Delayed emergency myelopoiesis following polymicrobial sepsis in neonates

Abstract

Neonates have increased susceptibility to infection, which leads to increased mortality. Whether or not this as a result of implicit deficits in neonatal innate immune function or recapitulation of innate immune effector cell populations following infection is unknown. Here, we examine the process of emergency myelopoiesis whereby the host repopulates peripheral myeloid cells lost following the initial infectious insult. As early inflammatory responses are often dependent upon NF-κB and type I IFN signaling, we also examined whether the absence of MyD88, TRIF or MyD88 and TRIF signaling altered the myelopoietic response in neonates to polymicrobial sepsis. Following neonatal polymicrobial septic challenge, hematopoietic stem cell (HSC) expansion in bone marrow and the spleen were both attenuated and delayed in neonates compared with adults. Similar reductions in other precursors were observed in neonates. Similar to adult studies, the expansion of progenitor stem cell populations was also seen in the absence of MyD88 and/or TRIF signaling. Overall, neonates have impaired emergency myelopoiesis in response to sepsis compared with young adults. Despite reports that this expansion may be related to TLR signaling, our data suggest that other factors may be important, as TRIF(-/-) and MyD88(-/-) neonatal HSCs are still able to expand in response to polymicrobial neonatal sepsis.

Keywords: LSK; Mice; infection; neutrophil.

© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Figures

Figure 1

Delayed restoration of neonatal bone marrow and splenic granulocytes following sepsis. (A) Bone marrow and (B) splenic cell suspensions were harvested at 0, 1, 3, 5 and 7 d post-sepsis and stained for granulocyte cell surface markers (n=4–5/group/time point). In neonates, sepsis is associated with the early and sustained depletion of granulocytes from bone marrow and spleen, as well as delayed reconstitution. (A) *P<0.001, **P=0.019, ***P<0.013. (B) *P<0.001, **P=0.007 by Student’s t-test.

Figure 2

Decreased expansion of neonatal LSKs following sepsis. (A) Bone marrow and (B) Splenic LSKs were determined at baseline (0 h), 36 h and 7 d after CS. The expansion of bone marrow LSKs was markedly attenuated in neonates compared with young adults. Extramedullary (splenic) expansion of LSKs was also attenuated in the neonates at 7 d post-sepsis (n=4–5/group/time point). *P<0.05, **P<0.001 by Student’s t-test.

Figure 3

Decreased expansion of neonatal CMPs and GMPs following sepsis. Bone marrow was harvested and stained for CMP and GMP markers at 0, 36 h and 7 d post-CS (n=4–5/group/time point). *P<0.05, **P<0.01 by Student’s t-test.

Figure 4

Poor in vitro production of CFUs of neonatal LSKs in response to myelopoietic growth factors. Bone marrow was harvested from neonates at baseline (0 h), 36 h and 7 d post-sepsis, and LSKs were sorted via flow cytometry. LSKs were then plated at a density of 500 cells/35-mm plate in the presence of G-CSF (100 ng/ml), M-CSF (50 ng/ml) and GM-CSF (10 ng/ml). Interestingly, the proliferative capacity of neonatal and young adult LSKs to various myeloid growth factors varied dramatically. (A) In response to G-CSF, neonatal LSKs had little baseline proliferative activity that was dramatically increased by sepsis, whereas young adult LSKs were greater at baseline and were unaffected by sepsis. (B, C) In contrast, the proliferative response to GM-CSF and M-CSF was attenuated in neonates after sepsis when compared with young adults (n=4–5/group/time point). *P<0.001, **P<0.01, ***P<0.05 by Student’s t-test.

Figure 5

Deletion of TLR signaling does not affect the expansion of neonatal LSKs following sepsis. LSKs were obtained from bone marrow of WT B6, MyD88−/−, TRIF− and both MyD88−/− TRIF−/−(DKO) at baseline (0 h), 36 h and 7 d after sepsis (n=4–5/group/time point). *P=0.036.