Altered distribution of NK cell subsets in children with Mycoplasma pneumoniae pneumonia

Background : Mycoplasma pneumoniae pneumonia (MPP) is a common respiratory illness in children, particularly those with allergic constitutions. NK cells and cytokines are thought to be involved; however, understanding of the immunopathology of MPP is incomplete. Methods : Peripheral blood samples were collected from 51 children hospitalized with with MPP, 26 with an allergic constitution and 25 without, and 29 healthy controls. NK cell subsets were analyzed by flow cytometry and the expression of interleukin (IL)-1 alpha and IL-18 was detected by ELISA. The relationship between NK cell subsets and the expression of IL-18 and IL-1 alpha was determined. Results : The number of CD3 − CD56 + NK cells and CD3 − CD56 dim CD16 bright NK cells in children with MPP was lower than in healthy controls (P < 0.05). The percentage of CD3 − CD56 + NK cells, CD3 − CD56 dim CD16 bright NK cells and the number of CD3 − CD56 dim CD16 bright NK cells in the MPP allergic group were lower than in the non-allergic group (P < 0.05). The expression of IL-18 was significantly increased in the MPP groups (P < 0.05), and the absolute number of CD3 − CD56 dim CD16 bright NK cells negatively correlated with IL-18 levels in the peripheral blood (P < 0.05). Conclusion : Reduced numbers of NK cell subsets were identified in children with MPP and MPP with an allergic predisposition compared with healthy controls. Concomitant increases in IL-18 in children with MPP suggest the


Background
Mycoplasma pneumoniae (MP) is a common pathogen associated with community-acquired pneumonia in children. It can cause wheezing, asthma attacks, and aggravation of pneumonia in children [1]. An allergic constitution refers to the abnormal increase of antigen-specific IgE and allergic inflammation of target organs such as the respiratory tract, gastrointestinal tract or skin after exposure to allergens. It is a complex immune disorder characterized by the infiltration of eosinophils and the production of cytokines due to the differentiation of Th2 cells. MP infection is an important factor leading to Th2 cell inflammatory responses in children with allergic constitutions, and induces airway hyper-responsiveness [2]. NK cells are an integral part of the innate immune system and can act as a bridge connecting innate and acquired immunity [3]. NK cells are divided into CD56 dim CD16 bright and CD56 bright CD16 dim subsets. In this study, we analyzed the distribution of NK cell subsets and cytokine levels in the blood of children with Mycoplasma pneumoniae pneumonia (MPP) and MPP with an allergic constitution to further understand the immunopathogenesis of MPP. MPP standard [4,5]: 1) pneumonia standard: respiratory symptoms, with or without fever, X-ray showed substantial lung infiltration; 2) Mycoplasma pneumoniae infection standard: nasopharynx aspirate (NPA) or bronchoalveolar lavage fluid (BALF) in MP-DNA-PCR. >1.0×10 3 copies/ml; single serum MP-IgM (> 1.1S/CO), double serum IgG and/or IgM increased 4 times or more than the first (7-14 days apart) and were diagnosed as acute Mycoplasma infection.

Objects of study
Any of the following criteria was defined as an allergic constitution [6]: 1) Clinical diagnosis of asthma, eczema, allergic rhinitis or atopic dermatitis; 2) History of drug or food allergy; 3) Blood allergen test or skin prick test had more than two positive allergens. Exclusion criteria: No asthma, eczema, allergic rhinitis or atopic dermatitis, no history of drug or food allergy, blood allergen test or skin prick test were negative.

Experimental methods
Within 2 hours of admission, 4 ml of venous blood was collected and 2 ml of whole blood was immediately analyzed by flow cytometry. Whole blood (2 ml) was collected in an anticoagulant tube and centrifuged for 5 minutes at 3500 rpm. The plasma obtained was stored at −80°C. 1) The percentage and absolute value of CD3 − CD56 + NK cells and CD3 − CD56 dim CD16 bright NK cells and CD3 − CD56 bright CD16 dim NK cells in the lymphocyte population were detected by flow cytometry; 2) The plasma cytokines interleukin (IL)-18 and IL-1α were measured by ELISA.

Statistical methods
Statistical data were analyzed using SPSS 21.0 software package. Normal distribution measurements were expressed as mean±standard deviation ( x ±s). T-tests were used for comparisons between groups. The measurement data of non-normal distribution were expressed as median (quartile spacing). Rank sum tests were used for comparison between the two groups. The counting data were expressed by ratio, and the comparison between groups was performed by Chi-square test or Fisher's exact probability method. The difference was deemed statistically significant when P < 0.05.

Patient information
The 51 children with MPP included 25 males and 26 females and the 29 healthy controls comprised 16 males and 13 females. There was no significant difference between the two groups. The average age of the children in the MPP group was 3.34±2.28 years, and the healthy control group was 3.29±2.17 years. There was no significant difference in age between the two groups (P > 0.05). See Table 1 Among the children with MPP, there were 26 cases in the allergic group, 13 males and 13 females, and 25 cases in the non-allergic group, 12 males and 13 females. There was no significant difference between the two groups. The average age of the allergic group was 3.26±2.19 and the non-allergic group was 3.43±2.40. There was no significant difference in age between the two groups (P > 0.05). See Table 1.

Distribution of NK cell subsets in the peripheral blood
Flow cytometry was used to detect NK cells and differentiate the subsets in the peripheral blood. The basic morphological differences of different cell groups were distinguished by scattering light signals, and then the lymphocyte groups were selected (Fig. 1A). NK cells were identified using basic morphological information, followed by positive expression of CD45, the common leukocyte antigen, and negative expression of CD3 (Fig. 1B) . In Fig. 1C, NK cell subsets were differentiated based on the expression of CD56 and CD16.

Distribution of NK cell subsets in peripheral blood of children with Mycoplasma pneumoniae pneumonia and allergic constitutions
The percentage of CD3 − CD56 dim CD16 bright NK cells from the peripheral blood of patients in the allergic group was 7.37±4.36%, compared with the non-allergic group 11.51±7.06%. The difference was statistically significant (t=2.527, P < 0.05). The  Table 3 and

Levels of IL-18 and IL-1α in the peripheral blood of children with Mycoplasma Pneumoniae pneumonia and allergic constitutions
The mean value of IL-18 was 118.95 pg/ml in the children with allergic constitution compared with 113.37 pg/ml in the children with non-allergic constitution.
IL-1α levels were 3.57 pg/ml in patients of an allergic constitution and 3.45 pg/ml in patients without an allergic constitution. There was no significant difference between the two groups (P > 0.05; Table 5).

Correlation analysis of NK cell subsets with IL-18 and IL-1α
The absolute value of CD3 − CD56 dim CD16 bright NK cells in peripheral blood of children with MPP was negatively correlated with the level of IL-18 (r=−0.892, P=0.000). The number of other NK cell subsets was not significantly correlated with the levels of IL-18 or IL-1α. The experimental results are shown in Figure 5.

Discussion
MPP is a common respiratory illness in children, particularly those with allergic constitutions; however, the immunopathogenesis of MPP is unclear. Animal models of MP infection show that the degree of lung injury caused by MP is related to the host immune response, not necessarily direct microbial damage [7]. MP infection can cause chronic lower respiratory tract inflammation by impairing mucociliary clearance and increasing the production of pulmonary mucus [8]. It can also induce the release of pro-inflammatory cytokines associated with chronic pulmonary diseases such as asthma attacks [9]. suggesting that children with allergic MPP have a higher risk of asthma [11].
NK cells have been studied in animal models of MP infection and MPP patients.

NK cells infected with MP can directly inhibit MP colonization in vivo and remove
MP from lungs after intravenous infusion [12]. MP infection can also cause serious immune damage and inflammatory cascades, leading to MPP-associated hemophagocytic syndrome (MPP-HPS) [13]. It is thought that the main pathogenesis of MPP-HPS is a decrease or complete lack of NK cell activity, which prevents the elimination of MP in a timely manner. Continuous stimulation leads to the proliferation of lymphocytes and histiocytes and the release of large quantities of cytokines; a cytokine storm, resulting in multiple organ hyperinflammation and tissue damage [14]. Early recruitment of NK cells is crucial to limiting MP infection. They release large amounts of IFN-γ and granulocyte-macrophage colony stimulating factor (GM-CSF), leading to macrophage activation [15].  [24]. The IL-1 cytokine superfamily is composed of 11 members, the most studied are IL-1α, IL-1β and IL-18 [25]. IL-18 is a pro-inflammatory cytokine, mainly produced by goblet cells, and monocytes/macrophages. It has many biological effects, including enhancing Th1 immune responses, inducing T cells and NK cells to produce IFN-γ [23], and promoting NK cell lysis.
The immunological pathogenesis of MPP involves many cytokines [26], and our results suggest that IL-18 is involved. IL-18 has many diverse functions including the induction of GM-CSF and up-regulation of intercellular adhesion molecule-1 in monocytes. IL-18 can also induce delayed-type hypersensitivity (DTH) in vivo. DTH responses to MP may be related to the severity of pneumonia, suggesting that IL-18 could directly affect the respiratory immune response induced by MP, IL-18 can also induce activated T cell proliferation and NK cell activation [27]. In this study, the expression of IL-18 and IL-1α in peripheral blood was significantly higher in the MPP patients than in the healthy controls, consistent with a previous report [28]. We found that the serum level of IL-18 was negatively correlated with the absolute value of

Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Review Board of Children's Hospital of Soochow University (project approval number 2012LW002) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Consent was provided by the parents or legal representatives of patients aged < 16 years and informed consent forms were archived.

Consent for publication
All authors have read and approved the content, and they agree to submit it for consideration for publication in the journal.