Munther Hussain

Functional Study of CD4+CD25+ Regulatory T Cells in Health and Autoimmune Hepatitis

Regulatory CD4+CD25+ T cells (Tregs) are defective numerically and functionally in autoimmune hepatitis (AIH). We have investigated and compared the mechanism of action of Tregs in healthy subjects and in AIH patients using Transwell experiments, where Tregs are cultured either in direct contact with or separated from their targets by a semipermeable membrane. We also studied Treg FOXP3 expression and effect on apoptosis. Direct contact is necessary for Tregs to suppress proliferation and IFN-γ production by CD4+CD25− and CD8+ T cells in patients and controls. Moreover, in both, direct contact of Tregs with their targets leads to increased secretion of regulatory cytokines IL-4, IL-10, and TGF-β, suggesting a mechanism of linked immunosuppression. Tregs/CD4+CD25− T cell cocultures lead to similar changes in IFN-γ and IL-10 secretion in patients and controls, whereas increased TGF-β secretion is significantly lower in patients. In contrast, in patients, Tregs/CD8+ T cell cocultures lead to a higher increase of IL-4 secretion. In AIH, Treg FOXP3 expression is lower than in normal subjects. Both in patients and controls, FOXP3 expression is present also in CD4+CD25− T cells, although at a low level and not associated to suppressive function. Both in patients and controls, addition of Tregs does not influence target cell apoptosis, but in AIH, spontaneous apoptosis of CD4+CD25− T cells is reduced. In conclusion, Tregs act through a direct contact with their targets by modifying the cytokine profile and not inducing apoptosis. Deficient CD4+CD25− T cell spontaneous apoptosis may contribute to the development of autoimmunity.

Reduced monocyte HLA‐DR expression: A novel biomarker of disease severity and outcome in acetaminophen‐induced acute liver failure

Acute liver failure (ALF) shares striking similarities with septic shock where a decrease in HLA‐DR expression on monocytes is associated with disease severity and predicts outcome. We investigated monocyte HLA‐DR expression in ALF in relation to inflammatory mediator levels and clinical outcome. Monocyte HLA‐DR expression was determined in 50 patients with acetaminophen‐induced ALF (AALF) and 20 non–acetaminophen‐induced ALF (NAALF). AALF patients were divided into dead/transplanted (AALF‐NS, n = 26) and spontaneous survivors (AALF‐S, n = 24). Fifty patients with chronic liver disease (CLD) and 50 healthy volunteers served as controls. Monocyte HLA‐DR expression was determined by double‐color flow‐cytometry with monoclonal antibodies detecting HLA‐DR and monocyte specific CD14. Serum levels of interleukin (IL) ‐4, ‐6, ‐10, tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ were concomitantly measured by ELISA. Compared to healthy volunteers (75%) and CLD (67%) monocyte HLA‐DR percentage expression was lower in AALF (15%, P < .001) and NAALF (22 %, P < .001). Compared to AALF‐S, AALF‐NS had lower monocyte HLA‐DR % (11% vs. 36%, P < .001) and higher levels of IL‐4, IL‐6, IL‐10 and TNF‐α (P < .001). HLA‐DR percentage negatively correlated with INR, blood lactate, pH and levels of encephalopathy (r = −0.8 to −0.5, P < .01), IL‐10 (r = −0.8, P < .0001), TNF‐α (r = −0.4, P = .02). HLA‐DR percentage level ≤15% has a 96% sensitivity and 100% specificity and 98% accuracy in predicting poor prognosis. In conclusion, the strong relationship of monocyte HLA‐DR expression with indices of disease severity, mediators of inflammation and outcome indicates a key role for this molecule as a biomarker of disease severity and prognosis. (HEPATOLOGY 2006;44:34–43.)

Source and characterization of hepatic macrophages in acetaminophen‐induced acute liver failure in humans

Acetaminophen‐induced acute liver failure (AALF) is associated with innate immunity activation, which contributes to the severity of hepatic injury and clinical outcome. A marked increase in hepatic macrophages (h‐mϕ) is observed in experimental models of AALF, but controversy exists regarding their role, implicating h‐mϕ in both aggravation and resolution of liver injury. The role of h‐mϕ in human AALF is virtually unexplored. We sought to investigate the role of chemokine (C‐C motif) ligand 2 (CCL2) in the recruitment of circulating monocytes to the inflamed liver and to determine how the h‐mϕ infiltrate and liver microenvironment may contribute to tissue repair versus inflammation in AALF. We evaluated circulating monocytes, their chemokine (C‐C motif) receptor 2 (CCR2) expression, and serum CCL2 levels in patients with AALF. Cell subsets and numbers of circulation‐derived (MAC387+) or resident proliferating (CD68/Ki67+) h‐mϕ in hepatic immune infiltrates were determined by immunohistochemistry. Inflammatory cytokine levels were determined in whole and laser microdissected liver tissue by proteome array. In AALF, circulating monocytes were depleted, with the lowest levels observed in patients with adverse outcomes. CCL2 levels were high in AALF serum and hepatic tissue, and circulating monocyte subsets expressed CCR2, suggesting CCL2‐dependent hepatic monocyte recruitment. Significant numbers of both MAC387+ and CD68+ h‐mϕ were found in AALF compared with control liver tissue with a high proportion expressing the proliferation marker Ki67. Levels of CCL2, CCL3, interleukin (IL)‐6, IL‐10, and transforming growth factor‐β1 were significantly elevated in AALF liver tissue relative to chronic liver disease controls. Conclusion: In AALF, the h‐mϕ population is expanded in areas of necrosis, both through proliferation of resident cells and CCL2‐dependent recruitment of circulating monocytes. The presence of h‐mϕ within an anti‐inflammatory/regenerative microenvironment indicates that they are implicated in resolution of inflammation/tissue repair processes during AALF. (HEPATOLOGY 2012)

A multifaceted imbalance of T cells with regulatory function characterizes type 1 autoimmune hepatitis

Immunotolerance is maintained by regulatory T cells (Tregs), including CD4+CD25hi, CD8+CD28−, γδ, and CD3+CD56+ [natural killer T (NKT)] cells. CD4+CD25hi cells are impaired in children with autoimmune hepatitis (AIH). Little is known about Tregs in adults with AIH. The aim of this study was to investigate the frequency and function of Treg subsets in adult patients with AIH during periods of active disease and remission. Forty‐seven AIH patients (16 with active disease and 31 in remission) and 28 healthy controls were studied. Flow cytometry was used to evaluate surface markers and function‐related intracellular molecules in γδ, CD8+CD28−, NKT, and CD4+CD25hi cells. CD4+CD25hi T cell function was determined by the ability to suppress proliferation and interferon gamma (IFN‐γ) production by CD4+CD25− target cells. Liver forkhead box P3–positive (FOXP3+) cells were sought by immunohistochemistry. In AIH patients, particularly during active disease, CD4+CD25hi T cells were fewer, expressed lower levels of FOXP3, and were less effective at inhibiting target cell proliferation versus healthy controls. Moreover, although the numbers of CD8+CD28− T cells were similar in AIH patients and healthy controls, NKT cells were numerically reduced, especially during active disease, and produced lower quantities of the immunoregulatory cytokine interleukin‐4 versus controls. In contrast, γδ T cells in AIH patients were more numerous versus healthy controls and had an inverted Vδ1/Vδ2 ratio and higher IFN‐γ and granzyme B production; the latter was correlated to biochemical indices of liver damage. There were few FOXP3+ cells within the portal tract inflammatory infiltrate. Conclusion: Our data show that the defect in immunoregulation in adult AIH is complex, and γδ T cells are likely to be effectors of liver damage. (HEPATOLOGY 2010)

Cytochrome P450IID6-specific CD8 T cell immune responses mirror disease activity in autoimmune hepatitis type 2†

Autoimmune hepatitis type 2 (AIH‐2) is a severe organ‐specific disorder characterized by liver kidney microsomal antibody type 1 targeting cytochrome P4502D6 (CYP2D6). Growing evidence implicates the involvement of CD8 T cell immune responses in its pathogenesis. We investigated CYP2D6‐specific CD8 T cell human leukocyte antigen (HLA)‐A2 restricted responses in AIH‐2 (20 patients, 11 HLA‐A2+). Binding affinity of CYP2D6 peptides to HLA‐A2 was predicted by the algorithm SYFPEITHI and assessed in vivo by T2 cell assays. CD8 T cell interferon (IFN)‐γ production was assessed via intracellular cytokine staining, cytotoxicity via chromium release assay, and frequency of circulating and intrahepatic CYP2D6‐specific CD8 T cells via tetramer staining. CYP2D6‐specific CD8 T cell reactivity was tested at diagnosis and during treatment and correlated with indices of disease activity. Seven CYP2D6 peptides with high HLA‐A2 binding affinity colocalizing with known B cell or CD4 T cell epitopes were selected. Five sequences inducing high levels of IFN‐γ were used for HLA‐A2 tetramer construction. Frequency, IFN‐γ production, and cytotoxicity of CYP2D6‐specific CD8 T cells were higher at diagnosis than during treatment. Intensity of CYP2D6‐specific CD8 T cell responses correlated with disease activity. Immune responses to CYP2D6245‐254 were the strongest both at diagnosis and during treatment. Conclusion: HLA‐A2–restricted, CYP2D6‐specific CD8 T cell immune responses vary according to disease stage and correlate with hepatocyte damage. CD8 T cell targets on CYP2D6—in particular CYP2D6245‐254—may be the focus of novel immune intervention in AIH‐2. (HEPATOLOGY 2007.)

Serum levels of CK18 M30 and leptin are useful predictors of steatohepatitis and fibrosis in paediatric NAFLD.

Background: With the alarming growth in prevalence of paediatric nonalcoholic fatty liver disease (NAFLD), there is a need for noninvasive methods of stratifying disease severity. Our aim was to evaluate a combination of serum biomarkers as a measure of disease activity in paediatric NAFLD. Patients and Methods: Forty-five children with biopsy-proven NAFLD were enrolled. Caspase-cleaved CK18 fragments (CK18 M30), hyaluronic acid, leptin, and adiponectin were measured in serum using enzyme-linked immunosorbent assays and high-sensitivity C-reactive protein using a colorimetric assay. Results: Median age was 12.7 years (55% boys). Median body mass index z score was 1.7. CK18 M30 levels were significantly higher in patients with NAFLD versus controls, median 288 IU/L versus 172 IU/L (P < 0.001), and in those with steatohepatitis, median 347 IU/L versus simple steatosis (NAFLD activity score < 3), median 191 IU/L (P = 0.006). Significant fibrosis (≥F2) could be differentiated from no/minimal fibrosis (<F2), median 393 IU/L versus 243 IU/L (P = 0.03). Leptin could distinguish <F2 from ≥F2; 28.9 ng/mL versus 70.1 ng/mL (P = 0.037). Adiponectin, hyaluronic acid, and high-sensitivity C-reactive protein did not achieve significance in predicting steatohepatitis nor significant fibrosis. Conclusions: The present study combines use of markers for different processes in the development of steatohepatitis. Serum biomarkers, especially CK18 M30, are useful in stratifying disease severity in paediatric NAFLD.

Serial circulating markers of inflammation in biliary atresia—Evolution of the post‐operative inflammatory process

Biliary atresia (BA) may be characterized as an occlusive cholangiopathy affecting both intra‐ and extra‐hepatic parts of the biliary tree, together with a pronounced inflammatory response consisting of hepatic infiltration of (predominantly) CD4+ lymphocytes and macrophages. Soluble cellular adhesion molecules are also known to be raised at the time of portoenterostomy, presumably reflecting intrahepatic disease. We investigated this measurable inflammatory component longitudinally by studying a panel of cellular adhesion molecules (soluble intercellular adhesion molecule‐1 [sICAM‐1], soluble vascular cell adhesion molecule‐1 [sVCAM‐1]) and soluble proinflammatory mediators (T helper 1 [interleukin {IL}‐2 and interferonγ] and T helper 2 [IL‐4 and IL‐10]) cytokines and macrophage markers (tumor necrosis factor [TNF] α and IL‐18) in 21 consecutive infants with BA post‐Kasai portoenterostomy (KP). The levels of all adhesion molecules and cytokines (except IL‐10) increased progressively by 6 months post‐portoenterostomy. The response was non‐polarized but with 100‐fold increases in IL‐2, TNFα and IL‐18 particularly but only modest elevations in IL‐10. When proinflammatory profiles were related to outcome, we found poor discrimination if assessed as clearance of jaundice but markedly higher values for IL‐2, interferonγ, IL‐4, IL‐10, TNFα and sICAM‐1 for those who would be transplanted by 1 year. Using ROC curve analysis for sICAM‐1 levels at 1 month post‐KP, a cutoff level of 1,779 ng/ml was determined to predict the need for transplantation at 1 year with 92% specificity and 87% sensitivity. Conclusion: The early circulating inflammatory process in BA is persistent, progressive and involves a non‐polarized T cell, macrophage and cell adhesion molecule response only partially ameliorated by KP. (HEPATOLOGY 2007;46:180–187.)

Autoantigen-specific regulatory T cells, a potential tool for immune-tolerance reconstitution in type-2 autoimmune hepatitis†

Effector CD4 and CD8 T cell immune responses to cytochrome P450IID6 (CYP2D6), the autoantigen of autoimmune hepatitis type 2 (AIH‐2), are permitted by a numerical and functional impairment of CD4posCD25high regulatory T cells (T‐regs). We aimed to investigate whether T‐regs specific for CYP2D6 immunodominant regions and restricted by the appropriate human leukocyte antigen (HLA)‐DR molecule can be generated in patients with AIH‐2 and can control CD4 and CD8 T cell effectors targeting identical or overlapping CYP2D6 regions. CYP2D6‐specific regulatory T cells (CYP2D6 T‐regs) were obtained from peptide‐pulsed monocyte‐depleted peripheral blood mononuclear cells of 17 patients with AIH‐2, who were positive for the predisposing HLA‐DR7 and/or HLA‐DR3 alleles. Their antigen specificity was assessed by cytofluorimetry using HLA class II tetramers and their cytokine profile by intracellular staining. T‐reg ability to suppress was ascertained by measuring reduction of CD4posCD25neg cell proliferation/effector cytokine secretion and of CD8 T cell cytotoxicity. The most efficient suppression of effector T cell proliferation, inflammatory cytokine release, and cytotoxicity was obtained by coculturing T‐regs with CYP2D6‐peptide‐loaded semimature dendritic cells (smDCs), and smDC‐CYP2D6 T‐regs also expressed high levels of FOXP3 (forkhead box P3). Possession of the appropriate HLA‐DR molecule and recognition of the CYP2D6 autoantigenic sequence were critical to the synergistic smDC‐CYP2D6 T‐reg immunoregulatory functions, and lack of either element led to poor control of responder cell proliferation and cytokine secretion. Moreover, interferon‐γ neutralization significantly boosted the suppressive ability of CYP2D6 T‐regs. Conclusion: T‐regs generated under CYP2D6‐specific conditions and cocultured with smDCs are highly effective at controlling autoreactive T cells, thus providing the basis for a powerful and tailored form of immunotherapy for AIH‐2. (HEPATOLOGY 2011;53:536‐547)

Vigorous Activation of Monocytes in Juvenile Autoimmune Liver Disease Escapes the Control of Regulatory T-Cells

Interface hepatitis, the histological lesion typical of autoimmune hepatitis (AIH), is composed of CD4 and CD8 T lymphocytes and of innate immunity cells, particularly monocytes. Studies in AIH have focused on autoreactive CD4 and CD8 T cells and impairment of CD4+CD25+ regulatory T cells (T‐regs), whereas little is known about the role of monocytes and their relationship with T‐regs. We have investigated 51 patients with autoimmune liver disease (AILD) and 27 healthy subjects, finding that monocytes were higher in number (P = 0.044), had a more vigorous spontaneous migration (P < 0.0005 in patients with inactive disease [ID], and P < 0.001 in those with active disease [AD]), displayed a higher tumor necrosis factor alpha (TNF‐α) over interleukin (IL)‐10 production (P = 0.07 in ID and P = 0.0005 in AD), and expressed higher levels of Toll‐like receptor (TLR) 4 (P = 0.048 in ID and P = 0.03 in AD). Addition of conventional T‐regs (cT‐regs) in AILD enhanced monocyte migration (P = 0.05 in ID and P = 0.08 in AD), magnified TNF‐α over IL‐10 production (P = 0.0005 in ID and P = 0.006 in AD), and markedly increased TLR4 expression levels (P = 0.01 in ID and P = 0.004 in AD), whereas in normal subjects it either restrained or left unchanged monocyte function. Because a CD127‐negative subpopulation within CD4+CD25+ T cells exerts the strongest regulatory activity, we performed additional experiments using purified CD4+CD25+CD127− T cells (true T‐regs [tT‐regs]). Addition of tT‐regs to monocytes decreased monocyte migration (P = 0.03) and promoted IL‐10 production (P = 0.009), leaving unchanged TLR4 expression in healthy subjects, whereas in patients with AILD it induced only a marginal increase in IL‐10 production (P = 0.045 in ID and P = 0.13 in AD). Conclusion: Monocyte overactivation and inability of cT‐regs and tT‐regs to restrain it may contribute to the loss of immune tolerance and perpetuation of the autoimmune attack in AILD. (HEPATOLOGY 2009.)

Cytokine overproduction in healthy first degree relatives of patients with IDDM

Summary Healthy family members of patients with insulin-dependent diabetes mellitus (IDDM) are known to share a number of immunological abnormalities with their affected relatives. Since monocyte and type 1 T-cell-derived cytokines contribute to the pathogenesis of IDDM, we studied the production of these cytokines in the healthy first degree relatives of 29 children with IDDM. We report that circulating tumour necrosis factor-α (TNF-α) and soluble interleukin-2 (sIL-2) receptor were present in increased amounts in non-diabetic family members at levels similar to those found in the diabetic children (duration of disease 3 months–5 years). Furthermore, marked hypersecretion of IL-1α and TNF-α by mitogen-stimulated peripheral blood mononuclear cells was found in both diabetic and healthy family members. Abnormalities of cytokine production in healthy relatives did not correlate with the presence of islet cell antibodies or with HLA DR type. These data indicate that healthy family members of patients with IDDM exhibit overproduction of a number of cytokines that have been implicated in diabetogenesis. [Diabetologia (1998) 41: 343–349]