Dietary fat differentially modulates the response of bone marrow-derived macrophages to TLR4 and NOD2 agonists.

Consumption of diets high in fat has been linked to the development of obesity and related metabolic complications. Such associations originate from the enhanced, chronic, low-grade inflammation mediated by macrophages in response to translocated bacteria, bacterial products, or dietary constituents such as fatty acids (FAs). Nucleotide-binding Oligomerization Domain 2 (NOD2) senses muramyl dipeptide (MDP), a component of bacterial peptidoglycan. The inability to sense peptidoglycan through NOD2 has been demonstrated to lead to dysbiosis, increased bacterial translocation, inflammation and metabolic dysfunction. Currently, it is unknown how consumption of HFDs with different FA compositions might influence NOD2-dependent responses. In this study, we subjected WT mice to a control diet or to HFDs comprised of various ratios of unsaturated to saturated fats and determined the macrophage response to TLR4 and NOD2 agonists. A HFD with equal ratios of saturated and unsaturated fats enhanced subsequent responsiveness of macrophages to LPS but not to MDP. However, a high-unsaturated fat diet (HUFD) or a high-saturated fat diet (HSFD) both decreased the responsiveness to NOD2 agonists compared to that observed in control diet (CD) fed mice. These data suggest that dietary fatty acid composition can influence the subsequent macrophage responsiveness to bacterial products.

LMAN1 is a receptor for house dust mite allergens.

Development of therapies with the potential to change the allergic asthmatic disease course will require the discovery of targets that play a central role during the initiation of an allergic response, such as those involved in the process of allergen recognition. We use a receptor glycocapture technique to screen for house dust mite (HDM) receptors and identify LMAN1 as a candidate. We verify the ability of LMAN1 to directly bind HDM allergens and demonstrate that LMAN1 is expressed on the surface of dendritic cells (DCs) and airway epithelial cells (AECs) in vivo. Overexpression of LMAN1 downregulates NF-κB signaling in response to inflammatory cytokines or HDM. HDM promotes binding of LMAN1 to the FcRγ and recruitment of SHP1. Last, peripheral DCs of asthmatic individuals show a significant reduction in the expression of LMAN1 compared with healthy controls. These findings have potential implications for the development of therapeutic interventions for atopic disease.

Intrarectal Xyloglucan Administration Reduces Disease Severity in the Dextran Sodium Sulfate Model of Mouse Colitis.

BACKGROUND: The pathophysiology of inflammatory bowel diseases remains poorly understood and treatment remains suboptimal for many patients. We hypothesize that the inflammatory milieu secondarily prolongs the injury and attenuates healing. We propose primary or adjuvant therapy with biocompatible adhesives to restore a barrier to protect submucosal structures, particularly stem cells. METHODS: We used the well-described mouse dextran sodium sulfate (DSS) model of colitis resembling human ulcerative colitis to test the therapeutic efficacy of intrarectal administration of the tamarind plant-derived xyloglucan (TXG) polymer adhesive which underwent extensive analytic characterization. Mice in control, DSS-only, TXG-only, and DSS + TXG groups were studied for gross (weight, blood in stool, length of colon) and multiple histologic parameters. RESULTS: Compared to DSS-only mice, TXG prevented the weight loss, occurrence of blood in the stool and colon shortening, with all those parameters not being statistically different from treatment naïve animals. Histologically, there was dramatic and highly statistically significant reduction in the total inflammatory index and protection from goblet cell loss, cellular infiltration, crypt abscess formation, epithelial erosion, granulation tissue, epithelial hyperplasia crypt irregularity and crypt loss. The TXG purity and characterization were established by nuclear magnetic resonance, infrared spectroscopy, differential scanning calorimetry, and texture analysis. CONCLUSION: The striking attenuation of disease severity by intrarectal TXG use warrants future investigations of natural bioadhesives with well-established high safety profiles, and which could potentially be derivatized to include therapeutically active moieties for local drug delivery.

Immune Modulation of Allergic Asthma by Early Pharmacological Inhibition of RIP2.

Exposure to house dust mite (HDM) is highly associated with the development of allergic asthma. The adaptive immune response to HDM is largely Th2 and Th17 dominant, and a number of innate immune receptors have been identified that recognize HDM to initiate these responses. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a cytosolic sensor of peptidoglycan, which is important for Th2 and Th17 polarization. NOD2 mediates its signaling through its downstream effector kinase, receptor-interacting serine/threonine protein kinase 2 (RIP2). We have previously shown that RIP2 promotes HDM-associated allergic airway inflammation and Th2 and Th17 immunity, acting early in the HDM response and likely within airway epithelial cells. However, the consequences of inhibiting RIP2 during this critical period has not yet been examined. In this study, we pharmacologically inhibited RIP2 activity during the initial exposure to allergen in an acute HDM model of asthma and determined the effect on the subsequent development of allergic airway disease. We show that early inhibition of RIP2 was sufficient to reduce lung histopathology and local airway inflammation while reducing the Th2 immune response. Using a chronic HDM asthma model, we demonstrate that inhibition of RIP2, despite attenuating airway inflammation and airway remodeling, was insufficient to reduce airway hyperresponsiveness. These data demonstrate the potential of pharmacological targeting of this kinase in asthma and support further development and optimization of RIP2-targeted therapies.

RIP2 promotes FcγR-mediated reactive oxygen species production.

Receptor-interacting protein 2 (RIP2) is a kinase that mediates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. Genetic loss or pharmaceutical inhibition of RIP2 has been shown to be beneficial in multiple inflammatory disease models with the effects largely attributed to reducing proinflammatory signaling downstream of peptidoglycan recognition. However, given the widespread expression of this kinase and its reported interactions with numerous other proteins, it is possible that RIP2 may also function in roles outside of peptidoglycan sensing. In this work, we show that RIP2 undergoes tyrosine phosphorylation and activation in response to engagement of the Fc γ receptor (FcγR). Using bone marrow-derived macrophages from WT and RIP2-KO mice, we show that loss of RIP2 leads to deficient FcγR signaling and reactive oxygen species (ROS) production upon FcγR cross-linking without affecting cytokine secretion, phagocytosis, or nitrate/nitrite production. The FcγR-induced ROS response was still dependent on NOD2, as macrophages deficient in this receptor showed similar defects. Mechanistically, we found that different members of the Src family kinases (SFKs) can promote RIP2 tyrosine phosphorylation and activation. Altogether, our findings suggest that RIP2 is functionally important in pathways outside of bacterial peptidoglycan sensing and that involvement in such pathways may depend on the actions of SFKs. These findings will have important implications for future therapies designed to target this kinase.

Frontline Science: RIP2 promotes house dust mite-induced allergic airway inflammation.

House dust mites (HDMs) are one of the most significant environmental allergens in the establishment of the so-called "Atopic March." It is known that the immune response to HDM is Th2 dominant, but the innate mechanisms leading to HDM-induced type 2 responses are still not completely understood. A number of innate immune receptors have been implicated in the response to HDM including toll-like receptors, C-type lectin receptors, and protease activated receptors. NOD2 is a member of the NOD-like receptor family, which has been reported to be involved in the establishment of type 2 immunity and in blocking respiratory tolerance. NOD2 mediates its effects through its downstream effector kinase, receptor interacting protein (RIP2). It has not been shown if RIP2 is involved in the innate response to HDM and in the resulting generation of type 2 immunity. Furthermore, the role of RIP2 in modulating allergic airway inflammation has been controversial. In this study, we show that RIP2 is activated in airway epithelial cells in response to HDM and is important for the production of CCL2. Using a murine HDM asthma model, we demonstrate that lung pathology, local airway inflammation, inflammatory cytokines, HDM-specific IgG1 antibody production, and HDM-specific Th2 responses are all reduced in RIP2 knockout mice compared to WT animals. These data illustrate that RIP2 can be activated by a relevant allergic stimulus and that such activation can contribute to allergic airway inflammation. These findings also suggest that RIP2 inhibitors might have some efficacy in down-regulating the inflammatory response in type 2 dominated diseases.

In vivo inhibition of RIPK2 kinase alleviates inflammatory disease.

The RIPK2 kinase transduces signaling downstream of the intracellular peptidoglycan sensors NOD1 and NOD2 to promote a productive inflammatory response. However, excessive NOD2 signaling has been associated with numerous diseases, including inflammatory bowel disease (IBD), sarcoidosis and inflammatory arthritis, making pharmacologic inhibition of RIPK2 an appealing strategy. In this work, we report the generation, identification, and evaluation of novel RIPK2 specific inhibitors. These compounds potently inhibit the RIPK2 tyrosine kinase activity in in vitro biochemical assays and cellular assays, as well as effectively reduce RIPK2-mediated effects in an in vivo peritonitis model. In conjunction with the development of these inhibitors, we have also defined a panel of genes whose expression is regulated by RIPK2 kinase activity. Such RIPK2 activation markers may serve as a useful tool for predicting settings likely to benefit from RIPK2 inhibition. Using these markers and the FDA-approved RIPK2 inhibitor Gefitinib, we show that pharmacologic RIPK2 inhibition drastically improves disease in a spontaneous model of Crohn Disease-like ileitis. Furthermore, using novel RIPK2-specific inhibitors, we show that cellular recruitment is inhibited in an in vivo peritonitis model. Altogether, the data presented in this work provides a strong rationale for further development and optimization of RIPK2-targeted pharmaceuticals and diagnostics.

Innate immune-directed NF-κB signaling requires site-specific NEMO ubiquitination.

While the I kappa kinase (IKK) scaffolding protein NF-κB essential modulator (NEMO) binds to polyubiquitin chains to transmit inflammatory signals, NEMO itself is also ubiquitinated in response to a variety of inflammatory agonists. Although there have been hints that polyubiquitination of NEMO is essential for avoiding inflammatory disorders, the in vivo physiologic role of NEMO ubiquitination is unknown. In this work, we knock in a NEMO allele in which two major inflammatory agonist-induced ubiquitination sites cannot be ubiquitinated. We show that mice with a nonubiquitinatable NEMO allele display embryonic lethality. Heterozygous females develop inflammatory skin lesions, decreased B cell numbers, and hypercellular spleens. Embryonic lethality can be complemented by mating onto a TNFR1(-/-) background, at the cost of severe steatohepatitis and early mortality, and we also show that NEMO ubiquitination is required for optimal innate immune signaling responses. These findings suggest that NEMO ubiquitination is crucial for NF-κB activity in response to innate immune agonists.

A discrete ubiquitin-mediated network regulates the strength of NOD2 signaling.

Dysregulation of NOD2 signaling is implicated in the pathology of various inflammatory diseases, including Crohn's disease, asthma, and sarcoidosis, making signaling proteins downstream of NOD2 potential therapeutic targets. Inhibitor-of-apoptosis (IAP) proteins, particularly cIAP1, are essential mediators of NOD2 signaling, and in this work, we describe a molecular mechanism for cIAP1's regulation in the NOD2 signaling pathway. While cIAP1 promotes RIP2's tyrosine phosphorylation and subsequent NOD2 signaling, this positive regulation is countered by another E3 ubiquitin ligase, ITCH, through direct ubiquitination of cIAP1. This ITCH-mediated ubiquitination leads to cIAP1's lysosomal degradation. Pharmacologic inhibition of cIAP1 expression in ITCH(-/-) macrophages attenuates heightened ITCH(-/-) macrophage muramyl dipeptide-induced responses. Transcriptome analysis, combined with pharmacologic inhibition of cIAP1, further defines specific pathways within the NOD2 signaling pathway that are targeted by cIAP1. This information provides genetic signatures that may be useful in repurposing cIAP1-targeted therapies to correct NOD2-hyperactive states and identifies a ubiquitin-regulated signaling network centered on ITCH and cIAP1 that controls the strength of NOD2 signaling.

Ubiquitination and phosphorylation in the regulation of NOD2 signaling and NOD2-mediated disease.

The immune system is exquisitely balanced. It has the ability to effectively respond to and control infections while at the same time preventing inappropriate responses to self and environmental antigens. When this response goes awry, either through a failure to activate the immune response, or failure to terminate it, inflammatory pathology results. Posttranslational modifications (PTMs) such as ubiquitination and phosphorylation help ensure that the delicate balance underlying immune signal transduction is maintained. Ubiquitination and phosphorylation affect localization, activity, stability, and interactions of various components of the immune signal transduction machinery. Moreover, ubiquitination and phosphorylation are tightly linked, with one PTM affecting the other. Therefore, in order to find potential therapies for many immune-related pathologies, it is necessary to understand not only how the immune response is activated by ubiquitination and phosphorylation, but also how it is regulated by these PTMs at different stages of the response. An excellent system to study such activation and regulation is the NOD2 pathway. Dysregulation of NOD2 signaling is involved in the pathogenesis of a variety of inflammatory disorders including Crohn's disease, early onset sarcoidosis, and Blau syndrome. More recently NOD2 has been implicated in the development of autoimmune disease, allergy and asthma. This review will focus on what is currently known about how ubiquitination and phosphorylation regulate NOD2 signaling with particular emphasis on novel in vitro substrates which may serve as potential in vivo therapeutic targets for hyperactive NOD2 states. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Inhibition of RIP2's tyrosine kinase activity limits NOD2-driven cytokine responses.

Upon intracellular bacterial exposure, the Crohn's disease and sarcoidosis susceptibility protein NOD2 (nucleotide oligomerization domain protein 2) binds to the protein kinase RIP2 (receptor-interacting protein 2) to coordinate NF-κB (nuclear factor κ B)-mediated cytokine responses. While RIP2 clearly has kinase activity, the function of its kinase domain has been enigmatic. Although originally classified as a serine-threonine kinase based on homology scans, we find that RIP2 also has tyrosine kinase activity. RIP2 undergoes autophosphorylation on Tyr 474 (Y474). This phosphorylation event is necessary for effective NOD2 signaling and does not occur in the presence of the most common Crohn's disease-associated NOD2 allele. Given this tyrosine kinase activity, a small-molecule inhibitor screen designed to identify pharmacologic agents that inhibit RIP2's tyrosine kinase activity was performed. At nanomolar concentrations, the EGFR (epidermal growth factor receptor) tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) were found to inhibit both RIP2 tyrosine phosphorylation and MDP (muramyl dipeptide)-induced cytokine release in a variety of NOD2 hyperactivation states. This effect is specific for RIP2 and does not depend on EGFR. The finding that RIP2 has tyrosine kinase activity and the finding that gefitinib and erlotinib, two agents already used clinically for cancer chemotherapy, can inhibit this activity suggest that RIP2's tyrosine kinase activity could be targeted specifically in the treatment of inflammatory diseases.

Th1 responses to beta-amyloid in young humans convert to regulatory IL-10 responses in Down syndrome and Alzheimer's disease.

A beta(1-42)-specific antibodies and T-cell proliferation point to the existence of a memory response to A beta(1-42) in humans. Using ELISPOT, we studied A beta(1-42)-specific T cells in individuals of various ages, and in subjects with Trisomy 21 or Alzheimer's disease. We show for the first time that A beta(1-42)-specific Th1-type T-cell memory is present in young humans, producing high levels of IFN-gamma and IL-2. With increasing age, the production of IFN-gamma and IL-2 decreases but is not discontinued in healthy subjects and is accompanied by a sharp rise in CD4(+) T-cell-derived regulatory IL-10 production. In contrast, individuals with Trisomy 21 and with Alzheimer's disease produce IL-10 only in the absence of any effector cytokine. This signifies a switch from a Th1 effector to an IL-10 mediated regulatory response.

Encephalitogenicity of complete Freund's adjuvant relative to CpG is linked to induction of Th17 cells.

For decades, CFA has been the classic adjuvant for the induction of experimental autoimmune encephalomyelitis (EAE). Its encephalitogenic activity has been originally linked to the induction of Th1 responses. CpG, which is also a potent Th1 inducer, has been suggested by some studies to be comparably encephalitogenic. In this study, using the SJL proteolipid protein (PLP) 139-151 peptide EAE model, we show that active immunizations using CFA but not CpG 1826/IFA as an adjuvant induced disease. Passive induction of EAE resulted in severe disease when cells were transferred from PLP in CFA-primed mice but resulted in only a mild, transient disease when cells originated from PLP in CpG 1826/IFA-primed mice. In accordance with these findings, immunizations using CFA but not CpG 1826/IFA as an adjuvant elicited a delayed-type hypersensitivity response. ELISPOT analysis revealed that CFA promoted the differentiation of much higher levels of PLP-specific, IL-17-secreting cells compared with CpG 1826/IFA. Both adjuvants induced comparable frequencies of PLP-specific, IFN-gamma-secreting cells and also induced Ag-specific proliferation to the same extent. The severity of EAE in PLP in CFA-immunized mice was reduced when IL-17 was neutralized in vivo, demonstrating the crucial role of this cytokine in disease induction. The data show that immunizations using the autoantigen in CpG 1826/IFA result in very low frequencies of Ag-specific IL-17 cells, suggesting a lower risk of Th17-mediated pathology when using this adjuvant.

Dissociated induction of cytotoxicity and DTH by CFA and CpG.

Vaccinations typically rely on immunization with live virus for eliciting protective CD8 T cell immunity. There is increasing interest to use subunit vaccination strategies to achieve such responses. Complete Freund's adjuvant (CFA) and unmethylated cytosine-guanine dinucleotide containing DNA are considered some of the most potent adjuvants for eliciting immunity. Whereas a wealth of information is available on how these adjuvants affect CD4 T cell responses, their effects on engaging CD8 T cell immunity are not completely understood. We immunized C57BL/6J mice with the class I restricted peptides Uty or SIINFEKL using these 2 adjuvants and tested for cytokine secretion, proliferation, in vivo cytotoxicity, and delayed-type hypersensitivity (DTH). Our data show that CFA-induced CD8 T cells to proliferate, mediate DTH, and to secrete interferon-gamma, interleukin (IL)-2 and IL-17. Despite these markers of CD8 T cell activation, CFA failed to induce an early cytotoxic CD8 T cell response. In contrast, unmethylated cytosine-guanine dinucleotide containing DNA promoted a vigorous cytolytic response without activating substantial cytokine production, proliferation or DTH. These data have implications for CD8 T cell subunit vaccine design in which cytotoxicity versus DTH plays a key role in host defense.