A Novel Transmembrane CXCR4 Variant That Expands the WHIM Genotype-Phenotype Paradigm.

Warts, Hypogammglobulinemia, Infections, Myelokathexis (WHIM) syndrome is a rare immunodeficiency disease that results from impaired leukocyte trafficking (myelokathexis) predominately caused by gain-of-function variants in C-X-C chemokine receptor type 4 (CXCR4). Clinical manifestations of WHIM syndrome can differ in familial forms or in people harboring identical CXCR4 variants. All known pathogenic CXCR4 variants associated with WHIM syndrome (CXCR4WHIM) to date are localized in the intracellular C-terminus of CXCR4. We identified 4 unrelated patients with variable WHIM-like clinical presentations harboring a novel heterozygous CXCR4 variant (c.250G>C; p.D84H) localized at a highly conserved position in the transmembrane domain of the receptor outside the C-terminus. Functional characterization of the CXCR4D84Hvariant (CXCR4D84H) using patient-derived peripheral blood mononuclear cells and in vitro cellular assaysshow decreased CXCR4 internalization and increased chemotaxis in response to CXCL12, similar to known CXCR4WHIM, but also revealed unique features of CXCR4D84H signaling to cAMP, Ca2+ mobilization and AKT/ERK pathways. These findings are consistent with molecular dynamics simulations that show disruption of the Na+ binding pocket by D84H, resulting in collapse of the hydrophobic gate above and destabilization of the inactive state of CXCR4. Mavorixafor, a CXCR4 antagonist being evaluated in clinical trials for chronic neutropenia and WHIM syndrome, normalized CXCL12-mediated chemotaxis of CXCR4D84H patient lymphocytes ex vivo and improved WBC and subset counts in 1 patient with CXCR4D84H enrolled in the chronic neutropenia phase 1b clinical trial (NCT04154488). The present study expands the current understanding of CXCR4 function and genotype-phenotype correlations in WHIM syndrome and in people with WHIM-like phenotypes.

Phase 3 randomized trial of mavorixafor, CXCR4 antagonist, in WHIM syndrome.

We investigated efficacy and safety of mavorixafor, an oral CXCR4 antagonist for participants with Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome, a rare immunodeficiency caused by CXCR4 gain-of-function variants. This randomized (1:1), double-blind, placebo-controlled, phase 3 trial enrolled participants aged ≥12 years with WHIM syndrome and absolute neutrophil count (ANC) ≤400/µL. Participants received once-daily mavorixafor or placebo for 52 weeks. Primary endpoint was time (hours) above ANC threshold ≥500/µL (TATANC; over 24 hours). Secondary endpoints included TAT absolute lymphocyte count ≥1000/µL (TATALC; defined similar to TATANC); absolute changes in white blood cell (WBC), ANC, and ALC from baseline; annualized infection rate; infection duration and total infection score (combined infection number/severity). In 31 participants (mavorixafor, n=14; placebo, n=17), mavorixafor least squares (LS) mean TATANC was 15.0 hours, placebo 2.8 hours (P<0.001). Mavorixafor LS mean TATALC was 15.8 hours, placebo 4.6 hours (P<0.001). Higher absolute WBC, ANC, and ALC levels were seen with mavorixafor than placebo at each timepoint assessed. Annualized infection rates were 60% lower with mavorixafor versus placebo (LS mean 1.7 versus 4.2; nominal P=0.007) and total infection scores were 40% lower (7.4 [95% CI, 1.6-13.2] versus 12.3 [95% CI, 7.2-17.3]). Treatment with mavorixafor reduced infection frequency, severity, duration, and antibiotic use. No discontinuations occurred due to treatment-emergent adverse events (TEAEs); no related serious TEAEs were observed. Overall, mavorixafor-treated participants showed significant increases in LS mean TATANC and TATALC, reduced infection frequency, severity/duration. Mavorixafor was well tolerated in participants with WHIM syndrome. Trial was registered at ClinicalTrials.gov NCT03995108.

Treatment with Elapegademase Restores Immunity in Infants with Adenosine Deaminase Deficient Severe Combined Immunodeficiency.

PURPOSE: Patients with adenosine deaminase 1 deficient severe combined immunodeficiency (ADA-SCID) are initially treated with enzyme replacement therapy (ERT) with polyethylene glycol-modified (PEGylated) ADA while awaiting definitive treatment with hematopoietic stem cell transplant (HSCT) or gene therapy. Beginning in 1990, ERT was performed with PEGylated bovine intestinal ADA (ADAGEN®). In 2019, a PEGylated recombinant bovine ADA (Revcovi®) replaced ADAGEN following studies in older patients previously treated with ADAGEN for many years. There are limited longitudinal data on ERT-naïve newborns treated with Revcovi. METHODS: We report our clinical experience with Revcovi as initial bridge therapy in three newly diagnosed infants with ADA-SCID, along with comprehensive biochemical and immunologic data. RESULTS: Revcovi was initiated at twice weekly dosing (0.2 mg/kg intramuscularly), and monitored by following plasma ADA activity and the concentration of total deoxyadenosine nucleotides (dAXP) in erythrocytes. All patients rapidly achieved a biochemically effective level of plasma ADA activity, and red cell dAXP were eliminated within 2-3 months. Two patients reconstituted B-cells and NK-cells within the first month of ERT, followed by naive T-cells one month later. The third patient reconstituted all lymphocyte subsets within the first month of ERT. One patient experienced declining lymphocyte counts with improvement following Revcovi dose escalation. Two patients developed early, self-resolving thrombocytosis, but no thromboembolic events occurred. CONCLUSION: Revcovi was safe and effective as initial therapy to restore immune function in these newly diagnosed infants with ADA-SCID, however, time course and degree of reconstitution varied. Revcovi dose may need to be optimized based on immune reconstitution, clinical status, and biochemical data.

Aged G Protein-Coupled Receptor Kinase 3 (Grk3)-Deficient Mice Exhibit Enhanced Osteoclastogenesis and Develop Bone Lesions Analogous to Human Paget's Disease of Bone.

Paget's Disease of Bone (PDB) is a metabolic bone disease that is characterized by dysregulated osteoclast function leading to focal abnormalities of bone remodeling. It can lead to pain, fracture, and bone deformity. G protein-coupled receptor kinase 3 (GRK3) is an important negative regulator of G protein-coupled receptor (GPCR) signaling. GRK3 is known to regulate GPCR function in osteoblasts and preosteoblasts, but its regulatory function in osteoclasts is not well defined. Here, we report that Grk3 expression increases during osteoclast differentiation in both human and mouse primary cells and established cell lines. We also show that aged mice deficient in Grk3 develop bone lesions similar to those seen in human PDB and other Paget's Disease mouse models. We show that a deficiency in Grk3 expression enhances osteoclastogenesis in vitro and proliferation of hematopoietic osteoclast precursors in vivo but does not affect the osteoclast-mediated bone resorption function or cellular senescence pathway. Notably, we also observe decreased Grk3 expression in peripheral blood mononuclear cells of patients with PDB compared with age- and gender-matched healthy controls. Our data suggest that GRK3 has relevance to the regulation of osteoclast differentiation and that it may have relevance to the pathogenesis of PDB and other metabolic bone diseases associated with osteoclast activation.

Combinatorial depletions of G-protein coupled receptor kinases in immune cells identify pleiotropic and cell type-specific functions.

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts, which eventually blocks signal propagation and often induces receptor internalization. However, there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2, GRK3, GRK5, GRK6) simultaneously, but we have very limited knowledge about their interplay in primary immune cells. In particular, we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type, and (b) one specific GPCR between several immune cell subsets. To address this issue, we generated mouse models of single, combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils, T cells, B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes, many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands, whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types, and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly, we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis, which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together, our findings demonstrate the complexity of GRK functions in immune cells, which go beyond GPCR desensitization in specific leukocyte types. Furthermore, they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset.

The Role of T Helper Type 2 (Th2) Cytokines in the Pathogenesis of Eosinophilic Granulomatosis with Polyangiitis (eGPA): an Illustrative Case and Discussion.

PURPOSEOF REVIEW: The pathogenesis of eosinophilic granulomatosis with polyangiitis (eGPA) is driven largely by CD4 + type 2 helper T cells (Th2), B cells, and eosinophils. Interleukin (IL)-4 and IL-13 are critical cytokines in Th2 cell-mediated inflammation; however, inhibition of IL-4 and IL-13 does not reduce serum eosinophil counts and has even been associated with hypereosinophilia. This review explores the role of IL-4, IL-5, and IL-13 in Th2-mediated inflammation to consider the potential clinical consequences of inhibiting these individual cytokines in eGPA. RECENT FINDINGS: Treatments for eosinophilic granulomatosis with polyangiitis (eGPA) are rapidly evolving through using biologic therapies to modulate the Th2 inflammatory response via eosinophil inhibition. While IL-4, IL-5, IL-13, and IL-25 can all affect eosinophils, only IL-5 inhibition has demonstrated therapeutic benefit to-date. In this review, we report a clinical vignette of a patient with adult-onset asthma who developed severe manifestations of eGPA after switching from mepolizumab (an IL-5 inhibitor) to dupilumab (an inhibitor of IL-4 and IL-13). By understanding the role of IL-4, IL-5, and IL-13 in Th2-mediated vasculitis, we can start to understand how eGPA might respond differently to focused cytokine inhibition.

Light-Triggered Drug Release from Red Blood Cells Suppresses Arthritic Inflammation.

Arthritis is a leading cause of disability in adults, which can be intensely incapacitating. The location and intensity of the pain is both subjective and challenging to manage. Consequently, patient-directed delivery of anti-inflammatories is an essential component of future therapeutic strategies for the management of this disorder. We describe the design and application of a light responsive red blood cell (RBC) conveyed dexamethasone (Dex) construct that enables targeted drug delivery upon illumination of the inflamed site. The red wavelength (650 nm) responsive nature of the phototherapeutic was validated using tissue phantoms mimicking the light absorbing properties of various skin types. Furthermore, photoreleased Dex has the same impact on cellular responses as conventional Dex. Murine RBCs containing the photoactivatable therapeutic display comparable circulation properties as fluorescently labelled RBCs. In addition, a single dose of light-targeted Dex delivery is 5-fold more effective in suppressing inflammation than the parent drug, delivered serially over multiple days. These results are consistent with the notion that the circulatory system be used as an on-command drug depot, providing the means to therapeutically target diseased sites both efficiently and effectively.

Practical guidance for the diagnosis and management of secondary hypogammaglobulinemia: A Work Group Report of the AAAAI Primary Immunodeficiency and Altered Immune Response Committees.

Secondary hypogammaglobulinemia (SHG) is characterized by reduced immunoglobulin levels due to acquired causes of decreased antibody production or increased antibody loss. Clarification regarding whether the hypogammaglobulinemia is secondary or primary is important because this has implications for evaluation and management. Prior receipt of immunosuppressive medications and/or presence of conditions associated with SHG development, including protein loss syndromes, are histories that raise suspicion for SHG. In patients with these histories, a thorough investigation of potential etiologies of SHG reviewed in this report is needed to devise an effective treatment plan focused on removal of iatrogenic causes (eg, discontinuation of an offending drug) or treatment of the underlying condition (eg, management of nephrotic syndrome). When iatrogenic causes cannot be removed or underlying conditions cannot be reversed, therapeutic options are not clearly delineated but include heightened monitoring for clinical infections, supportive antimicrobials, and in some cases, immunoglobulin replacement therapy. This report serves to summarize the existing literature regarding immunosuppressive medications and populations (autoimmune, neurologic, hematologic/oncologic, pulmonary, posttransplant, protein-losing) associated with SHG and highlights key areas for future investigation.

G protein-coupled receptor kinase 3 modulates mesenchymal stem cell proliferation and differentiation through sphingosine-1-phosphate receptor regulation.

BACKGROUND: The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling. METHODS: Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures. RESULTS: GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of ß-arrestin-2 in sphingosine-1-phosphate receptor internalization. CONCLUSIONS: Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting ß-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.

eConsults' Impact on Care Access and Wait Times in Rheumatology.

BACKGROUND/OBJECTIVE: A growing number of health systems have implemented eConsults to improve access to specialty advice, but few studies have described their use in rheumatology or impact on visit wait times. We evaluated the uptake of an eConsult program and its impact on wait times for in-person rheumatology visits. METHODS: In this quality improvement project, we analyzed electronic health record data from 4 intervention clinics and 4 comparison clinics, 12 months before and after implementation of an eConsult program. We compared median wait time for rheumatology appointments using a pre-post difference-in-differences analysis and quantile regression, adjusting for patient age, race, sex, clinic pair, and primary insurance payer. We also interviewed 11 primary care providers from the intervention clinics and conducted a rheumatology provider focus group (n = 4) to elucidate experiences with the program. RESULTS: Rheumatologists recommended management in primary care or referral to another specialty for 41% of eConsults, reducing initial demand for in-person visits. The median wait times dropped in the intervention and the comparison clinics (42 and 25 days, respectively). Intervention clinic median wait time dropped 17 days more than comparison clinics, and this was nonstatistically significant (p = 0.089). eConsults fit provider care tasks best for triage or initial workup for diagnosis, and less well when tests required interpretation, or when back and forth communication was needed to manage the patient's condition. CONCLUSIONS: Implementation of eConsults for rheumatology was associated with reduced wait times for rheumatology appointments and supported primary care providers in the triage and workup for a substantial portion of patients.

Neutrophils self-limit swarming to contain bacterial growth in vivo.

Neutrophils communicate with each other to form swarms in infected organs. Coordination of this population response is critical for the elimination of bacteria and fungi. Using transgenic mice, we found that neutrophils have evolved an intrinsic mechanism to self-limit swarming and avoid uncontrolled aggregation during inflammation. G protein-coupled receptor (GPCR) desensitization acts as a negative feedback control to stop migration of neutrophils when they sense high concentrations of self-secreted attractants that initially amplify swarming. Interference with this process allows neutrophils to scan larger tissue areas for microbes. Unexpectedly, this does not benefit bacterial clearance as containment of proliferating bacteria by neutrophil clusters becomes impeded. Our data reveal how autosignaling stops self-organized swarming behavior and how the finely tuned balance of neutrophil chemotaxis and arrest counteracts bacterial escape.

Paget's Disease of Bone: Osteoimmunology and Osteoclast Pathology.

PURPOSE OF REVIEW: The purpose of this review is to recognize clinical features of Paget's disease of bone and to describe how the osteoclast, a myeloid-derived cell responsible for bone resorption, contributes to the disease. RECENT FINDINGS: Recent studies have identified several variants in SQSTM1, OPTN, and other genes that may predispose individuals to Paget's disease of bone; studies of these genes and their protein products have elucidated new roles for these proteins in bone physiology. Understanding the pathologic mechanisms in the Pagetic osteoclast may lead to the identification of future treatment targets for other inflammatory and autoimmune diseases characterized by abnormal bone erosion and/or osteoclast activation.

A Mimic of Ankylosing Spondylitis, Ochronosis: Case Report and Review of the Literature.

PURPOSE OF REVIEW: Ochronosis and alkaptonuria are manifestations of the same condition-a rare autosomal recessive disorder resulting from a constitutional lack of homogentisate 1,2-dioxygenase (HGD) with the consequent accumulation of homogentisic acid (HGA). In ochronosis, HGA undergoes autoxidation as well as enzymatic oxidation to form an ochronotic pigment that accumulates in cartilage and connective tissues. In the beginning, there is homogentisic aciduria and pigmentation of cartilages and other connective tissues. In later years, generalized osteoarthritis of the spine and large joints, termed ochronotic arthropathy, develops. RECENT FINDINGS: The diagnosis is confirmed by quantitative measurement of HGA in urine and mutation analysis of the HGD gene. One of the differential diagnoses for the skin findings is exogenous ochronosis, a limited hyperpigmentation of skin caused by some chemicals. As for the lumbar spine findings, there can be radiographic similarities with ankylosing spondylitis (AS) including reduced intervertebral disc spaces and loss of lumbar lordosis; however, ochronosis will spare the sacroiliac joint, and the lumbar spine will show dense, wafer-like disk calcification with a vacuum disc phenomenon and broad syndesmophytes. Here, we present a case of a patient with probable ochronosis that was treated many years as ankylosing spondylitis without response, and we provide a review of the current literature on ochronosis pathogenesis, diagnosis, and treatment.

Hydroxychloroquine and COVID-19: a Rheumatologist's Take on the Lessons Learned.

PURPOSE OF REVIEW: Told from the viewpoint of rheumatologists, this review tells the story of hydroxychloroquine and its swift ascent to become a household name as a therapeutic strategy against the novel SARS-CoV-2 virus. This review describes the history, mechanisms, pharmacokinetics, therapeutic applications, and safety profile of hydroxychloroquine as an immunomodulatory and antiviral agent. It also summarizes the major studies that launched and assessed the use of hydroxychloroquine against COVID-19 infection. RECENT FINDINGS: More recent literature calls into question the long-held dogma that endolysosomal alkalinization is the primary mode of action of hydroxychloroquine. Ongoing uncertainty about the multiple potential mechanisms contributing to the therapeutic effect of hydroxychloroquine in rheumatic and viral disease led to a natural avenue for exploration in the treatment of COVID-19. Taken as a whole, the literature does not support utilizing hydroxychloroquine to treat or prevent infection from the SARS-CoV-2 virus. This is, at least in part, due to the wide variability in hydroxychloroquine pharmacokinetics between patients and difficulty achieving adequate target tissue concentrations of hydroxychloroquine without encountering unacceptable toxicities. Hydroxychloroquine continues to be a routinely prescribed, well-tolerated, effective, and low-cost treatment for rheumatic disease. Its therapeutic versatility has led to frequent repurposing for other conditions, most recently as an investigative treatment against the SARS-CoV-2 virus. Despite overall negative findings, the intense study of hydroxychloroquine against COVID-19 infection has enhanced our overall understanding of how hydroxychloroquine operates in autoimmune disease and beyond.

Taking phototherapeutics from concept to clinical launch.

More than four decades have passed since the first example of a light-activated (caged) compound was described. In the intervening years, a large number of light-responsive derivatives have been reported, several of which have found utility under a variety of in vitro conditions using cells and tissues. Light-triggered bioactivity furnishes spatial and temporal control, and offers the possibility of precision dosing and orthogonal communication with different biomolecules. These inherent attributes of light have been advocated as advantageous for the delivery and/or activation of drugs at diseased sites for a variety of indications. However, the tissue penetrance of light is profoundly wavelength-dependent. Only recently have phototherapeutics that are photoresponsive in the optical window of tissue (600-900 nm) been described. This Review highlights these recent discoveries, along with their limitations and clinical opportunities. In addition, we describe preliminary in vivo studies of prospective phototherapeutics, with an emphasis on the path that remains to be navigated in order to translate light-activated drugs into clinically useful therapeutics. Finally, the unique attributes of phototherapeutics is highlighted by discussing several potential disease applications.

Mesenchymal stromal cells reprogram monocytes and macrophages with processing bodies.

Mesenchymal stromal cells (MSCs) are widely used in clinical trials because of their ability to modulate inflammation. The success of MSCs has been variable over 25 years, most likely due to an incomplete understanding of their mechanism. After MSCs are injected, they traffic to the lungs and other tissues where they are rapidly cleared. Despite being cleared, MSCs suppress the inflammatory response in the long term. Using human cord tissue-derived MSCs (hCT-MSCs), we demonstrated that hCT-MSCs directly interact and reprogram monocytes and macrophages. After engaging hCT-MSCs, monocytes and macrophages engulfed cytoplasmic components of live hCT-MSCs, then downregulated gene programs for antigen presentation and costimulation, and functionally suppressed the activation of helper T cells. We determined that low-density lipoprotein receptor-related proteins on monocytes and macrophages mediated the engulfment of hCT-MSCs. Since a large amount of cellular information can be packaged in cytoplasmic RNA processing bodies (p-bodies), we generated p-body deficient hCT-MSCs and confirmed that they failed to reprogram monocytes and macrophages in vitro and in vivo. hCT-MSCs suppressed an inflammatory response caused by a nasal lipopolysaccharide challenge. Although both control and p-body deficient hCT-MSCs were engulfed by infiltrating lung monocytes and macrophages, p-body deficient hCT-MSCs failed to suppress inflammation and downregulate MHC-II. Overall, we identified a novel mechanism by which hCT-MSCs indirectly suppressed a T-cell response by directly interacting and reprogramming monocytes and macrophages via p-bodies. The results of this study suggest a novel mechanism for how MSCs can reprogram the inflammatory response and have long-term effects to suppress inflammation.

Diagnostic interpretation of genetic studies in patients with primary immunodeficiency diseases: A working group report of the Primary Immunodeficiency Diseases Committee of the American Academy of Allergy, Asthma & Immunology.

Genetic testing has become an integral component of the diagnostic evaluation of patients with suspected primary immunodeficiency diseases. Results of genetic testing can have a profound effect on clinical management decisions. Therefore clinical providers must demonstrate proficiency in interpreting genetic data. Because of the need for increased knowledge regarding this practice, the American Academy of Allergy, Asthma & Immunology Primary Immunodeficiency Diseases Committee established a work group that reviewed and summarized information concerning appropriate methods, tools, and resources for evaluating variants identified by genetic testing. Strengths and limitations of tests frequently ordered by clinicians were examined. Summary statements and tables were then developed to guide the interpretation process. Finally, the need for research and collaboration was emphasized. Greater understanding of these important concepts will improve the diagnosis and management of patients with suspected primary immunodeficiency diseases.

On Command Drug Delivery via Cell-Conveyed Phototherapeutics.

Herein, the use of red blood cells (RBCs) as carriers of cytoplasmically interned phototherapeutic agents is described. Photolysis promotes drug release from the RBC carrier thereby providing the means to target specific diseased sites. This strategy is realized with a vitamin B12-taxane conjugate (B12-TAX), in which the drug is linked to the vitamin via a photolabile CoC bond. The conjugate is introduced into mouse RBCs (mRBCs) via a pore-forming/pore-resealing procedure and is cytoplasmically retained due to the membrane impermeability of B12. Photolysis separates the taxane from the B12 cytoplasmic anchor, enabling the drug to exit the RBC carrier. A covalently appended Cy5 antenna sensitizes the conjugate (Cy5-B12-TAX) to far red light, thereby circumventing the intense light absorbing properties of hemoglobin (350-600 nm). Microscopy and imaging flow cytometry reveal that Cy5-B12-TAX-loaded mRBCs act as drug carriers. Furthermore, intravital imaging of mice furnish a real time assessment of circulating phototherapeutic-loaded mRBCs as well as evidence of the targeted photorelease of the taxane upon photolysis. Histopathology confirms that drug release occurs in a well resolved spatiotemporal fashion. Finally, acoustic angiography is employed to assess the consequences of taxane release at the tumor site in Nu/Nu-tumor-bearing mice.

Chemerin-activated functions of CMKLR1 are regulated by G protein-coupled receptor kinase 6 (GRK6) and ß-arrestin 2 in inflammatory macrophages.

Chemerin receptor (CMKLR1) is a G protein-coupled receptor (GPCR) implicated in macrophage-mediated inflammation and in several forms of human arthritis. Analogous to other GPCR, CMKLR1 is likely regulated by G protein-coupled receptor kinase (GRK) phosphorylation of intracellular domains in an activation-dependent manner, which leads to recruitment and termination of intracellular signaling via desensitization and internalization of the receptor. The ubiquitously expressed GRK family members include GRK2, GRK3, GRK5, and GRK6, but it is unknown which GRK regulates CMKLR1 cellular and signaling functions. Our data show that activation of CMKLR1 by chemerin in primary macrophages leads to signaling and functional outcomes that are regulated by GRK6 and ß-arrestin 2. We show that arrestin recruitment to CMKLR1 following chemerin stimulation is enhanced with co-expression of GRK6. Further, internalization of endogenous CMKLR1, following the addition of chemerin, is decreased in inflammatory macrophages from GRK6- and ß-arrestin 2-deficient mice. These GRK6- and ß-arrestin 2-deficient macrophages display increased migration toward chemerin and altered AKT and Extracellular-signal Related Kinase (ERK) signaling. Our findings show that chemerin-activated CMKLR1 regulation in inflammatory macrophages is largely GRK6 and ß-arrestin mediated, which may impact innate immunity and have therapeutic implications in rheumatic disease.