Acute inflammatory response via neutrophil activation protects against the development of chronic pain

Abstract

The transition from acute to chronic pain is critically important but not well understood. Here, we investigated the pathophysiological mechanisms underlying the transition from acute to chronic low back pain (LBP) and performed transcriptome-wide analysis in peripheral immune cells of 98 participants with acute LBP, followed for 3 months. Transcriptomic changes were compared between patients whose LBP was resolved at 3 months with those whose LBP persisted. We found thousands of dynamic transcriptional changes over 3 months in LBP participants with resolved pain but none in those with persistent pain. Transient neutrophil-driven up-regulation of inflammatory responses was protective against the transition to chronic pain. In mouse pain assays, early treatment with a steroid or nonsteroidal anti-inflammatory drug (NSAID) also led to prolonged pain despite being analgesic in the short term; such a prolongation was not observed with other analgesics. Depletion of neutrophils delayed resolution of pain in mice, whereas peripheral injection of neutrophils themselves, or S100A8/A9 proteins normally released by neutrophils, prevented the development of long-lasting pain induced by an anti-inflammatory drug. Analysis of pain trajectories of human subjects reporting acute back pain in the UK Biobank identified elevated risk of pain persistence for subjects taking NSAIDs. Thus, despite analgesic efficacy at early time points, the management of acute inflammation may be counterproductive for long-term outcomes of LBP sufferers.

Fig. 1.. Differential expression of genes in study design contrasts.

Study design pictograms (left) are juxtaposed to volcano plots (middle and right). The pictogram highlights the two contrasted conditions (large dots). The volcano plot shows statistical significance (y axis) as a function of fold change (x axis); each dot is a gene. Genes that would end up outside of the plot are squeezed inside. Vertical gray line indicates null fold change. Genes reaching statistical significance at the FDR 10% level (blue horizontal line) are highlighted in pink. Numbers in bold indicate counts of significantly differentially expressed genes that are down-regulated (lower left corner) or up-regulated (lower right corner). Volcano plots are shown uncorrected (middle column) and corrected (right column) for blood cell-type fractions. (A and B) Contrast between patients with persistent (P; orange) and resolved (R; green) pain outcomes (A) at t₀ or (B) at t₁. (C and D) Contrast between t₁ and t₀ in P patients (C) or in R patients (D).

Fig. 2.. Blood cell-type fraction trajectories in time, in subjects with persistent or resolved pain.

(A) Study design pictograms, showing contrasts in time in those with persistent pain (P; orange, column I), and in those with resolved pain (R; green, column II). (B to E) Box-and-whisker plots (columns I and II) showing the distributions of percent cell-type fraction at t₀ and at t₁. Cell-type fraction estimates inferred by CIBERSORT from transcriptomics data. P value obtained from logistic regression between the two time points shown on top; not significant (n.s.) when P > 0.05. Volcano plots (column III) for R patients, showing genes highly expressed in the corresponding row’s cell type (pink) versus all other genes (tan). (B) Neutrophils. (C) CD8⁺ T cells. (D) Natural killer (NK) cells, resting. (E) Mast cells, resting.

Fig. 3.. Functional differences between the resolved and persistent pain groups.

Functional differences assessed with selected pathways in Gene Ontology’s (GO) biological processes. Statistically significant pathways at the FDR 10% level are highlighted in nongray colors; blue at t₀ (column I), orange for group P (column III), and green for group R (column IV). Assessment performed between the groups with resolved (R) and persistent (P) pain, at the first visit (column I), and at the second visit (column II). Functional trajectories between t₀ and t₁ are also shown for the P group (column III) and for the R group (column IV). (A) Pathways under inflammatory response (GO:0006954). (B) Pathways under leukocyte activation (GO:0045321). (C) Pathways under leukocyte degranulation (GO:0043299).

Fig. 4.. Prolongation of neuropathic and inflammatory pain by early anti-inflammatory treatment.

(A) Mechanical pain thresholds before and after chronic constriction injury (CCI) of the sciatic nerve in mice treated from days 0 to 6 with saline or dexamethasone (DEXA). Symbols represent means ± SEM hind paw withdrawal threshold (g). (B) Percentage of maximum possible allodynia (% allodynia) on day 6 after drug; see Materials and Methods for calculation details. Error bars represent SEM. (C) Days required to return to baseline thresholds; see Materials and Methods. Error bars represent SEM. Mice not returning to baseline by day 70 were assigned a value of 80. (D) Mechanical pain thresholds before and after hind paw injection of nerve growth factor (NGF) into the muscles of the low back in mice treated with saline or DEXA. Symbols as in (A). (E) Percent allodynia on day 6 after drug. (F) Days to return to baseline; mice not returning to baseline on day 50 were assigned a value of 60. (G) Mechanical pain thresholds before and after hind paw injection of complete Freund’s adjuvant (CFA) in mice treated with saline or DEXA. Symbols as in (A). (H) Percent allodynia on day 6 post-drug. (I) Days to return to baseline; mice not returning to baseline on day 120 were assigned a value of 140. (J) Mechanical pain thresholds before and after CFA in mice treated with saline, diclofenac, gabapentin, or lidocaine. Symbols as in (A). (K) Percent allodynia on day 4 after drug. (L) Days to return to baseline; mice not returning to baseline on day 40 were assigned a value of 50. *P < 0.05, **P < 0.01, and ***P < 0.001, compared to the corresponding saline group.

Fig. 5.. Involvement of neutrophils and neutrophil-released proteins S100A8 and S100A9 in pain resolution in the mouse.

(A) Mechanical pain thresholds before and after CFA in mice treated with anti-Ly6G antibody or its isotype control (vehicle). Symbols represent means ± SEM hind paw withdrawal threshold (g). (B) Percent allodynia on day 6 after drug; see Materials and Methods for calculation details. (C) Days to return to baseline; see Materials and Methods. Mice not returning to baseline on day 120 were assigned a value of 140. (D) Mechanical pain thresholds before and after CFA in mice treated with DEXA (or saline), plus a hind paw injection of vehicle, isolated neutrophils, S100A8, or S100A9 on days 3 and 5 after DEXA. Symbols as in (A). (E) Percent allodynia on day 6 after DEXA. *P < 0.05, **P < 0.01, and ***P < 0.001, compared to the corresponding vehicle/no DEXA group.

Fig. 6.. Impact of drug class on the development of chronic pain in humans.

Forest plots track odds ratios (OR; log₂ scaled) for the acute-to-chronic transition of back pain and several factors, including analgesic drug classes (up-pointing triangle; green), demographics (down-pointing triangle; brown), possible confounding factors (circle; blue), and percent of neutrophils (lozenge; pink). Models 1 to 3 test each analgesic drug class separately (gray background when factor is not included in the model), whereas model 4 tests all analgesic drug classes simultaneously. Model 5 takes into account possible confounding factors for analgesic drug intake. Model 6 includes neutrophil percentages. Vertical gray bars indicate OR = 1 [log₂(OR) = 0]. ORs are indicated by dots inside shapes, whereas the 95% confidence intervals for OR are indicated by horizontal bars. Horizontal bars are filled when P < 0.05. *P < 5 × 10⁻² and **P < 5 × 10⁻³.