Granulocytes, Monocytes and Macrophages
Oral and Poster Abstracts
201. Granulocytes, Monocytes and Macrophages: Poster I
Hall A, Level 2
(Orange County Convention Center)
Michael Garbati, PhD, BS1*, Winifred Keeble, BS1*, Wenjin Yang, PhD2* and Grover C. Bagby Jr., MD, MACP1
1Oregon Health & Science University, Portland, OR
2Aldea Pharmaceuticals, Redwood City, CA
Replication and survival of FA-deficient hematopoietic
stem and progenitor cells (HSPC) are highly suppressed by exposure to inflammatory
cytokines including TNFα, IL-1β, IFNγ, and MIP-1α. Additionally,
FA macrophages exposed to specific toll-like receptor (TLR) ligands overproduce
the very inflammatory cytokines that exhaust the stem cell pool. Recent
evidence using double knockout mice suggests that aldehyde dehydrogenases (ALDH)
may play a role in protecting FA HSPC but the mechanisms involved are unclear. We
tested the hypothesis that the TLR-dependent overproduction of inflammatory
cytokines in FA macrophages results from a loss of FA protein-dependent ALDH
function. Control (T-shNT) or FANCC-deficient (T-shFC) THP-1 human monocytic
leukemia cells were treated with Alda-1 (a small molecule ALDH agonist known to
enhance the activity of both ALDH1A1 and ALDH2), before exposing them to the
TLR-7/8 agonist R848 and quantifying the production of TNFα (ELISA of 24-
culture supernatants). While R848 alone induced a 6-fold increase in TNFα
production by the FANCC-deficient cells, Alda-1 suppressed TLR-induced TNFα
production in a dose-dependent manner, by both T-shNT and T-shFC cells (Fig. 1).
To identify the ALDH isoform that played a role in the suppression of TNFα
production, we ectopically expressed ALDH1A1 or ALDH2 in T-shNT
and T-shFC cells. Overexpression of ALDH2 had little effect on
R848-induced TNFα production, but ALDH1A1 overexpression completely
suppressed TNFα production by FANCC-deficient cells and suppressed (by
approximately 50%) the production of TNFα by control cells (Fig. 2).
Likewise, in loss-of-function studies, siRNA knockdown of ALDH1A1 (but
not ALDH2) enhanced R848-induced production of TNFα (1.8-fold) in
T-shNT cells but did not enhance TNFα overproduction in either
FANCC-deficient (T-shFC) or FANCA-deficient (T-shFA [FANCA knockdown]) cells. Additionally,
ALDH1A1 (but not ALDH2) mRNA was highly inducible in both THP-1
cells (by R848) and in Lin-Sca-1+Kit+ murine
marrow cells (by TNFα). While ALDH1A1 mRNA is expressed similarly
in both T-shNT and T-shFC cells, our results indicate that in normal
macrophages ALDH1A1 plays a role in the constraining TLR-induced TNF production
but is significantly less functional in FANCC-deficient macrophages. In
summary, (1) either pharmacological enhancement of ALDH function with Alda-1 or
overexpression of ALDH1A1 is sufficient to restore the TLR-suppressive function
of ALDH1A1 in FANCC-deficient macrophages, and (2) specific suppression of ALDH1A1,
(but not ALDH2) induces an FA-like phenotype in control macrophages. We
conclude that (1) optimal function of ALDH1A1 is FANCC- and FANCA-dependent in normal
macrophages, (2) TLR-dependent overproduction of inflammatory cytokines by
FA-deficient macrophages may result either from an increase in aldehyde load or
the loss of a non-canonical signal-suppressive function of ALDH1A1, and (3)
enhancement of ALDH activity using small molecule agonists such as Alda-1 may
alleviate the FA macrophage/cytokine phenotype and thereby protect HSC from
inflammation-induced exhaustion.
Disclosures: No relevant conflicts of interest to declare.
*signifies non-member of ASH