MicroRNA-148a facilitates inflammatory dendritic cell differentiation and autoimmunity by targeting MAFB

Con-sequently, either miR-148a knockout or miR-148a inhibition by intradermal administration with antag-omir-148a prevented the development of moDCs and psoriasis-like inflammation in the IMQ-induced psoriasis-like mouse model. The mechanism research revealed that MAFB was a bona fide direct target of miR-148a , which was transcriptionally regulated by PU.1. Therefore, these results identify a pivotal role for the PU.1-miR-148a-MAFB pathway in the differentiation of moDCs and suggest a potential therapeutic avenue for inflammatory moDCs mediated autoimmune diseases. The separated proteins were transferred to an Immobilon-P PVDF membrane (Millipore), which was subsequently blocked with SuperBlock T20 PBS blocking buffer (Thermo Fisher Scientific). Final-ly, the membrane was incubated with rabbit antibodies against MAFB, 1:1000 (Abcam, ab66506), PU.1, 1:1000 (Cell Signaling Technology, 2258), and β -ACTIN, 1:2000 (Cell Signaling Technology, 4967). at specific time points, of IL-23, IL-17, IL-1 , IL-6, and TNF- α were determined with BD Cytometric Bead Array Soluble Protein Master Buffer Kit Flex Sets, to the instruction manual provided by manufacture. Briefly, the phycoerythrin-conjugated detection antibodies mixed with the specific capture beads and then incubated with recombinant protein standards or samples to generate sandwich complexes. Subsequently, the sandwich complexes of each sample ran on a flow cytometer. Standard curves for all analyses were generated through adopting recombinant protein standards to run one standard mixture. Finally, the sample results were analyzed by FCAP Array software (BD Biosciences).


Introduction
DCs comprise a heterogeneous family of professional antigen-presenting cells that maintain the homeostasis of the immune system (1)(2)(3)(4). DC populations in the skin have been well investigated in mice (5)(6)(7). In the steady state, two major subsets of conventional DCs (cDCs) have been characterized in the mouse nonlymphoid tissues, CD103 + CD11b -cDCs and CD103 -CD11b + cDCs, whereas the epidermis contains Langerhans cells. In the inflammatory state, peripheral monocytes are rapidly recruited to inflammatory sites and give rise to monocyte-derived DCs (moDCs) (5)(6)(7). Moreover, an increased frequency and activation status of plasmacytoid DCs (pDCs), a potent type I IFN-producing DC subset, have been documented in early psoriatic lesions (8,9). Both cDCs and pDCs originate from DC precursors in a Fms-related tyrosine kinase 3 ligand (Flt3L)-dependent manner, whereas the generation of moDCs is GM-CSF dependent (1,2).
Psoriasis is a common, chronic autoinflammatory skin disease characterized by demarcated, red, and scaly plaques. Environmental and genetic factors trigger the hyperproliferation and disturbed differentiation of keratinocytes, thickening of the epidermis, formation of new blood vessels, and accumulation of leukocytes in epidermis and dermis, of which T cells and DCs are the most critical (21,22). Psoriatic skin contains large numbers of activated DCs, which produce IL-23, IL-12, IL-6, and TNF-α.
Monocyte-derived DCs (moDCs) have been implicated in the pathogenesis of autoimmunity, but the molecular pathways determining the differentiation potential of these cells remain unclear. Here, we report that microRNA-148a (miR-148a) serves as a critical regulator for moDC differentiation. First, miR-148a deficiency impaired the moDC development in vitro and in vivo. A mechanism study showed that MAFB, a transcription factor that hampers moDC differentiation, was a direct target of miR-148a. In addition, a promoter study identified that miR-148a could be transcriptionally induced by PU.1, which is crucial for moDC generation. miR-148a ablation eliminated the inhibition of PU.1 on MAFB. Furthermore, we found that miR-148a increased in monocytes from patients with psoriasis, and miR-148a deficiency or intradermal injection of antagomir-148a immensely alleviated the development of psoriasis-like symptoms in a psoriasis-like mouse model. Therefore, these results identify a pivotal role for the PU.1-miR-148a-MAFB circuit in moDC differentiation and suggest a potential therapeutic avenue for autoimmunity.
They activate and polarize autoaggressive Th cells toward Th17, Th1, and Th22 cell subsets defined by the production of IL-17A, IL-17F, IFN-γ, and IL-22, respectively (9,23,24). Recently, the involvement of different skin DC subsets in regulating psoriatic plaque formation has been studied extensively. pDCs and cDCs are dispensable for the local skin inflammation in psoriasis, since E2-2 -/mice and Flt3L -/mice presented no significant difference in ear swelling and epidermal thickening compared with WT mice (25,26). The Ccr2 -/mice, which have profound monocytopenia due to a defect in monocyte egress from BM, showed a significant reduction in skin pathological phenotype (26). Depletion of monocytes in vivo through the use of antibody also greatly relieved the symptoms (26). The function of Langerhans cells is controversial because Lan-DTR mice developed a degree and course of psoriasiform skin disease similar to those of WT mice in an imiquimod-induced (IMQ-induced) model (25) but showed a certain degree of improvement in an IL-23-induced model (26). The finding that locally increased expression of GM-CSF and IL-23 was exclusively produced by Langerin -DCs in vivo, which further supports moDCs as being the most important DC subset in psoriasis (25).
miRNAs are short (~22 nt), evolutionarily conserved, single-stranded RNAs that control the expression of complementary target mRNAs, leading to their transcript destabilization, translational inhibition, or both (27). miRNAs are critical regulators of immune cell development and function (28). In this study, we used the psoriasis disease model to identify miR-148a as a paramount regulator for autoimmune-related moDC differentiation. miR-148a deficiency led to decreased pathogenic moDCs. Consequently, either miR-148a knockout or miR-148a inhibition by intradermal administration with antagomir-148a prevented the development of moDCs and psoriasis-like inflammation in the IMQ-induced psoriasis-like mouse model. The mechanism research revealed that MAFB was a bona fide direct target of miR-148a, which was transcriptionally regulated by PU.1. Therefore, these results identify a pivotal role for the PU.1-miR-148a-MAFB pathway in the differentiation of moDCs and suggest a potential therapeutic avenue for inflammatory moDCs mediated autoimmune diseases.

Results
miR-148a is indispensable for moDC differentiation. Proinflammatory moDC infiltration is critical for the progression of several autoimmune diseases, including systemic lupus erythematosus (SLE), psoriasis, inflammatory bowel disease, and so on (25,29,30). A comprehensive analysis of our previous data in patients with SLE (31) or psoriasis revealed an increase in the expression of miR-148a-3p (referred to as miR-148a here) (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.133721DS1). Other groups also reported similar elevation in autoimmune diseases (32). Therefore, we hypothesized that miR-148a might affect the development or function of pathogenic moDCs in autoimmune diseases.
Before evaluating the function of miR-148a in psoriatic inflammation, we first checked the effect on hematopoietic differentiation in the steady state (Supplemental Figure 2, A-D). Accordingly, different cell subsets in the spleen and skin from miR-148a -/and WT littermate mice were detected (Supplemental Figure 2, A and C). Consequently, there was no difference in the number of lymphoid or myeloid cells (Supplemental Figure 2, B and D), indicating that miR-148a was dispensable for hematopoietic differentiation in the steady state.
In the inflammatory state, Ly6C hi monocytes migrate to the lesion sites and then differentiate into moDCs. To validate whether the DC differentiation from monocytes was affected by miR-148a, Ly6C hi monocytes were isolated from BM of miR-148a -/and WT mice and then cultured with GM-CSF and IL-4. It has been demonstrated that Ly6C hi monocytes can give rise to both CD11c + MHCII hi CD11b int DC and CD11c + MHCII int CD11b hi macrophages (10). The conclusion was confirmed in the culture system that sorted CD11c + MHCII hi CD11b int cells exhibited DC morphology and CD11c + MHCII int CD11b hi cells had a typical macrophage morphology (Figure 1, A and B), allowing us to evaluate the differentiation potential of monocytes toward macrophages and DCs in the same culture system.
There was no difference of the expanding potential of monocytes between control and miR-148a deficiency, since the number of live cells was similar ( Figure 1C). The monocytes of the miR-148a -/group had considerably less moDCs (about 70% reduction) than those of controls ( Figure 1, A and C). In contrast, both the percentage and the number of macrophages increased in the miR-148a -/group ( Figure  1, A and C), indicating a critical role for miR-148a in promoting the differentiation of monocytes toward inflammatory DCs at the expense of macrophage.
Previous studies reported that miR-148a was involved in antiapoptosis and frequently upregulated in primary and tumor cells (33,34). To confirm whether the decrease in the proportion and number of miR-148a -/-moDCs was a result of the increased apoptosis, the entire cells were stained with annexin V and propidium iodide. Consequently, the examination of cell apoptosis showed no difference between miR-148a -/and WT throughout the inducing process, suggesting that miR-148a did not affect the moDC survival ( Figure 1D).
MAFB is a direct target of miR-148a. To gain insight into the molecular mechanism and thus facilitate the differentiation of moDCs for miR-148a, we performed RNA-sequencing analysis of monocytes isolated from WT mice or miR-148a -/mice in the presence of GM-CSF and IL-4, or not. Gene Ontology (GO) analysis showed that autoimmune disease-related pathways were enriched with obviously changed genes (SLE, RA, etc.), suggesting that miR-148a was important in autoimmune response ( Figure 2A). Since miRNA promotes the degradation of its target gene mRNAs, the mRNA abundance of miR-148a target gene should be higher in miR-148a -/monocytes than in WT monocytes. The threshold was set at ±0.2 CT, since the degrading potential of target mRNA by miRNA is much weaker than siRNA. Then, we identified 990 protein-coding genes upregulated in their mRNA abundance in miR-148a -/monocytes on day 0 and 1036 genes in miR-148a -/monocytes on day 3 ( Figure 2B). Among them, we found that 136 genes were upregulated on both day 0 and day 3 in miR-148a -/monocytes. Finally, bioinformatic analysis helped confirm that 7 of 136 genes contained predicted conserved miR-148a-binding sites ( Figure  2B). Targets previously implicated in hematopoietic differentiation were of special interest because the results suggested a role for miR-148a in regulating moDC generation. Of these 7 genes, MAFB has been reported to be increased during the differentiation from monocytes to macrophages and decreased during the differentiation from monocytes to moDCs (18,20). Overexpression of MAFB in myeloid progenitor cells is proposed to induce macrophage differentiation at the expense of moDC differentiation (19). These resulted in the selection of MAFB for further study ( Figure 2C).
The RNA-sequencing results were validated by qRT-PCR (Supplemental Figure 3A), and the protein expression encoded by MAFB was examined by immunoblot ( Figure 2D; see complete unedited blots in the supplemental material). Both the mRNA and protein levels of MAFB increased in miR-148a -/monocytes. We transfected human primary monocytes with antagomir-148a as well. The results showed that knockdown of miR-148a led to the increase of MAFB expression (Supplemental Figure 3D; see complete unedited blots in the supplemental material).
To test the direct binding of miR-148a with MAFB mRNA, we performed the RNA-binding protein immunoprecipitation (RIP) assay using an antibody against Argonaute-2 (Ago2), a component of the RNA-induced silencing complex that mediates miRNA-directed gene silencing. The results showed that overexpression of miR-148a significantly enriched the association of MAFB mRNA with the Ago2-containing complex, suggesting that the MAFB mRNA was directly bound by miR-148a ( Figure 2E). Furthermore, to confirm the effect of miR-148a on the MAFB 3′UTR, dual-luciferase reporter assays were conducted. Gene fragments encoding 3′UTR region of MAFB were cloned into the expression vector psiCHECK2, which encodes the renilla luciferase controlled by the cloned 3′UTR. These constructs were cotransfected in HEK 293T cells with miR-148a mimic. Data showed that miR-148a overexpression significantly reduced reporter protein expression through the 3′UTR region of MAFB ( Figure 2F). When the predicted binding site for miR-148a was mutated, the reduction was abolished ( Figure 2F). Therefore, miR-148a can suppress the expression of MAFB through its cognate binding site in 3′UTR.
Since miR-148a directly regulates MAFB expression, it is crucial to confirm that the higher protein level of MAFB mediates the miR-148a -/phenotype. To this end, miR-148a -/and WT monocytes were transfected with specific siRNA for MAFB and then cultured with GM-CSF and IL-4. Knockdown of MAFB promoted the moDC differentiation potential of miR-148a -/monocytes such that it was comparable to that of the WT monocytes ( Figure 2G and Supplemental Figure 3, B and C; see complete unedited blots in the supplemental material). These data have therefore demonstrated that MAFB mediated the phenotype of miR-148a deficiency and confirmed that MAFB was a bona fide direct target of miR-148a. miR-148a is involved in the inhibition of MAFB expression by PU.1. Considering that miR-148a can regulate moDC differentiation, we then explored how this miRNA was transcriptionally regulated. Previous studies have identified the transcriptional start sites (TSSs) of miR-148a (35). Intriguingly, a conserved PU.1 binding sites was identified in the promoter ( Figure 3A). To validate whether PU.1 can modulate miR-148a expression, we transfected primary monocytes with PU.1 siRNA. Knockdown of PU.1 led to the reduction of miR-148a expression ( Figure 3B and Supplemental Figure 4, A and B; see complete unedited blots in the supplemental material). To further explore the physiological relevance of PU.1-dependent regulation of miR-148a and subsequent MAFB expression, we measured the dynamic changes of PU.1, miR-148a, and MAFB during moDC differentiation. As expected, the miR-148a level paralleled the increase in PU.1 mRNA and protein levels, while the miR-148a level showed an opposite trend compared with the MAFB mRNA and protein levels (  (E) RNA immunopurification assay. Human primary monocytes were transfected with control (NC) or Agomir-148a. QRT-PCR analysis of enriched MAFB mRNA levels after Ago2 immunoprecipitation. IgG was used as negative control (n = 3). (F) Dual-luciferase reporter assays showing direct interaction of miR-148a with its cognate binding sites in the 3′UTR of MAFB. MAFB 3′UTR mutant refers to construct with mutated cognate binding sites for miR-148a. The renilla luciferase (Renilla) signal regulated by the target genes 3′UTR was normalized by the firefly luciferase (Firefly). The Renilla/Firefly ratio was set as 1 for the control psiCHECK2 plasmid (n = 3). (G) Knockdown of MAFB reduced the difference in the differentiation of moDCs between WT and miR-148a -/monocytes. Monocytes derived from WT and miR-148a -/mice were transfected with control (NC) or MAFB siRNA and then cultured with GM-CSF and IL-4 for indicated days. The differentiation of moDCs was analyzed (n = 3). Statistical analysis was performed with 2-tailed unpaired t test or 1-way ANOVA with Bonferroni's post hoc test. Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). miR-148a, microRNA-148a. miR-148a was indispensable for the inhibition of MAFB expression by PU.1.
PU.1 directly activates miR-148a promoter. To investigate the role of PU.1 in directly regulating miR-148a expression, we generated luciferase reporter constructs containing a 200-bp region of the human miR-148a promoter. As shown in Figure 3F, overexpression of PU.1 enhanced miR-148a promoter activity compared with that for cells transfected with an empty vector. Additionally, deletion of the PU.1 binding site significantly attenuated the promoter activity.
To determine whether PU.1 binds to this site in monocytes, we performed the ChIP assays. PU.1chromatin complexes were immunoprecipitated from monocytes with a PU.1 monoclonal antibody, and qPCR with site-specific primers was designed to analyze for enrichment of the identified site. Clear PU.1 occupancy was observed for the putative binding site within the promoter region of miR-148a ( Figure 3G).
In summary, these researches demonstrated that PU.1 inhibited MAFB expression through transcriptional activation of miR-148a directly.
miR-148a deficiency alleviates the development of psoriasis. To investigate the functional relevance of miR-148a in the development of moDCs, we subjected miR-148a -/mice to the IMQ-induced psoriasis-like mouse model. Repetitive application of IMQ onto WT mouse skin led to psoriasiform inflammation with significant thickening, redness, and scaling caused by keratinocyte hyperproliferation and leukocyte infiltration into the skin (Figure 4, A-F). Compared with WT mice, miR-148a -/mice . P values compare the indicated groups using a 2-tailed unpaired t test or 1-way ANOVA with Bonferroni's post hoc test. Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). miR-148a, microRNA-148a; moDC, monocyte-derived DC; quantitative real-time PCR, qRT-PCR.
developed much less skin inflammation; exhibited less thickening, redness, and scaling ( Figure 4A); displayed a greater reduction in ear thickness ( Figure 4B); and had a lower Psoriasis Area and Severity Index (PASI) score ( Figure 4C). H&E-stained back skin sections of miR-148a -/mice displayed a reduction in dermal thickness and acanthosis (Figure 4, D-F) during the course of disease. On day 6, the peak of psoriasis-like changes, we observed that the splenomegaly of mice in the miR-148a -/group was much less obvious than that in the control group (Supplemental Figure 5, A and B). In accordance with this, the reduced mRNA of IL23, TNFα, IL6, IL1β, and IL17a was observed in the skin lesions of the miR-148a -/group ( Figure 4G). Meanwhile, the protein levels of IL-23, IL-1β, and IL-17a were also found to be remarkably decreased in the serum of miR-148a -/mice ( Figure 4G).
To further determine the inflammatory cell infiltration in dermis from the miR-148a -/group, we obtained dermal single-cell suspensions and analyzed the percentage of different immune cell subsets. The percentage of CD45 + immune cells, including moDCs (CD45 + MHCII hi Ly6C -CD64 -CD-11c + CD11b + ) and IL-17 + γδT cells (CD45 + CD3 + γδTCR + IL-17A + ), was greatly reduced in dermis (Supplemental Figure 5C, and Figure 4, H and I); in contrast, macrophages (CD45 + MHCII + Ly6C -CD64 + ) increased (Figure 4, H and I). Similarly, the number of splenocytes, B cells, Ly6C hi monocytes, moDCs, CD4 + T cells, CD8 + T cells, pDCs, and CD8α -cDCs in spleen decreased in the miR-148a -/group with IMQ application (Supplemental Figure 5, B and D). Meanwhile, both CD8α + cDCs and neutrophils in spleen were not affected (Supplemental Figure 5D). To determine whether moDCs reduced in the skin lesions of miR-148a -/mice, we also performed the IHC assay on skin sections using an antibody specific for mouse CD11c. The results showed that moDCs decreased in the dermis of miR-148a -/mice with respect to those of WT mice (Supplemental Figure 5E). Furthermore, we also detected the expression of MAFB and found that MAFB decreased in this model (Supplemental Figure 5F).
In conclusion, these results suggested a reduction of systemic inflammation in the miR-148a -/group during the psoriasis.
miR-148a in moDC contributes to inflammation. To confirm whether miR-148a exerted an effect on psoriasis through dependence on immune cells, rather than other skin tissue cells, WT or miR-148a -/-BM cells were transplanted into sublethally irradiated CD45.1 + recipients, to generate BM chimeric mice. Six weeks after transplantation, these mice were subjected to the IMQ-induced psoriasis-like mouse model (Supplemental Figure 6A). Compared with WT mice, miR-148a -/chimeric mice developed much less skin inflammation; exhibited less thickening, redness, and scaling (Supplemental Figure 6B) and had lower PASI score and displayed a greater reduction in ear thickness (Supplemental Figure 6C). H&Estained back skin sections of miR-148a -/chimeric mice displayed a reduction in acanthosis and dermal thickness (Supplemental Figure 6, D and E) compared with controls. Meanwhile, we observed that splenomegaly was much less obvious in the miR-148a -/chimeric group than the control group (Supplemental Figure 6, F-H). FACS analysis of dermis showed that miR-148a -/chimeric mice had much less infiltration of CD45 + immune cells, including moDCs and IL-17 + γδT cells (Supplemental Figure 6I). Similarly, the reduced mRNA levels of IL23a, TNFα, and IL17a were also observed in the skin lesions of the miR-148a -/chimeric group (Supplemental Figure 6J).
To further assess that the role of miR-148a was moDC intrinsic, we adopted antibody-mediated depletion of monocytes in both miR-148a -/and WT mice. The available antibodies allowed us to deplete Ly6C hi monocytes plus neutrophils or neutrophils alone ( Figure 5A and Supplemental Figure 7A). Depleting neutrophils alone had little effect on the inflammatory response and psoriasis progression (Figure 5, B-G), which was consistent with a previous report (26). The additional depletion of Ly6C hi monocytes led to a partial loss of infiltrating monocytes and moDCs ( Figure 5G and Supplemental Figure 7A), accompanied by a significant decrease in ear swelling, PASI score ( Figure 5C), dermal thickness, and acanthosis ( Figure 5, D and E) as well as in mRNA levels of IL23a, TNFα, IL6, and IL17a ( Figure 5F). More crucial, there was no difference in the severity of psoriasis-like skin inflammation between IMQ-painted miR-148a -/and WT mice after Ly6C hi monocyte depletion, since they showed a similar reduction in ear and epidermal thickness ( Figure 5, B-E) and in moDC and IL-17 + γδT cell infiltration ( Figure 5G). The difference in the mRNA level of the inflammatory cytokines also greatly diminished ( Figure 5F). In accordance with these findings, the difference in the splenomegaly and moDC number between WT and miR-148a -/mice disappeared in the monocyte depletion group (Supplemental Figure 7, B and C). Consequently, the fact that the depletion of monocytes eliminated the acceleratory effects of miR-148a on the development of psoriasis suggested that the role of miR-148a was more likely moDC intrinsic.
asis-like mice. The above-mentioned results indicated that elevated miR-148a expression could promote psoriasis pathogenesis. Therefore, we next sought to evaluate the efficacy and potential application of a miR-148a-based therapy for psoriasis. Antagomir-148a or antagomir-NC was intradermally injected 4 times from the beginning to the third day to test the inhibitory effect on disease development in this model ( Figure 6A). miR-148a expression in skin lesions was dramatically reduced (Supplemental Figure  8A). The results displayed a significant improvement in both clinical and pathological characteristics on day 5 after antagomir-148a treatment (Figure 6, B-E), along with alleviated disease severity ( Figure 6C) and splenomegaly (Supplemental Figure 8, B and C). In addition, IMQ-treated mice injected with antago- . P values were determined by a 2-tailed unpaired t test or by 1-way ANOVA with Bonferroni's post hoc test. Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). moDC, monocyte-derived DC; miR-148a, microRNA-148a; PASI, Psoriasis Area and Severity Index.
mir-148a manifested lower levels of IL23a, TNFα, IL6, IL1β, and IL17a mRNA in skin lesions ( Figure 6F). We also assessed the effect of antagomir-148a in vivo on moDCs and IL-17 + γδT cells in psoriasis-like mouse models. Silencing of miR-148a significantly reduced the percentage of moDCs and IL-17 + γδT cells in skin lesions ( Figure 6, G and H). In accordance with the decrease of MAFB in the IMQ-induced model (Supplemental Figure 5F), mice with antagomir-148a administration showed higher levels of MAFB than mice with antagomir-NC (Supplemental Figure 8D). Because it has been demonstrated that miR-148a could regulate moDC differentiation by directly targeting MAFB in vitro, it is crucial to confirm the mechanism in vivo. Accordingly, miR-148a -/and WT mice were administered specific siRNA for MAFB or NC and painted with IMQ for 6 days. Then, the knockdown efficiency was verified (Supplemental Figure 9A). Compared with mice with NC siRNA, those with MAFB siRNA showed a significant aggravation in both clinical and pathological characteristics (Supplemental Figure 9, B-E), along with exacerbated disease severity (Supplemental Figure 9C) and splenomegaly (Supplemental Figure 9F). Furthermore, MAFB knockdown weakened the effect of miR-148a deficiency, because the difference between WT and miR-148a -/mice narrowed in the group treated with MAFB siRNA, compared with groups treated with NC (Supplemental Figure 9, B-F). Thus, MAFB partially mediated the function of miR-148a in vivo.
Finally, to examine the relevance of the expression level of miR-148a to psoriasis disease, we detected the expression level of miR-148a in peripheral monocytes from patients with psoriasis. Compared with those from healthy controls, there was an increase in mRNA levels of IL23a, IL1β, and IL17a in PBMCs from psoriatic patients ( Figure 7A). Monocytes from patients with psoriasis showed an increased expression level of miR-148a ( Figure 7B). The expression level of miR-148a positively correlated with the mRNA levels of IL23a and IL17a ( Figure 7C), supporting that miR-148a was crucial in psoriasis. To confirm the PU.1-miR-148a-MAFB circuit in psoriasis in vivo, we detected the expression of PU.1 and MAFB in peripheral monocytes from patients with psoriasis. The results demonstrated that monocytes from patients with psoriasis showed a decreased expression level of MAFB but an increased expression level of PU.1 ( Figure  7, D and E). Moreover, the expression level of miR-148a positively correlated with the mRNA level of PU.1 ( Figure 7E) and negatively correlated with MAFB level ( Figure 7D).
Consequently, these results indicated that the inhibition of miR-148a expression in vivo could prevent the immunopathological changes in psoriasis and effectively ameliorate disease severity, suggesting a novel treatment strategy for psoriasis and other autoimmune diseases.

Discussion
The transcriptional regulation of moDC differentiation has been extensively studied (2,13). The balance of MAFB and PU.1 was considered to specify alternative macrophage or DC fate (18). The differentiation of monocytes to DCs results in a rapid increase of PU.1, achieving high levels that precede the phenotypic changes, while MAFB expression is decreased during the differentiation. Inhibited MAFB expression in monocytes appears to be required for DC specification, since constitutive MAFB expression inhibits DC differentiation (18). In addition, MAFB deficiency is found to specifically enhance sensitivity to M-CSF and cause activation of the myeloid master regulator PU.1 in hematopoietic stem cells in vivo (19). One possible mechanism for the PU.1 regulation of MAFB function is that PU.1 can directly bind to MAFB and inhibit MAFB transactivation capacity. In this research, we found that miR-148a mediated the inhibition of MAFB expression through PU.1 regulation, suggesting a new mechanism for regulating the balance of MAFB and PU.1 and, thereby, the differentiation of moDCs. Interestingly, a recent study reported that PU.1 could directly bind to the superenhancer of miR-148a and facilitate its maturation in mouse primary B cells (36). Whether this regulation exists in primary monocytes and moDCs remains to be made clear.
miRNA is generally regarded as a multifunctional molecule and can exert diverse roles in different immune cells by targeting different molecules intracellularly (28). Increased expression of miR-148a, which occurs frequently in patients with lupus and lupus-prone mice, impaired B cell tolerancethrough promoting the survival of immature B cells upon B cell receptor engagement via suppressing the expression of GADD45a, PTEN, and BCL2l11 -and facilitated the development of lethal autoimmune disease (33). miR-148a could also considerably reduce the DNMT1 protein level, which was one of the major epigenetic components that linked to DNA hypomethylation in T cells in SLE (31). There was no significant difference in the number of B cell and T cell subsets between miR-148a -/mice and WT mice in the steady state. Hence, miR-148a might affect the survival and function of both autoreactive B and T cells in the inflammatory state. To confirm the cell-intrinsic role of miR-148a in moDCs, we designed two experiments. First, we constructed BM chimeric mice and found that miR-148a -/chimeric mice demonstrated less psoriatic skin inflammation, indicating that miR-148a takes effect only on immune cells, rather than other skin tissue cells during the process. Second, monocytes were depleted in vivo through the use of antibodies before and during IMQ painting. Monocyte depletion greatly alleviated the severity Data are shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant). IMQ, imiquimod; PASI, Psoriasis Area and Severity Index.
of the psoriasiform skin inflammation. In addition, there was no significant difference in the symptoms between miR-148a -/mice and WT mice, indicating that miR-148a in moDCs is more likely to be critical for the IMQ-induced psoriasis-like inflammation.
It has also been reported that miR-148a regulates the innate immune response. It has been demonstrated that miR-148a negatively regulates the TLR4-triggered functional maturation of moDCs by targeting CaMKIIa (34), and inhibits macrophage function by directly targeting several upstream regulators of NF-κB and STAT3 signaling, including GP130, IKKα, IKKβ, IL1R1, and TNFR2 (37). We also found that miR-148a -/-moDCs could produce increased levels of TNF-α and IL-6 in response to LPS stimulation (data not shown). However, a less than 10% increase was rather limited compared to the decreased potential of moDC differentiation both in vitro (about 70% reduction) and in vivo (a more than 1-fold reduction), as shown in Figure 1C, Figure 4I, and Figure 6H. Therefore, miR-148a in monocytes exerted deleterious effects during psoriasis.
In addition to miR-148a, several other miRNAs have been reported to regulate the differentiation and . P values compare the indicated groups using nonparametric Mann-Whitney U test or Spearman's test. Data are shown as mean ± SEM (*P < 0.05, ***P < 0.001, n.s., not significant). miR-148a, microRNA-148a.
As molecule fine-tuning gene expression, these miRNA targets generate a complex regulatory network that comprise different levels, including miR-21/WNT1 and miR-34a/JAG1 at the ligand/receptor level, miR-155/SOCS1 at the signal transduction level, and miR-148a/MAFB at the transcriptional factor level. Interestingly, miR-21 was found to be abnormally elevated in psoriasis, and the inhibition has been shown to have a beneficial effect on the treatment of psoriasis (42). In addition, miR-155 is also upregulated in psoriatic biopsy samples and responsible for maintaining the inflammation in psoriasis (43).
In conclusion, the present study provides evidence that miR-148a, which could be transcriptionally activated by PU.1, promotes moDC differentiation through directly targeting MAFB, thereby causing the immune imbalance and inflammatory response in skin lesions of psoriasis ( Figure 8). Since inflammatory DCs are pathogenic in various autoimmune diseases (SLE, RA, etc.), this research suggests that miR-148a could serve as a potential target for the treatment of not only psoriasis but also other autoimmune diseases.

Methods
Human subjects. A total of 34 psoriatic patients, who were diagnosed with psoriasis vulgaris, were recruited from outpatient clinics at Huashan Hospital of Fudan University. Psoriasis disease activity was assessed using PASI score. The patient information is shown in Supplemental Table 1. In addition, a total of 51 sexand age-matched healthy controls were recruited from Renji Hospital of SJTUSM.
Mice. The C57BL/6J mice were acquired from Slack Company. miR-148a-knockout mice B6/JNju-Mir148a em2Cd262in1 /Nju on a [C57BL/6] background were provided by Model Animal Research Center, Nanjing University. In addition, male mice at 6-8 weeks of age were used to carry out all the following experiments. All mice were bred and housed under the conditions of specific pathogen-free circumstance.
Scoring severity of skin inflammation in psoriasis-like mouse model. An objective scoring system was developed based on the clinical PASI for assessing the patients with psoriasis. Accordingly, erythema, thickening, and scaling were each scored according to the scope (0-4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked). The overall score was obtained by calculating a sum of the 3 index scores (score 0-12).
Cell isolation, flow cytometry analysis, and cell culture. Skin cells were prepared on the basis of previous research with minor modifications (45). In brief, the epidermis and dermis were separated by adopting the dispase (25 U/ml in Dulbecco's DPBS) and then the dermal cell suspension was acquired using collagenase and hyaluronidase. Splenocytes were prepared by digesting the tissue fragments with collagenase type IV and DNAse I. Antibodies used for cell staining were presented in Supplemental Table 2. Cells were analyzed using Fortessa (BD Biosciences) and FlowJo software.
PBMCs were separated from the peripheral blood of healthy controls and patients with psoriasis by density gradient centrifugation (GE Healthcare). Human and mouse monocytes were isolated with the Human Monocyte Isolation Kit II and Mouse Monocyte Isolation Kit (BM) (Miltenyi Biotec), respectively. Then, the cells were cultured in RPMI 1640 medium (Gibco) with 10% FBS (Gibco) at 37°C and 5% CO 2 , or collected for subsequent experiments. HEK 293T cells and HeLa cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco). To obtain moDCs, BM cells or Ly6C hi monocytes separated from BM were cultured with 100 ng/mL GM-CSF and 100 ng/mL IL-4 (Peprotech) for indicated days. The Annexin V Apoptosis Detection Kit (BioLegend) was used for apoptosis assays according to the manufacturer's protocol.
Histology. As for H&E staining, the histological analysis was conducted as Lai et al. reported (46). First, mouse skin tissues were fixed in formalin and embedded in paraffin and then stained with H&E. Finally, to assess dermal thickness, we measured the thickness from the stratum papillare to the stratum reticulare and the epidermal hyperplasia from the basal layer to the stratum corneum,using Case Viewer software (3DHISTECH Ltd). The measurement of skin thickness was conducted on 6 randomly selected areas per ×10 field from 3-5 fields per section. The obtained skin thickness was first averaged per mouse and then used to calculate the average per treatment group for the following statistical analysis. As for immunochemistry, the mouse skin paraffin sections were stained with hematoxylin and mouse monoclonal anti-CD11c antibody (GB11059, Servicebio).
Depletion of neutrophils and monocytes. The depletion in vivo was conducted based on the report by Singh et al. (26). In brief, as for the depletion of neutrophils alone, the mice were intraperitoneally administered 500 mg antibody anti-Ly6G (clone 1A8, BioXCell) on days 0, 1, 2, 3, and 4. Meanwhile, to deplete monocytes and neutrophils, the mice were injected intraperitoneally with 500 mg of antibody anti-Gr1 (clone RB6-8C5, BioXCell) at the same time points as above.
mRNA-Seq and miRNA-Seq. All the RNA was first extracted by using the miRNeasy Micro Kit (QIA-GEN) and determined with Bioanalyzer 4200 (Agilent). Then, the preparation for the next-generation mRNA libraries was performed with VAHTS mRNA-Seq v2 Library Prep Kit for Illumina (Vazyme), whereas the preparation for the miRNA libraries was performed with NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB) and then purified by QIAquick PCR Purification Kit (QIAGEN). Afterward, the acquisitions from above were subsequently put into the gel prep for size selection. In addition, the quality of the library was determined using Bioanalyzer 4200 (Agilent). Then, the mRNA-Seq libraries were run in HiSeq X10 system (Illumina) on a paired-end 150-bp run, and the miRNA-Seq libraries were sequenced on an Illumina HiSeq 2500 next-generation sequencer using single-read 50-bp sequencing. Among the compared groups, those with more than 1.2-fold differences of the geometrical mean expression and a statistically significant P value of less than 0.05 based on analysis of DESeq2 were selected as the differentially expressed genes. To determine statistically enriched GO categories, we conducted the