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Background COVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed. Methods We evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays’ performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10–40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence. Results Combined IgG + IgM sensitivities ranged from 33.9 to 94.6%, while combined specificities ranged from 92.6 to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG + IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG + IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 μg/mL), followed by a similar LOD of 1.5 μg/mL for CareHealth, Cellex, KHB, and Vivachek. Conclusion We provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Gout is a common inflammatory arthritis caused by precipitation of monosodium urate (MSU) crystals in individuals with hyperuricemia. Acute flares are accompanied by secretion of proinflammatory cytokines, including interleukin-1β (IL-1β). Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition predisposing to hematologic cancers and cardiovascular disease. CHIP is associated with elevated IL-1β, thus we investigated CHIP as a risk factor for gout. To test the clinical association between CHIP and gout, we analyzed whole exome sequencing data from 177 824 individuals in the MGB Biobank (MGBB) and UK Biobank (UKB). In both cohorts, the frequency of gout was higher among individuals with CHIP than without CHIP (MGBB, CHIP with variant allele fraction [VAF] ≥2%: odds ratio [OR], 1.69; 95% CI, 1.09-2.61; P = .0189; UKB, CHIP with VAF ≥10%: OR, 1.25; 95% CI, 1.05-1.50; P = .0133). Moreover, individuals with CHIP and a VAF ≥10% had an increased risk of incident gout (UKB: hazard ratio [HR], 1.28; 95% CI, 1.06-1.55; P = .0107). In murine models of gout pathogenesis, animals with Tet2 knockout hematopoietic cells had exaggerated IL-1β secretion and paw edema upon administration of MSU crystals. Tet2 knockout macrophages elaborated higher levels of IL-1β in response to MSU crystals in vitro, which was ameliorated through genetic and pharmacologic Nlrp3 inflammasome inhibition. These studies show that TET2-mutant CHIP is associated with an increased risk of gout in humans and that MSU crystals lead to elevated IL-1β levels in Tet2 knockout murine models. We identify CHIP as an amplifier of NLRP3-dependent inflammatory responses to MSU crystals in patients with gout.

Objective The patient’s needs and expectations can be assessed through satisfaction surveys, adverse event declarations and records of complaints. By cross-referencing individual complaints, satisfaction surveys and the adverse events received, we could get valuable information. The objective is to identify common elements of work between these different sources to improve care. Methods A retrospective analysis of patients’ complaints, surveys and adverse events was carried out in order to highlight common improvement items between these three sources of information. Results A satisfaction survey was given to the patients at the end of their treatment, who filled it out and left it in the ‘ad hoc’ letterbox. At the end of December 2019, 4695 questionnaires had been collected (response rate 37%). In addition, since 2014, 1369 patients (∼20 patients per month) have been interviewed ‘face to face’ by the research nurse who assesses their satisfaction using open questions. At the same time, a collection of complaints and adverse events was carried out. All these data have been analysed and cross-checked in order to highlight areas for improvement and strengthen the safety and quality of care in our department. Conclusions Collect and analyse satisfaction surveys, unexpected events and complaints constitute in our opinion, an effective tool to achieve patient empowerment. We aim for the patients to become a real player in their safety and involve in the overall effort to improve the quality of their radiotherapy treatment by reporting what does not meet their expectations.

Neutrophils transit through megakaryocytes in a process termed emperipolesis, but it is unknown whether this interaction is a single type of cell-in-cell interaction or a set of distinct processes. Using a murine in vitro model, we characterized emperipolesis by live-cell spinning disk microscopy and electron microscopy. Approximately half of neutrophils exited the megakaryocyte rapidly, typically in 10 minutes or less, displaying ameboid morphology as they passed through the host cell (fast emperipolesis). The remaining neutrophils assumed a sessile morphology, most remaining within the megakaryocyte for at least 60 minutes (slow emperipolesis). These neutrophils typically localized near the megakaryocyte nucleus. By ultrastructural assessment, all internalized neutrophils remained morphologically intact. Most neutrophils resided within emperisomes, but some could be visualized exiting the emperisome to enter the cell cytoplasm. Neutrophils in the cytoplasm assumed close contact with the platelet-forming demarcation membrane system or the perinuclear endoplasmic reticulum. These findings reveal that megakaryocyte emperipolesis reflects at least 2 distinct processes differing in transit time and morphology, fast and slow emperipolesis, suggesting divergent physiologic functions.

In emperipolesis, neutrophils transit through megakaryocytes, but it is unknown whether this interaction represents a single type of cell-in-cell interaction or a set of distinct processes. Using an in vitro model of murine emperipolesis, we characterized neutrophils entering megakaryocytes using live-cell spinning disk microscopy and electron microscopy. Approximately half of neutrophils exited the megakaryocyte rapidly, typically in 10 minutes or less, displaying ameboid morphology as they passed through the host cell (fast emperipolesis). The remaining neutrophils assumed a sessile morphology, most remaining within the megakaryocyte for at least 60 minutes (slow emperipolesis). These neutrophils typically localized near the megakaryocyte nucleus. By ultrastructural assessment, all internalized neutrophils remained morphologically intact. Most neutrophils resided within emperisomes, but some could be visualized exiting the emperisome into the cell cytoplasm. Neutrophils in the cytoplasm assumed close contact with the platelet-forming demarcation membrane system or with the perinuclear endoplasmic reticulum, as confirmed by immunofluorescence microscopy. Together, these findings reveal that megakaryocyte emperipolesis reflects at least two processes, fast and slow emperipolesis, each with its own characteristic transit time, morphology, and intracellular localization, suggesting distinct functions.Key PointsNeutrophil passage through megakaryocytes, termed emperipolesis, diverges into fast and slow forms that differ in transit time, morphology, and intracellular localizationDuring emperipolesis, neutrophils can reside in vacuoles (emperisomes) or escape into the cell cytoplasm to assume positions near the megakaryocyte’s demarcation membrane system, endoplasmic reticulum, or nucleus.

BackgroundCOVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed.MethodsWe evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays’ performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence.ResultsCombined IgG+IgM sensitivities ranged from 33.9% to 94.6%, while combined specificities ranged from 92.6% to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG+IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG+IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 μg/mL), followed by a similar LOD of 1.5 μg/mL for CareHealth, Cellex, KHB, and Vivachek.ConclusionWe provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Platelets play well-recognized roles in inflammation, but their cell of origin—the megakaryocyte—is not typically considered an immune lineage. Megakaryocytes are large polyploid cells most commonly identified in bone marrow. Egress via sinusoids enables migration to the pulmonary capillary bed, where elaboration of platelets can continue. Beyond receptors involved in hemostasis and thrombosis, megakaryocytes express receptors that confer immune sensing capacity, including TLRs and Fc-γ receptors. They control the proliferation of hematopoietic cells, facilitate neutrophil egress from marrow, possess the capacity to cross-present antigen, and can promote systemic inflammation through microparticles rich in IL-1. Megakaryocytes internalize other hematopoietic lineages, especially neutrophils, in an intriguing cell-in-cell interaction termed emperipolesis. Together, these observations implicate megakaryocytes as direct participants in inflammation and immunity.

Genome-wide association studies (GWAS) have identified many disease-associated noncoding variants, but cannot distinguish functional single-nucleotide polymorphisms (fSNPs) from others that reside incidentally within risk loci. To address this challenge, we developed an unbiased high-throughput screen that employs type IIS enzymatic restriction to identify fSNPs that allelically modulate the binding of regulatory proteins. We coupled this approach, termed SNP-seq, with flanking restriction enhanced pulldown (FREP) to identify regulation of CD40 by three disease-associated fSNPs via four regulatory proteins, RBPJ, RSRC2 and FUBP-1/TRAP150. Applying this approach across 27 loci associated with juvenile idiopathic arthritis, we identified 148 candidate fSNPs, including two that regulate STAT4 via the regulatory proteins SATB2 and H1.2. Together, these findings establish the utility of tandem SNP-seq/FREP to bridge the gap between GWAS and disease mechanism.

Background: Gout is a highly prevalent arthritis associated with debilitating joint pain and functional impairment. It is caused by elevated serum uric acid levels (hyperuricemia) and triggered by precipitation of urate crystals in and around joints. Urate crystals are ingested by macrophages and provoke an innate immune response with subsequent secretion of inflammatory cytokines including interleukin 1 beta (IL-1B). Clonal hematopoiesis of indeterminate potential (CHIP) is a precursor to hematologic malignancies defined by somatic mutations in hematopoietic cells that drive clonal expansion and inflammation. Specifically, CHIP is associated with an increased risk of cardiovascular events and can accelerate atherosclerosis. Mutations in TET2, one of the most commonly mutated genes in CHIP, lead to increased expression of IL-1B through inflammasome activation. Here we investigate the role of CHIP in the development of gout using a combination of human genetic studies and mouse models of CHIP. Methods: To determine the clinical association between CHIP and gout, we analyzed exome sequencing and clinical data from >50,000 individuals included in the UK Biobank (UKB) and Mass General Brigham Biobank (MGBB). To test whether mutant blood cells can promote gout, Tet2- and Dnmt3a-deficient mouse models were used. Results: CHIP was more prevalent in individuals with gout than without gout (MGBB: 12.3% vs. 7.9%, P=0.017; UKB: 8.2% vs. 5.8%, P=0.011) and individuals with CHIP were at increased risk of developing gout (UKB: hazard ratio [HR], 1.59; 95% confidence interval [CI], 1.27-2.00; P Conclusion: CHIP is associated with an increased risk of having and developing gout in human cohorts and distinct mouse models confirm a direct influence of mutant hematopoietic cells on gout-induced inflammation and arthropathy. CHIP may provide a mechanistic explanation for the heterogeneity in clinical symptoms and inflammation due to gout. Our findings substantiate the biologic rationale for interventional strategies directed at CHIP-associated inflammatory conditions beyond cardiovascular disease and thereby define a path for clinical evaluation of targeted therapies for patients with CHIP-positive gout. Disclosures Miller: Foundation Medicine: Consultancy. Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Other: Stock ownership. Natarajan: Amgen: Research Funding; Apple: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Boston Scientific: Research Funding; Blackstone Life Sciences: Consultancy; Genentech: Consultancy; Foresite Labs: Consultancy. Rao: Janssen: Honoraria, Research Funding; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; GlaxoSmithKline: Honoraria; Merck: Honoraria; Scipher Medicine: Honoraria.

The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell–deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1–dependent manner. Transfer of WT but not IL-1–deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1–driven systemic inflammatory disease.

Histology of bone marrow routinely identifies megakaryocytes that enclose neutrophils and other hematopoietic cells, a phenomenon termed emperipolesis. Preserved across mammalian species and enhanced with systemic inflammation and platelet demand, the nature and significance of emperipolesis remain largely unexplored. Recent advances demonstrate that emperipolesis is in fact a distinct form of cell-in-cell interaction. Following integrin-mediated attachment, megakaryocytes and neutrophils both actively drive entry via cytoskeletal rearrangement. Neutrophils enter a vacuole termed the emperisome which then releases them directly into the megakaryocyte cytoplasm. From this surprising location, neutrophils fuse with the demarcation membrane system to pass membrane to circulating platelets, enhancing the efficiency of thrombocytogenesis. Neutrophils then egress intact, carrying megakaryocyte membrane and potentially other cell components along with them. In this review, we summarize what is known about this intriguing cell-in-cell interaction and discuss potential roles for emperipolesis in megakaryocyte, platelet and neutrophil biology.

Diffuse alveolar hemorrhage (DAH) is a life-threatening pulmonary complication associated with systemic lupus erythematosus, vasculitis, and stem cell transplant. Little is known about the pathophysiology of DAH, and no targeted therapy is currently available. Pristane treatment in mice induces systemic autoimmunity and lung hemorrhage that recapitulates hallmark pathologic features of human DAH. Using this experimental model, we performed high-dimensional analysis of lung immune cells in DAH by mass cytometry and single-cell RNA sequencing. We found a large influx of myeloid cells to the lungs in DAH and defined the gene expression profile of infiltrating monocytes. Bone marrow-derived inflammatory monocytes actively migrated to the lungs and homed adjacent to blood vessels. Using 3 models of monocyte deficiency and complementary transfer studies, we established a central role of inflammatory monocytes in the development of DAH. We further found that the myeloid transcription factor interferon regulatory factor 8 is essential to the development of both DAH and type I interferon-dependent autoimmunity. These findings collectively reveal monocytes as a potential treatment target in DAH.

Antibody ligation of the murine neutrophil surface protein Ly6G disrupts neutrophil migration in some contexts but not others. We tested whether this variability reflected divergent dependence of neutrophil migration on β2 integrins, adhesion molecules that interact with Ly6G at the neutrophil surface. In integrin-dependent murine arthritis, Ly6G ligation attenuated joint inflammation, even though mice lacking Ly6G altogether developed arthritis normally. By contrast, Ly6G ligation had no impact on integrin- independent neutrophil migration into inflamed lung. In peritoneum, the role of β2 integrins varied with stimulus, proving dispensable for neutrophil entry in E. coli peritonitis but contributory in IL-1-mediated sterile peritonitis. Correspondingly, Ly6G ligation attenuated only IL-1 peritonitis, disrupting the molecular association between integrins and Ly6G and inducing cell-intrinsic blockade restricted to integrin-dependent migration. Consistent with this observation, Ly6G ligation impaired integrin-mediated postadhesion strengthening for neutrophils arresting on activated cremaster endothelium in vivo. Together, these findings identify selective inhibition of integrin- mediated neutrophil emigration through Ly6G ligation, highlighting the marked site and stimulus specificity of β2 integrin dependence in neutrophil migration.KEY POINTSThe contribution of β2 integrins to neutrophil migration into inflamed tissues varies with site and stimulus.Ligation of Ly6G, a GPI-linked neutrophil surface protein, selectively attenuates β2 integrin-dependent neutrophil migration in vivo.Blockade correlates with disrupted interaction between Ly6G and β2 integrins and impaired integrin-mediated postadhesion strengthening.

SummaryBone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the β2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated marrowin vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of inflammation associated with platelet overproduction, contributing to platelet productionin vitroandin vivo.These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.

Bone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the β2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated murine marrow in vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of murine inflammation associated with platelet overproduction, contributing to platelet production in vitro and in vivo. These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.

Monocytes are derived from hematopoietic stem cells through a series of intermediate progenitor stages, but the factors that regulate this process are incompletely defined. Using a Ccr2/Cx(3)cr1 dual-reporter system to model murine monocyte ontogeny, we conducted a small molecule screen that identified an essential role of mechanistic target of rapamycin complex 1 (mTORC1) in the development of monocytes and other myeloid cells. Confirmatory studies using mice with inducible deletion of the mTORC1 component Raptor demonstrated absence of mature circulating monocytes, as well as disruption in neutrophil and dendritic cell development, reflecting arrest of terminal differentiation at the granulocyte-monocyte progenitor (GMP) stage. Conversely, excess activation of mTORC1 through deletion of the mTORC1 inhibitor tuberous sclerosis complex 2 (Tsc2) promoted spontaneous myeloid cell development and maturation. Inhibitor studies and stage-specific expression profiling identified failure to downregulate the transcription factor Myc by the mTORC1 target ribosomal S6 kinase 1 (S6K1) as the mechanistic basis for disrupted myelopoiesis. Together, these findings define the mTORC1-S6K1-Myc pathway as a key checkpoint in terminal myeloid development.

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177pos and CD177neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration.

Understanding the implications of genome-wide association studies (GWAS) for disease biology requires both identification of causal variants and definition of how these variants alter gene function. The non-coding triallelic dinucleotide polymorphism CCR6DNP is associated with risk for rheumatoid arthritis, and is considered likely causal because allelic variation correlates with expression of the chemokine receptor CCR6. Using transcription activator-like effector nuclease (TALEN) gene editing, we confirmed that CCR6DNP regulates CCR6. To identify the associated transcription factor, we applied a novel assay, Flanking Restriction Enhanced Pulldown (FREP), to identify specific association of poly (ADP-ribose) polymerase 1 (PARP-1) with CCR6DNP consistent with the established allelic risk hierarchy. Correspondingly, manipulation of PARP-1 expression or activity impaired CCR6 expression in several lineages. These findings show that CCR6DNP is a causal variant through which PARP-1 regulates CCR6, and introduce a highly efficient approach to interrogate non-coding genetic polymorphisms associated with human disease., Author Summary Genome-wide association studies (GWAS) identify loci associated with human disease risk, but bridging the gap between locus and mechanism has proven particularly difficult in cases where associated variants do not alter coding. We aimed to develop a generalizable approach to this problem. Previously, a dual nucleotide polymorphism within the first intron of CCR6 (termed the CCR6DNP) had been associated with risk for rheumatoid arthritis, but the pathway by which this variant altered gene expression could not be determined. Here, we employed sequence perturbation to confirm a regulatory role for the CCR6DNP. Next, using a new technique termed Flanking Restriction Enhanced Pulldown (FREP), we identified PARP-1 as the protein that regulates CCR6 expression through allelic association with the CCR6DNP, a finding confirmed by chromatin immunoprecipitation and functional assays. These findings reveal an unexpected regulatory pathway for CCR6 implicated in rheumatoid arthritis and other disease by human genetics, and more generally introduce a novel approach to identifying regulatory protein-DNA interactions.

Increasing evidence suggests that neutrophils may participate in the regulation of adaptive immune responses, and can reach draining lymph nodes and cross-prime naive T cells. The aim of this study was to identify the mechanism(s) involved in the migration of neutrophils to the draining lymph nodes. We demonstrate that a subpopulation of human and mouse neutrophils express CCR7. CCR7 is rapidly expressed at the membrane upon stimulation. In vitro, stimulated human neutrophils migrate in response to the CCR7 ligands CCL19 and CCL21. In vivo, injection of complete Freund adjuvant induces a rapid recruitment of neutrophils to the lymph nodes in wild-type mice but not in Ccr7−/− mice. Moreover, intradermally injected interleukin-17–and granulocyte-macrophage colony-stimulating factor–stimulated neutrophils from wild-type mice, but not from Ccr7−/− mice, migrate to the draining lymph nodes. These results identify CCR7 as a chemokine receptor involved in the migration of neutrophils to the lymph nodes.

The nervous system influences immune responses through the release of neural factors such as neuropeptides. Among them, the tachykinin substance P (SP) signals via the neurokinin 1 receptor (NK-1R), which is expressed by various immune cells. We thereby analyzed in this paper whether tachykinins may participate in human CD4+ Th cell polarization. We report that SP and hemokinin-1 (HK-1) upregulate IL-17A and IFN-γ production by human memory CD4+ T cells without affecting IL-4 and IL-10 production. SP and HK-1 switch non–Th17-committed CD4+ memory T cells into bona fide Th17 cells and Th1/Th17 cells. In contrast, SP and HK-1 do not modulate the polarization of naive CD4+ T cells. SP- and HK-1–induced Th17 cell generation is mediated through NK-1R and requires the presence of monocytes. SP and HK-1 trigger IL-1β, IL-6, and TNF-α production, upregulate IL-23 production, and enhance TNF-like 1A expression on monocyte surface. Neutralization experiments demonstrated that IL-1β, IL-23, and TNF-like 1A are involved in the SP- and HK-1–induced Th17 cell. The other members of the tachykinin family, neurokinins A and B, have no effect on the differentiation of naive and memory T cells. These results thereby show that SP and HK-1 are novel Th17 cell-inducing factors that may act locally on memory T cells to amplify inflammatory responses.

Background Mast cells are joint-resident immune sentinels that have been implicated in the pathogenesis of experimental arthritis, through reconstitution experiments in mast cell-deficient W/Wv animals as well as in studies using mice lacking mast cell-specific proteins. This work has identified mast cells as a key source of IL-1 and of specific pro-inflammatory proteases. Yet certain mast cell-deficient strains, such as Wsh, remain susceptible to disease. This discrepancy contributes to uncertainty about the role of mast cells in arthritis. Objectives We sought to define factors mediating differential susceptibility to K/BxN serum transfer arthritis in mast cell-deficient W/Wv and Wsh mice. Methods We assessed the sensitivity of Wsh mice to titered doses of arthritogenic K/BxN serum, and tested whether hematopoietic aberrancy in this strain contributed to disease susceptibility. We then employed criss-cross bone marrow transplantation and adoptive transfer studies to define the contribution of hematopoietic and non-hematopoietic factors to differential arthritis susceptibility in W/Wv and Wsh mice. Results Wsh mice were susceptible to full-dose K/BxN arthritis but exhibited partial resistance at submaximal serum doses. We could identify no baseline pro-inflammatory state in this strain that might obviate the need for mast cells. Lethally irradiated Wsh transplanted with W/Wv marrow became resistant to arthritis, while irradiated W/Wv recipients of either Wsh or wild-type (WT) marrow became susceptible, confirming a primary role for the hematopoietic system in arthritis susceptibility and showing that repair of arthritis resistance in W/Wv mice does not require mast cells. Interestingly, transplanted W/Wv showed lower intensity of disease than either Wsh or WT donors, suggesting a contributory role for non-hematopoietic tissues to W/Wv arthritis resistance. To identify the hematopoietic lineage capable of “patching” arthritis resistance in W/Wv, we employed adoptive transfer. Whole marrow restored arthritis susceptibility to unirradiated W/Wv within 2 weeks, but IL-1-/- marrow could not, implicating a rapidly-engrafting IL-1-producing lineage. Surprisingly, adoptive transfer of neutrophils did not correct resistance, while GFI-1-/- marrow incapable of generating neutrophils could do so, excluding neutrophils as the IL-1 source. Studies to define the responsible lineage are in progress. Conclusions Arthritis susceptibility in W/Wv and Wsh mice is a function of the K/BxN serum dose and of both hematopoietic and non-hematopoietic factors. Engraftment studies implicate a non-neutrophil hematopoietic source for the IL-1 normally contributed by mast cells to early joint inflammation. These data support the concept that mast cells are immune amplifiers whose role in arthritis varies with the strength of the arthritogenic stimulus and with a complex variety of strain-specific factors that underlie disease susceptibility. Disclosure of Interest None Declared