eNAMPT/TLR4 inflammatory cascade activation is a key contributor to SLE Lung vasculitis and alveolar hemorrhage.

Rationale: Effective therapies to reduce the severity and high mortality of pulmonary vasculitis and diffuse alveolar hemorrhage (DAH) in patients with systemic lupus erythematosus (SLE) is a serious unmet need. We explored whether biologic neutralization of eNAMPT (extracellular nicotinamide phosphoribosyl-transferase), a novel DAMP and Toll-like receptor 4 ligand, represents a viable therapeutic strategy in lupus vasculitis. Methods: Serum was collected from SLE subjects (n = 37) for eNAMPT protein measurements. In the preclinical pristane-induced murine model of lung vasculitis/hemorrhage, C57BL/6 J mice (n = 5-10/group) were treated with PBS, IgG (1 mg/kg), or the eNAMPT-neutralizing ALT-100 mAb (1 mg/kg, IP or subcutaneously (SQ). Lung injury evaluation (Day 10) included histology/immuno-histochemistry, BAL protein/cellularity, tissue biochemistry, RNA sequencing, and plasma biomarker assessment. Results: SLE subjects showed highly significant increases in blood NAMPT mRNA expression and eNAMPT protein levels compared to healthy controls. Preclinical pristane-exposed mice studies showed significantly increased NAMPT lung tissue expression and increased plasma eNAMPT levels accompanied by marked increases in alveolar hemorrhage and lung inflammation (BAL protein, PMNs, activated monocytes). In contrast, ALT-100 mAb-treated mice showed significant attenuation of inflammatory lung injury, alveolar hemorrhage, BAL protein, tissue leukocytes, and plasma inflammatory cytokines (eNAMPT, IL-6, IL-8). Lung RNA sequencing showed pristane-induced activation of inflammatory genes/pathways including NFkB, cytokine/chemokine, IL-1ß, and MMP signaling pathways, each rectified in ALT-100 mAb-treated mice. Conclusions: These findings highlight the role of eNAMPT/TLR4-mediated inflammatory signaling in the pathobiology of SLE pulmonary vasculitis and alveolar hemorrhage. Biologic neutralization of this novel DAMP appears to serve as a viable strategy to reduce the severity of SLE lung vasculitis.

Oxidative DNA Damage Accelerates Skin Inflammation in Pristane-Induced Lupus Model.

Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disease in which type I interferons (IFN) play a key role. The IFN response can be triggered when oxidized DNA engages the cytosolic DNA sensing platform cGAS-STING, but the repair mechanisms that modulate this process and govern disease progression are unclear. To gain insight into this biology, we interrogated the role of oxyguanine glycosylase 1 (OGG1), which repairs oxidized guanine 8-Oxo-2'-deoxyguanosine (8-OH-dG), in the pristane-induced mouse model of SLE. Ogg1-/- mice showed increased influx of Ly6Chi monocytes into the peritoneal cavity and enhanced IFN-driven gene expression in response to short-term exposure to pristane. Loss of Ogg1 was associated with increased auto-antibodies (anti-dsDNA and anti-RNP), higher total IgG, and expression of interferon stimulated genes (ISG) to longer exposure to pristane, accompanied by aggravated skin pathology such as hair loss, thicker epidermis, and increased deposition of IgG in skin lesions. Supporting a role for type I IFNs in this model, skin lesions of Ogg1-/- mice had significantly higher expression of type I IFN genes (Isg15, Irf9, and Ifnb). In keeping with loss of Ogg1 resulting in dysregulated IFN responses, enhanced basal and cGAMP-dependent Ifnb expression was observed in BMDMs from Ogg1-/- mice. Use of the STING inhibitor, H151, reduced both basal and cGAMP-driven increases, indicating that OGG1 regulates Ifnb expression through the cGAS-STING pathway. Finally, in support for a role for OGG1 in the pathology of cutaneous disease, reduced OGG1 expression in monocytes associated with skin involvement in SLE patients and the expression of OGG1 was significantly lower in lesional skin compared with non-lesional skin in patients with Discoid Lupus. Taken together, these data support an important role for OGG1 in protecting against IFN production and SLE skin disease.

Evaluation of the Effectiveness and Tolerability of Mycophenolate Mofetil and Mycophenolic Acid for the Treatment of Morphea.

Importance: First-line systemic therapy for morphea includes methotrexate with or without systemic corticosteroids. When this regimen is ineffective, not tolerated, or contraindicated, a trial of mycophenolate mofetil (MMF) or mycophenolic acid (MPA)-referred to herein as mycophenolate-is recommended; however, evidence to support this recommendation remains weak. Objective: To evaluate the effectiveness and tolerability of mycophenolate for the treatment of morphea. Design, Setting, and Participants: A retrospective cohort study was conducted from January 1, 1999, to December 31, 2018, among 77 patients with morphea from 8 institutions who were treated with mycophenolate. Main Outcomes and Measures: The primary outcome was morphea disease activity, severity, and response at 0, 3 to 6, and 9 to 12 months of mycophenolate treatment. A secondary outcome was whether mycophenolate was a well-tolerated treatment of morphea. Results: There were 61 female patients (79%) and 16 male patients (21%) in the study, with a median age at disease onset of 36 years (interquartile range, 16-53 years) and median diagnostic delay of 8 months (interquartile range, 4-14 months). Generalized morphea (37 [48%]), pansclerotic morphea (12 [16%]), and linear morphea of the trunk and/or extremities (9 [12%]) were the most common subtypes of morphea identified. Forty-one patients (53%) had an associated functional impairment, and 49 patients (64%) had severe disease. Twelve patients received initial treatment with mycophenolate as monotherapy or combination therapy and 65 patients received mycophenolate after prior treatment was ineffective (50 of 65 [77%]) or poorly tolerated (21 of 65 [32%]). Treatments prior to mycophenolate included methotrexate (48 of 65 [74%]), systemic corticosteroids (42 of 65 [65%]), hydroxychloroquine (20 of 65 [31%]), and/or phototherapy (14 of 65 [22%]). After 3 to 6 months of mycophenolate treatment, 66 of 73 patients had stable (n = 22) or improved (n = 44) disease. After 9 to 12 months of treatment, 47 of 54 patients had stable (n = 14) or improved (n = 33) disease. Twenty-seven patients (35%) achieved disease remission at completion of the study. Treatments received in conjunction with mycophenolate were frequent. Mycophenolate was well tolerated. Gastrointestinal adverse effects were the most common (24 [31%]); cytopenia (3 [4%]) and infection (2 [3%]) occurred less frequently. Conclusions and Relevance: This study suggests that mycophenolate is a well-tolerated and beneficial treatment of recalcitrant, severe morphea.

Flotillin and AP2A1/2 Promote IGF-1 Receptor Association with Clathrin and Internalization in Primary Human Keratinocytes.

IGF-1 receptor (IGF1R) signaling promotes keratinocyte proliferation, migration, and survival. However, the mechanism of IGF1R endocytosis in normal keratinocytes remains unclear. Confocal, super resolution structured illumination microscopy, total internal reflection fluorescence microscopy, and coimmunoprecipitation studies reveal that IGF1R associates with flotillin-1 (Flot-1), which currently has no known role in normal receptor tyrosine kinase endocytosis, under basal conditions in monolayer keratinocyte cultures. Ligand stimulation of IGF1R promotes its clathrin-dependent endocytosis, mediated by two distinct adaptors, Flot-1 in noncaveolar lipid rafts and the AP2A1/2 complex in clathrin vesicles. Concurrent, but not individual, short hairpin RNA knockdown of FLOT1/2 and AP2A1/2 reduced IGF1R association with clathrin, internalization, and pathway activation by more than 50% (of phosphorylated IGF1R, phosphorylated protein kinase B, and phosphorylated MAPK kinase), suggesting the complementarity of these two adaptor-specific pathways. The Flot-1 pathway is more responsive to low IGF-1 concentrations, whereas the AP2A1/2 pathway predominates at higher IGF-1 concentrations. Selective association of IGF1R-Flot-1-clathrin with Rab4, but IGF1R-AP2A1/2-clathrin with Rab11, implicates Flot-1 as the adaptor for faster recycling and AP2A1/2 as the adaptor for slower IGF1R recycling. These dual pathways, particularly flotillin-dependent, clathrin-mediated endocytosis, provide a new avenue for drug targeting in disorders with aberrant regulation of IGF1R signaling.

Targeting the IL-17 Receptor Using Liposomal Spherical Nucleic Acids as Topical Therapy for Psoriasis.

The activation of T helper 17 signaling plays a critical role in psoriasis pathogenesis, and systemically-administered IL-17 inhibitors are highly effective therapy for moderate-to-severe disease. We generated topically-delivered gene-regulating nanoconstructs, comprised of spherically-arrayed antisense DNA (liposomal spherical nucleic acids [L-SNAs]), which are able to penetrate human skin to knock down cutaneous gene targets. Topically-applied L-SNAs targeting the gene encoding the mouse IL-17A receptor (Il17ra) reversed the development of psoriasis clinically, histologically, and transcriptionally in imiquimod-treated psoriasis-like mouse skin. Il17ra L-SNAs reduced the modified PASI by 74% versus controls and decreased epidermal thickness by 56%. Il17ra L-SNA reduced Il17ra protein expression by 75% and significantly decreased the mRNA expression of psoriasis markers, including Defb4, Il17c, S100a7, Pi3, Krt16, and Tnfa versus scrambled spherical nucleic acid (Scr SNA) controls. A human IL17RA L-SNA penetrates 3-dimensional cultures and normal human explants to knock down IL17RA mRNA by 63% and 66%, respectively. After topical application to psoriatic 3-dimensional rafts, anti-human IL17RA L-SNAs reduced the expression of IL17RA (by 72%) and the IL-17-induced genes IL17C (by 85%), DEFB4 (by 83%), TNFA (by 77%), and PI3 (by 65%) versus scrambled L-SNA and vehicle controls (all P < 0.001). Taken together, these data suggest that targeted suppression of IL17RA is a promising new topical treatment strategy for psoriasis.

Health care barriers and quality of life in central centrifugal cicatricial alopecia patients.

Central centrifugal cicatricial alopecia (CCCA) is known for delayed presentation and treatment. Identifying care barriers may shorten delays to treatment and improve outcomes. Understanding how CCCA impacts quality of life (QOL) also is important. Objectives of this pilot study were to elucidate the diagnostic experience for CCCA patients and understand how CCCA affects QOL. Patients completed a novel, 53-item, cross-sectional survey (CCCA Barriers to Care and Quality of Life Survey) that was developed by study investigators. The survey was completed in person or by telephone for English-speaking, adult, black female patients diagnosed with CCCA at the Northwestern University Department of Dermatology (Chicago, Illinois) between 2011 and 2017. The importance of the physician's experience with black hair and CCCA, the degree to which respondents were bothered by their CCCA, and median QOL score were noted. Central centrifugal cicatricial alopecia presents a unique set of challenges to obtain care. Lack of physician experience with black hair and CCCA is a barrier to care for many with this disease. Self-esteem of CCCA patients is affected by hair loss.

Supramolecular Nanofibers Enhance Growth Factor Signaling by Increasing Lipid Raft Mobility.

The nanostructures of self-assembling biomaterials have been previously designed to tune the release of growth factors in order to optimize biological repair and regeneration. We report here on the discovery that weakly cohesive peptide nanostructures in terms of intermolecular hydrogen bonding, when combined with low concentrations of osteogenic growth factor, enhance both BMP-2 and Wnt mediated signaling in myoblasts and bone marrow stromal cells, respectively. Conversely, analogous nanostructures with enhanced levels of internal hydrogen bonding and cohesion lead to an overall reduction in BMP-2 signaling. We propose that the mechanism for enhanced growth factor signaling by the nanostructures is related to their ability to increase diffusion within membrane lipid rafts. The phenomenon reported here could lead to new nanomedicine strategies to mediate growth factor signaling for translational targets.

Cell death versus cell survival instructed by supramolecular cohesion of nanostructures.

Many naturally occurring peptides containing cationic and hydrophobic domains have evolved to interact with mammalian cell membranes and have been incorporated into materials for non-viral gene delivery, cancer therapy or treatment of microbial infections. Their electrostatic attraction to the negatively charged cell surface and hydrophobic interactions with the membrane lipids enable intracellular delivery or cell lysis. Although the effects of hydrophobicity and cationic charge of soluble molecules on the cell membrane are well known, the interactions between materials with these molecular features and cells remain poorly understood. Here we report that varying the cohesive forces within nanofibres of supramolecular materials with nearly identical cationic and hydrophobic structure instruct cell death or cell survival. Weak intermolecular bonds promote cell death through disruption of lipid membranes, while materials reinforced by hydrogen bonds support cell viability. These findings provide new strategies to design biomaterials that interact with the cell membrane.