Interleukin 10 suppresses lysosome-mediated killing ofBrucella abortus in cultured macrophagesReceived for publication, July 5, 2017, and in revised form, November 6, 2017 Published, Papers in Press, January 4, 2018, DOI 10.1074/jbc.M117.805556

Huynh Tan Hop‡, Alisha Wehdnesday Bernardo Reyes‡, Tran Xuan Ngoc Huy‡, Lauren Togonon Arayan‡,WonGi Min‡, Hu Jang Lee‡, Man Hee Rhee§, Hong Hee Chang¶, and Suk Kim‡¶1

From the ‡Institute of Animal Medicine, College of Veterinary Medicine, and ¶Institute of Agriculture and Life Science, GyeongsangNational University, Jinju 52828, Republic of Korea and §College of Veterinary Medicine, Kyungpook National University,Daegu 41566, Republic of Korea

Edited by Luke O’Neill

Brucella abortus is a Gram-negative zoonotic pathogen forwhich there is no 100% effective vaccine. Phagosomes inB. abortus–infected cells fail to mature, allowing the pathogento survive and proliferate. Interleukin 10 (IL10) promotesB. abortus persistence in macrophages by mechanisms that arenot fully understood. In this study, we investigated the regula-tory role of IL10 in the immune response to B. abortus infection.B. abortus–infected macrophages were treated with either IL10siRNA or recombinant IL10 (rIL10), and the expression ofphagolysosome- or inflammation-related genes was evaluatedby qRT-PCR and Western blotting. Phagolysosome fusion wasmonitored by fluorescence microscopy. We found that the syn-thesis of several membrane-trafficking regulators and lysosomalenzymes was suppressed by IL10 during infection, resulting in asignificant increase in the recruitment of hydrolytic enzymes byBrucella-containing phagosomes (BCPs) when IL10 signalingwas blocked. Moreover, blocking IL10 signaling also enhancedproinflammatory cytokine production. Finally, concomitanttreatment with STAT3 siRNA significantly reduced the sup-pression of proinflammatory brucellacidal activity but notphagolysosome fusion by rIL10. Thus, our data provide the firstevidence that clearly indicates the suppressive role of IL10 onphagolysosome fusion and inflammation in response to B. abor-tus infection through two distinct mechanisms, STAT3-inde-pendent and -dependent pathways, respectively, in murinemacrophages.

Brucella spp. are facultative intracellular Gram-negative bac-teria that cause brucellosis in a variety of mammalian hosts;particularly, they cause more than 500,000 new human casesannually (1). They can prevent phagosome maturation by notfully understood mechanisms, leading to successful survivalwithin professional and non-professional phagocytes (2, 3).Vaccination seems to be a predominant manner for the controlof infectious diseases; however, there is no 100% efficacious

vaccine for brucellosis so far. Thus, identification of hostdefense mechanisms is essential to design rational approachesto eliminate brucellosis (4).

Interleukin 10 (IL10) is a pleiotropic immunomodulatorycytokine that is mainly produced by activated Th2 cells, mono-cytes, macrophages, and B cells. It was reported earlier to be akey inhibitor of inflammation and Th1-dependent cell-medi-ated immunity, especially production of proinflammatory cyto-kines such as interleukin 1� (IL1�), tumor necrosis factor(TNF), interleukin 6 (IL6), granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokines such as macrophageinflammatory peptide 1� (MIP-1�), generation of nitric oxide(NO), and up-regulation of surface antigen expression (MHCclass II, CD80, and CD86) in LPS-activated macrophages (5, 6).Additionally, various studies have also shown that IL10 plays asuppressive role in host response to Brucella abortus when thesurvival of intracellular B. abortus was markedly decreased inthe absence of IL10 (7, 8). A recent study indicated that IL10 isbeneficial for avoiding the phagolysosome fusion of intracellu-lar Brucella, resulting in prolonged persistence of Brucella inmacrophages (9). However, the underlying mechanisms of howIL10 could suppress the lysosome-mediated killing of Brucellain host cells still need to be clarified.

Phagolysosome fusion with possibly hundreds of proteins isthe most crucial effector in host responses against bacterialinfection. To date, lysosomal membrane glycoproteins 1 and 2(LAMP1 and LAMP2)2 and members of the RAS oncogenefamily (RABs) were the only known regulators to be importantin controlling this process (10 –12). Thus, our aim in the pres-ent study was to identify which regulators are controlled byIL10 during infection. Furthermore, expression of differenthydrolytic enzymes was also evaluated because sufficientrecruitment of lysosomal enzymes is essential to restrict bacte-ria within phagosomes. Our findings revealed that IL10 inhibitslysosome-mediated killing through governing a variety ofimportant proteins such as the RAB family, LAMP1, LAMP2,and the cathepsin (CTS) family through a STAT3-independentpathway in B. abortus–infected macrophages.This work was supported by Korea Health Technology Research and Devel-

opment (R&D) Project Grant HI16C2130 through the Korea Health IndustryDevelopment Institute funded by the Ministry of Health and Welfare,Republic of Korea. The authors declare that they have no conflicts of inter-est with the contents of this article.

This article contains Fig. S1.1 To whom correspondence should be addressed. Tel.: 82-55-772-2359; Fax:

82-55-772-2349; E-mail: kimsuk@gnu.ac.kr.

2 The abbreviations used are: LAMP, lysosomal membrane glycoprotein;rIL10, recombinant IL10; qRT-PCR, quantitative RT-PCR; BCP, Brucella-containing phagosome; CTS, cathepsin; BMM, bone marrow– derivedmacrophage.

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Results

IL10 represses brucellacidal activity through promotingbacterial ability to avoid late endosomes in macrophages

To monitor the impact of B. abortus infection on IL10expression, we first used RT-PCR to evaluate the transcrip-tional profile of Il10 mRNA in macrophages at differenttime points of infection. Interestingly, as shown in Fig. 1A,B. abortus infection markedly induced the transcription ofIl10 �2.2-, 1.8-, and 4.5-fold at 4, 24, and 48 h postinfection,respectively, as compared with uninfected cells. Addition-ally, the production of IL10 protein measured by indirectELISA was also consistently obtained with a marked in-crease of intracellular and secreted IL10 after infection(Fig. 1B).

To investigate the roles of IL10 in the brucellacidal activity,RAW 264.7 cells were pretreated with IL10 siRNA prior toinfection with B. abortus. As shown in Fig. 1C, inhibition ofIL10 signaling caused a significant decrease of intracellular Bru-cella survival that paralleled the observation of colocalization ofLAMP1 and Brucella-containing phagosomes (BCPs). In theIL10-blocked cells, colocalization was �1.4 times higher ascompared with control cells (Fig. 1, D and E). Taken together,these data clearly indicated that IL10 prevents the recruitmentof lysosomes by BCPs, leading to the persistence of bacteriawithin macrophages.

Interference of IL10 signaling alters normal acquisition ofmembrane-trafficking regulators by B. abortus phagosomes

Fusion of phagosomes with late endocytic organelles (lyso-somes) is a central effector of antimicrobial immunity thatrequires hundreds of proteins, and this process is suppressed byIL10; thus, identification of regulators mediated by IL10 couldprovide insights into its underlying mechanism during Brucellainfection. Based on previous reports on phagolysosome regula-tion (12, 13) and our previous study on microarray analysis ofgene expression profiling of B. abortus-infected macrophages,330 trafficking regulators of interest were selected. These tran-scripts were then assessed by RT-PCR in cells with or withouttreatment with IL10 siRNA at different phases of infection. Asshown in Fig. 2A, inhibition of IL10 signaling caused the induc-tion of Lamp1, Lamp2, and Rab34 mRNA at the early phase;however, no influence of IL10 on these genes was obtained at24 h postinfection (Fig. 2B). Interestingly, transcripts of Lamp1,Lamp2, Rab5a, Rab7, Rab20, Rab22a, Rab34, and Stx11 werefound to be remarkably increased in IL10-deficient cells incomparison with the controls at late infection (Fig. 2C).

To complement these data, we checked the expression ofselected proteins by Western blotting at 4 and 48 h postinfec-tion. As expected, the expression of LAMP2, RAB34, and

3 H. T. Hop, A. W. B. Reyes, T. X. N. Huy, L. T. Arayan, W. Min, H. J. Lee, M. H. Rhee,H. H. Chang, and S. Kim, unpublished data.

Figure 1. IL10 promotes B. abortus survival by suppressing phagolysosome fusion. Macrophages were infected with B. abortus, and the transcriptionaland translational profile of Il10 was examined by qRT-PCR (A) and sandwich ELISA (B). Bacterial intracellular growth was evaluated in cells with or without IL10siRNA treatment (C). The colocalization of BCPs with LAMP1 was analyzed at 48 h postinfection. LAMP1-positive (arrows) and -negative bacteria (arrowheads)were visualized by fluorescence microscopy (D), and the percentage of LAMP1 colocalized with BCPs in 100 cells was determined (E). Data represent themean � S.D. of triplicate experiments. Error bars represent S.D. Asterisks indicate significant differences (p � 0.05). Scale bar, 5 �m.

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RAB22A proteins was shown to be consistent with RT-PCRresults when blocking IL10 signaling significantly increasedthese proteins compared with the control (Fig. 2D). Further-more, evaluation of acquisition of these trafficking regulatorsby B. abortus phagosomes at 48 h postinfection revealed thatinhibition of IL10 in macrophages significantly induced thefraction of Brucella phagosomes labeled for LAMP2 (Fig. 3, Aand B) and RAB22A (Fig. 3, C and D) at late infection. Alto-gether, our findings suggest that the effect of suppression ofIL10 on phagolysosome fusion might be through inhibition ofLAMP1, LAMP2, RAB5A, RAB7, RAB20, RAB22A, RAB34,and STX11 during B. abortus infection.

IL10 mediates the expression and recruitment of hydrolyticenzymes during B. abortus infection in macrophages

The acquisition of acidic lysosomal enzymes by pathogen-containing phagosomes and their activation result in efficientkilling of intracellular pathogens (12, 14). Thus, we hypothe-sized that IL10 also manipulates the expression of these hydro-lytic enzymes. To address this hypothesis, transcriptional pro-filing of 30 lysosomal enzymes was initially assessed by RT-PCRat different times. Intriguingly, the expression of all enzymeswas independent of the deficiency of IL10 at early and middlephases of infection (Fig. 4, A and B); however, a number ofgenes, including Hexb, Gla, Ctsa, Ctsd, Ctsl, Man1a, andMan2a1, were uncovered to be negatively controlled by IL10 atthe late stage (Fig. 4C). To validate these data, we next evaluatedthe expression of proteins encoded by these genes by Westernblotting at 4 and 48 h postinfection. Consistent with the obser-vation from RT-PCR, the marked induction of HEXB andCTSD but not CTSZ proteins in IL10-deficient cells was onlyobserved at 48 h postinfection, whereas no difference wasobserved during earlier infection as compared with the control(Fig. 4D).

The observation that inhibition of IL10 induced the phagoly-sosome fusion suggested that not only the expression but thedelivery of lysosomal enzymes to Brucella phagosomes mightalso be increased in IL10-lacking cells. Therefore, we moni-tored the fraction of B. abortus phagosomes that could belabeled for CTSA and CTSD markers. At 48 h after infection,the colocalization of B. abortus phagosomes with both CTSA(Fig. 5, A and B) and CTSD (Fig. 5, C and D) was notably ele-vated in IL10-deficient cells relative to controls, suggesting thatIL10 controls the recruitment of CTSA and CTSD by BCPs.However, these results also raised the question whether thiseffect is general to all lysosomal enzymes, including those thatwere not altered by IL10. To answer this question, colocaliza-tion of CTSZ was observed by microscopy. However, the per-centage of colocalization for these proteins was not influencedby IL10 (Fig. 5, E and F).

To clarify whether IL10 could also have an inhibitory effecton the expression of phagolysosome-related genes in normalconditions, we treated RAW 264.7 cells with IL10 siRNA fol-lowed by incubation for 2 days. The expression of 14 represen-tative proteins was assessed by qRT-PCR; however, our datashowed that no difference between IL10 siRNA–treated cellsand control was observed (Fig. S1A), suggesting that the inhib-itory effect of IL10 only occurs in the B. abortus–infected con-dition. Furthermore, the above data suggested that IL10 mightplay a similar role in primary mouse macrophages. To addressthis question, we collected and differentiated bone marrow–derived macrophages (BMMs) from BALB/c mice and treatedthem with recombinant IL10 (rIL10) during B. abortus infec-tion. As expected, treatment with rIL10 induced B. abortus per-sistence within BMM cells at late infection (Fig. S1B). In paral-lel, down-regulation of trafficking regulators and lysosomalenzymes was also observed in rIL10-treated cells at 4 h postin-

Figure 2. IL10 significantly regulates the expression of membrane-trafficking regulators. Macrophages were treated with IL10 siRNA prior to B. abortusinfection. Total RNA content was isolated, and the expressions of representative membrane-trafficking proteins were evaluated by RT-PCR at 4 (A), 24 (B), and48 h (C) postinfection. The expression of LAMP2, RAB22A, and RAB34 proteins was checked at 4 and 48 h postinfection by Western blotting (D). Data representthe mean � S.D. of triplicate experiments. Error bars represent S.D. Asterisks indicate significant differences (p � 0.05).

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fection (pi) compared with control (Fig. S1, C and D). More-over, reduced colocalization of LAMP1 with BCPs wasobserved when IL10 signaling was enhanced (Fig. S1E), suggest-ing that IL10 also suppresses a phagolysosome fusion event topromote bacterial survival in primary mouse macrophages.

However, the target phagolysosomal genes of IL10 signalingwere different between BMM and RAW 264.7 cells, which mayresult from the different regulatory mechanisms activated inthe responses to pathogens in these cells (15). In summary,these results are the first evidence that clearly shows the regu-

Figure 3. IL10 inhibits the acquisition of trafficking regulators in B. abortus–infected macrophages. Macrophages were treated with IL10 siRNA prior toB. abortus infection. The fractions of BCPs that could be labeled for LAMP2 (A) and RAB22A (C) were evaluated by immunofluorescence assay. Arrows andarrowheads indicate marker-positive or -negative bacteria, respectively. At least 100 cells were counted to determine the percentage of colocalization ofLAMP2 (B) and RAB22A (D). Data represent the mean � S.D. of triplicate experiments. Error bars represent S.D. Asterisks indicate significant differences (p �0.05). Scale bar, 5 �m.

Figure 4. IL10 contributes to controlling the synthesis of lysosomal hydrolases. Macrophages were treated with IL10 siRNA and then infected withB. abortus. Total RNA was isolated at the indicated times. The expression of representative lysosomal hydrolases was evaluated by RT-PCR at 4 (A), 24 (B), and48 h (C) postinfection. Total cellular content of CTSD, CTSZ, and HEXB proteins was checked at 4 and 48 h postinfection by Western blotting (D). Data representthe mean � S.D. of triplicate experiments. Error bars represent S.D. Asterisks indicate significant differences (p � 0.05).

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latory role of IL10 in the lysosomal-mediated killing of B. abor-tus in murine macrophages.

Repressive effect of IL10 on the phagolysosome fusion inBrucella-infected macrophages is through STAT3-independentpathway

Binding of IL10 to the extracellular domain of IL10 receptoractivates various subsequent signaling pathways; however,JAK1/STAT3 is the best understood pathway to be mainlyresponsible for subsequent transduction (16, 17). Thus, wehypothesized that the suppressive effect of IL10 on the phagoly-sosome event is through the JAK1/STAT3 pathway. To test thishypothesis, we first used fluorescence microscopy to monitorthe translocation of STAT3 to the nucleus upon treatment ofB. abortus–infected RAW 264.7 cells with either rIL10 or IL10siRNA. Interestingly, the translocation of STAT3 into thenucleus was remarkably enhanced when the infected cells weretreated with rIL10, whereas the reverse was observed with IL10siRNA treatment (Fig. 6, A and B). Likewise, the results of eval-uation of the activation of STAT3 by Western blot assay werealso consistent with the microscopy observation (Fig. 6C).

To determine the role of the JAK1/STAT3 pathway in IL10signaling, the infected cells were concomitantly treated withrIL10 and STAT3 siRNA, and the transcriptional levels of IL10-regulated trafficking regulators and hydrolytic enzymes werethen assessed at 48 h postinfection. Surprisingly, the transcrip-tional levels of all IL10-regulated trafficking regulators andhydrolytic enzymes were found to be unchanged when theSTAT3 pathway was blocked (Fig. 6, D and E). In addition, theacquisition of LAMP2 by BCP was not different betweenSTAT3-blocked and -producing rIL10-treated cells (Fig. 6F),suggesting that the JAK1/STAT3 pathway is not responsible forthe suppressive effect of IL10 on phagolysosome fusion inB. abortus–infected macrophages.

IL10 represses expression of proinflammatory cytokines byup-regulating Sosc3 expression during B. abortus infection

Although our data clearly indicated that the inhibitory role ofIL10 on lysosomally mediated killing is independent of theJAK1/STAT3 pathway, we still assessed STAT3 function in theinflammatory response upon infection when the IL10 pathwaywas inhibited. For this, we evaluated the expression of Socs3 and

Figure 5. IL10 suppresses normal phagolysosome fusion during B. abortus infection. Macrophages were treated with IL10 siRNA prior to B. abortusinfection. The fractions of BCPs that could be labeled for CTSA (A), CTSD (C), and CTSZ (E) were evaluated by immunofluorescence assay. At least 100 cells werecounted to determine the percentage of colocalization of CTSA (B), CTSD (D), and CTSZ (F). Data represent the mean � S.D. of triplicate experiments. Error barsrepresent S.D. Asterisks indicate significant differences (p � 0.05). Scale bar, 5 �m.

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proinflammatory cytokines (Il6, Tnf, Mcp1, Il1a, and Il1b) byRT-PCR and indirect ELISA in infected cells concomitantlytreated with rIL10 and STAT3 siRNA. Interestingly, the addi-tion of rIL10 caused a significant increase of Socs3 that wasaccompanied by a decrease of Il6, Tnf, and Il1a but not Mcp1and Il1b mRNA levels at 48 h postinfection. However, this con-sequence was blocked when the cells were concomitantlytreated with STAT3 siRNA (Fig. 7A). In parallel, the presence ofsecreted cytokines in culture supernatant was also shown to beconsistent with the observation by RT-PCR (Fig. 7B). Thesefindings suggest that IL10 mediated the activation of STAT3/SOCS3 that inhibits the inflammatory response in B. abortus–infected macrophages.

Our findings showed two distinct regulatory mechanismsof IL10 in Brucella-infected macrophages that are throughSTAT3-dependent and -independent pathways, leading to the

question of what actual role STAT3 plays in anti-Brucella sup-pression by IL10. For this, we concomitantly treated RAW264.7 cells with rIL10 and STAT3 siRNA and then evaluatedbacterial survival. Surprisingly, treatment with STAT3 siRNAmarkedly reduced the rIL10-promoted bacterial persistence inRAW 264.7 cells (Fig. 7C), suggesting that STAT3 is requiredfor the antimicrobial suppression of IL10 during B. abortusinfection in macrophage cells. Taken together, our data clearlyindicate that IL10 regulates the proinflammatory anti-Brucellaimmunity through controlling the STAT3/SOCS3 pathway inRAW 264.7 macrophages.

Discussion

B. abortus, a causative agent of brucellosis, is one of thepathogens that have acquired the ability to survive and replicatewithin host cells by mechanisms that still needed to be eluci-

Figure 6. IL10 suppresses phagolysosome fusion by STAT3-independent pathway during B. abortus infection. Infected RAW 264.7 cells were treatedwith either rIL10 or IL10 siRNA and subjected to fluorescence microscopy observation of STAT3 translocation (A). The quantitative kinetic analysis of STAT3activation was determined in at least 100 cells in each experiment (B). The activation of STAT3 protein was evaluated by Western blot assay using anti-phospho-STAT3 antibody (C). To check the role of STAT3 in IL10 signaling, RAW 264.7 cells were concomitantly treated with rIL10 and STAT3 siRNA. Total RNA was thenextracted, and quantification of representative trafficking regulators (D) and hydrolytic enzymes (E) was measured by qRT-PCR. The fraction of BCPs that couldbe labeled for LAMP2 was evaluated by immunofluorescence assay (F). At least 100 cells were counted to determine the percentage of colocalization of LAMP2(G). Scale bar, 5 �m. Arrow and arrowhead indicate the marker-negative and -positive bacteria. Data represent the mean � S.D. of triplicate experiments. Errorbars represent S.D. Asterisks indicate significant differences (p � 0.05).

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dated. To date, several studies have shown that the fusion ofbacteria-containing phagosomes with late endosomes/lyso-somes is the most important innate immune effector againstintracellular Brucella; however, how this process is carried outby the host and subverted by bacteria is not fully understood.Thus, identification of potential molecules that may controlthis process will provide insights into rational therapeuticdesign for brucellosis elimination.

In agreement with previous observations, we also showedthat B. abortus infection markedly induces expression of IL10(7, 9). In this study, we proved that this induction is beneficialfor survival of intracellular Brucella because suppression ofIL10 signaling by siRNA treatment enhanced killing andrestricted bacteria within macrophages. Consistent with a pre-vious study (9), our study also showed that IL10 promotes sur-vival of intracellular Brucella by increasing bacterial ability toprevent the recruitment of lysosomes by BCPs, and this regula-tory role of IL10 was found at both early and late stages ofinfection.

It has been shown that IL10 negatively regulates the expres-sion of three regulators, Lamp1, Lamp2, and Rab34, at the earlyphase, whereas regulators at the late stage were Lamp1, Lamp2,Rab5a, Rab7, Rab20, Rab22a, Rab34, and Stx11. The observedup-regulation at the mRNA level resulted in a higher content ofthese proteins, suggesting that IL10 mediates these proteins tocontrol the fusion of phagosomes with late endosomes/lyso-somes during B. abortus infection. These data could be ratio-nalized by the findings that eight potent membrane-traffickingmolecules are subverted by IL10 during infection.

To date, LAMP1 and RAB7 were the only regulators thathave been proven to be crucial in regulating the fusion of BCPswith lysosomes (18), whereas the roles of other regulators areunknown. Our results showed that IL10 negatively regulatesRAB5A, RAB20, and RAB22A during infection; however, theseproteins have been found to be mainly associated to phago-somes containing intracellular pathogens such as Listeria andMycobacterium and to stimulate the maturation of these pha-gosomes at early infection (13), leading to the question of

Figure 7. STAT3/SOCS3 play a major role in anti-inflammatory effect of IL10. Infected RAW 264.7 cells were concomitantly treated with rIL10 and STAT3siRNA. The transcriptional and translational profiles of SOCS3 and proinflammatory cytokines were determined at 48 h postinfection by RT-PCR (A) andsandwich ELISA (B), respectively. RAW 264.7 cells were concomitantly treated with rIL10 and STAT3 siRNA, and bacterial survival was evaluated (C). Datarepresent the mean � S.D. of triplicate experiments. Error bars represent S.D. Asterisks indicate significant differences (p � 0.05).

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whether these functional proteins also play undescribed roles inlate Brucella infection. The other particularly interesting resultis that LAMP2, RAB34, and STX11 are also highly likely toregulate the fusion of phagosomes and lysosomes in macro-phages. LAMP2 was demonstrated to have overlapping func-tions with LAMP1 in recruitment of RAB7, movement towardthe microtubule-organizing center, and subsequent fusionwith lysosomes (11). Likewise, RAB34 was also reported to berequired for the fusion of phagosomes and lysosomes because adeletion mutant of this gene markedly reduced fusion ability(13, 19). STX11 is another membrane-trafficking regulator thatis suppressed by IL10; however, the general function of thisprotein in the phagolysosome event is still unknown. Takentogether, these findings and our results showing that IL10 sub-verts these proteins followed by inhibition of phagolysosomefusion suggest the potential of these trafficking regulators togovern phagolysosome fusion.

Likewise, several hydrolytic enzymes, including HEXB, GLA,CTSA, CTSD, CTSL, MAN1A, and MAN2A1, were clearlyshown to increase when IL10 signaling was inhibited at the latestage of infection. Different cathepsins, including CTSB, CTSD,CTSG, CTSL, and CTSS, are known to interact and contributeto killing intracellular Mycobacterium tuberculosis (20, 21),Mycobacterium bovis (22), Streptococcus pneumoniae (23), andListeria monocytogenes (24). Additionally, in macrophages,HEXB was also proven to protect them against Mycobacteriummarinum (25). Thus, the observation of an induced fraction ofphagosomes that are labeled by HEXB and CTSD in BCPs inparallel with an elevated fraction of phagosomes labeled withLAMP1 suggests that IL10 may mediate these enzymes toinhibit lysosome-mediated killing of Brucella within macro-phages, and this also opens up the discussion on the roles ofthese lysosomal enzymes in killing Brucella.

Conversely, our observation is similar to previous reports onthe anti-inflammatory role of IL10 in which blocking of IL10up-regulated production of inflammatory cytokines duringinfection. However, to date, only TNF has been recently provento participate in brucellacidal activity (26). The actual roles ofother proinflammatory cytokines in IL10 signaling and brucel-lacidal immunity have yet to be investigated. Additionally, oneof the striking findings in our study is that IL10 inhibitsphagolysosome fusion and proinflammatory brucellacidalactivity through two distinct signaling mechanisms in whichSTAT3 importantly uses the proinflammatory brucellacidalsuppression effect without phagolysosome interference. Thus,further investigations of the JAK/STAT pathways might revealthe relationship of inflammation and anti-Brucella activity,which could be useful for further understanding host resistanceto Brucella infection.

In summary, our findings reveal a possible novel role of IL10to suppress the synthesis and delivery of molecules involved inphagolysosome fusion, which prevents killing of intracellularB. abortus in macrophages. In addition, further investigation ofour identified molecules (eight trafficking regulators and sevenlysosomal enzymes regulated by IL10) might provide insightsinto how this process operates.

Experimental procedures

Reagents

Mouse IL10 and STAT3 siRNAs and rat anti-LAMP1 and-LAMP2; mouse anti-RAB34, -RAB22A, and -CTSZ; and goatanti-CTSA antibodies were obtained from Santa Cruz Biotech-nology. Goat anti-CTSD antibody was obtained from R&D Sys-tems. Texas Red-rabbit anti-goat IgG antibodies and themouse IL10 ELISA kit were purchased from Abcam. MouseIL10 recombinant protein and rabbit phospho-STAT3 poly-clonal, rabbit anti-HEXB, and FITC-goat anti-mouse IgGantibodies were obtained from Thermo Fisher Scientific.Texas Red-goat anti-rat IgG antibody and LipofectamineRNAiMAX were purchased from Life Technologies. FITC-conjugated goat anti-rabbit IgG antibody was obtained fromSigma-Aldrich.

Bacterial strain and cell culture

B. abortus 544 biovar 1 strain was routinely cultured inBrucella broth (BD Biosciences) at 37 °C until stationaryphase. The murine macrophage RAW 264.7 cells were grownat 37 °C in 5% CO2 atmosphere in RPMI 1640 mediumcontaining 10% heat-inactivated fetal bovine serum (FBS)with or without 100 units/ml penicillin and 100 �g/mlstreptomycin.

Bone marrow– derived macrophage preparation

BMMs from female BALB/c mice were prepared as describedpreviously (27). Briefly, bone marrow cells were collected andcultured in BMM medium containing L-cell– conditionedmedium for 5 days at 37 °C in 5% CO2. An equal volume of freshBMM medium without antibiotics was added, and the cellswere incubated further for 5 days. After 10 days of incubation,BMMs were washed three times with PBS and incubated withfresh RPMI 1640 medium containing 10% (v/v) heat-inacti-vated FBS for further experiments.

Bacterial infection and intracellular replication assay

This assay was performed as described previously (3). Briefly,macrophages (106 cells) were seeded in a 96-well plate andincubated for 24 h at 37 °C in 5% CO2. The cells were theninfected with 107 cfu of the virulent B. abortus for 1 h at 37 °C in5% CO2. RPMI 1640 medium containing 10% (v/v) FBS andgentamicin (30 �g/ml) were subsequently added to killextracellular bacteria. At 2, 24, and 48 h postinfection, cellswere lysed and plated on Brucella agar plates for cfu deter-mination. Additionally, the culture supernatant and totalproteins or RNA from macrophages were also obtained atdifferent time points.

RNA interference

RAW 264.7 cells were grown to 50% confluence on 6-, 12-, or96-well plates and transfected with siRNA directed against IL10using Lipofectamine RNAiMAX. The cells were incubatedfor 24 h at 37 °C and 5% CO2 prior to performing the intra-cellular growth assay or protein or RNA isolation. Thesame concentrations of negative control siRNAs were usedthroughout as controls. Knockdown efficiency was quanti-fied using RT-PCR.

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RNA extraction

Total RNA was isolated from RAW 264.7 cells (uninfectedor infected with B. abortus) at different time points using aQiagen RNeasy kit. DNA was removed before final elution ofthe RNA sample using the Qiagen on-column DNase diges-tion protocol.

RT-PCR

Real-time PCR analysis was performed as described previ-ously (12). Briefly, a mixture of SYBR Green PCR Master Mix(Applied Biosystems) and different pairs of 10 pM primers (Table1) were denatured at 95 °C for 10 min followed by 40 PCR cycles of95 °C for 15 s, 55 °C for 30 s, and 60 °C for 32 s. The mRNA expres-

Table 1List of primer sequences used for RT-PCR

Gene Common name Forward primer Reverse primer

b-actin �-Actin 5�-CGCCACCAGTTCGCCATGGA-3� 5�-TACAGCCCGGGGAGCATCGT-3Il1b Interleukin 1� 5�-CAACCACACAAGTGATATTC-3� 5�-GGATCCACACTCTCCAGCTG-3Il1a Interleukin 1� 5�-CCTTCTATGATGCAAGCTA-3� 5�-GTTGCTGATACTGTCACCC-3�Mcp1 CC motif chemokine 2 5�-GAGGTCTGTGCTGACCCCA-3� 5�-GCTTCAGATTTACGGGTCAA-3�Il6 Interleukin 6 5�-TCCAGTTGCCTTCTTGGGAC-3� 5�-GTACTCCAGAAGACCAGAG-3�Tnf Tumor necrosis factor 5�-CACAGAAAGCATGATCCGCG-3� 5�-CGGCAGAGAGGAGGTTGACT-3�Il10 Interleukin 10 5�-TGGCCCAGAAATCAAGGAGC-3� 5�-CAGCAGACTCAATACACACT-3�Socs3 Suppressor of cytokine Signaling 3 5�-GACCAGCGCCACTTCTTCACG-3� 5�-GTTCCGTGGGTGGCAAAGAA-3�Rab1 Rab1 5�-CCTTCAATAACGTTAAACAGT-3� 5�-TAGTCTACTACTTTCTTTGTGG-3�Rab5a Rab5a 5�-GTACTACCGAGGAGCACAAG-3� 5�-AAGCTGTTGTCATCTGCATAG-3�Rab5b Rab5b 5�-GACTAGCAGAAGTACAGCCAG-3� 5�-CAATGGTGCTTTCCTGGTATTC-3�Rab7 Rab7 5�-CCTCTAGGAAGAAAGTGTTGC-3� 5�-TTCTTGACCGGCTGTGTCCCA-3�Rab9 Rab9 5�-GCCCATGCAGATTTGGGACAC-3� 5�-GCCGGCTTGGGCTTCTTCTGTA-3�Rab10 Rab10 5�-GCCGAATGTTACTAGGGAACAAG-3� 5�-GCCGCCTCCTCCACTGCTGATA-3�Rab11 Rab11 5�-GAGCAGTAGGTGCCTTATTGG-3� 5�-GAACTGCCCTGAGATGACGTA-3�Rab14 Rab14 5�-GCCGGAGCTACTATAGAGGAGCT-3� 5�-GCCGTTCTGATAGATTTTCTTGG-3�Rab20 Rab20 5�-CTGCTGCAGCGCTACATGGAGCG-3� 5�-CTCCGCGGCAGTACAGGGAGC-3�Rab22a Rab22a 5�-GCCGACAAGAACGATTTCGTGCA-3� 5�-GCCGACTTCTCTGACATCAGTA-3�Rab24 Rab24 5�-GCGCGGGTGAGCACCGCAGGGC-3� 5�-GCCTCAGACCCCAACCCCAAG-3�Rab31 Rab31 5�-GCCCAGAAAACATTGTGATGGCG-3� 5�-GGCATTCTTCGCGCTGGTCTCC-3�Rab32 Rab32 5�-GCCGAGTATACTATAAGGAAGCTC-3� 5�-GCCCTGGGAAGGACTCTGGCTG-3�Rab34 Rab34 5�-GCAAAGTGACCCCGTGTGGCGGG-3� 5�-GGGCGTCCCGAAGACCACTCGG-3�Eea1 Early endosome antigen 1 5�-GCCCAATGAAGAGTCAGCAAGTC-3� 5�-GCCCACCTTGAGATGCTGGCGC-3�Rilp Rab-interacting lysosomal protein 5�-CAGGAACAGCTACAGCGCCTCCT-3� 5�-CTGAGGTTGCCGCATCAGGTTC-3�Sort1 Sortilin1 5�-GGGGAGCTGCGGACGGCCTTTTG-3� 5�-GGAGGCGCGGGCGGCGGCGGC-3�Lamp1 Lysosomal membrane glycoprotein 1 5�-GGCCGCTGCTCCTGCTGCTGCTG-3� 5�-ATATCCTCTTCCAAAAGTAATTG-3�Lamp2 Lysosomal membrane glycoprotein 2 5�-AGGGTACTTGCCTTTATGCAGAAT-3� 5�-GTGTCGCCTTGTCAGGTACTGC-3�Stx2 Syntaxin 2 5�-TGCCGTGGCAGCGCCTGCCCG-3� 5�-GGTCCCGCATCCCCACCGGC-3�Stx3 Syntaxin 3 5�-GATGACACGGACGAGGTTGAGAT-3� 5�-GTTGTGAGCTGTTCAAGGTCATC-3�Stx4a Syntaxin 4A 5�-CCCACGAGTTGAGGCAGGGGG-3� 5�-GGCGTGGCCAGGATGGTGACC-3�Stx5a Syntaxin 5A 5�-CGGGATCGGACCCAGGAGTTC-3� 5�-CAAAGAGGGACTTGCGCTTTG-3�Stx6 Syntaxin 6 5�-GTCAACACTGCCCAAGGATTGTTT-3� 5�-GTTTCATCGAGGTCCTCCAGATCC-3�Stx7 Syntaxin 7 5�-GGAAGCCGGCGAGGTCAGGGTGA-3� 5�-CATTGTGTGATCTTTTGGATGTTAG-3�Stx8 Syntaxin 8 5�-GGGCGGAGACTGCACCATGGCCCC-3� 5�-GTCTTCGATCCCCCTCCAGTTGTG-3�Stx11 Syntaxin 11 5�-GCTTCAAGAATTGTCCAGGAGCT-3� 5�-ATGGACGTGAGGAAGCGGACGTT 3�Stx12 Syntaxin 12 5�-CCGGTCTCTGCTCACTGTCATGTC-3� 5�-GTGGCTTGGCTGATCCGCTGGATG-3�Stxbp1 Syntaxin-binding protein 1 5�-CGGAGCCCGAAGACTCGAAGAACG-3� 5�-CAGCAGGAGGACAGCATCCTCATG-3�Stxbp2 Syntaxin-binding protein 2 5�-CCTCAGGGGAAGATGGCGCCCTTG-3� 5�-CAACAGGATGACAAGATTCGCATG-3�Lyz1 Lysozyme 1 5�-CTCTCCTGACTCTGGGACTCCTCC-3� 5�-CTGAGCTAAACACACCCAGTCAGC-3�Lyz2 Lysozyme 2 5�-GGCCAAGGTCTACAATCGTTGTG-3� 5�-GCAGAGCACTGCAATTGATCCCA-3�Hexa Hexosaminidase A 5�-GCCGGCTGCAGGCTCTGGGTTTC-3� 5�-GCGCGGCCGAACTGACATGGTAC-3�Hexb Hexosaminidase B 5�-CCCGGGCTGCTGCTGCTGCAGGC-3� 5�-GTGGAATTGGGACTGTGGTCGATG-3�Hexdc Hexosaminidase D 5�-CCACGCCATTTAAGATGAGATTAG-3� 5�-GGCCCTCAGCAGCCTCAGGTGGCC-3�Gla Galactosidase, � 5�-GGCCATGAAGCTTTTGAGCAGAG-3� 5�-AGTCAAGGTTGCACATGAAACGTT-3�Glb1 Galactosidase, �1 5�-GGAGGTGCAGCGGCTGGCCAGAGC-3� 5�-GGTGACATTATAGATGCCGTGCGC-3�Glb1l Galactosidase, �1-like 5�-GTGACGGGTGGGAAAGCCCTCACC-3� 5�-CTGTCATGTTCCCGATCCACAACG-3�Lpl Lipoprotein lipase 5�-CAGACATCGAAAGCAAATTTGCCC-3� 5�-GTCCATCCATGGATCACCACGAAG-3�Ctsa Cathepsin A 5�-GCCCTCCCCGGCCTGGCCAAGCAG-3� 5�-GCCGGCTGGATCAGAAAGGGGCCG-3�Ctsb Cathepsin B 5�-GCCGTGGTGGTCCTTGATCCTTCTT-3� 5�-GCCCCTCACCGAACGCAACCCTTC-3�Ctsc Cathepsin C 5�-GCCGCCACACAGCTATCAGTTACTG-3� 5�-GCCCCTGGAGACCTCCAAGATGTGC-3�Ctsd Cathepsin D 5�-CGTCTTGCTGCTCATTCTCGGCCTC-3� 5�-CACTGGCTCCGTGGTCTTAGGCGAT-3�Ctse Cathepsin E 5�-GGAGCAGAGTGAGAGAGAAGCTAC-3� 5�-GGGCCCGTAGTTTCTTCCGAAGGG-3�Ctsf Cathepsin F 5�-GCCGCAGGCTCCGCCTCG-3� 5�-GCCGCTCCTAGCACGGCC-3�Ctsg Cathepsin G 5�-CCTGTGCACACCTGTATCTACATAA-3� 5�-CTGTGTACCGAGTCACCGTACACGC-3�Ctsh Cathepsin H 5�-CTGAGAACCCTTCTTCCCAAGAGC-3� 5�-AGCAGCCAGGCCCCAGCGCACAGC-3�Ctsk Cathepsin K 5�-GGATGAAATCTCTCGGCGTTTAAT-3� 5�-GTCTCCCAAGTGGTTCATGGCCAG-3�Ctsl Cathepsin L 5�-GCCCCTTTTGGCTGTCCTCTGCTT-3� 5�-GCCCTCCATGGAAAAGCCGTGC-3�Ctso Cathepsin O 5�-GCCCGCAGTTGGTGAACCTCTTGCT-3� 5�-GCCGTCCTTCTGCTGGGTATCTGGG-3�Ctss Cathepsin S 5�-GCCGACTACCATTGGGATCTCTGGA-3� 5�-GCCGTCTCCCATATCGTTCATGCCC-3�Ctsz Cathepsin Z 5�-GGCGTCGTCGGGGTCGGTGCAGCA-3� 5�-CTGCGCCCCAGCAGAGCCAGCTG-3�Man1a Mannosidase 1, � 5�-CAAGCTGCTCAGCGGGGTCCTGTT-3� 5�-GCGGATCCTGGCTAAGTTGTCTTC-3�Man1a2 Mannosidase 1, �2 5�-GAAACTAGGTCCGGAGTCATTCAAG-3� 5�-CTTCCCAGCCCCACTGCCTGTATC-3�Man2a1 Mannosidase 2, �1 5�-GCTACAGACATTTTGTGCCATATG-3� 5�-CTGGGGGAACTCCCCAGGGACAAC-3�Man2a2 Mannosidase 2, �2 5�-GGATAGAACAGCTGGAACAACTGC-3� 5�-CCCCGTCCCCCCAAAGCAAACTGG-3�Man2b1 Mannosidase 2, � B1 5�-G TGATGTTCAGCACGCATCTGTTC-3� 5�-CGTACAGCGTCCTGGGTTGCACTG-3�Man2b2 Mannosidase 2, � B2 5�-CCGTCTTCCCAGAGCCACCCCCAG-3� 5�-CAGAGGACGTGGGGCGTCCGGAAC-3�Man1c1 Mannosidase � Class 1C Member1 5�-GAGGCCATAGAGACCTATCTCGTG-3� 5�-CATGGCACGTCCTGGTGATCTGGG-3�Man2c1 Mannosidase � Class 1C Member1 5�-GTAGCCTGCAATGGGCTTCT GGGG-3� 5�-CAACAGCTCCAGGTCCACCAGGAG-3�

Functional characterization of IL10 in B. abortus infection

3142 J. Biol. Chem. (2018) 293(9) 3134 –3144

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sion profiles were normalized with respect to �-actin. -Foldincrease of each gene was calculated using the 2���CT method.

Western blot assays

The lysates of cells were identified by Western blot assay asdescribed previously (4, 12). Briefly, the proteins were boiled for5 min at 100 °C in 2� SDS buffer and subjected to 10% SDS-PAGE. Separated proteins were then transferred onto Immo-bilon-P membranes (Millipore) using a semidry electroblotassembly (Bio-Rad). Membranes were blocked with 5% skimmilk (Difco) and subsequently incubated with primary antibod-ies (1:5,000 –1:1,000 dilution) in blocking buffer. After washingwith PBS with 0.05% Tween 20, membranes were incubatedwith HRP-conjugated goat anti-mouse IgG antibody (1:10,000dilution; Sigma) in blocking buffer. The proteins were detectedwith ECL solution (Thermo Fisher Scientific).

LAMP1, LAMP2, RAB22A, CTSA, CTSD, and CTSZ staining

Colocalization of BCPs with LAMP1, LAMP2, RAB22A,CTSA, CTSD, and CTSZ was performed as reported previously(26, 28). Briefly, RAW 264.7 cells were treated with IL10 siRNAprior to infection. The infected cells were incubated for 2(LAMP1) or 48 h (LAMP2, RAB22A, CTSA, CTSD, and CTSZ),fixed with 4% paraformaldehyde, permeabilized with 0.1% Tri-ton X-100, and blocked with blocking buffer (2% goat serum inPBS). The samples were stained with primary antibodies thatwere diluted 1:100 in blocking buffer followed by secondaryincubation with Texas Red-goat anti-rat IgG or Texas Red-rab-bit anti-goat IgG (1:1,000) in blocking buffer. The samples werestained with anti-B. abortus rabbit serum and FITC-conjugatedanti-rabbit IgG to identify the bacteria and placed in mountingmedium. Fluorescence images were captured using a laser-scanning confocal microscope (Olympus FV1000, Japan) andprocessed using FV10-ASW Viewer 3.1 software. 100 cells wererandomly selected, and the percentage of colocalization ofthese proteins with the BCPs was determined.

ELISA

The levels of IL10, TNF, IL6, IL1�, IL1�, and MCP1 in cul-ture supernatants were determined by sandwich ELISA per-formed in accordance with the manufacturer’s instructions(Thermo Fisher Scientific).

Statistical analysis

The data are expressed as the mean � (S.D. Student’s t testwas used to statistically compare the groups. Results with p �0.05 were considered significantly different.

Author contributions—H. T. H., H. J. L., W. M., M. H. R., H. H. C.,and S. K. designed the study. H. T. H. performed the experiments,analyzed the data, and wrote the manuscript. A. W. B. R, T. X. N. H.,and L. T. A. revised the manuscript. All the authors approved thefinal version of the manuscript.

Acknowledgments—We thank the members of Laboratory of Virologyand Laboratory of Biochemistry, Gyeongsang National University,Korea for technical supports.

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Functional characterization of IL10 in B. abortus infection

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KimTogonon Arayan, WonGi Min, Hu Jang Lee, Man Hee Rhee, Hong Hee Chang and Suk Huynh Tan Hop, Alisha Wehdnesday Bernardo Reyes, Tran Xuan Ngoc Huy, Lauren

macrophages in culturedBrucella abortusInterleukin 10 suppresses lysosome-mediated killing of

doi: 10.1074/jbc.M117.805556 originally published online January 4, 20182018, 293:3134-3144.J. Biol. Chem. 

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