Tutorial 2: Innate immunce cells Flashcards
from which 2 sources do macrophages originate?
Monocytes that arise in the bone marrow → mainly during inflammation.
Resident macrophage precursor cells → mainly during homeostasis.
Macrophages containing ink particles have been detected in the lymph nodes. Briefly explain (1-2 sentences) why macrophages would migrate to a lymph node after ingesting the ink particles; which immunological function do the macrophages exert there?
In the lymph nodes, the macrophages perform antigen presentation. Macrophages phagocytose ink particles and travel to the lymph node to present the foreign particles to T-cells.
Even for non-immunogenic materials, T cells play an important role in the macrophage response to a biomaterial and the foreign-body response. Briefly describe the function of T cells during the foreign body response and explain how this affects macrophage function. In your answer, name the type of T cells and at least 2 cytokines that play a dominant role.
During the FBR, the function of macrophages and multinucleated giant cells depends on the local cytokines that are secreted by other cells. Most important: T helper cells. TH1 cells secrete cytokines (e.g. INF-y) which promote a more pro-inflammatory, M1-like macrophage phenotype. TH2 cells secrete cytokines (IL-4) which promote a more pro-fibrogenic M2-like phenotype in macrophages.
Other cells that play a role are regulatory T cells (Treg) which secrete large amounts of IL-10 to stop inflammation and myofibroblast activation. IL-10 also leads to an M2-like macrophage, but with a more regulation function when compared to IL-4 and induced M2 macrophages.
Liver cirrhosis is a form of severe liver damage caused by excess exposure to toxins, for example, alcohol abuse. The onset of liver cirrhosis is characterized by chronic inflammation, which eventually leads to fibrotic scarring, hampering liver function. Kupffer cells (liver macrophages) are thought to play an important role in the progression of liver cirrhosis. Given the characteristics of cirrhosis, which macrophage phenotype(s) would you expect to find in the diseased tissue during disease progression and why?
Chronic inflammation is characterized by pro-inflammatory cytokines and macrophages, so phenotype M1. They are trying to remove the ingested toxins. If this persists for a long time, the macrophages are thought to attain a more pro-repair M2 phenotype. These cells promote fibrous tissue formation (via TGF-b production) and eventually fibrosis.
Various drugs are being evaluated in clinical trials to fight liver cirrhosis, each with varying degrees of success. Two examples are infliximab and prednisone. Infliximab is a TNF-a antagonist, while prednisone is a synthetic glucocorticoid analog. For both drugs, make an argument as to why or why not it is a good idea to administer it for the treatment of liver cirrhosis, and based on that, explain which of the two drugs you would prescribe to a patient.
Infliximab inhibits TNF-a, therefore it has the potential to inhibit the pro-inflammatory environment that characterizes the onset of cirrhosis. However, inhibition of TNF-a does not directly lead to resolution and it does not inhibit the pro-repair M2 macrophages. If this is administered in a late stage of the disease, it is unlike that it will stop the fibrotic response/ lead to resolution.
Prednisone is a stimulus for macrophage polarization to a pro-resolution M2 type. These cells secrete a lot of IL-10, which can inhibit both the pro-inflammatory macrophages and the pro-fibrotic macrophages.
Prescribe: prednisone, since it can target both inflammation and fibrosis, which infliximab is likely to inhibit mainly chronic inflammation.
List and briefly explain (1-2 sentences) two different mechanisms by which M2-like TAMs can promote tumor progression.
M2-like TAMs (1) promote angiogenesis and this gives more oxygen and nutrients to the tumor. (2) Also, they inhibit inflammation so that an immune attack is not happening.
A potential new treatment strategy that is currently being explored for treatment of pancreatic cancer makes use of a CCR2 antagonist. CCR2 is the receptor for MCP-1. Explain the rationale behind this treatment
MCP-1 (monocyte chemoattractant protein 1) is a pro-inflammatory cytokine that maintains inflammation and attracts monocytes. When an atagonist is used, this means that CCR2 cannot bind to MCP-1 and thus, MCP-1 can keep doing its pro-inflammatory function. The treatment aims to cut off the supply of monocytes via MCP-1 to the tumor, and this take away the supply of new TAMs to the tumor.
Briefly describe (1-2 sentences) what it is that triggers those immune cells to migrate from the vasculature to that wound site? In other words, how do the cells know where to go?
The cells know where to go because of the cytokine gradient from the site of injury (so chemotaxis).
Briefly describe two processes that happen to the endothelial cells in the microvasculature close to a wound site, which enables the extravasation of immune cells into the underlying tissue.
(1) In response to pro-inflammatory cytokines, the endothelial cells upregulate surface receptors (e.g. selectins and ICAM-1) to which circulating immune cells in the vasculature can bind.
(2) Histamine release (by mast cells) leads to the validation & disruption of endothelial-endothelial junctions, making the endothelial barrier permeable to immune cells.
Suppose that you get a small metal splinter in your finger and that splinter has some bacteria on it. Briefly describe (1-2 sentences) via which mechanism your innate immune cells recognize (i) the splinter, and (ii) the bacteria as being foreign bodies.
(1) The immune cells cannot directly interact with the splinter. Instead, the splinter will be covered in proteins, forming a protein layer with which the immune cells can interact (e.g. via integrins).
(2) Bacteria are ‘recognized’ by the pattern recognition receptors on the innate immune cells.
If the foreign body persists without being cleared, this will eventually lead to the foreign body response, which is dominated by foreign body giant cells. Briefly describe the process of macrophage fusion (2-3 sentences). In your answer, name which cytoskeletal component plays a major role and name at least 2 cytokines that are crucial for fusion. (max 3 points in total)
When there are particles > 5-10 um that macrophages cannot eat, they fuse together into giant cells. Fusion starts with IL-4 and IL-13 stimulation (via IL-4R). This triggers a pathway. The other trigger is cell-cell contact. You get a macrophage that is fusion-competent. This means they upregulate several membrane proteins that facillitate cell-cell binding (such as e-cadherin). This fusion-competent macrophage has to find another fusion-competent macrophage. They form a bridge (through actin-rich protrusions, or filopodia) and fuse together, leading to cytoskeletal rearrangement, including podosome rearrangement. Important: protrusion. They start to poke each other with podosomes and more bridges are formed in the fuse.
Also MCP-1 is thought to be important because it is a chemotactic signal for one macrophage to move the MCP-1 secreting macrophage. Actin is one of the cytoskeletal components that play a major role.
A research group is developing a new type of degradable scaffold for bone regeneration. In order to modulate the macrophage behavior upon implantation, the researchers functionalize their scaffolds with IFN-y, IL-4, or a combination of both. After having validated the efficacy of the functionalities via in vitro experiments, the researchers implant the scaffolds subcutaneously in rats to evaluate the host response to the different bioactive scaffolds.
Explain what could be the rationale behind this dual functionalization strategy.
The rationale could be to mimic the normal wound healing response, starting with a boost of IFN-y to ignite the inflammatory response (the first step in the wound healing cascade), followed by IL-4 secretion to polarize the recruited cells into a pro-regenerative M2/TH2-like profile.
INF-y is a pro-inflammatory cytokine. It keeps the macrophages in a pro-inflammatory state, recruiting more immune cells to the scene. IL-4 is a pro-inflammatory cytokine, but more reparative. Together they balance the formation of granulation tissue.
The researchers aim to characterize the macrophage phenotype via immunohistochemical analysis using antibodies for inducible Nitric Oxide Synthase (iNOS), Arginase 1 (Arg1), and F4/80 (pan-macrophage marker). Which marker expression (high/low) would you expect for each type of scaffold after 14 days in vivo?
IFN-y only scaffolds: pro-inflammatory factor which stimulates M1 polarization, which in turn amplifies the attraction of more macrophages to the implant. Consequently: a high expression of F4/80 and iNOS (M1 marker in rodents) and low expression of Arg1 (M2 marker in rodents).
IL-4 only scaffolds: stimulus for M2 polarization, so the expression of Arg1 is expected to be high, and expression of iNOS low. The production of chemokines is typically lower by these cells, so we may expect a lower expression F4/80 (less macrophages overall) than in the IFN-y loaded scaffolds.
Combination scaffold: a quick release of IFN-y will lead to a strong attraction of total macrophages in the first week or so after implantation. Therefore, we can expect a high expression of F4/80 after 14 days. Since IL-4 is the dominant factor around this time point, the macrophages will be stimulated to switch to an M2 phenotype, so we expect a high expression of Arg1 and a low expression of iNOS.
Unfortunately, based on these markers there appears to be no clear distinction according to the M1-M2 paradigm possible (Figure 1). Assuming that the activated scaffolds do in fact have a differential effect on macrophage polarization, explain why the researchers were unable to pick that up with this analysis and give an example of what would be a better read-out parameter instead of cell surface markers to characterize the macrophage phenotypes in the scaffolds?
The M1-M2 paradigm is highly simplified and highly idealized, with many shades of grey. It is likely that macrophage polarization has a mixed phenotype in which cells express all these markers to some extent. It would be best to perform functional read-outs to these stainigs. E.g. cytokine secretion, vascularization, ECM formation.
Using their controlled release methodology, the researchers are able to tune the release rate of each cytokine individually. For each cytokine, reason what you would recommend as an optimal release rate: 1 day, 1 week, 1 month, or 1 year?
Start with IFN-y to kick-off the inflammatory response (1 day), followed by IL-4 to polarize the cells into M2. However, this is also a risk factor for fibrosis and promotes macrophage fusion. So we wouldn’t want it to be present for too long as this may induce fibrosis (1 week).
