Nonalcoholic fatty liver disease as a risk factor for severity of acute cholangitis

Background: Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver disorders worldwide. Acute cholangitis (AC) is a serious and life-threatening illness. We aimed to determine whether NAFLD is a risk factor for severity of AC. Methods: We retrospectively studied hospitalized patients with diagnosis of AC over the span of 5 years. Subjects were divided into a NAFLD group and Non NAFLD group. We compared between the two groups in terms of demographic characteristics, comorbidities, laboratory data, and severity of AC (including Charlson Comorbidity index and Tokyo Consensus meeting criteria). Results: 298 of 419 patients who had AC and were hospitalized met the inclusion criteria and were included in the study. Of them, 73/298 (24.5%) were included in NAFLD group. NAFLD group patients were younger, diabetic and more obese than non NAFLD group. Subjects in the NAFLD presented with higher serum C-reactive protein and higher liver enzymes (p<0.05, for each parameter) and suffered more events of organ dysfunction(p<0.001) and bacteremia (p<0.005). Regarding the severity of AC according to Tokyo classication, among the NAFLD group more subjects presented with Grade II (39.7 Vs 33.3%, p <0.001) and Grade III (23.3 vs 18.3, p<0.001) cholangitis, as opposed more Grade I cholangitis among the non-NAFLD group (48.4 vs 37%, p<0.001). Multivariate logistic regression analysis showed that NAFLD is independently associated with severe AC, grade III (OR 3.25, 95% CI 1.65-6.45, p=0.038). Conclusions: NAFLD is as an independent risk factor for severity of AC.


Background
Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver disorders worldwide [1]. This entity encompasses a wide spectrum of hepatic damage in which steatosis with in ammation progresses to non-alcoholic steatohepatitis (NASH), brosis, cirrhosis, and, ultimately, hepatocellular carcinoma [1,2]. Biliary and pancreatic manifestations have been reported in patients with NAFLD. NAFLD has been positively found to be associated with pancreatitis and its severity [3][4][5][6][7][8].
NAFLD is associated with metabolic syndrome and is considered the hepatic manifestation of the metabolic syndrome. Moreover, NAFLD is linked with several components of the metabolic syndrome, primarily diabetes mellitus and obesity [9,10]. Importantly, a pathogenetic link between NAFLD and imbalance of the gut microbiome has been observed. Recent studies have indicated that changes in gut microbiome is associated with NAFLD and progression to NASH [11,12].
Acute cholangitis (AC) is a clinical syndrome characterized by fever, jaundice, and abdominal pain that develops as a result of stasis and infection in the biliary tract. It is also referred to as ascending cholangitis. Cholangitis was rst described by Charcot as a serious and life-threatening illness; however, it is now recognized that the severity can range from mild to life-threatening [13] .
As in NAFLD gut microbiome play a crucial role in the pathogenesis of acute cholangitis via an interesting recently described Gut-Liver Axis and In ammasome Activation in Cholangiocyte Pathophysiology [14] .
There are no data on the incidence, clinical course and outcomes of Cholangitis in this fast-growing NAFLD population. The question remains, how does gut microbiome change in NAFLD impact cholangitis susceptibility. The aim of this study is to determine whether NAFLD is a risk factor for severity of AC.

Study population, data collection and protocol design
This was a retrospective cohort study conducted at the Sharee Zedek Medical Center (SZMC) University Hospital located in Jerusalem, Israel. We reviewed the hospital records of all consecutive adult patients hospitalized over a ve-year period (January 2015 to December 2019) at SZMC. Included were adult patients (≥ 18 years) hospitalized for acute cholangitis (The clinical diagnosis of acute cholangitis was made on the basis of the clinical ndings, such as Charcot's triad, in combination with the laboratory data and imaging ndings) in subjects who underwent an abdominal ultrasonography (US) examination during hospitalization. Patients diagnosed with gastrointestinal infection other than Cholangitis were excluded. Furthermore, patients with known history of heavy alcohol use (de ned as subjects who had a history of alcohol consumption of more > 30 gram a day for at least 5 years) and those diagnosed with chronic viral hepatitis or patients with history of other known liver disease were excluded. Subjects with other hepatic pathology or autoimmune phenotypes (such as alcoholic liver disease, drug-induced liver injury, autoimmune hepatitis, viral hepatitis, cholestatic liver disease and metabolic/genetic liver disease) were also excluded using speci c clinical, laboratory, radiological and/or histological criteria/tests. The current study received ethical approval from the local hospital ethical committee and was conducted according to the Helsinki declaration and its subsequent amendments. Data were coded in order to preserve the anonymity of the patients. Informed consent was waived because of the non-interventional study design.
De nitions NAFLD was diagnosed by abdominal ultrasonography based on the presence of fatty liver (hepatic parenchymal brightness, visualization of portal and hepatic borders, liver-to-kidney contrast, deep beam attenuation, and bright vessel walls) [15] .
Charlson Comorbidity Index (CCI) predicts 10-year survival/mortality of patients with several comorbidities based on CCI scoring system. The CCI score is composed from 17 categories including (age, myocardial infarction, congestive heart failure, peripheral vascular disease, stroke or transient ischemic ischemia, dementia, chronic obstructive pulmonary disease, connective tissue disease, peptic ulcer disease, liver disease, diabetes, hemiplegia, chronic kidney disease, solid tumors, leukemia, lymphoma, and AIDS) [16] . Acute cholangitis was de ned according to the new diagnostic criteria and severity assessment of acute cholangitis in revised Tokyo Guidelines as: A. Systemic in ammation A-1: Fever and/or shaking chills; A-2: Laboratory data: evidence of in ammatory response. B. Cholestasis B-1: Jaundice B-2: abnormal liver function tests. C. Imaging C-1: Biliary dilatation C-2: Evidence of the etiology on imaging (stricture, stone, stent etc.). Suspected diagnosis: one item in A + one item in either B or C De nite diagnosis: one item in A, one item in B and one item in C. Tokyo Consensus Meeting de nes severity of AC in 3 grades: grade I, mild, with no onset of organ dysfunction and with a good response to initial antibiotic treatment. Grade II, moderate, with no onset of organ dysfunction but without response to initial antibiotic treatment, and grade III, severe de nes as with onset of organ dysfunction and without response to initial antibiotic treatment [17,18] .

Statistical analysis
Before commencing any statistical processing and analysis, data were visually inspected and checked for outliers. The analysis was performed using the commercial software Statistical Package for Social Science (SPSS version 24.0, IBM, Chicago, IL, USA). Categorial variables were tested using the chi-square test or Fisher's exact test, as appropriate. Continuous variables were examined using the student's t test if normally distributed and Man-Whitney test if not. To identify variables associated with the severity of AC, univariate analysis was performed. Variables that were signi cantly associated (p < 0.1) with the primary outcome (severe cases of AC) were entered into the multivariate logistic regression model. Adjusted odds-ratios (ORs) and 95% con dence intervals (CIs) for the severity of AC were calculated.
Statistical signi cance was set at p-value less than 0.05.

Results
Of the 419 patients aged ≥ 18 years hospitalized in the studied period, a total of 121 patients were excluded: 97 did not meet inclusion criteria (inde nite diagnosis of cholangitis or presence of other gastrointestinal active infection on or in ammation) and 24 patients had exclusion criteria (signi cant alcohol intake in 7, chronic viral hepatitis in 6, cirrhosis in 2 and hepatotoxic medications in 9 patients). Consequently, 298 patients were included in the study. Based upon the results of abdominal ultrasound, the patients were divided into two groups: Subjects with NAFLD (73, 24.5%) and subjects without NAFLD (225, 75.5%).
The demographic, clinical, and laboratory data are shown in Table 1. The NAFLD group were younger and predominant of male gender (53.4% vs 50.2%, p = 0.07) with a median age of 71.8 ± 20.9 years versus 73.6 ± 19.3 years in the non-NAFLD group (p = 0.035). Obesity and Diabetes mellitus were more frequent among NAFLD group (the mean BMI was 29.7 ± 7.1 vs. 26.3 ± 6.3, p = 0.020; and 29 (39.7%) versus 74 (32.8%), p = 0.049 respectively). There were no signi cant differences in other comorbidities between the two study groups.  Fig. 1 below.
With regards to laboratory features, NAFLD group subjects had higher serum CRP levels compared to the non-NAFLD group (37.8 mg/dl versus 19.6 mg/dl, p = 0.019). NAFLD subjects also had higher serum To identify potential risk factors for severity of AC, multivariate logistic regression analysis was performed (

Discussion
Our study results indicate that NAFLD is strongly associated with severe cholangitis (de ned as Tokyo score Grade III). To the best of our knowledge, no previous studies have studied the relationship between NAFLD and severe cholangitis, although a wide array of pancreatobiliary manifestations are commonly found among patients with NAFLD [19] .
Although the exact mechanism is yet to be revealed, alteration of the immune system function with involvement of the dynamic gut-liver axis seems like the most plausible explanation, presumably through the NLRP3 in ammasome activation and its recently described role in Cholangiocyte pathophysiology [14]. Other possible contributors within this recently described axis are the gut barrier dysfunction and intestinal immune defects that may overburden the liver's defense mechanisms, allowing bacteria to freely enter the circulation [24]. Indeed, more cases of Bacteremia and organ dysfunction were documented among the NAFLD group when compared to the non-NAFLD group in our study.
The mechanisms by which NAFLD may induce severe cholangitis have not been elucidated so far. An increasing body of evidence points towards an increased level of proin ammatory cytokines in patients with NAFLD and non-alcoholic steatohepatitis, with higher oxidative stress and abnormal lipoprotein metabolism also implicated [25]. Furthermore, NAFLD is closely associated with metabolic syndrome and obesity, which is now seen as a chronic low-grade in ammatory state as the adipocytes have been shown to secrete a variety of cytokines like IL-6 and TNF-alpha which promote in ammation [26]. Interestingly, in our study, NAFLD have been shown to be independently related to the occurence of severe cholangitis, regardless of obesity. NAFLD have also been shown to independently correlate with the risk of developing severe pancreatitis, presumably through similar mechanisms [3].
In our study, subjects in the NAFLD group presented with signi cantly higher CRP levels. This nding is consistent with the results reported from earlier studies where a positive correlation been CRP and NAFLD have been demonstrated [27]. Furthermore, subjects in the NAFLD group had higher Charlson comorbidity index scores, lower albumin levels and higher GGT levels which are all predictors of short-term and longterm mortality [16,28,29]. NAFLD has been well established as a predictor of increased all-cause mortality in a recently conducted comprehensive meta-analysis [30]. Similarly, Nseir et al demonstrated an increase in the all-cause 30-day mortality among NAFLD subjects who presented with Communityacquired pneumonia [21].
Our study has several limitations, including the inherent limitations of a retrospective design, a single center study, and its relatively small sample size. Another main limitation is the absence of liver biopsies for the diagnosis of NAFLD. Thus, we didn't correlate with the degree of in ammation in NAFLD subjects and no distinction was made between hepatic steatosis or NASH. On the other hand, this is the rst study to describe the association between NAFLD and severe cholangitis as we are not aware of any similar studies published elsewhere in the literature.

Conclusion
In conclusion, patients with NAFLD have been shown to be at an increased risk for developing severe cholangitis with higher incidence of bacteremia and organ dysfunction. Given the high prevalence of NAFLD and the substantial morbidity and mortality attributed to severe cholangitis, more rigorous studies are urgently needed to further characterize this important association and effectively reduce morbidity and mortality in this susceptible patient population.

Declarations
Ethics approval and consent to participate -The current study received ethical approval from the Helsinki Committee of Shaare Zedek Medical Center (reference number SZMC-19-0379) and was conducted according to the Helsinki declaration and its subsequent amendments Consent for publication -Not applicable Availability of data and materials -The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests -The authors declare that they have no competing interests Funding -No funding was received by the authors for the completion of this work