Female C3H/HeN, C3H/HeJ (Tlr4^Lps-d), and C57BL/6 mice, 6–8 weeks, were procured from the Institute of Microbial Technology, Chandigarh, India. All the animal experiments were approved and complied by the Institutional Animal Ethics Committee (IAEC) and Regulatory Guidelines issued by the Committee for the Purpose of Supervision of Experiments on Animals (CPCSEA) (No. 55/1999), Ministry of Environment and Forest, Government of India.

Recombinant GM-CSF, IL-4, and naïve CD4 T cell enrichment kit, Abs to IL-12p40/70, TNF-α, IL-6, IFN-γ, IL-17, PE-Cy7-CD11c, FITC-CD80, PE-CD86, efluor-MHCII, APC-CD40, STAT-1, pSTAT-1, STAT-4, pSTAT-4, JNK, and pJNK were purchased from BD Biosciences (San Diego, CA, USA). Biotinylated anti-CD83 and APC-conjugated CCR7 Abs were purchased from eBioscience (San Diego, CA, USA). Abs against actin were from Sigma (St. Louis, MO, USA), SOCS-3 from Abcam (Cambridge, MA, USA) and anti-pERK and ERK Abs from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The endotoxin removal column was purchased from Thermo Scientific (Rockford, IL, USA).

Bone marrow cells were collected from femurs and tibia. The BMCs (2 × 10⁶/well) were cultured in media containing GM-CSF (2 ng/ml) + IL-4 (4 ng/ml) either with Acr1 (AcrDC^pre) or without Acr1 (AcrDC^post). On the third day, both cultures were replenished with media containing GM-CSF and IL-4, with Acr1 added only to the AcrDC^pre culture. On the sixth day, the AcrDC^post were stimulated with Acr1 (0–18 µg/ml) for 24 h, while the BMCs incubated with GM-CSF + IL-4 were used as controls (cDCs).

AcrDC^pre and AcrDC^post were harvested, washed, and lysed in lysis buffer (RIPA buffer, protease, and phosphatase inhibitor cocktail). The supernatants (SNs) of the lysates were estimated and equal amount of lysates were subjected to SDS-PAGE. After transfer to nitrocellulose membrane and subsequent blocking, the membranes were immunoblotted with their respective Abs against SOCS-3 or phosphorylated STAT-1, STAT-4, and ERK. Afterward, blot was stripped off to determine the expression of ERK and actin, and then developed using chemiluminescence kit (Lumigen, Inc., Southfield, MI, USA). Blots were scanned with the help of Image Quant LAS 4000 (GE Healthcare, Pittsburgh, PA, USA). Densitometric analysis was done using Scion image software.

The cytokines in the culture SNs of AcrDC^post and AcrDC^pre were monitored by coating the ELISA plates overnight at 4°C with rat anti-mouse IFN-γ (2 µg/ml), IL-12 (1 µg/ml), TNF-α (2 µg/ml), IL-6 (1 µg/ml), and IL-10 (2 µg/ml) Abs in phosphate buffer (pH 9.6 and pH 6, 0.05 M). The unbound sites were blocked with BSA (1%) in PBS for 2 h at 37°C. The plates were washed with PBS-Tween-20 (0.05%) and culture SNs (50 µl) were added overnight at 4°C. Respective biotinylated Abs diluted in dilution buffer (1:1 solution of PBS-Tween-20 and blocking buffer) were added into plates and incubated for 2 h at 37°C. Thereafter, avidin-HRP (1:1,000) was added to plates and incubated at 37°C for 1 h. The usual procedures of washing and incubation were carried at each step. The color developed, due to H₂O₂-OPD substrate-chromogen, was stopped by H₂SO₄ (7%) and the plates were read at 492 nm. Serial dilutions of recombinant cytokines were used to prepare the standard curves to quantify the cytokines in the SNs.

AcrDC^post and AcrDC^pre were harvested on the sixth day, washed and treated with IFN-γ and LPS. After 30 min, nuclear extracts were prepared as mentioned elsewhere (6, 11). An equal amount of nuclear extract (10 µg) from each sample was incubated for 30 min/RT and labeled with P³²-duplex oligonucleotides containing binding site for NF-κB in binding buffer. The DNA–protein complexes were resolved by electrophoresis on a native PAGE (7%). After electrophoresis, the gel was dried and exposed to screen at for 12 h/RT. The screen was scanned by phosphoimager (Fujifilm, Tokyo, Japan).

Naïve T cells were purified by MACS, as per manufacturer’s instructions (BD Biosciences, San Diego, CA, USA). Briefly, after preparing single cell suspension, splenocytes were treated with CD4 T cell enrichment cocktail supplemented with biotinylated anti-CD44 and CD25 Abs. Later, same volume of streptavidin-magnetic beads was added and cells were isolated using BD IMagnet. The purity of naïve T cells was >97%, as ascertained by flow cytometry. Naïve CD4 T cells (3 × 10⁵/well, 24 well plate) were poured on anti-CD3 Ab (2 µg/ml) coated plate and cocultured with AcrDC^post and AcrDC^pre under Th1 (IL-12: 10 ng/ml, anti-IL-4 Ab: 5 µg/ml, IL-2: 100 U/ml), Th2 (IL-4: 5 ng/ml, anti-IFN-γ Ab: 5 µg/ml, anti-IL-12 Ab: 5 µg/ml) and Th17 (IL-6: 30 ng/ml, TGF-β: 5 ng/ml, anti-IL-4 Ab: 5 µg/ml, anti-IFN-γ Ab: 5 µg/ml, anti-IL-2 Ab: 5 µg/ml) polarization conditions.

MACS purified naïve T cells (2 × 10⁵/well) of C57BL/6 mice were cocultured with AcrDC^post and AcrDC^pre of C3H/HeN in a 1:10 ratio (DC: T cells). After 3 days, the SNs were collected and estimated for IFN-γ and IL-17 by ELISA.

Mice (n = 8) were injected subcutaneously (s.c.) with OVA (100 µg) in alum (2 mg) dissolved in PBS (0.2 ml). Ten days later, AcrDC^post and AcrDC^pre incubated for 2 h with OVA (100 µg/ml) were adoptively transferred s.c. into OVA-primed mice. On the seventh day, the mice were sacrificed and LNs were isolated and single cell suspension was prepared. These cells were then cultured with OVA (100 µg/ml) for 48 h. Later, polarization of T cells was monitored by quantification of IFN-γ and IL-17 in the SNs by ELISA.

Mice (n = 8) were sensitized s.c. with OVA (100 µg). After 7 days, T cells were purified from LNs by MACS. T cells were cocultured with OVA-pulsed AcrDC^post and AcrDC^pre at a ratio of 1:10 (DC: T cells) for 5 days. Later, IFN-γ and IL-17 were estimated in SNs by ELISA.

Mice (n = 8) were s.c. immunized with OVA in alum. After 10 days, T cells were isolated from LNs by MACS. The T cells were labeled with CFSE (1 µM) by incubating in PBS for 8 min at 37°C. The cells were washed 3× and cocultured with OVA-pulsed AcrDC^post and AcrDC^pre for 48 h. Proliferation was assayed by CFSE-dye dilution assay using a flowcytometer. The histogram data (stimulation index: SI) are represented as a bar diagram, SI = AcrDC^pre/postcDCs. The flowcytometric analysis was done using BD FACS Diva software.

AcrDC^post and AcrDC^pre were labeled with either CFSE (2 µM) or efluor (2 µM) and adoptively transferred i.v. into mice (n = 8). After 2 days, animals were sacrificed. The presence of CFSE or efluor-positive cells in the spleen was monitored by FACSAria flowcytometer. The flowcytometric analysis was done using FACS Diva software.

RNA was isolated using Trizol reagent; according to the manufacturer’s instruction (Invitrogen, Carisbad, CA). Briefly, RNA was quantified by NanoDrop spectrophotometer. A260/A280 ratio of samples was in the range of 1.90–2.00. Intactness of RNA samples was determined by formaldehyde denaturing agarose gel electrophoresis. DNA contamination of RNA samples was removed by amplification grade DNase. Later, DNase activity was terminated by adding stop solution. Further, the samples were heated to 70°C for 10 min to inactivate DNase. Analysis was done by comparative Ct method. RT-qPCR and data analysis were done by Realplex Mastercycler (Eppendorf, Hamburg, Germany). The following primers were used for the quantification.

Statistical analysis was carried out using non-parametric Student’s t-test using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

The optimum expression of MHC and costimulatory molecules is considered to be quite crucial in deciding the activation or anergy in T cells. Costimulatory molecules like CD80, CD86, CD40, and CD83 are regarded as maturation markers for DCs. Importantly, we noticed that Acr1 upregulates the CD80 molecule on AcrDC^post but downregulates CD80 on AcrDC^pre. The change was noted in a dose-dependent manner (Figure 1A). Similarly, we observed the augmented expression of MHCII, CD86, CD40, and CD83 on AcrDC^post, while downregulation in the levels of these molecules on AcrDC^pre (Figure 1B). Thus, substantiating the observed disparity in the expression of CD80 on AcrDC^pre and AcrDC^post. Since, optimum expression of CD80 was induced at a dose of 9 µg/ml of Acr1, subsequently in all the experiments this concentration was used. In order to validate that the downregulation of costimulatory molecules in AcrDC^pre is not due to cell death, we stained AcrDC^pre and AcrDC^post with PI and no change in the viability of AcrDC^post and AcrDC^pre was observed (Figure 1C).

We next conducted experiments to identify the mechanism responsible for the distinct behavior of AcrDC^post and AcrDC^pre. The STAT-1 is a critical transcription factor responsible for the activation and maturation of DCs (16). Therefore, we investigated the status of STAT-1 in AcrDC^post and AcrDC^pre. Intriguingly, substantial enhancement in the level of STAT-1 phosphorylation in AcrDC^post but decline in AcrDC^pre was observed (Figures 1D,E). The SOCS-3 is a suppressor of cytokine signaling that negatively regulates STAT-1 mediated maturation of DCs (17–19). SOCS-3 has also been linked with the inhibition of CD4 T cells activation and proliferation (20). Intriguingly, we observed an inverse correlation between STAT-1 and SOCS-3 (Figures 1D–G). AcrDC^pre induced higher SOCS-3, whereas AcrDC^post displayed lower level. These results indicate that the mechanism responsible for the phenotypic and functional difference in AcrDC^post and AcrDC^pre is mediated through SOCS-3, which in turn regulates the STAT-1 mediated maturation of DCs.

The cytokines released by DCs are indispensable for the differentiation of T cells. We observed significant (p < 0.001) increase in the secretion of IL-12 by C3H/HeN AcrDC^post, as compared to AcrDC^pre (Figures 2A,B). A similar trend in the secretion of IL-12 was noted in C57BL/6 strain, thereby illustrating that the impact of Acr1 is independent of any genetic diversity in the mice. IL-12 is a key regulator for the differentiation of Th1 cells. Like IL-12, a similar trend was noted in the case of TNF-α (Figure 2C). TNF-α plays a vital role in restricting the growth of Mtb (21). The results of AcrDC^pre are in consistent with our previous observation (6), we noticed substantial (p < 0.01) inhibition in the yield of IL-12 when BMCs were matured in the presence of Acr1. Further, as compared to AcrDC^post, we observed significantly (p < 0.05) higher levels of TGF-β in AcrDC^pre (Figure 2D). Overall, these results indicate that Acr1 exposure inhibits the maturation of DC precursors during early stage of differentiation, whereas the post-maturation encounter of Acr1 potentiates DCs development.

To establish the specificity of Acr1 and its observed effect is not due to any artifact, we digested Acr1 with proteinase K and cultured it with AcrDC^pre and AcrDC^post and studied the secretion of IL-12. Interestingly, no change in the release of IL-12 was observed by AcrDC^pre and AcrDC^post cultured with proteinase K digested Acr1, but undigested Acr1 retained its modulatory function (Figure 2E), explicitly confirming the specificity of Acr1 in modulating DCs functionality. No change was noticed when AcrDC^pre and AcrDC^post cultures were supplemented with OVA in lieu of Acr1, thereby further validating the specificity of Acr1.

Next, we elucidated the mechanism by which Acr1 elicited the IL-12 production. The MAPK pathway is critical in regulating the expression of IL-12. Therefore, we investigated the status of ERK and JNK activation. We observed augmented phosphorylation of ERK in AcrDC^pre (Figure 3A). In contrast, suppression was noted in AcrDC^post. No change was discernible in JNK phosphorylation (Figure 3B), indicating that Acr1 mediates the regulation of IL-12 through the ERK pathway.

The trafficking of DCs is critical for the activation of T cells. To observe this phenomenon, we adoptively transferred CFSE-labeled AcrDC^post and AcrDC^pre into mice and monitored their migration to the spleen. We noted a remarkable increase (p < 0.01) in the movement of AcrDC^post, as compared to AcrDC^pre (Figure 4A). Interestingly, we observed higher expression of CCR7 on AcrDC^post that may explain their greater trafficking capability (Figure 4B). Furthermore, we investigated whether AcrDC^post exhibited enhancement in their potential to activate T cells. We observed that AcrDC^post showed greater ability to help T cells, as evidenced by improved (p < 0.01) proliferation of T cells (Figures 4C,D). In contrast, AcrDC^pre significantly (p < 0.01) inhibited the proliferation of T cells, as compared to control DCs (cDCs).

We next examined why AcrDC^pre expressed inhibitory activity. Recently, it has been reported that DCs with accumulated lipids exhibit dysfunction in their activity (22). This prompted us to check whether the malfunctioning of AcrDC^pre can be attributed due to their lipid content. We observed that AcrDC^pre accumulated a substantially larger amount of lipids than AcrDC^post (p < 0.05) or cDCs (p < 0.01) (Figures 4E,F; Figure S1 in Supplementary Material). Consequently, higher lipid content may be one of the reasons for the suppressive nature of AcrDC^pre.

We noticed the discrete role of AcrDC^post and AcrDC^pre in modulating the secretion of IL-12 (Figure 2). DCs play a cardinal role in the activation and differentiation of naïve CD4 T cells. IL-12 is a differentiation factor for Th1 cells. Therefore, it was worth to monitor the influence of AcrDC^post and AcrDC^pre in the generation of Th1 cells. AcrDC^post and AcrDC^pre were cultured with either anti-CD3 Ab stimulated naïve CD4 T cells under Th1 differentiation conditions (Figure 5A) or allogeneic naïve CD4 T cells (Figure 5B) or antigen specific T cells (Figure 5C). Intriguingly, AcrDC^post promoted but AcrDC^pre retarded the differentiation of Th1 cells, as evidenced by higher expression of IFN-γ by AcrDC^post. This change was detected in both antigen-specific and allogeneic CD4 T cells (Figures 5A–C). Furthermore, we also adoptively transferred OVA-pulsed AcrDC^post and AcrDC^pre into the mice that were primed with OVA. Like the in vitro results, it was noticed in vivo, as well that AcrDC^post potentiated while AcrDC^pre inhibited the differentiation of Th1 cells (Figure 5D).

STAT-4 activation is responsible for the IFN-γ-inducible genes (23). Therefore, we further validated AcrDC^post mediated differentiation of Th1 cells by examining the activation of STAT-4. It was noticed that AcrDC^post showed higher phosphorylation of STAT-4 (Figure 5E). Contrary to this, AcrDC^pre exhibited substantial decline in STAT-4. This is, therefore, indicative of the role of STAT-4 pathway in promoting the differentiation of Th1 cells by AcrDC^post.

Th17 cells play a dual role not only in protection but also in the pathogenesis of the diseases, and TGF-β and IL-6 cytokines are necessary for their differentiation (24). We observed disparate production of TGF-β (Figure 2D) and IL-6 (Figure 6A) by AcrDC^post and AcrDC^pre. Consequently, we were curious to know whether AcrDC^post and AcrDC^pre can distinctly regulate Th17 cells. AcrDC^post and AcrDC^pre were cultured with anti-CD3 Ab stimulated naïve CD4 T cells under Th17 differentiation conditions (Figure 6B), allogeneic naïve CD4 T cells (Figure 6C), and antigen-specific T cells (Figure 6D). Like Th1 cells, AcrDC^post facilitated, whereas AcrDC^pre impeded the differentiation of Th17 cells (Figures 6B–D). No change was observed in Th2 cells differentiation by AcrDC^post and AcrDC^pre (Figure 6E).

Mycobacterium tuberculosis is an extremely successful pathogen because it can thwart its host’s immune system and thrives in dormant state in a hostile environment of one-third of world’s population. Acr1 is predominantly expressed by Mtb during latency (8, 25). The current study illustrates that Acr1 alters the function of AcrDC^post and AcrDC^pre, so we wanted to investigate their abilities to restrict the growth of Mtb. Importantly, as compared to AcrDC^pre, AcrDC^post notably (p < 0.01) constrained the survival of Mtb (Figure 7A). However, Mtb showed unobstructed growth in AcrDC^pre; which also supports our earlier study (6). These results suggest the dual role of Acr1 in controlling the stage-specific activity of DCs.

It is reported that Acr1 binds to TLR-4 (15). Therefore, we thought it would be prudent to evaluate the role of TLR-4 in Acr1 mediated modulation of DCs. Interestingly, AcrDC^post derived from TLR-4 mutant mice (C3H/HeJ) showed abrogation in the secretion of IL-6 and IL-12 (Figures 7B,C). Wild-type mice (C3H/HeN), on the other hand, exhibited augmented production of these cytokines. These results indicate that the modulatory activity of Acr1 on DCs may be dependent on the extent of TLR-4 signaling. Triggering through TLR-4 is reported to induce both activation and inhibition in the function of DCs (19). Consequently, this study validates our results.

NF-κB plays a key role in orchestrating the immune response upon microbial infection. NF-κB translocation to the nucleus leads to upregulation of various cytokines, chemokines, and inflammatory mediators, which are required for the migration of immune cells to the site of infection. This study suggests the stimulatory phenotype of AcrDC^post (CD80^hi, CD86^hi, CD40^hi, CD83^hi, MHCII^hi, IL-6^hi, IL-12^hi, TNF-α^hi, TGF-β^lo) and suppressive phenotype of AcrDC^pre(CD80^lo, CD86^lo, CD40^lo, CD83^lo, MHCII^lo, IL-6^lo, IL-12^lo, TNF-α^lo, TGF-β^hi). Therefore, we investigated the NF-κB activation in AcrDC^post and AcrDC^pre. AcrDC^post exhibited significant enhancement but AcrDC^pre showed impediment in the nuclear translocation of NF-κB (Figure 7D), indicating that the differential responses detected in AcrDC^pre and AcrDC^post related to costimulatory molecules, cytokines, and proliferation are coordinated through NF-κB signaling.