Characterization of Transcriptional Regulatory Networks that Promote and Restrict Identities and Functions of Intestinal Innate Lymphoid Cells.

Innate lymphoid cells (ILCs) promote tissue homeostasis and immune defense but also contribute to inflammatory diseases. ILCs exhibit phenotypic and functional plasticity in response to environmental stimuli, yet the transcriptional regulatory networks (TRNs) that control ILC function are largely unknown. Here, we integrate gene expression and chromatin accessibility data to infer regulatory interactions between transcription factors (TFs) and genes within intestinal type 1, 2, and 3 ILC subsets. We predicted the "core" TFs driving ILC identities, organized TFs into cooperative modules controlling distinct gene programs, and validated roles for c-MAF and BCL6 as regulators affecting type 1 and type 3 ILC lineages. The ILC network revealed alternative-lineage-gene repression, a mechanism that may contribute to reported plasticity between ILC subsets. By connecting TFs to genes, the TRNs suggest means to selectively regulate ILC effector functions, while our network approach is broadly applicable to identifying regulators in other in vivo cell populations.

Immunomodulatory effects of cytokine-induced expansion of cytotoxic lymphocytes in a mouse model of lupus-like disease.

BACKGROUND AIMS: Certain therapies (e.g., daclizumab) that promote expansion of natural killer (NK) cells are associated with clinical amelioration of disease in the context of multiple sclerosis and associated mouse models. The clinical benefits are putatively attributable to an enhanced capacity of NK cells to kill activated pathogenic T cells. Whether a parallel approach will also be effective in systemic lupus erythematosus (lupus), a multi-organ autoimmune disease driven by aberrant responses of self-reactive T and B cells, is unclear. METHODS: In the present study, the authors assess the therapeutic impact of IL-2- and IL-15-based strategies for expanding NK cells on measures of lupus-like disease in a mouse model. RESULTS: Unexpectedly, cytokine-mediated expansion of cytotoxic lymphocytes aggravated immunological measures of lupus-like disease. Depletion studies revealed that the negative effects of these cytokine-based regimens can largely be attributed to expansion of CD8 T cells rather than NK cells. CONCLUSIONS: These results provoke caution in the use of cytokine-based therapeutics to treat co-morbid cancers in patients with lupus and highlight the need for new methods to selectively expand NK cells to further assess their clinical value in autoimmune disease.

Application of directed evolution and back-to-consensus algorithms to human alpha1-antitrypsin leads to diminished anti-protease activity and augmented anti-inflammatory activities.

Primarily known as an elastase inhibitor, human alpha1-antitrypsin also exerts anti-inflammatory and immunomodulatory effects, both in vitro and in vivo. While the anti-protease mechanism of alpha1-antitrypsin is attributed to a particular protein domain coined the reactive center loop, anti-inflammatory and immunomodulatory loci within the molecule remain to be identified. In the present study, directed evolution and back-to-consensus algorithms were applied to human alpha1-antitrypsin. Six unique functional candidate sites were identified on the surface of the molecule; in manipulating these sites by point mutations, a recombinant mutant form of alpha1-antitrypsin was produced, depicting a requirement for sites outside the reactive center loop as essential for protease inhibition, and displaying enhanced anti-inflammatory activities. Taken together, outcomes of the present study establish a potential use for directed evolution in advancing our understanding of site-specific protein functions, offering a platform for development of context- and disease-specific alpha1-antitrypsin-based therapeutics.

Exploration of α1-antitrypsin treatment protocol for islet transplantation: dosing plan and route of administration.

Life-long weekly infusions of human α1-antitrypsin (hAAT) are currently administered as augmentation therapy for patients with genetic AAT deficiency (AATD). Several recent clinical trials attempt to extend hAAT therapy to conditions outside AATD, including type 1 diabetes. Since the endpoint for AATD is primarily the reduction of risk for pulmonary emphysema, the present study explores hAAT dose protocols and routes of administration in attempt to optimize hAAT therapy for islet-related injury. Islet-grafted mice were treated with hAAT (Glassia™; i.p. or s.c.) under an array of clinically relevant dosing plans. Serum hAAT and immunocyte cell membrane association were examined, as well as parameters of islet survival. Results indicate that dividing the commonly prescribed 60 mg/kg i.p. dose to three 20 mg/kg injections is superior in affording islet graft survival; in addition, a short dynamic descending dose protocol (240→120→60→60 mg/kg i.p.) is comparable in outcomes to indefinite 60 mg/kg injections. While hAAT pharmacokinetics after i.p. administration in mice resembles exogenous hAAT treatment in humans, s.c. administration better imitated the physiological progressive rise of hAAT during acute phase responses; nonetheless, only the 60 mg/kg dose depicted an advantage using the s.c. route. Taken together, this study provides a platform for extrapolating an islet-relevant clinical protocol from animal models that use hAAT to protect islets. In addition, the study places emphasis on outcome-oriented analyses of drug efficacy, particularly important when considering that hAAT is presently at an era of drug-repurposing towards an extended list of clinical indications outside genetic AATD.

Acute phase protein α1-antitrypsin reduces the bacterial burden in mice by selective modulation of innate cell responses.

BACKGROUND: Severe bacterial infection can cause sepsis, multiple organ dysfunction syndrome (MODS), and death. Human α1-antitrypsin (hAAT) is an antiinflammatory, immune-modulating, and tissue-protective circulating serine-protease inhibitor, with levels that increase during acute-phase responses. It is currently being evaluated as a therapeutic agent for individuals with diabetes and graft-versus-host disease. However, the concern of opportunistic bacterial infections has yet to be addressed. Therefore, we investigated host immune cell responses during acute bacterial infections under conditions of elevated hAAT levels. METHODS: Peritonitis and sepsis models were created using wild-type mice and hAAT-transgenic mice. Bacterial loads, MODS, leukopenia, neutrophil infiltration, immune cell activation, circulating cytokine levels, and survival rates were then assessed. RESULTS: hAAT significantly reduced infection-induced leukopenia and liver, pancreas, and lung injury, and it significantly improved 24-hour survival rates. Unexpectedly, bacterial load was reduced. Levels of early proinflammatory mediators and neutrophil influx were increased by hAAT soon after infection but not during sterile peritonitis. CONCLUSIONS: hAAT reduces the bacterial burden after infection. Since hAAT does not block bacterial growth in culture, its effects might rely on host immune cell modulation. These outcomes suggest that prolonged hAAT treatment in patients without hAAT deficiency is safe. Additionally, hAAT treatment may be considered a preemptive therapeutic measure for individuals who are at risk for bacterial infections.

Progressive accumulation of hyperinflammatory NKG2Dlow NK cells in early childhood severe atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic comorbidities remain ill-defined. Here, analysis of circulating NK cells in a longitudinal early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D, which was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aeroallergens, a risk factor for development of asthma. Individual-level longitudinal analysis in a subset of children revealed coincident reduction of NKG2D on NK cells with acquired or persistent sensitization, and this was associated with impaired skin barrier function assessed by transepidermal water loss. Low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine tumor necrosis factor-α. These observations provide important insights into a potential mechanism underlying the development of allergic comorbidity in early life in children with AD, which involves altered NK cell functional responses, and define an endotype of severe AD.

Progressive accumulation of hyperinflammatory NKG2D low NK cells in early childhood severe atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic co-morbidities remain ill-defined. Herein, analysis of circulating NK cells in a longitudinal early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D, which was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aero allergens, a risk factor for development of asthma. Individual-level longitudinal analysis in a subset of children revealed co-incident reduction of NKG2D on NK cells with acquired or persistent sensitization, and this was associated with impaired skin barrier function assessed by transepidermal water loss. Low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine TNF-α. These observations provide important insights into a potential mechanism underlying the development of allergic co-morbidity in early life in children with AD which involves altered NK-cell functional responses, and define an endotype of severe AD.

The Effect of Unconventional Cytokine Combinations on NK-Cell Responses to Viral Infection.

Cytokines are soluble and membrane-bound factors that dictate immune responses. Dogmatically, cytokines are divided into families that promote type 1 cell-mediated immune responses (e.g., IL-12) or type 2 humoral responses (e.g., IL-4), each capable of antagonizing the opposing family of cytokines. The discovery of additional families of cytokines (e.g., IL-17) has added complexity to this model, but it was the realization that immune responses frequently comprise mixtures of different types of cytokines that dismantled this black-and-white paradigm. In some cases, one type of response may dominate these mixed milieus in disease pathogenesis and thereby present a clear therapeutic target. Alternatively, synergistic or blended cytokine responses may obfuscate the origins of disease and perplex clinical decision making. Most immune cells express receptors for many types of cytokines and can mediate a myriad of functions important for tolerance, immunity, tissue damage, and repair. In this review, we will describe the unconventional effects of a variety of cytokines on the activity of a prototypical type 1 effector, the natural killer (NK) cell, and discuss how this may impact the contributions of these cells to health and disease.

Tumor Microenvironment-Derived R-spondins Enhance Antitumor Immunity to Suppress Tumor Growth and Sensitize for Immune Checkpoint Blockade Therapy.

Natural killer (NK) cells and T cells are key effectors of antitumor immune responses and major targets of checkpoint inhibitors. In multiple cancer types, we find that the expression of Wnt signaling potentiator R-spondin genes (e.g., RSPO3) is associated with favorable prognosis and positively correlates with gene signatures of both NK cells and T cells. Although endothelial cells and cancer-associated fibroblasts comprise the R-spondin 3-producing cells, NK cells and T cells correspondingly express the R-spondin 3 receptor LGR6 within the tumor microenvironment (TME). Exogenous expression or intratumor injection of R-spondin 3 in tumors enhanced the infiltration and function of cytotoxic effector cells, which led to tumor regression. NK cells and CD8+ T cells independently and cooperatively contributed to R-spondin 3-induced control of distinct tumor types. The effect of R-spondin 3 was mediated in part through upregulation of MYC and ribosomal biogenesis. Importantly, R-spondin 3 expression enhanced tumor sensitivity to anti-PD-1 therapy, thereby highlighting new therapeutic avenues. SIGNIFICANCE: Our study identifies novel targets in enhancing antitumor immunity and sensitizing immune checkpoint inhibition, which provides a rationale for developing new immunotherapies against cancers. It also offers mechanistic insights on Wnt signaling-mediated modulation of anticancer immunity in the TME and implications for a putative R-spondin-LGR6 axis in regulating NK-cell biology. This article is highlighted in the In This Issue feature, p. 2945.

IL-33 promotes type 1 cytokine expression via p38 MAPK in human NK cells.

This study tests the hypothesis that activation of MAPK by physiologically relevant concentrations of IL-33 contributes to enhanced cytokine expression by IL-12 stimulated human NK cells. While IL-33 canonically triggers type 2 cytokine responses, this cytokine can also synergize with type 1 cytokines like IL-12 to provoke IFN-γ. We show that picogram concentrations of IL-12 and IL-33 are sufficient to promote robust secretion of IFN-γ by human NK cells that greatly exceeds resposes to either cytokine alone. Nanogram doses of IL-33, potentially consistent with levels in tissue microenvironments, synergize with IL-12 to induce secretion of additional cytokines, including TNF and GM-CSF. IL-33-induced activation of the p38 MAPK pathway in human NK cells is crucial for enhanced release of IFN-γ and TNF in response to IL-12. Mechanistically, IL-33-induced p38 MAPK signaling enhances stability of IFNG transcripts and triggers A disintegrin and metalloproteinase domain 17 (ADAM17) mediated cleavage of TNF from the cell surface. These data support our hypothesis and suggest that altered sensitivity of NK cells to IL-12 in the presence of IL-33 may have important consequences in diseases associated with mixed cytokine milieus, like asthma and chronic obstructive pulmonary disease.

Dynamic Changes in Natural Killer Cell Subset Frequencies in the Absence of Cytomegalovirus Infection.

Individuals lacking functional natural killer (NK) cells suffer severe, recurrent infections with cytomegalovirus (CMV), highlighting the critical role of NK cells in antiviral defense. Therefore, ongoing attempts to develop an efficacious vaccine to prevent CMV infection should potentially aim to elicit NK-cell antiviral responses as an accessory to conventional T- and B-cell based approaches. In this regard, CMV infection provokes marked phenotypic and functional differentiation of the NK-cell compartment, including development of adaptive NK cells that exhibit enhanced antiviral activity. We examined longitudinal blood samples collected from 40 CMV-seronegative adolescents to ascertain whether a CMV glycoprotein B (gB) vaccine in the absence of CMV infection can stimulate differentiation or expansion of CMV-associated subsets of NK cells. Study participants uniformly lacked the CMV-dependent NKG2C+ subset of NK cells, suggesting that an adjuvanted CMV gB vaccine alone is an inadequate stimulus for sustained expansion of these cells. In contrast, we observed unexpected dynamic fluctuations in the frequency of NK cells lacking FcRγ, EAT-2, and SYK, which were independent of vaccination or CMV infection. Whereas, FcRγneg NK cells in CMV infection are reported to express increased levels of the maturation marker CD57, the FcRγneg NK cells observed in our CMV-negative vaccine cohort express less CD57 than their FcRγ+ counterparts. The FcRγneg NK cells in CMV-negative individuals were also functionally distinct from this subset in CMV infection, exhibiting comparable IFN-γ production and degranulation as FcRγ+ NK cells in response to cytokine or antibody-dependent stimuli. These results suggest that frequencies of some NK cell subsets may increase in response to unknown environmental or inflammatory cues distinct from that which occurs after CMV infection. Greater understanding of the nature of the signals driving CMV-independent accumulation of these subsets should permit development of mechanisms to facilitate vaccine-driven expansion of CMV-reactive NK cells.

Point Mutation of a Non-Elastase-Binding Site in Human α1-Antitrypsin Alters Its Anti-Inflammatory Properties.

Introduction: Human α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357-366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro, and in vivo. Methods: His-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo. Results: Compared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding. Conclusion: Our data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes.

Experimental Support for the Ecoimmunity Theory: Distinct Phenotypes of Nonlymphocytic Cells in SCID and Wild-Type Mice.

Immune tolerance toward "self" is critical in multiple immune disorders. While there are several mechanisms to describe the involvement of immune cells in the process, the role of peripheral tissue cells in that context is not yet clear. The theory of ecoimmunity postulates that interactions between immune and tissue cells represent a predator-prey relationship. A lifelong interaction, shaped mainly during early ontogeny, leads to selection of nonimmune cell phenotypes. Normally, therefore, nonimmune cells that evolve alongside an intact immune system would be phenotypically capable of evading immune responses, and cells whose phenotype falls short of satisfying this steady state would expire under hostile immune responses. This view was supported until recently by experimental evidence showing an inferior endurance of severe combined immunodeficiency (SCID)-derived pancreatic islets when engrafted into syngeneic immune-intact wild-type (WT) mice, relative to islets from WT. Here we extend the experimental exploration of ecoimmunity by searching for the presence of the phenotypic changes suggested by the theory. Immune-related phenotypes of islets, spleen, and bone marrow immune cells were determined, as well as SCID and WT nonlymphocytic cells. Islet submass grafting was performed to depict syngeneic graft functionality. Islet cultures were examined under both resting and inflamed conditions for expression of CD40 and major histocompatibility complex (MHC) class I/II and release of interleukin-1α (IL-1α), IL-1ß, IL-6, tumor necrosis factor-α (TNF-α), IL-10, and insulin. Results depict multiple pathways that appear to be related to the sculpting of nonimmune cells by immune cells; 59 SCID islet genes displayed relative expression changes compared with WT islets. SCID cells expressed lower tolerability to inflammation and higher levels of immune-related molecules, including MHC class I. Accordingly, islets exhibited a marked increase in insulin release upon immunocyte depletion, in effect resuming endocrine function that was otherwise suppressed by resident immunocytes. This work provides further support of the ecoimmunity theory and encourages subsequent studies to identify its role in the emergence and treatment of autoimmune pathologies, transplant rejection, and cancer.

Correspondence of Neutralizing Humoral Immunity and CD4 T Cell Responses in Long Recovered Sudan Virus Survivors.

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections.

Immune memory to Sudan virus: comparison between two separate disease outbreaks.

Recovery from ebolavirus infection in humans is associated with the development of both cell-mediated and humoral immune responses. According to recent studies, individuals that did not survive infection with ebolaviruses appear to have lacked a robust adaptive immune response and the expression of several early innate response markers. However, a comprehensive protective immune profile has yet to be described. Here, we examine cellular memory immune responses among survivors of two separate Ebolavirus outbreaks (EVDs) due to Sudan virus (SUDV) infection in Uganda-Gulu 2000-2001 and Kibaale 2012. Freshly collected blood samples were stimulated with inactivated SUDV, as well as with recombinant SUDV or Ebola virus (EBOV) GP (GP1-649). In addition, ELISA and plaque reduction neutralization assays were performed to determine anti-SUDV IgG titers and neutralization capacity. Cytokine expression was measured in whole blood cultures in response to SUDV and SUDV GP stimulation in both survivor pools, demonstrating recall responses that indicate immune memory. Cytokine responses between groups were similar but had distinct differences. Neutralizing, SUDV-specific IgG activity against irradiated SUDV and SUDV recombinant proteins were detected in both survivor cohorts. Furthermore, humoral and cell-mediated crossreactivity to EBOV and EBOV recombinant GP1-649 was observed in both cohorts. In conclusion, immune responses in both groups of survivors demonstrate persistent recognition of relevant antigens, albeit larger cohorts are required in order to reach greater statistical significance. The differing cytokine responses between Gulu and Kibaale outbreak survivors suggests that each outbreak may not yield identical memory responses and promotes the merits of studying the immune responses among outbreaks of the same virus. Finally, our demonstration of cross-reactive immune recognition suggests that there is potential for developing cross-protective vaccines for ebolaviruses.

α1-antitrypsin increases interleukin-1 receptor antagonist production during pancreatic islet graft transplantation.

Although islet transplantation for individuals with type 1 diabetes has been shown to yield superior blood glucose control, it remains inadequate for long-term control. This is partly due to islet injuries and stresses that can lead to beta cell loss. Inhibition of excess IL-1ß activity might minimize islet injuries, thus preserving function. The IL-1 receptor antagonist (IL-1Ra), an endogenous inhibitor of IL-1ß, protects islets from cytokine-induced necrosis and apoptosis. Therefore, an imbalance between IL-1ß and IL-1Ra might influence the courses of allogeneic and autoimmune responses to islets. Our group previously demonstrated that the circulating serine-protease inhibitor human alpha-1-antitrypsin (hAAT), the levels of which increase in circulation during acute-phase immune responses, exhibits anti-inflammatory and islet-protective properties, as well as immunomodulatory activity. In the present study, we sought to determine whether the pancreatic islet allograft-protective activity of hAAT was mediated by IL-1Ra induction. Our results demonstrated that hAAT led to a 2.04-fold increase in IL-1Ra expression in stimulated macrophages and that hAAT-pre-treated islet grafts exhibited a 4.851-fold increase in IL-1Ra transcript levels, which were associated with a moderate inflammatory profile. Unexpectedly, islets that were isolated from IL-1Ra-knockout mice and pre-treated with hAAT before grafting into wild-type mice yielded an increase in intragraft IL-1Ra expression that was presumably derived from infiltrating host cells, albeit in the absence of hAAT treatment of the host. Indeed, hAAT-pre-treated islets generated hAAT-free conditioned medium that could induce IL-1Ra production in cultured macrophages. Finally, we demonstrated that hAAT promoted a distinct phosphorylation and nuclear translocation pattern for p65, a key transcription factor required for IL-1Ra expression.

Human α1-Antitrypsin Binds to Heat-Shock Protein gp96 and Protects from Endogenous gp96-Mediated Injury In vivo.

The extracellular form of the abundant heat-shock protein, gp96, is involved in human autoimmune pathologies. In patients with type 1 diabetes, circulating gp96 is found to be elevated, and is bound to the acute-phase protein, α1-antitrypsin (AAT). The two molecules also engage intracellularly during the physiological folding of AAT. AAT therapy promotes pancreatic islet survival in both transplantation and autoimmune diabetes models, and several clinical trials are currently examining AAT therapy for individuals with type 1 diabetes. However, its mechanism of action is yet unknown. Here, we examine whether the protective activity of AAT is related to binding of extracellular gp96. Primary mouse islets, macrophages, and dendritic cells were added recombinant gp96 in the presence of clinical-grade human AAT (hAAT, Glassia™, Kamada Ltd., Israel). Islet function was evaluated by insulin release. The effect of hAAT on IL-1ß/IFNγ-induced gp96 cell-surface levels was also evaluated. In vivo, skin transplantation was performed for examination of robust immune responses, and systemic inflammation was induced by cecal puncture. Endogenous gp96 was inhibited by gp96-inhibitory peptide (gp96i, Compugen Ltd., Israel) in an allogeneic islet transplantation model. Our findings indicate that hAAT binds to gp96 and diminishes gp96-induced inflammatory responses; e.g., hAAT-treated gp96-stimulated islets released less pro-inflammatory cytokines (IL-1ß by 6.16-fold and TNFα by 2.69-fold) and regained gp96-disrupted insulin release. hAAT reduced cell activation during both skin transplantation and systemic inflammation, as well as lowered inducible surface levels of gp96 on immune cells. Finally, inhibition of gp96 significantly improved immediate islet graft function. These results suggest that hAAT is a regulator of gp96-mediated inflammatory responses, an increasingly appreciated endogenous damage response with relevance to human pathologies that are exacerbated by tissue injury.