Sivakumar Periasamy

Programmed Death 1 and Cytokine Inducible SH2-Containing Protein Dependent Expansion of Regulatory T Cells Upon Stimulation With Mycobacterium tuberculosis

We previously found that CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) expand in response to Mycobacterium tuberculosis infection in individuals who are healthy tuberculin reactors, but not in tuberculin-negative individuals. We also found that the M. tuberculosis mannose-capped lipoarabinomannan and prostaglandin E2 produced by monocytes are involved in Treg expansion. In this study, we found that Tregs expanded from CD4(+)CCR4(+) cells but not from CCR4(-) cells. However, introduction of CCR4 small interfering RNA (siRNA) into CD4(+) cells only marginally reduced expansion of Tregs. Using siRNA and neutralizing antibodies, we found that expansion of Tregs by M. tuberculosis required expression of programmed death1 (PD-1) and expression of the signaling molecule, cytokine inducible SH2-containing protein (CISH). Anti-PD-1 siRNA inhibited expression of CISH by expanded Tregs. M. tuberculosis-expanded Tregs produced transforming growth factor β and interleukin 10 and reduced the frequency of interferon γ-producing autologous CD8(+) cells. We conclude that M. tuberculosis infection induces development of Tregs from CCR4(+) cells through a process that depends on PD-1and CISH.

Development of tolerogenic dendritic cells and regulatory T cells favors exponential bacterial growth and survival during early respiratory tularemia

Tularemia is a vector‐borne zoonosis caused by Ft, a Gram‐negative, facultative intracellular bacterium. Ft exists in two clinically relevant forms, the European biovar B (holarctica), which produces acute, although mild, self‐limiting infections, and the more virulent United States biovar A (tularensis), which is often associated with pneumonic tularemia and more severe disease. In a mouse model of tularemia, respiratory infection with the virulence‐attenuated Type B (LVS) or highly virulent Type A (SchuS4) strain engenders peribronchiolar and perivascular inflammation. Paradoxically, despite an intense neutrophilic infiltrate and high bacterial burden, Th1‐type proinflammatory cytokines (e.g., TNF, IL‐1β, IL‐6, and IL‐12) are absent within the first ∼72 h of pulmonary infection. It has been suggested that the bacterium has the capacity to actively suppress or block NF‐κB signaling, thus causing an initial delay in up‐regulation of inflammatory mediators. However, our previously published findings and those presented herein contradict this paradigm and instead, strongly support an alternative hypothesis. Rather than blocking NF‐κB, Ft actually triggers TLR2‐dependent NF‐κB signaling, resulting in the development and activation of tDCs and the release of anti‐inflammatory cytokines (e.g., IL‐10 and TGF‐β). In turn, these cytokines stimulate development and proliferation of Tregs that may restrain Th1‐type proinflammatory cytokine release early during tularemic infection. The highly regulated and overall anti‐inflammatory milieu established in the lung is permissive for unfettered growth and survival of Ft. The capacity of Ft to evoke such a response represents an important immune‐evasive strategy.

An Immature Myeloid/Myeloid-Suppressor Cell Response Associated with Necrotizing Inflammation Mediates Lethal Pulmonary Tularemia.

Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but the mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that in contrast to overwhelming bacterial burden or cytokine production, an overt innate cellular response to Ft drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature and die, leading to subsequent necrotic lung damage, loss of pulmonary function, and host death that is partially dependent upon immature Ly6G+ cells. Acceleration of this process may account for the rapid lethality seen with Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection.

Inflammasome-Independent NLRP3 Restriction of a Protective Early Neutrophil Response to Pulmonary Tularemia

Francisella tularensis (Ft) causes a frequently fatal, acute necrotic pneumonia in humans and animals. Following lethal Ft infection in mice, infiltration of the lungs by predominantly immature myeloid cells and subsequent myeloid cell death drive pathogenesis and host mortality. However, following sub-lethal Ft challenge, more mature myeloid cells are elicited and are protective. In addition, inflammasome-dependent IL-1β and IL-18 are important for protection. As Nlrp3 appears dispensable for resistance to infection with Francisella novicida, we considered its role during infection with the virulent Type A strain SchuS4 and the attenuated Type B live vaccine strain LVS. Here we show that both in vitro macrophage and in vivo IL-1β and IL-18 responses to Ft LVS and SchuS4 involve both the Aim2 and Nlrp3 inflammasomes. However, following lethal infection with Francisella, IL-1r-, Caspase-1/11-, Asc- and Aim2-deficient mice exhibited increased susceptibility as expected, while Nlrp3-deficient mice were more resistant. Despite reduced levels of IL-1β and IL-18, in the absence of Nlrp3, Ft infected mice have dramatically reduced lung pathology, diminished recruitment and death of immature myeloid cells, and reduced bacterial burden in comparison to wildtype and inflammasome-deficient mice. Further, increased numbers of mature neutrophil appear in the lung early during lethal Ft infection in Nlrp3-deficient mice. Finally, Ft infection induces myeloid and lung stromal cell death that in part requires Nlrp3, is necrotic/necroptotic in nature, and drives host mortality. Thus, Nlrp3 mediates an inflammasome-independent process that restricts the appearance of protective mature neutrophils and promotes lethal necrotic lung pathology.

FcγR mediates TLR2- and Syk-dependent NLRP3 inflammasome activation by inactivated Francisella tularensis LVS immune complexes

IgG (mAb)‐opsonized, inactivated Francisella tularensis LVS (iFt‐mAb) enhances TLR2‐dependent IL‐6 production by macrophages via Fcγ receptors (FcγR). In mice, vaccination with iFt‐mAb provides IgA‐dependent protection against lethal challenge with Ft LVS. Because inflammasome maturation of IL‐1β is thought important for antibody‐mediated immunity, we considered the possibility that iFt‐mAb elicits an FcγR‐dependent myeloid cell inflammasome response. Herein, we find that iFt‐mAb enhances macrophage and dendritic cell IL‐1β responses in a TLR2‐ and FcγR‐dependent fashion. Although iFt‐mAb complexes bind FcγR and are internalized, sensing of cytosolic DNA by absent in melanoma 2 (AIM2) is not required for the IL‐1β response. In contrast, ASC, caspase‐1, and NLR family pyrin domain‐containing 3 (NLRP3) are indispensable. Further, FcγR‐mediated spleen tyrosine kinase (Syk) signaling is required for this NLRP3‐dependent IL‐1β response, but the alternative IL‐1β convertase caspase‐8 is insufficient. Finally, iFt‐mAb‐vaccinated wild‐type mice exhibit a significant delay in time to death, but IL‐1R1– or Nlrp3‐deficient mice vaccinated in this way are not protected and lack appreciable Francisella‐specific antibodies. This study demonstrates that FcγR‐mediated Syk activation leads to NLRP3 inflammasome‐dependent IL‐1β production in macrophages and suggests that an Nlrp3‐ and IL‐1R–dependent process contributes to the IgA response important for protection against Ft LVS. These findings extend our understanding of cellular responses to inactivated pathogen‐opsonized vaccine, establish FcγR‐elicited Syk kinase‐mediated NLRP3 inflammasome activation, and provide additional insight toward understanding crosstalk between TLR and FcγR signals.

Interleukin 1α (IL-1α) Promotes Pathogenic Immature Myeloid Cells and IL-1β Favors Protective Mature Myeloid Cells During Acute Lung Infection

Bacterial pneumonia is a common risk factor for acute lung injury and sepsis-mediated death, but the mechanisms underlying the overt inflammation and accompanying pathology are unclear. Infiltration of immature myeloid cells and necrotizing inflammation mediate severe pathology and death during pulmonary infection with Francisella tularensis. However, eliciting mature myeloid cells provides protection. Yet, the host factors responsible for this pathologic immature myeloid cell response are unknown. Here, we report that while the influx of both mature and immature myeloid cells is strictly MyD88 dependent, the interleukin 1 (IL-1) receptor mediates an important dual function via its ligands IL-1α and IL-1β. Although IL-1β favors the appearance of bacteria-clearing mature myeloid cells, IL-1α contributes to lung infiltration by ineffective and pathologic immature myeloid cells. Finally, IL-1α and IL-1β are not the sole factors involved, but myeloid cell responses during acute pneumonia were largely unaffected by lung levels of interleukin 10, interleukin 17, CXCL1, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor.

A prevalence study of typhoid fever and convalescent phase asymptomatic typhoid carriers among the schoolchildren in the northern part of Tamil Nadu

AimThis study reports, for the first time, the prevalence of typhoid fever and convalescent phase asymptomatic typhoid carriers among the schoolchildren in Vellore district of Tamil Nadu state in India.Subjects and methodsBlood and stool samples (n = 500) were collected from the schoolchildren, who were admitted to District Government hospitals for treatment of typhoid fever or other enteric disorders. Early morning stool and blood samples (n = 700) were collected from the schoolchildren, who had the previous history of typhoid fever. The blood and stool samples were subjected to immunological and bacteriological examination to confirm the cases of typhoid fever and typhoid carriers. Further, S. Typhi isolates from the typhoid patients and asymptomatic carriers were serogrouped by standard protocols at WHO Reference laboratory for Salmonella typing in India.ResultsA total of 360/500 (72%) children were positive for typhoid fever; while a total of 147/700 (21%) children were confirmed as convalescent phase asymptomatic typhoid carriers. All the S. Typhi isolates obtained from children positive from typhoid fever or typhoid carriers in this region were found to be serovars 9, 12, Vi: d and 4, 12: i: 1, 2.ConclusionThis study highlights the prevalence of typhoid fever among the schoolchildren and a possible role of convalescent phase asymptomatic typhoid carriers as a risk factor for the spread of infection in this locality.

Pyrin-only protein 2 limits inflammation but improves protection against bacteria

Pyrin domain-only proteins (POPs) are recently evolved, primate-specific proteins demonstrated in vitro as negative regulators of inflammatory responses. However, their in vivo function is not understood. Of the four known POPs, only POP2 is reported to regulate NF-κB-dependent transcription and multiple inflammasomes. Here we use a transgenic mouse-expressing POP2 controlled by its endogenous human promotor to study the immunological functions of POP2. Despite having significantly reduced inflammatory cytokine responses to LPS and bacterial infection, POP2 transgenic mice are more resistant to bacterial infection than wild-type mice. In a pulmonary tularaemia model, POP2 enhances IFN-γ production, modulates neutrophil numbers, improves macrophage functions, increases bacterial control and diminishes lung pathology. Thus, unlike other POPs thought to diminish innate protection, POP2 reduces detrimental inflammation while preserving and enhancing protective immunity. Our findings suggest that POP2 acts as a high-order regulator balancing cellular function and inflammation with broad implications for inflammation-associated diseases and therapeutic intervention.

Necroptotic debris including damaged mitochondria elicits sepsis-like syndrome during late-phase tularemia

Infection with Francisella tularensis ssp. tularensis (Ft) strain SchuS4 causes an often lethal disease known as tularemia in rodents, non-human primates, and humans. Ft subverts host cell death programs to facilitate their exponential replication within macrophages and other cell types during early respiratory infection (⩽72 h). The mechanism(s) by which cell death is triggered remains incompletely defined, as does the impact of Ft on mitochondria, the host cell’s organellar ‘canary in a coal mine’. Herein, we reveal that Ft infection of host cells, particularly macrophages and polymorphonuclear leukocytes, drives necroptosis via a receptor-interacting protein kinase 1/3-mediated mechanism. During necroptosis mitochondria and other organelles become damaged. Ft-induced mitochondrial damage is characterized by: (i) a decrease in membrane potential and consequent mitochondrial oncosis or swelling, (ii) increased generation of superoxide radicals, and (iii) release of intact or damaged mitochondria into the lung parenchyma. Host cell recognition of and response to released mitochondria and other damage-associated molecular patterns engenders a sepsis-like syndrome typified by production of TNF, IL-1β, IL-6, IL-12p70, and IFN-γ during late-phase tularemia (⩾72 h), but are absent early during infection.

Oxidized and Original article degraded mitochondrial polynucleotides (DeMPs), especially RNA, are potent immunogenic regulators in primary mouse macrophages

ABSTRACT Certain mitochondrial components can act as damage‐associated molecular patterns (DAMPs) or danger signals, triggering a proinflammatory response in target (usually immune) cells. We previously reported the selective degradation of mitochondrial DNA and RNA in response to cellular oxidative stress, and the immunogenic effect of this DNA in primary mouse astrocytes. Here, we extend these studies to assess the immunogenic role of both mitochondrial DNA and RNA isolated from hydrogen peroxide (HP) treated HA1 cells (designated “DeMPs” for degraded mitochondrial polynucleotides) using mouse bone marrow derived macrophages (BMDMs), a conventional immune cell type. DeMPs and control mitochondrial DNA (cont mtDNA) and RNA (cont mtRNA) were transfected into BMDMs and cell‐free media analyzed for the presence of proinflammatory cytokines (IL‐6, MCP‐1, and TNF&agr;) and Type I interferon (IFN‐&agr; and IFN‐&bgr;). Cont mtDNA induced IL‐6 and MCP‐1 production, and this effect was even greater with DeMP DNA. A similar response was observed for Type I interferons. An even stronger induction of proinflammatory cytokine and type 1 interferons was observed for cont mtRNA. However, contrary to DeMP DNA, DeMP RNA attenuated rather than potentiated the cont mtRNA cytokine inductions. This attenuation effect was not accompanied by an IL‐10 or TGF&bgr; anti‐inflammatory response. All DeMP effects were observed at multiple oxidant concentrations. Finally, DeMP production and immunogenicity overlaps with cellular adaptive response and so may contribute to cellular oxidant protection. These results provide new insight into the immunogenicity of mitochondrial polynucleotides, and identify new roles and selective consequences of cellular oxidation. HIGHLIGHTSControl mtDNA and mtRNA induce proinflammatory cytokines and type 1 interferon.These inductions are greater for oxidized degraded mitochondrial DNA (DeMPs).Conversely, DeMP RNA attenuates cytokine inductions by mtRNA.Effects occur at multiple oxidant concentrations and overlap with adaptive response.Control and DeMP mtRNAs are especially immunomodulatory.