P126 Effects of Histoplasma capsulatum infection on activation and proliferation of hematopoietic stem cells
Authors: Rodriguez-Echeverri Carolina , Gomez Beatriz L. , Gonzalez Angel 
Published: September 2022

Abstract

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM

Background
Hematopoietic stem cells (HSCs) are considered a multipotent population with high proliferative potential, and are widely used in the treatment of leukemias, multiple myeloma, and some lymphomas. In the context of infectious diseases, some microorganisms have been reported to induce changes in the expression of surface markers in HSCs by a direct effect or through the induction of cytokines. Systemic infections are characterized by inducing stress on the bone marrow, which is reflected in an increase or decrease in leukocytes and platelets in peripheral blood, a process known as ‘emergency hematopoiesis’. Histoplasmosis is a systemic mycosis caused by Histoplasma spp., which occurs mainly in immunosuppressed individuals; this mycosis can present a severe clinical picture with dissemination to various organs, including the bone marrow, and is associated with anemia and pancytopenia. So far, the effect of a possible interaction of Histoplasma with HSCs is unknown.

Objectives
To evaluate, in vitro, the effects of Histoplasma capsulatum infection on activation and proliferation of HSCs.

Methods
HSCs were obtained from bone marrow of C57BL/6 male mice; after isolation and purification, they were characterized by flow cytometry. Later, the basal expression of toll-like receptor (TLR)-2, TLR4, and Dectin-1 was determined using flow cytometry. HSCs were infected with H. capsulatum yeasts in a multiplicity of infection (MOI) of 5 and incubated for 24 h. In addition, some of the co-cultures were previously treated with specific blocking antibodies for TLR2 and TLR4 or with a blocking peptide specific for Dectin-1 (CLEC7A). Furthermore, phagocytosis, microbicidal, and cell proliferation assays were done, and the expression of the genes encoding the cytokines IL-1β, IL-6, IL-10, IL-17, TNF-α, and TGF-β as well as arginase-1 and iNOS were assessed.

Results
We observed that H. capsulatum has the capability to adhere and internalize within these HSCs; nonetheless, this process did not affect the survival of the fungus. The interaction of H. capsulatum with HSCs induced a significantly increased expression of TLR2 and Dectin-1 but not TLR4. In addition, this fungal interaction significantly induced an augmented expression of IL-6, IL-1β, IL-10, IL-17, TNF-α, TGF-β, as well as the immune mediators Arg-1 and iNOS. Interestingly, blockade of these receptors significantly decreased the phagocytosis process as well the expression of all inflammatory mediators evaluated, especially when blocking TLR4 and Dectin-1. Of note, H. capsulatum induced apoptosis but did not inhibit the proliferation of these stem cells.

Conclusions
These results indicate that HSCs are capable of phagocytosing H. capsulatum but do not affect its survival; moreover, this fungal pathogen could induce changes in the expression of pattern-recognition receptors (PRRs), especially TLR2 and Dectin-1, and could subsequently activate the HSCs leading to the expression of inflammatory mediators as well as affecting the viability of these stem cells. Altogether, these findings indicate that H. capsulatum could affect the hematopoiesis process as reflected in an increase or decrease in leukocytes, erythrocytes, and platelets as observed in patients with severe and disseminated disease, especially in those with dissemination to bone marrow.