David M. Cauvi

Definition of IFN-γ-related pathways critical for chemically-induced systemic autoimmunity

IFN-γ is essential for idiopathic and murine mercury-induced systemic autoimmunity (mHgIA), and heterozygous IFN-γ(+/-) mice also exhibit reduced disease. This suggests that blocking specific IFN-γ-related pathways that may only partially inhibit IFN-γ production or function will also suppress autoimmunity. To test this hypothesis, mice deficient in genes regulating IFN-γ expression (Casp1, Nlrp3, Il12a, Il12b, Stat4) or function (Ifngr1, Irf1) were examined for mHgIA susceptibility. Absence of either Ifngr1 or Irf1 resulted in a striking reduction of disease, while deficiency of genes promoting IFN-γ expression had modest to no effect. Furthermore, both Irf1- and Ifng-deficiency only modestly reduced the expansion of CD44(hi) and CD44(hi)CD55(lo) CD4(+) T cells, indicating that they are not absolutely required for T cell activation. Thus, there is substantial redundancy in genes that regulate IFN-γ expression in contrast to those that mediate later signaling events. These findings have implications for the therapeutic targeting of IFN-γ pathways in systemic autoimmunity.

Interaction of heat shock protein 70 with membranes depends on the lipid environment

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.

Cathepsin B Regulates the Appearance and Severity of Mercury-Induced Inflammation and Autoimmunity

Susceptibility and resistance to systemic autoimmunity are genetically regulated. This is particularly true for murine mercury-induced autoimmunity (mHgIA) where DBA/2J mice are considered resistant to disease including polyclonal B cell activation, autoantibody responses, and immune complex deposits. To identify possible mechanisms for the resistance to mHgIA, we exposed mHgIA sensitive B10.S and resistant DBA/2J mice to HgCl2 and assessed inflammation and pro-inflammatory responses at the site of exposure and subsequent development of markers of systemic autoimmunity. DBA/2J mice showed little evidence of induration at the site of exposure, expression of proinflammatory cytokines, T cell activation, or autoantibody production, although they did exhibit increased levels of total serum IgG and IgG1. In contrast B10.S mice developed significant inflammation together with increased expression of inflammasome component NLRP3, proinflammatory cytokines IL-1β, TNF-α, and IFN-γ, hypergammaglobulinemia, splenomegaly, CD4(+) T-cell activation, and production of autoantibodies. Inflammation in B10.S mice was associated with a selective increase in activity of cysteine cathepsin B but not cathepsins L or S. Increased cathepsin B activity was not dependent on cytokines required for mHgIA but treatment with CA-074, a cathepsin B inhibitor, led to transient reduction of local induration, expression of inflammatory cytokines, and subsequent attenuation of the systemic adaptive immune response. These findings demonstrate that sensitivity to mHgIA is linked to an early cathepsin B regulated inflammatory response which can be pharmacologically exploited to abrogate the subsequent adaptive autoimmune response which leads to disease.

Transport of the IgE Receptor α-Chain Is Controlled by a Multicomponent Intracellular Retention Signal

The human high affinity IgE receptor (FcϵRI) is a central component of the allergic response and is expressed as either a trimeric αγ2 or tetrameric αβγ2 complex. It has been previously described that the cytoplasmic domain (CD) of the α-chain carries a dilysine motif at positions -3/-7 from the C terminus that functions in intracellular retention prior to assembly with other FcϵRI subunits. In this report we have further explored the role of the -3/-7 dilysine signal in controlling steady-state α-chain transport by mutational analysis and found little surface expression of a -3/-7 dialanine α-chain mutant but significant Golgi localization. We compared the transport properties of a series of α-chain cytoplasmic domain truncation mutants and observed that truncation mutants lacking 23 or more C-terminal residues showed a dramatic increase in steady-state transport suggesting a role for the membrane-proximal CD sequence in α-chain retention. By performing alanine-scanning mutagenesis we identified a dilysine sequence (Lys212-Lys216) proximal to the transmembrane domain (TMD) that is important for both α-chain cell-surface expression and intracellular stability. Furthermore, co-mutation of the Lys212-Lys216 residues with the -3/-7 dilysine signal produced a dramatic increase in α-chain surface expression that was further increased by co-mutation of the lone charged residue (Asp192) in the TMD thereby defining three regions that function to regulate α-chain transport and in a highly synergistic manner.

The hormonogenic tyrosine 5 of porcine thyroglobulin is sulfated

Our previous results showed that sulfated tyrosines of thyroglobulin (Tg), the molecular support of thyroid hormonosynthesis, are involved in the hormonogenic process. Moreover, the consensus sequence required for tyrosine sulfation is present in most of the hormonogenic sites. These observations suggest that tyrosine sulfation might play a critical role in the hormonogenic process. In this paper we studied the putative sulfation of tyrosine 5 contained in the preferential hormonogenic site. Porcine thyrocytes were cultured with thyrotropin but without iodide to preserve the sulfation state of tyrosine 5 and then incubated or not with [35S]sulfate. Secreted Tg was purified and submitted to peptide sequence analysis which confirmed the known peptide sequence of the NH(2) extremity of Tg:NIFEYQV. The treatment of [35S]sulfate-labeled Tg by leucine aminopeptidase, which sequentially digested its amino-terminal extremity, released the same amino acids and further analysis by thin layer chromatography showed that the tyrosine was sulfated. We concluded that tyrosine 5 is sulfated but the role of sulfate group in the hormonogenic process remains to be elucidated.

Itraconazole, a commonly used antifungal, inhibits Fcγ receptor-mediated phagocytosis: alteration of Fcγ receptor glycosylation and gene expression.

ABSTRACT Itraconazole (ICZ) is commonly used for the treatment of fungal infections, particularly in immunocompromised patients. In addition, ICZ has been recently found to have antiangiogenic effects and is currently being tested as a new chemotherapeutic agent in several cancer clinical trials. We have previously shown that ICZ impaired complex N-linked glycosylation processing, leading to the accumulation of high-mannose glycoproteins on the surface of macrophages (Møs). This investigation was directed at determining the effects of ICZ on phagocytosis as a major function of Møs. We found a significant decrease in the phagocytosis of opsonized bacterial particles in ICZ-treated murine Møs in comparison with nontreated Møs. Furthermore, the impairment of phagocytosis was associated with a decrease in cell surface expression of Fc&ggr; receptors (Fc&ggr;Rs) as well as alteration of their glycosylation pattern. Concomitantly, a reduction in all three isoforms of the Fc&ggr;R family (i.e., Fcgr1, Fcgr2, and Fcgr3) mRNA levels was observed after incubation with ICZ. The effect of ICZ on phagocytosis and Fc&ggr;R expression was reversed by addition of low-density lipoprotein. These studies indicate that ICZ treatment certainly has a dramatic effect on macrophage function, which could result in a potential impairment of the immune system’;s ability to respond to pathogens and may lead to an elevated incidence of infections.

Deletion of scavenger receptor A gene in mice resulted in protection from septic shock and modulation of TLR4 signaling in isolated peritoneal macrophages

Scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a surface glycoprotein preferentially present in macrophages that plays a primary role in innate immunity. Previous studies have shown that Sra is a modifier gene for the response to bacterial LPS in mice at the level of IL-10 production, in particular. In the present study, we found that Sra(−/−) mice are more resistant to septic shock induced by cecal ligation and puncture than wild-type C57BL/6 J (B6) mice. In addition, Sra(−/−) mice displayed initial elevated high density lipoprotein (HDL) circulating levels. Naïve peritoneal macrophages (PMϕs) were isolated from Sra(−/−) mice to understand the possible protective mechanism. Incubation of these cells with LPS was found to modulate TLR4 signaling, leading to a reduction in IL-10 and IL-6 mRNA levels, but not TNF-α expression, at low concentrations of LPS in comparison with PMϕs isolated from B6 mice. No differences were found in LPS binding between PMϕs derived from Sra(−/−) or B6 mice. The lack of Sra binding to LPS was confirmed after transfection of Chinese hamster ovary (CHO) cells with the Sra gene. The contribution of Sra to the outcome of sepsis may be a combination of changes in TLR4 signaling pathway and elevated levels of HDL in circulation, but also LPS toxicity.

Tumor necrosis factor expression is ameliorated after exposure to an acidic environment

BACKGROUND It has been well established that laparoscopic surgery presents several clinical benefits, including reduced pain and a shorter hospital stay. These effects have been associated with a decrease in the inflammatory response. Previous studies have demonstrated that reduced inflammation after laparoscopic surgery is the product of carbon dioxide insufflation, which decreases peritoneal pH. The objective of this study was to investigate the cellular and molecular mechanisms responsible for the reduced response after exposure to acidic environments. MATERIALS AND METHODS A murine macrophage line (J744) was incubated in culture medium at pH 6.0 or pH 7.4 for 3 h at 37°C. Then, cells were stimulated with lipopolysaccharide (LPS) at pH 7.4, the expression of TNF-α (qRT-PCR or enzyme-linked immunosorbent assay (ELISA) and intracellular pH were measured. In addition, CD14 and Toll-like receptor 4 expression and NF-κB nuclear translocation were analyzed. RESULTS A significant decrease in LPS-induced TNF-α expression levels was observed in cells pre-incubated at pH 6.0 in comparison with cells at neutral pH conditions. This decrease in TNF-α levels was not associated with a reduction in cell surface expression of CD14 and Toll-like receptor 4. Exposure to an extracellular acidic environment resulted in a reduction of IκB phosphorylation and NF-κB nuclear translocation, secondary to a significant drop in cytosolic pH. CONCLUSIONS These observations provide a potential mechanism for the reduced expression of TNF-α after exposure to low extracellular pH, which may be related to acidification after CO(2) insufflation during laparoscopic surgery. In addition, extracellular acidic pH environments could emerge as an important regulator of macrophage function.

Period of Irreversible Therapeutic Intervention during Sepsis Correlates with Phase of Innate Immune Dysfunction

Background: Sepsis is a major health problem that can be investigated in experimental animal models. Results: Its etiology is divided into an early therapeutically reversible phase displaying a robust inflammatory response followed by a late therapeutically irreversible stage with reduced innate immune response. Conclusion: Mortality is associated with a late immune dysfunction. Significance: This study provides information for the potential window for treatment. Sepsis is a major health problem in the United States with high incidence and elevated patient care cost. Using an animal model of sepsis, cecum ligation, and puncture, we observed that mice became rapidly hypothermic reaching a threshold temperature of 28 °C within 5–10 h after initiation of the insult, resulting in a reliable predictor of mortality, which occurred within 30–72 h of the initial procedure. We also observed that the inflammatory gene expression in lung and liver developed early within 1–2 h of the insult, reaching maximum levels at 6 h, followed by a decline, approaching basal conditions within 20 h. This decrease in inflammatory gene expression at 20 h after cecal ligation and puncture was not due to resolution of the insult but rather was an immune dysfunction stage that was demonstrated by the inability of the animal to respond to a secondary external inflammatory stimulus. Removal of the injury source, ligated cecum, within 6 h of the initial insult resulted in increased survival, but not after 20 h of cecal ligation and puncture. We concluded that the therapeutic window for resolving sepsis is early after the initial insult and coincides with a stage of hyperinflammation that is followed by a condition of innate immune dysfunction in which reversion of the outcome is no longer possible.

Decay-accelerating factor 1 (Daf1) deficiency exacerbates xenobiotic-induced autoimmunity

Absence of decay‐accelerating factor 1 (Daf1) has been shown to enhance T‐cell responses and autoimmunity via increased expression of specific cytokines, most notably interferon (IFN)‐γ. To determine if Daf1 deficiency can exacerbate IFN‐γ‐dependent murine mercury‐induced autoimmunity (mHgIA), C57/BL6 Daf1+/+ and Daf1−/− mice were exposed to mercuric chloride (HgCl2) and examined for differences in cytokine expression, T‐cell activation and features of humoral autoimmunity. In the absence of Daf1, mHgIA was exacerbated, with increased serum immunoglobulin G (IgG), anti‐nuclear autoantibodies (ANAs) and anti‐chromatin autoantibodies. This aggravated response could not be explained by increased T‐cell activation but was associated with increased levels of IFN‐γ, interleukin (IL)‐2, IL‐4 and IL‐10 but not IL‐17 in Daf1‐deficient mice. Anti‐CD3/anti‐CD28 costimulation of Daf1−/− CD4+ T cells in vitro was also found to increase cytokine expression, but the profile was different from that of mHgIA, suggesting that the cytokine changes observed in Daf1 deficiency reflect a response to mercury. The role of Daf1 in influencing cytokine expression was further examined by stimulation of CD4+ T cells in the presence of anti‐CD3 and CD97, a molecular partner for Daf1. This resulted in increased IL‐10, decreased IL‐17 and IL‐21 and decreased IFN‐γ. These findings demonstrate that the absence of Daf1 exacerbates mHgIA, with changes in the profile of expressed cytokines. Interaction between Daf1 and its molecular partner CD97 was found to modify expression of mHgIA‐promoting cytokines, suggesting a possible approach for the suppression of overaggressive cytokine production in autoimmunity.