Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Shigella

• Macrophages

• Pathogen-host interactions

Main Findings

Shigellosis is a highly contagious and severe inflammatory gastrointestinal infection caused by Shigella species. While a small number of bacteria can cause disease in humans, mice are naturally resistant. However, mice deficient in NAIP-NLRC4 and Caspase-11 (Nlrc4-/-Casp11-/-) have recently emerged as a genetically tractable model for human shigellosis. Although Shigella infection recruits myeloid cells, the roles of macrophages and neutrophils remain understudied.

Eislmayr et al (not peer-reviewed) have investigated the role of neutrophils and macrophages in limiting Shigellainfection and in disease pathogenesis in Nlrc4-/-Casp11-/- mice. They demonstrated that IFNg neutralization leads to severe shigellosis, characterized by increased bacteria in intestinal epithelial cells (IECs), increased bacterial translocation to mesenteric lymph nodes and spleen, and elevated pro-inflammatory cytokines in lamina propria. Similarly, compared to Nlrc4-/-Casp11-/- mice, Nlrc4-/-Casp11-/- mice lacking Ifngr (Ifngr-/-Nlrc4-/-Casp11-/-)exhibited more severe disease, underscoring IFNg’s role in restricting Shigellareplication and host protection.

The authors utilized reciprocal bone marrow (BM) chimeras between Nlrc4-/-Casp11-/- mice and Ifngr-/-Nlrc4-/-Casp11-/- mice to determine whether hematopoietic or non-hematopoietic cells are important to restrict Shigella. Regardless of donor cell origin, bone marrow recipients lacking Ifngr lost more weight and had increased bacterial burden in IECs after Shigella infection, indicating that non-hematopoietic cells are essential to respond to IFNg to control shigellosis.

To elucidate the contribution of myeloid cells in control of Shigella infection, the authors generated Lyz2Cre/+iDTRlsl/lslNlrc4-/-Casp11-/- mice where Cre-inducible diphtheria toxin (DT) receptor gene is introduced to Nlrc4-/-Casp11-/-mice. Administration of DT resulted in depletion of macrophages and neutrophils and upon Shigella infection, these animals presented with more severe disease. Interestingly, selective neutrophil depletion by aLy6G antibodies or Mrp8Cre/+iDTRlsl/lslBM chimeras did not alter the disease course. In contrast, BM reconstitution and DT treatment of CD64Cre/+iDTRlsl/lsl mice phenocopied Lyz2Cre/+iDTRlsl/lsl reconstitution, indicating macrophages but not neutrophils are important in controlling the pathogenesis of Shigellainfection.

Finally, IL-12 neutralization increased bacterial burden, weight loss, and significantly reduced IFNg in the lamina propria. However, anti-IL-12 treatment in Ifngr-/-Nlrc4-/-Casp11-/-mice did not affect bacterial control, confirming that IL-12 acts upstream of IFNg.

This study redefines the innate immune response to Shigella, highlighting macrophages, rather than neutrophils, as the key players in bacterial control and positioning IL-12 as a crucial regulator of IFNg responses.

Limitations

● Lack of direct evidence for IFNg action on IECs. Utilization of Villin Cre mice, crossed to Ifngr1 fl/fl would have made this study much stronger.

● Administration of exogenous IFNg would be a good complementary experiment to IFNg neutralization studies.

● The authors suggest that bystander macrophages may sense Shigella through TLR ligands released extracellularly. The preprint offers significant evidence on the role of macrophages on Shigella control and the role of IL-12 on inducing IFNg. However, there is no experimental data to support that bystander cells produce IL-12.

● Considering that Shigella is an intracellular pathogen, T cells might be important to control Shigella. It would be interesting to also investigate the effect of IL-12 on T cells as well as the role of T cells as producers of IFNg.

● Almost all parameters are measured at 48 hours post-infection. Even though the data presented in the preprint is convincing for the early days of infection, the role of neutrophils and macrophages, or IL-12-IFNg axis has not been elucidated beyond 48 hours.

Significance/Novelty

This study investigates the role of different innate immune cells in Shigella control and challenges a long-standing assumption that neutrophils are the primary immune cells for Shigella clearance. With utilization of various novel mouse models, bone marrow transplantation studies and antibody-mediated neutralization of key cytokines, the authors suggest that macrophages, via IL-12-IFNg axis play an important role in controlling Shigellainfection. This study may put macrophage activation to the focus of Shigellatreatment and can enable novel therapies in the future.

Credit

Reviewed by Didem Ağaç Çobanoğlu as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute, the University of Toronto and MD Anderson Cancer Center.

The author declares no conflict of interests in relation to their involvement in the review.