Impaired T cell-mediated hepatitis in peroxisome proliferator activated receptor alpha (PPARα)-deficient mice
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Peroxisome proliferator activated receptor alpha (PPARα), a regulator of enzymes involved in β oxidation, has been reported to influence lymphocyte activation. The purpose of this study was to determine whether PPARα plays a role in T cell-mediated hepatitis induced by Concanavalin A (ConA).
Wild type (wt) or PPARα-deficient (PPARα−/−) mice were treated with ConA (15 mg/kg) by intravenous injection 0, 10 or 24 h prior to sacrifice and serum and tissue collection for analysis of tissue injury, cytokine response, T cell activation and characterization.
Ten and 24 h following ConA administration, wt mice had significant liver injury as demonstrated by serum transaminase levels, inflammatory cell infiltrate, hepatocyte apoptosis, and expression of several cytokines including interleukin 4 (IL4) and interferon gamma (IFNγ). In contrast, PPARα−/− mice were protected from ConA-induced liver injury with significant reductions in serum enzyme release, greatly reduced inflammatory cell infiltrate, hepatocellular apoptosis, and IFNγ expression, despite having similar levels of hepatic T cell activation and IL4 expression. This resistance to liver injury was correlated with reduced numbers of hepatic natural killer T (NKT) cells and their in vivo responsiveness to alpha-galactosylceramide. Interestingly, adoptive transfer of either wt or PPARα−/− splenocytes reconstituted ConA liver injury and cytokine production in lymphocyte-deficient, severe combined immunodeficient mice implicating PPARα within the liver, possibly through support of IL15 expression and/or suppression of IL12 production and not the lymphocyte as the key regulator of T cell activity and ConA-induced liver injury.
Taken together, these data suggest that PPARα within the liver plays an important role in ConA-mediated liver injury through regulation of NKT cell recruitment and/or survival.
KeywordsInflammation Cytokines T helper phenotype Interferon gamma
peroxisome proliferator activated receptor alpha
natural killer T cell
acyl CoA oxidase
nuclear factor kappa B
terminal UTP nick end labeling
enzyme linked immunosorbent assay
T cell receptor
severe combined immunodeficient
phosphate buffered saline
T box transcription factor expressed in T cells
fatty acid binding protein
Growing experimental and clinical data highlight a complex interaction among lipids, immune cells, and the hepatic inflammatory responses [1, 2, 3, 4]. Accumulation of lipid leads to inflammatory cell infiltration and activation which promotes secondary tissue injury and organ dysfunction . Key aspects in the regulation of this process remain unclear, particularly the intersection of lipid metabolism and immune cell function whether it be direct or indirect through hepatocellular stress/damage. Peroxisome proliferator activated receptor alpha (PPARα) is a nuclear hormone receptor associated with proliferation of peroxisomes in the hepatocytes of rodents in response to a number of naturally occurring as well as synthetic compounds . PPARα is also a regulator of the production of a number of enzymes including acyl Coenzyme A oxidase (AOX) involved in the metabolism of fatty acids within the liver [6, 7]. As a result, mice deficient in this AOX present with an age dependent increase in the accumulation of hepatocellular fat or steatosis.
PPARα also plays a prominent role in inflammatory response [8, 9, 10]. For example, foam cell formation is reduced by the ligand-specific activation of PPARα in a model of hypercholesterolemia induced atherosclerosis . Human monocyte-derived macrophages have also shown sensitivity to PPARα ligand activation with increased levels of apoptosis [9, 10, 12]. Further investigation has revealed an inhibitory effect of PPARα on the pro-inflammatory transcription factor nuclear factor kappa B (NFκB), a possible mechanism for its anti-inflammatory actions . Jones et al. also report the presence of PPARα in CD4+ T lymphocytes in rodents . As with macrophages, PPARα in T lymphocytes appears to regulate the activity of NFκB suggesting a common mechanism and role in immune cell function . Interestingly, studies have also demonstrated a dysregulation of cytokine production in T lymphocytes from PPARα-deficient (PPARα−/−) mice whereby deficient cells produce significantly larger quantities of interferon gamma (IFNγ) in response to anti-CD3/anti-CD28 activation . Such data would suggest that PPARα is capable of modulating the function and immunological response of a variety of immune cells from macrophages to T cells and therefore may play a significant role in the determination of T cell responsiveness in vivo.
Concanavalin A (ConA) is a plant lectin capable of inducing severe T cell mediated hepatitis in the mouse . ConA activates CD1d-dependent intrahepatic natural killer T (NKT) cells to produce a number of pro-inflammatory mediators including tumor necrosis factor alpha, interleukin 4 (IL4), and IFNγ [17, 18, 19]. Given the presence of PPARα in T cells, its apparent regulation of T cell and macrophage activation, and its influence on hepatocellular lipid metabolism, PPARα lies at the unique nexus of lipid metabolism and immunological function. The current study was thus aimed at understanding the impact of PPARα in the complex setting of T cell-mediated hepatitis. To this end, we have administered ConA to wild type and PPARα−/− mice and revealed a surprising and profoundly protective effect of PPARα deficiency on ConA-mediated, T cell-dependent liver injury, a protection likely related to reductions in hepatic NKT cell number and function.
Eight to twelve week old male C57Bl/6 mice, PPARα-deficient (PPARα−/−) mice , or severe combined immunodeficient (SCID) mice on a C57Bl/6 background were purchased from Jackson Laboratories (Bar Harbor, ME). All animals were housed in specific pathogen free conditions with 12 h light/dark cycles and free access to food and water. All subsequent procedures described were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill and complied with the “Guide for the Care and Use of Laboratory Animals”.
Male mice, either wild type or PPARα−/−, were administered lipopolysaccharide (LPS; 1 mg/kg, Sigma, St. Louis, MO) by intraperitoneal injection in 200 μl of normal saline or saline alone as control 6 h prior to sacrifice.
Concanavalin A (ConA) mediated hepatitis
Male mice, either wild type or PPARα−/−, were administered Concanavalin (ConA; Sigma, St. Louis, MO) at a dose of 15 mg/kg in sterile saline via tail vein injection as has previously been described . Mice were then anesthetized with ketamine and xylazine (100 and 10 mg/kg respectively) 10 or 24 h following injection, the diaphragm severed to effect euthanasia, and serum and tissue collected.
α-Galactosylceramide (αGal) treatment
Male mice, either wild type or PPARα−/−, were administered αGal (Funakoshi, Tokyo, Japan) by intravenous injection at a dose of 10 μg/mouse through the tail vein as previously reported . Mice were then euthanized 12 h as described above to assess liver injury and cytokine production.
Liver enzyme assessment
Blood was collected from the inferior vena cava from anesthetized mice 10 h following ConA administration into sterile microcentrifuge tubes. Blood was allowed to clot on ice for a period of 10 min after which it was centrifuged at 12,000×g allowing for collection of serum. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by the Clinical Chemistry Laboratory at the University of North Carolina at Chapel Hill using standard techniques.
Histopathology and immunohistochemistry
Liver tissue was collected at the time of sacrifice and placed in 10% buffered formalin (Thermo-Fisher Scientific, Waltham, MA) at 4 °C for 24 h. After fixation, the tissue was embedded in paraffin and 7 μm thick sections cut. Sections were then deparaffinized, rehydrated, and stained with hematoxylin and eosin. Additionally, some sections were stained for the T cell marker, CD3ε (Thermo-Fisher Scientific), as previously described . Sections were examined under routine light microscopy at 100× and 400× magnification and images captured using an Olympus DP70 digital camera.
Terminal UTP nick end labeling (TUNEL) staining
To assess liver cell death, deparaffinized sections were stained for DNA fragmentation using a commercially available kit (In situ cell death detection kit, Roche, Indianapolis, IN, Cat# 11684795910) according to the manufacturer’s recommendations as previously described . Stained sections were viewed by fluorescent microscopy and images capture with an Olympus DP70 digital camera. Five random high powered fields were observed and positive cells counted.
Hepatic triglyceride quantification
Liver triglycerides were quantified using kit from Sigma (Triglyceride Reagent, Cat.# T2449, St. Louis MO) according to the manufacturer’s recommendations as previously described by our group . Triglyceride content was normalized to wet weight of tissue used in the assay.
Real time polymerase chain reaction
Primer sequences used for quantitative PCR analysis
Tumor necrosis factor alpha (TNFα)
Interferon γ (IFNγ)
Interleukin 12 p40 (IL12p40)
Interleukin 4 (IL4)
Interleukin 5 (IL5)
Interleukin 10 (IL10)
Acyl-CoA oxidase (AOX)
Cluster of differentiation 1d (CD1d)
Peroxisome proliferator activated receptor alpha (PPARα)
Liver fatty acid binding protein (LFABP)
Liver mononuclear cells and total splenocytes were obtained as described previously [2, 21]. Isolated cells were stained for the immune cell markers T cell receptor beta (TCRβ; BD Pharmingen, San Jose, CA), CD4 (Thermo-Fisher), pan natural killer cell (DX5; Thermo-Fisher), and the activation marker, CD69 (Thermo-Fisher) at a 1:100 dilution for 30 min at room temperature. For spleen cells, whole spleens were homogenized between glass slides centrifuged at 500×g, and filtered through a 30 μm sterile filter followed by staining with the above listed antibodies. Again, cells were stained with the above listed antibodies. Cells were then analyzed and relative numbers expressed by % of total mononuclear cells and/or % of total liver TCRβ+ cells in liver mononuclear cell fraction.
Enzyme-linked immunosorbent assay
Serum and/or tissue culture media IL12, IFNγ, or IL4 protein was determined using a kit from R&D systems (IL12, Cat#M1270; IFNγ, Cat#MIF00; and IL4, Cat#M4000B) per manufacturer’s instruction as previously described . Samples were compared to a standard curve and values expressed per mg of liver protein.
In vitro ConA activation
Wild type or PPARα−/− mononuclear cells were isolated as described previously. For activation studies, 1 × 105 liver mononuclear cells or spleen cells were incubated in a 96 well plate in 300 μl of RPMI media (Invitrogen) in the presence or absence of 1 μg/ml ConA (Sigma) for 72 h at 37 °C and 5% CO2. Following incubation, media was collected and assessed for IFNγ and IL4 protein by ELISA as described above.
SCID lymphocyte reconstitution
Total splenocytes (2 × 107) were isolated as described above from wild type and PPARα−/− mice. Red blood cells were removed by incubation in red blood cell lysis solution for 10 min at room temperature. Cell viability and number were assessed by trypan blue exclusion. Splenocytes (2 × 107) were resuspended in 100 μl of PBS and injected intravenously into SCID recipients through the tail vein. SCID mice administered PBS alone served as controls for these experiments. Seven days following reconstitution, animals were administered ConA (15 mg/kg). Ten hours later, serum and tissue were collected to assess T cell reconstitution, liver injury, and cytokine expression.
Data are presented as mean ± standard error of the mean (SEM) of 4 or more animals per group. Data were analyzed using the non-parametric Mann–Whitney Rank Sum Test or analysis of variance where significance was set at p < 0.05.
Characterization of PPARα−/− mice
Deficiency in PPARα inhibits Concanavalin A (ConA)-mediated hepatitis
ConA has also been shown to induce liver injury through the induction of hepatocellular apoptosis via a Fas-dependent mechanism [25, 26, 27]. To determine if the ConA-induced apoptotic cell death was also disrupted in PPARα−/− mice, liver sections from wild type and PPARα−/− mice were subjected to the TUNEL assay to assess DNA fragmentation, a marker of apoptotic cell death. Consistent with serum enzyme measures and histopathological signs of liver damage, wild type mice given ConA had increased numbers of TUNEL positive cell number when compared to their untreated controls at 10 and 24 h post-injection (Fig. 2d). In contrast, PPARα−/− livers were resistant to ConA-induced increases in hepatocellular apoptosis, a finding consistent with an absence of liver injury. Taken together, these data suggest that PPARα may be involved in the early development of ConA-induced, T cell mediated, liver injury in mice.
Splenic and hepatic T cells are activated in wild type and PPARα−/− mice in response to ConA
Deficiency in PPARα alters ConA-induced cytokine expression in liver
It is becoming increasingly apparent that certain transcription factors play important roles in the differentiation of T cells towards Th1 or Th2 phenotypes [31, 32, 33]. T-bet, a T box transcription factor primarily expressed in T cells, is associated with the expression of Th1-type cytokines including IFNγ . Furthermore, activation of T-bet has been shown to be crucial to the development of ConA-mediated hepatitis . Given the reduction in expression of IFNγ in PPARα−/− mice following ConA when compared to ConA-treated wild type controls, we tested the hypothesis that PPARα positively regulates expression of this Th1-associated transcription factor. T-bet expression is strongly up-regulated in the livers of wild type mice 10 h following ConA administration (Fig. 4g). In contrast, deficiency in PPARα prevents the up-regulation of this Th1-associated transcription factor in the liver (Fig. 4g). Together, these data, in conjunction with the reductions in cytokine expression, suggest that PPARα does indeed play a role, either directly or indirectly, in the activation of the Th1-associated transcription factor T-bet following ConA administration.
PPARα−/− mice have reduced numbers of liver NKT cells
To further evaluate the functionality of NKT cells directly, wild type mice or PPARα−/− mice were administered alpha galactosylceramide (αGal), a potent and specific activator of NKT cells . Twelve hours following injection, mice were sacrificed and serum and tissue collected for liver enzyme release and pro-inflammatory cytokine production. As shown in Fig. 6b, absence of PPARα resulted in reduced αGal-induced liver injury as assessed by serum ALT levels as well as a reduction in IFNγ gene expression (Fig. 6c) following αGal administration when compared to similarly treated wild type mice. These data further confirm the dysfunction of hepatic NKT cells within PPARα−/− mice.
PPARα−/− splenocytes are capable of restoring ConA-dependent liver injury in SCID mice
PPARα-deficiency does not affect lipopolysaccharide-induced liver injury
PPARα-deficiency reduces hepatic IL15 expression
T cell-dependent liver injury represents an important component of a number of liver pathologies including autoimmune and viral hepatitis [41, 42, 43]. Defining the mechanisms by which lymphocytes exert their damaging effects represents an important area of scientific investigation. To this end, the current series of studies have identified PPARα as a potential regulator of hepatic T cells. Specifically, data here demonstrate the importance of PPARα in the recruitment and/or survival of NKT cells, independent of its function within these cells. The ability of PPARα to regulate the immune cell composition of the liver and lymphocyte responses may have important clinical implications in the treatment of a number of T cell-dependent liver pathologies.
ConA-mediated liver injury is a well-described model of T cell dependent acute hepatitis in rodents . NKT cells are activated by ConA in a CD1d-dependent manner to produce IFNγ and IL4 which serve to further activate this cell population as well as recruit and activate additional inflammatory cells including macrophages, thereby acting as a bridge between the innate and adaptive immune response [17, 18, 19, 24, 44, 45]. Recent studies by Li et al. as well as studies from our laboratory have drawn a strong correlation between the presence of hepatocellular lipid, absence or reduction in hepatic NKT cells and the production of a shifted Th1-type cytokine response in the liver [2, 46]. The results of the current study suggest that the loss of PPARα leads to a similar depletion of hepatic NKT cells which likely contributes to the reduced hepatocellular injury observed following both ConA administration as well as αGal treatment. Importantly, the reduced responsiveness to αGal supports the flow cytometric data indicating reduced NKT cells as numerous reports have shown a potential downregulation of defining cell surface markers, particularly NK1.1 and/or CD49b. Together, these data highlight the deficiency in NKT cells, both in phenotypic appearance and functionality in PPARα deficient mice, a key regulatory immune cell within the normal liver but stop short of defining the mediators of this hepatic immune phenotype.
Hepatic NKT cells are regulated by a variety of factors, both membrane bound as well as secreted. Loss of CD1d, reduced production of supportive cytokines such as IL15 or over-production of inflammatory mediators have all been associated with their depletion . Likewise, activation itself may reduce NKT cell function, alter their cell surface phenotype, or induce cell death. Multiple models of fatty liver have shown interactions with many of these factors. Leptin-deficient ob/ob mice have reduced NKT cell numbers which correlates with reduced hepatic CD1d expression as well as blunted IL15 production [40, 47]. Loss of PPARα did lead to a small but significant reduction in tissue IL15 expression but had no effect on CD1d tissue expression suggesting that PPARα-deficiency, or the accumulation of hepatic lipid that occurs as a result, may influence hepatic production of this important supportive signal as has been noted in other models of fatty liver disease [1, 4, 40]. Choline-deficient diet feeding leads to a time-dependent increase in lipid accumulation and hepatic macrophage IL12 production which inversely correlates with NKT cell numbers . Moreover, genetic deletion of IL12p40 restores the hepatic NKT cell population independent of changes in hepatosteatosis. Within the current study, loss of PPARα leads to a mild microvesicular lipid deposition which correlates with a small but significant increase in serum IL12 production at baseline (Fig. 8). Such data highlight a consistent IL12 response in the presence of excess hepatic lipid accumulation though the mechanism for this upregulation remains unclear. Previous studies reported the ability of PPARα activation to suppress NFκB activation in macrophages limiting their production of a number of inflammatory cytokines [9, 10]. Likewise, loss of PPARα interrupts normal lipid and cholesterol metabolism in macrophages similar to that seen in hepatocytes . Altered lipid homeostasis can have a profound effect on macrophage function, promoting inflammatory cytokine production. Loss of fatty acid binding protein 5 (FABP5) promotes LPS-induced IL12 production in vitro and in vivo from hepatic macrophages further supporting an interaction among lipid, macrophages, and their production of IL12 . The link between IL12 and PPARα at the level of the macrophage remains unclear but is likely related to lipid accumulation and subsequent inflammatory transcription factor activation.
The current series of studies are limited by the global loss of PPARα. Adoptive transfer experiments of lymphocyte populations allow for more selective examination of the effects of this transcription factor. Data from this approach support the notion that loss of PPARα leads to a hepatic microenvironment which is not conducive to NKT cell survival. Supporting this idea, reconstitution of PPARα sufficient, lymphocyte deficient SCID mice with either wild type or PPARα deficient lymphocytes restored ConA-induced tissue injury and cytokine production in these mice. In fact, reconstitution of SCID mice with PPARα-deficient lymphocytes caused a 4 fold enhancement in liver injury when compared to wild type lymphocyte reconstitution. The reasons for this enhancement in tissue injury are not clear. Previous studies have demonstrated the impact of PPARα deficiency on lymphocyte responsiveness [14, 15]. Loss of PPARα exaggerated IFNγ production by CD4+ T cells in vitro upon stimulation with CD3 and CD28. Pilot studies confirmed this enhancement in IFNγ production by PPARα deficient lymphocytes (data not shown). In vivo examination of IFNγ production did not, however, reveal significant increases in PPARα reconstituted mice when compared to wild type mice though IL4 levels were doubled. Further examination of the time-course of IFNγ expression is warranted in this setting to better understand its role though data from this study support a function for PPARα independent of the lymphocyte in the regulation of NKT cell function and ConA responsiveness.
Interestingly, in the current study, accumulation of lipid reduces NKT cell numbers and function but does not promote an enhanced Th1 response. This is in contrast to previous studies but may be related to the degree of lipid accumulation as well as the extent of NKT cell depletion. Indeed, previous studies have shown significantly higher levels of lipid accumulation as compared to the current results while also showing significantly higher numbers of hepatic NKT cells remaining following lipid accumulation [37, 40]. It may also be that PPARα regulates the function of other cells with respect to their ability to produce Th1-type cytokines. Data presented in Fig. 8 highlight the ability PPARα-deficiency to enhance lipopolysaccharide-induced IL12 production likely from macrophages but interestingly impair hepatic production of IFNγ. It is clear that macrophages contribute to ConA-induced liver injury as their depletion reduced hepatocellular injury in part through reductions in pro-inflammatory cytokine expression . The involvement of macrophages in the current paradigm remains unclear and reduced IFNγ production following ConA exposure may result from impaired Kupffer cell responses. In vitro studies and αGal administration support a defective NKT cell response but further study is needed to determine the specific source(s) of Th1 cytokines in this and other models and the relative contribution of these cells to overall ConA-induced liver injury. It is clear that loss of PPARα leads to a significant reduction in hepatic NKT cell number and function and limits ConA-induced and αGal stimulated cytokine responses and associated tissue injury.
As discussed above, activation of PPARα stimulates peroxisome proliferation and transcription of a number of lipid metabolizing enzymes in rodents . In humans, PPARα is present at low levels within the liver and does not appear to transactivate genes involved in peroxisomal β oxidation . As such, chronic treatment with PPARα activators does not activate peroxisome or hepatocellular proliferation in humans as it does in rodents. Recent studies have demonstrated that activation of PPARα in human T lymphocytes results in strong reductions in the activation-induced expression of a number of cytokines including IFNγ, a finding consistent with its overall anti-inflammatory effects and its function in this immune cell population . The role that PPARα plays in specific lymphocyte subpopulations as well as in tissue specific localization of these lymphocyte populations in humans has not, however, been explored. Given the results of the present study, modulation of PPARα function within the liver may indirectly modulate the immune response in humans. Additional investigation will be required to determine how PPARα affects lymphocyte function within the human liver.
In conclusion, data derived from the current series of studies demonstrates the importance of PPARα in the recruitment and/or survival of NKT cells within the liver. Consistent with these reductions in NKT cells, PPARα−/− mice shown strong resistance to ConA-activated and αGal stimulated cytokine production, specifically IFNγ, and subsequent liver damage. The role that other cell populations play in this process, particularly macrophages, cannot be fully addressed in the current paradigm. Further study is required to determine the exact mechanism by which PPARα regulates the localization and/or survival of NKT cells to the liver including the absolute importance of IL15 in this process and the direct contribution of macrophages both in NKT cell survival and tissue injury following ConA exposure. Understanding the mechanisms involved in PPARα-dependent regulation of hepatic immune cell populations may prove useful in the design of therapies to modulate the immunological response of the liver.
INH conceived the current study, conducted animal experiments and wrote and revised manuscript. MK conducted animal studies and in vitro work and assisted in original study design. SMM carried out gene expression profiling studies and contributed to manuscript synthesis. MDW performed flow cytometric studies and was a major contributor to manuscript editing and revision. All authors read and approved the final manuscript.
We would like to acknowledge Mr. Jameson Milton for his assistance in animal colony maintenance in support of the current studies.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Consent for publication
Ethics approval and consent to participate
All studies involving animal subjects were approved by the institutional animal care and use committees at the University of North Carolina or East Carolina University.
These studies were funded by the National Institutes of Health Grants AA016563 (to I.N.H.) and AA019559 (to M.D.W.) as well as startup funding from the Division of Research at East Carolina University.
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