Zhenping Wang

Skin Mast Cells Protect Mice against Vaccinia Virus by Triggering Mast Cell Receptor S1PR2 and Releasing Antimicrobial Peptides

Mast cells (MCs) are well-known effectors of allergic reactions and are considered sentinels in the skin and mucosa. In addition, through their production of cathelicidin, MCs have the capacity to oppose invading pathogens. We therefore hypothesized that MCs could act as sentinels in the skin against viral infections using antimicrobial peptides. In this study, we demonstrate that MCs react to vaccinia virus (VV) and degranulate using a membrane-activated pathway that leads to antimicrobial peptide discharge and virus inactivation. This finding was supported using a mouse model of viral infection. MC-deficient (Kitwsh−/−) mice were more susceptible to skin VV infection than the wild type animals, whereas Kitwsh−/− mice reconstituted with MCs in the skin showed a normal response to VV. Using MCs derived from mice deficient in cathelicidin antimicrobial peptide, we showed that antimicrobial peptides are one important antiviral granule component in in vivo skin infections. In conclusion, we demonstrate that MC presence protects mice from VV skin infection, MC degranulation is required for protecting mice from VV, neutralizing Ab to the L1 fusion entry protein of VV inhibits degranulation apparently by preventing S1PR2 activation by viral membrane lipids, and antimicrobial peptide release from MC granules is necessary to inactivate VV infectivity.

Mast Cells Are Key Mediators of Cathelicidin-Initiated Skin Inflammation in Rosacea

Rosacea is a chronic inflammatory skin disease whose pathophysiological mechanism is still unclear. However, it is known that mast cell (MC) numbers is 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. Following 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 a MC stabilizer significantly decreased the expressions of Mmp9 and Cxcl2 (p<0.01). Our data was confirmed on Erythematotelangiectatic rosacea subjects that showed a decrease in MMP activity (p<0.05), after eight weeks of topical cromolyn treatment. We conclude that MCs play a central role in the development of inflammation subsequent to Cath LL-37 activation and that down regulation of activated MCs may be a therapy for rosacea treatment.

Commensal Bacteria Lipoteichoic Acid Increases Skin Mast Cell Antimicrobial Activity against Vaccinia Viruses

Mast cells (MCs) are considered sentinels in the skin and mucosa. Their ability to release antimicrobial peptides, such as cathelicidin, protects against bacterial infections when the epithelial barrier is breached. We recently described that MCs defend against bacterial and viral infections through the release of cathelicidin during degranulation. In this study, we hypothesize that cathelicidin expression is induced in MCs by the activation of TLR2 from bacterial products (lipoteichoic acid) produced by commensal bacteria at the epithelial surface. Our research shows that signaling through TLR2 increases the production and expression of cathelicidin in mast cells, thereby enhancing their capacity to fight vaccinia virus. MCs deficient in cathelicidin were less efficient in killing vaccinia virus after lipoteichoic acid stimulation than wild-type cells. Moreover, the activation of TLR2 increases the MC recruitment at the skin barrier interface. Taken together, our findings reveal that the expression and control of antimicrobial peptides and TLR signaling on MCs are key in fighting viral infection. Our findings also provide new insights into the pathogenesis of skin infections and suggest potential roles for MCs and TLR2 ligands in antiviral therapy.

Skin microbiome promotes mast cell maturation by triggering stem cell factor production in keratinocytes.

Background: Mast cell (MC) progenitors leave the bone marrow, enter the circulation, and settle in the skin and other tissues. Their maturation in tissues is influenced by the surrounding microenvironment. Objective: We tested the hypothesis that environmental factors play a role in MC maturation in the skin. Methods: MCs were numerically, phenotypically, and functionally compared between germ‐free (GF), specific pathogen‐free, and GF mice reconstituted with microbiota. The maturity of MCs was then correlated with skin levels of stem cell factor (SCF), a critical MC differentiation factor, and lipoteichoic acid (LTA), a Toll‐like receptor 2 ligand. MCs were also evaluated in mice with keratinocyte‐specific deletion of Scf. Results: We found that GF mice express abnormally low amounts of SCF, a critical MC differentiation factor, and contain MCs that are largely undifferentiated. Reconstituting the GF microbiota reverted this MC phenotype to normal, indicating that the phenotype is related to ongoing interactions of the microbiota and skin. Consistent with the immaturity of GF MCs, degranulation‐provoking compound 48/80 induced less edema in the skin of GF mice than in conventional mice. Our results show that the skin microbiome drives SCF production in keratinocytes, which triggers the differentiation of dermal MCs. Because the skin microbiome is a rich source of LTA, a Toll‐like receptor 2 ligand, we mimicked the GF microbiomes effect on MCs by applying LTA to the skin of GF mice. We also demonstrated that MC migration within the skin depends exclusively on keratinocyte‐produced SCF. Conclusion: This study has revealed a novel mechanism by which the skin microbiota signals the recruitment and maturation of MCs within the dermis through SCF production by LTA‐stimulated keratinocytes. Graphical abstract Figure. No Caption available.

Vaccinia virus binds to the scavenger receptor MARCO on the surface of keratinocytes

Patients with altered skin immunity, such as individuals with atopic dermatitis (AD), can have a life-threatening disruption of the epidermis known as eczema vaccinatum (EV) after vaccinia virus (VV) infection of the skin. Here, we sought to better understand the mechanism(s) by which VV associates with keratinocytes. The class A scavenger receptor known as MARCO (macrophage receptor with collagenous structure) is expressed on human and mouse keratinocytes and found to be abundantly expressed in the skin of patients with AD. VV bound directly to MARCO, and overexpression of MARCO increased susceptibility to VV infection. Furthermore, ligands with affinity for MARCO, or excess soluble MARCO, competitively inhibited VV infection. These findings indicate that MARCO promotes VV infection and highlights potential new therapeutic strategies for prevention of VV infection in the skin.

Interaction of primary mast cells with Borrelia burgdorferi ( sensu stricto ): role in transmission and dissemination in C57BL/6 mice

BackgroundBorrelia burgdorferi (sensulato), the causative agent of Lyme borreliosis is a bacterium transmitted by hard ticks, Ixodes spp. Bacteria are injected into the host skin during the tick blood meal with tick saliva. There, Borrelia and saliva interact together with skin cells such as keratinocytes, fibroblasts, mast cells and other specific immune cells before disseminating to target organs.MethodsTo study the role of mast cells in the transmission of Lyme borreliosis, we isolated mouse primary mast cells from bone marrow and incubated them in the presence of Borrelia burgdorferi (sensu stricto) and tick salivary gland extract. We further analyzed their potential role in vivo, in a mouse model of deficient in mast cells (Kitwsh−/− mice).ResultsTo our knowledge, we report here for the first time the bacteria ability to induce the inflammatory response of mouse primary mast cells. We show that OspC, a major surface lipoprotein involved in the early transmission of Borrelia, induces the degranulation of primary mast cells but has a limited effect on the overall inflammatory response of these cells. In contrast, whole bacteria have an opposite effect. We also show that mast cell activation is significantly inhibited by tick salivary gland extract. Finally, we demonstrate that mast cells are likely not the only host cells involved in the early transmission and dissemination of Borrelia since the use of mast cell deficient Kitwsh−/− mice shows a limited impact on these two processes in the context of this mouse genetic background.ConclusionsThe absence of mast cells did not change the replication rate of Borrelia in the skin. However, in the absence of mast cells, Borrelia dissemination to the joints was faster. Mast cells do not control skin bacterial proliferation during primary infection and the establishment of the primary infection, as shown in the C57BL/6 mouse model studied. Nevertheless, the Borrelia induced cytotokine modulation on mast cells might be involved in long term and/or repeated infections and protect from Lyme borreliosis due to the development of a hypersensitivity to tick saliva.

A Study and Review of Effects of Botulinum Toxins on Mast Cell Dependent and Independent Pruritus

Pruriceptive itch originates following activation of peripheral sensory nerve terminals when pruritogens come in contact with the skin. The ability of botulinum neurotoxins (BoNTs) to attenuate transmitter release from afferent terminals provides a rationale for studying its effect on pruritus. This study investigated the effects of BoNT/A1 and BoNT/B1 on mast cell dependent (Compound 48/80:48/80) and independent (Chloroquine:CQ) scratching. C57Bl/6 male mice received intradermal injection of 1.5 U of BoNT/A1, BoNT/B1 or saline 2, 7, 14 and 21 days prior to ipsilateral 48/80 or CQ at the nape of the neck. Ipsilateral hind paw scratching was determined using an automated recording device. The effect of BoNTs on 48/80 mediated mast cell degranulation was analyzed in human and murine mast cells and the presence of SNAREs was determined using qPCR, immunostaining and Western blot. Pre-treatment with BoNT/A1 and BoNT/B1 reduced 48/80 and CQ induced scratching behavior starting on day 2 with reversal by day 21. Both serotypes inhibited 48/80 induced mast cell degranulation. qPCR and immunostaining detected SNAP-25 mRNA and protein, respectively, in mast cells, however, Western blots did not. This study demonstrates the long-lasting anti-pruritic effects of two BoNT serotypes, in a murine pruritus model using two different mechanistically driven pruritogens. These data also indicate that BoNTs may have a direct effect upon mast cell degranulation.

Effects of opioid and nonopioid analgesics on canine wheal formation and cultured human mast cell degranulation

ABSTRACT Mast cell (MC) degranulation has been implicated in the side effect profile of a variety of clinically useful agents. Thus, after intrathecal delivery, formation of space‐occupying, meningeally‐derived masses may be related to local MC degranulation. We systematically characterized degranulating effects of opioid and nonopioid analgesics on cutaneous flares in the dog and in primary human MC (hMC) cultures. Methods: Dogs were anesthetized with IV propofol and received intradermal (ID) injections (50 &mgr;L). Flare diameters were measured at 30 min. Drugs showing flare responses were tested after intramuscular (IM) cromolyn (10 mg/kg), a MC stabilizer. Human primary MCs (human cord blood CD34+/CD45+ cells) were employed and &bgr;‐hexosaminidase in cell‐free supernatants were measured to assess degranulation. Results: A significant skin flare for several classes of agents was observed including opioids, ziconotide, ketamine, ST‐91, neostigmine, adenosine, bupivacaine, lidocaine, MK‐801 and 48/80. Tizanidine, fentanyl, alfentanil, gabapentin and baclofen produced no flare. Flare produced by all ID agents, except adenosine, bupivacaine and lidocaine, was reduced by cromolyn. Naloxone had no effect upon opiate or 48/80 evoked flares. In hMC studies, 48/80 resulted in a concentration‐dependent release of &bgr;‐hexosaminidase. The rank order of drug‐induced hMC &bgr;‐hexosaminidase release was similar to that for flares. Conclusions: A variety of therapeutically useful drugs degranulate MCs. This action may account for side effects such as the intrathecal granuloma resulting from spinally‐delivered opioids. This degranulating effect may be useful in predicting potential intrathecal toxicity in the development of novel agents. HighlightsDrugs are delivered spinally for pain or spasticity.Several agents, such as morphine, can produce intrathecal masses.These masses result from meningeal mast cell degranulation.Molecules were assessed for degranulation in human mast cells and dog skin.Covariance between degranulation potency and mass formation was noted.

Skin commensal bacteria Staphylococcus epidermidis promote survival of melanocytes bearing UVB-induced DNA damage, while bacteria Propionibacterium acnes inhibit survival of melanocytes by increasing apoptosis

Skin commensal bacteria have been described to help orchestrate skin homeostasis, signaling through innate immunity pathways. This study for the first time aimed at studying the relationship between skin commensals and melanocytes after UVB exposure.

Mast Cells: Sentinels of Innate Skin Immunity

Since its discovery over a century ago, the mast cell has been considered an important effector cell of the innate immune system. From mediating various allergic responses to playing a direct role in eliminating pathogens from the skin, the mast cell carries out its effects through a highly coordinated process of degranulation. Following stimulation by pathogens or other host cells, the mast cell is able to release chemical mediators from its intracellular stores, which include pro-inflammatory cytokines, peptidases, and antimicrobial peptides. These mediators serve to alter the inflammatory environment and allow for the recruitment of other immune cells; however, overactivity of the mast cell response has been shown to lead to a variety of disease processes. As the main drivers of both type 1 hypersensitivity and inflammatory disorders, like rosacea, the mast cell has been studied as a target for many disease-altering therapies. Continued research in the field of mast cell biology has the potential to further unveil the coordinated workings of the innate immune system.