Masayuki Taniguchi

BACKGROUND: Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. METHODS: We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45⁺ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. RESULTS: CD4⁺ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7⁺ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)⁺ regulatory CD4⁺ T cells. The proportion of central memory CD4⁺ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4⁺ T cells into effector CD4⁺ T cells and that from naive CD4⁺ T cells into central memory CD4⁺ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4⁺ T cells. CONCLUSIONS: For the first time, we revealed single cell–level characteristics of CD4⁺ T cells in patients with ACS. CD4⁺ T cells could be therapeutic targets of ACS. REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.

Chronic stress causes emotional disturbances and is a risk factor for mental illnesses such as depression. It was reported that chronic social stress activates microglia in the medial prefrontal cortex (mPFC) through innate immune receptors TLR2/4, leading to depressive-like behavior. Since lipopolysaccharide, a TLR4 ligand, differently activates microglia isolated from left and right hemispheres, we analyzed chronic social stress-induced gene expression changes in mPFC microglia of each hemisphere using laser microdissection microscopy combined with RNA-seq. Given individual variability in stress susceptibility, we categorized the stressed mice into susceptible and resilient mice based on the level of social avoidance. We identified genes that increased or decreased in expression in mPFC microglia with chronic stress. Many of these genes responded to stress differently between left and right hemispheres and between susceptible and resilient mice. Notably, the genes altered in expression only in resilient mice showed hemispheric asymmetry in their stress responses. These findings demonstrate hemispheric asymmetry of chronic stress-induced microglial responses and suggest its relevance to stress resilience.

Stress is caused by various adverse environments, and often causes emotional changes. Using social defeat stress (SDS) in mice, we previously reported that repeated SDS activates microglia in the medial prefrontal cortex (mPFC), and then leads to social avoidance. Recently, we found that microglial activation in the mPFC occurs more rapidly and strongly with repetition of SDS. This finding led us to speculate that repetition of SDS induced persistent epigenomic changes of microglia in the mPFC. However, due to the limited sensitivity of chromatin immune precipitation sequencing (ChIP-seq), a brain region- and cell type-specific epigenomic analysis has been challenging. Here we optimized the protocol of ChIP-seq for epigenomic analyses of mPFC microglia. This protocol allows us to detect enrichment of active histone marks near microglia-specific genes in mPFC microglia. Our preliminary findings reveal that whole-genome epigenomic profiles of microglia reflected the experience of single SDS or repeated SDS. Interestingly, these epigenomic profiles in mPFC microglia were different from the profiles in the nucleus accumbens, indicating brain-region-specific epigenomic changes. We are currently investigating transcription factors related to regulation of mPFC microglial epigenomic changes associated with emotional changes.

Recent studies in mice started to reveal brain region-specific transcriptome and epigenome profiles of microglia. The heterogeneity of microglia might even exist across different layers of the cerebral cortex, since microglia are regulated by various receptors for neurotransmitters that are distributed preferentially in specific layers of the cerebral cortex. We also suspect the layer-dependent heterogeneity of microglia in the medial prefrontal cortex (mPFC), since repeated social defeat stress (R-SDS) induces microglial activation in the mPFC, leading to dendritic shrinkage of pyramidal neurons preferentially in its superficial layer. However, the heterogeneity of microglia across different cortical layers has not been elucidated, perhaps due to technical difficulty. To address this issue, here we developed a technique based on laser microdissection microscopy to perform transcriptome analyses with RNA-seq for microglia in the superficial and deep layers of the mPFC. Our preliminary findings suggest that mPFC microglia in adult mouse brains show layer-specific gene expression profiles even under the resting condition. We are currently investigating whether and how R-SDS affects layer-specific gene expression profiles of mPFC microglia.

Treatment with an angiotensin blocker (ARB) and an aldosterone blocker has been shown to have beneficial effects on cardiac remodeling in several cardiac diseases. It is still not clear whether the combination of these drugs is more effective against cardiac remodeling than the use of either agent alone. We examined the effects of combined treatment with valsartan, an ARB, and spironolactone, an aldosterone blocker, on cardiac remodeling in the renovascular hypertensive (RHT) rat. The RHT rats were divided into 4 groups administered valsartan (3 mg/kg/day, ARB group), spironolactone (4 mg/kg/day, SPRL group), both drugs at these doses (combined group), or neither drug (untreated RHT group). After 5 weeks, systolic blood pressure was significantly reduced in the 3 treatment groups, however, there were no significant differences in the extent of blood pressure reduction among the 3 treatment groups. The heart weight/body weight ratio in each of the 3 treatment groups was significantly lower than that in the untreated RHT group. The degree of cardiac and perivascular fibrosis in the SPRL group and the combined group were significantly lower than that in the untreated RHT group. Myocyte remodeling in the ARB group and in the combined group was significantly smaller than that in the untreated RHT group. These results suggest that SPRL treatment prevents cardiac and perivascular fibrosis and ARB treatment suppresses the cellular hypertrophy of myocytes, and that, therefore, combined treatment with both drugs prevents cardiac remodeling by acting against both myocyte hypertrophy and cardiac fibrosis in RHT rats.

We investigated the effects of an angiotensin-converting enzyme inhibitor and an angiotensin II type 1 receptor blocker on cardiac hypertrophy in rats with renovascular hypertension. Renovascular hypertensive (Goldblatt) rats were surgically prepared from Wistar rats. Four weeks later, the rats showed a significant increase in blood pressure. At high doses, both the perindopril (1 mg/kg/day) and the candesartan (2 mg/kg/day) decreased the systolic pressure in these rats to the level of control Wistar rats. At low doses (perindopril 0.1 mg/kg/day and candesartan 0.1 mg/kg/day), these drugs lowered blood pressure to 85% of that in hypertensive rats. Echocardiographic and morphological studies revealed severe cardiac hypertrophy and fibrosis in untreated Goldblatt rats. High-dose treatment with both drugs suppressed the progression of hypertrophy and fibrosis. Also, low-dose perindopril prevented cardiac hypertrophy and fibrosis. In contrast, at the same levels of blood-pressure reduction, low-dose candesartan did not prevent cardiac fibrosis nor the upregulation of cardiac collagen types I and III mRNA observed in untreated Goldblatt rats. Atrial natriuretic peptide mRNA was up-regulated in untreated Goldblatt rats. These changes were significantly decreased by both doses of perindopril or the high dose of candesartan. Serum levels of angiotensin II and aldosterone were significantly higher in untreated Goldblatt rats. Both doses of perindopril inhibited activation of the renin-angiotensin system, whereas candesartan had weaker effects. In particular, serum aldosterone was 347 ± 20 pg/ml in low-dose perindopril versus 1796 ± 324 pg/ml in low-dose candesartan. These results suggest that there were no differences between the cardioprotective actions of perindopril and candesartan at high dosages. On the other hand, low-dose treatment with perindopril was more effective in preventing cardiac fibrosis than was low-dose treatment with candesartan, despite similar changes in blood pressure. It is possible that changes in aldosterone secretion are related to this difference.