Salmonella typhimurium infection triggers dendritic cells and macrophages to adopt distinct migration patternsin vivo

Salmonella typhimurium infection triggersdendritic cells and macrophages to adopt distinctmigration patterns in vivo

Chunfang Zhao1, Michael W. Wood2, Edouard E. Galyov2, Uta E. H�pken2,Martin Lipp3, Helen C. Bodmer1, David F. Tough1 and Robert W. Carter1

1 The Edward Jenner Institute for Vaccine Research, Compton, Newbury, UK2 Institute for Animal Health, Compton, Newbury, UK3 Department for Molecular Tumour Genetics and Immunogenetics, Max-Delbr�ckCentre for Molecular Medicine, Berlin, Germany

The presence of an anti-bacterial T cell response and evidence of bacterial products ininflamed joints of reactive arthritis patients suggests an antigen transportation role inthis disease for macrophages and dendritic cells. We have investigated the functionalproperties and in vivomigration of macrophages and DC after infection with Salmonellaenterica serovar Typhimurium (S. typhimurium). BM-derived macrophages and DCdisplayed enhanced expression of costimulatory molecules (CD40 and CD86) andincreased production of pro-inflammatory cytokines (TNF-alpha, IL-6 and IL-12p40)and nitric oxide after infection. Upon adoptive transfer into mice, infected DC migratedto lymphoid tissues and induced an anti-Salmonella T cell response, whereas infectedmacrophages did not. Infection of DC with S. typhimurium was associated with strongup-regulation of the chemokine receptor CCR7 and acquisition of responsiveness tochemokines acting through this receptor. Moreover, S. typhimurium-infected CCR7-deficient DC were unable to migrate to lymph nodes after adoptive transfer, althoughthey did reach the spleen. Our data demonstrate distinct roles for macrophages and DCas antigen transporters after S. typhimurium infection and a dependence on CCR7 formigration of DC to lymph nodes after bacterial infection.

Introduction

Salmonella enterica serovar Typhimurium (S. typhimur-ium) has been extensively used to infect mice to studythe immune response to gastrointestinal infections [1].After infection, macrophages and DC are the major cell

types to be infected, and these cells transport thebacteria to the liver and spleen, the major sites ofbacterial replication [2–4]. Both cell types are crucial tothe immune response to Salmonella infection, withmacrophages controlling bacterial growth in the earlyphases of infection and DC responsible for the initiationof the T cell response [5, 6].

The in vivomigration of immune cells is controlled bya tightly regulated system of chemokines and chemokinereceptors [7]. For DC, two chemokine receptors thatplay an important role in regulating movement betweenperipheral tissues and secondary lymphoid organs areCCR6 and CCR7. Immature DC isolated from areas ofantigen encounter, such as the epithelia of the skin orgut, express CCR6 and are retained in the tissue by the

Correspondence: Robert W. Carter, The Edward Jenner In-stitute for Vaccine Research, Compton, Newbury, Berkshire,RG20 7NN, UKFax: +44-1635-577901e-mail: [email protected]

Received 19/4/06Revised 7/8/06

Accepted 13/9/06

[DOI 10.1002/eji.200636179]

Key words:Chemokines

� Dendritic cells� Macrophages

� SalmonellaMigration

Abbreviations: BMMac: BM macrophages � HEV: high endo-thelial venules � MLN: mesenteric LN � NP: nucleoprotein �TS: temperature sensitive

Eur. J. Immunol. 2006. 36: 2939–2950 Immunity to infection 2939

f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

presence of the CCR6 ligand CCL20 [8–10]. Uponencounter with antigen and subsequent maturation, DCexpress CCR7, leading to migration to draining lymphnodes via the CCR7 ligands CCL19 and CCL21 [8, 11].

The HLA-B27-associated spondyloarthropathy reac-tive arthritis is a well-described example of anautoimmune disease that is triggered by a bacterialinfection, such as an enteric infection with Salmonella[12]. In patients with reactive arthritis, HLA-B27-restricted bacteria-specific T cell responses can bedetected, as can the presence of Salmonella LPS in thejoints, supporting the notion that the infection may bethe driving force for the disease [13, 14]. At present,however, the mechanisms responsible for initiation ofthe anti-Salmonella T cell response and the transporta-tion of bacteria or bacterial products to the joints isunclear.

Since macrophages and DC are critical componentsof innate and acquired anti-bacterial responses, wesought to study their function after infection with S.typhimurium. Utilising HLA-B27 transgenic mice andT cells recognising HLA-B27-restricted, S. typhimurium-expressed antigens, we demonstrate multiple differ-ences in the functional properties of infected DC andmacrophages. The relevance of our findings to thepathogenesis of reactive arthritis is discussed.

Results

Activation of BM macrophages and BMDC afterinfection with S. typhimurium

Macrophage (BMMac) and DC (BMDC) were generatedin vitro from the BM of HLA-B27 transgenic mice. Afterculture, cells were phenotyped using cell surfacemarkers, with BMMac defined as CD11c-/CD11b+/classIIlo and BMDC as CD11c+/CD11b+/class IIhi. Forinfection, we initially used a temperature-sensitive(TS) mutant of the C5 strain of S. typhimurium(previously described by Hormaeche et al. [15]), whichwas able to grow at 30�C but not at 37�C. Because of theinability of the bacteria to grow at the core temperatureof mice, higher doses of bacteria can be used forinfection. Exposure of both BMMac and BMDC to C5(TS) for a 2-h infection period at an MOI of 10 led tocellular activation that was similar to that induced byexposure of cells to the TLR4 agonist LPS (Fig. 1). Up-regulation of costimulatory molecules CD40 and CD86was observed after infection, and as expected levels ofexpression were higher on BMDC than BMMac (Fig. 1a).Production of the proinflammatory cytokines TNF-a,IL-6 and IL-12p40 was also increased after either LPStreatment or C5 (TS) infection as shown by intracellularcytokine staining (Fig. 1b). In addition, high concentra-

tions of nitrite, an indicator of nitric oxide (NO)production, were present in the supernatant of BMMacand BMDC cultures after C5 (TS) infection (Fig. 1c).

BMDC but not BMMac migrate to lymphoid tissueafter S. typhimurium infection

The fate of S. typhimurium-infected BMMac and BMDCin vivo was tested by adoptive transfer of CFSE-labelledcells into HLA-B27 transgenic mice. At 48 h after i.p.injection of labelled cells, cells from the peritoneallavage, spleen and mesenteric LN (MLN) were isolatedand the numbers of CFSE+ cells determined by flowcytometry (Fig. 2). Populations of transferred CFSEBMMac and BMDC were detected in mice that receiveduninfected (not shown) or C5 (TS)-infected cells(Fig. 2a) as compared to mice that received no celltransfer (Fig. 2a, inset). Analysis of data from a total ofup to 15 mice showed that there was a significantdecrease in the number of CFSE+ cells in the peritoneallavage of mice receiving infected BMMac or BMDC ascompared to their uninfected counterparts (Fig. 2b,upper panel). There was no difference in the numbers ofBMMac recovered from the spleen or MLN between theuninfected and C5 (TS)-infected groups. In contrastsignificantly more transferred BMDC were observed inthe spleen and MLN of mice receiving infected vs.uninfected transferred BMDC (Fig. 2b).

Transferred BMDC are able to inducean anti-Salmonella T cell response in vivo

To examine the function of S. typhimurium-infectedBMDC, their ability to induce an anti-Salmonella T cellresponse was tested. For this purpose, we utilised astrategy inwhich Tcell epitopes frommodel antigens areexpressed as fusion proteins with type III secretedSalmonella proteins, a method which has been usedpreviously to induce anti-Salmonella CD8+ T cells [16].Two epitopes from the influenza nucleoproteinantigen (NP366–374, H-2Db restricted and NP383–391,HLA-B27 restricted) and one from the bacterial proteinb-galactosidae (BG876–884 H-2L

d restricted) were clonedinto the bluescript plasmid as part of a fusion proteinwith the type III secreted Salmonella gene sopE [17]. Theplasmids were transformed into the attenuated aroAmutant strain of S. typhimurium SL3261 and T cellresponses tested. All antigens were able to stimulatetheir respective T cell response both in vitro and in vivo(data not shown).

Antigen-experienced HLA-B27-restricted NP383–391-specific CD8+ T cells were generated ex vivo fromsplenocytes of HLA-B27 transgenic mice infected withrecombinant vaccinia virus expressing NP, by culture for10 days with peptide and irradiated splenocytes fol-

Chunfang Zhao et al. Eur. J. Immunol. 2006. 36: 2939–29502940


lowed by a repeat stimulation for another 10 days. Thesecells were then used as responding cells to test if BMMacor BMDC could present Salmonella antigens. UsingIFN-c production as a readout, CD8+ T cells respondedto both BMMac and BMDC infected with SL3261expressing the specific NP383–391 epitope but not theirrelevant BG876–884 epitope (Fig. 3a).

We recently developed a novel TCR transgenic mouseon theHLA-B27 background, called GRb, inwhich Tcellsare specific for HLA-B27-restricted NP383–391 [18]. Aninterestingpropertyof theTcell specificityof thesemice isthatCD4+aswell as CD8+Tcells are able to recognise theclass I-restricted antigen, making it a potentially usefulmodel to studyHLA-B27-restricted CD4+Tcells thatmayplay a role in the spondylarthropathies [19]. Using na�veGRb CD4+ T cells, BMDC but not BMMac were able to

induce proliferation of the GRb T cells in an antigen-specific manner (Fig. 3b). This was observed if peptide-pulsed cells (NP383; Fig. 3b, left panel) or recombinantS. typhimurium-infected cells (NP383 SL3261; Fig. 3b,right panel) were used as APC. In addition, BMDCinfected with SL3261 expressing NP383–391, but not thenegative control peptideNP366-374,were able to stimulatethe proliferation of both na�ve CFSE-labelled CD8+ andCD4+ GRb T cells, as shown by the loss of CFSE stainingover a 4-day period of culture (Fig. 3c). Importantly, theT cell response was also stimulated in vivo by BMDC(Fig. 3d). Thus, BMDC infected with SL3261 NP383–391,but not BMDC infected with SL3261 NP366–374 oruninfected BMDC, were able to stimulate the prolifera-tion of CFSE-labelled GRb CD8+ T cells after adoptivetransfer into HLA-B27 transgenic mice.

Figure 1. BMMac and BMDC are activated after infection with a TS mutant of S. typhimurium. Macrophages and DC were culturedfrom the BM of HLA-B27 transgenic mice in the presence of M-CSF or GM-CSF, respectively. Cells were then either left untreated,treated with LPS (1 lg/mL) or infected with C5 (TS) S. typhimurium at 10 MOI for 2 h. After 24 h, cells were harvested and analysedfor markers of activation. (A) Cell surface expression of co-stimulatory molecules CD40 and CD86. (B) Percentage of cellsexpressing the cytokines TNF-a, IL-6 and IL-12p40, measured by intracellular antibody staining and flow cytometry. Thepercentage positive cells expressing cytokine was determined by comparison to cells stained with isotype-matched antibody. (C)Concentration of nitrite in the supernatant of cultured cells as measured by the Griess assay. All data is representative of aminimum of three experiments.



Figure 2. Migration of BMDC to lymphoid organs and BMMac to inflamed limbs after infection with S. typhimurium. Macrophagesand DCwere either left untreated or infectedwith C5 (TS) (10 MOI) for 2 h. After 24 h, cells were harvested, labelledwith CFSE and107 cells transferred via an i.p. injection into HLA-B27 transgenic mice. (A) Representative cell density plots showing detection ofCFSE+ cells in the peritoneal lavage, spleen and MLN of mice injected with C5 (TS)-infected BMMac or BMDC. Inset shows theprofile ofmice that received no transferred cells. (B) Box andwhisker plots showingnumbers of recoveredCFSE+ BMMac (n=14) andBMDC (n=15) in indicated tissues. Boxes represent the interquartile range of databetween the 25th and 75th percentile andwhiskersrepresent the upper and lower limits of the data. The dividing line in the box represents themedian,means are indicated by a solidcircle and outlier points are represented by an asterisk. Statistical significance, as shown, was calculated by two sample t-testusing Mini-Tab statistical software.



Up-regulation of CCR7 on S. typhimurium infectedBMDC leads to their migration to LN in vivo

Chemokine receptors CCR6 and CCR7 are expressed byimmature and mature DC respectively, controlling themovement between peripheral tissues (immature DC)and LN (mature DC) [8–11]. As BMDC but not BMMacmigrated preferentially to lymphoid tissues after infec-tion with S. typhimurium, expression of the chemokinereceptors CCR7 and CCR6 by these cells was analysed.When expression was measured at the mRNA level, itwas evident that maturation, induced by either LPStreatment or infection with C5 (TS), stimulated up-regulation of CCR7 and down-regulation of CCR6; forCCR7, we further showed that maturation was asso-ciated with greatly increased cell surface expression ofthe receptor (Fig. 4a and b). Qualitatively similarchanges were observed upon activation of BMMac (i.e.,up-regulation of CCR7 and down-regulation of CCR6).However, the absolute expression levels, particularly forCCR7, weremuch lower onmacrophages (Fig. 4a and b).The functional significance of increased CCR7 expres-sion on BMDC after C5 (TS) infection was borne out byan increased migration of these cells towards the CCR7ligand CCL19 (Fig. 4c). Migration of uninfected BMDCtowards the CCR6 ligand CCL20 was low, and this wasfurther decreased after C5 (TS) infection, reflecting thechanges in expression detected at the mRNA level(Fig. 4c).

Since C5 (TS)-infected BMDC migrated in greaternumber than uninfected cells to the spleen and LN afteradoptive transfer, the role of CCR7 in this migration wasinvestigated using BMDC prepared from CCR7 knockout(KO) mice. As shown in Fig. 4d, very few C5 (TS)-infected CCR7 KO BMDC reached the LN after i.p.injection. By contrast, similar numbers of C5 (TS)-infected WT BALB/c and CCR7 KO were recovered fromthe spleen (Fig. 4d). Therefore, migration of C5 (TS)-infected DC to the LN was highly dependent on CCR7,while movement of these cells to the spleen did notrequire CCR7 expression.

Discussion

In this study, the functional consequences of S. typhi-murium infection of macrophages and DC has beencompared. These two APC exhibited a number ofqualitatively similar responses to infection, includingup-regulation of costimulatory molecules, production ofinflammatory cytokines and NO, down-regulation ofCCR6 and up-regulation of CCR7. However, themagnitude of these changes differed, and this wasassociated with the acquisition of distinct functionalabilities.

Macrophages and DC, as part of the innate immunesystem, receive signals for activation upon encounterwith bacteria or bacterial products. This was clearlyshown to occur in BMMac and BMDC cultures afterinfection with S. typhimurium, with levels of bothcostimulatory molecules and cytokines reaching levelssimilar to those induced by exposure to LPS. The similarconsequences of infection and LPS treatment in thisrespect are consistent with previous observations, whichshowed that bacterial contact but not internalisationwasrequired for activation [6, 20–22]. In agreement withthis, we observed that addition of heat killed bacteria orinfection with the TS mutant at the non-replicatingtemperature of 37�C led to activation of cells (data notshown). Of note, our finding that BMDC as well asBMMac produce NO after infection contrasts with theresults of a previous study using murine BMDC [20].However, DC have been shown to produce NO afterinfection with S. typhimurium [23]. These discrepanciesmay be related to the time after infection whenresponses were studied and/or the number of bacteriathat are used for infection. In support of the latter idea,higher levels of infection, as can be attained using ourTS mutant of S. typhimurium C5 (TS), have been shownto induce DC production of NO [24].

Clear differences in the in vivo migration of S. typhi-murium-infected BMMac and BMDC were observedafter adoptive transfer. The migration of BMDC but notBMMac to the LN was likely linked to the much higherexpression of CCR7 on BMDC. Cheminay et al. [25]described the induction of CCR7 on BMDC afterinfection with S. typhimurium, which, similar to ourfindings, led to increased migration to the chemokineCCL19. We have extended these findings by showingthat this up-regulation of CCR7 is functional in vivo.Moreover, a crucial role for CCR7 in the migration ofinfected BMDC to the MLN and inguinal LN was shownby the inability of CCR7 KO BMDC to reach theselocations. These results add to previous reportsimplicating CCR7 as a key molecule controlling DCmigration to LN. Thus, a lack of CCR7 expression by DCresults in an inability to migrate from tissues, such asskin, to draining lymph nodes [26, 27]. In addition, DCinduced to mature by LPS were shown to migrate to LNin a CCR7-dependent manner [11]. Interestingly, ourresults showed that CCR7 is not required for migrationof S. typhimurium-infected BMDC to the spleen,implying that alternative chemokine receptors controlthis process or that cells enter passively through thebloodstream. In this respect, it would be of interest todetermine if CCR7 is required for the localisation oftransferred infected DC within specific areas of thespleen. Of note, we cannot rule out the possibility that aproportion of the labelled cells detected by FACS couldrepresent endogenous cells that have taken up CFSE+





transferred cells. However, we believe that this isunlikely to be the case for the majority of cells detectedin our experiments for the following two reasons. Firstly,the intensity of CFSE-labelling among cells detected inrecipients after transfer of macrophages or dendriticcells was at a similar level to that observed in cells pre-transfer. A host cell that has taken up CFSE dye from atransferred cell would likely have lower levels of CFSEfluorescence. Secondly, the notion that transferred cellsactively migrate to LN is strongly supported by the CCR7dependence of this process. If host cells were transport-ing label (from internalised cells or cell fragments) toLN, expression of CCR7 by the injected cells should beirrelevant.

It is clear that the migration of DC to lymphoid tissueafter adoptive transfer into the peritoneal cavity isdependent on CCR7. How these cells get to the LN is lessclear, but there are several possibilities. Firstly, it hasbeen shown that direct drainage from the peritonealcavity to the MLN can occur [28, 29], even though theMLN are not thought to be the principle LN that drainthis site [30, 31]. Secondly, DC could access MLN viadirect uptake through lymphatic stomata, a mechanismthat is enhanced by NO [32]. Thirdly, DC may enter LNfrom the bloodstream via high endothelial venules(HEV). In support of this idea, the presence of DC in thespleen demonstrates that the adoptively transferred cellsmigrate through the blood, and immigration from theblood would account for the appearance of i.p. injectedDC in distant nodes such as the inguinal LN. Althoughsome studies have failed to find evidence for DC entryinto LN through HEV [33, 34], this has been shown tooccur after infection of mice with mouse mammarytumour virus [35]. Whether Salmonella-infected DCgain this ability is an issue of interest for furtherinvestigation.

A consistent finding for infected BMMac and BMDCwas the disappearance of transferred cells from the

peritoneal cavity. For BMDC this could be partlyexplained by their movement into lymphoid tissue.However, the paucity of BMMac in LN and spleenindicates that they may be resident elsewhere; onepossibility is that they migrate into non-lymphoid tissue.Although we did not look extensively in other tissues, wewere able to detect the presence of adoptivelytransferred S. typhimurium infected BMMac but notBMDC in the inflamed joints of mice induced using aSalmonella-induced arthritis model, lending some sup-port to this notion (data not shown). We can not rule outthe possibility that the disappearance of infected cellsfrom the peritoneal cavity could be due to increased celldeath. However, when apoptosis of BMMac and BMDCwas examined in vitro, we observed little evidence forincreased death in macrophages after infection and onlya twofold increase with infected DC (data not shown).Since macrophages were more resistant than DC toSalmonella-induced apoptosis in vitro, it seems unlikelythat increased cell death could account for the lowerrecovery of macrophages than DC after i.p. injection. Inthis respect, our choice of C5 (TS) as the attenuatedstrain of S. typhimurium to be used in our experimentswas based on its ability to be used at a high MOI with noadverse effects on cell viability.

BMDC also down-regulated CCR6 after they becameactivated by infection with S. typhimurium or exposureto LPS. Expression of CCR6 on DC is thought to beimportant for localisation of DC in non-lymphoid tissueswhere antigen encounter can occur and such cells wouldhave an immature phenotype [8, 9]. The relevance ofCCR6 expression to anti-Salmonella immunity is evidentfrom the ability of Salmonella to induce CCR6 ligandCCL20 production by intestinal epithelial cells afterinfection [36]. This was shown to stimulate themigration of immature DC in a CCL20-dependentmanner. The low levels of both CCR6 and CCR7expression on BMMac suggest that other chemokine

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Figure 3. Stimulation of S. typhimurium-specific T cell responses by BMDC in vitro and in vivo. S. typhimurium (strain SL3261)engineered to express the class I-restricted T cell epitopes NP383–391, NP366–374 or BG876–884 was used to infect BMDC and BMMac.Infected cells were cultured with TCR transgenic T cells isolated from the GRb mouse, which have specificity for the HLA-B27-restricted epitopeNP383–391. (A) IFN-c production, asmeasured by ELISpot, by antigen experienced CD8+ GRbT cells after co-culturewith BMDC and BMMac pulsed with 1 lM of either NP383–391 or BG876–884 peptide (left panel) or infected with recombinant SL3261NP383–391 or BG876–884 (right panel). Bars represent themean� SD of triplicatewells. (B) Proliferation of na�ve GRb CD4+ T cells afterco-culture with BMDC and BMMac pulsed with 1 lM of either NP383–391 or BG876–884 peptide (left panel) or infected withrecombinant SL3261NP383–391 or BG876–884 (right panel). Proliferationwasmeasured after 72 h of culture by uptake of [3H]thymidine(cpm). Bars represent themean� SD of triplicatewells. (C) Proliferation of na�ve GRb CD4+ (circles) and CD8+ (squares) T cells afterco-culturewith recombinant SL3261NP383–391 (solid symbols) or NP366–374 (open symbols) infected BMDC. T cellswere labelledwithCFSE prior to co-culture and the loss of CFSE (% CFSE lo) used as an indicator of a proliferative response andmeasured over a 4-dayperiod. Histograms show representative CFSE profiles as measured on day 2. A total of 50 000 events were acquired on the flowcytometer. (D) Antigen-specific proliferation of GRb CD8+ T cells in response to BMDC in vivo. GRb CD8+ T cells were labelled withCFSE and adoptively transferred into HLA-B27 transgenicmice. After 1 day, BMDC, either uninfected or infectedwith recombinantSL3261NP383–391 or NP366–374, were adoptively transferred into themice. After 3 days, spleenswere removed and the loss of CFSE onGRb CD8+ T cellsmeasured as amarker of proliferation (% CFSE lo) (n=3; 500 000 total eventswere acquired on the flow cytometer).All data represent one experiment from a minimum of two experiments.





receptors may be responsible for their movement in vivo.Of particular note is the study by Nanki et al. [37], inwhich splenic macrophages were shown to migrate tothe inflamed synovium in a collagen induced arthritismouse model. This was dependent on the chemokinereceptor CX3CR1, which mediated the effects of thechemokine fractalkine (CX3CL1), expressed by syno-viocytes. Further work will be required to determinewhether this receptor is also involved in the migration ofSalmonella infected BMMac to the joints of mice withSalmonella-induced arthritis.

We have developed further a system previouslydescribed by R�ssmann et al. [16], to express T cellepitopes in Salmonella via a protein secreted through thetype III secretion system. Our unpublished observationshave shown that using our recombinant bacteria, we areable to induce specific anti-Salmonella immune re-sponses, either in vitro or after oral or systemic infectionin vivo. Both BMMac and BMDC could stimulate antigen-experienced T cells in vitro. The ability of bothmacrophages and DC to present Salmonella antigensto T cells supports previous findings, whereby infectionin vitro with Salmonella engineered to express modelantigens can stimulate a response from T cell hybrido-mas [38–40]. However, we found that only BMDC couldstimulate a response from na�ve T cells, with BMMacunable to stimulate na�ve T cells even after pulsing withpeptide. This is not surprising, given the higherexpression of MHC class I and II and costimulatorymolecules, especially after infection, on BMDC and thelong established role of DC in priming T cell responses[41]. BMDC also induced the na�ve Tcell response in vivoas observed in experiments involving co-adoptivetransfer of GRb TCR transgenic T cells, confirming thesignificance of their migration to lymphoid tissue afteradoptive transfer and the findings of others [42].Nevertheless, it should be mentioned that our data donot distinguish between direct antigen presentation bythe transferred DC and indirect presentation by hostcells that have acquired antigen from the injected DC.Such amechanism of antigen transfer has been proposed

to occur between migrating DC and lymphoid residentDC under physiological conditions [43].

The significance of BMMac stimulating only antigen-experienced Tcells and not na�ve Tcells and its relevanceto the immune response to Salmonella infection in vivo isunclear. It is possible that macrophages are not requiredfor the Tcell response, although they do form a source ofSalmonella antigen for bystander DC after they havedied as a consequence of their infection [44]. Con-versely, macrophages could function as APC in inflamedtissues, presenting antigen to infiltrating activatedT cells. Hence, one consequence of the presence of S.typhimurium-infected macrophages in inflamed jointsmight be the in situ stimulation of primed T cells;however, understanding the role of such a mechanism inreactive arthritis is an area for future study.

We have previously shown that T cells from the GRbTCR transgenic mouse are responsive to virally infectedcells in vivo and we have extended this to a bacterialinfection system [18]. The ability to study an HLA-B27-restricted, Salmonella-induced T cell response in vivoshould aid in elucidating the role of this response inreactive arthritis. Based on the results of the presentstudy, it will be of interest to further investigate thecontributions of different antigen presenting in thissetting.

Materials and methods

Animals

All mice were bred in the specific pathogen-free facility at theInstitute for Animal Health (Compton, UK). B27b2 M(BALB/c) mice expressing HLA-B2705 and human b2 m wereoriginally obtained from E. Weiss [45]. GRb � B27b2 M TCRtransgenicmice, expressing the a and b chains of a TCR specificfor influenza NP have been described previously [18]. BM fromCCR7 KOmice was kindly provided byM. Lipp [26]. All animalexperimentationwas conducted with the approval of the HomeOffice and the ethical review committee of the Institute forAnimal Health.

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Figure 4. S. typhimurium-induced up-regulation of CCR7 on BMDC leads tomigration to lymph nodes after adoptive transfer. BMDCand BMMacwere either left uninfected, treatedwith LPS (1 lg/mL) or infectedwith S. typhimurium [strain C5 (TS)] and the levels ofCCR7 and CCR6measured. (A) Real time RT-PCR analysis of CCR6 and CCR7mRNA levels (represented as the fold change over thelevels in uninfected BMMac. (B) Flow cytometry histograms showing expression of CCR7 protein on BMMac and BMDC asmeasured by the binding of the CCR7 ligand, CCL19. A fusion protein of CCL19 and the Fc portion of human IgG, followed by anantibody to human IgG were used to facilitate detection of ligand binding. Staining using CCL19-Ig (shown in grey) is compared tostaining in the absence of CCL19-Ig fusionprotein (solid line) as a negative control. (C) Transwell assay to detect functional CCR7 onBMDC. The percentage of total cells passing through the transwell in response to medium alone or medium containing CCL19(0.3 lM) or CCL20 (100 ng/mL) was assessed 4 h after addition to the transwell. The data represent one experiment from a total ofthree independent experiments. (D) Numbers of CFSE adoptively transferred cells recovered per 2 � 106 total cells from spleen,MLNand inguinal LN, 48 h after i.p. injection of 107WTBALB/c or CCR7KOBMDC into BALB/cmice. Histograms represent themean� SD of four mice and data are representative of two experiments.



Generation of BMMac and BMDC from murine BM

BMwas isolated from the femurs and tibiae of the hind limbs ofmice. A single cell suspension was prepared and red blood cellslysed using lysis buffer (Sigma, Poole, UK). For culture ofmacrophages, 2 � 107 cells were resuspended in 20 mL cellculture medium [IMDM (Gibco, Paisley, UK) supplementedwith 10% FCS (Harlan Sera-lab, Loughborough, UK) 50 lM2-ME (Sigma), 100 U/mL penicillin (Gibco) and 100 lg/mLstreptomycin (Gibco)] supplemented with 20% M-CSF con-taining Ladmac cell tissue culture supernatant, final concen-tration equivalent to 10 ng/mL M-CSF, in a T75 tissue cultureflask. Cells were cultured for 3 weeks with feeding every3 days at 37�C in a 5% CO2 in air incubator; after this timeadherent cells were harvested by cell scraping. For culture ofDC, 2 � 106 BM cells were resuspended in cell culturemedium, supplemented with 20 ng/mL GM-CSF (R&Dsystems, Abingdon, UK) and cultured in a T75 flask for 7 daysat 37�C in a 5%CO2 in air incubator, with feeding at days 3 and6 with media supplemented with 20 ng/mL and 10 ng/mLGM-CSF, respectively. On day 8 adherent cells were obtainedusing accutase (Sigma, Poole, UK).

Infection of cells with S. typhimurium

The attenuated strains SL3261 (aroAmutant) [46] and C5 (TS)(temperature-sensitive mutant growing at 30�C) [15] wereused in this study. Mid-log phase growth bacteria (3 h) werecultured from a static culture in Luria-Bertani media. BMDCand BMMac were infected at a multiplicity of infection of 10(C5 (TS) infection at 30�C) or 1 (SL3261 infection at 37�C) for2 h in the tissue culture flask. Cells were washed three timeswith PBS and supplemented with culture media containing50 lg/mL gentamicin (Sigma). Cells were then cultured for afurther 24 h before harvesting for further experiments. As acontrol for maturation after infection, cells were incubated for24 h in media containing 1 lg/mL Salmonella LPS (Sigma).

Generation of recombinant S. typhimurium expressingT cell epitopes

The DNA sequence for the type III secreted S. typhimuriumprotein SopE and its promoter area was amplified by PCR andcloned in pBluescript using BglI and XhoI cloning sites.Subsequently, the DNA sequences encoding the CD8+ T cellepitopes for influenza NP366–374 (ASNENMDAM, H-2Db

restricted), NP383–391 (SRYWAIRTR, HLA-B27 restricted) andb-galactosidase BG876–884 (TPHPARIGL, H-2L

d restricted) weresub-cloned into the sopE sequence via a SalI and EcoRVrestriction sites to produce in-frame fusions. The plasmid wasthen transformed into the attenuated S. typhimurium strainSL3261 and bulk recombinant strains selected for by resistanceto ampicillin.

Flow cytometry

For extracellular staining, 2 � 105 cells were incubated for10 min at 4�C with anti-CD16/CD32 Fc block (1 lg/mL). Thiswas then followed by addition of fluorochrome-conjugatedanti-CD40, anti-CD86 and isotype-matched controls (BD

Pharmingen, Oxford, UK). After 30 min incubation at 4�C,cells were washed in wash buffer (PBS/1% FCS/0.1% sodiumazide) and resuspended in 0.4 mL 1% paraformaldehyde. Forintracellular cytokine staining, S. typhimurium-infected cellswere incubated for 6 h with culture media containing 10 lg/mL brefeldin-A (Sigma) at 37�C/5% CO2 in air incubator. Cellswere then washed and extracellular molecules stained asdescribed. Following this, cells were fixed in 2% paraform-aldehyde containing 10 lg/mL brefeldin-A for 20 min at roomtemperature. Cells were then washed twice with wash bufferand permeabilised by incubation with 0.5% Saponin in washbuffer. Cells were resuspended in 50 lL antibody to fluor-ochrome-conjugated IL-6, TNF-a, IL-12p40 or isotype-matched control antibodies (BD Pharmingen) diluted in0.5% saponin. After 30 min, cells were washed twice in0.5% saponin, then twice in wash buffer, followed by fixationin 0.4 mL 1% paraformaldehyde. CCR7 expression wasdetected by binding of a CCL19-human Ig fusion protein(4 lg/mL; kindly provided by J.G. Cyster [47]), followed bydetection with a goat-anti-human Ig PE conjugate (10 lg/mL;Jackson ImmunoResearch laboratories, Stratech, Soham, UK).All stained cells were acquired on a FACSCalibur flowcytometer and data analysed using WinMDI software.

Griess assay

To detect the production of reactive nitrogen intermediatessuch as NO generated by the L-arginine oxidation pathway,nitrite (NO2

–), the stable end product of L-arginine oxidationby inducible NO synthase (iNOS), was quantified using theGriess diazotization reaction. Solution 1 (2% sulphanilamide,5% H3PO4 in distilled water) and Solution 2 (0.2%naphthylethylenediamine in PBS) were prepared and addedin a 1:1 ratio to obtain a colourless solution. Following this,standards of sodium nitrite were prepared in PBS by seventwofold serial dilutions of a 100 lM solution, using wells withno NaNO2 as a negative control. A 75-lL aliquot of supernatantor standard to be tested was added to the wells of a 96-wellMicroTest III plate in triplicate, and 25 lL of the colourlesssolution mixture was then added to each test well. The platewas incubated with shaking for 5 min on a plate shaker; theabsorbance of the colour reaction was measured at 540 nmwavelength in a microtiter plate reader.

T cell isolation

NP383–391-specific CD8+ and CD4+ T cells were isolated fromGRb TCR transgenic mice. Single cell suspensions of spleencells were obtained and incubated with mAb against B220,class II and either CD4 or CD8, followed by anti-rat IgG beads(Dynal) to remove unwanted cells. Purified cells achievedgreater than 95% purity after isolation, as deemed by antibodystaining and flow cytometry.

Adoptive transfer

BMMac, BMDC and T cells were labelled with the fluorescentprobe CFSE (Molecular Probes-Invitrogen, Paisley, UK) totrack their location after adoptive transfer in vivo. Cells wereresuspended to a density of 107/mL in PBS/0.1% BSA



containing 5 lMCFSE and incubated for 10 min at 37�C. Afterincubation, labelled cells were washed twice with 50 mL coldPBS/5% FCS. Cells were passed through 40-lm filterresuspended to appropriate cell density in PBS and 200 lLinjected into mice. The i.p. route was used for BMMac andBMDC transfer and i.v. route for T cell transfer.

IFN-c ELISpot

A 96-well ELISpot plate (Millipore, Watford, UK) wasincubated overnight at 4�C with a rat anti-mouse IFN-ccapture antibody (final concentration 10 lg/mL; BD Pharmin-gen) in sterile PBS. On the following day, the plate was washedwith PBS and blockedwith cell culturemedia (RPMI 1640,10%FCS, 50 lM 2-ME, 100 U/mL penicillin, 100 lg/mL strepto-mycin and 1 mM sodium pyruvate) for 2 h at 37�C. Respondercells (105/well) in 50 lL media were added to each well, towhich 103–105 stimulators, either BMMac or BMDC, eitherpulsed with 1 lM peptide or infected with recombinantSalmonella, were added. After 24 h of incubation at 37�C/5%CO2 in air incubator, wells were washed PBS/0.01% Tween20.Antibody binding was then detected with a biotinylated ratanti-IFN-c detection antibody (2 lg/mL; BD Pharmingen),followed by streptavidin-HRP. Spots were developed byincubation with 3-amino-9-ethylcarbazole (AEC) HRP sub-strate (Calbiochem, Nottingham, UK).

Proliferation assay

Briefly, 105 T cells (responder cells) and to which 103–105

stimulators, either BMMac or BMDC, either pulsed with 1 lMpeptide or infected with recombinant Salmonella, were addedand cultured in 200 lL cell culture medium in 96-well round-bottom tissue culture plates. After 72 h, cultures were pulsedwith 0.5 lCi [3H]thymidine per well and incubated for afurther 18 h. After this time, wells were harvested ontofiltermats and thymidine incorporation measured by b scin-tillation.

Transwell migration assay

The in vitro migration of BMDC in response to CC chemokineswas assessed in a 24-well transwell cell culture chamber with5.0-lm pore size polycarbonate filters (Corning Costar, HighWycombe, UK). Cell culture medium (100 lL, containing2 � 105 cells) was added to the upper chamber of thetranswell. Media alone, CCL19 (0.3 lM) or CCL20 (100 ng/mL) (both R&D systems) in a volume of 600 lL was added tothe lower chamber. Cells were incubated at 37�C/5% CO2 for4 h. The cells that hadmoved through the polycarbonate filtersinto the lower chamber were harvested, stained with anti-CD11c and anti-I-A/I-E antibodies and numbers acquired byflow cytometry. The percentage of migrated cells wasdetermined by the number of cells in the lower chamberdivided by the number seeded in the upper chamber.

Real time RT-PCR

Total RNA was isolated from cells using the RNeasy mini kit(Qiagen, Crawley, UK) as according to the manufacturer's

protocol. Samples were then subjected to real-time RT-PCRanalysis using the Reverse Transcriptase qPCRTM Master Mixkit (Oswel Research Products Ltd, Southampton, UK). Primers(Sigma-Genosys, Poole, UK) and probes (Eurogentec, Romsey,UK) were as follows: 28S RNA (positive control; forwardprimer 50-CGCCGCTAGAGGTGAAATTCT-30; reverse primer 50-CATTCTTGGCAAATGCTTTCG-30; Probe FAM-ACCGGCGCAA-GACGCACCAG-TAMRA), CCR7 (forward primer 50-CCC-AAAACGACAGCCAAAA-30; reverse primer 50-GGCCCCACAT-CCCTCACT-30; Probe FAM-AAAGTGAGAGGCTGCCACACT-TTCCG –TAMRA) and CCR6 (forward primer 50-TTG-GTGCAGGCCCAGAA-30; reverse primer 50-CACGAGAACCA-CAGCGATCA-30; Probe FAM-CCAAGAGGCACAGAGCAAT-CCGAGTC–TAMRA). Reaction conditions consisted of30 min at 48�C and 10 min at 95�C for the reversetranscription reaction, followed by an amplification profileconsisting of 40 cycles of 15 s at 95�C, 1 min at 60�C.

Statistical analysis

Statistical significance was calculated using one-way analysisof variance followed by a two-sample t-test, using Mini-Tabstatistical software (version 13.1).

Acknowledgements: We are grateful to JagadeeshBayry for the critical reading of the manuscript. Thiswork was supported by The Edward Jenner Institute forVaccine Research. This is publication 115 from TheEdward Jenner Institute for Vaccine Research.

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Salmonella typhimurium infection triggers dendritic cells and macrophages to adopt distinct migration patternsin vivo