IL-10 KO Mice Research Articles

GPR120, a potential therapeutic target for experimental colitis in IL-10 deficient mice.

It has been proved that interleukin-10-knockout (IL-10 KO) mice display the most similar characteristics to that of human Crohn's disease (CD). Docosahexaenoic acid (DHA) has well established beneficial effects on human and animal models health with potent anti-inflammatory effects with poorly understood mechanisms. This study was aimed at figuring out whether DHA could ameliorate the Crohn's colitis by activating GPR120 and whether GPR120 could be a potential therapeutic target for CD.16 week-old mice included in our present study were divided into three groups, WT group, IL-10 KO group and DHA group(IL-10 KO mice with DHA treatment, i.g., 35.5mg/kg/d), containing 8 mice in each group. The severity of colitis, pro-inflammatory cytokines concentrations, the expression/distribution of protein GPR120 and TAK1/IKK-α/IkB-α/p65 pathway in the proximal colons were evaluated at the end of the experiment. Administration of DHA showed promising results in the experimental chronic colitis (demonstrated by reduced infiltration of inflammatory cells, lowered inflammation scores, decreased pro-inflammatory cytokines) and body weight loss improvement. Moreover, in the DHA-treated mice, enhanced expression and improved distribution integrity of protein GPR120 were observed, which was probably associated with the regulation of TAK1/IKK-α/IkB-α/p65 pathway. Our results indicated that triggering GPR120 via the inhibition of TAK1/IKK-α/IkB-α/p65 pathway might be an important target for Crohn's colitis.

Metformin Improves Ileal Epithelial Barrier Function in Interleukin-10 Deficient Mice.

Background and aimsThe impairment of intestinal epithelial barrier is the main etiologic factor of inflammatory bowel disease. The proper intestinal epithelial proliferation and differentiation is crucial for maintaining intestinal integrity. Metformin is a common anti-diabetic drug. The objective is to evaluate the protective effects of metformin on ileal epithelial barrier integrity using interleukin-10 deficient (IL10KO) mice.MethodsWild-type and IL10KO mice were fed with/without metformin for 6 weeks and then ileum was collected for analyses. The mediatory role of AMP-activated protein kinase (AMPK) was further examined by gain and loss of function study in vitro.ResultsCompared to wild-type mice, IL10KO mice had increased proliferation, reduced goblet cell and Paneth cell lineage differentiation in the ileum tissue, which was accompanied with increased crypt expansion. Metformin supplementation mitigated intestinal cell proliferation, restored villus/crypt ratio, increased goblet cell and Paneth cell differentiation and improved barrier function. In addition, metformin supplementation in IL10KO mice suppressed macrophage pro-inflammatory activity as indicated by reduced M1 macrophage abundance and decreased pro-inflammatory cytokine IL-1β, TNF-α and IFN-γ expressions. As a target of metformin, AMPK phosphorylation was enhanced in mice treated with metformin, regardless of mouse genotypes. In correlation, the mRNA level of differentiation regulator including bmp4, bmpr2 and math1 were also increased in IL10KO mice supplemented with metformin, which likely explains the enhanced epithelial differentiation in IL10KO mice with metformin. Consistently, in Caco-2 cells, metformin promoted claudin-3 and E-cadherin assembly and mitigated TNF-α-induced fragmentation of tight junction proteins. Gain and loss of function assay also demonstrated AMPK was correlated with epithelial differentiation and proliferation.ConclusionsMetformin supplementation promotes secretory cell lineage differentiation, suppresses inflammation and improves epithelial barrier function in IL10KO mice likely through activation of AMPK, showing its beneficial effects on gut epithelial.

Induction of Alternative Proinflammatory Cytokines Accounts for Sustained Psoriasiform Skin Inflammation in IL-17C+IL-6KO Mice

IL-6 inhibition has been unsuccessful in treating psoriasis, despite high levels of tissue and serum IL-6 in patients. In addition, de novo psoriasis onset has been reported after IL-6 blockade in patients with rheumatoid arthritis. To explore mechanisms underlying these clinical observations, we backcrossed an established psoriasiform mouse model (IL-17C+ mice) with IL-6-deficient mice (IL-17C+KO) and examined the cutaneous phenotype. IL-17C+KO mice initially exhibited decreased skin inflammation; however, this decrease was transient and reversed rapidly, concomitant with increases in skin Tnf, Il36α/β/γ, Il24, Epgn, and S100a8/a9 to levels higher than those found in IL-17C+ mice. A comparison of IL-17C+ and IL-17C+KO mouse skin transcriptomes with that of human psoriasis skin revealed significant correlation among transcripts of skin of patients with psoriasis and IL-17C+KO mouse skin, and confirmed an exacerbation of the inflammatory signature in IL-17C+KO mice that aligns closely with human psoriasis. Transcriptional analyses of IL-17C+ and IL-17C+KO primary keratinocytes confirmed increased expression of proinflammatory molecules, suggesting that in the absence of IL-6, keratinocytes increase production of numerous additional proinflammatory cytokines. These preclinical findings may provide insight into why patients with arthritis being treated with IL-6 inhibitors develop new onset psoriasis and why IL-6 blockade for the treatment of psoriasis has not been clinically effective.

Nuclear expression of IL-33 in epidermal keratinocytes promotes wound healing in mice

BackgroundSkin is the outermost tissue of the human body, and works as a mechanical, chemical, and biological barrier. The epidermis is the uppermost layer of the skin, and keratinocytes constitute the majority of epidermal cells. Wounds are disruptions of skin integrity, and cause tremendous disadvantages to humans; accordingly, rapid wound healing is very important. Interleukin (IL)-33 is expressed in barrier tissue cells, such as epithelial and endothelial cells. Upon injury, IL-33 is released to stimulate immune cells, functioning as an “alarmin.” ST2 is a receptor for IL-33; its soluble form (s)ST2 acts as a decoy receptor and competes for IL-33 binding. ObjectivesWe aimed to clarify the role of IL-33 in wound healing. Materials and methodsWild-type (WT), IL-33 knockout (IL33 KO) mice, and sST2 transgenic (Tg) mice were wounded with a 4-mm punch, and the wound healing process was compared. Immunohistochemical analyses were performed to detect macrophages, neutrophils, and mast cells. Total RNA was extracted from the skin samples and real-time PCR was performed. An in vitro scratch wound assay was performed. ResultsWound healing was delayed in IL33 KO mice compared to WT mice, while wound healing in sST2 Tg mice was comparable to that of WT mice. A histological examination showed delayed elongation of the epidermal tongue in IL-33 KO mice. An immunohistochemical study revealed prolonged neutrophilic infiltration at a later stage in IL-33 KO mice. IL-6, IL-1β, and CXCL1 transcripts were more abundant in the wounds of IL-33 KO mice than WT mice. Intraperitoneal administration of an NFκB inhibitor to IL-33 KO mice normalized the delayed wound healing and the enhanced expression of IL-6 in IL-33 KO mice. Epidermal keratinocytes from IL-33 KO mice showed delayed wound closure compared to those from WT mice. ConclusionOur results indicate that nuclear IL-33, but not IL-33 as a cytokine, has beneficial effects on wound healing in mice, probably by suppressing NFκB to inhibit excessive inflammation and by maintaining keratinocyte proliferation or migration for epithelialization.

IL-10 plays an important role in the control of inflammation but not in the bacterial burden in S. epidermidis CNS catheter infection.

BackgroundShunt infection is a frequent and serious complication in the surgical treatment in hydrocephalus. Previous studies have shown an attenuated immune response to these biofilm-mediated infections. We proposed that IL-10 reduces the inflammatory response to Staphylococcus epidermidis (S. epidermidis) CNS catheter infection.MethodsIn this study, a murine model of catheter-associated S. epidermidis biofilm infection in the CNS was generated based on a well-established similar model for S. aureus. The catheters were pre-coated with a clinically derived biofilm-forming strain of S. epidermidis (strain 1457) which were then stereotactically implanted into the lateral left ventricle of 8-week-old C57BL/6 and IL-10 knockout (IL-10 knockout) mice. Bacterial titers as well as cytokine and chemokine levels were measured at days 3, 5, 7, and 10 in mice implanted with sterile and S. epidermidis-coated catheters.ResultsCultures demonstrated a catheter-associated and parenchymal infection that persisted through 10 days following infection. Cytokine analysis of the tissue surrounding the catheters revealed greater levels of IL-10, an anti-inflammatory cytokine, in the infected group compared to the sterile. In IL-10 KO mice, we noted no change in bacterial burdens, showing that IL-10 is not needed to control the infection in a CNS catheter infection model. However, IL-10 KO mice had increased levels of pro-inflammatory mediators in the tissues immediately adjacent to the infected catheter, as well as an increase in weight loss.ConclusionsTogether our results indicate that IL-10 plays a key role in regulating the inflammatory response to CNS catheter infection but not in control of bacterial burdens. Therefore, IL-10 may be a useful therapeutic target for immune modulation in CNS catheter infection but this should be used in conjunction with antibiotic therapy for bacterial eradication.

Lipocalin 2 prevents intestinal inflammation by enhancing phagocytic bacterial clearance in macrophages.

Lipocalin 2 (Lcn2), also called neutrophil gelatinase B-associated lipocalin (NGAL), is an anti-microbial peptide originally identified in neutrophil granules. Although Lcn2/NGAL expression is increased in the inflamed intestinal tissues of patients with inflammatory bowel disease, the role of Lcn2/NGAL in the development of intestinal inflammation remains unclear. Here we investigated the role of Lcn2/NGAL in intestinal inflammation using a spontaneous mouse colitis model, interleukin-10 knock out (IL-10 KO) mice. Lcn2 expression in the colonic tissues of IL-10 KO mice increased with the development of colitis. Lcn2/IL-10 double-KO mice showed a more rapid onset and development of colitis compared to IL-10 KO mice. Lcn2 enhanced phagocytic bacterial clearance in macrophages in vitro after infection with Escherichia coli. Transfer of Lcn2-repleted macrophages prevented the development of colitis in Lcn2/IL-10 double-KO mice in vivo. Our findings revealed that Lcn2 prevents the development of intestinal inflammation. One crucial factor seems to be the enhancement of phagocytic bacterial clearance in macrophages by Lcn2.

The role of recipient derived interleukin-17A in a murine orthotopic lung transplant model of restrictive chronic lung allograft dysfunction

The single most important cause of late mortality after lung transplantation is chronic lung allograft dysfunction (CLAD). However, the pathological development of CLAD was not as simple as previously presumed and subclassification phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD), have been introduced. We want to re-investigate how CLAD manifests in the murine orthotopic lung transplant model and investigate the role of interleukin 17A (IL-17A) within this model.Orthotopic LTx was performed in CB57BL/6, IL-17 WT and IL-17 KO mice. In a first experiment, CB57BL/6 mice receiving an isograft (CB57BL/6) or allograft (BALB/C) were compared. In a second experiment IL-17 WT and IL-17 KO mice (both CB57BL/6 background) received an allograft (BALB/C). Mice received daily immunosuppression with steroids and cyclosporine and were sacrificed 10weeks after transplantation for histopathological analysis by an experienced lung pathologist.After murine orthotopic lung transplantation, the allograft histopathologically presented features of human rCLAD (i.e. overt inflammation, pleural/parenchymal fibrosis and obliterative bronchiolitis). In the IL-17A KO group, less inflammation in the bronchovascular axis (p=0.03) was observed and a non-significant trend towards less bronchovascular fibrosis, pleural/septal inflammation and fibrosis, and parenchymal inflammation and fibrosis when compared to WT mice.The major mismatch orthotopic lung transplant model resembles features of human rCLAD. IL-17A mediated immunity is involved in the inflammatory component, but had little influence on the degree of fibrosis. Further mechanistic and therapeutic studies in this mouse model are needed to fully understand the mechanisms in rCLAD.

Endogenous IL-17 as a factor determining the severity of Clostridium difficile infection in mice.

Clostridium difficile infection (CDI) is a toxin-mediated intestinal disease. Toxin A, toxin B and binary toxin are believed to be responsible for the pathogenesis of CDI, which is characterized by massive infiltration of neutrophils at the infected intestinal mucosa. IL-17 is one of the cytokines that play critical roles in several inflammatory and immunological diseases through various actions, including promoting neutrophil recruitment. The aim of this study was to examine the role of this cytokine in CDI by employing IL-17 A and F double knockout (IL-17 KO) mice for the CDI model. We demonstrated that IL-17 KO mice were more resistant to CDI than WT mice using several factors, such as diarrhoea score, weight change and survival rate. Although the bacterial numbers of C. difficile in faeces were not different, the inflammatory mediator levels at the large intestine on day 3 post-infection were attenuated in IL-17 KO mice. Finally, we showed that infiltration of neutrophils, but not macrophages, in the large intestine was significantly decreased in IL-17 KO mice compared to WT mice. In conclusion, the data demonstrate that endogenous IL-17 may be a factor determining the severity of CDI in mice. Although the mechanism is totally unknown, IL-17-mediated inflammatory responses, such as cytokine/chemokine production and neutrophil accumulation, may be plausible targets for future investigations.

Dermatophagoides pteronyssinus group 2 allergen bound to 8-OH modified adenine reduces the Th2-mediated airway inflammation without inducing a Th17 response and autoimmunity.

8-OH modified adenine bound to Dermatophagoides pteronyssinus group 2 (nDer p2-Conj), a novel allergen-TLR7 agonist conjugate, improves murine airway inflammation in priming and therapeutic settings, however no data are known on the activity of this construct on Th17 cells. The aim of the study was to evaluate if nDer p2-Conj elicited in vivo Th17 cells and Th17-driven autoimmune responses, by using both short- and long-term priming and therapeutic protocols in a nDer p2-driven model of murine airway inflammation. The conjugate induced the in vitro production of cytokines favouring the Th17 polarization by bone marrow-derived dendritic cells. In short-term protocols, the priming or treatment with the conjugate ameliorated the airway inflammation by shifting Th2 allergen-specific cells into T cells producing IFN-γ, IL-10, but not IL-17A. Similar results were found in long-term protocol where the conjugate down-regulated airway inflammation without any evidence of autoimmune response and B cell compartment expansion. nDer p2-Conj also failed to shorten the spontaneous onset of diabetes on conjugates-primed NOD/LtJ mice. We found that neutrophils in BALF, ROR-γt and IL-17A expression in lungs were increased in conjugate-treated IL-10KO mice. These data emphasize the role of conjugate-driven IL-10 production, which can regulate the activity of memory Th17 cells and prevent the onset of autoimmune response.

Abstract 99: IL10-inhibits Fibroblast Progenitor Cell-mediated Cardiac Fibrosis in Pressure-overloaded Myocardium

Background: Activated fibroblasts (myoFBs) play critical role in cardiac fibrosis, however, their origin in diseased heart remains uncertain. Recent studies suggest the contribution of bone marrow fibroblasts progenitor cells (BM-FPC) in pressure overload (PO)-induced cardiac fibrosis. Previously we have shown that interleukin-10 suppress PO-induced cardiac fibrosis, however, its role on inhibition of BM-FPC-mediated fibrosis is not known. Thus, we hypothesized that IL-10 inhibits PO-induced homing and transition of BM-FPC to myoFBs and therefore, attenuates cardiac fibrosis. Methods and Results: Cardiac fibrosis was induced in Wild-type (WT) and IL-10-knockout (KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice. Systemic recombinant IL-10 administration markedly improved LV function and inhibited PO-induced cardiac fibrosis. PO-enhanced FPC (Prominin1 + cells) mobilization and homing in IL-10 KO mice compared to WT mice. Furthermore, bone marrow transplantation (BMT) experiment was performed wherein WT marrow from GFP mice was repopulated in IL-10 KO mice. FPC mobilization was significantly reduced in BMT-IL10 KO mice compared to IL-10 KO mice after TAC. Furthermore, immunofluorescence result in BMT mice showed that subsets of myoFBs are derived from BM after TAC. To identify the molecular mechanism, wild type BM-FPC were treated with TGFβ 2 with or without IL10. IL10 treatment significantly inhibits TGFβ 2 -induced FPC to myoFBs transition. As miRNAs are key players in cardiac fibrosis, next we performed fibrosis-associated miRNA profiling using miRNA array kit. TGFβ 2 -induced miR-208, 155, 21 and 145 expression was markedly inhibited by IL-10. Conclusion: Taken together, our findings suggest that both reduced homing to heart and transition of FPC to myofibroblasts mediate anti-fibrotic effect of IL10 during PO-induced heart failure. Ongoing investigations using molecular approaches will provide a better understanding on the mechanistic and therapeutic aspects of IL10 on PO-induced cardiac fibrosis and heart failure.

Abstract 4055: Interleukin-12 deficiency increases modulation of epigenetic regulators and silencing of tumor suppressor genes in UVB-exposed mouse skin and skin tumors

Abstract Ultraviolet (UV) radiation has variety of deleterious effects on mammalian skin, and triggers several causative factors such as DNA damage, inflammation, immune suppression and genetic and epigenetic alterations which leads to the development of skin cancer. Earlier, we have shown that the deficiency of interleukin (IL)-12, an immunoregulatory cytokine, enhances photocarcinogenesis in mice through inhibition of repair of damaged DNA. In further studies we have tried to explore and examine the effect of IL-12 on epigenetic regulators in UV-exposed skin and UV-induced skin tumors using IL-12 knock out (IL-12 KO) mice as an in vivo animal model. The exposure of IL-12 KO mice with UVB (200 mJ/cm2) radiation resulted in higher levels of DNA methylation, DNA methyltransferase (Dnmt) activity, Ten-eleven translocations (TETs), a family of methylcytosine dioxygenase, histone deacetylases (HDAC), and lower levels of histone acetylase (HAT) activity than their wild type counterparts. To verify that IL-12 deficiency is associated with DNA hypermethylation and higher HDAC activity in UVB exposed skin, IL-12-KO mice were treated with endotoxin-free murine rIL-12 (50 ng/mouse/100 μl PBS; subcutaneous injection) 3 h before UVB exposure. Our results indicate that significant reduction in the levels of global DNA methylation, Dnmt and HDAC activity were observed compared with non-rIL-12 treated mice. Conversely, UVB exposed IL-12 wild type mice were treated with anti-IL-12 antibody (500 ng/mouse/100 μl PBS; subcutaneous injection). This treatment resulted in increased levels of DNA methylation, Dnmt and HDAC activities. These observations were also noted and compared between skin tumors from IL-12 KO mice and their wild type counterparts. To further explore the role of IL-12 deficiency on epigenetic modification-mediated photocarcinogenesis, we determined its effect on methylated and unmethylated levels of tumor suppressor genes, such as p16INK4a and RASSF1A, in UV-exposed skin and UV-induced skin tumors. It was observed that the levels of methylated tumor suppressor genes were higher in IL-12 KO mice skin than their wild type counterparts. Together, these findings indicate that IL-12 deficiency promotes: (i) DNA hypermethylation, (ii) HDAC activity and (iii) silencing of tumor suppressor genes in UV exposed mice and that makes the mice more susceptible to UV radiation-induced skin cancer risk. Together, these observations suggest that augmentation of IL-12 should be considered as a strategy for the prevention and treatment of UV radiation-induced skin cancer risk. Citation Format: Ram Prasad, Tripti Singh, Santosh K. Katiyar. Interleukin-12 deficiency increases modulation of epigenetic regulators and silencing of tumor suppressor genes in UVB-exposed mouse skin and skin tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4055.

The Role of Neutrophils in the Induction of Specific Th1 and Th17 during Vaccination against Tuberculosis.

Mycobacterium tuberculosis causes tuberculosis (TB), a disease that killed more than 1.5 million people worldwide in 2014, and the Bacillus Calmette Guérin (BCG) vaccine is the only currently available vaccine against TB. However, it does not protect adults. Th1 and Th17 cells are crucial for TB control, as well as the neutrophils that are directly involved in DC trafficking to the draining lymph nodes and the activation of T lymphocytes during infection. Although several studies have shown the importance of neutrophils during M. tuberculosis infection, none have shown its role in the development of a specific response to a vaccine. The vaccine mc2-CMX was shown to protect mice against M. tuberculosis challenge, mainly due to specific Th1 and Th17 cells. This study evaluated the importance of neutrophils in the generation of the Th1- and Th17-specific responses elicited by this vaccine. The vaccine injection induced a neutrophil rich lesion with a necrotic central area. The IL-17 KO mice did not generate vaccine-specific Th1 cells. The vaccinated IL-22 KO mice exhibited Th1- and Th17-specific responses. Neutrophil depletion during vaccination abrogated the induction of Th1-specific responses and prohibited the bacterial load reduction observed in the vaccinated animals. The results show, for the first time, the role of neutrophils in the generation of specific Th1 and Th17 cells in response to a tuberculosis vaccine.

Dysfunction of mitochondria and deformed gap junctions in the heart of IL-18-deficient mice.

Interleukin-18 (IL-18) was discovered as an interferon-γ-inducing factor and has been regarded as a proinflammatory cytokine. However, IL-18 is ubiquitously expressed both in immune/inflammatory cells and in nonimmune cells, and its biological roles have not been sufficiently elucidated. Here, we demonstrate that IL-18-deficient [IL-18 knockout (KO)] mice have heart abnormalities that may be related to impaired autophagy. In endurance running tests, IL-18KO mice ran significantly shorter distances compared with wild-type (WT) mice. Echocardiographs indicated disability in the systolic and diastolic functions of the IL-18KO mouse heart. Immunostaining of connexin 43 showed heterogeneous localization of gap junctions in the lateral membranes of the IL-18KO cardiac myocytes. Western blotting analysis revealed decreased phosphorylated connexin 43 in the IL-18KO heart. Electron microscopy revealed unusual localization of intercalated disks, swollen or damaged mitochondria, and broad, indistinct Z-lines in the IL-18KO heart. In accordance with the morphological observation, mitochondrial respiratory function, including that of complexes I and IV, was impaired, and production of reactive oxygen species was augmented in IL-18KO hearts. Notably, levels of LC3-II were markedly lower in the IL-18KO hearts than in WT hearts. In the culture of cardiac myocytes of IL-18KO neonates, exogenous IL-18 upregulated LC3-II and increased the number of intact mitochondria with high mitochondrial membrane potential. These results indicated that IL-18 has roles apart from those as a proinflammatory cytokine in cardiac myocytes and suggested that IL-18 contributes to the homeostatic maintenance of mitochondrial function and gap-junction turnover in cardiac myocytes, possibly by upregulating autophagy.

Phosphatase Wip1 Masters IL-17-producing Neutrophil-mediated Colitis in Mice.

Wild-type p53-induced phosphatase 1 (Wip1) is currently believed to be a promising drug target for cancer therapy. Our recent studies showed that deletion of Wip1 remarkably promoted neutrophil inflammatory response. Whether Wip1 is involved in the regulation of inflammatory bowel disease is unknown. In the present study, we found that Wip1 knockout (KO) mice were more susceptible to colitis induced by dextran sulphate sodium (DSS) than wild-type mice as substantiated by the lower mouse survival ratio, rapid bodyweight loss, increased disease activity index, shorter colon length, and more severe pathology of colons in Wip1KO mice. Using full bone marrow chimera mouse models, we demonstrated that Wip1 intrinsically controls inflammatory response of immune cells. Deletion of IL-17 (Wip1/IL-17 double KO mice) significantly rescued the pathology in Wip1KO mice. Neutrophils of DSS-treated wild-type and Wip1KO mice expressed significantly higher IL-17. After adoptive transfer of sorted Wip1KO or double KO neutrophils into IL-17KO mice, mice receiving double KO neutrophils were more resistant to DSS-induced colitis than mice receiving Wip1KO neutrophils. These data collectively indicate that Wip1 modulates host sensitivity to colitis by intrinsically regulating immune cells. The enhanced IL-17 expression in neutrophils contributed to the increased sensitivity and severity of colitis in Wip1KO mice. Thus, Wip1 may be used as a drug target to treat colitis.

A novel PAK1/Notch-1 axis contributes to intestinal inflammation and cancer

Background: PAK1 belongs to a family of serine-threonine kinase and contributes to various oncogenic pathways like MAPK and Wnt-β catenin. Our lab recently linked activation of p21-activated kinase (PAK1) to IBD as well as colitis-associated cancer (Khare V, Inflamm Bowel Dis 2015). Here we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL-10 KO mice. Methods: IL-10 KO mice were crossed with Pak1 KO mice to generate double-knockout mice (DKO). Inflammation was triggered with piroxicam (200ppm mixed to chow for 14 d). After 12 wks mice were euthanized, intestines were prepared and length was measured. Paraffin embedded Swiss rolls were stained with HE MAPK pathway activation, as shown by increased pERK1/2 staining and an increase in γH2AX positive nuclei, indicating more DNA double-strand breaks. A reduced differentiation of goblet cells and Paneth cells was marked by PAS- and Lysozyme staining, together with an expansion of the stem cell compartment (shown by increased activated Notch1 and LGR5 staining) in the colonic crypts of DKOs. Interestingly NFκB was found to be reduced in DKO mice. Conclusion: Our data suggest a novel interaction of PAK-1 with the Notch pathway in regulating crypt cell stemness, proliferation and differentiation. Lack of mature goblet cells and mucus can exacerbate colitis in IL-10 KO mice. Hyperproliferation may promote dysplastic lesions independent of NFκB.

Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway.

Clinical and experimental studies have shown that estradiol (E2) confers protection against HIV and other sexually transmitted infections. Here, we investigated the underlying mechanism. Better protection in E2-treated mice, immunized against genital HSV-2, coincided with earlier recruitment and higher proportions of Th1 and Th17 effector cells in the vagina post-challenge, compared to placebo-treated controls. Vaginal APCs isolated from E2-treated mice induced 10-fold higher Th17 and Th1 responses, compared to APCs from progesterone-treated, placebo-treated, and estradiol-receptor knockout mice in APC-T cell co-cultures. CD11c+ DCs in the vagina were the predominant APC population responsible for priming these Th17 responses, and a potent source of IL-6 and IL-1β, important factors for Th17 differentiation. Th17 responses were abrogated in APC-T cell co-cultures containing IL-1β KO, but not IL-6 KO vaginal DCs, showing that IL-1β is a critical factor for Th17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1β in vaginal DCs, and addition of IL-1β restored Th17 induction by IL-1β KO APCs in co-cultures. Finally, we examined the role of IL-17 in anti-HSV-2 memory T cell responses. IL-17 KO mice were more susceptible to intravaginal HSV-2 challenge, compared to WT controls, and vaginal DCs from these mice were defective at priming efficient Th1 responses in vitro, indicating that IL-17 is important for the generation of efficient anti-viral memory responses. We conclude that the genital mucosa has a unique microenvironment whereby E2 enhances CD4+ T cell anti-viral immunity by priming vaginal DCs to induce Th17 responses through an IL-1-dependent pathway.

The lack of NK and mCD8+ T cells in IL-15KO mice confers resistance to septic shock

Abstract Septic shock remains one of the leading causes of death in the USA. However, the immunological mechanisms of septic shock are still not well understood. Interleukin (IL)-15 is an essential mediator for natural killer (NK) and memory (m) CD8+T cell survival, as indicated by the deficits of these cells in IL-15 knockout (KO) mice. As NK and CD8+T cells are implicated in the pathogenesis of septic shock, we hypothesized that IL-15KO mice will be resistant to septic shock due to their lack of NK and mCD8+T cells. IL-15KO mice showed a significant survival advantage over wild type (WT) control mice during septic shock caused by cecal ligation and puncture (CLP) or LPS challenge. IL-15KO mice displayed less sepsis-induced hypothermia and attenuated plasma IL-6 production in our sepsis models. Treatment with low-dose IL-15 superagonist (SA) regenerated NK and mCD8+T cells in IL-15KO mice and reestablished mortality in these mice during CLP- and LPS-induced septic shock. However, if NK and CD8+T cell regeneration was prevented by anti-asialoGM1 and anti-CD8α prior to IL-15 SA treatment, IL-15 SA failed to reestablish sepsis-induced mortality in IL-15 KO mice. When given to WT mice 4 days prior to CLP or LPS challenge, M96, an IL-15 neutralizing IgG, caused 80.8% depletion of splenic NK cells and conferred protection against septic shock. However, when given shortly prior to CLP or LPS challenge, M96 did not deplete splenic NK cells and failed to confer protection against septic shock. In conclusion, IL-15 contributes to the lethality of septic mice by maintaining NK and mCD8+ T cell populations, but does not play a direct role in septic shock. Thus, NK and mCD8+ T cells may represent effective therapeutic targets for septic shock.

IL-6 influences the balance between M1 and M2 macrophages in a mouse model of irritant contact dermatitis

Abstract Irritant contact dermatitis (ICD) is an acute inflammatory response that is ranked the 2nd most prevalent occupational injury associated with workman’s compensation. Interleukin-6 (IL-6), a pro-inflammatory cytokine produced in the skin, is closely associated with healing and decreased levels are associated with more severe ICD. The phenotypic nature of the macrophage infiltrate during skin inflammation can greatly influence damage and healing during ICD. The pro-inflammatory, classically activated (M1) macrophage produce cytokines such as IL-6, IL-1, and TNFα, while alternatively activated (M2) macrophages produce IL-10 and TGFβ, and are thought to be associated with tissue repair. IL-6 may play a regulatory role in mediating the M1 versus M2 phenotypic change in skin. To examine the role of IL-6 in ICD, IL-6KO and C57 mice were exposed to benzoalkonium chloride (BKC) for 7 days. IL-6KO mice displayed decreased expression of CD206 (M2) as compared to CD86 (M1) and the ration of CD86:CD206 was significantly increased in IL-6KO mice compared to control. Multiplex protein analysis showed expression of multiple M1-related cytokines such IFN-g, CCL3, CCL4, CCL5, and CCL7 significantly modulated in IL6-KO BKC treated mice compared to C57. Additionally, M2 related cytokine/chemokine receptors IL-4R and CCR1 were significantly dysregulated in IL-6KO BKC treated mice. IL-6 treatment of RAW cells enhanced the expression of CD206 in manner similar to IL-4. Overall, these results further confirm a role for IL-6 in macrophage polarization and could eventually result in the development of therapies for dermatitis based on this cytokines activity.

Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis

Abstract Lipocalin 2 (Lcn2) is a multifunctional innate immune protein whose expression closely correlates with extent of intestinal inflammation. However, whether Lcn2 plays a role in the pathogenesis of gut inflammation is unknown. Herein, we investigated the extent to which Lcn2 regulates inflammation and gut bacterial dysbiosis in mouse models of IBD. Lcn2 expression was monitored in murine colitis models and upon microbiota ablation/restoration. WT and Lcn2 knockout (Lcn2KO) mice were analyzed for gut bacterial load, composition by 16S rRNA gene pyrosequencing and, their colitogenic potential by co-housing with Il-10KO mice. Acute (dextran sodium sulfate) and chronic (IL-10R neutralization) colitis was induced in WT and Lcn2KO mice with or without antibiotics. Lcn2 expression was dramatically induced upon inflammation and was dependent upon presence of a gut microbiota and MyD88 signaling. Use of bone-marrow chimeric mice revealed non-immune cells are the major source of circulating Lcn2. Lcn2KO mice exhibited elevated levels of entA-expressing gut bacteria burden and, moreover, a broadly distinct bacterial community relative to WT littermates. Lcn2KO mice developed highly colitogenic T-cells and exhibited exacerbated colitis upon exposure to DSS or neutralization of IL-10. Such exacerbated colitis could be prevented by antibiotic treatment. Moreover, exposure to the microbiota of Lcn2KO mice, via cohousing, resulted in severe colitis in Il-10KO mice. Lcn2 is a bacterially-induced, MyD88-dependent, protein that play an important role in gut homeostasis and a pivotal role upon challenge. Hence, therapeutic manipulation of Lcn2 levels may provide a strategy to help manage diseases driven by alteration of the gut microbiota.

Immune phenotype and IL-6 deficiency modulate the inflammatory response in a mouse model of Irritant Contact Dermatitis

Abstract Skin diseases are the second most common occupational illness with associated per annum costs exceeding $1 billion. Irritant contact dermatitis (ICD), the most common occupational skin disease, is characterized as an acute inflammatory response that manifests as a result of topical irritant exposure. Immune phenotype, such as Th1- and Th2- dominance, is postulated to contribute to the variability seen between patients. In addition, interleukin 6, which is known to have variably both Th1 and Th2 properties, has been shown to play a key role in ICD. C57BL/6J (“Th1 dominant”), Balb/c (“Th2 dominant”), and IL-6KO mice were utilized to investigate ICD induced by exposure to the occupational irritants, benzoalkonium chloride (BKC) and JP-8 jet fuel. Histopathology revealed epidermal thickening and dermal cellular infiltration in dermatitis lesions, with IL-6KO skin exhibiting the most severe damage. Analysis of skin cytokine and chemokine protein expression showed that IL-1β, TNF-α, TGF-β, CCL2, CCL3 and CXCL1, were elevated in mice exposed to BKC regardless of immune phenotype. In contrast, IL-1β, TNF-α, TGF-β, and CXCL1 varied between mouse strains when JP-8 was compared to control exposure. Flow cytometric analysis and immunohistochemistry indicated neutrophil and monocyte presence within dermatitis lesions; however, IL-6KO showed a greater amount of neutrophils following BKC exposure and decreased neutrophil presence after JP-8 exposure when compared to C57 mice. Overall, it appears that immune phenotype and IL-6 deficiency alters the skin inflammatory response in ICD via differential cytokine and chemokine expression that influence the type of inflammatory cells that infiltrate into the skin.