The junctional adhesion molecule-like protein (JAML) is important for the inflammatory response during contact hypersensitivity.

BACKGROUND: The junctional adhesion molecule-like protein (JAML) plays important roles in wound healing and activation of epidermal γδ T cells in mice. Whether JAML plays a role in contact hypersensitivity (CHS), the animal model of allergic contact dermatitis (ACD), is not known. METHODS: To examine the role of JAML in CHS, we used various mouse models of CHS in JAML knockout (KO) and wild-type (WT) mice. Furthermore, the expression of the JAML ligand coxsackievirus and adenovirus receptor (CXADR) on keratinocytes was accessed in vitro and in vivo. RESULTS: JAML KO mice had a diminished inflammatory response during both the sensitization and elicitation phase of CHS and had reduced numbers of CD8+ and CD4+ T cells in the epidermis. Furthermore, interferon γ (IFNγ), interleukin 1ß (IL-1ß) and CXCL10 production were significantly reduced in JAML KO mice during the elicitation phase. We found that CD8+ T cells express JAML and that JAML is essential for rapid flare-up responses to contact allergens. Finally, we show that keratinocytes up-regulate the JAML ligand CXADR following exposure to contact allergens. CONCLUSION: Our study is the first to show a central role of JAML in CHS and reveals a potential new target for the treatment of ACD in humans.

CD100 boosts the inflammatory response in the challenge phase of allergic contact dermatitis in mice.

BACKGROUND: Allergic contact dermatitis (ACD) is an inflammatory disease with a complex pathophysiology in which epidermal-resident memory CD8+ T (TRM ) cells play a key role. The mechanisms involved in the activation of CD8+ TRM cells during allergic flare-up responses are not understood. METHODS: The expression of CD100 and its ligand Plexin B2 on CD8+ TRM cells and keratinocytes before and after allergen exposure was determined by flow cytometry and RT-qPCR. The role of CD100 in the inflammatory response during the challenge phase of ACD was determined in a model of ACD in CD100 knockout and wild-type mice. RESULTS: We show that CD8+ TRM cells express CD100 during homeostatic conditions and up-regulate it following re-exposure of allergen-experienced skin to the experimental contact allergen 1-fluoro-2,4-dinitrobenzene (DNFB). Furthermore, Plexin B2 is up-regulated on keratinocytes following exposure to some contact allergens. We show that loss of CD100 results in a reduced inflammatory response to DNFB with impaired production of IFNγ, IL-17A, CXCL1, CXCL2, CXCL5, and IL-1ß and decreased recruitment of neutrophils to the epidermis. CONCLUSION: Our study demonstrates that CD100 is expressed on CD8+ TRM cells and is required for full activation of CD8+ TRM cells and the flare-up response of ACD.

Lack of Both Nucleotide-Binding Oligomerization Domain-Containing Proteins 1 and 2 Primes T Cells for Activation-Induced Cell Death.

Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2-/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2-/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2-/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation.

The Bacterial Peptidoglycan-Sensing Molecules NOD1 and NOD2 Promote CD8+ Thymocyte Selection.

Nucleotide-binding and oligomerization domain (NOD)-like receptors NOD1 and NOD2 are cytosolic innate immune receptors that recognize microbial peptidoglycans. Although studies have addressed the role of NOD proteins in innate immune responses, little attention has been given to their impact on the developing adaptive immune system. We have assessed the roles of NOD1 and NOD2 deficiency on T cell development in mice. Our results demonstrate that NOD1 and NOD2 promote the positive selection/maturation of CD8 single-positive thymocytes in a thymocyte-intrinsic manner. TCR-mediated ERK phosphorylation is significantly reduced in the absence of NOD proteins, but receptor-interacting protein 2 is not involved in CD8 single-positive thymocyte selection or ERK signaling. Commensal bacteria-free animals have thymocyte maturation defects, and exogenous NOD ligands can enhance thymocyte maturation in culture. These results raise the intriguing possibility that abnormal lymphocyte responses observed in NOD-dependent inflammatory diseases are not driven solely by microbial signals in the gut, but may also involve intrinsic lymphocyte defects resulting from impaired CD8 T cell thymic development.

Pregnancy in chronic kidney disease and kidney transplantation.

Chronic kidney disease (CKD) affects up to 6% of women of childbearing age in high income countries, and is estimated to affect 3% of pregnant women. Advanced renal dysfunction, proteinuria, hypertension, and poorly controlled underlying primary renal disease are all significant risks for adverse maternal, fetal, and renal outcomes. In order to achieve the best outcomes, it is therefore of paramount importance that these pregnancies are planned, where possible, to allow the opportunity to counsel women and their partners in advance and to optimize these risks. These pregnancies should be deemed high risk and they require close antenatal monitoring from an expert multidisciplinary team. We discuss the effect of pregnancy on CKD, and also current guidelines and literature with specific reference to transplantation, autoimmune disease, and medication use in pregnancy. We also discuss the benefits of prepregnancy counseling and give practical recommendations to advise pregnant women with renal disease.

Urine Fibrosis Markers and Risk of Allograft Failure in Kidney Transplant Recipients: A Case-Cohort Ancillary Study of the FAVORIT Trial.

BACKGROUND: Kidney tubulointerstitial fibrosis marks risk for allograft failure in kidney transplant recipients, but is poorly captured by estimated glomerular filtration rate (eGFR) or urine albumin-creatinine ratio (ACR). Whether urinary markers of tubulointerstitial fibrosis can noninvasively identify risk for allograft failure above and beyond eGFR and ACR is unknown. STUDY DESIGN: Case-cohort study. SETTING & PARTICIPANTS: The FAVORIT (Folic Acid for Vascular Outcome Reduction in Transplantation) Trial was a randomized double-blind trial testing vitamin therapy to lower homocysteine levels in stable kidney transplant recipients. We selected a subset of participants at random (n=491) and all individuals with allograft failure during follow-up (cases; n=257). PREDICTOR: Using spot urine specimens from the baseline visit, we measured 4 urinary proteins known to correlate with tubulointerstitial fibrosis on biopsy (urine α1-microglobulin [A1M], monocyte chemoattractant protein 1 [MCP-1], and procollagen type III and type I amino-terminal amino pro-peptide). OUTCOME: Death-censored allograft failure. RESULTS: In models adjusted for demographics, chronic kidney disease risk factors, eGFR, and ACR, higher concentrations of urine A1M (HR per doubling, 1.73; 95% CI, 1.43-2.08) and MCP-1 (HR per doubling, 1.60; 95% CI, 1.32-1.93) were strongly associated with allograft failure. When additionally adjusted for concentrations of other urine fibrosis and several urine injury markers, urine A1M (HR per doubling, 1.76; 95% CI, 1.27-2.44]) and MCP-1 levels (HR per doubling, 1.49; 95% CI, 1.17-1.89) remained associated with allograft failure. Urine procollagen type III and type I levels were not associated with allograft failure. LIMITATIONS: We lack kidney biopsy data, BK titers, and HLA antibody status. CONCLUSIONS: Urine measurement of tubulointerstitial fibrosis may provide a noninvasive method to identify kidney transplant recipients at higher risk for future allograft failure, above and beyond eGFR and urine ACR.

Inflammation in AKI: Current Understanding, Key Questions, and Knowledge Gaps.

Inflammation is a complex biologic response that is essential for eliminating microbial pathogens and repairing tissue after injury. AKI associates with intrarenal and systemic inflammation; thus, improved understanding of the cellular and molecular mechanisms underlying the inflammatory response has high potential for identifying effective therapies to prevent or ameliorate AKI. In the past decade, much knowledge has been generated about the fundamental mechanisms of inflammation. Experimental work in small animal models has revealed many details of the inflammatory response that occurs within the kidney after typical causes of AKI, including insights into the molecular signals released by dying cells, the role of pattern recognition receptors, the diverse subtypes of resident and recruited immune cells, and the phased transition from destructive to reparative inflammation. Although this expansion of the basic knowledge base has increased the number of mechanistically relevant targets of intervention, progress in developing therapies that improve AKI outcomes by modulation of inflammation remains slow. In this article, we summarize the most important recent developments in understanding the inflammatory mechanisms of AKI, highlight key limitations of the commonly used animal models and clinical trial designs that may prevent successful clinical application, and suggest priority approaches for research toward clinical translation in this area.

Ischemia as a factor affecting innate immune responses in kidney transplantation.

PURPOSE OF REVIEW: Ischemic injury inevitably occurs during the procurement of organs for transplantation, and the injury is worsened by inflammation following reperfusion. The purpose of this review is to describe the role of the innate immune system in ischemia-induced renal injury in kidneys procured for transplantation. The key role of pattern recognition receptors in immune responses to ischemia is described. Innate immune receptors are emerging novel targets for the amelioration of ischemic injury of donor kidneys. RECENT FINDINGS: Several families of pattern recognition receptors are direct mediators of early injurious events during kidney procurement, and also innate and adaptive immune responses after transplantation. The deleterious events associated with the activation of the innate immune system in donor kidneys significantly contribute to short and long-term allograft outcomes. SUMMARY: Although a number of therapies have been proposed to decrease ischemic donor kidney injury, targeting the innate immune system is an exciting new area that is gaining significant interest in transplantation. As we learn more about how these important receptors are regulated by ischemia, strategies will likely evolve to allow their modulation in ischemic renal injury.

Assessment and management of hypertension in transplant patients.

Hypertension in renal transplant recipients is common and ranges from 50% to 80% in adult recipients and from 47% to 82% in pediatric recipients. Cardiovascular morbidity and mortality and shortened allograft survival are important consequences of inadequate control of hypertension. In this review, we examine the epidemiology, pathophysiology, and management considerations of post-transplant hypertension. Donor and recipient factors, acute and chronic allograft injury, and immunosuppressive medications may each explain some of the pathophysiology of post-transplant hypertension. As observed in other patient cohorts, renal artery stenosis and adrenal causes of hypertension may be important contributing factors. Notably, BP treatment goals for renal transplant recipients remain an enigma because there are no adequate randomized controlled trials to support a benefit from targeting lower BP levels on graft and patient survival. The potential for drug-drug interactions and altered pharmacokinetics and pharmacodynamics of the different antihypertensive medications need to be carefully considered. To date, no specific antihypertensive medications have been shown to be more effective than others at improving either patient or graft survival. Identifying the underlying pathophysiology and subsequent individualization of treatment goals are important for improving long-term patient and graft outcomes in these patients.

Regulation of TLR2 and NLRP3 in primary murine renal tubular epithelial cells.

Pattern recognition receptors (PRRs) are now recognized to be key triggers of injury in a variety of renal diseases. Several families of these receptors are present in the kidney, and recent data suggest that they are differentially expressed and regulated in the kidney. This study evaluated the interaction between two distinct PRRs that are expressed in the kidney, i.e. TLR2 (Toll-like receptor 2) and the NLRP3 inflammasome. The regulation and activation of these receptors in primary renal tubular epithelial (RTE) cells from murine kidneys were evaluated. RTE cells were extracted from WT and NLRP3-mutant mice and treated ex vivo with ligands specific for TLR2 or NLRP3. We found that TLR2 upregulated NLRP3 as well as its substrate IL-1ß, and that signaling through the NLRP3 inflammasome induced RTE cell necrosis. The results of this study suggest a previously unknown interaction between TLR2 and NLRP3 in primary RTE cells and highlight the importance of the cross talk that occurs in kidney-related PRRs. Understanding how PRRs are regulated is important for the design of rationale therapeutic strategies to modulate these receptors in renal disease.

Human pulmonary microvascular endothelial cells respond to DAMPs from injured renal tubular cells.

Acute kidney injury (AKI) causes distant organ dysfunction through yet unknown mechanisms, leading to multiorgan failure and death. The lungs are one of the most common extrarenal organs affected by AKI, and combined lung and kidney injury has a mortality as high as 60%-80%. One mechanism that has been implicated in lung injury after AKI involves molecules released from injured kidney cells (DAMPs, or damage-associated molecular patterns) that promote a noninfectious inflammatory response by binding to pattern recognition receptors (PRRs) constitutively expressed on the pulmonary endothelium. To date there are limited data investigating the role of PRRs and DAMPs in the pulmonary endothelial response to AKI. Understanding these mechanisms holds great promise for therapeutics aimed at ameliorating the devastating effects of AKI. In this study, we stimulate primary human microvascular endothelial cells with DAMPs derived from injured primary renal tubular epithelial cells (RTECs) as an ex-vivo model of lung injury following AKI. We show that DAMPs derived from injured RTECs cause activation of Toll-Like Receptor and NOD-Like Receptor signaling pathways as well as increase human primary pulmonary microvascular endothelial cell (HMVEC) cytokine production, cell signaling activation, and permeability. We further show that cytokine production in HMVECs in response to DAMPs derived from RTECs is reduced by the inhibition of NOD1 and NOD2, which may have implications for future therapeutics. This paper adds to our understanding of PRR expression and function in pulmonary HMVECs and provides a foundation for future work aimed at developing therapeutic strategies to prevent lung injury following AKI.

Innate immunity in donor procurement.

PURPOSE OF REVIEW: Ischaemic kidney injury occurs during organ procurement and can lead to delayed graft function or nonviable grafts. The innate immune system is a key trigger of inflammation in renal ischaemia. This review discusses the components of innate immunity known to be involved in renal ischaemic reperfusion injury (IRI). Understanding how inflammatory damage is initiated in renal IRI is important for the development of targeted therapies aimed at preserving the donor organ. RECENT FINDINGS: Much remains to be determined about the role of innate immune signalling in renal ischaemia/reperfusion injury. Recently, discoveries about complement receptors, Toll-like receptors (TLRs), NOD-like receptors (NLRs) and inflammasomes have opened new avenues of exploration. We are also now learning that macrophages, complement and TLR activation may have additional roles in renal repair following IRI. SUMMARY: A greater understanding of the mechanisms that contribute to innate immune-mediated renal ischaemic damage will allow for the development of therapeutics targeted to the donor organ. New data suggest that treatment limited to specific receptors on specific cells, or localized to specific regions within the kidney, may provide novel approaches to maximize our use of donor organs, particularly those that may have been discarded due to prolonged preimplantation ischaemia.

Intracellular pattern recognition receptors and renal ischemia.

Renal ischemia is a common cause of acute kidney injury in hospitalized patients. In certain settings renal ischemia is unavoidable, such as in kidneys harvested for transplantation. The molecular and cellular mechanisms that lead to the syndrome of ischemic renal injury are complicated and involve multiple cell types within the kidney, including renal epithelium and vasculature. Although it has been difficult to define pharmacologic targets for AKI, emerging information about a newly discovered host defense system is providing hope for novel pharmacologic targets to prevent and treat AKI. Molecular initiators of damage associated with hypoxia involve a phylogenically conserved host defense system called the innate immune system. Data point to an essential role for receptors of the innate immune system, particularly the membrane-bound Toll-like receptors and the intracellular nucleotide-binding oligomerization domain-like receptors. These receptors have been identified in human and rodent kidneys, and many investigators have shown that their deletion protects from experimental ischemia/reperfusion injury (a model for ischemic acute kidney injury). This review details current information about the innate immune system and the ischemic kidney with a focus on the emerging role of intracellular innate immune receptors.

Nod1 and nod2 are expressed in human and murine renal tubular epithelial cells and participate in renal ischemia reperfusion injury.

Nucleotide-binding oligomerization domain (Nod) 1 and Nod2 are members of a family of intracellular innate sensors that participate in innate immune responses to pathogens and molecules released during the course of tissue injury, including injury induced by ischemia. Ischemic injury to the kidney is characterized by renal tubular epithelial apoptosis and inflammation. Among the best studied intracellular innate immune receptors known to contribute to apoptosis and inflammation are Nod1 and Nod2. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in Nod1, Nod2, Nod(1 x 2), and in their downstream signaling molecule receptor-interacting protein 2. We found that Nod1 and Nod2 were present in renal tubular epithelial cells in both mouse and human kidneys and that the absence of these receptors in mice resulted in protection from kidney ischemia reperfusion injury. Significant protection from kidney injury was seen with a deficiency of Nod2 and receptor-interacting protein 2, and the simultaneous deficiency of Nod1 and Nod2 provided even greater protection. We conclude that the intracellular sensors Nod1 and Nod2 play an important role in the pathogenesis of acute ischemic injury of the kidney, although possibly through different mechanisms.

An inflammasome-independent role for epithelial-expressed Nlrp3 in renal ischemia-reperfusion injury.

Cytoplasmic innate immune receptors are important therapeutic targets for diseases associated with overproduction of proinflammatory cytokines. One cytoplasmic receptor complex, the Nlrp3 inflammasome, responds to an extensive array of molecules associated with cellular stress. Under normal conditions, Nlrp3 is autorepressed, but in the presence of its ligands, it oligomerizes, recruits apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and triggers caspase 1 activation and the maturation of proinflammatory cytokines such as IL-1ß and IL-18. Because ischemic tissue injury provides a potential source for Nlrp3 ligands, our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in components of the Nlrp3 inflammasome (Nlrp3(-/-) and Asc(-/-) mice). To examine the role of the inflammasome in renal ischemia-reperfusion injury (IRI) we also tested its downstream targets caspase 1, IL-1ß, and IL-18. Both Nlrp3 and Asc were highly expressed in renal tubular epithelium of humans and mice, and the absence of Nlrp3, but not Asc or the downstream inflammasome targets, dramatically protected from kidney IRI. We conclude that Nlrp3 contributes to renal IRI by a direct effect on renal tubular epithelium and that this effect is independent of inflammasome-induced proinflammatory cytokine production.

CD14 blockade to prevent ischemic injury to donor organs.

The purpose of this review is to highlight the potential role for the cluster of differentiation protein 14 (CD14), a co-receptor for toll-like receptor (TLR) signals and as a proximal target for innate immune signals induced during procurement of solid organs for transplantation. CD14 facilitates the detection of multiple pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) by various TLRs. All solid organs used for transplantation are exposed to PAMPs and DAMPs generated during the course of procurement that inevitably trigger injurious inflammatory responses in the donor organ. Multiple experimental animal studies and observations in human organs have provided a solid rationale to consider CD14 blockade as a therapeutic target. CD14 has been recognized for over three decades to play an essential role in innate immune signals associated with sepsis. More recent data now show that genetic deletion or antibody blockade of CD14 can modify ischemic tissue injury in the kidney, liver, heart and lung. Thus, data presented in this review suggest that anti-CD14 directed therapies might be applied to organ preservation strategies in solid organ transplantation.

DAMPs Released From Injured Renal Tubular Epithelial Cells Activate Innate Immune Signals in Healthy Renal Tubular Epithelial Cells.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) predictably causes acute kidney injury after shock and major cardiovascular procedures in all kidneys procured for transplantation. The earliest events of IRI are triggered by molecules released from injured cells, damage-associated molecular patterns (DAMPs), that bind pattern recognition receptors (PRRs) constitutively expressed on many cells within the kidney. Activation of PRR signaling leads to production of proinflammatory molecules, which incite a cascade of inflammatory events leading to acute kidney injury. Renal tubular epithelial cells (RTECs) are particularly susceptible to ischemic injury, and proximal RTEC injury is pathognomonic of renal IRI. To better understand how injured RTECs contribute to the cycle of deleterious inflammation in the setting of renal IRI, this study asked whether DAMPs released from injured RTECs induced PRR signals in healthy RTECs. METHODS: Human RTECs were necrosed ex vivo to release intracellular DAMPs and resulting necrotic supernatant used to stimulate healthy RTECs, T lymphocytes, and monocytes. RESULTS: DAMPs released from necrosed RTECs upregulated PRRs known to be associated with renal IRI and activated mitogen-activated protein kinase signaling pathways. Proinflammatory cytokines were upregulated in response to necrotic supernatant, and this upregulation was abrogated by MEK-1 inhibition. The RTEC-derived DAMPs were also potent inducers of T-cell activation/proliferation and monocyte migration. CONCLUSIONS: This is the first study to our knowledge to show that endogenous DAMPs released from injured RTECs directly activate PRR signaling in healthy RTECs. These findings provide new insights directed to therapeutics for renal IRI.

Blockade of T cell activation induced by the simultaneous absence of Nod1 and Nod2 is bypassed by TLR2 signals.

Pattern recognition receptors (PRRs) trigger adaptive inflammatory responses and as such are attractive targets for therapeutic manipulation of inflammation. In order to develop effective therapies however we need to understand the complexities of PRR signaling and clarify how individual PRRs contribute to an inflammatory response in a given cell type. Data from our lab and others have shown that cross-talk occurs between different PRR family members that directs T cell responses to a particular stimuli. It is well-established that the cell surface toll-like receptor 2 (TLR2) provides a potent costimulatory signal for TCR-stimulated T cell activation. We have shown that signaling through the intracellular nucleotide-binding oligomerization domain-containing proteins 1 and 2 (Nod1 and Nod2) also provides important signals for T cell activation, and that when both Nod1 and Nod 2 are deleted stimulated T cells undergo activation-induced cell death. This study found that TLR2 costimulation could bypass the defect induced by the simultaneous absence of Nods1 and 2 in both antibody- and antigen-stimulated T cells. Since blocking one set of PRR-mediated responses can be overcome by signaling through another PRR family member, then effective therapeutic immune blockade strategies will likely require a multi-pronged approach in order to be effective.

Defects in skin gamma delta T cell function contribute to delayed wound repair in rapamycin-treated mice.

Disruptions in the normal program of tissue repair can result in poor wound healing, which perturbs the integrity of barrier tissues such as the skin. Such defects in wound repair occur in transplant recipients treated with the immunosuppressant drug rapamycin (sirolimus). Intraepithelial lymphocytes, such as gammadelta T cells in the skin, mediate tissue repair through the production of cytokines and growth factors. The capacity of skin-resident T cells to function during rapamycin treatment was analyzed in a mouse model of wound repair. Rapamycin treatment renders skin gammadelta T cells unable to proliferate, migrate, and produce normal levels of growth factors. The observed impairment of skin gammadelta T cell function is directly related to the inhibitory action of rapamycin on mammalian target of rapamycin. Skin gammadelta T cells treated with rapamycin are refractory to IL-2 stimulation and attempt to survive in the absence of cytokine and growth factor signaling by undergoing autophagy. Normal wound closure can be restored in rapamycin-treated mice by addition of the skin gammadelta T cell-produced factor, insulin-like growth factor-1. These studies not only reveal that mammalian target of rapamycin is a master regulator of gammadelta T cell function but also provide a novel mechanism for the increased susceptibility to nonhealing wounds that occurs during rapamycin administration.