Project title: The role of immune semaphorins in NAFLD and sepsis, SepsisFAT

Principal investigator: Neven Papić, MD, PhD

Host institution: School of Medicine, University of Zagreb, Zagreb, Croatia

 

Collaborative institutions:

University Hospital for Infectious Diseases “Dr. Fran Mihaljević“, Mirogojska 8, 10000 Zagreb

University Hospital Center Zagreb, Kišpatićeva 12, 10000 Zagreb

University Hospital Merkur, Zajčeva 19, 10000 Zagreb

Funding: Croatian Science Foundation, Installation Research Project, (UIP-2019-04-7194)

Researchers

Neven Papić, MD, PhD, assistant professor

Ivana Knežević Štromar, MD, PhD, assistant professor

Marko Kutleša, MD, PhD, associate professor

Vladimir Krajinović, MD, PhD, assistant professor

Iva Butić, MD, PhD

Maja Mijić, MD

Branimir Gjurašin, MD

Juraj Krznarić, MD

Nina Vrsaljko, MD

Martina Vargović, MD

Viktor Kotarski, MD

Anita Atelj, MD

Karlo Jeličić, MD

Lara Šamadan, mag.biol.mol.

Leona Radmanić, mag.biol.mol.

 

STUDY DESCRIPTION AND AIMS

Sepsis and non-alcoholic fatty liver disease (NAFLD) are both inflammation-related entities and major disease burdens with a high impact on modern healthcare systems. Sepsis is a heterogeneous syndrome, with an outcome dependent on the culprit pathogen, infection site and host factors (e.g. comorbidities, genetic factors, immunosuppression, including the de- gree and types of the host immune response). Only recently has the role of the liver in sepsis begun to be revealed. Evidence has shown that liver dysfunction in sepsis in patients without pre-existing liver disease is an independent predictor of mortality (54-68%), higher than the mortality rates of sepsis complicated with respiratory or kidney failure. On the other hand, chronic liver disease is a risk factor for the progression of infection to sepsis. Patients with liver cirrhosis are exposed to an increased risk of developing bacterial infections and have ten times more common bloodstream infections with a fourfold increase in mortality in comparison to patients without cirrhosis. Progression to septic shock in these patients is associated with an in-hospital mortality rate as high as 70%. These clinical findings highlight the critical role of the liver as an immunomodulatory organ in sepsis.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease. Although the pathogenesis of NAFLD is linked with systemic changes in immune response, the question remains whether patients with NAFLD are more prone to bacterial infections and what is the impact of persistent local inflammation on the systemic response to infection, sepsis course and outcome.

There were only several clinical studies that examined the role of NAFLD in infectious diseases in the pre-COVID era. It was shown that the post-transplant risk for infection is significantly higher in patients transplanted due to non-alcoholic steatohepatitis (NASH) when compared to other transplant indications. NAFLD was associated with an increase in all-cause mortality in patients with community-acquired pneumonia even in the absence of advanced fibrosis. Bacteraemia of gastrointestinal origin was more common in patients with NAFLD.

Therefore, the impact of NAFLD on severe bacterial infections, sepsis and pneumonia has not been established so far. Although there is an abundance of data on the gut-liver axis and changes in gut microbiome shown to be associated with NAFLD and progression to NASH, there are no data on the incidence, clinical course and outcome of gastrointestinal infections in this population, specifically the risk for Clostridiodes difficile infection. In addition, there is a question if the NAFLD gut microbiome profile contributes to the colonization with MDR bacteria. Therefore, significant gaps in our understanding of the role of NAFLD in infectious diseases exist.

In 2020 we started the Installation Research Project entitled „The role of immune semaphorins in NAFLD and sepsis”, SepsisFAT, financed by the Croatian Science Foundation, to provide insight into these clinical questions. We planned to conduct the first comprehensive interdisciplinary clinical, microbiological, immunological, and biomarker study of the impact of NAFLD on sepsis. In addition, we planned to examine new biomarkers semaphorins in these conditions. Semaphorins were recently recognized as one of the key regulators of immune responses; while some suppress immune cell activation, proliferation and production of inflammatory cytokines, others stimulate immune responses. We have previously shown that semaphorins are associated with the pathogenesis of viral hepatitis and the progression of fibrosis. However, their role in NAFLD and sepsis is unknown. One of the hypotheses of this project is that semaphorins are regulators of inflammation in patients with NAFLD that have an impact on sepsis outcome.

The main aims of the project are:

(a) to identify semaphorins that are associated with NAFLD;

(b) to analyze the impact of NAFLD on sepsis outcomes;

(c) to analyze changes in semaphorin concentration according to steatosis grade in patients with sepsis and its impact on clinical outcomes;

(d) to describe the correlation of semaphorins with the profile of pro- and anti-inflammatory cytokines in patients with sepsis;

(e) to analyze the changes in semaphorin concentration and the profile of immune response in patients with severe community-acquired pneumonia;

(f) to identify semaphorin gene polymorphism associated with NAFLD and its impact on sepsis outcomes.

RESEARCH METHODOLOGY

This study is designed as a prospective, non-interventional study.

The project is divided into 3 consecutive phases:

PHASE 1. IDENTIFICATION OF SEMAPHORINS AS BIOMARKERS OF NAFLD.

To goal was to identify semaphorins that are associated with NAFLD and to investigate their relationship with variable degrees of steatosis and fibrosis. The degree of steatosis was estimated using the controlled attenuation parameter (CAP). Routine laboratory tests and anthropometric measures were collected, including the components of metabolic syndrome. Semaphorin concentrations were measured in patient sera by ELISA. The correlation between serum semaphorin concentration and other non-invasive markers of fibrosis and steatosis, laboratory and anthropometric measures were analyzed. In addition, immunohistochemistry was performed on liver biopsy specimens diagnosed and staged with NAFLD according to NAFLD activity score. To identify semaphorins’ gene polymorphisms associated with NAFLD, three genes with the highest association with NAFLD were sequenced.

PHASE 2. ANALYSIS OF SERUM SEMAPHORIN CONCENTRATION IN PATIENTS WITH SEPSIS AND NAFLD IN COMPARISON TO PATIENTS WITH SEPSIS AND WITHOUT LIVER DISEASE.

Adult patients diagnosed with sepsis were offered to participate in the study. Patients were screened for the presence of NAFLD using non-invasive procedures (US, CAP, TE, and APRI, FIB-4 and NAFLD score). Routine demographic, clinical, microbiological, treatment and laboratory parameters, including the disease severity scores (APACHE, SAPP, SOFA), were collected and patients were followed until discharge. Serum semaphorins’ concentration and the panel of pro-inflammatory and anti-inflammatory markers were measured by flow cytometer microsphere-based assay.

PHASE 3. ANALYSIS OF SEMAPHORINS IN PATIENTS WITH SEVERE COMMUNITY-ACQUIRED PNEUMONIA AND NAFLD.

The goals are (a) to investigate the impact of NAFLD on the course and outcome of severe pneumonia as a source of sepsis; (b) to describe the systemic and local immune response to infection; (c) to describe the association of semaphorins with disease severity and outcome. Serum samples and bronchoalveolar fluid (BAL) will be collected and concentrations of semaphorins and pro- and anti-inflammatory cytokines will be measured.

 

SepsisFAT RESULTS

SepsisFAT study so far yielded a plethora of interesting results.

Phase I of the study was completed in July 2022 and results were published in November 2022. In this prospective, case-control study, serum semaphorin concentrations (SEMA3A, -3C, -4A, -4D, -5A and -7A) were measured in 95 NAFLD patients and 35 healthy controls. Significantly higher concentrations of SEMA3A, -3C and -4D and lower concentrations of SEAMA5A and -7A were found in NAFLD. While there was no difference according to steatosis grades, SEMA3C and SEMA4D significantly increased and SEMA3A significantly decreased with fibrosis stages and had better accuracy in predicting fibrosis compared to the FIB-4 score. Immunohistochemistry confirmed higher expression of SEMA4D in hepatocytes, endothelial cells and lymphocytes in NAFLD livers. The SEMA5A rs1319222 TT genotype was more frequent in the NAFLD group and was associated with higher liver stiffness measurements. To conclude, in Phase 1 we provide the first evidence of the association of semaphorins with fibrosis in patients with NAFLD.

Unfortunately, the phase II study was marked with the COVID-19 pandemic, and we could not start the recruitment of patients with community-acquired sepsis as initially planned. Therefore, we started a spin-off of the SepsisFAT study on hospitalized patients with COVID-19 (CovidFAT). Firstly, we showed that patients with NAFLD had higher disease severity assessed by a 7-category ordinal scale, more frequently required high-flow nasal cannula or noninvasive ventilation, had longer duration of hospitalization, and more frequently had pulmonary thromboembolism. Next, we examined the specificities of cytokine profiles in patients with severe COVID-19 and NAFLD and identified different cytokine signatures; interleukins-6, -8, and -10 and CXCL10 were significantly higher, while IFN-γ was lower in NAFLD patients. Finally, we identified semaphorins associated with COVID-19 severity and outcomes. SEMA3C, SEMA3F and SEMA7A serum concentrations were significantly higher in patients with COVID-19, while SEMA3A was significantly lower than in healthy controls. Furthermore, SEMA3A and SEMA3C decreased with COVID-19 severity, while SEMA3F and SEMA7A increased. Serum semaphorin levels show better predictive value than CRP, IL-6 and LDH for differentiating critical from moderate/severe COVID-19. SEMA3F and SEMA7A serum concentrations were associated with time to recovery, requirement of invasive mechanical ventilation, development of pulmonary thrombosis and nosocomial infections, as well as with in-hospital mortality.

Next, we performed a series of retrospective cohort studies as a preparation for phase II. These showed a significant association between NAFLD and infections. Two studies showed an association of NAFLD with both the development of nosocomial Cl. difficile associated disease and CDAD recurrence in elderly population. We have shown that invasive Group B Streptococcus (GBS) disease in patients with NAFLD is associated with higher mortality than in patients without NAFLD and this appears to be independent of other components of metabolic syndrome, such as obesity and diabetes mellitus. Nosocomial infections and acute renal failure were more common in the NAFLD group. In a retrospective cohort study that included 138 consecutively hospitalized adult patients with severe CAP admitted to the ICU (80 patients with NAFLD and 58 controls), patients with NAFLD more frequently presented with ARDS and required invasive mechanical ventilation, respiratory ECMO, and continuous renal replacement therapy. Mortality was significantly higher in the NAFLD group, and the time from hospital admission to death was significantly shorter. In survival analysis, NAFLD was associated with mortality independently of other components of metabolic syndrome.

In 2022 we finally started recruitment of patients with severe community-acquired bacterial infections. So far, 378 patients with community-acquired sepsis were recruited. Preliminary results show a high prevalence of NAFLD (46%) and metabolic syndrome (41%) in hospitalized patients with sepsis. Importantly, in-hospital mortality was significantly higher in the NAFLD group (18.4% vs 9.8%). NAFLD was associated with mortality independently of other components of metabolic syndrome. Patients with NAFLD more frequently developed ARDS and acute kidney injury, and more frequently required invasive mechanical ventilation or renal replacement therapy.

 

Selected published SepsisFAT results

	Study design	Study methodology	Main findings	Comment
Bacterial community acquired pneumonia
Gjurašin et al. Life (Basel) 2022	retrospective cohort study	138 patients with severe CAP treated at the ICU	– the prevalence of NAFLD was 57.9% – the mortality of the NAFLD group was 50% and in controls 20.7%	NAFLD (HR 2.21, 95%CI 1.03–5.06) was associated with mortality independently of other components of metabolic syndrome
COVID-19
Vrsaljko et al. Open Forum Infect Dis (2022)	prospective observational  cohort study	216 adult patients hospitalized with severe COVID were also assessed for NAFLD	–  NAFLD is linked to worse COVID-19 outcomes, more pulmonary thromboses, and worse COVID-19 severity	patients with NAFLD typically required noninvasive ventilation or high-flow nasal cannulas more frequently, spent longer time in hospitals
Papic et al. Life (Basel) 2022	prospective observational case-control study	94 adult patients hospitalized with severe COVID	NAFLD group had higher CRP, PCT, ALT, LDH, and fibrinogen. Interleukins-6, -8, and -10 and CXCL10 were significantly higher, while IFN-γ was lower in NAFLD group	IL-8 and IL-10 were associated with time to recovery in NAFLD group
Susak et al. Microorganisms (2023)	prospective observational case-control study	60 adult patients hospitalized with severe COVID (30 with NAFLD)	NAFLD was associated with higher serum TGF-β1 concentrations that increased with disease severity	Admission TGF-β1 concentrations showed good discriminative accuracy in predicting the development of critical disease and COVID-19 complications
Močibob et al J Clin Med (2022)	prospective observational cohort study	575 adult patients with COVID-19 who underwent CTPA	PT was diagnosed in 178 (30.9%) patients	PT was not associated with in-hospital mortality D-dimer ≥ 1.0 mg/L and O2 ≥ 15L were associated with PT
Vargović et al IDweek 2023	prospective observational cohort study	80 adult patients with COVID-19 (42 with NAFLD)	NAFLD group had significantly higher SEMA3C and SEMA3F serum concentrations	SEMA3F and SEMA7A serum concentrations were associated requirement of IMV, PT, nosocomial infections and in-hospital mortality
Clostridioides difficile-associated disease
Papić et al. Eur J Clin Microbiol Infect Dis (2020)	retrospective cohort study	314 patients ≥ 65 years, treated with antimicrobial therapy ≥24hrs, and hospitalized ≥72hrs in a 36-month period	– 16% of the patients with NAFLD and 7.4% of patients in the control group developed in-hospital CAD	– NAFLD is an independent predictor of CAD
Šamadan et al. Antibiotics (2021)	retrospective cohort study	329 patients ≥ 60 years hospitalized with CAD, outcome: rCAD within 3 months of hospital discharge	– age > 75 years, NAFLD, CACI > 6, chronic kidney disease, statins and immobility were associated with rCAD	– NAFLD is a possible host-related risk factor associated with recurrent CAD
Bacteremia and bacterial infections
Gjurašin et al. CJI (2021)	retrospective cohort study	102 patients with invasive GBS	– in-hospital mortality was higher in patients with NAFLD	– NAFLD is associated with higher mortality in patients suffering from invasive GBS disease
Krznarić et al. IHEPCRO 2023	prospective observational cohort study	378 patients with community-acquired sepsis	– the prevalence of NAFLD was 46 % – the mortality of the NAFLD group was 18.9% and in controls 9.8%	– NAFLD was associated with mortality independently of other components of MetS – Patients with NAFLD more frequently developed ARDS, AKI, required IMV and CRRT
Vrsaljko et al IDweek 2023	prospective observational case-control study	80 patients with community-acquired sepsis (40 with NAFLD)	On admission, patients with NAFLD had higher TGF-β1 that decreased on day 5th of hospitalization	Patients without NAFLD had lower TGF-β1 concentrations on admission that significantly increased in a compensatory anti-inflammatory phase of the sepsis
Chronic hepatitis C
Radmanić et al IHEPCRO 2023	prospective observational cohort study	56 patients with CHC (28 with SLD)	Patients with SLD had higher EGF, SCF, VEGF and ANG serum concentrations	Presence of SLD significantly changed GF concentrations independently of fibrosis during DAA treatment
NAFLD and semaphorins
Šamadan et al Biomedicines (2022)	prospective case-control study	95 patients with NAFLD and 35 controls	NAFLD group significantly higher SEMA3A, -3C and -4D and lower SEAMA5A and -7A serum concentrations	SEMA3C and SEMA4D significantly increased and SEMA3A significantly decreased with fibrosis stages. SEMA5A rs1319222 TT genotype was more frequent in the NAFLD group.