Open Access

RIP2 Contributes to Expanded CD4+ T Cell IFN-γ Production during Efferocytosis of Streptococcus pneumoniae–Infected Apoptotic Cells

Victoria Eugenia Niño-Castaño, Letícia de Aquino Penteado, Ludmilla Silva-Pereira, Júlia Miranda Ribeiro Bazzano, Allan Botinhon Orlando, Ana Carolina Guerta Salina, Naiara Naiana Dejani, Vânia L. D. Bonato, C. Henrique Serezani and Alexandra Ivo Medeiros
ImmunoHorizons July 1, 2022, 6 (7) 559-568; DOI: https://doi.org/10.4049/immunohorizons.2200001

Abstract

Apoptotic cell clearance by professional and nonprofessional phagocytes in the process of efferocytosis is critical to preserve tissue homeostasis. Uptake of apoptotic cells by dendritic cells generates regulatory T cells and induces immunologic tolerance against self-antigens. In contrast, ingestion of infected apoptotic cells promotes activation of TLR4/MyD88-dependent bone marrow–derived dendritic cells (BMDCs) and triggers Th17 cell differentiation. In this study, we evaluated the impact of Streptococcus pneumoniae–infected apoptotic cell efferocytosis by BMDCs derived from C57BL/6 mice on differentiation and expansion of CD4+ T cell subsets, as well as the role of TLR2/4 and receptor-interacting protein 2 (RIP2) receptors in recognizing intracellular pathogens during efferocytosis. We demonstrated that BMDC-mediated efferocytosis of S. pneumoniae–infected apoptotic cells induced Th1 cell differentiation and expansion. Although TLR2/4 and RIP2 deficiency in BMDCs did not affect Th1 cell differentiation during efferocytosis, the absence of RIP2 decreased IFN-γ production by CD4 T cells during the expansion phase. These findings suggest that RIP2-mediated IL-1β production during efferocytosis of S. pneumoniae–infected apoptotic cells partially supports a Th1-mediated IFN-γ production microenvironment.

Introduction

Engulfment of apoptotic cells by phagocytes (termed efferocytosis) is essential to prevent tissue damage (1). In uninfected conditions, efferocytosis induces the production of anti-inflammatory mediators, including TGF-β, IL-10, and PGE2 (2, 3), that can either inhibit macrophage effector functions or promote regulatory T cell differentiation (2, 4). During infections, pathogens can promote cell death and tissue damage (5), and phagocytosis of infected apoptotic cells (iACs) has a critical impact during innate and adaptive immune cell activation. Indeed, in an influenza infection model, newly infiltrated inflammatory monocytes are a significant phagocyte group with a vital role in clearing apoptotic infected neutrophils at an early stage of infection. At this early stage, apoptotic neutrophils release epidermal growth factor that promotes the differentiation of tissue-resident inflammatory monocytes into APCs, needed to activate CD8+ T cells through Ag cross-presentation (6). Efferocytosis of Mycobacterium tuberculosis–infected macrophages impairs bacterial survival, allowing activation of cross-primed CD8+ T cells that have a critical function as cytotoxic cells in addition to producing cytokines such as IFN-γ, TNF-α, and IL-2 that kill M. tuberculosis within macrophages (7, 8). Furthermore, recognition of Escherichia coli–infected apoptotic neutrophils by bone marrow–derived dendritic cells (BMDCs) induces proinflammatory and anti-inflammatory cytokine production and Th17 cell differentiation (9, 10). Interestingly, efferocytosis of E. coli-iACs induces high levels of PGE2 while also increasing BMDC maturation (10, 11) and migration in a CCR7-dependent manner (11). PGE2 is a lipid mediator derived from arachidonic acid metabolism via cyclooxygenase-1 or -2 and PGE2 synthase activation (12). This prostanoid is reported to have pleiotropic roles in the effector function of innate and adaptive immune cells. Despite the well-known function of PGE2 in CD4+ T cell development (13), Th1 differentiation, and Th17 expansion (14–16), few studies have directly linked PGE2 to the differentiation of distinct T cell subsets during efferocytosis of infected cells (17). We have demonstrated that high PGE2 levels, produced during efferocytosis of E. coli-iACs, inhibit Th17 differentiation in a manner dependent on the E prostanoid (EP) receptor 4 (EP4)–protein kinase A signaling pathway (17). However, whether this depends on the pathogen inside the apoptotic cells or is a general consequence of efferocytosis in infectious conditions remains to be addressed.

Streptococcus pneumoniae exhibits several pathogen-associated molecular patterns (PAMPs) that can be sensed by a variety of pattern recognition receptors (PRRs). For example, pneumolysin and lipoteichoic acid are ligands for TLR4 and TLR2, respectively, and induce TNF-α, IL-1, IL-8, IL-10, and G-CSF production from human whole blood or peritoneal macrophages (18, 19). Bacteria and pneumolysin can also escape from phagolysosomes and access the cytoplasm (20), where they favor recognition by nucleotide-binding oligomerization domain-containing protein (NOD) 1 and NOD2 (21, 22). NOD1 and NOD2 bind the adapter molecule receptor-interacting protein 2 (RIP2), resulting in NF-κB activation and expression of proinflammatory mediators, including IL-1β, TNF-α, IL-6, and MCP-1 (23, 24). Consequently, recognizing S. pneumoniae promotes high levels of IL-1β, TNF-α, and PGE2 (25–27).

Th1 and Th17 both play important roles during protection against S. pneumoniae. Previous studies have described that the depletion of CD4+ T cells or administration of IL-17A neutralizing Ab in S. pneumoniae–infected mice impairs bacterial clearance, which seems to be associated with deficient macrophage and neutrophil recruitment into the nasal spaces (28). In additional studies, IL-12p40 knockout mice or wild-type (WT) mice treated with neutralizing anti–IFN-γ have shortened survival and diminished bacterial clearance (29, 30). However, the mechanism by which S. pneumoniae inside apoptotic cells is sensed by dendritic cells (DCs) and drives differentiation of specific CD4+ T cells subsets is still unknown. We found that the inflammatory milieu produced by BMDCs during efferocytosis of S. pneumoniae–infected cells leads to differentiation and expansion of Th1 cells. Moreover, RIP2 signaling seems to be important for IL-1β production during efferocytosis, which, along with other mediators, contributes to IFN-γ released during the expansion of effector CD4+ T cells.

Materials and Methods

Animals

All mice were bred and maintained under specific pathogen-free conditions. Female Wistar rats were purchased from the Animal Facility of the Ribeirão Preto Pharmaceutical School, University of São Paulo. C57BL/6 female mice were purchased from the Animal Facility of the Multidisciplinary Center for Biological Research, University of Campinas (CEMIB/UNICAMP). TLR4−/−, TLR2−/−, and RIP2−/− mice were from the Animal Facility of the Ribeirão Preto Medical School, University of São Paulo. RIP2−/− mice were kindly provided by Dr. D. Zamboni. Mice used in this study were on a C57BL/6 background. The animals were maintained with controlled temperature, humidity, airflow, and dark/light cycle with free access to sterilized water and food. All animal experiments performed were approved by the Institutional Animal Care and Use Committee from the School of Pharmaceutical Sciences CEUA/FCF/CAr no. 02/2016, São Paulo State University.

Generation of noninfected or infected apoptotic neutrophils

Wistar rats were infected i.p. with 108 CFUs of S. pneumoniae in 3 ml of 4% thioglycolate (BD Biosciences, Franklin Lakes, NJ). To recruit noninfected neutrophils, we injected mice with 4% thioglycolate alone. After 13 h, neutrophils obtained from peritoneal lavage were submitted to 1 mJ of UVC irradiation for apoptosis induction. The percentage of infected neutrophils was confirmed using S. pneumoniae FITC conjugated, according to protocol previously described (31), with a few adjustments. FITC+ cells were evaluated by flow cytometry. Apoptosis rate was confirmed by Annexin V and 7-aminoactinomycin D staining (BD Biosciences) (Supplemental Fig. 1).

Generation of BMDCs

DCs were differentiated from bone marrow precursor cells of C57BL/6 WT, RIP2−/−, TLR4−/−, or TLR2−/− mice according to the protocol described by Lutz et al. (32), with a few adjustments. DCs were cultured in 100 × 20-mm tissue culture plates (Falcon; BD Biosciences, Mountain View, CA) with 10 ml of complete RPMI-1640 medium (Lonza Walkersville, Rockville, MD) supplemented with 10% fetal bovice serum and 10 µg/ml gentamicin (Life Technologies, Grand Island, NY) and containing 40 ng/ml GM-CSF (PeproTech, Rocky Hill, NJ). The medium was replaced on the third, sixth, and eighth days for fresh complete RPMI-1640 medium containing 40 ng/ml GM-CSF. On day 10, the supernatant was discharged, and BMDCs were resuspended in the RPMI-1640 medium.

Conditioned medium generation

BMDCs were cocultured with apoptotic neutrophils noninfected or infected with S. pneumoniae ATCC 49619 (ratio, 1 BMDC:3 apoptotic cells/iACs) in RPMI serum-free condition, in a 24-well plate, during 18 h. After 18 h, supernatant (conditioned medium [CM]) was collected for cytokines/PGE2 quantification assay, as well as naive CD4+ T cell differentiation. The percentage of apoptotic cells infected with S. pneumoniae (Supplemental Fig. 1A, 1B) and the ability of BMDCs to uptake those iACs were evaluated.

Differentiation of effector CD4+ T cells

Naive CD4+ T cells (CD4+CD62L+) were isolated from the spleen of C57BL/6 mice according to the manufacturer's instructions (CD4+CD62L+ T Cell Isolation Kit II, mouse Catalog No. 130-093-227; Miltenyi Biotec). Naive CD4+ T cells were differentiated in the presence of CM obtained from coculture of WT, RIP2−/−, TLR4−/−, or TLR2−/− BMDCs with iACs. In brief, 1.5 × 105 naive CD4+ T cells were stimulated with anti-CD3 and anti-CD28 (2 µg/ml; BD Pharmingen), in a 96-well plate in the presence of the CM. As positive control, cells were stimulated with Th17 polarizing conditions (2.5 ng of TGF-β; 20 ng/ml IL-6 [PeproTech]; 10 µg/ml anti–IL-2 [S4B6 clone], anti–IFN-γ [H22 clone], and anti–IL-4 [BVD4-1D11 clone] [all from BD Pharmingen]) or Th1 polarizing conditions (50 ng/ml rIL-12; 20 ng/ml rIL-2 [PeproTech], and 1 µg/ml anti–IL-4 [BVD4-1D11 clone; BD Pharmingen]) in IMDM (Lonza) complete medium (10% fetal bovine serum, 100 µg/ml gentamicin, 25 µM 2 β-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, and nonessential amino acids [all from Sigma]). After 72 h, T cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml; Sigma), calcium ionophore (1 µg/ml; A23187), and monensin (10 µg/ml; BD) during 4–6 h. Th1 or Th17 phenotype was evaluated by extracellular stain with anti-mouse CD4 (KG1.5 clone) and intracellular cytokine detection using the Fixation/Permeabilization Buffer, BD Cytofix/Cytoperm (BD Biosciences) and anti-mouse IFN-γ (XMG1.2 clone) and anti-mouse IL-17-A (N49-653 clone). Cells were assessed by flow cytometry (FACSCanto; Becton & Dickinson, San Diego, CA) and analyzed by FCS express 4 (De Novo Software, Thornhill, ON, Canada). As a gating strategy, cells were pregated on CD4+ cells before evaluation of intracellular IFN-γ and IL-17A.

CD4+ T cell expansion assay

CD4+ T cells were labeled with CFSE (0.5 μM CellTrace; Thermo Fisher Scientific) and cultured for 3 d (primary culture) under CM obtained from coculture of WT BMDCs or RIP2-deficient BMDCs with apoptotic S. pneumoniae–infected cells, as previously described earlier in the Differentiation of effector CD4+ T cells section. For the expansion assay, after 3 d of differentiation, the cells were washed and restimulated for another 3 d (secondary culture) with anti-CD3 (1 μg/ml) and rIL-2 (20 U/ml) in the presence of WT CM or RIP2 CM with or without 500 pg/ml rIL-1β (BD). When indicated, we added 13.6 µg/ml anti–IL-1β Abs (B122 clone) to the WT CM. After 72 h, T cells were stimulated with PMA plus calcium ionophore and treated with monensin as previously described. Then cells were labeled with fixable viability dye (FVS 780; eBioscience, San Diego, CA), anti-CD3 (17A2 clone), anti-CD44 (IM7 clone), and anti–IFN-γ (XMG1 2 clone) (all from BD Pharmingen); assessed by flow cytometry (FACSCanto Becton & Dickinson, San Diego, CA); and analyzed by FCS express 4 (De Novo Software). Live cells were pregated on CD3+CD44+ cells before evaluation of IFN-γ+ cells. Dead cells were excluded from analysis using FVS 780.

ELISA

All cytokines and PGE2 present in the CM were determined using the immunoenzymatic technique. The kits used in our studies were Rat Anti-Mouse IL-6 Clone MP5-20F3 (Catalog No. 555240), Hamster Anti-Mouse IL-1β Clone B122 (Catalog No. 559603), Rat Anti-Mouse IL-10 Clone JES5-16E3 (Catalog No. 555252), Rat Anti-Mouse TNF Clone MP6-XT22 (Catalog No. 558534), and Rat Anti-Mouse IL-12 (p40/p70) Clone C15.6 (Catalog No. 555256). The clone reported from R&D System is Rat Anti-Mouse TGF-β Clone 860206 (Catalog No. DY1679). The minimum detectable concentrations are 62.5 pg/ml for IL-12 (BD Pharmingen) and 31.25 pg/ml for IL-10 and IL-17A (BD Pharmingen); 15.6 pg/ml for IL-1β, IL-6, TGF-β, and TNF-α (DuoSet ELISA; BD Pharmingen and R&D Systems); 7.8 pg/ml for PGE2 (PGE2 EIA Kit; Cayman Chemicals); and 3.1 pg/ml for IFN-γ (R&D Systems). All procedures were performed according to the manufacturer's instructions.

Statistical analysis

For comparison between two groups, Student t test was applied for independent samples. One-way ANOVA analysis was performed to compare three or more experimental groups, followed by Tukey’s multiple comparison test in parametric conditions and Dunn’s multiparametric comparison test in nonparametric conditions. Statistically significant differences were indicated for p values ≤0.05. All data were analyzed by Prism 6.0 (GraphPad Software, San Diego, CA).

Data availability statement

The data that support the findings of this study are available from the corresponding author on reasonable request. The corresponding author had full access to all data in the study, and all authors take responsibility for data integrity and analysis.

Results

Efferocytosis of S. pneumoniae–infected apoptotic cells results in Th1 cell differentiation

Initially, we determined the production of BMDC mediator in the presence of S. pneumoniae–infected apoptotic cells (Sp-iACs) and its impact on CD4+ T cell subset differentiation. CM derived from efferocytosis of Sp-iACs drove CD4+ T cell differentiation to Th1 cells (Fig. 1A, 1B). These findings were supported by increased levels of IFN-γ produced by CD4+ T cells in the presence of CM from Sp-iACs (Fig. 1C). We also detected higher levels of IL-17 in the iACs condition than in controls; however, IL-17 production was considerably lower than IFN-γ levels (Fig. 1B, 1C).

FIGURE 1.
FIGURE 1.

Efferocytosis of Sp-iACs drives naive CD4+ T cells into specific Th1 effector subsets.

Naive CD4+ T cells obtained from spleen of C57BL/6 mice were stimulated with anti-CD3 and anti-CD28 in the presence of CM from C57BL/6 mouse–derived BMDCs cocultured with Sp-iACs obtained from rat peritoneal lavage for 72 h, and as a control, BMDCs (CT) were cultured in RPMI alone. (A and B) The representative dot plot and bar graphs of the percentage of IFN-γ+– and IL-17A+–producing CD4+ T cells from CM of CT or Sp-iACs. (C) IL-17A and IFN-γ+ produced by CD4+ T cells differentiated in CM from CT or CM from Sp-iACs were measured by ELISA. (D) BMDCs were cocultured with Sp-iACs, at the ratio of 1:3, for 18 h. IL-10, TGF-β, IL-6, TNF-α, IL-1β, and IL-12 (p70) concentrations in the culture supernatant were evaluated by ELISA according to the manufacturer’s instructions. (A and B) Data are from at least five independent experiments. (C and D) One of two independent experiments is plotted. The values are shown as mean ± SEM. *p < 0.05.

To determine whether T cell division impacts the percentage of Th1/Th17 cells, we examined the proliferative response using CFSE dilution. Cell divisions of Th1 and Th17 were similar when cultured in CM derived from efferocytosis of Sp–iACs (Supplemental Fig. 2).

Because CD4+ T cell differentiation induced by CM derived from efferocytosis of Sp-iACs had a Th1 phenotype, we investigated the pattern of soluble mediators produced by BMDCs after efferocytosis of Sp-iACs. IL-1β, TNF-α, IL-10, and IL-6 were produced at higher levels after phagocytosis of Sp–iACs than in resting BMDCs. Interestingly, the conditions evaluated did not affect IL-12 (p70) production (Fig. 1D). We also confirmed that in the presence of uninfected apoptotic cells, BMDCs had a slight inhibitory effect on TNF-α and IL-6 production (Supplemental Fig. 1C), suggesting that residual bacteria inside the apoptotic cell are recognized and capable of modulating BMDC cytokine production. Moreover, culturing BMDCs with S. pneumoniae alone induced higher levels of IL-1β, IL-6, and IL-10 than BMDCs in the presence of RPMI. Interestingly, S. pneumoniae on its own did not induce TNF-α production (Supplemental Fig. 1D).

In addition to cytokines, PGE2 has also been implicated in Th1 differentiation (13), and we have previously shown that PGE2 resulting from efferocytosis of E. coli-iACs inhibits the Th17 response (17). Therefore, we next asked whether PGE2 is produced by efferocytosis of S. pneumoniae–infected cells and could play an essential role during Th1 cell differentiation. Although efferocytosis of Sp-iACs triggered PGE2 production by BMDCs, the results strongly suggest that it is not required for Th1 cell differentiation. The presence of EP1, EP2, and EP4 antagonists, the most important EP receptors expressed in naive CD4+ T cells (17), did not impair Th1 cell differentiation (Fig. 2). These results demonstrate that efferocytosis of S. pneumoniae–infected cells drives BMDCs to create an environment favorable for Th1 cell differentiation using a mechanism independent of PGE2.

FIGURE 2.
FIGURE 2.

PGE2 has no effect on Th1 differentiation in the context of efferocytosis of Sp-iACs.

C57BL/6 mouse-derived BMDCs were cocultured with rat-derived Sp-iACs and after 18 h, PGE2 production was measured in the supernatant according to the manufacturer’s instructions. C57BL/6 mice naive CD4 T cells were stimulated with anti-CD3 and anti-CD28 and differentiated during 3 d in the presence of CM from BMDCs with Sp-iACs and 5 or 10 μM EP1 antagonist (SC 19220 or SC 51089) (B), EP2 antagonist (AH6809 or PF04418948) (C), or EP4 antagonist (AH23848 or L161,982) (D). PGE2 production by BMDCs after coculture with Sp-iAC. Data are from five independent experiments (A). (B–D) Bar graphs represent the fold change of CD4+IFN-γ+ T cells in percentage for each antagonist treatment relative to CM without treatment. Data are from three independent experiments. Data represent mean ± SEM. *p < 0.05.

TLR2 and RIP2 deficiency compromise BMDC cytokine production during efferocytosis of Sp-iACs without impacting BMDC maturation or Th1 differentiation

We next sought to evaluate the role of TLR2, TLR4, and RIP2 in BMDC activation and cytokine production during efferocytosis of Sp-iACs, because these PRRs are involved in recognizing S. pneumoniae and activating phagocytes (23).

BMDCs from WT, RIP2, TLR4, and TLR2 knockout mice were cocultured with apoptotic Sp-iACs, and expression of molecules associated with BMDC maturation (e.g., CD86 and CCR7) in previously selected CD11c and MHC class II–positive cells was evaluated. The absence of RIP2, TLR4, and TLR2 did not affect the CD86 or CCR7 expression (data not shown). Interestingly, when we evaluated whether the absence of these PRRs would influence BMDC efferocytosis, we found that TLR2 deficiency had a modest inhibitory effect on engulfment of apoptotic Sp-iACs compared with WT BMDCs (Supplemental Fig. 3).

Efferocytosis of Sp-iACs by RIP2-deficient BMDCs showed less IL-1β, TGF-β, IL-6, and TNF-α production than did WT BMDCs (Fig. 3A, 3B, 3D, 3F). Indeed, RIP2-deficient BMDCs produced less IL-1β and TNF-α than did TLR4- and TLR2-deficient BMDCs (Fig. 3A, 3B). Moreover, the absence of TLR2 inhibited IL-6 and TGF-β production (Fig. 3D, 3F), whereas TLR4-deficient BMDCs had a modest increase in TNF-α production over that in WT BMDCs (Fig. 3E). However, the absence of those receptors did not impact IL-10 and IL-12 production (Fig. 3C, 3F).

FIGURE 3.
FIGURE 3.

TLR2 and RIP2 modulate cytokine production during efferocytosis of Sp-iACs.

BMDCs obtained from WT, RIP2−/−, TLR4−/−, and TLR2−/− C57BL/6 mice were cocultured with Sp-iACs at the ratio of 1:3 for 18 h. Cytokines concentration in the culture supernatant was evaluated by ELISA according to the manufacturer’s instructions. The dot plot graphic represents the production of IL-1β (A), TGF-β (B), IL-12 (p70) (C), IL-6 (D), TNF-α (E), and IL-10 (F). One experiment is representative of three independent experiments. Data represent mean ± SEM of sextuplicate. *p < 0.05.

Because Sp-iACs induced predominantly Th1 cell differentiation, we next asked whether reduced levels of BMDC cytokines from TLR4, TLR2, and RIP2 knockout mice affect Th1 differentiation. CM from efferocytosis of Sp-iACs by BMDCs from WT, TLR4, TLR2, and RIP2-deficient mice or from BMDCs cultured in RPMI alone (CT) had similar levels of intracellular IFN-γ–producing Th cells (Th1) (Fig. 4A, Supplemental Fig. 4). Although the RIP2 deficiency in BMDCs partially inhibited IL-1β, TNF-α, IL-6, and TGF-β during efferocytosis, no significant difference in the percentage of Th1 cell differentiation and IFN-γ production was observed (Fig. 4).

FIGURE 4.
FIGURE 4.

The absence of TLR2 and RIP2 has a modest effect on CD4+/IFN-γ+ T differentiation.

C57BL/6 mice naive CD4+ T cells were stimulated with anti-CD3 and anti-CD28 and differentiated for 3 d in the presence of CM from WT, RIP2−/−, TLR4−/−, or TLR2−/− BMDCs cocultured with Sp-iACs. The percentage of CD4+IFN-γ+ T cells was determined by flow cytometry (A), and IFN-γ and IL-17A concentrations in the culture supernatant were evaluated by ELISA according to the manufacturer’s instructions (B). (A) These data are representative of three independent experiments performed in sextuplicate. (B) One representative experiment measured in 12 replicates from three independent experiments. Data represent mean ± SEM.

RIP2 is required for IFN-γ production during CD4+ T cell expansion during efferocytosis of S. pneumoniae–infected cells

IL-1β is involved in Th17 differentiation (33) and in expansion of Th1 and Th17 effector cells in vitro and in vivo (34, 35). Because the absence of RIP2 in BMDCs inhibited IL-1β production during uptake of Sp-iACs, but did not affect Th1 differentiation, we next asked whether lower levels of IL-1β in CM from RIP2-deficient BMDCs affected IFN-γ release from expanded Th1 effector cells.

To evaluate the expansion of CD4+ T cells, we labeled naive T cells with CFSE and cultured for 3 d under CM obtained by coculturing WT BMDCs or RIP2-deficient BMDCs with Sp-iACs (primary culture), as previously described. After differentiation, cells were washed and restimulated for another 3 d (secondary culture) with anti-CD3 and rIL-2 in the presence of WT CM or RIP2−/− CM with or without 500 pg/ml rIL-1β. After 3 d of restimulation, the expansion capacity was determined on CD3+CD44+IFN-γ+ cells (shown in (Fig. 5A). Interestingly, based on the percentage of CD3+CD44+IFN-γ+ cells, CD4+ T cell expansion was slightly affected by CM from RIP2-deficient BMDCs (RIP2−/−) (Fig. 5C). Nonetheless, less IFN-γ was produced in RIP2-deficient BMDCs treated with CM than from WT BMDCs (Fig. 5B). Considering this, we hypothesized that, although diminished IL-1β production by RIP2-deficient BMDCs did not affect Th1 cell differentiation, IL-1β might be important for IFN-γ production during the expansion of effector CD4+ T cells.

FIGURE 5.
FIGURE 5.

RIP2 deficiency in BMDCs derived from C57BL/6 mice affected IFN-γ production by expanded CD4+ T cells through IL-1β production during efferocytosis of Sp-iACs.

C57BL/6 mice naive CD4+ T cells were labeled with CFSE (0.5 μM CellTrace; Thermo Fisher Scientific) and cultured for 3 d (primary culture) under CM obtained from coculture of WT BMDCs or RIP2-deficient BMDCs with apoptotic S. pneumoniae–infected cells. For the expansion assay, after 3 d of differentiation, the cells were washed and restimulated for another 3 d (secondary culture) with anti-CD3 (1 μg/ml) and rIL-2 (20 U/ml) in the presence of WT CM or RIP2 CM with or without 500 pg/ml rIL-1β (BD). Where indicated, we added 13.6 µg/ml anti–IL-1β Abs (BD) to the WT CM. After 72 h, T cells were stimulated with PMA plus calcium ionophore and treated with monensin. The percentage of live CD3+CD44+IFN-γ+ T cells was determined by flow cytometry, and IFN-γ concentration in the culture supernatant was evaluated by ELISA according to the manufacturer’s instructions. Representative scheme of CD4+ T cell expansion in the presence of CM from WT or RIP2−/− BMDCs (A). The IFN-γ production (B) and the percentage of CD3+CD44+IFN-γ+ T cells (C). These data are representative of two independent experiments performed in four replicates. Data represent mean ± SEM. *p < 0.05.

To determine whether reduced IL-1β production contributes to diminished IFN-γ production, we supplemented CM from RIP2-deficient BMDCs with rIL-1β (Fig. 5A). The addition of rIL-1β to CM from RIP2-deficient BMDCs increased IFN-γ levels in CM from RIP2-deficient BMDCs (Fig. 5B). In addition, we have performed a different approach by adding anti–IL-1β Abs to CM from WT BMDCs. Unexpectedly, we observed only a partial decrease in IFN-γ production from CD4+ T expanded cells, suggesting that besides IL-1β, other mediators are likely playing a role in inducing IFN-γ production by CD4+ T cells (Fig. 5B). These results suggest that IL-1β and other mediators, produced via adaptor protein RIP2, may contribute to IFN-γ production from CD4+ expanded T cells during efferocytosis of Sp-iACs by BMDCs.

Discussion

This study demonstrated that engulfment of Sp-iACs by BMDCs promoted a microenvironment suitable for Th1 differentiation. TLR2/4 and RIP2 deficiency in BMDCs partially inhibited IL-1β, TNF-α, IL-6, and TGF-β production during efferocytosis; however, this inhibition did not affect Th1 cell differentiation. Interestingly, RIP2 deficiency in BMDCs during efferocytosis of Sp-iACs compromised IFN-γ production during effector CD4+ T cell expansion.

The cytokine milieu produced during efferocytosis of Sp-iACs favors Th1 differentiation, suggesting that recognition of pathogen components inside apoptotic cells by BMDCs triggers distinct subsets of CD4+ T cells. Previous reports have shown that efferocytosis of E. coli–infected cells by BMDCs induces high levels of IL-6 and TGF-β via TLR4, favoring the Th17 subtype (9, 17). In contrast, we demonstrated that uptake of apoptotic S. pneumoniae–infected cells by BMDCs promoted secretion of IL-1β, TNF-α, IL-6, and IL-10 cytokines that led to Th1 differentiation. Even though TLR2 and TLR4 recognize S. pneumoniae ligands, these data provide evidence that bacteria-derived PAMPs within the apoptotic cell critically affect BMDCs effector mechanisms. Although no significant IL-12 was detected at 18 h of BMDC coculture with Sp-iACs, TNF-α and IL-1β production by BMDCs might favor Th1 induction in those conditions. Consistent with these results, in the presence of IL-1β or IL-6, TNF-α has been shown to increase IFN-γ production and Th1 cell proliferation (34). Furthermore, psoriasis treatment with anti–TNF-α Ab decreases the percentage of Th1 cells in the blood (36, 37). However, the relative contribution of individual cytokines to Th1 differentiation remains to be determined. In our study, we used rat neutrophils as a source of infected and uninfected apoptotic cells, whereas BMDCs and T cells were obtained from C57BL/6 mice. Although this is not a physiological condition, the main focus of our study was to evaluate the ability of soluble mediators produced during efferocytosis (CM) to affect CD4 T cell differentiation. Using apoptotic neutrophils from rats rather than mice provides some advantages, such as avoiding the potential that rat-derived cytokines could affect BMDCs or T cell function, because apoptotic cells are an important source of the anti-inflammatory mediators IL-10 and TGF-β (38, 39).

PGE2 is known to be produced during phagocytosis of infected and uninfected apoptotic cells (3, 10), reaching considerably higher levels during the efferocytosis of E. coli-iACs (10, 17). In this context, previous results from our group have shown that PGE2 production by BMDCs during efferocytosis inhibits Th17 cell differentiation (17). However, even though efferocytosis of Sp-iACs induces PGE2 production by BMDCs, the presence of this prostanoid does not affect Th1 differentiation, probably because the low levels of PGE2 produced were not sufficient to modulate Th1 differentiation. Consistent with this result, a high PGE2 concentration (100 nM) in polarizing conditions has a synergistic effect of inducing both Th1 and Th17 differentiation. In contrast, lower concentrations of this prostanoid do not affect T cell differentiation (16).

Efferocytosis of S. pneumoniae–infected cells by RIP2-deficient BMDCs induced low levels of IL-1β, IL-6, and TNF-α production, suggesting that this adaptor protein might be a critical component for S. pneumoniae recognition and activation of pathways involved in IL-1β production during efferocytosis. These findings are consistent with previous studies showing that RIP2 is involved in production of IL-6 and TNF-α after DC and macrophage stimulation with muramyl dipeptide, a component of the S. pneumoniae bacterial cell wall (40). RIP2 deficiency in DCs and macrophages reduces phosphorylation of p38, IκB-α, ERK, and JNK, resulting in reduced NF-κB activation and, therefore, less IL-6 and TNF-α production (40, 41). S. pneumoniae cell wall components can also access the cytosol after enzymatic degradation in macrophage phagolysosomes (20), where they form inflammasomes and activate NLRP3 to promote pro–IL-1β cleavage by caspase-1 (25, 42). Reduced IL-1β production was observed in RIP2- and NOD2-deficient macrophages in response to muramyl dipeptide after LPS priming (22). We detected low amounts of IL-6 in CM from TLR2-deficient DCs. Indeed, expression of lipoteichoic acid, another wall component of S. pneumoniae, in TLR2 knockout animals leads to production of low IL-6 levels in the lung of these animals (43). Our results showed that mainly IL-6 synthesis is reduced in the absence of TLR2 in BMDCs, while TNF-α production is not affected. This result suggests that TLR2 signaling in BMDCs has an important role in IL-6 production after Sp-iAC recognition.

Although TLR2 and RIP2 deficiency in BMDCs compromised IL-1β, IL-6, TNF-α, and TGF-β production, Th1 differentiation was not affected. Furthermore, the lack of TLR4, TLR2, and RIP2 on BMDCs did not affect BMDC maturation during efferocytosis of Sp-iACs (data not shown). Moreover, in the absence of TLR2, the efferocytosis capacity of BMDCs was less than that of WT BMDCs, even though the ability of macrophages to uptake apoptotic cells depends on TLR3 and TLR9 expression, but not TLR2 (44, 45).

We also demonstrated that RIP-2 deficiency affects IL-1β production, indicating that IL-1β may be important for Th1 cell expansion during efferocytosis of Sp-iACs. However, the presence of anti–IL-1β Abs to CM from WT BMDCs promoted a modest decrease in IFN-γ production from CD4+ T expanded cells, suggesting that besides IL-1β, other mediators can also play a key role in modulating IFN-γ production by expanded CD4+ T cells (Fig. 5B).

Nonetheless, additional approaches will be required to conclusively determine the mechanism by which RIP2 plays a role to maintain IFN-γ production during CD4+ effector T cell expansion. Although IL-1β is known to control S. pneumoniae colonization (46), we observed that other mediators contribute to maintain the Th1 microenvironment and immunity against S. pneumoniae during the efferocytosis of infected cells. Future efforts will also be needed to validate our findings in relevant primary human cells.

In summary, our data demonstrate that during clearance of Sp-iACs, bacterial-derived PAMPs inside the apoptotic corpse dictate the pattern of soluble mediators produced by BMDCs. These PAMPs also drive Th1 cell differentiation because, unlike the recognition of E. coli-iACs that trigger the Th17 response, engulfment of Sp-iACs favored Th1 cell differentiation. Moreover, our findings suggest that the RIP2 pathway helps to recognize S. pneumoniae inside apoptotic bodies, resulting in IL-1β and other mediators production by BMDCs, which seems to contribute to IFN-γ production during CD4+ effector T cell expansion. These findings bring new insights into efferocytosis that connect innate and adaptive immunity and highlight the role of different bacterial sources inside apoptotic bodies in engaging particular pathogen recognition pathways and specific T cell immunity.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Dario Simões Zamboni from the University of São Paulo for kindly providing the RIP2−/− mice.

Footnotes

  • This work was supported by São Paulo Research Foundation (FAPESP) and FAPESP fellowships (11/17611-7, 12/23580-0, 16/10964-5, 17/04786-0, 18/19638-9, 20/09327-6, 17/21629-5); the PAEDEX/AUIP/UNESP (School of Pharmaceutical Sciences, São Paulo State University) 2012 program for the awarding of a scholarship to V.E.N.-C.; the Brazilian National Council for Scientific and Technological Development–CNPq (471945/2012-9, 306363/2013-5, and 307109/2016-0); and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) Finance Code 001 and PROPG-UNESP (6/2021).

  • Conceived and designed the experiments: V.E.N.-C., N.N.D., L.d.A.P., A.C.G.S., A.I.M., V.L.D.B., and C.H.S.; performed the experiments: V.E.N.-C., N.N.D., L.d.A.P., A.B.O., J.M.R.B., A.C.G.S., and L.S.-P.; analyzed the data: V.E.N.-C., L.d.A.P., N.N.D., L.S.-P., A.B.O., J.M.R.B., and A.I.M.; wrote the paper: V.E.N.-C., L.d.A.P., N.N.D., J.M.R.B., A.C.G.S., L.S.-P., A.I.M., V.L.D.B., and C.H.S.; provided reagents: V.L.D.B. and C.H.S.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article

    BMDC
    bone marrow–derived dendritic cell
    CM
    conditioned medium
    EP
    E prostanoid
    EP4
    E prostanoid receptor 4
    iAC
    infected apoptotic cell
    NOD
    nucleotide-binding oligomerization domain
    PAMP
    pathogen-associated molecular pattern
    PMA, phorbol 12-myristate 13-acetate; PRR
    pattern recognition receptor
    RIP2
    receptor-interacting protein 2
    Sp-iAC
    Streptococcus pneumoniae–infected apoptotic cell
    WT
    wild-type

  • Received January 11, 2022.
  • Accepted June 29, 2022.

This article is distributed under the terms of the CC BY-NC 4.0 Unported license.

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