The role of itaconate in host defense and inflammation.

Macrophages exposed to inflammatory stimuli including LPS undergo metabolic reprogramming to facilitate macrophage effector function. This metabolic reprogramming supports phagocytic function, cytokine release, and ROS production that are critical to protective inflammatory responses. The Krebs cycle is a central metabolic pathway within all mammalian cell types. In activated macrophages, distinct breaks in the Krebs cycle regulate macrophage effector function through the accumulation of several metabolites that were recently shown to have signaling roles in immunity. One metabolite that accumulates in macrophages because of the disturbance in the Krebs cycle is itaconate, which is derived from cis-aconitate by the enzyme cis-aconitate decarboxylase (ACOD1), encoded by immunoresponsive gene 1 (Irg1). This Review focuses on itaconate's emergence as a key immunometabolite with diverse roles in immunity and inflammation. These roles include inhibition of succinate dehydrogenase (which controls levels of succinate, a metabolite with multiple roles in inflammation), inhibition of glycolysis at multiple levels (which will limit inflammation), activation of the antiinflammatory transcription factors Nrf2 and ATF3, and inhibition of the NLRP3 inflammasome. Itaconate and its derivatives have antiinflammatory effects in preclinical models of sepsis, viral infections, psoriasis, gout, ischemia/reperfusion injury, and pulmonary fibrosis, pointing to possible itaconate-based therapeutics for a range of inflammatory diseases. This intriguing metabolite continues to yield fascinating insights into the role of metabolic reprogramming in host defense and inflammation.

Irg1/itaconate metabolic pathway is a crucial determinant of dendritic cells immune-priming function and contributes to resolute allergen-induced airway inflammation.

Itaconate is produced from the mitochondrial TCA cycle enzyme aconitase decarboxylase (encoded by immune responsive gene1; Irg1) that exerts immunomodulatory function in myeloid cells. However, the role of the Irg1/itaconate pathway in dendritic cells (DC)-mediated airway inflammation and adaptive immunity to inhaled allergens, which are the primary antigen-presenting cells in allergic asthma, remains largely unknown. House dust mite (HDM)-challenged Irg1-/- mice displayed increases in eosinophilic airway inflammation, mucous cell metaplasia, and Th2 cytokine production with a mechanism involving impaired mite antigen presentations by DC. Adoptive transfer of HDM-pulsed DC from Irg1-deficient mice into naïve WT mice induced a similar phenotype of elevated type 2 airway inflammation and allergic sensitization. Untargeted metabolite analysis of HDM-pulsed DC revealed itaconate as one of the most abundant polar metabolites that potentially suppress mitochondrial oxidative damage. Furthermore, the immunomodulatory effect of itaconate was translated in vivo, where intranasal administration of 4-octyl itaconate 4-OI following antigen priming attenuated the manifestations of HDM-induced airway disease and Th2 immune response. Taken together, these data demonstrated for the first time a direct regulatory role of the Irg1/itaconate pathway in DC for the development of type 2 airway inflammation and suggest a possible therapeutic target in modulating allergic asthma.

Itaconate controls the severity of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease in which airway macrophages (AMs) play a key role. Itaconate has emerged as a mediator of macrophage function, but its role during fibrosis is unknown. Here, we reveal that itaconate is an endogenous antifibrotic factor in the lung. Itaconate levels are reduced in bronchoalveolar lavage, and itaconate-synthesizing cis-aconitate decarboxylase expression (ACOD1) is reduced in AMs from patients with IPF compared with controls. In the murine bleomycin model of pulmonary fibrosis, Acod1−/− mice develop persistent fibrosis, unlike wild-type (WT) littermates. Profibrotic gene expression is increased in Acod1−/− tissue-resident AMs compared with WT, and adoptive transfer of WT monocyte-recruited AMs rescued mice from disease phenotype. Culture of lung fibroblasts with itaconate decreased proliferation and wound healing capacity, and inhaled itaconate was protective in mice in vivo. Collectively, these data identify itaconate as critical for controlling the severity of lung fibrosis, and targeting this pathway may be a viable therapeutic strategy.

Immune-responsive gene 1 (IRG1) and dimethyl itaconate are involved in the mussel immune response.

Immune-responsive gene 1 (irg1) is a gene that is well-conserved among different taxa and is highly expressed in the mussel Mytilus galloprovincialis at the constitutive level. The expression of this gene increases after a bacterial infection, primarily in haemocytes. irg1 catalyses the production of itaconic acid from cis-aconitic acid in the Krebs cycle. Recently, itaconate has been revealed as an immune metabolite involved in macrophage polarization. In this work, we studied the effects of exogenous dimethyl itaconate (DI) on mussels in vitro and in vivo at relevant previously described endogenous concentrations and in mussels infected with Vibrio splendidus. DI did not have adverse effects on the haemocytes viability, apoptotic cells, proliferation and phagocytic activity; however, haemocyte size, velocity and accumulated distance were decreased. The antibacterial activity of DI in vitro and in vivo was observed with high concentrations of DI, that is, 30 and 50 mM, respectively. Furthermore, DI inhibited total ROS, increased mitochondrial ROS and modulated antioxidant genes, such as SOD and CAT, related to Nrf2 activation. In this research, we have demonstrated some important pathways in haemocytes in which itaconate can be involved after its production in a bacterial infection.

Salmonella enterica's "Choice": Itaconic Acid Degradation or Bacteriocin Immunity Genes.

Itaconic acid is an immunoregulatory metabolite produced by macrophages in response to pathogen invasion. It also exhibits antibacterial activity because it is an uncompetitive inhibitor of isocitrate lyase, whose activity is required for the glyoxylate shunt to be operational. Some bacteria, such as Yersinia pestis, encode enzymes that can degrade itaconic acid and therefore eliminate this metabolic inhibitor. Studies, primarily with Salmonella enterica subspecies enterica serovar Typhimurium, have demonstrated the presence of similar genes in this pathogen and the importance of these genes for the persistence of the pathogen in murine hosts. This minireview demonstrates that, based on Blast searches of 1063 complete Salmonella genome sequences, not all Salmonella serovars possess these genes. It is also shown that the growth of Salmonella isolates that do not possess these genes is sensitive to the acid under glucose-limiting conditions. Interestingly, most of the serovars without the three genes, including serovar Typhi, harbor DNA at the corresponding genomic location that encodes two open reading frames that are similar to bacteriocin immunity genes. It is hypothesized that these genes could be important for Salmonella that finds itself in strong competition with other Enterobacteriacea in the intestinal tract-for example, during inflammation.

Cytokine-like Roles for Metabolites in Immunity.

Metabolites have functions in the immune system independent of their conventional roles as sources or intermediates in biosynthesis and bioenergetics. We are still in the pioneering phase of gathering information about the functions of specific metabolites in immunoregulation. In this review, we cover succinate, itaconate, α-ketoglutarate, and lactate as examples. Each of these metabolites has a different story of how their immunoregulatory functions were discovered and how their roles in the complex process of inflammation were revealed. Parallels and interactions are emerging between metabolites and cytokines, well-known immunoregulators. We depict molecular mechanisms by which metabolites prime cellular and often physiological changes focusing on intra- and extra-cellular activities and signaling pathways. Possible therapeutic opportunities for immune and inflammatory diseases are emerging.

Itaconate: the poster child of metabolic reprogramming in macrophage function.

Itaconate is one of the best examples of the consequences of metabolic reprogramming during immunity. It is made by diverting aconitate away from the tricarboxylic acid cycle during inflammatory macrophage activation. The main reason macrophages exhibit this response currently appears to be for an anti-inflammatory effect, with itaconate connecting cell metabolism, oxidative and electrophilic stress responses and immune responses. A role for itaconate in the regulation of type I interferons during viral infection has also been described, as well as in M2 macrophage function under defined circumstances. Finally, macrophage-specific itaconate production has also been shown to have a pro-tumour effect. All of these studies point towards itaconate being a critical immunometabolite that could have far-reaching consequences for immunity, host defence and tumorigenesis.

Biochemistry of proinflammatory macrophage activation.

In the last decade, metabolism has been recognized as a major determinant of immunological processes. During an inflammatory response, macrophages undergo striking changes in their metabolism. This metabolic reprogramming is governed by a complex interplay between metabolic enzymes and metabolites of different pathways and represents the basis for proper macrophage function. It is now evident that these changes go far beyond the well-known Warburg effect and the perturbation of metabolic targets is being investigated as a means to treat infections and auto-immune diseases. In the present review, we will aim to provide an overview of the metabolic responses during proinflammatory macrophage activation and show how these changes modulate the immune response.

Synthetic Toll-like receptor 7 ligand inhibits porcine reproductive and respiratory syndrome virus infection in primary porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV), a common viral pathogen, causes huge annual economic losses to the swine industry worldwide. After triggering by specific ligands, the Toll-like receptor 7 (TLR7), a type of pattern-recognition receptor (PRR), induces antiviral cytokines production. Previously, we synthesized an adenine analog, designated SZU101, a TLR7-specific ligand. In this study, we assessed the inhibitory effect of SZU101 on PRRSV infection in vitro. SZU101 significantly suppressed PRRSV infection in primary porcine alveolar macrophages (PAMs) in a dose-dependent manner. Moreover, SZU101-induced inhibition involved NF-κB pathway activation in PAMs to initiate expression of TLR7-mediated cytokines and induce expression of downstream signaling IFN-stimulated genes (ISGs). Chloroquine, a TLR7 inhibitor, and BAY 11-7082, an NF-κB inhibitor, reversed both the SZU101-induced antiviral effect and induction of cytokine genes and ISGs expression. Therefore, SZU101 antiviral effects depend at least in part on TLR7-NF-κB signaling pathway. Additionally, administration of SZU101 enhanced the humoral and cell-mediated immune responses against PRRSV antigens in mice. Given these results, SZU101 holds promise as an antiviral agent and a vaccine adjuvant to prevent PRRSV infection in pigs.

Efficiency of pH-sensitive fusogenic polymer-modified liposomes as a vaccine carrier.

The usefulness of pH-sensitive fusogenic polymer-(succinylated poly(glycidol)-(SucPG-) modified liposomes as a vaccine carrier in the induction of immune responses was evaluated. Mice were intraperitoneally immunized with ovalbumin- (OVA-) containing SucPG-modified liposomes. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, OVA-specific IgG1 antibody responses were noted in mice immunized with OVA-containing polymer-unmodified liposomes, whereas immunization with OVA-containing SucPG-modified liposomes resulted in the induction of OVA-specific IgG1, IgG2a, and IgG3 Ab responses. In spleen lymphocytes from mice immunized with OVA-containing SucPG-modified liposomes, both IFN-γ-(Th1-type-) and IL-4-(Th2 type-) specific mRNA were detected. Moreover, substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from OVA-containing SucPG-modified liposomes in vitro. These results suggest that the pH-sensitive fusogenic polymer-(SucPG-) modified liposomes would serve effectively as an antigen delivery vehicle for inducing Th1 and Th2 immune responses.

Immediate-type hypersensitivity to succinylated corticosteroids.

BACKGROUND: Despite their frequent use, systemic corticosteroids have rarely elicited immediate-type reactions. OBJECTIVE: We report two male patients, aged 26 and 70 years, respectively, with severe immediate-type hypersensitivity secondary to the administration of corticosteroids esterified with succinate. METHODS: Skin tests, basophil activation tests and challenge tests were performed for diagnostic evaluation. RESULTS: In both patients, immediate-type skin test reactions were found to methylprednisolone sodium hemisuccinate (MSH) and prednisolone sodium hemisuccinate (PSH). In contrast, nonsuccinylated corticosteroids (including methylprednisolone and prednisolone in one patient) yielded no test reactions. Basophils from one patient exhibited a stimulated expression of the activation marker CD63 upon in vitro incubation with PSH or hydrocortisone sodium succinate, but not with hydrocortisone. Skin tests and basophil activation tests were negative in controls. One patient was challenged with the incriminated drugs. He developed flush, conjunctivitis, tachycardia and dyspnea 2 min after injection of MSH, and dyspnea shortly after intravenous administration of PSH. Oral and intravenous challenge tests with nonsuccinylated corticosteroids were tolerated well by both patients. CONCLUSIONS: These case reports should alert clinicians to rare, but severe immediate-type reactions to corticosteroids, related to the succinate moiety in our patients. In case of allergic reactions to corticosteroids, it is mandatory to identify the causative agent and find safe alternatives.

Dioxin immunosensor using anti-2,3,7,8-TCDD antibody which was produced with mono 6-(2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl succinate as a hapten.

To detect dioxin using a quartz crystal microbalance (QCM) immunosensor, anti-2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) monoclonal antibodies (MAbs) were produced as types of IgG1 and IgM, with mono 6-(2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl succinate (as a hapten) conjugated with bovine serum albumin (dioxin-BSA). Furthermore, ScFv was generated from hybridoma-producing IgG1 MAb. Among these antibodies, ScFv showed excellent capability for dioxin detection using QCM immunosensors.

Preparation of specific antisera to digoxin by using digoxin C-3' and C-3" hemisuccinate-bovine serum albumin conjugates.

The preparation and antigenic properties of digoxin C-3' and C-3" hemisuccinate-bovine serum albumin (BSA) conjugates are described. The antisera were prepared by immunizing rabbits with each of the digoxin-BSA conjugates, and properties of the antisera were characterized by RIA with 3H-labeled digoxin. The antidigoxin antiserum from immunization with digoxin 3'-hemisuccinate-BSA conjugate possessed high specificity for digoxin, exhibiting fairly low cross-reactions with dihydrodigoxin (2.1%), digoxigenin monodigitoxoside (0.9%), digoxigenin bisdigitoxoside (0.6%), and digoxigenin (0.1%).

Production and characterization of monoclonal antibodies against two haptenic derivatives of 1 alpha,25-dihydroxyvitamin D3 conjugated with bovine serum albumin through the C-3 or C-24 position.

To develop the immunochemical methods for determining 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in clinical samples, a variety of monoclonal anti-1,25(OH)2D3 antibodies have been generated. Two kinds of hapten-carrier conjugates, 25-hydroxyvitamin D3 3-hemisuccinate (hapten 3-HS) and 1 alpha-hydroxy-25,26,27-trinorvitamin D3 24-oic acid (hapten 24-OA) conjugated with bovine serum albumin, were used for immunization. Spleen cells from SD rats or BALB/c mice, each immunized with the conjugate of hapten 3-HS or 24-OA, were fused with P3/NS1/1-Ag4-1 myeloma cells. After screening by ELISA employing beta-galactosidase-labeled haptens, seven kinds of hybridomas secreting anti-1,25(OH)2D3 antibodies were established. Binding characteristics of these antibodies (Ka 0.73-20 x 10(9) M-1) were investigated by an RIA using tritium-labeled 1,25(OH)2D3. The data suggested that the rat monoclonal antibody 3R-1 derived from the hapten 3-HS and the mouse monoclonal antibody 24M-3 from the hapten 24-OA would be available for developing practical analytical systems.

Immunorecognition of ring skeleton of taxanes by chicken egg yolk antibodies.

Anti-10 deacetylbaccatin III (DAB) antibodies (IgY) were elicited in hens immunized with a succinyl-DAB/BSA conjugate and extracted from egg yolk. As shown by indirect competitive inhibition enzyme immunoassay (CIEIA), the addition of free-DAB competitively inhibited the binding of affinity purified anti-DAB IgY to DAB/BSA solid phase conjugated antigen. The assay enabled the detection of DAB in concentrations as low as 7.5ng/ml (13.7 nM DAB), whereas anti-DAB IgY did not react with taxol even at a concentration a thousand times higher. The structural requirements of the diterpenoid nucleus for binding to IgY were considered on the basis of the levels of cross-reaction found with 10 authentic taxanes. The results indicate that anti-DAB IgY represents the first high affinity antibody produced capable of recognizing the ring skeleton of taxol precursors.

Synthesis and characterization of gentiobiose heptaacetate conjugate vaccines that produce endotoxin-neutralizing antibodies.

We have prepared aminoethyl (AE), aminopropyl (AP), and aminopentyl (APT) derivatives of gentiobiose heptaacetate (GH). These spacer compounds (AEGH, APGH, APTGH) have been coupled to succinylated diphtheria toxoid (Suc.DT) to produce conjugate vaccines. These conjugates all bind to the anti-lipid A human monoclonal antibody A6(H4C5) in an ELISA binding assay. Rabbits immunized with the APGH conjugate vaccine in either Freund's complete adjuvant or aluminum hydroxide gel produced antibody levels of 5120 and 3600 ELISA units, respectively, compared to an antibody level of less than 20 ELISA units for the prebleed sera. Sera from mice immunized with either the aminopropyl or the aminopentyl conjugate had antibody levels of 5120 and 2560 ELISA antibody units, respectively. These antibodies neutralized endotoxin in a Limulus lysate neutralization assay. Protection against the local Shwartzman reaction was demonstrated (p less than 0.05) in eight out of nine rabbits immunized with the Suc-DT-APGH conjugate vaccine compared to three out of 10 rabbits immunized with the carrier protein Suc-DT. Passive transfer experiments demonstrated that four out of five rabbits receiving immune serum were protected from Shwartzman reaction compared to one out of five rabbits receiving normal serum (p less than 0.1). These results indicated that epitopes contained in gentiobiose heptaacetate when properly presented as conjugate vaccines were capable of inducing neutralizing antibodies against endotoxin.

A rapid and sensitive screening method for the detection of anti-2-acetylaminofluorene immunoglobulins.

A method is described in which anti-2-acetylaminofluorene immunoglobulins may be detected using a simple and sensitive screening procedure. The method is based on immunoglobulin binding of an 125I derivatized 2-aminofluorene radiotracer. Tracer binding is not isotype specific, and thus the method is useful for the detection of either IgG or IgA. Competitive binding experiments with the radiotracer were used to determine the specificity of immunoglobulin response by measurement of cross-reactivity with related ligands. This method allows quantitation of the immune response to the carcinogen in serum and other biological fluids (i.e., intestinal secretions).

Instability of succinyl ester linkages in O2'-monosuccinyl cyclic AMP-protein conjugates at neutral pH.

Chromatographic and immunological evidence is presented regarding the hydrolysis of the ester linkage of O2'-monosuccinyl cyclic AMP in neutral solutions. Such hydrolysis occurs whether the nucleotide derivative is present in free form in solution or conjugated through its succinyl carboxyl group via an amide bond to proteins. The latter process apparently occurs when succinyl cyclic AMP is conjugated to human serum albumin for use as an immunogen in the production of anti-cyclic AMP antibodies and when the derivative is coupled to the enzyme glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49). The enzyme conjugate has been used in developing a homogeneous enzyme immunoassay for cyclic AMP. Inhibition of the catalytic activity of enzyme-cyclic AMP conjugates by anti-cyclic AMP antibody decreases with time, apparently due to the loss of cyclic AMP from enzyme-cyclic AMP conjugates stored in neutral solutions. In addition, the ability of free cyclic AMP to completely reverse the inhibition process decreases with time because of the presence of antibodies in the anti-cyclic AMP sera that apparently inhibit enzyme activity because of their binding specificity for the residual succinate-protein determinant sites of the enzyme conjugates. Lyophilization of the conjugates immediately after preparation helps to overcome the problem; however, in vivo hydrolysis of immunogens prepared with the succinyl cyclic AMP derivative may always occur. The consequence of this hydrolysis reaction and the subsequent formation of anti-succinyl-protein antibodies will be discussed with regard to existing RIAs for cyclic AMP and a new homogeneous enzyme immunoassay for the nucleotide.

Investigation of the possible involvement of IgE anti-salicyloyl antibodies in patients with urticaria.

19 aspirin-sensitive and 14 non-aspirin-sensitive patients with chronic urticaria were tested for the presence of specific anti-salicyloyl-IgE antibodies by prick testing, RAST and in vitro histamine release using an aspirin-polylysine conjugate. Polylysine succinate served as a control. Negative results were obtained in all cases with the exception of 1 patient who gave a positive prick test response. However, this patient reacted to the control conjugate as well as aspirin polylysine. Skin prick test responses to the battery of environmental allergens revealed that the majority (90%) of the aspirin-sensitive patients were non-atopic. The findings suggest that the clinical symptoms and signs in patients with chronic urticaria associated with aspirin sensitivity are not mediated by specific IgE antibodies.