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Trafficking of Streptococcus pneumoniae in the lymph node
This project focuses on how S. pneumoniae serotype 2 strain D39 is transported in the lymph node. Clearance of S. pneumoniae is mediated through opsonization by Igs and by Ig-independent components(Neufeld, 1904) now known as complement(Ward and Enders, 1933). Receptor mediated binding of complement opsonins leads to microbial clearance of pathogens by phagocytes(Fearon and Wong, 1983), B-cell stimulation(Carroll, 1998) and retention of antigens on follicular dendritic cells (FDC)(Pozdnyakova et al., 2003). This makes complement a major player in the immune response and the generation of neutralizing antibodies against S. pneumoniae. We try to elucidate the interplay of complement components with immune cells of the lymph node that are responsible for a proper antibody response. We have shown that complement components as well as dendritic cells are crucial for the transport to the FDC as well as for an appropriate antibody response. We try to elucidate the exact mechanism of interplay between complement and DC.
Regulation of B cell tolerance to lupus antigen
Autoimmune diseases arise through the failure to keep immune cell activation in check. The cellular events that underlie such anti-self immune responses are poorly understood but one major factor is the early complement system. For example, humans (and mice) deficient in complement C4 almost always develop systemic lupus erythematosus (SLE). To understand how complement protects from SLE, one model we are using is an Ig-transgenic mouse strain, termed 564 Igi, that was engineered to produce anti-self B cells that recognize nuclear auto-antigens (Berland et al., 2006 Immunity). In the absence of complement, we found a dramatic increase in the frequency of mature self-reactive B cells. We hypothesize that in the absence of complement C4, the excess load of apoptotic debris chronically stimulates myeloid cells to produce elevated levels of survival factors such as type I interferons and BAFF; therefore, enhancing escape of autoreactive B cells from elimination. To test our hypothesis, we are using a combination of approaches including tracking of auto-reactive B cells by 2-photon imaging in anesthetized mice combined with blockade of survival factors. Understanding how complement C4 is protective against SLE could prove highly beneficial for treatment of patients where excess apoptotic debri triggers disease. FIGURE LEGEND: Figure 1: Myeloid cell derived factors released in excess in C4-deficient 564Igi mice help in autoreactive B cell survival and maturation. In the absence of C4, excess apoptotic debris activates myeloid cells and leads to increased production of factors, like BAFF, that can lead to survival of self-reactive B cell clones that should have ideally been deleted if C4 was present. Thus, in C4-deficient mice, there is a greater proportion of mature anti-self B cells in the periphery and a higher proportion of self-reactive germinal centers.
Lymph node dendritic cells transport influenza vaccine into B cell compartment
In order to investigate the role of lymph node resident dendritic cells in large antigen capture, multi-photon microscopy has proved an essential tool as a method of live imaging the popliteal node. Using this technology in combination with various strains of transgenic mice, we are making strides in understanding the kinetics of dendritic cell transfer of antigen to the B cell follicle. While multi-photon imaging has given us great insight into the fine details of antigen arrival within the node and the dendritic cell response, it has become clear that a broader understanding of lymph node architecture is essential for pinning down dendritic cell phenotypes to specific locations within the node. As a result, we have worked to develop a system for the complete reconstruction of popliteal nodes using the same fluorescence technology used for live imaging. By serially thick sectioning the lymph node, we have successfully developed reproducible systems that allow us to compare entire populations of dendritic cells between mice. This new application of existing technology has allowed us to gain a better understanding of the gross architecture of the lymph node, and will guide our experiments surrounding antigen arrival and transport going forward.
Role of migratory DCs in the immune response against influenza
Influenza is a global health problem with over 500 million infections annually and high mortality rates. Studies in mouse models have shown that both humoral and CD8 T cell immunity are important in viral clearance and host protection. Respiratory dendritic cells (RDCs) are critical to the transport of viral antigen (Ag) from the lung to the lung-draining lymph nodes (LNs). The mechanisms governing transport of influenza antigen (Ag) by RDCs to the LNs are unknown. Moreover, once RDCs arrive in the LN it is not known whether they present viral Ag directly to B and T cells or if they transfer viral Ag to resident DCs. Studies with influenza A/H1N1 strain Puerto Rico 8 (PR8) demonstrated a role for complement C3 in CD8 and humoral immunity correlating with viral clearance. We have observed higher mortality rates in C3-deficient mice infected with influenza, and lower levels of IL-12 and TNF-α in the lung-draining LNs in comparison with WT mice. Ongoing studies are investigating the role of complement in RDC-mediated initiation of the immune response to influenza, and shed light on the involvement of RDCs in Ag presentation in the LN.
Does reperfusion injury represent an autoimmune response?
When circulation is blocked to an area of tissue (ischemia), such as during trauma, stroke or heart attack, an acute inflammatory response is initiated with the return of circulation (reperfusion). Although the mechanism that contributes to such a response is poorly understood, it has been shown that non-muscle myosin heavy chain II (NMHC-II) is the target self-antigen for circulating natural IgM. We propose that oxidative stress induces increased exposure of NMHC-II on the cell surface of endothelium providing a target for IgM and activation of the complement pathway. In order to visualize the early events in the pathway and to test our hypothesis, we have set-up the cremaster muscle model for imaging in real time by 2-photon microscopy. Initial experiments are underway to determine if inflammation is dependent on the NMHC-natural IgM pathway. As a read out for induction of acute inflammation, we quantitate infiltration of leukocytes using two different approaches. The first is detection of neutrophils (CD45+Gr-1+Ly-6G+) by flow cytometry analysis of post-reperfusion extracts. Alternatively, we quantitate infiltration of GFP-labeled leukocytes (Lys-M-Cre-GFP reporter mice) by analysis of static images after 3 hrs post-reperfusion. To test a role for NMHC-II in induction of injury, we administer (40 uM) a peptide mimetope (N2 peptide blocks IgM binding) immediately prior to reperfusion. By blocking pathogenic IgM with N2 mimetope, we expect to reduce the activation of complement and mast cells thus limiting recruitment of neutrophils. Preliminary results from flow cytometry analysis support a role for NMHC-II pathway in induction of inflammation. In summary, we have established two-photon imaging of the murine cremaster muscle model to visualize early events following reperfusion. Preliminary results show the feasibility of the model and suggest that induction of inflammation is dependent on the NMHC-II- natural IgM pathway.
Areas of Research

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