Early-life inflammation, immune response and ageing

Abstract

Age-related diseases are often attributed to immunopathology, which results in self-damage caused by an inappropriate inflammatory response. Immunopathology associated with early-life inflammation also appears to cause faster ageing, although we lack direct experimental evidence for this association. To understand the interactions between ageing, inflammation and immunopathology, we used the mealworm beetle Tenebrio molitor as a study organism. We hypothesized that phenoloxidase, an important immune effector in insect defence, may impose substantial immunopathological costs by causing tissue damage to Malpighian tubules (MTs; functionally equivalent to the human kidney), in turn accelerating ageing. In support of this hypothesis, we found that RNAi knockdown of phenoloxidase (PO) transcripts in young adults possibly reduced inflammation-induced autoreactive tissue damage to MTs, and increased adult lifespan. Our work thus suggests a causative link between immunopathological costs of early-life inflammation and faster ageing. We also reasoned that if natural selection weakens with age, older individuals should display increased immunopathological costs associated with an immune response. Indeed, we found that while old infected individuals cleared infection faster than young individuals, possibly they also displayed exacerbated immunopathological costs (larger decline in MT function) and higher post-infection mortality. RNAi-mediated knockdown of PO response partially rescued MTs function in older beetles and resulted in increased lifespan after infection. Taken together, our data are consistent with a direct role of immunopathological consequences of immune response during ageing in insects. Our work is also the first report that highlights the pervasive role of tissue damage under diverse contexts of ageing and immune response.

Keywords: Malpighian tubules; ageing; immunopathology; infection; inflammation.

Figure 1.

Impact of RNAi-mediated knockdown of prophenoloxidase transcripts on (a) phenoloxidase (PO) response (n = 16 beetles per treatment) and (b) Malpighian tubule (MT) activity (n = 20–28 beetles per treatment), serving as a proxy for immunopathological damage, measured at day 4 post-immune challenge (i.e. day 15 post-emergence). PO activity was measured as V_max of the enzymatic reaction with l-DOPA substrate. MT activity was measured as the rate of fluid secretion. Significantly different groups are indicated by distinct alphabets (based on Tukey's HSD/Steel–Dwass test). Control = unhandled control beetles; early inflammation = early immune challenged control beetles; early inflammation + RNAi-PO1 = RNAi of PO1 transcript followed by an early inflammation; early inflammation + RNAi-PO2 = RNAi of PO2 transcript followed by an early inflammation.

Figure 2.

(a) Survival curves of adults after an early immune challenge that induced inflammation (n = 30 beetles per treatment). (b) Impact of phenoloxidase (PO) knockdown on median lifespan. Survival of each experimental group was compared with the unhandled control group using an accelerated failure time model. The c-parameter denotes the effect of the immune challenge treatment on survival, averaged over total survival time. Error bars represent 95% confidence intervals. (c) Impact of PO knockdown on maximum lifespan. ‘Ageing acceleration’ on the y-axis denotes the reduction in maximum lifespan caused by immune challenge treatments (the percentage of survivors to the 90th percentile survival time in the full control group/the percentage of survivors to the 90th percentile survival time in the experimental group). Control = unhandled control beetles; sham inflammation = procedural control for early life immune challenge; early inflammation = early life immune challenged control beetles; early inflammation + RNAi-PO1 = RNAi of PO1 transcript followed by an early inflammation; early inflammation + RNAi-PO2 = RNAi of PO2 transcript followed by an early inflammation.

Figure 3.

(a–f) Impact of age on (a) bacterial clearance at different time points after infection (n = 9–11 beetles per age group per treatment per time point); (b) haemolymph anti-S. aureus activity of infected beetles at different time points (n = 9–13 beetles per age group per treatment per time point); (c) phenoloxidase (PO) response of naive beetles (n = 30 beetles per age group); (d) expression of antimicrobial peptide genes attacin and tenecin-1 in naive beetles relative to an internal control (rpl27a) (n = 5–6 pairs per age group per gene); (e) post-infection survival (n = 14–15 beetles per age group per treatment) and (f) Malpighian tubule (MT) activity after bacterial infection (n = 11–14 beetles per age-group per infection status); (g,h) Effect of RNA interference of PO1 transcript on (g) immunopathological damage to MTs (n = 12–16 beetles per RNAi treatment) and (h) post-infection survival in older beetles (n = 15–18 beetles per RNAi treatment). Bacterial clearance was measured as log₁₀ of colony-forming units (CFUs) recovered from perfused haemolymph after injection of S. aureus cells into each beetle. Haemolymph antibacterial activity was measured as the number of S. aureus cells killed during 2 h of exposure to beetle haemolymph. PO response and immunopathology were measured as described in figure 1. Significantly different groups are indicated by distinct alphabets (based on Tukey's HSD/Steel–Dwass test). For panels (a,b,d), alphabet assignments are meaningful only within each time point (or gene) (partitioned by dashed vertical lines), and are not comparable across time points (or genes). Y = young beetles; O = old beetles; YSI = young sham-infected beetles; OSI = old sham-infected beetles; YI = young infected beetles; OI = old infected beetles; PC = sham infection with insect ringer; I = S. aureus infection; RNAi + I = RNAi followed by S. aureus infection.