Dysfunctional neutrophil effector organelle mobilization and microbicidal protein release in alcohol-related cirrhosis.

Patients with alcohol-related cirrhosis (ALD) are prone to infection. Circulating neutrophils in ALD are dysfunctional and predict development of sepsis, organ dysfunction, and survival. Neutrophil granules are important effector organelles containing a toxic array of microbicidal proteins, whose controlled release is required to kill microorganisms while minimizing inflammation and damage to host tissue. We investigated the role of these granular responses in contributing to immune disarray in ALD. Neutrophil granular content and mobilization were measured by flow cytometric quantitation of cell-surface/intracellular markers, [secretory vesicles (CD11b), secondary granules (CD66b), and primary granules (CD63; myeloperoxidase)] before and after bacterial stimulation in 29 patients with ALD cirrhosis (15 abstinent; 14 actively drinking) compared with healthy controls (HC). ImageStream Flow Cytometry characterized localization of granule subsets within the intracellular and cell-surface compartments. The plasma cytokine environment was analyzed using ELISA/cytokine bead array. Circulating neutrophils were primed in the resting state with upregulated surface expression of CD11b (P = 0.0001) in a cytokine milieu rich in IL-8 (P < 0.001) and lactoferrin (P = 0.035). Neutrophils showed exaggerated mobilization to the cell surface of primary granules at baseline (P = 0.001) and in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine (P = 0.009) and Escherichia coli (P = 0.0003) in ALD. There was no deficit in granule content or mobilization to the cell membrane in any granule subset observed. Paradoxically, active alcohol consumption abrogated the hyperresponsive neutrophil granular responses compared with their abstinent counterparts. Neutrophils are preprimed at baseline with augmented effector organelle mobilization in response to bacterial stimulation; neutrophil degranulation is not a mechanism leading to innate immunoparesis in ALD.NEW & NOTEWORTHY Neutrophil granule release is dysregulated in patients with alcohol-related cirrhosis (ALD) with augmented effector organelle mobilization and microbiocidal protein release. Neutrophil granules are upregulated in ALD at baseline and demonstrate augmented responses to bacterial challenge. The granular responses in ALD did not contribute to the observed functional deficit in innate immunity but rather were dysregulated and hyperresponsive, which may induce bystander damage to host tissue. Paradoxically, active alcohol consumption abrogated the excessive neutrophil granular responses to bacterial stimulus compared with their abstinent counterparts.

Systemic inflammation and ammonia in hepatic encephalopathy.

Infection and inflammation have been associated with the development of delirium for many centuries and there is a rapidly growing evidence base supporting the role of inflammation in exacerbating the neurological manifestations of both acute and chronic liver failure. Inflammation in the context of hepatic encephalopathy (HE) can arise directly within the brain itself resulting in astrocytic, microglial and neuronal dysfunction, impacting on the development of 'brain failure'. Inflammation may also develop systemically and indirectly influence brain function. Systemic inflammation develops following liver injury, resulting in hyperammonemia and a 'cytotoxic soup' of pro-inflammatory mediators which are released into the circulation and modulate the impact of ammonia on the brain. The aim of this review is to summarise the current evidence base supporting the synergistic role of systemic inflammation and hyperammonemia in the pathogenesis of hepatic encephalopathy. Systemic inflammation and ammonia induce neutrophil degranulation and release reactive oxygen species into the peripheral circulation that may ultimately cross the blood brain barrier. Circulating endotoxin arising from the gut (bacterial translocation), superimposed sepsis, and hyperammonemia upregulate the expression of microbial pattern recognition receptors such as Toll-like receptors. The early recognition and management of systemic inflammation may not only facilitate improved outcomes in HE but supports the development of novel therapeutic strategies that reduce circulating endotoxemia and immune cell dysfunction.

CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi) /CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo) /CD16(pos) monocytes in acute liver failure.

Changes in monocytes and their subsets (CD14(hi)/CD16(neg), CD14(hi)/CD16(pos) and CD14(lo)/CD16(pos)) have been described in several diseases. The combination of CD14, CD16 and HLA-DR has been suggested to discriminate monocytes from the CD16(pos) /HLA-DR(neg) NK-cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA-DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66b(pos) neutrophils, CD56(pos) NKcells, CD19(pos) B-cells, and CD3(pos) T-cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA-DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA-DR expression]. Bland-Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14(hi)/CD16(pos) monocytes was higher (7% vs 4%) whilst the percentage of CD14(lo)/CD16(pos) was lower (1.9% vs. 7%) (P ≤ 0.001); HLA-DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA-DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody-fluorochrome "backbone" targeting HLA-DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody-fluorochrome "backbone" of HLA-DR, CD14, and CD16. CD16(pos) monocytes continue to constitutively express HLA-DR even in conditions where HLA-DR is pathologically reduced on CD14(hi)/CD16(neg) monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico-pathological diseases may allow the development of novel biomarkers or therapeutic strategies.