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1.  Safety, tolerability, and biomarkers of the treatment of mice with aerosolized Toll-like receptor ligands 
We have previously discovered a synergistically therapeutic combination of two Toll-like receptor ligands, an oligodeoxynucleotide (ODN) and Pam2CSK4. Aerosolization of these ligands stimulates innate immunity within the lungs to prevent pneumonia from bacterial and viral pathogens. Here we examined the safety and tolerability of this treatment in mice, and characterized the expression of biomarkers of innate immune activation. We found that neutrophils appeared in lung lavage fluid 4 h after treatment, reached a peak at 48 h, and resolved by 7 days. The peak of neutrophil influx was accompanied by a small increase in lung permeability. Despite the abundance of neutrophils in lung lavage fluid, only rare neutrophils were visible histopathologically in the interstitium surrounding bronchi and veins and none were visible in alveolar airspaces. The cytokines interleukin 6 (IL-6), tumour necrosis factor, and Chemokine (C-X-C motif) ligand 2 rose several hundred-fold in lung lavage fluid 4 h after treatment in a dose-dependent and synergistic manner, providing useful biomarkers of lung activation. IL-6 rose fivefold in serum with delayed kinetics compared to its rise in lavage fluid, and might serve as a systemic biomarker of immune activation of the lungs. The dose–response relationship of lavage fluid cytokines was preserved in mice that underwent myeloablative treatment with cytosine arabinoside to model the treatment of hematologic malignancy. There were no overt signs of distress in mice treated with ODN/Pam2CSK4 in doses up to eightfold the therapeutic dose, and no changes in temperature, respiratory rate, or behavioral signs of sickness including sugar water preference, food disappearance, cage exploration or social interaction, though there was a small degree of transient weight loss. We conclude that treatment with aerosolized ODN/Pam2CSK4 is well tolerated in mice, and that innate immune activation of the lungs can be monitored by the measurement of inflammatory cytokines in lung lavage fluid and serum.
PMCID: PMC3915096  PMID: 24567720
pneumonia; innate immunity; Toll-like receptor; oligodeoxynucleotide; lipopeptide; aerosol; myeloablation
2.  Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells 
Biochemical Journal  2012;446(Pt 3):383-394.
Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.
PMCID: PMC3430001  PMID: 22694344
exocytosis; mast cell; mucin; mucus; Munc18; secretion; AB-PAS, Alcian Blue/periodic acid/Schiff reagent; bHLH, basic helix–loop–helix; CCSP, Clara cell secretory protein; Clca3, chloride channel, calcium-activated, family member 3; CRE, cAMP-response element; DNP, 2,4-dinitrophenol; FBS, fetal bovine serum; FcϵRIα, high-affinity IgE receptor, α subunit; FRT, flippase recognition target; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRE, glucocorticoid-response element; HA, haemagglutinin; HSA, human serum albumin; HRP, horseradish peroxidase; IL-3, interleukin-3; INR, initiator; ISH, in situ hybridization; MC, mast cell; mBMMC, mouse bone-marrow-derived MC; mClca3, mouse Clca3; MFI, mean fluorescent intensity; mtCC, mouse transformed Clara cell; NK, natural killer; OCT, optimal cutting temperature compound; PAFS, periodic acid/fluorescent Schiff reagent; PBST, PBS containing 0.05% Tween 20; PGD2, prostaglandin D2; PGK, phosphoglucokinase; SCF, stem cell factor; SM, Sec1/Munc18; SNAP, soluble N-ethylmaleimide-sensitive factor-attachment protein; SNARE, SNAP receptor; Stxbp2, syntaxin-binding protein 2; TK, thymidine kinase; TNFα, tumour necrosis factor α; WT, wild-type; YFP, yellow fluorescent protein
3.  Stimulated Innate Resistance of Lung Epithelium Protects Mice Broadly against Bacteria and Fungi 
Pneumonia is a serious problem worldwide. We recently demonstrated that innate defense mechanisms of the lung are highly inducible against pneumococcal pneumonia. To determine the breadth of protection conferred by stimulation of lung mucosal innate immunity, and to identify cells and signaling pathways activated by this treatment, mice were treated with an aerosolized bacterial lysate, then challenged with lethal doses of bacterial and fungal pathogens. Mice were highly protected against a broad array of Gram-positive, Gram-negative, and class A bioterror bacterial pathogens, and the fungal pathogen, Aspergillus fumigatus. Protection was associated with rapid pathogen killing within the lungs, and this effect was recapitulated in vitro using a respiratory epithelial cell line. Gene expression analysis of lung tissue showed marked activation of NF-κB, type I and II IFN, and antifungal Card9-Bcl10-Malt1 pathways. Cytokines were the most strongly induced genes, but the inflammatory cytokines TNF and IL-6 were not required for protection. Lung-expressed antimicrobial peptides were also highly up-regulated. Taken together, stimulated innate resistance appears to occur through the activation of multiple host defense signaling pathways in lung epithelial cells, inducing rapid pathogen killing, and conferring broad protection against virulent bacterial and fungal pathogens. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value for protection of patients with neutropenia or impaired adaptive immunity against opportunistic pneumonia, and for defense of immunocompetent subjects against a bioterror threat or epidemic respiratory infection.
PMCID: PMC2809220  PMID: 19329554
innate immunity; pneumonia; immunocompromised host; lung epithelium
4.  Stimulation of Lung Innate Immunity Protects against Lethal Pneumococcal Pneumonia in Mice 
Rationale: The lungs are a common site of serious infection in both healthy and immunocompromised subjects, and the most likely route of delivery of a bioterror agent. Since the airway epithelium shows great structural plasticity in response to inflammatory stimuli, we hypothesized it might also show functional plasticity.
Objectives: To test the inducibility of lung defenses against bacterial challenge.
Methods: Mice were treated with an aerosolized lysate of ultraviolet-killed nontypeable (unencapsulated) Haemophilus influenzae (NTHi), then challenged with a lethal dose of live Streptococcus pneumoniae (Spn) delivered by aerosol.
Measurements and Main Results: Treatment with the NTHi lysate induced complete protection against challenge with a lethal dose of Spn if treatment preceded challenge by 4 to 24 hours. Lesser levels of protection occurred at shorter (83% at 2 h) and longer (83% at 48–72 h) intervals between treatment and challenge. There was also some protection when treatment was given 2 hours after challenge (survival increased from 14 to 57%), but not 24 hours after challenge. Protection did not depend on recruited neutrophils or resident mast cells and alveolar macrophages. Protection was specific to the airway route of infection, correlated in magnitude and time with rapid bacterial killing within the lungs, and was associated with increases of multiple antimicrobial polypeptides in lung lining fluid.
Conclusions: We infer that protection derives from stimulation of local innate immune mechanisms, and that activated lung epithelium is the most likely cellular effector of this response. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value.
PMCID: PMC2427056  PMID: 18388354
innate immunity; pneumonia; immunocompromised host; lung epithelium
5.  An intramolecular t-SNARE complex functions in vivo without the syntaxin NH2-terminal regulatory domain 
The Journal of Cell Biology  2006;172(2):295-307.
Membrane fusion in the secretory pathway is mediated by SNAREs (located on the vesicle membrane [v-SNARE] and the target membrane [t-SNARE]). In all cases examined, t-SNARE function is provided as a three-helix bundle complex containing three ∼70–amino acid SNARE motifs. One SNARE motif is provided by a syntaxin family member (the t-SNARE heavy chain), and the other two helices are contributed by additional t-SNARE light chains. The syntaxin family is the most conformationally dynamic group of SNAREs and appears to be the major focus of SNARE regulation. An NH2-terminal region of plasma membrane syntaxins has been assigned as a negative regulatory element in vitro. This region is absolutely required for syntaxin function in vivo. We now show that the required function of the NH2-terminal regulatory domain (NRD) of the yeast plasma membrane syntaxin, Sso1p, can be circumvented when t-SNARE complex formation is made intramolecular. Our results suggest that the NRD is required for efficient t-SNARE complex formation and does not recruit necessary scaffolding factors.
PMCID: PMC2063558  PMID: 16401725
6.  Sec1p directly stimulates SNARE-mediated membrane fusion in vitro 
The Journal of Cell Biology  2004;167(1):75-85.
Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.
PMCID: PMC2172507  PMID: 15466482

Results 1-6 (6)