Importance of PLD2 in an IL-23 driven psoriasiform dermatitis model and potential link to human psoriasis.

Phospholipase D2 (PLD2), a major isoform of the PLD family, has been reported to regulate inflammatory responses. Thus far, the relevance of PLD2 in psoriasis, an inflammatory skin disease, has not been explored. In the current study, we examined PLD2 expression in the skin of psoriasis patients and the role of PLD2 in an interleukin (IL)-23-induced mouse model of psoriasiform dermatitis. Both in situ hybridization and bulk RNA sequencing showed PLD2 gene expression is significantly higher in lesional relative to non-lesional skin of psoriasis patients or the skin of healthy subjects. PLD2 expression is also enriched in residual lesions from patients on biologic therapies. Murine in vivo studies showed that PLD2 deficiency significantly reduced psoriasiform inflammation in IL-23-injected ears, as reflected by decreases in ear thickness, expression of defensin beta 4A and the S100 calcium binding protein A7A, macrophage infiltrate, and expression of CXCL10 and IL-6. However, the expression of type 17 cytokines, IL-17A and IL-17F, were not reduced. Dual knockout of PLD1 and PLD2 offered little additional protection compared to PLD2 knockout alone in the IL-23 model. In addition, pharmacological inhibition with a pan-PLD1/PLD2 inhibitor also suppressed IL-23-induced psoriasiform dermatitis. Bone-marrow-derived macrophages from wild type (WT) and PLD2 knockout (KO) mice exhibited little difference in viability and sensitivity to lipopolysaccharide and/or interferon gamma, or resiquimod (R848). PLD2 deficiency did not alter the differentiation and function of Th17 cells in an ex vivo study with splenocytes isolated from WT and PLD2 KO mice. Overall, these data suggest that PLD2 may play a role in the pathophysiology of psoriasis. Reducing macrophage infiltrate and cytokine/chemokine production might contribute to an anti-inflammatory effect observed in PLD2 knockout mice. Further studies are required to better understand the mechanisms by which PLD2 contributes to skin lesions in psoriasis patients and psoriasiform dermatitis models.

Identification and structure-based drug design of cell-active inhibitors of interleukin 17A at a novel C-terminal site.

Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein-protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (< 50 nM) and showed functional inhibition of IL17A-induced cellular signaling (IC50~1 µM). We also describe a fluorescence-based probe molecule suitable to discern/screen for additional molecules binding in this C-terminal site.

ACT1 Is Required for Murine IL-23-Induced Psoriasiform Inflammation Potentially Independent of E3 Ligase Activity.

Psoriasis is a debilitating skin disease characterized by epidermal thickening, abnormal keratinocyte differentiation, and proinflammatory immune cell infiltrate into the affected skin. IL-17A plays a critical role in the etiology of psoriasis. ACT1, an intracellular adaptor protein and a putative ubiquitin E3 ligase, is essential for signal transduction downstream of the IL-17A receptor. Thus, IL-17A signaling in general, and ACT1 specifically, represent attractive targets for the treatment of psoriasis. We generated Act1 knockout and Act1 L286G knockin (ligase domain) mice to investigate the potential therapeutic effects of targeting ACT1 and its U-box domain, respectively. Act1 knockout, but not Act1 L286G knockin, mice were resistant to increases in CXCL1 plasma levels induced by subcutaneous injection of recombinant IL-17A. Moreover, in a mouse model of psoriasiform dermatitis induced by intradermal IL-23 injection, Act1 knockout, but not Act1 L286G knockin, was protective against increases in ear thickness, keratinocyte hyperproliferation, expression of genes for antimicrobial peptides and chemokines, and infiltration of monocytes and macrophages. Our studies highlight the critical contribution of ACT1 to proinflammatory skin changes mediated by the IL-23/IL-17 signaling axis and illustrate the need for further insight into ACT1 E3 ligase activity.

The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation.

The Krebs cycle-derived metabolite itaconate is highly upregulated in inflammatory macrophages and exerts immunomodulatory effects through cysteine modifications on target proteins. The NLRP3 inflammasome, which cleaves IL-1ß, IL-18, and gasdermin D, must be tightly regulated to avoid excessive inflammation. Here we provide evidence that itaconate modifies NLRP3 and inhibits inflammasome activation. Itaconate and its derivative, 4-octyl itaconate (4-OI), inhibited NLRP3 inflammasome activation, but not AIM2 or NLRC4. Conversely, NLRP3 activation was increased in itaconate-depleted Irg1-/- macrophages. 4-OI inhibited the interaction between NLRP3 and NEK7, a key step in the activation process, and "dicarboxypropylated" C548 on NLRP3. Furthermore, 4-OI inhibited NLRP3-dependent IL-1ß release from PBMCs isolated from cryopyrin-associated periodic syndrome (CAPS) patients, and reduced inflammation in an in vivo model of urate-induced peritonitis. Our results identify itaconate as an endogenous metabolic regulator of the NLRP3 inflammasome and describe a process that may be exploited therapeutically to alleviate inflammation in NLRP3-driven disorders.

Small Molecule and Pooled CRISPR Screens Investigating IL17 Signaling Identify BRD2 as a Novel Contributor to Keratinocyte Inflammatory Responses.

Interleukin-17A (IL17A) plays a critical role in the development of numerous autoimmune diseases, including psoriasis. The clinical success of IL17A neutralizing biologics in psoriasis has underlined its importance as a drug discovery target. While many studies have focused on the differentiation and trafficking of IL17A producing T-helper 17 cells, less is known about IL17A-initiated signaling events in stromal and parenchymal cells leading to psoriatic phenotypes. We sought to discover signaling nodes downstream of IL17A contributing to disease pathogenesis. Using IL17A and tumor necrosis factor α (TNF) to stimulate primary human epidermal keratinocytes, we employed two different phenotypic screening approaches. First, a library of ∼22000 annotated compounds was screened for reduced secretion of the pro-inflammatory chemokine IL8. Second, a library of 729 kinases was screened in a pooled format by utilizing CRISPR-Cas9 and monitoring IL8 intracellular staining. The highest-ranking novel hits identified in both screens were the bromodomain and extra-terminal domain (BET) family proteins and bromodomain-containing protein 2 (BRD2), respectively. Comparison of BRD2, BRD3, and BRD4 silencing with siRNA and CRISPR confirmed that BRD2 was responsible for mediating IL8 production. Pan-BRD inhibitors and BRD2 knockout also reduced IL17A/TNF-mediated CXC motif chemokines 1/2/6 (CXCL1/2/6) and granulocyte colony stimulating factor (G-CSF) production. In RNA-Seq analysis, 438 IL17A/TNF dependent genes were reduced in BRD2-deficient primary keratinocytes. KEGG pathway analysis of these genes showed enrichment in TNF signaling and rheumatoid arthritis relevant genes. Moreover, a number of genes important for keratinocyte homeostasis and cornification were dysregulated in BRD2-deficient keratinocytes. In IL17A/TNF/IL22 stimulated three-dimensional organotypic raft cultures, pan-BRD inhibition reduced inflammatory factor production but elicited aberrant cornification, consistent with RNA-Seq analysis. These studies highlight a novel role for BRDs and BRD2 in particular in IL17A-mediated inflammatory signaling.

Dual Blockade of Interleukin-1ß and Interleukin-17A Reduces Murine Arthritis Pathogenesis but Also Leads to Spontaneous Skin Infections in Nonhuman Primates.

Despite the efficacy of biologics for treatment of rheumatoid arthritis (RA), many patients show inadequate responses and likely require neutralization of multiple mediators. Neutralization of both interleukin (IL)-1ß and IL-17A with monoclonal antibodies showed greater efficacy than either agent alone in a mouse arthritis model with cooperative inhibition of key inflammatory factors, IL-6, granulocyte colony-stimulating factor (G-CSF), and CXC chemokine ligand (CXCL)1. Given the potential clinical benefit in RA, we generated a human dual variable domain antibody Ig, ABBV-615, capable of simultaneous binding and neutralization of IL-1ß and IL-17A. ABBV-615 was characterized and evaluated in cynomolgus monkeys for pharmacokinetics and toxicity to enable clinical development. ABBV-615 exhibited affinities (KD) of 12 and 3 pM on human IL-1ß and IL-17A, respectively, and potencies (IC50) of 3 and 58 pM, respectively, as well as excellent drug-like properties. ABBV-615 pharmacokinetics in cynomolgus monkeys was dose proportional from 20 to 100 mg/kg with a mean half-life of 16 days. However, a 13-week repeat-dose toxicity study in cynomolgus monkeys revealed time-dependent spontaneous infections exclusively in skin at all doses tested and not historically seen with single-agent anti-IL-1α/ß or anti-IL-17A. Consistent with reduced resistance to skin infections, IL-1ß- and IL-17A-stimulated human keratinocytes demonstrate cooperative or compensatory production of key antibacterial and inflammatory mediators such as lipocalin-2, G-CSF, CXCL1, IL-8, tumor necrosis factor, and IL-6, which aid in defense against skin bacterial infections. These results illustrate the skin-specific antimicrobial mechanisms of IL-1ß and IL-17A and highlight the importance of understanding unique combinatorial effects of biologic agents.

Inhibition of COX1/2 alters the host response and reduces ECM scaffold mediated constructive tissue remodeling in a rodent model of skeletal muscle injury.

Extracellular matrix (ECM) has been used as a biologic scaffold material to both reinforce the surgical repair of soft tissue and serve as an inductive template to promote a constructive tissue remodeling response. Success of such an approach is dependent on macrophage-mediated degradation and remodeling of the biologic scaffold. Macrophage phenotype during these processes is a predictive factor of the eventual remodeling outcome. ECM scaffolds have been shown to promote an anti-inflammatory or M2-like macrophage phenotype in vitro that includes secretion of downstream products of cycolooxygenases 1 and 2 (COX1/2). The present study investigated the effect of a common COX1/2 inhibitor (Aspirin) on macrophage phenotype and tissue remodeling in a rodent model of ECM scaffold treated skeletal muscle injury. Inhibition of COX1/2 reduced the constructive remodeling response by hindering myogenesis and collagen deposition in the defect area. The inhibited response was correlated with a reduction in M2-like macrophages in the defect area. The effects of Aspirin on macrophage phenotype were corroborated using an established in vitro macrophage model which showed a reduction in both ECM induced prostaglandin secretion and expression of a marker of M2-like macrophages (CD206). These results raise questions regarding the common peri-surgical administration of COX1/2 inhibitors when biologic scaffold materials are used to facilitate muscle repair/regeneration. STATEMENT OF SIGNIFICANCE: COX1/2 inhibitors such as nonsteroidal anti-inflammatory drugs (NSAIDs) are routinely administered post-surgically for analgesic purposes. While COX1/2 inhibitors are important in pain management, they have also been shown to delay or diminish the healing process, which calls to question their clinical use for treating musculotendinous injuries. The present study aimed to investigate the influence of a common NSAID, Aspirin, on the constructive remodeling response mediated by an ECM scaffold (UBM) in a rat skeletal muscle injury model. The COX1/2 inhibitor, Aspirin, was found to mitigate the ECM scaffold-mediated constructive remodeling response both in an in vitro co-culture system and an in vivo rat model of skeletal muscle injury. The results presented herein provide data showing that NSAIDs may significantly alter tissue remodeling outcomes when a biomaterial is used in a regenerative medicine/tissue engineering application. Thus, the decision to prescribe NSAIDs to manage the symptoms of inflammation post-ECM scaffold implantation should be carefully considered.

Solubilized extracellular matrix from brain and urinary bladder elicits distinct functional and phenotypic responses in macrophages.

Extracellular matrix (ECM) derived from a variety of source tissues has been successfully used to facilitate tissue reconstruction. The recent development of solubilized forms of ECM advances the therapeutic potential of these biomaterials. Isolated, soluble components of ECM and matricryptic peptides have been shown to bias macrophages toward a regulatory and constructive (M2-like) phenotype. However, the majority of studies described thus far have utilized anatomically and morphologically similar gastrointestinal derived ECMs (small intestine, esophagus, urinary bladder, etc.) and a small subset of macrophage markers (CD206, CD86, CCR7) to describe them. The present study evaluated the effect of solubilized ECM derived from molecularly diverse source tissues (brain and urinary bladder) upon primary macrophage phenotype and function. Results showed that solubilized urinary bladder ECM (U-ECM) up-regulated macrophage PGE2 secretion and suppressed traditional pro-inflammatory factor secretion, consistent with an M2-like phenotype. The hyaluronic acid (HA) component in solubilized U-ECM played an important role in mediating this response. Brain ECM (B-ECM) elicited a pro-inflammatory (M1-like) macrophage response and contained almost no HA. These findings suggest that the molecular composition of the source tissue ECM plays an important role in influencing macrophage function and phenotype.

Effects of biologic scaffolds on human stem cells and implications for CNS tissue engineering.

Biologic scaffolds composed of mammalian extracellular matrix (ECM) promote constructive remodeling of tissues via mechanisms that include the recruitment of endogenous stem/progenitor cells, modulation of the host innate immune response, and influence of cell fate differentiation. Such scaffold materials are typically prepared by decellularization of source tissues and are prepared as sheets, powder, or hydrogels. It is plausible that ECM derived from an anatomically distinct tissue would have unique or specific effects on cells that naturally reside in this same tissue. The present study investigated the in vitro effect of a soluble form of ECM derived from central nervous system (CNS) tissue, specifically the spinal cord or brain, versus ECM derived from a non-CNS tissue; specifically, the urinary bladder on the behavior of neural stem cells (NSCs) and perivascular stem cells. All forms of ECM induce positive, mitogenic, and chemotactic effects at concentrations of approximately 100 µg/mL without affecting stem cell viability. CNS-derived ECMs also showed the ability to differentiate NSCs into neurons as indicted by ßIII-tubulin expression in two-dimensional culture and neurite extension on the millimeter scale after 24 days of three-dimensional cultures in an ECM hydrogel. These results suggest that solubilized forms of ECM scaffold materials may facilitate the postinjury healing response in CNS tissues.

Defining a key receptor-CheA kinase contact and elucidating its function in the membrane-bound bacterial chemosensory array: a disulfide mapping and TAM-IDS Study.

The three core components of the ubiquitous bacterial chemosensory array - the transmembrane chemoreceptor, the histidine kinase CheA, and the adaptor protein CheW - assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate kinase activity. Both the regulatory domain of the kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor-CheA kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium, and other bacteria. Here we employ disulfide mapping and tryptophan and alanine mutation to identify docking sites (TAM-IDS) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA kinase binding in the array and a regulatory function central to transmission of the activation signal from receptor to CheA kinase. On-off switching alters the disulfide formation rates of specific Cys pairs at the interface, but not most Cys pairs, indicating that signaling perturbs localized regions of the interface. The findings suggest a simple model for the rearrangement of the interface triggered by the attractant signal and for longer range transmission of the signal in the chemosensory array.

Isolated bacterial chemosensory array possesses quasi- and ultrastable components: functional links between array stability, cooperativity, and order.

Bacteria utilize a large multiprotein chemosensory array to sense attractants and repellents in their environment. The array is a hexagonal lattice formed from three core proteins: a transmembrane receptor, the His kinase CheA, and the adaptor protein CheW. The resulting, highly networked array architecture yields several advantages including strong positive cooperativity in the attractant response and rapid signal transduction through the preformed, integrated signaling circuit. Moreover, when isolated from cells or reconstituted in isolated bacterial membranes, the array possesses extreme kinetic stability termed ultrastability (Erbse and Falke (2009) Biochemistry 48:6975-87) and is the most long-lived multiprotein enzyme complex described to date. The isolated array retains kinase activity, attractant regulation, and its bound core proteins for days or more at 22 °C. The present work quantitates this ultrastability and investigates its origin. The results demonstrate that arrays consist of two major components: (i) a quasi-stable component with a lifetime of 1-2 days that decays due to slow proteolysis of CheA kinase in the lattice and (ii) a truly ultrastable component with a lifetime of ~20 days that is substantially more protected from proteolysis. Following proteolysis of the quasi-stable component the apparent positive cooperativity of the array increases, arguing the quasi-stable component is not as cooperative as the ultrastable component. Introduction of structural defects into the array by coupling a bulky probe to a subset of receptors reveals that modification of only 2% of the receptor population is sufficient to abolish ultrastability, supporting the hypothesis that the ultrastable component requires a high level of array spatial order. Overall, the findings are consistent with a model in which the quasi- and ultrastable components arise from distinct regions of the array, such that the ultrastable regions possess more extensive, better-ordered, multivalent interconnectivities between core components, thereby yielding extraordinary stability and cooperativity. Furthermore, the findings indicate that the chemosensory array is a promising platform for the development of ultrastable biosensors.

Evidence that opioids may have toll-like receptor 4 and MD-2 effects.

Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll-like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically-employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

A peptide antagonist of the TLR4-MD2 interaction.

Toll-like receptors are an integral part of innate immunity in the central nervous system (CNS); they orchestrate a robust defense in response to both exogenous and endogenous danger signals. Recently, toll-like receptor 4 (TLR4) has emerged as a therapeutic target for the treatment of CNS-related diseases such as sepsis and chronic pain. We herein report a chemical biology approach by using a rationally designed peptide inhibitor to disrupt the TLR4-MD2 association, thereby blocking TLR4 signaling.

Design, synthesis, and evaluation of biotinylated opioid derivatives as novel probes to study opioid pharmacology.

A generally applicable strategy of chemically labeling (-)-morphine (1) is described. The synthesis starts from commercially available starting materials and can be completed in two steps with an overall yield of 23%. In silico simulation and NMR results show that the binding of (-)-morphine to one of its molecular targets, toll-like receptor 4 (TLR4), was not affected by the modification. Secreted embryonic alkaline phosphatase (SEAP) reporter assay results demonstrate that C(3) biotinylated and unmodified (-)-morphine show similar biological activities in live cells. To our knowledge, these studies provide the first practical and concise method to label various opioid derivatives, a group of important therapeutics in pain management, for biochemical/pharmacological studies.

Peptide probes for protein transmembrane domains.

Much current interest in chemical biology focuses on the transmembrane domains of proteins, which have emerged as targets for the development of novel diagnostics and therapeutics. Integral membrane proteins are a group of important biomolecules that play pivotal roles in many cellular activities. Previous studies primarily focused on the extra- and/or intracellular domains of membrane proteins. However, the importance of transmembrane regions in the regulation of protein complexes is beginning to emerge. As such, a number of methods for designing and testing novel exogenous peptides that recognize transmembrane targets and modulate cellular functions have been developed. This Review outlines current methodologies for developing these transmembrane probes that may provide useful tools to study a variety of biological phenomena in the membrane.