MISSION

flagellin, released from commensal and enteroinvasive microbes in the human gut, stimulates Toll-like
receptor 5 (TLR5) leading to proinflammatory responses and activation of PI3K in colonic epithelial cells.
However, the molecular mechanisms by which TLR5 activates PI3K have not been well investigated. In
this study, Dr. Rhee determined the signaling pathways involved in flagellin/TLR5-induced PI3K
activation in human colonocytes. In order to determine PI3K activation in vitro, expression of TLR5 or its
adaptor molecule, MyD88, was silenced by stable transfection of TLR5 or MyD88 siRNA constructs,
respectively, in non-transformed NCM460 human colonocytes. Several TLR5 mutants harboring pointmutated
tyrosine residues, including SH-2-domain binding motif ‘YXXM’ in the cytoplasmic TIR domain
of TLR5, were generated by site-directed mutagenesis. Co-immunoprecipitation assays were performed to
determine the association between TLR5 and MyD88 or PI3K.
The investigators showed that flagellin exposure to NCM460 cells transiently activated PI3K within 10-
30 min, while blocking PI3K reduced IL-8 production in colonocytes. TLR5 silencing blocked PI3K
activation by flagellin, indicating that TLR5 specifically mediates PI3K activation in colonocytes.
Immunoprecipitation studies showed that TLR5 recruits the p85 regulatory subunit of PI3K to its
cytoplasmic TIR domain following flagellin stimulation, and MyD88 interacted with TLR5 in a timedependent
manner. Additional immunoprecipitation studies with TLR5 mutant constructs demonstrated
that the SH-2 binding ‘YXXM’ motif in the TIR domain of TLR5 is not involved in p85 recruitment,
implying that p85 is indirectly recruited to TLR5. Moreover, silencing MyD88 expression in colonocytes
disrupted PI3K activation and blocked the association between TLR5 and the p85 regulatory subunit.
Thus, PI3K activation in TLR5-associated signaling in human colonocytes is MyD88-dependent.
ROLE OF SERPINB1 IN CIGARETTE SMOKE-INDUCED DEFECTIVE ANTIMICROBIAL DEFENSE
Charaf Benarafa, DVM, PhD; Immune Disease Institute; YCSA 2008
Oxidative damage induced by SHS leads to cellular injury and inflammation in the lung and these events
are associated with increased risk for pulmonary infection. The direct effects of repeated smoke exposure
and the additional burden produced by recruited leukocytes that release proteases, oxidants and
inflammatory mediators on the lungs, can cause dysregulation of the immune response to pathogens.
SERPINB1 (also known as monocyte neutrophil elastase inhibitor, MNEI) is an ancestral member of the
serpin (SERine Protease INhibitor) family and one of the most potent inhibitors of the neutrophil serine
proteases: elastase, cathepsin G and proteinase-3. Dr. Benarafa and collaborators have recently developed a
model of protease-antiprotease imbalance by deleting the mouse gene. Serpinb1-deficient (serpinb1-/-)
mice have a late-onset defective immune response to acute Pseudomonas aeruginosa infection and fail to
clear the infection due to increased neutrophil death and proteolysis of surfactant protein-D (SP-D). Dr.
Benarafa hypothesizes that SERPINB1 participates in the protection of cellular and molecular antimicrobial
mechanisms dysregulated by the oxidative and inflammatory damage induced by cigarette smoke. To test
this hypothesis, the scientists are investigating: 1) whether oxidative damage induced by exposure to
cigarette smoke affects SERPINB1 expression in lung cells in vitro and in vivo; 2) whether the
combination of oxidative injury of cigarette smoke and the antiprotease defect of serpinb1-/- mice
increases the susceptibility to subacute bacterial lung infection; and 3) whether recombinant SERPINB1
preserves the innate immune response to P. aeruginosa infection in wild-type mice exposed to cigarette
smoke. These studies are to define a role for SERPINB1 in regulating damage caused by cigarette smoke
components, and protecting molecular and cellular components of the healthy lung innate immune
response. Importantly, inhaled recombinant SERPINB1 may be considered as a possible adjunct treatment
in cases of pulmonary exacerbation caused by bacterial infection in COPD patients.
THE INFLUENCE OF SECOND HAND CIGARETTE SMOKE ON THE INNATE IMMUNE FUNCTION
OF NASAL EPITHELIAL CELLS.
James A. Jukosky, PhD; Dartmouth College; YCSA 2008
Adults and children exposed to SHS have an elevated risk of developing chronic sinus and bronchial
infections, asthma, and allergies. This suggests that cigarette smoke (CS) has adverse effects on immune
defenses. The goal of Dr. Jukosky’s study is to understand which innate immune responses are altered by
CS exposure. Epithelial cells are the key to innate immunity and the first line of defense against infection.
Airway epithelial cells form a physical barrier, but also respond to the presence of microbes by secreting
antimicrobials, cytokines and chemokines. These molecules can neutralize infectious microorganisms
and/or provide signals critical to the initiation of adaptive immune responses. Preliminary results show
that aspects of innate immune protection provided by nasal epithelial cells are altered by CS exposure. Dr.
Jukosky’s data demonstrate that nasal epithelial cell secretion of CCL20, (a protein that has antimicrobial
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