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.