Th1 Responses Are More Susceptible to Infliximab-Mediated Immunosuppression Than Th17 Responses
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Treatment with infliximab, a chimeric anti-tumor necrosis factor (TNF)-α antibody, is highly efficient in patients with inflammatory bowel disease (IBD). It neutralizes soluble TNF-α and induces the apoptosis of transmembrane TNF-α-positive macrophages and T cells in the gut. Recently, T helper (Th)17, as well as Th1, responses have been implicated in the pathogenesis of IBD.
To clarify the effects of infliximab on Th1 and Th17 responses in vitro.
Naive CD4+ T cells isolated from the peripheral blood of healthy volunteers were stimulated under Th1- or Th17-inducing conditions in the presence of 10 μg/ml of infliximab or control immunoglobulin (Ig)G1. The concentrations of interferon (IFN)-γ, interleukin (IL)-17, and TNF-α in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Th1 and Th17 cells were immunostained with infliximab or control IgG1 and transmembrane TNF-α-positive cells were analyzed by flow cytometry. Annexin V staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assays were conducted in order to analyze the percentage of apoptotic cells.
Both Th1 and Th17 cells expressed soluble and transmembrane TNF-α abundantly. Although infliximab suppressed IFN-γ secretion by Th1 cells and IL-17 secretion by Th17 cells, the level of the former was more profound than the latter. Infliximab increased annexin V- and TUNEL-positive apoptotic cells under Th1-inducing conditions, but not under Th17-inducing conditions.
Infliximab suppressed Th1 and Th17 differentiation in vitro; however, IFN-γ production by Th1 cells was more profoundly suppressed than IL-17 secretion by Th17 cells. Th1 responses were more susceptible to infliximab-mediated apoptosis than Th17 responses. Our results clarify a new mechanism of action of infliximab.
KeywordsInfliximab Tumor necrosis factor-α T helper 1 T helper 17 Apoptosis
Human inflammatory bowel diseases (IBDs), which include Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders of the intestinal tract with unknown etiologies, for which no current cures exist. Over the past few years, it has become evident that both CD and UC are caused by excessive immune reactivity in the gut . Classically, T helper (Th) cell responses have been divided into two distinct categories according to their characteristic cytokine profiles, namely, Th1 responses with increased interferon (IFN)-γ production and Th2 responses with increased expression of interleukin (IL)-4 and IL-13. In the era of the Th1/Th2 paradigm, CD was considered to be a Th1-dominant disorder, while UC was thought to be associated with augmented Th2 responses . However, recent studies have revealed a novel class of CD4+ effector T cells with a distinct cytokine expression profile that does not fall into either category. These cells are named Th17 cells [2, 3], because of their characteristic production of the proinflammatory cytokine IL-17. IL-12, which induces Th1 differentiation, and IL-23, which is important for the maintenance of Th17 cells, are both heterodimeric molecules and share a common component, the p40 subunit [4, 5]. As a consequence, some Th17-mediated disorders have been considered to be Th1 diseases [6, 7, 8, 9].
CD has been considered to be Th1-mediated diseases . However, recent studies have indicated a link between IBD and Th17 responses. Hölttä et al.  reported that the mRNA expression of the Th17 cytokines IL-17 and IL-23 is increased in CD. Kobayashi et al.  reported that the mRNA expression of IL-17 and IFN-γ is upregulated in both CD and UC, although IL-17 upregulation is more prominent in UC, while IFN-γ upregulation is more marked in CD . In some experimental murine models of IBD, Th17 cells rather than Th1 cells have been shown to be the major effector cells [12, 13]. Both Th1 and Th17 responses seem augmented in IBDs and further studies are awaited in order to address which responses are important in their pathogenesis.
Infliximab is a chimeric monoclonal antibody (mAb) against tumor necrosis factor (TNF)-α and is one of the major therapeutic agents for CD [14, 15, 16]. It has also been demonstrated to be efficacious as a treatment for UC [17, 18, 19]. Infliximab acts not only by neutralizing TNF-α but also by inducing the apoptosis of TNF-α-producing cells that express transmembrane TNF-α on their surface through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) [20, 21]. The production of TNF-α by intestinal mucosal mononuclear cells is increased in CD , and treatment with infliximab induces the apoptosis of both macrophages and T cells [23, 24, 25, 26]. Th17 cells have been reported to produce TNF-α [8, 27]. Given the implication of Th1 and/or Th17 responses in the pathogenesis of IBD, it is of importance to establish whether Th1 and Th17 cells are susceptible to infliximab-mediated apoptosis. To the best of our knowledge, the effects of infliximab on these cells have not been clarified. Therefore, we carried out this study in order to examine the effects of infliximab treatment on human Th1 and Th17 responses in vitro.
Antibodies and Reagents
Infliximab was a kind gift from Tanabe-Mitsubishi Pharmaceutical (Tokyo, Japan). Human immunoglobulin (Ig)G1 as a relevant isotype control was obtained from Ikwan (Bangkok, Thailand). Recombinant human IL-2, IL-12, IL-1β, IL-6, and IL-23, and mAbs against human IL-12 and IL-4 were purchased from R&D Systems (Minneapolis, MN). Anti-human CD3 and CD28 mAbs were purchased from BD PharMingen (San Diego, CA).
Samples and Isolation of Naive CD4+ T cells
Heparinized peripheral blood was drawn from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were collected by density-gradient centrifugation using Lymphocyte Separation Media (MP Biomedicals, Santa Ana, CA). The cells were separated magnetically using the Naive CD4+ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) to isolate naive CD4+ T cells.
Naive CD4+ T cells were stimulated with beads bearing anti-CD2, anti-CD3, and anti-CD28 Abs (T Cell Activation/Expansion Kit; Miltenyi Biotec) at a ratio of 1 bead/cell, according to the manufacturer’s instructions, at a density of 1 × 106 cells/ml in RPMI 1640 medium (Sigma, St. Louis, MO) containing 10% heat-inactivated fetal bovine serum (FBS) (Cell Culture Technologies, Gravesano, Switzerland), 1% l-glutamine (Sigma), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA), 1% HEPES buffer (Sigma), and 1% nonessential amino acids (Invitrogen). To induce Th1 differentiation, recombinant human IL-2 (20 ng/ml), recombinant human IL-12 (10 ng/ml), and anti-human IL-4 mAb (5 μg/ml) were added to the culture medium. To induce Th17 differentiation, recombinant human IL-1β (10 ng/ml), recombinant human IL-6 (20 ng/ml), recombinant human IL-23 (100 ng/ml), anti-human IL-12 mAb (5 μg/ml), and anti-human IL-4 mAb (5 μg/ml) were added to the culture medium as previously described . Cells were cultured in the presence of infliximab (10 μg/ml) or control human IgG1 (10 μg/ml), or without an immunoglobulin.
To examine the infliximab-induced apoptosis of TNF-α-bearing cells, cells were cultured under ADCC conditions. CD56+ natural killer cells were enriched from PBMCs using MACS CD56 microbeads and MACS-positive selection columns (Miltenyi Biotec) in accordance with the manufacturer’s protocol. Naive CD4+ T cells and CD56+ cells were incubated at a ratio of 5:2 in the presence of 10% heat-inactivated FBS.
Enzyme-Linked Immunosorbent Assay
Concentrations of IFN-γ, IL-17, and TNF-α in the culture supernatants were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) technique using the Human IFN-γ ELISA Ready-SET-Go! Reagent Set (eBioscience, San Diego, CA), the Human IL-17 DuoSet (R&D Systems), and the Human TNF-alpha/TNFSF1A DuoSet (R&D Systems), respectively. IFN-γ and IL-17 secretions were assessed after the stimulation of cells with T cell-activating beads as described above for 3 days in the presence or absence of infliximab. For the measurement of the TNF-α concentration, the cells were stimulated with T cell-activating beads for 7 days in the presence or absence of infliximab. The cells were then washed extensively and restimulated with 10 μg/ml of immobilized anti-CD3 mAb and 3 μg/ml of soluble anti-CD28 mAb for 48 h without the addition of infliximab, because the presence of infliximab interfered with the ELISA reaction for TNF-α.
Detection of Cell Surface TNF-α
Naive CD4+ T cells were cultured under Th1- or Th17-inducing conditions with anti-CD3 and anti-CD28 mAbs for 0, 48, 60, 72, and 96 h. Activated T cells were washed and incubated with infliximab or control human IgG1 as a primary Ab for 30 min. The cells were then washed and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG F(ab’)2 fragment (Jackson ImmunoResearch, West Grove, PA) as a secondary Ab. The cells were analyzed by flow cytometry using FACSCalibur (BD Biosciences, San Jose, CA) and CellQuest software (BD Biosciences).
To measure the effects of infliximab on the apoptosis of Th1 and Th17 cells, two different methods, namely, annexin V staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assays, were performed. For annexin V assays, the cells were stained with annexin V and 7-amino-actinomycin D (7-AAD) using the Annexin V-Phycoerythrin Apoptosis Detection Kit (BD PharMingen), according to the manufacturer’s protocol. The cells were washed in phosphate-buffered saline (PBS) and labeled at room temperature in the dark for 15 min with 5 μl of staining solution/100 μl cell suspension. After washing, the percentage of annexin V-positive 7-AAD-negative cells was determined by flow cytometry within 1 h of staining.
TUNEL assays were performed using an Apo-Direct kit (BD PharMingen), according to the manufacturer’s instructions. At the end of the culture period, the cells were fixed with 1% paraformaldehyde for 1 h on ice and stored in 70% (v/v) ethanol at −20°C for 12–18 h. The fixed cells were washed with PBS and incubated in a staining solution containing TdT enzyme and dUTP-FITC at 37°C for 1 h. After washing, the percentage of dUTP-FITC-positive cells was determined by flow cytometry.
The Mann–Whitney U-test was used to compare the numerical data between two groups. The Kruskal–Wallis H-test was applied for the comparison of numerical data among three groups. When statistical significance among three groups was reached, the Mann–Whitney U-test with Bonferroni correction was used to compare each category. Statistical analysis was carried out using JMP Software (SAS Institute, Cary, NC). P values < 0.05 were considered to be statistically significant. For the Mann–Whitney U-test with Bonferroni correction, P < 0.05/no. of groups compared was considered to be statistically significant.
Th1 and Th17 Cells Produce Soluble and Membrane-Bound TNF-α
Infliximab Suppresses Th1 and Th17 Cytokine Secretion
These results indicate that infliximab suppressed both Th1 and Th17 differentiation. However, the levels of suppression were more profound for Th1 induction than for Th17 induction. Although the suppressions of Th1 and Th17 differentiation were partial, treatment with infliximab completely blocked the generation of TNF-α-producing cells during the induction of Th1 and Th17 differentiation.
Infliximab Induces the Apoptosis of Th1 Cells but Not Th17 Cells
IL-12 and IL-23 are key cytokines for Th1 and Th17 responses, respectively. As they are both heterodimeric molecules and share a common p40 subunit [4, 5], some Th17-mediated diseases were previously considered to be Th1-induced disorders, until the recent discovery of the Th17 cell subset. Notably, rheumatoid arthritis and multiple sclerosis were originally considered to be Th1-mediated diseases, but are now considered to be induced by Th17 . CD is considered to be a Th1-mediated disorder . However, evidence for a connection between IBD and Th17 has recently been accumulating. Hölttä et al.  reported that IL-17 and IL-23 are increased in CD, suggesting that Th17 responses are involved in its pathogenesis. Kobayashi et al.  reported that the mRNA expressions for IL-17 and IFN-γ are upregulated in CD and UC, although IL-17 upregulation is more prominent in UC and IFN-γ upregulation is more marked in CD . It is, therefore, probable that Th1 and Th17 responses are both augmented in IBDs, and may be involved in their pathogenesis.
TNF-α is a pivotal inflammatory cytokine in the pathogenesis of IBD [1, 29]. Infliximab, a chimeric mAb against TNF-α, is highly efficacious against the induction and maintenance of moderate to severely active CD [14, 15, 16]. It has also been demonstrated to be efficacious against refractory UC [17, 18, 19]. It blocks the actions of TNF-α not only by neutralizing TNF-α, but also by inducing the apoptosis of TNF-α-producing cells that bear transmembrane TNF-α on their surface [20, 21]. In the gut of patients with IBD, TNF-α is prominently produced by immune cells [22, 29]. It is known that treatment with infliximab induces the apoptosis of macrophages and T cells in CD [23, 24, 25, 26]. Generally, macrophages produce much more TNF-α than T cells. However, animal models have suggested the importance of TNF-α produced by lymphocytes in the pathogenesis of IBD. Specifically, mice lacking TNF-α AU-rich elements, in which immune cells produce abundant TNF-α, develop spontaneous colitis and arthritis . Double-mutant mice with a TNF-α AU-rich element mutation introduced into a recombination-activating gene-1-deficient background develop arthritis but not colitis , suggesting that lymphocyte-derived TNF-α is essential for the development of IBD. Consequently, it is of interest to establish whether Th1 and Th17 cells produce TNF-α, and whether infliximab is capable of blocking the actions of TNF-α produced by Th1 or Th17 cells.
Although it has been reported that Th17 cells produce TNF-α [8, 27], data regarding TNF-α production by Th1 cells are relatively sparse. In this study, we have shown that human Th1 and Th17 cells produced soluble and transmembrane TNF-α. Treatment with infliximab strongly reduced IFN-γ production by Th1 cells, but only partially blocked IL-17 production by Th17 cells. These results suggest that infliximab suppresses both Th1 and Th17 differentiation. However, Th17 responses are less sensitive to infliximab-mediated suppression than Th1 responses.
As mentioned above, infliximab induces the apoptosis of TNF-α-producing cells through CDC or ADCC mechanisms , as well as neutralizing soluble TNF-α. The treatment of Th1 cells with infliximab increased the percentages of annexin V-positive and TUNEL-positive apoptotic cells. Surprisingly, although some Th17 cells expressed transmembrane TNF-α similarly to Th1 cells, infliximab did not increase the percentages of annexin V-positive and TUNEL-positive cells among Th17 cells. These results indicate that infliximab inhibits Th1 responses at least partially by inducing the apoptosis of Th1 cells. In contrast, Th17 cells appear to be relatively resistant to infliximab-mediated cytotoxicity, and the neutralization of TNF-α by infliximab suppressed the differentiation of Th17 cells without apoptosis. These findings suggest that TNF-α is important for Th17 differentiation, in accordance with a previous study in which TNF-α was used to induce Th17 differentiation . These differences in the susceptibilities to infliximab-mediated apoptosis may explain the differences in the sensitivities of Th1 and Th17 differentiation to infliximab-mediated suppression. Although infliximab failed to induce the apoptosis of Th17 cells bearing TNF-α on their membranes, it completely blocked TNF-α production by Th17 cells, as well as that by Th1 cells in vitro. However, in vivo, the ability of infliximab to elicit apoptosis in target cells may be important for its efficacy in the treatment of CD. Among several anti-TNF-α agents that are efficacious for rheumatoid arthritis, some have failed to show efficacy for the treatment of CD . The superiority of infliximab for CD treatment may be explained by the difference in its abilities to exert cytotoxicity toward transmembrane TNF-α-positive cells [25, 33]. Indeed, it has been reported that infliximab treatment increases the number of apoptotic T cells in the lamina propria of CD patients [24, 25, 26]. Further studies are awaited in order to establish whether apoptotic Th1 or Th17 cells are increased in the gut of patients with IBD after infliximab therapy.
Whether Th1 or Th17 response is pathogenically important may be different between CD and UC. In patients with CD or UC, cytokine expression pattern may be different among individuals. In this study, we showed that the sensitivity of Th1 and Th17 cells to infliximab-mediated suppression is different. It is, therefore, suggested that a certain proportion of IBD patients who are resistant to infliximab may be due to Th17 cells perpetuating the inflammation. The analysis of cytokine expression in the gut may be useful for the prediction of responses to certain therapies, including anti-TNF agents.
In this study, we have shown that Th1 and Th17 cells produce abundant TNF-α in soluble and transmembrane forms. Infliximab suppressed Th1 and Th17 differentiation in vitro; however, IFN-γ production by Th1 cells was more profoundly suppressed than IL-17 secretion by Th17 cells. Th1 responses were more susceptible to infliximab-mediated apoptosis than Th17 responses. The results of our study clarify a new mechanism of action of infliximab.
The authors thank Drs. Takahiro Mizutani and Hiromi Muta for their help in conducting the experiments.
Conflicts of interest
The authors have no conflicts of interest to disclose.