Abstract
The dysregulation of the M1/M2 macrophage balance plays a pivotal role
in autoimmune diseases. However, the interplay between microRNAs
(miRNAs) and N6‐methyladenosine (m6A) modulation in regulating this
balance remains poorly understood. Here, a significant reduction in
miR‐31‐5p levels is observed in the lacrimal glands of rabbit
autoimmune dacryoadenitis and the peripheral blood mononuclear cells
(PBMCs) of Sjögren's syndrome (SS) dry eye patients. Overexpression of
miR‐31‐5p exhibits preventive and therapeutic effects on rabbit
autoimmune dacryoadenitis. Further investigation revealed that
miR‐31‐5p overexpression significantly restored the M1/M2 macrophage
balance both in vivo and in vitro. Mechanistically, miR‐31‐5p directly
targets the P2x7 receptor (P2RX7), leading to the inactivation of p38
mitogen‐activated protein kinases (MAPK) signaling and reduced
expression of M1 markers. Furthermore, methylated RNA
immunoprecipitation and luciferase reporter assays demonstrated that
fat mass and obesity‐associated protein (FTO)‐mediated m6A
demethylation, which sustains pri‐miR‐31 stability, is responsible for
the decreased miR‐31‐5p levels in autoimmune dry eye. Notably, PBMC
samples from SS dry eye patients further support the link between
reduced miR‐31‐5p levels and M1 macrophage activation observed in
rabbits. Overall, these data highlight the critical role of the
FTO/miR‐31‐5p/P2RX7/p38 MAPK axis in autoimmune inflammation,
suggesting their potential as therapeutic targets for autoimmune dry
eye.
Keywords: m6A, macrophage polarization, miR‐31‐5p, p2x7 receptor,
sjogren's syndrome dry eye
__________________________________________________________________
The upregulation of FTO in autoimmune dry eye suppresses the
recognition of pri‐miR‐31 by DGCR8, thereby inhibiting the maturation
of miR‐31‐5p and reducing its inhibitory effect on the target gene
P2RX7. The activation of P2RX7 subsequently triggers the p38 MAPK
signaling pathway and increases the level of M1‐related genes, thus
exacerbating the development of autoimmune dry eye. Conversely,
overexpression of miR‐31‐5p converts M1 macrophages into an M2
phenotype, which secretes anti‐inflammatory mediators and alleviates
the symptoms of autoimmune dry eye.
graphic file with name ADVS-12-2415341-g008.jpg
1. Introduction
Sjogren's syndrome (SS) dry eye is an intractable autoimmune disease
characterized by lymphocyte infiltration and dysfunction in the
lacrimal glands (LGs), which may ultimately lead to visual
impairment.^[ [44]^1 , [45]^2 ^] Although autoreactive T cells play an
important role in SS dry eye pathogenesis,^[ [46]^2 , [47]^3 ^] the
implication of macrophages in the development of SS dry eye has been
gaining ground recently.^[ [48]^4 ^] Indeed, the number of macrophages
aggregating in LGs was found to be positively correlated with the
infiltration grade and biopsy focus scores in SS patients.^[ [49]^5 ,
[50]^6 ^]
Macrophages exhibit different phenotypes that exert distinct functions
on the development of autoimmune diseases. M1 macrophages, marked by
nitric oxide synthase 2 (NOS2), exert pro‐inflammatory effects through
promoting the production of inflammatory factors, including interleukin
(IL)‐1β and tumour necrosis factor (TNF)‐α.^[ [51]^7 , [52]^8 ^] On the
contrary, M2 macrophages, which highly express arginase 1 (Arg1) and
mannose receptor (CD206), can facilitate the resolution of inflammation
and tissue repair via secreting anti‐inflammatory mediators, such as
IL‐10 and transforming growth factor (TGF)‐β.^[ [53]^7 , [54]^9 ^]
Recent studies have demonstrated that M1/M2 imbalance plays a
pathogenic role in SS pathogenesis.^[ [55]^10 , [56]^11 ^] Significant
increases in M1 macrophages and associated pro‐inflammatory cytokines
have been found in the peripheral blood of SS patients, potentially
fueling the chronic inflammatory process within the exocrine glands.^[
[57]^11 ^] Our published works have verified that skewing macrophages
to an M2 phenotype can contribute to alleviating autoimmune
dacryoadenitis.^[ [58]^4 , [59]^12 ^] However, the molecular mechanisms
regulating the M1/M2 balance in autoimmunity remain largely unknown.
Named after their small size of ≈21–25 nucleotides, microRNA (miRNAs)
are non‐coding RNAs that negatively regulate gene expression at the
post‐transcriptional level.^[ [60]^13 ^] A miRNA can target a large
network of mRNAs to affect diverse aspects of physical and pathological
processes, including macrophage polarization.^[ [61]^14 ^] By
conducting miRNAs sequencing analysis of LGs between a rabbit dry eye
model and normal controls, we found that miR‐31‐5p was significantly
downregulated in the LGs of dry eye rabbits. Emerging evidence has
highlighted the controversial role of miR‐31 in autoimmune disorders.
miR‐31 promoted epidermal hyperplasia and exacerbated psoriatic
inflammation in psoriasis,^[ [62]^15 ^] worsened experimental
autoimmune encephalomyelitis by repressing G protein‐coupled receptor 5
(GPRC5).^[ [63]^16 ^] However, overexpression of miR‐31 has been found
to attenuate murine allergic rhinitis via suppressing IL‐13‐induced
nasal epithelial inflammatory responses,^[ [64]^17 ^] and to relieve
neuropathic pain by inhibiting TNF receptor associated factor 6
(TRAF6)‐mediated neuroinflammation.^[ [65]^18 ^] However, to our
knowledge, no prior study has addressed whether and how miR‐31
regulates the pathogenesis of autoimmune dry eye.
N6‐methyladenosine (m6A) methylation, a ubiquitous RNA modification of
eukaryotic RNA, participates in RNA transcription, translation, and
stability.^[ [66]^19 , [67]^20 ^] m6A is dynamically regulated by
methyltransferases (methyltransferase like (METTL)3 and METTL14),
demethylases (fat mass and obesity‐associated protein (FTO) and AlkB
homolog 5 (ALKBH5)), and methylated reading proteins (YTH
domain‐containing (YTHDC)1/2 and YTH domain‐containing family protein
(YTHDF)1/2/3).^[ [68]^20 , [69]^21 ^] Aberrant expression of these m6A
regulators has been implicated in the pathogenesis of autoimmune
disorders, including SS.^[ [70]^22 , [71]^23 , [72]^24 ^] In recent
years, m6A modification in miRNAs targeting oncogenes or tumor
suppressors has been reported to play a pivotal role in cancer
progression.^[ [73]^25 ^] Through repressing immunoregulators, such as
dual‐specificity phosphatase 16 (DUSP16) and protein kinase
AMP‐Activated catalytic subunit alpha 2 (PRKAA2), m6A‐regulated miRNAs
have been found to aggravate experimental autoimmune uveitis (EAU) and
osteoarthritis.^[ [74]^26 , [75]^27 ^] Nevertheless, the role of m6A
modification in SS dry eye remains unclear. Moreover, the function of
m6A‐regulated miRNAs in autoimmune dry eye needs further exploration.
In this study, we demonstrated that FTO‐regulated miR‐31‐5p,
significantly downregulated in peripheral blood mononuclear cells
(PBMCs) of SS dry eye patients and in LGs of rabbit autoimmune
dacryoadenitis, an animal model of autoimmune dry eye which can closely
mimic human SS dry eye disease,^[ [76]^28 , [77]^29 ^] was critical for
autoimmune dry eye progression. By targeting the p2x7 receptor (P2RX7),
miR‐31‐5p inactivated p38 mitogen‐activated protein kinases (MAPK)
signaling, leading to suppressed M1 activation and alleviated
autoimmune dacryoadenitis.
2. Results
2.1. miR‐31‐5p is Significantly Decreased Both in LGs of Rabbit Autoimmune
Dacryoadenitis and in PBMCs of Human SS Dry Eye Patients
Using global miRNA sequencing of LGs of rabbit autoimmune
dacryoadenitis, we found that 6 miRNAs (miR‐381‐3p, miR‐31‐5p,
miR‐656‐3p, miR‐432‐5p, miR‐34a‐5p and miR‐219a‐5p) were significantly
down‐regulated at least 1.5‐fold in LGs of rabbit autoimmune
dacryoadenitis compared to normal controls (Figure [78]1A,B).
Measurement of these downregulated miRNAs in PBMCs samples from SS dry
eye patients showed that the levels of miR‐656‐3p, miR‐34a‐5p, and
miR‐219a‐5p were all below the detection thresholds. Notably, only the
expression of miR‐31‐5p was significantly lower in SS dry eye patients
compared to healthy individuals, as depicted in Figure [79]1C. More
importantly, Further correlation analysis revealed that the level of
miR‐31‐5p was positively associated with tear production (r = 0.8347, p
= 0.0387) and tear break‐up time (BUT) (r = 0.7346, p = 0.0242), while
negatively correlated with the corneal epithelial damage scores (r =
−0.8728, p = 0.0021) of SS dry eye patients (Figure [80]1D), suggesting
the level of miR‐31‐5p is closely related to the severity of SS dry eye
disease. Subsequently, this miRNA was chosen for functional studies.
Then, the lower levels of miR‐31‐5p in both LGs and PBMCs of a separate
cohort of rabbits with autoimmune dacryoadenitis relative to normal
controls were verified by real‐time quantitative RT‐PCR (qRT‐PCR)
(Figure [81]1E).
Figure 1.
Figure 1
[82]Open in a new tab
miR‐31‐5p is decreased in LGs of rabbit autoimmune dacryoadenitis and
PBMCs of human SS dry eye patients. A) Small RNA sequencing workflow
schematic. Autoimmune dacryoadenitis in rabbits was induced by
transferring activated PBLs. At 6 weeks post‐induction, LGs of normal
and model group rabbits were collected for small RNA sequencing (n =
3/group). B) Small RNA‐sequencing‐based scatter plot showing the
differentially expressed miRNAs in LGs isolated from rabbit autoimmune
dacryoadenitis relative to those from normal controls. Red dots
indicate upregulated miRNAs and blue dots represent downregulated
miRNAs C) Dysregulated miRNAs expression level in PBMCs from SS dry eye
patients and healthy controls (n = 11 per group). D) Correlation
between miR‐31‐5p expression and dry eye severity of SS dry eye
patients was calculated by the Pearson correlation test. E) Real‐time
qRT‐PCR analysis of miR‐31‐5p expression in LGs and PBMCs from model
rabbits and normal controls (n = 3 per group). LG, lacrimal gland;
pLGECs, purified lacrimal gland epithelial cells; PBLs: peripheral
blood lymphocytes; FC: fold change; HC, healthy controls; SS, Sjogren's
syndrome; BUT, tear break‐up time; CFS, corneal fluorescein staining.
Data was shown as mean ± SD, and the differences were analyzed by
Unpaired Student's t‐test. ^* p < 0.05, ^*** p < 0.001.
2.2. Overexpression of miR‐31‐5p Prevents the Development of Rabbit
Autoimmune Dacryoadenitis
To investigate whether there is a causative link between reduced
miR‐31‐5p and autoimmune dry eye, a lentiviral vector containing the
pre‐miR‐31‐5p sequence was constructed, and the lentiviruses were
subsequently packaged (Figure [83]2A). Then, rabbits were
subconjunctivally injected with a single dose of
miR‐31‐5p‐overexpressing lentivirus at the time of disease induction
(day 1 post transfer) (Figure [84]2B). Using real‐time qRT‐PCR, we
detected significantly increased miR‐31‐5p expression in LGs of rabbits
injected with miR‐31‐5p‐overexpressing lentivirus relative to the
control group, indicating the successful overexpression of miR‐31‐5p in
vivo (Figure [85]S1A, Supporting Information). The severity of dry eye
was evaluated by assessing tear production, BUT and corneal epithelial
integrity every 2 weeks after transfer. As shown in Figure [86]2C–E,
miR‐31‐5p overexpressing rabbits displayed significantly increased tear
production, prolonged tear BUT, and milder corneal epithelial damage
compared with control group rabbits. After 8 weeks of adoptive
transfer, the LGs and conjunctivas were collected, and the inflammatory
infiltration was assessed by H&E staining. As shown in Figure [87]2F,G,
miR‐31‐5p overexpressing rabbits showed a significantly decreased
inflammatory cell infiltration and tissue damage in the LGs and
conjunctivas relative to control diseased rabbits. All together, these
data suggest that miR‐31‐5p significantly attenuates the disease
severity of the induced rabbit autoimmune dry eye.
Figure 2.
Figure 2
[88]Open in a new tab
miR‐31‐5p overexpression prevents the development of rabbit autoimmune
dacryoadenitis. A) Lentiviral vector plasmids used to overexpress
miR‐31‐5p. B) Schematic diagram illustrating LV‐miR‐31‐5p
administration at the early stage (day 1 post transfer) of rabbit
autoimmune dacryoadenitis (2 × 10^7 transducing units/eye). C)
Representative corneal fluorescein staining images. D,E) Tear
production, tear break‐up time and corneal fluorescein staining scores
of each group of rabbits (n = 6/group). F,G) Representative H&E
staining photographs and scores of LGs and conjunctivas in each group
of rabbits (n = 6/group). Scale bars, 100 and 50 µm. Arrows indicate
infiltrating lymphocytes. PBLs, peripheral blood lymphocytes; The data
were shown as mean ± SD. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.
D,E) two‐way ANOVA; G) one‐way ANOVA.
2.3. Overexpression of miR‐31‐5p Restores the M1/M2 Macrophage Balance In
Vivo
Given that increased M1 macrophage activation and defective M2
macrophage polarization contribute to the development of autoimmune
dacryoadenitis,^[ [89]^4 , [90]^12 ^] we next investigate the effects
of miR‐31‐5p on macrophage polarization. As shown in Figure [91]3A,B,
in comparison with those in the control group, the expression of M1
macrophage marker NOS2 and M1 related pro‐inflammatory cytokines, such
as TNF‐α and IL‐1β, were significantly decreased, whereas the M2
macrophage markers such as Arg1 and CD206, and anti‐inflammatory
cytokines TGF‐β were dramatically increased in LGs of
miR‐31‐5p‐overexpressing rabbits. Consistently, increased Arg1 and
reduced NOS2 protein levels were observed in the LGs of
miR‐31‐5p‐overexpressing rabbits (Figure [92]3C). Together, these data
indicate that miR‐31‐5p can suppress M1 macrophage activation and
promote M2 macrophage polarization in response to chronic inflammation
in the LGs.
Figure 3.
Figure 3
[93]Open in a new tab
Overexpression of miR‐31‐5p restores the M1/M2 macrophage balance in
vivo. LGs were collected from miR‐31‐5p overexpressing‐rabbits or
control rabbits at week 8 after adoptive transfer of activated PBLs,
and then subjected to real‐time qRT‐PCR or Western blot analysis. A)
The relative expression of M1‐related genes. B) The mRNA expression of
M2‐associated genes. C) The protein levels of Arg1 and NOS2.
LV‐miR‐31‐5p, miR‐31‐5p‐overexpressing rabbits; LV‐Ctrl, control
rabbits. Data was from at least three independent experiments and
presented as mean ± SD. Data was analyzed by one‐way ANOVA. ^* p
< 0.05, ^** p < 0.01.
2.4. miR‐31‐5p Inhibits M1 Activation and Facilitates Macrophages into M2
Phenotype In Vitro
We next investigate the effects of miR‐31‐5p on macrophage polarization
in vitro. To address this, PBMCs from model rabbits were transfected
with miR‐31‐5p mimics or control mimics, washed, and then stimulated
with irradiated purified lacrimal gland epithelial cells (pLGECs)
(Figure [94]4A). As shown in Figure [95]4B,C, the mRNA levels of
M1‐associated genes including NOS2, IL‐1β and TNF‐α were significantly
decreased, whereas the transcript levels of M2‐related genes, such as
Arg1, CD206, and IL‐10 were dramatically increased in miR‐31‐5p
overexpressing group cells relative to controls.
Figure 4.
Figure 4
[96]Open in a new tab
miR‐31‐5p inhibits M1 macrophage activation and facilitates macrophages
into M2 phenotype in vitro. A) Scheme of experimental procedures to
evaluate the role of miR‐31‐5p on macrophage polarization. B,C) PBMCs
isolated from model rabbits were transfected with miR‐31‐5p mimics of
control mimics (300 nmol) and cocultured with irradiated pLGECs for
48h. Relative expression of M1 markers and M2‐related genes were
analyzed by real‐time qRT‐PCR. D–I) Macrophage derived from THP‐1 cells
were transfected with miR‐31‐5p mimics, miR‐31‐5p inhibitors or their
negative controls for 24 h, and then stimulated with LPS and IFN‐γ to
induce M1 macrophage polarization. D–G) Real‐time qRT‐PCR analysis of
M1 and M2 macrophage‐related gene expression. H,I) Western blot
analysis of Arg1 and NOS2 protein level. J,K) Representative confocal
images of Arg1 (red) and NOS2 (green) immunofluorescence staining in
macrophages transfected with miR‐31‐5p mimics or control mimics. Scale
bars, 20 µm. Data were representative of at least three independent
experiments and were analyzed by Unpaired Student's t‐test or
Mann–Whitney U test. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ns, not
significant.
To further confirm the macrophage‐intrinsic role of miR‐31‐5p on
macrophage phenotype, THP‐1‐derived macrophages were transfected with
miR‐31‐5p mimics, miR‐31‐5p inhibitor or their negative controls for
24 h, and then stimulated with LPS + IFN‐γ to induce M1 macrophage
polarization (Figure [97]4A). real‐time qRT‐PCR analysis showed that
miR‐31‐5p overexpression markedly decreased the mRNA levels of
M1‐associated genes and upregulated M2‐related gene expression
(Figure [98]4D,E), whereas blocking miR‐31‐5p had the opposite effect
(Figure [99]4F,G). Meanwhile, significantly increased Arg1 protein
expression and dramatically decreased NOS2 protein levels were observed
in miR‐31‐5p mimics‐transfected macrophages (Figure [100]4H,I).
Furthermore, immunofluorescence staining showed that the expression of
M2‐related marker Arg1 was dramatically higher in
miR‐31‐5p‐overexpressing macrophages relative to controls, whereas the
level of M1‐associated gene NOS2 was significantly decreased in
macrophages overexpressing miR‐31‐5p (Figure [101]4J,K). Collectively,
these data indicate that miR‐31‐5p inhibits M1 macrophage activation
and facilitates macrophages into an anti‐inflammatory M2 phenotype in
vitro.
2.5. miR‐31‐5p Suppresses M1 Polarization by Targeting P2RX7
To define the molecular mechanisms by which miR‐31‐5p modulates
macrophage polarization, we used miRanda database to identify the
potential targets of miR‐31‐5p. Further, GO category analysis focusing
on immune system processes using Cytoscape software identified 15
candidate genes that were significantly enriched in immune responses
(Figure [102]S2, Supporting Information). Among these, P2RX7 garnered
our attention owing to its established role in modulating
macrophage‐mediated inflammation.^[ [103]^30 ^] As shown in Figure
[104]5A, bioinformatics analysis showed the putative miR‐31‐5p seed
sequences in the 3^′ UTR of P2RX7, and further luciferase assay showed
that miR‐31‐5p suppressed the luciferase activity of a reporter with
the wild‐type (WT), but not the mutant (Mut) 3^′UTR of P2RX7
(Figure [105]5B), suggesting that miR‐31‐5p specifically targets P2RX7
and regulates its expression. Indeed, a gain and loss function assay
showed that P2RX7 mRNA and protein levels were significantly reduced in
macrophages transfected with miR‐31‐5p mimics, whereas knockdown of
miR‐31‐5p dramatically increased P2RX7 expression (Figure [106]5C–E).
In addition, we observed a significantly increased P2RX7 mRNA level in
LGs of model rabbits (Figure [107]5F), whereas the level of P2RX7 was
markedly decreased in LGs of miR‐31‐5p‐overexpressing rabbits
(Figure [108]5G). These results indicate that P2RX7 is a functional
target of miR‐31‐5p.
Figure 5.
Figure 5
[109]Open in a new tab
P2RX7 is a functional target of miR‐31‐5p. A) Sequence alignment
between miR‐31‐5p and its potential binding sites (in red letters) in
the 3^′UTR of R2RX7 mRNA. The mutation of the miR‐31‐5p binding sites
is shown in bule. B) Luciferase activity analysis of reporter carrying
the wild‐type or mutant P2RX7 3^′UTR co‐transfected into HEK293T cells
with miR‐31‐5p mimics or control mimics. C–E) Macrophage derived from
THP‐1 cells were transfected with indicated mimics or inhibitors, and
the expression of P2RX7 was measured by real‐time qRT‐PCR and western
blot. F,G) Real‐time qRT‐PCR analysis of P2RX7 expression in LGs of
each group of rabbits. H–N) Macrophage derived from THP‐1 cells were
transfected with indicated siRNA or inhibitors for 24 h, and then
stimulated with LPS+IFN‐γ to induce M1 macrophage polarization. H)
Real‐time qRT‐PCR analysis of P2RX7 expression in each group of cells.
I–N) The relative expression of M1 and M2 macrophage associated genes,
detected by real‐time qRT‐PCR or western blot, is shown. Data were
shown as mean ± SD from at least three independent experiments. ^* p
< 0.05, ^** p < 0.01, ^*** p < 0.001, ns, not significant. (B‐K)
Unpaired Student's I‐test; L–N) one‐way ANOVA.
To determine whether P2RX7 is important for miR‐31‐5p‐mediated
macrophage polarization, THP‐1‐derived macrophages were transfected
with P2RX7 siRNA (Figure [110]5H) or control siRNA, and then stimulated
with LPS + IFN‐γ. As shown in Figure [111]5I, the transcript levels of
M1 markers were significantly decreased in P2RX7‐knockdown macrophages
compared with the controls, whereas no remarkable differences were
observed in the mRNA levels of M2‐related genes between the two groups.
Consistently, western blot analysis showed a dramatic decrease in the
protein levels of M1 marker NOS2 in P2RX7‐silenced macrophages
(Figure [112]5J,K). Notably, knockdown of P2RX7 partially abolished the
upregulation of M1‐related genes induced by miR‐31‐5p inhibitor
(Figure [113]5L–N). These data suggest that P2RX7 is a functional
target mediating reduced M1 polarization caused by miR‐31‐5p.
2.6. miR‐31‐5p/P2RX7 Inhibits M1 Polarization by Suppressing p38 MAPK
Signaling
To elucidate the downstream pathway by which miR‐31‐5p/P2RX7 modulates
M1 macrophage polarization, we performed KEGG analysis of miR‐31‐5p
target gene using the DAVID database. As shown in Figure [114]6A, the
target genes of miR‐31‐5p are top enriched in MAPK pathway. Given that
MAPK signaling is critical for M1 activation,^[ [115]^31 ^] and that
P2RX7 has been reported to activate the MAPK pathway in Osteoclasts,^[
[116]^32 ^] we next examined whether the MAPK pathway is involved in
the action of miR‐31‐5p on macrophage polarization. To this end,
macrophages transfected with miR‐31‐5p mimics, P2RX7 siRNA, or their
negative controls were stimulated to polarize toward M1 macrophages,
and the phosphorylation levels of p38, JNK and ERK were monitored 48 h
later. As shown in Figure [117]6B–D, both miR‐31‐5p overexpression and
P2RX7 knockdown dramatically reduced the phosphorylation levels of p38,
while the phosphorylation level of JNK and ERK showed no difference
between the two groups. Notably, silencing P2RX7 partially reversed the
increased levels of p‐p38 induced by miR‐31‐5p inhibitor
(Figure [118]6E,F). These data suggest that miR‐31‐5p/P2RX7 might
suppress M1 macrophage polarization by regulating the p38 MAPK pathway.
Figure 6.
Figure 6
[119]Open in a new tab
miR‐31‐5p/P2RX7 inhibits M1 macrophage polarization by suppressing p38
MAPK signaling. A) KEGG analysis on target genes of miR‐31‐5p. The top
10 pathways are summarized. B–F) THP‐1 derived macrophages were
transfected with indicated mimics, siRNA or inhibitors, and then
stimulated with LPS + IFN‐γ to induce M1 macrophage polarization. The
phosphorylation level of p38, JNK and ERK were detected by western blot
48 h later. G) THP‐1‐derived macrophages were pretreated with 10 µm
SB203580 p38 inhibitor or DMSO for 1 h prior to stimulation with LPS
and IFN‐γ to induce M1 macrophage polarization. The relative mRNA
expression of M1‐related genes was then examined by real‐time qRT‐PCR.
I) THP‐1‐derived macrophages were transfected with miR‐31‐5p inhibitor
or Ctrl inhibitor following pretreatment with 10 µm SB203580 p38
inhibitor or DMSO for 1h. The levels of M1‐associated genes were
analyzed. Data were shown as mean ± SD from at least three independent
experiments. ^* p < 0.05, ^** p < 0.01, ns, not significant. (C‐D and
G) Unpaired Student's t‐test or Mann–Whitney U test; (F and H) one‐way
ANOVA.
To further investigate the role of p38 MAPK pathway in modulating
macrophage polarization, we pretreated THP1‐derived macrophages with
the p38 inhibitor SB203580 for 1 h prior to stimulating them with LPS
and IFN‐γ to induce M1 macrophage polarization, and we found that
inhibiting the p38 MAPK signaling pathway significantly decreased the
transcript levels of M1 markers, including NOS2, IL‐1β and TNF‐α
(Figure [120]6G), suggesting the important role of the p38 MAPK pathway
in modulating the function and phenotype of macrophages. Notably,
inhibition of p38 MAPK signaling partially reversed the upregulation of
M1‐related genes induced by the miR‐31‐5p inhibitor (Figure [121]6H),
indicating that the p38 MAPK pathway is closely involved in
miR‐31‐5p‐mediated macrophage polarization.
2.7. miR‐31‐5p is Regulated by m6A Methylation
m6A, the most prevalent modification of mRNAs and noncoding RNAs, plays
an important role in miRNA biogenesis through modulating pri‐miRNA
processing and miRNA maturation.^[ [122]^33 ^] The m6A modification
mainly occurs at the consensus motif “RRACH” (R = G or A; H = A, C or
U).^[ [123]^34 ^] To explore why miR‐31‐5p is decreased in autoimmune
dry eye, we first sought for the m6A sites on pri‐miR‐31 sequence using
SRAMP. As shown in Figure [124]7A, we found two potential m6A sites
located near the splicing site of pri‐miR‐31, suggesting that m6A may
play a role in the regulation of miR‐31‐5p during dry eye.
Figure 7.
Figure 7
[125]Open in a new tab
m6A methylation regulates the expression of miR‐31‐5p in autoimmune dry
eye. A) The sequences of pri‐miR‐31, miR‐31‐5p, and the potential m6A
motif (GAACU) were highlighted with different colors. B) Relative
expression of m6A enzymes (METTL3, METTL14, WTAP and FTO) in LGs of
normal and model rabbits were detected by real‐time qRT‐PCR. C,D) PBMCs
from model rabbits were transfected with indicated FTO siRNA. The
relative expression of FTO, pri‐miR‐31‐5p and miR‐31‐5p was detected by
real‐time qRT‐PCR. E) Pri‐miR‐31 levels in FTO knockdown and negative
control PBMCs after actinomycin D treatment at the indicated times. F)
Flow diagram of MeRIP‐qPCR assays. G) MeRIP‐qPCR analysis of the m6A
levels of pri‐miR‐31 in FTO silenced PBMCs and control cells. H)
Luciferase activity analysis was performed on a reporter carrying the
wild‐type or mutant sequence of pri‐miR‐31, which was co‐transfected
into HEK293T cells with either FTO siRNA or control siRNA. I) Total
protein from HEK293T and THP‐1 cells was immunoprecipitated with an
anti‐DGCR8 antibody. Western blots for FTO and DGCR8 are shown. J)
Detection of the abundance of pri‐miR‐31 binding to DGCR8 in HEK293T
cells by immunoprecipitation with an anti‐DGCR8 antibody. Data were
shown as mean ± SD, and the differences were analyzed by one‐way ANOVA,
Unpaired Student's t‐test or Mann–Whitney U test. ^* p < 0.05, ^** p
< 0.01, ns, not significant.
To determine m6A enzymes involved in the regulation of miR‐31‐5p
expression in autoimmune dry eye, major m6A writers (METTL3, METTL14,
and WTAP) and erasers (FTO) expression levels in LGs of model rabbits
and normal controls were evaluated by real‐time qRT‐PCR. As shown in
Figure [126]7B, the m6A demethylase FTO, but not other enzymes, was
significantly elevated in LGs of model rabbits, suggesting that FTO may
be involved in the dysregulation of miR‐31‐5p in autoimmune dry eye.
Indeed, using siRNA‐mediated knockdown of FTO in PBMCs from model
rabbits (Figure [127]7C), we found that silencing FTO significantly
decreased pri‐miR‐31 expression, whereas miR‐31‐5p levels were
dramatically upregulated (Figure [128]7D), indicating that FTO may
regulate the processing of miR‐31‐5p via m6A. This was further
confirmed by the observation that depletion of FTO dramatically
decreased the half‐life of pri‐miR‐31 in PBMCs from model rabbits
(Figure [129]7E). Importantly, Methylated RNA immunoprecipitation qPCR
(MeRIP‐qPCR) analysis uncovered that m6A was highly enriched within the
pri‐miR‐31 sequence, and the enrichment m6A level was significantly
upregulated following the knockdown of FTO (Figure [130]7F,G). To
further validate the impact of FTO on pri‐miR‐31 processing through
m6A, we constructed dual‐luciferase reporters containing WT (containing
potential m6A sites) or Mut forms (the adenosine base in m6A consensus
sequences replaced with thymine to abolish the m6A modification) of
pri‐miR‐31. As shown in Figure [131]7H, FTO knockdown significantly
decreased the luciferase activity in the WT group but not in the Mut
group, further supporting the m6A modification in pri‐miR‐31
processing. The m6A modification has been proved to facilitate the
recognition of pri‐miRNA hairpin by DiGeorge syndrome critical region 8
(DGCR8), thus promoting miRNAs maturation.^[ [132]^19 ^] We next asked
whether FTO regulated pri‐miR‐31 processing in a DGCR8‐dependent
manner. To this end, total protein from HEK293T and THP‐1 cells, where
both FTO and DGCR8 were expressed, was immunoprecipitated with an
anti‐DGCR8 antibody. As presented in Figure [133]7I, we found that FTO
was not co‐precipitated with DGCR8 in either HEK293T or THP‐1 cell
lines, indicating that FTO did not directly interact with DGCR8.
Further RNA immunoprecipitation (RIP) experiments showed that knockdown
of FTO significantly increased the enrichment of pri‐miR‐31 in the
complex precipitated with antibody against DGCR8 (Figure [134]7J),
suggesting that FTO modulates miR‐31‐5p processing by influencing the
recognition of pri‐miR‐31 by DGCR8. Together, these data indicate that
FTO mediated m6A demethylation, which sustained pri‐miR‐31 stability,
was responsible for decreased miR‐31‐5p.
2.8. Overexpression of miR‐31‐5p at the Developed Stage Efficiently
Alleviates Autoimmune Dacryoadenitis in Rabbits
The induced rabbit autoimmune dacryoadenitis typically exhibits evident
clinical dry eye symptoms starting from the 2 weeks following the
adoptive transfer of activated peripheral blood lymphocytes (PBLs).^[
[135]^4 , [136]^35 ^] To determine whether overexpression of miR‐31‐5p
had therapeutic effects on rabbit autoimmune dacryoadenitis, a single
dose of miR‐31‐5p‐overexpressing lentivirus was injected
subconjunctivally into rabbits after disease onset (day 15 after
transfer), as illustrated in Figure [137]8A. Clinically, miR‐31‐5p
overexpression (Figure [138]S1B, Supporting Information) significantly
alleviated dry eye symptoms, as evidenced by increased tear secretion,
prolonged tear BUT, and decreased corneal fluorescein staining (CFS)
scores compared to the control group (Figure [139]8B,C).
Histologically, the infiltration of inflammatory cells in LGs and
conjunctivas was significantly attenuated in miR‐31‐5p‐overexpressing
rabbits (Figure [140]8D). Together, the administration of
miR‐31‐5p‐overexpressing lentivirus effectively attenuated the severity
of autoimmune dry eye in rabbits at the developed stage of the disease.
Figure 8.
Figure 8
[141]Open in a new tab
Overexpression of miR‐31‐5p at the developed stage efficiently
alleviates autoimmune dacryoadenitis in Rabbits. A) Schematic diagram
illustrating LV‐miR‐31‐5p administration (2 × 10^7 transducing
units/eye) at the developed stage (day 15 post transfer) of rabbit
autoimmune dacryoadenitis. B) Representative corneal fluorescein
staining images. C) Tear production, tear break‐up time and corneal
fluorescein staining scores of each group of rabbits (n = 5/group). D)
Representative specimens of H&E staining in LGs and conjunctivas. Scale
bars, 100 and 50 µm. Arrows indicate infiltrating lymphocytes. PBLs,
peripheral blood lymphocytes. Data was shown as mean ± SD and analyzed
by two‐way ANOVA. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.
2.9. Downregulation of miR‐31‐5p is Associated with Increased M1 Macrophage
Activation in SS Dry Eye Patients
As demonstrated by the results obtained both in vivo and in vitro,
miR‐31‐5p is implicated in the pathogenesis of autoimmune dry eye by
facilitating M1 macrophage polarization via targeting P2RX7, we next
aim to validate these results using samples from SS dry eye patients.
To this end, PBMCs isolated from both healthy controls and SS dry eye
patients were collected and analyzed (Figure [142]9A). As shown in
Figure [143]9B, we first noted a significant upregulation of M1‐related
genes, including NOS2, TNF‐α, and IL‐1β, in SS dry eye patients,
whereas no statistically significant differences were observed in the
expression of M2 markers, such as Arg1, pointing to M1 macrophage
activation in the progression of SS dry eye. Specifically, we
discovered that the decreased miR‐31‐5p level (Figure [144]1C)
significantly correlated with higher NOS2 expression (r = −0.7056, p =
0.0337) in SS dry eye patients (Figure [145]9C). These data indicated
that reduced miR‐31‐5p may contribute to M1 macrophage activation and
autoinflammation in autoimmune dry eye. Additionally, we observed a
dramatic increase in FTO expression in SS dry eye patients, as shown in
Figure [146]9D. Based on our in vitro data, the upregulation of FTO may
impede the maturation of miR‐31‐5p and reduce its expression, thereby
dampening the inhibitory effect of miR‐31‐5p on its target gene, P2RX7.
Of note, a negative correlation was observed between FTO and miR‐31‐5p
levels (r = −0.7924, p = 0.019) in SS dry eye patients
(Figure [147]9E). Furthermore, SS dry eye patients exhibited a markedly
elevated P2RX7 level, which displayed a negative trend, albeit not
statistically significant, in correlation with miR‐31‐5p (r = −0.4097,
p = 0.2734) (Figure [148]9F,G). Together, these data support the
existence of potential crosstalk between the FTO‐miR‐31‐5p‐P2RX7 axis
and M1 macrophage activation in human autoimmune dry eye.
Figure 9.
Figure 9
[149]Open in a new tab
Downregulation of miR‐31‐5p is associated with increased M1 macrophage
activation in SS dry eye patients. A) Schematic outline of experimental
procedures designed to assess gene expression in patients with SS dry
eye patients. B) Expression levels of macrophage‐related genes in PBMCs
from SS dry eye patients and controls subjects. C) Correlation analysis
between miR‐31‐5p expression and NOS2 levels in PBMCs of SS dry eye
patients. D,E) Quantification of FTO mRNA levels in PBMCs of SS dry eye
patients and its correlation with miR‐31‐5p expression. F,G) Expression
of P2RX7 in PBMCs of SS dry eye patients and its association with
miR‐31‐5p level. H,I) PBMC‐derived macrophages from SS dry eye patients
were transfected with miR‐31‐5p mimics or negative controls and
subsequently polarized into M1 macrophages. The expression of M1 and M2
macrophage‐related gene was measured by real‐time qRT‐PCR. Data was
shown as mean ± SD and analyzed by Unpaired Student's t‐test or
Mann–Whitney U test. ^* p < 0.05, ^** p < 0.01.
To further investigate the clinical significance of miR‐31‐5p,
PBMC‐derived macrophages from SS dry eye patients were transfected with
miR‐31‐5p mimics or negative controls and subsequently polarized into
M1 macrophages (Figure [150]9H). As shown in 9I, the overexpression of
miR‐31‐5p significantly reduced the mRNA expression of M1‐related genes
(NOS2, TNF‐α) while upregulating the levels of M2‐associated genes
(Arg1, CD206). Taken together, our data strongly suggest that miR‐31‐5p
is involved in modulating macrophage polarization in SS dry eye
patients.
3. Discussion
Through miRNA sequencing of LGs from model rabbits, together with
validation in PBMCs of SS dry eye patient, we characterized miR‐31‐5p
as a potential immunoregulator of autoimmune dry eye. Importantly,
using the rabbit autoimmune dacryoadenitis model, we demonstrated that
miR‐31‐5p can protect rabbits against autoimmune dacryoadenitis by
modulating M1/M2 balance through suppressing P2RX7 and inactivating p38
MAPK signaling (Figure [151]10 ).
Figure 10.
Figure 10
[152]Open in a new tab
Schematic depicting the mechanisms by which miR‐31‐5p regulates M1/M2
balance in autoimmune dry eye. The upregulation of FTO in autoimmune
dry eye suppresses the recognition of pri‐miR‐31 by DGCR8, thereby
inhibiting the maturation of miR‐31‐5p and reducing its inhibitory
effect on the target gene P2RX7. The activation of P2RX7 subsequently
triggers the p38 MAPK signaling pathway and increases the level of
M1‐related genes, thus exacerbating the development of autoimmune dry
eye. Conversely, overexpression of miR‐31‐5p converts M1 macrophages
into an M2 phenotype, which secretes anti‐inflammatory mediators and
alleviates the symptoms of autoimmune dry eye.
Previous studies, including our own, have demonstrated the pivotal role
of M1/M2 macrophages in regulating autoimmune dry eye, among which M1
macrophages are closely involved in the progression of autoimmune dry
eye lesions, while M2 is critical for resolving chronic inflammation
and slowing down disease progression.^[ [153]^4 , [154]^12 ^] miR‐31‐5p
has been linked to macrophage function. In RAW264.7 macrophages,
overexpression of miR‐31‐5p obviously downregulated the expression of
LPS‐induced inflammatory factors including TNF‐α, IL‐6, and IL‐1β.^[
[155]^36 ^] Nonetheless, little is known about the role of miR‐31‐5p in
macrophage polarization. In this study, we for the first time reported
that overexpression of miR‐31‐5p dramatically augmented the expression
of M2 markers (Arg1 and CD206), while it markedly decreased M1 marker
NOS2 expression both in vivo and in vitro, suggesting a critical role
of miR‐31‐5p in modulating M1/M2 balance. In addition, using
THP‐1‐derived macrophages, we further demonstrated that miR‐31‐5p
possessed the ability to shift M1 macrophage to an anti‐inflammatory M2
phenotype, underscoring the pivotal role of macrophage‐intrinsic
miR‐31‐5p in regulating macrophage phenotypes. Increased M2 macrophages
induced by miR‐31‐5p may result in the attenuated autoimmune
dacryoadenitis observed in miR‐31‐5p overexpressing model rabbits.
Considering that M1 activation in inflamed LGs induced tissue
destruction in autoimmune dry eye, targeting miR‐31‐5p may be a
promising therapeutic strategy.
P2RX7, a ligand‐gated ion channel that is abundantly expressed in
macrophages, plays a pivotal role in the initiation and progression of
inflammatory and autoimmune disorders.^[ [156]^37 ^] Here, utilizing
the miRanda database and luciferase reporter assays, we identified
P2RX7, which has been documented to be upregulated in PBMCs of SS dry
eye patients,^[ [157]^38 ^] was a novel target of miR‐31‐5p in
macrophages. Using LPS/IFN‐γ‐induced M1 macrophages, we found that
knockdown of P2RX7 significantly reduced the expression of M1‐related
genes in vitro. This finding is further supported by a previous report
showing that P2RX7 drives the secretion of M1 macrophage cytokines in
human monocyte‐derived macrophages.^[ [158]^39 ^] Importantly, we
demonstrated that the knockdown of P2RX7 dramatically rescued the
increased M1 markers in macrophages induced by miR‐31‐5p knockdown,
indicating an important regulatory role of P2RX7 in miR‐31‐5p mediated
macrophage polarization in autoimmune dry eye. Nonetheless, the
knockdown of P2RX7 had no obvious effect on M2‐related genes. This
suggests that miR‐31‐5p may modulate macrophage polarization through
other additional mechanisms beyond targeting P2RX7, which need further
exploration. miR‐31 has been proven to exert pro‐inflammatory or
immune‐suppressive effects in various autoimmune disorders by
interacting with a broad spectrum of molecular targets in specific
tissues and cellular contexts. Through targeting signal transducer and
activator of transcription 1 (STAT1)^[ [159]^40 ^] and carcinoembryonic
antigen‐related cell adhesion molecule 1 (CEACAM1),^[ [160]^41 ^]
miR‐31 has been found to blunt Th1 responses and enhance Treg cell
differentiation, thereby exerting a protective effect during HIV
infection and the progression of systemic lupus erythematosus.
Conversely, by suppressing SH2 domain containing 1A (SH2D1A)^[ [161]^42
^] and GPRC5A,^[ [162]^16 ^] miR‐31 promotes Th1 cytokine transcription
but suppresses Treg generation, thus promoting autoimmunity in sepsis
and experimental autoimmune encephalomyelitis. Our findings here
uncover a previously unrecognized functional interplay between
miR‐31‐5p and P2RX7 in macrophage polarization, which adds another
layer to the complicated regulatory network of miR‐31‐5p in
autoimmunity.
MAPKs, including p38, ERK, and JNK, are a group of highly conserved
serine/threonine protein kinases in eukaryotes, orchestrating various
intracellular activities such as inflammation and innate immunity.^[
[163]^43 , [164]^44 ^] P2RX7 has been linked to MAPK pathways in an
intranigral lipopolysaccharide rat model of Parkinson's disease^[
[165]^45 ^] and in septic mice,^[ [166]^46 ^] but according to
bioinformatic analysis and validation through in vitro experiments, we
are the first to find the interaction between P2RX7 and MAPK signaling
in macrophage polarization. We found that silencing P2RX7 selectively
inhibited the activation of p38, but not ERK and JNK, in macrophages.
Previous studies have documented the crucial role of p38 MAPK signaling
in the transcriptional activation of M1 macrophage‐related genes.^[
[167]^47 , [168]^48 ^] In line with these observations, we found that
inhibiting p38 MAPK activity using SB203580 led to a substantial
decrease in the transcript levels of M1 marker NOS2, as well as the
polarizing cytokines IL‐1β and TNF‐α. Importantly, we demonstrated that
blocking p38 MAPK antagonized increased M1 related molecules caused by
silencing miR‐31‐5p, and knockdown of P2RX7 rescued the increased
phosphorylation level of p38 MAPK induced by the miR‐31‐5p inhibitor.
These results suggested that the P2RX7‐p38 axis is an important
downstream mechanism involved in the action of miR‐31‐5p on M1
activation.
m6A is a critical post‐transcriptional regulator that can modulate
pri‐miRNA processing and miRNA maturation, thereby functioning in a
variety of diseases such as cancer and autoimmune disorders.^[ [169]^25
, [170]^26 , [171]^49 ^] Our previous research has uncovered that
METTL3‐induced miR‐338‐3p promoted pathogenic Th17 cell responses and
retinal inflammation in EAU.^[ [172]^26 ^] By altering the m6A
modification status of pri‐miRNA, FTO has been reported to block the
maturation of miR‐17‐5p in triple‐negative breast cancer (TNBC) cells
and decrease miR‐3591‐5p expression in chondrocytes, thereby
suppressing the progression of TNBC and osteoarthritis.^[ [173]^27 ,
[174]^50 ^] However, the functions of m6A modified miRNAs in autoimmune
dry eye remain elusive. In this study, we identified the m6A
modification at the pri‐miR‐31 sequence by a series of experiments.
Importantly, using MeRIP‐qPCR, Luciferase reporter assay, and RNA
stability analysis, we uncovered that knockdown of FTO dramatically
upregulated the m6A level on pri‐miR‐31 transcripts and shortened their
half‐life, indicating that FTO‐mediated m6A demethylation may maintain
the stability of pri‐miR‐31, thereby reducing its expression in
autoimmune dry eye. RNA‐binding protein DGCR8 plays an important role
in miRNA maturation. FTO has been reported to suppress the binding of
DGCR8 to pri‐miRNA through m6A demethylation, thus restraining
miR‐138‐5p and miR‐3591‐5p maturation.^[ [175]^27 , [176]^51 ^]
Consistent with these findings, we also observed that knockdown of FTO
significantly increased the interaction between pri‐miR‐31 and DGCR8,
suggesting that FTO‐mediated m6A demethylation affects the recognition
of pri‐miR‐31 by DGCR8, thereby inhibiting the maturation of miR‐31‐5p.
However, contrasting evidence also exists. For example, m6A
modification on pri‐miRNA‐374c has been shown to impede the maturation
of miRNA‐374c‐5p in T‐47D cells.^[ [177]^52 ^] Different cell types and
disease contexts may explain the discrepancy among these studies.
LGs samples of SS dry eye patients are very difficult to obtain. To
provide an alternative clinical link of our findings with SS dry eye,
we have measured the levels of miR‐31‐5p in PBMCs from SS dry eye
patients, and we found that levels of miR‐31‐5p were significantly
lower in SS dry eye patients than those in healthy controls, which were
consistent with miR‐31‐5p downregulation in LGs of rabbit dry eye
models. Furthermore, we observed that miR‐31‐5p not only significantly
reduced the expression of M1‐related markers while upregulating the
levels of M2‐associated genes in PBMCs from SS dry eye patients, but
also were closely associated with clinical parameters of SS dry eye
patients, suggesting the clinical importance of miR‐31‐5p. Thus,
miR‐31‐5p in PBMCs may be a novel biomarker for SS dry eye patients.
Despite the promising findings, this study is limited by its focus on
examining the role of miR‐31‐5p in the macrophages. It remains to be
determined whether and how miR‐31‐5p affects other immune cells that
also play important roles in autoimmune dry eye, such as CD4^+ T cells.
Moreover, miRNAs can target many gene transcripts, so our study may
miss some related targets that are also involved in the action of
miR‐31‐5p. Furthermore, studies involving more clinical samples,
including PBMCs, tears, and serum from patients at different disease
stages and with different treatment responses, would be of great
importance to prove the relationship between miR‐31‐5p and disease
characteristics in depth. In addition, we first paid attention to the
downregulated miRNAs in this study. Given that both upregulated and
downregulated miRNAs may contribute to the development of autoimmune
dry eye disease, more studies are needed to investigate the role of
those significantly upregulated miRNAs in SS dry eye.
In summary, our data demonstrated that m6A modified miR‐31‐5p can act
by selectively suppressing P2RX7‐p38 pathway to restore M1/M2 balance,
thereby alleviating autoimmune dry eye. These findings shed new light
on autoimmune dry eye mechanisms and may provide a promising
therapeutic target for the treatment of autoimmune dry eye.
4. Experimental Section
Animals
One‐year‐old female New Zealand white rabbits, weighing between 3.5 and
4 kg, were purchased from Vital River Laboratory Animal Technology
(Beijing, China). All rabbits were housed in a pathogen‐free
environment at Tianjin Medical University Eye Hospital, where the
temperature was controlled at 25 °C ± 2 °C, the relative humidity
ranged from 50% to 75%, and a 12‐h light‐dark cycle (from 8 am to 8 pm)
was maintained. The research adhered strictly to the guidelines of the
ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research, and the experimental procedures were approved by the
Institutional Animal Care and Use Committee of Tianjin Medical
University Eye Hospital (Permit Number: TJYY2023120210).
Human Samples
PBMC samples from eleven SS dry eye patients (female; mean age,
57.5±13.5 years) and eleven age‐matched healthy controls (female; mean
age, 55.7±7.9 years) were collected from Tianjin Medical University Eye
Hospital biobank and used to evaluate gene expression. All SS dry eye
patients were diagnosed based on the American College of
Rheumatology/European League Against Rheumatism (ACR/EULAR) 2016
criteria. The study adhered to the Helsinki Declaration and was
approved by the Ethics Committee of Tianjin Medical University Eye
Hospital (Ethical batch number: 2021KY‐17). Written informed consent
was obtained from all participants. Dry eye examination was conducted
for SS dry eye patients, including measurement of tear BUT, Schirmer I
test, and assessment of CFS scores.
Rabbit Autoimmune Dacryoadenitis Induction and Evaluation
Autoimmune dacryoadenitis was induced in rabbits using the protocol
described by Wei et al.^[ [178]^53 ^] Briefly, left inferior LGs and
peripheral blood were harvested from each rabbit for isolating pLGECs
and PBLs, respectively. After two days of separate cultivation, the
pLGECs were irradiated (25 Gy) and subsequently cocultured with an
equal number of PBLs (1 × 10^6/well) for a duration of 5 days.
Autoimmune dacryoadenitis was induced by injecting the activated
lymphocytes (2 × 10^6 cells) back into rabbits through ear margin
veins.
To monitor the progression of autoimmune dacryoadenitis following the
injection of activated PBLs, dry eye clinical evaluations were
conducted biweekly, encompassing assessments of tear production, tear
BUT, and CFS scores. Histopathological evaluation of LGs and
conjunctivas was performed by hematoxylin and eosin (H&E) staining at
the end of the experiment. In brief, the rabbits were sacrificed and
the left eyeball and the right inferior LGs were surgically removed.
Then the eyeball and half of the LGs were fixed in 10% formalin,
embedded in paraffin, routinely stained with H&E, and then photographed
using a BX51 microscope (Olympus Corporation, Tokyo, Japan). The number
of focus (aggregates of >50 lymphocytes) per 4 mm^2 in LGs and
conjunctivas was recorded by two experienced technicians in a blinded
manner.
Plasmid Construction, Lentivirus Preparation and Administration
miRNAs originate from pri‐miRNAs in the nucleus, which are then cleaved
into pre‐miRNAs. These pre‐miRNAs are subsequently exported to the
cytoplasm, where they undergo final processing into mature miRNAs.^[
[179]^54 ^] To construct a lentiviral vector (LV‐miR‐31‐5p) that
overexpresses miR‐31‐5p, an 86 bp fragment containing the rabbit
pre‐miR‐31‐5p sequence was amplified and cloned into the PLL3.7 plasmid
(Addgene, Watertown, USA) using Hpa I and Xho I restriction enzymes
(Takara, Kusatsu, Japan). The recombinant plasmid was verified by
sequencing and the lentivirus was produced according to the
manufacturer's instructions. For administration in rabbits, a single
dose of lentiviruses (2 × 10^7 transducing units) was injected
subconjunctivally following the adoptive transfer of activated PBLs
(day 1) or after the onset of disease (2 weeks after transfer). The
following sequences of primers for pre‐miR‐31‐5p amplification were
used: forward primer 5^′‐TGTGGAGAGGAGGCAAGATGCTGGC‐3^′ and reverse
primer 5^′‐ CCGCTCGAGAAAAAAAAGATGGCAATATGTTGGC‐3^′.
THP‐1 Cell Culture and PBMC Derived Macrophages Differentiation
The Human monocytic THP‐1 cell line (Stem Cell Bank, Chinese Academy of
Sciences) was cultured in Gibco RPMI 1640 medium supplemented with 10%
FBS (Gibco, USA), 1% Penicillin/Streptomycin (Gibco, USA), and 45 µm
β‐mercaptoethanol (Gibco, USA) at 37 °C in a 5% CO[2] incubator. To
generate M0 macrophages, 1 × 10^6 ml^−1 THP‐1 cells (third to seventh
passage) were seeded in a 24‐well plate and treated with 160 ng ml^−1
PMA (Sigma–Aldrich, Saint Louis, MO, USA) for 12h. Subsequently, the
PMA‐stimulated THP‐1 cells were further stimulated with 100 ng ml^−1
LPS and 50 ng ml^−1 IFN‐γ (R&D Systems, Minneapolis, MN, USA) for an
additional 48 h to obtain M1 macrophages.
PBMCs were isolated from freshly obtained peripheral blood of SS dry
eye patients using gradient centrifugation techniques. These PBMCs were
then plated onto a 24‐well plate at a density of 3 × 10^6 ml^−1 and
cultured in RPMI 1640 medium supplemented with 10% FBS and 100 ng ml^−1
M‐CSF (PeproTech, USA, cat. 300–25) for 7 days to obtain macrophages.
Following this, the macrophages were polarized into M1 phenotype by
stimulation with 100 ng ml^−1 LPS and 20 ng ml^−1 IFN‐γ.
Transfection
The siRNA, miR‐31‐5p mimics, inhibitors, and their corresponding
negative controls were designed and synthesized by Gene Pharma (Suzhou,
China). PBMCs and THP‐1 derived macrophages were transfected with these
RNAs at a final concentration of 300 and 100 nmol, respectively, using
Lipofectamine 2000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA)
following the manufacturer's instructions. At 24 h post‐transfection,
THP‐1 derived macrophages and PBMCs from SS dry eye patients were
stimulated with LPS and IFN‐γ to induce M1 macrophage polarization, and
PBMCs from model rabbits were collected and cocultured with irradiated
pLGECs for subsequent experiments. All sequence information used in
this study was shown in Table [180]1 .
Table 1.
List of oligonucleotides used in this study.
Name Sence Sequence (5^′–3^′) Antisence Sequence (5^′–3^′)
Ctrl mimics UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT
Ctrl inhibitor CAGUACUUUUGUGUAGUACAA
Ctrl siRNA UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT
miR‐31‐5p‐mimics (rabbit) AGGCAAGAUGCUGGCAUAGCUGU
AGCUAUGCCAGCAUCUUGCCUUU
FTO siRNA‐1 (rabbit) GCUGAAAUAGGUGCUGCUUTT AAGCAGCACCUAUUUCAGCTT
FTO‐siRNA‐2 (rabbit) GCCAGUGUGCAUGGCAGAATT UUCUGCCAUGCACACUGGCTT
miR‐31‐5p‐mimics (human) AGGCAAGAUGCUGGCAUAGCU CUAUGCCAGCAUCUUGCCUUU
P2RX7 siRNA (human) CGAUGGACUUCACAGAUUU AAAUCUGUGAAGUCCAUCG
miR‐31‐5p‐inhibitor (human) AGCUAUGCCAGCAUCUUGCCU
FTO siRNA (human) AAAUAGCCGCUGCUUGUGAGA UCUCACAAGCAGCGGCUAUUU
[181]Open in a new tab
Real‐Time qRT‐PCR and Western Blot
Total RNA from LGs or cells was extracted using EZ‐press RNA
Purification Kit (EZBioscience, USA) according to the manufacturer's
instructions. The first‐strand cDNA was synthesized with the reverse
transcription kit (Thermo Fisher Scientific, USA) and the real‐time
qRT‐PCR was performed using SYBR Green Master Mix (Thermo Fisher
Scientific, USA) with a Roche LightCycler 480 II Analyzer. The relative
gene expression was calculated using the 2 ^[ΔCt(control)–ΔCt(target)]
method. Gene‐specific primers are listed in Tables [182]2 , [183]3 ,
[184]4 .
Table 2.
Sequences of primers in this study for real‐time qRT‐PCR (rabbit).
Gene Forward Primer Sequence (5^′–3^′) Reverse Primer Sequence
(5^′–3^′)
GAPDH GGGTGGTGGACCTCATGGT CGGTGGTTTGAGGGCTCTTA
Arg1 GAAGTAACTCGAACGGTGAACACA TCCCGAGCAACTCCAAAAGA
CD206 CTGATAGATGGAGGGTGAGGTACA CCAGATAGACGCATGCTGACTTC
TGF‐β CAAGGACCTGGGCTGGAA AGGCAGAAGTTGGCGTGGTA
IL‐10 GGCTGAGGCTGCGACAAT TGCCTTGCTCTTGTTTTCACA
NOS2 TCCACCAGGAGATGCTCAACT TGGGTTTTCCACGCCTCTAC
IL‐1β CTCCTGCCAACCCTACAACAA TCCAGAGCCACAACGACTGA
TNF‐α AGCTTCTCGGGCCCTGAGT CCACTTGCGGGTTTGCTACT
P2RX7 GCAGAAAGGGATGGATGGAC CCTCGTGGTGTAGTTGTGGC
METTL3 ATTGAGGTAAAGCGAGGTCTCC GCTTGGAATGGTCAGCATAGGT
METTL14 AACAATCCTGGCAAGACAA CATCTGTGCTACGCTTCA
WTAP CAACAACAGCAGGAGTCT AGTCGCATTACAAGGATGT
FTO TATCTCGCATCCTCATTGG TTGACAAGCAGCACCTATT
pri‐miR‐31 TTAACTTGGATCTGGAGAGG AACACATGGAGGAATGGTAT
[185]Open in a new tab
Table 3.
Sequences of primers in this study for real‐time qRT‐PCR (human).
Gene Forward Primer Sequence (5^′–3^′) Reverse Primer Sequence
(5^′–3^′)
GAPDH CTGGGCTACACTGAGCACC AAGTGGTCGTTGAGGGCAATG
Arg1 GCGCCAAGTCCAGAACCA CGTGGCTGTCCCTTTGAGAA
CD206 CGCTACTAGGCAATGCCAATG GCAATCTGCGTACCACTTGTTT
IL‐10 TGAGAACAGCTGCACCCACTT TCGGAGATCTCGAAGCATGTTA
NOS2 CCCCTTCAATGGCTGGTACA GCGCTGGACGTCACAGAA
IL‐1β TCAGCCAATCTTCATTGCTCAA TGGCGAGCTCAGGTACTTCTG
TNF‐α GCAGGTCTACTTTGGGATCATTG GCGTTTGGGAAGGTTGGA
P2RX7 AGGGCGGAATAATGGGC GGAAACTGTATTTGGGACGG
FTO GTTCACAACCTCGGTTTAGTTC CATCATCATTGTCCACATCGTC
[186]Open in a new tab
Table 4.
Gene‐specific primers used for stem‐loop q‐PCR.
primer sequence(5^′‐3^′)
Name Reverse transcription Forward Reverse
ocu‐miR‐31‐5p GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGCT
CGAGGCAAGATGCTGGCAT AGTGCAGGGTCCGAGGTATT
hsa‐miR‐31‐5p GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGCTAT
GCGAGGCAAGATGCTGGC AGTGCAGGGTCCGAGGTATT
hsa‐miR‐381‐3p GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGAG
CGCGTATACAAGGGCAAGCT AGTGCAGGGTCCGAGGTATT
hsa‐miR‐656‐3p GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGAGGT
GCGCGCGAATATTATACAGTCA AGTGCAGGGTCCGAGGTATT
hsa‐miR‐432‐5p GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCACCC
CGCGTCTTGGAGTAGGTCATT AGTGCAGGGTCCGAGGTATT
hsa‐miR‐34a‐5p GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAACC
CGCGTGGCAGTGTCTTAGCT AGTGCAGGGTCCGAGGTATT
hsa‐miR‐219‐5p GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGAATT
CGCGTGATTGTCCAAACGC AGTGCAGGGTCCGAGGTATT
U6 TTCACGAATTTGCGTGTCATC CGCTTCGGCAGCACATATAC TTCACGAATTTGCGTGTCATC
[187]Open in a new tab
For western blot, cells or frozen tissues were lysed with RIPA lysis
buffer (Solarbio, Beijing, China) containing protease inhibitor PMSF
(Solarbio) and phosphatase protease inhibitor cocktail (Cell Signaling
Technology, Danvers, Massachusetts, USA), and the protein concentration
was quantified using the Bicinchoninic Acid Assay Kit (Solarbio). Equal
amounts of proteins were separated by SDS‐PAGE and transferred onto
polyvinylidene difluoride (PVDF) membrane. After blocking with 5%
non‐fat milk, membranes were incubated with antibodies specific for
Arg1 (1:500, Abcam, UK, cat. ab239731), NOS2 (1:500, R&D Systems,
MAB9502‐SP, for rabbit sample detection; 1:1000, Cell Signaling
Technology, USA, cat. #20609, for human sample detection), phospho‐p38,
phospho‐JNK, phospho‐ERK (1:2000, Cell Signaling Technology, USA, cat.
#9215, #9106, #9255), P2RX7 (1:1000, Cell Signaling Technology, USA,
cat. #13809), DGCR8 (1:1000, Abcam, UK, cat. ab191875), FTO (1:1000,
Cell Signaling Technology, USA, cat. # 31687) or β‐actin (1:2000,
ZSGB‐BIO, China, cat. TA‐09) and detected using Tanon 4800
Multispectral Imaging System (Tanon Science & Technology, China).
Luciferase Reporter Assay
First, the P2RX7 3^′UTR‐ WT fragment or P2RX7 3^′UTR‐ Mut fragment was
amplified and cloned into pMIR‐Report vector (Promega, Madison, USA) as
we described previously.^[ [188]^55 , [189]^56 ^] The sequences of
primers used were as follows: P2RX7 3^′UTR‐WT forward,
5^′‐GGACTAGTCCCGTTTGGAGTCAGGATTTG‐3^′; P2RX7 3^′UTR‐WT reverse, 5^′‐
CCCAAGCTTCAAACAGTCAAGCTAAACAAGAAT‐3^′; P2RX7 3^′UTR‐Mut forward, 5^′‐
GGACTAGTCTGTGTCTGTGTAGACCGTTTTCAACTACTGCCTAAAGT‐3^′; P2RX7 3^′UTR‐Mut
reverse, 5^′‐ CCCAAGCTTTCCTGGGCGGTTACCACGGTCAGAG‐3^′. The
dual‐luciferase reporter plasmids for both the WT and Mut forms of
pri‐miR‐31 were purchased from Hanbio. Specifically, pri‐miR‐31
sequence was cloned into a Pmir‐GLO dual luciferase expression vector.
In the Mut plasmid, the adenosine base in the m6A consensus sequences
was replaced with thymine to abolish the m6A modification. For
luciferase reporter assay, HEK293T cells were cultured in the 96‐well
plates at a density of 2.5 × 10^4 cells/well. 24 h later, HEK293T cells
were co‐transfected with indicated plasmids and oligonucleotides using
Lipofectamine 2000 reagent (Thermo Fisher Scientific). 48 h later, the
samples were collected, and the relative luciferase activity was
detected by the Dual‐Luciferase Reporter Assay System (Promega)
following the manufacturer's instruction.
Immunofluorescence Staining
miR‐31‐5p mimics or control mimics transfected macrophages were fixed
with 4% paraformaldehyde (Solarbio) for 15 min at room temperature.
After blocking with 5% goat serum (Solarbio) for 1 h, cells were
incubated with mouse anti‐Arg1 primary antibody (1:100, Abcam, UK, cat.
ab239731) or mouse anti‐NOS2 primary antibody (1:250, R&D Systems,
MAB9502‐SP) overnight at 4 °C. The next day, the cells were washed and
incubated with goat anti‐mouse Alexa fluor647 (IgG H&L) secondary
antibodies (1:500, Abcam, UK) and DAPI (0.5ug ml^−1, Sigma–Aldrich,
Merck) at room temperature for 1h. Fluorescence images were captured
using a confocal fluorescence microscope (LSM800, Zeiss, Oberkochen,
Germany).
Bioinformatic Analysis
The miRanda database was used to identify the potential targets of
miR‐31‐5p. The m6A sites of pri‐miR‐31 were analyzed using SRAMP
([190]http://www.cuilab. cn/sramp). The DAVID database
([191]https://david.ncifcrf.gov) was employed for KEGG pathway
enrichment analysis. The target genes of miR‐31‐5p were mapped for the
GO category: immune system process using Cytoscape software v3.7.0.
MeRIP‐qPCR
Total RNA was extracted from FTO‐knockdown PBMCs and negative controls
using the Universal RNA Purification Kit (EZBioscience, USA), and the
Magna MeRIP m6A Kit (Millipore, Billerica, MA, USA; cat, 17–10499) was
employed to determine the enrichment of m6A modifications on the
pri‐miR‐31 transcript according to the manufacturer's instructions.
Briefly, the anti‐m6A antibody was conjugated to protein A/G magnetic
beads and mixed with 80 µg of total RNA in IP buffer. Then, the
m6A‐modified RNA was eluted twice with 20 mm N6‐methyladenosine
5ʹ‐monophosphate sodium salt at 4 °C for 1h. Finally, real‐time qRT‐PCR
was performed to quantify the abundance of m6A enrichment on
pri‐miR‐31.
RNA Stability Assay
PBMCs isolated from model rabbits were transfected with FTO siRNA or
its negative controls for 24h. Following this, the cells were exposed
to Actinomycin D (MedChemExpress, Monmouth Junction, NJ, USA) for 0, 3,
6, and 9 h at a final concentration of 5 µg ml^−1. Subsequently, total
RNA was extracted, and the relative level of pri‐miR‐31 in each group
at the indicated time was analyzed by real‐time qRT‐PCR. The
degradation rate (k) and half‐life (t[1/2] ) of pri‐miR‐31 were
estimated by the following equation:
[MATH: lnAtA0
=−kt
:MATH]
(1)
[MATH:
t1/2
=ln2<
/mrow>k :MATH]
(2)
where At and A0 represent the concentration of pri‐miR‐31 at time t and
time 0, and t is the transcription inhibition time.
Co‐IP and RIP Analysis
Co‐IP experiments were performed in HEK293T and THP‐1 cell lines using
Pierce Classic Magnetic IP/Co‐IP Kit (cat. 88 804; Thermo Fisher
Scientific) according to the manufacturer's protocol. Briefly, whole
cell lysates were incubated overnight at 4 °C with gentle shaking,
using either 8 µg of anti‐DGCR8 antibody (Abcam, UK; cat. ab191875) or
anti‐IgG antibody (Proteintech Group, Chicago, IL; cat. 30000‐0‐AP).
Subsequently, the complex was incubated with protein A/G magnetic beads
for 1 h at room temperature. After washing, the immune complex bound to
the beads was dissociated using a low‐pH buffer for subsequent western
blot analysis.
The Magna RIP kit (Millipore, Billerica, MA, USA; cat, 17–700) was
utilized to conduct the RIP experiment. In briefly, HEK293T cells
transfected with either FTO siRNA or negative controls were collected
and lysed in RIP lysis buffer that contained both protease and RNase
inhibitors. Then the lysate was incubated with magnetic beads coupled
with the anti‐DGCR8 antibody at 4 °C overnight. Subsequently, the
coprecipitated RNAs were isolated and subjected to real‐time qRT‐PCR
analysis.
Statistical Analysis
Data from at least three independent experiments were presented as mean
± SD and analyzed using GraphPad Prism 9.0 software. The normality of
the data was evaluated by the Shapiro–Wilk test. Statistical
significance between groups was determined using the Unpaired Student's
t‐test, Mann–Whitney test, or one‐way/two‐way ANOVA, as appropriate.
The indicated p values <0.05 were considered statistically significant.
The correlation analysis was calculated by the Pearson correlation
test. A correlation coefficient (r value) > 0 indicates a positive
correlation between the two variables, whereas an r value < 0 signifies
a negative correlation.
Ethics Approval Statement
The human participants in this study were reviewed and approved by the
Ethics Committee of Tianjin Medical University Eye Hospital. Written
informed consent was obtained from all subjects. The animal study was
reviewed and approved by the Animal Care and Use Committee of the
Tianjin Medical University Eye Hospital.
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
L.Z. and X.L. contributed equally to this work. L.Z., X.J.L., and M.G.
performed the experiments, X.L., J.C.Z., and R.X.L. assisted with data
analysis and provided technical assistance. L.L., B.Y.M., and B.D.
collected samples from clinical patients and analyzed the data. L.Z.
wrote the manuscript. HN and RHW designed the research, supervised the
overall project, wrote the manuscript, and provided financial support.
All authors contributed to the article and approved the submitted
version.
Supporting information
Supporting Information
[192]ADVS-12-2415341-s001.docx^ (659.7KB, docx)
Acknowledgements