THE ACTIN CYTOSKELETON CONTROLS NADPH OXIDASE ACTIVATION AND G PROTEIN RECRUITMENT MEDIATED BY NEUTROPHIL Gαq-COUPLED RECEPTORS

Signaling by formyl peptide receptor 1 (FPR1), the prototype G protein -coupled receptor (GPCR) expressed in neutrophil leukocytes, is initiated by an activation of a G protein containing a G ai subunit. FPR1 activation results in an increase in the cytosolic concentration of free calcium ions ( [Ca2+]i), and an activation of the superoxide anion producing NADPH oxidase. Receptor downstream signals generated by the danger molecule ATP recognized by the purinergic receptor P2Y2 are transduced by a G protein containing a Gαq subunit. The neutrophil response induced by ATP also includes a transient rise in [Ca2+]i, but the downstream signals do not activate the NADPH oxidase . ATP can, however, activate th is enzyme system through a receptor transactivation mechanism dependent not only on the ATP receptor but also on the free fatty acid receptor FFA2R, provided that this receptor is allosterically modulated . This occurs through a novel mechanism whereby FFA2R is activated from the cytosolic side of the plasma membrane by Gαq transduced signals generated by the ATP receptor. Furthermore, in neutrophils with a disrupted actin cytoskeleton, ATP (as well as platelet activating factor; recognized by the Gαq-coupled PAFR) becomes a potent NADPH oxidase activating agonist . At high concentrations of the actin cytoskeleton disrupting drug latrunculin A the activation was only partly reduced by Gαq inhibition. More importantly , this response was also partly inhibited by pertussis toxin. The effects on the ATP-induced NADPH oxidase activity, of the Gαq inhibitor and pertussis toxin were more and less pronounced, respectively, when the concentration of latrunculin A was reduced. Taken together, we show that in primary human neutrophils the actin cytoskeleton is part of the regulatory machinery that determines the activation of NADPH oxidase activation and the G protein recruitment profile downstream of activated of Gαq-coupled GPCRs. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 5

Neutrophils; actin cytoskeleton; NADPH oxidase; G protein-coupled receptors; GPCRs; biased signaling; functional selectivity; G protein recruitment; P2Y2R; PAFR .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 6

Professional phagocytes such as neutrophil granulocytes and monocytes express an NADPH oxidase with the capacity to transport electrons over a membrane using electrons from NADPH, produced in the hexose monophosphate shunt . The electrons reduce molecular oxygen to superoxide anions (O 2-; [1, 2]). The membrane component of the NADPH oxidase is made up by one larger (NOX2; gp91phox ) and one smaller protein (p22 phox ) and this heterodimer (cytochrome b 558) contains two heme groups, and, together with a redox coenzyme (FAD), the cytochrome functions as the catalytic center of the electron transporting enzyme. In resting (naïve) neutrophils several phagocyte oxidase factors (p40phox , p47 phox , p67phox , Rac1/2) are cyto solic and for an activation of NADPH oxidase , these protein s must first be recruited to the b cytochrome containing membrane [3]. It has long been well established that signals generated in neutrophils by chemoattractant receptors such as formyl peptide receptor 1 (FPR1), trigger an assembly and activat ion of the NADPH oxidas e in the plasma membrane [4]. As a consequence, t he O2- produced by neutrophils activated by the FPR1 agonist fMLF are not retained inside the cells but instead released/secreted extracellularly where other reactive oxygen species ( ROS) are formed with O2- as the starting component (see [5, 6]). Although signaling by activated FPRs has been intensively studied for long, the precise signals and molecular mechanisms that regulate the NADPH oxidase activation process have not yet been identified [7]. Neutrophil receptors and the ligands that regulate their functions constitute the basis for the ability of our innate immune system to recognize both pathogen -associated molecular patterns (PAMPs) and danger -associated molecular patterns (DAMPs). The neutr ophil activities induced by such PAMPs and DAMPs are of importance for .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 7 our ability to combat invading microbes and to repair d amaged tissues [8]. Many of the neutrophil plasma membrane -localized receptor s belong, just as FPR1, to the family of G protein coupled receptors ( GPCRs, also known as seven-transmembrane receptors [7TM receptors ] [9]). FPR1 is usually denoted as the prototype for the neutrophil GPCRs and i n accordance with name and receptor group affiliation , this receptor is activated by N -formyl methionyl containing peptides and signaling rely on coupling to, and activation of, a heterotrimeric G protein containing a Gα i subunit . Basically, the majority of the initial GPCR activities that are important for receptor downstream signals are mediated by G protein complexes containing a GTP/GDP - binding α subunit with GTPase activity and a dimeric β/γ subunit. In the non - activated and low -signaling state of the receptor, GDP (guanosine diphosphate) is bound to the α subunit, and when the receptor is activated, GTP replaces GDP, and this binding triggers a dissociation of the α subunit from the β/γ subunit. The two dissociated G protein subunits then transfer the information from the activated GPCR to a second messenger cascade specifically characterized by the receptor, the receptor specific ligand, the G protein involved in the response and , the cell in which the receptor is expressed [9, 10]. The dissociated β/γ subunit downstream of FPR1 activates PLC (phospholipase C), an enzyme that catalyzes the hydrolysis of PIP 2 (phosphatidyl -inositol -4,5-bisphosphate) to IP 3 (inositol trisphosphate) and DAG (diacylglycerol). The latter is an activator of PKC (protein kinase C). The IP 3 that is released to the cytoplasm is recognized by IP 3-receptors on the Ca 2+ storing endoplasmic reticulum (ER), and when these receptors are occupied, stored Ca 2+ is released from the ER and the intracellular concentration of free Ca 2+ ([Ca2+]i) is increased. Our genome encodes many different G protein subunits, including around 20 α-subunits divided into four groups (Gα s, Gαq, Gαi/o and Gα 12/13 ). These subunits .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 8 combine with a β/γ dimer generated from the 5 and 12 subunit genes encoding β and γ subunits, respectively [11]. The literature suggests the presence of at least 30 distinct G protein complexes, with the possibility of additional complexes yet to be identified. Studies involving the overexpression of GPCRs in conjunction with various G proteins in cell lines have r evealed that certain GPCRs exhibit selective coupling to a limited number of G proteins, while other s exhibit promiscuity by coupling to several different G proteins [12]. This promiscuity is expected to increase as more data become available . It is also reasonable to assume that GPCRs previously identified as promiscuous may exhibit a more selective G protein coupling profile when activated in primary cells. The recruitment profile may be contingent not only on the cell in which the receptor is expressed but also on the agonist that activates the receptor [13, 14]. However, the regulatory repertoire is also open to other novel mechanisms that restrict the recruitment of different G proteins. [15] In addition to FPRs, neutrophils express GPCRs that recognize platelet activating factor (PAF; recognized by PAFR), generated by diverse cells through lipid remodeling , short chain fatty acids ( SCFAs, recognized by FFARs) generated by bacteria , and adenosine trisphosphate (ATP; recognized by the purinoreceptor P2Y2R) generated by host cell mitochondria [7, 16-19]. Like the FPRs, FFA2R (one of the neutrophil FFARs) couple to a G protein containing a Gα i subunit, and the activated dissociated β/γ subunit activates the PLC-PIP2-IP3-Ca2+ pathway. This signaling pathway is activated also by ATP and PAF but the rise in [Ca2+]i downstream of their cognate receptors is mediated by the Gα subunit of a Gαq containing G protein [7]. Despite the signaling similarities, but in contrast to the activation profiles of several neutrophil GPCRs coupled to Gα i containing G proteins, the agonist occupied P2Y2R in naïve neutrophils do not generate signals that trigger an assembly and activation of the ROS producing NADPH oxidase [20]. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 9 In the context of ATP, we have previously shown that the ATP/P2Y2R complex can activate the ROS producing NADPH oxidase by two different mechanisms ; i) in TNF -primed neutrophils, ATP triggers such an activation in the presence of an FFA2R selective allosteric modulator, and this activation is achieved through a novel receptor transactivation (cross- talk) mechanism that results in an activation of the allosterically modulated FFA2R [7], and ii) ATP triggers this activation also in neutrophils in which the integrity of the actin cytoskeleton has been dis rupted by an actin binding toxin [20]. The precise signaling triggered by ATP leading to an activation of the NADPH oxidase in neutrophils is still poorly understood. We now show that that ATP also activates the NADPH oxidase in neutrophils that have been primed with GM-CSF (granulocyte -macrophage colony - stimulating factor) through a transactivation mechanism involving also FFA 2R. The transactivating signals are generated downstream of a Gαq containing G protein. Furthermore, in the context of neutrophils with a disrupted actin cytoskeleton, the activation of the NADPH oxidase by ATP occurs through signals generated both with and without the involvement of a Gαq containing G protein. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 10

Chemicals and reagents Dextran T500 was from Pharmacosmos and Cytiva Ficoll-PaqueTM Plus Medium was from Fischer Scientific . Fura -2-acetoxymethyl ester (AM) was from Invitrogen. Isoluminol, bovine serum albumin ( BSA), the actin cytoskeleton disrupter latrunculin A , the Gαi inhibitor pertussis toxin (PTX), the P2Y2R agonist ATP, the FFA2R agonist propionic acid (propionate), the FFA2R allosteric modulator Cmp58 ((S)-2-(4-chlorophenyl)-3,3- dimethyl-N-(5-phenylthiazol-2-yl)butanamide), the FPR1 agonist fMLF and the GPR84 agonist ZQ16 were from Sigma-Aldrich. The P2Y2R antagonist AR-C118925 ((5-[[5-(2,8- dimethyl-5Hdibenzo[a,d]cyclohepten-5-yl)-3,4-dihydro-2-oxo-4-thioxo-1(2H)- pyrimidinyl]methyl]-N-2H-tetrazol-5-yl-2-furan-carboxamide)) and the FFA2R antagonist CATPB ((S) -3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)phenyl) butanoic acid) were from Tocris. The PAFR agoni st PAF was from Avanti Research. The Gαq inhibitor YM-254890 was acquired from FUJIFILM Wako Pure Chemical Corporation and GM-CSF was purchased from PeproTech. All stock solutions were dissolved in DMSO and subsequent dilutions performed in Krebs-Ringer glucose phosphate buffer (KRG, 120 mM NaCl, 4.9 mM KCl, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, 1.5 mM MgSO4, 10 mM glucose, and 1 mM CaCl2 in dH2O, pH 7.3). Ethics statement The present study comprises buffy coats obtained from healthy human blood donors at the blood bank at Sahlgrenska University Hospital in Gothenburg, Sweden. All buffy coats were obtained anonymously and therefore no ethical approval was required according to the Swedish legislation section code 4§ 3p SFS 2003:460 (Law on Ethical Testing of Research Relating to People). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 11 Isolation of human neutrophils Neutrophils were isolated from buffy coats of human healthy blood donors as previously described [21, 22] using dextran sedimentation followed by Ficoll -Paque gradient centrifugation. Erythrocytes were lysed by hypotonicity, and the remaining pellet was washed after which the purity was measured on a Sysmex KX-21 N Hematology Analyzer (Sysmex Corporation). All pellets comprised ≥ 90% neutrophils and these were resuspended in KRG to a final neutrophil concentration of 1x107/mL and kept on ice until further analysis the same day. To amplify the activation signals of the NADPH oxidase, the freshly isolated neutrophils were incubated with GM-CSF (2 nM, 20 min, 37°C) at a concentration of 106 neutrophils/mL and then kept on ice until further analysis the same day (these neutrophils are referred to as GM-CSF-primed neutrophils from herein). Neutrophil NADPH-oxidase activity The release of superoxide anions (O2-) by the neutrophil NADPH oxidase was monitored using an isoluminol-enhanced chemiluminescence (CL) technique as previously described [23, 24]. In short, each 900 μL reaction mix which contained 105/mL neutrophils, 0.2 μM isoluminol and 4 Units/mL HRP was incubated for five min at 37°C prior to the addition of an agonist (100 μL) The NADPH oxidase activity was measured using a six -channel Biolumat LB 9505 (Berthold Co., Wildbad, Germany). In case s were the effect s of certain ligands (inhibitor, antagonist, allosteric modulator , latrunculin A), were studied, these reagents were added either five (inhibitor, antagonist, allosteric modulator) or two minutes (latrunculin A) prior stimulation with the agonist. In experiments were the effect of PTX was studied, GM-CSF-primed neutrophils were .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 12 incubated without or with PTX (500 ng/mL ) for 30 min at 37°C, followed by addition of latrunculin A and stimulation with the agonist. The O2- production was recorded continuously over time and expressed in Mega counts per minute (Mcpm). The peak O2- released was used for summary analyses of NADPH oxidase activity. Determination of changes in concentration of free calcium ions in the cytosol Fura-2 AM (2 μM) was added to neutrophils (2 x 107/mL) resuspended in Ca2+-free KRG with 0.1% BSA. The cells were incubated in the dark for 30 min followed by washing and resuspension in KRG (2 x 107/mL). The rise in the cytosolic concentration of free Ca 2+ ([Ca2+]i) was then measured using a Perkin Elmer fluorescence spectrophotometer (LC50), with excitation wavelengths of 340 nm and 380 nm and emission wavelength of 509 nm. A ratio of the values obtained for 340 nm and 380 nm excited Fura -2 fluorescence over time was calculated and used to express the increase in [Ca2+]i. Data analysis Data analysis and processing was performed with GraphPad Prism 10.4. 1 (GraphPad Software, San Diego, CA, USA). All statistical tests were performed using the peak values of the raw data including the statistically significant differences found in data set where the

are shown as percent of control. The specific statistical test used are described in the figure legends and statistically significant differences are denoted by the following p-values: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (* ***). Non -statistically significant differences are denoted by ns, i.e., p ≥ 0.05. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 13

The P2Y2R agonist ATP is a functional selective neutrophil activator. In neutrophils, ATP induced a transient elevation in the cytosolic concentration of free Ca2+ ([Ca2+]i). The rise in [Ca2+]i was rapidly initiated and reached a peak within the first minute after ATP addition (Fig 1A). This response was inhibited by the specific P2Y2R antagonist AR-C118925 (Fig 1A and [25, 26]), consistent with the receptor’s preference for ATP in neutrophils. P2Y2R belongs to the family of G protein-coupled receptors (GPCRs), and the [Ca2+]i rise induced by GPCRs may be attributed to an activated Gαq subunit as well as a βγ dimer separated from its Gαi subunit in the receptor -coupled G protein [27, 28]. The inhibitory effect of the Gαq inhibitor YM -254890 demonstrated that the ATP -activated purine receptor P2Y2R couples to a Gαq-containing G protein (Fig 1 A). In contrast, the transient rise in [Ca2+]i induced by the formylated peptide fMLF, specifically recognized by the Gαi-coupled FPR1, was unaffected by YM -254890 (Fig 1B). Figure 1. ATP triggers a transient increase in intracellular Ca²⁺ ([Ca²⁺] i) but does not activate the NADPH oxidase in human neutrophils. A-B. Fura-2-loaded neutrophils were pre -incubated (10 min, 37°C) with either buffer, the P2Y₂R-specific antagonist AR-C118925 (AR-C, 1 μM), or the Gαq inhibitor YM-254890 (YM, 200 nM). Thereafter A. ATP (5 μM) or B. fMLF (10 nM) was added (arrow indicates time of addition) and the transient rise in [Ca²⁺] i, was measured over time. Representative traces from an individual experiment are shown. C. Superoxide anion (O2-) production measured in GM-CSF-primed neutrophils stimulated with ATP (50 μM) or fMLF (25 nM) over time (arrow indicates time of addition). Inset. The peak O₂⁻ production after stimulation with ATP (n=18) or fMLF (n=4) are summarized in the bar graph (mean ± SD). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 14 The binding of fMLF to its neutrophil receptor, FPR1, was associated with an assembly of the NADPH oxidase and by that a generation of superoxide anions (Fig 1C). In contrast, ATP did not induce this activation (Fig 1C), consistent with previous findings that there is no direct correlation between an increase in [Ca2+]i and NADPH oxidase activation [7]. GM-CSF primes the NADPH oxidase activity in human neutrophils. In accordance with earlier findings [29], the NADPH oxidase activity triggered by fMLF in neutrophils preincubated with GM -CSF w as primed, i.e., the response was substantially increased compared to the response induced in naïve neutrophils (Fig 2). The ROS production was also augmented in neutrophils pre -treated with GM -CSF compared to naïve neutrophils stimulated with either propionate (FFA2R agonist) combined with the allosteric FFA2R modulator Cmp58, ZQ16 (GPR84 agonist), or PAF (a PAFR agonist) (Fig. 2). Like the inability for ATP to activate the NADPH oxidase in naïve neutrophils, ATP on its own did not trigger any NADPH oxidase activity in GM -CSF- primed neutrophils (Fig 3). Figure 2. GM-CSF primes neutrophils -- the NADPH oxidase activity in response to various GPCR agonists is increased. Neutrophils incubated with or without GM-CSF (2 nM, 20 min, 37°C) were activated with either the FPR1 agonist fMLF (25 nM), the FFA2R agonist propionate (25 µM, combined with in the allosteric FFA2R modulator Cmp58, 1 µM), the GPR84 agonist ZQ16 (1 μM), or the PAFR agonist PAF (100 nM) and the O2- production was monitored over time. Representative traces for each agonist are shown. Insets. The ratio of the peak O2- production in GM-CSF-primed neutrophils (+ GM-CSF) versus unprimed cells (- GM- CSF) are shown (mean ± SD, n=5). The dotted horizontal line in each bar graph represents a ratio of 1. Statistically significant differences between GM-CSF-primed and unprimed neutrophils were evaluated by a paired Student’s t-test for each agonist separately (*p < 0.05, **p < 0.01). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 15 Taken together, as compared to untreated (naïve) neutrophils, the cytokine GM-CSF-primed neutrophils to induce increased ROS production in response to several GPCR agonists . Based on these results, GM -CSF priming was used to amplify the NAPDH oxidase activation signals in response to GPCR agonists for the rest of the experiments in this study. Allosteric modulation of FFA2R turns ATP into an NADPH oxidase activating agonist. As mentioned, ATP on its own did not trigger any NADPH oxidase activity in GM -CSF primed neutrophils (Fig 3A). However, the inability of the signals from P2Y 2R to activate the neutrophil NADPH oxidase was not absolute. In the presence of the positive allosteric Figure 3. Allosteric modulation of FFA2R converts ATP into an activator of the neutrophil NADPH oxidase . The O2- production was measured in GM -CSF-primed neutrophils pre -incubated (5 min, 37°C) with either buffer, the allosteric FFA2R modulator Cmp58 (1 μM), the Gαq inhibitor YM-254890 (YM, 200 nM), the P2Y₂R-specific antagonist AR-C118925 (AR- C, 1 μM), or the FFA2R -specific antagonist CATPB (100 nM) prior stimulation with ATP (50 μM; arrows indicate time of addition). A. Left: A representative trace of the O 2- production induced by ATP in the absence or presence of Cmp58 is shown. Right: Summary of the peak O₂ ⁻ production (mean ± SD, n=18). B. Left: A representative trace of the O 2- production induced by ATP in the presence of Cmp58 alone or in combination with YM, AR -C or CATPB is shown. Right: Summary of the peak O 2- production (mean ± SD, n=5). Statistically significant differences in A. were evaluated by a paired Student’s t-test and in B. with a repeated measures one-way ANOVA followed by Dunnett’s multiple comparisons test to Cmp58 alone control (*p < 0.05, ****p < 0.0001). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 16 FFA2R modulator Cmp58 , ATP induced a release of superoxide anions (O2-) from neutrophils (Fig 3A). This ATP -induced response was blocked by the specific FFA2R antagonist CATPB, confirming the involvement of FFA2R (Fig 3B). As anticipated, the response was also inhibited by the P2Y 2R-specific antagonist AR -C118925 and the Gαq- selective inhibitor YM-254890 (Fig 3B). Our model for how this activation is achieved assumes that Gαq dependent signals induced by ATP transactivates FFA2R from the cytosolic side of the plasma membrane and the transactivated receptor subsequently activate the NADPH oxidase. Latrunculin A turns ATP into an activator of the NADPH oxidase and concentration- dependently renders the response insensitive to Gαq-inhibition. Latrunculin A is a research tool -toxin that binds actin monomers and prevent s them from participating in the polymerization process; binding of the toxin, thus, results in a disruption of the actin filaments of the cell cytoskeleton [30]. When Cmp58 was replaced by latrunculin A, ATP was converted into an NADPH oxidase activating agonist and the effect of a depolymerized actin cytoskeleton was clearly more dramatic (Fig 4). At latrunculin A concentrations above 25 ng/m L, there was essentially no further increase in the ATP- induced NADPH oxidase activity (data not shown). The NADPH oxidase activity was dependent on the concentration of ATP and the effect was most pronounced with a latrunculin A concentration of 25 ng/mL (Fig 4). Based on the inhibitory effect of the Gαq- selective inhibitor YM-254890 on the downstream signaling of the activated P2Y 2R, we assumed that the inhibitor would potently reduce the response induced by ATP in the presence of latrunculin A. However, our data show that the inhibition mediated by YM- 254890, was not very pronounced when a high concentration of latrunculin A was added to the system (Fig 4A) . This lack of inhibition was ev ident irrespectively of the ATP .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 17 concentration used to activate the cells. With ATP concentrations of 50 µM and 1 µM the inhibition was around 25% and 50%, respectively (Fig 4A). At latrunculin A concentrations below 25 ng/mL, the level of NADPH oxidase activity was gradually reduced in relation to the concentration of the toxin (Fig 4B and C). In contrast, the inhibitory effects of the Gαq- selective inhibitor YM-254890 on the ATP induced activation of the NADPH oxidase, was gradually increased in relation to the concentration of latrunc ulin A, reaching a level of around 90% with a latrunculin A concentration of 5 ng/mL (Fig 4C). Taken together, these data show that ATP has the capacity to activate the neutrophil NADPH oxidase by signals generated downstream of a Gαq-containing G protein as well as signals generated independent of such a G protein. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 18 Figure 4. Latrunculin A converts ATP into an activator of the neutrophil NADPH oxidase and changes the sensitiv ity of the response to Gαq-inhibition. ATP induced superoxide anion (O 2-) production measured in GM -CSF-primed neutrophils pre -incubated with different concentrations of latrunculin A (LA) in the absence or presence of the Gαq inhibitor YM-254890 (YM, 200 nM). A. Left: Representative traces of the O2- production induced by ATP (50 μM; arrow indicates time of addition) in the presence of 25 ng/mL LA with or without YM. Inset: The graph displays peak O 2- values triggered by different concentrations of ATP (1, 5, 10, or 50 μM) in the presence of 25 ng/mL LA. Right: The bar graph shows the remaining O 2- activity after ATP (1, 5, 10, or 50 μM) activation of neutrophils in the presence of YM and LA (25 ng/mL). B–C. The same experimental setup as in panel A, but with 10 ng/mL (B) or 5 ng/mL ( C) LA, respectively. Insets and bar graphs are represented as mean ± SD (n=5). Statistically significant differences in bar graphs were evaluated by repeated measures one -way ANOVA followed by Šídák's multiple comparisons test for analysis of each ATP concentration in the absence or presence of YM, separately ( *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 19 Latrunculin A affects the sensitivity to Gαq inhibition also in neutrophils activated by PAF. A comparison of the neutrophil -expressed GPCRs that recognize ATP and PAF reveals both similarities and differences [7]. One difference being that PAF is a n activator of the NADPH oxidase also in neutrophils with an intact actin cytoskeleton. A similarity is that they both couple to a Gαq containing G protein, and in accordance with this , the PAF- induced response was sensitive to Gαq inhibition (Fig 5 A). Regarding the impact of a disruption of the actin cytoskeleton, a similar pattern as that described for the ATP induced response (Fig 4), also characterized the response induced by PAF. That is, the NADPH oxidase activity was substantially increased in the presence of latrunculin A and at high concentrations of the toxin (10 and 25 ng/mL, respectively) the response was only partly reduced by the Gαq inhibitor (Fig 5B and C). However, when the latrunculin A concentration was reduced to 5 ng/mL, the PAF -induced response was once again dependent on Gαq- signaling as the Gαq inhibitor reduced the response by ≈ 95% (Fig 5D). In summary, these data show that not only ATP but also PAF, has the capacity to activate the neutrophil NADPH oxidase via both Gαq-dependent and Gαq-independent signals. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 20 Pertussis toxin sensitivity reveals Gαi involvement in the ATP induced NADPH oxidase activation. To further elucidate the intracellular signaling pathway in Gαq-independent ATP induced activation of the NADPH oxidase , we employed pertussis toxin (PTX) which, although the G protein subtype selectivity of PTX has been questioned [7], is an established Figure 5. Latrunculin A potentiates the PAF-induced neutrophil NADPH oxidase activity and changes the sensitivity of the response to Gαq-inhibition. PAF induced O 2- production measured in GM -CSF-primed neutrophils incubated without or with different concentrations of latrunculin A (LA) in the absence or presence of the Gαq inhibitor YM-254890 (YM, 200 nM) prior stimulation with PAF (100 nM; arrow indicates time of addition). A. A representative trace of the neutrophil O2- production when activated by PAF (100 nM, arrow indicates time of addition) without any LA and in the absence or presence of YM. Inset: The bar graph shows the remaining NADPH oxidase activity induced in neutrophils by PAF in the presence of YM. B–D. The same experimental setup as in panel A, but with the PAF-induced NADPH oxidase activity in the presence of 25 ng/mL (B), 10 ng/mL (C) or 5 ng/mL (D) LA. Insets: Inhibition of the response by YM expressed as remaining activity in the presence of the inhibitor (mean ± SD, n=5). Statistically significant differences in the bar graphs were evaluated by a paired Student’s t-test (*p < 0.05, **p < 0.01). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 21 inhibitor of signaling by GPCRs that couple to Gαi-containing G proteins [31]. Neutrophils treated with latrunculin A at a concentration of 25 ng/mL and 10 ng/mL, exhibited approximately 40% and 60% remaining ATP response (relative to control cells not treated with PTX, Fig 6A and B). Conversely, PTX lacked inhibitory effect on the ATP induced neutrophil response when the latrunculin A concentration was reduced to 5 ng/mL (Fig 6C). Taken together, t he data presented clearly show that in neutrophils treated with high concentrations of latrunculin A, the downstream signals that activates the NADPH oxidase utilizes signals that depend on Gαq and a switch to a Gαi initiated pathway. Conversely, no G protein switch occurs when the concentration of latrunculin A is reduced, yet signaling proceeding exclusively via Gαq still activates the NADPH oxidase. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 22 Figure 6. The ATP -induced neutrophil NADPH oxidase activity is sensitive to pertussis toxin in the presence of high latrunculin A concentrations. Superoxide anion (O2-) production was measured in GM- CSF-primed neutrophils, incubated without (control) or with pertussis toxin (PTX, 500 ng/mL; 30 min at 37°C). These neutrophils were activated by ATP (50µM; arrow indicates time of addition) in the presence of different concentrations of latrunculin A ( LA). A. Left: Representative traces of the O 2- production in PTX treated and control neutrophils activated by ATP (50µM) in the presence of 25 ng/mL LA. Inset: Peak values of the ATP induced NADPH oxidase activity in PTX treated and control neutrophils (mean±SD, n=7). Right: Inhibitory effect of PTX, expressed as remaining NADPH oxidase activity induced by ATP in neutrophils treated with PTX. B–C. The same experimental setup as in panel A, but with 10 ng/mL ( B) or 5 ng/mL ( C) LA, respectively. Statistically significant differences in the bar graphs were evaluated by a paired Student’s t-test (**p < 0.01, ***p < 0.001, ns = not significant). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 23

The receptor downstream signals generated in neutrophils by t he agonist/receptor pair ATP/P2Y2R fulfills the basic characteristic of an agonist that through binding to its receptor induces a “functional selective” response that is the result of a “biased receptor signaling cascade” [32]. This means that in contrast to a balanced GPCR agonists such as the FPR1 agonist fMLF , which facilitates signaling that regulates several different neutrophil functions, the activated ATP receptor transfers signals that result in a functional profile that is circumcised. Agonist binding both to P2Y 2R and FPR1 results in coupling of the two receptors to G proteins, which activate phospholipase C and this activation gives directly rise to a production of inositol trisphosphate (IP 3), which is linked to an increase in the cytosolic concentration of f ree calcium ions ([ Ca2+]i,) [20, 33]. Although t his response is induced by both receptors it is transduced by different G proteins; whereas FPR1 couples to a Gαi containing G protein, the signaling G protein partner of P2Y2R contains an αq subunit (this study and [7]). Despite th e signaling similarit y, the functional outcome in naïve neutrophils differs. The s ignals that trigger an assembly and activation of the superoxide anion generating NADPH oxidase are not generated by the ATP activated P2Y 2R, clearly showing that the downstream signals generated are not alone sufficient to activate the oxidase. The incapacity of ATP to activate the NADPH oxidase may be due either to that no NADPH oxidase activating signals can be produced by the Gαq-coupled P2Y2R, or that such signals can be produced also by this receptor and its G protein partner, but that this signaling pathway is (in one way or another) blocked in naïve neutrophils. The primary signaling mechanism downstream of the activated P2Y 2R involves coupling to the G αq- containing G protein and this signaling synergize with other neutrophil receptors such as the free fatty acid 2 receptor (FFA2R) . Our data show that in the presence of an FFA2R specific positive allosteric modulator, the P2Y2R agonist ATP activates the neutrophil .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 24 NADPH oxidase. We have earlier shown that PAF shares the basic primary signaling mechanism with ATP, involving coupling to Gαq and activation of the neutrophil NADPH oxidase by signals that synergize with FFA2R in the presence of a positive allosteric FFA2R modulator [34]. The actin-rich cortex of neutrophils is, together with several different actin binding proteins, closely associated with the cytosolic side of the plasma membrane. I t can therefore be deduced that an interaction between cytoskeleton proteins and integral plasma membrane proteins such as GPCR s may affect the function of the latter. In neutrophils, t his has primarily been studied with FPR1 as a model for GPCRs . The basic regulatory function of the actin cytoskeleton in FPR1 signaling was described over 30 years ago in some excellent publications by Algirdas Jesaitis and co -workers [35-39]. Little notice has been given to these early observation s, which have been confirmed and extended to also include the closely related FPR2 [7, 15]. Based on the results obtained in these studies, a working model that describes how FPR signaling is regulated has been developed; signaling by FPRs is terminated by actin cytoskeleton dependent receptor desensitization, a process in which a continuous G protein recruitment to the activated FPRs becomes physically blocked when the actin cytoskeleton binds to the activated receptor . The molecular mechanism through which the cytoskeletal proteins bind to the cytosolic parts of the FPRs remains to be elucidated, but the results obtained in studies of the neutrophil NADPH oxidase activity, induced by FPR agonists in the presence of inhibitors of actin polymerization , such as latrunculin A, provide strong support for the suggested model [7, 15]. However, a somewhat divergent regulatory profile emerges when the model is applied to analyze the data obtained when ATP is used as the activating agonist instead of FPR agonist s. In naïve neutrophils, the NADPH oxidase activating signals are evidently not generated by the activated ATP receptor unless the actin cytoskeleton first has been disrupted. This suggests that in .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 25 neutrophils, the signaling pathway leading to an activation of the NADPH oxidase is blocked by the cytoskeleton whereas the pathway resulting in a transient rise in [Ca2+]i is accessible. Furthermore, the prolongation of the response, characterizing the response induced by FPR agonists in neutrophils with a disrupted actin cytoskeleton [40-42], is absent in the response to ATP, suggesting that this response is terminated without the involvement of the actin cytoskeleton. More importantly, however, that the inhibitory effect of the G aq inhibitor diminishes with increasing concentrations of latrunculin A. These data suggest on the one hand, that NADPH oxidase activating signals are generated through an involvement of a Gaq-containing G protein and, on the other hand, that such signals are generated also by Gaq-independent signals . In agreement with this , the ATP induced response in the presence of high latrunculin A concentrations is part ly reduced by pertussis toxin, an inhibitor of Gai-mediated signaling. Conversely, the ATP induced response in the presence of low latrunculin A concentrations is entirely dependent on Gaq, thereby evading inhibition by PTX. The effect of a disruption of the actin cytoskeleton on the response induced by PAF, an agonist recognized by a receptor (PAFR) that , like P2Y 2R, couples to a Gaq containing G protein, is analogous to that described for the ATP-induced response, thereby adding another functional similarity for these two neutrophil-expressed GPCRs. Conclusions. A disruption of the actin cytoskeleton affects G protein involvement in ATP- induced signals that activate the neutrophil NADPH oxidase; the Gaq signals that activate the NADPH oxidase are apparently blocked in neutrophils with an intact actin cytoskeleton, whereas such signals are generated when this block is removed by the actin cytoskeleton disrupting drug latrunculin A. In addition, a disrupted cytoskeleton allows for a change of the G protein recruitment pattern. As such, also Gai dependent signals activate the NADPH oxidase in ATP activated neutrophils with a disrupted cytoskeleton. The G protein coupling .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 26 maps for a multitude of GPCRs reveal that, whereas some GPCRs selectively activate a limited number of Ga proteins, others have the capacity to couple promiscuously to a large number of different Ga subtypes [12]. The purinergic receptor P2Y2R belongs to the group of promiscuous GPCRs , capable of recruiting multiple G proteins, whereas the PAFR displays a more restricted coupling profile that has been shown not to engage Gaq [12]. Nevertheless, it is evident from the data presented, that in neutrophils the immediate signals generated, when ATP as well as PAF bind to the ir respective receptor, are primarily mediated by a Gaq-containing G protein. This raises questions concerning the determinants of such a single-faceted selectivity profile. The molecular basis for the G protein selectivity could of course be related to the fact that cells/tissues differ in their G protein expression profile, but other mechanisms may regulate the G protein recruitment and signaling processes, mechanisms that allow responding cells to alter intracellular signaling pathways and the functional outcome of an agonist-induced activation. It has been shown that binding of an allosteric modulator to the prostaglandin 2 receptor EP4, occupying a receptor site localized close to the binding site for the recruited G protein complex, affects the G protein recruitment profile. That is, binding of a positive allosteric modulator to an intracellular receptor site in EP4, converts the signaling from being Gas-dependent to instead use Gai as the signaling partner [43]. However, G protein selectivity most likely relies on a multitude of other molecular mechanisms, including structural conformation alterations in intracellular G protein binding receptor interfaces , induced from within the cell by regulatory proteins that dynamically facilitate or selectively block G protein binding. Collectively, s uch mechanisms ensure that GPCRs engage the appropriate G protein signaling pathway in response to different stimuli and in neutrophils the actin cytoskeleton is suggested to be part of this regulatory machinery. It is reasonable to assume that other binding partners present on the cytosolic side of the receptor-expressing plasma membrane .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 27 may also have the capacity to be unbound or bound to a receptor, thereby regulating GPCR signaling through an interaction with the parts of a receptor that are accessible from the cytosolic side of the plasma membrane . Although the precise mechanisms by which the GPCR mediated recruitment of G proteins is regulated in neutrophils are unknown, it is evident that the actin cytoskeleton has a regulatory role that is crucial for the signaling and activation profile in ATP-activated neutrophils. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 28 Author contributions Neele K. Levin: Conducted experiments, analyzed and interpreted data, and designed new experiments; Writing - review and editing. Claes Dahlgren: Supervised, analyzed and interpreted data, and designed new experiments; Writing - original draft preparation. Huamei Forsman: Supervised, analyzed and interpreted data, and designed new experiments; Writing - review and editing. Martina Sundqvist : Conceptualization - original idea ; Supervised, analyzed and interpreted data, and designed new experiments; Writing - review and editing. Declaration of competing interest The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 29 Funding sources The work was supported by grants from the Åke Wiberg Foundation (M21-0025, M23-0193 and M24-0227), the Swedish state under the agreement between the Swedish government and the county councils, the ALF -agreement (ALFGBG 78150), , the Swedish Medical Research Council (2018-02848 and 2022-00624), the Swedish Rheumatism Association (R- 995669, R-1013370 and R-995361), the Rune and Ulla Almlövs Foundation (2023 -418), the Mary von Sydow foundation (2023-4723 and 2024-163), the King Gustaf the V 80-year foundation (FAI-2021-0804 and FAI-2022-0873), the Magnus Bergwall foundation (2024- 1434), the Health & Medical Care Committee of the Region Västra Götaland (VGFOUREG-979715, VGFOUREG-995348) and the Wilhelm and Martina Lundgren Science Fund (2024-SA-4605). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 30

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It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 37 FIGURE LEGENDS Figure 1. ATP triggers a tra nsient increase in intracellular Ca²⁺ ([Ca²⁺] i) but does not activate the NADPH oxidase in human neutrophils. A-B. Fura-2-loaded neutrophils were pre-incubated (10 min, 37°C) with either buffer, the P2Y₂R -specific antagonist AR- C118925 (AR-C, 1 μM), or the Gαq inhibitor YM-254890 (YM, 200 nM). Thereafter A. ATP (5 μM) or B. fMLF (10 nM) was added (arrow indicates time of addition) and the transient rise in [Ca²⁺]i, was measured over time. Representative traces from an individual experiment are shown. C. Superoxide anion (O2-) production measured in GM-CSF-primed neutrophils stimulated with ATP (50 μM) or fMLF (25 nM) over time (arrow indicates time of addition). Inset. The peak O₂⁻ production after stimulation with ATP (n=18) or fMLF (n=4) are summarized in the bar graph (mean ± SD). Figure 2. GM-CSF primes neutrophils -- the NADPH oxidase activity in response to various GPCR agonists is increased. Neutrophils incubated with or without GM-CSF (2 nM, 20 min, 37°C) were activated with either the FPR1 agonist fMLF (25 nM), the FFA2R agonist propionate (25 µM, combined with in the allosteric FFA2R modulator Cmp58, 1 µM), the GPR84 agonist ZQ16 (1 μM), or the PAFR agonist PAF (100 nM) and the O2- production was monitored over time. Representative traces for each agonist are shown. Insets. The ratio of the peak O 2- production in GM-CSF-primed neutrophils (+ GM-CSF) versus unprimed cells (- GM-CSF) are shown (mean ± SD, n=5). The dotted horizontal line in each bar graph represents a ratio of 1. Statistically significant differences between GM-CSF-primed and unprimed neutrophils were evaluated by a paired Student’s t-test for each agonist separately (*p < 0.05, **p < 0.01). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 38 Figure 3. Allosteric modulation of FFA2R converts ATP into an activator of the neutrophil NADPH oxidase. The O2- production was measured in GM -CSF-primed neutrophils pre -incubated (5 min, 37°C) with either buffer, the allosteric FFA2R modulator Cmp58 (1 μM), the Gαq inhibitor YM-254890 (YM, 200 nM), the P2Y₂R-specific antagonist AR-C118925 (AR-C, 1 μM), or the FFA2R -specific antagonist CATPB (100 nM) prior stimulation with ATP (50 μM; arrows indicate time of addition). A. Left: A representative trace of the O 2- production induced by ATP in the absence or presence of Cmp58 is shown. Right: Summary of the peak O₂⁻ production (mean ± SD, n=18). B. Left: A representative trace of the O2- production induced by ATP in the presence of Cmp58 alone or in combination with YM, AR -C or CATPB is shown. Right: Summary of the peak O2- production (mean ± SD , n=5 ). Statistically significant differences in A. were evaluated by a paired Student’s t-test and in B. with a repeated measures one-way ANOVA followed by Dunnett’s multiple comparisons test to Cmp58 alone control (*p < 0.05, ****p < 0.0001). Figure 4. Latrunculin A converts ATP into an activator of the neutrophil NADPH oxidase and changes the sensitivity of the response to Gαq-inhibition ATP induced superoxide anion (O2-) production measured in GM-CSF-primed neutrophils pre-incubated with different concentrations of latrunculin A (LA) in the absence or presence of the Gαq inhibitor YM-254890 (YM, 200 nM). A. Left: Representative traces of the O2- production induced by ATP (50 μM; arrow indicates time of addition) in the presence of 25 ng/mL LA with or without YM. Inset: The graph displays peak O 2- values triggered by different concentrations of ATP (1, 5, 10, or 50 μM) in the presence of 25 ng/mL LA. Right: The bar graph shows the remaining O2- activity after ATP (1, 5, 10, or 50 μM) activation of .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 39 neutrophils in the presence of YM and LA (25 ng/mL). B–C. The same experimental setup as in panel A, but with 10 ng/mL (B) or 5 ng/mL (C) LA, respectively. Insets and bar graphs are represented as mean ± SD (n=5). Statistically significant differences in bar graphs were evaluated by repeated measures one -way ANOVA followed by Šídák's multiple comparisons test for analysis of each ATP concentration in the absence or presence of YM, separately ( *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant). Figure 5. Latrunculin A potentiates the PAF-induced neutrophil NADPH oxidase activity and changes the sensitivity of the response to Gαq-inhibition. PAF induced O2- production measured in GM -CSF-primed neutrophils incubated without or with different concentrations of latrunculin A (LA) in the absence or presence of the Gαq inhibitor YM-254890 (YM, 200 nM) prior stimulation with PAF (100 nM; arrow indicates time of addition). A. A representative trace of the neutrophil O2- production when activated by PAF (100nM, arrow indicates time of addition) without any LA and in the absence or presence of YM. Inset: The bar graph shows the remaining NADPH oxidase activity induced in neutrophils PAF in the presence of YM. B–D. The same experimental setup as in panel A, but with the PAF-induced NADPH oxidase activity in the presence of 25 ng/mL (B), 10 ng/mL (C) or 5 ng/mL (D) LA. Insets: Inhibition of the response by YM expressed as remaining activity in the presence of the inhibitor ( mean ± SD , n=5). Statistically significant differences in the bar graphs were evaluated by a paired Student’s t-test (*p < 0.05, **p < 0.01). Figure 6. The ATP-induced neutrophil NADPH oxidase activity is sensitive to pertussis toxin in the presence of high latrunculin A concentrations. Superoxide anion (O 2-) production was measured in GM -CSF-primed neutrophils, incubated without (control) or .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint 40 with pertussis toxin (PTX, 500 ng/mL ; 30 min at 37°C). These neutrophils were activated by ATP (50µM; arrow indicates time of addition) in the presence of different concentrations LA. A. Left: Representative trace s of the O 2- production in PTX treated and control neutrophils activated by ATP (50µM) in the presence of 25 ng/mL latrunculin A ( LA). Inset: Peak values of the ATP induced NADPH oxidase activity in PTX treated and control neutrophils (mean±SD, n=7) . Right: Inhibitory effect of PTX , expressed as remaining NADPH oxidase activity induced by ATP in neutrophils treated with PTX. B–C. The same experimental setup as in panel A, but with 10 ng/mL (B) or 5 ng/mL (C) LA, respectively. Statistically significant differences in the bar graphs were evaluated by a paired Student’s t- test (**p < 0.01, ***p < 0.001, ns = not significant). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 2, 2025. ; https://doi.org/10.1101/2025.05.30.656946doi: bioRxiv preprint