TNFα and Anti-IFNγ Rescues Chronic Anemia of Inflammation Phenotype in Tnfαko Mice Treated with Heat-Killed Brucella Abortus

In this study, we investigated the role of TNFα in the development of anemia of inflammation (AI) in a TNFα knockout (KO) mouse model of AI, which is induced via one intraperitoneal (i.p.) injection of heat-killed Brucella Abortus (BA) (Gardenghi et al., 2014; Kim et al., 2014). Due to reports of amelioration of anemia in patients with autoimmune disorders as secondary outcomes (Kalliolias et al., 2016) when treated with anti-TNFα drugs, we hypothesized that TNFαKO mice would show a less severe form of AI when challenged with BA.

Our results were both unexpected and astonishing. TNFαKO-BA mice developed leukocytosis, with elevated macrophages and T-lymphocytes, and an irreversible macrocytic, hyperchromic anemia, which resulted in death after 10 weeks. Serum analysis showed that erythropoietin, iron and hepcidin were elevated in TNFαKO-BA, 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.

Chronic inflammation is well known to increase proliferative stress of hematopoietic stem cells (HSC) and contribute to their accelerated exhaustion. Increased IFNγ during chronic inflammatory stress negatively affects HSC homeostasis by skewing HSC towards differentiation, impeding self-renewal and resulting in HSC exhaustion (Morales-Matilla and King, 2018). In 2019, Yamashita & Passegue, showed that TNFα plays a protective role in HSC regeneration during inflammatory stress. Therefore, we analyzed the HSC compartment of TNFαKO -PBS or -BA treated mice at 8 weeks. We found a dramatic increase in HSCs and common lymphoid progenitors (CLP), suggesting that TNFαKO-BA mice might die due to bone marrow failure caused by chronic inflammation.

We questioned if TNFα served an anti-inflammatory role in a feedback mechanism between IL-12p40 and IFNγ secreting cells. In the absence of TNFα, we hypothesized, HSCs would be unable to return to quiescence and would be skewed towards the CLP lineage, resulting in pronounced anemia due to decreased erythroid progenitors because of this skewing. To test if concurrent loss of IFNγ would correct the observed phenotype, we crossed TNFαKO with IFNγKO mice (DKO). Indeed, IFNγKO and DKO mice challenged with BA showed a mild anemic phenotype compared to WT-BA and TNFαKO-BA. Additionally, serum levels of IL-12p40 were normalized by 4 weeks in IFNγKO-BA and DKO-BA compared to TNFαKO-BA mice. Analysis of livers and SPLs of BA treated mice by immunohistochemistry using an anti-CD3 antibody shows 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 at 8 weeks.

Lastly, retro-orbital injection of TNFαKO-BA mice between 2-8 weeks with an anti-IFNγ antibody (0.25mg) did not correct the phenotype. However, combination treatment with an anti-IFNγ antibody (0.25mg) in combination with recombinant TNFα (0.02mg), rescued the anemia phenotype. Current experiments are assessing the effects of TNFα and anti-IFNγ antibody combination treatment on the immune and HSC compartments of TNFαKO-BA mice.

Currently, treatments that block TNFα are central for the management of autoimmune diseases. However, some patients do not respond to TNFα inhibitor treatment. Others show exacerbation of a pre-existing autoimmune disease or the onset of a new autoimmune condition following anti-TNFα treatment (Salomon 2021). This finding in patients reveals an anti-inflammatory role most likely explained by the downstream effects of the TNF receptor 2, which activate pro-survival signals via the NFkB signaling pathway (Salomon 2021). In this study we demonstrate that in the absence of TNFα, injection with BA induces uncontrolled elevation of IL12p40 and IFNγ, producing a phenotype which included lymphocytic infiltration, thymic atrophy and possible HSC exhaustion. Administering anti-IFNγ antibody alone was not sufficient to rescue the TNFαKO-BA phenotype. Administration of TNFα was essential in rescuing TNFαKO-BA mice. This work supports the notion that both inflammatory and regulatory roles of TNFα and associated pathways need to be better understood to develop better and safer anti-TNFα therapies to treat autoimmune disorders.

Rivella:BVF Partners L.P: Consultancy; Cambridge Healthcare Res: Consultancy; Catenion: Consultancy; Celgene: Consultancy; Disc Medicine: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; First Manhattan Co: Consultancy; FORMA: Consultancy; Ghost Tree Capita: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Keros Therapeutics: Consultancy; MeiraGTx: Consultancy; Noble insight: Consultancy; Protagonist Therapeutics: Consultancy; Rallybio, LLC: Consultancy; Sanofi Aventis U.S: Consultancy; Slingshot Insight: Consultancy; Techspert.io: Consultancy; venBio Select LLC: Consultancy; Vifor: Membership on an entity's Board of Directors or advisory committees.