Expression of von Willebrand factor, pulmonary intravascular macrophages, and Toll-like receptors in lungs of septic foals

Foals, especially those with failure of passive transfer of maternal antibodies, are highly susceptible to bacterial infections and endotoxemia; however, the mechanisms underlying such susceptibilities are not fully understood [19, 25]. Non-specific intestinal absorption, hypoglycaemia, omphalophlebitis, unsanitary environmental conditions, and abnormal gestational lengths are common factors that predispose foals to further complications following birth, including septicaemia [16, 20, 25]. Sepsis is a systemic bacterial invasion that commonly results in systemic inflammatory response syndrome, which can lead to numerous sequelae such as coagulopathies and immune system dysfunction [25]. Indeed, septic shock can result in hypotension, organ hypoperfusion, and multiple organ dysfunction syndrome, which may lead to death of foals, especially neonates [19, 20]. Mortality rates of septic neonatal foals generally range from 40 to 55% [25].

The majority of research on human sepsis and its effects on lung inflammation has focused on acute lung injury and/or acute respiratory distress syndrome [4, 30]. Indeed there is very little data available regarding lung injury and inflammation in foals suffering from sepsis [16, 18, 20, 25]. Because of the increased awareness of acute lung injury in veterinary species, a consensus statement was released to establish a veterinary specific definition [29]. The defining criteria for acute lung injury in veterinary patients, including foals, are similar to those used in human medicine and include acute onset, known association with systemic inflammation or sepsis, signs of pulmonary capillary leakage in the absence of pulmonary hypertension, and evidence of inefficient gas exchange or hypoxemia. A fifth optional factor is evidence of diffuse pulmonary inflammation seen on radiographs [29]. While septicaemic foals are commonly diagnosed with pulmonary inflammation, most clinical attention has focused on sequelae other than lung inflammation, such as haemostasis, arthritis, or organ dysfunction involving mainly the liver, spleen, and gastrointestinal tract [19, 25, 26]. Previously, pneumonia was diagnosed either clinically or at post-mortem in 28 to 50% of septic foals [20]. Interestingly, necropsy data have highlighted lung pathologies in septic foals, even when little or no ante-mortem pulmonary signs were observed [18, 25]. Regardless of these findings, precise descriptions and information describing the mechanisms of lung inflammation in septic foals are lacking.

Fortunately, studies of other animal species and humans have provided a general mechanistic framework for systemic inflammatory response syndrome and its effects on the mammalian lung. This dysregulated inflammatory response involves activation of innate immune cells such as macrophages and neutrophils through signalling via various pattern recognition receptors, including toll-like receptors (TLRs), which engage and identify conserved molecular patterns of bacteria and other danger signals [15]. For example, Toll-like receptor (TLR) 4 recognizes lipopolysaccharide, while TLR9 recognizes cytosine guanine phosphodiester DNA patterns, which are most common to gram-negative and gram-positive bacteria, respectively [15]. The recognition of pathogenic material by immune cells leads to the release of pro-inflammatory cytokines and chemokines, which induce adhesion phenotype changes in epithelial and endothelial cells to activate the recruitment of neutrophils [13, 15]. Neutrophils secrete many cytotoxic molecules, such as free oxygen radicals, which cause cellular damage and increase vascular permeability [5]. In addition, circulating endotoxin promotes activation of the coagulation cascade via tissue factor production [7]. In conjunction, there is increased expression of a glycoprotein known as von Willebrand factor (vWF) on endothelial cells and platelets that promotes cell-endothelial adhesion and platelet activation [14]. The acquisition of a pro-coagulant phenotype by the endothelium may eventually lead to disseminated intravascular coagulation and potentially irreversible vascular damage [19]. However, no molecular characterizations of lung inflammation in septic foals have been conducted to date.

This study was conducted to compare lung tissue from septic and healthy control foals using histological and immunohistological techniques. Through this examination, we tested the hypothesis that lung inflammation is present in septic foals.

Although pulmonary pathology is a common sequela to septicaemia in humans and other veterinary species, there is still very little data available regarding the occurrence and characterization of lung inflammation in septic foals. Here, we report the first data to show vascular inflammation, an increase in septal monocytes and macrophages, and increased expression of TLR4 and TLR9 on mononuclear cells in the lungs of septic foals. These data will help improve our understanding of pulmonary complications found in foals experiencing septicaemia.

While the medical records regarding the origin of sepsis in some of the foals were incomplete, the majority of foals had sepsis originating remote from the lung. Therefore, it is likely that foals experience lung inflammation as a sequel to sepsis, similar to humans, as has been previously reported [6]. Based on these findings, foals should be evaluated and treated for pulmonary complications associated with non-pulmonary origin septicaemia.

We observed lung inflammation based on increased migration of cells in the alveolar septa, thickening of the alveolar septa, and through semi-quantitative scoring of the lung sections for MAC387 positive cell infiltration. Lung inflammation is characterized by recruitment of inflammatory cells, such as neutrophils and monocytes, and by increased vascular permeability [23, 27]. The recruitment of cells such as platelets, neutrophils, and monocytes is facilitated through activation of the endothelium by cytokines leading to the expression of adhesion molecules. vWF, an adhesive and coagulation protein, is stored in endothelial cytoplasmic Weibel-Palade bodies, which are exocytosed upon endothelial cell activation [14]. The recruitment of platelets, which is facilitated by vWF endothelial expression and other proteins such as selectins, is considered one of the earliest events in acute inflammation [13]. Here, we report the expression of vWF in alveolar septa in lung sections from foals with septicaemia, where it is normally not expressed. It is generally believed that lung microvascular endothelium lacks Weibel-Palade bodies that store preformed vWF. Therefore, the vWF expression in the alveolar septa could be due to accumulation of vWF-containing platelets, or the release and spread of vWF from the endothelium in larger blood vessels, which normally express this protein, into the alveolar capillaries. The increased expression of vWF and accumulation of septal mononuclear cells may suggest a role of vWF in their recruitment. Nevertheless, the increased expression of vWF provides molecular evidence of microvascular inflammation, including a coagulative phenotype of the endothelium in the lungs of septic foals.

Our immunohistochemical data demonstrated mostly monocytes and macrophages in the alveolar septa of septic foals. We previously used calprotectin-binding antibody, MAC387, to identify equine lung macrophages with light and electron microscopy [17, 21]. MAC387 can also stain neutrophils; however, we eliminated neutrophils from our counts based on their nuclear morphology. Moreover, we found very few neutrophils in the septa of normal or septic foals. The presence of large numbers of septal monocytes and macrophages, but fewer neutrophils, in the lungs of septic foals is intriguing. In animal species such as the mouse, endotoxin-induced lung inflammation results in initial recruitment of neutrophils followed by infiltration of monocytes. The newly arriving monocytes are known to express anti-inflammatory cytokines such as transforming growth factor beta and interleukin-10 to hasten resolution of inflammation [3]. However, there is evidence that newly recruited intravascular monocytes/macrophages increase the risk of pulmonary inflammation in response to secondary endotoxin challenges [8, 9, 24]. Preliminary electron microscopic observations of tissues derived from paraffin sections also suggest there are numerous monocytes and macrophages located in the septal capillaries (B. Singh et al., unpublished data). Interestingly, the normal foals in our study had very few intravascular/septal mononuclear cells that stained positive for MAC387, which is similar to the findings from the day old pig [31]. These findings indicate that, similar to pigs, foals may also recruit pulmonary intravascular mononuclear cells such as PIMs during their post-natal life, which may increase host susceptibility for lung inflammation and injury [1, 17].

Our study does not address the function of newly recruited septal monocytes/macrophages, but we speculate that the newly recruited macrophages are pro-inflammatory. This speculation is based on our data showing expression of TLR4 and TLR9 on septal mononuclear cells, but not on airway epithelium and endothelium in lungs from septic foals. It is also consistent with previous results showing reduced TLR4 staining in airways and large blood vessels during pulmonary inflammation with increased TLR4 expression in mononuclear cells [27]. However, data describing pulmonary monocytes in rats suggest that after exposure to pro-inflammatory endotoxins, TLR4 expression is initially increased, but then decreases significantly 36 hours after exposure [11]. Previous data has shown increased TLR9 expression on both PIMs and epithelial/endothelial surfaces of adult horses treated with LPS [22]. Since sepsis in foals is most often caused by gram-negative bacteria and their associated endotoxins [26], the increased numbers of septal mononuclear cells expressing TLR4 and TLR9 may lead to more pronounced inflammation in response to circulating endotoxins and other bacterial molecules. Therefore, we cannot concretely attribute the increased staining of TLR4/TLR9 to the recruitment of PIMs in the septic foal. However, it is possible that increased expression of vWF may have contributed to the increased numbers of monocytes and macrophages in lung septa, which may have led to an appearance of increased expression of TLR4 and TLR9. Further data using transmission electron microscopy and immune-gold TLR labeling should be conducted to improve our understanding of lung inflammation in septic foals.

These data, which are the first to characterize inflammation in the lungs of septic foals, show histological inflammation including thickened alveolar septa, increased septal mononuclear cells expressing TLR4 and TLR9, and signs of microvascular inflammation. These cellular and molecular changes in lungs of septic foals suggest association of lung injury with sepsis. Since significant recruitment of septal monocytes and macrophages, most likely PIMs, may increase lung injury, further study is required to better understand their function.