Relevance of N-terminus amino acids in CCL28 mediated murine airway hyper-reactivity

RATIONALE: CCL28 is a chemokine associated with human asthma; we have demonstrated it is critical for development of post-viral asthma in a mouse model. Interestingly, administration of CCL28 alone (without a viral infection) is sufficient to drive development of airway hyper-reactivity (AHR). In CC chemokines, the N terminus is thought to interact with the chemokine receptor. We undertook this study to examine the importance of N terminal amino acids in CCL28’s ability to drive AHR.

METHODS: C57BL/6 mice were inoculated intranasally daily for 3 days with 3, 1, 0.3, and 0.1 µg/ml of CCL28 or a CCL28 variant lacking the first 3 amino acids of CCL28 (del3-CCL28). On day 4 a non-invasive, two-chamber plethysmography system was used to measure AHR to increasing doses of methacholine. Unfolded versions of the proteins were used as negative controls.

RESULTS: 3 µg/mL CCL28 significantly increased sRaw and decreased sGaw (p<0.0001 versus unfolded CCL28 for both; n≥6); however, no other doses led to increased AHR. Removing the first 3 amino acids (del3-CCL28) led to marked increase in potency, with significantly increased sRaw (p=0.001 versus unfolded del3-CCL28; n≥4) and reduced sGaw (p=0.011) at only the 0.3 µg/ml dose.

CONCLUSIONS: CCL28 drives AHR in the absence of a viral infection. The first 3 N terminal amino acids decrease potency of this effect by a log-fold, suggesting these amino acids may interfere with the most efficient binding of CCL28 to its receptor. This effect could be utilized to develop novel therapeutics for asthma.