Pollution of mycological surfaces in hospital emergency departments correlates positively with blood NKT CD3⁺ 16⁺ 56⁺ and negatively with CD4⁺ cell levels of their staff

Introduction

This is the second part of our studies on the immunological status of medical emergency workers exposed for many years to hazardous biological agents and on the immune system of their corresponding controls. Previously, we reported the existence of a negative correlation between the number of phagocytising monocytes in the blood of hospital emergency departments (HED) staff and the number of fungi (colony-forming units – CFU) collected by imprinting method from the surfaces. Surprisingly, the similar study performed in control offices and their staff revealed positive correlation between above parameters [1]. It was difficult to explain this discrepancy. Persons from both examined groups had been working for at least five years at the present location and, at least at the moment of comparative sampling, mycological pollution of surfaces was similar and generally low. Moreover, the number of phagocytising monocytes in the blood of personnel did not differ between HED and office locations. However, the obtained results might suggest the existence of some intrinsic disturbances in innate defence cell subsets.
In this paper we present the results of the examination of natural killer (NK) cell CD4⁺ and natural killer T lymphocyte (NKT) levels in the blood of these HED workers and their corresponding controls from control offices. NK and NKT lymphocytes belong to the group of cells participating in the mechanisms of innate cell-mediated immunity, together with granulocytes, monocytes, macrophages, and dendritic cells. Cells from this group recognise microbial patterns without prior sensitization and react rapidly to the pathogen. Natural killer cells are cytotoxic against neoplastic and pathogen-infected host cells [2]. In response to stimuli, they produce interferon-(IFN-) and tumor necrosis factor  (TNF-) and also act directly against some extracellular pathogens.
Natural killer T cells are innate-like T lymphocytes restricted by the CD1d molecule that presents self and exogenous glycolipids. NKT after activation can immediately secrete a high amount of IFN- and other cytokines and are able to activate NK cells, T cells, B cells, and dendritic cells (DCs). They are possibly responsible for induction or activation of innate as well as adaptive immunity and play an important role in protecting the host against infectious pathogens [2-4].

Material and methods

The study of mycological contamination was conducted as previously described [1]. Briefly, the samples were collected from 10 Warsaw hospital emergency departments (HED) and in 10 control locations (office spaces), and included imprints of floors and walls. 90 samples were collected from 10 hospitals, and 90 samples from 10 control offices. In the imprinted method a special applicator with media (Count Tack CT SI – Biomerieux) was dedicated for fungi. These samples were taken from the flat surfaces of walls and floors. Next the biological material was put into a plastic container and transported to the microbiological laboratory.
Environmental samples culture and identification of fungi were performed as described previously [1].
Blood for immunological investigation was taken from 40 men, 26 to 53 years old, health care workers of HED departments, who had been working for at least five years in their current positions and from 36 corresponding controls, who worked in control offices, without a history of systemic, inflammatory, or immunological diseases. Evaluation of blood leukocyte subpopulations was done by haematological analyser and cytometry analysis.
Informed consent from study participants and permission from the Local Ethical Committee were obtained.
The total number of white blood cells was measured in EDTA blood samples using a haematological analyser in accordance with the manufacturer’s protocol (Exigo Vet, Boule Medical AB, Sweden). Subsequently, the cytometry assay was performed.
For examination of NK, NKT, and CD4 subsets, samples were labelled with CD3 FITC and CD16/56 PE or CD4 FITC antibodies (IMK Test, BD Biosciences) as previously described [5]. The samples were acquired by flow cytometry (FACS Calibur) and analysed in CellQuest Software (BD Biosciences). The NK, NKT, and CD4⁺ results were presented as percentage of lymphocytes population (R1). Typical analysis of NK/NKT cell populations is shown in Fig. 1.
Statistical evaluation of the results was done by column statistics, Spearman correlation analysis, and Mann-Whitney test (GraphPadPrism).

Results

Fungi examination. The results of fungi quantitative analysis collected by imprint method from walls and floors are presented in Fig. 2 (walls) and Fig. 3 (floors). No statistically significant differences were observed between the values obtained for HED and office surfaces (Table 1 and 2).
The qualitative analysis of the surfaces and air samples revealed a prevalence of genus Aspergillus spp. and Penicillium spp. The most common species were: Aspergillus niger, Aspergillus fumigatus, Penicillium expansum, and Trichoderma koningii. In two samples, one collected from a hospital emergency department and the other from a control location, Aspergillus fumigatus was found. One emergency department was a source of samples containing Aspergillus flavus. Furthermore, various species belonging to the Candida spp. were found in hospital emergency rooms as well as Aspergillus niger.

Immunological study

As was described previously [1], there were no statistical differences between emergency and office workers with respect to age, leukocytes number, and percentage of phagocytising granulocytes and monocytes. Presently, we found lower numbers of NK (CD3–CD16⁺CD56⁺) lymphocytes in the blood of HED workers (median value 11.87) than in the blood of control office staff (median value 15.49) (Fig. 4). No difference was observed between the percentage of NKT (CD3⁺CD16⁺CD56⁺) cells in the blood of HED workers (median value 3.87%) and respective value of control office staff (4.26%).
Spearman correlation analysis revealed significant positive correlation (r = 0.4677; p = 0.002) between the percentage of NKT cells in the blood of HED workers and the number of CFU/25cm2 obtained from the walls and floors of hospital emergency departments (Fig. 5). No correlation was found in the case of control offices (Fig. 6) and their staff (r = –0.0251; p = 0.8841). Negative correlation was found between total number of fungi on surfaces in HED and % of CD4⁺ lymphocytes in the blood of heath care workers (r = –0.3688; p = 0.019, Fig. 7) and no such correlation in office workers (Fig. 8).

Discussion

Disruptions in immune system response may result in severe diseases, i.e. autoimmune diseases, hypersensitivity syndromes, or cancer. Such disruption may be a result of malnutrition [6], drug action [7], or chronic inflammatory conditions caused by microorganisms [8]. It is well known how the human immune system recognises and combats bacterial and fungal infection. However, there is still little information about the long-term effect of fungi exposure on the function and reactivity of the immune system. Pioneering experiments by Mierzwińska et al. revealed impairment of cell-mediated immune responses in patients with fungal prosthetic stomatopathies during long-term oral candidiasis. In these patients eradication of Candida infection resulted in normalisation of immunological cellular responses [9, 10].
Fungi are a heterogeneous group of microorganisms. They are able to colonise both internal and external environments of the human body. The range of immune interactions with fungi is not fully recognised and is multidirectional. More than 400 mould species live in indoor environments [11]. Many of them are pathogenic in humans. Penicillium, Aspergillus, and Candida species secrete proteases disturbing anti-fungal immunity as well as immunotoxic and cancerogenic mycotoxins. In mycoses nonspecific innate and acquired immunity play important roles in host defence. It involves neutrophils, monocytes, macrophages, dendritic and mast cells, as well as lymphocytes [18]. NK and NKT cells play a specific and exceptional role in this process.
NK cells participate in anti-mycotic response by direct or indirect mechanism. NK cells are able to induce apoptosis by Fas ligand or tumour necrosis factor ligands. Moreover, NK cells may release from granule content constitutively expressed proteins such as perforin, granzymes and granulysin. The released proteins can perforate the cell membrane (which leads to water influx and cell lysis) and trigger apoptosis [19]. NK cells may damage fungal cells also by a direct mechanism. They are able to produce several important cytokines, i.e. GM-CSF, RANTES, or IFN-, which enhances migration, adherence phagocytosis of neutrophils and macrophages, maturation of dendritic cells, and proliferation of T-cell [20]. Additionally, NK cells may induce CD4⁺ T-cell response by MHC II class activation [21]. Fungi are able to disturb the response of immune system cells mainly by modulation of the regulatory network. This is connected with mycotoxin activity, which can inhibit phagocytosis and ROS produce in neutrophils, increase apoptosis in monocytes, and attenuate T-cell response [22]. It has been demonstrated that the level of NK cells plays an important role in fungal infection. Fungal mycotoxin may impair NK functionality. It has been presented that mycotic infection (i.e. Aspergillus fumigatus or Candida albicans) downregulates level of INF-, GM-CSF, and TNF- produced by NK cells [20]. In the present work we did not study the functionality of NK cells isolated from HED and office staff;, however to fully understand the effect of long-time exposure on mycotic pollution this study should be made.
Studies pertaining to immune response to fungi in residential houses have shown increases in lymphocyte B and T levels (including CD4) with associated decreases in NK levels in exposed residents [23, 24]. The role of NKT lymphocytes in anti-tumour immune defence is widely known. Except for secretion cytokines characteristic for Th2 in humoral defence or INF- and TNF, they show direct cytolytic activity eliminating cancer target cells [25-27]. In the present work we examined the effect of mycological pollution in work areas on the level of NK cells in the blood of their staff. The study was conducted in 10 Warsaw hospital emergency departments (potentially with increased levels of dangerous pathogens) and in 10 control offices. The analysis of general fungal pollution did not reveal significant differences between colony-forming unit numbers in HED and control offices. These fungi were able to produce mycotoxins floating in the air, which may affect immune response in exposed personnel. Interestingly, despite the similar fungal contamination in control (offices) and study (hospital) environments, we found a positive correlation between the fungal contamination in the study environment (hospital) and NKT levels in the study group (rescue personnel) (Fig. 5), while there was no such correlation in the control group (Fig. 6). It was also shown that levels of NK cells were lower in the study group than in the control group (Fig. 4). We noticed a negative correlation between CD4 lymphocyte levels in hospital personnel and fungal contamination levels but no such relation in the control group (Figs. 7 and 8).
CD4 lymphocytes play a key role in antifungal protection [28]. Differentiation of naive CD4 to Th1 and Th17 strengthen this response. CD4 lymphocytes take an active part in induction of allergic reaction by fungi Helper T lymphocytes are engaged in cell-type reaction to massive and long-term exposures to fungi. Many fungi are intracellular pathogens and trigger cell-type response (Th1-associated). On the other hand, immune response insufficiency with relevant decrease in CD4 count may cause susceptibility to fungal infection, e.g. in AIDS patients. Th1-associated response plays a pivotal role in fungal infection. Th1 lymphocytes secrete INF- that activates phagocytes (neutrophils, macrophages) [29]. One could expect an increase in CD4⁺ count after fungal exposure or at least its positive correlation with CD4⁺. A possible explanation of our findings is that exposure to pathogenic fungi together with other unidentified factors, prolonged for many years, may change this trend. Our observations may be partly dependent on other confounding variable modifying rescuers’ immune response, altering the response to protracted fungal exposure. Taking into consideration the specificity of the life-saver’s job, the authors suspect the influence of chronic stress and fatigue.

The authors declare no conflict of interest.
This article was supported by project number II.P.19 (contract number 42/2014/PW-PB) titled “Identification of biological hazards in rescue operations and their impact on the competence of the immune system in the perspective of health consequences” carried out under the Program titled “Improving safety and working conditions” led by the Central Institute for Labour Protection – National Research Institute.

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