IL-10 KO Mice Research Articles

CD44 and Bak Expression in IL-6 or TNF-alpha Gene Knockout Mice After Whole Lung Irradiation

To understand the molecular mechanisms that underlie radiation pneumonitis, we examined whether knockout of the TNF or the IL-6 gene could give mice an inherent resistance to radiation in the acute phase of alveolar damage after thoracic irradiation. The temporal expression of inflammation (CD44) and apoptosis (Bak) markers in lung after thoracic irradiation was measured to determine the degree of alveolar damage. At 4 weeks post-irradiation (10 Gy), small inflammatory foci were observed in all mice, but there were no obvious histological differences between control (C57BL/6JSlc), TNF-alpha knockout (TNF KO), and IL-6 knockout (IL-6 KO) mice. However, immunohistochemical analysis of CD44 and Bak expression over a time course of 2 weeks highlighted significant differences between the three groups. C57BL/6JSlc and TNF KO mice had increased numbers of both CD44-positive and Bak-positive cells after irradiation, while the IL-6 KO mice showed stable levels of CD44 and Bak. In conclusion, the radioresistant status of IL-6 KO mice in the acute phase of alveolar damage after irradiation suggested an important role for IL-6 in radiation pneumonitis.

VSL#3 Probiotic Upregulates Intestinal Mucosal Alkaline Sphingomyelinase and Reduces Inflammation

Alkaline sphingomyelinase, an enzyme found exclusively in bile and the intestinal brush border, hydrolyzes sphingomyelin into ceramide, sphingosine and sphingosine-1-phosphate, thereby inducing epithelial apoptosis. Reduced levels of alkaline sphingomyelinase have been found in premalignant and malignant intestinal epithelia and in ulcerative colitis tissue. Probiotic bacteria can be a source of sphingomyelinase. To determine the effect of VSL#3 probiotic therapy on mucosal levels of alkaline sphingomyelinase, both in a mouse model of colitis and in patients with ulcerative colitis. Interleukin-10 gene-deficient (IL10KO) and wild type control mice were treated with VSL#3 (10(9) colony-forming units per day) for three weeks, after which alkaline sphingomyelinase activity was measured in ileal and colonic tissue. As well, 15 patients with ulcerative colitis were treated with VSL#3 (900 billion bacteria two times per day for five weeks). Alkaline sphingomyelinase activity was measured through biopsies and comparison of ulcerative colitis disease activity index scores obtained before and after treatment. Lowered alkaline sphingomyelinase levels were seen in the colon (P=0.02) and ileum (P=0.04) of IL10KO mice, as compared with controls. Treatment of these mice with VSL#3 resulted in upregulation of mucosal alkaline sphingomyelinase activity in both the colon (P=0.04) and the ileum (P=0.01). VSL#3 treatment of human patients who had ulcerative colitis decreased mean (+/- SEM) ulcerative colitis disease activity index scores from 5.3+/-1.8946 to 0.70+/-0.34 (P=0.02) and increased mucosal alkaline sphingomyelinase activity. Mucosal alkaline sphingomyelinase activity is reduced in the intestine of IL10KO mice with colitis and in humans with ulcerative colitis. VSL#3 probiotic therapy upregulates mucosal alkaline sphingomyelinase activity.

The JAK-3 inhibitor CP-690550 is a potent anti-inflammatory agent in a murine model of pulmonary eosinophilia

Janus kinase 3 (JAK-3) is a tyrosine kinase that has been shown to participate in the signaling of several cytokines that are believed to play a role in allergic airway disease, e.g. IL-2, 4 and 9. The current study describes the immunosuppressive effects of CP-690550, a novel, small molecule inhibitor of JAK-3, in a murine model of allergic pulmonary inflammation. In vitro, CP-690550 potently inhibited IL-4 induced upregulation of CD23 (IC 50 = 57 nM) and class II major histocompatibility complex (MHCII) expression (IC 50 = 71 nM) on murine B cells. Repeat aerosol exposure to ovalbumin in wild-type mice sensitized to the antigen resulted in preferential recruitment of Th2-like cells (IL-4+ and IL-5+) into bronchoalveolar lavage fluid (BAL). The importance of IL-4 in the development of pulmonary eosinophilia was supported by a marked (90%) reduction in the influx of these cells in IL-4KO mice similarly sensitized and ovalbumin exposed. Animals dosed with CP-690550 (15 mg/kg/d) during the period of antigen sensitization and boost demonstrated marked reductions in BAL eosinophils and levels of IL-13 and eotaxin following ovalbumin aerosol exposure. The JAK-3 inhibitor (1.5–15 mg/kg/d) also effectively reduced the same parameters when administered during the period of antigen challenge. In contrast, the calcineurin inhibitor tacrolimus (10 mg/kg) was effective only when administered during the period of ovalbumin aerosol exposure. These data support the participation of JAK-3 in processes that contribute to pulmonary eosinophilia in the allergic mouse model. CP-690550 represents an intriguing novel therapy for treatment of allergic conditions associated with airway eosinophilia including asthma and rhinitis.

A Crucial Role of Interleukin-6 in the Pathogenesis of Thyrotoxicosis-related Disturbances of Bone Turnover in Mice

Interleukin-6 (IL-6) has been shown to be involved in the pathogenesis of several bone diseases characterized by a negative balance between bone resorption and formation. The aim of the study was to estimate serum markers of bone turnover: osteoclast-derived tartrate-resistant acid phosphatase form 5a (TRACP 5b) reflecting resorption, and osteocalcin as a marker of bone formation in IL-6 knock-out mice to assess the role of IL-6 in the pathogenesis of thyrotoxicosis-related disturbances of bone metabolism. The study was performed on forty, 14-15 weeks old, female mice: C57BL/6J (wild-type; WT) and C57BL/6J (IL6-/-Kopf) (IL-6 knock-out; IL6KO). The mice were randomly divided into 4 groups with 10 mice in each one: 1. WT mice in thyrotoxicosis (WT-thx), 2. WT controls (WT-ctrl), 3. IL6KO mice with thyrotoxicosis (IL6KO-thx), and 4. IL6KO controls. Experimental model of hyperthyroidism was induced by intraperitoneal injection of levothyroxine at a dose of 1 microg/g, daily over 21 days. The serum levels of TRACP 5b and osteocalcin were determined by ELISA. Serum concentration of TRACP 5b (median and interquartile ranges) were significantly increased in both groups of mice with thyrotoxicosis: WT [28.2(18.8-41.6) U/l] and IL6KO [26.4(23.0-31.2) U/l] as compared to the respective controls. Osteocalcin serum levels in IL6KO-thx mice [111.9(103.1-175.6) ng/ml] were significantly elevated in comparison to WT-thx animals [46.1(32.5-58.9) ng/ml]. In summary, the results of the present study suggest that IL-6 plays a crucial role in thyrotoxicosis-related disturbances of bone turnover in mice, determining the imbalance between bone resorption and bone formation caused by excess of thyroid hormones predominantly by inhibition of bone formation.

IL‐15 exacerbates collagen‐induced arthritis with an enhanced CD4+ T cell response to produce IL‐17

IL-15 is thought to be involved in the pathogenesis of rheumatoid arthritis (RA). We found that IL-15 plays an important role in the development of murine collagen-induced arthritis (CIA). The incidence and severity of CIA were slightly decreased in IL-15 KO mice but were increased in IL-15 Tg mice compared with wild-type (WT) mice. The levels of type II collagen (CII)-specific IL-17 production were significantly increased in IL-15 Tg mice compared with WT mice with CIA. Expression of IL-23R was up-regulated in CD4(+) T cells in IL-15 Tg mice but down-regulated in IL-15 KO mice compared with WT mice. In correlation with the expression levels of IL-23R, IL-17 production by CD4(+) T cells in response to exogenous IL-23 was increased in IL-15 Tg mice compared with WT mice. Furthermore, exogenous IL-15 synergized with IL-23 to induce CII-specific IL-17 production by CD4(+) T cells in vitro. Taken together, these results indicate that IL-15 plays an important role in the progression of CIA through increasing antigen-specific IL-17 production by CD4(+) T cells.

Host-Mediated Inflammation Disrupts the Intestinal Microbiota and Promotes the Overgrowth of Enterobacteriaceae

While the normal microbiota has been implicated as a critical defense against invading pathogens, the impact of enteropathogenic infection and host inflammation on intestinal microbial communities has not been elucidated. Using mouse models of Citrobacter rodentium, which closely mimics human diarrheal pathogens inducing host intestinal inflammation, and Campylobacter jejuni infection, as well as chemically and genetically induced models of intestinal inflammation, we demonstrate that host-mediated inflammation in response to an infecting agent, a chemical trigger, or genetic predisposition markedly alters the colonic microbial community. While eliminating a subset of indigenous microbiota, host-mediated inflammation supported the growth of either the resident or introduced aerobic bacteria, particularly of the Enterobacteriaceae family. Further, assault by an enteropathogen and host-mediated inflammation combined to significantly reduce the total numbers of resident colonic bacteria. These findings underscore the importance of intestinal microbial ecosystems in infectious colitis and noninfectious intestinal inflammatory conditions, such as inflammatory bowel disease.

IL-6-dependent and -independent pathways in the development of interleukin 17-producing T helper cells.

CD4(+) T cells producing IL-17 [T helper (Th)17], as distinct from Th1 or Th2 cells, have recently been shown to be associated with autoimmunity, but it is not entirely clear how Th17 cells are generated from naïve T cells. We demonstrate here that IL-6, but not TNF-alpha or IL-1beta, can, in combination with TGF-beta, induce Th17 cell generation from naïve T cells and inhibit TGF-beta-induced Foxp3 expression. Moreover, conditioned medium from lipopolysaccharide-stimulated bone marrow-derived dendritic cells (DCCM) can induce IL-17 production in naïve T cells. Interestingly, IL-17 was produced by DCCM even with the addition of anti-gp130 antibody or DCCM from IL-6 KO mice. The combination of IL-6 and TGF-beta could maintain activation of signal transducer and activator of transcription (Stat)3, but not of Stat1. IL-27 or IFN-gamma suppressed the induction of Th17 cells by TGF-beta plus IL-6 and maintained Stat1 activation under these conditions. In contrast, both Stat1 and Stat3 remained to be activated in naïve T cells cultured with DCCM. These findings represent a different basis for Th17 differentiation from naïve T cells.

48 Oncostatin M Receptor Signalling Regulates Monocytic Cell Trafficking during Acute Inflammation

Although the interleukin (IL)-6 related cytokine oncostatin-M (OSM) affects a variety of inflammatory events associated with disease progression, the function of OSM in the face of an inflammatory challenge remains unclear. In this current report a peritoneal model of acute inflammation has been used to define the influence of OSM on chemokine-mediated leukocyte recruitment. When compared to wild type mice (WT) the induction of peritoneal inflammation in oncostatin-M receptor-β deficient mice (OSMR-KO) resulted in enhanced monocytic cell trafficking. No difference in neutrophil and lymphocyte migration was however noted suggesting that OSM control of leukocyte recruitment is functionally distinct from that of IL-6. Subsequent in vitro studies using human peritoneal mesothelial cells and an in vivo appraisal of inflammatory chemokine expression following peritoneal inflammation inferred that OSM regulation of CCL5 might account for the observed difference in monocytic cell trafficking. Indeed through comparative analysis of inflammatory events triggered in OSMR-KO and IL-6KO mice it is evident that certain chemokines commonly regulated in vitro by both IL-6 and OSM are preferentially regulated in vivo by one prevailing factor. In this respect, it is evident that IL-6 acts as a more prominent in vivo regulator of CCL2 expression than OSM. In contrast, OSM was found to uniquely inhibit the IL-1β driven expression of CCL5. This was substantiated in vivo where induction of peritoneal inflammation in OSMR-KO mice resulted in significantly raised CCL5 levels, as compared to WT and IL-6KO mice. No difference in CCL3 and CCL4 was however noted. Mechanistically, these studies inferred a hitherto unidentified interplay between OSM-mediated STAT signaling and NF-κB activation. In this respect, EMSA analysis of nuclear extracts from peritoneal membranes isolated during course of the inflammatory response showed that OSMR-KO mice display an enhanced profile of NF-κB activation as compared to WT mice. These findings suggest that activation of gp130 by IL-6 and OSM trigger distinct inflammatory responses to affect individual aspects of leukocyte trafficking. The outcome of such a response may ultimately rely on a defined ability to modulate NF-κB signaling.

Endogenous Interleukin-10 Produced by Antigen-Irrelevant Cells Promotes the Development of Experimental Murine Allergic Conjunctivitis

Interleukin (IL)-10 is known to act as an immunoregulatory cytokine in both T helper cell 1 (Th1)- and Th2-mediated immune responses. Here, we ask whether IL-10 regulates the development of experimental allergic conjunctivitis (EC), a Th2-mediated inflammatory disease. Wild-type (WT) and IL-10 knockout (IL-10 KO) mice were immunized with ragweed (RW) and then repeatedly challenged with RW in eye drops. Twenty-four hours after the final challenge, conjunctivas were harvested for histological analysis, while the blood and spleens were used to determine the RW-specific immunoglobulin levels in serum and proliferation or cytokine responses and splenocyte transfer, respectively. The IL-10 KO mice had significantly less severe EC (as determined by conjunctival eosinophil infiltration) than the WT mice and evinced greater RW-specific splenocyte proliferation and cytokine production. However, the RW-specific immunoglobulin levels of the two strains did not differ. When the splenocytes from RW-primed WT mice were transferred into IL-10 KO or WT mice, the IL-10 KO mice showed significantly less conjunctival eosinophil infiltration. In contrast, when the splenocytes from RW-primed IL-10 KO or WT mice were transferred into WT mice, both splenocyte populations generated equivalent severe EC. These data indicate that IL-10 does not serve as an immunoregulatory cytokine in the development of EC. Instead, it appears that IL-10 produced by antigen-irrelevant cells acts in the effector phase to promote the development of EC.

Interleukin-6 (IL-6) modulates migration and matrix metalloproteinase function in dermal fibroblasts from IL-6KO mice

Interleukin-6-deficient (IL-6KO) mice display significantly delayed cutaneous wound healing characterized by decreased re-epithelialization, granulation tissue and wound closure. Dermal fibroblasts are one of the principal cell types found in granulation tissue and mediate numerous processes during healing. To investigate the effects that IL-6 might have on granulation tissue formation and fibroblast motility. As fibroblast motility is associated with matrix metalloproteinase (MMP) activity, the expression of MMP-2 and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2 were assessed. Punch biopsies (4 mm) were performed in the skin of IL-6KO and C57BL/6 mice. The expression of MMP-2, TIMP-1 and -2 in wound tissue was monitored over time. Cellular infiltration and granulation tissue formation was monitored by subcutaneous implantation of polyvinyl alcohol (PVA) sponges. A free-floating collagen lattice model was also used to investigate the direct effects of IL-6 treatment on isolated IL-6KO fibroblasts. The expression of MMP-2, and the inhibitors TIMP-1 and -2, were assessed via real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. IL-6KO wounds showed impaired granulation tissue formation 5 days postwounding and fewer fibroblasts had populated the PVA matrices 7 days after implantation in IL-6KO mice compared with wild-type C57BL/6. The mRNA and protein expression of MMP-2 and TIMP-2 mRNA was increased in IL-6KO mice compared with wild-type mice beyond 1 day postwounding, while the expression of TIMP-1 mRNA was transiently higher in IL-6KO only 3 days postwounding. Treatment of collagen lattices with various concentrations of rmIL-6 again showed a dose-response decrease in mRNA and protein expression of MMP-2 and TIMP-2 protein expression, compared with saline control, while TIMP-1 did not appear to be significantly modulated. These results indicate that IL-6 influences the function of fibroblasts in wounds, and one mechanism of this regulation may be through the modulation of MMP-2 and TIMP proteins.

Interleukin-12 Deficiency Is Permissive for Angiogenesis in UV Radiation-Induced Skin Tumors

We have shown previously that endogenous deficiency of interleukin (IL)-12 promotes photocarcinogenesis in mice. To characterize the role of IL-12 deficiency in tumor angiogenesis, we developed IL-12p35 knockout (IL-12 KO) mice on a C3H/HeN background. IL-12 KO mice and their wild-type (WT) counterparts were subjected to a photocarcinogenesis protocol. When tumor yield was stabilized, samples of tumor and tumor-uninvolved UVB-exposed skin were collected and subjected to immunohistochemistry, gelatinolytic zymography, real-time PCR, and Western blot analysis of angiogenic factors. We found that the protein, mRNA expression and/or activity of the matrix metalloproteinases (MMP)-2, MMP-3, MMP-7, and MMP-9, and basic fibroblast growth factor, which play crucial roles in tumor growth, were significantly higher in UVB-exposed skin and tumors of IL-12 KO mice compared with WT mice. With respect to the tumor vasculature, the expression of CD31-positive cells and the expression of vascular endothelial growth factor were higher in the tumors of IL-12 KO mice than WTs. The proliferative capacity of tumor cells of the IL-12 KO mice was significantly higher than their WT counterparts when determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and by analyzing the expression of cyclin D1. The level of the proinflammatory cytokine IL-6 and the expression of IL-23 in tumors of IL-12 KO mice were markedly higher than in the tumors of WT mice. IL-23 has been shown to promote tumor growth. Together, these data indicate for the first time that IL-12 deficiency promotes proangiogenic stimuli in UVB-induced skin tumors and suggest that endogenous enhancement of IL-12 levels may be effective in the prevention and treatment of UV-induced skin cancers.

Stress induced morphological microglial activation in the rodent brain: Involvement of interleukin-18

The present study investigated the possibility that acute stress might activate microglial cells. Wistar rats were exposed to 2 h period of restraint combined with water immersion stress prior to brain analysis by immunohistochemistry with OX-42, a marker of complement receptor CR3. A single session of stress provoked robust morphological microglial activation in the thalamus, hypothalamus, hippocampus, substantia nigra and central gray. These effects appeared as early as at 1 h of exposure and were further intensified at 2 h. Morphological activation was not accompanied with changes in markers of functional activation or of inflammation including interleukin-1β (IL-1β), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS). Similar results were obtained with mice where the effects of stress were compared in animals null for interleukin-18 (IL-18 KO), a cytokine previously demonstrated to be modulated by stress and to contribute to microglia activation. The results demonstrated significant reduction of stress-induced microglial activation in IL-18 KO mice. The present study reports evidence that physical/emotional stress may induce morphological microglial activation in the brain and this activation is in part mediated by interleukin-18.

IL-23 and IL-17 in pathogenesis of experimental ocular autoimmunity: requirement for IL–23 may extend beyond its role in sustaining the IL-17 effector response (129.26)

Abstract Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans. Here we examined the role of the IL-23/IL-17 pathway in EAU. We immunized IL-23p19, IL-12p35, IL-12p40, IFN-γ and IL-17 KO mice with the uveitogenic Ag IRBP. Alternatively, we treated wild type mice immunized for EAU with Abs to IL-23p19 or to IL-17 throughout the disease course (prevention), or only during the effector phase (reversal). IL-23p19 KO were resistant to EAU, showing reduced Ag-specific DTH, IL-2, IFN-γ, IL-17, IL-1β, IL-6 and IL-5. In contrast, IL-12p35 and IFN-γ KO mice developed exacerbated EAU, DTH and IL-17 responses. Treatment with anti-IL-23p19 protected from EAU and reduced immunological responses, but was unable to reverse disease, indicating that EAU is dependent on early availability of IL-23. In contrast, anti-IL-17 prevented as well as reversed EAU. Interestingly, severe EAU could be induced with a Th1 cell line that does not produce IL-17. In vivo cytokine neutralization revealed requirement for IFN-γ and TNF-α, but not for IL-17, suggesting that under some conditions an antigen-specific Th1 effector is sufficient to induce EAU. In keeping with this, IL-17 KO mice were still susceptible to disease. These data raise the possibility that the nonredundant need for IL-23 in EAU may extend beyond its role in promoting the Th17 effector response and point to IL-23 and IL-17 as new therapeutic targets for uveitis.

Role of TNF‐α in the dependency of Angiotensin II hypertension on IL‐6

Our laboratory has reported that chronic Angiotensin II (Ang II) infusion increases plasma concentration of IL-6, and that preventing the increase in IL-6, by using IL-6 KO mice, attenuates Ang II hypertension. The mechanism by which Ang II increases IL-6 has yet to be determined. However, the proinflammatory cytokine TNF-alpha is an important stimulus for IL-6 secretion. Therefore, this study aimed to determine the role of TNF-alpha in the dependency of Ang II hypertension on IL-6. Mice were divided randomly into 4 groups: WT, TNF-alpha KO, WT + ANG, and TNF-alpha KO + ANG, and were implanted with blood pressure biotelemetry devices and allowed to recover for 5–7 days. After the baseline period (4 days) the animals were placed on a 4% NaCl diet for a 4-day high-salt control period. The AngII animals then had Ang II (90 ng/min) 14-day minipumps implanted subcutaneously under brief isoflurane anesthesia. High salt diet alone did not change MAP in either group. Ang II increased mean arterial pressure similarly by ~30 mmHg over the 14-day infusion period in both the WT and KO groups. AngII infusion also increased plasma IL-6 in the TNF-KO mice from < 5 to 27+/− 8 pg/ml, thus suggesting that TNF was not required for the effect of AngII to stimulate IL-6 secretion. These results suggest that neither the IL-6 response nor the hypertensive response to chronic AngII-salt is dependent on TNF-alpha.

Interleukin 6 modulates genes associated with fibroblast migration and function (101.10)

Abstract Interleukin 6 deficient mice (IL-6KO) display significantly delayed cutaneous wound healing, characterized by decreased granulation tissue formation. Dermal fibroblasts are one of the principal cell types found in granulation tissue and mediate numerous processes during healing including collagen deposition. Polyvinyl alcohol sponge matrices implanted seven days in IL-6KO mice showed far fewer infiltrating fibroblasts as compared to wild-type C57. It was also found that full thickness wounds from IL-6KO mice showed higher expression of MMP-2 and TIMP-2 as compared to wild-type from 1 to 5 days post wounding. Treatment of IL-6KO fibroblast populated free-floating collagen lattices with various concentrations of rmIL-6 showed a dose response decrease in MMP-2 TIMP-2 mRNA and protein expression. The expression of TGFβ’s 1, and 3 as well as TGFβRII, which can mediate collagen formation, were increased in IL-6KO mouse skin treated intradermally with rmIL-6 in a dose response manner. Expression of TGFβ1, 3, and RII were induced as well in IL-6KO fibroblast collagen lattice cultures in a similar manner. These results indicate that IL-6 may influence on collagen deposition during granulation tissue formation in skin wounds. The mechanism of this may involve both the inhibition of MMP function, and the induction of TGFβ genes. This research was supported by a grant from NIH/NIGMS (5R01GM067745-03).

FcγRIII mediates IgG-induced IL-10 and is required for chronic Leishmania lesions (51.2)

Abstract Chronic disease caused by the protozoan parasite Leishmania mexicana, requires IL-10 and the common γ chain (FcRγ) shared by the activating IgG receptors FcγRI, III, and IV and by FcαR and FcεR. FcRγ can also take the place of CD3ζ in some T cells. Which pathway is responsible for IL-10 production and suppression of a protective immune response is unknown. We recently showed that FcγRIII KO mice, like IL-10 and FcRγ KO mice, resolve L. mexicana lesions with a stronger Th1 response and much lower parasite burdens (by 4–5 logs) than WT mice. We now show that FcγRIII KO macrophages have a severe IL-10 defect, when induced by IgG-opsonized parasites, and that this defect is comparable to FcRγ KO macrophages. Thus FcγRIII appears to contribute most of the IgG-induced IL-10, supporting the in vivo findings. We also found that anti-Leishmania IgG responses (IgG1 and IgG2a) were not altered in FcγRIII KO mice compared to WT mice, consistent with the model that parasite-bound IgG alters T cell development and thus may determine the strength of the Th1 response rather than the reverse. Furthermore, depletion of CD25+ T cells did not change the chronic nature of L. mexicana lesions, nor was T cell IL-10 production from FcγRIII KO mice lower than from B6 mice. When taken together these findings support a model in which macrophage FcγRIII, rather than Treg cells, provides the IL-10 that suppresses a protective immune response to L. mexicana.

IL-4 promotes experimental drug-induced hepatitis by reducing immune responses to haptenated proteins and inhibiting regulatory responses to native CYP2E1 (129.38)

Abstract Susceptible persons develop drug-induced hepatitis (DIH) following administration of halogenated anesthetics or similar drugs. IL-4 worsens autoimmune hepatitis by promoting Th2 responses while other possible mechanisms for this observation have not been fully investigated. Using our experimental model of anesthetic DIH created by immunizing mice with hepatic proteins covalently linked to the TFA hapten (TFA-S100) formed from CYP2E1 metabolism of anesthetics, we found that IL-4 deficient (KO) mice developed significantly less hepatitis than BALB/c (WT) mice. Moreover, ex vivo, antigen specific T cells to CYP2E1 and TFA from IL-4 KO mice proliferated significantly less than those from WT mice. To uncover additional IL-4-dependent mechanisms for the development of anesthetic DIH we measured supernatant cytokines from splenocytes stimulated in vitro with TFA or CYP2E1 and obtained from WT and KO mice following TFA-S100 immunization. KO mice had less IL-1β (p &amp;lt; 0.01), IL-2, IFN-γ, TNF-α, (p &amp;lt; 0.05), IL-13 (p &amp;lt; 0.05) and IL-6 (p &amp;lt; 0.05) in response to TFA when compared to WT mice, suggesting that IL-4 had a critical role in the development of immune responses to TFA-haptenated proteins. Ex vivo, antigen specific T cells to CYP2E1 had higher levels of IL-10 (p &amp;lt; 0.01) suggesting that IL-4 promotes anesthetic DIH by diminishing regulatory responses to CYP2E1. Supported by R21DK075828.

Protection of CD8+ T cells from activation-induced cell death by IL-18

Role of IL-18 on proliferation and survival of CD8+ T cells, activated by immobilized anti-CD3 antibody (anti-CD3), was examined. Proliferation and survival of activated T cells, especially that of CD8+ T cells, were impaired by IL-18 deficiency [IL-18 knockout (KO)]. After 3 days of culture with anti-CD3, the number of living CD8+ T cells from IL-18KO mice was approximately 25% of that from wild-type (WT) mice but was increased to the same level as WT cells by the addition of IL-18. The expression of IL-18 receptors (IL-18Rs), particularly IL-18Rbeta chain, in naïve CD8+ T cells was very low but elevated after stimulation with anti-CD3. Blockade of IL-18R by anti-IL-18R antibody on activated WT CD8+ T cells resulted in reduction of living cells, suggesting that IL-18 promotes survival of proliferating CD8+ T cells. Levels of Bcl-2 in activated IL-18KO CD8+ T cells were lower than those in WT cells but were raised by exogenous IL-18. Blockade of IL-18R on WT CD8+ T cells decreased the expression of surface markers CD122 and CD94, which are related to cell viability, and the expression of these markers was increased by exogenous IL-18 in IL-18KO cells. These results suggest that IL-18 acts directly on activated CD8+ T cells through IL-18Rs and promotes their survival to expand the population.

Activation of toll-like receptor 3 protects against DSS-induced acute colitis

Mimetics of bacterial DNA, given orally or subcutaneously, protect mice from experimental colitis via a toll-like receptor (TLR)-9-dependent mechanism. The goal of the study was to define whether synthetic viral RNA, polyinosinic acid:cytidylic acid [poly(I:C)], which is also a potent immunomodulator, might also affect murine colitis and, if so, define whether such effects were mediated by TLR3, which is one of at least 4 known receptors for this viral RNA analog. Mice (C57BL6, IL-10KO, or TLR3 KO) were administered 1.5% dextran sodium sulfate (DSS) in drinking water for 7 days. Two hours before treatment with DSS, mice were given phosphate-buffered saline (PBS) or poly(I:C) 20 mug subcutaneously (s.c.), or 100 mug intragastrically (i.g.). In wildtype mice s.c. administration of poly(I:C) dramatically protected against DSS-induced colitis as assessed by every parameter analyzed, which included body weight, rectal bleeding, colonic myeloperoxidase, histopathology, serum keratinocyte-derived chemokine, serum amyloid A, and lipocalin-2. In contrast, i.g. administration of poly(I:C) offered no protection in this colitis model nor did its administration activate the innate immune system as assessed by serologic parameters. Subcutaneous poly(I:C) protected against DSS-induced colitis equally well in C57BL6 and IL-10KO mice, indicating that this antiinflammatory cytokine is not required for such protection. Protection against colitis given by poly(I:C) treatment was ablated in TLR3 KO, indicating that the protective action of this viral RNA analog was mediated by this receptor. Activation of TLR3 on cells that are accessible by systemic, but not oral, administration of synthetic viral RNA results in protection against the acute inflammation that can ensue upon damage of the gut epithelium. Thus, this viral RNA analog, which is under clinical trials for other inflammatory disorders (e.g., lupus), may also have therapeutic value for inflammatory bowel disease.

IL-15 and IL-15Rα gene deletion: Effects on T lymphocyte trafficking and the microglial and neuronal responses to facial nerve axotomy

IL-15 is a potent T cell chemoattractant, and this cytokine and its unique α subunits, IL-15Rα, can modify immune cell expression of several T cell chemokines and their receptors. Facial nerve axotomy in mice leads to T cell migration across an intact blood–brain-barrier (BBB), and under certain conditions T cells can provide neuroprotection to injured neurons in the facial motor nucleus (FMN). Although chemokines and chemoattractant cytokines are thought to be responsible for T cell migration to the injured cell bodies, data addressing this question are lacking. This study tested the hypothesis that T cell homing to the axotomized FMN would be impaired in knockout (KO) mice with the IL-15 and IL-15Rα genes deleted, and sought to determine if microglial responsiveness and motoneuron death are affected. Both IL-15KO and IL-15RαKO mice exhibited a marked reduction in CD3 + T cells and had fewer MHC2 + activated microglia in the injured FMN than their respective WT controls at day 14 post-axotomy. Although there was a relative absence of T cell recruitment into the axotomized FMN in both knockout strains, IL-15RαKO mice had five times more motoneuron death (characterized by perineuronal microglial clusters engulfing dead motoneurons) than their WT controls, whereas dead neurons in IL-15KO did not differ from their WT controls. Further studies are needed to dissect the mechanisms that underlie these observations (e.g., central vs. peripheral immune contributions).