Autophagy Induced by Palmitic Acid: a Brake in NAFLD Neutrophils

Innate immune suppression and high blood fatty acid levels are the pathological basis of multiple metabolic diseases. Neutrophil vacuolation is an indicator of the immune status of patients, which is associated with autophagy-dependent granule degradation. Vacuolated neutrophils are observed in ethanol toxicity and septicemia patients due to the changes in their blood constituents, but how about the neutrophils in nonalcoholic fatty liver disease (NAFLD) patient is unknown. Here, we confirmed that an adhesion deficiency and an increased autophagy level existed in NAFLD neutrophils, and the three neutrophil granule subunits, namely, the azurophil granules, specific granules and gelatinase granules, could be engulfed by autophagosomes for degradation, and these autophagy-triggered granule degradation events were associated with vacuolation in palmitic acid (PA)-treated and NAFLD neutrophils. Concordantly, the adhesion-associated molecules CD11a, CD11b, CD18 and Rap1 on the three granule subunits were degraded during PA induced autophagy. Moreover, the cytosolic CD11a, CD11b, CD18 and Rap1 were targeted by Hsc70 and then delivered to lysosomal-like granules for degradation. Notably, in vitro and ex vivo, PA induced autophagy by inhibiting the p-PKCα/PKD2 pathway. Overall, we showed that high blood PA level inhibited the p-PKCα/PKD2 pathway to induce NAFLD neutrophil autophagy, which promoted the degradation of CD11a, CD11b, CD18 and Rap1 and further decreased the adhesion of neutrophils, thereby impairing the neutrophil function of NAFLD patients. This theory provides a new therapeutic strategy to improve the immune deficiency in NAFLD patients. Visual Abstract Key Points Vacuolation and adhesion deficiency of NAFLD neutrophils are associated with autophagy-dependent granule degradation PA inhibits p-PKCα/PKD2 to induce autophagy, which induces the degradation of CD11a, CD11b, CD18 and Rap1 and decreases neutrophil adhesion


Introduction 30
Neutrophils are the most abundant immune cells in human circulation, ranging from 40 to 70% of our circulating 31 leukocytes 1 . Generally, neutrophils roll along the vessel walls to conduct immune surveillance. When they sense a 32 chemotactic cue, the circulating neutrophils roll slowly to adhere to the venular endothelium, extravasate from the 33 bloodstream and are rapidly recruited to infectious sites to provide the first line of defense against invading 34 pathogens. Once there, neutrophils phagocytose the pathogen, release their anti-microbial content normally kept 35 intracellularly in granules, and can release neutrophil extracellular traps to kill and prevent the dissemination of 36 microbes. There are three neutrophil granules types: azurophil granules (AGs), specific granules (SGs) and 37 gelatinase granules (GGs), each of which contains a specific array of microbicidal proteins, adhesion molecules, 38 and various enzymes 2 . 39 In order for neutrophils to efficiently migrate to the site of infection, they express numerous adhesion 40 molecules. Many β2 integrins 3 , including CD11a/CD18, CD11b/CD18, CD11c/CD18 and CD11d/CD18, are 41 differentially expressed in neutrophils, either spatially or temporally (located in granules, plasma membranes and 42 trafficked vesicles). These integrins mediate the firm adhesion between neutrophils and the endothelium by binding 43 with intercellular adhesion molecule-1 (ICAM) and -2 after neutrophil activation 4-6 . CD11a/CD18 and 44 CD11b/CD18 are the most abundant and critical β2 integrins during this process 7 . The small GTPase Rap1 is a key 45 β2 integrin activity regulator 8 . Impaired Rap1 or excessive degradation of the β2 integrin subunits (α-chain or β-46 chain) leads to neutrophil adhesion and diapedesis deficiency, inducing a decrease in immune function 47 protein (Hsc70) have been shown to mediate the fusion of EEs and MVBs with autophagosomes 16-18 , while 60 integrins were located on Rab-positive EEs and MVBs 19,20 . In addition, molecular chaperone proteins, β2 integrins 61 and Rap1 were found on the three lysosome-like granules 2 . Accordingly, we speculated that chaperone-mediated 62 autophagy (CMA) mediated the degradation of ubiquitylated β2 integrins and Rap1 in neutrophils and further 63 influenced neutrophil adhesion and migration. 64 Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent condition which affects 25% of the 65 population worldwide. NAFLD has been associated with obesity, insulin resistance, type 2 diabetes mellitus 66 (T2DM), hypertension, hyperlipidemia, and metabolic syndromes. High blood palmitic acid (PA) levels are a major 67 pathological hallmark of NAFLD and have been shown to be a direct activator of autophagy via the downregulation 68 of protein kinase C α subunit (PKCα) 21 . A recent study revealed that knockout or pharmacological inhibition of 69 PKCα dramatically increased autophagy 22,23 , suggesting that PKCα is a negative regulator of autophagy. We 70 speculated that in NAFLD patients, when cells are exposed to high blood concentrations of PA, autophagy in 71 neutrophils could contribute to dampening neutrophil function, thus alleviating inflammatory functions 21 . However, 72 the mechanism via which PA influences neutrophil autophagy, adhesion and diapedesis in NAFLD patients is 73

unknown. 74
Here, we identified a mechanism in which the three neutrophil granule subsets of neutrophils were degraded 75 by autophagy. Ex vivo and in vitro, PA treatment induced autophagy via the p-PKCα/PKD2 pathway, decreasing the 76 accumulation of CD11a, CD11b, CD18 and Rap1, and leading to deficiencies in neutrophil adhesion and 77 diapedesis. In summary, here we present evidence that autophagy plays a bridging role between metabolic diseases, 78 such as fatty liver disease and deficiency in neutrophil adhesion and diapedesis .  79   80  81  82  83  84  85  86  87  88  89  90  91  92  93

HL-60 125
The HL-60 cell line was purchased from Keygentec (Nanjing, China) and was cultured in IMDM medium 126 supplemented with 20% FBS. The HL-60 cells were differentiated to a neutrophil-like phenotype with a final 127 concentration of 1.3% DMSO for 6 d. 128 primary antibodies were cross-linked to protein A/G plus agarose. The precleared lysate was added to the primary 187 antibody-crosslinked resin in the column overnight at 4°C. The unbound proteins were washed away with IP 188 lysis/wash buffer. Then, the immunoprecipitated proteins were eluted. The eluate concentrations were determined 189 using the BCA Protein Assay Kit (Pierce, IL, USA). The protein complexes were analyzed by SDS-PAGE, and the 190 gel was stained with Coomassie blue. HL-60 cells were differentiated to a neutrophil-like phenotype with a final concentration of 1.3% DMSO for 6 d. 198 Stably knocking down or overexpressing cell lines were selected with puromycin (5 μg/ml) and identified by qRT-199 PCR, western blotting and immunofluorescence. The wild-type and the relevant empty lentivectors cells were used 200 as negative control.  Human database (156639 sequences, downloaded on January 5, 2017). For protein identification and quantification, 220 the parameters were selected as previously described 33 . The median protein ratio should be 1 after the 221 normalization. 222

Quantification and statistical analysis 223
Statistical analysis was carried out using PRISM 6 (GraphPad). The unpaired t-test (when comparing 2 groups), 224 One-way ANOVA test (when comparing with a single group), and two-way ANOVA (when comparing multiple

Autophagy-dependent Vacuolation and Adhesion Deficiency Existed in NAFLD Neutrophils 259
Neutrophil vacuolation is an indicator of the immune status of patients 34 . An increase in vacuolated neutrophils are 260 commonly observed in ethanol toxicity and septicemia patients 35,36 , which have been associated with autophagy-261 triggered granule degradation 12,37 , Whether vacuolization of neutrophils also contribute in NAFLD patients is 262 currently unknown. To investigate the immune status of NAFLD patients, neutrophils were obtained from normal 263 individuals (n=12) and NAFLD patients (n=12). NAFLD patients were diagnosed by liver biopsy and hepatic HE 264 staining ( Figure S1). The clinical parameters of the subjects are listed in Table 1. Four consecutive stages of or granules: early autophagic vacuoles (AVi), degradative autophagic vacuoles (AVd), glycogen vacuoles, and 267 vacuoles. The four stages of autophagic vacuoles were observed in NAFLD neutrophils, with increased total 268 vacuole number and autophagic vacuole to neutrophil area ratio, compared to normal neutrophils ( Figure 1A and 269 Figure 1B). The lipidation level of LC3B was also markedly increased and the accumulation of p62 decreased in 270 NAFLD neutrophils ( Figure 1C). These results indicated that the autophagic process was more prominent in 271 NAFLD neutrophils. Interestingly, the number of granules in NAFLD neutrophils was significantly reduced (Figure 272 1D). In the AVi and AVd stages, a significant number of granules were engulfed by autophagic vacuoles (Figure 273 1A). To evaluate whether granule-associated adhesion molecules were degraded with granules in NAFLD 274 neutrophils, the total protein levels and the surface expression of CD11a, CD11b CD18 and Rap1 were assessed by 275 immunoblotting and by flow cytometry, respectively. As expected, the total protein level and surface expression of 276 CD11a, CD11b and CD18 were all significantly lower in patients compared to the healthy controls ( Figure 1C, 277 Figure 1E and Figure S2). The reduced expression of the adhesion molecules correlated with an impaired level of 278 adhesion of NAFLD neutrophils in vitro compared to healthy neutrophils ( Figure 1F). These results showed that 279 NAFLD neutrophils had an increased number of autophagic vacuoles correlated to a decreased adhesion deficiency, 280 suggesting that autophagy inhibited the motility of neutrophils in NAFLD patients. 281

PA Enhanced Autophagy and Degraded the Granules in Neutrophils 282
PA, a major pathological hallmark of NAFLD, can induce autophagy in mouse embryonic fibroblasts 21 . We 283 investigated the effect of 0.25 mM PA, a pathological concentration in NAFLD, on autophagy in neutrophils. 284 Immunoblotting results showed that neutrophil autophagy was induced between 4 and 8 hours of PA treatment 285 ( Figure S3). PA also strongly triggered neutrophil vacuolation ( Figure?). Consistent with our ex vivo findings, the 286 four stages of autophagic vacuoles were observed in PA-treated neutrophils ( Figure 2A). The number of autophagic 287 vacuoles and the ratio of autophagic vacuole area to neutrophil area were significantly higher in PA-treated 288 neutrophils than in control neutrophils ( Figure 2B). The lipidation levels of LC3B were significantly higher in PA-289 treated neutrophils, while p62 were significantly reduced ( Figure 2C). Furthermore, the number of granules 290 significantly decreased in PA-treated neutrophils ( Figure 2D). To make sure autophagy mediates the decrease of 291 granules triggered by PA, Rapamycin (RAP) was used to activate autophagy. Expectedly, the number of granules 292 decreased in RAP treated neutrophils ( Figure 2D). Moreover, when autophagy induced by PA was blocked by 293 bafilomycin A (BafA1) or hydroxychloroquine sulfate (CQ), the number of granules significantly increased (Figure 294 observed in granule numbers compared to the unstimulated groups ( Figure S4B). These results suggests that PA 297 induced autophagy plays an important role in neutrophil granule homeostasis. ATG5 knockdown lentivirus were 298 infected the neutrophil-like differentiated (dHL-60) cells to deficient autophagy to further reiterate the effect of PA-299 induced autophagy. PA-treated ATG5-KD dHL-60 cells showed a similar recovery of granule number ( Figure 2E). 300 These results indicate that PA strongly enhances neutrophil autophagy and vacuolation, which is associated with 301 granule degradation. 302 Organelles delivery to lysosomes for degradation is dependent on the autophagic receptor p62 38 . To investigate 303 whether the decrease in granule levels was associated with p62-mediated granule degradation, immunogold 304 electron microscopy was performed. The p62 electron-dense gold particles were predominantly localized on the 305 AVi and AVd, but rarely on the vacuoles in PA-treated neutrophils ( Figure 2F). This data motivated us to speculate 306 that p62 might mediate the degradation of granules. AGs, SGs and GGs can be distinguished according to their size 307 and electron density 27 . In addition, myeloperoxidase (MPO), lactoferrin and gelatinase (MMP-9) are markers of 308 AG, SG, and GG, respectively 28-30 . To investigate whether all three granule types could be degraded by autophagy,

PA-induced Autophagy Decreased Neutrophil Adhesion 318
In control neutrophils, CD11a, CD11b, CD18 and Rap1 electron-dense gold particles were present on the granules, 319 secretory vesicles, and the plasma membrane as well as in the cytoplasmic matrix ( Figure 3A). In PA-treated 320 neutrophils, the CD11a, CD11b, CD18 and Rap1 gold particles were primarily present on AVi and AVd, but 321 sparsely on vacuoles ( Figure 3A). AGs, SGs and GGs were distributed with Rap1 in neutrophils 2 . We next 322 investigated whether CD11a, CD11b and CD18 were also detected on AGs, SGs and GGs using double-labeling 323 (CD11a, CD11b and CD18 colocalized with MPO, lactoferrin and MMP-9, respectively) and the morphological 324 analysis of the three granule subtypes. We found that the gold particles corresponding to CD11a, CD11b and CD18 325 were all present on AGs ( Figure 3B), SGs (Figure 3C) and GGs ( Figure 3D), and few located on vacuoles ( Figure 3B, Figure 3C and Figure 3D). These results suggested that the degradation of the three granules could possibly be 327 mediated by autophagy. Consistent with this observation, the protein levels of CD11a, CD11b, CD18 and Rap1 328 were significantly lower in PA-treated neutrophils than in control neutrophils ( Figure 3E). Furthermore, surface 329 expression of CD11a, CD11b, and CD18 were greatly decreased in PA-treated neutrophils (Figure 3F   showed that the adhesion of PA-treated neutrophils was significantly impaired compared to control neutrophils 335 ( Figure 3G). This was not due to a cytotoxic effect of PA on neutrophils at the concentration used in our 336 experiments (0.25 mM) ( Figure S7). Taken together, the results showed that PA induced autophagy triggered AG, 337 SG and GG degradation and was accompanied by the degradation of CD11a, CD11b, CD18, and Rap1 in 338 neutrophils, significantly decreasing neutrophil adhesion. 339

Hsc70-Dependent CD11a, CD11b, CD18 and Rap1 Degradation by Autophagy Reduced Neutrophil Adhesion 340
Ubiquitination is a prerequisite for protein degradation by autophagy 39 . We initially investigated whether 341 CD11a, CD11b, CD18 and Rap1 could be ubiquitinated. The data showed that polyubiquitin was reciprocally 342 coimmunoprecipitated with CD11a, CD11b, CD18 and Rap1 in neutrophils, which suggested that the IP complexes 343 of CD11a, CD11b, CD18 and Rap1 could be polyubiquitinated ( Figure 4A). The accumulation of CD11a, CD11b, 344 CD18 and Rap1 was greatly decreased when autophagy was induced by PA, while protein levels were increased 345 significantly when autophagy was blocked by BafA1 or CQ in PA-treated neutrophils ( Figure 4B and Figure S8A). 346 Similarly, inhibition of PA-induced autophagy increased neutrophil adhesion ( Figure 4C and Figure S8B). To 347 further confirm that PA-induced autophagy decreased neutrophil adhesion by promoting CD11a, CD11b, CD18, 348 and Rap1 degradation, HL-60 cells were transduced with shRNAs specific for ATG5 ( Figure 4D, Figure S9A, 349 Figure S9B and Figure S9C). ATG5 knockdown attenuated PA-induced vacuolation ( Figure S9A and Figure S9B), 350 reduced the degradation of CD11a, CD11b, CD18 and Rap1 ( Figure 4D and Figure S9C) and partially restored cell 351 adhesion ( Figure 4E and Figure S9D). 352 To identify the molecules involved in the degradation of the adhesion molecules and Rap1, proteins present in 353 IP complexes of CD11a, CD11b, CD18 and Rap1 were identified by a shotgun analysis. A total of 415 proteins 354 interacted with CD11a (Table S1); 217 proteins were identified in CD11b complexes (Table S2); 236 proteins were CD11a, CD11b, CD18 and Rap1 nor peptides of p62 were detected. This suggested that CD11a, CD11b, CD18 and 357 Rap1 were not directly ubiquitinated and recognized by p62. Unexpectedly, a total of 27 common proteins 358 interacted with CD11a, CD11b, CD18 and Rap1 (Table S5), including the molecular chaperone Hsc70. Hsc70 is 359 known to target and then deliver cytosolic proteins to lysosomes for degradation via the chaperone molecular 360 autophagy (CMA) pathway 40 . We confirmed the interaction as Hsc70 was reciprocally coimmunoprecipitated with 361 CD11a, CD11b, CD18 and Rap1 ( Figure 4F). In addition, immunogold electron microscopy results showed that 362 Hsc70 gold particles were present on the granules in control neutrophils ( Figure S10). However, Hsc70 363 immunogold signal was observed on AVi and AVd but rarely present on the vacuoles in PA-treated neutrophils 364 ( Figure S10). This suggested that Hsc70 was targeting the adhesion molecules and Rap1 for lysosomal degradation.  Integrins are required for cancer cell matrix adhesion and firm adhesion of neutrophils 45,46 . Autophagy 406 decreases cancer cell matrix adhesion and facilitates tumor metastasis by degrading β1 integrins 47 . However, it is 407 unknown whether autophagy decreases the firm adhesion of neutrophils by degrading β2 integrins. In metabolic 408 diseases, neutrophils are exposed to abnormal metabolite levels, such as high blood levels of fatty acids, which 409 exhibit lipotoxicity and can impair neutrophil immune function. In this study, we found that the three neutrophil 410 granule types, namely, AGs, SGs and GGs, could be engulfed by autophagosomes for degradation in NAFLD 411 neutrophils. Furthermore, CD11a, CD11b, CD18 and Rap1 in the neutrophils were targeted by Hsc70 and degraded 412 via autophagy. Consequently, neutrophil adhesion was significantly decreased. Notably, we found that PA inhibited 413 the p-PKCα/PKD2 pathway to induce autophagy. In neutrophils, autophagic vacuoles exhibit morphological 414 diversity, and the classification of these vacuoles is not well standardized. Many appellations, such as phagocytic We first divided the neutrophil autophagic vacuoles according to four consecutive stages, namely, AVi, AVd, 417 glycogen vacuole and vacuole, depending on the degree of degradation of the engulfed granules or other cytosolic 418 cargoes. The autophagy receptorp62 mediates the degradation of the damaged mitochondria in energy cells 51 . We 419 found that most AGs, SGs and GGs colocalized with p62 and MPO, lactoferrin and MMP-9, respectively, were 420 located on the AVi and AVd of neutrophil autophagic vacuoles. Little to no signal was observed on the glycogen 421 vacuole and vacuole stages, which indicated that p62 might deliver the damaged AGs, SGs and GGs to lysosomes 422 for degradation via autophagy. Interestingly, granules are also considered as the lysosomes of neutrophils 52,53 . 423 This continuous autophagic flux contributed to neutrophil vacuolation. As mentioned above, the immunity of 429 patients with severe vacuolated neutrophils is decreased 34 . Interestingly, we found that autophagic vacuoles also 430 existed in NAFLD neutrophils, hinting at a reduced immunity of NAFLD patients. Interestingly, CD11a, CD11b 431 and CD18 were all observed on the AGs, SGs and GGs. These β2 integrins protein levels were lowered 432 concomitantly with the number of the three granule types. The decreased protein level of the adhesion molecules 433 and the upstream signaling molecule Rap1 was dependent on autophagy, and impaired neutrophil adhesion. Rap1, a 434 β2 integrin activity regulator 8 . These findings suggested that autophagy decreases neutrophil adhesion by both 435 degrading CD11a, CD11b and CD18 and reducing the activity of these proteins by facilitating Rap1 degradation. 436 Although we confirmed that the CD11a, CD11b, CD18 and Rap1 could be degraded through autophagy and 437 autophagy plays an important role in neutrophil adhesion, we also showed that the proteasome inhibitor MG132 438  , Table S1 and Table S2 and Table S4) and E3 ubiquitin-protein ligases (Q76N89, 447 O76064, Q86UK7, Table S1; Q86Y13, H7C3Z1 Table S3; Q9NQC1, A0A096LP02, Q96T88, Table S4) were 448 identified in the IP complexes, suggesting that these four proteins might form polyubiquitin-protein conjugates and 449 might be degraded by p62-dependent autophagy. The α5β1 integrin could be degraded in a ligand (fibronectin)-450 dependent manner 58 . However, no β2 integrin ligands (FGA, FGB and FGG) and p62 were observed in the mass 451 spectrometry results, which indicated that the degradation of CD11a, CD11b, CD18 and Rap1 was not dependent 452 on ligands or p62 in neutrophils. Notably, Hsc70 (P11142) was identified from four IP complexes and could be 453 reciprocally coimmunoprecipitated with CD11a, CD11b, CD18 and Rap1. Furthermore, CD11b, CD18, and AG, 454 SG, and GG marker proteins (MPO: P05164, lactoferrin: P02788 and MMP-9: P14780, respectively) were also 455 observed in the IP complexes of Hsc70 by the shotgun approach (Table S7). Coincidentally, MPO, lactoferrin and 456 MMP-9 were also identified in the IP complexes of CD11a, CD11b, CD18 and Rap1. In addition, Hsc70 457 colocalized with MPO, lactoferrin and MMP-9 on AGs, SGs and GGs, respectively. These results suggested that 458 CD11a, CD11b, CD18 and Rap1 were delivered by Hsc70 to lysosomes for degradation. However, the special 459 motif (KFERQ) was not found in peptides of the CD11a, CD11b, CD18 and Rap1. Hsc70 might a interact with a 460 partner protein of the adhesion molecules with the motif and delivered them to the lysosome for degradation. 461 Notably, Hsc70 knockdown attenuated autophagy-mediated degradation of CD11a, CD11b, CD18 and Rap1, 462 thereby increasing the adhesion of HL-60 cells. Taken together, the results showed that CD11a, CD11b, CD18 and 463

Rap1 could be degraded via autophagy. 464
Our data demonstrated that autophagy induced by PA decreased the adhesion of PA-treated and NAFLD 465 neutrophils. However, the underlying mechanism was unclear. Interestingly, using quantitative proteomic analysis, 466 we found that a downstream target of PKCα, namely, PKD2 59 , a key regulatory protein of autophagy 60 , was 467 significantly downregulated. PKCα is a negative regulator of autophagy in neuroepithelial cells 23 . Our data showed 468 that PKCα/PKD2 was indeed inhibited in PA-treated and NAFLD neutrophils. Moreover, pharmacological 469 inhibition of PKCα/PKD2 by GO6983 strongly induced neutrophil autophagy, vacuolation and decreased 470 neutrophil adhesion, while PKD2 overexpression significantly attenuated the PA-induced autophagy, vacuolation 471 and decrease in adhesion. These results indicated that PKCα/PKD2 pathway was involved in PA-induced 472 autophagy and then caused neutrophil vacuolation and a decrease in adhesion. A previous study indicated that PKC 473 inhibitors dramatically induced autophagy 22 , which further support our conclusion. further decreased neutrophil adhesion, thereby impairing neutrophil immunity. Notably, we found that the three 477 neutrophil granule subunits, namely, AGs, SGs and GGs, were degraded by autophagy. This phenomenon might be 478 termed "granulophagy" (a combination of "granule" and "autophagy"). In addition to adhesion-associated proteins, 479 proteins associated with endocytosis, phagocytosis, phagosomes, chemotaxis, microbicidal substances, 480 cytoskeleton remodeling, etc. are also present on AGs, SGs, and GGs 2 , and these proteins might be degraded by 481 autophagy. It still remains to be determined if, besides adhesion, the ability of neutrophils to migrate and to kill 482 microbes is also affected by PA or is impaired in NAFLD PMNs. Autophagy might be an immune switch for 483 neutrophils that control the above biological functions. Understanding the mechanisms of autophagy-induced 484 degradation of intracellular immune-associated proteins is fundamental to the identification of new therapeutic 485 strategies against metabolic disease-induced innate immune deficiency. Neutrophil vacuolation, an indicator of the 486 immune status of patient 34 , is associated with autophagy resulting from changes in blood constituents. Therefore, 487 further research is needed to investigate the effects of the metabolic disorders on the autophagy and vacuolation of 488 neutrophils, which will provide a new therapeutic strategy to improve the immune deficiency resulting from the 489 above diseases.   NAFLD neutrophils for transmission electron microscope were directly fixed after isolated from the patient's blood 649 without incubation, for adhesion assay, normal and NAFLD neutrophils were incubated with the subjects serum.  (C) Immunoblot for LC3B, p62, CD11a, CD11b, CD18, and Rap1 in normal and NAFLD neutrophils. ACTB was 656 used as a loading control (n = 3). Data represent the mean ± s.e.m. (* P < 0.05 and ** P < 0.01 versus the control  Table S1. The list of proteins interacting with CD11a identified by the shotgun 1039 Table S2. The list of proteins interacting with CD11b identified by the shotgun 1040 Table S3. The list of proteins interacting with CD18 identified by the shotgun 1041 Table S4. The list of proteins interacting with Rap1 identified by the shotgun 1042 Table S5. The list of common proteins interacting with CD11a, CD11b, CD18 and Rap1 identified by the shotgun 1043 Table S6. The list of differentially expressed proteins between PA treatment group and control group by iTRAQ 1044