IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

CREB and AP‐1 activation regulates MKP‐1 induction by LPS or M‐CSF and their kinetics correlate with macrophage activation versus proliferation

MAPK phosphatase‐1 (MKP‐1) is a protein phosphatase that plays a crucial role in innate immunity. This phosphatase inactivates ERK1/2, which are involved in two opposite functional activities of the macrophage, namely proliferation and activation. Here we found that although macrophage proliferation and activation induce MKP‐1 with different kinetics, gene expression is mediated by the proximal promoter sequences localized between ?380 and ?180 bp. Mutagenesis experiments of the proximal element determined that CRE/AP‐1 is required for LPS‐ or M‐CSF‐induced activation of the MKP‐1 gene. Moreover, the results from gel shift analysis and chromatin immunoprecipitation indicated that c‐Jun and CREB bind to the CRE/AP‐1 box. The distinct kinetics shown by M‐CSF and LPS correlates with the induction of JNK and c‐jun, as well as the requirement for Raf‐1. The signal transduction pathways that activate the induction of MKP‐1 correlate kinetically with induction by M‐CSF and LPS.     

Pannexin‐1‐dependent caspase‐1 activation and secretion of IL‐1β is regulated by zinc

Inflammatory processes induced by IL‐1β are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase‐1 activation and IL‐1β secretion. One of the endogenous mediators of IL‐1β secretion is adenosine triphosphate, acting via the P2X7‐receptor and caspase‐1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre‐treatment with the zinc chelator N,N,N′,N′‐tetrakis‐(2‐pyridylmethyl) ethylene diamine (TPEN). These effects on IL‐1β secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase‐1 activity, and upstream of caspase‐1 activation. TPEN did however inhibit the activity of pannexin‐1, a hemi‐channel critical for adenosine triphosphate and nigericin‐induced IL‐1β release. These data provide new insights into the mechanisms of caspase‐1 activation and how zinc metabolism contributes to inflammatory mechanisms.     

The CDK domain of p21 is a suppressor of IL‐1β‐mediated inflammation in activated macrophages

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21^(WAF1/CIP1), an established suppressor of cell cycle progression, is a inhibitor of IL‐1β synthesis in macrophages. Mice deficient in p21 (p21^?/?) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL‐1β. Administration of IL‐1 receptor antagonist reduces LPS‐induced lethality in p21^?/? mice. Analysis of isolated macrophages, which are one of the central producers of IL‐1β, reveals that deficiency for p21 led to more IL‐1β mRNA and pro‐protein synthesis following TLR ligation. The increase in IL‐1β pro‐protein is associated with elevated secretion of active IL‐1β by p21^?/? macrophages. siRNA‐mediated knockdown of p21 in human macrophages results in increased IL‐1β secretion as well. A peptide mapping strategy shows that the cyclin‐dependent‐kinase (CDK)‐binding domain of p21 is sufficient to reduce the secretion of IL‐1β by p21^?/? macrophages. These data suggest a novel role for p21 and specifically for the CDK‐binding domain of p21^(WAF1/CIP1) in inhibiting inflammation.     

Th2 cell‐derived IL‐4/IL‐13 promote ILC2 accumulation in the lung by ILC2‐intrinsic STAT6 signaling in mice

Infection of mice with the gastrointestinal helminth Nippostrongylus brasiliensis elicits profound local proliferation and accumulation of type 2 innate lymphoid cells (ILC2s) in the lung. The regulation of ILC2 proliferation and accumulation in the lung is poorly understood. Using T cell‐specific IL‐4/IL‐13‐deficient mice, we demonstrate that IL‐4/IL‐13 secretion from Th2 cells promotes proliferation and expansion of the ILC2 population in the lung of N. brasiliensis‐infected mice. Competitive mixed BM chimeras containing normal and STAT6‐deficient ILC2s further indicated that ILC2s have to respond directly to IL‐4/IL‐13 for this effect while STAT6 is not required for IL‐13 production in ILC2s. In addition, expression of a constitutively active form of STAT6 in ILC2s was sufficient to promote their proliferation in uninfected mice. The expression of MHC class II in ILC2s appeared to be enhanced by STAT6 signaling supporting the concept that Th2 cells and ILC2s can communicate in an antigen‐dependent manner resulting in a Th2‐regulated accumulation of ILC2s in the lung during an acute type 2 immune response. Based on our observations, targeting the STAT6 pathway in ILC2s could help to develop new treatments to dampen ILC2 proliferation in the lung and thereby ameliorate ILC2‐mediated allergic inflammation.     

NR4A1‐dependent Ly6Clow monocytes contribute to reducing joint inflammation in arthritic mice through Treg cells

Monocytes are central to the physiopathology of arthritis, but their roles in progression and resolution of the disease remain to be clarified. Using NR4A1^?/? mice, which lack patrolling lymphocyte antigen 6C (Ly6C^low) monocytes, we found that inflammatory Ly6C^high monocytes contribute to rapid development of arthritis in a serum transfer‐induced arthritis (STIA) model. Our experiments suggest that patrolling monocytes do not promote the initiation and progression of arthritis in mice, as severity of symptoms was amplified in NR4A1^?/? mice. Moreover, we show that treatment of arthritic wild type (WT) mice with cytosporone B (Csn‐B), a NR4A1‐specific agonist, significantly reduces severity of disease. Effects of Csn‐B were absent in monocyte‐depleted mice treated with clodronate until Ly6C^low monocytes were restored. Adoptive transfer of Ly6C^low monocytes in arthritic NR4A1^?/? mice treated with Csn‐B reduces joint inflammation, supporting the regulatory role of Ly6C^low subset on disease development. Our results also reveal that administration of Csn‐B to arthritic mice enhances levels of circulating CD4⁺CD25⁺FoxP3⁺ Treg cells, a process requiring the presence of Ly6C^low monocytes. Together, these data indicate that Ly6C^high monocytes are involved in the initiation and progression of arthritis and Ly6C^low monocytes contribute to reduce joint inflammation through the mobilization of Treg cells.     

ORIGINAL ARTICLE: Expression of IL‐6, IL‐8, TNF‐α, IL‐10, HSP‐60, Anti‐HSP‐60 Antibodies,and Anti‐sperm Antibodies,in Semen of Men with Leukocytes and/or Bacteria

Citation Martínez‐Prado E, Bermúdez MIC. Expression of IL‐6, IL‐8, TNF‐α, IL‐10, HSP‐60, anti‐HSP‐60 antibodies, and anti‐sperm antibodies, in semen of men with leukocytes and/or bacteria. Am J Reprod Immunol 2010; 63: 233–243 Problem Different cellular and biochemical markers have been proposed as indicators of infection‐inflammation of male genital tract. Method of study Semen samples from 80 men attending an andrologic clinic were evaluated to determine the presence of leukocyte, bacteria, antibodies against Chlamydia trachomatis, levels of IL‐6, IL‐8, IL‐10, and TNF‐α, HSP‐60, anti‐HSP‐60 antibodies, and anti‐sperm antibodies. Results Leukocytes in semen significantly correlated with an increase in IL‐6, IL‐8, and TNF‐α. The simultaneous presence of pathogens and leukocytes was associated with high levels of IL‐8 and TNF‐α, whereas IL‐6 was more associated with the presence of leukocytes. Anti‐HSP‐60 antibodies positively correlated with IL‐6 and IL‐8. The presence of anti‐sperm antibodies highly associated with an increase in anti‐HSP‐60 antibodies. Conclusions The type of cytokines present in the semen will depend on the single or simultaneous presence of leukocytes and/or pathogens. Chronic male genital tract infections could be associated with the development of anti‐HSP‐60 antibodies and anti‐sperm antibodies.     

LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis,selectively induces pro‐inflammatory cytokines and IL‐10 and is regulated by TNF‐α, IL‐10 and IFN‐γ

Leukocyte immunoglobulin‐like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane‐bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross‐linking of LILRA5 on monocytes induced production of pro‐inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common γ chain of the FcR and LILRA5 cross‐linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro‐inflammatory cytokines as well as IL‐10. LILRA5 mRNA and protein expression was tightly regulated by TNF‐α, IL‐10 and IFN‐γ. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.     

IL‐12 and IL‐4 activate a CD39‐dependent intrinsic peripheral tolerance mechanism in CD8+ T cells

Immune responses to protein antigens involve CD4⁺ and CD8⁺ T cells, which follow distinct programs of differentiation. Naïve CD8 T cells rapidly develop cytotoxic T‐cell (CTL) activity after T‐cell receptor stimulation, and we have previously shown that this is accompanied by suppressive activity in the presence of specific cytokines, i.e. IL‐12 and IL‐4. Cytokine‐induced CD8⁺ regulatory T (Treg) cells are one of several Treg‐cell phenotypes and are Foxp3^? IL‐10⁺ with contact‐dependent suppressive capacity. Here, we show they also express high level CD39, an ecto‐nucleotidase that degrades extracellular ATP, and this contributes to their suppressive activity. CD39 expression was found to be upregulated on CD8⁺ T cells during peripheral tolerance induction in vivo, accompanied by release of IL‐12 and IL‐10. CD39 was also upregulated during respiratory tolerance induction to inhaled allergen and on tumor‐infiltrating CD8⁺ T cells. Production of IL‐10 and expression of CD39 by CD8⁺ T cells was independently regulated, being respectively blocked by extracellular ATP and enhanced by an A2A adenosine receptor agonist. Our results suggest that any CTL can develop suppressive activity when exposed to specific cytokines in the absence of alarmins. Thus negative feedback controls CTL expansion under regulation from both nucleotide and cytokine environment within tissues.     

Critical role of p38 MAPK in IL‐4‐induced alternative activation of peritoneal macrophages

Alternative activation of macrophages plays an important role in a range of physiological and pathological processes. This alternative phenotype, also known as M2 macrophages, is induced by type 2 cytokines such as IL‐4. The binding of IL‐4 to its receptor leads to activation of two major signaling pathways: STAT‐6 and PI3K. However, recent studies have described that p38 MAPK might play a role in IL‐4‐dependent signaling in some cells, although its role in macrophages is still controversial. In this study, we investigated whether p38 MAPK plays a role in the polarization of macrophages in mice. Our results reveal that IL‐4 induces phosphorylation of p38 MAPK in thioglycollate‐elicited murine peritoneal macrophages, in addition to STAT‐6 and PI3K activation. Furthermore, p38 MAPK inactivation, by gene silencing or pharmacological inhibition, suppressed IL‐4‐induced typical M2 markers, indicating the involvement of p38 MAPK in the signaling of IL‐4 leading to M2‐macrophage polarization. Moreover, p38 MAPK inhibition blocked phosphorylation of STAT‐6 and Akt, suggesting that p38 MAPK is upstream of these signaling pathways. Finally, we show that in an in vivo model of chitin‐induced M2 polarization, p38 MAPK inhibition also diminished activation of M2 markers. Taken together, our data establish a new role for p38 MAPK during IL‐4‐induced alternative activation of macrophages.     

Differential expression of constitutive and inducible proteasome subunits in human monocyte‐derived DC differentiated in the presence of IFN‐α or IL‐4

Several studies strongly suggest that DC differentiated in vitro in the presence of type I IFN acquire more potent immune stimulatory properties, compared with DC differentiated in vitro with IL‐4. However, little is known about the molecular mechanisms underlying this phenomenon. To address this question, we compared the Ag‐processing machinery (APM) profile in human DC grown in the presence of IFN‐α (_IFNDC) or IL‐4 (_IL‐4DC). Using a panel of APM component‐specific mAb in Western blot experiments, we found that _IFNDC preferentially express inducible proteasome subunits (LMP2, LMP7, and MECL1) both at immature and mature stages. In contrast, immature _IL‐4DC co‐express both constitutive (β1, β2, and β5) and inducible subunits, as shown by Western blotting analysis. In addition, immature _IFNDC express higher levels of TAP1, TAP2, calnexin, calreticulin, tapasin, and HLA class I molecules than _IL‐4DC. The different proteasome profiles of _IFNDC and _IL‐4DC were associated with a greater ability of _IFNDC to present an immunodominant epitope that requires LMP7 expression for its processing. In general, these data show the impact of cytokines on APM component expression and hence the Ag‐processing ability of DC.     

Early onset wheeze associated with enhanced combined IL‐1β, IL‐6, and IL‐12/IL‐23p40 in LPS‐stimulated cord blood mononuclear cells

Background Neonates with a family history of atopy are at higher risk for developing wheezing in early life. Objective From a birth cohort of at risk infants (first‐degree family with atopic disease), we evaluated the influence of distinct intrinsic immunologic risk factors on wheezing disorders in the first 2 years of life. Methods Cord blood samples were collected from 195 eligible subjects of a birth cohort of 253 subjects. The subjects studied were those who developed wheezing (n=34) or eczema (n=29) in the first 2 years of life, and 65 healthy control infants. At the time of thawing the viability of the cells were median 70% (range 67.5%–72.5%). Cytokines from lipopolysaccharide (LPS)‐stimulated mononuclear cells were analysed using fluorescent‐activated cell sorting‐array and their profiles were evaluated using factor analysis. Results Infants with wheeze were significantly associated with enhanced combined LPS stimulated IL‐1β, IL‐6, and IL‐12/IL‐23p40 compared with healthy controls (P=0.003). This profile was also associated with the increased risk for wheeze at 2 years of age (OR=2.45; 95% CI=1.50–3.93, P=0.001). LPS‐stimulated cytokine IL‐8 was also significantly higher in the wheeze group compared with healthy controls and eczema (P=0.003). Intracellular staining showed that monocytes are main producers of IL‐6 and IL‐8 from cord blood mononuclear cells. Most of the subjects were non‐atopic with 3/34 (9%) wheeze and 9/29 (31%) eczema subjects sensitized to the common dietary or inhalant allergens. Conclusion and Clinical Relevance In infants at genetic risk of atopy, wheeze but not eczema in the first 2 years of life is associated with intrinsic hyperresponsive innate cytokine responses which might predispose infants to wheeze development. Distinct pre‐symptomatic hyperresponsive innate immune responses risk factors were found to be associated with early onset wheeze disorders, but not eczema. Cite this as: P. L. Quah, I‐C Kuo, C. H. Huang, L. P‐C Shek, B. W. Lee and K. Y. Chua, Clinical & Experimental Allergy, 2011 (41) 970–978.     

CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL‐17 in a STAT3‐dependent manner

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell‐based clinical therapy. However, human Treg cells are “plastic”, and are able to produce IL‐17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL‐17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL‐17 in vitro when activated in the presence of IL‐1β, but not IL‐6. “IL‐17 potential” is restricted to population III (CD4⁺CD25^hiCD127^loCD45RA^?) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL‐17 induction. Importantly, we find that IL‐17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL‐17. Finally, we show that CD161⁺ population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL‐17‐producing Treg‐cell population at these sites. As IL‐17 production from this Treg‐cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.