of biomaterial integration and wound healing require different macrophage functions mediated by distinct macrophage subsets. During the initial phase of healing, pro-inflammatory M1 macrophages (MΦ1) are required to clear the wound from microbes and debris; however, their unopposed, persistent activation often leads to disturbed integration of biomaterials and perturbed wound healing. Here we investigated whether pro-inflammatory macrophage functions are affected by immunomodulatory biomaterials based on artificial extracellular matrices (aECM). To address this issue, we tested the capacity of two-dimensional aECM consisting of collagen I and hyaluronan or sulfated derivatives of hyaluronan to affect functions of in vitro polarized human pro-inflammatory MΦ1. The aECM containing high-sulfated hyaluronan substantially decreased inflammatory macrophage functions, including pathogen uptake and release of the pro-inflammatory cytokines tumor necrosis factor alpha and interleukin-12 due to impaired activation of nuclear factor "kappa-light-chain-enhancer" of activated B-cells. Moreover, these macrophages secreted immunregulatory IL-10 and showed reduced activity of the transcription factors signal transducer and activator of transcription 1 and interferon-regulating factor 5, both controlling macrophage polarization to MΦ1 subsets. Our data reveal that the collagen I matrix containing high-sulfated hyaluronan possesses immunomodulating properties and dampens inflammatory macrophage activities by impeding signaling pathways crucial for polarization of pro-inflammatory MΦ1. We therefore suggest this aECM as a promising coating for biomaterials to modulate inflammatory macrophage functions during the healing response and recommend its further testing as a three-dimensional construct and in in vivo models.

Artificial extracellular matrices composed of collagen I and high sulfated hyaluronan modulate monocyte to macrophage differentiation under conditions of sterile inflammation.

Integration of biomaterials into tissues is often disturbed by unopposed activation of macrophages. Immediately after implantation, monocytes are attracted from peripheral blood to the implantation site where they differentiate into macrophages. Inflammatory signals from the sterile tissue injury around the implanted biomaterial mediate the differentiation of monocytes into inflammatory M1 macrophages (M1) via autocrine and paracrine mechanisms. Suppression of sustained M1 differentiation is thought to be crucial to improve implant healing. Here, we explore whether artificial extracellular matrix (aECM) composed of collagen I and hyaluronan (HA) or sulfated HA-derivatives modulate this monocyte differentiation. We mimicked conditions of sterile tissue injury in vitro using a specific cytokine cocktail containing MCP-1, IL-6 and IFNγ, which induced in monocytes a phenotype similar to M1 macrophages (high expression of CD71, HLA-DR but no CD163 and release of high amounts of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, IL-12 and TNFα). In the presence of aECMs containing high sulfated HA this monocyte to M1 differentiation was disturbed. Specifically, pro-inflammatory functions were impaired as shown by reduced secretion of IL-1ß, IL-8, IL-12 and TNFα. Instead, release of the immunregulatory cytokine IL-10 and expression of CD163, both markers specific for anti-inflammatory M2 macrophages (M2), were induced. We conclude that aECMs composed of collagen I and high sulfated HA possess immunomodulating capacities and skew monocyte to macrophage differentiation induced by pro-inflammatory signals of sterile injury toward M2 polarization suggesting them as an effective coating for biomaterials to improve their integration.

The NF-kappaB signalling pathway is involved in the LPS/IL-2-induced upregulation of FoxP3 expression in human CD4+CD25high regulatory T cells.

Regulatory T cells (Treg) have been found to be central for host defense regulation against microbial antigens, the prevention of allergic and autoimmune diseases and the suppression of effective tumor immune responses. However, the influence of the microenvironment and the mechanisms leading to their activation in the periphery still remain unclear. In vitro infection models revealed that survival and suppressive function of Treg is improved when they are confronted with lipopolysaccharide (LPS). Because LPS initiates signalling via the receptor Toll-like receptor 4 (TLR4) and the consequent activation of the transcription factor nuclear factor-kappaB (NF-kappaB), we investigated TLR4 expression and NF-kappaB regulation in human Treg. We demonstrated that LPS in combination with IL-2 induces human CD25(+)FoxP3(+) T cells in vitro. FoxP3 expression of purified natural Treg increased and suppressive capacity was markedly improved compared with unstimulated Treg upon stimulation with LPS/IL-2. Furthermore, blockade of the NF-kappaB pathway by a selective inhibitor of IkappaB kinase (IKK)beta abrogated the upregulation of FoxP3 expression. Taken together, our results suggest an important role of the NF-kappaB signalling pathway for the induction and modulation of suppressive function of natural Treg, if they are confronted with TLR4-stimulating agents such as Gram-negative bacteria.