Why do neutrophils need to die after pathogen phagocytosis?

Why do neutrophils need to die after pathogen phagocytosis?

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From referenced article below, neutrophils need to be removed because its granule contents and oxygen metabolites (used for killing phagocytosed pathogen) are harmful to the surrounding tissue. Thus, the process used for this is that neutrophils undergo apoptosis, then are phagocytosed by macrophages.

However, I do not see why neutrophils need to first die to be phagocytosed by macrophages. Do macrophages not phagocytose living cells?


Once the pathogens have been dealt with, and to completely resolve inflammation, neutrophils need to be cleared from the tissue… For removal, neutrophils firstly need to die.

Macrophages can certainly phagocytose living cells, as you can read in Sompayrac's "How the Immune System Works", Chapter 1 and 2, but they do need an "eat me" signal of some kind in order to do it. For a bacterium that might be lipopolysaccharide and mannose. For endogenous cells, there is a balance of "eat me" and "don't eat me" signals. Apoptosis, in addition to neatly packaging up potentially toxic intracellular components, involves downregulating the don't eat me signals and upregulating the eat me signals. Macrophages can phagocytose live cells that seem to have some sort of problem (for example, tumor cells), but they are much more efficient at phagocytosing apoptotic cells.

As far as why the neutrophil has to die, in addition to making them more efficient targets for phagocytosis, they die pretty quickly anyway. There is some debate about the exact length of their life span, but from the time they're released by the bone marrow, the average circulating life span is probably less than 24 hours, regardless of whether they exit the blood stream as part of an inflammatory process or not. The exact function of this short lifespan is the source of some speculation, but we do know that, after being recruited in the inflammatory response, apoptotic neutrophils help suppress that inflammation, playing an important role in regulating the processes that sent them out into the tissue in the first place.

Sompayrac's book is a good overview, but if you want to read the gory details this is a good review.


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Phagocytosis, process by which certain living cells called phagocytes ingest or engulf other cells or particles. The phagocyte may be a free-living one-celled organism, such as an amoeba, or one of the body cells, such as a white blood cell. In some forms of animal life, such as amoebas and sponges, phagocytosis is a means of feeding. In higher animals phagocytosis is chiefly a defensive reaction against infection and invasion of the body by foreign substances (antigens).

Pathogen recognition

When a pathogen enters the body, cells in the blood and lymph detect the specific pathogen-associated molecular patterns (PAMPs) on the pathogen&rsquos surface. PAMPs are carbohydrate, polypeptide, and nucleic acid &ldquosignatures&rdquo that are expressed by viruses, bacteria, and parasites, but which differ from molecules on host cells. These PAMPs allow the immune system to recognize &ldquoself&rdquo from &ldquoother&rdquo so as not to destroy the host.

The immune system has specific cells with receptors that recognize these PAMPs. A macrophage is a large, phagocytic cell that engulfs foreign particles and pathogens. Macrophages recognize PAMPs via complementary pattern recognition receptors (PRRs). PRRs are molecules on macrophages and dendritic cells which are in contact with the external environment and can thus recognize PAMPs when present. A monocyte, a type of leukocyte (white blood cell) that circulates in the blood and lymph, differentiates into macrophages after it moves into infected tissue. Dendritic cells bind molecular signatures of pathogens, promoting pathogen engulfment and destruction.

Figure (PageIndex<1>): Blood cells related to the innate immune response: Cells of the blood include (1) monocytes, (2) lymphocytes, (3) neutrophils, (4) red blood cells, and (5) platelets. Leukocytes (1, 2, 3) are white blood cells that play an important role in the body&rsquos immune system. Figure (PageIndex<1>): Cells involved in the innate immune system: The immune system has specific cells whose job is to recognize pathogen-associated molecular patterns. The characteristics and location of cells involved in the innate immune system are described in this chart.

Once a pathogen is detected, the immune system must also track whether it is replicating intracellularly (inside the cell, as with most viruses and some bacteria) or extracellularly (outside of the cell, as with other bacteria, but not viruses). The innate immune system must respond accordingly by identifying the extracellular pathogen and/or by identifying host cells that have already been infected.

What are neutrophils and what do they do?

Neutrophils are a type of white blood cell that helps heal damaged tissues and resolve infections. Neutrophil blood levels increase naturally in response to infections, injuries, and other types of stress. They may decrease in response to severe or chronic infections, drug treatments, and genetic conditions.

Neutrophils help prevent infections by blocking, disabling, digesting, or warding off invading particles and microorganisms. They also communicate with other cells to help them repair cells and mount a proper immune response.

The body produces neutrophils in the bone marrow, and they account for 55–70 percent of all white blood cells in the bloodstream. A normal overall white blood cell level in the bloodstream for an adult is somewhere between 4,500 and 11,000 per millimeters cubed (mm3).

When there is an infection or another source of inflammation in the body, special chemicals alert mature neutrophils, which then leave the bone marrow and travel through the bloodstream to the site in need.

Unlike some other cells or blood components, neutrophils can travel through junctions in the cells that line blood vessel walls and enter into tissues directly.

In this article, we look at the reasons for high or low neutrophil levels, how doctors can test these levels, and what normal neutrophil levels are for different groups.

There are many different reasons why a person may have higher or lower than normal levels of neutrophils in their blood.

High levels

Share on Pinterest Neutrophils are a type of white blood cell.

Having an abnormally high level of neutrophils in the blood is known as neutrophilic leukocytosis, also known as neutrophilia.

Rises in neutrophil levels usually occur naturally due to infections or injuries. However, neutrophil blood levels may also increase in response to:

  • some medications, such as corticosteroids, beta-2-agonists, and epinephrine
  • some cancers
  • physical or emotional stress
  • surgery or accidents
  • smoking tobacco
  • pregnancy
  • genetic conditions, such as Down syndrome
  • surgical removal of the spleen

Some inflammatory conditions can increase neutrophil levels, including rheumatoid arthritis, inflammatory bowel disease, hepatitis, and vasculitis.

Low levels

An abnormally low blood level of neutrophils is a condition called neutropenia.

A drop in neutrophil blood levels typically occurs when the body uses immune cells faster than it produces them or the bone marrow is not producing them correctly.

An enlarged spleen may also cause a decrease in neutrophil levels because the spleen traps and destroys neutrophils and other blood cells.

Some conditions and procedures that cause the body to use neutrophils too quickly include:

  • severe or chronic bacterial infections
  • allergic disorders
  • certain drug treatments
  • autoimmune disorders

Some specific conditions, procedures, and drugs that interfere with neutrophil production include:

  • cancer
  • viral infections, such as influenza
  • bacteria infections, such as tuberculosis
  • myelofibrosis, a disorder that involves bone marrow scarring B-12 deficiency involving bone marrow
  • phenytoin and sulfa drugs medications
  • toxins, such as benzenes and insecticides
  • aplastic anemia, when the bone marrow stops producing enough blood cells
  • severe congenital neutropenia, a group of disorders where neutrophils cannot mature
  • cyclic neutropenia, which causes cell levels to rise and fall
  • chronic benign neutropenia, which causes low cell levels for no apparent reason

Doctors can identify changes in neutrophil levels from a blood test called a complete blood count (CBC) with differential, which identifies specific groups of white blood cells.

A doctor may order a CBC test when someone is experiencing a range of symptoms related to infection, chronic illness, and injury, such as fever, pain, and exhaustion. A nurse or technician will draw a small amount of blood from the arm and send it off for evaluation.

If the initial test shows a higher or lower number of white blood cells than normal, the doctor will likely repeat the test to confirm the results. If the initial results are confirmed, a doctor will perform a physical exam, ask questions about the person’s lifestyle, and review their medical history.

If there is no apparent reason for changes in white blood cell levels, the doctor will order a more specific test. Laboratory specialists will look for specific white blood cells, such as immature neutrophils called myeloblasts. During an infection or chronic illness, these cells emerge from the bone marrow and mature in the blood instead of the bone marrow.

If myeloblasts or other white blood cells appear in significant levels in the blood, the doctor will request a bone marrow sample.

Bone marrow collection involves inserting a long needle into part of the pelvis near the back of your hip. The procedure can be very painful, and a doctor will typically take the sample in a hospital setting with at least a local anesthetic.

Experts will examine the bone marrow sample to see if neutrophils and other blood cells are developing correctly and are in regular supply.

If the cause of the high or low neutrophil levels is still uncertain, the doctor will order other tests to try to pinpoint the cause of the changes, such as:

Changes in neutrophil levels are often a sign of more significant changes in white blood cell levels.

The amount and proportion of white blood cells in the bloodstream change over time with age and other events, such as pregnancy. While everyone’s normal range is slightly different, some commonly used ranges include:

  • Newborn: 13,000 to 38,000 per mm3
  • Infant 2 weeks of age: 5,000 to 20,000 per mm3
  • Adult: 4,500 to 11,000 per mm3
  • Pregnant female (third trimester): 5,800 to 13,200 per mm3

In non-pregnant adults, a white blood cell blood count over 11,000 per mm3 is known as leukocytosis, which is an elevated white blood cell count. Neutrophilic leukocytosis occurs when a person has over 7,000 per mm3 mature neutrophils in their bloodstream.

The lower blood level limit for neutrophils in human blood is 1,500 per mm3. When a person’s levels of neutrophils are low, it is known as neutropenia. The lower the level of neutrophils circulating in the blood, the more severe neutropenia. Neutropenia levels are:

  • Mild neutropenia: 1,000 to 1,500 per mm3
  • Moderate neutropenia: 500 to 999 per mm3
  • Severe neutropenia: 200-499 per mm3
  • Very severe neutropenia: below 200 per mm3

Minor changes in neutrophil or white blood cell levels are typically nothing to worry about as long as they are temporary. A raised white blood cell count often means the body is responding to infection, injury, or stress.

Some people have naturally lower levels of white blood cells and neutrophils than other people due to a range of factors, including congenital conditions.

If neutrophil or white blood cell levels are significantly altered for no apparent reason or remain raised or lowered, a doctor will order more tests to determine the cause.

Severely high or low levels of white blood cells often require emergency care and monitoring. People with severe neutropenia will have an inadequate defense against infection.

People with severe neutrophilia typically have a life-threatening type of infection or other inflammatory illness that requires treatment, such as cancer.

The best way to correct abnormal neutrophil levels is to address and treat the underlying cause.

Antibiotics can treat bacterial infections, while antifungal medicine treats fungal infections. People can treat certain viral infections with medications that slow viral activity. Otherwise, supportive therapies, such as fluids and rest, may be part of the treatment plan.

People with altered neutrophil levels caused by medications or procedures may need to stop or adjust treatments.

People with chronic conditions that disrupt adequate neutrophil production or maturation may need to take drugs that allow the body to raise neutrophil production, such as:

  • colony-stimulating factors
  • corticosteroids
  • anti-thymocyte globulin
  • bone marrow or stem cell transplantation

People with severely low levels of neutrophils often require monitoring, antibiotic therapy, and hospitalization to reduce the risk of severe infection.

This period of intensive care helps keep people with weakened immune systems away from potentially harmful microorganisms. It also supports the body, giving it time to produce more white blood cells.

One of the causes of low neutrophil blood levels is a vitamin B-12 deficiency. Eating foods rich in B-12 may help improve low neutrophil blood levels. Examples of foods rich in vitamin B-12 include:

  • eggs
  • milk and other dairy products
  • meat
  • fish
  • poultry
  • many fortified breakfast cereals and bread products
  • fortified nutritional yeast products

To help reduce the risk of high or low neutrophil levels, people may want to try the following tips:

  • Try not to over-exercise or exercise beyond comfort levels.
  • Reduce stress levels and treat chronic or severe stress.
  • Seek medical attention for signs of infection, such as fever, weakness, fatigue, or pain, and treat infections exactly as prescribed.
  • Follow a healthful, balanced diet.
  • Eat enough protein.
  • Treat chronic conditions, such as genetic or inflammatory conditions, exactly as prescribed.

However, people with only minor or mild changes in their neutrophil blood levels often show no symptoms and do not require any treatment.


Macrophages are not required for the control of E. coli infection in the notochord

We previously showed that K12 Escherichia coli cells injected in the notochord of zebrafish embryos cannot be reached by phagocytes, yet are killed in one day [21]. We confirmed the physical separation of freshly injected K12 from phagocytes by the notochord collagen matrix (S1A and S1B Fig). To verify that this is not a quirk of this laboratory strain, we first compared enteric adherent invasive E. coli strains, E. coli AIEC LF82 and its mutant, LF82-ΔlpfA, E. coli JM83-ΔmsbB strain and laboratory K12 strain in our notochord infection model. We observed that they behaved similarly (S1C and S1D Fig). We therefore went on using the laboratory K12 strain. To investigate the role of macrophages in the observed bacterial clearance, we injected liposome-encapsulated clodronate (Lipo-clodronate) that kills phagocytic macrophages [22,23]. At 1 day post-fertilization (dpf), macrophage/neutrophil dual reporter embryos, tg(mpeg1:mCherry-F)/tg(mpx:GFP), or macrophage reporter embryos, tg(mpeg1:mCherry-F), were injected with 10 nl of Lipo-Clodronate in the posterior caudal vein (intravenous, i.v.). As previously described [22] 24 h after Lipo-Clodronate injection, macrophages were efficiently eliminated without affecting the neutrophil population, nor inducing unspecific toxicity (Fig 1A and 1B). This was correlated with the decrease of mpeg1 mRNA expression in Lipo-Clodronate treated larvae compared to Lipo-PBS controls, as shown by RT-qPCR (Fig 1C). To further confirm the efficiency of lipo-clodronate to suppress macrophage population, we generated another macrophage reporter line with microfibrillar-associated protein 4 (mfap4) promoter whose expression is strong and stable in zebrafish macrophages [24], i.e. the tg(mfap4:mCherry-F) line. Injection of Lipo-clodronate in tg(mfap4:mCherry-F) induced a dramatic reduction in the number of mfap4 + cells (Fig 1D and 1E), showing the suitability of this approach to deplete macrophages. Macrophage depleted larvae were selected and injected in the notochord with fluorescent E. coli. We observed that bacteria were cleared within the first 24 hours post infection (hpi) in both, macrophage-depleted larvae, as well as in control Lipo-PBS injected larvae, as revealed by fluorescence microscopy and CFU counts (Fig 1F and 1G). Importantly, upon notochord infection, neutrophils were normally recruited around the infected notochord regardless of the presence or absence of macrophages (Fig 1H).

(A) Experimental scheme. One dpf tg(mpeg1:mCherry-F/mpx:GFP) or tg(mfap4:mCherry-F) or tg(mpeg1:mCherry-F) embryos were i.v. injected with Lipo-Clodronate (L-clo) or Lipo-PBS (L-PBS). Correctly depleted larvae were selected based on the loss of red fluorescent macrophages, and GFP or DsRed expressing E. coli were injected within their notochord at 2 dpf. The infection outcome was analyzed at 1 and 2 dpi using fluorescence microscopy. (B) Lipo-Clodronate efficiently depletes macrophages without affecting neutrophil population. Experiments were performed as described in (A) on tg(mpeg1:mCherry-F/mpx:GFP). GFP (neutrophils) and mCherry (macrophages) were analysed by fluorescence microscopy at 2 dpf. (C) qRT-PCR measurement of mpeg1 mRNA relative to ef1a in Lipo-PBS and Lipo-clodronate conditions in whole larvae at 3 dpf (pool of 10 larvae, mean values ± Standard Error of the Mean (SEM), three experiments, Mann Whitney test, one tailed, *P<0.05). (D) Tg(mfap4:mCherry-F) were treated with Lipo-Clodronate or Lipo-PBS as described in (A). mCherry (macrophages) was analysed by fluorescence microscopy at 2 dpf. Representative fluorescence overlaid with brightfield images show macrophage depletion in Lipo-Clodronate treated larvae. (E) Macrophage counts (mfap4 + cells) at 2 dpf in indicated conditions (horizontal lines indicate the mean ± SEM, Student test, one-tailed, ***p<0.001). (F) E. coli-GFP infections in the notochord of tg(mpeg1:mCherry-F) embryos are cleared in macrophage-depleted embryos. GFP (E. coli) and mCherry (macrophages) were imaged repeatedly in individual larvae using fluorescence microscopy at 6 hpi and 1 dpi. In both Lipo-PBS and Lipo-clodronate conditions, E. coli-GFP are present in the notochord at 6 hpi (white arrows) but are cleared at 1 dpi (NL-PBS = 5 and NL-clo = 9). Arrowhead shows the recruitment of macrophage in Lipo-PBS injected larvae. Asterisks show the auto-fluorescence of the yolk. (G) CFU counts at 1 dpi in notochord infected of Lipo-PBS and Lipo-Clodronate treated larvae (mean number of CFU per larva ± SEM, NL-PBS = 9 and NL-clo = 5, Mann Whitney test, two tailed, p>0.05, ns = not significant). (H) E. coli infections in the notochord of tg(mpx:GFP) embryos after macrophage depletion with Lipo-Clodronate. GFP (Neutrophils) was imaged in larvae using fluorescence microscopy at 2 dpi (NL-PBS = 25 and NL-clo = 24). Scale bars: 400 μm.

To confirm, that macrophages are not fundamental for bacterial clearance in notochord infection model, we ablate macrophages using tg(mpeg1:Gal4 / UAS:nfsB-mCherry) embryos in which macrophage express gene 1 promoter indirectly drives the expression of E. coli nitroreductase enzyme in macrophages. Treatment of tg(mpeg1:Gal4/UAS:nfsB-mCherry) embryos with the pro-drug metronidazole (MTZ) at 30 hpf (hours post-fertlilization) specifically decreased macrophage number at 1 and 2 days post-treatment (dpT) (S2A and S2B Fig). Tg(mpeg1:Gal4/UAS:nfsB-mCherry) were then infected with E. coli-GFP at 2 dpf in the notochord. MTZ-mediated macrophage depletion did not impact the bacterial burden at 1 dpi (day post-infection) as shown by Fluorescent Pixel Counts (FPC) (S2C and S2D Fig). Altogether, these data show that macrophages are not required for bacterial clearance in this model.

Neutrophils are essential for the control of notochord infection by E. coli

To investigate the role of neutrophils in bacterial clearance, we ablated neutrophils by two independent approaches. First, we specifically inhibited neutrophil development and function by knocking down the G-CSF/GCSFR pathway using a morpholino oligonucleotide (MO) specifically blocking gcsfr/csf3r translation (MO csf3r) [25,26]. Injection of MO csf3r in the neutrophil reporter embryos, tg(mpx:GFP), led to approximately 70% reduction in the total number of neutrophils as compared to larvae injected with a control morpholino (MO CTRL) at 3 dpf (Fig 2A, 2C and 2D). We infected these morphants with 2500 CFUs fluorescent E. coli. Bacteria disappeared in the control larvae (Fig 2B and 2E) while they proliferated in neutrophil-depleted embryos (Fig 2B and 2F). The bacterial proliferation correlated with a further dramatic reduction in neutrophil number at 1 and 2 dpi (days post infection), suggesting neutrophil death (Fig 2D). Subsequently, infected csf3r morphants died between 2 and 3 dpi (Fig 2G) with overwhelming bacterial proliferation and neutropenia (S3B Fig).

Tg(mpx:GFP) embryos were injected at the one cell stage with either csf3r morpholino (MO csf3r) to induce neutrophil depletion or a control morpholino (MO CTRL). (A) Steady-state neutrophil populations were imaged repeatedly in individual morphants using GFP fluorescence in both MO csf3r and control conditions between 2 and 4 dpf. (B) Fluorescent E. coli-DsRed were injected in the notochord of csf3r and CTRL morphants. GFP (Neutrophils) and DsRed (E. coli) fluorescence were imaged at indicated time points. E. coli-DsRed (red) disappeared from 1 dpi in control embryos (left panels), while it increased in csf3r morphants at 1 and 2 dpi (white arrowheads) with a concomitant decrease in neutrophil number (green). Scale bars: 400 μm. (C, D) Quantification of total neutrophils in CTRL (C) and csf3r (D) morphants at the indicated time points following PBS (light grey columns) or E. coli (dark grey columns) injections (mean number of cell per larva ± SEM, Mann-Whitney test, two-tailed, **p<0.005, ***p<0.001, Nlarvae = 7–16 per condition, from two independent experiments). (E, F) E. coli log counts (CFU) in CTRL (E) and csf3r morphants (F) (mean number of CFU per larva ± SEM, Mann-Whitney test, two-tailed, ***p<0.001, Nlarvae = 3–4 per condition). (G) Survival curve of MO csf3r and MO CTRL larvae infected with E. coli from 0 to 3 dpi (Nlarvae is indicated in the figure, log rank test, p<0.001, from two independent experiments).

We also ablated neutrophils, using tg(mpx:Gal4/UAS:nfsB-mCherry) embryos in which the myeloperoxidase promoter (mpx) indirectly drives the expression of nitroreductase in neutrophils. Treatment of tg(mpx:Gal4/UAS:nfsB-mCherry) embryos with metronidazole at 40 hpf specifically depleted neutrophils at 1 and 2 days post-treatment (Fig 3A). Since macrophages are required to clear apoptotic cells, we asked whether neutrophil death in MTZ treatment alters macrophage number or distribution in the triple transgenic line tg(mpx:Gal4/UAS:nfsB-mCherry/ mpeg1:GFPcaax). At 1 dpT, MTZ treatment did not affect the number of macrophages and they were similarly distributed throughout the larva to the control (Fig 3B and 3C). Larvae were then infected with E. coli-crimson and 4 hours after E. coli injection, macrophages were recruited to the infected notochord in both MTZ and DMSO conditions, showing that ablation of neutrophil using nfsB/MTZ system does not impair macrophage response (Fig 3D). Infection outcome was then analysed in tg(mpx:Gal4/UAS:nfsB-mCherry) larvae infected with fluorescent E. coli-GFP. Similarly to csf3r morphants, bacteria were cleared in control larvae (nfsB + DMSO and nfsB - MTZ), while bacteria proliferated in embryos with low neutrophil density (nfsB + MTZ), as shown by fluorescent microscopy and by quantification of bacterial burden (Fig 3E and 3F). These experiments demonstrate that neutrophils are essential for the control of notochord infection by E. coli.

(A-B-C-D) Tg(mpx:Gal4/UAS:nfsB-mCherry/mpeg1:GFPcaax) embryos were treated with DMSO or MTZ at 40 hpf and imaged at 0, 1 and 2 days post-treatment (dpT) with fluorescence microscopy. (A) Quantification of total neutrophils in DMSO and MTZ treated larvae at 0 and 1 and 2 dpT (mean number of neutrophils per larva ± SEM, Student’s test, one-tailed, *p<0.05, ***p<0.001, NDMSO = 21, NMTZ = 13–23). (B) Quantification of total macrophages in DMSO and MTZ treated larvae at 1 dpT (horizontal lines indicate mean values ± SEM, two independent experiments, Student’s test, two-tailed, ns: not significant, p>0.05, NDMSO = 15, NMTZ = 19). (C-D) Transgenic embryos were infected with E. coli-crimson in the notochord one day after MTZ treatment and imaged (C) before infection and (D) at 4 hpi with Spinning Disk confocal microscopy. (C) Representative overlay of maximum projections of montage acquisitions (mCherry and GFPcaax) with transmitted light images show neutrophil and macrophage distribution in DMSO and MTZ treated larvae before infection and (D) macrophage recruitment (arrowheads) at 4 hpi to the notochord (n). White boxes are zoomed areas. Similar results were obtained with 5 and 10 mM MTZ. (E) Tg(mpx:Gal4/UAS:nfsB-mCherry) embryos were treated with MTZ at 40 hpf and, at 3 dpf, larvae were injected either with PBS or E. coli-GFP in the notochord. The outcome of the infection was analysed by fluorescent microscopy. Larva images are representative overlays of fluorescence and transmitted light images at 2 dpi. In the absence of MTZ, neutrophils are massively recruited to the notochord and E. coli is cleared (white arrowheads). In MTZ-treated larvae, E. coli (green arrowheads) grow heavily. Scale bars: 400 μm. (F) Bacterial load quantification by Fluorescent Pixel Count (FPC) in MTZ treated Tg(mpx:Gal4/UAS:nfsB-mCherry) (nfsB + MTZ) at 0, 1 and 2 dpi showing significant differences in the bacterial load with control groups (Tg(mpx:Gal4/UAS:nfsB-mCherry) treated with DMSO referred as nfsB + DMSO and non transgenic siblings treated with MTZ referred as nfsB - MTZ) (horizontal bars indicate the median, Kruskall-Wallis test with Dunn’s post-test, **p<0.01, ***p<0.001, NnfsB+ DMSO = 9–12, NnfsB- MTZ = 8–9, NnfsB+ MTZ = 7–12).

We further investigated the relationship between neutrophil supply and bacterial disappearance in the notochord. Normal neutrophil levels were able to eliminate small amounts of bacteria (S3A Fig), but embryos with depressed neutrophil populations did not survive low bacterial loads (S3B Fig), while a higher bacterial inoculum overcame larvae with a normal neutrophil population (S3C Fig). However, by artificially increasing neutrophil density in the developing embryo through overexpression of gcsfa, we observed that increasing neutrophil density allow the embryo to cope with even higher amounts of injected bacteria (S3D Fig and S4A and S4C Fig). Similar results were observed by overexpressing gcsfb (S4 Fig). Our data reveals that the balance of neutrophils versus bacteria is instrumental for the outcome of the infection and that neutrophil populations are limiting in fighting the infection. To evaluate cell death, Sytox Green, a vital dye which labels DNA of dying cells, was injected into the vein of infected tg(lyz:DsRed) larvae. While PBS and low dose E. coli induced few cell death around the notochord, embryos experiencing neutropenia (i.e. infected with high dose E. coli) displayed increased cell death including dead neutrophils (S5 Fig). This suggests that when the neutrophil versus bacteria balance is not correct, neutrophils die by apoptosis. Of note, by contrast to neutrophil, macrophage number did not decrease, but instead increased 2 days after high dose infection (S6 Fig). These results are reminiscent to what happen in mammals in which neutrophil/bacteria ratio is fundamental for host defence [27].

Neutrophil myeloperoxidase is not required to control notochord infection

Our previous study revealed that approximately one-third of recruited neutrophils degranulate around infected notochords [21]. We therefore investigated the role of the neutrophil-specific myeloperoxidase (Mpx) that is present in the azurophilic granules, in bacterial clearance. We introduced the mpx:GFP transgene in the mpx-null mutant ‘spotless’ [28] to generate tg(mpx:GFP)/mpx-/- offspring in which neutrophils express the eGFP but lack Mpx activity. Active MPX in neutrophil granules can be visualized in zebrafish embryos using Sudan black staining [29]. Sudan Black staining confirmed that neutrophils did not carry Mpx activity in tg(mpx:GFP)/mpx-/- while in tg(mpx:GFP)/mpx+/- siblings, neutrophils contained active Mpx in their granules (Fig 4A). A low dose of fluorescent E. coli was injected in the notochord of 2 dpf tg(mpx:GFP)/mpx-/- embryos neutrophils were normally recruited along the notochord, and the injected E. coli were cleared at 1 dpi as in the wild type (Fig 4B). Mpx is therefore not required for the clearance of E. coli in the notochord.

(A) Tg(mpx:GFP)/mpx +/- (A1, A2) and tg(mpx:GFP)/mpx -/- (A3, A4) embryos were infected with E. coli in the notochord. Sudan Black staining and immuno-detection of neutrophils (anti-GFP) were performed in whole embryos at 1 dpi. The top right panel shows the regions imaged by confocal microscopy in the larvae in A1 and A3 (green box) and in A2 and A4 (red box). Representative transmitted light images, overlaid with a maximal projection of confocal fluorescence images show the presence of black granules in the neutrophils (white arrows) of tg(mpx:GFP)/mpx +/- embryos. MPX granules are absent in neutrophils (white arrowheads) of tg(mpx:GFP)/mpx -/- embryos. Scale bars: 10 μm and white dotted lines outline neutrophils. (B) Tg(mpx:GFP)/mpx -/- embryos were infected with E. coli-DsRed in the notochord. Neutrophils (GFP) and E. coli (DsRed) were imaged repeatedly in individual larvae using fluorescent microscopy at 6 hpi and 1 dpi. While E. coli locates in the notochord at 6 hpi (arrowheads), it disappears at 1 dpi. (Nmpx+/- = 9, Nmpx-/- = 8 embryos per condition, from two independent experiments). Scale bar: 400 μm.

Superoxide is produced in neutrophils of notochord-infected embryos

Neutrophils use different diffusible molecules to fight infections, including NO and ROS. We investigated NO production by neutrophils during the course of notochord infections using the NO reporter fluorescent probe DAF-FM-DA. We used Salmonella infected embryos as positive controls to detect NO production in neutrophils within the Aorta-Gonad-Mesonephros (AGM) (S7A Fig) [30]. As described [31], the notochord itself was labelled by DAF-FM-DA in uninfected embryos, but we could not observe any evidence of NO production by neutrophils in our notochord infection model (S7B Fig). L-NAME was previously shown to specifically inhibit NO synthases in zebrafish larvae [30]. To block NO production in our system, we thus treated larvae with L-NAME and injected E. coli into the notochord. We did not observe any difference in the outcome of the infection between L-NAME-treated larvae and controls (DMSO) (S7C Fig).

The phagocyte NADPH oxidase and ROS production play a key role in the elimination of engulfed bacteria [4]. To detect intracellular ROS accumulation in the form of superoxide anions in tg(mpx:GFP) embryos infected with E. coli, we used Dihydroethidium (DHE), a cell permeable probe that fluoresces in red after reacting with superoxide within the cell [32,33]. First, we imaged the injection site, where some bacteria initially leaked from the pierced notochord and got engulfed by neutrophils and observed that these phagocytosing leukocytes, abundantly produced superoxide in intracellular compartments harboring bacteria, which are most probably phagosomes (Fig 5A and 5B). Green fluorescent E. coli were rapidly lysed within 20 minutes in the putative phagosome (Fig 5B and 5C and S1 Video). We then imaged the upstream region, where bacteria are separated from the recruited neutrophils by the notochord collagen sheath. Interestingly, these recruited neutrophils also produced large amounts of superoxide, even though they had not phagocytosed bacteria (Fig 5D). DHE was also detected at a basal level in notochord surrounding tissues (Fig 5E). To test the specificity of DHE staining in detecting superoxide anions we treated infected embryos with N-acetyl-cysteine (NAC), a broad-specificity ROS scavenger. We observed a general decrease of DHE staining within cells of the trunk and more particularly a decrease of DHE + recruited cells (Fig 5E and 5F) and of DHE + recruited neutrophils (Fig 5E and 5G) around the infected notochord while the number of recruited neutrophils was unchanged by the treatment (Fig 5E and 5H), confirming that DHE probe specifically detects ROS in this model.

(A-D) Two dpf tg(mpx:GFP) embryos were either injected with PBS (A) or infected with E. coli-GFP in the notochord (B, C, D). At 6 hpi, superoxide was detected in living animals using Dihydroethidium (DHE, red) and neutrophils were visualized using GFP fluorescence (green). (A) Representative transmitted light images, overlaid with a maximal projection of confocal fluorescence images show that superoxide is lightly produced in the recruited neutrophil at the injection site. (B) White boxes in the larva image show the regions imaged by high resolution confocal microscopy and green arrowhead shows the injection site. (C) Representative time-lapse maximum projections starting 6 hpi during 16 min, show superoxide presence in phagosomes (white arrows) bearing bacteria (yellow stars: E. coli-GFP, Green) in recruited neutrophils at the injection site. Time is in minutes. (D) Representative transmitted light images, overlaid with a maximum projection of confocal fluorescence images show superoxide in neutrophils (white arrowheads) over the E. coli (yellow stars) infected notochord. Scale bars: 15 μm, dotted lines encase the notochord (NC). (E) Tg(mpx:GFP) larvae were infected with E. coli-GFP in the notochord and treated either with DMSO or NAC. Trunk images are representative maximum projections of single fluorescence (DHE and GFP) and merge channels using confocal microscopy. Scale bar = 50 μm. (F-H) Quantification of recruited DHE + cells (F), recruited DHE + MPX + cells (G), and recruited neutrophils (H) in indicated conditions (mean number of cell/larva ± SEM, ***p<0.001, ns: non significant, NDMSO = 16–17 and NNAC = 13–14, from three independent experiments). The diagrams represent the regions selected for the counting.

NADPH oxidase activity is essential for bacterial killing at a distance and larva survival to notochord infection

To investigate whether this superoxide production could be involved in bacterial killing, we used Apocynin, a NADPH oxidase (NOX) inhibitor [34,35]. Upon notochord infection, Apocynin-treated embryos had reduced number of superoxide producing cells, including recruited DHE + neutrophils at the inflammation site, as compared to DMSO-treated larvae (Fig 6A and 6B), showing the efficiency of Apocynin as a NOX inhibitor in zebrafish. To test whether Apocynin alters the steady state of neutrophils, tg(mpx:GFP) larvae were treated with this drug at 2 dpf. Apocynin treatment decreased the total number of neutrophils after 6 or 24 h of treatment, but by less than 15% (Fig 6C and 6D), showing that this approach is suitable to test the role of NOX in zebrafish neutrophils. Therefore, we infected tg(lyz:DsRed) embryos with a very low dose of E. coli (<1000 CFUs) in the notochord. Even with the very low dose infection, 80% of Apocynin-treated embryos failed to clear the bacteria, while all bacteria were efficiently killed in DMSO-control embryos (Fig 6E). Apocynin-treated embryos displayed unrestricted bacterial growth in the notochord at 1 dpi, as demonstrated with fluorescence microscopy (Fig 6E and 6F). This was correlated with neutropenia and eventually death at 2–3 dpi (Fig 6F and 6G). The effect was specific to the clearance of bacteria in this notochord infection model since Apocynin treatment did not interfere with the clearance of bacteria injected in the muscle, where phagocytosis occurs (S8 Fig). Similar results were obtained using another NOX inhibitor [36], VAS2870 (VAS) (S9 Fig).

(A-B) E. coli-GFP were injected in the notochord of 2 dpf tg(mpx:GFP) embryos in DMSO or Apocynin treatment conditions. (A) At 1 dpi, superoxide production was visualised using DHE (red), neutrophils and E. coli were detected using GFP. Notochord images are representative maximum projection of fluorescence confocal images overlaid with transmitted light images. Pink arrowheads show DHE + neutrophils, white arrows show DHE - neutrophils and white arrowheads: E. coli, scale bars: 30 μm. (B) Quantification of DHE-positive cells in DMSO and Apocynin treated larvae (mean ±SEM, Nlarvae = 5 per condition, Mann-Whitney test, one-tailed, * p<0.05). (C, D) Tg(mpx:GFP) embryos were treated with Apocynin (APO) or DMSO at 2 dpf. Neutrophils (GFP) were imaged using fluorescent microscopy at 6 hours post-treatment (hpT) and 24 hpT. (C) Representative fluorescent images of Apocynin or DMSO treated larvae at 24 hpT. Scale bar: 400 μm. (D) Corresponding counts of total neutrophil population in indicated conditions (mean ± SEM, NDMSO = 31 and NAPO = 29, Mann-Whitney test, two-tailed, * p<0.05, representative of 2 independent experiments). (E, F, G) Two dpf tg(lyz:DsRed) embryos were infected in the notochord with E.coli-GFP and treated with Apocynin. (E) Neutrophils (DsRed) and E. coli (GFP) were imaged repeatedly in individual larvae using fluorescent microscopy at 6 hpi and 1 dpi. Bacteria (white arrowheads) were present at 6 hpi in both DMSO- and Apocynin-treated embryos. At 1 dpi, bacteria disappeared in DMSO-treated embryos (arrows) while their number increased in Apocynin-treated embryos (white arrowheads). (F) Infection outcome of E. coli infected embryos after in DMSO or Apocynin treatments were scored from 0 to 3 dpi (the number of larvae is indicated in the columns). (G) Survival curves of larvae uninfected and infected with E. coli from 0 to 3 dpi in DMSO or Apocynin treatments. (Nlarvae is indicated in the figure, log rank test, p<0.001, from two independent experiments). (H) Two dpf mpx +/+ or mpx -/- embryos were infected in the notochord with E. coli-GFP and treated either with DMSO or Apocynin (APO). Infection outcome of E. coli infected embryos were scored from 0 to 2 dpi (the absolute number of larvae is indicated in the columns). (I) Survival curves of mpx +/+ or mpx -/- larvae infected with E. coli from 0 to 2 dpi in DMSO or Apocynin treatments (Nlarvae is indicated in the figure, log rank test, p<0.01, from two independent experiments).

Interestingly, in mammals, Apocynin activity requires that target cells do express an active Mpx [35]. Therefore, we compared the results of Apocynin treatment in mpx -/- and mpx +/+ infected embryos, and observed that Apocynin increased susceptibility to notochord infection only in the presence of Mpx (Fig 6H and 6I). Thus, Apocynin action is also dependent on Mpx in zebrafish, and thus specifically acts on neutrophils. Overall, these data thus strongly suggest that inhibition of superoxide production in neutrophils increases susceptibility to notochord infection.

To further examine the role of phagocyte NOX, morpholino-mediated gene knockdown was used. Injection of p47 phox MO in tg(mpx:GFP) did not induce noticeable morphological defects, but, as expected, decreased superoxide production in neutrophils following infection compared to control morpholino (CTRL MO) (S10 Fig). To address the effect p47 phox MO on the development and the recruitment of neutrophil, we analyzed tg(mpx:GFP) p47 phox morphants before and after E. coli infection in the notochord at 2 dpf. Although p47 phox morphants displayed 20% less neutrophils than in control morphants, (Fig 7A and 7B) these leukocytes were recruited in normal numbers to the notochord at 4 hpi and 1 dpi (Fig 7C), showing that p47 phox morphants can mobilize neutrophils properly during the infection. Then, p47 phox morphants were infected in the notochord with E. coli-GFP. P47 phox MO induced higher bacterial burden as evidenced by fluorescence microscopy (Fig 7D) and CFUs counts (Fig 7E). This was correlated with an increase in the severity of infection (Fig 7F).

(A-C) Tg(mpx:GFP) embryos were injected at the one cell stage with either p47 phox morpholino (MO p47 phox ) or a control morpholino (MO CTRL). Steady-state neutrophil populations were imaged in 2 dpf morphants using fluorescence microscopy. Scale bar: 400 μm. (B) Neutrophil counts in whole larvae (mean number of neutrophils per larva ± SEM, NMO CTRL = 47 and NMO P47 = 47, Mann-Whitney test, two-tailed, **p<0.005, from three independent experiments). (C) At 2 dpf, p47 phox and CTRL morphants were infected with E. coli-GFP in the notochord and imaged using fluorescence microscopy at 4 hpi and 1 dpi. Graph represents mean number of recruited neutrophils per larva ± SEM in the notochord region (NMO CTRL = 10–14 and NMO P47 = 12–16, Mann-Whitney test, one-tailed, p>0.05 ns: non significant, from two independent experiments). (D) p47 phox and CTRL morphants were infected with E. coli-GFP in the notochord at 2 dpf and GFP fluorescence (bacteria) was imaged repeatedly in individual larva, fluorescence was overlaid with transmitted light images at 1 hpi and 1 dpi. Black arrowheads indicate the infection site, scale bar: 400 μm (NMO CTRL = 19/21 and NMO p47 = 16/21). (E) The CFU counts at 1 dpi in notochord infected of p47 phox and CTRL morphants (mean number of CFU per larva ± SEM, NMO CTRL = 13 and NMO P47 = 8, Mann-Whitney test, one-tailed, **p<0.01, from three independent experiments). (F) Survival curves of p47 phox and CTRL morphants that have been injected with PBS or E. coli in the notochord from 0 to 3 dpi (N is indicated in the figure, log rank test, ***p<0.001, from 4 independent experiments).

As neutrophils are instrumental for larva survival and bacterial clearance during notochord infection and as pharmacological (apocynin and VAS2870) and genetic (p47 phox morpholino) inhibition caused a slight decrease of neutrophil numbers, we tested whether inducing high neutrophil number in the context of NADPH incompetence could restore survival of the infected larvae. One-cell stage tg(lyz:DsRed) embryos were thus injected with gcsfa expressing plasmid and 2 days later were treated either with DMSO or VAS2870 (Fig 8A). Beside the fact that gcsfa forced expression increased the number of neutrophils compared to controls (Fig 8B), it did not restore a better survival of the infected larvae in the presence of Nox inhibitor VAS2870 (Fig 8C).

(A) Diagram shows the experimental strategy to induce high neutrophil number during NOX inhibition in zebrafish larvae. To increase neutrophil density, tg(lyz:DsRed) embryos were injected with the gcsfa over-expressing plasmid at one cell stage. At 2 dpf, larvae were treated with VAS2870 to inhibit NOX enzyme. Then, NOX incompetent larvae were infected with E. coli-GFP for monitoring of the survival. (B) Tg(lyz:DsRed) larvae were imaged 5 h after treatment with DMSO or VAS2870 using fluorescence microscopy. The plot shows quantification of total neutrophils in indicated conditions (horizontal lines indicate mean number of neutrophils ± SEM, from two independent experiments, ANOVA with Tukeys’ post-test, **p<0.01 and ***p<0.001). (C) One hour after treatment (at 2 dpf) larvae were infected in the notochord with E. coli-GFP. Survival curves of larvae in indicated conditions from 0 to 3 dpi (Nlarvae is indicated in the figure, log rank test, *p>0.05, ns: not significant).

Altogether these data show that NOX-induced superoxide is necessary for bacteria elimination at a distance by neutrophils.

Intracellular Mediators of the Cell Death of Neutrophils

Free Radicals

In a normal process and under the appropriate stimuli, neutrophils are capable of releasing cytotoxic mediators, such as ROS and RNS, generating damage to pathogens but also to the host's tissues. After the elimination of the proinflammatory stimuli, repaiment of the damaged tissue is necessary to return the tissue to a homeostasis state. At this point, anti-inflammatory signals start to be released contributing to the resolution of inflammation, and neutrophils in the tissue should enter apoptosis and be ingested by macrophages in order to clean the inflamed area. Apoptosis of neutrophils is regulated by intracellular mediators and extracellular signals among the intracellular mediators are the ROS, mainly produced by the NADPH oxidase of the activated neutrophils (161, 162), although some reports show that ROS can be generated by mechanisms that are independent of the NADPH oxidase. Thus, it was reported that the production of superoxide and hydrogen peroxide can be mediated by low-conductance, calcium-activated potassium channels known as SK (small conductance) channels (163, 164). Another way of generating ROS in the neutrophil is through the accumulation of electrons of the respiratory chain, linked to a low activity of complex IV due to the low levels of cytochrome c (16, 165�). Decreasing intracellular ROS levels by reduced glutathione (GSH), as well as by catalase, inhibits the death of neutrophils (39, 168, 169).

ROS have the capacity to damage DNA generating cell death through direct or indirect activation of p53 (170). ROS can also induce activation of the inflammasome, formed by NLRs, an adaptor molecule, and caspase-1 (171, 172). However, ROS can directly alter the activity of the intracellular signaling pathways implicated in the death and survival of neutrophils, such as NF-㮫 and MAPK (mitogen-activated protein kinases) (173, 174). In fibroblasts, ROS cause oxidation and inhibition of JNK-inactivating phosphatases, promoting JNK activation, release of cytochrome c, and the subsequent activation of caspase-3 (175, 176). Similarly, it has been demonstrated that the accumulation of ROS results in the grouping of death receptors in lipid rafts and the activation of caspase-8, independently of the Fas ligand (29). The generation of intracellular (but not phagosomal) ROS, caused by neutrophil activation, triggers the process of apoptosis, while the production of intraphagosomal ROS during phagocytosis does not have this effect. This suggests that the location of the production of ROS is fundamental for inducing the death of neutrophils (177). Among the molecules involved in the regulation of ROS production after cell activation, is Bruton's tyrosine kinase (Btk). The absence of this kinase induces a greater production of ROS by hyper phosphorylation and activation of phosphatidylinositol-3-OH-kinase (PI3K) and protein tyrosine-kinases (TKPs) (178). In addition, the levels of free radicals can be elevated by nitric oxide synthase (NOS) through the production of nitric oxide (NO) (177, 179). In recent years, it has been demonstrated that NO has also the capacity to induce apoptosis in neutrophils (180). It has been shown that the derivative of oxatriazol-5-amine, PGE 3162, and SIN-1 increase the rate of apoptosis in human neutrophils, correlating with data of the greater DNA fragmentation and cell death in neutrophils treated with exogenous NO (181, 182). Interestingly, high levels of ROS or RNS in neutrophils inhibits the activity of the caspases, suggesting the existence of an alternative, caspase-independent cell-death pathway (177, 179, 183, 184).

Caspases and Calpains Involved in Apoptosis

Most stimuli that lead to apoptosis converge in the mitochondria inducing the permeabilization of their external membrane. With permeabilization, a series of proteins are released that activate the caspases (185), which carry out the majority of the proteolytic events of apoptosis and are considered as the ultimate responsible for cell death. Caspases are localized in the form of procaspases in the cytoplasm and the intermembrane space of the mitochondria. Procaspases are activated by cleavage and act as executors cleaving cellular survival molecules triggering processes that induce cell death. Caspases are regulated at the post-translational level and possess a classic structure consisting of a prodomain (N-terminal), a small subunit (p10) in the C-terminal, and a large subunit (p20) that contains the active center with cysteine within a QACXG-conserved motif (186, 187).

The initiator caspases possess prodomains larger than effector caspases. The latter contain caspase recruitment domains (CARD), as in the case of caspase-2 or caspase-9, or cell death effector domains (DED), as in the case of caspase-8 and caspase-10, which permits them to interact with other molecules that regulate their activation. The stimulus for cell death induces the grouping of the initiator caspases (Scaffold-Mediated Activation), allowing them to transactivate by cross-cleavage and, thus, proceed to activate the effector caspases. In all the studied cases, the mature enzyme is a heterotetramer that contains two p20/p10 heterodimers and two active centers (186). In neutrophils, caspases 3 and 10 play an important role in the induction of cell death (188). In Helicobacter pylori-infected neutrophils, the activity of caspase-1 is increased, promoting its association into inflammasomes to participate in the triggering of pyroptosis (189). In neutrophils, the activity of caspase-9 also increases drastically during apoptosis (167).

The calpains are calcium-dependent cysteine proteases that can be divided into two subfamilies according to the cation concentrations necessary for their activation: micromolar for the μ-calpains and millimolar for the m-calpains. They play an important role in cell proliferation, progression of the cell cycle, cell migration, and programmed cell death (190). Calpains are present in the cytosol as inactive enzymes that are activated in response to increases in the cellular concentration of Ca 2+ ions. Once activated, they can modify transcription factors, cytoskeletal proteins, kinases, and proapoptotic proteins, such as Bid and Bax, into fragments incapable of interacting with antiapoptotic proteins, inducing the release of cytochrome c and activation of caspase-3 leading to apoptosis (191, 192). In human neutrophils, calpains do not only play a role in apoptosis, but calpains are also involved in the adhesion of TNF-α-stimulated cells, and play a role in migration regulated by the cytosolic Ca ʲ concentration (193, 194).

Bcl2 and IAP (Apoptosis-Regulatory Proteins)

The process of apoptosis in human cells is regulated by a family of diverse pro-apoptotic and anti-apoptotic proteins, with protein Bcl-2 as the prototype protein. Members of this family are grouped into three subfamilies as follows: the antiapoptotic proteins (Bcl-2, Bcl-Xl, Mcl-1, and others), the “multidomain”-type proapoptotic proteins (Bax and Bak), and the 𠇋H3-only”-type proapoptotic proteins (Bid, Bim, Bad, among others). The balance among these three groups determines susceptibility to cell death or survival as shown by the increased resistance to apoptosis of certain tumors related to the overexpression of the antiapoptotic proteins.

Another family of proteins with the ability to inhibit apoptosis (denominated IAPs) regulate the cytochrome c-caspase pathway. In humans, the following three members have been characterized: XIAP: c-IAP 1, and c-IAP 2, which bind to caspase-9 preventing its activation. In neutrophils, caspase-9 increases its activity during apoptosis, even though the levels of cytochrome c in these cells are very low (165�, 195). Data obtained by Murphy et al. (196) suggest that neutrophils possess a lower threshold of cytochrome c for the assembly of functional apoptosomes and their low content of cytochrome c can be compensated for by the elevated expression of apoptotic protease-activating factor 1 (Apaf-1). These further suggest that neutrophils retain a low expression of cytochrome c for the assembly of functional apoptosomes rather than for oxidative phosphorylation.

Neutrophils contain few mitochondria and these are found as tubular networks, and are grouped and depolarized under apoptotic conditions. Despite the relatively low levels of cytochrome c in these cells, the mitochondrial death pathway is functional (167, 197). In neutrophils, the expression of antiapoptotic molecules, such as Mcl-1 and Bcl-xl, is preferential in comparison with those of the proapoptotic protein family (198).

One of the mechanisms that regulate the transcription of the antiapoptotic proteins in neutrophils is stimulation with GM-CSF and TNF-α, which prevents the time-dependent nuclear localization of Mcl-1, and an increase in the transcription and translocation of Bcl-Xl, via stimulation of the NF-㮫 pathway. However, these cytokines also participate in the increase and maintenance of RNAm levels of BH3-only proapoptotic proteins. GM-CSF is a survival factor for neutrophils that promotes the transcription of Bim and the expression of BimeL (199). During neutrophil cell death by apoptosis, Mcl-1 levels diminish gradually, leading to the release of Bax and to its later translocation to the mitochondrial membrane (200). The amount of Mcl-1 can be regulated at the transcriptional level through NF-㮫 and PI3K (201). Another mechanism of regulation of the neutrophil mitochondrial integrity is the flow of potassium (K). A high concentration of extracellular K + promotes the survival of neutrophils through the prevention of mitochondrial dysfunction and the release of proapoptotic factors.


ROS production in phagocytes serves multiple purposes, from cell signaling to microbial killing. Numerous reactive species over a wide range of concentrations are involved. Timing and location of ROS production, diffusion, and scavenging are critical for the outcome. To illustrate this complexity, we referred the reader to reviews that deal with fundamental aspects of phagocyte ROS production wherever possible, and we apologize to those whose work is not mentioned explicitly. The phagocyte community needs new probes for high-level (phagosomal) ROS detection, as well as low-level (signaling) detection. The recent interest in redox biology has pushed the development of new detection methods with improved specificity, better sensitivity, and new ways to localize the detector with cells. The harsh conditions of the phagosome (pH, proteases, level, and diversity of ROS) are particularly challenging to any detection method, and the specificity of any new dye should be tested under these conditions. Subcellular targeting of FPs and the SNAP-tag technology for organic dyes are good candidates to improve the spatial resolution of ROS detection. The end of ROS production may be addressed with nanoparticles. The spatiotemporal correlation of ROS production with signaling events will be addressed by combining dyes of different color that become available now. We are convinced that new organic compounds, as well as FPs, will increase our choice to explore in more detail the why, when, and where of phagocyte ROS production.

Why do neutrophils need to die after pathogen phagocytosis? - Biology

Phagocytosis is the process by which a cell takes in particles such as bacteria, parasites, dead host cells, and cellular and foreign debris. It involves a chain of molecular processes. Phagocytosis occurs after the foreign body, a bacterial cell, for example, has bound to molecules called “receptors” that are on the surface of the phagocyte. The phagocyte then stretches itself around the bacterium and engulfs it. Phagocytosis of bacteria by human neutrophils takes on average nine minutes to occur. Once inside the phagocyte, the bacterium is trapped in a compartment called a phagosome. Within one minute the phagosome merges with either a lysosome or a granule, to form a phagolysosome. The bacterium is then subjected to an overwhelming array of killing mechanisms and is dead a few minutes later. Dendritic cells and macrophages, on the other hand, are not so fast, and phagocytosis can take many hours in these cells. Macrophages are slow and untidy eaters they engulf huge quantities of material and frequently release some undigested material back into the tissues. This debris serves as a signal to recruit more phagocytes from the blood. 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. However, if they receive a signal directly from an invader, they become “hyperactivated”, stop proliferating, and concentrate on killing. 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.

When an infection occurs, a chemical “SOS” signal is given off to attract phagocytes to the site. 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. Chemotaxis is the process by which phagocytes follow the cytokine “scent” to the infected spot. 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.

Killer Skills of a Neutrophil

I'd like to tell you a secret. I am a superhero. I can devour my enemies whole, release my own chemical weapons and trap and kill my prey in nets spun from my own DNA. And I don’t even need to wear my pants on the outside.

I am a neutrophil, and several billion of me are made in your bone marrow every day.

Neutrophils are the most abundant white blood cell in the human body. They play a vital role in an ancient, rapid response called the innate immune system which is our first line of defense against disease-causing microbes. This system can mount a protective offense within minutes of the body being invaded, before the nature of the attack is known. This buys time for the body to produce a tailored response. The neutrophil is at the heart of the action, a killing machine that destroys unwanted intruders.

The neutrophil has many enemies. Perhaps you have a snot-filled toddler, a slobbery dog, or a propensity for paper cuts, but there will be something that exposes you to infection. Within minutes of infection invading your body the damaged tissue releases a chemical distress signal that attracts neutrophils out of the blood stream and activates them.

Activated neutrophils employ three key killing strategies. First, they can engulf and devour microbes. This process, called phagocytosis, was first described over one hundred years ago by Mechnikov who won the Nobel Prize for Medicine in 1908. In his Nobel lecture he described “white corpuscles of the blood. which absorb the microbes and destroy them” [1]. The process of cell devouring is directed by molecular tags called opsonins which are produced by the body and stick to microbes. Imagine the microbe is a cookie: opsonins are like chocolate chips which make the cookie that much more appealing to the hungry neutrophil. Once consumed, the microbe is exposed to enzymes which kill and digest it.

The neutrophil's second strategy, called degranulation, kills microbes occupying the local area. The neutrophil releases packets of enzymes which attack the outside of the microbe. This is like pouring boiling oil on invaders crude but effective. Unfortunately this can cause collateral damage to the very tissue the neutrophils are meant to protect. The damage is limited because the neutrophils are designed to die 24-48 hours after moving into the tissue. As the dead neutrophils accumulate we can see evidence of them in the form of pus.

Even in death the neutrophil works to bring down enemy forces through its third killer creation: Neutrophil Extracellular Traps (NETs). NETs are a relatively recent discovery, outlined in 2004 by Brinkmann and colleagues. NETs are created once the neutrophil's self-destruct programme has been engaged. DNA, proteins and hostile enzymes mingle within the cell which bursts open in a final kamikaze act that unleashes a web which can trap and kill bacteria. This works against an array of different bacteria, from Shigella, which causes dysentery, to Salmonella, which is responsible for typhoid fever.

Understanding this triad of killer skills is an important area of biomedical research. Neutrophils are designed to be part of a hard-and-fast response. If their assault is abnormally prolonged or excessive it can cause more harm than good. This process contributes to common autoimmune diseases including rheumatoid arthritis and emphysema. By understanding how neutrophils cause damage we hope to design new anti-inflammatory drugs to tone down the response and tackle these crippling conditions.

Yet we must not forget that we need neutrophils. Without their killer skills you couldn't go for a stroll in a park or kiss someone without risking death by infection. This is a reality for people on certain chemotherapy regimes which decimate neutrophil numbers. Doctors can try to protect patients by putting them in dedicated isolation rooms and using stringent hygiene controls. However these are short term measures and what patients really need are their neutrophils. We can stimulate recovery of neutrophil numbers using medications that promote their production and therefore give patients their freedom back.

We've all heard of the villains – superbugs, anthrax, flesh-eating bacteria. We've celebrated medicine's pharmacological victories like penicillin. It's time we recognise the remarkable feats happening inside each and everyone of us every day. To uncover these mysteries it is imperative that we keep funding research in this tough economic climate – it took almost 100 years between finding phagocytosis and NETs. There is so much more to be found, and to find it we have to keep looking. Forget space, forget the ocean floor, the human body is a veritable treasure trove for scientific explorers and the spoils – improved quality and quantity of life – can be enjoyed by all. So next time you see some pus, take a second to marvel at those millions of superheroes and the scientists helping us to understand them.

[1]. Ilya Mechnikov's Nobel Lecture (Accessed 23/4/12)

Image: Neutrophil engulfing anthrax bacteria, taken with a Leo 1550 scanning electron microscope. Scale bar is 5 micrometers.From "Neutrophil engulfing Bacillus anthracis". PLoS Pathogens 1 (3): Cover page. DOI:10.1371. November 2005.

The views expressed are those of the author(s) and are not necessarily those of Scientific American.

(NETs). A web of chromatin and granule proteins that are expelled from neutrophils during a unique form of cell death called ‘NETosis’ . The biological role of NETs is still debated.

The phenomenon of increased O2 consumption on neutrophil activation. It is primarily due to a non-mitochondrial mechanism through the activity of the neutrophil NADPH oxidase NOX2.

A subset of circulating granulocytes with unusually low density that appear in the mononuclear fraction during density gradient separation of leukocytes. Low-density granulocytes are abundant in certain autoimmune diseases, such as systemic lupus erythematosus. Their origin and functional importance in disease pathogenesis are poorly understood.

G protein-coupled receptors recognizing N-formylated peptides of bacterial or mitochondrial origin during bacterial infection or tissue damage, respectively.

The phenomenon of massive focal accumulation of neutrophils at sites of infection or tissue injury. It is likely mediated by positive-feedback amplification of neutrophil recruitment signals.

A member of a family of transmembrane enzyme complexes leading to the generation of superoxide (O2 .– ) radicals. They are involved in reactive oxygen species generation by neutrophils (through NOX2), as well as several other redox signalling processes.

A member of a family of enzymes involved in the citrullination of proteins (that is, the conversion of arginine into citrulline residues). Besides a number of biological functions, citrullination is also thought to generate neoantigens during autoimmune diseases.

An adapter protein linking immune receptors to nuclear factor-κB activation in myeloid cells during fungal infection and other inflammatory processes.

An intracellular tyrosine kinase mediating immunoreceptor tyrosine-based activation motif (ITAM)-based signalling by B cell receptors, Fc receptors and certain C-type lectins. SYK has diverse roles in immunity and inflammation.

(JAK). A member of a family of intracellular tyrosine kinases mediating signalling by most (but not all) cytokine receptors through activation of signal transducer and activator of transcription (STAT)-family transcription factors. The JAK family consists of JAK1, JAK2, JAK3 and TYK2.

An antiapoptotic member of the BCL-2 family present in various immune cells and overexpressed in certain tumours. MCL1 blocks the intrinsic apoptotic programme of neutrophils, and therefore MCL1 deficiency leads to severe neutropenia.

Resolution of inflammation

An active process of restoring normal tissue structure and function after an acute inflammatory insult. Defective resolution is thought to lead to chronic inflammation.

Myeloid-derived suppressor cell

(MDSC). A diverse subset of myeloid cells that promote tumour development by suppressing antitumour immunity. MDSCs may phenotypically be similar to monocytes (monocytic MDSCs) or granulocytes (granulocytic or polymorphonuclear MDSCs).

Plasmacytoid dendritic cells

A unique circulating subset of dendritic cells capable of producing large amounts of type I interferons. Besides their role in antimicrobial host defence, they likely contribute to autoimmune diseases such as systemic lupus erythematosus.

Anti-citrullinated peptide autoantibodies

(ACPAs). Autoantibodies against various citrullinated autoantigens present in a subset of patients with rheumatoid arthritis. It is still unclear how ACPAs participate in the pathogenesis of rheumatoid arthritis.

An immune signalling pathway whereby IL-23 leads to IL-17 production by T helper 17 cells. Besides its role in antimicrobial host defence, the IL-23–IL-17 axis also participates in the development of various autoimmune and inflammatory diseases, such as psoriasis, and serves as a regulator of granulopoiesis.

Neutrophils accumulating within the tumour tissue as one of the dominant tumour-infiltrating immune cell types in certain tumours. Tumour-associated neutrophils may exert either antitumoural (N1) or pro-tumoural (N2) effects.

Local environments in distant secondary organs that promote the engraftment and colonization by primary tumour cells, leading to metastasis formation. Preparation of premetastatic niches begins long before the actual translocation of primary tumour cells.

A process whereby immune cells extract membrane fragments and cytoplasm from target cells by mechanically tearing out parts of the target cell. Neutrophils use trogocytosis to kill cancer cells in a process called ‘trogoptosis’.

(AAV). Small-vessel vasculitis co-occurring with circulating antibodies against neutrophil components (anti-neutrophil cytoplasmic antibodies (ANCAs)). It is generally believed that ANCAs and ANCA-mediated neutrophil activation play a pathogenetic role in AAV.

The acute exacerbation of gouty arthritis, characterized by massive inflammation caused by deposition of monosodium urate crystals. Neutrophils are believed to be involved in the inflammation process during gout flares.

Endoplasmic reticulum stress

The accumulation of misfolded or unfolded proteins in the endoplasmic reticulum, for example, during prion diseases or on mutations leading to folding defects. Endoplasmic reticulum stress triggers a process called ‘unfolded protein response’ and may lead to apoptosis of the cell.

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