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. 2021 Aug:96:28-39.
doi: 10.1016/j.bbi.2021.05.004. Epub 2021 May 12.

Moderately pathogenic maternal influenza A virus infection disrupts placental integrity but spares the fetal brain

Adrienne M Antonson  1 , Adam D Kenney  2 , Helen J Chen  3 , Kara N Corps  4 , Jacob S Yount  2 , Tamar L Gur  5
Affiliations

Moderately pathogenic maternal influenza A virus infection disrupts placental integrity but spares the fetal brain

Adrienne M Antonson et al. Brain Behav Immun. 2021 Aug.

Abstract

Maternal infection during pregnancy is a known risk factor for offspring mental health disorders. Animal models of maternal immune activation (MIA) have implicated specific cellular and molecular etiologies of psychiatric illness, but most rely on pathogen mimetics. Here, we developed a mouse model of live H3N2 influenza A virus (IAV) infection during pregnancy that induces a robust inflammatory response but is sublethal to both dams and offspring. We observed classic indicators of lung inflammation and severely diminished weight gain in IAV-infected dams. This was accompanied by immune cell infiltration in the placenta and partial breakdown of placental integrity. However, indications of fetal neuroinflammation were absent. Further hallmarks of mimetic-induced MIA, including enhanced circulating maternal IL-17A, were also absent. Respiratory IAV infection did result in an upregulation in intestinal expression of transcription factor RORγt, master regulator of a subset of T lymphocytes, TH17 cells, which are heavily implicated in MIA-induced etiologies. Nonetheless, subsequent augmentation in IL-17A production and concomitant overt intestinal injury was not evident. Our results suggest that mild or moderately pathogenic IAV infection during pregnancy does not inflame the developing fetal brain, and highlight the importance of live pathogen infection models for the study of MIA.

Keywords: Fetal development; Fetal neuroinflammation; Gestational infection; Infection during pregnancy; Influenza A virus; Influenza infection; Maternal immune activation; Maternal inflammation; Maternal viral infection; T helper 17 cells.

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Conflict of interest statement

Declarations of Interest. The authors declare no interests.

Figures

Figure 1.
Figure 1.. Gestational respiratory IAV infection induces robust respiratory and systemic inflammation.
IAV inoculation at gestational day (GD)10 (A) suppressed body weight gain beginning three days post-inoculation (repeated measures ANOVA: time x infection, p < 0.001, Bonferroni multiple comparisons correction, *** = adj p < 0.001, ** = adj p < 0.01, * = adj p < 0.05), resulting in substantial reduction in maternal body weight compared to control dams at 7 dpi. (B) Relative to body weight, maternal spleen mass at 7 dpi was increased due to IAV infection. Con: n = 17, IAV: n = 20. (C) Presence of IAV in the lung was confirmed through PCR, using a cycle threshold of ≤ 32 cycles as a confirmed infection (dotted line). (D) Inflammatory cytokines associated with innate and adaptive anti-viral immune response were increased in the lungs of IAV infected dams at 7 dpi, concomitant with (E) upregulated immune cell marker genes (Cd4: T helper cells, Cd8a1: cytotoxic T cells, Itgam: neutrophils and monocytes, Ly6c1: inflammatory monocytes), indicating immune cell infiltration. (F) IAV infection increased concentrations of IL-6, TNFα, and IFN-γ in circulation. Lung cytokines: Con n = 8, IAV n = 6; lung PCR: Con n = 17, IAV n = 20; plasma cytokines: n = 13/group. IAV = influenza A virus; dpi = days post-inoculation; ND = not detectable; *** = p < 0.001, ** = p < 0.01, * = p < 0.05. Data are presented as mean ± SEM, with each individual dot representing one dam.
Figure 2.
Figure 2.. Maternal respiratory IAV-infection increases placental necrosis, mineralization and inflammation.
Representative photomicrographs of H&E-stained placental sections and lesion score quantification from Control and IAV-infected dams. While Con dams had small foci of necrosis only in the maternal portion of the placenta and lacked an overt inflammatory response (A), all IAV-infected dams had larger, multifocal to coalescing areas of necrosis in the maternal layers (B) and 2 of 6 had necrosis in the fetal layers. Necrosis in IAV dams was frequently accompanied by neutrophilic inflammation, and scattered neutrophils were present in all layers of the maternal tissue (C, arrows). Control dams had small foci of mineralization in the fetal vascular labyrinth, a frequent and expected finding (D, arrow). In contrast, IAV-infected dams had numerous scattered foci of mineral throughout the fetal vascular labyrinth that were occasionally accompanied by neutrophilic inflammation or focal necrosis (E, arrows). Increased overall basophilic staining was also evident (B, E) and is caused by a combination of increased circulating inflammatory cells, particularly mononuclear cells and multifocal neutrophils, and mild-to-moderate mineralization of capillary walls. Inflammatory lesion scores are quantified in (F); scoring criteria is listed in the Methods. Two sections each from two representative placentas per litter were scored and averaged; Con: n = 8 litters, IAV: n = 6 litters. A-B: 10X magnification, scale bar = 100 μm; C-E: 20X magnification, scale bar = 50 μm. IAV = influenza A virus; *** = p < 0.001, * = p < 0.05. Group scores are presented as mean ± SEM.
Figure 3.
Figure 3.. Maternal IAV infection increases placental inflammatory signaling and bactericidal responses.
(A) Pooled placental protein extracts from control (n = 8) and IAV-infected (n = 6) dams were assayed on the Proteome Profiler Cytokine Array, suggesting enhanced immune responses due to maternal IAV infection. (B) Upregulation of Ifng, bactericidal C-type lectin Reg3b, and Gzmb (Granzyme B) expression in placentas from IAV-infected dams (n = 9–13/group). IAV = influenza A virus; ** = p < 0.01, # = p ≤ 0.10. Proteome profiler data are presented as mean pixel density across the two spots (no error bars). Gene expression data are presented as mean ± SEM.
Figure 4.
Figure 4.. Fetal neuroinflammation is not overtly apparent during mild maternal IAV infection.
(A) Pooled fetal brain protein extracts from litters of control (n = 7) and IAV-infected (n = 4) dams assayed on the Proteome Profiler Cytokine Array revealed only two detectable signaling molecules, suggesting limited inflammatory response to maternal IAV infection in the fetal brain. Proteome profiler data are presented as mean pixel density across the two spots (no error bars). (B) Independent qPCR was carried out on fetal brain tissue to confirm and extend upon the NanoString Neuroinflammation Panel data. PCR data are represented as a heatmap of delta Ct values (compared to housekeeping control gene = Rpl19) of select genes involved in neuroimmune responses, blood brain barrier integrity, and neurodevelopment within the fetal brain. Purple indicates low expression compared to Rpl19 (i.e. higher cycle threshold); red indicates high expression compared to Rpl19 (i.e. lower cycle threshold). A modest but significant downregulation in Il17ra and Il1b expression was observed due to maternal IAV-infection, but no other genes were differentially expressed. Mean ± SEM and p-values for all genes are listed in Supp. Table S5. IAV = influenza A virus. ** = p < 0.01, * = p < 0.05.
Figure 5.
Figure 5.. Downstream inflammatory impacts of gestational IAV infection on the maternal intestine.
IAV-infected dams displayed (A) reduced colon lengths (Con: n = 17, IAV: n = 20) concurrent with upregulation in colonic expression of transcription factor Rorc, Ifng, and tight junction proteins Cldn1 and Ocln (n = 6–13/group), while there were no changes in Cldn2 and Cldn5 expression or IL-17 protein within colon protein extracts (n = 9/group). (B) Intraepithelial lymphocytes (IEL; CD45+ fraction) isolated from the small intestine upregulated Rorc expression (n = 5–8/group). (C) Relative expression of most antimicrobial response genes was dysregulated in intestinal epithelial cells (IEC; CD45− fraction) of IAV-infected dam small intestine, while TH17-related genes were unchanged (n = 5–8/group). (D) The plasma concentration of LPS-binding protein, a proxy for disrupted intestinal barrier integrity, was not impacted by IAV infection (p = 0.52; Con: n = 12, IAV: n = 14). IAV = influenza A virus; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, # = p ≤ 0.10. Lines indicate mean ± SEM, with each individual dot representing one dam. Gene expression data are presented as mean ± SEM.
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