Host responses in Fas-deficient autoimmune-prone lpr mice upon Salmonella Typhimurium infection

Abstract

Autoimmune diseases can potentially pre-dispose an individual to infections. On the other hand, infections can trigger, exacerbate or ameliorate autoimmunity. Autoimmune Lymphoproliferative Syndrome (ALPS) is a rare genetic disorder, often leading to a systemic autoimmune disease, and arises due to mutations in genes encoding the Fas-death pathway. lpr (lymphoproliferation) mice, widely used as autoimmune disease models for systemic autoimmune diseases like SLE, bear clinical and genotypic resemblance to ALPS. lpr mice are mutants for Fas and develop an age-dependent progressive systemic autoimmune disease that is associated with spontaneous lymphoproliferation. ALPS patients have been reported to exhibit a pre-disposition to post splenectomy related sepsis. The dynamics of other infections in ALPS remain widely unexplored. The lpr mouse model, on the other hand, has been used to study a wide range of infection outcomes, with an increased susceptibility observed for certain infections, but an increased clearance from others. At the same time, certain infections accelerate the autoimmune disease progression in lpr mice while a few reduce it. However, the role of Salmonella infection in this context remains largely unknown.

Salmonella spp. are Gram-negative, intracellular pathogens and one of the most common causative agents of foodborne diseases globally. Salmonella Typhi, a human restricted pathogen, causes Typhoid (enteric fever). Salmonella Typhimurium, a zoonotic pathogen causes gastroenteritis in humans and a systemic typhoid-like disease in susceptible mice strains (mouse typhoid model). S. Typhimurium mutants of certain genes, such as rpoS, are compromised in virulence, exhibiting increased survival in mice. RpoS is an alternate sigma factor of bacterial RNA polymerase that plays an important role during stationary phase and regulation of various stress responses as well as virulence. The objective of the first part of the study was to confirm the rpoS gene deletion in Salmonella Typhimurium ΔrpoS and functionally characterise and validate the strain. S. Typhimurium ΔrpoS exhibited reduced growth during stationary phase, increased susceptibility to oxidative stress and inefficient entry and intra-cellular replication in macrophages in comparison to the WT strain. All these results are consistent with known traits of the ΔrpoS strain.

Once the ΔrpoS strain was characterised, the objective of the second part of the study was to compare and understand the outcome of infection with a virulent (WT) and an attenuated (ΔrpoS) S. Typhimurium strain in autoimmune-prone lpr mice, along with the congenic C57BL/6 (B6) mice, using the mouse typhoid model. S. Typhimurium WT caused an acute and lethal infection in B6 and lpr mice, via the intraperitoneal (i.p.) as well as the per-oral route. In contrast, S. Typhimurium ΔrpoS led to a prolonged survival in B6 mice post i.p. as well as per-oral infection. In lpr mice, S. Typhimurium ΔrpoS caused acute lethality post i.p. infection whereas complete lethality after prolonged survival post per-oral infection. The prolonged survival of both B6 and lpr mice post per-oral infection with S. Typhimurium ΔrpoS was exploited to compare the acute and late stages of infection to understand the disease progression. Consequently, all further analyses were performed post per-oral infection. The S. Typhimurium ΔrpoS strain was found to be competent in establishing infection but compromised in sustaining it, as observed by the similar organ bacterial loads in WT and ΔrpoS infected mice during acute stage of infection, and a progressive reduction in bacterial load of ΔrpoS infected mice during the later stages of infection. During the late stage of infection, organ bacterial load and serum IFNγ levels were higher in the ΔrpoS strain infected lpr mice compared to B6 mice. The ΔrpoS strain infected lpr mice also exhibited greater bacterial faecal shedding and greater tissue histopathological changes denoting infection induced damage in colon and liver. Higher brain bacterial load was also detected in WT-infected lpr mice than WT-infected B6 mice during acute infection. Interestingly, ΔrpoS-infected B6 mice showed minimal microbial load in the brain; however, ΔrpoS-infected lpr mice displayed sustained brain bacterial load. Overall, lpr mice were observed to be less efficient in controlling the attenuated infection with ΔrpoS. The findings from this study demonstrate that a genetic pre-disposition to autoimmunity maybe sufficient for a greater host susceptibility to S. Typhimurium infection.

S. Typhimurium WT infection has been shown to induce thymic atrophy in mice resulting in a loss in cellularity of the thymus. The thymus is a primary lymphoid organ crucial for the differentiation, selection and maturation of T cells. The thymus undergoes atrophy under various stressful conditions. S. Typhimurium infection induced thymic atrophy in mice is a well-established model in our laboratory using which susceptibility of the different thymocyte sub-populations has been studied. However, the effects on thymic atrophy and sub-population of thymocytes post infection with an attenuated strain of S. Typhimurium has not been studied. Thus, the third part of the current study was designed to examine changes in lymphocyte populations in the mesenteric lymph node (MLN) and thymocyte sub-populations using the long-term infection model established with S. Typhimurium ΔrpoS in B6 and lpr mice. These results were compared to the acute infection model with S. Typhimurium WT. Extensive loss of thymocytes or thymic atrophy was observed upon infection, with greater loss in WT infected lpr mice than WT infected B6 mice. WT infected mice exhibited greater thymic atrophy than ΔrpoS infected mice during the acute infection stage. During late infection, ΔrpoS infected lpr mice exhibited greater thymic atrophy than B6 mice. Infection also led to a moderate drop in MLN cell numbers, with a greater drop in ΔrpoS infected lpr mice than B6 mice during late infection. Previous studies from the lab have shown the roles of IFNγ and cortisol during acute thymic atrophy. Consistent with these studies, higher levels of serum cortisol and IFNγ correlated with the greater thymic atrophy in WT infected mice during acute infection and ΔrpoS infected lpr mice during late infection in the current study. The MLN lymphocyte populations and the different thymocyte sub-populations in B6 and lpr mice post infection with S. Typhimurium WT and ΔrpoS were analysed using high end multi-colour flow cytometry, the details of which are discussed subsequently.

Overall, the study highlights the heightened susceptibility of the autoimmune-prone lpr mice to Salmonella Typhimurium infection, reflected in a greater mortality with the attenuated strain and a more severe disease outcome. The greater mortality appears to be the consequence of multiple inter-connected factors including reduced bacterial clearance from infection sites, heightened tissue damage and a higher inflammatory environment during the late infection stages. In addition, the study demonstrates lpr mice to be more susceptible to brain colonisation with the otherwise non-neurotropic S. Typhimurium. Furthermore, the study highlights the greater sensitivity of lpr mice to S. Typhimurium infection induced thymic atrophy, reflected not only in the greater depletion of immature thymocytes but also of some otherwise resistant mature thymocyte sub-populations. This is despite the normal thymic development in uninfected lpr mice. In conclusion, either the general genetic pre-disposition to autoimmunity and/or the absence of a functional Fas-death pathway renders the lpr mice more sensitive to S. Typhimurium infections. Further mechanisms need to be explored to better understand the various manifestations in the autoimmune-prone lpr mice upon S. Typhimurium virulent as well as attenuated infections.