Neutrophil extracellular traps involvement in corneal fungal infection

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: Neutrophils release neutrophil extracellular traps (NETs) when defending against invading microorganisms. We investigated the existence of NETs in fungal keratitis.

Methods: Fourteen patients with unilateral fungal keratitis were included. Detailed information about each patient was recorded, including (1) patient history (onset of symptoms and previous therapy), (2) ocular examination findings by slit-lamp biomicroscopy, (3) laboratory findings from direct smear examination and culture of corneal scrapings, (4) NET formation, and (5) treatment strategy and prognosis. Immunofluorescence staining was used to evaluate the existence of NETs on corneal scrapings. The relationship between the quantification of NETs and the clinical character of the fungal keratitis was identified.

Results: NETs were identified in all 14 patients. Patients with a higher grade of NET formation and fewer fungal hyphae always showed a good treatment response and a short course of infection. NETs were consistently found mixed with fungal hyphae in the corneal scrapings from infected patients. No statistical significance was found between the grade of NETs formed and the course of infection before presentation, and no relationship between the quantification of NETs and the size of the ulcer was found.

Conclusions: The results suggest that NETs are involved in fungal keratitis. The number of NETs in infected corneas may provide a tool for evaluating the prognosis for fungal keratitis.

Fungal keratitis is a sight-threatening eye disease that requires timely and appropriate treatment. The predominant causes of fungal keratitis are Aspergillus, Acremonium, Penicillium, and Fusarium solani, which are all non-pigmented fungi [1]. Although Aspergillus and Fusarium clearly dominate the list, causative agents vary in different geographic areas. Xie et al. [2] reported that Fusarium species accounted for 73.3% of isolated fungal keratitis with Fusarium solani as the most common in northern China. Moharram et al. [3] reported that Fusarium species accounted for 13.8% of isolated fungal keratitis with Fusarium solani as the most common in Upper Egypt. Moreover, since June 2005, a global increase in the incidence of keratomycosis from Fusarium species has appeared in contact lens wearers who use Bausch & Lomb (Rochester, New York, NY) ReNu® with MoistureLoc or ReNu® Multiplus [4]. To avoid permanent vision loss and other related complications, antifungal treatment must be promptly started based on clinical findings and microbial examination [5]. Neutrophils are one of the first lines of defense against invading pathogenic microorganisms and play an important role in host defense against fungal pathogens. Neutrophils are shown to defend against the invasion of microorganisms by phagocytosis, degranulation, production of reactive oxygen species (ROS) [6-8], production of antimicrobial peptides [9], and extrusion of neutrophil extracellular traps (NETs) [10]. Neutrophils are capable of phagocytic clearance of unicellular microbes but are also reported to be challenged by fungal hyphae that are too large to be ingested. NETs are a newly discovered extracellular structure composed of the chromatin associated mainly with histones and many granular antimicrobial proteins, which trap, disarm, and destroy bacteria [11], as well as fungi [12]. However, the relationship between the existence of NETs and fungal keratitis is still unknown.

The early neutrophil response to fungal invasion is poorly understood but is critically important in corneal infection, which could be highly related to the prognosis for keratitis. In the present study, we investigated the existence of NETs in fungal keratitis and explored the relationship between the quantification of NETs and prognosis. Our results indicated that NETs are involved in the immune response following fungal infection and the number of NETs was highly related to the prognosis for this disease.

All patients agreed to the treatment before the smear examination. Patient information is shown in Table 1. The results of the microbial tests and clinical therapy confirmed that 14 patients had fungal keratitis. Thirteen cases were identified from the microbial culture, and one fungal keratitis case was identified with confocal microscopy.

NETs were identified in all 14 patients with fungal keratitis (Figure 1). The NETs and fungal hyphae (12/14) were often found in corneal scrapings of fungal keratitis with immunofluorescence staining (Figure 2 and Figure 3). The existence of NETs was detected at different stages of infection from day 2 to day 22, suggesting that the formation of NETs may have a crucial role during the entire stage of infectious keratitis. NETs were counted in ten consecutive fields (400X magnification) of each specimen. The number of NETs according to the proportion of DNA-releasing neutrophils is shown in Figure 2. We found that the relationship between the number of NETs and the size of the ulcer is variable (Figure 3). The patients’ responses to antifungal therapy showed a close relationship to the number of NETs formed (r = –0.813, p<0.001). Patients with a higher grade of NET formation and fewer fungal hyphae always showed a good treatment response and a short course of infection (Table 1 and Figure 4A–C). Different NET formation and clinical courses among fungal species are shown in Figure 5A and Figure 5B. However, statistical analysis was not performed among the fungal species as the sample was too small. No statistical significance was found between the course of treatment and the course of fungal keratitis before presentation (r = 0.479, p = 0.051). No statistical significance was found between the course of fungal keratitis before presentation and the grade of NET formation (r = –0.321, p = 0.157).

The present study showed that neutrophils are activated in fungal keratitis to produce NETs, thus pointing to a possible role of NETs in fungal keratitis. Previously, most studies in infectious keratitis have been limited to studying the role of T-lymphocytes, macrophages, nature kill cells, and dendritic cells. Less attention has been given to the role of neutrophils, especially NETs, which serve as the first line of defense against infectious microorganisms.

NETs are web-like structures released extracellularly from activated neutrophils during infection and are composed of DNA, histones, and neutrophil elastase [7,8]. In the context of infectious disease, the formation of NETs has been shown to play a more important role in antifungal defense [11,12]. Different from bacteria, pathogenic fungi are dimorphic and switch between conidia and hyphal forms [16]. The conidia can be cleared by phagocytosis and intracellular degranulation, but the hyphal forms are too large to be internalized by this classic mechanism of antimicrobial host defense [11]. Instead, released NETs provide the host with an effective extracellular antifungal defense. These large extracellular web-like structures provide a physical barrier that prevents microbial dissemination and increases the local concentration of antimicrobial substances [10,11,17]. Our results show that the existence of NETs can be detected at different stages of infection from day 2 to day 22, suggesting that the formation of NETs may have a crucial role during the whole stage of fungal keratitis. In contrast to most other tissues, the initial immune response to infectious pathogens may be critically important, as the cornea is avascular. Therefore, neutrophil-derived NETs could be an important mechanism for defending against fungal infection in the cornea.

Our findings of NETs in all 14 fungal keratitis patients suggested a higher sensitivity of NETs than in other routine microbial investigations. The detection of NETs in the fungi-infected cornea may represent a promising appending method to standard microbial testing for rapid detection of causative organisms, particularly in cases where standard microbial test results are negative. Unfortunately, the discrimination of NET formation in fungal keratitis from that in other types of infectious keratitis, such as bacterial keratitis, is difficult. Until now, the different characteristics of NETs among different types of infectious keratitis have not been exploited, a topic that merits more research.

Shan et al. analyzed the contribution of NETs to the mediation of protection during ocular keratitis. Their results suggest that a balance between NET production and NET degradation is needed to achieve maximal NET immunity [18]. The course of fungal keratitis is always longer than 20 days [19]; therefore, NETs may play a crucial role in combatting fungal infection of the cornea. Previously, the prognosis for fungal keratitis was difficult to evaluate clinically, and the therapy could be modified only in accordance with the clinical response [20]. Previous reports presented that phagocytosis is the main mechanism for clearing microbes during the first 60 to 120 min following infection after which neutrophils’ phagocytic capacity is restricted [19]. Afterward, active neutrophils display a high ratio of coiling versus conventional phagocytosis [21], which leads to nuclear and granular components mingling in the cytoplasm and then rapidly being released as NETs when the neutrophils finally die. NETs can then bind, trap, and kill microbes by providing a high local concentration of antimicrobial peptides. In this vein, the NETs act as soldiers to protect the cornea from fungi invasion, and a higher number of NETs may lead to a better prognosis. In the present study, we explored the relationship between the number of NETs and prognosis. Although the number of NETs was not related to the course of the infection or the size of the ulcer, NETs were related to the prognosis for fungal keratitis. A higher number of NETs was closely related to a better treatment response and a shorter course of infection. These data suggest that measuring host immune markers of NET formation could prove useful for in vivo monitoring of treatment responses. However, the discrimination of NET formation in different types of fungus is difficult. In the present study, four fungal species were examined in the experiment, and in particular, for Alternaria sp., there was only one case; thus, as the total sample was small, statistical analysis could not be performed. Nonetheless, we still observed that the number of NETs of Candida albicans and Alternaria sp. was higher than that of Fusarium and Aspergillus, and the clinical course of treatment for the former was shorter than that for the latter. More samples and further studies are needed to elucidate the different NET formation and clinical courses among various fungal species.

In conclusion, this study provided in vivo data on the release of NETs during fungal keratitis. We presumed that neutrophils first performed phagocytosis of the fungi and then released NETs to defend against fungal infection. The number of NETs may be used to evaluate the prognosis for infectious keratitis. Further studies to analyze other functional roles of NETs in infectious keratitis are warranted.