Basic Mechanisms of Immunometabolites in Shaping the Immune Response

Background: Innate immune cells play a crucial role in responding to microbial infections, but their improper activation can also drive inflammatory disease. For this reason, their activation state is governed by a multitude of factors, including the metabolic state of the cell and, more specifically, the individual metabolites which accumulate intracellularly and extracellularly. This relationship is bidirectional, as innate immune cell activation by pathogen-associated molecular patterns causes critical changes in cellular metabolism. Summary: In this review, we describe the emergence of various “immunometabolites.” We outline the general characteristics of these immunometabolites, the conditions under which they accumulate, and their subsequent impact on immune cells. We delve into well-studied metabolites of recent years, such as succinate and itaconate, as well as newly emerging immunometabolites, such as methylglyoxal. Key Messages: We hope that this review may be used as a framework for further studies dissecting the mechanisms by which immunometabolites regulate the immune system and provide an outlook to harnessing these mechanisms in the treatment of inflammatory diseases

Human‐mediated dispersal and disturbance shape the metapopulation dynamics of a long‐lived herb

As anthropogenic impacts on the natural world escalate, there is increasing interest in the role of humans in dispersing seeds. But the consequences of this Human‐Mediated Dispersal (HMD) on plant spatial dynamics are little studied. In this paper, we ask how secondary dispersal by HMD affects the dynamics of a natural plant metapopulation. In addition to dispersal between patches, we suggest within‐patch processes can be critical. To address this, we assess how variation in local population dynamics, caused by small‐scale disturbances, affects metapopulation size. We created an empirically based model with stochastic population dynamics and dispersal among patches, which represented a real‐world, cliff‐top metapopulation of wild cabbage Brassica oleracea. We collected demographic data from multiple populations by tagging plants over eight years. We assessed seed survival, and establishment and survival of seedlings in intact vegetation vs. small disturbances. We modeled primary dispersal by wind using field data and used experimental data on secondary HMD by hikers. We monitored occupancy patterns over a 14‐yr period in the real metapopulation. Disturbance had large effects on local population growth rates, by increasing seedling establishment and survival. This meant that the modeled metapopulation grew in size only when the area disturbed in each patch was above 35%. In these growing metapopulations, although only 0.2% of seeds underwent HMD, this greatly enhanced metapopulation growth rates. Similarly, HMD allowed more colonizations in declining metapopulations under low disturbance, and this slowed the rate of decline. The real metapopulation showed patterns of varying patch occupancy over the survey years, which were related to habitat quality, but also positively to human activity along the cliffs, hinting at beneficial effects of humans. These findings illustrate that realistic changes to dispersal or demography, specifically by humans, can have fundamental effects on the viability of a species at the landscape scale

Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation

The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1β and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1−/− mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation.We would like to acknowledge the following grants: the National Health and Medical Research Council of Australia (NHMRC) is thanked for Project Grant APP1124673 to P.G.B., M.G.C., and L.A.J.O.; Principal Research Fellowship 1117602 to J.B.B.; and NHMRC Project Grant APP1156455 to J.B.B., P.G.B., and M.G.C. The O’Neill laboratory acknowledges the following grant support: European Research Council (ECFP7-ERC-MICROINNATE) and Science Foundation Ireland Investigator Award (SFI 12/IA/1531)

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-β production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses

The Immunomodulatory Metabolite Itaconate Regulates NLRP3 Inflammasome Activation and Type I Interferon Signalling

The NLRP3 inflammasome is a multi-protein complex which activates caspase-1 for the cleavage and release of mature IL-1? and IL-18. Caspase-1 also cleaves gasdermin D, driving a process called pyroptosis. Results from clinical trials have demonstrated the utility of anti-IL-1? therapy in reducing the incidence of cardiovascular events and lung cancer, and blocking IL-1? has shown clinical utility in the treatment of classically IL-1?-driven pathologies such as cryopyrinassociated periodic syndromes (CAPS). There is therefore a strong interest in targeting NLRP3 with small molecules, several of which are in development. Indeed, murine models have implicated the NLRP3 inflammasome in various autoinflammatory diseases, including Alzheimer?s disease, Parkinson?s disease and type I diabetes. Itaconate has emerged as a prominent metabolite in macrophages activated with the gram-negative bacterial product lipopolysaccharide (LPS). It has been shown to elicit immunomodulatory effects via modification of target cysteines and activation of the transcription factor NRF2. Here I have investigated the effect of itaconate on NLRP3 inflammasome activation. I have shown that itaconate is an endogenous inhibitor of NLRP3 inflammasome activation. The cell-permeable itaconate derivative 4-octyl itaconate (4-OI) blocked NLRP3 inflammasome-induced IL-1? release, pyroptosis and ASC oligomerisation in murine macrophages, but had little effect on AIM2 and NLRC4 inflammasome activation. Irg1-/- macrophages, which lack endogenous itaconate, exhibited heightened NLRP3 inflammasome activation. Conversely, Irg1 overexpression in an inflammasome reconstitution model reduced inflammasome activation. These data suggest that endogenous itaconate also inhibits inflammasome activation. These effects were not dependent on NRF2. 4-OI modified a specific cysteine (C548) on NLRP3 and inhibited the NLRP3-NEK7 interaction which is required for inflammasome activation to take place. 4-OI also blocked inflammasome activation in human PBMCs isolated from both healthy volunteers and CAPS patients, suggesting that itaconate may be harnessed therapeutically for the treatment of NLRP3-driven disorders. I also examined the non-canonical inflammasome, which activates caspase-11. Caspase-11 is induced by LPS via type I interferon signalling and directly senses intracellular LPS to promote pyroptosis. I have found that 4-OI, but not unmodified itaconate, inhibits type I interferon release, signalling and subsequent caspase-11 expression. 4-OI inhibited non-canonical inflammasome-induced pyroptosis by blocking the upregulation of caspase-11 in response to LPS. As caspase-11 is an interferon-stimulated gene (ISG), reduced caspase-11 expression with 4-OI was due to reduced type I interferon release in response to LPS. Interestingly, itaconic acid treatment actually boosted LPS-induced IFN-? production and Irg1-/- macrophages released less IFN-? upon LPS stimulation. These results reveal a clear difference between 4-OI and unmodified itaconate. 4-OI also blocked caspase-11 induction following IFN-? stimulation, which I found was due to reduced JAK/STAT signalling. ACE2, the host cell entry receptor for SARS-CoV- 2, is also an ISG and its upregulation in airway epithelial cells was similarly blocked by 4-OI. Taken together, these results provide further evidence of the immunoregulatory role of itaconate. I have found evidence of metabolic regulation of the NLRP3 inflammasome, and in 4-OI I have described a specific pharmacological inhibitor of NLRP3 activation. 4-OI also inhibits type I interferon release and signalling, a property not shared by unmodified itaconate. These results in particular highlight some of the current issues associated with the study of itaconate biology and further our knowledge of this important immunometabolite, derivatives of which might have potential in the treatment of autoinflammatory diseases

Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation

The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1? and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1-/- mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation

The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation

The Krebs cycle-derived metabolite itaconate is highly upregulated in inflammatory macrophages and exerts immunomodulatory effects through cysteine modifications on target proteins. The NLRP3 inflammasome, which cleaves IL-1?, IL-18, and gasdermin D, must be tightly regulated to avoid excessive inflammation. Here we provide evidence that itaconate modifies NLRP3 and inhibits inflammasome activation. Itaconate and its derivative, 4-octyl itaconate (4-OI), inhibited NLRP3 inflammasome activation, but not AIM2 or NLRC4. Conversely, NLRP3 activation was increased in itaconate-depleted Irg1-/- macrophages. 4-OI inhibited the interaction between NLRP3 and NEK7, a key step in the activation process, and "dicarboxypropylated" C548 on NLRP3. Furthermore, 4-OI inhibited NLRP3-dependent IL-1? release from PBMCs isolated from cryopyrin-associated periodic syndrome (CAPS) patients, and reduced inflammation in an in vivo model of urate-induced peritonitis. Our results identify itaconate as an endogenous metabolic regulator of the NLRP3 inflammasome and describe a process that may be exploited therapeutically to alleviate inflammation in NLRP3-driven disorders