DataSheet1_Development of a novel human triculture model of non-alcoholic fatty liver disease and identification of berberine as ameliorating steatosis, oxidative stress and fibrosis.docx

Objectives: Non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) and hepatocarcinoma is a serious and growing problem. However, the development of new therapies is severely hindered by a lack of high-throughput assays for drug testing.Methods: We have developed a simple transwell assay comprised of HepG2 hepatocytes, hepatic LX-2 stellate cells, and differentiated THP-1 cells. The cells were incubated with an activating mixture containing the NASH-associated risk factors, glucose, insulin, free fatty acids (FFAs), and lipopolysaccharide (LPS) for 72 h. We compared different combinations of culture conditions to obtain a model system that recapitulates the main features of NAFLD/NASH, i.e., increased steatosis, reactive oxygen species (ROS), secretion of pro-inflammatory cytokines/chemokines, and presence of fibrosis. To confirm the usefulness of the optimized model system, we screened for compounds that inhibit steatosis in the hepatocytes and evaluated the most effective compound in the triculture model system.Results: The activating mixture stimulated HepG2 cells in this triculture to accumulate more fat and produce higher levels of reactive oxygen species (ROS) than HepG2 cells in monocultures. As well, higher levels of inflammatory cytokines and chemokines (IL-8, IL-6, MIP-1α, etc.) were produced in this triculture compared to monocultures. In addition, in all LX-2 monocultures and cocultures, exposure to the activating mixture increased markers of fibrosis. A major strength of our triculture system is that it makes possible the simultaneous monitoring of 4 main features of NASH, i.e., steatosis, oxidative stress, inflammation and fibrosis. Screening potential modulators that may reduce steatosis in HepG2 cells revealed the protective effects of the isoalkaloid, berberine. Tested using this novel triculture assay, treatment with 5 µM berberine decreased steatosis and ROS in HepG2 hepatocytes, reduced inflammatory cytokine production and inhibited collagen production from LX-2 cells.Conclusion: This simple triculture model recapitulates the main features of NAFLD/NASH and should be useful for high-throughput preclinical drug discovery. In this model, berberine showed promising results in decreasing steatosis and ROS and protection against fibrosis.</p

Table_4_A low carbohydrate diet high in fish oil and soy protein delays inflammation, hematopoietic stem cell depletion, and mortality in miR-146a knock-out mice.DOCX

Since our previous studies found a low carbohydrate (CHO) diet containing soy protein and fish oil (15%Amylose/Soy/FO) significantly reduced lung and breast cancer in mice we asked herein if this low CHO diet could also delay the onset of myeloid malignancies. To test this we employed a miR-146a knock-out (KO) mouse model and found the 15%Amylose/Soy/FO diet increased their median lifespan by 8.5 month, compared to these mice on a Western diet. This was associated with increased lymphocytes and reduced monocytes, granulocytes, blood glucose and insulin levels. Inflammatory cytokine/chemokine studies carried out with 6-month-old mice, before any signs of illness, revealed the 15%Amylose/Soy/FO diet significantly reduced pro-inflammatory cytokines. This low CHO diet also led to an increase in plasma β-hydroxybutyrate and in liver fatty acid synthase levels. This, together with higher liver carnitine palmitoyltransferase I levels suggested that the 15%Amylose/Soy/FO diet was causing a systemic metabolic shift from glucose to fatty acids as an energy source. Lastly, we found the 15%Amylose/Soy/FO diet resulted in significantly higher numbers of primitive hematopoietic stem cells (HSCs) in the bone marrow of 6-month-old mice than those fed a Western diet. Taken together, these results suggest a 15%Amylose/Soy/FO diet reduces chronic inflammation and increases fatty acid oxidation and that this, in turn, may prevent HSC proliferation and exhaustion, thereby delaying myeloid malignancy-induced death of miR-146a KO mice. We suggest a low CHO diet containing soy protein and fish oil could be beneficial in reducing the risk of myeloid malignancies in patients with low miR-146a levels.</p

SHIP+/+ and −/− GM-CSF-derived DCs suppress via a contact-dependent mechanism.

<p><b>A)</b> The indicated number of SHIP<b>+/+</b> and −/− GM-DCs were plated in the bottom chamber of a 0.4 µm 96 well transwell plate and WT splenocytes (2×10⁵ ) were stimulated with soluble αCD3+ αCD28 Abs and plated in the top chamber. Proliferation was determined after 72 hrs by incorporation of ³H-thymidine for the last 18 hrs. Data shown are the mean ± SEM of triplicate cultures and are representative of 3 independent experiments. <b>B)</b> Left panel, relative percent suppression with the addition of agents that reduce the presence of ROS (2 mM NAC, 100 U/ml catalase, 200 U/ml SOD). Right panel, relative percent suppression with the addition of blocking antibodies to CTLA4 (10 µg/ml), LFA1+ mac1 (5 µg/ml each) and PD-L2 (10 µg/ml). Data shown are the mean ± SEM of triplicate cultures and are representative of at least 2 independent experiments with the exception of PD-L2 which was only performed once. <b>C)</b> SHIP+/+ and −/− GM-DCs were cultured for 4 days with WT sorted conventional T cells at a ratio of 1∶2 DCs to T cells and analyzed for Treg induction by flow cytometry. Data shown are the mean ± SEM of two independent experiments.</p

SHIP+/+ and −/− GM-DCs suppress T cell activation.

<p>2×10^{5<b> </b>}WT splenocytes were stimulated with soluble αCD3+ αCD28 antibodies and incubated with the indicated number of SHIP+/+ or −/− <b>A)</b> CD11c⁺ splenic DCs, FL- or GM-DCs. Proliferation was determined after 72 hrs by incorporation of ³H-thymidine for the last 18 hrs. Data shown are the mean ± SEM of triplicate cultures and are representative of more than 3 independent experiments. Supernatants were collected after 72 hrs from <b>B)</b> Splenic (25×10³) <b>C)</b> Flt3L-derived (25×10³) and <b>D)</b> GM-derived DCs (50×10³) co-cultures and subjected to cytokine ELISAs or Griess assays for NO determination. Data shown are the mean ± SEM of triplicate cultures and are representative of 2–3 independent experiments. *p<0.05 relative to stimulated splenocytes in the absence of DCs (Ctrl).</p

Presentation_1_A low carbohydrate diet high in fish oil and soy protein delays inflammation, hematopoietic stem cell depletion, and mortality in miR-146a knock-out mice.pdf

Since our previous studies found a low carbohydrate (CHO) diet containing soy protein and fish oil (15%Amylose/Soy/FO) significantly reduced lung and breast cancer in mice we asked herein if this low CHO diet could also delay the onset of myeloid malignancies. To test this we employed a miR-146a knock-out (KO) mouse model and found the 15%Amylose/Soy/FO diet increased their median lifespan by 8.5 month, compared to these mice on a Western diet. This was associated with increased lymphocytes and reduced monocytes, granulocytes, blood glucose and insulin levels. Inflammatory cytokine/chemokine studies carried out with 6-month-old mice, before any signs of illness, revealed the 15%Amylose/Soy/FO diet significantly reduced pro-inflammatory cytokines. This low CHO diet also led to an increase in plasma β-hydroxybutyrate and in liver fatty acid synthase levels. This, together with higher liver carnitine palmitoyltransferase I levels suggested that the 15%Amylose/Soy/FO diet was causing a systemic metabolic shift from glucose to fatty acids as an energy source. Lastly, we found the 15%Amylose/Soy/FO diet resulted in significantly higher numbers of primitive hematopoietic stem cells (HSCs) in the bone marrow of 6-month-old mice than those fed a Western diet. Taken together, these results suggest a 15%Amylose/Soy/FO diet reduces chronic inflammation and increases fatty acid oxidation and that this, in turn, may prevent HSC proliferation and exhaustion, thereby delaying myeloid malignancy-induced death of miR-146a KO mice. We suggest a low CHO diet containing soy protein and fish oil could be beneficial in reducing the risk of myeloid malignancies in patients with low miR-146a levels.</p

Table_1_A low carbohydrate diet high in fish oil and soy protein delays inflammation, hematopoietic stem cell depletion, and mortality in miR-146a knock-out mice.DOCX

Since our previous studies found a low carbohydrate (CHO) diet containing soy protein and fish oil (15%Amylose/Soy/FO) significantly reduced lung and breast cancer in mice we asked herein if this low CHO diet could also delay the onset of myeloid malignancies. To test this we employed a miR-146a knock-out (KO) mouse model and found the 15%Amylose/Soy/FO diet increased their median lifespan by 8.5 month, compared to these mice on a Western diet. This was associated with increased lymphocytes and reduced monocytes, granulocytes, blood glucose and insulin levels. Inflammatory cytokine/chemokine studies carried out with 6-month-old mice, before any signs of illness, revealed the 15%Amylose/Soy/FO diet significantly reduced pro-inflammatory cytokines. This low CHO diet also led to an increase in plasma β-hydroxybutyrate and in liver fatty acid synthase levels. This, together with higher liver carnitine palmitoyltransferase I levels suggested that the 15%Amylose/Soy/FO diet was causing a systemic metabolic shift from glucose to fatty acids as an energy source. Lastly, we found the 15%Amylose/Soy/FO diet resulted in significantly higher numbers of primitive hematopoietic stem cells (HSCs) in the bone marrow of 6-month-old mice than those fed a Western diet. Taken together, these results suggest a 15%Amylose/Soy/FO diet reduces chronic inflammation and increases fatty acid oxidation and that this, in turn, may prevent HSC proliferation and exhaustion, thereby delaying myeloid malignancy-induced death of miR-146a KO mice. We suggest a low CHO diet containing soy protein and fish oil could be beneficial in reducing the risk of myeloid malignancies in patients with low miR-146a levels.</p

Secreted cytokines are not responsible for T cell suppression.

<p><b>A)</b> WT splenocytes (2×10⁵) were stimulated with soluble αCD3+ αCD28 Abs and co-incubated with SHIP+/+ or −/− GM-DCs (50×10³) containing isotype control Ab (iso) or the indicated neutralizing cytokine Ab (10 µg/ml) or LAP (250 ng/ml). <b>B)</b> WT splenocytes were stimulated with soluble αCD3+ αCD28 Abs and incubated with the indicated number of SHIP+/+ and −/− GM-DCs and IL-2 (100 U) was added as indicated and proliferation determined after 72 hrs. Data shown are the mean ± SEM of triplicate cultures and is representative of at least 2 independent experiments. <b>C)</b> CD4⁺ T cells from SHIP+/+ and −/− GM-DC (50×10³) co-cultures were analyzed for expression of CD25 by flow cytometry. Splenocyte control  =  grey fill, WT GM-DCs  =  black line and SHIP−/− GM-DCs  =  grey line. Data shown are representative of 2 independent experiments.</p

Model of SHIP+/+ and −/− GM-DC-induced T cell suppression.

<p>SHIP+/+ and −/− GM-DCs both suppress T cell proliferation in a contact-dependent manner. αCD3+ αCD28-stimulated T cells secrete IFNγ, which acts on WT GM-DCs to upregulate iNOS and secrete NO. This NO then suppresses T cell proliferation. SHIP−/− GM-DCs express Arg 1 and do not produce NO, but may use an alternate direct mechanism of suppression or induce the expansion or differentiation of a regulatory cell, likely not Tregs, to suppress T cell proliferation. If a second cell type is involved in SHIP−/− GM-DC-induced suppression, its induction or activation is contact-dependent.</p

SHIP−/− GM-DCs express Arg 1.

<p><b>A)F</b> Day 8 SHIP+/+ and −/− GM- and FL-DCs were subjected to Western analysis using Abs to SHIP, Arg1 and Grb2 as a loading control. Data shown are representative of at least 3 independent experiments. <b>B)</b> mRNA expression of <i>Arg 1</i>, <i>Arg 2</i>, <i>Nos2</i>, and <i>Indo</i> in SHIP+/+ and SHIP−/− GM- and FL-DCs. Data shown is mean ± SEM of duplicate determinations from 2–3 independent experiments. *p<0.05 relative to SHIP+/+. <b>C)</b> WT splenocytes were stimulated with soluble αCD3+ αCD28 Abs and incubated with SHIP+/+ or −/− GM-DCs (50×10³) ±100 µM of the arginase inhibitor, Bec, 2 mM L-arginine (L-Arg), 1 µM of the IDO inhibitor, exiguamine A (Exi) or 200 µM L-tryptophan (L-Tryp). Data shown are the mean ± SEM of triplicate determinations and are representative of 2 independent experiments.</p

Table_2_A low carbohydrate diet high in fish oil and soy protein delays inflammation, hematopoietic stem cell depletion, and mortality in miR-146a knock-out mice.DOCX

Since our previous studies found a low carbohydrate (CHO) diet containing soy protein and fish oil (15%Amylose/Soy/FO) significantly reduced lung and breast cancer in mice we asked herein if this low CHO diet could also delay the onset of myeloid malignancies. To test this we employed a miR-146a knock-out (KO) mouse model and found the 15%Amylose/Soy/FO diet increased their median lifespan by 8.5 month, compared to these mice on a Western diet. This was associated with increased lymphocytes and reduced monocytes, granulocytes, blood glucose and insulin levels. Inflammatory cytokine/chemokine studies carried out with 6-month-old mice, before any signs of illness, revealed the 15%Amylose/Soy/FO diet significantly reduced pro-inflammatory cytokines. This low CHO diet also led to an increase in plasma β-hydroxybutyrate and in liver fatty acid synthase levels. This, together with higher liver carnitine palmitoyltransferase I levels suggested that the 15%Amylose/Soy/FO diet was causing a systemic metabolic shift from glucose to fatty acids as an energy source. Lastly, we found the 15%Amylose/Soy/FO diet resulted in significantly higher numbers of primitive hematopoietic stem cells (HSCs) in the bone marrow of 6-month-old mice than those fed a Western diet. Taken together, these results suggest a 15%Amylose/Soy/FO diet reduces chronic inflammation and increases fatty acid oxidation and that this, in turn, may prevent HSC proliferation and exhaustion, thereby delaying myeloid malignancy-induced death of miR-146a KO mice. We suggest a low CHO diet containing soy protein and fish oil could be beneficial in reducing the risk of myeloid malignancies in patients with low miR-146a levels.</p