A sprinkle of salt in the pressure cooker of innate immunity and inflammation

Innate immune cells, including macrophages, employ a plethora of detection systems to sense and respond to danger signals that are indicative of perturbed homeostasis. Pattern recognition receptors, such as the Toll-like receptors, have been most widely studied in this context. However, macrophages can sense and respond to many other environmental challenges. Mechanical forces, such as tension and compression, as well as dietary factors, such as salt (NaCl), can all regulate macrophage functions, immune responses and inflammation-associated pathology. In this issue of Immunology & Cell Biology, Schröder et al.¹ assess the impacts of both mechanical strain and salt on macrophage inflammatory responses, as well as the involvement of the nuclear factor of activated T cells 5 (NFAT5) transcription factor, which senses and responds to osmotic stress,² in these processes. Overlapping but distinct effects of tensile force, compressive force and salt on specific inflammatory mediators are identified, with NFAT5 having complex and divergent roles in these processes. The authors speculate on the implications of their findings to orthodontic treatments, but the reported phenomena may also have relevance to other physiological and pathophysiological processes.

Cells and tissues of our bodies have the capacity to sense and respond to mechanical stress, such as tensile and compressive strain. Mechanobiology, which refers to the study of how such physical forces regulate cell and tissue functions, is particularly important in both bone homeostasis and its dysregulation.³ Orthodontic appliances (such as aligners), used to correct misplaced teeth, rely on mechanical force to achieve orthodontic tooth movement. This results in tensile and pressure zones, which are respectively linked to bone deposition and resorption, as well as inflammation.^{4, 5} Dietary factors, including salt, can also influence inflammatory responses and the formation of bone-resorbing osteoclasts.^{6, 7} However, we have a limited understanding of the combinatorial effects of physical forces and salt on inflammatory responses of cells that influence oral health, including periodontal ligament fibroblasts, lymphocytes and innate immune cells, such as macrophages. Given that inflammation underlies the pathology of gingivitis and other oral health conditions, such knowledge is clearly important. As a step toward generating such an understanding, Schröder et al. investigated the effects of both tensile and compressive strain on macrophage functional responses in vitro, with production of tumor necrosis factor (TNF), interleukin (IL)-6 and prostaglandin E₂ (PGE₂) being selected as relevant immunological read-outs (Figure 1).

In general, Schröder et al. found that mechanical strain (both tensile and compressive) induced all three mediators to some extent in the mouse macrophage populations examined (RAW 264.7 macrophage-like cells and/or bone marrow-derived macrophages). However, compressive strain had a much more pronounced effect than tensile strain in inducing Ptgs2 messenger RNA expression and/or PGE₂ secretion. Given that pressure is associated with bone resorption⁵ and that PGE₂ is one of many mediators that contributes to bone resorption in periodontitis,⁸ the selective regulation of this inflammatory mediator by pressure may be important. In contrast to the effects of mechanical strain, salt tended to increase production of TNF and PGE₂, but not IL-6. However, the effects of salt were somewhat variable between the two experimental systems used to elicit tensile versus compressive strain. It is possible that this relates to differences in the specific types of plastic that cells were cultured on before delivering these opposing forces (for tensile strain, cells were grown on specialized tissue culture plates made from silicone elastomer, in contrast to the polystyrene plates used for compressive strain). Moreover, most of the presented data focused on a single time point of 4 h post-treatment, so more detailed kinetic studies may help to tease out the potential intricacies of these responses. Nonetheless, one consistent observation was that salt suppressed mechanical strain-mediated production of IL-6. This was apparent when either tension or compression was used as the inducing agent, although the effect was most pronounced for the latter. IL-6 has complex roles in regulating immune responses and inflammation, which partly reflects its capacity to initiate both conventional signaling in IL-6 receptor-expressing cells and soluble IL-6 receptor-dependent trans-signaling in cells that do not themselves express the receptor. For this reason, the significance of the suppressive effect of salt on mechanical strain-induced IL-6 production is difficult to predict. However, a recent study showed that, following tooth extraction in an animal model, the IL-6 receptor antagonist tocilizumab reduced osteoclast numbers and bone resorption, but increased rates of bacterial infection.⁹ Thus, it is possible that the suppressive effect of salt on IL-6 production from macrophages may similarly reduce bone resorption and/or increase susceptibility to infection during periodontal disease.

NFAT5, otherwise known as tonicity-responsive enhancer binding protein (TonEBP), is a member of the Rel family of transcription factors. It has been extensively studied for its role in cellular adaptation to hypertonicity and responses to salt.² In more recent years, it has become apparent that NFAT5 also controls macrophage and T-cell functions, influencing inflammatory responses both through its actions as a sensor of hypertonicity and as a downstream target of various inflammatory signaling pathways.¹⁰ Consequently, NFAT5 has a number of important roles in macrophages, including coordinating host defense¹¹ and driving Toll-like receptor-mediated gene expression.¹² Not surprisingly, NFAT5 has also been linked to many pathological processes, including experimental autoimmune encephalomyelitis (a mouse model of multiple sclerosis),¹³ obesity and insulin resistance,¹⁴ rheumatoid arthritis¹⁵ and oral squamous cell carcinoma.¹⁶ Thus, the contributions of NFAT5 to biological responses affected by mechanical strain and salt were next examined by the authors, using both gain- and loss-of-function approaches.

The first notable finding was that compressive, but not tensile, strain induced expression of NFAT5 protein in macrophages. By contrast, pressure had no effect on Nfat-5 messenger RNA. This may reflect differences in kinetics of the response, given that the authors examined only a single 4-h time point for both messenger RNA and protein. Alternatively, it is possible that pressure stabilizes the NFAT5 protein, without affecting gene expression. Whether pressure influences nuclear translocation of NFAT5 is unknown, given that protein expression was only examined in whole-cell lysates. This could be given some attention in the future. Subsequent gain- and loss-of-function approaches revealed complex roles for NFAT5 in responses to both pressure and salt. Broadly, NFAT5 appears to positively regulate TNF and PGE₂ production in macrophages, whereas it suppresses IL-6 expression. As anticipated, this transcription factor was particularly important for salt-mediated responses in macrophages, including induction of TNF and PGE₂, and repression of pressure-induced IL-6. However, the presented data also support a partial role for NFAT5 in compressive strain-inducible inflammatory mediator production in macrophages, particularly PGE₂. A previous study revealed that NFAT5 is required for optimal Toll-like receptor-inducible expression of a number of genes in macrophages, including Tnf, Il6 and Ptgs2.¹² Hence, it is possible that signaling responses converge downstream of both Toll-like receptors and compressive strain to enable NFAT5-dependent inflammatory gene expression. Further studies aimed at delineating the molecular mechanisms by which pressure increases NFAT5 protein expression and/or NFAT5-dependent transcriptional responses could address this possibility. Interestingly, TNF, along with IL-1 and transforming growth factor-β, have been shown to upregulated NFAT5 expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis,^{17, 18} suggesting reciprocal regulation of these factors in inflammation. Intriguingly, gene silencing studies suggested a role for NFAT5 in pressure-induced IL-6 production, whereas it inhibited basal IL-6 expression and contributed to the repressive effect of salt on pressure-induced IL-6 production. This suggests that NFAT5 might have divergent roles in regulating the same gene, depending on the nature of the environmental challenge. Consistent with this, others have shown that salt also inhibits Toll-like receptor-inducible IL-6 production in macrophages.¹⁹ That study implicated different sources of reactive oxygen species in NFAT5-mediated activation versus repression of the Il6 gene.

Together, the findings of Schröder et al.¹ reveal a complex, but potentially important role for NFAT5 in the regulation of macrophage inflammatory responses to both mechanical stress and exposure to salt (Figure 1). The data add further weight to previous studies highlighting a role for salt in regulating macrophage inflammatory responses.^{6, 20} Effects on host defense pathways against infectious organisms are likely to be complex. One study demonstrated an antimicrobial effect of salt in the skin, accompanied by an increase in NFAT5 expression in cutaneous macrophages.²⁰ However, the inhibitory effect of salt on IL-6 production could potentially impair antimicrobial responses.⁹ Previous work by these authors has also shown that salt promotes the release of PGE₂ and RANK-L by periodontal fibroblasts, thus increasing osteoclastogenesis.⁶ As bone remodeling is key to orthodontic tooth movement, this might suggest that high salt concentrations would accelerate this process. So, would a high-salt diet mean less time wearing that aligner? Well, hands off that saltshaker for the moment. Aside from the many other well-documented detrimental effects of a high-salt diet on health, including hypertension and osteopenia, this and other studies suggest that salt might promote uncontrolled inflammation and osteoclastogenesis, leading to dental root resorption and/or periodontal bone loss. Further work is needed to better understand the mechanisms at play, as well as the full range of immunomodulatory effects of mechanical stress in this setting. In this regard, only a limited number of mediators (TNF, IL-6 and PGE₂) were assessed in this study. Unbiased gene expression profiling of macrophages responding to pressure and salt (alone or in combination) could be used in the future to generate a more complete picture of how salt influences pathways and inflammatory mediators relevant to pressure-induced bone remodeling. Most importantly, the implications of this in vitro study to oral health in vivo, including during chronic disease and response to infection, await elucidation. It would also be interesting to know whether these responses change with age and how this might inform future orthodontic procedures. Hopefully, the intriguing findings presented by Schröder et al. will inspire such studies.

Conflict of Interest

The authors have no conflicts of interest.

Author Contributions

Matthew J Sweet: Conceptualization; writing-original draft; writing-review & editing. Sarah A Jones: Conceptualization; writing-original draft; writing-review & editing. James Harris: Conceptualization; writing-original draft; writing-review & editing.