Expression of PKM2 in wound keratinocytes is coupled to angiogenesis during skin repair in vivo and in HaCaT keratinocytes in vitro.

An injured skin is rapidly restored in a manner of wound healing. We have previously shown that intact insulin signaling and glucose uptake are fundamental to proper wound closure. Consequently, under exacerbated inflammation, compromised insulin action and glucose uptake lead to impaired healing. However, in spite of the increased attention to cell metabolism during tissue regeneration, metabolic mediators that govern cellular and physiological processes throughout skin repair remained largely elusive. Through assessment of mRNA using real-time PCR and protein blot analysis, we report that healing of cutaneous wounds comprise a boosted expression of genes involved in glycolysis, oxidative phosphorylation, pentose phosphate shunt, and glutamine anaplerosis. We further focused on the functional role of pyruvate kinase M (PKM) isoenzymes that catalyze the final and rate-limiting step of glycolysis. Whereas the expression of the metabolic constitutively active Pkm1 isozyme remained almost unchanged, Pkm2 is augmented during the inflammatory phase of healing. The immunohistochemistry and RNA in situ hybridization analysis showed a confined Pkm2 expression to keratinocytes of the hyperproliferative epithelium and, to a lesser extent, infiltrating neutrophils and monocytes as well as later on in macrophages. Notably, the expression of Pkm2 in keratinocytes facing the wound bed side colocalized with VEGF expression. The in vitro knockdown of PKM2 in HaCaT keratinocytes using small interfering (si) RNA confirmed an acute role for PKM2 in facilitating the complete induction of VEGF mRNA and protein expression in keratinocytes; this function is mainly HIF-1α independent. KEY MESSAGES: • Wound healing involves activation of glycolysis, oxidative phosphorylation, pentos-phosphate shunt, and replenishment of tri-carboxylic acid (TCA) cycle through glutamine anaplerosis. • The pyruvate kinase M2 (PKM2) isoform is upregulated during the inflammatory phase of cutaneous healing, mainly in keratinocytes of hyperproliferative epithelia. • In vivo, the expression of VEGF in wound keratinocytes is colocalized with PKM2. • PKM2 is required for full induction of VEGF in HaCaT keratinocytes in vitro.

Reciprocal abrogation of PKM isoforms: contradictory outcomes and differing impact of splicing signal on CRISPR/Cas9 mediates gene editing in keratinocytes.

The healing of wounded skin is a highly organized process involving a massive cell in- and outflux, proliferation and tissue remodelling. It is well accepted that metabolic constraints such as diabetes mellitus, overweight or anorexia impairs wound healing. Indeed, wound inflammation involves a boost of overall metabolic changes. As wound healing converges inflammatory processes that are also common to transformation, we investigate the functional role of the pro-neoplastic factor pyruvate kinase (PK) M2 and its metabolic active splice variant PKM1 in keratinocytes. Particularly, we challenge the impact of reciprocal ablation of PKM1 or two expression. Here, CRISPR/Cas9 genome editing of the PKM gene in HaCaT reveals an unexpected mutational bias at the 3'SS of exon 9, whereas no preference for any particular kind of mutation at exon 10 3' splice, despite the close vicinity (400 nucleotides apart) and sequence similarity between the two sites. Furthermore, as opposed to transient silencing of PKM2, exclusion splicing of PKM2 via genome editing mutually increases PKM1 mRNA and protein expression and compensates for the absence of PKM2, whereas the reciprocal elimination of PKM1 splicing reduces PKM2 expression and impedes cell proliferation, thus unveiling an essential role for PKM1 in growth and metabolic balance of HaCaT keratinocytes.

3'mRNA sequencing reveals pro-regenerative properties of c5ar1 during resolution of murine acetaminophen-induced liver injury.

Murine acetaminophen-induced acute liver injury (ALI) serves as paradigmatic model for drug-induced hepatic injury and regeneration. As major cause of ALI, acetaminophen overdosing is a persistent therapeutic challenge with N-acetylcysteine clinically used to ameliorate parenchymal necrosis. To identify further treatment strategies that serve patients with poor N-acetylcysteine responses, hepatic 3'mRNA sequencing was performed in the initial resolution phase at 24 h/48 h after sublethal overdosing. This approach disclosed 45 genes upregulated (≥5-fold) within this time frame. Focusing on C5aR1, we observed in C5aR1-deficient mice disease aggravation during resolution of intoxication as evidenced by increased liver necrosis and serum alanine aminotransferase. Moreover, decreased hepatocyte compensatory proliferation and increased caspase-3 activation at the surroundings of necrotic cores were detectable in C5aR1-deficient mice. Using a non-hypothesis-driven approach, herein pro-regenerative/-resolving effects of C5aR1 were identified during late acetaminophen-induced ALI. Data concur with protection by the C5a/C5aR1-axis during hepatectomy and emphasize the complex role of inflammation during hepatic regeneration and repair.

Interleukin 10 Restores Lipopolysaccharide-Induced Alterations in Synaptic Plasticity Probed by Repetitive Magnetic Stimulation.

Systemic inflammation is associated with alterations in complex brain functions such as learning and memory. However, diagnostic approaches to functionally assess and quantify inflammation-associated alterations in synaptic plasticity are not well-established. In previous work, we demonstrated that bacterial lipopolysaccharide (LPS)-induced systemic inflammation alters the ability of hippocampal neurons to express synaptic plasticity, i.e., the long-term potentiation (LTP) of excitatory neurotransmission. Here, we tested whether synaptic plasticity induced by repetitive magnetic stimulation (rMS), a non-invasive brain stimulation technique used in clinical practice, is affected by LPS-induced inflammation. Specifically, we explored brain tissue cultures to learn more about the direct effects of LPS on neural tissue, and we tested for the plasticity-restoring effects of the anti-inflammatory cytokine interleukin 10 (IL10). As shown previously, 10 Hz repetitive magnetic stimulation (rMS) of organotypic entorhino-hippocampal tissue cultures induced a robust increase in excitatory neurotransmission onto CA1 pyramidal neurons. Furthermore, LPS-treated tissue cultures did not express rMS-induced synaptic plasticity. Live-cell microscopy in tissue cultures prepared from a novel transgenic reporter mouse line [C57BL/6-Tg(TNFa-eGFP)] confirms that ex vivo LPS administration triggers microglial tumor necrosis factor alpha (TNFα) expression, which is ameliorated in the presence of IL10. Consistent with this observation, IL10 hampers the LPS-induced increase in TNFα, IL6, IL1ß, and IFNγ and restores the ability of neurons to express rMS-induced synaptic plasticity in the presence of LPS. These findings establish organotypic tissue cultures as a suitable model for studying inflammation-induced alterations in synaptic plasticity, thus providing a biological basis for the diagnostic use of transcranial magnetic stimulation in the context of brain inflammation.

Increase of cystathionine-γ-lyase (CSE) during late wound repair: Hydrogen sulfide triggers cytokeratin 10 expression in keratinocytes.

The gaseous mediators nitric oxide (NO), carbon monoxide (CO) and lately also hydrogen sulfide (H2S) have been described to contribute to the interplay of protein type- and lipid mediators in the regulation of wound healing. In particular, the recently reported role of H2S in skin repair remains largely unresolved. Therefore we assessed the expressional kinetics of potential H2S-producing enzymes during undisturbed skin repair: the cystathionine-γ-lyase (CSE), the cystathionine-ß-synthase (CBS) and the 3-mercaptopyruvate sulfurtransferase (MPST). All three enzymes were not transcriptionally induced upon wounding and remained silent through the acute inflammatory and proliferative phase of skin repair. By contrast, CSE expression started to increase significantly at the later stages of healing, when cellular proliferation ceases within the granulation tissue and neoepidermis. The importance of H2S production in late healing phases was supported by a strong induction of otherwise not-induced CBS to complement the loss of CSE function in CSE-deficient mice. Immunohistochemistry revealed hair follicle keratinocytes and basal keratinocytes of the neo-epidermis covering the wound area as sources of CSE expression. Subsequent in vitro studies implicated a role of CSE-derived H2S for keratinocyte differentiation: the H2S-donor GYY4137 markedly increased the Ca2+-triggered expression of the early keratinocyte differentiation markers cytokeratin 10 (CK10) and involucrin (IVN) in cultured human keratinocytes. Here, GYY4137-derived H2S strongly enhanced CK10 expression by increasing the binding of RNA polymerase II to the CK10 promoter.

A heterogeneous Ly-6B2+ leukocyte population consists of yet undescribed iNOS-expressing cell types in murine skin wounds.

The gaseous mediator nitric oxide (NO) is a central regulatory molecule during the inflammatory phase of cutaneous tissue repair. The inducible NO-synthase (iNOS) represents the main isoform of the three NO producing enzymes at the wound site. In particular, keratinocytes and macrophages are described as main sources of iNOS-derived NO in skin wounds. Here we provide experimental evidence that Ly-6B2+ leukocytes are an additional cellular source of iNOS-derived NO in wounds. As wound iNOS protein expression temporally coincides with both macrophage and neutrophil infiltration, we used immunohistochemistry (IHC) and fluorescence-activated cell sorting (FACS) to address iNOS expression in both macrophages and neutrophil subsets. IHC analyses excluded F4/80+ macrophages as iNOS producers, but indicated Ly-6G/C (Gr-1)+ neutrophils to express iNOS in wound granulation tissue. A subsequent FACS-based analysis from cellular wound tissue preparations revealed an iNOS-expressing fraction of Ly-6B2-determined leukocytes that consisted of Ly-6G+ and Ly-6G- cells, meaning that mainly mature neutrophils (Ly-6B2+/Ly-6G+) as well as inflammatory monocytes (Ly-6B2+/Ly-6G-) are dominant iNOS-expressing cell types in the developing granulation tissue of acute wounds.

Salt supplementation ameliorates developmental kidney defects in COX-2-/- mice.

Deficiency of cyclooxygenase-2 (COX-2) activity in the early postnatal period causes impairment of kidney development leading to kidney insufficiency. We hypothesize that impaired NaCl reabsorption during the first days of life is a substantial cause for nephrogenic defects observed in COX-2-/- mice and that salt supplementation corrects these defects. Daily injections of NaCl (0.8 mg·g-1·day-1) for the first 10 days after birth ameliorated impaired kidney development in COX-2-/- pups resulting in an increase in glomerular size and fewer immature superficial glomeruli. However, impaired renal subcortical growth was not corrected. Increasing renal tubular flow by volume load or injections of KCl did not relieve the renal histomorphological damage. Administration of torsemide and spironolactone also affected nephrogenesis resulting in diminished glomeruli and cortical thinning. Treatment of COX-2-/- pups with NaCl/DOCA caused a stronger mitigation of glomerular size and induced a slight but significant growth of cortical tissue mass. After birth, renal mRNA expression of NHE3, NKCC2, ROMK, NCCT, ENaC, and Na+/K+-ATPase increased relative to postnatal day 2 in wild-type mice. However, in COX-2-/- mice, a significantly lower expression was observed for NCCT, whereas NaCl/DOCA treatment significantly increased NHE3 and ROMK expression. Long-term effects of postnatal NaCl/DOCA injections indicate improved kidney function with normalization of pathologically enhanced creatinine and urea plasma levels; also, albumin excretion was observed. In summary, we present evidence that salt supplementation during the COX-2-dependent time frame of nephrogenesis partly reverses renal morphological defects in COX-2-/- mice and improves kidney function.

Angiotensin II-AT1-receptor signaling is necessary for cyclooxygenase-2-dependent postnatal nephron generation.

Deletion of cyclooxygenase-2 (COX-2) causes impairment of postnatal kidney development. Here we tested whether the renin angiotensin system contributes to COX-2-dependent nephrogenesis in mice after birth and whether a rescue of impaired renal development and function in COX-2-/- mice was achievable. Plasma renin concentration in mouse pups showed a birth peak and a second peak around day P8 during the first 10 days post birth. Administration of the angiotensin II receptor AT1 antagonist telmisartan from day P1 to P3 did not result in cortical damage. However, telmisartan treatment from day P3 to P8, the critical time frame of renal COX-2 expression, led to hypoplastic glomeruli, a thinned subcapsular cortex and maturational arrest of superficial glomeruli quite similar to that observed in COX-2-/- mice. In contrast, AT2 receptor antagonist PD123319 was without any effect on renal development. Inhibition of the renin angiotensin system by aliskiren and enalapril caused similar glomerular defects as telmisartan. Administration of the AT1 receptor agonist L162313 to COX-2-/- pups improved kidney growth, ameliorated renal defects, but had no beneficial effect on reduced cortical mass. L162313 rescued impaired renal function by reducing serum urea and creatinine and mitigated pathologic albumin excretion. Moreover, glomerulosclerosis in the kidneys of COX-2-/- mice was reduced. Thus, angiotensin II-AT1-receptor signaling is necessary for COX-2-dependent normal postnatal nephrogenesis and maturation.

Inhibition of cyclooxygenase-1 and -2 activity in keratinocytes inhibits PGE2 formation and impairs vascular endothelial growth factor release and neovascularisation in skin wounds.

Inhibition of cyclooxygenase (Cox) enzymatic activity by non-steroidal anti-inflammatory drugs (NSAIDs) provides the molecular basis of analgesia following wounding or surgery. This study investigated the role of Cox activity in the regulation of vascular endothelial growth factor (VEGF) expression in keratinocytes and the formation of new blood vessels in acute wounds in mice. To this end, human HaCaT keratinocytes were stimulated with epidermal growth factor (EGF). EGF increased Cox-1 mRNA in the presence of the constitutively expressed Cox-1 protein in keratinocytes. EGF coinduced Cox-2 and VEGF165 mRNA and protein expression and an accumulation of prostaglandin E2 (PGE2 ) in cell culture supernatants. Inhibition of Cox isozyme activity by Cox-1 and -2 siRNA or ibuprofen reduced PGE2 and VEGF165 release from keratinocytes. In a mouse model of excisional wound healing, Cox-2 and VEGF165 expression were colocalized in the granulation tissue of acute wounds. Oral treatment of mice with the Cox-1 and -2 inhibitor diclofenac was associated with reduced levels of VEGF165 protein and an impaired blood vessel formation in acute wound tissue. In summary, our data suggest that a reduction of PGE2 -triggered VEGF165 protein expression in wound keratinocytes is likely to contribute to the observed impairment of wound neovascularisation upon Cox inhibition.

Anti-Inflammatory Effects of Rosiglitazone in Obesity-Impaired Wound Healing Depend on Adipocyte Differentiation.

Combined diabetes-obesity syndromes severely impair regeneration of acute skin wounds in mouse models. This study assessed the contribution of subcutaneous adipose tissue to exacerbated wound inflammatory conditions. Genetically obese (ob/ob) mice showed an increased expression of positive transcriptional effectors of adipocyte differentiation such as Krüppel-like factor (KLF)-5 and peroxisome proliferator-activated receptor (PPAR)-γ and an associated expression of leptin and fatty acid-binding protein (FABP)-4, but also CXCL2 in isolated subcutaneous fat. This observation in obese mice is in keeping with differentially elevated levels of KLF-5, PPAR-γ, leptin, FABP-4 and CXCL2 in in vitro-differentiated 3T3-L1 adipocytes. Notably, CXCL2 expression restrictively appeared upon cytokine (IL-1ß/TNF-α) stimulation only in mature, but not immature 3T3-L1 adipocytes. Of importance, the critical regulator of adipocyte maturation, PPAR-γ, was merely expressed in the final phase of in-vitro induced adipocyte differentiation from 3T3-L1 pre-adipocytes. Consistently, the PPAR-γ agonist rosiglitazone suppressed cytokine-induced CXCL2 release from mature adipocytes, but not from early 3T3-L1 adipocyte stages. The inhibitory effect of PPAR-γ activation on CXCL2 release appeared to be a general anti-inflammatory effect in mature adipocytes, as cytokine-induced cyclooxygenase (Cox)-2 was simultaneously repressed by rosiglitazone. In accordance with these findings, oral administration of rosiglitazone to wounded obese mice significantly changed subcutaneous adipocyte morphology, reduced wound CXCL2 and Cox-2 expression and improved tissue regeneration. Thus, our data suggest that PPAR-γ might provide a target to suppress inflammatory signals from mature adipocytes, which add to the prolonged wound inflammation observed in diabetes-obesity conditions.

Uptake of neutrophil-derived Ym1 protein distinguishes wound macrophages in the absence of interleukin-4 signaling in murine wound healing.

The determination of regenerative wound-healing macrophages as alternatively activated macrophages is currently questioned by the absence of IL-4 in wound tissue. Yet, murine wound tissue expressed high levels of Ym1 (chitinase 3-like 3), an established marker of the IL-4-induced alternatively activated macrophage phenotype. Ym1 was expressed in wound neutrophils but not in macrophages. Initially, Ym1-free wound-healing macrophages, invading from the wound margins, became gradually positive for the protein in the absence of IL-4 signaling and Stat6 activation, as they entered the neutrophil-populated wound regions. IL-4 failed to induce Ym1 protein in ex vivo-cultured wound tissue explants containing wound-healing macrophages. Recombinant Ym1 protein was selectively taken up by macrophages but not by keratinocytes and endothelial cells. Cultured macrophages lost the ability to take up the recombinant protein when four highly conserved residues and the 70-amino acid small α+ß domain essential for Ym1 function were removed. The data suggest that the IL-4/Stat6-independent presence of Ym1 protein in wound-healing macrophages is of exogenous origin, with Ym1 taken up from wound neutrophils as the cellular source. The data suggest that in situ determination of wound-healing macrophages, often defined by Ym1, might not essentially describe an IL-4-dependent macrophage phenotype. Consequently, wound-healing macrophages should not be classified by the established categories of the well-accepted but simplified paradigm of M1/M2 macrophage activation.

Deregulated unfolded protein response in chronic wounds of diabetic ob/ob mice: a potential connection to inflammatory and angiogenic disorders in diabetes-impaired wound healing.

Type-2 diabetes mellitus (T2D) represents an important metabolic disorder, firmly connected to obesity and low level of chronic inflammation caused by deregulation of fat metabolism. The convergence of chronic inflammatory signals and nutrient overloading at the endoplasmic reticulum (ER) leads to activation of ER-specific stress responses, the unfolded protein response (UPR). As obesity and T2D are often associated with impaired wound healing, we investigated the role of UPR in the pathologic of diabetic-impaired cutaneuos wound healing. We determined the expression patterns of the three UPR branches during normal and diabetes-impaired skin repair. In healthy and diabetic mice, injury led to a strong induction of BiP (BiP/Grp78), C/EBP homologous protein (CHOP) and splicing of X-box-binding protein (XBP)1. Diabetic-impaired wounds showed gross and sustained induction of UPR associated with increased expression of the pro-inflammatory chemokine macrophage inflammatory protein (MIP)2 as compared to normal healing wounds. In vitro, treatment of RAW264.7 macrophages with tunicamycin, and subsequently stimulation with lipopolysaccharide (LPS) and interferon (IFN)-γ enhances MIP2 mRNA und protein expression compared to proinflammatory stimulation alone. However, LPS/IFNγ induced vascular endothelial growth factor (VEGF) production was blunted by tunicamycin induced-ER stress. Hence, UPR is activated following skin injury, and functionally connected to the production of proinflammatory mediators. In addition, prolongation of UPR in diabetic non-healing wounds aggravates ER stress and weakens the angiogenic phenotype of wound macrophages.

Application of interleukin-22 mediates protection in experimental acetaminophen-induced acute liver injury.

Acetaminophen (APAP, paracetamol)-induced hepatotoxicity, although treatable by timely application of N-acetylcysteine, can be fatal. Because it is among the common causes of acute liver failure in intensive care units and in light of its gradually increasing incidence, the need for novel therapeutic strategies aimed at severe intoxication is apparent. Recently, it has been shown that IL-22, a STAT3-activating cytokine, has the capability to mediate liver protection. Herein, the protective potential of IL-22 in murine APAP-induced hepatotoxicity was assessed. Intravenous administration of prophylactic IL-22 significantly reduced serum alanine aminotransferase levels and histopathologic damage in APAP-induced liver injury, a process that coincided with increased hepatocyte proliferation in vivo. Concomitant gene expression analysis revealed hepatic induction of genes prototypically up-regulated by the IL-22/STAT3 axis, among others suppressor of cytokine signaling-3, lipocalin-2, and α1-antichymotrypsin. Notably, in a translational setting of therapeutic treatment 2 hours after APAP, IL-22 supported protection in the context of suboptimal N-acetylcysteine dosing. IL-22 likewise connected to augmented hepatocyte proliferation in this experimental setting. As detected by analysis of inflammatory cytokine production, systemically applied IL-22 did not display acute immunomodulation/stimulation in otherwise untreated or endotoxemic mice. Those latter observations clearly confirm acute tolerability of systemically applied IL-22. Observations presented altogether suggest that therapeutic IL-22 administration is a conceivable tissue-protective regimen aimed at hard-to-treat patients with severe APAP-induced hepatotoxicity.

Protective properties of inhaled IL-22 in a model of ventilator-induced lung injury.

High-pressure ventilation induces barotrauma and pulmonary inflammation, thus leading to ventilator-induced lung injury (VILI). IL-22 has both immunoregulatory and tissue-protective properties. Functional IL-22 receptor expression is restricted to nonleukocytic cells, such as alveolar epithelial cells. When applied via inhalation, IL-22 reaches the pulmonary system directly and in high concentrations, and may protect alveolar epithelial cells against cellular stress and biotrauma associated with VILI. In A549 lung epithelial cells, IL-22 was able to induce rapid signal transducer and activator of transcription (STAT)-3 phosphorylation/activation, and hereon mediated stable suppressor of cytokine signaling (SOCS) 3 expression detectable even 24 hours after onset of stimulation. In a rat model of VILI, the prophylactic inhalation of IL-22 before induction of VILI (peak airway pressure = 45 cm H(2)O) protected the lung against pulmonary disintegration and edema. IL-22 reduced VILI-associated biotrauma (i.e., pulmonary concentrations of macrophage inflammatory protein-2, IL-6, and matrix metalloproteinase 9) and mediated pulmonary STAT3/SOCS3 activation. In addition, despite a short observation period of 4 hours, inhaled IL-22 resulted in an improved survival of the rats. These data support the hypothesis that IL-22, likely via activation of STAT3 and downstream genes (e.g., SOCS3), is able to protect against cell stretch and pulmonary baro-/biotrauma by enhancing epithelial cell resistibility.

Early production of IL-22 but not IL-17 by peripheral blood mononuclear cells exposed to live Borrelia burgdorferi: the role of monocytes and interleukin-1.

If insufficiently treated, Lyme borreliosis can evolve into an inflammatory disorder affecting skin, joints, and the CNS. Early innate immunity may determine host responses targeting infection. Thus, we sought to characterize the immediate cytokine storm associated with exposure of PBMC to moderate levels of live Borrelia burgdorferi. Since Th17 cytokines are connected to host defense against extracellular bacteria, we focused on interleukin (IL)-17 and IL-22. Here, we report that, despite induction of inflammatory cytokines including IL-23, IL-17 remained barely detectable in response to B. burgdorferi. In contrast, T cell-dependent expression of IL-22 became evident within 10 h of exposure to the spirochetes. This dichotomy was unrelated to interferon-γ but to a large part dependent on caspase-1 and IL-1 bioactivity derived from monocytes. In fact, IL-1ß as a single stimulus induced IL-22 but not IL-17. Neutrophils display antibacterial activity against B. burgdorferi, particularly when opsonized by antibodies. Since neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection, protective and antibacterial properties of IL-22 may close this gap and serve essential functions in the initial phase of spirochete infection.

Wound healing in mice with high-fat diet- or ob gene-induced diabetes-obesity syndromes: a comparative study.

In the past, the genetically diabetic-obese diabetes/diabetes (db/db) and obese/obese (ob/ob) mouse strains were used to investigate mechanisms of diabetes-impaired wound healing. Here we determined patterns of skin repair in genetically normal C57Bl/6J mice that were fed using a high fat diet (HFD) to induce a diabetes-obesity syndrome. Wound closure was markedly delayed in HFD-fed mice compared to mice which had received a standard chow diet (CD). Impaired wound tissue of HFD mice showed a marked prolongation of wound inflammation. Expression of vascular endothelial growth factor (VEGF) was delayed and associated with the disturbed formation of wound margin epithelia and an impaired angiogenesis in the reduced granulation tissue. Normal wound contraction was retarded and disordered. Wound disorders in obese C57Bl/6J mice were paralleled by a prominent degradation of the inhibitor of NFκB (IκB-α) in the absence of an Akt activation. By contrast to impaired wound conditions in ob/ob mice, late wounds of HFD mice did not develop a chronic inflammatory state and were epithelialized after 11 days of repair. Thus, only genetically obese and diabetic ob/ob mice finally developed chronic wounds and therefore represent a better suited experimental model to investigate diabetes-induced wound healing disorders.

The suppressor of cytokine signaling (SOCS)-3 determines keratinocyte proliferative and migratory potential during skin repair.

Recently, the suppressor of cytokine signaling (SOCS)-3 has been shown to be expressed in disturbed wound margin epithelia during diabetes-impaired wound healing in mice. To functionally connect a potential contribution of SOCS-3 expression to the control of wound keratinocyte behavior in skin repair, we created a transgenic mouse (tsgn-K5/SOCS3) overexpressing SOCS-3 in keratinocytes using the bovine keratin 5 promoter. Tsgn-K5/SOCS3 mice showed a constitutive expression of SOCS-3 in the basal layer of skin epidermis. Keratinocytes of tsgn-K5/SOCS3 mice showed full inhibition of signal transducer and activator of transcription (STAT)-3 phosphorylation. Tsgn-K5/SOCS3 keratinocytes also showed a strong inhibition of migratory and proliferative potential in vitro. In addition, tsgn-K5/SOCS3 keratinocytes co-expressed the differentiation marker loricrin in the basal layer of nonwounded skin in vivo. Upon wounding, wound tissues of tsgn-K5/SOCS3 mice showed an impairment of wound closure characterized by strongly atrophied wound margin epithelia. Atrophied epithelia of tsgn-K5/SOCS3 mice exhibited a marked reduction in proliferating cells and reduced total keratinocyte numbers. In summary, this study suggests that the presence of SOCS-3 in keratinocytes strongly disturbs epithelial repair of cutaneous wounds by interfering with keratinocyte proliferation and migration.

Epithelial overexpression of SOCS-3 in transgenic mice exacerbates wound inflammation in the presence of elevated TGF-beta1.

The suppressor of cytokine signaling (SOCS)-3 has been shown to impair proliferation and migration of keratinocytes. To assess the functional dependency among wound inflammation, SOCS-3 induction in keratinocytes, and the outcome of healing, we generated a transgenic mouse that specifically overexpresses SOCS-3 in keratinocytes. Acute wound healing in transgenic mice was severely impaired. Keratinocyte-specific overexpression of SOCS-3 led to atrophied wound-margin epithelia and augmented the inflammatory response of wound keratinocytes by an increase in chemokine (MIP-2) and inflammatory enzyme (COX-2 and iNOS) expression. In addition, wound tissue of transgenic mice showed a prolonged persistence of neutrophils and macrophages. Remarkably, impaired wounds showed elevated levels of transforming growth factor (TGF)-beta1, which appeared to interfere with healing, as its neutralization markedly improved wound closure in transgenic mice. Interestingly, administration of a TGF-beta-neutralizing antibody increased wound inflammation in nontransgenic mice but not in transgenic littermates. This study suggests that SOCS-3-driven disturbances in wound keratinocytes are sufficient to induce inflamed wound conditions that resemble characteristics of chronic wounds in mice.

A transgenic mouse model of inducible macrophage depletion: effects of diphtheria toxin-driven lysozyme M-specific cell lineage ablation on wound inflammatory, angiogenic, and contractive processes.

Whether the wound macrophage is a key regulatory inflammatory cell type in skin repair has been a matter of debate. A transgenic mouse model mediating inducible macrophage depletion during skin repair has not been used to date to address this question. Here, we specifically rendered the monocyte/macrophage leukocyte lineage sensitive to diphtheria toxin by expressing the lysozyme M promoter-driven, Cre-mediated excision of a transcriptional STOP cassette from the simian DT receptor gene in mice (lysM-Cre/DTR). Application of diphtheria toxin to lysM-Cre/DTR mice led to a rapid reduction in both skin tissue and wound macrophage numbers at sites of injury. Macrophage-depleted mice revealed a severely impaired wound morphology and delayed healing. In the absence of macrophages, wounds were re-populated by large numbers of neutrophils. Accordingly, macrophage-reduced wound tissues exhibited the increased and prolonged persistence of macrophage inflammatory protein-2, macrophage chemoattractant protein-1, interleukin-1beta, and cyclooxygenase-2, paralleled by unaltered levels of bioactive transforming growth factor-beta1. Altered expression patterns of vascular endothelial growth factor on macrophage reduction were associated with a disturbed neo-vascularization at the wound site. Impaired wounds revealed a loss of myofibroblast differentiation and wound contraction. Our data in the use of lysM-Cre/DTR mice emphasize the pivotal function of wound macrophages in the integration of inflammation and cellular movements at the wound site to enable efficient skin repair.

Tight spatial and temporal control in dynamic basal to distal migration of epithelial inflammatory responses and infiltration of cytoprotective macrophages determine healing skin flap transplants in mice.

OBJECTIVE: We aimed to elucidate to date unknown molecular patterns of dynamic inflammatory tissue responses during uncomplicated healing of caudally pedicled skin flap transplants in mice. SUMMARY BACKGROUND DATA: Distal skin flap ischemic necrosis is a well-known complication in surgery. To improve ischemic conditions in impaired skin flaps, recent work attempted to increase insufficient vascularity by application of angiogenic growth factors or pluripotent cells. Wound inflammation is in the center of tissue repair, but its temporal and spatial regulation remains nearly unstudied in conditions of transplanted skin flap tissue. METHODS: RNase protection assay, quantitative real-time polymerase chain reaction, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and immunoblot techniques were used to determine expression and cellular localization of central inflammation-related chemokines, cytokines, enzymes and cell types upon skin flap transplantation. RESULTS: We observed a marked keratinocyte-driven inflammation that moved from the caudal base to distal flap regions during healing. Keratinocytes of the skin flap epithelium expressed increasingly large amounts of chemokines (MIP-2, MCP-1) and cyclooxygenase (Cox)-2 particularly in distal portions of the transplant. The underlying wound bed did not appear to contribute essentially to the inflammatory response. Despite strong attracting chemokine signals, distal flap tissue was not infiltrated by excess numbers of neutrophils and macrophages. Moreover, infiltrating macrophages exhibited an anti-inflammatory phenotype characterized by the absence of NFkappaB activation and Cox-2 in the presence of a marked heme oxygenase (HO)-1 expression in surviving skin flap tissue. CONCLUSION: Survival of skin flap tissue might be determined by a cytoprotective type of wound macrophage in the presence of an intense epithelium-derived inflammation.