One.0056004.g(CKPV)2 Inhibits Candida albicans VaginitisFigure 4. (CKPV)2 inhibits macrophages phagocytosis of Candida albicans. (A) The macrophage cells (wide arrow) were polygonal shape stretching and Candida albicans (tiny arrow) were dyed bright blue by Wright-Ji Musa in inverted microscope. (Magnification: 40). The number of cells and engulfed microorganism were recorded in 10 random views under the microscope. (B) The phagocytosis index (number of engulfed fungus/100 cells)of macrophages with indicated treatment. *p,0.01 (ANOVA). Experiments in this figure were repeated three times and similar results were obtained. doi:10.1371/journal.pone.0056004.gsignificantly augmented in primary cultured macrophages after (CKPV)2 treatment. To demonstrate the role of MC1R in this process, we applied target get AZ876 siRNAs to effectively knockdown MC1R in macrophages (Fig. 5A). Results showed that knockdown of MC1R by target siRNAs (S1, S2 and S3) (see RNAi sequence in Tab. 1) significantly reduced (CKPV)2induced cAMP production in macrophages (Fig. 5A). However, no cAMP was induced in MC1R-null COS-7 cells after (CKPV)2 exposure. On the other hand, cAMP level was increased in COS-7 cells exogenously expressing MC1R.(Fig. 5B). These results tert-Butylhydroquinone cost together suggest that (CKPV)2 promotes cAMP production via MC1R.(CKPV)2 Induces Macrophage M1 to M2 PolarizationMacrophage M1 to M2 polarization involves a reduction in expression of pro-inflammatory cytokines, together with an increase of IL-10 secretion and arginase activity [40]. Here, we 
observed that LPS/IFN-c-induced TNF-a production was almost blocked by co-administration with (CKPV)2 in primary cultured macrophages, as the cytotoxicity of (CKPV)2-treated macrophages(CKPV)2 Inhibits Candida albicans VaginitisFigure 5. (CKPV)2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV)2-induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV)2 mRNA expression in MC1R cDNA-transfected COS-7 cells 
(COS-7/MC1R) or nonsensecDNA-transfected control cells (COS-7). Lower panel: (CKPV)2-induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNAtransfected COS-7 cells. *p,0.01 (ANOVA). Experiments in this figure were repeated three times and similar results were obtained. doi:10.1371/journal.pone.0056004.gsupernatant to L929 cells decreased significantly (Fig. 6A). The secretion of inflammatory cytokines including IL-1b and IL-6 was also inhibited after (CKPV)2 administration (Fig. 6C and D). Importantly, macrophage IL-10 production and arginase activity, 1326631 the indicators of M2 polarization, were significantly increased by (CKPV)2 (Fig. 6B and E). Notably, (CKPV)2’s effects on cytokinesproduction were almost blocked by MC1R siRNA knockdown, suggesting that MC1R was required for (CKPV)2 effects on macrophages (Fig. 6C ). It should be noted that a-MSH had similar effects on macrophages as (CKPV)2 (Fig. 6). Based on these data, we summarized that (CKPV)2 inhibited pro-inflammatory cytokines (TNF-a, IL-1b and IL-6) production while increasing(CKPV)2 Inhibits Candida albicans VaginitisFigure 6. (CKPV)2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1b, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LP.One.0056004.g(CKPV)2 Inhibits Candida albicans VaginitisFigure 4. (CKPV)2 inhibits macrophages phagocytosis of Candida albicans. (A) The macrophage cells (wide arrow) were polygonal shape stretching and Candida albicans (tiny arrow) were dyed bright blue by Wright-Ji Musa in inverted microscope. (Magnification: 40). The number of cells and engulfed microorganism were recorded in 10 random views under the microscope. (B) The phagocytosis index (number of engulfed fungus/100 cells)of macrophages with indicated treatment. *p,0.01 (ANOVA). Experiments in this figure were repeated three times and similar results were obtained. doi:10.1371/journal.pone.0056004.gsignificantly augmented in primary cultured macrophages after (CKPV)2 treatment. To demonstrate the role of MC1R in this process, we applied target siRNAs to effectively knockdown MC1R in macrophages (Fig. 5A). Results showed that knockdown of MC1R by target siRNAs (S1, S2 and S3) (see RNAi sequence in Tab. 1) significantly reduced (CKPV)2induced cAMP production in macrophages (Fig. 5A). However, no cAMP was induced in MC1R-null COS-7 cells after (CKPV)2 exposure. On the other hand, cAMP level was increased in COS-7 cells exogenously expressing MC1R.(Fig. 5B). These results together suggest that (CKPV)2 promotes cAMP production via MC1R.(CKPV)2 Induces Macrophage M1 to M2 PolarizationMacrophage M1 to M2 polarization involves a reduction in expression of pro-inflammatory cytokines, together with an increase of IL-10 secretion and arginase activity [40]. Here, we observed that LPS/IFN-c-induced TNF-a production was almost blocked by co-administration with (CKPV)2 in primary cultured macrophages, as the cytotoxicity of (CKPV)2-treated macrophages(CKPV)2 Inhibits Candida albicans VaginitisFigure 5. (CKPV)2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV)2-induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV)2 mRNA expression in MC1R cDNA-transfected COS-7 cells (COS-7/MC1R) or nonsensecDNA-transfected control cells (COS-7). Lower panel: (CKPV)2-induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNAtransfected COS-7 cells. *p,0.01 (ANOVA). Experiments in this figure were repeated three times and similar results were obtained. doi:10.1371/journal.pone.0056004.gsupernatant to L929 cells decreased significantly (Fig. 6A). The secretion of inflammatory cytokines including IL-1b and IL-6 was also inhibited after (CKPV)2 administration (Fig. 6C and D). Importantly, macrophage IL-10 production and arginase activity, 1326631 the indicators of M2 polarization, were significantly increased by (CKPV)2 (Fig. 6B and E). Notably, (CKPV)2’s effects on cytokinesproduction were almost blocked by MC1R siRNA knockdown, suggesting that MC1R was required for (CKPV)2 effects on macrophages (Fig. 6C ). It should be noted that a-MSH had similar effects on macrophages as (CKPV)2 (Fig. 6). Based on these data, we summarized that (CKPV)2 inhibited pro-inflammatory cytokines (TNF-a, IL-1b and IL-6) production while increasing(CKPV)2 Inhibits Candida albicans VaginitisFigure 6. (CKPV)2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1b, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LP.
