A Genetic Model of Constitutively Active CD11b/CD18 Laisel Martinez, Xiaobo Li, Gioser Ramos-Echazabal, Hafeez Faridi, Zachary M. Zigmond, Nieves Santos Falcon, This information is current as Diana R. Hernandez, Serene A. Shehadeh, Omaida C. of September 27, 2021. Velazquez, Vineet Gupta and Roberto I. Vazquez-Padron J Immunol published online 16 September 2020 http://www.jimmunol.org/content/early/2020/09/14/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 16, 2020, doi:10.4049/jimmunol.1901402 The Journal of Immunology

A Genetic Model of Constitutively Active Integrin CD11b/CD18

Laisel Martinez,*,1 Xiaobo Li,†,1 Gioser Ramos-Echazabal,* Hafeez Faridi,‡ Zachary M. Zigmond,x Nieves Santos Falcon,* Diana R. Hernandez,* Serene A. Shehadeh,* Omaida C. Velazquez,* Vineet Gupta,† and Roberto I. Vazquez-Padron*

Pharmacological activation of integrin CD11b/CD18 (aMb2, Mac-1, and CR3) shows anti-inflammatory benefits in a variety of animal models of human disease, and it is a novel therapeutic strategy. Reasoning that genetic models can provide an orthogonal and direct system for the mechanistic study of CD11b agonism, we present in this study, to our knowledge, a novel knock-in model of constitutive active CD11b in mice. We genetically targeted the Itgam (which codes for CD11b) to introduce a point mutation that results in the I332G substitution in the . The I332G mutation in CD11b promotes an active, higher- affinity conformation of the ligand-binding I/A-domain (CD11b aA-domain). In vitro, this mutation increased adhesion of Downloaded from knock-in to fibrinogen and decreased to a formyl–Met–Leu–Phe gradient. In vivo, CD11bI332G animals showed a reduction in recruitment of neutrophils and in a model of sterile peritonitis. This genetic activation of CD11b also protected against development of atherosclerosis in the setting of hyperlipidemia via reduction of recruitment into atherosclerotic lesions. Thus, our animal model of constitutive genetic activation of CD11b can be a useful tool for the study of integrin activation and its potential contribution to modulating leukocyte recruitment and alleviating different inflammatory diseases. The Journal of Immunology, 2020, 205: 000–000. http://www.jimmunol.org/

lso known as aMb2, Mac-1, and CR3, CD11b/CD18 CD11b/CD18 is among the most versatile of all , with is the predominant b2 integrin in polymorphonuclear more than 40 reported ligands (4). As a result, it plays an im- A leukocytes and is abundantly expressed in , portant role in a diversity of immunological processes, from leu- macrophages, and dendritic cells (1, 2). A heterodimer of the kocyte recruitment to the sites of tissue injury to the resolution of CD11b (aM) and CD18 (b2) subunits encoded by the ITGAM and inflammation (5–7). ITGB2 , respectively, it exists on the cell surface in two key The affinity of CD11b/CD18 to multiple endothelial ligands

conformational states: 1) an inactive, low-affinity, closed confor- (e.g., ICAM-1, fibrinogen, and endothelial protein C by guest on September 27, 2021 mation, and 2) an active, high-affinity, open conformation (3). [EPCR]) (8–10) has made it a promising target for the treatment of inflammatory diseases. Pharmacological blockade or genetic *DeWitt Daughtry Family Department of Surgery, Leonard M. Miller School of deletion of CD11b improved restenosis (11), cerebral and renal Medicine, University of Miami, Miami, FL 33136; †Department of Internal Medi- ischemic injury (12, 13), glomerulonephritis (14, 15), and throm- cine, Rush University Medical Center, Chicago, IL 60612; ‡Department of Pharma- ceutical Sciences, College of Pharmacy, Chicago State University, Chicago, IL bosis (16) in animal models. Based on these findings, it was ini- 60612; and xDepartment of Molecular and Cellular Pharmacology, Leonard M. Miller tially thought that inhibition of ligand binding to CD11b/CD18 School of Medicine, University of Miami, Miami, FL 33136 would be a potential therapeutic strategy for vascular inflamma- 1L.M. and X.L. contributed equally to the manuscript. tion. However, several such anti-integrin therapeutic antagonists ORCIDs: 0000-0002-3302-8478 (X.L.); 0000-0002-3205-1369 (H.F.); 0000-0001- failed to meet efficacy end points in clinical trials (17). 6987-2550 (V.G.). Similarly, large-scale, genome-wide association studies have Received for publication November 25, 2019. Accepted for publication August 23, shown that mutations that reduce activity of CD11b or render it 2020. inactive are pathogenic (18–23). These studies have identified This work was supported by National Heart, Lung, and Blood Institute/National Institutes of Health Grants R01-HL125672 (to R.I.V.-P. and O.C.V.) and K08- single-nucleotide polymorphisms in the human ITGAM gene that HL151747 (to L.M.), National Institute of Diabetes and Digestive and Kidney result in the production of functionally deficient CD11b/CD18 but Diseases/National Institutes of Health Grants R01-DK121227 (to R.I.V.-P.) and without change in the level of surface expression (22, 24). The R01-DK107984 and R01-DK084195 (to V.G.), and American Heart Association Predoctoral Fellowship 18PRE34030314 (to Z.M.Z.). This project was also supported detected variants are associated with increased susceptibility to in part by Bears Care, the Department of Internal Medicine at Rush University systemic erythematosus and lupus nephritis (18, 20, 21, 25). Medical Center, and the National Cancer Institute/National Institutes of Health Grant R01-CA244938 (to V.G.). Animal models of autoimmunity also confirmed the relationship Address correspondence and reprint requests to Vineet Gupta or Roberto I. Vazquez- between Mac-1 deficiency and end-organ damage (26, 27). To- Padron, Department of Internal Medicine, Rush University, Rush Medical College, gether, these studies suggested that integrin activation may be an 1735 W Harrison Street, Suite 222, Chicago, IL 60612 (V.G.) or Division of Vascular alternate and highly promising therapeutic approach for these Surgery, University of Miami, Miller School of Medicine, 1600 NW 10th Avenue, RMSB 1048, Miami, FL 33136 (R.I.V.-P.). E-mail addresses: [email protected] pathological conditions. (V.G.) or [email protected] (R.I.V.-P.) Toward that, we were among the first, to our knowledge, to The online version of this article contains supplemental material. develop novel small-molecule agonists of CD11b, and have + Abbreviations used in this article: CD11bWT, CD11b wild-type; ES, embryonic stem; shown that such agents reduce recruitment of CD11b cells and KI, knock-in; KO, knockout; SILEN, socket for isoleucine; SMA, smooth muscle have therapeutic efficacy in a variety of disease models (28–30). actin; WT, wild-type. These include experimental models of vascular injury (28, 30), Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 nephritis (28), organ allograft vasculopathy (31), fibrosis

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901402 2 A MOUSE MODEL OF GENETICALLY ACTIVE CD11b

(32), hyperoxic lung injury (33), autoimmunity (23), and pan- procedures were performed according to the National Institutes of Health creatic cancer (34). Small-molecule CD11b agonists bind to an guidelines (Guide for the Care and Use of Laboratory Animals) and approved allosteric site in the aA-domain (also known as aI-domain) of by the respective Institutional Animal Care and Use Committees at the Uni- versity of Miami Miller School of Medicine and Rush University Medical CD11b, where they stabilize a more active conformation of the Center. receptor and prime it for ligand binding (23). We and others have also shown that CD11b activation can inhibit proinflammatory Peritonitis model TLR pathways (23, 35). Peritonitis was induced in 14–16-wk-old CD11bWT and CD11bI332G mice To fully understand the therapeutic potential of CD11b activation, of both sexes by injecting 3 ml of a sterile 3% Brewer’s thioglycollate as well as to elucidate the molecular pathways involved, alternative, broth (Sigma-Aldrich, St. Louis, MO). To analyze cell recruitment, leu- orthologous approaches are needed. In this study, we present, to our kocyte cell suspensions were obtained by lavage of the peritoneal cavity with 10 ml of PBS at 4 and 48 h after injection. Cells were collected by knowledge, a novel knock-in (KI) mouse model expressing a con- centrifugation for 10 min at 250 3 g and washed twice with PBS before stitutively active mutant CD11b. In this model, the Itgam gene was analysis. genetically targeted to introduce a mutation (I332G) that promotes a Atherosclerosis model higher-affinity conformation of this integrin subunit. The ligand- binding aA-domain of CD11b contains an allosteric site, known Hypercholesterolemia was induced in 12-wk-old CD11bWT and CD11bI332G as socket for isoleucine (SILEN) (36) or I-domain allosteric site mice of both sexes after administration of a single tail-vein injection of the recombinant rAAV8-D377Y-mPCSK9 virus (University of North Carolina (IDAS) (37), that modulates its conformation and binding affinity. 3 10 a at Chapel Hill Vector Core, Chapel Hill, NC) (2.0 10 copies per Mutagenesis of isoleucine 332 to glycine (I332G) shifts the A- mouse) according to the published protocol (39). The exogenous expres- domain into its open, ligand-competent conformation. We intro- sion of PCSK9 enhances internalization and degradation of hepatic low- Downloaded from duced this mutation in murine CD11b to generate CD11bI332G mice density lipoprotein receptors. One week after the virus injection, mice (KI mice). Our investigations show that this activating mutation started feeding on a cocoa butter Teklad research custom diet (catalog decreases inflammatory cell recruitment and vascular inflammation TD.88051; Envigo, Somerset, NJ) for 12 wk ad libitum. Cholesterol levels were measured 1 wk after diet initiation and at the end of the 12 wk diet in models of sterile peritonitis and atherosclerosis, respectively. To using cholesterol test strips by PTS Panels (Indianapolis, IN). After our knowledge, this novel experimental model can be used to gain completion of the diet, nonfasting blood was submitted to the University of deeper insight into mechanisms that can be further therapeutically Miami Division of Comparative Pathology for complete blood count and http://www.jimmunol.org/ targeted in inflammation and atherosclerosis. blood chemistry, and whole aortas were submitted to Eehscience (Pick- erington, OH) for independent determination of atherosclerotic burden by Materials and Methods Oil Red O staining. Serial cross-sections of the brachiocephalic trunk were I332G obtained for plaque characterization. Lesion size and macrophage infil- Construction of the CD11b targeting vector tration were quantified at three positions along the brachiocephalic artery ∼ ∼ ∼ m The CD11bI332G targeting vector was engineered by Cyagen (Santa Clara, ( 550, 450, and 350 m from the aortic arch). 9 9 CA) as portrayed in Fig. 1A. Briefly, the 5 and 3 homology arms including Adhesion assay exons 7–12 of the Itgam gene were amplified by PCR from bacterial artificial clones RP23-343J4 and RP23-323J3 from the C57BL/6J library. Peritoneal neutrophils were isolated 4 h after sterile 3% Brewer’s thio- The I332G mutation (ATC to GGC) was introduced by site-directed muta- glycollate injection. Cell purity was assessed by flow cytometry. Cell ad- by guest on September 27, 2021 genesisintoexon9inthe39 homology arm. The homology arms were cloned hesion assays were performed as previously described (40) and used into the targeting vector using the NotI and BsiWI (59 arm) and XhoI and NruI immobilized fibrinogen as Mac-1 ligand. Briefly, 384-well microplates (39 arm) restriction sites. An Neo cassette flanked by two Frt sites was inserted (Corning, Corning, NY) were coated with 15 mg/ml fibrinogen in PBS between the homology arms. A diphtheria toxin A cassette cloned down- containing 1 mM of each Ca2+ and Mg2+ by incubating overnight at 4˚C. 9 stream of the 3 homology arm was used for negative selection. The above The nonspecific sites were blocked with 1% gelatin in TBS, followed by cloning strategy was confirmed by PCR, restriction digestion, and sequencing. washing three times with TBS. Cells were suspended in TBS containing 2+ 2+ 2+ Generation of CD11bI332G KI mice 1 mM of each Ca and Mg (TBS buffer) or in TBS containing 1 mM Mn2+ in the presence or absence of an anti-CD11b blocking Ab (clone M1/ CD11bI332G KI mice were generated by Cyagen. Briefly, the targeting vector 70, 10 mg/ml). Cells were incubated in the ligand-coated wells (3000 cells was linearized with NotI and electroporated into C57BL/6 embryonic stem per well) for 10 min at 37˚C. The assay plates were then gently inverted (ES) cells. Ninety-three G418-resistant clones were screened by PCR, of and kept in the inverted position for 30 min at room temperature to dis- which 23 positive clones were sent to sequencing. Fourteen ES clones were lodge nonadherent cells. The remaining adherent cells were fixed using 4% positive by sequencing, six of which were expanded for Southern blot formaldehyde and quantified by automated imaging microscopy as previ- confirmation using KpnI and EcoRI digestion, followed by hybridization ously described (41). Assays were performed in triplicate wells and at least with a probe complementary to the Neo cassette (Supplemental Fig. 1). three independent times. Clones 1B6 and 1C9 were selected for blastocyst injection and chimera production. Chimeras were bred with C57BL/6 females to test germline Chemotaxis assay transmission. Three pups from clone 1B6 and three from clone 1C9 were identified as positive germline F1 founders by PCR and sequencing. F1 Peritoneal neutrophils were obtained as above. Neutrophil chemotaxis on founders were crossed with homozygous ROSA26::FLPe KI mice (38) planar surfaces was performed using 48-well Zigmond chambers (Neuro Probe, Gaithersburg, MD) as previously described (40). Briefly, cells were (stock no. 003946; The Jackson Laboratory, Bar Harbor, ME) at the Uni- m versity of Miami to excise the Neo cassette. Mac-1I332G heterozygotes and preincubated for 10 min on fibrinogen-coated (25 g/ml) glass coverslips negative for the Neo insertion were then backcrossed with C57BL/6 mice to in a humidified chamber with RPMI 1640 supplemented with 1% FBS. The remove the ROSA26::FLPe allele. Once achieved, mice were further back- coverslips were then placed on top of Zigmond chambers, and fMLF crossed into the C57BL/6 background for six to eight generations. Primers (Sigma-Aldrich) gradient was created by placing the assay buffer (RPMI m 59-AAGGAATATCTTTTGCTGGACGTACG-39 and 59-CAGAAGGCA- 1640 with 1% FBS) in one well of the chamber and 10 M fMLF in assay GCTTAGAACAGGGC-39 were used to screen for the excision of Neo, buffer in the other well. The migration of neutrophils toward fMLF was whereas 59-CACTGATATTGTAAGTAGTTTGC-39 and 59-CTAGTGC- recorded at 5–30-s intervals for a period of 25 min using a Nikon Eclipse GAAGTAGTGATCAGG-39 were used to detect the ROSA26::FLPe allele. 90i microscope, as previously described (40, 42). Images were acquired The Mac-1I332G colony is maintained as heterozygotes, from which CD11bI332G using a Nikon DS camera with a PLAN APO 320 differential interference homozygotes and CD11b wild-type (CD11bWT) littermates are obtained for contrast microscopy objective and the Nikon Imaging software. Analysis experiments. Genotyping of the Itgam wild-type (WT) and I332G alleles is of neutrophil migration was performed with the motile population that had performed with primers 59-ATTTAGCTTTGGCTCCTTGGCAAC-39 and 59- moved more than 10 mm using the ImageJ software (National Institutes of TGGAGCAAGTCAGACCCAAATGTC-39, followed by enzymatic digestion Health, Bethesda, MD) with manual cell tracking using the chemotaxis and with BglII. This strategy results in 107- and 162-bp bands for the WT allele and migration tool plugins (Ibidi) for ImageJ. Data from 30 neutrophils per the undigested 269-bp band for the I332G variant (Fig. 1B). All animal mouse strain were quantified in at least three independent experiments. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

I332G FIGURE 1. Generation of CD11b KI mice. (A) Schematic of the targeting strategy. From top to bottom, the diagrams indicate the genomic structure of by guest on September 27, 2021 the WT allele, the targeting vector, and the targeted KI allele. Confirmatory Southern blots used a probe complementary to the Neo cassette and DNA digested with EcoRI or KpnI (see Supplemental Fig. 1). (B) Genotyping of the WT and KI alleles in mice by PCR, followed by BglII digestion. The I332G mutation disrupts a BglII recognition site in exon 9 of the Itgam gene, generating a 269-bp band in KI homozygotes and 107- and 162-bp bands for the WT allele. (C) Partial amino acid alignment of the human, WT mouse, and mutant (I332G) mouse CD11b subunits of integrin CD11b/CD18. The mutant position is highlighted in black. The boxed sequence delineates the CD11b A-domain, with asterisks indicating amino acid residues that form a hydrophobic coordination socket (known as SILEN) for WT Ile332. Amino acid numbering is based on the full-length in GenBank accession numbers NP_001139280.1 (https://www.ncbi.nlm. nih.gov/protein/NP_001139280.1) and NP_001076429.1 (https://www.ncbi.nlm.nih.gov/protein/NP_001076429.1) for human and mouse, respectively.

ELISA the slides were incubated with Alexa Fluor 546 goat anti-rat Ab (1:1000, no. A11081; Thermo Fisher Scientific, Waltham, MA) and Alexa Fluor 488 goat Primary macrophages were collected by peritoneal lavage from 14- to 16- WT I332G anti-mouse Ab (1:1000, no. A11029; Thermo Fisher Scientific) for 45 min. wk-old CD11b and CD11b mice of both sexes, 4 d after injecting Sections were counter stained with 300 nM DAPI solution (no. D1306; 3 ml of sterile 3% Brewer’s thioglycollate broth (Sigma-Aldrich). Cells Thermo Fisher Scientific) in PBS for 3 min and mounted in DABCO anti- were adherence purified for 1 h, followed by a wash with PBS to remove fading polyvinyl alcohol mounting medium (no. 10981; Sigma-Aldrich). nonadherent cells. Adherent cells were suspended in DMEM supplemented Sections were examined in an Olympus Ix71 inverted microscope and with 10% FBS and counted. One million cells per well were plated in photographed using the Olympus cellSens Standard software. Image anal- 12-well plates and allowed to settle down for 2 d before treatment. Cells ysis was performed with ImageJ (National Institutes of Health). were stimulated with 50 ng/ml of LPS from Escherichia coli K12 (Invi- voGen, San Diego, CA), and supernatants were collected at baseline and at Flow cytometry 2, 4, and 8 h after treatment. Levels of secreted IL-1b, IL-6, and TNF-a All Abs and reagents were purchased from BioLegend except when in- were quantified by ELISA following the manufacturer’s protocols (R&D dicated otherwise. Quantification of integrin subunit expression used the Systems, Minneapolis, MN). Abs CD11a PE (no. 101107), CD11b PerCP (no. 101230), CD11c BV650 Immunofluorescence (no. 564079; BD Biosciences), CD18 APC (no. 562828; BD Biosciences), CD115 BV605 (catalog no. 135577), and Gr-1 PE/Dazzle 594 (no. 108452). Three brachiocephalic artery cross-sections per animal, corresponding to Briefly, cells were washed with PBS twice and counted, and Fc receptors positions ∼550, ∼450, and ∼350 mm from the aortic arch were costained were blocked with anti-mouse CD16/32 Ab for 10 min at 4˚C. Cells were with anti–galectin-3 (Mac2) and smooth muscle actin (SMA) Abs to washed with PBS, and True-Stain Blocker was added before identify macrophages and smooth muscle cells, respectively. Briefly, Ag labeling with eBioscience Fixable Viability Dye eFluor 660 (Thermo retrieval was performed in 10 mM sodium citrate, 0.05% Tween 20 Fisher Scientific) for 30 min. Cells were washed with FACS buffer with (pH 6) solution for 30 min in 95˚C water, followed by treatment with 3% 0.1% sodium azide (flow buffer), and True-Stain Monocyte Blocker was hydrogen peroxide and TNB blocking solution (no. FP1020; Perki- added again, followed by 30-min incubation with the remaining labeling nElmer, Waltham, MA). Then, slides were incubated with rat anti-mouse Abs. Excess Abs were washed, and cells were fixed with flow cytometry Mac2 Ab (1:50, no. 125402; BioLegend, San Diego, CA) and mouse anti- buffer supplemented with 1% paraformaldehyde prior to flow cytometric human SMA Ab (1:200, no. M0851; Dako) overnight at 4οC. The next day, analyses. Flow cytometry data were read using a BD LSRFortessa High 4 A MOUSE MODEL OF GENETICALLY ACTIVE CD11b

Table I. Complete blood count analysis of peripheral blood collected from 16-wk-old WT and CD11bI332G KI mice

WT (n = 8) CD11bI332G KI (n =8) p Value Complete Blood Count WBC (1 3 103/ml) 3.2 6 1.5 2.2 6 0.5 0.09 RBC (1 3 106/ml) 7.8 6 0.5 7.4 6 0.6 0.11 Hemoglobin (g/dl) 10.9 6 0.5 10.5 6 0.5 0.16 Hematocrit (%) 37.3 6 2.3 35.3 6 2.5 0.12 MCV (fl) 47.5 6 1.2 47.9 6 1.6 0.61 MCH (pg) 14.0 (13.3–14.0) 14.0 (14.0–14.8) 0.50 MCHC (%) 29.5 6 1.6 30.0 6 0.8 0.44 Segs (1 3 103/ml) 0.2 (0.1–0.3) 0.2 (0.1–0.2) 0.24 Bands (1 3 103/ml) N.D. N.D. — Lymphocytes (1 3 103/ml) 2.4 6 1.0 1.7 6 0.4 0.09 Monocytes (1 3 103/ml) 0.4 6 0.3 0.3 6 0.1 0.09 Eosinophils (1 3 103/ml) 0.03 (0.0–0.04) 0.02 (0.0–0.03) 0.41 (1 3 103/ml) N.D. N.D. — NRBC N.D. N.D. — Values are presented as mean 6 SD or median (interquartile range). Dashes indicated p values could not be calculated. MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; N.D., not detected; NRBC, nucleated RBC; Seg, segmented neutrophils. Downloaded from Throughput Sampler analyzer (BD Biosciences) and processed with the agreement with the activating role of this mutation (36), neutro- FlowJo software (Ashland, OR). phils from CD11bI332G mice showed increased static adhesion to Statistics fibrinogen than those from WT controls under physiological conditions (1 mM Ca2+ and 1 mM Mg2+; Fig. 2). The enhanced Statistical analyses were performed in GraphPad Prism 5 (San Diego, CA). adhesion of KI neutrophils was CD11b dependent and could be Normally distributed values are presented as mean 6 SEM and compared using a two-tailed Student t test or one-way ANOVA. Nonnormally dis- neutralized with an anti-CD11b blocking Ab (M1/70). Both WT http://www.jimmunol.org/ tributed data are presented as median 6 interquartile range and compared and CD11bI332G neutrophils reached similar levels of adhesion in using the Mann–Whitney U test. A p value ,0.05 was considered the presence of the activating cation Mn2+ (Fig. 2) (43). significant. Firmer adhesion of neutrophils is expected to result in impaired chemotaxis in response to an fMLF gradient (28), and indeed, we Results observed that KI neutrophils showed significantly reduced che- I332G Generation of CD11b KI mice motaxis compared with WT cells (Figs. 3, 4A). As with chemo- Isoleucine 332 in CD11b, corresponding to residue 316 after taxis of neutrophils upon pharmacologic CD11b activation (28), processing the N-terminal signal peptide, has been identified as a the chemotaxing KI neutrophils showed prevalence of elongated, key residue in stabilizing the inactive low-affinity conformation of trailing uropods compared with those from WT animals (Fig. 4B). by guest on September 27, 2021 the CD11b/CD18 integrin receptor (36). The regulatory role of this These results suggest increased adhesion and reduced detachment residue relies on its hydrophobic interactions with the SILEN from the substrate as the reasons for their impaired motility. (Fig. 1C). The I332G substitution weakens these interactions and Effect of integrin activation in acute inflammation favors the active, high-affinity, open conformation of the CD11b aA-domain (36). However, consequences of such activation of The CD11b/CD18 integrin plays a fundamental role in the re- CD11b in animals are currently not fully understood. Therefore, to cruitment of neutrophils and monocytes to sites of inflammation I332G investigate the role of active CD11b in vivo, we generated mice (5, 44, 45). Therefore, we tested the effects of the CD11b harboring the I332G-activating CD11b mutation following the mutation in inflammatory cell recruitment in vivo in the setting of strategy delineated in Fig. 1. Briefly, a genetic construct was thioglycollate-induced peritonitis. The number of infiltrated neu- designed to replace the WT exon 9 in the Itgam gene with the trophils and macrophages in the peritoneal cavity was measured at mutant exon 9 downstream of an Frt-flanked Neo cassette (Fig. 1A). The construct was inserted by homologous recombination in ES cells, followed by antibiotic selection, PCR screening, and Southern blot confirmation (Supplemental Fig. 1). After blastocyst injection and chimera production, heterozygous founders were crossed with ROSA26::FLPe mice to generate Neo-deleted CD11bI332G KI animals (KI animals), and backcrossed successively into the C57BL/6 background. Fig. 1B presents the genotyping analysis of CD11bI332G KI and littermate WT mice. CD11bI332G KI mice are normal in size and in reproductive and social behavior. Their complete blood count at 16 wk showed no significant differences from littermate WT mice (Table I). Surface expression of the integrin subunits CD11b and CD18, and of other a subunits (CD11a and CD11c) from the b2 integrin family, in peripheral blood neutrophils and monocytes was also similar be- FIGURE 2. Adhesion of neutrophils from CD11b WT and KI (I332G) tween KI and WT animals (Supplemental Fig. 2). mice. Adhesion of peritoneal neutrophils to fibrinogen in the presence of 2+ 2+ Adhesion and chemotaxis of CD11bI332G neutrophils in vitro physiological divalent cations (1 mM Ca and 1 mM Mg ), the activating cation Mn2+ (1 mM), and the anti-CD11b blocking Ab clone M1/70. Bars The functional effect of the I332G mutation was tested in peritoneal represent the mean 6 SEM, n = 4 per group in three independent exper- neutrophils using adhesion and chemotaxis assays (Figs. 2–4). In iments. N.S., not significant. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 3. Chemotaxis of neutrophils from CD11b WT and KI (I332G) mice. (A) Spider plots delineating the chemotaxis of peritoneal neutrophils in by guest on September 27, 2021 response to an fMLF gradient. (B–D) Quantification of Euclidean distance (B), accumulated distance (C), and cell velocity (D) in 30 migration tracks from CD11bWT and CD11bI332G neutrophils as shown in (A). Bars represent the mean 6 SEM, n = 30 per group in three independent experiments. two time points after thioglycollate injection (Fig. 5A, 5B). (5, 7, 48). We previously showed that pharmacologic activation of Neutrophil counts were significantly lower in KI versus WT mice CD11b reduces influx of macrophages after arterial injury (28). To 4 h after injection but equivalent at 48 h in both animal groups investigate whether activation of CD11b is also atheroprotective, (Fig. 5A). Similarly, there was a significant decrease in peritoneal we used KI animals in a model of atherosclerosis. Hyperlipidemia macrophage infiltration in CD11bI332G mice compared with WT was induced in cohorts of CD11bI332G and WT mice via injection animals at the 48-h time point (Fig. 5B). These results indicate a of the recombinant rAAV8-D377Y-mPCSK9 virus, followed by 12 reduction and/or delay in leukocyte recruitment in KI mice in the wk of high-fat diet, a recently established model of atherosclerosis setting of acute sterile inflammation. Cytokine secretion by CD11bI332G macrophages Primary macrophages were obtained from the peritoneal cavity of CD11bI332G and WT animals at 4 d after thioglycollate-induced peritonitis. Count-normalized cells were plated and cultured for 2 d in the absence of CD11b-specific ligands. After this pre- treatment period, cells were stimulated with LPS, and cytokine secretion was quantified at three time points (Fig. 5C–E). CD11bI332G cells showed significantly lower IL-1b and TNF-a secretion starting at 2 h after treatment (Fig. 5C, 5E). Significantly reduced IL-6 levels were also observed starting at 4 h poststim- ulation (Fig. 5D). These results parallel published reports with pharmacological CD11b-activating agents (23, 46) and are further supported by the opposite pattern in CD11b knockout (KO) ani- mals (47). Effect of genetic activation of CD11b in atherosclerosis FIGURE 4. Time-lapse video and photography of chemotaxing neu- trophils from CD11b WT and KI (I332G) mice. (A) Time-lapse videos of CD11b is a key mediator of vascular inflammation and athero- representative peritoneal neutrophils during chemotaxis toward an fMLF sclerosis by regulating leukocyte adhesion and extravasation across gradient. (B) Microphotographs of representative chemotaxing neutrophils the endothelium via its ligands ICAM-1, lipoprotein(a), and others from CD11bWT and CD11bI332G mice in response to an fMLF stimulus. 6 A MOUSE MODEL OF GENETICALLY ACTIVE CD11b Downloaded from http://www.jimmunol.org/

FIGURE 5. Inflammatory cell recruitment and cytokine secretion in thioglycollate-induced peritonitis. (A and B) Cell counts of neutrophils (A) and macrophages (B) in the peritoneum of CD11bWT and CD11bI332G mice by flow cytometry at 4 and 48 h after sterile thioglycollate injection. Neutrophils were selected as Gr-1hi, whereas macrophages were identified as F4/80+. Bars represent the mean 6 SEM, n = 7–9 mice per group. (C–E) Secretion of inflammatory cytokines by peritoneal macrophages isolated at 4 d after thioglycollate injection. Cells were count normalized and stimulated with LPS for increasing periods of time in vitro. Bars represent the mean 6 SEM, n = 4 mice per group in three independent experiments. N.S., not significant. by guest on September 27, 2021

(39). Both experimental groups had overt hypercholesterolemia at models of inflammation. In vitro studies with KI, neutrophils the end point (Supplemental Fig. 3A, 3B). However, CD11bI332G showed impaired chemotaxis and pronounced uropod elongation mice showed significantly lower atherosclerotic burden than WT in response to an fMLF gradient. Directional motility of neutro- animals, particularly in the thoracic and abdominal aorta (Fig. 6). phils requires a tightly coordinated and self-organizing cell polarity. These results were confirmed in cross-sections of the brachioce- Redistribution of b2 integrins and regulation of their ligand-binding phalic artery (Fig. 7). Lesion size (as percentage of total wall area) affinity along the cell body is one of the mechanisms by which was significantly smaller in CD11bI332G mice compared with their neutrophils regulate polarized adhesion (52). Ultimately, after WT counterparts (Fig. 7B). In addition, the number of infiltrated initial adhesion and cell elongation, the trailing edge of the cell macrophages per section was significantly lower in the former (uropod) needs to be able to detach and retract to promote for- (Fig. 7C). This suggests that CD11b activation has a protective ward protrusion of the leading edge (pseudopod) in the direction role in atherosclerosis in great part because of a reduction of of the chemoattracting agent (52, 53). In migrating neutrophils, macrophage recruitment to atherosclerotic lesions. CD11b/CD18 redistributes to the uropod, where it stabilizes microtubules and regulates myosin L chain–mediated uropod Discussion contractility (54–57). The morphology of chemotaxing neutro- Integrin CD11b/CD18 is essential for a diversity of functions in phils from CD11bI332G KI mice is indicative of impaired uropod immune cells (2, 4). In addition to regulating , mi- detachment as a result of CD11b/CD18 integrin activation. In vivo, gration, and , CD11b/CD18 modulates pro- and anti- both CD11b+ neutrophils and monocytes from CD11bI332G mice inflammatory signaling (23, 49–51). Our recent studies show that showed a significant reduction in inflammatory cell recruitment in CD11b acts as a negative regulator of proinflammatory TLR the setting of peritonitis. These data confirm our previous findings signaling and of autoreactivity (23) and that pharmacologic that CD11b activation can efficiently regulate neutrophil and mono- activation of CD11b reduces inflammation and injury (28–31, 33). cyte infiltration (28, 29). However, our current knowledge of CD11b functions and down- Finally, using a model of chronic vascular inflammation, we stream signaling pathways is largely based on pharmacological show that genetic activation of CD11b results in a significant agents and CD11b KO models, whereas a genetic activation model decrease in aortic and brachiocephalic plaque burden in hyper- was missing. In this study, to our knowledge, we present a novel lipidemic KI mice compared with WT controls. Our results suggest genetic model of constitutive CD11b activation to study the role of that a reduction in macrophage infiltration is one of the mechanisms active integrin CD11b/CD18 in inflammatory injury in vivo. behind CD11b-mediated atheroprotection. Nonetheless, future Our results show that global, constitutive CD11bI332G KI ani- studies will help determine whether other mechanisms contribute to mals developed reduced inflammatory injury in two different this effect, including lower macrophage retention or proliferation in The Journal of Immunology 7 Downloaded from

FIGURE 6. Atherosclerosis development in the aorta of CD11b WT and KI (I332G) mice. (A) Heatmap and percentage of plaque burden by area in http://www.jimmunol.org/ whole aortas from CD11bWT (n = 10) and CD11bI332G hyperlipidemic mice (n = 13). (B–D) Quantification of plaque burden in the aortic arch (B), the thoracic aorta (C), and the abdominal aorta (D) of hyperlipidemic mice. Error bars represent the median and interquartile range, n = 10–13 per group. N.S., not significant. plaques, and/or a regulatory role in macrophage activation and CD11b activation in which to test these findings in vivo. polarization. Previous studies have associated CD11b/CD18 Similarly, CD11b KO mice have provided conflicting data (60, surface upregulation and activation in macrophages with in- 61), likely because of compensatory effects by other integrins, creased macrophage egression from atherosclerotic plaques among other factors. Despite the previously reported athero- by guest on September 27, 2021 (58) and the peritoneum (6). Others have proposed that active protective role of CD11b in females, but not males (61), we CD11b suppresses the alternative activation of macrophages observed a similar trend of decreased plaque burden in both and decreases foam cell formation in vitro through the down- sexes. Unfortunately, our study was not adequately powered regulation of lipid peroxidation mechanisms and scavenger for gender effects. Therefore, this is a question that deserves receptors (49, 59). However, a clear understanding of these further attention. Future investigations on the role of activated mechanisms is lacking because of the use on nonselective CD11b on controlling atherogenesis and/or enhancing ath- integrin activating agents and the absence of a murine model of erosclerosis regression are warranted.

FIGURE 7. Atherosclerosis development in the brachiocephalic trunk of CD11b WT and KI (I332G) mice. (A) Representative cross-sections of advanced atherosclerotic lesions in the brachiocephalic artery of CD11bWT and CD11bI332G hyperlipidemic mice. Galectin-3 (Mac2)+-positive macrophages/foam cells are stained in yellow, SMA+ cells are in green, and nuclei are counter stained in blue (DAPI). Scale bar, 100 mm. (B and C) Quantification of lesion size as percentage of total wall area (B)andMac2+ macrophage counts per section (C) in brachiocephalic artery sections of CD11bWT and CD11bI332G mice. Values represent the average of three positions in the artery (at ∼550, ∼450, and ∼350 mmfromthe aortic arch). Error bars represent the median and interquartile range, n = 7 per group. 8 A MOUSE MODEL OF GENETICALLY ACTIVE CD11b

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Figure 4A – Video 1. Time-lapse video of chemotaxing neutrophils from CD11b wild type

(WT) mice. Time-lapse video of representative peritoneal neutrophils during chemotaxis towards a formyl-Met-Leu-Phe (fMLF) gradient.

Figure 4A – Video 2. Time-lapse video of chemotaxing neutrophils from CD11b knock-in

(I332G) mice. Time-lapse video of representative peritoneal neutrophils during chemotaxis towards a formyl-Met-Leu-Phe (fMLF) gradient.