Rosacea is a chronic inflammatory skin disease whose pathophysiological mechanism is still unclear. However, it is known that mast cell (MC) numbers are increased in the dermis of rosacea patients. ...MC proteases not only recruit other immune cells, which amplify the inflammatory response, but also cause vasodilation and angiogenesis. MCs are also one of the primary sources of cathelicidin LL-37 (Cath LL-37), an antimicrobial peptide that has been shown to be an enabler of rosacea pathogenesis. Here, we demonstrate that MCs are key mediators of cathelicidin-initiated skin inflammation. After Cath LL-37 injection into the dermis, MC-deficient B6.Cg−KitW−sh/HNihrJaeBsmJ (KitW−sh) mice did not develop rosacea-like features. Conversely, chymase (P<0.001), tryptase, and Mmp9 (P<0.01) mRNA levels were significantly higher in C57BL/6 wild-type (WT) mice. Treating WT mice with an MC stabilizer significantly decreased the expressions of Mmp9 and Cxcl2 (P<0.01). Our data were confirmed on erythematotelangiectatic rosacea subjects who showed a decrease in matrix metalloproteinase activity (P<0.05), after 8 weeks of topical cromolyn treatment. We conclude that MCs have a central role in the development of inflammation subsequent to Cath LL-37 activation and that downregulation of activated MCs may be a therapy for rosacea treatment.
Inflammation under sterile conditions is not well understood despite its importance in trauma and autoimmune disease. To investigate this process we established mouse models of sterile injury and ...explored the role of hyaluronan in mediating inflammation following injury. The response of cultured monocytes to hyaluronan was different than the response to lipopolysaccharide (LPS) despite both being dependent on Toll-like receptor 4 (TLR4). Cultured cells exposed to hyaluronan showed a pattern of gene induction that mimics the response seen in mouse skin after sterile injury with an increase in molecules such as transforming growth factor-β2 and matrix metalloproteinase-13. These factors were not induced by LPS despite the mutual dependence of both hyaluronan and LPS on TLR4. Explanation for the unique response to hyaluronan was provided by observations that a lack of TLR4 or CD44 in mice diminished the response to sterile injury, and together with MD-2, was required for responsiveness to hyaluronan in vitro. Thus, a unique complex of TLR4, MD-2, and CD44 recognizes hyaluronan. Immunoprecipitation experiments confirmed the physical association of TLR4 and CD44. Taken together, our results define a previously unknown mechanism for initiation of sterile inflammation that involves recognition of released hyaluronan fragments as an endogenous signal of tissue injury.
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•Chronic aerobic exercise (AE) downregulated the HPA axis by compensatory and/or supercompensatory mechanisms.•Treadmill-running regular AE, when performed in accordance with its ...basic principles produced no chronic stress.•Lifelong AE reduced the stress response and positively impacted on the stress resilience.•Aging had an anxiogenic effect that was mitigated by AE.•Aging reduced the adrenal sensitivity and altered the neuroendocrine feedback.
The aim of this study was to analyze the effects of lifelong aerobic exercise (AE) on the adaptive response of the stress system in rats. It is well known that hypothalamic–pituitary–adrenal axis (HPA) activity differs when triggered by voluntary or forced exercise models. Male Wistar rats belonging to exercise (E) or control (C) groups were subjected to chronic AE, and two cutoff points were established at 8 (middle age) and 18 months (old age). Behavioral, biochemical and histopathological studies were performed on the main components/targets of the stress system.
AE increased adrenal sensitivity (AS), brain corticosterone (CORT) and corticotropin-releasing factor (CRF), but had no effect on the thymus, adrenal glands (AGs) weight or plasma CORT. In addition, AE exerted no effect on the sympathetic tone, but significantly reduced anxiety-related behavior and emotionality. Aging decreased AS and deregulated neuroendocrine feedback, leading to an anxiogenic state which was mitigated by AE. Histopathological and morphometric analysis of AGs showed no alterations in middle-aged rats but adrenal vacuolization in approximately 20% old rats. In conclusion, lifelong AE did not produce adverse effects related to a chronic stress state. On the contrary, while AE upregulated some components of the HPA axis, it generated an adaptive response to cumulative changes, possibly through different compensatory and/or super compensatory mechanisms, modulated by age. The long-term practice of AE had a strong positive impact on stress resilience so that it could be recommended as a complementary therapy in stress and depression disease.
Innate immune defense against microbial pathogens occurs by physical barriers, by recruitment of cells such as neutrophils, NK cells, and macrophages, and by secretion of molecules with antimicrobial ...activity. Such molecules are produced by various epithelia including skin. The importance of antimicrobial peptides has been shown in cathelicidin-deficient mice, which have increased susceptibility to skin infection by Streptococcus. Although keratinocytes increase cathelicidin expression upon injury, their role relative to neutrophil cathelicidin and their sites of peptide storage and activation have not been elucidated. Herein, it is reported that cathelicidin predominantly resides in granules of the superficial epidermis and partially localizes in lamellar bodies as determined by immunogold electron microscopy and immunoblot of lamellar bodies isolated from mice. In cultured keratinocytes, cathelicidin displays a granular distribution and partially localizes within the Golgi apparatus. Cathelicidin processing can be observed by western blot analysis in keratinocyte extracts but not in conditioned media. Further, fluorescent bacteria colocalize with cathelicidin in granules both intracellularly and at the cell surface. These observations illustrate the immune defense potential of keratinocytes acting directly through storage and processing of antimicrobial peptides.
Many biochemical processes proceed through the formation of functionally significant intermediates. Although the identification and characterization of such species can provide vital clues about the ...mechanisms of the reactions involved, it is challenging to obtain information of this type in cases where the intermediates are transient or present only at low population. One important example of such a situation involves the folding behaviour of small proteins that represents a model for the acquisition of functional structure in biology. Here we use relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy to identify, for two mutational variants of one such protein, the SH3 domain from Fyn tyrosine kinase, a low-population folding intermediate in equilibrium with its unfolded and fully folded states. By performing the NMR experiments at different temperatures, this approach has enabled characterization of the kinetics and energetics of the folding process as well as providing structures of the intermediates. A general strategy emerges for an experimental determination of the energy landscape of a protein by applying this methodology to a series of mutants whose intermediates have differing degrees of native-like structure.
Cathelicidins (caths) are peptides that are expressed at high levels in neutrophils and some epithelia and can act as natural antibiotics by directly killing a wide range of microorganisms. We ...hypothesized that caths are expressed in mast cells (MCs), because these cells have been previously associated with inherent antimicrobial activity. Cultured murine MCs contained abundant amounts of cathelin-related antimicrobial peptide (AMP), the murine cath, and this expression was inducible by LPS or lipoteichoic acid. Human skin MCs also expressed cath as detected by immunohistochemical analysis for the human cath LL-37. The functional significance of this expression was shown by comparing MCs cultured from normal mice to MCs from littermates deficient in the cathelin-related AMP gene (Cnlp(-)). MCs derived from Cnlp(-/-) animals had a 50% reduction in their ability to kill group A STREPTOCOCCUS: These MCs expressed equivalent amounts of mRNA for murine beta-defensin-4, a beta-defensin AMP. Thus, different antimicrobials can be identified in MCs, and the presence of cath is necessary for efficient bacterial killing. These observations suggest that the presence of cath is vital to the ability of mammalian MCs to participate in antimicrobial defense.
Production of antimicrobial peptides by epithelia is an essential defense against infectious pathogens. In this study we evaluated whether the commensal microorganism Staphylococcus epidermidis may ...enhance production of antimicrobial peptides by keratinocytes and thus augment skin defense against infection. Exposure of cultured undifferentiated human keratinocytes to a sterile nontoxic small molecule of <10kDa from S. epidermidis conditioned culture medium (SECM), but not similar preparations from other bacteria, enhanced human β-defensin 2 (hBD2) and hBD3 mRNA expression and increased the capacity of cell lysates to inhibit the growth of group A Streptococcus (GAS) and S. aureus. Partial gene silencing of hBD3 inhibited this antimicrobial action. This effect was relevant in vivo as administration of SECM to mice decreased susceptibility to infection by GAS. Toll-like receptor 2 (TLR2) was important to this process as a TLR2-neutralizing antibody blocked induction of hBDs 2 and 3, and Tlr2-deficient mice did not show induction of mBD4. Taken together, these findings reveal a potential use for normal commensal bacterium S. epidermidis to activate TLR2 signaling and induce antimicrobial peptide expression, thus enabling the skin to mount an enhanced response to pathogens.
The normal microflora of the skin includes staphylococcal species that will induce inflammation when present below the dermis but are tolerated on the epidermal surface without initiating ...inflammation. Here we reveal a previously unknown mechanism by which a product of staphylococci inhibits skin inflammation. This inhibition is mediated by staphylococcal lipoteichoic acid (LTA) and acts selectively on keratinocytes triggered through Toll-like receptor 3(TLR3). We show that TLR3 activation is required for normal inflammation after injury and that keratinocytes require TLR3 to respond to RNA from damaged cells with the release of inflammatory cytokines. Staphylococcal LTA inhibits both inflammatory cytokine release from keratinocytes and inflammation triggered by injury through a TLR2-dependent mechanism. To our knowledge, these findings show for the first time that the skin epithelium requires TLR3 for normal inflammation after wounding and that the microflora can modulate specific cutaneous inflammatory responses.
Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to ...peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.
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