Cutting Edge: Neutrophils License the Maturation of Monocytes into Effective Antifungal Effectors

Neutrophils are well known for their essential role in defense against fungal pathogens and are rapidly recruited to the site of infection (1). Neutrophils can directly kill fungal pathogens via multiple mechanisms, including phagocytosis, the release of proteases, neutrophil extracellular traps, and the generation of reactive oxygen species (ROS) (2). The enhanced susceptibility of neutropenic patients to invasive fungal infection has thus been primarily understood as a failure of pathogen control due to the absence of these important effector cells. Emerging studies suggest that neutrophils can shape the response of other immune cell subsets, and can be relevant sources of cytokines and chemokines (3–7). Whether neutrophils have more nuanced contributions in antifungal protection that go beyond their traditional role as effectors remains to be determined.

In addition to neutrophils, CCR2⁺ inflammatory monocytes (CCR2⁺Mos) are innate cell precursors to macrophage and dendritic cell subsets that are crucial for defense against multiple pathogens, including fungi (8–15). In previous studies, we established that CCR2⁺Mos are essential for defense against invasive aspergillosis (IA) (14, 16). Upon infection with Aspergillus fumigatus, CCR2⁺Mos help activate antifungal neutrophils (16), and they differentiate into CCR2⁺CD11b⁺CD11c⁺MHC class II (MHC II)⁺ cells (monocyte-derived dendritic cells [Mo-DCs], same definition used throughout) (10, 14) that mediate fungal killing, produce cytokines, and help recruit plasmacytoid DCs, thus promoting optimal defense against IA (14, 17, 18).

Despite the important, antifungal functions of CCR2⁺Mos and Mo-DCs, these cells are not able to protect against IA when neutrophils are depleted (14). Why Mo-DCs are unable to protect against IA in the absence of neutrophils is unclear. In this study, we set out to examine the impact of neutrophil depletion on the antifungal response of CCR2⁺Mos.

CCR2 reporter (CCR2-GFP), CCR2^Cre, and STAT1^fl/fl mice were generated as previously described (19–21). Studies were performed in specific pathogen-free conditions according to Institutional Animal Care and Use Committee–approved protocols. Fungal infections were done with A. fumigatus-DsRed (22), A. fumigatus 293, or FLARE (fluorescent Aspergillus reporter) conidia (23). FLAREs were generated and analyzed as previously described and shown in Supplemental Fig. 1 (23). Fungal culture, mouse infections, tissue collection, and fungal growth assessments on infected lungs were done as previously described (14, 16).

Mice were injected daily with 500 μg i.p. and 250 μg intratracheally of 1A8 (anti-Ly6G) mAb (Bio X Cell). Treatments were started at day −1. Mouse monoclonal IgG2a (2A3) was used as a control (Bio X Cell). For IFN treatment (Fig. 3), neutrophil-depleted mice were treated with 1.0 μg i.p. of murine IFN-γ (PeproTech) on the day of infection and day +1. For survival experiments (Fig. 4F), mice were injected with IFN-γ (1.0 μg i.p) on day 0 and every other day.

Single-cell suspensions from lung samples were done and analyzed as previously described (14, 16). Bronchoalveolar lavage (BAL) cells from infected mice were cultured with 1.0 μM CM-H2DCFA (Life Technologies) for 60 min at 37°C for ROS detection. All flow cytometry was done in a BD LSRFortessa X-20 and analyzed with FlowJo software.

RNA from lungs was extracted with TRIzol (Invitrogen). Gene expression was calculated using the ΔΔCt method relative to naive samples using TaqMan probes (Applied Biosystems) and normalized to GAPDH. IFN-γ, IFN-λ, and TNF-α cytokines in lung homogenates were measured by ELISAs (Thermo Fisher Scientific and R&D Systems).

Live CCR2^GFP+CD45⁺CD11b⁺NK1.1⁻Ly6C⁺Ly6G⁻ (CCR2⁺Mos) were isolated from the lung of A. fumigatus–infected CCR2-GFP mice (48 h) to >98.9% purity using a BD FACSAria II cell sorter. The transcriptional profile of CCR2⁺Mos was examined by RNA sequencing (RNA-seq). The transcriptional profile of CCR2⁺Mos was examined by RNA-seq. RNA processing for library generation and sequencing on an Illumina HiSeq instrument was done by the Genomic Research Core facility as described (14, 16). For each group, three different biological replicates were examined, and differentially regulated gene clusters were identified. RNA-seq data have been deposited in BioProject metadata under number PRJNA847069 (available at: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA847069). Neutrophils (CD45⁺, Siglec F⁻, CD11b⁺, Ly6C^int, Ly6G⁺), monocytes (CD45⁺, Siglec F⁻, CD11b⁺, Ly6C^hi, Ly6G⁻, CCR2⁺, NK1-1⁻), and alveolar macrophages (CD45⁺, Siglec F⁺, CD11c⁺) were sorted from A. fumigatus–infected (48 h postinfection) and naive lung; 1 × 10⁵ cells were cultured overnight and examined for cytokine secretion in supernatants by ELISA.

To examine the impact of neutrophils on global antifungal immunity, we examined the response of A. fumigatus–infected mice after neutrophil depletion (anti-Ly6G). Consistent with previous studies, neutrophil depletion resulted in increased mortality (Fig. 1A), increased fungal burden, and susceptibility to IA (Fig. 1B, 1C). We confirmed the efficient depletion of neutrophils in the lung at 36 h postinfection (Fig. 1D). The recruitment of CCR2⁺Mos to the lung was increased in neutrophil-depleted mice (Fig. 1E). This observation is consistent with previous studies in neutropenic mice (24). Although the number of recruited cells was increased, Mo-DCs failed to upregulate MHC II expression (Fig. 1F), showed diminished killing of fungal conidia (Fig. 1G), and had reduced ROS (Fig. 1H) (a critical antifungal effector mechanism) (25, 26). These observations suggest that in the absence of neutrophils, monocytes are impaired in their ability to become efficient antifungal effectors. These findings suggest that susceptibility to IA in neutropenic mice is likely a combination of lack of fungal conidia inactivation by neutrophils as well as a failure of monocytes to become competent antifungal effector cells.

To test whether neutrophils shape the response of CCR2⁺Mos, we performed a transcriptional analysis of CCR2⁺Mos isolated from the lung of A. fumigatus–infected mice in the presence or absence of neutrophils. Neutrophil depletion altered the response of CCR2⁺Mos (Fig. 2A, Supplemental Table I). Ingenuity Pathway Analysis (IPA) suggested that in the context of neutropenia, CCR2⁺Mos had impaired activation of genes in canonical pathways associated with DC maturation (Fig. 2B). Furthermore, Ingenuity Pathway Analysis for upstream regulators of differentially expressed genes (threshold set at fold changes >2.5, (Fig. 2C) predicted altered IFN activation as a potential basis for dysfunctional responses of CCR2⁺Mos in A. fumigatus–infected, neutropenic mice (Fig. 2D).

In previous studies, we uncovered that CCR2⁺Mos are required for the proper activation of IFN responses that are necessary for the activation of antifungal neutrophils (16). The transcriptional analysis of CCR2⁺Mos suggests that neutrophils might also be required to trigger the production of IFNs, and that these cytokines may act on monocytes to promote their maturation into antifungal effectors. We previously determined that all IFNs are produced in response to A. fumigatus infection with distinct kinetics (16). Thus, we examined IFN expression at their respective peaks of expression in neutropenic mice. We observed that neutrophil depletion in A. fumigatus infection resulted in reduced expression of all IFNs examined (Fig. 3A–C). In contrast to IFNs, neutrophil depletion led to an increase in TNF production (Fig. 3D), an observation consistent with previous studies (24, 27). In aggregate, our data suggest that neutrophils are required for optimal expression of IFNs during pulmonary aspergillosis, but are not linked to a global dysregulation of inflammatory responses.

To determine whether the impaired maturation of Mo-DCs in neutrophil-depleted mice was linked to defective IFN production, we tested whether the antifungal response of CCR2⁺Mos could be rescued by IFN administration. We chose to test the role of IFN-γ on this response based on the previously reported importance of this cytokine in the activation of Mo-DCs (28). We observed that neutrophil-depleted mice treated with IFN-γ had improved CCR2⁺Mo responses that included increased ROS production (Fig. 3E), increased MHC II expression (Fig. 3F), and improved control of fungal burden (Fig 3G). Importantly, neutrophils sorted from the lung of A. fumigatus–infected mice secreted significant amounts of IFN-γ as compared with neutrophils from naive mice (Fig. 3H). Monocytes and alveolar macrophages sorted from the same mice also secreted some IFN-γ as compared with their naive counterparts, but to a lesser extent than what was measured in neutrophils (Fig. 3H). Altogether, these data support the idea that defective IFN production in neutrophil-depleted mice is linked to CCR2⁺Mo dysfunction during A. fumigatus infection.

A limitation of this approach is that exogenous IFN-γ may have improved antifungal CCR2⁺Mo function via indirect effects. Thus, we employed a genetic loss-of-function model to test the impact of IFN–STAT1 signaling directly on CCR2⁺Mos. STAT1 is critical in the signaling cascade of all IFNs (29, 30). We thus generated mice with conditional knockout of the STAT1 gene in CCR2⁺ cells (CCR2^creSTAT1^fl/fl mice) (20). CCR2⁺Mo (Fig. 4A) and neutrophil (Fig. 4B) recruitment to the lung was not affected in CCR2^creSTAT1^fl/fl mice. In contrast, control of fungal burden was diminished in A. fumigatus–infected CCR2^creSTAT1^fl/fl mice (Fig. 4E). Functionally, monocytes displayed diminished Mo-DC differentiation (Fig. 4C) and reduced production of ROS (Fig. 4D). Importantly, CCR2^creSTAT1^fl/fl mice succumbed to infection to A. fumigatus (Fig. 4F), which was linked to IA (Fig. 4G, 4H). Administration of IFN-γ to CCR2^creSTAT1^fl/fl mice was not able to rescue A. fumigatus–infected mice (Fig. 4F), thus supporting the importance of IFN-γ acting directly on CCR2⁺Mos. In aggregate, these observations suggest that IFNs trigger a STAT1-dependent transcriptional program on CCR2⁺Mos that is necessary for the full activation of antifungal responses by CCR2⁺Mos and their derivative effectors.

Our observations support the idea that beyond their role as antifungal effectors, neutrophils are important for the regulation of antifungal CCR2⁺Mos. Our findings suggest that one of the important ways in which neutrophils influence antifungal monocytes is by promoting the production of IFNs. The mechanisms of neutrophil-dependent IFN production could be as a direct cellular source and/or by regulating IFN production in other cells. Monocytes are known to be sensitive to the effects of both type I and II IFNs, and previous studies have found an important role for IFNs in the local differentiation of monocytes into Mo-DCs (28, 31, 32). Furthermore, in a model of toxoplasmosis, neutrophils were found to be important, direct sources of IFN-γ (33, 34). Similarly, we observed that neutrophils sorted from the lung of A. fumigatus–infected mice secreted IFN-γ to a significant extent. Thus, during A. fumigatus infection, neutrophils contribute to IFN-γ levels, at least in part, via direct production. Additional contributions to global IFN levels during A. fumigatus infection could be indirect via effects on other cells such as NK cells (35, 36). In turn, IFNs then help license CCR2⁺Mo differentiation and acquisition of Mo-DC antifungal functions. The current study adds to our understanding of innate immune cell cross-talk during aspergillosis by placing neutrophils as both targets and regulators of antifungal monocytes. The observation that STAT1 is an important regulator of antifungal monocytes also suggests the expanded, therapeutic potential of IFNs in the treatment of fungal infections by activating essential antifungal effector responses in both neutrophils and monocytes.

The authors have no financial conflicts of interest.