Unique B Cell and Germinal Center Responses in Mice with Severe versus Mild Orientia tsutsugamushi Infection

Abstract

The intracellular bacterium Orientia tsutsugamushi (Ot) is the causative agent of scrub typhus, an emerging and neglected tropical disease. Immunity against Ot infection in patients is known to be short-lived, as circulating antibodies can wane around one-year post-infection. However, its underlying mechanisms remain unclear; host immune signatures to clinically prevalent Ot strains are also undefined. In this study, we infected C57BL/6 mice with two clinically prevalent Ot strains with distinct virulence to examine splenic B cell and germinal center (GC) responses. While highly virulent Karp strain-infected mice had 50% mortality rates, intermediate virulent strain Gilliam-infected mice survived, gained body weight, and maintained relatively low bacterial burden levels during infection. Yet, Gilliam infection resulted in strong splenic B cell responses, as judged by absolute cell numbers of total splenic B cells, follicular B cells, and marginal zone (MZ) B cells alongside with significantly higher titers of total IgG and IgM antibodies at the late stage of infection. While altered splenic architecture with increased distance between white pulp regions was detectable in both strains, GC disorganization/collapse and MZ abrogation were only detected in Karp-infected spleens. To define the driving force of GC loss, we compared RNAseq profiles between spleens infected with the Karp or Gilliam strain, and found significantly upregulated inflammation biomarkers (Ccl2, Il33, Ifng) and severe scrub typhus-related inflammatory pathways (Il1, Il6, Tnf, and Ifng) in Karp infection at day 4 (D4, early stage of infection). IPA analysis further showed upregulated neutrophil degranulation and defense response pathways in Karp-infected spleens, while Gilliam-infected spleens showed upregulation of phagocytosis signaling pathways. Flow cytometry confirmed a marked influx of activated myeloid cell subsets (neutrophils, M1 macrophages, and inflammatory monocytes) in the spleen during Karp infection as compared to Gilliam infection, indicating excessive inflammatory infiltration in highly virulent Ot strain infection. Collectively, our findings establish several Ot strain-related immune signatures which help fill the knowledge gap of deficiencies in the host adaptive immune response during acute scrub typhus. Author SummaryThe intracellular bacterium O. tsutsugamushi (Ot) is the etiologic agent of the neglected tropical disease scrub typhus. It is well-known that immunity against Ot infection is short-lived, yet no in-depth studies have examined or compared adaptive immunity or deficiencies at cellular and molecular levels during acute Ot infection with clinically relevant Ot strains. In this study, we used two clinically prevalent Ot strains that cause differential diseases outcomes in the C57BL/6 murine model to examine B cell and germinal center (GC) responses. In sharp contrast to Karp infection, which caused 50% mortality rates by D12, Gilliam infection elicited self-limiting infection with low tissue splenic bacterial burdens. Yet, Gilliam-infected mice demonstrated robust humoral immune responses, as demonstrated by strong IgM and IgG antibody titers at D12 and absolute numbers of splenic B cell subsets at D8 and D12. While both Ot strains altered typical splenic white pulp architecture, only Karp infection abrogated GC and MZ structures. Comparison of RNAseq profiles of Karp-vs. Gilliam-infected spleens revealed several unique features, including Karp infection-associated upregulation of inflammation genes (Ccl2, Il33, Ifng) and inflammatory pathways (Il1, Il6, Tnf, Ifng) at D4, as well as upregulated leukocyte recruitment, neutrophil degranulation, and defense response pathways. In sharp contrast, Gilliam infection upregulated pathways involved in phagocytosis immunoregulatory interactions. Flow cytometry confirmed significant influx and activation of myeloid cell subsets (neutrophils, macrophages, M1 macrophages, monocytes) during Karp infection. Our study reveals unique patterns in the differential immune responses to two clinically prevalent Ot strains and helps understand potential mechanisms of immune alterations during severe scrub typhus.