Enrico Velardi

Interleukin-22 Protects Intestinal Stem Cells from Immune-Mediated Tissue Damage and Regulates Sensitivity to Graft versus Host Disease

Little is known about the maintenance of intestinal stem cells (ISCs) and progenitors during immune-mediated tissue damage or about the susceptibility of transplant recipients to tissue damage mediated by the donor immune system during graft versus host disease (GVHD). We demonstrate here that deficiency of recipient-derived IL-22 increased acute GVHD tissue damage and mortality, that ISCs were eliminated during GVHD, and that ISCs as well as their downstream progenitors expressed the IL-22 receptor. Intestinal IL-22 was produced after bone marrow transplant by IL-23-responsive innate lymphoid cells (ILCs) from the transplant recipients, and intestinal IL-22 increased in response to pretransplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of ISCs, and loss of epithelial integrity. Our findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISCs during inflammatory intestinal damage.

Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5+ ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.

Nrf2 regulates haematopoietic stem cell function

Coordinating the balance between haematopoietic stem cell (HSC) quiescence and self-renewal is crucial for maintaining haematopoiesis lifelong. Equally important for haematopoietic function is modulating HSC localization within the bone marrow niches, as maintenance of HSC function is tightly controlled by a complex network of intrinsic molecular mechanisms and extrinsic signalling interactions with their surrounding microenvironment. In this study we demonstrate that nuclear factor erythroid 2-related factor 2 (Nfe2l2, or Nrf2), well established as a global regulator of the oxidative stress response, plays a regulatory role in several aspects of HSC homeostasis. Nrf2 deficiency results in an expansion of the haematopoietic stem and progenitor cell compartment due to cell-intrinsic hyperproliferation, which was accomplished at the expense of HSC quiescence and self-renewal. We further show that Nrf2 modulates both migration and retention of HSCs in their niche. Moreover, we identify a previously unrecognized link between Nrf2 and CXCR4, contributing, at least partially, to the maintenance of HSC function.

Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice

Treating neutropenic fever with broad-spectrum antibiotics after allogeneic hematopoietic stem cell transplant is associated with an increase in graft-versus-host disease in mice and humans. Antibiotics for allogeneic transplant—A double-edged sword Patients undergoing allogeneic hematopoietic stem cell transplantation often receive antibiotics for infections, which can also unfortunately kill intestinal bacteria. These symbiotic bacteria in the gut generally do not cause disease and are thought to suppress inflammation. In a new study, Shono et al. examined the records of 857 transplant patients and found that certain antibiotics were linked with development of graft-versus-host disease (GVHD), which can cause severe intestinal inflammation. Using a mouse model, the authors showed that these antibiotics may select for bacteria that consume intestinal mucus and lead to loss of this important layer of protection for the gut, thus exacerbating GVHD in the intestine. This study suggests that not all antibiotic regimens are appropriate for treating transplant patients. Intestinal bacteria may modulate the risk of infection and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT recipients often develop neutropenic fever, which is treated with antibiotics that may target anaerobic bacteria in the gut. We retrospectively examined 857 allo-HSCT recipients and found that treatment of neutropenic fever with imipenem-cilastatin and piperacillin-tazobactam antibiotics was associated with increased GVHD-related mortality at 5 years (21.5% for imipenem-cilastatin–treated patients versus 13.1% for untreated patients, P = 0.025; 19.8% for piperacillin-tazobactam–treated patients versus 11.9% for untreated patients, P = 0.007). However, two other antibiotics also used to treat neutropenic fever, aztreonam and cefepime, were not associated with GVHD-related mortality (P = 0.78 and P = 0.98, respectively). Analysis of stool specimens from allo-HSCT recipients showed that piperacillin-tazobactam administration was associated with perturbation of gut microbial composition. Studies in mice demonstrated aggravated GVHD mortality with imipenem-cilastatin or piperacillin-tazobactam compared to aztreonam (P < 0.01 and P < 0.05, respectively). We found pathological evidence for increased GVHD in the colon of imipenem-cilastatin–treated mice (P < 0.05), but no difference in the concentration of short-chain fatty acids or numbers of regulatory T cells. Notably, imipenem-cilastatin treatment of mice with GVHD led to loss of the protective mucus lining of the colon (P < 0.01) and the compromising of intestinal barrier function (P < 0.05). Sequencing of mouse stool specimens showed an increase in Akkermansia muciniphila (P < 0.001), a commensal bacterium with mucus-degrading capabilities, raising the possibility that mucus degradation may contribute to murine GVHD. We demonstrate an underappreciated risk for the treatment of allo-HSCT recipients with antibiotics that may exacerbate GVHD in the colon.

Glucocorticoid-induced leucine zipper (GILZ)/NF-κB interaction: role of GILZ homo-dimerization and C-terminal domain

Glucocorticoid-induced leucine zipper (GILZ) is a 137 amino acid protein, rapidly induced by treatment with glucocorticoids (GC), characterized by a leucine zipper (LZ) domain (76–97 amino acids), an N-terminal domain (1–75 amino acids) and a C-terminal PER domain (98–137 amino acids) rich in proline and glutamic acid residues. We have previously shown that GILZ binds to and inhibits NF-κB activity. In the present study we used a number of mutants with the aim of defining the GILZ molecular domains responsible for GILZ/p65NF-κB interaction. Results, obtained by in vitro and in vivo co-immunoprecipitation (Co-IP) and by transcriptional activity experiments, indicate that GILZ homo-dimerization, through the LZ domain, as well as the C-terminal PER domain, particularly the 121–123 amino acids, are both necessary for GILZ interaction with NF-κB, inhibition of transcriptional activity and of IL-2 synthesis.

Peroxisome Proliferator-Activated Receptor-α Contributes to the Anti-Inflammatory Activity of Glucocorticoids

Glucocorticoids (GCs) are effective anti-inflammatory agents widely used in the therapeutic approach to treatment of acute and chronic inflammatory diseases. Previous results suggest that peroxisome proliferator-activated receptor-α (PPAR-α), an intracellular transcription factor activated by fatty acids, plays a role in the control of inflammation. With the aim of characterizing the role of PPAR-α in GC-mediated anti-inflammatory activity, we tested the efficacy of dexamethasone (DEX), a synthetic GC specific for glucocorticoid receptor, in an experimental model of lung inflammation, carrageenan-induced pleurisy, comparing mice lacking PPAR-α (PPAR-αKO) with wild-type (WT) mice. We also tested the possible synergism of combined treatment with DEX and clofibrate, a PPAR-α agonist. Results indicate that DEX-mediated anti-inflammatory activity is weakened in PPAR-αKO mice compared with WT controls, and that is increased in WT mice when combined with PPAR-α agonist treatment. In particular, DEX was less effective in PPAR-αKO, compared with WT mice, as evaluated by inhibition of NF-κB, of TNF-α production, of cell migration, of cycloxygenase-2 (COX-2) and inducible nitric-oxide synthase activation. Interestingly enough, macrophages from PPAR-αKO were less susceptible to DEX-induced COX-2 inhibition in vitro compared with WT mice. However, PPAR-α transfection in PPAR-αKO macrophages, with consequent receptor expression, resulted in reconstitution of susceptibility to DEX-induced COX-2 inhibition to levels comparable with that obtained in WT macrophages. It is noteworthy that the DEX effect on macrophages in vitro was significantly increased in WT cells when combined with PPAR-α agonist treatment. These results indicate that PPAR-α can contribute to the anti-inflammatory activity of GCs.

Silymarin suppress CD4+ T cell activation and proliferation: effects on NF-κB activity and IL-2 production.

Silymarin, a mixture of bioactive flavonolignans isolated from Silybum marianum, exhibits anti-carcinogenic, anti-inflammatory and cytoprotective effects. In this study, the in vitro immunomodulatory activity of silymarin was investigated using CD4+ splenocytes from C57/Bl6 mice. Proliferation assay revealed that silymarin, at 50 microM concentration, significantly inhibited CD4+ cells proliferation. ELISA analyses indicated that silymarin significantly inhibited IL-2 and IFN-gamma production. Immunofluorescence staining performed on the mouse hybridoma T cell line (3DO) revealed a block of nuclear translocation of transcription factor kappaB (NF-kappaB), which is known to be responsible for IL-2 transcriptional activation. Moreover, silymarin inhibited p65/NF-kappaB phosphorylation in CD4+ T cell. These results suggest that silymarin is able to inhibit T cell activation and proliferation, notably acting on pathways of NF-kappaB activation/translocation.

Donor CD19 CAR T cells exert potent graft-versus-lymphoma activity with diminished graft-versus-host activity

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT remain major impediments to the success of allo-HSCT. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells. CD19 is an attractive CAR target, which is expressed in most B cell malignancies, as well as in healthy B cells. Clinical trials using autologous CD19-targeted T cells have shown remarkable promise in various B cell malignancies. However, the use of allogeneic CAR T cells poses a concern in that it may increase risk of the occurrence of GVHD, although this has not been reported in selected patients infused with donor-derived CD19 CAR T cells after allo-HSCT. To understand the mechanism whereby allogeneic CD19 CAR T cells may mediate anti-lymphoma activity without causing a significant increase in the incidence of GVHD, we studied donor-derived CD19 CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19 CARs experience enhanced stimulation, resulting in the progressive loss of both their effector function and proliferative potential, clonal deletion, and significantly decreased occurrence of GVHD. Concurrently, the other CAR T cells that were present in bulk donor T cell populations retained their anti-lymphoma activity in accordance with the requirement that both the T cell receptor (TCR) and CAR be engaged to accelerate T cell exhaustion. In contrast, first-generation and 4-1BB-costimulated CAR T cells increased the occurrence of GVHD. These findings could explain the reduced risk of GVHD occurring with cumulative TCR and CAR signaling.

Long Glucocorticoid-induced Leucine Zipper (L-GILZ) Protein Interacts with Ras Protein Pathway and Contributes to Spermatogenesis Control

Background: Understanding how spermatogenesis occurs in mammals is not yet fully understood. Results: L-GILZ deficiency in germ cells leads to complete loss of germ cell lineage resulting in male sterility. Conclusion: Our study identifies L-GILZ as an important factor for spermatogenesis. Significance: Identification of genes critical for maintenance of spermatogenesis is pivotal for diagnosis and treatment of male infertility. Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.

Reduction of ischemic brain injury by administration of palmitoylethanolamide after transient middle cerebral artery occlusion in rats

Stroke is the third leading cause of death and the leading cause of long-term disability in adults. Current therapeutic strategies for stroke, including thrombolytic drugs, such as tissue plasminogen activator offer great promise for the treatment, but complimentary neuroprotective treatments are likely to provide a better outcome. To counteract the ischemic brain injury in mice, a new therapeutic approach has been employed by using palmitoylethanolamide (PEA). PEA is one of the members of N-acyl-ethanolamine family maintain not only redox balance but also inhibit the mechanisms of secondary injury on ischemic brain injury. Treatment of the middle cerebral artery occlusion (MCAo)-induced animals with PEA reduced edema and brain infractions as evidenced by decreased 2,3,5-triphenyltetrazolium chloride (TTC) staining across brain sections. PEA-mediated improvements in tissues histology shown by reduction of lesion size and improvement in apoptosis level (assayed by Bax and Bcl-2) further support the efficacy of PEA therapy. We demonstrated that PEA treatment blocked infiltration of astrocytes and restored MCAo-mediated reduced expression of PAR, nitrotyrosine, iNOS, chymase, tryptase, growth factors (BDNF and GDNF) and GFAP. PEA also inhibited the MCAo-mediated increased expression of pJNK, NF-κB, and degradation of IκB-α. PEA-treated injured animals improved neurobehavioral functions as evaluated by motor deficits. Based on these findings we propose that PEA would be useful in lowering the risk of damage or improving function in ischemia-reperfusion brain injury-related disorders.