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Complement Proteins in Milk Promote Healthy Gut Microbiome

    A mother stares down at her infant lovingly. A new study reveals that milk complement proteins can protect infants from gastro-intestinal infections on their own.

    Written by: Lauren Milligan Newmark, Ph.D. | Issue # 120 | 2024

    • Human and some mammalian milks contain complement proteins, but it was not clear what role they played in infant immunity.
    • A new study in a mouse model demonstrates for the first time that milk complement proteins prevent infections by selectively eliminating bacteria from the infant gut.
    • In mice, and presumably humans, milk complement proteins work independently of antibodies to help establish a protective gut microbiome in infants.

     

    Complement proteins were so named because they act as immunological sidekicks to antibodies and other immune cells. Like Robin creating a diversion to help Batman take down the villain, complement proteins from the bloodstream help antibodies identify and attack bacteria. Unlike Robin, however, complement proteins can occasionally destroy bacteria or other pathogens on their own. As a part of the innate, or non-specific, arm of the immune system, complement proteins are one of the body’s first lines of defense in preventing infection—no superhero antibodies needed. 

    In addition to circulating in the blood, complement proteins are also passed from human and other mammalian mothers to their infants in milk. However, the influence of these acquired complement proteins on the infant’s immune system is not well understood. Do maternal complement proteins assist milk antibodies? Are they on the front lines attacking foreign microbes on their own? Or do they serve another immunological role in infants? A new study [1] demonstrates for the first time that milk complement proteins, without the help of antibodies, protect infants from gastro-intestinal infections by modifying the composition of the infant’s gut microbiome. 

    Milk complement protects infants

    To zero in on the immunological role of maternal complement proteins among the multitude of immune factors present in milk, a team of researchers from Johns Hopkins University disabled or “knocked out” the sections of DNA coding for complement proteins in a mouse model [1]. Without genetic instructions for making complement proteins in their liver cells, mouse mothers (aka dams) could not pass complement to their nursing pups in milk.

    The study authors hypothesized that infants receiving complement in their milk would fare better during an immunological challenge than those without milk complement [1]. To test this, their study design had two experimental mouse groups: one with a knocked out C1 protein and another with a knocked out C3 protein. There are over 30 different complement proteins, but these two proteins were selected because of their key roles in activating other complement proteins and immune cells in both innate and adaptive immune responses [1,2]. 

    To tease out the influence of maternal (milk) complement versus infant-produced complement, the researchers designed a cross-fostering system. C1 knockout dams (C1-), C3 knockout dams (C3-), and control (or wild type, WT) dams were bred to produce litters on the same day. Then, pups were moved so that C1- nursed either C1- or WT pups, C3- nursed either C3- or WT pups, and WT nursed WT, C1- or C3- pups. After 21 days of nursing, the pups were removed from their foster mother and challenged with Citrobacter rodentium (CR), a pathogen that causes infection in the guts of mice.

    As predicted, milk complement proteins offered a protective effect to nursing mice pups [1]. Pups fostered by C1- and C3- dams demonstrated signs of infection from CR, including diarrhea, reductions in body growth, and even death, whereas pups fostered by WT dams had limited or no signs of infection, regardless of their genetic make-up [1]. With no noted differences across knockout and WT dams in the concentration of milk nutrients, total milk energy, or important milk antibodies (e.g., secretory immunoglobulin A), susceptibility to CR was presumed to be mediated by the presence of milk C1 and C3 complement proteins [1]. 

    Milk complement influences the infant’s gut microbiome

    Now came the tricky part: determining how milk complement proteins prevent CR infection. Studies on milk complement from human cell cultures suggested these proteins selectively killed or inhibited the growth of certain types of commensal gut bacteria, including Escherichia coli and Helicobacter pylori—could milk complement behave the same way in a living mouse pup? 

    To investigate this, the research team first profiled the gut microbiomes of WT, C1-, and C3- pups. They applied a sequencing technique called 16s ribosomal RNA that identifies specific types of gut microbes and found that each pup type had a distinct microbiome composition. Of particular interest were the elevated levels of Staphylococcus bacteria in the guts of pups from C1- and C3- fosters compared with pups from WT fosters [1].

    Intrigued by this difference, the researchers gave C1- and C3- dams a broad-spectrum antibiotic that would kill gut Staphylococcus bacteria in both mothers and nursing pups. The antibiotic treatment changed the composition of the gut microbiome by reducing the abundance of Staphylococcus in C1- and C3- pups while also reducing symptoms associated with CR infection [1]. The exact mechanism by which Staphylococcus promotes CR infection is still unknown, but the study team did observe that the reduction of Staphylococcus bacteria was associated with a decrease in infant gut inflammation. This finding suggests that a non-inflammatory gut environment may prevent the growth of pathogenic microbes such as CR [1].

    Milk complement selectively kills bacteria

    The study team had demonstrated an association between milk complement proteins and CR susceptibility and between Staphylococcus abundance and CR susceptibility, but still needed to link them functionally. To find the smoking gun—evidence that milk complement proteins were directly involved in reducing Staphylococcus populations in the infant gut—they turned to in vitro experiments. 

    The whey fraction from milk from WT, C1-, and C3- dams was applied to cell cultures on agar plates containing a particular species of Staphylococcus, S. lentus. The plates with C1- and C3- milk had continued growth whereas the plate with WT milk stopped the growth of S. lentus. They then repeated this experiment using the whey fraction from human milk and found that it also stopped the growth of S. lentus. The time it took for both human and mouse milk complement proteins to stop the growth of S. lentus was indictive of bactericidal activity—complement was killing the bacteria by poking holes in their cell membrane [1]. And the complement continued to kill the S. lentus bacteria even after the researchers removed antibodies such as immunoglobulin G (IgG) and IgA from the whey fraction—milk complement is nobody’s sidekick. 

    Altering the Gut Microbe Environment

    This study demonstrated that milk complement proteins protect infants against infection, but also went to the trouble to demonstrate how they do this, down to the molecular level. And in doing so, the researchers identified a novel molecular role for maternal complement proteins of selectively killing gut bacteria, independent of antibodies. The benefits of passive immunity almost exclusively focus on the role maternally derived antibodies, but this study is a good reminder that innate immune factors in milk, like complement, can be equally important in protecting infants from infection. 

    References

    1. Xu D, Zhou S, Liu Y, Scott AL, Yang J, Wan F. Complement in breast milk modifies offspring gut microbiota to promote infant health. Cell. 2024 Feb;187(3): 750-63.
    2. Ricklin D, Reis ES, Mastellos DC, Gros P, Lambris JD. Complement component C3–The “Swiss Army Knife” of innate immunity and host defense. Immunological reviews. 2016 Nov;274(1): 33-58.