Cheryl Chan

Mast Cells Augment Adaptive Immunity by Orchestrating Dendritic Cell Trafficking through Infected Tissues

Mast cells (MCs) are best known for eliciting harmful reactions, mostly after primary immunity has been established. Here, we report that, during footpad infection with E. coli in MC-deficient mice, as compared to their MC-sufficient counterparts, the serum antibody response is significantly diminished and less protective following passive immunization in a urinary tract infection (UTI) model in wild-type mice. MCs were found to recruit large numbers of dendritic cells (DCs) into the infected tissue site, which eventually migrated into draining lymph nodes (DLNs) during a prolonged time course. This pattern of trafficking was facilitated by MC-generated TNF, which increased the expression of E-selectin on local blood vessels. Antibody blockade of E-selectin inhibited DC recruitment into the site of infection and DLNs and consequently impaired the primary humoral immune response. Thus, during infection, resident MCs contribute to the primary protective adaptive response through recruitment of DCs from the circulation into infected sites.

Mast cell interleukin-10 drives localized tolerance in chronic bladder infection.

The lower urinary tracts virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected in combination with clinical observations that antibody responses after bladder infections are not detectable suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells (MCs). This involves localized production of interleukin-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of MCs orchestrating the early innate immunity during bladder infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of MC-maintained immune privilege.

Synthetic mast-cell granules as adjuvants to promote and polarize immunity in lymph nodes

Granules of mast cells (MCs) enhance adaptive immunity when, on activation, they are released as stable particles. Here we show that submicrometre particles modelled after MC granules augment immunity when used as adjuvants in vaccines. The synthetic particles, which consist of a carbohydrate backbone with encapsulated inflammatory mediators such as tumour necrosis factor, replicate attributes of MCs in vivo including the targeting of draining lymph nodes and the timed release of the encapsulated mediators. When used as an adjuvant during vaccination of mice with haemagglutinin from the influenza virus, the particles enhanced adaptive immune responses and increased survival of mice on lethal challenge. Furthermore, differential loading of the particles with the cytokine IL-12 directed the character of the response towards Th1 lymphocytes. The synthetic MC adjuvants replicate and enhance the functions of MCs during vaccination, and can be extended to polarize the resulting immunity.

Plasticity in mast cell responses during bacterial infections.

Mast cells (MCs) have been implicated in orchestrating the hosts early innate immune and adaptive immune responses in several models of acute bacterial infections. Most of this activity results in early clearance of the bacteria and timely resolution of infection. However, during chronic infections because of the prolonged nature of MC-bacterial interactions, the role of the MC in determining the fate of infection is markedly more complex. Depending on the nature of the pathogen, severity of infection, and its association with a preexisting inflammatory disease, MCs may promote rather than contain chronic infections and exacerbate their pathological sequellae.

Altered Binding Site Selection of p53 Transcription Cassettes by Hepatitis B Virus X Protein

ABSTRACT The key cellular regulator p53 is a common target of viral oncoproteins. However, the mechanism by which p53 transcription regulation is modulated by hepatitis B virus X protein (HBx), a transcription cofactor implicated in hepatitis B virus-associated hepatocellular carcinoma (HCC), is poorly understood. By integrating p53 chromatin immunoprecipitation (ChIP)-on-chip and expression profiling of an HBx-expressing cell culture system, we report that HBx alters p53 binding site selectivity in the regulatory regions of genes, and this is associated with their aberrant expression. Using an HBx-deregulated gene, p53AIP1, as a model, we show that HBx aberrantly increases p53AIP1 expression by conferring p53 selectivity for a more conserved binding site in its regulatory region. We further demonstrate that HBx-deregulated increased p53AIP1 expression is relevant in HCC livers and define a functional role for p53AIP1 in mediating HBx-induced apoptosis in vitro. Significantly, we provide evidence that specific p53-associated transcription cofactors and coregulators are differentially recruited in the presence of HBx, effecting a PCAF-mediated “p53 Lys320 acetylation switch” that results in altered binding site selection of distinct p53 transcription cassettes. The findings here clarify the role of HBx in modulating p53 transcription regulation and provide a novel mechanistic insight into this deregulation.

Disruption of FAT10–MAD2 binding inhibits tumor progression

Significance FAT10, a ubiquitin-like modifier, is an oncogene that interacts with mitotic arrest-deficient 2 (MAD2) and confers cellular malignancy. Here we identified the MAD2-binding residues of FAT10 and determined the first solution structure, to our knowledge, of the first FAT10 ubiquitin-like domain. Importantly, we demonstrated the proof-of-mechanism for a novel and specific drug-targeting strategy that entails the specific inhibition of the pathological activity of a therapeutic target but not its reported physiological function, thus minimizing undesirable side effects: Abrogation of the FAT10–MAD2 interaction curtailed tumor progression without affecting FAT10’s interaction with its other known physiological binding partners. This study presents a paradigm for drug targeting and paves the way for the development of a novel small-molecule anticancer inhibitor targeting the MAD2-binding interface of FAT10. FAT10 (HLA-F-adjacent transcript 10) is a ubiquitin-like modifier that is commonly overexpressed in various tumors. It was found to play a role in mitotic regulation through its interaction with mitotic arrest-deficient 2 (MAD2). Overexpression of FAT10 promotes tumor growth and malignancy. Here, we identified the MAD2-binding interface of FAT10 to be located on its first ubiquitin-like domain whose NMR structure thus was determined. We further proceeded to demonstrate that disruption of the FAT10–MAD2 interaction through mutation of specific MAD2-binding residues did not interfere with the interaction of FAT10 with its other known interacting partners. Significantly, ablation of the FAT10–MAD2 interaction dramatically limited the promalignant capacity of FAT10, including promoting tumor growth in vivo and inducing aneuploidy, proliferation, migration, invasion, and resistance to apoptosis in vitro. Our results strongly suggest that the interaction of FAT10 with MAD2 is a key mechanism underlying the promalignant property of FAT10 and offer prospects for the development of anticancer strategies.

Loss of Bladder Epithelium Induced by Cytolytic Mast Cell Granules.

Programmed death and shedding of epithelial cells is a powerful defense mechanism to reduce bacterial burden during infection but this activity cannot be indiscriminate because of the critical barrier function of the epithelium. We report that during cystitis, shedding of infected bladder epithelial cells (BECs) was preceded by the recruitment of mast cells (MCs) directly underneath the superficial epithelium where they docked and extruded their granules. MCs were responding to interleukin-1β (IL-1β) secreted by BECs after inflammasome and caspase-1 signaling. Upon uptake of granule-associated chymase (mouse MC protease 4 [mMCPT4]), BECs underwent caspase-1-associated cytolysis and exfoliation. Thus, infected epithelial cells require a specific cue for cytolysis from recruited sentinel inflammatory cells before shedding.

Involvement of dynamin-2 in formation of discoid vesicles in urinary bladder umbrella cells

Umbrella cells (UCs) of the epithelium of the urinary bladder have the capacity to control bladder volume by regulating exocytosis/endocytosis of their intracellular discoid vesicles (DVs). Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. We have examined whether Dyn2, the most abundant Dyn form, is expressed in UCs and contributes to their endocytic actions. A specific antibody against Dyn2 was used to localize Dyn2 in human and rodent UCs by immunohistochemistry. To clarify the functional roles of Dyn2, mouse bladders were treated with a Dyn-GTPase inhibitor, dynasore, and its effects on their UC structure were assessed. Since uropathogenic Escherichia coli can be encased into UCs during infection, we used immunohistochemistry to determine whether bacteria-encasing compartments in the infected UCs were also enriched with Dyn2. Light microscopy showed that Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method confirmed these findings and revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Dynasore treatment of bladders markedly reduced the number of DVs in UCs. In infected UCs, E. coli was encased in compartments enriched in Dyn2. Therefore, Dyn2 is highly enriched in UCs and mostly associated with membranes of DVs and microfilaments in the UCs. Pretreatment of bladders with dynasore inhibits E. coli invasion of UCs. Dyn2 thus contributes to the structural integrity of DVs and to the endocytic activity of UCs.

Global re-wiring of p53 transcription regulation by the hepatitis B virus X protein

The tumour suppressor p53 is a central player in transcription regulation and cell fate determination. By interacting with p53 and altering its sequence‐specific binding to the response elements, the hepatitis B virus X protein (HBx) was reported to re‐direct p53 regulation of some genes.