TNFα Plays Essential Role in Resolution of Anemia of Inflammation in Tnfαko Mice Treated with Heat-Killed Brucella Abortus
Authors: Lo Presti Vania , Chen Ding-wen , Martins Ana , Sharma Pankaj , Ginzburg Yelena , Castruccio Castracani Carlo , Casu Carla , Gupta Ritama , Gozzelino Raffaella , Behrens Edward , Bennett Laura F , Paulson Robert , Kurre Peter , Rivella Stefano , Guerra Amaliris 
Published: December 2024

Anemia of inflammation (AI) is the second most prevalent form of anemia, and is common in patients with chronic inflammatory states, such as infection, autoimmunity, and cancer. There are no targeted treatments available for AI, strategies typically focus on treating the underlying disease. Inhibiting tumor necrosis factor-α (TNFα) has become the gold standard for treating autoimmune disorders. However, some patients show exacerbations or onsets of new autoimmune conditions following treatment. We became interested in the effects of TNFα on erythropoiesis due to reports of TNFα blocking antibodies (Ab) improving anemia as a secondary treatment outcome.

We started our studies using a well-established model of AI induced by injection of heat-killed Brucella Abortus (BA) in germline TNFα knockout (TNFαKO) mice. Our findings show that TNFαKO+BA mice developed a macrocytic hyperchromic anemia, leukocytosis, and abnormally increased myeloid and lymphocytes populations in the bone marrow (BM) and spleens, which resulted in death after 10-weeks. Serum cytokine analysis of TNFαKO+BA mice displayed sustained elevations of interleukin (IL)12p40 and interferon-γ (IFNγ) levels. Given the reports of improvements in anemia in patients treated with TNFα blocking agents, these surprising findings made us question whether TNFα played an unknown anti-inflammatory role which impacted erythropoiesis.IFNγ's role in RBC lifespan, erythrophagocytosis, and BM failure is well-established. We hypothesized that TNFα played an important anti-inflammatory role in modulating IFNγ. IFNγKO+BA mice showed normal TNFα serum levels, a reduced inflammation profile, normal red blood cell (RBC) counts and minimal perturbations to erythropoiesis at time points tested. Additionally, a TNFαKO/IFNγKO double knock-out (DKO) mouse line was generated and challenged with BA to test if lack of IFNγ would correct the TNFαKO+BA phenotype. Indeed, DKO+BA mice had a phenotype closer to that of IFNγKO+BA and WT+BA than TNFαKO+BA. We tested if administration of recombinant TNFα (rTNFα) would correct the chronic AI phenotype in TNFαKO+BA. TNFαKO+BA treated with rTNFα died shortly after administration. We also tested if reduction of IFNγ by an anti-IFNγ Ab would rescue TNFαKO+BA mice. We found that treatment with anti-IFNγ Ab partially corrected the anemia phenotype but reverted after 4 weeks. However, rTNFα in combination with anti-IFNγ Ab reversed the hyper-inflammatory phenotype, rescued erythropoiesis, and prevented death in TNFαKO+BA mice.

We next sought to identify the cause of death in TNFαKO+BA. 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. We analyzed the HSC compartment of TNFαKO+BA at 8 weeks and found a dramatic increases in HSCs, myeloid and the lymphoid compartment, suggesting that HSCs in TNFαKO+BA exhibit elevated proliferation and differentiation activities. To assess whether the elevated HSC cycling phenotype in TNFαKO+BA leads to HSC exhaustion, we transplanted whole BM from untreated-WT, untreated-TNFαKO, WT+BA (8 weeks) or TNFαKO+BA (8 weeks) into myeloblated CD45.1 mice. Our preliminary data showed that recipient mice transplanted with whole BM of TNFαKO+BA died or had to be sacrificed due to low red blood cell (RBC) counts by 4-weeks post BMT, while the other groups had normal RBC levels. Interestingly, CD45.1 recipients with donor TNFαKO+BA cells showed no splenomegaly. Additionally, untreated-TNFαKO whole BM CD45.1 recipients displayed elevations in lymphocyte counts a 4-week post BMT compared to controls. These experiments are ongoing.

TNFα has been demonstrated to provide essential pro-survival signals to HSCs for the resolution of inflammation. We are currently working towards understanding the direct effects of TNFα on erythroid populations by focusing on the pathways regulated by the TNFα receptors during inflammation. Our work supports an essential role for TNFα in resolving the inflammatory response, characterizes a new model to investigate the role of TNFα in the etiology of AI, and provides potential clues for exacerbations in patients treated with TNFα inhibitors.