Mice lacking cyclophilin B, but not cyclophilin A, are protected from the development of NASH in a diet and chemical-induced model.

Cyclophilins are a diverse family of peptidyl-prolyl isomerases (PPIases) of importance in a variety of essential cellular functions. We previously reported that the pan-cyclophilin inhibitor drug reconfilstat (CRV431) decreased disease in mice under the western-diet and carbon tetrachloride (CCl4) non-alcoholic steatohepatitis (NASH) model. CRV431 inhibits several cyclophilin isoforms, among which cyclophilin A (CypA) and B (CypB) are the most abundant. It is not known whether simultaneous inhibition of multiple cyclophilin family members is necessary for the observed therapeutic effects or if loss-of-function of one is sufficient. Identifying the responsible isoform(s) would enable future fine-tuning of drug treatments. Features of human liver fibrosis and complete NASH can be reliably replicated in mice by administration of intraperitoneal CCl4 alone or CCl4 in conjunction with high sugar, high cholesterol western diet, respectively. Here we show that while wild-type (WT) and Ppia-/- CypA KO mice develop severe NASH disease features under these models, Ppib-/- CypB KO mice do not, as measured by analysis of picrosirius red and hematoxylin & eosin-stained liver sections and TNFα immuno-stained liver sections. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NASH. In this study, mice without CypB, but not mice without CypA, were significantly protected from the development of the characteristic features of NASH. These data suggest that CypB is necessary for NASH disease progression. Further investigation is necessary to determine whether the specific role of CypB in the endoplasmic reticulum secretory pathway is of significance to its effect on NASH development

Set 3 CypB KO mice did not exhibit chronically activated ER stress markers relative to Set 3 WT mice.

Set 3 CypB KO mice did not exhibit chronically activated ER stress markers relative to Set 3 WT mice.</p

Body and liver weights of CypA and CypB KO mice were reduced relative to WT controls while liver to body weight ratios were mostly unchanged.

Body and liver weights of CypA and CypB KO mice were reduced relative to WT controls while liver to body weight ratios were mostly unchanged.</p

CypB KO mice and not CypA KO mice develop less liver fibrosis after administration of a CCl₄ and WD model to replicate NAFLD/NASH.

WT, CypB KO, CypA KO, and CypD KO mice were nourished with western diet chow and sugar solution while also administered CCl4 twice weekly via intraperitoneal (IP) injection for twenty weeks. WT and CypA mice developed significant interlobular branching fibrosis, as determined by histological analysis of picrosirius red-stained liver tissue sections. CypB KO mice however, displayed significantly less stained area relative to WT. The area of stained tissue relative to the total area was quantified with ImageJ software over several fields per sample. Representative images are shown here. ***p≤0.001 significance between a condition and WT control by unpaired students t-test.</p

NASH scoring criteria.

Cyclophilins are a diverse family of peptidyl-prolyl isomerases (PPIases) of importance in a variety of essential cellular functions. We previously reported that the pan-cyclophilin inhibitor drug reconfilstat (CRV431) decreased disease in mice under the western-diet and carbon tetrachloride (CCl4) non-alcoholic steatohepatitis (NASH) model. CRV431 inhibits several cyclophilin isoforms, among which cyclophilin A (CypA) and B (CypB) are the most abundant. It is not known whether simultaneous inhibition of multiple cyclophilin family members is necessary for the observed therapeutic effects or if loss-of-function of one is sufficient. Identifying the responsible isoform(s) would enable future fine-tuning of drug treatments. Features of human liver fibrosis and complete NASH can be reliably replicated in mice by administration of intraperitoneal CCl4 alone or CCl4 in conjunction with high sugar, high cholesterol western diet, respectively. Here we show that while wild-type (WT) and Ppia-/- CypA KO mice develop severe NASH disease features under these models, Ppib-/- CypB KO mice do not, as measured by analysis of picrosirius red and hematoxylin & eosin-stained liver sections and TNFα immuno-stained liver sections. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NASH. In this study, mice without CypB, but not mice without CypA, were significantly protected from the development of the characteristic features of NASH. These data suggest that CypB is necessary for NASH disease progression. Further investigation is necessary to determine whether the specific role of CypB in the endoplasmic reticulum secretory pathway is of significance to its effect on NASH development.</div

CypB KO livers, but not CypA KO livers, show clearly less inflammatory TNFα staining relative to WT controls after administration of a CCl₄ and WD model to replicate NAFLD/NASH.

WT, CypB KO, and CypA KO mice were nourished with western diet chow and sugar solution while also administered CCl4 twice weekly via intraperitoneal (IP) injection for twenty weeks. WT and CypA mice exhibited extensive staining for the inflammatory cytokine TNFα as determined by immunohistochemical staining of liver tissue sections. CypB KO mice however, displayed qualitatively less TNFα staining relative to WT. Representative images are shown here. Blue staining reflects nuclei stained with hematoxylin. Brown coloration from the DAB chromagen reflects areas of reactivity with secondary antibody and thus the presence of TNFα protein. DAB color intensity was not quantified because there is not a linear relationship between antigen concentration and absorbance.</p

CypB KO mice measurements and data.

Raw data from Set 1, 2 and 3 CypB KO mice were taken immediately after sacrifice. Histological data are also included. Averages, standard deviation, and standard error are listed for each set. (TIF)</p

CypB KO mice exhibit differential gene expression compared to WT mice undergoing a NAFLD/NASH model.

WT and CypB KO mice were nourished with western diet chow and sugar solution while also administered CCl4 twice weekly via intraperitoneal (IP) injection for twenty weeks. RNA isolated from the 8 WT livers and 5 CypB KO livers were analyzed in PCR arrays for genes relevant to hepatotoxicity or liver fibrosis. Green dots represent gene transcripts induced in Set 3 CypB KO mouse livers, while red dots represent decreased expression. Notably induced genes are summarized in the accompanying tables.</p

CypB KO mice and not CypA KO mice do not develop essential features of NAFLD/NASH after administration of a CCl₄ and WD model to replicate NAFLD/NASH.

WT, CypB KO, and CypA KO mice were nourished with western diet chow and sugar solution while also administered CCl4 twice weekly via intraperitoneal (IP) injection for twenty weeks. WT and CypA mice developed significant steatosis, inflammation, and hepatocyte ballooning, as determined by histological analysis of H&E stained liver tissue sections. NAS were significantly higher for these groups than non-diseased mice. CypB KO mice however, displayed significantly lower NAS relative to WT. Representative images are shown here. ***p≤0.001 significance between a condition and WT control by unpaired students t-test.</p

Neither WT nor CypA or CypB KO Set 1 or Set 2 mice showed significant increases in NAS scoring.

Neither WT nor CypA or CypB KO Set 1 or Set 2 mice showed significant increases in NAS scoring.</p