Administration of an AAV vector coding for a P2X7-blocking nanobody-based biologic ameliorates colitis in mice.

BACKGROUND: The pro-inflammatory ATP-gated P2X7 receptor is widely expressed by immune and non-immune cells. Nanobodies targeting P2X7, with potentiating or antagonistic effects, have been developed. Adeno-associated virus (AAV)-mediated gene transfer represents an efficient approach to achieve long-term in vivo expression of selected nanobody-based biologics. This approach (AAVnano) was used to validate the relevance of P2X7 as a target in dextran sodium sulfate (DSS)-induced colitis in mice. RESULTS: Mice received an intramuscular injection of AAV vectors coding for potentiating (14D5-dimHLE) or antagonistic (13A7-Fc) nanobody-based biologics targeting P2X7. Long-term modulation of P2X7 activity was evaluated ex vivo from blood samples. Colitis was induced with DSS in mice injected with AAV vectors coding for nanobody-based biologics. Severity of colitis, colon histopathology and expression of chemokines and cytokines were determined to evaluate the impact of P2X7 modulation. A single injection of an AAV vector coding for 13A7-Fc or 14D5-dimHLE efficiently modulated P2X7 function in vivo from day 15 up to day 120 post-injection in a dose-dependent manner. An AAV vector coding for 13A7-Fc significantly ameliorated DSS-induced colitis and significantly reduced immune cell infiltration and expression of chemokines and proinflammatory cytokines in colonic tissue. CONCLUSIONS: We have demonstrated the validity of AAVnano methodology to modulate P2X7 functions in vivo. Applying this methodological approach to a DSS-induced colitis model, we have shown that P2X7 blockade reduces inflammation and disease severity. Hence, this study confirms the importance of P2X7 as a pharmacological target and suggests the use of nanobody-based biologics as potential therapeutics in inflammatory bowel disease.

Icos gene disruption in non-obese diabetic mice elicits myositis associated with anti-troponin T3 autoantibodies.

AIMS: Idiopathic inflammatory myopathies (IIM) are autoimmune inflammatory disorders leading to skeletal muscle weakness and disability. The pathophysiology of IIM is poorly understood due to the scarcity of animal disease models. Genetic deletion of Icos or Icosl (inducible T cell co-stimulator/ligand) in non-obese diabetic (NOD) mice leads to muscle disease. Our aim was to characterise Icos-/- NOD myopathy and to search for novel autoantibodies (aAbs) in this model. METHODS: Diabetes, weight, myopathy incidence/clinical score and grip strength were assessed over time. Locomotor activity was analysed with the Catwalk XT gait analysis system. Muscle histology was evaluated in haematoxylin/eosin and Sirius red-stained sections, and immune infiltrates were characterised by immunofluorescence and flow cytometry. 2D gel electrophoresis of muscle protein extracts and mass spectrometry were used to identify novel aAbs. NOD mice were immunised with troponin T3 (TNNT3) in incomplete Freund's adjuvant (IFA) and R848. An addressable laser bead immunoassay (ALBIA) was developed to measure aAb IgG serum levels. RESULTS: Icos-/- NOD mice did not exhibit diabetes but developed spontaneous progressive myositis with decreased muscle strength and altered locomotor activity. Muscle from these mice exhibited myofibre necrosis, myophagocytosis, central nuclei, fibrosis and perimysial and endomysial cell infiltrates with macrophages and T cells. We identified anti-TNNT3 aAbs in diseased mice. Immunisation of NOD mice with murine TNNT3 protein led to myositis development, supporting its pathophysiological role. CONCLUSIONS: These data show that Icos-/- NOD mice represent a spontaneous model of myositis and the discovery of anti-TNNT3 aAb suggests a new autoantigen in this model.

Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase.

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA.

Systemic administration of orexin A ameliorates established experimental autoimmune encephalomyelitis by diminishing neuroinflammation.

BACKGROUND: Orexins (hypocretins, Hcrt) A and B are GPCR-binding hypothalamic neuropeptides known to regulate sleep/wake states and feeding behavior. A few studies have shown that orexin A exhibits anti-inflammatory and neuroprotective properties, suggesting that it might provide therapeutic effects in inflammatory and neurodegenerative diseases like multiple sclerosis (MS). In MS, encephalitogenic Th1 and Th17 cells trigger an inflammatory response in the CNS destroying the myelin sheath. Here, we investigated the effects of peripheral orexin A administration to mice undergoing experimental autoimmune encephalomyelitis (EAE), a widely used model of MS. METHODS: Mice were subcutaneously immunized with myelin oligodendrocyte glycoprotein peptide (MOG)35-55 in CFA. Mice were treated intraperitoneally for five consecutive days with either PBS or 300 µg of orexin A starting at a moderate EAE score. Molecular, cellular, and histological analysis were performed by real-time PCR, ELISA, flow cytometry, and immunofluorescence. RESULTS: Orexin A strongly ameliorated ongoing EAE, limiting the infiltration of pathogenic CD4+ T lymphocytes, and diminishing chemokine (MCP-1/CCL2 and IP-10/CXCL10) and cytokine (IFN-γ (Th1), IL-17 (Th17), TNF-α, IL-10, and TGF-ß) expressions in the CNS. Moreover, orexin A treatment was neuroprotective, decreasing demyelination, astrogliosis, and microglial activation. Despite its strong local therapeutic effects, orexin A did not impair peripheral draining lymph node cell proliferation and Th1/Th17 cytokine production in response to MOG35-55 in vitro. CONCLUSIONS: Peripherally-administered orexin A ameliorated EAE by reducing CNS neuroinflammation. These results suggest that orexins may represent new therapeutic candidates that should be further investigated for MS treatment.

VPAC1 receptor (Vipr1)-deficient mice exhibit ameliorated experimental autoimmune encephalomyelitis, with specific deficits in the effector stage.

BACKGROUND: Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two highly homologous neuropeptides. In vitro and ex vivo experiments repeatedly demonstrate that these peptides exert pronounced immunomodulatory (primarily anti-inflammatory) actions which are mediated by common VPAC1 and VPAC2 G protein-coupled receptors. In agreement, we have shown that mice deficient in PACAP ligand or VPAC2 receptors exhibit exacerbated experimental autoimmune encephalomyelitis (EAE). However, we observed that VIP-deficient mice are unexpectedly resistant to EAE, suggesting a requirement for this peptide at some stage of disease development. Here, we investigated the involvement of VPAC1 in the development of EAE using a VPAC1-deficient mouse model. METHODS: EAE was induced in wild-type (WT) and VPAC1 knockout (KO) mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55), and clinical scores were assessed continuously over 30 days. Immune responses in the spinal cords were determined by histology, real-time PCR and immunofluorescence, and in the draining lymph nodes by antigen-recall assays. The contribution of VPAC1 expression in the immune system to the development of EAE was evaluated by means of adoptive transfer and bone marrow chimera experiments. In other experiments, VPAC1 receptor analogs were given to WT mice. RESULTS: MOG35-55-induced EAE was ameliorated in VPAC1 KO mice compared to WT mice. The EAE-resistant phenotype of VPAC1 KO mice correlated with reduced central nervous system (CNS) histopathology and cytokine expression in the spinal cord. The immunization phase of EAE appeared to be unimpaired because lymph node cells from EAE-induced VPAC1 KO mice stimulated in vitro with MOG exhibited robust proliferative and Th1/Th17 responses. Moreover, lymph node and spleen cells from KO mice were fully capable of inducing EAE upon transfer to WT recipients. In contrast, WT cells from MOG-immunized mice did not transfer the disease when administered to VPAC1 KO recipients, implicating a defect in the effector phase of the disease. Bone marrow chimera studies suggested that the resistance of VPAC1-deficient mice was only minimally dependent on the expression of this receptor in the immunogenic/hematopoietic compartment. Consistent with this, impaired spinal cord inductions of several chemokine mRNAs were observed in VPAC1 KO mice. Finally, treatment of WT mice with the VPAC1 receptor antagonist PG97-269 before, but not after, EAE induction mimicked the clinical phenotype of VPAC1 KO mice. CONCLUSIONS: VPAC1 gene loss impairs the development of EAE in part by preventing an upregulation of CNS chemokines and invasion of inflammatory cells into the CNS. Use of VPAC1 antagonists in WT mice prior to EAE induction also support a critical role for VPAC1 signaling for the development of EAE.

VPAC2 (vasoactive intestinal peptide receptor type 2) receptor deficient mice develop exacerbated experimental autoimmune encephalomyelitis with increased Th1/Th17 and reduced Th2/Treg responses.

Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two structurally-related neuropeptides with widespread expression in the central and peripheral nervous systems. Although these peptides have been repeatedly shown to exert potent anti-inflammatory actions when administered in animal models of inflammatory disease, mice deficient in VIP and PACAP were recently shown to exhibit different phenotypes (ameliorated and exacerbated, respectively) in response to experimental autoimmune encephalomyelitis (EAE). Therefore, elucidating what are the specific immunoregulatory roles played by each of their receptor subtypes (VPAC1, VPAC2, and PAC1) is critical. In this study, we found that mice with a genetic deletion of VIPR2, encoding the VPAC2 receptor, exhibited exacerbated (MOG35-55)-induced EAE compared to wild type mice, characterized by enhanced clinical and histopathological features, increased proinflammatory cytokines (TNF-α, IL-6, IFN-γ (Th1), and IL-17 (Th17)) and reduced anti-inflammatory cytokines (IL-10, TGFß, and IL-4 (Th2)) in the CNS and lymph nodes. Moreover, the abundance and proliferative index of lymph node, thymus and CNS CD4(+)CD25(+)FoxP3(+) Tregs were strikingly reduced in VPAC2-deficient mice with EAE. Finally, the in vitro suppressive activity of lymph node and splenic Tregs from VPAC2-deficient mice was impaired. Overall, our results demonstrate critical protective roles for PACAP and the VPAC2 receptor against autoimmunity, promoting the expansion and maintenance of the Treg pool.

Vasoactive intestinal peptide-deficient mice exhibit reduced pathology in trinitrobenzene sulfonic acid-induced colitis.

OBJECTIVES: Vasoactive intestinal peptide (VIP) is an immunomodulatory neuropeptide with therapeutic properties in multiple murine models of inflammatory disease including the trinitrobenzene-sulfonic acid (TNBS)-colitis model of Crohn's disease. Understanding the spectrum of biological actions of endogenously produced VIP may help us dissect the complex and multifactorial pathogenesis of such inflammatory diseases. Our goal was to determine the contribution of endogenously produced VIP to TNBS-colitis by using VIP knockout (KO) mice. METHODS: TNBS was intracolonically administered to wild-type (WT) and VIP KO mice, and weight loss and colitis were assessed over time. Colon histopathological changes and myeloperoxidase activities were analyzed and the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in colon and serum quantified. The proliferative response in vitro of splenocytes from TNBS WT and VIP KO administered mice to anti-CD3 and anti-CD28 was determined. RESULTS: VIP KO mice did not exhibit the predicted exacerbated response to TNBS. Instead, they developed a milder clinical profile than WT mice, with lower TNF-α and IL-6 levels. Such potential defects seem selective, because other parameters such as the histopathological scores and the cytokine levels in the colon did not differ between the two strains of mice. Moreover, splenocytes from TNBS-treated VIP KO mice exhibited an enhanced proliferative response to anti-CD3/CD28 stimulation in vitro. CONCLUSION: Chronic loss of VIP in mice leads to a disruption of certain but not all immunological compartments, corroborating recent findings that VIP KO mice exhibit reduced mortality in the lipopolysaccharide-induced endotoxemia model and attenuated clinical development of experimental autoimmune encephalomyelitis while developing robust T-cell responses.

Neuropeptide PACAP in mouse liver ischemia and reperfusion injury: immunomodulation by the cAMP-PKA pathway.

UNLABELLED: Hepatic ischemia and reperfusion injury (IRI), an exogenous antigen-independent local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The immune system and the nervous system maintain extensive communication and mount a variety of integrated responses to danger signals through intricate chemical messengers. This study examined the function and potential therapeutic potential of neuropeptide pituitary adenylate cyclase-activating polypeptides (PACAP) in a murine model of partial liver "warm" ischemia (90 minutes) followed by reperfusion. Liver IRI readily triggered the expression of intrinsic PACAP and its receptors, whereas the hepatocellular damage was exacerbated in PACAP-deficient mice. Conversely, PACAP27, or PACAP38 peptide monotherapy, which elevates intracellular cyclic adenosine monophosphate/protein kinase A (cAMP-PKA) signaling, protected livers from IRI, as evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture. The liver protection rendered by PACAP peptides was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and selectively augmented hepatic interleukin (IL)-10 expression. Strikingly, PKA inhibition readily restored liver damage in otherwise IR-resistant, PACAP-conditioned mice. In vitro, PACAP treatment not only diminished macrophage tumor necrosis factor alpha/IL-6/IL-12 levels in a PKA-dependent manner, but also prevented necrosis and apoptosis in primary mouse hepatocyte cultures. CONCLUSION: Our novel findings document the importance of PACAP-mediated cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. Because the enhancement of neural modulation differentially regulates local inflammation and prevents hepatocyte death, these results provide the rationale for novel approaches to manage liver inflammation and IRI in transplant patients.

IFNγ causes mitochondrial dysfunction and oxidative stress in myositis.

Idiopathic inflammatory myopathies (IIMs) are severe autoimmune diseases with poorly understood pathogenesis and unmet medical needs. Here, we examine the role of interferon γ (IFNγ) using NOD female mice deficient in the inducible T cell co-stimulator (Icos), which have previously been shown to develop spontaneous IFNγ-driven myositis mimicking human disease. Using muscle proteomic and spatial transcriptomic analyses we reveal profound myofiber metabolic dysregulation in these mice. In addition, we report muscle mitochondrial abnormalities and oxidative stress in diseased mice. Supporting a pathogenic role for oxidative stress, treatment with a reactive oxygen species (ROS) buffer compound alleviated myositis, preserved muscle mitochondrial ultrastructure and respiration, and reduced inflammation. Mitochondrial anomalies and oxidative stress were diminished following anti-IFNγ treatment. Further transcriptomic analysis in IIMs patients and human myoblast in vitro studies supported the link between IFNγ and mitochondrial dysfunction observed in mice. These results suggest that mitochondrial dysfunction, ROS and inflammation are interconnected in a self-maintenance loop, opening perspectives for mitochondria therapy and/or ROS targeting drugs in myositis.

Vasoactive intestinal peptide loss leads to impaired CNS parenchymal T-cell infiltration and resistance to experimental autoimmune encephalomyelitis.

The neuropeptide vasoactive intestinal peptide (VIP) has been shown to inhibit macrophage proinflammatory actions, promote a positive Th2/Th1 balance, and stimulate regulatory T-cell production. The fact that this peptide is highly efficacious in animal models of inflammatory diseases such as collagen-induced arthritis and experimental autoimmune encephalomyelitis (EAE) suggests that the endogenous peptide might normally provide protection against such pathologies. We thus studied the response of VIP-deficient (i.e., VIP KO) mice to myelin oligodendrocyte protein-induced EAE. Surprisingly, VIP KO mice were almost completely resistant to EAE, with delayed onset and mild or absent clinical profile. Despite this, flow cytometric analyses and antigen-rechallenge experiments indicated that myelin oligodendrocyte protein-treated VIP KO mice exhibited robust Th1/Th17 cell inductions and antigen-specific proliferation and cytokine responses. Moreover, adoptive transfer of lymphocytes from immunized VIP KO mice to WT recipients resulted in full-blown EAE, supporting their encephalitogenic potential. In contrast, transfer of encephalitogenic WT cells to VIP KO hosts did not produce EAE, suggesting that loss of VIP specifically affected the effector phase of the disease. Histological analyses indicated that CD4 T cells entered the meningeal and perivascular areas of VIP-deficient mice, but that parenchymal infiltration was strongly impaired. Finally, VIP pretreatment of VIP KO mice before immunization was able to restore their sensitivity to EAE. These results indicate that VIP plays an unanticipated permissive and/or proinflammatory role in the propagation of the inflammatory response in the CNS, a finding with potential therapeutic relevance in autoimmune neuroinflammatory diseases such as multiple sclerosis.

Pituitary adenylyl cyclase-activating polypeptide is an intrinsic regulator of Treg abundance and protects against experimental autoimmune encephalomyelitis.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a widely expressed neuropeptide originally discovered in the hypothalamus. It closely resembles vasoactive intestinal peptide (VIP), a neuropeptide well known to inhibit macrophage activity, promote Th2-type responses, and enhance regulatory T cell (Treg) production. Recent studies have shown that administration of PACAP, like VIP, can attenuate dramatically the clinical and pathological features of murine models of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis. However, specific roles (if any) of endogenous VIP and PACAP in the protection against autoimmune diseases have not been explored. Here, we subjected PACAP-deficient mice to myelin oligodendrocyte glycoprotein (MOG(35-55))-induced EAE. MOG immunization of PACAP-deficient mice triggered heightened clinical and pathological manifestations of EAE compared to wild-type mice. The increased sensitivity was accompanied by enhanced mRNA expression of proinflammatory cytokines (TNFalpha, IL-6, IFN-gamma, IL-12p35, IL-23p19, and IL-17), chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, and RANTES/CCL5), and chemotactic factor receptors (CCR1, CCR2, and CCR5), but downregulation of the anti-inflammatory cytokines (IL-4, IL-10, and TGF-beta) in the spinal cord. Moreover, the abundance of CD4(+)CD25(+)FoxP3(+) Tregs in lymph nodes and levels of FoxP3 mRNA in the spinal cord were also diminished. The reduction in Tregs was associated with increased proliferation and decreased TGF-beta secretion in lymph node cultures stimulated with MOG. These results demonstrate that endogenous PACAP provides protection in EAE and identify PACAP as an intrinsic regulator of Treg abundance after inflammation.

Evaluation of nanobody-based biologics targeting purinergic checkpoints in tumor models in vivo.

Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. ATP promotes tumor growth but also anti-tumor immune responses notably via the P2X7 receptor. ATP can also be catabolized by CD39 and CD73 ecto-enzymes into immunosuppressive adenosine. P2X7, CD39 and CD73 have attracted much interest in cancer as targets offering the potential to unleash anti-tumor immune responses. These membrane proteins represent novel purinergic checkpoints that can be targeted by small drugs or biologics. Here, we investigated nanobody-based biologics targeting mainly P2X7, but also CD73, alone or in combination therapies. Blocking P2X7 inhibited tumor growth and improved survival of mice in cancer models that express P2X7. P2X7-potentiation by a nanobody-based biologic was not effective alone to control tumor growth but enhanced tumor control and immune responses when used in combination with oxaliplatin chemotherapy. We also evaluated a bi-specific nanobody-based biologic that targets PD-L1 and CD73. This novel nanobody-based biologic exerted a potent anti-tumor effect, promoting tumor rejection and improving survival of mice in two tumor models. Hence, this study highlights the importance of purinergic checkpoints in tumor control and open new avenues for nanobody-based biologics that may be further exploited in the treatment of cancer.

Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates.

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.

Evaluation of P2X7 Receptor Function in Tumor Contexts Using rAAV Vector and Nanobodies (AAVnano).

Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. Extracellular ATP is known to signal through plasma membrane receptors of the P2Y and P2X families. Among the P2X receptors, P2X7 has attracted increasing interest in the field of inflammation as well as in cancer. P2X7 is expressed by immune cells and by most malignant tumor cells where it plays a crucial yet complex role that remains to be clarified. P2X7 activity has been associated with production and release of pro-inflammatory cytokines, modulation of the activity and survival of immune cells, and the stimulation of proliferation and migratory properties of tumor cells. Hence, P2X7 plays an intricate role in the tumor microenvironment combining beneficial and detrimental effects that need to be further investigated. For this, we developed a novel methodology termed AAVnano based on the use of Adeno-associated viral vectors (AAV) encoding nanobodies targeting P2X7. We discuss here the advantages of this tool to study the different functions of P2X7 in cancer and other pathophysiological contexts.

Orexins as Novel Therapeutic Targets in Inflammatory and Neurodegenerative Diseases.

Orexins [orexin-A (OXA) and orexin-B (OXB)] are two isoforms of neuropeptides produced by the hypothalamus. The main biological actions of orexins, focused on the central nervous system, are to control the sleep/wake process, appetite and feeding, energy homeostasis, drug addiction, and cognitive processes. These effects are mediated by two G protein-coupled receptor (GPCR) subtypes named OX1R and OX2R. In accordance with the synergic and dynamic relationship between the nervous and immune systems, orexins also have neuroprotective and immuno-regulatory (i.e., anti-inflammatory) properties. The present review gathers recent data demonstrating that orexins may have a therapeutic potential in several pathologies with an immune component including multiple sclerosis, Alzheimer's disease, narcolepsy, obesity, intestinal bowel diseases, septic shock, and cancers.

Induction of colitis and rapid development of colorectal tumors in mice deficient in the neuropeptide PACAP.

Pituitary adenylyl cyclase activating peptide (PACAP) is expressed in central, sensory, autonomic, and enteric neurons. Although it classically acts as a neurotransmitter/neuromodulator, recent studies indicate that PACAP can also regulate immune function. To this effect, PACAP has been shown to reduce clinical symptoms and inflammation in mouse models of human immune-based diseases such as rheumatoid arthritis, Crohn's Disease, septic shock and multiple sclerosis. Despite these findings, the role of the endogenous peptide in regulating immune function is unknown. To determine if endogenous PACAP plays a protective role in inflammatory bowel disease (IBD) and IBD-associated colorectal cancer in mice, PACAP-deficient (KO) mice were subjected to 3 cycles of dextran sulfate sodium (DSS) in drinking water over 2 months, an established mouse model for colitis. Compared to wild type (WT) controls, PACAP KO mice exhibited more severe clinical symptoms of colitis and had significantly higher colonic inflammation on pathological examination. Moreover, 60% of the PACAP KO mice developed colorectal tumors with an aggressive-appearing pathology. Consistent with published data, DSS-treated WT mice did not develop such tumors. The results demonstrate a new mouse model which rapidly develops inflammation-associated colorectal cancer in the absence of a carcinogen.

Mice deficient in both pituitary adenylyl cyclase-activating polypeptide and vasoactive intestinal peptide survive, but display growth retardation and sex-dependent early death.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two closely related neuropeptides exhibiting overlapping activities which have actions on almost every organ system of the body. To determine if these peptides exert essential but redundant functions, we interbred VIP- and PACAP-deficient mice to obtain VIP/PACAP double knockout (DKO) mice. DKO mice had normal birth weights and survived to weaning, but exhibited a dramatic postnatal growth rate reduction. Analyses at postnatal day 16 indicated that all organs examined except the brain were reduced in mass by 40-70% compared to mixed background controls, with the thymus and spleen most profoundly affected. Brain size was also significantly reduced, but by only 10%. The reduced growth rate of DKO mice was associated with reduced serum concentrations of insulin-like growth hormone-1 (IGF-1), but unchanged levels of growth hormone. Despite the normal survival of DKO mice up to the weaning stage, many subsequently experienced early sudden death, with only 48% of females and 82% of males surviving past 6 months. The results indicate that a significant percentage of mice deficient in both VIP and PACAP survive to adulthood, but their growth rate is profoundly affected, and that females in particular exhibit high rate of mortality after about 3 months of age.

Vasoactive intestinal peptide is critical for circadian regulation of glucocorticoids.

BACKGROUND/AIMS: Circadian control of behavior and physiology is a central characteristic of all living organisms. The master clock in mammals resides in the hypothalamus, where the suprachiasmatic nucleus (SCN) synchronizes daily rhythms. A variety of recent evidence indicates that the neuropeptide vasoactive intestinal peptide (VIP) is critical for normal functioning of the SCN. The aim of our study was to examine the possible role of VIP in driving circadian rhythms in the hypothalamic-pituitary-adrenal axis. METHODS: Circulating ACTH and corticosterone concentrations were determined by round-the-clock sampling under diurnal and circadian conditions. The responsive aspects of the hypothalamic-pituitary-adrenal axis were tested by application of acute stress by footshock and light. RESULTS: We demonstrate that the circadian rhythms in ACTH and corticosterone are lost in VIP-deficient mice. The ability of light to induce a corticosterone response was also compromised in the mutant mice, as was photic induction of Per1 in the adrenal glands. In contrast, the acute stress response was apparently unaltered by the loss of VIP. CONCLUSION: Thus, our data demonstrate that VIP is essential for the circadian regulation of an otherwise intact hypothalamic-pituitary-adrenal axis.

Immunomodulatory Roles of PACAP and VIP: Lessons from Knockout Mice.

A bidirectional cross-talk is established between the nervous and immune systems through common mediators including neuropeptides, neurotransmitters, and cytokines. Among these, PACAP and VIP are two highly related neuropeptides widely distributed in the organism with purported immunomodulatory actions. Due to their well-known anti-inflammatory properties, administration of these peptides has proven to be beneficial in models of acute and chronic inflammatory diseases. Nevertheless, the relevance of the endogenous source of these peptides in the modulation of immune responses remains to be elucidated. The development of transgenic mice with specific deletions in the genes coding for these neuropeptides (Vip and Adcyap1) or for their G-protein-coupled receptors VPAC1, VPAC2, and PAC1 (Vipr1, Vipr2, Adcyap1r1) has allowed to address this question, underscoring the complexity of the immunoregulatory properties of PACAP and VIP. The goal of this review is to integrate the existing information on the immune phenotypes of mice deficient for PACAP, VIP, or their receptors, to provide a global view on the roles of these endogenous neuropeptides during immunological health and disease.