Recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF) and recombinant murine interleukin 4 (rmIL-4) were purchased from Peprotech (Rocky Hill, NJ, USA). LPS was purchased from Sigma (St. Louis, MO, USA). β-mercaptoethanol was obtained from MP Biomedicals LLC (Solon, OH, USA). Pam3csk4, poly (I:C), and CpG ODN1826 were purchased from InvivoGen (San Diego, CA, USA). FITC-, APC-, and PE-conjugated anti-murine CD4, CD11c, CD40, CCR7, CD80, CD86, and MHC-II antibodies were purchased from BioLegend (San Diego, CA, USA) and eBiosciences (New York, NY, USA). Antibodies against CD11c, TLR4, MyD88, and IκB were purchased from Biosynthesis Biotechnology Co. Ltd. (Beijing, China). Antibodies against p65 and p38 were obtained from Zhongshan Golden Bridge Co. Ltd. (Beijing, China). Antibodies against phospho-p65, phospho-IRF3, phospho-ERK1/2, and IRF3 were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phospho-p38 and ERK1/2 were purchased from Bioworld Technology (Nanjing, China). Anti-Tmod1 antibody was prepared by AbMax Biotechnology Co., Ltd. (Beijing, China). Antibodies against GAPDH and β-actin were obtained from Santa Cruz Biotech. (Santa Cruz, CA, USA). Tmod1 adenovirus and control adenovirus were constructed by SinoGeneMax Co. Ltd. (Beijing, China).

A Tmod1 knockout mouse model was first created by disrupting exon 1 in Dr. L. Amy Sung’s laboratory at the University of California, San Diego (31). While Tmod1^-/- mice died at embryonic day 9.5, the heterozygous Tmod1^+/- mice were able to mate with a cardiac specific Tmod1 overexpressing transgenic (TOT) mice (37). Interbreeding between their offsprings resulted in TOT/Tmod1^-/- mice where the overexpression of Tmod1 in the heart rescued the homozygosity’s lethality (38). TOT/Tmod1^-/- mice were transferred from Dr. L. Amy Sung’s lab to the Peking University Health Science Center and maintained in SPF animal room. C57BL/6J (Tmod1^+/+) mice were purchased from the animal department of the Peking University Health Science Center. The animal study was approved by the ethics committee of the Peking University Health Science Center. BMDCs were obtained from 8 week-old female TOT/Tmod1^-/- and Tmod1^+/+ mice according to the method developed by Roney (39). Briefly, bone marrow cells were flushed out from freshly dissected femurs and tibias. Following lysis of the red blood cells in lysis buffer, the remaining cells were resuspended in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 0.1% β-mercaptoethanol, and differentiated into imDCs in the presence of 20 ng/ml rmGM-CSF and 10 ng/ml rmIL-4 for 7 days. The imDCs were then matured into mDCs by adding 100 ng/ml LPS for another 2 days. The imDCs were also treated with Pam3csk4 (100 ng/ml), poly (I:C) (25 μg/ml), and CpG ODN 1826 (1 μM), respectively, for 1–2 days.

ImDCs and mDCs were fixed with 4% paraformaldehyde and labeled with FITC-, PE-, or APC-conjugated antibodies to mouse CD11c, CD86, MHC-II, CD80, CD40, and CCR7, and the corresponding isotype controls. The cells were then analyzed with a BD FACS Calibur™ flow cytometer (BD Biosciences, San Diego, CA, USA).

ImDCs and mDCs were harvested and lysed in RIPA buffer containing protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). Total proteins (100 μg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a nitrocellulose membrane. After blocking with 5% nonfat milk, the membrane was incubated with antibodies against Tmod1, GAPDH, CD11c, p65, p-p65, p38, p-p38, ERK1/2, p-ERK1/2, and TLR4 overnight at 4°C. After washing, the membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature (25°C). The signals were visualized using an enhanced chemiluminescence (ECL) detection kit (Evergreen, Beijing, China) and exposed to X-ray films.

Total RNA was extracted from imDCs and mDCs using RNAtrip reagent (Applygen Biotechnologies Inc., Beijing, China) and then reverse transcribed into cDNA using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time quantitative PCR (RT-qPCR) was performed on a Mx3000 Multiplex Quantitative PCR system (Stratagene, La Jolla, CA, USA) using EvaGreen qPCR Master Mix-low Rox (Applied Biological Materials (abm) Inc., Richmond, BC, Canada). The sequences of the primers are listed in Table 1. GAPDH was used as an internal control. A relative fold change in the gene expression was calculated using the method of 2^-△△Ct method.

About 5 × 10⁵ TOT/Tmod1^-/- imDCs were infected with Tmod1 adenovirus (Ad-Tmod1) or control adenovirus (Ad-Null) at 200 MOI (multiplicity of infection) for 6 h. Equal number of TOT/Tmod1^-/- imDCs without adenovirus treatment were used as the negative control. The cells were further treated with LPS (100 ng/mL) for 24 h before cells and culture media were collected.

Culture media of imDCs, mDCs, and OVA peptide-treated spleen cells were collected. The levels of cytokines and chemokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), interferon-γ (IFN-γ), interferon-β (IFN-β), and interleukin-10 (IL-10), were measured by flow cytometry using LEGENDplex™ bead-based immunoassay kits (BioLegend). The levels of TNF-α and IL-10 in the culture media of adenovirus-infected and LPS-treated TOT/Tmod1^-/- imDCs and TLR agonist-treated imDCs were measured using Enzyme linked immunosorbent assay (ELISA) kits (BOSTER Biotechnology, Wuhan, China).

Approximately 5 × 10⁵ imDCs were incubated with 500 μg/ml FITC-dextran particles (43.2 kDa, Sigma) in 1 ml of RPMI 1640 at 4°C or 37°C (40). After 0.5 or 2 h of incubation, the cells were washed three times with PBS to remove the un-endocytosed particles and the endocytosed particles were analyzed using flow cytometry. The differences in the fluorescence intensities between 37°C and 4°C represented the endocytosis abilities of imDCs.

Migration of imDCs and mDCs was measured using a modified Boyden chamber (5-μm pore size). A total of 2 × 10⁵ cells in 200μl volume was plated in the upper compartment of the chamber, and 600μl of complete medium supplemented with or without 200 ng CCL19 was added to the lower compartment. The chamber was incubated for 12 h at 37°C, following which the migrated cells were counted under a light microscope.

Mixed lymphocyte reaction (MLR) assay was used to determine the T-cell activation abilities of the mDCs (40). Allogeneic T cells were obtained from the spleen cells of C57BL/6J mice by passing the spleen cells through a nylon wool column. mDCs (1 × 10⁵, 1 × 10⁴, and 1 × 10³) were mixed with 1 × 10⁵ T cells, respectively, in a total volume of 200μl, and the mixtures were incubated in a CO₂ incubator for 48 h. Then, 20μl CCK8 solution (Enhanced Cell Counting Kit-8, Beyotime Biotech., Shanghai, China) was added to each mixture for 4 h. The proliferation of the T cells was quantified by measuring the optical density at 450 nm in a microplate reader (BioRad, Hercules, CA, USA).

Mature DCs were incubated with 10 μg/ml OVA peptide (323–339) (Alpha Diagnostic Intl Inc., San Antonio, TX, USA) at 37°C for 2 h. The cells were washed twice and resuspended in serum-free RPMI 1640 medium. Treated mDCs (1 × 10⁶) were injected intravenously into C57BL/6J mice on days 1, 3, and 5. On day 8, the mice were sacrificed, and their spleen cells were collected, and re-stimulated with 10 μg/ml OVA peptide (323–339) at 37°C for 3 days (41). Then, the spleen cells were washed with PBS, stained with FITC-conjugated anti-mouse CD4 antibody, and further analyzed by flow cytometry. The fluorescence intensity indicated the proliferation of CD4⁺ T cells. The concentrations of IFN-γ and IL-10 in culture medium of OVA-peptide-treated spleen cells were measured by flow cytometry using LEGENDplex™ bead-based immunoassay kits (BioLegend).

Mature DCs were pulsed with 20 μg/ml MOG35-55 peptide (Sigma) for 1 h at 37°C (41). The pulsed cells (1.2 × 10⁶) were injected intravenously into female C57BL/6J mice twice (on days 0 and 4). Experimental autoimmune encephalomyelitis (EAE) was induced by subcutaneous injection of 100 μg of MOG35-55 peptide emulsified in complete Freund’s adjuvant (Chondrex, Redmond, WA, USA) with 100 μl of 4 mg/ml Mycobacterium tuberculosis (Chondrex) on days 7 and 14. In addition, 200 ng of pertussis toxin (Invitrogen, Carlsbad, CA, USA) was injected intraperitoneally on days 7 and 9. The mice were observed and scored on the scale of 0 to 5: 0, no symptoms; 1, flaccid tail; 2, hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, moribund state (41).

ImDCs and mDCs were fixed in 4% paraformaldehyde and washed twice with PBS. After permeabilization with 0.1% Triton X-100, the cells were blocked with 1% bovine serum albumin (BSA) for 30 min at room temperature. The cells were then stained with 0.165 μM rhodamine phalloidin (Invitrogen) in the dark for 20 min. The cells were washed, resuspended in PBS, and analyzed using a BD FACS Calibur (BD Bioscience). The mean fluorescence intensity represented the F-actin content in the cells.

ImDCs and mDCs were cultured on poly L-lysine-treated coverslips and stained with rhodamine phalloidin as mentioned above. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime Biotech., Shanghai, China) for 5 min. After washing with PBS, the cells were observed under a laser scanning confocal microscope (Leica TCS SP8 MP FLIM, Wetzlar, Germany).

ImDCs and mDCs were cultured on poly-L-lysine-treated coverslips. The petri dish containing the coverslip was placed on an inverted microscope equipped with a nanoindenter (Piuma Chiaro, Optic II, Amsterdam, Netherlands). A probe with spring constant of 0.18 N/m and a spherical tip radius of 9-μm was mounted on the cantilever. During indentation, the tip was brought into contact with the cell surface and load-indentation and load-time data were recorded. The indentation depth was 10µm and the loading and unloading time was set to 2 s. The loading and unloading curves were fitted with Hertzian contact model and Young’s modulus was calculated. The mean Young’s modulus for each specimen was generated from at least 20 to 30 cells from three independent preparations.

All experimental procedures were repeated at least three times and are presented as mean ± standard error of mean (SEM). GraphPad Prism 7.0 software was used for the normal distribution test, homogeneity of variance test, statistical analysis, and plotting. Paired or unpaired Student’s t-tests were used to compare the results of the two groups, and analysis of variance was used between the groups. P < 0.05 was considered statistically significant.

To define the immune functions of Tmod1, we first examined whether Tmod1 was expressed in DCs. We isolated bone marrow cells from C57BL/6J (Tmod1^+/+) mice and differentiated them into imDCs following treatment with IL-4 and GM-CSF. LPS was used to induce DC maturation. qPCR data showed that compared with imDCs, Tmod1 was upregulated by approximately 4-fold in mDCs (Figure 1A, p < 0.05). Western blotting data further confirmed that Tmod1 was present in imDCs and its expression level increased significantly upon DC maturation (Figure 1B, p < 0.05). We wondered whether agonists for other TLRs could also promote Tmod1 expression. Therefore, imDCs of Tmod1^+/+ mice were treated with TLR2 agonist Pam3csk4 and TLR3 agonist poly (I:C). Un-treated and LPS-treated imDCs served as a negative and positive control. Both qPCR and western blotting data showed that neither Pam3csk4 nor poly (I:C) could stimulate Tmod1 expression (Figures S1A, B). This suggests that LPS is the strongest stimulation for Tmod1 expression among these TLR agonists.

We also cultured DCs of TOT/Tmod1^-/- mice and analyzed Tmod1 expression in these cells. However, no Tmod1 could be detected, suggesting the complete absence of Tmod1 in TOT/Tmod1^-/- DCs (Figure 1B). In addition to Tmod1, three homologs of similar size exist in Tmod family, Tmod2 (neuronal specific), Tmod3 (ubiquitous), and Tmod4 (skeletal muscle specific) (42). Compensatory effect is observed among some of the homologs (43). To examine the expression of other Tmod homologs in Tmod1-deficient DCs, we performed qPCR on imDCs and LPS-induced mDCs of Tmod1^+/+ and TOT/Tmod1^-/- mice. Results showed that the expression levels of Tmod2, Tmod3, and Tmod4 were all comparable between the two genotypes (Figure S2). This suggests that other Tmod homologs did not compensate for the loss of Tmod1.

Since Tmod1 is expressed in both imDCs and mDCs, we sought to determine whether Tmod1 plays a role in DC differentiation. We measured the expression level of the DC marker, CD11c, on the surface of imDCs and mDCs from Tmod1^+/+ and TOT/Tmod1^-/- mice. Flow cytometry data showed that CD11c levels were comparable in Tmod1^+/+ and TOT/Tmod1^-/- DCs, regardless of their state of maturation (immature or mature; Figures 1C, D). We also analyzed CD11c levels in the cell lysates of Tmod1^+/+ and TOT/Tmod1^-/- DCs using western blotting, but did not detect any difference (Figure S3). These results suggest that Tmod1 is not essential for the differentiation of BMDCs.

The finding that Tmod1 was upregulated upon maturation suggests that Tmod1 may be involved in the maturation process and the functional changes related to maturation. DC maturation is characterized by increased surface expression of costimulatory molecules and chemokine receptors as well as the secretion of cytokines. Therefore, we examined the expression of MHC-II, CD80, CD86, CD40, and CCR7 on Tmod1-deficient DCs using flow cytometry. The results showed that Tmod1-deficient imDCs had considerably lower expression of MHC-II and CCR7 than Tmod1^+/+ imDCs (Figures 2A, B). However, the expression of CD80, CD86, and CD40 was similar between the two groups. Following LPS induction, the expression of MHC-II, CD80, CD86, CD40, and CCR7 increased markedly on both Tmod1^+/+ and TOT/Tmod1^-/- mDCs. However, the expression levels of these molecules were significantly lower on TOT/Tmod1^-/- mDCs than on Tmod1^+/+ mDCs (Figures 2A, B), suggesting that TOT/Tmod1^-/- mDCs were phenotypically less mature than Tmod1^+/+ mDCs. We also compared the levels of cytokines secreted by Tmod1^+/+ and TOT/Tmod1^-/- mDCs. The data showed that compared with Tmod1^+/+mDCs, TOT/Tmod1^-/- mDCs secreted noticeably less TNF-α, IFN-γ, and IL-12 (p70) (p < 0.05, Figure 2C). IL-6 secretion was similar in both groups. These results indicate that Tmod1 deficiency retarded LPS-induced DC maturation by reducing surface marker expression and cytokine secretion.

The decreased expression of multiple surface markers and secretion of several cytokines suggests that Tmod1 deficiency may affect the activities of upstream signaling pathways. TLR4 is the receptor for LPS and its downstream MyD88-dependent pathway activates NF-κB and MAPK pathways, which in turn are responsible for the expression of costimulatory molecules and inflammatory cytokines (44, 45). Therefore, we compared the activities of these two pathways in LPS-treated Tmod1^+/+ and TOT/Tmod1^-/- imDCs using western blotting. We found that IκB, which retains p65 in the cytosol, started to degrade within 5 min of LPS treatment and was almost undetectable by 30–60 min in Tmod1^+/+ imDCs (Figure 3A). However, in TOT/Tmod1^-/- imDCs, although it started to become degraded within 5 min, it was still present in large amounts (1.7-fold relative to Tmod1^+/+) at 30–60 min, suggesting that the degradation of IκB was retarded (Figures 3A, B). Correspondingly, p65 phosphorylation increased in 5 min and reached the highest level at 30 min in both Tmod1^+/+ and TOT/Tmod1^-/- imDCs, but the phosphorylation level was considerably lower in TOT/Tmod1^-/- imDCs than in Tmod1^+/+ imDCs (80% less at 5 min, Figures 3A, B). Although LPS-induced phosphorylation kinetics of p38 MAPK was similar between Tmod1^+/+ and TOT/Tmod1^-/- imDCs, its phosphorylation level in TOT/Tmod1^-/- imDCs was considerably lower than that in Tmod1^+/+ imDCs (30% less at 5 min, Figures 3A, B). No difference was found in the phosphorylation levels of ERK1/2 MAPK between the two groups (Figure S4). Our results indicate that in the absence of Tmod1, LPS-induced phosphorylation of the key signaling molecules, NF-κB and p38 MAPK, is significantly compromised.

Next, we examined the mRNA expression of costimulatory molecules and inflammatory cytokines. The data showed that Tmod1^+/+ and TOT/Tmod1^-/- imDCs had low and comparable expression levels of MHC-II, CD80, CD40, and CCR7. However, the expression of CD86 was significantly lower in TOT/Tmod1^-/- imDCs compared to Tmod1^+/+ imDCs (p < 0.001, Figure 3C). LPS treatment induced the upregulation of all the maturation markers in the two genotypes. The expression levels of CD80 and CD86 were significantly lower in TOT/Tmod1^-/- mDCs than in Tmod1^+/+ mDCs (p < 0.05, Figure 3C), while expression levels of MHC-II, CD40, and CCR7 were similar between the two groups. In terms of the mRNA expression of cytokines, we found that the expression of Il6 and Ifng in TOT/Tmod1^-/- mDCs was about 50% less than that in Tmod1^+/+ mDCs (p < 0.05, Figure 3D), but the expression of Tnfa and Il12 was not significantly different between the two groups. These data suggest that Tmod1 deficiency has different and complex effects on the expression of costimulatory molecules and inflammatory cytokines.

In addition to the MyD88-dependent pathway, the binding of LPS with TLR4 also triggers the activation of the TRIF-dependent pathway, resulting in the phosphorylation of IRF3 and the expression of type I interferons (46, 47). We treated imDCs of the two genotypes with LPS for 2, 4, and 6 h, and analyzed the phosphorylation of IRF3 using western blotting. The data showed that, following LPS treatment, IRF3 phosphorylation gradually increased and reached the highest level by 6 h in Tmod1^+/+ DCs. However, in TOT/Tmod1^-/- DCs, the phosphorylation level of IRF3 was significantly higher than that in Tmod1^+/+ DCs at 2 and 4 h (p < 0.01) and remained high up to 6 h (Figures 4A, B). Ifnb and Il10 are the two major downstream genes of IRF3 (9). We performed qPCR to compare their expression levels in LPS-induced Tmod1^+/+ and TOT/Tmod1^-/- mDCs. We found that the expression of Ifnb and Il10 was about 6- and 2- fold higher, respectively, in TOT/Tmod1^-/- mDCs compared to that in Tmod1^+/+ mDCs (Figure 4C). Consistently, the concentrations of IFN-β and IL-10 secreted in the culture medium of TOT/Tmod1^-/- mDCs were 318.8 ± 13.8 pg/ml and 70.4 ± 23.6 pg/ml, respectively, which were markedly higher than those of Tmod1^+/+ mDCs (280.6 ± 10.5 pg/ml and 11.7 ± 1.2 pg/ml, respectively) (Figure 4D). These data suggest that Tmod1 deficiency promotes the activation of the TRIF-dependent pathway and the expression of type I interferons.

Besides TLR4, other TLRs also signal via MyD88 or IRF or both, such as TLR2 agonist Pam3csk4 (MyD88-NF-κB pathway), TLR3 agonist poly (I:C) (TRIF-IRF3 pathway), and TLR9 agonist CpG ODN 1826 (MyD88-NF-κB and -IRF7 pathway) (48). To examine whether Tmod1 was required for signaling through other TLRs, we stimulated Tmod1^+/+ and TOT/Tmod1^-/- imDCs with LPS, Pam3csk4, poly (I:C), and CpG. We observed a significant decrease in Il6 mRNA level and markedly augmentation in Il10 and Ifnb mRNA levels by LPS in TOT/Tmod1^-/- imDCs (Figure S5A, C, D). But Pam3csk4, poly (I:C), and CpG failed to induce changes in Il6, Tnfa, Il10, and Ifnb mRNA expression in TOT/Tmod1^-/- imDCs (Figure S5A–D). Consistently, TNF-α secretion was significantly inhibited and IL-10 secretion was considerably enhanced by LPS in TOT/Tmod1^-/- imDCs, while other TLR agonists did not show much effect (Figure S5E, F). These data suggest that Tmod1 may have a unique function on TLR4 signaling.

Since Tmod1 deficiency resulted in inhibited TLR4/MyD88 signaling and enhanced TLR4/TRIF/IRF3 signaling, we next sought to explore whether exogenous Tmod1 could rescue these effects. We introduced exogenous Tmod1 into TOT/Tmod1^-/- imDCs by adenovirus infection and then treated cells with LPS. The expressions of MyD88 and TRIF/IRF3 downstream genes were measured by qPCR and/or ELISA. qPCR data confirmed the re-expression of Tmod1 in Ad-Tmod1 infected cells (Figure 5A, left panel). Compared with Ad-Null infected mDCs, the expression of maturation marker, CD86, was increased to some extent in Ad-Tmod1 infected mDCs (Figure 5A, second panel). Strikingly, the expressions of Il6, Tnfa, and Il12 were all significantly elevated, while the expression of Il10 was greatly reduced (p < 0.05, Figure 5A, right panels). Furthermore, ELISA data showed that TNF-α secretion in Ad-Tmod1 infected mDCs was increased a little bit but did not show significance (Figure 5B). Yet, IL-10 secretion was significantly decreased (p < 0.05, Figure 5C). These data suggest that rescue with exogenous Tmod1 expression in Tmod1-deficient DCs made MyD88-dependent pathway more activated and TRIF-dependent pathway more inhibited, thus, it reversed the effect of Tmod1 deficiency on TLR4 signaling.

Endocytosis of antigens, migration, and antigen presenting abilities are the most important immune functions of DCs. Therefore, we investigated whether Tmod1 deficiency affects these immune functions. ImDCs, residing in the peripheral tissues, engulf pathogens and antigens by endocytosis. We incubated Tmod1^+/+ and TOT/Tmod1^-/- imDCs with FITC-labeled dextran for 0.5 and 2 h and detected their fluorescence intensities using flow cytometry. The data showed that the mean fluorescence intensities at 0.5 h were comparable between the two genotypes (82.7 ± 20.0 vs. 69.5 ± 13.5, Figure 6A). The mean fluorescence intensities increased considerably at 2 h, suggesting that more endocytosis occurred, but there was no difference between the two groups (Figure 6B). This indicates that Tmod1 deficiency did not affect the endocytosis ability of imDCs.

ImDCs patrol in the periphery by random migration, while mDCs undergo directional migration to lymph nodes because of high CCR7 expression (49, 50). Therefore, we used a modified Boyden chamber to measure the random migration of imDCs and mDCs, and chemotactic migration of mDCs in the two genotypes. We found that TOT/Tmod1^-/- imDCs and mDCs showed significantly slower random migration compared to their Tmod1^+/+ counterparts (p < 0.01 and p < 0.05, respectively, Figure 6C, left panel). When we measured the chemotaxis of mDCs toward CCL19, we found that the percentage of migrated TOT/Tmod1^-/- mDCs was half of that of migrated Tmod1^+/+ mDCs (Figure 6C, right panel). These data suggest that knockout of Tmod1 impaired the migration abilities of both imDCs and mDCs.

mDCs are mainly responsible for presenting antigens to naïve T cells and priming them to produce inflammatory cytokines such as IFN-γ and IL-10, and then mediating immune responses (15). Therefore, we sought to determine whether Tmod1 deficiency affects the T-cell stimulatory abilities of mDCs in vitro and in vivo. Allogeneic MLR data showed that Tmod1^+/+ mDCs substantially promoted the proliferation of T cells, while the proliferation of T cells mixed with TOT/Tmod1^-/- mDCs was significantly reduced (p < 0.05, Figure 7A). To further validate this data, an in vivo assay was performed (41), in which C57BL/6J mice were transfused with OVA peptide-loaded Tmod1^+/+ and TOT/Tmod1^-/- mDCs, respectively, and their spleen cells were collected and re-stimulated with OVA peptide for 3 days. The spleen cells were stained with FITC-conjugated CD4 antibody and subjected to flow cytometry analysis. We found that the percentage of CD4⁺ T cells in the spleen cells from TOT/Tmod1^-/- mDC transfused mice was 22.02 ± 1.80%, which was significantly lower than that in spleen cells from Tmod1^+/+ mDC transfused mice (36.23 ± 2.05%) (p < 0.01, Figures 7B, C). We also measured the concentrations of IFN-γ and IL-10 in the culture medium of the spleen cells by flow cytometry. The data showed that the spleen cells from TOT/Tmod1^-/- mDC transfused mice produced markedly less IL-10 compared to those from Tmod1^+/+ mDC transfused mice (3062.2 ± 311.1 pg/ml vs. 6021.7 ± 1032.7 pg/ml, p < 0.05) (Figure 7D). However, the production of IFN-γ was similar between the two groups (Figure 7D). These data suggest that TOT/Tmod1^-/- mDCs were defective in T cell activation and priming. Moreover, Tmod1-deficient mDCs impaired the production of IL-10-producing T cells, but not IFN-γ-producing T cells. The results also indicate that Tmod1-deficient mDCs may be able to induce peripheral tolerance.

To test this hypothesis, we compared the abilities of Tmod1^+/+ and TOT/Tmod1^-/- mDCs to prevent the development of EAE in mice, which was induced through subcutaneous injection of an encephalitogenic MOG peptide. The data showed that the mean disease score in mice injected with Tmod1^+/+ mDCs increased gradually from day 14 to day 35 (0 to 2.0), and then dropped after day 35. However, the mean disease score in mice injected with TOT/Tmod1^-/- mDCs remained < 0.5 from day 14 to day 44 (Figure 7E), indicating that three prior injections of MOG peptide-pulsed TOT/Tmod1^-/- mDCs offered almost completely protection against the neuropathological symptoms associated with EAE. This suggests that Tmod1-deficient mDCs induced tolerance to the MOG peptides.

The immune functions of DCs rely on their cytoskeletal structure and biomechanical properties. It has been shown that F-actin is increased in LPS-stimulated DCs (21). Since Tmod1 regulates the polymerization of actin filaments, it may participate in LPS-induced F-actin remodeling. Therefore, we analyzed the F-actin content in imDCs and mDCs of Tmod1^+/+ and TOT/Tmod1^-/- mice using flow cytometry and fluorescence microscopy. As shown in Figures 8A, B, the F-actin content was significantly increased in Tmod1^+/+ mDCs compared to Tmod1^+/+ imDCs (p < 0.01). However, no difference was found in the F-actin contents of TOT/Tmod1^-/- imDCs and mDCs. We further measured the cell stiffness of imDCs and mDCs using a nanoindenter, in which the cells were indented with a probe and the loading (blue) and unloading (green) curves were drawn (Figure 8C). Young’s modulus was calculated by fitting the curves with the Hertzian contact model. We found that Tmod1^+/+ mDCs had greater Young’s moduli than Tmod1^+/+ imDCs, while TOT/Tmod1^-/- imDCs and mDCs had similar Young’s moduli (Figure 8D). Our data suggest that Tmod1 deficiency diminished LPS-induced F-actin formation and cell stiffening in the DCs.