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.
Article: The pathophysiologic role of monocytes and macrophages in systemic lupus erythematosus: a reappraisal.[show abstract] [hide abstract]
ABSTRACT: To review current developments, regarding the pathophysiologic role of monocytes and macrophages in systemic lupus erythematosus (SLE). We searched Medline for articles written in the English language using the following terms: monocyte(s) or macrophage(s) and lupus. Although our search spanned the years 1971 to 2008, the majority of the short-listed articles belonged to the period 2000 to 2008. Published literature on phenotypic and functional properties of monocytes/macrophages (Mo/Mphi) in SLE was reviewed. References from identified articles were also selected. Currently available experimental data and their relevance to the pathogenesis of SLE are critically discussed. It has traditionally been held that impaired phagocytosis by monocytes and macrophages in SLE allows for the accumulation of apoptotic debris leading to a sequel of autoimmune phenomena. Recent paradigms derived from animal models of systemic autoimmunity, however, has broadened our understanding regarding the possible pathophysiologic roles of Mo/Mphi in SLE. Data derived from studies in patients with SLE show multiple aberrations in activation status and secretory functions of circulating and tissue-infiltrating Mo/Mphi. Such aberrations may be associated with dysregulation of T-cell function and autoantibody production in SLE. Moreover, emerging evidence suggests that phagocytic capacity and antigen-presenting properties of Mo/Mphi are enhanced in some patients with SLE. While defective phagocytosis represents a distinctive feature of monocyte function in some patients with SLE, aberrant activation of the Mo/Mphi system may be a more appropriate concept to encompass the broad spectrum of Mo/Mphi disorders in SLE. Aberrant function of lupus Mo/Mphi appears to play a dynamic role in the initiation and perpetuation of the systemic autoimmune response and organ damage. Delineation of the altered biology of lupus Mo/Mphi could provide possible future therapeutic targets for patients with SLE.Seminars in arthritis and rheumatism 02/2009; 39(6):491-503. · 4.72 Impact Factor
Article: Aberrant phenotype and function of myeloid dendritic cells in systemic lupus erythematosus.[show abstract] [hide abstract]
ABSTRACT: Systemic lupus erythematosus (SLE) is characterized by a systemic autoimmune response with profound and diverse T cell changes. Dendritic cells (DCs) are important orchestrators of immune responses and have an important role in the regulation of T cell function. The objective of this study was to determine whether myeloid DCs from individuals with SLE display abnormalities in phenotype and promote abnormal T cell function. Monocyte-derived DCs and freshly isolated peripheral blood myeloid DCs from lupus patients displayed an abnormal phenotype characterized by accelerated differentiation, maturation, and secretion of proinflammatory cytokines. These abnormalities were characterized by higher expression of the DC differentiation marker CD1a, the maturation markers CD86, CD80, and HLA-DR, and the proinflammatory cytokine IL-8. In addition, SLE patients displayed selective down-regulation of the maturation marker CD83 and had abnormal responses to maturation stimuli. These abnormalities have functional relevance, as SLE DCs were able to significantly increase proliferation and activation of allogeneic T cells when compared with control DCs. We conclude that myeloid DCs from SLE patients display significant changes in phenotype which promote aberrant T cell function and could contribute to the pathogenesis of SLE and organ damage.The Journal of Immunology 12/2006; 177(9):5878-89. · 5.79 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Over the last decade, the role of dendritic cells (DCs) in the immunopathogenesis of systemic lupus erythematosus (SLE) has become apparent. As unique mediators of both tolerance and immunity, aberrant myeloid and plasmacytoid DC function can promote autoimmune responses via a number of mechanisms and proinflammatory pathways. This review provides an overview of DC function, the potential role of DCs in promoting autoimmune responses in SLE, and how other abnormalities in lupus can lead to an enhanced engagement of DCs in immune responses. How medications used to treat SLE and other autoimmune conditions may exert effects on DCs is also explored.Immunologic Research 02/2007; 37(2):135-45. · 3.03 Impact Factor
TNF-a and TGF-b Counter-Regulate PD-L1
Expression on Monocytes in Systemic
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,
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
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.
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.
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.
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
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
T cells1–3. The mechanism which leads to dysregulated lymphocyte activation in SLE has not been
11 October 2011
6 February 2012
2 March 2012
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SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
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.
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
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
Correlations were analyzed by the Spearman’s rank correlation test.
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
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
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
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
We next tested the direct effect of cytokines on isolated CD141
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.
ofPD-L1onmonocytes inSLEPBMCtreatedfor24 hours orculturedinmediaalone.InductionofPD-L1proteinbycytokinetreatmentswastestedfor
significance by the Wilcoxon signed-rank test. Results were derived from multiple independent experiments.
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
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-
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.
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
cells, and tested for significance by the Wilcoxon signed-rank test.
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
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-
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
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
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
contribute to disease. These studies underline the complex role of
TNF-a in autoimmunity, with different effects at distinct cellular
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
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.
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295