Transcriptomic Analysis of Splenic Leukocytes Reveals a Norepinephrine‐Mediated Dynamic Immune Cell Deployment Mechanism in Heart Failure Rat
Authors: Li Hanzeng , Yu Yang , Sun Luyang , Chen Qing , Li Mingxuan , Md Irfan Nafis , Xue Baojian , Weiss Robert , Dang Weiwei , Wei Shun‐Guang 
Published: May 2022

Spleen plays an important role in promoting inflammation in myocardial infarction (MI)‐induced heart failure (HF). Spleen may have the temporal and/or permanent changes to accommodate the neurohumoral compensatory responses in the context of HF. However, the mechanisms underlying the immune cell deployment from the spleen in HF remain elusive. To gain unbiased insights into how splenic cells are transcriptionally adapted to the neurohumoral alterations in HF, we performed RNA‐seq analysis on spleen‐derived leukocytes from HF rats or sham‐operated (Sham) rats. Male Sprague‐Dawley rats underwent coronary artery ligation to induce HF (n=3) or Sham (n=4). HF was confirmed by echocardiography. Splenic leukocytes were prepared two weeks after MI. RNA were extracted, sequenced and analyzed with standard RNA sequencing procedure. We found 466 upregulated and 113 downregulated genes in splenic leukocytes of HF rats (|log2FoldChanges|>0.2, p<0.05). The genes associated with erythropoiesis (e.g Trim10, Dmtn, and Spta1) and cell cycles regulatory factors such as Ki‐67, and polo‐like kinase2 (plk2) were transcriptionally activated and the enhanced expression was validated using RT‐qPCR, indicating that active myeloid proliferation and differentiation are present within the spleen in the development of HF. Unexpectedly, the expression of inflammation‐related genes such as chemokine ccl3, JUN, JUNB, and fosB were reduced in HF. Moreover, pathway enrichment analysis also displayed a suppression of the immune‐related pathways including cytokine‐cytokine interaction pathway, IL‐17 signaling, tumor necrosis factor signaling, and rheumatoid arthritis. To determine whether the suppression of these pathways is resulted from the egress of immune cells in spleen, we performed flow cytometry profiling of splenic cells. We found that CD3+T cell (21% vs. 27%*), especially CD8+ T cells (7.5 % vs 12%*) were significantly (*p<0.05) reduced in HF compared with Sham animals. However, the immune cells in other categories such as monocytes (M1, CD43low;HIS48high: 3% vs. 2%*; M2, CD43high;HIS48low: 5% vs. 3%*), NK cells (5% vs. 4%*), and neutrophiles (2.5% vs. 1%*) were significantly elevated in HF. The B cells remain unchanged. Flow cytometrical analysis of immune cell subtypes in the blood of HF showed a similar trend with those of splenic leukocytes. We proposed that the sympathetic activation in HF may contribute to the dynamic changes of splenocytes. To test this hypothesis, norepinephrine (NE) was injected (0.1mg/kg, IP) in normal rats (n=4) for 3 consecutive days followed by flow cytometry analysis of splenic leukocytes. The results indicated that, while all other immune cell subtypes remain unchanged, both M1 (2% vs. 1.4%*) and M2 (5% vs. 2%*) monocytes are decreased in NE‐treated rats, implying that elevated NE level at early stage of HF can robustly trigger the infiltration of the spleen‐derived monocytes into injured cardiac tissues. Taken together, these data provided an informative perspective to understand splenic modulations in HF. The dynamic changes of splenocytes composition in HF is most likely a combined result of splenic cell deployment and compensatory proliferation/differentiation. Our data also suggest that the sympathetic overactivity may play an important role in the migration of spleen‐resident leukocytes and inflammatory regulation in HF.