FTIR spectra of <i>L. monocytogenes</i> re-isolated from challenged mice.

<p>(A) Average FTIR spectra derived from 129/Sv (dotted), Tyk2−/− (dashed) and C57BL/6 (solid) isolates are shown from the whole spectral range (B) Subtraction spectra were generated from second derivative, vector-normalized, average FTIR spectra of the three different mouse genotypes. Spectra from C57BL/6 mice were subtracted from 129/Sv isolates (black), spectra from C57BL/6 mice were subtracted from Tyk2−/− (red) and spectra from Tyk2−/− were subtracted from 129/Sv isolates (blue). Most pronounced differences were observed in the spectral range of 1700–1500 cm⁻¹ (protein region) and can be assigned to amide I (1670–1625 cm⁻¹) and amide II (1560–1530 cm⁻¹).</p

Deciphering Host Genotype-Specific Impacts on the Metabolic Fingerprint of <i>Listeria monocytogenes</i> by FTIR Spectroscopy

<div><p>Bacterial pathogens are known for their wide range of strategies to specifically adapt to host environments and infection sites. An in-depth understanding of these adaptation mechanisms is crucial for the development of effective therapeutics and new prevention measures. In this study, we assessed the suitability of Fourier Transform Infrared (FTIR) spectroscopy for monitoring metabolic adaptations of the bacterial pathogen <i>Listeria monocytogenes</i> to specific host genotypes and for exploring the potential of FTIR spectroscopy to gain novel insights into the host-pathogen interaction. Three different mouse genotypes, showing different susceptibility to <i>L. monocytogenes</i> infections, were challenged with <i>L. monocytogenes</i> and re-isolated bacteria were subjected to FTIR spectroscopy. The bacteria from mice with different survival characteristics showed distinct IR spectral patterns, reflecting specific changes in the backbone conformation and the hydrogen-bonding pattern of the protein secondary structure in the bacterial cell. Coupling FTIR spectroscopy with chemometrics allowed us to link bacterial metabolic fingerprints with host infection susceptibility and to decipher longtime memory effects of the host on the bacteria. After prolonged cultivation of host-passaged bacteria under standard laboratory conditions, the host's imprint on bacterial metabolism vanished, which suggests a revertible metabolic adaptation of bacteria to host environment and loss of host environment triggered memory effects over time. In summary, our work demonstrates the potential and power of FTIR spectroscopy to be used as a fast, simple and highly discriminatory tool to investigate the mechanism of bacterial host adaptation on a macromolar and metabolic level.</p></div

Host imprint on mice passaged <i>L. monocytogenes</i> isolates monitored be FTIR spectroscopy.

<p>LDA was carried out on second derivative, vector-normalized FTIR spectra from <i>L. monocytogenes</i> re-isolated from challenged 129/Sv (X), Tyk2−/− (O) and C57BL/6 (+) mice derived from the two independent experiments.</p

Potential host memory effects on mice passaged <i>L. monocytogenes</i> isolates monitored by FTIR spectroscopy.

<p>PCA on second derivative, vector-normalized FTIR spectra from <i>L. monocytogenes</i> isolates derived from challenged 129/Sv (X), Tyk2−/− (O) and C57BL/6 (+) mice from one representative experiment (A) directly after passage in mice and after consecutive sub-cultivations at 37°C on laboratory standard media for one week (B) and for 6 weeks (C).</p

Survival rates of different mouse genotypes challenged with <i>L. monocytogenes</i>.

<p>Mice of the indicated genotypes (n = 8 mice/group) were infected with 5×10⁴ cfu bacteria intraperitoneally and survival was monitored over a 12-day period.</p

ANOVA-pairwise comparison to assess significant differentiation among the three genotypes (<i>p</i>-values).

<p>ANOVA-pairwise comparison to assess significant differentiation among the three genotypes (<i>p</i>-values).</p

Type I Interferons Promote Fatal Immunopathology by Regulating Inflammatory Monocytes and Neutrophils during <em>Candida</em> Infections

<div><p>Invasive fungal infections by <em>Candida albicans</em> (Ca) are a frequent cause of lethal sepsis in intensive care unit patients. While a contribution of type I interferons (IFNs-I) in fungal sepsis remains unknown, these immunostimulatory cytokines mediate the lethal effects of endotoxemia and bacterial sepsis. Using a mouse model lacking a functional IFN-I receptor (<em>Ifnar1^−/−</em>), we demonstrate a remarkable protection against invasive Ca infections. We discover a mechanism whereby IFN-I signaling controls the recruitment of inflammatory myeloid cells, including Ly6C^hi monocytes and neutrophils, to infected kidneys by driving expression of the chemokines CCL2 and KC. Within kidneys, monocytes differentiate into inflammatory DCs but fail to functionally mature in <em>Ifnar1^−/−</em> mice, as demonstrated by the impaired upregulation of the key activation markers PDCA1 and iNOS. The increased activity of inflammatory monocytes and neutrophils results in hyper-inflammation and lethal kidney pathology. Pharmacological diminution of monocytes and neutrophils by treating mice with pioglitazone, a synthetic agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ), strongly reduces renal immunopathology during Ca infection and improves mouse survival. Taken together, our data connect for the first time the sepsis-promoting functions of IFNs-I to the CCL2-mediated recruitment and the activation of inflammatory monocytes/DCs with high host-destructing potency. Moreover, our data demonstrate a therapeutic relevance of PPAR-γ agonists for microbial infectious diseases where inflammatory myeloid cells may contribute to fatal tissue damage.</p> </div

Reduced immunopathology protects <i>Ifnar1^−/−</i> mice from kidney injury.

<p>Mice of the indicated genotype were injected with a lethal dose of 1×10⁵ cfus Ca. At indicated time points, serum and kidneys were collected. (A) Histopathology of the cortical part of kidneys at day 1, 3 and 7 post infection. Longitudinal sections of paraffin-embedded organs were stained with periodic acid-Schiff (PAS) to visualize fungal cells. Counterstaining was performed with hematoxylin. (n = 3–4 mice per group) (B) Kidney total RNA was analysed for gene expression of kidney injury marker-1 (<i>Kim-1</i>). Data presented show the mean ± SEM of three independent experiments (n = 8–10 mice per group). (C) Urea concentration in serum. Data presented show the mean ± SEM of two independent experiments (n = 7–9 mice per group).</p

IFNs-I regulate detrimental Ly6C^hi monocyte and neutrophil activity.

<p>Model of IFN-I-mediated monocyte/neutrophil recruitment and activation of inflammatory DC during invasive Ca infections. Ca recognition triggers an IFN response, which controls the production of various chemokines, including CCL2 and KC, at different anatomical body sites (BM and kidneys). In response to local CCL2 in the BM, Ly6C^hi monocytes exit into the blood stream and migrate towards the target organ where they differentiate into inflammatory DCs. To fully functionally mature and become iNOS-producing cells, DCs require signaling through IFNAR1. The high presence and activity of inflammatory DCs and neutrophils in the kidney during the early infection phase promotes a secondary strong influx of neutrophils culminating in lethal immunopathology. The suppressive action of pioglitazone on Ly6C^hi monocyte/neutrophil recruitment and function ameliorates hyper-inflammation and kidney pathology.</p

IFN-I signaling promotes hyper-inflammatory immune responses.

<p>Mice of the indicated genotype were injected with a lethal dose of 1×10⁵ cfus Ca. At indicated time points, serum/whole blood and kidneys were collected. (A) Sera concentrations of IL-6 and TNF-α were measured using a multiplex bead array system. Data presented show the mean ± SEM of four independent experiments (n = 7–12 mice per group). (B) Blood cell populations were analysed by an automated blood counter. Data presented show the mean ± SEM of two independent experiments (n = 6–8 mice per group). Plotted are the absolute numbers of leukocytes, granulocytes, and lymphocytes expressed as cell number ×10⁹/l. (C) IL-6 concentrations in kidney supernatants were measured using a multiplex bead array system. Gene expression levels of <i>Icam-1</i> and <i>P-Selectin</i> were quantified by qPCR in kidney total RNA. Data presented show the mean ± SEM of three independent experiments (n = 7–12 mice per group). (D) Kidney leukocytes were enriched and characterized by multi-label flow cytometry. Graphs show absolute numbers of leukocytes (CD45⁺) and myeloid cells (CD11b⁺) per total mouse kidneys. Data presented is one representative of two independent experimental repeats (n = 3–5 mice per group).</p