Kurt Q. Lu

Skin-Infiltrating Monocytes/Macrophages Migrate to Draining Lymph Nodes and Produce IL-10 After Contact Sensitizer Exposure to UV-Irradiated Skin

Low-dose UVB exposure induces antigen-specific unresponsiveness to antigen(s) introduced through UV-irradiated skin (tolerance). Analysis of cytokine expression in murine draining lymph nodes (DLNs) revealed that IL-12p40 mRNA and protein expression as well as IL-12p70 protein were upregulated after application of the contact sensitizer 2,4 dinitro-1-fluorobenzene (DNFB) to normal skin. The cellular source of IL-12p40 mRNA was CD11c+ cells. By contrast, following DNFB application to UV-irradiated skin (UV+DNFB), IL-12p40 mRNA was not upregulated, and DLN IL-12p40 and p70 proteins were reduced. UVB irradiation alone did not upregulate IL-10 mRNA, but UV+DNFB upregulated IL-10 mRNA as early as 3-6 hours after DNFB application, immediately preceding a decrease of IL-12p40 mRNA from the level induced by UVB. The infiltration of F4/80+ cells into UV-irradiated skin was followed by a rapid and remarkable increase of F4/80+CD11c(-) cells in DLN 3 hours following DNFB application. FITC/DNFB skin painting and subsequent enzyme-linked immunospot assay demonstrated that flow-sorted FITC+F4/80+CD11c(-) cells from the DLN produce IL-10. Thus, monocytes/macrophages that infiltrated into the skin following UVB exposure migrate to the DLN triggered by contact sensitizers. Production of IL-10 by migrating macrophages, in conjunction with IL-12 inhibition in the DLN, likely reflects a role as mobile suppressive mediators for locally induced UV tolerance.

Suppression of Hyperactive Immune Responses Protects against Nitrogen Mustard Injury

DNA alkylating agents like nitrogen mustard (NM) are easily absorbed through the skin and exposure to such agents manifest not only in direct cellular death but also in triggering inflammation. We show that toxicity resulting from topical mustard exposure is mediated in part by initiating exaggerated host innate immune responses. Using an experimental model of skin exposure to NM we observe activation of inflammatory dermal macrophages that exacerbate local tissue damage in an inducible nitric oxide synthase (iNOS)-dependent manner. Subsequently these activated dermal macrophages reappear in the bone marrow to aid in disruption of hematopoiesis and contribute ultimately to mortality in an experimental mouse model of topical NM exposure. Intervention with a single dose of 25-hydroxyvitamin D3 (25(OH)D) is capable of suppressing macrophage-mediated iNOS production resulting in mitigation of local skin destruction, enhanced tissue repair, protection from marrow depletion, and rescue from severe precipitous wasting. These protective effects are recapitulated experimentally using pharmacological inhibitors of iNOS or by compounds that locally deplete skin macrophages. Taken together, these data highlight a critical unappreciated role of the host innate immune system in exacerbating injury following exposure to NM and support the translation of 25(OH)D in the therapeutic use against these chemical agents.

Hyper-Inflammation and Skin Destruction Mediated by Rosiglitazone Activation of Macrophages in IL-6 Deficiency

Injury initiates recruitment of macrophages to support tissue repair; however, excessive macrophage activity may exacerbate tissue damage causing further destruction and subsequent delay in wound repair. Here we show that the peroxisome proliferation–activated receptor-γ agonist, rosiglitazone (Rosi), a medication recently reintroduced as a drug to treat diabetes and with known anti-inflammatory properties, paradoxically generates pro-inflammatory macrophages. This is observed in both IL-6-deficient mice and control wild-type mice experimentally induced to produce high titers of auto-antibodies against IL-6, mimicking IL-6 deficiency in human diseases. IL-6 deficiency when combined with Rosi-mediated upregulation of suppressor of cytokine signaling 3 leads to an altered ratio of nuclear signal transducer and activator of transcription 3/NF-κB that allows hyper-induction of inducible nitric oxide synthase (iNOS). Macrophages activated in this manner cause de novo tissue destruction, recapitulating human chronic wounds, and can be reversed in vivo by recombinant IL-6, blocking macrophage infiltration, or neutralizing iNOS. This study provides insight into an unanticipated paradoxical role of Rosi in mediating hyper-inflammatory macrophage activation significant for diseases associated with IL-6 deficiency.

Cutaneous Complications in Hematopoietic Cell Transplant Recipients: Impact of Biopsy on Patient Management

The utility of cutaneous biopsies in directing the management of post-hematopoietic cell transplantation (HCT) eruptions remains uncertain. We retrospectively analyzed 439 consecutive HCT procedures for malignant hematologic disorders performed at our institution between January 2005 and December 2012; 192 patients underwent 430 cutaneous biopsies. The clinical and dermatopathologic diagnosis differed in 240 cases (56%). Biopsy results led to a change in therapy in 69 (16%) episodes. Seventeen of 69 management changes occurred in response to a clinical diagnosis of graft-versus-host disease and resulted in augmentation of systemic immunosuppression. The management was modified with similar frequencies with respect to concordance or discordance between the clinical and histopathologic diagnosis (P = .51). We used classification and regression tree (CART) analysis, a decision-modeling technique, to predict the biopsy yield as expressed by impact on clinical management in the allogeneic and autologous setting. The models were cross-validated and then tested against a validation subset, and they maintained a high negative predictive value and high specificity. Although skin biopsies may not be mandatory for either diagnostic or therapeutic reasons, in carefully chosen circumstances, this procedure can yield extremely important data. We believe a prospective study should be undertaken to evaluate current practice data and to validate our decision tree models.

Defining the timing of 25(OH)D rescue following nitrogen mustard exposure

Abstract Objective: Mass exposure to alkylating agents such as nitrogen mustard (NM), whether accidental or intentional as during warfare, are known to cause systemic toxicity and severe blistering from cutaneous exposure. Thus, establishing the timing and appropriate dose of any potential drug designed to reverse or impede these toxicities is critical for wound repair and survival. Our previous data demonstrates that a single intraperitoneal injection of low-dose 25-hydroxyvitamin D3 (25(OH)D) given as early as 1 h following NM exposure is sufficient to rescue mice from pancytopenia and death. However, the duration of time following exposure where intervention is still effective as a countermeasure is unknown. In this study, we sought to assess the maximal time permissible following NM exposure where 25(OH)D still affords protection against NM-induced cutaneous injury. Additionally, we determined if a higher dose of 25(OH)D would be more efficacious at time interval where low dose 25(OH)D is no longer effective. Methods: Low (5 ng) and high (50 ng) doses of 25(OH)D were administered intraperitoneally to mice following exposure to topical NM to assess wound resolution and survival. Mice were imaged and weighed daily to measure wound healing and to monitor systemic toxicity. Results: We demonstrated that 5 ng 25(OH)D administered as early as 1 h and as late as 24 h post-NM exposure is able to achieve 100% recovery in mice. In contrast, intervention at and beyond 48 h of NM exposure failed to achieve full recovery and resulted in ≥60% death between days 6 and 12, demonstrating the critical nature of timely intervention with 25(OH)D at each respective dose. In order to circumvent the observed failure at >48 h exposure, we provided two consecutive doses of 5 ng or 50 ng of 25(OH)D at 48 h and 72 h post-NM exposure. Repeat dosing with 25(OH)D at 48 h and beyond led to marked improvement of lesion size with 75% recovery from mortality. Conclusions: The opportunity to use 25(OH)D as a medical countermeasure for NM-induced toxicity has a finite of window for intervention. However, modifications such as repeat dosing can be an effective strategy to extend the intervention potential of 25(OH)D.

Evaluation of O6-Benzylguanine−Potentiated Topical Carmustine for Mycosis Fungoides: A Phase 1-2 Clinical Trial

Importance In a phase 1 trial, single-dose O6-benzylguanine with topical carmustine for patients with early stage (stage IA through stage IIA) cutaneous T-cell lymphoma, mycosis fungoides (MF) type, resulted in clinical responses proportional to inhibition of O6-alkylguanine–DNA alkyltransferase activity, but a maximum tolerated dose (MTD) was not reached. Objective To determine whether dose escalation of carmustine in combination with dual-dose O6-benzylguanine to prolong alkyltransferase inhibition could reach an MTD. Design, Setting, and Participants A single-arm, phase 1-2 clinical trial conducted at a university teaching hospital enrolled 17 adults with stage IA through stage IIA cutaneous T-cell lymphoma, MF type, to evaluate treatment using topical carmustine plus 2 subsequent daily doses of intravenous O6-benzylguanine, administered every 2 weeks for up to 24 weeks (12 cycles). All patients who received treatment were included in an intent-to-treat analysis of the response rate. The study was conducted from February 17, 2010, to April 8, 2014. Data analysis was performed from May 1, 2014, to December 1, 2015. Interventions Topical carmustine and intravenous O6-benzylguanine. Main Outcomes and Measures Clinical disease response was assessed by the Severity-Weighted Assessment Tool (score range, 0-400; higher score indicates worse disease). Safety data were acquired by review of adverse events at study visits. Results Of the 17 patients enrolled, 12 (71%) were men; mean (SD) age was 45.2 (14.6) years. There were 7 complete responses and 8 partial responses to combination carmustine and O6-benzylguanine treatment. The overall clinical response rate was 88%, with a mean (SD) duration of complete response of 14.43 (6.6) months. The MTD was 20 mg of carmustine applied once in combination with 2 daily doses of 120 mg/m2 of O6-benzylguanine. Most adverse events (112 [67%]) were grade I. Of 15 patients with dermatitis, 5 individuals (33%) demonstrated grade II dermatitis that was unresponsive to topical corticosteroid therapy. The dermatitis was characterized by high levels of macrophage activation, and clearance was associated with vitamin D3 administration. Conclusions and Relevance Compared with single-dose O6-benzylguanine and carmustine, dual-dose O6-benzylguanine resulted in higher overall response rates and reduced total carmustine doses but was associated with more cutaneous adverse events. The MTD for dual-dose O6-benzylguanine plus carmustine was also ascertained. Trial Registration clinicaltrials.gov Identifier: NCT00961220

Vitamin D as a Therapeutic Option for Sunburn: Clinical and Biologic Implications

Vitamin D3 (cholecalciferol) is a fat-soluble steroid hormone obtained from food sources and produced locally in the skin from the ultraviolet radiation (UVR)-dependent conversion of cholesterol precursors to the inactive form of vitamin D3 (Bikle, 2011). In addition to its classically described functions related to the regulation of calcium homeostasis and bone metabolism, vitamin D3 has numerous nonclassical effects, including the ability to modulate immune responses (Mora et al., 2008; Wobke et al., 2014). Specifically, vitamin D3 induces monocyte to macrophage differentiation, enhances antimicrobial activity, stimulates autophagy, and suppresses the production of proinflammatory cytokines (Liu et al., 2006; Fabri et al., 2011; Di Rosa et al., 2012; Zhang et al., 2014). However, it was previously unknown whether vitamin D3 was capable of modulating acute inflammation in humans. In a randomized, double-blinded, placebo-controlled pilot study, our group recently demonstrated that vitamin D3 reduces skin inflammation when administered to humans 1 h after sunburn (Scott et al., 2017). This commentary will attempt to outline the major clinical and biologic implications of using vitamin D as an anti-inflammatory therapeutic agent.

Early indicators of survival following exposure to mustard gas: Protective role of 25(OH)D

The use of sulfur mustard (SM) as a chemical weapon for warfare has once again assumed center stage, endangering civilian and the military safety. SM causes rapid local skin vesication and late-onset systemic toxicity. Most studies on SM rely on obtaining tissue and blood for characterizing burn pathogenesis and assessment of systemic pathology, respectively. However the present study focuses on developing a non-invasive method to predict mortality from high dose skin SM exposure. We demonstrate that exposure to SM leads to a dose dependent increase in wound area size on the dorsal surface of mice that is accompanied by a progressive loss in body weight loss, blood cytopenia, bone marrow destruction, and death. Thus our model utilizes local skin destruction and systemic outcome measures as variables to predict mortality in a novel skin-based model of tissue injury. Based on our recent work using vitamin D (25(OH)D) as an intervention to treat toxicity from SM-related compounds, we explored the use of 25 (OH)D in mitigating the toxic effects of SM. Here we show that 25(OH)D offers protection against SM and is the first known demonstration of an intervention that prevents SM-induced mortality. Furthermore, 25 (OH)D represents a safe, novel, and readily translatable potential countermeasure following mass toxic exposure.

The Virtues of Oxygenation: Low Tissue Oxygen Adversely Affects the Killing of Leishmania

Hypoxia contributes to the persistence of infections through altered immune responses. Studies examining skin O2 changes at the site of a lesion are limited. The prevailing methods require the use of electrochemical O2 sensors or radiolabeled electrodes that utilize O2 and may interfere with the precision at low O2 levels. In this issue, Mahnke et al. (2014) demonstrate, using a novel fluorescence-based imaging technology, that low oxygen tension (pO2) impairs NO-mediated anti-leishmanial immunity, leading to increased parasite burden. Replenishing tissue oxygen profoundly enhanced NO-mediated leishmanial killing, underscoring the need to accurately assess oxygenation in infected tissues as a novel strategy to challenge intracellular infection. The technology presented here may have clinical-translational potential in noninvasively assessing disease burden and response to treatment.

Seemed Like Cellulitis: Intravascular Large B-Cell Lymphoma

PRESENTATION Further investigation was spurred after an ostensible case of bilateral cellulitis did not improve with a 2-week course of intravenous antibiotics. The patient, a 50-year-old white woman, reported a 6-month history of leg swelling and a 2month history of painful nodules on the thighs and buttocks. She also described subjective fevers, chills, night sweats, malaise, and an unintentional 20-pound weight loss over the previous 6 months.