Cathelicidins and the regulation of the innate immune system

Abstract

While antibiotics have been indispensible for the treatment of bacterial infections, the increase in antibiotic resistance and lack of new antibiotic compounds greatly limits the available treatment options. Therefore, development of novel anti-infective therapies is crucial for the treatment of bacterial infections in the future. In the search for new anti-infective templates, host defense peptides, such as cathelicidins, have gained a lot of interest. These short cationic peptides have been shown to elicit broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria and are thought to have beneficial immunomodulatory functions as well. Of these cathelicidins, the chicken cathelicidin-2 (CATH-2) has been shown to be an interesting template, with strong antimicrobial activity against a wide variety of bacterial pathogens. However, much remains to be learned about how cathelicidins, such as CATH-2, affect immune activation. Cathelicidins have been suggested to exert various functions in innate immune activation, however, most of these functions have been described in the context of the human LL-37. To determine whether CATH-2 also influences immune activation 12 cathelicidins from 6 different species, including CATH-2 and LL-37, were compared for various previously described cathelicidin functions. This comparison showed that most cathelicidins show antimicrobial activity against E. coli and MRSA and many show LPS and/or LTA-neutralization. However, little to no change in chemokine expression was observed. CATH-2 was among the most potent peptides, with strong antimicrobial activity and LPS/LTA-neutralization. In addition, CATH-2 strongly enhanced DNA-induced activation of both mammalian and avian macrophages. This was caused by direct complex formation between CATH-2 and DNA, which enhances DNA internalization into endosomes. Interestingly, once internalized, CATH-2 degradation was essential to free the DNA and allow activation of the avian TLR21. To further determine whether the effects on immune activation, such as LPS-neutralization, also occur under more complex conditions comprising viable bacteria, macrophages were incubated with live E. coli or S. enteritidis in combination with CATH-2. By analyzing both bacterial viability and macrophage activation, it was shown that CATH-2, but also other cathelicidins such as LL-37 and CRAMP, are able to inhibit macrophage activation by E. coli or S. enteritidis, but only when bacterial viability is lost, either due to cathelicidin-mediated killing or other types of killing. Furthermore, this inhibition was caused by neutralization of LPS and lipoproteins from the bacterial outer membrane, which prevented TLR4 and TLR2 activation, respectively. To determine whether the “silent killing” of Gram-negatives by CATH-2 would also prevent in vivo inflammation, CATH-2-killed P. aeruginosa was instilled in murine lungs, after which cell recruitment and cytokine release were determined in the bronchoalveolar lavage fluid (BALF). Treatment of these mice with CATH-2-killed P. aeruginosa strongly limited inflammation in comparison to heat-killed or gentamicin-killed P. aeruginosa, demonstrating the “silent killing” capacity of CATH-2 in vivo. Overall, these results show the potential of CATH-2 as a novel anti-infective with strong antimicrobial activity against Gram-positive and Gram-negative bacteria. In addition, by inhibiting inflammation against non-viable Gram-negatives, CATH-2 can dampen inflammation when the bacterial threat has been neutralized to prevent unnecessary tissue damage and sepsis.