Immunoregulation via Cell Density and Quorum Sensing-like Mechanisms: An Underexplored Emerging Field with Potential Translational Implications.

Quorum sensing (QS) was historically described as a mechanism by which bacteria detect and optimize their population density via gene regulation based on dynamic environmental cues. Recently, it was proposed that QS or similar mechanisms may have broader applications across different species and cell types. Indeed, emerging evidence shows that the mammalian immune system can also elicit coordinated responses on a population level to regulate cell density and function, thus suggesting that QS-like mechanisms may also be a beneficial trait of the immune system. In this review, we explore and discuss potential QS-like mechanisms deployed by the immune system to coordinate cellular-level responses, such as T cell responses mediated via the common gamma chain (γc) receptor cytokines and the aryl hydrocarbon receptors (AhRs). We present evidence regarding a novel role of QS as a multifunctional mechanism coordinating CD4+ and CD8+ T cell behavior during steady state and in response to infection, inflammatory diseases, and cancer. Successful clinical therapies such as adoptive cell transfer for cancer treatment may be re-evaluated to harness the effects of the QS mechanism(s) and enhance treatment responsiveness. Moreover, we discuss how signaling threshold perturbations through QS-like mediators may result in disturbances of the complex crosstalk between immune cell populations, undesired T cell responses, and induction of autoimmune pathology. Finally, we discuss the potential therapeutic role of modulating immune-system-related QS as a promising avenue to treat human diseases.

Inhibition of IL-6 in the LCWE Mouse Model of Kawasaki Disease Inhibits Acute Phase Reactant Serum Amyloid A but Fails to Attenuate Vasculitis.

Objective: Kawasaki disease (KD) is the most common cause of acquired pediatric heart disease in the developed world. 10% of KD patients are resistant to front-line therapy, and no interventions exist to address secondary complications such as myocardial fibrosis. We sought to identify proteins and pathways associated with disease and anti-IL-1 treatment in a mouse model of KD. Methods: Vasculitis was induced via Lactobacillus casei cell wall extract (LCWE) injection in 5-week-old male mice. Groups of mice were injected with LCWE alone, LCWE and IL-1 receptor antagonist anakinra, or saline for controls. Upper heart tissue was assessed by quantitative mass spectrometry analysis. Expression and activation of STAT3 was assessed by immunohistochemistry, immunofluorescence and Western blot, and IL-6 expression by RNA-seq and ELISA. A STAT3 small molecular inhibitor and anti-IL-6R antibody were used to evaluate the role of STAT3 and IL-6 in disease development. Results: STAT3 was highly expressed and phosphorylated in cardiac tissue of LCWE-injected mice, and reduced following anakinra treatment. Il6 and Stat3 gene expression was enhanced in abdominal aorta of LCWE-injected mice and reduced with Anakinra treatment. IL-6 serum levels were enhanced in LCWE-injected mice and normalized by anakinra. However, neither inhibition of STAT3 nor blockade of IL-6 altered disease development. Conclusion: Proteomic analysis of cardiac tissues demonstrates differential protein expression between KD-like, control and anakinra treated cardiac tissue. STAT3 and IL-6 were highly upregulated with LCWE and normalized by anakinra treatment. However, both STAT3 and IL-6 were dispensable for disease development indicating they may be bystanders of inflammation.

Limitations of cell-lineage-specific non-dynamic gene recombination in CD11c.Cre+ITGA4fl/fl mice.

BACKGROUND: The Cre-lox system is a non-dynamic method of gene modification and characterization. Promoters thought to be relatively cell-specific are utilized for generation of cell-lineage-specific gene modifications. METHODS: CD11c.Cre+ITGA4fl/fl mice were generated to abolish the expression of ITGA (α4-integrin) in CD11c+ cells. Ex vivo flow cytometry studies were used to assess the expression of cellular surface markers in different lymphoid compartments and leukocytes subsets after Cre-mediated recombination. RESULTS: A significant reduction of α4-integrin expression among CD11c+- cells was achieved in CD11c.Cre+ITGA4fl/fl mice in primary and secondary lymphoid tissues. A similar reduction in the expression of α4-integrin was also observed in CD11c- cells. CONCLUSION: Cre-lox-mediated cell lineage-specific gene deletion is limited by the transient expression of recombination regulating sequences in hematopoietic cell lines. These methodological issues indicate the need to consider when to employ non-dynamic DNA recombination models in animal models of CNS autoimmunity. An experimental algorithm to address the biological complexities of non-dynamic gene recombination is provided.

TNFR2 limits proinflammatory astrocyte functions during EAE induced by pathogenic DR2b-restricted T cells.

Multiple sclerosis (MS) is an autoimmune neuroinflammatory disease where the underlying mechanisms driving disease progression have remained unresolved. HLA-DR2b (DRB1*15:01) is the most common genetic risk factor for MS. Additionally, TNF and its receptors TNFR1 and TNFR2 play key roles in MS and its preclinical animal model, experimental autoimmune encephalomyelitis (EAE). TNFR2 is believed to ameliorate CNS pathology by promoting remyelination and Treg function. Here, we show that transgenic mice expressing the human MHC class II (MHC-II) allele HLA-DR2b and lacking mouse MHC-II and TNFR2 molecules, herein called DR2bΔR2, developed progressive EAE, while disease was not progressive in DR2b littermates. Mechanistically, expression of the HLA-DR2b favored Th17 cell development, whereas T cell-independent TNFR2 expression was critical for restraining of an astrogliosis-induced proinflammatory milieu and Th17 cell responses, while promoting remyelination. Our data suggest the TNFR2 signaling pathway as a potentially novel mechanism for curtailing astrogliosis and promoting remyelination, thus providing new insights into mechanisms limiting progressive MS.

The role of B cells in multiple sclerosis: Current and future therapies.

While it was long held that T cells were the primary mediators of multiple sclerosis (MS) pathogenesis, the beneficial effects observed in response to treatment with Rituximab (RTX), a monoclonal antibody (mAb) targeting CD20, shed light on a key contributor to MS that had been previously underappreciated: B cells. This has been reaffirmed by results from clinical trials testing the efficacy of subsequently developed B cell-depleting mAbs targeting CD20 as well as studies revisiting the effects of previous disease-modifying therapies (DMTs) on B cell subsets thought to modulate disease severity. In this review, we summarize current knowledge regarding the complex roles of B cells in MS pathogenesis and current and potential future B cell-directed therapies.

The role of glial-neuronal metabolic cooperation in modulating progression of multiple sclerosis and neuropathic pain.

While the etiology of multiple sclerosis (MS) remains unclear, research from the clinic and preclinical models identified the essential role of inflammation and demyelination in the pathogenesis of MS. Current treatments focused on anti-inflammatory processes are effective against acute episodes and relapsing-remitting MS, but patients still move on to develop secondary progressive MS. MS progression is associated with activation of microglia and astrocytes, and importantly, metabolic dysfunction leading to neuronal death. Neuronal death also contributes to chronic neuropathic pain. Metabolic support of neurons by glia may play central roles in preventing progression of MS and chronic neuropathic pain. Here, we review mechanisms of metabolic cooperation between glia and neurons and outline future perspectives exploring metabolic support of neurons by glia.

Glucan-Chitin Particles Enhance Th17 Response and Improve Protective Efficacy of a Multivalent Antigen (rCpa1) against Pulmonary Coccidioides posadasii Infection.

Developing an effective and safe recombinant vaccine requires microbe-specific antigens combined with an adjuvant/delivery system to strengthen protective immunity. In this study, we designed and expressed a multivalent recombinant Coccidioides polypeptide antigen (rCpa1) that consists of three previously identified antigens (i.e., Ag2/Pra, Cs-Ag, and Pmp1) and five pathogen-derived peptides with high affinity for human major histocompatibility complex class II (MHC-II) molecules. The purified rCpa1 was encapsulated into four types of yeast cell wall particles containing ß-glucan, mannan, and chitin in various proportions or was mixed with an oligonucleotide (ODN) containing two methylated dinucleotide CpG motifs. This multivalent antigen encapsulated into glucan-chitin particles (GCP-rCpa1) showed significantly greater reduction of fungal burden for human HLA-DR4 transgenic mice than the other adjuvant-rCpa1 formulations tested. Among the adjuvants tested, both GCPs and ß-glucan particles (GPs) were capable of stimulating a mixed Th1 and Th17 response. Mice vaccinated with GCP-rCpa1 showed higher levels of interleukin 17 (IL-17) production in T-cell recall assays and earlier lung infiltration by activated Th1 and Th17 cells than GP-rCpa1-vaccinated mice. Both C57BL/6 and HLA-DR4 transgenic mice that were vaccinated with the GCP-rCpa1 vaccine showed higher survival rates than mice that received GCPs alone. Concurrently, the GCP-rCpa1 vaccine stimulated greater infiltration of the injection sites by macrophages, which engulf and process the vaccine for antigen presentation, than the GP-rCpa1 vaccine. This is the first attempt to systematically characterize the presentation of a multivalent coccidioidomycosis vaccine encapsulated with selected adjuvants that enhance the protective cellular immune response to infection.

Targeting "Retired Antigens" for Cancer Immunoprevention.

Identification of immune targets for cancer immunoprevention, or immunotherapy, has historically focused on tumor-associated (self) antigens or neoantigens expressed on malignant cells. For self-antigens, overcoming tolerance can be a difficult challenge. Neoantigens do not suffer from this limitation, but the lack of recurrent mutations yielding common neoantigens that can be exploited in vaccines is a problem for many tumor types. Targeting "retired antigens," a specialized type of self-antigen, may have considerable advantages. Antigens no longer expressed in mature or aged individuals should pose reduced risk of autoimmune sequelae. Indeed, self-tolerance of these antigens may have naturally faded. Thus, when the retired antigens are highly expressed in cancer cells, it may be easier to overcome the remaining tolerance. Women who are BRCA1/2 carriers may be among the first to benefit as candidate retired antigens have been identified as highly expressed in ovarian and breast cancer cells. Although there is good preclinical data supporting this immune targeting concept, additional research is needed to understand the underlying immune phenomena and optimize the vaccine strategy. Cancer Prev Res; 10(11); 607-8. ©2017 AACRSee related article by Mazumder et al., p. 612.

Identification of Iguratimod as an Inhibitor of Macrophage Migration Inhibitory Factor (MIF) with Steroid-sparing Potential.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in a broad range of inflammatory and oncologic diseases. MIF is unique among cytokines in terms of its release profile and inflammatory role, notably as an endogenous counter-regulator of the anti-inflammatory effects of glucocorticoids. In addition, it exhibits a catalytic tautomerase activity amenable to the design of high affinity small molecule inhibitors. Although several classes of these compounds have been identified, biologic characterization of these molecules remains a topic of active investigation. In this study, we used in vitro LPS-driven assays to characterize representative molecules from several classes of MIF inhibitors. We determined that MIF inhibitors exhibit distinct profiles of anti-inflammatory activity, especially with regard to TNFα. We further investigated a molecule with relatively low anti-inflammatory activity, compound T-614 (also known as the anti-rheumatic drug iguratimod), and found that, in addition to exhibiting selective MIF inhibition in vitro and in vivo, iguratimod also has additive effects with glucocorticoids. Furthermore, we found that iguratimod synergizes with glucocorticoids in attenuating experimental autoimmune encephalitis, a model of multiple sclerosis. Our work identifies iguratimod as a valuable new candidate for drug repurposing to MIF-relevant diseases, including multiple sclerosis.

IFN-γ ameliorates autoimmune encephalomyelitis by limiting myelin lipid peroxidation.

Evidence has suggested both a pathogenic and a protective role for the proinflammatory cytokine IFN-γ in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying the protective role of IFN-γ in EAE have not been fully resolved, particularly in the context of CNS antigen-presenting cells (APCs). In this study we examined the role of IFN-γ in myelin antigen uptake by CNS APCs during EAE. We found that myelin antigen colocalization with APCs was decreased substantially and that EAE was significantly more severe and showed a chronic-progressive course in IFN-γ knockout (IFN-γ-/-) or IFN-γ receptor knockout (IFN-γR-/-) mice as compared with WT animals. IFN-γ was a critical regulator of phagocytic/activating receptors on CNS APCs. Importantly, "free" myelin debris and lipid peroxidation activity at CNS lesions was increased in mice lacking IFN-γ signaling. Treatment with N-acetyl-l-cysteine, a potent antioxidant, abolished lipid peroxidation activity and ameliorated EAE in IFN-γ-signaling-deficient mice. Taken together the data suggest a protective role for IFN-γ in EAE by regulating the removal of myelin debris by CNS APCs and thereby limiting the substrate available for the generation of neurotoxic lipid peroxidation products.

Small molecule inhibitor of antigen binding and presentation by HLA-DR2b as a therapeutic strategy for the treatment of multiple sclerosis.

The strong association of HLA-DR2b (DRB1*1501) with multiple sclerosis (MS) suggests this molecule as prime target for specific immunotherapy. Inhibition of HLA-DR2b-restricted myelin-specific T cells has the potential to selectively prevent CNS pathology mediated by these MHC molecules without undesired global immunosuppression. In this study, we report development of a highly selective small molecule inhibitor of peptide binding and presentation by HLA-DR2b. PV-267, the candidate molecule used in these studies, inhibited cytokine production and proliferation of myelin-specific HLA-DR2b-restricted T cells. PV-267 had no significant effect on T cell responses mediated by other MHC class II molecules, including HLA-DR1, -DR4, or -DR9. Importantly, PV-267 did not induce nonspecific immune activation of human PBMC. Lastly, PV-267 showed treatment efficacy both in preventing experimental autoimmune encephalomyelitis and in treating established disease. The results suggest that blocking the MS-associated HLA-DR2b allele with small molecule inhibitors may be a promising therapeutic strategy for the treatment of MS.

Anti-CRLF2 Antibody-Armored Biodegradable Nanoparticles for Childhood B-ALL.

B-precursor acute lymphoblastic leukemia (B-ALL) lymphoblast (blast) internalization of anti-cytokine receptor-like factor 2 (CRLF2) antibody-armored biodegradable nanoparticles (AbBNPs) are investigated. First, AbBNPsaere synthesized by adsorbing anti-CRLF2 antibodies to poly(D,L-lactide- co -glycolide) (PLGA) nanoparticles of various sizes and antibody surface density (Ab/BNP) ratios. Second, AbBNPs are incubated with CRLF2-overexpressing (CRLF2+) or control blasts. Third, internalization of AbBNPs by blasts is evaluated by multicolor flow cytometry as a function of receptor expression, AbBNP size, and Ab/BNP ratio. Results from these experiments are con-firmed by electron microscopy, fluorescence microscopy, and Western blotting. The optimal size and Ab/BNP for internalization of AbBNPs by CRLF2+ blasts is 50 nm with 10 Ab/BNP and 100 nm with 25 Ab/BNP. These studies show that internalization of AbBNPs in childhood B-ALL blasts is AbBNP size-and Ab/BNP ratio-dependent. All AbBNP combinations are non-cytotoxic. It is also shown that CD47 is very slightly up-regulated by blasts exposed to AbBNPs. CD47 is "the marker of self" overexpressed by blasts to escape phagocytosis, or "cellular devouring", by beneficial macrophages. The results indicate that precise engineering of AbBNPs by size and Ab/BNP ratio may improve the internalization and selectivity of future biodegradable nanoparticles for the treatment of leukemia patients, including drug-resistant minority children and Down's syndrome patients with CRLF2+B-ALL.

Regulation of adaptive immunity by the fractalkine receptor during autoimmune inflammation.

Fractalkine, a chemokine anchored to neurons or peripheral endothelial cells, serves as an adhesion molecule or as a soluble chemoattractant. Fractalkine binds CX3CR1 on microglia and circulating monocytes, dendritic cells, and NK cells. The aim of this study is to determine the role of CX3CR1 in the trafficking and function of myeloid cells to the CNS during experimental autoimmune encephalomyelitis (EAE). Our results show that, in models of active EAE, Cx3cr1(-/-) mice exhibited more severe neurologic deficiencies. Bone marrow chimeric mice confirmed that CX3CR1 deficiency in bone marrow enhanced EAE severity. Notably, CX3CR1 deficiency was associated with an increased accumulation of CD115(+)Ly6C(-)CD11c(+) dendritic cells into EAE-affected brains that correlated with enhanced demyelination and neuronal damage. Furthermore, higher IFN-γ and IL-17 levels were detected in cerebellar and spinal cord tissues of CX3CR1-deficient mice. Analyses of peripheral responses during disease initiation revealed a higher frequency of IFN-γ- and IL-17-producing T cells in lymphoid tissues of CX3CR1-deficient as well as enhanced T cell proliferation induced by CX3CR1-deficient dendritic cells. In addition, adoptive transfer of myelin oligodendrocyte glycoprotein35-55-reactive wild-type T cells induced substantially more severe EAE in CX3CR1-deficient recipients when compared with wild-type recipients. Collectively, the data demonstrate that besides its role in chemoattraction, CX3CR1 is a key regulator of myeloid cell activation contributing to the establishment of adaptive immune responses.

Mast cells: multitalented facilitators of protection against bacterial pathogens.

Mast cells are crucial effector cells evoking immune responses against bacterial pathogens. The positioning of mast cells at the host-environment interface, and the multitude of pathogen-recognition receptors and preformed mediator granules make these cells potentially the earliest to respond to an invading pathogen. In this review, the authors summarize the receptors used by mast cells to recognize invading bacteria and discuss the function of immune mediators released by mast cells in control of bacterial infection. The interaction of mast cells with other immune cells, including macrophages, dendritic cells and T cells, to induce protective immunity is highlighted. The authors also discuss mast cell-based vaccine strategies and the potential application in control of bacterial disease.

Tumor necrosis factor alpha production from CD8+ T cells mediates oviduct pathological sequelae following primary genital Chlamydia muridarum infection.

The immunopathogenesis of Chlamydia trachomatis-induced oviduct pathological sequelae is not well understood. Mice genetically deficient in perforin (perforin(-/-) mice) or tumor necrosis factor alpha (TNF-α) production (TNF-α(-/-) mice) displayed comparable vaginal chlamydial clearance rates but significantly reduced oviduct pathology (hydrosalpinx) compared to that of wild-type mice. Since both perforin and TNF-α are effector mechanisms of CD8(+) T cells, we evaluated the role of CD8(+) T cells during genital Chlamydia muridarum infection and oviduct sequelae. Following vaginal chlamydial challenge, (i) mice deficient in TAP I (and therefore the major histocompatibility complex [MHC] I pathway and CD8(+) T cells), (ii) wild-type mice depleted of CD8(+) T cells, and (iii) mice genetically deficient in CD8 (CD8(-/-) mice) all displayed similar levels of vaginal chlamydial clearance but significantly reduced hydrosalpinx, compared to those of wild-type C57BL/6 mice, suggesting a role for CD8(+) T cells in chlamydial pathogenesis. Repletion of CD8(-/-) mice with wild-type or perforin(-/-), but not TNF-α(-/-), CD8(+) T cells at the time of challenge restored hydrosalpinx to levels observed in wild-type C57BL/6 mice, suggesting that TNF-α production from CD8(+) T cells is important for pathogenesis. Additionally, repletion of TNF-α(-/-) mice with TNF-α(+/+) CD8(+) T cells significantly enhanced the incidence of hydrosalpinx and oviduct dilatation compared to those of TNF-α(-/-) mice but not to the levels found in wild-type mice, suggesting that TNF-α production from CD8(+) T cells and non-CD8(+) cells cooperates to induce optimal oviduct pathology following genital chlamydial infection. These results provide compelling new evidence supporting the contribution of CD8(+) T cells and TNF-α production to Chlamydia-induced reproductive tract sequelae.

Non-FcεR bearing mast cells secrete sufficient interleukin-4 to control Francisella tularensis replication within macrophages.

Mast cells have classically been implicated in the triggering of allergic and anaphylactic reactions. However, recent findings have elucidated the ability of these cells to selectively release a variety of cytokines leading to bacterial clearance through neutrophil and dendritic cell mobilization, and suggest an important role in innate host defenses. Our laboratory has established a primary bone marrow derived mast cell-macrophage co-culture system and found that mast cells mediated a significant inhibition of Francisella tularensis live vaccine strain (LVS) uptake and replication within macrophages through contact and the secreted product interleukin-4 (IL-4). In this study, we utilized P815 mast cells and J774 macrophages to further investigate whether mast cell activation by non-FcεR driven signals could produce IL-4 and control intramacrophage LVS replication. P815 supernatants collected upon activation by the mast cell activating peptide MP7, as well as P815 cells co-cultured with J774 macrophages, exhibited marked inhibition of bacterial uptake and replication, which correlated with the production of IL-4. The inhibition noted in vitro was titratable and preserved at ratios relevant to cellular infiltration events following pulmonary challenge. Collectively, our data suggest that both primary mast cell and P815 mast cell (lacking FcεR) secreted IL-4 can control intramacrophage Francisella replication.

Therapeutic enzyme deimmunization by combinatorial T-cell epitope removal using neutral drift.

A number of heterologous enzymes have been investigated for cancer treatment and other therapeutic applications; however, immunogenicity issues have limited their clinical utility. Here, a new approach has been created for heterologous enzyme deimmunization whereby combinatorial saturation mutagenesis is coupled with a screening strategy that capitalizes on the evolutionary biology concept of neutral drift, and combined with iterative computational prediction of T-cell epitopes to achieve extensive reengineering of a protein sequence for reduced MHC-II binding propensity without affecting catalytic and pharmacological properties. Escherichia coli L-asparaginase II (EcAII), the only nonhuman enzyme approved for repeated administration, is critical in treatment of childhood acute lymphoblastic leukemia (ALL), but elicits adverse antibody responses in a significant fraction of patients. The neutral drift screening of combinatorial saturation mutagenesis libraries at a total of 12 positions was used to isolate an EcAII variant containing eight amino acid substitutions within computationally predicted T-cell epitopes--of which four were nonconservative--while still exhibiting k(cat)/K(M) = 10(6) M(-1) s(-1) for L-Asn hydrolysis. Further, immunization of HLA-transgenic mice expressing the ALL-associated DRB1*0401 allele with the engineered variant resulted in significantly reduced T-cell responses and a 10-fold reduction in anti-EcAII IgG titers relative to the existing therapeutic. This significant reduction in the immunogenicity of EcAII may be clinically relevant for ALL treatment and illustrates the potential of employing neutral drift screens to achieve large jumps in sequence space as may be required for the deimmunization of heterologous proteins.

Anaphylaxis and mortality induced by treatment of mice with anti-VLA-4 antibody and pertussis toxin.

Ab-mediated blockade of the adhesion molecule VLA-4 has been shown to ameliorate disease in human multiple sclerosis patients and experimental autoimmune encephalomyelitis (EAE) animal models. We wanted to determine whether anti-VLA-4 Ab treatment affected the function and persistence of autoreactive T cells in mice with EAE. Unexpectedly, we observed a high level of mortality in anti-VLA-4 mAb (PS/2)-treated mice with actively induced EAE despite decreased disease severity. Investigation of the underlying mechanism showed that injection of PS/2 mAb in combination with pertussis toxin resulted in anaphylaxis and mortality. Furthermore, the data showed that CD4(+) T cells were required for this effect and suggested a role for IL-1ß and TNF-α in the underlying pathology. The results reveal a previously not appreciated deleterious effect of anti-VLA-4 Ab treatment in combination with exposure to pertussis toxin.

Prosurvival and proapoptotic functions of ERK1/2 activation in murine thymocytes in vitro.

The extracellular signal-regulated kinases 1/2 (ERK1/2) are serine/threonine-selective protein kinases involved in proliferation and differentiation of cells, including thymocytes. The requirement of ERK1/2 for thymocyte differentiation and maturation has been well established; however, their role in regulating thymocyte survival and apoptosis has not been resolved. Here, we asked whether ERK1/2 affected thymocyte survival in vitro in response to apoptotic stimuli. The results show that phorbol 12-myristate 13-acetate (PMA) treatment (with or without ionomycin) and serum starvation (s/s) induced sustained ERK1/2 activation in murine thymocytes. Importantly, pharmacological treatment of thymocytes with the MEK inhibitor UO126 revealed that PMA-induced ERK1/2 activation was proapoptotic, whereas serum starvation-induced ERK1/2 activation inhibited apoptosis and promoted cell survival. While basal MEK activity was required for both s/s- and PMA-induced ERK1/2 activation, MEK activity increased only in response to PMA. The results show that the suppression of ERK1/2 phosphatases was responsible for s/s-induced sustained ERK1/2 activation. Unexpectedly, neither s/s-induced proapoptotic nor PMA-induced anti-apoptotic functions of ERK1/2 depended on the Bcl-2 family phosphoprotein Bim(EL), which was previously implicated in thymocyte apoptosis. Lastly, etoposide treatment of immature thymocytes induced both p53 and ERK1/2 activation, but ERK1/2 activity did not affect the phosphorylation and stabilization of p53. Thus, ERK1/2 has a dual role in promoting cell survival and cell death in thymocytes in the context of different stimuli.

Mast cells inhibit intramacrophage Francisella tularensis replication via contact and secreted products including IL-4.

Francisella tularensis is an intracellular, Gram-negative bacterium that is the causative agent of pulmonary tularemia. The pathogenesis and mechanisms related to innate resistance against F. tularensis are not completely understood. Mast cells are strategically positioned within mucosal tissues, the major interface with the external environment, to initiate innate responses at the site of infection. Mast cell numbers in the cervical lymph nodes and the lungs progressively increased as early as 48 h after intranasal F. tularensis live vaccine strain (LVS) challenge. We established a primary bone marrow-derived mast cell-macrophage coculture system and found that mast cells significantly inhibit F. tularensis LVS uptake and growth within macrophages. Importantly, mice deficient in either mast cells or IL-4 receptor displayed greater susceptibility to the infection when compared with corresponding wild-type animals. Contact-dependent events and secreted products including IL-4 from mast cells, and IL-4 production from other cellular sources, appear to mediate the observed protective effects. These results demonstrate a previously unrecognized role for mast cells and IL-4 and provide a new dimension to our understanding of the innate immune mechanisms involved in controlling intramacrophage Francisella replication.