Tumor necrosis factor α (TNF) is a proinflammatory cytokine with established roles in host defense and immune system organogenesis. We studied TNF function and found a previously unidentified physiological function that extends its effect beyond the host into the developing offspring. A partial or complete maternal TNF deficit, specifically in hematopoietic cells, resulted in reduced milk levels of the chemokines IP-10, MCP-1, MCP-3, MCP-5 and MIP-1β, which in turn augmented offspring postnatal hippocampal proliferation, leading to improved adult spatial memory in mice. These effects were reproduced by the postpartum administration of a clinically used anti-TNF agent. Chemokines, fed to suckling pups of TNF-deficient mothers, restored both postnatal proliferation and spatial memory to normal levels. Our results identify a TNF-dependent 'lactrocrine' pathway that programs offspring hippocampal development and memory. The level of ambient TNF is known to be downregulated by physical activity, exercise and adaptive stress. We propose that the maternal TNF–milk chemokine pathway evolved to promote offspring adaptation to post-weaning environmental challenges and competition.
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We thank S. Nedospasov (Lomonosov Moscow State University) for providing the TnfloxP/loxP mouse strain and for discussions on inducible knockout and TNF neutralization strategies. We thank D. Jing (Weill Cornell Medical College) for providing Golgi staining reagents and technical support. We would like to thank C. Dipace and K. Rosania for the ELISA-based assays of TNF, and C. Pang for counting BrdU+ cells. This work was supported by US National Institute of Mental Health grant 1RO1MH080194 to M.T.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Breeding strategy to generate TNF+/+ and TNF−/− mice born to and raised by TNF+/+ (WT), TNF+/− (H), TNF−/− (KO) parents. (b) Design of the MWM experiment to test the spatial reference memory of offspring of TNF mutant parents. (c) Second probe trial following extensive training in MWM. While a second round of training improved the recall of the platform location of WT(WT) mice in probe trial 2, offspring of mutant parents still spent more time in the NW target quadrant (quadrant F3,148=172.69, P<10-5; quadrant x group, F9,148=2.84, P=0.004; LSD posthoc; N= 8,11,12,13/group). (d) Design of the contextual and cued fear conditioning experiments with the offspring of TNF mutant parents.
(a) Generation of male WT offspring of mothers with hematopoietic (mx-cre) or brain (nestin-cre) specific TNF deletion. SP=spleen, BM=bone marrow. (b) Hematopoietic system specificity of mx-cre mediated recombination demonstrated by the cre-reporter Gt(ROSA)26Sortm1Sor/J strain. LacZ immunostaining in the macrophage rich white pulp (WP), including the marginal zone (MZ), and in the red blood cells and macrophages rich red pulp (RP) in mx-Cre/ Gt(ROSA)26Sortm1Sor/J mice indicates recombination. No apparent immunoreactivity in cortex (Ctx) and hippocampus (Hip). Bar represents 50 μm.
Supplementary Figure 3 Performance in the MWM and conditional fear test of the offspring born to conditional TNF−/− mothers.
(a) All offspring improved in finding the platform during the first (Repeated measures of ANOVA: session, F4,244=45.10, P<10-5, N=7,9/group and F4,324=28.63, P <10-5, N=9,13 for mx-cre and nestin-cre, respectively) and second training periods (F4,240=6.71, P<10-5 and F4,128=6.02, P <10-5). There was also a genotype effect in the mx-cre (F1,62=6.23, P=0.015 and F1,60=9.15, P=0.004 for the first and second training trial, respectively; LSD posthoc *P<0.05) but not in the nestin-cre group comparison. (b) Performance of the offspring born to mx-cre and nestin-cre mothers in probe trial 2 of MWM. A second period of 5 training sessions increased spatial memory in all groups in probe trial 2, eliminating the difference caused by the maternal hematopoietic deletion of TNF-α, seen in probe trial 1 (ANOVA: effect of quadrant for mx-cre and nestin-cre; F3,56=68.35, P<10-5, N=7,9 and F3,28=19.77, P<10-5, N=9,13; no group x location interaction). (c) PolyIC and the mx transgene do not confound the behavior in MWM. In WT mice, e.g. in the absence of floxed TNF alleles, poly IC and the mx-cre transgene in mothers have no effect on offspring learning and memory. The behavior of the offspring of mx-cre+ polyIC; TNF+/+ mothers is indistinguishable from the behavior of the control offspring of mx-cre-; TNFflox/flox mothers (control data are same as shown in Fig. 2c). (d) A temporal increase in freezing in the offspring of mx-cre+ but not nestin-cre+ mothers during tone-shock pairing (Repeated measures ANOVA; session effect F4,96=53.34, P<10-5, N=13,13/group and F4,60=23.80, P<10-5, N=7,10/group for mx-cre and nestin-cre, respectively; group effect F1,24=11.49, P=0.002 and F1,15=0.26, P=0.6; LSD posthoc. *P<0.05, **<0.005). The difference between the groups disappeared at the end of the training.
(a) Total number of cells (F3,14=3.95, P=0.03, N=4,5,5,5; *p<0.05, #p=0.05-0.1), (b) correlation between total and BrdU positive cells, and (c) the fraction of BrdU positive cells in percent of all cells (F3,14=0.52, P=0.67, N=4,5,5,5) in the GCL at P14. (d,e) No change in proliferation in the d-hilus, SVZ, and RMS at P14 (N=4,5,5,5). (f) No significant effect of the maternal genotype on the number of surviving BrdU positive cells in the DG, 3 weeks after pulse labeling with BrdU at P14 (P>0.05, N=5,5,5,6).
(a-h) No volumetric changes in neurogenic areas in the offspring of TNF deficient mothers. Volume of GCL (a, c, g, h), hilus (b, d), SVZ (e), RMS (f) at P5 (a and b), P14 (c-f) and adolescent/adult age (g,h). The maternal effect has no significant impact on the volume at any region and age (N=5 per group). (i-k) No significant effect of the maternal genotype on the number of GFAP+ astrogia and Iba1+ microglia, in the stratum moleculare (mol), stratum oriens (ori), and hilus of the hippocampus and in the cortex of P14 pups (P>0.05, N=4,6).
(a,b) Repeated GCV administration results in a cumulative dose dependent reduction in P14 proliferation (ANOVA and LSD postoc test: F6,112=6.85, P<10-3; N=4,4,6,8,4,5/per group; **P<0.005, ***P<0.0005). GCV administered at P5+6 is sufficient to reduce proliferation by approximately 30%. (c) Postnatal administration of GCV did not alter weigh gain, except in the P5+6+7+8 group at P14 (weight of animals in this group was not followed further) (Repeated measures ANOVA: F3,14=3.88, P=0.03, *P=0.013, N=7,7,4,7/group). (d,f) GCV (P5+6) reduces ANP proliferation in P14 DG (t test, p< 0.05, N=5/group). Box-whisker plots represent the first three quartiles (25%, median and 75%) and values 1.5× the interquartile range below the first quartile (lower horizontal line) and above the third quartile (upper horizontal line). Representative micrographs showing BrdU+/Tbr2+ ANPs in the DG of GCV treated TK+ and TK- mice. Bar=50μm. (e,g) GCV (P5+6) does not alter QNP proliferation in P14 DG. Representative confocal images with orthogonal views of SGZ showing BrdU-labeled Sox-2 and GFAP positive QNPs in the DG of GCV treated TK+ and TK- mice. Arrows indicate triple stained QNPs. Bar=20μm.
Supplementary Figure 7 No long term consequence of GCV administration on proliferation and reactive gliosis.
(a) GCV (P5+6) resulted in no change in adult DG proliferation. (b) GCV (P5+6) resulted in no change in GFAP positive cell number in the neocortex (Ctx), hilus and molecular layer (ML) of the DG in P14 animals. Representative confocal images with orthogonal views of cortex showing no obvious loss of GFAP positive cells in 2x GCV injected mice at P14. Bar=20μm.
(a) (a,b) Arched-back nursing (ABN) of TNF+/− mothers during the light and dark periods, respectively. Data show an effect of time (F4,73=3.13, P=0.02 and F4,78=4.58, P=0.02) but no group effect (n=7). (c,d) Licking/grooming (LG) of TNF+/− mothers during the light and dark periods, respectively. Effect of time only in the dark period (F4,77=34, p=0.013) but no group effect. (e) No difference between WT and TNF+/− mothers in the latency to pup retrieval. Effect of time (F2,24= 4.87 P=0.017). (f) No difference in nest quality between WT and TNF+/− mothers.
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Liu, B., Zupan, B., Laird, E. et al. Maternal hematopoietic TNF, via milk chemokines, programs hippocampal development and memory. Nat Neurosci 17, 97–105 (2014). https://doi.org/10.1038/nn.3596
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