Neonatal immune cells have heightened responses following in-utero exposure to chorioamnionitis or COVID-19

Abstract

Background: Chorioamnionitis alters neonatal immune responses. Gestational COVID-19 infection is associated with adverse pregnancy outcomes, but its impact on neonatal immunity is unclear. We hypothesized that gestational COVID-19 exposure would result in exaggerated neonatal immune responses, similar to chorioamnionitis-exposed neonates.

Methods: Term umbilical cord blood mononuclear cells (CBMCs) were isolated from neonates exposed to chorioamnionitis, gestational COVID-19 or unexposed controls. CBMCs were cultured and stimulated with heat-killed Escherichia coli, Streptococcus agalactiae or Staphylococcus epidermidis. A multiplexed protein assay was used to measure cytokine levels in cell culture supernatants and flow cytometry was used to evaluate cellular-level cytokine expression.

Results: Both chorioamnionitis-exposed and COVID-19 exposed CBMCs demonstrated upregulation of IL-1β and IL-6 compared to unexposed CBMCs, while only COVID-19 exposure resulted in IL-8 upregulation. There were no differences between chorioamnionitis-exposed and COVID-19 exposed CBMCs when these groups were directly compared. Flow cytometry demonstrated immune cell subset specific differences in cytokine expression between the exposure groups.

Conclusion: The fetal/neonatal response to maternal inflammation differed based on immune cell subset and etiology of inflammation, but the global neonatal cytokine responses were similar between exposure groups. This suggests that targeting perinatal inflammation rather than the specific etiology may be a possible therapeutic approach.

Impact: Neonatal immune cells have similar pathogen-associated global cytokine responses, but different cell-level immune responses, following in-utero exposure to chorioamnionitis or COVID-19. This is the first study to directly compare immune responses between neonates exposed to chorioamnionitis and COVID-19. This suggests that the fetal/neonatal cellular response to perinatal inflammation differs based on the etiology and severity of maternal inflammation, but still results in a similar overall inflammatory profile regardless of the cause of perinatal inflammation.

Figure 1.. Chorioamnionitis-exposed CBCMs have elevated IL-1β and IL-6 levels after heat-killed bacterialstimulation compared to unexposed CBMCs.

Cord blood mononuclear cells were isolated from umbilical cord blood afterdelivery. CBMCs were then cultured and stimulated with heat-killed E. coli, S. agalactiae, and S. epidermidis at an MOI of 10:1.Cytokine levels were evaluated using multiplexed protein assay for unexposed CBMCs and chorioamnionitis-exposed CBMCs. ●= unexposed CMBC (n=23), ○ = chorioamnionitis-exposed CBMC (n=9). (A) IL-1β, IL-6, IL-8, IL-10, IP-10, and MCP-1 levelswith no stimulation, (B) IL-1β, IL-6, IL-8, IL-10, IP-10, and MCP-1 levels after E. coli stimulation, (C) IL-1β, IL-6, IL-8, IL-10, IP-10, and MCP-1 levels after S. agalactiae stimulation, (D) IL-1β, IL-6, IL-8, IL-10, IP-10, and MCP-1 levels after S. epidermidisstimulation. Bars represent the mean cytokine level (pg/mL) and error bars represent SEM with individual data points shown.*p<0.05, **p<0.01. Data was analyzed using Welch ANOVA test following log transformation of the data.

Figure 2.. COVID-19 exposed CBMCs demonstrate elevated IL-1β, IL-6, and IL-8 levels after heat-killed bacterialstimulation compared to unexposed CBMCs.

Cord blood mononuclear cells were isolated from umbilical cord blood afterdelivery. CBMCs were then cultured and stimulated with heat-killed E. coli, S. agalactiae, and S. epidermidis. Cytokine levelswere evaluated using multiplexed protein assay for unexposed CBMCs and COVID-19 exposed CBMCs in each trimester ofpregnancy. ● = unexposed CMBCs (n=23), ○ (gray) = COVID-19 exposed CBMCs in any trimester (n=36). (A) IL-1β, IL-6, IL-8,IL-10, IP-10, and MCP-1 levels after no stimulation, (B) IL-1β, IL-6, IL-8, IL-10, IP-10, and MCP-1 levels after E. colistimulation, (C) IL-1β, IL-6, IL-8, IL-10, IP-10, and MCP-1 levels after S. agalactiae stimulation, (D) IL-1β, IL-6, IL-8, IL-10, IP-10, and MCP-1 levels after S. epidermidis stimulation. Bars represent the mean cytokine level (pg/mL) and error bars representSEM with individual data points shown. *p<0.05, **p<0.01. Data was analyzed using Welch ANOVA test following log transformation of the data.

Figure 3.. There are no differences in cytokine profiles between chorioamnionitis and COVID-19 exposed CBMCs.Cord blood mononuclear cells were isolated from umbilical cord blood after delivery.

CBMCs were then cultured and stimulatedwith heat-killed E. coli, S. agalactiae, and S. epidermidis. Cytokine levels were evaluated using multiplexed protein assay forchorioamnionitis-exposed CBMCs, COVID-19 exposed CBMCs in any trimester of pregnancy, and both chorioamnionitis-exposedand COVID-19 exposed CBMCs. ○ = chorioamnionitis-exposed CBMCs (n=9), ○ (gray) = COVID-19 exposed CBMCs in anytrimester (n=36), ● = chorioamnionitis and COVID-19 exposed CBMCs in any trimester (n=7). (A) IL-1β, IL-6, IL-8, IL-10, IP-10, MCP-1 levels after no stimulation, (B) IL-1β, IL-6, IL-8, IL-10, IP-10, MCP-1 levels after E. coli stimulation, (C) IL-1β, IL-6,IL-8, IL-10, IP-10, MCP-1 levels after S. agalactiae stimulation, (D) IL-1β, IL-6, IL-8, IL-10, IP-10, MCP-1 levels after S.epidermidis stimulation. Bars represent the mean cytokine level (pg/mL) and error bars represent SEM with individual datapoints shown. Data was analyzed using the Brown-Forsythe and Welch ANOVA test following log transformation of the data.