Acute Influenza A Virus Infection Affects Cell Cycle and Lineage Output of Hematopoietic Stem Cells

Long-term hematopoietic stem cells (LT-HSC) persist in quiescence to maintain their hematopoietic potential throughout life. In the case of need LT-HSC can be activated to replenish the pool of blood cells.

We investigated the impact of acute influenza A virus (IAV) infection on hematopoiesis in C57Bl/6N mice, focusing on the most immature HSC and progenitors. Mice were infected with a lethal dose of IAV PR/8/1934 H1N1 (humane endpoints reached within 6 days post infection (dpi)). In two further groups, mice were treated daily with oseltamivir (antiviral neuraminidase inhibitor, dpi 0-4) or were vaccinated with single-cycle vesicular stomatitis virus replicon particles expressing a miss-matched neuraminidase from influenza A virus Yamaguchi/7/2004 H5N1 four weeks prior to infection. Both treatments rescued mice from infection-induced mortality. Every day 6-9 mice were analyzed for differences in the bone marrow (BM) and blood by flow cytometry and multiplex cytokine assays as well as in the lung to determine viral tissue titers and histopathology. HSC functionality was analyzed in a competitive BM transplantation of infected and non-infected mice.

Irrespective of the treatment, high IAV lung tissue titers (≥5x10⁶ tissue culture infectious dose 50) in the first days post infection (dpi 1-5) were associated with activation of HSC into the cell cycle. LT-HSCs (LSK, CD150+, CD34- and CD48-) were 50% less quiescent and shifted into the G1/S-G2-M phase (dpi 2-6) and returned to quiescence state after virus clearance (dpi 10). Furthermore, we detected 1.5-fold increase in proliferation of phenotypic LT-HSC. Differentiation was increased towards lymphoid progenitors (≥3-fold more compared to non-infected mice) during the acute phase of infection in untreated and oseltamivir treated mice and myeloid progenitors were reduced ~50% in all groups (dpi 4-8). We found the inflammatory cytokines IFNγ, IL-1α, IL-6, and TNFα to be significantly upregulated in the BM of untreated and oseltamivir treated mice but less in vaccinated animals (dpi 2-4). IL-1α or IL-6 stimulation of LT-HSCs was sufficient to initiate proliferation in cell culture. In all groups the initial drop of the peripheral platelet count (~30% lower compared to non-infected mice, dpi 2) was replenished with an excessive production of platelets (~45% increased, dpi 8-15). Histopathology and electron microscopy revealed the sequestration and accumulation of platelets in pulmonary capillaries and vessels. Subsequently, we detected twice as many mature megakaryocytes in the BM (dpi 2-4) and elevated CD41 expression on HSCs (LSK, CD150+ and CD34-; 3-fold more compared to non-infected mice dpi 2-6) indicating a myeloid/platelet-biased HSC compartment in response to infection. Competitive whole BM transplantation with activated LT-HSCs from the acute phase of infection vs non-infected mice showed delayed reconstitution of T-cells but a preferential differentiation towards platelets in recipient mice.

Taken together, local IAV infection in the lung substantially affected LT-HSC quiescence and differentiation by inflammatory cytokines with systemic consequences and a myeloid/platelet-biased lineage output.