Midon, L. M. | bioRxiv Science

bioRxiv2025

Complement C3 Deficiency Enhances Renal Leptospiral Load and Inflammation While Impairing T Cell Differentiation During Chronic Leptospira interrogans Infection

Leptospirosis is a neglected zoonotic disease caused by pathogenic Leptospira spp., affecting an estimated of one million people annually and resulting in approximately 60,000 fatalities. The disease can lead to hepatic, renal, and pulmonary dysfunctions and may contribute to the development of chronic kidney disease. The Complement System plays a crucial role in eliminating bacteria by generating opsonins, anaphylotoxins, that degranulate mastocytes and basophils, and attract immune cells to the infection site, among other important functions. This study aimed to investigate the role of C3, the central protein of the Complement System, in vivo during chronic infection of L interrogans serovar Copenhageni strain FIOCRUZ L1-130 (LIC). C57BL/6 wild-type (WT) and C3 knockout (C3KO) mice were infected with 108 LIC and monitored at 15, 30, 60, 90 and 180 days post-infection (d.p.i.). LIC-infected C3KO mice exhibited a significantly higher leptospiral load in the kidneys compared to WT counterparts. Both groups showed local inflammation at 15 and 30 d.p.i., but only C3KO LIC-infected mice had a higher number of Leptospira DNA copies at 30 d.p.i. At the same time point, C3KO LIC-infected mice developed a larger fibrotic area than WT mice. Interestingly, independent of C3, mice pre-treated with a nephrotoxic drug increased the renal inflammatory response; however, this pretreatment did not affect the local leptospiral load in infected mice. Additionally, levels of specific IgG2b and IgG3 antibodies were significantly higher in LIC-infected C3KO mice compared to WT mice. Proteomic analysis showed lower levels of C5/C5a in WT and C3KO LIC-infected mice, as well as in C3KO control mice. M-CSF and SDF-1 cytokine levels were reduced in both LIC-infected groups. Furthermore, naive T lymphocytes count (both CD4+ and CD8+) were higher in LIC-infected C3KO mice, whereas effector CD8+ T lymphocyte numbers declined during LIC infection - a phenomenon more pronounced in C3KO mice. Overall, this study demonstrates that during LIC infection, the absence of C3 does not affect mouse survival but leads to increased renal leptospiral load and fibrosis. Additionally, it highlights the crucial role of C3 in supporting the maturation and differentiation of T lymphocytes into pre-effector cells, underscoring its importance as a key link between the innate and adaptive immune responses in leptospirosis. Author SummaryLeptospirosis is an infectious disease with approximately one million new cases annually and is responsible for about 5% of deaths, particularly in underdeveloped countries. Our objective is to understand the immune response in leptospirosis, specifically the role of the Complement C3 protein. In this study, we observed that C3 deficiency is associated with a higher renal leptospiral load and an increased incidence of renal fibrosis after one month of chronic infection. Notably, C3 also influences the differentiation of helper and cytotoxic T lymphocytes into effector cells, potentially contributing to the increased severity of chronic leptospirosis observed in C3-deficient animals.

immunology↗

bioRxiv2025

C3H-HeN as a TLR4 competent mouse model of sublethal leptospirosis

AbstractMice are slowly being accepted as alternative models for investigation of leptospiral infection. The strain most used to analyse sublethal disease (C3H-HeJ) expresses a tlr4 gene in its immune cells that is hyporesponsive to leptospiral LPS and thus the model is deemed immunocompromised. To help resolve this valid scientific concern we did a study in which we compared infection with Leptospira interrogans serovar Copenhageni Fiocruz in mice expressing fully competent tlr4 (C3H-HeN, C57BL6) versus tlr4 hyporesponsive mice (C3H- HeJ) over a period of two weeks. We found that the C3H-HeJ mouse was the only strain that sustained lethal infection, thus confirming the association between tlr4 function and acute disease. We found that the two mouse strains with a fully functional tlr4 gene (C3H-HeN and C57BL/6) also developed clinical and molecular signs of sublethal leptospirosis nearly on par with C3H-HeJ, as quantified by weight loss, survival curves, presence of Leptospira 16S rRNA in blood and urine, and burden of viable spirochetes in kidney. Regarding kidney markers of inflammation and fibrosis, C3H-HeN and C57BL/6 produced less IL-1 beta, iNOS and ColA1 than C3H-HeJ, which is consistent with increased resilience to infection expected of the tlr4 competent strains. Regarding Leptospira-specific antibody, all mouse strains produced IgM and T-cell independent IgG3. Interestingly, in contrast to C57BL/6, both C3H strains produced circulating IgG1 but only C3H-HeN produced IgG2a to L. interrogans two weeks post infection. Thus, TLR4-competent C3H-HeN and C57BL/6 may be more appropriate mouse models of sublethal leptospirosis, whereas C3H-HeJ can be used to further establish a mouse model of lethal leptospirosis. Our data strongly suggests that TLR4 function is important but not sufficient to cause susceptibility to leptospirosis. Author summaryPrevious studies by different laboratories have shown C3H-HeJ to be a susceptible mouse strain to sublethal and lethal leptospirosis. The goal of our study was to evaluate if low tlr4 responsiveness to leptospiral LPS in C3H-HeJ mice is the factor that drives susceptibility to infection with pathogenic Leptospira. We did a comparative study using mouse strains immunocompetent and hyporesponsive to tlr4. The data shows that although tlr4 does affect the virulence of leptospirosis (only C3H-HeJ succumbed to infection), the tlr4 competent strains (C3H-HeN and C57BL/6) also developed measurable sublethal leptospirosis. Thus, competent recognition of leptospiral-LPS by murine TLR4 plays a role but it is not sufficient to determine susceptibility to leptospirosis. Furthermore, the data suggests that T-cell dependent responses could be involved in susceptibility to leptospirosis in the C3H models of infection.

microbiology↗