Carole A. Parent

The HL60 promyelocytic leukemia cell line is widely used to investigate neutrophil biology due to its genetic tractability and accessibility. HL60 cells can be differentiated into neutrophil-like cells using all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO) treatment. However, these approaches produce cells that lack critical features of mature granulocytes, such as robust chemotaxis with ATRA treatment or multilobed nuclear morphology with DMSO treatment. To overcome these limitations, we developed a sequential differentiation protocol—ATRA for 1 d followed by DMSO for 4 d (A1D4)—which yields neutrophil-like cells that more faithfully recapitulate the morphology and functionality of mature human neutrophils. A1D4-differentiated HL60 cells display segmented nuclei with low lamin A/C expression and demonstrate strong chemotactic, oxidative burst, and phagocytic activity. This protocol combines the advantages of treatment with the individual compounds, producing cells that closely mimic primary human neutrophils in both phenotype and function.

The stress-induced keratin 16 intermediate filament gene, KRT16 , and K16 protein are spatially restricted to the suprabasal layers of the epidermis and widely used as biomarkers in psoriasis, hidradenitis suppurativa, atopic dermatitis, and other inflammatory disorders. Pathogenic variants in KRT16 cause pachyonychia congenita (PC), a rare genetic condition in which tissue homeostasis is disrupted in palmoplantar epidermis, nail, oral mucosa, and other KRT16 -expressing epithelial appendages. How K16 may regulate the molecular mechanisms underlying these conditions is poorly understood. Here, we show that K16 negatively regulates type I interferon (IFN) signaling and innate immune responses in skin. Clinically, the signature palmoplantar keratoderma lesions in individuals with PC showed enhanced type I IFN signaling. In mouse skin in vivo, loss of Krt16 led to exacerbation of imiquimod-induced psoriasiform disease and heightened recruitment of neutrophils in a phorbol ester–induced model of acute sterile inflammation. In KRT16 -null human keratinocytes, loss of K16 amplified IFN signaling including phosphorylated interferon regulatory factor 7 (IRF7) and interferon-stimulated gene 15 (ISG15) after treatment with synthetic dsRNA poly(I:C). Mechanistically, K16 interacted with effectors of the RIG-I–like receptor (RLR) pathway, including 14-3-3ɛ, and inhibited the 14-3-3ɛ:RIG-I interaction upstream of IFN activation in mouse and immortalized human keratinocytes ex vivo. Topical application of the Janus kinase inhibitor cream ruxolitinib to palmoplantar keratoderma lesions of Krt16 -null mice reduced lesion severity. These findings uncover a previously unrecognized paradigm for keratin-dependent regulation of innate immunity, with implications for our understanding of inflammatory skin diseases and potential treatments for PC.

As solid tumors progress, the tumor microenvironment (TME) becomes increasingly immunosuppressive, impairing cytotoxic T-cell activity and limiting the efficacy of the immune checkpoint blockade. However, the mechanistic drivers of this immunosuppression remain poorly understood. Here, we identify a tumor-derived lipid–neutrophil–adenosine axis as a critical regulator of immune suppression in advanced colorectal cancer (CRC). We show that fatty acids enriched in tumor interstitial fluid reprogram neutrophils to generate adenosine via PPARα activation, leading to T-cell suppression. Using AB928, a dual A2aR/A2bR adenosine receptor antagonist currently in clinical trials, we restored T-cell proliferation, effector function, and tumor-killing capacity in vitro and in vivo. Importantly, AB928 synergized with anti-PD-1 therapy to enhance survival in an autochthonous model of metastatic CRC. Our findings define a metabolic immune evasion mechanism in the TME and provide a rationale for targeting neutrophil-derived adenosine signaling to improve immunotherapy responses in CRC and other solid tumors.

Directional persistence is essential for efficient immune cell migration in tissues, yet how cytoskeletal systems stabilize migration in complex three-dimensional environments remains unclear. Using intravital subcellular microscopy and quantitative analysis of membrane dynamics, we identify two spatially distinct architectures of non-muscle myosin II (NMII) that coordinate protrusion dynamics during neutrophil migration. In vivo and in collagen matrices, NMII assembles at the leading edge into lattice-like structures that are structurally and functionally distinct from rear contractile actomyosin bundles. Protrusion-resolved analyses reveal that directional persistence correlates strongly with protrusion lifetime and sustained NMII engagement, with rear NMII load showing the strongest association with protrusion persistence. Strikingly, directional migration is not determined by the abundance of favorable protrusions but by their temporal organization during migration. Pharmacological perturbations that redistribute NMII activity disrupt this temporal organization and alter migration trajectories. Together, these findings reveal that spatially distinct NMII architectures coordinate protrusion dynamics across time to stabilize directional migration in complex environments. Highlights: Migrating neutrophils assemble spatially distinct myosin II architectures at the leading edge and rear Protrusion dynamics and directional persistence are linked to sustained myosin II engagement Directional migration emerges from the temporal organization of protrusion states rather than their abundance

Efficient neutrophil chemotaxis requires the integration of mechanical forces and lipid-mediated signaling. While the signaling lipid leukotriene B4 (LTB ₄ ) reinforces cellular polarity, how mechanical cues regulate its production remains unclear. We now show that cytosolic phospholipase A2α (cPLA ₂ α), which is essential for the synthesis of LTB ₄ , functions as a nuclear curvosensor. cPLA ₂ α responds to nuclear squeezing by localizing to ceramide-rich inner nuclear membrane microdomains and incorporating onto the exofacial surface of nuclear envelope–derived exosomes. This unique topology enables localized LTB ₄ synthesis, which synchronizes calcium spikes, promotes myosin light chain II phosphorylation, and sustains polarity and directional persistence after constriction. In neutrophils passing through tight spaces, cPLA ₂ α activity drives the chemotactic response to nuclear squeezing by promoting exosomal LTB ₄ production and persistence after constriction. These findings uncover a cPLA ₂ α-dependent mechanochemical axis linking nuclear architecture to chemotactic efficiency and offer alternative strategies to modulate inflammatory responses.

Most adhesion G protein–coupled receptors (AGPCRs) are activated by the intramolecular binding of a tethered agonist, which is exposed by shear force–induced dissociation of the N- and C-terminal fragments of AGPCRs. The decrypted tethered agonist binds to its orthosteric site in the C-terminal fragment to stabilize the active state of the AGPCR. Corticosteroids have been proposed to be orthosteric agonists for GPR97/ADGRG3. Here, we showed that GPR97/ADGRG3 is activated by the canonical tethered agonist mechanism. In cell-based luciferase reporter assays and receptor/G protein reconstitution assays, GPR97/ADGRG3 was stimulated by agonist peptidomimetics and by 3-acetoxydihydrodeoxygeduin (3-α-DOG), a partial agonist of the ADGRG subfamily, but not by corticosteroids. We showed that GPR97 was a promiscuous AGPCR that coupled to G ₁₃ , G _s , and G _i but not to G _q . GPR97 is abundant in neutrophils, which undergo cell shape changes and polarization and migrate upon activation. GPR97 activation by tethered agonist peptidomimetics or 3-α-DOG robustly stimulated cAMP production, polarization, and chemotaxis in human and mouse neutrophils. These effects in mouse neutrophils required GPR97 and were not mimicked by the corticosteroid beclomethasone in either cell type. Together, our results demonstrate that GPR97/ADGRG3 uses a tethered agonist mechanism to activate G protein signaling and induce neutrophil polarization and migration.

The HL60 promyelocytic leukemia cell line is widely used to investigate neutrophil biology due to its genetic tractability and accessibility. HL60 cells can be differentiated into neutrophil-like cells using all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO) treatment. However, these approaches produce cells that lack critical features of mature granulocytes, such as robust chemotaxis with ATRA treatment or multilobed nuclear morphology with DMSO treatment. To overcome these limitations, we developed a sequential differentiation protocol - ATRA for one day followed by DMSO for four days (A1D4) - which yields neutrophil-like cells that more faithfully recapitulate the morphology and functionality of mature human neutrophils. A1D4-differentiated HL60 cells display segmented nuclei with low lamin A/C expression and demonstrate strong chemotactic, oxidative burst, and phagocytic activity. This protocol combines the advantages of individual compounds, producing cells that closely mimic primary neutrophils in both phenotype and function.

Supplemental Figure 3: Liver metastases augment neutrophils to promote cancer progression.

Supplemental Figure 4: Liver metastases augment neutrophils to promote cancer progression.