- Email: [email protected]
Overexpression of MHCII by hepatocytes in alcoholic hepatitis (AH) compared to non-alcoholic steatohepatitis (NASH) and normal controls
Journal Pre-proof Overexpression of MHCII by hepatocytes in Alcoholic Hepatitis (AH) compared to Non-alcoholic Steatohepatitis (NASH) and normal controls Jiajie G. Lu, Askalu Iyasu, Barbara French, Brittany Tillman, Samuel W. French, Samuel W. French, M.D PII:
S0741-8329(19)30067-9
DOI:
https://doi.org/10.1016/j.alcohol.2019.08.008
Reference:
ALC 6939
To appear in:
Alcohol
Received Date: 13 April 2019 Revised Date:
21 August 2019
Accepted Date: 30 August 2019
Please cite this article as: Lu J.G., Iyasu A., French B., Tillman B., French S.W., French S.W. & M.D Overexpression of MHCII by hepatocytes in Alcoholic Hepatitis (AH) compared to Nonalcoholic Steatohepatitis (NASH) and normal controls, Alcohol (2019), doi: https://doi.org/10.1016/ j.alcohol.2019.08.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Overexpression of MHCII by hepatocytes in Alcoholic Hepatitis (AH) compared to Non-alcoholic Steatohepatitis (NASH) and normal controls.
Jiajie G. Lu, Askalu Iyasu, Barbara French, Brittany Tillman, Samuel W. French
Samuel W. French, M.D. Harbor-UCLA Medical Center 1000 W Carson Street Torrance, CA 90502 310-222-2643 Telephone 310-222-8002 Fax [email protected]
Highlights: -
There is little characterization of lymphocytic response in alcoholic hepatitis. In human liver biopsies, we show aberrant MHCII expression in hepatocytes affected by alcoholic hepatitis, at greater levels than in non-alcoholic steatohepatitis (NASH) and normal controls. Further, we show CD4 positive lymphocytes associated with these hepatocytes and eating them piece by piece Expression levels of IL-1α are higher in alcoholic hepatitis compared to NASH and controls. These results show the importance of autoimmune mechanisms in alcoholic hepatitis.
Abstract: Previously we have shown that in autoimmune hepatitis CD4 positive lymphocytes form an immunologic synapse with hepatocytes, leading to gradual diminishing and elimination of the hepatocyte. We wondered whether a similar mechanism may occur in alcoholic hepatitis (AH) and non-alcoholic steatohepatitis (NASH). We conducted immunofluorescence studies of expression of MHCII, the binding partner of CD4, on patient liver biopsies of AH, NASH, and normal controls. In cases of alcoholic hepatitis, there was prominent sinusoidal expression of MHC II; In NASH biopsies there was comparatively lower expression of MHC II, but still more than control tissue. Immunohistochemical stain for CD4 showed CD4 positive lymphocytes closely associated with hepatocytes in AH biopsies. Furthermore, expression levels of the multifunctional cytokine IL-1α was higher in AH compared to NASH and control biopsies. These results underlie the more severe nature of alcoholic hepatitis and underscore the autoimmune mechanisms involved in the liver damage found in alcoholic hepatitis. Key words: alcoholic hepatitis, non-alcoholic steatohepatitis, MHCII, CD4, immune synapse Introduction
Alcoholic hepatitis (AH) is characterized histologically by hepatocyte balloon cell change, Mallory-Denk body formation, and neutrophilic infiltration (Kumar et al., 2015). It is often accompanied by steatosis and fibrosis. The mechanism of liver damage in alcoholic hepatitis is multifactorial, including lipid peroxidation, acetaldehyde-protein adduct formation, reactive oxygen species production, and inflammatory response (Wang et al., 2012). Recently we showed that in autoimmune hepatitis, CD4 positive lymphocytes infiltrate the parenchyma and gradually nibble away the hepatocytes (French and Lu, 2018). Electron microscopy showed a lymphocyte forming a 15-20 µm immunological synapse with a hepatocyte. We questioned whether a similar phenomenon occurs in AH or non-alcoholic hepatitis (NASH). We examined liver biopsies of de-identified patients with AH, NASH, or no liver disease and examined the level of expression of MHC II, the binding partner of CD4, in these biopsies. Methods Human formalin-fixed paraffin-embedded liver biopsies from 10 alcoholic hepatitis, 3 NASH, and 3 normal controls were obtained from Harbor UCLA hospital archives and clinical trials to treat alcoholic hepatitis at VA Hospital Long Beach, USC, and UC Davis. Liver biopsy sections were cut 4 µm thick. The study was carried out in accordance with the Declaration of Helsinki and designated as exempt by our institutional review board. Each liver slide was double stained with protein-specific antibodies for MHCII and CAM5.2, followed by secondary fluorescence antibodies. In a separate experiment, each liver slide was double stained with antibodies for IL-1α and ubiquitin, followed by secondary fluorescence antibodies. The biopsies were all stained at the same time in order to allow comparison of fluorescence intensities between cases. Rabbit anti-MHCII antibody was obtained from Abcam, Cambridge MA and mouse anti-CAM5.2 antibody from Sigma Aldrich, Carlsbad, CA. The slides were also nuclear stained by DAPI. For each protein, we measured the intensity of the fluorescent staining in three different areas of the biopsies with 40x magnification and 800ms standard exposure time, using a Nikon 400 fluorescent microscope. On each slide area, 10 peak fluorescence intensities were measured. The Nikon morphometric system was used to quantitate the fluorescent intensity of liver cells and sinusoids for each individual antibody. The mean, standard error, and statistical differences of data achieved from the Nikon were analyzed by Sigma Pletand statistical software. Controls vs ASH, controls vs NASH, and ASH vs NASH were compared by unpaired t-test with a p-value of <0.05 considered statistically significant. Results We investigated the expression of MHCII in liver biopsies showing alcoholic hepatitis (AH) compared to nonalcoholic steatohepatitis (NASH) and controls. In AH biopsies there was prominent expression of MHCII in the hepatic sinusoids and on hepatocytes (Figure 1A-B). Expression of MHCII in the NASH biopsies also occurred in a sinusoidal pattern (Figure
1C) but was much less intense compared to ASH. Normal liver controls showed virtually no expression of MHC II (Figure 1D). Analysis of MHCII fluorescence intensity showed statistically significant differences in AH, NASH, and normal livers (Figure 2). MHCII fluorescent intensity in AH was much higher than in NASH (196.0 ± 24.7 vs. 41.8 ± 15.2, p < 0.001), while MHCII immunofluorescence in NASH was somewhat higher than in the normal controls (41.8 ± 15.2 vs. 31.3 ± 10.8, p = 0.003). We performed immunohistochemical stains of CD4 on selected liver biopsies. The AH biopsies showed large numbers of CD4 positive lymphocytes and strong staining of CD4 in the hepatic sinusoids, compared to the lower numbers in NASH and none in control tissue (Figure 3). Electron microscopy of an AH biopsy shows sinusoidal lymphocytes binding to hepatocytes (Figure 3D). Finally, we examined the expression of the cytokine interleukin-1α (IL-1α) on AH, NASH, and control biopsies (Figure 4). AH biopsies show increased IL-1α fluorescence intensity compared to NASH (183.1 ± 26.8 vs. 157.5 ± 50.1, p = 0.015) and controls (183.1 ± 26.8 vs. 104.7 ± 12.5, p < 0.001). Discussion Alcoholic hepatitis is associated with an immune response that targets hepatocytes. The nature of this immune response is complex. For instance, two studies report formation of autoantibodies against CYP2E1 and CYPEA4 in alcoholic hepatitis (Lytton et al., 1998) (Albano et al., 1998). Autoantibodies can also develop to hydroxyethyl radical protein adducts (Clot et al., 1995) (Albano et al., 1999) and acetaldehyde binding to cellular proteins (Worrall et al., 1989). Other mechanisms include lipopolysaccharide-induced acute inflammation due to increased gut epithelial membrane permeability (Rao 2009), direct response to free fatty acids (Mavrelis et al., 1983), and reactive oxygen species formation (Lakshminarayanan et al., 1997). In total these responses induce a primarily acute inflammatory response including neutrophils and macrophages. Lymphocyte response in alcoholic hepatitis is less prominent and less well described (Wang et al., 2012). Furthermore, direct cytotoxic effects to hepatocytes by lymphocytes in alcoholic hepatitis, to our knowledge, have not been described. Our cases of alcoholic hepatitis showed overexpression of MHCII on hepatocellular membranes and Kupffer cells. It has been shown that human hepatocytes can be induced to express MHCII by interferon gamma (Franco et al., 1988). Further, in mice, hepatocytes that constitutively express MHCII are able to activate CD4 positive lymphocytes (Herkel et al., 2003). As MHCII loaded with antigen is the binding partner of CD4, we suspect that this overexpression would allow targeting of hepatocytes by CD4 positive lymphocytes; in fact, we observed the close association of CD4 positive lymphocytes to hepatocytes in biopsies with alcoholic hepatitis (Figure 4). Although CD4 cells are traditionally considered helper T cells, a subset of CD4 positive cells have cytotoxic activity (Takeuchi et al., 2017), including in viral hepatitis (Aslan et al., 2006). The CD4 positive cytotoxic T cells can derive from any subset of T helper cells but usually
arise from Th1 cells, which secrete interferon-ƴ and promote a cell-mediated response (Appay et al., 2002). We previously showed in a case of autoimmune hepatitis that CD4 positive lymphocytes appeared to infiltrate and eliminate the hepatocytes piece by piece (French and Lu, 2018). In addition, in that case of autoimmune hepatitis, electron microscopy also showed the formation of immunologic synapses between the plasma membranes of lymphocytes and hepatocytes. This observed phenomenom is similar to a recent report of neutrophils killing antibody-opsonized cancer cells by “trogoptosis” (Matlung et al., 2018). The liver is generally a site that promotes immune tolerance (Horst et al., 2016), as it must tolerate oral intake ingested of cells and their metabolites (Jenne and Kubes, 2013). Locally there are high levels of the immunoregulatory cytokine IL10 (Di Marco et al., 1999). However, aberrant expression of MHCII on hepatocytes may bypass the normal mechanisms of immune tolerance and lead to autoimmune response towards hepatocytes in alcoholic hepatitis. In support of this is that interferon gamma, which triggers a pro-inflammatory Th1 response, can also induce hepatocyte MHCII expression in vitro (Franco et al., 1988). Whether hepatocyte presentation of MHCII-antigen complexes leads to a Th1 or other helper T cell type response remains to be investigated. Interleukin-1α (IL-1α) is a potent inflammatory cytokine which acts via two mechanisms. First, in response to oxidative or metabolic stressors, cytosolic IL-1α translocates and binds to a plasma membrane, which activates IL-1R1 and production of chemokines and cytokines; this results in further recruitment of hematopoietic responders (Di Paolo and Shayakhmetov, 2016). Second, necrosis of hepatocytes leads release of IL-1α which directly activates IL-1R1 on neighboring cells (Rider et al., 2013). The downstream effects of IL-1R1 include recruitment of neutrophils and macrophages (Rider et at., 2011), and enhancing the activity of antigen-activated CD4+ T lymphocytes (Lichtman et al., 1988 and Pape et al., 1997). Interestingly, loss of expression of the IL-1α receptor, IL1-R1, lessens cellular damage and death from noxious stimuli (Gehrke et al., 2018). Thus, the higher level of IL-1α expression in AH compared to NASH in our study corresponds to a more severe inflammatory response in AH. Overall, one can envision a process where autoantibodies are formed in the context of alcoholic hepatitis, leading to a Th1 response and release of interferon-ƴ. Interferon-ƴ release causes hepatocytes to aberrantly express MHCII, leading to their targeting by CD4 positive cytotoxic lymphocytes which form immunological synapses with hepatocytes. As the hepatocytes are destroyed they release IL-1α, further enhancing CD4+ T cells in a positive feedback loop. (Figure 5) Our data shows that the MHCII-based immune response is more severe in AH than in NASH. The NASH biopsy shows much lower expression of MHC II in hepatocytes and in Kupffer cells compared to AH, yet still higher than the controls. This is consistent with animal studies showing that MHCII-knockdown mice can still develop NASH; thus the MHCII pathway is not required to develop NASH (Willemin et al., 2013). Thus, these new data sheds further light on the importance of the lymphocytic inflammatory response in alcoholic hepatitis. Acknowledgement
This study was supported by a grant by NIH/NIAAA # UO-21898-05.
References Albano E, French SW, and Ingelman-Sundberg M, 1998. Cytochrome P4502E1, hydroxyethyl free radicals, and immune reactions associate with alcoholic liver disease. Alcoholism: Clinical Exp Res: 22:739-742. Albano E, French SW, and Ingelman-Sundberg M, 1999. Hydroxyethyl radicals in ethanol hepatotoxicity. Frontiers Bio Science 4: dA533-540. Appay V, Zaunders JJ, Papagno L, Sutton J, Jaramillo A, Waters A, et al., 2002. Characterization of CD4(+) CTLs ex vivo. J Immunol 168:5954–8 Aslan N, Yurdaydin C, Wiegand J, Greten T, et al., 2006. Cytotoxic CD4 T cells in viral hepatitis. J Viral Hepat. 13:505-14. Bommireddy R, Ball C, Cherukuri D, et al., 2011. SMAD3 deficiency causes immune dysregulation, colitis and colon tumorigenesis. J Immunol 186: 66.13 Clot P, Bellomo G, Tabone M, et al., 1995. Detection of antibodies against proteins modified by hydroxyethyl free radicals in patients with alcoholic cirrhosis. Gastroenterology 108: 201-207. Di Marco R, Xiang M, Zaccone P, et al., 1999. Concanavalin A-induced hepatitis in mice is prevented by interleukin (IL)-10 and exacerbated by endogenous IL-10 deficiency. Autoimmunity 31: 75–83. Di Paolo NC and Shayakhmetov DM, 2016. Interleukin 1α and the inflammatory process. Nat Immunol. 17: 906-913. Rider P, Carmi Y, Voronov E, and Apte RN, 2013. Interleukin-1α. Semin Immunol. 2013; 25: 430-438. Franco A, Barnaba V, Natali P, et al., 1988. Expression of class I and class I major histocompatibility complex antigens of human hepatocytes. Hepatology 8: 449-54. French SW and Lu JG, 2018. Piecemeal necrosis is due to the immunologic synapse formation and internalization of intact TCR-MHCII complexes by CD4 T cells. Exp Mol Pathol; 105: 150-152 Gehrke N, Hovelmeyer N, Waisman A, et al., 2018. Hepatocyte-specific deletion of IL1-RI attenuates liver injury by blocking IL-1 driven autoinflammation. J. Hepatol. 68, 986–995. Herkel J, Jagemann B, Wiegard C, et al., 2003. MHC Class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes. Hepatology 37: 1079-1085. Horst AK, Neumann K, Diehl L, et al., 2016. Modulation of liver tolerance by conventional and nonconventional antigenpresenting cells and regulatory immune cells. Cellular and Molecular Immunol 13: 277-292. Jenne CN and Kubes P, 2013. Immune surveillance by the liver. Nat Immunol 14: 996–1006. Kumar, V, Abbas, AK, & Aster, JC, 2015. Robbins and Cotran pathologic basis of disease (Ninth edition.). Philadelphia, PA: Elsevier/Saunders, p.842-844. Lakshminarayanan V, Beno DW, Costa RH, and Roebuck KA, 1997. Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2O2 and tumor necrosis factor-alpha in endothelial and epithelial cells. J Biol Chem 272: 32910-8.
Lichtman AH, Chin J, Schmidt JA, and Abbas AK, 1988. Role of interleukin 1 in the activation of T lymphocytes. Proc Natl Acad Sci USA 85: 9699-9703. Lytton SD, Helander A, Zhang-Gouillon, ZZ, et al., 1999. Autoantibodies against cytochromes P-4502E1 and P-4503A in alcoholics. Molecular Pharmacology 55:223-33. Matlung HL, Babes L, Zhao XW, et al., 2018. Neutrophils kill antibody-opsonized cancer cells by trogoptosis. Cell Reports 23: 3946-59. Mavrelis PG, Ammon HV, Gleysteen JJ, Komorowski RA, et al, 1983. Hepatic free fatty acids in alcoholic liver disease and morbid obesity. Hepatology 3: 226-31. Pape KA, Khoruts A, Mondino A, and Jenkins MK, 1997. Inflammatory cytokines enhance the in vivo clonal expansion and differentiation of antigen-activated CD4+ T cells. J Immunol 159: 591-598. Petkau K, Ferguson M, Guntermann S, et al., 2017. Constitutive immune activity promotes tumorigenesis in Drosophila intestinal progenitor cells. Cell Reports 22: 1784-93. Rao R, 2009. Endotoxemia and gut barrier dysfunction in alcoholic liver disease. Hepatology 50: 638-44. Rider P, Carmi Y, Guttman O, et al., 2011. IL-1α and IL-1B recruit different myeloid cells and promote different stages of sterile inflammation. J Immunol 187: 1-9. Rider P, Carmi Y, Voronov E, and Apte RN, 2013. Interleukin-1α. Semin Immunol 25: 430-438. Takeuchi A and Saito T, 2017. CD4 CTL, a cytotoxic subset of CD4+ T cells, their differentiation and function. Front Immunol 8: 194. Wang HJ, Gao B, Zakhari S, and Nagy LE, 2012. Inflammation in Alcoholic Liver Disease. Annu Rev Nutr 32: 343-368. Willemin G, Roger C, Bauduret A, et al., 2013. Major histocompatibility class II pathway is not required for the development of nonalcoholic fatty liver disease in mice. Int J Endocrinol 972962. Worrall S, De Jersey J, Shanley BC, et al., 1989. Ethanol induces the production of antibodies to acetaldehyde-modifed epitopes in rats. Alcohol 24, 217-223.
Figure 1:
AA
B
C
D
Figure 1: (A) Aberrant MHC II production (green) in a liver biopsy with alcoholic hepatitis. Note the Mallory body (arrow) which stains positively for CAM5.2 (red) in the hepatocyte coated with MHC II. Nuclei are stained with DAPI (blue). 780x magnification. (B) Same biopsy with only MHC II (green); note the pericellular expression of MHC II in the hepatocyte with Mallory body. (C) In a biopsy with nonalcoholic steatohepatitis (NASH) there are lower levels of MHC II (green, arrow) compared to alcoholic hepatitis but still increased compared to normal control (D).
A
B
Figure 2:
A
B MHCII Intensity Measurements 250
CONTROL vs ASH (p=<0.001) CONTROL vs NASH (p=0.003) ASH vs NASH (p=<0.001)
Intensity
200
C
150
100
50
D 0 CONTROL
ASH
NASH
Figure 2: Quantification of immunofluorescence intensity for liver biopsies showing alcoholic hepatitis (A), non-alcoholic hepatitis (B) and normal control (C). On each biopsy, three fields are selected, a line drawn through the middle, and the ten highest immunofluorescence intensities are measured. (D) Graph showing average MHCII peak fluorescence intensities for control, AH, and NASH. Levels of MHCII expression are significantly higher in AH compared to control and NASH (p < 0.001) and in NASH compared to control (p = 0.003).
Figure 3:
A
A
B
B
H L
C
L
D
D C Figure 3: Immunohistochemical stains of CD4 in liver biopsies with alcoholic hepatitis (A), non-alcoholic steatohepatitis (B), and normal control (C). CD4 positive lymphocytes closely associate with hepatocytes in AH (arrows). The extent of lymphocytic infiltrate is less in NASH (arrows) but still greater than normal control. Images A-C are at 800x magnification. (D) Electron microscopy of the alcoholic hepatitis biopsy at 1570x magnification shows sinusoidal lymphocytes (“L”) binding to hepatocytes (“H”). (E). Immunofluorescence double stain of MHC II (red) and CD4 (green) shows CD4 positive lymphocytes (arrows) closely associating with MHC II producing hepatocytes. Some lymphocytes also produce MHC II and thus produce a co-localization yellow signal.
E
Figure 4: INTENSITY MEAUREMENT OF IL-1α
INTENSITY MEAUREMENT OF IFN-γ
A
Figure 4: Quantifying fluorescence intensity of IL-1α (A) and IFN-γ (B) in alcoholic hepatitis, NASH, and control tissues. Levels of both IL-1α and IFN-γ are higher on alcoholic hepatitis compared to NASH and control.
Figure 5:
IFN- ƴ
Macrophage
Alcohol-liver protein adducts Damaged Hepatocyte
Hepatocyte
Autoantibodies
Aberrant MHCII expression
Immunologic Synapse
IFN- ƴ
Th1 CD4+ Cell
IL-1α
CD4+ cytotoxic lymphocyte
CD4+ cytotoxic lymphocyte
?
Figure 5: Model for autoimmune mechanisms in alcoholic hepatitis. Auto-antibodies to alcohol-liver protein adducts lead to a Th1 response, leading to secretion of IFN-ƴ. IFN-ƴ causes aberrant MHC II production on hepatocytes, leading to targeting by CD4 positive cytotoxic lymphocytes. The CD4 positive lymphocyte internalizes the MHC II / CD 4 complex in lysosomes and eats the hepatocyte piece by piece. As hepatocytes are destroyed they release IL-1α, which, among other effects, further enhances antigen-activated CD4+ T cell response, creating a positive feedback loop. IL-1α also induces macrophages to secrete IFN-ƴ leading to further hepatocyte MHC II production.
S0741-8329(19)30067-9
DOI:
https://doi.org/10.1016/j.alcohol.2019.08.008
Reference:
ALC 6939
To appear in:
Alcohol
Received Date: 13 April 2019 Revised Date:
21 August 2019
Accepted Date: 30 August 2019
Please cite this article as: Lu J.G., Iyasu A., French B., Tillman B., French S.W., French S.W. & M.D Overexpression of MHCII by hepatocytes in Alcoholic Hepatitis (AH) compared to Nonalcoholic Steatohepatitis (NASH) and normal controls, Alcohol (2019), doi: https://doi.org/10.1016/ j.alcohol.2019.08.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Overexpression of MHCII by hepatocytes in Alcoholic Hepatitis (AH) compared to Non-alcoholic Steatohepatitis (NASH) and normal controls.
Jiajie G. Lu, Askalu Iyasu, Barbara French, Brittany Tillman, Samuel W. French
Samuel W. French, M.D. Harbor-UCLA Medical Center 1000 W Carson Street Torrance, CA 90502 310-222-2643 Telephone 310-222-8002 Fax [email protected]
Highlights: -
There is little characterization of lymphocytic response in alcoholic hepatitis. In human liver biopsies, we show aberrant MHCII expression in hepatocytes affected by alcoholic hepatitis, at greater levels than in non-alcoholic steatohepatitis (NASH) and normal controls. Further, we show CD4 positive lymphocytes associated with these hepatocytes and eating them piece by piece Expression levels of IL-1α are higher in alcoholic hepatitis compared to NASH and controls. These results show the importance of autoimmune mechanisms in alcoholic hepatitis.
Abstract: Previously we have shown that in autoimmune hepatitis CD4 positive lymphocytes form an immunologic synapse with hepatocytes, leading to gradual diminishing and elimination of the hepatocyte. We wondered whether a similar mechanism may occur in alcoholic hepatitis (AH) and non-alcoholic steatohepatitis (NASH). We conducted immunofluorescence studies of expression of MHCII, the binding partner of CD4, on patient liver biopsies of AH, NASH, and normal controls. In cases of alcoholic hepatitis, there was prominent sinusoidal expression of MHC II; In NASH biopsies there was comparatively lower expression of MHC II, but still more than control tissue. Immunohistochemical stain for CD4 showed CD4 positive lymphocytes closely associated with hepatocytes in AH biopsies. Furthermore, expression levels of the multifunctional cytokine IL-1α was higher in AH compared to NASH and control biopsies. These results underlie the more severe nature of alcoholic hepatitis and underscore the autoimmune mechanisms involved in the liver damage found in alcoholic hepatitis. Key words: alcoholic hepatitis, non-alcoholic steatohepatitis, MHCII, CD4, immune synapse Introduction
Alcoholic hepatitis (AH) is characterized histologically by hepatocyte balloon cell change, Mallory-Denk body formation, and neutrophilic infiltration (Kumar et al., 2015). It is often accompanied by steatosis and fibrosis. The mechanism of liver damage in alcoholic hepatitis is multifactorial, including lipid peroxidation, acetaldehyde-protein adduct formation, reactive oxygen species production, and inflammatory response (Wang et al., 2012). Recently we showed that in autoimmune hepatitis, CD4 positive lymphocytes infiltrate the parenchyma and gradually nibble away the hepatocytes (French and Lu, 2018). Electron microscopy showed a lymphocyte forming a 15-20 µm immunological synapse with a hepatocyte. We questioned whether a similar phenomenon occurs in AH or non-alcoholic hepatitis (NASH). We examined liver biopsies of de-identified patients with AH, NASH, or no liver disease and examined the level of expression of MHC II, the binding partner of CD4, in these biopsies. Methods Human formalin-fixed paraffin-embedded liver biopsies from 10 alcoholic hepatitis, 3 NASH, and 3 normal controls were obtained from Harbor UCLA hospital archives and clinical trials to treat alcoholic hepatitis at VA Hospital Long Beach, USC, and UC Davis. Liver biopsy sections were cut 4 µm thick. The study was carried out in accordance with the Declaration of Helsinki and designated as exempt by our institutional review board. Each liver slide was double stained with protein-specific antibodies for MHCII and CAM5.2, followed by secondary fluorescence antibodies. In a separate experiment, each liver slide was double stained with antibodies for IL-1α and ubiquitin, followed by secondary fluorescence antibodies. The biopsies were all stained at the same time in order to allow comparison of fluorescence intensities between cases. Rabbit anti-MHCII antibody was obtained from Abcam, Cambridge MA and mouse anti-CAM5.2 antibody from Sigma Aldrich, Carlsbad, CA. The slides were also nuclear stained by DAPI. For each protein, we measured the intensity of the fluorescent staining in three different areas of the biopsies with 40x magnification and 800ms standard exposure time, using a Nikon 400 fluorescent microscope. On each slide area, 10 peak fluorescence intensities were measured. The Nikon morphometric system was used to quantitate the fluorescent intensity of liver cells and sinusoids for each individual antibody. The mean, standard error, and statistical differences of data achieved from the Nikon were analyzed by Sigma Pletand statistical software. Controls vs ASH, controls vs NASH, and ASH vs NASH were compared by unpaired t-test with a p-value of <0.05 considered statistically significant. Results We investigated the expression of MHCII in liver biopsies showing alcoholic hepatitis (AH) compared to nonalcoholic steatohepatitis (NASH) and controls. In AH biopsies there was prominent expression of MHCII in the hepatic sinusoids and on hepatocytes (Figure 1A-B). Expression of MHCII in the NASH biopsies also occurred in a sinusoidal pattern (Figure
1C) but was much less intense compared to ASH. Normal liver controls showed virtually no expression of MHC II (Figure 1D). Analysis of MHCII fluorescence intensity showed statistically significant differences in AH, NASH, and normal livers (Figure 2). MHCII fluorescent intensity in AH was much higher than in NASH (196.0 ± 24.7 vs. 41.8 ± 15.2, p < 0.001), while MHCII immunofluorescence in NASH was somewhat higher than in the normal controls (41.8 ± 15.2 vs. 31.3 ± 10.8, p = 0.003). We performed immunohistochemical stains of CD4 on selected liver biopsies. The AH biopsies showed large numbers of CD4 positive lymphocytes and strong staining of CD4 in the hepatic sinusoids, compared to the lower numbers in NASH and none in control tissue (Figure 3). Electron microscopy of an AH biopsy shows sinusoidal lymphocytes binding to hepatocytes (Figure 3D). Finally, we examined the expression of the cytokine interleukin-1α (IL-1α) on AH, NASH, and control biopsies (Figure 4). AH biopsies show increased IL-1α fluorescence intensity compared to NASH (183.1 ± 26.8 vs. 157.5 ± 50.1, p = 0.015) and controls (183.1 ± 26.8 vs. 104.7 ± 12.5, p < 0.001). Discussion Alcoholic hepatitis is associated with an immune response that targets hepatocytes. The nature of this immune response is complex. For instance, two studies report formation of autoantibodies against CYP2E1 and CYPEA4 in alcoholic hepatitis (Lytton et al., 1998) (Albano et al., 1998). Autoantibodies can also develop to hydroxyethyl radical protein adducts (Clot et al., 1995) (Albano et al., 1999) and acetaldehyde binding to cellular proteins (Worrall et al., 1989). Other mechanisms include lipopolysaccharide-induced acute inflammation due to increased gut epithelial membrane permeability (Rao 2009), direct response to free fatty acids (Mavrelis et al., 1983), and reactive oxygen species formation (Lakshminarayanan et al., 1997). In total these responses induce a primarily acute inflammatory response including neutrophils and macrophages. Lymphocyte response in alcoholic hepatitis is less prominent and less well described (Wang et al., 2012). Furthermore, direct cytotoxic effects to hepatocytes by lymphocytes in alcoholic hepatitis, to our knowledge, have not been described. Our cases of alcoholic hepatitis showed overexpression of MHCII on hepatocellular membranes and Kupffer cells. It has been shown that human hepatocytes can be induced to express MHCII by interferon gamma (Franco et al., 1988). Further, in mice, hepatocytes that constitutively express MHCII are able to activate CD4 positive lymphocytes (Herkel et al., 2003). As MHCII loaded with antigen is the binding partner of CD4, we suspect that this overexpression would allow targeting of hepatocytes by CD4 positive lymphocytes; in fact, we observed the close association of CD4 positive lymphocytes to hepatocytes in biopsies with alcoholic hepatitis (Figure 4). Although CD4 cells are traditionally considered helper T cells, a subset of CD4 positive cells have cytotoxic activity (Takeuchi et al., 2017), including in viral hepatitis (Aslan et al., 2006). The CD4 positive cytotoxic T cells can derive from any subset of T helper cells but usually
arise from Th1 cells, which secrete interferon-ƴ and promote a cell-mediated response (Appay et al., 2002). We previously showed in a case of autoimmune hepatitis that CD4 positive lymphocytes appeared to infiltrate and eliminate the hepatocytes piece by piece (French and Lu, 2018). In addition, in that case of autoimmune hepatitis, electron microscopy also showed the formation of immunologic synapses between the plasma membranes of lymphocytes and hepatocytes. This observed phenomenom is similar to a recent report of neutrophils killing antibody-opsonized cancer cells by “trogoptosis” (Matlung et al., 2018). The liver is generally a site that promotes immune tolerance (Horst et al., 2016), as it must tolerate oral intake ingested of cells and their metabolites (Jenne and Kubes, 2013). Locally there are high levels of the immunoregulatory cytokine IL10 (Di Marco et al., 1999). However, aberrant expression of MHCII on hepatocytes may bypass the normal mechanisms of immune tolerance and lead to autoimmune response towards hepatocytes in alcoholic hepatitis. In support of this is that interferon gamma, which triggers a pro-inflammatory Th1 response, can also induce hepatocyte MHCII expression in vitro (Franco et al., 1988). Whether hepatocyte presentation of MHCII-antigen complexes leads to a Th1 or other helper T cell type response remains to be investigated. Interleukin-1α (IL-1α) is a potent inflammatory cytokine which acts via two mechanisms. First, in response to oxidative or metabolic stressors, cytosolic IL-1α translocates and binds to a plasma membrane, which activates IL-1R1 and production of chemokines and cytokines; this results in further recruitment of hematopoietic responders (Di Paolo and Shayakhmetov, 2016). Second, necrosis of hepatocytes leads release of IL-1α which directly activates IL-1R1 on neighboring cells (Rider et al., 2013). The downstream effects of IL-1R1 include recruitment of neutrophils and macrophages (Rider et at., 2011), and enhancing the activity of antigen-activated CD4+ T lymphocytes (Lichtman et al., 1988 and Pape et al., 1997). Interestingly, loss of expression of the IL-1α receptor, IL1-R1, lessens cellular damage and death from noxious stimuli (Gehrke et al., 2018). Thus, the higher level of IL-1α expression in AH compared to NASH in our study corresponds to a more severe inflammatory response in AH. Overall, one can envision a process where autoantibodies are formed in the context of alcoholic hepatitis, leading to a Th1 response and release of interferon-ƴ. Interferon-ƴ release causes hepatocytes to aberrantly express MHCII, leading to their targeting by CD4 positive cytotoxic lymphocytes which form immunological synapses with hepatocytes. As the hepatocytes are destroyed they release IL-1α, further enhancing CD4+ T cells in a positive feedback loop. (Figure 5) Our data shows that the MHCII-based immune response is more severe in AH than in NASH. The NASH biopsy shows much lower expression of MHC II in hepatocytes and in Kupffer cells compared to AH, yet still higher than the controls. This is consistent with animal studies showing that MHCII-knockdown mice can still develop NASH; thus the MHCII pathway is not required to develop NASH (Willemin et al., 2013). Thus, these new data sheds further light on the importance of the lymphocytic inflammatory response in alcoholic hepatitis. Acknowledgement
This study was supported by a grant by NIH/NIAAA # UO-21898-05.
References Albano E, French SW, and Ingelman-Sundberg M, 1998. Cytochrome P4502E1, hydroxyethyl free radicals, and immune reactions associate with alcoholic liver disease. Alcoholism: Clinical Exp Res: 22:739-742. Albano E, French SW, and Ingelman-Sundberg M, 1999. Hydroxyethyl radicals in ethanol hepatotoxicity. Frontiers Bio Science 4: dA533-540. Appay V, Zaunders JJ, Papagno L, Sutton J, Jaramillo A, Waters A, et al., 2002. Characterization of CD4(+) CTLs ex vivo. J Immunol 168:5954–8 Aslan N, Yurdaydin C, Wiegand J, Greten T, et al., 2006. Cytotoxic CD4 T cells in viral hepatitis. J Viral Hepat. 13:505-14. Bommireddy R, Ball C, Cherukuri D, et al., 2011. SMAD3 deficiency causes immune dysregulation, colitis and colon tumorigenesis. J Immunol 186: 66.13 Clot P, Bellomo G, Tabone M, et al., 1995. Detection of antibodies against proteins modified by hydroxyethyl free radicals in patients with alcoholic cirrhosis. Gastroenterology 108: 201-207. Di Marco R, Xiang M, Zaccone P, et al., 1999. Concanavalin A-induced hepatitis in mice is prevented by interleukin (IL)-10 and exacerbated by endogenous IL-10 deficiency. Autoimmunity 31: 75–83. Di Paolo NC and Shayakhmetov DM, 2016. Interleukin 1α and the inflammatory process. Nat Immunol. 17: 906-913. Rider P, Carmi Y, Voronov E, and Apte RN, 2013. Interleukin-1α. Semin Immunol. 2013; 25: 430-438. Franco A, Barnaba V, Natali P, et al., 1988. Expression of class I and class I major histocompatibility complex antigens of human hepatocytes. Hepatology 8: 449-54. French SW and Lu JG, 2018. Piecemeal necrosis is due to the immunologic synapse formation and internalization of intact TCR-MHCII complexes by CD4 T cells. Exp Mol Pathol; 105: 150-152 Gehrke N, Hovelmeyer N, Waisman A, et al., 2018. Hepatocyte-specific deletion of IL1-RI attenuates liver injury by blocking IL-1 driven autoinflammation. J. Hepatol. 68, 986–995. Herkel J, Jagemann B, Wiegard C, et al., 2003. MHC Class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes. Hepatology 37: 1079-1085. Horst AK, Neumann K, Diehl L, et al., 2016. Modulation of liver tolerance by conventional and nonconventional antigenpresenting cells and regulatory immune cells. Cellular and Molecular Immunol 13: 277-292. Jenne CN and Kubes P, 2013. Immune surveillance by the liver. Nat Immunol 14: 996–1006. Kumar, V, Abbas, AK, & Aster, JC, 2015. Robbins and Cotran pathologic basis of disease (Ninth edition.). Philadelphia, PA: Elsevier/Saunders, p.842-844. Lakshminarayanan V, Beno DW, Costa RH, and Roebuck KA, 1997. Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2O2 and tumor necrosis factor-alpha in endothelial and epithelial cells. J Biol Chem 272: 32910-8.
Lichtman AH, Chin J, Schmidt JA, and Abbas AK, 1988. Role of interleukin 1 in the activation of T lymphocytes. Proc Natl Acad Sci USA 85: 9699-9703. Lytton SD, Helander A, Zhang-Gouillon, ZZ, et al., 1999. Autoantibodies against cytochromes P-4502E1 and P-4503A in alcoholics. Molecular Pharmacology 55:223-33. Matlung HL, Babes L, Zhao XW, et al., 2018. Neutrophils kill antibody-opsonized cancer cells by trogoptosis. Cell Reports 23: 3946-59. Mavrelis PG, Ammon HV, Gleysteen JJ, Komorowski RA, et al, 1983. Hepatic free fatty acids in alcoholic liver disease and morbid obesity. Hepatology 3: 226-31. Pape KA, Khoruts A, Mondino A, and Jenkins MK, 1997. Inflammatory cytokines enhance the in vivo clonal expansion and differentiation of antigen-activated CD4+ T cells. J Immunol 159: 591-598. Petkau K, Ferguson M, Guntermann S, et al., 2017. Constitutive immune activity promotes tumorigenesis in Drosophila intestinal progenitor cells. Cell Reports 22: 1784-93. Rao R, 2009. Endotoxemia and gut barrier dysfunction in alcoholic liver disease. Hepatology 50: 638-44. Rider P, Carmi Y, Guttman O, et al., 2011. IL-1α and IL-1B recruit different myeloid cells and promote different stages of sterile inflammation. J Immunol 187: 1-9. Rider P, Carmi Y, Voronov E, and Apte RN, 2013. Interleukin-1α. Semin Immunol 25: 430-438. Takeuchi A and Saito T, 2017. CD4 CTL, a cytotoxic subset of CD4+ T cells, their differentiation and function. Front Immunol 8: 194. Wang HJ, Gao B, Zakhari S, and Nagy LE, 2012. Inflammation in Alcoholic Liver Disease. Annu Rev Nutr 32: 343-368. Willemin G, Roger C, Bauduret A, et al., 2013. Major histocompatibility class II pathway is not required for the development of nonalcoholic fatty liver disease in mice. Int J Endocrinol 972962. Worrall S, De Jersey J, Shanley BC, et al., 1989. Ethanol induces the production of antibodies to acetaldehyde-modifed epitopes in rats. Alcohol 24, 217-223.
Figure 1:
AA
B
C
D
Figure 1: (A) Aberrant MHC II production (green) in a liver biopsy with alcoholic hepatitis. Note the Mallory body (arrow) which stains positively for CAM5.2 (red) in the hepatocyte coated with MHC II. Nuclei are stained with DAPI (blue). 780x magnification. (B) Same biopsy with only MHC II (green); note the pericellular expression of MHC II in the hepatocyte with Mallory body. (C) In a biopsy with nonalcoholic steatohepatitis (NASH) there are lower levels of MHC II (green, arrow) compared to alcoholic hepatitis but still increased compared to normal control (D).
A
B
Figure 2:
A
B MHCII Intensity Measurements 250
CONTROL vs ASH (p=<0.001) CONTROL vs NASH (p=0.003) ASH vs NASH (p=<0.001)
Intensity
200
C
150
100
50
D 0 CONTROL
ASH
NASH
Figure 2: Quantification of immunofluorescence intensity for liver biopsies showing alcoholic hepatitis (A), non-alcoholic hepatitis (B) and normal control (C). On each biopsy, three fields are selected, a line drawn through the middle, and the ten highest immunofluorescence intensities are measured. (D) Graph showing average MHCII peak fluorescence intensities for control, AH, and NASH. Levels of MHCII expression are significantly higher in AH compared to control and NASH (p < 0.001) and in NASH compared to control (p = 0.003).
Figure 3:
A
A
B
B
H L
C
L
D
D C Figure 3: Immunohistochemical stains of CD4 in liver biopsies with alcoholic hepatitis (A), non-alcoholic steatohepatitis (B), and normal control (C). CD4 positive lymphocytes closely associate with hepatocytes in AH (arrows). The extent of lymphocytic infiltrate is less in NASH (arrows) but still greater than normal control. Images A-C are at 800x magnification. (D) Electron microscopy of the alcoholic hepatitis biopsy at 1570x magnification shows sinusoidal lymphocytes (“L”) binding to hepatocytes (“H”). (E). Immunofluorescence double stain of MHC II (red) and CD4 (green) shows CD4 positive lymphocytes (arrows) closely associating with MHC II producing hepatocytes. Some lymphocytes also produce MHC II and thus produce a co-localization yellow signal.
E
Figure 4: INTENSITY MEAUREMENT OF IL-1α
INTENSITY MEAUREMENT OF IFN-γ
A
Figure 4: Quantifying fluorescence intensity of IL-1α (A) and IFN-γ (B) in alcoholic hepatitis, NASH, and control tissues. Levels of both IL-1α and IFN-γ are higher on alcoholic hepatitis compared to NASH and control.
Figure 5:
IFN- ƴ
Macrophage
Alcohol-liver protein adducts Damaged Hepatocyte
Hepatocyte
Autoantibodies
Aberrant MHCII expression
Immunologic Synapse
IFN- ƴ
Th1 CD4+ Cell
IL-1α
CD4+ cytotoxic lymphocyte
CD4+ cytotoxic lymphocyte
?
Figure 5: Model for autoimmune mechanisms in alcoholic hepatitis. Auto-antibodies to alcohol-liver protein adducts lead to a Th1 response, leading to secretion of IFN-ƴ. IFN-ƴ causes aberrant MHC II production on hepatocytes, leading to targeting by CD4 positive cytotoxic lymphocytes. The CD4 positive lymphocyte internalizes the MHC II / CD 4 complex in lysosomes and eats the hepatocyte piece by piece. As hepatocytes are destroyed they release IL-1α, which, among other effects, further enhances antigen-activated CD4+ T cell response, creating a positive feedback loop. IL-1α also induces macrophages to secrete IFN-ƴ leading to further hepatocyte MHC II production.