TNFα Controls the Delicate Balance between Erythropoiesis and Stem Cell Exhaustion during Inflammatory Stress
Authors: Guerra Amaliris , Lo Presti Vania , Martins Ana Catarina , Castruccio Castracani Carlo , Gupta Ritama , Oikonomidou Rea , O'Hara Emir , Casu Carla , Bennett Laura , Paulson Robert , Kurre Peter , Rivella Stefano , Gozzelino Raffaella 
Published: November 2021

Abstract
Anemia of Inflammation (AI) is prevalent in patients with chronic inflammatory states, such as infection, autoimmunity, or cancer. Induced expression of hepcidin by pro-inflammatory cytokines results in iron-restricted anemia. In particular, abnormally elevated levels of the cytokine Tumor Necrosis Factor-α (TNFα) is a hallmark of AI, however its contribution to the pathophysiology of AI is not well understood.
In this study, we investigated the role of TNFα in the development of anemia in a TNFα knockout (TNFαKO) mouse model of AI, which is induced with a single intraperitoneal (i.p.) injection of heat-killed Brucella Abortus (BA) (Kim et al. Blood). We hypothesized that TNFαKO mice would show a less severe form of AI compared to WT animals when challenged with BA.
Our results showed that WT-BA mice developed severe anemia within 2 weeks, which was resolved by 8 weeks, whereas the TNFαKO mice developed leukocytosis and an irreversible macrocytic, hyperchromic anemia. Serum analysis at 8 weeks showed that erythropoietin (EPO) and iron parameters were elevated in TNFαKO compared to WT mice, which ruled out iron-restriction as the cause for the persistent anemia. However, serum cytokine measurements of TNFαKO mice at 4 weeks showed continual elevation of interleukin (IL)-12p40 and Interferon-γ (IFNγ) compared to WT-BA controls. We hypothesized that TNFα served an anti-inflammatory role that restrained prolonged inflammation after BA, and in its absence, pro-inflammatory macrophages continuously secreted IL-12p40 levels and induced the proliferation of IFNγ secreting cells.
To test if concurrent loss of IFNγ would correct the inflammatory phenotype, we crossed TNFαKO with IFNγKO mice (DKO). Indeed, IFNγKO and DKO mice challenged with BA showed complete reversal of the anemic phenotype present in WT-BA and TNFαKO-BA at 2 weeks. Additionally, serum levels of IL-12p40 were normalized by 4 weeks in IFNγKO-BA and DKO-BA compared to TNFαKO-BA mice. Flow cytometry analysis of the bone marrow (BM) and spleen (SPL) at 8 weeks showed T-lymphocytes and macrophages were markedly expanded, whereas erythrocytes and B-lymphocytes were reduced in TNFαKO-BA mice. However, only modest differences in erythrocytes, macrophage, T- and B-lymphocyte in WT-BA, IFNγKO-BA and DKO-BA in the BM and SPL were detected. Additionally, we performed immunohistochemistry using an anti-CD3 antibody in SPL and livers of BA treated mice sacrificed at 8 weeks. We found complete disorganization of the white pulp in the SPL and infiltration of T-Lymphocytes in livers of TNFαKO-BA but not in WT-BA, IFNγKO-BA or DKO-BA animals.
These results led us to question if lack of TNFα skewed the BM towards T-Lymphocytes. We investigated the hematopoietic stem cell (HSC) LSK, multipotent myeloid progenitors (MPP) LK, and common lymphoid progenitor (CLP) compartments by flow cytometry. Shockingly, the TNFαKO had over a 30% increase of Lin-cKit+Sca1+ LSKs, compared to TNFαKO controls, while the Lin-cKit+Sca1- LK population was drastically reduced. By contrast, the Lin-CD217-cKit+Sca1+ CLP population was expanded by more than 40% in the TNFαKO-BA compared to TNFαKO controls.
Considering this data, it is difficult to disentangle the effects of TNFα in AI in vivo from its role in regulating upstream stem and progenitor cell differentiation using a germline KO strategy. However, our observations reinforce the link between iron homeostasis and HSC self-renewal and provide a new model to study inflammation associated bone marrow failure. The TNFα-BA mice displayed severe anemia, which seems to result from persistent IFNγ elevation. A recent study identified TNFα as a major pro-survival and pro-regeneration factor for HSCs (Yamashita & Passegue, Cell Stem Cell). Other studies have shown that IFNγ restricts HSCs self-renewal (Chen et al. Blood). Our results in the TNFαKO-BA treated mice suggest that TNFα preserves balanced progenitor output by countering the action of IFNγ at the HSC level. Ongoing work aims to understand the relationship between TNFα and IFNγ in regulating HSC quiescence, self-renewal, and overall pool size.

Disclosures
Paulson: Forma Therapeutics: Consultancy. Rivella: Meira GTx: Consultancy; Ionis Pharmaceuticals: Consultancy.