Identification of dendritic cell subsets responding to genital infection by Chlamydia muridarum

Authors

  • Raymond J. Moniz,

    1. Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
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  • Ann M. Chan,

    1. Department of Physiological Science, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
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  • Kathleen A. Kelly

    1. Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
    2. California NanoSystems Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
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  • Editor: Svend Birkelund

Correspondence: Kathleen A. Kelly, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA. Tel.: +1 310 206 5562; fax: +1 310 794 4863; e-mail: kkelly@mednet.ucla.edu

Abstract

Dendritic cells (DCs) are central for the induction of T-cell responses needed for chlamydial eradication. Here, we report the activation of two DC subsets: a classical CD11b+ (cDC) and plasmacytoid (pDC) during genital infection with Chlamydia muridarum. Genital infection induced an influx of cDC and pDC into the genital tract and its draining lymph node (iliac lymph nodes, ILN) as well as colocalization with T cells in the ILN. Genital infection with C. muridarum also stimulated high levels of costimulatory molecules on cDC central for the activation of naïve T cells in vivo. In contrast, pDC expressed low levels of most costimulatory molecules in vivo and did not secrete cytokines associated with the production of T helper (Th)1 cells in vitro. However, pDC upregulated inducible costimulatory ligand expression and produced IL-6 and IL-10 in response to chlamydial exposure in vitro. Our findings show that these two DC subsets likely have different functions in vivo. cDCs are prepared for induction of antichlamydial T-cell responses, whereas pDCs have characteristics associated with the differentiation of non-Th1 cell subsets.

Introduction

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide (WHO, 2001). Inflammation of the reproductive tract following infection with Chlamydia can result in severe sequelae, which include pelvic inflammatory disease and infertility (WHO, 2001). Recent examinations of the clinical diagnosis and treatment of genital chlamydial infections have revealed that attenuation of infection through antimicrobial treatment increases the reinfection rate in humans and may exacerbate immunopathology within the reproductive tract (Brunham & Rekart, 2008). These findings highlight the need for an effective vaccine against genital chlamydial infection. Recent efforts to design a vaccine against Chlamydia have been ineffective due, in part, to the elusiveness of chlamydial antigens and conditions that induce a long-lasting, protective response. An additional obstacle for vaccine design against Chlamydia is the implication of the immune response in the genital pathology associated with infection (Roan & Starnbach, 2008). Together, these findings emphasize the importance of discrimination between protective and pathological host immune responses in the design of an effective vaccine against genital infection by C. trachomatis.

The culmination of work to date examining the immune response to genital infection by Chlamydia has indicated that T cells are central to the clearance of infection (Brunham & Rey-Ladino, 2005). Among T-cell subsets, an interferon (IFN) γ-producing CD4+ subset [T helper (Th)1] has been shown to be dominant and to be required for clearance of genital infection. In addition to protection, T cells have also been implicated in prolonged inflammation thought to promote adverse reproductive outcomes (Roan & Starnbach, 2008). This possibility, as well as the central role that T cells play in the clearance of infection, emphasizes the need for a more clear delineation of the events that initiate and regulate T-cell responses to chlamydial infections.

The induction of effector T-cell responses is controlled by dendritic cells (DCs) (Steinman & Hemmi, 2006). Recent examination of the potential for DC to induce protective and nonpathological T-cell responses has been demonstrated in a mouse model of chlamydial infection (Su et al., 1998). Adoptive transfer of bone marrow (BM)-derived DC pulsed with inactivated Chlamydia muridarum was capable of inducing a protective Th1 response and did not induce immunopathology within the genital tract (GT) following a challenge with C. muridarum. Subsequent work utilizing BM-derived DC has shown that varying the chlamydial antigens used to pulse DC in vitro can affect the level of protection conferred following adoptive transfer (Rey-Ladino et al., 2005). Despite these efforts, little has been demonstrated as to the activity of DC subsets in vivo during genital chlamydial infection. DCs are comprised of a heterogeneous family of cells that exhibit an extensive plasticity of function. Dependent on the immune context, different DC subsets have been shown to induce contrasting T-cell responses (Kapsenberg, 2003). Recent examination of DC subsets in a lung model of chlamydial infection demonstrated that distinct DC subsets have divergent effects on the differentiation of T-cell subsets (Bilenki et al., 2006). Therefore, the existence of multiple DC subsets may account for the varying T-cell responses seen during chlamydial genital infection.

This study was designed to determine whether distinct DC subsets respond to chlamydial genital infection. Our efforts have focused on those DCs that may account for the varying T-cell responses demonstrated to be induced during genital infection, with particular focus on the potential of DCs to induce or alter the protective Th1 response. To examine DC subset activity in vivo, we have examined the influx, localization, and costimulatory molecule expression of DC subsets both in the GT and its draining lymph node (iliac lymph node, ILN).

Materials and methods

Chlamydia muridarum propagation and infection

Chlamydia muridarum (Nigg strain) was grown, purified, and titered in McCoy cells as described previously (Maxion et al., 2004). Elementary bodies (EB) and reticulate bodies isolated from McCoy cells were frozen in sucrose-phosphate-buffered saline (SPS) at −80 °C until use. Female BALB/c mice, aged 5–6 weeks, were treated with progesterone (Depo-Provera, 2.5 mg in 100 μL sterile PBS) 1 week before infection. Following anesthetization, mice were inoculated with 1 × 105 infection-forming units intravaginally. For determination of bacterial load, mice were swabbed vaginally every 3 days after infection. Swabs were placed in 200 μL SPS and frozen at −80 °C. Bacterial load was determined in McCoy cells as described previously (Maxion et al., 2004). For all experiments, mice were housed and treated in accordance with the American Association of Accreditation of Laboratory Animal Care guidelines. Experimental procedures were approved by the UCLA Institutional Animal Care and Use Committee.

DC isolation and characterization from C. muridarum-infected mice

DC isolation was performed as described previously, with modifications (Nakano et al., 2001). Briefly, tissues of interest were excised from five mice, minced, pooled, and cultured in the presence of collagenase (1 mg mL−1, Type I, Sigma) and DNAse (0.4 mg mL−1, Sigma) in Hank's balanced salt solution with Ca2+ and Mg2+. Spleens and ILN were incubated for 20 min and GTs for 60 min at 37 °C. Following incubation, single-cell suspensions were obtained by mincing tissue through a 70-μm filter (Falcon) in HBS with 5 mM EDTA and 0.5% BSA. Cells were washed twice and maintained in a solution containing 5 mM EDTA to minimize cell adhesion. Cell suspensions were applied to freshly prepared Nycodenz (Accurate Chemical) gradients [15% w/v in RPMI supplemented with 10% v/v heat-inactivated fetal calf serum (FCS)] and spun for 20 min at 450 g. Low-density cells were removed from the interface, washed, and resuspended in the appropriate buffer.

DC subset identification was performed by fluorescence-activated cell sorting (FACS) as described previously (Liu & Kelly, 2008). Briefly, low-density fractions were incubated on ice in the presence of Fc-Block (BD Pharmingen) for 20 min, washed, and incubated with antibodies specific for surface molecules. Fluorochrome or biotin-conjugated antibodies for CD11c (N418), CD45R (RA3-62B), CD11b (M1/70), CD3 (145-2C11), CD19 (MB19-1), and Ly6G/C (RB6-865) were purchased from eBioscience. The pDC-specific antibodies, mPDCA-1 and 120G8, were purchased from Miltenyi Biotec or received as a gift from Schering Plough Research Institute, respectively. DC subsets were defined as low-density cells that were CD11c+CD3−CD19− and expressed either CD11b or CD45R (see results for DC subset phenotyping). Costimulatory molecules were measured on DC subsets by staining for the surface molecules: CD40 (1C10), CD80 (16-10A1), CD86 (GL1), ICOSL (HK5.3), and were purchased from eBioscience. Antibodies to I-Ad (AMS-32.1) were purchased from BD Pharmingen. FACS acquisition was performed on a FACSCaliber (Becton Dickinson) at the UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility. FACS analysis was performed using fcs express (Denovo software).

Immunofluorescent localization of DC subsets

ILN were harvested 3 days after infection, placed in optimal cutting temperature freezing media (Tissue Tek), flash frozen, and stored at −80 °C. Tissue sections (5 μm) were cut by the UCLA Tissue Procurement Laboratory. Consecutive sections were stained sequentially with unconjugated CD3 (145-2C11, eBioscience), goat-anti-rat ALEXA 546 (Invitrogen), CD11c-biotin (HL3, BD Pharmingen) or BST2-biotin (conjugated inhouse using FluorReporter Biotin-XX, Invitrogen), and streptavidin-ALEXA 488, and mounted using Prolong Gold antifade reagent with 4′,6′-diamino-2-phenylindole (Invitrogen). Sections were visualized using a Leica TCS-SP MP at the UCLA Brain Research Institute Microscopy Core Facility. Images were taken using a × 20 objective and resized for publication using photoshop cs3 (Adobe).

In vitro differentiation and stimulation of pDC

pDCs were differentiated from BM precursors as described previously, with modifications (Naik et al., 2005). Briefly, the femurs and tibias were excised from 8–10-week-old female BALB/c mice and BM extracted by flushing with RPMI supplemented with 10% FCS. After RBC lysis, cells were cultured in complete media (RPMI 1640 with 10 mM HEPES, 10 mM sodium pyruvate, 2 mM l-glutamine, 10 mM nonessential amino acids, 10% v/v FCS, and 100 μg mL−1 penicillin and streptomycin) supplemented with recombinant FLT3L (0.2 μg mL−1; R&D Systems) in 100-mm bacteriological petri dishes (Becton Dickinson) at 2 × 106 cells mL−1. Half of the culture media was removed and replaced with fresh FLT3L containing complete media every 3 days. On day 10 of culture, nonadherent cells were removed by pipetting and enriched on a Nycodenz gradient. The BM cells were further enriched for pDC by depleting CD11b+ cells from the low-density fraction using magnetic bead isolation and CD11b-biotin (M1/70, eBioscience), followed by streptavidin microbeads (Miltenyi Biotec). pDCs were routinely purified to >90% CD11c+CD45R+120G8+CD11b− cells.

Purified pDCs were stimulated in complete media (2 × 106 cells mL−1) without penicillin and streptomycin in the presence of renograffin-purified live C. muridarum EBs (a multiplicity of infection of 1), CpG-C (5 μg mL−1, 5′-TCGTCGAACGTTCGAGATGAT-3′), or media alone for 48 h. For CpG motifs, bold letters indicate phosphorothioate linkages. After stimulation, cell culture supernatants were collected for enzyme-linked immunosorbent assay (ELISA) analysis and the remaining cell pellet was resuspended, washed, and analyzed by FACS as described above. ELISA analyses of culture supernatants were carried out using DUO-KIT sets for IFNα, interleukin (IL)-6, IL-10, and IL-12p70 according to the manufacturer's protocol (R&D Systems).

Results

Two distinct DC subsets dominate the response to genital C. muridarum infection

To determine the DC subsets responding to genital C. muridarum infection, we examined both the GT and its primary draining lymph node, the ILN. We focused our initial examination of DC subsets responding to infection on day 7 postinfection when bacterial burden was high in GT tissues (Fig. 1a) and the T-cell response was developing in the ILN (data not shown). This time point ensured that the DC subsets examined would be those most likely to interact with T cells responsible for clearance of infection. Our classification of DC subsets focused on the distinct expression of surface molecules by individual DC subsets (Ardavin, 2003). All DC subsets in mice express CD11c and can be broadly divided into two subsets: classical DC (cDC) and plasmacytoid DC (pDC). cDCs encompass a heterogeneous group of cells classified by surface expression of the molecules CD8 or CD11b. Expressions of CD11b and CD8 on cDCs in the mouse are mutually exclusive. pDCs are unique among DCs in their expression of CD45R (B220) and have been shown to express variable levels of CD8 (O'Keeffe et al., 2002). Therefore, to determine the composition of DC responding to chlamydial infection, we utilized a four-color FACS-staining regimen, which identified CD11c+, CD11b+, CD45R+ cells plus a single channel for excluding T (CD3+) and B (CD19+) cells that have been shown to express low levels of CD11b and high levels of CD45R (Nakano et al., 2001). In the ILN and GT, we found that two DC subsets dominated the response to infection. These included a CD11c+CD11b+(cDC) and a CD11c+CD45R+(pDC) subset (Fig. 1b). The pDC subset was further confirmed by the expression of antigens found on pDC, but not cDC: BST2 (Blasius et al., 2006) and Gr-1 (Ly6C/G) (Nakano et al., 2001) (Fig. 1c). Expression of CD8 by DC subsets was limited to heterogeneous levels on only pDC (data not shown), indicating that the CD11c+CD8+DC described in a lung model of C. muridarum infection does not respond to genital C. muridarum infection (Bilenki et al., 2006). Together, these data demonstrate that two DC subsets respond to genital C. muridarum infection: a CD11b+cDC and a pDC subset.

Figure 1.

 Identification of DC subsets responding to genital infection by Chlamydia muridarum. (a) Shedding of C. muridarum was determined every 3 days after vaginal inoculation with 1 × 105 infection-forming units. Data points represent the SEM from three mice. (b) FACS plots of DC subsets from the ILN and GT of day 7 infected mice. Plots represent CD11c+CD3−CD19− cells, and are representative of three independent experiments. (c) Phenotyping of pDC was determined as in (b), but gated on CD11c+CD3−CD19−CD45R+ cells. Black histograms indicate staining for the indicated marker and gray histograms represent isotype controls. For both (b) and (c), tissues from five mice were pooled and treated as described in Material and methods.

cDC and pDC show comparable recruitment to the draining lymph node, but not the site of infection during genital C. muridarum infection

DCs acquire the antigen at sites of infection and home to the draining lymph node of the infected tissues to present the acquired antigen to naïve T cells (Randolph et al., 2008). Our finding that two DC subsets respond to C. muridarum in the GT and ILN raises the possibility that both subsets could be responsible for the induction of effector T cells. Additionally, T-cell responses to C. muridarum have been shown to vary during the course of genital infection (Kelly, 2003) and hence we sought to determine whether the variability in the distribution of DC subsets responding to infection might account for the variability in T-cell responses. Because naïve T-cell differentiation in the draining lymph node is essential for clearance of chlamydial infection, we first examined the distribution of DC in the ILN. As others have found in similar models (Asselin-Paturel et al., 2003), pDCs dominate the distribution of DC subsets in the naïve ILN (Fig. 2a). Alternatively, cDCs represent a small percentage of the DC present in the naïve ILN. Following infection, this distribution shifts to comparable proportions of both subsets between days 7 and 10 postinfection. The DC distribution in the ILN begins to revert to the naïve state as the bacterial load in the GT decreases (Fig. 1a), and by day 21 postinfection, the DC distribution is nearly equivalent to that of the naïve ILN (Fig. 2a). In contrast to the ILN, the distribution of DC subsets in the GT is dominated by cDC throughout the course of infection (Fig. 2b) and persists beyond detectable bacterial shedding on day 21 (Fig. 1a). Together, these data indicate that both cDC and pDC respond comparably in their influx to the ILN, but that cDCs show a dominance in the distribution of DC in the GT throughout infection.

Figure 2.

 DC subset distribution and total numbers in the ILN and GT of Chlamydia muridarum-infected mice. The distribution of DC subsets in the ILN (a) and GT (b) was determined as the percentage of DC (CD11c+ CD3−CD19−) for cDC (CD11b+ DC) or pDC (CD45R+ DC). Error bars represent the SEM of three independent experiments in which the DC subset distribution was determined by pooling of ILNs or GT from five mice. (c) DC subset numbers in naïve (mock) and infected tissues were determined by FACS as in (a) and (b). Naïve mice were inoculated with SPB without C. muridarum (CM). Infected groups represent the combined analysis of DC subsets on days 7 and 10 postinfection. Data points represent the mean value +/− SEM from three independent experiments in which DC were examined from the pooled tissues of five mice. *P>0.05 vs. uninfected, **P>0.05 between DC subsets by Student's t-test. ND, not detected.

To determine the distribution of DC in the ILN and GT during infection more precisely, we compared absolute numbers of cDC and pDC on days 7 and 10 postinfection. As mentioned and indicated in the above data, this is the approximate peak of both DC (Fig. 2a) and T cells (not shown) in the ILN and GT. We found that cDC and pDC are present in comparable numbers in the ILN of infected mice (Fig. 2c). Alternatively, there are nearly threefold greater numbers of cDC in the infected GT. These data further demonstrate that both DC subsets respond comparably to infection by their influx to the ILN, whereas cDCs dominate the DC population in the GT throughout the course of infection.

Both cDC and pDC colocalize with T cells in the ILN during infection

The disparity in DC subset numbers and distribution in the ILN and GT raises the question as to whether these DC subsets have the same potential for interaction with naïve T cells. Recent work has shown that the trafficking patterns of cDC and pDC differ. cDCs accumulate in tissues and enter lymph nodes through the afferent lymphatics. In contrast, pDCs primarily enter peripheral lymph nodes (PLN) from the blood through high endothelial venules (HEV) (Randolph et al., 2008). This would indicate that during genital C. muridarum infection, pDC could influx to the ILN without encountering antigen in the GT. Furthermore, pDC may adhere to HEV in the ILN without interacting with T cells (Diacovo et al., 2005). The above data, demonstrating a strong pDC influx into the ILN, but not the GT, are consistent with this possibility. To examine whether pDCs have the potential to interact with T cells in the ILN, we histologically examined DC localization in the ILN on day 3 postinfection. The differential expression of CD11c on cDC and pDC allows for discrimination between these DC subsets histologically (Asselin-Paturel et al., 2005). Upon examination of DC subsets in situ with immunofluorescence, pDCs appear negative or very dim for CD11c, whereas cDCs appear bright. pDCs express high levels of BST2 and are stained brightly when using the antibody 120G8. We therefore examined the localization of pDC and cDC in the ILN by examination of BST2 and CD11c bright cells, respectively. As shown in Fig. 3, cDC and pDC are present in the T-cell zone and not in the B-cell zone of the ILN on day 3 postinfection. We found similar localization of DC through day 10 postinfection (not shown). Therefore, the accumulation of cDC and pDC in the T-cell zone of the ILN suggests that both DC subsets have the potential to interact with naïve T cells during genital infection by C. muridarum.

Figure 3.

 Localization of DC subsets in the ILN on day 3 postinfection. ILNs were extracted from mice on day 3 postinfection, flash frozen, and stored until sectioning as described in the Materials and methods. Sections were stained sequentially for T cells (CD3, red), pDC (BST2, green) or cDC (CD11c, green), and nuclei (4′,6′-diamino-2-phenylindole postinfection, blue). Images represent consecutive 5-μm sections examining cDC (a, c, and e) or pDC (b, d, and f) at × 40 magnification objective.

cDC and pDC exhibit differential expression of costimulatory molecules in the ILN

The above data suggest that cDC and pDC have the potential for T-cell interaction in the ILN. However, the capacity of pDC to enter inflamed lymph nodes without becoming activated in tissues suggests that pDC may enter the ILN without prior exposure to C. muridarum (Diacovo et al., 2005). Microbial stimulus has been shown to increase the expression of both major histocompatibility complex (MHC) and costimulatory molecules on DC, and is considered essential for DC-mediated activation and differentiation of naïve T cells into effector T cells such as Th1 cells (Kapsenberg, 2003). To examine each subset's activation level and thus potential for CD4+ T helper cell activation, we examined the expression of MHC2 and costimulatory markers on each DC subset in the ILN on day 7 postinfection. The costimulatory molecules examined were chosen for their indication of DC activation (CD80, CD86, and ICOSL) as well as their implication in T-cell interaction (CD40). To control for baseline levels of MHC2 and costimulatory expression, we examined the expression of these molecules on DC isolated from pooled PLN of naïve mice (mesenteric, cervical, and iliac). During infection, cDCs within the ILN expressed high levels of MHC2 and the costimulatory molecules examined (Fig. 4a). However, cDCs in the ILN only markedly upregulated CD80 in response to chlamydial genital infection compared with basal levels of costimulatory molecules measure in naïve PLN (Fig. 4a). In contrast, pDCs in the ILN express lower levels of MHC2, CD40, CD80, and CD86 (Fig. 4b), indicating that pDCs inherently express lower levels of costimulatory molecules, which are not upregulated in response to C. muridarum infection. Interestingly, higher expression levels of ICOSL were found in pDC from infected ILN compared with the basal expression measured on pDC from PLN. These data indicate that cDC in the ILN are well prepared for the activation of naïve T cells. Alternatively, pDCs express a pattern of costimulatory molecules distinct from cDCs, suggesting that they do not directly induce T-cell activation.

Figure 4.

 DC subset expression of costimulatory molecules in the ILN. DC subsets were examined by FACS as in Fig. 1, and further examined for surface expression of costimulatory molecules. Histograms represent gating on cDC (a) or pDC (b) from pooled naïve PLN (gray), day 7 ILN (black), and isotype controls (dotted, open) for expression of the indicated protein. Data are representative of at least three independent experiments, and represent the pooled ILNs of five mice.

Chlamydia muridarum does not activate pDC for Th1 cell activation in vitro

The above findings indicate that pDC influx into and colocalize with T cells in the ILN, but lack the requisite surface expression of molecules for T-cell activation (Kapsenberg, 2003). These data raise the question as to whether pDCs can be activated following exposure to C. muridarum. To confirm the unique expression of costimulatory molecules on pDC and investigate cytokine production in response to Chlamydia, we utilized an in vitro model using pDC differentiated from BM precursors (Naik et al., 2005). We examined the production of cytokines central in the differentiation (IL-12) (Szabo et al., 2003) or inhibition (IL-10) of Th1 differentiation (Couper et al., 2008) and cytokines associated with innate functions of pDC (IFNα and IL-6) (Liu, 2005). We exposed pDC to CpG as a positive control of both activation and cytokine expression by pDC (Liu, 2005). Upon exposure to C. muridarum in vitro, pDC upregulated the expression of MHC2, but at lower levels compared with stimulation with the CpG control. As seen in vivo, pDC upregulated ICOSL in response to chlamydial exposure (Fig. 5a). Upon examination of cytokine expression (Fig. 5b), we found that pDC did express small amounts of IL-6 and IL-10, but not IL-12 or IFNα. These data indicate that immature pDCs respond mildly to chlamydial exposure. Likewise, these data indicate that pDCs only show a mild innate response to C. muridarum as compared with CpG. This is consistent with previous work demonstrating that toll-like receptor 9 (TLR9), the receptor for CpG, which is highly expressed by pDC (Liu, 2005), does not play a role in innate responses to chlamydial infection (Rothfuchs et al., 2004). Finally, the lack of expression of IL-12, but mild expression of IL-10 and upregulation of ICOSL suggest that pDCs do not contribute toward inducing a protective Th1 response.

Figure 5.

 pDC reactivity to Chlamydia muridarum in vitro. pDC were differentiated from BM precursors and purified as described in Materials and methods. (a) Expressions of the indicated markers were examined by FACS on pDC as in Fig. 4 following a 48-h exposure to live C. muridarum (CM) EBs (black histograms), CpG (hashed histograms), or media alone (gray histograms). Dotted, open histograms were incubated with CM and stained with isotype control for the indicated protein. Data are representative of two independent experiments. (b) Cytokine expression by pDC was determined by ELISA from the tissue culture supernatant of the cultures in (a). Data represent the mean values of duplicate measurements from one experiment, and are representative of two independent experiments.

Discussion

The involvement of multiple DC subsets responding to microbial insult has been described in several models of infection (Bilenki et al., 2006; GeurtsvanKessel et al., 2008; Hao et al., 2008; Pepper et al., 2008; Smit et al., 2008). This work is the first to describe the extent of the involvement of distinct DC subsets in a model of chlamydial genital infection. The involvement of two distinct DC subsets in this model has particular significance as DCs are central in the induction of T-cell responses, and clearance of chlamydial infection requires T cell-mediated immunity. The importance of DCs in the induction of a protective, nonpathological T-cell response has been shown by others through the adoptive transfer of C. muridarum-exposed DC before genital challenge (Su et al., 1998). This work utilized BM-derived DC in cultures supplemented with granulocyte macrophage colony-stimulating factor and IL-4, a method that effectively induces the differentiation of activated cDC, but not pDC (Xu et al., 2007). Our findings are consistent with this work in that we also found that a cDC subset is activated as a result of chlamydial infection. Furthermore, we demonstrate that this cDC subset is well prepared for the induction of chlamydial-specific T-cell responses through its influx into the site of infection, increased expression of costimulatory molecules, and localization in the T-cell zones of the ILN as a result of infection. Therefore, the cDC we describe in this work may be the in vivo equivalent of the DC utilized in previous studies (Su et al., 1998; Rey-Ladino et al., 2005). This indicates that the primary DC subset involved in the induction of antichlamydial T-cell responses to genital chlamydial infection is a CD11b+ cDC.

It is interesting to note that we did not detect any CD8+ cDC in either the GT or the ILN during infection. In a recent examination of a lung model of chlamydial infection, it was shown that a CD8+ cDC subset increased in the spleen of mice infected with C. muridarum (Bilenki et al., 2006). Our study did not examine the DC subsets in the spleens of infected mice, but rather focused on more immediate sites of T-cell induction (Randolph et al., 2008). The findings of this study and those of chlamydial infection in the lung indicate that compartmentalization of DC subsets may occur during chlamydial infection. In the lung model, a splenic CD8+ cDC subset is the prominent inducer of a protective T-cell response. Because of the constraints of the DC numbers found in the ILN, we were unable to directly examine the T cell-inducing capacity of individual DC subsets. However, our findings indicate that during genital infection, a CD11b+ cDC is well prepared for the induction of a protective T-cell response against C. muridarum. The significance of this discrepancy between models of chlamydial infection may be indicative of both a compartmentalization of DC subsets to specific tissues as well as the variability of DC subsets in their capacity to induce protective responses to Chlamydia in different tissues. Both of these factors must therefore be taken into account in future studies targeting specific DCs for vaccination against chlamydial infection in specific tissues.

The influx of two distinct DC subsets into both the GT and the ILN during infection suggests that the T-cell response to C. muridarum would be dictated by these subsets. Interestingly, our data indicate that there is a division of labor between cDC and pDC. Our work demonstrates that the cDC subset resembles DCs that induce a protective Th1 cell response (Su et al., 1998; Rey-Ladino et al., 2005), while pDCs seem to be incapable of inducing such responses. In other models of genital infection, pDCs play a significant role in the innate response to viral infection through the production of IFNα (Lund et al., 2006). In contrast, we demonstrate that pDCs do not respond robustly to C. muridarum in vitro (Fig. 5b), producing no IFNα and only small amounts of IL-6 that might contribute to innate responses in the GT. This indicates that pDCs are inherently not capable of a strong innate response to C. muridarum, and is consistent with the demonstration that TLR9, a key protein in pDC activation, is not involved in the innate response to Chlamydia (Rothfuchs et al., 2004). Alternatively, the disparity between DC subsets may in part be attributed to the inability of pDCs to harbor a viable chlamydial infection. Our current efforts are focused on determining this possibility. Together, our findings indicate that pDCs do not contribute to the innate response in the GT during chlamydial infection. Instead, the role of pDCs in the GT may be to modulate the function of other cells responding to infection (Liu, 2005).

The influx of pDCs into the ILN during infection, and yet its apparent inability to directly induce a protective T-cell response, raises the question as to the role of pDC in the ILN. The ability of pDCs to enter inflamed lymph nodes through HEV may explain how pDCs enter the ILN despite an inability to react to C. muridarum (Diacovo et al., 2005). To our knowledge, no data exist demonstrating that pDCs acquire antigen in the periphery for presentation in the draining lymph node. Instead, it appears that pDCs do not traffic through the afferent lymph (Yrlid et al., 2006). It is possible that pDCs acquire chlamydial antigen in the ILN in a secondary manner, without acquisition in the GT. However, the lack of costimulatory molecule expression and in particular CD40 expression (Fig. 4b) by pDC in the ILN indicates that pDCs do not function in an antigen-specific manner (Quezada et al., 2004). Therefore, our findings and work by others would suggest that pDCs in the ILN are acting in an antigen-independent manner and are not directly responsible for the induction of antichlamydial T cells.

Our data indicate that pDCs upregulate the expression of ICOSL in response to C. muridarum in vivo (Fig. 4b) and in vitro (Fig. 5a). Additionally, pDCs express IL-10 upon exposure to C. muridarum in vitro (Fig. 5b). Previous work examining the role of ICOS during chlamydial infection demonstrated that ICOS acts to dampen the Th1 response to chlamydial infection through induction of IL-10 and expansion of regulatory T cells (Treg) (Marks et al., 2007). Therefore, our observations that pDCs express ICOSL and IL-10 in response to C. muridarum indicate that pDCs may promote the expansion of regulatory responses during chlamydial infection. This possibility is consistent with recent work demonstrating that pDCs have a propensity for induction of Treg cells (Kuwana et al., 2001; Martin et al., 2002; Bilsborough et al., 2003). In preliminary studies, we have found Treg to be present in the ILN and GT during the course of infection in a pattern that mimics the presence of pDC in these tissues. Therefore, our current efforts are focused on determining whether pDCs have a role in the regulation of the immune response to C. muridarum infection through the induction of Treg subsets.

In summary, we have demonstrated that two distinct DC subsets respond in a genital model of chlamydial infection. Of these subsets, a CD11b+ cDC is the most well prepared for the induction of the requisite Th1 response for clearance of genital chlamydial infection. Alternatively, a pDC subset also responds to infection, but does not demonstrate the requisite characteristics for the induction of an antichlamydial effector T-cell response. Although pDCs have been shown to activate CD4+ T cells (Sapoznikov et al., 2007), our findings indicate that pDCs do not activate T cells during genital C. muridarum infection. Instead, pDCs seem well prepared for the induction of Treg responses during genital chlamydial infection. This has particular relevance in the rational design of DC-targeting vaccines, indicating that cDC and not pDC should be the focus for induction of antichlamydial T-cell responses.

Acknowledgements

We would like to thank Micheal Gunn and Ken Shortman for their guidance in the isolation of DC and Carine Asselin-Paturel for assistance with utilization of the 120G8 antibody. This work was supported by NIH grants RO1 IA-026328 (K.A.K., R.J.M., and A.M.C.), T32 AI107126-28 (R.J.M.), and T32 AI52031 (R.J.M.). The UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility are supported by National Institutes of Health awards CA-16042 and AI-28697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School of Medicine at UCLA.

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