The many functions of autophagy in neutrophil extracellular trap formation

Kelsey Rogers

Human Genetics Preliminary Exam EIHG Room 6205

May 29, 2013 at 2pm

Kelsey  Rogers  

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Abstract

Neutrophils are essential innate immune cells, and among the first cells to respond to an

infection1. They participate in the immune response through the release of toxic granule proteins,

signaling to other immune cells, and intracellular killing of microbes by phagocytosis2. Recently

it was discovered that neutrophils can also kill pathogens extracellularly, through the release of

neutrophil extracellular traps (NETs) composed of chromatin and antimicrobial proteins3. The

unique cell death program leading to NET formation, termed NETosis, is an evolutionarily

conserved process observed in granulocytes of humans, mice, and fish4,5. NETosis involves

drastic cell morphological changes, but the molecular mechanisms regulating the complex

membrane trafficking events are largely unknown. NETosis could thus serve as a model to

examine complicated cell biological and membrane trafficking events. Recent studies have

implicated autophagy, a ubiquitous lysosomal degradation and recycling process, in the

membrane changes observed during NETosis6,7. I hypothesize that autophagy is necessary for

NETosis, mediating changes in lysosome integrity and the secretion of proinflammatory factors.

Specific Aims

NETosis is known to involve autophagy8, leading in some cases to massive vacuolization

of cellular contents6 and in others to the sequestration of proinflammatory factors7. It is unknown

whether autophagy is specifically required for NETosis, and what effect the formation of

autophagosomes has on cargo or lysosome integrity. I propose that autophagy is necessary to

induce NETosis, mediates changes in lysosome integrity, and in severe inflammatory conditions,

is also required for secretion of the proinflammatory factor high mobility group box 1 (HMGB1).

To test this hypothesis, I will use a genetic approach to examine the necessity of autophagy for

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NETosis (Aim 1). Additionally, I will test whether

autophagy mediates changes in lysosome integrity

during NETosis (Aim 2). Finally, I will investigate

whether HMGB1 secretion is mediated by secretory

autophagy during NETosis (Aim 3). These

experiments will help to illuminate the molecular

mechanisms regulating complex membrane events

in NETosis (Figure 1).

Aim 1. Determine if autophagy is necessary for

NETosis, specifically during the early

vacuolization of the cell. NETosis is characterized by extreme morphological changes that

occur within the neutrophil. These changes involve massive vacuolization of cellular contents,

decondensation of chromatin, and finally plasma membrane rupture and NET release9. During

vacuolization, many of the newly formed vesicles in NETosis display hallmarks of

autophagosomes, such as a double membrane and the membrane-associated protein LC36.

Further supporting a required role for autophagy, pharmacological inhibition of autophagy

blocks NET release6,7. However, these inhibitors target entire classes of phosphatidylinositide 3-

kinases (PI3K)10, and thus are not restricted to autophagy per se. Direct evidence is lacking for

autophagy-dependent NETosis. Therefore I propose to establish a genetic system to determine

the necessity of autophagy in NETosis. I will determine whether neutrophils lacking the essential

autophagy gene Atg5 are capable of generating NETs (Aim 1.1). Using this system, I will also

examine whether autophagy is necessary for the initial vacuolization step of NETosis (Aim 1.2).

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Aim 2. Determine if NETosis alters lysosome integrity, either through changes in

acidification or membrane stability. The typical endpoint for an autophagosome is fusion with

the acidic lysosome, followed by degradation of vesicle contents11. The lysosome is an essential

organelle for cellular homeostasis because of its recycling functions, but perturbations in

lysosome integrity can instead lead to cell death12. Specifically, lysosome membrane

permeabilization (LMP) results in the release of active enzymes capable of inducing apoptosis

and necrosis13. LMP has been previously observed in neutrophils in response to inflammatory

stimuli14, but has yet to be addressed in the context of NETosis. It is unknown whether LMP or

any changes in lysosome integrity occur during this cell death program. However, NETosis is

known to induce autophagy and generate reactive oxygen species8, both of which can trigger

LMP15. To identify defects in lysosomes during NETosis, I will determine whether the pH of the

lysosome increases, as lysosome enzyme activity depends upon an acidic environment (Aim

2.1). I will further examine whether LMP occurs during NETosis, as another possible change in

lysosome integrity (Aim 2.2).

Aim 3. Determine if the proinflammatory factor HMGB1 released during NETosis is

actively secreted via autosecretion. HMGB1 is actively secreted by neutrophils in response to

inflammatory conditions16, and acts as a proinflammatory mediator when present

extracellularly17. This is significant during extreme inflammatory conditions like sepsis, when

HMGB1 serum levels correlate with disease severity18. Understanding the mechanisms driving

HMGB1 secretion during sepsis can elucidate pathways for therapeutic intervention. Neutrophils

from patients with sepsis generate NETs that contain HMGB17, but it is still unclear whether this

is due to active secretion or passive release. In support of active secretion are the findings that

HMGB1 localizes to autophagosomes7 and is detected extracellularly prior to NET release19.

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These data are consistent with the proposed autophagy-based secretion pathway for HMGB1 in

response to inflammation20,21. However, it has not been directly tested whether autosecretion is

the mechanism of HMGB1 secretion in neutrophils, or whether active secretion is the main

source of NET-associated HMGB1. As acetylation of HMGB1 is required for secretion20, I will

examine levels of acetylated HMGB1 isolated from NETs (Aim 3.1). Additionally, I will

investigate whether secretory autophagy is required for the extracellular release of HMGB1

during NETosis (Aim 3.2).

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Introduction

Neutrophil Extracellular Traps: Neutrophils are the most abundant immune cells in the human

body and the first cells to migrate to sites of infection. These  essential  innate  immune  cells  act  

as   phagocytes,   signal   to   other   immune   cells,   and   can   also   kill   pathogens   extracellularly,  

through  the  release  of  neutrophil  extracellular  traps  (NETs)1.  NETs  are  composed  of  DNA,  

histones,   and   antimicrobial   proteins3.   NET   release   results   in   death   of   the   neutrophil  

independent   of   apoptosis   and   necrosis,   and   this   unique   form   of   cell   death   is   termed  

NETosis9.   Within   NETs,   the   decondensed   chromatin   traps   pathogens   to   prevent  

dissemination22,  accompanied  by  antimicrobial  activity  mediated  by  the  high  concentration  

of   histones23   and   other   NET-­‐associated   proteins24.   NETosis   is   activated   in   response   to  

infection   by   bacteria,   fungi,   and   viruses,   the   protein   kinase   C   agonist   phorbol   myristate  

acetate   (PMA),   and   in   certain   inflammatory   conditions   such   as   sepsis,   systemic   lupus  

erythematosus   (SLE),   cystic   fibrosis,   and   preeclampsia25,26.   NET   components   can   both  

enhance   host   defenses   and   induce   inflammatory   injury27,   highlighting   the   necessity   for  

tight  control  of  NETosis.  Understanding  the  intricate  cellular  processes  regulating  NETosis  

may  lead  to  novel  methods  for  treating  a  diverse  set  of  inflammatory  diseases.

  Neutrophils  undergo  multiple  morphological  changes  during  NETosis  (Figure  3),  but  

the  molecular  mechanisms  driving   these  changes  are   largely  unknown.  After   stimulation,  

neutrophils  initially  display  massive  vacuolization,  followed  by  chromatin  decondensation  

and  loss  of  lobulated  nuclei9.  Subsequently,  the  nuclear  envelope  disintegrates  into  distinct  

vesicles  and  granules  disappear,  presumably  allowing  cellular  contents  to  mix.  Finally,  the  

plasma   membrane   ruptures,   releasing   the   NET.   Chromatin   decondensation   and   NET  

release  are  dependent  on  NADPH-­‐oxidase-­‐mediated  superoxide  production,  as  neutrophils  

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from   chronic   granulomatous   disease   patients   with   mutations   in   the   NADPH   oxidase  

complex   fail   to   generate   NETs9.   Autophagy   has   also   been   implicated   in   driving   the  

morphological   changes   observed   in   NETosis,   as   many   of   the   vesicles   formed   during  

NETosis   possess   hallmarks   of   autophagosomes6.   However,   it   is   currently   unknown   how  

autophagy  drives  NETosis.  

Degradative   Autophagy:   Autophagy is a ubiquitous degradative and recycling process in

eukaryotic cells. Autophagy is required for normal cell homeostasis, and is induced by stress

conditions such as starvation and reactive oxygen species11. Autophagy can also lead to cell

death28, depending on the severity or duration of induction29. During autophagy, cytoplasmic

contents are packaged into double-membraned autophagosomes and transported to the lysosome

for degradation11. These events are mediated by autophagy-related genes (Atgs), most of which

are conserved from yeast to mammals30. The molecular events of degradative autophagy can be

conceptually divided into seven discrete steps. These steps are: cargo recognition and packaging,

vesicle nucleation, expansion, completion, fusion with the lysosome, degradation, and recycling

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of macromolecules31. Vesicle formation is an essential step mediated by both Atg proteins and

the class III phosphatidylinositide 3-kinase (PI3K) complex11. Thus inhibitors of class III PI3Ks

are often used to inhibit autophagy10.  

The final steps of autophagy are dependent on the lysosome for breakdown of atuophagic

contents and recycling of macromolecules back into the cytoplasm11. The lysosome is an acidic

organelle that contains over fifty acid hydrolases32. These hydrolase enzymes include proteases,

of which the cathepsin family is the best studied33. Cathepsins and the other lysosomal enzymes

are active within the acidic pH of the lysosome, and compartmentalization thus serves to

maintain activity32. Separation of these enzymes also protects other cellular components from

degradation, and indeed massive release of the hydrolytic enzymes into the cytosol leads to

necrosis12. It now generally accepted that a limited release of cathepsins into the cytosol is often

associated with apoptosis, and thus lysosomal membrane permeabilization (LMP) is a common

occurrence in various types of cell death13. It has not been addressed whether LMP also occurs

during the NETosis cell death program.

Secretory Autophagy: Recent evidence supports an additional function of autophagy separate

from the classic degradation and recycling of cellular contents. Studies in yeast34,35,

Dictyostelium discoideum36, and mammalian cells21 suggest that autophagy also participates in

unconventional protein trafficking and secretion. Proteins lacking the typical secretion signal for

endoplasmic reticulum to Golgi transport are secreted via a Golgi-independent pathway, and

until recently the mechanism for their secretion was unclear. The secretory autophagy pathway,

also termed autosecretion, is dependent on autophagy factors and the Golgi reassembly and

stacking protein (GRASP)37. In mammalian cells, the proinflammatory cytokine IL-1β first

localizes to autophagosomes, which may undergo further maturation through fusion with

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endosomes, multivesicular bodies, and/or lysosomes, but eventually fuse with the plasma

membrane to release IL-1β into the extracellular milieu21. Another proinflammatory factor

proposed to follow the secretory autophagy pathway is HMGB1, which also lacks a conventional

secretion signal38.

HMGB1:  Neutrophils  are  one  of  the  few  cell  types  to  secrete  HMGB116,  and  NET-associated

HMGB1 has been identified using neutrophils from patients with systemic lupus erythematosus

(SLE)19, gout39, and sepsis7. HMGB1 has distinct functions dependent on its cellular localization.

HMGB1 modulates transcriptional activity within the nucleus, but is a proinflammatory factor

when present extracellularly17. The proinflammatory properties of HMGB1 were first

demonstrated in mice as a mediator of endotoxin lethality18. In the aforementioned study,

injection of HMGB1 into mice was lethal, but addition of antibodies against HMGB1 prevented

endotoxemic death. Serum levels of HMGB1 correlate with the severity of sepsis in both mice

and human patients18, and HMGB1 polymorphisms are associated with increased risk of

mortality in patients with systemic inflammatory response syndrome and sepsis40. Extracellular

HMGB1 is passively released by necrotic cells, and actively secreted by neutrophils and

macrophages in inflammatory conditions like sepsis41. Understanding the molecular mechanisms

of active HMGB1 secretion could lead to the discovery of new therapeutic targets for the

treatment of sepsis or other inflammatory diseases.  

The proposed pathway for HMGB1 active secretion is through secretory autophagy.

Inflammatory signals induce the acetylation of lysine residues within HMGB1 to mask nuclear

localization signals, causing its accumulation in the cytosol17. Once in the cytoplasm, HMGB1 is

incorporated into secretory vesicles20. In macrophages, HMGB1 secretion is dependent on Atg5,

implicating autophagosomes and autophagy in the process21. It remains unclear if HMGB1

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secretion follows the autosecretion pathway, as the role of GRASP has not been addressed.

GRASP is the only mammalian protein known to delineate the autosecretion pathway from other

secretion pathways42. During NETosis, HMGB1 localizes to autophagosomes7, and extracellular

HMGB1 can be detected prior to NET release19. These data support an autosecretion pathway for

HMGB1 release during NETosis, but definitive evidence is lacking.

Summary: Neutrophils are essential innate immune cells that undergo massive cellular changes

during the unique form of cell death leading to NET formation. NETosis provides a model for

better understanding the membrane trafficking events leading to cell death during inflammation.

Elucidating the function of autophagy during this program will not only add to the molecular

understanding of NETosis, but may also reveal means of ameliorating neutrophil-mediated

inflammatory diseases.

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Research Plan and Methods

Specific Aim 1: Determine if autophagy is necessary for NETosis, specifically during the

early vacuolization of the cell.

Rationale: Autophagy has been implicated in the membrane trafficking events during NETosis8.

Massive vacuolization of cellular contents occurs within minutes of stimulation9, and many of

the vesicles in PMA-stimulated neutrophils were identified as autophagosomes6. Pre-treatment of

neutrophils with the autophagy inhibitors 3-MA or wortmannin prevents cellular vacuolization

and NET release6,7, supporting a crucial role for autophagy in the membrane events during

NETosis. These inhibitors block autophagy by targeting class III PI3Ks, which are required for

vesicle expansion10. However, both inhibitors can also target class I PI3Ks that are involved in a

number of cellular processes, including TLR signaling and survival10. Thus, to more definitively

determine whether autophagy is necessary for the membrane changes in NETosis and therefore

NET release, I propose to use a genetic means of inhibiting autophagy.

Aim 1.1: Determine whether Atg5 is necessary for NET release.

Experimental design and expected outcomes: A number of autophagy genes (Atgs) are required

for autophagosome formation, and thus mice with homozygous mutations in the essential gene

Atg5 are autophagy defective43. However, autophagy is an essential cellular process, and indeed

Atg5-/- mice die shortly after birth43. Thus I will utilize the Atg5fl/fl-LyzM-Cre mouse, which

contain the Atg5 deletion only in the myeloid cell lineage44. Neutrophils isolated from this mouse

are autophagy defective44, and I will use these cells to determine whether autophagy is necessary

for NET release. Hematopoietic cells will be collected from femurs of Atg5fl/fl-LyzM-Cre

(Atg5KO) and control Atg5fl/fl (Cre-) mice, followed by percoll gradient centrifugation to isolate

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neutrophils as previously described4. Hematoxylin-eosin staining and fluorescence-activated cell

sorting (FACS) for the neutrophil marker Ly6G (Abcam) will be used to assess neutrophil purity.

NETosis will be induced through stimulation of neutrophils with 100nM PMA9 or 6% serum

from septic patients7 for 180 minutes. Neutrophil NET release will be quantified by fluorescence

microscopy to detect extracellular DNA using the cell impermeable dye sytox green45. Results

will be confirmed by fluorescence microscopy of extracellular DNA and neutrophil elastase, a

common procedure for detecting NETs26.

If Atg5 is necessary for NET release, I expect that NET formation will occur in Cre-

neutrophils, but not in Atg5KO neutrophils or unstimulated cells of either genotype. The results

will be the same for PMA- and septic serum-stimulated neutrophils, if there is not a stimulation-

specific response. Atg5 is required for autophagy, so this result would support previous data,

implicating autophagy as a necessary process for NETosis. Following this result, I would repeat

the experiment using siRNA to knock down Atg5 in human neutrophils, to confirm a conserved

necessity for autophagy in NETosis from mice to humans. Alternatively, if Atg5 is not required

for NETosis, I expect NETs to form from both Atg5KO and Cre- neutrophils in response to

stimulation. This result would suggest that the pharmacological inhibitors of autophagy used in

previous studies have off-target effects that prevent NET release independent of autophagy. This

could be tested using inhibitors specific for class I PI3Ks, to determine if the block in NETosis is

independent of class III PI3Ks.

Potential pitfalls and alternative approaches: It is possible that autophagosomes are formed

independent of Atg5 during NETosis, and thus autophagy is still functional in the Atg5KO

neutrophils. To test for an Atg5-independent autophagy pathway, I would generate a LyzM

conditional knockout mouse for a different essential autophagy gene, Beclin146, and determine

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whether neutrophils lacking Beclin1 generate NETs. Alternatively, rendering neutrophils

autophagy defective may alter the lifespan of the cells, which has been proposed to influence

NET release8. To test for an indirect loss of NET release due to changes in lifespan, I would

measure the death rate of Atg5KO compared to Cre- neutrophils.

Aim 1.2: Determine whether autophagy is necessary for vacuolization during NETosis.

Experimental design and expected outcomes: Phase-contrast and transmission electron

microscopy (TEM) of NETing neutrophils reveals numerous intracellular vesicles filling the

cytoplasm within the first hour after stimulation9. The vesicles have a double phospholipid

bilayer, and analysis of PMA-stimulated neutrophils revealed the formation of vesicles to

coincide with punctate staining of LC3, the membrane marker of autophagosomes6. It is

unknown whether formation of vesicles in response to endogenous stimuli is also due to

autophagy. PMA induces the typical features of NETosis, but differs from physiologically

relevant stimuli in some of the NET components and the number of responding neutrophils47. To

test if autophagy induces vacuolization during NETosis with an endogenous stimulus, I will

utilize neutrophils from Atg5KO and Cre- mice stimulated with serum from septic patients as

before. I will use fluorescence microscopy staining for LC3 (Sigma-Aldrich) in combination

with phase-contrast microscopy every 15 minutes following stimulation to follow

autophagosome and vesicle formation, respectively. LC3 is diffuse in resting neutrophils6, and

formation of puncta is indicative of autophagosome formation. Neutrophils after 15, 30, 60, and

90 minutes post-stimulation will also be fixed and prepared for gold-labeled immunodetection of

LC3 by TEM.

If autophagy is necessary for vacuolization during NETosis, I expect Cre- neutrophils but

not Atg5KO neutrophils to display vesicle formation by phase-contrast microscopy within the

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first hour of stimulation. If the vesicles observed are autophagosomes, vesicle formation should

be concomitant with LC3 punctate staining. I also expect TEM analysis of vesicles following

stimulation to display double-membraned vesicles immunolabeled with LC3. Such a result

following the physiologically relevant septic stimulus would be consistent with previous data

using PMA stimulation, and more definitively implicate autophagy in the early events of

NETosis. Alternatively, if both Cre- and Atg5KO neutrophils display vacuolization following

stimulation, this suggests that autophagy is not required for vesicle formation. LC3 punctate

staining and TEM analysis of LC3-positive autophagosomes would indicate an Atg5-

independent mechanism for autophagosome formation, which would be further tested with a

Beclin1 conditional knockout as above.

Potential pitfalls and alternative approaches: It is possible that neither Atg5KO nor Cre-

neutrophil vesicles will display LC3 staining indicative of autophagosome formation following

sepsis serum stimulation. This result would indicate that sepsis serum induces different

membrane events than PMA, which would identify an important difference between NET-

inducing stimuli. Vesicle origin could be identified in this case by staining for markers of other

cellular membranes, such as the nuclear envelope or endoplasmic reticulum.

Specific Aim 2: Determine if NETosis alters lysosome integrity, either through changes in

acidification or membrane stability.

Rationale: Autophagy typically results in degradation of cellular components in the lysosome11.

Oxidative stress can cause lysosome membrane permeabilization (LMP) in neutrophils14, while

also generating reactive oxygen species that are critical for NETosis25. LMP facilitates apoptotic

and necrotic cell death in many cell types, partly because lysosomal contents are capable of

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indeterminately damaging cellular contents13,48. Lysosome function and integrity have not been

addressed during NETosis, even though LMP is a common occurrence during cell death12. I

propose to determine whether the lysosome undergoes alterations during NETosis, through

changes in acidification or membrane integrity.

Aim 2.1: Determine whether lysosomal pH increases during NETosis.

Experimental design and expected outcomes: Lysosomes contain various hydrolytic enzymes

that are active within the acidic (pH 4.6-5.0) lumen of the lysosome49. Therefore, neutralization

of the organelle inactivates many of the lysosomal enzymes. Intracellular membranes largely

disappear during the late stages of NETosis 9, and an increase in lysosomal pH would be

protective if lysosomal contents were released. Furthermore, no lysosomal enzymes were

identified in proteomic studies of NET-associated proteins8,50. Thus the fate of the lysosome

during NETosis is unknown. To test whether lysosomal pH is altered during NETosis, I will use

human neutrophils isolated from the peripheral blood of healthy donors using density

centrifugation as previously described51. Neutrophils will be stimulated with serum from septic

patients to induce NETosis. I will use fluorescence microscopy of neutrophils over the course of

NETosis to track changes in lysosomal pH, using the lysosomotropic probe LysoTracker

(Invitrogen), which accumulates within lysosomes and fluoresces only in an acidic pH.

Methylamine (Sigma-Aldrich) will be used as a positive control because it induces neutralization

of the lysosome.

If NETosis causes a loss of lysosomal integrity through neutralization, I expect

stimulated neutrophils to display a decrease in LysoTracker fluorescence over the course of

NETosis, similar to cells treated with methylamine, while unstimulated cells should maintain

fluorescence. This result would elucidate the fate of the lysosome, suggesting the inactivation of

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lysosomal enzymes during the NETosis cell death program. Alternatively, if there is no change

in lysosomal pH, I expect LysoTracker fluorescence to remain constant after stimulation. This

result would suggest that lysosomal enzymes are functional throughout NETosis.

Potential pitfalls and alternative approaches: Neutralization of the lysosome may be a gradual or

rapid process. If the lysosome suddenly becomes neutralized, especially if it occurs just before

NET release, this would be very difficult to detect by fluorescence microscopy. The experiment

also does not directly test whether lysosomal enzymes are inactivated by a change in pH,

although this has been well established based on the chemistry of the enzyme active sites12. To

investigate these issues, I would utilize subcellular fractionation to isolate neutrophil lysosomes

at different times following stimulation, and test for enzymatic activity of lysosomal cathepsins.

Aim 2.2: Determine whether NETosis causes lysosome membrane permeabilization.

Experimental design and expected outcomes: LMP is observed in both apoptosis and necrosis13,

but it is unknown whether LMP occurs during NETosis. The exact triggers of LMP are still

being discovered, but it is known that reactive oxygen species and autophagy cause LMP15, both

of which are involved in NETosis6. To test if NETosis causes LMP, I will measure cytosolic

cathepsin D activity and changes in acridine orange fluorescence in human neutrophils

stimulated with septic serum. Cathepsin D is a lysosomal enzyme that is only active in the

cytoplasm if the lysosomal membrane has become permeabilized52. Cytosolic and whole cell

extracts will be collected every 30 minutes post-stimulation, and assayed for cathepsin D activity

using a fluorogenic substrate (Calbiochem). Acridine orange (AO) is a lysosomotropic

metachromatic fluorochrome that emits red fluorescence at high concentrations (within the

lysosome), and green fluorescence at low concentrations (in the cytosol). Neutrophils will be

stained with AO every 30 minutes post-stimulation, followed by flow cytometry for green

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fluorescence. Treatment of neutrophils with the bacterial toxin pyocyanin, which is known to

induce LMP, will serve as a positive control53, and no treatment will serve as a negative control.

If NETosis induces LMP, I expect cytosolic activity of cathepsin D in stimulated

neutrophils, similar to pyocyanin-treated cells. I also expect an increase in green fluorescence of

AO-stained neutrophils in septic serum-stimulated neutrophils and pyocyanin-treated

neutrophils, but not in unstimulated cells. This result would be consistent with other forms of cell

death in which LMP is known to occur. I would then analyze the effects of LMP during NETosis

using inhibitors and activators of LMP, to determine whether perturbations in LMP promote or

inhibit NETosis. I would also test whether LMP is dependent upon autophagy during NETosis,

using the Atg5KO mouse. Alternatively, a lack of cytosolic cathepsin D activity and no change

in AO fluorescence following stimulation would indicate that LMP is not induced during

NETosis, and the lysosome is intact throughout NETosis. This result would further separate

NETosis from other cell death programs that include LMP.

Potential pitfalls and alternative approaches: If lysosomal acidity is lost prior to LMP (Aim 2.2),

measuring cathepsin D activity is not a good measure for LMP. Neutralization of the lysosome

would cause inactivation of cathepsin D, so I would not expect to detect either cytoplasmic or

whole cell extract activity. With this result, I would still be able to use AO staining to detect

LMP, as AO fluorescence is not dependent upon acidity like the LysoTracker dye. Alternatively,

I would transfect in fluorescent dextrans to measure their release into the cytoplasm as a measure

of LMP.

Specific Aim 3: Determine if the proinflammatory factor HMGB1 released during NETosis

is actively secreted via autosecretion.

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Rationale: HMGB1 as a NET protein is

unique in that it does not possess antimicrobial

properties like most other NET-associated

proteins. HMGB1 is found on NETs from

neutrophils in inflammatory disease

conditions, but not normal conditions19. One

explanation for this difference is that

inflammation triggers the extracellular

localization of HMGB1. Indeed, it has

previously been shown that HMGB1 is

actively secreted from particular cell types,

including neutrophils, in response to

inflammatory stimuli41. Serum levels of

HMGB1 correlate with disease severity in

sepsis18, a disease in which neutrophils are a key player54. Release of HMGB1 from NETing

neutrophils during sepsis could thus contribute to disease. Extracellular   HMGB1   is   detected  

before   plasma  membrane   rupture   in   NETosis19,   supporting   a  model   in   which   HMGB1   is  

actively  secreted  during  this  process.  HMGB1  lacks  a  conventional  secretion  signal,  and  is  

proposed   to   utilize   the   secretory   autophagy   pathway   for   active   secretion17   (Figure   4).  

Determining   whether   HMGB1   is   released   via   autosecretion   during   NETosis   will   explain  

why  HMGB1   is   only   detected   under   inflammatory   conditions   on  NETs,   and   demonstrate  

that  neutrophils  utilize  the  secretory  autophagy  pathway  under  stress.  Identification  of  the  

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secretory   route   for   HMGB1   from   neutrophils   under   septic   conditions   may   also   open  

avenues  for  therapeutic  intervention.

Aim  3.1:  Determine  whether  NET-­‐associated  HMGB1  has  the  molecular  signature  of  

secretion.  

Experimental design and expected outcomes: Secreted HMGB1 is molecularly different from

passively released HMGB1, because acetylation of lysine residues within HMGB1 is required

for secretion20. To test if the HMGB1 found on NETs in septic conditions is secreted, I will

analyze whether NET-associated HMGB1 is acetylated. I will use purified human neutrophils

from healthy donors, and stimulate with serum from septic patients to induce NETosis. I will use

the cytokines IL-1β, TNF-α, and IL-8 as a positive control for HMGB1 secretion from

neutrophils16, and PMA as a negative control, since HMGB1 is not found on PMA-induced

NETs19. Three hours post-stimulation, cells will be treated with DNaseI to liberate NET-

associated proteins from chromatin, and centrifuged to remove whole cells, as previously

described50. Protein samples will be immunoprecipitated with anti-HMGB1 antibody (Abcam),

and immunoblotted with anti-acetyl-lysine (Cell Signal)55. I will also use the reciprocal co-

immunoprecipitation, using anti-acetyl-lysine for immunoprecipitation and anti-HMGB1 for

immunoblot. Whole cell lysate co-immunoprecipitation will serve as a control for total

acetylated HMGB1, and immunoblot of NET proteins for HMGB1 as a control for total NET-

associated HMGB1.

If HMGB1 on NETs is secreted, I expect to detect acetylation of HMGB1 lysine residues.

Thus I predict that HMGB1 will be detected in NETs after treatment with septic serum and

cytokines, but not PMA. In addition, the NET-associated HMGB1 will be acetylated after serum

and cytokine treatment, indicative of the secreted form of HMGB1. This is consistent with

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previous work in which extracellular HMGB1 was detected prior to NET release from systemic

lupus erythematosus (SLE) neutrophils19. To determine when HMGB1 is secreted during

NETosis following this result, I would use enzyme-linked immunosorbent assay (ELISA) to test

supernatants from septic serum-treated neutrophils at different timepoints before NET release for

extracellular HMGB1. Alternatively, if HMGB1 is not secreted during NETosis, I expect the

HMGB1 isolated from NETs after serum treatment will not be acetylated. With this result, total

NET protein immunoblot will detect HMGB1, but there will be no co-immunoprecipitation with

the acetyl-lysine antibody. This result suggests that HMGB1 is passively released during

NETosis under septic conditions. However, this would not explain either the extracellular release

of HMGB1 from SLE neutrophils, or why PMA-stimulated neutrophils do not release HMGB1

on NETs.

Potential pitfalls and alternative approaches: The HMGB1 isolated from NETs may be

comprised of both actively secreted and passively released forms of the protein. Though

unlikely, as HMGB1 is mostly cytoplasmic even in resting neutrophils39, it would be difficult to

detect acetylated HMGB1 if it is a small proportion of the NET-associated protein. I would

increase the number of neutrophils in the co-immunoprecipitation experiment to increase yield.

The use of different NET-inducing stimuli may alter HMGB1 trafficking, and I would test for

acetylated HMGB1 under different inflammatory conditions like those found in SLE19 or gout39.  

Aim 3.2: Determine whether autosecretion is necessary for the extracellular release of

HMGB1 during NETosis.

Experimental design and expected outcomes: Autosecretion in mammalian cells utilizes

autophagy factors like Atg5, and is distinguished from other secretion pathways through the use

of GRASP proteins21. HMGB1 secretion from macrophages is Atg5-dependent21. It is unknown

Kelsey  Rogers  

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whether secretion is also dependent upon GRASP, or whether either factor is necessary for

secretion from neutrophils. I will isolate neutrophils from C57/BL6 (WT), Atg5KO, or Cre-

mice, and induce NETosis with serum from septic patients. I will knockdown the murine

paralogues GRASP55 and GRASP65 by nucleoporation56 of siRNAs into WT neutrophils. I will

include two separate siRNAs for each GRASP as a control for off-target effects, and scrambled

siRNA as a negative control. I will also use western blot of each GRASP to determine the degree

of siRNA knockdown. To quantify secretion, I will use ELISA and immunofluorescence with

antibodies against murine HMGB1. For ELISA, protein will be collected from NETs by DNaseI

treatment followed by centrifugation to remove whole cells and debris.

If extracellular HMGB1 levels are significantly reduced in Atg5KO and GRASP

knockdown cells compared to controls, this supports the model of HMGB1 autosecretion in

response to septic conditions. I expect that Atg5KO neutrophils will not release NETs, as

described in Aim 1, but this experiment will test whether HMGB1 is secreted in an Atg5-

dependent manner from neutrophils. A decrease in extracellular HMGB1 in GRASP knockdown

cells would specifically implicate autosecretion of HMGB1, as GRASP is so far the only

mammalian protein that delineates the autosecretion pathway42. However, it is unclear if one or

both GRASP paralogues are required for autosecretion in mammalian cells. The two paralogues

may be functionally redundant in autosecretion. To account for this possibility, I will add

siRNAs against both GRASPs, and measure their combined effect on HMGB1 secretion. If there

is no change in extracellular HMGB1 levels in Atg5 or GRASP deficient cells, this would

suggest that HMGB1 is not secreted by an autosecretory mechanism. In this case, I would test for

other mechanisms of unconventional secretion, such as direct translocation across the plasma

Kelsey  Rogers  

21  

membrane or microvesicle shedding57. In either case, I would next repeat the experiment using

siRNAs in human neutrophils to assess the conservation of the pathway from mice to humans.

Potential pitfalls and alternative approaches: Thus far, Atg5 and GRASP are among the few

molecules identified in the mammalian autosecretion pathway. It is possible that HMGB1 is

being secreted via an autophagy-based pathway independent of Atg5 and/or GRASP. There is

one study implicating Rab8a and the exocyst complex in the autosecretion of IL-1β21. While

these components are not seen in other models of autosecretion, I would test for the necessity of

Rab8a and exocyst in the secretion of HMGB1 during NETosis.

Autophagosomes are characterized by their double phospholipid bilayer, and it is debated

whether autosecretion cargo is released in an exosome or not. This depends on whether

autophagosomes fuse directly with the plasma membrane, or whether vesicle maturation induces

loss of the inner membrane prior to fusion38. Poliovirus induces membranes with characteristics

of autophagosomes for non-lytic release of virions, so this may be a system in which to study

double-membrane topology58,59. Lipases and reactive oxygen species may be involved in

degrading the inner autophagosome membrane during vesicle maturation30, or low pH outside

the cell could rupture exosomes38.

Discussion

The process of NETosis as a distinct form of cell death is a complex program of cell

biological and membrane trafficking events. It is clear that multiple pathways are involved in the

induction and control of NET formation, but the mechanisms are largely unknown. The

experiments in this proposal aim to define the functions of autophagy during NETosis, in testing

whether autophagy is specifically required for NETosis, and what the consequences are for

Kelsey  Rogers  

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lysosomal integrity and protein secretion. Furthermore, different stimuli induce NETosis with a

different subset of proteins associated, suggesting multiple routes of NET formation. In this

proposal I chose to focus on the release of HMGB1 during NETosis in extreme inflammatory

conditions, possibly elucidating autosecretion as a new level of regulation for the release of

proinflammatory factors. Many other questions remain as to the relative in vivo contribution of

NETs during infection and inflammation, and mechanistic studies may elucidate a means of

modulating NET formation without affecting other neutrophil functions to begin to investigate

these issues.

Kelsey  Rogers  

23  

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