Differential impact of the dual CCR2/CCR5 inhibitor cenicriviroc on migration of monocyte and lymphocyte subsets in acute liver injury

<div><p>A hallmark of acute hepatic injury is the recruitment of neutrophils, monocytes and lymphocytes, including natural killer (NK) or T cells, towards areas of inflammation. The recruitment of leukocytes from their reservoirs bone marrow or spleen into the liver is directed by chemokines such as CCL2 (for monocytes) and CCL5 (for lymphocytes). We herein elucidated the impact of chemokine receptor inhibition by the dual CCR2 and CCR5 inhibitor cenicriviroc (CVC) on the composition of myeloid and lymphoid immune cell populations in acute liver injury. CVC treatment effectively inhibited the migration of bone marrow monocytes and splenic lymphocytes (NK, CD4 T-cells) towards CCL2 or CCL5 <i>in vitro</i>. When liver injury was induced by an intraperitoneal injection of carbon tetrachloride (CCl₄) in mice, followed by repetitive oral application of CVC, flow cytometric and unbiased t-SNE analysis of intrahepatic leukocytes demonstrated that dual CCR2/CCR5 inhibition <i>in vivo</i> significantly decreased numbers of monocyte derived macrophages in acutely injured livers. CVC also reduced numbers of Kupffer cells (KC) or monocyte derived macrophages with a KC-like phenotype, respectively, after injury. In contrast to the inhibitory effects <i>in vitro</i>, CVC had no impact on the composition of hepatic lymphoid cell populations <i>in vivo</i>. Effective inhibition of monocyte recruitment was associated with reduced inflammatory macrophage markers and moderately ameliorated hepatic necroses at 36h after CCl₄. In conclusion, dual CCR2/CCR5 inhibition primarily translates into reduced monocyte recruitment in acute liver injury <i>in vivo</i>, suggesting that this strategy will be effective in reducing inflammatory macrophages in conditions of liver disease.</p></div

CVC attenuates CCL2 and CCL5 dependent leukocyte chemotaxis <i>in vitro</i>.

<p>(A) Representative FACS plots displaying transwell migration chemotaxis assays (at 2 hours) of CCL2-induced monocyte migration from total bone marrow of c57bl/6 wildtype mice. CVC was used at a concentration of 1μM. Normalized chemotaxis ratio towards CCL2 (5nM) or CCL5 (5nM) of bone-marrow derived monocytes compared to vehicle (Vhc). (B) Same analysis for NK cells from bone marrow. (C) Transwell migration assays using lymphocytes from mouse spleen. Normalized chemotaxis ratio of lymphocytes towards CCL2 (5nM) or CCL5 (5 nM). Data are presented as mean ± SD based on n≥3 per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student <i>t</i> test).</p

CVC modulates monocyte-dependent liver inflammation without directly interfering with macrophage polarization.

<p>(A) RNA from whole liver tissue was subjected to quantitative gene expression analysis (NanoString kit, covering 72 genes). Gene expression analysis of macrophage (CCR2) and Kupffer cell (CD68) markers, hepatocyte function (albumin) as well as chemokines (CCL2) and inflammation associated genes (S100a8, S100a9). (B) Demonstration of Log2-fold change in gene expression of 19 chosen candidates comparing Vhc and CVC treated livers. (C) Bone marrow derived macrophages (BMDM) were cultured for 7 days and then stimulated for 24h with IFNγ (M1 phenotype), IL-4 (M2) or LPS (pathogen recognition), either in presence or without CVC (1μM). Representative phase contrast images of BMDM 24h after stimulation, taken at 10x, scale bar 100μm. Gene expression analysis of macrophage polarization markers and effector cytokines. Data are presented as mean ± SD based on n≥6 mice per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student <i>t</i> test).</p

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NanoString gene analysis raw data file

CVC inhibits hepatic monocyte infiltration in acute liver injury.

<p>All results were obtained from c57bl/6 wildtype mice 36h after CCl₄ challenge. (A) Representative FACS plots showing monocyte-derived macrophages (MoMF, dashed) and Kupffer cells (KC, solid) as well as corresponding quantification in percent of liver leukocytes. (B) Total numbers of blood monocytes and the Ly-6C positive subset were analyzed in parallel. (C+D) Unbiased t-SNE analysis of myeloid liver (C) or blood (D) cells from treatment groups (n = 6) illustrate myeloid immune cell populations, which are (mostly) unique in vehicle treated (yellow) or CVC treated mice (blue). Mixed cell population that are equally found in both treatment groups are displayed in dark-green (liver) or grey-blue (blood). Single cell clusters were further characterized by relative myeloid surface marker expression of treatment groups and compared to total liver or blood cells (representative histograms). Data are presented as mean ± SD based on n≥6 mice per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student <i>t</i> test).</p

CVC reduces the accumulation of hepatic macrophages in CCl₄-induced acute liver injury.

<p>(A) Acute liver injury was conducted by a single IP injection of CCl₄. The <i>in vivo</i> effects of CVC on immune cell migration into acutely injured liver was assessed at 12h, 24h and 36h after CCl₄, and after one to three doses of CVC. (B) Liver histology (H&E staining) of representative liver sections for control and treatment groups. Hepatic injury is assessed by necrotic area fraction and serum alanine transaminase (ALT) levels. (C) Representative F4/80 immunohistochemical staining of liver sections and the corresponding F4/80 positive area fraction demonstrate reduced macrophage numbers in CVC treated livers. Data are presented as mean ± SD based on n≥3 per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student <i>t</i> test).</p

CVC inhibits hepatic monocyte infiltration in acute liver injury.

<p>All results were obtained from c57bl/6 wildtype mice 36h after CCl₄ challenge. (A) Representative FACS plots showing monocyte-derived macrophages (MoMF, dashed) and Kupffer cells (KC, solid) as well as corresponding quantification in percent of liver leukocytes. (B) Total numbers of blood monocytes and the Ly-6C positive subset were analyzed in parallel. (C+D) Unbiased t-SNE analysis of myeloid liver (C) or blood (D) cells from treatment groups (n = 6) illustrate myeloid immune cell populations, which are (mostly) unique in vehicle treated (yellow) or CVC treated mice (blue). Mixed cell population that are equally found in both treatment groups are displayed in dark-green (liver) or grey-blue (blood). Single cell clusters were further characterized by relative myeloid surface marker expression of treatment groups and compared to total liver or blood cells (representative histograms). Data are presented as mean ± SD based on n≥6 mice per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student <i>t</i> test).</p

Dose response curves for cenicriviroc-dependent inhibition (A) and maraviroc-dependent inhibition (B).

<p>R5, dual and X4-tropic HIV-2 strains are depicted in continuous, dashed and dotted black lines, respectively. HIV-1 R5-tropic strain is depicted using the continuous gray line.</p

Table_1_APOBEC3G/3A Expression in Human Immunodeficiency Virus Type 1-Infected Individuals Following Initiation of Antiretroviral Therapy Containing Cenicriviroc or Efavirenz.docx

<p>Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family members are cytidine deaminases that play crucial roles in innate responses to retrovirus infection. The mechanisms by which some of these enzymes restrict human immunodeficiency virus type 1 (HIV-1) replication have been extensively investigated in vitro. However, little is known regarding how APOBEC3 proteins affect the pathogenesis of HIV-1 infection in vivo and how antiretroviral therapy influences their expression. In this work, a longitudinal analysis was performed to evaluate APOBEC3G/3A expression in peripheral blood mononuclear cells of antiretroviral-naive HIV-1-infected individuals treated with cenicriviroc (CVC) or efavirenz (EFV) at baseline and 4, 12, 24, and 48 weeks post-treatment follow-up. While APOBEC3G expression was unaffected by therapy, APOBEC3A levels increased in CVC but not EFV arm at week 48 of treatment. APOBEC3G expression correlated directly with CD4⁺ cell count and CD4⁺/CD8⁺ cell ratio, whereas APOBEC3A levels inversely correlated with plasma soluble CD14. These findings suggest that higher APOBEC3G/3A levels may be associated with protective effects against HIV-1 disease progression and chronic inflammation and warrant further studies.</p

Table_2_APOBEC3G/3A Expression in Human Immunodeficiency Virus Type 1-Infected Individuals Following Initiation of Antiretroviral Therapy Containing Cenicriviroc or Efavirenz.docx

<p>Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family members are cytidine deaminases that play crucial roles in innate responses to retrovirus infection. The mechanisms by which some of these enzymes restrict human immunodeficiency virus type 1 (HIV-1) replication have been extensively investigated in vitro. However, little is known regarding how APOBEC3 proteins affect the pathogenesis of HIV-1 infection in vivo and how antiretroviral therapy influences their expression. In this work, a longitudinal analysis was performed to evaluate APOBEC3G/3A expression in peripheral blood mononuclear cells of antiretroviral-naive HIV-1-infected individuals treated with cenicriviroc (CVC) or efavirenz (EFV) at baseline and 4, 12, 24, and 48 weeks post-treatment follow-up. While APOBEC3G expression was unaffected by therapy, APOBEC3A levels increased in CVC but not EFV arm at week 48 of treatment. APOBEC3G expression correlated directly with CD4⁺ cell count and CD4⁺/CD8⁺ cell ratio, whereas APOBEC3A levels inversely correlated with plasma soluble CD14. These findings suggest that higher APOBEC3G/3A levels may be associated with protective effects against HIV-1 disease progression and chronic inflammation and warrant further studies.</p