The Neuropeptide Y Y2 Receptor Is Coexpressed with Nppb in Primary Afferent Neurons and Y2 Activation Reduces Histaminergic and IL-31-Induced Itch.

Itch stimuli are detected by specialized primary afferents that convey the signal to the spinal cord, but how itch transmission is regulated is still not completely known. Here, we investigated the roles of the neuropeptide Y (NPY)/Y2 receptor system on scratch behavior. The inhibitory Y2 receptor is expressed on mouse primary afferents, and intrathecal administration of the Y2 agonist peptide YY (PYY)3-36 reduced scratch episode frequency and duration induced by compound 48/80, an effect that could be reversed by intrathecal preadministration of the Y2 antagonist BIIE0246. Also, scratch episode duration induced by histamine could be reduced by PYY3-36 In contrast, scratch behavior induced by α-methyl-5HT, protease-activated receptor-2-activating peptide SLIGRL, chloroquine, topical dust mite extract, or mechanical itch induced by von Frey filaments was unaffected by stimulation of Y2 Primary afferent neurons expressing the Npy2r gene were found to coexpress itch-associated markers such as natriuretic peptide precursor b, oncostatin M receptor, and interleukin (IL) 31 receptor A. Accordingly, intrathecal PYY3-36 reduced the scratch behavior induced by IL-31. Our findings imply that the NPY/Y2 system reduces histaminergic and IL-31-associated itch through presynaptic inhibition of a subpopulation of itch-associated primary afferents. SIGNIFICANCE STATEMENT: The spinal neuropeptide Y system dampens scratching behavior induced by histaminergic compounds and interleukin 31, a cytokine involved in atopic dermatitis, through interactions with the Y2 receptor. The Y2 receptor is expressed by primary afferent neurons that are rich in itch-associated neurotransmitters and receptors such as somatostatin, natriuretic peptide precursor b, and interleukin 31 receptors.

Nonclinical safety assessment of a human interleukin-22FC IG fusion protein demonstrates in vitro to in vivo and cross-species translatability.

Although Interleukin-22 (IL-22) is produced by various leukocytes, it preferentially targets cells with epithelial origins. IL-22 exerts essential roles in modulating various tissue epithelial functions, such as innate host defense against extracellular pathogens, barrier integrity, regeneration, and wound healing. Therefore, IL-22 is thought to have therapeutic potential in treating diseases associated with infection, tissue injury or chronic tissue damage. A number of in vitro and in vivo nonclinical studies were conducted to characterize the pharmacological activity and safety parameters of UTTR1147A, an IL-22 recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of a human immunoglobulin. To assess the pharmacological activity of UTTR1147A, STAT3 activation was evaluated in primary hepatocytes isolated from human, cynomolgus monkey, minipig, rat, and mouse after incubation with UTTR1147A. UTTR1147A activated STAT3 in all species evaluated, demonstrating that all were appropriate nonclinical species for toxicology studies. The nonclinical safety profile of UTTR1147A was evaluated in rats, minipigs, and cynomolgus monkeys to establish a safe clinical starting dose for humans in Phase I trials and to support clinical intravenous, subcutaneous and/or topical administration treatment regimen. Results demonstrate the cross-species translatability of the biological response in activating the IL-22 pathway as well as the translatability of findings from in vitro to in vivo systems. UTTR1147A was well tolerated in all species tested and induced the expected pharmacologic effects of epidermal hyperplasia and a transient increase in on-target acute phase proteins. These effects were all considered to be clinically predictable, manageable, monitorable, and reversible.

IL-31-induced pruritus in dogs: a novel experimental model to evaluate anti-pruritic effects of canine therapeutics.

BACKGROUND: Pruritus is a characteristic clinical sign of allergic skin conditions including atopic dermatitis (AD) in the dog. IL-31 is a cytokine found in the serum of some dogs with AD and can induce pruritic behaviours in laboratory beagle dogs. HYPOTHESIS/OBJECTIVES: The objectives were to characterize an IL-31-induced pruritus model by evaluating the efficacy of prednisolone, dexamethasone and oclacitinib, and to compare the speed of anti-pruritic effects of oclacitinib against those of prednisolone and dexamethasone. ANIMALS: Purpose-bred beagle dogs were used in all studies. METHODS: Randomized, blinded, placebo-controlled studies were designed to evaluate and compare the anti-pruritic properties of prednisolone, dexamethasone and oclacitinib following a single intravenous injection of recombinant canine IL-31. Video surveillance was used to monitor and score pruritic behaviours in study animals. RESULTS: Prednisolone [0.5 mg/kg, per os (p.o.)] reduced IL-31-induced pruritus when given 10 h prior to observation. When the time interval between drug treatment and observation was shortened to 1 h, dexamethasone (0.2 mg/kg, intramuscular) but not prednisolone (0.25 or 0.5 mg/kg, p.o.) reduced IL-31-induced pruritus. Oclacitinib (0.4 mg/kg, p.o.) reduced pruritus when given 1, 6, 11 and 16 h prior to the observation period, and the anti-pruritic activity of oclacitinib was greater when compared to prednisolone and dexamethasone at all time points assessed. CONCLUSION AND CLINICAL IMPORTANCE: The efficacy of prednisolone, dexamethasone and oclacitinib in the IL-31-induced pruritus model gives confidence that this may be a relevant model for acute pruritus associated with allergic dermatitis including AD and can be used to evaluate novel compounds or formulations.

Interleukin-22 exacerbates airway inflammation induced by short-term exposure to cigarette smoke in mice.

AIM: Interleukin-22 (IL-22) exhibits both proinflammatory and anti-inflammatory properties in various biological processes. In this study we explored the effects of exogenous recombinant IL-22 (rIL-22) on cigarette smoke (CS)-induced airway inflammation in mice. METHODS: Male C57BL/6 mice were divided into groups: (1) CS group exposed to tobacco smoke for 3 consecutive days, (2) rIL-22 group received rIL-22 (100 mg/kg, ip), and (3) CS plus rIL-22 group, received rIL-22 (100 mg/kg, ip) before the CS exposure. The airway resistance (Rn), lung morphology, inflammatory cells in the airways, and inflammatory cytokines and CXCR3 ligands in both bronchoalveolar lavage (BAL) fluids and lung tissues were analyzed. RESULTS: CS alone significantly elevated IL-22 level in the BAL fluid. Both CS and rIL-22 significantly augmented airway resistance, an influx of inflammatory cells into the airways and lung parenchyma, and significantly elevated levels of pro-inflammatory cytokines (TGFß1 and IL-17A) and CXCR3 chemokines (particularly CXCL10) at the mRNA and/or protein levels. Furthermore, the effects of rIL-22 on airway resistance and inflammation were synergistic with those of CS, as demonstrated by a further increased Rn value, infiltration of greater numbers of inflammatory cells into the lung, higher levels of inflammatory cytokines and chemokines, and more severe pathological changes in CS plus rIL-22 group as compared to those in CS group. CONCLUSION: Exogenous rIL-22 exacerbates the airway inflammatory responses to CS exposure in part by inducing expression of several proinflammatory cytokines and CXCR3 ligands.

Integrative biology approach identifies cytokine targeting strategies for psoriasis.

Cytokines are critical checkpoints of inflammation. The treatment of human autoimmune disease has been revolutionized by targeting inflammatory cytokines as key drivers of disease pathogenesis. Despite this, there exist numerous pitfalls when translating preclinical data into the clinic. We developed an integrative biology approach combining human disease transcriptome data sets with clinically relevant in vivo models in an attempt to bridge this translational gap. We chose interleukin-22 (IL-22) as a model cytokine because of its potentially important proinflammatory role in epithelial tissues. Injection of IL-22 into normal human skin grafts produced marked inflammatory skin changes resembling human psoriasis. Injection of anti-IL-22 monoclonal antibody in a human xenotransplant model of psoriasis, developed specifically to test potential therapeutic candidates, efficiently blocked skin inflammation. Bioinformatic analysis integrating both the IL-22 and anti-IL-22 cytokine transcriptomes and mapping them onto a psoriasis disease gene coexpression network identified key cytokine-dependent hub genes. Using knockout mice and small-molecule blockade, we show that one of these hub genes, the so far unexplored serine/threonine kinase PIM1, is a critical checkpoint for human skin inflammation and potential future therapeutic target in psoriasis. Using in silico integration of human data sets and biological models, we were able to identify a new target in the treatment of psoriasis.

IL-22 regulates iron availability in vivo through the induction of hepcidin.

Iron is a trace element important for the proper folding and function of various proteins. Physiological regulation of iron stores is of critical importance for RBC production and antimicrobial defense. Hepcidin is a key regulator of iron levels within the body. Under conditions of iron deficiency, hepcidin expression is reduced to promote increased iron uptake from the diet and release from cells, whereas during conditions of iron excess, induction of hepcidin restricts iron uptake and movement within the body. The cytokine IL-6 is well established as an important inducer of hepcidin. The presence of this cytokine during inflammatory states can induce hepcidin production, iron deficiency, and anemia. In this study, we show that IL-22 also influences hepcidin production in vivo. Injection of mice with exogenous mouse IgG1 Fc fused to the N terminus of mouse IL-22 (Fc-IL-22), an IL-22R agonist with prolonged and enhanced functional potency, induced hepcidin production, with a subsequent decrease in circulating serum iron and hemoglobin levels and a concomitant increase in iron accumulation within the spleen. This response was independent of IL-6 and was attenuated in the absence of the IL-22R-associated signaling kinase, Tyk2. Ab-mediated blockade of hepcidin partially reversed the effects on iron biology caused by IL-22R stimulation. Taken together, these data suggest that exogenous IL-22 regulates hepcidin production to physiologically influence iron usage.

Glia maturation factor induces interleukin-33 release from astrocytes: implications for neurodegenerative diseases.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and Multiple sclerosis (MS) involve activation of glial cells and release of inflammatory mediators leading to death of neurons. Glia maturation factor (GMF) is up-regulated in the central nervous system (CNS) in these neurodegenerative diseases. Interleukin-33 (IL-33) is highly expressed constitutively in the CNS. We have treated mouse astrocytes, mixed culture with glial cells and neurons, and only neurons with GMF and/or IL-33 in vitro. Both GMF and IL-33-induced chemokine (C-C motif) ligand 2 (CCL2) release in a dose and time-dependent manner. We report that GMF induced IL-33 release, and that IL-33 augments GMF-induced tumor necrosis factor-alpha (TNF-α) release from mouse astrocytes. IL-33 induces CCL2, TNF-α and nitric oxide release through phosphorylation of ERK in mouse astrocytes. Incubation of mixed culture containing glial cells and neurons or only neuronal culture with IL-33 reduced the number of neurons positive for microtubule-associated protein 2. In conclusion, IL-33 augments GMF-mediated neuroinflammation and may provide a new drug target for neurodegenerative and autoimmune diseases.

IL-33: a Janus cytokine.

Interleukin (IL) 33, a member of the IL-1 family, is the ligand of ST2 that is expressed mainly on activated Th2 cells and mast cells. IL-33 can skew a predominantly Th1 cell population to a mainly Th2 cells phenotype in vivo. IL-33 messenger RNA is expressed early during infection of the intestinal-dwelling nematode Trichuris muris in mice. IL-33 treatment enhances resistance to Trichuris infection. IL-33 also effectively attenuates sepsis by mobilising the innate cells, neutrophils, to the site of infection, helping to clear the pathogens. Thus, IL-33 may be evolutionally preserved for the host defence against infections. IL-33 can reduce an ongoing atherosclerosis in ApoE(-/-) mice and attenuate adipocytes mainly by inducing the production of type II cytokines. In contrast, IL-33 can also exacerbate allergy and the inflammation in collagen-induced or serum-induced arthritis. Hence, IL-33 is a double-edged sword, and targeting IL-33 should be approached with caution.

A CC' loop decoy peptide blocks the interaction between Act1 and IL-17RA to attenuate IL-17- and IL-25-induced inflammation.

Interleukin-17 (IL-17) and IL-25 signaling induce the expression of genes encoding inflammatory factors and are implicated in the pathology of various inflammatory diseases. Nuclear factor κB (NF-κB) activator 1 (Act1) is an adaptor protein and E3 ubiquitin ligase that is critical for signaling by either IL-17 or IL-25, and it is recruited to their receptors (IL-17R and IL-25R) through heterotypic interactions between the SEFIR [SEF (similar expression to fibroblast growth factor genes) and IL-17R] domain of Act1 and that of the receptor. SEFIR domains have structural similarity with the Toll-IL-1 receptor (TIR) domains of Toll-like receptors and IL-1R. Whereas the BB' loop of TIR is required for TIR-TIR interactions, we found that deletion of the BB' loop from Act1 or IL-17RA (a common subunit of both IL-17R and IL-25R) did not affect Act1-IL-17RA interactions; rather, deletion of the CC' loop from Act1 or IL-17RA abolished the interaction between both proteins. Surface plasmon resonance measurements showed that a peptide corresponding to the CC' loop of Act1 bound directly to IL-17RA. A cell-permeable decoy peptide based on the CC' loop sequence inhibited IL-17- or IL-25-mediated signaling in vitro, as well as IL-17- and IL-25-induced pulmonary inflammation in mice. Together, these findings provide the molecular basis for the specificity of SEFIR-SEFIR versus TIR-TIR domain interactions and consequent signaling. Moreover, we suggest that the CC' loop motif of SEFIR domains is a promising target for therapeutic strategies against inflammatory diseases associated with IL-17 or IL-25 signaling.

IL-33 induces skin inflammation with mast cell and neutrophil activation.

Psoriasis is a common chronic autoimmune condition of the skin characterized by hyperplasia of epidermal keratinocytes associated with pro-inflammatory cytokines. IL-33 is a new member of the IL-1 superfamily that signals through the ST2 receptor and was originally defined as an inducer of T helper 2 (Th2) cytokines. Recently, broader immune activatory potential has been defined for IL-33 particularly via mast cell activation and neutrophil migration. Here, we show that ST2(-/-) mice exhibit reduced cutaneous inflammatory responses compared with WT mice in a phorbol ester-induced model of skin inflammation. Furthermore, injections of IL-33 into the ears of mice induce an inflammatory skin lesion. This inflammatory response was partially dependent on mast cells as mast cell-deficient mice (Kit(W-sh/W-sh) ) showed delayed responses to IL-33. IL-33 also recruited neutrophils to the ear, an effect mediated in part by increased production of the chemokine KC (CXCL1). Finally, we show that IL-33 expression is up-regulated in the epidermis of clinical psoriatic lesions, compared with healthy skin. These results therefore demonstrate that IL-33 may play a role in psoriasis-like plaque inflammation. IL-33 targeting may provide a new treatment strategy for psoriasis.

IL-33 exacerbates autoantibody-induced arthritis.

Rheumatoid arthritis pathogenesis comprises dysregulation in both innate and adaptive immunity. There is therefore intense interest in the factors that integrate these immunologic pathways in rheumatoid arthritis. In this paper, we report that IL-33, a novel member of the IL-1 family, can exacerbate anti-glucose-6-phosphate isomerase autoantibody-induced arthritis (AIA). Mice lacking ST2 (ST2(-/-)), the IL-33 receptor alpha-chain, developed attenuated AIA and reduced expression of articular proinflammatory cytokines. Conversely, treatment of wild-type mice with rIL-33 significantly exacerbated AIA and markedly enhanced proinflammatory cytokine production. However, IL-33 failed to increase the severity of the disease in mast cell-deficient or ST2(-/-) mice. Furthermore, mast cells from wild-type, but not ST2(-/-), mice restored the ability of ST2(-/-) recipients to mount an IL-33-mediated exacerbation of AIA. IL-33 also enhanced autoantibody-mediated mast cell degranulation in vitro and in synovial tissue in vivo. Together these results demonstrate that IL-33 can enhance autoantibody-mediated articular inflammation via promoting mast cell degranulation and proinflammatory cytokine production. Because IL-33 is derived predominantly from synovial fibroblasts, this finding provides a novel mechanism whereby a host tissue-derived cytokine can regulate effector adaptive immune response via enhancing innate cellular activation in inflammatory arthritis.

Correction of humoral derangements from mutant superoxide dismutase 1 spinal cord.

OBJECTIVE: We sought to define molecular and cellular participants that mediate motor neuron injury in amyotrophic lateral sclerosis using a coculture system. METHODS: We cocultured embryonic stem cell-derived motor neurons with organotypic slice cultures from wild-type or SOD1G93A (MT) mice. We examined axon lengths and cell survival of embryonic stem cell-derived motor neurons. We defined and quantified the humoral factors that differed between wild-type and MT organotypic cultures, and then corrected these differences in cell culture. RESULTS: MT spinal cord organotypic slices were selectively toxic to motor neurons as defined by axonal lengths and cell survival. MT spinal cord organotypic slices secreted higher levels of nitric oxide, interleukin (IL)-1beta, IL-6, and IL-12p70 and lower levels of vascular endothelial growth factor. The toxicity of MT spinal cord organotypic cultures was reduced and axonal lengths were restored to near normal by coculturing in the presence of reactive oxygen species scavenger, vascular endothelial growth factor, and neutralizing antibodies to IL-1beta, IL-6, and IL-12. INTERPRETATION: MT spinal cord organotypic cultures overexpress certain factors and underexpress others, creating a nonpermissive environment for cocultured motor neurons. Correction of these abnormalities as a group, but not individually, restores axonal length to near normal. Such a "cocktail" approach to the treatment of amyotrophic lateral sclerosis should be investigated further.

Tratamento coadjuvante do câncer do reto: III: Imunoterapia / Adjuvant treatment in rectal cancer: III: Immunoterapy

Apesar dos resultados ainda pouco convincentes, a imunoterapia tem sido incluída em muitos ensaios clínicos como nova e promissora modalidade adjuvante de terapêutica do câncer colorreta, näo só para eliminar micro focos implantados de células neoplásicas como também para destruir as células cancerosas, circulantes. Embora haja muito para ser elucidado a respeito a inter-relaçäo do sistema imunológico com o câncer, os conhecimentos atuais sobre a resposta imune, seus modificadores biológicos e a possibilidade de produçäo laboratorial dessas substâncias naturais, por meio da engenharia genética e de técnicas de hibridoma, criam amplas perspectivas de se poder agir no sistema imunológico e na antienicidade tumoral criando, assim, uma nova, potente e específica arma auxiliar na terapêutica do câncer humano, mormente do câncer colorretal. Nesse campo, a imunoterapia cai em três principais categorias: 1) modificadores de resposta imune, 2) anticorpos monoclonais e 3) vacinas. Os modificadores da resposta imune säo substâncias intrínsecas (modificadores biológicos) tais como as interleucinas, os interferons e o fator de necrose tumoral, que exercem influências em diferentes estágios da resposta imune; ou extrínsecos, como o BCG, C. parvum, endotoxinas, outros produtos bacterianos e o levamisol, que, também, agem estimulando, de maneira específica, a resposta imune. Os anticorpos monoclonais säo anticorpos puros que podem, produzidos em grande quantidade, ser dirigidos, de forma específica, contra antígenos tumorais. Ligados à isótopos radioativos, säo usados para localizar grupos de células cancerosas em qualquer parte do organismo e, portanto, cumprem papel no diagnóstico; assim como, quando ligados às drogas anticancerosas ou às toxinas naturais, eles podem servir como carregador dessas substâncias até aos focos neoplásicos , no desempenho de auxílio à terapêutic. As vacinas, em fase inicial de aquisiçäo de conhecimentos, estäo sendo desenvolvidas e testadas para prevenir a recidiva de câncer em pacientes previamente tratados, ou para estimular a resposta imune do paciente a um tipo específico de câncer

Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2.

Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin-15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2.

Interleukin 12, interferon gamma, and tumor necrosis factor alpha are the key cytokines of the generalized Shwartzman reaction.

The Shwartzman reaction is elicited by two injections of lipopolysaccharide (LPS) in mice. The priming LPS injection is given in the footpad, whereas the lethal LPS challenge is given intravenously 24 h later. The injection of interferon gamma (IFN-gamma) or interleukin 12 (IL-12) instead of the LPS priming injection induced the lethal reaction in mice further challenged with LPS. Antibodies against IFN-gamma when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS, IL-12, or IFN-gamma. Antibodies against IL-12, when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS or IL-12 but not with IFN-gamma. These results strongly suggest that LPS induces the release of IL-12, that IL-12 induces the production of IFN-gamma, and that IFN-gamma is the cytokine that primes macrophages and other cell types. Upon LPS challenge, the lethal Shwartzman reaction is induced by a massive production of inflammatory cytokines that act on the target sites already sensitized by IFN-gamma. If mixtures of TNF and IL-1 or mixtures of TNF and IFN-gamma are used to challenge mice previously primed with IFN-gamma or IL-12, mortality is induced. In the same conditions, the individual cytokines or a mixture of IL-1 and IFN-gamma do not replace the LPS challenge. When the mice are primed with LPS, the combination of TNF, IL-1, and IFN-gamma induced only a partial mortality incidence suggesting that the involvement of other LPS-induced factors.

Studies on intraocular inflammation produced by intravitreal human interleukins in rabbits.

Recombinant human interleukin-1 alpha, interleukin-1 beta (10, 80 or 200 units), interleukin-8 (10 or 40 units) or endotoxin was injected intravitreally into rabbit eyes. Twenty-four hours thereafter aqueous humor protein, leukocyte number, prostaglandin E2, leukotriene B4 and rabbit interleukin-1 beta were measured. In addition, synthesis of prostaglandin E2 and leukotriene B4 in iris-ciliary body and myeloperoxidase (MPO) activity were determined. Recombinant human interleukins 1 alpha and 1 beta, but not interleukin-8 induced signs of uveitis, i.e. protein and leukocytic infiltration into aqueous humor. At 200 unit activities, human interleukin-1 beta was significantly greater than interleukin-1 alpha in causing leukocyte infiltration response. Interleukin-1 alpha did not stimulate the release of prostaglandin E2 or leukotriene B4. In fact, interleukin-1 beta significantly inhibited the synthesis of prostaglandin E2 in iris-ciliary body. Both of these human interleukins caused a release of rabbit interleukin-1 beta in aqueous achieving a level significantly higher than observed after endotoxin injection. This study demonstrates that intravitreal injections of human IL-1 alpha and IL-1 beta induce uveitis by releasing rabbit interleukin-1 beta within the eye.

Interleukins. Clinical pharmacokinetics and practical implications.

Interleukins and tumour necrosis factor (TNF) are a complex group of proteins and glycoproteins able to exert pleiotropic effects with respect to a number of different target cells. In physiological conditions, they are induced and released in basal amounts only in restricted microenvironments where they have paracrine activity. Any small amounts reaching the circulation do not disturb homoeostasis. During therapy, particularly when these cytokines are administered via conventional routes, it has become apparent that their presence in nonphysiological plasma concentrations and their unselective action cause toxic effects with small benefits. The pharmacokinetics of interleukins-1, -2, -3 and -6 and TNF have been evaluated, and their disappearance from plasma after intravenous administration is very rapid (i.e. the distribution half-life is measured in minutes; the elimination half-life is several hours). The efficiency of catabolic pathways such as renal filtration and/or liver uptake is interpreted as a salutary mechanism for extracting proteins that should not be in the circulation. However, because these cytokines are very potent immunomodulatory agents there is a need to improve their therapeutic index, and to this end a number of possible formulations and routes of administration are now available and may eventually be of practical use.