Browsing by Author "Abraham, Soman N"

Bacterial bladder infection, also known as cystitis, is one type of urinary tract infections (UTIs). These infections typically initiate when uropathogens, especially uropathogenic E. coli (UPEC), invade into the bladder through the urethra. Those bacteria could attach to the uroepithelium of the bladder and invade into bladder epithelial cells (BECs). The bladder is critical for the control of these bacteria by activating BECs and recruiting immune cells. If bacteria are not promptly cleared in the bladder, they will further invade ureters and kidneys causing pyelonephritis, which is another common form of UTIs. Nowadays, bacterial bladder infection is one of the most common bacterial infections and a big clinical burden. Its annual incidence rate among human population was estimated to be around 3%-12.6% in female and 0.5%-3% in male. Besides the high incidence rate, the recurrence rate is also very high, up to 44%. As a comparison, the recurrence rate of bacterial infections in the respiratory tract is only around 10% and that rate in the gastrointestinal tract is about 1.5% to 12%. The high incidence rate and high recurrence rate pinpoint the inefficiency of immunity in bladder. However, it is unclear which component of the bladder immunity is inefficient in clearing bacteria and preventing recurrence. By using cutting edge models and techniques, such as genetic knock-out mice, newly developed cytokine reporter mice, optimized flow cytometry and microscopy, I identified that the bladder immunity, especially CD4 T cell mediated adaptive immunity, is focusing on repairing damaged uroepithelium rather than clearing UPEC. This response is modulated by antigen presenting cells (APCs) in bladder. This abnormal bladder immunity also leads to bladder dysfunction featured by urinary frequency. By applying a vaccination strategy, I successfully improved the anti-bacteria ability of CD4 mediated bladder adaptive immunity in mouse model. In summary, this study identified that an abnormal adaptive immunity induced by the uroepithelium-APC-T cell signaling axis is responsible for the suboptimal clearance of bacteria and infection recurrence in bladder. With proper vaccination, the adaptive immunity in bladder can be tuned to be protective against UPEC infections.

Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.

We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.

In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.

Lymph nodes are organs of efficiency. Once activated, they essentially function to optimize and accelerate the production of the adaptive immune response, which has the potential to determine survival of the host during an initial infection and protect against repeated infections, should specific and appropriate immunological memory be sufficiently induced. We now have an understanding of the fundamental structure of lymph nodes and many of the interactions that occur within them throughout this process. Yet, lymph nodes are dynamic and malleable organs and much remains to be investigated with regards to their responses to various types of challenges. In this work, we examined multiple inflammatory scenarios and sought to understand the complex ways that lymph nodes can be externally targeted to impact immunity. First, we outline a novel mechanism of cellular communication, where cytokine messages from the periphery are delivered to draining lymph nodes during inflammation. These signals are sent as particles, released by mast cells, and demonstrate the ability of the infected tissue to communicate to lymph nodes and shape their responses. Based on these interactions, we also explored the ability to therapeutically or prophylactically modulate lymph node function, using bioengineered particles based on mast cell granules, containing encapsulated cytokines. When we used these particles as a vaccine adjuvant, we were able to polarize adaptive immune responses, such as to promote a Th1 phenotype, or enhance a specific attribute of the immune response, such as the production of high avidity antibodies. We then explore three examples of lymph node-targeting pathogens: Salmonella typhimurium, Yersinia pestis and Dengue virus. Each of these pathogens has a well-characterized lifecycle including colonization of draining lymph node tissue. In the case of S. typhimurim, we report that the virulence this pathogen depends on a specific shut down of the chemotactic signals in the lymph node that are required to maintain appropriate cellular localization within it. Our results demonstrate that these architecture changes allow S. typhimurim to target the adaptive immune process in lymph nodes and contribute to its spread in vivo and lethality to the host. With Y. pestis, similar targeting of cellular trafficking pathways occurs through the modulation of chemokine expression. Y. pestis appears to use the host's cellular trafficking pathways to spread to lymph nodes in two distinct waves, first exploiting dendritic cell movement to lymph nodes and then enhancing monocyte chemoattractants to replicate within monocytes in draining lymph nodes. These processes also promote bacterial spread in vivo and we further demonstrate that blocking monocyte chemotaxis can prolong the host's survival. In the third example of pathogen challenge, we report for the first time that mast cells can contribute functionally to immunosurveillance for viral pathogen, here, promoting cellular trafficking of innate immune cells, including NK cells, and limiting the spread of virus to draining lymph nodes. For each of these three examples of lymph node targeting by microbial pathogens, we provide data that modulation of cellular trafficking to and within lymph nodes can drastically influence the nature of the adaptive immune response and, therefore, the appropriateness of that response for meeting a unique infectious challenge. Cumulatively this work highlights that a balance exists between host and pathogen-driven modulation of lymph nodes, a key aspect of which is movement of cells within and into this organ. Cytokine and chemokine pathways are an area of vulnerability for the host when faced with host-adapted pathogens, yet the lymph node's underlying plasticity and the observation that slight modulations can be beneficial or detrimental to immunity also suggests the targeting of these pathways with therapeutic intentions and during vaccine design.

The mammalian urinary bladder is a highly specialized organ that must be able to withstand considerable amounts of osmotic pressure at its mucosal surface, in addition to maintaining an impenetrable barrier against potential pathogens. The lower urinary tract's virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected with uropathogenic E.coli (UPEC) in combination with clinical observations that antibody responses following 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. This mast cell-dependent phenomenon involves localized production of IL-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of mast cells orchestrating the early innate immune responses in the bladder during 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 mast cell-maintained immune privilege.

Interestingly, though the bladder is not capable of initiating an effective adaptive immune response during bladder infections, we have generated data showing that it was possible to circumvent the immune limitations of the bladder to provoke a strong adaptive and protective immune response by vaccinating against UPEC at an alternate mucosal site. We reasoned that by immunizing the nasal regions of mice with a vaccine formulation comprising of FimH adhesin, a highly conserved adhesive moiety of type 1 fimbriae expressed on UPEC, and an effective mucosal adjuvant we would evoke protective immunity against UPEC infections. We found that a FimH vaccine coupled with either a mast cell activating adjuvant c48/80 or CpG oligodeoxynucleotide, a TLR9 agonist, evoked high levels of FimH specific IgG antibody in the serum and IgA in the urine of immunized mice. We also observed that following UPEC challenge, these FimH/adjuvant immunized mice exhibited significantly reduced bacterial load in the bladders compared to mice challenged with just FimH. These studies reveal that immunization of nasal regions with a FimH vaccine is an effective strategy to overcome the limitation in adaptive immunity observed in the bladder.

The key components of innate immune defense to pathogens are various migratory as well as tissue resident innate immune cells, however, their interactions with pathogens as well as their immune-orchestrating roles are often poorly understood. While immune cells encounter pathogens at barrier sites and mount the first line of defense, pathogens are well adapted to bypass, inactivate and even exploit the functions of these cells. Better understanding of the interactions between pathogens and innate immune cells can teach us how pathogens avoid or exploit immune cells and how to overcome these mechanisms of pathogenesis by therapeutic interventions. In this work, we examined two scenarios of pathogen invasion and sought to understand the complex ways of external targeting of innate immunocytes that can either benefit the pathogen or the host.

First, we studied the migratory innate immunocytes in draining lymph nodes upon entry of Yersinia pestis via the skin and identified how this plague-causing bacterium coopted host cell death pathways of infiltrated mononuclear phagocytes. By employing time-lapse microscopy and flow cytometry, we demonstrated that within the confines of infected lymph nodes, bacteria-triggered necroptotic cell death resulted in the release of intracellular bacteria into the extracellular environment and attracted neighboring phagocytic cells, promoting their infection by these recently released bacteria. This expansion of bacteria-bearing immune cells which eventually migrate to secondary lymph nodes, enables large numbers of Y. pestis to disseminate from one node to the next via the lymphatic system. We show this mechanism of dissemination being essential for the transition of plague from a bubonic to septicemic stage and demonstrate immunotherapeutic potential of necroptosis inhibitors.

Next, we focused on mast cells, a resident innate immunocyte in the context of skin infection by Staphylococcus aureus. We showed that connective tissue mast cells promoted recruitment of neutrophils at the early stage and CD301b+ dendritic cells at the later stages of infection, which played critical roles in infection control and repair, respectively. We further demonstrated that exogenous activation of skin mast cells via a mast cell-specific G protein-coupled receptor controlled infection as well as enhanced mobilization of dendritic cells to draining lymph nodes in a mast-cell dependent manner and protected mice from re-infection. Therefore, selective activation of mast cells appears to orchestrate immunomodulation integrating both the innate and adaptive immune arms.

These studies reveal the yin and yang of innate immune cells in two very different infectious settings. They emphasize how different strategies to target these cells at the immune checkpoints can be beneficial for host-directed therapy against bacterial infections.

Urinary tract infections (UTIs) are typically caused by bacteria that colonize different regions of the urinary tract, mainly the bladder and the kidney. Approximately 25% of women that suffer from UTIs experience a recurrent infection within 6 months of the initial bout, making UTIs a serious economic burden resulting in more than 10 million hospital visits and $3.5 billion in healthcare costs in the United States alone. Type-1 fimbriated Uropathogenic E. coli (UPEC) is the major causative agent of UTIs, accounting for almost 90 % of bacterial UTIs. The unique ability of UPEC to bind and invade the superficial bladder epithelium allows the bacteria to persist inside epithelial niches and survive antibiotic treatment. Persistent, intracellular UPEC are retained in the bladder epithelium for long periods, making them a source of recurrent UTIs. Hence, the ability of UPEC to persist in the bladder is a matter of major health and economic concern, making studies exploring the underlying mechanism of UPEC persistence highly relevant.

In my thesis, I will describe how intracellular Uropathogenic E.coli (UPEC) evade host defense mechanisms in the superficial bladder epithelium. I will also describe some of the unique traits of persistent UPEC and explore strategies to induce their clearance from the bladder. I have discovered that the UPEC virulence factor Alpha-hemolysin (HlyA) plays a key role in the survival and persistence of UPEC in the superficial bladder epithelium. In-vitro and in-vivo studies comparing intracellular survival of wild type (WT) and hemolysin deficient UPEC suggested that HlyA is vital for UPEC persistence in the superficial bladder epithelium. Further in-vitro studies revealed that hemolysin helped UPEC persist intracellularly by evading the bacterial expulsion actions of the bladder cells and remarkably, this virulence factor also helped bacteria avoid t degradation in lysosomes.

To elucidate the mechanistic basis for how hemolysin promotes UPEC persistence in the urothelium, we initially focused on how hemolysin facilitates the evasion of UPEC expulsion from bladder cells. We found that upon entry, UPEC were encased in “exocytic vesicles” but as a result of HlyA expression these bacteria escaped these vesicles and entered the cytosol. Consequently, these bacteria were able to avoid expulsion by the cellular export machinery.

Since bacteria found in the cytosol of host cells are typically recognized by the cellular autophagy pathway and transported to the lysosomes where they are degraded, we explored why this was not the case here. We observed that although cytosolic HlyA expressing UPEC were recognized and encased by the autophagy system and transported to lysosomes, the bacteria appeared to avoid degradation in these normally degradative compartments. A closer examination of the bacteria containing lysosomes revealed that they lacked V-ATPase. V-ATPase is a well-known proton pump essential for the acidification of mammalian intracellular degradative compartments, allowing for the proper functioning of degradative proteases. The absence of V-ATPase appeared to be due to hemolysin mediated alteration of the bladder cell F-actin network. From these studies, it is clear that UPEC hemolysin facilitates UPEC persistence in the superficial bladder epithelium by helping bacteria avoid expulsion by the exocytic machinery of the cell and at the same time enabling the bacteria avoid degradation when the bacteria are shuttled into the lysosomes.

Interestingly even though UPEC appear to avoid elimination from the bladder cell their ability to multiple in bladder cells seem limited.. Indeed, our in-vitro and in-vivo experiments reveal that UPEC survive in superficial bladder epithelium for extended periods of time without a significantly change in CFU numbers. Indeed, we observed these bacteria appeared quiescent in nature. This observation was supported by the observation that UPEC genetically unable to enter a quiescence phase exhibited limited ability to persist in bladder cells in vitro and in vivo, in the mouse bladder.

The studies elucidated in this thesis reveal how UPEC toxin, Alpha-hemolysin plays a significant role in promoting UPEC persistence via the modulation of the vesicular compartmentalization of UPEC at two different stages of the infection in the superficial bladder epithelium. These results highlight the importance of UPEC Alpha-hemolysin as an essential determinant of UPEC persistence in the urinary bladder.

Antimicrobial peptides (AMPs) are part of the innate immune system that is widely distributed in nature, acting as a defense mechanism against invading microorganisms. AMPs have potent antimicrobial activity against a range of microorganisms including fungi, bacteria and viruses. In view of growing multidrug resistance, AMPs are increasingly being viewed as potential therapeutic agents with a novel mechanism of action. Mastoparan is a natural, highly positively charged AMP derived from the venom of wasps. It was originally of interest based on its inherent mast cell degranulation activity. Previously, mastoparan has been shown to exhibit antimicrobial activity in vitro however these studies have been limited in scope. Here we hypothesize that mastoparan possess the capacity to be a potent broad spectrum antibacterial agent including activity against multidrug resistant bacteria.

We examined the scope of antibacterial activity exhibited by mastoparan using a variety of antimicrobial susceptibility tests and have utilized a bacterial skin infection (S. aureus) model to determine the potential of mastoparan to serve as a therapeutic agent. We tested mastoparan against 4 Gram-positive clinical isolates (e.g., S. aureus, and E. faecium), 9 Gram-negative clinical isolates (e.g., E. coli, P. aeruginosa, and B. cepacia), and 4 multidrug resistant clinical isolates (e.g., MRSA, ESBL E.coli, and ESBL K. pneumonia). These studies reveal that mastoparan exhibits broad spectrum activity against both Gram-negative (MIC: 1.9 - 125 &mug/ml) and Gram-positive (MIC: 15.6 - 125 &mug/ml) bacteria and against multidrug resistant bacteria (MIC: 7.8 - 125 &mug/ml). We also demonstrated that mastoparan disrupts the bacterial membrane, exhibits fast acting antibacterial activity and is highly effective against both multiplying and non-multiplying bacteria. Furthermore, we have shown that mastoparan demonstrates efficacy as a topical antimicrobial agent reducing lesion size by up to 79% and the amount of bacteria recovered from skin lesions by up to a 98% reduction. Based on these results we conclude that mastoparan is a highly effective antibacterial agent and is therefore a potential alternative to currently antibiotics. Mastoparan offers a promising new therapeutic option for treating bacterial infections.

Urinary tract infections (UTIs) are the second most common type of infection identified in the clinical setting and disproportionately afflict women. UTIs most frequently manifest in the form of infection of the lower urinary tract, involving the bladder. Uropathogens, particularly uropathogenic E. coli, progressively colonize the urethra and ascend to the bladder, where they initiate cystitis. In some cases, infection further ascends through the ureters and reaches the kidneys, where it causes pyelonephritis. Infection of both the upper and lower urinary tract can have serious ramifications for the host, and this is in large part due not to infection itself but to host-directed responses to bacterial insults.

In this thesis, I will describe and discuss two distinct aspects of UTIs. In the first study, in vivo work in a mouse model of urinary tract infection revealed a novel role for mast cells, which are tissue-resident granulated innate immune cells, in directing the detachment and death of epithelial cells during cystitis, facilitating the clearance of bacteria from the bladder. An ex vivo porcine bladder infection model suggested a specific role for mast cell granules and the proteases contained therein, which was corroborated with in vitro experiments utlizing isolated mast cell granules and human epithelial cells to demonstrate granule-induced exfoliation and cell death. From this work, it is clear that mast cells play a highly targeted role in modulating urothelial integrity during bladder infection by mediating host-directed epithelial loss.

In the second study described in this dissertation, the synergistic roles of both pyelonephritis and vesico-ureteric reflux (VUR), a congenital urinary tract defect that results in the improper backflow of urine from the bladder to the kidney, in the development of reflux nephropathy, a fibrotic host response characterized by renal scar formation, were elucidated in a series of in vivo experiments. Specifically, the C3H mouse, which is naturally susceptible to VUR, was utilized to characterize the dynamics of kidney infection and the onset of reflux nephropathy. Renal scarring was dependent on the presence of sustained kidney infection and the accompanying inflammatory response due to VUR, while neither transient infection nor reflux alone were sufficient to provoke nephropathy. Thus, the development of reflux nephropathy is dependent upon the confluence of both infection and VUR.

This body of work reveals the double-edged sword of the host inflammatory response to urinary tract infection. In the bladder, mast cell activation and degranulation leads to granule-induced epithelial exfoliation and consequently a reduction in the bacterial burden in the bladder. However, the sustained inflammatory response that accompanies pyelonephritis in vesico-ureteric reflux-affected individuals results in significant damage to the kidney without any accompanying reduction in infection. These findings highlight the dueling roles of the host inflammatory response to infection in the upper and lower urinary tract and strongly suggest that differential clinical approaches to cystitis and pyelonephritis are necessary to promote an effective mast cell in the bladder in the former and facilitate the clearance of renal infection while mitigating tissue damage in the latter.

Bladder Pain Syndrome (BPS) is a broad-spectrum pelvic pain disorder, characterized by pain and at least one underlying lower urinary tract symptom, that affects millions in the US. Despite its prevalence, the underlying etiology is unknown. Several clinical findings point to a potential role of peripheral nerves in the bladder, such as hyperinnervation and increased urinary nerve growth factor (NGF) in BPS patients. In addition, mast cell (MC) hyperplasia and a history of recurrent urinary tract infections (recUTIs) is also observed. Based on the similar findings of elevated urinary NGF in recUTI patients and the prominent role of MCs in bladder immunity to UTI, we examined whether mice that experience multiple UTIs develop BPS symptoms. We found that mice subjected to multiple bacterial bladder infections experienced both pelvic hypersensitivity and bladder frequency, assayed by mechanical stimulation of the pelvic region and cystometric analysis, respectively. These pathological outcomes were linked to extensive hyperinnervation, specifically in sensory nociceptive nerves in the bladder lamina propria, induced by recruited NGF-producing CCR2+ Ly6Chigh inflammatory monocytes and to the presence of activated MCs near sprouting nerves. Notably, both CCR2-/- and MC-deficient mice each were protected from developing BPS symptoms after multiple infections. Lastly, the MC product histamine was sufficient to trigger both hypersensitivity and frequency in a TRPV1 dependent manner. Taken together, these findings reveal chronic MC activity in a hyperinnervated bladder, triggered by past infections, could be an underlying basis for BPS.

Mast cells, best known for their pathologic role in allergy, have recently been shown to have key roles in the initiation of adaptive immune responses. These cells are located throughout the body just beneath barriers separating host from environment, possess multiple pathogen recognition systems, and store large quantities of fully active inflammatory mediators. These key features make them uniquely situated to act as sentinels of immunity, releasing the very earliest alarm signals when a pathogen is present. As a testament to the importance of these cells, mast cell-deficient mice have suboptimal immune responses, and mast cell activators can act as potent adjuvants for experimental immunizations. Specifically, mast cells have been shown to enhance the number of naive lymphocytes in infection site-draining lymph nodes, and to encourage the migration of dendritic cells to responding lymph nodes.

Although infections usually occur at peripheral sites, adaptive immune responses are initiated in distant lymph nodes. Despite the distance, signals from the site of infection result in dramatic, rapid reorganization of the node, including massive recruitment of naive lymphocytes from the circulation and extensive vascular restructuring to accommodate the increase in size. How such signals reach the lymph node is not well understood.

When mast cells degranulate, in addition to releasing soluble mediators such as histamine, they expel large, stable, insoluble particles composed primarily of heparin and cationic proteins. The work presented herein demonstrates that these particles act as extracellular chaperones for inflammatory mediators, protecting them from dilution into the interstitial space, degradation, and interaction with non-target host cells and molecules. The data show clearly that mast cells release such particles, that they are highly stable, that they contain tumor necrosis factor (a critically important immunomodulator), and that they can traffic from peripheral sites to draining lymph nodes via lymphatic vessels. Furthermore, extensive biochemical characterization of purified mast cell-derived particles was performed. Finally, evidence is presented that such particles can elicit lymph node enlargement, an infection-associated phenomenon that favors the development of adaptive immunity, by delivering peripheral TNF to draining lymph nodes.

This signaling concept, that particles may chaperone signals between distant sites, also has important implications for adjuvant design. The evidence presented here shows that encapsulation of TNF into synthetic particles similar to mast cell-derived particles greatly enhances its potency for eliciting lymph node enlargement, an indication that adaptive immunity may be improved. This delivery system should ensure that more adjuvant arrives in the draining lymph node intact, where it would lead to changes favorable to the development of the immune response. Such a system would also facilitate the delivery of multi-component adjuvants that would act synergistically at the level of the lymph node when gradually released from microparticle carriers. An additional advantage of microparticle encapsulation is that vaccine formulations of this type may require much lower doses of expensive antigen and adjuvants.

The delivery of inflammatory mediators to lymph nodes during immune responses may be an important general feature of host defense. Although the action of mediators of peripheral origin on draining lymph nodes has been described before, this is the first demonstration of a specific adaptation to deliver such mediators. Not only is the characterization of mast cell-derived particles important to basic immunology, but mimicking this adaptation may also lead to improved therapeutics.

Due to its close proximity to the gastrointestinal tract, the human urinary tract is

subjected to constant barrage by gut-­associated bacteria. However, for the most part, this tract has resisted infection by various microbes. The impregnability of the urinary tract to microbial colonization is attributable to the ability of the bladder to promptly sense and mount robust responses to microbial challenge. A powerful mechanism for the elimination of invading bacteria was recently described in bladder epithelial cells, involving non-­lytic ejection of intracellular bacteria back into the extracellular milieu. In spite of the effectiveness of this defense strategy, much of the underlying mechanisms surrounding how this powerful cellular defense activity detects intracellular UPEC and shuttles them from their intracellular location to the plasma membrane of BECs to be exported remains largely a mystery.

Here, we describe uropathogenic E.coli (UPEC) expelled from infected bladder

epithelium cells (BECs) within membrane-­bound vesicles as a distinct cellular defense

response. Examination of the intracellular UPEC revealed that intracellular bacteria were

initially processed via autophagy, the conventional degradative pathway, then delivered

into multivesicular bodies (MVBs) and encapsulated in nascent intraluminal vesicle membrane. We further show the bacterial expulsion is triggered when intracellular UPEC follow the natural degradative trafficking pathway and reach lysosomes and attempt to neutralize its pH to avoid degradation. This pathogen-­mediated activity is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized on lysosomes, which spontaneously initiates lysosome exocytosis resulting in expulsion of exosome-­encased bacteria. These studies reveal a cellular default system for lysosome homeostasis and also, how it is coopted by the autonomous defense program to clear recalcitrant pathogens.

Mast cells (MCs) are long-lived hematopoietic cells located within tissues. These cells are densely packed with granules containing preformed bioactive components that are released within seconds to minutes upon activation in a process called degranulation. MCs have beneficial roles in pathogen clearance and wound healing but are most widely associated with their deleterious effects in allergic diseases. Importantly, MCs have been shown to reform granules following degranulation in vitro. This capacity for multiple cycles of degranulation and regranulation is thought to contribute to chronic allergic diseases such as asthma and atopic dermatitis, however, MC regranulation has not been previously demonstrated in vivo. Additionally, how MCs regulate regranulation has not been previously shown. In this study, we demonstrate that following anaphylaxis, peritoneal MCs from mice can undergo regranulation. Additionally, using inducible Raptor knockout mice, we show that mTORC1, a well-known mediator of cellular metabolism, is necessary for MC regranulation in vitro and in vivo. Using a microarray approach, we determined that mTORC1 activity is regulated by Slc37a2. This glucose-6-phosphate transporter is necessary for increased glucose-6-phosphate and ATP concentrations during regranulation, two upstream signals of mTOR. Additionally, Scl37a2 was found localized to endosomes during regranulation, where it concentrated extracellular cargo which are trafficked into newly formed granules. Thus, MC regranulation is regulated by a metabolic reprogramming that requires the interaction of the glucose-6-phosphate transporter Slc37a2 and the nutrient sensor mTORC1.

A great number of people suffer from allergic disorders, some of which can be serious and life threatening. Unfortunately, there are limited ways to treat or prevent these maladies, in part due to our limited understanding. This dissertation addresses a novel mechanism of rapid desensitization, a procedure used to achieve temporary tolerance to allergens to prevent anaphylaxis, a severe allergic reaction involving mast cell degranulation. Specifically, we found that desensitization results in the aberrant reorganization of the actin cytoskeleton in mast cells, preventing calcium mobilization and inhibiting degranulation. Additionally, we found that a bacterial-derived phosphatase, YopH-TAT, could be potentially used for the treatment of allergic diseases, as it efficiently blocked mast cell activation by inhibiting tyrosine phosphorylation.