The gut microbiome is a central regulator of overall health, and establishing mutually beneficial relationships between the host and resident gut bacteria is important for preventing the later development of pathologies such as ulcerative colitis, metabolic dysregulation, and colon cancer. While microbiota-reactive maternal IgA antibodies in the breastmilk have historically been considered to be a primary regulator of host-microbiota interactions, maternal IgG antibodies have historically been associated with the protection of neonates against pathogens. However, our lab has identified a novel function for microbiota-reactive maternal IgG2b and IgG3 antibodies in preventing dysregulated adaptive immune responses and reinforcing intestinal homeostasis in early life. To investigate how these maternal antibodies function, we created a mouse strain (IgG3^-/-) lacking IgG3 antibodies. Using IgG3^-/- dams, we were able to selectively prevent the transfer of maternal IgG3 antibodies to their pups while maintaining normal transfer of the other antibody isotypes. Compared to the pups born to wild type (i.e. antibody replete) dams, these pups displayed reduced weight gain, increased inflammatory gene expression, dysregulated T cell immunity, and increased susceptibility to intestinal colitis induced by dextran sodium sulfate (DSS). With our data suggesting an integral role for maternal IgG3 antibodies in limiting neonatal immune responses toward gut microbes, my current experiments aim to elucidate the underlying mechanisms by which these antibodies function. Using pups born to C1q deficient and FcγR deficient dams, which lack the ability to activate the complement pathway and FcγRs, respectively, I am aiming to test the roles of these antibody “sensing” pathways in maternal IgG3-mediated suppression of neonatal intestinal immunity. Defining these mechanisms not only gives us a clearer picture of the ways in which neonatal health is regulated; it also advances our ability to manipulate neonatal immunity and early life gut microbiome-targeted treatments to improve human health.

Breast milk is essential to the health and development of a child, containing antibodies that protect infants from common illnesses. However, exclusive breastfeeding is not always possible, and no infant formula substitutes for maternal antibodies. Previous studies on mice showed high germinal center (GC) B cell levels in response to the absence of maternal antibodies from breast milk early in life. Germinal centers B cells are involved in the adaptive immune system by secreting high affinity antibodies. However, little is known about the consequence of this, making characterizing the isotype, location, and duration of the antibody response in neonates (newborns) lacking maternal breast milk antibodies essential. For this project, I designed and optimized a tissue preparation and flow cytometry panel to assess memory B cells, GC B cells, and plasma cells (cells that secrete antibodies to fight infections and disease). The flow panel uses the cell markers CD138 and B220 to identify plasma cells by isolating the cells that are positive for CD138 and negative for B220. However, the marker CD138 can be sensitive to collagenase, resulting in the potential failure to identify plasma cells successfully. For this reason, I tested a range of collagenases, including Collagenase A, D, and IV. Concluding that CD138 was being cleaved off by all tested collagenases, I then used TACI as a new type of plasma cell differentiation marker. I evaluated these protocols by the viability of cells and the plasma cells' frequency. This protocol allows for the determination of localization, persistence, and isotype of early life B cells activated in the absence of breast milk.

We are currently investigating how neonates establish a symbiotic relationship between their developing immune system and the microbes colonizing their gut immediately post birth, and what role factors (e.g. antibodies) from the mother play in establishing this relationship. Lactobacillus is a genus of commensal bacteria that is commonly found in the neonatal intestine of both mice and humans. To track immune responses in the gut, I will create a fluorescent Lactobacillus species by transforming (genetically altering a cell through uptake and incorporation of outside DNA) a plasmid encoding a fluorescent protein into the bacterial cell. By engineering a fluorescent commensal species, we can track a normal, healthy immune-commensal interaction in vivo (in an animal model). Using this novel tool, we will study and compare how neonatal mice that do and do not receive antibodies from the mother post-birth differ in their immune response against these commensal species.