Persistently high and rising bilirubin is very suggestive of an unremitting inflammation, likely to be unresponsive to steroids.

Whether assessment of the same parameters which form the Lille score can reliably predict non-response at day 4 rather than day 7, was evaluated. The data showed that Lille score on day 4 was as good as day 7 to predict 3-month mortality and reduces unnecessary steroid exposure [17]. However, these results have not been validated by other workers. In our own large experience (unpublished data), day 4 is not a very reliable indicator of response to steroids, and legitimacy to continue therapy for 7 days.

A pre-therapy liver biopsy helps in stratification of SAH patients not only to assess the severity of liver disease, but also to predict response to therapy. Liver biopsy showing ballooning hepatocytes and bilirubinostasis, indicates a poor response to steroids. In one study, non-responders had higher ballooning degeneration (BD) (mean, 3.87 [standard deviation (SD), -0.91] vs. 2.92 [SD, -1.33], P=0.013) with increased density of Mallory-Denk bodies (MD) (mean, 2.27 [SD, -0.79] vs. 1.69 [SD, -0.97], P=0.028). A score derived using BD and MD (range, 0–8) had high sensitivity and specificity (81.2% and 64%, respectively), and negative predictive value (91.4%) to identify non-responders to steroids at baseline if the score is >5 [18].

Patients with SAH have a unique metabolic phenotype, one with severe inflammatory milieu and an impaired immune status. Whether baseline metabolic phenotype could identify non-responders and those with unfavorable outcome was investigated [3]. Baseline urine metabolome of SAH patients was subjected to ultra-high performance liquid chromatography and high-resolution mass spectrometry. Nine urinary metabolites linked to mitochondrial functions significantly discriminated non-responders, most importantly the increased acetyl-L-carnitine (12-fold) levels. A baseline urinary acetyl-L-carnitine level predicted non-response with a receiver operating characteristic of 0.96 (95% confidence interval [CI], 0.85–0.99) and a hazard ratio of 3.5 (95% CI, 1.5–8.3) for the prediction of mortality in patients with SAH. Acetyl-L-carnitine levels of >2,500 ng/mL reliably segregated survivors from non-survivors (P<0.01, log-rank test) in the study cohort [3]. This low cost test, if validated in other studies, could be of help in selection of patients for steroid therapy.

Microvesicles (MVs) reflect the cellular stress and the disease conditions. In one study, pre-therapy peripheral plasma MV levels of haematopoietic stem-cells (CD45+ CD34+; 116.8 vs. 13.4 MV/μL; P=0.0001) and hepatocytes (ASGPR+; 470 vs. 361 MV/μL; P=0.01) were higher in steroid non-responders, compared to responders (Fig. 1). In fact, the two together could predict steroid non-response in 94% patients at baseline in peripheral plasma. The basis for these observations lies in the fact that the MVs from SAH patients, trigger more (P=0.04) reactive oxygen species (ROS) generation, tumor necrosis factor-α production (P=0.04) and upregulate pro-inflammatory cytokine related genes in neutrophils in vitro [19]. The source of these MVs was also validated with higher levels in hepatic vein plasma of non-responders. The MVs from hematopoietic cells suggests that the bone marrow cells do migrate to liver, but probably, a fair proportion is unable to induce effective regeneration due to an unfavorable milieu and, hence possibly perish as assessed by high MV levels in hepatic vein in non-responders [19]. More such biomarkers are needed for proper patient selection for steroid therapy.

Whether variation to steroid response in SAH patients has a genetic basis, is largely unknown. This was explored by investigating the hepatic transcriptome in patients originating in different countries [20]. Though at presentation the patients were similar, the responders and non-responders had distinct patterns of gene expression. There were >1,100 genes that were overexpressed (>2-fold, P<0.05) in non-responders in comparison to responders. Importantly, the glucocorticoid receptor (NR3C1) expressed a significantly reduced amount of the alpha isoform in non-responders. Additionally, in one population the BCL2 associated athanogene-1 (BAG1) was significantly increased in non-responders. BAG1 is known to cleave the glucocorticosteroid receptor (Fig. 2) [21]. This data shows BAG1 as a promising candidate to be explored in a larger population to segregate steroid non-responders at baseline.

Circulatory albumin is a potent ROS scavenger. However, structural modifications in albumin may modulate its antioxidant and immune‐regulatory properties. Such alterations in circulating albumin in SAH patients along with their contribution to neutrophil activation, intracellular stress, and alteration in associated molecular pathways have been assessed [22]. The 3 isoforms of circulating albumin - human mercaptalbumin (HMA) and human non-mercaptalbumin (HNA1 and 2) were measured in SAH patients. Albumin isoforms showed that total HNA1 was significantly (P<0.05) higher in SAH (44.5%) patients compared to healthy controls (28.6%). Additional modifications like glycosylated HMA was also higher in SAH than controls (17.4% vs. 10.6%, P<0.05, respectively).

G-CSF (also known as CSF3) is a colony stimulating factor which stimulates bone-marrow precursor cells to produce granulocytes and stem cells, and release them into the blood [23].

Inflammation and impaired liver repair and regeneration are the major factors responsible for liver failure in patients with AH. A state of hyper-inflammatory response leading to progressive hepatocyte damage is accompanied by skewed and deficient immune system in SAH patients. Agents that can ameliorate gut-derived infection and inflammation, and enhance native liver regeneration including bone marrow response are needed. The maximal level of CD34⁺ cells are released on day 5 after daily G-CSF administration, and the levels fall rapidly on subsequent days despite a continued rise in white blood cell counts [24].

G-CSF regulates innate immune cell response by modulating the production, maturation, migration, and function of neutrophils, monocytes and dendritic cells (DCs). Excessive alcohol consumption impairs this granulopoiesis and the functionality of granulocytes resulting in defects in immune defense and susceptibility to serious infections [25]. Despite, peripheral neutrophilia, AH patients are susceptible to infections, due to neutrophil dysfunction in the form of neutrophil resting oxidative burst greater than or equal to 55% and reduced phagocytic capacity of <42% due to persistent endotoxemia [26]. This is partly due to the defects in the interleukin (IL)-33/suppression of tumorigenicity-2 pathway which is involved in sepsis control and is associated with decreased C-X-C motif chemokine receptor 2 expression on the cell surface and lower neutrophil migration capacity [27]. It is therefore important to identify patients in the golden window [28], the time between the inflammatory response and the onset of sepsis, as the ideal time for therapeutic interventions for SAH. It is probably the most suitable time to introduce the G-CSF therapy, to prevent infections and enhance liver regeneration.

Locally produced G-CSF within the injured tissue also affects the function of recruited neutrophils and DCs. The frequencies of intrahepatic myeloid DCs and plasmacytoid DCs are lower in non-survivors compared to survivors. Intravenously given G-CSF has been shown to enhance the recruitment of plasmacytoid DCs, nearly 12-folds, resulting in reduction in the intrahepatic gamma interferon production and amelioration of hepatic injury (Fig. 5) [29].

G-CSF mobilizes bone marrow hematopoietic stem cells into the circulation by reduced C-X-C motif chemokine ligand 12 levels and adhesive action and facilitates their release into the circulation. G-CSF also stimulates the peripheral sympathetic nervous system, increasing catecholamine concentrations [30]. G-CSF facilitates liver regeneration by migration of bone marrow-derived progenitors to the liver and by enhancing hepatic progenitor cell proliferation by its chemoattractant and mitogenic effects [31].

In a randomized trial of patients with acute-on-chronic liver failure, mostly due to alcohol, G-CSF was able to reduce the incidence of new infections, hepatorenal syndrome and hepatic encephalopathy with an improved 28-day survival compared to placebo (69% vs. 29%, P<0.001) [32]. Also, CD34⁺ hematopoietic cells were increased in liver biopsies with post G-CSF therapy. The drug was given as 5 mg/kg/day subcutaneously for 5 days and then once on alternate day for a period of 28 days (total 12 doses). It was well tolerated and no major side effects were reported.

G-CSF (given as 5 mg/kg, twice daily for 5 days) in another study on 46 SAH patients with a mean MDF of 85, was found to improve CD34⁺ cells in peripheral blood at the end of therapy along with improved 90-day survival (78.3% vs. 30.4%, P<0.001) [33].

These reports are mainly from Asian countries. In one study [34] on 58 decompensated alcohol-related cirrhotics, isolated bone marrow derived mononuclear cells including CD34⁺ hematopoietic cell infusion in the hepatic artery did not show survival benefit or better regeneration on repeat liver biopsy. Similarly, when given in compensated cirrhotics, G-CSF therapy for 5 days followed by leukapheresis and intravenous infusion of CD133-positive hematopoietic stem cells on day 5, 30, and 60 did not show benefit. An important aspect to be noted is that the studies from Europe, had included entirely different set of patients, not those with active AH, and with many having had sepsis. A recent meta-analysis has highlighted the differences in various studies [35]. Based on multiple randomized control trials (RCTs), it can be concluded that in carefully selected patients of AH, G-CSF therapy can help in reducing inflammation, incidence of infection and sepsis, reducing liver severity scores and possibly improve the survival (Fig. 6).

For steroid non-responders, there are few choices. In the only available study, G-CSF was evaluated in steroid non-responsive SAH patients. Shasthry et al. [6] in a group of 32 steroid non-responders, showed that G-CSF administration reduced the MDF and MELD scores and in-turn the mortality, compared to the placebo (35.7% vs. 71.4%, P=0.04).

Patients with severe AH, not amenable to steroids treatment or steroid non-responders can be considered for G-CSF administration. However, as noted in most of the RCTs, patients with MDF >90, hemoglobin <8 g/dL, high ferritin levels, macrophage activation syndrome (MAS), evidence of iron overload, hypersplenism, ascites, total leucocyte count of >40,000/mm³, infections, culture positive sepsis, acute kidney injury [36], hemodynamic instability, human immunodeficiency virus seropositivity, high adenomatous colorectal polyps or suspicion of malignancy, should be excluded from G-CSF therapy [6,8]. In fact, it is safer and the response is more likely, if the MELD is <15 [37]. A good bone marrow is very helpful in stimulating the hepatic regeneration. Patients with severe sarcopenia, osteoporosis and anemia are less likely to have a responsive bonemarrow [38]. Also, a careful screening for the presence of infections should be undertaken. The efficacy of G-CSF is mainly in prevention of infection, rather than in the management of sepsis in SAH patients, where systemic inflammatory response syndrome (SIRS) can get exaggerated.

The gut microbiome (GM) plays a major role in liver disease. A chronically altered and unhealthy microbiome contributes to the development and perpetuation of liver disease, and this is probably true for SAH. The GM digests the ingested alcohol, with an increase in acetaldehyde concentrations in the gut lumen, and disruption of the mucosal barrier, permitting translocation of viable bacteria and their products, into portal circulation. Locally in the gut, the overgrowth of pathogenic species changes the bile acid composition, enteral metabolites, and entero-hepatic circulation [39]. Resultant immune dysregulation makes the host susceptible to infections and continued hepatic injury [40,41].

Patients with AH, have a pathobiont of their own [40]. This alcohol pathobiont represents altered bacteria, their metabolites, and imbalanced cytokine milieu. Transferring intestinal microbiota from a patient with severe AH into mice, can lead to the development of severe liver inflammation in the animal [42].

Quantitative and qualitative variations in fecal lipid metabolites like the short-chain fatty acids butyrate and propionate have been reported in rats following chronic ethanol administration [43]. Lactobacillus species use saturated long chain-fatty acids (LCFAs) as a source of energy, which are consequently reduced, resulting in tight junction barrier disruption. In mice models, saturated LCFA supplementation has been shown to restore eubiosis, stabilize the intestinal gut barrier, and reduce ethanol-mediated liver disease (Fig. 7). Ethanol exerts a direct effect on the saturated fatty acid (FA) biosynthetic gene in intestinal bacteria, independently of the host [44]. Hepatocyte synthesized bile acids are secreted via bile duct into the duodenum facilitating digestion, and absorption of lipids, and lipid soluble vitamins. The bile acids modulate the gut microbiota through direct and indirect mechanisms [45]. The gut microbiota then metabolizes primary bile acids into secondary bile acids [44]. More than 95% bile acids actively taken up by enterocytes in the terminal ileum are exported through the basolateral membrane into the portal vein. Bile acids bind to the farnesoid X nuclear receptor and induce its target gene, the fibroblast growth factor (FGF)-19 in humans (or in mice, FGF-15). Both, FGF19/15 are released into the portal circulation, inhibiting hepatic bile acid synthesis and exerting beneficial effects on hepatic lipid metabolism [46]. A significant increase of serum FGF19, total and conjugated bile acids is observed AH patients.

AH-related dysbiosis is associated with an increase in Bifidobacteria, Streptococci, Enterobacteria with concomitant decrease in anti-inflammatory Clostridium leptum or Faecalibacterium prausnitzii. The population density of these protective strains inversely correlates with clinical parameters such as bilirubin [42]. Fecal microbiota transplantation (FMT) studies showed that there is a distinct microbiota signature associated with severe AH, and that a specific alcohol ‘pathobiont’ possibly exists [47]. Identification of these microbial species would identify new targets and therapies.

Alteration in the GM of a patient can be achieved by using probiotics, FMT or using genetically altered bacteria [36]. While the later has not reached the stage of clinical application, the first two approaches have been evaluated in small sets of patients.

Probiotics have been shown in a small randomized trial to be of benefit in cirrhotic patients, including those with hepatic encephalopathy [48]. No large clinical studies are available of the use of probiotics in SAH patients.

FMT has been used in multiple clinical conditions in gastroenterology. Its use was evaluated in a pilot study of eight steroidineligible SAH patients, and was compared to anecdotal controls. FMT showed significant survival benefit at day 90 with the donor microbiota co-existing in the recipients till 12 months [47].

It is known that subjects in the same household, living and eating the same food, generally share common bacterial taxa. We therefore preferred using FMT material from a related healthy individual living in the same household, with a presumption that the bacteria could adapt readily in the patient.

Recently, concerns have been raised about the safety of FMT therapy as fecal instillate may serve as a medium to carry pathogens [49]. Infection with pathogenic Escherichia coli and development of adverse events in immunocompromised and immunocompetent patients indicates need for additional caution [50]. However, risk of bacterial and fungal infections is high after steroid therapy in SAH patients, and may add to mortality [47].

We have completed a large study on 112 SAH patients, comparing the safety and efficacy in 90 day-survival in those receiving steroids with healthy donor FMT. We serially assessed the recipients for the improvement in clinical and biochemical parameters, changes in their GM, occurrence of adverse events, new complications, and infections. Since Lille score has been validated only for steroid therapy, new scores for assessing the therapeutic response to emerging therapies have also been developed. The initial results of the FMT in SAH patients are quite encouraging. Attempts have been made to inhibit the effects of pro-inflammatory IL-1 by using antagonist to its receptor, anakinra. In a clinical trial comparing anakinra efficacy in combination with pentoxyfylline and zinc against methylprednisolone, the results suggest that long term survival was similar between the two groups (66.8%, anakinra group vs. 52.8%, prednisolone group, P=0.26) [51]. Although the inflammation was shown to be reduced, and gut barrier function was improved. Targeted studies are required to identify the patients suitable for such inhibitors and combinations thereof.