Phagocytes have voracious appetites; scientists have even fed macrophages with iron filings and then used a small magnet to separate them from other cells. All phagocytes, and especially macrophages, exist in degrees of readiness. Macrophages are usually relatively dormant in the tissues and proliferate slowly. In this semi-resting state, they clear away dead host cells and other non-infectious debris and rarely take part in antigen presentation.
But, during an infection, they receive chemical signals—usually interferon gamma—which increases their production of MHC II molecules and which prepares them for presenting antigens. In this state, macrophages are good antigen presenters and killers. Their size and rate of phagocytosis increases—some become large enough to engulf invading protozoa.
In the blood, neutrophils are inactive but are swept along at high speed. When they receive signals from macrophages at the sites of inflammation, they slow down and leave the blood. In the tissues, they are activated by cytokines and arrive at the battle scene ready to kill.
Neutrophils : Neutrophils move through the blood to the site of infection. These chemical signals may include proteins from invading bacteria, clotting system peptides, complement products, and cytokines that have been given off by macrophages located in the tissue near the infection site.
Another group of chemical attractants are cytokines that recruit neutrophils and monocytes from the blood. To reach the site of infection, phagocytes leave the bloodstream and enter the affected tissues. Signals from the infection cause the endothelial cells that line the blood vessels to make a protein called selectin, which neutrophils stick to when they pass by.
Other signals called vasodilators loosen the junctions connecting endothelial cells, allowing the phagocytes to pass through the wall. Neutrophils travel across epithelial cell-lined organs to sites of infection, and although this is an important component of fighting infection, the migration itself can result in disease-like symptoms.
During an infection, millions of neutrophils are recruited from the blood, but they die after a few days. These blood vessels are surrounded by thicker, multilayer protective walls, in contrast to the thin single-cell-layer walls of capillaries.
Furthermore, the blood flow in arteries is too turbulent to allow for rolling adhesion. Also, some leukocytes tend to respond to an infection more quickly than others. The first to arrive typically are neutrophils , often within hours of a bacterial infection. By contract, monocytes may take several days to leave the bloodstream and differentiate into macrophages. Figure 1. Click for a larger image. Damaged cells and macrophages that have ingested pathogens release cytokines that are proinflammatory and chemotactic for leukocytes.
In addition, activation of complement at the site of infection results in production of the chemotactic and proinflammatory C5a. Leukocytes exit the blood vessel and follow the chemoattractant signal of cytokines and C5a to the site of infection.
Granulocytes such as neutrophils release chemicals that destroy pathogens. They are also capable of phagocytosis and intracellular killing of bacterial pathogens. As described in the previous section, opsonization of pathogens by antibody; complement factors C1q, C3b, and C4b; and lectins can assist phagocytic cells in recognition of pathogens and attachment to initiate phagocytosis.
However, not all pathogen recognition is opsonin dependent. Phagocytes can also recognize molecular structures that are common to many groups of pathogenic microbes. Such structures are called pathogen-associated molecular patterns PAMPs. Common PAMPs include the following:. Like numerous other PAMPs, these substances are integral to the structure of broad classes of microbes.
Figure 2. Phagocytic cells contain pattern recognition receptors PRRs capable of recognizing various pathogen-associated molecular patterns PAMPs. These PRRs can be found on the plasma membrane or in internal phagosomes.
When a PRR recognizes a PAMP, it sends a signal to the nucleus that activates genes involved in phagocytosis, cellular proliferation, production and secretion of antiviral interferons and proinflammatory cytokines, and enhanced intracellular killing. Many TLRs and other PRRs are located on the surface of a phagocyte, but some can also be found embedded in the membranes of interior compartments and organelles Figure 2.
These interior PRRs can be useful for the binding and recognition of intracellular pathogens that may have gained access to the inside of the cell before phagocytosis could take place. Viral nucleic acids, for example, might encounter an interior PRR, triggering production of the antiviral cytokine interferon.
PRRs on macrophages also respond to chemical distress signals from damaged or stressed cells. This allows macrophages to extend their responses beyond protection from infectious diseases to a broader role in the inflammatory response initiated from injuries or other diseases. The Gram-negative bacteria Coxiella burnetti , the causative agent of Q fever, resides inside a large phagolysosome-like vesicle known as parasitophorous vacuole This modified phagosome concentrates Rab5 on the membrane and avoids lysosome fusion Figure 6.
The fungi A. In the case of A. For Leishmania , the promastigote is efficiently internalized by receptor-mediated phagocytosis Complement and mannose receptors participate in macrophage ingestion Once internalized, promastigotes insert lipophosphoglycan LPG into the phagosome membrane.
LPG inhibits depolymetization of F-actin , and in consequence prevents lysosome fusion Figure 6. This allows enough time for the promastigote to transform into the other life-cycle form, the amastigote, which can then replicate inside the phagosome. In addition to preventing phagolysosome formation, pathogens also possess various mechanisms to resist the microbial components found in the phagolysosome lumen. A prominent example is S. These bacteria express the enzyme O -acetyltransferase A OatA , which causes O-acetylation of the peptidoglycan.
This modification makes the peptidoglycan resistant to the muramidase activity of lysozyme , Figure 7. Second, bacteria alter the composition of its membrane. Phosphatidylglycerol is modified with l -lysine, causing a reduction in the negative charge of the membrane In addition, the cell wall is also modified by incorporation of teichoic acids and lipoteichoic acids , making it more positively charged.
Third, the metalloprotease aureolysin can degrade LL, an antimicrobial peptide with potent activity against staphylococci Figure 7.
Figure 7. Resistance of Staphylococcus aureus to phagolysosome contents. The bacteria S. In addition, S. The bacterial urease catalyzes the hydrolysis of urea to form ammonia, resulting in pH neutralization Finally, S.
Also, several pathogens express urease, an enzyme that catalyzes the hydrolysis of urea to form ammonia, resulting in the pH neutralization of the phagosome Figure 7.
Important examples of microorganisms using this strategy to survive in the phagosome are S. The oxidative environment of the phagolysosome is also very damaging to most microorganisms. For example, S. Also, the protein SOK surface factor promoting resistance to oxidative killing , that is expressed on the bacteria surface, was recently described as a virulence factor that blocks the effects of ROS Similarly, M.
A novel glycosylated and surface-localized lipoprotein, Lprl can inhibit the lytic activity of lysozyme Figure 8. These effects seem to depend on the N -acetyltransferase domain of the Eis protein Figure 8.
In both cases, apoptosis is inhibited, but the mechanisms are different. In the case of NDH-1, apoptosis is dependent on caspase-3 and caspase-8 , while for Eis, apoptosis seems to be caspase independent The mechanism for iNOS inhibition is not completely elucidated, but it seems to involve both having less iNOS on the membrane and blocking its enzymatic activity.
The way M. Figure 8. Resistance of Mycobacterium tuberculosis to phagolysosome contents. The bacterial lipoprotein, Lprl, can inhibit the lytic activity of lysozyme Also, M. In addition, M. The enzymes isocitrate lyases ICLs allow bacteria survival on even acetate and odd propionate chain fatty acids in lipid bodies Streptococcus agalactiae Group B Streptococcus is an important cause of pneumonia and meningitis in neonatal humans The latter two compounds functions as ROS scavengers , Similarly, the yeast C.
The fungus C. In all these pathogens, the expression of these enzymes and virulent factors effectively reduces the levels of ROS and RNS within the phagosome.
Yet, very little is known about the mechanisms that induce expression of these virulent factors in each pathogen and the molecular details by which they inhibit NADPH oxidase and iNOS enzymes. The phagolysosome is a place where microbial nutrients are eliminated to arrest pathogen growth. In response to this, several microorganisms have evolved mechanisms to retain these important nutrients.
These siderophores are very efficient because they avoid detection by the phagocyte siderophore-binding protein lipocalin 96 , The type VII secretion system Esx-3 contributes to siderophore production and release from these bacteria , Figure 8. Recently, another siderophore export system was identified in M.
The MmpS4 and MmpS5 transporters are required for biosynthesis and secretion of siderophores Figure 8. Because a M. Inactivation of the irtAB system decreases the ability of M. Similarly, other microorganisms such as A. Intracellular bacteria have also evolved various means to take nutrients from the host cell. Lipids are important building blocks for bacterial membrane formation and an energy source Upon infection, M.
A virulent factor was identified within the gene cluster, mce4, because it was specifically required for bacterial survival during prolonged infection. It was found that mce4 encodes a cholesterol import system that enables these bacteria to derive both carbon and energy from this lipid in host membranes Figure 8. This foamy phenotype is caused by bacterial manipulation of host cellular metabolism to divert the glycolytic pathway toward ketone body synthesis This deregulation results in feedback activation of the anti-lipolytic G protein-coupled receptor GPRA, causing changes in lipid homeostasis and accumulation of lipid bodies in the cell.
ESAT-6, a secreted M. Another strategy used by M. These ICLs are catalytically bifunctional isocitrate and methylisocitrate lyases that allow bacteria survival on even acetate and odd propionate chain fatty acids Figure 8.
Moreover, the miR induced by M. Alveolar macrophages are not only responsible for phagocytosis of these bacteria but also for catabolizing lung surfactant, a lipid—protein complex that lines the alveolar spaces.
Recently, it was found that the scavenger receptor CD36 is redistributed to the macrophage cell membrane following exposure to surfactant lipids and participated in surfactant lipid uptake by these cells Figure 5. These macrophages also supported better bacterial growth in a CDdependent manner Thus, it seems that CD36 mediates surfactant lipid uptake by human macrophages and that M.
In addition to resisting all the microbial effectors within a phagolysosome, several pathogens such as C. By getting out of the phagosome, these microorganisms can in the cytoplasm travel to other cell sites and finally leave the host cell. As mentioned earlier, the fungus C. In addition, it can subsequently escape the cell by a non-lytic tactic known as vomocytosis , Vomocytosis allows for the pathogen escape leaving the phagocytic cell alive Although the molecular details of vomocytosis are not completely described, the process involves an exocytic fusion of the phagosome with the plasma membrane, thus releasing the fungus Figure 9.
Vomocytosis also involves microtubules, but apparently not actin polymerization. These actin structures actually prevent vomocytosis. Yet, fungus strains with high rates of vomocytosis induce more actin flashes, suggesting that these flashes are a reaction from the cell to contain the phagosome. Still, at the end, the fungal phagosome is fused with the cell membrane and the pathogen is liberated Also, the secreted phospholipase B1 PLB 1 is required for vomocytosis It is thought that PLB 1 helps permeabilizing the fungal phagosome to neutralize its lumen and to allow nutrients to come in , Although vomocytosis is a unique escape function known only for cryptococci, a similar process has recently been described for C.
Figure 9. Escape from the phagosome. Several pathogens escape from the phagosome to persist in the less harsh environment of the cytoplasm. Here, the phagosome fuses with the cell membrane with assistance of the secreted phospholipase B1 PLB 1 , leaving the pathogen free and the phagocytic cell alive.
Another intracellular pathogen capable of escaping from the phagosome and then from the infected cell is L. LLO is a pore-forming toxin that permeabilizes the phagosome membrane. It is a potent toxin capable of also degrading the cell membrane, thus its expression and activity are strictly regulated.
In addition, several phospholipases are activated to completely degrade the phagosomal membrane and allow the bacterial escape Once in the cytosol, the bacterium is propelled by the formation of actin tails that push it across the cell. Bacteria from the genus Rickettsia are obligate intracellular pathogens that can also escape phagosomes.
Rickettsia uses a secreted phospholipase A2 to disturb the phagosome membrane Once in the cytosol, Rickettsia produce actin tails that allow them direct cell to cell transfer. Another microorganism that seems capable of phagosome escaping from neutrophils but not macrophages is S. These bacteria produce phenol soluble modulins PSMs , which are peptides with lytic activity toward many mammalian cells Other bacteria, such as M.
After escaping the phagosome into the cytosol, M. The M. Thus, it was thought that all mycobacteria could escape from phagosomes using the pore-forming activity of ESX However, this has to be formally proven experimentally. Therefore, there is no doubt about the capacity of mycobacteria to escape into the cytosol but the significance of this phenomenon is still a matter of debate.
A simple idea is that bacteria need to leave the phagosome to replicate and then leave the cell. However, bacilli escape the phagosome at later times of infection and this is followed by cell lysis and release of bacilli In consequence, escaping from the vacuole is not a requirement for either survival or growth of M.
Instead, it was proposed that the escape from the vacuole represents a transient state that could be critical to the rapid expansion of the bacterial population If this is the case, then escaping from the phagosome is just an important step in the pathology that accompanies progression of tuberculosis infection to active disease. How, mycobacteria kill the cell to allow its release is not clear.
Yet, recently, it was reported that the M. CpnT consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain.
This secreted portion is also named tuberculosis-necrotizing toxin However, the mechanism for this cell lysis remains to be elucidated. Clearly, CpnT has a dual function in M. It is used for uptake of nutrients within the phagosome and for induction of host cell lysis in the cytosol.
The regulation of CpnT functions becomes then a topic of important research for controlling M. Another M. The unique cell wall lipid phthiocerol dimycocerosates greatly augmented the bacteria escape from its intracellular vacuole , by a process not well understood. The mechanism for phagosome lysis is clearly complex as indicated by the fact that host molecules are also recruited by the bacteria to aid in its escape. Activation of host cytosolic phospholipase A2 rapidly led to phagosome lysis for bacteria moving into the cytoplasm of the host cell The study of the many mechanisms used by microbial pathogens to control phagocytosis provides opportunity for detecting novel potential targets of clinical intervention.
Promising therapeutic approaches will be designed based on our new understanding of the tactics pathogens use to interfere with phagocytosis.
For example, studies with miRNA in mycobacteria infections identified TLR2 as a potential target to prevent the blockage of phagosome maturation Figure 5. This explained the increased risk for mycobacterial infections associated with the use of glucocorticoids.
Moreover, this group also found that giving imatinib, a tyrosine kinase inhibitor, to glucocorticoid-treated macrophages induced lysosome acidification and antimicrobial activity without reversing the anti-inflammatory effects of glucocorticoids Thus, an improved therapy would be to administer glucocorticoids together with drugs that induce phagosome acidification.
In another recent report, a phagosome maturation assay using confocal microscopy in THPderived macrophages infected with an attenuated M. Saxifragifolin D a pentacyclic triterpenoid compound first isolated from the rockjasmine Androsace umbellata reduced the inhibition of phagosome maturation.
Using assays of this type, new potential drugs can be tested for future therapies. Another potential therapeutic approach would be to modulate macrophage function to improve their antimicrobial potential against bacterial infections.
The feasibility of such an approach has been suggested in a recent report of macrophage phagocytosis of L. In this study, the engagement of receptor T cell immunoglobulin mucin-3 Tim-3 on macrophages inhibited phagocytosis of L.
In contrast, inhibition of Tim-3 augmented phagocytosis Thus, modulating the Tim-3 pathway to alter macrophage function is a promising tool for treating infectious diseases, such as Listeria infections. Phagocytosis of opsonized particles is, in general, more efficient and more efficacious in eliminating microorganisms. The idea to generate opsonizing antibodies for controlling infections is another promising area of opportunity for novel therapeutics. The value of this approach has been suggested in studies where opsonizing antibodies improve elimination of bacteria.
In a study with five apparently healthy Indian donors having high titers of serum antibodies against M. Another study showed that antibodies directed at the R domain of S.
This coagulase activates host prothrombin and generates fibrin fibers that cover the bacteria and prevent phagocytosis. These antibodies directed the fibrin-covered bacteria to phagocytes and also protected mice against lethal bloodstream infections caused by methicillin-resistant S. Yet, another study, showed that a monoclonal antibody mAb directed at the Protein A could protect neonatal mice against S. A humanized version of this mAb was developed, and it is proposed as a potential new therapy for S.
These reports encourage the development of novel vaccines that favor the formation of opsonizing antibodies against bacterial antigens to activate phagocyte innate immunity. Phagocytosis is a fundamental biological process that in multicellular organisms is required for proper homeostasis and for fighting infections 1 , 2. Therefore, it is not surprising that many microbial pathogens have mechanisms to counteract phagocytosis.
As we have discussed here, for some model pathogens, namely S. For many other microbial pathogens, their tactics for interfering with phagocytosis are only beginning to be defined. Despite the tremendous amount of published studies on microbial phagocytosis or knowledge on microbial control of this biological process is still incipient and fragmented.
We know that some pathogens block phagocytosis at one step or another, but no information is available on how this blockage is accomplished. Some molecules have been identified but their mechanisms of action are not yet described. Future research will serve to fill these gaps and will provide clues on how to improve antimicrobial therapeutics.
An important element for future research is the implementation of novel techniques. Great advances have been achieved by application of proteomics analysis to phagosomes formed under different infection conditions Earlier studies on M.
The effect of a particular protein of the phagocytic machinery identified by proteomics can then also be tested by RNA-mediated interference By comparing the protein profile of phagosomes formed with virulent and avirulent variants of a pathogen, relevant molecules for pathogenesis can be identified.
For example, comparing phagosomes containing highly virulent L. Similarly, comparing macrophage phagosomes formed after triggering different receptors, it was found that phagosome outcome was regulated by the individual receptors triggered for phagocytosis This is in agreement with recent findings that indicate particular FcRs promote particular cell responses on neutrophil phagocytes Thus, phagocytosis is clearly modified according to the receptor involved.
We have a good understanding on how opsonic phagocytic receptors signal, but very little is known about the signaling pathways activated by other phagocytic receptors. This is an area of research that needs much further exploration in the future. Other techniques that have been instrumental for our present understanding of phagocytosis are fluorescence microscopy coupled to particular probes to measure phagosome pH , to describe phospholipid dynamics during phagosome formation , and to quantify antibody-dependent phagocytosis Together with these, the use of confocal microscopy coupled to fluorescence resonance energy transfer-based assays has been helpful to investigate the mechanisms of L.
Equally important, the use of novel microbial readouts of bacterial fitness have been developed to probe the host cell environments that promote or control bacterial growth In particular, M. These assays will be very useful in future studies on phagocytosis of other microbial pathogens. To implement these assays, the proper fluorescent probes will need to be developed. During phagocytosis, both the phagocyte and the microorganism adapt to fight and overcome each other. These changes, important to the final outcome of an infection, can be studied by modern techniques such as transcriptional analysis via RNA sequencing RNA-seq.
Changes in pathogen phenotype under various conditions are revealed when the total transcriptome is analyzed. For example, it is known that cigarette smoke predisposes exposed individuals to respiratory infections by enhancing the virulence of pathogenic bacteria.
A recent study on the effect of cigarette smoke on S. A similar comparative transcriptome study with RNA-seq of Brucella melitensis grown in normal-medium culture and in acid-medium pH 4. Among these genes, a two-component response regulator gene in the transcriptional regulation pathway was identified as important for acid resistance and virulence of Brucella Also, an analysis of RNA-seq data from in vivo and in vitro cultures of Cryptococcus gattii identified highly expressed genes and pathways of amino acid metabolism that would enable these bacteria to survive and proliferate in vivo Hence, particular genes expressed under particular conditions can be identified as potential therapeutic targets for controlling infections.
Likewise, changes in cell phenotype can be analyzed by RNA-seq. For example, increased susceptibility to bacterial pneumonia is found after influenza infections. Therefore, a promising strategy for controlling postinfluenza bacterial pneumonia would be to increase MARCO expression by targeting Nrf2 and Akt signaling in alveolar macrophages.
Another example of RNA-seq analysis of macrophages in two different conditions, namely infection with virulent or avirulent strains of M. The product of one such gene, RAB8B that is required for phagosome maturation, was reduced due to elevated levels of truncated RAB8B variants in cells with virulent mycobacteria Alternative splicing is a new mechanism that M.
The molecular details of this mechanism are not known and will certainly become an area of interesting research in the near future. We have described phagocytosis as a general model based mainly on macrophages. However, there are important differences among diverse types of phagocytes and even between phenotypes of the same phagocyte. As indicated earlier, environmental cues can alter the functioning of a phagocyte, and no much is known about the mechanisms involved in these cell changes.
Hence, this is an area of great interest, as shown by some recent studies. Metabolic conditions can alter macrophage function , and in the case of diabetes mellitus it was found that phagocytosis was reduced This disease is also associated with increased tuberculosis risk and severity. Recently, it was also reported that alveolar murine macrophages from diabetic mice have a reduced expression of MARCO The lack of this receptor could be the reason for inefficient phagocytosis in diabetic cells.
Future research should determine whether other phagocytic receptors are also altered in diabetic macrophages. Nothing is known about the metabolic mechanisms that control phagocyte receptor expression. The role of other phagocytes besides macrophages in controlling some intracellular bacterial infections is just beginning to be appreciated.
For example, neutrophils also participate in controlling M. In addition, other cells such as dendritic cells can also perform phagocytosis by mechanisms that are different from those of macrophages The particular role of these various phagocytic cells in different infection settings will also become an area of fruitful research in the future.
Macrophages not only perform phagocytosis of microbial pathogens but also ingest dead and dying host cells. The process of engulfing apoptotic cells is called efferocytosis, and it has an important role in the resolution of inflammation Although efferocytosis of M. Understanding how macrophages, neutrophils, and dendritic cells process pathogens within a dying cell is another area for future research. Discoveries in this field should lead to novel therapeutics that simultaneously suppress inflammation and promote pathogen clearance.
Elimination of pathogens by macrophages and neutrophils is an essential function of our innate defenses. These phagocytic leukocytes clear microorganisms from tissues via phagocytosis. Once inside the phagocyte, the microorganism is destroyed by a series of degrading mechanisms inside the phagosome.
Despite this, many pathogens have evolved means to prevent phagocytosis or to resist its effects inside the phagocytic cells. Thus, these pathogens remain a considerable health threat. We have presented the main mechanisms phagocytes have for eliminating microbes and then we discussed the strategies used by some pathogens to interfere with each step of the phagocytic process. Our list of pathogens is not complete, since there are many microorganisms capable of resisting phagocytosis in ways, we do not completely recognize.
Technical advances have allowed us to make significant advances toward understanding the molecular details of the interaction between some pathogens and phagocytes, but important questions remain. Future research in this area will certainly bring us interesting surprises that will help us conceive novel therapeutic approaches that could render pathogens more susceptible to phagocyte attack.
CR and EU-Q both equally conceived the issues, which formed the content of the manuscript, prepared the figures, and wrote the manuscript. The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Diversity and versatility of phagocytosis: roles in innate immunity, tissue remodeling, and homeostasis. Front Cell Infect Microbiol Rosales C, Uribe-Querol E. Phagocytosis: a fundamental process in immunity.
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Phagocytes surround any pathogens in the blood and engulf them. They are attracted to pathogens and bind to them. The phagocytes membrane surrounds the pathogen and enzymes found inside the cell break down the pathogen in order to destroy it.
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