Macrophages control intracellular pathogens like Salmonella by using two caspase enzymes at different times during infection.

Host-pathogen interactions involve two critical strategies: resistance, whereby hosts clear invading microbes, and tolerance, whereby hosts carry high pathogen burden asymptomatically. Here, we investigate mechanisms by which Salmonella-superspreader (SSP) hosts maintain an asymptomatic state during chronic infection. We found that regulatory T cells (Tregs) are essential for this disease-tolerant state, limiting intestinal immunopathology and enabling SSP hosts to thrive, while facilitating Salmonella transmission. Treg depletion in SSP mice resulted in decreased survival, heightened gut inflammation, and impairment of the intestinal barrier, without affecting Salmonella persistence. Colonic Tregs from SSP mice exhibited a unique transcriptomic profile characterized by the upregulation of type 1 inflammatory genes, including the transcription factor T-bet. In the absence of Tregs, we observed robust expansion of cytotoxic CD4+ T cells, with CD4+ T cell depletion restoring homeostasis. These results uncover a critical host strategy to establish disease tolerance during chronic enteric infection, providing novel insights into mucosal responses to persistent pathogens and chronic intestinal inflammation.

Salmonella enterica serovar Typhimurium must adapt to rapid environmental shifts, including those encountered upon entry and during replication to survive within macrophages during pathogenesis. Despite extensive RNA-seq-based investigations, questions remain regarding the range, timing and magnitude of response dynamics. Here we constructed a comprehensive GFP-reporter strain library representing 2,901 computationally identified Salmonella promoter regions to study time-resolved Salmonella transcriptional responses. Promoter activity was measured during in vitro growth and during intracellular infection of RAW 264.7 macrophages. Using bulk measurements and single-cell imaging, we uncovered condition-specific transcriptional regulation and population-level heterogeneity in SPI2-related promoter activity. We also discovered previously unidentified transcriptional activity from 234 promoters. These analyses revealed metabolic shifts including requirements for mntS expression to support manganese homeostasis and expression of Entner-Doudoroff pathway-associated genes to support growth within macrophages. Our library and datasets, made available through the online tool SalComKinetics, provide resources for systems-level interrogation of Salmonella transcriptional dynamics.

Salmonella enterica can persist asymptomatically within tissues for extended periods. This is achieved through intricate host-pathogen interactions in immune cell aggregates called granulomas, wherein Salmonella establish favourable cellular niches to exploit while the host limits its expansion and tissue dissemination. Here, using a mouse model of persistent Salmonella infection, we identify a host-protective role for eosinophils in the control of Salmonella Typhimurium (STm) infection within the mesenteric lymph nodes, the main lymphoid tissue of STm persistence. Combining spatial transcriptomics and experimental manipulations, we found that monocytes and macrophages responding to STm infection recruited eosinophils in a C-C motif chemokine ligand 11 (CCL11)-dependent manner and enhanced their activation. The protein major basic protein, primarily expressed by eosinophils, was associated with altered macrophage polarization and bacterial control. Thus, eosinophils play a vital role in restraining Salmonella exploitation of granuloma macrophages at a key site of bacterial persistence.

Researchers have advocated elevating mouse housing temperatures from the conventional ~22 °C to the mouse thermoneutral point of 30 °C to enhance translational research. However, the impact of environmental temperature on mouse gastrointestinal physiology remains largely unexplored. Here we show that mice raised at 22 °C exhibit whole gut transit speed nearly twice as fast as those raised at 30 °C, primarily driven by a threefold increase in colon transit speed. Furthermore, gut microbiota composition differs between the two temperatures but does not dictate temperature-dependent differences in gut motility. Notably, increased stress signals from the hypothalamic-pituitary-adrenal axis at 22 °C have a pivotal role in mediating temperature-dependent differences in gut motility. Pharmacological and genetic depletion of the stress hormone corticotropin-releasing hormone slows gut motility in stressed 22 °C mice but has no comparable effect in relatively unstressed 30 °C mice. In conclusion, our findings highlight that colder mouse facility temperatures significantly increase gut motility through hormonal stress pathways.

Macrophages provide a crucial environment for Salmonella enterica serovar Typhi (S. Typhi) to multiply during typhoid fever, yet our understanding of how human macrophages and S. Typhi interact remains limited. In this study, we delve into the dynamics of S. Typhi replication within human macrophages and the resulting heterogeneous transcriptomic responses of macrophages during infection. Our study reveals key factors that influence macrophage diversity, uncovering distinct immune and metabolic pathways associated with different stages of S. Typhi intracellular replication in macrophages. Of note, we found that macrophages harboring replicating S. Typhi are skewed towards an M1 pro-inflammatory state, whereas macrophages containing non-replicating S. Typhi exhibit neither a distinct M1 pro-inflammatory nor M2 anti-inflammatory state. Additionally, macrophages with replicating S. Typhi were characterized by the increased expression of genes associated with STAT3 phosphorylation and the activation of the STAT3 transcription factor. Our results shed light on transcriptomic pathways involved in the susceptibility of human macrophages to intracellular S. Typhi replication, thereby providing crucial insight into host phenotypes that restrict and support S. Typhi infection.

Salmonella enterica is comprised of genetically distinct 'serovars' that together provide an intriguing model for exploring the genetic basis of pathogen evolution. Although the genomes of numerous Salmonella isolates with broad variations in host range and human disease manifestations have been sequenced, the functional links between genetic and phenotypic differences among these serovars remain poorly understood. Here, we conduct high-throughput functional genomics on both generalist (Typhimurium) and human-restricted (Typhi and Paratyphi A) Salmonella at unprecedented scale in the study of this enteric pathogen. Using a comprehensive systems biology approach, we identify gene networks with serovar-specific fitness effects across 25 host-associated stresses encountered at key stages of human infection. By experimentally perturbing these networks, we characterize previously undescribed pseudogenes in human-adapted Salmonella. Overall, this work highlights specific vulnerabilities encoded within human-restricted Salmonella that are linked to the degradation of their genomes, shedding light into the evolution of this enteric pathogen.

Salmonella enterica serovar Typhi ( S. Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the S. Typhi Type 3 secretion systems (T3SSs) encoded on Salmonella Pathogenicity Islands (SPI) -1 (T3SS-1) and -2 (T3SS-2) during human macrophage infection. We found that mutants of S . Typhi deficient for both T3SSs were defective for intramacrophage replication as measured by flow cytometry, viable bacterial counts, and live time-lapse microscopy. T3SS-secreted proteins PipB2 and SifA contributed to S. Typhi replication and were translocated into the cytosol of human macrophages through both T3SS-1 and -2, demonstrating functional redundancy for these secretion systems. Importantly, an S . Typhi mutant strain that is deficient for both T3SS-1 and -2 was severely attenuated in the ability to colonize systemic tissues in a humanized mouse model of typhoid fever. Overall, this study establishes a critical role for S. Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. Importance:Salmonella enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate S. Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit spread of this pathogen. While S. Typhimurium replication in murine models has been studied extensively, there is limited information available about S. Typhi replication in human macrophages, some of which directly conflicts with findings from S. Typhimurium murine models. This study establishes that both of S. Typhi's two Type 3 Secretion Systems (T3SS-1 and -2) contribute to intramacrophage replication and virulence.

Salmonella Typhimurium is an enteric pathogen that causes gastroenteritis in humans and Typhoid fever-like symptoms in mice. Our lab has shown that oral infection of 129X1/SvJ mice with Salmonella Typhimurium results in ~25% of mice becoming persistent high-shedders (>10 8CFU/g feces). Because these mice can transmit infection to a naïve host, we refer to them as super-spreaders (SS). Unexpectedly, SS mice are asymptomatic despite a persistent high-pathogen burden in the gut. We hypothesized that SS mice are disease resistant due to a gut-specific immune state that facilitates homeostasis between host and pathogen. To examine this, we performed extensive immuno-phenotyping of SS mice, which revealed a distinct type-1 immune state in the gut. We found that IFNγ producing effector CD4 T cells were the dominant population to accumulate in SS guts. Additionally, a population of Tbet+ Tregs were found only in the gut of SS mice, suggesting these cells may enable the SS asymptomatic state. Accordingly, specific ablation of Tregs resulted in a dramatic weight loss, gut inflammation, and a compromised epithelial barrier in SS mice. Bacterial burden was unchanged in Treg depleted mice, demonstrating that morbidity was not caused by Salmonella burden but likely due to immune pathology. Indeed, depletion of effector CD4+ T cells in Treg-deficient SS mice reversed this morbidity, confirming the disease state was directly caused by immune pathology. These results demonstrate that in SS mice Tregs play an essential tolerogenic role maintaining intestinal homeostasis. We are currently investigating the immunological mechanisms triggering this effect and whether this new immune-adapted state may impact infections with other enteric pathogens.

Macrophages function as tissue-immune sentinels and mediate key antimicrobial responses against bacterial pathogens. Yet, they can also act as a cellular niche for intracellular bacteria, such as Salmonella entenca, to persist in infected tissues. Macrophages exhibit heterogeneous activation or polarization, states that are linked to differential antibacterial responses and bacteria permissiveness. Remarkably, recent studies demonstrate that Salmonella and other intracellular bacteria inject virulence effectors into the cellular cytoplasm to skew the macrophage polarization state and reprogram these immune cells into a permissive niche. Here, we review mechanisms of macrophage reprogramming by Salmonella and highlight manipulation of macrophage polarization as a shared bacterial pathogenesis strategy. In addition, we discuss how the interplay of bacterial effector mechanisms, microenvironmental signals, and ontogeny may shape macrophage cell states and functions. Finally, we propose ideas of how further research will advance our understanding of macrophage functional diversity and immunobiology.

Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica . Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures composed of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent S. enterica serovar Typhimurium ( S Tm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using an S Tm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin-converting enzyme (ACE) defines a granuloma macrophage population that is nonpermissive for intracellular bacteria, and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE + macrophages in infected tissues. Thus, ACE + macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.

The authors have identified an error in the figure legend for fig. 3A in this manuscript. Currently the figure legend states (A) Western blot analysis demonstrating Drp1 levels in mitochondrial-enriched fractions in 3 conditions (control, LPS treated, LPS ± P110 treated) after 24 h. (n = 3 biological replicates). VDAC was used as a loading control and protein levels were quantified and represented as a ratio. The timeline for the western blot is 4 h (not 24 h) while the remainder of the experiment in the in this fig. (3B–D) were performed at 24 h following LPS. We would like to correct this to state the following (A) Western blot analysis demonstrating Drp1 levels in mitochondrial-enriched fractions in 3 conditions (control, LPS treated, LPS ± P110 treated) after 4 h. (n = 3 biological replicates, representative blot for control and LPS also demonstrated in fig. 2b). VDAC was used as a loading control and protein levels were quantified and represented as a ratio. The authors would like to apologise for any inconvenience caused. Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathyJournal of Molecular and Cellular CardiologyVol. 130PreviewMitochondrial dysfunction is a key contributor to septic cardiomyopathy. Although recent literature implicates dynamin related protein 1 (Drp1) and its mitochondrial adaptor fission 1 (Fis1) in the development of pathologic fission and mitochondrial failure in neurodegenerative disease, little is known about the role of Drp1/Fis1 interaction in the context of sepsis-induced cardiomyopathy. Our study tests the hypothesis that Drp1/Fis1 interaction is a major driver of sepsis-mediated pathologic fission, leading to mitochondrial dysfunction in the heart. Full-Text PDF

Salmonella enterica is one of the most widespread bacterial pathogens found worldwide, resulting in approximately 100 million infections and over 200 000 deaths per year. Salmonella isolates, termed 'serovars', can largely be classified as either nontyphoidal or typhoidal Salmonella, which differ in regard to disease manifestation and host tropism. Nontyphoidal Salmonella causes gastroenteritis in many hosts, while typhoidal Salmonella is human-restricted and causes typhoid fever, a systemic disease with a mortality rate of up to 30% without treatment. There has been considerable interest in understanding how different Salmonella serovars cause different diseases, but the molecular details that underlie these infections have not yet been fully characterized, especially in the case of typhoidal Salmonella. In this review, we highlight the current state of research into understanding the pathogenesis of both nontyphoidal and typhoidal Salmonella, with a specific interest in serovar-specific traits that allow human-adapted strains of Salmonella to cause enteric fever. Overall, a more detailed molecular understanding of how different Salmonella isolates infect humans will provide critical insights into how we can eradicate these dangerous enteric pathogens.

The molecular understanding of host-pathogen interactions in the gastrointestinal (GI) tract of superspreader hosts is incomplete. In a mouse model of chronic, asymptomatic Salmonella enterica serovar Typhimurium (S. Tm) infection, we performed untargeted metabolomics on the feces of mice and found that superspreader hosts possess distinct metabolic signatures compared with non-superspreaders, including differential levels of L-arabinose. RNA-seq on S. Tm from superspreader fecal samples showed increased expression of the L-arabinose catabolism pathway in vivo. By combining bacterial genetics and diet manipulation, we demonstrate that diet-derived L-arabinose provides S. Tm a competitive advantage in the GI tract, and expansion of S. Tm in the GI tract requires an alpha-N-arabinofuranosidase that liberates L-arabinose from dietary polysaccharides. Ultimately, our work shows that pathogen-liberated L-arabinose from the diet provides a competitive advantage to S. Tm in vivo. These findings propose L-arabinose as a critical driver of S. Tm expansion in the GI tracts of superspreader hosts.

The molecular understanding of host-pathogen interactions in the guts of superspreader hosts is incomplete. In a mouse model of chronic, asymptomatic Salmonella enterica serovar Typhimurium (S. Tm) infection, we performed untargeted metabolomics on the feces of mice and found superspreader hosts possess distinct metabolic signatures compared to non-superspreaders, including differential levels of L-arabinose. RNA-seq on S. Tm in superspreader fecal samples showed increased expression of the L-arabinose catabolism pathway in vivo. By combining bacterial genetics and diet manipulation, we demonstrate that diet-derived L-arabinose provides S. Tm a competitive advantage in the gut. We show that expansion of S. Tm in the gut requires a previously uncharacterized alpha-N-arabinofuranosidase that can liberate L-arabinose from dietary polysaccharides. Ultimately, we demonstrate that pathogen-liberated L-arabinose from the diet provides a competitive advantage to S. Tm in vivo and propose L-arabinose as a critical driver of S. Tm expansion. Funding Information: Research reported in this publication was supported by grants R01-AI116059 and R01AI095396 from the National Institute of Allergy and Infectious Diseases, United States (D.M.M.), Paul Allen Stanford Discovery Center on Systems Modeling of Infection (to D.M.M.), Gates Grand Challenge Grant from Bill & Melinda Gates Foundation (to D.M.M.), and the Graduate Research Fellowship Program funded by the National Science Foundation (S.R.). Declaration of Interests: The authors declare no competing interests Ethics Approval Statement: Experiments involving animals were performed in accordance with NIH guidelines, the Animal Welfare Act, and US federal law. All animal experiments were approved by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC) and overseen by the Institutional Animal Care and Use Committee (IACUC) under Protocol ID 12826.

OBJECTIVES:Recent publications have shown that mitochondrial dynamics can govern the quality and quantity of extracellular mitochondria subsequently impacting immune phenotypes. This study aims to determine if pathologic mitochondrial fission mediated by Drp1/Fis1 interaction impacts extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis. DESIGN:Laboratory investigation. SETTING:University laboratory. SUBJECTS:C57BL/6 and BALB/C mice. INTERVENTIONS:Using in vitro and murine models of endotoxin tolerance (ET), we evaluated changes in Drp1/Fis1-dependent pathologic fission and simultaneously measured the quantity and quality of extracellular mitochondria. Next, by priming mouse macrophages with isolated healthy mitochondria (MC) and damaged mitochondria, we determined if damaged extracellular mitochondria are capable of inducing tolerance to subsequent endotoxin challenge. Finally, we determined if inhibition of Drp1/Fis1-mediated pathologic fission abrogates release of damaged extracellular mitochondria and improves macrophage response to subsequent endotoxin challenge. MEASUREMENTS AND MAIN RESULTS:When compared with naïve macrophages (NMs), endotoxin-tolerant macrophages (ETM) demonstrated Drp1/Fis1-dependent mitochondrial dysfunction and higher levels of damaged extracellular mitochondria (Mitotracker-Green + events/50 μL: ETM = 2.42 × 106 ± 4,391 vs NM = 5.69 × 105 ± 2,478; p < 0.001). Exposure of NMs to damaged extracellular mitochondria (MH) induced cross-tolerance to subsequent endotoxin challenge, whereas MC had minimal effect (tumor necrosis factor [TNF]-α [pg/mL]: NM = 668 ± 3, NM + MH = 221 ± 15, and NM + Mc = 881 ± 15; p < 0.0001). Inhibiting Drp1/Fis1-dependent mitochondrial fission using heptapeptide (P110), a selective inhibitor of Drp1/Fis1 interaction, improved extracellular mitochondrial function (extracellular mitochondrial membrane potential, JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0; p < 0.001) and subsequently improved immune response in ETMs (TNF-α [pg/mL]; ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4; p < 0.0001). Similarly, P110-treated endotoxin tolerant mice had lower amounts of damaged extracellular mitochondria in plasma (represented by higher extracellular mitochondrial membrane potential, TMRM/MT-G: endotoxin tolerant [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02; p = 0.03) and improved immune response to subsequent endotoxin treatment as well as cecal ligation and puncture. CONCLUSIONS:Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improved macrophage function and immune response in both in vitro and in vivo models of ET. This benefit is mediated, at least in part, by decreasing the release of damaged extracellular mitochondria, which contributes to endotoxin cross-tolerance. Altogether, these data suggest that alterations in mitochondrial dynamics may play an important role in sepsis-induced immunoparalysis.

Human epithelial organoids-3D spheroids derived from adult tissue stem cells-enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules. One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). Our protocol establishes apical-out polarity rapidly (24-48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology-specifically the impact of host-microbe interactions on epithelial function. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research.

ABSTRACT Superspreader hosts carry out most pathogen transmission events and are often disease tolerant since they remain asymptomatic despite high pathogen burdens. Here we describe the superspreader immune state that allows for disease tolerance. In a model of Salmonella infection, superspreader mice develop colitis with robust CD4 + and CD8 + T-cell responses, however, they remain asymptomatic. We found that superspreaders have significantly more regulatory T cells (Tregs) in the distal gut compared to non-superspreader infected hosts. Surprisingly, the depletion of Tregs did not induce pathogen clearance but rather exacerbated weight loss, increased gut inflammation, and compromised epithelial intestinal barrier. This loss of tolerance correlated with dramatic increases in cytotoxic CD4 + and CD8 + T cells. Interestingly, CD4 neutralization in Tregs-depleted superspreaders was sufficient to rescue tolerance. Our results indicate that Tregs play a crucial role in maintaining immunologic tolerance in the guts of superspreader mice by suppressing cytotoxic CD4 + and CD8 + T-cell activities. AUTHOR SUMMARY Superspreader hosts are the main cause of disease transmission and a very important public health concern. Here, we evaluated the immunological tolerance of the Salmonella infected superspreaders in a mouse model. By manipulating Tregs, we demonstrated the immunological mechanism from the host to maintain health status and high pathogen burden. Tregs depletion in the superspreaders led to severe disease, with damage of the intestinal epithelia, and high morbidity without having any effect on shedding and systemic Salmonella burden. Furthermore, we demonstrated that the damage of the intestinal epithelia was related to cytotoxic activity of T cells. When Tregs were depleted, CD8 + T cells produced high levels of granzyme B and perforin. CD8 + T cells neutralization in Tregs depleted mice led to increased cytotoxic CD4 + T cells. Interestingly, neutralization of CD4 + T cells in the Tregs depleted mice led to a reduction in the CD8 + T cells producing granzyme B and it was sufficient to rescue host tolerance in this model. We demonstrate for the first time that cytotoxic CD4 + T cells damage the epithelial intestinal barrier and contribute to loss of tolerance in the context of a superspreader host. These findings open new perspectives to understand mechanisms of tolerance in the intestine of a superspreader host.