Kihong Lim

Neutrophil trails guide influenza-specific CD8+ T cells in the airways

Neutrophils lay down the tracks T cells constantly circulate throughout the body until an invading pathogen calls them into action. Microbes often cause localized infections, so how do T cells know where to go? Lim et al. explore this question in a mouse model of influenza infection and find that immune cells called neutrophils help guide the way (see the Perspective by Kiermaier and Sixt). Upon infection, neutrophils quickly traffic to the trachea. There, they lay down “tracks” enriched in proteins called chemokines, especially the chemokine CXCL12, which guide CD8+ T cells to the infected organ. Mice whose neutrophils could not lay down such tracks exhibited defects in CD8+ T cell recruitment and viral clearance. Science, this issue 10.1126/science.aaa4352; see also p. 1055 Trails of chemokines left behind by neutrophils guide T cells to sites of viral infection. [Also see Perspective by Kiermaier and Sixt] INTRODUCTION Influenza virus infects the epithelial cells that line the respiratory tract. Therefore, cytotoxic CD8+ T cells must traffic to this site to eliminate infected cells. The functions of antiviral CD8+ T cell effector at tissue sites require a successful and early innate immune response. Neutrophils are an immune cell subset that helps organs initiate and maintain immune reactions and shapes the overall immune response by signaling to multiple immune cell types, including T cells. Under most inflammatory conditions, neutrophils are the first cell type that crosses the blood vessel endothelium into the tissue, often preceding a subsequent wave of effector T cells. Although neutrophils are known to recruit T cells into infected sites during both bacterial and viral infections and in chronic inflammatory diseases, the molecular mechanisms that link neutrophil and T cell migration remain unknown. RATIONALE The chemokine receptor family is the most potent tissue-specific family of homing receptors for T cells and is subset-selective. Therefore, it is widely assumed that the distinct migratory properties and distribution patterns of different subsets of specialized T cells result from the differential expression of the chemokines and their receptors. Although this idea has been verified experimentally in some settings, multiple chemokine receptors expressed on the effector T cells and the redundancy in their signaling pathways suggest the presence of a more complex mechanism that can confer specificity and selectivity to T cell recruitment. Furthermore, less is known about how chemokines released from newly recruited leukocytes act together with the local chemokines produced within the inflamed tissue. To address this, we performed intravital multiphoton microscopy imaging of the influenza-infected mouse trachea and explored how neutrophil-derived chemokines cooperate with the tissue-specific inflammatory cues to finely control the recruitment of CD8+ T cells to the influenza-infected trachea. RESULTS Here, we show that optimal CD8+ T cell–mediated immune protection requires the early recruitment of neutrophils into influenza-infected trachea. In particular, the relative motility of virus-specific CD8+ T cells in the trachea was determined by their localization to the epithelium, which was governed by the presence of neutrophils during early infection. Both in vitro and in vivo imaging showed that migrating neutrophils leave behind long-lasting trails from their elongated uropods (a protrusion at the rear of a cell) that are prominently enriched in the chemokine CXCL12. We observed that CXCL12 derived from the epithelial cells remained close to the epithelium, whereas CXCL12 derived from neutrophils was the main source of CXCL12 in the tissue interstitium during infection. Experiments with granulocyte-specific CXCL12 conditionally depleted (knockout) mice and a CXCR4 antagonist revealed that CXCL12 derived from neutrophil trails is critical for virus-specific CD8+ T cell recruitment and antiviral effector functions. CONCLUSION The data presented here demonstrate that migrating neutrophils leave behind chemoattractant-containing trails, which result in the local accumulation of neutrophil-derived chemoattractant signals in inflamed tissues. As chemokines are small, diffusible molecules, perhaps these trails function to package the chemoattractant so that it can be preserved and survive severe mechanical perturbation during inflammation. Otherwise, the chemoattractant would be present only transiently, or it would immediately diffuse away from the site. Neutrophils trails guide virus-specific CD8+ T cell migration. In the influenza-infected trachea, tissue-infiltrating neutrophils (pink) deposit chemokine (CXCL12)–containing trails, which may serve like breadcrumbs or long-lasting chemokine depots to provide both chemotactic and haptotactic cues for efficient virus-specific CD8+ T cell migration and localization in the infected tissues. During viral infections, chemokines guide activated effector T cells to infection sites. However, the cells responsible for producing these chemokines and how such chemokines recruit T cells are unknown. Here, we show that the early recruitment of neutrophils into influenza-infected trachea is essential for CD8+ T cell–mediated immune protection in mice. We observed that migrating neutrophils leave behind long-lasting trails that are enriched in the chemokine CXCL12. Experiments with granulocyte-specific CXCL12 conditionally depleted mice and a CXCR4 antagonist revealed that CXCL12 derived from neutrophil trails is critical for virus-specific CD8+ T cell recruitment and effector functions. Collectively, these results suggest that neutrophils deposit long-lasting, chemokine-containing trails, which may provide both chemotactic and haptotactic cues for efficient CD8+ T cell migration and localization in influenza-infected tissues.

Optogenetic control of chemokine receptor signal and T-cell migration

Significance Precise regulation of chemokine signal is critical for directional migration of cells. In the study of complex cell behavior, it remains difficult to manipulate chemokine activity at precise times and places within living animals, and it is not possible to study different chemokine effects on defined cell types over a range of timescales. Furthermore, a given chemokine can activate multiple chemokine receptors and vice versa. Here we developed a photoactivatable chemokine receptor that can induce highly specific chemokine signals and guide cell migration toward the light stimulation. This work will advance our understanding of the cell migration process with a number of previously unidentified findings. Clinically, our photoactivatable chemokine receptor approach may have broad applications for adoptive cell transfer therapy. Adoptive cell transfer of ex vivo-generated immune-promoting or tolerogenic T cells to either enhance immunity or promote tolerance in patients has been used with some success. However, effective trafficking of the transferred cells to the target tissue sites is the main barrier to achieving successful clinical outcomes. Here we developed a strategy for optically controlling T-cell trafficking using a photoactivatable (PA) chemokine receptor. Photoactivatable-chemokine C-X-C motif receptor 4 (PA-CXCR4) transmitted intracellular CXCR4 signals in response to 505-nm light. Localized activation of PA-CXCR4 induced T-cell polarization and directional migration (phototaxis) both in vitro and in vivo. Directing light onto the melanoma was sufficient to recruit PA-CXCR4–expressing tumor-targeting cytotoxic T cells and improved the efficacy of adoptive T-cell transfer immunotherapy, with a significant reduction in tumor growth in mice. These findings suggest that the use of photoactivatable chemokine receptors allows remotely controlled leukocyte trafficking with outstanding spatial resolution in tissues and may be feasible in other cell transfer therapies.

O-linked N-acetylglucosamine suppresses thermal aggregation of Sp1

We demonstrate that O‐linked N‐acetylglucosamine (O‐GlcNAc), a ubiquitous protein modification in eukaryotes, suppresses thermal inactivation of Sp1 transcription factor. 6‐Diazo‐5‐oxonorleucine treatment or O‐GlcNAcase overexpression, which reduced O‐GlcNAc levels on Sp1, deteriorated thermal stability of Sp1 and O‐GlcNAc modified molecules of Sp1 resist thermal aggregation in vitro. We also showed that heat‐induced elevation of heat shock protein 70 was facilitated by Sp1 but blunted under low O‐GlcNAc levels, suggesting that O‐GlcNAc might upregulate the expression of heat shock protein 70 through thermoprotection of Sp1, which eventually enhanced cellular thermotolerance.

O-GlcNAc inhibits interaction between Sp1 and Elf-1 transcription factors.

The novel protein modification, O-linked N-acetylglucosamine (O-GlcNAc), plays an important role in various aspects of cell regulation. Although most of nuclear transcription regulatory factors are modified by O-GlcNAc, O-GlcNAc effects on transcription remain largely undefined yet. In this study, we show that O-GlcNAc inhibits a physical interaction between Sp1 and Elf-1 transcription factors, and negatively regulates transcription of placenta and embryonic expression oncofetal protein gene (Pem). These findings suggest that O-GlcNAc inhibits Sp1-mediated gene transcription possibly by interrupting Sp1 interaction with its cooperative factor.

Sepsis lethality via exacerbated tissue infiltration and TLR-induced cytokine production by neutrophils is integrin α3β1-dependent

Integrin-mediated migration of neutrophils to infected tissue sites is vital for pathogen clearance and therefore host survival. Although β2 integrins have been shown to mediate neutrophil transendothelial migration during systemic and local inflammation, relatively little information is available regarding neutrophil migration in sepsis beyond the endothelial cell layer. In this study, we report that integrin α3β1 (VLA-3; CD49c/CD29) is dramatically upregulated on neutrophils isolated from both human septic patients and in mouse models of sepsis. Compared with the α3β1 (low) granulocytes, α3β1 (high) cells from septic animals displayed hyperinflammatory phenotypes. Administration of a α3β1 blocking peptide and conditional deletion of α3 in granulocytes significantly reduced the number of extravasating neutrophils and improved survival in septic mice. In addition, expression of α3β1 on neutrophils was associated with Toll-like receptor-induced inflammatory responses and cytokine productions. Thus, our results show that α3β1 is a novel marker of tissue homing and hyperresponsive neutrophil subtypes in sepsis, and blocking of α3β1 may represent a new therapeutic approach in sepsis treatment.

O-GlcNAc inhibits interaction between Sp1 and sterol regulatory element binding protein 2.

O-linked N-acetylglucosamine (O-GlcNAc), a monosaccharide N-acetylglucosamine on the serine and threonine residues of nucleocytoplasmic proteins, is a novel protein modification that is ubiquitous among eukaryotes and implicated in cell regulation. Recent evidence indicates that O-GlcNAc regulates protein-protein interactions. Here we provide evidence that O-GlcNAc interrupts a known interaction between Sp1 and sterol regulatory element binding protein 2 (SREBP2), thereby inhibiting expression of the gene encoding acetyl-CoA synthetase 1, which is involved in lipid synthesis. This study suggests a novel mechanism in which lipid biosynthesis may be regulated by O-GlcNAc.

O-GlcNAc modification of Sp1 inhibits the functional interaction between Sp1 and Oct1.

MINT‐6803470, MINT‐6803484: Sp1 (uniprotkb:P08047) physically interacts (MI:0218) with Oct1 (uniprotkb:P14859) by anti bait coimmunoprecipitation (MI:0006)

Opposing roles for RhoH GTPase during T-cell migration and activation

T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and then quickly recirculate through the bloodstream to another lymph node. Therefore, regulation of a T-cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients (“go” signal) as well as to receive appropriate T-cell receptor (TCR) activation signals upon cognate antigen recognition (“stop” signal). However, the mechanisms by which T cells regulate these go and stop signals remain unclear. We found that overexpression of the hematopoietic-specific RhoH protein in the presence of chemokine signals resulted in decreased Rap1–GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell chemotaxis; while in the presence of TCR signals, there were enhanced and sustained Rap1–GTP and LFA-1 activation as well as prolonged T:APC conjugates. RT-PCR analyses of activated CD4+ T cells and live images of T-cell migration and immunological synapse (IS) formation revealed that functions of RhoH took place primarily at the levels of transcription and intracellular distribution. Thus, we conclude that RhoH expression provides a key molecular determinant that allows T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals.

O-GlcNAcylation of Sp1 interrupts Sp1 interaction with NF-Y

O-linked N-acetylglucosamine (O-GlcNAc), a monosaccharide N-acetylglucosamine addition on nucleocytoplasmic proteins, is abundant in transcription regulators and has been implicated in gene regulation. Sp1 transcription factor is multiply modified by O-GlcNAc within its serine/threonine-rich region and glutamine-rich transactivation domain. In the present study, we show that O-GlcNAc of Sp1 serine/threonine-rich region interrupts a physical interaction between Sp1 and NF-YA, thus inhibiting Sp1-NF-Y cooperative activation of gene transcription. Our results strengthen the notion that O-GlcNAc regulates gene transcription by modulating the protein-protein interaction network among transcription regulatory proteins.

Extravasating Neutrophil-derived Microparticles Preserve Vascular Barrier Function in Inflamed Tissue

Emerging evidence suggests that gap formation and opening of the endothelial junctions during leukocyte extravasation is actively controlled to maintain the integrity of the vascular barrier. While the role for endothelial cells to this process has been well defined, it is not clear whether leukocytes are also actively contributing to endothelial barrier function. We have recently showed that extravasating leukocytes deposit microparticles on the subendothelium during the late stages of extravasation, which is LFA-1 dependent. Using multiphotonintravital microscopy (MP-IVM) of mouse cremaster muscle vessels in the current work, we show that microparticle formation and deposition maintains the integrity of the microvascular barrier during leukocyte extravasation. Inhibition of neutrophil-derived microparticle formation resulted in dramatically increased vascular leakage. These findings suggest that deposition of microparticles during neutrophil extravasation is essential for maintaining endothelial barrier function and may result in temporal difference between neutrophil extravasation and an increase in vascular leakage.