Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress

<p>Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an <i>in vivo</i> stress-response mouse strain expressing the <i>Gadd45β</i>-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene <i>Fanca</i> or <i>Fancc</i> persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of <i>Fanca</i> deficiency almost completely abolished the persistent oxidative stress-induced G₂/M arrest and DNA damage response <i>in vivo</i>. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress.</p

Enhanced survival following oral and systemic <i>Salmonella enterica</i> serovar Typhimurium infection in polymeric immunoglobulin receptor knockout mice

<div><p>Background</p><p>Polymeric immunoglobulin receptor (pIgR) transport of secretory immunoglobulin A (SIgA) to mucosal surfaces is thought to promote gut integrity and immunity to <i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>. <i>Typhimurium</i>), an invasive pathogen in mice. To elucidate potential mechanisms, we assessed intestinal barrier function and both oral and systemic S. <i>Typhimurium</i> virulence in pIgR knockout (KO) and wildtype (WT) mice.</p><p>Methods</p><p>In uninfected animals, we harvested jejunal segments for Ussing chamber analyses of transepithelial resistance (TER); mesenteric lymph nodes (mLN) for bacterial culture; and serum and stool for IgA. Separately, we infected mice either orally or intravenously (IV) with <i>S</i>. <i>Typhimurium</i> to compare colonization, tissue dynamics, and inflammation between KOs and WTs.</p><p>Results</p><p>Uninfected KOs displayed decreased TER and dramatically increased serum IgA and decreased fecal IgA vs. WT; however, KO mLNs yielded fewer bacterial counts. Remarkably, WTs challenged orally with <i>S</i>. <i>Typhimurium</i> exhibited increased splenomegaly, tissue colonization, and pro-inflammatory cytokines vs. pIgR KOs, which showed increased survival following either oral or IV infection.</p><p>Conclusions</p><p>Absence of pIgR compromises gut integrity but does not exacerbate bacterial translocation nor <i>S</i>. <i>Typhimurium</i> infection. These findings raise the possibility that immune adaptation to increased gut permeability and elevated serum IgA in the setting of SIgA deficiency provides compensatory protection against invasive gut pathogens.</p></div

Serum pro-inflammatory cytokines TFNα, IL-1β, and IL-6 are significantly lower in pIgR KO mice vs wildtype mice 7 days post oral <i>S</i>. <i>Typhimurium</i> infection.

<p>C57BL/6 mice showed significantly increased (A) TNFα (<i>P</i><0.0001, Mann-Whitney), (B) IL-1β (<i>P</i><0.005, Mann-Whitney test), and (C) IL-6 (<i>P</i><0.0001, Mann-Whitney) compared to pIgR KO. (D) No difference in IFNγ levels between groups.</p

Increased survival of pIgR KO following high-dose oral <i>S</i>. <i>Typhimurium</i> challenge.

<p>Mice were gavaged with 10⁹ CFU of <i>S</i>. <i>Typhimurium</i> following a 4-hour fast and followed for 2 weeks. Mean survival for C57BL/6 mice was statistically significantly shorter than the pIgR KO mice (<i>P</i><0.05, log-rank Mantel-Cox test). Data are representative of 3 experiments, with equivalent infectious doses between KO and WT in each experiment.</p

Intestinal and systemic indicators of <i>S</i>. <i>Typhimurium</i> infection intensity are reduced in pIgR KO mice following oral challenge.

<p>(A) Spleen weight was significantly elevated in C57BL/6 mice 7 days post-oral infection compare to both uninfected C57BL/6 (<i>P</i><0.001) and infected pIgR KO (<i>P</i><0.01, Kruskal-Wallis with Dunn multiple comparison test). (B) Cecum weight was significantly diminished in C57BL/6 mice compared to pIgR KO mice at 7 days post-oral infection (<i>P</i><0.05, Kruskal-Wallis with Dunn multiple comparison test). Data are representative of 3 experiments. Baseline weights of spleen and cecum did not differ in uninfected C57BL/6 and pIgR KO mice.</p

Small intestinal transepithelial resistance is impaired, but translocation of bacteria is diminished, in pIgR KO mice.

<p>Ussing chamber studies of mid-jejunal sections revealed a modest decrease in (A) transepithelial resistance in pIgR knockout mice as compared to C57BL/6 mice (<i>P</i><0.05, by t-test), and no difference in measures of (B) short circuit current, or (C) FITC-dextran flux. Data are representative of 3 experiments. (D) Fewer aerobic bacterial colonies were recovered from homogenized mesenteric lymph nodes of pIgR KO mice compared to C57BL/6 mice (<i>P</i><0.05, Mann-Whitney). Data are representative of 6 experiments.</p

Increased survival of pIgR KO following intravenous <i>S</i>. <i>Typhimurium</i> challenge.

<p>Mice were injected IV via the tail vein with 3.2x10² CFU of <i>S</i>. <i>Typhimurium</i> and followed for 26 days. Mean survival for C57BL/6 mice was statistically significantly shorter than the pIgR KO mice (<i>P</i><0.0001, log-rank Mantel-Cox test). Data are representative of 2 experiments.</p

Elevated serum IgA, decreased stool IgA, and decreased IgA coating of fecal bacteria in pIgR KO mice.

<p>(A) Serum IgA is significantly increased in pIgR KO mice compared to C57BL/6 mice (<i>P</i><0.0001, Mann-Whitney). (B) No significant difference in the serum IgG between pIgR KO and C57BL/6 mice. (C) Stool IgA is significantly decreased in pIgR KO mice compared to C57BL/6 mice (<i>P</i><0.0001, Mann-Whitney). (D) pIgR KO mice showed a 3-fold reduction in the percentage of stool bacteria coated with IgA (<i>P</i><0.0001, Mann-Whitney).</p