Anti-Inflammatory Effects of Moxifloxacin with and without Anti Toll-Like Receptors and Protein-Tyrosine-Kinase Inhibitors in Human Monocytes Stimulated with Aspergillus fumigatus.
Authors: Fabian Ina , Levitov Avital , Haite Debby , Halperin Drora , Romano Jacob , Osherov Nir , Shalit Itamar 
Published: April 2020

Abstract
Invasive aspergillosis is a life-threatening disease in immunocompromised patients. Studies have shown that conidia from Aspergillus fumigatus (AF) induce the release of proinflammatory cytokines from leukocytes and provoke an inflammatory response. Although cytokines such as TNF-α and IL-8 are critical for optimal host defenses, in some patients with invasive aspergillosis such as patients with neutropenia or allergic bronchopulmonary aspergillosis, limiting the immune response by decreasing cytokine release could prove beneficial. We have shown that the fluoroquinolone moxifloxacin (MXF) conferred protective anti-inflammatory effects in a murine model of Candida pneumonia in immune suppressed animals (AAC46: 2442, 2002). In the present study, we investigated the effect of MXF on cytokine response and signal transduction mechanisms in human peripheral blood monocytes (PBMN) of healthy individuals stimulated with conidia of AF. PBMN incubated with 2% autologous serum were stimulated for 6 h with AF (1.5x106/ml) and cytokine secretion was measured with and without the addition of MXF. Secretion of IL-8, IL-1β and TNF-α was significantly enhanced by AF (4.3, 8.2 and 7-fold respectively), while MXF (5–20μg/ml) inhibited the cytokines secretion in a dose dependent manner up to 46%, 72% and 73% respectively (p&lt;0.05). Recognition of invading microorganisms by the innate immune system is a first step in their successful elimination. In the mouse, toll-like receptor (TLR) 2 and 4 were found to be essential for AF-induced activation of macrophages while conflicting data have been published regarding human macrophages. We treated PBMN with blocking antibodies (Ab) directed against TLR2 and TLR4 and found that both TLRs are involved in cellular sensing of AF. Blocking of PBMN with a combination of anti-TLR2 and 4 Ab before exposure to AF resulted in 42%, 46% and 61% decrease in IL-8, IL-1β and TNF-α, secretion, respectively.
Pre-incubation of the cells with MXF (20 μg/ml) and the combined anti-TLR2 and 4 Ab resulted in an additional decrease in the cytokines secretion up to 60%, 68% and 80%, respectively. Previous studies have implicated activation of protein tyrosine kinases (PTK) in LPS-induced NFkB activation and TNF-α production. In-vitro studies performed previously by us showed that MXF inhibits NFkB and MAPK activation and the synthesis of proinflammatory cytokines in activated human monocytic cells (AAC48:1974, 2004). In the present study we stimulated PBMN with AF, with and without the PTK inhibitor, genistein (Gen) in the presence and absence of MXF. Gen (5–40 μM) inhibited in a dose dependent manner IL-8 secretion up to 25%. Pre-incubation of PBMN with MXF (20 μg/ml) and Gen resulted in an additional decrease in IL-8 secretion up to 49%. Our results indicate that the anti-inflammatory effect of MXF in AF-stimulated PBMN does not signal via TLR2 or TLR4, nor via PTK. Further studies in invasive aspergillosis models are needed to evaluate the importance of these immunomodulatory effects.