Salivary scavenger and agglutinin SALSA in innate immunity

To live a healthy life, humans need to co-exist with foreign organisms. These consist of the thousands of different types of microbes that colonize the human body. But also, in the case of a pregnant woman, the fetus can be viewed as a foreign organism. To avoid disease, the barriers of the human body, e.g. the mucosal surfaces, must be maintained. Here the innate immune defense system plays an important role. The salivary scavenger and agglutinin (SALSA), also known as gp340, DMBT1 and SAG, is a molecule found at most mucosal surfaces. SALSA is associated with the epithelium or secreted into the lining fluids, such as tears, saliva and mucus in the respiratory tract. SALSA is known to bind and agglutinate a broad spectrum of bacteria, as well as viruses, and thus play a role in the innate immune defense against invading microbes. The effect of SALSA is mediated in concert with several other defense molecules such as IgA, surfactant proteins A and D, and the complement component C1q. These have all been shown to be ligands of SALSA. Alongside the role of SALSA in innate immunity, evidence for a function in epithelial and stem cell differentiation has emerged. This thesis work has addressed the function of SALSA in innate immunity, especially in early life. SALSA was found in the amniotic fluid and in meconium and feces of newborns. In fact, SALSA was among the most abundant proteins in the intestines of newborn children. By comparing the SALSA protein in the different samples we found size polymorphisms, varying from one individual to another, but also from compartment to compartment within the same individual. These differences were found to alter the ability of SALSA to bind known endogenous and bacterial ligands. SALSA was also found to be expressed in the human placental and decidual tissues. In the 1st trimester of pregnancy, SALSA was detected sporadically in maternal decidual capillaries. Closer to term SALSA was found to be expressed by the syncytiotrophoblast layer of the placental villous trees. In certain sites, e.g. at disrupted and damaged areas of the syncytium, SALSA was found deposited into fibrinoid formations. It partially co-localized with the fibrinoid component fibronectin. Complement activation has been observed at the feto-maternal interface of both healthy and complicated pregnancies. SALSA had previously been found to bind C1q. Thus, it was of interest to investigate the ability of SALSA to interact directly with the complement system. We found that SALSA bound to both mannan-binding lectin and to some extent to all three ficolins (H, L and M). SALSA activated complement, when it was bound to a surface. In contrast, fluid-phase SALSA was able to inhibit the deposition of complement on SALSA non-binding microbial surfaces. It thus acted in dual fashion to target complement attack. In the human placenta we observed C1q-targeting of the SALSA-positive fibrinoid formations. C1q and complement are known to function in the clearance of apoptotic cells and debris. Thereby SALSA and complement probably have a cooperative function in the containment and clearance of the injured structures, thus linking its innate immune activity with the maintenance of tissue homeostasis.

The Salivary Scavenger and Agglutinin (SALSA) in Healthy and Complicated Pregnancy

Pre-eclampsia is a leading cause of maternal and perinatal morbidity and mortality worldwide. The etiology is not clear, but an immune attack towards components of placenta or fetus has been indicated. This involves activation of the complement system in the placenta. We have previously described the presence of the complement-regulating protein salivary scavenger and agglutinin (SALSA) in amniotic fluid. In this study we investigated the potential role of SALSA in pregnancy by analyzing its presence in amniotic fluid and placental tissue during healthy and complicated pregnancies. SALSA levels in amniotic fluid increased during pregnancy. Before 20 weeks of gestation the levels were slightly higher in patients who later developed pre-eclampsia than in gestation age-matched controls. In the placenta of pre-eclamptic patients syncytial damage is often followed by the formation of fibrinoid structures. SALSA was found clustered into these fibrinoid structures in partial co-localization with complement C1q and fibronectin. In vitro analysis showed direct protein binding of SALSA to fibronectin. SALSA binds also to fibrin/fibrinogen but did not interfere with the blood clotting process in vitro. Thus, in addition to antimicrobial defense and epithelial differentiation, the data presented here suggest that SALSA, together with fibronectin and C1q, may be involved in the containment of injured placental structures into fibrinoids.Peer reviewe

Intestinal SALSA/dmbt1 levels are decreased in prematurely born infants

The first months of life represent a crucial time period for an infant. Alongside establishing the early microbiome, the mucosal immunological homeostasis is being developed. Both processes may be perturbed in prematurely born infants. The glycoprotein SALSA plays a role in mucosal inflammation and microbial clearance. It is one of the most abundant molecules on the intestinal mucosal surfaces in early life. SALSA binds to many types of microbes and host defence molecules like IgA, C1q and collectin molecules. We here describe the development in faecal SALSA levels during the first three months of life. During these 90 days, the median SALSA level in full-term babies decreased from 1100 mu g/mL (range 49-17 000 mu g/mL) to 450 mu g/mL (range 33-1000 mu g/mL). Lower levels of SALSA were observed in prematurely born infants in the same time period. Our novel observation thus indicates an impact of prematurity on an important component of the infant intestinal immune system. Changes in SALSA in early life may have an effect on the early establishment of the human microbiome.Peer reviewe

Binding of SALSA to the fibrinoid component fibronectin.

<p>Purified SALSA (1 μg/ml) was added to Maxisorp plate wells coated with fibronectin (1–10 μg/ml) and binding was monitored using monoclonal anti-SALSA antibodies. A clear calcium- and dose-dependent binding of SALSA to fibronectin was observed. Averages ± SD’s from three different experiments are shown.</p

Targets for SALSA in human placenta.

<p>To determine further binding sites for SALSA in the placenta, SALSA containing AF was added as an overlay to frozen placental sections prior to detection of SALSA by fluorescence immunohistochemistry. The addition of the SALSA containing overlay (A) revealed a strong binding of SALSA to numerous additional structures in the placenta which were not observed without the overlay (B). SALSA binds to the endothelium of most capillaries (Ca) and larger vessels (Ve) as well as to most of the syncytiotrophoblast layer (A). Without overlay SALSA staining is much more sporadic or lacking in these structures (B). The strong binding after <i>ex vivo</i> addition of SALSA suggests that the focal staining observed in the absence of overlay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147867#pone.0147867.g002" target="_blank">Fig 2</a>) is a result of limited availability of SALSA rather than the lack of interacting tissue components. 400× magnifications.</p

Immunofluorescence microscopy detection of SALSA localization in human placenta.

<p>Paraffin-embedded tissue from healthy (panels A-D) and pre-eclamptic (PE) (panels E-F) placentas. Sections were stained with an anti-SALSA antibody (Hyb 213–06) and Alexa 546-conjugated goat anti-mouse IgG. Red: SALSA, blue: DAPI. * denotes the center of the villus structures. M denotes the intervillous space/maternal tissue. Panels A to F show a widespread but focal staining of SALSA in the human placenta. Based on the morphology and localization of the placental structures SALSA appears to be present in fibrinoid formations, especially in relation to a disruption of the syncytiotrophoblast layer. (A) shows the expression of SALSA found intracellularly in the syncytiotrophoblast layer of some, but not all, villi (white arrowhead). SALSA was observed abundantly in fibrinoid structures at the edge of the villi. A breach of the syncytiotrophoblast layer accompanied by formation of SALSA-positive fibrinoid in the intervillous space can be observed (white arrowhead, panel B). (C) shows SALSA in a necrotic villus with fibrinoid formation. (D) shows a villus with disrupted syncytiotrophoblast layer (white arrowhead). Here the disruption may have led to the influx of maternal blood, and SALSA-positive fibrinoid can be observed separating the syncytiotrophoblast layer from the underlying basement membrane. The fibrinoid may deposit all the way around the exposed villus structure, thus forming a ring structure. Syncytial damage and fibrinoid formation is observed more frequently in PE. (E) and (F) show examples of a necrotic villus and a ring formation in PE placentas. 200× magnifications.</p

Immunohistochemistry analysis of SALSA in placenta.

<p>Paraffin-embedded placental tissue from healthy and pre-eclamptic (PE) pregnancies were stained for SALSA. Both healthy (A) and (B) and PE (C) and (D) sections are displayed with corresponding negative IgG-controls (Neg). Based on the morphology and location of the positively staining structures, SALSA primarily stains fibrin-type fibrinoids. (A) and (D) show staining of specific focal fibrinoid formations in apparently necrotic villous structures. Panel B shows an example of fibrinoid “gluing” of villi. (D) shows a larger necrotic area with massive fibrinoid formation. 200× magnifications.</p

Analysis of the effect of SALSA on coagulation.

<p>A pool of citrated plasma was added to SALSA-coated wells (1–5 μg/ml) and coagulation was initiated by adding BC Thrombin reagent at time point 0. The coagulation was followed by absorbance at 405 nm and compared to coagulation in wells without SALSA coating. We did not observe an effect of SASLA on coagulation. Displayed are averages ± SD’s from 5 different wells. A similar result was obtained when soluble SALSA (1–5 μg/ml) was added to the plasma prior to the coagulation test (data not shown).</p

Immunohistochemical analysis of SALSA in paraffin-embedded healthy first trimester decidua.

<p>SALSA staining (A), (C) and (E) was compared to the endothelial marker CD34 (B) and (D) and the epithelial marker cytokeratin 7 to identify uterine glands and extravillous trophoblasts (F). In the 1^st trimester decidua, SALSA was still found abundantly. However less fibrinoid structures were observed (A) and (C). Instead, SALSA was found to co-localize with CD34 (B) and (D). The SALSA staining pattern of the endothelium is scattered, and found in both small capillaries and larger vessels. Thus, it may be related to the activation state of the endothelium. SALSA was not found to co-localize with cytokeratin 7, and is thus not likely to be produced in the uterine glands (E) and (F). 200× magnifications.</p