M1 macrophage activation in severe Plasmodium falciparum malaria patients with pulmonary oedema

Pulmonary oedema (PE) is a serious complication of severe P. falciparum malaria which can lead to acute lung injury in severe cases. Lung macrophages are activated during malaria infection due to a complex host-immune response. The molecular basis for macrophage polarisation is still unclear but understanding the predominant subtypes could lead to new therapeutic strategies where the diseases present with lung involvement. The present study was designed to study the polarisation of lung macrophages, as M1 or M2 macrophages, in the lungs of severe P. falciparum malaria patients with and without evidence of PE. Lung tissue samples, taken from patients who died from severe P. falciparum malaria, were categorised into severe malaria with PE and without PE (non-PE). Expression of surface markers (CD68- all macrophages, CD40- M1 macrophage and CD163- M2 macrophage) on activated lung macrophages was used to quantify M1/M2 macrophage subtypes. The study polarisation lung

characterisation in malaria infection may provide new insights into therapeutic approaches that could be deployed to reduce lung damage in severe P. falciparum malaria.

Background
Pulmonary oedema (PE) is one of the major complications and therapeutic challenges in severe P. falciparum malaria. This condition is associated with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [1]. PE occurs in approximately 21% of cerebral malaria patients and 40% of these develop into ARDS [2]. Furthermore, substantial fatality (70%) is observed in malaria patients with PE who subsequently progress to develop ARDS [2,3]. In P. falciparum malaria, PE is associated with inflammatory infiltrates consisting of mainly mononuclear cells, hemozoin deposit, the accumulation of macrophages, as well as parasite sequestration [4]. Recruitment of macrophages to the lung signifies an important immune response in the pathogenesis of ALI/ARDS [5]. Activated macrophages have been described as two functional subsets, namely classically activated macrophages (M1) and alternatively activated macrophages (M2) [6,7]. M1 macrophages are considered as pro-inflammatory macrophages that produce pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin-6 (IL-6) and other mediators. M2 macrophages are typically anti-inflammatory macrophages and produce anti-inflammatory cytokines, such as IL-10 and secrete growth factor such as transforming growth factor beta 1 (TGF-β1) for tissue repair [7].
The polarisation of M1 and M2 macrophages are important for disease regulation.
Previous studies have documented that activated macrophages are prominent in lung infection with bacteria [8] and viruses [9][10][11] as well as in lungs from smokers and chronic obstructive pulmonary disease (COPD) [12]. The main purpose of this study was to investigate the status of lung macrophages in severe P. falciparum malaria patients with and without PE. The outcome of the work highlighted the role of macrophages on PE/ALI and serve as a basis for drug adjuvant therapy for lung complications in severe P. falciparum malaria.

Tissue specimens
Embedded human lung tissues from severe P. falciparum malaria infected patients and non-infected controls were retrieved from the Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. Lung tissues were embedded and prepared for histopathological evaluation. Based on histopathological findings of oedematous fluid in the lung, lung tissues were divided into severe P. falciparum malaria with PE, non-PE and control lung tissues. Normal control lung tissues were obtained from patients who died from accidents, and showed no pathological changes in the lungs. The study protocol was reviewed and ethical clearance was obtained from the Ethics Committee of Faculty of Tropical Medicine, Mahidol University (MUTM 2017-054-01).

Histopathology and evaluation
Lung tissues were re-embedded with new paraffin medium, sectioned at 4 μm in thickness and routinely stained with haematoxylin and eosin (H&E). The pathological changes of lung tissues were interpreted based on eight histological criteria in twenty low power fields (LPF) (200X) per slide, namely septal congestion, alveolar haemorrhage, alveolar oedema, hyaline membrane formation, parasitised red blood cell (PRBC) sequestration, malarial pigment, lung macrophages and infiltration of inflammatory cells [13]. Each variable was graded on a scale based on percentage of severity based on a previous study with modifications, as follows: no injury = 0, injury £ 25% /LPF = 1, injury > 25% and £ 50%/ LPF = 2, injury > 50% and £ 75%/ LPF = 3, and injury > 75% LPF = 4 [14]. Lung macrophages and white blood cells (WBC) were determined using a standard Z-score from the mean of cell count/high power field (HPF): -1.5 SD = 0, -0.5 SD = 1, mean = 2, +0.5 SD = 3 and +1.5 SD = 4. Subsequently, a lung injury score ranging from 0 to 32 points was calculated by adding the sum of each variable to determine the overall histopathological changes in lung tissues from malaria patients with P. falciparum. A score of 0 means the absence of histopathological changes while a score of 32 signified the most severe histopathological changes. All histopathological parameters were examined in a blinded manner, without prior knowledge of the patients' clinical status.

Immunohistochemical evaluation of lung macrophages
The expressions of macrophage surface markers (CD68, CD40, and CD163) were detected by immunohistochemical staining [15,16]. Lung sections of 4 μm thickness were placed on adhesive slides coated with poly-L-lysine and de-paraffinised through a series of xylene and re-hydrated through graded alcohol solutions. Mayer's haematoxylin, mounted with a coverslip and evaluated under a light microscope. Spleen was used as a positive control for CD68, CD40 and CD163 [17].
The anti-CD68 within the tissue sections were used as positive internal controls.
Positive cells stained brown pattern with different degrees of immunoreactive intensity.

Evaluation of immunohistochemistry staining
Quantitative assessments of macrophage subtypes were based on the number of positive stained cells for CD68, CD40 and CD163. Macrophages in lung tissues were evaluated randomly in twenty fields per slide, in both the septal area and within the alveoli under high magnification (400X). The numbers of surface markers specific for each macrophage subtype were counted and expressed as percentage of positive cells. The intensity of positive stained cells was graded as follows: 0 = negative; 1 = weak staining; 2 = moderate staining; 3 = strong staining. The total score (TS) for this immunohistochemical study was determined by obtaining the product of the percentage of positive cells and the intensity of the staining [18,19].
The sections were examined in a blinded manner, without prior knowledge of the patients, clinical status.

Statistical analysis
All quantitative data were expressed as means ± standard error of the mean (SEM).
Statistical analysis was performed using SPSS version 18.0 software (SPSS, IL, USA).
Data was tested with Kolmogorov-Smirnov test for normality of distribution.
Difference of M1/M2 expression between groups was analysed by the Mann-Whitney U test. The correlations between M1/M2 expression and histological/clinical data were analysed by Spearman's correlation. Statistical differences at p < 0.05 were considered to be statistically significant.

Summary of clinical and laboratory data from malaria patients
Malarial lung tissues were obtained from 17 patients, 9 cases with PE and 8 cases with non-PE. Six normal lung tissues samples were used as control. No difference in clinical complications of severe malaria (ie. cerebral malaria, acidosis, hypoglycaemia, shock, disseminated intravascular coagulopathy) was observed between the two groups, except for the presence of acute kidney injury (p = 0.03).
The demographic data from the severe P. falciparum malaria patients is documented in Table 1. There was a significant difference in the patient haematocrit between non-PE and PE (p = 0.04). No significant difference in age, blood urea nitrogen (BUN), creatinine, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, globulin, total bilirubin, direct bilirubin, WBC counts, and parasitaemia (all p > 0.05) was observed.

Histopathological changes in the lungs of P. falciparum malaria patients
Common histopathological changes in the lungs of severe P. falciparum malaria included the presence of septal congestion and alveolar haemorrhage (Fig. 1A), alveolar oedema (Fig. 1B), hyaline membrane formation (Fig. 1C), PRBC sequestration in pulmonary capillaries (Fig. 1D), malarial pigment and an increase in the number of macrophages in alveolar spaces and septal area (interstitial area) ( Fig. 1E) (all p < 0.05, compared to control lung, Fig. 1F). Mixed inflammatory cells are occasionally seen within the alveoli. Comparing non-PE and PE groups, significant difference in histological findings were noted in the presence of alveolar haemorrhage, accumulation of malarial pigment and the number of lung macrophages (all p < 0.05). The calculated lung injury score (based on histopathological criteria) was significantly higher in the PE group (19.22±0.90), compared to non-PE group (12.13±0.09, p = 0.001) (Fig. 2).

Expression of CD68
CD68 expressing cells were detected as fine brown color in the cytoplasm of lung macrophages in both the septal area and within the alveoli (Fig 3, Panel A)

Expression of CD40
CD40 was used to detect activated M1 subtype lung macrophages. Positive cells expressed a fine brown color on the cell membrane of macrophages. The expression of CD40 was prominent in the group of severe P. falciparum malaria with PE (97.88±1.71%) compared to non-PE (72.81±7.17%, p = 0.001) and the control group (19.83±11.81%, p = 0.003) (Fig. 3, Panel B). Macrophages expressing CD40 in the alveolar septal area and within the alveoli show similar trends between PE and non-PE groups, as depicted in Fig. 4A.

Expression CD163
CD163 was used as a marker for the M2 subtype. Positive cells express a fine brown color in the cytoplasm of macrophages. Lung macrophages in severe P. falciparum malaria patients with PE and non-PE show an increase in CD163 expression, compared to the control group (p < 0.01). No significant difference in the mean percentage of CD163 positive cells was observed between PE and non-PE groups ( Fig. 3, Panel C, Fig. 4B). PE is a common finding in ALI and can cause the lungs to develop into acute respiratory distress syndrome (ARDS) [20]. The activation of lung macrophages and release of cytokines are important mechanisms contributing to lung damage in patients with ARDS [15,21]. Under various triggers, lung macrophages can be polarised into either classically activated (or M1) or alternatively activated (or M2) subtypes [22]. M1 macrophages can be stimulated by interferon (IFN) phenotype to the anti-inflammatory M2 phenotype. The switching of macrophage polarity has been associated with transcriptional control [3]. M2 macrophages have an important role in lung tissue repair by limiting the levels of proinflammatory cytokines in the cellular space and producing anti-inflammatory cytokines such as IL-10 and IL-1 receptor antagonist [5]. M2 macrophages also remove necrotic cells and debris through phagocytic activity, which can be demonstrated in the malaria lung tissues as lung macrophages engulf hemozoin and fragmented PRBCs.

Correlations between M1/M2 surface markers and histopathological changes
Pulmonary macrophages are important factor in causing lung pathology in malaria.
The study showed M1 macrophage activation in ALI in severe P. falciparum malaria, which indicates disease severity. As M1 polarisation prevails, more lung damage can occur. Further investigation on the pathways of the malaria-induced M1 phenotype is necessary to idenitfy specific activators for M1 polarisation that could be attenuated and factors that promote the anti-inflammatory M2 phenotype, in order to suppress the inflammation and improve clinical ALI in severe malaria patients.   Figure 1 Histopathological changes of lungs in P. falciparum malaria: A-alveolar congestion and haem Lung injury score in malaria patients. A significant difference was observed between PE and The immunohistochemical staining of lung macrophages in P. falciparum malaria. Expression Positive correlations between M1/M2 surface markers and histopathological changes (A-F)