Deciphering the role of mitogen-activated protein kinases in host cell death induced by Mycobacterium tuberculosis

The emergence and prevalence of Mycobacterium tuberculosis (Mtb) resistant strains requires the development of alternative therapeutic strategies, such as host-directed therapies (HDT), to treat tuberculosis (TB) infections. These HDT act on the host- response to a pathogen rather than directly on the pathogen itself. A HDT already applied to TB patients are corticosteroids, such as dexamethasone, which are combined with antibiotic treatment in a subset of patients. The exact mechanism of action of corticosteroids in TB remains elusive. Mtb is a highly adapted pathogen that continuously exploits the immune system of the host to ensure its own survival. A major mechanism of pathogenesis in TB is the induction of host cell death. Host cell death leads to the escape of Mtb from the phagocyte and results in dissemination of the disease. However, the exact cell death pathway induced by Mtb as well as the key regulators remain unknown. In-depth understanding of this cell death pathway and the protective mechanism of dexamethasone would provide valuable targets for HDT and may allow for a tailored therapy in patients with extensive tissue necrosis and inflammation. In this thesis, I decipher a novel host cell death pathway triggered by Mtb, which can be inhibited by dexamethasone. Infection of macrophages (Mᴓ) with Mtb induces the phosphorylation of p38 mitogen-activated protein kinase (MAPK). I show that p38 MAPK signaling triggers necrosis rather than apoptosis in Mtb-infected phagocytes. The activation of p38 MAPK promotes the dissociation of hexokinase II (HKII) from mitochondria and allows the opening of the mitochondrial permeability transition pore (mPTP). The opening of the mPTP results in adenosine triphosphate (ATP) depletion and finally in necrosis of the host cell. I can show that dexamethasone inhibits this pathway by activating MAPK phosphatase 1 (MKP-1) to downregulate p38 MAPK activity. Moreover, a direct inhibition of p38 MAPK by the specific p38 MAPK inhibitor doramapimod has similar effects on host cell survival. Since corticosteroids are anti-inflammatory drugs, which among others inhibit tumor necrosis factor (TNF) signaling, I further characterized TNF and necroptosis in Mtb-infected cells using mixed lineage kinase domain-like pseudokinase (MLKL) and TNF receptor 1 (TNFR1) knockout Mᴓ. I could demonstrate that the underlying mechanism of dexamethasone is independent from TNF and necroptosis. Thus, my results link p38 MAPK inhibition by corticosteroids or p38 MAPK inhibitors to the abrogation of mitochondria-mediated host cell death in TB infection and provides new opportunities for research on novel HDT concepts

Corticosteroids inhibit Mycobacterium tuberculosis-induced necrotic host cell death by abrogating mitochondrial membrane permeability transition

Corticosteroids are host-directed drugs that enhance survival of tuberculosis patients through unclear mechanisms. Here, Gräb et al. show that corticosteroids inhibit necrotic death of cells infected with Mycobacterium tuberculosis by facilitating MKP-1-dependent dephosphorylation of p38 MAPK

<i>H</i>. <i>polygyrus</i> infection in the late phase of colitis-associated colon cancer does not impact tumor growth.

<p>(A) Schematic time schedule of <i>H</i>. <i>polygyrus</i> (<i>H</i>.<i>poly</i>) infection and the induction of colitis-associated colon cancer (CAC) in BALB/c mice. After an intraperitoneal injection of the procarcinogen azoxymethane (AOM), 3 cycles of dextran sulfate sodium (DSS) were given via the drinking water. At week 8, mice were infected with 200 stage-three larvae (L3) <i>H</i>. <i>polygyrus</i> by oral gavage, and tumor development was analyzed at week 12. (B) Weight change of naïve mice (unfilled circles), AOM/DSS-treated mice (CAC, black circles) and AOM/DSS-treated <i>H</i>. <i>polygyrus</i> infected mice (CAC+<i>H</i>.<i>poly</i>, grey rectangles) relative to initial body weight during the course of the experiment. The graph shows data from 4 independent experiments (naïve, n = 12; CAC, n = 17; CAC+<i>H</i>.<i>poly</i>, n = 17). (C) Murine endoscopy was performed to obtain representative endoscopic images from the distal colon of CAC and CAC+<i>H</i>.<i>poly</i> mice and to determine tumor scores. Bars represent the mean±SEM of data from 4 experiments (CAC, n = 17; CAC+<i>H</i>.<i>poly</i>, n = 17). (D) Colon from naïve mice, <i>H</i>. <i>polygyrus</i> infected mice, CAC mice and CAC+<i>H</i>.<i>poly</i> mice were prepared to calculate colon weight to length ratios. Data from 4 independent experiments are shown (naïve, n = 12; naïve+<i>H</i>.<i>poly</i>, n = 16; CAC, n = 17; CAC+<i>H</i>.<i>poly</i>, n = 17). Statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test (***, p≤ 0.001).</p

<i>H</i>. <i>polygyrus</i> infection in the early inflammatory phase of colitis-associated colon cancer increases tumor development.

<p>(A) Schematic time schedule of <i>H</i>. <i>polygyrus</i> (<i>H</i>.<i>poly</i>) infection and the induction of colitis-associated colon cancer (CAC) in BALB/c mice. One day after intraperitoneal injection of the procarcinogen azoxymethane (AOM), mice were infected with 200 stage-three larvae (L3) <i>H</i>. <i>polygyrus</i> by oral gavage. After 6 days, dextran sulfate sodium (DSS) was given via the drinking water. DSS administration was repeated twice, and mice were analyzed at week 12. (B) Weight change of naïve mice (unfilled circles), AOM/DSS-treated mice (CAC, black circles) and AOM/DSS-treated <i>H</i>. <i>polygyrus</i> infected mice (CAC+<i>H</i>.<i>poly</i>, grey rectangles) relative to initial body weight during the course of the experiment. The graph shows data from 5 independent experiments (naïve, n = 12; CAC, n = 24; CAC+<i>H</i>.<i>poly</i>, n = 25). Statistical significance was calculated using two-way ANOVA and Bonferroni posttests (**, p≤ 0.01). (C) Murine endoscopy was performed to obtain representative endoscopic images from the distal colon of CAC and CAC+<i>H</i>.<i>poly</i> mice and to determine tumor scores. Bars represent the mean±SEM of data from 5 experiments (CAC, n = 24; CAC+<i>H</i>.<i>poly</i>, n = 25). Statistical analyses were performed by unpaired t test (**, p≤ 0.01). (D) At week 12, Colon from naïve mice, <i>H</i>. <i>polygyrus</i> infected mice, CAC mice and CAC+<i>H</i>.<i>poly</i> mice were prepared to calculate colon weight to length ratios. LPLs from the colons were isolated and stained for the expression of CD4, CD8, and intracellular Foxp3. Frequencies of (E) CD4⁺Foxp3⁺ Tregs, (F) Foxp3⁺ CD8⁺ Tregs and (G) CD8⁺ T cells were determined by flow cytometry and absolute numbers were calculated. Graphs represent the mean±SEM of data from 5 experiments (naïve, n = 12; naïve+<i>H</i>.<i>poly</i>, n = 16; CAC, n = 24; CAC+<i>H</i>.<i>poly</i>, n = 25). Statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test (*, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001).</p

Colitis-associated colon cancer induction impacts the course of <i>H</i>. <i>polygyrus</i> infection.

<p>(A) Schematic time schedule of <i>H</i>. <i>polygyrus</i> (<i>H</i>.<i>poly</i>) infection and CAC induction in BALB/c mice. One day after i.p. injection of azoxymethane (AOM), mice were infected with 200 stage-three larvae (L3) <i>H</i>. <i>polygyrus</i>. After 6 days, dextran sulfate sodium (DSS) was given via the drinking water. DSS administration was repeated twice, and mice were analyzed at week 10 to 12. (B) Fecal egg counts were measured weekly during the course of the experiment until mice were sacrificed at week 10. Mice were graded as recovered when no eggs could be counted in their feces. The graph shows the percentage of recovered mice from 3 experiments with n = 10 <i>H</i>. <i>polygyrus</i> infected mice (open rectangles) and n = 19 CAC+<i>H</i>. <i>polygyrus</i> infected mice (grey rectangles) as survival curve. Statistical significance was calculated using log-rank test (*, p≤0.05). (C) At week 12, LPLs from the colons were isolated and stained for the expression of CD4 and intracellular IL-4. Frequencies of IL-4⁺ CD4⁺ T cells were determined by flow cytometry. (D) Colon biopsies were incubated <i>in vitro</i> for 6 hours in culture medium. Levels of IL-6 and CXCL1 in the supernatants were determined by Luminex and cytokines per milligram tissue were calculated. Bars represent the mean±SEM of data from 2–3 experiments (naïve, n = 4; naïve+<i>H</i>.<i>poly</i>, n = 6; CAC, n = 10–15; CAC+<i>H</i>.<i>poly</i>, n = 10–15). Statistical significance was calculated using one-way ANOVA followed by Dunn’s or Tukey's Multiple Comparison Test (*, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001).</p

<i>H</i>. <i>polygyrus</i> infection activates colonic effector T cells during DSS-induced acute colitis.

<p>BALB/c mice were infected with <i>H</i>. <i>polygyrus</i> and subjected to acute DSS colitis 6 days later. At day 15, LPLs from the colons of naïve mice, <i>H</i>. <i>polygyrus</i> infected mice, DSS mice and DSS+<i>H</i>. <i>poly</i> mice were isolated and stained for the expression of CD4, CD8, CD62L, and intracellular Ki67 and Foxp3. Frequencies of (A) CD4⁺ or CD8⁺ T cells and (B) expression of CD62L and Ki67 were assessed among Foxp3^-CD4⁺ T cells. (C) Frequencies of Foxp3⁺ among CD4⁺ T cells or CD8⁺ T cells were determined by flow cytometry. Bars represent the mean±SEM of data from 3 experiments (naïve, n = 6; naïve+<i>H</i>.<i>poly</i>, n = 7; DSS, n = 9; DSS+<i>H</i>.<i>poly</i>, n = 8). Statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test (*, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001).</p

<i>H</i>. <i>polygyrus</i> infection leads to the expansion of regulatory T cells in the colon.

<p>BALB/c mice were infected with 200 stage-three larvae (L3) <i>H</i>. <i>polygyrus</i> by oral gavage, and at indicated time points post infection mice were sacrificed. (A) At day 14, representative tissue sections of the colon and the small intestine from naïve mice and <i>H</i>. <i>polygyrus</i> (<i>H</i>.<i>poly</i>) infected mice were fixed and stained with haematoxylin and eosin (H&E) or periodic acid Schiff (PAS) to show pathologic changes. Images show magnification at x200. (B) Goblet cells in PAS stained sections were counted and referred to villi length. Bars represent the mean±SEM of data from one experiment (naïve, n = 2; naïve+<i>H</i>.<i>poly</i>, n = 3). Statistical significance was calculated using unpaired t test (**, p≤ 0.01). (C) At indicated days post infection (dpi), colon from naïve mice and <i>H</i>. <i>polygyrus</i> infected mice were prepared and colon length was measured. Bars represent the mean±SEM of data from 2 independent experiments (naïve, n = 10; naïve+<i>H</i>.<i>poly</i>, n = 5). (D, E) At indicated dpi, mLNs and LPLs of naïve mice and <i>H</i>. <i>polygyrus</i> infected mice were isolated and stained for the expression of CD4, CD103 and intracellular Foxp3. Bars represent the mean±SEM of data from 3 independent experiments (naïve, n = 14; naïve+<i>H</i>.<i>poly</i>, n = 8). (F) To determine the suppressive capacity of Treg <i>in vitro</i>, Foxp3/eGFP mice were infected with 200 L3 <i>H</i>. <i>polygyrus</i>, and at day 10 post infection, CD4⁺eGFP⁺ (Foxp3⁺) T cells (Treg) from mLN of infected or naive mice were sorted. Tregs were co-cultured at a ratio of 1:1 with eFluor-labeled CD4⁺ responder T cells (Tresp) and with antigen-presenting cells in the presence of a-CD3. Proliferation of Tresp was measured by loss of eFluor dye. Bar diagram represents the proliferation as mean±SEM of 2 independent experiments (naïve, n = 7; <i>H</i>.<i>poly</i>, n = 7). Statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test (*, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001).</p

Gene silencing of IL-6 and CXCL1 ameliorates colonic inflammation in <i>H</i>. <i>polygyrus</i> infected DSS treated animals.

<p>(A) Schematic time schedule of <i>H</i>. <i>polygyrus</i> (<i>H</i>.<i>poly</i>) infection and siRNA treatment during the induction of dextran sulfate sodium colitis (DSS) in BALB/c mice. At day 1, mice were infected with 200 stage-three larvae (L3) <i>H</i>. <i>polygyrus</i> by oral gavage. Six days later, DSS was given via the drinking water for 7 days and mice were sacrificed on day 14. Eight μg siRNA-loaded CaP/PLGA nanoparticles directed against IL-6 and CXCL1 were applied intrarectally daily during DSS treatment until day 13. (B) Disease activity index (DAI) of DSS-treated mice (DSS, black circles), DSS-treated <i>H</i>. <i>polygyrus</i> infected mice (DSS+<i>H</i>.<i>poly</i>, grey rectangles) and siRNA+DSS-treated <i>H</i>. <i>polygyrus</i> infected mice (DSS+<i>H</i>.<i>poly</i>+siRNA, red rectangles) during the course of the experiment. The graph shows data from 1 experiment. Statistical significance was calculated using two-way ANOVA and Bonferroni posttests (**, p≤ 0.01). (C) Representative tissue sections of colon samples from DSS, DSS+<i>H</i>. <i>poly</i> and DSS+<i>H</i>.poly+siRNA mice were fixed and stained with haematoxylin and eosin to show pathologic changes. Images show magnification at x200. (D) At day 14 colons were prepared and biopsies from colon samples were cultured <i>in vitro</i> for 6 hours in culture medium. Levels of IL-6 and CXCL1 in the supernatants were determined by Luminex. Bars show the mean ± SEM of cytokines per milligram tissue from 1 experiment (DSS, n = 5; DSS+<i>H</i>.<i>poly</i>, n = 6; DSS+<i>H</i>.<i>poly</i>+siRNA, n = 5). Statistical significance was calculated using Mann Whitney test (*, p≤ 0.05).</p