Role of the Skin Immune System in Wound Healing.

Wound healing is a dynamic and complex process, characterized by the coordinated activities of multiple cell types, each with distinct roles in the stages of hemostasis, inflammation, proliferation, and remodeling. The cells of the immune system not only act as sentinels to monitor the skin and promote homeostasis, but they also play an important role in the process of skin wound repair. Skin-resident and recruited immune cells release cytokines and growth factors that promote the amplification of the inflammatory process. They also work with non-immune cells to remove invading pathogens and debris, as well as guide the regeneration of damaged host tissues. Dysregulation of the immune system at any stage of the process may lead to a prolongation of the inflammatory phase and the development of a pathological condition, such as a chronic wound. The present review aims to summarize the roles of different immune cells, with special emphasis on the different stages of the wound healing process.

Functional TRPA1 Channels Regulate CD56dimCD16+ NK Cell Cytotoxicity against Tumor Cells.

Transient receptor potential ankyrin 1 (TRPA1) channels are expressed on the surface of different cell types, including immune cells. However, TRPA1's role in the context of innate and adaptive immune responses has not been fully elucidated so far. In this study, we aimed at investigating the expression and function of TRPA1 channels on NK cells. Among NK cells, TRPA1 was highly expressed by the CD56dimCD16+ subpopulation, but not by CD56brightCD16- cells, as detected by FACS. TRPA1 activation with the potent ligand allyl isothiocyanate (AITC) induces intracellular calcium flux in CD56dimCD16+ cells, which was prevented by the TRPA1 antagonist HC-030031. AITC treatment increased the membrane around NKp44 and strongly decreased CD16 and CD8 expression, while CD158a, CD159a, NKG2d, NKp46 were substantially unaffected. Importantly, AITC increased the granzyme production and CD107 expression and increased NK cell-mediated cytotoxicity towards the K562 cell line and two different melanoma cell lines. In parallel, TRPA1 activation also plays regulatory roles by affecting the survival of NK cells to limit uncontrolled and prolonged NK cell-mediated cytotoxicity. Our results indicate that the activation of TRPA1 is an important regulatory signal for NK cells, and agonists of TRPA1 could be used to strengthen the tumor response of the immune system.

Platelet Derivatives and the Immunomodulation of Wound Healing.

Besides their primary role in hemostasis, platelets contain a plethora of immunomodulatory molecules that profoundly affect the entire process of wound repair. Therefore, platelet derivatives, such as platelet-rich plasma or platelet lysate, have been widely employed with promising results in the treatment of chronic wounds. Platelet derivatives provide growth factors, cytokines, and chemokines targeting resident and immigrated cells belonging to the innate and adaptive immune system. The recruitment and activation of neutrophils and macrophages is critical for pathogen clearance in the early phase of wound repair. The inflammatory response begins with the release of cytokines, such as TGF-ß, aimed at damping excessive inflammation and promoting the regenerative phase of wound healing. Dysregulation of the immune system during the wound healing process leads to persistent inflammation and delayed healing, which ultimately result in chronic wound. In this review, we summarize the role of the different immune cells involved in wound healing, particularly emphasizing the function of platelet and platelet derivatives in orchestrating the immunological response.

The phosphoinositide-3-kinase (PI3K)-delta inhibitor seletalisib impairs monocyte-derived dendritic cells maturation, APC function, and promotes their migration to CCR7 and CXCR4 ligands.

PI3K pathway plays a crucial role in dendritic cells (DCs) functions, as it regulates different cellular processes, such as maturation and cytokines production. However, the specific role of PI3K p110δ isoform in human DCs has not been thoroughly addressed. In this study, we analyze the effects of seletalisib, a potent and specific inhibitor of PI3K p110δ, on phenotype and antigen-presenting functions of monocyte-derived DCs undergone maturation via LPS. Seletalisib treatment reduced membrane HLA-DR as well as CD83 and CD40 costimulatory molecules, whereas CD80 and CD86 expression was only partially affected. Additionally, DCs cultures showed reduced TNF-α, IL-10, and IL-12 and increased IL-23 secretion levels. This resulted in a reduced capacity of DCs to prime allogeneic T cells, with a strong decrease of Th1 differentiation. On the other hand, PI3K p110δ inhibitor seletalisib increased CXCR4 and CCR7 expression and augmented the DCs migration toward CCL19 and CXCL12 ligands. At molecular level, inhibition of PI3K p110δ isoform by seletalisib significantly down-regulated the phosphorylation of AKT and other downstream signaling molecules, such as ribosomal protein S6, 4E-BP1, and NF-κB p65. In contrast, seletalisib did not affect p38 MAP kinase phosphorylation or TLR-associated adapter molecule TIRAP in DCs. Our results indicate that PI3K p110δ can serve as an important regulatory signal for DCs, and selective inhibition of PI3K p110δ isoform by seletalisib could be used for the prevention of exaggerated and harmful immune responses occurring in pathologic conditions, such as autoimmune disorders.

Reduced Interleukin-17-Expressing Cells in Cutaneous Melanoma.

Characterization of tumor associated lymphocytes (TILs) in tumor lesions is important to obtain a clear definition of their prognostic value and address novel therapeutic opportunities. In this work, we examined the presence of T helper (Th)17 lymphocytes in cutaneous melanoma. We performed an immunohistochemical analysis of a small cohort of primary melanomas, retrospectively selected. Thereafter, we isolated TILs from seven freshly surgically removed melanomas and from three basal cell carcinomas (BCC), as a comparison with a non-melanoma skin cancer known to retain a high amount of Th17 cells. In both studies, we found that, differently from BCC, melanoma samples showed a lower percentage of Th17 lymphocytes. Additionally, TIL clones could not be induced to differentiate towards the Th17 phenotype in vitro. The presence or absence of Th17 cells did not correlate with any patient characteristics. We only observed a lower amount of Th17 cells in samples from woman donors. We found a tendency towards an association between expression by melanoma cells of placenta growth factor, angiogenic factors able to induce Th17 differentiation, and presence of Th17 lymphocytes. Taken together, our data indicate the necessity of a deeper analysis of Th17 lymphocytes in cutaneous melanoma before correlating them with prognosis or proposing Th17-cell based therapeutic approaches.

Bevacizumab Arrests Osteoarthritis Progression in a Rabbit Model: A Dose-Escalation Study.

Cartilage neoangiogenesis holds a prominent role in osteoarthritis (OA) pathogenesis. This study aimed to assess the efficacy bevacizumab, an antibody against vascular endothelial growth factor and inhibitor of angiogenesis, in a rabbit OA model. Animals were divided into four groups: one receiving a sham intra-articular knee injection and three groups undergoing 5, 10, and 20 mg intra-articular bevacizumab injections. The effect of the antibody on articular cartilage and synovium was assessed through histology and quantified with the Osteoarthritis Research Society International (OARSI) scores. Immunohistochemistry was performed to investigate type 2 collagen, aggrecan, and matrix metalloproteinase 13 (MMP-13) expression. Bevacizumab treatment led to a significant reduction of cartilage degeneration and synovial OA changes. Immunohistochemistry revealed significantly lower cartilage MMP-13 expression levels in all experimental groups, with the one receiving 20 mg bevacizumab showing the lowest. The antibody also resulted in increased production of aggrecan and type 2 collagen after administration of 5, 10, and 20 mg. The group treated with 20 mg showed the highest levels of type 2 collagen, while aggrecan content was even higher than in the healthy cartilage. Intra-articular bevacizumab has been demonstrated to effectively arrest OA progression in our model, with 20 mg being the most efficacious dose.

Platelet lysate promotes the expansion of T regulatory cells that favours in vitro wound healing by increasing keratinocyte migration and fibroblast production of extracellular matrix components.

BACKGROUND: Platelet lysate (PL) contains a cocktail of growth factors and cytokines that promote tissue repair and regeneration. In vitro studies have shown that PL may affect the reparative function of keratinocytes and fibroblasts, but little is known about the effect of PL on immune cells involved in wound healing. OBJECTIVES: To analyse the effects of PL on T cells involved in the wound repair process. MATERIALS AND METHODS: The effect of PL on T cell proliferation, activation, and cytokine production was measured by ELISA and cytofluorometry and regulatory function based on cytofluorometry and Foxp3 RNA expression. Using an in vitro model of wound healing, we investigated the effect of PL-treated T cells on fibroblast proliferation and production of fibronectin and type-1 collagen as well as keratinocyte migration. RESULTS: PL induced T lymphocyte proliferation and CD69 expression, and promoted a transient upregulation of IFN-γ and TNF-α. However, later on, PL enhanced the number of CD25+ T cells releasing TGF-ß and expressing Foxp3 RNA, which was accompanied by a suppression in the level of type 1 cytokines. In the in vitro model, supernatants of PL-treated T cells positively affected the reparative capacity of human keratinocytes and induced fibroblast proliferation and production of fibronectin and type-1 collagen. CONCLUSION: These results indicate that PL temporally regulates T cells during the healing process, enhancing protective cytokines in the early phase, followed by a prominent expansion of TGF-ß+ T regulatory cells that promote tissue regeneration and dampen the inflammatory response to prevent excessive tissue damage.

Paradoxical psoriasis induced by TNF-α blockade shows immunological features typical of the early phase of psoriasis development.

Immunomodulation with anti-TNF-α is highly effective in the treatment of various immune-mediated inflammatory diseases, including hidradenitis suppurativa (HS). However, this may be responsible for unexpected paradoxical psoriasiform reactions. The pathogenic mechanisms underlying the induction of these events are not clear, even though the involvement of innate immune responses driven by plasmacytoid dendritic cells (pDC) has been described. In addition, the genetic predisposition to psoriasis of patients could be determinant. In this study, we investigated the immunological and genetic profiles of three HS patients without psoriasis who developed paradoxical psoriasiform reactions following anti-TNF-α therapy with adalimumab. We found that paradoxical psoriasiform skin reactions show immunological features common to the early phases of psoriasis development, characterized by cellular players of innate immunity, such as pDC, neutrophils, mast cells, macrophages, and monocytes. In addition, IFN-ß and IFN-α2a, two type I IFNs typical of early psoriasis, were highly expressed in paradoxical skin reactions. Concomitantly, other innate immunity molecules, such as the catheledicin LL37 and lymphotoxin (LT)-α and LT-ß were overproduced. Interestingly, these innate immunity molecules were abundantly expressed by keratinocytes, in addition to the inflammatory infiltrate. In contrast to classical psoriasis, psoriasiform lesions of HS patients showed a reduced number of IFN-γ and TNF-α-releasing T lymphocytes. On the contrary, IL-22 immunoreactivity was significantly augmented together with the IL-36γ staining in leukocytes infiltrating the dermis. Finally, we found that all HS patients with paradoxical reactions carried allelic variants in genes predisposing to psoriasis. Among them, SNPs in ERAP1, NFKBIZ, and TNFAIP genes and in the HLA-C genomic region were found.

Novel stepwise model of intervertebral disc degeneration with intact annulus fibrosus to test regeneration strategies.

Novel preclinical models that do not damage the annulus fibrosus (AF) of the intervertebral disc are required to study the efficacy of new regenerative strategies for the nucleus pulposus (NP). The aim of the study was to characterize a preclinical ovine model of intervertebral disc degeneration (IDD) induced by endplate (EP) damage and repair via the transpedicular approach, with or without partial nucleotomy, while keeping the AF intact. Twelve adult sheep were used. By the transpedicular approach, a 2 mm tunnel was drilled to the NP through the EP. A partial-nucleotomy was performed. The tunnel was sealed using a polyurethane scaffold. Lumbar discs were assigned to different groups: L1-2: nucleotomy; L2-3: EP tunnel; L3-4: nucleotomy + EP repair; L4-5: EP tunnel + repair; L5-6: control. X-Ray and MRI were performed at 0, 1, 3, and 6 months after surgery. Disc height and MRI indexes were calculated. Macro- and micro-morphology were analyzed. Pfirrmann and Thompson grades were assigned. The treated discs exhibited a progressive decrease in NP signal intensity and MRI index, displaying specific grades of degeneration based on the surgical treatment. According to Pfirrmann and Thompson grades different procedures were staged as: EP tunnel + repair: grade-II; EP tunnel: grade-III, nucleotomy + EP repair: grade-IV; nucleotomy: grade-V. A new stepwise model of IDD to study and test safety and efficacy of novel strategies for NP regeneration has been characterized. The different degrees of IDD have been observed similar to Pfirrmann and Thompson grading system. The intact AF allows for loading studies and eliminating the need for AF closure. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2460-2468, 2018.

Clinically relevant hydrogel-based on hyaluronic acid and platelet rich plasma as a carrier for mesenchymal stem cells: Rheological and biological characterization.

Intervertebral disc regeneration is quickly moving towards clinical applications. However, it is still missing an ideal injectable hydrogel to support mesenchymal stem cells (MSC) delivery. Herein, a new injectable hydrogel composed of platelet rich plasma (PRP) and hyaluronic acid (HA) blended with batroxobin (BTX) as gelling agent, was designed to generate a clinically relevant cell carrier for disc regeneration. PRP/HA/BTX blend was tested for rheological properties. Amplitude sweep, frequency sweep, and rotational measurements were performed and viscoelastic properties were evaluated. Human MSC encapsulated in PRP/HA/BTX hydrogel were cultured in both growing medium and medium with or without TGF-ß1 up to day 21. The amount of glycosaminoglycan was evaluated. Quantitative gene expression evaluation for collagen type II, aggrecan, and Sox 9 was also performed. Rheological tests showed that the hydrogel jellifies in 15 min 20°C and in 3 min at 37°C. Biological test showed that MSCs cultured in the hydrogel maintain high cell viability and proliferation. Human MSC within the hydrogel cultured with or without TGF-ß1 showed significantly higher GAG production compared to control medium. Moreover, MSCs in the hydrogel underwent differentiation to chondrocyte-like cells with TGF-ß1, as shown by histology and gene expression analysis. This novel hydrogel improves viability and proliferation of MSCs supporting the differentiation process toward chondrocyte-like cells. Rheology tests showed optimal gelation kinetics at room temperature for manipulation and faster gelation after transplantation (37°C). The clinical availability of all components of the hydrogel will allow a rapid translation of this regenerative approach into the clinical scenario. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2109-2116, 2017.

Platelet Rich Plasma and Hyaluronic Acid Blend for the Treatment of Osteoarthritis: Rheological and Biological Evaluation.

INTRODUCTION: Osteoarthritis (OA) is the most common musculoskeletal disease. Current treatments for OA are mainly symptomatic and inadequate since none results in restoration of fully functional cartilage. Hyaluronic Acid (HA) intra-articular injections are widely accepted for the treatment of pain associated to OA. The goal of HA viscosupplementation is to reduce pain and improve viscoelasticity of synovial fluid. Platelet-rich plasma (PRP) has been also employed to treat OA to possibly induce cartilage regeneration. The combination of HA and PRP could supply many advantages for tissue repair. Indeed, it conjugates HA viscosupplementation with PRP regenerative properties. The aim of this study was to evaluate the rheological and biological properties of different HA compositions in combination with PRP in order to identify (i) the viscoelastic features of the HA-PRP blends, (ii) their biological effect on osteoarthritic chondrocytes and (iii) HA formulations suitable for use in combination with PRP. MATERIALS AND METHODS: HA/PRP blends have been obtained mixing human PRP and three different HA at different concentrations: 1) Sinovial, 0.8% (SN); 2) Sinovial Forte 1.6% (SF); 3) Sinovial HL 3.2% (HL); 4) Hyalubrix 1.5% (HX). Combinations of phosphate buffered saline (PBS) and the four HA types were used as control. Rheological measurements were performed on an Anton PaarMCR-302 rheometer. Amplitude sweep, frequency sweep and rotational measurements were performed and viscoelastic properties were evaluated. The rheological data were validated performing the tests in presence of Bovine Serum Albumin (BSA) up to ultra-physiological concentration (7%). Primary osteoarthritic chondrocytes were cultured in vitro with the HA and PRP blends in the culture medium for one week. Cell viability, proliferation and glycosaminoglycan (GAG) content were assessed. RESULTS: PRP addition to HA leads to a decrease of viscoelastic shear moduli and increase of the crossover point, due to a pure dilution effect. For viscosupplements with HA concentration below 1% the viscoelasticity is mostly lost. Results were validated also in presence of proteins, which in synovial fluid are more abundant than HA. Chondrocytes proliferated overtime in all different culture conditions. The proliferation rate was higher in chondrocytes cultured in the media containing PRP compared to the cultures with different HA alone. GAG content was significantly higher in chondrocytes cultured in PRP and HL blend. DISCUSSION: We investigated the rheological and biological properties of four different HA concentrations when combined with PRP giving insights on viscoelastic and biological properties of a promising approach for future OA therapy. Our data demonstrate that PRP addition is not detrimental to the viscosupplementation effect of HA. Viscosupplements containing low HA concentration are not indicated for combination with PRP, as the viscoelastic properties are lost. Although having the same rheological behavior of SF and HX, HL was superior in stimulating extracellular matrix production in vitro.

The E3 ligase Itch knockout mice show hyperproliferation and wound healing alteration.

The HECT-type E3 ubiquitin ligase Itch is absent in the non-agouti-lethal 18H or Itchy mice, which develop a severe immunological disease. Several of the known Itch substrates are relevant for epidermal development and homeostasis, such as p63, Notch, c-Jun and JunB. By analysing Itchy mice before the onset of immunological alterations, we investigated the contribution of Itch in skin development and wound healing. Itchy newborn mice manifested hyperplastic epidermis, which is not present in adulthood. Itch(-/-) cultured keratinocytes showed overexpression of proliferating markers and increased capability to proliferate, migrate and to repair a scratch injury in vitro. These data correlated with improved in vivo wound healing in Itchy mice, at late time points of the repair process when Itch is physiologically upregulated. Despite healing acceleration, epidermal remodelling was delayed in the scars of Itch(-/-) mice, as indicated by enhanced epidermal thickening, keratinocyte proliferation and keratin 6 expression, and retarded keratin 14 polarization to the basal layer. Itch(-/-) keratinocyte prolonged activation was not associated with increased immune cell persistence in the scars. Our in vitro and in vivo results indicate that Itch plays a role in epidermal homeostasis and remodelling and this feature does not seem to depend on immunological alterations.

miR-181b as a therapeutic agent for chronic lymphocytic leukemia in the Eµ-TCL1 mouse model.

The involvement of microRNAs (miRNAs) in chronic lymphocytic leukemia (CLL) pathogenesis suggests the possibility of anti-CLL therapeutic approaches based on miRNAs. Here, we used the Eµ-TCL1 transgenic mouse model, which reproduces leukemia with a similar course and distinct immunophenotype as human B-CLL, to test miR-181b as a therapeutic agent.In vitro enforced expression of miR-181b mimics induced significant apoptotic effects in human B-cell lines (RAJI, EHEB), as well as in mouse Eµ-TCL1 leukemic splenocytes. Molecular analyses revealed that miR-181b not only affected the expression of TCL1, Bcl2 and Mcl1 anti-apoptotic proteins, but also reduced the levels of Akt and phospho-Erk1/2. Notably, a siRNA anti-TCL1 could similarly down-modulate TCL1, but exhibited a reduced or absent activity in other relevant proteins, as well as a reduced effect on cell apoptosis and viability. In vivo studies demonstrated the capability of miR-181b to reduce leukemic cell expansion and to increase survival of treated mice.These data indicate that miR-181b exerts a broad range of actions, affecting proliferative, survival and apoptotic pathways, both in mice and human cells, and can potentially be used to reduce expansion of B-CLL leukemic cells.

Expression of vascular endothelial growth factor-C in primary cutaneous melanoma predicts sentinel lymph node positivity.

BACKGROUND: Vascular endothelial growth factor-C (VEGF-C), a lymphatic vessel growth factor, has been involved in the formation of lymph nodal metastases in different tumor types. Early evidences indicate that VEGF-C expression in human primary melanoma could be predictive of lymph nodal metastases, whereas the role of lymphangiogenesis is still controversial. METHODS: By immunohistochemical analysis, we investigated VEGF-C or CC chemokine receptor 7 expression, together with the lymphatic and blood vessel network, in 36 patients with primary skin melanomas and metastases at the sentinel lymph node biopsy (SLN-positive), and 26 melanoma patients with negative SLN biopsy (SLN-negative). RESULTS: We found that VEGF-C expression in primary melanoma specimens was significantly associated with SLN-positive (p < 0.001), particularly in thin melanomas. An association between augmented peritumoral lymphatic vessel area and SLN-positive (p < 0.02) was also seen. Conversely, no association between either expression of the CC chemokine receptor 7 in the primary tumor, or intratumoral lymphatic vessel or peritumoral and intratumoral blood vessel area, and SLN-positive was found. CONCLUSIONS: Our results, taking into account the expression of either VEGF-C or related histopathological markers, indicated the possibility to use VEGF-C immunohistochemistry as a marker of metastatic progression, especially in thin cutaneous melanomas.