Claire B. Larmonier

Doxorubicin eliminates myeloid-derived suppressor cells and enhances the efficacy of adoptive T-cell transfer in breast cancer.

Myeloid-derived suppressor cells (MDSC) expand in tumor-bearing hosts and play a central role in cancer immune evasion by inhibiting adaptive and innate immunity. They therefore represent a major obstacle for successful cancer immunotherapy. Different strategies have thus been explored to deplete and/or inactivate MDSC in vivo. Using a murine mammary cancer model, we demonstrated that doxorubicin selectively eliminates MDSC in the spleen, blood, and tumor beds. Furthermore, residual MDSC from doxorubicin-treated mice exhibited impaired suppressive function. Importantly, the frequency of CD4(+) and CD8(+) T lymphocytes and consequently the effector lymphocytes or natural killer (NK) to suppressive MDSC ratios were significantly increased following doxorubicin treatment of tumor-bearing mice. In addition, the proportion of NK and cytotoxic T cell (CTL) expressing perforin and granzyme B and of CTL producing IFN-γ was augmented by doxorubicin administration. Of therapeutic relevance, this drug efficiently combined with Th1 or Th17 lymphocytes to suppress tumor development and metastatic disease. MDSC isolated from patients with different types of cancer were also sensitive to doxorubicin-mediated cytotoxicity in vitro. These results thus indicate that doxorubicin may be used not only as a direct cytotoxic drug against tumor cells, but also as a potent immunomodulatory agent that selectively impairs MDSC-induced immunosuppression, thereby fostering the efficacy of T-cell-based immunotherapy.

Imatinib Mesylate Inhibits CD4+CD25+ Regulatory T Cell Activity and Enhances Active Immunotherapy against BCR-ABL− Tumors

Imatinib mesylate (Gleevec, STI571), a selective inhibitor of a restricted number of tyrosine kinases, has been effectively used for the treatment of Philadelphia chromosome-positive leukemias and gastrointestinal stromal tumors. Imatinib may also directly influence immune cells. Suppressive as well as stimulating effects of this drug on CD4+ and CD8+ T lymphocytes or dendritic cells have been reported. In the current study, we have investigated the influence of imatinib mesylate on CD4+CD25+FoxP3+ regulatory T cells (Treg), a critical population of lymphocytes that contributes to peripheral tolerance. Used at concentrations achieved clinically, imatinib impaired Treg immunosuppressive function and FoxP3 expression but not production of IL-10 and TGF-β in vitro. Imatinib significantly reduced the activation of the transcription factors STAT3 and STAT5 in Treg. Analysis of Treg TCR-induced signaling cascade indicated that imatinib inhibited phosphorylation of ZAP70 and LAT. Substantiating these observations, imatinib treatment of mice decreased Treg frequency and impaired their immunosuppressive function in vivo. Furthermore, imatinib mesylate significantly enhanced antitumor immune responses to dendritic cell-based immunization against an imatinib-resistant BCR-ABL negative lymphoma. The clinical applications of imatinib mesylate might thus be expanded with its use as a potent immunomodulatory agent targeting Treg in cancer immunotherapy.

Dendritic Cell-Specific Disruption of TGF-β Receptor II Leads to Altered Regulatory T Cell Phenotype and Spontaneous Multiorgan Autoimmunity

In vitro data and transgenic mouse models suggest a role for TGF-β signaling in dendritic cells (DCs) to prevent autoimmunity primarily through maintenance of DCs in their immature and tolerogenic state characterized by low expression of MHC class II (MHCII) and costimulatory molecules and increased expression of IDO, among others. To test whether a complete lack of TGF-β signaling in DCs predisposes mice to spontaneous autoimmunity and to verify the mechanisms implicated previously in vitro, we generated conditional knockout (KO) mice with Cre-mediated DC-specific deletion of Tgfbr2 (DC-Tgfbr2 KO). DC-Tgfbr2 KO mice die before 15 wk of age with multiorgan autoimmune inflammation and spontaneous activation of T and B cells. Interestingly, there were no significant differences in the expression of MHCII, costimulatory molecules, or IDO in secondary lymphoid organ DCs, although Tgfbr2-deficient DCs were more proinflammatory in vitro and in vivo. DC-Tgfbr2 KO showed attenuated Foxp3 expression in regulatory T cells (Tregs) and abnormal expansion of CD25−Foxp3+ Tregs in vivo. Tgfbr2-deficient DCs secreted elevated levels of IFN-γ and were not capable of directing Ag-specific Treg conversion unless in the presence of anti–IFN-γ blocking Ab. Adoptive transfer of induced Tregs into DC-Tgfbr2 KO mice partially rescued the phenotype. Therefore, in vivo, TGF-β signaling in DCs is critical in the control of autoimmunity through both Treg-dependent and -independent mechanisms, but it does not affect MHCII and costimulatory molecule expression.

Tumor Necrosis Factor and Interferon-γ Down-regulate Klotho in Mice With Colitis

BACKGROUND & AIMS Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. METHODS We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10(-/-)), and colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-gamma on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). RESULTS Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-gamma. The combination of TNF and IFN-gamma increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-gamma was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-gamma, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. CONCLUSIONS The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.

Modulation of neutrophil motility by curcumin: Implications for inflammatory bowel disease

Background: Neutrophils (PMN) are the first cells recruited at the site of inflammation. They play a key role in the innate immune response by recognizing, ingesting, and eliminating pathogens and participate in the orientation of the adaptive immune responses. However, in inflammatory bowel disease (IBD) transepithelial neutrophil migration leads to an impaired epithelial barrier function, perpetuation of inflammation, and tissue destruction via oxidative and proteolytic damage. Curcumin (diferulolylmethane) displays a protective role in mouse models of IBD and in human ulcerative colitis, a phenomenon consistently accompanied by a reduced mucosal neutrophil infiltration. Methods: We investigated the effect of curcumin on mouse and human neutrophil polarization and motility in vitro and in vivo. Results: Curcumin attenuated lipopolysaccharide (LPS)‐stimulated expression and secretion of macrophage inflammatory protein (MIP)‐2, interleukin (IL)‐1β, keratinocyte chemoattractant (KC), and MIP‐1α in colonic epithelial cells (CECs) and in macrophages. Curcumin significantly inhibited PMN chemotaxis against MIP‐2, KC, or against conditioned media from LPS‐treated macrophages or CEC, a well as the IL‐8‐mediated chemotaxis of human neutrophils. At nontoxic concentrations, curcumin inhibited random neutrophil migration, suggesting a direct effect on neutrophil chemokinesis. Curcumin‐mediated inhibition of PMN motility could be attributed to a downregulation of PI3K activity, AKT phosphorylation, and F‐actin polymerization at the leading edge. The inhibitory effect of curcumin on neutrophil motility was further demonstrated in vivo in a model of aseptic peritonitis. Conclusions: Our results indicate that curcumin interferes with colonic inflammation partly through inhibition of the chemokine expression and through direct inhibition of neutrophil chemotaxis and chemokinesis. Inflamm Bowel Dis 2011

Limited Effects of Dietary Curcumin on Th-1 Driven Colitis in IL-10 Deficient Mice Suggest an IL-10 Dependent Mechanism of Protection

Curcumin (diferulolylmethane) demonstrates profound anti-inflammatory effects in intestinal epithelial cells (IEC) and in immune cells in vitro and exhibits a protective role in rodent models of chemically induced colitis, with its presumed primary mechanism of action via inhibition of NF-kappaB. Although it has been demonstrated effective in reducing relapse rate in ulcerative colitis patients, curcumins effectiveness in Crohns disease (CD) or in Th-1/Th-17 mediated immune models of CD has not been evaluated. Therefore, we investigated the effects of dietary curcumin (0.1-1%) on the development of colitis, immune activation, and in vivo NF-kappaB activity in germ-free IL-10(-/-) or IL-10(-/-);NF-kappaB(EGFP) mice colonized with specific pathogen-free microflora. Proximal and distal colon morphology showed a mild protective effect of curcumin only at 0.1%. Colonic IFN-gamma and IL-12/23p40 mRNA expression followed similar pattern ( approximately 50% inhibition at 0.1%). Secretion of IL-12/23p40 and IFN-gamma by colonic explants and mesenteric lymph node cells was elevated in IL-10(-/-) mice and was not decreased by dietary curcumin. Surprisingly, activation of NF-kappaB in IL-10(-/-) mice (phospho-NF-kappaBp65) or in IL-10(-/-);NF-kappaB(EGFP) mice (whole organ or confocal imaging) was not noticeably inhibited by curcumin. Furthermore, we demonstrate that IL-10 and curcumin act synergistically to downregulate NF-kappaB activity in IEC and IL-12/23p40 production by splenocytes and dendritic cells. In conclusion, curcumin demonstrates limited effectiveness on Th-1 mediated colitis in IL-10(-/-) mice, with moderately improved colonic morphology, but with no significant effect on pathogenic T cell responses and in situ NF-kappaB activity. In vitro studies suggest that the protective effects of curcumin are IL-10 dependent.

Changes in mucosal homeostasis predispose NHE3 knockout mice to increased susceptibility to DSS-induced epithelial injury

BACKGROUND & AIMS NHE3 is a target of inhibition by proinflammatory cytokines and pathogenic bacteria, an event contributing to diarrhea in infectious and idiopathic colitis. In mice, NHE3 deficiency leads to mild diarrhea, increased intestinal expression of interferon (IFN)-gamma, and distal colitis, suggesting its role in epithelial barrier homeostasis. Our aim was to investigate the role of NHE3 in maintaining mucosal integrity. METHODS Control or dextran sulfate sodium (DSS)-treated, 6- to 8-week-old wild-type (WT) and NHE3(-/-) mice were used for the experiments. Small intestines were dissected for further analysis. RESULTS NHE3(-/-) mice have elevated numbers of CD8alpha(+) T and natural killer cells in the intraepithelial lymphocytes and lamina propria lymphocytes compartments, representing the source of IFN-gamma. NHE3(-/-) mice display alterations in epithelial gene and protein expression patterns that predispose them to a high susceptibility to DSS, with accelerated mortality resulting from intestinal bleeding, hypovolemic shock, and sepsis, even at a very low DSS concentration. Microarray analysis and intestinal hemorrhage indicate that NHE3 deficiency predisposes mice to DSS-induced small intestinal injury, a segment never reported as affected by DSS, and demonstrate major differences in the colonic response to DSS challenge in WT and NHE3(-/-) mice. In NHE3(-/-) mice, broad-spectrum oral antibiotics or anti-asialo GM1 antibodies reduce the expression of IFN-gamma and iNOS to basal levels and delay but do not prevent severe mortality in response to DSS treatment. CONCLUSIONS These results suggest that NHE3 participates in mucosal responses to epithelial damage, acting as a modifier gene determining the extent of the gut inflammatory responses in the face of intestinal injury.

Myeloid-derived suppressor cells from tumor-bearing mice impair TGF-β-induced differentiation of CD4+CD25+FoxP3+ Tregs from CD4+CD25−FoxP3− T cells

MDSCs and Tregs play an essential role in the immunosuppressive networks that contribute to tumor‐immune evasion. The mechanisms by which tumors promote the expansion and/or function of these suppressive cells and the cross‐talk between MDSC and Treg remain incompletely defined. Previous reports have suggested that MDSC may contribute to Treg induction in cancer. Herein, we provide evidence that tumor‐induced gr‐MDSCs, endowed with the potential of suppressing conventional T Lc, surprisingly impair TGF‐β1‐mediated generation of CD4+CD25+FoxP3+ iTregs. Furthermore, gr‐MDSCs impede the proliferation of nTregs without, however, affecting FoxP3 expression. Suppression of iTreg differentiation from naïve CD4+ cells by gr‐MDSC occurs early in the polarization process, requires inhibition of early T cell activation, and depends on ROS and IDO but does not require arginase 1, iNOS, NO, cystine/cysteine depletion, PD‐1 and PD‐L1 signaling, or COX‐2. These findings thus indicate that gr‐MDSCs from TB hosts have the unanticipated ability to restrict immunosuppressive Tregs.

Reduced colonic microbial diversity is associated with colitis in NHE3-deficient mice

Chronic inflammation and enteric infections are frequently associated with epithelial Na(+)/H(+) exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3(-/-) mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3(-/-) mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3(-/-) mice compared with WT. Rederivation of NHE3(-/-) mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3(-/-) mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3(-/-) mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.

Natural killer cells play a key role in the antitumor immunity generated by chaperone-rich cell lysate vaccination

Tumor derived chaperone‐rich cell lysate (CRCL) when isolated from tumor tissues is a potent vaccine that contains at least 4 of the highly immunogenic heat shock proteins (HSP) such as HSP70, HSP90, glucose related protein 94 and calreticulin. We have previously documented that CRCL provides both a source of tumor antigens and danger signals triggering dendritic cell (DC) activation. Immunization with tumor derived CRCL elicits tumor‐specific T cell responses leading to tumor regression. In the current study, we further dissect the mechanisms by which CRCL simulates the immune system, and demonstrate that natural killer (NK) cells are required for effective antitumor effects to take place. Our results illustrate that CRCL directly stimulates proinflammatory cytokine and chemokine production by NK cells, which may lead to activation and recruitment of macrophages at the tumor site. Thus, this report provides further insight into the function of CRCL as an immunostimulant against cancer.