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Macrolide antibiotic azithromycin is widely used in clinical practice to treat respiratory tract infections and inflammatory diseases. However, its mechanism of action is not fully understood. Given the involvement of the CD27 pathway in the pathophysiology of various T-lymphocyte-mediated inflammatory, autoimmune, and lymphoproliferative diseases, we examined the impact of AZM on CD27 regulation and potential consequences on CD4+ and CD8+ T-cell phenotypes. Using cellular immunology approaches on healthy donors' peripheral blood mononuclear cells, we demonstrate AZM-mediated downregulation of surface CD27 expression as well as its extracellular release as soluble CD27. Notably, AZM-exposed CD27high (hi) cells were defective in their ability to expand compared to CD27intermediate (Int) and CD27low (lo) subsets. The defective CD27hi subset expansion was found to be associated with impaired cell proliferation and cell division. At the molecular level, the CD27hi subset exhibited lower mTOR activity than other subsets. Functionally, AZM treatment resulted in marked depletion of helper CD4+ (Th1) and cytotoxic CD8+ T-lymphocyte (Tc1)-associated CXCR3+CD27hi effector cells and inhibition of inflammatory cytokine IFN-γ production. These findings provide mechanistic insights on immunomodulatory features of AZM on T-lymphocyte by altering the CD27 pathway. From a clinical perspective, this study also sheds light on potential clinical benefits observed in patients on prophylactic AZM regimens against various respiratory diseases and opens avenues for future adjunct therapy against Th1- and Tc1-dominated inflammatory and autoimmune diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ansari, Jayakumar, Ahmad, Venkatachalam, Salameh, Unnikannan, Raheed, Mohammed, Mahboub, Al-Ramadi, Hamid, Steinhoff and Hamoudi.)

Manuka honey (MH) has garnered much attention due to its remarkable antimicrobial, anticancer, immunomodulatory and wound-healing properties. This study compared the antiproliferative effects of raw and powdered MH (pMH) on various human and murine cancer cell lines. A detailed metabolomics analysis was also carried out using untargeted ultrahigh-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS) to compare the constituents in raw MH and pMH. The results of the viability studies showed that both raw MH and pMH caused a dose-dependent inhibition of tumor cell growth at concentrations of > 1% w/v (equivalent to ~ 10 mg/ml). A differential susceptibility to MH was observed among the cell lines with the human MDA-MB-231 and A549 cells and murine B16.F10 cells being relatively resistant to MH while the murine MC38 colorectal adeno-carcinoma cells showing the most sensitivity. The effect of raw MH and pMH on cell viability was validated using 2 indepndent assays. Metabolomics analysis detected 2440 compounds, out of which 833 were successfully identified. Among these, 90 phytochemical compounds, predominantly comprising terpenoids, flavonoids, coumarins and derivatives, and phenylpropanoic acids, and 79 lipids were identifiable. Significant differences in 5 metabolite classes, including flavonoids, phenols, terpenoids, carbohydrates, and organic acids were observed between the raw and pMH. Moreover, several altered metabolic pathways were identified in pMH compared to raw MH, such as energy metabolism, amino acid metabolism, and various other pathways that collectively influence biological functions associated with cellular growth, signaling, and stress response., (© 2024. The Author(s).)

The gastrointestinal tract (GI) is the largest immune organ whose function is controlled by a complex network of neurons from the enteric nervous system (ENS) as well as the sympathetic and parasympathetic system. Evolving evidence indicates that cross-communication between gut-innervating neurons and immune cells regulates many essential physiological functions including protection against mucosal infections. We previously demonstrated that following paraoxon treatment, 70 % of the mice were able to survive an oral infection with S. typhimurium , a virulent strain of Salmonella enterica serovar Typhimurium. The present study aims to investigate the effect that rivastigmine, a reversible AChE inhibitor used for the treatment of neurodegenerative diseases, has on the murine immune defenses of the intestinal mucosa. Our findings show that, similar to what is observed with paraoxon, administration of rivastigmine promoted the release of secretory granules from goblet and Paneth cells, resulting in increased mucin layer. Surprisingly, however, and unlike paraoxon, rivastigmine treatment did not affect overall mortality of infected mice. In order to investigate the mechanistic basis for the differential effects observed between paraoxon and rivastigmine, we used multi-color flowcytometric analysis to characterize the immune cell landscape in the intraepithelial (IE) and lamina propria (LP) compartments of intestinal mucosa. Our data indicate that treatment with paraoxon, but not rivastigmine, led to an increase of resident CD3 + CD8 + T lymphocytes in the ileal mucosa (epithelium and lamina propria) and CD11b - CD11c + dendritic cells in the LP. Our findings indicate the requirement for persistent cholinergic pathway engagement to effect a change in the cellular landscape of the mucosal tissue that is necessary for protection against lethal bacterial infections. Moreover, optimal protection requires a collaboration between innate and adaptive mucosal immune responses in the intestine., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

Background: To investigate the potential of Manuka honey (MH) as an immunomodulatory agent in colorectal cancer (CRC) and dissect the underlying molecular and cellular mechanisms., Methods: MH was administered orally over a 4 week-period. The effect of MH treatment on microbiota composition was studied using 16S rRNA sequencing of fecal pellets collected before and after treatment. Pretreated mice were implanted with CRC cells and followed for tumor growth. Tumors and lymphoid organs were analyzed by flow cytometry (FACS), immunohistochemistry and qRT-PCR. Efficacy of MH was also assessed in a therapeutic setting, with oral treatment initiated after tumor implantation. We utilized IFNγ-deficient mice to determine the importance of interferon signaling in MH-induced immunomodulation., Results: Pretreatment with MH enhanced anti-tumor responses leading to suppression of tumor growth. Evidence for enhanced tumor immunogenicity included upregulated MHC class-II on intratumoral macrophages, enhanced MHC class-I expression on tumor cells and increased infiltration of effector T cells into the tumor microenvironment. Importantly, oral MH was also effective in retarding tumor growth when given therapeutically. Transcriptomic analysis of tumor tissue highlighted changes in the expression of various chemokines and inflammatory cytokines that drive the observed changes in tumor immunogenicity. The immunomodulatory capacity of MH was abrogated in IFNγ-deficient mice. Finally, bacterial 16S rRNA sequencing demonstrated that oral MH treatment induced unique changes in gut microbiota that may well underlie the IFN-dependent enhancement in tumor immunogenicity., Conclusion: Our findings highlight the immunostimulatory properties of MH and demonstrate its potential utilization in cancer prevention and treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Masad, Idriss, Mohamed, Al-Sbiei, Bashir, Al-Marzooq, Altahrawi, Fernandez-Cabezudo and Al-Ramadi.)

The gastrointestinal (GI) tract of multicellular organisms, especially mammals, harbors a symbiotic commensal microbiota with diverse microorganisms including bacteria, fungi, viruses, and other microbial and eukaryotic species. This microbiota exerts an important role on intestinal function and contributes to host health. The microbiota, while benefiting from a nourishing environment, is involved in the development, metabolism and immunity of the host, contributing to the maintenance of homeostasis in the GI tract. The immune system orchestrates the maintenance of key features of host-microbe symbiosis via a unique immunological network that populates the intestinal wall with different immune cell populations. Intestinal epithelium contains lymphocytes in the intraepithelial (IEL) space between the tight junctions and the basal membrane of the gut epithelium. IELs are mostly CD8 + T cells, with the great majority of them expressing the CD8αα homodimer, and the γδ T cell receptor (TCR) instead of the αβ TCR expressed on conventional T cells. γδ T cells play a significant role in immune surveillance and tissue maintenance. This review provides an overview of how the microbiota regulates γδ T cells and the influence of microbiota-derived metabolites on γδ T cell responses, highlighting their impact on immune homeostasis. It also discusses intestinal neuro-immune regulation and how γδ T cells possess the ability to interact with both the microbiota and brain.

Inflammation of the GI tract leads to compromised epithelial barrier integrity, which increases intestine permeability. A compromised intestinal barrier is a critical event that leads to microbe entry and promotes inflammatory responses. Inflammatory bowel diseases that comprise Crohn's disease (CD) and ulcerative colitis (UC) show an increase in intestinal permeability. Nerolidol (NED), a naturally occurring sesquiterpene alcohol, has potent anti-inflammatory properties in preclinical models of colon inflammation. In this study, we investigated the effect of NED on MAPKs, NF-κB signaling pathways, and intestine epithelial tight junction physiology using in vivo and in vitro models. The effect of NED on proinflammatory cytokine release and MAPK and NF-κB signaling pathways were evaluated using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. Subsequently, the role of NED on MAPKs, NF-κB signaling, and the intestine tight junction integrity were assessed using DSS-induced colitis and LPS-stimulated Caco-2 cell culture models. Our result indicates that NED pre-treatment significantly inhibited proinflammatory cytokine release, expression of proteins involved in MAP kinase, and NF-κB signaling pathways in LPS-stimulated RAW macrophages and DSS-induced colitis. Furthermore, NED treatment significantly decreased FITC-dextran permeability in DSS-induced colitis. NED treatment enhanced tight junction protein expression (claudin-1, 3, 7, and occludin). Time-dependent increases in transepithelial electrical resistance (TEER) measurements reflect the formation of healthy tight junctions in the Caco-2 monolayer. LPS-stimulated Caco-2 showed a significant decrease in TEER. However, NED pre-treatment significantly prevented the fall in TEER measurements, indicating its protective role. In conclusion, NED significantly decreased MAPK and NF-κB signaling pathways and decreased tight junction permeability by enhancing epithelial tight junction protein expression.

The use of immune checkpoint inhibitors to treat cancer resulted in unprecedented and durable clinical benefits. However, the response rate among patients remains rather modest. Previous work from our laboratory demonstrated the efficacy of using attenuated bacteria as immunomodulatory anti-cancer agents. The current study investigated the potential of utilizing a low dose of attenuated Salmonella typhimurium to enhance the efficacy of PD-L1 blockade in a relatively immunogenic model of colon cancer. The response of MC38 tumors to treatment with αPD-L1 monoclonal antibody (mAb) was variable, with only 30% of the mice being responsive. Combined treatment with αPD-L1 mAb and Salmonella resulted in 75% inhibition of tumor growth in 100% of animals. Mechanistically, the enhanced response correlated with a decrease in the percentage of tumor-associated granulocytic cells, upregulation in MHC class II expression by intratumoral monocytes and an increase in tumor infiltration by effector T cells. Collectively, these alterations resulted in improved anti-tumor effector responses and increased apoptosis within the tumor. Thus, our study demonstrates that a novel combination treatment utilizing attenuated Salmonella and αPD-L1 mAb could improve the outcome of immunotherapy in colorectal cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Al-Saafeen, Al-Sbiei, Bashir, Mohamed, Masad, Fernandez-Cabezudo and al-Ramadi.)

Manuka honey (MH) is known for its wound-healing, anti-microbial, anti-oxidant and anti-tumor properties. However, there is conflicting evidence regarding the role of MH in inflammatory responses, with some studies highlighting its pro-inflammatory capacity and others showing that it has a predominantly anti-inflammatory activity. The current study is aimed at characterizing the immunomodulatory capacity of MH using both in vitro and in vivo approaches, focusing on the underlying mechanisms. Treatment of RAW 264.7 macrophages with 1% MH (w/v) resulted in a significant increase in the gene expression (~26-fold) and secretion (~27-fold) of tumor necrosis factor-alpha (TNF-α). Similarly, an increase was observed in the gene expression of other inflammatory cytokines including interleukin-1β ( IL-1β) , interleukin-6 ( IL-6 ), and inducible nitric oxide synthase (i NOS ), as well as the chemokines; (C-X-C motif) ligand 2 ( CXCL2 ) and (C-C) motif ligand 2 ( CCL2 ). Using an in vivo model, intraperitoneal (i.p.) administration of MH in C57BL/6 mice elicited a peritoneal response characterized by a significant expansion in the number of peritoneal exudate cells (PECs), which was mainly due to a 35-fold increase in the recruitment of neutrophils. Importantly, this response was evident in toll-like receptor 4 (TLR4)-defective C3H/HeJ mice, indicating that the observed stimulatory effect occurs independently of TLR4 and unlikely to be mediated by any lipopolysaccharide (LPS) contaminant. MH administration also led to changes in the phenotypic expression and functional maturation of peritoneal macrophages, as evidenced by a shift towards the CD11b lo F4/80 lo phenotype and an increase in the expression of major histocompatibility complex (MHC) class II proteins. In contrast, the MH-initiated peritoneal response was largely abrogated in mice deficient in myeloid differentiation primary response 88 (MyD88) protein, a critical adaptor of most TLR signaling pathways. Thus, the current findings help to characterize the immunostimulatory properties of MH and their dependence on TLR signaling, and highlight the potential utility of MH as an immunomodulatory agent in a variety of disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Masad, Nasser, Bashir, Mohamed, Al-Sbiei, Al-Saafeen, Fernandez-Cabezudo and Al-Ramadi.)

Neutrophils are produced in the BM in a process called granulopoiesis, in which progenitor cells sequentially develop into mature neutrophils. During the developmental process, which is finely regulated by distinct transcription factors, neutrophils acquire the ability to exit the BM, properly distribute throughout the body, and migrate to infection sites. Previous studies have demonstrated that CD40 ligand (CD40L) influences hematopoiesis and granulopoiesis. Here, we investigate the effect of CD40L on neutrophil development and trafficking by performing functional and transcriptome analyses. We found that CD40L signaling plays an essential role in the early stages of neutrophil generation and development in the BM. Moreover, CD40L modulates transcriptional signatures, indicating that this molecule enables neutrophils to traffic throughout the body and to migrate in response to inflammatory signals. Thus, our study provides insights into the complex relationships between CD40L signaling and granulopoiesis, and it suggests a potentially novel and nonredundant role of CD40L signaling in neutrophil development and function.

Current modalities of cancer treatment have limitations related to poor target selectivity, resistance to treatment, and low response rates in patients. Accumulating evidence over the past few decades has demonstrated the capacity of several strains of bacteria to exert anti-tumor activities. Salmonella is the most extensively studied entity in bacterial-mediated cancer therapy, and has a good potential to induce direct tumor cell killing and manipulate the immune components of the tumor microenvironment in favor of tumor inhibition. In addition, Salmonella possesses some advantages over other approaches of cancer therapy, including high tumor specificity, deep tissue penetration, and engineering plasticity. These aspects underscore the potential of utilizing Salmonella in combination with other cancer therapeutics to improve treatment effectiveness. Herein, we describe the advantages that make Salmonella a good candidate for combination cancer therapy and summarize the findings of representative studies that aimed to investigate the therapeutic outcome of combination therapies involving Salmonella . We also highlight issues associated with their application in clinical use.

Cell death is a vital event in life. Infections and injuries cause lytic cell death, which gives rise to danger signals that can further induce cell death, inflammation, and tissue damage. The mevalonate (MVA) pathway is an essential, highly conserved and dynamic metabolic pathway. Here, we discover that farnesyl pyrophosphate (FPP), a metabolic intermediate of the MVA pathway, functions as a newly identified danger signal to trigger acute cell death leading to neuron loss in stroke. Harboring both a hydrophobic 15-carbon isoprenyl chain and a heavily charged pyrophosphate head, FPP leads to acute cell death independent of its downstream metabolic pathways. Mechanistically, extracellular calcium influx and the cation channel transient receptor potential melastatin 2 (TRPM2) exhibit essential roles in FPP-induced cell death. FPP activates TRPM2 opening for ion influx. Furthermore, in terms of a mouse model constructing by middle cerebral artery occlusion (MCAO), FPP accumulates in the brain, which indicates the function of the FPP and TRPM2 danger signal axis in ischemic injury. Overall, our data have revealed a novel function of the MVA pathway intermediate metabolite FPP as a danger signal via transient receptor potential cation channels., Competing Interests: The authors have declared that no competing interests exist.

Honey has exerted a high impact in the field of alternative medicine over many centuries. In addition to its wound healing, anti-microbial and antioxidant properties, several lines of evidence have highlighted the efficiency of honey and associated bioactive constituents as anti-tumor agents against a range of cancer types. Mechanistically, honey was shown to inhibit cancer cell growth through its pro-apoptotic, anti-proliferative and anti-metastatic effects. However, the potential of honey to regulate anti-tumor immune responses is relatively unexplored. A small number of in vitro and in vivo studies have demonstrated the ability of honey to modulate the immune system by inducing immunostimulatory as well as anti-inflammatory effects. In the present review, we summarize the findings from different studies that aimed to investigate the immunomodulatory properties of honey and its flavonoid components in relation to cancer. While these studies provide promising data, additional research is needed to further elucidate the immunomodulatory properties of honey, and to enable its utilization as an adjuvant therapy in cancer.

Several studies reported a central role of the endothelin type A receptor (ETAR) in tumor progression leading to the formation of metastasis. Here, we investigated the in vitro and in vivo anti-tumor effects of the FDA-approved ETAR antagonist, Ambrisentan, which is currently used to treat patients with pulmonary arterial hypertension. In vitro, Ambrisentan inhibited both spontaneous and induced migration/invasion capacity of different tumor cells (COLO-357 metastatic pancreatic adenocarcinoma, OvCar3 ovarian carcinoma, MDA-MB-231 breast adenocarcinoma, and HL-60 promyelocytic leukemia). Whole transcriptome analysis using RNAseq indicated Ambrisentan's inhibitory effects on the whole transcriptome of resting and PAR2-activated COLO-357 cells, which tended to normalize to an unstimulated profile. Finally, in a pre-clinical murine model of metastatic breast cancer, treatment with Ambrisentan was effective in decreasing metastasis into the lungs and liver. Importantly, this was associated with a significant enhancement in animal survival. Taken together, our work suggests a new therapeutic application for Ambrisentan in the treatment of cancer metastasis.

Despite the much improved therapeutic approaches for cancer treatment that have been developed over the past 50 years, cancer remains a major cause of mortality globally. Considerable epidemiological and experimental evidence has demonstrated an association between ingestion of food and nutrients with either an increased risk for cancer or its prevention. There is rising interest in exploring agents derived from natural products for chemoprevention or for therapeutic purposes. Honey is rich in nutritional and non-nutritional bioactive compounds, as well as in natural antioxidants, and its potential beneficial function in human health is becoming more evident. A large number of studies have addressed the anti-cancer effects of different types of honey and their phenolic compounds using in vitro and in vivo cancer models. The reported findings affirm that honey is an agent able to modulate oxidative stress and has anti-proliferative, pro-apoptotic, anti-inflammatory, immune-modulatory and anti-metastatic properties. However, despite its reported anti-cancer activities, very few clinical studies have been undertaken. In the present review, we summarise the findings from different experimental approaches, including in vitro cell cultures, preclinical animal models and clinical studies, and provide an overview of the bioactive profile and bioavailability of the most commonly studied honey types, with special emphasis on the chemopreventive and therapeutic properties of honey and its major phenolic compounds in cancer. The implications of these findings as well as the future prospects of utilising honey to fight cancer will be discussed.

Almost 70 years after establishing the concept of primary immunodeficiency disorders (PIDs), more than 320 monogenic inborn errors of immunity have been identified thanks to the remarkable contribution of high-throughput genetic screening in the last decade. Approximately 40 of these PIDs present with autoimmune or auto-inflammatory symptoms as the primary clinical manifestation instead of infections. These PIDs are now recognized as diseases of immune dysregulation. Loss-of function mutations in genes such as FOXP3, CD25, LRBA, IL-10, IL10RA, and IL10RB , as well as heterozygous gain-of-function mutations in JAK1 and STAT3 have been reported as causative of these disorders. Identifying these syndromes has considerably contributed to expanding our knowledge on the mechanisms of immune regulation and tolerance. Although whole exome and whole genome sequencing have been extremely useful in identifying novel causative genes underlying new phenotypes, these approaches are time-consuming and expensive. Patients with monogenic syndromes associated with autoimmunity require faster diagnostic tools to delineate therapeutic strategies and avoid organ damage. Since these PIDs present with severe life-threatening phenotypes, the need for a precise diagnosis in order to initiate appropriate patient management is necessary. More traditional approaches such as flow cytometry are therefore a valid option. Here, we review the application of flow cytometry and discuss the relevance of this powerful technique in diagnosing patients with PIDs presenting with immune dysregulation. In addition, flow cytometry represents a fast, robust, and sensitive approach that efficiently uncovers new immunopathological mechanisms underlying monogenic PIDs., (Copyright © 2019 Cabral-Marques, Schimke, de Oliveira, El Khawanky, Ramos, Al-Ramadi, Segundo, Ochs and Condino-Neto.)

Aberrantly high levels of tyrosine-phosphorylated signal transducer and activator of transcription 3 (p-STAT3) are found constitutively in ~50% of human lung and breast cancers, acting as an oncogenic transcription factor. We previously demonstrated that Manuka honey (MH) inhibits p-STAT3 in breast cancer cells, but the exact mechanism remained unknown. Herein, we show that MH-mediated inhibition of p-STAT3 in breast (MDA-MB-231) and lung (A549) cancer cell lines is accompanied by decreased levels of gp130 and p-JAK2, two upstream components of the IL-6 receptor (IL-6R) signaling pathway. Using an ELISA-based assay, we demonstrate that MH binds directly to IL-6Rα, significantly inhibiting (~60%) its binding to the IL-6 ligand. Importantly, no evidence of MH binding to two other cytokine receptors, IL-11Rα and IL-8R, was found. Moreover, MH did not alter the levels of tyrosine-phosphorylated or total Src family kinases, which are also constitutively activated in cancer cells, suggesting that signaling via other growth factor receptors is unaffected by MH. Binding of five major MH flavonoids (luteolin, quercetin, galangin, pinocembrin, and chrysin) was also tested, and all but pinocembrin could demonstrably bind IL-6Rα, partially (30-35%) blocking IL-6 binding at the highest concentration (50 μM) used. In agreement, each flavonoid inhibited p-STAT3 in a dose-dependent manner, with estimated IC 50 values in the 3.5-70 μM range. Finally, docking analysis confirmed the capacity of each flavonoid to bind in an energetically favorable configuration to IL-6Rα at a site predicted to interfere with ligand binding. Taken together, our findings identify IL-6Rα as a direct target of MH and its flavonoids, highlighting IL-6R blockade as a mechanism for the anti-tumor activity of MH, as well as a viable therapeutic target in IL-6-dependent cancers.

Type I diabetes (T1D) is a T cell-driven autoimmune disease that results in the killing of pancreatic β-cells and, consequently, loss of insulin production. Using the multiple low-dose streptozotocin (MLD-STZ) model of experimental autoimmune diabetes, we previously reported that pretreatment with a specific acetylcholinesterase inhibitor (AChEI), paraoxon, prevented the development of hyperglycemia in C57BL/6 mice. This correlated with an inhibition of T cell infiltration into the pancreatic islets and a reduction in pro-inflammatory cytokines. The cholinergic anti-inflammatory pathway utilizes nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs, respectively) expressed on a variety of cell types. In this study, we carried out a comparative analysis of the effect of specific antagonists of nAChRs or mAChRs on the development of autoimmune diabetes. Co-administration of mecamylamine, a non-selective antagonist of nAChRs maintained the protective effect of AChEI on the development of hyperglycemia. In contrast, co-administration of atropine, a non-selective antagonist of mAChRs, mitigated AChEI-mediated protection. Mice pretreated with mecamylamine had an improved response in glucose tolerance test (GTT) than mice pretreated with atropine. These differential effects of nAChR and mAChR antagonists correlated with the extent of islet cell infiltration and with the structure and functionality of the β-cells. Taken together, our data suggest that mAChRs are essential for the protective effect of cholinergic stimulation in autoimmune diabetes.

Introduction: CD40 ligand (CD40L) deficiency or X-linked Hyper-IgM syndrome is a severe primary immunodeficiency caused by mutations in the CD40L gene. Despite currently available treatments, CD40L-deficient patients remain susceptible to life-threatening infections and have poor long term survival. Areas covered: Here, we discuss clinical and immunological characteristics of CD40L deficiency as well as current therapeutic strategies used for patient management. This review highlights that beyond B cell defects, patients' susceptibility to opportunistic pathogens might be due to impaired T cell and innate immune responses. In this context, we discuss how better knowledge of CD40L function and regulation may result in the development of new treatments. Expert opinion: Despite the introduction of hematopoietic stem-cell transplantation, immunoglobulin replacement, granulocyte colony-stimulating factor (G-CSF) administration, and prophylactic antibiotic therapies, life-threatening infections still cause high morbidity and mortality among CD40L-deficient patients. The reasons for this inadequate response to current therapies remains poorly understood, but recent reports suggest the involvement of CD40L-CD40 interaction in early stages of the innate immune system ontogeny. The development of novel gene therapeutic approaches and the use of redirected immunotherapies represent alternative treatment methods that could offer reduced morbidity and mortality rates for patients with CD40L deficiency.

Autoantibodies have been associated with autoimmune diseases. However, studies have identified autoantibodies in healthy donors (HD) who do not develop autoimmune disorders. Here we provide evidence of a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCR) in HD compared to patients with systemic sclerosis, Alzheimer's disease, and ovarian cancer. Sex, age and pathological conditions affect autoantibody correlation and hierarchical clustering signatures, yet many of the correlations are shared across all groups, indicating alterations to homeostasis. Furthermore, we identify relationships between autoantibodies targeting structurally and functionally related molecules, such as vascular, neuronal or chemokine receptors. Finally, autoantibodies targeting the endothelin receptor type A (EDNRA) exhibit chemotactic activity, as demonstrated by neutrophil migration toward HD-IgG in an EDNRA-dependent manner and in the direction of IgG from EDNRA-immunized mice. Our data characterizing the in vivo signatures of anti-GPCR autoantibodies thus suggest that they are a physiological part of the immune system.

Background: Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches., Objectives: We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the in vitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function., Methods: We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function., Results: Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved in vitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients., Conclusion: Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved in vitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Growing evidence indicates a link between persistent infections and the development of autoimmune diseases. For instance, the inability to control Salmonella infection due to defective toll-like receptor (TLR)/myeloid differentiation primary response 88 (MyD88) signaling has linked the development of persistent infections to a breakdown in B cell tolerance. However, the extent of immune dysregulation in the absence of TLR-MyD88 signaling remains poorly characterized. Here, we show that MyD88 -/- mice are unable to eliminate attenuated Salmonella enterica serovar Typhimurium, even when challenged with a low-dose inoculum (200 CFUs/mouse), developing a persistent and progressive infection when compared to wild-type (MyD88 +/+ ) animals. The splenic niche of MyD88 -/- mice revealed increased counts of activated, Sca-1-positive, myeloid subpopulations highly expressing BAFF during persistent Salmonella infection. Likewise, the T cell compartment of Salmonella -infected MyD88 -/- mice showed increased levels of CD4 + and CD8 + T cells expressing Sca-1 and CD25 and producing elevated amounts of IL-4, IL-10, and IL-21 in response to CD3/CD28 stimulation. This was associated with increased Tfh cell differentiation and the presence of CD4 + T cells co-expressing IFN-γ/IL-4 and IFN-γ/IL-10. Noteworthy, infected MyD88 -/- mice had enhanced serum titers of both anti- Salmonella antibodies as well as autoantibodies directed against double-stranded DNA, thyroglobulin, and IgG rheumatoid factor, positive nuclear staining with HEp-2 cells, and immune complex deposition in the kidneys of MyD88 -/- mice infected with live but not heat-killed Salmonella . Infection with other microorganisms ( Acinetobacter baumanii, Streptococcus agalactiae , or Escherichia coli ) was unable to trigger the autoimmune phenomenon. Our findings suggest that dysregulation of the immune response in the absence of MyD88 is pathogen-dependent and highlight potentially important genotype-environmental factor correlations.

The use of attenuated bacteria as cancer therapeutic tools has garnered increasing scientific interest over the past 10 years. This is largely due to the development of bacterial strains that maintain good anti-tumor efficacy, but with reduced potential to cause toxicities to the host. Because of its ability to replicate in viable as well as necrotic tissue, cancer therapy using attenuated strains of facultative anaerobic bacteria, such as Salmonella , has several advantages over standard treatment modalities, including chemotherapy and radiotherapy. Despite some findings suggesting that it may operate through a direct cytotoxic effect against cancer cells, there is accumulating evidence demonstrating that bacterial therapy acts by modulating cells of the immune system to counter the growth of the tumor. Herein, we review the experimental evidence underlying the success of bacterial immunotherapy against cancer and highlight the cellular and molecular alterations in the peripheral immune system and within the tumor microenvironment that have been reported following different forms of bacterial therapy. Our improved understanding of these mechanisms should greatly aid in the translational application of bacterial therapy to cancer patients.

Background: Combined immunodeficiencies (CIDs) are diseases of defective adaptive immunity with diverse clinical phenotypes. Although CIDs are more prevalent in the Middle East than Western countries, the resources for genetic diagnosis are limited., Objectives: This study aims to characterize the categories of patients with CIDs in Iran clinically and genetically., Methods: Clinical and laboratory data were obtained from 696 patients with CIDs. Patients were subdivided into those with syndromic (344 patients) and nonsyndromic (352 patients) CIDs. Targeted DNA sequencing was performed on 243 (34.9%) patients., Results: The overall diagnostic yield of the 243 sequenced patients was 77.8% (189 patients). The clinical diagnosis of hyper-IgE syndrome (P < .001), onset of disease at greater than 5 years (P = .02), and absence of multiple affected family members (P = .04) were significantly more frequent in the patients without a genetic diagnosis. An autosomal recessive disease was found in 62.9% of patients, reflecting the high rate of consanguinity in this cohort. Mutations impairing VDJ recombination and DNA repair were the most common underlying causes of CIDs. However, in patients with syndromic CIDs, autosomal recessive mutations in ataxia-telangiectasia mutated (ATM), autosomal dominant mutations in signal transducer and activator of transcription 3 (STAT3), and microdeletions in 22q11.21 were the most commonly affected genomic loci. Patients with syndromic CIDs had a significantly lower 5-year survival rate rather than those with nonsyndromic CIDs., Conclusions: This study provides proof of principle for the application of targeted next-generation sequencing panels in countries with limited diagnostic resources. The effect of genetic diagnosis on clinical care requires continued improvements in therapeutic resources for these patients., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Inflammation is a crucial defense mechanism that protects the body from the devastating effects of invading pathogens. However, an unrestrained inflammatory reaction may result in systemic manifestations with dire consequences to the host. The extent of activation of the inflammatory response is tightly regulated through immunological and neural pathways. Previously, we demonstrated that cholinergic stimulation confers enhanced protection in experimental animals orally infected with virulent Salmonella enterica serovar Typhimurium. In this study, we investigated the mechanism by which this enhanced protection takes place. Cholinergic stimulation was induced by a 3-week pretreatment with paraoxon, a highly specific acetylcholinesterase (AChE) inhibitor. This treatment enhanced host survival following oral-route infection and this correlated with significantly reduced bacterial load in systemic target organs. Enhanced protection was not due to increased gut motility or rapid bacterial clearance from the gastrointestinal tract. Moreover, protection against bacterial infection was not evident when the animals were infected systemically, suggesting that acetylcholine-mediated protective effect was mostly confined to the gut mucosal tissue. In vivo imaging demonstrated a more localized infection and delay in bacterial dissemination into systemic organs in mice pretreated with paraoxon. Morphological analysis of the small intestine (ileum) showed that AChE inhibition induced the degranulation of goblet cells and Paneth cells, two specialized secretory cells involved in innate immunity. Our findings demonstrate a crucial pathway between neural and immune systems that acts at the mucosal interface to protect the host against oral pathogens.

Treatment of osteomyelitis by conventional antibiotics has proven to be challenging due to limited accessibility to this unique location. Inorganic routes against bacterial infection have been reported for external and topical applications, however in vivo application of these antimicrobials has not been fully explored. Targeted delivery of metallic nanoparticles with inherent antimicrobial activity represents an alternative means of overcoming the challenges posed by multidrug-resistant bacteria and may potentially reduce overall morbidity. In this study we utilized silver-copper-boron composite nanoparticles in an attempt to eradicate S. aureus bone infection in mice. Our results demonstrate effective response when nanoparticles were administered via i.v. or i.m. route (1mg/kg dose) where 99% of bacteria were eliminated in an induced osteomyelitis mouse model. The 1mg/kg dose was neither toxic nor produced any adverse immune response, hence it is believed that metallic nanoparticles present an alternative to antibiotics for the treatment of bone infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

There is renewed interest in the potential use of natural compounds in cancer therapy. Previously, we demonstrated the anti-tumor properties of manuka honey (MH) against several cancers. However, the underlying mechanism and molecular targets of this activity remain unknown. For this study, the early targets of MH and its modulatory effects on proliferation, invasiveness, and angiogenic potential were investigated using two human breast cancer cell lines, the triple-negative MDA-MB-231 cells and estrogen receptor-positive MCF-7 cells, and the non-neoplastic breast epithelial MCF-10A cell line. Exposure to MH at concentrations of 0.3-1.25% (w/v) induced a dose-dependent inhibition of the proliferation of MDA-MB-231 and MCF-7, but not MCF-10A, cells. This inhibition was independent of the sugar content of MH as a solution containing equivalent concentrations of its three major sugars failed to inhibit cell proliferation. At higher concentrations (>2.5%), MH was found to be generally deleterious to the growth of all three cell lines. MH induced apoptosis of MDA-MB-231 cells through activation of caspases 8, 9, 6, and 3/7 and this correlated with a loss of Bcl-2 and increased Bax protein expression in MH-treated cells. Incubation with MH induced a time-dependent translocation of cytochrome c from mitochondria to the cytosol and Bax translocation from the cytosol into the mitochondria. MH also induced apoptosis of MCF-7 cells via the activation of caspases 9 and 6. Low concentrations of MH (0.03-1.25% w/v) induced a rapid reduction in tyrosine-phosphorylated STAT3 (pY-STAT3) in MDA-MB-231 and MCF-7 cells. Maximum inhibition of pY-STAT3 was observed at 1 h with a loss of >80% and coincided with decreased interleukin-6 (IL-6) production. Moreover, MH inhibited the migration and invasion of MDA-MB-231 cells as well as the angiogenic capacity of human umbilical vein endothelial cells. Our findings identify multiple functional pathways affected by MH in human breast cancer and highlight the IL-6/STAT3 signaling pathway as one of the earliest potential targets in this process.

Type I diabetes (T1D) results from T cell-mediated damage of pancreatic β-cells and loss of insulin production. The cholinergic anti-inflammatory pathway represents a physiological link connecting the central nervous and immune systems via vagus nerve, and functions to control the release of proinflammatory cytokines. Using the multiple low-dose streptozotocin (MLD-STZ) model to induce experimental autoimmune diabetes, we investigated the potential of regulating the development of hyperglycemia through administration of paraoxon, a highly specific acetylcholinesterase inhibitor (AChEI). We demonstrate that pretreatment with paraoxon prevented hyperglycemia in STZ-treated C57BL/6 mice. This correlated with a reduction in T cell infiltration into pancreatic islets and preservation of the structure and functionality of β-cells. Gene expression analysis of pancreatic tissue revealed that increased peripheral cholinergic activity prevented STZ-mediated loss of insulin production, this being associated with a reduction in IL-1β, IL-6, and IL-17 proinflammatory cytokines. Intracellular cytokine analysis in splenic T cells demonstrated that inhibition of AChE led to a shift in STZ-induced immune response from a predominantly disease-causing IL-17-expressing Th17 cells to IFNγ-positive Th1 cells. Consistent with this conclusion, inhibition of AChE failed to prevent STZ-induced hyperglycemia in IFNγ-deficient mice. Our results provide mechanistic evidence for the prevention of murine T1D by inhibition of AChE and suggest a promising strategy for modulating disease severity.

Nanoparticles (NPs) are increasingly being commercialized for use in biomedicine. NP toxicity following acute or chronic exposure has been described, but mechanistic insight into this process remains incomplete. Recent evidence from in vitro studies suggested a role for NLRP3 in NP cytotoxicity. In this study, we investigated the effect of systemic administration of composite inorganic NP, consisting of Ag:Cu:B (dose range 1-20 mg/kg), on the early acute (4-24 h post-exposure) and late phase response (96 h post-exposure) in normal and NLRP3-deficient mice. Our findings indicate that systemic exposure (≥2 mg/kg) was associated with acute liver injury due to preferential accumulation of NP in this organ and resulted in elevated AST, ALT and LDH levels. Moreover, within 24 h of NP administration, there was a dose-dependent increase in intraperitoneal neutrophil recruitment and upregulation in gene expression of several proinflammatory mediators, including TNF-α, IL-1β and S100A9. Histological analysis of liver tissue revealed evidence of dose-dependent hepatocyte necrosis, increase in sinusoidal Kupffer cells, lobular granulomas and foci of abscess formation which were most pronounced at 24 h following NP administration. NP deposition in the liver led to a significant upregulation in gene expression of S100A9, an endogenous danger signal recognition molecule of phagocytes, IL-1β and IL-6. The extent of proinflammatory cytokine activation and hepatotoxicity was significantly attenuated in mice deficient in the NLRP3 inflammasome, demonstrating the critical role of this innate immune system recognition receptor in the response to NP.

Despite major advances in early detection and prognosis, chemotherapy resistance is a major hurdle in the battle against breast cancer. Identifying predictive markers and understanding the mechanisms are key steps to overcoming chemoresistance. Methylation-controlled J protein (MCJ, also known as DNAJC15) is a negative regulator of mitochondrial respiration and has been associated with chemotherapeutic drug sensitivity in cancer cell lines. Here we show, in a retrospective study of a large cohort of breast cancer patients, that low MCJ expression in breast tumors predicts high risk of relapse in patients treated with chemotherapy; however, MCJ expression does not correlate with response to endocrine therapy. In a prospective study in breast cancer patients undergoing neoadjuvant therapy, low MCJ expression also correlates with poor clinical response to chemotherapy and decreased disease-free survival. Using MCJ-deficient mice, we demonstrate that lack of MCJ is sufficient to induce mammary tumor chemoresistance in vivo. Thus, loss of expression of this endogenous mitochondrial modulator in breast cancer promotes the development of chemoresistance.

Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.

Introduction: Regulatory T cells (Tregs) have been characterised in different cancers. They accumulate in peripheral blood and tumour microenvironments where they suppress tumour-specific immune responses, enabling tumours to develop without challenge. This tumour immune evasion represents a major obstacle to successful cancer therapies. Whilst Tregs are generally divided into thymic-derived and peripherally induced, Tregs exhibit a wide spectrum of phenotypes and functional capacity dependent on microenvironment. This phenotypic diversity is also reflected in tumour-infiltrating Treg (TI Treg) populations, which may explain the variable impact of Treg accumulation on prognosis in different cancers. Identifying TI Treg subsets is critical to understand TI Treg biology and for developing effective immunotherapies., Areas Covered: This review discusses current and potential markers, and the modulation of these markers in cancer. In addition, we systematically review the clinical impact of Tregs in cancer and their potential as a therapeutic target, with a focus on TI Tregs., Expert Opinion: TI Tregs represent dynamic and diverse subsets that are key in promoting tumour progression through their suppressive activities. Targeting specific TI Treg subpopulations and functional TI Treg markers represents a feasible therapeutic strategy that might allow reestablishment of antitumour immune responses without affecting physiological immune regulation.

The effectiveness of attenuated Salmonella in inhibiting tumor growth has been demonstrated in many therapeutic models, but the precise mechanisms remain incompletely understood. In this study, we show that the anti-tumor capacity of Salmonella depends on a functional MyD88-TLR pathway and is independent of adaptive immune responses. Since myeloid suppressor cells play a critical role in tumor growth, we investigated the consequences of Salmonella treatment on myeloid cell recruitment, phenotypic characteristics, and functional activation in spleen and tumor tissue of B16.F1 melanoma-bearing mice. Salmonella treatment led to increased accumulation of splenic and intratumoral CD11b(+)Gr-1(+) myeloid cells, exhibiting significantly increased expression of various activation markers such as MHC class II, costimulatory molecules, and Sca-1/Ly6A proteins. Gene expression analysis showed that Salmonella treatment induced expression of iNOS, arginase-1 (ARG1), and IFN-γ in the spleen, but down-regulated IL-4 and TGF-β. Within the tumor, expression of iNOS, IFN-γ, and S100A9 was markedly increased, but ARG1, IL-4, TGF-β, and VEGF were inhibited. Functionally, splenic CD11b(+) cells maintained their suppressive capacity following Salmonella treatment, but intratumoral myeloid cells had significantly reduced suppressive capacity. Our findings demonstrate that administration of attenuated Salmonella leads to phenotypic and functional maturation of intratumoral myeloid cells making them less suppressive and hence enhancing the host's anti-tumor immune response. Modalities that inhibit myeloid suppressor cells may be useful adjuncts in cancer immunotherapy.

C3H/HeJ mice carry a mutated allele of TLR4 gene (TLR4 ( d )) and thus are hyporesponsive to the lethal effects of lipopolysaccharide (LPS). Characteristically, however, the mice are also hypersusceptible to infections, particularly by Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) and are known to be difficult to vaccinate against virulent exposure. This is observed despite the expression of wild-type allele of Nramp1 gene, another important determinant of Salmonella susceptibility. In contrast, C3H/HeN mice (TLR4 ( n ) Nramp1 ( n )) express a functional TLR4 protein and are resistant to infection, even by virulent strains of S. typhimurium. In the present study, we describe the immune system-enhancing properties of an attenuated strain of S. typhimurium engineered to express murine IFN-γ. This strain (designated GIDIFN) was able to modulate immune responses following systemic inoculation by upregulating the production of inflammatory mediators (IL-6 and IL-12) and anti-bacterial effector molecules (nitric oxide; NO). Consequently, this led to a more effective control of bacterial proliferation in systemic target organs in both C3H/HeJ and C3H/HeN mice. Although evidence for the enhancement in immune responses could be observed as early as few hours post-inoculation, sustained improvements required 2-3 days to manifest. Vaccination of C3H/HeJ mice with GIDIFN strain, even at low doses, conferred a significantly higher degree of protection against challenge with virulent Salmonella in susceptible C3H/HeJ mice. Our data demonstrate that IFNγ-expressing Salmonella are immunogenic and confer excellent protection against virulent challenge in susceptible C3H/HeJ mice; in addition they may be used as an effective mucosal delivery vectors against virulent infection and for boosting immune responses in immunodeficient hosts.

Manuka honey has been recognized for its anti-bacterial and wound-healing activity but its potential antitumor effect is poorly studied despite the fact that it contains many antioxidant compounds. In this study, we investigated the antiproliferative activity of manuka honey on three different cancer cell lines, murine melanoma (B16.F1) and colorectal carcinoma (CT26) as well as human breast cancer (MCF-7) cells in vitro. The data demonstrate that manuka honey has potent anti-proliferative effect on all three cancer cell lines in a time- and dose-dependent manner, being effective at concentrations as low as 0.6% (w/v). This effect is mediated via the activation of a caspase 9-dependent apoptotic pathway, leading to the induction of caspase 3, reduced Bcl-2 expression, DNA fragmentation and cell death. Combination treatment of cancer cells with manuka and paclitaxel in vitro, however, revealed no evidence of a synergistic action on cancer cell proliferation. Furthermore, we utilized an in vivo syngeneic mouse melanoma model to assess the potential effect of intravenously-administered manuka honey, alone or in combination with paclitaxel, on the growth of established tumors. Our findings indicate that systemic administration of manuka honey was not associated with any alterations in haematological or clinical chemistry values in serum of treated mice, demonstrating its safety profile. Treatment with manuka honey alone resulted in about 33% inhibition of tumor growth, which correlated with histologically observable increase in tumor apoptosis. Although better control of tumor growth was observed in animals treated with paclitaxel alone or in combination with manuka honey (61% inhibition), a dramatic improvement in host survival was seen in the co-treatment group. This highlights a potentially novel role for manuka honey in alleviating chemotherapy-induced toxicity.

Activation of the innate immune system is a prerequisite for the induction of adaptive immunity to both infectious and non-infectious agents. TLRs are key components of the innate immune recognition system and detect pathogen-associated molecular patterns. Most TLRs utilize the MyD88 adaptor for their signaling pathways. In the current study, we investigated innate and adaptive immune responses to primary as well as secondary Salmonella infections in MyD88-deficient (MyD88(-/-)) mice. Using i.p. or oral route of inoculation, we demonstrate that MyD88(-/-) mice are hypersusceptible to infection by an attenuated, double auxotrophic, mutant of Salmonella enterica serovar Typhimurium (S. typhimurium). This is manifested by 2-3 logs higher bacterial loads in target organs, delayed recruitment of phagocytic cells, and defective production of proinflammatory cytokines in MyD88(-/-) mice. Despite these deficiencies, MyD88(-/-) mice developed Salmonella-specific memory Th1 responses and produced elevated serum levels of anti-Salmonella Abs, not only of Th1-driven (IgG2c, IgG3) but also IgG1 and IgG2b isotypes. Curiously, these adaptive responses were insufficient to afford full protection against a secondary challenge with a virulent strain of S. typhimurium. In comparison with the high degree of mortality seen in MyD88(-/-) mice following i.p. inoculation, oral infections led to the establishment of a state of long-term persistence, characterized by continuous bacterial shedding in animal feces that lasted for more than 6 months, but absence from systemic organs. These findings suggest that the absent expression of MyD88 affects primarily the innate effector arm of the immune system and highlights its critical role in anti-bacterial defense., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Previously, we demonstrated that cell-cell communications via the CD40-CD154 pathway play a critical role in the induction of type 1 cytokine responses, including IL-12 and IFN-γ, which in turn greatly influence the response to Salmonella infections. Mice genetically deficient in the expression of CD154 exhibited markedly increased susceptibility to infection by an attenuated, double auxotrophic (aroA-aroD-) strain, designated BRD509, of Salmonella enterica Serovar Typhimurium. In the present study, we used a strain of Salmonella engineered to express murine IFN-γ, designated GIDIFN, in order to assess its potential to enhance the host's immune response in CD154-deficient animals. We demonstrate that infection of animals with GIDIFN results in markedly enhanced anti-bacterial response, as evidenced by the significant reduction in bacterial loads in target organs and decreased animal mortality. This was associated with a more robust proinflammatory cytokine response, including IL-6, IL-12, TNF-α and IFN-γ. In protection studies, GIDIFN strain was demonstrably superior than the BRD509 strain in affording protection against virulent Salmonella challenge in naïve CD154-/- mice. Interestingly, however, infection with GIDIFN failed to correct the isotype switching defect in CD154-/- mice, suggesting that the enhanced immunity triggered by GIDIFN strain occurs independently of humoral immune responses. These findings demonstrate that GIDIFN has immunopotentiating effects on the host's immune response and provide direct evidence for the utility of IFN-γ-expressing attenuated Salmonella in enhancement of immune responsiveness in immunodeficient hosts., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Summary: The cholinergic nervous system has been demonstrated to attenuate the inflammatory response during sepsis via the inhibitory action of acetylcholine (ACh) on macrophages. These findings were largely based on experimental sepsis models using endotoxin as the inducing agent. Herein, however, we report that the specific inhibition of acetylcholinesterase (AChE) renders animals more resistant to infection by a virulent strain of Salmonella enterica serovar Typhimurium, a Gram-negative enteric pathogen. Inhibition of AChE was induced by a subchronic exposure to paraoxon, a potent anti-cholinesterase metabolite of the organophosphorous compound parathion. Our findings indicate that inhibition of AChE enhanced survival of infected mice in a dose-dependent fashion and this correlated with efficient control of bacterial proliferation in target organs. Immunologically, inhibition of AChE enabled the animals to mount a more effective inflammatory anti-microbial response, and to secrete higher levels of interleukin-12, a key T helper type 1-promoting cytokine. The ACh-induced enhancement in resistance to infection was abrogated by co-administration of an oxime which can reactivate AChE. Hence, in a model of Gram-negative bacterial infection, cholinergic stimulation is shown to enhance the anti-microbial immune response leading to effective control of bacterial proliferation and enhanced animal survival.

Salmonella enterica serovar Typhimurium (hereafter S. typhimurium) stains have been shown to exert a potent inhibitory effect on the growth of human and mouse tumors in experimental models. Our laboratory has previously demonstrated that an attenuated strain of S. typhimurium engineered to express IL2 (designated strain GIDIL2) has demonstrable immunopotentiating properties, particularly affecting the innate arm of the immune system. In the present study, we wished to explore the properties of IL2-expressing Salmonella as an oncolytic agent in the highly tumorigenic B16F1 melanoma mouse model and shed light on its mechanism of action. Our data demonstrate that the systemic administration of a single dose of GIDIL2, two weeks post B16F1 implantation, had a significantly superior effect than its parental, non cytokine-expressing, strain (known as BRD509E). The improved response, which was dependent on the bacterial dose used, was observed in terms of stronger inhibition of tumor growth as well as enhanced host survival. The GIDIL2-induced anti-tumor response was correlated with decreased angiogenesis and increased necrosis within the tumor tissue. A treatment regimen involving multiple low doses of GIDIL2 was more efficacious than a single high dose regimen, resulting in extension of animal survival well beyond the normal 30 day post implantation period typically observed in this aggressive melanoma tumor model. This supports the notion of using cytokine-expressing attenuated Salmonella organisms in cancer therapy.

We previously reported that the intraperitoneal administration of recombinant strains of Salmonella enterica serovar Typhimurium, engineered to express murine IL-2 (designated GIDIL2) or IFN-gamma (GIDIFNgamma), induced a cytokine-specific modulation of the host innate immune response. Interestingly, the bacteria-expressed cytokines were not secreted, but instead were associated with the bacterial cytosol. To understand the mechanism by which these two transfectants influence immune cells, we investigated their effect on two macrophage populations, J774A.1 cell line and ex vivo isolated peritoneal macrophages (PM). The parental, cytokine-negative, Salmonella strain (designated BRD509E), was used as a control. The capacity of the bacterial strains to activate macrophages was assessed by modulation of surface expression of costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2) and activation marker Ly-6A/E, and by induction of cytokine production. Our data revealed that GIDIFNgamma was the only strain capable of upregulating the expression of cell-surface markers. Moreover, infection of macrophages with GIDIFNgamma induced a stronger cytokine response in comparison with BRD509E or GIDIL2 strain, as demonstrated by the production of TNF-alpha, IL-6, IL-12/IL23p40 and NO. The ability of GIDIL2 and GIDIFNgamma strains to activate macrophages was not due to enhanced invasiveness, as their cellular invasion rates were 2-fold lower than the parental strain. Further investigation of cytokine expression by GIDIL2 and GIDIFNgamma strains showed that while the cytokines were not secreted, they were expressed on the bacterial surface suggesting that their effect on macrophages could be through a direct interaction with their receptors on target cells. This was confirmed by showing that cytochalasin D-treated macrophages, a treatment which effectively inhibited bacterial invasion, could be induced to secrete high levels of cytokines by GIDIFNgamma organisms. Our data demonstrate that cytokine-expressing bacteria modulate macrophage activation independently of their entry into cells and may explain the rapid action of these bacterial strains when injected systemically into susceptible mice.

Paraoxon is the bioactive metabolite of the organophosphate pesticide parathion. Desulphuration of parathion by liver enzymes or sunlight results in the formation of paraoxon which inhibits acetylcholine esterase (AChE) activity. In the present study, we analyzed the effect of a 6-week, subchronic treatment with two different daily intraperitoneal doses (30 or 40 nmol) of paraoxon on the immune system of BALB/c mice. At a dose of 30 nmol/day, body weight of treated animals was unchanged compared to the controls. In contrast, the higher dose (40 nmol/day) induced a reduction in body growth, particularly in the first 3 weeks of treatment, peaking at week 2 when the saline group showed a 14.2-fold increase in body weight gain compared to paraoxon-treated animals. Moreover, mice treated with either dose of paraoxon had a >50% reduction in AChE activity during the first 3 weeks of treatment, but by the end of the treatment (week 6), AChE activity returned to normal. With regard to immunological parameters, there was no significant difference in either total spleen weight or in the ratios of various spleen cell populations between control and paraoxon-treated animals. Furthermore, no changes were observed in mitogen-induced cytokine secretion from splenocytes of paraoxon-treated mice. Finally, subchronic exposure to paraoxon did not alter mortality of mice exposed to a bacterial infection with Salmonella typhimurium. These data suggest that although subchronic exposure to paraoxon induced a transient inhibition in AChE activity, it had no demonstrable effect on the host immune system.

Despite the great strides made in understanding the basic biology of cancer and the multiple approaches to cancer therapy that have been utilized, cancer remains a major cause of death worldwide. The two properties that define the most successful tumors are low antigenicity, enabling cancer cells to escape immune system recognition, and high tumorigenicity, allowing the cells to proliferate aggressively and metastasize to other tissues. The development of novel anticancer therapies is aimed at enhancing the antigenicity of tumors and/or increasing the functional efficiency of various effector immune system cells. The use of obligate/facultative anaerobic bacteria, which preferentially replicate within tumor tissue, as an oncolytic agent is one of the innovative approaches to cancer therapy. Over the past several years, we have studied the properties of attenuated strains of Salmonella typhimurium, a facultative anaerobe, genetically engineered to express murine cytokines. Previously, we demonstrated that cytokine-expressing strains have the capacity to modulate immunity to infection. Given the preferential tumor-homing properties of attenuated Salmonella bacteria, the potential capacity of a cytokine-encoding Salmonella strain to retard the growth of experimental melanomas was investigated. Mice pre-implanted with melanoma cells were treated with an attenuated strain of S. typhimurium or with one of its derivatives expressing IL-2. Our data demonstrate that IL-2-encoding Salmonella organisms were superior in suppressing tumor growth as compared to the parental noncytokine-expressing strain. This supports the notion of using cytokine-expressing attenuated Salmonella organisms in cancer therapy.

Persistent exposure to inorganic lead (Pb) is known to adversely affect the immune system. In the present study, we assessed the effect of chronic Pb exposure on susceptibility to infection by the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. Mice were exposed to 10 mM Pb-acetate in drinking water for approximately 16 weeks, resulting in a significant level of Pb in the blood (106.2+/-8.9 microg/dl). Pb exposure rendered mice susceptible to Salmonella infection, manifested by increased bacterial burden in target organs and heightened mortality. Flow cytometric analysis of the splenic cellular composition in normal and Pb-exposed mice revealed no gross alteration in the ratios of B and T lymphocytes or myeloid cells. Similarly, the capacity of B and T cells to upregulate the expression of activation antigens in response to mitogenic or inflammatory stimuli was not hindered by Pb exposure. Analysis of the ability of ex vivo-cultured splenocytes to secrete cytokines demonstrated a marked reduction in IFN-gamma and IL-12p40 production associated with Pb exposure. In contrast, secretion of IL-4 by splenocytes of Pb-treated mice was 3- to 3.6-fold higher than in normal mice. The increased capacity to produce IL-4 correlated with a shift in the in vivo anti-Salmonella antibody response from the protective IgG2a isotype to the Th2-induced IgG1 isotype. We conclude that chronic exposure to high levels of Pb results in a state of immunodeficiency which is not due to an overt cytotoxic or immunosuppressive mechanism, but rather is largely caused by a shift in immune responsiveness to Th2-type reactions.

The immunomodulatory activities of recombinant human interleukin-11 (rhIL-11) were investigated in a clinical trial among patients with hematological malignancy, randomized to either rhIL-11 or placebo throughout chemotherapy. Daily serum concentrations of sTNFRI, IL-6, IL-8, TNFalpha, and CRP were measured. Higher sTNFRI levels [mean pg/ml (95% CI)] were detected in patients receiving rhIL-11 compared to placebo [1749.7 (1626-1882.9) versus 1038.5 (953.3-1131.3)] respectively (P = 0.01) for all 898 observations and during febrile days [2327.6 (2142.6-2528.2) versus 1308.9 (1163-1473.2), P = 0.12] and during days without infection [1406.6 (1266.1-1563) versus 871.3 (774.9-979.6), P < 0.001]. A similar pattern in CRP concentrations was observed. Multivariate analysis indicated rhIL-11 was associated with elevated sTNFRI or CRP independent of infectious episodes and other factors. 7 patients (all receiving placebo) of 40 had elevated TNFalpha levels. IL-6 and IL-8 levels were not substantially affected by rhIL-11. Bacteremia, fungal infections, and fever of unknown origin (FUO) were reduced in rhIL-11-treated patients. Given the role of sTNFRI in dampening the deleterious effects of a hyperactive TNFalpha environment, rhIL-11-induced upregulation of sTNFRI shedding is a potentially important mechanism for modulating immune and inflammatory responses in humans.