TNF-α and TGF-β Counter-Regulate PD-L1 Expression on Monocytes in Systemic Lupus Erythematosus.

Abstract

Monocytes in patients with systemic lupus erythematosus (SLE) are hyperstimulatory for T lymphocytes. We previously found that the normal program for expression of a negative costimulatory molecule programmed death ligand-1 (PD-L1) is defective in SLE patients with active disease. Here, we investigated the mechanism for PD-L1 dysregulation on lupus monocytes. We found that PD-L1 expression on cultured SLE monocytes correlated with TNF-α expression. Exogenous TNF-α restored PD-L1 expression on lupus monocytes. Conversely, TGF-β inversely correlated with PD-L1 in SLE and suppressed expression of PD-L1 on healthy monocytes. Therefore, PD-L1 expression in monocytes is regulated by opposing actions of TNF-α and TGF-β. As PD-L1 functions to fine tune lymphocyte activation, dysregulation of cytokines resulting in reduced expression could lead to loss of peripheral T cell tolerance.

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TNF-a and TGF-b Counter-Regulate PD-L1
Expression on Monocytes in Systemic
Lupus Erythematosus
Jing-Ni Ou1, Alice E. Wiedeman2& Anne M. Stevens1,3
1Seattle Children’s Research Institute,2Department of Immunology,3Department of Pediatrics, University of Washington, Seattle,
WA.
Monocytes in patients with systemic lupus erythematosus (SLE) are hyperstimulatory for T lymphocytes.
We previously found that the normal program for expression of a negative costimulatory molecule
programmed death ligand-1 (PD-L1) is defective in SLE patients with active disease. Here, we investigated
the mechanism forPD-L1dysregulation on lupusmonocytes.Wefound that PD-L1expressiononcultured
SLE monocytes correlated with TNF-a expression. Exogenous TNF-a restored PD-L1 expression on lupus
monocytes.Conversely,TGF-binverselycorrelatedwithPD-L1inSLEandsuppressedexpressionofPD-L1
on healthy monocytes. Therefore, PD-L1 expression in monocytes is regulated by opposing actions of
TNF-a and TGF-b. As PD-L1 functions to fine tune lymphocyte activation, dysregulation of cytokines
resulting in reduced expression could lead to loss of peripheral T cell tolerance.
M
elucidated, but most likely depends on differential levels of positive and negative costimulatory molecules
expressed on antigen presenting cells (APCs)4,5. PD-L1 (B7-H1 or CD274) functions as a critical regulatory
protein to maintain T cell self-tolerance6, and could play a major role in determining monocyte activity in SLE.
PD-L1, expressed on hematopoietic and parenchymal cells, binds to programmed death-1 (PD-1) to inhibit T
cell receptor-mediated proliferation and induce T cell anergy6. Engagement of the PD-1 pathway is essential in
suppressing autoimmunity, as originally demonstrated in mice lacking PD-1 expression that developed a disease
similar to SLE7. Blockade of PD-1 has been shown to affect disease activity in a lupus mouse model8,9. PD-L1
deficiency does not by itself lead to SLE, but exacerbates systemic autoimmunity in lupus-prone mice6,10. The
mechanistic link between PD-1 or PD-L1 expression and the pathogenesis of human SLE is not well understood.
Polymorphisms in the PD-1 gene are associated with SLE susceptibility in some populations of adults and
children11–15. However, PD-L1 gene polymorphisms were not associated with SLE16,17.
PD-L1expressionisloworabsentonmonocytesexvivo18,19.WhenPBMCareculturedovernightintheabsence
ofstimulation,PD-L1expressionisup-regulatedonhealthymonocytesandmyeloidDCs.Incontrast,wefounda
diminutiveinductionofPD-L1onAPCsfrompatientswithactiveSLE.Cellsfrompatientsinremission regained
PD-L1expression,suggestingareversiblemechanismforregulationofPD-L119.Theexperimentalculturesystem
encompasses two abnormal processes that occur in SLE: the APC response to apoptotic cells and lymphocyte
response to autologous antigens. The failure of APCs to up-regulate PD-L1 may occur in vivo and contribute to
the breakdown of self-tolerance in SLE patients.
Cytokines abnormally expressed in SLE have been implicated in the regulation of PD-L118,20–26. Specifically,
TNF-a has been associated with increased PD-L1 expression on synovial and peripheral macrophages derived
from patients with rheumatoid arthritis24, whereas TGF-b inhibits PD-L1 expression on renal tubular cells21.
Therefore,thereducedexpressionofPD-L1onlupusmonocytesmaybetheresultofalteredcytokineproduction
ex vivo.
In this report we investigated the role of cytokines in regulating expression of PD-L1 in SLE. During active
disease, the overexpression of TGF-b correlated with decreased levels of PD-L1 surface protein on lupus mono-
cytes. Deficient PD-L1 expression could be restored in vitro by TNF-a, a factor required to induce PD-L1
expressiononhealthymonocytes.PD-L1wasnotinducedonisolatedSLEmonocytesormyeloidDCs,indicating
that PD-L1 expression was not inhibited by SLE lymphocytes. TNF-a induced expression of PD-L1 mRNA in
onocytes and dendritic cells from SLE patients display aberrant phenotypes, namely abnormal cytokine
production,defectivephagocytosisofapoptoticcells,andhyperstimulatoryactivityforallogeneicCD41
T cells1–3. The mechanism which leads to dysregulated lymphocyte activation in SLE has not been
SUBJECT AREAS:
CYTOKINES
AUTOIMMUNITY
MEDICAL RESEARCH
ANTIGEN PRESENTATION
Received
11 October 2011
Accepted
6 February 2012
Published
2 March 2012
Correspondence and
requests for materials
should be addressed to
A.M.S. (amsteve@u.
washington.edu)
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lupus cells, while TGF-b suppressed induction of the mRNA in
healthy control cells, suggesting opposing transcriptional regulation
by these two cytokines. These findings demonstrate that abnormal
cytokine production may lead to poor PD-L1 expression on mono-
cytes, contributing to the hyperstimulatory phenotype found in SLE.
Results
Aberrant expression of TNF-a and TGF-b correlates with PD-L1
levels on SLE monocytes. To investigate the mechanism for dys-
regulation of PD-L1 expression in SLE, PBMC from patients and
healthy controls were cultured for 24 hours without stimulation.
PD-L1 surface protein on monocytes was assayed by flow cyto-
metry (Supplemental Figure 1). We observed no PD-L1 expression
at the initiation of culture on either monocytes or myeloid DC from
SLEpatientsorcontrols.PD-L1proteinwasfirstdetectedby8 hours,
increased at 24 hours, and remained high until day five (Sup-
plemental Figure 2). Deficient PD-L1 expression on cultured SLE
monocytes during active disease was not related to specific me-
dications, and is unlikely to be a gene defect, as most patients were
able to restore PD-L1 protein during remission19. Hence we
postulated that dysregulated cytokine production in SLE may lead
to decreased expression of PD-L1 on SLE APCs. Supernatants from
control and SLE PBMC cultures were assayed for expression of
cytokines known to associate with the severity of SLE, including
IFN-c, IFN-a, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-a, and TGF-b.
The most significant differences between SLE patients and healthy
controls were found in the expression of TNF-a and TGF-b.
Interestingly, TNF-a, reported to be increased in SLE patient
serum25, was expressed at 2.7-fold higher levels by PBMC from
healthy controls compared to SLE patients in our experiments
(mean, 129.4 pg ml21vs. 44.2 pg ml21, Figure 1A). Though
production of TNF-a was restored in some SLE patients during
remission, the mean was still significantly reduced compared to
controls (81.7 pg ml21). In contrast, TGF-b was induced to higher
levelsinbothactiveSLE(2393 pgml21)andremission(3914 pgml21)
compared to controls (1684 pg ml21, Figure 1B). Among other
cytokines tested, IFN-c, IL-4, IL-10, IFN-a and IL-2 were
undetectable in supernatants from most subjects, an expected result
considering that T lymphocytes were unstimulated. Moreover,
expression of neither IL-6 nor IL-8 was significantly different in
controls compared to SLE patients (data not shown).
We then tested for correlations between TNF-a, TGF-b and PD-L1
expression. The expression of PD-L1 significantly correlated with
TNF-a during disease remission (Figure 1C), suggesting that express-
ion of TNF-a may be required to restore PD-L1 expression on lupus
monocytes. In contrast, high expression of TGF-b was significantly
correlated with low PD-L1 levels during active disease (Figure 1D). In
PBMC from healthy controls, there was no correlation between
Figure 1 | PD-L1 levels correlated with TNF-a in remission and with TGF-b in patients with active disease. (A) and (B) Levels of TNF-a and TGF-b
detected in supernatants from PBMC cultured overnight without stimulation. Horizontal lines represent mean values. Cytokine levels between healthy
controls and SLE patient groups were compared using the Wilcoxon-Mann-Whitney test. Significance was assigned where p,0.05; N.S., not significant.
(C) PD-L1 protein levels on monocytes in the same culture was assayed by flow cytometry; TNF-a positively correlated with PD-L1 expression on
monocytesinSLEpatientsinremission(dashedline).(D)TGF-bnegativelycorrelatedwithPD-L1proteininSLEpatientswithactivedisease(solidline).
Correlations were analyzed by the Spearman’s rank correlation test.
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cytokines and PD-L1 expression (data not shown), as most cells
expressed PD-L1. Thus, correlation analyses suggested that TGF-b
suppresses PD-L1 expression during active SLE, while TNF-a induces
PD-L1 expression during remission and in healthy controls.
PD-L1 surface protein is induced by TNF-a and down-regulated
by TGF-b. We have previously shown that cells from patients in
remission restored PD-L1 expression, suggesting a reversible me-
chanism for regulation of PD-L1 during the disease course of
SLE19. Therefore, we tested the hypothesis that expression of PD-
L1 on SLE cells can be restored by TNF-a. To determine whether
TNF-a is normally required for induction of PD-L1, we examined
the change of PD-L1 expression on healthy cells treated with a TNF-
a neutralizing antibody. Blocking TNF-a resulted in significantly
decreased expression of PD-L1 surface protein on monocytes from
healthy controls (Figure 2A). Likewise, treatment with TGF-b
significantly suppressed PD-L1 surface protein on healthy mono-
cytes. Addition of neither recombinant TNF-a nor TGF-b block-
ing antibody affected PD-L1 expression on healthy cells.
We found that TNF-a significantly induced PD-L1 protein ex-
pression on SLE remission monocytes (Figure 2B). However, induc-
tion of PD-L1 by TNF-a was not significant in patients with active
disease. Of note, TNF-a treatment resulted in little induction of PD-
L1 on myeloid DCs (data not shown), indicating that expression of
PD-L1 is differentially regulated on monocytes and myeloid DCs.
Blocking TGF-b with a mAb was not sufficient to restore PD-L1 on
SLEmonocytes(Figure2B).ThedatasuggeststhatrestorationofPD-
L1 expression on SLE monocytes may require TNF-a and other
positive regulatory factors in addition to blocking inhibitory TGF-
b. TNF-a is normally required for PD-L1 induction, whereas TGF-b
may oppose the induction in active SLE. As SLE monocytes express
low levels of PD-L1, inhibition of TNF-a by mAb or addition of
recombinant TGF-b was not able to reduce the protein expression
further (Figure 2B).
PD-L1 expression is induced by monocyte-derived factors.
Cytokines that dysregulate expression of PD-L1 can be derived
from either aberrant SLE myeloid or lymphoid cells2,27. To identify
other cell populations affecting PD-L1 expression on monocytes, we
compared the expression of PD-L1 on isolated CD141cells cultured
alonewiththatonmonocytesintotalPBMCcultures.Noexpression
ofPD-L1wasdetectedonisolatedmonocytespriortocultureattime
0 (Supplemental Figure 2). PD-L1 was induced on isolated healthy
CD141monocytes cultured overnight without stimulation, indi-
cating that monocyte-intrinsic factors are sufficient for induction
of surface PD-L1 (Figure 3A and B). However, the depletion of
CD142cells decreased expression of PD-L1 on myeloid DCs and
monocytes from healthy donors that had higher PD-L1 expression
(Figure 3B), suggesting that induction of PD-L1 could be enhanced
by lymphocytes. Both regulatory T cells and NKT cells have been
previously reported to induce PD-L1 expression, and may play roles
in defective PD-L1 induction in SLE28,29. There was no change in
the level of PD-L1 expression on either isolated SLE monocytes or
myeloid DCs as compared to total PBMCs (Figure 3C and D),
demonstrating that PD-L1 expression is not suppressed by SLE
lymphocytes. Rather, SLE APCs lack intrinsic factors required for
PD-L1 induction.
We next tested the direct effect of cytokines on isolated CD141
monocytesandmyeloidDCs.Theinduction ofPD-L1expressionon
monocytes after treatment of total PBMCs with TNF-a that we
showed in Figure 2 was similarly demonstrated in isolated SLE
monocytes, where expression of PD-L1 was significantly induced
in four out of five patients (Figure 3E). Blocking TGF-b did not
changed expression of PD-L1 protein on isolated SLE monocytes
(Figure 3F), whereas blocking TNF-a significantly decreased PD-
L1 expression on isolated healthy monocytes (Figure 3G).
Moreover, TGF-b treatment of isolated monocytes resulted in sig-
nificant repression of PD-L1 in healthy donors (Figure 3H). These
findings support the hypothesis that intrinsic defects in SLE mono-
cyte cytokine expression result in poor PD-L1 induction.
Expression of PD-L1 correlated with TNF-a in monocyte culture
supernatants. In order to determine whether or not PD-L1 is
induced by monocyte-intrinsic factors, we assayed levels of TNF-a
and TGF-b in purified monocyte culture supernatants. Increased
TNF-a was detected in supernatants from healthy monocytes
(mean, 157 pg/ml) compared to those from patients with active
disease (Figure 4A, p 5 0.027). TNF-a expression was higher in
SLE remission (109 pg/ml) compared to active disease (37 pg/ml),
but the difference was not statistically significant (p 5 0.067).
Expression of PD-L1 on isolated monocytes from remission pa-
tients also significantly correlated with TNF-a (Figure 4B). These
Figure 2 | PD-L1 surface protein on SLE APCs is differentially regulated by TNF-a and TGF-b. (A) Control PBMC were treated for 24 hours.
ExpressionofPD-L1onCD14highCD11c1monocyteswasassayedbyflowcytometry,andisexpressedasfoldinductionoveruntreatedcells.(B)Induction
ofPD-L1onmonocytes inSLEPBMCtreatedfor24 hours orculturedinmediaalone.InductionofPD-L1proteinbycytokinetreatmentswastestedfor
significance by the Wilcoxon signed-rank test. Results were derived from multiple independent experiments.
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data provide evidence that monocytes produce TNF-a required to
induce PD-L1. Conversely, substantial expression of TGF-b was
detected in monocyte supernatants (Figure 4C). Isolated mono-
cytes from SLE patients produce higher level of TGF-b compared
to healthy controls (active disease 5 3527 pg/ml; remission 5
2189 pg/ml; healthy controls 5 1446 pg/ml), but the difference was
insignificant. No correlation was found between PD-L1 protein level
andTGF-b(Figure 4D).Thesedata suggestthatcelltypesother than
monocytes can also produce TGF-b to counter-regulate level of PD-
L1 on monocytes.
Opposing effects of TNF-a and TGF-b on PD-L1 mRNA
expression. PD-L1 expression has been reported to be regulated at
both transcriptional and translational levels in different model
systems30–32. PD-L1 mRNA expression was assayed in cultured
PBMC from SLE patients and healthy donors. We found that PD-
L1mRNAwasexpressed incellsfromallcontrolsubjects(median6
S.E.M.50.2260.16,Figure5A),andwasonaverageten-foldhigher
thaninSLEpatients(0.01860.09foractivedisease,0.02460.08for
remission), although the differences were not significant. Parallel
flow cytometry assays demonstrated that some patients with active
disease and high PD-L1 mRNA expression had little to no PD-L1
protein on the surface of monocytes, myeloid DCs, or lymphocytes.
Moreover, there was no correlation between PD-L1 mRNA and pro-
tein expression in any subject group (data not shown), indicating that
restoration of PD-L1 surface protein was not entirely dependent upon
mRNA expression, but may require additional translational signals, as
demonstrated in other biological models30,31.
The signaling pathways mediated by TNF-a and TGF-b are well
documented33,34. However, it is unclear how these distinct pathways
converge to counter-regulate PD-L1 expression. We next demon-
strated that PD-L1 mRNA is differentially regulated by TGF-b and
TNF-a. PD-L1 mRNA was significantly induced in SLE cells treated
with TNF-a (Figure 5B), whereas blocking TNF-a prevented induc-
tion of PD-L1 mRNA in three of six healthy donors (Figure 5C).
Similarly, TGF-b significantly inhibited expression of PD-L1 mRNA
in control PBMC (Figure 5D). Overall, these data support a model
in which PD-L1 gene expression is tightly regulated by the action
of two opposing cytokines, TNF-a and TGF-b.
Discussion
Although functional defects in monocytes and DCs are well known
in SLE, the underlying molecular mechanisms are not fully under-
stood. The present study addresses the mechanism for regulating
Figure 3 | ExpressionofPD-L1onisolatedCD141cellsisnotentirelydependentonlymphocytes. (A)and(C)Representativehistogramsdemonstrate
PD-L1 induction on total PBMC or isolated CD141cells from a control subject and an active SLE patient. Protein expression was assayed by flow
cytometry after culturing cells for 24 hours without stimulation. (B) and (D) PD-L1 expression on monocytes gated from total PBMC compared to
isolated monocytes and myeloid DCs in eight healthy controls and eight SLE patients. (E) and (F) Fold induction of PD-L1 MFI on isolated CD141SLE
monocytes treated withTNF-a or withanti-TGF-bmAb. (G)and(H) Foldinduction ofPD-L1 MFI onisolated CD141monocytes from healthy donors
treatedwithanti-TNF-amAborwithrecombinantTGF-b.InductionwasquantifiedbydividingtheMFIofcytokine-treatedsamplesbythatofuntreated
cells, and tested for significance by the Wilcoxon signed-rank test.
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PD-L1,acriticalimmunoregulatoryproteinthatdeterminesthepro-
versus anti-inflammatory activity of APCs. PD-L1 expression is
regulated by a balance of cytokines with known importance in
SLE21,35. In other inflammatory diseases including rheumatoid arth-
ritis, infection, and malignancies, PD-L1 expression has been
reported to be up-regulated by inflammatory cytokines36,37. In con-
trast, we previously demonstrated deficient PD-L1 surface express-
ion on monocytes and myeloid DCs with active SLE19. We have
shown here that the defect on SLE monocytes can be attributed
partially to overexpression of TGF-b, which inhibits PD-L1 express-
ion,andpartlytodecreasedproductionofTNF-arequiredforinduc-
tion of PD-L1 mRNA and surface protein expression.
In the current study, some patients with active SLE produced less
TNF-a, a trait reported to correlate with SLE-associated HLA-DR
alleles38–40. Whether children with SLE expressing less TNF-a in
correlation with decreased PD-L1 expression on monocytes carry
the polymorphisms leading to low TNF-a expression is not known.
Expression of PD-L1 may also be influenced by the availability of
receptorsandinduciblesignalingmoleculesandtranscriptionfactors
in the TNF receptor signaling pathway. TNF receptor expression is
reportedly normal in SLE, though decreased expression of TNF-a
signaling proteins in SLE patients has been reported41,42, and could
accountforthedecreasedPD-L1inductionobservedinunresponsive
SLE monocytes from some patients, although most patient mono-
cytes respond well to TNF-a. Anti-TNF-a therapy is effective in
treatingrheumatoid arthritis, auto-inflammatory diseases, and some
SLE patients. However, TNF-a blockers have been shown to trigger
lupus in some patients, and induce lupus-related autoantibody pro-
duction. The onset of autoimmunity triggered by TNF-a blockers
may be in part through activation of plasmacytoid dendritic cells,
which lead to enhanced production of IFN-a43. Our data suggests
thatlackofnormalTNF-ainductionofPD-L1expression couldalso
contribute to disease. These studies underline the complex role of
TNF-a in autoimmunity, with different effects at distinct cellular
targets.
The role of TGF-b in the pathogenesis of SLE remains unclear.
Previous studies have shown decreased TGF-b production by SLE
lymphocytes44, in contrast to our increase in TGF-b production by
SLEPBMCwithconcurrentsuppressionofPD-L1.However,TGF-b
therapies in lupus-prone mice have yielded mixed results45, perhaps
because TGF-b contributes to the development of both proinflam-
matory Th17 cells and regulatory T cells (Treg)46. TGF-b is essential
for differentiation of murine Treg, but its role in human Treg is less
well defined. PD-L1 has been shown to induce Treg in mice and
humans28,47, suggesting defective induction of PD-L1 may lead to
reduced number of functional Treg observed in SLE patients48.
Figure 4 | MonocytesproduceTNF-atoinduceexpressionofPD-L1. (A)TNF-awasassayedinsupernatantsfromisolatedCD141monocytescultured
overnight without stimulation. Horizontal lines represent mean values. (B) PD-L1 protein levels on monocytes in the same culture was assayed by flow
cytometry; TNF-a positively correlated with PD-L1 expression on monocytes in SLE patients in remission. (C) TGF-b protein levels in monocyte
supernatants. (D) Correlation betweenTGF-bandPD-L1 protein levelson monocytes. Cytokine levelsbetweenhealthy controls and SLEpatient groups
were compared using the Wilcoxon-Mann-Whitney test. Correlations were determined by the Spearman’s rank correlation test.
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