Patent Application
20230241185
The present invention is in the field of treatment of immunosuppression, in particular in the field of treatment of post sterile traumatic immunosuppression, more particularly wherein said immunosuppression occurs after an immunoactivation. The present invention generally relates to a composition comprising a DNA-degrading enzyme for use in a method for the treatment of such immunosuppression. Further, the present invention relates to the composition for the use of the present invention, wherein the DNA-degrading enzyme is e.g. a nuclease, which is administered after acute tissue injury and/or in the course of the treatment of acute tissue injury.
Acute tissue injuries such as stroke (Vogelgesang et al., 2008), myocardial infarction (Kohsaka et al., 2005) and burn injury (Xu et al., 2016) induce both local and systemic inflammatory responses. These immune perturbations are characterized by an acute proinflammatory response, followed by an immunosuppressive phase, which can be acute and which predisposes patients to infections. Secondary immune-mediated complications such as acute cytokine-induced comorbidities and infections reportedly cause more patient deaths than the primary injury (Dantzer et al., 2008; Vermeij et al., 2009; D'Avignon et al., 2010). The actual mediators and underlying mechanism of the brain-immune communication are so far unknown.
For example, De Meyer et al. (Arterioscler. Thromb. Vasc. Biol., 2012) describes extracellular chromatin as an important mediator of ischemic stroke in mice.
Mcllroy et al. (J. Trauma Acute Care Surg., 2018) suggests that reduced deoxyribonuclease enzyme activity might provide a therapeutic target for Systemic Inflammatory Response Syndrome.
Though it has been demonstrated that soluble mediators derived from the brain are responsible for the development and progression of the systemic immune response after stroke (Roth et al., 2018), wherein similar findings have been reported for burns and traumatic injuries (Hazeldine et al., 2015; Manson et al., 2012), the identity of these mediators as well as the mechanism linking acute immune activation and subsequent immunosuppression were unclear so far.
Consequently, the inventors of the present invention have established possibilities for treating a new clinical scenario, namely post sterile traumatic immunosuppression. It was not able to provide a composition for the use of the treatment thereof so far as the causal reasons, namely how the immunosuppression after acute tissue injury was triggered, where unidentified. Those have not been known so far in the prior art.
Thus, there has been a drastical need to provide such a composition for the use in the treatment for the described clinical scenario.
According to the present invention, the inventors have found that a DNA-degrading enzyme, for example an enzyme which degrades nuclear DNA and possibly additionally mtDNA can be used in a method for the treatment of the clinical scenario post sterile traumatic immunosuppression. In other words, the inventors demonstrate that different tissue injuries induce a uniform and systemic activation of the inflammasome by sensing cell-free nucleic acids released from injured tissues. In this context, the inflammasome is a multi-protein complex in peripheral monocytes which accumulates and orchestrates caspase-1 cleavage upon activation of a wide range of danger signals sensed by the inflammasome. Inflammasome activation is primarily described as an innate response to bacterial and viral non-self molecules. Yet, the inventors observed that cell-free self-DNA activates the inflammasome in peripheral monocytes via the nucleic acid-sensing AIM2-inflammasome. In other words, it was shown that the inflammasome was triggered by AIM2 in myeloid cells, which sense cell-free DNA released after an acute tissue injury/damage. It was further demonstrated that monocytic inflammasome activation then drives overexpression of FasL on monocytes, subsequently inducing caspase-8-dependent apoptosis in T cells. The induction of FasL-expressing monocytes and preferably consecutive lymphopenia is in a more detailed embodiment of the invention driven by inflammasome-dependent IL-1 secretion. Consequently, the inventors provide a mechanistic understanding for a common, yet thus far elusive, clinical observation: the biphasic systemic immune response to sterile tissue injuries. With these findings the inventors provide further studies involving novel therapeutic strategies against post-injury immune alterations, thereby preventing the medical burden of inflammatory comorbidities in a wide range of acute tissue injuries. In sum, it has been demonstrated that inflammasome-dependent monocyte activation is the cause of T cell death after injury, and challenges the current paradigms of post-injury lymphopenia. Thus, the present invention provides new therapeutic targets for the pathway identified here along the events of increased cf-dsDNA concentration after acute tissue injury, inflammasome activation, IL-1β secretion, and Fas-mediated T cell death. This reduces the medical burden of postinjury immunosuppression and secondary infections. For the majority of the experiments in the present invention an experimental stroke model as a prototypic tissue injury model was applied. Key findings from the stroke model were also generalizable to a second tissue injury model of burn lesions. Thus, the extension to other tissue injuries/damages has been plausibly presented by the inventors.
Thus, the present invention provides a composition comprising a DNA-degrading enzyme for use in a method for the treatment of post sterile traumatic immunosuppression.
In one embodiment of the composition for the use of the present invention, an immunoactivation before the immunosuppression occurs.
According to one embodiment of the composition for the use of the present invention, the post sterile traumatic immunosuppression is characterized by an early systemic immune response syndrome and subsequent lymphocyte death.
In one further embodiment of the composition for the use of the present invention, the lymphocyte death is caused by apoptosis.
According to one embodiment of the composition for the use of the present invention, the post sterile traumatic immunosuppression is associated with systemic immune response syndrome (SIRS).
In one further embodiment of the composition for the use of the present invention, the post sterile traumatic immunosuppression is triggered by acute tissue injury.
According to one embodiment of the composition for the use of the present invention, the acute tissue injury is triggered by a physical, chemical, or metabolic noxious stimulus.
In one specific embodiment of the composition for the use of the present invention, the acute tissue injury is selected from stroke, myocardial infection, haemorrhagic shock, ischemia, ischemia reperfusion injury, chronic inhalation of irritants (e.g. asbestos, silica), atherosclerosis, gout, pseudogout, trauma, non-penetrating polytrauma (multiple bone fractures), and thermal trauma.
In one further embodiment of the composition for the use of the present invention, the post sterile traumatic immunosuppression is associated with a secondary infectious disease.
According to one embodiment of the composition for the use of the present invention, the DNA-degrading enzyme is a nuclease. In one specific embodiment thereof, the nuclease is an exonuclease or endonuclease. In one further embodiment of the composition for the use of the present invention, the endonuclease is a deoxyribonuclease. In a preferred embodiment of the composition for the use of the present invention, the deoxyribonuclease is DNase I.
In one further embodiment of the composition for the use of the present invention, the nuclease is administered after the acute tissue injury and/or in the course of the treatment of the acute tissue injury.
According to one further embodiment of the composition for the use of the present invention, the nuclease is administered parenterally, preferably intravenously or by inhalation.
The present invention provides a composition comprising a DNA-degrading enzyme for use in a method for the treatment of post sterile traumatic immunosuppression.
As used within the context of the present invention, the term “DNA-degrading enzyme” means any enzyme that is able to degrade DNA into its individual nucleotide components. Various events may cause the degradation of DNA into nucleotides, e.g. DNA degradation is one of the final consequences of activation of the apoptotic cascade, and can be measured by quantification of free 3′-hydroxyl groups in tissue sections. If DNA is not properly degraded, this may cause various diseases.
The composition of the present invention is for use in a method for the treatment of a wide variety of different diseases and disorders characterized by the post sterile traumatic immunosuppression. Thus, the invention envisages the composition to be for use in a method for the treatment of a subject in need thereof. The subject is typically a mammal, e.g., a human. In some embodiments the subject is a non-human animal that serves as a model for a disease or disorder that affects humans. The animal model may be used, e.g., in preclinical studies, e.g., to assess efficacy and/or to determine a suitable dose. In some embodiments, inventive compositions may be used prophylactically, e.g., may be used for a subject who does not exhibit signs or symptoms of the disease or disorder (but may be at increased risk of developing the immunosuppression or is expected to develop the immunosuppression). Thus, the term “for the treatment” as used herein may comprise “for the prevention” as well. In some embodiments, an inventive composition is for use in a method of treatment of a subject who has developed one or more signs or symptoms of immunosuppression, e.g., the subject has been diagnosed as having immunosuppression. It is preferred that the composition for use is administered to the subject in need thereof in a therapeutically effective amount. By “therapeutically effective amount” is meant an amount of the composition of the present invention that elicits a desired therapeutic effect. The exact amount dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for age, body weight, general health, sex, diet, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. Further, it is also comprised herein a method of treating post sterile traumatic immunosuppression as defined elsewhere herein, the method comprising administering a therapeutically effective amount of a composition comprising a DNA-degrading enzyme to a subject in need thereof as defined elsewhere herein. In addition, the present invention also comprises the use of a composition comprising a DNA-degrading enzyme for the manufacture of a medicament for the treatment of post sterile traumatic immunosuppression. Each definition made herein may also be applicable to the method of treatment and the Swiss type format.
The term “post sterile traumatic immunosuppression” refers to the medical condition which is known to a person skilled in the art (see f.e. Islam et al. Sterile post-traumatic immunosuppression”, Clin Transl Immunology (2016)) Said term is encompassed by the term “immunosuppression after acute tissue injury” as it is also used herein. Thus, the definitions which apply to the term “immunosuppression after acute tissue injury” also apply to the term “post sterile traumatic immunosuppression” and vice versa. The term “immunosuppression after acute tissue injury”, as used within the context of the present invention, may be used synonymously with the terms “immune suppression after acute tissue injury”, “immune changes after acute tissue injury”, “immune alterations after acute tissue injury”, and “systemic immune consequences after acute tissue injury”. These terms may also encompass “immunosuppression after sterile tissue damage/injury”. Preferably, said term(s) encompass(es) the medical condition “post sterile traumatic immunosuppression”. “Sterile trauma(tic)” refers to tissue damage/injury devoid of primary wound infection. Thus, “sterile trauma(tic)” may include “sterile tissue injury” or “sterile tissue damage” in the absence of microbial infection. Inflammation following sterile trauma without exposure to microbial pathogens is termed “sterile inflammation”: Immunosuppression followed by this sterile inflammation is termed as “sterile immunosuppression”. Thus, “post sterile traumatic immunosuppression” is an immunosuppression after an inflammation following sterile trauma, the latter being caused without exposure to microbial pathogens. Indeed, the present invention demonstrates that due to T cell apoptosis, an immunosuppression occurs. This predisposes patients with local tissue injuries/tissue damages to systemic infections as defined elsewhere herein, which may be a major cause of death after such injuries/damages. Without being bound by theory, it is assumed that DNA, e.g. nuclear DNA and/or mtDNA causes the native immune system to trigger apoptosis in T cells due to the interaction of cells of the native immune system with cells of the adaptive immune system, e.g. lymphocytes, preferably T cells via apoptosis-inducing receptor/ligand interactions. This has been proven by the present invention as described elsewhere herein. Accordingly, the term “post sterile traumatic immunosuppression” preferably encompasses “post sterile traumatic lymphopenia”, more preferably “post sterile traumatic T cell cytopenia”.
As used herein, the term “immunosuppression” means any form of a reduction of the activation or efficacy of the immune system. Thus, immunosuppression is the suppression of the endogenous defense system. It refers to a process of repressing the immunological activity of the humoral and/or cellular immune system. This can be an undesirable consequence of an effect from the inanimate environment, of an infection, of a malignant suffering, of a disease caused by another condition, of a mental or physical overload, or due to an undesired consequence of a medical diagnosis or a consequence of a medical treatment. Some portions of the immune system itself have immunosuppressive effects on other parts of the immune system, and immunosuppression may occur as an adverse reaction to treatment of other conditions. The immunosuppression may comprise decreased capacity to neutralize external organisms, which may result in repeated, more severe, or prolonged infections, as well as an increased susceptibility to cancer development. As used within the context of the present invention, an immunosuppression may be present, when one or more of the following cell types are suppressed with regard to their activity or reduced in their cell count, consisting of myeloid cells (including granulocytes, monocytes, macrophages, dendritic cells and mast cells) or lymphocytes (including T cells, B cells, Plasma cells, NK cells and NKT cells) or wherein the subject diagnosed with a suppressed immunosystem may develop infections by opportunistic pathogens (for example Pneumocystis or cytomegalovirus). The presence of the immunosuppression may be investigated in reference to a state which does not comprise an acute tissue injury/damage as defined herein or an immunosuppression of other cause.
“Acute tissue injury”, (also called “acute tissue damage”) as used within the context of the present invention, means an injury/damage with a sudden onset, for example being characterized by cell death concerning one or more organs in a certain time range, e.g. within 24 hours. Such an injury is not limited to an organ or any noxae. During such an acute tissue injury an organ or a part thereof can be affected, where cell function and integrity is lost within less than 24 h due to an insult. This insult can be ischemia (lack of blood flow) to the organ, a mechanical tissue trauma, the effect of a toxic agent or a thermal injury, for example. Examples for acute tissue injury are stroke, myocardial infarction, trauma, ischemia-reperfusion injuries to limbs or kidneys, burn injury or pharmacological toxicities such as acute liver failure due to various medication overuses. Acute tissue injuries include local, tissue-specific inflammatory and repair mechanisms that contribute to wound healing and scar formation. Besides these localized tissue-specific effects, acute tissue injuries also have a substantial and uniform impact on systemic immunity. The initial incidence of tissue damage acutely induces a pronounced local immune response and systemic proinflammatory activation, which is characterized by a rapid increase in circulating leukocytes pro-inflammatory cytokine levels (Offner et al., 2006; Emsley et al., 2003). After this early activation has resolved, a subsequent immune deficient phase follows. This immune deficiency is characterized by increased levels of circulating immature monocytes and systemic lymphopenia (Offner et al., 2006; Howard et al., 1974), which predisposes patients with local tissue injuries to systemic bacterial infections. In fact, infections are a major cause of death after acute tissue injuries such as stroke, trauma and burn injury.
The term “acute” as used within the term “acute tissue injury” as defined above, is a term, which may be understood in contrast to chronic diseases, leading to tissue injuries. Chronic diseases in general are slowly progressing, while the definition of “slowly progressing” depends on the specific disease entity, but may be generally over several weeks or months. For example, chronic vascular impairment may be in contrast to an acute ischemic injury to the brain (stroke) or the heart (myocardial infarction). An acute disease onset is clinically defined by the rapid onset of clinical symptoms. Acute diseases—in contrast to a chronic disease progression—are often more severe and require urgent medical attention. In some cases, an acute condition, e.g. a myocardial infarction or stroke, might lead to chronic conditions, such as chronic heart failure or immobility, respectively.
In one embodiment of the composition for use of the present invention, an immunoactivation before the immunosuppression after acute tissue injury occurs. Immunoactivation in general comprises all forms of activation of the immune system and the subsequent immune response. As used herein, “immunoactivation”, “immune activation” or “activation of the immune system” refers to an increase in the number and/or function of immune system cells, such as lymphocytes or myeloid cells, and/or an increase in humoral function of the immune system, involved in plasma cell and antibody production along with cytokine production. An immunoactivation may be, for example, present, when one or more of the following cell/cells is/are activated with regard to their activity/activities, which is/are selected from the group consisting of myeloid cells (including granulocytes, monocytes, macrophages, dendritic cells and mast cells) or lymphocytes (including T cells, B cells, plasma cells, NK cells and NKT cells) or wherein the subject diagnosed with an activated immunosystem may have the following clinical symptoms or parameters: increased blood cytokine levels, increased number of immune cells as specified above, increase in blood concentration of acute phase proteins (including C-reactive protein), fever and clinical signs of cytokine-induced sickness behavior (reduced appetite, apathy, sleeping disorder, reduced motivation and depressed mood). The presence of an immunoactivation may be investigated in reference to a healthy control population or the presence of the immunoactivation may be investigated in reference to a state which does not comprise an acute tissue injury as defined herein or an immunoactivation of other cause.
In one specific embodiment of the composition for use of the present invention, the immunosuppression after acute tissue injury is characterized by lymphocyte death. In one specific embodiment of the composition for use of the present invention, “immunosuppression after acute tissue injury” can be used synonymously to “lymphocyte death”. A lymphocyte is one of the subtypes of a white blood cell in a vertebrate's immune system. Lymphocytes include natural killer cells (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity), which is well known to a person skilled in the art. They are the main type of cell found in lymph, which prompted the name “lymphocyte”. The term “lymphocyte death”, as used within the context of the present invention, means the drop in or reduction of the lymphocyte count, e.g. in the blood of a subject, which can be, for example, prompted by lymphocyte apoptosis. The “lymphocyte death” may also comprise “lymphopenia”, which is the reduction in the numbers of lymphocytes in the blood. Lymphocyte death may also occur by both death receptor and mitochondrial-mediated apoptosis, so that there may be multiple triggers for lymphocyte death. Thus, in one specific embodiment of the composition for use of the present invention, the lymphocyte death is caused by apoptosis. In one further specific embodiment of the composition of the present invention, the lymphocyte death comprises or is characterized by or is caused by T cell death. In one further specific embodiment of the composition for use of the present invention, the lymphocyte death comprises or is characterized by or is caused by T cell cytopenia. Cytopenia is a reduction in the number of mature blood cells. In one further preferred embodiment of the composition for use of the present invention, the T cell cytopenia or the T cell death is caused by T cell apoptosis. “Apoptosis” is the programmed cell death that occurs in multicellular organisms, which is known by a person skilled in the art. In one further specific embodiment of the composition of the present invention, the lymphocyte death comprises or is characterized by or is caused by B cell death. In this regard, it is referred to
According to one embodiment of the composition for use of the present invention, the immunosuppression after acute tissue injury is associated with systemic immune response syndrome (SIRS). “Systemic immune response syndrome” (SIRS) is an inflammatory state affecting the whole body. It is the body's response to an infectious or non-infectious insult. Although SIRS may refer to an “inflammatory” response, it actually has pro- and anti-inflammatory components. According to the foundings of the inventors, immunosuppression may contribute to SIRS development or accompanies the SIRS symptoms, while SIRS is characterized by the clinical parameters of dysregulated body temperature, elevated heart rate, tachypnea, a decreased or increased number of blood leukocytes and a high number of immature innate immune cells. For example, manifestations of SIRS for adults may include, but are not limited to, a body temperature less than 36 C or greater than 38 C, a heart rate greater than 90 beats per minute, a tachypnea (high respiratory rate) with greater than 20 breaths per minute or an arterial partial pressure of carbon dioxide less than 4.3 kPa and a white blood cell count less than 4000 cells/mm3 (4×109 cells/L) or greater than 12,000 cells/mm3 (12×109 cells/L) or the presence of greater than 10% immature neutrophils. When two or more of these criteria are met with or without evidence of infection, patients may be diagnosed with “SIRS”. Patients with SIRS and acute organ dysfunction may be termed “severe SIRS”.
In one further embodiment of the composition for use of the present invention, the immunosuppression is triggered by acute tissue injury. The “acute tissue injury” is used in this embodiment as defined herein above. The terms “triggers”, “triggered” or “triggering”, as used within any embodiment of the present invention, means to cause something to start leading to a specific outcome or condition. For example, the inventors of the present invention have found that a clinical condition like an acute tissue injury leads to or results in immunosuppression, which can be detected in the subject who has experienced the acute tissue injury.
According to one embodiment of the composition for use of the present invention, the acute tissue injury is triggered by a physical, chemical, or metabolic noxious stimulus. The stimulus is any kind of change in substances or in happenings, occurring in the surrounding of a living thing that bring about any kind of response from it. The term “physical stimulus”, as used within the context of the present invention, means such a stimulus that directly affects one of the five senses. A chemical stimulus might be a stimulus that is caused by a chemical (liquid, gaseous, or solid) substance that is capable of evoking a response, e.g. in a subject exposed to said chemical stimulus. A noxious stimulus is an actually or potentially tissue damaging event. Noxious stimuli can either be mechanical (e.g. pinching or other tissue deformation), chemical (e.g. exposure to acid or irritant), or thermal (e.g. high or low temperatures).
In one specific embodiment of the composition for use of the present invention, the acute tissue injury is selected from stroke, myocardial infarction, haemorrhagic shock, ischemia, ischemia reperfusion injury, chronic inhalation of irritants (e.g. asbestos, silica), atherosclerosis, gout, pseudogout, trauma, non-penetrating polytrauma (multiple bone fractures), and thermal trauma.
Stroke is known to a person skilled in the art to be a medical condition with a sudden onset due to a vascular injury to the brain. There may be two main types of stroke: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding.
The term “myocardial infarction”, as used within the context of the present invention, refers to tissue death (infarction) of the heart muscle (myocardium) caused by ischaemia, that is the lack of oxygen delivery to myocardial tissue. It is a type of acute coronary syndrome, which describes a sudden or short-term change in symptoms related to blood flow to the heart.
The term “haemorrhagic shock”, as used within the context of the present invention, means a shock resulting from reduction of the volume of blood in the body due to hemorrhage. It is also known as a hypovolemic shock resulting from acute hemorrhage, characterized by hypotension, tachycardia, pale, cold, and clammy skin, and oliguria.
The term “ischemia” or “ischaemia”, as used within the context of the present invention, is a restriction in blood supply to tissues, causing a shortage of oxygen that is needed for cellular metabolism (to keep tissue alive). Ischemia is generally caused by problems with blood vessels, with resultant damage to or dysfunction of tissue.
The term “ischemia reperfusion injury”, also known as reperfusion injury or reoxygenation injury, refers to tissue damage caused when blood supply returns to tissue (re-+perfusion) after a period of ischemia or lack of oxygen (anoxia or hypoxia). The absence of oxygen and nutrients from blood during the ischemic period creates a condition, in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than (or along with) restoration of normal function.
The term “chronic inhalation of irritants”, as used within the context of the present invention, means, for example, chronic exposure to asbestos or tobacco, which may result when being inhaled, into many airway diseases. Thus, many of the irritants can cause harm to the lungs or other parts of the airways, leading to a range of different inhalation disorders. However, possible is also that the chronic inhalation of irritants comprises irritant gas inhalation injuries. Irritant gases are those which, when being inhaled, dissolve in the water of the respiratory tract mucosa and cause an inflammatory response, usually due to the release of acidic or alkaline radicals. Irritant gas exposures predominantly affect the airways, causing tracheitis, bronchitis, and bronchiolitis. Other inhaled agents may be directly toxic (e.g., cyanide or carbon monoxide) or may cause harm simply by displacing oxygen and causing asphyxia (e.g., methane or carbon dioxide).
The term “atherosclerosis”, as used within the context of the present invention, refers to a process of progressive thickening and hardening of the walls of medium-sized and large arteries as a result of fat deposits on their inner lining. Risk factors for atherosclerosis include high blood pressure (hypertension), smoking, diabetes and a genetic family history of atherosclerotic disease. Atherosclerosis can cause a heart attack if it completely blocks the blood flow in the heart (coronary) arteries. It can cause a stroke if it completely blocks the brain (carotid) arteries. Atherosclerosis can also occur in the arteries of the neck, kidneys, thighs, and arms, causing kidney failure or gangrene and amputation.
The term “gout”, as used within the context of the present invention, refers to a metabolic disease marked by a painful inflammation of the joints, deposits of urates in and around the joints, and usually an excessive amount of uric acid in the blood. The tendency to develop gout and elevated blood uric acid level (hyperuricemia) is often inherited and can be promoted by obesity, weight gain, alcohol intake, high blood pressure, abnormal kidney function, and drugs. The most reliable diagnostic test for gout is the identification of crystals in joints, body fluids and tissues.
The term “pseudogout”, as used within the context of the present invention, instead refers to an arthritic condition, which resembles gout, but is characterized by the deposition of crystalline salts other than urates in and around the joints. Specifically, it is characterized by an inflammation of the joints that is caused by deposits of calcium pyrophosphate crystals, resulting in arthritis, most commonly of the knees, wrists, shoulders, hips, and ankles. Pseudogout has sometimes been referred to as calcium pyrophosphate deposition disease or CPPD. Pseudogout is clearly related to aging as it is more common in the elderly and is associated with degenerative arthritis. Acute attacks of the arthritis of pseudogout can be caused by dehydration.
The term “trauma”, as used within the context of the present invention, means any injury caused by a mechanical or physical agent. The term “non-penetrating polytrauma”, as used within the context of the present invention, means there may be an impact, but the skin is not necessarily wounded. In contrast thereto, a penetrating polytrauma is an injury that occurs when an object enters a tissue of the body and creates an open wound. Conversely, the term “thermal trauma”, as used within the context of the present invention, may include any burn-related injury as well as any cold/freeze-related skin injury that can potentially lead to serious outcomes. There are various causes of thermal trauma, including fire, radiant heat, radiation, chemical, or electrical contact that can affect a person in many ways based on factors from anatomical and physiological factors.
In one further embodiment of the composition for use of the present invention, the immunosuppression after acute tissue injury is associated with a secondary infectious disease. Such a secondary infectious disease is a disease that may occur as a result of another disease, herein, preferably, as a result of the acute tissue injury. Such may be, for example pneumonia (infection of the lung), urinary tract infections or sepsis. The infections may be caused by bacteria, viruses or fungi.
According to one embodiment of the composition for use of the present invention, the DNA-degrading enzyme is a nuclease. The term “nuclease”, as used within the context of the present invention, refers to any of various enzymes that promote the hydrolysis of nucleic acids. Specifically, a nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. In living organisms, they are essential machineries for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency.
In one specific embodiment of the composition for use of the present invention, the nuclease is an exonuclease or endonuclease. Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′- or the 5′-end occurs. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 1. 5′ to 3′-exonuclease (Xrn1), which is a dependent decapping protein; 2. 3′- to 5′-exonuclease, an independent protein; and 3. poly(A)-specific 3′- to 5′-exonuclease. Endonucleases instead are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively non-specifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. Thus, endonucleases differ from exonucleases, which, as described above, cleave the ends of recognition sequences instead of the middle (endo) portion. Some enzymes known as “exo-endonucleases”, however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like.
In one further embodiment of the composition for use of the present invention, the endonuclease is a deoxyribonuclease, preferably DNase I. DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5′-phosphate-terminated polynucleotides with a free hydroxyl group on position 3′, on average producing tetranucleotides. It acts on single-stranded DNA, double-stranded DNA, and chromatin.
In one further embodiment of the composition for use of the present invention, the nuclease is administered after the acute tissue injury and/or in the course of the treatment of the acute tissue injury.
According to one further embodiment of the composition for use of the present invention, the nuclease is administered parenterally, preferably intravenously or by inhalation.
The term “parenterally”, as used within the context of the present invention, refers to an administration route other than through the alimentary canal, such as by subcutaneous, intramuscular, intrasternal, or intravenous injection. Intravenously administration means an administration of a fluid performed or occurred within or entering by way of a vein. By inhalation means the act or an instance of inhaling a substance.
It is noted that as used herein, the singular forms “a”, “an” and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. Additionally, for example, a reference to “a host cell” includes one or more of such host cells, respectively. Similarly, for example, a reference to “methods” or “host cells” includes “a host cell” or “a method”, respectively.
Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”. For example, A, B and/or C means A, B, C, A+B, A+C, B+C and A+B+C.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. When used herein “consisting of” excludes any element, step, or ingredient not specified.
The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.
The term “about” or “approximately” as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes also the concrete number, e.g., about 20 includes 20.
Further, in describing representative embodiments of the present invention, the specification may have presented the method and/or process of the present invention as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process of the present invention should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the present invention.
A better understanding of the present invention and of its advantages will be gained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.
Hereinafter, the present invention is explained in detail through examples. The following examples are intended merely to illustrate the present invention, to which the scope of the present invention is not restricted.
Experimental Design of Animal Experiments
All animal experiments were performed in accordance with the guidelines for the use of experimental animals and were approved by the government committee of Upper Bavaria (Regierungspraesidium Oberbayern, #175-2013, Rhineland Palatinate Landesuntersuchungsamt Koblenz, #G-19-07-41). Wild type C57BL6/J mice, Rag-1−/− (NOD.129S7(B6)-Rag-1tm1Mom/J), Fas−/− (MRL/MpJ-Faslpr/J), Casp1−/− (B6N.129S2-Casp1<tm1Flv>/J) were bred and housed at the animal core facility of the Center for Stroke and Dementia Research (Munich, Germany). The ASC-Citrine reporter mice (B6. Cg-Gt(ROSA)26Sortm1.1(CAG-Pycard/mCitrine*,-CD2)Dtg/J) and Faslpr (MRL/MpJ-Faslpr/J) were obtained from Jackson Laboratories (Bar Harbor, USA). Aim2−/− mice (Aim2<tm1.2Arte>) where bred at the Institute for Innate Immunity, University Bonn (Germany). Asc−/− mice (B6.129S5-Pycardtm1Vmd) and Pycard−/− mice (B6.129S5-Pycardtm1Vmd) were bred at the Gene Center of the LMU University Munich (Germany). Monocyte-specific ASC-knockout mice (LysM-Cre×Asc−/−; Lyz2-cre×Pycardfl/fl) mice were bred at the Institute for Clinical Chemistry and Pathobiochemistry (Technical University Munich, Germany). Cx3Cr1GFP/+ mice were purchased from Jackson Laboratory (Bar Harbor, USA) and bred at the animal core facility of Lanzhou University. All mice were housed with free access to food and water at a 12 h dark-light cycle.
A priori sample size calculation was based upon the criteria of 1) variance and effect size from previous studies or 2) preliminary pilot experiments performed during the study. Data was excluded from all mice that died during surgery. Detailed exclusion criteria are described below. Animals were randomly assigned to treatment groups and all analyses were performed by investigators blinded to group allocation. All animal experiments were performed and reported in accordance with the ARRIVE guidelines (Kilkenny et al., 2010).
Transient Ischemia-Reperfusion Stroke Model
Mice were anaesthetized with isoflurane delivered in a mixture of 30% O2 and 70% N2O. An incision was made between the ear and the eye in order to expose the temporal bone. Mice were placed in supine position, and a laser Doppler probe was affixed to the skull above the middle cerebral artery (MCA) territory. The common carotid artery and left external carotid artery were exposed via midline incision and further isolated and ligated. A 2-mm silicon-coated filament (Doccol) was inserted into the internal carotid artery, advanced gently to the MCA until resistance was felt, and occlusion was confirmed by a corresponding decrease in blood flow (i.e., a decrease in the laser Doppler flow signal by 80%. After 60 minutes of occlusion, the animals were re-anesthetized, and the filament was removed. After recovery, the mice were kept in their home cage with ad libitum access to water and food. Sham-operated mice received the same surgical procedure, but the filament was removed in lieu of being advanced to the MCA. Body temperature was maintained at 37° C. throughout surgery in all mice via feedback-controlled heating pad. The overall mortality rate of animals subjected to MCA occlusion was approximately 20%. All animals in the sham group survived the procedure. Exclusion criteria: 1. Insufficient MCA occlusion (a reduction in blood flow to >20% of the baseline value). 2. Death during the surgery. 3. Lack of brain ischemia as quantified post-mortem by histological analysis.
Germfree (GF) Mouse Handling
All surgeries, housing and post-operative animal handling were performed under sterile conditions as previously described (Singh et al., 2018). In brief, stroke and sham surgeries have been performed under sterile conditions in a microbiological safety cabinet, animals received sterilized water and irradiated food and animals were kept in sterile gnotocage mini-isolators. All surgical procedures and post-surgical care were otherwise performed as stated above.
Experimental Thermal Trauma Model
Male C57Bl/6J mice (Charles River, Freiburg, Germany), aged 7-8 weeks, received a 35% total body surface area (TBSA) full thickness scald burn to the back through 10 seconds immersion in 98° C. water under deep anesthesia with 2% isoflurane and analgesia with 0.1 mg kg−1 buprenorphine. Immediately after burn injury, the mice were resuscitated with 2 ml of lactated Ringer's solution (Baxter, Unterschleißheim, Germany) via i.p. injection as previously described (Hundeshagen et al., 2018; Bohannon et al., 2008; Toliver-Kinsky et al., 2005). Animals in the sham burn group were subjected to identical treatment except for water temperature during immersion being 36 C. Following burn injury or sham burn, mice were singly housed at room temperature (21° C.).
Transient Hind Limb Ischemia-Reperfusion Injury
Mice were anaesthetized with isoflurane delivered in a mixture of 30% O2 and 70% N2O. An incision was made between the ear and the eye in order to expose the temporal bone. Mice were placed in supine position, and a laser Doppler probe was affixed to the skull above the middle cerebral artery (MCA) territory. The common carotid artery and left external carotid artery were exposed via midline incision and further isolated and ligated. A 2-mm silicon-coated filament (Doccol) was inserted into the internal carotid artery, advanced gently to the MCA until resistance was felt, and occlusion was confirmed by a corresponding decrease in blood flow (i.e., a decrease in the laser Doppler flow signal by ≥80%. After 60 minutes of occlusion, the animals were re-anesthetized, and the filament was removed. After recovery, the mice were kept in their home cage with ad libitum access to water and food. Sham-operated mice received the same surgical procedure, but the filament was removed in lieu of being advanced to the MCA. Body temperature was maintained at 37° C. throughout surgery in all mice via feedback-controlled heating pad. The overall mortality rate of animals subjected to MCA occlusion was approximately 20%. All animals in the sham group survived the procedure. Exclusion criteria: 1. Insufficient MCA occlusion (a reduction in blood flow to >20% of the baseline value). 2. Death during the surgery. 3. Lack of brain ischemia as quantified post-mortem by histological analysis.
Parabiosis
Parabiosis experiments were performed at the Gansu Key Laboratory in Lanzhou, China. Pairs of weight-matched wild type C57Bl6/J and heterozygous Cx3Cr1GFP/+ mice were subjected to parabiotic surgery (Wright et al., 2001; Li et al., 2013). Animals were anesthetized by intraperitoneal injection of 20 mg/ml ketamine and 2 mg/ml xylazine. The flanks were shaved and sterilized. An incision from behind the ear to the hip was made on the opposing sides of two mice. Opposing posterior muscles were joined with a 5-0 suture. The scapular region was conjoined then dorsal and ventral skin edges were sutured with a 4-0 suture. Mice were kept at 37° C. in a recovery box until completely recovered from anesthesia. During the first 7 days after surgery, Tylenol is mixed in the food for analgesic purposes. Food and water were provided ad libitum. The optimized procedure had a survival rate of ≥75%.
Intranasal Bacterial Infection
Pneumococcal infection experiments were performed at the Helmholtz Centre for Infection Research (HZI) in Braunschweig, Germany. Mice were anesthetized with isoflurane delivered in a mixture of 30% O2 and 70% N2O. The inoculum (106 CFU of TIGR4, a serotype 4 S. pneumoniae strain (Tettelin et al., 2001) or 2×105 CFU of K. pneumoniae subsp. pneumoniae (ATCC 43816) (Wu et al., 2020) in a total volume of 25 μl PBS) was administered with a pipette onto the nostrils of the mice.
Drug Administration
Anti-IL1β: Mice received two injections of antagonizing anti-IL-1β in sterile saline (clone: B122, InVivoMab, BioXcell, US), 1 hour prior to and 1 hour after surgery. Anti-IL-1β or the corresponding IgG control (Armenian hamster IgG, InVivoMab, BioXcell, US) was injected i.p. at a dose of 4 mg kg−1 body weight in a final volume of 200 μl.
Anti-FasL: Mice received two injections of antagonizing anti-FasL in sterile saline (clone: MFL3, InVivoMab, BioXcell, US), 1 hour prior to and 1 hour after surgery. Anti-FasL or the corresponding IgG control (Armenian hamster IgG, InVivoMab, BioXcell, US) was injected i.p. at a dose of 4 mg kg−1 body weight in a final volume of 100 μl.
Human recombinant DNase (hrDNase): 1000 U of human recombinant DNase (Roche, Switzerland) dissolved in incubation 1× buffer (40 mM Tris-HCl, 10 mM NaCl, 6 mM MgCl2, 1 mM CaCl2, pH 7.9, diluted in PBS, Roche) was injected i.v. in the tail vein 1 hour after surgery in a final volume of 100 μl. The control group was administered vehicle injections at the same volume, route, and timing as experimental animals.
Caspase-1 inhibitor (VX-765): The caspase-1 inhibitor VX-765 in DMSO dissolved in PBS (Belnacasan, Invivogen, US) was injected i.p. 1 hour prior to surgery at a dose of 100 mg kg−1 body weight at a final volume of 300 μl. The control group was administered vehicle injections at the same volume, route, and timing as experimental animals.
Caspase-8 inhibitor (Z-IETD-FMK) & Caspase-9 inhibitor (Z-LEHD-FMK): The apoptosis inhibitors Z-IETD-FMK and Z-LEHD-FMK (R&D systems, US) in DMSO dissolved in PBS and injected i.p. 30 minutes after surgery. Z-LEHD-FMK was injected at a dose of 0.8 μM kg−1 body weight at a final volume of 200 μl. Z-IETD-FMK was injected at a dose of 0.8 mg kg−1 body weight at a final volume of 100 μl. The control groups were administered vehicle injections at the same volume, route, and timing as experimental animals.
Selective Beta2-adrenoreceptor inhibitor (ICI-118,551): The β2-adrenoreceptor inhibitor ICI118,551 (Sigma, Germany) was dissolved in PBS and administered 1 hour prior to and 1 hour after surgery at a dose of 4 mg kg−1 body weight at a final volume of 200 μl. The control group was administered vehicle injections at the same volume, route, and timing as experimental animals.
Murine recombinant IL-1β: Recombinant IL-1β (401-ML, R&D systems, US) was diluted in sterile PBS and administered intraperitoneally at a dose of 100 or 1000 ng per mouse in a total volume of 100 μl. The control group was administered vehicle injections at the same volume, route and timing as experimental animals.
Adoptive T Cell Transfer in Rag-1−/− Recipient Mice
Donor animals (C57BL6/J, Casp1−/−, Asc−/−) were euthanized and spleens were collected in Dulbecco's Modified Eagle Medium (DMEM+GlutaMax). Spleens were homogenized and filtered through 40 μm cell strainers. T cells were enriched using a negative selection kit for CD3+ T cells (MagniSort, Thermo Fisher). After washing and quantification, cells were injected i.p. into Rag-1−/− recipient mice (4×106CD3+ T cells per mouse) in a total volume of 200 μl saline. Mice were maintained for 4 weeks in order to establish a functional T cell niche, and then assigned to the surgery groups.
T Cell Isolation and Culture
Round-bottom tissue culture-treated 96-well plates were coated with 100 μl of PBS containing a mixture of 0.5 mg/mL purified NA/LE hamster anti-mouse CD3e (clone: 145-2C11, BD Pharmingen) and 0.5 mg/mL anti-mouse CD28 (clone: 37.51, Invitrogen), and then incubated overnight at 37° C. with 5% CO2. Spleens (wild type, Casp1−/−) isolated from mice were homogenized into single splenocyte suspensions by using a 40 μm cell strainer followed by erythrolysis as described above. T cells were purified from splenocytes using a negative selection kit (MagniSort, Thermo Fisher) according to the manufacturer's instructions. Purity was reliably ≥90% as assessed by flow cytometry. Cells were resuspended in complete RPMI1640 (Gibco) and supplemented with 10% FBS, 1% penicillin/streptomycin and 10 μM β-mercaptoethanol. T cells were seeded into the CD3/CD28 coated plates at a density of 300,000 cells per well in a total volume of 200 μl.
Organ and Tissue Processing
Mice were deeply anaesthetized with ketamine (120 mg/kg) and xylazine (16 mg/kg) and blood was drawn via cardiac puncture in 50 mM EDTA (Sigma-Aldrich). Plasma was isolated by centrifugation at 3,000 g for 10 minutes and stored at −80° C. until further use. The blood pellet was resuspended in DMEM and erythrocytes were lysed using isotonic ammonium chloride buffer. Immediately following cardiac puncture, mice were transcardially perfused with normal saline for dissection of bone marrow and spleen. Spleen and bone marrow were transferred to tubes containing Hank's balanced salt solution (HBSS), homogenized and filtered through 40 μm cell strainers to obtain single cell suspensions. Homogenized spleens were subjected to erythrolysis using isotonic ammonium chloride buffer.
Bacterial Culture and CFU Counts
S. pneumoniae TIGR4, an encapsulated strain of serotype 4, was grown overnight on Columbia blood agar plates (37° C., 5% CO2), single colonies were cultured in Todd-Hewitt broth with 1% yeast extract to mid-logarithmic phase (OD600 nm: 0.35), washed, and diluted in sterile PBS to the desired concentration. Klebsiella pneumoniae subsp. pneumoniae was grown in Mueller Hinton broth to mid-logarithmic growth phase (OD600 nm: 0.7), washed and diluted in PBS. 14 h post bacterial infection, mice were euthanized, tracheas and lungs were aseptically removed and mechanically homogenized in PBS. Serial dilutions of lung and tracheal tissue homogenates were plated onto blood agar plates and CFU were determined after 16 h of incubation.
Fluorescence-Activated Cell Sorting (FACS) Analysis
The anti-mouse antibodies listed below (see Table 1) were used for surface marker staining of CD45+ leukocytes, CD45+CD11b+ monocytes (+FasL+ expression), CD3+ T cells, CD3+CD4+ Thelper cells, CD3+CD8+ Tcytotox cells and CD19+ B cells (for representative gating strategy, see
Dimensionality Reduction Analysis for FACS Data
FACS data acquired with FACSVerse was pre-analyzed with FlowJo software. To normalize the data, each sample was down-scaled (“DownSample” plugin FlowJo) to 3,000 CD45+ cells per individual mouse. After concatenating the individual samples into a batch, t-distributed stochastic neighboring embedding (t-SNE) analysis was conducted (Parameters: Iterations 550; Perplexity 30; Eta learning rate 200) using the “t-SNE plugin” of the FlowJo software (V10.6).
FAM FLICA Caspase-1 Staining for FACS
To detect the active forms of caspase-1 in blood, spleen, and bone marrow samples, cell suspensions were stained with the fluorescent inhibitor probe FAM-YVAD-FMK (FAM FLICA, BioRad, Germany) for 30 minutes at 37° C. according to the manufacturer's instructions. After washing, the cells were stained for CD45+CD3+ T cells and CD45+Cd11b+ monocytes. The flow cytometry data was acquired on a BD FACSVerse (for representative gating strategy, see
FACS Imaging of ASC-Citrine Reporter Mice
Spleens from ASC-citrine reporter mice were dissected and single splenocyte suspensions were prepared using a 40 μm cell strainer, then subjected to erythrolysis as described above. Splenocytes were then stained with FACS antibodies against CD45, CD3 and CD11b as described above. Cells were resuspended at a concentration of 107 cells/ml for FACS imaging using the Flowsight Imaging flow cytometer (Amnis). The results were analyzed using the IDEAS software (Amnis) (Tzeng et al., 2016). For the speck analysis, cells were pre-gated for CD45+CD11b+ or CD45+CD3+ and then gated for citrine+. Citrine+ cells were randomly selected (50 CD45+CD11b+ citrine+ cells per mouse) and the numbers of specks per cells was analyzed.
FAM FLICA Caspase-1 Staining on Fresh Frozen Spleen Sections
Mice were deeply anesthetized and euthanized as described above. Spleens were immediately removed, embedded in cryotech solution (OCT, tissue-tek) and cryosectioned sagittally (20 μm thickness). FAM-YVAD-FMK (FAM FLICA, BioRad, Germany) solution was prepared as indicated in the manufacturer's instruction and sections were incubated for 1 hour at 37 C. Sections were then washed with PBS, stained with DAPI (1:5,000; Dako), and mounted (Aqueous mounting medium, Dako). Epifluorescence images were acquired at 20× magnification (Axio Imager 2, Carl Zeiss).
Whole Splenocyte Culture
Spleens from naïve wild type mice were dissected and single splenocyte suspensions were prepared using a 40 μm cell strainer, then subjected to erythrolysis as described above. Cells were washed three times with PBS, then cell number and viability was assessed using an automated cell counter (BioRad) and Trypan blue solution (Merck). Required viability threshold was 80%. Cells were cultured (complete RPM11640, 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, 10 μM β-mercaptoethanol) overnight for 16 hours on a 96 well flat bottom (anti-CD3/CD28 coated) plate at a density of 105 cells per 100 μl in a final volume of 200 μl. Cells were then stimulated by 12-hour incubation with serum from either stroke or sham operated mice at a concentration of 25% total well volume. After the stimulation, cell death and activation status were analyzed via FACS as described above.
Bone Marrow-Derived Macrophages (BMDM) Isolation and Cell Culture
BMDMs were generated from tibia and femur of transcardially perfused mice. After careful isolation and dissection of tibia and femur, bone marrow was flushed out of the bones through a 40 μm strainer using a plunger and 1 ml syringe filled with sterile 1×PBS. Strained bone marrow cells were washed with PBS, and resuspended in DMEM+GlutaMAX-1 (Gibco, US), supplemented with 10% FBS and 1% Gentamycin (Thermo Fisher Scientific) and counted. 5×107 cells were plated onto 150 mm culture dishes. Cells were differentiated into BMDMs over the course of 8-10 days. For the first 3 days after isolation, cells were supplemented with 20 L929 cell-conditioned media (LCM), as a source of M-CSF. Cultures were then maintained at 37° C. with 5% CO2 until 90% confluency.
BMDM—T Cell Co-Culture Assays
Differentiated BMDMs were cultured for 8-10 days, then harvested, washed, counted, and seeded in flat-bottom tissue-culture treated 96-well plates at a density of 100,000 cells per well in a total volume of 200 μl, and then cultured overnight for 16 h (see
For the kinetic analysis of T cell death, we used either eGFP-actin+ T cells (see
Western Blotting
Spleens were harvested from deeply anaesthetized mice, and the whole organs were processed into single cell suspensions as described above. Single cell suspensions were lysed with RIPA lysis/extraction buffer with added protease/phosphatase inhibitor (Thermo Scientific, US). The total protein content of each sample was measured via bicinchoninic acid assay (Thermo Fisher Scientific, USA). Whole cell extracts were fractionated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (BioRad, Germany). After blocking for 1 hour in TBS-T (TBS with 0.1% Tween 20, pH 8.0) containing 4% skim milk powder (Sigma), the membrane was washed with TBS-T and incubated with the primary antibodies against caspase-1 (1:1000; AdipoGen), IL-1β (1:500; R&D systems) and β-actin (1:1000; Sigma). Membranes were washed three times with TBS-T and incubated for 1 hour with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:5,000, Dako) at room temperature. Membranes were washed three times with TBS-T, developed using ECL substrate (Millipore) and acquired via the Vilber Fusion Fx7 imaging system.
Clinical Stroke Study Population
Ischemic stroke patients were recruited within 24 hours of symptom onset through the emergency department at the LMU University Hospital Munich (Germany), a tertiary level hospital. All patients had a final diagnosis of ischemic stroke as defined by 1) an acute focal neurological deficit in combination with a diffusion weighted imaging-positive lesion on magnetic resonance imaging, or 2) a new lesion on a delayed CT scan. Age-matched control patients were recruited in the neurological outpatient clinic. The study was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki as well as institutional guidelines. Written and informed consent was obtained from all patients.
For analysis of secondary infections (see
Thermal Injury Patients
Patients with severe burn injury encompassing more than 40% of total body surface area (TBSA) were recruited through BG Trauma Center Ludwigshafen (Germany). TBSA was assessed on admission by the attending burn surgeon using Lund-Browder charts and serum samples were collected at 24 hours post burn. Age- and sex-matched control patients were recruited in the trauma center outpatient clinic. The study was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki as well as institutional guidelines. Written and informed consent was obtained from all patients.
Human Monocyte Culture Stimulation with Patient's Serum
Human Monocytes cells (3×105/well) were seeded in 96 flat bottom plates with 50 ng/ml recombinant human M-CSF in RPMI 1640 (Gibco) supplemented with 2.5% (v/v) human serum (Sigma-Aldrich), Penicillin-Streptomycin (100 Thermo Fisher Scientific), Pyruvate (1 mM, Gibco) and HEPES (10 mM, Sigma-Aldrich) overnight to adjust the cells. Next day, cells were replaced with fresh medium (without M-CSF) in presence or absence of Pam3CSK4 (2.5 μg/ml) for 2 hours. Next, cells were stimulated with either control serum or stroke serum (1:4 dilution). After 2 hours, medium was gently removed and cells were washed once with PBS and replaced with fresh medium (150 μl each well) for 6 hours. Nigericin (Sigma) was used as positive control at final concentration 6.5 μM, stimulated for 6 hours. For each condition, 5 wells were stimulated. Supernatants were collected and 50 μl from each well was used for human IL-1β ELISA (BD) while remaining supernatants were combined and used for Western blot analysis after precipitating with methanol/chloroform. Cells were directly lysed in 1×SDS Laemmli buffer and lysates were combined from 5 wells for each condition. Samples were heated at 95° C. with 1100 rpm and loaded on SDS-PAGE gel (5% stacking gel and 12% separating gel; BioRad). Afterward, proteins were transferred on nitrocellulose membrane (GE healthcare) for 1 h. Membranes were blocked for another 60 min in 3% milk in PBST (PBS containing 0.05% Tween 20). All primary antibodies of caspase-1 (1:1000; AdipoGen), IL-1β (1:500; R&D systems) were incubated at least overnight in 1% milk in PBST at 4° C. Next day, membranes were incubated for at least 1 h in secondary antibody (Santa Cruz) and washed gently in PBST buffer for further 30-60 min. Loading control β-actin-HRP antibody was purchased from Santa Cruz (1:3000). Chemiluminescent signal was recorded with CCD camera in Fusion SL (PEQLAB). If needed, the whole image was contrast-enhanced in a linear fashion.
Free Nucleic Acid Quantification
Cell-free nucleic acids (RNA, single strand (ss) DNA and double strand (ds) DNA) levels in the plasma of mice and human patients was assessed with a Qubit 2.0 fluorometer (Invitrogen) using specific fluorescent dyes which bind either ssDNA (ssDNA Assay Kit, Thermo Fisher Scientific) or dsDNA (HS dsDNA Assay kit, Thermo Fisher Scientific). Dilutions and standards were generated following the manufacturer's instructions (Thermo Fisher scientific).
Enzyme Linked Immunosorbent Assay (ELISA)
Total IL-1β and caspase-1 levels from patient plasma samples (diluted 1:10 in sterile PBS) were obtained using commercial assay kits according to the manufacturers instructions (Quantikine ELISA human IL-1β, Quantikine ELISA human caspase-1 R&D systems). Total IL-1β and IL-18 levels from murine plasma samples were measured using the Duoset ELISA IL-1β and the Duoset ELISA IL-18 kit according to the manufacturer's instructions (R&D systems).
Quantitative RT-PCR
Total RNA was purified from naïve splenic CD11b+ monocytes and CD3+ T cells using the RNeasy Mini Kit (Qiagen). RNA from each sample was used for cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The quantitative expression of different cytokines was measured by quantitative real-time PCR with the LightCycler 480 II (Roche) and RT2 qPCR Primer Assays and SYBR Green ROX qPCR Mastermix (Qiagen).
Multiplex Mouse Cytokine Quantification
Plasma samples from mice were used to assess cytokine and chemokine levels (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, Eotaxin, G-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, Tnf-α) using a Luminex-100 system following the instructions in the manufacturer's manual (Bio-Plex23 Pro Mouse Cytokine Grp1, BioRad).
Infarct Volumetry
Mice were euthanized by overdose of ketamine-xylazine and perfused intracardially with normal saline. Brains were removed and immediately frozen in powdered dry ice. Frozen brains were fixed in cryotech solution (OCT, tissue-tek) and 20 μm coronal sections were collected at 400 μm intervals. Sections were stained with cresyl violet and scanned at a resolution of 600 dpi. Infarct area of each section was assessed by ImageJ software (NIH). The Swanson method was employed to measure the infarct area and to correct for cortical swelling: [ischemic area]=[area of the contralateral hemisphere]−[non-ischemic area of the ipsilateral hemisphere]. The total infarct volume was determined by integrating measured areas and distances between sections.
Statistical Analysis
Data were analyzed using GraphPad Prism version 6.0. All summary data are expressed as the mean±standard deviation (s.d.). All data sets were tested for normality using the Shapiro-Wilk normality test. The groups containing normally distributed independent data were analyzed using a two-way Student's t-test (for 2 groups) or ANOVA (for >2 groups). Normally distributed dependent data (i.e. in vitro co-culture kinetics) were analyzed using a 2-way ANOVA. The remaining data were analyzed using the Mann-Whitney U test (for 2 groups) or Kruskal-Wallis Test (H-test, for >2 groups). Similar variance was assured for all groups, which were statistically compared. P-values were adjusted for comparison of multiple comparisons using Bonferroni correction or Dunn's multiple comparison tests. A p value<0.05 was considered to be statistically significant.
For statistical analysis of human patient data (see
Experimental tissue injury of different etiologies and organs such as stroke (brain), burn injury (skin) or hindlimb ischemia (skeletal muscle) all result in subacute immunosuppression, characterized by a massive T cell death with approx. 50% loss within less than 24 h after the injury (see
IL-1β cleavage of the pro-form to the mature cytokine and its extracellular release are tightly regulated by caspase-1, the central effector enzyme of the inflammasome (Lopez-Castejon et al., 2011; Bauernfeind et al., 2011). The inflammasome is a multi-protein complex which accumulates and orchestrates caspase-1 cleavage upon activation of a wide range of danger signals sensed by the inflammasome. The inventors were able to identify systemic inflammasome activation after local tissue injury in the brain by several lines of evidence: they observed an increase of both pro-caspase-1 as well as its active cleavage isoforms in spleens by western blot (see
Likewise, pharmacological inhibition of caspase-1 using the small molecule inhibitor VX-765 improved T cell survival and spleen cellularity after stroke as well as burn injury in mice (see
Inflammasome subtypes are defined by the sensor molecule which determines the specificity for different activation signals, such as non-self proteins, ion flux or nucleic acids (Latz et al., 2013; Hornung et al., 2009). Most, but not all, inflammasome subtypes require the ASC adaptor protein for oligomerization and caspase-1 activation using its caspase activation and recruitment domain (CARD) (Hoss et al., 2017). The inventors observed a significantly improved T cell survival after stroke in ASC-deficient mice, indicating an ASC-dependent inflammasome activation in T cell death after tissue injury (see
The inventors identified in this Example the upstream mediator leading to systemic inflammasome activation. They detected a significant increase in cell free double strand DNA (cf-dsDNA) after stroke and burn lesions in mice as well as in patients (see
Notably, while the used genetic and pharmacological models to block AIM2 inflammasome activation efficiently prevented myeloid FasL upregulation, the primary lesion size was unaffected by these approaches. These results underscore the notion that the inflammasome pathway impacts on the systemic, immunological events following stroke rather than modulating lesion severity (see
Taken together, the inventors have observed that acute tissue injury increases cf-dsDNA blood concentrations and that cf-dsDNA is a potent and sufficient activator of the AIM2 inflammasome, leading to T cell death.
The inventors have also identified the mechanisms by which inflammasome activation in monocytes results in T cell death after tissue injury. First, the inventors tested whether cell-cell contact is necessary or soluble mediators released by monocytes are sufficient for this interaction. Therefore, the inventors established an in vitro co-culture model of BMDMs and T cells with or without cell contact (see
Additionally, the inventors found corresponding findings for the mechanism of B cell death as above for T cells. Experimental tissue injury (stroke and burn injury) results as well in a massive B cell death with approx. 40-50% loss within less than 24 h after the injury (see
The inventors aimed to test the hypothesis that soluble mediators released after injury are a potential cause for T cell apoptosis. First, the inventors confirmed a pronounced and general T cell death across subpopulations after experimental stroke which occurred even under sterile (germfree) conditions, hence, cannot be attributed to potential concomitant microbial infections (see
Treatment of mixed splenocytes—which allows an unbiased ex vivo analysis of all splenic leukocyte subpopulations and their potential interactions—with stroke serum in vitro revealed a close temporal association between the monocytic FasL upregulation and T cell death (see
Patients with severe tissue injuries after stroke, trauma, or burn have a high susceptibility to infections, which contribute substantially to secondary mortality. Therefore, after identifying the mechanism of T cell death by a bystander mechanism to inflammasome activation in monocytes, the inventors aimed to test the relevance of this pathway for post-injury infections. They analyzed 174 patients with ischemic stroke for which complete information was available for serum concentrations of dsDNA and IL-1β at hospital admission (d0; mean time after symptom onset: 4.9 hours), their blood lymphocyte counts on the subsequent day (d1) and the occurrence of infections (requiring antibiotic treatment and CRP>30 mg/I and/or radiographic confirmation) between days 2-7 after stroke onset (see
Summary: Dysregulation of systemic immune homeostasis is a common consequence of local tissue injuries. The inventors have identified a surprising mechanism by which systemic activation of the AIM2 inflammasome links an immediate pro-inflammatory response with subsequent immunosuppression after various types of acute injuries in mice and human patients (see
Therefore, in addition to a detailed understanding of the immunological mechanisms, the present invention also provides several novel therapeutic targets to ameliorate the diverse immunological consequences of tissue injuries. The identified pathway along the events of increased cf-dsDNA concentrations, inflammasome activation, IL-1β secretion and Fas-mediated T cell death provides several druggable, therapeutic targets—for which available drugs could even be repurposed. The most promising therapeutic target seems to be the pathological initiator of this immunological cascade, the increase in circulating cf-dsDNA. The inventors have shown the efficient degradation of cf-dsDNA and reduction of the immunological consequences by use of human recombinant DNAse. Inhaled hrDNAse is already in clinical use for patients with cystic fibrosis, however, its systemic administration and immunological effects in tissue injury have so far not been tested. Additionally, the key effector molecule of the inflammasome, the IL-1β cytokine, represents another promising drug target. The inventors have identified IL-1β secretion to be important in mediating the downstream cell-cell contact-dependent T cell death after tissue injury. Hence, neutralization of circulating IL-1β by monoclonal antibodies might paradoxically improve systemic immunocompetence after tissue injury by preventing T cell death despite being currently used as an immunosuppressive drug. Indeed, while IL-1β blockade has initially been developed for rare autoimmune disorders, this approach has recently been tested also for patients with myocardial infarction. IL-1β blockade significantly lowered recurrent local cardiovascular events in a large clinical trial (Ridker et al., 2017) and its local anti-inflammatory effects might reduce development of heart failure (Panahi et al., 2018).
Taken together, the present invention identified a surprising systemic activation of the inflammasome as the linking mechanisms between a systemic immune response and subsequent immunosuppression after various local tissue injuries. Inhibiting the inflammasome-IL-1β-Fas pathway is therefore important for preventing secondary immunosuppression in patients with acute tissue injury.
The following items also characterize the present invention:
| Number | Date | Country | Kind |
|---|---|---|---|
| 20171271.8 | Apr 2020 | EP | regional |
This application claims priority to International Application No. PCT/EP2021/060664, filed Apr. 23, 2021, which claims the benefit of priority of EP Patent Application No. 20171271.8 filed 24 Apr. 2020, the contents of which are hereby incorporated by reference in their entireties for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/060664 | 4/23/2021 | WO |
