Fig 2: Pattern of the hepatic CD8+ and CD4+ phenotypes and Th1, Th2, and Th17 cells during the progression of or a rescue from HCCFemale or male DIAMOND mice were fed a CD or a WD for 24 weeks (24FCD, 24FWD, 24MCD, and 24MWD) and 40 weeks, starting at 10 weeks of age. A group of female or male mice set aside to receive a WD for 48 weeks (48FWD or 48MWD) or put back on a CD at 36 weeks of being on a WD and continued on a CD for an additional 12 weeks through week 48 (48MWDR and 48FWDR) is shown.(A and B) FVS- viable hepatic CD8+ or CD4+ T cells were analyzed for percentage of Te (CD44+CD62L-), Tem (blue line, CD44+CD62Llow), Tcm (red line, CD44+CD62Lhigh), and Tn (CD44—CD62L+) cells.(C) FVS- viable hepatic cells gated for CD4+ T cells were analyzed for the percentage of Th1 (CD3+CD4+CXCR3+CCR5-), Th2 (CCR8+IL-3Ra-), or Th17 (CD3+CD4+CCR6+CD161-) cells after 24 weeks of being on a CD or a WD.(D) The hepatic Th1 cells were analyzed for the expression of TIM-3.(E) FVS- viable hepatic cells gated for CD4+ T cells were analyzed for the percentage of Th1 or Th17 cells after 40 weeks of being on a CD or a WD.(F) The hepatic Th1 cells were analyzed for the expression of TIM-3.(G) FVS- viable hepatic cells gated for CD4+ T cells were analyzed for the percentage of Th1, Th2, or Th17 cells after 48 weeks of being on a WD or during an RD.(H) The hepatic Th1 cells were analyzed for the expression of TIM-3, after 48 weeks of being on a WD or during an RD. Data represent three to five mice/group.Error bars are SEM. *p % 0.01, **p % 0.001, and ***p % 0.0001.

Fig 3: Pattern of systemic inflammation in DIAMOND mice as they age on a regular chow diet(A) Sera were collected from DIAMOND mice at weeks 19, 24, or 40 of being on a CD or WD three to five mice/group) and analyzed for 44 cytokines and chemokines in association with time using multivariate linear regression. The overall time trend, summarized across subjects, is presented for all cytokines and chemokines, which have a significant time trend during a CD. Data represent five to nine mice/group.(B) FVS− viable splenocytes were gated for CD3+ T cells to show the proportion of CD8+ T cells and CD4+ T cells in males or females (three to five mice/group) being on a CD for 24 weeks (24MCD or 24FCD) and 40 weeks (40MCD or 40FCD) and were analyzed for M1 (F4/80+CD68+CD206−) and M2 macrophages (F4/80+CD68+/−CD206+), NK cells (CD3−CD4−CD8−CD49b+), NKT cells CD3+CD4−CD8−CD49b+), and CD11b+Gr1+ MDSCs.(C) FVS− viable splenocytes gated for CD3+CD4+ T cells were analyzed for the percentage of Th1 (CXCR3+CCR5−), Th2 (CCR8+IL-3Ra−), or Th17 (CCR6+CD161−) cells: percentage of Th1 cells that do not express TIM-3 was determined.(D) FVS− viable splenic CD8+ or CD4+ T cells were analyzed for percentage of T cell phenotypes, including Te (CD44+CD62L−), Tem (blue line, CD44+CD62Llow), Tcm (red line, CD44+CD62Lhigh), and Tn (CD44−CD62L+) cells.Error bars are SEM.

Fig 4: Females tend to modulate the hepatic inflammation better than males as they age on a regular chow diet(A) Female or male DIAMOND mice received a CD for 24 and 40 weeks, starting at 10 weeks of age (three to five mice/group). Livers were collected after 24 and 40 weeks of being on a CD and subjected to an H&E staining followed by enumeration of inflammatory cells using a machine learning (ML) model trained to detect and quantitate lobular inflammation on whole-slide histology images. ML lobular inflammation is shown as blue color.(B) FVS− viable cells gated for CD3+CD4+ T cells were analyzed for the percentage of Th1 (CXCR3+CCR5−), Th2 (CCR8+IL-3Ra−), or Th17 (CCR6+CD161−) cells: percentage of Th1 cells that do not express TIM-3 was determined.(C) FVS− viable cells were gated for CD3+ T cells to show the proportion of CD8+ T cells and CD4+ T cells in males or females being on a CD for 24 weeks (24MCD or 24FCD) and 40 weeks (40MCD or 40FCD), were analyzed for M1 (F4/80+CD68+CD206−) and M2 macrophages (F4/80+CD68+/−CD206+), and were analyzed for NK cells (CD3−CD4−CD8−CD49b+) and NKT cells (CD3+CD4−CD8−CD49b+). Percent total cells were analyzed for the ratio of CD8+ T cells to MDSC (CD8/MDSC) or CD4+ T cells to MDSC (CD4/MDSC).(D) FVS− viable splenic CD8+ or CD4+ T cells were analyzed for percentage of T cell phenotypes, including Te (CD44+CD62L−), Tem (blue line, CD44+CD62Llow), Tcm (red line, CD44+CD62Lhigh), and Tn (CD44−CD62L+) cells.Error bars are SEM. *p ≤ 0.01, **p ≤ 0.001, and ***p ≤ 0.0001.