XIAP restrains TNF-driven intestinal inflammation and dysbiosis by promoting innate immune responses of Paneth and dendritic cells.

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In Xiap−/− mice, we observed spontaneous terminal ileitis and microbial dysbiosis characterized by a reduction of Clostridia species. We showed that in inflamed mice, both TNF receptor 1 and 2 (TNFR1/2) cooperated in promoting ileitis by targeting TLR5-expressing Paneth cells (PCs) or dendritic cells (DCs). Using intestinal organoids and in vivo modeling, we demonstrated that TLR5 signaling triggered TNF production, which induced PC dysfunction mediated by TNFR1. TNFR2 acted upon lamina propria immune cells. scRNA-seq identified a DC population expressing TLR5, in which Tnfr2 expression was also elevated. Thus, the combined activity of TLR5 and TNFR2 signaling may be responsible for DC loss in lamina propria of Xiap−/− mice. Consequently, both Tnfr1−/−Xiap−/− and Tnfr2−/−Xiap−/− mice were rescued from dysbiosis and intestinal inflammation. Furthermore, RNA-seq of ileal crypts revealed that in inflamed Xiap−/− mice, TLR5 signaling was abrogated, linking aberrant TNF responses with the development of a dysbiosis. Evidence for TNFR2 signaling driving intestinal inflammation was detected in XLP2 patient samples. Together, these data point toward a key role of XIAP in mediating resilience of TLR5-expressing PCs and intestinal DCs, allowing them to maintain tissue integrity and microbiota homeostasis.

Angiotensin II-induced renal angiotensinogen formation is enhanced in mice lacking tumor necrosis factor-alpha type 1 receptor.

In hypertension induced by angiotensin II (AngII) administration with high salt (HS) intake, intrarenal angiotensinogen (AGT) and tumor necrosis factor-alpha (TNF-α) levels increase. However, TNF-α has been shown to suppress AGT formation in cultured renal proximal tubular cells. We examined the hypothesis that elevated AngII levels during HS intake reduces TNF-α receptor type 1 (TNFR1) activity in the kidneys, thus facilitating increased intrarenal AGT formation. The responses to HS diet (4% NaCl) with chronic infusion of AngII (25 ng/min) via implanted minipump for 4 weeks were assessed in wild-type (WT) and knockout (KO) mice lacking TNFR1 or TNFR2 receptors. Blood pressure was measured by tail-cuff plethysmography, and 24-h urine samples were collected using metabolic cages prior to start (0 day) and at the end of 2nd and 4th week periods. The urinary excretion rate of AGT (uAGT; marker for intrarenal AGT) was measured using ELISA. HS +AngII treatment for 4 weeks increased mean arterial pressure (MAP) in all strains of mice. However, the increase in MAP in TNFR1KO (77 ± 2 to 115 ± 3 mmHg; n = 7) was significantly greater (p < 0.01) than in WT (76 ± 1 to 102 ± 2 mmHg; n = 7) or in TNFR2KO (78 ± 2 to 99 ± 5 mmHg; n = 6). The increase in uAGT at 4th week was also greater (p < 0.05) in TNFR1KO mice (6 ± 2 to 167 ± 75 ng/24 h) than that in WT (6 ± 3 to 46 ± 16 ng/24 h) or in TNFR2KO mice (8 ± 7 to 65 ± 44 ng/24 h). The results indicate that TNFR1 exerts a protective role by mitigating intrarenal AGT formation induced by elevated AngII and HS intake.

Tumor Necrosis Factor Receptor-1 (p55) Deficiency Attenuates Tumor Growth and Intratumoral Angiogenesis and Stimulates CD8+ T Cell Function in Melanoma.

The role of tumor necrosis factor-α (TNF-α) in shaping the tumor microenvironment is ambiguous. Consistent with its uncertain role in melanoma, TNF-α plays a dual role, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. TNF-α signals via two cognate receptors, namely TNFR1 (p55) and TNFR2 (p75), which mediate divergent biological activities. Here, we analyzed the impact of TNFR1 deficiency in tumor progression in the B16.F1 melanoma model. Tumors developed in mice lacking TNFR1 (TNFR1 knock-out; KO) were smaller and displayed lower proliferation compared to their wild type (WT) counterpart. Moreover, TNFR1 KO mice showed reduced tumor angiogenesis. Although no evidence of spontaneous metastases was observed, conditioned media obtained from TNFR1 KO tumors increased tumor cell migration. Whereas the analysis of tumor-associated immune cell infiltrates showed similar frequency of total and M2-polarized tumor-associated macrophages (TAMs), the percentage of CD8+ T cells was augmented in TNFR1 KO tumors. Indeed, functional ex vivo assays demonstrated that CD8+ T cells obtained from TNFR1KO mice displayed an increased cytotoxic function. Thus, lack of TNFR1 attenuates melanoma growth by modulating tumor cell proliferation, migration, angiogenesis and CD8+ T cell accumulation and activation, suggesting that interruption of TNF-TNFR1 signaling may contribute to control tumor burden.

A Study of Cecal Ligation and Puncture-Induced Sepsis in Tissue-Specific Tumor Necrosis Factor Receptor 1-Deficient Mice.

Sepsis is a complex syndrome resulting from a dysregulated immune response to an infection. Due to the high prevalence, morbidity, and mortality, there is a lot of interest in understanding pathways that play a role in sepsis, with a focus on the immune system. Tumor necrosis factor (TNF) is a pleiotropic pro-inflammatory cytokine and a master regulator of the immune system but clinical trials with TNF blockers in sepsis have failed to demonstrate significant protection. Since TNF stimulates two different receptors, TNF receptor 1 (TNFR1) and TNFR2, pan-TNF inhibition might be suboptimal since both receptors have opposite functions in polymicrobial sepsis. Therefore, we hypothesized that TNF has a dual role in sepsis, namely a mediating and a protective role, and that protection might be obtained by TNFR1-specific inhibition. We here confirmed that TNFR1-/- mice are protected in the sterile endotoxemia model, whereas TNFR1 deficiency did not protect in the cecal ligation and puncture (CLP)-induced polymicrobial sepsis model. Since whole body TNFR1 blockage might be deleterious because of the antibacterial function of TNF/TNFR1 signaling, we focused on the potential devastating role of TNF/TNFR1 signaling in specific cell types. We were interested in the gut epithelium, the endothelium, and hepatocytes using conditional TNFR1-/- mice, as these cell types have been shown to play a role in sepsis. However, none of these conditional knockout mice showed improved survival in the CLP model. We conclude that cell-specific targeting of TNFR1 to these cell types has no therapeutic future in septic peritonitis.

TNF-α is responsible for the contribution of stromal cells to osteoclast and odontoclast formation during orthodontic tooth movement.

Compressive force during orthodontic tooth movement induces osteoclast formation in vivo. TNF-α plays an important role in mouse osteoclast formation and bone resorption induced by compressive force during orthodontic tooth movement. Stromal cells, macrophages and T cells take part in TNF-α-induced osteoclast formation in vitro. Root resorption caused by odontoclasts is a major clinical problem during orthodontic tooth movement. In this study, we determined the cell type targeted by TNF-α during compressive-force-induced osteoclast and odontoclast formation to elucidate the mechanism of bone and root resorption in vivo. An orthodontic tooth movement mouse model was prepared with a nickel-titanium closed coil spring inserted between the maxillary incisors and the first molar. Using TNF receptor 1- and 2-deficient (KO) mice, we found that osteoclast and odontoclast formation was mediated by TNF-α in orthodontic tooth movement. We generated four types of chimeric mice: wild-type (WT) bone marrow cells transplanted into lethally irradiated WT mice (WT>WT), KO bone marrow cells transplanted into lethally irradiated WT mice (KO>WT), WT bone marrow cells transplanted into lethally irradiated KO mice (WT>KO), and KO marrow cells transplanted into lethally irradiated KO mice (KO>KO). Using anti-CD4 and anti-CD8 antibodies, T cells were eliminated from these mice. We subjected these chimeric mice to orthodontic tooth movement. Orthodontic tooth movement was evaluated and tartrate-resistant acid phosphatase-positive cells along the alveolar bone (osteoclasts) and along the tooth root (odontoclasts) were counted after 12 days of tooth movement. The amount of orthodontic tooth movement, and the number of osteoclasts and odontoclasts on the compression side were significantly lower in WT>KO and KO>KO mice than in WT>WT and KO>WT mice. According to these results, we concluded that TNF-α-responsive stromal cells are important for osteoclast and odontoclast formation during orthodontic tooth movement.

Reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55 deficient mice.

Endometriosis is a chronic gynecological disease, characterized by growth of endometrial tissue in ectopic sites due to alteration of peritoneal homeostasis and deregulation of apoptosis. Here we have examined whether TNFRp55 deficiency modulates the pro-inflammatory state and the reinnervation of endometriotic-like lesions in mice. Two-month-old female C57BL/6 mice, eight wild type (WT) and eight TNFRp55-/- (KO) were used in the study. Endometriotic-like lesions were induced experimentally. The right uterine horn was removed from the animal, divided longitudinally, cut in three square pieces and sutured to the intestine mesentery. After 4 weeks, the lesions and the peritoneal fluid were collected. The level of TNFα in the peritoneal fluid was evaluated by enzyme-linked immunosorbent assay (EIA). The expressions of COX2, GRα and GRß were evaluated in the lesions by western blot and immunohistochemistry. ß-III TUBULIN, BDNF and NGF protein concentrations were evaluated in the lesions by western blot. Gene expression of Pgp 9.5, SP and Th was analyzed by RT-PCR, whereas relative concentrations of TRKA, NTRp75, phosphorylated NFκB (pNFκB) and total NFκB in lesions were measured by EIA. Compared with the WT group, the KO mice showed lower TNFα levels in the peritoneal fluid and lower numbers of COX2 immunoreactive cells along with increased expression of GRα, ß-III TUBULIN, Pgp 9.5, SP, Th, BDNF, NGF, NTRp75 and pNFκB in the lesions. Future histological studies will be necessary to confirm the sensory/sympathetic imbalance in the endometriotic-like lesions of the KO mice. Our results suggest that a reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55-/- mice. Chronic deregulation of TNF receptors can have serious consequences for women with advanced endometriosis.

Deletion of tumour necrosis factor α receptor 1 elicits an increased TH17 immune response in the chronically inflamed liver.

Tumour necrosis factor α receptor 1 (TNFR1) activation is known to induce cell death, inflammation, and fibrosis but also hepatocyte survival and regeneration. The multidrug resistance protein 2 knockout (Mdr2-/) mice are a model for chronic hepatitis and inflammation-associated hepatocellular carcinoma (HCC) development. This study analysed how the absence of TNFR1 mediated signalling shapes cytokine and chemokine production, immune cell recruitment and ultimately influences liver injury and fibrotic tissue remodelling in the Mdr2-/- mouse model. We show that Tnfr1-/-/Mdr2-/- mice displayed increased plasma levels of ALT, ALP, and bilirubin as well as a significantly higher collagen content, and markers of fibrosis than Mdr2-/- mice. The expression profile of inflammatory cytokines (Il1b, Il23, Tgfb1, Il17a), chemokines (Ccl2, Cxcl1, Cx3cl1) and chemokine receptors (Ccr6, Cxcr6, Cx3cr1) in livers of Tnfr1-/-/Mdr2-/- mice indicated TH17 cell infiltration. Flow cytometric analysis confirmed that the aggravated tissue injury in Tnfr1-/-/Mdr2-/- mice strongly correlated with increased hepatic recruitment of TH17 cells and enhanced IL-17 production in the injured liver. Moreover, we observed increased hepatic activation of RIPK3 in Tnfr1-/-/Mdr2-/- mice, which was not related to necroptotic cell death. Rather, frequencies of infiltrating CX3CR1+ monocytes increased over time in livers of Tnfr1-/-/Mdr2-/- mice, which expressed significantly higher levels of Ripk3 than those of Mdr2-/- mice. Overall, we conclude that the absence of TNFR1-mediated signalling did not improve the pathological phenotype of Mdr2-/- mice. It instead caused enhanced infiltration of TH17 cells and CX3CR1+ monocytes into the injured tissue, which was accompanied by increased RIPK3 activation and IL-17 production.

Endometriosis progression in tumor necrosis factor receptor p55-deficient mice: Impact on oxidative/nitrosative stress and metallomic profile.

The present study was conducted to investigate whether the deficiency of tumor necrosis factor receptor p55 (TNFRp55) modulates oxidative/nitrosative stress and metallomic profile into the peritoneal cavity during the experimental endometriosis progression in mice. Female C57BL/6 mice, wild-type (WT) and TNFRp55 knockout (KO) of two months were used. Endometriosis was induced experimentally by autotransplanting three pieces of the right uterine horn to the intestinal mesentery. After four weeks, endometriotic-like lesions and peritoneal lavage fluid were collected. The obtained peritoneal fluid was analyzed for nitrite levels using the Griess method and trace elements concentrations by ICP-MS. Both endometriotic-like lesions and cells isolated from peritoneal lavage were analyzed for the following oxidative/nitrosative stress markers: inducible nitric oxide synthase (iNOS) expression by Western Blot; total antioxidant capacity (TAC), the activity of two antioxidant enzymes (CAT and GPX) and thiobarbituric acid-reactive substances (TBARS) concentration, by spectrophotometric method; and protein carbonyl content and nitrotyrosine presence by ELISA. In comparison to WT group, KO mice exhibited larger lesion volume; higher levels of nitrite, copper (Cu) and strontium (Sr) in the peritoneal fluid; increased TAC, CAT, and GPX in peritoneal lavage cells; decreased concentration of TBARS in lesions and protein carbonyl in peritoneal lavage cells. Significant positive correlations between Cu and lesion volume, Sr and lesion volume, and Cu and Sr were obtained. Our results suggest that the TNFRp55 deficiency increases antioxidant protection and promotes high Cu-Sr concentrations in the peritoneal cavity, which favors the progression of experimental endometriosis.

Contribution of the TNF-α (Tumor Necrosis Factor-α)-TNF-Rp55 (Tumor Necrosis Factor Receptor p55) Axis in the Resolution of Venous Thrombus.

Objective- Deep vein thrombosis results from a combination of risk factors including genetic conditions, obesity, drugs, pregnancy, aging, and malignancy. We examined pathophysiological roles of the TNF-α (tumor necrosis factor-α)-TNF-Rp55 (tumor necrosis factor receptor p55) axis in thrombus resolution using Tnfrp55-/- (TNF-Rp55-deficient) mice. Approach and Results- On ligating the inferior vena cava of wild-type (WT) mice, venous thrombi formed and grew progressively until 5 days but shrunk to <50% of the thrombus weight at day 14. Concomitantly, inferior vena cava ligation enhanced intrathrombotic gene expression of Tnfa and Tnfrp55, and intrathrombotic macrophages expressed both TNF-α and TNF-Rp55 proteins. In Tnfrp55-/- mice treated with the same manner, thrombus formed at a similar rate for 5 days, but shrinking was delayed compared with WT mice. Moreover, the blood flow recovery in thrombosed inferior vena cava was suspended in Tnfrp55-/- mice compared with WT mice. Intrathrombotic Plau (urokinase-type plasminogen activator), Mmp2 (matrix metalloproteinase 2), and Mmp9 (matrix metalloproteinase 9) mRNA expression was significantly reduced in Tnfrp55-/- mice, compared with WT ones. Supportingly, the administration of anti-TNF-α antibody or TNF-α inhibitor (etanercept) delayed the thrombus resolution in WT mice. Furthermore, TNF-α treatment enhanced gene expression of Plau, Mmp2, and Mmp9 in WT macrophages but not Tnfrp55-/- macrophages. These effects were significantly suppressed by ERK (extracellular signal regulated kinase) and NF-κB (nuclear factor-kappa B) inhibitors. Therefore, the lack of TNF-Rp55 has detrimental roles in the thrombus resolution by suppressing PLAU, MMP-2, and MMP-9 expression. In contrast, TNF-α administration accelerated thrombus resolution in WT but not Tnfrp55-/- mice. Conclusions- The TNF-α-TNF-Rp55 axis may have essential roles in the resolution of venous thrombus in mice.

TNFR1 Signaling Contributes to T Cell Anergy During Staphylococcus aureus Sepsis.

Early research on sepsis has focused on the initial hyper-inflammatory, cytokine mediated phase of the disorder whereas the events that govern the concomitant and subsequent anti-inflammatory compensatory response are not completely understood. In this context, the putative participation of TNFR1-mediated signaling in the immunosuppressive phase of Staphylococcus aureus sepsis has not been elucidated. The aim of this study was to determine the role of TNFR1 in directing the immune dysfunction during S. aureus sepsis and the potential contribution of MDSC to this process. Using a model of sepsis of peritoneal origin and tnfr1-/- mice, we demonstrated that during staphylococcal sepsis CD4+ T cell anergy is significantly dependent on TNFR1 expression and that signaling through this receptor has an impact on bacterial clearance in the spleen. MDSC played a major role in the generation of anergic CD4+ T cells and their accumulation in the spleen during S. aureus sepsis correlated with IL-6 induction. Although TNFR1 signaling was not required for MDSC accumulation and expansion in the spleen, it determined the in vivo expression of Arginase 1 and iNOS, enzymes known to participate in the suppressive function of this population. Moreover, our data indicate that TNFR1-mediated IL-10 production may modulate MDSC function during staphylococcal sepsis. Taken together these results indicate that TNFR1 plays a critical role on T cell dysfunction during S. aureus sepsis by regulating immunomodulatory mediators in MDSC. The role of TNFR1-mediated signaling during the immunosuppressive phase of staphylococcal sepsis should be considered when designing novel alternative therapeutic approaches.

TNF-α/TNFR1 Signaling is Required for the Full Expression of Acute and Chronic Itch in Mice via Peripheral and Central Mechanisms.

Increasing evidence suggests that cytokines and chemokines play crucial roles in chronic itch. In the present study, we evaluated the roles of tumor necrosis factor-alpha (TNF-α) and its receptors TNF receptor subtype-1 (TNFR1) and TNFR2 in acute and chronic itch in mice. Compared to wild-type (WT) mice, TNFR1-knockout (TNFR1-KO) and TNFR1/R2 double-KO (DKO), but not TNFR2-KO mice, exhibited reduced acute itch induced by compound 48/80 and chloroquine (CQ). Application of the TNF-synthesis inhibitor thalidomide and the TNF-α antagonist etanercept dose-dependently suppressed acute itch. Intradermal injection of TNF-α was not sufficient to evoke scratching, but potentiated itch induced by compound 48/80, but not CQ. In addition, compound 48/80 induced TNF-α mRNA expression in the skin, while CQ induced its expression in the dorsal root ganglia (DRG) and spinal cord. Furthermore, chronic itch induced by dry skin was reduced by administration of thalidomide and etanercept and in TNFR1/R2 DKO mice. Dry skin induced TNF-α expression in the skin, DRG, and spinal cord and TNFR1 expression only in the spinal cord. Thus, our findings suggest that TNF-α/TNFR1 signaling is required for the full expression of acute and chronic itch via peripheral and central mechanisms, and targeting TNFR1 may be beneficial for chronic itch treatment.

Lack of TNF-α signaling through p55 makes the mice more susceptible to acute infection but does not alter state of latency and reactivation of HSV-1.

TNF-α has been shown to play an important role in pathogenesis and latency of HSV-1 infections. TNF-α signals through TNFR1 (p55) and TNFR2 (p75), and signaling through p55 generally results in gene activation leading to induction of inflammatory responses. Here, we studied the role of TNF-α signaling in latent virus reactivation in p55-knock out (KO) mouse model of ocular HSV-1 infection. We found that KO mice are more susceptible to HSV-1 infection compared to wild type C57Bl/6 mice. While the absence of TNFRI signaling enhanced the ganglion latent DNA content by two folds, there was no difference in the maintenance and reactivation of latent HSV-1. Strikingly, interfering with inflammatory responses through PGE2 synthesis by treating latently infected wild type mice with indomethacin (COX inhibitor) prior to UV-exposure prevented HSV-1 reactivation. These results suggest that reactivation of latent HSV-1 might result from the cumulative effects of a cascade of inflammatory cytokines including TNF-α.

TNFR1 inhibition with a Nanobody protects against EAE development in mice.

TNF has as detrimental role in multiple sclerosis (MS), however, anti-TNF medication is not working. Selective TNF/TNFR1 inhibition whilst sparing TNFR2 signaling reduces the pro-inflammatory effects of TNF but preserves the important neuroprotective signals via TNFR2. We previously reported the generation of a Nanobody-based selective inhibitor of human TNFR1, TROS that will be tested in experimental autoimmune encephalomyelitis (EAE). We specifically antagonized TNF/TNFR1 signaling using TROS in a murine model of MS, namely MOG35-55-induced EAE. Because TROS does not cross-react with mouse TNFR1, we generated mice expressing human TNFR1 in a mouse TNFR1-knockout background (hTNFR1 Tg), and we determined biodistribution of 99mTc-TROS and effectiveness of TROS in EAE in those mice. Biodistribution analysis demonstrated that intraperitoneally injected TROS is retained more in organs of hTNFR1 Tg mice compared to wild type mice. TROS was also detected in the cerebrospinal fluid (CSF) of hTNFR1 Tg mice. Prophylactic TROS administration significantly delayed disease onset and ameliorated its symptoms. Moreover, treatment initiated early after disease onset prevented further disease development. TROS reduced spinal cord inflammation and neuroinflammation, and preserved myelin and neurons. Collectively, our data illustrate that TNFR1 is a promising therapeutic target in MS.

Integrin beta 1 inhibition alleviates the chronic hyperproliferative dermatitis phenotype of SHARPIN-deficient mice.

SHARPIN (Shank-Associated RH Domain-Interacting Protein) is a component of the linear ubiquitin chain assembly complex (LUBAC), which enhances TNF-induced NF-κB activity. SHARPIN-deficient (Sharpincpdm/cpdm) mice display multi-organ inflammation and chronic proliferative dermatitis (cpdm) due to TNF-induced keratinocyte apoptosis. In cells, SHARPIN also inhibits integrins independently of LUBAC, but it has remained enigmatic whether elevated integrin activity levels in the dermis of Sharpincpdm/cpdm mice is due to increased integrin activity or is secondary to inflammation. In addition, the functional contribution of increased integrin activation to the Sharpincpdm/cpdm phenotype has not been investigated. Here, we find increased integrin activity in keratinocytes from Tnfr1-/- Sharpincpdm/cpdm double knockout mice, which do not display chronic inflammation or proliferative dermatitis, thus suggesting that SHARPIN indeed acts as an integrin inhibitor in vivo. In addition, we present evidence for a functional contribution of integrin activity to the Sharpincpdm/cpdm skin phenotype. Treatment with an integrin beta 1 function blocking antibody reduced epidermal hyperproliferation and epidermal thickness in Sharpincpdm/cpdm mice. Our data indicate that, while TNF-induced cell death triggers the chronic inflammation and proliferative dermatitis, absence of SHARPIN-dependent integrin inhibition exacerbates the epidermal hyperproliferation in Sharpincpdm/cpdm mice.

Chronic Brucella Infection Induces Selective and Persistent Interferon Gamma-Dependent Alterations of Marginal Zone Macrophages in the Spleen.

The spleen is known as an important filter for blood-borne pathogens that are trapped by specialized macrophages in the marginal zone (MZ): the CD209+ MZ macrophages (MZMs) and the CD169+ marginal metallophilic macrophages (MMMs). Acute systemic infection strongly impacts MZ populations and the location of T and B lymphocytes. This phenomenon has been linked to reduced chemokine secretion by stromal cells. Brucella spp. are the causative agent of brucellosis, a widespread zoonotic disease. Here, we used Brucella melitensis infection as a model to investigate the impact of chronic stealth infection on splenic MZ macrophage populations. During the late phase of Brucella infection, we observed a loss of both MZMs and MMMs, with a durable disappearance of MZMs, leading to a reduction of the ability of the spleen to take up soluble antigens, beads, and unrelated bacteria. This effect appears to be selective as every other lymphoid and myeloid population analyzed increased during infection, which was also observed following Brucella abortus and Brucella suis infection. Comparison of wild-type and deficient mice suggested that MZ macrophage population loss is dependent on interferon gamma (IFN-γ) receptor but independent of T cells or tumor necrosis factor alpha receptor 1 (TNF-αR1) signaling pathways and is not correlated to an alteration of CCL19, CCL21, and CXCL13 chemokine mRNA expression. Our results suggest that MZ macrophage populations are particularly sensitive to persistent low-level IFN-γ-mediated inflammation and that Brucella infection could reduce the ability of the spleen to perform certain MZM- and MMM-dependent tasks, such as antigen delivery to lymphocytes and control of systemic infection.

TNFRp55 deficiency promotes the development of ectopic endometriotic-like lesions in mice.

Endometriosis is an inflammatory disease depending on estradiol, with TNF-α being one of the most representative cytokines involved in its pathogenesis. TNF-α acts through its bond to the TNFRp55 and TNFRp75 membrane receptors. The aim of this study was to analyze the effect of the TNFRp55 deficiency on the development of ectopic endometriotic-like lesions. Endometriosis was induced surgically in mice of the C57BL/6 strain, wild type (WT) and TNFRp55-/- (KO). After four weeks, the peritoneal fluid was collected and the lesions were counted, measured with a caliper, removed, weighed, fixed or kept at -80°C. We evaluated the cell proliferation by proliferating cell nuclear antigen (PCNA) immunohistochemistry and apoptosis by TUNEL technique in the ectopic lesions. MMP-2 and MMP-9 activities (factors involved in invasiveness) were measured by zymography in the peritoneal fluid; estradiol and progesterone levels were measured by radioimmunoassay in the lesions and in the peritoneal fluid. We found that in KO animals the mean number of lesions established per mouse, the lesion volume, weight and cell proliferation increased and apoptosis decreased. In addition, the activity of MMP-2 and the estradiol level increased, whereas the progesterone level was not significantly modified. In conclusion, the deficiency of TNFRp55 promoted the establishment and development of endometriosis through an increase in the lesion size and high levels of estradiol which correlate with an increase in the MMP-2 activity. This is evidence of the possible association of the deregulation of the TNFRp55 expression and the survival of the endometriotic tissue in ectopic sites.

Mechanisms of Retinal Damage after Ocular Alkali Burns.

Alkali burns to the eye constitute a leading cause of worldwide blindness. In recent case series, corneal transplantation revealed unexpected damage to the retina and optic nerve in chemically burned eyes. We investigated the physical, biochemical, and immunological components of retinal injury after alkali burn and explored a novel neuroprotective regimen suitable for prompt administration in emergency departments. Thus, in vivo pH, oxygen, and oxidation reduction measurements were performed in the anterior and posterior segment of mouse and rabbit eyes using implantable microsensors. Tissue inflammation was assessed by immunohistochemistry and flow cytometry. The experiments confirmed that the retinal damage is not mediated by direct effect of the alkali, which is effectively buffered by the anterior segment. Rather, pH, oxygen, and oxidation reduction changes were restricted to the cornea and the anterior chamber, where they caused profound uveal inflammation and release of proinflammatory cytokines. The latter rapidly diffuse to the posterior segment, triggering retinal damage. Tumor necrosis factor-α was identified as a key proinflammatory mediator of retinal ganglion cell death. Blockade, by either monoclonal antibody or tumor necrosis factor receptor gene knockout, reduced inflammation and retinal ganglion cell loss. Intraocular pressure elevation was not observed in experimental alkali burns. These findings illuminate the mechanism by which alkali burns cause retinal damage and may have importance in designing therapies for retinal protection.

Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways.

Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF- and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM-induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.

Sensitizing leukemia stem cells to NF-κB inhibitor treatment in vivo by inactivation of both TNF and IL-1 signaling.

We previously reported that autocrine TNF-α (TNF) is responsible for JNK pathway activation in a subset of acute myeloid leukemia (AML) patient samples, providing a survival/proliferation signaling parallel to NF-κB in AML stem cells (LSCs). In this study, we report that most TNF-expressing AML cells (LCs) also express another pro-inflammatory cytokine, IL1ß, which acts in a parallel manner. TNF was produced primarily by LSCs and leukemic progenitors (LPs), whereas IL1ß was mainly produced by partially differentiated leukemic blasts (LBs). IL1ß also stimulates an NF-κB-independent pro-survival and proliferation signal through activation of the JNK pathway. We determined that co-inhibition of signaling stimulated by both TNF and IL1ß synergizes with NF-κB inhibition in eliminating LSCs both ex vivo and in vivo. Our studies show that such treatments are most effective in M4/5 subtypes of AML.

Regulation of myocardial stromal cell-derived factor 1α/CXCL12 by tumor necrosis factor signaling.

BACKGROUND: Global myocardial ischemia-reperfusion (I/R) occurs during cardiac operations. This I/R injury leads to increased production of tumor necrosis factor α (TNF) instantly and upregulated expression of stromal cell-derived factor 1 α (SDF-1). On the basis of the published data from our laboratory and other groups, locally produced TNF contributes to cardiac dysfunction mainly via binding to its receptor (tumor necrosis factor receptor 1 [TNFR1]), whereas ischemia-induced myocardial SDF-1 mediates cardioprotection. Although TNF has been shown to work as an upstream initiator for induction of other cytokines and chemokines, there is no information regarding the interaction among TNF, TNFRs, and myocardial SDF-1 expression. In this study, given that TNF downregulated SDF-1 in vascular endothelial cells, we therefore hypothesized that TNF would have a negative effect on myocardial SDF-1 production, which is attributable to TNFR-initiated actions. METHODS: Using a Langendorff model, isolated male mouse hearts were infused with TNF for 45 min. Male adult mouse hearts from wild type, TNFR1 knockout (TNFR1KO), TNFR2KO, and TNFR1/2KO were subjected to global I/R. H9c2 cells with small interfering RNA transfection were used as an in vitro model. The levels of SDF-1 (protein and messenger RNA) were detected by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction . Protein kinases of IκB (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α) and c-jun N-terminal kinase were also determined using Western blot assay. RESULTS: TNF infusion downregulated myocardial SDF-1 production in a dose-dependent manner in the hearts. In addition, using TNF significantly decreased SDF-1 expression in cardiomyoblasts (H9c2 cells), which was associated with reduced IκB level. Knockdown of TNFR1 or TNFR2 by small interfering RNAs neutralized TNF-suppressed SDF-1 in H9c2 cells. Furthermore, deletion of TNFR1/2 or TNFR2 increased SDF-1 production in the hearts after I/R. CONCLUSIONS: Our study represents the initial evidence showing that TNF plays an inhibitory role in modulating myocardial SDF-1 production and blockade of TNF signaling by ablation of TNFR1 and TNFR2 genes increased SDF-1 expression in the heart. These data expand on TNF signaling-initiated mechanisms in myocardium, which may lend a more complete understanding of SDF-1 and TNFR-derived actions in hopes of advancing ischemic heart injury treatments.