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.

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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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Chitin is a polysaccharide that provides structure and rigidity to the cell walls of fungi and insects. Mammals possess multiple chitinases, which function to degrade chitin, thereby supporting a role for chitinases in immune defense. However, chitin degradation has been implicated in the pathogenesis of asthma. Here, we determined the impact of acidic mammalian chitinase (AMCase) () deficiency on host defense during acute exposure to the fungal pathogen as well as its contribution to-associated allergic asthma. We demonstrate that chitin in the fungal cell wall was detected at low levels in conidia, which emerged at the highest level during hyphal transition. In response to acute challenge, mice unexpectedly demonstrated lower lung burdens at 2 days postchallenge. The lower fungal burden correlated with decreased lung interleukin-33 (IL-33) levels yet increased IL-1β and prostaglandin E (PGE) production, a phenotype that we reported previously to promote the induction of IL-17A and IL-22. During chronic exposure, AMCase deficiency resulted in lower dynamic and airway lung resistance than in wild-type mice. Improved lung physiology correlated with attenuated levels of the proallergic chemokines CCL17 and CCL22. Surprisingly, examination of inflammatory responses during chronic exposure revealed attenuated IL-17A and IL-22 responses, but not type 2 responses, in the absence of AMCase. Collectively, these data suggest that AMCase functions as a negative regulator of immune responses during acute fungal exposure and is a contributor to fungal asthma severity, putatively via the induction of proinflammatory responses.

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Humans are constantly exposed to the opportunistic mold and disease caused by this pathogen is often determined by the magnitude of local and systemic immune responses. We have previously shown a protective role for IL-22 after acute exposure. Here, employing IL22R26R reporter mice, we identified iNKT cells, γδ T cells and ILC3 cells as lung cell sources of IL-22 in response to acute exposure. As these cells often utilize common γ-chain cytokines for their development or maintenance, we determined the role of IL-7, IL-21 and IL-15 on lung IL-22 induction and lung clearance. We observed that IL-7, IL-21 and IL-15 were essential, partially required or negatively regulated the production of IL-22, respectively. Deficiency in IL-7 and IL-21, but not IL-15R, resulted in impaired fungal clearance. Surprisingly however, the absence of IL-7, IL-21 or IL-15R signaling had no effect on neutrophil recruitment. The levels of IL-1α, an essential anti- proinflammatory cytokine, were increased in the absence of IL-7 and IL-15R, but decreased in the absence of IL-21. IL-7 was responsible for maintaining lung iNKT cells and γδ T cells whereas IL-21 was responsible for maintaining lung iNKT cells and ILC3s. In contrast, IL-15R deficiency had no effect on the absolute numbers of any IL-22 cell source, rather resulting in enhanced per cell production of IL-22 by iNKT cells and γδ T cells. Collectively, these results provide insight into how the IL-22 response in the lung is shaped after acute exposure.