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Comment
. 2024 Aug 13;57(8):1713-1715.
doi: 10.1016/j.immuni.2024.07.008.

The effect of transient, constricted migration on neutrophil intracellular bacteria-killing capability

Peng Wu  1 , Alan Y Hsu  2 , Ting Peng  3 , Wenxue Zhao  3 , Fei Liu  2 , Zhaoran Zhang  1 , Laxman Ghimire  2 , Arumugam Balasubramanian  2 , Xuemei Xie  2 , Jiajia Li  2 , Shin-Young Park  2 , Fengxia Ma  1 , Cheng Li  3 , Hongbo R Luo  4
Affiliations
Comment

The effect of transient, constricted migration on neutrophil intracellular bacteria-killing capability

Peng Wu et al. Immunity. .
No abstract available

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Fig. 1.
Fig. 1.. Constricted migration does not alter gene expression or bacterial killing capability in neutrophils.
(A) Schematic outlining the method for assessing gene expression in neutrophils following constricted migration. Neutrophils were isolated from the peripheral blood of 10-week-old male C57BL/6 mice, 6 hr after the induction of peritonitis using thioglycolate (TG). This model was selected to simulate the inflammation-induced mobilization of neutrophils. The neutrophils were purified using a standard Percoll method. Libraries for transcriptome profiling were constructed using the Single Cell 3’ Library Kit V3 from 10x Genomics. The transcriptome profiles of individual cells were then determined through droplet sequencing based on the 10x Genomics technology. The indexed complementary DNA (cDNA) libraries, once prepared, were sequenced using paired-end reads on an Illumina NovaSeq 6000 system. (B) Uniform manifold approximation and projection (UMAP) of unbiased clustering in the unstimulated fresh neutrophil sample (6181 cells). Neutrophil subtypes were defined based on signature genes characterized in (6). (C) UMAP visualization of neutrophil single-cell transcriptomes in the indicated samples. Unstimulated (7048 cells), fMLP-PMNs (9443 cells), 8 μm-PMNs (8657 cells), and 3 μm-PMNs (8030 cells) were mapped onto the reference data set generated in (B). (D) Fraction of neutrophil subtypes in indicated samples. (E) Heatmap showing row-scaled expression of Differentially Expressed Genes (DEGs) across indicated samples. DEGs were defined by Wilcoxon rank-sum test with an average expression fold-change threshold >2. (F) Scatter plots comparing gene expression profiles of fMLP-PMNs, 8 μm-PMNs, and 3 μm-PMNs based on average expression of all genes. Red lines indicate the linear regression function. R indicates Pearson’s correlation coefficient. 0.8 < R < 1 is considered strong positive correlation. (G) MA plots displaying DEGs in each neutrophil subtype after constricted migration. Dashed lines denote fold change thresholds used when identifying DEGs (fold-change threshold>2, Padj <0.05). Padj, adjusted Pvalue. Blue: Down-regulated DEGs. No Up-regulated DEGs were identified. (H) Violin plot of apoptosis score (GO: 0043065), phagocytosis score (GO:0006911), chemotaxis score (GO:0030593), ROS production score (reactive oxygen species biosynthetic process, GO:1903409) for each indicated sample. Scores are defined as the average normalized expression of corresponding genes in indicated pathways. For the box plot within each violin plot, middle lines indicate median values, boxes range from the 25th to 75th percentiles. Significance was determined by Wilcoxon rank-sum test. ns, not significant (Padj > 0.05); * Padj <0.05, ** Padj <0.01,*** Padj < 0.001. Padj, adjusted Pvalue. (I) Schematic of the experimental approach used to assess the intracellular and total bacteria-killing function of neutrophils following constricted migration in vitro. Neutrophils were isolated using a standard Percoll gradient from the bone marrow of 10-week-old male C57BL/6 mice. The cells underwent transwell migration (Millipore Millicell® Hanging Cell Culture Insert) for 2 hours with 1μM fMLP in the lower chamber. The entire transwell process was conducted in 2% FBS/RPMI. After migration, the cells were collected and allowed to settle for 30 minutes before the addition of E. coli (ATCC #25922GFP) as indicated. (J) The total bacterial killing by neutrophils presented as the number of bacteria (CFU) and the percentage of killing compared to bacteria alone at 3 hours. Data are presented as mean ± SD (n=5); ns, not significant (P > 0.05). (K) The intracellular bacteria-killing capability of neutrophils presented as the bacterial count (CFU) at indicated time points and the killing percentage at 2 hr compared to 0 hr post-gentamicin treatment. Data are presented as mean ± SD (n=4); ns, not significant (P > 0.05).
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