Mark J. Miller, PhD
Associate Professor of Medicine
Antigen capture and presentation in the spleen
Understanding how the spleen’s structure and diverse mix of resident cells (Mebius and Kraal, 2005) collaborate during host defense provides insight into bacterial pathogenesis and guides live attenuated vaccine development. Our goal is to understand how bacterial antigen is captured and presented to T cells in the spleen during bacterial infection (Aoshi, 2008; Konjufca and Miller, 2009b). This work uses the well characterized Listeria monocytogenes (Listeria) infection model in mice (reviewed by Pamer, 2004). In published work we’ve shown that early host-pathogen interactions in the splenic marginal zone induce bactericidal responses in marginal zone macrophages (MZ MACs) to control the initial bacterial burden (Aoshi, 2009), whereas dendritic cells (DCs) provide a temporary niche for bacterial proliferation and transport Listeria to the periarteriolar lymphoid sheath (PALS) for antigen presentation (Aoshi, 2008).
The entry of Listeria into the PALS has been interpreted as immune evasion by others, however we found that when DC migration was inhibited by pre-treating mice with pertussis toxin (PTx), Listeria failed to enter the PALS and antigen presentation to Listeria-specific WP11.12 T cells was inhibited (Aoshi, 2008). These studies suggest that DC trafficking from MZ to the PALs plays a crucial antigen transport function analogous to DC migration from peripheral tissues to draining lymph nodes during infection (Aoshi, 2008). More recently, we used the Batf3 knockout mouse lacking CD8a+ DCs (Edelson, 2011) and the PTEN-CKO mouse (Sathaliyawala, 2010), with increased numbers of CD8a+ DCs, to show that the CD8a+ DC subset in the spleen is responsible for the transport and growth of Listeria in the PALS and for antigen presentation to CD8 T cells. We are following up on these studies by examining the role of CCR7 and other chemokine receptors in orchestrating DC-mediated antigen transport in the spleen. In addition, we are working to determine the location and timing of antigen presentation to polycolonal CD8 and CD4 T cells during the course of infection using a novel CD69-Y-pet BAC reporter mouse (Andrey Shaw, WUSM).
Antigen capture and T cell outcomes in the small intestine
The intestinal immune system must protect a large environmentally exposed mucosal surface from infection, while simultaneously preventing inappropriate inflammatory responses to commensal microbiota and food antigens. Peyer’s patches are well recognized portals for antigen delivery to DCs. However, several DC subsets reside in the villus lamina propria (LP) of the small intestine (Fries and Griebel, 2011) with distinct capacities for promoting tolerance and inflammation suggesting that the balance between tolerance and immunity may rest upon which LP-DC subtype acquires antigen, as well as the inflammatory context in which antigen is presented to T cells. In collaboration with Rodney Newberry (WUSM, GI), we are examining the function of CD103+CD11b+ and CD103-CD11b+ LP-DC subsets (del Rio, 2010) during mucosal tolerance and immunity in the gut. Our goal is to identify the cellular mechanisms by which LP DCs acquire antigen from food material, non-pathogenic bacteria (E. coli) and pathogenic bacteria (Salmonella) and present these antigens to T cells. To study theses processes in vivo, we have developed an intravital gut imaging approach that maintains epithelial viability and allows host-cell dynamics in the Peyer’s patches and lamina propria of the small intestine to be examined in real-time (Erdman, 2009; Miller, 2010b, McDole, 2012). Our work demonstrates that luminal antigen is preferentially delivered to LP-DCs in the steady-state by a novel mechanism that we have termed goblet cell-associated antigen passages (GAPs). GAPs are abundant in the small intestine and constitutively pass low molecular weight material across the villus epithelium without compromising barrier function. GAPs deliver antigens preferentially to tolerogenic CD103+CD11b+ DCs and therefore may play an important role in the maintenance of peripheral tolerance (McDole, 2012). Our central hypothesis is that depending on the DC subset and the antigen acquisition pathway, LP-DCs serve as either stimulators of effector T cell responses in the gut during infection, or as inducers of peripheral regulatory T cell development and homeostatic immune responses.
Multi-scale modeling of T cell activation and clonal expansion kinetics
Mathematical modeling is important for understanding phenomena that occur over temporal and spatial scales that defy direct experimental observation. Moreover, modeling often provides tremendous insight into how complex systems (such as the immune system) operate. In a series of papers, we proposed that T cell receptor (TCR) repertoire scanning is a stochastic process (or agent based system) that occurs through the dynamic behavior of DC dendrites and robust T cell motility, which mediates numerous random TCR-pMHC interactions (Miller, 2002; Miller, 2003; Miller, 2004a; Miller, 2004b, Aoshi, 2008). In collaboration with computational biologists, we are using in vivo cell tracking data from 2P imaging studies to create agent based models (ABMs) of infection and immunity (Beltman, 2007, Riggs, 2008, Linderman, 2010). Our work focuses on identifying key parameters that affect the efficiency of adaptive immune responses and thus provide insight into vaccine strategies and pathogen immune evasion mechanisms (Mirsky, 2011).
Regulation of leukocyte recruitment
In other work, we are using in vivo imaging to define the cellular and molecular mechanisms that regulate leukocyte recruitment mouse models of inflammation. In collaboration with Dan Kreisel’s group (WUSM, Surgery), we are investigating how polymorphonuclear neutrophil (PMN) recruitment in the lung is regulated in response to transplant-mediated ischemia reperfusion injury. A key aspect to these studies is that we are using an intravital lung imaging preparation to document leukocyte dynamics in vivo during acute lung injury. Using a combination of bone marrow chimera, Ab-mediated depletion and KO mouse approaches, imaging will allow us to determine precisely which trafficking steps require specific chemokines and chemokine receptors. Recently our lung imaging work has uncovered a role for circulating monocytes in regulating PMN extravasation during acute lung injury (Kriesel, 2010). We are currently working to assess the contribution of different monocyte subsets (CCR2+and Cx3CR1+) and identify the mechanism(s) they use to enhance PMN transendothelial migration. Our hope is that identifying key regulatory check points in leukocyte recruitment and activation will lead to new therapies to control ischemia induced primary graft dysfunction and perhaps provide insight into other pulmonary inflammatory diseases such as asthma and chronic obstructive pulmonary disease.
In a second project, we are collaborating with Paul Allen’s group (WUSM, Pathology) to evaluate the impact chemokine receptor blockade on leukocyte recruitment and histopathology in a mouse arthritis model (Wang et al., 2012). To study PMN trafficking to the joints in real-time, we developed an accelerated K/B×N serum transfer arthritis model (aSTA) optimized for two-photon 2P imaging (Wang, 2010, Wang, 2012). In our model, arthritogenic serum is injected subcutaneously (s.c.) into one hind footpad to produce a local arthritis with synchronized PMN recruitment. Our studies have revealed that CCR2+ monocytes are required for efficient PMN transendothelial migration during arthritis induction, but not in response to bacterial challenge. These findings suggest that anti-inflammatory therapies targeting monocytes may act in part through antagonizing PMN extravasation at sites of aseptic inflammation. Moreover, this implies that PMN recruitment during infection is inherently more robust or involves different signaling pathways than those that operate during aseptic inflammation. In collaboration with Mike Caparon (WUSM, Molecular Micro), we are working to identify the key factors that control PMN recruitment following infection with Group A Streptococcus (Streptococcus pyogenes) (Lin, 2009). Our intent is to identify mediators of cell recruitment that operate specifically during aseptic inflammation that could be targeted to treat inflammatory diseases without increasing susceptibility to bacterial infection.