MMP-9 cleaves SP-D and abrogates its innate immune functions in vitro

Possession of a properly functioning innate immune system in the lung is vital to prevent infections due to the ongoing exposure of the lung to pathogens. While mechanisms of pulmonary innate immunity have been well studied, our knowledge of how these systems are altered in disease states, leading to increased susceptibility to infections, is limited. One innate immune protein in the lung, the pulmonary collectin SP-D, has been shown to be important in innate immune defense, as well as clearance of allergens and apoptotic cells. MMP-9 is a protease with a wide variety of substrates, and has been found to be dysregulated in a myriad of lung diseases ranging from asthma to cystic fibrosis; in many of these conditions, there are decreased levels of SP-D. Our results indicate that MMP-9 is able to cleave SP-D in vitro and this cleavage leads to loss of its innate immune functions, including its abilities to aggregate bacteria and increase phagocytosis by mouse alveolar macrophages. However, MMP-9-cleaved SP-D was still detected in a solid-phase E. coli LPS-binding assay, while NE-cleaved SP-D was not. In addition, MMP-9 seems to cleave SP-D much more efficiently than NE at physiological levels of calcium. Previous studies have shown that in several diseases, including cystic fibrosis and asthma, patients have increased expression of MMP-9 in the lungs as well as decreased levels of intact SP-D. As patients suffering from many of the diseases in which MMP-9 is over-expressed can be more susceptible to pulmonary infections, it is possible that MMP-9 cleavage of SP-D may contribute to this phenotype

The Human IL-22 Receptor Is Regulated through the Action of the Novel E3 Ligase Subunit FBXW12, Which Functions as an Epithelial Growth Suppressor

Interleukin- (IL-) 22 signaling is protective in animal models of pneumonia and bacteremia by Klebsiella pneumoniae and mediates tissue recovery from influenza and Staph aureus infection. We recently described processing of mouse lung epithelial IL-22 receptor (IL-22R) by ubiquitination on the intracellular C-terminal. To identify cellular factors that regulate human IL-22R, we screened receptor abundance while overexpressing constituents of the ubiquitin system and identify that IL-22R can be shuttled for degradation by multiple previously uncharacterized F-box protein E3 ligase subunits. We observe that in human cells IL-22R is destabilized by FBXW12. FBXW12 causes depletion of endogenous and plasmid-derived IL-22R in lung epithelia, binds the E3 ligase constituent Skp-1, and facilitates ubiquitination of IL-22R in vitro. FBXW12 knockdown with shRNA increases IL-22R abundance and STAT3 phosphorylation in response to IL-22 cytokine treatment. FBXW12 shRNA increases human epithelial cell growth and cell cycle progression with enhanced constitutive activity of map kinases JNK and ERK. These findings indicate that the heretofore-undescribed protein FBXW12 functions as an E3 ligase constituent to ubiquitinate and degrade IL-22R and that therapeutic FBXW12 inhibition may enhance IL-22 signaling and bolster mucosal host defense and infection containment

Azithromycin decreases NALP3 mRNA stability in monocytes to limit inflammasome-dependent inflammation

Abstract Background Azithromycin, an antibiotic used for multiple infectious disorders, exhibits anti-inflammatory effects, but the molecular basis for this activity is not well characterized. Azithromycin inhibits IL-1β-mediated inflammation that is dependent, in part, on inflammasome activity. Here, we investigated the effects of azithromycin on the NACHT, LRR, and PYD domains-containing protein 3 (NALP3) protein, which is the sensing component of the NALP3 inflammasome, in human monocytes. Methods THP-1 cells were treated with azithromycin alone, LPS alone, or both. NALP3 and IL-1β protein levels were determined by immunoblotting. NLRP3 gene (encoding NALP3) transcript levels were determined by quantitative qPCR. In order to measure NLRP3 transcript decay, actinomycin D was used to impair gene transcription. THP-1 Lucia cells which contain an NF-κB responsive luciferase element were used to assess NF-κB activity in response to azithromycin, LPS, and azithromycin/LPS by measuring luminescence. To confirm azithromycin’s effects on NLRP3 mRNA and promoter activity conclusively, HEK cells were lipofected with luciferase reporter constructs harboring either the 5’ untranslated region (UTR) of the NLRP3 gene which included the promoter, the 3’ UTR of the gene, or an empty plasmid prior to treatment with azithromycin and/or LPS, and luminescence was measured. Results Azithromycin decreased IL-1β levels and reduced NALP3 protein levels in LPS-stimulated THP-1 monocytes through a mechanism involving decreased mRNA stability of the NALP3 – coding NLRP3 gene transcript as well as by decreasing NF-κB activity. Azithromycin accelerated NLRP3 transcript decay confirmed by mRNA stability and 3’UTR luciferase reporter assays, and yet the antibiotic had no effect on NLRP3 promoter activity in cells containing a 5’ UTR reporter. Conclusions These studies provide a unique mechanism whereby azithromycin exerts immunomodulatory actions in monocytes by destabilizing mRNA levels for a key inflammasome component, NALP3, leading to decreased IL-1β-mediated inflammation

Cleaved SP-D Fails to Increase Phagocytosis by MH-S Cells.

<p>Examination and quantitation of phagocytosis of <i>E. coli</i> by MH-S cells was performed using (A) a gentamicin protection assay and (B and C) flow cytometry. For (A), all conditions are significantly different (p<0.05) from 1 µg/mL intact SP-D. An asterisk (*) denotes p≤0.001 when compared to 1 µg/mL intact SP-D. Columns marked with # are significantly different when compared to 0.5 µg/mL intact SP-D (p<0.05). For (B), intact SP-D causes MH-S cells to have significantly higher mean fluorescence intensity (MFI) than all other conditions (p≤0.001). Cleaved SP-D is significantly different when compared to both MMP-9 and PBS controls (p≤0.001). For (C), flow data was gated on forward and side scatter to select for MH-S cells. For both (A) and (B), error bars represent standard deviation.</p

MMP-9 Cleaves SP-D in a Dose- and Time-dependent Manner.

<p>(A) Dose and (B) time course digestions of SP-D by MMP-9. For the dose course (A), the reaction loaded onto lane 2 contained 62 ng/mL MMP-9 and the concentration was raised 3-fold serially to 5 µg/mL MMP-9 at lane 6. Reactions were incubated for 4 hours. For the time course (B), the length of digestion in minutes is listed above the lane. SP-D concentration was 20 µg/mL for both experiments, and MMP-9 concentration was 5 µg/mL for the time course. For the Native PAGE (C), lane 1 contains intact SP-D and lane 2 contains cleaved SP-D. An arrow indicates the band corresponding to MMP-9.</p

Cleaved SP-D Retains its Ability to Bind <i>E. coli</i> LPS.

<p>Examination of the ability of SP-D to bind to LPS-coated plates. In (A), the ELISA was performed using 2-fold serially diluted SP-D samples. In (B), 1 µg/mL intact or cleaved SP-D was analyzed in triplicate with MMP-9 as a control and NE-cleaved SP-D as a negative control. The average of PBST alone ran in triplicate was subtracted from all values. While intact and MMP-9-cleaved SP-D in PBST are significantly different from PBST alone (p≤0.001 and p<0.05, respectively) and NE-cleaved SP-D (p≤0.001 and p<0.05, respectively), MMP-9 along with intact and cleaved SP-D in PBST with maltose were not significantly different from PBST or PBST with maltose. For (B), error bars represent standard deviation.</p

Mortality factor 4 like 1 protein mediates epithelial cell death in a mouse model of pneumonia

Unchecked epithelial cell death is fundamental to the pathogenesis of pneumonia. The recognition of unique signaling pathways that preserve epithelial cell viability may present new opportunities for interventional strategies. We describe that mortality factor 4 like 1 (Morf4l1), a protein involved in chromatin remodeling, is constitutively expressed at low levels in the lung because of its continuous degradation mediated by an orphan ubiquitin E3 ligase subunit, Fbxl18. Expression of Morf4l1 increases in humans with pneumonia and is up-regulated in lung epithelia after exposure to Pseudomonas aeruginosa or lipopolysaccharide. In a mouse model of pneumonia induced by P. aeruginosa, Morf4l1 is stabilized by acetylation that protects it from Fbxl18-mediated degradation. After P. aeruginosa infection of mice, overexpression of Morf4l1 resulted in lung epithelial cell death, whereas its depletion restored cell viability. Using in silico modeling and drug-target interaction studies, we identified that the U.S. Food and Drug Administration–approved thrombin inhibitor argatroban is a Morf4l1 antagonist. Argatroban inhibited Morf4l1-dependent histone acetylation, reduced its cytotoxicity, and improved survival of mice with experimental lung injury at doses that had no anticoagulant activity. These studies uncover a previously unrecognized biological mechanism whereby pathogens subvert cell viability by extending the life span of a cytotoxic host protein. Morf4l1 may be a potential molecular target for non-antibiotic pharmacotherapy during severe pulmonary infection