Browsing by Subject "Chemokine"

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.

G protein-coupled receptors (GPCRs) are the most common transmembrane receptors in the human genome and the target of approximately one-third of all approved drugs. GPCRs interact with many transducers like G proteins and β-arrestins. Some GPCRs preferentially activate specific signaling transducers over others, leading to unique signaling profiles – a phenomenon called biased signaling. The chemokine system, a subfamily of GPCRs, serves as an endogenous example of biased signaling where over 50 different chemokines and 20 receptors interact promiscuously. While previous research has shown that chemokines which activate the same receptor can produce different physiologic responses, the mechanisms underlying these findings remain unclear. Using the three endogenous chemokines of the chemokine receptor CXCR3, we investigated two mechanisms underlying biased signaling at GPCRs. First, using mass spectrometry and cell-based assays, we determined that the chemokines induce different amounts and patterns of GPCR phosphorylation which direct CXCR3 engagement with different transducers. Second, we determined that biased signaling is dependent on the specific location of CXCR3, and subcellular signaling regulates inflammation in a mouse model of contact hypersensitivity. Together, we conclude that differential receptor phosphorylation and subcellular signaling are two mechanisms underlying the biased signaling observed at GPCRs.

G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and one of the most common drug targets. It is now well-established that GPCRs can signal through multiple transducers, including heterotrimeric G proteins, G protein receptor kinases, and beta-arrestins. Certain ligands can preferentially activate certain signaling cascades while inhibiting others, a phenomenon referred to as biased signaling. While biased signaling is observed in many ex-vivo assays, the physiological relevance of biased signaling is not well established. Using the chemokine receptor CXCR3, a receptor that regulates T cell function, and its endogenous chemokines CXCL9, CXCL10, and CXCL11, I established that endogenous biased signaling exists at CXCR3. After identifying small molecule biased CXCR3 agonists using cell-based assays, I utilized human samples and mouse models of T cell movement and inflammation to determine that differential activation of either the G protein or beta-arrestin signaling pathways downstream of CXCR3 produces distinct functional differences. I identified that beta-arrestin regulated-Akt signaling appears critical for full efficacy chemotaxis. I conclude that biased signaling at CXCR3 produces distinct physiological responses.

Many microbial pathogens have evolved to replicate within host cells. While a number of these pathogens reside within vacuolar compartments, others escape from host endosomal pathways to replicate intracytosolically. To counter microbial invasion, host cells employ numerous defense proteins to limit microbial growth and mediate pathogen destruction. Among these host defense proteins are a number of dynamin-like GTPases expressed in response to the cytokine Interferon-gamma, including the p65 Guanylate Binding Proteins (GBPs). Murine GBPs have previously been shown to target both vacuolar and cytosolic pathogens to mediate pathogen destruction and potentiate host inflammatory responses via both the canonical (caspase-1) and noncanonical (caspase-11) inflammasomes. However, whether these functions are conserved among the human orthologs of murine GBPs has remained unclear.

To determine whether the ability to physically target pathogens is conserved among the human GBPs, I monitored the localization of all seven human GBPs within cells infected with the cytosol-resident Gram-negative bacterium Shigella flexneri, the causative agent of bacillary dysentery. Among the human GBP paralogs, I identified the unique ability of GBP1 to physically associate with S. flexneri, and showed that GBP1-targeting extends to a second cytosolic Gram-negative bacterium, Burkholderia thailandensis, but not to the cytosolic Gram-positive bacterium Listeria monocytogenes. Using mutational analysis, I determine that GBP1 targeting is directed by a C-terminal Polybasic Motif (PBM) centered around three arginine residues, and further relies on a lipidated CaaX motif and protein oligomerization via the GBP1 Large GTPase domain. Among the human GBP paralogs, the combination of a PBM and CaaX motif is unique to GBP1. Furthermore, I found that rough lipopolysaccharide (LPS) mutants of S. flexneri co-localize with GBP1 less frequently than wildtype S. flexneri, suggesting that host recognition of O-antigen promotes GBP1 targeting to Gram-negative bacteria. GBP1-targeting to S. flexneri led to co-recruitment of four additional human GBP paralogs (GBP2, GBP3, GBP4, and GBP6).

S. flexneri and a number of other cytosolic bacteria promote bacterial dissemination by hijacking host actin cytoskeleton machinery to form actin comet tails which emanate from one pole of the bacterium and provide mechanical force to propel bacterium-containing extensions into neighboring cells. I found that while GBP1-targeted bacteria remain viable, they replicate within intracellular aggregates and fail to form actin comet tails. Accordingly, wildtype but not a PBM-deficient GBP1 mutant restricts S. flexneri cell-to-cell spread in plaque assays. I also found that S. flexneri counters GBP1-mediated host defenses using a secreted effector, IpaH9.8. Accordingly, human-adapted S. flexneri, through the action of IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis.

Finally, I examined the role of human GBP1 in shaping the host cell transcriptional response in S. flexneri infected cells, and found that GBP1 promotes the expression of several chemokines, including CXCL1, CXCL9, CXCL10, and CCL2, which act as chemoattractants for professional immune cells. This role in chemokine expression was independent of the GBP1 PBM and CaaX motif necessary for bacterial targeting, and extended not only to B. thailandensis, but also L. monocytogenes, which is untargeted by GBP1. Furthermore, GBP2 could functionally substitute for GBP1 to support expression of CXCL10, implicating other GBPs in the process.

Together, the work encompassed in this dissertation sheds light on the role of the human GBPs in host cell defense against intracellular pathogens, and identifies previously unknown roles for the GBPs in precluding bacterial actin-based motility and shaping the host transcriptional response to pathogens.