Vibrio cholerae-Induced Inflammation in the Neonatal Mouse Cholera Model

Abstract

Vibrio cholerae is the causative agent of the acute diarrheal disease of cholera. Innate immune responses to V. cholerae are not a major cause of cholera pathology, which is characterized by severe, watery diarrhea induced by the action of cholera toxin. Innate responses may, however, contribute to resolution of infection and must be required to initiate adaptive responses after natural infection and oral vaccination. Here we investigated whether a well-established infant mouse model of cholera can be used to observe an innate immune response. We also used a vaccination model in which immunized dams protect their pups from infection through breast milk antibodies to investigate innate immune responses after V. cholerae infection for pups suckled by an immune dam. At the peak of infection, we observed neutrophil recruitment accompanied by induction of KC, macrophage inflammatory protein 2 (MIP-2), NOS-2, interleukin-6 (IL-6), and IL-17a. Pups suckled by an immunized dam did not mount this response. Accessory toxins RtxA and HlyA played no discernible role in neutrophil recruitment in a wild-type background. The innate response to V. cholerae deleted for cholera toxin-encoding phage (CTX) and part of rtxA was significantly reduced, suggesting a role for CTX-carried genes or for RtxA in the absence of cholera toxin (CTX). Two extracellular V. cholerae DNases were not required for neutrophil recruitment, but DNase-deficient V. cholerae caused more clouds of DNA in the intestinal lumen, which appeared to be neutrophil extracellular traps (NETs), suggesting that V. cholerae DNases combat NETs. Thus, the infant mouse model has hitherto unrecognized utility for interrogating innate responses to V. cholerae infection. Vibrio cholerae is the causative agent of cholera, which remains endemic in many regions of Africa and Asia (1). Sporadic outbreaks can devastate immunologically naïve populations, such as occurred in Haiti in 2010 (2). The O1 serogroup and, to a lesser degree, O139 are the major causes of cholera, and El Tor is currently the circulating O1 biotype (1). Cholera toxin (CTX), encoded by a lysogenic phage (3), is the major cause of the severe secretory diarrhea that is typical of cholera. Without rehydration therapy, cholera results in 25 to 50% mortality, but if treated, cholera will resolve in most patients (4). Unlike diseases such as shigellosis and Salmonella-induced gastroenteritis (5, 6), the pathology of cholera is not immune driven. However, innate immune responses have been observed in cholera patients during the acute phase of infection (7) and likely play a direct and/or indirect role in the resolution of disease. The majority of individuals who survive a bout of cholera will become immune to subsequent infection for at least 3 years (8-10), and some aspects of the observed innate immune response must be required to initiate this protective immunity.Many live attenuated vaccine candidate strains of V. cholerae have been developed in which ctxA, the gene encoding the catalytic subunit of cholera tox...

Comparative Proteomic Analysis Reveals Activation of Mucosal Innate Immune Signaling Pathways during Cholera

“…6). Our proposed model is also supported by gene expression studies of biopsy specimens in acute-phase cholera (2,4,6), in vitro studies of immune sensing of Gram-negative bacteria (11,12), and in vitro stimulation of immune cells with V. cholerae antigens (5,13,17,18). In this model, LPS in the cell membrane of V. cholerae O1 binds to TLR4, which activates NF-B and leads to production of inflammatory cytokines (IL-6, IL-12␤, IL-18, and IL-1␤).…”

“…We observed differential abundance of these proteins in acutephase cholera biopsy specimens, and their known activity in neutrophils (41)(42)(43) suggests the possible importance of activated neutrophils in controlling the growth of V. cholerae O1 in the gut. In fact, during cholera, neutrophils are present in high numbers in the lamina propria, and in mouse models of cholera, both neutrophil recruitment and expression of innate defense proteins are reported during the acute phase (2,18,44). In addition, there is also an increase of neutrophils in the systemic circulation at the acute phase in cholera (2).…”

“…Neutrophil recruitment plays a role in controlling the spread of V. cholerae to extraintestinal organs during infection of mice (17,18). Furthermore, V. cholerae infection induces production of inflammatory cytokines and chemokines in the mouse gut, such as nitric oxide (NO), IL-6, and IL-17A; this response was reduced in V. cholerae strains lacking CT (18).…”

“…Neutrophil recruitment plays a role in controlling the spread of V. cholerae to extraintestinal organs during infection of mice (17,18). Furthermore, V. cholerae infection induces production of inflammatory cytokines and chemokines in the mouse gut, such as nitric oxide (NO), IL-6, and IL-17A; this response was reduced in V. cholerae strains lacking CT (18). Murine and cell culture models have been useful for determining the role of NLRP3, NF-B, and proinflammatory cytokines in mediating the inflammatory response to V. cholerae; however, given the disparity between human and mouse inflammasome regulation (19,20) and the fact that murine models do not fully replicate the clinical manifestation of cholera (21), these models may be insufficient for understanding the human innate immune response in cholera patients.…”

Phenotypic Analysis Reveals that the 2010 Haiti Cholera Epidemic Is Linked to a Hypervirulent Strain

“…This finding suggests that the successful AET could have emerged as the predominant global clone due to positive selection of increased virulence, ultimately outcompeting other strains through repeated cycles of human transmission. Such a selection could be enhanced by an ability to significantly increase and to more successfully defend against V. cholerae-stimulated inflammation in the mouse intestine (26,(46)(47)(48).…”