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Blood, 1 February 2008, Vol. 111, No. 3, pp. 1437-1447. Prepublished online as a Blood First Edition Paper on November 8, 2007; DOI 10.1182/blood-2007-07-100404.
IMMUNOBIOLOGY A common pathway mediated through Toll-like receptors leads to T- and natural killer–cell immunosuppression1 Lautenberg Center for General and Tumor Immunology, Hebrew University Hadassah Medical School, Jerusalem; 2 Institute of Animal Science, Agricultural Research Organization, Volcani Center; and 3 Department of Pathology, Hebrew University Hadassah Medical School, Ein Kerem Campus, Kiryat Hadassah, Jerusalem, Israel
T- and natural killer (NK)–cell immunosuppression associated with  -chain down-regulation has been described in cancer, autoimmune, and infectious diseases. However, the precise stimuli leading to this bystander phenomenon in such different pathogen-dependent and sterile pathologies remained unresolved. Here, we demonstrate that Toll-like receptors (TLRs) play a major role in the induction of innate and adaptive immune system suppression; repetitive administration of single TLR 2, 3, 4, or 9 agonists, which do not exhibit any virulent or immune invasive properties, was sufficient to induce a bystander NK- and T-cell immunosuppression associated with -chain down-regulation mediated by myeloid suppressor cells, as observed in the course of active pathologies. We identified a 35-amino acid (aa) region within the -chain as being responsible for its degradation under TLR-mediated chronic inflammation. Furthermore, we provide evidence that -chain levels could serve as a biomarker for chronic inflammation-dependent immunosuppression. Thus, although acute TLR-mediated activation could be beneficial in clearing pathogens or may serve as an immune adjuvant, such activation could be detrimental under sustained conditions.
During a sustained response to inactivated pathogens, and in the course of cancer, infections, or autoimmune diseases, T and natural killer (NK) cells can become -chain deficient and immunologically nonfunctional (reviewed in1). In our previous studies, we suggested that the causative link between these different pathologies is chronic inflammation and the ensuing accumulation of myeloid suppressor cells (MSCs), which lead to decreased -chain expression and impaired T-cell function.2,3 However, several critical questions remain unresolved such as how is a similar immunosuppressed state induced during sterile and pathogen-dependent diseases, and what is the nature of the stimuli leading to the generation of massive inflammation resulting in such generalized immunosuppression? We raised the hypothesis that in the course of the above-mentioned pathologies, chronic stimulation of the innate immune system via Toll-like receptors (TLRs) would lead to the induction of immunosuppression. TLRs recognize conserved pathogen-associated molecular patterns, which serve as TLR agonists/ligands (TLRLs).4–9 Moreover, recent studies suggest that endogenous, host-derived components, including fibrinogen, heat shock proteins, β-defensins, RNA, and DNA could also serve as TLRLs.5,10 TLRs are primarily expressed on cells of the innate immune system (myeloid and NK cells) and by some cells of the adaptive immune system (regulatory and activated T cells). Upon activation of any of the TLR signaling pathways, a primarily Th1-inflammatory response ensues, as reflected by the production of proinflammatory cytokines, chemokines, nitric oxide (NO), and other factors.1,11 Thus, TLRs could be activated during infections as well as in the course of sterile pathologies. It is well established that TLR-mediate acute activation is beneficial to the host because it leads to a critical response responsible for the clearance of pathogens and abnormal or damaged self-tissues. However, based on our observations, when TLR-mediated stimulation is sustained and chronic inflammation ensues, the appearance of immunosuppression is expected.
In the current study, we tested whether chronic exposure to a single TLRL, which does not exhibit any virulent or immune invasive properties, could induce immunologic nonresponsiveness characterized by reduced
We demonstrate that chronic exposure to a single TLRL specific for TLR 2, 3, 4, or 9 is sufficient to induce T- and NK-cell immunosuppression mediated by MSCs, which is associated with
Mice
Female BALB/c, C3H/HeN, C3H/HeJ, B10.A, and C57BL/6 mice, 6 to 8 weeks of age, were bred at the Hebrew University specific pathogen-free facility. Transgenic mice encoding full-length (FL) Antigen, TLRLs, and immunization
Antigens and immune modulators were obtained as follows: ovalbumin grade 3, Zymosan A, poly(I:C), lipopolysaccharide (LPS) from Escherichia coli strain 055:B5 (cat.L-4524), all purchased from Sigma-Aldrich (St Louis, MO). Endotoxin-free ( Immunostaining and fluorescence-activated cell sorter analysis
Antibodies used for cell surface labeling were purchased from BD PharMingen (San Diego, CA) and included fluorescein isothiocyanate (FITC)–labeled anti-Thy-1.2, anti-Gr-1, and anti-bromodeoxyuridine (BrdU), phycoerythrin-labeled anti-CD4, anti-CD3, and anti-NK1.1. Biotinylated anti-CD3 6-Carboxyfluorescein succinimidyl ester staining and ex vivo proliferation assay
Splenocytes (20 x 106/mL) were incubated in phosphate-buffered saline (PBS) without Ca2+/Mg2+ containing 5 µM 6-carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes) for 8 minutes at 25°C. At the end of this incubation, fetal calf serum (FCS) was added and the cells were washed in RPMI/8% FCS serum. CFSE-labeled splenocytes were activated with anti-CD3 Intracellular BrdU staining Splenocytes were activated as described in the previous section. They were then surface labeled with antiThy1.2, fixed as described previously,2 and stained with FITC-conjugated anti-BrdU antibodies. The cells were washed and analyzed by FACS. Nonpulsed activated cells were used as a control for the anti-BrdU staining. Cell isolation and separation For magnetic column separation, splenocytes were first labeled (30 minutes at 4°C) with biotin-conjugated antibodies. Negative selection using anti-CD11b and anti-B220 antibodies was performed for T-cell isolation ( > 90% purity), and positive selection was performed for the separation of Gr-1+Mac-1+ cells using anti-Gr-1 antibodies ( > 98% purity). The cells were then washed, labeled (45 minutes at 4°C) with anti-biotin antibodies conjugated to magnetic microbeads (Miltenyi Biotec, Auburn, CA), washed, and loaded onto a column placed in a magnetic field (Miltenyi Biotec). Coincubation experiments
Hybridoma T cells or T cells isolated from the spleen were suspended in complete growing medium and coincubated for 16 hours at 37°C with a Gr1+Mac-1+–enriched cell population obtained from the spleen of LPS-treated mice. The cells were then harvested and the level of Cytotoxicity assays For the in vitro assay, splenocytes were harvested from control or LPS-treated mice 18 hours on intraperitoneal injection of 200 µg poly(I:C) or PBS, and their killing activity was examined against target cells (YAC-1) that were pre-labeled with [35S]methionine. Various effector/target ratios were used in a cytotoxicity assay of 5-hour incubation at 37°C. Specific killing was calculated as described18 and normalized according to NK1.1+ cell percentage. For the in vivo assay, a published fluorescence labeling method19 was used. Briefly, splenocytes from BALB/C and C57BL/6 mice were stained with CFSE (Invitrogen, Carlsbad, CA) at a final concentration of 0.5 µM (CFSElow) and 5 µM (CFSEhigh), respectively. Cells (5 x 106) of each type were mixed and injected intravenously into recipient C57BL/6 mice (control, LPS-treated, and NK1.1-depleted mice). Peripheral blood lymphocytes (PBLs), lymph node, and spleen cells were harvested 18 hours after injection, and the ratio between the CFSEhigh and CFSElow populations was determined by FACS. The index of specific allogeneic cell clearance was calculated according to the formula: Index of specific allogeneic cell clearance = 1– BALB/C splenocytes (CFSElow) cells of total CFSE+ cells/ B6 splenocytes (CFSEhigh) cells of total CFSE+ cells. Influenza virus infection and mice survival Mice were infected by intranasal inoculation with the LD50 dose of influenza virus (A/PR/8/34) 2 days before the last LPS injection and mice survival was followed. LPS treatment itself did not affect mice survival. Spleen immunohistochemistry Five-micrometer, formalin-fixed, and paraffin-embedded sections from the spleens of control and LPS-treated mice were deparaffinized and rehydrated. After endogenous peroxidase quenching (H2O2), antigens were retrieved by boiling the sections in 10 mM citrate buffer (pH 6.0) or after pronase digestion. Immunostaining was then performed using the specific anti-Gr1 (clone RB6-8C5) or anti-CD3 antibodies, and the appropriate second reagents were applied (Zymed Laboratories, South San Francisco, CA) to visualize the labeled cells. Images of immunohistochemical staining were viewed with a Zeiss Axioplan 2 microscope and acquired with a Zeiss AxioCam digital camera (Zeiss, Oberkochen, Germany), using a 10x/0.30 NA Plan neofluar (Figures 4D,5Ci-iv) or a 20x/0.50 NA Plan neofluar (Figure 5Cv-vi) objective lens. Digitized images were processed using Adobe Photoshop 7 image processing and manipulation software (Adobe Systems, San Jose, CA). Measurement of serum cytokines
Serum was obtained from control and LPS-treated (1 day after the third LPS injection) mice. Interleukin-4 (IL-4), IL-6, interferon- Constructs and transfection
FL and proximal
Repeated LPS treatment induces TLR4-dependent impairment of T-cell function associated with -chain down-regulationWe first analyzed the effect of sustained in vivo TLR4 activation on the adaptive immune system (T cells). This TLR is commonly activated because of its ability to recognize bacterial compounds, such as LPS, as well as endogenous proteins that are released from damaged tissues.20 In addition, TLR4 agonists are currently used as vaccine-adjuvants,21 and assessing their effect on the immune system is therefore critical. To this end, Tlr4 wild-type (C3H/HeN) and Tlr4 defective (C3H/HeJ) mice were repeatedly injected with highly purified LPS from E coli, encapsulated in MLVs (LPS-treated) to reduce its toxicity and enable its slow release, or injected with empty MLVs (control; Figure 1A), as described in "Antigen, TLRLs, and immunization." We initially assessed the effect of this treatment on T-cell function. The ex vivo proliferation of splenic T cells from control and LPS-treated mice on TCR-mediated activation was evaluated by measuring cell divisions of gated CFSE-labeled T cells (Figure 1B) or BrdU incorporation (Figure 1C). Treatment with LPS was sufficient to induce significantly decreased TCR-mediated proliferation in splenic T cells of C3H/HeN-treated mice compared with that of cells derived from C3H/HeJ-treated mice or untreated control mice (Figure 1Bi-iii,C). However, when splenocytes derived from LPS-treated C3H/HeN mice were stimulated with a combination of PMA and Ca+2 ionophore, which bypasses TCR signaling, the T-cell proliferative response was partially rescued (up to 70%) relative to those isolated from the LPS-treated C3H/HeJ or control mice (Figure 1Biv-vi,C). Although both TCR-dependent and -independent signaling events are impaired in T cells derived from LPS-treated C3H/HeN mice, the effect on the TCR-mediated activation was by far more pronounced.
We next assessed whether the impaired TCR-mediated signaling function is associated with an abnormal TCR structure. Intracellular staining of splenic T cells (Figure 1D) and immunoblot analysis (Figure 1E) showed a reduced -chain expression induced by LPS in C3H/HeN (Figure 1Di,E) but not in C3H/HeJ (Figure 1Diii,E) mice. Despite the loss of -chain in splenic T cells from LPS-treated C3H/HeN mice, these cells expressed normal total levels of the CD3 -chain (Figure 1Dii) as well as of the surface CD3 and TCR  β subunits (data not shown). The expression level of the protein tyrosine kinase, -associated protein-70 (ZAP-70), a T-cell protein that is unrelated to the TCR subunits, was unaffected under these conditions (Figure 1E). Thus, persistent activation via a single TLR can induce the entire immunosuppressive milieu affecting all T cells similarly to that induced by an intact inactivated pathogen (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). The bystander T-cell immunosuppression was observed in both CD4+ and CD8+ subpopulations (data not shown). The results showing that both TCR-dependent and -independent signaling pathways are affected on chronic LPS treatment suggest that the abnormal TCR structure is not the sole cause for the observed T-cell dysfunction. Reduced antiviral response and impaired NK-cell function in LPS-treated mice Because the immune response to viral infections involves a complex coordination between the innate and adaptive systems, we next examined the in vivo antiviral immunity of LPS-treated mice relative to controls. Mice were infected with an LD50 dose of influenza virus and monitored daily for mortality. The results revealed a reduced survival rate of LPS-treated mice relative to controls (Figure 2A). Because of the kinetics of an antiviral immune response in which NK-cell activity precedes the T-cell response, the expedited death of LPS-treated mice, 5 to 7 days after the viral infection, suggests that mortality is primarily attributable to an impaired NK-mediated innate immune response, although the initial involvement of T cells could not be excluded. These results demonstrate the suppressed antiviral activity of the innate and adaptive immune systems on repeated treatment of mice with LPS.
We next assessed the effect of sustained activation of TLR4 by LPS on the innate immune system, focusing on NK cells, given that the -chain also plays a crucial role in the function of NK-cell activating receptors. In this set of experiments, we used C57B/6 mice because NK cells could be detected by anti-NK1.1 antibodies. To study the effect of LPS treatment on in vivo NK-cell function, we followed their capacity to clear fluorescently labeled allogeneic cells.19 To verify that allogeneic cell clearance depends on NK-cell activity, naive C57B/6 mice were first depleted of NK cells using anti-NK1.1 antibody or nondepleted (Figure 2B) and then were intravenously injected with a mixture of labeled allogeneic (CFSElow) and syngeneic (CFSEhigh) cells. The index of allogeneic cell clearance within the cell population of different lymphatic organs was then calculated. The results confirm that rejection of allogeneic splenocytes is mediated primarily by NK cells because the clearance index of the allogeneic cells was significantly reduced in mice that were depleted of NK1.1-expressing cells. Based on the results showing that the most pronounced effect was observed in the spleen (Figure 2C), we then analyzed the function of NK cells in the spleens of LPS-treated mice. The results revealed significantly impaired NK-cell function within LPS-treated mice, manifested by reduced clearance of allogeneic cells (Figure 2D, top and bottom panels). Furthermore, the dysfunction of NK cells isolated from LPS-treated mice was also apparent by their reduced in vitro cytotoxic activity (Figure 2E). Poly(I:C) treatment of mice 24 hours before cell harvest induced recovery of the NK-cell function (Figure 2F). As in the case of T cells, the impaired NK-cell function in the LPS-treated mice was associated with a dramatic loss of -chain expression (Figure 2G). The approximate 5-fold increase in specific lysis activity of splenic NK cells derived from control or LPS-treated mice observed on treatment with poly(I:C) is mostly attributable to the in vivo priming of NK cells by type 1 IFNs22 and could result from activation of NK-cell receptors that do not require -chain expression. Thus, chronic exposure to LPS immunosuppressed the innate immune system, as reflected by the impaired killing function of NK cells and the reduction in -chain expression levels.
Repetitive treatment with TLR 2, 3, or 9 ligands affect T-cell function and
To evaluate whether TLRLs other than the TLR4L, LPS, could induce an immunosuppressive effect in our in vivo model, we repeatedly exposed normal mice to TLR2, 3, or 9 ligands encapsulated in MLVs. To evaluate the function of splenic T cells derived from these mice, ex vivo proliferation assays were performed using intracellular staining for BrdU. We found that the ex vivo T-cell proliferation in the TLRL-treated mice after TCR-mediated activation was impaired, whereas activation with PMA and Ca2+ ionophore partially overcame this defect (Figure 3A). Impaired NK-cell function within the TLRL-treated mice was also observed (data not shown). The immunosuppression was associated with
LPS treatment leads to an inflammatory response associated with abnormal spleen architecture
LPS treatment is known to induce an inflammatory response as reflected by the secretion of high levels of proinflammatory cytokines such as TNF-
-chain as a biomarker for chronic inflammation-dependent immunosuppression induced by MSCs
We next characterized the pathway leading to
To evaluate whether the -chain expression levels could serve as a biomarker for sensing changes in chronic inflammation-dependent immunosuppression, its emergence and disappearance, we performed kinetic experiments comparing the time frame of both processes (Figure 5D). Gradual -chain down-regulation was observed as the inflammatory response progressed during the different intervals (1 to 3 LPS injections), with the most pronounced effect after the third LPS injection (Figure 5E). Interestingly, 8 days after the third LPS injection, a recovery of -chain expression was observed relative to day + 2 (Figure 5E), correlating with normal percentages of MSCs (Figure 5F) and recovered in vitro TCR-mediated T-cell proliferation (Figure 5G). Thus, -chain expression levels are reversible and are modified according to the inflammatory environment, suggesting its possible use as a biomarker to monitor chronic inflammation-dependent immunosuppression.
The mechanism for
We next assessed at which level
Our next goal was to locate the region within the -chain that is responsible for its targeting to degradation. To this end, we used transgenic mice expressing either FL -chain (FL-) or -chain variants truncated in the intracytoplasmic domain, one containing only a single distal ITAM (distal) and the second containing no ITAMs (TL).15,16 These mice were treated with LPS as described in Materials and methods. Chronic inflammation was generated in all LPS-treated transgenic mice as shown by the elevation of MSCs within their spleen relative to the nontreated mice (Figure 6C left panel). Interestingly, analysis of -chain expression in these mice revealed that although down-regulation was induced in the FL- and distal transgenic mice after LPS treatment, -chain expression levels in the TL mice were not altered in response to LPS (Figure 6C right panel). These results indicate that the region spanning 35 aa within the -chain intracytoplasmic domain (115-150 aa; Figure 6D) is responsible for its targeting to degradation under the LPS-induced immunosuppressive conditions. To support this conclusion, -deficient hybridoma T cells were stably transfected with FL or with a 35-aa truncated -chain (proximal; Figure 6D) and cocultured with MSCs derived from LPS-treated syngeneic mice. The transfected cells were analyzed for total CD3 and -chain expression levels by FACS. The results revealed that whereas cells expressing FL -chain are sensitive to the immunosuppressive environment and down-regulate the -chain, cells expressing the proximal -chain are resistant to the immunosuppressive environment and the truncated protein remains stable (Figure 6E). Thus, lack of the identified -chain 35-aa region protects -chain from degradation mediated by the MSCs derived from LPS-treated mice.
In the current study, we highlight the role of TLRs in the induction of chronic inflammation-dependent immunosuppression, as observed during infections and sterile pathologies. We demonstrate that antigen-independent sustained in vivo activation through single TLRs, which does not involve virulent or immune invasive mechanisms, is sufficient to induce immunosuppression of the adaptive (T cells) and innate (NK cells) immune systems associated with -chain down-regulation, as observed during chronic infections, cancer, and autoimmune disorders. Moreover, we provide evidence supporting the possible use of -chain expression as a biomarker for chronic inflammation-induced immunosuppression.
The role of individual TLRs in the induction of a bystander immunosuppression in the course of chronic inflammation was tested by subjecting normal mice to a repeated administration of a single TLRL encapsulated in negatively charged MLVs.17 TLR2, 3, 4, or 9 ligands were encapsulated in MLVs to reduce their toxicity and enable their slow release and efficient entry into the cells, thus activating intracellular TLRs including TLRs 3 and 9. The TLRLs used in the current study are derived from or are constituents of various potential pathogens, including yeast (Zymosan A), viruses [Poly (I:C)] and bacteria (LPS and CpG-ODNs). We demonstrate that the sustained exposure of mice to each of the TLRLs resulted in the induction of both local and systemic inflammatory immune responses associated with a bystander T- and NK-cell immunosuppression, similar to those observed in the course of a variety of chronic pathologies. Moreover, we show that the induced immunosuppression is the consequence of a Th1-dependent chronic inflammatory response associated with a dramatic enlargement and disrupted architecture of the spleen attributable to the abnormal accumulation of MSCs, the cells responsible for
The chronic inflammatory conditions generated on the TLRLs treatment not only affects
The strong and consistent association between
Based on our data and the results of others, we propose a model (Figure 7) explaining the TLR-mediated T- and NK-cell immunosuppression and its association with
Because of the key role of TLRLs in activation of the innate immune system leading mostly to a Th1-dependent inflammatory response, these agents are currently being tested as adjuvants for antimicrobial, antiallergic, and anticancer immunotherapy.5,33 However, based on our results, the feasibility of using TLRLs in a given therapy must be carefully investigated; the duration of such a treatment will dictate whether its effect is beneficial or harmful to the patient. Our description of the immunosuppressive process of the adaptive and innate immune systems triggered upon sustained activation of individual TLRs may allow a range of challenges in immunology and medicine to be addressed, with the goal of developing modalities that will block the inflammatory environment specifically induced by TLRLs. Identification of specific TLRs involved in the induction of chronic inflammation in a given pathology (eg, TLR9 in Lupus, TLR4 in LPS-mediated septic shock) could enable the development of controlled and disease-specific immunotherapy. Moreover, the mouse model system that we used in the current study might serve as a platform for testing new or currently used drugs for their ability to reduce chronic inflammation and to avoid complications resulting from the associated immunosuppression.
Contribution: I.V. designed and performed research and wrote the paper; L.B., R.G., and E.S. performed research; L.W. contributed vital new reagents; E.P. contributed vital new reagents or analytical tools and analyzed data; M.P. contributed vital new reagents or analytical tools; and M.B. designed research and wrote the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Dr Michal Baniyash, Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel; e-mail: baniyash{at}cc.huji.ac.il.
We gratefully acknowledge the support of the Society of Research Associates of the Lautenberg Center, the Concern Foundation of Los Angeles, and the Harold B. Abramson Chair in Immunology. Thanks to Dr Elizabeth Shores from the FDA, National Institutes of Health, Bethesda, MD, for providing us with -transgenic and –/– mice. We thank Dr Eyal Raz from the University of California, San Diego, for providing the CpG-ODNs and (m)CpG-ODNs. Thanks also to Prof Eli Kedar and Dr Aviva Joseph for helping us with MLV preparation and to Prof Zichria Zakay-Rones and Roi Gazit for providing the influenza virus. Thanks to Dr Tali Nave for helping with Northern blot analysis. Thanks also to Efrat Manaster, Dr Shelley Shwarzbaum, Dr Steve Caplan, and Prof Rachel Ehrlich for critical reading of the manuscript. This study was supported by the Israel Academy of Sciences and Humanities, the Israeli Ministry of Health, the Israel Cancer Association, the Joint German-Israeli Research Program (DKFZ), the Israel Cancer Research Fund (ICRF), and by the Joseph and Matilda Melnick Funds.
Submitted July 10, 2007; accepted November 5, 2007.
Prepublished online as Blood First Edition Paper, November 8, 2007
DOI: 10.1182/blood-2007-07-100404
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Abstract
Introduction
1 ng/mg DNA) phosphorothioate CpG-ODNs (5'-TGA CTGTGAACGTTCGAGATGA-3'), and the mutant (m)CpG-ODNs (5'-TGACTGTGAAGG TTAGAGATGA-3') were kindly provided by Dr Eyal Raz from the University of California, San Diego. Mice were injected subcutaneously 3 times with specific TLR ligands (LPS [70 µg], Zymosan [200 µg], CpG-ODNs [70 µg], and Poly (I:C) [150 µg]) encapsulated in MLVs as described previously.
(IFN-
) relative to TLRL-treated mice (
) after stimulation with anti-CD3 and anti-CD28 antibodies or with PMA and Ca2+ ionophore. The results are presented as the mean values of 3 independent experiments, and standard deviations are shown. *P < .005 (Student t test). (B) Equal numbers of splenic T cells from Zymosan, poly (I:C), and CpG-treated and control mice were lysed and subjected to Western blot analysis. Expression of the protein kinase ZAP-70, CD3
