No abstract

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-1β, 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-1β 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.

SignificanceThe molecular clock provides an anticipatory mechanism, allowing organisms to prepare and respond to daily changes in the external environment. The response of the innate immune system to pathogenic threats is dependent on time of day; however, the molecular mechanisms underlying this have yet to be fully uncovered. We observe that the core molecular clock component, BMAL1, is crucial in promoting an antioxidant response in myeloid cells. Deletion of Bmal1 in macrophages disrupts NRF2 activity, facilitating accumulation of reactive oxygen species and the proinflammatory cytokine, IL-1β. Thus the molecular clock directly controls NRF2 transcriptional activity and antioxidant capacity to regulate IL-1β in myeloid cells.

PGE has been shown to increase the transcription of pro-IL-1β. However, recently it has been demonstrated that PGE can block the maturation of IL-1β by inhibiting the NLRP3 inflammasome in macrophages. These apparently conflicting results have led us to reexamine the effect of PGE on IL-1β production. We have found that in murine bone marrow-derived macrophages, PGE via the cAMP/protein kinase A pathway is potently inducing IL-1β transcription, as well as boosting the ability of LPS to induce IL-1β mRNA and pro-IL-1β while inhibiting the production of TNF-α. This results in an increase in mature IL-1β production in macrophages treated with ATP. We also examined the effect of endogenously produced PGE on IL-1β production. By blocking PGE production with indomethacin, we made a striking finding that endogenous PGE is essential for LPS-induced pro-IL-1β production, suggesting a positive feedback loop. The effect of endogenous PGE was mediated by EP2 receptor. In primary human monocytes, where LPS alone is sufficient to induce mature IL-1β, PGE boosted LPS-induced IL-1β production. PGE did not inhibit ATP-induced mature IL-1β production in monocytes. Because PGE mediates the pyrogenic effect of IL-1β, these effects might be especially relevant for the role of monocytes in the induction of fever. A positive feedback loop from IL-1β and back to PGE, which itself is induced by IL-1β, is likely to be operating. Furthermore, fever might therefore occur in the absence of a septic shock response because of the inhibiting effect of PGE on TNF-α production.

No abstract

SummaryHumans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.

The unintentional generation of amorphous character in crystalline active pharmaceutical ingredients (APIs) is an adverse consequence of mechanical activation during dosage form manufacture. In this study, we assess and compare the ability of low glass transition temperature (Tg) dicarboxylic acids to mitigate amorphisation of a model API, salbutamol sulphate (SS), on both co-milling and co-mixing. SS processed alone, as well as co-milled and co-mixed composites of the API with glutaric acid (GA), adipic acid (AA) and pimelic acid (PA) were characterised by powder X-ray diffraction (pXRD), differential scanning calorimetry (DSC) and dynamic vapour sorption (DVS). Milling and dry mixing of SS both resulted in pXRD amorphous materials. No amorphous content of SS was detected by DVS on co-milling with 50% (w/w) GA, while amorphisation was more than halved, relative to the API milled alone, on co-milling with 50% (w/w) AA and PA, respectively. Co-mixing with each excipient also resulted in a decrease in API amorphicity, although the extent of reduction was considerably less compared to the co-milling experiments. The solubility (Solexcipient) of each excipient in amorphous SS was determined by thermal methods. No further reduction in API amorphisation was achieved on co-mixing with 50% (w/w) excipient, compared to concentrations corresponding to the solubility of each excipient in the amorphous API (SolGA=36%, SolAA=21%, SolPA=22%). PXRD confirmed gradual dissolution over time of GA in amorphous SS on co-mixing. In contrast to co-mixing, co-milling SS at excipient weight fractions above their respective solubilities in the amorphous drug resulted in further reductions in API amorphisation. This is thought to be due to the generation of a molecular dispersion of amorphous API, supersaturated with excipient, thereby leading to a more pronounced composite Tg lowering effect. The results indicate that co-processing with low Tg excipients is an effective strategy at minimising amorphisation of an API on mechanical activation.

Prostaglandins (PGs) are important proinflammatory lipid mediators, the significance of which is highlighted by the widespread and efficacious use of non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment of inflammation. 4-Octyl itaconate (4-OI), a derivative of the Krebs cycle-derived metabolite itaconate, has recently garnered much interest as an anti-inflammatory agent. Here we show that 4-OI limits PG production in macrophages stimulated with the Toll-like receptor 1/2 (TLR1/2) ligand Pam3CSK4. This decrease in PG secretion is due to a robust suppression of COX2 expression by 4-OI, with both mRNA and protein levels decreased. Dimethyl fumarate (DMF), a fumarate derivative used in the treatment of multiple sclerosis (MS), with properties similar to itaconate, replicated the phenotype observed with 4-OI. We also demonstrate that the decrease in COX2 expression and inhibition of downstream prostaglandin production occurs in an NRF2-independent manner. Our findings provide a new insight into the potential of 4-OI as an anti-inflammatory agent and also identifies a novel anti-inflammatory function of DMF.