Macrophage Depletion Reduces Disease Pathology in Factor H-Dependent Immune Complex-Mediated Glomerulonephritis.

Complement factor H (FH) is a key regulator of the alternative pathway of complement, in man and mouse. Earlier, our studies revealed that the absence of FH causes the C57BL6 mouse to become susceptible to chronic serum sickness (CSS) along with an increase in the renal infiltration of macrophages compared to controls. To understand if the increased recruitment of macrophages (Mϕs) to the kidney was driving inflammation and propagating injury, we examined the effect of Mϕ depletion with clodronate in FH knockout mice with CSS. Eight-week-old FHKO mice were treated with apoferritin (4 mg/mouse) for 5 wks and with either vehicle (PBS) or clodronate (50 mg/kg ip, 3 times/wk for the last 3 weeks). The administration of clodronate decreased monocytes and Mϕs in the kidneys by >80%. Kidney function assessed by BUN and albumin remained closer to normal on depletion of Mϕs. Clodronate treatment prevented the alteration in cytokines, TNFα and IL-6, and increase in gene expression of connective tissue growth factor (CTGF), TGFß-1, matrix metalloproteinase-9 (MMP9), fibronectin, laminin, and collagen in FHKO mice with CSS (P < 0.05). Clodronate treatment led to relative protection from immune complex- (IC-) mediated disease pathology during CSS as assessed by the significantly reduced glomerular pathology (GN) and extracellular matrix. Our results suggest that complement activation is one of the mechanism that regulates the macrophage landscape and thereby fibrosis. The exact mechanism remains to be deciphered. In brief, our data shows that Mϕs play a critical role in FH-dependent ICGN and Mϕ depletion reduces disease progression.

An explanation of the pathogenesis of several autoimmune diseases in immuno-compromised individuals.

Some pathogen infections and immune system deficiencies have been linked to a few autoimmune diseases. However, the pathogenesis of most autoimmune diseases is unknown. An explanatory hypothesis for the pathogenesis of infection-initiated autoimmune diseases is provided. Virulent pathogen infections create extensive pathogen antigens that frequently require antibodies. These antibodies create extensive antigen-antibody immune complexes, which some immuno-compromised individuals will not adequately eliminate. This will cause inflammatory type III hypersensitivity symptoms, including protease releases that destroy epithelium, mesothelium and endothelium basement membranes, express new immunogenic antigens from previously sequestered basement membrane constituents, and ultimately induce new autoantibodies. This can continue after the infection ends, if the first wave of protease attacks on basement membranes induces new autoantibodies that cause new uncleared antigen-antibody immune complexes and type III hypersensitivity reactions. The secreted proteases and other enzymes will have preferred substrates and these proteases or other enzymes by themselves, or by their processed protein substrates, can express immunogenic antigens that induce new autoantibodies and initiate various autoimmune diseases. In summary, several autoimmune diseases can be initiated in immuno-compromised individuals during extensive pathogen infections, if these individuals have two immune problems: (a) slow or weak initial immune responses that result in a reliance on antibodies and (b) an inability to eliminate the resulting antigen-antibody immune complexes by phagocytosis. These two immune problems and the resulting immune system type III hypersensitivity reaction can explain the causation of several autoimmune diseases, including the most common and the rarest autoimmune diseases, both their differences and their similarities.

A viral infection explanation for Kawasaki disease in general and for COVID-19 virus-related Kawasaki disease symptoms.

SARS-CoV-2, a new virus that appeared in Wuhan, China, in 2019 has approximately an 80% genomic match to the Severe Acute Respiratory Symptom (SARS) virus, which is known to come from a bat virus. Symptoms of Kawasaki disease in general and incomplete Kawasaki disease have been seen in a subset of pediatric patients having a current or previous infection of SARS-CoV-2. A viral infection, such as a SARS-CoV-2 virus infection, could result in extensive antigen-antibody immune complexes that cannot be quickly cleared in a subset of patients and thus create a type III hypersensitivity immune reaction and cause Kawasaki disease or Kawasaki disease symptoms (also known as multisystem inflammatory syndrome) in a subset of patients. Extensive binding of antibodies to viral antigens can create antigen-antibody immune complexes, which, if not eliminated in certain individuals having dysfunctional complement systems, can start inflammatory type III hypersensitivity symptoms, including protease releases that can disrupt epithelium, mesothelium, and endothelium basement membranes, and induce pervasive inflammation throughout the body. This could continue after SARS-CoV-2 infections end if the first wave of protease attacks on basement membranes created new secondary autoantibodies and new uncleared antigen-antibody immune complexes.

Type 3 hypersensitivity in COVID-19 vasculitis.

Coronavirus Disease 2019 (COVID-19) is an ongoing public health emergency and new knowledge about its immunopathogenic mechanisms is deemed necessary in the attempt to reduce the death burden, globally. For the first time in worldwide literature, we provide scientific evidence that in COVID-19 vasculitis a life-threatening escalation from type 2 T-helper immune response (humoral immunity) to type 3 hypersensitivity (immune complex disease) takes place. The subsequent deposition of immune complexes inside the vascular walls is supposed to induce a severe inflammatory state and a cytokine release syndrome, whose interleukin-6 is the key myokine, from the smooth muscle cells of blood vessels.

Immune complex disease in a chronic monkey study with a humanised, therapeutic antibody against CCL20 is associated with complement-containing drug aggregates.

Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.

Low-dose 17α-ethinyl estradiol (EE) exposure exacerbates lupus renal disease and modulates immune responses to TLR7/9 agonists in genetically autoimmune-prone mice.

Estrogens have been shown to regulate the immune system and modulate multiple autoimmune diseases. 17α-ethinyl estradiol (EE), a synthetic analog of 17ß-estradiol, is prescribed commonly and found in oral contraceptives and hormone replacement therapies. Surprisingly, few studies have investigated the immunoregulatory effects of exposure to EE, especially in autoimmunity. In this study, we exposed autoimmune-prone female MRL/lpr mice to a human-relevant dose of EE through the oral route of exposure. Since lupus patients are prone to infections, groups of mice were injected with viral (Imiquimod, a TLR7 agonist) or bacterial (ODN 2395, a TLR9 agonist) surrogates. We then evaluated autoimmune disease parameters, kidney disease, and response to in vivo TLR7/9 pathogenic signals. EE-exposed mice had increased proteinuria as early as 7 weeks of age. Proteinuria, blood urea nitrogen, and glomerular immune complex deposition were also exacerbated when compared to controls. Production of cytokines by splenic leukocytes were altered in EE-exposed mice. Our study shows that oral exposure to EE, even at a very low dose, can exacerbate azotemia, increase clinical markers of renal disease, enhance glomerular immune complex deposition, and modulate TLR7/9 cytokine production in female MRL/lpr mice. This study may have implications for EE-exposure risk for genetically lupus-prone individuals.

Hepatitis B-related glomerulonephritis and optimization of treatment.

Introduction: Multiple studies have revealed a strong relationship between the development of nephropathy and hepatitis B virus (HBV) infection. The underlying pathogenesis of hepatitis B-related glomerulonephritis (HBV-GN) involves immune complexes, which can be isolated from kidney tissues. Clearance of HBV antigenemia improves renal impairment and proteinuria in HBV-GN patients.Areas covered: In this review, we present our current understanding of the epidemiology, pathogenesis, pathology, diagnosis, and treatment of HBV-GN. We discuss the advantages and disadvantages of oral nucleoside/nucleotide analogs (NAs), and the main pharmaceutical treatment for hepatis B.Expert opinion: Currently, antiviral agents are the main HBV-GN therapeutic agents. Although no randomized controlled clinical trials have compared the efficacy of interferon (IFN) and NA, we suggest IFN treatment for pediatric patients (IFN-α in patients ≥1 year; pegIFN-α in patients ≥3 years) considering treatment duration and absence of resistance. Novel NAs have brought about promising treatment options involving high efficacy viral suppression and low resistance rates. NAs with a high barrier to resistance (e.g. entecavir) are recommended as first-line therapy of HBV-GN. Immunosuppression monotherapy, such as corticosteroids, is of little benefit and potentially harmful to HBV-GN patients due to the possibility of viral reactivation.

Common Mechanisms of Viral Injury to the Kidney.

Viral infections in an immunocompetent host can cause both acute and chronic kidney diseases, either by direct damage to the infected kidney cells or as a consequence of systemic immune responses that impact the kidneys' function. Viruses have evolved mechanisms to hijack signaling pathways of the infected cell, including the mammalian target of rapamycin pathway to support viral replication, and to evade antiviral immune responses such as those mediated by miR-155 via microRNA mimetics expressed by the virus. At both the cellular and systemic levels, the host has also evolved mechanisms to counter the viral subversion strategies in the evolutionary battle for mutual survival. In the era of genomic medicine, understanding individual genetic variations that lead to differences in susceptibilities to infection and variabilities in immune responses may open new avenues for treatment, such as the recently described functions of apolipoprotein L1 risk alleles in HIV-associated nephropathy. In addition, state-of-the-art high-throughput sequencing methods have discovered new viruses as the cause for chronic diseases not previously attributed to an infection. The potential application of these methods to idiopathic kidney diseases may reveal similar occult infections by unknown viruses. Precision medicine objectives to optimize host-directed and pathogen-directed therapies for kidney diseases associated with infectious causes will only be achieved through detailed understanding of genetic susceptibility associated with immune responses and viral tropism.

The Changing Face of Human Immunodeficiency Virus-Mediated Kidney Disease.

In nearly 40 years since human immunodeficiency virus (HIV) first emerged, much has changed. Our understanding of the pathogenesis of HIV infection and its effect on the cells within each kidney compartment has progressed, and the natural history of the disease has been transformed. What was once an acutely fatal illness is now a chronic disease managed with oral medications. This change is largely due to the advent of antiretroviral drugs, which have dramatically altered the prognosis and progression of HIV infection. However, the success of antiretroviral therapy has brought with it new challenges for the nephrologist caring for patients with HIV/acquired immune deficiency syndrome, including antiretroviral therapy-induced nephrotoxicity, development of non-HIV chronic kidney disease, and rising incidence of immune-mediated kidney injury. In this review, we discuss the pathogenesis of HIV infection and how it causes pathologic changes in the kidney, review the nephrotoxic effects of select antiretroviral medications, and touch upon other causes of kidney injury in HIV cases, including mechanisms of acute kidney injury, HIV-related immune complex glomerular disease, and thrombotic microangiopathy.

Immune complex-mediated rapidly progressive glomerulonephritis in a patient with family history of systemic lupus erythematosus.

We describe here a case of a 53-year-old Saudi female who presented to the emergency room with shortness of breath progressive in nature for the previous one month, associated with a cough and occasional greenish sputum. She turned out to be a case of immunocomplex mediated glomerulonephritis presenting as rapidly progressive glomerulonephritis in a female patient with a history of systemic lupus erythematosus (SLE) in her daughter.

Immunogenicity and Immune Complex Disease in Preclinical Safety Studies.

This article summarizes a continuing education presentation on immunogenicity that was part of a continuing education course entitled, "Clinical Pathology of Biotherapeutics." Immunogenicity of a biotherapeutic can have diverse impacts including altered systemic exposure and pharmacologic responses and, in a fraction of the cases, safety concerns including cross-reactive neutralization of endogenous proteins or sequela related to immune complex disease (ICD). In most cases, immune complexes are readily cleared from circulation; however, based on physiochemical properties, insoluble complexes form, activate complement, and deposit in tissues. Using published information and personal experience, a set of repeat-dose monkey toxicity studies with manifestations suggestive of ICD was reviewed to summarize the spectrum of clinical and pathology findings. The most common live-phase observation linked to ICD was an acute postdosing reaction following multiple dose administrations characterized by generalized collapse and attributed to acute complement activation. Less common live-phase observations were related to syndromes such as a consumptive coagulopathy or a protein losing nephropathy. The most common histologic change attributed to ICD was multi-organ vascular/perivascular inflammation followed by glomerulonephritis. The presentation concluded with a description of the challenges in assessing the relevance of immunogenicity-related reaction in monkey to human clinical use.

Complement Activation in Inflammatory Skin Diseases.

The complement system is a fundamental part of the innate immune system, playing a crucial role in host defense against various pathogens, such as bacteria, viruses, and fungi. Activation of complement results in production of several molecules mediating chemotaxis, opsonization, and mast cell degranulation, which can contribute to the elimination of pathogenic organisms and inflammation. Furthermore, the complement system also has regulating properties in inflammatory and immune responses. Complement activity in diseases is rather complex and may involve both aberrant expression of complement and genetic deficiencies of complement components or regulators. The skin represents an active immune organ with complex interactions between cellular components and various mediators. Complement involvement has been associated with several skin diseases, such as psoriasis, lupus erythematosus, cutaneous vasculitis, urticaria, and bullous dermatoses. Several triggers including auto-antibodies and micro-organisms can activate complement, while on the other hand complement deficiencies can contribute to impaired immune complex clearance, leading to disease. This review provides an overview of the role of complement in inflammatory skin diseases and discusses complement factors as potential new targets for therapeutic intervention.

Overactivity of Alternative Pathway Convertases in Patients With Complement-Mediated Renal Diseases.

Overactivation of the alternative pathway of the complement system is associated with the renal diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). C3 nephritic factors (C3NeF) play an important role in C3G pathogenesis by stabilizing the key enzymatic complex of complement, the C3 convertase. However, the reliability of assays detecting these autoantibodies is limited. Therefore, in this study, we validated and optimized a prototype hemolytic method for robust detection and characterization of factors causing convertase overactivity in large patient cohorts. The assay assesses convertase activity directly in the physiological milieu of serum and therefore is not restricted to detection of stabilizing autoantibodies such as C3NeF but may also reveal genetic variants resulting in prolonged convertase activity. We first defined clear cutoff values based on convertase activity in healthy controls. Next, we evaluated 27 C3G patient samples and found 16 positive for prolonged convertase activity, indicating the presence of factors influencing convertase stability. In three patients, the overactive convertase profile was persistent over disease course while in another patient the increased stability normalized in remission. In all these four patients, the convertase-stabilizing activity resided in the purified immunoglobulin (Ig) fraction, demonstrating the autoantibody nature. By contrast, the Igs of a familial aHUS patient carrying the complement factor B mutation p.Lys323Glu did not reveal convertase stabilization. However, in serum prolonged convertase activity was observed and segregated with the mutation in both affected and unaffected family members. In conclusion, we present a robust and reliable method for the detection, characterization, and evaluation over time of factors prolonging convertase activity (C3NeF or certain mutations) in patient cohorts. This assay may provide new insights in disease pathogenesis and may contribute to the development of more personalized treatment strategies.

The atypical chemokine receptor 2 limits renal inflammation and fibrosis in murine progressive immune complex glomerulonephritis.

The atypical chemokine receptor 2 (ACKR2), also named D6, regulates local levels of inflammatory chemokines by internalization and degradation. To explore potential anti-inflammatory functions of ACKR2 in glomerulonephritis, we induced autologous nephrotoxic nephritis in C57/BL6 wild-type and Ackr2-deficient mice. Renal ACKR2 expression increased and localized to interstitial lymphatic endothelium during nephritis. At two weeks Ackr2-/-mice developed increased albuminuria and urea levels compared to wild-type mice. Histological analysis revealed increased structural damage in the glomerular and tubulointerstitial compartments within Ackr2-/- kidneys. This correlated with excessive renal leukocyte infiltration of CD4+ T cells and mononuclear phagocytes with increased numbers in the tubulointerstitium but not glomeruli in knockout mice. Expression of inflammatory mediators and especially markers of fibrotic tissue remodeling were increased along with higher levels of ACKR2 inflammatory chemokine ligands like CCL2 in nephritic Ackr2-/- kidneys. In vitro, Ackr2 deficiency in TNF-stimulated tubulointerstitial tissue but not glomeruli increased chemokine levels. These results are in line with ACKR2 expression in interstitial lymphatic endothelial cells, which also assures efflux of activated leukocytes into regional lymph nodes. Consistently, nephritic Ackr2-/- mice showed reduced adaptive cellular immune responses indicated by decreased regional T-cell activation. However, this did not prevent aggravated injury in the kidneys of Ackr2-/- mice with nephrotoxic nephritis due to simultaneously increased tubulointerstitial chemokine levels, leukocyte infiltration and fibrosis. Thus, ACKR2 is important in limiting renal inflammation and fibrotic remodeling in progressive nephrotoxic nephritis. Hence, ACKR2 may be a potential target for therapeutic interventions in immune complex glomerulonephritis.

Type III hypersensitivity reactions to a B cell epitope antigen are abrogated using a depot forming vaccine platform.

Peptide antigens are combined with an adjuvant in order to increase immunogenicity in vivo. The immunogenicity and safety of a RSV vaccine formulated in a novel oil-based platform, DepoVax™ (DPX), was compared to an alum formulation. A peptide B cell epitope derived from RSV small hydrophobic ectodomain (SHe) served as the antigen. Both vaccines induced SHe-specific antibodies after immunization of mice. A single dose of the DPX-based formulation resulted in anti-SHe titres for up to 20 weeks. Boosting with Alum-SHe, but not with DPX-SHe, led to unexpected clinical signs such as decreased activity, cyanosis and drop in body temperature in mice but not in rabbits. The severity of adverse reactions correlated with magnitude of SHe-specific IgG immune responses and decreased complement component 3 plasma levels, indicating a type III hypersensitivity reaction. By RP-HPLC analysis, we found that only 8-20% of the antigen was found to be adsorbed to alum in vitro, indicating that this antigen is likely released systemically upon injection in vivo. Clinical signs were not observed in rabbits, indicating the response correlates with peptide dose relative to size of animal. These results suggest that peptide antigens targeted to produce B cell mediated response may result in increased incidence of type III hypersensitivity reactions when delivered in non-depot forming vaccines. The DPX formulation induced strong antibody titres to the antigen without causing adverse events, likely due to the strength of the depot in vivo, and demonstrates the potential safety and immunogenicity of this platform for B cell peptide antigens.

Ubiquitin C-Terminal Hydrolase L1 is required for regulated protein degradation through the ubiquitin proteasome system in kidney.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1-deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1-deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury.