IL-10 KO Mice Research Articles

IL-4 Plays a Pivotal Role in Regulating the Development of Th2 and Th17 Responses in the Airways

RATIONALE: IL-33 is produced by tissue cells, such as airway epithelial cells and endothelial cells, and has been implicated in the development of Th2-type inflammatory responses in mucosal organs. However, the roles for IL-4 in the development of IL-33-mediated airway immunity are unknown. METHODS: Naïve 4get, BALB/cJ (WT) and IL-4 knockout (IL-4 KO) mice were exposed intranasally to a model antigen, endotoxin-free ovalbumin (OVA), with or without IL-33. In vitro splenocyte recall responses to OVA and intracellular expression of cytokines, as well as in vivo airway responses to intranasal challenge with OVA, were studied. RESULTS: Four days after exposure to OVA plus IL-33, naïve 4get mice showed IL-4 expression by CD4+ T cells. After 2 weeks, this response was followed by robust development of OVA antigen-specific, Th2-type CD4+ T cells. Intranasal OVA challenge of WT mice, previously exposed to OVA plus IL-33, provoked airway Th2 cytokine production and eosinophilia. In contrast, intranasal OVA challenge of IL-4 KO mice, previously exposed to OVA plus IL-33, partially impaired the development of OVA-specific Th2-type CD4+ T cells, but enhanced the development of Th17-type CD4+ T cells. These mice also developed airway neutrophilia accompanied by increased airway production of IL-12p40, IL-17 and keratinocyte-derived chemokine. CONCLUSIONS: IL-33 may serve as a potent airway mucosal adjuvant to induce adaptive T cell responses to inhaled antigens. When airways are exposed to IL-33, IL-4 may play a pivotal role to direct to the development of either Th2- or Th17-type CD4+ T cells.

Local pulmonary immunotherapy with siRNA targeting TGFβ1 enhances antimicrobial capacity in Mycobacterium tuberculosis infected mice

In this study we demonstrate that it is possible to shift the immune system during a chronic infection with Mycobacterium tuberculosis. TGFβ and IL10 cytokines inhibit the Th1 response during chronic pulmonary infection with M. tuberculosis. We show that intrapulmonary delivery of siRNA targeting TGFβ1 is able to reduce the pulmonary bacillary load in mice chronically infected with M. tuberculosis: an effect that appears to be partly dependent on IL10 expression. To demonstrate this, we induced gene silencing of tgfβ1 in the lungs of wild type and IL10 knockout mice using a non-invasive aerosolized intrapulmonary delivery of siRNA targeting TGFβ1. Five days after the last treatment with siRNA, the levels of tgfb1 transcripts and TGFβ1 protein were reduced when compared with control groups treated with RNase-free water or non-targeting siRNA. Mice treated with siRNA also had increased expression of the antimicrobial mediators (NO and iNOS) which effectively reduced the bacterial load by 0.17 and 0.47 log(10) in C57BL/6 and IL-10 KO mice respectively when compared with their respective control mice. More importantly, the bacterial load in siRNA treated IL-10 KO mice four weeks after the last treatment remained 0.32 log(10) lower than in control mice.

Role of heat shock protein 47 in intestinal fibrosis of experimental colitis

Background and aims Intestinal fibrosis is a clinically important issue of inflammatory bowel disease (IBD). It is unclear whether or not heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in intestinal fibrosis. The aim of this study is to investigate the role of HSP47 in intestinal fibrosis of murine colitis. Methods HSP47 expression and localization were evaluated in interleukin-10 knockout (IL-10KO) and wild-type (WT, C57BL/6) mice by immunohistochemistry. Expression of HSP47 and transforming growth factor-β1 (TGF-β1) in colonic tissue was measured. In vitro studies were conducted in NIH/3T3 cells and primary culture of myofibroblasts separated from colonic tissue of IL-10KO (PMF KO) and WT mice (PMF WT) with stimulation of several cytokines. We evaluated the inhibitory effect of administration of small interfering RNA (siRNA) targeting HSP47 on intestinal fibrosis in IL-10KO mice in vivo. Results Immunohistochemistry revealed HSP47 positive cells were observed in the mesenchymal and submucosal area of both WT and IL-10 KO mice. Gene expressions of HSP47 and TGF-β1 were significantly higher in IL-10KO mice than in WT mice and correlated with the severity of inflammation. In vitro experiments with NIH3T3 cells, TGF-β1 only induced HSP47 gene expression. There was a significant difference of HSP47 gene expression between PMF KO and PMF WT. Administration of siRNA targeting HSP47 remarkably reduced collagen deposition in colonic tissue of IL-10KO mice. Conclusions Our results indicate that HSP47 plays an essential role in intestinal fibrosis of IL-10KO mice, and may be a potential target for intestinal fibrosis associated with IBD.

The Role of Interleukin-6 and Bone-Marrow Derived Cell Production of Hepcidin In Anemia of Inflammation

AbstractAbstract 167Anemia of inflammation is the second most common form of anemia in the general population, and its impact on patient well-being is largely underestimated. Anemia cause by inflammation is multi-factorial and includes hepcidin-induced iron restricted erythropoiesis as well as direct cytokine effects on the bone marrow, erythropoietin production and efficacy, and on the lifespan of red cells. Many murine models of anemia of inflammation are unreliable or cumbersome, but a new model introduced by Sasu et al (Blood, 2010) using a single intraperitoneal injection of heat-killed brucella abortus antigen (HKBA) has proven reproducible and robust. We have used this model to explore the role of interleukin-6 and bone marrow derived cell production of hepcidin in anemia of inflammation (AI). First, we sought to explore the effect this model of AI in wild type mice, iterleukin-6 knockout mice (IL6-KO) and hepcidin knockout mice (Hamp-KO) (n≥15 for each group). We followed these mice for 7 weeks with weekly CBC's to observe the severity and time to recovery from anemia. Wild type mice were most affected 2 weeks after injection and slowly recovered over 7 weeks (HgB at 2 week = 6.4g/dl ± 1.2). IL6-KO mice were equally affected initially, with similar hemoglobin values at 2 weeks (6.9g/dl ± 1.3) and recovered by 6 weeks. Hamp-KO mice were less affected throughout the course of anemia, with hemoglobin values of 10.8g/dl ± 0.7 at 2 weeks with resolution by week 4. IL6-KO mice began to recover more quickly than wild type mice by week three, with hemoglobin values of 10.9g/dl ± 1.5 at that time, compared to wild type mice at 3 weeks with hemoglobin values of 7.4g/dl ± 0.7 (p= 0.0001). We believe that this demonstrates that interleukin-6 and hepcidin do coordinate to contribute to anemia of inflammation, but that there may be independent effects or additional factors. To address these questions, we are currently evaluating iron-related gene expression in these groups of mice as well as evaluating iron stores at multiple time points. We also evaluated serum cytokine levels in each of these groups of mice. We found similar elevations TNF-alpha and interferon gamma in all three groups at 6 and 24 hours. We found similar elevations of IL-6 in wild type and Hamp-KO mice at 6 and 24 hours. Bone marrows and spleens form each group of mice were evaluated at 2 weeks by flow cytometry using ter119 and CD44 to evaluate specific effects on erythroid maturation. This evaluation demonstrated a a profound inhibitory effect on erythropoiesis and, in particular, on the production of erythroid progenitor cells, showing a similar profile by flow cytometry between the three groups. In vitro studies have suggested that macrophage production of hepcidin is important in the development of AI (Theurl et al 2008). We evaluated the importance of bone marrow derived cell production of hepcidin on the development of AI using bone marrow chimeras. Using 600cGy × 2 as a preparative regimen, we transplanted wild type mice with bone marrow from Hamp-KO mice. We also irradiated Hamp-KO mice and transplanted them with wild type marrow. We injected these two groups of mice as well as wild type and Hamp-KO controls, we followed them for a period of 4 weeks with weekly CBC's to evaluate the degree of anemia. Hemoglobin values of wild type mice transplanted with Hamp-KO marrow were statistically indistinguishable from those of non-transplanted wild type mice during the follow-up period (HgB values at 1 week = 6.8g ± 0.7 vs 7.29g ± 1.1; at 2 weeks = 7.3 ± 0.6 vs 6.4 ± 1.2; at 3 weeks = 8.5 ± 1.8 vs 7.4 ± 0.5; at 4 weeks = 9.1 v 1.9 vs 8.6 ± 0.5; p<0.02 for all time points). Hamp-KO mice with wild-type bone marrow were statistically indistinguishable from non-transplanted Hamp-KO mice (Hgb values at 1 week = 9.9 ± 2.4 vs 10.8 ± 0.7; at 2 weeks = 10.6 ± 1.5 vs 10.3 ± 1.0; at 3 weeks = 12.8 ± 1.2 10.8 ± 0.7; at 4 weeks 12.6 ± 1.2 vs. 13.6 ± 1.0; p<0.02 for all time points). This suggests that the production of hepcidin by bone marrow derived cells dose not play a physiologically important role in the development of anemia of inflammation. Disclosures:No relevant conflicts of interest to declare.

Interleukin-6 is an important mediator for mitochondrial DNA repair after alcoholic liver injury in mice

We investigated the hypothesis that a prominent effect of chronic ethanol consumption is mitochondrial DNA (mtDNA) injury and compared this injury in IL-6 knockout (KO) and wild-type (WT) mice. Ethanol feeding for 4 weeks resulted in steatosis and oxidative mtDNA damage (8-OHdG) in both IL-6KO and WT mice. However, the WT mice were able to repair the injury by increased production of mtDNA repair enzymes (OGG-1, Neil 1) and check point (p21, p53) proteins and avoid the mtDNA mutations. By contrast the IL-6 KO mice were unable to repair mtDNA resulting in deletions and diminished transcription of the mtDNA encoded protein cytochrome c oxidase subunit-I (COI). The mitochondrial injury was reflected by decreased membrane potential, reduced levels of ATP, and apoptosis-inducing factor (AIF)-induced apoptosis. IL-6 plays a critical role in allowing the liver to recover from significant mtDNA oxidation caused by alcohol. The data suggests that IL-6 activates mtDNA repair enzymes and induces cell cycle arrest allowing time for mtDNA repair.

Cellular and molecular mechanisms regulating the hepatic erythropoietin expression during acute-phase response: a role for IL-6

The source of circulating erythropoietin (EPO), the mediators and the mechanisms involved in the upregulation of EPO gene expression during acute-phase reaction are still poorly understood. Acute-phase reaction was induced by either intramuscular turpentine oil (TO) or intraperitoneal lipopolysaccharide (LPS) administration into wild-type and interleukin (IL)-6 knockout (KO) mice. Animals were killed at different time points and blood, liver and muscle tissue were collected. Serum levels of EPO were measured by enzyme-linked immunoadsorbent assay; liver and injured muscle samples were processed for RNA isolation and for protein analysis. EPO, hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) mRNA were analyzed by RT–PCR and the protein levels were analyzed by western blot and electrophoretic mobility shift assay. HIF-1α and HIF-2α localization was performed through immunofluorescence staining. EPO, HIF-1 and HIF-2 gene and protein expression levels were also analyzed in isolated mouse hepatocytes after stimulation with IL-6. In the wild-type animals, EPO serum levels increased dramatically at 12 h after the insults together with the hepatic gene expression. In TO-treated animals, the EPO gene expression reached an 8.2-fold increase at 12 h, and in LPS-treated mice a similar induction was recorded at 6 h (about 4.5-fold increase). In the IL-6KO strain, the upregulation after the inflammatory stimuli was much lower (only 2.0-fold increase). A progressive upregulation of HIF-1α and HIF-2α was detectable until 6 h after the insults, but only HIF-1α upregulation was reduced in IL-6KO mice. In isolated hepatocytes, stimulation with a single dose of IL-6 induced a nuclear accumulation of HIF-1α, in parallel with an increase of EPO mRNA. No effect on HIF-2α expression was found. IL-6 appears to be the main regulator of EPO gene expression and a major contributor for HIF-1α induction in hepatocytes and Kupffer cells during acute-phase response. The increase of HIF-2α, predominantly expressed in endothelial cells and fibroblast-like cells, seems not to be affected by the lack of IL-6.

New molecular mechanisms of the unexpectedly complex role of VEGF in ulcerative colitis

The effects of VEGF on endothelial cells are mediated by different intracellular signaling cascades (e.g., Erk1/2, Akt, Src). VEGF plays a recently recognized role in ulcerative colitis (UC) pathogenesis, mostly by increasing vascular permeability and promoting the infiltration of inflammatory cells. We hypothesized that the excessive activation of signal transduction pathways, which is responsible for VEGF/VEGFR-2-mediated endothelial permeability (Src, Akt), is a new element in the pathogenesis of chronic UC. We demonstrated increased expression of pro-angiogenic growth factor VEGF and its receptor VEGFR-2 in colonic tissue during acute 6% iodoacetamide-induced UC in rats and chronic spontaneously developed UC in IL-10 knockout mice (IL-10 KO). Development of acute 6% iodoacetamide-induced UC in rats was accompanied by activation of Erk1/2 and Src kinase, while expression of total proteins Erk1/2 and Src was unchanged. During chronic colitis phosphorylation (i.e., activation) of Erk1/2 was significantly decreased in IL-10 KO mice vs. wild-type mice. Levels of total Erk1/2 proteins were unchanged, but the expression of total Src protein as well as its phosphorylated form was significantly increased in IL-10 KO vs. wild-type mice. There were no changes in total Akt proteins, while levels of activated Akt (pAkt) were slightly increased in IL-10 KO vs. wild-type mice. We conclude that VEGF/VEGFR-2-associated signal transduction pathways, that mediate increased vascular permeability (Src, Akt), might play a central role in perpetuation of chronic experimental UC.

Acute ANIT Toxicity in Male IL-10 Knockout and Wild-type Mice

The innate immune response is known to modify hepatocellular injury induced by toxicants. To assess the role of IL-10, a component of the innate immune response, in toxicant-induced injury of biliary epithelium, wild-type (WT) and IL-10 knockout mice (KO) were given a single toxic dose (50 mg/kg) of alpha-napthylisothiocyanate (ANIT) and assessed at twenty-four-hour intervals for four days following treatment. Clinical signs of toxicity were greater in WT mice. Unexpectedly, over the course of the study, there was a consistent tendency for ANIT-treated IL-10 KO mice to have less hepatocellular injury than WT mice. However, changes in the biliary epithelium differed in that there was more histologic evidence of inflammation and necrosis on days 2 and 3, respectively, in ANIT-treated IL-10 KO mice compared with WT mice. Proliferation of biliary epithelium and hepatocytes was greater and/or occurred earlier in the ANIT-treated IL-10 KO mice compared with the ANIT-treated WT mice, suggesting a greater reparative response was needed for recovery after toxicant injury in the IL-10 KO mice. Overall, our data suggest that IL-10 KO mice have less hepatocellular injury than WT mice following a toxic dose of ANIT and that biliary epithelial injury is accentuated in the KO mice.

Filaria-induced IL-10 suppresses murine cerebral malaria

Filarial nematodes achieve long survival in their hosts due to their capacity to modulate immune responses. Therefore, immunomodulation by filarial nematodes may alter responses to concomitant infections such as malaria. Cerebral malaria (CM), a severe complication of Plasmodium falciparum infections, is triggered as a consequence of the immune response developed against malaria parasites. The question arises whether prior infection with helminth parasites is beneficial against CM. In the present work a murine model for subsequent has been used to assess this hypothesis. C57BL/6 mice were infected with the rodent filarial parasite Litomosoides sigmodontis and the murine model parasite for CM, Plasmodium berghei ANKA. Previously filaria-infected C57BL/6 mice showed significantly reduced CM rates. CD8 + T cell recruitment to the brain, a hallmark for CM development, was reduced in protected mice. Furthermore, in contrast to P. berghei single-infected animals, filaria-infected mice had significantly higher levels of circulating IL-10. The requirement for IL-10 in CM protection was demonstrated by the lack of protection in IL-10 KO mice. This suggests that the anti-inflammatory IL-10 elicited by filarial nematodes is able to suppress the overwhelming inflammatory reaction otherwise triggered against malaria parasites in C57BL/6 mice, preventing full progress to CM.

Abstract 5702: Soluble epoxide hydrolase: unique biomarker and chemopreventive target of chronic colitis-induced carcinogenesis

Abstract Chronic ulcerative colitis (UC) is a well-recognized risk factor of colon cancer; aberrant arachidonic acid (AA) metabolism in UC is a key event leading to cancer development. Epoxyeicosatrienoic acids (EETs) are AA metabolites with anti-inflammatory activity; under physiological conditions, EETs are quickly inactivated as they are converted to their corresponding dihydroxyeicosatrienoic acids (DHETs) by the enzyme soluble epoxide hydrolase (sEH). The inhibition of sEH retards hydrolysis of the epoxide, reducing the conversion of EETs to DHETs. Thus, sEH would be a unique target for colitis. However, the role of sEH in chronic colitis and its associated carcinogenesis is not known. In the present study, we have determined if the sEH over-expresses in human ulcerative colitis and its associated dysplasia and carcinoma. Tissue microarrays contained 180 patients (4 tissue samples/patient) with UC (72 patients), UC-dysplasia (54 patients), and UC-carcinoma (54 patients) were established. Using specific anti-sEH antibody and immunohistochemistry approach, distinct expression of sEH was found in the microarray samples. Normal colon displayed weak positivity in 39.1% (n=79), mainly in the focally reactive epithelia. In UC, 74.6% (n=72) displayed positivity, extensively expressed in the hyperplastic epithelia. 88.2% (n=54) of colitis-induced dysplasia and 93.9% (n=54) of colitis-induced carcinoma displayed positivity. Markedly increased sEH staining intensity was observed in UC-induced dysplasia and carcinoma. Secondly, the effect of highly selective and potent sEH inhibitor, trans/−4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), on chronic colitis-induced carcinoma development was determined in IL-10 knockout mice. t-AUCB was administered to IL-10 KO mice in the drinking fluid for three months with the doses of 1.25mg/kg and 2.5 mg/kg body weight (n=10 mice per group). Both low and high doses of t-AUCB significantly suppressed body weight reduction caused by colitis. Histopathologic analysis revealed that there were 80% tumor incidence (8/10 mice) in IL-10 colitis control mice; low and high doses of t-AUCB significantly inhibited the colitis-induced carcinoma development with tumor incidence 10% (1/10 mice) and 40% (4/10 mice), respectively. The tumor volume was markedly reduced (0.062 ± 0.019 cm3 in IL-10 KO colitis mice versus 0.026 ± 0.008 and 0.014 ± 0.012 cm3 in IL-10 KO colitis mice treated with low and high doses of t-AUCB, respectively). The results indicate that sEH is a crucial enzyme over-expressed in human colitis-induced carcinogenesis, and sEH inhibitor is an efficient approach for chemoprevention. (Supported by NIH CA137467 grant). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5702.

Loss of Interleukin‐6 Blunts Glucagon Secretion During Hypoglycemia

We have shown that the glucagon response to lipopolysaccharide was blunted in interleukin 6 knock‐out (IL6 KO) mice. In this study, we investigated the effects of a non‐inflammatory stimulus on the glucagon response in IL6 KO mice. Chronically catheterized conscious male mice (C57BL6/j; 8–10 weeks old) were subjected to a 5‐hr fast, after which hypoglycemic clamps were performed. Insulin (10mU/kg/min) and glucose were infused to maintain arterial blood glucose between 50–70 mg/dl. Initial glucagon response was blunted in the IL6 KO mice compared to the wild‐type (WT) (213±74 vs. 867±294, pg/ml). Epinephrine was significantly higher in the IL6 KO mice compared to WT (1049±298 vs. 308±123, pg/ml). There was no significant difference in the insulin concentration, corticosteroids, and norepinephrine response. Perifused islets were also stimulated in vitro using hypoglycemia, epinephrine and arginine. There was no significant difference in the glucagon response between the islets from WT and IL6 KO mice. In summary, IL6 KO mice have an impaired glucagon response when subjected to a non‐ inflammatory stimulus in vivo, but the blunted response is not observed in islets in vitro. Since the epinephrine response to hypoglycemia was not blunted, IL6 may play a role in augmenting pancreatic autonomic tone during hypoglycemia. Funding: DK043748, DK078188 and DK059637

Role of MKP-1 in an IL-10 knockout murine inflammatory bowel disease model (47.7)

Abstract IBD is a chronic intestinal inflammatory disease that often has extra-intestinal manifestations. We examined the role of the MAPK phosphatase Mkp-1 in a mouse model of IBD. Mkp-1+/+/Il-10+/+, Mkp-1-/-/Il-10+/+, Mkp-1+/+/Il-10-/-, and Mkp-1-/-/Il-10-/- (dKO) mice on a 129 background were housed in a specific pathogen-free environment. Colitis signs were evaluated using a clinical scoring system, histological examination, and cytokine analysis. Most dKO mice developed severe rectal prolapse, peri-ocular lesions, and high clinical scores, signs generally not seen in Il-10 KO mice. dKO colons had extensive chronic and neutrophilic colitis, mucosal hyperplasia, and higher histological IBD scores than Il-10 KO colons. dKO eyelids and conjunctival mucosa were thickened and had neutrophilic and lymphoplasmocytic conjunctivitis. After LPS treatment, splenocytes and lymph node cells isolated from dKO mice had more robust production of Th-1 cytokines than cells from Il-10 KO mice. dKO colons contained higher levels of Th-1 cytokines and IL-17, and exhibited greater p38 and ERK activation, than did Il-10 KO colons. These data indicate that loss of Mkp-1 skews host immunity towards an exaggerated Th-1/Th-17 response and provide novel insights on the interaction between Mkp-1 and IL-10 in colitis models. Our studies support a pivotal role of Mkp-1 as a brake in the mucosal immune response to limit IBD development.

IL-4Rα signaling may enhance resolution of parasite-induced lung damage by inhibiting inflammation and mediating acute wound healing (37.8)

Abstract The parasitic nematode Nippostrongylus brasiliensis (Nb) third-stage larvae (L3) migrate to the host lung at about 24 to 48 hrs after infection, causing lung damage, inflammation and a potent Th2-type response. Yet, little is known about the mechanism causing lung damage and whether components of the Th2-type immune response contribute to acute lung wound repair. In this study we report that migrating Nb larvae (L3) induce lung inflammation that results in increased hemorrhaging in addition to mechanical damage in the lung. Neutrophil depletion reduced lung hemorrhage and both hemorrhaging and neutrophil infiltration were decreased in Nb-infected IL-17R KO mice. Resolution of acute hemorrhage and inflammation was dependent on either IL-4 or IL-13 as IL-4Rα KO mice, but not IL-13Rα or IL-4 deficient mice, induced significantly higher hemorrhaging, neutrophil influx, and IL-17 expression. Elevations in Arg1, IGF-1, and IL-10 were also inhibited in IL-4RαKO mice. In IL-10KO mice, resolution of inflammation was also blocked, control of hemorrhaging was partly suppressed, and Arg1 and IGF-1 elevations were not affected. These data suggest that the Th2-type immune response mediates acute wound healing and control of IL-17-dependent inflammation during helminth infection.

Down‐regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin‐10 knockout mice

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappaB ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.

A role for IL‐18 in protective immunity against Mycobacterium tuberculosis

Tuberculosis remains the most hazardous bacterial infection worldwide. The causative agent, Mycobacterium tuberculosis, is a facultative intracellular pathogen of resting MPhi. IFN-gamma secreted by natural killer, CD4 Th 1 and CD8 T cells upon instruction by IL-12 and -18 activates MPhi to restrict mycobacterial growth. Production of both cytokines is induced by TLR signalling in DC and MPhi. Mice deficient for the TLR adaptor, MyD88, are highly susceptible to M. tuberculosis infection. Shared usage of MyD88 by signalling cascades for TLR and receptors for IL-1 and IL-18 prompted us to revisit the role of IL-18 during experimental infection with M. tuberculosis. We show that mice deficient for IL-18 and MyD88 but not for IL-18 receptor promptly succumbed to M. tuberculosis infection in contrast to WT or TLR-2/-4 double KO mice indicating that lack of IL-18 contributes to the high susceptibility of MyD88 KO mice to M. tuberculosis. Without IL-18, the protective Th1 response was decreased and hence, mycobacterial propagation was favoured. Neutrophil-driven lung immunopathology concomitant with unrestrained growth of tubercle bacilli are most likely responsible for the premature death of IL-18 KO mice. Thus, IL-18 plays a decisive role in protective immunity against tuberculosis.

Aggravated experimental autoimmune encephalomyelitis in IL-15 knockout mice

IL-15 initially identified as a T proliferating cytokine has several structural and biological similarities with IL-2 and has been associated with a number of autoimmune diseases. Because of the scarcity of information available on the role of IL-15 in MS pathogenesis, we have investigated how the absence of IL-15 affected the development of experimental autoimmune encephalomyelitis, a mouse model of MS. Following immunization of IL-15 −/− and C57BL/6 mice with MOG 35–55, we observed a more severe neurological impairment in the IL-15 knockout mice than in the wild-type group. The enhanced disease severity in IL-15 −/− mice was associated with greater demyelination in the spinal cord, increased immune cell infiltration and inflammation. These events may be related to the higher CD4/CD8 ratio and the almost absent NK cell activity, congenital immune features of IL-15KO mice. Moreover, we found that the fractalkine receptor CX3CR1 was overexpressed in the spinal cord of IL-15 −/− mice, mainly localized on infiltrating CD8 + T cells. How these findings are contributing to the aggravated EAE development in IL-15 KO mice remain unclear and need to be further investigated.

Interleukin-6 Contributes to Age-Related Alteration of Cytokine Production by Macrophages

Here, we studied in vitro cytokine production by splenic macrophages obtained from young and aged BALB/c wild type (WT) and IL-6 knockout (IL-6 KO) mice. Relative to macrophages obtained from young WT mice given lipopolysaccharide (LPS), those from aged WT mice had decreased production of proinflammatory cytokines. In contrast, when compared to macrophages from young IL-6 KO mice, LPS stimulation yielded higher levels of these cytokines by cells from aged IL-6 KO mice. Aging or IL-6 deficiency did not affected the percentage of F4/80+ macrophages, or the surface expression of Toll-like receptor 4 (TLR4) and components of the IL-6 receptor. Overall, our results indicate that IL-6 plays a role in regulating the age-related defects in macrophages through alteration of proinflammatory cytokines, adding to the complexity of IL-6-mediated impairment of immune cell function with increasing age.

The role of sex hormones in the development of Th2 immunity in a gender‐biased model of Trichuris muris infection

Trichuris muris infection is an ideal model for defining T-cell-driven immunity, and also provides essential insights that may impact on potential helminth therapies currently in development. Conflicting host variables determine the efficiency of such treatments and we have identified host-derived sex steroid hormones as key factors in the development of immunity. The female-associated hormone 17-β estradiol (E2) significantly enhanced the generation of a Th2 response in vitro; however, this stimulatory effect was found to be dispensable for the generation of immunity to Trichuris in the gender-biased IL-4KO mouse model. In contrast, the male-associated hormone dihydrotestosterone significantly inhibited the T-cell stimulatory capacity of DC and directly suppressed the immune response of male IL-4KO mice, with worm expulsion restored following castration. This finding was associated with dramatically reduced IL-18 mRNA expression suggesting androgens may act via this cytokine to suppress Th2 immunity to Trichuris. This study has critical implications for the development and efficacy of potential helminth therapeutics and identifies host gender – specifically sex hormones – as important factors in the development of Th2 immunity in susceptible and immunocompromised mice.

IL-12 deficiency suppresses 12- O -tetradecanoylphorbol-13-acetate-induced skin tumor development in 7,12-dimethylbenz( a )anthracene-initiated mouse skin through inhibition of inflammation

Interleukin (IL)-12 deficiency exacerbates tumorigenesis in ultraviolet (UV) radiation-induced skin. Here, we assessed the effects of IL-12 deficiency on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated mouse skin. Using this two-stage chemical carcinogenesis protocol, we found that the development of DMBA/TPA-induced skin tumors was diminished in IL-12p40-knockout mice than in their wild-type counterparts. At the termination of the experiment (at 24 weeks), the skin tumor incidence and tumor multiplicity were significantly lower (P < 0.005) in interleukin-12-knockout (IL-12 KO) mice than in their wild-type counterparts, as was the malignant transformation of DMBA/TPA-induced papillomas to carcinomas (P < 0.01). Analysis of samples collected at the termination of the experiments for biomarkers of inflammation by immunohistochemical analysis, western blotting, enzyme-linked immunosorbent assay and real-time polymerase chain reaction revealed significantly lower levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E(2), proliferating cell nuclear antigen, cyclin D1 and the proinflammatory cytokines (tumor necrosis factor-alpha, IL-1beta and IL-6) in the DMBA/TPA-treated tumors and tumor-uninvolved skin of IL-12 KO mice than the skin and tumors of DMBA/TPA-treated wild-type mice. Analysis of the skin 6 h after TPA treatment showed that the TPA-induced promotion of skin edema, inflammatory leukocyte infiltration, COX-2 expression and PGE(2) production was significantly lower in the skin of the IL-12-KO mice than their wild-type counterparts. These results indicate that DMBA/TPA-induced skin tumor development differs from UVB-induced skin tumor development in that endogenous IL-12 acts to inhibit UVB-induced skin tumor development and malignant progression of the skin tumors to carcinoma. In the case of DMBA/TPA-induced skin tumor development, the endogenous IL-12 modulates the tumor promoter stimulation of inflammatory responses.

Adoptive transfer of protective immunity from Cryptosporidium parvum-infected interferon-γ and interleukin-12-deficient mice to naive recipients

We investigated the possibility of transfer immunity from Cryptosporidium parvum-infected interferon-γ (GKO) and interleukin-12p40 (IL-12KO) deficient C57BL/6 mice to naive mice by transfer of intraepithelial lymphocytes (IELs) and CD4 + T cells from spleen and mesenteric lymph nodes (MLNs). Three days after the transfer recipients were infected with C. parvum. IELs isolated from GKO donor mice after resolution of infection (day 15) but not at the peak of infection (day 8) significantly reduced the parasite load in recipient mice. In IL-12KO mice, IELs and also CD4 + T cells isolated from the spleen and MLNs of donor mice at the peak of infection (day 5) and after resolution (day 15) significantly reduced the parasite excretion, emphasizing the role of interferon-γ in the host–parasite interaction. However, after resolution of infection, interferon-γ-independent mechanisms have evolved that render GKO IELs capable of protecting mice from severe infection.