Neutrophil-like cells derived from the HL-60 cell-line as a genetically-tractable model for neutrophil degranulation.

Research on neutrophil biology has been limited by the short life span and limited genetic manipulability of these cells, driving the need for representative and efficient model cell lines. The promyelocytic cell line HL-60 and its subline PLB-985 can be differentiated into neutrophil-like cells (NLCs) and have been used to study neutrophil functions including chemotaxis, phagocytosis, endocytosis, and degranulation. Compared to neutrophils derived from hematopoietic stem cells, NLCs serve as a cost-effective neutrophil model. NLCs derived from both HL-60 and PLB-985 cells have been shown to perform degranulation, an important neutrophil function. However, no study has directly compared the two lines as models for degranulation including their release of different types of mobilizable organelles. Furthermore, Nutridoma, a commercially available supplement, has recently been shown to improve the chemotaxis, phagocytosis, and oxidative burst abilities of NLCs derived from promyelocytic cells, however it is unknown whether this reagent also improves the degranulation ability of NLCs. Here, we show that NLCs derived from both HL-60 and PLB-985 cells are capable of degranulating, with each showing markers for the release of multiple types of secretory organelles, including primary granules. We also show that differentiating HL-60 cells using Nutridoma does not enhance their degranulation activity over NLCs differentiated using Dimethyl Sulfoxide (DMSO) plus Granulocyte-colony stimulating factor (G-CSF). Finally, we show that promyelocytic cells can be genetically engineered and differentiated using these methods, to yield NLCs with a defect in degranulation. Our results indicate that both cell lines serve as effective models for investigating the mechanisms of neutrophil degranulation, which can advance our understanding of the roles of neutrophils in inflammation and immunity.

Complement receptor 3 is required for maximum in vitro trogocytic killing of the parasite Trichomonas vaginalis by human neutrophil-like cells.

Trichomonas vaginalis (Tv) is a parasite that causes trichomoniasis, a prevalent sexually-transmitted infection. Neutrophils are found at the site of infection, and can rapidly kill the parasite in vitro, using trogocytosis. However, the specific molecular players in neutrophil killing of Tv are unknown. Here, we show that complement proteins play a role in Tv killing by human neutrophil-like cells (NLCs). Using CRISPR/Cas9, we generated NLCs deficient in each of three complement receptors (CRs) known to be expressed on human neutrophils: CR1, CR3, and CR4. Using in vitro trogocytosis assays, we found that CR3, but not CR1 or CR4 is required for maximum trogocytosis of the parasite by NLCs, with NLCs lacking CR3 demonstrating ~40% reduction in trogocytosis, on average. We also observed a reduction in NLC killing of Tv in CR3 knockout, but not CR1 or CR4 knockout NLCs. On average, NLCs lacking CR3 had ~50% reduction in killing activity. We also used a parallel approach of pre-incubating NLCs with blocking antibodies against CR3, which similarly reduced NLC killing of parasites. These data support a model in which Tv is opsonized by the complement protein iC3b, and bound by neutrophil CR3 receptor, to facilitate trogocytic killing of the parasite.

Bovine neutrophils kill the sexually-transmitted parasite Tritrichomonas foetus using trogocytosis.

The protozoan parasite Tritrichomonas foetus (T. foetus) is the causative organism of bovine trichomonosis (also referred to as trichomoniasis), a sexually-transmitted infection that reduces fertility in cattle. Efforts to control trichomonosis on cattle farms are hindered by the discouragement of antibiotic use in agriculture, and the incomplete, short-lived protection conferred by the current vaccines. A more complete mechanistic understanding of what effective immunity to T. foetus entails could enable the development of more robust infection control strategies. While neutrophils, the primary responders to infection, are present in infected tissues and have been shown to kill the parasite in vitro, the mechanism they use for parasite killing has not been established. Here, we show that primary bovine neutrophils isolated from peripheral blood rapidly kill T. foetus in vitro in a dose-dependent manner, and that optimal parasite killing is reduced by inhibitors of trogocytosis. We also use imaging to show that bovine neutrophils surround T. foetus and trogocytose its membrane. These findings are consistent with killing via trogocytosis, a recently described novel neutrophil antimicrobial mechanism.

Trichomonas vaginalis adherence phenotypes and extracellular vesicles impact parasite survival in a novel in vivo model of pathogenesis.

Trichomonas vaginalis is a human infective parasite responsible for trichomoniasis-the most common, non-viral, sexually transmitted infection worldwide. T. vaginalis resides exclusively in the urogenital tract of both men and women. In women, T. vaginalis has been found colonizing the cervix and vaginal tract while in men it has been identified in the upper and lower urogenital tract and in secreted fluids such as semen, urethral discharge, urine, and prostatic fluid. Despite the over 270 million cases of trichomoniasis annually worldwide, T. vaginalis continues to be a highly neglected organism and thus poorly studied. Here we have developed a male mouse model for studying T. vaginalis pathogenesis in vivo by delivering parasites into the murine urogenital tract (MUT) via transurethral catheterization. Parasite burden was assessed ex-vivo using a nanoluciferase-based gene expression assay which allowed quantification of parasites pre- and post-inoculation. Using this model and read-out approach, we show that T. vaginalis can be found within MUT tissue up to 72 hrs post-inoculation. Furthermore, we also demonstrate that parasites that exhibit increased parasite adherence in vitro also have higher parasite burden in mice in vivo. These data provide evidence that parasite adherence to host cells aids in parasite persistence in vivo and molecular determinants found to correlate with host cell adherence in vitro are applicable to infection in vivo. Finally, we show that co-inoculation of T. vaginalis extracellular vesicles (TvEVs) and parasites results in higher parasite burden in vivo. These findings confirm our previous in vitro-based predictions that TvEVs assist the parasite in colonizing the host. The establishment of this pathogenesis model for T. vaginalis sets the stage for identifying and examining parasite factors that contribute to and influence infection outcomes.

Human neutrophil-like cells demonstrate antimicrobial responses to the chronic cyst form of Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii infects approximately 2.5 billion people worldwide. Infection induces a rapid dissemination of parasites throughout the body followed by the formation of lifelong cysts within neurons of the host brain. Both stages require a dynamic immune response comprised of both innate and adaptive cells. Neutrophils are a primary responding cell to acute infection and have been observed in the brain during murine chronic infection. Previous studies investigating human neutrophils found that invasion by Toxoplasma tachyzoites inhibits apoptosis of neutrophils, prolonging their survival under inflammatory conditions. Here, we demonstrate the differentiation of two distinct subsets following exposure of human neutrophil-like-cells (HNLC) to Toxoplasma cysts. In vitro stimulation and imaging studies show cyst-specific induction of cytokines and cyst clearance by HNLCs. Further testing demonstrates that aged HNLCs perform less phagocytosis of cysts compared to non-aged HNLCs. In conclusion, this study identifies a novel response of HNLCs to Toxoplasma cysts and may indicate a role for neutrophils in the clearance of cysts during human infection with Toxoplasma.

Neutrophil interactions with the sexually transmitted parasite Trichomonas vaginalis: implications for immunity and pathogenesis.

Trichomoniasis is the third most common sexually transmitted infection in humans and is caused by the protozoan parasite, Trichomonas vaginalis (Tv). Pathogenic outcomes are more common in women and generally include mild vaginitis or cervicitis. However, more serious effects associated with trichomoniasis include adverse reproductive outcomes. Like other infectious agents, pathogenesis from Tv infection is predicted to be the result of both parasite and host factors. At the site of infection, neutrophils are the most abundant immune cells present and probably play key roles in both parasite clearance and inflammatory pathology. Here, we discuss the evidence that neutrophils home to the site of Tv infection, kill the parasite, and that in some circumstances, parasites possibly evade neutrophil-directed killing. In vitro, the parasite is killed by neutrophils using a novel antimicrobial mechanism called trogocytosis, which probably involves both innate and adaptive immunity. While mechanisms of evasion are mostly conjecture at present, the persistence of Tv infections in patients argues strongly for their existence. Additionally, many strains of Tv harbour microbial symbionts Mycoplasma hominis or Trichomonasvirus, which are both predicted to impact neutrophil responses against the parasite. Novel research tools, especially animal models, will help to reveal the true outcomes of many factors involved in neutrophil-Tv interactions during trichomoniasis.

Active Learning and Technology Approaches for Teaching Immunology to Undergraduate Students.

Immunology is a fascinating and extremely complex field, with natural connections to many disciplines both within STEM and beyond. Teaching an undergraduate course in immunology therefore provides both opportunities and challenges. Significant challenges to student learning include mastering the volume of new vocabulary and figuring out how to think coherently about a physiological system that is so anatomically disseminated. More importantly, teaching immunology can be complicated because it requires students to integrate knowledge derived from prior introductory courses in a range of fields, including cell biology, biochemistry, anatomy and genetics. However, this also provides an opportunity to use the study of the immune system as a platform on which students can assemble and integrate foundational STEM knowledge, while also learning about a new and exciting field. Pedagogical theory has taught us that students learn best by engaging with complicated questions and by thinking metacognitively about how to approach solutions. Building this skill set in today's students, who now hail from a broad demographic and who are accustomed to acquiring their knowledge from a variety of different media, requires a new set of teaching tools. Using perspectives from four different immunology educators, we describe a range of student-centered, active learning approaches that have been field-tested in a number of different immunology classrooms and that are geared to a variety of learning styles. In this paper, we explore the hypothesis that active learning approaches to immunology improve comprehension and retention by increasing student engagement in class and their subsequent mastery of complex topics.

Targeting the Achievement Gap: Strategies Toward Removing Inequities in Undergraduate Immunology Education.

A diverse student body enriches the classroom with lived experiences, varied skillsets, community and cultural knowledge, resiliency, and altruistic interests, all critical attributes that benefit both the classroom and the STEM field at large. However, a persistent disparity in academic and educational attainment exists between under-represented minority (URM) and non-URM students in STEM fields. This achievement gap discourages talented URM students from entering STEM professions, threatening the potential, expertise, and perspective of these professions. Here we describe the factors that contribute to the achievement gap and present strategies, utilized in our Immunology classrooms, for combating each factor. We discuss project-based learning pedagogy to give students increased agency and feelings of empowerment. We also highlight concrete practices to foster students' science identities and sense of community, factors that highly promote STEM retention. The dynamic subject of Immunology provides myriad opportunities to implement a curriculum committed to equity, as we outline below.

Trichomonas vaginalis: Pathogenesis, Symbiont Interactions, and Host Cell Immune Responses.

The parasite Trichomonas vaginalis (Tv) causes a highly prevalent sexually transmitted infection. As an extracellular pathogen, the parasite mediates adherence to epithelial cells to colonize the human host. In addition, the parasite interfaces with the host immune system and the vaginal microbiota. Modes of Tv pathogenesis include damage to host tissue mediated by parasite killing of host cells, disruption of steady-state vaginal microbial ecology, and eliciting inflammation by activating the host immune response. Recent Tv research has uncovered new players that contribute to multifactorial mechanisms of host-parasite adherence and killing, and has examined the relationship between Tv and vaginal bacteria. Mechanisms that may lead to parasite recognition and killing, or the evasion of host immune cells, have also been revealed.

Neutrophils kill the parasite Trichomonas vaginalis using trogocytosis.

T. vaginalis, a human-infective parasite, causes the most common nonviral sexually transmitted infection (STI) worldwide and contributes to adverse inflammatory disorders. The immune response to T. vaginalis is poorly understood. Neutrophils (polymorphonuclear cells [PMNs]) are the major immune cell present at the T. vaginalis-host interface and are thought to clear T. vaginalis. However, the mechanism of PMN clearance of T. vaginalis has not been characterized. We demonstrate that human PMNs rapidly kill T. vaginalis in a dose-dependent, contact-dependent, and neutrophil extracellular trap (NET)-independent manner. In contrast to phagocytosis, we observed that PMN killing of T. vaginalis involves taking "bites" of T. vaginalis prior to parasite death, using trogocytosis to achieve pathogen killing. Both trogocytosis and parasite killing are dependent on the presence of PMN serine proteases and human serum factors. Our analyses provide the first demonstration, to our knowledge, of a mammalian phagocyte using trogocytosis for pathogen clearance and reveal a novel mechanism used by PMNs to kill a large, highly motile target.

Leukocyte Lysis and Cytokine Induction by the Human Sexually Transmitted Parasite Trichomonas vaginalis.

Trichomonas vaginalis (Tv) is an extracellular protozoan parasite that causes the most common non-viral sexually transmitted infection: trichomoniasis. While acute symptoms in women may include vaginitis, infections are often asymptomatic, but can persist and are associated with medical complications including increased HIV susceptibility, infertility, pre-term labor, and higher incidence of cervical cancer. Heightened inflammation resulting from Tv infection could account for these complications. Effective cellular immune responses to Tv have not been characterized, and re-infection is common, suggesting a dysfunctional adaptive immune response. Using primary human leukocyte components, we have established an in vitro co-culture system to assess the interaction between Tv and the cells of the human immune system. We determined that in vitro, Tv is able to lyse T-cells and B-cells, showing a preference for B-cells. We also found that Tv lysis of lymphocytes was mediated by contact-dependent and soluble factors. Tv lysis of monocytes is far less efficient, and almost entirely contact-dependent. Interestingly, a common symbiont of Tv, Mycoplasma hominis, did not affect cytolytic activity of the parasite, but had a major impact on cytokine responses. M. hominis enabled more diverse inflammatory cytokine secretion in response to Tv and, of the cytokines tested, Tv strains cleared of M. hominis induced only IL-8 secretion from monocytes. The quality of the adaptive immune response to Tv is therefore likely influenced by Tv symbionts, commensals, and concomitant infections, and may be further complicated by direct parasite lysis of effector immune cells.

Differentiation of IL-17-producing effector and regulatory human T cells from lineage-committed naive precursors.

A subset of human regulatory T cells (Tregs) secretes IL-17 and thus resembles Th17 effector cells. How IL-17(+) Tregs differentiate from naive precursors remains unclear. In this study, we show that IL-17-producing T cells can differentiate from CCR6(+) naive T cell precursors in the presence of IL-2, IL-1ß, TGF-ß, and IL-23. CCR6(+) naive T cells are present in adult peripheral and umbilical cord blood and in both conventional T naive and FOXP3(+) naive Treg subsets. IL-17(+) cells derived from CCR6(+) naive Tregs (referred to as IL-17(+) Tregs) express FOXP3 but not HELIOS, another Treg-associated transcription factor, and these cells display suppressor capacity and a surface phenotype resembling memory Tregs. Remarkably, the IL-17(+) Treg compartment was preferentially reduced relative to the canonical Th17 and Treg compartments in a subset of HIV(+) subjects, suggesting a specific perturbation of this subset during the course of disease. Our findings that CCR6(+) naive precursors contain a predetermined reservoir to replenish IL-17-secreting cells may have implications in balancing the Th17 and IL-17(+) Treg compartments that are perturbed during HIV infection and potentially in other inflammatory diseases.

Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses.

The human-infective parasite Trichomonas vaginalis causes the most prevalent nonviral sexually transmitted infection worldwide. Infections in men may result in colonization of the prostate and are correlated with increased risk of aggressive prostate cancer. We have found that T. vaginalis secretes a protein, T. vaginalis macrophage migration inhibitory factor (TvMIF), that is 47% similar to human macrophage migration inhibitory factor (HuMIF), a proinflammatory cytokine. Because HuMIF is reported to be elevated in prostate cancer and inflammation plays an important role in the initiation and progression of cancers, we have explored a role for TvMIF in prostate cancer. Here, we show that TvMIF has tautomerase activity, inhibits macrophage migration, and is proinflammatory. We also demonstrate that TvMIF binds the human CD74 MIF receptor with high affinity, comparable to that of HuMIF, which triggers activation of ERK, Akt, and Bcl-2-associated death promoter phosphorylation at a physiologically relevant concentration (1 ng/mL, 80 pM). TvMIF increases the in vitro growth and invasion through Matrigel of benign and prostate cancer cells. Sera from patients infected with T. vaginalis are reactive to TvMIF, especially in males. The presence of anti-TvMIF antibodies indicates that TvMIF is released by the parasite and elicits host immune responses during infection. Together, these data indicate that chronic T. vaginalis infections may result in TvMIF-driven inflammation and cell proliferation, thus triggering pathways that contribute to the promotion and progression of prostate cancer.

The metalloprotease ADAM12 regulates the effector function of human Th17 cells.

A key modulator of immune homeostasis, TGFß has an important role in the differentiation of regulatory T cells (Tregs) and IL-17-secreting T cells (Th17). How TGFß regulates these functionally opposing T cell subsets is not well understood. We determined that an ADAM family metalloprotease called ADAM12 is specifically and highly expressed in both Tregs and CCR6+ Th17 cells. ADAM12 is induced in vitro upon differentiation of naïve T cells to Th17 cells or IL-17-secreting Tregs. Remarkably, silencing ADAM12 expression in CCR6+ memory T cells enhances the production of Th17 cytokines, similar to suppressing TGFß signaling. Further, ADAM12 knockdown in naïve human T cells polarized towards Th17/Treg cells, or ectopically expressing RORC, greatly enhances IL-17-secreting cell differentiation, more potently then inhibiting TGFß signals. Together, our findings reveal a novel regulatory role for ADAM12 in Th17 cell differentiation or function and may have implications in regulating their aberrant responses during immune pathologies.

Probing the effector and suppressive functions of human T cell subsets using antigen-specific engineered T cell receptors.

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8(+) and CD4(+) T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression.

Expression and function of TNF and IL-1 receptors on human regulatory T cells.

Regulatory T cells (Tregs) suppress immune activation and are critical in preventing autoimmune diseases. While the ability of Tregs to inhibit proliferation of other T cells is well established, it is not yet clear whether Tregs also modulate inflammatory cytokines during an immune response. Here, we show that the expression of inflammatory cytokine receptors IL-1R1 and TNFR2 were higher on resting mature Tregs compared to naïve or memory T cells. While upon activation through the T cell receptor (TCR), expression of IL-1R1 and TNFR2 were upregulated on all T cell subsets, IL-1R1 maintained significantly higher expression on activated Tregs as compared to other T cell subsets. The decoy receptor for IL-1 (IL-1R2) was not expressed by any of the resting T cells but was rapidly upregulated and preferentially expressed upon TCR-stimulation on Tregs. In addition, we found that Tregs also expressed high levels of mRNA for IL-1 antagonist, IL-1RA. TCR-stimulation of naïve T cells in the presence of TGFbeta, which induces FOXP3 expression, however did not result in upregulation of IL-1R1 or IL-1R2. In addition, ectopic expression of FOXP3 in non-Tregs, while causing significant upregulation of IL-1R1 and IL-1R2, did not achieve the levels seen in bona fide Tregs. We also determined that resting human Tregs expressing IL-1R1 did not have higher suppressive capacity compared to IL-1R1- Tregs, suggesting that IL-1R1 does not discriminate suppressive resting Tregs in healthy individuals. Functionally, activated human Tregs displayed a capacity to neutralize IL-1beta, which suggests a physiological significance for the expression of IL-1 decoy receptor on Tregs. In conclusion, our findings that human Tregs preferentially express receptors for TNF and IL-1 suggest a potential function in sensing and dampening local inflammation.

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFbeta, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP- cells. Remarkably, CD25+GARP- T cells expanded in culture contained 3-5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25-GARP- cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) -infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation.

The biology of FoxP3: a key player in immune suppression during infections, autoimmune diseases and cancer.

The Transcription factor FoxP3 belongs to the forkhead/winged-helix family of transcriptional regulators and shares general structural features with other FoxP family members. FoxP3 functions as a master of transcription for the development of regulatory T-cells (Treg cells) both in humans and in mice. Natural genetic mutations ofFoxP3 that disrupt its function in humans result in an autoimmune syndrome called Immune Polyendocrinopathy, Enteropathy, X-linked (IPEX) and in mice, its deletion causes the Scurfy phenotype, with similar pathology. The finding that FoxP3 is required for the development and function of Tregs has led to an explosion of research in determining its regulation and function in the immune system. Understanding the biological properties of FoxP3 has a wide range of implications for immune tolerance, autoimmune disorders, inflammation and immune response to infectious diseases and cancer.