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  • Imaging reveals novel innate immune responses in lung, liver, and beyond. Immunological reviews Neupane, A. S., Kubes, P. 2021


    Highly dynamic immune responses are generated toward pathogens or injuries, in vivo. Multiple immune cell types participate in various facets of the response which leads to a concerted effort in the removal and clearance of pathogens or injured tissue and a return to homeostasis. Intravital microscopy (IVM) has been extensively utilized to unravel the dynamics of immune responses, visualizing immune cell behavior in intact living tissues, within a living organism. For instance, the phenomenon of leukocyte recruitment cascade. Importantly, IVM has led to a deep appreciation that immune cell behavior and responses in individual organs are distinct, but also can influence one another. In this review, we discuss how IVM as a tool has been used to study the innate immune responses in various tissues during homeostasis, injury, and infection.

    View details for DOI 10.1111/imr.13040

    View details for PubMedID 34816440

  • Patrolling Alveolar Macrophages Conceal Bacteria from the Immune System to Maintain Homeostasis CELL Neupane, A., Willson, M., Chojnacki, A., Castanheira, F., Morehouse, C., Carestia, A., Keller, A., Peiseler, M., DiGiandomenico, A., Kelly, M., Amrein, M., Jenne, C., Thanabalasuriar, A., Kubes, P. 2020; 183 (1): 110-+


    During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing in vivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P. aeruginosa and S. aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.

    View details for DOI 10.1016/j.cell.2020.08.020

    View details for Web of Science ID 000576772900011

    View details for PubMedID 32888431

  • Invariant natural killer T-cell development and function with loss of microRNA-155. Immunology Frias, A. B., Buechel, H. M., Neupane, A., D'Cruz, L. M. 2018; 153 (2): 238-245


    Invariant natural killer T (iNKT) cells are adaptive T cells with innate-like characteristics including rapid cytokine production and a proliferative response to stimulation. Development of these cells in the thymus is dependent on expression of the microRNA (miRNA) processing enzyme Dicer, indicating that iNKT cells probably have distinct miRNA requirements for gene regulation during development. The miRNA miR-155 has previously been shown to have numerous roles in T cells, including regulation of proliferation and differentiation, and positive modulation of interferon-γ expression. We examined the role of miR-155 in the development and function of iNKT cells. Using germline-deficient miR-155 mice, we showed that loss of miR-155 resulted in unchanged iNKT cell frequency and cell number. Although miR-155 was up-regulated in iNKT cells upon activation with α-galactosylceramide, loss of miR-155 did not affect cytokine production or proliferation by iNKT cells. Hence, cytokine production occurs in iNKT cells independently of miR-155 expression.

    View details for DOI 10.1111/imm.12836

    View details for PubMedID 28892129

    View details for PubMedCentralID PMC5765382

  • Bispecific antibody targets multiple Pseudomonas aeruginosa evasion mechanisms in the lung vasculature. The Journal of clinical investigation Thanabalasuriar, A., Surewaard, B. G., Willson, M. E., Neupane, A. S., Stover, C. K., Warrener, P., Wilson, G., Keller, A. E., Sellman, B. R., DiGiandomenico, A., Kubes, P. 2017; 127 (6): 2249-2261


    Pseudomonas aeruginosa is a major cause of severe infections that lead to bacteremia and high patient mortality. P. aeruginosa has evolved numerous evasion and subversion mechanisms that work in concert to overcome immune recognition and effector functions in hospitalized and immunosuppressed individuals. Here, we have used multilaser spinning-disk intravital microscopy to monitor the blood-borne stage in a murine bacteremic model of P. aeruginosa infection. P. aeruginosa adhered avidly to lung vasculature, where patrolling neutrophils and other immune cells were virtually blind to the pathogen's presence. This cloaking phenomenon was attributed to expression of Psl exopolysaccharide. Although an anti-Psl mAb activated complement and enhanced neutrophil recognition of P. aeruginosa, neutrophil-mediated clearance of the pathogen was suboptimal owing to a second subversion mechanism, namely the type 3 secretion (T3S) injectisome. Indeed, T3S prevented phagosome acidification and resisted killing inside these compartments. Antibody-mediated inhibition of the T3S protein PcrV did not enhance bacterial phagocytosis but did enhance killing of the few bacteria ingested by neutrophils. A bispecific mAb targeting both Psl and PcrV enhanced neutrophil uptake of P. aeruginosa and also greatly increased inhibition of T3S function, allowing for phagosome acidification and bacterial killing. These data highlight the need to block multiple evasion and subversion mechanisms in tandem to kill P. aeruginosa.

    View details for DOI 10.1172/JCI89652

    View details for PubMedID 28463232

    View details for PubMedCentralID PMC5451222

  • iNKT Cell Emigration out of the Lung Vasculature Requires Neutrophils and Monocyte-Derived Dendritic Cells in Inflammation. Cell reports Thanabalasuriar, A., Neupane, A. S., Wang, J., Krummel, M. F., Kubes, P. 2016; 16 (12): 3260-3272


    iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against bacterial infections, including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand α-galactosylceramide or S. pneumoniae infection. In untreated mice, the major fraction of iNKT cells resided in the vasculature, but a small critical population resided in the extravascular space in proximity to monocyte-derived DCs. Administration of either α-GalCer or S. pneumoniae induced CD1d-dependent rapid recruitment of neutrophils out of the vasculature. The neutrophils guided iNKT cells from the lung vasculature via CCL17. Depletion of monocyte-derived DCs abrogated both the neutrophil and subsequent iNKT cell extravasation. Moreover, impairing iNKT cell recruitment by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for the influx of iNKT cells in host defense.

    View details for DOI 10.1016/j.celrep.2016.07.052

    View details for PubMedID 27653688

    View details for PubMedCentralID PMC5318399

  • Expression of NALPs in adipose and the fibrotic progression of non-alcoholic fatty liver disease in obese subjects. BMC gastroenterology Mehta, R., Neupane, A., Wang, L., Goodman, Z., Baranova, A., Younossi, Z. M. 2014; 14: 208


    Visceral obesity is often accompanied by non-alcoholic fatty liver disease (NAFLD). Activation of NACHT, LRR and PYD domains-containing proteins (NALPs) may contribute to the release of pro-inflammatory cytokines by adipose and the obesity-associated progression of NAFLD to non-alcoholic steatohepatitis (NASH).We analyzed visceral adipose expression of various NALPs and its downstream effectors caspase-1, ASC (Apoptosis-associated speck-like protein containing a CARD), IL-18 (Interleukin-18) and IL-1β (Interleukin- 1Beta) in obese subjects (BMI ≥ 35) with biopsy proven NAFLD.In adipose samples collected from NASH and pericellular fibrosis patients cohorts, expression levels of NALPs and IL-1β were lower than that in non-NASH patients. In portal fibrosis, the levels of mRNA encoding anti-inflammatory NALP6 were upregulated. The expression levels of all NALPs were significantly co-correlated. Circulating IL-18 levels were associated with increased liver injury markers AST and ALT and portal fibrosis.Our observations point at a possible shift in inflammation and fibrotic response from adipose tissue to liver and a possible negative feedback regulation of tissue inflammation that may instigate NAFLD severity.

    View details for DOI 10.1186/s12876-014-0208-8

    View details for PubMedID 25512222

    View details for PubMedCentralID PMC4279907

  • Expression of inflammation-related genes is altered in gastric tissue of patients with advanced stages of NAFLD. Mediators of inflammation Mehta, R., Birerdinc, A., Neupane, A., Shamsaddini, A., Afendy, A., Elariny, H., Chandhoke, V., Baranova, A., Younossi, Z. M. 2013; 2013: 684237


    Obesity is associated with chronic low-grade inflammation perpetuated by visceral adipose. Other organs, particularly stomach and intestine, may also overproduce proinflammatory molecules. We examined the gene expression patterns in gastric tissue of morbidly obese patients with nonalcoholic fatty liver disease (NAFLD) and compared the changes in gene expression in different histological forms of NAFLD. Stomach tissue samples from 20 morbidly obese NAFLD patients who were undergoing sleeve gastrectomy were profiled using qPCR for 84 genes encoding inflammatory cytokines, chemokines, their receptors, and other components of inflammatory cascades. Interleukin 8 receptor-beta (IL8RB) gene overexpression in gastric tissue was correlated with the presence of hepatic steatosis, hepatic fibrosis, and histologic diagnosis of nonalcoholic steatohepatitis (NASH). Expression levels of soluble interleukin 1 receptor antagonist (IL1RN) were correlated with the presence of NASH and hepatic fibrosis. mRNA levels of interleukin 8 (IL8), chemokine (C-C motif) ligand 4 (CCL4), and its receptor chemokine (C-C motif) receptor type 5 (CCR5) showed a significant increase in patients with advanced hepatic inflammation and were correlated with the severity of the hepatic inflammation. The results of our study suggest that changes in expression patterns for inflammatory molecule encoding genes within gastric tissue may contribute to the pathogenesis of obesity-related NAFLD.

    View details for DOI 10.1155/2013/684237

    View details for PubMedID 23661906

    View details for PubMedCentralID PMC3626032