PIAS1 and STAT‐3 impair the tumoricidal potential of IFN‐γ‐stimulated mouse dendritic cells generated with IL‐15

Primarily defined by their antigen‐presenting property, dendritic cells (DCs) are being implemented as cancer vaccines in immunotherapeutic interventions. DCs can also function as direct tumor cell killers. How DC cytotoxic activity can be efficiently harnessed and the mechanisms controlling this nonconventional property are not fully understood. We report here that the tumoricidal potential of mouse DCs generated from myeloid precursors with GM‐CSF and IL‐15 (IL‐15 DCs) can be triggered with the Toll‐like receptor (TLR) 4 ligand lipopolysaccharide to a similar extent compared with that of their counterparts, conventionally generated with IL‐4 (IL‐4 DCs). The mechanism of tumor cell killing depends on the induction of iNOS expression by DCs. In contrast, interferon (IFN)‐γ induces the cytotoxic activity of IL‐4 but not IL‐15 DCs. Although the IFN‐γ‐STAT‐1 signaling pathway is overall functional in IL‐15 DCs, IFN‐γ fails to induce iNOS expression in these cells. iNOS expression is negatively controlled in IFN‐γ‐stimulated IL‐15 DCs by the cooperation between the E3 SUMO ligase PIAS1 and STAT‐3, and can be partially restored with PIAS1 siRNA and STAT‐3 inhibitors.     

Tumour‐associated macrophage‐mediated survival of myeloma cells through STAT3 activation

Overcoming drug resistance is one of the greatest challenges in the treatment of multiple myeloma (MM). The interaction of myeloma cells with the bone marrow (BM) microenvironment is a major factor contributing to drug resistance. Tumour‐associated macrophages (TAMs) with different polarization states constitute an important component of this microenvironment. Previous studies have revealed a role of TAMs in MM cell survival and drug resistance; however, the impact of macrophage polarization (anti‐tumoural ‘M1’ versus pro‐tumoural ‘M2’‐like phenotype) in this process has not yet been described. Here, the presence of TAMs was confirmed in BM sections from MM patients, both at diagnosis and relapse, with two M2 markers, CD163 and CD206. By following different TAM subpopulations during disease progression in the syngeneic murine 5T33MM model, we demonstrated a decrease in the number of inflammatory monocytes and an increase in the number of M2‐oriented TAMs in BM. Co‐culture experiments demonstrated that macrophages provide a survival benefit to myeloma cells that is maintained after treatment with several classes of anti‐myeloma agent (melphalan and bortezomib); the greatest effect was observed with M2‐polarized macrophages. The pro‐survival effect was associated with activation of the STAT3 pathway in 5T33MM cells, less cleavage of caspase‐3, and thus less apoptosis. AZD1480, an ATP‐competitive JAK2 inhibitor, abrogated the observed TAM‐mediated MM cell survival, and partially inhibited resistance to bortezomib. Despite having only a small quantitative impact on myeloid cells in vivo, AZD1480 treatment alone and in combination with bortezomib significantly reduced tumour load. In conclusion, M2 TAMs are present in the MM microenvironment, and contribute to MM cell survival and protection from drug‐induced apoptosis. As a result of TAM‐induced activation of the STAT3 pathway, 5T33MM cells are sensitized to AZD1480 treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

FCRL3 promotes TLR9‐induced B‐cell activation and suppresses plasma cell differentiation

Fc receptor‐like (FCRL) molecules are preferentially expressed by B lymphocytes and possess tyrosine‐based immunoregulatory function. Although they generally inhibit B‐cell receptor signaling, their influence on other activation pathways remains largely unexplored. In humans, FCRL3 encodes a type I transmembrane protein harboring both cytoplasmic ITAM and ITIM elements that can repress B‐cell receptor activation. Despite this inhibitory property, mounting associations for FCRL3 with autoimmune and lympho‐proliferative disorders imply a role for it in promoting B‐cell pathogenesis. Here, we explore the influence of FCRL3 on B‐cell responses to innate TLR9 stimulation. A detailed survey of blood B‐cell populations found that FCRL3 expression increased as a function of differentiation and was higher among memory subsets with innate‐like features. FCRL3 ligation augmented CpG oligodeoxynucleotide TLR9‐mediated B‐cell proliferation, activation, and survival, but surprisingly, abrogated plasma cell differentiation and antibody production. Although FCRL3 amplified the NF‐κB and mitogen‐activated protein kinase signaling cascades, it halted CpG triggered BLIMP1 induction in an ERK‐dependent fashion. These findings indicate that FCRL3 differentially modulates innate signaling in B cells and provide new insight into the potential of this disease‐associated receptor to counter‐regulate adaptive and innate immunity.     

Lactobacillus crispatus strain SJ‐3C‐US induces human dendritic cells (DCs) maturation and confers an anti‐inflammatory phenotype to DCs

Lactobacillus crispatus is one of the most predominant species in the healthy vagina microbiota. Nevertheless, the interactions between this commensal bacterium and the immune system are largely unknown. Given the importance of the dendritic cells (DCs) in the regulation of the immunity, this study was performed to elucidate the influence of vaginal isolated L. crispatus SJ‐3C‐US from healthy Iranian women on DCs, either directly by exposure of DCs to ultraviolet‐inactivated (UVI) and heat‐killed (HK) L. crispatus SJ‐3C‐US or indirectly to its cell‐free supernatant (CFS), and the outcomes of immune response. In this work we showed that L. crispatus SJ‐3C‐US induced strong dose‐dependent activation of dendritic cells and production of high levels of IL‐10, whereas IL‐12p70 production was induced at low level in an inverse dose‐dependent manner. This stimulation skewed T cells polarization toward CD4⁺ CD25⁺ FOXP3⁺ Treg cells and production of IL‐10 in a dose‐dependent manner in mixed leukocyte reaction (MLR) test. The mode of bacterial inactivation did not affect the DCs activation pattern, upon encounter with L. crispatus SJ‐3C‐US. Moreover, while DCs stimulated with CFS showed moderate phenotypic maturation and IL‐10 production, it failed to skew T cells polarization toward CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg) and production of IL‐10. This study showed that L. crispatus SJ‐3C‐US confers an anti‐inflammatory phenotype to DCs through up‐regulation of anti‐inflammatory/regulatory IL‐10 cytokine production and induction of CD4⁺ CD25⁺ FOXP3⁺ T cells at optimal dosage. Our findings suggest that L. crispatus SJ‐3C‐US could be a potent candidate as protective probiotic against human immune‐mediated pathologies, such as chronic inflammation, vaginitis or pelvic inflammatory disease (PID).     

Serum amyloid A3 exacerbates cancer by enhancing the suppressive capacity of myeloid‐derived suppressor cells via TLR2‐dependent STAT3 activation

Myeloid‐derived suppressor cells (MDSCs), which suppress diverse innate and adaptive immune responses and thereby provide an evasion mechanism for tumors, are emerging as a key population linking inflammation to cancer. Although many inflammatory factors that induce MDSCs in the tumor microenvironment are known, the crucial components and the underlying mechanisms remain elusive. In this study, we proposed a novel mechanism by which serum amyloid A3 (SAA3), a well‐known inflammatory factor, connects MDSCs with cancer progression. We found that SAA3 expression in BALB/c mice increased in monocytic MDSCs (Mo MDSCs) with tumor growth. The induction of SAA3 by apo‐SAA treatment in Mo MDSCs enhanced their survival and suppressive activity, while it inhibited GM‐CSF‐induced differentiation. Endogenous SAA3 itself contributed to the increase in the survival and suppressive activity of Mo MDSCs. We demonstrated that SAA3 induced TLR2 signaling, in turn increasing the autocrine secretion of TNF‐α, that led to STAT3 activation. In addition, activated STAT3 enhanced the suppressive activity of Mo MDSCs. Furthermore, SAA3 induction in Mo MDSCs contributed to accelerating tumor progression in vivo. Collectively, these data suggest a novel mechanism by which Mo MDSCs mediate inflammation through SAA3‐TLR2 signaling and thus exacerbate cancer progression by a STAT3‐dependent mechanism.     

Properdin and factor H production by human dendritic cells modulates their T‐cell stimulatory capacity and is regulated by IFN‐γ

Dendritic cells (DCs) and complement are both key members of the innate and adaptive immune response. Recent experimental mouse models have shown that production of alternative pathway (AP) components by DCs strongly affects their ability to activate and regulate T‐cell responses. In this study we investigated the production and regulation of properdin (fP) and factor H (fH) both integral regulators of the AP, by DCs and tolerogenic DCs (tolDCs). Both fP and fH were produced by DCs, with significantly higher levels of both AP components produced by tolDCs. Upon activation with IFN‐γ both cells increased fH production, while simultaneously decreasing production of fP. IL‐27, a member of the IL‐12 family, increased fH, but production of fP remained unaffected. The functional capacity of fP and fH produced by DCs and tolDCs was confirmed by their ability to bind C3b. Inhibition of fH production by DCs resulted in a greater ability to induce allogenic CD4⁺ T‐cell proliferation. In contrast, inhibition of fP production led to a significantly reduced allostimulatory capacity. In summary, this study shows that production of fP and fH by DCs, differentially regulates their immunogenicity, and that the local cytokine environment can profoundly affect the production of fP and fH.     

Imbalance of different types of CD4+forkhead box protein 3 (FoxP3)+ T cells in patients with new‐onset systemic lupus erythematosus

The aim of this study was to examine the numbers of CD4⁺CD25⁻forkhead box protein 3 (FoxP3)⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells in patients with new-onset systemic lupus erythematosus (SLE). The numbers of CD4⁺CD25⁻FoxP3⁺, CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and the concentrations of serum interleukin (IL)-10 in 23 patients and 20 healthy controls (HC) were measured. The potential correlations between CD4⁺FoxP3⁺ T cells, serum IL-10 and clinical measures in SLE patients were analysed. In comparison with that in the HC, significantly reduced numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells, but increased numbers of CD4⁺CD25⁻FoxP3⁺ T cells, were detected, accompanied by significantly lower levels of serum IL-10 in the patients. Stratification analysis indicated the numbers of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CXCR5⁺FoxP3⁺ T cells and serum IL-10 levels in the patients with seropositive anti-dsDNA were significantly less than that in those with seronegative anti-dsDNA. Treatment with the anti-SLE therapy, particularly with prednisone, leflunomide and methotrexate, significantly improved the imbalance of these types of FoxP3⁺ T cells and increased the concentrations of serum IL-10 in the drug-responding patients. The numbers of CD4⁺CD25⁺FoxP3⁺ T cells were correlated negatively with the values of SLE disease activity index (SLEDAI), whereas the numbers of CD4⁺CD25⁻FoxP3⁺ T cells were correlated positively with the values of SLEDAI, erythrocyte sedimentation rate (ESR) and serum C3. In addition, the concentrations of serum IL-10 were correlated positively with the numbers of CD4⁺CD25⁺FoxP3⁺ T cells, but negatively with the values of SLEDAI, serum C3, CRP and ESR in these patients. Our data indicate that the imbalance of different types of FoxP3⁺CD4⁺ T cells may contribute to the development of SLE in Chinese patients.     

TLR2 engagement on memory CD8+ T cells improves their cytokine‐mediated proliferation and IFN‐γ secretion in the absence of Ag

Persistence of memory CD8⁺ T cells is known to be largely controlled by common gamma chain cytokines, such as IL‐2, IL‐7 and IL‐15. However, other molecules may be involved in this phenomenon. We show here that TLR2^?/? mice have a decreased frequency of memory phenotype CD8⁺ T cells when compared with WT mice. This prompted us to investigate the role of TLR2 in the homeostasis of memory CD8⁺ T cells. We describe here a new TLR2‐dependent mechanism which, in the absence of specific antigen, directly controls memory CD8⁺ T‐cell proliferation and IFN‐γ secretion. We demonstrate that TLR2 engagement on memory CD8⁺ T cells increases their proliferation and expansion induced by IL‐7 both in vitro and in vivo. We also show that TLR2 ligands act in synergy with IL‐2 to induce IFN‐γ secretion in vitro. Both conclusions are obtained with spontaneously arising memory phenotype and antigen‐specific memory CD8⁺ T cells. Altogether, our data support the idea that continuous TLR2 signaling in response to microbial stimuli or endogenous danger signals might directly contribute to the maintenance of the diversity memory CD8⁺ T cells in the organism.     

Dietary gluten alters the balance of pro‐inflammatory and anti‐inflammatory cytokines in T cells of BALB/c mice

Several studies have documented that dietary modifications influence the development of type 1 diabetes. However, little is known about the interplay of dietary components and the penetration of diabetes incidence. In this study we tested if wheat gluten is able to induce differences in the cytokine pattern of Foxp3⁺ regulatory T cells, as well as Foxp3⁻ T cells, isolated from intestinal mucosal lymphoid tissue and non-mucosal lymphoid compartments in BALB/c mice. The gluten-containing standard diet markedly changed the cytokine expression within Foxp3⁻ T cells, in all lymphoid organs tested, towards a higher expression of pro-inflammatory interferon-γ (IFN-γ), interleukin-17 (IL-17) and IL-2. In Foxp3⁺ regulatory T cells, gluten ingestion resulted in a mucosal increase in IL-17 and IL-2 and an overall increase in IFN-γ and IL-4. The gluten-free diet induced an anti-inflammatory cytokine profile with higher proportion of transforming growth factor-β (TGF-β)⁺ Foxp3⁻ T cells in all tested lymphoid tissues and higher IL-10 expression within non-T cells in spleen, and a tendency towards a mucosal increase in TGF-β⁺ Foxp3⁺ regulatory T cells. Our data shows that the gluten-containing standard diet modifies the cytokine pattern of both Foxp3⁻ T cells and Foxp3⁺ regulatory T cells towards a more inflammatory cytokine profile. This immune profile may contribute to the higher type 1 diabetes incidence associated with gluten intake.     

Caspase‐4 mediates non‐canonical activation of the NLRP3 inflammasome in human myeloid cells

Inflammasome activation culminates in activation of caspase‐1, which leads to the maturation and subsequent release of cytokines of the interleukin 1 (IL‐1) family and results in a particular form of cell death known as pyroptosis. In addition, in the murine system, a so‐called non‐canonical inflammasome involving caspase‐11 has been described that directly responds to cytosolic LPS. Here, we show that the human monocytic cell line THP1 activates the inflammasome in response to cytosolic LPS in a TLR4‐independent fashion. This response is mediated by caspase‐4 and accompanied by caspase‐1 activation, pyroptosis, and IL‐1β maturation. In addition to caspase‐4, efficient IL‐1β conversion upon intracellular LPS delivery relies on potassium efflux, NLRP3, ASC, and caspase‐1, indicating that although caspase‐4 activation alone is sufficient to induce pyroptosis, this process depends on the NLRP3 inflammasome activation to drive IL‐1β maturation. Altogether, this study provides evidence for the presence of a non‐canonical inflammasome in humans and its dependence on caspase‐4.     

Interferon‐γ enhances both the anti‐bacterial and the pro‐inflammatory response of human mast cells to Staphylococcus aureus

Human mast cells (huMCs) are involved in both innate and adaptive immune responses where they release mediators including amines, reactive oxygen species (ROS), eicosanoids and cytokines. We have reported that interferon‐γ (IFN‐γ) enhances FcγR‐dependent ROS production. The aim of this study was to extend these observations by investigating the effect of IFN‐γ on the biological responses of huMCs to Staphylococcus aureus. We found that exposure of huMCs to S. aureus generated intracellular and extracellular ROS, which were enhanced in the presence of IFN‐γ. IFN‐γ also promoted bacteria killing, β‐hexosaminidase release and eicosanoid production. Interferon‐γ similarly increased expression of mRNAs encoding CCL1 to CCL4, granulocyte–macrophage colony‐stimulating factor (GM‐CSF), tumour necrosis factor‐α and CXCL8 in S. aureus‐stimulated huMCs. The ability of IFN‐γ to increase CXCL8 and GM‐CSF protein levels was confirmed by ELISA. Fibronectin or a β₁ integrin blocking antibody completely abrogated IFN‐γ‐dependent S. aureus binding and reduced S. aureus‐dependent CXCL8 secretion. These data demonstrate that IFN‐γ primes huMCs for enhanced anti‐bacterial and pro‐inflammatory responses to S. aureus, partially mediated by β₁ integrin.     

Flt3 ligand‐eGFP‐reporter expression characterizes functionally distinct subpopulations of CD150+ long‐term repopulating murine hematopoietic stem cells

The pool of hematopoietic stem cells (HSCs) in the bone marrow is a mixture of resting, proliferating, and differentiating cells. Long‐term repopulating HSCs (LT‐HSC) are routinely enriched as Lin^?Sca1⁺c‐Kit⁺CD34^?Flt3^?CD150⁺CD48^? cells. The Flt3 ligand (Flt3L) and its receptor Flt3 are important regulators of HSC maintenance, expansion and differentiation. Using Flt3L‐eGFP reporter mice, we show that endogenous Flt3L‐eGFP‐reporter RNA expression correlates with eGFP‐protein expression. This Flt3L‐eGFP‐reporter expression distinguishes two LT‐HSC populations with differences in gene expressions and reconstituting potential. Thus, Flt3L‐eGFP‐reporter^low cells are identified as predominantly resting HSCs with long‐term repopulating capacities. In contrast, Flt3L‐eGFP‐reporter^high cells are in majority proliferating HSCs with only short‐term repopulating capacities. Flt3L‐eGFP‐reporter^low cells express hypoxia, autophagy‐inducing, and the LT‐HSC‐associated genes HoxB5 and Fgd5, while Flt3L‐eGFP‐reporter^high HSCs upregulate genes involved in HSC differentiation. Flt3L‐eGFP‐reporter^low cells develop to Flt3L‐eGFP‐reporter^high cells in vitro, although Flt3L‐eGFP‐reporter^high cells remain Flt3L‐eGFP‐reporter^high. CD150⁺Flt3L‐eGFP‐reporter^low cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L‐eGFP‐reporter^high cells do express EPCR but not CD41. Thus, FACS‐enrichment of Flt3/ Flt3L‐eGFP‐reporter negative, Lin^?CD150⁺CD48^? EPCR⁺CD41⁺ HSCs allows a further 5‐fold enrichment of functional LT‐HSCs.