Odette M. Smith

Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation

GVHD is associated with significant shifts in the composition of the intestinal microbiota in human and mouse models; manipulating the microbiota can alter the severity of GVHD in mice.

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.

IL-17 contributes to CD4-mediated graft-versus-host disease

CD4(+) interleukin-17 (IL-17)(+) T cells (Th17 cells) have been implicated in allograft rejection of solid organs and several autoimmune diseases. However, the functional role of Th17 cells in the development of acute graft-versus-host disease (GVHD) has not been well-characterized. We detected significant numbers of alloreactive CD4(+) donor T cells expressing IL-17, IL-17F, or IL-22 in the lymphoid organs of recipients of an allogeneic bone marrow transplant. We found no differences in GVHD mortality or graft-versus-tumor (GVT) activity between wild type (WT) and IL-17(-/-) T-cell recipients. However, upon transfer of murine IL-17(-/-) CD4(+) T cells in an allogeneic BMT model, GVHD development was significantly delayed behind recipients of WT CD4(+) T cells, yet overall GVHD mortality was unaffected. Moreover, recipients of IL-17(-/-) CD4(+) T cells had significantly fewer Th1 cells during the early stages of GVHD. Furthermore, we observed a decrease in the number of IFN-gamma-secreting macrophages and granulocytes and decreased production of proinflammatory cytokines (interferon [IFN]-gamma, IL-4, and IL-6) in recipients of IL-17(-/-) CD4(+) T cells. We conclude that IL-17 is dispensable for GVHD and GVT activity by whole T cells, but contributes to the early development of CD4-mediated GVHD by promoting production of proinflammatory cytokines.

Interleukin-22 Drives Endogenous Thymic Regeneration in Mice

IL-22 Protects the Thymus One of the side effects associated with radiation treatment and some types of chemotherapy is damage to the thymus. Immunological T cells develop in the thymus, and so damage to this organ results in immunodeficiency and increased susceptibility to infectious disease. Although the organ eventually recovers, therapies that speed this recovery process are of interest. Dudakov et al. (p. 91, published online 1 March; see the Perspective by Bhandoola and Artis) now show in mice that interleukin-22 (IL-22) production in the thymus is increased in response to radiation damage and that this cytokine promotes thymic repair. After radiation treatment, IL-23 production by thymic dendritic cells induced IL-22 secretion by a population of radio-resistant innate lymphoid cells. IL-22 appeared to mediate its effects by promoting the survival and proliferation of thymic epithelial cells. Damage to the thymus caused by infection or radiation is reversed by a cytokine. Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence after stress, infection, or immunodepletion. However, the mechanisms governing this regeneration remain poorly understood. We detail such a mechanism, centered on interleukin-22 (IL-22) and triggered by the depletion of CD4+CD8+ double-positive thymocytes. Intrathymic levels of IL-22 were increased after thymic insult, and thymic recovery was impaired in IL-22–deficient mice. IL-22, which signaled through thymic epithelial cells and promoted their proliferation and survival, was up-regulated by radio-resistant RORγ(t)+CCR6+NKp46– lymphoid tissue inducer cells after thymic injury in an IL-23–dependent manner. Administration of IL-22 enhanced thymic recovery after total body irradiation. These studies reveal mechanisms of endogenous thymic repair and offer innovative regenerative strategies for improving immune competence.

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation.

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell–depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1–derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1–derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1–derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1–derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.

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.

NOD2 regulates hematopoietic cell function during graft-versus-host disease

Nucleotide-binding oligomerization domain 2 (NOD2) polymorphisms are independent risk factors for Crohns disease and graft-versus-host disease (GVHD). In Crohns disease, the proinflammatory state resulting from NOD2 mutations have been associated with a loss of antibacterial function of enterocytes such as paneth cells. NOD2 has not been studied in experimental allogeneic bone marrow transplantation (allo-BMT). Using chimeric recipients with NOD2−/− hematopoietic cells, we demonstrate that NOD2 deficiency in host hematopoietic cells exacerbates GVHD. We found that proliferation and activation of donor T cells was enhanced in NOD-deficient allo-BMT recipients, suggesting that NOD2 plays a role in the regulation of host antigen-presenting cells (APCs). Next, we used bone marrow chimeras in an experimental colitis model and observed again that NOD2 deficiency in the hematopoietic cells results in increased intestinal inflammation. We conclude that NOD2 regulates the development of GVHD through its inhibitory effect on host APC function.

Abrogation of donor T-cell IL-21 signaling leads to tissue-specific modulation of immunity and separation of GVHD from GVL

IL-21 is a proinflammatory cytokine produced by Th17 cells. Abrogation of IL-21 signaling has recently been shown to reduce GVHD while retaining graft-versus-leukemia/lymphoma (GVL) responses. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL remain incompletely understood. In a murine MHC-mismatched BM transplantation model, we observed that IL-21 receptor knockout (IL-21R KO) donor T cells mediate decreased systemic and gastrointestinal GVHD in recipients of a transplant. This reduction in GVHD was associated with expansion of transplanted donor regulatory T cells and with tissue-specific modulation of Th-cell function. IL-21R KO and wild-type donor T cells showed equivalent alloactivation, but IL-21R KO T cells showed decreased infiltration and inflammatory cytokine production within the mesenteric lymph nodes. However, Th-cell cytokine production was maintained peripherally, and IL-21R KO T cells mediated equivalent immunity against A20 and P815 hematopoietic tumors. In summary, abrogation of IL-21 signaling in donor T cells leads to tissue-specific modulation of immunity, such that gastrointestinal GVHD is reduced, but peripheral T-cell function and GVL capacity are retained. IL-21 is thus an exciting target for therapeutic intervention and improvement of clinical transplantation outcomes.