https://openworks.mdanderson.org/sumexp22/1151/thumbnail.jp

Garcia, Samantha Rose

Ramesh, Ashwin

Sharma, Atul

Sharma, Jyotika

English

OpenWorks @ MD Anderson

• Chronic Granulomatous Disease (CGD) is aprimary immunodeficiency of phagocyte functiondue to defective NADPH oxidase resulting in a lackof reactive oxygen species (ROS) generationamong innate immune cells such as neutrophils.• ROS generation is a major antimicrobial defensemechanism of neutrophils and other phagocyticcells, and its impairment leads to susceptibilitytowards life-threatening pyogenic infections andinflammatory granulomas.• Granulibacter bethesdensis (Gb) is a recentlydiscovered bacterial pathogen known to causerecurrent and occasionally fatal infections inimmune compromised CGD patients, owing to itsability to persist in a latent form in phagocytes.• Very little is known regrading hostimmune responses required for efficient clearanceof this pathogen in lung microenvironment, aprimary site of recurrent infections in CGD patients.• Using the NOX2-deficient mouse model of X-linkedCGD (XCGD), we have established a pulmonaryinfection model of Gb infection to study immune cellfunctions, disease progression and infectionoutcome following intranasal infection with a clinicalisolate of Gb.(A) Mice Studies6–8-week-old mice were infected intranasally with2x106 CFU of Gb and monitored for clinical signsof infection for up to 16 days.Upon euthanasia, Blood, Bronchioalveolarlavage, and lungs were collected.(B) Bronchioalveolar lavage fluid (BALF) isolation(C) Preparation of cytospin slides(D) Geimsa Staining(E) Microscopy to determine cell counts.Compared to WT mice, XCGD mice were highlysusceptible to infection and succumbed by day 12-14 post-infection. WT mice displayed a transient increase in clinicalscore correlating with moderate increase in local andsystemic bacterial burdens characteristic of a mild,resolving infection. XCGD mice, on the otherhand, exhibited a marked increase in clinical scores andbacterial burdens which remained high through Day 12-p.i., the time at which 100% of these mice becamemoribund.Light Microscopy revealed that BAL cells primarilycomprised of macrophages and neutrophils with thenumbers mirroring the clinical severity of infection amongWT and XCGD mice. We observed high numbers ofmacrophages and neutrophils at Day 1 among both groupsof mice with the cell numbers returning to basal levels byDay 4 among WT mice. An increase in neutrophil countswas observed in XCGD mice at Day 4 and remained highat Day 12 indicating a continued state of inflammationwithin the lung microenvironment.ReferencesTitle of the Poster Presentation Goes HereAuthors of the Poster Presentation Goes HereInstitutional and/or Graduate School of Biomedical Sciences Affiliation Goes Here Immunopathogenesis of Granulibacter bethesdensis, an opportunistic pathogen causing recurrent infection in immunocompromised PatientsAtul Sharma2, Ashwin Ramesh2, Samantha Garcia1, Jyotika Sharma21Partnership for Careers in Cancer Science and Medicine, Department of Faculty Diversity, Equity & Inclusion, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030.2Department of Critical Care, University of Texas MD Anderson Cancer Center, Houston, Texas 77030.ResultsBackground• A comparative Kinetic assessment of immune cellinfiltrates in lungs of Gb infected wildtype (WT)and XCGD mice will provide important insightsinto detrimental and protective immuneresponses to help tailor future therapeuticstrategies against this infection.HypothesisExperiment MethodsFigure 1: XCGD mice infected with Gb resulted in increased bacterial burden in lungs and blood leading to severe lung pathology. (A) Kaplan-Meyer survival analysis of Gb infection in WT and XCGD mice. (B) Lung tissues isolated from WT and XCGD mice upon euthanasia at Day 12-p.i. (C) The average clinical scores and the bacterial burden in (D) lungs and (E) blood are depicted as bar graphs. Paired T-test (p<0.0001)log CFUs/mLWT XCGD012345BLOODlog CFUs/mLWT XCGD(A) (B)(D) (E)(C)1) Christine Deffert; Stephanie Carnesecchi; Huiping Yuan; Anne-Laure Rougemont; Tiina Kelkka; Rikard Holmdahl; Karl-Heinz Krause; Michela G Schäppi. Hyperinflammation of chronic granulomatous disease is abolished by NOX2 reconstitution in macrophages and dendritic cells. JOP. 2012. 341-350.2) D E Morgenstern; M A Gifford; L L Li; C M Doerschuk; M C Dinauer. Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response to Aspergillus fumigatus. J Exp Med. 1997.3) Jessica Chu; Margery G. Smelkinson; David W. Dorward; Kol A. Zarember; John I. Gallin. Early Intracellular Trafficking of Granulibacter bethesdensis in Human Macrophages. ASM-II. 2017.4) Atul Sharma; Katelyn McCann; Jitendra Kumar Tripathi; Pooja Chauhan; Christa Zerbe; Bibhuti B Mishra; Steven M Holland; Jyotika Sharma. Tamoxifen restores extracellular trap formation in neutrophils from patients with chronic granulomatous disease in a reactive oxygen species-independent manner. J Allergy Clin Immunol. 2019.Figure 3: Bar graph showing the percentage of (A) macrophage and (B) neutrophils identified from BAL cells at Day 1, Day 4, and Day 12 p.i. The average numbers of cells counted within each frame are noted at the bottom of each bar for perspective.Future DirectionsResults & ConclusionsMacrophages & MonocytesMacrophages and monocytes were identified as large cells with granular pattern across the cytoplasm. Tissue-resident macrophages differentiate from blood monocytes upon the presence of pro-inflammatory signals.NeutrophilsNeutrophils are identified within tissues as a response infections. They perform a variety of functions to combat infection. These cells characteristically contain a multi-lobed nucleus that come in a variety of different shapes and sizes (poly-morpho-nuclear cells).Identification of Different BAL cell types by Wright-Giemsa StainingFigure 2: Wright-Giemsa staining of BALF cells representing different cellpopulations and their morphologies. Different colored arrows indicate thedifferent types of nucleated cells identified. Red: Macrophage/Monocyte;Orange: Neutrophil; Blue: Lymphocyte (Giemsa, original magnificationx200)(B)(A)The rise of multi-drug resistant bacteria has forced us toreconsider the use of antibiotics. Shifting focus towards theuse of alternative therapeutic strategies capable of boostingthe immune system would strongly benefitimmunocompromised patients inherently susceptible tosevere infections. Understanding the key playersresponsible for triggering severe inflammatory responsesand reducing their effects on resident cell populationswould allow us to control the outcomes of debilitatinginfections.

Immunopathogenesis of Granulibacter bethesdensis, an opportunistic pathogen causing recurrent infection in immunocompromised patients

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1151&context=sumexp22

Immunopathogenesis of Granulibacter bethesdensis, an opportunistic pathogen causing recurrent infection in immunocompromised patients

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