Abstract

Tumor necrosis factor alpha (TNF-α) is a critical cytokine in systemic inflammation and a potent inducer of apoptotic cell death. Type 1 cytokines are involved in the pathogenesis of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. Overproduction of interferon gamma (IFN-γ) and TNF-α from activated T cells or bone marrow cells has been proposed as a mechanism of BM failure. We hypothesized that inhibiting the production of TNF-α or blockade of the TNF-α signaling pathway would attenuate hematopoietic cell destruction in murine models of immune BM failure. Therefore, we tested the role of TNF-α in several mouse models of human AA, using C57BL/6 (B6) mice carrying germline deletions of TNF-α (TNF-α-/-) or its receptors including TNF-α receptor superfamily member 1a (TNF-rsf1a-/-), TNF-rsf1b-/-, and TNF-rsf1a-/-1b-/-as donors or recipients, respectively.

Lymph node (LN) cells from both TNF-α-/- and TNF-α receptor knock out mice induced fatal BM failure with efficacy similar to LN cells from wild-type B6 donors when infused into CByB6F1 recipients that had received 5 Gy of total body irradiation (TBI), indicating that expression of TNF-α or TNF-α receptors on effector cells did not affect the development of BM failure.

However, TNF-α-/- mice as recipients unexpectedly showed resistance to immune-mediated BM failure. Infusion of 5 million LN cells from MHC-mismatched FVB donors to 6.5 Gy TBI pre-conditioned TNF-α-/- recipients did not induce AA: the animals had normal or increased levels of WBC, RBC, platelets, and total BM cells relative to TBI-only TNF-α-/- mice; control wild-type B6 recipient mice developed severe pancytopenia and BM failure after LN infusion at 10 days (Fig. 1A&B). This result suggests that TNF-α production in the recipients had a role in immune attack by effector cells. When residual BM cells were cultured in vitro in cytokine-supplemented methylcellulose media, cells from wild-type BM failure mice, but not from TNF-α-/- recipient mice, showed a significant reduction in colony forming units relative to their TBI controls. By flow cytometry, Fas+ cells and Annexin V-binding cells were increased in BM of wild-type marrow failure mice relative to wild-type TBI controls, but no increase in Fas+ cells and Annexin V-binding cells was observed in TNF-α-/- mice that had received LN infusion. In parallel experiments, injection of FVB LN cells into sub-lethally-irradiated TNF-rsf1a-/-, TNF-rsf1b-/-, or TNF-rsf1a-/-1b-/- mice resulted in BM failure with declines in WBC and platelets as observed in wild-type B6 mice after FVB LN infusion. Plasma levels of both IFN-γ and TNF-α were markedly increased in all the wild-type B6 and TNF-α receptor knock out mice that developed BM failure, but not in TNF-α-/- recipient mice. These data suggest that depletion of TNF-α receptors could not attenuate immune-mediated BM destruction and that TNF-α did not exert its destructive effect on hematopooetic targets through the TNF-α/TNF-rsf1a and 1b pathways.

We hypothesized that TNF-α might modulate T cell IFN-γ production to affect immune-mediated BM destruction. TNF-α is mainly produced by activated macrophages, while IFN-γ is mainly secreted by activated T cells. Blockade of either IFN-γ in effector cells or IFN-γ receptor in target cells ameliorates the development of BM failure (Chen J, Blood.2015;126:2621). To test this hypothesis, we first depleted macrophages from CByB6F1 recipient mice with liposomal clodronate before BM failure induction with TBI and B6 LN cell injection. Indeed, removal of macrophages attenuated BM destruction relative to BM failure mice pre-injected with PBS-loaded liposomes without macrophage depletion (Figure 1C). When we injected recombinant TNF-α daily i.v. for 1 week into TNF-α-/- recipients following TBI and FVB LN cell infusion, TNF-α-injected mice had decreased BM cellularity relative to mice that received TBI and LN infusion without TNF-α (Figure 1D). Remarkably, TNF-α injection increased intracellular IFN-γ expression in BM T cells as determined by flow cytometry, indicating that TNF-α was capable of stimulating T cells to secrete IFN-γ.

In summary, our study reveals a critical role of TNF-α in target cells but not in effector cells, in the pathogenesis of immune-mediated BM failure by cell-cell "crosstalk" modulation of IFN-γ secretion.

Disclosures

Young: Novartis/GSK to institute: Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.