The effect of expressive writing on wound healing: Immunohistochemistry analysis of skin tissue two weeks after punch biopsy wounding.

OBJECTIVE: To investigate the effects of expressive writing and its timing (pre or post wounding) on re-epithelialisation and leucocyte subsets within healing tissue. We previously showed expressive writing pre-wounding improved re-epithelialisation. Here we investigate cellular processes in the wound. METHODS: In a 2(writing content) x 2(writing timing) randomized trial, 122 participants were randomized to perform either expressive or control writing, before or after a 4 mm punch biopsy wound. On day 14 post-wounding, participants had a 5 mm punch biopsy of the initial wound. Seven of 16 primary registered outcomes were analysed, including re-epithelialisation from two photographs of the 4 mm biopsy (previously reported). This paper reports immunohistochemistry analysis of five primary outcomes - Langerhans cells, immune cell activation (HLA and CD3+), and macrophages (CD68 and MPO) - in the 5 mm biopsies in a random sample of 96 participants. RESULTS: Participants who performed either writing task pre-wounding had greater Langerhans cell infiltration, than those who wrote post-wounding (F(1,85) = 7.86, p = .006, ηp2 = 0.08). Those who performed expressive writing also had greater Langerhans cell infiltration than those who performed control writing (F(1,85) = 4.00, p = .049, ηp2 = 0.04). There were no significant group or interaction effects on immune cell activation or macrophages. Healed wounds on day 10 had lower levels of macrophages (z = -1.96, p = .050), and CD3+ cells (z = -1.99, p = .046) than non-healed wounds. CONCLUSION: Langerhans cells in the healing skin are affected by the timing and topic of writing. More research is needed to further explore timing and corroborate these results. CLINICAL TRIALS REGISTRATION: Registered at https://www.anzctr.org.au/ (Trial ID: ACTRN12614000971639).

Stress-related changes to immune cells in the skin prior to wounding may impair subsequent healing.

Higher psychological stress is associated with slower dermal wound healing, but the immunological mechanisms behind this effect are only partially understood. This paper aims to investigate whether immune cells present in the skin prior to wounding can affect subsequent healing in high-stress and low-stress participants. Two studies are presented in which skin biopsies were analysed using immunohistochemistry for numbers of macrophages and Langerhans cells, and immune cell activation (Study 2 only). Immune cells were related to perceived stress levels and subsequent healing. Study 1 included 19 healthy older adults and showed that higher stress was associated with significantly fewer macrophages in the skin. Study 2 included 22 younger adults and showed that higher stress was associated with significantly lower activation of immune cells in the skin. Furthermore, lower activation of immune cells (as measured by human leukocyte antigen (HLA expression)) and fewer Langerhans cells were associated with slower healing. Together these studies show the first preliminary evidence that the number and activation of immune cells in the skin prior to wounding are affected by stress and can impact healing. Larger studies are needed to confirm these effects.

IL-13 promotes collagen accumulation in Crohn's disease fibrosis by down-regulation of fibroblast MMP synthesis: a role for innate lymphoid cells?

BACKGROUND: Fibrosis is a serious consequence of Crohn's disease (CD), often necessitating surgical resection. We examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment. METHODS: Factors potentially modulating collagen deposition were examined in intestinal tissue samples from fibrotic (f) CD and compared with cancer control (C), ulcerative colitis (UC) and uninvolved (u) CD. Mechanisms attributable to IL-13 were analysed using cell lines derived from uninvolved muscle tissue and tissue explants. RESULTS: In fCD muscle extracts, collagen synthesis was significantly increased compared to other groups, but MMP-2 was not co-ordinately increased. IL-13 transcripts were highest in fCD muscle compared to muscle from other groups. IL-13 receptor (R) α1 was expressed by intestinal muscle smooth muscle, nerve and KIR(+) cells. Fibroblasts from intestinal muscle expressed Rα1, phosphorylated STAT6 in response to IL-13, and subsequently down-regulated MMP-2 and TNF-α-induced MMP-1 and MMP-9 synthesis. Cells with the phenotype KIR(+)CD45(+)CD56(+/-)CD3(-) were significantly increased in fCD muscle compared to all other groups, expressed Rα1 and membrane IL-13, and transcribed high levels of IL-13. In explanted CD muscle, these cells did not phosphorylate STAT6 in response to exogenous IL-13. CONCLUSIONS: The data indicate that in fibrotic intestinal muscle of Crohn's patients, the IL-13 pathway is stimulated, involving a novel population of infiltrating IL-13Rα1(+), KIR(+) innate lymphoid cells, producing IL-13 which inhibits fibroblast MMP synthesis. Consequently, matrix degradation is down-regulated and this leads to excessive collagen deposition.

Mechanisms of chronic skin ulceration linking lactate, transforming growth factor-beta, vascular endothelial growth factor, collagen remodeling, collagen stability, and defective angiogenesis.

Up to one million people suffer from chronic skin ulcers in the US. Little is known of the mechanisms leading to tissue breakdown, although inadequate circulation and ischemia are common elements in most dermal ulcers. Collagen is the principal source of mechanical strength in most tissues, and its molecular and fibrillar stability is dependent on adequate oxygen supply. In wound repair, localized ischemia leads to fibrogenic responses culminating in elevated collagen synthesis and remodeling. This study examines factors influencing collagen turnover and stabilization before ulceration in "at risk" patients. Severely ischemic but uninjured ischemic skin (IS) was compared with patient- and site-matched non-ischemic skin. Biochemical mechanisms of tissue repair were activated in IS, with increased lactate, transforming growth factor-beta, vascular endothelial growth factor, collagen synthesis and matrix metalloproteinases (MMPs)-1 and 2. The absence of MMP-9 and inflammatory cells confirmed that this upregulation was inappropriate and not in response to injury. Molecular stability of collagen was reduced in IS, and there was increased susceptibility to enzymic degradation. In conclusion, chronic ischemia and long-term hypoxia result in elevated collagen remodeling in an oxygen-poor environment. Unstable collagen molecules are synthesized together with upregulated MMPs, resulting in collagen denaturation, defective angiogenesis, weaker skin, and predisposition to ulceration.

Abnormal extracellular matrix metabolism in chronically ischemic skin: a mechanism for dermal failure in leg ulcers.

Extracellular matrix (ECM) metabolism and homeostasis is sensitive to changes in oxygen tension manifest in ischemia. We hypothesize that in chronically ischemic limbs, abnormalities in uninjured skin, secondary to hypoxia, predispose to dermal breakdown. Paired biopsies of uninjured distal ischemic and proximal non-ischemic skin were harvested at below knee amputation from 14 patients with peripheral vascular disease following quantification of ischemia. Age- and site-matched controls were taken at total knee replacement (TKR) and varicose vein (VV) operations. Matrix metalloproteinase (MMP)-2 and -9 expression was determined using gelatin zymography, MMP-1 by western blotting and ELISA and tissue inhibitor of MMP (TIMP) by reverse zymography. Collagen content was measured by determining hydroxyproline levels, and collagen type I synthesis by ELISA. Collagen type I synthesis was upregulated in ischemic tissue compared with non-ischemic matched pairs (p<0.001) and both TKR and VV controls, however, there was no increase in collagen deposition. Levels of MMP-2 (p<0.0005) and TIMP-2 (p<0.01), were elevated in ischemic samples. MMP-9 was unaltered, signifying no inflammatory changes. Tissue ischemia was linked to elevated ECM turnover, associated with matrix failure when compounded with problems of matrix stabilization, likely in ischemia. This represents a potential mechanism for ulcer formation.

Dietary n-3 polyunsaturated fatty acids reduce disease and colonic proinflammatory cytokines in a mouse model of colitis.

BACKGROUND: n-3 polyunsaturated fatty acids (PUFAs) reduce the severity of chronic inflammatory bowel disease, probably by means of reduction of immune cell activation or enhancement of the epithelial barrier. Using the severe combined immunodeficient (SCID) mouse model of colitis, this study examined the effect of dietary n-3 PUFAs on development of colitis and on immunologic, epithelial, and matrix parameters in the intestines of control and colitic animals. METHODS: SCID mice were fed n-3-enriched or control diet for 3 weeks before colitis induction by transplantation of CD45RB T cells and maintained on the same diet for 4 to 8 weeks. Phenotype of infiltrating cells, epithelial ZO-1 protein, and mucosal type I collagen were assessed by immunohistology and tissue cytokines by ELISA. RESULTS: Transplanted n-3-fed animals had significantly reduced pathology scores, colonic tumor necrosis factor-alpha, interleukin-12, and interleukin-1beta compared with animals fed standard diet. Proinflammatory cytokines were reduced despite a similar level of immune cell infiltration by T cells, CD11c cells, and CD11b cells. Neutrophil infiltration was significantly reduced in n-3-fed control and colitic mice, and other myeloid populations were reduced in mice on the n-3 diet. Epithelial ZO-1 expression was increased, and myofibroblast activation significantly decreased in transplanted n-3-fed animals compared with standard diet mice. Submucosal collagen synthesis was enhanced in n-3-fed mice. CONCLUSIONS: Dietary n-3 PUFAs reduced clinical colitis and colonic immunopathology in this model of colonic inflammation by decreasing proinflammatory cytokine synthesis, reducing myeloid cell recruitment and activation, and enhancing epithelial barrier function and mucosal wound healing mechanisms.

Abnormal mucosal extracellular matrix deposition is associated with increased TGF-beta receptor-expressing mesenchymal cells in a mouse model of colitis.

Transforming growth factor-beta (TGF-beta) depresses mucosal inflammation and upregulates extracellular matrix (ECM) deposition. We analyzed TGF-beta receptors RI and RII as well as ECM components using the CD4(+) T-cell-transplanted SCID mouse model of colitis. The principal change in colitis was an increased proportion of TGF-beta RII(+) mucosal mesenchymal cells, predominantly alpha-smooth muscle actin (SMA)(+) myofibroblasts, co-expressing vimentin and basement membrane proteins, but not type I collagen. TGF-beta RII(+) SMA(-) fibroblasts producing type I collagen were also increased, particularly in areas of infiltration and in ulcers. Type IV collagen and laminin were distributed throughout the gut lamina propria in disease but were restricted to the basement membrane in controls. In areas of severe epithelial damage, type IV collagen was lost and increased type I collagen was observed. To examine ECM production by these cells, mucosal mesenchymal cells were isolated. Cultured cells exhibited a similar phenotype and matrix profile to those of in vivo cells. The data suggested that there were at least two populations of mesenchymal cells responsible for ECM synthesis in the mucosa and that ligation of TGF-beta receptors on these cells resulted in the disordered and increased ECM production observed in colitic mucosa.