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.

Keywords: RORγt; Ruxolitinb; SR1001; Stattic; keratitis.

Figure 1.. p-STAT3 in vivo.

(A–D) Bone marrow neutrophils. (E, F) Neutrophils from infected corneas. (A) Representative histograms and Wright-Giemsa staining of murine NIMP-R14⁺ bone marrow neutrophils recovered 3 d after subcutaneous injection of heat-killed, swollen A. fumigatus conidia (primed) and from naïve C57BL/6 mice. (B) Percent intracellular STAT3 and p-STAT3 NIMP-R14⁺ bone marrow cells from naïve and primed C57BL/6 mice. (C) Western blot of total neutrophil lysates from the bone marrows of naïve and primed C57BL/6 mice. Membranes were probed with antibodies reactive with STAT3 (total STAT3), p-STAT3 (Tyr⁷⁰⁵), or β-actin. (D) Il17a gene expression in bone marrow neutrophils from naïve and primed C57BL/6 mice, showing products from qPCR. Actb (which encodes β-actin) served as the control for gel loading. (E) Representative flow cytometry of IL-17+ve Ly6G⁺ neutrophils from corneas 24 h after infection with live A. fumigatus conidia (cells were pooled from 10 corneas) and gated on p-STAT-3+ve cells. (F) Percent of p-STAT3⁺/IL-17⁺ neutrophils in infected and primed, infected corneas of C57BL/6 mice in 3 separate experiments (Exp.). CT scores from each experiment are in Supplemental Table 3.

Figure 2.. p-STAT3 requirement to regulate Aspergillus hyphal growth in vivo.

Ruxolitinib (Ruxo) or vehicle control was administered by oral gavage twice/d for 5 d, primed with heat-killed, swollen conidia, and infected intrastromally with live A. fumigatus conidia. Bone marrow neutrophils and cells from infected corneas were examined after 24 h. (A) Flow cytometry of intracellular p-STAT3 and total STAT3 in bone marrow neutrophils. (B) Total NIMP-R14⁺ neutrophils in infected corneas of mice given either systemic Ruxolitinib or vehicle control (mean ± sd of 5 mice/group). (C) IL17a gene expression in total, homogenized corneas. (D) Total cells gated for NIMP-R14⁺ neutrophils in infected corneas. (E) Intracellular IL-17A and p-STAT3 in NIMP-R14⁺ neutrophils from infected corneas. (F) In vivo growth of RFP-expressing A. fumigatus hyphae in corneas of primed mice treated with Ruxolitinib. Total RFP was quantified by image analysis (data points represent individual corneas). Original magnification of representative, infected corneas, ×20. Results are combined from 3 separate experiments. (Individual experiments are shown in Supplemental Fig. 1). (G) MMP9 activity in infected corneas (mean ± sd) from 3 separate experiments.

Figure 3.. Neutrophil regulation of hyphal growth and ROS production in vitro.

(A and B) Growth of dsRed expressing A. fumigatus after 18 h incubation with bone marrow neutrophils from C57BL/6 mice (A) or with human peripheral blood neutrophils (B) in the presence of Stattic, Ruxolitinib, or SR1001. Fungal mass was measured by fluorimetry of dsRed and represented as RFU. Controls are medium only [no neutrophils (no neuts)] and neutrophils that were not stimulated with IL-6/23. (C and D) ROS production (intracellular CFDA) by IL-6/23-stimulated murine and human neutrophils. MFI, Mean fluorescence intensity. Controls are unstimulated neutrophils (no hyphae). All wells are the means ± sd of 3 wells/experimental condition from neutrophils pooled from 3 mice/group (A and C) or from a single donor (B and D). Data are representative of 2 separate experiments. *P < 0.01.

Figure 4.. p-STAT3 and RORγt nuclear translocation in human neutrophils.

(A–D) Western blots of total cell lysates and nuclear extracts from IL-6/23-stimulated peripheral blood human neutrophils in the presence of Stattic or Ruxolitinib. Blots were probed with antibodies to p-STAT3, STAT3, RORγt, and either β-actin as a loading control for total extracts or TBP for nuclear extracts. (E) Representative confocal images of intracellular RORγt (red) in unstimulated (unstim) and IL-6/23-stimulated human neutrophils in the presence of Stattic or Ruxolitinib. Cell nuclei were visualized using DAPI (blue). (F) Quantitative analysis (using ImageStream) showing percent of IL-6/23-stimulated neutrophils in which RORγt was detected in the nucleus. Original magnification, ×1000.

Figure 5.. JAK/STAT and RORγt regulation of IL-17 production by murine and human neutrophils.

(A, C, and E) Bone marrow neutrophils from C57BL/6 mice. (B, D, and F) Human peripheral blood neutrophils. (A and B) Il17a gene expression in IL-6/23-stimulated neutrophils in the presence of Stattic, Ruxolitinib, or the RORγt inhibitor SR1001. Actb and GAPDH were loading controls. (C and D) Quantification of IL-17A protein in mouse and human neutrophils by ELISA. (E and F) Intracellular IL-17A production assessed by flow cytometry. Three experiments were performed with similar results.

Figure 6.. JAK/STAT-dependent elastase and MMP9 activity in human neutrophils.

Human peripheral blood neutrophils were incubated with AspHE or IL-6/23 in the presence of Stattic, Ruxolitinib, or SR1001. Supernatants were assayed for elastase (A) and MMP9 (B) activity. *P < 0.01; **P < 0.001.

Figure 7.. JAK/STAT-dependent cell-surface expression of MMP9 in human neutrophils.

Neutrophils were incubated with anti-human MMP9 antibody to detect extracellular MMP9 or were permeabilized to detect intracellular MMP9. (A) Representative flow cytometry profiles and (B) combined results. Data are representative of 2 repeat experiments.