Enhanced Inducible Costimulator Ligand (ICOS-L) Expression on Dendritic Cells in Interleukin-10 Deficiency and Its Impact on T-Cell Subsets in Respiratory Tract Infection
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An association between inducible costimulator ligand (ICOS-L) expression and interleukin (IL)-10 production by dendritic cells (DCs) has been commonly found in infectious disease. DCs with higher ICOS-L expression and IL-10 production are reportedly more efficient in inducing regulatory T cells (Tregs). Here we use the Chlamydia muridarum(Cm) lung infection model in IL-10 knockout (KO) mice to test the relationship between IL-10 production and ICOS-L expression by DCs. We examined ICOS-L expression, the development of T-cell subsets, including Treg, Th17 and Th1 cell, in the background of IL-10 deficiency and its relationship with ICOS-L/ICOS signaling after infection. Surprisingly, we found that the IL-10 KO mice exhibited significantly higher ICOS-L expression by DCs. Moreover, IL-10 KO mice showed lower Tregs but higher Th17 and Th1 responses, but only the Th17 response depended on ICOS signaling. Consistently, most of the Th17 cells were ICOS+, whereas most of the Th1 cells were ICOS− in the infected mice. Furthermore, neutralization of IL-17 in IL-10 KO mice significantly exacerbated lung infection. The data suggest that ICOS-L expression on DC may be negatively regulated by IL-10 and that ICOS-L expression on DC in the presence or absence of IL-10 costimulation may promote Treg or Th17 response, without significant impact on Th1.
In addition to T-cell receptor (TCR) engagement, the initiation of optimal T-cell response requires costimulatory signals. The molecular mechanisms underlying the development of different T-cell subsets remain unclear. Inducible costimulator (ICOS) and its ligand (ICOS-L) are recently recognized costimulatory molecules belonging to the CD28 superfamily (1, 2, 3). ICOS is not expressed on naive T cells but can be upregulated quickly when the cells are activated (3,4). ICOS can be expressed by different T-cell subsets, but appears more on Th2 and regulatory T cells (5, 6, 7, 8, 9, 10, 11). The only ligand for ICOS is B7RP-1 (ICOS-L), which is found on dendritic cells (DCs), macrophages and some epithelial cells (12). DCs are the most important antigen-presenting cell (APC) in activation and direction of T cells. Expression of surface costimulatory molecules and production of cytokines by DCs are critically important for the modulating effect of DCs on T-cell function and development of T-cell subsets. Akbari et al. (13) reported the importance of ICOS-ICOS-L interaction in the development of regulatory T cells (Tregs). They found that both the development and the inhibitory function of regulatory cells depended on the presence of interleukin (IL)-10 and on ICOS-ICOS-L interactions. More recently, we reported that chlamydial lung infection and systemic mycobacterial infection can generate DCs that exhibit increased surface expression of ICOS-L and increased IL-10 production (14,15). In addition, we found that the DCs, which coexpressed higher ICOS-L and IL-10 after infection, were more efficient in inducing Tregs to allergen exposure (14,15).
A newly identified CD4+ T-cell subset (Th17) is characterized by its predominant production of IL-17. Th17 cell is distinct from Th1 and Th2 in its developmental pathway and function. Certain cytokines are particularly important for Th17 response (17, 18, 19, 20, 21). There is overlap in the required signaling of the cell surface marker and cytokine environments for Treg and Th17 development (22, 23, 24, 25, 26, 27). In particular, ICOS-ICOS-L interaction appears highly associated with both Treg and Th17 responses (22,23). Th17 was initially reported to be pathological in inflammatory autoimmune diseases (28,29) but was later found to be involved in host defense against extracellular bacterial and fungal infections (rev. in 30). More recently, the involvement of Th17/IL-17 in protective immunity against intracellular bacterial infections was also reported (31, 32, 33, 34). In particular, we and others reported that IL-17 is important in host defense against chlamydial lung infection (31,34). Inconsistencies on the role of ICOS-ICOS-L interaction in Th17 responses have been reported (35, 36, 37, 38). One study found that ICOS knockout (KO) mice had reduced Th17 cells (37), whereas other studies showed increased Th17 cells in the condition of ICOS or ICOS-L deficiency (36,38).
Chlamydia, a genus of obligate intracellular bacteria, consists of human pathogenic species such as Chlamydia trachomatis and Chlamydophila pneumoniae, causing a diverse spectrum of human diseases involving ocular, genital and respiratory systems (39,40). No human vaccine is available to prevent chlamydial infections. Better understanding of the mechanisms related to protection and pathogenesis of chlamydial infections is essential for rational development of a safe and effective vaccine. Chlamydia muridarum (Cm), an adapted chlamydial strain in mouse, has been widely used in mouse models for studying the mechanisms of host defense and chlamydial pathogenesis (41). Previous studies have demonstrated that Th1 cells are critically important for host defense against chlamydial infections (42, 43, 44, 45, 46, 47, 48, 49, 50). However, we recently found that ICOS-L KO mice showed exacerbated chlamydial infection, even with an enhanced Th1 response (51).
Given the demonstrated involvement of ICOS-ICOS-L signaling in both Treg and Th17 responses and the reported importance of Th1 in host defense against chlamydial infections, we planned to further dissect the condition for the development of these T-cell subsets by using a lung infection model. In addition, considering the reported close association of IL-10 production and ICOS-L expression on DCs, we wanted to specifically test if ICOS-L expression on DCs depends on IL-10 production. We used IL-10 KO and wild-type (WT) C57BL/6 mice to address these questions. We found that DCs in IL-10 KO mice expressed significantly higher levels of ICOS-L than WT mice after chlamydial lung infection. Moreover, we found that IL-10 deficiency led to reduced Tregs but higher Th17 and Th1 responses. However, only Th17 cells depended on ICOS-ICOS-L signaling, and most of Th17 but not Th1 cells expressed ICOS. Further, blockade of IL-17 in IL-10 KO mice led to enhanced chlamydial infection in vivo. The results suggest a negative regulatory role of IL-10 on ICOS-L expression and a key role of ICOS-ICOS-L interaction in Th17 development, which is critical for host defense against an intracellular bacterial infection.
Materials and Methods
Female IL-10 KO mice (IL-10−/−; C57BL/6-Il-10tm1Cgn) and ICOS KO mice (ICOS−/−, C57BL/6.129P2-Icostm1mak/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Age- and sex-matched WT C57BL/6 mice were purchased from The Jackson Laboratory, Charles River Canada (St. Constant, Canada) or the University of Manitoba breeding facility (Winnipeg, Manitoba, Canada). The mice were hosted under specific-pathogen-free conditions in the University of Manitoba animal facility for at least 2 wks before the experiment started and during the whole experimental period. No difference was noted among the WT mice from different sources after Cm infection. Six- to eight-week-old mice were used in the study. All mouse experiments were performed in accordance with the guidelines issued by the Canadian Council on Animal Care. The animal experimental protocol was approved by the ethical committee of University of Manitoba
Mice Treatment and Quantitation of Chlamydial Growth In Vivo
Cm was grown in HeLa 229 cells and purified by discontinuous density gradient centrifugation as described previously (46). Infectivity of the purified Cm elementary bodies was titrated in HeLa cell culture and demonstrated as inclusion-forming units (IFUs) as described (49). The same batch of Cm preparation was used throughout the study. IL-10 KO, ICOS KO and WT mice were inoculated intranasally (i.n.) with Cm (1), 000 IFUs) in 40 µL sterile, proteinfree sucrose-phosphate-glutamic acid buffer as described (46,49). In the designated experiments, IL-17 activity in IL-10 KO mice was neutralized by using monoclonal antibodies (mAbs) as described (34). Briefly, 10 µg anti-mouse IL-17 mAbs (R&D, Minneapolis, MN, USA) in 40 µL phosphate-buffered saline (PBS) were administered i.n. to IL-10 KO mice 2 h after inoculation of Cm and was repeatedly administered every 48 h until mice were killed at d 7 after infection. The mice were monitored daily for body weight changes. The growth of Cm in the lung was determined as described (46,49).
Lung Mononuclear Cell Preparation
Lung leucocytes were prepared by collagenase XI and DNase digestion of the lung tissue and Percoll gradient isolation (34). Briefly, the lung tissues were minced into small pieces and incubated in digestive buffer (containing 2 mg/mL collagenase type XI and 100 µg/mL DNase [Sigma-Aldrich, St. Louis, MO, USA]) for 60 min at 37°C. The cell population was purified by centrifugation through a Percoll gradient. Cell suspension was gently mixed with 35% Percoll and centrifuged for 20 min at 750g. The pellet was collected and resuspended in flow cytometry buffer (FAC) buffer (1× Dulbecco’s phosphate-buffered saline [DPBS], 2% fetal bovine serum [FBS], 0.02% NaN3, 5 mmol/L ethylenedi-aminetetraacetic acid [EDTA]). The erythrocytes were lysed with ammonium-chloride-potassium (ACK) lysis buffer (150 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA) followed by two washes in RPMI 1640 with 5% fetal calf serum and resuspended in complete RPMI 1640 medium (RPMI, 10% FBS, 100 U penicillin).
In Vitro Restimulation Assays and Cytokine Measurement
Mice treated with different approaches were killed at d 7 after infection. Spleen and lungs were aseptically removed. To analyze cytokine production, single-cell suspensions were prepared from spleen and lungs as described previously (53,54). The cells were cultured at a concentration of 7.5 × 106 cells/mL (splenocytes) or 5.0 × 106 cells/mL (lung cells), respectively, in complete culture medium with or without stimulation of ultraviolet-inactivated Cm (105 IFU/mL). Culture supernatants were harvested at 72 h, and cytokine concentrations in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA) by using antibodies purchased from eBioscience (San Diego, CA, USA).
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
To analyze the expression of Foxp3 retinoic acid-related orphan receptor γ (ROR-γt) transcripts, the mRNA was prepared from lung tissues by using TRIzol reagent protocol (Invitrogen/Life Technologies, Carlsbad, CA, USA) (52). Briefly, total cellular RNA was extracted from lung tissues using phenol-guanidinium followed by ethanol precipitation. The first-strand cDNA was synthesized from 1.2 µg RNA by using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen/Life Technologies) and oligo(dT) primer. One microliter of cDNA was used for each PCR. β-Actin was used as a loading control. GeneAmp PCR System 2400 (PerkinElmer, Waltham, MA, USA) was used at 36 cycles for Foxp3, forward 5′-AACATCTGGAACCAC GGGCACTA-3′, reverse: 5′-GCATTGCTTG AGGCTGCGTATGAT-3′; β-actin, forward: 5′-GTGGGCCGCCCTAGGCACCA-3′, reverse: 5′-CTCTTTGATGTCACGCACGA TTTC-3′. Real-time PCR was conducted on the MJ Mini Thermal Cycler (Bio-Rad, Mississauga, Ontario, Canada) by using iQ™ SYBR Green Supermix.
DCs and CD4 T-Cell Purification and DC/T-Cell Coculture
C57BL/6 mice were immunized with UV-killed Cm (1 × 105) by intraperitoneal injection (IP) and boosted 2 wks later with the same dose. Cm-specific CD4+ T cells were isolated from the spleen of Cm-immunized mice by using a CD4 magnetic cell sorting buffer (MACS)-positive selection column (Miltenyi Biotec, Auburn, CA, USA) as described (54). DCs were isolated from spleen of WT or IL-10 KO mice by using magnetic CD11c columns from MACS. The purities of the purified CD4+ cells and DCs (based on CD11c) were >96%. The isolated CD4+ T cells were cocultured with purified DCs in the presence of ultraviolet-inactivated Cm as described (54). In designated experiments, anti-ICOS-L mAbs (10 µg/mL, clone HK5.3; eBioscience) were added to the co-culture wells to block ICOS-L signal pathway (51). The cells harvested after 48-h DC:CD4+ T-cell coculture were washed twice with cold PBS and further analyzed by intracellular cytokine staining.
To evaluate the expression of CD11c and ICOS-L on the surface of DCs, fluorescein isothiocyanate-conjugated anti-CD11c (anti-CD11c-FITC) and phycoerythrin-conjugated anti-ICOS ligand (anti-ICOS-L-PE) antibodies and corresponding isotype controls were purchased from eBioscience and used for flow cytometric analysis. The freshly isolated DCs were stained by using labeled antibodies as previously described (54). Analyses were performed by using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and Cell Express (De Novo, Los Angeles, CA, USA). Standard FACS buffer (1× DPBS, 2% FBS, 0.02% NaN3, 5 mmol/L EDTA) was used.
For intracellular cytokine analysis, freshly isolated splenocytes and lung mononuclear cells and cells collected from DC:CD4+ T-cell coculture were analyzed as described (54). For splenocytes, erythrocytes were first depleted. For cells from DC:CD4+ T-cell coculture, cells were collected after 48 h coculture. All the cells were washed twice and then stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/mL; Sigma-Aldrich) and ionomycin (1 µg/mL; Sigma-Aldrich) and incubated for 6 h in complete RPMI-1640 medium at 37°C. For the last 3 h of incubation, brefeldin A (Sigma-Aldrich) was added to accumulate cytokines intracellularly. After the stimulation, cells (2 × 106) were washed twice and incubated with Fc receptor (FcR) block antibodies (anti-CD16/CD32; eBioscience) for 15 min at 4°C to block nonspecific staining. Cell surface markers (CD3ε and CD4) were stained first. The cells were then fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Jose, CA, USA) and stained for intracellular cytokines with allophycocyanin-conjugated anti-IFNγ (anti-interferon-γ) (XMG 1.2) and allophycocyanin-conjugated anti-IL-17 (17B7; eBioscience) or with corresponding isotope control antibodies. Finally, the cells were washed, resuspended in PBS containing 2% fetal calf serum and analyzed by flow cytometry for intracellular cytokines by gating on CD3γ+ and CD4+ or CD8+ T cells, respectively.
One-way analysis of variance (ANOVA) and Newman-Keuls test or unpaired t test were used to determined statistical significance among groups. IFUs of Cm were converted to logarithmic values and analyzed using ANOVA. The value of p < 0.05 was considered significant.
DCs from IL-10 KO Mice Show Significantly Higher ICOS-L Expression after Cm Infection
IL-10 KO Mice Show Reduced Tregs but Increased Th17 and Th1 Responses after Cm Lung Infection
Expansion of Th17, but Not Th1, Cells Depends on ICOS-ICOS-L Interaction
Most Cm-Activated Th17 Cells Are ICOS Positive, Whereas Most Th1 Cells Are ICOS Negative
ICOS KO Mice Show Reduced Th17 but Increased Th1 Response after Cm Lung Infection
Blockade of Th17 Function Reverses the Better Protection of IL-10 KO Mice against Cm Infection
This study provides clear evidence that ICOS-L expression on DCs does not depend on IL-10 production and that IL-10 may even inhibit ICOS-L expression. More importantly, the study demonstrates that ICOS-ICOS-L interaction is critical for Th17, but not Th1, responses in chlamydial lung infection. Notably, this is the first report showing enhanced ICOS-L expression in IL-10 KO mice, which is associated with enhanced protective Th17 response in a respiratory tract infection model.
The finding of the increased ICOS-L expression in IL-10 KO mice is rather surprising but interesting. Given the reported association of ICOS-L expression by DCs with the induction of Tregs, and immune tolerance, one would expect knockout of IL-10 is likely to reduce the expression of ICOS-L. Indeed, our and others previous studies have shown that the DCs, which are more efficient in inducing Tregs, express higher ICOS-L and produce higher IL-10 (14,15,56,57). However, the data in the present study suggest the possibility that IL-10 may play a negative regulatory role in ICOS-L expression. Therefore, the increase of ICOS-L and IL-10 in tolerogenic DCs is likely independently regulated. On the other hand, the increased ICOS-L expression, on DCs of IL-10 KO mice, may reflect a compensation effect for the reduction of Treg caused by IL-10 deficiency. The exact mechanism remains unclear, but our data at least suggest that ICOS-L expression on DCs does not depend on, and indeed is reduced by, IL-10 production. It should be pointed out that the method used for DC isolation in this study was based on the expression of CD11c, which could also be expressed in other cells such as macrophages. Although this is a commonly used method for DC isolation, because it mainly isolates higher CD11c cells, which are mostly DCs, the contamination of other cells in the DC preparation cannot be completely excluded.
The most interesting finding in the study is the distinction of Th17 and Th1 cells in dependence on ICOS-ICOS-L signaling. Notably, although the functional distinction of different CD4+ T-cell subsets have been well documented, the molecular basis for determining the skewing of the different T-cell subsets remains unclear. The present data show that ICOS-ICOS-L interaction was particularly important for Th17 response, but not for Th1 response, in both in vitro coculture and in vivo infection experiments. Interestingly, IL-10 KO mice showed enhanced response of both Th17 and Th1 cells, but only Th17 response is related to increased ICOS-L expression. The blocked ICOS-ICOS-L interaction significantly reduced Th17, but not Th1, in the DC:CD4+ T-cell coculture. The specific role of ICOS-ICOS-L signaling in Th17 response is further confirmed by the fact that ICOS KO mice showed dramatically reduced Th17 cells but not Th1 cells. In addition, the analysis of ICOS expression by CD4 T cells showed that most of the Th17 cells in the spleen and lung of infected mice were ICOS positive (70–80%), whereas most of the Th1 cells in the infected mice were ICOS negative in lung tissues. The results were consistent with our recent finding that ICOS-L KO mice mounted significantly lower Th17, but higher Th1, response after chlamydial lung infection (51).
The exact mechanism for ICOS-ICOS-L signaling to enhance Th17 response remains unclear. The importance of ICOS-ICOS-L signaling for Th17 response is reflected not only by its effect on increasing IL-17-producing cells, but by its influence on the capacity of individual Th17 cells to produce IL-17. We not only found that most Th17 cells were ICOS+, but also found that the ICOS+ Th17 cells were higher producers of IL-17. Although some ICOS− Th17 cell were found, the data showed that the feature of the ICOS− and ICOS+ Th17 cells was different, when measured by their capacity to produce IL-17. Indeed, the MFI of IL-17 in ICOS+ Th17 cells was significantly higher than ICOS− Th17 cells in the Cm-infected IL-10 KO and WT mice (Figure 5E). It is likely that the higher ICOS-L surface expression (in percentage and MFI) made the IL-10 KO DCs not only more efficient for inducing Th17 cells, but also more efficient for inducing/promoting ICOS+ Th17 cells, which are higher IL-17 producers after chlamydial lung infection.
Better protection of IL-10 KO mice in chlamydial infection has been reported previously (46). However, the better protection was attributed to the higher Th1 response in these mice. The present study has confirmed the higher Th1 response in IL-10 KO mice and further demonstrated higher Th17 response in these mice. More importantly we showed that blockade of IL-17 in vivo abolished the better protection in IL-10 KO mice. Notably, the role of Th17 cells in host defense against chlamydial lung infection has been recently realized. In particular, we recently reported that IFNγ and IL-17 can work synergistically to inhibit chlamydial growth (58). Therefore, the better protection in IL-10 KO mice is likely the synergistic effect of Th17 and Th1, which are both increased in these mice.
In summary, we found that the IL-10 KO mice exhibited significantly higher ICOS-L expression by DCs, which is associated with lower Tregs but higher Th17 and Th1 cells. Although both Th17 and Th1 responses are higher in IL-10 KO mice, only the Th17 response is found to depend on ICOS signaling. The data suggest that ICOS-L and IL-10 are discordantly regulated and that the coexistence of ICOS-L with or without IL-10 can determine the polarization of CD4 T cells to Tregs or Th17 cells in bacterial infection.
ICOS-L expression on DCs may be negatively regulated by IL-10, and the ICOS-L expression of DCs in the presence or absence of IL-10 costimulation may, respectively, promote Treg or Th17 response, without significant impact on Th1, in intracellular bacterial infection.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by grants from the Canadian Institutes of Health Research, Manitoba Health Research Council and Manitoba Institute for Child Health (to X Yang). X Gao was a trainee in the Canadian Institutes of Health Research National Training Program in Allergy and Asthma and a recipient of a Manitoba Health Research Council/Manitoba Institute for Child Health Graduate Studentship. X Yang is the Canada Research Chair in Infection and Immunity. The authors thank Grant McClarty for help editing the manuscript.
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