Offspring born to influenza A virus infected pregnant mice have increased susceptibility to viral and bacterial infections in early life

Abstract

Influenza during pregnancy can affect the health of offspring in later life, among which neurocognitive disorders are among the best described. Here, we investigate whether maternal influenza infection has adverse effects on immune responses in offspring. We establish a two-hit mouse model to study the effect of maternal influenza A virus infection (first hit) on vulnerability of offspring to heterologous infections (second hit) in later life. Offspring born to influenza A virus infected mothers are stunted in growth and more vulnerable to heterologous infections (influenza B virus and MRSA) than those born to PBS- or poly(I:C)-treated mothers. Enhanced vulnerability to infection in neonates is associated with reduced haematopoetic development and immune responses. In particular, alveolar macrophages of offspring exposed to maternal influenza have reduced capacity to clear second hit pathogens. This impaired pathogen clearance is partially reversed by adoptive transfer of alveolar macrophages from healthy offspring born to uninfected dams. These findings suggest that maternal influenza infection may impair immune ontogeny and increase susceptibility to early life infections of offspring.

Fig. 1. 1st hit maternal influenza virus infection and immune activation.

a The first hit was performed during early pregnancy by application of either poly(I:C) or infection with IAV. b Pregnant C57BL/6 mice were treated intraperitoneally (i.p.) with 4 mg/kg poly(I:C) (n = 24) or infected intranasally (i.n.) with 10³ PFU of IAV (n = 24) on day 5.5 of gestation. Phosphate-buffered saline (PBS)-treated (i.n.) dams were used as controls (n = 20). Weight development was monitored for 14 days post infection (d p.i.), p = 0.0003 (1 d p.i.), p < 0.0001 (3-12 d p.i.). c Weight of non-pregnant C57BL/6 mice treated with PBS, poly(I:C) or infected with IAV (n = 10), p = 0.0003 (1 d p.i.), p < 0.0001 (3–10 d p.i.), p = 0.0044 (13 d p.i.). d Representative immunohistology of IAV NP antigen in maternal lungs at 3 days post infection, Scale bar = 500 µm. e IAV titers as plaque forming units (PFU) per gram were measured in whole lung homogenates from pregnant dams treated with PBS (n = 4, 5, 6 [1, 3, 6 d p.i.], poly(I:C) (n = 6, 4, 6 [1, 3, 6 d p.i.] or infected with IAV (n = 6, 6, 4 [1, 3, 6 d p.i.] by plaque assay. f–h Lung cytokines IL-6, p < 0.0001 (3 d p.i.), p = 0.0010 (6 d p.i.) (f); TNF, p = 0.0001 (3 d p.i.), p = 0.0103 (6 d p.i.) (g); and MCP-1, p = 0.0195 (3 d p.i.), p = 0.0058 (6 d p.i.) (h) detected by Luminex assay in lungs of pregnant mice treated with PBS (IL-6, n = 10, 10, 7 [1, 3, 6 d p.i.]); (TNF, n = 11, 10, 7 [1, 3, 6 d p.i.]); (MCP-1, n = 6, 5, 7 [1, 3, 6 d p.i.]), poly(I:C) (IL-6, n = 10, 8, 9 [1, 3, 6 d p.i.]); (TNF, n = 10, 8, 9 [1, 3, 6 d p.i.]) and (MCP-1, n = 11, 9, 10 [1, 3, 6 d p.i.]) or infected with IAV (IL-6, n = 11, 9, 6 [1, 3, 6 d p.i.]), (TNF, n = 10, 9, 6 [1, 3, 6 d p.i.]); (MCP-1, n = 5, 5, 6 [1, 3, 6 d p.i.]) at E5.5. i–k Plasma cytokines IL-6, p = 0.0343 (i); TNF, p = 0.0218 poly(I:C), p = 0.0429 (j) and MCP-1, p = 0.0329 poly(I:C) (k) measured by Luminex assay in plasma of pregnant mice treated with PBS (IL-6, n = 6, 4, 7 [1, 3, 6 d p.i.]); (TNF, n = 6, 5, 7 [1, 3, 6 d p.i.]; (MCP-1, n = 6, 5, 7 [1, 3, 6 d p.i.]); poly(I:C) (IL-6, n = 9, 7, 9 [1, 3, 6 d p.i.]); (TNF, n = 8, 7, 9 [1, 3, 6 d p.i.]); (MCP-1, n = 9, 7, 9 [1, 3, 6 d p.i.]) or infected with IAV (IL-6, n = 5, 6, 6 [1, 3, 6 d p.i.]; (TNF, n = 6, 6, 6 [1, 3, 6 d p.i.]); (MCP-1, n = 6, 6, 6 [1, 3, 6 d p.i.]) at E5.5. Values are normalized to organ weight if applicable. l, m Hormones (corticosterone, p = 0.0456 (l) and progesterone, p = 0.0247 (1 d p.i.), p = 0.0254 (3 d p.i.) (m) measured by ELISA in plasma of pregnant mice treated with PBS (n = 9, 11, 3 [1, 3, 6 d p.i.]; poly(I:C) (n = 7, 7, 6 [1, 3, 6 d p.i.] or infected with IAV (n = 11, 11, 11 [corticosterone 1, 3, 6 d p.i.], n = 11, 10, 3 [progesterone, 1, 3, 6 d p.i.] at E5.5. All data are presented as mean and SD. Different groups are depicted in dark circles (PBS), medium squares (poly(I:C)) or light triangles (IAV) in violet colors. Cytokine levels that were below detection limit were set to the kit’s lower detection limit of 1 pg/g. The statistical significance was calculated by Welch’s t test (two-tailed) (*p < 0.05, **p < 0.01, ***p < 0.001). PBS-treated groups were used as a reference and compared to IAV-infected groups in all statistical analyses unless stated otherwise. Non-significant comparisons are not depicted within the respective figures. n. d. not detectable. Source data are provided as a Source Data file.

Fig. 2. 1st hit maternal influenza and reproductive outcome.

a Pregnancy outcome was assessed at embryonic day 17.5 (E17.5) or post partum (pp) with 2-week-old offspring. b Gestational length in days (d) of dams that were treated with phosphate-buffered saline (PBS) (n = 66), treated with 4 mg/kg poly(I:C) (n = 65), or infected with 10³ PFU IAV (n = 61). c Litter size of PBS-treated dams at E17.5 (n = 14) and 2 w p.p. (n = 53), poly(I:C)-treated dams at E17.5 (n = 6) and 2 w p.p. (n = 48) and IAV-infected dams at E17.5 (n = 13) and 2 w p.p. (n = 43). Only viable fetuses were counted. d Percentage of male fetuses per litter for PBS-treated dams at E17.5 (n = 10) and 2 w p.p. (n = 53), poly(I:C)-treated dams at E17.5 (n = 6) and 2 w p.p. (n = 48) and IAV-infected dams at E17.5 (n = 13) and 2 w p.p. (n = 43). Only viable fetuses were counted. Fetal sex was determined by PCR, sex of 2-week-old offspring was determined by physiological examination. e Placenta weight in grams (g) from dams treated with PBS (n = 72), treated with poly(I:C) (n = 23, p < 0.0001) or infected with IAV (n = 43, p = 0.0062) at E17.5. f Relative mRNA expression of nutrient supply genes of growth inhibitory gene Grb10 (growth factor receptor bound protein 10); growth stimulatory gene Igf2 (insulin-like growth factor II), p = 0.0441 poly(I:C); Slc38a1 (solute carrier family 38, member 1), p = 0.049 and Slc38a2 (solute carrier family 38, member 2), p = 0.0059 (poly(I:C), p = 0.0148 (IAV) of placentas from fetuses from PBS (n = 5), poly(I:C) (n = 8) or IAV (n = 6) infected dams. The relative expression in the PBS group was set to 1 for each gene after normalization to the housekeeping gene Ywhaz. g Fetuses were weighed on E17.5 for PBS-treated dams (n = 29 males, 26 females), poly(I:C)-treated dams (n = 20 males, p < 0.0001, 29 females; p < 0.0001), and IAV-infected dams (n = 23 males, p < 0.0001, 35 females, p < 0.0001). Only viable fetuses were included. h Offspring weight at 2 weeks of age (2 weeks post partum; 2 wpp) born to mothers treated with PBS (n = 54 males; n = 56 females), poly(I:C) (n = 47 males, p = 0.8791; n = 49 females, p = 0.2665), and IAV (n = 59 males, p < 0.0001; n = 67 females, p < 0.0001). i Offspring weight at 6 weeks of age (6 wpp) born to mothers treated with PBS (n = 48 males, n = 57 females), poly(I:C) (n = 36 males, p = 0.9430; n = 46 females, p = 0.9831), and IAV (n = 69 males, p = 0.0064; n = 57 females, p = 0.039). j Offspring born to PBS-treated mothers was exchanged with offspring born to IAV-infected mothers immediately after birth and weight gain was observed for each animal individually during the first 2 weeks of life. Each group consisted of two litters with eight pups each. All data are presented as mean and SD. Different groups of mothers are depicted in dark circles (PBS), medium squares (Poly(I:C)), or light triangles (IAV) in violet colors. Different groups of male (♂) offspring are depicted in dark circles (PBS), medium squares (Poly(I:C)), or light triangles (IAV) in blue colors and female (♀) offspring in red colors. The statistical significance was calculated by two-tailed ANOVA (*p < 0.05, **p < 0.01, ***p < .001). PBS-treated groups were used as a reference in all statistical analyses. Source data are provided as a Source Data file.

Fig. 3. 2nd hit susceptibility of offspring from influenza infected dams to early life infections.

a Second-hit experiments were performed in 2-week-old offspring using influenza B virus (IBV) or Methicillin-resistant Staphylococcus aureus (MRSA) for a respiratory challenge. b Survival of 2-week-old male offspring born to IAV-infected dams (n = 11 [day 3], n = 9 [day 6], n = 7 [day 14]) or born to control treated dams with polyinosinic:polycytidylic acid (poly(I:C))-treated (n = 11 [day 3], n = 9 [day 6], n = 7 [day 14]) or PBS (n = 9 [day 3], n = 7 [day 6], n = 5 [day 14]) after infection with 10⁵ plaque forming units (PFU) of IBV; p = 0.0355. c Survival of 2-week-old female offspring born to IAV-infected dams (n = 13 [day 3], n = 11 [day 6], n = 9 [day 14]) or born to control treated dams with poly(I:C)-treated (n = 11 [day 3], n = 9 [day 6], n = 7 [day 14]) or PBS (n = 8 [day 3], n = 6 [day 6], n = 4 [day 14]) after infection with 10⁵ PFU of IBV. Survival was monitored in three independent experiments. d 2-week-old offspring (both sexes) born to poly(I:C)-treated (n = 21 [day 1], 14 [day 3], 7 [day 6]) or IAV-infected (n = 55 [day 1], 34 [day 3], 16 [day 6]) dams were infected with 10⁸ CFU of MRSA; p = 0.0167. Offspring born to PBS-treated dams (n = 38 [day 1], 25 [day 3], 12 [day 6]) were used as controls. Survival was monitored on day 1, 3 or 6 p.i. in three independent experiments. e–g Cytokines (TNF (e), IL-6 (f), and IL-2 (g)), p = 0.0034 poly(I:C), p = 0.0219 measured by Luminex assay in lungs of 2-week-old offspring (both sexes) born to PBS- or poly(I:C)-treated or IAV-infected offspring (n = 4) after infection with 10⁵ PFU of IBV measured at 1, 3, or 6 d p.i. h–j Cytokines (TNF (h), IL-6 (i), and IL-2 (j)) measured by Luminex assay in lungs of 2-week-old offspring (both sexes) born to PBS- (n = 10) or poly(I:C)-treated (n = 7) or IAV-infected offspring (n = 11) after infection with 10⁸ CFU of MRSA measured at 1, 3, or 6 d p.i. k IBV lung titer in 2-week-old offspring (both sexes) born to PBS-, poly(I:C)-treated, or IAV-infected (n = 4) dams, measured on day 1, 3 (p = 0.0270), and 6 p.i. is shown as fold-change in lung titers compared to the respective PBS group of the indicated time point. l MRSA lung titer in 2-week-old offspring (both sexes) born to PBS- (n = 10 [1 d p.i.], 10 [3 d p.i.], 7 [6 d p.i.]), poly(I:C)-treated (n = 7 [1 d p.i.], 7 [3 d p.i.], 4 [6 d p.i.]) or IAV-infected (n = 11 [1 d p.i.], 13 [3 d p.i., p = 0.0189], 9 [6 d p.i., p = 0.0223]) dams, measured on days 1, 3, and 6 p.i. is shown as fold-change in lung titers compared to the respective PBS group of the indicated time point. All n represent number of male and/or female offspring from respective groups. Groups were merged for clarity if no difference between males and females was observed. Data in (e–j) are presented as mean and SD. Different groups of male offspring are depicted in dark circles (PBS, medium squares (Poly(I:C)) or light triangles (IAV) in blue colors and female offspring in red colors. Different groups of offspring that were not stratified by sex are depicted in orange colors with the same icons. Cytokine levels that were below detection limit were set to the kit’s lower detection limit of 1 pg/g. The statistical significance was calculated by Welch’s t test (two-tailed) (e–j, k, l) without multiple comparison or Log Rank test (b–d) (*p < 0.05, **p < 0.01). PBS-treated groups were used as a reference and compared to IAV-infected groups in all statistical analyses unless stated otherwise. Source data are provided as a Source Data file.

Fig. 4. Altered hematopoiesis of offspring from influenza infected dams.

a Markers used to define respective stem (HSC) and progenitor cell populations by flow cytometry in this study. b–f Frequency of Lin^- Sca⁺ cKit⁺ HSC, p = 0.0236 (b); Lin⁻ Sca^low cKit⁺ progenitor cells, p = 0.0121 (c); Lin⁻ Sca^low cKit⁺ CD16/32^low CD34⁺ common myeloid progenitor (CMP) cells, p = 0.0035 (d); Lin⁻ Sca^low cKit⁺ CD16/32⁺ CD34⁺ granulocyte–monocyte progenitor (GMP) cells, p = 0.0518 (e); and Lin⁻ Sca^low cKit⁺ CD16/32⁻ CD34⁻ megakaryocyte-erythrocyte progenitor (MEP) cells (f) as percentage of Lin⁻ DAPI⁻ events in the bone marrow of 2-week-old offspring (n = 8) born to early gestational phosphate-buffered saline (PBS)-treated, polyinosinic:polycytidylic acid (poly(I:C))-treated or influenza A virus (IAV)-infected dams, as assessed by flow cytometry. g–l Frequency of B220⁺ B cells (g and j, p = 0.114 poly(I:C), p < 0.0001), CD49b⁺ natural killer (NK) cells (h, p = 0.0008 poly(I:C), p < 0.0001 and k, p = 0.0167), CD3⁺ CD4⁺ CD25⁺ FoxP3⁺ regulatory T ⁽T_Reg) cells (I, p = 0.0710 and l, p = 0.0059) as percentage of alive leukocytes in spleens (g–i) or lungs (j–l) of 2-week-old offspring (n = 8) born to early gestational PBS-treated, poly(I:C)-treated or IAV-infected dams, as assessed by flow cytometry. m Frequency of CD11b^- CD11⁺ CD45⁺ SiglecF⁺ alveolar macrophages as percentage of CD45⁺ cells in lung of 2-week-old offspring born to early gestational PBS-treated (n = 18), poly(I:C)-treated (n = 16) or IAV-infected (n = 16, p = 0.0287) dams, as assessed by flow cytometry. All n represent number of male and/or female offspring from respective groups. Groups were merged for clarity as no difference between males and females was observed. All data are presented as mean and SD. Different groups of offspring are depicted in dark circles (PBS), medium squares (Poly(I:C)), or light triangles (IAV) in orange colors. The statistical significance was calculated by Welch’s t test (two-tailed) (*p < 0.05, **p < 0.01, ***p < 0.001). PBS-treated groups were used as a reference and compared to IAV-infected groups in all statistical analyses unless stated otherwise. Source data are provided as a Source Data file.

Fig. 5. Functionally impaired alveolar macrophages in offspring of influenza infected dams.

a Adoptive transfer experiments were performed in 2-week-old offspring followed by influenza B virus (IBV) infection 16 h post transfer (h p.t.). b, e Alveolar macrophages (AM) of 2-week-old male, p = 0.0058 (b) and female (e) offspring born to polyinosinic:polycytidylic acid (poly(I:C))-treated mothers (n = 10 males, 10 females) or influenza A virus (IAV)-infected mothers (n = 11 males, 14 females) were pooled, and 10⁵ AMs were transferred intranasally into offspring of the same sex (n = 5 [male, poly(IC)], 6 [male, IAV], 8 [female, poly(I:C)], 7 [female, IAV]) born to phosphate-buffered saline (PBS)-treated mothers with subsequent infection of the sentinel-offspring 16 h post transfer with 10⁵ plaque forming units (PFU) of IBV. c, d, f, g Alveolar macrophages of 2-week-old male (c, d) and female (f, g) offspring born to PBS-treated mothers (n = 22 males, 29 females) were pooled and 10⁵ AMs were transferred intranasally into offspring of the same sex (n = 7 [male, poly(I:C)], 8 [female, poly(I:C)], 6 [male IAV, p = 0.0115 (1 dp.i.), p = 0.0050 (2d p.i.)], 6 [female IAV, p = 0.008 (1d p.i.), p = 0.0068 (2d p.i.), p = 0.0071 (3d p.i.)]) born to poly(I:C)-treated (c, f) or IAV-infected (d, g) mothers with subsequent infection of the sentinel-offspring 16 h post transfer with 10⁵ PFU of IBV. Weight development was observed until 3 d p.i. h IBV lung titer from offspring born to PBS-treated dams and after transfer of AMs from poly(I:C)- or IAV-offspring with subsequent IBV challenge 12 h p.t. at 3 d p.i. (n = 10). Dotted lines indicate reference titers from IBV infected offspring without AM transfer (shown in Fig. 3). i IBV lung titer from offspring born to poly(I:C)-treated (n = 10) or IAV-infected, p = 0.089 (n = 9) dams and after transfer of AMs from PBS-offspring with subsequent IBV challenge 12 h p.t. at 3 d p.i. All experiments were also performed with PBS-treatment of the offspring post AM transfer but data are not shown to enhance clarity and all PBS-treated offspring were negative for reduced weight gain or virus titers, as expected. All data are presented as mean and SD. Different groups of male (♂) offspring are depicted in dark circles (PBS, medium squares (Poly(I:C)) or light triangles (IAV) in blue colors and female (♀) offspring in red colors. Different groups of offspring that were not stratified by sex are depicted in orange colors with the same icons. The statistical significance was calculated by two-tailed t-tests using the Bonferroni-Dunn multiple testing correction (b–g) or by Welch’s t test (two-tailed) (h, i) (*p < 0.05, **p < 0.01, ***p < 0.001). Non-significant comparisons are not depicted within the respective figures. PBS-treated groups were used as a reference and compared to IAV-infected groups in all statistical analyses unless stated otherwise. Source data are provided as a Source Data file.