European Review for Medical and Pharmacological Sciences

2019; 23: 1641-1651

Interaction between bone marrow-derived
dendritic cells and miR-21 of tubular renal
epithelial cells under hypoxia
Y.-R. LIANG1, T. ZHANG1, P. JIA1, X.-L. XU1, Y. FANG1,2,3,4, X.-Q. DING1,2,3,4
Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China
Shanghai Medical Center of Kidney, Shanghai, China
3
Shanghai Key Laboratory of Kidney and Blood Purification, Shanghai, China
4
Shanghai Institute of Kidney and Dialysis, Shanghai, China
1
2

Abstract. – OBJECTIVE: To investigate the intracellular response and role of microRNA 21 in
the regulation of dendritic cell maturation andfunction.
MATERIALS AND METHODS: Bone marrow-derived DCs (BMDCs) isolated from male
C57BL/6J mice and primary renal tubular epithelial cells were used as primary cells to perform
this study. Flow cytometry was used to determine BMDCs and analyze the apoptosis effect.
Transmission electron microscopy was used forthe identification of the diameter of exosomes.
Reverse transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting were used
to detect the effect after cells were transfectedwith oligo. ELISA was used to determine the tumor necrosis factor-α (TNF-α), interleukin-12 (IL12), and IL-1beta in DC supernatants.
RESULTS: We found that the upregulation of microRNA 21 in dendritic cells induced by physical hypoxia contributed to decreased expressions of CD80
(cluster of differentiation 80), CD86 (cluster of differentiation 86), and MHCII (major histocompatibility complex class II molecules) of dendritic cells andsuppressed secretion of inflammatory cytokinesand chemokine receptor type 7. Co-culture with tubular epithelial cells or hypoxia-pretreated tubularepithelial cell-derived conditional medium promoted
bone marrow-derived dendritic cell maturation. Exosomes purified from the supernatant of cultured marrow-derived dendritic cells showed upregulated microRNA 21 under hypoxia, whereas anti-microRNA
21 treated tubular epithelial cells promoted co-cultured marrow-derived dendritic cell maturation.
CONCLUSIONS: Both oxygen concentrationand tubular epithelial cells participate in regulating dendritic cell maturation, directly or indirectly through the microRNA 21 signal pathway.
Key Words:
Dendritic cells, Hypoxia, Ischemic kidney injury, MiR21, Tubular epithelial cells.

Introduction
Acute kidney injury (AKI), a common clinical complication, has a reported prevalence rate
ranging from 3.49% to 23.2%1-3. Despite the rapid
development of blood purification techniques, themortality of critically ill AKI patients remains
unchanged, with one reason being the lack of effective interventions.
Ischemic injury is one of the major pathological mechanisms of AKI1. Ischemia directly leads to hypoxia and renal tubular epithelial cell
(TEC) injury, whereas the inflammatory immune response secondary to renal TEC injury leads
to a variety of events triggering ischemic AKI1.
Danger-associated molecular patterns (DAMPs)
released by injured TECs trigger the downstream
signaling pathways, resulting in the activation ofvarious immune cells such as macrophages, dendritic cells, and neutrophils4. An adaptive immune
response can be further triggered by an overwhelming immuno-inflammatory imbalance5.
Dendritic cells (DCs) are the strongest antigen-presenting cells and relate to the innate andadaptive immune responses6. They develop frommyeloid progenitors and undergo a maturation
process via migration to the secondary lymph nodes. This is stimulated by inflammatory signals,
such as DAMPs, with upregulation of costimulatory and migration molecules7 (e.g., when DCs in
the peripheral tissue come into contact with microbial components, such as lipopolysaccharides(LPS), they are activated and migrate to the draining lymph nodes)7,8. DCs can be licensed to T
helper (Th)1-type or Th2-type immune response
depending on the type and dose of the stimulation9-11. Kidney intrinsic DCs originate from theCorresponding Authors: Yi Fang, MD; e-mail: fang.yi@zs-hospital.sh.cn
Xiaoqiang Ding, MD; e-mail: ding.xiaoqiang@zs-hospital.sh.cn

1641

Y.-R. Liang, T. Zhang, P. Jia, X.-L. Xu, Y. Fang, X.-Q. Dingbone marrow and spleen. Under physiologicalconditions, most intrinsic DCs are located in therenal interstitial cells12.
DCs travel via lymphatic vessels to transportantigens and present them to T cells in the lymph nodes. They directionally move toward lymphatic vessels by C-C chemokine receptor type 7(CCR7)13. MicroRNAs (miRNAs), small noncoding RNAs that modulate gene expression, bindto the 3′-untranslated region of target messenger
RNAs14. Reportedly, a bundle of miRNAs regulates DC functions15-17. A previous study18 showedthat CCR7 was the target gene of miR-21. In our
precedent work, we showed that miR-21 wasupregulated under hypoxia in HK2 cells and supernatant of HK2 cells19. Thus, we suggested thatthe phenotype and function of kidney intrinsic
DCs can be regulated by miR-21 via a paracrine
pathway20. Therefore, in this study, we aimed toinvestigate the intracellular response and the roleof miR-21 in the regulation of DC-mediated inflammatory responses secondary to hypoxia.

Materials and Methods
Culture of Bone Marrow-Derived DCs(BMDCs)This study was approved by the Animal Ethics
Committee of Fudan University Animal Center.
BMDCs were isolated from male C57BL/6J mice,as previously reported21, and cultured in Roswell
Park Memorial Institute-1640 (RPMI-1640) medium (HyClone, South Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand
Island, NY, USA) and supplemented with 10 ng/
mL recombinant mouse granulocyte-macrophagecolony-stimulating factor and 10 ng/mL IL-4 (PeproTech Inc., Rocky Hill, NJ, USA). After 48 h,
non-adherent cells were removed from the dish,and the remaining cluster was cultured; the medium was changed every 48 h. On day 6, the cells
were collected for treatment with hypoxia-pretreated TEC-derived conditional medium (HCM)or normal medium. The phenotypes of BMDCs
were determined based on the protein levels of
CD11c, CD80 (cluster of differentiation 80), CD86
(cluster of differentiation 86), MHCII (major histocompatibility complex class II molecules), andCCR7 (eBioscience, Frankfurt, Germany).
Primary Murine Renal TEC Culture
Primary renal TECs were bought from AllCells (Allcells, LLC, Berkeley, CA, USA) and cultu1642

red in a specific medium (AllCells, Berkeley, CA,
USA). After reaching 80% confluency, the TECs
were solely cultured or co-cultured with BMDCs.
Hypoxia (1% O2, 5% CO2, and 94% N2) was set
up in an oxygen control incubator (Thermo Fisher
Scientific Inc, Waltham, MA, USA), and the indicated cells were cultured under hypoxia for 6-48 h.
BMDC Analysis by Flow Cytometry
In this work, we used LPS to stimulate mature DCs (mDCs), and no-LPS-stimulated DCs
were defined as immature DCs (imDCs). BMDCs were analyzed by flow cytometry for surface marker expression using antibodies against
CD80, CD86, MHCII, and CCR7 (eBioscience,Frankfurt, Germany).
Exosome Purification
BMDCs were cultured in RPMI-1640 medium without serum. Further, the medium wascollected to purify exosomes after 24 h exposure
to hypoxia. Supernatant fractions collected fromthe cell cultures were subjected to exosome extraction. Briefly, the supernatant was centrifugedat 300 rpm for 15 min to remove cells, followedby filtration through 0.22 μm filter to remove celldebris. Exosomes in the medium were precipitated with ExoQuick TC (System Biosciences, Palo
Alto, CA, USA), according to the manufacturer’s
instructions, and exosomes pellet were resuspended in 100 μL phosphate-buffered saline (PBS)
and stored at -80°C until further use.
Transmission Electron Microscopy
For transmission electron microscopy (TEM)morphology analysis, 5 μL exosome pellets wereplaced on Formvar/carbon-coated 200-mesh copper electron microscopy grids. The grids were incubated for 10 min at room temperature, followed
by standard uranyl acetate staining. Then, thegrids were washed with three changes of PBS andallowed to semi-dry at room temperature before
observation under TEM (Hitachi H7500, Tokyo,
Japan). The obtained micrographs were used toquantify the diameter of the exosomes.
Transfection of Anti-MiR Oligo
The synthesized anti-miR-21 and anti-scramble(Exiqon, Vedbaek, Denmark) were transfected
into TECs with Lipofectamine (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s
instructions. Briefly, TECs (106 per well) wereplated onto 24-well plates and allowed to grow for
12 h. Anti-miR-21 (50 nM) or anti-scramble with

TEC promotes kidney DC maturation via miR-21 regulation

1 μL Lipofectamine were added to the cells. Aftertransfection, the cells were incubated at 37°C
for 24-72 h; next, the cells were cultured underhypoxia or co-cultured with BMDCs. The cells
were harvested for analysis at the indicated time
points as described before; proteins and RNA
were assessed using Western blot assay and Real-Time reverse-transcriptase-Polymerase Chain
Reaction (RT-PCR) assay, respectively.
Western Blot Analysis
The cells or exosomes were lysed, and the total
cell protein was extracted. The protein concentration was estimated by a bicinchoninic acid assay
reagent (Beyotime, Shanghai, China). Then, theproteins were resolved on a 10% sodium dodecyl
sulfate bis-tris gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore,
Billerica, MA, USA). The membrane was blockedwith 5% non-fat milk in TBST buffer, followed
by incubation with rabbit anti-CD63 (1:1000) (Abcam, Cambridge, MA, USA), PDCD4 (1:1000)(Cell Signaling Technology [CST], Danvers, MA,
USA), CCR7 (1:1000) (CST, Danvers, MA, USA),
pp65 (1:1000, CST, Danvers, MA, USA), andp65 (1:1000, CST, Danvers, MA, USA) overnightat 4°C, washed with TBST, and incubated with
HRP-linked goat anti-rabbit IgG (1:10000) (CST,
Danvers, MA, USA). Further, the protein bands
were visualized using an automatic imager (General Electric, Fairfield, CT, USA).
RNA Extraction and MiRNA Real-Time
RT-PCR
The cells were washed with ice-cold phosphate buffer, and total RNA was extracted using the
TRIzol agent (Invitrogen, Carlsbad, CA, USA),according to the manufacturer’s instructions.
Complementary deoxyribose nucleic acid (cDNA)
was synthesized using one-step prime script miRNA cDNA Synthesis kit (PrimeScript RT Reagent
Kit) (TaKaRa, Otsu, Shiga, Japan). miRNA-specific primers were designed according to the manufacturer’s instructions and synthesized at Applied
Biosystems (Foster City, CA, USA). U6 small
nuclear RNA was used as endogenous control
for miRNA. The level of miR-21 expression wasquantified using Real-Time RT-PCR with TaqMan
chemistry (Applied Biosystems, Foster City, CA,
USA) and monitored by a 7500 RT-PCR system(Life Technologies, Grand Island, NY, USA). The
samples were analyzed in duplicate. The levelof miRNA expression was calculated according
to the 2−∆∆Ct method. The relative gene levelis expressed as ratio to control. Primer sequences used in this study were as follows: PCAT6,
F: 5’-CAGAATTGCCCAGGCTTTTA-3’, R:
5’-AATCCATGAGGCAAGGTGAC-3’; U6: F:
5’-GCTTCGGCAGCACATATACTAAAAT-3’,
R: 5’-CGCTTCAGAATTTGCGTGTCAT-3’.
Apoptosis Analysis by Flow Cytometry
The cells were harvested by ice-cold PBS fromthe culture dishes. The apoptosis was detectedusing Annexin V-FITC/PI staining kit (Vazyme,
Nanjing, China), according to the manufacturer’s instructions. Before starting flow cytometry,the cells were again rinsed twice with phosphate buffer. Flow cytometry was conducted using
FACSCalibur flow cytometer (Thermo Scientific,
Waltham, MA, USA).
Enzyme-Linked Immunosorbent Assay
The concentration of tumor necrosis factor-α(TNF-α), interleukin-12 (IL-12), and IL-1beta in
DC supernatants was measured using specifickits purchased from eBioscience, in accordancewith the manufacturer’s instructions.
Statistical Analysis
The Student’s t-test was used while comparing the difference between the two groups.
Data are presented as mean ± standard error of
the mean. Statistical analyses were conductedusing Statistical Product and Service Solutions(SPSS) Version 16.0 (Chicago, IL, USA). Oneway analysis of variance with Tukey’s multiple
comparison tests was used to analyze multiple
group comparisons. p-value <0.05 was considered statistically significant.

Results
In Vitro Culture of Immature DCs (imDCs)
and Mature DCs (mDCs)
CD11c+MHCII+ double-positive cells havebeen identified as BMDCs that differ from bonemarrow-derived macrophages22 (Figure 1A).
Based on whether the toll-like receptor 4 of the
BMDCs had been stimulated by LPS, our research aim included in vitro culture of imDCs
and mDCs23. Flow cytometry revealed elevatedexpressions of MHCII (mDCs vs. imDCs, 1493 ±44 vs. 1259 ± 59, p<0.05), CD80 (mDCs vs. imDCs, 624 ± 11 vs. 386 ± 31, p<0.01), and CD86
(mDCs vs. imDCs, 427 ± 11 vs. 311 ± 10, p<0.01)
in mDCs (Figure 1B).
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Y.-R. Liang, T. Zhang, P. Jia, X.-L. Xu, Y. Fang, X.-Q. Ding

Figure 1. Hypoxia had a suppressive effect on the maturation of BMDCs. A, Induction rate of BMDCs gated from
CD11c+MHCII+ double-positive cells. B, LPS stimulation elevated antigen-presenting marker expression of BMDC. C-E,
Hypoxia exposure suppressed maturation marker CD80, CD86, and CCR7 expressions of BMDCs at indicated time points,
tested by flow cytometry. *p< 0.05, #p< 0.01, vs. normoxia group. N = 4. F, Exposure to hypoxia for 24–48 h upregulated themiR-21 levels in BMDCs. G, 24-h LPS stimulation suppressed miR-21 levels in BMDCs. H and I, Time-dependent apoptosis
rate of BMDCs at 1% O2 concentration. J, Successful transfection of anti-miR-21 to TECs. K-M, Transfection of anti-miR-21
to BMDC elevated the levels of CD80, CD86, and CCR7 MFI of BMDCs under normoxia or hypoxia.

Hypoxia Inhibited BMDC Maturation
ImDCs were exposed to normoxia at 21% O2(N-imDCs) or hypoxia at 1% O2 (H-imDCs). In
H-imDCs, expressions of CD80 (H-imDCs 24
1644

h vs. N-imDCs 24 h, 1377 ± 60 vs. 2107 ± 128,p<0.05) (Figure 1C), CD86 (H-imDCs 24 h vs.
N-imDCs 24 h, 3182 ± 314 vs. 6033 ± 140, p<0.05)(Figure 1D), and CCR7 (H-imDCs 24 h vs. N-im-

TEC promotes kidney DC maturation via miR-21 regulation

DCs 24 h, 1022 ± 23 vs. 1489 ± 102, p<0.05) (Figure 1E) were significantly lower than those in
N-imDCs.
After exposure to hypoxia for 24-48 h, H-imDCs expressed a two-fold level of miR-21 expression compared with N-imDCs (Figure 1F). Further, mDCs expressed lower level of miR-21
expression than imDCs (Figure 1G). Furthermore, we measured the apoptosis rate of imDCs
using flow cytometry. Compared with N-imDCs,
H-imDCs showed a significantly increased apoptosis rate, which was aggravated by a longer
exposure to hypoxia (6-h H-imDCs vs. N-imDCs, 13.6% ± 0.9% vs. 7.2% ± 1.5%, p<0.05; 12-h
H-imDCs vs. N-imDCs, 17.2% ± 1.4% vs. 13.1%± 0.7%, p<0.05; 24-h H-imDCs vs. N-imDCs,
24.1% ± 1.4% vs. 15.5% ± 0.6%, p<0.05; and 48-h
H-imDCs vs. N-imDCs, 44.0% ± 2.9% vs. 19.9%± 0.7%, p<0.05) (Figure 1H, 1I).
MiR-21 Negatively Regulated
DC Maturation
The knockdown of miR-21 (Figure 1J) augmented the expression of maturation molecules suchas CD80 (hypoxia anti-miR-21 vs. anti-scramble, 683 ± 10 vs. 642 ± 8, p<0.05), CD86 (normoxia anti-miR-21 vs. anti-scramble, 8759 ± 143
vs. 6902 ± 279, p<0.01; hypoxia anti-miR-21 vs.
anti-scramble, 3746 ± 61 vs. 3445 ± 33, p<0.05),
and CCR7 (normoxia anti-miR-21 vs. anti-scramble, 1391 ± 21 vs. 1328 ± 8, p<0.05; hypoxia anti-miR-21 vs. anti-scramble, 719 ± 8 vs. 671 ± 11,
p<0.05) (Figure 1K-N). Compared with imDCs
under normoxia, the level of miR-21 expressed by
the imDCs under hypoxia increased by more than
two-fold (Figure 1F). These results suggest thatthe lower antigen presentation ability of H-imDCs
could be attributed to the negative regulatory roleof miR-21 in DC maturation.
Supernatant of TEC Cultured Under
Hypoxia Promoted DC Maturation
The link of kidney intrinsic and inflammatory
cells may function via miR-21 signaling. To test
this hypothesis, we first pretreated TECs under
1% O2 for 24 h. Then, we collected the supernatant(also regarded as a pretreated TEC-derived HCM)
to further incubate BMDCs under normoxia for
24 h. As opposed to the negative effects of direct
hypoxia on the maturation markers expression of
BMDCs (Figure 1C–1E), the BMDCs cultured
with HCM exhibited an upregulated expression of
CD80 and MHCII median fluorescence intensity
levels compared with the control group (Figure

2A, B). Compared with a control group (normal
TEC-derived conditional medium, NCM), HCM
upregulated the pro-inflammatory cytokine secretion of BMDCs, which was verified by IL-12
overexpression (Figure 2C) and IL-4 downregulation in the supernatant of BMDCs (Figure 2D).
Notably, we studied imDCs in this part, rather
than the mature DCs.
Transwell Co-Culture System Mimics
the Interaction Between DCs and TECs
Under Hypoxia
To better study the relationship between TECs
and DCs, we used a transwell co-culture systemwith BMDCs cultured in the upper room (approximate cell amount, 105/well), whereas TECs were
cultured in the bottom room (106/well). First, weobserved that in the co-cultured groups, co-culture with TEC resulted in a significant upregulationof CD80 and CD86 expressions under hypoxia(Figure 3A) or normoxia (Figure 3B). Second,
after 48-h co-culturing, BMDCs secreted more
pro-inflammatory cytokine IL-1β (BMDC + TEC
vs. BMDC, 248.0 ± 26.41 ng/mL vs. 87.82 ± 4.89
ng/mL, p<0.05) (Figure 3C) and TNF-α (BMDC+ TEC vs. BMDC, 2695 ± 48.22 ng/mL vs. 2403
±20.43 ng/mL, p<0.05) (Figure 3D), but it had no
significant effect on the secretion of IL-12 (Figure3E). Third, hypoxia had significantly suppressedmDC maturation, such as of CD80 at each indicated time point (48-h hypoxia vs. normoxia, 11060± 108.7 vs. 21080 ± 316.5, p<0.05; 24-h hypoxia
vs. normoxia, 6958 ± 101.3 vs. 10120 ± 82.30,
p<0.05; 12-h hypoxia vs. normoxia, 5840 ± 420.7
vs. 6334 ± 732.7, p<0.05; and 6-h hypoxia vs. normoxia, 4680 ± 17.87 vs. 5515 ± 106.6, p< 0.05)(Figure 3F) and migration molecule CCR7 (48-hhypoxia vs. normoxia, 1506 ± 12.15 vs. 3359 ±
96.70, p<0.05; 24-h hypoxia vs. normoxia, 1155 ±
14.77 vs. 1702 ± 10.35, p<0.05; and 6-h hypoxia vs.
normoxia, 960.5 ± 7.963 vs. 1211 ± 10.85, p<0.05)(Figure 3G). These results indicate that co-culture
with TECs promoted BMDCs antigen-presenting
function, migration, and pro-inflammatory activity under hypoxia.
Effect of Transwell Co-Culture on the
PDCD4-CCR7 Signaling Pathway
of BMDCs
Co-culture with TECs or LPS stimulation
showed a slight but insignificant elevation of
PDCD4 protein expression in BMDCs (Figure
4A, B). An efficient transfection of anti-miR-21 to
BMDCs upregulated CCR7 and PDCD4 expres1645

Y.-R. Liang, T. Zhang, P. Jia, X.-L. Xu, Y. Fang, X.-Q. Ding

Figure 2. Hypoxia-pretreated TEC-derived conditional medium promoted the maturation marker expression and secretionof inflammatory cytokines by BMDCs. A, and B, Hypoxia-pretreated TEC-derived conditional medium (HCM) upregulated
MHCII and CD80 expression of BMDCs (N = 4, *p<0.05, #p<0.01 vs. NCM-cultured BMDCs). C, HCM treatment promoted
IL-12 secretion of BMDCs. D, HCM treatment suppressed the secretion of IL-4 of BMDCs supernatant (*p<0.05, #p<0.01, vs.
NCM group. N=4).

sions (Figure 4C, D). Co-culture with anti-miR-21
transfected TECs had no significant effect on
PDCD4 expression of BMDCs (Figure 4E, F).

normoxia (Figure 5E). Overall, these data indicate that TECs can indirectly regulate the functionof DCs.

Exosomes May Act as the Potential
Determinant Between the Interactionof TECs and BMDCs
We investigated the BMDC-derived exosome
miR-21 under hypoxia. BMDC-derived exosomes (Figure 5A, B) showed upregulated miR-21
expression under 1% O2 concentration (Figure
5C). Conversely, inhibiting miR-21 levels in TECs
with anti-miR-21 promoted the median fluorescence intensity level of CD80 (anti-miR-21 vs.
anti-scramble: 683.7 ± 10.48 vs. 642.3 ± 7.881, p<0.05), CD86 (anti-miR-21 vs. anti-scramble: 3746
± 61.28 vs. 3445 ± 33.39, p< 0.05), and migration
molecule CCR7 (anti-miR-21 vs. anti-scramble:
719.0 ± 8.386 vs. 671.0 ± 11.27, p< 0.05) of theco-cultured mDCs under hypoxia (Figure 5D) or

Discussion

1646

Pathogenesis of ischemia-reperfusion (IR) andthe related molecular mechanisms that regulate
inflammatory injury in local ischemic tissue haverecently gained much attention24. Tissue hypoxia
is regarded as an important factor in the pathophysiology of AKI and chronic kidney disease25,26. In
recent years, inflammatory reactions have beenshown to play key roles in the pathophysiology
of organ damage following IR 27. DAMPs are released by intrinsic kidney cells, such as TECs, endothelial and parenchymal cells, which are subsequently presented to antigen-presenting cells; thisfurther triggers downstream adaptive immune
cell activity and pro-inflammatory cytokine se-TEC promotes kidney DC maturation via miR-21 regulation

cretion, which, in response, causes an exaggerated renal injury28. DCs are the strongest specific
APC and have anti- and pro-inflammatory effects
depending on their maturation state29. In thisstudy, we developed an in vitro hypoxic modelthat mimicked an in vivo IR microenvironment.
With our model, we validated the DC phenotypes
and function variations when DCs were underhypoxia, either incubated alone or with TECs.
Because kidney DCs migrate from the spleen
and bone marrow, BMDCs were cultured from thebone marrow cells induced by IL-4 and GM-CSF
in this experiment. CD11c+MHCII+ double-positive cells were identified as BMDCs. LPS stimulation of the toll-like receptor 4 of BMDCs’
completely activated BMDCs to mDCs. This effect was evidenced by the upregulation of CD80,
CD86, and CCR7.

In this study, we first found that hypoxia suppressed imDC maturation and upregulated miR21 expressions. mDCs showed lower-level miR-21
expressions compared with imDCs. These datasuggest an opposite tendency for BMDC maturation and miR-21 levels. The transfection ofanti-miR oligos further verified this hypothesis.
Second, we used hypoxia-pretreated TEC-derived medium or employed a transwell co-culture
system at 1% O2 to study the interaction between
DCs and TECs and to mimic the AKI microenvironment in vitro. We found that a co-cultureof HCM and TECs contributed to the promotionof imDC maturation, which was evidenced by
the upregulation of DC maturation markers andwas accompanied by elevated pro-inflammatory
cytokine secretion. Lastly, anti-miR treatment of
co-cultured TECs further clarified the negative

Figure 3. Transwell co-culture with TEC elevated the inflammatory activity of BMDCs under hypoxia. A, and B, Whether
exposed to hypoxia (A) or normoxia (B), co-culture with TECs elevated the levels of CD80 MFI and CCR7 MFI of imDCs
and mDCs. *p<0.05, vs. sole DCs culture group. C-E, 48-h transwell co-culture with TECs under hypoxia promoted inflammatory cytokine secretion of BMDCs compared with those in the sole DC culture. *p<0.05, vs. sole DCs culture group. F andG, Co-culture with TECs under hypoxia suppressed the expression of BMDC maturation markers compared with normoxic
co-culture group. *p<0.05, vs. normoxia group. N = 4.

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Y.-R. Liang, T. Zhang, P. Jia, X.-L. Xu, Y. Fang, X.-Q. Ding

Figure 4. Protein expressions of PDCD4 in BMDCs under different conditions. A, and B, Western blot analysis revealed thatco-culture with TECs under hypoxia or LPS treatment had no significant effect on PDCD4 expression of BMDCs. C, and D,
Anti-miR-21 treatment upregulated the protein expression of CCR7 and PDCD4 in BMDCs (*p<0.05, vs. anti-scramble group.
N=4). E and F, Co-culture with anti-miR-21 transfected TECs had no significant effect on the PDCD4 expression of BMDCs.

role of miR-21 in the relationship between BMDCs and TECs. The PDCD4-CCR7 signaling pathway was negatively regulated by miR-21, which
was subsequently attributed to the phenotypic
profiles of BMDCs.
Several miRNAs have been reported as important regulators of IR immune responses, suchas miR-146a, miR142, miR-155, and miR-150.
Wu et al30-32 reported that miR-181a overexpression downregulated CD40 and CD83 and suppressed the necrotic myocyte-induced upregulation of CD40, CD83, and CD864. MiR-21 hasemerged as one of the most upregulated mRNAs
after ischemia injury in some organs, such as thebrain33, the heart34, and the liver35. In our in vitro
study, we found that hypoxia-induced the upregulation of miR-21 in BMDCs, but suppressed
the expression of CD80, CD86, and CCR7, when
imDCs were solely cultured, which are consistent
with the previous finding that miR-21 alleviated1648

hypoxic injury36. In addition, we found that the
inhibition of miR-21 in TECs or imDC promoted
the maturation of BMDCs, with overexpressionof CD80, CD86, and CCR7. Additionally, CCR7
was proven to be one of the miR-21 targets; the reciprocal suppression between miR-21 and CCR7
in our study further proved that hypoxia-induced
upregulation of miR-21 and contributed to the immature state of BMDCs37.
Hypoxia, a common condition related to theischemic environment secondary to AKI, triggers
renal intrinsic cell and immune cell apoptosis38.
We investigated the role of miR-21 in BMDCs
apoptosis, and our study showed that hypoxia solely increased the apoptosis rate of BMDCs under hypoxia (at 1% O2) for 6 h. Additionally, theapoptosis rate kept increasing with the increase in
exposure time. The apoptosis was also aggregatedin miR-21−/− BMDCs under normoxia (data notshown). These data provided additional evidence

TEC promotes kidney DC maturation via miR-21 regulation

to support the renoprotective effect of miR-21 in a
hypoxic microenvironment, which was present in
the DC-mediated inflammatory response. However, the precise mechanism by which miR-21 modulates apoptosis in renal intrinsic and immune
cells is yet to be elucidated. Thus, future studies
should aim to identify relevant targets of miR-21,
such as PDCD4 that participates in response tohypoxia and inflammation.
Further, we investigated the source of miR-21 of
BMDCs. Exosomes have been a hotspot of studies
about cell-to-cell interaction. We primarily observed the upregulated miR-21 in BMDC-derived
exosomes from the supernatant of hypoxia-cultured BMDCs. Conversely, inhibiting miR-21 levels
in TECs promoted maturation of the co-cultured
mDCs, which also indicated that TECs regulates
the function of DCs in a paracrine way.

This work has certain relevant limitations.
First, the effect of DCs on the differentiation of
T cells was not investigated. Second, this was anin vitro study, and the findings need to be further
validated for clinical application. Our team hasrecently published a paper reporting that delayedischemic preconditioning protected against renal
IR injury by inhibiting DC maturation in a mousemodel39; however, many questions remain to beanswered. Third, we were not able to yield conclusive evidence showing the downstream regulationof CCR7 by miR-21. Lastly, compared with previous investigations, our research showed some
controversial results such as the effect of hypoxia
on the CCR7 expression due to different cell models. Therefore, further research is warranted toclarify the specific mechanisms involved in theinteraction between TECs and DCs.

Figure 5. Anti-miR-21 treatment of TEC promoted maturation of co-cultured BMDCs. A, Electronic microscopy image of
the BMDC supernatant-derived exosomes (red arrow), scale bars, 100 nm. B, Vesicle content of exosome marker CD63 confirmed by Western blot. C, MiR-21 levels in exosomes extracted from the supernatant of cultured mDCs. D and E, Whether
exposed to normoxia or hypoxia, co-culture with transfected TEC elevated CD80, CD86, and CCR7 MFI of imDCs. *p<0.05,**
p<0.01, N = 4.

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Y.-R. Liang, T. Zhang, P. Jia, X.-L. Xu, Y. Fang, X.-Q. Ding

Conclusions
We revealed the novel effect of miR-21 in DC
inflammatory responses against hypoxia and TEC
stimulation. Further studies and clinical trials thatcan provide valuable insight into the mechanisms
involved in DC maturation and its clinical application are needed.

10)

11)

Conflict of Interest
The Authors declare that they have no conflict of interest.

12)

Acknowledgments

Key Project of National Natural Science Foundation of China(81430015); Science and Technology Commission of Shanghai Municipality (14DZ2260200, the Project of Shanghai Key
Laboratory of Kidney and Blood Purification).

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