Chemokine Receptor CXCR4 Research Articles

Phase 1 Results of the First-in-Class CXCR1/2 Inhibitor SX-682 in Patients with Hypomethylating Agent Failure Myelodysplastic Syndromes

Background: SX-682 is an oral dual allosteric inhibitor of the CXCR1 and CXCR2 (CXCR1/2) chemokine receptors, a signaling pathway involved in the recruitment of immunosuppressive MDSCs and autocrine growth of disease-initiating leukemic stem cells (LSCs) in AML and myelodysplastic syndromes (MDS). CXCR2 overexpression is predictive of transfusion dependence in MDS. CXCR2 inhibition reduces proliferation of leukemic cell lines and primary MDS/AML patient (pt) samples in vitro and impairs leukemic growth in an in vivo AML animal model. CXCR2 is also involved in normal neutrophil egress from the bone marrow and thus absolute neutrophil count (ANC) serves as a pharmacodynamic (PD) marker for CXCR1/2 inhibition by SX-682. In pts with normal bone marrow, 25-400 mg SX-682 twice-daily (BID) dose-dependently reduces ANC, with maximum ANC reduction (-66% of baseline) obtained at >150 mg BID, consistent with maximum CXCR1/2 inhibition above this dose. ANC rapidly (24 hrs) normalizes with dose-holding. This NHLBI funded trial (HL142389, NCT04245397) explored the safety and efficacy of SX-682 monotherapy in pts with hypomethylating agent (HMA) failure MDS. Methods: This Phase 1 dose-escalation with expansion study treated five cohorts of HMA failure MDS pts with escalating 25-400 mg BID doses of SX-682 in six continuous 28-day cycles with responding pts continuing treatment. Steady-state pharmacokinetics (PK) were obtained. Response assessment was at the end of cycles 1, 3 and 6 and every third cycle thereafter using IWG criteria (Cheson 2006). This interim analysis after dose-escalation to select the expansion dose was pre-specified. Results:17 MDS pts received SX-682 (median 1 prior therapies; range 1-4) with 6 pts in the 200 mg cohort, 3 pts in each of the 25, 50 and 100 cohorts and 2 pts in the 400 mg cohort (Table). Median age was 76, and pts were low (n=1), intermediate-1 (n=10), intermediate-2 (n=5) and high (n=1) risk by the International Prognostic Scoring System (IPSS). All pts were transfusion-dependent and all had failed prior HMA and 24% lenalidomide. Steady-state PK exhibits increasing drug plasma concentration with dose comparable to pts with solid tumors. The ANC PD marker exhibited a dose-dependent decrease from baseline (ΔANC) that plateaued in the 200 and 400 mg dose cohorts consistent with maximum receptor inhibition (Table). Marrow MDSCs and LSCs were reduced after initiation of SX-682. SX-682 was well-tolerated with no maximally tolerated dose and no pt discontinued treatment for adverse events. Treatment-emergent adverse events > grade 3 were most common in the 200 and 400 mg dose cohorts, and those related to neutrophils are consistent with being related to SX-682. Effects on neutrophils reversed on cessation of drug dosing. There was a dose-dependent increase in overall response rate (ORR) from 0% at 25 mg BID to 50% at 200 mg BID consistent with the observed clinical benefit being related to CXCR1/2 inhibition by SX-682 (Table). Four of the five responders had mutant genes (MGs) involving splicing factors (60% of responders; SF3B1, SRSF2) and/or epigenetic regulation (60% of responders; TET2, ASXL1, IDH2). Across all dose cohorts, 8 of 17 (47%) pts had a reduction in marrow blasts after initiating SX-682 (Figure). The 200 mg dose yielded the most rapid and deep reduction in blasts with 2 of 6 pts (33%) achieving a marrow complete remission (mCR) in keeping with this dose providing maximum receptor inhibition. There was hematologic improvement (HI) in 3 pts with one having a durable near-CR response (on SX-682 >500 days) consisting of trilineage HI (hemoglobin > 10 g/dL) and transfusion independence for over 4 months (pt required 16 transfusions in the 7 weeks before starting SX-682 therapy). Based on ANC PD, ORR and marrow blast responses, the 200 mg BID dose was selected for the expansion phase of the trial. Conclusions: SX-682 is a novel therapy in HMA failure MDS with dual activity against LSCs and MDSCs. SX-682 was well tolerated with encouraging efficacy with a biologically effective oral dose selected at 200 mg BID. An expansion cohort in both lower and higher risk MDS pts is ongoing and will additionally include HMA naïve patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

IMMU-21. GLIOMA-DERIVED FACTORS RECRUIT AND INDUCE AN IMMUNE SUPPRESSIVE PHENOTYPE IN BONE MARROW-DERIVED CCR2+ MYELOID CELLS

Abstract INTRODUCTION Infiltrating immune-suppressive myeloid cells represent a tumor supportive population that contributes to immune checkpoint inhibitor resistance and poor survival in Glioblastoma (GBM) patients. We have previously characterized monocytic-myeloid derived suppressor cells (M-MDSCs) based on their dual expression of chemokine receptors CCR2 and CX3CR1(CCR2+/CX3CR1+). Genetic and pharmacologic targeting of CCR2, in combination with PD-1 blockade, reduced the percentage of M-MDSCs in the glioma-microenvironment and slowed the progression of KR158 and 005GSC murine gliomas. Additional studies are needed to investigate the chemokines responsible for the tumor recruitment of CCR2+/CX3CR1+ cells and the impact of glioma derived factors on their immune suppressive phenotype. OBJECTIVE Evaluate the effect of glioma derived factors on the migration and suppression of bone marrow CCR2+/CX3CR1+ myeloid cells. METHODS A transwell migration assay was utilized to determine the migratory ability of CCR2+/CX3CR1+ cells to KR158B conditioned in the presence of CCL2 and CCL7 neutralizing antibodies. Ly6G-/GR1+ cells were isolated from bone marrow cultured with KR158B conditioned media and co-cultured with freshly isolated T-cells to examine their immune-suppressive phenotype. RESULTS KR158B gliomas differentially upregulate cytokines including CCL2, IL6, G-CSF, GM-CSF as compared to healthy naive brains. KR158B conditioned media increased the percentage of bone marrow-derived CCR2+/CX3CR1+ cells that are CD11b+, Ly6Chi, and Ly6G-. Bone marrow-derived CCR2+/CX3CR1+ cells expanded in KR158B condition media suppress both CD4+ and CD8+ T cell proliferation. Bone marrow-derived CCR2+/CX3CR1+ cells migrate to recombinant CCL2 and CCL7 as well as KR158B glioma conditioned media. Migration to conditioned media is completely inhibited by the combination of CCL2 and CCL7 neutralizing antibodies. High CCL2 and CCL7 are associated with poor prognosis in human GBM. CONCLUSION Glioma-derived CCL2 and CCL7 mediate migration of CCR2+ myeloid cells into the tumor microenvironment in a redundant manner. Additional glioma-derived factors induce CCR2+/CX3CR1+ myeloid cells to a CD4/8+ T cell suppressive state.

Effect of azithromycin on migration of peripheral blood NK cells from patients with chronic obstructive pulmonary disease

Currently, no drugs have been identified that could slow progression of chronic obstructive pulmonary disease (COPD), or have a significant impact on patient mortality. Therefore, research continues aimed at studying the mechanisms of COPD development and searching for drugs that affect its molecular pathogenesis. The aim of our work was to determine the ability of azithromycin combined with corticosteroids to affect the migration of peripheral blood NK cells from the COPD patients. In the present study, we have measured expression of chemokine receptors CCR5, CCR6, CCR7, CXCR3, CXCR4, CXCR6 on the surface of peripheral blood NK cells (CD3- CD56+) by means of flow cytometry in 54 smoking patients with COPD, 21 healthy smokers, and 20 healthy non-smokers. Moreover, the effect of azithromycin (10 µg/mL) and budesonide (10 nM) on the migration of NK cells from COPD patients (n = 8) towards CCL5 (10 nM) and CXCL10 (10 nM) was determined. We found that the percentage of NK cells expressing CXCR3 and CCR5 chemokine receptors was increased in smoking patients with COPD compared with healthy smokers and healthy non-smokers. However, the proportion of these NK cell subsets did not differ between healthy smokers and healthy non-smokers. There were no significant differences in the percentage of NK cells expressing CXCR4, CXCR6, CCR6, CCR7 chemokine receptors between the three groups of subjects. Addition of budesonide to the cell suspensions decreased the migration of blood NK cells towards CCL5 and CXCL10. Azithromycin was also shown to suppress the migration of blood NK cells towards these chemokines. The combination of azithromycin and budesonide was more potent at inhibiting NK cell chemotaxis towards CCL5 and CXCL10 than any of these drugs added alone. Our results demonstrate a change in the chemokine receptor profile of NK cells in COPD patients and indicate the advantages of the combined use of corticosteroids and azithromycin for COPD treatment.

MODL-27. PRECLINICAL INTERROGATION OF IMMUNOTHERAPY TREATMENT STRATEGIES IN GLIOBLASTOMA (GBM) USING A CLINICALLY RELEVANT DISEASE MODEL

Abstract Clinically faithful pre-clinical models are essential for screening therapies and studying resistance mechanisms. Historically models have failed to predict response in the clinical setting. Current models seldom incorporate surgical resection, and commonly use young animals whose immune contexture differs from older patients. Here, we have established an orthotopic model, employing the syngeneic, mesenchymal-NFpp10a-cell line, in both young and aged mice. We have characterised the model's response to standard of care resection and temozolomide (TMZ) treatment. We have further studied response to anti-PD1 therapy (adjuvant and neoadjuvant). NFpp10a-Luc2 expressing cells were orthotopically implanted into C57BL/6 mice (aged { > 18months} and young {6-8weeks}) and weekly bioluminescence imaging (BLI) performed to monitor tumour growth. Several therapeutic interventions were assessed to study tumour growth and survival: (1)surgical resection, (2)TMZ, (3)anti-PD1 (4)neoadjuvant anti-PD1. RNAseq, murine microenvironment cell population (mMCP) counter and gene ontology analyses characterised treatment related changed in the TME. We demonstrated survival advantage in aged mice undergoing resection (Resection:33.5 days vs Non-Resection:18 days;p=0.0166). Subsequently, we observed that TMZ and anti-PD1 monotherapy had no impact on NFpp10a-Luc2 growth (TMZ-overall:p=0.9001, and anti-PD1-overall:p = 0.7933) or survival (TMZ-overall:p = 0.3035, anti-PD1-overall:p= 0.6328). Neoadjuvant anti-PD1 treated mice demonstrated no survival advantage compared to IgG control (33-days vs 35-days:p = 0.9429). or BLI signal (p = 0.1703). NFpp10a-Luc2 forms immune-cold TME relative to commonly employed models. mMCP counter analysis demonstrates neoadjuvant anti-PD1 induced influx of CD8+-T cells, B cells, and monocytes into the TME compared to IgG control, and was associated with significantly upregulated CXCR3 chemokine receptor binding pathway on gene ontology analysis (p = 0.0045). Overall, we have, for the first time established and characterised response of the NFpp10a-Luc2-C57BL/6 model to resection, TMZ and anti-PD1 therapies. NFpp10a-Luc2 tumours demonstrate similar therapeutic resistance and TME profile compared to human mesenchymal GBM. The model may therefore be employed in future pre-clinical studies to guide clinical trials in mesenchymal GBM.

Computationally designed GPCR quaternary structures bias signaling pathway activation

Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can be readily characterized, receptor associations into quaternary structures are challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed binding interactions, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers activated the G protein Gi, but only specific dimer structures also recruited β-arrestins. Overall, we demonstrate that quaternary structures represent an important unforeseen mechanism of receptor biased signaling and reveal the existence of a bias switch at the dimer interface of several G protein-coupled receptors including CXCR4, mu-Opioid and type-2 Vasopressin receptors that selectively control the activation of G proteins vs β-arrestin-mediated pathways. The approach should prove useful for predicting and designing receptor associations to uncover and reprogram selective cellular signaling functions.

Alternate-Day Fasting Ameliorates Newly Established Sjögren's Syndrome-like Sialadenitis in Non-Obese Diabetic Mice.

Intermittent fasting confers protections to various diseases including autoimmune disorders, but the specific effects of intermittent fasting on Sjögren's syndrome (SS) remains inconclusive. The present study was undertaken to determine the specific impact of alternate-day fasting (ADF) on newly established SS-like sialadenitis using non-obese diabetic (NOD) mice. Female NOD mice were deprived of food every other day from 10 to 13 weeks of age, the early stage of established SS, and then analyzed for the disease characteristics. Mice in the ADF group had higher salivary flow rate and attenuated submandibular gland (SMG) inflammation, compared to the control mice fed with standard chow ad libitum. The improvements were accompanied with a decrease in the total leukocytes, T and B lymphocytes and activated CD4 and CD8 T cells, and a down-regulation of pro-inflammatory cytokines IFN-γ and IL-17, chemokine receptor CXCR3 and its ligands CXCL9 and CXCL11 in the SMGs. ADF also led to elevated mRNA levels of water channel protein aquaporin 5 and tight junction protein claudin-1, two factors crucial for normal salivary secretion in the SMGs. In addition, ADF reduced the proportion of IFN-γ- and IL-17- expressing CD4 T cells and diminished mRNA levels of IFN-γ, TNF-α, and IL-17 in the total submandibular draining lymph node cells. Taken together, ADF is effective in ameliorating newly established SS-associated salivary gland exocrinopathy in NOD mice.

Relevant biomarkers of kidney allograft rejection

This review focuses on the new relevant biomarkers proposed for the diagnosis of different types of allograft rejections. The immune response against the transplanted tissues can lead to rejection. Kidney allograft rejection occurs when the recipient component's immune system reacts against the donor's cells. MicroRNAs, dd-cf DNA, CD103 markers, CXCR3 chemokine receptor, IP-10, KIR genes, HLA antibodies, the perforin and granzyme B molecules - the constant assessment of all these parameters could prevent acute rejection episodes and kidney injuries. In this way, both immune response and tissue destruction biomarkers are essential for the long-term survival of kidney-transplanted patients. They also contribute to personalizing treatments, precisely personalized immunosuppressive regiments.

PIM3 kinase promotes tumor metastasis in hepatoblastoma by upregulating cell surface expression of chemokine receptor cxcr4.

Patients presenting with metastatic hepatoblastoma have limited treatment options and survival rates as low as 25%. We previously demonstrated that Proviral Integration site in Maloney murine leukemia virus 3 (PIM3) kinase promotes tumorigenesis and cancer cell stemness in hepatoblastoma. In this study, we assessed the role of PIM3 kinase in promoting hepatoblastoma metastasis. We utilized a tail vein injection model of metastasis to evaluate the effect of CRISPR/Cas9-mediated PIM3 knockout, stable overexpression of PIM3, and pharmacologic PIM inhibition on the formation of lung metastasis. In vivo studies revealed PIM3 knockout impaired the formation of lung metastasis: 5 out of 6 mice injected with wild type hepatoblastoma cells developed lung metastasis while none of the 7 mice injected with PIM3 knockout hepatoblastoma cells developed lung metastasis. PIM3 overexpression in hepatoblastoma increased the pulmonary metastatic burden in mice and mechanistically, upregulated the phosphorylation and cell surface expression of CXCR4, a key receptor in the progression of cancer cell metastasis. CXCR4 blockade with AMD3100 decreased the metastatic phenotype of PIM3 overexpressing cells, indicating that CXCR4 contributed to PIM3's promotion of hepatoblastoma metastasis. Clinically, PIM3 expression correlated positively with CXCR4 expression in primary hepatoblastoma tissues. In conclusion, we have shown PIM3 kinase promotes the metastatic phenotype of hepatoblastoma cells through upregulation of CXCR4 cell surface expression and these findings suggest that targeting PIM3 kinase may provide a novel therapeutic strategy for metastatic hepatoblastoma.

A circulating subset of iNKT cells mediates antitumor and antiviral immunity.

Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244-CXCR6-), C1 (CD244-CXCR6+), or C2 (CD244+CXCR6+) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell-like features, whereas C1 iNKT cells showed more T cell-like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244+CXCR6+ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell-like properties distinct from conventional tissue-resident iNKT cells.

DOCK2 and phosphoinositide-3 kinase δ mediate two complementary signaling pathways for CXCR5-dependent B cell migration.

Naive B cells use the chemokine receptor CXCR5 to enter B cell follicles, where they scan CXCL13-expressing ICAM-1+ VCAM-1+ follicular dendritic cells (FDCs) for the presence of antigen. CXCL13-CXCR5-mediated motility is mainly driven by the Rac guanine exchange factor DOCK2, which contains a binding domain for phosphoinositide-3,4,5-triphosphate (PIP3) and other phospholipids. While p110δ, the catalytic subunit of the class IA phosphoinositide-3-kinase (PI3K) δ, contributes to CXCR5-mediated B cell migration, the precise interdependency of DOCK2, p110δ, or other PI3K family members during this process remains incompletely understood. Here, we combined in vitro chemotaxis assays and in vivo imaging to examine the contribution of these two factors during murine naïve B cell migration to CXCL13. Our data confirm that p110δ is the main catalytic subunit mediating PI3K-dependent migration downstream CXCR5, whereas it does not contribute to chemotaxis triggered by CXCR4 or CCR7, two other chemokine receptors expressed on naïve B cells. The contribution of p110δ activity to CXCR5-driven migration was complementary to that of DOCK2, and pharmacological or genetic interference with both pathways completely abrogated B cell chemotaxis to CXCL13. Intravital microscopy of control and gene-deficient B cells migrating on FDCs confirmed that lack of DOCK2 caused a profound migration defect, whereas p110δ contributed to cell speed and directionality. B cells lacking active p110δ also displayed defective adhesion to ICAM-1; yet, their migration impairment was maintained on ICAM-1-deficient FDCs. In sum, our data uncover two complementary signaling pathways mediated by DOCK2 and p110δ, which enable CXCR5-driven naïve B cell examination of FDCs.

CXCR2 intrinsically drives the maturation and function of neutrophils in mice.

Neutrophils play a major role in the protection from infections but also in inflammation related to tumor microenvironment. However, cell-extrinsic and -intrinsic cues driving their function at steady state is still fragmentary. Using Cxcr2 knock-out mice, we have evaluated the function of the chemokine receptor Cxcr2 in neutrophil physiology. We show here that Cxcr2 deficiency decreases the percentage of mature neutrophils in the spleen, but not in the bone marrow (BM). There is also an increase of aged CD62Llo CXCR4hi neutrophils in the spleen of KO animals. Spleen Cxcr2-/- neutrophils display a reduced phagocytic ability, whereas BM neutrophils show an enhanced phagocytic ability compared to WT neutrophils. Spleen Cxcr2-/- neutrophils show reduced reactive oxygen species production, F-actin and α-tubulin levels. Moreover, spleen Cxcr2-/- neutrophils display an altered signaling with reduced phosphorylation of ERK1/2 and p38 MAPK, impaired PI3K-AKT, NF-κB, TGFβ and IFNγ pathways. Altogether, these results suggest that Cxcr2 is essential for neutrophil physiology.

A humanized mouse model for in vivo evaluation of invariant Natural Killer T cell responses.

Invariant natural killer T (iNKT) cells mediate immune responses when stimulated by glycolipid agonists presented by CD1d. In extensive studies of synthetic analogues of α-galactosyl ceramides, we identified numerous examples of significant differences in the recognition of specific glycolipids in wild type mice versus human iNKT cell clones or PBMC samples. To predict human iNKT cell responses more accurately in a mouse model, we derived a mouse line in which compound genetic modifications were used to express a human-like iNKT cell TCR along with human CD1d in place of the endogenous mouse proteins. Detailed transcriptional and phenotypic profiling demonstrated that these partially humanized mice developed an expanded population of T cells recognizing CD1d-presented glycolipid antigens, among which a subset characterized by expression of chemokine receptor CXCR6 had features characteristic of authentic iNKT cells. Responses to iNKT cell activating glycolipids in these mice generated cytokine production in vitro and in vivo that showed a pattern of fine specificity that closely resembled that of cultured human iNKT cell clones. Anti-tumor responses to variants of α-galactosyl ceramide in VαKI mice also correlated with their potency for stimulating human iNKT cells. This genetically modified mouse line provides a practical model for human presentation and recognition of iNKT cell activators in the context of a normally functioning immune system, and may furnish valuable opportunities for preclinical evaluation of iNKT cell-based therapies.

The chemokine receptor CXCR4 regulates satellite cell activation, early expansion, and self-renewal, in response to skeletal muscle injury.

Acute skeletal muscle injury is followed by satellite cell activation, proliferation, and differentiation to replace damaged fibers with newly regenerated muscle fibers, processes that involve satellite cell interactions with various niche signals. Here we show that satellite cell specific deletion of the chemokine receptor CXCR4, followed by suppression of recombination escapers, leads to defects in regeneration and satellite cell pool repopulation in both the transplantation and in situ injury contexts. Mechanistically, we show that endothelial cells and FAPs express the gene for the ligand, SDF1α, and that CXCR4 is principally required for proper activation and for transit through the first cell division, and to a lesser extent the later cell divisions. In the absence of CXCR4, gene expression in quiescent satellite cells is not severely disrupted, but in activated satellite cells a subset of genes normally induced by activation fail to upregulate normally. These data demonstrate that CXCR4 signaling is essential to normal early activation, proliferation, and self-renewal of satellite cells.

Nutritional immunomodulation of Atlantic salmon response to Renibacterium salmoninarum bacterin.

We investigated the immunomodulatory effect of varying levels of dietary ω6/ω3 fatty acids (FA) on Atlantic salmon (Salmo salar) antibacterial response. Two groups were fed either high-18:3ω3 or high-18:2ω6 FA diets for 8 weeks, and a third group was fed for 4 weeks on the high-18:2ω6 diet followed by 4 weeks on the high-18:3ω3 diet and termed “switched-diet”. Following the second 4 weeks of feeding (i.e., at 8 weeks), head kidney tissues from all groups were sampled for FA analysis. Fish were then intraperitoneally injected with either a formalin-killed Renibacterium salmoninarum bacterin (5 × 107 cells mL−1) or phosphate-buffered saline (PBS control), and head kidney tissues for gene expression analysis were sampled at 24 h post-injection. FA analysis showed that the head kidney profile reflected the dietary FA, especially for C18 FAs. The qPCR analyses of twenty-three genes showed that both the high-ω6 and high-ω3 groups had significant bacterin-dependent induction of some transcripts involved in lipid metabolism (ch25ha and lipe), pathogen recognition (clec12b and tlr5), and immune effectors (znrf1 and cish). In contrast, these transcripts did not significantly respond to the bacterin in the “switched-diet” group. Concurrently, biomarkers encoding proteins with putative roles in biotic inflammatory response (tnfrsf6b) and dendritic cell maturation (ccl13) were upregulated, and a chemokine receptor (cxcr1) was downregulated with the bacterin injection regardless of the experimental diets. On the other hand, an inflammatory regulator biomarker, bcl3, was only significantly upregulated in the high-ω3 fed group, and a C-type lectin family member (clec3a) was only significantly downregulated in the switched-diet group with the bacterin injection (compared with diet-matched PBS-injected controls). Transcript fold-change (FC: bacterin/PBS) showed that tlr5 was significantly over 2-fold higher in the high-18:2ω6 diet group compared with other diet groups. FC and FA associations highlighted the role of DGLA (20:3ω6; anti-inflammatory) and/or EPA (20:5ω3; anti-inflammatory) vs. ARA (20:4ω6; pro-inflammatory) as representative of the anti-inflammatory/pro-inflammatory balance between eicosanoid precursors. Also, the correlations revealed associations of FA proportions (% total FA) and FA ratios with several eicosanoid and immune receptor biomarkers (e.g., DGLA/ARA significant positive correlation with pgds, 5loxa, 5loxb, tlr5, and cxcr1). In summary, dietary FA profiles and/or regimens modulated the expression of some immune-relevant genes in Atlantic salmon injected with R. salmoninarum bacterin. The modulation of Atlantic salmon responses to bacterial pathogens and their associated antigens using high-ω6/high-ω3 diets warrants further investigation.

68Ga]Ga-Pentixafor and Sodium [18F]Fluoride PET Can Non-Invasively Identify and Monitor the Dynamics of Orthodontic Tooth Movement in Mouse Model.

The cellular and molecular mechanisms of orthodontic tooth movement (OTM) are not yet fully understood, partly due to the lack of dynamical datasets within the same subject. Inflammation and calcification are two main processes during OTM. Given the high sensitivity and specificity of [68Ga]Ga-Pentixafor and Sodium [18F]Fluoride (Na[18F]F) for inflammation and calcification, respectively, the aim of this study is to assess their ability to identify and monitor the dynamics of OTM in an established mouse model. To monitor the processes during OTM in real time, animals were scanned using a small animal PET/CT during week 1, 3, and 5 post-implantation, with [68Ga]Ga-Pentixafor and Na[18F]F. Both tracers showed an increased uptake in the region of interest compared to the control. For [68Ga]Ga-Pentixafor, an increased uptake was observed within the 5-week trial, suggesting the continuous presence of inflammatory markers. Na[18F]F showed an increased uptake during the trial, indicating an intensification of bone remodelling. Interim and end-of-experiment histological assessments visualised increased amounts of chemokine receptor CXCR4 and TRAP-positive cells in the periodontal ligament on the compression side. This approach establishes the first in vivo model for periodontal remodelling during OTM, which efficiently detects and monitors the intricate dynamics of periodontal ligament.

Interplay between cellular changes in the knee joint, circulating lipids and pain behaviours in a slowly progressing murine model of osteoarthritis.

Synovial inflammation has known contributions to chronic osteoarthritis (OA) pain, but the potential role in transitions from early to late stages of OA pain is unclear. The slowly progressing surgical destabilization of the medial meniscus (DMM) murine OA model and sham control, was used in male C57BL/6J mice to investigate the interplay between knee inflammation, plasma pro- and anti-inflammatory oxylipins and pain responses during OA progression. Changes in joint histology, macrophage infiltration, chemokine receptor CX3CR1 expression, weight bearing asymmetry, and paw withdrawal thresholds were quantified 4, 8 and 16 weeks after surgery. Plasma levels of multiple bioactive lipid mediators were quantified using liquid chromatography with tandem mass-spectrometry (LC-MS/MS). Structural joint damage was evident at 8 weeks post-DMM surgery onwards. At 16 weeks post-DMM surgery, synovial scores, numbers of CD68 and CD206 positive macrophages and pain responses were significantly increased. Plasma levels of oxylipins were negatively correlated with joint damage and synovitis scores at 4 and 8 weeks post-DMM surgery. Higher circulating levels of the pro-resolving oxylipin pre-cursor 17-HDHA were associated with lower weight bearing asymmetry at week 16. The transition to chronic OA pathology and pain is likely influenced by both joint inflammation and plasma oxylipin mediators of inflammation and levels of pro-resolution molecules. Using a slow progressing surgical model of osteoarthritis we show how the changing balance between local and systemic inflammation may be of importance in the progression of pain behaviours during the transition to chronic osteoarthritis pain.

Heterocomplexes between the atypical chemokine MIF and the CXC-motif chemokine CXCL4L1 regulate inflammation and thrombus formation

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine–chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.

A comparison of four leukapheresis methods to harvest an optimal dose of CD34+ cells: A single center experience

Chemokine receptor CXCR4 antagonist plerixafor (Px) as well as high volume (HV) leukapheresis have been shown to reduce hematopoietic stem progenitor cell (HSPC) mobilization failure rates. However, no direct comparisons of such methods currently exists. We compared the HSPC collection yield based on basal peripheral blood CD34+ cell numbers in patients diagnosed with multiple myeloma or non-Hodgkin's lymphoma undergoing autologous stem cell transplantation in a retrospective chart review. The leukapheresis methods used included HV versus regular volume (RV) with or without Px. There were 116 patients in the study group while the historical control group had 34 patients. Control group underwent RV leukapheresis without Px. Addition of Px or HV in the study group failed to display significant improvement in CD34+ cell collection yield; however, when basal CD34+ cell numbers were taken into account, both Px + RV and HV without Px increased CD34+ cell collection yield. The collection failure rates of HV without Px group were comparable to Px + RV when the basal CD34+ cell numbers were over 20/μl. Of interest, multivariate linear regression analysis did not detect any significant difference between HV versus Px + RV or other leukapheresis methods in CD34 yields or collection failure rates from a single collection after controlling for other factors (sex, age, or underlying disease). In multivariate analysis, pre apheresis CD34+ cell number was significantly and positively associated with the CD34+ cell yields from a single apheresis. In our studies, the majority of patients can be rescued without Px by HV alone as a potential cost saving approach. In summary, trend in our studies reflects that both Px and HV are capable of reducing the mobilization failure rates except the poorest mobilizers, which will need to be validated in larger studies.

Interleukin 4 Controls the Pro-Tumoral Role of Macrophages in Mammary Cancer Pulmonary Metastasis in Mice.

Simple SummaryMetastasis is the main cause of death from breast cancer. In mouse models of breast cancer lung metastasis, macrophages enhance metastasis by promoting tumor cell seeding and persistent growth. Here, we show that interleukin-4 (IL4) is required for this process as IL4 receptor (IL4rα)-null mice develop fewer and smaller lung metastases. This deficiency is partially rescued by adoptive transfer of wild-type monocytes. IL4 signaling in macrophages upregulates the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. In addition, expression of several other genes already causally associated with lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1 are upregulated in macrophages. High-resolution intravital imaging at the time of metastatic seeding showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages, showing the dependence on IL4. We conclude that IL4 signaling in monocytes and macrophages is important during seeding and growth of breast metastasis in the lung.Metastasis is the systemic manifestation of cancer and the main cause of death from breast cancer. In mouse models of lung metastases, recruitment of classical monocytes from blood to the lung and their differentiation to metastasis-associated macrophages (MAMs) facilitate cancer cell extravasation, survival and growth. Ablation of MAMs or their monocytic progenitors inhibits metastasis. We hypothesized that factors controlling macrophage polarization modulate tumor cell extravasation in the lung. We evaluated whether signaling by Th1 or Th2 cytokines in macrophages affected transendothelial migration of tumor cells in vitro. Interferon gamma and LPS inhibited macrophage-dependent tumor cell extravasation while the Th2 cytokine interleukin-4 (IL4) enhanced this process. We demonstrated that IL4 receptor (IL4rα)-null mice developed fewer and smaller lung metastasis in E0771-LG mammary cancer models of this disease. Adoptive transfer of wild-type monocytes to IL4rα-deficient mice partially rescued this phenotype. IL4 signaling in macrophages controlled the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. Furthermore, IL4 signaling in macrophages regulated the transcript abundance of several other genes already causally associated with mammary cancer lung metastasis including Ccl2, Csf1, Ccr1, Hgf and Flt1. The central role of IL4 signaling in MAMs was confirmed by high-resolution intravital imaging of the lung in mice at the time of metastatic seeding, which showed reduced physical interaction between tumor cells and IL4rα-deficient macrophages. This interaction with wild-type MAMs enhanced tumor cell survival and seeding, which was lost in the IL4rα mice. These data indicate that IL4 signaling in monocytes and macrophages is key during seeding and growth of breast metastasis in the lung, as it regulates pro-tumoral paracrine signaling between cancer cells and macrophages.