JAK/STAT Signaling Predominates in Human and Murine Fungal Post-infectious Inflammatory Response Syndrome.

Post-infection inflammatory syndromes have been increasingly recognized as a cause of host damage in a variety of infectious diseases including tuberculosis, bacterial meningitis, and COVID-19. Recently, a post-infectious inflammatory response syndrome (PIIRS) was described in non-HIV-infected cryptococcal fungal meningoencephalitis (CM) as a major cause of mortality. Inflammatory syndromes are particularly severe in neurological infections due to the skull's rigid structure which limits unchecked tissue expansion from inflammatory-induced edema. In the present studies, neurologic transcriptional pathway analysis utilizing a murine PIIRS model demonstrated a predominance of Janus kinase/signal transducer and activator of transcription (JAK/STAT) activation. JAK/STAT inhibitor treatment resulted in improvements in CNS damage markers, reductions in intrathecal CD44hiCD62lo CD4+ effector CD4+ T-cells and MHC II+ inflammatory myeloid cells, and weight gains in mice, the latter after treatment with antifungals. Based on these data, pathway-driven steroid-sparing human treatment for steroid-refractory PIIRS was initiated using short courses of the JAK/STAT inhibitor ruxolitinib. These were well tolerated and reduced activated HLA-DR+ CD4+ and CD8+ cells and inflammatory monocytes as well as improved brain imaging. Together, these findings support the role of JAK/STAT in PIIRS as well as further study of JAK/STAT inhibitors as potential adjunctive therapy for PIRS and other neural inflammatory syndromes.

Magic bubbles: utilizing histotripsy to modulate the tumor microenvironment and improve systemic anti-tumor immune responses.

Focused Ultrasound (FUS) is emerging as a promising primary and adjunct therapy for the treatment of cancer. This includes histotripsy, which is a noninvasive, non-ionizing, non-thermal ultrasound guided ablation modality. As histotripsy has progressed from bench-to-bedside, it has become evident that this therapy has benefits beyond local tumor ablation. Specifically, histotripsy has the potential to shift the local tumor microenvironment from immunologically 'cold' to 'hot'. This is associated with the production of damage associated molecular patterns, the release of a selection of proinflammatory mediators, and the induction of inflammatory forms of cell death in cells just outside of the treatment zone. In addition to the induction of this innate immune response, histotripsy can also improve engagement of the adaptive immune system and promote systemic anti-tumor immunity targeting distal tumors and metastatic lesions. These tantalizing observations suggest that, in settings of widely metastatic disease burden, selective histotripsy of a limited number of accessible tumors could be a means of maximizing responsiveness to systemic immunotherapy. More work is certainly needed to optimize treatment strategies that best synergize histotripsy parameters with innate and adaptive immune responses. Likewise, rigorous clinical studies are still necessary to verify the presence and repeatability of these phenomena in human patients. As this technology nears regulatory approval for clinical use, it is our expectation that the insights and immunomodulatory mechanisms summarized in this review will serve as directional guides for rational clinical studies to validate and optimize the potential immunotherapeutic role of histotripsy tumor ablation.

Spatiotemporal local and abscopal cell death and immune responses to histotripsy focused ultrasound tumor ablation.

Introduction: Histotripsy is a novel focused ultrasound tumor ablation modality with potent immunostimulatory effects. Methods: To measure the spatiotemporal kinetics of local andabscopal responses to histotripsy, C57BL/6 mice bearing bilateral flank B16 melanoma or Hepa1-6 hepatocellular carcinoma tumors were treated with unilateral sham or partial histotripsy. Treated and contralateral untreated (abscopal) tumors were analyzed using multicolor immunofluorescence, digital spatial profiling, RNA sequencing (RNASeq), and flow cytometry. Results: Unilateral histotripsy triggered abscopal tumor growth inhibition. Within the ablation zone, early high mobility group box protein 1 (HMGB1) release and necroptosis were accompanied by immunogenic cell death transcriptional responses in tumor cells and innate immune activation transcriptional responses in infiltrating myeloid and natural killer (NK) cells. Delayed CD8+ T cell intratumoral infiltration was spatiotemporally aligned with cancer cell features of ferroptosis; this effect was enhanced by CTLA-4 blockade and recapitulated in vitro when tumor-draining lymph node CD8+ T cells were co-cultured with tumor cells. Inoculation with cell-free tumor fractions generated by histotripsy but not radiation or freeze/thaw conferred partial protection from tumor challenge. Discussion: We propose that histotripsy may evoke local necroptotic immunogenic cell death, priming systemic adaptive immune responses and abscopal ferroptotic cancer cell death.

Caveolin-initiated macropinocytosis is required for efficient silica nanoparticles' transcytosis across the alveolar epithelial barrier.

Removal of particulate materials that would otherwise cumulate within the airspace and hinder the gas exchange is one of the central processes of maintaining lung homeostasis. While the importance of the particle uptake by alveolar macrophages and their expulsion via the airways mucociliary escalator is well established, very little is known about the alternative route for removing the particles via direct crossing the lung epithelium for transfer into the pulmonary lymph and bloodstream. This study dissected sequential mechanisms involved in nanoparticle transcytosis through the alveolar epithelial cell layer. By a combination of live cell, super resolution, and electron microscopy and RNA interference study, we have dissected temporal steps of nanoparticle transcytosis through alveolar epithelium. Our study revealed that caveolin is essential for the firm adhesion of the silica nanoparticle agglomerates to the apical membrane and their subsequent rapid internalization with the help of macropinocytic elements C-terminal-binding protein1 and Rabankyrin-5 but not dynamin. Actin, but not microtubules, played a major role in nanoparticle uptake and subsequent transportation. The compartments with nanoparticles were tethered to trans-Golgi network to be jointly transported along actin stress fibers across the cytoplasm, employing a myosin-dependent mechanism. The trans-Golgi nanoparticle transport machinery was positive to Rab6A, a marker linked to vesicle exocytosis. Exocytosis was primarily occurring at the basolateral plane of the alveolar epithelial cells. The high-proficiency novel caveolin and Rabankyrin-5 associated uptake and transcellular transport of nanoparticles across the AEC barrier supports its importance in clearance of amorphous silica and other types of non-inflammatory nanoparticles that are rapidly removed from the lungs following their inhalation.

Impact of Histotripsy on Development of Intrahepatic Metastases in a Rodent Liver Tumor Model.

Histotripsy has been used for tumor ablation, through controlled, non-invasive acoustic cavitation. This is the first study to evaluate the impact of partial histotripsy ablation on immune infiltration, survival outcomes, and metastasis development, in an in vivo orthotopic, immunocompetent rat HCC model (McA-RH7777). At 7−9 days post-tumor inoculation, the tumor grew to 5−10 mm, and ~50−75% tumor volume was treated by ultrasound-guided histotripsy, by delivering 1−2 cycle histotripsy pulses at 100 Hz PRF (focal peak negative pressure P− >30 MPa), using a custom 1 MHz transducer. Complete local tumor regression was observed on MRI in 9/11 histotripsy-treated rats, with no local recurrence or metastasis up to the 12-week study end point, and only a <1 mm residual scar tissue observed on histology. In comparison, 100% of untreated control animals demonstrated local tumor progression, developed intrahepatic metastases, and were euthanized at 1−3 weeks. Survival outcomes in histotripsy-treated animals were significantly improved compared to controls (p-value < 0.0001). There was evidence of potentially epithelial-to-mesenchymal transition (EMT) in control tumor and tissue healing in histotripsy-treated tumors. At 2- and 7-days post-histotripsy, increased immune infiltration of CD11b+, CD8+ and NK cells was observed, as compared to controls, which may have contributed to the eventual regression of the untargeted tumor region in histotripsy-treated tumors.

Subcellular localization of hippocampal ryanodine receptor 2 and its role in neuronal excitability and memory.

Ryanodine receptor 2 (RyR2) is abundantly expressed in the heart and brain. Mutations in RyR2 are associated with both cardiac arrhythmias and intellectual disability. While the mechanisms of RyR2-linked arrhythmias are well characterized, little is known about the mechanism underlying RyR2-associated intellectual disability. Here, we employed a mouse model expressing a green fluorescent protein (GFP)-tagged RyR2 and a specific GFP probe to determine the subcellular localization of RyR2 in hippocampus. GFP-RyR2 was predominantly detected in the soma and dendrites, but not the dendritic spines of CA1 pyramidal neurons or dentate gyrus granular neurons. GFP-RyR2 was also detected within the mossy fibers in the stratum lucidum of CA3, but not in the presynaptic terminals of CA1 neurons. An arrhythmogenic RyR2-R4496C+/- mutation downregulated the A-type K+ current and increased membrane excitability, but had little effect on the afterhyperpolarization current or presynaptic facilitation of CA1 neurons. The RyR2-R4496C+/- mutation also impaired hippocampal long-term potentiation, learning, and memory. These data reveal the precise subcellular distribution of hippocampal RyR2 and its important role in neuronal excitability, learning, and memory.

Inhibition of DNA-PK may improve response to neoadjuvant chemoradiotherapy in rectal cancer.

Treatment of locally advanced rectal cancer includes chemoradiation and surgery, but patient response to treatment is variable. Patients who have a complete response have improved outcomes; therefore, there is a critical need to identify mechanisms of resistance to circumvent them. DNA-PK is involved in the repair of DNA double-strand breaks caused by radiation, which we found to be increased in rectal cancer after treatment. We hypothesized that inhibiting this complex with a DNA-PK inhibitor, Peposertib (M3814), would improve treatment response. We assessed pDNA-PK in a rectal cancer cell line and mouse model utilizing western blotting, viability assays, γH2AX staining, and treatment response. The three treatment groups were: standard of care (SOC) (5-fluorouracil (5FU) with radiation), M3814 with radiation, and M3814 with SOC. SOC treatment of rectal cancer cells increased pDNA-PK protein and increased γH2AX foci, but this was abrogated by the addition of M3814. Mice with CT26 tumors treated with M3814 with SOC did not differ in average tumor size but individual tumor response varied. The clinical complete response rate improved significantly with the addition of M3814 but pathological complete response did not. We investigated alterations in DNA repair and found that Kap1 and pATM are increased after M3814 addition suggesting this may mediate resistance. When the DNA-PK inhibitor, M3814, is combined with SOC treatment, response improved in some rectal cancer models but an increase in other repair mechanisms likely diminishes the effect. A clinical trial is ongoing to further explore the role of DNA-PK inhibition in rectal cancer treatment.

Murine Inducible Nitric Oxide Synthase Expression Is Essential for Antifungal Defenses in Kidneys during Disseminated Cryptococcus deneoformans Infection.

Disseminated cryptococcosis has a nearly 70% mortality, mostly attributed to CNS infection, with lesser-known effects on other organs. Immune protection against Cryptococcus relies on Th1 immunity with M1 polarization, rendering macrophages fungicidal. The importance of M1-upregulated inducible NO synthase (iNOS) has been documented in pulmonary anticryptococcal defenses, whereas its role in disseminated cryptococcosis remains controversial. Here we examined the effect of iNOS deletion in disseminated (i.v.) C. deneoformans 52D infection, comparing wild-type (C57BL/6J) and iNOS-/- mice. iNOS-/- mice had significantly reduced survival and nearly 100-fold increase of the kidney fungal burden, without increases in the lungs, spleen, or brain. Histology revealed extensive lesions and almost complete destruction of the kidney cortical area with a loss of kidney function. The lack of fungal control was not due to a failure to recruit immune cells because iNOS-/- mice had increased kidney leukocytes. iNOS-/- mice also showed no defect in T cell polarization. We conclude that iNOS is critically required for local anticryptococcal defenses in the kidneys, whereas it appears to be dispensable in other organs during disseminated infection. This study exemplifies a unique phenotype of local immune defenses in the kidneys and the organ-specific importance of a single fungicidal pathway.

CCR2 Signaling Promotes Brain Infiltration of Inflammatory Monocytes and Contributes to Neuropathology during Cryptococcal Meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is a leading cause of central nervous system (CNS) infection-related mortality worldwide, with surviving patients often developing neurological deficiencies. While CNS inflammation has been implicated in the pathogenesis of CM, little is known about the relative contribution of the specific inflammatory/immune pathways to CNS pathology versus fungal clearance. Increased cerebrospinal fluid level of C-C chemokine receptor 2 (CCR2) ligand CCL2 is associated with disease deterioration in patients with CM. Using a murine model, we investigated the role of the CCR2 pathway in the development of CNS inflammation and pathology during CM. We found that CCR2-deficient mice exhibited improved 28-day survival and alleviated neurological disease scores despite a brain fungal burden higher than that of the WT mice. Reduced CM pathology in CCR2-deficient mice was accompanied by markedly decreased neuronal cell death around cryptococcal microcysts and restored expression of genes involved in neurotransmission, connectivity, and neuronal cell structure in the brains. Results show that CCR2 axis is the major pathway recruiting CD45hiCD11b+Ly6C+ inflammatory monocyte to the brain and indirectly modulates the accumulation of CD4+ T cells and CD8+ T cells. In particular, CCR2 axis promotes recruitment of interferon gamma (IFN-γ)-producing CD4+ T cells and classical activation of myeloid cells. In this context, CCR2 deletion limits the immune network dysregulation we see in CM and attenuates neuropathology. Thus, the CCR2 axis is a potential target for interventions aimed to limit inflammatory CNS pathology in CM patients. IMPORTANCE Cryptococcal meningoencephalitis (CM) causes nearly 200,000 deaths worldwide each year, and survivors frequently develop long-lasting neurological sequelae. The high rate of mortality and neurologic sequelae in CM patients indicate that antifungal therapies alone are often insufficient to control disease progression. Here, we reveal that CM disease progression in mice is accompanied by inflammatory monocytes infiltration at the periphery of the infected foci that overlap locally perturbed neuronal function and death. Importantly, we identified that CCR2 signaling is a critical pathway driving neuroinflammation, especially inflammatory monocyte recruitment, as well as CNS pathology and mortality in CM mice. Our results imply that targeting the CCR2 pathway may be beneficial as a therapy complementary to antifungal drug treatment, helping to reduce CNS damage and mortality in CM patients.

LINC00671 suppresses cell proliferation and metastasis in pancreatic cancer by inhibiting AKT and ERK signaling pathway.

Long noncoding RNAs (lncRNAs) represent an emerging field of tumor biology, playing essential roles in cancer cell proliferation, invasion, and metastasis. However, the overall functional and clinical significance of most lncRNAs in pancreatic cancer is not thoroughly understood. Here, we described most of the lncRNAs with aberrant expression patterns in pancreatic cancer as detected by microarray. Quantitative real-time polymerase chain reaction further verified that the expression of LINC00671 was decreased in pancreatic cancer cell lines and patient samples. Furthermore, lower LINC00671 expression was associated with reduced tumor differentiation, aggressiveness, and poor prognosis. Functionally, LINC00671 overexpression inhibited pancreatic cancer cell proliferation, invasion, and migration in vitro, and reduced tumor growth in vivo. LINC00671 is mainly located in the cytoplasm. RNA sequencing and bioinformatics analyses indicated that LINC00671 binds to multiple miRNAs and therefore could be involved in multiple tumor-associated pathways, such as the AMPK signaling pathway and PI3K-Akt signaling pathway. Western blotting and immunohistochemistry further confirmed that LINC00671 overexpression suppressed the AKT, ERK, and epithelial-mesenchymal transition pathways. Overall, these results indicated that LINC00671 acts as a novel tumor suppressor in pancreatic cancer. Our findings may provide a new potential target for the treatment of pancreatic cancer.

Phagosomal F-Actin Retention by Cryptococcus gattii Induces Dendritic Cell Immunoparalysis.

Cryptococcus gattii is a major cause of life-threatening mycosis in immunocompetent individuals and responsible for the ongoing epidemic outbreak of cryptococcosis in the Pacific Northwest of North America. This deadly fungus is known to evade important host immune responses, including dendritic cell (DC) maturation and concomitant T cell immunity, via immune evasion mechanisms that remain unclear. Here, we demonstrate that primary human DCs phagocytose C. gattii but the maturation of phagosomes to phagolysosomes was blocked as a result of sustained filamentous actin (F-actin) that entrapped and concealed the phagosomes from recognition. Superresolution structured illumination microscopy (SR-SIM) revealed that the persistent phagosomal F-actin formed a cage-like structure that sterically hindered and functionally blocked the fusion of lysosomes. Blocking lysosome fusion was sufficient to inhibit phagosomal acidification and subsequent intracellular fungal killing by DCs. Retention of phagosomal F-actin by C. gattii also caused DC immunoparalysis. Disrupting the retained F-actin cage with cytochalasin D not only restored DC phagosomal maturation but also promoted DC costimulatory maturation and robust T cell activation and proliferation. Collectively, these results reveal a unique mechanism of DC immune evasion that enhances intracellular fungal pathogenicity and may explain suppressed cell-mediated immunity.IMPORTANCECryptococcus yeast species typically display characteristics of opportunistic pathogens, with the exception of C. gattii, which can cause life-threatening respiratory and disseminated brain infections in otherwise healthy people. The pathogenesis of C. gattii is not well understood, but an important characteristic is that C. gattii is capable of evading host cell-mediated immune defenses initiated by DCs. Here, we report that when virulent C. gattii becomes ingested by a DC, the intracellular compartment containing the fungi is covered by a persistent protein cage structure consisting of F-actin. This F-actin cage acts as a barrier to prevent interaction with other intracellular compartments, and as a result, the DC fails to kill the fungi and activate important cell-mediated immune responses. We propose that this unique immune evasion mechanism permits C. gattii to remain unchallenged within host cells, leading to persistent infection.

Chemokine receptor CXCR3 is required for lethal brain pathology but not pathogen clearance during cryptococcal meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is the major cause of infection-related neurological death, typically seen in immunocompromised patients. However, T cell-driven inflammatory response has been increasingly implicated in lethal central nervous system (CNS) immunopathology in human patients and murine models. Here, we report marked up-regulation of the chemokine receptor CXCR3 axis in human patients and mice with CM. CXCR3 deletion in mice improves survival, diminishes neurological deficits, and limits neuronal damage without suppressing fungal clearance. CD4+ T cell accumulation and TH1 skewing are reduced in the CNS but not spleens of infected CXCR3-/- mice. Adoptive transfer of WT, but not CXCR3-/- CD4+ T cells, into CXCR3-/- mice phenocopies the pathology of infected WT mice. Collectively, we found that CXCR3+CD4+ T cells drive lethal CNS pathology but are not required for fungal clearance during CM. The CXCR3 pathway shows potential as a therapeutic target or for biomarker discovery to limit CNS inflammatory damages.

Beta3-Tubulin is Critical for Microtubule Dynamics, Cell Cycle Regulation, and Spontaneous Release of Microvesicles in Human Malignant Melanoma Cells (A375).

Microtubules (MTs), microfilaments, and intermediate filaments, the main constituents of the cytoskeleton, undergo continuous structural changes (metamorphosis), which are central to cellular growth, division, and release of microvesicles (MVs). Altered MTs dynamics, uncontrolled proliferation, and increased production of MVs are hallmarks of carcinogenesis. Class III beta-tubulin (ß3-tubulin), one of seven ß-tubulin isotypes, is a primary component of MT, which correlates with enhanced neoplastic cell survival, metastasis and resistance to chemotherapy. We studied the effects of ß3-tubulin gene silencing on MTs dynamics, cell cycle, and MVs release in human malignant melanoma cells (A375). The knockdown of ß3-tubulin induced G2/M cell cycle arrest, impaired MTs dynamics, and reduced spontaneous MVs release. Additional studies are therefore required to elucidate the pathophysiologic and therapeutic role of ß3-tubulin in melanoma.

Non-thermal histotripsy tumor ablation promotes abscopal immune responses that enhance cancer immunotherapy.

BACKGROUND: Developing the ability to use tumor-directed therapies to trigger potentially therapeutic immune responses against cancer antigens remains a high priority for cancer immunotherapy. We hypothesized that histotripsy, a novel non-invasive, non-thermal ablation modality that uses ultrasound-generated acoustic cavitation to disrupt tissues, could engender adaptive immune responses to tumor antigens. METHODS: Immunocompetent C57BL/6 mice inoculated with flank melanoma or hepatocellular carcinoma tumors were treated with histotripsy, thermal ablation, radiation therapy, or cytotoxic T lymphocyte-associated protein-4 (CTLA-4) blockade checkpoint inhibition. Lymphocyte responses were measured using flow cytometric and immunohistochemical analyses. The impact of histotripsy on abscopal immune responses was assessed in mice bearing bilateral tumors, or unilateral tumors with pulmonary tumors established via tail vein injection. RESULTS: Histotripsy ablation of subcutaneous murine melanoma tumors stimulated potent local intratumoral infiltration of innate and adaptive immune cell populations. The magnitude of this immunostimulation was stronger than that seen with tumor irradiation or thermal ablation. Histotripsy also promoted abscopal immune responses at untreated tumor sites and inhibited growth of pulmonary metastases. Histotripsy was capable of releasing tumor antigens with retained immunogenicity, and this immunostimulatory effect was associated with calreticulin translocation to the cellular membrane and local and systemic release of high mobility group box protein 1. Histotripsy ablation potentiated the efficacy of checkpoint inhibition immunotherapy in murine models of melanoma and hepatocellular carcinoma. CONCLUSIONS: These preclinical observations suggest that non-invasive histotripsy ablation can be used to stimulate tumor-specific immune responses capable of magnifying the impact of checkpoint inhibition immunotherapy.

CARD9 Is Required for Classical Macrophage Activation and the Induction of Protective Immunity against Pulmonary Cryptococcosis.

Caspase recruitment domain-containing protein 9 (CARD9) is a critical adaptor molecule triggered by the interaction of C-type lectin receptors (CLRs) with carbohydrate motifs found in fungi. Consequently, clinical and animal studies indicate that CARD9 is an important regulator of protective immunity against fungal pathogens. Previous studies suggest that CARD9 is important for the induction of protection against Cryptococcus neoformans, an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system in immunocompromised patients. However, the effect of CARD9 deficiency on the induction of protective immune responses against C. neoformans is unknown. Immunization with a C. neoformans mutant that overexpresses the transcription factor zinc finger 2, denoted LW10, results in protection against an otherwise lethal challenge with wild-type (WT) C. neoformans Our results showed that CARD9 is essential for the induction of vaccine-mediated immunity against C. neoformans infection. We observed significant decreases in interleukin-17 (IL-17) production and significant increases in Th2-type cytokine (IL-4, IL-5, and IL-13) production in CARD9-deficient mice after inoculation with strain LW10. While leukocyte infiltration to the lungs of CARD9-deficient mice was similar in LW10 and WT C. neoformans-infected mice, macrophages derived from CARD9-deficient mice inherently skewed toward an M2 activation phenotype, were unable to contain the growth of LW10, and failed to produce nitric oxide in response to infection with LW10 or stimulation with lipopolysaccharide. These results suggest that CARD9-mediated signaling is required for M1 macrophage activation and fungicidal activity necessary for the induction of vaccine-mediated immunity against C. neoformansIMPORTANCECryptococcus neoformans is a fungal pathogen that is found throughout the environment and can cause life-threatening infections of the lung and central nervous system in severely immunocompromised individuals. Caspase recruitment domain-containing protein 9 (CARD9) is a critical molecule that is activated after interactions of C-type lectin receptors (CLRs) found on the surfaces of specific immune cells, with carbohydrate structures associated with fungi. Patients with defects in CARD9 are significantly more susceptible to a multitude of fungal infections. C. neoformans contains several carbohydrate structures that interact with CLRs on immune cells and activate CARD9. Consequently, these studies evaluated the necessity of CARD9 for the induction of protective immunity against C. neoformans infection. These results are important, as they advance our understanding of cryptococcal pathogenesis and host factors necessary for the induction of protective immunity against C. neoformans.

In vivo clearance of nanoparticles by transcytosis across alveolar epithelial cells.

Nanoparticles in polluted air or aerosolized drug nanoparticles predominantly settle in the alveolar lung. Here, we describe a novel, highly effective pathway for the particles to cross the alveolar epithelium and reach the lymph and bloodstream. Amorphous silica nanoparticles, suspended in perfluorocarbon, were instilled into the lungs of mice for intravital microscopy. Particles formed agglomerates that settled on the alveolar wall, half of which were removed from the lung within 30 minutes. TEM histology showed agglomerates in stages of crossing the alveolar epithelium, in large compartments inside the epithelial cells and crossing the basal membrane into the interstitium. This pathway is consistent with published kinetic studies in rats and mice, using a host of (negatively) charged and polar nanoparticles.

Granule-Dependent NK Cell Killing of Cryptococcus Requires Kinesin to Reposition the Cytolytic Machinery for Directed Cytotoxicity.

Cryptococcus is the most important cause of fungal meningitis in immunocompromised individuals. Host defense against Cryptococcus involves direct killing by NK cells. That NK cells from HIV-infected patients fail to polarize perforin to the microbial synapse and kill C. neoformans led us to explore the mechanisms used to reposition and polarize the cytolytic granules to the synapse. Using live-cell imaging, we observed microtubule and granule movements in response to Cryptococcus that revealed a kinesin-dependent event. Eg5-kinesin bound to perforin-containing granules and was required for association with the microtubules. Inhibition of Eg5-kinesin abrogated dynein-dependent granule convergence to the MTOC and granule and MTOC polarization to the synapse and suppressed NK cell killing of Cryptococcus. In contrast, Eg5-kinesin was dispensable for tumor killing. This reveals an alternative mechanism of MTOC repositioning and granule polarization, not used in tumor cytotoxicity, in which Eg5-kinesin is required to initiate granule movement, leading to microbial killing.

Elastin Shapes Small Molecule Distribution in Lymph Node Conduits.

The spatial and temporal Ag distribution determines the subsequent T cell and B cell activation at the distinct anatomical locations in the lymph node (LN). It is well known that LN conduits facilitate small Ag distribution in the LN, but the mechanism of how Ags travel along LN conduits remains poorly understood. In C57BL/6J mice, using FITC as a fluorescent tracer to study lymph distribution in the LN, we found that FITC preferentially colocalized with LN capsule-associated (LNC) conduits. Images generated using a transmission electron microscope showed that LNC conduits are composed of solid collagen fibers and are wrapped with fibroblastic cells. Superresolution images revealed that high-intensity FITC is typically colocalized with elastin fibers inside the LNC conduits. Whereas tetramethylrhodamine isothiocyanate appears to enter LNC conduits as effectively as FITC, fluorescently-labeled Alexa-555-conjugated OVA labels significantly fewer LNC conduits. Importantly, injection of Alexa-555-conjugated OVA with LPS substantially increases OVA distribution along elastin fibers in LNC conduits, indicating immune stimulation is required for effective OVA traveling along elastin in LN conduits. Finally, elastin fibers preferentially surround lymphatic vessels in the skin and likely guide fluid flow to the lymphatic vessels. Our studies demonstrate that fluid or small molecules are preferentially colocalized with elastin fibers. Although the exact mechanism of how elastin fibers regulate Ag trafficking remains to be explored, our results suggest that elastin can be a potentially new target to direct Ag distribution in the LN during vaccine design.

Dynamic and Irregular Distribution of RyR2 Clusters in the Periphery of Live Ventricular Myocytes.

Cardiac ryanodine receptors (RyR2s) are Ca2+ release channels clustering in the sarcoplasmic reticulum membrane. These clusters are believed to be the elementary units of Ca2+ release. The distribution of these Ca2+ release units plays a critical role in determining the spatio-temporal profile and stability of sarcoplasmic reticulum Ca2+ release. RyR2 clusters located in the interior of cardiomyocytes are arranged in highly ordered arrays. However, little is known about the distribution and function of RyR2 clusters in the periphery of cardiomyocytes. Here, we used a knock-in mouse model expressing a green fluorescence protein (GFP)-tagged RyR2 to localize RyR2 clusters in live ventricular myocytes by virtue of their GFP fluorescence. Confocal imaging and total internal reflection fluorescence microscopy was employed to determine and compare the distribution of GFP-RyR2 in the interior and periphery of isolated live ventricular myocytes and in intact hearts. We found tightly ordered arrays of GFP-RyR2 clusters in the interior, as previously described. In contrast, irregular distribution of GFP-RyR2 clusters was observed in the periphery. Time-lapse total internal reflection fluorescence imaging revealed dynamic movements of GFP-RyR2 clusters in the periphery, which were affected by external Ca2+ and RyR2 activator (caffeine) and inhibitor (tetracaine), but little detectable movement of GFP-RyR2 clusters in the interior. Furthermore, simultaneous Ca2+- and GFP-imaging demonstrated that peripheral RyR2 clusters with an irregular distribution pattern are functional with a Ca2+ release profile similar to that in the interior. These results indicate that the distribution of RyR2 clusters in the periphery of live ventricular myocytes is irregular and dynamic, which is different from that of RyR2 clusters in the interior.

Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy.

Dendritic cells are targeted by regulatory T (T reg) cells, in a manner that operates as an indirect mode of T cell suppression. In this study, using a combination of single-cell force spectroscopy and structured illumination microscopy, we analyze individual T reg cell-DC interaction events and show that T reg cells exhibit strong intrinsic adhesiveness to DCs. This increased DC adhesion reduces the ability of contacted DCs to engage other antigen-specific cells. We show that this unusually strong LFA-1-dependent adhesiveness of T reg cells is caused in part by their low calpain activities, which normally release integrin-cytoskeleton linkage, and thereby reduce adhesion. Super resolution imaging reveals that such T reg cell adhesion causes sequestration of Fascin-1, an actin-bundling protein essential for immunological synapse formation, and skews Fascin-1-dependent actin polarization in DCs toward the T reg cell adhesion zone. Although it is reversible upon T reg cell disengagement, this sequestration of essential cytoskeletal components causes a lethargic state of DCs, leading to reduced T cell priming. Our results reveal a dynamic cytoskeletal component underlying T reg cell-mediated DC suppression in a contact-dependent manner.