Memory CD8+ T Cells Balance Pro- and Anti-inflammatory Activity by Reprogramming Cellular Acetate Handling at Sites of Infection.

Serum acetate increases upon systemic infection. Acutely, assimilation of acetate expands the capacity of memory CD8+ T cells to produce IFN-γ. Whether acetate modulates memory CD8+ T cell metabolism and function during pathogen re-encounter remains unexplored. Here we show that at sites of infection, high acetate concentrations are being reached, yet memory CD8+ T cells shut down the acetate assimilating enzymes ACSS1 and ACSS2. Acetate, being thus largely excluded from incorporation into cellular metabolic pathways, now had different effects, namely (1) directly activating glutaminase, thereby augmenting glutaminolysis, cellular respiration, and survival, and (2) suppressing TCR-triggered calcium flux, and consequently cell activation and effector cell function. In vivo, high acetate abundance at sites of infection improved pathogen clearance while reducing immunopathology. This indicates that, during different stages of the immune response, the same metabolite-acetate-induces distinct immunometabolic programs within the same cell type

Robust microbe immune recognition in the intestinal mucosa.

The mammalian mucosal immune system acts as a multitasking mediator between bodily function and a vast diversity of microbial colonists. Depending on host-microbial interaction type, mucosal immune responses have distinct functions. Immunity to pathogen infection functions to limit tissue damage, clear or contain primary infection, and prevent or lower the severity of a secondary infection by conferring specific long-term adaptive immunity. Responses to nonpathogenic commensal or mutualistic microbes instead function to tolerate continuous colonization. Mucosal innate immune and epithelial cells employ a limited repertoire of innate receptors to program the adaptive immune response accordingly. Pathogen versus nonpathogen immune discrimination appears to be very robust, as most individuals successfully maintain life-long mutualism with their nonpathogenic microbiota, while mounting immune defense to pathogenic microbe infection specifically. However, the process is imperfect, which can have immunopathological consequences, but may also be exploited medically. Normally innocuous intestinal commensals in some individuals may drive serious inflammatory autoimmunity, whereas harmless vaccines can be used to fool the immune system into mounting a protective anti-pathogen immune response. In this article, we review the current knowledge on mucosal intestinal bacterial immune recognition focusing on TH17 responses and identify commonalities between intestinal pathobiont and vaccine-induced TH17 responses

Author Correction: Uncoupling of invasive bacterial mucosal immunogenicity from pathogenicity.

ISSN:2041-172

Innate immunity restricts <i>Citrobacter rodentium</i> A/E pathogenesis initiation to an early window of opportunity

<div><p><i>Citrobacter rodentium</i> infection is a mouse model for the important human diarrheal infection caused by enteropathogenic <i>E</i>. <i>coli</i> (EPEC). The pathogenesis of both species is very similar and depends on their unique ability to form intimately epithelium-adherent microcolonies, also known as “attachment/effacement” (A/E) lesions. These microcolonies must be dynamic and able to self-renew by continuous re-infection of the rapidly regenerating epithelium. It is unknown whether sustained epithelial A/E lesion pathogenesis is achieved through re-infection by planktonic bacteria from the luminal compartment or local spread of sessile bacteria without a planktonic phase. Focusing on the earliest events as <i>C</i>. <i>rodentium</i> becomes established, we show here that all colonic epithelial A/E microcolonies are clonal bacterial populations, and thus depend on local clonal growth to persist. In wild-type mice, microcolonies are established exclusively within the first 18 hours of infection. These early events shape the ongoing intestinal geography and severity of infection despite the continuous presence of phenotypically virulent luminal bacteria. Mechanistically, induced resistance to A/E lesion de-novo formation is mediated by TLR-MyD88/Trif-dependent signaling and is induced specifically by virulent <i>C</i>. <i>rodentium</i> in a virulence gene-dependent manner. Our data demonstrate that the establishment phase of <i>C</i>. <i>rodentium</i> pathogenesis <i>in vivo</i> is restricted to a very short window of opportunity that determines both disease geography and severity.</p></div

Uncoupling of invasive bacterial mucosal immunogenicity from pathogenicity.

There is the notion that infection with a virulent intestinal pathogen induces generally stronger mucosal adaptive immunity than the exposure to an avirulent strain. Whether the associated mucosal inflammation is important or redundant for effective induction of immunity is, however, still unclear. Here we use a model of auxotrophic Salmonella infection in germ-free mice to show that live bacterial virulence factor-driven immunogenicity can be uncoupled from inflammatory pathogenicity. Although live auxotrophic Salmonella no longer causes inflammation, its mucosal virulence factors remain the main drivers of protective mucosal immunity; virulence factor-deficient, like killed, bacteria show reduced efficacy. Assessing the involvement of innate pathogen sensing mechanisms, we show MYD88/TRIF, Caspase-1/Caspase-11 inflammasome, and NOD1/NOD2 nodosome signaling to be individually redundant. In colonized animals we show that microbiota metabolite cross-feeding may recover intestinal luminal colonization but not pathogenicity. Consequent immunoglobulin A immunity and microbial niche competition synergistically protect against Salmonella wild-type infection

Dynamics of <i>Citrobacter rodentium</i> infection in SPF and germ-free wild-type mice.

<p>(<b>A</b>) Schematic of possible routes of epithelial infection shaping A/E lesion development and renewal in the face of continuous epithelial regeneration. Black arrows indicate direction of epithelial cell migration and luminal exfoliation. Green arrows indicate possible routes of bacterial infection, with or without luminal planktonic stage. (<b>B-D</b>) Luminal colonization quantitated by bacterial plating from feces of SPF mice (panel B; n = 12–21 per group and time point) and germ-free mice (panel C; n = 3–15 per group and time point) following inoculation with 10¹⁰ (red squares) and 10⁴ (blue circles) CFU/mouse, respectively. (D) Early colonization in mice (n = 4 per group) sampled every hour during the first 12 hours after gavage. Fitted exponential curves were used to extrapolate the time to reach levels of 2.5x10⁹ CFU/g in the animals inoculated with 10⁴ CFU. The average of these 4 values is represented by the vertical dotted line (at 15.5 h). (<b>E-H</b>) Representative fluorescent microscopy images of distal colon cross sections of SPF (E and G) and germ-free (F and H) mice infected with 10¹⁰ (E and F) and 10⁴ (G and H) CFU/mouse of <i>C</i>. <i>rodentium</i> analyzed on day 7 post infection. All individual mice depicted were infected with a 1:1 mixture of bacteria carrying a mCherry (red) or GFP (green) fluorescent protein expression plasmid. Grey, F-actin stained with phalloidin; green, GFP-expressing <i>C</i>. <i>rodentium</i>; red, mCherry-expressing <i>C</i>. <i>rodentium</i>. Inset indicates area shown in higher magnification panel. Scale bars: 100 μm. (<b>I</b>) Numbers of A/E microcolonies in the distal colon of SPF mice infected with either 10⁴ (blue circles) or 10¹⁰ (red squares) CFU of <i>C</i>. <i>rodentium</i> quantified over a time course of 10 days (n = 3–6 per group and time point, data pooled from 2 independent experiments). Connecting lines indicate means. (<b>J</b>) Numbers of A/E microcolonies in the distal colon of germ-free mice infected with either 10⁴ (blue circles) or 10¹⁰ (red squares) CFU of <i>C</i>. <i>rodentium</i> quantified over a time course of 22 days (n = 2–6 per group and time point, data pooled from 3 independent experiments). Connecting lines connect means; horizontal dotted lines indicate detection limit; ****, p < 0.0001 (Student`s t-test); F-test, Fisher’s test.</p

Ongoing A/E lesion induction in mice deficient for innate signaling through MyD88 and Trif.

<p>(<b>A</b>) Colonic colonization dynamics in MyD88^-/-Trif^lps/lps germ-free mice consecutively infected, first with mCherry⁺ Kan^R Tet^S <i>C</i>. <i>rodentium</i> wild type (WT) or an isogenic Δ<i>ler</i> mutant (10¹⁰ CFU, red squares and triangles), and 18 hours later superinfected with mCherry^- Kan^S Tet^R <i>C</i>. <i>rodentium</i> wild type (10⁴ CFU, blue circles). Total <i>C</i>. <i>rodentium</i> counts are depicted as black symbols. Connecting lines connect means; error bars indicate standard deviation; horizontal dotted lines indicate detection limit. (<b>B and C</b>) Representative fluorescent microscopy images of distal colon of MyD88^-/-Trif^lps/lps mice shown in panel A pre-infected with wild type (B) and Δ<i>ler</i> (C) <i>C</i>. <i>rodentium</i>, respectively. Grey, F-actin/phalloidin; green, anti-<i>Citrobacter</i> O-antigen antibody (pre-infection strain and superinfecting strain); red, mCherry-expressing <i>C</i>. <i>rodentium</i> (pre-infection strain only; none detectable). Scale bars: 50 μm.</p

Early <i>C</i>. <i>rodentium</i>-induced innate immune response.

<p>(<b>A</b>) Lipocalin-2 quantification by ELISA in feces of germ-free mice infected for 18 h with 10⁴ CFU <i>C</i>. <i>rodentium</i> (blue circles), 10¹⁰ CFU <i>C</i>. <i>rodentium</i> (red squares), 10¹⁰ CFU <i>C</i>. <i>rodentium</i> Δ<i>ler</i> (green triangles), 10¹⁰ <i>E</i>. <i>coli</i> HS (orange hexagons) or left uninfected (grey diamonds). MyD88^-/-Trif^lps/lps germ-free mice were infected for 18 h with 10⁴ CFU <i>C</i>. <i>rodentium</i> (open blue circles), 10¹⁰ CFU <i>C</i>. <i>rodentium</i> (open red squares), or left uninfected (open grey diamonds). N = 4–13 per group. Data were pooled from 3 independent experiments. (<b>B and C</b>) Gene expression levels of <i>Cxcl1</i> (B) and <i>Nos2</i> (C) in the distal colon of infected mice as determined by qPCR. (<b>D-G</b>) Cytokine protein levels in the distal colon of infected mice as determined by Luminex technology. (<b>H and I</b>) Leukocyte analysis by flow cytometry of peripheral blood. Monocytes were defined as CD45⁺, CD11b⁺, CD115⁺ population (H). Neutrophils were defined as CD45⁺, CD11b⁺, Ly6G⁺ population. Quantities are expressed as absolute numbers of cells per mL blood. (<b>J and K</b>) Large intestinal leukocyte analysis by flow cytometry. Infiltrating monocytes were defined as CD45⁺, CD11b⁺, CD64^-, LygG^-, Ly6C^high, and MHCII^- population (J). Neutrophils were defined as CD45⁺, CD11b⁺, Ly6G⁺ population (K). Quantities are expressed as percentages of leukocytes (of CD45⁺ population). Dotted lines represent detection limit. ns, statistically not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; groups were compared with the matching uninfected control group; statistical tests: Kruskal-Wallis (A), and 1-way ANOVA (B-K).</p

High-dose <i>C</i>. <i>rodentium</i> infection is associated with reduced severity of disease in germ-free wild-type mice.

<p>(<b>A</b>) Body weight of germ-free mice infected with either 10⁴ (blue circles) or 10¹⁰ (red squares) CFU/mouse of <i>C</i>. <i>rodentium</i>, (n = 15 until day 10). (<b>B</b>) Survival curve of the animals shown in A. (<b>C</b>) Splenic bacterial loads of mice shown in A infected with 10⁴ (blue circles) or 10¹⁰ (red squares) CFU/mouse of <i>C</i>. <i>rodentium</i> for 10–13 days; n = 10–12 per group. (<b>D</b>) Histopathological scores of mice shown in panel A (Endpoint criterion: weight loss of >20 %). Mice were scored for epithelial hyperplasia and integrity, infiltration of PMNs, submucosal edema, and loss of goblet cells. Graph shows combined score (= sum of 5 individual scores). (<b>E</b>) Representative images of colonic histopathology scored in panel D (H&E staining) of mice infected for 13 days with 10⁴ (top) and 10¹⁰ (bottom) CFU. Arrows in panel D indicate the individuals depicted. Scale bar: 100 μm; Sm, submucosa; Cr, crypts; Ep, epithelium; Lp, lamina proria; (<b>F</b>) Lipocalin-2 measurement by ELISA in feces from infected mice (n = 5 per group, same animals as shown in A; after day 13 n = 3 survivors in 10⁴ group). Error bars indicate standard deviation. Dotted lines indicate detection limit; ns, statistically not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; statistical tests: Student`s t-test (A), Mantel-Cox test (B), Mann-Whitney U test (C, D, F).</p

An early window of opportunity for A/E microcolony induction in wild-type mice.

<p>(<b>A</b>) Colonic luminal colonization trajectories of the pre-infection strain (red open symbols: circles, 10⁴ CFU Wild type <i>C</i>. <i>rodentium</i> [WT]; squares, 10¹⁰ CFU WT; inverted triangles; 10¹⁰ CFU Δ<i>ler</i> mutant), the super-infection strain (blue filled symbols: circles, 10⁴ CFU WT; squares, 10¹⁰ CFU WT), and total bacteria (pre-infection + super-infection strain; black triangles). Connecting lines connect means; error bars indicate standard deviations. Horizontal dotted lines indicate the lower detection limit. (<b>B</b>) Microcolony formation of superinfection strain after 7 days. N = 6–7 per group, pooled from 3 independent experiments. (<b>C-G</b>) Representative confocal fluorescent microscopy images of distal colon of mice shown in panel B. Grey, F-actin/phalloidin; green, anti-O-antigen Antibody (<i>C</i>. <i>rodentium</i> total); red, mCherry-expressing <i>C</i>. <i>rodentium</i> (pre-infection strain only); Scale bars: 200 μm and 100 μm as indicated; White squares indicate origin of higher-magnification image; *, p < 0.05; **, p < 0.01; ***, P < 0.001; ns, not significant (p ≥ 0.05); statistical test: one-way ANOVA.</p