Inflammation is increasingly recognized as an important contributor to a host of CNS disorders; however, its regulation in the brain is not well delineated. Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing 3 (NLRP3) is a key component of the inflammasome complex, which also includes ASC (apoptotic speck-containing protein with a card) and procaspase-1. Inflammasome formation can be triggered by membrane P2X 7 R engagement leading to cleavage-induced maturation of caspase-1 and interleukin-1␤ (IL-1␤)/IL-18. This work shows that expression of the Nlrp3 gene was increased Ͼ100-fold in a cuprizone-induced demyelination and neuroinflammation model. Mice lacking the Nlrp3 gene (Nlrp3 Ϫ/Ϫ ) exhibited delayed neuroinflammation, demyelination, and oligodendrocyte loss in this model. These mice also showed reduced demyelination in the experimental autoimmune encephalomyelitis model of neuroinflammation. This outcome is also observed for casp1 Ϫ/Ϫ and IL-18 Ϫ/Ϫ mice, whereas IL-1␤ Ϫ/Ϫ mice were indistinguishable from wild-type controls, indicating that Nlrp3-mediated function is through caspase-1 and IL-18. Additional analyses revealed that, unlike the IL-1␤ Ϫ/Ϫ mice, which have been previously shown to show delayed remyelination, Nlrp3 Ϫ/Ϫ mice did not exhibit delayed remyelination. Interestingly, IL-18Ϫ/Ϫ mice showed enhanced remyelination, thus providing a possible compensatory mechanism for the lack of a remyelination defect in Nlrp3 Ϫ/Ϫ mice. These results suggest that NLRP3 plays an important role in a model of multiple sclerosis by exacerbating CNS inflammation, and this is partly mediated by caspase-1 and IL-18. Additionally, the therapeutic inhibition of IL-18 might decrease demyelination but enhance remyelination, which has broad implications for demyelinating diseases.

Outer surface proteins (Osp) A and C of the Lyme disease spirochete (Borrelia burgdorferi) are selectively produced and of functional significance in the tick vector and mammalian host, respectively. Some studies indicate a simple, reciprocal relationship where the signals and pathways that turn on ospC also turn off ospA. Other studies indicate a more complex regulation where many spirochetes produce both proteins and others produce one of the proteins or neither protein. Here, we have used flow cytometry to characterize ospA and ospC transcript and protein levels in individual bacterial cells grown in culture. The results support a simple, reciprocal model where, at the level of single cells, the transcription of ospC is linked to the repression of ospA. We also demonstrate that under conditions conducive for OspC production, spirochetes display an "all or none" response, with some cells displaying high levels of ospC transcription and others demonstrating little or no transcription. Despite the reciprocal regulation of ospA and ospC at the single-cell level, we propose that spirochetes display an array of phenotypes due to stochasticity in the pathways that regulate osp expression and the slow turnover of outer surface proteins.Borrelia burgdorferi, the spirochete responsible for Lyme disease, is transmitted by Ixodes ticks (3). B. burgdorferi persistently colonizes the gut of ticks. When infected ticks feed, the spirochetes multiply within the gut, migrate to the tick's salivary glands, and infect the vertebrate host (1). Within the feeding tick, the spirochetes alter the expression of many genes in preparation for transmission and infection of the new host (1, 26). B. burgdorferi outer surface proteins (Osp) A and C have served as a paradigm for understanding the regulation of bacterial gene expression within feeding ticks. In ticks, ospA is predominantly expressed before the blood meal, whereas ospC is induced during the blood meal (9,17,22,23). The functions of these two proteins are consistent with their pattern of expression, where OspA is required for colonizing the vector and OspC is required for infecting the host (12,19,29).Temperature, pH, and cell density act as signals for regulating the expression of ospA and -C in culture, and these signals are likely to play a role in the feeding tick as well (26). Proteomic and microarray studies with cultured spirochetes grown in "tick-like" (low temperature, high pH) or "host-like" (high temperature, low pH) conditions have led to the identification of large subsets of Borrelia proteins and genes with "OspAlike" or "OspC-like" patterns of expression (18,20). The bacterial signaling pathway regulating the expression of ospC and ospC-like genes has been characterized in some detail (6,7,14,24,27,28). The pathway is activated by a two-component system consisting of a sensor with a histidine kinase domain (HK2) and a cytoplasmic response regulator protein (Rrp2) (14, 27). Activated Rrp2, together with the alternative sigma factor RpoN, induces the expression of...

Lyme disease is caused by the tick-borne spirochete, Borrelia burgdorferi. It has been documented that B. burgdorferi form aggregates within ticks and during in vitro culture. However, Borrelia aggregates remain poorly characterized, and their functional significance is unknown. Here we have characterized Borrelia aggregates using microscopy and flow cytometry. Borrelia aggregation was temperature, pH, and growth phase dependent. Environmental conditions (high temperature, low pH, and high cell density) favorable for aggregation were similar to the conditions that increased the expression of B. burgdoferi genes, such as outer surface protein C (ospC), that are regulated by the RpoN/RpoS sigma factors. Experiments were conducted to determine if there is a relationship between aggregation and gene regulation through the RpoN/RpoS pathway. ospC Transcript levels were similar between aggregates and free cells. Moreover, no differences were observed in aggregate formation when null mutants of rpoS, rpoN, or ospC were compared to wild-type spirochetes. These results indicated that, despite the similar external signals that promoted aggregation and the RpoN/RpoS pathway, the two processes were not linked at the molecular level. The methods developed here to study B. burgdorferi aggregates will be useful for further studies on spirochete aggregates.

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