We focus on biochemistry and molecular biology of the human inflammatory response and its control.
Main achievements include the following discoveries:
- Potent activation of the stress response kinase PKR by short intragenic RNA elements, resulting in eIF2α phosphorylation, controls expression of the key inflammatory cytokine genes encoding immune interferon and tumor necrosis factor at mRNA translation and splicing, respectively, and is used more widely within the human genome, in particular, by the human globin genes;
- Pro-inflammatory signaling leading to a lethal inflammatory cytokine storm during infections is regulated - and can be contained - through a novel checkpoint, the homodimer interfaces of the principal costimulatory receptors. A therapeutic molecule is currently advancing in a Phase 3 clinical trial against polymicrobial sepsis. Novel molecules that protect broadly from lethal infections are being designed and researched.
Regulation of Inflammatory Cytokine Gene Expression through RNA Activators of PKR
Our lab discovered, within inflammatory cytokine genes, a novel class of intragenic elements that by evoking a cellular stress response, regulate expression of the gene carrying that element at either mRNA splicing or mRNA translation. Once activated by double-helical RNA, the interferon-inducible RNA-dependent stress protein kinase PKR phosphorylates translation initiation factor eIF2α, to inhibit translation, critical for coping with stress. We revealed short, 100-200 nucleotide elements within cellular genes that, once transcribed, fold into RNA structures that potently activate PKR in the vicinity of the RNA and thereby tightly regulate expression in cis. Intragenic RNA activators of PKR can (a) attenuate translation of the encoded mRNA by over an order of magnitude by activating PKR and inducing eIF2α phosphorylation, exemplified by the immune interferon gene (IFN-γ) that through this negative control, avoids hyperinflammation; or (b) potently enhance pre-mRNA splicing efficiency by activating PKR and inducing nuclear eIF2α phosphorylation, thus enabling efficient early-stage spliceosome assembly, exemplified by adult and fetal globin genes and tumor necrosis factor (TNF-α) that activates PKR through an RNA pseudoknot conserved from teleost fish to humans. Our work revealed a novel function for eIF2α phosphorylation in control of mRNA splicing that clarifies the need for PKR activation, extending its role from negative control of translation to positive control of splicing. These opposite outcomes considerably expand the scope of gene regulation enabled by these elements. Our findings explain why genes vital to survival that must be expressed highly efficiently, exemplified by IFN-γ and TNF-α, key mediators of protective immunity and the antitumor response, and the globin genes, indispensable for life, acquired intragenic RNA activators of PKR. Current research focuses on the characterization and role of intragenic RNA activators of PKR in regulating expression of the inflammatory response modulator, suppressor of cytokine signaling (SOCS3), and human immunodeficiency virus (HIV).
Inflammatory Cytokine Gene Regulation and Biodefense
Kaempfer Lab studied the molecular biology of cytokine gene expression from its outset. Early focus on regulation of inflammatory cytokine gene expression also allowed us to investigate how it is disturbed in diseases. This laid the foundation for our later work on superantigen toxins and cytokine storm. The Pentagon recruited Ray into biodefense research based on this expertise which provided essential tools. Currently, our biodefense research focuses on wound infections by multidrug-resistant organisms, a major challenge today, at the end of the antibiotics era. Based on detailed insight into molecular mechanisms of human inflammatory signaling, we are designing novel, broad-spectrum therapeutics that protect from lethal infections.
Control of Inflammatory Cytokine Storm Triggered by Infections
During severe bacterial or viral infections, disease and death are often caused by an overly strong immune response of the human host (‘cytokine storm’). Acute toxic shock is induced by superantigen toxins, a diverse set of proteins secreted by Gram-positive bacterial strains that overstimulate inflammatory gene expression by orders of magnitude. The need to protect from superantigen toxins led to our discoveries that the principal costimulatory receptor, CD28, and its coligand, B7-2 (CD86), previously thought to have only costimulatory function, are actually critical superantigen receptors. Binding of the superantigen into the homodimer interfaces of these costimulatory receptors triggers B7-2/CD28 costimulatory receptor engagement, leading to excessive pro-inflammatory signaling. This finding led to the design of receptor dimer interface mimetic peptides that block binding of superantigen and thus protect from death. It then turned out that such peptides will protect also from Gram-negative bacterial infections and polymicrobial sepsis. One such CD28 mimetic peptide is now advancing in a US Phase 3 clinical trial to protect from lethal necrotizing wound infections by flesh-eating bacteria. Based on fundamental insight into signaling by costimulatory receptors and their structure, we are designing and developing novel therapeutics that show even greater efficacy. These molecules are host-oriented therapeutics that target the human immune system itself, protecting thereby against a broad range of pathogens while avoiding emergence of resistance.
Relieving Tumor-induced Repression of the Human Inflammatory Response
A new project in the cancer field with promising pilot results. Tumor cells evade immune surveillance by silencing expression of human inflammatory genes that provide the natural response against cancer. Antibodies currently used to offset this immune suppression cause severe side effects in patients. We are developing a novel molecular approach that allows for relief of inflammatory silencing while maintaining a normal inflammatory response.
Collaborations with research teams in:
- Baltimore, Maryland, USA (animal models of live infection, protection from death)
- Ness Ziona, Israel (biodefense against priority pathogens)
- Strasbourg, France (structure of RNA elements)
- Amsterdam, Netherlands (HIV gene regulation)
- Logan, Utah, USA (protection from lethal virus infection)
- Magdeburg, Germany (control of inflammatory signaling, SOCS3)
We are currently recruiting MSc and PhD students!
Klepsch O, Namer LS, Kaempfer R, Dittrich A, Schaper F. Intragenic regulation of SOCS3 isoforms. Cell Commun Signal 2019, in final revision.
Kaempfer R, Namer LS, Osman F, Ilan L. Control of mRNA splicing by noncoding intragenic RNA elements that evoke a cellular stress response. Int J Biochem Cell Biol 2018 Dec;105:20-23. Link
Kaempfer R. Bacterial superantigen toxins, CD28, and drug development. Toxins 2018;10, 459 (6 pages). Link
Namer LS, Osman F, Banai Y, Masquida B, Jung R, Kaempfer R. An ancient pseudoknot in TNF-α pre-mRNA activates PKR, inducing eIF2α phosphorylation that potently enhances splicing. Cell Reports 2017 Jul 5;20(1):188-200. Link
Kaempfer R. Ribosome cycle emerges from DNA replication. Nat Rev Mol Cell Biol. 2017 Aug;18(8):470. Link
Ilan L, Osman F, Namer LS, Eliahu E, Cohen-Chalamish S, Ben-Asouli Y, Banai Y, Kaempfer R. PKR activation and eIF2α phosphorylation mediate human globin mRNA splicing at spliceosome assembly. Cell Research 2017 May;27(5):688-704. Link
Kaempfer R, Popugailo A, Levy R, Arad G, Hillman D, Rotfogel Z. Bacterial superantigen toxins induce a lethal cytokine storm by enhancing B7-2/CD28 costimulatory receptor engagement, a critical immune checkpoint. Receptors Clin Investig 2017;4(1). pii: e1500. Link
Levy R, Rotfogel Z, Hillman D, Popugailo A, Arad G, Supper E, Osman F, Kaempfer R. Superantigens hyperinduce inflammatory cytokines by enhancing the B7-2/CD28 costimulatory receptor interaction. Proc Natl Acad Sci USA 2016 Oct 18;113(42):E6437-E6446. Link
Ramachandran G, Kaempfer R, Chung CS, Shirvan A, Chahin AB, Palardy JE, Parejo NA, Chen Y, Whitford M, Arad G, Hillman D, Shemesh R, Blackwelder W, Ayala A, Cross AS, Opal SM. CD28 homodimer interface mimetic peptide acts as a preventive and therapeutic agent in models of severe bacterial sepsis and gram-negative bacterial peritonitis. J Infect Dis 2015 Mar 15;211(6):995-1003. Link
Bulger EM, Maier RV, Sperry J, Joshi M, Henry S, Moore FA, Moldawer LL, Demetriades D, Talving P, Schreiber M, Ham B, Cohen M, Opal S, Segalovich I, Maislin G, Kaempfer R, Shirvan A. A novel drug for treatment of necrotizing soft-tissue infections: A randomized clinical trial. JAMA Surg 2014 Jun;149(6):528-536. Link
Mirzoeva S, Paunesku T, Wanzer MB, Shirvan A, Kaempfer R, Woloschak GE, Small W Jr. Single administration of p2TA (AB103), a CD28 antagonist peptide, prevents inflammatory and thrombotic reactions and protects against gastrointestinal injury in total-body irradiated mice. PLoS One 2014 Jul 23;9(7):e101161. Link
Ramachandran G, Tulapurkar ME, Harris KM, Arad G, Shirvan A, Shemesh R, Detolla LJ, Benazzi C, Opal SM, Kaempfer R*, Cross AS*. A peptide antagonist of CD28 signaling attenuates toxic shock and necrotizing soft-tissue infection induced by Streptococcus pyogenes. J Infect Dis 2013 Jun 15;207(12):1869-1877. Link
Kaempfer R, Arad G, Levy R, Hillman D, Nasie I, Rotfogel Z. CD28: direct and critical receptor for superantigen toxins. Toxins 2013 Sep 9;5(9):1531-1542. Link
Arad G, Levy R, Nasie I, Hillman D, Rotfogel Z, Barash U, Supper E, Shpilka T, Minis A, Kaempfer R. Binding of superantigen toxins into the CD28 homodimer interface is essential for induction of cytokine genes that mediate lethal shock. PLoS Biol 2011 Sep;9(9):e1001149. Link
Cohen-Chalamish S, Hasson A, Weinberg D, Namer LS, Banai Y, Osman F, Kaempfer R. Dynamic refolding of IFN-gamma mRNA enables it to function as PKR activator and translation template. Nature Chemical Biology 2009 Dec;5(12):896-903. Link
Arad G, Hillman D, Levy R, Kaempfer R. Broad-spectrum immunity against superantigens is elicited in mice protected from lethal shock by a superantigen antagonist peptide. Immunol Lett 2004 Feb 15;91(2-3):141-145. Link
Kaempfer R. Peptide antagonists of superantigen toxins. Molec Diversity 2004;8(2):113-120. PubMed PMID: 15209162.
Kaempfer R, Arad G, Levy R, Hillman D. Defense against biologic warfare with superantigen toxins. Isr Med Assoc J 2002 Jul;4(7):520-523. Link
Ben-Asouli Y, Banai Y, Pel-Or Y, Shir A, Kaempfer R. Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR. Cell 2002 Jan 25;108(2):221-232. Link
Arad G, Levy R, Hillman D, Kaempfer R. Superantigen antagonist protects against lethal shock and defines a new domain for T-cell activation. Nature Medicine 2000 Apr;6(4):414-421. Link
Osman F, Jarrous N, Ben-Asouli Y, Kaempfer R. A cis-acting element in the 3'-untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR. Genes & Development 1999 Dec 15;13(24):3280-3293. Link