Development of tissue inflammation accompanied by NLRP3 inflammasome activation in rabbits infected with Treponema pallidum strain Nichols

The T. pallidum Nichols strain was kindly provided by Lorenzo Giacani, Ph.D. (University of Washington, Seattle) and was propagated via intra-testicular serial passage in New Zealand white rabbits to maintain virulence in our laboratory as previously described [12]. Forty-five male New Zealand white rabbits (purchased from the Xiamen University Laboratory Animal Center, weighing approximately three kilograms each) with negative results in both the reactive rapid plasma reagin and T. pallidum particle agglutination tests, were randomly assigned to two groups, a blank group (n = 15) and an infection group (n = 30). The latter was divided into the no benzathine penicillin G (BPG) treatment subgroup (n = 15) and the BPG treatment subgroup (n = 15). The animals were housed individually at 16 to 18 °C and were fed with antibiotic-free food and water. Rabbits in the infection group were injected intradermally with 0.1 mL of a 10⁷ treponeme/mL suspension at 10 marked sites along the back, while rabbits in the blank group were injected with normal saline. The backs of the rabbits were meticulously kept free of fur by daily clipping throughout the experiment. Rabbits in the BPG treatment subgroup received 200,000 U of BPG administered intramuscularly twice, at 14 d and 21 d post-infection.

One representative site of each animal was selected separately and biopsied (4-mm punch biopsies obtained under local lidocaine anesthesia) for RNA extraction at 1, 4, 7, 10, 14, 18, 21, 28, 35 and 42 d post-infection. One representative site on each animal was dedicated exclusively for the observation of lesion appearance and development up to 42 d post-infection; the diameter of the lesion was measured using a vernier caliper. Three animals were randomly selected for euthanasia in the two groups at 7, 14, 21, 28, and 42 d post-infection, and the kidney, liver, spleen, lung, and testis organs were then harvested for experimental analysis. Blood was collected at 1, 4, 7, 10, 14, 18, 21, 28, 35 and 42 d post-infection, and serum was isolated and frozen at − 80 °C until analysis of the IL-1β concentration. All protocols involving animals were approved in advance by the animal experimental ethics committee of the Medical College of Xiamen University.

To assess the expression of mRNA, total RNA from lesions/tissues was isolated using the RNeasy Kit (Qiagen Inc., Valencia, CA) and was reverse transcribed using a high-capacity cDNA reverse transcription kit (Takara Inc., Dalian, China). The generated cDNA was amplified using quantitative PCR assays and the SYBR Advantage PCR Premix (Takara Inc., Dalian, China) with the 7500 Real Time PCR System (Applied Biosystems, Carlsbad, USA). The following primer pairs were used: NLRP3, (5’-CCACTTCCCCAGAATCGAGA-3′ and 5’-TGGACGTGAGACAGGAGTTC-3′); Caspase-1, (5’-CAAGTCTCAAGCTTTGCCCG-3′ and 5’-TAATGAGGGCAAGACGGGTG-3′); IL-1β, (5’-GGATGACGGCCTGAGAACTT-3′ and 5’-TACGTGCCAGACAACACCAA-3′); and GAPDH, (5’-GCTTCTTCTCGTGCAGTGCA-3′ and 5’-ATGACCAGCTTCCCGTTCTC -3′). After amplification, Ct values were normalized to GAPDH as an internal control, and the relative copy number was determined using the standard 2^-△△Ct method [13]. A commercial enzyme-linked immunosorbent assay kit (Cloud-Clone Inc., USA) was used to measure the IL-1β levels in the rabbit serum samples according to the manufacturer’s instructions.

In the infected rabbits, cutaneous lesions began to develop at 4 d post-infection and then reached a peak at 16 d in the BPG treatment subgroup and at 18 d in the no BPG treatment subgroup (Fig. 1a, b). In the BPG treatment subgroup, the lesions gradually began to shrink at 16 d (2 d after the first BPG treatment) and subsequently disappeared at 28 d post-infection. The cutaneous lesions disappeared at an earlier time point in the BPG treatment subgroup than that in the no BPG treatment subgroup (28 d vs. 42 d). The lesions were barely detectable at 35 d and disappeared at 42 d in the no BPG treatment subgroup. The cutaneous lesions in the no BPG treatment subgroup were significantly larger than those in the BPG treatment subgroup at 18, 21, 24, 28 and 35 d post-infection (Fig. 1c) (P < 0.05). No lesions developed in the blank group (data not shown).

In the infected rabbit group, the NLRP3 mRNA levels showed a trend in elevation to decline and reached a peak at 18 d post-infection in the BPG treatment subgroup and at 21 d in the no BPG treatment subgroup. In the BPG treatment subgroup, NLRP3 mRNA expression was suppressed at 18 d post-infection (4 d after the first BPG treatment) and returned to “normal” levels [(i.e., not significantly different from the blank group (P > 0.05)] at 42 d post-infection. Notably, the level of NLRP3 mRNA exhibited a reduction at an earlier time point in the BPG treatment subgroup (18 d) than that in the no BPG treatment subgroup (21 d). The expression of NLRP3 mRNA in the no BPG treatment subgroup was significantly higher than that in the BPG treatment subgroup at 21, 28, and 35 d post-infection (P < 0.05) (Fig. 2a). However, the expression of caspase-1 and IL-1β mRNA showed a “saddle pattern” of change over time post-infection; caspase-1 expression reached an initial peak at 7 d and a second peak at 28 d, while IL-1β mRNA reached a first peak at 14 d and a second peak at 28 d in both the BPG treatment and no BPG treatment subgroups (Fig. 2b, c). Notably, despite different trends in the expression of NLRP3, caspase-1, and IL-1β mRNAs in cutaneous lesions during infection, at 42 d post-infection, the expression of all three mRNAs returned to “normal” levels [i.e., were not significantly different from the blank group levels (P > 0.05)] in both the BPG treatment and no BPG treatment subgroups. The expression of NLRP3, caspase-1, and IL-1β mRNAs was maintained at a low level and showed no fluctuations in the blank group during the experimental period (Fig. 2).

Except at 1 d and 42 d post-infection, the IL-1β concentration in the infection group was significantly higher than that in the blank group, which maintained a low level with no fluctuations. The dynamic tendency of the serum IL-1β concentration in the BPG treatment subgroup was similar to that in the no BPG treatment subgroup, which presented a trend of increasing early and a later decrease (Fig. 3). The serum IL-1β level reached a peak at 18 d in the BPG treatment subgroup and at 21 d in the no BPG treatment subgroup, before returning to normal levels [i.e., not significantly different from the blank group level (P > 0.05)] at 42 d post-infection.

Additionally, the dynamics of NLRP3, caspase-1, and IL-1β mRNAs were monitored in five organs: the kidney, liver, spleen, lung, and testis. The results showed that NLRP3, caspase-1, and IL-1β mRNAs had different expression levels in the different organs of infected rabbits. The NLRP3 mRNA expression levels in the infection group showed a trend in elevation to decline in all five organs but was still higher than “normal” at the endpoint of the study (vs. the blank group, P < 0.05) (Fig. 4a-e). Similar to the trend in NLRP3, caspase-1 mRNA showed an initial increase and then a decreasing trend later in four organs (kidney, liver, lung, and testis). The expression of caspase-1 mRNA in the kidney showed a “saddle pattern” of change over time post-infection, which was different from that in the other three organs (liver, lung, and testis) and remained higher than that in the blank group at the endpoint of the study (P < 0.05). However, caspase-1 mRNA level in the spleen was not different among the BPG treatment subgroup, the no BPG treatment subgroup, and the blank group (Fig. 4f-j). Regarding the expression level of IL-1β mRNA, there was no difference in the testes among the BPG treatment subgroup, the no BPG treatment subgroup, and the blank group. However, IL-1β mRNA expression showed an earlier increase and later decrease in the kidney, liver, spleen, and lung and returned to normal at 21 d in the kidney and 42 d in the liver [vs. the blank group (P > 0.05)]. IL-1β mRNA was still expressed in the lung and spleen (vs. the blank group, P < 0.05) at the endpoint of the study (42 d post-infection) (Fig. 4k-o).