Neutrophils are essential for immune defense and can respond to infection by releasing chromatin in the form of neutrophil extracellular traps (NETs). Here we show that NETs are induced by mitogens and accompanied by induction of cell-cycle markers, including phosphorylation of the retinoblastoma protein and lamins, nuclear envelope breakdown, and duplication of centrosomes. We identify cyclin-dependent kinases 4 and 6 (CDK4/6) as essential regulators of NETs and show that the response is inhibited by the cell-cycle inhibitor p21 Cip . CDK6, in neutrophils, is required for clearance of the fungal pathogen Candida albicans. Our data describe a function for CDK4/6 in immunity.

One of the most distinctive features of NETosis is the breakdown of the nuclear envelope. This characteristic sets it apart from apoptosis, and is highly reminiscent of nuclear envelope disintegration during mitosis in dividing cells. This observation led us to postulate that NET induction is linked to mitogenic reactivation of cell-cycle regulators. We show that neutrophils, which are terminally differentiated cells, upregulate Ki-67, phosphorylate retinoblastoma protein (pRb), and nuclear lamins, and separate centrosomes without replicating DNA or undergoing cytokinesis. We demonstrate that core members of the cell-cycle machinery, cyclin-dependent kinases 4 and 6 (CDK4/6), are required for NET formation. Taken together, the data presented here show that, in neutrophils, cell-cycle pathways are repurposed for controlling NETosis.

NETosis is a type of neutrophil cell death, distinct from apoptosis or necrosis, which remains poorly characterized. It is an active process characterized by internal breakdown of nuclear and granular membranes, the mixing of the contents of these compartments in the cytosol and finally, their extracellular release via plasma membrane rupture (). Although alternative pathways have been reported (), the most studied NETosis program is the one in response to C. albicans and the mitogen phorbol myristate acetate (PMA). It requires signaling through mitogen-activated protein kinases (MAPKs) () and the production of ROS by the enzyme NADPH oxidase (NOX2) (). ROS are required for the permeabilization of granules by a protein complex called the azurosome, which allows translocation of a protease, neutrophil elastase, into the nucleus where it cleaves histones and leads to chromatin decondensation (). Other mechanistic aspects remain unknown, although some forms of NET formation are associated with citrullination of histones, catalyzed by the enzyme protein arginine deiminase 4 (PAD4) ().

Neutrophils kill microbes by phagocytosis, production of reactive oxygen species (ROS), and by release of various microbicidal proteins. Before activation, these antimicrobials are kept in intracellular storage vesicles called granules. When the cell senses pathogens, the antimicrobials can be released by a process called degranulation, whereby the granules fuse with the plasma membrane and release their contents. An additional antimicrobial strategy has been described, which entraps pathogens in neutrophil extracellular traps (NETs) (). These consist of extruded chromatin bound to various antimicrobials from the granules. By associating with NETs, the antimicrobial molecules are physically limited from diffusing away from their site of action, which may enhance their killing capacity, as well as limit collateral damage to host tissues. NETs are triggered by large pathogens, such as hyphal forms of Candida albicans (). Furthermore, excessive NET release is associated with a growing list of inflammatory and autoimmune diseases, including systemic lupus erythematosus (SLE) (), atherosclerosis (), diabetes (), vasculitis (), thrombosis (), sepsis (), and cancer (). Understanding the molecular mechanism of NET formation is thus crucial for developing therapeutics in the context of immune defense and inflammatory diseases.

Neutrophils are the most abundant immune cells in the bloodstream. They are essential for survival; in humans a lack of neutrophils results in severe immunodeficiency and a drastically reduced lifespan, as for example in individuals with mutations causing congenital neutropenias (). Neutrophils are produced in the bone marrow from dividing progenitors. After maturing and exiting the cell cycle they move to the circulatory system where they are thought to have a short lifespan. Upon infection or injury they migrate into affected tissues where they directly kill pathogens, as well as shaping the ensuing adaptive immune response ().

Cdk6 is broadly expressed in hematopoietic and some non-hematopoietic mouse tissues (), so its contribution to proper immune defenses could derive from non-myeloid cell activity. To investigate if Cdk6 is required in neutrophils, we restored expression of the kinase in knockout animals by breeding Cdk6mice with animals expressing Cre recombinase (Cre) under the control of a lysozyme M promoter (LysM-Cre). This driver leads to expression of Cre in cells of the myeloid lineage, including neutrophils, monocytes, and macrophages. Expression of Cre in neutrophils led to excision of the floxed STOP cassette and restored Cdk6 to similar levels as in WT mice ( Figure S6 C). Importantly, genetic rescue of Cdk6 in myeloid cells of knockout mice restored the ability of these mice to mount proper immune responses against C. albicans ( Figure 6 C), demonstrating a role for Cdk6 in antimicrobial defense.

CDK6 – a review of the past and a glimpse into the future: from cell-cycle control to transcriptional regulation.

Consistent with previous descriptions of sublethal C. albicans infections (), histological analysis of infected kidneys demonstrated that, 5 days post-infection, the fungus produced hyphal filaments, which are associated with tissue destruction, and that these hyphae were collecting in excretory lesions in the papillae of the renal pelvis ( Figure 6 D). Immunofluorescence analysis revealed that hyphal masses were more abundant in knockout animals: 90% of Cdk6mice had detectable hyphae in the renal papillae, compared with 25% of WT mice.

We next infected mice with a sublethal dose of C. albicans and compared fungal loads in the kidneys 5 days post-infection. We found a strikingly elevated C. albicans fungal load in knockout animals ( Figure 6 C). This effect was not due to differences in peripheral blood counts of neutrophils ( Figure S6 A) or other immune cells ( Figure S5 C). Cdk6 was previously implicated in regulating cytokine production (), but we found equivalent, or even elevated numbers of neutrophils infiltrating the kidneys of Cdk6animals on day 5 post-infection ( Figure S6 B), indicating that migratory capacity was unaffected. The higher rates of neutrophil accumulation in knockout mice was likely a reflection of higher fungal burdens in these animals. Furthermore, bone marrow-derived macrophages from WT and Cdk6 null mice produced pro- and anti-inflammatory mediators at similar rates ( Figure S6 D).

To test the effect of Cdk6 and NET deficiency in infections, we used the murine C. albicans sepsis model, which closely reflects the human disease (). In this model, C. albicans colonizes the kidneys leading to renal failure (). The host anti-Candida response is mediated primarily by neutrophils (), making this a good model for investigating the effect of Cdk6 and NET deficiency on immunity. Mice were intravenously injected with live C. albicans and monitored over the course of the infection. Cdk6animals lost weight more rapidly than WT animals ( Figure 6 A) and also succumbed to the disease more rapidly than WT animals ( Figure 6 B). Nox2-deficient animals, which are similarly impaired in NET production, also showed reduced survival in response to C. albicans challenge ( Figure 6 B).

(D) C. albicans (red) and neutrophils (Ly6G, green) in papillae of renal pelvis (visualized by DNA stain/blue) of WT and Cdk6mice, day 5 post-infection. Representative image of nine analyzed kidneys per genotype. Scale bar, 200 μm. See also Figure S6

(C) C. albicans CFU expressed per gram of kidney tissue in WT, Cdk6 −/− , and conditional rescue Cdk6 −/− ; LysM-Cre + mice, 5 days post-infection with 1 × 10 5 CFU. Lines indicate median. ∗∗ p < 0.01; ∗ p < 0.05 Mann-Whitney U test. ns, not significant.

(A) Weights (g) of WT and Cdk6 −/− mice before and 2 days post-infection with 5 × 10 5 colony-forming unit (CFU) C. albicans, boxplots show means; whiskers, 5–95th percentiles; n = 13 mice per group. ∗ p < 0.05, Mann-Whitney U test. ns, not significant.

Neutrophil depletion increases susceptibility to systemic and vaginal candidiasis in mice, and reveals differences between brain and kidney in mechanisms of host resistance.

CDK4/6 double knockout mice die at late stages of embryonic development due to severe anemia (). The kinases have, however, been knocked out individually. Cdk6 knockout mice are viable and have only mild hematopoietic disturbances (). We used a Cdk6mouse strain in which expression of the gene was interrupted by insertion of a floxed transcriptional stop cassette in the first exon (). We purified neutrophils from the peritoneum of Cdk6and WT mice ( Figure S5 A) and obtained similar yields and purities ( Figure S5 B). Like human neutrophils, mouse neutrophils expressed Cdk4 as well as Cdk6 ( Figure S5 A). Despite this redundancy, Cdk6-deficient neutrophils were impaired in NET production in response to both PMA and C. albicans hyphae ( Figures 5 H and S5 C). We obtained similar results with bone marrow-purified neutrophils ( Figure S5 D). The oxidative burst was unaffected in the knockout animals ( Figure 5 I), similar to the result with the CDK4/6 inhibitor. There was no difference in peritoneal neutrophil maturity between the two genotypes, as revealed by expression level of the surface marker Ly6G ( Figure S5 E) and nuclear morphology ( Figure S5 F). Likewise, we observed no difference in cytokine production ( Figure 5 J), indicating that Cdk6 deficiency specifically affects NET production, rather than generally modulating inflammatory pathways.

To test if these G1 kinases are involved in NET formation, we incubated human neutrophils with a CDK4/6 pharmacological inhibitor (abemaciclib/LY2835219), which efficiently blocked NETs in a dose-dependent manner ( Figures 5 D, S4 D, and S4E), but did not inhibit the oxidative burst ( Figure 5 F), phagocytosis ( Figure 5 G), or degranulation ( Figure S4 F). This was confirmed with a second Cdk4/6 inhibitor (palbociclib, Figure 5 E). Both inhibitors blocked ConA-induced NETs at lower concentrations than those required for PMA, consistent with ConA being a weaker stimulus. CDK4/6 inhibition blocked translocation of elastase to the nucleus ( Figure S4 G), a requirement for NET release ().

CDK4/6 are implicated in phosphorylation of pRb () and in centrosome separation () both of which occur during NET formation ( Figures 2 C and 2F). Furthermore, CDK4/6 activity can be induced via MAPK signaling and is a central link between extracellular growth signals and induction of proliferation (). CDK6 localizes throughout the cytoplasm in unstimulated neutrophils and transiently accumulates in the nucleus of PMA-stimulated cells ( Figure 5 B). We could not determine the localization of CDK4. Neutrophils express the CDK4/6 regulatory co-factor cyclin D2 and low levels of cyclin D3 ( Figure 5 C).

CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly.

The early stages of NET formation display various cell-cycle markers and can be blocked by the CDK binding domain of the p21 cell-cycle inhibitor. CDKs and their regulatory subunits, the cyclins, are the major drivers of the mammalian cell cycle ( Figure S1 A). The CDKs are a large group of kinases, many of which have functions unrelated to the cell cycle (). Of the canonical cell-cycle regulatory kinases, the close homologs CDKs 4 and 6 regulate the transition from Gto Gphase and are considered to be largely redundant (). CDK2 drives progression through S phase, while CDK1 is essential for mitosis, although much functional overlap has been reported ( Figure S1 A;). We examined protein expression of the cell-cycle CDKs in human neutrophils and were able to detect CDK4/6 ( Figure 5 A). CDK4 was present at the protein level, as well as induced at the transcriptional level, after NET induction ( Figure S4 A), while CDK6 was only present at the protein level ( Figure 5 A). Neutrophils did not express CDK2 or CDK1 ( Figures 5 A and S4 A), in agreement with previous reports ().

(J) Mip1α production in WT and Cdk6mouse neutrophils (n = 3 per group), in response to LPS (200 ng/mL). (F–J) Graph shows means ± SEM, unpaired Student's t test. See also Figures S4 and S5

(H) NET formation in WT and Cdk6 −/− mouse peritoneal neutrophils after 5 hr of stimulation with PMA or heat-killed C. albicans hyphae. n = 4 per group. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001.

(F) Oxidative burst in human neutrophils treated with Cdk4/6 inhibitor or vehicle. Representative data from three independent repeats. Bars = mean and SD.

(D and E) NET inhibition with the Cdk4/6 inhibitor abemaciclib (LY2835219) (D) and palbociclib (PD-0332991) after 4 hr of PMA or ConA stimulation (E). Combined data from four donors. Graph shows means and SEM.

(C) Expression of cyclin D in human neutrophils (neutro) and control PLB-985 cells (PLB) by western blot. (A–C) Representative blots or image from three independent experiments with different donors.

(B) Left: image showing subcellular localization of CDK6 in naive and PMA-stimulated human neutrophils. DNA is shown in blue and CDK6 in green. Scale bars, 5 μm. Right: Quantification of enrichment of CDK6 in neutrophil nuclei, n = 3 donors, bars show means and SEM. ∗∗ p < 0.01, unpaired Student's t test.

End-stage differentiation of neutrophil granulocytes in vivo is accompanied by up-regulation of p27kip1 and down-regulation of CDK2, CDK4, and CDK6.

Human neutrophils are refractory to transfection and gene editing because of their short lifespans. As an initial approach to determine whether NETosis is dependent on the cell cycle, we used cell-penetrating peptides. In dividing cells, one class of negative cell-cycle regulators is a family of small proteins termed cip/kip (CDK interacting protein/Kinase inhibitory protein). The prototype of this family is p21(p21), a protein that blocks the cell cycle at several different points ( Figure S1 A) by inhibiting cyclin-CDK complexes. We synthesized a peptide mimic corresponding to the CDK inhibitory domain of p21 (), which was previously shown to inhibit proliferation by interfering with the activity of CDKs. We coupled this peptide to the cell-penetrating tat sequence (). A scrambled peptide served as the control. Both peptides penetrated neutrophils with similar efficiencies ( Figure S3 A), and neither were toxic to the cells ( Figure S3 B). Transduction of the p21 inhibitory peptide (p21inh) into human neutrophils completely blocked NET formation, while transduction of the control scrambled peptide (p21ctrl) had little effect ( Figures 4 A and 4B ). Interference with CDKs thus leads to inhibition of NET release, indicating that these cell-cycle regulators are necessary for NETosis. We also examined NET formation in p21 knockout mice, whose cells have higher proliferative capacities due to increased CDK activity and loss of cell-cycle control (). As expected, peritoneal neutrophils from p21 knockout mice made more NETs than wild-type (WT) cells ( Figure 4 C), consistent with p21 being a regulator of both cell-cycle and NET formation. ROS production, however, was similar in both strains ( Figure 4 D).

(D) Oxidative burst in WT and p21mouse peritoneal neutrophils in response to PMA. Bars show means of maximum luminescence intensity, ± SEM. n = 4; ns, non-significant, unpaired Student's t test. See also Figure S3

(B) Representative immunofluorescence images of PMA-stimulated cells pre-incubated with p21 peptide or scrambled control, from three independent repeats. DNA is shown in blue and chromatin in red. Scale bars, 10 μm.

(A) Human neutrophils were incubated with a peptide corresponding to the CDK inhibitory domain of the p21 protein (p21inh), or a scrambled control peptide (p21ctrl), at a concentration of 20 μM before activation of NETs with PMA. The graph shows means ± SEM of NET formation after 4 hr. n = 3 donors. ∗∗ p < 0.01, unpaired Student's t test.

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

We also examined human histological sections from patients with brain fungal abscesses, identified by periodic acid-Schiff staining ( Figure S2 B). We observed NETs in these sections by detecting neutrophil elastase co-localizing with DNA ( Figure S2 C). Next, we co-stained sections with Ki-67 and the neutrophil marker calgranulin, and indeed were able to observe Ki-67-positive neutrophil infiltrates ( Figure 3 B). We confirmed this finding using immunohistochemical methods with a second Ki-67 antibody ( Figure S2 D), as well as immunofluorescent-based detection of a second neutrophil marker (CD66b; Figure S2 E). Neutrophils were only Ki-67 positive when they had intact nuclei (NET precursors), as was the case with Ki-67 detection in purified neutrophils ( Figure 2 B). Remarkably, we could also detect duplicated centrosomes in these cells ( Figure 3 C), demonstrating the induction of mitotic pathways in neutrophils in vivo and confirming our results with isolated human cells.

Induction of cell-cycle markers in neutrophils was surprising since they are terminally differentiated cells. To verify that this also occurs in vivo, we induced NETs by inoculating mice with C. albicans via the orotracheal route. We then co-stained lung sections with antibodies against neutrophil markers (calgranulin A or Ly6G) and the cell division marker Ki-67. Interestingly, we identified many Ki-67-positive neutrophils in the infected lungs ( Figures 3 A and S2 A ).

(A and B) Immunostaining of mouse lung infected with C. albicans (A) or human brain fungal abscess (B) showing neutrophils labeled with calgranulin (red) expressing Ki-67 (green) in the nucleus. Representative image from six mice or four different human specimens.

To test whether NET formation involves induction of other mitotic markers, we visualized microtubule dynamics by immunofluorescence microscopy. During mitosis, microtubule-based structures called centrosomes, which were duplicated in the preceding S phase, separate and migrate to opposing poles of the cell where they nucleate formation of the mitotic spindle (). In unstimulated neutrophils, immunostaining with an antibody directed against both α- and β-tubulin demonstrated a “skeletal” or scaffold-like staining pattern with several filaments extending from a central microtubule-organizing center ( Figures 2 E and S1 F). After 30 min of PMA stimulation these extended filaments were lost and rearranged into single or double dot-like structures. Strikingly, 60 min post-stimulation, these dot-like structures had duplicated and migrated away from each other. The structures were most fully separated from each other at 90 min post-stimulation and were no longer present by 120 min, which coincided with breakdown of the nuclear envelope. Immunofluorescence detection of the centrosome marker γ-tubulin confirmed that these structures were bona fide centrosomes ( Figures 2 F and 2G). Treatment with the control stimulus fMLP did not induce microtubule rearrangement ( Figure S1 G). Centrosome separation, together with phosphorylation of lamins and histone H3S10, demonstrate that markers of a mitotic program are present just before neutrophils die and release NETs.

Our initial observation of nuclear envelope breakdown during NETosis ( Figure 1 C) argued that mitotic pathways are also activated. In mitosis (M phase), nuclear envelope disintegration is triggered by phosphorylation of lamins, intermediate filament proteins that form a sheet underneath the envelope called the nuclear lamina (). Phosphorylation of the lamina disrupts the structural rigidity of the nucleus, allowing the nuclear envelope to disintegrate. We tested if NET formation is also associated with phosphorylation of lamins. Remarkably, treatment with the NET-inducing stimuli PMA, ConA, or C. albicans hyphae all led to robust phosphorylation of lamin A/C ( Figure 2 C). This was confirmed by immunofluorescence ( Figures S1 D and S1E). Furthermore, NETosis was accompanied by induction of a second mitotic marker: phosphorylation of histone H3 at serine 10 (H3S10). Neither lamin nor H3S10 phosphorylation were induced by the control stimulus fMLP ( Figure 2 C), which, as mentioned above, does not induce NETs.

Despite activation of G1 kinases, we did not detect incorporation of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) ( Figure 2 D), indicating that DNA synthesis, the hallmark of S phase, did not occur. As a control for EdU incorporation we used continuously cycling HEK293 cells ( Figure 2 D) as well as T lymphocytes, which can be induced to proliferate by treatment with PMA, analogously to our stimulation of neutrophils. T cells stimulated with PMA/ionomycin successfully incorporated EdU, but this required a longer period of stimulation than the 3 hr it took neutrophils to make NETs ( Figure S1 B), highlighting an important difference in the temporal dynamic between NETosis and cell-cycle re-entry. Similarly, we observed no induction of the S-phase transcriptional program in neutrophils making NETs. In cycling cells, phosphorylation of pRb leads to the derepression of E2F transcription factors and expression of S-phase genes, but these were not induced in NETosis as measured by qPCR analysis ( Figure S1 C) and whole-genome microarray analysis (data not shown).

We examined a series of other cell-cycle markers, in the order in which they are induced in dividing cells. Neutrophils are typically considered to be in the Gstage, like other terminally differentiated and quiescent cells ( Figure S1 ). Cells in Gcan resume cycling after mitogenic stimulation; this is associated with phosphorylation of pRb by CDK4/6 (). Notably, neutrophils stimulated to make NETs also exhibited robust phosphorylation of pRb, indicating that activation of G1 kinases was occurring ( Figure 2 C). Importantly, pRb phosphorylation was not only triggered by PMA and ConA, but also by infection with C. albicans hyphae ( Figure 2 C). Stimuli that activate neutrophils but do not induce NETs, such as bacterial peptide analog N-formylmethionyl-leucyl-phenylalanine (fMLP), did not lead to pRb phosphorylation.

We first examined cell-cycle markers in neutrophils induced to make NETs. The nuclear antigen Ki-67 is expressed in all cycling cells, irrespective of the stage of the cell cycle they are in, while it is absent in non-cycling, quiescent, or senescent cells. Resting neutrophils were negative for Ki-67 but, surprisingly, cells stimulated to undergo NETosis transiently reactivated expression of this cell division marker, visualized by immunofluorescence ( Figures 2 A and 2B ).

(G) Quantification of separated centrosomes from (F). Graph shows means ± SEM from combined data of four donors. See also Figure S1

(F) Nonstimulated and PMA-treated (1 hr) human neutrophils stained for γ-tubulin (red) and DNA (blue). (E and F) Representative images from three different independent experiments, with cells from different donors. Scale bars 5 μm.

(E) Immunofluorescence detection of microtubules with an antibody against α- and β-tubulin (red), in a time course experiment of human neutrophils stimulated with PMA.

(D) Immunofluorescence analysis of EdU incorporation in control HEK cells (left) and human neutrophils making NETs (right). Neutrophils were incubated with nucleotide analog EdU for 1 hr before PMA stimulation and were then fixed at indicated times. HEK cells (nonstimulated) were incubated with EdU or vehicle control for 1 hr before fixing.

(C) Western blot of G1/S and M phase markers in lysates prepared from human neutrophils simulated with NET inducers (PMA, ConA, and C. albicans) and a control stimulus that does not induce NETs (fMLP). “P-” indicates phosphorylation. Representative blot from three or more repeats with different donors. Molecular weights are indicated in kDa.

(B) Quantification of nuclear Ki-67 staining during PMA time course stimulation. The percentage of Ki67-positive cells (gray) is plotted along with the percentage of SYTOX-positive cells (black). The graph shows means ± SEM from combined data of four healthy donors.

Neutrophils are terminally differentiated cells that have withdrawn from the cell cycle and lost their capacity to divide. Indeed, mature neutrophils downregulate most of the genes involved in cell-cycle regulation (), a finding we confirmed by genome-wide expression analysis of peripheral blood neutrophils from healthy donors ( Table S1 ). Despite this, mitogenic stimulation of neutrophils leads to nuclear envelope breakdown as it does in the case of mitosis in dividing cells. Transmission electron microscopy comparing mitosis in the myeloid cell line PLB-985, and NETosis in neutrophils, revealed that the vesiculation of the nuclear envelope is common to both processes ( Figure 1 C;). We were struck by this similarity and postulated that common cellular pathways are employed during NETosis and mitosis.

End-stage differentiation of neutrophil granulocytes in vivo is accompanied by up-regulation of p27kip1 and down-regulation of CDK2, CDK4, and CDK6.

Fungi and the protein kinase C agonist PMA activate a similar pathway to make NETs. Both induce MAPK signaling (), the activation of NOX2, and the production of ROS (). PMA is also a strong mitogen, a chemical with the ability to trigger cell division. We tested if other mitogens also lead to NET release. Concanavalin A (ConA) and phytohaemagglutinin, two plant lectins commonly used to induce proliferation of lymphocytes, both induced NETs, distinguishable by their morphology and the colocalization of nuclear and granular markers ( Figures 1 A and 1B ). Importantly, NET induction with mitogens was also dependent on ROS production, since neutrophils isolated from chronic granulomatous disease (CGD) patients, who have deficiencies in NOX2, or those treated with diphenyleneiodonium, a NOX2 chemical inhibitor, did not undergo NETosis ( Figures 1 A and 1B).

(C) Electron micrograph comparing nuclear envelope breakdown in mitosis and NETosis. Left: PLB-985 cells were synchronized in G2 and then released into mitosis. In G2 the nuclear envelope is intact but disintegrates in the prophase of mitosis, concurrently with appearance of condensed chromosomes (darker staining territories in the nucleus). In anaphase, chromosomes have separated and, eventually the nuclear envelope begins to reform. Right: nonstimulated neutrophils have a lobulated nucleus, but this lobulation is lost as they prepare to release NETs. Approximately 2 hr after PMA stimulation, the nuclear envelope starts to disintegrate, allowing nuclear material to mix with the contents of the granules and the cytoplasm before being released into the extracellular space. The bottom row shows magnified details from corresponding top panels. Arrows indicate vesiculation of the nuclear envelope. Neutrophil images are representative from at least five different embeddings. Scale bars, 5 μm (upper panels; overview) and 1 μm (lower panels; detailed views).

(B) Quantification of NET induction with mitogens and C. albicans, and inhibition with the NOX2 inhibitor diphenyliodonium (DPI, 5 μM). Representative result of three independent repeats, means ± SD.

(A) Human peripheral blood neutrophils isolated from a healthy donor or CGD patient (n = 2) with a genetic deficiency in NOX2, stimulated with PMA (50 nM) or ConA (50 μg/mL) for 5 hr. Both stimuli lead to colocalization of chromatin (red), DNA (blue), and the granule protein neutrophil elastase (green). Representative image from three independent experiments. Nonstim, nonstimulated. Top row, merge; bottom row, DNA. Scale bars, 5 μm.

Discussion

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De Larranaga G. Neutrophil extracellular traps in sepsis. We identify a role for Cdk6 in innate immune defense. Mice lacking this kinase are highly susceptible to infection with the fungal pathogen C. albicans. In acute systemic candidiasis, the immune response is predominantly mediated by neutrophils and not by T cell responses, since mice lacking T cells do not show any increase in susceptibility to disease (). CDK6 was shown to regulate cytokine production in HeLa cells (), but we failed to detect a defect in neutrophil recruitment to infected organs. Macrophages, the major producers of cytokines during infections, produced normal levels of these inflammatory mediators despite lacking CDK6. Elevated kidney fungal loads are thus likely a reflection of a defect in neutrophil function, specifically one in NET formation, although the use of the LysM-Cre driver in the rescue experiment means we cannot rule out other impairments in myeloid cells. Cdk6 was shown to regulate macrophage adhesion in response to lipopolysaccharide LPS, and knockout mice are protected against LPS-induced sepsis (). This finding is in line with a role for Cdk6 in NET formation, since NETs are also implicated in the pathology of sepsis ().

Raychaudhuri et al., 2008 Raychaudhuri S.

Remmers E.F.

Lee A.T.

Hackett R.

Guiducci C.

Burtt N.P.

Gianniny L.

Korman B.D.

Padyukov L.

Kurreeman F.A.

et al. Common variants at CD40 and other loci confer risk of rheumatoid arthritis. Tigan et al., 2015 Tigan A.S.

Bellutti F.

Kollmann K.

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Sexl V. CDK6 – a review of the past and a glimpse into the future: from cell-cycle control to transcriptional regulation. Demers et al., 2012 Demers M.

Krause D.S.

Schatzberg D.

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Fuchs T.A.

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Wagner D.D. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Rickles et al., 1992 Rickles F.R.

Levine M.

Edwards R.L. Hemostatic alterations in cancer patients. NETs are important for immune defense, but are also dysregulated in many autoimmune diseases. Intriguingly, a genetic polymorphism in CDK6 has been associated with rheumatoid arthritis (). CDK6 expression levels are also elevated in various types of cancer, and inhibition of this kinase appears to be a promising new antitumor therapy (). It is of note that malignancies such as chronic myelogenous leukemia are associated with high levels of NET-induced thrombosis (). The contribution of CDK6 to cancer may thus be 2-fold: cell-cycle dysregulation contributing to tumorigenesis as well as increased NET formation by myeloid cell precursors leading to thrombosis, the second most common cause of death in cancer patients (). Use of CDK4/6 inhibitors in cancer therapy may consequently lower rates of thrombosis, but also dampen immune responses in patients.

Herrup and Yang, 2007 Herrup K.

Yang Y. Cell cycle regulation in the postmitotic neuron: oxymoron or new biology?. Busser et al., 1998 Busser J.

Geldmacher D.S.

Herrup K. Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer's disease brain. Herrup and Yang, 2007 Herrup K.

Yang Y. Cell cycle regulation in the postmitotic neuron: oxymoron or new biology?. Interestingly, other instances exist where cell-cycle signaling in postmitotic cells has been linked to disease (). In the brain, neurons can inappropriately re-activate the cell cycle, which leads to cell death just as it does in neutrophils. Cell death resulting from neuron cell-cycle re-entry has been linked to several neurodegenerative diseases, including Alzheimer's disease and amyotrophic lateral sclerosis (). Dysregulation of cell-cycle proteins in postmitotic neurons and neutrophils can thus be viewed as a unifying disease principle in neurodegenerative and inflammatory disorders.

In summary, NET formation, an antimicrobial form of cell death, is controlled by the activation of the cell-cycle kinases CDK4/6. During infection, lymphocytes activate the cell cycle to rapidly increase their numbers and thus fulfill their memory or cytotoxic functions. We show that, in neutrophils, similar pathways are used for regulating the NETotic cell death program, with essential consequences for immunopathology and defense against pathogens. These findings open up avenues for therapeutic interventions in NET-related diseases.