Semaphorin 3E Regulates the Response of Macrophages to Lipopolysaccharide-Induced Systemic Inflammation

This information is current as Ashfaque Mohammed, Ifeoma Okwor, Lianyu Shan, of September 28, 2021. Chukwunonso Onyilagha, Jude E. Uzonna and Abdelilah S. Gounni J Immunol published online 27 November 2019 http://www.jimmunol.org/content/early/2019/11/26/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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 27, 2019, doi:10.4049/jimmunol.1801514 The Journal of Immunology

Semaphorin 3E Regulates the Response of Macrophages to Lipopolysaccharide-Induced Systemic Inflammation

Ashfaque Mohammed,1 Ifeoma Okwor,1 Lianyu Shan, Chukwunonso Onyilagha, Jude E. Uzonna, and Abdelilah S. Gounni

Semaphorin 3E (Sema3E) is a secreted protein that was initially discovered as a neuronal guidance cue. Recent evidence showed that Sema3E plays an essential role in regulating the activities of various immune cells. However, the exact role of Sema3E in macrophage function, particularly during inflammation, is not fully understood. We studied the impact of Sema3E gene deletion on macrophage function during the LPS-induced acute inflammatory response. We found that Sema3E-deficient (Sema3e2/2) mice were better protected from LPS-induced acute inflammation as exemplified by their superior clinical score and effective temperature control compared with their wild-type littermates. This superior control of inflammatory response in Sema3e2/2

mice was associated with significantly lower phosphorylation of ERK1/2, AKT, STAT3, and NF-kB, and a concomitant reduction Downloaded from in inducible NO synthase expression and production of TNF and IL-6 compared with their Sema3e+/+ littermates. Sema3e2/2 mice also contained significantly higher numbers of activated macrophages compared with their Sema3e+/+ littermates at both baselines and after LPS challenge. In vivo–specific deletion of the Sema3E high-affinity , plexinD1, on macrophages led to the improvement in clinical disease following exposure to a lethal dose of LPS. Collectively, our data show that Sema3E plays an essential role in dampening the early inflammatory response to LPS by regulating macrophage function, suggesting an essential

role of this pathway in macrophage inflammatory response. The Journal of Immunology, 2020, 204: 000–000. http://www.jimmunol.org/

acrophages are critical innate immune cells that process such as Chlamydia pneumoniae, thus playing an essential role in and present Ags to cells of the adaptive immune systems immune evasion (7). In the murine model of sepsis, pentamethox- M (1). They are the primary source of inflammatory/anti- yflavanone was shown to have a protective effect by its ability to inflammatory mediators such as chemokines, , oxygen- polarize macrophages to mostly M1 phenotype, and the adoptive free radicals, and lipid mediators (2), which help in destroying the transfer of M1 macrophages increased survival and improved dis- invading pathogens (1). ease in septic mice (8). Studies looking at the impact of sepsis on Depending on the prevailing microenvironment, macrophages monocytes/macrophages suggest that there is a suppression of the by guest on September 28, 2021 can be classically (M1) or alternatively (M2) activated. The dif- innate immune system following repeated LPS stimulation in vitro ferentiation into M1 or M2 is a well-coordinated process involving and the development of an M2 phenotype characterized by reduced many transcriptional, posttranslational, and signal transduction proinflammatory production (9, 10). Furthermore, blood pathways (3). Although M1 macrophages produce proinflammatory monocytes from septic patients show reduced ability to produce cytokines, M2 macrophages produce anti-inflammatory cytokines proinflammatory cytokines such as TNF, IL-16, and IL-12 (11), (4, 5), and the imbalance in these two populations may lead to suggesting an M2 phenotype. various diseases (6). The impact of M1 and M2 macrophages have Sema3E is a secreted protein initially known as a regulator of been studied in different disease models. M2 macrophages are the axonal growth of neurons (12). However, it has been shown to preferred cell type for the proliferation of intracellular pathogens exert various physiological and pathological roles in immune re- sponse, including cell migration (13), proliferation (14), and cytokine release via the binding to its receptor, plexinD1 (15). Department of Immunology, Max Rady College of Medicine, Rady Faculty of Health + + Sciences, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada Sema3E modulates the migration of CD4 CD8 T cells in the 1 thymus via inhibition of CCL25-CCR9 chemokine signaling, A.M. and I.O. are cofirst authors. which is a critical inducer of thymocyte migration from the cortex ORCID: 0000-0003-1265-6560 (A.S.G.). into the medulla (16), and through dynamic control of b1 Received for publication December 21, 2018. Accepted for publication October 28, 2019. adhesion (16). Sema3E deficiency is associated with a significant reduction in macrophage numbers and their production of proin- This work was supported by the Manitoba Research Chair (to A.S.G.). A.M. was supported by a Children’s Hospital Research Institute of Manitoba studentship. flammatory cytokines (15). Furthermore, M1 macrophages stim- I.O. was supported by the International American Association of Immunologists ulated with oxidized low-density lipoprotein (LDL), LPS, and Fellowship. under hypoxic conditions upregulated Sema3E expression (17), Address correspondence and reprint requests to Dr. Abdelilah S. Gounni, Department and Sema3E was shown to attract and convert monocytes into of Immunology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, 419 Apotex Centre, 750 McDermot Avenue, Winnipeg, MB proinflammatory macrophages via the induction of p53 in adipose R3E 0T5, Canada. E-mail address: [email protected] tissue (15). These observations suggest that Sema3E could impact The online version of this article contains supplemental material. macrophage function and phenotype in various disease conditions. Abbreviations used in this article: BMDM, bone marrow–derived macrophage; iNOS, However, the role of Sema3E–plexinD1 interaction in LPS-induced inducible NO synthase; M1, classically activated; M2, alternatively activated; WT, acute inflammatory conditions and sepsis is unknown. Therefore, in wild-type. this study, we evaluated how Sema3E–plexinD1 pathway modulates Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 macrophage function and phenotype during LPS-induced systemic

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801514 2 Sema3E AND SYSTEMIC INFLAMMATION inflammatory response. Our data show that deficiency of Sema3E (Qiagen, Mississauga, ON, Canada). High-Capacity cDNA Reverse Tran- leads to downregulation of the phosphorylation of key molecules scription Kit (Applied Biosystems) was used to synthesize cDNA from RNA involved in LPS-signaling pathways in macrophages leading to a according to the manufacturer’s instruction. Expression of TNF, IL-6, and housekeeping genes was analyzed by real-time PCR. The primer sequences concomitant reduction in the production of proinflammatory cy- were as follows: TNF forward 59-ATGAGCACAGAAAGCATGATC-3, tokines by these cells. Sema3E deficiency also resulted in reduced reverse 59-TACAGGCTTGTCACTCGAAT-39;IL-6forward59- phagocytosis and Ag-processing ability of peritoneal and bone GAGGATACCACTCCCAACAGA. marrow–derived macrophages (BMDMs), as well as in a higher CC-39, reverse 59-AAGTGCATCATCGTTGTTCATACA-39;GAPDH forward 59-AACTTTGGCATTGTGGAAGG-39, reverse 59-ACACATT- number of M2 macrophages. Specific deletion of the Sema3E GGGGGTAGGAACA-39. high-affinity receptor, plexinD1, on macrophages led to an im- The PCR reaction was run using BioRad CFX with protocol, initial one provement in clinical disease following exposure to a lethal dose cycle denaturation step: 10 min at 95˚C, 40 cycles of PCR (95˚C for 15 s, of LPS. Overall, our data show a previously undescribed role for 60˚C for 35 s and 72˚C for 35 s), one cycle of melting, and one cooling Sema3E/plexinD1 pathway in macrophage functions and re- cycle. Real-time RT-PCR data were quantified as Cq values that were inversely related to the amount of starting template. Results were nor- sponses to LPS and its role in the pathogenesis of LPS-induced malized to an internal control reference gene, as described previously (23). acute inflammatory response and sepsis. Flow cytometry Materials and Methods PL cells were collected from LPS or saline-treated mice, as previously Animal housing and breeding described (22). The PL cells were stained with fluorochrome-labeled Abs 2/2 against the given surface molecules: CD11b (allophycocyanin or PE.cy.7, Sema3e mice (129 P2) used in this study were kindly provided by clone M1/70), F4/80 (FITC, PE, clone BM8), NOS2 (allophycocyanin, Dr. F. Mann Developmental Biology Institute of Marseille, Luminy, clone CNXFT), TLR4 (PE.cy.7, clone HTA125), CD206 (PE.Cy.7, clone Downloaded from Universite De la Mediterranee, Marseille, France (18). PlexinD flox/flox tm1.1Tmj/J MR5D3) for flow cytometry. All Abs were obtained from eBioscience mice (B6;129-Plxnd1 )(19)werekindlyprovidedbyDr.T.M. except for (F4/80 FITC), which was purchased from BioLegend. Samples Jessell, Columbia University/Howard Hughes Medical Institute, (cre/ERT2)Jung/J were acquired with FACSCanto II (BD Biosciences). Data were subse- New York, and crossed to B6.129P2(C)-Cx3cr1-tm2.1 mice quently analyzed with FlowJo software (FlowJo, Ashland, OR). (The Jackson Laboratory) that express Cre recombinase under the con- trol of tamoxifen (TAM)–inducible Cx3cr1 promoter (20) to generate Sandwich ELISA Cx3Cr1Cre+/2ERT2:PlexinD1flox/flox mice. http://www.jimmunol.org/ The Cx3Cr1Cre+ERT2:PlexinD1flox/flox mice were treated with TAM (Sigma) The levels of TNF, IL-6, and CCl2 (MCP-1) in PL and BMDM super- dissolvedinCanolaoil(Fluka)toafinalconcentrationof40mg/ml(20).Mice natant fluids were measured by sandwich ELISA (BioLegend, San Diego, received three doses of TAM (8 mg each) at days 0, 1, and 3 by oral gavage as CA) according to the manufacturer’s suggested protocols. Detection limit previously described (20) and were used for the experiment on day 14 after was 7.8 pg.ml, 7.8, and 62.5 pg/ml for TNF, IL-6, and CCL2 (MCP-1), the initiation of treatment. Cx3Cr1Cre-ERT2:PlexinD1flox/flox mice were respectively. used as controls. All experimental animals were males and were main- tained in a specific pathogen-free environment at the Central Animal In vitro stimulation of cells Care Services, University of Manitoba. All procedures were performed For Western blot analysis, BMDMs from Sema3e+/+ and Sema3e2/2 mice according to the guidelines stipulated by the Canadian Council for Animal were serum deprived for 6 h. After the deprivation period, cells were Care and approved by the University of Manitoba Animal Care and Use washed and replaced with complete RPMI media. Cells were stimulated Committee (protocol no. 13-016/3). with 100 ng/ml LPS and at 0, 1, 5, 15, 30, 60, and 120 min, media by guest on September 28, 2021 Clinical score and body temperature assessment was removed, and the signaling reaction was stopped by adding ice-cold 13 PBS. In another experiment, BMDMs were serum starved and treated Groups of 8–10-wk-old mice were injected (5 or 25 mg/kg) with LPS from with LPS at indicated time, and supernatants and cell lysate were collected Escherichia coli 0111: B4 (Sigma-Aldrich, Mississauga, ON, Canada) or for cytokine and RNA quantification, respectively. saline by i.p. route. Body temperature was taken every 3 h after LPS in- jection. Mice were also observed for movement, body condition, and Lysate preparation and Western blotting alertness. A clinical score of 4 was used as the humane end point, as Cells were lysed with lysis buffer containing M-PER (Thermo Fisher previously described (21). In brief, disease severity was scored as follows: Scientific) and protease inhibitors (Roche). Cell lysates were subjected to 0, no abnormal clinical sign; 1, ruffled fur but lively; 2, ruffled fur, moving centrifugation at 10,000 rpm for 10 min at 4˚C. The supernatant was slowly, hunched, and sick; 3, ruffled fur, squeezed eye, hardly moving, collected and stored at 2800˚C for Western blot analysis. Protein con- down, and very sick; 4, moribund; and 5, dead. A clinical score of 5 was centration was determined using BCA assay (Thermo Fisher Scientific). not permitted by our institutional ethical regulation. Protein lysates (10 mg) were loaded on 10% SDS-PAGE, transferred to Sample collection and preparation PVDF membranes, blocked, and then incubated overnight at 4˚C with Abs specific for phosphorylated ERK1/2 (T202/Y204), AKT (S473), NF-kB At the indicated time points, the animals were euthanized using isofluorane p65 (S536), and STAT3 (Y705). After washing, the blots were incubated gas exposure (3%), and the peritoneal lavage (PL) and blood were collected. with HRP-conjugated secondary Abs, and bands were revealed with Blood was allowed to clot at room temperature and then subjected to ECL reagents. Total anti-ERK1/2, Akt, STAT3, and NF-kBwereusedas centrifugation at 1000 rpm for 10 min at 4˚C to obtain serum, which was loading controls. stored at 280˚C until use. The PL was collected after injecting 3 ml of RPMI 1640 media in the abdominal cavity, as previously described (22). In vivo phagocytosis assay The supernatant was stored in 280˚C for cytokines estimation, and the cell pHrodo E. coli Green fluorescent bioparticles (Thermo Fisher Scientific, pellet was processed for flow cytometry. Burlington, ON) were reconstituted following the manufacturer’s instruction, Generation of BMDMs and 100 ml of particles were injected i.p. into mice. Mice were sacrificed 2 h later and PL cells collected as previously described (22). PL cells were Bone marrow cells were flushed from the femur and tibia of Sema3e+/+ and stained with allophycocyanin-conjugated CD11b (clone M1/70) and PE- 2 2 Sema3e / mice. The cells were differentiated to macrophages in complete conjugated F4/80 (clone BM8) Abs and processed for flow cytometry. RPMI 1640 medium (Life Technologies) containing 10% FBS, penicillin/ streptomycin (100 U/ml each), which was supplemented with 30% L929 In vitro Ag-processing assay

(National Collection of Type Cultures clone 929; American Type Culture 2/2 +/+ Collection) cell culture supernatant as previously described (22). Seven-day BMDM from Sema3e and Sema3e mice were resus- pended in 3 ml of complete RPMI at a concentration of 500 3 105 and RNA isolation and real-time quantitative PCR then pulsed with DQ-OVA (Thermo Fisher Scientific) at a final concen- (primer sequences) tration of 1 mg/ml and incubated for 2 h at 37˚C or 4˚C. After incubation, cells were washed three times with PBS containing 5% FBS and stained PL cells and BMDMs were stimulated with LPS (100 ng/ml). At the end of with anti-mouse CD11b allophycocyanin-conjugated Ab (clone M1/70) the stimulation period, total RNA was extracted using the RNeasy mini kit and acquired with a flow cytometer. The Journal of Immunology 3

Statistical analysis Reduced proinflammatory cytokine levels in BMDM from 2/2 All results were expressed as mean 6 SEM. Potential significant differ- LPS-treated Sema3e mice ences in clinical signs, rectal temperature, and Western blot data were Among innate cells, macrophages are the primary responders to determined through two-way ANOVA. Multiple comparisons between the LPS in vivo and are the major producers of TNF and IL-6 groups were made by Tukey posttest. The significance of other sets of data were determined using one-way ANOVA. Data were analyzed using GraphPad following LPS exposure (24). TNF and IL-6 are eventually Prism 5 (GraphPad Software, La Jolla, CA), and a was set at 0.05. transported to the hypothalamus, where they regulate the temperature threshold (25). Therefore, we tested the hypothesis Results that the difference in hypothermia between Sema3e2/2 and Sema3E deficiency impacts early clinical score, body Sema3e+/+ mice following LPS challenge was due to differ- temperature, and cytokine expression in the LPS-induced acute ences in the production of IL-6 and TNF by macrophages. inflammatory response BMDMs from Sema3e2/2 and Sema3e+/+ mice were stimu- lated with LPS (100 ng/ml) and analyzed at various time points. We determined the impact of the Sema3E deficiency on LPS- 2/2 induced inflammatory response by injecting (i.p.) Sema3e+/+ At2and6hafterstimulation,BMDMsfromSema3e mice , and Sema3e-/- mice with sublethal (5 mg/kg) dose of LPS and showed significantly (p 0.05) lower level of mRNA and 2/2 protein for TNF (Fig. 2A, 2C) and IL-6 (Fig. 2B, 2D) com- monitoring them for clinical score and hypothermia. Sema3e +/+ mice had significantly (p , 0.05) lower change in rectal tem- pared with those from Sema3e mice. Together, our data perature (Fig. 1A) and clinical score (Fig. 1B) relative to the show that the absence of Sema3E leads to a reduced proin- +/+ flammatory cytokine response in vitro and in vivo and may baseline compared with the Sema3e mice. Interestingly, the Downloaded from difference in clinical scores and temperature was transient, as it play a role in subsequent regulation of hypothermia in LPS- disappeared by 12 h after treatment (Fig. 1A, 1B). induced inflammatory response. Next, we evaluated the impact of Sema3E on systemic cytokine production following LPS exposure. The serum level of IL-6, Sema3E deficiency leads to reduced inducible NO synthase TNF, and CCL2 were significantly (p , 0.05–0.01) lower in expression in PL macrophages in response to LPS the Sema3e2/2 mice compared with their wild-type (WT) lit- NO is a critical inflammatory mediator released by macro- http://www.jimmunol.org/ termates at 8 h after LPS injection (Fig. 1C–E). Similarly, phages under the control of inducible NO synthase (iNOS) (26). PL level of CCL2 and IL-6 showed a significant reduction The expression of iNOS in macrophages is directly propor- in Sema3e2/2 compared with Sema3e+/+ mice (Fig. 1F, 1G). tional to the extent of macrophage activation during acute in- Overall, the deficiency in Sema3E led to early but transient flammation (27). iNOS is typically absent in resting cells and protection from LPS-induced hypothermia and clinical dis- is expressed in response to proinflammatory signals, including ease, suggesting that Sema3E may play a role in regulating the LPS and cytokine (TNF and IFN-g) (26) stimulation. Because early systemic inflammatory response. we observed significant differences in the level of proinflammatory by guest on September 28, 2021

FIGURE 1. Initial transient protec- tion from LPS-induced hypothermia in mice lacking Sema3e. Sema3e+/+ and Sema3e2/2 were injected with 5 mg/kg LPS. Mice were observed for rectal temperature (A) and clinical signs (B). Mice were sacrificed 8 h later, and the level of serum IL-6 (C), TNF (D), and CCL2 (E), the level of PL IL-6 (F) and CCL2 (G) were determined by ELISA. Results are expressed in mean 6 SEM (n =6). *p , 0.05, **p , 0.01. 4 Sema3E AND SYSTEMIC INFLAMMATION

FIGURE 2. BMDM Sema3e2/2 mice release lower amount of proinflammatory cytokines in response to LPS. BMDMs from Sema3e+/+ and Sema3e2/2 mice were treated with 100 ng/ml LPS, and the mRNA ex- pression and protein level of TNF (A and C) and IL-6 (B and D) were determined by real-time quantitative PCR and ELISA, respectively. Results are expressed in mean 6 SEM. *p , 0.05, **p , 0.01. Downloaded from http://www.jimmunol.org/ responses both in vitro and in vivo (Figs. 1, 2), we sought to de- after LPS injection (n =6,p , 0.01, Fig. 3). At 24 h, the level of termine if Sema3E also regulated iNOS expression. In agreement iNOS expression on peritoneal macrophages was not different with the increased proinflammatory cytokines levels, the level of between Sema3e2/2 and WT mice and WT controls. Taken to- iNOS expression on peritoneal macrophages was lower in the gether, the data presented in this study suggest that Sema3E Sema3e2/2 mice compared with the Sema3e+/+ mice (n =6,p , regulates the early activation of macrophages under systemic in- 0.01). The difference in iNOS expression was observed up to 12 h flammatory conditions. by guest on September 28, 2021

FIGURE 3. Reduced iNOS expression on peritoneal macrophages in the absence of Sema3E. Sema3e+/+ and Sema3e2/2 mice were injected with a sublethal dose of LPS (5 mg/kg, i.p) and sacrificed at 8 h after LPS injection. Gating strategy (A), iNOS expression (B and C) on PL macrophages were determined by flow cytometry. Results are expressed in mean 6 SEM (n = 6). *p , 0.05, ***p , 0.001. The Journal of Immunology 5

Sema3E regulates the phenotype and function of irrespective of strain (Fig. 4B). However, at 2 and 8 h post LPS peritoneal macrophages injection, the percentage and the absolute number of CD11b+iNOS+ 2/2 Under different microenvironments, two types of macrophages M1 macrophages in the Sema3e mice were significantly lower +/+ can develop: the M1 macrophages that produce mostly proin- compared with the Sema3e mice (Fig. 4B, 4C). Interestingly, the + + flammatory cytokines and express a high level of iNOS, and the M2 percentage of CD11b CD206 cells was significantly higher in the +/+ macrophages that produce mostly anti-inflammatory mediators Sema3e mice at 2 h post LPS treatment (p , 0.05). However, at and express a low level of iNOS (27, 28). Because we found a 8 h post LPS treatment, the percentages and absolute numbers of differential iNOS expression by macrophages from Sema3e2/2 and CD11b+CD206+ cells were significantly higher (p , 0.01– 2 2 Sema3e+/+ mice, we then characterized the phenotype of mac- 0.001) in the Sema3e / mice compared with the Sema3e+/+ rophages in PL fluid from Sema3e2/2 and Sema3e+/+ mice after mice (Fig. 4D, 4E). LPS treatment. Based on the gating strategy shown in Fig. 4A, there Because it is known that phagocytosis is a crucial function was no iNOS expression on PL macrophages from naive mice of macrophages, we wondered whether the absence of Sema3E Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Sema3E regulates PL macrophage phenotype and function. Sema3e+/+ and Sema3e2/2 mice were injected with a sublethal dose of LPS (5 mg/kg) or saline (i.p.) and sacrificed at 2 and 8 h after LPS injection. Gating strategy (A). CD11b+iNOS+ M1 cells (B and C) as well as CD11b+ CD206+ cells (D and E) were determined by flow cytometry. In a separate study, Sema3e2/2 and Sema3e+/+ were injected with pHrodo Green (F). Percentage of E. coli–positive cells (G and H) were determined by flow cytometry. In another experiment, PL macrophages from Sema3e+/+ and Sema3e2/2 mice were cocultured with FITC DQ-OVA at 370˚C for 2 h. FITC-DQ-OVA–positive cells (I) were determined by flow cytometry. Results are expressed in mean 6 SEM (n =6).*p , 0.05, **p , 0.01, ***p , 0.001. 6 Sema3E AND SYSTEMIC INFLAMMATION impacted the phagocytic ability of these cells. Sema3e2/2 and regulate the phosphorylation of proteins involved in the LPS Sema3e+/+ mice were injected with pHrodo Green E. coli parti- signaling pathway in macrophages. cles, and the PL cells were collected 2 h later and analyzed by Deficiency of PlexinD1 on macrophages recapitulates the early flow cytometry for phagocytic ability. Data presented in Fig. 4G 2 2 protection of Sema3e / mice from LPS-induced endotoxemia and 4H show that there was a significant (p , 0.05) decrease in the phagocytic ability of Sema3e2/2 PL macrophages compared Several immune cells contribute to the pathogenesis of LPS- with those from the Sema3e+/+ mice. We next tested whether induced acute inflammatory response in mice. Our data strongly Sema3E regulated the Ag-processing ability of macrophages using suggest that Sema3E regulates early macrophage function and OVA-DQ model. Our data show lower mean fluorescence intensity phenotype and could be the critical cell that mediates the early 2/2 of FITC DQ-OVA in PL macrophages from Sema3e2/2 compared protection observed in Sema3e mice following LPS challenge. with the cells from the Sema3e+/+ mice (Fig. 4I). Overall, these To further validate the role of Sema3E/plexinD1–macrophage axis observations showed that the absence of Sema3E led to a reduced in this early inflammation, we used an inducible Cre-Lox sys- phagocytic and Ag-processing ability of macrophages, suggesting tem to delete the high-affinity receptor for Sema3E, specif- flox/flox a putative role of this pathway in macrophage phenotype and ically, plexinD1, on macrophages. We crossed PlexinD1 function. mice with inducible Cx3cr1 promoter-driven Cre recombinase (Cx3cr1Cre+ERT2) mice (20) resulting in offspring with specific Deficiency of Sema3E leads to impaired MAP kinase and STAT deletion of PlexinD1 on macrophages following treatment with signaling in BMDM after LPS stimulation TAM (Fig. 6A). As expected, this treatment did not alter the

So far, we have shown that Sema3e gene deletion causes down- percentage of T cells, B cells, and NK cells in these mice Downloaded from regulation of proinflammatory cytokine production and iNOS (Supplemental Fig. 1). expression in macrophages following LPS challenge. Because Upon LPS exposure, Cx3cr1Cre+ERT2:PlexinD1flox/flox TAM-treated intracellular signaling proteins like MAPK, STATs, AKT, and NF- mice showed significantly (p , 0.05) lower clinical score (better kß, which are downstream of TLR4 signaling, regulate the pro- outcome) compared with the Cx3cr1Cre+ERT2:PlexinD1flox/flox duction of proinflammatory cytokines in macrophages, we next non–TAM-treated control group (Fig. 6B). In line with this, the + flox/flox

investigated whether the absence of Sema3E in BMDMs had any Cx3cr1 Cre ERT2:PlexinD1 Tam had a lower degree of http://www.jimmunol.org/ impact on these signaling proteins. BMDMs from Sema3e2/2 and hypothermia (Fig. 6C) and survival rate (Fig. 6D) than the Sema3e+/+ were stimulated with LPS (100 ng/ml), and cell lysates Cx3cr1Cre-ERT2:PLexinD1flox/flox mice. Further, the serum levels were collected at 0, 1, 5, 15, 30, 60, and 120 min and assessed by of proinflammatory cytokines such as TNF (Fig. 6E) and Western blot for these key signaling molecules. Our data show that IL-6 (Fig. 6F) were significantly (p , 0.05) lower in the the absence of Sema3E impaired phosphorylation of ERK1/2, Cx3cr1Cre+ERT2:PlexinD1flox/flox TAM-treated mice compared AKT, STAT3, and NF-kB (p65) in BMDMs following LPS with the Cx3cr1Cre+ERT2:PlexinD1flox/flox non–TAM-treated con- stimulation (Fig. 5A–D). These data suggest that Sema3E may trol group. Overall our data suggest that Sema3E/PlexinD1 by guest on September 28, 2021

FIGURE 5. Sema3e2/2 BMDMs showed re- duced phosphorylation of key molecules in LPS signaling pathways. BMDMs from Sema3e+/+ and Sema3e2/2 mice were treated with 100 ng/ml LPS. Cell lysates were collected at different time points, andtotalandphosphorylatedERK1/2(A), AKT (B), STAT3 (C), and NF-kBweremeasuredby Western blotting. The results shown are of three independent experiments and are expressed as mean 6 SEM (n =5).*p , 0.05, **p , 0.01. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Macrophages play an important role in early immune response and outcome of LPS-induced sepsis. Cx3Cr1Cre+ERT2:PlexinD1flox/flox mice were treated with TAM, and the expression of plexinD1 on CD11b+ peritoneal macrophages (A) (dark solid line: Cx3Cr1Cre-ERT2:PlexinD1flox/flox, dotted gray line: Cx3Cr1Cre+ERT2:PlexinD1flox/flox Tam and solid gray line: unstained cells) were determined by flow cytometry. Cx3Cr1Cre-ERT2:PlexinD1flox/flox non- and TAM-treated mice group were exposed to a lethal dose (25 mg/kg), and clinical score (B), rectal temperature (C), and percent survival (D) were determined. Serum levels of TNF (E) and IL-6 (F) were measured by ELISA. Results are expressed in mean 6 SEM (n = 4). *p , 0.05. pathway on macrophages plays a significant role in shaping the Furthermore, Sema3E-deficient macrophages exhibited overall early immune response and subsequent outcome of LPS-induced lower inflammatory response following exposure to LPS that was sepsis. exemplified by reduced phosphorylation of critical molecules that are involved in LPS-signaling pathways such as ERK1/2, AKT, Discussion STAT3, and NF-kB. Selective deletion of the Sema3E receptor In this study, we investigated the impact of Sema3E deletion on plexinD1 on macrophages led to improved clinical disease and macrophage function during LPS-induced inflammation, and we survival in LPS-induced sepsis. Collectively, our data provide show that Sema3E regulates macrophage function both in vivo and novel evidence that Sema3E regulates macrophage function in vitro. In contrast to their WT littermates, Sema3E-deficient mice during LPS-induced inflammation. showed transient protection from LPS-induced hypothermia. We Hypothermia is a common feature of sepsis in both humans and also observed that Sema3E plays an important role in peritoneal mice and is an indicator of severe disease. However, recent re- macrophage phenotype and function; deletion of Sema3E led ports indicated that hypothermia might be protective in a mouse to a higher percentage of M2 peritoneal macrophages as well as model of sepsis (29, 30), and accompanying mild hypothermia their reduced phagocytic ability and lower iNOS expression. was associated with reduced mortality (31). Previous reports 8 Sema3E AND SYSTEMIC INFLAMMATION show that TNF and IL-6 are key cytokines that play a role in endotoxemia and acute lung injury (42), and inflammatory bowel temperature regulation during septic inflammation (32), and disease (43, 44). mortality in sepsis is thought to be due to excessive production Phagocytosis by cells of the innate immune system like mac- of proinflammatory cytokines and chemokines (30). Early on, rophages leads to the clearance of invading microorganisms and following LPS injection, Sema3e-deficient mice displayed mild the subsequent resolution of inflammation. Many studies showed hypothermia, and lower levels of serum proinflammatory cyto- that dysfunction in the phagocytic ability of macrophages and kines such as TNF, IL-6, and MCP1 (CCL2) compared with polymorphonuclear cells leads to impaired pathogen clearance their WT littermates. These data suggest that Sema3E play a (45–47). Impaired FcgR-mediated phagocytosis has been linked pathogenic role in early LPS-induced inflammation by its with Src kinases such as lyn, Hck, and Fgr downstream activa- ability to regulate early proinflammatory cytokine production tion (48). However, more studies are needed to determine the and/or temperature. Further studies are warranted to clarify exact mechanism of how Sema3E regulates phagocytosis. Simi- these events. lar to our finding in the Sema3e2/2 mice, specific deletion of Macrophages are an essential component of innate immunity and plexinD1 from macrophages led to better disease outcome com- are the early controllers of both innate and adaptive immunity (3). pared with the intact mice, thus confirming that the Sema3E/ They limit infections through their ability to phagocytose as well plexinD1 pathway in macrophages may contribute to enhanced as release inflammatory mediators (33). The presence of hypo- disease in early LPS-induced inflammation. In conclusion, the responsive macrophages or total absence of macrophages leads data presented in this manuscript reveal a previously unidentified to a reduced immune response against LPS (34, 35) and can role for Sema3E in the regulation of innate immune system be attributed to the inability of hyporesponsive macrophages during sepsis and suggest that Sema3E could be a strong ther- Downloaded from to produce high amounts of TNF and NO (36). Sema3E at- apeutic target for sepsis and other inflammatory diseases. tracts proinflammatory monocytes/macrophages in adipose tis- sue, where they produce proinflammatory cytokines (IL-6 and Acknowledgments TNF) (16). This finding is in line with our data showing that PL We thank Dr. C. Zhang and Dr. H. Movassagh for helpful discussion. macrophages and BMDMs from Sema3E-deficient mice had reduced iNOS expression and produced low levels of proin- Disclosures http://www.jimmunol.org/ flammatory cytokine (TNF and IL-6) levels. 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