A Retrospective Analysis of Leadership, Awardees, and Member Gender Representation of the Canadian Society for Immunology.

The Canadian Society for Immunology (CSI) established a formal Equity, Diversity, and Inclusion (EDI) Committee with the goal of providing EDI advocacy and leadership within the CSI, as well as in the broader scientific community. A first task of this committee was to review the publicly available historical data on gender representation within the CSI's membership, leadership, award recipients, and conference chairs/presenters as a step in establishing a baseline reference point and monitoring the trajectory of future success in achieving true inclusion. We found that, except for overall membership and a specific subset of awards, all categories showed a historical bias toward men, particularly prior to 2010. Bias persists in various categories, evident even in recent years. However, we note an encouraging trend toward greater gender parity, particularly in the roles of President, symposium presenters, and workshop chairs, especially from 2017 onward. We present these findings as well as our recommendations to enhance inclusivity. These include a more comprehensive collection and secure storage of self-identification data, emphasis on EDI as an essential component of all annual meeting activities, and innovative measures of outreach, collaboration, and leadership with the aim of making the CSI a model for improving EDI in other professional research societies.

Trickle infection with Heligmosomoides polygyrus results in decreased worm burdens but increased intestinal inflammation and scarring.

Introduction: Intestinal roundworms cause chronic debilitating disease in animals, including humans. Traditional experimental models of these types of infection use a large single-dose infection. However, in natural settings, hosts are exposed to parasites on a regular basis and when mice are exposed to frequent, smaller doses of Heligmosomoides polygyrus, the parasites are cleared more quickly. Whether this more effective host response has any negative consequences for the host is not known. Results: Using a trickle model of infection, we found that worm clearance was associated with known resistance-related host responses: increased granuloma and tuft cell numbers, increased levels of granuloma IgG and decreased intestinal transit time, as well as higher serum IgE levels. However, we found that the improved worm clearance was also associated with an inflammatory phenotype in and around the granuloma, increased smooth muscle hypertrophy/hyperplasia, and elevated levels of Adamts gene expression. Discussion: To our knowledge, we are the first to identify the involvement of this protein family of matrix metalloproteinases (MMPs) in host responses to helminth infections. Our results highlight the delicate balance between parasite clearance and host tissue damage, which both contribute to host pathology. When continually exposed to parasitic worms, improved clearance comes at a cost.

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System.

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

The global response to the COVID-19 pandemic: how have immunology societies contributed?

The COVID-19 pandemic is shining a spotlight on the field of immunology like never before. To appreciate the diverse ways in which immunologists have contributed, Nature Reviews Immunology invited the president of the International Union of Immunological Societies and the presidents of 15 other national immunology societies to discuss how they and their members responded following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Intravital imaging of eosinophils: Unwrapping the enigma.

Eosinophils are traditionally associated with allergic and parasitic inflammation. More recently, eosinophils have also been shown to have roles in diverse processes including development, intestinal health, thymic selection, and B-cell survival with the majority of these insights being derived from murine models and in vitro assays. Despite this, tools to measure the dynamic activity of eosinophils in situ have been lacking. Intravital microscopy is a powerful tool that enables direct visualization of leukocytes and their dynamic behavior in real-time in a wide range of processes in both health and disease. Until recently eosinophil researchers have not been able to take full advantage of this technology due to a lack of tools such as genetically encoded reporter mice. This mini-review examines the history of intravital microscopy with a focus on eosinophils. The development and use of eosinophil-specific Cre (EoCre) mice to create GFP and tdTomato fluorescent reporter animals is also described. Genetically encoded eosinophil reporter mice combined with intravital microscopy provide a powerful tool to add to the toolbox of technologies that will help us unravel the mysteries still surrounding this cell.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Synthesis of Sialyl LewisX Glycomimetics Bearing a Bicyclic 3- O,4- C-Fused Galactopyranoside Scaffold.

Reported herein is the synthesis of sialyl LewisX analogues bearing a trans-bicyclo[4.4.0] dioxadecane-modified 3- O,4- C-fused galactopyranoside scaffold that locks the carboxylate pharmacophore in either the axial or equatorial position. This novel series of bicyclic galactopyranosides are prepared through a stereocontrolled intramolecular cyclization reaction that has been evaluated both experimentally and by density functional theory calculations. The cyclization precursors are obtained from ß-d-galactose pentaacetate in a nine-step sequence featuring a highly diastereoselective equatorial alkynylation and Cu(I) catalyzed formation of the acetylenic α-ketoester moiety. Preliminary biological evaluations indicate improved activity as P-selectin antagonists for the axially configured analogues as compared to their equatorial counterparts.

Intravital imaging allows real-time characterization of tissue resident eosinophils.

Eosinophils are core components of the immune system, yet tools are lacking to directly observe eosinophils in action in vivo. To better understand the role of tissue resident eosinophils, we used eosinophil-specific CRE (eoCRE) mice to create GFP and tdTomato reporters. We then employed intravital microscopy to examine the dynamic behaviour of eosinophils in the healthy GI tract, mesentery, liver, lymph node, skin and lung. Given the role of eosinophils in allergic airway diseases, we also examined eosinophils in the lung following ovalbumin sensitization and challenge. We were able to monitor and quantify eosinophilic behaviours including patrolling, crawling, clustering, tissue distribution and interactions with other leukocytes. Thus, these reporter mice allow eosinophils to be examined in real-time in living animals, paving the way to further understanding the roles eosinophils play in both health and disease.

Inhibiting focal adhesion kinase (FAK) blocks IL-4 induced VCAM-1 expression and eosinophil recruitment in vitro and in vivo.

Leukocyte recruitment plays a critical role during both normal inflammation and chronic inflammatory diseases, and ongoing studies endeavor to better understand the complexities of this process. Focal adhesion kinase (FAK) is well known for its role in cancer, yet it also has been shown to regulate aspects of neutrophil and B16 melanoma cell recruitment by rapidly influencing endothelial cell focal adhesion dynamics and junctional opening. Recently, we found that FAK related non-kinase (FRNK), a protein that is often used as a FAK dominant negative, blocked eosinophil transmigration by preventing the transcription of vascular cell adhesion molecule-1 (VCAM-1) and eotaxin-3 (CCL26). Surprisingly, the blocking occurred even in the absence of endogenous FAK. To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vitro and in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment.

An orientation to wellness for new faculty of medicine members: meeting a need in faculty development.

OBJECTIVES: To evaluate the format, content, and effectiveness of a newly developed orientation to wellness workshop, and to explore participants' overall perceptions. METHODS: This was a mixed methods study. Participants consisted of 47 new faculty of medicine members who attended one of the four workshops held between 2011 and 2013. Questionnaires were used to evaluate workshop characteristics (10 survey items; response scale 1=unacceptable to 7=outstanding), intention to change behavior (yes/no), and retrospective pre/post workshop self-efficacy (4 survey items; response scale 1=no confidence to 6=absolute confidence). Mean scores and standard deviations were calculated for the workshop characteristics. Pre/post workshop self-efficacy scores were compared using a Wilcoxon signed-rank test. Participants' written qualitative feedback was coded using an inductive strategy to identify themes. RESULTS: There was strong support for the workshop characteristics with mean scores entirely above 6.00 (N=42). Thirty-one of 34 respondents (91%) expressed intention to change their behavior as a result of participating in the workshop. The post workshop self-efficacy scores (N=38 respondents) increased significantly for all four items (p<0.0001) compared to pre workshop ratings. Participants perceived the key workshop elements as the evidence-based content relevant to academic physicians, incorporation of practical tips and strategies, and an atmosphere conducive to discussion and experience sharing. CONCLUSIONS:   Participants welcomed wellness as a focus of faculty development. Enhancing instruction around wellness has the potential to contribute positively to the professional competency and overall functioning of faculty of medicine members.

Abnormal cannabidiol attenuates experimental colitis in mice, promotes wound healing and inhibits neutrophil recruitment.

BACKGROUND: Non-psychotropic atypical cannabinoids have therapeutic potential in a variety of inflammatory conditions including those of the gastrointestinal tract. Here we examined the effects of the atypical cannabinoid abnormal cannabidiol (Abn-CBD) on wound healing, inflammatory cell recruitment and colitis in mice. METHODS: Colitis was induced in CD1 mice by a single intrarectal administration of trinitrobenzene sulfonic acid (TNBS, 4 mg/100 µl in 30 % ethanol) and Abn-CBD and/or the antagonists O-1918 (Abd-CBD), AM251 (CB1 receptor) and AM630 (CB2 receptor), were administered intraperitoneally (all 5 mg/kg, twice daily for 3 days). The degree of colitis was assessed macro- and microscopically and tissue myeloperoxidase activity was determined. The effects of Abn-CBD on wound healing of endothelial and epithelial cells (LoVo) were assessed in a scratch injury assay. Human neutrophils were employed in Transwell assays or perfused over human umbilical vein endothelial cells (HUVEC) to study the effect of Abn-CBD on neutrophil accumulation and transmigration. RESULTS: TNBS-induced colitis was attenuated by treatment with Abn-CBD. Histological, macroscopic colitis scores and tissue myeloperoxidase activity were significantly reduced. These effects were inhibited by O-1918, but not by AM630, and only in part by AM251. Wound healing of both HUVEC and LoVo cells was enhanced by Abn-CBD. Abn-CBD inhibited neutrophil migration towards IL-8, and dose-dependently inhibited accumulation of neutrophils on HUVEC. CONCLUSIONS: Abn-CBD is protective against TNBS-induced colitis, promotes wound healing of endothelial and epithelial cells and inhibits neutrophil accumulation on HUVEC monolayers. Thus, the atypical cannabinoid Abn-CBD represents a novel potential therapeutic in the treatment of intestinal inflammatory diseases.

Peloruside A is a microtubule-stabilizing agent with exceptional anti-migratory properties in human endothelial cells.

Peloruside A is a novel antimitotic drug originally isolated from the marine sponge Mycale hentschieli. Previous studies showed that peloruside A stabilizes microtubules by binding to a site on tubulin distinct from paclitaxel, another microtubule stabilizing drug. Peloruside A blocks mitosis, but little is known about the effects on other cellular activities. Here we report that peloruside A is the most potent microtubule inhibitor yet tested for its ability to block endothelial cell migration. Quantitative analysis indicated that it inhibits microtubule dynamics and endothelial cell migration at 1/200(th) of the concentration needed to inhibit cell division (the cytotoxic concentration), indicating that it could potentially have a large margin of safety when used to specifically target angiogenesis. By comparison, paclitaxel, a well-known cancer therapeutic drug, suppresses cell migration at 1/13(th) of its cytotoxic concentration; and vinblastine suppresses cell migration at just slightly below its cytotoxic antimitotic concentration. Thus, different microtubule targeted drugs have varying relative potencies for inhibition of cell migration versus cell division. The results suggest that peloruside A may be an especially useful agent for anti-angiogenesis therapy and point to the likelihood that other antimitotic drugs might be found with an even larger potential margin of safety.

FRNK negatively regulates IL-4-mediated inflammation.

Focal adhesion kinase (FAK)-related nonkinase (PTK2 isoform 6 in humans, hereafter referred to as FRNK) is a cytoskeletal regulatory protein that has recently been shown to dampen lung fibrosis, yet its role in inflammation is unknown. Here, we show for the first time that expression of FRNK negatively regulates IL-4-mediated inflammation in a human model of eosinophil recruitment. Mechanistically, FRNK blocks eosinophil accumulation, firm adhesion and transmigration by preventing transcription and protein expression of VCAM-1 and CCL26. IL-4 activates STAT6 to induce VCAM-1 and CCL26 transcription. We now show that IL-4 also increases GATA6 to induce VCAM-1 expression. FRNK blocks IL-4-induced GATA6 transcription but has little effect on GATA6 protein expression and no effect on STAT6 activation. FRNK can block FAK or Pyk2 signaling and we, thus, downregulated these proteins using siRNA to determine whether signaling from either protein is involved in the regulation of VCAM-1 and CCL26. Knockdown of FAK, Pyk2 or both had no effect on VCAM-1 or CCL26 expression, which suggests that FRNK acts independently of FAK and Pyk2 signaling. Finally, we found that IL-4 induces the late expression of endogenous FRNK. In summary, FRNK represents a novel mechanism to negatively regulate IL-4-mediated inflammation.

Microtubule dynamics control tail retraction in migrating vascular endothelial cells.

Drugs that target microtubules are potent inhibitors of angiogenesis, but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; however, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish, rather than elongated, cells, rapid but nondirectional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells, whereas more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay.

Studying leukocyte recruitment under flow conditions.

Leukocyte recruitment from the vasculature occurs under conditions of haemodynamic shear stress. The parallel plate flow chamber apparatus is an in vitro system that is widely used to study leukocyte recruitment under shear conditions. The flow chamber is a versatile tool for examining adhesive interactions, as it can be used to study a variety of adhesive substrates, ranging from monolayers of primary cells to isolated adhesion molecules, and a variety of adhesive particles, ranging from leukocytes in whole blood to antibody-coated latex beads. We describe here methods for studying leukocyte recruitment to cytokine-stimulated, transfected or transduced endothelial cells using both whole blood and isolated leukocyte suspensions. These methods enable multiple parameters to be measured, including the total number of recruited leukocytes, the percentage of leukocytes that are rolling or firmly adherent, and the percentage of leukocytes that have transmigrated. Although these methods are described for interactions between leukocytes and endothelial cells, they are broadly applicable to the study of interactions between many combinations of adhesive substrates and adhesive particles.

The role of microtubules and their dynamics in cell migration.

Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds, but the movement became more random. We conclude that microtubules act both to restrain cell movement and to establish directionality.

Isolation of human umbilical vein endothelial cells and their use in the study of neutrophil transmigration under flow conditions.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes. This method focuses on neutrophil transmigration across human endothelial cells. One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC). Neutrophil isolation has been described visually elsewhere; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature. The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass. Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi µslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

Endothelial paxillin and focal adhesion kinase (FAK) play a critical role in neutrophil transmigration.

During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins ß1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.

Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo.

The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin-binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1ß [IL-1ß]). Endothelial domes were significantly reduced in Lsp1(-/-) compared with wild-type (WT) mice. Lsp1(-/-) animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.