Hypertonic saline attenuates the cytokine-induced pro-inflammatory signature in primary human lung epithelia

Abstract

Trauma/hemorrhagic shock is a complex physiological phenomenon that leads to dysregulation of many molecular pathways. For over a decade, hypertonic saline (HTS) has been used as an alternative resuscitation fluid in the setting of trauma/hemorrhagic shock. In addition to restoring circulating volume within the vascular space, studies have shown a positive immunomodulatory effect of HTS. Targeted studies have shown that HTS affects the transcription of several pro-inflammatory cytokines by inhibiting the NF-κB-IκB pathway in model cell lines and rats. However, few studies have been undertaken to assess the unbiased effects of HTS on the whole transcriptome. This study was designed to interrogate the global transcriptional responses induced by HTS and provides insight into the underlying molecular mechanisms and pathways affected by HTS. In this study, RNA sequencing was employed to explore early changes in transcriptional response, identify key mediators, signaling pathways, and transcriptional modules that are affected by HTS in the presence of a strong inflammatory stimulus. Our results suggest that primary human small airway lung epithelial cells (SAECS) exposed to HTS in the presence and absence of a strong pro-inflammatory stimulus exhibit very distinct effects on cellular response, where HTS is highly effective in attenuating cytokine-induced pro-inflammatory responses via mechanisms that involve transcriptional regulation of inflammation which is cell type and stimulus specific. HTS is a highly effective anti-inflammatory agent that inhibits the chemotaxis of leucocytes towards a pro-inflammatory gradient and may attenuate the progression of both the innate and adaptive immune response.

Fig 1. HTS inhibits the expression of several key inflammatory mediators.

SAECS in P3 were grown in triplicate to 80% confluence in 12-well dishes, stimulated with TNFα, IL-1β, and IFNγ (cytomix) in the presence and absence of hypertonic saline (HTS) for 4 hrs. RNA was harvested and RNAseq performed. HTS decreases the expression of (a) key chemokines and cytokines, (b) Transporters and (c) Kinases which are important effectors of the inflammatory signaling cascade.

Fig 2. HTS inhibits the expression of chemokines and cytokines that regulate immune cell trafficking and polarization.

SAECS in P4 were treated with cytomix in the presence and absence of HTS for 4 and 8hrs, and RNA isolated for qPCR analysis. Validation of the RNAseq shows that (a) chemokines that affect T-cell, Dendritic cell, and NK cell interaction/migration, (b) monocyte trafficking, (c) IL-1 family cytokines, and Th17 response are all increased due to cytokine treatment, and attenuated by HTS; whereas HTS-specific genes such as IL-11 are upregulated in response to HTS. (*p< 0.05; **p< 0.01; ***p< 0.001; ****p< 0.0001).

Fig 3. HTS inhibits the transcriptional activation of IRF1 and STAT1 dependent genes.

In the Ingenuity Pathway Analysis of cytomix vs cytomix+HTS, IRF1 and STAT1 were inferred to regulate the expression of several downstream molecules in this study. (a) 12 of 18 genes have expression consistent with upstream inhibition of IRF1 by HTS (Z-score: -2.564; p = 1.05E-10). (b) 26 of 32 genes have expression consistent with STAT1 inhibition when cytomix is added to cells treated with HTS (Z-score: -4.638; p = 3.85E-16). (c) HTS attenuates cytomix-induced luciferase expression in SAECS transiently transfected with IRF1 and STAT1 reporter expression plasmids treated with cytomix for 4hrs in the presence and absence of HTS. (d) The expression of CCL5, a downstream endogenous target of IRF1 and STAT1, is also attenuated by HTS at 4, 6 and 8hrs as determined by ELISA. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Fig 4. HTS inhibits chemotaxis of neutrophils and splenocyte-derived T-cells.

SAECS in P3 were cultured for 24 hrs with cytomix in the presence and absence of HTS and the supernatants were harvested and applied to the bottom chamber of the chemotaxis plate. Chemotaxis of neutrophils and splenocyte derived T cells towards the chemotactic gradient was assayed using Incucyte Zoom imaging system. The results show (a) supernatant from cytomix treated cells elicit maximal migration of neutrophils, and this migration was attenuated in supernatants from cytomix + HTS treated cells. Similarly, in a separate experiment (b), HTS inhibited the migration of splenocyte derived T-cells through the matrigel towards the chemotactic gradient. [solid lines indicate the loss of surface area previously occupied by the neutrophils or splenocytes derived T-cells on the top of membrane as they migrate to the reservoir containing the chemoattractant at the bottom of the membrane; dashed lines indicate p values for pairwise comparison of migration of neutrophils or splenocyte at each time point (control vs HTS, control vs cytomix, control vs cyto-HTS: Fig 4a and Fig 4b main panel; cytomix vs cyto–HTS: Fig 4a and Fig 4b inset). FMLP and CCL5 were used as positive control for the migration assays for neutrophil and splenocyte derived T cells respectively.

Fig 5. HTS inhibits CCL5 expression in cytokine-pretreated cells.

SAECS in P3 were co-treated or pretreated with cytomix ± HTS for 8hrs and HTS was added at 30, 60, 120, 240, and 360 minutes after cytokine addition. The data shows (a) HTS inhibits the expression of CCL5 mRNA and (b) protein in both pre-treatment and co-treatment, as well as when HTS is applied in incremental time points after cytomix pretreatment. Although maximal inhibition was seen when HTS was applied 30mins or earlier after cytokine pre-incubation, the inhibitory effect remains significant when applied even 6 hrs after initial cytokine challenge. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Fig 6. The anti-inflammatory effects of HTS on CCL5 expression are reversible.

SAECS in P4 were cultured in the presence of HTS for 4hrs and allowed to rest for 2 and 4hrs in isotonic media before being subjected to a subsequent cytomix challenge. Cells previously treated by HTS can respond to a subsequent cytokine challenge after a minimal recovery period (2-4hrs), with CCL5 levels comparable to initial controls treated with cytomix.