Pneumocystis pneumonia (PCP) remains a serious complication of AIDS.1-5 Pc thrives during impaired lymphocytic immunity, leading to respiratory failure.3,6-8 To better understand host responses during CD4 deficiency, we studied host innate immunity in PCP, demonstrating central roles for alveolar macrophages (AMS) in Pc clearance.9 We also defined activity of Pc associated inflammation from AMS and alveolar epithelial cells (AECs) leading to respiratory impairment.10,11 Interactions of Pc ?-glucans with the C-Type Lectin Receptor (CLR) Dectin-1 and interactions of Pc MSG with the CLR Mincle initiate clearance of Pc, but also trigger damaging lung inflammation.4,12-16 Understanding mechanisms to restore balance between Pc clearance and inflammation holds potential benefit for treating severe PCP. Since the last submission, we have compelling new data demonstrating central roles across the CLR family during PCP both in CD4 depleted and in immunocompetent hosts using CARD9-/- mice. CARD9, a Caspase Recruitment Domain (CARD) adaptor protein, is the central molecule through which Dectin-1, Mincle, and other CLRs signal.17 Hence, CARD9-/- mice exhibit a deficiency of most CLR immunity. Notably, CD4-deficient CARD9-/- mice had impaired killing of Pc, with markedly greater Pc burdens and concurrently a dramatic reduction of inflammation compared to wild-type mice. Immune competent CARD9-/- mice also had significantly impaired Pc clearance. Despite this phenotype, the molecular events mediated by CARD9 during PCP are unknown. We hypothesize that impairment of CARD9, a central regulator of major CLR function, leads to broad deficiency critical for Pc clearance and inflammatory responses. This will be addressed through three independent but interrelated Aims.
In Aim 1, we will evaluate the role of CARD9 in driving Pc clearance and inflammation following interactions with Pc. We will study uptake and killing of Pc, cytokine release, polarization of macrophages, and the cell signaling mechanisms in CARD9-/- versus wild type AMS, AECs and dendritic cells. Next, in Aim 2, we will determine the impact of CARD9 on Pc clearance, inflammation, and mortality in immune competent and CD4 deficient CARD9-/- mice during PCP. We will examine the major causes of lethality during PCP, namely Pc burden and lung inflammation by performing kinetic studies of Pc-infected CARD9-/- mice using both CD4 depleted and immunocompetent mice. Finally, in Aim 3, we will explore cell based treatments targeting CARD9 as adjunctive therapies for PCP. While cell based treatment strategies are not needed for mild to moderate PCP, such therapies may be beneficial in addition to antibiotics in severe PCP with mortality over 80%.5,18 We have demonstrated that transfer of M2 polarized macrophages during PCP signficantly enhances Pc clearance and improves lung inflammation.19 Therefore, we will test the utlility of macrophages driven to overexpress CARD9 and CLR as adjuctive therapy during severe PCP. These studies will define the activity of the important CLR/CARD9 system in host defense during PCP, which may be exploited for novel therapies for Pc infection.

Public Health Relevance

Pneumocystis pneumonia (PCP) remains a major cause of illness and death in patients with impaired CD4 lymphocytic immunity, particularly in patients with AIDS. Host C-Type Lectin Receptors (CLRs; Dectin-1 and Mincle) on macrophages and other lung cells interact with Pneumocystis and activate the central CARD9 signaling molecule initiating killing and clearance of Pneumocystis and triggering lung inflammatory responses, which are of particular importance in hosts lacking CD4 cell immunity. This proposal will evaluate how CARD9 and the related CLRs act to eliminate Pc organisms and regulate lung inflammatory responses during PCP, and how this system can be exploited for cell-based therapies given in addition to traditional antibiotic therapy for severe PCP.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062150-27
Application #
9840922
Study Section
AIDS-associated Opportunistic Infections and Cancer Study Section (AOIC)
Program Officer
Zhou, Guofei
Project Start
1993-07-10
Project End
2022-12-31
Budget Start
2020-01-01
Budget End
2020-12-31
Support Year
27
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Kottom, Theodore J; Hebrink, Deanne M; Jenson, Paige E et al. (2018) Dectin-2 Is a C-Type Lectin Receptor that Recognizes Pneumocystis and Participates in Innate Immune Responses. Am J Respir Cell Mol Biol 58:232-240
Cheon, In Su; Son, Young Min; Jiang, Li et al. (2018) Neonatal hyperoxia promotes asthma-like features through IL-33-dependent ILC2 responses. J Allergy Clin Immunol 142:1100-1112
Burnham-Marusich, Amanda R; Hubbard, Breeana; Kvam, Alexander J et al. (2018) Conservation of Mannan Synthesis in Fungi of the Zygomycota and Ascomycota Reveals a Broad Diagnostic Target. mSphere 3:
Kottom, Theodore J; Hebrink, Deanne M; Jenson, Paige E et al. (2017) Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species. A New Potential Target for Therapy. Am J Respir Cell Mol Biol 56:213-222
Nandakumar, Vijayalakshmi; Hebrink, Deanne; Jenson, Paige et al. (2017) Differential Macrophage Polarization from Pneumocystis in Immunocompetent and Immunosuppressed Hosts: Potential Adjunctive Therapy during Pneumonia. Infect Immun 85:
Kottom, Theodore J; Hebrink, Deanne M; Jenson, Paige E et al. (2017) The Interaction of Pneumocystis with the C-Type Lectin Receptor Mincle Exerts a Significant Role in Host Defense against Infection. J Immunol 198:3515-3525
Kottom, Theodore J; Limper, Andrew H (2016) Evidence for a Pneumocystis carinii Flo8-like transcription factor: insights into organism adhesion. Med Microbiol Immunol 205:73-84
Calero-Bernal, Maria L; Martin-Garrido, Isabel; Donazar-Ezcurra, Mikel et al. (2016) Intermittent Courses of Corticosteroids Also Present a Risk for Pneumocystis Pneumonia in Non-HIV Patients. Can Respir J 2016:2464791
Kottom, Theodore J; Hebrink, Deanne M; Jenson, Paige E et al. (2015) Evidence for Proinflammatory ?-1,6 Glucans in the Pneumocystis carinii Cell Wall. Infect Immun 83:2816-26
Ali, Mohamed F; Driscoll, Christopher B; Walters, Paula R et al. (2015) ?-Glucan-Activated Human B Lymphocytes Participate in Innate Immune Responses by Releasing Proinflammatory Cytokines and Stimulating Neutrophil Chemotaxis. J Immunol 195:5318-26

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