Background
Malaria is an infectious disease that is caused by the genus
Plasmodium sp. transmitted through the bite of Anopheles mosquitoes that are infected with protozoan parasites and is a major public health problem 40 % or more of the global population is at risk for malaria [
1]. The pathogenesis of malaria is multi-factorial, with both host and
Plasmodium sp. factors playing critical roles [
2,
3]. Nevertheless, the mechanisms responsible for the high morbidity and mortality of severe cases of malaria remain poorly understood. In endemic areas, many infections in semi-immune population present as an uncomplicated febrile illness. In more severe cases, non-immune individuals may exhibit a number of syndromes including severe anaemia (SA), cerebral malaria (CM) or respiratory distress syndrome [
4,
5].
Lung involvement in malaria has been described most often in non-immune individuals, with infection by
Plasmodium falciparum [
6‐
9],
Plasmodium ovale [
7,
10,
11],
Plasmodium vivax and
Plasmodium malariae [
6,
7,
12‐
16]. While the alveoli and airways can also be involved in mild infection [
17], acute alveolar injury and acute respiratory distress syndrome (ARDS) are major sequelae of severe malaria and have significant morbidity and mortality [
3,
17‐
19]. Malaria-associated ARDS (MA-ARDS) has been reported in infection with all human malarial parasites, although the greatest number of cases is caused by
P. falciparum and
P. vivax [
17,
20,
21]. Patients with alveolar involvement in malaria have classically been reported to have pulmonary oedema, with recent recognition that the alveolar oedema is due to increased pulmonary capillary permeability [
21‐
24]. In addition to altered alveolar-capillary barrier function with leak of protein-rich oedema fluid [
3,
22‐
24], which is a cardinal manifestation of acute alveolar inflammation, alveolar involvement in human malaria has other significant inflammatory components, including leukocyte accumulation [
17,
21,
25‐
29]. Alveolar inflammation is also a fundamental feature of ARDS induced by bacterial sepsis, infectious pneumonia, aspiration of gastric contents, major trauma, and other common ‘triggers’ [
30]. A difference is that, in these more common etiologies of ARDS, alveolar injury is thought to be primarily caused by neutrophil- and platelet-dependent damage to endothelial and epithelial barriers of the alveolar-capillary membrane [
30,
31], whereas in MA-ARDS monocytes and macrophages dominate in the inflammatory infiltrate [
21,
25‐
29]. MA-ARDS has been modelled in studies utilizing mice and other animals, yielding mechanistic insights and experimental correlates [
21,
32‐
37]. There is evidence that lung injury in the murine model
Plasmodium berghei ANKA strain [
33,
34,
37,
38] is associated with intravascular sequestration of parasitized red blood cells [
33,
39], suggesting that it is a useful surrogate for human malarial disease [
26,
40].
Integrins are cell surface heterodimers formed by non-covalent association of α and β polypeptide chains (sub-units). Integrins are widely expressed on mammalian cells and have multiple activities in cellular adhesion, migration, signalling, and fate [
41,
42]. A sub-family of integrins, termed the β
2, CD18, or leukocyte integrins, share a common β
2 sub-unit and are expressed on circulating and tissue leukocytes [
43,
44]. Four α chains pair with the β
2 peptide sub-unit to yield four leukocyte-restricted integrins: α
Lβ
2 (CD11a/CD18, LFA-1), α
Mβ
2 (CD11b/CD18, MAC-1, CR3), α
Xβ
2 (CD11c/CD18), and α
Dβ
2 (CD11d/CD18) [
43,
44]. Leukocyte β
2 integrins are required for host defence against many pathogens and for tissue surveillance and repair, as demonstrated by deficiency syndromes that cause recurrent infections and impaired wound healing in humans and animals [
44,
45]. In contrast, however, β
2 integrin-mediated activities of leukocytes also contribute to tissue injury in a variety of inflammatory syndromes [
46].
Integrin α
Dβ
2, the most recently identified β
2 integrin, is expressed on human and murine leukocytes, although its basal expression is different in man and mouse [
47‐
51]. Integrin α
Dβ
2 is expressed on tissue leukocytes in human inflammatory syndromes, including atherosclerosis [
48], arthritis [
52], and ARDS [
51]. In rodents, there is evidence that α
Dβ
2 can be induced on macrophages or monocytes in the spleen and liver [
50], lung [
53], and blood [
54] in response to inflammatory challenge, and that α
Dβ
2 contributes to inflammatory tissue damage in experimental spinal cord and brain injury [
55‐
57]. Previously, Miyazaki et al. [
50] found that genetic deletion of α
D in mice, leading to deficiency of α
Dβ
2, alters survival and systemic cytokine levels in mice infected with
P. berghei without altering parasitaemia or anaemia. α
Dβ
2 influences the pathogenesis of experimental cerebral malaria in
P. berghei infection (unpublished studies). In this work was examined α
Dβ
2 in lung involvement in
P. berghei -infected animals and found that it influences key features of acute lung injury in this model of experimental MA-ARDS.
Discussion
Complicated malaria is a major challenge in management of malarial infections, which are dominant global public health problems [
3]. Pulmonary complications are among the most serious and potentially lethal consequences of malaria [
18,
19,
22], and it is clear that they occur in human malarial infections caused by parasite species in addition to
P. falciparum, including
P. vivax and
Plasmodium knowlesi [
17,
20,
21,
26,
28,
29]. MA-ARDS is the most fulminant syndrome of lung involvement in malaria [
18,
19,
21] and, like ARDS induced by other infectious and non-infectious causes [
30,
31,
68], is characterized by generation of pro-inflammatory cytokines, acute alveolar inflammation, disrupted alveolar capillary membrane barrier function, and increased permeability pulmonary oedema [
19,
21]. Alveolar involvement in MA-ARDS may be due in part to organ-specific, local intravascular inflammation and unique events such as release of toxins from parasitized RBCs sequestered in the lung [
37,
69,
70]. Mouse models have the potential to reveal key mechanistic features and common and divergent organ-specific responses in MA-ARDS, cerebral malaria, and other complicated malarial syndromes [
21,
40,
69]. The present study provides additional evidence that acute alveolar injury in the
Plasmodium berghei ANKA model of MA-ARDS has features similar to those in humans with clinical MA-ARDS [
19,
21], including increased permeability pulmonary oedema, vascular and interstitial inflammation with accumulation of alveolar monocytes and macrophages, focal parenchymal haemorrhages, and pulmonary generation of cytokines. In addition, AHR and obstruction are elements of lung involvement in the
P. berghei model, as they are in some patients with complicated and uncomplicated clinical malaria [
6,
17,
19]. Moreover,
P. berghei infection alters respiratory system elastic components, consistent with alveolar and airway inflammation. It is important to note that changes in PV relationships are late responses to progressive pulmonary involvement, and with a more extended time course this tends to worsen. Together, these measurements indicate that
P. berghei infection induces substantial alterations in physiologic lung and airway function that accompanies lung inflammation and oedema. Finally, this model was used to examine regulation of key events in
P. berghei-induced lung inflammation by an important leukocyte integrin, α
Dβ
2.
β
2 integrins have critical activities in leukocyte biology, including regulation of adhesion, targeting and accumulation in infected or injured tissue sites, apoptosis, activation and inflammatory signalling, and immune interactions [
42,
44,
45,
71]. Thus, β
2 integrins are members of a complex fabric of effector molecules that regulate leukocyte participation in infectious, inflammatory and immune host responses. Integrin α
Dβ
2 is the most recently identified β
2 integrin [
44,
48], and its specific contributions to infectious and inflammatory pathologies are largely unexplored. Experiments in this study indicate that it has major effector activities in experimental MA-ARDS induced by
P. berghei. Expression of transcripts coding for the α
D subunit were increased in the lungs of mice infected with
P. berghei. Increased
α
D
transcripts may have been due to accumulation of
α
D
-expressing monocytes or other
α
D
-expressing leukocytes from the blood [
54], induction of
α
D
expression in resident alveolar macrophages and other lung myeloid leukocytes [
50,
53], or both mechanisms. While this issue remains to be resolved, increased expression of
α
D
in the lungs of animals with experimental MA-ARDS implies upregulation of α
Dβ
2 and that α
Dβ
2 on lung leukocytes has important activities in this condition. Of interest, α
Dβ
2 is increased on the surfaces of leukocytes in the lungs of human subjects who died of ARDS triggered by other causes [
51].
This study used mice with genetic deletion of
α
D
and consequent absence of α
Dβ
2 [
50] to examine its contributions to the pathophysiology of
P. berghei-induced MA-ARDS. Specific blocking antibodies against the α
D subunit or α
Dβ
2 are not commercially or generally available, making a genetic approach essential to target α
Dβ
2 while leaving other leukocyte integrins intact [
42,
44]. This study shows that key determinants of acute lung injury were improved in α
D
−/−
animals at 7 days after infection. The seven-day time point was chosen because, in previous study of severe malaria caused by
P. berghei, survival curves for wild type and α
D
−/−
mice began to diverge at 7 days [
50]. In the current experiments, lung leukocyte accumulation was reduced in α
D
−/−
at 7 days. This finding is consistent with published evidence that α
Dβ
2 mediates leukocyte accumulation and monocyte migration in tissue inflammation and injury in vivo [
55,
57,
72], and that it mediates macrophage adhesion to tissue ligands [
50] and purified protein targets [
73] in vitro. In parallel, pulmonary vascular leak, evaluated by Evans Blue Dye extravasation [
21,
62,
74], and increased permeability alveolar oedema, assayed by BALF protein concentration and lung weight [
21,
62], were ameliorated in α
Dβ
2-deficient animals. Thus, key determinants of the pathology and pathophysiology of acute alveolar injury in experimental and clinical ARDS [
30,
62,
68] were improved in α
D
−/−
animals in comparison to assessment of these variables in wild type mice. Furthermore, there was a similar pattern of improvement in airway function in α
Dβ
2-deficient compared to wild type mice. AHR has not previously been examined in rodent models of malaria-induced pulmonary involvement. While airway hyperreactivity is not a prominent feature of ARDS triggered by sepsis and other common causes of ARDS [
31,
68], cough, other symptoms of airway obstruction, and physiologic evidence for small airway narrowing and reactivity have been detected in patients with malaria [
6,
7,
17,
19]. These findings suggest that airway dysfunction may contribute to the pathophysiology of MA-ARDS. Improvement in airway function in α
D
−/−
mice indicates that airway inflammation is a mechanism of airway dysfunction in the
P. berghei model. A trend in improvement was found in PV relationships in α
D
−/−
mice, although this was not statistically significant.
Chemokine and cytokine levels were also reduced in the lungs of infected α
D
−/−
mice compared to the levels in pulmonary tissue from wild type mice with MA-ARDS. This may be a key mechanism in improved alveolar inflammation, reduced alveolar-capillary leak, and reduction in alveolar oedema in α
Dβ
2-deficient animals. Pro-inflammatory cytokines, including IL-1β, TNF, IL-8/KC, and others, are synthesized by alveolar macrophages and monocytes and are thought to drive alveolar inflammation and injury in experimental and clinical ARDS [
30,
31,
68]. Cytokine imbalance is proposed to be a feature of the pathophysiology of clinical MA-ARDS [
19]. In the studies reported here, intrapulmonary IL-1β, TNF, and KC were substantially reduced in infected α
D
−/−
mice. Similarly, IL-12 and RANTES, which have pleiotropic activities in inflammation, were also reduced, as was MCP-1, which recruits monocytes to the lungs [
75,
76]. Thus, reduced pro-inflammatory cytokines in infected α
D
−/−
mice may in part account for reductions in the vascular and interstitial inflammation in these animals. Reductions in key cytokines may also account for improvement in alveolar-capillary barrier integrity and reduced leak of protein and fluid into the alveolar spaces of α
Dβ
2-deficient mice compared to wild type mice with MA-ARDS. IL-1β and TNF are major agonists for endothelial VE cadherin internalization and endothelial barrier disruption in models of inflammatory injury, including experimental ARDS [
77‐
79]. TNF and IL-1β were reduced to baseline levels in the lungs of infected α
D
−/−
mice, potentially contributing to improved barrier function. Of note, a reduced systemic cytokine levels was also found in α
Dβ
2-deficient mice infected with
P. berghei in earlier analysis of this model [
50]. The molecular events that mediate altered chemokine and cytokine levels in α
Dβ
2-deficient mice are not yet completely defined. Nevertheless, in studies with human monocytes were found that engagement of α
Dβ
2 with activating antibodies or specific ligands induces outside-in signaling to chemokine and cytokine synthetic pathways [
51]. These evolving observations indicate that α
Dβ
2, like other β
2 integrins [
80,
81], is a key regulator of chemokine and cytokine gene expression in myeloid leukocytes.
Conversely, the release of cytokines, leads to cell activation and increased expression of adhesion molecules such as integrins and immunoglobulins superfamily members [
82‐
84]. Several studies show the role of integrins in development pulmonary oedema, including beta integrins [
85]. The main functions of CD11/CD18 integrins are adhesion and migration [
86,
87], and previous studies showed that VCAM-1 is an important adhesion receptor in models of experimental [
88] and human malaria [
89‐
91] and is a potential ligand for leukocytes and PRBC, in cytoadherence processes that lead to obstruction of the microcirculation. This work demonstrate that the absence of CD11d integrin does not interfere in VCAM-1 expression, and suggest that the effects described in this work are due to an increased expression of CD11d integrin and not by a lack of its ligand. This study with VCAM-1 suggests increased endothelial activation during
P. berghei infection, however, more experiments are necessary to better characterize this activation.
Authors’ contributions
IGAQ and AVA designed and performed the experiments, discussed the results, analysed the data, and wrote the manuscript. ACF, DON, AMS, TPTF, TMG, and GMR designed and performed the experiments, discussed the results and analysed the data. RAC and ARC discussed the results and analysed the data. IGAQ, AVA, GAZ, and HCCFN conceived and designed the study, discussed the results and wrote the manuscript. PTB, PMRS and GAZ reviewed the manuscript. AVA, PMRS, PTB, GAZ, and HCCFN funded this work. All authors read and approved the final manuscript.