TIPE2 protein serves as a negative regulator of phagocytosis and oxidative burst during infection
TIPE2protein serves as a negative regulator of phagocytosis and oxidative burst during infection
Zhaojun Wang a,b,Svetlana Fayngerts a,Peng Wang a,Honghong Sun a,Derek S.Johnson a,Qingguo Ruan a,Wei Guo c, and Youhai H.Chen a,1
a Department of Pathology and Laboratory Medicine,University of Pennsylvania School of Medicine,Philadelphia,PA19104;
b Department of Microbiology and Parasitology,Shanghai Jiaotong University School of Medicine,Shanghai20025,China;and
c Department of Biology,University of Pennsylvania, Philadelphia,PA19104
Edited by Michael J.Lenardo,National Institute of Allergy and Infectious Diseases,National Institutes of Health,Bethesda,MD,and accepted by the Editorial Board August11,2012(received for review March15,2012)
Phagocytosis and oxidative burst are two major effector arms of innate immunity.Although it is known that both are activated by Toll-like receptors(TLRs)and Rac GTPases,how their strengths are controlled in quiescent and TLR-activated cells is not clear.We re-port here that TIPE2(TNFAIP8L2)serves as a negative regulator of innate immunity by linking TLRs to Rac.TLRs control the expression levels of TIPE2,which in turn dictates the strengths of phagocytosis and oxidative burst by binding to and blocking Rac GTPases.Con-sequently,TIPE2knockout cells have enhanced phagocytic and bac-tericidal activities and TIPE2knockout mice are resistant to bacterial infection.Thus,TIPE2sets the strengths of phagocytosis and oxi-dative burst and may be targeted to effectively control infections. P hagocytosis and oxidative burst(or respiratory burst)are two fundamental effector mechanisms of innate immunity that
work in concert to eliminate infectious microbes(1,2).Phago-
cytosis allows the phagocytes of the immune system(monocytes
and granulocytes)to engulf infectious microbes and to contain
them in a special vacuole called a phagosome.Oxidative burst in turn injects into the vacuole reactive oxygen species(ROS)(e.g., superoxide radical and hydrogen peroxide)that kill the microbes. De?ciency in either of these innate immune mechanisms leads to immune de?ciency and uncontrolled infections(3–6).
Both phagocytosis and oxidative burst are controlled by the Rac
proteins of the Ras small GTPase superfamily(1–4).There are
three mammalian Rac GTPases,which are designated as Rac1,
Rac2,and Rac3.Small GTPases are enzymes that hydrolyze GTP.
They are active when bound to GTP and inactive when bound to
GDP and serve as molecular“on-and-off”switches of signaling
pathways that control a wide variety of cellular processes including
growth,motility,vesicle traf?cking,and death(7).Rac GTPases
control phagocytosis by promoting actin polymerization through
their effector proteins such as p21-activated kinases(PAKs),
WASP family Verprolin homology domain-containing protein
(WAVE),and IQ motif containing GTPase-activating protein-1
(IQGAP1)(1).Rac GTPases also mediate ROS production by
binding and activating the NADPH oxidase complex through the p67(Phox)protein(1).Rac GTPase de?ciency in mice and humans leads to an immune-de?cient syndrome,which is characterized by defective phagocytosis and oxidative burst,recurrent infection,and granulomas(3–6).
Although quiescent phagocytes are capable of phagocytosis
and ROS production,their levels are low.Toll-like receptor
(TLR)activation or microbial infection signi?cantly up-regulates
these innate immune processes(8–11).However,the mechanisms
whereby microbes promote them are not well understood.TIPE2,
or tumor necrosis factor-α–induced protein8(TNFAIP8)-like2
(TNFAIP8L2),is a member of the TNFAIP8family,which is
preferentially expressed in hematopoietic cells(12–18).It is sig-
ni?cantly down-regulated in patients with infectious or autoim-
mune disorders(15,19).The mammalian TNFAIP8family
consists of four members:TNFAIP8,TIPE1,TIPE2,and TIPE3,
whose functions are largely unknown(14,20).We recently generated TIPE2-de?cient mice and discovered that TIPE2plays a crucial role in immune homeostasis(14).We report here that TIPE2controls innate immunity by targeting the Rac GTPases. Results and Discussion
TLR Stimulation and Bacterial Infection Markedly Diminish TIPE2 Expression.TIPE2is constitutively expressed at high levels in my-eloid cells(14).To explore the relationship between TIPE2and innate immunity,we examined its expression in murine bone marrow-derived macrophages(BMDMs)before and after stimu-lation with different TLR ligands/agonists.Upon stimulation with lipopolysaccharide(LPS)(the TLR4ligand),Poly(I:C)(the TLR3 agonist),and Zymosan A(the TLR2ligand),the expression of cytokine genes(IL-6,TNFα,and IFNβ1)signi?cantly increased;by contrast,TIPE2expression was signi?cantly diminished(Fig.1A). Because TLRs play essential roles in pathogen recognition and innate immunity to microbes,we next challenged the BMDMs with the bacteria Escherichia coli and Listeria monocytogenes.Both bacteria signi?cantly augmented cytokine gene expression,but inhibited that of TIPE2(Fig.1B).Similar effects were observed in the murine macrophage cell line RAW264.7(Fig.1C).However, unlike the RAW cell line that exhibited transient TIPE2down-regulation(21),primary BMDMs expressed markedly reduced TIPE2mRNA(Fig.1D)and protein(Fig.1E and F)even16h after LPS or bacterial challenge.These results indicate that TIPE2 may regulate the early phase of innate immune responses.
TIPE2Binds to Rac GTPases Through Their C-Terminal CAAX Motif.To identify TIPE2-interacting proteins,we performed large-scale coimmunoprecipitation and mass spectrometry screenings using RAW264.7cells.Among the peptides identi?ed,three were shared by the small GTPases Rac1and Rac2,members of the Rho family.Both Rac1and Rac2are expressed in neutrophils and macrophages.They share92%identity in primary sequences and perform crucial functions in innate immune responses(2). To determine whether TIPE2indeed interacts with the Rac GTPases in mammalian cells,we undertook four complementary approaches.First,we expressed Flag-tagged TIPE2and Myc-tagged Rac1or Rac2in293T cells and,by coimmunoprecipita-tion(co-IP)analyses,we found that TIPE2interacted with both Rac1and Rac2(Fig.2A).Second,we determined whether en-dogenous TIPE2interacts with endogenous Rac.Murine RAW 264.7macrophage extracts were used to immunoprecipitate Rac-
Author contributions:Z.W.and Y.H.C.designed research;Z.W.,P.W.,H.S.,D.S.J.,and Q.R. performed research;S.F.and W.G.contributed new reagents/analytic tools;Z.W.and Y.H.C.analyzed data;and Z.W.and Y.H.C.wrote the paper.
The authors declare no con?ict of interest.
This article is a PNAS Direct Submission.M.J.L.is a guest editor invited by the Editorial Board.
1To whom correspondence should be addressed.E-mail:yhc@https://www.wendangku.net/doc/666862779.html,.
This article contains supporting information online at https://www.wendangku.net/doc/666862779.html,/lookup/suppl/doi:10. 1073/pnas.1204525109/-/DCSupplemental.
https://www.wendangku.net/doc/666862779.html,/cgi/doi/10.1073/pnas.1204525109PNAS|September18,2012|vol.109|no.38|15413–15418I M M U N O L O G Y
binding proteins with anti-Rac1/2/3polyclonal antibodies.Upon blotting with anti-TIPE2antibody,a strong TIPE2signal was detected in the precipitates (Fig.2B ),indicating that TIPE2is
constitutively associated with Rac proteins in nonstimulated immune cells.By contrast,in the same RAW cells,we did not detect any interaction between endogenous TIPE2and several other GTPases,including Cdc42,RhoA,RalA,and HRas,sug-gesting that the TIPE2?Rac interaction is speci ?c.Third,we tested whether TIPE2and Rac might directly interact with each other.TIPE2and Rac1proteins were synthesized separately,using an in vitro transcription –translation system.After mixing them together,we found that TIPE2physically associated with Rac1,indicating that the two proteins might directly bind to each other (Fig.2C and D ).Finally,using both GDP and GTP forms of Rac1(22,23),we showed that TIPE2?Rac interaction was not affected by the Rac1-activating status;i.e.,TIPE2binds to both GDP and GTP forms of Rac1(Fig.2E ).
To map the region within Rac that is responsible for TIPE2interaction,we generated full-length Rac1(amino acids 1–192)and a series of deletion constructs in frame with an HA tag (Fig.3A ).Among ?ve deletion Rac1mutants we generated,two (with amino acids 48–123deletion and amino acids 136–161deletion)were unstable in cells and could not be further studied.By contrast,Rac1mutants without the N terminus (amino acids 1–47),the in-sert region (amino acids 124–135),or the C terminus (amino acids 162–192)were stable and therefore were used in this study.By co-IP,we found that the C terminus,but not the N terminus or the insert region,was required for TIPE2interaction (Fig.3B ).
The C terminus of Rac contains a polybasic region (PBR)and a CAAX motif.To determine which of these elements is involved in TIPE2binding,we made two additional Rac1mutants,one without the PBR and one without the CAAX motif.By co-IP,we found that the CAAX motif,but not the PBR,was required for TIPE2interaction (Fig.3C ).The cysteine residue,Cys-189,in the
C-terminal CAAX motif is crucial for the posttranslational modi-?cation and function of Rac (23).Using a point mutant form of Rac1,Rac1C189S,in which Cys-189is replaced by serine,we found that TIPE2binding required Cys-189(Fig.3D ).
TIPE2Inhibits Rac Membrane Translocation,Rac Activation,and Downstream Rac Signaling.The hydrophobic C-terminal region of
Rac is responsible for targeting and anchoring Rac to the plasma membrane.Our ?nding that TIPE2binds to the C terminus of Rac suggests that it may regulate membrane translocation of Rac.To test this possibility,we transiently expressed increasing amounts of TIPE2in 293T cells.At 8h after transfection,TIPE2did not in-duce a signi ?cant amount of cell death (Fig.S1),but reduced the membrane-bound,not the cytosolic,endogenous Rac in a dose-dependent manner (Fig.3E ).The TIPE2N-terminal lysine or ar-ginine residues,Lys-15,Lys-16,and Arg-24,are important for TIPE2–Rac1interaction because replacing them with glutamine or alanine markedly reduced the Rac1binding (Fig.3F ).By contrast,Lys-20mutation did not affect the binding.As a consequence,the TIPE2K15/16Q and R24A mutants were not as effective as wild-type TIPE2in inhibiting Rac membrane translocation (Fig.3G ).As stated above,LPS markedly reduced TIPE2levels in macro-phages.This reduction was associated with increased Rac1mem-brane translocation in LPS-treated cells (Fig.3H ),indicating that TIPE2regulates Rac membrane translocation.
Because plasma membrane translocation of small GTPases is required for their activation,these results indicate that TIPE2may regulate Rac activation.To test this hypothesis,we undertook three complementary approaches.First,using confocal microscopy and an antibody that recognized GTP-Rac but not GDP-Rac,we ex-amined the levels of membrane-bound GTP-Rac in 293T cells that did or did not receive TIPE2transfection.We found that 293T cells exhibited prominent endogenous GTP-Rac staining on the plasma membrane,which was signi ?cantly blocked in cells transfected with
Fig.1.TLR stimulation and bacterial infection markedly diminish TIPE2expression.(A )Wild-type (WT)bone marrow-derived macrophages (BMDMs)were stimulated with lipopolysaccharide (100ng/mL),Poly(I:C)(10μg/mL),and Zymosan A (100μg/mL)for 2h.TIPE2,IL-6,TNF α,and IFN β1mRNA levels were de-termined by real-time PCR.(B and C )WT BMDMs (B )or RAW 264.7cells (C )were infected with Escherichia coli or Listeria monocytogenes at a multiplicity of infection (MOI)of 10for 2h.The mRNA levels of indicated genes were determined by real-time PCR.(D and E )BMDMs from WT mice were treated with LPS for the indicated times.TIPE2mRNA (D )and protein (E )levels were analyzed by real-time PCR and Western blot,respectively.BMDMs from TIPE2-de ?cient mice (Tipe2?/?)were used as a control.(F )WT BMDMs were infected with or without the indicated bacteria for 8or 16h.TIPE2protein levels were analyzed by Western blot.Statistics were performed on pooled data from three independent experiments.Error bars represent the SDs of the means.*P <0.05,**P <0.01.
15414|https://www.wendangku.net/doc/666862779.html,/cgi/doi/10.1073/pnas.1204525109
Wang et al.
TIPE2,but not in cells transfected with a control GFP plasmid (Fig.4A ).Second,using a GST pull-down assay that speci ?cally recog-nized active GTP-bound Rac,we tested the TIPE2effect in 293T cells that did or did not express a constitutive active form of HRas (HRas12V).We found that TIPE2transfection decreased the Rac-GTP levels in both cell types (Fig.4B ).Third,we tested the effect of TIPE2de ?ciency on Rac activation in macrophages.We found that resting TIPE2-de ?cient murine macrophages exhibited an increase in active Rac levels over wild-type controls.The difference was more dramatic upon the activation of the Rac pathway with ?-bronectin (Fig.4C ).The total Rac levels were not affected by TIPE2overexpression or de ?ciency,indicating that TIPE2may not regulate Rac activity through protein expression or degradation.A number of studies have demonstrated that Rac can activate both the c-Jun N-terminal kinase (JNK)and the PAK pathways (24).In 293T cells,we found that constitutively active Rac expression
did induce JNK and PAK activation as re ?ected by increased phosphorylation;this effect was blocked by TIPE2in a dose-dependent manner (Fig.4D ).Active Rac1can induce the assembly of a ?lamentous (F)-actin structure through PAK-dependent and -independent signals (24).Because polymerization of F-actin can be induced by LPS,we next examined the effect of TIPE2de ?ciency on LPS-induced F-actin polymerization.We found that in TIPE2-de ?cient BMDMs,the F-actin polymerization rate was signi ?cantly enhanced compared with that in wild-type cells (Fig.4E ).A similar phenotype was observed in TIPE2-de ?cient splenocytes (21).
TIPE2Inhibits the Basal Level of Phagocytosis Through Rac,and TIPE2Down-Regulation Contributes to TLR-Induced Augmentation of Phagocytosis.Rac proteins are key molecular switches of phagocy-
tosis (2).To determine the roles of TIPE2in phagocytosis,we used both TIPE2loss-of-function and gain-of-function approaches.Thus,macrophages were ?rst generated from WT and Tipe2-de ?cient murine bone marrow.They showed no differences in surface
marker
Fig.2.TIPE2interacts with Rac GTPase.(A )293T cells were transfected with pRK5vector or expression plasmids for Flag-tagged TIPE2and Myc-tagged Rac1or Rac2as indicated.Eighteen hours later,cell lysates were prepared and immunoprecipitated with anti-Flag,anti-Myc,or control Ig.The precipitates and lysates were subjected to Western blotting with antibodies for the in-dicated antigens.(B )Total cell lysates of RAW 264.7cells were immunopre-cipitated with rabbit polyclonal anti-Rac1/2/3or control rabbit Ig.The immunoprecipitates and cell lysates were then blotted with anti-TIPE2or anti-Rac1/2/3.(C )[35S]Methionine-labeled TIPE2and unlabeled Myc-tagged Rac1were synthesized in vitro,using the TNT transcription –translation system.The mixture was immunoprecipitated with anti-Myc or Ig control and then ana-lyzed by SDS/PAGE and Western blotting.(Upper )Autoradiograph of the immunoprecipitates on SDS/PAGE;(Lower )anti-Myc immunoblotting result.(D )In vitro synthesized [35S]methionine-labeled Rac1and Flag-tagged TIPE2mixture were immunoprecipitated with anti-Flag or control Ig and analyzed as in C .(E )293T cells were transiently transfected with Flag-TIPE2,Myc-tag-ged Rac1-17N,or Rac1-61L.Eighteen hours later,lysates of Myc-Rac1-17N –and Myc-Rac1-61L –transfected cells were loaded with GDP and GTP,re-spectively,and mixed with lysates of Flag-TIPE2–transfected cells.After im-munoprecipitation with anti-Flag or control Ig,the samples were subjected to Western blotting with antibodies for the indicated antigens.The experiments were repeated at least three times with similar
results.
Fig.3.TIPE2binding to Rac requires the C-terminal CAAX motif,and TIPE2inhibits Rac membrane translocation.(A )Schematic diagram of the Rac1protein.(B –D )Lysates of 293T cells transiently transfected with HA-Rac1constructs and Flag-TIPE2construct were immunoprecipitated with anti-Flag and subjected to Western blotting.(E )293T cells were transiently trans-fected with increasing amounts of TIPE2plasmids for 8h.Cell lysates were separated into membrane and cytosolic fractions.Equal amounts of cytosolic and membrane materials (3μg)were separated by SDS/PAGE and immu-noblotted for integrin β1,β-actin,and Rac.(F )Lysates of 293T cells tran-siently transfected with or without HA-Rac1construct and Flag-TIPE2constructs were immunoprecipitated and subjected to Western blotting.(G )293T cells were transiently transfected with or without the indicated TIPE2plasmids.Equal amounts of cytoplasmic and membrane protein fractiona-tions were subjected to Western blotting.(H )WT BMDMs were treated with LPS for up to 16h.Cytosolic and membrane protein fractionations were subjected to Western blotting.The experiments were repeated at least three times with similar results.
Wang et al.
PNAS |September 18,2012|vol.109|no.38|15415
I M M U N O L O G Y
expression (Fig.S2).They were then fed with apoptotic thymocytes from GFP transgenic mice.Thirty minutes later,the engulfment of GFP-positive cells was measured by ?ow cytometry.Remarkably,TIPE2de ?ciency increased the basal phagocytosis rate by ap-proximately twofold (Fig.5A ).Moreover,the mean ?uorescence intensity of Tipe2-de ?cient GFP-positive cells was also signi ?cantly increased.Addition of the actin polymerization inhibitor,cyto-chalasin B,markedly reduced phagocytosis in both WT and Tipe2?/?groups,indicating that actin remodeling is required and that TIPE2may regulate internalization rather than adherence (Fig.5A ).The increased phagocytic activity of Tipe2-de ?cient cells was not re-stricted to apoptotic cells,because Tipe2-de ?cient macrophages were signi ?cantly more active in engul ?ng ?uorescently labeled 2-μm beads (Fig.5B )and live or dead bacteria (Fig.5C ).The increase in phagocytosis of Tipe2-de ?cient cells was not affected by the culture media used [with or without 10%(vol/vol)fetal bovine sera or freshly prepared murine sera that contain different amounts of Ig and/or complements],indicating that the TIPE2effect on phagocytosis may
not be dependent on Fc or complement receptor use (Fig.S3).In contrast to phagocytosis,endocytosis of ?uorescence-labeled dextran was not affected by TIPE2de ?ciency (Fig.S4).
To directly test the effect of TIPE2on phagocytosis,we over-expressed TIPE2in WT and Tipe2?/?macrophages.In Tipe2?/?cells,ectopic TIPE2reduced phagocytosis by more than fourfold,whereas in WT cells that constitutively expressed high levels
of
Fig.4.TIPE2inhibits the activation of Rac and its effectors.(A )Confocal microscopy of 293T cells expressing TIPE2-GFP or GFP alone.To detect active Rac,cells were stained with anti –GTP-Rac and Alexa Fluor 555-labeled goat anti-mouse Ig (red).Images are representative of three independent experi-ments.(Right )The percentages of GTP-Rac –positive cells in total GFP positive cells.A total of 70cells were counted for each condition (B )293T cells were transiently transfected with TIPE2and/or HRas12V as indicated.Cell lysates were subjected to pull-down,using PAK-GST protein beads.Rac from the pull-down and total Rac in the lysates were detected by Western blot.(C )WT and Tipe2?/?BMDMs were plated on uncoated or ?bronectin-coated dishes for 15min at 37°C.Active and total Rac levels in the cells were determined as in B .(D )293T cells were transfected with or without Myc-Rac161L (2μg/6-cm dish)and increasing amounts of Flag-TIPE2constructs (1–2μg/6-cm dish)for 8h,and the levels of phospho(p)-JNK,total JNK,phospho(p)-PAK,total PAK,Myc,and Flag were determined by Western blot.Bar graphs show relative p-JNK and p-PAK levels as determined by densitometry.(E )WT and Tipe2?/?BMDMs were treated with LPS (100ng/mL)for the indicated times and F-actin level was measured as described in Methods .The “%increase in F-actin ”=[(F-actin in LPS-treated cells ?F-actin in untreated cells)/F-actin in untreated cells]×100.Results are means ±SEM.Statistics were performed on pooled data from three independent experiments.*P <0.05,**P <
0.01.
Fig.5.TIPE2controls the strength of phagocytosis through Rac.(A )BMDMs from WT and Tipe2?/?mice were incubated with or without apoptotic GFP thymocytes (GFP-Thy)for 30min at a ratio of 1:5.One group of BMDMs was also pretreated with 5μM Cytochalasin B (Cyto B)for 5min as indicated.After incubation,cells were ?xed,stained with an antibody to the macro-phage marker F4/80,and analyzed by ?ow cytometry.Only cells in the macrophage gates set based on the FSC/SSC values are shown.The number in each contour plot represents the percentage of GFP +F4/80+cells (macro-phages with ingested thymocytes)in the gated area.Solid bar,WT BMDMs;open bar,Tipe2?/?BMDMs.Phagocytic index =the percentage of gated ?uorescent macrophages ×mean ?uorescence intensity of gated macro-phages.(B and C )WT (solid bars)and Tipe2?/?BMDMs (open bars)were fed with 2-μm ?uorescent beads at the indicated ratio with or without Cyto B (B )or CFSE-labeled live or heat-killed L.monocytogenes (Lm)at a ratio of 1:10(C )and incubated for 20min.Phagocytic indexes were determined as in A .(D )WT and Tipe2?/?BMDMs were infected with retroviruses that carried either nerve growth factor receptor (NGFR)or NGFP plus TIPE2cDNAs (TIPE2)as described in Methods .Phagocytic indexes were determined as in A .Only data from NGFR-positive macrophages are shown.(E )WT (solid bars)and Tipe2?/?BMDMs (open bars)were infected with retroviruses that carried NGFR or NGFP plus wild-type Rac1(Rac1-WT),constitutively active Rac1(Rac1-61L),or dominant-negative Rac1(Rac1-17N)cDNAs.Phagocytic in-dexes were determined as in A .Only data from NGFR-positive macrophages are shown.(F )WT (solid bars)and Tipe2?/?BMDMs (open bars)were pre-treated with Rac1inhibitor NSC24766at the indicated concentrations for 24h and incubated with GFP-Thy for 30min at a ratio of 1:5.Phagocytosis index was determined as in A .(G )WT (solid bars)and Tipe2?/?BMDMs (open bars)were treated with LPS (100ng/mL)for the indicated times.After the treatment,cells were subjected to the phagocytosis assay as in A .The fold increase in the phagocytic index is shown,with the value of the untreated cells set as 1.Results shown are means ±SEM.Statistics were performed on pooled data from three independent experiments.**P <0.01.
15416|https://www.wendangku.net/doc/666862779.html,/cgi/doi/10.1073/pnas.1204525109
Wang et al.
TIPE2it had a much smaller effect (Fig.5D and Fig.S5).Im-portantly,ectopic TIPE2completely eliminated the augmented phagocytic activity of Tipe2?/?cells compared with WT cells.To determine whether TIPE2regulates phagocytosis through Rac,we manipulated the Rac activity using Rac mutants and chemical blockers.Expression of a constitutively active Rac (61L)dramatically increased phagocytosis in both WT and Tipe2?/?macrophages and abolished the difference between them.On the other hand,dominant-negative Rac1(17N)signi ?cantly reduced phagocytosis in Tipe2?/?cells and eliminated the difference be-tween WT and Tipe2?/?cells (Fig.5E and Fig.S6).Similarly,the Rac1antagonist NSC23766depressed the ability of macrophages to uptake apoptotic cells and reduced the difference between WT and Tipe2?/?groups in a dose-dependent manner (Fig.5F ).
The basal level of phagocytosis is signi ?cantly increased upon TLR activation (8,9).Because TLRs down-regulate TIPE2ex-pression,we asked whether TLRs augment phagocytosis through TIPE2.WT and Tipe2?/?macrophages were therefore stimu-lated with LPS and their phagocytic ability was tested.As reported,LPS stimulation enhanced phagocytosis of WT cells in a time-dependent manner.However,this enhancement was sig-ni ?cantly reduced in Tipe2?/?cells (Fig.5G and Fig.S7).These results indicate that TIPE2down-regulation contributes to the TLR-induced increase in phagocytosis.
TIPE2Inhibits Oxidative Burst,and Its Down-Regulation Contributes to TLR-Induced Augmentation of Bactericidal Activity.Because Rac
proteins are crucial for assembling the NADPH complex and for producing ROS,we next investigated whether TIPE2regulates ROS production and bacterial killing.We found that Tipe2?/?macrophages exhibited enhanced bacterial clearance within 2h of bacterial infection.They killed L.monocytogenes and E.coli more ef ?ciently than WT macrophages.Similar differences were observed between Tipe2?/?and WT neutrophils (Fig.6A ).
We then assessed ROS production in bone marrow neutrophils of WT and Tipe2?/?mice,using a horseradish peroxidase (HRP)-de-pendent chemiluminescence assay.N -formyl-methionyl-leucyl-phe-nylalanine (fMLP),a bacterial peptide,and L.monocytogenes were used to induce ROS https://www.wendangku.net/doc/666862779.html,pared with WT cells,Tipe2?/?neutrophils exhibited signi ?cantly enhanced ROS production
after
Fig. 6.TIPE2controls the oxidative burst and bactericidal activity.(A )BMDMs and neutrophils from WT and Tipe2?/?mice were infected with wild-type (hly +)or hly mutant (hly ?)L.monocytogenes (Lm),E.coli ,or S.aureus ,at a ratio of 1:1for 20min.After washing,cells were incubated for an ad-ditional 2h.Bacterial numbers in the cells were determined by the colony-forming unit assay both before and after the 2-h incubation period.(B )Neutrophils from WT (solid squares)and Tipe2?/?mice (open squares)were stimulated with 5μM fMLP or L.monocytogenes (Lm)at MOI of 10.Total ROS responses were measured by the chemiluminescence assay as described in Methods .Data shown are pro ?les of the kinetics of ROS production,from one representative experiment of four.RU,relative units.The differences at the peak light production time points between the two groups are statisti-cally signi ?cant as determined by Student ’s t test (P <0.01).(C )Neutrophils from WT (solid bars)and Tipe2?/?mice (open bars)were treated with GM-CSF (10ng/mL)and fMLP (1μM)or infected with L.monocytogenes at a ratio of 1:5.Intracellular ROS levels were measured by DCFDA (DCFH)staining.(D )BMDMs from WT (solid bars)and Tipe2?/?mice (open bars)were treated with or without LPS (100ng/mL)for 24h.After the treatment,bactericidal activity was measured as in A .Results shown are means ±SEM and are pooled from three to six independent experiments.*P <0.05,**P <
0.01.
Fig.7.Tipe2?/?mice are resistant to L.monocytogenes and S.aureus in-fection.(A )Survival rate of WT and Tipe2?/?mice (n =7)infected with 2×105cfus of L.monocytogenes (Lm)via the tail vein.The difference between the two groups is statistically signi ?cant (P <0.01).(B )The numbers of bacteria in the spleen and liver of individual mice were determined on day 3by colony-forming unit assay.(C )Levels of serum aspartate transaminase (AST)and alanine transaminase (ALT)on day 3.(D )One day after the in-fection,serum cytokines were determined by ELISA.Error bars represent the SDs of the means.(E )Survival rate of WT and Tipe2?/?mice (n =9)infected with S.aureus (6×107cfus/mouse)via the tail vein.(F )Bacterial titers were assayed at 24h after i.v.inoculation of S.aureus (2×107cfus/mouse).Sta-tistics were performed on pooled data from two independent experiments.*P <0.05,**P <0.01.
Wang et al.
PNAS |September 18,2012|vol.109|no.38|15417
I M M U N O L O G Y
stimulation(Fig.6B).Intracellular ROS levels were then mea-sured in the dichloro?uorescin diacetate(DCFDA)assay(Fig. 6C).GM-CSF priming and fMLP stimulation triggered signi?-cantly more ROS production in Tipe2?/?cells than in WT cells. Similarly,when cells were challenged with Listeria,more ROS were detected in Tipe2?/?neutrophils than in WT neutrophils. Intracellular ROS production was also tested by luminol-de-pendent luminescence assay in the absence of HRP.Again,sig-ni?cant differences in the ROS production between Tipe2?/?and WT neutrophils were observed(Fig.S8).
TLR activation increases the bactericidal activity of macrophages (25,26).We next investigated whether TIPE2down-regulation contributes to LPS-induced enhancement of bacterial killing.We found that LPS stimulation signi?cantly enhanced the killing ability of WT macrophages.By contrast,the LPS effect was markedly re-duced in Tipe2?/?cells,indicating that TIPE2is involved in the LPS-induced regulation of bacterial killing(Fig.6D).
TIPE2-De?cient Mice Are Resistant to Bacterial Infection.In vitro, down-regulation of TIPE2is associated with increased phagocy-tosis and bacterial killing.To assess the in vivo relevance of these ?ndings,we infected Tipe2?/?and WT mice with a lethal dose of L. monocytogenes(Fig.7A).More than80%of WT mice succumbed within7d after infection,whereas all Tipe2?/?mice survived(P< 0.01).The increased mortality of WT mice was associated with enhanced bacteria growth,tissue damage,and cytokine pro-duction.At day3postinfection,the Listeria titer in the liver and spleen of WT mice was signi?cantly higher than that in Tipe2?/?mice(Fig.7B).The blood alanine transaminase(ALT)and aspartate transaminase(AST)levels are indicators of hepatic in-jury.After Listeria infection,the blood ALT and AST levels were signi?cantly higher in WT than in Tipe2?/?mice(Fig.7C).Fewer serum in?ammatory cytokines were detected in Tipe2?/?mice compared with WT mice(Fig.7D).Additionally,we also chal-lenged the Tipe2?/?and WT mice with Staphylococcus aureus. Consistent with the Listeria experiment,Tipe2?/?mice survived for a longer time and carried signi?cantly fewer bacteria in their liver and spleen(Fig.7E and F).Taken together,these results dem-onstrate that TIPE2inhibits immunity to bacteria.
Methods
Mice.C57BL/6mice that carry a Tipe2gene null mutation were generated by backcrossing Tipe2?/?129mice to C57BL/6(B6)mice for12generations.
Methods.The following methods are described in SI Methods:plasmid con-structs;cell culture and transfection;macrophage generation and neutrophil isolation;gene transfer in primary macrophages;microbial strains and in-fection;real-time quantitative PCR;protein extraction,cell subcellular frac-tionation,and immunoblotting;cell lysate preparation,protein in vitro translation,and immunoprecipitation;loading of Rac1with GDP and GTP; immuno?uorescence and confocal microscopy;PBD pull-down assay;F-actin determination;phagocytosis assay;determination of bactericidal activity; detection of ROS;ELISA;and statistical analyses.
ACKNOWLEDGMENTS.The authors thank Drs.H.Shen and Y.Paterson (University of Pennsylvania)for providing L.monocytogenes and Dr.Y.Fan (Shandong University)for rabbit anti-TIPE2.This work was supported by National Institutes of Health Grants AI-077533,AI-050059,and GM-085112.
1.Diebold BA,Bokoch GM(2005)Rho GTPases and the control of the oxidative burst in
polymorphonuclear leukocytes.Curr Top Microbiol Immunol291:91–111.
2.Niedergang F,Chavrier P(2005)Regulation of phagocytosis by Rho GTPases.Curr Top
Microbiol Immunol291:43–60.
3.Ambruso DR,et al.(2000)Human neutrophil immunode?ciency syndrome is associ-
ated with an inhibitory Rac2mutation.Proc Natl Acad Sci USA97:4654–4659.
4.Roberts AW,et al.(1999)De?ciency of the hematopoietic cell-speci?c Rho family
GTPase Rac2is characterized by abnormalities in neutrophil function and host de-fense.Immunity10:183–196.
5.Heyworth PG,Cross AR,Curnutte JT(2003)Chronic granulomatous disease.Curr Opin
Immunol15:578–584.
6.Assari T(2006)Chronic granulomatous disease;fundamental stages in our un-
derstanding of CGD.Med Immunol5:4.
7.Colicelli J(2004)Human RAS superfamily proteins and related GTPases.Sci STKE2004:
RE13.
8.Blander JM,Medzhitov R(2004)Regulation of phagosome maturation by signals from
toll-like receptors.Science304:1014–1018.
9.Takeda K,Akira S(2005)Toll-like receptors in innate immunity.Int Immunol17:1–14.
10.Kong L,Ge BX(2008)MyD88-independent activation of a novel actin-Cdc42/Rac
pathway is required for Toll-like receptor-stimulated phagocytosis.Cell Res18: 745–755.
11.Tricker E,Cheng G(2008)With a little help from my friends:Modulation of phago-
cytosis through TLR activation.Cell Res18:711–712.
12.Ahn SH,et al.(2010)Two genes on A/J chromosome18are associated with suscep-
tibility to Staphylococcus aureus infection by combined microarray and QTL analyses.
PLoS Pathog6:e1001088.
https://www.wendangku.net/doc/666862779.html,libertéB,et al.(2010)TNFAIP8:A new effector for Galpha(i)coupling to reduce cell
death and induce cell transformation.J Cell Physiol225:865–874.
14.Sun H,et al.(2008)TIPE2,a negative regulator of innate and adaptive immunity that
maintains immune homeostasis.Cell133:415–426.15.Li D,et al.(2009)Down-regulation of TIPE2mRNA expression in peripheral blood
mononuclear cells from patients with systemic lupus erythematosus.Clin Immunol 133:422–427.
16.Zhang X,et al.(2009)Crystal structure of TIPE2provides insights into immune ho-
meostasis.Nat Struct Mol Biol16:89–90.
17.Zhang G,et al.(2010)Tissue-speci?c expression of TIPE2provides insights into its
function.Mol Immunol47:2435–2442.
18.Zhang S,et al.(2010)Expression and regulation of a novel identi?ed TNFAIP8family
is associated with diabetic nephropathy.Biochim Biophys Acta1802:1078–1086. 19.Xi W,et al.(2011)Roles of TIPE2in hepatitis B virus-induced hepatic in?ammation in
humans and mice.Mol Immunol48:1203–1208.
20.Zhang C,et al.(2006)Role of SCC-S2in experimental metastasis and modulation of
VEGFR-2,MMP-1,and MMP-9expression.Mol Ther13:947–955.
21.Gus-Brautbar Y,et al.(2012)The anti-in?ammatory TIPE2is an inhibitor of the on-
cogenic Ras.Mol Cell45:610–618.
22.Hirshberg M,Stockley RW,Dodson G,Webb MR(1997)The crystal structure of human
rac1,a member of the rho-family complexed with a GTP analogue.Nat Struct Biol4: 147–152.
23.Kreck ML,Uhlinger DJ,Tyagi SR,Inge KL,Lambeth JD(1994)Participation of the small
molecular weight GTP-binding protein Rac1in cell-free activation and assembly of the respiratory burst oxidase.Inhibition by a carboxyl-terminal Rac peptide.J Biol Chem 269:4161–4168.
24.Kreis P,Barnier JV(2009)PAK signalling in neuronal physiology.Cell Signal21:
384–393.
25.West AP,et al.(2011)TLR signalling augments macrophage bactericidal activity
through mitochondrial ROS.Nature472:476–480.
26.Henneke P,et al.(2002)Cellular activation,phagocytosis,and bactericidal activity
against group B streptococcus involve parallel myeloid differentiation factor88-de-pendent and independent signaling pathways.J Immunol169:3970–3977.
15418|https://www.wendangku.net/doc/666862779.html,/cgi/doi/10.1073/pnas.1204525109Wang et al.