Wasteosomes (corpora amylacea) of human brain can be phagocytosed and digested by macrophages

Time-lapse study of the phagocytosis of wasteosomes by THP-1 macrophages

To study the phagocytosis of wasteosomes by THP-1 macrophages, different sets of experiments were performed using time-lapse imaging.

In the first set of experiments, wasteosomes obtained from the CSF were stained with ConA-Rhod and added to a culture of THP-1 macrophages that had been previously stained with the vital tracer CFDA-SE. From that point on and for each ROI (i.e., regions that contain wasteosomes), an image was taken every 2 min over a period of a minimum of 15 h. The sequence of images was then put into a video format to see the interaction between the macrophages and wasteosomes at each ROI during this period.

In all the ROIs, we observed that the macrophages did interact with wasteosomes. Additional file 1: Video 1A, summarized in Fig. 1A, shows the encounter of one macrophage with a wasteosome as well as several steps of phagocytosis. Firstly, a lamellipodium extends from the macrophage until it reaches the wasteosome. Once the lamellipodium has attached to the wasteosome, it pulls it towards the body of the macrophage and the macrophage completely engulfs the wasteosome. Once the wasteosome has been phagocytosed, the red fluorescence signal progressively spreads inside the macrophage, indicating that the wasteosome (or its ConA-Rhod protein fraction) has been digested. After the engulfment and digestion, some of the fluorescence appears on the surface of the macrophage. This process was corroborated by the 3D reconstructions from the confocal images obtained in the last moment of some sequences (Fig. 1B, Additional file 2: Video 1B). This suggests that parts of the stained wasteosome were exposed on the surfaces of THP-1 macrophages. Thus, THP-1 macrophages could act as APCs.

Fig. 1figure 1

A Sequence of images from a time-lapse recording showing how a THP-1 macrophage (green arrow) extends a lamellipodium (empty green arrow) to a wasteosome opsonized with ConA (red arrow) and pulls it towards the body of the macrophage, triggering the engulfment of the wasteosome. The phagocytosed wasteosome become later digested and fragmented (yellow arrows). Empty yellow arrow: wasteosome is out of the focus plane. See video for details. At 1202 min, confocal images were taken and the 3D reconstruction was made. B Sequence of images showing the 360° rotation of the 3D reconstruction. Images permit to observe the location of the remains of the wasteosome (red and yellow) at the macrophage. Some dots of red or yellow fluorescence appear on the surface of the macrophage, suggesting antigen presentation. The big green spot in the lower region corresponds to a macrophage that is in contact with the one that has phagocytosed and digested the wasteosome

In other cases, the wasteosomes were too big to be engulfed by the macrophages, but an interaction between the wasteosomes and macrophages could be observed. Figure 2A and Additional file 3: Video 2A show different macrophages making contact with a large wasteosome. The wasteosome is eroded by these macrophages and a gradual increase in the red fluorescence signal can be seen inside the macrophages. Moreover, some spots of fluorescence are also present on the surface of the macrophages and some interchanges of these spots of fluorescence between the different macrophages can be appreciated. Additional file 4: Video 2B and Fig. 2B show three different wasteosomes, of which two are just eroded by the macrophages and the remaining one is not just eroded but fragmented, with the resulting fragments digested by several macrophages.

Fig. 2figure 2

A Sequence of images from a time-lapse recording showing two THP-1 macrophages (green arrows) eroding a wasteosome opsonized with ConA (red arrow). Some spots of red fluorescence become incorporated into the macrophages. In some cases, the fluorescence is transferred from one macrophage to another one (empty green arrow). See video for details. B Sequence of images showing different macrophages interacting with three different wasteosomes (arrows). One of the wasteosomes (yellow arrow) become digested and fragmented. Empty arrows indicate that the wasteosome is out of the focus plane

From the results of this first set of experiments, we deduced that THP-1 macrophages can engulf and digest wasteosomes when these are opsonized by ConA-Rhod. Moreover, these macrophages might act as APCs by presenting the fluorescent component (i.e., ConA-Rhod or some of its fragments containing Rhod) on their surface.

In the second set of experiments, wasteosomes obtained from the CSF were stained with AF555-NHS and added to a culture of THP-1 macrophages that had been stained with CFDA-SE. In contrast to the first set of experiments, where we used an external protein (ConA-Rhod) that binds to the sugar components of wasteosomes, in this second set of experiments we used the AF555-NHS probe, which directly stains the proteins contained in the wasteosomes, thus enabling the determination of whether these proteins were also digested and presented on the surface of macrophages.

In this second set of experiments, we also observed that some macrophages interacted with the wasteosomes stained with the AF555-NHS dye. Additional file 5: Video 3A and Fig. 3A show a macrophage making contact with a wasteosome at different times. Although the wasteosome is not entirely phagocytosed, some spots of red fluorescence are translocated from the wasteosome to the macrophage. When the macrophage detach from the wasteosome, the spots of fluorescence remain at the macrophage, indicating that the fluorescence signal from the wasteosome has been incorporated into the macrophage. The same process can be seen in Additional file 6: Video 3B and Fig. 3B. In this case, some spots of fluorescence can be observed on the surface of the macrophage, indicating possible antigen presentation. Thus, this second set of experiments indicated that the proteins contained in the wasteosomes can be phagocytosed by macrophages and that these proteins can later be presented on the surface of macrophages.

Fig. 3figure 3

A Sequence of images showing a THP-1 macrophage (green arrow) eroding a wasteosome opsonized with AF555-NHS (red arrow). Note that the spots of red fluorescence, corresponding to stained proteins, become incorporated into the macrophage. A similar process can be observed in B. See the corresponding videos for details

In the third set of experiments, wasteosomes obtained from the CSF were stained with the PAS technique and added to a culture of THP-1 macrophages that had been stained with CFDA-SE. In contrast to the first and second set of experiments, the fluorescence was generated here by the PAS staining, specifically related to the carbohydrate constituents of wasteosomes. This protocol allowed to determine if the carbohydrate components of wasteosomes are also digested and/or presented on the macrophage surface.

In this case, we observed that macrophages also interacted with wasteosomes. A representative sequence of images is presented in Additional file 7: Video 4A and Fig. 4A. Initially, a lamellipodium from a distant macrophage making contact with the wasteosome can be observed. Thereafter, the macrophage shrinks over the wasteosome and both structures displace together. However, unlike in the previous sets of experiments, digestion of the wasteosomes could not be observed in most of these experiments. In only a few cases, as that shown in Fig. 4B and Additional file 8: Video 4B, small amounts of fluorescence were observed to detach from the wasteosomes and spread inside the macrophages. In any case, the processes of phagocytosis, digestion and antigen presentation seemed to be reduced in this set of experiments, which indicates that the processing of the polyglucosan structure of wasteosomes differs from that of the protein fraction.

Fig. 4figure 4

A Sequence of images showing a THP-1 macrophage (green) contacting a wasteosome stained with PAS (red). Initially, a lamellipodium from a distant macrophage making contact with the wasteosome can be observed. Thereafter, the macrophage shrinks over the wasteosome and both structures displace together. However, unlike in the previous sets of experiments, the digestion of the PAS stained wasteosomes could not be observed in most of the experiments. B In only a few cases, as the one shown here, small amounts of fluorescence were observed to detach from the wasteosomes and spread inside the macrophages. See videos for details

Identification of phagocytic receptors on THP-1 macrophages that interact with wasteosomes

Since the time-lapse studies demonstrated that THP-1 macrophages interact with wasteosomes and can phagocytose them, the next step was to shed light on the mechanisms triggering this phagocytosis. In this regard, the study of the phenotype or, more specifically, of the phagocytic receptors expressed by THP-1 macrophages was determining. We postulated that the phagocytosis of wasteosomes could be mediated by CD206, CD35 and/or FAIM3. Thus, we isolated wasteosomes from the CSF and stained them with ConA-Fl or opsonized them with IgM before adding them to THP-1 macrophage cultures. The cultures were then fixed and processed for immunofluorescence analysis using anti-CD68, anti-CD206, anti-CD35 and anti-FcμR (directed against FAIM3) antibodies, and adding the AF488 anti-IgM antibody in the cases of wasteosomes that had been opsonized with IgM. This protocol allowed not only the detection of the abovementioned markers in THP-1 macrophages, but also the detection of these markers in wasteosomes-interacting macrophages. CD68 is a transmembrane glycoprotein that is highly expressed by human monocytes and macrophages [44]. It was used here to identify macrophages in the cultures, observe the interactions between macrophages and wasteosomes, and validate the effectiveness of the immunofluorescence method. As shown in Fig. 5a, THP-1 macrophages stained with the anti-CD68 antibody were observed to make contact with ConA-Fl-stained wasteosomes. Using the same protocol, we searched for the presence of the other indicated markers on macrophages that made contact with wasteosomes. CD206, also known as the mannose receptor, is a C-lectin that recognizes mannose residues, as well as N-acetylglucosamine and fucose residues [45]. It is normally expressed on M2, but not on M1 macrophages [46]. Figure 5b shows a representative image of CD206-positive THP-1 macrophage encircling and making contact with a ConA-Fl-stained wasteosome. These results, which are consistent with those obtained previously [40], suggest that macrophages that phagocytose wasteosomes are non-inflammatory or of the M2 subtype. Regarding the possible CD35-mediated phagocytosis, we stained THP-1 macrophages with the anti-CD35 antibody. CD35, also known as Complement Receptor type 1 (CR1), is a protein that binds to C3b/C4b-opsonized substances that are tagged for phagocytosis [47]. Figure 5c and d indicate that THP-1 macrophages that phagocytose wasteosomes express CD35, suggesting that these macrophages might also recognize some complement proteins in wasteosomes. Since wasteosomes are recognized by natural IgMs [10], complement activation could be triggered through the classical complement pathway, which would lead to wasteosomes opsonization by C3b and the induction of their CD35-mediated phagocytosis. However, as IgMs do not cross the blood–brain barrier, this process might occur in the lymphatic system or beyond, but not inside the brain. Since wasteosomes may contain mannose and N-acetylglucosamine, which are both targets of MBL [48], wasteosomes could also activate the complement system through the lectin pathway. The alternative pathway is the third biochemical pathway of the complement cascade and should also be considered. This pathway is based on the spontaneous hydrolysis of C3 into C3b. It has been reported that C3b binds to glucose oligomers [49, 50]. Since glucose has been described to be the main component of wasteosomes, C3b could probably bind to this carbohydrate. Given that natural IgMs recognize wasteosomes, we also considered FAIM3-mediated phagocytosis as well. FAIM3, also known as FcμR, is an IgM receptor found in some macrophages and dendritic cells that are associated with phagocytic processes [51,52,53]. Accordingly, we stained THP-1 macrophages with the anti-FcµR antibody, but these cells did not stain with this antibody, thus ruling out this pathway in the phagocytosis of wasteosomes by THP-1 macrophages.

Fig. 5figure 5

THP-1 macrophages which make contact with wasteosomes are CD68 + , CD206 + and CD35 + . a A CD68 + THP-1 macrophage (red) in contact with a wasteosome stained with ConA (green). b A CD206 + THP-1 macrophage (red) attached to a wasteosome (ConA, green). c A CD35 + THP-1 macrophage (red) encircling a wasteosome stained with ConA (green). d A CD35 + THP-1 macrophage (red) encircling a wasteosome immunostained with IgM (green). Nuclei are stained with Hoechst (blue). Scale bar: 25 µm

The presence of opsonins on the wasteosomes surface

Wasteosomes have a polyglucosan structure based on polymerized hexoses, which are mainly glucose [1] although not exclusively. As previously mentioned, MBL binds to several hexoses such as mannose and N-acetylglucosamine, while C3b can bind to glucose [48,49,50]. Accordingly, we explored the CD35- or complement-mediated phagocytosis and analyzed if the wasteosomes from the CSF could be opsonized by MBL or C3b. After incubating the wasteosomes with human plasma, some aliquots were immunostained with the anti-C3b antibody (directed against C3b) while others were immunostained with the anti-MBL antibody. As shown in Fig. 6A, wasteosomes were stained by both the anti-C3b and anti-MBL antibodies, indicating that wasteosomes are opsonized by MBL and C3b. However, and surprisingly, wasteosomes from the control samples (where the wasteosomes were incubated with PBS instead of human plasma) were also stained with the anti-C3b or anti-MBL antibody, indicating that wasteosomes from the CSF are opsonized by MBL and C3b. It is of interest, as commented in the discussion section, that wasteosomes located in the brain parenchyma are not opsonized by these proteins (Fig. 6B).

Fig. 6figure 6

A Wasteosomes from CSF have opsonins on their surface. a wasteosomes purified from CSF and incubated with human plasma become stained with anti-MBL (green). b wasteosomes from CSF and incubated with human plasma become immunostained with anti-C3b (red). c wasteosomes purified from CSF and incubated with PBS instead of human plasma also become immunostained with anti-MBL (green). d wasteosomes purified from CSF and incubated with PBS instead of human plasma also become immunostained with anti-C3b (red). B Hippocampal wasteosomes do not have the opsonins on their surface. a When the hippocampal tissue is double-immunostained with anti-MBL (green) and anti-C3b (red), the wasteosomes do not become immunostained and are observed as a black circle. In this case, one wasteosome can be observed in the center of the image. b When the hippocampal tissue is immunostained with anti-GS and anti-C3b, wasteosomes become stained with anti-GS (green) but not by anti-C3b (red). Scale bars: 25 µm

Wasteosomes and macrophage interactions at central nervous system interfaces

After observing that different mechanisms might be involved in the phagocytosis of wasteosomes by THP-1 macrophages in vitro, we ascertained whether this phagocytosis also happens in vivo when wasteosomes are released from the brain parenchyma into the CSF. As mentioned above, wasteosomes accumulate mainly in the perivascular, periventricular and subpial regions of the brain. When they are expelled from these regions, they may encounter perivascular macrophages, choroid plexus macrophages and meningeal macrophages. Therefore, double immunostaining of human hippocampal sections was performed with the anti-p62 antibody, a protein marker that allows the localization of wasteosomes, together with the anti-CD206, anti-CD35, anti-FAIM3 or anti-CD68 antibody, which are associated with phagocytosis or phagocytic cells. Immunostaining with the anti-CD206 antibody revealed some CD206-positive macrophages that were in contact with wasteosomes. Figure 7a1, exhibiting a section including a part of the hippocampus and the lateral ventricle, shows a CD206-positive choroid plexus macrophage making contact with a wasteosome that has been released from the brain tissue into the CSF in the lateral ventricle. The choroid plexus macrophage attached to the wasteosome can be clearly observed in the magnification of this image shown in Fig. 7a2. Figure 7a3 and 7a4 show several wasteosomes that have been released from the bordering regions of the hippocampus into the subarachnoid space making contact with CD206-positive meningeal macrophages. A magnification of Fig. 7a4 is shown in Fig. 7a5, where the staining of wasteosome is digitally intensified to illustrate the presence of a wasteosome with two encircling meningeal macrophages. Staining with the anti-CD35 antibody also revealed some positive cells located at the border of the brain parenchyma surrounding several wasteosomes (Fig. 7b). As astrocytes in the glia limitans of brain cavities can be positive for CD35 [54, 55], we tested the possible colocalization of CD35 with GFAP, which is a specific marker of astrocytes. As shown in Fig. 7c, CD35 staining colocalized with GFAP staining, indicating that the cells containing the wasteosomes are not macrophages in this case, but astrocytes. The staining with the anti-FAIM3 antibody did not show any positive cells in the hippocampal sections (Fig. 7d). Finally, the staining with the anti-CD68 antibody stained some cells located in the brain parenchyma. For their localization, these cells are presumably microglial cells, although it cannot be discarded possible infiltrating macrophages. In any case, these CD68 positive cells do not contact with wasteosomes (Fig. 7e). As expected, microglia or macrophages did not reach the wasteosomes within the brain parenchyma since in this region wasteosomes are intracellular astrocytic structures.

Fig. 7figure 7

Some macrophages at central nervous system interfaces interact with wasteosomes. a1, a2, a3, a4 and a5 wasteosomes from human hippocampal sections immunostained with anti-p62 (red) and interface macrophages immunostained with anti-CD206 (green). a1 A choroid plexus macrophage in contact with a wasteosome released from the brain tissue to the ventricular CSF. a2 inset of a1, where the macrophage attached to the wasteosome is magnified. a3 and a4 wasteosomes released from hippocampus to the subarachnoid space in contact with meningeal macrophages. a5 inset of a4, where the red staining is digitally intensified to evidence the presence of a wasteosome, in this case surrounded by two macrophages (white arrowheads). b within the brain parenchyma, wasteosomes become immunostained with anti-p62 (red) and are surrounded by CD35 + cells (green). c CD35 staining (red) colocalize with GFAP staining (green, white arrowheads), indicating that cells that surround wasteosomes are CD35 + astrocytes. d wasteosomes from human hippocampus immunostained with anti-p62. FAIM3 positive cells are not found in the hippocampal sections (green). e wasteosomes from human hippocampal tissue immunostained with IgMs (green) are not contacted by CD68 + cells (red, marked with white arrowheads). For their localization, these CD68 + cells are presumably microglial cells, although possible infiltrating macrophages cannot be discarded. In any case, and as expected, microglia or macrophages did not reach the wasteosomes within the brain parenchyma since in this region wasteosomes are intracellular astrocytic structures. Scale bar in a1: 200 µm; scale bars in a2, a3 and a4: 25 µm; other scale bars: 50 μm. Hoechst (blue) was used for nuclear staining

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