Previous anti-inflammatory strategies against sepsis, a leading cause of death in hospitals, had limited efficacy in clinical trials, in part because they targeted single cytokines and the experimental models failed to mimic clinical settings1-3. Neuronal networks represent physiological mechanisms selected by evolution to control inflammation that can be exploited for the treatment of inflammatory and infectious disorders3. Here, we report that sciatic nerve activation with electroacupuncture controls systemic inflammation and rescues mice from polymicrobial peritonitis. Electroacupuncture at the sciatic nerve controls systemic inflammation by inducing a vagal activation of DOPA decarboxylase leading to the production of dopamine in the adrenal medulla. Experimental models with adrenolectomized animals mimic clinical adrenal insufficiency4, increase the susceptibility to sepsis, and prevent the anti-inflammatory potential of electroacupuncture. Dopamine inhibits cytokine production via dopaminergic type-1 receptors. Dopaminergic D1-agonists suppress systemic inflammation and rescue mice from polymicrobial peritonitis in animals with adrenal insufficiency. Our results suggest a novel anti-inflammatory mechanism mediated by the sciatic and the vagus nerves modulating the production of catecholamines in the adrenal glands. From a pharmacological perspective, selective dopaminergic agonists mimic the anti-inflammatory potential of electroacupuncture and can provide therapeutic advantages to control inflammation in infectious and inflammatory disorders.

Classically, sympathetic and parasympathetic systems act in opposition to maintain the physiological homeostasis. In this article, we report that both systems work together to restrain systemic inflammation in life-threatening conditions such as sepsis. This study indicates that vagus nerve and cholinergic agonists activate the sympathetic noradrenergic splenic nerve to control systemic inflammation. Unlike adrenalectomy, splenectomy and splenic neurectomy prevent the anti-inflammatory potential of both the vagus nerve and cholinergic agonists, and abrogate their potential to induce splenic and plasma norepinephrine. Splenic nerve stimulation mimics vagal and cholinergic induction of norepinephrine and re-establishes neuromodulation in α7 nicotinic acetylcholine receptor (α7nAChR)-deficient animals. Thus, vagus nerve and cholinergic agonists inhibit systemic inflammation by activating the noradrenergic splenic nerve via the α7nAChR nicotinic receptors. α7nAChR represents a unique molecular link between the parasympathetic and sympathetic system to control inflammation.

The nervous system is classically organized into sympathetic and parasympathetic systems acting in opposition to maintain physiological homeostasis. Here, we report that both systems converge in the activation of β2-adrenoceptors of splenic regulatory lymphocytes to control systemic inflammation. Vagus nerve stimulation fails to control serum TNF levels in either β2-knockout or lymphocyte-deficient nude mice. Unlike typical suppressor CD25(+) cells, the transfer of CD4(+)CD25(-) regulatory lymphocytes reestablishes the anti-inflammatory potential of the vagus nerve and β2-agonists to control inflammation in both β2-knockout and nude mice. β2-Agonists inhibit cytokine production in splenocytes (IC(50)≈ 1 μM) and prevent systemic inflammation in wild-type but not in β2-knockout mice. β2-Agonists rescue wild-type mice from established polymicrobial peritonitis in a clinically relevant time frame. Regulatory lymphocytes reestablish the anti-inflammatory potential of β2-agonists to control systemic inflammation, organ damage, and lethal endotoxic shock in β2-knockout mice. These results indicate that β2-adrenoceptors in regulatory lymphocytes are critical for the anti-inflammatory potential of the parasympathetic vagus nerve, and they represent a potential pharmacological target for sepsis.

The role of STAT3 in infectious diseases remains undetermined, in part because unphosphorylated STAT3 has been considered an inactive protein. Here, we report that unphosphorylated STAT3 contributes to cholinergic anti-inflammation, prevents systemic inflammation, and improves survival in sepsis. Bacterial endotoxin induced STAT3 tyrosine phosphorylation in macrophages. Both alpha7 nicotinic receptor (alpha7nAChR) activation and inhibition of JAK2 blunt STAT3 phosphorylation. Inhibition of STAT3 phosphorylation mimicked the alpha7nAChR signaling, inhibiting NF-κB and cytokine production in macrophages. Transfection of macrophages with the dominant-negative mutant STAT3F, to prevent its tyrosine phosphorylation, reduced TNF production but did not prevent the alpha7nAChR signaling. However, inhibition of STAT3 protein expression enhanced cytokine production and abrogated alpha7nAChR signaling. Alpha7nAChR controls TNF production in macrophages through a mechanism that requires STAT3 protein expression, but not its tyrosine phosphorylation. In vivo, inhibition of STAT3 tyrosine phosphorylation by stattic prevented systemic inflammation and improved survival in experimental sepsis. Stattic also prevented the production of late mediators of sepsis and improved survival in established sepsis. These results reveal the immunological implications of tyrosine-unphosphorylated STAT3 in infectious diseases.

Sepsis, a leading cause of death in hospitalized patients, is characterized by lethal systemic inflammatory responses. JAK2 is an essential tyrosine kinase modulating immune responses. However, the implications of JAK2 in infectious disorders remain undetermined. Here, we report that JAK2 inhibitors rescue animals from polymicrobial sepsis in a clinically relevant time frame. JAK2 inhibition with AG490 prevents NF-κB activation, modulates macrophage activation, and restrains the production of inflammatory cytokines. The inhibition of JAK2 blunted TNF production in both macrophages and splenocytes in a concentration-dependent manner. JAK2 inhibition specifically prevents LPS-induced STAT3 tyrosine phosphorylation without affecting serine phosphorylation in macrophages. JAK2 inhibitor prevents the activation of the canonical p65RelA/p50NF-κB1 pathway but not the other NF-κB proteins. In vivo, JAK2 inhibition restrains serum TNF levels by modulating TNF production in the lung and the spleen and protects mice from lethal endotoxemia in a concentration-dependent manner. AG490 also inhibits extracellular release of HMGB1 from macrophages and prevents an increase in serum HMGB1 levels during sepsis. JAK2 inhibition started at 24 h after the onset of sepsis rescued the mice from polymicrobial sepsis. Our study is the first experimental evidence that JAK2 inhibitors may provide a pharmacological advantage for the treatment of sepsis in a clinically relevant time frame.

Many anti-inflammatory strategies successful in healthy animals fail in clinical trials for sepsis, in part, because sepsis normally involves immunocompromised patients, and massive lymphocyte apoptosis prevents immunomodulation. Here we report a new set of regulatory lymphocytes able to reestablish the cholinergic anti-inflammatory modulation and to provide therapeutic advantages in sepsis. Vagus nerve controls inflammation in healthy, but not in septic mice. Likewise, vagus nerve and cholinergic agonists fail to control inflammation in splenectomized and nude animals. Unlike typical suppressor CD25+ cells, CD4+CD25− lymphocytes reestablish the anti-inflammatory potential of the vagus nerve and cholinergic agonists in immunocompromised and septic animals. These cholinergic lymphocytes reestablish splenic protection and the potential of cholinergic agonists to rescue immunocompromised animals from established sepsis. These results reveal these new regulatory lymphocytes as the first known physiological target for neuromodulation of the innate immune responses, and a potential therapeutic target for sepsis.

Interactions of Toll-like receptors (TLR) with non-microbial factors plays a major role in the pathogenesis of early trauma-hemorrhagic shock (T/HS)-induced organ injury and inflammation. Thus, we tested the hypothesis that TLR4 mutant (TLR4mut) mice would be more resistant to T/HS-induced gut injury and neutrophil (PMN) priming than their wild-type (WT) littermates and found that both were significantly reduced in the TLR4mut mice. Additionally, the in vivo and ex vivo PMN priming effect of T/HS intestinal lymph observed in the WT mice was abrogated in TLR4mut mice as well the TRIFmut deficient mice and partially attenuated in Myd88-/- mice suggesting that TRIF activation played a more predominant role than MyD88 in T/HS lymph-induced PMN priming. PMN depletion studies showed that T/HS lymph-induced acute lung injury (ALI) was PMN-dependent, since lung injury was totally abrogated in PMN-depleted animals. Since the lymph samples were sterile and devoid of endotoxin or bacterial DNA, we investigated whether the effects of T/HS lymph was related to endogenous non-microbial TLR4 ligands. HMGB1, heat shock protein (Hsp)-70, Hsp27 and hyaluronic acid, since all have been implicated in ischemia-reperfusion-induced tissue injury. None of these ‘danger’ proteins appeared to be involved, since their levels were similar between the sham and shock lymph samples. In conclusion, TLR4 activation is important in T/HS-induced gut injury and in T/HS lymph-induced PMN priming and lung injury. However, the T/HS-associated effects of TLR4 on gut barrier dysfunction can be uncoupled from the T/HS lymph-associated effects of TLR4 on PMN priming.

Females have a longer lifespan and better general health than males. Considerable number of studies also demonstrated that, after trauma and sepsis, females present better outcomes as compared to males indicating sex-related differences in the innate immune response. The current notion is that differences in the immuno-modulatory effects of sex hormones are the underlying causative mechanism. However, the field remains controversial and the exclusive role of sex hormones has been challenged. Here, we propose that polymorphic X-linked immune competent genes, which are abundant in the population are important players in sex-based immuno-modulation and play a key role in causing sex-related outcome differences following trauma or sepsis. We describe the differences in X chromosome (ChrX) regulation between males and females and its consequences in the context of common X-linked polymorphisms at the individual as well as population level. We also discuss the potential pathophysiological and immune-modulatory aspects of ChrX cellular mosaicism, which is unique to females and how this may contribute to sex-biased immune-modulation. The potential confounding effects of ChrX skewing of cell progenitors at the bone marrow is also presented together with aspects of acute trauma-induced de novo ChrX skewing at the periphery. In support of the hypothesis, novel observations indicating ChrX skewing in a female trauma cohort as well as case studies depicting the temporal relationship between trauma-induced cellular skewing and the clinical course are also described. Finally, we list and discuss a selected set of polymorphic X-linked genes, which are frequent in the population and have key regulatory or metabolic functions in the innate immune response and, therefore, are primary candidates for mediating sex-biased immune responses. We conclude that sex-related differences in a variety of disease processes including the innate inflammatory response to injury and infection may be related to the abundance of X-linked polymorphic immune-competent genes, differences in ChrX regulation, and inheritance patterns between the sexes and the presence of X-linked cellular mosaicism, which is unique to females.