Local Chemokine Research Articles

Adeno-associated virus delivered CXCL9 sensitizes glioblastoma to anti-PD-1 immune checkpoint blockade

There are numerous mechanisms by which glioblastoma cells evade immunological detection, underscoring the need for strategic combinatorial treatments to achieve appreciable therapeutic effects. However, developing combination therapies is difficult due to dose-limiting toxicities, blood-brain-barrier, and suppressive tumor microenvironment. Glioblastoma is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment and activation. Herein, we develop a recombinant adeno-associated virus (AAV) gene therapy that enables focal and stable reconstitution of the tumor microenvironment with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for lymphocytes. By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by cytotoxic lymphocytes, sensitizing glioblastoma to anti-PD-1 immune checkpoint blockade in female preclinical tumor models. These effects are accompanied by immunologic signatures evocative of an inflamed tumor microenvironment. These findings support AAV gene therapy as an adjuvant for reconditioning glioblastoma immunogenicity given its safety profile, tropism, modularity, and off-the-shelf capability.

B-cells in pulmonary arterial hypertension: friend, foe or bystander?

There is an unmet need for new therapeutic strategies that target alternative pathways to improve the prognosis of patients with pulmonary arterial hypertension (PAH). As immunity has been involved in the development and progression of vascular lesions in PAH, we review the potential contribution of B-cells in its pathogenesis and evaluate the relevance of B-cell-targeted therapies. Circulating B-cell homeostasis is altered in PAH patients, with total B-cell lymphopenia, abnormal subset distribution (expansion of naïve and antibody-secreting cells, reduction of memory B-cells) and chronic activation. B-cells are recruited to the lungs through local chemokine secretion, and activated by several mechanisms: 1) interaction with lung vascular autoantigens through cognate B-cell receptors; 2) costimulatory signals provided by T follicular helper cells (interleukin (IL)-21), type 2 T helper cells and mast cells (IL-4, IL-6 and IL-13); and 3) increased survival signals provided by B-cell activating factor pathways. This activity results in the formation of germinal centres within perivascular tertiary lymphoid organs and in the local production of pathogenic autoantibodies that target the pulmonary vasculature and vascular stabilisation factors (including angiotensin-II/endothelin-1 receptors and bone morphogenetic protein receptors). B-cells also mediate their effects through enhanced production of pro-inflammatory cytokines, reduced anti-inflammatory properties by regulatory B-cells, immunoglobulin (Ig)G-induced complement activation, and IgE-induced mast cell activation. Precision-medicine approaches targeting B-cell immunity are a promising direction for select PAH conditions, as suggested by the efficacy of anti-CD20 therapy in experimental models and a trial of rituximab in systemic sclerosis-associated PAH.

Cannabidiol Reduces Systemic Immune Activation in Experimental Acute Lung Injury.

Background: The underlying pathomechanism of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is the immune response to inflammation or infection within the pulmonary microcirculation. Systemic spread of pathogens, activated immune cells, and inflammatory mediators contributes significantly to mortality in patients with ARDS. Objective: The endogenous cannabinoid system is a major modulator of the immune response during inflammation and infection. Phytocannabinoids, such as cannabidiol (CBD), have shown promising anti-inflammatory effects in several pathologies. The overall objective of this study was to evaluate the effects of CBD on local and systemic inflammation in endotoxin-induced ALI in mice. Materials and Methods: ALI was induced by pulmonary endotoxin challenge. Four groups of male C57BL/6 mice were randomized in this study: control, ALI, ALI with CBD treatment, and control with CBD treatment. Analyses of local and systemic cytokine levels, lung histology, and leukocyte activation as visualized by intravital microscopy of the intestinal and pulmonary microcirculation were performed 6 h following intranasal endotoxin administration. Results: Pulmonary endotoxin challenge induced significant inflammation evidenced by local and systemic cytokine and chemokine release, lung histopathology, and leukocyte adhesion. Intraperitoneal CBD treatment resulted in a significant decrease in systemic inflammation as shown by reduced leukocyte adhesion in the intestinal microcirculation and reduced plasma cytokine and chemokine levels. Pulmonary chemokine levels were decreased, while pulmonary cytokine levels were unchanged. Surprisingly, the ALI score was slightly increased by CBD treatment in a manner driven by enhanced neutrophil infiltration of the alveoli. Conclusion: In this model of experimental ALI, CBD administration was associated with reduced systemic inflammation and heterogeneous effects on pulmonary inflammation. Future studies should explore the mechanisms involved as they relate to neutrophil infiltration and proinflammatory mediator production within the lungs.

Unveiling the role of neutrophils in chronic spontaneous urticaria: Beyond mast cells.

Mast cells and eosinophils are considered pivotal contributors to the pathogenesis of chronic spontaneous urticaria (CSU). However, emerging evidence suggests that neutrophils also play a central role. Cutaneous mast cells and macrophages orchestrate the recruitment of neutrophils through the regulation and activation of diverse processes, including heightened local vascular permeability and chemokine release. Studies have demonstrated increased activation and elevated levels of neutrophil-related cytokines in CSU patients. Moreover, neutrophils have been proposed as antigen-presenting cells during the late-phase reaction of immunoglobulin E-mediated allergy and have been associated with the expression of calcitonin gene-related protein and vascular endothelial growth factor in CSU. Histopathological analysis of lesional skin in CSU patients revealed significantly higher eosinophil and neutrophil counts than unaffected skin. However, the extent of neutrophil infiltration in the skin does not appear to correlate with the number of neutrophils in peripheral blood. The utility of the neutrophil-lymphocyte ratio as a marker for disease activity or remission in CSU remains inconclusive. Neutrophil-targeted therapy may confer benefits for CSU patients who exhibit resistance to antihistamines. Omalizumab has demonstrated its ability to reduce neutrophil counts, the neutrophil-lymphocyte ratio, and the neutrophil-monocyte ratio in peripheral blood. While dapsone and colchicine are recommended as alternative treatment options for CSU, their evidential support from published studies remains limited. Inhibitors targeting interleukin-1 and neutrophil-related cytokines have been proposed as potential therapeutic interventions for patients exhibiting neutrophil predominance. Further research is warranted to gain deeper insights into the involvement of neutrophils in CSU and to explore potential therapeutic interventions.

Polarization Behavior of Bone Macrophage as Well as Associated Osteoimmunity in Glucocorticoid-Induced Osteonecrosis of the Femoral Head.

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a disabling disease with high mortality in China but the detailed molecular and cellular mechanisms remain to be investigated. Macrophages are considered the key cells in osteoimmunology, and the cross-talk between bone macrophages and other cells in the microenvironment is involved in maintaining bone homeostasis. M1 polarized macrophages launch a chronic inflammatory response and secrete a broad spectrum of cytokines (eg, TNF-α, IL-6 and IL-1β) and chemokines to initiate a chronic inflammatory state in GIONFH. M2 macrophage is the alternatively activated anti-inflammatory type distributed mainly in the perivascular area of the necrotic femoral head. In the development of GIONFH, injured bone vascular endothelial cells and necrotic bone activate the TLR4/NF-κB signal pathway, promote dimerization of PKM2 and subsequently enhance the production of HIF-1, inducing metabolic transformation of macrophage to the M1 phenotype. Considering these findings, putative interventions by local chemokine regulation to correct the imbalance between M1/M2 polarized macrophages by switching macrophages to an M2 phenotype, or inhibiting the adoption of an M1 phenotype appear to be plausible regimens for preventing or intervening GIONFH in the early stage. However, these results were mainly obtained by in vitro tissue or experimental animal model. Further studies to completely elucidate the alterations of the M1/M2 macrophage polarization and functions of macrophages in glucocorticoid-induced osteonecrosis of the femoral head are imperative.

Heparin Specifically Interacts with Basic BBXB Motifs of the Chemokine CCL21 to Define CCR7 Signaling.

Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21-in contrast to CCL19-is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner.

Inflammation Self-Limiting Electrospun Fibrous Tape via Regional Immunity for Deep Soft Tissue Repair.

Overexpression of inflammatory cytokines and chemokines occurs at deep soft tissue injury sites impeding the inflammation self-limiting and impairing the tissue remodeling process. Inspired by the electrostatically extracellular matrix (ECM) binding property of the inflammatory signals, an inflammation self-limiting fibrous tape is designed by covalently modifying the thermosensitive methacrylated gelatin (GelMA) and negatively charged methacrylated heparin (HepMA) hydrogel mixture with proper ratio onto the electrospun fibrous membrane by mild alkali hydrolysis and carboxyl-amino condensation reaction to restore inflammation self-limiting and promote tissue repair via regional immunity regulation. While the GelMA guarantees cell compatibility, the negatively charged HepMA successfully adsorbs the inflammatory cytokines and chemokines by electrostatic interactions and inhibits immune cell migration in vitro. Furthermore, in vivo inflammation self-limiting and regional immunity regulation efficacy is evaluated in a rat abdominal hernia model. Reduced local inflammatory cytokines and chemokines in the early stage and increased angiogenesis and ECM remodeling in the later phase confirm that the tape is an approach to maintain an optimal regional immune activation level after soft tissue injury. Overall, the reported electrospun fibrous tape will find its way into clinical transformation and solve the challenges of deep soft tissue injury.

Initial TK-deficient HSV-1 infection in the lip alters contralateral lip challenge immune dynamics

Primary infection with herpes simplex type 1 (HSV-1) occurring around the mouth and nose switches rapidly to lifelong latent infection in sensitive trigeminal ganglia (TG) neurons. Sporadic reactivation of these latent reservoirs later in life is the cause of acute infections of the corneal epithelium, which can cause potentially blinding herpes simplex keratitis (HSK). There is no effective vaccine to protect against HSK, and antiviral drugs provide only partial protection against recurrences. We previously engendered an acute disease-free, non-reactivating latent state in mice when challenged with virulent HSV-1 in orofacial mucosa, by priming with non-neurovirulent HSV-1 (TKdel) before the challenge. Herein, we define the local immune infiltration and inflammatory chemokine production changes after virulent HSV-1 challenge, which were elicited by TKdel prime. Heightened immunosurveillance before virulent challenge, and early enhanced lymphocyte-enriched infiltration of the challenged lip were induced, which corresponded to attenuation of inflammation in the TG and enhanced viral control. Furthermore, classical latent-phase T cell persistence around latent HSV-1 reservoirs were severely reduced. These findings identify the immune processes that are likely to be responsible for establishing non-reactivating latent HSV-1 reservoirs. Stopping reactivation is essential for development of efficient vaccine strategies against HSV-1.

Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.

Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Influence of Local Chemokine Delivery and Postoperative Peripheral Blood Mobilization on Achilles Tendon Repair

Category:Basic Sciences/Biologics; Sports; TraumaIntroduction/Purpose:Mesenchymal stem cells (MSCs) have demonstrated promise in augmenting the surgical repair of full thickness tendon tears. Despite this promise, obtaining a therapeutic concentration of MSCs is time-, labor- and resource intensive, and is subject to ever-tightening governmental regulations. Our group recently demonstrated that postoperative mobilization of MSCs from the marrow compartment and into peripheral blood via subcutaneous granulocyte- colony stimulating factor administration enhanced tendon-bone healing in a rat model of surgical repair of a full-thickness supraspinatus tendon tear. We hypothesized that combining postoperative stem cell mobilization with local delivery of a chemokine that stimulates MSC chemotaxis would enhance the surgical repair of a mid-substance Achilles tendon transection.Methods:Following IACUC approval, Achilles tendons were unilaterally frayed via multiple longitudinal scalpel blade nicks in 24 Lewis rats. Tendons were then surgically transected, followed by repair with braided non-absorbable suture in a modified-Kessler pattern. Based on randomization (n=6/group), the tendon anastomosis was coated with a resorbable chitosan hydrogel containing 100 ng of the chemokine, CCL5 (RANTES), or hydrogel without RANTES. Skin closure and anesthesia recovery were then performed; rats were allowed ad libitum activity. Beginning 24 hours postoperatively, rats received a daily subcutaneous injection of either a peripheral blood mobilizing agent, AMD3100 (5 mg/kg), or saline; dosing was repeated at 48 and 72 hours. At the 4- week endpoint, Achilles tendons harvested en bloc. Decalcified histologic sections were stained with H&E and Alcian Blue as well as immunostained for Collagen III, CD163, and CD68. Slides were digitized (40x magnification) and graded by blinded reviewers via modified-Bonar score to assess tendon integrity.Results:Rats that received local application of RANTES-loaded chitosan hydrogel had worse (higher) modified-Bonar subscores with respect to tenocyte morphology, collagen orientation, and ground substance staining, though none of these comparisons were statistically significant (p=0.068, p=0.672, and p=0.407, respectively). Animals that received subcutaneous AMD3100 alone, or in conjunction with RANTES-loaded chitosan hydrogel displayed the best (lowest) total modified Bonar scores, though these comparisons were also not statistically significant (p = 0.498). No significant differences were found with respect to CD163 (p=0.132) and CD68 (p=0.104) staining. Despite a lack of statistical significance, animals that received AMD3100 displayed a higher number of both macrophage subtypes. No differences were found with respect to Collagen III immunostaining (p=0.808).Conclusion:Tendon repair and regeneration remains a significant clinical challenge, which may be aided by MSC transplantation. The mobilization of endogenous marrow-derived MSCs into peripheral blood and subsequent recruitment via local chemokine delivery may be an efficacious way to enhance tendon repair without the need for ex vivo processing. We failed to demonstrate significant effects of stem cell mobilization via AMD3100 injections, and chemokine-directed recruitment, though trends were present indicating that AMD3100 alone may be beneficial. Additional work is required to draw any translatable conclusions from this research.

The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:β2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

Strong Association of Polymorphism in SPRED2 Gene with Disease Susceptibility and Clinical Characteristics of Rheumatoid Arthritis in the Iranian Population.

Background:The high heritability of Rheumatoid Arthritis (RA) has been estimated from different studies. Recently, Genome-Wide Association Studies (GWAS) show a large number of Single Nucleotide Polymorphisms (SNPs) loci affecting susceptibility to RA. The rs934734 polymorphism in the SPRED2 gene is one of these loci. Studies have shown that the SPRED2 gene is involved in the regulation of inflammatory response, leukocyte infiltration, and local chemokine production. In the current study, the possible association between SNP rs934734 (intronic variant) in the SPRED2 gene with RA risk in the Iranian population was evaluated.Methods:One hundred fourteen RA patients and 120 healthy counterparts were recruited in this case-control study to evaluate rs934734 genotypes using the real-time PCR High Resolution Melting method (HRM).Results:Logistic regression analysis demonstrated that GG and AG genotypes compared with AA genotype increase the risk of RA (GG vs. AA; OR=4.61; 95%CI [2.21–9.35]; p<0.001 and AG vs. AA; OR=2.54; 95%CI [1.36–4.76]; p=0.004). Furthermore, subjects with allele G were more frequently affected with RA than subjects with A allele (OR=2.33; 95%CI [1.61–3.38]; p<0.001). Besides, in the patient group, there was a significant correlation between Erythrocyte Sedimentation Rate (ESR) and C-reactive protein (CRP) concentration with rs934734 polymorphism (p<0.05).Conclusion:Our findings suggest that rs934734 in SPRED2 strongly underlies RA development and is associated with clinicopathological characteristics of this disease.

Endometriotic Peritoneal Fluid Stimulates Recruitment of CD4+CD25highFOXP3+ Treg Cells.

Endometriosis is a common gynecological disorder characterized by the presence of endometrial-like tissue outside the uterus. The disease is associated with disturbed local and systemic immunity. It has been reported that the proportion of CD4+CD25highFOXP3+ Treg cells may be significantly increased in the peritoneal fluid of patients with endometriosis. Therefore, the aim of our study was to investigate whether the proportions of Treg cells in the peritoneal cavity of patients with endometriosis are related to the chemotactic and stimulatory activity of the local peritoneal milieu. The peritoneal fluid was collected from 13 women with ovarian endometriosis and 12 control women without the disease. T cell populations were analyzed by flow cytometry, cytokines and chemokines were evaluated using the cytometric bead kit, and cell chemotaxis was studied by cell migration assay. We confirmed that the proportions of Treg cells are increased in the peritoneal fluid of women with endometriosis as compared to the control women. Endometriosis was also associated with elevated concentrations of IL-6, IL-10, and TGF-β1/2 as well as CCL20, CXCL8, CXCL9, and CXCL10. We did not reveal any changes in the proportion of peritoneal Th17 cells and concentrations of IL-17A. Peritoneal Treg cells positively correlated with concentrations of TGF-β, IL-10, and CCL20. Endometriotic peritoneal fluid stimulated chemotaxis of both CD4+ and Treg cells. This chemotactic activity positively correlated with concentrations of CCL20. CCL20 stimulated the migration of Treg cells, and the chemotactic activity of the endometriotic peritoneal fluid was inhibited by neutralizing anti-CCL20 antibodies. These results imply that increased proportions of the peritoneal Treg cells in women with endometriosis may result from attraction and activation by local chemokines and cytokines, especially CCL20 and TGF-β. Since Treg cells contribute to the immunopathogenesis of endometriosis, their chemotaxis and activation may be considered as a target for therapeutic intervention.

Oral Cavity as a Source of Mesenchymal Stem Cells Useful for Regenerative Medicine in Dentistry

The use of mesenchymal stem cells (MSCs) for regenerative purposes has become common in a large variety of diseases. In the dental and maxillofacial field, there are emerging clinical needs that could benefit from MSC-based therapeutic approaches. Even though MSCs can be isolated from different tissues, such as bone marrow, adipose tissue, etc., and are known for their multilineage differentiation, their different anatomical origin can affect the capability to differentiate into a specific tissue. For instance, MSCs isolated from the oral cavity might be more effective than adipose-derived stem cells (ASCs) for the treatment of dental defects. Indeed, in the oral cavity, there are different sources of MSCs that have been individually proposed as promising candidates for tissue engineering protocols. The therapeutic strategy based on MSCs can be direct, by using cells as components of the tissue to be regenerated, or indirect, aimed at delivering local growth factors, cytokines, and chemokines produced by the MSCs. Here, the authors outline the major sources of mesenchymal stem cells attainable from the oral cavity and discuss their possible usage in some of the most compelling therapeutic frontiers, such as periodontal disease and dental pulp regeneration.

Association of the CXCL9-CXCR3 and CXCL13-CXCR5 axes with B-cell trafficking in giant cell arteritis and polymyalgia rheumatica

ObjectiveB-cells are present in the inflamed arteries of giant cell arteritis (GCA) patients and a disturbed B-cell homeostasis is reported in peripheral blood of both GCA and the overlapping disease polymyalgia rheumatica (PMR). In this study, we aimed to investigate chemokine-chemokine receptor axes governing the migration of B-cells in GCA and PMR. MethodsWe performed Luminex screening assay for serum levels of B-cell related chemokines in treatment-naïve GCA (n = 41), PMR (n = 31) and age- and sex matched healthy controls (HC, n = 34). Expression of chemokine receptors on circulating B-cell subsets were investigated by flow cytometry. Immunohistochemistry was performed on GCA temporal artery (n = 14) and aorta (n = 10) and on atherosclerosis aorta (n = 10) tissue. ResultsThe chemokines CXCL9 and CXCL13 were significantly increased in the circulation of treatment-naïve GCA and PMR patients. CXCL13 increased even further after three months of glucocorticoid treatment. At baseline CXCL13 correlated with disease activity markers. Peripheral CXCR3+ and CXCR5+ switched memory B-cells were significantly reduced in both patient groups and correlated inversely with their complementary chemokines CXCL9 and CXCL13. At the arterial lesions in GCA, CXCR3+ and CXCR5+ B-cells were observed in areas with high CXCL9 and CXCL13 expression. ConclusionChanges in systemic and local chemokine and chemokine receptor pathways related to B-cell migration were observed in GCA and PMR mainly in the CXCL9-CXCR3 and CXCL13-CXCR5 axes. These changes can contribute to homing and organization of B-cells in the vessel wall and provide further evidence for an active involvement of B-cells in GCA and PMR.

Dysregulation of the Immune Environment in the Airways During HIV Infection.

HIV-1 increases susceptibility to pulmonary infection and disease, suggesting pathogenesis in the lung. However, the lung immune environment during HIV infection remains poorly characterized. This study examined T cell activation and the cytokine milieu in paired bronchoalveolar lavage (BAL) and blood from 36 HIV-uninfected and 32 HIV-infected participants. Concentrations of 27 cytokines were measured by Luminex, and T cells were phenotyped by flow cytometry. Blood and BAL had distinct cytokine profiles (p=0.001). In plasma, concentrations of inflammatory cytokines like IFN-γ (p=0.004) and TNF-α (p=0.004) were elevated during HIV infection, as expected. Conversely, BAL cytokine concentrations were similar in HIV-infected and uninfected individuals, despite high BAL viral loads (VL; median 48,000 copies/ml epithelial lining fluid). HIV-infected individuals had greater numbers of T cells in BAL compared to uninfected individuals (p=0.007); and BAL VL positively associated with CD4+ and CD8+ T cell numbers (p=0.006 and p=0.0002, respectively) and CXCL10 concentrations (p=0.02). BAL T cells were highly activated in HIV-infected individuals, with nearly 2-3 fold greater frequencies of CD4+CD38+ (1.8-fold; p=0.007), CD4+CD38+HLA-DR+ (1.9-fold; p=0.0006), CD8+CD38+ (2.8-fold; p=0.0006), CD8+HLA-DR+ (2-fold; p=0.022) and CD8+CD38+HLA-DR+ (3.6-fold; p<0.0001) cells compared to HIV-uninfected individuals. Overall, this study demonstrates a clear disruption of the pulmonary immune environment during HIV infection, with readily detectable virus and activated T lymphocytes, which may be driven to accumulate by local chemokines.

Erythrocyte-mediated systemic immunotherapy

The restoration of a local chemokine gradient by nanoparticles non-covalently anchored on the surface of systemically administered red blood cells delays cancer progression in mouse models of lung metastasis.

Temporal dynamics of intradermal cytokine response to tuberculin in Mycobacterium bovis BCG-vaccinated cattle using sampling microneedles

Bovine tuberculosis (bTB) is a disease of livestock with severe and worldwide economic, animal welfare and zoonotic consequences. Application of test-and-slaughter-based control polices reliant on tuberculin skin testing has been the mainstay of bTB control in cattle. However, little is known about the temporal development of the bovine tuberculin skin test response at the dermal sites of antigen injection. To fill this knowledge gap, we applied minimally-invasive sampling microneedles (SMNs) for intradermal sampling of interstitial fluid at the tuberculin skin test sites in Mycobacterium bovis BCG-vaccinated calves and determined the temporal dynamics of a panel of 15 cytokines and chemokines in situ and in the peripheral blood. The results reveal an orchestrated and coordinated cytokine and local chemokine response, identified IL-1RA as a potential soluble biomarker of a positive tuberculin skin response, and confirmed the utility of IFN-γ and IP-10 for bTB detection in blood-based assays. Together, the results highlight the utility of SMNs to identify novel biomarkers and provide mechanistic insights on the intradermal cytokine and chemokine responses associated with the tuberculin skin test in BCG-sensitized cattle.

The mast cell/S1P axis is not linked to pre-lesional male skin remodeling in a mouse model of eczema.

Atopic dermatitis (AD, eczema) is an inflammatory skin condition whose histopathology involves remodeling. Few preclinical AD studies are performed using male mice. The histopathological mechanisms underlying AD development were investigated here in male mice at a pre-lesional stage using a human AD-like mouse model. Hypodermal cellular infiltration without thickening of skin layers was observed after one epicutaneous exposure to antigen ovalbumin (OVA), compared to controls. In contrast to our previous report using female mice, OVA treatment did not activate skin mast cells (MC) or elevate sphingosine-1-phosphate (S1P) levels while increasing systemic but not local levels of CCL2, CCL3 and CCL5 chemokines. In contrast to the pathogenic AD mechanisms we recently uncovered in female, S1P-mediated skin MC activation with subsequent local chemokine production is not observed in male mice, supporting sex differences in pre-lesional stages of AD. We are proposing that differential involvement of the MC/S1P axis in early pathogenic skin changes contributes to the well documented yet still incompletely understood sex-dimorphic susceptibility to AD in humans.

Abstract A48: Quantifying stroma-tumor cell interactions in three-dimensional cell culture systems

Abstract Background: In cancer research, studying cell motility is fundamental to investigate cancer invasion and drug resistance. In solid tumors constituted by a huge stroma component, such as pancreatic ductal adenocarcinoma (PDAC), the ability to quantify cell movement and interactions is mandatory to better understand the complex crosstalk between cancer and stroma cells. In PDAC, the pancreatic stellate cells (PSCs) are the principal source of dense fibrotic stroma: these closely interact with the PDAC cells to create a facilitating tumor microenvironment that supports local and distant tumor progression through the secretion and/or the response to a number of cytokines that globally increase cancer invasiveness; moreover, the dense tumor microenvironment contributes to resistance to chemotherapy and radiation therapy. Recent studies have established that targeting the stromal compartment in PDAC may lead to promising outcomes. Unfortunately, significant improvements in the overall survival of patients have not been realized in more than four decades, in part because of the lack of relevant preclinical models. Methods: To infer interactions existing between stromal and cancer cells in complex 3D environments, we developed a novel platform that combines time-lapse fluorescence microscopy, automated image detection, and extensive statistical analysis. To better mimic in situ PDAC, we created a hydrogel-based (Matrigel, collagen) model that allows cell tracking in 4 dimensions (x, y, z, t), not possible in 2D, using a mixed population of L3.6PL pancreatic cancer cells and PSCs at a ratio of 25% and 75%, respectively. To precisely identify the two populations and facilitate automatic cell detection, cells were transfected with GFP and mCherry vectors. Once in the 3D conformation, the cocultures were monitored through time-lapse confocal microscopy (CLSM) in controlled conditions, and the dataset containing the temporal evolution of the cells was processed by statistical tools. Results: Key to our approach was the generation of new machine learning-driven automated inferential protocols that resulted in the high-resolution imaging of the strength of the interactions between cells, as well as the potential presence of local chemokine gradients. In our ongoing studies, both commercial PDAC cell lines and pancreatic stellate cells were used to establish the model system. We are currently adopting the model to take full advantage of patient-derived PDAC cells established using the conditionally reprogrammed cells technique, mixed with patient-matched stellate cells. Since cell migration is a hallmark of cancer, we believe that this platform could be used as a reliable and reproducible approach for studying single-cell migration and invasion also in patient-derived models. The research leading to these results received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 759959, ERC-StG “INTERCELLMED”). Citation Format: Marta Maria Cavo, Francesco Alemanno, Donatella Delle Cave, Eliana D'Amone, Adriano Barra, Enza Lonardo, Erika Parasido, Chris Albanese, Loretta Laureana del Mercato. Quantifying stroma-tumor cell interactions in three-dimensional cell culture systems [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A48.