Antifolates, folate analogs that inhibit vitamin B 9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.isothermal titration calorimetry | targeted drug delivery
Asthmatics sensitized to fungi are reported to have more severe asthma, yet the immunopathogenic pathways contributing to this severity have not been identified. In a pilot assessment of human asthmatics, those subjects sensitized to fungi demonstrated elevated levels of the common γ-chain cytokine IL-7 in lung lavage fluid, which negatively correlated with the lung function measurement PC20. Subsequently, we show that IL-7 administration during experimental fungal asthma worsened lung function and increased the levels of type 2 cytokines (IL-4, IL-5, IL-13), proallergic chemokines (CCL17, CCL22) and proinflammatory cytokines (IL-1α, IL-1β). Intriguingly, IL-7 administration also increased IL-22, which we have previously reported to drive immunopathogenic responses in experimental fungal asthma. Employing IL22R26R reporter mice, we identified γδ T cells, iNKT cells, CD4 T cells and ILC3s as sources of IL-22 during fungal asthma; however, only iNKT cells were significantly increased after IL-7 administration. IL-7-induced immunopathogenesis required both type 2 and IL-22 responses. Blockade of IL-7Rα in vivo resulted in attenuated IL-22 production, lower CCL22 levels, decreased iNKT cell, CD4 T-cell and eosinophil recruitment, yet paradoxically increased dynamic lung resistance. Collectively, these results suggest a complex role for IL-7 signaling in allergic fungal asthma.
Members of the IL-1 family play protective and regulatory roles in immune defense against the opportunistic mold Aspergillus fumigatus. Here, we investigated the IL-1 family member IL-33 in lung defense against A. fumigatus. IL-33 was detected in the naïve lung, which further increased after exposure to A. fumigatus in a Dectin-1 independent manner. Mice deficient in the receptor for IL-33 (Il1rl1−/−) unexpectedly demonstrated enhanced lung clearance of A. fumigatus. IL-33 functioned as a negative regulator of multiple inflammatory cytokines, as IL-1α, IL-1β, IL-6, IL-17A and IL-22 were significantly elevated in fungal-exposed Il1rl1−/− mice. Subsequently, IL-33 administration to normal mice attenuated fungal-induced IL-17A and IL-22, but not IL-1α, IL-1β and IL-6 production. IL-33 mediated regulation of IL-17A and IL-22 did not involve the modulation of IL-23 but rather prostaglandin E2 (PGE2); PGE2 was significantly increased in fungal-exposed Il1rl1−/− mice and normal mice produced less PGE2 after fungal exposure when administered IL-33, suggesting that IL-33 mediated regulation of IL-17A and IL-22 occurred at the level of PGE2. This was confirmed by in vivo cyclooxygenase 2 (COX-2) inhibition, which attenuated fungal-induced IL-17A and IL-22, as well as IL-1α, IL-1β and IL-6 production, in Il1rl1−/− mice resulting in impaired fungal clearance. We also show that a PGE2 receptor agonist increased, whereas a PGE2 synthase inhibitor decreased, the levels of IL-17A and IL-22, but not IL-1α, IL-1β and IL-6. This study establishes novel mechanisms of induction of innate IL-17A/IL-22 production via PGE2 and regulation of the PGE2-IL-17A-IL-22 axis via IL-33 signaling during lung fungal exposure.
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