Varying susceptibility exists among mammalian species to the development of potentially fatal endotoxemia due to gram-negative bacteria molecular component, lipopolysaccharide (LPS). Toll-like receptor 4 (TLR4) is responsible for LPS-associated immune response and is expressed on numerous immune cells including B lymphocytes. TLR4 is expressed in a functional form on mouse B lymphocytes, a species much less susceptible to endotoxemia compared with humans who are highly sensitive to endotoxin. Humans possess B lymphocytes that are not responsive to LPS. Likewise, horses are highly susceptible to endotoxemia but the expression and function of TLR4 on horse B lymphocytes is not known. Colic, the major cause of mortality in horses, is often complicated by resultant endotoxemia. The objective of this study was to determine the expression and function of TLR4 on equine B lymphocytes. Lymphocytes were isolated from peripheral blood mononuclear cells that were collected from six horses, and the expression and function of TLR4 was analyzed for each horse. Flow cytometry results indicate TLR4 is expressed on horse B lymphocytes but stimulation with LPS did not alter this expression (P = 0.99) compared with unstimulated B lymphocytes after 24 h. After 72 h of in vitro LPS stimulation, analysis of cell proliferation dye by flow cytometry demonstrated that equine B lymphocytes did not proliferate, while mouse B lymphocytes predictably did. Furthermore, the total number of LPS stimulated equine B lymphocytes did not significantly differ from unstimulated cells after 72 h of culture (P = 0.92). Horse lymphocytes exhibited no significant differences in the measured TLR4 signaling pathway genes (TLR4, IL-10, IL-6, IFNβ, and TNFα) when expression was compared with LPS stimulated vs. unstimulated cells. In conclusion, while TLR4 is expressed on horse B lymphocytes, it appears minimally responsive to LPS in vitro, similar to results seen in human B lymphocytes. While further studies are still needed, our work reveals a potential link between B lymphocyte TLR4 expression and endotoxin sensitivity.

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Toll-like receptor 4 (TLR4) is a well-documented Toll-like receptor that recognizes bacterial lipopolysaccharide (LPS) as its ligand. Expression of TLR4 has been well described in murine and human B cells. Murine B lymphocytes have relatively constant TLR4 expression, where in humans, expression is present, but low. However, systemically mice show low sensitivity to LPS and humans show high sensitivity. Horses share this high sensitivity to LPS with humans. Here, we look at TLR4 presence and activation in equine B lymphocytes. Research in this area has been hampered due to the lack of quality B lymphocyte markers. Anti-human CD21 was previously found to mark peripheral equine B cells. We show CD45R (B220) also accurately labels equine B lymphocytes. We also demonstrate TLR4 is expressed on equine B lymphocytes using the TLR4 antibody, clone 76B357.1. By using the density gradient, Lympholyte, equine lymphocytes can be separated out of peripheral blood and stimulated with LPS. We demonstrate equine B lymphocytes fail to proliferate under LPS challenge as opposed to highly proliferative mouse B lymphocytes. This lack of response is similar to that seen in humans and may be a clue to LPS sensitivity shared by horses and humans.

Systemic lupus erythematosus (SLE) is an autoimmune disease mediated mainly by autoantibodies reactive to nuclear acids and proteins. These autoantibodies are mostly of IgG isotype, indicating that class-switch recombination (CSR) may play an important role in the development of SLE. CSR is initiated by the enzyme, activation-induced cytidine deaminase (AID) which induces double strand DNA breaks then repaired by homologous recombination using the RAD51 complex. We hypothesize that therapeutic targeting of CSR will retard the development of SLE by inducing cell death in AID-expressed B cells and reducing pathogenic IgG antibodies. The BXSB/MpJ mouse is a well characterized model of human SLE, exhibiting a robust germinal center response and copious production of immunoglobulins. 4,4’-diisothiocyanatostilbene-2-2’-disulfonic acid (DIDS) is a molecule which impairs homologous recombination by inhibiting the binding of RAD51 complex to DNA. In this study, male BXSB/MpJ mice were injected with DIDS or control. Treated mice showed a reduction in the germinal center B cell population and a corresponding decrease in follicular T helper cells. T1 B cell and follicular B cell populations were significantly decreased in DIDS treated mice but no changes were found in marginal zone B cells. Sera were analyzed for the immunoglobulin levels and antinuclear antibodies. These findings demonstrate that targeting CSR by DIDS reduces potentially pathogenic B lymphocyte populations and may be a viable novel therapeutic option in the treatment of SLE.

The debilitating and potentially fatal neurologic disease, Equine Protozoal Myoencephalitis (EPM), is one of the most common neurologic diseases seen in the equine population of the United States. Disease develops as a result of unintentional ingestion of the pathogenic parasite, Sarcocystis neurona. Treatment options consist of the anticoccidial drug Diclazuril and numerous other antiprotozoal drugs. Weeks to months after cessation of antiprotozoal drug treatment and corresponding neurologic improvement, horses may present with clinical disease symptoms again. Little is known whether this reoccurrence of symptoms is from relapse or reinfection. We sought to understand if relapse was possible following appropriate treatment with diclazuril, a commonly used medication for the treatment of EPM. Using a mouse model of equine EPM, we subjected infected mice to treatment with or without diclazuril for 30 and 60 days. All untreated mice developed neurologic symptoms consistent with S. neurona infection within 30 days post infection. All diclazuril treated mice developed neurologic symptoms cessation of treatment. Cerebellum samples were examined for lesions characteristic of those associated with S. neurona infection and immunohistochemically for presences S. neurona. Sera immunoglobulin levels were analyzed to evaluate treatment’s effect on humoral immunity. In conclusion horses treated appropriately with 60 days of diclazuril may be at risk for relapse and more effective treatment options should be explored.