The Gut Microbiome Regulates FVIII Immune Responses

The development of FVIII inhibitory antibodies (inhibitors) is a significant complication not only in FVIII protein replacement therapy but also in gene therapy for hemophilia A. Accumulating evidence has demonstrated that the intestinal microbiota is involved in maintaining health and shaping the immune system. The interactions between the intestinal microbiota and the immune system and disruption of the intestinal environment may influence immune response outcomes. Here, we explored how intestinal microbiota influences FVIII immune responses using two models, germ-free and antibiotic-treated models.

In the germ-free model, when animals were immunized with recombinant human FVIII (rhFVIII, Kogenate), at a dose of 200 U/kg/week by intravenous (IV) injection for 4 weeks, only 4 of 9 mice developed detectable inhibitors, with a titer of 2.7±4.6 BU/ml. In contrast, in conventional mice (normal mice) under the same immunization protocol, all animals developed inhibitors (145.6±82.6 BU/ml, n=8), which was significantly higher than in the germ-free group (P<0.001). When germ-free mice were immunized with 600 U/kg in the presence of Incomplete Freund’s adjuvant via intraperitoneal (IP) administration twice with a 3-week interval, 7 of 8 germ-free mice developed inhibitors with a titer of 57.9±52.1 BU/ml, which was 29-fold lower than in normal mice (1697±1382 BU/ml, n=14, P<0.01). The titers of anti-FVIII total IgG in the germ-free mice were 267±160 in the IV group and 8675±9330 in the IP group, which were 17- and 99-fold lower (P<0.0001) than the titers developed in normal mice under the same immunization protocol. In germ-free mice, the percentage of germinal center marker GL7⁺ T follicular helper (GC⁺-Tfh) cells and T follicular regulatory (GL7⁺-Tfr) cells, the ratio of GL7⁺ Tfh/Tfr, as well as GL7⁺germinal center B (GC-B) cells in the spleen, were significantly higher in the IP group compared to the IV and naïve unimmunized groups, but there was no difference between the IV and naïve groups.

For the antibiotic-treated model, mice were immunized with rhFVIII at 200 U/kg/week for 4 weeks by IV injection and animals were supplemented with drinking water containing a cocktail of vancomycin, neomycin, ampicillin, and metronidazole (VNAM) for 3 weeks before and during immunization. Plasma samples were collected 5-7 days after the last immunization for Bethesda assay to determine the titers of FVIII inhibitors. Splenocytes were isolated for flow cytometry analysis to determine the changes in immune cells upon FVIII infusion. Among 6 animals treated with VNAM, none developed a titer of inhibitors greater than 20 BU/ml upon FVIII immunization. Three of the 6 did not develop detectable inhibitors; one developed a titer of 18 BU/ml, and the other two developed low titers of inhibitors of 1.2 BU/ml and 4.2 BU/ml, respectively. In contrast, all mice with drinking water containing glucose as a vehicle control developed FVIII inhibitors with titers greater than 20 BU/ml, ranging from 21-112.5 BU/ml (53.6±33.0 BU/ml, n=7). The incidence of animals that developed high titers (>20 BU/ml) and the inhibitor titers in the VNAM group were significantly lower than in the vehicle control group (P<0.001) under the same immunization protocol. The ratio of Tfh/Tfr and the percentage of GC-B and marginal zone B (MZ-B) cells in the spleen of the VNAM group was significantly lower than in the vehicle control group upon rhFVIII immunization. The total numbers of Tfh and Tfr cells per spleen in the VNAM group were (4.0±1.2) x10⁴ and (3.8±1.2) x10⁴, which were 7- and 4-fold lower than those in the vehicle control group [(28.8±4.5) x10⁴ and (15.6±3.9) x10⁴ after rhFVIII immunization.

In summary, our studies showed that germ-free mice produce significantly lower FVIII inhibitors and total anti-FVIII IgG antibodies than conventional mice under the same immunization protocol. Intravenous FVIII immunization doesn’t induce GC-B cell, effector, or Tfh CD4 T cell expansion in the spleen of germ-free mice. VNAM-treated mice developed lower FVIII inhibitor titers and had lower frequencies and numbers of activated Tfh and Tfr cells, as well as MZ- and GC-B cells upon FVIII immunization. In conclusion, the gut microbiota governs FVIII immune responses.