Graphical Abstract

   graphic file with name fx1.jpg
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Highlights

     * •
       NCoR1 directly represses tolerogenic program in mouse cDCs
     * •
       Depletion of NCoR1 in cDCs enhanced Treg development ex vivo and
       in vivo
     * •
       NCoR1 masks PU.1-bound super-enhancers on tolerogenic genes in cDCs
     * •
       NCoR1^DC−/− animals depicted enhanced Treg frequency and infection
       load
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   Molecular Mechanism of Gene Regulation; Immunology; Immune Response;
   Transcriptomics

Introduction

   Dendritic cells (DCs) are specialized antigen-presenting cells (APCs)
   that link innate to adaptive immunity ([44]Steinman, 2006). Upon
   encounter with pathogens, they get activated resulting in maturation
   and migration to draining lymph nodes. Primed DCs then polarize naive
   and memory T helper (Th) cells into various effector subtypes such as
   Th1, Th2, Th17, or Tregs ([45]Hochweller et al., 2010, [46]Kapsenberg,
   2003). The differentiation of these different Th subsets depends on the
   DC maturation status and the secreted cytokines. DCs are thus
   considered as major players in fine-tuning immunity versus immune
   tolerance.

   DCs express a range of pattern recognition receptors such as Toll-like
   receptors (TLRs) to identify a wide plethora of pathogens ([47]Takeuchi
   and Akira, 2010). Here in this study, we have focused mostly on CD8^+
   cDC1 DCs. cDC1 DCs are the main antigen cross-presenting DCs for
   intracellular pathogens detected through TLR3 and TLR9 ([48]Reizis,
   2011, [49]Schnorrer et al., 2006). Upon ligation of these receptors,
   Myd88 and TRIF signaling pathways are stimulated, consequently
   activating transcription factors (TFs) like nuclear factor (NF)-κB and
   interferon regulatory factors (IRFs) and a variety of protein kinases
   ([50]Kawasaki and Kawai, 2014, [51]O'Neill et al., 2013). It has been
   reported that in DCs canonical NF-κB signaling through IkBα activates
   both RelA and RelB, whereas in other cells like fibroblasts only
   non-canonical signaling results in RelB activation ([52]Shih et al.,
   2012). RelA signaling is important for pro-inflammatory response and
   cell survival ([53]Shih et al., 2012). On the other side, RelB is
   reported to be involved in DC maturation and induction of tolerance
   ([54]Azukizawa et al., 2011, [55]Thomas, 2013). Therefore depending on
   the stimulus and a fine balance of NF-κB activity, DCs exhibit either
   immunogenic or regulatory phenotype ([56]Vendelova et al., 2018). The
   activation stimulus results in signal-specific expression of a large
   number of DC response genes including cytokines (interleukin [IL]-6,
   IL-10, IL-12) and co-stimulatory markers (CD40, CD80, and CD86)
   ([57]Matsushima et al., 2004). Depending on the strength and
   specificity of these DC responses, naive Th cells differentiate into
   different effector subtypes ([58]Hochweller et al., 2010, [59]Kaiko
   et al., 2008). The inflammatory cytokine IL-12 with strong
   co-stimulatory signals results in Th1, whereas anti-inflammatory
   signals IL-4, IL-5, and IL-13 induce Th2 development ([60]Liu et al.,
   2005b) ([61]MacDonald and Maizels, 2008). Similarly, the cytokine
   IL-10, with strong or weak co-stimulatory signals, respectively,
   generates Th2 or Tregs ([62]Couper et al., 2008, [63]Saraiva and
   O'Garra, 2010). Thus, it has been widely proposed that DC responses can
   be modulated to treat a variety of diseases, such as cancer and
   autoimmunity ([64]Moreau et al., 2017).

   We know that DCs receive multiple stimulatory cues to generate
   signal-specific responses, and it appears improbable that TFs alone can
   integrate such a vast number of regulatory signals. One of the
   well-characterized mechanisms how nuclear receptors (NRs) regulate
   transcriptional responses is through the recruitment of transcriptional
   co-regulators (co-repressors or co-activators) ([65]McKenna and
   O'Malley, 2002, [66]Mouchiroud et al., 2014, [67]Ng et al., 2011).
   Recent reports show that DC treatment with high-affinity NR ligands
   such as rosiglitazone and vitamin D modulates expression of
   co-stimulatory molecules and cytokine genes, which perturbs their
   functional responses ([68]Agrawal et al., 2016, [69]Farias et al.,
   2013, [70]Thompson et al., 2007). Analysis of published NCoR1 chromatin
   sequencing (ChIP-seq) data from macrophages revealed NCoR1 binding on
   genes accountable for antigen recognition, co-stimulation, and T cell
   polarization in DCs. This strongly suggested the functional involvement
   of NR co-repressors like NCoR1 in DCs, as they were identified in
   complexes with unliganded NRs such as peroxisome proliferator activated
   receptor gamma (PPARγ) and thyroid receptor (TR) ([71]Cohen et al.,
   1998, [72]Huang et al., 2009, [73]Li et al., 2013, [74]Mottis et al.,
   2013, [75]Yu et al., 2005). These co-repressors inhibit gene expression
   through chromatin compaction by recruiting histone deacetylases
   ([76]Huang et al., 2009, [77]Perissi et al., 2008). In contrast, we
   reported NCoR1 enrichment at open chromatin regions marked by
   H3K27ac/H3K4me1 ([78]Raghav et al., 2012). Despite several reports
   indicating so, a role of NCoR1 in DC function has not been directly
   evaluated in a systematic study.

   Here, we employed an immunogenomics approach to elucidate the role of
   NCoR1 in DC function. We demonstrated NCoR1 as a master regulator of
   the tolerogenic program in cDCs. We found that direct repression of
   tolerogenic genes by NCoR1 upon DC activation is important to induce an
   optimal immunogenic response. In addition, we explored the underlying
   molecular mechanisms and in vivo physiological impact of
   NCoR1-modulated DC responses in parasite-infected animals.

Results

NCoR1 KD cDC Line Showed Enhanced Tolerogenic Responses upon Activation

   To determine the role of NCoR1 in DCs, we have developed a stable NCoR1
   knockdown (KD) and matched empty vector-transduced control cells using
   lentiviral short hairpin RNA (shRNA) approach in a CD8α^+ cDC1 DC line
   that mimics remarkably the ex vivo isolated cDC1 DCs ([79]Fuertes
   Marraco et al., 2012, [80]Smita et al., 2018). We found that NCoR1
   transcript levels were significantly reduced (≥85%) in NCoR1
   shRNA-transduced DCs ([81]Figures 1A and [82]S1A). We first performed
   qPCR-based immune profiling of control and NCoR1 KD DCs. We found that
   NCoR1 depletion significantly increased transcripts of several
   immune-modulatory genes including Il-10, Il-12p40, Il-27, Ido1, and
   Pdl1 upon 2-, 6-, and 12-h CpG challenge ([83]Figures 1A and [84]S1A).
   The luminex assay showed significantly increased secretion of IL-2,
   IL-6, and IL-10 cytokines in the culture supernatants of 6-h
   CpG-activated NCoR1 KD DCs, whereas the IL-12 cytokine was
   insignificantly increased ([85]Figures 1B and [86]S1B). The median
   fluorescence intensity (MFI) shifts showed a significant decrease of
   CD80 in unstimulated conditions, whereas upon CpG activation CD80,
   CD86, major histocompatibility complex (MHC)-I, and PDL1 expression
   were significantly increased ([87]Figures 1C and [88]S1C). CD40 and
   MHC-II remain unchanged in both the conditions ([89]Figures 1C and
   [90]S1C). Moreover, the intracellular expression of IL-6, IL-10, IDO1,
   and IL-27 cytokines showed significantly increased levels in 6-h
   CpG-challenged NCoR1 KD DCs, whereas IL-12p40 showed an insignificant
   increase ([91]Figures 1D and [92]S1D). The expression of IL-10 is
   predominantly dependent on Erk kinase activity ([93]Saraiva and
   O'Garra, 2010). We found significantly increased pErk^+IL-10^+pSTAT3^-
   cells in activated NCoR1 KD DCs ([94]Figure S1E).

Figure 1.

   [95]Figure 1
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   NCoR1 KD Enhances Tolerogenic Responses in Conventional DCs upon CpG
   Challenge

   (A) Bar plot depicting the transcript expression of selected
   immunogenic and tolerogenic response genes in 6 h CpG-stimulated NCoR1
   KD cDC1 DCs relative to control cells as quantified by qPCR (n = 3).

   (B) Bio-Plex quantitation of the secreted cytokines IL-2 and IL-10 in
   the culture supernatants of 6 h CpG-challenged NCoR1 KD and control DCs
   (n = 6).

   (C) Graph demonstrating the MFI for DC activation and co-stimulation
   markers CD80, CD86, MHC-II, and PDL1 in NCoR1 KD and control cDC1 DCs
   before and after 6 h CpG challenge. Corresponding histogram plot is a
   representative plot for MFI shifts observed for respective markers (n =
   4–8).

   (D) Scatterplot showing the percentage positive cells for intracellular
   expression of IL-10, IDO1, and IL-27 in 0 h and 6 h CpG-stimulated
   NCoR1 KD and control cDC1 line. Corresponding bar plot and histogram
   show the MFI shifts observed for the respective genes (n = 4).

   p values are calculated using two-tailed paired Student's t test; error
   bars represent SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. See also
   [97]Figures S1 and [98]S2.

   Moreover, we also developed a stable NCoR1 KD CD11b^+ cDC2 DC line
   ([99]Pigni et al., 2018). Similar to cDC1 DCs, we identified a
   significantly increased percentage of positive cells for IL-10, IL-27,
   PDL1, IL-6, and IL-12p40 cytokines after CpG activation in
   NCoR1-depleted DCs ([100]Figure S1F). Lipopolysaccharide challenge
   showed similar trends ([101]Figure S1F).

   At the same time, to identify if any strong stimulation renders similar
   responses in NCoR1-depleted cDCs, we challenged NCoR1 KD and matched
   control cDC1 DCs with pIC (TLR3) and CpG + pIC (TLR3 and TLR9 together)
   simultaneously. We found that NCoR1 depletion enhanced expression of
   IL-10, IL-27, IDO-1, and PDL1 along with IL-6 and IL-12p40 in cDC1 DC
   line with both these stimulations ([102]Figure S2A). Moreover,
   simultaneous activation showed stronger DC responses ([103]Figure S2A).
   We also challenged NCoR1 KD and control CD8α^+cDC1 DCs with
   gram-positive (Mycobacterium smegmatis, B. subtilis and Staphylococcus
   aureus), and gram-negative (Vibrio cholerae, Shigella dysenteriae, and
   Salmonella typhimurium) bacteria. Interestingly, we found a profound
   increase of IL-10, IL-27, IDO1, CTLA4, and PDL1 in NCoR1 KD DCs in all
   these stimulations ([104]Figures S2B and S2C).

   Indoleamine 2,3-dioxygenase (IDO) enzyme metabolizes L-tryptophan to
   L-kynurenine resulting in the inhibition of effector Th cell
   differentiation ([105]Yan et al., 2010). We found a significantly
   increased IDO activity in the culture supernatants of NCoR1 KD DCs when
   compared with controls ([106]Figure S1G). This further substantiated
   our observation that NCoR1 directly controls the tolerogenic program in
   cDCs upon activation. Several recent reports showed an enhanced
   expression of IL-10 along with PDL1, IL-27, and IDO1 in tolerogenic DCs
   ([107]Kowalczyk et al., 2014, [108]Mellor and Munn, 2004,
   [109]Tsoumakidou et al., 2014, [110]Yoo and Ha, 2016). Collectively, we
   demonstrated that NCoR1 depletion induces strong tolerogenic response
   upon activation in cDCs.

NCoR1 KD cDC1 DCs Increased Frequency of Treg Development Ex Vivo

   To access the impact of NCoR1 depletion in cDCs on Th cell
   differentiation, we co-cultured NCoR1 KD and control cDC1 DCs with
   naive CD4^+ Th cells isolated from OT-II mice ([111]Figure 2A). We
   found profoundly increased proliferation of Th cells co-cultured with
   CpG-pulsed NCoR1 KD DCs when compared with control cells
   ([112]Figure 2B). In addition, the differentiation profile showed a
   significantly higher number of CD25^+FoxP3^+ Tregs in NCoR1-depleted
   conditions ([113]Figure 2C). Tbet and interferon (IFN)-γ-positive cells
   were significantly reduced, whereas GATA3 showed an increasing trend
   ([114]Figure 2D) The MFI shifts showed similar trends ([115]Figure 2C).
   In unstimulated conditions, no changes were observed ([116]Figure 2D).
   These results confirmed the tolerogenic potential of activated NCoR1 KD
   cDC1 DCs.

Figure 2.

   [117]Figure 2
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   NCoR1 Depletion Enhances Differentiation of Naive CD4^+ Th Cells into
   Tregs

   (A) Experimental outline used to assess the effects of NCoR1 KD on
   T cell polarization ex vivo.

   (B) Representative histograms showing the MFI shifts observed for
   proliferation of T helper cells co-cultured with unstimulated or
   CpG-activated NCoR1 KD and control cDC1 DCs (n = 3).

   (C) Histogram plots demonstrating the representative MFI shifts
   observed for FoxP3, Tbet, IFNγ, and GATA3 in helper T cells co-cultured
   with NCoR1 KD and control cDC1 DCs for 5 days (n = 5).

   (D) Scatterplots from flow cytometric analysis showing the percentage
   positive T helper cells expressing CD25^+FoxP3^+, Tbet, IFNγ, and GATA3
   after 5 days of co-culture with activated NCoR1 KD and control DCs (n =
   5).

   p values are calculated using two-sample unpaired Student's t test;
   error bars represent SEM. *p ≤ 0.05, ***p ≤ 0.001.

Conventional DCs from NCoR1^DC-/- Mice Showed Increased Tolerogenic Responses
upon CpG Challenge

   To validate the findings in vivo, we developed DC-specific conditional
   NCoR1 knockout mice by crossing CD11c-Cre strains with floxed NCoR1
   mice ([119]Yamamoto et al., 2011). Genotyping PCR confirmed NCoR1
   ablation in CD11c^+ cDCs ([120]Figure S3A). To obtain sufficient
   numbers of DCs ex vivo from NCoR1^DC−/- and wild-type (WT) mice, we
   treated them with serum from FLT3L transgenic mice (equivalent to 50 μg
   FLT3L/mouse/day) for eight consecutive days ([121]Baerenwaldt et al.,
   2016). First, we profiled DC subtype composition (cDCs and pDCs) after
   FLT3L treatment. We found that the frequencies of CD8^+ (cDC1) and
   CD11b^+ cDCs (cDC2) were profoundly increased in FLT3L-treated mice
   ([122]Figures S3B and S3C). Then, splenocytes from NCoR1^DC−/- and WT
   mice were stimulated for 6 h with or without CpG and analyzed by
   fluorescence-activated cell sorting (FACS) to identify the impact of
   NCoR1 ablation on different subsets of primary CD11c^+ cDCs
   ([123]Figure S3D). We found that NCoR1 loss in CD11c^highCD8^+ cDC1 DCs
   significantly enhanced the percentage positive cells of IL-10, IL-27,
   and IDO1 after activation, whereas IL-6 showed a marginal but
   insignificant increase ([124]Figure 3A). The MFI shifts showed similar
   trend ([125]Figure 3A). In addition, CD11b^+ cDC2 DCs also showed a
   significant increase of IL-27 and IDO1 in NCoR1^DC−/- when compared
   with WT mice, whereas IL-6 and IL-10 exhibited an increasing trend
   ([126]Figures 3B and [127]S3E). Surface expression of CD80 was
   significantly decreased upon NCoR1 loss of function in both CD8^+ and
   CD11b^+ primary cDCs in unstimulated conditions ([128]Figures 3C and
   3D). No significant differences were observed for other co-stimulatory
   genes CD40, CD86, and MHC-II ([129]Figures S3F and S3G). As we found an
   increased expression of IL-2 cytokine in DC line, we analyzed it in
   primary DCs using FACS. We found significantly higher IL-2-positive
   cells in both cDC1 (CD8^+) and cDC2 DCs (CD11b^+) in CpG-challenged
   NCoR1-ablated condition ([130]Figure S3I).

Figure 3.

   [131]Figure 3
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   Conventional DCs from NCoR1^DC−/- Mice Show Increased Tolerogenic
   Behavior upon Activation

   Splenocytes from NCoR1^DC−/- and WT mice were treated with and without
   CpG for 6 h and the impact of NCoR1 ablation on different DC subsets
   was analyzed by flow cytometry.

   (A) Scatterplots depicting the percentage positive cells for IL-10,
   IL-27, IDO1, and IL-6 in primary cDC1 DCs from NCoR1^DC−/- and WT mice.
   Corresponding histogram plots depict the MFI shifts (n = 6).

   (B) Scatterplots demonstrating the percentage positive cells for IL-10,
   IL-27, IDO1, and IL-6 in primary cDC2 DCs from NCoR1^DC−/- and WT mice
   (n = 6).

   (C) and (D) Scatterplot showing the percentage of CD80-positive cells
   in primary cDC1 and cDC2 DCs respectively from NCoR1^DC−/- and WT mice
   before and after 6 h CpG activation. Representative histograms depict
   the MFI shift.

   (E) Experimental outline depicting the method employed to identify the
   in vivo impact of NCoR1 depletion on CD4^+ T cell polarization in
   NCoR1^DC−/- and WT mice.

   (F) Dot plots showing the percentage of CD4^+CD44^+FoxP3^+-expressing
   effector T cells from the draining inguinal lymph nodes of NCoR1^DC−/-
   and WT mice vaccinated with CpG and OVA for 1 month (n = 15).

   p values are calculated using two-tailed unpaired Student's t test;
   error bars represent SEM. *p ≤ 0.05, **p ≤ 0.01. See also [133]Figures
   S3 and [134]S4.

OVA Immunization Enhances Tregs Frequency in NCoR1^DC-/- Mice

   To access the in vivo impact of NCoR1 deletion in cDCs on Th cell
   differentiation, we vaccinated NCoR1^DC−/- and WT mice with ovalbumin
   (OVA) and CpG at D0 followed by a booster immunization after 30 days
   ([135]Figure 3E). Three days after the booster injection, draining
   lymph nodes were harvested and restimulated with PMA/ionomycin.
   CD3^+CD4^+CD44^+ effector Th cells were analyzed to assess the
   differentiation patterns (IFNγ for Th1, IL-13 for Th2, FoxP3 for Tregs,
   and IL-17 for Th17). We found significantly higher percentages of
   FoxP3^+ cells in NCoR1^DC−/- when compared with WT mice
   ([136]Figure 3F). On the other side, we did not find any change in the
   IFNγ-, IL-13-, and IL-17-expressing cells in the analyzed effector Th
   population ([137]Figure S3H). To further check the OVA-specific T cell
   responses, we also restimulated lymph node cells with OVA-pulsed DCs
   for 72 h. We found an increased proliferation of effecter Th cells in
   both WT and NCoR1^DC−/- mice when compared with PBS controls
   ([138]Figure S4A). At the same time, we found a robust and
   significantly increased FoxP3^+ Treg population in CD3^+CD4^+CD44^+ Th
   cells from NCoR1^DC−/- when compared with WT mice ([139]Figures S4A and
   S4B). These experiments confirmed the development of OVA-specific
   immune responses in both WT and NCoR1^DC−/--immunized animals.

   Moreover, we performed ELISA to evaluate OVA-specific antibody titers
   in immunized NCoR1^DC−/- and WT animals to confirm the development of
   OVA-specific responses. We assayed OVA-specific total IgG and its
   subtypes IgG1, IgG2a, IgG2b, and IgG3. We found high titers of
   OVA-specific total IgG in both WT and NCoR1^DC−/- animals immunized
   with OVA + CpG when compared with PBS controls ([140]Figure S4C). We
   did not observe any significant difference in total IgG levels as well
   as IgG1, IgG2a, IgG2b, and IgG3 between WT and NCoR1^DC−/- mice
   ([141]Figure S4C).

NCoR1 Loss in cDC1 DCs Enhances Treg Development and Parasite Burden

   To explore the in vivo implications, we further investigated if NCoR1
   KD activated DCs can be adoptively transferred at precise time points
   to skew T cell polarization into Tregs and the associated disease
   phenotypes. We employed a well-established helminth infection
   (Heligmosomoides polygyrus) model in C57BL/6 mice ([142]Filbey et al.,
   2014). It is reported that the enhanced Treg population in mesenteric
   lymph nodes (MLNs) increases worm loads in the intestine ([143]Taylor
   et al., 2012).

   Adoptive transfer of CpG-pulsed NCoR1 KD cDC1 DC line at (Day 10) D10
   post helminth infection in mice resulted in significantly increased egg
   counts at D13 and D14 post-infection ([144]Figure 4A). The excreted egg
   numbers were found to be consistently higher as measured until D18. In
   addition, the intestinal worm load was also observed to be
   significantly higher in NCoR1-depleted cDC1-treated animals
   ([145]Figures 4B and [146]S5A). Besides, NCoR1 KD cDC-treated mice
   depicted a profound increase in FoxP3^+ effector Th cells in the MLNs
   when compared with controls ([147]Figures 4C and [148]S5B). No
   differences were observed in Tbet^+ cells, whereas GATA3^+ cells were
   also significantly increased ([149]Figure S5C).

Figure 4.

   [150]Figure 4
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   NCoR1 Loss in cDCs Enhances Treg Frequency In Vivo Leading to Increased
   Disease Burden in Mice

   (A) Line graph demonstrating the egg loads in the feces of
   H. polygyrus-infected C57BL/6J mice at different days post-helminth
   infection after treatment with CpG-pulsed NCoR1 KD and control CD8α^+
   cDC1 DCs. The eggs were counted until D18 post-infection (n = 8).

   (B) Bar graph showing the intestinal worm counts from activated NCoR1
   KD and control cDC1 DC-treated C57BL/6J mice 15 days post-infection
   (n = 5).

   (C) Scatterplot showing the increased effector CD25^+FoxP3^+ Treg
   population in MLN of CpG-pulsed NCoR1 KD DC-treated mice compared with
   control cell-treated animals.

   (D) Line graph demonstrating the egg loads in the feces of
   H. polygyrus-infected NCoR1^DC−/- and WT C57BL/6J mice at different
   days post-helminth infection after CpG treatment. The eggs were counted
   until D17 of infection, and then animals were dissected for intestinal
   worm counting and T cell profiling from MLNs (n = 5).

   (E) Bar plot showing the helminth worm counts from the intestines of
   CpG-treated NCoR1^DC−/- and control C57BL/6J mice on D17 post infection
   (n = 5).

   (F) Bar graph depicting the effector CD25^+FoxP3^+ Treg cells in the
   MLNs of CpG-treated NCoR1^DC−/- and WT mice 17 days post-helminth
   infection (n = 5).

   (G) Line graph demonstrating the paw inflammation in L. major-infected
   NCoR1^DC−/- and WT mice before and after CpG treatment. The CpG
   treatment was given on D18 after infection. Five animals were used in
   each group.

   (H) Bar plot showing the increase in percentage positive FoxP3 Th cells
   in draining lymph nodes of NCoR1^DC−/- and WT mice at D24 after
   infection. Five mice were used in each group.

   p values are calculated using two-tailed unpaired Student's t test;
   error bars represent SEM. *p ≤ 0.05, **p ≤ 0.01. See also
   [152]Figure S5.

   To validate our adoptive transfer observations in vivo in NCoR1^DC−/-
   animals, we infected the NCoR1^DC−/- and WT mice with helminth larvae
   and treated the animals with CpG at D10 post-infection. We found a
   significantly increased helminth egg load at D17 in the feces of
   NCoR1^DC−/- mice compared with WT animals ([153]Figure 4D). The
   intestinal worm counts were also increased ([154]Figure 4E). In
   addition, the effector CD44^+ Th cell profiling from MLNs of infected
   animals showed an increased FoxP3^+ population in NCoR1-ablated mice
   ([155]Figure 4F). We did not find any significant differences in the
   GATA3 and Tbet-expressing Th cells ([156]Figure S5D). We also developed
   a Leishmania major parasitic infection model. After subcutaneous
   injection of parasite in the right footpad of NCoR1^DC−/- and WT mice,
   paw thickness was monitored before and after CpG challenge. As
   cross-presenting DCs are essential for developing immune protection
   against L. major between 17 and 19 days ([157]Ashok et al., 2014), mice
   were treated with CpG at day 18 post-infection. We observed a
   significantly increased (p value ≤ 0.020) foot inflammation at day 21
   post infection in NCoR1^DC−/- animals when compared with WT mice
   ([158]Figure 4G). Although the NCoR1^DC−/- mice had thicker paws than
   the WT mice, the differences disappeared 2 weeks after the CpG
   injection ([159]Figure 4G). Draining lymph nodes from NCoR1^DC−/- mice
   also showed significant increase in FoxP3^+ Th cells ([160]Figure 4H).
   These results indicated a strong potential of NCoR1 in inducing Th cell
   polarization toward Tregs in vivo and modulating the underlying disease
   phenotype. Next, to assess the specificity of antigen presentation of
   DCs, we injected carboxyfluorescein succinimidyl ester (CFSE) labeled
   CD45.1^+ OT-II T cells intravenously in CD45.2^+ helminth-infected WT
   and NCoR1^DC−/- mice at D12 of infection. Next day, we injected WT DCs
   pulsed with or without OVA peptide intravenously, and the animals were
   kept further for 3 days. Then we harvested the splenocytes to check the
   OVA-specific proliferation of CD45.1-gated cells. We observed no
   difference in proliferation of CD45.1^+ OT-II T cells analyzed from WT
   and NCoR1^DC−/- mice suggesting there were similar levels of antigen
   presentation in both WT and NCoR1^DC−/- mice ([161]Figure S5E).

Global Profiling of NCoR1 Identifies Its Direct Control on DC Tolerogenicity

   To characterize the molecular mechanisms underlying NCoR1 depletion
   mediated tolerogenic behavior in cDC1 DCs, we performed RNA sequencing
   (RNA-seq) analysis of NCoR1 KD and control cDC1 DC line at 0h and 6h
   after CpG challenge. Besides, we also performed ChIP-seq profiling of
   NCoR1 in WT cDC1 DC line in similar conditions. We identified ≈13,000
   genomic regions bound by NCoR1 at both 0 and 6 h after CpG activation
   ([162]Table S1). In addition, we performed ChIP-qPCR for 10 randomly
   selected NCoR1 peaks to validate the ChIP-seq data ([163]Figure S6A,
   [164]Table S1). Transcriptome profiling of NCoR1 KD cDC1 line
   identified 1,099 upregulated in contrast to 537 downregulated genes
   when compared with control DCs (q-value ≤ 0.05 and 2-fold)
   ([165]Figure 5A, [166]Table S2). On the other side, in unstimulated
   conditions, only 390 and 360 genes were up- and downregulated,
   respectively, in NCoR1 KD DCs ([167]Figure 5A, [168]Table S3). Overlap
   with ChIP-seq data revealed 658 up- and 224 downregulated genes as
   direct targets of NCoR1 upon CpG activation ([169]Figure 5A, [170]Table
   S2). The numbers of NCoR1 directly bound and upregulated genes were
   much higher than the downregulated genes, confirming NCoR1's role as a
   global transcriptional co-repressor. Pathway enrichment analysis for
   NCoR1 direct target genes in CpG-activated DCs showed significant
   enrichment of IL-10 signaling (p value ≤ 10^−7) and Tregs mediated
   modulation of APC functions (p value ≤ 10^−6) ([171]Figure 5B,
   [172]Table S4). In contrast, NCoR1-unbound and NCoR1-regulated genes
   showed pathway enrichments like “Immune response IFN-alpha/beta
   signaling via MAPKs” and “Cytoskeleton remodeling TGF and WNT and
   cytoskeletal remodeling” ([173]Table S5). Next, we examined the
   expression patterns of NCoR1 direct target genes that are
   differentially regulated in CpG-activated NCoR1 KD cDC1 DCs.
   Interestingly, cluster 2 revealed a wide variety of tolerogenic genes
   such as Lag3, Kmo, Vdr, Cd83, and Tgfbr1 along with Ctla4, Ido1, Ido2,
   Il-10, Pdl1, and Il-27 ([174]Schinnerling et al., 2015)
   ([175]Figure 5C, [176]Table S2). integrative genomics viewer (IGV)
   snapshots and FPKM (fragment counts/kb/million reads) plots from
   RNA-seq data showed that these genes are highly upregulated after CpG
   activation in NCoR1-depleted DCs ([177]Figures 5D and 5E). This global
   analysis further confirmed our in vitro and in vivo observations that
   NCoR1 KD cDCs develop strong tolerogenic behavior upon activation.

Figure 5.

   [178]Figure 5
   [179]Open in a new tab

   Global Profiling of NCoR1 in cDC1 DC Line Depicts Its Direct Control on
   DC Tolerance

   (A) Scatterplot demonstrating the global RNA-seq data of NCoR1 KD and
   control CD8α^+ cDC1 DC line after 6-h CpG challenge. Red and blue dots
   indicate the significantly up- and downregulated genes, respectively.
   Genes of interest are marked in bold. The inset bar graph shows the
   number of genes directly or indirectly controlled by NCoR1 (n = 2), see
   also [180]Table S2.

   (B) Top biological pathways significantly enriched for the list of
   direct target genes identified from RNA-seq analysis of 6 h
   CpG-activated NCoR1 KD DCs. See also [181]Table S4.

   (C) Heatmap depicting the clusters observed for the list of genes
   directly regulated by NCoR1 at 0 h and 6 h after CpG challenge.
   Important DC immune tolerance genes regulated by NCoR1 are marked in
   bold. See also [182]Tables S2 and [183]S3.

   (D) IGV snapshots showing the NCoR1 binding and RNA-seq tag density
   observed at Il-10, Ahr, Pdl1, and Ido2 tolerogenic genes.

   (E) FPKM (fragment counts/kb/million reads) plots demonstrating the
   levels of transcript expression for important DC tolerogenicgenes
   identified to be directly regulated by NCoR1 in CD8α^+ cDC1 DCs. See
   also [184]Table S2.

NCoR1 Strongly Represses the PU.1-Bound Enhancers on Tolerogenic Genes

   To identify the molecular mechanism underlying NCoR1-mediated DC
   tolerance, we have performed an integrative genomic analysis. Our de
   novo motif prediction within NCoR1 ChIP-seq peaks identified PU.1 as
   highly enriched motifs (55%–60% with p value ≤10^−3) in both
   unstimulated and CpG-challenged conditions ([185]Figures 6A and 6B).
   Furthermore, RUNX1, BATF, and IRF: BATF motifs were enriched in
   unstimulated DCs, whereas RUNX1, FRA1, NF-κB, ATF7, and PU.1: IRF
   motifs were enriched in CpG condition ([186]Figures 6A and 6B). To
   substantiate our motif predictions, we overlapped NCoR1 binding in cDC1
   DC line with publically available TF ChIP-seq data from primary bone
   marrow-derived dendritic cells (BMDCs) ([187]Garber et al., 2012). We
   found strong overlap of NCoR1 with PU.1 (50%–55%), NF-–kB (35%–38%),
   IRF1 (20%–25%), IRF4 (19%–23%), Junb (18%–22%), and the active enhancer
   mark H3K27ac (60%–65%). Moreover, we performed ChIP-seq and ChIP-qPCR
   for PU.1 in cDC1 DC line to validate our predictions. We found a strong
   overlap of PU.1 (≥60%) with NCoR1 peaks, confirming it to be the most
   putative NCoR1-recruiting factor in DCs ([188]Figures 6C, [189]S6B, and
   S6C).

Figure 6.

   [190]Figure 6
   [191]Open in a new tab

   NCoR1 Strongly Represses PU.1-Bound Super-Enhancers Present on
   Tolerogenic Genes after CpG Activation in CD8α^+ cDCs

   (A) Top significantly enriched de novo predicted DNA motifs of TFs in
   NCoR1 ChIP-seq peaks of unstimulated CD8α^+ cDC1 DC line.

   (B) Top significantly enriched DNA motifs of TFs in NCoR1 ChIP-seq
   peaks of 6-h CpG-stimulated CD8α^+ cDC1 DCs.

   (C) Bar graph showing the percentage overlap of NCoR1 with PU.1
   ChIP-seq peaks before and after 6 h CpG activation.

   (D) SeqMINER clustering showing clusters I-II depicting increased NCoR1
   binding after CpG activation in cDC1 DC line when compared with
   unstimulated cells. Corresponding PU.1 ChIP-seq data lanes show its
   overlap with NCoR1-bound regions. Corresponding density plot shows the
   differential enrichment of reads in CpG compared with unstimulated at
   clusters I-II genomic regions. See also [192]Table S6.

   (E) Distribution of PU.1 ChIP-seq signals across the total PU.1-bound
   regulatory regions in CpG-activated CD8α^+ DCs. Bound regions were
   ranked by the tag counts within the PU.1-enriched peaks to identify the
   super-enhancer regulatory regions or genes. Tolerogenic genes enriched
   in the PU.1 super-enhancer regions are marked. See also [193]Table S7.

   (F and G) Distribution of NCoR1 ChIP-seq signals across the total
   NCoR1-bound regulatory regions in unstimulated and CpG-activated CD8α^+
   DCs. Bound regions were ranked by the tag counts within the
   NCoR1-enriched peaks to identify the regulatory regions or genes
   super-repressed by NCoR1. Tolerogenic genes enriched in the NCoR1
   super-repressed regions are marked. See also [194]Table S7.

   (H) Metagene plot showing the RNA Pol-II enrichment in unstimulated and
   6-h CpG-stimulated control and NCoR1 KD DCs within the gene-body
   regions of the genes that are annotated to NCoR1 bound cluster I-II
   genomic regions.

   (I) Metagene plot depicting the RNA Pol-II enrichment profile in
   unstimulated and 6 h CpG-stimulated control and NCoR1 KD DCs within the
   gene-body regions of the genes that are upregulated in 6 h
   CpG-activated NCoR1 KD DCs in RNA-seq data.

   (J) IGV snapshots showing the ChIP-seq enrichments of NCoR1, PU.1, and
   RNA Pol-II at tolerogenic genes Il-10 and Pdl1 at 0 h and 6 h
   CpG-stimulated CD8α^+ cDC1 DC line. RNA-Pol-II enrichment in
   CpG-activated control and NCoR1 KD DCs demonstrate the real-time
   transcription of these representative tolerogenic genes. The horizontal
   bars above the plot show the PU.1-bound super-enhancer (SE) regions
   that are strongly repressed (SR) by NCoR1 after CpG activation.

   (K) Representative western blot picture for NF-κB subunits RelA and
   RelB depicting their nuclear translocation at 0 h, 2 h, and 6 h after
   CpG activation in control and NCoR1 KD CD8α^+ cDC1 line. Corresponding
   densitometric analysis bar plots shows the relative intensity of RelA
   and RelB from four biological replicates (n = 4).

   (L) Electrophoretic mobility shift assay (EMSA) demonstrating the
   binding activity of RelA and RelB at 0 h, 2 h, and 6 h after CpG
   activation. RelA and RelB bands are indicated by the marks.

   (M) Fragment counts/kb/million read counts plots from control and NCoR1
   KD RNA-seq data showing the differential expression of NF-κB negative
   regulators of NF-κB canonical signaling.

   (N) RelA ChIP-qPCR depicting the enrichment of RelA at 10 randomly
   selected genomic regions when compared with negative control genomic
   regions.

   p values are calculated using two-tailed paired Student's t test; fold
   change error is depicted as SEM. *p ≤ 0.05. See also [195]Figures S6
   and [196]S7.

   To identify the mechanistic control of gene regulation by NCoR1, we did
   SeqMINER ([197]Ye et al., 2011) clustering to identify the regulatory
   regions differentially bound by NCoR1 in CpG-activated cDCs compared
   with unstimulated conditions ([198]Figure S6B). Two of these genomic
   clusters (clusters I-II) showed increased NCoR1 binding, whereas
   clusters III-XI showed similar enrichment after activation
   ([199]Figures 6D and [200]S7A). Interestingly, these regions were
   annotated to 214 genes that were significantly upregulated in NCoR1 KD
   DCs upon CpG activation, which includes major tolerogenic genes Il-10,
   Pdl1, Ido2, Vdr, and Cd83 ([201]Table S6). The cluster I-II genomic
   regions were also enriched for TFs PU.1, IRF1, IRF4, Junb, and H3K27ac
   marks, whereas RelA and RelB were enriched only after activation
   ([202]Figure S6B). Thus, these regulatory regions appear to be the
   hotspots where several of these activating TFs appear to form
   complexes.

   Moreover, to identify if enhancers at tolerogenic genes are strongly
   repressed by NCoR1 after CpG challenge, we ran the super-enhancer
   discovery program to rank PU.1- and NCoR1-bound signals ([203]Table
   S7). We found that PU.1 super-enhancers on tolerance-inducing genes
   like Il-10, Pdl1, Cd83, Ctla4, and Tgfβr1 were strongly repressed by
   NCoR1 after CpG activation ([204]Figures 6E–6G). Our ChIP-qPCR analysis
   further confirmed the enrichment of PU.1 at these NCoR1-bound genomic
   regions ([205]Figure S7B). This suggested that NCoR1 masks the
   PU.1-bound enhancers after DC activation to prevent induction of the
   major tolerogenic genes. We performed RNA Pol-II ChIP-seq and ChIP-qPCR
   in control and NCoR1 KD cDC line before and after CpG activation to
   estimate the changes in transcription rate of differentially regulated
   genes ([206]Figure S7C). We observed profoundly increased gene body RNA
   Pol-II tag counts for the cluster I-II annotated genes that are
   upregulated in NCoR1-depleted cDCs ([207]Figures 6H–6J). The
   downregulated genes showed a decreased Pol-II tag density ([208]Figures
   S7C and S7D). Representative IGV snapshots depicted the normalized
   ChIP-seq and RNA-seq tag counts at major tolerogenic genes Il-10 and
   Pdl1 ([209]Figure 6J).

   Collectively, these results demonstrated that NCoR1 directly represses
   the tolerogenic enhancers after DC activation. Hence upon NCoR1
   depletion, these genes are derepressed leading to their enhanced
   transcription and concomitant mRNA expression after DC activation. This
   NCoR1-mediated repression of the tolerogenic program appears to be a
   very important event to prevent the cDCs from going into tolerogenic
   modality under the strong inflammatory stimulus.

NCoR1 Depletion Perturbs NF-κB Activity on Tolerogenic Genes

   We found a significant enrichment of NF-κB DNA motif in NCoR1-bound
   regions, and a fine balance of NF-κB activity controlling the
   immunogenic versus regulatory phenotype in DCs has been widely reported
   ([210]Dohler et al., 2017, [211]Thomas, 2013, [212]Vendelova et al.,
   2018). Therefore, two mechanisms could be suggested: (1) NCoR1 directly
   represses PU.1-regulated tolerogenic genes and (2) it directly
   represses inhibitors of RelA (NF-κB), which provides for immunogenic
   functions. Therefore, we probed NF-κB nuclear translocation (RelA and
   RelB) and binding activity in NCoR1 KD and control cDC1 DCs before and
   after CpG activation. We found significantly inhibited RelA
   translocation and activity in NCoR1-depleted DCs at 2 h after
   activation, whereas RelB activity was unaffected ([213]Figures 6K and
   6L). We found increased RelA nuclear localization and binding at 2 h
   when compared with 6 h, whereas RelB comes up at 6 h after CpG
   activation. Interestingly, NCoR1 had little, if any, impact on RelB
   activity, which is known for immune tolerance in DCs. In addition, we
   also observed significantly increased expression of negative regulators
   of NF-κB signaling, i.e., Tnfaip3, Nfkbia, and Traf1 along with
   cytokine-signaling regulators Socs1 and Socs3 in NCoR1 KD DCs
   ([214]Figure 6M). RelA ChIP-qPCR further confirmed an overall decreased
   RelA enrichment on selected tolerogenic/immunogenic genes except Il-10
   and Il-23a in activated NCoR1-depleted DCs ([215]Figure 6N). This
   indicated toward a role of NCoR1 in perturbing NF-κB activity to
   fine-tune DCs immunogenic versus tolerogenic state.

Discussion

   DCs link innate to adaptive immunity and regulate a fine balance of
   inflammatory and tolerogenic responses to prevent immune pathology.
   Although the signaling pathways underlying immunogenic and tolerogenic
   program are explored, the mechanisms underlying fine control of this
   balance in DCs is of paramount interest for therapeutic approaches and
   are largely unknown. Using in vitro, ex vivo, and in vivo models, we
   revealed that NCoR1 is a master repressor of the tolerogenic program in
   cDC1 DCs and its depletion renders DCs tolerogenic, irrespective of
   activation by any strong TLR ligand or microbe. NCoR1-depleted DCs have
   strong potential to polarize Th cells into Tregs ex vivo and in vivo
   with a concomitant increase in disease phenotype. Moreover, we
   demonstrated using integrative genomic analyses that NCoR1 strongly
   represses tolerogenic genes and its KD modulates NF-κB activity in
   activated DCs that could shift immunogenic program toward tolerance.

   A number of reports suggested that treatment of DCs with a variety of
   strong NR ligands such as vitamin-D and dexamethasone induces DC
   tolerance by suppressing their activation along with an increased IL-10
   production ([216]Anderson et al., 2017). NRs are well known to control
   their target genes by switching from co-repressor to co-activator
   complexes upon stimulation ([217]McKenna and O'Malley, 2002,
   [218]Mouchiroud et al., 2014). Recently, NCoR1 co-repressor has been
   reported to interact with non-NR TFs such as NF-κB and AP1 that are
   crucial for DC function ([219]Barish et al., 2012, [220]Huang et al.,
   2009, [221]Li et al., 2013). Activation signals remove NCoR1 from
   co-repressor complex through recruitment of activating TFs like NF-κB
   or through complex destabilization by kinases such as p38 and pErk
   ([222]Ghisletti et al., 2009). In DCs CpG stimulation activates
   canonical NF-κB signaling along with phosphorylation of p38 and Erk
   kinases ([223]O'Neill et al., 2013, [224]Yu et al., 2004). Activation
   of Erk kinase is predominantly reported to induce IL-10 expression,
   whereas p38 largely induces inflammatory cytokines like IL-12
   ([225]Saraiva and O'Garra, 2010, [226]Yu et al., 2004). We found a
   profoundly increased pErk activity and concomitantly increased IL-10
   levels in activated NCoR1 KD DCs. At the same time PDL1, IL-27, and
   IDO1 genes were upregulated, which are also reported to induce strong
   tolerogenic responses ([227]Raker et al., 2015, [228]Shiokawa et al.,
   2009, [229]Sumpter and Thomson, 2011, [230]Yoo and Ha, 2016). The IL-10
   cytokine further induces Pdl1 through STAT3 signaling ([231]Wolfle
   et al., 2011). The expression of major tolerogenic genes like Ido1,
   Pdl1, and Il-10 are also induced by IL-27 cytokine, an IL-12 family
   member cytokine through STAT1 and STAT3 signaling ([232]Carbotti
   et al., 2015, [233]Murugaiyan et al., 2009). Both these STATs were
   increased in NCoR1 KD DCs ([234]Carbotti et al., 2015), which are known
   to induce regulatory Tr1 cells by inhibiting Th17 cell development
   ([235]Kushwah and Hu, 2011). The increased IL-2 could also enhance the
   secretion of IL-27-induced IL-10 ([236]Murugaiyan et al., 2009).
   Studies suggest that STAT3 is also phosphorylated via both IL-6 and
   IL-10 signaling in immune cells ([237]Niemand et al., 2003). It is
   unlikely that increased IL-6 is responsible for this STAT3 activation
   in NCoR1 KD cells as the IL-6rα expression was significantly decreased
   after CpG stimulation. There is a concomitant increase in Socs3 as
   well, which strongly inhibits STAT3 phosphorylation leaving open the
   IL-10 signaling pathway ([238]Ahmed and Ivashkiv, 2000). Socs2 and
   Socs3 are also reported to suppress uncontrolled inflammatory cytokine
   expression and were highly elevated in tolerogenic-state mature DCs
   ([239]Connor et al., 2017, [240]Yoshimura et al., 2005).

   Contrary to the reported suppression of IL-12 by enhanced IL-10
   signaling in DCs ([241]Ma et al., 2015), we noticed a marginal increase
   of IL-12 in NCoR1 KD DCs when compared with the robust increase of
   IL-10 production. Last but not the least, IL-12p40 homo-dimers are
   inhibitory and IL-12p70 is not significantly upregulated in NCoR1 KD
   DCs. Type-I IFN signaling is also suggested to stimulate IL-10 and
   suppress IL-12 and IL-23 cytokines in DCs ([242]Yen et al., 2015).
   Interestingly, we found Ifnβ1 and its signaling intermediate Stat2
   significantly increased in CpG-stimulated NCoR1 KD DCs. IFNβ1 is also
   known to represses IL-12 expression ([243]Zhao et al., 2015).

   Some of the surface markers and secreted factors that showed a marginal
   increase after NCoR1 KD are often considered inflammatory.
   Nevertheless, co-stimulation and antigen presentation by MHC class-II
   are required for Treg generation ([244]Price et al., 2015). Therefore,
   an increase of co-stimulatory molecules can also contribute to Treg
   differentiation. As Tregs express CD25 (IL-2Rα), increased IL-2
   secreted by NCoR1 KD DCs could induce Treg proliferation and clonal
   expansion and further contribute to tolerogenicity. In RNA-seq, we find
   upregulation of IL-12p35 and this molecule has inflammatory (IL-12p70)
   as well as anti-inflammatory/tolerogenic properties (IL-35 composed of
   IL-12p35 and EBI3) ([245]Collison et al., 2010).

   Furthermore, we characterized the direct and indirect target genes of
   NCoR1 for mechanistic insight. We found a strong overlap of PU.1
   binding with NCoR1, suggesting it as the most putative TF-tethering
   NCoR1 in DCs. Integrative genomics demonstrated that NCoR1 masks the
   PU.1-bound super-enhancers at the major tolerogenic genes including
   Il-10, Cd83, Ctla4, and Pdl1. Therefore, upon NCoR1 depletion these
   genes are derepressed leading to enhanced transcription as evident by
   increased gene body RNA Pol-II counts after activation. We also
   observed a significant enrichment of NF-κB at NCoR1 peaks in
   CpG-activated condition, which suggested involvement of NF-κB TF in
   NCoR1-mediated effects. Recent reports also indicated that
   perturbations in the activity of different NF-κB subunits like RelA and
   RelB modulate DCs' inflammatory versus tolerogenic phenotype
   ([246]Azukizawa et al., 2011). We identified that RelA-binding activity
   was profoundly reduced after activation, whereas RelB was unaffected.
   In addition, NCoR1 directly represses negative regulators of canonical
   NF-κB signaling such as Tnfaip3 (A20), Traf1, and Pias1, which are well
   reported to repress inflammatory responses by inhibiting RelA activity
   ([247]Kool et al., 2011, [248]Liu et al., 2005a, [249]Sen and Smale,
   2010, [250]Vereecke et al., 2009). We also noticed increased RelB
   translocation and activity from 2 to 6 h after CpG activation in CD8α^+
   cDCs, whereas the RelA activity was reduced. It would be further
   interesting to characterize how this switch of NF-κB activity regulates
   DC responses. In addition, we showed that increased NCoR1 biding after
   CpG activation overlaps with binding of other activating TFs.
   Therefore, it is highly probable that other activating TFs may also
   bind at these tolerogenic enhancers after NCoR1 depletion. Treatment
   with live bacteria showed even more pronounced tolerogenic effects
   compared with CpG challenge, which suggests that multiple strong
   stimulations further enhance tolerogenicity in NCoR1 KD DCs.
   Altogether, our integrative genomic analysis identified NCoR1 as a
   master regulator of the tolerogenic program in DCs.

Limitations of the Study

   In this study we have majorly used cDC1 mutuDC line and validated the
   findings ex vivo and in vivo using primary DCs. It is important to
   mention that from a single WT C57BL/6 mice one can get nearly 100,000
   cDC1 DCs in good condition from splenocytes, using magnetic bead-based
   kits. Therefore, using primary DCs is a major limitation for
   high-throughput genomic analysis such as ChIP-seq and bulk RNA-seq that
   we reported here.

Acknowledgments