Reciprocal Inhibition of Adiponectin and Innate Lung Immune Responses to Chitin and Aspergillus fumigatus

Chitin is a structural biopolymer found in numerous organisms, including pathogenic fungi, and recognized as an immune-stimulating pathogen associated molecular pattern by pattern recognition molecules of the host immune system. However, programming and regulation of lung innate immunity to chitin inhalation in the context of inhalation of fungal pathogens such as Aspergillus fumigatus is complex and our understanding incomplete. Here we report that the systemic metabolism-regulating cytokine adiponectin is decreased in the lungs and serum of mice after chitin inhalation, with a concomitant decrease in surface expression of the adiponectin receptor AdipoR1 on lung leukocytes. Constitutive lung expression of acidic mammalian chitinase resulted in decreased inflammatory cytokine gene expression and neutrophil recruitment, but did not significantly affect lung adiponectin transcription. Exogenous recombinant adiponectin specifically dampened airway chitin-mediated eosinophil recruitment, while adiponectin deficiency resulted in increased airway eosinophils. The presence of adiponectin also resulted in decreased CCL11-mediated migration of bone marrow-derived eosinophils. In contrast to purified chitin, aspiration of viable conidia from the high chitin-expressing A. fumigatus isolate Af5517 resulted in increased neutrophil recruitment and inflammatory cytokine gene expression in adiponectin-deficient mice, while no significant changes were observed in response to the isolate Af293. Our results identify a novel role for the adiponectin pathway in inhibition of lung inflammatory responses to chitin and A. fumigatus inhalation

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori's modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking γδ T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosi

Caspofungin Increases Fungal Chitin and Eosinophil and γδ T Cell-Dependent Pathology in Invasive Aspergillosis

The polysaccharide-rich fungal cell wall provides pathogen-specific targets for antifungal therapy and distinct molecular patterns that stimulate protective or detrimental host immunity. The echinocandin antifungal caspofungin inhibits synthesis of cell wall β-1,3-glucan and is used for prophylactic therapy in immune-suppressed individuals. However, breakthrough infections with fungal pathogen Aspergillus fumigatus are associated with caspofungin prophylaxis. In this study, we report in vitro and in vivo increases in fungal surface chitin in A. fumigatus induced by caspofungin that was associated with airway eosinophil recruitment in neutropenic mice with invasive pulmonary aspergillosis (IA). More importantly, caspofungin treatment of mice with IA resulted in a pattern of increased fungal burden and severity of disease that was reversed in eosinophil-deficient mice. Additionally, the eosinophil granule proteins major basic protein and eosinophil peroxidase were more frequently detected in the bronchoalveolar lavage fluid of lung transplant patients diagnosed with IA that received caspofungin therapy when compared with azole-treated patients. Eosinophil recruitment and inhibition of fungal clearance in caspofungin-treated mice with IA required RAG1 expression and γδ T cells. These results identify an eosinophil-mediated mechanism for paradoxical caspofungin activity and support the future investigation of the potential of eosinophil or fungal chitin-targeted inhibition in the treatment of IA

Differential abundance of CK1α provides selectivity for pharmacological CK1α activators to target WNT-dependent tumors

Constitutive WNT activity drives the growth of various human tumors, including nearly all colorectal cancers (CRCs). Despite this prominence in cancer, no WNT inhibitor is currently approved for use in the clinic largely due to the small number of druggable signaling components in the WNT pathway and the substantial toxicity to normal gastrointestinal tissue. We have shown that pyrvinium, which activates casein kinase 1α (CK1α), is a potent inhibitor of WNT signaling. However, its poor bioavailability limited the ability to test this first-in-class WNT inhibitor in vivo. We characterized a novel small-molecule CK1α activator called SSTC3, which has better pharmacokinetic properties than pyrvinium, and found that it inhibited the growth of CRC xenografts in mice. SSTC3 also attenuated the growth of a patient-derived metastatic CRC xenograft, for which few therapies exist. SSTC3 exhibited minimal gastrointestinal toxicity compared to other classes of WNT inhibitors. Consistent with this observation, we showed that the abundance of the SSTC3 target, CK1α, was decreased in WNT-driven tumors relative to normal gastrointestinal tissue, and knocking down CK1α increased cellular sensitivity to SSTC3. Thus, we propose that distinct CK1α abundance provides an enhanced therapeutic index for pharmacological CK1α activators to target WNT-driven tumors