The first major finding of this study is that delirium and/or the severity of delirium symptoms are significantly associated with the IRS/CIRS ratio, namely positively with M1 (i.e. IL-6, CXCL8 and TNF-α), Th1 and Th17 activation and T cell growth (positively), and inversely with IL-4 and sIL-1RA. These findings extend those of previous papers showing that alterations in peripheral levels of IRS cytokines, namely IL-1β, IL-6, CXCL8, IL-10, and TNF-α, and also C-reactive protein (CRP) and NLR are associated with the onset of delirium [3, 18, 19, 39, 40].
M1-associated cytokines including IL-1β, IL-6, CXCL8, and TNF-α, play a key role in the immune response to injuries. Danger associated molecular patterns, endogenous molecules released from death cells, induce local monocytes and macrophages to secrete IL-1β , which is a major pro-inflammatory cytokine secreted from sterile injurious areas . Increased IL-1β signaling is involved in many medical and psychiatric conditions such as tissue damage , sepsis , rheumatoid arthritis , as well as schizophrenia , and mood disorders . IL-6 produced by locally damaged tissue and macrophages pleiotropically regulates CD4 + T cell differentiation including Th17 proliferation and Treg inhibition . At the local injurious site, macrophages also secrete CXCL8 to promote further local and systemic inflammatory processes including neutrophil stimulation . Accumulation and migration of mononuclear and polymorphonuclear cells (such as neutrophils, macrophages, lymphocytes, monocytes) to the fracture site are observed after a traumatic injury event . Consequently, these immune cells and the injured tissues secrete several pro-inflammatory cytokines and chemokines which expand the inflammatory/anti-inflammatory processes from the local to the systemic level and then to the brain . In this respect, TNF-α is one of these potent pro-inflammatory markers which orchestrate acute inflammatory cascades throughout the body and the central nervous system as well [51, 52]. It is important to note that the above inflammatory markers are all consistently associated with delirium [53, 54]. [39, 59–61]. Unsurprisingly, our study also observed significantly increased levels the Th1 (combination of IL-2, IFN-γ and IL-12) and T cell growth and activation factors (a combination of IL-4, IL-7, IL-9, IL-12, IL-15, GM-CSF) in the delirious hip fracture patients compared to the non-delirious group. As such, activated cell-mediated immune pathways are associated with the severity of delirium symptoms in elderly subject with post-surgery hip fracture.
Based on the above results we may conclude that delirium is accompanied by a cascade of early inflammatory mechanisms which extend from local tissue injury to inflammatory cell activation to cytokine release with increased CRP production and an increased NLR, and neuroinflammation . In response to the acute phase of trauma and inflammation, locally and systematically increased IL-6 secreted from macrophages and T-cells signal the hepatocytes to produce positive acute phase proteins including CRP [55, 56]. Here we report that the NLR is a highly significant biomarker of delirium which strongly reflects IRS, M1, Th2, Th17, and IRS/CIRS activation and Tcell growth as well. As such, the present study extends previous research that high serum and CSF CRP and an increased NLR in peripheral blood are consistently reported as biomarkers or predictive risk factors of post-operative delirium [19, 57–60]. Nevertheless, the immune profiles assayed here better predict delirium symptoms than NLR.
IL-17 is another inflammatory mediator that is produced by CD4 + Th17 cells stimulated by IL-6 and IL-1β and by CD8 T cells and neutrophils. IL-17 plays a key role in chronic inflammatory disorders and autoimmunity, and this cytokine stimulates chemokines (e.g., CXCL1, CXCL2 and CXCL8) and granulopoiesis . Due to its neurotoxic effects, increased IL-17 levels play a role in first episode psychosis and schizophrenia [62, 63], and mood , and neurodegenerative disorders .
Importantly, CXCL8 was significantly associated with the severity of delirium as assessed with the DRS-R-98 scale. Increased levels of peripheral IL-8 are reported in many neuropsychiatric disorders such as major depression, schizophrenia, bipolar disorder, autistic spectrum disorder, and Alzheimer’s disease [66–68]. In response to intracerebral pro-inflammatory stimuli, IL-8 is released from microglia and consequently attracts neutrophils and leucocytes to expand the neuro-inflammation, which may lead to a poorer CNS outcome after a traumatic brain injury or bacterial meningitis . Although increased IL-15 levels in peripheral blood and the CNS play a crucial role in neuro-inflammatory disorders including multiple sclerosis, encephalomyelitis, and intracerebral hemorrhage [70–73], this study could not find any increases in this cytokine in delirium. A study in 2009 found that high IFN-γ was significantly associated with more severe delirium  and IFN-γ participates in the macrophage stimulating process during acute inflammation [73, 75].
The second major finding of this study is that the CIRS index and IL-4 and sIL-RA, which display anti-inflammatory effects, are inversely associated with delirium and/or the severity of delirium symptoms. The sIL-RA is secreted by activated macrophages and inhibits pro-inflammatory IL-1β signaling  and, therefore, the decrease in sIL-RA levels as seen the current and previous studies [74, 77], suggests lowered negative feedback on pro-inflammatory IL-1 signaling . IL-4, which is secreted by Th2 lymphocytes, basophils, mast cells, and eosinophils, leads to the modification and proliferation of lymphocytes, macrophages, fibroblasts and endothelial cells and promotes anti-inflammatory and immunoregulatory processes, as well as healing of tissues . Interestingly, IL-4 function is associated with neuro-restorative effects after cerebral ischemic and traumatic brain injury events, while lower IL-4 signaling is associated with cognitive impairments in schizophrenia . Moreover, relative lower CIRS activity in first episode psychosis predicts a worse outcome following treatment with antipsychotics . Previous research showed that the anti-inflammatory cytokine IL-10, which is produced by different cells but especially by Treg cells, may regulate the initial pro-inflammatory response in delirium [82, 83]. Overall, lowered anti-inflammatory defenses through lowered CIRS functions appear to contribute to delirium.
The third major finding of this study is that the neurotoxic immune profile comprising neurotoxic cytokines and chemokines was not associated with delirium. This contrasts with the increased neurotoxic profiles established in schizophrenia, cognitive impairments in schizophrenia, mood disorders, and suicide attempts and ideation [84–86]. At the phenomenological level, post-hip fracture operative delirium has an acute onset and is frequently presenting with more positive psychotic features  contrasting with the chronic nature of schizophrenia and mood disorders. Thus, the acute increase in IRS (M1, Th1 and Th17) cytokines in patients with attenuated CIRS functions appears to be the most important factor in delirium.
It is important to note that the increased cytokine levels produced at the traumatic site or peripheral vascular and lymphatic system may pass through a damaged blood brain barrier or circumventricular organs to signal inflammatory cascades in the central nervous system [88, 89]. For example, significantly elevated levels of peripheral cytokines including IL-1β, IL-6, IL-8, TNF-α, and IFN-ɣ are involved in the peripheral to central inflammatory signal transduction [52, 90]. Peripheral inflammatory processes which translate peripheral inflammatory signals into central neuro-inflammation and microglial activation are described in delirium  and unipolar depression  and psychosis .
The fourth major finding of this study is that higher pain scores in post-surgery patients are positively associated with CXCL8 and CCL3, and inversely with IL-4 and sIL-1RA. The pain scores were not related to delirium or severity of delirium which contrasts with previous studies which identified increased pain scores as a major contributing factor to postoperative delirium [92, 93]. Almost all of the patients in our cohort received an ultrasound-guided fascia iliaca block pre-operatively [94, 95] which may contribute to attenuated peri-operative pain experiences. Similar to our findings, increased CXCL8 and CCL3 levels, and decreased IL-4 and IL-1RA level were previously reported to be associated with neuropathic pain .
The results of the present study should be discussed with regard to its limitations. A first limitation is that we did not distinguish between delirium subtypes including the hyperactive, hypoactive, and mixed phenotypes. The different delirium subtypes may have a different pathogenesis and may represent different immune-inflammatory and neurochemical pathways . Second, the biomarkers analyzed in this study reflect the peripheral part of the IRS response and not the neuro-inflammatory changes in the CNS which should be examined by cerebrospinal fluid analyses or brain imaging techniques .