T cell receptor (TCR) complexes of conventional CD4+ and CD8+ T cells are known to generate essential biochemical signals to initiate T cell immunity upon recognizing antigenic peptides derived from lysosomal or proteosomal proteolytic processing. Antigenic peptides are presented by major histocompatibility complex (MHC) molecules widely expressed on professional antigen presenting cells (APCs) including dendritic cell (DCs), macrophages, and B cells (1, 2). In contrast to conventional T cell activation, invariant natural killer T (iNKT) cells can recognize lipid or glycolipid antigens presented on CD1d, an MHC class I (MHC-I)-like molecule, and show prolonged cytokine production upon activation (3). CD1d is a cell surface glycoprotein comprising a heavy chain in non-covalent association with a β2-microglobulin light chain. It is broadly expressed in lym-phoid and myeloid cells (4). iNKT cells can be distinguished based on their TCR expression. They share some markers such as CD161 (NK1.1 in mice) and NKR-P1 that are characteristics of natural killer cells (3). iNKT cells can produce a wide variety of cytokines including proinﬂammatory and anti-inﬂammatory cytokines with multiple effects on the outcome of immune reactions (5). iNKT cells can also be activated in the absence of foreign microbial challenges, suggesting that they might occupy some immunological niches under immunologically quiet time and inflammatory condition (6).
During the past several years, regulatory and autoimmune roles of iNKT cells have been characterized. However, contrasting results have been observed using various approaches per-taining to iNKT cell-targeted treatments. It is currently unclear whether their effects are beneficial or detrimental to the host (5, 7, 8). Differing effects of iNKT cells in various systems reflect their ability to inform or influence functions of APCs (6). It has been recently shown that iNKT cells can reverse suppressive types of regulatory APCs known as myeloid-derived suppressor cells (MDSCs) into DCs to stimulate Th1 T cell responses (9). However, repeated administration of α-Galcer can result in an exhausted phenotype of iNKT cells that provides altered signals to DC and induces regulatory DC phenotypes that can prevent the onset of autoimmunity and silence autopathogenic T cells (10). Other studies have shown that CD1d-dependent iNKT cells play crucial roles in reducing joint inflammation (11). These effects were correlated with other autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) (12).
Macrophages play pivotal roles in rheumatoid arthritis (RA). They are prevalent in inflamed synovial membranes and at the cartilage–pannus junction. These cells possess broad proinflammatory, destructive, and remodeling capacities that contribute to acute and chronic phases of RA. Previously, it has been demonstrated that augmented recruitment and enhanced function of APCs are key steps associated with innate and adaptive immunity (13).
We have previously reported that TGFβ-treated tolerogenic pMφ from CD1d+/− mice, but not from CD1d KO (iNKT cell-deficient) mice, can facilitate APC-mediated suppression of CIA (14). In this study, we expanded these initial findings and investigated capabilities of pMφ. We found that their characteristics as tolerogenic APCs to suppress CIA were mostly cell-intrinsic rather than caused by TGFβ treatment.
iNKT cells not only can regulate local immune effector functions, but also can promote or inhibit priming of adaptive im-mune responses by releasing cytokines to induce APCs toward immunogenic or tolerogenic phenotypes (10). Tolerogenic effects of TGF-β2-treated APCs (Tol-APCs) require iNKT cells to mediate the suppression of CIA (14). Based on these findings, we ini-tially determined whether pMφ from CD1d+/− mice and CD1d−/− (iNKT cell-deficient) mice possessed intrinsic variances that might contribute to differential responses of CIA. Results validated previous findings, demonstrating that transfer of pMφ from CD1d+/− mice lowered incidences and clinical CIA scores compared to transfer of pMφ from CD1d−/− mice (Fig. 1). These results suggest that iNKT cells might be involved in the tolerogenicity of CD1d+/− pMφ. Therefore, we further assessed characteristics of these cells from CD1d+/− and CD1d−/− mice by observing changes in costimulatory molecule expression with or without LPS stimulation. Results showed that CD86 levels were significantly lower in pMφ from CD1d+/− mice. However, there was no significant difference in MHC II level (Fig. 2A). Other markers such as CD11c, CD80, CD206, F4/80, PD-L1, and PD-L2 showed no significant difference in their expression between pMφ from CD1d+/− mice and pMφ from CD1d−/− mice (Supplementary Fig. 1). There were no significant differences in the expression of CD1d, MHC II, CD11b, CD80, or CD86 either between pMφ and Tol-pMφ (data not shown). Secretion of inflammatory cytokines such as TNF-α and IL-6 was markedly reduced in pMφ from CD1d+/− mice compared to that in pMφ from CD1d−/− mice after LPS stimulation (Fig. 2B). In contrast, secretion of anti-inflammatory IL-10 in pMφ from CD1d+/− mice was significantly higher than that in pMφ from CD1d−/− mice (Fig. 2B), further suggesting that differences in pMφ between CD1d+/− and CD1d−/− mice were probably associated with the presence or absence of iNKT cells during pMφ maturation.
To confirm tolerogenic phenotypes of pMφ from CD1d+/− mice,
Differential CD4 T cell responses were observed upon stimulation with antigen-loaded pMφ matured in environments with differential CD1d expression. Therefore, we determined whether DCs from peritoneum/macrophages and DCs from other tissues had similar differential characteristics depending on CD1d expression. Splenic DCs and splenic Mφ showed no significant differences in CD1d-dependent CD80 or CD86 expression (data not shown). CD1d−/− peritoneal dendritic cells (pDCs) showed no increase in CD4 T cell stimulation compared to CD1d+/− pDCs either in CFSE dilution assays. These results suggest that iNKT cell-mediated tolerogenicity of APCs is CD1d-dependent and uniquely evident in pMφ.
We have previously shown that iNKT cells are critical for the induction of Tol-APC-mediated suppression of CIA (14). Thus, we further investigated whether the tolerizing potential was an intrinsic character of pMφ rather than an acquired phenotype following interactions with iNKT cells during immune responses. To exclude iNKT cells’ involvement, we adoptively transferred CD1d+/− Tol-pMφ or CD1d−/− Tol-pMφ cells into CIA-induced CD1d KO DBA/1 mice that lacked iNKT cells. When disease progression in mice was compared, treatments by CD1d+/− Tol-pMφ showed significantly reduced percentages of incidence (Fig. 4A, n = 4-5 mice) and clinical scores (4.8 ± 1.8 vs. 7.4 ± 2.0 at day 48) compared to treatments by CD1d−/− Tol-pMφ (Fig. 4A). These data validated the ability of CD1d+/− Tol-pMφ to ameliorate CIA by attenuating CD4+ T cell activation. In parallel, we compared effects of pMφ in CD1d+/− and CD1d−/− mice with CIA. CD1d+/− pMφ showed significantly lower incidence and severity of arthritis than those of CD1d−/− pMφ (6.0 ± 1.1 vs. 9.7 ± 0.7 at day 50; Fig. 4B). Taken together with data from experiments using Tol-pMφ, these results clearly demonstrate that CD1d-expressing pMφ have intrinsic immunosuppressive functions, suggesting that iNKT cells possibly can confer these tolerogenic activities during the development of pMφ.
Selective moderation of macrophage activation remains an attractive therapeutic approach to diminish local and systemic inflammation for preventing irreversible joint damage because the activation of monocytic lineage is not locally restricted, but extended to systemic parts of the mononuclear phagocyte system (16). Activation of APCs following interactions with iNKT cells may occur during immune activation and quiescence. Thus, interactions of iNKT cells with APCs might have proinflammatory or tolerizing outcomes, suggesting that effector and regulatory iNKT cells can coexist (6).
Induction of antigen-specific tolerance is critical for preventing autoimmunity and maintenance of immune tolerance. TGF-β2-treated Tol-APCs are known to induce anterior chamber-associated immune deviation (ACAID)-like tolerance (17). ACAID is a peripheral tolerance that protects eye tissues from destructive inflammation. It is mainly mediated by eye-derived APCs and B cells (18), αβ T cells (19), and NKT cells (20). Earlier data have shown that ACAID tolerance can be induced by Tol-APCs by inhibiting CIA and its related systemic immune responses in murine arthritis models following a single injection of Tol-APCs where iNKT cells are associated with a shift from Th1 to Th2 responses of CII-specific T and B cells (14).
CD1d antigen presentation is defective in some patients with RA, showing reduced iNKT cell numbers and altered functions (21). APCs express functional CD1d molecules on their surface to retain suppressive capacities following burn injury-induced immune suppression (22). B cells expressing CD1d are also required for iNKT cells to facilitate enhanced antibody production (23). The ensuing mechanism is probably restricted to T-independent antigens (24). Sonoda
In summary, our results revealed that CD1d-expressing pMφ suppressed CD4+ T cell proliferation after coculture, leading to down-regulation of Ag-specific IFN-γ production by CD4 T cells. We also observed lower CIA severity both in CD1d+/− and CD1d−/− recipient mice after adoptive transfer of CD1d+/− pMφ, but not by such transfer of CD1d−/− pMφ. These observations suggest that CIA suppression is mediated by CD1d-expressing pMφ and that tolerogenicity of pMφ is a cell-intrinsic property probably conferred by iNKT cells during pMφ development. Emerging evidence indicates that environmental factors can shape the identity of tissue resident macrophages. Therefore, earlier education by NKT cells potentially contributes to tolerogenic programs of pMφ, thus influencing the course of the CIA.
WT C57BL/6 mice were purchased from Orient Bio (Seongnam-si, Gyeonggi-do, Korea). C57BL/6 CD1d−/− mice used in this study were provided by Albert Bendelac’s lab (3). All animal experiment protocols adapted in this study were approved by the Institutional Animal Care and Use Committee of Korea University (KUIACUC-2018-25). DBA/1 mice were purchased from Charles River Laboratories (Japan) and backcrossed more than eight times with C57BL/6 CD1d−/− mice to generate DBA/1CD1d−/− mice. Mice with DBA background were used for
Peritoneal exudate cells (PECs) were prepared after collecting peritoneal washes of C57BL/6 or DBA/1 mice at three days after intraperitoneal (i.p.) injections of 3 ml of 3% thioglycolate solution (Sigma-Aldrich). Isolated PECs (pMφ) were then cultured overnight in a serum-free medium. For the generation of Tol-pMφ, 5 ng/ml TGF-β2 (R&D systems, Minneapolis, MN, USA) was added into the culture medium. After culture, pMφ and Tol-pMφ were washed three times with phosphate buffer saline (PBS). Remaining adherent cells were subjected to cold stress at 4°C in PBS for 2 h and then collected by vigorous pipetting. Cells were then washed three times with PBS and resuspended in PBS to cell density of 1 × 106 cells/ml (17). Subsequently, cells were stained with anti-CD11b and CD11c antibodies. Typical pMφ phenotypes (CD11b+CD11c+) were confirmed in more than 90% of cells. To transfer pMφ into CIA-induced mice, 100 μl of cell suspensions containing 1 × 106 cells CII in incomplete Freund’s adjuvant (IFA; Sigma-Aldrich) was injected into tail veins at 7 days after the second immunization.
DBA/1 mice were immunized intradermally (i.d.) at the base of the tail with 100 μg of chicken type CII (Sigma-Aldrich) emulsified with an equal volume (50 μl) of CFA (Sigma-Aldrich) according to a standard method (25). Mice were boosted by i.d. injections with 100 μg of CII emulsified in IFA on day 21. Seven days later, mice received intravenous (i.v.) injections of either pMφ or Tol-pMφ at 1 × 106 cells/mouse. Mice were then monitored on alternate days for the development of arthritis until the end of the experiment. Arthritis severity was graded as follows: 0 = normal paws; 1 = edema and erythema in only one digit; 2 = slight edema or erythema in multiple digits; 3 = slight edema involving the entire paw; 4 = moderate edema and erythema involving the entire paw; and 5 = severe edema and erythema involving the entire paw and subsequent ankylosis. Cumulative values were determined for all paws, with a maximum score of 20. Average macroscopic scores were then calculated.
Effector CD4+ T cells were obtained from OT-II transgenic mice immunized subcutaneously (s.c.) using 100 μg of OVA protein in CFA. After two weeks, primed CD4+ T cells were sorted using antibody-coated magnetic beads and labeled with 5 μM CFSE. Purified CD4+ T cells (5 × 105 cells/well) were then added into 24-well plates containing OVA-loaded pMφ or Tol-pMφ (5 × 104 cells/well). After three days, culture supernatants were collected and analyzed for cytokines using enzyme-linked immunosorbent assay (ELISA) kits. Cultured cells were then harvested for proliferation assays using CFSE dilution.
In addition to pMφ, splenic macrophages and dendritic cells were isolated using anti CD11b- and CD11c-magnetic beads through magnetic-activated cell sorting (MACS). Expression levels of CD11c, CD11b, F4/80, CD80 (B7-1), CD86 (B7-2), CD1d (1B1), CD206, and MHC II (IAb) were analyzed using a FACS Verse flow cytometer (BD). Culture supernatants were then assayed for IL-6, IL-10, and TNF-α levels using relevant enzyme-linked immunoassay kits (BD Pharmingen, San Diego, CA, USA) after stimulation with LPS from
Cells were stained with anti-FcR-γ mAb (2.4G2) at 4°C for 20 min in FACS staining buffer (PBS containing 0.1% BSA and 0.01% sodium azide). Cells were then stained with the following mAbs (BD Biosciences) for an additional 30 min: TCRβ (H57), CD4 (RM4-5), CD8α (53-6.7), IFN-γ (XMG1.2), and IL-4 (11B11). Stained cells were then analyzed using a FACS Calibur or FACS Verse and analyzed with FlowJo program.
Differences in clinical data between groups were assessed by Kruskal-Wallis test followed by Dunn’s Multiple comparison post-test (clinical score) or Student’s t-test using Prism 7 software (GraphPad Software, La Jolla, CA, USA). Statistical significance was considered at P < 0.05.
This work was supported by a grant (NRF-2018R1A2A2A050 23297) of the Basic Science Research Program of the National Research Foundation of Korea and a grant (K2010761) from Korea University. We thank crews of Geyrim Experimental Animal Resource Center for their assistance in animal handling and maintenance.
The authors have no conflicting interests.