Ali Shawki

Anemia is the most common extraintestinal manifestation of inflammatory bowel disease (IBD). Iron deficiency is the most frequent cause of anemia in IBD; however, the mechanisms involved are still poorly understood. Here, we investigated the role of the IBD risk gene, protein tyrosine phosphatase non-receptor type 2 (PTPN2), in regulating iron homeostasis. Proteomic analyses were performed on serum from IBD patients genotyped for the IBD-associated loss-of-function rs1893217 PTPN2 variant. Constitutive Ptpn2 wild type (WT), heterozygous (Het), and knockout (KO) mice were analyzed for iron content, blood parameters, and expression of iron handling proteins. Iron absorption was assessed through radiotracer assays. Serum proteomic analyses revealed that the “iron homeostasis signaling pathway” was the main pathway downregulated in Crohn’s disease (CD) patients carrying the PTPN2 risk allele, independent of disease activity. Ptpn2-KO mice showed characteristics of anemia, including reduced hemoglobin concentrations along with serum and tissue iron deficiency and elevated serum hepcidin levels vs. Ptpn2-WT and Het mice. ⁵⁵Fe absorption via oral gavage was significantly impaired in Ptpn2-KO mice. Correspondingly, Ptpn2-KO mice showed reduced apical membrane expression of the iron transporter DMT1. CD patients with the PTPN2 loss-of-function rs1893217 variant display alterations in serum iron handling proteins. Loss of Ptpn2 in mice caused features of anemia, including iron deficiency associated with reduced apical membrane expression of DMT1. These findings identify an important role for PTPN2 in regulating systemic iron homeostasis.

Macrophages intimately interact with intestinal epithelial cells, but the consequences of defective macrophage–epithelial cell interactions for protection against enteric pathogens are poorly understood. Here, we show that in mice with a deletion in protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in macrophages, infection with Citrobacter rodentium , a model of enteropathogenic and enterohemorrhagic E . coli infection in humans, promoted a strong type 1/IL-22–driven immune response, culminating in accelerated disease but also faster clearance of the pathogen. In contrast, deletion of PTPN2 specifically in epithelial cells rendered the epithelium unable to upregulate antimicrobial peptides and consequently resulted in a failure to eliminate the infection. The ability of PTPN2-deficient macrophages to induce faster recovery from C . rodentium was dependent on macrophage-intrinsic IL-22 production, which was highly increased in macrophages deficient in PTPN2. Our findings demonstrate the importance of macrophage-mediated factors, and especially macrophage-derived IL-22, for the induction of protective immune responses in the intestinal epithelium, and show that normal PTPN2 expression in the epithelium is crucial to allow for protection against enterohemorrhagic E . coli and other intestinal pathogens.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.

Inflammatory bowel disease (IBD) pathogenesis involves significant contributions from genetic and environmental factors. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene increase IBD risk and are associated with altered microbiome population dynamics in IBD. Expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in IBD pathogenesis as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation – suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. We aimed to determine if PTPN2 deficiency alters intestinal microbiome composition to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2, we identified increased abundance of a novel mouse AIEC (mAIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages, to the IBD-associated AIEC, LF82. Furthermore, mAIEC caused disease when administered to mice lacking segmented-filamentous bacteria (SFB), and in germ-free mice but only when reconstituted with a microbiome, thus supporting its classification as a pathobiont, not a pathogen. Moreover, mAIEC infection increased the severity of, and prevented recovery from, induced colitis. Although mAIEC genome sequence analysis showed >90% similarity to LF82, mAIEC contained putative virulence genes with >50% difference in gene/protein identities from LF82 indicating potentially distinct genetic features of mAIEC. We show for the first time that an IBD susceptibility gene, PTPN2, modulates the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD and identifies a new model to study AIEC-host interactions.

Divalent metal‐ion transporter‐1 (DMT1) and the high‐affinity copper transporter‐1 (CTR1) play essential roles in the intestinal absorption of iron and copper respectively. Both transporters are potential therapeutic targets in disorders of metal metabolism. Disagreement remains over whether the metal‐ion specificities of the two transporters overlap. For example, others have concluded that DMT1 transports Cu ⁺ and CTR1 transports Fe ²⁺ based on the results of shRNA knockdown of gene expression in Caco‐2 cells [Espinoza A et al (2012) Biol Trace Elem Res 146, 281]. Since these observations contradicted our published data regarding the substrate profile of DMT1 in vitro [Illing AC et al (2012) J Biol Chem 287, 30485] and in vivo [Shawki A et al (2015) Am J Physiol Gastrointest Physiol 309, G635] or of CTR1 in vitro [Lee J et al (2002) J Biol Chem 277, 4380], we have re‐examined metal‐ion transporter substrate profiles. We tested the hypothesis that DMT1 can transport copper and that CTR1 can transport iron by using radiotracer assays and the voltage clamp in RNA‐injected oocytes expressing human DMT1 or CTR1. Expression of CTR1 in oocytes stimulated up to 20‐fold the uptake of 2 μM ⁶⁴ Cu ⁺ (pH 5.5) but did not stimulate the uptake of ⁶⁴ Cu ²⁺ or ⁵⁵ Fe ²⁺ . Expression of DMT1 in oocytes stimulated 80‐fold the uptake of 2 μM ⁵⁵ Fe ²⁺ (pH 6.0) but transport of ⁶⁴ Cu ⁺ or ⁶⁴ Cu ²⁺ did not differ from that in control oocytes. We found that Cu ⁺ inhibited DMT1‐mediated ⁵⁵ Fe ²⁺ uptake in a competitive fashion ( K _i ≈ 58 μM). In DMT1‐expressing oocytes, Cu ⁺ evoked inward currents; however, such currents were also observed in control oocytes and their I / V relationships did not resemble those for DMT1 substrates Fe ²⁺ and Cd ²⁺ . Addition of Fe ²⁺ or Cd ²⁺ abolished the presteady‐state (transient) currents observed in the absence of metal ion whereas addition of Cu ⁺ or Cu ²⁺ did not. We found, however, that Cu ²⁺ accelerated the decay of the transient currents. We conclude that (1) whereas Cu ⁺ and Cu ²⁺ are capable of interacting with DMT1, neither are transported substrates; and (2) CTR1 mediates transport of Cu ⁺ but not of Cu ²⁺ or Fe ²⁺ . We therefore find no evidence of overlapping metal‐ion specificities in DMT1 and CTR1. Support or Funding Information: PHS grants R01‐DK080047, R01‐DK107309, R01‐DK074192, and R25‐HL115473; and the American Physiological Society

Inflammatory bowel diseases (IBD) involve genetic and environmental factors that play major roles in disease pathogenesis. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 ( PTPN2 ) gene increase the risk of IBD and are associated with altered microbiome population dynamics in IBD. Moreover, expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in the pathogenesis of IBD as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. The aim of this study was to identify if PTPN2 deficiency disturbs the composition of the intestinal microbiome to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2 we identified increased abundance of a novel adherent-invasive E. coli (AIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages to the IBD associated AIEC, LF82. Furthermore, we confirmed this novel mouse AIEC ( m AIEC) caused disease when administered to germ-free and mice lacking segmented-filamentous bacteria (SFB). Moreover, m AIEC infection increased severity of and prevented recovery from dextran-sodium sulfate (DSS)-induced colitis. m AIEC genome sequence analysis showed >90% similarity to LF82. Interestingly, m AIEC contained distinct attachment genes not found in LF82 thereby also demonstrating the novelty of this AIEC. We show here for the first time that an IBD susceptibility gene, PTPN2 , plays a key role in modulating the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD.

Divalent metal-ion transporter-1 (DMT1), the principal mechanism by which nonheme iron is taken up at the intestinal brush border, is energized by the H⁺-electrochemical potential gradient. The provenance of the H⁺gradient in vivo is unknown, so we have explored a role for brush-border Na⁺/H⁺exchanger (NHE) isoforms by examining iron homeostasis and intestinal iron handling in mice lacking NHE2 or NHE3. We observed modestly depleted liver iron stores in NHE2-null (NHE2^−/−) mice stressed on a low-iron diet but no change in hematological or blood iron variables or the expression of genes associated with iron metabolism compared with wild-type mice. Ablation of NHE3 strongly depleted liver iron stores, regardless of diet. We observed decreases in blood iron variables but no overt anemia in NHE3-null (NHE3^−/−) mice on a low-iron diet. Intestinal expression of DMT1, the apical surface ferrireductase cytochrome b reductase-1, and the basolateral iron exporter ferroportin was upregulated in NHE3^−/−mice, and expression of liver Hamp1 (hepcidin) was suppressed compared with wild-type mice. Absorption of⁵⁹Fe from an oral dose was substantially impaired in NHE3^−/−compared with wild-type mice. Our data point to an important role for NHE3 in generating the H⁺gradient that drives DMT1-mediated iron uptake at the intestinal brush border.