Phillip T. Marucha

Mucosal wound healing is impaired by examination stress.

Objective Impairment of wound healing is a well-recognized sequelae of conditions that alter immune function, including diabetes, jaundice, and advanced age. There is also growing evidence that psychological stress has adverse consequences for immune function. This study addressed the effects of a commonplace stressor on wound healing. Method Two punch biopsy wounds were placed on the hard palate of 11 dental students. The first wound was timed during summer vacation, whereas the second was placed on the contralateral side 3 days before the first major examination of the term; thus, each student served as her or his own control. Two independent methods assessed healing (daily photographs and a foaming response to hydrogen peroxide). Results Students took an average of 3 days longer to completely heal the 3.5-mm wound during examinations, ie, 40% longer to heal a small, standardized wound. Production of interleukin 1 [small beta, Greek] (IL-1 [small beta, Greek]) messenger RNA (mRNA) declined by 68% during examinations, providing evidence of one possible immunological mechanism. These differences were quite reliable: No student healed as rapidly or produced as much IL-1 [small beta, Greek] mRNA during examinations as during vacation. Conclusions These data suggest that even something as transient, predictable, and relatively benign as examination stress can have significant consequences for wound healing.

Restraint Stress Slows Cutaneous Wound Healing in Mice

The impact of stress on cutaneous wound healing was assessed in a murine model. Female, hairless SKH-1 mice, 6-8 weeks of age were subjected to restraint stress (RST) 3 days before and for 5 days following dorsal application of a 3.5-mm sterile punch wound. Control mice were wounded, but not restrained. Using photography and image analysis, the rate of wound healing was compared between the two groups. Wounds on control mice healed on average 3.10 days sooner than RST-treated mice. In addition, cross-sectional, morphometric analysis of the dermal and epidermal layers revealed reduced inflammation surrounding wounds from RST mice at 1, 3, and 5 days after wounding. In the RST group, serum corticosterone levels averaged 162.5 ng/ml compared to 35.7 ng/ml in the controls. Treatment of RST-stressed animals with the glucocorticoid receptor antagonist RU40555 resulted in healing rates comparable to those of control animals. Thus, the reduction in inflammation and delayed healing correlated with serum corticosterone levels and suggest that disruption of neuroendocrine homeostasis modulates wound healing.

Social stress increases the susceptibility to endotoxic shock

The influence of social disruption stress (SDR) on the susceptibility to endotoxic shock was investigated. SDR was found to increase the mortality of mice when they were challenged with the bacterial endotoxin lipopolysaccharide (LPS). Histological examination of SDR animals after LPS injection revealed widespread disseminated intravascular coagulation in the brain and lung, extensive meningitis in the brain, severe hemorrhage in the lung, necrosis in the liver, and lymphoid hyperplasia in the spleen, indicating inflammatory organ damage. In situ hybridization histochemical analysis showed that the expression of the glucocorticoid receptor mRNA was down-regulated in the brain and spleen of SDR animals while the ratio of expression of AVP/CRH-the two adrenocorticotropic hormone secretagogue, increased. After LPS injection, the expression of pro-inflammatory cytokines, IL-1beta and TNF-alpha, was found significantly higher in the lung, liver, spleen, and brain of the SDR mice as compared with the LPS-injected home cage control animals. Taken together, these results show that SDR stress increases the susceptibility to endotoxic shock and suggest that the development of glucocorticoid resistance and increased production of pro-inflammatory cytokines are the mechanisms for this behavior-induced susceptibility to endotoxic shock.

Characterization of cytokines present in middle ear effusions

Retention of inflammatory mediators and cells in the middle ear cleft during chronic otitis media with effusion (COME), results in ongoing inflammation with the potential for pathologic changes and hearing loss. Cytokines are glycoproteins produced by macrophages and other cells. Activities of cytokines include fever production, osteoclast, fibroblast, phagocyte and cytotoxic cell activation, regulation of antibody formation, and inhibition of cartilage, bone and endothelial cell growth. Using enzyme-linked immunospecific assays we measured levels of six cytokines in middle ear effusions (MEE) from children with COME. Significant levels of four cytokines: interleukin-1-beta (greater than 50 pg/ml), interleukin-2 (greater than 300 pg/ml), tumor necrosis factor-alpha (greater than 40 pg/ml), and gamma-interferon (greater than 6.25 pg/ml) were found in 51%, 54%, 63%, and 19% of MEE, respectively. In contrast, levels of a fifth cytokine, granulocyte-macrophage colony-stimulating factor, and a sixth cytokine, interleukin-4, were undetectable. Age was observed to have a significant effect on the levels of specific cytokines. Interleukin-1 (IL-1) correlated inversely (P less than .02) with age such that the younger the child, the higher the level of IL-1 in MEE. Tumor necrosis factor-alpha (TNF) correlated directly (P less than .005) with age such that the older the child, the higher the level of TNF in MEE. Children undergoing tympanostomy on multiple occasions had average MEE TNF levels (234.2 +/- 109.1 pg/mg total protein) that were nearly 14 times higher (P less than .005) than those from children undergoing their first tympanostomy (16.9 +/- 3.0 pg/mg total protein). Thus IL-1 correlated with the early stages of COME, while TNF correlated with persistence of disease. The presence of these cytokines in MEE may be responsible for the mucosal damage, bone erosion, fibrosis, and resulting hearing loss seen in some cases of COME.

Acute stress evokes selective mobilization of T cells that differ in chemokine receptor expression: A potential pathway linking immunologic reactivity to cardiovascular disease

T lymphocytes and monocytes/macrophages are the most abundant cells found in the atherosclerotic plaque. These cells can migrate towards the activated endothelium through the local release of chemotactic cytokines, or chemokines. Given the important role of leukocyte migration in atherosclerosis and the role of stress in mediating leukocyte trafficking, the present study examined the effects of an acute stressor on the redistribution of T cells (CD3+) and monocytes that express the chemokine receptors CCR5, CCR6, CXCR1, CXCR2, CXCR3, and CXCR4. Forty-four undergraduate students underwent a public speaking task. The acute stressor induced sympathetic cardiac activation, parasympathetic cardiac withdrawal, lymphocytosis, and monocytosis (all p<.001). Although the total number of T lymphocytes did not change, there was a selective increase in the number of circulating T cells expressing CXCR2, CXCR3, and CCR5. The ligands of these receptors are chemokines known to be secreted by activated endothelial cells. Analyses of individual differences in stress-induced responses demonstrated a positive relationship between sympathetic cardiac reactivity and mobilization of the various T cell subsets (.35<r<.56;p<.05). For the monocytes, all sub-populations increased in parallel with total monocyte numbers, with no relation to changes in sympathetic cardiac drive. These results indicate that acute stress induces a mobilization of T cells that are primed to respond to inflamed endothelium. Acute stressors may thus promote the recruitment of circulating immune cells into the sub-endothelia, and therefore accelerate atherosclerotic plaque formation and potentially contribute to the complications that follow acute stressful events. This mechanism may help explain the link between stress, reactivity, and cardiovascular disease.

Positional differences in the wound transcriptome of skin and oral mucosa

BackgroundWhen compared to skin, oral mucosal wounds heal rapidly and with reduced scar formation. Recent studies suggest that intrinsic differences in inflammation, growth factor production, levels of stem cells, and cellular proliferation capacity may underlie the exceptional healing that occurs in oral mucosa. The current study was designed to compare the transcriptomes of oral mucosal and skin wounds in order to identify critical differences in the healing response at these two sites using an unbiased approach.ResultsUsing microarray analysis, we explored the differences in gene expression in skin and oral mucosal wound healing in a murine model of paired equivalent sized wounds. Samples were examined from days 0 to 10 and spanned all stages of the wound healing process. Using unwounded matched tissue as a control, filtering identified 1,479 probe sets in skin wounds yet only 502 probe sets in mucosal wounds that were significantly differentially expressed over time. Clusters of genes that showed similar patterns of expression were also identified in each wound type. Analysis of functionally related gene expression demonstrated dramatically different reactions to injury between skin and mucosal wounds. To explore whether site-specific differences might be derived from intrinsic differences in cellular responses at each site, we compared the response of isolated epithelial cells from skin and oral mucosa to a defined in vitro stimulus. When cytokine levels were measured, epithelial cells from skin produced significantly higher amounts of proinflammatory cytokines than cells from oral mucosa.ConclusionsThe results provide the first detailed molecular profile of the site-specific differences in the genetic response to injury in mucosa and skin, and suggest the divergent reactions to injury may derive from intrinsic differences in the cellular responses at each site.

Differential mobilization of functionally distinct natural killer subsets during acute psychologic stress

Objective and Methods: Two functionally distinct natural killer (NK) subsets can be identified according to surface CD56 expression: CD56lo cells compose the majority of NK cells and function as cytotoxic cells, whereas CD56hi cells have an immunomodulatory function through the secretion of cytokines. These NK subsets also differ in the expression levels of adhesion molecules such as CD62L and CD11a, indicating distinct potentials to migrate to lymphoid and nonlymphoid tissues. We investigated whether NK cell mobilization during acute stress varies according to these functional and phenotypic distinctions. Methods and Results: Fifty-three undergraduate students performed a public-speaking task and 21 students participated in a control session. The task increased heart rate and catecholamines. No change was observed for the immunoregulatory CD56hi NK subset, whereas the number of cytotoxic CD56lo NK cells tripled. In line with the observation that NK mobilization is related to cytotoxic function, we found larger increases in NK cells that express higher levels of CD16 (a receptor that mediates antibody-dependent cytotoxicity). Consistent with known subset differences in adhesion molecule expression, we also found larger stress-induced increases for NK cells that were CD62L-negative and CD11ahi. Plasma levels of soluble CD62L remained unaltered, suggesting that the increase in CD62L-negative NK cells did not result from CD62L shedding. Regression analyses demonstrated independent contributions of epinephrine and norepinephrine to NK subset mobilization. Conclusion: The marked specificity and robustness of these effects support the idea that NK cell mobilization is a functionally relevant response that is aimed at protecting the organism during acutely stressful situations. ANOVA = analysis of variance; CD = cluster of differentiation; ECG = electrocardiogram; ELISA = enzyme-linked immunosorbent assay; Hb = hemoglobin; HPLC = high-pressure liquid chromatography; Htc = hematocrit; NK = natural killer; POMS = Profile of Mood States; sCD62L = soluble CD62L; SEM = standard error of mean.

MicroRNA-99 Family Targets AKT/mTOR Signaling Pathway in Dermal Wound Healing

Recent studies suggest that microRNAs play important roles in dermal wound healing and microRNA deregulation has been linked with impaired wound repair. Here, using a mouse experimental wound healing model, we identified a panel of 63 differentially expressed microRNAs during dermal wound healing, including members of miR-99 family (miR-99a, miR-99b, miR-100). We further demonstrated that miR-99 family members regulate cell proliferation, cell migration, and AKT/mTOR signaling. Combined experimental and bioinformatics analyses revealed that miR-99 family members regulate AKT/mTOR signaling by targeting multiple genes, including known target genes (e.g., IGF1R, mTOR) and a new target (AKT1). The effects of miR-99 family members on the expression of IGF1R, mTOR and AKT1 were validated at both the mRNA and protein levels. Two adjacent miR-99 family targeting sites were identified in the 3′-UTR of the AKT1 mRNA. The direct interaction of miR-100 with these targeting sites was confirmed using luciferase reporter assays. The microRNA-100-directed recruitment of AKT1 mRNA to the RNAi-induced silencing complex (RISC) was confirmed by a ribonucleoprotein-IP assay. In summary, we identified a panel of differentially expressed microRNAs which may play important roles in wound healing. We provide evidence that miR-99 family members contribute to wound healing by regulating the AKT/mTOR signaling.

Depressive symptoms predict mucosal wound healing

Objective: There is mounting evidence that psychosocial stress can delay wound healing, but this literature almost exclusively pertains to dermal wound healing. Many surgical procedures involve damage to mucosal tissues and the time course and the role of repair processes, such as inflammation, in the healing of these tissues are markedly different from those in dermal healing. Feelings of depression and social isolation are common among surgical patients, and the present study therefore investigated if these factors predict the rate of mucosal wound healing. Methods: Undergraduate students were invited to participate in the study if they reported high or low levels of loneliness or depressive symptoms, corresponding to the upper or lower quintile of their peer group. The UCLA loneliness scale and the Beck Depression Inventory [short form] were used for this screening. A sample of 193 healthy young adults (age range 18–31 years) received a 3.5-mm circular wound on the oral hard palate, under local anesthesia. Healing was monitored by daily videographs of the wound. Results: The median healing rate was 7 days. High dysphoric participants were, however, more likely to heal slower than this median healing rate (odds ratio 3.57 (1.58–8.07); p < .001). This association remained robust after correction for a broad range of demographic and behavioral variables, including gender, age, ethnicity, and health behaviors. High dysphoric individuals also exhibited significantly larger average wound sizes from day 2 post wounding onward. Loneliness and diurnal cortisol secretion (measured over 5 days) were unrelated to healing. Conclusion: Depressive symptoms predict the rate of mucosal wound healing in healthy young adults. We discuss potential pathways that warrant further investigation. HPA = hypothalamic-pituitary-adrenal; BDI-sf = Beck Depression Inventory short form; UCLA-R = Revised UCLA Loneliness Scale; OR = odds ratio; AUC = area under the curve.

Experimental Models of Stress and Wound Healing

Experimental animal models have been used to examine stress-induced interactions among the nervous, endocrine, and immune systems. Generally, the response to stress results in a body-wide set of physiologic adaptations, mediated through the activation of neuroendocrine pathways that intersect and modulate inflammatory and immune responses. These interacting responses modulate diverse physiological processes including the initiation of tissue repair and wound healing. Two different stressors were used to activate neuroendocrine responses to study their impact on wound healing: restraint (RST) and social disruption (SDR). Previous studies showed that both stressors activate the hypothalamic-pituitary-adrenal (HPA) axis, resulting in elevated plasma levels of the corticosterone (cort) response and modulation of pro-inflammatory cytokine response. To test the effects of stress-induced HPA activation on inflammatory responses and wound healing, a 3.5-mm cutaneous punch biopsy wound was placed on the dorsal surface of control and stressed (RST or SDR) C57BL/6 male mice and the kinetics of wound healing were studied over 10 days. RST slowed wound healing in inbred C57BL/6 mice, whereas the wounds on SDR mice healed in a fashion similar to the non-stressed, home cage controls.