NLRP12 Inflammasome Expression in the Rat Brain in Response to LPS during Morphine Tolerance

Morphine, an effective but addictive analgesic, can profoundly affect the inflammatory response to pathogens, and long-term use can result in morphine tolerance. Inflammasomes are protein complexes involved in the inflammatory response. The nucleotide-binding oligomerization domain-like receptor (NLR) Family Pyrin Domain Containing (NLRP) 12 (NLRP12) inflammasome has been reported to have anti-inflammatory activity. In this study, we examined the expression of NLRP12 inflammasome related genes in the adult F344 rat brain in response to the bacterial endotoxin lipopolysaccharide (LPS) in the presence and absence of morphine tolerance. Morphine tolerance was elicited using the 2 + 4 morphine-pelleting protocol. On Day 1, the rats were pelleted subcutaneously with 2 pellets of morphine (75 mg/pellet) or a placebo; on Days 2 and 4 pellets were given. On Day 5, the animals were randomly assigned to receive either 250 µg/kg LPS or saline (i.p.). The expression of 84 inflammasome related genes in the rat brain was examined using a Ploymerase Chain Reaction (PCR) array. In response to LPS, there was a significant increase in the expression of the pro-inflammatory cytokine/chemokine genes interleukin-1 beta (Il-1β), interleukin-6 (Il-6), C-C motif chemokine ligand 2 (Ccl2), C-C motif chemokine ligand 7 (Ccl7), C-X-C motif chemokine ligand 1 (Cxcl1), and C-X-C motif chemokine ligand 3 (Cxcl3) and a significant decrease in the anti-inflammatory NLRP12 gene in both morphine-tolerant and placebo-control rats compared to saline-treated rats, although the changes were greater in the placebo-control animals. The Library of Integrated Network-Based Cellular Signatures’ (LINCS) connectivity map was used to analyze the list of affected genes to identify potential targets associated with the interactions of LPS and morphine tolerance. Our data indicate that, in the morphine tolerant state, the expression of NLRP12 and its related genes is altered in response to LPS and that the Vacuolar protein-sorting-associated protein 28 (VPS28), which is involved in the transport and sorting of proteins into sub-cellular vesicles, may be the key regulator of these alterations.


Introduction
Morphine is a potent analgesic that is widely used clinically for pain management. However, long-term use of morphine can lead to morphine tolerance and addiction [1]. In addition, morphine can profoundly and detrimentally affect the body's immune system, at both the cellular and molecular levels. Morphine suppresses lymphocyte trafficking; decreases natural killer cell activity; inhibits the production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and

PCR Array Data Analysis
The expression of each gene was normalized to housekeeping genes and calculated using the ∆∆Ct method. The threshold and baseline values were set manually, and the resulting threshold cycle values (Ct) were analyzed using the PCR array data analysis template supplied on the manufacturer's website [25]. The mean fold change in mRNA expression from 3 to 5 biological replicates was considered significant at p < 0.05. The gene profile signatures were created for every two groups compared.

LINCS Analysis
The differentially expressed genes were input into the Query App (apps.lincscloud.org/query), as described previously [26,27]. Based on the LINCS database, LincsCloud utilized gene profile signatures generated from the PCR array to generate a report, including probability outcomes in terms of gene knockdown effects and drug mimics. The scores given in the report evaluated how much a Brain Sci. 2017, 7, 14 4 of 14 particular set of gene regulation features (named pertubagens) was likely to be connected with the genes listed in the LINCS report. Positive readings in the Consensus Knockdown Connections in the report indicate that knockdown of the genes listed in the LINCS report would match the gene changes input into the Query App, and thus the genes with high scores represent potential target genes for the experimental treatment.

Expression Profile of Inflammasome-Related Genes Following an LPS Challenge, with and without Morphine Tolerance
Alterations in gene expression were measured in rats challenged with LPS, with and without morphine tolerance, using a PCR array containing 84 genes related to inflammasome activation and function. NLRP12 expression was significantly decreased in response to LPS in both the morphine-tolerant (morphine-tolerant + LPS) and control (placebo-control + LPS) rats, compared to the rats given saline (morphine-tolerant + saline and placebo-control + saline) ( Figure 1, Table 1). However, the decrease was greater in the placebo-control rats (−7.3 fold; p < 0.01) than in the morphine-tolerant rats (−4.5 fold; p < 0.05).
Brain Sci. 2017, 7, 14 4 of 15 much a particular set of gene regulation features (named pertubagens) was likely to be connected with the genes listed in the LINCS report. Positive readings in the Consensus Knockdown Connections in the report indicate that knockdown of the genes listed in the LINCS report would match the gene changes input into the Query App, and thus the genes with high scores represent potential target genes for the experimental treatment.

Expression Profile of Inflammasome-Related Genes Following an LPS Challenge, with and without Morphine Tolerance
Alterations in gene expression were measured in rats challenged with LPS, with and without morphine tolerance, using a PCR array containing 84 genes related to inflammasome activation and function. NLRP12 expression was significantly decreased in response to LPS in both the morphinetolerant (morphine-tolerant + LPS) and control (placebo-control + LPS) rats, compared to the rats given saline (morphine-tolerant + saline and placebo-control + saline) ( Figure 1, Table 1). However, the decrease was greater in the placebo-control rats (−7.3 fold; p < 0.01) than in the morphine-tolerant rats (−4.5 fold; p < 0.05).

Figure 1.
Inflammasome-related gene expression in the rat brain in response to lipopolysaccharide (LPS), with and without morphine tolerance. The expression of the inflammasome-related NOD-like receptor (NLR) genes (Naip2, Nlrp12, Nlrp5, Nlrc4, Nlrp1a, Nlrp3, Nlrp6, And Nlrx1) in the brains of rats given an i.p. injection of either 250 µg/kg LPS or saline, with and without morphine tolerance (n = 3-5 rats per group), was determined using a PCR array. Data were calculated using the ΔΔCT method, relative to the control group (placebo-control +saline), and are represented as a fold change. * p < 0.05, ** p < 0.01. Naip2: NLR family, apoptosis inhibitory protein 6  Inflammasome-related gene expression in the rat brain in response to lipopolysaccharide (LPS), with and without morphine tolerance. The expression of the inflammasome-related NOD-like receptor (NLR) genes (Naip2, Nlrp12, Nlrp5, Nlrc4, Nlrp1a, Nlrp3, Nlrp6, And Nlrx1) in the brains of rats given an i.p. injection of either 250 µg/kg LPS or saline, with and without morphine tolerance (n = 3-5 rats per group), was determined using a PCR array. Data were calculated using the ∆∆CT method, relative to the control group (placebo-control +saline), and are represented as a fold change. * p < 0.05, ** p < 0.01. Naip2: NLR family, apoptosis inhibitory protein 6 Table 1. Expression profile of inflammasomes and NLR genes in the rat brain in response to lipopolysaccharides (LPS), with and without morphine tolerance.

Expression Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, ith and Without Morphine Tolerance
With a few exceptions, there were no significant changes in expression of the downstream gnaling genes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or e control animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + line and placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), important regulator gene involved in the downstream effects of inflammasomes, was significantly creased in response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p 0.01) groups, compared to the rats given saline ( Figure 2, Table 2). In addition, NF-Kappa-B hibitor Alpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS orphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) (Figure 2, Table  . Figure 2. Inflammasome-related downstream gene expression in the rat brain in response to Lipopolysaccharides (LPS), with and without morphine tolerance. The expression of the inflammasome-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and NF-Kappa-B Inhibitor Alpha (Nfkbia) in the brains of rats given an i.p. injection of either 250 µg/kg LPS or saline, with and without morphine tolerance (n = 3-5 rats per group), was determined using a Polymerase Chain Reaction (PCR) array. The data were calculated using the ΔΔCT method relative to the control group (placebo-control + saline) and are represented as fold change.

Expression Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, and Without Morphine Tolerance
With a few exceptions, there were no significant changes in expression of the downstream aling genes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or ontrol animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + e and placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), portant regulator gene involved in the downstream effects of inflammasomes, was significantly ased in response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p 1) groups, compared to the rats given saline ( Figure 2, Table 2). In addition, NF-Kappa-B bitor Alpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS rphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) ( Figure 2, Table   Figure 2. Inflammasome-related downstream gene expression in the rat brain in response to Lipopolysaccharides (LPS), with and without morphine tolerance. The expression of the inflammasome-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and NF-Kappa-B Inhibitor Alpha (Nfkbia) in the brains of rats given an i.p. injection of either 250 µg/kg LPS or saline, with and without morphine tolerance (n = 3-5 rats per group), was determined using a Polymerase Chain Reaction (PCR) array. The data were calculated using the ΔΔCT method relative to the control group (placebo-control + saline) and are represented as fold change.

Expression Profile of Inflammasome-Related Down With and Without Morphine Tolerance
With a few exceptions, there were no signi signaling genes in response to LPS in either the mo the control animals (placebo-control + LPS), comp saline and placebo-control + saline) ( Table 1). How an important regulator gene involved in the downs increased in response to LPS in both the morphine < 0.01) groups, compared to the rats given salin Inhibitor Alpha (Nfkbia), an inhibitor protein of (morphine-tolerant + LPS, 3.4 fold, p < 0.01; placeb 2).

Expression Profile of Inflammasome-Related Downst With and Without Morphine Tolerance
With a few exceptions, there were no signific signaling genes in response to LPS in either the mor the control animals (placebo-control + LPS), compar saline and placebo-control + saline) ( Table 1). Howev an important regulator gene involved in the downstr increased in response to LPS in both the morphine-t < 0.01) groups, compared to the rats given saline Inhibitor Alpha (Nfkbia), an inhibitor protein of N (morphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo 2). 1. Expression profile of inflammasomes and NLR genes in the rat brain in response to lysaccharides (LPS), with and without morphine tolerance. sion Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, ithout Morphine Tolerance a few exceptions, there were no significant changes in expression of the downstream enes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), nt regulator gene involved in the downstream effects of inflammasomes, was significantly n response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p ups, compared to the rats given saline ( Figure 2, Table 2). In addition, NF-Kappa-B lpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS -tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) ( Figure 2, Table   2. Inflammasome-related downstream gene expression in the rat brain in response to lysaccharides (LPS), with and without morphine tolerance. The expression of the masome-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and ppa-B Inhibitor Alpha (Nfkbia) in the brains of rats given an i.p. injection of either 250 µg/kg saline, with and without morphine tolerance (n = 3-5 rats per group), was determined using a erase Chain Reaction (PCR) array. The data were calculated using the ΔΔCT method relative control group (placebo-control + saline) and are represented as fold change. Expression profile of inflammasomes and NLR genes in the rat brain in response to saccharides (LPS), with and without morphine tolerance. n Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, hout Morphine Tolerance few exceptions, there were no significant changes in expression of the downstream es in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or nimals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + acebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), regulator gene involved in the downstream effects of inflammasomes, was significantly response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p ps, compared to the rats given saline ( Figure 2, Table 2). In addition, NF-Kappa-B ha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS lerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) ( Figure 2, Table   . Inflammasome-related downstream gene expression in the rat brain in response to saccharides (LPS), with and without morphine tolerance. The expression of the asome-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and a-B Inhibitor Alpha (Nfkbia) in the brains of rats given an i.p. injection of either 250 µg/kg line, with and without morphine tolerance (n = 3-5 rats per group), was determined using a ase Chain Reaction (PCR) array. The data were calculated using the ΔΔCT method relative ntrol group (placebo-control + saline) and are represented as fold change. xpression profile of inflammasomes and NLR genes in the rat brain in response to charides (LPS), with and without morphine tolerance.

rofile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, ut Morphine Tolerance
w exceptions, there were no significant changes in expression of the downstream in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or als (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + ebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), gulator gene involved in the downstream effects of inflammasomes, was significantly ponse to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p , compared to the rats given saline ( Figure 2, Table 2). In addition, NF-Kappa-B a (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS rant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) ( Figure 2, Table   nflammasome-related downstream gene expression in the rat brain in response to ccharides (LPS), with and without morphine tolerance. The expression of the me-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and B Inhibitor Alpha (Nfkbia) in the brains of rats given an i.p. injection of either 250 µg/kg e, with and without morphine tolerance (n = 3-5 rats per group), was determined using a Chain Reaction (PCR) array. The data were calculated using the ΔΔCT method relative ol group (placebo-control + saline) and are represented as fold change.

Expression Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, With and Without Morphine Tolerance
With a few exceptions, there were no significant changes in expression of the downstream signaling genes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or the control animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + saline and placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), an important regulator gene involved in the downstream effects of inflammasomes, was significantly increased in response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p < 0.01) groups, compared to the rats given saline ( Figure 2, Table 2). In addition, NF-Kappa-B Inhibitor Alpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS (morphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) ( Figure 2, Table 2).

Expression Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, With and Without Morphine Tolerance
With a few exceptions, there were no significant changes in expression of the downstream signaling genes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or the control animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + saline and placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), an important regulator gene involved in the downstream effects of inflammasomes, was significantly increased in response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p < 0.01) groups, compared to the rats given saline ( Figure 2, Table 2). In addition, NF-Kappa-B Inhibitor Alpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS (morphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) ( Figure 2, Table  2). Inflammasome-related downstream gene expression in the rat brain in response to Lipopolysaccharides (LPS), with and without morphine tolerance. The expression of the inflammasome-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and NF-Kappa-B Inhibitor Alpha (Nfkbia) in the brains of rats given an i.p. injection of either 250 µg/kg LPS or saline, with and without morphine tolerance (n = 3-5 rats per group), was determined using a Polymerase Chain Reaction (PCR) array. The data were calculated using the ΔΔCT method relative to the control group (placebo-control + saline) and are represented as fold change. Inflammasome-related downstream gene expression in the rat brain in response to Lipopolysaccharides (LPS), with and without morphine tolerance. The expression of the inflammasome-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and NF-Kappa-B Inhibitor Alpha (Nfkbia) in the brains of rats given an i.p. injection of either 250 µg/kg LPS or saline, with and without morphine tolerance (n = 3-5 rats per group), was determined using a Polymerase Chain Reaction (PCR) array. The data were calculated using the ∆∆CT method relative to the control group (placebo-control + saline) and are represented as fold change. Table 2. Expression profile of inflammasome-related downstream signaling genes in the rat brain in response to lipopolysaccharides (LPS), with and without morphine tolerance. Full name of the genes were provided in Table A1.

Expression Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, With and Without Morphine Tolerance
With a few exceptions, there were no significant changes in expression of the downstream signaling genes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or the control animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + saline and placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), an important regulator gene involved in the downstream effects of inflammasomes, was significantly increased in response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p < 0.01) groups, compared to the rats given saline (Figure 2, Table 2). In addition, NF-Kappa-B Inhibitor Alpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS (morphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) ( Figure 2, Table  2).

Expression Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, h and Without Morphine Tolerance
With a few exceptions, there were no significant changes in expression of the downstream naling genes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or control animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + ne and placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), important regulator gene involved in the downstream effects of inflammasomes, was significantly reased in response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p .01) groups, compared to the rats given saline (Figure 2, Table 2). In addition, NF-Kappa-B ibitor Alpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS rphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) (Figure 2, Table

Expression Profile of Inflammasome-Related Down With and Without Morphine Tolerance
With a few exceptions, there were no signi signaling genes in response to LPS in either the m the control animals (placebo-control + LPS), comp saline and placebo-control + saline) ( Table 1). How an important regulator gene involved in the down increased in response to LPS in both the morphine < 0.01) groups, compared to the rats given sali Inhibitor Alpha (Nfkbia), an inhibitor protein of (morphine-tolerant + LPS, 3.4 fold, p < 0.01; placeb 2).

Expression Profile of Inflammasome-Related Downst With and Without Morphine Tolerance
With a few exceptions, there were no signific signaling genes in response to LPS in either the mor the control animals (placebo-control + LPS), compar saline and placebo-control + saline) ( Table 1). Howev an important regulator gene involved in the downstr increased in response to LPS in both the morphine-t < 0.01) groups, compared to the rats given saline Inhibitor Alpha (Nfkbia), an inhibitor protein of N (morphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo 2). 1. Expression profile of inflammasomes and NLR genes in the rat brain in response to lysaccharides (LPS), with and without morphine tolerance.   (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), nt regulator gene involved in the downstream effects of inflammasomes, was significantly n response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p ups, compared to the rats given saline (Figure 2, Table 2). In addition, NF-Kappa-B lpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS -tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) (Figure 2, Table   : 6-10 fold increase;

, 14 5 of 15
Expression profile of inflammasomes and NLR genes in the rat brain in response to saccharides (LPS), with and without morphine tolerance. n Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, hout Morphine Tolerance few exceptions, there were no significant changes in expression of the downstream es in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or nimals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + acebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), regulator gene involved in the downstream effects of inflammasomes, was significantly response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p ps, compared to the rats given saline (Figure 2, Table 2). In addition, NF-Kappa-B ha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS lerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) (Figure 2, Table   : 11-30 fold increase; xpression profile of inflammasomes and NLR genes in the rat brain in response to charides (LPS), with and without morphine tolerance.

Expression Profile of Inflammasome-Related Chemokine and Cytokine Genes after an LPS Challenge, with and without Morphine Tolerance
Cytokine and chemokine gene expression in response to LPS was greater in the control rats (placebo-control + LPS) compared to the morphine-tolerant animals (morphine-tolerant + LPS) ( Table 3). The cytokines IL-1β and IL-6 were significantly increased 7-and 12-fold, respectively (p < 0.01-0.001), in the control rats given LPS (placebo-control + LPS), whereas in the morphine-tolerant group (morphine-tolerant + LPS) the fold changes were not statistically significant (3-and 7-fold, respectively) compared to the rats given saline (Figure 3, Table 3). Table 3. Expression profile of the cytokine and chemokine genes in the rat brain in response to LPS, with and without morphine tolerance.

Expression Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, ith and Without Morphine Tolerance
With a few exceptions, there were no significant changes in expression of the downstream gnaling genes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or e control animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + line and placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), important regulator gene involved in the downstream effects of inflammasomes, was significantly creased in response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p 0.01) groups, compared to the rats given saline (Figure 2, Table 2). In addition, NF-Kappa-B hibitor Alpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS orphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) (Figure 2, Table .

Expression Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, and Without Morphine Tolerance
With a few exceptions, there were no significant changes in expression of the downstream aling genes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or ontrol animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + e and placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), portant regulator gene involved in the downstream effects of inflammasomes, was significantly ased in response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p 1) groups, compared to the rats given saline (Figure 2, Table 2). In addition, NF-Kappa-B bitor Alpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS rphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) (Figure 2, Table   Figure 2. Inflammasome-related downstream gene expression in the rat brain in response to Lipopolysaccharides (LPS), with and without morphine tolerance. The expression of the inflammasome-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and

Expression Profile of Inflammasome-Related Down With and Without Morphine Tolerance
With a few exceptions, there were no signi signaling genes in response to LPS in either the mo the control animals (placebo-control + LPS), comp saline and placebo-control + saline) ( Table 1). How an important regulator gene involved in the downs increased in response to LPS in both the morphine < 0.01) groups, compared to the rats given salin Inhibitor Alpha (Nfkbia), an inhibitor protein of (morphine-tolerant + LPS, 3.4 fold, p < 0.01; placeb 2).

Expression Profile of Inflammasome-Related Downst With and Without Morphine Tolerance
With a few exceptions, there were no signific signaling genes in response to LPS in either the mor the control animals (placebo-control + LPS), compar saline and placebo-control + saline) ( Table 1). Howev an important regulator gene involved in the downstr increased in response to LPS in both the morphine-t < 0.01) groups, compared to the rats given saline Inhibitor Alpha (Nfkbia), an inhibitor protein of N (morphine-tolerant + LPS, 3.4 fold, p < 0.01; placebo 2). 1. Expression profile of inflammasomes and NLR genes in the rat brain in response to lysaccharides (LPS), with and without morphine tolerance. sion Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, ithout Morphine Tolerance a few exceptions, there were no significant changes in expression of the downstream enes in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or animals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + placebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), nt regulator gene involved in the downstream effects of inflammasomes, was significantly n response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p ups, compared to the rats given saline (Figure 2, Table 2). In addition, NF-Kappa-B lpha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS -tolerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) (Figure 2, Table   2. Inflammasome-related downstream gene expression in the rat brain in response to lysaccharides (LPS), with and without morphine tolerance. The expression of the masome-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and Expression profile of inflammasomes and NLR genes in the rat brain in response to saccharides (LPS), with and without morphine tolerance. n Profile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, hout Morphine Tolerance few exceptions, there were no significant changes in expression of the downstream es in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or nimals (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + acebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), regulator gene involved in the downstream effects of inflammasomes, was significantly response to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p ps, compared to the rats given saline (Figure 2, Table 2). In addition, NF-Kappa-B ha (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS lerant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) (Figure 2, Table   . Inflammasome-related downstream gene expression in the rat brain in response to saccharides (LPS), with and without morphine tolerance. The expression of the asome-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and xpression profile of inflammasomes and NLR genes in the rat brain in response to charides (LPS), with and without morphine tolerance. rofile of Inflammasome-Related Downstream Signaling Genes Following an LPS Challenge, ut Morphine Tolerance w exceptions, there were no significant changes in expression of the downstream in response to LPS in either the morphine-tolerant rats (morphine-tolerant + LPS) or als (placebo-control + LPS), compared to the rats given saline (morphine-tolerant + ebo-control + saline) (Table 1). However, Baculoviral IAP Repeat-Containing 3 (Birc3), gulator gene involved in the downstream effects of inflammasomes, was significantly ponse to LPS in both the morphine-tolerant (21.5 fold; p < 0.05) and control (21 fold; p , compared to the rats given saline (Figure 2, Table 2). In addition, NF-Kappa-B a (Nfkbia), an inhibitor protein of NF-κB, was increased in both groups given LPS rant + LPS, 3.4 fold, p < 0.01; placebo-control + LPS, 3.9 fold, p < 0.01) (Figure 2, Table   nflammasome-related downstream gene expression in the rat brain in response to ccharides (LPS), with and without morphine tolerance. The expression of the me-related downstream signaling genes Baculoviral IAP Repeat-Containing 3 (Birc3) and  . Cytokine gene expression in the rat brain in response to lipopolysaccharides (LPS), with and without morphine tolerance. Gene expression of interleukins Interleukin (Il)-1β, Il-6, Il-12a, Il-12b, Il-18, and Il-33 in the brains of rats, with and without morphine tolerance, following an i.p. injection of either 250 µg/kg LPS or saline (n = 3-5 rats per group) was determined using a Polymerase Chain Reaction (PCR) array. Data were calculated using the ∆∆CT method relative to the control group (placebo-control + saline) and are represented as a fold change. * p < 0.05, ** p < 0.01, *** p < 0.001 Similarly, Ccl2, Ccl7, Cxcl1, and Cxcl3 chemokine expression was significantly increased 24-, 11-, 37-, and 8-fold, respectively (p < 0.01-0.001), in response to LPS in the control rats (placebo-control + Brain Sci. 2017, 7, 14 8 of 14 LPS), whereas in the morphine-tolerant (morphine-tolerant + LPS) group the fold changes (9-, 7-, 14-, and 3-fold, respectively) were not statistically significant ( Figure 4, Table 3). Figure 3. Cytokine gene expression in the rat brain in response to lipopolysaccharides (LPS), with and without morphine tolerance. Gene expression of interleukins Interleukin (Il)-1β, Il-6, Il-12a, Il-12b, Il-18, and Il-33 in the brains of rats, with and without morphine tolerance, following an i.p. injection of either 250 µg/kg LPS or saline (n = 3-5 rats per group) was determined using a Polymerase Chain Reaction (PCR) array. Data were calculated using the ΔΔCT method relative to the control group (placebo-control + saline) and are represented as a fold change. * p < 0.05, ** p < 0.01, *** p < 0.001 Figure 4. Chemokine gene expression in the rat brain in response to Lipopolysaccharides (LPS), with and without morphine tolerance. Gene expression of the chemokines C-C motif chemokine ligand (Ccl)2, Ccl5, Ccl7, Ccl11, Ccl12, C-X-C motif chemokine ligand (Cxcl)1, and Cxcl3 in the brains of rats with and without morphine tolerance, following an i.p. injection of either 250 µg/kg LPS or saline (n = 3-5 rats per group), was determined using a Polymerase Chain Reaction (PCR) array. Data were calculated using the ΔΔCT method relative to the control group (placebo-control + saline) and are represented as a fold change. * p < 0.05, ** p < 0.01, *** p < 0.001

LINCS Analysis of the Differentially Expressed Genes
Differentially expressed genes in the morphine-tolerant + saline versus morphine-tolerant + LPS rats and in the placebo-control + saline versus placebo-control + LPS rats as well as gene changes in rats the placebo-control + saline versus morphine-tolerant + saline rats were input into the Query App . Chemokine gene expression in the rat brain in response to Lipopolysaccharides (LPS), with and without morphine tolerance. Gene expression of the chemokines C-C motif chemokine ligand (Ccl)2, Ccl5, Ccl7, Ccl11, Ccl12, C-X-C motif chemokine ligand (Cxcl)1, and Cxcl3 in the brains of rats with and without morphine tolerance, following an i.p. injection of either 250 µg/kg LPS or saline (n = 3-5 rats per group), was determined using a Polymerase Chain Reaction (PCR) array. Data were calculated using the ∆∆CT method relative to the control group (placebo-control + saline) and are represented as a fold change. * p < 0.05, ** p < 0.01, *** p < 0.001

LINCS Analysis of the Differentially Expressed Genes
Differentially expressed genes in the morphine-tolerant + saline versus morphine-tolerant + LPS rats and in the placebo-control + saline versus placebo-control + LPS rats as well as gene changes in rats the placebo-control + saline versus morphine-tolerant + saline rats were input into the Query App (apps.lincscloud.org/query). One report was generated by LINCS for each set of genes input. The genes with a high positive score in Consensus Knockdown Connections were considered to be potential gene targets (Table 4). In the placebo-control + saline versus placebo-control + LPS report, VPS28, protein C receptor (PROCR), and charged multivesicular body protein 2A (CHMP2A) were the top three with the highest scores. VPS28 is an ESCRT-I complex subunit that functions in the transport and sorting of proteins into sub-cellular vesicles. PROCR is endothelial protein C receptor involved in the blood coagulation pathway. CHMP2A is a component of the endosomal sorting complex required for transport III, which is involved in the degradation of surface receptor proteins and the formation of endocytic multivesicular bodies.
In the placebo-control + saline versus morphine-tolerant + saline report, SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily e, member 1 (SMARCE1), aryl-hydrocarbon receptor repressor (AHRR), and glutathione peroxidase 7 (GPX7) were the most likely targets predicted by LINCS. SMARCE1 is required for the transcriptional activation of genes normally repressed by chromatin. AHRR mediates dioxin toxicity and is involved in the regulation of cell growth and differentiation. GPX7 is involved with cellular senescence and insulin secretion.
In the morphine-tolerant + saline versus morphine-tolerant + LPS group, AHR (aryl hydrocarbon receptor), UBE2L6 (ubiquitin-conjugating enzyme E2L 6), and PAFAH1B3 (platelet-activating factor acetylhydrolase 1b, Catalytic Subunit 3) were the top three candidates. AHR is involved in the regulation of biological responses to planar aromatic hydrocarbons; UBE2L6 targets abnormal or short-lived proteins for degradation; and PAFAH1B3 functions in brain development and is associated with mental retardation, ataxia, and atrophy of the brain. The predicted potential targets in each group were different from those in other groups, both in targets and their possibility rankings. VPS28 was the only one that appeared in both the Top 100 lists of placebo-control + saline versus placebo-control + LPS (No. 1 in Table 4) and morphine-tolerant + saline versus morphine-tolerant + LPS (No. 4 in Table 4). Table 4 shows the top three potential target genes from each set of gene comparisons. There was no similarity in the gene rankings in the three sets of gene comparisons.  Table A2.

Discussion
Inflammasomes recognize a variety of pathogen-associated molecular patterns (PAMPs), including endotoxins such as LPS. Depending on the NLR proteins that constitute inflammasomes, an inflamasome can be pro-inflammatory or anti-inflammatory in nature [28]. For pro-inflammatory inflammasomes such as NLRP3, in vitro studies have shown that the activation and release of pro-inflammatory cytokines requires two signals. The first signal, triggered by PAMPs, leads to the activation of inflammasomes, which then provide the second signal. The activated inflammasomes, through caspase 1 activation, promote the production of the pro-inflammatory cytokines, IL-1β and IL-18. However, the signaling pathways during infection or inflammation in vivo are not yet completely defined [29], and the characteristics of anti-inflammatory inflammasomes such as NLRP12 have not yet been extensively investigated. To our knowledge, our study is one of the first to report the modulation of NLRP12 expression in response to LPS and morphine in vivo.
Recently, NLRP12 was designated as an anti-inflammatory NLR inflammasome protein. It is believed to be a negative regulator of the NF-κB signaling pathway by inhibiting downstream signaling of TLRs, particularly IRAK-1 [28,30]. Our results showed that NLRP12 expression decreased in the brains of both the control (placebo-control + LPS) and morphine-tolerant (morphine-tolerant + LPS) rats in response to an LPS challenge, indicating that one of the mechanisms by which LPS induces an inflammatory response is by inhibiting the expression of the anti-inflammatory NLRP12 inflammasome.
Although NLRP12 expression was decreased in both groups given LPS, the decrease was significantly greater in the control rats than in the morphine-tolerant rats, which suggests that the LPS-induced NLRP12 decrease is countered during morphine tolerance. Hence morphine may also modulate NLRP12 activity, directly or indirectly, thereby exerting its immunosuppressive effects and opposing the LPS-induced decrease in NLRP12 in the presence of morphine tolerance.
Birc3, a downstream regulator of inflammasome signaling, is essential for controlling the synthesis of cytokines and chemokines in the inflammatory Mapk and NF-κB pathways. It is also required for inflammasome activation, subsequent caspase 1 activity, and IL-1β formation [31]. In our study, Birc3 expression was significantly increased in both the placebo-control and morphine-tolerant rats in response to LPS, indicating that LPS is able to induce an inflammatory response through Birc3 activity, following inhibition of the anti-inflammatory NLRP12. However, in response to LPS, Birc3 expression in the morphine-tolerant rats did not change in comparison to the placebo-control rats. This indicates that morphine may not be able to modulate Birc3 expression, and therefore there is no change, increase or decrease, in its expression in the morphine tolerant state.
NF-κB is important in the activation of inflammatory mediators such as cytokines and chemokines [16]. Previous studies have reported that NLRP12 inhibits both canonical and non-canonical NF-κB activation [16,28] and that Nfkbia, a downstream regulator of inflammasomes, inhibits the activity of dimeric NF-κB/Rel complexes [32]. In our study, Nfkbia was significantly increased in response to LPS in both the placebo-control and morphine-tolerant rats. During an inflammatory response, one would expect the expression and activity of a positive regulator of inflammation to be increased, whereas that of a negative regulator would be decreased. However, from a physiological standpoint, there is a constant effort to balance pro-and anti-inflammatory activity [33,34]. This quest to balance the pro-and anti-inflammatory responses could be one of the reasons for an increase in Nfkbia, which is known to inhibit the activity of the pro-inflammatory dimeric NF-κB/REL, thus reducing the production of pro-inflammatory mediators.
As expected, we found that the expression of pro-inflammatory cytokines (IL-1β and IL-6) and chemokines (Ccl2, Ccl7, Cxcl1, and Cxcl3) was increased in response to LPS in the placebo-control rats [35][36][37]. In the morphine-tolerant rats, however, the LPS-induced cytokine and chemokine expression levels were lower, suggesting that NLRP12 inhibition in response to LPS may be opposed or subdued in the morphine tolerant state.
In a previous study, we observed that, in peripheral immune organs such as the spleen, NLRP3 expression, but not NLRP12 expression, is altered in response to LPS, with and without morphine tolerance [38], suggesting that the mechanism(s) of inflammasome activation in response to pathogens may be different in peripheral immune organs, compared to the central nervous system. During morphine tolerance, the LPS-induced expression of NLRP3, as well as that of cytokines and chemokines, is reduced in comparison to the placebo-control rats given LPS [38]. These observations are consistent with previous studies showing that immune activation, including an inflammatory response, is diminished during morphine tolerance [39]. Therefore, the data from the present study, as well as from our previous report [38], collectively indicate that morphine may exert its effects through both pro-and anti-inflammatory inflammasomes.
LINCS analysis is able to predict potential target genes based on a certain treatment and the gene profile signatures in its database. In our study, LINCS was able to generate a report of potential targets with a p value of <0.05 from the list of genes with altered expression in response to LPS in control rats (placebo-control + saline versus placebo-control + LPS) but not from the other two comparisons (placebo-control + saline versus morphine-tolerant + saline, morphine-tolerant + saline versus morphine-tolerant + LPS), because there were not enough significant gene features in those two groups. When enlarging the set of gene features for the comparison of placebo-control + saline versus placebo-control + LPS to a p-value of <0.1, LINCS generated a report with similar potential targets. Thus, the gene features were then studied with a p-value of <0.1 on all three sets of comparisons. VPS28, PROCR, and CHMP2A were the top three with the highest scores in LINCS report generated based on placebo-control + saline versus placebo-control + LPS gene alternations, suggesting that Vps28, Procr and Chmp2a were potential targets of LPS. In the report for placebo-control + saline versus morphine-tolerant + saline, SMARCE1, AHRR, and GPX7 were the most likely targets altered in morphine tolerance predicted by LINCS. In the morphine-tolerant + saline versus morphine-tolerant + LPS report, AHR, UBE2L6, and PAFAH1B3 were the top three candidates that potentially responsible for the LPS-induced immune responses during morphine tolerance in rats. In the LINCS reports, the listed potential targets had different rankings using the different sets of gene features. This confirms that the response to LPS by those inflammosome-related genes could be affected by morphine tolerance.
Moreover, among the targets listed above, while VPS28 was No. 1 in the control rats in response to LPS, it was No. 4 in morphine-tolerant rats ( Table 4). The VPS28 protein functions in transporting and sorting proteins into sub-cellular vesicles. In our study, LINCS analysis suggests that the actions of VPS28 in response to LPS could be dampened during morphine tolerance.

Conclusions
The results from our study indicate that, in the rat brain, LPS-induced inflammation involves both the inhibition of the NLRP12 anti-inflammatory inflammasome and the stimulation of downstream regulators such as Birc3, thereby increasing the expression of pro-inflammatory chemokines and cytokines. However, in the morphine tolerant state, the response to LPS is dampened, as indicated by the reduced expression of inflammasome-related genes. LINCS analysis confirmed that the response to LPS is altered during morphine tolerance and indicated that VPS28 may be one of the genes responsible for the alterations associated with morphine tolerance.