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  1. R. A. Dimmitt,
  2. T. Soltau,
  3. G. Chuang,
  4. R. G. Lorenz
  1. Departments of Pediatrics and Pathology, University of Alabama at Birmingham, Birmingham, AL


Background The intestinal innate mucosal immune function is important in health and disease. Specifically, Toll-like receptors (TLRs) are cell surface molecules that recognize distinct molecular patterns with a resultant inflammatory response. It is generally accepted that the acquisition of the intestinal microbiota occurs within the first few days of life. The role of this colonization on the expression of TLR in the developing intestine is not well understood. We hypothesized that the acquisition of commensal bacteria would result in increased expression of gastrointestinal TLR and the associated inflammatory response.

Methods Conventionally housed C57BL/6 mice were sacrificed at different time points (E 20.5, 1 day, 3 days, 1 week, 2 weeks, 3 weeks, and 6 weeks). The gastrointestinal tract was divided into stomach, small intestine, and cecum/colon. RNA was isolated using the Trizol® method (Invitrogen, Inc) and cDNA was synthesized using the First Stand Transcriptor Kit® (Roche, Inc). Real-time PCR was performed to measure the expression of TLR2, TLR4, TLR5, TLR9, and MIP-2 (murine IL-8) using Assay on Demand® (Applied Biosystems). The crossing threshold of each TLR was normalized to 18S ribosomal expression. Fold change in expression was compared to the 6-week-old animal levels. Statistical significance was set at > 4-fold change or < 0.25-fold change.

Results TLR levels at E 20.5, 1 day, 3 days, and 1 week were not different than 6-week-old expression. In contrast, expression of gastric and colonic TLR2, -4, -5, and -9 were significantly greater in the 2-week-old mice. Intestinal TLR4 and -5 were also greater at 2 weeks. This increased expression was dampened by 3 weeks of age. Mirroring the TLR expression, MIP-2 was also significantly greater at 2 weeks.

Conclusions TLR expression is greatest in the time period associated with the acquisition of commensal mirobiota. Additionally, there is a local inflammatory response as measured by MIP-2 levels. This peak in expression is not maintained and diminishes by 6 weeks of age. Implications: The transient increase in TLR expression, coinciding with bacterial colonization, likely recruits effector cells into the mucosal immune system. This process may also play a role in the pathogenesis of necrotizing enterocolitis (NEC), a disease of premature infants. Since the median age of onset of NEC is approximately 3 weeks, the inflammatory response to the colonization of intestinal commensal microbiota could be an initiating factor in the disease.

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