Frontiers in Immunology | www.frontiersin.org March 2020 | Volume 11 | Article 397Gonzalez-Cotto et al. Treml4 in Macrophages and AtherosclerosisMetabolomic analysis confirmed that Treml4 deficiency may promote a beneficial relationship between iron homeostasis and glucose metabolism. Together, our results suggest that Treml4 plays a role in the development of cardiovascular disease, as indicated by Treml4-dependent dysregulation of macrophage inflammatory pathways, macrophage metabolism and promotion of vulnerability features in advanced lesions.

Disability after traumatic spinal cord injury (TSCI) results from physical trauma and from "secondary mechanisms of injury" such as low metabolic energy levels, oxidative damage and lipid peroxidation. In order to prove if early metabolic reactivation is a better therapeutic option than antioxidant therapy in the acute phase of TSCI, spinal cord contusions were performed in adult rats using a well-characterized weight drop technique at thoracic 9 level. After TSCI, pyrophosphate of thiamine or non-degradable cocarboxylase (NDC) enzyme was used to maintain energy levels, antioxidants such as superoxide dismutase and catalase (ANT) were used to decrease oxidative damage and methylprednisolone (MP), which has both therapeutic properties, was used as a control. Rats were divided into one sham group and six with TSCI; one of them received no treatment, and the rest were treated with NDC, MP, NDC + MP, NDC + ANT or ANT. The ANT group decreased lactate and creatine phosphokinase levels and increased the amount of preserved tissue (morphometric analysis) as well as functional recovery (Basso, Beattie and Bresnahan or BBB motor scale). In contrast, NDC treatment increased lipid peroxidation, measured through thiobarbituric acid reactive substances (TBARS) levels, as well as spinal cord tissue destruction and functional deficit. Early metabolic reactivation after a TSCI may be deleterious, while natural early metabolic inhibition may not be a "secondary mechanism of injury" but a "secondary neuroprotective response". While increased antioxidant defence after a TSCI may currently be an ideal therapeutic strategy, the usefulness of metabolic reactivation should be tested in the sub-acute or chronic phases of TSCI and new strategies must continue to be tested for the early ones.

Study design: This work is an experimental and prospective study in adult, female, Long-Evans rats. Objectives: The aim of this study was to probe the effect of metabolic inhibition after an acute traumatic spinal cord injury (TSCI) using a standardized contusion model (NYU impactor) to know whether the metabolic inhibition is a 'secondary mechanism of injury' or a mechanism of protection. Setting: All experimental procedures were carried out in the Mexico City. Methods: Animals were divided into five groups: one sham and four with TSCI, including no treatment, rotenone (inhibitor of mitochondrial complex I), sodium azide (inhibitor of mitochondrial complex IV) and pyrophosphate of thiamine or non-degradable cocarboxylase as a metabolic reactivator. Results: After TSCI, the metabolic inhibition with sodium azide treatment diminished the lipid peroxidation process (malondialdehyde levels by spectrophotometric procedures) and the damage to the spinal cord tissue (morphometric analysis), and increased the activity of creatine kinase and lactate dehydrogenase enzymes (Po0.05) (measured by spectrophotometric procedures 24 h after TSCI as well as after the functional recovery of the hind limb (evaluated weekly for 2 months by the BBB (Basso, Beattie and Bresnahan) scale)) when compared with the TSCI group without treatment. Conclusion:The results show that the partial and transitory inhibition of the aerobic metabolism after an acute TSCI could be a self-protection mechanism instead of being a 'secondary mechanism of injury'.

While it is known that high blood cholesterol levels are associated with increased risk and poor outcomes during cardiovascular disease (CVD), all of the mechanisms that regulate cholesterol levels remain unclear. Recent studies on the Triggering Receptor Expressed in Myeloid (TREM) like transcript (TLT)-1 null mice demonstrate changes in cholesterol regulation. TLT-1 was identified as a platelet specific immunoreceptor prompting our studies on TLT-1’s potential involvement in CVD. We crossed our treml1-/- mice on to the apoe-/- background and placed them on high fat diet (HFD). To our surprise, the treml1-/- /apoe-/- (DKO) mice had total cholesterol levels 1.5 fold higher compared to apoE controls. These changes can be seen in treml1-/- mice that are not on the apoE background as well. There are no cholesterol differences in mice not on HFD, demonstrating that these changes are diet-induced. These differences led us to investigate, “why the treml1-/- mouse has higher cholesterol?” Serum chemistry panels reveal high triglycerides, increased glucose levels, and potential liver damage in the treml1-/- mice. Histological evaluation of liver sections revealed large lipid-filled vacuoles and ballooning of DKO and treml1-/- hepatocytes suggesting direct liver involvement in regulating the aforementioned cholesterol levels. What brings these findings together is that we have identified an alternate transcript of TLT-1, TLT-1s, associated with mitochondrial fractions of hepatocytes. Immunohistochemistry of liver sections demonstrate a unique TLT-1s expression in the perivascular hepatocytes of the central vein. Preliminary metabolic analysis of mice livers confirm the hyperlipidemia and demonstrate significant changes in protein metabolism as well as Krebs cycle intermediates. Interestingly, treml1-/- mice have smaller lesions in the aortic sinus which may be explained by less reactive treml1-/- platelets (Gonzalez et al.). Our data suggests that treml1 gene products may affect energy metabolism and mutations in TLT-1 may affect energy use in platelets. This uncanny hypothesis potentially explains how we could have high cholesterol levels, with smaller atherosclerotic lesions and less reactive platelets in the face of hyperlipidemia.

Atherosclerosis is a chronic inflammatory process of the vessel wall driven by an inflammatory state caused by the involvement of different cell populations, including platelets. The receptor Triggering Receptor Expressed on Myeloid Cells (TREM)-like transcript (TLT)-1 is prepacked in platelet α-granules and brought to the surface upon activation. TLT-1 has both membrane-bound and soluble forms (sTLT-1) and the latter has been found to enhance platelet activation as well as platelet-endothelial cell interactions. TLT-1 null mice (treml1-/-) have reduced platelet aggregation, are significantly more susceptible to lipopolysaccharide challenge compared to their wild type counterparts, and accordingly, hemorrhage in response to an acute inflammatory challenge. These results suggest that TLT-1 is a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms and in light of this, we hypothesized that TLT-1 plays an important role in atherosclerosis progression. To address this, we generated apoE-/-/treml1-/- double knockout mice [DKO]. We found that DKO mice fed an atherogenic diet (HFD) showed significantly increased weight gain when compared to apoE-/-. Accordingly, DKO mice showed increased total cholesterol and triglycerides compared to controls. Surprisingly, assessment of lesion size revealed that DKO mice have significantly smaller lesions in the aortic sinus at four and 12 weeks after HFD compared to apoE-/-. At 20 weeks, lesion differences are no longer significant, but compositional analysis revealed that DKO atherosclerotic lesions are less vulnerable as seen by decreased lesion calcification and increased smooth muscle cell content. Furthermore, q-rtPCR analysis of atherosclerosis-related genes revealed that TLT-1 differentially affects genes related to lipid metabolism (apo AI and B) and vascular inflammation (thrombospondin 4, PAI-1 and VGFR-2). While our data suggests that the smaller lesion size may be explained by lower platelet reactivity, differences in cholesterol levels suggest altered lipid metabolism which is supported by our metabolomics studies (González, M. et.al). Taken together TLT-1 plays a dual role in the progression of atherosclerosis.