Microtubules and Dynein Regulate Human Neutrophil Nuclear Volume and Hypersegmentation During H. pylori Infection.

Stephanie L Silva-Del Toro,Lee-Ann H Allen

doi:10.3389/fimmu.2021.653100

Stephanie L Silva-Del Toro, Lee-Ann H Allen

Open Access

https://doi.org/10.3389/fimmu.2021.653100

Copy DOI

Abstract

Neutrophils (also called polymorphonuclear leukocytes, PMNs) are heterogeneous and can exhibit considerable phenotypic and functional plasticity. In keeping with this, we discovered previously that Helicobacter pylori infection induces N1-like subtype differentiation of human PMNs that is notable for profound nuclear hypersegmentation. Herein, we utilized biochemical approaches and confocal and super-resolution microscopy to gain insight into the underlying molecular mechanisms. Sensitivity to inhibition by nocodazole and taxol indicated that microtubule dynamics were required to induce and sustain hypersegmentation, and super-resolution Stimulated Emission Depletion (STED) imaging demonstrated that microtubules were significantly more abundant and longer in hypersegmented cells. Dynein activity was also required, and enrichment of this motor protein at the nuclear periphery was enhanced following H. pylori infection. In contrast, centrosome splitting did not occur, and lamin B receptor abundance and ER morphology were unchanged. Finally, analysis of STED image stacks using Imaris software revealed that nuclear volume increased markedly prior to the onset of hypersegmentation and that nuclear size was differentially modulated by nocodazole and taxol in the presence and absence of infection. Taken together, our data define a new mechanism of hypersegmentation that is mediated by microtubules and dynein and as such advance understanding of processes that regulate nuclear morphology.

Highlights

Neutrophils or polymorphonuclear leukocytes (PMNs) are the most abundant white blood cell in circulation, are the first leukocytes to be recruited to sites of infection, and are essential for innate host defense [1,2,3]
To test the hypothesis that MTs are required for H. pyloriinduced nuclear hypersegmentation PMNs were treated with taxol or nocodazole at time zero in the presence and absence of bacteria and nuclear morphology was assessed at 24 h using Hema-3 staining and light microscopy
Little is known about the mechanisms that control neutrophil nuclear morphology

Summary

Introduction

Neutrophils or polymorphonuclear leukocytes (PMNs) are the most abundant white blood cell in circulation, are the first leukocytes to be recruited to sites of infection, and are essential for innate host defense [1,2,3]. PMNs were thought to be a homogeneous population of cells with a singular phenotype. Mechanisms of Neutrophill Hypersegmentation antigen presenting cells involved in adaptive immunity, to bacterial and parasitic infections and sepsis as well as autoimmune disease, a spectrum of pro-inflammatory, regulatory and suppressive PMNs have been described. Thought to occur only in response to in vivo cues, we discovered that infection of human neutrophils with the ulcer and gastric cancer-causing Gram-negative bacterium Helicobacter pylori can induce N1-like subtype differentiation of human neutrophils in vitro that is notable for profound nuclear hypersegmentation, proinflammatory cytokine secretion, a CD62Ldim, CD16bright, CD11bbright, CD66bbright, CD63bright surface phenotype and a 72 h lifespan [16]. Ohms et al [17] attempted in vitro polarization of human neutrophils, but their analysis did not include examination of nuclear morphology

Methods

Results

Conclusion