JAK/STAT regulation of Aspergillus fumigatus corneal infections and IL-6/23-stimulated neutrophil, IL-17, elastase, and MMP9 activity

Abstract

IL-6 and IL-23 (IL-6/23) induce IL-17A (IL-17) production by a subpopulation of murine and human neutrophils, resulting in autocrine IL-17 activation, enhanced production of reactive oxygen species, and increased fungal killing. As IL-6 and IL-23 receptors trigger JAK1, -3/STAT3 and JAK2/STAT3 phosphorylation, respectively, we examined the role of this pathway in a murine model of fungal keratitis and also examined neutrophil elastase and gelatinase (matrix metalloproteinase 9) activity by IL-6/23-stimulated human neutrophils in vitro. We found that STAT3 phosphorylation of neutrophils in Aspergillus fumigatus-infected corne as was inhibited by the JAK/STAT inhibitor Ruxolitinib, resulting in impaired fungal killing and decreased matrix metalloproteinase 9 activity. In vitro, we showed that fungal killing by IL-6/23-stimulated human peripheral blood neutrophils was impaired by JAK/STAT inhibitors Ruxolitinib and Stattic, and by the retinoic acid receptor-related orphan receptor γt inhibitor SR1001. This was also associated with decreased reactive oxygen species, IL-17A production, and retinoic acid receptor-related orphan receptor γt translocation to the nucleus. We also demonstrate that IL-6/23-activated neutrophils exhibit increased elastase and gelatinase (matrix metalloproteinase 9) activity, which is inhibited by Ruxolitinib and Stattic but not by SR1001. Taken together, these observations indicate that the regulation of activity of IL-17-producing neutrophils by JAK/STAT inhibitors impairs reactive oxygen species production and fungal killing activity but also blocks elastase and gelatinase activity that can cause tissue damage.

Cytokine production by human neutrophils: Revisiting the “dark side of the moon”

“…Since studies reporting neutrophil positivity to IL‐17A continue to be published, ignoring either the previously mentioned information, or the few works showing the contrary, recently we decided to perform additional investigations . Using multiple methodological approaches (RT‐qPCR, ChIP‐seq, ELISA, intracellular staining and immunoblotting), we could confirm that highly purified populations of human neutrophils, either resting, or activated by a variety of stimulatory conditions, such as TLR and dectin ligands, fungal PAMPs and cytokines, including 20 μg/mL IL‐6 plus 2 μg/mL IL‐23, neither express IL‐17A, IL‐17F, IL‐17B, IL‐17C, IL‐17D and IL‐17E mRNA, nor produce IL‐17A, IL‐17F, IL‐17A/F and IL‐17B in vitro .…”

“…41 Similarly, 4 different groups 40,[42][43][44][45] have shown that IL-6 plus IL-23 efficiently induce IL-17A, IL-17F and IL-17RC mRNA expression and protein production by human neutrophils. Curiously, 2 of these groups have specified that concentrations lesser than 20 lg/mL IL-6 plus 2 lg/mL for IL-23 are not effective in terms of IL-17A induction, [42][43][44] while another group found 20 ng/mL IL-6 and/or 20 ng/mL IL-23 as perfectly functional in neutrophils from asthmatic subjects, 45 as well as up to 100 ng/ mL in control neutrophils. Whatever the case is, the most numerous evidence for the expression of IL-17 by human neutrophils derives from IHC and/or IF studies documenting IL-17A + -neutrophils in tissue specimens from a variety of pathological conditions (see Table 1).…”

“…7 Moreover, immunoprecipitation experiments followed by mass spectrometry have also uncovered that both AF-317-NA and #41802 antibodies actually recognize several proteins expressed in the granules and cytoskeleton of neutrophils, while other anti-human IL-17A antibodies (sc-6077) were found to non-specifically recognize multiple proteins in immunoblotting experiments of neutrophil lysates. 7 Since studies reporting neutrophil positivity to IL-17A continue to be published, 40,41,[43][44][45][46][47][48][49][50][51][52][53] ignoring either the previously mentioned information, or the few works showing the contrary, 7,54-57 recently we decided to perform additional investigations. 9 Using multiple methodological approaches (RT-qPCR, ChIP-seq, ELISA, intracellular staining and immunoblotting), we could confirm that highly purified populations of human neutrophils, either resting, or activated by a variety of stimulatory conditions, such as TLR and dectin ligands, fungal PAMPs and cytokines, including 20 lg/mL IL-6 plus 2 lg/mL IL-23, neither express IL-17A, IL-17F, IL-17B, IL-17C, IL-17D and IL-17E mRNA, nor produce IL-17A, IL-17F, IL-17A/F and IL-17B in vitro.…”

Innate Lung Defense during Invasive Aspergillosis: New Mechanisms

“…Recent studies have now extended this role to the eye, as neutrophils have been identified as a critical source of IL-17A during fungal keratitis [53] . Assessment of JAK/ STAT pathway inhibition in this model was shown to have a negative effect on ROS production and fungal killing activity, but also blocked the activity of elastase and gelatinase that is associated with tissue damage [54] . CD4 and γδ T cells may also produce IL-17A after A. fumigatus challenge.…”

Immune responses to invasive aspergillosis: new understanding and therapeutic opportunities

“…S tat3 -defective human neutrophils inhibit the growth of A. fumigatus swollen conidia and germlings and have normal chemotactic activity [60], consistent with the model that invasive aspergillosis susceptibility in these patients stems from anatomic lung defects associated with recurrent bacterial infections. Ruxolitinib, a small molecule JAK/STAT inhibitor, reduced Aspergillus killing by IL-6-stimulated and IL23-stimulated human neutrophils [61]. These data support the notion that JAK/STAT signaling contributes to neutrophil-mediated Aspergillus clearance though the precise mechanisms remain to be defined.…”