Histatin-1 Attenuates LPS-Induced Inflammatory Signaling in RAW264.7 Macrophages

Abstract

Macrophages play a critical role in the inflammatory response to environmental triggers, such as lipopolysaccharide (LPS). Inflammatory signaling through macrophages and the innate immune system are increasingly recognized as important contributors to multiple acute and chronic disease processes. Nitric oxide (NO) is a free radical that plays an important role in immune and inflammatory responses as an important intercellular messenger. In addition, NO has an important role in inflammatory responses in mucosal environments such as the ocular surface. Histatin peptides are well-established antimicrobial and wound healing agents. These peptides are important in multiple biological systems, playing roles in responses to the environment and immunomodulation. Given the importance of macrophages in responses to environmental triggers and pathogens, we investigated the effect of histatin-1 (Hst1) on LPS-induced inflammatory responses and the underlying molecular mechanisms in RAW264.7 (RAW) macrophages. LPS-induced inflammatory signaling, NO production and cytokine production in macrophages were tested in response to treatment with Hst1. Hst1 application significantly reduced LPS-induced NO production, inflammatory cytokine production, and inflammatory signaling through the JNK and NF-kB pathways in RAW cells. These results demonstrate that Hst1 can inhibit LPS-induced inflammatory mediator production and MAPK signaling pathways in macrophages.

Keywords: adaptive; anti-microbial peptide; histatin; immune; inflammation; innate; lipopolysaccharide; macrophage.

Figure 1

Effects of Hst1 on cell viability, nitric oxide production and iNOS expression in murine macrophage RAW264.7 cells. (A) Cell viability in HST1-treated cells was evaluated using the WST-1 assay. RAW264.7 cells were incubated with 10 to 200 μM of Hst1 for 18 h. The results are displayed as a percentage of control samples. The lack of Hst1 induced cell toxicity up to 200 μM is notable. Each value represents the mean ± SD and is representative of results obtained from three independent experiments. (B) Nitrite level was measured by the Griess assay. RAW264.7 cells were pre-treated with Hst1 at the indicated concentrations (50 and 100 μM) for 2 h and then exposed to Hst1 with LPS (10 ng/mL) or LPS alone for 16 h. The culture supernatants were subsequently isolated and analyzed for nitrite levels. Results reveal a statistically significant and dose-dependent reduction in LPS-induced nitrite production in Hst1-treated cells. The values for nitrite are the mean ± SD from three independent experiments. ** p < 0.01 compared to treatment with LPS alone. (C) For the qRT-PCR of iNOS gene expression, RAW264.7 cells were treated by the same procedure for Griess assay. The levels of iNOS mRNAs were determined by qRT-PCR analysis and GAPDH mRNA served as the internal control for the normalization of iNOS mRNA expression. The dose-dependent and statistically significant reduction in iNOS gene expression in cells treated with Hst1 is notable. (D) For the Western blotting of iNOS protein, lysates were prepared from RAW264.7 cells and treated by the same procedure for Griess assay. Total cellular proteins (40 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes and detected with specific antibodies against murine iNOS and β-Actin served as the internal control for the normalization of iNOS protein expression. These results are displayed in fold of LPS alone samples. Results indicate that Hst1 treatment reduces protein levels of iNOS induced by LPS in a dose-dependent and statistically significant manner. Each value represents the mean ± SD and is representative of results obtained from three independent experiments. ** p < 0.01 compared to treatment with LPS alone.

Figure 2

Effects of Hst1 on production of pro-inflammatory cytokines in LPS-stimulated RAW264.7 cells. (A) The mRNA levels of IL-6, IL-1β, and TNF-α were determined by using qRT-PCR. RAW264.7 cells were pre-treated with Hst1 at the indicated concentrations (50 and 100 μM) for 2 h and then exposed to Hst1 with LPS (10 ng/mL) or LPS alone for 16 h. The expression level of GAPDH mRNA served as the internal control for the normalization of IL-6, IL-1β, and TNF-α mRNAs expression. These results are displayed in fold of LPS alone samples. Gene expression analysis revealed a statistically significant reduction in inflammatory cytokine gene expression by treatment with Hst1. Each value represents the mean ± SD and is representative of results obtained from three independent experiments. * p < 0.05 and ** p < 0.01 compared to treatment with LPS alone. (B) RAW264.7 cells were treated by the same procedure for qRT-PCR. The production levels of IL-6, IL-1β, and TNF-α in the culture medium were determined by ELISA in triplicate samples according to the manufacturer’s instruction. The statistically significant, dose-dependent reduction of all inflammatory cytokine protein production by treatment with Hst1 is notable. Data are shown as the mean values ± SD (n = 3). * p < 0.05 and ** p < 0.01 compared to treatment with LPS alone.

Figure 3

Effects of Hst1 on LPS-induced phosphorylation of MAPKs in RAW264.7 cells RAW264.7 cells were starved for 6 h and then pre-treated with HST1 at the indicated concentrations (50 and 100 μM) for 2 h and then exposed to HST1 with LPS (10 ng/mL) or LPS alone for 2 h. Whole-cell lysates were analyzed by Western blot analysis using specific phospho-JNK and phospho-p38 antibodies. The blot was stripped and reprobed with an antibody against non-phospho antibodies. Non phospho-JNK and p38 served as the control for the normalization of each MAPKs protein expression. These results are displayed in fold of LPS alone samples. The results of this analysis indicate that Hst1 treatment reduces LPS-induced phosphorylation of JNK and p38 in RAW cells by Hst1, in a dose-dependent manner. Each value represents the mean ± SD and is representative of results obtained from three independent experiments. * p < 0.05 and ** p < 0.01 compared to treatment with LPS alone.

Figure 4

Effects of the Hst1 on the phosphorylation of IκB-α and nuclear translocation of NF-κB LPS-induced RAW264.7 macrophage cells. RAW264.7 cells were starved for 6 h, pre-treated with Hst1 at the indicated concentrations (50 and 100 μM) for 2 h and then exposed to Hst1 with LPS (10 ng/mL) or LPS alone for 2 h. (A) Whole-cell lysates were analyzed by Western blot analysis using a specific anti-phospho-IκB-α antibody. The blot was stripped and reprobed with an antibody against β-Actin. β-Actin was used as the internal control. Analysis reveals that p-IκB-α induction by LPS is reduced by treatment with Hst1 in RAW cells. (B) Nuclear extracts were prepared from RAW264.7 cells. RAW264.7 cells were treated by the same procedure for whole-cell lysates Western blot analysis and were analyzed using an anti-p65 antibody. The blot was stripped and reprobed with an antibody against PCNA to verify equal loading of proteins in each lane. These results are displayed in fold of LPS alone samples. Results indicate that the increased ratio of p65 to PCNA levels induced by LPS in RAW cells was statistically significantly reduced by treatment with Hst1. Each value represents the mean ± SD and is representative of results obtained from three independent experiments. * p < 0.05 and ** p < 0.01 compared to treatment with LPS alone.