ASB <i>E. coli</i> isolate 2–12 rapidly attenuates UTI visceral pain of diverse uropathogens.

<p>A) Allodynia was quantified daily in groups of sham– or NU14-infected mice that were then treated at PID1 with saline, a three-day course of ciprofloxacin, or a single dose of intravesical or intravaginal 2–12 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109321#pone-0109321-g002" target="_blank">Figure 2A</a> (n = 8 in all groups except the 2–12 intravaginal group where n = 6). NU14-infected mice exhibited a significant decrease in pelvic pain 24 hours following treatment with 2–12 via the bladder or vaginal introitus (P<0.05). Allodynia was quantified in mice infected via transureathral catheter with <i>Proteus mirabilis</i> (PM1), <i>Enterocccus faecalis</i> (EF1) and <i>Klebsiella pneumoniae</i> (KP1) and then treated with a three-day course of ciprofloxacin initiated at PID1 or a single intravesical dose of 2–12 (n = 5). B) PM1 induced pelvic allodynia that was significantly attenuated on PID3 and PID4 by 2–12 treatment, relative to the ciprofloxacin or saline groups (P<0.05). C) EF1 induced allodynia that was significantly attenuated on PID2-PID4 by 2–12 treatment, relative to ciprofloxacin or saline groups (P<0.05). Allodynia was also significantly reduced on PID5–6 compared to the saline group (P<0.05). D) KP1 induced allodynia that was significantly attenuated on PID2 by 2–12 treatment, relative to ciprofloxacin or saline groups (P<0.05).</p

ASB strains differentially attenuate UTI visceral pain.

<p>Modulation of UTI visceral pain by a panel of 16 ASB <i>E. coli</i> strains. A) Mice were infected with NU14 and then instilled with saline or ASB <i>E. coli</i> at PID1, and allodynia was quantified through PID7 (n = 6 for all groups). For clarity, timecourses of allodynia for all 17 conditions were divided among 3 panels. All 16 ASB strains attenuated NU14-induced pelvic pain compared to the saline treated group. B) The relative analgesic activity of ASB strains was arbitrary grouped according to the magnitude of analgesia: strains with analgesic relative to saline but increased allodynia from PID1 (black bars), those that exhibited no increase in allodynia from PID1 to less than 75% reduction in visceral pain from PID1 (white bars), and those that exhibited greater than 75% reduction (gray bars). C) Representative ASB strains from each group in (B) were used in serial infections at two-week intervals. Serial infection did not induce allodynia. Data are reported as the mean ± SEM.</p

<i>E. coli</i> 83972 attenuates NU14 bacteriuria.

<p>A) Experimental scheme for assessing efficacy of a single administration of ASB therapy relative to 3-day course of ciprofloxacin (n = 5 for all groups). B) Mice infected with NU14 exhibited a significant decrease in NU14 bacteriuria 24 hours after initiation of ciprofloxacin (group C, back triangles, PID2, 4, 10), relative to saline-treated mice (group S, white triangles, *P<0.05). A single instillation of 83972 (group ASB, grey inverted triangles) also resulted in a significant decrease in NU14 bacteriuria after 24 hours (PID2, 4, 10, *P<0.05). C) Urinary 83972 for all three groups during the experiment. D and E) Bladder colonization on PID20 was not significantly different between the groups tested. Dashed lines represent limits of detection.</p

<i>E. coli</i> 83972 attenuates NU14-induced bacteriuria and pelvic pain.

<p>A) Referred visceral hyperalgesia was measured as responses to mechanical stimulation of the pelvic region with von Frey filaments of 5 intensities. Responses were quantified at baseline, PID1 following NU14 infection, and 24 hours following of saline (n = 5) or 83972 (n = 10) instillation (PID2). B) NU14-infected mice exhibited a significant decrease in allodynia 24 hours following 83972 instillation. C) Percent increase in allodynia from PID1 was 146.2% in saline-treated mice, but decreased by over 68.9% in 83972-treated mice (P<0.01). D) Mice treated with 83972 exhibited a significantly less NU14 bacteriuria compared to saline-treated mice (P<0.01). Dashed line represents limit of detection. E) At PID1, NU14-infected mice were instilled with saline into the bladder or 2% lidocaine in the bladder, vaginal introitus or colon. Allodynia was significantly reduced at 1 h after lidocaine into any compartment, relative to bladder saline (*P<0.05; n = 9 saline, n = 10 bladder lidocaine, n = 11 colon lidocaine, n = 12 vaginal lidocaine). F) NU14-infected mice exhibited increased allodynia 24 hours after bladder instillation of saline, relative to PID1 (n = 10). Allodynia was significantly decreased at 24 hours following 83972 instillation into the bladder, colon or vaginal introitus (*P<0.05, n = 10 all groups). Data are reported as the mean ± SEM (A–C, E & F).</p

O-Antigen Modulates Infection-Induced Pain States

<div><p>The molecular initiators of infection-associated pain are not understood. We recently found that uropathogenic <em>E. coli</em> (UPEC) elicited acute pelvic pain in murine urinary tract infection (UTI). UTI pain was due to <em>E. coli</em> lipopolysaccharide (LPS) and its receptor, TLR4, but pain was not correlated with inflammation. LPS is known to drive inflammation by interactions between the acylated lipid A component and TLR4, but the function of the O-antigen polysaccharide in host responses is unknown. Here, we examined the role of O-antigen in pain using cutaneous hypersensitivity (allodynia) to quantify pelvic pain behavior and using sacral spinal cord excitability to quantify central nervous system manifestations in murine UTI. A UPEC mutant defective for O-antigen biosynthesis induced chronic allodynia that persisted long after clearance of transient infections, but wild type UPEC evoked only acute pain. <em>E. coli</em> strains lacking O-antigen gene clusters had a chronic pain phenotype, and expressing cloned O-antigen gene clusters altered the pain phenotype in a predictable manner. Chronic allodynia was abrogated in TLR4-deficient mice, but inflammatory responses in wild type mice were similar among <em>E. coli</em> strains spanning a wide range of pain phenotypes, suggesting that O-antigen modulates pain independent of inflammation. Spinal cords of mice with chronic allodynia exhibited increased spontaneous firing and compromised short-term depression, consistent with centralized pain. Taken together, these findings suggest that O-antigen functions as a rheostat to modulate LPS-associated pain. These observations have implications for an infectious etiology of chronic pain and evolutionary modification of pathogens to alter host behaviors.</p> </div

O-antigen modulates pain states.

<p>Mice were infected with NU14Δ<i>wz</i>* bearing a deletion of the O-antigen gene clusters and harboring like or heterologous complementation constructs. (<b>A</b>) NU14 smooth colony morphology (i) is rough in the NU14Δ<i>wz</i>* mutant with a human X chromosome plasmid (ii) or a 83972 <i>wz</i>* plasmid (iv) but is rescued by an NU14<i>wz</i>* plasmid (iii). (<b>B</b>) Tactile allodynia of mice in response to sequential infection with NU14 or NU14Δ<i>wz*</i> containing plasmids with the wz* cluster of 83972, NU14, or a fragment of the human X chromosome (n = 10). (<b>C</b>) Summary of O-antigen modulation of pain responses.</p

<i>E. coli</i> with differential pain phenotypes do not elicit differential inflammation.

<p>(<b>A</b>) Hematoxylin-eosin stained sections of bladders from mice instilled with saline, 83972, NU14, Δ<i>waaL</i>, and SΦ874 appeared similar at 6 hours and 14 days. Calibration mark is 100 µm. (<b>B</b>) Inflammation that was scored by a blinded reviewer and expressed as arbitrary units (AU) was significantly elevated for 83972-, NU14-, Δ<i>waaL-</i>, and SΦ874-infected bladders harvested at 6 hours, relative to saline (P<0.001) but was not significantly different among <i>E. coli</i>. (<b>C</b>) Inflammation scores were not significantly different for 83972-, NU14-, Δ<i>waaL-</i>, and SΦ874-infected bladders harvested at 14 days (P = 0.11). (<b>D</b>) Myeloperoxidase (MPO) was quantified in mouse urine by ELISA. Urines were collected at 6 h and 14 d following instillation of saline, 83972, or SΦ874. (<b>E</b>) MPO was quantified in mouse urines obtained at baseline or at 6 h, 24 h, and 14 d following serial instillation of saline (—, n = 4), NU14 (N, n = 4), or Δ<i>waaL</i> (Δ, n = 7). *P<0.05. No significant differences were observed in urinary MPO of mice with treated with NU14 or Δ<i>waaL.</i></p

Purified LPS mimics effects of intact <i>E. coli</i>, and chronic pain is TLR4-dependent.

<p>(<b>A</b>) Mice (n = 8) were instilled 25 µl of 2 µg/ml of LPS purified from NU14, 83972, Δ<i>waaL</i>, or SΦ874 and then evaluated for pelvic allodynia. (<b>B</b>) SΦ874-induced pain in +/+ mice is reduced in TLR4^−/− mice (n = 5; P<0.05 Days 4–14). (<b>C</b>) +/+ mice (C3H/HeJOuJ, “OuJ") or TLR4-deficient mice (C3H/HeJ, “HeJ") were used as bone marrow donors for γ-irradiated recipients; legend arrow indicates donor bone marrow into recipient (n = 9, 9, 14 and 15 respectively). C3H/HeJOuJ recipients exhibited SΦ874-induced pain that was reduced in C3H/HeJ recipients (P<0.01 Days 3–14).</p

TLR4 mediates chronic pain.

<p>UTI was induced in female mice by instilling 10⁸<i>E. coli</i> into the bladder, and tactile allodynia and bladder colonization were quantified. (<b>A</b>) Mice were instilled repeatedly with saline, NU14, or Δ<i>waaL</i> (n = 9). NU14 induced resolving acute pain (P<0.001 Days 2–5, P<0.01 Days 1 and 6, P<0.05 Days 7–10), but Δ<i>waaL</i> induced chronic pain following the second infection. (<b>B</b>) Bladders from mice in (A) had detectable NU14 colonization but not Δ<i>waaL</i> colonization (P<0.044). (<b>C</b>) SΦ874 pain (n = 10) was abrogated in mice infected with SΦ874 pWQ288 (n = 10). (<b>D</b>) SΦ874 is cleared rapidly from the bladder; open circle indicates inoculum (n = 5 Days 1–14, n = 10 Day 35).</p

Pelvic pain behavior is associated with sacral spinal cord excitability.

<p>Spontaneous action potentials and evoked potentials were quantified in sacral spinal cords ex vivo at ventral roots S1–S3. (<b>A</b>) A sacral spinal cord is mounted in a recording chamber (upper panel), and spontaneous activity is recorded from the ventral root (lower panel). (<b>B</b>) Representative action potentials from spontaneous firing of individual neurons identified by pCLAMP. (<b>C</b>) Firing activity in sacral spinal cords is higher in Δ<i>waaL</i> at 14 d (n = 5, P = 0.0099), SΦ874 at 20 days (n = 9, P = 0.0001), and NU14 mice at 2 d (n = 6, P = 0.0298) than in saline controls (n = 7) or resolved NU14 (n = 5). (<b>D</b>) Evoked ventral root responses to dorsal root current at 2× current intensity for spinal cord of saline mouse at 2 d (upper trace) and Δ<i>waaL</i>-infected mouse after serial infection. (<b>E</b>) Normalized responses at P2–P5 relative to P1 in ventral roots of mice instilled with saline (n = 23), NU14 (n = 26), SΦ874 (n = 21), or Δ<i>waaL</i> (n = 12). *P<0.05 and **P<0.01 by Student <i>t</i> test relative to saline. (<b>F</b> and <b>G</b>) Responses across stimulus intensities at P3 and P4, respectively.</p