Exploratory mass cytometry analysis reveals immunophenotypes of cancer treatment-related pneumonitis.

Anticancer treatments can result in various adverse effects, including infections due to immune suppression/dysregulation and drug-induced toxicity in the lung. One of the major opportunistic infections is Pneumocystis jirovecii pneumonia (PCP), which can cause severe respiratory complications and high mortality rates. Cytotoxic drugs and immune-checkpoint inhibitors (ICIs) can induce interstitial lung diseases (ILDs). Nonetheless, the differentiation of these diseases can be difficult, and the pathogenic mechanisms of such diseases are not yet fully understood. To better comprehend the immunophenotypes, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid from patients with PCP, cytotoxic drug-induced ILD (DI-ILD), and ICI-associated ILD (ICI-ILD) using two panels containing 64 markers. In PCP, we observed an expansion of the CD16+ T cell population, with the highest CD16+ T proportion in a fatal case. In ICI-ILD, we found an increase in CD57+ CD8+ T cells expressing immune checkpoints (TIGIT+ LAG3+ TIM-3+ PD-1+), FCRL5+ B cells, and CCR2+ CCR5+ CD14+ monocytes. These findings uncover the diverse immunophenotypes and possible pathomechanisms of cancer treatment-related pneumonitis.

[RECOMMENDATION FOR ITCH ASSESSMENT FROM THE ATOPIC ITCH CONSENSUS MEETING (AICOM)].

BACKGROUND: Itch is the most troublesome symptom of atopic dermatitis, and it is important to assess it appropriately for optimal treatment. We discussed issues regarding itch and the most appropriate methods of assessment at the Atopic Itch Consensus Meeting (AICOM), attended by physicians and researchers with expertise in itch treatment and research. METHODS: The AICOM participants prepared a draft consensus statement that addressed the most appropriate itch assessment methods for age groups <2 years, 2-6 years, 7-14 years, and ≥15 years. Consensus was defined as agreement by ≥80% of the participants. RESULTS: Votes were cast by 20 participants (8 dermatologists, 7 pediatricians, and 5 researchers), and a consensus on the best current methods of itch assessment was reached with 95% agreement. For infants and preschool children, because subjective evaluation is difficult, a checklist for itch assessment was developed for caregivers. CONCLUSION: For itch assessment, we recommend subjective evaluation by the patient using a rating scale. For infants and preschoolers, evaluation should be done by the caregiver using a checklist, combined with objective evaluation (of skin lesions, for example) by a physician. We anticipate that more objective itch assessment indices will be established in the future.

The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses.

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating ß2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and ß2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs.

Altered macrophage phenotypes in a case of autoimmune pulmonary alveolar proteinosis.

Mass cytometry of BALF cells from a pulmonary alveolar proteinosis patient, positive for anti-GM-CSF antibodies, suggests potential impairment in human alveolar macrophage differentiation https://bit.ly/45JHUrz.

Rap1 organizes lymphocyte front-back polarity via RhoA signaling and talin1.

Lymphocyte trafficking requires fine-tuning of chemokine-mediated cell migration. This process depends on cytoskeletal dynamics and polarity, but its regulation remains elusive. We quantitatively measured cell polarity and revealed critical roles performed by integrin activator Rap1 in this process, independent of substrate adhesion. Rap1-deficient naive T cells exhibited impaired abilities to reorganize the actin cytoskeleton into pseudopods and actomyosin-rich uropods. Rap1-GTPase activating proteins (GAPs), Rasa3 and Sipa1, maintained an unpolarized shape; deletion of these GAPs spontaneously induced cell polarization, indicative of the polarizing effect of Rap1. Rap1 activation required F-actin scaffolds, and stimulated RhoA activation and actomyosin contractility at the rear. Furthermore, talin1 acted on Rap1 downstream effectors to promote actomyosin contractility in the uropod, which occurred independently of substrate adhesion and talin1 binding to integrins. These findings indicate that Rap1 signaling to RhoA and talin1 regulates chemokine-stimulated lymphocyte polarization and chemotaxis in a manner independent of adhesion.

Expansion of ST2-expressing macrophages in a patient with bronchiolitis obliterans syndrome.

This case study of a patient with BOS after HSCT found increased ST2+CD64+ macrophages in BALF, a potential therapeutic target for treatment-refractory BOS, and reduced CCR2+CD14+ monocytes compared to other lung disorders https://bit.ly/406Uyy9.

Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis.

Introduction: Rac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens. Methods: Here, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control. Results: All the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis. Discussion: Collectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils.

Cholesterol sulfate limits neutrophil recruitment and gut inflammation during mucosal injury.

During mucosal injury, intestinal immune cells play a crucial role in eliminating invading bacteria. However, as the excessive accumulation of immune cells promotes inflammation and delays tissue repair, it is essential to identify the mechanism that limits the infiltration of immune cells to the mucosal-luminal interface. Cholesterol sulfate (CS) is the lipid product of the sulfotransferase SULT2B1 and suppresses immune reactions by inhibiting DOCK2-mediated Rac activation. In this study, we aimed to elucidate the physiological role of CS in the intestinal tract. We found that, in the small intestine and colon, CS is predominantly produced in the epithelial cells close to the lumen. While dextran sodium sulfate (DSS)-induced colitis was exacerbated in Sult2b1-deficient mice with increased prevalence of neutrophils, the elimination of either neutrophils or intestinal bacteria in Sult2b1-deficient mice attenuated disease development. Similar results were obtained when the Dock2 was genetically deleted in Sult2b1-deficient mice. In addition, we also show that indomethacin-induced ulcer formation in the small intestine was exacerbated in Sult2b1-deficient mice and was ameliorated by CS administration. Thus, our results uncover that CS acts on inflammatory neutrophils, and prevents excessive gut inflammation by inhibiting the Rac activator DOCK2. The administration of CS may be a novel therapeutic strategy for inflammatory bowel disease and non-steroidal anti-inflammatory drug-induced ulcers.

Mass cytometry identifies characteristic immune cell subsets in bronchoalveolar lavage fluid from interstitial lung diseases.

Immune cells have been implicated in interstitial lung diseases (ILDs), although their phenotypes and effector mechanisms remain poorly understood. To better understand these cells, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF), connective-tissue disease (CTD)-related ILD, and sarcoidosis, using two panels including 64 markers. Among myeloid cells, we observed the expansion of CD14+ CD36hi CD84hiCCR2- monocyte populations in IPF. These CD14+ CD36hi CD84hi CCR2- subsets were also increased in ILDs with a progressive phenotype, particularly in a case of acute exacerbation (AEx) of IPF. Analysis of B cells revealed the presence of cells at various stages of differentiation in BALF, with a higher percentage of IgG memory B cells in CTD-ILDs and a trend toward more FCRL5+ B cells. These FCRL5+ B cells were also present in the patient with AEx-IPF and sarcoidosis with advanced lung lesions. Among T cells, we found increased levels of IL-2R+ TIGIT+ LAG3+ CD4+ T cells in IPF, increased levels of CXCR3+ CD226+ CD4+ T cells in sarcoidosis, and increased levels of PD1+ TIGIT+ CD57+ CD8+ T cells in CTD-ILDs. Together, these findings underscore the diverse immunopathogenesis of ILDs.

Structural basis for the dual GTPase specificity of the DOCK10 guanine nucleotide exchange factor.

Dedicator of cytokinesis 10 (DOCK10), an evolutionarily conserved guanine nucleotide exchange factor (GEF) for Rho GTPases, has the unique specificity within the DOCK-D subfamily to activate both Cdc42 and Rac, but the structural bases for these activities remained unknown. Here we present the crystal structures of the catalytic DHR2 domain of mouse DOCK10, complexed with either Cdc42 or Rac1. The structures revealed that DOCK10DHR2 binds to Cdc42 or Rac1 by slightly changing the arrangement of its two catalytic lobes. DOCK10 also has a flexible binding pocket for the 56th GTPase residue, allowing a novel interaction with Trp56Rac1. The conserved residues in switch 1 of Cdc42 and Rac1 showed common interactions with the unique Lys-His sequence in the ß5/ß6 loop of DOCK10DHR2. However, the interaction of switch 1 in Rac1 was less stable than that of switch 1 in Cdc42, due to amino acid differences at positions 27 and 30. Structure-based mutagenesis identified the DOCK10 residues that determine the Cdc42/Rac1 dual specificity.

DOCK2 regulates MRGPRX2/B2-mediated mast cell degranulation and drug-induced anaphylaxis.

BACKGROUND: Drug-induced anaphylaxis is triggered by the direct stimulation of mast cells (MCs) via Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MRGPRB2). However, the precise mechanism that links MRGPRX2/B2 to MC degranulation is poorly understood. Dedicator of cytokinesis 2 (DOCK2) is a Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 regulates migration and activation of leukocytes, its role in MCs remains unknown. OBJECTIVE: We aimed to elucidate whether-and if so, how-DOCK2 is involved in MRGPRX2/B2-mediated MC degranulation and anaphylaxis. METHODS: Induction of drug-induced systemic and cutaneous anaphylaxis was compared between wild-type and DOCK2-deficient mice. In addition, genetic or pharmacologic inactivation of DOCK2 in human and murine MCs was used to reveal its role in MRGPRX2/B2-mediated signal transduction and degranulation. RESULTS: Induction of MC degranulation and anaphylaxis by compound 48/80 and ciprofloxacin was severely attenuated in the absence of DOCK2. Although calcium influx and phosphorylation of several signaling molecules were unaffected, MRGPRB2-mediated Rac activation and phosphorylation of p21-activated kinase 1 (PAK1) were impaired in DOCK2-deficient MCs. Similar results were obtained when mice or MCs were treated with small-molecule inhibitors that bind to the catalytic domain of DOCK2 and inhibit Rac activation. CONCLUSION: DOCK2 regulates MRGPRX2/B2-mediated MC degranulation through Rac activation and PAK1 phosphorylation, thereby indicating that the DOCK2-Rac-PAK1 axis could be a target for preventing drug-induced anaphylaxis.

Pharmacological intervention of cholesterol sulfate-mediated T cell exclusion promotes antitumor immunity.

Effective cancer immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute special microenvironments that exclude T cells and resist immunotherapy. Cholesterol sulfate (CS) is a product of sulfotransferase SULT2B1b and acts as an endogenous inhibitor of DOCK2, a Rac activator essential for migration and activation of lymphocytes. We have recently shown that cancer-derived CS prevents tumor infiltration by effector T cells. Therefore, SULT2B1b may be a therapeutic target to dampen CS-mediated immune evasion. Here, we identified 3ß-hydroxy-5-cholenoic acid (3ß-OH-5-Chln) as a cell-active inhibitor of SULT2B1b. 3ß-OH-5-Chln inhibited the cholesterol sulfotransferase activity of SULT2B1b in vitro and suppressed CS production from cancer cells expressing SULT2B1b. In vivo administration of 3ß-OH-5-Chln locally reduced CS level in murine CS-producing tumors and increased infiltration of CD8+ T cells. When combined with immune checkpoint blockade or antigen-specific T cell transfer, 3ß-OH-5-Chln suppressed the growth of CS-producing tumors. These results demonstrate that pharmacological inhibition of SULT2B1b can promote antitumor immunity through suppressing CS-mediated T cell exclusion.

Cancer-derived cholesterol sulfate is a key mediator to prevent tumor infiltration by effector T cells.

Effective tumor immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute a specialized microenvironment that excludes T cells from the vicinity of cancer cells, and its underlying mechanisms are still poorly understood. DOCK2 is a Rac activator critical for migration and activation of lymphocytes. We herein show that cancer-derived cholesterol sulfate (CS), a lipid product of the sulfotransferase SULT2B1b, acts as a DOCK2 inhibitor and prevents tumor infiltration by effector T cells. Using clinical samples, we found that CS was abundantly produced in certain types of human cancers such as colon cancers. Functionally, CS-producing cancer cells exhibited resistance to cancer-specific T-cell transfer and immune checkpoint blockade. Although SULT2B1b is known to sulfate oxysterols and inactivate their tumor-promoting activity, the expression levels of cholesterol hydroxylases, which mediate oxysterol production, are low in SULT2B1b-expressing cancers. Therefore, SULT2B1b inhibition could be a therapeutic strategy to disrupt tumor immune evasion in oxysterol-non-producing cancers. Thus, our findings define a previously unknown mechanism for tumor immune evasion and provide a novel insight into the development of effective immunotherapies.

The molecular basis for IL-31 production and IL-31-mediated itch transmission: from biology to drug development.

Atopic dermatitis (AD) is one of the most prevalent chronic inflammatory skin diseases in the world. It is characterized by recurrent eczematous lesions and intense itch, and many cytokines are involved in the pathogenesis of AD. Among them, much attention has been paid to interleukin 31 (IL-31) as an AD-associated itch mediator. IL-31 is mainly produced by CD4+ helper T cells and transmits the signals via a heterodimeric receptor composed of IL-31 receptor A (IL-31RA) and oncostatin M receptor (OSMR), both of which are expressed in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms of how IL-31 is produced in helper T cells upon stimulation and transmits the itch sensation to the brain were largely unknown. Recently, by using original mouse models of AD, we have identified endothelial PAS domain 1 (EPAS1) and neurokinin B (NKB) as key molecules critical for IL-31 production and IL-31-mediated itch transmission, respectively. These molecules could be novel drug targets for AD-associated itch. This review highlights our recent findings, which show the functional significance of these molecules in the IL-31-induced itch sensation, referring to their application to drug development.

DOCK8 deficiency causes a skewing to type 2 immunity in the gut with expansion of group 2 innate lymphoid cells.

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor (GEF) for Cdc42. In humans, homozygous or compound heterozygous deletions in DOCK8 cause a combined immunodeficiency characterized by various allergic diseases including food allergies. Although group 2 innate lymphoid cells (ILC2s) contribute to the development of allergic inflammation by producing interleukin (IL)-5 and IL-13, the role of ILC2s in DOCK8 deficiency has not been fully explored. With the use of cytometry by time-of-flight (CyTOF), we performed high-dimensional phenotyping of intestinal immune cells and found that DOCK8-deficient (Dock8-/-) mice exhibited expansion of ILC2s and other leukocytes associated with type 2 immunity in the small intestine. Moreover, IL-5- and IL-13-producing cells markedly increased in Dock8-/- mice, and the majority of them were lineage-negative cells, most likely ILC2s. Intestinal ILC2s expanded when DOCK8 expression was selectively deleted in hematopoietic cells. Importantly, intestinal ILC2 expansion was also observed in Dock8VAGR mice having mutations in the catalytic center of DOCK8, thereby failing to activate Cdc42. Our findings indicate that DOCK8 is a negative regulator of intestinal ILC2s to inhibit their expansion via Cdc42 activation, and that deletion of DOCK8 causes a skewing to type 2 immunity in the gut.

Targeted inhibition of EPAS1-driven IL-31 production by a small-molecule compound.

BACKGROUND: IL-31 is a major pruritogen associated with atopic dermatitis (AD). Although a specific antibody for IL-31 receptor has been shown to alleviate pruritus in patients with AD, therapeutic approaches to inhibition of IL-31 production remain unexploited. IL-31 production by TH cells critically depends on the transcription factor EPAS1, which mediates IL31 promoter activation in collaboration with SP1. OBJECTIVE: We aimed at developing small-molecule inhibitors that selectively block IL-31 production by TH cells. METHODS: We generated the reporter cell line that inducibly expressed EPAS1 in the presence of doxycycline to mediate Il31 promoter activation, and we screened 9600 chemical compounds. The selected compounds were further examined by using TH cells from a spontaneous mouse model of AD and TH cells from patients with AD. RESULTS: We have identified 4-(2-(4-isopropylbenzylidene)hydrazineyl)benzoic acid (IPHBA) as an inhibitor of IL31 induction. Although IPHBA did not affect nonspecific T-cell proliferation, IPHBA inhibited antigen-induced IL-31 production by TH cells from both an AD mouse model and patients with AD without affecting other cytokine production and hypoxic responses. In line with this, itch responses induced by adoptive transfer of IL-31-producing TH cells were attenuated when mice were orally treated with IPHBA. Mechanistically, IPHBA inhibited the association between EPAS1 and SP1, resulting in defective recruitment of both transcription factors to the specific sites of the IL31 promoter. We also determined the structure-activity relationship of IPHBA by synthesizing and analyzing 201 analogous compounds. CONCLUSION: IPHBA could be a potential drug leading to inhibition of EPAS1-driven IL-31 production.

A conserved PI(4,5)P2-binding domain is critical for immune regulatory function of DOCK8.

DOCK8 is a Cdc42-specific guanine-nucleotide exchange factor that is essential for development and functions of various subsets of leukocytes in innate and acquired immune responses. Although DOCK8 plays a critical role in spatial control of Cdc42 activity during interstitial leukocyte migration, the mechanism remains unclear. We show that the DOCK homology region (DHR)-1 domain of DOCK8 binds specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and is required for its recruitment to the plasma membrane. Structural and biochemical analyses reveal that DOCK8 DHR-1 domain consists of a C2 domain-like core with loops creating the upper surface pocket, where three basic residues are located for stereospecific recognition of phosphoinositides. Substitution of the two basic residues, K576 and R581, with alanine abolished PI(4,5)P2 binding in vitro, ablated the ability of DOCK8 to activate Cdc42 and support leukocyte migration in three-dimensional collagen gels. Dendritic cells carrying the mutation exhibited defective interstitial migration in vivo. Thus, our study uncovers a critical role of DOCK8 in coupling PI(4,5)P2 signaling with Cdc42 activation for immune regulation.

Expression of leukotriene B4 receptor 1 defines functionally distinct DCs that control allergic skin inflammation.

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a chemotactic G protein-coupled receptor expressed by leukocytes, such as granulocytes, macrophages, and activated T cells. Although there is growing evidence that BLT1 plays crucial roles in immune responses, its role in dendritic cells remains largely unknown. Here, we identified novel DC subsets defined by the expression of BLT1, namely, BLT1hi and BLT1lo DCs. We also found that BLT1hi and BLT1lo DCs differentially migrated toward LTB4 and CCL21, a lymph node-homing chemoattractant, respectively. By generating LTB4-producing enzyme LTA4H knockout mice and CD11c promoter-driven Cre recombinase-expressing BLT1 conditional knockout (BLT1 cKO) mice, we showed that the migration of BLT1hi DCs exacerbated allergic contact dermatitis. Comprehensive transcriptome analysis revealed that BLT1hi DCs preferentially induced Th1 differentiation by upregulating IL-12p35 expression, whereas BLT1lo DCs accelerated T cell proliferation by producing IL-2. Collectively, the data reveal an unexpected role for BLT1 as a novel DC subset marker and provide novel insights into the role of the LTB4-BLT1 axis in the spatiotemporal regulation of distinct DC subsets.