Cristina Bozzola

Adaptive immune responses triggered by oxidative stress contribute to hepatic inflammation in NASH

Previous studies have shown that human nonalcoholic steatohepatitis (NASH) is often associated with the presence of circulating antibodies against protein adducted by lipid peroxidation products. Here we used the methionine‐choline deficient (MCD) model of NASH to characterize the possible involvement of adaptive immunity in NASH. In mice fed up to 8 weeks with the MCD diet the extension of liver injury and lobular inflammation paralleled the development of immunoglobulin G (IgG) against malonyldialdehyde (MDA) and 4‐hydroxynonenal (4‐HNE)‐derived antigens as well as with the hepatic recruitment of CD4+ and CD8+ T‐lymphocytes responsive to the same antigens. Moreover, in these animals the individual IgG reactivity against MDA‐adducts positively correlated with transaminase release and hepatic tumor necrosis factor alpha (TNF‐α) expression. To substantiate the role of immune responses triggered by oxidative stress in the progression of NASH, mice were immunized with MDA‐adducted bovine serum albumin (MDA‐BSA) before feeding the MCD diet. MDA‐BSA immunization did not affect control mice livers, but further stimulated transaminase release, lobular inflammation, and the hepatic expression of proinflammatory cytokine in MCD‐fed mice. The increased severity of NASH in immunized MCD‐fed mice involved liver recruitment and the T helper (Th)‐1 activation of CD4+ T cells that, in turn, further stimulated macrophage M1 responses. Moreover, hepatic fibrosis was also evident in these animals in relation with an IL‐15‐mediated increase of natural killer T‐cells (NKT) and the up‐regulation in liver production of osteopontin by NKT cells and hepatic macrophages. Conclusion: These results indicate that oxidative stress can contribute to the progression of NASH by stimulating both humoral and cellular immune responses, pointing to the possible role of adaptive immunity in the pathogenesis of the disease. (Hepatology 2014;59:886–897)

Endogenous annexin A1 is a novel protective determinant in nonalcoholic steatohepatitis in mice

Annexin A1 (AnxA1) is an effector of the resolution of inflammation and is highly effective in terminating acute inflammatory responses. However, its role in chronic settings is less investigated. Because changes in AnxA1 expression within adipose tissue characterize obesity in mice and humans, we queried a possible role for AnxA1 in the pathogenesis of nonalcoholic steatohepatitis (NASH), a disease commonly associated with obesity. NASH was induced in wild‐type (WT) and AnxA1 knockout (AnxA1 KO) C57BL/6 mice by feeding a methionine‐choline deficient (MCD) diet up to 8 weeks. In MCD‐fed WT mice, hepatic AnxA1 increased in parallel with progression of liver injury. This mediator was also detected in liver biopsies from patients with NASH and its degree of expression inversely correlated with the extent of fibrosis. In both humans and rodents, AnxA1 production was selectively localized in liver macrophages. NASH in AnxA1 KO mice was characterized by enhanced lobular inflammation resulting from increased macrophage recruitment and exacerbation of the M1 phenotype. Consistently, in vitro addition of recombinant AnxA1 to macrophages isolated from NASH livers down‐modulated M1 polarization through stimulation of interleukin‐10 production. Furthermore, the degree of hepatic fibrosis was enhanced in MCD‐fed AnxA1 KO mice, an effect associated with augmented liver production of the profibrotic lectin, galectin‐3. Accordingly, AnxA1 addition to isolated hepatic macrophages reduced galectin‐3 expression. Conclusions: Macrophage‐derived AnxA1 plays a functional role in modulating hepatic inflammation and fibrogenesis during NASH progression, suggesting the possible use of AnxA1 analogs for therapeutic control of this disease. (Hepatology 2014;60:531–544)

NF-κB1 deficiency stimulates the progression of non-alcoholic steatohepatitis (NASH) in mice by promoting NKT-cell-mediated responses.

Growing evidence indicates that NF-κB (nuclear factor κB) activation contributes to the pathogenesis of NASH (non-alcoholic steatohepatisis). Among the NF-κB subunits, p50/NF-κB1 has regulatory activities down-modulating NF-κB-mediated responses. In the present study, we investigated the effects of NF-κB1 deficiency on the progression of NASH induced by feeding mice on an MCD (methionine/choline-deficient) diet. Following 4 weeks on the MCD diet, steatosis, ALT (alanine aminotransferase) release, hepatocyte apoptosis, lobular inflammation and TNFα (tumour necrosis factor α) production were higher in NF-κB1(-/-) (NF-κB1-knockout) mice than in WT (wild-type) mice. NF-κB1(-/-) mice also showed appreciable centrilobular collagen deposition, an increased number of activated hepatic stellate cells and higher type-I procollagen-α and TIMP-1 (tissue inhibitor of metalloproteases-1) mRNA expression. Although NF-κB p50 homodimers regulate macrophage activation, the number of hepatic macrophages and liver mRNAs for iNOS (inducible NO synthase), IL (interleukin)-12p40, CCL2 (CC chemokine ligand 2) and CXCL10 (CXC chemokine ligand 10) were comparable in the two strains. NASH was associated with an increase in liver infiltrating T-cells that was more evident in MCD-fed NF-κB1(-/-) than in similarly treated WT mice. Flow cytorimetry showed that T-cell recruitment involved effector CD8+ T-cells without changes in the helper CD4+ T-cell fraction. Furthermore, although NASH lowered hepatic NKT cells [NK (natural killer) T-cells] in WT mice, the NKT cell pool was selectively increased in the livers of MCD-fed NF-κB1(-/-) mice. Such NKT cell recruitment was associated with an early overexpression of IL-15, a cytokine controlling NKT cell survival and maturation. In the livers of MCD-fed NF-κB1(-/-) mice, but not in those of WT littermates, we also observed an up-regulation in the production of NKT-related cytokines IFN (interferon)-γ and osteopontin. Taken together, these results indicate that NF-κB1 down-modulation enhanced NASH progression to fibrosis by favouring NKT cell recruitment, stressing the contribution of NKT cells in the pathogenesis of NASH.

Renal outcome and monoclonal immunoglobulin deposition disease in 289 old patients with blood cell dyscrasias: A single center experience

Monoclonal components (MC) formed by chains/fragments of intact/truncated globulin components produced in different lymphoproliferative diseases are responsible for monoclonal immunoglobulin deposition disease (MIDD) and consequent tissue damage by organized (amyloid fibrils) or non-organized (amorphous) deposits. The kidneys are the most commonly affected organs in MIDD, and renal failure represents an important adverse factor for prognosis. The renal outcome and the role of renal pathology in diagnosing MIDD was evaluated in 289 elderly patients with multiple myeloma (MM, n=115) and monoclonal gammopathy (MGUS, n=174). Renal impairment was the only significant risk factor for patient death, while significant risk factors for renal impairment were diabetes (HR 3.65, 95% CI: 2.08-6.41), light chain (LC) proteinuria (HR 2.18; 95% CI: 1.10-4.32) and type of MC (p=0.0019). Renal pathology documented MIDD in 12/30 cases (40%): six cases of AL-amyloidosis, two of LC disease, one of heavy chain disease and three of cast nephropathy, as well as four cases of glomerulonephritis, eight of arteriolosclerosis and six of normal picture. Main conclusions are that diabetes, sharing common glomerular damage with LC disease, is the strongest risk factor for progression of renal disease, and glomerular proteinuria or heavy LC proteinuria should raise a strong suspicion index of MIDD and prompt pathology assessment to reach the correct diagnosis.

Heterozygous β -globin gene mutations as a risk factor for iron accumulation and liver fibrosis in chronic hepatitis c

Background: Iron accumulation is a well-known risk factor for the progression of chronic hepatitis C (CHC) to fibrosis. However, the profibrogenic role of the genes controlling iron homeostasis is still controversial. Aim: To evaluate the relative role of haemachromatosis (HFE), ferroportin and β-globin gene mutations in promoting iron accumulation and fibrosis in patients with CHC. Methods: Genetic analysis was performed together with the assessment of hepatic iron content and histology in 100 consecutive HIV-antibody and hepatitis B surface antigen-negative patients with biopsy-proven CHC. Results: Among the patients investigated, 12 were heterozygous for various β-globin gene mutations (39[C→T], IVS1.1[G→A], 22 7 bp deletion and IVS1.6[T→C]) and 29 carried HFE (C282Y, H63D and S65C) gene mutations. One further patient was heterozygous for both HFE (H63D) and β-globin (39[C→T]) variants, whereas 58 had the wild-type alleles of both the genes. Hepatic iron concentration (HIC) and hepatic stainable iron were significantly higher (p<0.05) in patients with CHC carrying β-globin mutations than in those with HFE mutations or the wild-type alleles. Multivariate analysis confirmed that the presence of β-globin mutations was independently associated with both HIC (p = 0.008) and hepatic-stainable iron (odds ratio (OR) 6.11; 95% CI 1.56 to 23.92; p = 0.009). Moderate/severe fibrosis or cirrhosis (Ishak’s score >2) was observed in 48 of 100 patients. Logistic regression demonstrated that age (OR 1.05; 95% CI 1.02 to 1.09; p<0.005) and β-globin mutations (OR 4.99; 95% CI 1.22 to 20.3; p = 0.025) were independent predictors of the severity of fibrosis. Conclusions: Heterozygosis for β-globin mutations is a novel risk factor for both hepatic iron accumulation and the progression to fibrosis in patients with CHC.

Decreased Kidney Function and Crystal Deposition in the Tubules After Kidney Transplant

Adenine phosphoribosyltransferase (APRT) deficiency is an autosomal recessive purine enzyme defect that results in the inability to utilize adenine, which consequently is oxidized by xanthine dehydrogenase to 2,8-dihydroxyadenine (2,8-DHA), an extremely insoluble substance eventually leading to crystalluria, nephrolithiasis, and kidney injury. We describe a case of APRT deficiency not diagnosed until the evaluation of a poorly functioning kidney transplant in a 67-year-old white woman. After the transplant, there was delayed transplant function, urine specimens showed crystals with unusual appearance, and the transplant biopsy specimen showed intratubular obstruction by crystals identified as 2,8-DHA using infrared spectroscopy. APRT enzymatic activity was undetectable in red blood cell lysates, and analysis of the APRT gene showed 1 heterozygous sequence variant, a duplication of T at position 1832. The patient was treated with allopurinol, 300 mg/d, and transplant function progressively normalized. Because patients with undiagnosed APRT deficiency who undergo kidney transplant may risk losing the transplant because of an otherwise treatable disease, increased physician awareness may hasten the diagnosis and limit the morbidity associated with this disease.

CX3CR1-expressing inflammatory dendritic cells contribute to the progression of steatohepatitis.

Liver monocytes play a major role in the development of NASH (non-alcoholic steatohepatitis). In inflamed tissues, monocytes can differentiate in both macrophages and dendritic cells. In the present study, we investigated the role of moDCs (monocyte-derived inflammatory dendritic cells) in experimental steatohepatitis induced in C57BL/6 mice by feeding on a MCD (methionine/choline-deficient) diet. The evolution of steatohepatitis was characterized by an increase in hepatic CD45+ / CD11b+ myeloid cells displaying the monocyte/macrophage marker F4-80(+). In the early phases (4 weeks of treatment), Ly6C(high)/CD11b(+)/F4-80(+) inflammatory macrophages predominated. However, their frequency did not grow further with the disease progression (8 weeks of treatment), when a 4-fold expansion of CD11b(+)/F4-80(+) cells featuring the fractalkine receptor (CX3CR1) was evident. These CX3CR1+ cells were also characterized by the combined expression of inflammatory monocyte (Ly6C, CD11b) and dendritic cell (CD11c, MHCII) markers as well as by a sustained TNFα (tumour necrosis factor α) production, suggesting monocyte differentiation into inflammatory moDCs. The expansion of TNFα-producing CX3CR1+ moDCs was associated with an elevation in hepatic and circulating TNFα level and with the worsening of parenchymal injury. Hydrogen sulfide (H2S) has been shown to interfere with CX3CR1 up-regulation in monocyte-derived cells exposed to pro-inflammatory stimuli. Treating 4-week-MCD-fed mice with the H2S donor NaHS while continuing on the same diet prevented the accumulation of TNFα-producing CX3CR1+ moDCs without interfering with hepatic macrophage functions. Furthermore, NaHS reduced hepatic and circulating TNFα levels and ameliorated transaminase release and parenchymal injury. Altogether, these results show that inflammatory CX3CR1+ moDCs contributed in sustaining inflammation and liver injury during steatohepatitis progression.

Pitfall in nephrology: contrast nephropathy has to be differentiated from renal damage due to atheroembolic disease.

INTRODUCTION The topic of contrast-induced nephropathy (CIN) has been receiving an enormous amount of interest in recent times; however, this review is not a review of what CIN is, but what it is not. METHODS We will review the main topics demonstrating that the post hoc ergo propter hoc assumption that renal impairment occurring after contrast medium (CM) infusion is necessarily because of it, is wrong, as we are dealing with different diseases, depending on the way the CM is administered and on the type of patient. RESULTS After >1,000 often repetitive papers, we must deal with an unacceptably wide range of incidences of CIN, with completely different prognoses and astonishingly conflicting results regarding the efficacy of preventive measures with the exception of hydration. So what went wrong? How to separate tares from wheat? When years ago we challenged the diagnosis of CIN, the words cholesterol embolism had never appeared in this setting. Now, we can split the possible renal dysfunctions following CM administration into CM-related hemodynamic and/or tubular damage, cholesterol embolism, ischemia from acute blood loss or hypotension/hypoperfusion and nephrotoxicity from concomitant drugs. CONCLUSIONS In a setting regarding millions of patients and millions of dollars/year, in order to clarify the true renal damage directly related to CM, we ask for prospective studies differentiating cohorts receiving intravenous and intra-arterial, transradial and transfemoral injections, and clinically relevant renal outcomes, thus avoiding the dangers that can come from the idolatry of a surrogate end point such an asymptomatic 25% transient increase of serum creatinine. To avoid that, patients may lose the possibility of a more useful radiological diagnosis, because of an exaggerated suspicion of risk.

A case-control histological study on the effects of phlebotomy in patients with chronic hepatitis C.

Objective The aim of this study was to assess the actual effectiveness of long-term phlebotomy by comparing histological improvement (HI) in 69 Caucasian HCV-RNA-positive CHC patients undergoing phlebotomy or receiving an interferon-based therapy without virological response [nonresponders to interferon therapy(IBT-NR)]. Methods HI was defined by at least one point reduction of the staging score or, in the case of unchanged stage, by at least two points reduction of the grading score (Knodel’s Activity Index) and was retrospectively evaluated by comparing two consecutive (56±28 months apart) liver biopsies from 30 phlebotomized and 39 IBT-NR patients. Results HI was observed in 15 of 30 (50%) patients treated with phlebotomy and in six of 39 (15%) IBT-NR subjects (P=0.002). Furthermore, AST, ALT, and GGT serum levels were significantly reduced only in phlebotomized patients (P⩽0.003) at the time of the second biopsy. Univariate and multivariate analysis showed that histological grading score before therapy (P=0.001) and phlebotomy (P=0.002) were independently predictors of HI. Conclusion HI induced by long-term phlebotomy effectively exceeds that spontaneously occurring in patients IBT-NR confirming the efficacy of iron depletion in attenuating CHC progression when other therapies have failed.

Fat-laden macrophages modulate lobular inflammation in nonalcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) is characterized by extensive hepatic monocyte infiltration and monocyte-derived macrophages have an important role in regulating the disease evolution. However, little is known about the functional changes occurring in liver macrophages during NASH progression. In this study, we investigated phenotypic and functional modifications of hepatic macrophages in experimental NASH induced by feeding C57BL/6 mice with a methionine-choline deficient (MCD) diet up to 8weeks. In mice with steatohepatitis liver F4/80-positive macrophages increased in parallel with the disease progression and formed small clusters of enlarged and vacuolated cells. At immunofluorescence these cells contained lipid vesicles positive for the apoptotic cell marker Annexin V suggesting the phagocytosis of apoptotic bodies derived from dead fat-laden hepatocytes. Flow cytometry revealed that these enlarged macrophages expressed inflammatory monocyte (CD11b, Ly6C, TNF-α) markers. However, as compared to regular size macrophages the enlarged sub-set was characterized by an enhanced production of arginase-1 and of the anti-inflammatory mediators IL-10 and annexin A1. Similar vacuolated macrophages producing annexin A1 were also evident in liver biopsies of NASH patients. In mice with NASH, the accumulation of enlarged F4/80(+) cells paralleled with a decline in the expression of the macrophage M1 activation markers iNOS, IL-12 and CXCL10, while the levels of M2 polarization markers arginase-1 and MGL-1 were unchanged. Interestingly, the lowering of IL-12 expression mainly involved the macrophage sub-set with regular size. We conclude that during the progression of NASH fat accumulation within liver macrophages promotes the production of anti-inflammatory mediators that influence hepatic inflammatory responses.