Evaluation of the role of secretory sphingomyelinase and bioactive sphingolipids as biomarkers in hemophagocytic lymphohistiocytosis

Abstract

Hemophagocytic lymphohistiocytosis (HLH) is a rare systemic inflammatory syndrome that results from unrestrained immune cell activation. Despite significant advances in the understanding of the pathophysiology of HLH, interventions remain limited for this often-fatal condition. Secretory sphingomyelinase (S-SMase) is a pro-inflammatory lipid hydrolase that is upregulated in several inflammatory conditions, including HLH. S-SMase promotes the formation of ceramide, a bioactive lipid implicated in several human disease states. However, the role of the S-SMase/ceramide pathway in HLH remains unexplored. To further evaluate the role of S-SMase upregulation in HLH, we tested the serum of patients with HLH (n = 16; primary = 3, secondary = 13) and healthy control patients (n = 25) for serum S-SMase activity with tandem sphingolipid metabolomic profiling. Patients with HLH exhibited elevated levels of serum S-SMase activity, with concomitant elevations in several ceramide species and sphingosine, while levels of sphingosine-1-phosphate were significantly decreased. Importantly, the ratio of C16 -ceramide:sphingosine was uniquely elevated in HLH patients that died despite appropriate treatment, but remained low in HLH patients that survived, suggesting that this ratio may be of prognostic significance. Together, these results demonstrate upregulation of the S-SMase/ceramide pathway in HLH, and suggest that the balance of ceramide and sphingosine determine clinical outcomes in HLH. .

Figure 1. Serum S-SMase activity in healthy controls and HLH patients

A) Sera prepared from healthy volunteers (Ctl; n=25) and patients with HLH (n=16, 66 total sera samples) were processed for in vitro Zn²⁺-dependent acid SMase activity, as described above (see Methods). (2-tailed, unpaired Man-Whitney test***p<.001). B) S-SMase activity was determined for patients with primary (n=3; 4 samples) and secondary (n=13, 62 samples). Kruskal-Wallis with Dunn’s post-test, **p<.01, ***p<.001.

Figure 2. Sphingolipid profile in serum of healthy controls and HLH patients

Sphingolipids were measured sera (100 μL) from healthy volunteers (Ctl; control) and patients with HLH, as described in Methods. A) Total sphingomyelin, B) total ceramide, C) sphingosine, and D) sphingosine-1-phosphate, (n=25 for control, n=66 for HLH; 2-tailed, unpaired Mann-Whitney test; ns – not statistically significant, *p<.05, **p<.01, ***p<.001).

Figure 3. Ceramide profile in serum of healthy controls and HLH patients

Sphingolipids were measured sera (100 μL) from healthy volunteers (Ctl; control) and patients with HLH, as described in Methods. A) C₁₆-ceramide, B) dihydro-C₁₆-ceramide, C) C₂₄-ceramide, and D) C₂₆-ceramide, (n=25 for control, n=66 for HLH; 2-tailed, unpaired Mann-Whitney test; ***p<.001).

Figure 4. C₁₆-ceramide:sphingoid base ratio and survival in HLH

A-B) Ratios of C16-Cer:S1P and C16-Cer:Sph were plotted for healthy volunteers (Ctl) and HLH patients (HLH) C-D) Ratios of C16-Cer:S1P and C16-Cer:Sph for healthy volunteers (Ctl), HLH patients that did passed away despite aggressive therapy (HLH-Died), and HLH patients that recovered following treatment (HLH-Recovered). Kruskal-Wallis with Dunn’s post-test analysis; ns – not statistically significant, **p<.01, ***p<.001). Note, that C16-Cer was evaluated (and not other Cer species) as levels of C16-Cer exhibited the greatest fold-change (see Table III).

Figure 5. Schematic of S-SMase/ceramide pathway in HLH

In HLH, dysregulation of the inflammatory response results in hypercytokinemia which in turn promotes upregulation of serum S-SMase. S-SMase is well positioned to act on serum SM (primarily bound to lipoproteins) driving production of select Cer species. Upon action of a downstream CDase, S-SMase-derived Cer can be deacylated to form Sph. Key changes in the serum SPL profile in patients with HLH, include elevations in total Cer and Sph, as well as decreased levels of S1P. C₁₆-Cer levels exhibited the most significant increase in HLH. As described in Figure 7, the ratio of C₁₆-Cer:Sph correlates very strongly with survival – HLH patients that died had higher serum levels of C₁₆-Cer relative to Sph, whereas the ratio of C₁₆-Cer:Sph was significantly lower HLH patients that survived.