Peptides targeting Toll-like receptor signalling pathways for novel immune therapeutics.

Toll-like receptors (TLRs) are a family of key proteins that permit mammals to detect microbes and endogenous molecules, which are present in body fluids, cell membranes and cytoplasm. They confer mechanisms to the host for maintaining homeostasis, activating innate immunity and inducing signals that lead to the activation of adaptive immunity. TLR signalling induces the expression of pro-inflammatory and anti-viral genes through different and intricate pathways. However, persistent signalling can be dangerous and all members of the TLR family are involved in the pathogenesis of acute and chronic inflammation, autoimmunity, allergy, cancer and aging. The pharmaceutical industry has begun intensive work developing novel immunotherapeutic approaches based on both activation and inhibition of TLR triggering. Further, clinical trials are pending to evaluate TLR agonists as novel vaccine adjuvants and for the treatment of infectious diseases, allergic diseases and asthma. Since systemic, metabolic and neuroendocrine changes are elicited by inflammation, TLR activity is susceptible of regulation by hormones and neuroendocrine factors. Neuroendocrine mediators are important players in modulating different phases of TLR regulation contributing to the endogenous control of homeostasis through local, regional and systemic routes. Vasoactive intestinal peptide (VIP) is an important signal molecule of the neuroendocrine-immune network that has recently emerged as a potential candidate for the treatment of inflammatory and autoimmune disorders by controlling innate and adaptive immunity. This review shows current advances in the understanding of TLR modulation by VIP that could contribute to the use of this natural peptide as a therapeutic tool.

Vasoactive intestinal peptide as a healing mediator in Crohn's disease.

The vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide (VIP/PACAP) system is considered as a paradigm for the use of a neuroendocrine-immune mediator in therapy. We review the role of VIP in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis as a murine model of Crohn's disease. VIP treatment led to the recovery of clinical factors, the amelioration of parameters related to the recruitment and traffic of cell populations, and the balance of inflammatory mediators derived from granulocytes, antigen-presenting cells and T lymphocytes including Th1, Th2 and Th17. Finally, the most recent data validate its therapeutic role through the modulation of TLR2 and 4 receptors.

Regulation of TLR expression, a new perspective for the role of VIP in immunity.

The contribution of VIP immune functions to the regulation of homeostasis and health is well known. Modulation of immune responses through new therapeutics is one of the main goals of physicians and scientists seeking to control inflammatory/autoimmune diseases in humans. Initial therapeutic strategies targeted adaptive immune responses; discovery of Toll-like receptors (TLR) has widened the horizon to include targeting the innate immune system. In this review we have summarized recent information about VIP modulation of TLR function, and we suggest that VIP represents a new therapeutic option in the management of several pathologies.

VIP-PACAP system in immunity: new insights for multitarget therapy.

Our research about VIP/PACAP and the immune system goes back to 1990 when our group described the expression of VIP on lymphocytes for the first time. Since this year, using three models of disease, septic shock, rheumathoid arthritis, and Crohn's disease, we are trying to contribute with new pieces to the puzzle of immunity to approach the use of VIP/PACAP system as a therapeutic agent. In 1999 we established that the first step in the beneficial effect of the VIP/PACAP system exerts consists in its potent anti-inflammatory action. Thus, VIP and PACAP inhibit the expression and release of proinflammatory cytokines and chemokines, and enhance the production of the anti-inflammatory factors. These effects were reported both in vitro and in vivo, are mediated by the presence of PAC1, VPAC1, and VPAC2 receptors, in the three models of diseases used. The next step was that the system favors Th2 responses versus Th1 contributing to the remission of illness as rheumatoid arthritis or Crohn's disease by blocking the autoimmune component of these diseases. Because it appears that inflammatory processes requires more than blockade of a single mediator, new therapies blocking several components of both the infection- and the autoimmunity-induced inflammation cascades should be an interesting focus of attention. In this sense, at present we are trying to dissect new aspects of the potential therapeutic of the VIP/PACAP system in the control of CC and CXC chemokine and their receptors, coagulation factors, adhesion molecules, acute phase proteins, and osteoclastogenesis mediators as well as in the modulation of the expression of Toll-like receptors. Our more recent data open a hopeful door for the therapeutic use of VIP/PACAP in humans.

VIP decreases TLR4 expression induced by LPS and TNF-alpha treatment in human synovial fibroblasts.

It has been demonstrated that VIP produces beneficial effects both in a murine model of rheumatoid arthritis and in human rheumatoid synovial fibroblasts through the modulation of proinflammatory mediators. Toll-like receptors (TLRs) play a key role in the immediate recognition of microbial surface components by immune cells prior to the development of adaptative microbe-specific immune responses. In this study, we demonstrate that VIP decreases lipopolysaccharide (LPS) and TNF-alpha-induced expression of TLR4 and its correlation with the production of CCL2 and CXCL8 chemokines in human synovial fibroblasts from patients with rheumatoid arthritis and osteoarthritis. Our results add a new step for the use of VIP, as a promising candidate, for the treatment of rheumatoid arthritis.

VIP and tolerance induction in autoimmunity.

Vasoactive intestinal peptide (VIP) is a potent anti-inflammatory agent with immunoregulatory properties, skewing the immune response to a Th2 pattern of cytokine production. Here, we studied the effect of treatment with VIP in the development of diabetes in nonobese diabetic (NOD) mice, an animal model of type 1 diabetes. Mice treated with VIP from 4 weeks of age did not develop diabetes and showed milder insulitis than nontreated mice. The protective mechanism of VIP was associated with a reduction in the circulating levels of Th1 cytokines. In the pancreas of VIP-treated animals, regulatory T cell markers predominate, as indicated by the upregulation of FoxP3 and transforming growth factor-beta (TGF-beta), and the downregulation of the transcription factor, T-bet. These findings indicate that VIP restores tolerance to pancreatic islets by promoting the local differentiation and function of regulatory T cells.

Defective signalling in salivary glands precedes the autoimmune response in the non-obese diabetic mouse model of sialadenitis.

The spontaneous non-obese diabetic (NOD) mouse model of Sjögren's syndrome provides a valuable tool to study the onset and progression of both the autoimmune response and secretory dysfunction. Our purpose was to analyse the temporal decline of salivary secretion in NOD mice in relation to the autoimmune response and alterations in various signalling pathways involved in saliva secretion within each salivary gland. A progressive loss of nitric oxide synthase activity in submandibular and parotid glands started at 12 weeks of age and paralleled the decline in salivary secretion. This defect was associated with a lower response to vasoactive intestinal peptide in salivary flow rate, cAMP and nitric oxide/cGMP production. No signs of mononuclear infiltrates or local cytokine production were detectable in salivary glands in the time period studied (10-16 weeks of age). Our data support a disease model for sialadenitis in NOD mice in which the early stages are characterized by defective neurotransmitter-mediated signalling in major salivary glands that precedes the autoimmune response.

Vasoactive intestinal peptide modulates proinflammatory mediator synthesis in osteoarthritic and rheumatoid synovial cells.

OBJECTIVE: Vasoactive intestinal peptide (VIP) has demonstrated beneficial effects in several murine models of immune-mediated inflammation by inhibiting both the inflammatory and the autoimmune components of the disease. We investigate its potential to modulate the release of proinflammatory cytokines and chemokines by human synovial cells from patients with rheumatoid arthritis (RA). METHODS: Fresh suspensions of synovial tissue cells (STC) or cultured fibroblast-like synoviocytes (FLS) were obtained from patients with RA or osteoarthritis (OA). The effects of VIP on basal or tumour necrosis factor alpha (TNF-alpha)-stimulated production of CCL2 (MCP-1, monocyte chemotactic protein 1), CXCL8 [interleukin (IL)-8], IL-6 and TNF-alpha were studied by specific ELISAs (enzyme-linked immunosorbent assays). The mRNAs for CCL2, CXCL8 and IL-6 in FLS were analysed by real-time reverse transcription-polymerase chain reaction. RESULTS: VIP at 10 nm down-regulated chemokine production by STC and FLS from RA and OA patients. VIP also down-regulated the expression of mRNAs for CCL2, CXCL8 and IL-6. The effects of VIP were more clearly detected in RA samples and after stimulation with TNF-alpha. CONCLUSION: Our observations confirm that the proposed anti-inflammatory actions of VIP in murine models also apply to human synovial cells ex vivo. Further studies are encouraged to evaluate the use of VIP as a potential therapy for chronic inflammatory joint diseases.

Vasoactive intestinal peptide in the immune system: potential therapeutic role in inflammatory and autoimmune diseases.

Vasoactive intestinal peptide (VIP), a neuropeptide that is produced by lymphoid as well as neural cells, exerts a wide spectrum of immunological functions, controlling the homeostasis of the immune system through different receptors expressed in various immunocompetent cells. In the last decade, VIP has been clearly identified as a potent anti-inflammatory factor, which acts by regulating the production of both anti- and pro-inflammatory mediators. In this sense, VIP has been described to prevent death by septic shock, an acute inflammatory disease with a high mortality. In addition, VIP regulates the expression of co-stimulatory molecules, this being an action that may be related to modulating the shift toward Th1 and Th2 differentiation. We have recently reported that VIP prevents the deleterious effects of an experimental model of rheumatoid arthritis, by downregulating both inflammatory and autoimmune components of the disease. Therefore, VIP has been proposed as a promising candidate alternative treatment for acute and chronic inflammatory and autoimmune diseases such as septic shock, arthritis, multiple sclerosis, Crohn disease, or autoimmune diabetes.

Pituitary adenylate cyclase-activating polypeptide inhibits collagen-induced arthritis: an experimental immunomodulatory therapy.

Rheumatoid arthritis is a chronic, systemic, autoimmune, and inflammatory disorder that affects the synovial lining of the joints. We describe the beneficial effects of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the collagen-induced arthritis experimental murine model being proposed as a novel therapeutic approach in the treatment of rheumatoid arthritis. PACAP greatly decreases arthritis frequency and severity in the studied mice by improving clinical symptoms, ameliorating joint damage, and blocking both the inflammatory and autoimmune mediators which are the main keys of the pathogenesis of this disease. With this study, PACAP emerges as a promising candidate for the treatment of a pathology with a high world incidence but currently no effective treatment.

Vasoactive intestinal peptide prevents experimental arthritis by downregulating both autoimmune and inflammatory components of the disease.

Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease of unknown etiology, characterized by chronic inflammation in the joints and subsequent destruction of the cartilage and bone. We describe here a new strategy for the treatment of arthritis: administration of the neuropeptide vasoactive intestinal peptide (VIP). Treatment with VIP significantly reduced incidence and severity of arthritis in an experimental model, completely abrogating joint swelling and destruction of cartilage and bone. The therapeutic effect of VIP was associated with downregulation of both inflammatory and autoimmune components of the disease. Our data indicate VIP as a viable candidate for the development of treatments for RA.

Immunology of VIP: a review and therapeutical perspectives.

Vasoactive intestinal peptide (VIP) is a neuropeptide with a broad distribution in the body that exerts very important pleiotropic functions in several systems. The present work reviews the immunology of VIP. Being daring, this neuropeptide could be included in the group of cytokines since it is produced and secreted by different immunocompetent cells in response to various immune signals, plays a broad spectrum of immunological functions, and exerts them, in a paracrine and/or autocrine way, through three different specific receptors. Although VIP has been classically considered as an immunodepressant agent, and its main described role has been as an anti-inflammatory factor, several evidences suggest that a better way to see this peptide is as a modulator of the homeostasis of the immune system. In the last decade, the pharmacology of VIP has spectacularly grown, and VIP itself, as well as more stable VIP-derived agents, have been used or proposed as efficient therapeutical treatments of several disorders, specially inflammatory and autoimmune diseases, such as septic shock, rheumatoid arthritis, multiple sclerosis, Crohn's disease and autoimmune diabetes. A broad field of perspectives is actually open, and further investigations will help us to definitively understand the immunology of this very important peptide.

Anti-inflammatory properties of the type 1 and type 2 vasoactive intestinal peptide receptors: role in lethal endotoxic shock.

Vasoactive intestinal peptide (VIP) is a neuropeptide that can modulate several immune aspects. Previous reports showed that VIP attenuates the deleterious consequences of septic shock by inhibiting the production of pro-inflammatory agents and stimulating the production of anti-inflammatory cytokines in activated macrophages. In this study, by using selective VIP agonists, we investigated the differential involvement of the VIP receptors in the anti-inflammatory role of VIP. Both the type 1 VIP receptor (VPAC1) agonist, [K(15), R(16), L(27)] VIP 1-7-GRF 8-27, and the type 2 VIP receptor (VPAC2) agonist, Ro25-1553, protected mice from lethal endotoxemia by inhibiting the macrophage-derived pro-inflammatory mediators IL-6, TNF-alpha, IL-12 and NO, and by stimulating the production of the anti-inflammatory cytokine IL-10. In addition, both VIP and VPAC1 agonist, but not the VPAC2 agonist, reduced in vitro and in vivo the expression of the co-stimulatory B7. 1/B7.2 molecules, and the subsequent stimulatory activity for T helper cells in stimulated macrophages. The higher effectiveness of the VPAC1 agonist compared with the VPAC2 agonist suggests that VPAC1 is the major mediator of the anti-inflammatory action of VIP. Since VIP and the two agonists appear to affect multiple cytokines and inflammatory factors, they might provide a more efficient therapeutical alternative to the use of specific cytokine antibodies or antagonists.

VIP and PACAP induce shift to a Th2 response by upregulating B7.2 expression.

VIP and PACAP, two structurally related neuropeptides produced within the lymphoid microenvironment, modulate several immunological functions. Although primarily anti-inflammatory in nature, VIP and PACAP also affect resting macrophages. In this study, we report on the role of VIP and PACAP on macrophage B7 expression and costimulatory function for antigen-primed CD4+ T cells, and on the macrophage-induced regulation of Th1/Th2 differentiation in vitro and in vivo. VIP and PACAP upregulate B7.2, but not B7.1, MHC class II, or ICAM-1 expression, and activate macrophages to stimulate the proliferation of naïve T cells in response to soluble anti-CD3 or allogeneic stimulation. The stimulatory effect is mediated through the specific receptor VPAC1, and involves the cAMP/PKA pathway as second messengers. The enhancement in B7.2 expression occurs at both mRNA and protein levels, and correlates with the VIP/PACAP induced upregulation of the costimulatory activity of macrophages for antigen-primed CD4+ T cells. VIP/PACAP-treated macrophages gain the ability to induce Th2-type cytokines such as IL-4 and IL-5, and to reduce Th1-type cytokines such as IFN gamma and IL-2. In vivo administration of VIP or PACAP in antigen-immunized mice reduces the numbers of IFN gamma-secreting cells, enhances the numbers of IL-4-secreting cells, and affects the pattern of antigen-specific Ig isotypes. The preferential differentiation into Th2 effector cells induced by VIP/PACAP-treated macrophages is mediated through the upregulation of B7.2 expression. Since Th1-dominated responses are associated with some autoimmune diseases and inflammatory reactions typical of cell-mediated immunity, the VIP/PACAP inhibition of Th1 development may represent an additional mechanism for the general anti-inflammatory activity of the two neuropeptides.

Receptors and transcriptional factors involved in the anti-inflammatory activity of VIP and PACAP.

VIP and PACAP modulate the function of inflammatory cells through specific receptors. VIP/PACAP inhibit the production of TNF alpha, IL-6, IL-12, and nitric oxide (NO), and stimulate IL-10 in peritoneal macrophages and Raw 264.7 cells. Here we report on the specific VIP/PACAP receptors, transduction pathways, and transcriptional factors involved in the regulation of these macrophage factors by VIP and PACAP. Both neuropeptides inhibit IL-6 production mainly through PAC1 binding, PKC activation, and the subsequent shedding of the LPS receptor CD14 in macrophages. However, the effects on TNF alpha, IL-10, IL-12, and NO are mostly mediated through the constitutively expressed VPAC1 receptor, although the inducible expressed VPAC2 may also participate. VIP/PACAP binding to VPAC1 induces both a cAMP-dependent and a cAMP-independent pathways that regulate cytokine and NO production at the transcriptional level. VIP/PACAP inhibit TNF alpha through reduction in NFkB binding and changes in the composition of CRE-binding complexes; they inhibit IL-12 through reduction in NFkB binding and changes in the composition of the ets-2 complexes. VIP/PACAP inhibit iNOS expression through reduction in NFkB and IRF-1 binding, and augment IL-10 by increasing CREB-binding. Whereas the inhibition of IRF-1 and CRE-binding complexes seems to be mediated through the cAMP-dependent pathway, VIP/PACAP inhibition of NFkB nuclear translocation is mediated through a reduction in IkB alpha degradation mediated by the cAMP-independent pathway. This study provides new evidence for the understanding of the molecular mechanism by means of which VIP and PACAP attenuate the inflammatory response.

VIP and PACAP differentially regulate the costimulatory activity of resting and activated macrophages through the modulation of B7.1 and B7.2 expression.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP), two structurally related neuropeptides produced and/or released within the lymphoid microenvironment, modulate numerous immune functions. Although primarily antiinflammatory in nature, VIP and PACAP also affect resting macrophages. In this study, we report on in vitro and in vivo dual effects of VIP/PACAP on the expression of B7.1 and B7.2 and on the costimulatory activity for T cells in unstimulated and LPS/IFN-gamma-activated macrophages. VIP and PACAP up-regulate B7.2, but not B7.1, expression and induce the capacity to stimulate the proliferation of naive T cells in response to soluble anti-CD3 or allogeneic stimulation. In contrast, both neuropeptides down-regulate B7.1/B7.2 expression on LPS/IFN-gamma-activated macrophages and inhibit the endotoxin-induced costimulatory activity for T cells. Interestingly, both the stimulatory and the inhibitory effects of VIP/PACAP are mediated through the specific receptor VPAC1 and involve the cAMP/protein kinase A transduction pathway. The dual effect on B7.1 and B7.2 expression occurs at both mRNA and protein level and correlates with the VIP/PACAP regulation of the macrophage costimulatory activity. Through their regulatory role for resting and activated macrophages, VIP and PACAP act as endogenous participants in the control of immune homeostasis. Their effects depend not only on the timing of their release, but also on the activation and differentiation state of the neighboring immune cells.

Shedding of membrane-bound CD14 from lipopolysaccharide-stimulated macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide.

Macrophage activation and deactivation play essential roles in the initiation and maintenance of a successful immune response. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP), two structurally related neuropeptides, act as macrophage deactivating factors. We reported previously that VIP and PACAP inhibit IL-6, IL-12, TNF alpha and NO production, and enhance IL-10 production, from lipopolysaccharide (LPS)-stimulated macrophages. In this study, we demonstrate that VIP and PACAP down-regulate the expression of CD14, the membrane-bound LPS receptor, by inducing its rapid shedding. The soluble CD14 released by VIP and PACAP corresponds in size to the soluble CD14 released by PMA. Neither VIP/PACAP nor PMA, affect the steady-state levels of CD14 mRNA. The CD14 shedding induced by VIP/PACAP is mediated through the PAC1 specific receptors and the major transduction pathway involves the protein kinase C (PKC). The VIP/PACAP inhibition of TNF alpha and NO occurs through both CD14-dependent and -independent mechanisms, whereas the inhibition of IL-6 production appears to be strictly CD14-dependent. The shedding of CD14 by VIP and PACAP represents an important mechanism by which these neuropeptides limit the macrophage inflammatory response.