Monday, July 4, 2011

Does Borrelia burgdorferi cause an inadequate antibody response by altering B cell activation in the lymph node?

One of the characteristic features of Lyme disease is lymphadenopathy or swollen lymph nodes.  It's not too surprising when a lymph node draining a site of infection swells.  However when investigators looked at the lymph node draining the inoculation site of Borrelia burgdorferi in mice, they found that the spirochete had somehow altered the course of activation of B cells producing the antibodies that targeted the spirochete.

Before discussing the findings in the paper, a review of how the antibody response evolves in the lymph node is in order.  An antibody response to microbial proteins is sparked when antigen from microbes breaching the skin layer flow into the draining lymph node or are carried to the lymph node by dendritic cellsLymph nodes are where naive B cells, upon recognition of antigen, differentiate into plasma cells, which secrete large amounts of antibody that target the invading microbe.  The antibodies which bind most tightly to protein antigens are made with T cell help in germinal centers, which emerge from the rare B cells in the lymph node that produce antibody capable of recognizing the antigen.  (I say "rare" here because each B cell in the lymph node produces antibodies with different antigenic specificities to ensure that any microbe that the host may possibly encounter will be recognized by antibodies displayed by at least a few B cells.)  Upon binding the antigen and reception of critical signals from T cells, the B cells migrate to areas in the lymph node containing fixed networks of follicular dendritic cells (FDCs), a type of immune cell with long branched processes that extend out from the body of the cell.  (FDCs differ from the dendritic cells that bring antigen to the lymph node.)  The B cells then start to proliferate wildly, doubling every 6 to 8 hours (faster than B. burgdorferi!).  As the B cell numbers surge, they form germinal centers, which can be identified easily by standard histological stains (see image below).  The lymph node may even swell, depending on how much the B cells proliferate.

Lymph node: (1) capsule; (2) subcapsular sinus; (3) germinal centers; (4) lymphoide nodule; (5) trabeculae.  Source

As the B cells multiply in the germinal center, a process called somatic hypermutation, which is promoted by signals received from T cells, causes a large number of mistakes to be made within the segment of DNA encoding the antigen binding portion of the antibody.  Consequently, the antibodies displayed by some germinal center B cells are no longer able to bind to the microbial antigen whereas those made by other B cells will bind better.  The FDC processes, whose surfaces are loaded with antigen, continuously probe the antibodies expressed by the newly arising B cells.  Since the new B cells are programmed to die unless they express antibody able to bind the antigen displayed by the FDCs, only B cells displaying antibody that bind most tightly to the antigen will survive.  This process by which B cells expressing the antibodies with the highest affinity for antigen are selected is called affinity maturation.  Somatic hypermutation and affinity maturation can only occur in germinal centers.  The B cells also undergo class switching, in which the class of antibody expressed by the B cells switches from IgM and IgD, which are expressed by naive B cells, to IgA, IgE, or one of the IgG subclasses.  The exact switch that occurs is governed by the cytokines that the B cells are exposed to.  Which cytokines are present depends on the nature of the infection.  Eventually the B cells expressing high-affinity antibodies of the appropriate class differentiate into antibody-secreting plasma cells, which are released from the lymph node to circulate throughout the body and fight the infection.

So what happens in the lymph nodes during a B. burgdorferi infection?  When the investigators inoculated B. burgdorferi into or underneath the skin of mice, the lymph node draining the site swelled considerably, enlarging by more than a factor of 10 by the tenth day of infection.

From Fig. 2 of Tunev et al., 2011.  Arrow points to lymph node draining the inoculation site.  Source

What the investigators saw when they looked at lymph node sections under the microscope was very different from the textbook description of T-cell dependent B cell activation that I gave above.  First of all, live B. burgdorferi was found in the lymph node draining the site of infection in mice.  This is unusual since phagocytes would normally greet and destroy any microbe that managed to find its way into the lymph node.

From Fig. 3 of Tunev et al., 2011.  Day 8 of infection.  The arrows point to intact extracellular B. burgdorferi in the subcapsular sinus of the lymph node, which was culture positive beginning on day 1 of infection.   Source

Second, massive proliferation of B cells accounted for lymph node swelling, but the expansion of B cell numbers wasn't occurring in well-defined germinal centers. The authors also noted that T cells were not increasing in number.  These observations suggested that T cells, which are required for germinal centers to form, were not fully participating in B-cell activation.  The lack of germinal centers suggested that somatic hypermutation and affinity maturation were not occurring.

From their observations, the authors speculated that B. burgdorferi somehow subverted B cell activation in the lymph node so that the end result was a large number of plasma cells secreting antibodies of poor quality.  By poor "quality," I assume that the authors meant that the affinity of the antibody for B. burgdorferi proteins was low and that the "wrong" subclasses of IgG antibodies were expressed.  The most abundant IgG subclasses being produced in the draining lymph node at its most swollen state were IgG2b and IgG3.  Whether other IgG subclasses would be more effective at clearing B. burgdorferi from the host and whether the affinities of the antibodies for B. burgdorferi proteins were poor still need to be determined experimentally.  Perhaps a classic T-cell dependent B cell response involving the formation of germinal centers accompanied by somatic hypermutation, affinity maturation, and appropriate class switching would have led to production of "high" quality antibodies.  If the authors are correct, they have revealed yet another means by which B. burgdorferi could persist in the host.

Featured paper

Tunev SS, Hastey CJ, Hodzic E, Feng S, Barthold SW, and Baumgarth N (May 2011).  Lymphadenopathy during Lyme borreliosis is caused by spirochete migration-induced specific B cell activation.  PLoS Pathogens 7(5):e1002066.  DOI: 10.1371/journal.ppat.1002066