Lyme disease, caused by the spirochete Borrelia burgdorferi, is an emerging infectious disease, and therefore, of major public health significance. The spirochete invades multiple organs and induces acute and chronic inflammatory conditions. Inflammation, as induced by the spirochete, plays a primary role in the pathogenesis of the disease. Our broad long-term goal is to identify the mechanism(s) required for the control of inflammation in Lyme disease. Recently we observed that B. burgdorferi alone or together with IL-10 additively induced the expression of the suppressor of cytokine signaling (SOCS) 1 and 3 in J774 macrophages. This correlated with the IL-10-mediated inhibition of the pro-inflammatory cytokines IL-6, IL-12p40, IL-18, TNF? and IL-1?. Our hypothesis is that expression of SOCS1/SOCS3 induced by B. burgdorferi alone or together with IL-10 in macrophages is functionally important in the control of inflammation during the course of Lyme disease; SOCS1/SOCS3 would act either as mediators of the IL-10 anti-inflammatory activity or as direct modulators of inflammatory cytokine signaling.
The specific aims are to: 1. Determine the effect of silencing socs1/socs3 gene expression in J774 macrophages using RNA interference (RNAi) (a) on the IL-10-mediated inhibition of inflammatory cytokines induced by B. burgdorferi and (b) on inflammatory cytokine signaling. We will silence socs genes by delivering small interfering RNA (siRNA) duplexes into macrophages, followed by stimulation with live spirochetes or spirochetal lipoproteins in the presence and absence of IL-10. In this fashion we will assess (i) the ability of IL-10 to inhibit pro-inflammatory cytokines (IL-6, IL-12p40, IL-18, TNF?, IL-1?) using quantitative real-time PCR (qPCR) and ELISAs and (ii) the effect of SOCS on inflammatory cytokine signaling by their ability to inhibit the activation and DNA binding activities of STAT proteins (in response to IL-6, IL-12, IFN?) and of NF?B (in response to IL-1 , IL-12, TNF?) using gene function assays. 2. Compare the expression of SOCS1/SOCS3 in primary macrophages and joint tissues from Lyme disease-resistant C57BL/6J and disease- susceptible C3H/HeJ mice before and during the course of a B. burgdorferi infection, and to correlate SOCS1/SOCS3 expression with IL-10 anti-inflammatory activity and with disease. We will employ macrophages derived from the bone marrow, lymph nodes and spleen from mice (and stimulate them as in Aim 1) to assess the expression levels of SOCS by qPCR, Western blot and confocal microscopy. We will correlate SOCS expression (i) in macrophages with the IL-10-mediated inhibition of pro-inflammatory cytokines (as in Aim 1) and (ii) in joint tissues with disease severity. If SOCS (or their downstream mediators) are differentially expressed in cells and tissues of mice from those two strains, and SOCS silencing results in the dysregulation of Lyme disease inflammatory responses in macrophages, then the SOCS pathway may dictate susceptibility and resistance to Lyme disease, as modeled in mice, and perhaps also in humans. Lyme disease, caused by the spirochete Borrelia burgdorferi, is an emerging infectious disease, and therefore, of major public health significance. Inflammation, as induced by the spirochete in multiple organs, plays a major role in disease pathogenesis, but its regulation is not understood. Achieving this proposal's goal will explain how inflammation is regulated, and help understand the basis of susceptibility and resistance to Lyme disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
7R21AI073356-03
Application #
7996833
Study Section
Special Emphasis Panel (ZRG1-IDM-A (90))
Program Officer
Breen, Joseph J
Project Start
2007-09-20
Project End
2010-08-31
Budget Start
2009-05-01
Budget End
2010-08-31
Support Year
3
Fiscal Year
2008
Total Cost
$75,878
Indirect Cost
Name
Alabama State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
040672685
City
Montgomery
State
AL
Country
United States
Zip Code
36101