A Curious Case of Cyclin-dependent Kinases in Neutrophils

Abstract

Neutrophils are terminally differentiated, short-lived white blood cells critical for innate immunity. Although cyclin-dependent kinases (CDKs) are typically related to cell cycle progression, increasing evidence has shown that they regulate essential functions of neutrophils. This review highlights the roles of CDKs and their partners, cyclins, in neutrophils, outside cell cycle regulation. CDK1–10 and several cyclins are expressed in neutrophils, albeit at different levels. Observed phenotypes associated with specific inhibition or genetic loss of CDK2 indicate its role in modulating neutrophil migration. CDK4 and 6 regulate neutrophil extracellular traps (NETs) formation, while CDK5 regulates neutrophil degranulation. CDK7 and 9 are critical in neutrophil apoptosis, contributing to inflammation resolution. In addition to the CDKs that regulate mature neutrophil functions, cyclins are essential in hematopoiesis and granulopoiesis. The pivotal roles of CDKs in neutrophils present an untapped potential in targeting CDKs for treating neutrophil-dominant inflammatory diseases and understanding the regulation of neutrophils.

Graphical Abstract

Introduction

Cyclin-dependent kinases (CDKs) are serine/threonine kinases generally maintained at relatively constant levels in the cytosol^1,2. Partnered with cyclins, CDKs are the main drivers in cell cycle entry and progression^3,4. Recent studies have shown novel CDK functions outside the context of the traditional cell cycle regulation. Several reports and reviews have highlighted the progress of this paradigm by summarizing noncanonical functions of CDKs⁵ and featured the overlapping functions of CDKs⁶ in regulating other processes such as apoptosis⁷, cell migration⁸, and cell signaling⁹. In addition, cyclins also present significant roles in growth and development¹⁰ and the proliferation of immune cells^11,12. These findings have encouraged studies involving CDKs in the context of the immune response, particularly, in neutrophils.

It was initially thought that CDKs might play a minimal role in neutrophils, which are terminally differentiated with limited proliferation ability. However, emerging evidence supports CDKs being essential for neutrophil development and functions. Whether certain CDKs are present in neutrophils is still debated. Since the discussions of CDKs in the context of neutrophils are currently limited, efforts to feature and relate their roles in neutrophils are essential to address the various views and contributions that prevail in the field.

Findings that expand the knowledge of CDKs from the classical view have opened avenues for targeting CDKs for various therapeutic use. The pan-CDK inhibitor roscovitine is in phase II clinical trial for colitis and cystic fibrosis, both neutrophil-mediated diseases¹³. CDK4/6 inhibitors, such as palbociclib, ribocilcib, and abemaciclib, are approved to treat metastatic breast cancer^14,15. Since neutrophils also play a prominent role in the progression of cancer¹⁶, it is possible that neutrophil suppression contributed to the therapeutic outcome.

This review introduces the general background of neutrophils, CDKs, and cyclins. We summarize the current findings on CDKs and cyclins concerning neutrophil development and functions. We outline the observed phenotypes and molecular mechanisms involved in specific inhibition or genetic loss of certain CDKs in neutrophils. Finally, we address several central questions and challenges in the field and potential future directions to utilize CDKs as therapeutic targets in alleviating over-inflammation as seen in neutrophil-dominant inflammation-related diseases.

Neutrophils and Inflammation in Human Diseases

Neutrophils are the most abundant leukocytes in humans and play an integral role in the innate immune system. They act as the first line of defense, attacking invading pathogens by carrying out various effector functions after they reach the infection site. Neutrophils develop from common myeloid progenitors that undergo granulopoiesis^17,18. Under homeostatic conditions, neutrophils are released from the bone marrow¹⁹ into the blood circulation, and this process further heightens during inflammation.

Signaling molecules such as chemokines generated during inflammation instruct neutrophils to extravasate and enter affected tissues via rolling, firm adhesion, and trans-endothelial migration. Various adhesion molecules on both endothelium cells and neutrophil surface²⁰ facilitate these processes. In tissue, neutrophils perform multiple functions to combat the invading pathogen, such as phagocytosis, degranulation, and releasing cytokines and reactive oxygen species (ROS). Neutrophils secrete acute-phase pro-inflammatory cytokines such as IL-8, IL-1α, and TNF-β, providing positive feedback and enhancing inflammation response²¹. Another function of neutrophils is their ability to release neutrophil extracellular traps, or NETs, composed of DNA, granules, and histones, to entrap and kill bacteria extracellularly²². When neutrophils reach wounds, they upregulate expressions of genes that can recruit other inflammatory cells, encourage angiogenesis, and promote keratinocyte and fibroblast proliferation which are critical responses needed for wound healing²³. After carrying out their functions at the wound site, neutrophils undergo apoptosis²⁴ to resolve inflammation. Macrophages will then engulf the apoptotic neutrophils, release signaling molecules to resolve inflammation, and initiate the phase of wound healing²⁵.

Neutrophil-dominant inflammation can be the primary driver of immunopathology in many human diseases. Although neutrophils produce relatively fewer cytokines than other leukocytes, their numbers during inflammation are significantly higher, contributing to a significant amount of local cytokines²⁶. Such neutrophil-derived cytokines mediate complex neutrophil engagement with other cells inside or outside the immune system. Together with the degree of neutrophilic inflammation, these interactions point to disease severity and autoimmunity^27–29. A chronic state of inflammation from increased ROS production causes endothelial dysfunction and tissue injury³⁰. Pathologic alterations that lead to autoimmune diseases³¹, such as systemic lupus erythematosus (SLE), may develop from the production of autoantibodies against components present in NETs³². Human neutrophils have a short lifespan of fewer than 24 hours in the bloodstream and are programmed to die^33,34. Neutrophil apoptosis is accelerated during an infection to resolve inflammation³⁵. Failure to do so can lead to chronic inflammation, as observed in cystic fibrosis, atherosclerosis, chronic obstructive pulmonary, and rheumatoid arthritis³⁶.

Together, neutrophil homeostasis and function must be tightly regulated to avoid over-inflammation^37,38 while maintaining normal immune functions. Due to the significant roles neutrophils play in inflammation pathology, they act as suitable targets for therapy³⁹. Neutrophil-targeted strategies to inhibit their functions may involve lowering neutrophil production by utilizing monoclonal antibodies to block granulocyte-colony stimulating factor (G-CSF) receptors⁴⁰ or using CDK inhibitors to promote apoptosis⁴¹. Neutrophil recruitment can also be reduced via selectin and integrin blockers^42,43 or chemokine receptor inhibitors^44,45. Neutrophil-derived mediators such as proteinase 3 and elastase have been targeted to block neutrophil degranulation⁴⁶. Formation of NETs can be inhibited by targeting peptidyl arginine deiminase 4 (PAD4)^47–49.

In addition to serving as therapeutic targets, due to their abundance of recruitment to inflammation sites, neutrophils are exploited as drug delivery systems⁵⁰. Nanoparticles encapsulating drugs are internalized by activated neutrophils⁵¹, presenting a promising avenue for delivering nanotherapeutics across blood vessel barriers to tissues. For example, intake of albumin nanoparticles loaded with pyropheophorbide-a (Ppa) by neutrophils inhibited tumor growth⁵². Besides being used as whole cells, neutrophils are disrupted via physical force to generate membrane-formed nanovesicles that retain surface receptors and adhesion molecules^53,54. When loaded with an anti-inflammation agent, these extracellular vesicle-like nanovesicles reduce lipopolysaccharide-induced acute lung inflammation⁵³.

CDKs and Cyclins

The mammalian CDK family members comprise 20 protein kinases (CDK1–20) grouped based on their sequences and functions⁵⁵. CDK1–9 and CDK11 require association with regulatory subunits for biological activity⁵⁶. CDK1–4 and CDK6 drive the cell cycle progression by partnering with cyclins among these heterodimeric kinases. Cyclins are classified by their amino acid sequence, and there are at least 15 different cyclins known from A to Y⁵⁷. The phases in the cell cycle are characterized by the oscillating expression of specific cyclins that sets up the stage-specific timing of CDK activitation⁵⁸. Cyclin-CDK complexes dictate the downstream substrates specificity⁵⁹ and phosphorylate substrates appropriate in particular cell cycle phases⁶⁰.

After binding cyclins, CDKs are phosphorylated at threonine residues, such as T172 in CDK4 and T160 in CDK2 in the T-loop CDK-activating kinase (CAK) and are activated⁴⁶. Dual specificity kinases such as WEE1 and MYT1 can inhibit CDK-cyclin complexes by phosphorylation of adjacent threonine and tyrosine residues within the ATP-binding sites (T14 and Y15). Dephosphorylation of these residues ensures proper ATP alignment, thus releasing the inhibition^9,61. The binding of cyclin-dependent kinase inhibitors (CKIs) to CDKs can restrain its activity. CKIs of the INK4 family can inactivate CDK4 and 6. The Cip and Kip families of CKIs are inhibitors of CDK2-cyclin A/E but are positive regulators of cyclin D-dependent kinases^62,63.

CDKs and Cyclins in the Cell Cycle

It is appreciated that specific CDK-cyclin complexes catalyze certain cell-cycle transitions⁶⁴. This canonical perspective is that cyclin D-CDK4/6 and cyclin E-CDK2 initiate the S phase, cyclin A with CDK1 or CDK2 allows the S phase’s progression and entry into mitosis, and the following cyclin B-CDK1 allow transition into mitosis⁶⁵.

The cell cycle G1 phase first involves synthesizing D-type cyclins that bind and transport CDK4/6 into the nucleus⁵⁶. They phosphorylate retinoblastoma (Rb) family proteins, including pRb, p107, and p130, that modulate the activity of E2F family transcription factors⁶⁶, inducing gene transcriptions that mediate S phase entry. Cyclin D-CDK4/6 phosphorylates Rb by docking at the alpha-helix in the C-term of Rb^67,68. CDK4 phosphorylates S807/S811 on Rb, and this partial phosphorylation is needed for CDK2 phosphorylation⁶⁹. The activation of CDK2 completes the G1 phase and transitions the cell cycle to the S phase. CDK2 forms active complexes with A-type cyclins, D-type cyclins⁴⁷, or cyclin B1⁷⁰. Unlike CDK4/6, phosphorylation by CDK2 with its partner, cyclin E, of Rb proteins inactivates them by allowing the release of E2F. Cyclin E-CDK2 targets the KxLxF sequence on Rb⁷¹. Cyclin C-CDK3 can also phosphorylate Rb at S807/811 and has shown to be involved in the cell cycle re-entry as it ensures G0 phase exit^72,73. Activation of E2F-1, E2F-2, and partially E2F-3 by CDK3 also contributes to the progression into S-phase⁷⁴.

During the S phase, A-type cyclins continue to accumulate and form complexes with CDK2 to phosphorylate multiple substrates involved in the completion of the S phase, such as regulators of cyclin A, transcription factors, and proteins involved in DNA replication and repair, and cell-cycle checkpoints^42,75. Cyclin A also forms complexes with CDK1 and share similar substrates to CDK2-cyclin A complexes. B-type cyclins are synthesized in the transition from S to G2 phase when cyclin A degrades via ubiquitin-mediated proteolysis. Mitosis is activated after the G2 phase when CDK1 is dephosphorylated by Cdc25 and forms complexes with cyclin B, specifically B1 and B2. These complexes then phosphorylate many other substrates that play essential roles in the progression of mitosis, for instance, kinesin, Golgi matrix components, and nuclear lamins⁷⁶.

Other CDK functions

Not all CDKs are involved in cell cycle regulation. In neurons, CDK5 mediates neuronal migration and synaptic transmission⁷⁷ with its partners p35 and p39. The p35/CDK5 complex mediates the synaptic integrity of the central nervous system but triggers neurodegeneration under abnormal conditions. D- and E-type cyclins also bind to CDK5, although neither activates CDK5 kinase activity⁷⁸. Cyclin E forms complexes with Cdk5 and restrains its activation, which is necessary for controlling synapse formation⁷⁹.

Several CDKs regulate transcription. The CAK complex, consisting of CDK7, Cyclin H, and MAT1 (ménage a Trois)⁸⁰, controls the activation of some CDKs such as CDK1, CDK2, CDK4, and CDK6. CDK7 is a core subunit of the general transcription factor TFIIH, important in promoter clearance and progression of transcription. CDK8/cyclin C is a component of the RNA polymerase II holoenzyme complex⁸¹, implicated in the regulation of transcription. CDK8/cyclin C phosphorylates cyclin H, repressing TFIIH kinase activity and transcription⁸². CDK9 also affects transcription by phosphorylating RNA polymerase II holoenzyme at the carboxy-terminal domain (CTD). T-type cyclins (T1 and T2) and cyclin K activate CDK9 by forming the Positive-Transcription Elongation Factors (P-TEFb), which regulates transcriptional elongation positively and negatively^83,84. CDK9 also interacts with TNF Receptor Associated Factor 2 (TRAF2) at a conserved domain in the TRAF-C region, a motif known to bind other kinases involved in TRAF-mediated signaling⁸⁵.

CDK 10 is a member of the Cdc2 family of kinases and regulates the G2-M phase of the cell cycle⁸⁶. In addition, CDK10 interacts with the N-terminus of the Ets2 transcription factor and inhibits Ets2 transactivation in mammalian cells⁸⁷. CDK11, CDK12, and CDK14 are involved in the G1-S transition phase, while CDK12 and CDK13 regulate transcription and RNA splicing⁵⁷. CDK11–16 and CDK19 are known to promote the progression of cancer⁸⁸. CDK12 has been shown to control the expression of DNA damage response genes^89,90. CDK15 can act as an anti-apoptotic protein by inducing BIRC5 phosphorylation at Thr-34, thus inhibiting TRAIL/TNSF10-induced apoptosis⁹¹.

CDKs and cyclins regulate hematopoiesis and granulopoiesis

The roles of these CDKs in hematopoiesis are expected, as blood generation depends on the cell cycle machinery. However, this is rarely the case with single CDK or cyclin deletion due to possible compensatory mechanisms⁹². CDK2 knockout mice are viable, albeit sterile and with more petite body sizes⁹³, despite the inhibition of CDK2 causing cell cycle arrest in vitro⁹⁴. CDK2 is essential only in the meiotic but not the somatic cell cycle in mice⁹². The only indispensable CDK is CDK1, where no cell division occurs when CDK1 is disrupted⁵⁹. CDK1 is present throughout the cell cycle, likely compensating for the loss of CDK2. CDK1 binds all classical cyclins, though sometimes with lower affinity than other CDKs. When CDK2 is absent in mice, CDK1 binds to cyclin E to facilitate the G1-S phase transition⁴⁸.

Like CDK2 knockout mice, CDK4 knockout mice display growth retardation and reproductive dysfunction⁶⁶. CDK4 knockout fibroblasts exhibited significant delays to S phase entry, possibly due to increased binding of the CDK2 inhibitor, p27, to cyclin E/CDK2, and decreased activation, leading to decreased CDK2 activation and Rb phosphorylation. A similar decline in Rb phosphorylation is also observed in CDK2 knockout fibroblasts⁹⁵. Mice with CDK2/4 double knockouts can develop up to embryonic day E16.5. The mice showed increased erythrocyte volume and died due to a lack of hematopoietic cells⁹⁶.

Similarly, CDK6 knockout mice showed slight impairment of hematopoiesis, while CDK4/6 double knockout mice die during late stages of embryonic development due to severe anemia⁹⁷. Embryonic fibroblasts of these CDK knockouts can proliferate in low serum conditions and respond to mitogenic stimuli, although at a lower rate. Hence, D-type cyclin-dependent kinases are required for hematopoiesis but are not essential for the proliferation of embryonic fibroblasts, highlighting the context-dependent mechanisms of cell cycle progression.

Besides CDKs, cyclin A is essential for the cell-cycle progression of hematopoietic and embryonic stem cells⁹⁸. Knocking out both cyclin A1 and A2 in bone marrow cells of mice results in anemia, as observed by decreased red blood cell and platelet counts. Cyclin A ablation causes a significant reduction of hematopoietic stem cells and their lineage-committed descendants, but not more differentiated cells. Interestingly, its function is dispensable in fibroblasts of cyclin A knockout mice as the cells can still proliferate⁹⁸. This observation highlights cyclin A’s functional specificity in the cell cycle of stem cells.

Mice lacking all D-cyclins develop up to E13.5 but die before E17.5 from cardiac abnormalities and severe anemia⁹⁹. Hematopoietic stem cells cannot proliferate, and their numbers reduce significantly. Mice lacking individual D-cyclins are viable but show tissue-specific defects, indicating that they are required for proliferation only in selected cell types¹⁰⁰. Deletion of cyclin D1 results in neuronal abnormality¹⁰. Cyclin D2 deletion impairs proliferation of B-lymphocytes¹¹. Cyclin D3 deletion affects the development of immature T-lymphocytes¹². Mice that express any one of the D type cyclins develop normally until late gestation, suggesting that these cyclins are interchangeable and functionally redundant to an extent¹⁰¹. In addition, D-cyclins can regulate the levels of p27 as the ablation of D-cyclins led to the downregulation of this CDK inhibitor.

Cyclin D3 plays a prominent role in granulopoiesis of neutrophils and is regulated by G-CSF signaling¹⁰². Mice lacking cyclin D3 are refractory to the stimulation by G-CSF, exhibiting significantly decreased numbers of mature granulocytes but increased numbers of immature granulocyte progenitors in the peripheral blood and the bone marrow. Consequently, cyclin D3 deficient mice are susceptible to bacterial infections due to the inability to undergo emergency granulopoiesis.

CDKs in neutrophil function

CDK and cyclin expression in neutrophils

Based on DNA staining, most mature neutrophils are in the G0/G1 phase. The activity of G1-phase CDKs peaks at the myeloblast and promyelocyte stage and decreases as neutrophils differentiate. Simultaneously, CDK2, 4, and 6 transcript levels decrease. This downregulation is also seen in the level of cyclins D1, D2, and D3. Cyclin E, however, is slightly upregulated during differentiation after the band cell stage, which is the stage before complete maturation of segmented neutrophils¹⁰³. The low expression of these CDKs and D-type cyclins results in a lack of hyperphosphorylation of pRb¹⁰³, leading to cell cycle arrest. In addition, the upregulation of the CKI p27 also contributes to cell cycle arrest in mature neutrophils.

Human neutrophils express CDK1 and CDK2 proteins^7,41, despite some earlier studies that failed to detect CDK1 or CDK2³⁵. In neutrophil precursor cell lines, HL60 and PLB-985 cells, both of which can undergo neutrophil-like differentiation in vitro, the levels of CDKs vary. CDK7 and CDK9 are expressed at similar levels before and after differentiation at transcriptional levels. CDK5 and CDK6 are expressed at relatively low levels in primary human neutrophils but higher in undifferentiated neutrophil-like cell lines¹⁰⁴. Levels of CDK and cyclin transcription and protein expression in primary human neutrophils and differentiated HL-60 cells are summarized in Table 1.

Table 1.

RNA and protein levels of CDK/cyclins in human neutrophils and HL60 cells.

CDK/cyclin	RNA level in human neutrophils133–139	Protein level
CDK1	−0.56	PMN: Detected41,49, not detected123. HL60: Detected123
CDK2	0.89	PMN: Detected41,49, not detected7,103,123. HL60: Detected7,123. Differentiated HL60: Detected109
CDK3	0.54	-
CDK4	0.49	PMN: Detected49,103,120,123, not detected123
CDK5	0.11	PMN: Detected120,121,123, HL60: Detected7,123
CDK6	0.16	PMN: Detected49,103, not detected123. HL60: Detected123
CDK7	1.16	PMN: Detected123. HL60: Detected7,123
CDK8	0.41	-
CDK9	1.58	PMN: Detected123. HL60: Detected7
CDK10	1.13	-
CDK11A	1.94	-
CDK11B	1.69	-
CDK12	1.45	-
CDK13	1.30	-
CDK14	1.16	-
CDK15	−0.71	-
CDK16	1.61	-
CDK17	1.35	-
CDK18	−1.04	-
CDK19	1.35	-
CDK20	−0.28	-
Cyclin A1	−1.33	PMN: Not detected103
Cyclin A2	−0.44
Cyclin B1	−0.20	-
Cyclin B2	−0.52	-
Cyclin B3	−3.04	-
Cyclin C	0.56	-
Cyclin D1	−0.68	PMN: Not detected49,103
Cyclin D2	0.73	PMN: Detected49,103
Cyclin D3	2.27	PMN: Detected49, not detected103
Cyclin E1	−0.43	PMN: Detected103
Cyclin E2	0.24
Cyclin F	−0.91	-
Cyclin G1	0.98	-
Cyclin G2	1.87	-
Cyclin H	1.62	HL60: Detected7
Cyclin I	2.42	-
Cyclin J	−0.10	-
Cyclin K	1.80	-
Cyclin L1	2.51	-
Cyclin L2	1.80	-
Cyclin O	−4.00	-
Cyclin T1	1.02	PMN: Detected123. HL60: Detected7
Cyclin T2	1.27	-
Cyclin Y	1.88	-

Note: Expression values are fragments per kilobase of transcript per million mapped reads. Undetected transcripts are assigned −4.00¹³³. Abbreviations: PMN = human PolyMorphoNuclear cells, HL60 = human leukemia cells.

Once neutrophils mature, genes involved in cell cycle regulation are mostly downregulated¹⁰³. Hence, the question lies in whether certain CDKs exert specific functions in neutrophils. Only a handful of studies specifically observe CDK functions in neutrophils. The following section describes studies using various models to characterize CDK functions in neutrophils outside cell cycle regulation.

CDK2 regulates neutrophil migration

Neutrophils sense extracellular cues from their surface receptors and respond accordingly by activating signal transduction pathways¹⁰⁵ to reach the site of infection. Chemokines and other chemoattractants initiate neutrophil migration by inducing cell polarization to generate a defined leading edge. Signaling molecules such as Rac^106,107 and PI3K¹⁰⁸ stimulate F-actin polymerization, pushing the membrane forward to develop the leading edge. Rho and its effector molecules regulate actomyosin fibers’ contraction in the trailing edge to retract the tail.

CDK2 was identified through a microRNA overexpression screen as a critical regulator for neutrophil migration. Hsu et al.¹⁰⁹ first discovered that miR-199 overexpression led to reduced neutrophil motility in zebrafish. One of its direct targets, CDK2, was then found to regulate neutrophil migration. CDK2 specific inhibitors NU6140 and CVT313, and the pan-CDK inhibitor, roscovitine, blocked zebrafish neutrophil motility and recruitment to infected ear and tail wound sites without causing neutrophil apoptosis. The kinase activity of Cdk2 in zebrafish is essential since overexpressing the catalytically dead dominant-negative form (DN) of Cdk2 disrupted cell polarity and actin dynamics. In addition, NU6140 inhibited primary human neutrophils chemotaxis towards the chemokine IL-8, with a decreased chemotaxis index. To get to the molecular level, Hsu et al.¹⁰⁹ used HL60 cell lines with CDK2 knockdown, which exhibited hampered chemotaxis and reduced cell polarization. After chemoattractant stimulation, the cells showed a decrease in PAK (P21 (RAC1) activated kinase) and AKT (protein kinase B) phosphorylation, indicating lower Rac activation and reduced level of phosphatidylinositol (3–5)-trisphosphate. For specificity, dHL60 with CDK2 knockdown did not affect other neutrophil functions such as NETosis. In addition, only CDK2, but not CDK1 or CDK5, is required for neutrophil migration. Remarkably, treating zebrafish with CDK2 inhibitors increased fish survival during infection and sterile inflammation conditions.

There are no human conditions associated with abnormal CDK2 function reported so far, possibly due to the overlapping roles of CDKs or the necessity of CDK2 for fertility. Given that CDK2 knockout mice are generally healthy and that neutrophil behavior can be affected through CDK2¹⁰⁹, the inhibition of this kinase can be utilized to control neutrophil numbers at infection or injury sites, thus potentially preventing neutrophil-mediated over-inflammation. Subsequent discoveries on substrates of CDK2 also carry therapeutic potential as these substrates can potentially serve as novel drug targets in neutrophils. Currently, CDK2 inhibitor CVT313 has reduced the progression of colorectal cancer¹¹⁰. A selective CDK2 degrader that significantly reduces CDK2 levels in cell lines via ubiquitin degradation also promises acute myeloid leukemia therapy¹¹¹.

CDK4/6 regulates the NET formation

A unique function of neutrophils in combating pathogens is the formation of NETs. Multiple stimuli induce NETs, including mitogen phorbol myristate (PMA)¹¹², Candida albicans, Group B Streptococcus, and others¹¹³. The NET formation is activated through NADPH oxidase and an increase in anti-apoptotic proteins in the Raf-MEK-ERK pathway¹¹⁴. Independent of the NADPH pathway, NETosis can be induced by calcium ionophores through mitochondrial ROS and the calcium-activated small conductance potassium channel, SK3¹¹⁵.

NETosis involves the nuclear envelope breakdown, a feature observed during mitosis, markedly different from apoptosis. Amulic et al.⁴⁹ demonstrated a mitogenic reactivation of cell-cycle regulators and a critical role of G1 kinases, CDK4 and 6, in NETs production. They first showed that cell division markers accompany NETosis in human neutrophils. Following NETosis induction, G1 kinase activation, as detected by the phosphorylation of pRb, is observed without the subsequent expression of S-phase genes. Phosphorylation of a mitotic marker, histone H3 at serine 10, and a marker for proliferating cells, the nuclear antigen Ki-67, are elevated. Phosphorylation of lamin A/C, which contributes to the nuclear envelope breakdown, is also upregulated. Inhibiting cyclin-CDK complexes using a synthesized peptide mimicking the cell cycle inhibitor p21cip1 (p21) interfered with the proper NET formation in human neutrophils. In neutrophils of p21 knockout mice, NETs were more abundant, supporting that G1 kinases (CDK4 and CDK6) activation is required to form NETs. Amulic et al.⁴⁹ used CDK4/6 pharmacological inhibitor (abemaciclib/LY2835219) and found that it dose-dependently blocked NET formation without compromising other functions in human neutrophils. Neutrophils from CDK6 knockout mice also display impaired NET formation. The significance of Cdk6 in NETosis also extends to provide better survival in murine C. Albicans sepsis model as Cdk6 knockouts exhibited higher fungal load than WT mice, whereas neutrophil recruitment was intact. When Cdk6 was rescued in myeloid cells of knockout mice, the fungal burden was decreased, indicating restoration of antimicrobial defense. Although this observation suggests neutrophil function defect, it does not rule out impairments in other blood cells.

Despite the importance of NETs in protecting against foreign microbes, NETs play a significant role in autoimmune diseases, including rheumatoid arthritis (RA), psoriasis, and SLE. In RA, infiltrated neutrophils in the synovial cavity of RA patients are more ready to form NETs⁹⁴, increasing inflammation due to autoantigens¹¹⁶. With the knowledge that the induction of NETs is dependent on CDK4 and 6, specific inhibitors can potentially be used to alleviate over-inflammation in RA and other autoimmune diseases.

CDK5 regulates neutrophil degranulation

Degranulation is when stored granules are released via exocytosis to fight off pathogens. Human neutrophils possess azurophilic granules containing myeloperoxidase and defensins. They also have specific granules containing lactoferrin, NADPH oxidase, and alkaline phosphatases¹¹⁷. The mechanism of degranulation actively involves the actin cytoskeleton and microtubule assembly¹¹⁸, elevated intracellular Ca²⁺, and hydrolysis of ATP and GTP. The GTP-dependent secretion mechanism involves a cytosolic factor that is not a member of the Rho or Rac families of GTPases but a complex with a GTP-binding protein¹¹⁹. Upon further investigation, the element was CDK5, and its activator, p35¹²⁰.

Due to the observed roles of Cdk5 in regulating secretion from neurons, Rosales et al.¹²⁰ sought to investigate the function of this kinase in neutrophil degranulation. Human neutrophil lysates contain active Cdk5 activity. Subsequent immunoprecipitation of the kinase from neutrophil subcellular fractions indicated that the Cdk5-p35 complex localizes primarily in the secretory granules and plays a vital function in its formation and secretion. The pan-CDK inhibitor roscovitine inhibited the GTP-stimulated lactoferrin secretion from specific granules. In mouse neutrophils, roscovitine reduced the expression of CD63, CD66b, markers of azurophil, and specific granules secretion. This partial inhibition is similarly observed in p35−/− mouse neutrophils, suggesting that Cdk5-p35 activity is required for maximum GTP-induced secretory response in neutrophils.

Upon characterization of the phosphorylated proteins by Cdk5, Lee et al.¹²¹ found Cdk5 phosphorylates vimentin explicitly at Ser56. Vimentin undergoes reorganization following GTP stimulation. Roscovitine blocked vimentin phosphorylation and partially inhibited neutrophil degranulation. Considering incomplete inhibition of roscovitine, other cellular proteins may regulate vimentin Ser56 phosphorylation, or other mechanisms may regulate vimentin activity. Furthermore, the authors used siRNA to knock down Cdk5 and found decreased secretion of β-hexosaminidase, lactoferrin, and matrix metalloprotease-9 from neutrophils stimulated with GTP. Together, Cdk5 is significantly involved in GTP-induced neutrophil degranulation, and its inhibition leads to a considerable decrease in the secretory process.

CDK7 and 9 regulate neutrophil apoptosis

Apoptotic neutrophils and their clearance by macrophages play a critical role in resolving inflammation¹²². Neutrophil apoptosis reduces overall circulating cytokines and further infiltration of neutrophils, thus promoting resolution³⁵. CDK7 and CDK9 phosphorylate RNA polymerase II at the CTD to upregulate inducible genes that control neutrophil apoptosis, such as the anti-apoptotic protein Mcl-1. Inhibition of these CDKs by roscovitine and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) (a specific inhibitor for CDK7 and 9) reduces the level of Mcl-1 and thus induces apoptosis in human neutrophils^7,41. Roscovitine induces caspase-dependent apoptosis and resolves neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice, highlighting its therapeutic potential^7,41. Other inhibitors that can cause human neutrophil apoptosis include NG75 and hymenialdisine⁴¹, which may also reduce Mcl-1.

The maintenance of this short-lived, anti-apoptotic protein Mcl-1 via CDK9 activity is confirmed by Wang et al.¹²³. CDK9 and its partner cyclin T1 form part of the P-TEFb complex involved in transcriptional regulation, and its activity declines as neutrophils age. A potent CDK9 inhibitor, flavopiridol (AT7519), causes a rapid decrease in Mcl-1. In zebrafish, pharmacological inhibition, morpholino-mediated knockdown, or CRISPR/cas9-mediated knockout of CDK9 induces neutrophil apoptosis and increases inflammation resolution after tailfin injury¹²⁴. The involvement of CDK9 with transcriptional regulation is confirmed with the knockdown of an endogenous negative regulator of the P-TEFb complex, La-related protein 7 (LaRP7), which leads to increasing neutrophilic inflammation at a wound site.

A small-molecule multi-CDK inhibitor, AT7519, inhibits RNA polymerase II phosphorylation carried out by CDK7 and 9¹²⁵. Kaveh et al.¹²⁶ have shown that in zebrafish, treatment with this inhibitor or flavopiridol resolves neutrophil infiltration by inducing reverse migration from the cardiac lesion in a cardiac injury model. AT7519 is found to be more selective to CDK9 than flavopiridol, and transient treatment with this inhibitor increases neutrophil resolution and improves the rate of myocardial wound closure through amplifying macrophage-dependent cardiomyocyte expansion. Further research involving the systemic inflammation model may help expand the roles of CDK7 and 9 in acute and chronic inflammatory diseases where neutrophil-dominant inflammation is widely present.

Concluding Remarks

In this review, we have summarized current findings that support the essential functions of CDKs and cyclins in not only neutrophil development but also their survival and immune function. These findings indicate a growing picture of the complexity CDKs may have in regulating innate immunity in response to challenges.

Current limitations and questions still exist in the neutrophil field regarding CDKs. First, the consensus of cyclins and CDKs present in neutrophils and their pairing for activation is lacking. Such discrepancies in results may arise from using different models like humans, mice, or zebrafish. The sharing of cyclins and co-factors between different CDKs due to similar binding motifs also complicates investigations into their specific interactions and roles^6,92. CDK4/6 inhibitors used in clinics have been shown to cross-react and also inhibit CDK2 activity through promoting p21 dissociation from CDK4¹²⁷. Indirect interactions of CDK and cyclins with CKI can also result in different phenotypes. Multiple knockouts of CDK, cyclin, and complex inhibitors may be needed to overcome compensatory effects and fully explore their specific interactions and functions. Specific CDK degraders can serve as a unique tool in understanding individual CDK functions. However, the possibility of compensation effects cannot be ruled out, and they may not be as potent as the pan-inhibitor of CDKs.

There are still knowledge gaps in how CDKs may facilitate other neutrophil functions not mentioned here, such as phagocytosis, cytokine release, ROS production, and immune presentation. Molecules related to CDK activation, their co-factors, and substrates in neutrophil function pathways have yet to be fully characterized. Since neutrophils play such a prominent role in inflammation, CDK substrates can be used as novel therapeutic targets to precisely control neutrophil behavior. For instance, targeting CDKs and their substrates involved in neutrophil ROS production can reduce tissue injury in chronic inflammation. Resolving inflammation by developing drugs that target CDKs functioning in neutrophil apoptosis can help alleviate many diseases, including rheumatoid arthritis and cystic fibrosis.

Although terminally differentiated, neutrophils display heterogeneous populations, due to their plasticity in properties, depending on multiple factors and conditions^128–130. In tumors, similar cytokines and effector signals have been shown to affect the pro- and anti-inflammatory properties of neutrophils, leading to either tumor progression or suppression¹³¹. In COVID19 patients, multiple neutrophil populations exhibited a correlation with disease severity¹³². Exploring neutrophil CDKs while considering their plasticity in response to various conditions will be beneficial in developing neutrophil-targeted therapies catered for disease-specific pathologies.

In summary, a detailed understanding of CDKs and cyclins and their co-factors in neutrophils opens a promising avenue in manipulating this prominent immune cell type.

Figure 1. Overview of CDKs involved in several neutrophil functions.

CDK2 phosphorylates molecules related to neutrophil migration as CDK2 inhibition hampered chemotaxis and cell polarization. CDK5 and p35 regulate neutrophil degranulation by phosphorylating vimentin upon GTP stimulation. CDK9 and CCNT1 form part of the P-ETFb complex, which LaRP7 negatively regulates. A decrease in complex activity will decrease Mcl-1 and induce apoptosis. A reduction of CDK7 activity will also reduce Mcl-1 levels and result in apoptosis. CDK4 and 6 phosphorylate Rb, histone H3 at serine 10 (H3S10), and lamin A/C, facilitating nuclear envelope breakdown and NETosis.

Table 2.

Functions of CDKs and cyclins in neutrophils and other cell types.

Function in neutrophils	CDK/Cyclin	Direct substrates	Description
Migration	CDK2	-	Regulation of cytoskeleton arrangement109
Apoptosis	CDK7/9 and CCNT1	Mcl1	Resolution of inflammation via neutrophil apoptosis7,123,124
Defense	CDK4/6	Rb, lamin A/C, H3S10	Formation and release of NETs49
	CDK5 and p53	Vimentin	Formation of secretory granules120,121
Function in other cell types	CDK/Cyclin	Direct substrates	Description
Hematopoiesis	CDK2/4	-	Regulation of erythrocyte formation96
	Cyclin D1	-	Promote neuronal differentiation10,140
	Cyclin D2	-	Development of B-lymphocytes11
	Cyclin D3	-	Progression of granulopoiesis102 and development of T-lymphocytes12
Migration	CDK5	Rac1	Regulation of endothelial cell migration and angiogenesis8
	CDK6	-	Increase macrophage adhesion141
	Cyclin A2	RhoA	Regulation of RhoA activity in fibroblasts142

Abbreviations

CDK

cyclin-dependent kinase

CKA

CDK-activating kinase

CKI

cyclin-dependent kinase inhibitors

CTD

carboxy-terminal domain

DN

dominant negative

DRB

D-ribofuranosylbenzimidazole

G-CSF

granulocyte-colony stimulating factor

IL

Interleukin

LaRP7

la-related protein 7

NETs

neutrophil extracellular traps

PAD4

peptidylarginine deiminase 4

PAK

p21-activated kinase

PMA

phorbol myristate

Ppa

pyropheophorbide-a

P-TEFb

positive-transcription elongation factors

RA

rheumatoid arthritis

Rb

retinoblastoma

ROS

reactive oxygen species

SLE

systemic lupus erythematosus

TRAF

TNF Receptor Associated Factor