Amplified centrosomes in dendritic cells promote immune cell effector functions

Centrosomes constitute structural elements organizing the mitotic spindle in animal cells for proper chromosome segregation. Centrosome numbers are tightly controlled and limited to one during interphase and two before a cell enters mitosis. Defects in regulating centrosome numbers lead to the presence of amplified centrosomes, which are a hallmark of malignant cells and sufficient to induce tumorigenesis. By contrast, amplified centrosomes are rarely observed in normal somatic cells and often removed during terminal differentiation. Here, we demonstrate the presence of amplified centrosomes in dendritic cells (DCs) during immune activation. Mature DCs accumulate centrosomes by mitotic defects and show high expression levels of polo-like kinase 2 (PLK2) leading to over-duplication of centrioles. During cell migration, amplified centrosomes tightly cluster and act as functional microtubule-organizing centers, which promote persistent locomotion. Moreover, DCs with amplified centrosomes show enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of amplified centrosomes in promoting the ability of leukocytes to enhance immune responses.


Introduction
Centrosomes are highly conserved cell organelles, consisting of two centrioles, which are surrounded by ordered layers of pericentriolar material (PCM) 1,2 . They are present as single copy in interphase and duplicate precisely once before a cell enters mitosis 3 . Centrosome abnormalities are found in virtually all types of human cancers and have been classified as structural or numerical aberrations. Numerical aberrations in cancer cells, such as centrosome amplification (CA), arise due to cell cycle dysregulation, and correlate with karyotype alterations, clinical aggressiveness and lymph node metastasis 4,5 . Mechanistically, amplified centrosomes promote the formation of lagging chromosomes and micronuclei during bipolar cell divisions 6,7 . Chromosomes in micronuclei often undergo defective and asynchronous DNA replication, resulting in DNA damage and extensive fragmentation 7 . These findings establish a causal link between centrosome amplification and chromosomal instability (CIN), which has recently been confirmed in vivo 8 . Of note, normal somatic cells poorly tolerate amplified centrosomes and lose them in the absence of selection 6 .
Dendritic cells (DCs) are leukocytes, which constitute critical cellular players that integrate innate immune signals and initiate adaptive immunity via antigen-presentation. They reside in peripheral tissues and are characterized by a stellate morphology, high expression of major histocompatibility class (MHC) II, as well as their capacity to sense antigens via Toll-like receptors (TLRs) 9 . Upon antigen recognition, DCs enter a maturation program, which triggers antigen uptake and processing and subsequent homing to secondary lymphoid organs. Within lymph nodes, DCs activate antigen-specific T cells, thereby initiating adaptive immune responses 10 . To efficiently navigate through complex three dimensional (3D) environments, DCs choose the path of least resistance and move without tightly adhering to the substrate, while being able to adapt their migration mode according to the environment [11][12][13] . Besides DCs, metastasizing cancer cells, which frequently exhibit amplified centrosomes 4 , use similar mechanisms of locomotion to move through interstitial tissues 14 . This motivated us to define the role of centrosomes in DC biology.

DCs show upregulation of centrosome numbers upon maturation
We first analyzed centrosome numbers in peripheral DCs isolated from mouse skin explants.
Split ear sheets were floated on culture medium supplemented with the chemokine CCL19, which allows emigration of dermal DCs into the culture medium 15 . After three days of emigration, we collected all non-adhering cells, which expressed high cell-surface levels of classical DC markers such as CD11c and MHC II ( Supplementary Fig. 1A) and efficiently migrated towards chemotactic gradients in 3D collagen matrices ( Supplementary Fig. 1B).
Immunostaining against the PCM component -tubulin and the centriolar marker acetylated (ac)-tubulin 16 revealed that a proportion of dermal DCs displayed four or more centrioles, corresponding to two or more centrosomes, which are all located in close proximity to the nucleus (Fig. 1a,b). To test whether upregulated centrosome numbers in dermal DCs are a 4 consequence of ongoing cell proliferation, we analyzed 5-ethynyl-2′-deoxyuridine (EdU) incorporation and pospho-Histone3 (pH3) levels as a marker for S-phase and G2-M transition, respectively. Dermal DCs were largely EdU-and pH3-negative, demonstrating that extra centrosomes are not a consequence of ongoing cell proliferation (Fig. 1c). Similar centrosome numbers were also observed in sorted splenic DCs, but not in other subtypes of the myeloid or lymphoid lineage that we analyzed ( Supplementary Fig. 1C). These findings indicate that in the DC compartment cells that reside in G1 of the cell cycle carry two or more centrosomes.
Due to the absence of proliferation markers, which would explain the presence of two centrosomes, we refer to this phenomenon as CA in DCs.
As DCs constitute a rare population of cells in vivo, accounting for only 1-2% of total cells in most tissues 10 , we used DCs from murine bone marrow-derived stem cells to study the underlying mechanisms of CA. We first analyzed centrosome numbers before and after stimulation with the bacterial cell wall component lipopolysaccharide (LPS). LPS treatment leads to activation of immature DCs into mature cells, which present antigens via MHC I and II complexes 17,18 . Similar to dermal DCs, mature bone marrow-derived DCs (BMDCs) showed amplified centrosome numbers while staining negative for pH3 or EdU (Fig. 1d,e and Supplementary Fig. 1D,E). In contrast to mature cells, immature DCs stain positive for pH3 and EdU indicating that these cells are actively proliferating. Yet, pH3-negative immature DCs typically showed only one centrosome (Fig. 1e).
We concluded that in primary DCs, centrosomes overamplify when cells become activated by inflammatory stimuli such as LPS.

DCs acquire amplified centrosomes via two different pathways
Two major mechanisms are known to contribute to CA in cancer cells: i) over-duplication of centrioles and ii) accumulation of centrosomes due to mitotic defects 19,20 . During the cell cycle, centrioles duplicate in S phase, using a semi-conservative replication mechanism. A new daughter centriole forms next to a pre-existing mother centriole, that elongates and matures until mitosis 21,22 . As a result, each cell contains exactly four centrioles at mitosis: two mature 5 mother and two daughter centrioles (Supplementary Fig. 2A). Consequently, the mother/daughter ratio is equal to one. During centriole over-duplication, multiple daughter centrioles are generated around a single mother centriole 19 leading to a mother/daughter ratio smaller than one. Mitotic defects such as cytokinesis failure yield in centrosome accumulation and simultaneously polyploidization. As centriole duplication occurs normally, the mother/daughter ratio in polyploid cells still equals one 23 . Thus, determination of DNA content, centriole numbers and their maturation state allows to differentiate between the two pathways of centrosome amplification 24 .
To simultaneously visualize centrosome numbers and DNA content in DCs, we made use of reporter mice, which constitutively express Centrin-2 (CETN2) fused to green fluorescent protein (GFP) as integral part of the centriole. CETN2-GFP expressing cells generally show two fluorescently labeled centrioles for every organ examined 25 . Similar to wildtype BMDCs and dermal DCs, mature CETN2-GFP expressing BMDCs exhibited amplified centrosomes upon LPS treatment (Fig. 2a). Analysis of DNA content revealed that 17% of mature CETN2-GFP expressing BMDCs showed a DNA profile of tetra-or even higher ploidy (Fig. 2b).
Fluorescence-activated cell sorting (FACS) based on DNA content allowed us to separate and enrich diploid (2N) from polyploid (≥4N) cells. The majority of ≥4N cells contained two or more centrosomes but stained negative for pH3, confirming again that amplified centrosomes are not a consequence of ongoing cell proliferation ( Fig. 2c and Supplementary Fig. 2B). Moreover, ≥4N cells showed abnormal mitotic figures, such as large chromosome bridges and binucleated cells indicating cytokinesis failure (Fig. 2d). Similar mitotic figures were also observed in dermal DCs ( Supplementary Fig. 2C). However, a proportion of 2N cells, which did not experience mitotic defects, also displayed amplified centrosomes (Fig. 2e), indicating that mitotic defects are not the only pathway leading to amplified centrosomes in DCs. To investigate whether over-duplication of centrioles occurs in DCs, we stained CETN2-GFP expressing BMDCs against a marker for maternal centrioles. Proteins such as CEP170 localize to subdistal appendages, which are exclusively present on mature mother centrioles 26 (Fig.   2f). We next characterized CEP170 signals in sorted 2N and ≥4N BMDCs. Most ≥4N cells 6 revealed two CEP170 signals, leading to a mother/daughter ratio of one (Fig. 2g). Similarly, within the 2N fraction, cells with only one centrosome (2N one ) displayed a mother/daughter ratio of one, while cells with amplified centrosomes (2N amplified ) showed less mother than daughter centrioles and consequently a ratio smaller than one. These data support the hypothesis that over-duplication of centrioles takes place in DCs and together with mitotic defects, account for amplified centrosome numbers.

PLK2 expression after LPS stimulations leads to centriole over-duplication in DCs
Over-duplication of centrioles typically results from increased expression of proteins controlling regular centrosome duplication. Two key players of centriole duplication are the polo-like kinases (PLK) 2 and 4 27,28 . mRNA levels of Plk2 (and to a lesser extent of Plk4) were highly upregulated in DCs upon LPS stimulation (Fig. 3a). To test whether high expression levels of Plk2 and/or Plk4 account for centriole over-duplication in DCs, we generated CRISPR/Cas9 knock-outs in precursor cell lines 29 and differentiated them into DCs (Fig. 3b,c). While PLK2deficient cells differentiated into the DC lineage and upregulated MHC II molecules upon LPS stimulation, PLK4-deficiency diminished cellular growth and differentiation, which is why we excluded these cells from further assays. Of note, Plk2 -/and control cells showed the same extent of polyploidization confirming that PLK2-deficiency does not interfere with cell cycle progression in DCs (Fig. 3c). Analysis of centrosome numbers revealed a significant reduction of amplified centrosomes in Plk2 -/cells compared to wildtype controls (Fig. 3d). These results demonstrate that PLK2 plays a major role in centriole over-duplication in mature DCs.

Amplified centrosomes promote directional locomotion
Rapid migration of DCs is a prerequisite for initiating adaptive immune responses. Directional locomotion requires actin-rich protrusions at the cell front and acto-myosin mediated retraction of the cell's rear. In DCs, the centrosome acts as major microtubule organizing center (MTOC), which determines the path of the cell body. Consequently, loss of microtubule (MT) cytoskeleton integrity leads to impaired persistent locomotion along gradients of chemotactic cues 30 . To investigate whether amplified centrosomes act as functional MTOCs, we first determined the amount of MT filaments emanating from individual centrosomes in migrating BMDCs by high-resolution microscopy. Cells were injected under a block of agarose and exposed to the chemokine CCL19 to attract mature DCs. Under these 2D conditions, cells form a flat lamellipodium at the front, which allows to monitor intracellular structures.
Immunostaining against -tubulin revealed that all centrosomes nucleated MT filaments along the axis of migration, implying that extra centrosomes act as functional MTOCs. Quantification of individual MT filaments showed that cells with amplified centrosomes contained a larger number of total MT filaments within the cytoplasm (Fig. 4a). Measuring intercentrosomal distances between pairs of centrioles unveiled that all centrosomes were located in close proximity to each other (Fig. 4b). A similar phenomenon of centrosomal clustering was recently reported for mitotic cancer cells in order to avoid spindle multipolarity 31 .
To determine the behavior of individual centrosomes and their impact on cell locomotion, we followed the dynamics of CETN2-GFP expressing BMDCs during 2D migration over time To address whether amplified centrosomes are causally linked to enhanced directional locomotion, we removed extra centrosomes during BMDC migration by laser ablation and measured migration velocity and persistence before and after the ablation process. To this aim, we first determined the settings required for efficient centriole ablation. Maximal Zprojections of CETN2-GFP signals before and after full centrosomal ablation revealed a loss of fluorescence signal at the irradiated region immediately after laser exposure (Fig. 5a). To test whether laser exposure can efficiently disrupt MTOC function, we stained MT filaments after full centrosomal ablation. Following laser exposure, less MT filaments nucleated from ablated centrioles compared to non-treated cells (Fig. 5b). Overall, MT filaments in ablated cells were shorter and showed a disorganized structure with a more bent configuration compared to control cells (Fig. 5c). From that we concluded that our laser set-up allows for efficient destruction of centrosomes and MT organization. To assess the impact of noncentrosome targeted laser exposure on cell behavior, we exposed random areas in close proximity to centrosomes with identical laser settings. Cells experiencing control, noncentrosome targeted ablations, retained their polarized shape and continued to migrate throughout the imaging period. Migration velocity was slightly decreased, whereas directional Altogether, our data unequivocally demonstrate that amplified centrosomes enhance persistent migration in DCs.

Enrichment of diploid cells with amplified centrosomes
Due to the beneficial role of amplified centrosomes during DC migration, we next sought to address the impact of extra centrosomes on adaptive immune responses. In contrast to our single cell approach for determining migration parameters, T cell activation assays require large amounts of cells. Due to the heterogeneity in centrosome numbers in DCs we aimed for separating BMDC subpopulations of different centrosomal content to directly compare cells with one and amplified centrosomes. CETN2-expressing BMDCs show a prominent centriole signal, while background fluorescence is low indicating that all CETN2-GFP is either incorporated into centrioles or degraded by the proteasome. This motivated us to separate and enrich cells with different numbers of centrosomes according to CETN2-GFP signal intensities.
Staining for DNA content allowed us to separate polyploid (≥4N) from diploid cells (2N) as described for Figure 2. The diploid fraction contains cells with amplified (2NCA) and one (2N2C) centrosome(s). As polyploid cells are generally larger than diploid cells due to their surplus of DNA and show gene-dosage dependent expression of a variety of proteins 32 , we focused exclusively on diploid cells containing either one or amplified centrosome(s). To separate those two populations, we used CETN2 signal intensities of polyploid cells for our gating strategy. We reasoned that due to the surplus of centrioles in polyploid cells, the CETN2-GFP signal should be comparable to the signal of 2NCA cells. By overlaying the intensity distribution of polyploid cells with the distribution of diploid cells we determined the gating areas for 2N2C and 2NCA cells, respectively (Fig. 6a). Re-analysis of sorted BMDC subpopulations indicated that CETN2-GFP signals were shifted to higher values in the 2NCA population, while both populations showed a diploid DNA profile (Fig. 6b). After cell sorting we analyzed centrosome numbers by confocal microscopy to assess the purity of individual subpopulations. We were able to enrich 2NCA cells by a factor of at least 1.5, ranging from 8-42% amplified centrosomes within the 2N2C population and 21-70% amplified centrosomes within the 2NCA population. Both diploid subpopulations expressed the same levels of classical DC markers and co-stimulatory molecules, while macrophage/granulocyte markers were absent (Fig. 6c) confirming that both subpopulations consisted of DCs.

Cells with amplified centrosomes are more potent in eliciting T cell responses
To induce T cell immunity, DCs present antigenic peptides by MHC complexes on their cell surface. Immature DCs sequester internalized antigens in lysosomes, process them into small peptides and load them on MHC II molecules for presentation to CD4 + T cells 33 To address whether increased T cell activation is a consequence of enhanced intracellular processing of antigens, we directly loaded sorted DC subpopulations with OVA329-337 peptide to bypass the processing step. Similar to OVA protein, we detected marked differences in IL-2 secretion and T cell proliferation ( Supplementary Fig. 4A,B), suggesting that enhanced T cell activation is not a consequence of improved intracellular antigen processing. Similarly, we excluded T cell co-stimulation via CD40, CD70 and CD86 as reason for optimized T cell activation as cell-surface levels were indistinguishable on both DC subpopulations (Fig. 7c).

Cells with amplified centrosomes show increased cytokine secretion
Recent data in macrophages suggest an important role of centrosomes for cytokine production in response to inflammatory stimuli 36 . Moreover, the extra centrosome-associated secretory pathway (ECASP) has been identified as a distinct secretory phenotype in cells with extra centrosomes 37 . In DCs, cytokines are stored in lysosomes and released via the secretory pathway 38 . MTs emanating from centrosomes act as main tracks, which orchestrate long-range intracellular vesicle trafficking of cargos to their destination compartment 39 . As MT numbers were increased in cells with amplified centrosomes (Fig. 4a), we investigated whether cytokine release is altered in cells with distinct centrosome numbers.
To quantify cytokine levels, we collected supernatants of sorted DC subpopulations and monitored cytokines by antibody arrays and ELISA, respectively. Both approaches revealed that cytokine levels were increased in supernatants harvested from cells with amplified centrosomes. In particular, chemokines which attract and activate naïve T cells and neutrophils such as CCL17, CCL5, IL-6 and CXCL1 [40][41][42][43] were elevated (Fig. 7d and Supplementary Fig.   4C). Except for CCL17, which induces T cell chemotaxis rather than stimulating T cells 44 , mRNA levels of CCL5, IL-6 and CXCL1 were indistinguishable in both diploid DC subpopulations (Fig. 7e), pointing out that the production of these cytokines is not affected by the presence of amplified centrosomes. Instead, our data suggest that rather trafficking and secretion of vesicles containing T cell stimulatory molecules account for enhanced activation of T cells. To further elaborate on this concept, we inhibited intracellular protein transport in sorted DC subpopulations using Monensin and Brefeldin A. Intracellular cytokine staining after blocking protein transport revealed increased levels of IL-6 and CCL5 in cells with amplified centrosome numbers (Fig. 7f). As both cytokines are produced at equal amounts in 2N2C and 2NCA cells, these findings support the notion that enhanced cytokine trafficking accounts for improved T cell activation in cells with amplified centrosomes.
Altogether, our findings demonstrate that DCs with amplified centrosomes show enhanced secretion of inflammatory cytokines and optimized T cell responses and revise the current view of amplified centrosomes to be present exclusively under pathological conditions.

Discussion
The presence of amplified centrosomes has been demonstrated to cause aneuploidy and constitutes a well appreciated hallmark of malignancy and cancer development. Several studies emphasis the detrimental effects and consequences of misregulating centrosome numbers: artificial induction of centrosome amplification in mice and flies by overexpression of the centriole replication protein PLK4/SAK is sufficient to initiate spontaneous tumorigenesis in certain tissues 8,45 . Moreover, CA has been described to confer advantageous features such as enhanced invasion to some tumor cells, thereby promoting metastasis and tumor progression 46 . Clinically, centrosome amplification is frequently observed in aggressive tumors and associated with lymph node metastasis and poor patient prognosis 4 .
Despite the adverse relationship between extra centrosomes and cell transformation, we provide evidence that amplified centrosomes can be part of regular cell physiology in postmitotic immune cells. DCs serve as first line defense against invading pathogens and represent the most potent antigen-presenting cells of the innate immune system. Our data demonstrate that DCs upregulate centrosome numbers upon exposure to inflammatory stimuli and subsequent maturation. In contrast to immature DCs, mature cells are non-proliferative and reside in G1 of the cell cycle. However, they often exhibit two or more centrosomes, which we refer to as CA. We further identified two pathways being responsible for excess centrosome numbers in DCs: accumulation of centrosomes due to mitotic defects such as cytokinesis failure and over-duplication of centrioles, which is caused by elevated PLK2 levels upon LPStreatment.
Functionally, amplified centrosomes ameliorate persistent locomotion along gradients of chemotactic cues and enhance the secretion of cytokines which facilitate T cell activationtwo fundamental tasks for a properly operating immune system.
In contrast to other terminally differentiated cells, which often show MT nucleation from noncentrosomal MTOCs such as the Golgi, the nucleus or the cell cortex 47 , DCs use the centrosome as major site for MT nucleation. This centrosome-derived MT array is required for executing DC effector functions: In most tissues, DCs reside in an immature state, unable to activate T cells. However, they are well equipped to capture antigens, which trigger full maturation and antigen presentation via MHC complexes. Mature DCs upregulate MHC class II molecules, which bind to peptides that are derived from proteins processed in the endocytic pathway 48 . Fast transport of MHC class II containing vesicles to the cell surface is accomplished along MT filaments 33,49 . During DC migration, MTs control pathfinding and coordination of multiple protrusions in complex 3D environments as well as maintenance of cell coherence 13,30 . Upon arrival in lymph nodes, DCs and T lymphocytes engage with each other at a structure termed immunological synapse, which allows transmission of different types of signals. The DC cytoskeleton undergoes major spatial redistributions during immune synapse formation, whereby the centrosome reorients toward the DC-T cell interface leading to polarized transport of soluble mediators of T cell activation along MT filaments 50 . Due to these multifaceted functional tasks, DCs need to be able to rapidly re-organize their MT array.
Amplifying centrosome numbers could be one strategy which allows the cells to adapt to various different environments such as the interstitium or the lymph node and fulfill distinct tasks depending on their location.