*3.3. Influenza A virus*

LL-37 therapeutic activity against influenza type A virus has been demonstrated in vivo and in vitro. It is likely that in vivo, IAV encounters LL-37 in the respiratory tract following innate immune responses against the virus and is secreted from neutrophils, macrophages, and epithelial cells [44,49]. Early studies assessed the antiviral activity of LL-37 in vivo using a mouse IAV strain [50]. Mice were nebulized with LL-37 (500 μg/mL) a day prior to infection with a lethal dose of IVA PR/8 mouse strain and survival and weight loss were monitored for 14 days following infection [50]. Initially, all mice exhibited weight loss, but weight loss ceased at day seven in mice treated with LL-37 or the IAV antiviral zanamivir. Mice treated with LL-37 and zanamivir exhibited 60% survival compared to the untreated group which succumbed to infection by day nine suggesting that therapeutic use of LL-37 reduces IAV infection severity in a manner comparable to zanamivir [50]. LL-37 also decreased expression of inflammatory cytokines particularly IL-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), keratinocytes chemoattractant (KC), and the chemotactic cytokine known as regulated on activation normal t-cell expressed and secreted (RANTES), in bronchoalveolar lavage fluid in mice infected with PR/8 at two days following LL-37 treatment as determined by immunoassay demonstrating the immunomodulatory properties of LL-37 [50]. In vitro plaque assays demonstrated one log inhibition of PR/8 when virus was pre-incubated with LL-37 (50 μg/mL) in Madin-Darby canine kidney (MDCK) cells [50].

During IAV infection, in vitro LL-37 treatment did not prevent viral uptake, cause viral aggregation, and was not associated with blocking of hemagglutinin (HA). Interestingly LL-37 inhibits IAV replication at post-entry steps prior to viral RNA or protein synthesis [44]. A reduction in viral load, direct antiviral effects in epithelial cells, and inflammatory cytokine production have all been linked to LL-37 activity [49]. LL-37 inhibits the NY01 strain of IAV with a significant reduction in uptake of virus into cells, and in a manner dependent on dosage [44,49]. For optimal anti-IAV activity the central helix of LL-37 is required, as evident by fragments of LL-37 containing the complete central sequence of the peptide demonstrating more robust antiviral responses as compared to fragments with shorter central fragments. At a concentration of <2 μM, NY01 was only partially inhibited; however, this inhibition was surprisingly lost at higher concentrations of LL-37 [44]. A strain containing only the pandemic HA (Mex 1:7); however, was inhibited by LL-37 at all concentrations tested (up to 10 μM). Consistent with previous studies, these results suggest the antiviral effects of LL-37 are not determined by direct interaction of LL-37 with the viral HA [44].

Experimental data also demonstrates the participation of LL-37 in host defenses against IAV through modulation of innate immune cells, particularly neutrophils. IAV infection induces a respiratory burst response in neutrophils, and this response is noticeably up-regulated by pre-incubation of LL-37 with IAV [49]. LL-37 alone does not stimulate this respiratory burst response; however, optimal enhancement of this antiviral response is achieved only when the virus is pre-incubated with LL-37 [49]. Furthermore, there is growing evidence that NET formations play an important role during IAV infections [49]. Recently, in vivo studies have provided evidence of NET formation in the lungs of IAV-infected mice [51]. On the other hand, in vitro evidence of binding of IVA to NETs suggests LL-37 induces an increase in NET formation in response to IAV, which may promote viral clearance in vivo [49,51]. Data also suggest that significant protection against IAV may be provided by therapeutic treatment of influenza infected individuals with LL-37 or by increasing natural cathelicidin expression in the IAV-infected lung [11]. Hence, to maximize anti-IAV functions, approaches, such as therapeutic administration of naturally-occurring cathelicidins, as well as increasing vitamin D levels to boost endogenous cathelicidin have been proposed [11].
