Lipoproteins, Not Lipopolysaccharide, Are the Key Mediators of the Proinflammatory Response Elicited by Heat-Killed Brucella abortus

Abstract

Inflammation is a hallmark of brucellosis. Although Brucella abortus, one of the disease’s etiologic agents, possesses cytokine-stimulatory properties, the mechanism by which this bacterium triggers a proinflammatory response is not known. We examined the mechanism whereby heat-killed B. abortus (HKBA), as well as its LPS, induces production of inflammatory cytokines in monocytes/macrophages. Polymyxin B, a specific inhibitor of LPS activity, did not inhibit the production of TNF-α- and IL-6-induced HKBA in the human monocytic cell line THP-1. HKBA induced the production of these cytokines in peritoneal macrophages of both C3H/HeJ and C3H/HeN mice, whereas B. abortus LPS only stimulated cells from C3H/HeN mice. Anti-TLR2 Ab, but not anti-TLR4 Ab, blocked HKBA-mediated TNF-α and IL-6 production in THP-1 cells. Because bacterial lipoproteins, a TLR2 ligand, have potent inherent stimulatory properties, we investigated the capacity of two B. abortus lipoproteins, outer membrane protein 19 (Omp19) and Omp16, to elicit a proinflammatory response. Lipidated (L)-Omp16 and L-Omp19, but not their unlipidated forms, induced the secretion of TNF-α, IL-6, IL-10, and IL-12 in a time- and dose-dependent fashion. Preincubation of THP-1 cells with anti-TLR2 Ab blocked L-Omp19-mediated TNF-α and IL-6 production. Together, these results entail a mechanism whereby B. abortus can stimulate cells from the innate immune system and induce cytokine-mediated inflammation in brucellosis. We submit that LPS is not the cause of inflammation in brucellosis; rather, lipoproteins of this organism trigger the production of proinflammatory cytokines, and TLR2 is involved in this process.

TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro

“…23 Different groups have investigated the contribution of TLRs in the host resistance to B. abortus infection. Studies using live B. abortus, 59 heat-killed B. abortus 40 or Brucella lipoproteins 86 demonstrated a role for TLRs. Indeed, the production of IL-12 is TLR2-dependent in heat-killed B. abortus-stimulated DCs.…”

A conserved and immunodominant lipoprotein of Francisella tularensis is proinflammatory but not essential for virulence

“…The proinflammatory nature of lipoproteins has been attributed to their attached lipid, and not the protein itself. Lipoproteins from Borrelia burgdorferi and Brucella abortus are able to induce inflammation in host cells only after they have been processed and modified with lipid [41,42,44]. However, lipoproteins display significant functional diversity that likely can be attributed to the variability in their associated protein moieties.…”

“…This vector then was sent to the Northeast Biodefense Center Protein Expression Core (Wadsworth Center, Albany, NY) for purification of LpnA by Triton X-114 phase partitioning [56]. This method has been previously employed to isolate functional, acylated proteins [44,56]. Potential contaminating E. coli LPS was removed from the LpnA preparations using Endotoxin Removal Affinity Resin (Cape Cod Associates, East Falmouth, MA).…”

Peptidoglycan-associated lipoprotein ofAggregatibacter actinomycetemcomitansinduces apoptosis and production of proinflammatory cytokines via TLR2 in murine macrophages RAW 264.7in vitro

“…This was seen for each tested AaPAL concentration (0.5, 1, and 2 µg/mL) at each time point (6, 24, and 48 h), although the production of IL-6 increased linearly with time with low (0.5 and 1.0 µg/mL) AaPAL concentrations, and the peak value for TNF-α production decreased toward 48 h. Higher TNF-α production compared to IL-6 has also been reported from the human macrophage cell line THP-1 stimulated with B. abortus PAL [12] and H. influenzae PAL-stimulated human macrophages [39]. Yet, stimulation of mouse macrophages with E. coli PAL induces comparable levels of TNF-a and IL-6 production [22].…”

“…For example, Escherichia coli PAL is released into the bloodstream and has a pathogenic role in Gram-negative sepsis in a mouse model [21], and a form of E. coli PAL which is released into human serum in vitro is able to induce pro-inflammatory cytokine production by mouse macrophages [22]. Furthermore, a Hemophilus ducrey PAL-deficient mutant strain was less virulent than its parental strain in a human infection model [23], and B. abortus PAL is one of the lipoproteins which causes production of pro-inflammatory cytokines in brucellosis [12]. Klebsiella pneumoniae PAL protects bacterial cells from serum killing, as well as phagocytosis, and is an important virulence factor in a mouse infection model [24].…”