Immune landscape of human placental villi using single-cell analysis.

Maintenance of a healthy pregnancy is reliant on a successful balance between the fetal and maternal immune systems. Although the maternal mechanisms responsible have been well studied, those used by the fetal immune system remain poorly understood. Using suspension mass cytometry and various imaging modalities, we report a complex immune system within the mid-gestation (17-23 weeks) human placental villi (PV). Consistent with recent reports in other fetal organs, T cells with memory phenotypes, although rare in abundance, were detected within the PV tissue and vasculature. Moreover, we determined that T cells isolated from PV samples may be more proliferative after T cell receptor stimulation than adult T cells at baseline. Collectively, we identified multiple subtypes of fetal immune cells within the PV and specifically highlight the enhanced proliferative capacity of fetal PV T cells.

Murine Type III interferons are functionally redundant and correlate with bacterial burden during influenza/bacterial super-infection.

BACKGROUND: Type III interferon, or interferon lambda (IFNλ) is a crucial antiviral cytokine induced by influenza infection. While IFNλ is important for anti-viral host defense, published data demonstrate that IFNλ is pathogenic during influenza/bacterial super-infection. It is known that polymorphisms in specific IFNλ genes affect influenza responses, but the effect of IFNλ subtypes on bacterial super-infection is unknown. METHODS: Using an established model of influenza, Staphylococcus aureus super-infection, we studied IFNλ3-/- and control mice to model a physiologically relevant reduction in IFNλ and to address its role in super-infection. RESULTS: Surprisingly, IFNλ3-/- mice did not have significantly lower total IFNλ than co-housed controls, and displayed no change in viral or bacterial clearance. Importantly, both control and IFNλ3-/- mice displayed a positive correlation between viral burden and total IFNλ in the bronchoalveolar lavage during influenza/bacterial super-infection, suggesting that higher influenza viral burden drives a similar total IFNλ response regardless of IFNλ3 gene integrity. Interestingly, total IFNλ levels positively correlated with bacterial burden, while viral burden and bronchoalveolar lavage cellularity did not. CONCLUSIONS: These data suggest IFNλ2 can compensate for IFNλ3 to mount an effective antiviral and defense, revealing a functional redundancy in these highly similar IFNλ subtypes. Further, the IFNλ response to influenza, as opposed to changes in cellular inflammation or viral load, significantly correlates with susceptibility to bacterial super-infection. Moreover, the IFNλ response is regulated and involves redundant subtypes, suggesting it is of high importance to pulmonary pathogen defense.

CD16+CD163+ monocytes traffic to sites of inflammation during necrotizing enterocolitis in premature infants.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using suspension and imaging mass cytometry coupled with single-cell RNA sequencing, we demonstrate severe inflammation in patients with NEC. NEC mucosa could be subtyped by an influx of three distinct neutrophil phenotypes (immature, newly emigrated, and aged). Furthermore, CD16+CD163+ monocytes/Mϕ, correlated with newly emigrated neutrophils, were specifically enriched in NEC mucosa, found adjacent to the blood vessels, and increased in circulation of infants with surgical NEC, suggesting trafficking from the periphery to areas of inflammation. NEC-specific monocytes/Mϕ transcribed inflammatory genes, including TREM1, IL1A, IL1B, and calprotectin, and neutrophil recruitment genes IL8, CXCL1, CXCL2, CXCL5 and had enrichment of gene sets in pathways involved in chemotaxis, migration, phagocytosis, and reactive oxygen species generation. In summary, we identify a novel subtype of inflammatory monocytes/Mϕ associated with NEC that should be further evaluated as a potential biomarker of surgical NEC and a target for the development of NEC-specific therapeutics.

In utero human intestine harbors unique metabolome, including bacterial metabolites.

Symbiotic microbial colonization through the establishment of the intestinal microbiome is critical to many intestinal functions, including nutrient metabolism, intestinal barrier integrity, and immune regulation. Recent studies suggest that education of intestinal immunity may be ongoing in utero. However, the drivers of this process are unknown. The microbiome and its byproducts are one potential source. Whether a fetal intestinal microbiome exists is controversial, and whether microbially derived metabolites are present in utero is unknown. Here, we aimed to determine whether bacterial DNA and microbially derived metabolites can be detected in second trimester human intestinal samples. Although we were unable to amplify bacterial DNA from fetal intestines, we report a fetal metabolomic intestinal profile with an abundance of bacterially derived and host-derived metabolites commonly produced in response to microbiota. Though we did not directly assess their source and function, we hypothesize that these microbial-associated metabolites either come from the maternal microbiome and are vertically transmitted to the fetus to prime the fetal immune system and prepare the gastrointestinal tract for postnatal microbial encounters or are produced locally by bacteria that were below our detection threshold.

Immune Cells in the Placental Villi Contribute to Intra-amniotic Inflammation.

Intra-amniotic (IA) inflammation is associated with significant morbidities for both the mother and the fetus. Prior studies have illustrated many of the effects of IA inflammation on the uterine lining (decidua) and membranous layers of the placenta at the fetal-maternal interface. However, much less is known about the immunological response occurring within the villous placenta. Using a rhesus macaque model of lipopolysaccharide (LPS)-induced IA inflammation, we showed that pregnancy-matched choriodecidua and villi have distinct immunological profiles in rhesus pregnancies. In the choriodecidua, we show that the abundance of neutrophils, multiple populations of antigen-presenting cells, and two populations of natural killer (NK) cells changes with prenatal IA LPS exposure. In contrast, in immune cells within the villous placenta we observed alterations in the abundance of B cells, monocytes, and CD8 T cells. Prior work has illustrated that IA inflammation leads to an increase in tumor necrosis factor alpha (TNFα) at the fetal-maternal interface. In this study, pretreatment with a TNFα blockade partially reversed inflammation in the placental villi. Furthermore, we report that immune cells in the villous placenta sensed LPS during our experimental window, and subsequently activated T cells to produce proinflammatory cytokines. Moreover, this study is the first report of memory T cells in third-trimester non-human primate placental villi and provides evidence that manipulation of immune cells in the villi at the fetal-maternal interface should be considered as a potential therapeutic target for IA inflammation.

Maturation of the Human Intestinal Immune System Occurs Early in Fetal Development.

There are limited data on fetal and early life development of human intestinal immunity. Using mass cytometry (CyTOF) and next-generation sequencing of B and T cell receptor (BCR and TCR) repertoires, we demonstrate complex intestinal immunity from 16 weeks' gestational age (GA). Both BCR and TCR repertoires are diverse with CDRH and CDR3ß length increasing with advancing GA. The difference-from-germline, CDR insertions and/or deletions, similarly occur in utero for TCR but not BCR, suggesting earlier mucosal T than B cell maturity. Innate immunity is dominated by macrophages, dendritic cells (DCs), innate lymphoid cells (ILCs), and natural killer (NK) cells. Follicular and transitional B cells are enriched in fetuses while CD69+IgM+ B cells are abundant in infants. Both CD4+ and CD8+ T cells are abundant, capable of secreting cytokines and are phenotypically of the tissue resident memory state in utero. Our data provide the foundation for a 2nd trimester and infant intestinal immune atlas and suggest that a complex innate and adaptive immune landscape exists significantly earlier than previously reported.

Interferon Lambda Inhibits Bacterial Uptake during Influenza Superinfection.

Influenza kills 30,000 to 40,000 people each year in the United States and causes 10 times as many hospitalizations. A common complication of influenza is bacterial superinfection, which exacerbates morbidity and mortality from the viral illness. Recently, methicillin-resistant Staphylococcus aureus (MRSA) has emerged as the dominant pathogen found in bacterial superinfection, with Streptococcus pneumoniae a close second. However, clinicians have few tools to treat bacterial superinfection. Current therapy for influenza/bacterial superinfection consists of treating the underlying influenza infection and adding various antibiotics, which are increasingly rendered ineffective by rising bacterial multidrug resistance. Several groups have recently proposed the use of the antiviral cytokine interferon lambda (IFN-λ) as a therapeutic for influenza, as administration of pegylated IFN-λ improves lung function and survival during influenza by reducing the overabundance of neutrophils in the lung. However, our data suggest that therapeutic IFN-λ impairs bacterial clearance during influenza superinfection. Specifically, mice treated with an adenoviral vector to overexpress IFN-λ during influenza infection exhibited increased bacterial burdens upon superinfection with either MRSA or S. pneumoniae Surprisingly, adhesion molecule expression, antimicrobial peptide production, and reactive oxygen species activity were not altered by IFN-λ treatment. However, neutrophil uptake of MRSA and S. pneumoniae was significantly reduced upon IFN-λ treatment during influenza superinfection in vivo Together, these data support the theory that IFN-λ decreases neutrophil motility and function in the influenza-infected lung, which increases the bacterial burden during superinfection. Thus, we believe that caution should be exercised in the possible future use of IFN-λ as therapy for influenza.