Jyotsna sharma

ID-53421
Date-11-09-2021
 Introduction
There are many IBD associated risk alleles identified by GWAS study .Some of the
IBD risk loci identified leads to loss of innate immune-associated outcomes across
broad range of PRRs thereby leading to reduced ability to clear microbes in
intestine causing chronic inflammation.

The rs6426833A a common noncoding variant and RNF186 A64T rare coding
mutation confers increased risk of ulcerative colitis.

RNF186 is E3 ubiquitine ligase which localise to ER and causes ER stress mediated

apoptosis in hela cells.RNF186 also promotes ER stress leading to imapired insulin
signalling in mouse hepatocytes

Despite understanding of above function, role of RNF186 in ER associated innate

immune outcome in myeloid cells in not known. Hence they wanted to understand
role of RNF186 in NOD2 stimulated ER stress mediated immune outcomes like NF
KB, MAPK signalling leading to cytokine production and bacterial clearance.
Amplification of pathogen immune responses via endoplasmic
reticulum (ER) stress and the unfolded protein response (UPR )
 Intersections of endoplasmic reticulum (ER) stress/unfolded protein
response (UPR) and immune signaling.

ER stress and the UPR activate multiple PRRs (purple) including stimulator of interferon gene
(STING), NLRP3, and other inflammasomes ,and the NOD1/2 receptors. Much of this
interaction occurs at the mitochondrial-ER interface, where released calcium (Ca2+) and ROS
feed into PRR activation.
 Results
Figure 1 :
RNF186 localization to the ER in hu macrophage and its regulation by PRR stimulation

RNF186 partially localizes to ER in

response to NOD2 stimulation by MDP
siRNA mediated KD of RNF186 in hu
monocyte derived MФ to check its
effect on UPR activation.
NOD2 stimulation led to PERK and IRE1 α activation in MDMs which is
significantly reduced in siRNF186.

MDMs KD siRNF186 treated with 100ug/ml MDP and 10micromol CPA.

NOD2 stimulation induced UPR
transcriptional programme
XBP1s,CHOP,ERdj4,Bip expression.
Which was reduced in SiRNF186 .

Transcript expression checked at 6

h
Whether UPR activation downstream to NOD2 stimulation needed for
downstream signalling,NF KB and MAPK pathways and cytokine production
UPR activation and associated NF KB and MAPK pathways as well as cytokine
production downstream to NOD2 stimulation impaired in RNF186 KD MDMs.CPA
treatment restored these outcomes in RNF186 KB MDMs
 Fig 2 NOD2 stimulation induces an RNF186 complex with UPR sensors
MDMs treated with 100ug/ml MDP

MDMs treated
with 100ug/ml
MDP for 30 min

NOD2 stimulation induces RIP2

localization to ER and formation of
complex with UPR sensors. RNF186 after
NOD2 stimulation localizes and forms
complex with RIP2 and UPR sensors.
MDMs KD RNF186 treated with
100ug/ml MDP for 30 min .

RNF186 is needed for optimal

formation of signalling complex
after NOD2 stimulation.
Figure 3. RNF186 localization to the ER is required for NOD2-induced UPR signaling
and downstream outcomes

HeLa cells were transfected with GFP-tagged RNF186-WT or RNF186-ΔDLE and

NOD2 and then treated with 100 μg/mL MDP for 30 minutes.
C)HeLa cells were transfected with myc-RNF186 WT or ΔDLE and NOD2 followed
by treatment with 100 μg/mL MDP for 30 minutes. Myc (RNF186) was
immunoprecipitated, and the recruitment of the indicated proteins was assessed
by immunoblotting .

In addition to MDMs, PRR stimulation regulated RNF186 localization ,and

interaction with UPR in epithelial cells also.
MDMs from rs6426833 AA low RNF186-expressing carriers (n = 6) were transfected
with empty vector (EV) or myc-tagged RNF186 WT or ΔDLE and then treated with 100
μg/mL MDP and checked for UPR activation and downstream signalling
 Figure 4. RNF186 promotes NOD2-induced ubiquitination of the ATF6 complex, and
RNF186 E3 ubiquitin ligase activity is required for the NOD2 induced UPR

MDMs were treated with 100 μg/mL

MDP for the indicated times. ATF6 was
immunoprecipitated, and
ubiquitinated proteins (Ubs) were
assessed by Western blot
B)MDMs were transfected with scrambled or RNF186 siRNA and then treated
with 100 μg/mL MDP for 30 minutes. ATF6 was immunoprecipitated, and Ubs
were assessed by Western blot.

C).In vitro ubiquitination was assessed as per Methods with purified HA-
ubiquitin with (+) or without (-) purified FLAG-ATF6 with/without purified myc-
RNF186 WT or ΔZnF.
In vitro ubiquitination assays.

Purified recombinant myc-RNF186 (750 ng) was incubated with FLAG-ATF6 (500
ng) in a 50 μL reaction mixture containing HA-ubiquitin (5 μg), UBE1 (100 nM),
UBE2N (Ubc13/Uev1a) (500 nM), 2 mM MgCl2, 2 mM ATP, and 1× reaction
buffer (all from Boston Biochem).

Reaction mixtures were incubated at 30°C for 1 hour and terminated by the
addition of 1× loading dye followed by heating to 95°C for 5 minutes. The
samples were probed by Western blot with antibodies to HA tag
MDMs were transfected with empty vector (EV) or myc-tagged RNF186
WT or ΔZnF. Transfected MDMs (n = 6) were treated with 100 μg/mL MDP.
Figure 5. Lysine 152 in ATF6 is critical for RNF186-dependent ubiquitination of
ATF6 and PRR-induced, RNF186-dependent downstream outcomes.

In vitro ubiquitination was assessed with purified HA-ubiquitin.Flag tag ATF6

WT and FLAG-ATF6 mutants purified from HEK and invitro ubiquitination
assay done with RNF186.
Whether the K152A residue in the ATF6 protein is required for PRR-
induced, RNF186-dependent outcomes in MDMs .

MDMs transfected with FLAG-ATF6 WT and FLAG-ATF6 mutants.

Stimulated with 100ug/ml MDP and checked UPR
activation,downstream signalling and cytokine production
ATF6-K152 residue was critical for RNF186-mediated ubiquitination and
was required for NOD2-induced activation of the UPR and downstream
signaling and cytokines.
Figure 6. The rare RNF186 A64T IBD risk variant demonstrates lower levels
of NOD2-induced, UPR-dependent outcomes

(A) In vitro ubiquitination with purified HA-ubiquitin with/without

purified FLAG-ATF6 with/without purified myc-RNF186-A64 or myc-
RNF186-T64
MDMs (rs6426833 AA low RNF186-expressing carriers) were transfected with
empty vector (EV) or myc-tagged RNF186-A64 (WT) or RNF186-T64 (risk variant).
NOD2 stimulation mediated UPR activation and down stream signalling as well as
cytokine production analysed in WT and RNF64T variant
Taken together, the rare disease risk variant RNF186-A64T in the RING domain of
RNF186 demonstrated a reduced ability to ubiquitinate ATF6 and activate the UPR
and, in turn, mediate UPR-dependent downstream signaling and cytokines upon
PRR stimulation.

Complementation of the UPR through CPA treatment in RNF186-T64– transfected

MDMs (Figure 6, D and E) restored downstream signaling and cytokine secretion
Figure 7. RNF186-dependent UPR signaling is required for NOD2-induced
bacterial clearance and antimicrobial pathways

NOD2 stimulation for 48h increased bacterial uptake and clearance in WT while
reduced in RNF deficient hu macrophage.
Complementation of UPR activation by CPA restored bacterial uptake and
clearance.
Bacterial clearance in hu MDMs checked by ROS,and autophgy which leads to
bacterial clearance after PRR stimulation. ROS production and autophagy was
altered in RNF186 deficient MDMs which is restored after CPA treatment.
Hu MDMs transfected with Flag-ATF6 WT, EV and FLAG-ATF6 lysine mutants to
check role of ATF6 K152 residue (ubiquitination target) in bacterial clearance.
MDMs (n = 6) (rs6426833 AA low RNF186-expressing carriers) were transfected
with EV or myc-tagged RNF186-A64 (WT) or RNF186-T64 (risk variant) and then
treated with 100 μg/mL MDP for 48 hours ± 10 μM CPA
Compared with RNF186-WT–transfected MDMs, NOD2-mediated induction of
bacterial uptake intracellular bacterial clearance and antimicrobial pathways)
were impaired in RNF186-T64– transfected MDMs but could be restored with
complementation of UPR signaling through CPA treatment
Figure 8. IBD risk rs6426833 AA disease risk carriers demonstrate reduced PRR-
induced UPR signaling

MDMs isolated from rs6426833 disease risk carrier were checked for UPR
activation after NOD2 stimulation.
UPR activation is less in risk variant MDMs
 Figure 9. Complementation of the UPR in MDMs from IBD risk rs6426833 AA carriers
increases NOD2-induced, RNF186-dependent outcomes to levels observed in GG
carrier MDMs

MDMs from rs6426833 GG, GA and

AA carriers were left untreated or
treated with 100μg/ml MDP.
UPR complementation by 10 micro molar CPA restored UPR activation in
AA risk variant to GG .
Supplementary

RNF186 expression in the intestinal myeloid cells from AA risk carrier

Myeloid cells from rs6426833 AA risk carrier expressing low RNF186 expression
were investigated for basal and salmonella induced UPR activation.

UPR activation is less in risk variant myeloid cells.

Gene Expression Omnibus
GEO is a public functional genomics data repository supporting MIAME-compliant data
submissions. Array- and sequence-based data are accepted. Tools are provided to help
users query and download experiments and curated gene expression profiles.
Figure S17. RNF186 promotes the UPR, cytokines, bacterial uptake and bacterial
clearance in colonic lamina propria macrophages

Mice (n=5) were administered scrambled or RNF186 siRNA i.p.

They checked salmonella induced UPR activation nd downstream outcomes in the
lamina propria macrophage as they respond poorly to PRR induction.

RNF186 ATF6 and UPR transcripts expression increased.

C57BL/6N mice were maintained in a specific pathogen–free facility.

15 μg/mouse ON-TARGETplus RNF186 (sequence per J-050361-12),

ATF6 (sequence as per J-044894-08)
siRNA, or control (sequence per D-001810-02) with UU overhang/in vivo design
(Dharmacon) was i.p. administered in Atelo- Gene Systemic Use (Cosmo Bio USA), and
then studies were conducted 48 hours later.

For in vivo studies extending beyond 4 days, a second dose of in vivo siRNA was i.p.
administered on day 4 of the disease model. Colonic lamina propria cells were isolated
as previously described (56) and CD11b+ cells were then purified (Miltenyi Biotec).
Intestinal lamina propria cell isolation Wu et al 2014
The proximal SI was cut longitudinally and then into ~1 mm pieces. SI pieces were washed
thoroughly (ice-cold PBS, 5% FCS) and then digested (PBS, 5 mMEDTA, 5% FCS) at 37°C in a
rotating incubator to remove epithelial cells.

The supernatants containing epithelial cells and intraepithelial lymphocytes were

discarded, and intestineswere washed twice in ice-cold PBS to remove residual EDTA. The
remaining tissue wasthen incubated for 1h at 37°C in a rotating incubator in collagenase
buffer consisting ofRPMI 1640, 10% FCS, 200 U/ml collagenase VIII (Sigma-Aldrich), 2 mM
glutamine, 0.1mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 50
μM 2-ME, 40μ;g/ml gentamicin, and 50 U/ml penicillin-50 μg/ml streptomycin.

The cells were then filtered through a 40 μM filter (BD Biosciences) and washed twice in
RPMI media. For intestinal stromal cells, intestinal lamina propria cells were plated
overnight. The next day, nonadherent cells were removed and the adherent fibroblast-like
cells were cultured for 7 days.
The intestinal stromal cells expressed α-SMA, vimentin, P4HA1, desmin and Myh10
consistent with a myofibroblast phenotype. In contrast, they were negative for
hematopoietic markers (e.g. CD45, CD11b, CD11c). They did not contain T cell or B cell
contamination as assessed by CD3 and CD19, respectively.
Figure 10. RNF186 promotes bacterially induced UPR pathways and cytokines and
bacterial clearance in mouse intestinal macrophages during intestinal injury.

(A-D) RNF exp ,UPR and cytokine checked in intestinal macrophage isolated
on 6 day of DSS which was high it was Cocultured with salmonella and
checked PRR stimulated immune outcomes.
 To access mechanism by which RNF186 regulated macrophage
outcomes,RNF186 KD done in mice and checked UPR activation after
coculture with salmonella.

E-SiRNF186 mice given DSS and colonic macrophage isolated on 4th day and
checked for UPR, cytokine with or without salmonella coculture.
Similar result was found in intestinal tissue of the mice.
 Figure 11. RNF186 promotes clearance of resident intestinal microbiota during
DSS-induced injury

To understand role of RNF186 regulated disease outcomes,RNF186 KD mice were

given DSS and disease progression as well as UPR activation and downstream
outcomes were evaluated in colonic tissue.
Colonic tissue was homogenized in
hexadecyltrimethylammonium
bromide (MilliporeSigma) buffer. MPO
was assayed using o-dianisidine
dihydrochloride (MilliporeSigma)
and H2O2 (change in optical density at
450 nm).
Frozen fecal sample reconstituted in PBS/0.1% tween20 lipocalin was assessed
by ELISA
RNF186 deficient mice after DSS administration develops more severe disease
despite decreased inflammatory mediators .
Both peripheral and intestinal macrophage has impaired bacterial uptake and
clearance ,similarly bacterial load is high in MLN and spleen.
Therefore, during intestinal injury, RNF186 promoted the UPR and bacterial
clearance mechanisms; optimal bacterial clearance is critical for limiting intestinal
injury.
 Figure 12. RNF186 is required for optimal clearance of S. Typhimurium upon oral
infection in mice

S. Typhimurium in vivo infection. Mice were

orally inoculated with streptomycin (20
mg/mouse) (VWR International) and 24 hours
later orally inoculated with 1 × 106 CFU of S.
Typhimurium (strain SL1344). Mice were
euthanized and organs collected in PBS.
RNF186 is required for regulating oral challenge with invasive bacteria in vivo
similar to DSS mice.
Figure 13. ATF6 promotes clearance of resident intestinal microbiota during
DSS-induced injury
Figure 14. ATF6 is required for optimal clearance of S. Typhimurium upon oral
infection in mice
Model of RNF186-mediated regulation of PRR-initiated UPR-dependent outcomes.
 Conclusion

Loss-of-function, immune-mediated disease risk variants in

RNF186 resulted in a reduced PRR-induced UPR in human
macrophages, which then resulted in reduction of multiple key
downstream outcomes, including microbial clearance
Isolation of lamina properia cells from mucosal biopsies of the human colon
Bowcutt et al 2015 J Immunol Methods.
1. Prepare collagenase-DNase digestion mix with 100 units/mL of collagenase and 150
μg/mL DNase in 10 mL complete RPMI per sample.
2.Transfer 10 mL of the collagenase-DNase digestion mix to a 50 mL conical tube.
3. Transfer biopsy tissue to the collagenase-DNAse digestion mix tube and shake
vigorously.
4. Incubate at 37°C for one hour.
5. During the one hour incubation, prepare the 100% Percoll solution (9 parts Percoll, 1
part 10X PBS). Dilute the 100% Percoll solution to a 40% solution and an 80% solution with
complete media. Note: By making the Percoll solution at this time, the solutions will then
be at RT by the time the Percoll step arrives.
6. Take out the 50 mL conical tube from the incubator and shake vigorously.
7. Place a 100 μm cell strainer over a new 50 mL conical tube.
8. Pour the collagenase-DNase digestion mix through the 100 μm cell strainer.
9. Mash any undigested tissue through the filter using the bottom of a 1 mL syringe.
.
10. Add 10 mL 1X PBS through the 100 μm cell strainer to wash out any cells stuck on the filter.
11. Spin tube for 10 minutes at 600 RCF at RT.
12. Aspirate supernatant carefully down to the pellet level.
13. Resuspend pellet in 5 mL of the 40% Percoll solution and pipet up and down to mix. Transfer
mix into a new 15 mL conical tube.
14. Very slowly, underlay 5 mL of the 80% Percoll solution below the 40% Percoll solution. Note: Do
not pipet out all of the solution from the pipet. Save a small amount of liquid inside the pipet, as
pipetting out the complete 5 mL solution will cause bubbles, which will interfere with the
interphase.
15. Spin tubes for 20 minutes at 600 RCF at RT with the brake off.
16. Aspirate out 3 mL of the top layer. With a 3 mL transfer pipet, extract the interphase and
transfer to a new 15 mL conical tube with 10 mL 1X PBS.
17. Spin the cells for 10 minutes at 600 RCF at 4°C.
18. Aspirate supernatant carefully down to the pellet level.
19. Resuspend pellet in 200 μL of complete RPMI