Discussion
Immune dysfunction is an important facet of poor bacterial clearance and
secondary infection in the critically ill surgical patient. The failure
to orchestrate an effective immune response is a result of both impaired
innate and adaptive systems [15]. Interferon-γ has been frequently
studied as an adjuvant in surgical patients to correct monocyte
impairment [14]. However, the effect of interferon-γ on the adaptive
system, and in particular, upon the PD-1-PD-L1 pathway involved in
lymphocyte anergy, is not fully understood.
Our data demonstrate agreement that an endotoxin challenge leads to an
increase in both the pro- and anti-inflammatory cytokine response and
leads to activation of monocytes with increased HLA-DR expression.
Interferon-γ treatment of LPS-exposed leukocytes promoted an “M1”
pro-inflammatory phenotype, by causing further increases in TNF-α with a
reciprocal decrease in the anti-inflammatory cytokine IL-10. Monocyte
function appeared to improve with an augmented HLA-DR expression, a
representative marker of antigen presentation. These findings are in
keeping with many previous studies on the effects of interferon-γ [9,
10, 36-39].
The presence of endotoxin stimulated the increase of monocyte PD-L1
expression. Although interferon-γ improved monocyte function, it
synergistically increased PD-L1 levels beyond that of the levels
associated with LPS alone. This is consistent with previous studies
[36, 40]. This increase in PD-L1 expression despite apparent
improvements in monocyte function conflicts with the concept of PD-L1
acting as a marker of monocyte dysfunction. Despite this increase in
monocyte PD-L1, a marker previously associated with monocyte impairment
and worse clinical outcomes, we found that T-cell function was preserved
with interferon-γ treatment. This finding is in keeping with clinical
observations of T-cell reactivity in patients undergoing interferon-γ
treatment in the context of both trauma and elective gastrointestinal
surgery [41].
In original trauma studies, there appeared to be a “rebound” of
decreased monocyte function after the cessation of adjuvant
interferon-γ. We hypothesized that this may be due an unrecognized PD-L1
induction, resulting in T-cell dysfunction and decreased endogenous
interferon-γ as a mechanism for this treatment failure. However, our
findings that elevated PD-L1 does not appear to reflect impaired
lymphocyte function, with preserved endogenous interferon-γ responses,
likely dispels this concern.
These findings are interesting mechanistically. One explanation may be
that interferon-γ leads to increased TNF-α, and that the upregulated
PD-L1 may in fact be a result of the TNF-α levels, rather than a direct
effect of interferon-γ. A previous study has shown that culture with
recombinant TNF-α increases monocyte PD-L1, which in turn can be
prevented with TNF–α inhibition [42]. More recently, a study of
various cell types found that both TNF-α and IFN-γ synergistically
increased PD-L1 expression, but then through induction of a miR-155
negative feedback loop, PD-L1 levels were then suppressed [43].
The cellular and clinical effects of interferon-γ in experimental and
clinical scenarios relating to the surgical patient are summarized in
Figure 5. Although this list is not exhaustive, both established as well
as recent findings of the effects of interferon-γ are highlighted. This
includes effects on both the adaptive as well as the innate immune
system. Although interferon-γ therapy upregulates PD-L1 expression on
circulating and fixed leukocytes, there is not a deleterious effect on
lymphocyte function. Given the increasing body of evidence toward T-cell
anergy and exhaustion in patients suffering from trauma and sepsis, the
addition of interferon-γ does not appear to exacerbate this important
defect. A recent study of interferon-γ therapy given to septic patients
with low monocyte HLA-DR expression demonstrated equivalent lymphocyte
populations after adjuvant treatment, other than a non-significant
decrease in NK cell numbers [44]. Many groups have moved away from
developing therapies targeting monocyte function, with a current focus
on the role of checkpoint inhibitors to reverse immune dysfunction.
Recent randomized controlled trials using IL-7 and anti-PD-L1 therapies
to correct T-cell defects appear well tolerated, with the latter study
also appearing to restore monocyte HLA-DR [45, 46]. Our findings
highlight that labelling PD-L1 as the critical link from monocyte to
lymphocyte dysfunction during septic immunopathology may be
oversimplifying such a complex leukocyte interaction. Furthermore, given
that our results demonstrate that interferon-γ increases PD-L1
expression but does not impact on T-cell function, our study sets the
scene for potential combination strategies with interferon-γ
complimenting anti-PD-L1 therapy.
This study has some limitations. It presents a small and
narrowly-focused set of experiments to address specific questions about
feedback mechanisms of potential interferon-γ-based therapy. Murine
models of sepsis often fail to translate to humans effectively and
single-cell culture does not preserve the important interaction among
immune cells, and in particular, innate and adaptive systems [47].
While this whole blood model allows for the study of the milieu of
diverse cellular subsets, it does not account for the role of fixed,
non-circulating leukocytes such as those in the hepatic and splenic
compartments. Due to the ex-vivo nature of whole blood, study beyond the
18 hour time period cannot be relied upon due to concerns about cell
viability beyond this time point. Finally, LPS was used to represent the
bacterial challenge with which cells were stimulated. Many surgical
patients will sustain gram negative bacteraemia due to a primary
gastrointestinal source of infection or endotoxin translocation in the
critically ill patient [48]. Some evidence has shown that the human
genomic response to LPS largely overlaps with that of trauma, sepsis and
even burn injury [15]. Nevertheless, the in-vivo response to
bacterial DAMPs and PAMPs and from polymicrobial infection will
presumably be more complex than this study represents. Nevertheless,
this established model accounts for some of the human variability in
responses between donors. It is static and reproducible in its findings.
We conclude that in the context of endotoxin-stimulated whole blood,
interferon-γ not only improves monocyte function, but also increases
monocyte surface PD-L1 expression. However, despite increases in PD-L1
expression, T-cell function is not affected. This could be reassuring
for clinician-scientists re-considering the use of interferon-γ as an
adjuvant in critically ill patients, and these findings expand the
knowledge on the role of PD-L1 in the growing field of immune checkpoint
inhibition in sepsis-related immunopathology.